UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE CIÊNCIAS EXATAS E DA TERRA INSTITUTO DE QUÍMICA PROGRAMA DE PÓS-GRADUAÇÃO EM QUÍMICA Nanopartículas de ouro e prata aplicadas a sistemas biológicos Heloiza Fernanda Oliveira da Silva Athayde Tese de Doutorado Natal/RN, novembro de 2019 Heloiza Fernanda Oliveira da Silva Athayde EFEITOS ANTI-INFLAMATÓRIO, ANTITUMORAL E ANALGÉSICO E DE NANO- PARTÍCULAS DE OURO E ANTIBACTERIANO DE NANOPARTÍCULAS DE PRATA Tese de doutorado submetida ao Programa de Pós- Graduação em Química do Instituto de Química da Universidade Federal do Rio Grande do Norte em cumprimento as exigências para a obtenção do título de Doutora em Química. Orientador: Prof. Dr. Luiz Henrique da Silva Gasparotto NATAL-RN 2019 Universidade Federal do Rio Grande do Norte - UFRN Sistema de Bibliotecas - SISBI Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial Prof. Francisco Gurgel De Azevedo - Instituto Química - IQ Athayde, Heloiza Fernanda Oliveira da Silva. Efeitos anti-inflamatório, antitumoral e analgésico de nanopartículas de ouro e antibacteriano de nanopartículas de prata / Heloiza Fernanda Oliveira da Silva Athayde. - Natal: UFRN, 2019. 160f.: il. Tese (Doutorado) - Universidade Federal do Rio Grande do Norte. Centro de Ciências Exatas e da Terra - CCET, Instituto de Química. Programa de Pós-Graduação em Química (PPGQ). Orientador: Dr. Luiz Henrique da Silva Gasparotto. 1. Nanopartículas de ouro - Tese. 2. HT-29 - Tese. 3. Nanopartículas de prata - Tese. 4. Escherichia coli - Tese. I. Gasparotto, Luiz Henrique da Silva. II. Título. RN/UF/BSQ CDU 54(043.2) Elaborado por FERNANDO CARDOSO DA SILVA - CRB-759/15 A meus pais, esposo e filho. AGRADECIMENTOS Agradeço ao Pai Celestial que sempre cumpre Suas promessas. Uma delas é este momento. Aos meus pais e aos demais familiares pelo apoio e oportunidades de estudo. Ao meu esposo por toda paciência em suportar com bom ânimo minha ausência em alguns momentos e por todo amor e incentivo para que eu conquistasse mais esse objetivo. E ao nosso filho Noah por mesmo me mostrar que sou capaz de muito mais do que já imaginei. Amo vocês! Ao professor Luiz Henrique da Silva Gasparotto pela orientação, pelas oportunidades, dedicação, pela confiança, apoio e paciência. A professora Mª Celeste de Melo por me receber em seu laboratório e pela oportunidade de aprendizado. Ao professor Raimundo Fernandes por também me receber em seu laboratório e me dar oportunidade de colaborar com seus alunos. Sou muito grata pela confiança! Ao professor Luiz Seixas das Neves pelos conselhos sempre edificantes. Aos meus queridos amigos. Vocês são os melhores! Gostaria de agradecer especialmente a:  Fernanda, Leomir e Camilo por sempre atenderem aos meus pedidos, tais como ficar até mais tarde, caronas, por me ouvirem após cada momento de frustração e de conquista, por suas palavras de incentivo, por pensar juntos a CHEMOVECTOR.  Rayane, Isadora, Rubens e Eryka pela companhia e ajuda com os experimentos;  Ana Luiza, Rômulo e Vinícius pela boa amizade e auxílio nas discussões dos ensaios biológicos;  Aos demais integrantes do Grupo de Química Biológica e Quimiometria - QBQ e Laboratório de Eletroquímica e Nanopartículas Aplicadas - LENA pela boa convivência e apoio. A técnica de laboratório Ana e ao professor Hugo pela ótima recepção e auxílio durante o treinamento no laboratório de cultura celular. Luíza e a professora Viviane pela recepção e confiança nas ideias que levei para discutirmos. Ao Celso pelas análises de TEM, ao técnico Joadir por sempre atender com atenção e confiança para o uso do fluorímetro, aos integrantes do Laboratório de Bioquímica pela água ultrapura, ao técnico Antônio Marcos por auxiliar nas análises de FTIR-ATR, ao professor Edgar Moraes por me ajudar com os questionamentos quimiométricos. “Se eu enxerguei mais longe, foi porque me apoiei sobre os ombros de gigantes”. Isaac Newton Por vezes sentimos que aquilo que fazemos não é senão uma gota de água no mar. Mas o mar seria menor se lhe faltasse uma gota. Madre Teresa de Calcutá RESUMO SILVA, H. F. O. Nanopartículas De Ouro E Prata Aplicadas A Sistemas Biológicos. [Gold and Silver Nanoparticles Applied to Biological Systems]. 2019. Tese (Doutorado em Química) – Instituto de Química, Universidade Federal do Rio Grande do Norte, Natal, 2019. A neoplasia e a resistência bacteriana estão entre os principais desafios na área biomédica atualmente, cenário este que demanda estudo e desenvolvimento de tratamentos alternativos. Nanopartículas metálicas tem se mostrado promissoras em aplicações dessa natureza. Esta tese apresenta aplicações de nanopartículas de ouro e prata (NanoAu e NanoAg, respectivamente) a sistemas biológicos. NanoAu são consideradas como potenciais plataformas para drug delivery. Entretanto, é essencial o entendimento da interação dessas partículas com os organismos vivos antes da sua utilização como nanocarreadores. Assim, o primeiro estudo apresenta a síntese e caracterização de NanoAu e a investigação dos efeitos anti-inflamatório, analgésico e antitumoral, além da biodistribuição das mesmas e o seu efeito sobre vários tipos de tecidos. As NanoAu foram sintetizadas por meio de uma rota de baixo impacto ambiental e caracterizadas com microscopia eletrônica de transmissão (TEM) e espectroscopia na região do UV-Visível (UV-vis). Posteriormente, células HT-29 foram expostas às NanoAu e avaliou-se a apoptose por meio da atividade da caspase-3. Em relação a ensaios in vivo, NanoAu foram administradas a camundongos Swiss fêmeas e ratos machos para posterior avaliação das suas propriedades anti-inflamatórias e analgésicas. A biodistribuição das NanoAu e seu impacto sobre os tecidos foram estudados por espectroscopia de UV-Vis e análise histopatológica, respectivamente. A apoptose das células foi dependente da dose para concentrações de NanoAu entre 40 g -1 -1 ml e 80 g ml (p < 0,05). A melhor atividade anti-inflamatória foi observada na dose -1 de 1500 g kg , o que ocasionou uma redução de 49,3% na migração de leucócitos. -1 NanoAu mostraram analgesia periférica na dose de 1500 g kg e foram encontradas no fígado, baço, rim e pulmão dos camundongos Swiss. O exame histopatológico revelou extravasamento de hemácias no pulmão. Já o segundo estudo, aborda a investigação do efeito sinérgico metabólico das NanoAg combinadas ao hiclato de doxiciclina. Para isso foi utilizada a bioespectroscopia ATR-FTIR de amostras bacterianas de Staphylococcus aureus. A PCA foi aplicada ao grupo de espectros mostrando um padrão de diferenciação entre as classes. Devido à complexidade dos dados obtidos (apresentarem bandas de sobreposição e bastante variância dentro da mesma classe), foi necessário o emprego da ferramenta de seleção de variáveis PLS-DA. Foram identificados que todos os antimicrobianos desse estudo interferem na síntese protéica. Valores de sensibilidade de 75%, 100% e 75 % para Controle, NanoAg e hiclato de doxiciclina respectivamente. Ainda foi determinado se havia ou não diferença estatística nos valores médios de absorbância, isso permitiu apontar em quais casos os biomarcadores eram mais expressos o que sugeriu a possível causa da sinergia do efeito antibacteriano das NanoAu combinadas com hiclato de doxiciclina. Os resultados obtidos nos dois estudos confirmam a potencialidade das nanopartículas sintetizadas em nosso grupo de pesquisa como possíveis nanocarreadores. Palavras-chave: Nanopartículas de ouro, HT-29,nanopartículas de prata, Escherichia coli. ABSTRACT SILVA, H. F. O. Gold and Silver Nanoparticles Applied to Biological Systems [Nanopartículas De Ouro E Prata Aplicadas A Sistemas Biológicos]. 2019. Tese (Doutorado em Química) – Instituto de Química, Universidade Federal do Rio Grande do Norte, Natal, 2019. Neoplasia and bacterial resistance are among the main challenges in the biomedical área. It requires study and development of alternative. Metal nanoparticles have shown promise in such applications. This thesis presents applications of gold and silver nanoparticles (NanoAu and NanoAg, respectively) to biological systems. NanoAu are considered as potential drug delivery platforms. However, understanding the interaction of these particles with living organisms before their use as nanocarriers is essential. Thus, the first study presents the synthesis and characterization of NanoAu and the investigation of anti- inflammatory, analgesic and antitumor effects, as well as their biodistribution and their effect on various tissue types. The NanoAu were synthesized by enviromental friendly route and characterized by transmission electron microscopy (TEM) and UV-Vis spectroscopy (UV-vis). Subsequently, HT-29 cells were exposed to NanoAu and apoptosis was evaluated by caspase-3 activity. Regarding in vivo assays, NanoAu were administered to female Swiss mice and male rats for further evaluation of their anti-inflammatory and analgesic properties. NanoAu biodistribution and its impact on tissues were studied by UV- Vis spectroscopy and histopathological analysis, respectively. Cell apoptosis was dose dependent for NanoAu concentrations between 40 µg ml -1 and 80 µg ml -1 (p <0.05). The best anti-inflammatory activity was observed at 1500 g kg-1, which led to a reduction of 49.3% in leukocyte migration. NanoAu showed peripheral analgesia at 1500 g kg-1 and were found in the liver, spleen, kidney and lung of Swiss mice. Histopathological examination revealed extravasation of red blood cells in the lung. The second study addresses the investigation of the metabolic synergistic effect of NanoAg combined with doxycycline hiclate, DO. ATR-FTIR biospectroscopy of bacterial samples of Staphylococcus aureus was used. PCA was applied to the spectrum group showing a pattern of differentiation between the classes. Due to the complexity of the obtained data (presenting overlapping bands and enough variance within the same class), it was necessary to use the PLS-DA variable selection tool. It was identified that all antimicrobials in this study interfere with protein synthesis. Sensitivity values of 75%, 100% and 75% for Control, NanoAg and doxycycline hyclate respectively. It was also determined whether or not there was a statistical difference in the mean absorbance values, which allowed us to indicate in which cases the biomarkers were more expressed, which suggested the possible cause of the synergy of the antibacterial effect of NanoAu combined with doxycycline hyclate. The results obtained in both studies confirm the potential of synthesized nanoparticles in our research group as possible nanocarriers. Keywords: Gold nanoparticles, HT-29, silver nanoparticles, Escherichia coli. LISTA DE ABREVIATURAS E SIGLAS ATR-FTIR – Espectroscopia de Reflectância Total Atenuada no infravermelho com transformada de Fourier (do inglês, Attenuated total reflectance Fourier transform infrared) BHI - Caldo de infusão de cérebro e coração. (do inglês, Brain Heart Infusion) CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior DNA – ácido desoxirribonucleico (do inglês, deoxyribonucleic acid) DO - Hiclato de doxiciclina E. coli - Escherichia coli FT – Transformada de Fourier (do inglês, Fourier transform) HAuCl4 – Ácido cloroáurico HT29 - Linhagem celular de câncer de cólon humano IHQ – Imuno-histoquímica IUPAC - União Internacional de Química Pura e Aplicada (do inglês, International Union of Pure and Applied Chemistry) MEC - Ministério da Educação MET - Microscopia Eletrônica de Transmissão MIR - Espectroscopia na região do infravermelho médio (do inglês, Medium Infrared Spectroscopy) Nano - Nanopartículas NanoAg - Nanopartículas de prata NanoAu - Nanopartículas de ouro NanoM - Nanopartículas metálicas PCA - Análise por componentes principais (do inglês, Principal Component Analysis) PLS- Mínimos quadrados parciais (do inglês partial least squares ) PLS-DA - Análise de discriminante pelos mínimos quadrados parciais (do inglês partial least squares discriminant analysis) PVP - Polivinilpirrolidona. RNA - Ácido Ribonucléico (do inglês, Ribonucleic acid) RPS - Ressonância de Plasmon de Superfície S. aureus - Staphylococcus aureus TSA - Ágar Triptona de Soja. (do inglês, Tryptic Soy Agar) UV-Vis - Espectroscopia na Região do UV-Visível SUMÁRIO Capítulo 1 INTRODUÇÃO GERAL .................................................................... 11 Capítulo 2 Anti-inflammatory, analgesic and anti-tumor properties of gold nanoparticles. R. F. de Araújo Júnior, A. A. de Araújo, J. B. Pessoa, F. P. Freire Neto, G. R. da Silva, A. L. C. S. L. Oliveira, T. G. de Carvalho, H. F. O. Silva, M. Eugênio, C. Sant‘Anna, L. H. S. Gasparotto. Pharmacological Reports, 2017, Vol. 69, 119- 129 ............................ 56 Capítulo 3 On the synergy between silver nanoparticles and doxycycline towards the inhibition of Staphylococcus aureus growth. H. F. O. Silva, R. P. de Lima, F. S. L. da Costa, E. P. Moraes, M. C. N. Melo, C.Sant‘Anna, M. Eugenio, L. H. S. Gasparotto. RSC Advances, 2018, Vol. ,8, 23578–23584 ....................................... 68 Capítulo 4 CONCLUSÕES E PERSPECTIVAS .................................................. 76 Apêndice A Environmentally compatible bioconjugated gold nanoparticles as efficient contrast agents for inflammation-induced cancer imaging Garcia, V. B.; de Carvalho, T.G.; da Silva Gasparotto, L. H.; da Silva, H. F. O.; de Araújo, A. A.; Guerra, G. C. B.; Schomann, T.; Cruz, L. J. ; Chan, A. B.; de Araújo Júnior, R. F.. Nanoscale Research Letters, v. 14, p.xx, 2019 ....................................77 Apêndice B Functionalization of gold nanoparticles with two aminoalcohol-based quinoxaline derivatives for targeting phosphoinositide 3-kinases (PI3K). J. Araújo, F. G. Menezes, H. F. O. Silva, D. S. Vieira, S. R. B. Silva, A.J. Bortoluzzi, C. Sant‘Anna, M. Eugenio, J. M. Neri, L. H. S. Gasparotto. New Journal of Chemistry, v. 43, p. 1803-1811, 2019 ....................... 90 Apêndice C Spherical neutral Gold nanoparticles improve anti-inflammatory response, oxidative stress and fibrosis in alcohol-methamphetamine- induced liver injuryin rats. T. G. Carvalho, V. B. Garcia, A. A. Araújo, L. H. S. Gasparotto, H. F. O. Silva, G. C. B. Guerra, E. C. Miguel, R. F. C. Leitão, D. V. S. Costa, L. J Cruz, A. B. Chan, R. F. de Araújo Jr. International Journal of Pharmaceutics, 2018, Vol. 548, 1–14 ....100 Apêndice D Lipopolysaccharides and peptidoglycans modulating the interaction of Au nanoparticles with cell membranes models at the air-water interface. da Silva RLCG, da Silva HFO, da Silva Gasparotto LH, Caseli L. Biophysical Chemistry, 2018,Vol.238, 22-29 ................................... 115 Apêndice E Apoptosis in human liver carcinoma caused by gold nanoparticles in combination with carvedilol is mediated via modulation of MAPK/Akt/mTOR pathway and EGFR/FAAD proteins. Araújo RF Jr, Pessoa JB, Cruz LJ, Chan AB, De Castro Miguel E, Cavalcante RS, Brito GAC, Silva HFO, Gasparotto LHS, Guedes PMM, Araújo AA. International Journal of Oncology, 2018, Vol. 52, 1, 189-200……. 124 Apêndice F How the Interaction of PVP-stabilized Ag Nanoparticles with Models of Cellular Membranes at the Air-Water Interface is Modulated by the Monolayer Composition R. L. C. G. Silva, H. F. O. Silva, L. H. S. Gasparotto, L. Caseli. Journal of Colloid and Interface Science, 2018, Vol. 512, 792-800. 137 Apêndice G Dual Role of a Ricinoleic Acid Derivative in the Aqueous Synthesis of Silver Nanoparticles. I. D. Costa, A. O. Wanderley Neto, H. F. O. Silva, E. P. Moraes, E. T. D. Nóbrega, C. Sant‘Anna, M. Eugenio, L. H. S. Gasparotto. Journal of Nanomaterials, 2017, Vol. 2017, 1-8 .............................. 147 Anexo I MINI-CURRICULUM ...................................................................... 157 Anexo II CARTA DE CONCLUSÃO ............................................................. 160 Capítulo 1 - Introdução geral 1 REVISÃO BIBLIOGRÁFICA. .................................................................. 11 2 ORGANIZAÇÃO DA TESE. ..................................................................... 34 3 OBJETIVOS ............................................................................................... 36 4 METODOLOGIAS EMPREGADAS ......................................................... 36 Referências. ................................................................................................ 46 1- REVISÃO BIBLIOGRÁFICA 1.1 Nanomedicina A utilização de nanopartículas em aplicações biomédicas é conhecida desde o final da década de 1970. Entretanto o termo nanomedicinasó passou a ser usado na virada do presente século. A Fundação Européia para a Ciência definiu o termo da seguinte forma: A nanomedicina utiliza ferramentas de tamanho nano para o diagnóstico, a prevenção e o tratamento de doenças e para aumentar a compreensão da complexa base fisiopatológica da doença. O objetivo final é melhorar a qualidade de vida [1]. De acordo com a definição acima, as aplicações da nanotecnologia na biomedicina podem ser divididas em três grandes áreas conforme exposto na tabela a seguir: Tabela 1: Aplicações da nanomedicina. Aplicações da nanomedicina Área I Diagnóstico, biosensores e próteses externas. Área II Agentes de imagem e técnicas de monitorização que atuem nas células. Área III Inovação de biomateriais para drug delivery (a administração pode ser ex vivo ou não, incluindo implantes) e engenharia tecidual. Fonte: Adaptada da Referência [5]. Resumidamente, as tecnologias são de diagnóstico, agentes de imagem e administração de medicamentos com nanopartículas [2,3]. A proposição de soluções para cada uma dessas áreas é a utilização da nanotecnologia na investigação de processos fisiopatológicos, ou seja, a obtenção de nanoestruturas com tamanho similares aos das moléculas biológicas com o objetivo de investigar sua interação com células humanas. Há estudos voltados para a melhoria das propriedades dos fármacos, exemplos disso são a solubilidade, a 1 1 estabilidade e a disponibilidade. Diversos estudos relatam a aplicação de nanoestruturas na administração de fármaco que vão desde a radiofreqüência a novos sistemas de drug delivery [4- 6]. Duncan e Gaspar [5] destacaram em seu trabalho de revisão que dependendo do tipo de material suas propriedades físico-químicas são bem distintas e isso influencia diretamente na faixa de tamanho a ser utilizada, e consequentemente no alvo da aplicação. Assim, torna-se essencial a compreensão dos tipos de nanomateriais que podem ser empregados. A Figura 1 apresenta um esquema dos principais nanomateriais e uma ideia dos tamanhos relativos de nanomedicamentos. Figura 1: Esquema mostrando as principais classes de nanomedicamentos de primeira geração em ensaio e uso clínico de rotina. A inserção dá uma ideia dos tamanhos relativos dos nanomedicamentos, já que os desenhos em cada região não estão em escala. Por exemplo, lipossomas, nanocristais, e algumas nanopartículas poliméricas são ≥ 100 nm, e algumas nanopartículas poliméricas, conjugados de polímero e os dendrímeros estão no intervalo 5-25 nm. Fonte: Adaptada da Referência [5]. Tais nanomedicamentos geralmente atendem a faixa de tamanho de 1 a 1000 nm [7]. Entretanto, nem todos os nanomateriais conservam o desempenho diferenciado em toda essa faixa. Cada material apresentará um tamanho crítico onde acima dele não são observadas as propriedades peculiares que os distinguem dos de maiores tamanhos, e consequentemente os efeitos biológicos. Em alguns casos, Toxicidades clínicas, incluindo efeitos colaterais, têm sido amplamente estudadas e algumas vezes apontam para a individualização do paciente. Ainda dentro da temática dos nanomedicamentos, um termo mais recente foi introduzindo que são os teranósticos, que remete a um nanomedicamento que apresenta as propriedades multimodais [7-9]. As nanopartículas de ouro (NanoAu) são representantes desse termo, 1 2 especialmente nas proposições relacionadas ao câncer [10,11]. Já as nanopartículas de prata (NanoAg) são mais utilizadas visando a diminuição da resistência bacteriana [12, 13]. Nesta tese apresentaremos um trabalho envolvendo o estudo de células HT-29 expostas às NanoAu (capítulo 2) e outro envolvendo NanoAg combinadas ao hiclato de doxiciclina (DO) em contato com células bacterianas do tipo Staphylococcus aureus (capítulo 3). Ambas nanopartículas foram obtidas via síntese química com condições que atendem a demanda da química verde, tais como redução/ eliminação do uso de reagentes tóxicos e energia. No próximo tópico apresentaremos algumas características das nanopartículas e as diferentes formas de obtenção das mesmas. 1.2 Métodos de obtenção das NanoAu e NanoAg De acordo com o Golden Book da IUPAC (1972) [14], um sistema contendo partículas ou moléculas que possuam uma de suas dimensões entre 1,0 nm e 1,0 m, ou que tenham descontinuidades com distâncias dessa ordem dispersas em um meio é chamado de colóide. Assim, podemos classificar a solução homogênea resultante da síntese de NanoAu e de NanoAg, que serão apresentadas neste trabalho, como uma solução coloidal. Partículas metálicas em regime nanométrico passam a apresentar propriedades físico-químicas bem diferentes do material bulk (sólido estendido), visto que sua razão superfície/volume ou fração de átomos superficiais é bem elevada quando comparado com o bulk do mesmo metal. Nanopartículas de metais nobres (ouro, prata e cobre), por exemplo exibem bandas na região visível do espectro eletromagnético, chamadas de bandas de plasmon. Tal fenômeno é atribuído à oscilação coletiva dos elétrons de condução quando estes são submetidos à radiação eletromagnética de comprimento de onda maior do que a partícula. Resumidamente, a incidência de um campo elétrico homogêneo sobre a nanopartícula metálica, resulta no deslocamento dos seus elétrons no sentido contrário ao do campo elétrico da onda incidente. Tal deslocamento das cargas promove a indução de um dipolo elétrico na nanopartícula. Por sua vez, o dipolo induzido promove o aparecimento de um campo elétrico restaurador na partícula, o qual tem a função de recompor o Figura 2: Ilustração esquemática do fenômeno de RLPS para nanopartículas esféricas. Fonte: Adaptado da Referência [17]. 1 3 equilíbrio dado pela distorção das cargas. Esta força restauradora e a indução do dipolo, quando vinculadas, geram a Ressonância Localizada de Plasmon de Superfície, RLPS. A Figura 2 ilustra o dipolo elétrico induzido e a força restauradora criada devido a separação de cargas na partícula. Com base nisso, pode-se concluir que a banda de LPS é fortemente dependente dos fatores: a) tamanho das partículas, b) forma (esfera, bastões, triângulos, pirâmides, cubos); c) distribuição de tamanho, d) meio no qual as NanoMs estão imersas (viscosidade, constante dielétrica, íons e/ou moléculas coordenadas a superfície). Uma propriedade das NanoMN é a cor característica que advém das oascilações coletivas coerentes dos elétrons livres na banda de condução. No caso das NanoM utilizadas neste trabalho (todas esféricas), a cor observada foi vermelho para as NanoAu e amarelo castanho para as NanoAg. A espectroscopia na região do Ultravioleta Visível, UV-Vis se torna uma ferramenta essencial no acompanhamento da obtenção de nanopartículas de metais nobres e nas inferências iniciais a respeito dos fatores e propriedades citados [16-20], As NanoM, de maneira geral, podem ser obtidas por dois métodos principais o top- down e botton-up (Figura 3). O primeiro consiste na manipulação do macromaterial e por meio de processos físicos, fragmenta-se o material até que estejam em escala nanométrica. E o segundo, fundamenta-se em reações químicas e se usa percursores moleculares ou atômicos para a aquisição de nanopartículas. Este método é o mais utilizado, visto que é mais simples e permite um controle maior dos parâmetros do processo tal como o tamanho e a forma das NanoM, além da possibilidade de obtenção de partículas muito menores que as obtidas pelo método top-down [21]. Dentro dos métodos bottom-up a síntese coloidal é a mais utilizada por ser mais barata. São muitos os trabalhos na literatura que funcionam como prova de conceito para tal [22-24]. Esta síntese baseia-se na formação de átomos do metal por meio da utilização de um agente químico redutor em solução. No instante que são formados, os átomos metálicos passam por processo de nucleação e posterior crescimento para só depois resultar em nanopartículas. É importante frisar que é necessária a presença de um agente químico estabilizante, visto que sistemas coloidais são essencialmente instáveis termodinamicamente devido à razão volume/superfície que influencia a agregação e posterior coalescência, gerando assim partículas maiores. Essa estabilização pode se dar de maneira eletrostática ou eletroestérica, são exemplos íons e polímeros, respectivamente [22,25]. 1 4 Figura 3: Principais métodos de obtenção de nanopartículas metálicas (NanoM). Metal bulk Fonte: Autor. As rotas clássicas utilizam como agentes redutores íons citrato, borohidreto e hidrazina [22,26]. No trabalho desenvolvido foi empregado o método desenvolvido por Gasparotto e colaboradores [27] que supera a utilização de agentes redutores e estabilizantes tão tóxicos como o íon borohidreto e também condições tão drásticas de temperatura. Esta rota utiliza glicerol em meio básico como agente redutor e a polivinilpirrolidona (PVP) como agente estabilizador sob temperatura ambiente. Este método se mostrou válido para a obtenção tanto das NanoAu, quanto das NanoAg que foram obtidas não só nos estudos que serão compartilhados aqui, mas também nos demais realizados em nosso grupo. 1.2.1 Nanopartículas de ouro Em 1856 o cientista Michel Faraday sintetizou uma solução de NanoAu. O interessante é que se deu de forma acidental enquanto ele estava na montagem de folhas finas de ouro em lâminas de microscópios. O mais incrível é que a solução coloidal de ouro obtida naquela época continua estável mesmo após mais de 160 anos. O que intriga é não ser possível saber o porquê de tamanha estabilidade, uma vez que não é possível abrir os frascos sem danificá-los. Isso aconteceu mesmo antes do termo ―nano‖ ser introduzido na ciência [28]. As NanoAu são certamente as nanopartículas mais estudadas. Seu uso tão expressivo é atribuído a fácil preparação de colóides, bem como sua bioafinidade. Como uma nanopartícula metálica, as NanoAu podem ser obtidas com diferentes formas, tamanhos e combinadas a outros materiais. Como exemplos, temos os nanobastões de ouro, nanoconchas de ouro-sílica, as nanocelas e as ocas [29, 30]. O estudo oficial utilizando as NanoAu já é reportado a bastante tempo, Horisberger mostrou que pelo fato das NanoAu possuírem a propriedade ópticas peculiares elas poderiam ser conjugadas a diferentes moléculas que seriam marcadores [29]. Roth publicou um trabalho de revisão cujo título era ―O aniversário de prata do ouro: 25 anos do sistema de marcação de ouro coloidal para imuno-histoquímica e histoquímica‖, neste trabalho de revisão ele aborda a conjugação de NanoAu a proteínas e lectinas que permite a localização de receptores [31]. Já no início dos anos 2000, a literatura mostrou que sais de ouro tem 1 5 açãosignificativa no tratamento de artrite reumatóide, em geral a via de administração indicada é a intramuscular [32]. De todos os estudos já realizados com as NanoAu, a terapia do câncer é a aplicação mais relatada. Fazendo o cruzamento no portal de periódicos da CAPES/MEC de “gold nanoparticles and cancer” até julho de 2019 vê-se que dos 200.935 artigos publicados envolvendo apenas tema “gold nanoparticles”, 48.727 (equivalendo a 24,2%) tinham o câncer como aplicação. Assim, vê-se que o desenvolvimento e estudo de NanoAu nessa área ainda é bastante relevantes. No primeiro trabalho que será compartilhado nesta tese (capítulo 2), foram utilizadas nanopartículas de ouro esféricas e estudados seus efeitos analgésico, anti- inflamatório e anticâncer, além de sua biodistribuição em diversos tecidos [33]. 1.2.2 Nanopartículas de prata Segundo relatos da literatura, nanopartículas de prata foram utilizadas sem nenhuma intenção de obter nanomateriais por aproximadamente 1000 anos. Elas eram apenas incorporadas em matrizes vítreas para obtenção de vitrais coloridos. Entretanto, a real descoberta das NanoAg se deu há mais de 120 anos [34]. Em 1889, Lea MC sintetizou a partir de citrato nanopartículas de prata com estabilidade coloidal e diâmetro médio para as partículas entre 7,0 e 9,0 nm. A prata coloidal era utilizada no tratamento de doenças e infecções antes da descoberta da penicilina, em 1928 [34-36]. Em 1954, nos EUA, foi registrada nanopartículas de prata como biocida e desde então este material tem sido utilizado com essa finalidade. A aplicação de partículas de prata em regime nano aumentou a eficácia no controle de microrganismos patógenos (bactérias e fungos), uma vez que passa a apresentar uma grande área superficial das nanopartículas e, consequentemente, maior contato com os microrganismos [37]. Ou seja, aumenta a disponibilidade de íons liberado que é tida como um dos fatores responsáveis pela inibição do crescimento e/ou morte celular [34]. Abordagens mais recentes apontam para uma possível sinergia do efeito bacteriano e/ou bactericida quando é utilizada NanoAg combinadas a antibióticos [38- 41]. Realizando busca semelhante ao item anterior no portal periódico da CAPES/MEC, com o cruzamento dos termos “silver nanoparticles and bacteria” até julho de 2019 vê-se que dos 132.290 artigos publicados envolvendo apenas tema “silver nanoparticles”, 25.776 (equivalendo a 19,5%) tinham bactérias como alvo da aplicação. Assim, nesse caso também se constata que o desenvolvimento e estudo de NanoAg nessa área ainda são bastante relevantes. No segundo trabalho que será compartilhado nesta tese (capítulo 3), foram utilizadas nanopartículas de prata esféricas, sintetizadas pela mesma rota utilizada nas NanoAu, combinadas ao DO e avaliada o efeito no metabolismo de bactérias do tipo Staphylococcus aureus quando expostas ao combinado [42]. 1 6 1.3 Problemáticas abordadas nos estudos 1.3.1 Estudo com as NanoAu (capítulo 2) 1.3.1.1 Câncer colorretal: uma ponderação Dentre os diagnósticos de neoplasias, o câncer de intestino grosso (câncer colorretal ou de cólon) é o terceiro mais comumente diagnosticado. Em 2018 o número de novos casos foi de 33.750, sendo 17.380 homens e 16.370 mulheres no Brasil [43]. Isso poderia ser modificado se a cultura da prevenção fosse adotada (tais como hábitos saudáveis de alimentação e realização de exames), já que a maioria dos casos é iniciada por meio de pólipos que são tumores pré- cancerosos no colón e no reto. Entretanto nem todos os pólipos desenvolverão câncer, mas retirando-os após identificação no processo de triagem garante uma prevenção eficaz. Além de que se detectado nos estágios iniciais o tratamento é mais eficiente [44]. O colón pode ser dividido em quatro partes, conforme mostrado na Figura a seguir: Figura 4: Anatomia do Cólon e Reto. Adaptada. Fonte: Adaptada da referência [44]. O cólon e o reto constituem o chamado intestino grosso. Voltando aos pólipos, o mais comum nesse tipo de segmento é o adenomatoso ou adenoma que nada mais é do que células glandulares que são responsáveis pela produção de muco para a lubrificação [45, 46]. Trabalhos na literatura apontam que pelo menos um terço da população irá desenvolver esse tipo de pólipo, mas menos de 10% se tornarão tumores malignos [47, 48]. O câncer será mais invasivo à medida que ele se desenvolva por meio de células glandulares denominadas adenocarcinoma que correspondem a 96% dos casos de câncer colorretal. Esse tipo de neoplasia é de desenvolvimento lento, geralmente na faixa de 10 a 20 anos [49] o que remete mais uma vez a 1 7 importância da prevenção e diagnóstico por meio de triagem. A Figura 5 apresenta os principais fatores que causam o câncer colorretal: Figura 5: Principais causas do câncer colorretal. Fonte: Autor. A maioria envolve nossas escolhas diárias, mas quando não podemos mais prevenir passamos a focalizar no que ocorre após o diagnóstico do câncer colorretal que é o tratamento. Em 2017, 18867 pessoas morreram vítimas desse tipo de câncer, dos quais 9207 eram homens e 9660 eram mulheres [43]. Isso nos alerta para a necessidade de estudos na área do tratamento dessa doença. Para esse tipo de neoplasia o tratamento indicado, na maioria dos casos, é a cirurgia para remoção do(s) tumor (es) e uma terapia adjuvante. Independente da ordem das etapas de tratamento, temos que a cirurgia, quimioterapia, radioterapia e a terapia-alvo proporcionam efeitos adversos [44]. Isso instiga a busca para o desenvolvimento e prospecção de novos materiais e métodos com o objetivo de proporcionar um tratamento menos agressivo e mais eficiente. Nosso trabalho apresentado no capítulo 2 desta tese se encaixa nessas iniciativas. Células HT29 Para o estudo da ação de diferentes candidatos a quimioterápicos são utilizados modelos tanto celulares quanto modelos animais. Com o objetivo de economia de ensaios com animais e pela conveniência de poder fazer testes em batelada com o controle das condições ambientais, os primeiros testes são realizados com modelos celulares [50]. Com relação aos testes para avaliação do efeito anticâncer de cólon, um dos modelos celulares mais utilizados é a linhagem HT-29 [51-54]. Fogh e Trempe isolaram tal linhagem de um carcinoma de cólon humano e foram mantidas tanto as características do tecido normal, quanto dos receptores hormonais [55, 1 8 56]. No estudo que será apresentado no capítulo 2 foi utilizada a linhagem celular de adenocarcinoma de cólon humano HT-29 para o estudo do efeito antitumoral das NanoAu sintetizadas pela rota ambientalmente correta utilizada em nosso laboratório. Nesse mesmo estudo também foram avaliados os efeitos analgésico e anti-inflamatório das NanoAu utilizando também modelo animal. 1.3.2 Estudo com as NanoAg (capítulo 3) 1.3.2.1 Resistência bacteriana e bactérias do tipo Staphylococcus aureus No mundo, 20% dos casos novos de câncer são associados a infecções virais e bacterianas. Esse fato já é o suficiente para justificar todo investimento em pesquisa e desenvolvimento de estratégias para o combate a resistência bacteriana. Entretanto, a problemática é preocupante, uma vez que de maneira geral o sistema de saúde tem enfrentado casos de infecções bacterianas cada vez mais resistentes [57]. Uma bactéria é considerada resistente quando níveis terapêuticos da droga perdem a eficácia no processo de morte ou controle do seu crescimento. O uso indiscriminado de antibióticos colabora para esse fenômeno, assim cepas cada vez menos sensíveis vêm surgindo. O governo brasileiro, por meio do ministério da saúde, vem investindo em ações contra resistência aos antimicrobianos mais incisivamente desde 2018. Mas esse problema não é só nacional, na realidade faz parte de um desafio de saúde pública mundial [58, 59, 60]. A resistência antibiótica tanto pode ser um processo natural (ou seja, algumas bactérias são naturalmente resistentes a determinados antibióticos) quanto pode ser induzida por meio de uma mutação genética ou contraindo resistência de outra bactéria. Tal fenômeno se dá quando uma bactéria assimila elementos genéticos móveis de outra bactéria, tais como os plasmídeos (DNAs separados do cromossomo, com habilidade de replicação) e os transposons (regiões de DNA que são móveis). Os processos dessas mutações sejam naturais ou adquiridas podem de dar das seguintes maneiras: (a) com redução da permeabilidade da membrana (produzindo o biofilme), evitando a entrada do composto na célula; (b) coma modificação do alvo do antibiótico; (c) com o desenvolvimento de uma via bioquímica resistente, que pode ser promovida por transferência genética; (d) com a transformação enzimática, produzindo enzimas que atuam na composição química dos medicamentos, inativando-os; (e) o efluxo que se trata da retirada dos antibióticos de dentro da célula [61, 62, 63]. Os antimicrobianos que a serem propostos necessitam contornar um ou mais mecanismos dos citados acima. 1 9 Bactérias e Staphylococcus aureus Trata-se de organismos unicelulares procariontes que podem ser encontrados em colônias ou isolados. A classificação procariótica é pelo fato desse tipo de célula não apresentar núcleo definido nem outras organelas ligadas à membrana. O inverso trata-se de células eucarióticas. Outras peculiaridades das bactérias é que sua reprodução é do tipo assexuado, por meio de mitoses sucessivas. Esses microrganismos apresentam tamanho na faixa de 1,0 a 5,0 m. Eles podem ser classificados como gram-positivos e gram-negativos. Tal atribuição é dada de maneira empírica, onde o resultado de um ensaio realizado por um método denominado de Gram o classificará como positivo ou negativo. Esse ensaio consiste no tratamento de bactérias com dois corantes, um violeta e outro rosa. Bactérias que absorvem ambos são classificadas como gram-positivas, e aquelas que absorvem apenas a rosa são ditas gram-negativas. A divergência nos testes é atribuída à composição da parede celular bacteriana. O diagrama simplificado do envelope celular de bactéria gram-positiva e gram-negativa apresentado na Figura 6 ajuda a compreender que as gram- positivas possuem uma camada espessa de peptídeoglicanos , enquanto as gram-negativas possuem uma camada mais fina envolta por uma segunda membrana lipoproteica [64, 65]. As representantes mais comuns são: Escherichia coli (E. coli), gram-negativa e a Staphylococcus aureus, (S. aureus), gram-positiva. No estudo que será apresentado no capítulo 3, foi avaliada a resposta metabólica de S. aureus frente às NanoAg combinadas ao DO. Essa bactéria é normalmente encontrada em indivíduos saudáveis, especifi- Figura 6: Diagrama simplificado do envelope celular de bactéria gram-positiva e gram-negativa. Fonte: Adaptado da Referência [57]. camente nas regiões da pele e das fossas nasais. S. aureus, cocos gram-positivos, enquanto patógenos são capazes de provocar desde pequenas infecções na pele até graves infecções que podem levar a óbito. 2 0 Algumas dessas infecções são: pneumonia, endocardite, osteomielite, septicemia, entre outros. Essa patogenia é atribuída ao uso indiscriminado de antibióticos, como discutido anteriormente quando tratamos das causas da resistência bacteriana, e também ao fato dessa bactéria passar a habitar tecidos que não lhe são comuns de habitar. Isso causa infecções muito graves, especialmente quando entra na corrente sanguínea a que geralmente leva a infecção generalizada (ou sepse) e a óbito. Com o desenvolvimento dos antibióticos, como discutimos, acreditava-se ter encontrado uma solução para o fim dessas infecções, no entanto, estudos mostram que o S. aureus é uma das bactérias que possui resistência a diversos antimicrobianos, o que tem estimulado novas pesquisas a fim de solucionar essa problemática [66-70]. 1.4 Técnicas Instrumentais 1.4.1 Técnicas Espectroscópicas O espectro completo da radiação eletromagnética (vide Figura 7) é o intervalo completo da radiação eletromagnética que contém as ondas de rádio, as micro-ondas, o infravermelho, os raios X, a radiação gama, os raios violeta e a luz visível ao olho humano. Diferentes substâncias Figura 7: Esquema do espectro eletromagnético. Fonte: Autor. interagem de forma distinta com a radiação eletromagnética, assim a compreensão desse espectro permite utilizar a radiação para o estudo de caracterização de diversos materiais 2 1 relacionando o que se deseja observar/estudar com a quantidade de energia radiante a ser utilizada. Com base nisso, diversos equipamentos instrumentais de análise e metodologias surgiram e continuam a ser aperfeiçoados. Dentre as técnicas de caracterizações utilizadas nos trabalhos dessa tese estão as espectroscopias. A IUPAC em seu Golden Book [71] define espectroscopia como: O estudo de sistemas físicos pela radiação eletromagnética com a qual eles interagem ou que eles produzem. Espectrometria é a medida de tais radiações como um meio de obter informações sobre os sistemas e seus componentes. Em certos tipos de espectroscopia óptica, a radiação origina-se de uma fonte externa e é modificada pelo sistema, enquanto que em outros tipos, a radiação se origina dentro do próprio sistema. As técnicas espectroscópicas utilizadas nesse trabalho foram: UV-Visível e ATR-FTIR. Mais a frente elas serão fundamentadas, e o seu uso em cada ensaio dos trabalhos justificado. Basicamente, cada substância apresentará um perfil espectral característico. Para aqueles obtidos na região do UV-Visível, isso acontece por causa dos grupos denominados cromóforos que é definido pela IUPAC [71] como: A parte (átomo ou grupo de átomos) de uma entidade molecular na qual a transição eletrônica responsável por uma determinada banda espectral é aproximadamente localizada. 1.4.1.1 Espectroscopia no UV-Visível A espectroscopia de UV-Vis baseia-se na absorção de radiação, por uma amostra, na região UV-Vis-IVpróximo (190-1100 nm). Certos compostos têm a capacidade de absorver radiação nessa faixa do espectro eletromagnético, passando para um estado excitado. Na figura 8 são apresentados dois espectros de absorção com os máximos de absorção, tanto de molécula(DO) como de nanopartículas metálica (NanoAg). Quando se trata de nanopartículas metálicas, esses máximos de absorção são definidos pelo fenômeno da Ressonância de Plasmons de Superfície (RPS) que foi apresentado no item 1.2. 2 2 Figura 8: Espectros no UV-Visível simulados de (A) Hiclato de doxiciclina e de (B) NanoAg, ambas em solução aquosa. A B Fonte: Autor. A Figura 9 apresenta um esquema da absorção de luz por uma solução de concentração C acondicionada numa cubeta. A absorção de radiação UV ou visível por uma espécie atômica Figura 9: Esquema da absorção de luz e as etapas de excitação e relaxação eletrônica. Fonte: Autor. ou molecular pode ser considerada como um processo que ocorre em duas etapas: excitação e relaxação, elas foram equacionados na figura acima. São três tipos de transições eletrônicas: a) elétrons p, s e n (moléculas e íons inorgânicos), b) elétrons d e f (íons de metais de transição), c) transferência de carga (complexos metal-ligante). Se N* sofrer decomposição ou formar novas espécies, o processo é chamado de reação fotoquímica. Como resposta, a técnica fornece o quanto de luz é absorvido pela amostra, por meio da medida da luz transmitida. Para a análise quantitativa da absorção de luz é utilizada a relação apresentada na Figura 10. 2 3 Figura 10: Esquema da absorção de luz por uma espécie atômica ou molecular e análise quantitativa por meio da relação da quantidade de luz absorvida e transmitida. Fonte: Autor. A absorbância é dependente da concentração da espécie absorvedora. Com base na relação acima, é possível verificar que quanto maior a concentração do analito, maior a absorbância. Entretanto, o valor dessa capacidade intrínseca também é dependente do espaço físico ocupado pela amostra (caminho óptico) e da absortividade molar de cada substância. A última nada mais é do que a habilidade que uma espécie apresenta em absorver uma radiação em determinada frequência. A relação entre absorção e concentração pode ser realizada com base na Lei de Lambert-Beer (vide esquema da Figura 11). Observações realizadas pelos cientistas Johann Heinrich Lambert (1728 – Figura 11: Relação entre absorção e concentração (Lei de Lambert-Beer) e relação linear entre Absorbância e Concentração se as medidas são feitas em condição de caminho óptico constante (aplicação da equação da reta para determinar o valor de  para obter o de . Fonte: Autor. 2 4 1777) e August Beer (1825 – 1863) deram origem a tal relação. O primeiro, em seus estudos observou que a intensidade da luz transmitida por um meio absorvedor proporcional à espessura do meio pelo qual a luz passava. E o segundo, observou que a intensidade da luz transmitida por um meio absorvedor era proporcional à concentração da espécie absorvedora. Como toda lei, essa também apresenta limitações e elas são: só é -1 válida para soluções diluídas (C < 0,01 mol L ), necessita de radiação monocromática e não devem estar presentes na mesma solução mais de uma substância absorvedora de luz [72-74]. Isso quando buscamos fazer a relação de proporcionalidade entre concentração e absorbância. Em nossos estudos utilizamos os dados obtidos via espectroscopia na região do UV-Visível de maneira qualitativa, onde se buscou observar o perfil espectral tanto das soluções de NanoAu quanto as de NanoAg a medida que a síntese progredia. Além de utilizar também para avaliar a estabilidade dessas soluções após o ajuste do pH do meio. Outro uso dessa técnica foi observar o resultado da combinação de DO com as NanoAg antes de expô-las às bactérias S. aureus. Como discutido, a espectroscopia UV-Visível (UV-Vis) mede a extinção (dispersão + absorção) da luz que passa através de uma amostra. As nanopartículas têm propriedades ópticas únicas que são sensíveis ao tamanho, forma, concentração, estado de aglomeração e índice de refração próximo à superfície das nanopartículas, o que torna o UV-Vis uma ferramenta valiosa para identificar, caracterizar e estudar nanomateriais. 1.4.1.2 Espectroscopia na infravermelho médio (MIR) O fundamento desta técnica é similar ao discutido no item anterior, diferenciando apenas na energia da radiação que é incidida na amostra. Nesse caso ela abrange a faixa de -1 -1 4000 cm a 400 cm do espectro eletromagnético (também conhecida como região fundamental ou infravermelho médio, MIR). Da mesma maneira que a espectroscopia no UV-Visível, essa técnica pode ser utilizada par relações quantitativas desde que respeite a Lei de Lambert-Beer. Os dados obtidos por essa técnica geram um gráfico de -1 Transmitância (%) X Número de onda (cm ), que pode ser facilmente convertido em -1 Absorbância (%) X Número de onda (cm ) conforme a relação apresentada anteriormente na figura 11. Esse espectro é obtido quando um feixe de radiação com energia na faixa mencionada é absorvida em frequências específicas por moléculas que possuam a capacidade de ter uma alteração no seu momento de dipolo. Essa alteração se dá pelo fato 2 5 de que as ligações químicas da espécie em análise vibram em frequências específicas. Isso está diretamente relacionado com os níveis de energia da molécula, nesse caso os níveis vibracionais. Essas vibrações específicas são diretamente dependentes da geometria da molécula, das massas dos átomos envolvidos, do formato da superfície de energia potencial da molécula e, algumas vezes do acoplamento vibrônico (também conhecido como efeito pseudo-Jahn-Teller). As ligações podem vibrar de seis modos: estiramento simétrico, estiramento assimétrico, tesoura, torção, balanço e rotação, conforme apresenta- do na Figura 12. Essas diversas possibilidades de vibração, que são dependentes da composição e estrutura química, fazem com que cada molécula exiba um espectro singular podendo ser feita uma analogia com a impressão digital. Para que os espectros sejam cada vez mais claros, a maior parte dos equipamentos tem utilizado um sistema óptico (chamado interferômetro de Michelson) aliado a transformações matemáticas (conhecida como transformada de Fourier, sigla em inglês FT). As análises podem se feitas em amostras nos estados: sólido, líquido e gasoso. A ressalva é que alguns equipamentos dependendo da fase em que a amostra se encontra, é necessário um pré-tratamento para garantir uma boa resolução espectral e reprodutibilidade. Com a finalidade de suplantar a necessidade de pré-tratar as amostras líquidas e sólidas (incluindo amostras de difícil manipulação) surgiu equipamentos de es- Figura 12: Tipos de vibrações das ligações. Fonte: Autor. 2 6 pectroscopia com transformada de Fourier aliada à análise por Reflectância Total Atenuada (sigla em inglês ATR). Brevemente, O modo em reflectância total atenuada consiste no uso de um cristal com índice de refração maior do que o da amostra e baixa absorção no infravermelho, como representado na Figura 13. Figura 13: Esquema do fundamento da técnica de Espectroscopia de Infravermelho por Reflectância Total Atenuada (ATR-FTIR). Fonte: Adaptado da Referência [75]. De acordo com o ângulo de incidência da radiação infravermelha, pode-se obter uma reflexão considerada completa. A partir desse ângulo crítico o feixe de luz incidente age como se penetrasse na amostra (gerado a onda evanescente), possibilitando assim medidas de absorção. O feixe que chega ao detector é atenuado, uma vez que ocorre o fenômeno da onda evanescente (penetração de 0,5 a 5,0 m) [76-78]. Com base no discutido até aqui, a técnica de ATR-FTIR se torna uma ferramenta bastante interessante quando se deseja analisar amostras biológicas, já que é menos predisposta à influência da água. Isso é extremamente relevante, uma vez que as amostras biológicas apresentam uma porção considerável de água em sua constituição. A análise de amostras biológicas por meio de técnicas espectroscópicas foi nomeada bioespectroscopia. Os primeiros trabalhos utilizando essa perspectiva buscavam por marcadores de doenças (carcinoma de bexiga, carcinoma mamário humano e fibroadenoma mamário humano). Utilizaram como amostras sangue e tecido leucêmico e os analisaram via espectroscopia na região do infravermelho [79, 80]. No trabalho desenvolvido no capítulo 3 utilizamos bioespectroscopia ATR-FTIR para analisar amostras de S. aureus após exposição aos antimicrobianos NanoAg, DO e o combinado dos dois. 2 7 1.4.2 Fluorescência Baseia-se num fenômeno denominado luminescência que tem como definição a emissão espontânea de radiação por uma espécie excitada eletronicamente. Basicamente, para que tal fenômeno ocorra é necessário que espécies que se encontram em seu estado fundamental absorvam energia suficiente para que atinjam estados eletrônicos de maior energia (excitado). Nesse caso, o tipo de radiação a ser absorvida é na faixa do ultravioleta (> 200 nm) e visível do espectro eletromagnético. Quando tais espécies são moléculas (especialmente as que possuem estruturas aromáticas, rígidas, planares e que possuem duplas ligações conjugadas) as transições envolvidas são entre os elétrons não-ligantes   (elétrons do tipo ―n”) e elétrons de ligações  (do tipo n e  ). Já quando se trata de nanopartículas metálicas o fenômeno da luminescência está relacionado à presença de pequenos clusters (aglomerados) de alguns átomos desse metal, que se adsorvem na superfície da partícula. Em ambos os casos, após o processo de excitação as espécies voltam ao estado de mais baixa energia emitindo parte da energia (regra de Stokes) que outrora foi absorvida na forma de luz [81-83]. O fenômeno da luminescência pode ser dividido em fluorescência e fosforescência. O que diferencia os dois processos é a natureza do estado excitado que a espécie atinge. No primeiro, o elétron que participa da transição para o estado excitado conserva a orientação do seu spin prosseguindo emparelhado com o elétron que permaneceu em seu orbital de origem, gerando o estado excitado do tipo singlete. Já o segundo caso é quando a orientação do spin do elétron não é conservada, gerando assim o estado triplete (vide Figura 14). Figura 14: Representação do estado fundamental e dos estados excitados singleto e tripleto Fonte: Autor O diagrama de Jablonski (apresentado na Figura 15) é comumente utilizado para expressar os processos de excitação e emissão envolvidos no fenômeno da luminescência [84,85]. 2 8 Figura 15: Diagrama de Jablonski para fluorescência e fluorescência retardada. Fonte: Autor Existem diversas técnicas de análise instrumental que utilizam o fenômeno da fluorescência como base. No trabalho que será apresentado no capítulo 2, utilizamos as técnicas da microscopia confocal de varredura a laser e citometria de fluxo. Essas técnicas são bastante utilizadas tanto para a detecção de espécies que possuem auto-fluorescência, quanto para espécies marcadas com fluoróforo. Quando se fala em metodologia de ―marcar‖ moléculas na área biológica, logo se remete a imuno-histoquímica (IHQ) que nada mais é do que uma metodologia que se baseia na identificação seletiva de antígenos (proteínas) em células presentes em uma sessão de tecido. Basicamente, o método é fundamentado no conhecimento de que anticorpos se ligam de maneira específica a antígenos. Albert Coons conceituou e implementou tal procedimento pela primeira vez em 1941 [86]. Os ensaios de IHQ são amplamente utilizados no diagnóstico de células anormais, como as encontradas em tumores cancerígenos. Marcadores moleculares específicos são característicos de eventos celulares específicos, como proliferação ou morte celular (apoptose) [87,88]. A IHQ também é vastamente utilizada em pesquisas básicas com a finalidade de compreender a distribuição e localização de biomarcadores e proteínas expressas de maneira diferencial em diversas partes de um tecido biológico. 2 9 1.4.2.1 Microscopia Confocal Essa técnica consiste na iluminação de uma amostra com raios de um determinado comprimento de onda (que sabemos ser na faixa do comprimento da luz ultravioleta) com o auxílio de um laser. Os dados obtidos originam uma imagem que é o resultado da emissão de radiação eletromagnética (denominada emissão secundária) com base no fenômeno da fluorescência que foi apresentado anteriormente. Trata-se de uma ferramenta de imagem óptica com a finalidade de aumentar a resolução óptica e o contraste de uma micrografia por meio do uso de um orifício espacial para bloquear a luz fora de foco na formação de imagens. A captura de várias imagens bidimensionais em profundidades distintas de uma amostra possibilita a reconstrução de estruturas tridimensionais (processo conhecido como corte óptico) dentro de um objeto. Esse processo também conhecido como corte ótico, se faz agrupando esses cortes por fim resultando numa imagem tridimensional da topografia de objetos complexos. Permite também a eliminação de informações fora de foco da imagem, o que favorece a análise de amostras mais espessas, como, estruturas fúngicas, biofilmes bacterianos, tecidos dentários e dentre outros tipos celulares. A microscopia confocal pode ser empregada para: (a) observação do estado fisiológico das células e tecidos, (b) construção de Imagens em 3D, (c) colocalização e (d) observação de células e tecidos marcados com fluorocromos por imunofluorescência [89, 90]. A última aplicação foi utilizada no estudo do capítulo 2 desse trabalho, onde foi avaliada por meio da ação da enzima Caspase-3 que o as NanoAu provocaram preferencialmente um processo apoptótico. 1.4.2.2 Citometria de fluxo Essa técnica também pode utilizar os princípios do processo de fluorescência, onde são utilizados marcadores fluocrômicos por meio de ensaios de IHQ. Brevemente, as células e partículas são analisadas enquanto fluem por meio de uma célula de fluxo muito estreita. Após ser marcada com um marcador fluorescente que se liga de maneira específica (vide item 1.4.2 onde se apresentou em que se baseiam os ensaios de IHQ) aos ácidos nucléicos. Posteriormente a amostra é constantemente transportada para a célula de fluxo e iluminada por um laser semicondutor, que consegue separar as células utilizando três sinais diretos que são eles: (a) dispersão frontal de luz (do inglês, forward scatter ou FSC), (b) dispersão lateral de luz (do inglês, side scatter ou SSC) e (c) fluorescência lateral (do inglês, side fluorescence ou SFL). Cada um dos sinais fornece informações distintas, onde FSC indica o volume celular, SSC indica elementos sobre o 3 0 conteúdo celular tais como grânulos e núcleos; e a SFL indica de maneira quantitativa DNA e RNA presentes na célula. Os resultados são expressos por meio de diagramas de dispersão que agrupam as células com propriedades físico-químicas semelhantes [91,92]. No estudo do capítulo 2, utilizamos o kit Anexina V-FITC como marcadores fluorescentes e o ensaio apoptótico das células de HT-29 após tratamento com as NanoAu e cisplatina foi realizado por meio dessa técnica. 1.4.3 Microscopia eletrônica de transmissão (MET) Esse tipo de microscopia se baseia na passagem de um feixe de elétrons da amostra que resulta em diversos tipos de espalhamentos. Essa diversidade está relacionada com as características da amostra. Essa técnica gera como resultado micrografias que nada mais são do que imagens em escala de cinza, onde as regiões mais claras são referentes aos elétrons pouco desviados e as mais escuras aos que são refratados [93]. Essa técnica foi utilizada para avaliação da forma e tamanho médio das nanopartículas obtidas nos dois estudos que serão apresentados nos capítulos 2 e 3. 1.4.4 Microscopia de contraste de fase Levando em conta o fato de que as células vivas não tratadas previamente com marcadores possuem baixa absorção de luz, e que isso leva a diferenças extremamente pequenas na distribuição de intensidade na imagem tornando-as quase imperceptíveis, ou invisíveis em um microscópio óptico simples. A microscopia de constraste de fase (realizado em nosso estudo com um microscopio invertido) surge como alternativa, uma vez que suplanta as interferências na imagem que são causadas pela proximidade das densidades ópticas e, índices de refração das diferentes partes do material biológico. Essa técnica consiste na conversão das diferenças do índice de refração em diferenças de intensidades que passem a ser visíveis. O que ocorre é que as ondas de luz que atravessam os componentes celulares (com densidades ópticas diferentes) assim fazem em diferentes velocidades. Assim, as ondas luminosas que atravessam núcleos, mitocôndrias e inclusões celulares emergirão em tempos e fases diferentes, de um elemento em relação ao outro possibilitando a observação. Como resposta tem imagens em escala de cinza [94,95]. No estudo apresentado no capítulo 2 essa técnica com o objetivo de observar alterações morfológicas associadas à apoptose nas células de HT-29 . 3 1 1.5 Análise Quimiométrica Com isso uma demanda que fez com que diversos cursos introduzissem uma nova disciplina da Química com a finalidade de formar profissionais capazes de relacionar as medidas de um sistema ou processo com o estado do sistema, via aplicação de métodos matemáticos e estatísticos [71]. A análise multivariada auxilia na análise de informações oriundas de resultados experimentais que envolvem muitas variáveis [96]. E dados espectroscópicos são exemplos desse tipo de dados multivariados. A classificação multivariada pode ser supervisionada e não supervisionada. No estudo do capítulo 3 foram aplicados os dois tipos de metodologia. 1.5.1 Análise por Componentes Principais (do inglês, PCA) Nessa técnica de reconhecimento de padrões não supervisionado não se tem o conhecimento a priori a que classes pertencem as amostras (ou pelo menos essa informação é omitida quando se constrói o modelo). Análise por Componentes Principais (do inglês, Principal Component Analysis, PCA) é uma representante desse tipo de método e é amplamente utilizada. A PCA admite a redução do número de dados representativos da informação original [97]. Brevemente, a PCA se vale de uma uma transformação linear ortogonal que transforma os dados para um novo sistema de coordenadas de forma que a maior variância por qualquer projeção dos dados fica ao longo da primeira coordenada (componente principal), a segunda maior variância fica ao longo da segunda coordenada, e assim sucessivamente. Essa diminuição expressiva no número de variáveis quando comparado ao conjunto original possibilita a representação de um conjunto por meio de escores e pesos. As semelhanças e diferenças são expressas normalmente por meio de um gráfico de escores de PC1 X escores de PC2, por exemplo. A decomposição de uma matriz espectral X se dá conforme a relação abaixo: t X= SL + R equação (1) onde os demais termos representam as matrizes de scores (S), loadings (L) e de resíduos (R) [98, 99]. No trabalho do capítulo 3 a PCA foi aplicada com a finalidade de análise não supervisionada, possibilitando assim a averiguação da existência ou não de grupos (também chamado clusters) após o tratamento de S. aureus com os antimicrobianos NanoAg, DO e o combinado dos dois. A matriz espectral original nesse caso foi obtida pela análise de ATR-FTIR. 3 2 1.5.2 Análise de Discriminante pelos Mínimos Quadrados Parciais (do inglês, PLS-DA) A PLS-DA é um exemplo de técnica de reconhecimento de padrões supervisionado. Basicamente, a PLS-DA utiliza a regressão de uma matriz espectral X contra um vetor de números (que pode ser 0, 1, n...) que representam o número de classes que as amostras podem se agrupar [100]. A PLS-DA é bastante válida quando se pretende analisar conjuntos de dados bioespectrais, já que esses dados possuem a característica de alta variância intraclasse e esse método atende bem a tal particularidade [101]. 2- ORGANIZAÇÃO DA TESE Esta tese foi organizada em ordem cronológica de desenvolvimento e está composta por publicações em que participei como co-autora e autora, que relatam estudos focados nos tema central proposto para o projeto de pesquisa - estudo e aplicação de nanopartículas de metais nobres (Au e Ag) a sistemas biológicos- essenciais para o desenvolvimento do doutorado. Ainda, nos apêndices são apresentados trabalhos realizados por meio de diferentes colaborações que ocorreram durante o período de doutoramento. Capítulo 2 –“Anti-inflammatory, analgesicandanti-tumorproperties of gold nanoparticles‖ (publicado no periódico Pharmacological Reports, DOI: 10.1016/j.pharep.2016.09.017) – Relata um rastreamento do efeito que nanopartículas de ouro (NanoAu), sintetizadas por uma rota ambientalmente correta, causam em células representativas do câncer colorretal (HT-29 ) e em sistemas in vivo (utilizando camundongos) nos quais pôde ser observados os efeitos anti-inflamatório, analgésico e antitumoral. Capítulo 3 – “On the synergy between silver nanoparticles and doxycycline towards the inhibition of Staphylococcus aureus growth” (publicado no periódico RSC Advances, DOI: 10.1039/C8RA02176G) - Relata uma aplicação de espectroscopia no infravermelho e análise de discriminante via PLS para identificação de sinergia de efeito inibitório de crescimento de Staphylococcus aureus após tratamento com o combinado NanoAg e DO. Capítulo 4 – Conclusões e Perspectivas - apresenta de forma resumida os principais resultados alcançados dentre os estudos, sua concordância com os objetivos propostos e a relevância para o cada subtema abordado, além de perspectivas para próximos trabalhos. Apêndice A - Environmentally compatible bioconjugated gold nanoparticles as efficient contrast agents for inflammation-induced cancer imaging (Nanoscale Research Letters, DOI: 10.1186/S11671-019-2986-Y)- Relata a eficiência das NanoAu conjugadas a 3 3 anticorpos primários no direcionamento específico de processos inflamatórios crônicos que podem levar ao câncer colorretal e carcinoma hepático, respectivamente. Apêndice B - Functionalization of gold nanoparticles with two aminoalcohol-based quinoxaline derivatives for targeting phosphoinositide 3-kinases (PI3K) (New Journal of Chemistry, DOI: 10.1039/C8NJ04314K) - Relata um estudo pioneiro a respeito da funcionalização de NanoAu com quinoxalinas tendo alvo a proteína PI3K . Apêndice C - Spherical neutral Gold nanoparticles improve anti-inflammatory response, oxidative stress and fibrosis in alcohol-methamphetamine-induced liver injury in rats. (International Journal of Pharmaceutics DOI: 10.1016/j.ijpharm.2018.06.008) - Relata os efeitos anti-inflamatórios, antioxidantes e antifibróticos das NanoAu em camundongos submetidos à lesão hepática com etanol e metanfetamina (METH). Apêndice D - Lipopolysaccharides and peptidoglycans modulating the interaction of Au nanoparticles with cell membranes models at the air-water interface. (Biophysical Chemistry, DOI: 10.1016/j.bpc.2018.04.007) - Apresenta um estudo de NanoAu estabilizadas com PVP nanopartículas dispersadas em água e incorporadas em monocamadas flutuantes de lipídios selecionados na interface água-ar como modelos de membrana celular. Apêndice E - Apoptosis in human liver carcinoma caused by gold nanoparticles in combination with carvedilol is mediated via modulation of MAPK/Akt/mTOR pathway and EGFR/FAAD proteins. (International Journal of Oncology, DOI: 10.3892/ijo.2017.4179) - Apresenta uma investigação da ação antitumoral e citoprotetora de NanoAu, caverdilol e o combinado dos dois. Apêndice F -How the Interaction of PVP-stabilized Ag Nanoparticles with Models of Cellular Membranes at the Air-Water Interface is Modulated by the Monolayer Composition (International Journal of Oncology, DOI: 10.1016/j.jcis.2017.10.091. ) – Apresenta um estudo com NanoAg (PVP como agente estabilizante) dispersas em meio aquoso e incorporadas em monocamadas de lipídios selecionados (modelo Langmuir) que atuaram como modelos de membrana celular Apêndice G -Dual Role of a Ricinoleic Acid Derivative in the Aqueous Synthesis of Silver Nanoparticles. (Journal of Nanomaterials, DOI: 10.1155/2017/1230467) – Apresenta um estudo em que o 9,10-epoxi-12-hidroxitetradecanoato de sódio (SEAR), um derivado epoxidado do ácido ricinoleico, atuou simultaneamente como agente redutor e estabilizante na síntese de NanoAg em meio aquoso alcalino. 3 4 3 OBJETIVOS 3.1 Objetivo Geral A presente tese de doutorado tem por avaliar os efeitos anti-inflamatório, antitumoral e analgésico de nanopartículas de ouro, bem como sua biodistribuição. E também, estudar via bioespectroscopia de ATR-FTIR aliada a ferramentas quimiométricas a sinergia do efeito antibacteriano de nanopartículas de prata combinadas ao hiclato de doxiciclina. 3.2 Objetivos Específicos  Determinar os efeitos analgésico, anti-inflamatório e anticâncer das NanoAu sintetizadas por uma rota de síntese ambientalmente mais branda.  Avaliar a biodistribuição das NanoAu após a administração das mesmas a camundongos.  Combinar NanoAg ao DO e avaliar, por meio da espectroscopia ATR-FTIR combinada a Análise por Componentes Principais (PCA), o perfil das respostas metabólicas das bactérias do tipo Staphylococcus aureus após serem tratadas com esse combinado  Utilizar o combinado mencionado no item anterior com o intuito de desenvolver um método complementar para confirmar sinergia em tratamentos com antimicrobianos combinados utilizando a espectroscopia ATR-FTIR combinada com o método de seleção de variáveis PLS-DA. 4 METODOLOGIAS EMPREGADAS 4.1 Obtenção das Nanopartículas e conjugado As sínteses tanto das NanoAu, quanto das NanoAg foram realizadas pelo método descrito por Gasparotto e colaboradores [27] que se baseia na mistura da solução de glicerol em NaOH (hidróxido de sódio) com outra solução contendo fonte do metal precursor (AgNO3, nitrato de prata ou HAuCl4, ácido cloroaurico) e PVP (polivinilpirrolidona). A Figura 17 apresenta uma ilustração desse processo para obtenção das NanoAu. Após a síntese as soluções coloidais tiveram seu meio ajustado para neutro, garantindo o encerramento da reação. 3 5 Figura 17- Esquema utilizado para obtenção de nanopartículas de ouro via rota verde. Fonte: Autor Para as NanoAu que foram administradas aos animais (capítulo 2), foi necessário uma purificação da solução por meio de diálise utilizando três ciclos com duração de 2 h cada, onde foram utilizadas membranas de celulose (Sigma-Aldrich) e água ultrapura. A Figura 18 mostra como as soluções coloidais de NanoAu estavam acondicionadas nas membranas de celulose antes de iniciar o primeiro ciclo de diálise. Figura 18: Aparato utilizado para submeter as NanoAu à diálise. Fonte: Autor. No caso das NanoAg utilizadas no estudo do capítulo 3, foi necessário conjugá-las ao DO e seguiu-se a metodologia empregada em trabalho anterior do nosso grupo [39]. O procedimento se dá vertendo uma solução de DO na solução NanoAg. O volume de DO adicionado variou em função da técnica de caracterização aplicada, para que contenha as concentrações desejadas de DO. 3 6 4.2 Coleta e preparação das amostras biológicas  Viabilidade celular- Células HT-29 A viabilidade celular foi determinada por meio de um ensaio de exclusão de azul de tripano. Resumidamente, alíquotas de células foram misturadas com 0,5% (m/v) de azul de tripano, e posteriormente foram incubadas à temperatura ambiente durante 5 min. O número de células viáveis foi calculado usando um hemocitômetro. O efeito do agente -1 estabilizante das nanopartículas de ouro, PVP (10 g L ) também foi testada. Esse método permite detectar células inviáveis, cuja membrana, por apresentar danos, permite a incorporação do corante e coram-se em azul; enquanto que as células viáveis, por apresentarem membrana íntegra, bloqueiam a passagem do corante, ficando transparente. O número total de células (células vivas e mortas) foi calculado segundo o método utilizado por Gorjão [102].  Animais Camundongos Swiss fêmeas (25-35 g) e ratos machos (200-250 g) de 90 dias de vida foram obtidos do biotério do Departamento de Biofísica e Farmacologia da Universidade Federal do Rio Grande Norte. Todos os animais foram alojados em gaiolas, sob condições laboratoriais padrão de 22 ± 2 °C e 12 h de luz/12 horas de ciclo escuro, e alimentados com ração peletizada e água. Foram aclimatados durante sete dias antes das experiências e submetidos a jejum antes do início dos testes. O bem-estar dos animais foi assegurado em conformidade com Revisão Institucional Conselho de Administração da Universidade Federal do Rio Grande do Norte que aprovou este estudo específico (protocolo aprovado nº053 / 2013), bem como os procedimentos experimentais adotados. Neste trabalho, os animais foram tratados de maneira adequada e com humanização.  Células de Staphylococcus aureus No preparo da amostra biológica foi utilizada a cepa padrão de S. aureus ATCC® 25923™. A ativação das bactérias foi realizada transferindo 100 μL da amostra padrão em aproximadamente 5 mL de caldo de infusão de cérebro e coração, do inglês Brain Heart Infusion (BHI) por 24 h a 37 ºC, na estufa. Em seguida, as bactérias ativadas foram semeadas. O processo consiste em inserir o swab na suspensão preparada e passá-lo de forma leve e rápida com movimentos horizontais da direita para a esquerda em outra placa de Petri contendo meio TSA, de modo a garantir uma semeadura uniforme. Sendo posteriormente incubadas por um período de 12 h a 37 ºC. Depois, com uma alça bacteriológica retirou-se massa de colônias da superfície da placa e homogeneizou-se em 3 7 solução salina estéril a 0,9 %. A quantidade retirada foi apenas o necessário para que a turbidez dessa solução apresentasse o mesmo padrão de turvação da solução padrão de 8 -1 MacFarland a 0,5, resultando em aproximadamente 1 x 10 UFC mL . Esse procedimento é baseado na percepção visual, que compara o padrão de turbidez entre a amostra e a solução padrão, com o auxílio de um cartão com linhas horizontais. Para a etapa de exposição das S. aureus aos antimicrobianos, seguiu-se o procedimento apresentação na figura a seguir: Figura 20– Esquema de ativação, semeadura, exposição aos antimicrobianos e finalizando com a aquisição espectral por meio da técnica de ATR-FTIR. Fonte: Autor. Levando em consideração a curva de crescimento das S. aureus ATCC® 25923™ fornecido na literatura [103, 104], optou-se por realizar a aplicação dos antimicrobianos após 12 h da incubação a 37 ºC. As S. aureus ficaram incubadas por mais 12 h após o tratamento, só então os espectros de ATR-FTIR das massas bacterianas foram coletados. Mais detalhes sobre a aquisição desses dados serão apresentados no item 4.3.1.2. 3 8 4.3 Aquisição de dados instrumentais  Técnicas Espectroscópicas 4.3..1 UV-Visível Os espectros UV-Vis obtidos nos dois estudos foram adquiridos com o auxílio do espectrofotômetro USB-650 Red Tide (OceanOptics), na região de 200 a 880 nm, em cubeta de quartzo com 1,0 cm de caminho ótico. Foram coletados espectros da solução de NanoAu e NanoAg antes e depois do ajuste do pH, e após a diálise para as NanoAu. Também foram adquiridos espectros para o combinado NanoAg + DO e posteriormente comparado com o espectro resultante do somatório matemático dos espectros das soluções isoladas. As soluções foram previamente diluídas para garantir a linearidade das medidas. Foi usado como branco a água ultrapura. 4.3..2 ATR-FTIR A aquisição dos espectros de ATR-FTIR foi realizada com o auxílio do espectrômetro Bruker VERTEX 70 FTIR (Bruker OpticsLtd., Coventry, UK) contendo um elemento reflexivo interno de cristal de diamante a um ângulo de incidência de 45º do feixe de infravermelho. Configurou-se o instrumento para realizar 16 scans com resolução -1 espectral de 4,0 cm para a referência e para a amostra. Em uma temperatura de 21 ± 1 ºC foram coletados os espectros, em um total de 480, com 5 réplicas para cada uma das 96 amostras, a saber: Controle (n=24), DO (n=24), NanoAg + DO (n=24 e NanoAg (n=24). O espectro de referência foi o ar e então, com uma alça metálica, a massa de bactéria foi removida e colocada no suporte do equipamento. Um pedaço de folha de alumínio foi colocado sobre a amostra para a coleta do espectro com o objetivo de minimizar a interferência externa, conforme descrito por Cui e colaboradores [105]. Para garantir a reprodutibilidade, a confiabilidade das medidas e a biosegurança, após cada coleta o cristal de diamante foi limpo com álcool etílico 70 %. Esperou-se evaporar e antes de colocar o próxima amostra um espectro foi adquirido para verificar se ainda havia impurezas e/ou água residual que interferissem nas medidas. 3 9 4.4 Técnicas Microscópicas  MET Para a análise por Microscopia Eletrônica de Transmissão, as soluções de NanoAu e NanoAg foram pingadas sobre grades de cobre recobertas com um filme de carbono. Após evaporação do solvente, as imagens foram adquiridas com um microscópio Tecnai Spirit Bio Twin 12 operando em 120 kV.  Microscopia de contraste de fase Para análise das células HT-29 que foram previamente tratadas com NanoAu (40 mM e 400 mM) e 100 mM de cisplatina por 48 h. As células que ficaram aderidas à placa foram levadas ao microscópio invertido e após a identificação das células e outros elementos de interesse ao estudo do capítulo 2, as imagens foram adquiridas.  Microscopia confocal Para essa análise, as células foram previamente semeadas em lâminas de vidro em 4 placas de 24 poços (5x10 células/poço). Passadas 24 h, se prosseguiu com o tratamento das mesmas com as NanoAu (nas concentrações 40 M - NanoAu40 e 400 M - NanoAu400) e cisplatina (100 M - Cis100) por um período de 48 h. As células foram então lavadas, fixadas com para formaldeído, permeabilizadas por Triton-X, e então incubadas com anticorpo policlonal de coelho anti-caspase-3 (Abcam, San Francisco, CA, EUA) diluído na proporção de 1:500 em tampão PBS contendo albumina de soro de bovino, BSA (5%; Life Technologies do Brasil Ltda., São Paulo, Brasil). A incubação se deu durante 1 h à temperatura ambiente numa atmosfera úmida. A marcação do anticorpo primário foi realizada com Alexa Fluor 488 que nada mais é do que um anticorpo secundário de cabra, anti-coelho (Abcam), e também foi utilizado o 4,6-diamidino-2- fenilindol (Life Technologies DO BRASIL Ltda.) com a finalidade de corar os núcleos. Um Microscópio de Varredura a Laser Confocal LSM 510 (Carl Zeiss, Jena, Alemanha) foi empregado para examinar as lâminas de vidro imunomarcadas. As imagens foram obtidas com o laser de excitação em 442 nm. Os controles positivos e negativos conhecidos foram incluídos em cada lote de amostras. As imagens obtidas pelo microscópio confocal foram utilizadas para determinar a reatividade de todos os grupos (controle, NanoAu40, NanoAu400 e Cis100) com o auxílio da análise de densitometria computadorizada. Para determinação dos valores densitométricos médios foi utilizado o software ImageJ (http://rsb.info.nih.gov/ij/). As medidas do índice de contraste foram obtidas com a seleção de cinco áreas x 100/área total após a remoção da rotulagem (azul ou 4 0 verde) posicionada entre as regiões de interesse. Cinco áreas foram analisadas por amostra, e cada grupo teve suas análises em triplicata.  Citometria de fluxo Para esta análise as células necessitaram ser marcadas previamente com o kit de Detecção de Apoptose Anexina V-FITC I que pode ser descrito como solução de coloração de iodeto de propídio. Cada teste requereu 5,0 L dessa solução. Esse kit atuou como uma sonda sensível para análise citométrica de fluxo das células de HT-29 que estavam sendo submetidas à apoptose. 4.5 Ensaios e testes biológicos  Avaliação do efeito anti-inflamatório Para os ensaios de avaliação do efeito anti-inflamatório, os camundongo s foram divididos em cinco grupos, num total de 6 animais por grupo. A Tabela abaixo apresenta a descrição de cada um dos grupos, bem como as dosagens utilizadas nesse ensaio: Tabela 2: Descrição dos grupos, com suas respectivas doses utilizadas no ensaio para avaliação do efeito antiinflamatório. Grupos Descrição Dose -1 700,0 g/kg Teste NanoAu -11.000 g/kg -1 1.500 g/kg Padrão Indometacina 10 mg kg-1 Controle positivo PVP 10mg kg-1 Fonte: Autor. As doses foram administradas via oral. Passados trinta minutos, todos os grupos receberam 0,25 mL de carragenina (1% em solução salina) por meio de injeção subcutânea. Depois de 4 h, os animais foram sacrificados usando tiopental (100 mg kg-1 ). A contagem total de leucócitos foi determinada em câmara de Neubauer. A percentagem da inibição de leucócitos foi calculada utilizando a equação: % de inibição de leucócitos = (1 - T / C) x 100 equação (2) Onde os termos representam a contagem de leucócitos do grupo tratado (T) e a contagem 4 1 de leucócitos do grupo de controle (C). O fluido peritoneal, armazenado a -70 °C após a extração foi homogeneizado e processado como descrito por Safieh-Garabedian e -1 colaboradores [106]. Os níveis de IL-1β (intervalo de detecção: 62,50 - 4000 pg mL ; -1 limite inferior de detecção: 12,50 ng mL de camundongo recombinante IL-1β) e TNF ( -1 -1 intervalo de detecção: 62,50 - 4000 pg mL ; limite inferior de detecção: 50 ng mL camundongo recombinante TNF-) foram determinadas usando kits de ELISA comercial (R & D Systems, Minneapolis, MN, EUA), como descrito por Kendall e colaboradores [107]. Todos os ensaios foram realizados no mínimo em triplicata e as diferenças significativas entre os grupos foram determinados com o auxílio da análise de variância (ANOVA) e o teste de Bonferroni. Os valores de p inferior a 0,05 foram considerados estatisticamente significativos.  Avaliação do efeito analgésico Para essa etapa foi utilizado o teste da placa quente que visa avaliar a atividade analgésica central. Apenas os camundongos que apresentaram respostas nociceptivas iniciais (lamber das patas dianteiras ou pular) entre 3,0 e 19 segundos foram utilizados para experiências adicionais. Os ensaios foram realizados a 55 ± 0,5 ºC com um aparelho de introspecção de placa quente (São Paulo, Brasil). Os camundongos selecionados foram pré- tratados conforme as seguintes substâncias e doses: Tabela 3: Descrição dos grupos, com suas respectivas doses utilizadas no ensaio para avaliação do efeito de analgesia central. Grupos Descrição Dose -1 700,0 g/kg Teste NanoAu -11.000 g/kg -1 1.500 g/kg -1 Padrão Morfina 10 mg kg 1 Controle positivo PVP 10 mg kg- Fonte: Autor. O tempo de exposição do camundongo à placa foi fixado em 30 segundos para minimizar os danos na pele do animal. O tempo de reação é contado como o intervalo para o animal começar a lamber as patas dianteiras ou saltar. As medidas foram nos tempos: 0 min, 30 min, 60 min, 90 min e 120 min a contar da administração. 4 2 O método de Koster e colaboradores [108] foi utilizado para inferir a respeito da atividade analgésica periférica. Os camundongos Swiss foram divididos em cinco grupos de seis camundongos e jejuaram durante uma noite. Os animais foram tratados por via oral conforme grupos e doses apresentados na Tabela a seguir: Tabela 4: Descrição dos grupos, com suas respectivas doses utilizadas no ensaio para avaliação do efeito de analgesia periférica. Grupos Descrição Dose -1 700,0 g/kg Teste NanoAu -11.000 g/kg -1 1.500 g/kg -1 Padrão indometacina 10 mg kg 1 Controle positivo PVP 10 mg kg- Fonte: Autor. Os camundongos foram tratados com ácido acético (0,6% v/v em solução salina, 10 mL kg- 1, administrada via intraperitoneal - ip) 30 minutos após o tratamento supracitado. O número de contorções foi contado por um período de 20 minutos e foi calculada a percentagem de inibição utilizando a relação a seguir: % Inibição = número de contorções (controle) - número de contorções (teste) x 100 equação (3) número de contorções (controle)  Biodistribuição histopatológica quantitativa O ensaio de biodistribuição das NanoAu seguiu o procedimento da Figura 21, onde se vê que foi realizada a eutanásia dos camundongos fêmeas (pesando na faixa de 30-40 g) com o auxílio de tiopental 2% (100 mg kg-1 ) nos tempos de 30 min, 60 min e 24 h após a -1 -1 administração das NanoAu (1500 mg kg ) ou de PVP (10 mg kg ) , seguido pela coleta do coração, pulmões, fígado, rins, baço, estômago, intestino. Posteriormente foi utilizado o hidróxido de tetrametilamônio, TMAH a 20% (solução a 25%, Sigma-Aldrich) para digestão por um período de 24 h para liberação das nanopartículas dos tecidos [109]. A técnica de UV-Vis foi utilizada para fazer a determinação da extração das NanoAu dos tecidos. 4 3 Figura 21: Fluxograma do ensaio de biodistribuição. Fonte: Autor. Para a análise histológica, amostras do fígado de cada camundongo foram cortadas imediatamente após a coleta, fixadas em formalina tamponada neutra (10%), desidratadas com etanol a diferentes graus e foram eles: 70, 80, 90, 95 e 100%. As amostras foram lavadas com xileno em dois ciclos, seguido por impregnação com cera de parafina fundida (2 mudanças), incorporado e bloqueado. As secções de parafina (4-5 hum) foram marcadas com hematoxilina e eosina de acordo com a literatura [110], e então foram adquiridas as imagens obtidas via microscopia. 4.6 Análise computacional Todo o tratamento computacional do trabalho apresentado no capítulo 3, desde a importação dos dados espectrais até a construção dos modelos multivariados, foi realizado no software MATLAB® R2014 (Mathworks, Natrick, USA) usando o PLS Toolbox version 7.9.3 (Eigen vector Research, Inc., Manson, USA). Os espectros brutos foram pré- -1 -1 processados selecionando entre 1800 cm e 900 cm (gerando um total de 468 números de -1 onda em resolução espectral de 4 cm ) e centralização média. A PLS-DA foi utilizada para a construção dos modelos multivariados de classificação. A figura 16 exibe um fluxograma das etapas seguidas nesse trabalho de classificação multivariada. 4 4 Figura 16: Fluxograma representando as etapas para entrada de dados, seleção de amostras pelo algoritmo, redução de dimensionalidade e/ou seleção de variáveis e classificação multivariada. Fonte: Autor. 4 5 Referências [1] European Science Foundation. Forward Look Nanomedicine: An EMRC Consensus Opinion 2005. Disponível em: http://www.esf.org (acessado em agosto de 2019) [2] Peer, D.; Karp, J.M.; Hong, S.; Farokhzad, O.C.; Margalit, R.; Langer, R. Nanocarriers as an emerging platform for cancer therapy. Nature Nanotechnology, v. 2, p. 751–760, 2007. Disponível em: https://www.nature.com/articles/nnano.2007.387. Acesso em setembro de 2019. [3] Duncan, R. Polymer conjugates as anticancer nanomedicines. Nature Review.Cancer, v. 6, p. 688–701, 2006. Disponível em: https://www.nature.com/articles/nrc1958 Acesso em setembro de 2019. [4] Lavan, D. A.; Langer, R. Small-scale systems for in vivo drug delivery. Nature Biotechnology, v.21, p. 1184–91, 2003.DOI:10.1038/nbt876. [5] Duncan, R.; Gaspar, R. Nanomedicine(s) under the microscope. Molecular Pharmaceutic, v. 8, p. 2101–2041, 2011. DOI: dx.doi.org/10.1021/mp200394t. [6] Parreira, D. B.; Eugénio, J. Nanopartículas para aplicação oncológica. INPI (2011). [7] Bardhan, R.; Lal, S.; Joshi, A.; Halas, N.J. Theranosctic Shells: From Probe Design to Imaging and Treatment of Cancer. Accounts of Chemical Research, v. 44, p. 936–946, 2011.DOI: 10.1021/ar200023x [8] Chen, F., Ehlerding, E.B.; Cai, W. Theranostic Nanoparticles. Journal of Nuclear Medicine.v.55, n. 12, p. 1919–1922, 2014.DOI:10.2967/jnumed.114.146019. [9] Bejarano, J.; Navarro-Marquez, M.; Morales-Zavala, F.; Morales, J. O.; Garcia- Carvajal, I.; Araya-Fuentes, E.; Flores, I.; Verdejo, H. E.; Castro,P. F.; Lavandero, S.; Kogan, M. J.Nanoparticles for diagnosis and therapy of atherosclerosis and myocardial infarction: evolution toward prospective theranostic approaches. Theranostics, v. 8: p. 4710-4732, 2018. DOI: 10.7150/thno.26284 [10] Priyanka Singh,1Santosh Pandit,2 V.R.S.S. Mokkapati,2 Abhroop Garg,1 Vaishnavi Ravikumar,1 and Ivan Mijakovic.Gold Nanoparticles in Diagnostics and Therapeutics for Human Cancer.Int J Mol Sci., v. 9, n. 7, p. 1979-1995, 2018.DOI: 10.3390/ijms19071979 [11] Yang,C.;Bromma,K.; Ciano‑ Oliveira, C.; Zafarana,G.vanProoijen,M.; Chithran, D. B.Gold nanoparticle mediated combined cancer therapy.Cancer Nano, v. 9, p.1-14, 2018. DOI:https://doi.org/10.1186/s12645-018-0039-3 [12] Qing,Y.;Cheng,L.;Li,R.;Liu,G.;Zhang,Y.; Tang, X.;Jincheng Wang,J. Liu H.;Qin,Y.Potential antibacterial mechanism of silver nanoparticles and the optimization of orthopedic implants by advanced modification technologies. Int J Nanomedicine, v..13,p. 3311–3327, 2018.DOI: 10.2147/IJN.S165125 [13] Tian, X.; Jiang, X.; Welch, C.;Croley, T.R.; Wong, T.Y.; Chen, C.; Fan, S.; Chong, Y.; Li, R.;Ge, C.; Chen, C.; Yin, J.J.Bactericidal Effects of Silver Nanoparticles on Lactobacilli and the Underlying Mechanism.ACS Appl Mater Interfaces, v. 10, n.10, p. 8443-8450., 29, 2018. DOI: 10.1021/acsami.7b17274. 4 6 [14] IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1972). Online version (2019-) created by S. J. Chalk. [15] KUMAR, C. S. S. R. UV-VIS and photoluminescence spectroscopy for nanomaterials characterization. Heidelberg: Springer 2013, p.3-4. [16] Garcia, M. A. Surface plasmons in metallic nanoparticles: fundamentals and applications. Journal of Physics D: Applied Physics, v. 45, p.389-501, 2012. [17] Jana, J.; Ganguly, M.; Pal, T. Enlightening surface plasmon resonance effect of metal nanoparticles for practical spectroscopic application. RSC Advances. v. 6, 86174-86211, 2016. DOI: 10.1039/C6RA14173K [18] Rycenga, M.; Cobley, C.M.; Zeng, J.; Li, W.; Moran, C. H.; Zhang, Q.; Qin, D.; Xia, Y. Controlling the Synthesis and Assembly of Silver Nanostructures for Plasmonic Applications. Chemical Reviews, v. 111, p. 3669-3712, 2011. [19] Ringe, E.; Langille, M.R.; Sohn, K.; Zhang, J.; Huang, J. ; Mirkin, C. A.; Duyne, R.P. V.; Marks, L. D. Plasmon Length: A Universal Parameter to Describe Size Effects in GoldNanoparticles. The Journal of Physical Chemistry Letters.v.3, p 1479−1483, 2012.DOI: 10.1021/jz300426p. [20] Xiang, H.; Zhang, X.; Neuhauser, D.; Lu, G. Size-Dependent Plasmonic Resonances from Large-Scale Quantum Simulations. The Journal of Physical Chemistry Letters.v.5, p 1163-1169, 2014.DOI: 10.1021/jz500216t. [21] Merkel, T. J.; Herlihy, K. P.;Nunes,J.; Orgel, R.M.;Rolland,J.P;DeSimone, J.M. Scalable, Shape-specific, Top-down Fabrication Methods for the Synthesis of Engineered Colloidal Particles.Langmuir, v.26, n.16, p.13086–13096, 2010.DOI: 10.1021/la903890h [22] Nogueira, A. F.; Gonçalves, M. C.; Santos, L. S. S.; Melo Jr., M. A. Preparação de nanopartículas de prata e ouro: um método simples para introdução da nanociência em laboratório de ensino. Química Nova, v. 35, n. 9, p. 1872-1878, 2012.DOI:http://dx.doi.org/10.1590/S0100-40422012000900030 [23] Alkilany, A.M.;Caravana, A.C.; Hamaly, M.A.; Lerner, K.T.; Thompson, L.B. Phase transfer of citrate stabilized gold nanoparticles using nonspecifically adsorbed polymers. Journal of Colloid and Interface Science, v. 461, p.39-44, 2016. DOI: 10.1016/J.JCIS.2015.09.010. [24] Hur, Y.E.; Park, Y. Vancomycin-Functionalized Gold and Silver Nanoparticles as an Antibacterial NanoplatformAgainst Methicillin-Resistant Staphylococcus aureus. J. Nanosci. Nanotechnol. v. 16, p. 6393–6399, 2016. DOI: 10.1166/jnn.2016.12393. [25] Kheybari S., Samadi N., Hosseini S.V., Fazeli A., Fazeli M.R.. Synthesis and antimicrobial effects of silver nanoparticles produced by chemical reduction method.DARU Journal Of PharmaceuticalSciences. v.18, p. 168-172, 2010. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/22615613Acesso em agosto de 2019. 4 7 [26] Mavani, K.; Mihir, S. Synthesis of silver nanoparticle by using sodium borohydride as a reducing agent. International Journal of Engineering Research & Technology, v.2, n. 3, p.1-5, 2013.DOI: 10.13140/2.1.3116.8648 [27] Gasparotto, L.H.S.; Garcia, A.C.; Gomes, J.F.; Tremiliosi-Filho, G. Electrocatalytic performance of environmentally friendly synthesized gold nanoparticles towards the borohydride electro-oxidation reaction. J. Power Sources. v.218, p.73-78, 2012.DOI: 10.1016/j.jpowsour.2012.06.064. [28] Faraday‘s notebooks: Gold colloids https://www.rigb.org/docs/faraday_notebooks_colloids_0.pdf [29] Horisberger, M. Colloidal gold: a cytochemical marker for light and fluorescent microscopy and for transmission and scanning electron microscopy. Gold Bulletin, v. 14, p. 9–31, 1981.DOI:10.1007/BF03216735 [30] Khlebtsov, N. &Dykman, L. Biodistribution and toxicity of engineered gold nanoparticles: a review of in vitro and in vivo studies. Chemical Society Reviews, v. 40, p. 1647–1671, 2011.DOI: 10.1039/c0cs00018c. [31] Roth, J. The silver anniversary of gold: 25 years of the colloidal gold marker system for immunocytochemistry and histochemistry.Histochemistry Cell Biology, v.106, n. 1, p. 1-8, 1996.https://www.ncbi.nlm.nih.gov/pubmed/8858362 [32] Kvien, T. K.; Hannonen, P.; Wollheim, F.; Forre, O.; Hafstrom, I.; Kaltw-Asser, J.; Leirisalo-Repo, M.; Manger, B.; Laasonen, L.; Prestele, H.; Kurki, P. Long term efficacy and safety of cyclosporin versus parenteral gold in early rheumatoid arthritis: a three year study of radiographic progression, renal function, and arterial hypertension. Annual of theRheumaticDiseases, v. 61, p. 511–516, 2002. DOI: 10.1136/ard.61.6.511. [33] Araújo, R.F. Júnior; de Araújo A. A.; Pessoa J. B.; Freire Neto F. P.; da Silva G.R.; Leitão Oliveira, A. L.; de Carvalho T .G.; Silva H. F.; Eugênio, M.; Sant'Anna, C.; Gasparotto, L. H.Anti-inflammatory, analgesicandanti-tumorproperties of gold nanoparticles.Pharmacology Reports, v. 69, n. 1, p. 119-129, 2017.DOI: 10.1016/j.pharep.2016.09.017. [34] Varner, K. U.S. Environmental Protection Agency Office of Research and Development. State of the science literature review: everything nanosilver and more. Scientific, technical, research, engineering and modeling support final report. Washington (DC), 2010. Disponível em: http://www.epa.gov/chemical-research/research- evaluating-nanomaterials-chemical-safety. Acesso em: agosto de 2019. [35] Rai, M.; Yadav, A.; Gade, A. Silver nanoparticles as a new generation of antimicrobials. Biotechnology Advances, v. 27, p. 76-83, 2009. DOI:https://doi.org/10.1016/j.biotechadv.2008.09.002 [36] Nowack, B.; Krug, H.F.; Height, M. 120 years of nanosilver history: implications for policy makers. Environmental Science & Technology, v. 45, p. 1177-1183, 2011.DOI: https://doi.org/10.1021/es103316q [37] Rai, M. K.; Deshmukh, S. D.; Ingle, A. P.; Gade, A. K. Silver nanoparticles: the powerful nanoweapon against multidrug-resistant bacteria. Journal of Applied Microbiology, V. 112, n. 5, p. 842-852, 2012.DOI: 10.1111/j.1365-2672.2012.05253.x 4 8 [38] Hari, N.; Thomas, T. K.; Nair, A. J. Comparative Study on the Synergistic Action of Garlic Synthesized and Citrate Capped Silver Nanoparticles with 𝛽-Penem Antibiotics. IRS Nanotechnology, V. 2013, p. 1-7, 2013DOI:http://dx.doi.org/10.1155/2013/792105 [39] Silva, H.F.O.; Lima, K.M.G.; Cardoso, M. B.; Oliveira, J.F.A.; Melo, M. C.N.; Sant‘anna, C.; Eugênio, M.; Gasparotto, L.H.S. Doxycycline conjugated with polyvinylpyrrolidone encapsulated silver nanoparticles: a polymer's malevolent touch against Escherichia coli. RSC Advances, v. 5, p. 66886-66893, 2015.DOI: 10.1039/C5RA10880B. [40] Katva, S.; Das, S.; Moti, H. S.; Jyoti, A.; Kaushik, S. Antibacterial Synergy of Silver Nanoparticles with Gentamicin and Chloramphenicol against Enterococcus faecalis. Pharmacognosy Magazine,v.13, p. 828–833.DOI:10.4103/pm.pm_120_17. [41] Kaura, A.; Rajesh Kumar, R. Enhanced bactericidal efficacy of polymer stabilized silver nanoparticles in conjugation with different classes of antibiotics. RCS Advances, v.9, p.1095–1105, 2019.DOI: 10.1039/c8ra07980c [42] Silva, H. F. O.; de Lima, R.P.;da Costa, F. S. L.; Moraes, E. P.; Melo,M C. N.;Sant‘Annac, C.; Eugênio, M.; Gasparotto, L. H. S.On the synergy between silver nanoparticles and doxycycline towards the inhibition of Staphylococcus aureus growth.RSC Advances, v. 8, p. 23578-23584, 2018. DOI:10.1039/C8RA02176G [43] INSTITUTO NACIONAL DO CÂNCER (INCA). Câncer colorretal. INCA (2016). Disponível em: http://www2.inca.gov.br/wps/wcm/connect/tiposdecancer/site/home/colorretal/definicao+. Acesso em: agosto de 2019. [44] American CancerSociety. ColorectalCancerFacts& Figures2017-2019. Atlanta: American Cancer Society, 2017. [45] Schatzkin, A.; Freedman, L. S.; Dawsey, S. M.; LanzaE. Interpreting precursor studies: what polyp trials tell us about large-bowel cancer. Journal of the National Cancer Institute, v. 86, p. 1053-1057, 1994. DOI: 10.1093/jnci/86.14.1053. [46] Bond, J. H. Polyp guideline: diagnosis, treatment, and surveillance for patients with colorectal polyps. Practice Parameters Committee of the American College of Gastroenterology. The American Journal of Gastroenterology, v. 95, p. 3053-3063, 2000. DOI:10.1111/j.1572-0241.2000.03434.x. [47] Levine, J. S.; Ahnen, D. J. Clinical practice. Adenomatous polyps of the colon. The New England Journal of Medicine, v. 355, p. 2551-2557, 2006. DOI: 10.1056/NEJMcp063038. [48] Risio, M. The natural history of adenomas. Best Practice & Research: Clinical Gastroenterology, v. 24, p. 271-280, 2010. DOI: 10.1016/j.bpg.2010.04.005. [49] Winawer, S. J.; Zauber, A. G. The advanced adenoma as the primary target of screening. Gastrointestinal Endoscopy Clinics of North America, v.12: 1-9, 2002. DOI: 10.1016/S1052-5157(03)00053-9. 4 9 [50] Sonali, K.; Shashikant, D; Dhawale, C. Alternatives to animal testing: A review. Saudi Pharmaceutical Journal, v. 23, n. 3, p. 223-229, 2015. DOI: https://doi.org/10.1016/j.jsps.2013.11.002. [51] Kitamura, H.; Cho, M. ; Lee, B. H.; Gum, J. R.; Siddiki, B.B.; , S.B.; Toribara, N.W.; Lesuffleur, T.; Zweibaum, A.; , Y.; Yonezawa, S.; Kim, Y.S. Alteration in mucin gene expression and biological properties of HT29 colon cancer cell subpopulations. European Journal of Cancer, v.32, n.10, p. 1788-1796, 1996. [52] Lay, M. M.; Karsani, S. A.; Malek, S. N. A. Induction of Apoptosis of 2,4′,6- Trihydroxybenzophenone in HT-29 Colon Carcinoma Cell Line. BioMed Research International, v. 2014, p. 1-12, 2014. DOI: http://dx.doi.org/10.1155/2014/468157 [53] Shabahang, M.; Buras, R. R.; Davoodi, F.; Schumaker, L. M.; Nauta, R. J.; Uskokovic, M. R.; Brenner, R. V.; Evans S. R. Growth inhibition of HT-29 human colon cancer cells by analogues of 1,25-dihydroxyvitamin D3. Cancer Research, v.1, n. 54, p. 4057-4064, 1994. Disponível em: https://cancerres.aacrjournals.org/content/54/15/4057.long Acesso em setembro de 2019. [54] Bundscherer, A. C.; Malsy, M.; Bitzinger, D. I.; Wiese, C. H.; Gruber, M. A.; Graf, B. M. Effects of Lidocaine on HT-29 and SW480 Colon Cancer Cells In Vitro. Anticancer Research, v. 37, n. 4, p. 1941-1945, 2017. DOI: 10.21873/anticanres.11534 [55] T-Johari, S.A.T.; Hshim, F.; Imail, W. I.; Ali, A. M. Combinatorial Cytotoxic Effects of Gelam Honey and 5-Fluorouracil against Human Adenocarcinoma Colon Cancer HT-29 Cells In Vitro. International Journal of Cell Biology, v. 2019, p. 1-10, 2019. DOI:https://doi.org/10.1155/2019/3059687 [56] Martínez-Maqueda, D.; Miralles, B; Recio, I. The Impact of Food Bioactives on Health Chapter: HT29 Cell Line, Springer, p 113-124. DOI: https://doi.org/10.1007/978-3-319-16104-4_11 [57] https://www.who.int [58] Tenover, F.C.; Mcgowan, J.E. Jr. Reasons for the Emergence of Antibiotic Resistance. Am. J. Med. Sci. v. 311, n. 1, p. 9-16, 1996.DOI: 10.1016/S0002-9629(15)41625-8. [59] Mota, R.A. et al. Utilização indiscriminada de antimicrobianos e sua contribuição a multirresitência bacteriana. Braz. J. Vet. Res. Anim. Sci. v. 42, p. 465-470, 2005. DOI: 10.11606/issn.1678-4456.bjvras.2005.26406. [60] Loureiro, R. J.; Roque, F.; Rodrigues, A. T.; Herdeiro, M. T.; Ramalheira, E. Use of antibiotics and bacterial resistances: Brief notes on its evolution. Revista Portuguesa de Saúde Pública, v. 32, n.1, p. 77-84, 2016. DOI: https://doi.org/10.1016/j.rpsp.2015.11.003 [61] Wright, G.D.; Sutherland, A.D. New strategies for combating multidrug-resistant bacteria.Trends Mol. Med. v.13, p. 260–267, 2007.DOI: 10.1016/j.molmed.2007.04.004. [62] Davies, J.; Davies, D. Origins and Evolution of Antibiotic Resistance. Microbiol.Mol. Biol. Rev. v. 74, p. 417-433, 2010.DOI: 10.1128/MMBR.00016-10. 5 0 [63] Lewis, K.; Shan, Y. Why tolerance invites resistance. Science.v. 335, p. 796, 2017. DOI: 10.1126/science.aam7926. [64] Tortora, G. J.; Funk, B. R.; Case, C. L. Microbiologia. Porto Alegre: Artmed Editora S.A., 2012. [65] Männik, J.; Driessen, R.;Galajda, P.; Keymer, J. E.; Dekker, C.Bacterial growth and motility in sub-micron constrictions. PNAS, v. 106, n. 35, p. 14861–14866, 2009. DOI:10.1073/pnas.0907542106 [66] Santos, A.L.; Santos, D. O.; Freitas, C. C.; Ferreira, B. L. A.; Afonso, I. F.; Rodrigues, C.R.; Castro, H. C. Staphylococcus aureus: visitando uma cepa de importância hospitalar. Jornal Brasileiro de Patologia e Medicina Laboratorial, v. 43, n.6,p. 413-423, 2007. [67] Chua, K.Y.;Stinear, T.P.; Howden, B. P. Functional genomics of Staphylococcus aureus. Briefings in Functional Genomics, v. 12, n.4, p. 305-315, 2013. DOI:10.1093/bfgp/elt006. [68] Chua, K.Y.; Howden, B.P.; Jiang, J.H.; Stinear, T.; Peleg, A. Population genetics and the evolution of virulence in Staphylococcus aureus. Infect Genet Evol., v. 21, p 554-562., 2014. DOI: 10.1016/j.meegid.2013.04.026. [69] AL-Haj, N, A.; Hauter, J. M.; Al-Bulili, N. H.; Al-Hotami, R. A.; Al-Horaibi, M. T.Nasal Carriage of Staphylococcus aureusAmong Students of Public Schools in Sana‘a, Yemen. Research Journal of Microbiology, v. 13, n. 1, p. 65-69, 2018.DOI:10.3923/jm.2018.65.69 [70] Hur YE, Park Y.Vancomycin-Functionalized Gold and Silver Nanoparticles as an Antibacterial NanoplatformAgainst Methicillin-Resistant Staphylococcus aureus. J Nanosci. Nanotechnol., v.16, n. 6, p. 6393-6399, 2016. doi:10.1166/jnn.2016.12393. [71] IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997). Online version (2019-) created by S. J. Chalk. DOI: https://doi.org/10.1351/goldbook. [72] Pavia, D. L.; Lampman, G. M.; Kriz, G. S.; Vyvyan, J. R. Introdução a espectroscopia. Tradução 5. ed. São Paulo: Cenage Learning, 2016. [73] Abbas, Q. Understanding the UV-Vis Spectroscopy for Nanoparticles. J Nanomater Mol Nanotechnol, v. 8, n. 3, p.1-3, 2019. DOI: 10.4172/2324-8777.1000268. [74] Douglas A. Skoog, Donald M. West, F. James Holler, Stanley R. Crouch Fundamentals of Analytical Chemistry. 8th Ed., 2003. [75] Perkinelmer, Life And Analytical Sciences. FT-IR Spectroscopy Attenuated Total Reflectance (ATR). Technical note. 2005. Disponível em: https://www.perkinelmer.com Acesso em: setembro de 2019. [76] Skoog, D. A.; Holler, F. J.; Nieman, T. A. Princípios de Análise Instrumental. 5ª ed., Mc, Madrid, 2001. 5 1 [77] Ramer, G.; Lendl, B. Attenuated Total Reflection Fourier Transform Infrared Spectroscopy, 2013. DOI: https://doi.org/10.1002/9780470027318.a9287 [78] Mourant, J. R.; Gibson, R. R.; Johnson, T. M.; Carpenter, S.; Short, K. W.; Yamada, Y. R.; Freyer, J. P. . Methods for measuring the infrared spectra of biological cells. Physics in Medicine and Biology, v. 48, p. 243–257, 2003. Disponível em: https://iopscience.iop.org/article/10.1088/0031-9155/48/2/307/pdf [79] Blout, E.R.; Mellors, R.C. Infrared spectra of tissues. Science. v. 110, p. 137–138, 1949. DOI: 10.1126/science.110.2849.137. [80] Woernley, D.L. IR absorption curves for normal and neoplastic tissues and related biological substances. Cancer Res. v. 12, p. 516–523, 1952. Disponível em: http://cancerres.aacrjournals.org/content/12/7/516.full-text.pdf. Acesso em: setembro 2017. [81] Albani, J. R. Structure and Dynamics of Macromolecules: Absorption and Fluorescence Studies. Elsevier, 2004. ISBN 0-444-51449-X. [82] Valeur, B.; Berberan-Santo, M. N. A Brief History of Fluorescence and Phosphorescence before the Emergence of Quantum Theory. Journal of Chemical Education, v.88, n. 6, p. 731–738, 2011. DOI:10.1021/ed100182h [83] Phillips, R.; Fluorometric techniques in clinical pathology. Progress in Clinical Pathology, Grune and Stratton, New York, 1965. [84] Sauer, M.; Hofkens, J. J.; Enderlein, J. Handbook of fluorescence spectroscopy and imaging: from esemble to single molecules. Wiley, 2011. ISBN: 978-3-527- 63352-4 [85] Lakowics, J. R. Principles of fluorescence Spectroscopy. 3rd Ed. New York : Springer, 2006. [86] Ramos-Vara, J. A.; Miller M. A. When tissue antigens and antibodies get along: revisiting the technical aspects of immunohistochemistry--the red, brown, and blue technique. Veterinary Pathology, v. 51, n. 1, p. 42–87, 2014. DOI: 10.1177/0300985813505879 [87] Coons, A.H.; Creech, H.J.; Jones, R.N. Immunological properties of an antibody containing a fluorescent group. Proc Soc Exp Biol Med, v. 47, p. 200-202, 1941. [88] Whiteside, G; Munglani, R (1998). "TUNEL, Hoechst and immunohistochemistry triple-labelling: an improved method for detection of apoptosis in tissue sections—an update". Brain Research Protocols. 3: 52–53. DOI:10.1016/s1385-299x(98)00020-8 [89] Minsky, M. Memoir on Inventing the Confocal Scanning Microscope. Published in Scanning, v.10, p. 128-138, 1988. DOI: https://doi.org/10.1002/sca.4950100403 [90] Tovey, S. C.; Brighton, P. J.; Bampton, E. T. W.; Huang, Y.; Willars, G. B. Confocal Microscopy: Theory and Applications for Cellular Signaling. Calcium Signaling Protocols, v. 937, p. 51-93, 2013. DOI: https://doi.org/10.1007/978-1- 62703-086-1_3 5 2 [91] Picot, J.; Guerin, C. L.; Le Van Kim, C.; Boulanger, C. M. Flow cytometry: retrospective, fundamentals and recent instrumentation. Cytotechnology, v. 64, n. 2, p. 109–130, 2012. DOI:10.1007/s10616-011-9415-0 [92] Givan, A. L. Flow Cytometry: An Introduction. Flow Cytometry Protocols. Methods in Molecular Biology, v. 699, p. 1–29, 2015. DOI:10.1007/978-1-61737-950- 5_1. [93] Mannheimer, W. A. Microscopia dos materiais: uma introdução. Rio de Janeiro: E-papers services editoriais, 2002. [94] F. Zernike. Phase contrast, a new method for the microscopic observation of transparent objects. Physica, 1942, part I: 10.1016/S0031-8914(42)80035-X, part II: DOI: 10.1016/S0031-8914(42)80079-8. [95] E. Horn, R Zantl. Phase-Contrast Light Microscopy of Living Cells Cultured in Small Volumes. Microsc Anal, v. 20, n. 3, p. 5–7, 2006. DOI: [96] Neto, J.M.M.; Moita, G.C. Uma introdução à análise exploratória de dados multivariados. Quim. Nova. v. 21, p. 467-469, 1997. DOI: 10.1590/S0100- 40421998000400016. [97] Gemperline, P. Practical Guide To Chemometrics. 2. ed. CRC Press, 2006. [98] Bro, R.; Smilde, A. K. Principal component analysis. Analytical Methods, v. 6, p. 2812-2831, 2014. DOI: 10.1039/c3ay41907j. [99] Jake Lever, J.; Krzywinski, M.; Altman, N. Points of significance Principal component analysis. Nature Methods, v. 14, p. 641–642, 2017. [100] Brereton, R. G.; Loyd, G. R. Partial least squares discriminant analysis: taking the magic away. Journal of Chemometrics, v. 28, p. 213-225, 2014. DOI: 10.1002/cem.2609. [101] Bylesjo, M.; Rantalainen, M.; Cloarec, O.; Nicholson, J. K.; Holmes, E.; Trygg, J. OPLS discriminant analysis: combining the strengths of PLS-DA and SIMCA classificationy. Journal of Chemometrics, v. 20, p. 341–351, 2006. DOI: 10.1002/cem.1006. [102] Gorjão, R. Contagem de Células. In: PERES, C. M & CURI, R. Como Cultivar Células. Rio de Janeiro: Guanabara Koogan, 2005, cap. 5, p. 22-24. [103] Romeo, L.; Lanza Cariccio, V.; Iori, R.; Rollin, P.; Bramanti, P.; Mazzon, E. The α-Cyclodextrin/Moringin Complex: A New Promising Antimicrobial Agent against Staphylococcus aureus. Molecules, v. 23, n. 9, p. 2097-2107, 2018. DOI: 10.3390/molecules23092097. [104] Vargas-Casanova, Y.; Rodríguez-Mayor, A. V.; Cardenas, K. J.; Leal-Castro, A. L.; Muñoz-Molina, L. C.; Fierro-Medina, R.; Rivera-Monroy, Z. J.; García-Castañeda, J. E. Synergistic bactericide and antibiotic effects of dimeric, tetrameric, or palindromic peptides containing the RWQWR motif against Gram-positive and Gram-negative strains. RSC Advances, v. 9, p. 7239-7245, 2019. DOI: 10.1039/C9RA00708C 5 3 [105] Cui, L.; Butler, H. J.; Martin-Hirsch, P. L.; Martin, F. L. Aluminium foil as a potential substrate for ATR-FTIR, transflection FTIR or Raman spectrochemical analysis of biological specimens. Analytical Methods. v. 8, p. 481-487, 2016. DOI: 10.1039/C5AY02638E. [106] Safieh-Garabedian B, Poole S, Allchorne A, Winter J, Woolf CJ. Contribution of interleukin-1 beta to the inflammation-induced increase in nerve growth factor levels and inflammatory hyperalgesia. Br J Pharmacol 1995;115:1265– 75. [107] Kendall C.; Ionescu-Matiu, I.; Dreesman, G.R. Utilization of the biotin/avidin system to amplify the sensitivity of the enzyme-linked immunosorbent assay (ELISA). J Immunol Methods 1983;56:329–39. [108] Koster, R.; Anderson, M.; Debeer, E. J. Acetic acid for analgesic screening. Fed Proc 1959;18:412–6. [109] Gray, E. P.; Coleman, J. G.; Bednar, A. J.; Kennedy, A. J.; Ranville, J. F.; Higgins, C. P. Extraction and Analysis of Silver and Gold Nanoparticles from Biological Tissues Using Single Particle Inductively Coupled Plasma Mass Spectrometry. Environmental Science & Technology, v. 47, p. 14315-14323, 2013. DOI: 10.1021/es403558c. [110] Abdelhalim, M. A. K.; Jarrar, B. M. Histological alterations in the liver of rats induced by different gold nanoparticle sizes, doses and exposure duration. Journal of Nanobiotechnology, v. 10, p. 5-5, 2012. DOI: 10.1186/1477-3155-10-5. 5 4 Capítulo 2 Anti-inflammatory, analgesic and anti-tumor properties of gold nanoparticles Raimundo Fernandes de Araújo Júnior, Aurigena Antunes de Araújo, Jonas Bispo Pessoa, Franscisco Paulo Freire Neto, Gisele Ribeiro da Silva, Ana Luiza C. S. Leitão Oliveira, Thaís Gomes de Carvalho, Heloiza F. O. Silva, Mateus Eugênio, Celso Sant‘Anna, Luiz H. S. Gasparotto. Pharmacological Reports, 2017, Vol. 69, 119- 129. Contribuição:  Realizei a síntese e purificação das NanoAu.  Realizei a caracterização das NanoAu.  Acompanhei parte dos ensaios e testes biológicos.  Auxiliei parte da escrita da primeira versão do manuscrito. Heloiza Fernanda O. da Silva Prof. Luiz Henrique da S. Gasparotto 5 5 TERMO DE AUTORIZAÇÃO AUTORAL Declaro para os devidos fins, que eu Raimundo Fernandes De Araújo Junior, matricula Siape sob o nº2329140, autorizo Heloiza Fernanda Oliveira da Silva Athayde, matrícula sob o nº20151020790, a utilizar o artigo, no qual sou primeiro autor, intitulado ―Anti- inflammatory, analgesic and anti-tumor properties of gold nanoparticles‖, como parte integrante em sua tese para conclusão do curso de Doutorado em Química pelo Programa de Pós-Graduação em Química da Universidade Federal do Rio Grande do Norte. Comprometendo-me, mediante a apresentação do presente termo, não autorizar outros e nem utilizar o referido artigo em outros trabalhos de conclusões. Natal, 28 de agosto de 2019. Raimundo Fernandes De Araújo Junior Professor Associado- Departamento de Morfologia Universidade Federal do Rio Grande do Norte 5 6 Pharmacological Reports 69 (2017) 119–129 Contents lists available at ScienceDirect Pharmacological Reports journal homepa ge: www.elsev ier .com/locate /pharep Original article Anti-inflammatory, analgesic and anti-tumor properties of gold nanoparticles Raimundo Fernandes de Araújo Júniora,b,c,**, Aurigena Antunes de Araújod, Jonas Bispo Pessoab, Franscisco Paulo Freire Netoe, Gisele Ribeiro da Silvad, Ana Luiza C.S. Leitão Oliveirac, Thaís Gomes de Carvalhoc, Heloiza F.O. Silvaf, Mateus Eugêniog, Celso Sant’Annag, Luiz H.S. Gasparottof,* aDepartment of Morphology, Federal University of Rio Grande do Norte, Natal 59072-970, RN, Brazil b Post Graduation Programme in Structural and Functional Biology, Federal University of Rio Grande do Norte, Natal 59072-970, RN, Brazil c Post Graduation Programme in Health Science, Federal University of Rio Grande do Norte, Natal 59072-970, RN, Brazil dDepartment of Biophysics and Pharmacology, Post Graduation Programme in Public Health, Post graduation programme in Pharmaceutical Science, Federal University of Rio Grande do Norte, Natal 59072-970, RN, Brazil eDepartment of Biochemistry, Federal University of Rio Grande do Norte, Natal 59072-970, RN, Brazil fGroup of Biological Chemistry and Chemometrics, Institute of Chemistry, Federal University of Rio Grande do Norte, Natal 59072-970, RN, Brazil g Laboratory of Biotechnology – Labio, National Institute of Metrology, Quality and Technology – Inmetro, Duque de Caxias 25250-020, RJ, Brazil A R T I C L E I N F O A B S T R A C T Article history: Background: Gold nanoparticles (GNPs) are regarded as potential platforms for drug delivery. However, Received 14 March 2016 their interaction with live organisms must be understood prior to their utilization as drug carriers. The Received in revised form 17 September 2016 present study reports the anti-inflammatory, analgesic and anti-tumor effects of GNPs. The Accepted 19 September 2016 biodistribution of GNPs and their effect on various tissues have also been studied. Available online 21 September 2016 Methods: GNPs were synthesized through an environmentally friendly route and characterized with TEM and UV–vis. After HT-29 cells had been exposed to GNPs, apoptosis was assessed with Annexin V and Keywords: propidium iodide staining and caspase-3 activity determined with a confocal laser scanning microscope. Gold nanoparticles Cancer cell apoptosis GNPs were administrated to male and female Swiss mice for posterior assessment of their anti- Anti-inflammatory activity inflammatory and analgesic properties. The biodistribution of GNPs and their impact on tissues were Peripheral analgesia studied with UV–vis and histopathological analysis, respectively. Results: Cell apoptosis was observed in a dose-dependent manner for GNPs concentrations ranging from 40 mg/mL to 80 mg/mL (p < 0.05). The best anti-inflammatory activity was observed at the dose of 1500 mg/kg, which caused a reduction of 49.3% in leukocyte migration. GNPs showed peripheral analgesia at the dose of 1500 mg/kg and have been found in liver, spleen, kidney and lungs. Histopathological examination revealed extravasation of red blood cells in lungs. Conclusion: The study draws attention to gold nanoparticles as a resource for technological innovation in the anti-inflammatory, analgesic and anti-tumor fields. GNPs have biological effects that deserve investigation to assess their full interaction with organic systems. © 2016 Published by Elsevier Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences. Background potential early detection, diagnosis, and targeted treatment of diseases [2,3]. However, for safe use of this relative new Metal nanoparticles have been employed in biomedicine since technology, it is imperative that its biological and toxicological the 1970s thanks to the conjugation of gold nanoparticles (GNP) effects on living systems be understood prior to its dissemination. with antibodies, opening the era of immune gold labeling [1]. Due to their minute dimensions and high surface-to-volume ratio, Nanotechnology has since then furthered its boundaries for nanomaterials may enter the circulatory and lymphatic systems producing irreversible injuries through exacerbate oxidative stress [4]. As an example, silver nanoparticles induced oxidative stress * Corresponding author. and genotoxicity in cultured cells and animal tissues [5]. On the ** Corresponding author. other hand, studies have shown that nanoparticles may be used as E-mail addresses: araujojr@cb.ufrn.br (R.F. de Araújo), lhgasparotto@ufrnet.br carriers of anti-inflammatory [6] and immunosuppressive [7] (L.H.S. Gasparotto). http://dx.doi.org/10.1016/j.pharep.2016.09.017 1734-1140/© 2016 Published by Elsevier Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences. 57 120 R.F. de Araújo et al. / Pharmacological Reports 69 (2017) 119–129 Fig. 1. (A) UV–vis spectrum of the colloidal GNPs, (B) TEM image of the GNPs. Inset: size distribution of the GNPs. drugs with mild acute citoxicity [8]. Gold or silver nanoparticles Synthesis and characterization of GNPs conjugated with heparin exhibited anti-inflammatory properties without any significant effect on systemic hemostasis [9]. Chen 100 mg of PVP are mixed with 2 mmol of HAuCl4 and ultrapure et al. [10] reported a reduction in TNFa and IL-6 mRNA levels when water was added to generate a 5-mL solution. In a separate flask, 21-nm gold nanoparticles were injected into mice, a result 0.20 mol of glycerol was mixed with 0.20 mol of NaOH and attributed to fat loss and inhibition of inflammatory effects. ultrapure water was added to generate a 5-mL solution. The Administration of nanoparticles of MnO2 caused accumulation of glycerol-NaOH solution was then added to the HAuCl4-PVP to yield Mn in brain, spinal cord and muscle tissues of rats [11], leading to 10 mL of GNPs solution. The GNPs colloidal solution had then its pH an impact on pain sensation. Finally, nanoparticles have also adjusted to 7 by addition of diluted HCl and was subjected to presented anti-tumor properties, which is quite interesting from dialysis for purification. UV–vis absorption spectra of the GNPs the viewpoint of formulating new drugs against cancer [12]. were acquired with an Evolution 60S UV–vis spectrophotometer The aim of the present study is to evaluate the anti- (Thermo Scientific). Transmission electron microscopy (TEM) inflammatory, analgesic, and anti-tumor properties of GNPs that images were acquired with a Tecnai Spirit BioTwin 12 microscope were synthesized through a low-toxicity chemical route. We operating at 120 kV. already showed that gold nanoparticles produced via the glycerol route [13] have catalytic [14] properties and can also be combined Cell culture with antibodies to generate an extremely efficient contrast agent for colorectal cancer cell imaging [15]. The latter study prompted A colorectal cancer cell line (HT-29) was purchased from the us to understand the effect of GNPs on cancer cell viability and Culture Collection of the Federal University of Rio de Janeiro (RJCB other effects in vivo, since the ultimate goal is to formulate, in the Collection, Rio de Janeiro, RJ). HT-29 cells were maintained in future, more efficient drugs that are based on nanoparticles. Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum. Gold nanoparticles and Materials and methods cisplatin solutions were filtered using a 0.22 mm membrane (EMD Millipore) and stored at 20  C. Reagents Cell viability The following reagents were purchased as indicated: Dulbec- co’s modified Eagle’s medium (Life Technologies, Grand Island, NY, HT-29 cell viability (1105 cells) was determined by Trypan USA), 10% (v/v) heat-inactivated fetal bovine serum (CULTILAB Blue exclusion assay 24 h after inoculation with different concen- LTDA/Brazil), trypsin/EDTA (Gibco BRL, Life Technologies, Grand trations of GNPs (5–400 mM in aqueous suspensions). Briefly, cell Island, NY, USA), cisplatin (citoplax, 50 mg, Bergamo Taboão da aliquots were mixed with 0.5% (w/v) Trypan Blue and incubated at Serra, SP, Brazil). Gold(III) chloride (30% wt in HCl), sodium room temperature for 5 min. The number of viable cells was hydroxide, glycerol, and polyvinylpyrrolidone (PVP, molecular calculated using a hemocytometer. The effect of stabilizing agent of weight = 10,000) were products of Sigma-Aldrich Co. gold nanoparticle (10 1 g l PVP) was also tested. Annexin V and propidium iodide staining The apoptotic assay was conducted according Araújo Jr et al. [16]. HT-29 cells were placed in 6-well plates (2  105 cells/well) with 2 ml medium/well. After 24 h, different concentrations of gold nanoparticles (10 mM, 20 mM and 40 mM) and cisplatin (50 mM and 100 mM) were added and allowed to react for 24 h and 48 h. In parallel, control cells were maintained in culture medium without gold nanoparticles or cisplatin. The cells were then assayed using the Annexin V-FITC Apoptosis Detection kit I (Biosciences Pharmingen, San Diego, USA). Annexin V-FITC and propidium iodide (PI) were added to the cellular suspension according to the manufacturer’s instructions. A total of 1.0106 cells from each Fig. 2. Half-maximal inhibition of HT-29 growth was observed for concentrations of GNPs ranging from 5mM to 40mM. PVP did not alter HT-29 growth. ***p 0.0001, # sample were then analyzed using a FACS Calibur cytometer (BD < p > 0.05. Bioscience, Franklin Lakes, NJ, USA) and FlowJo software (BD 58 R.F. de Araújo et al. / Pharmacological Reports 69 (2017) 119–129 121 Fig. 3. Effect of different doses of GNPs and cisplatin on early and late apoptosis in HT-29 cells. Bottom-right quadrants represent the Annexin V-FITC-positive/PI-negative cells in the early stages of apoptosis, while top-right quadrants include Annexin V-FITC-positive/PI-positive cells in late stages of apoptosis. Treatment with 50 mM and 100 mM cisplatin induced both early and late apoptosis in HT-29 cells at 24 h (B, C) and 48 h (H,I). 59 122 R.F. de Araújo et al. / Pharmacological Reports 69 (2017) 119–129 90-day-old male and female Swiss mice (25–35 g) and rats (200–250 g) were donated by the Department of Biophysics and Pharmacology at the Federal University of Rio Grande Norte. All animals were housed in cages under standard laboratory conditions  of 22  2 C and 12 h light/12 h dark cycle, and fed with pelleted food and water ad libitum. They were acclimatized for 7 days prior to the experiments and subjected to fasting before running the tests. Animal welfare and experimental procedures were in strict accordance with Institutional Review Board of the Federal University of Rio Grande do Norte that approved this specific study (approved protocol no 053/2013). In this work animals were handled in a humane and appropriate manner. For the anti-inflammatory experiments female rats were divided into five groups (n = 6/group). The testing group received GNPs orally at doses of 700 mg/kg, 1000 mg/kg and 1500 mg/kg. The Fig. 4. Apoptosis induced by GNPs and cisplatin in HT-29 cells detected by flow cytometry. ***p<0.001; **p<0.01; *p<0.05; #p> 0.05. anti-inflamatory standard group received indomethacin orally at a dose of 10 mg/kg. Finally, 10 mg/kg PVP (the vehicle) was administered to the positive control group. Thirty minutes later all groups were injected subcutaneously with 0.25 ml of carra- Biosciences). Annexin V-FITC- positive/PI-negative cells were geenan (1% in saline solution), and after 4 h the animals were identified as cells in the early stages of apoptosis, while Annexin euthanized with Thiopental (100 mg/kg). The total leukocyte count V-FITC-positive/PI-positive cells were identified as cells in late was determined in a Neubauer chamber. The percentage of the stages of apoptosis, or cells that were undergoing necrosis. The leukocyte inhibition was calculated with the following equation: experiment was run in triplicate. % leukocyte inhibition = (1  T/C)  100, Caspase-3 activity where T represents the treated-group leukocyte counts and C represents the control-group leukocyte counts. Cultured cells were plated on glass coverslips in 24-well plates  4 Peritoneal fluid, stored at 70  C after extraction, was (5 10 cells/well). After 24 h, they were treated with the 40 mM homogenized and processed as described elsewhere [17]. Levels and 400 mM GNPs and 100 mM cisplatin for 48 h. The cells were of IL-1b (detection range: 62.5–4000 pg/mL; lower limit of then washed, fixed with paraformaldehyde, permeabilized by detection: 12.5 ng/mL recombinant mouse IL-1b) and TNF-a Triton-X, and incubated with rabbit polyclonal anti-caspase-3 (detection range: 62.5–4000 pg/mL; lower limit of detection: (Abcam, San Francisco, CA, USA) diluted 1: 500 in PBS containing 50 ng/mL recombinant mouse TNF-a) were determined using bovine serum albumin (5%; Life Technologies do Brasil LTDA, commercial ELISA kits (R&D Systems, Minneapolis, MN, USA), as SâoPaulo, Brazil) for 1 h at RT in a humid atmosphere. The primary described previously [18]. All experiments were performed at least antibody was detected with Alexa Fluor 488 goat anti-rabbit in triplicate and significant differences between groups were secondary antibody (Abcam), and 4,6-diamidino-2-phenylindole calculated using analysis of variance (ANOVA) and Bonferroni's (Life Technologies do Brasil LTDA) was used for nuclear staining. test. Values of p lower than 0.05 were considered statistically The immunostained coverslips were examined under a LSM 510 significant. confocal laser scanning microscope (Carl Zeiss, Jena, Germany). The hot plate test was carried out to assess the central analgesic Images were obtained with laser excitation at 442 nm. Known activity promoted by GNPs. Only mice that showed initial positive and negative controls were included in each batch of nociceptive responses (licking of the forepaws or jumping) in samples. Cell reactivity in all groups (control, 40 mM GNPs, 400 mM the interval of 3–19 s were used for additional experiments. The GNPs and 100 mM Cisplatin) was assessed by computerized experiments were conducted at 55  0.5 C with an Insight hot densitometry analysis using digital images captured with the plate apparatus (São Paulo, Brazil). The selected mice were pre- confocal immunofluorescence microscope. Average densitometric treated with GNPs (700 mg/kg, 1000 mg/kg, 1500 mg/kg; po) and values were obtained using the Image J software (version 1.51f) 30 min later the tests were conducted. A morphine-treated group (http://rsb.info.nih.gov/ij/). Contrast index measurements were (10 mg/kg; ip) was included as a standard group. The vehicle (PVP, obtained from five selected areas 100/total area after removing 10 mg/kg, po) was included as a positive control group. The cut-off the labeling (blue or green) positioned across the regions of time was set at 30 s to minimize skin damages the time of latency interest. Five areas were analyzed per sample (triplicate for each was measured at intervals of 30 min. group).  5 The method of Koster et al. [19] was used to evaluate peripheralHT-29 cells were placed (5 10 cells/well in 2 mL medium) in analgesic activity. The female Swiss mice were divided in five six- six-well culture plates. After 24 h the cells were treated with GNPs mouse groups and fasted overnight. The animals were treated (40 mM and 400 mM) and 100 mM Cisplatin for 48 h. Afterwards, orally with indomethacin/standard Group (10 mg/kg, po), vehicle/ morphological changes associated with apoptosis, such as positive control group (10 ml/kg, po) and GNPs (700 mg/kg, membrane blebbing and formation of apoptotic bodies, were 1000 mg/kg and 1500 mg/kg, po). The mice were treated with observed with a phase-contrast microscope. acetic acid (0.6%, v/v in saline, 10 ml/kg, ip) 30 min after the above- mentioned treatment. The number of writhes was counted for Animals 20 min and the percentage of inhibition was calculated as follows: %Inhibition = number of writhes (control)  number of writhes The following chemicals were used: 195.8 mg/mL GNPs, (test)  100 number of writhes (control) thiopental (Thiopentax, 1.0 g, Cristália, São Paulo, Brazil), indo- methacin (INDOCID 25 mg Aspen Pharma), morphine sulphate Quantitative determination of the biodistribution of GNPs and (Dimorf 10mg/mL1ml/Cristália1 São Paulo, Brazil), histopathological examination were carried out according to the 60 R.F. de Araújo et al. / Pharmacological Reports 69 (2017) 119–129 123 Fig. 5. Detection of caspase-3 in HT-29 cells stained with DAPI (blue) and anti-caspase-3 antibodies (green). (A) Untreated HT-29 cells. Positive staining observed in HT-29 cells exposed to (B) 100 mM cisplatin, (C) 40 mM GNPs and weak staining (D) to 400 mM GNPs for 48 h, respectively. (E) Densitometric analysis confirmed a significant increased in caspase-3 immunoreactivity in HT-29 cells treated with 40 mM GNPs and 100 mM cisplatin for 48 h. However, there was a blockage of caspase-3 immunoreactivity in the case of 400 mM GNPs. Five areas were selected from each sample (three samples per treatment), **p < 0.01, *p < 0.05, Kruskal-Wallis test followed by Dunn’s test. Phase-contrast images of HT-29 cells (F1) without treatment (control), (F2) exposed to 100 mM cisplatin, (F3) exposed to 40 mM GNPs and (F4) exposed to 400 mM GNPs taken after 48 h. Magnification: 100. Symbols on the images indicate normal morphology (asterisk), bubbles on the membrane (light-blue arrows), apoptotic cells (dark- blue arrow); accumulation of GNPs inside the cell (blue circles), and blackened cluster corresponding to GNPs (triangle). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) following procedure. 2% thiopental (100 mg/kg) was employed to euthanize female mice 30 min, 60 min and 24 h after administra- tion of 1500 mg/kg GNPs or 10 mg/kg PVP, followed by harvesting of heart, lungs, liver, kidneys, spleen, stomach, and intestine with their posterior digestion in tetramethylammonium hydroxide 20% (TMAH solution 25%, Sigma-Aldrich) for 24 h to release the nanoparticles from the tissues [20]. For histophatological analysis, samples of the liver from each mice were cut immediately after harvesting, fixed in neutral buffered formalin (10%), and dehy- drated with ethanol of distinct grades (70, 80, 90, 95 and 100%). The samples were then cleared in 2 changes of xylene, followed by impregnation molten paraffin wax (2 changes), embedded and Fig. 6. Percentage of leucocyte inhibition by indomethacin and GNPs (*p < 0.05; blocked out. Paraffin sections (4–5 mM) were stained with **p < 0.01). hematoxylin and eosin according to [21]. 61 124 R.F. de Araújo et al. / Pharmacological Reports 69 (2017) 119–129 Fig. 8. Percentage of writing response inhibition for indomethacin (control) and for the following GNPs doses: 700 mg/kg, 1000 mg/kg and 1500 mg/kg. (***p < 0.001). with a half-maximal inhibiting concentration value (IC50) of 10 mM, 20 mM and 40 mM. Induction of apoptosis by GNPs evaluated using flow cytometry Cells were stained with Annexin-V-FITC and PI in order to determine whether cell death induced by GNPs was achieved through apoptosis. Using flow cytometry, early and late stages of apoptosis were detected based on the percentage of Annexin V- Fig. 7. Levels of IL-1b and TNF-a in peritoneal fluid (*p < 0.05; ***p < 0.001). FITC-positive cells/PI-negative cells and the percentage of Annexin V-FITC-positive/PI-positive cells (Fig. 3, lower right quadrant data Results versus top left quadrant data, respectively). For HT-29 cells, treatment with 10 mM, 20 mM and 40 mM of GNPs induced early Synthesis and characterization of GNPs and late apoptosis both at 24 h and 48 h after treatment (Fig. 3). When HT-29 cells were treated with 50 mM and 100 mM cisplatin, Fig. 1A shows a UV–vis spectrum of GNPs produced as result of both early and late stage apoptosis were detected 24 h and 48 h addition of glycerol/NaOH to HAuCl4-PVP at room temperature. after treatment. Comparison with cisplatin is important since this Red-colored solutionswere readily obtained uponmixing glycerol/ drug is used for a broad spectrum of cancer types. After 48 h NaOH with Au3+/PVP. The red color is due to the surface plasmon apoptosis was observed for all GNPs doses and cisplatin (Fig. 4). band (SPB) as a The GNPs dose of 40 mM GNPs (p < 0.001) also induced apoptosis result of the resonant coherent dipolar oscillations of the electron gas (electrons of the conduction band) at the after 24 h. surface of nanoparticles. The SPB is a valuable tool for inference about the size regime of some metal particles (e.g. Ag and Au) [14,22]. The Caspase-3 activity colloidal GNPs spectrum had a maximum absorbance (lMax) at 520 nm, a value typical for spherical gold nanoparticles [23–25]. Representative images of HT-29 cells following 40 mM and The TEM image illustrated that themajority of GNPswere spherical 400 mM GNPs treatment and 100 mM cisplatin are shown in Fig. 5. in shape, corroborating the UV–vis results. The mean particle size Strong positive staining (green) to caspase-3 indicated that HT-29 calculated through the TEM images was 7.4 nm2.8 nm (inset of cell death was mediated by an apoptotic process. Densitometric Fig. 1B). analysis confirmed a significant increase in caspase-3 immunore- activity in the HT-29 cells treated with 40 mM GNPs (p < 0.05) and Colorectal cancer cells viability 100 mM cisplatin (p < 0.01) for 48 h, which was weakly labeled in the case of 400 mM GNPs. HT-29 cells showed a decreased growth when they were Cell membrane blistering and apoptotic bodies formation submitted to the lowest concentrations (5mM, 10mM, 20mM and (Fig. 5F2) appeared upon treatment with 100 mM cisplatin for 40mM) of GNPs for 24h. According to Tryplan Blue assay results, 48 h. Exposure to 40 mM GNPs promoted similar changes in cells, the GNPs significantly impacted the cell viability in a concentra- with formation of bubbles on their membranes and apoptotic tion-dependent manner. The lowest mortality rate was obtained at bodies (Fig. 5F4). Moreover, GNPs were found inside the cells. On a nanoparticle concentration of 100mM, 200mM and 400mM the other hand, no significant alterations on cell morphology were (p> 0.05), whereas the highest mortality rate was obtained at observed upon treatment with 400 mM GNPs, with cellular aspect 40mM (p<0.05) (Fig. 2). The GNPs were cytotoxic to HT29 cells similar to that of the control group. In this case, the accumulation Table 1 Effect of GNPs and morphine on pain latency (hot plate test). Initial pain latency (s) Pain latency after drug administration observed * at distinct time. p < 0.05 and **p < 0.01 compared to saline-treated group. 0 min 30 min 60 min 90 min 120 min Control (DMSO) 10.0  5.3 4.0  1.7 6.5  3.1 8.5  1.6 6.3  0.8 10 mg/kg morphine 8.6  2.2 27.2  3.7 ** 24.4  5.9* 19.6  9.1* 18.5  3.4* 700 mg/kg GNPs 10  28.2 13  11.2 11.2  13.3 7.8  4.5 6.8  5.3 1000 mg/kg GNPs 20  6.1 12.4  5.3 9.6  9.0 13.2  7.4 10.4  8 1500 mg/kg GNPs 14  6.0 7.8  4.1 10.4  6.3 10.4  11.8 10.4  11.4 62 R.F. de Araújo et al. / Pharmacological Reports 69 (2017) 119–129 125 into the peritoneal cavity. The positive control group showed an increase in the numbers of leukocytes from peritoneal exudates, while the standard group (indomethacin) reduced the cell recruitment into the peritoneal cavity. The number of leukocytes was significantly reduced up to 4 h after carrageenan injection in peritoneal exudates by indomethacin (77.7%), GNPs 700 mg/kg (18.3%), GNPs 1000 mg/kg (25.2%) and GNPs 1500 mg/kg (49.3%) pretreatments, when compared to exudates from the positive control group (animal injected with carrageenan). The doses of 1000 mg/kg (p < 0.05) and 1500 mg/kg (p < 0.01) significantly reduced leukocytes migration as shown in Fig. 6. The mechanism of carrageenan action on peritonitis involves synergism among prostanoids, leukotriene B4, and other chemostactic agents such as C5a and IL-8 which promote leukocyte recruitment [26]. Effect of GNPs on cytocines IL-1b and TNF-a The group subjected to GNPs showed decreased levels of Fig. 9. Tissue distribution of 1500 mg/kg GNPs administered orally. proinflammatory cytokine IL-1b (700 and 1500 mg/kg, p < 0.05) and TNF- a (all doses, p < 0.001) compared to PVP (positive control of GNPs inside the cell was also visualized in parallel with their group) as shown in Fig. 7. aggregation on the membrane (Fig. 5F3). Hot plate test Tests with animals Nanoparticle GNPs administered at 700 mg/kg, 1000 mg/kg and 1500 mg/kg did not show analgesic activity at times of incubation GNPs inhibit carrageenan peritonitis in mice ranging from 30 min to 120 min (p > 0.05). Morphine caused The carrageenan-induced peritonitis test was used in order to significant anti-nociception during the course of experiment evaluate a possible inhibitory effect of GNPs for cells recruitment (p < 0.05) (Table 1). In the hot-plate model, nociceptive reaction Fig. 10. Microscopic images of liver (A) and lung (B) cells after GNPs administration of 1500 mg/kg GNPs. Magnification: 40. 63 126 R.F. de Araújo et al. / Pharmacological Reports 69 (2017) 119–129 Fig. 11. Microscopic images of kidney (A) and spleen (B) cells after administration of 1500 mg/kg GNPs. Magnification: 40. toward thermal stimuli in mice is a well-validated model for looking heart muscles of distinct heart muscle orientations with no detection of opiate analgesics as well as several types of analgesics sign of pathological processes such as inflammation or necrosis drugs from spinal origin [27]. (Fig. 12A). Microscopic images of lungs tissue of control groups (saline medium and exposed to PVP) revealed well-formed and Acetic acid writhing reflex open alveoli with normal spate, few scattered small lymphocytes, The effects of GNPs and indomethacin on the writhing response and minimal eosinophils (Fig. 10B), whereas the GNPs-treated in mice are shown in Fig. 8. The dose of 1500 mg/kg inhibited the group presented extravasation of red blood cells that became more acetic acid induced writhing response. The percentage of evident with time (Fig. 10B). The GNPs have not caused appreciable contraction inhibition by the GNPs at the dose of 1500 mg/kg pathological alterations in stomach and intestine (Fig. 12B and C, was reached close to the standard drug percentage (68.8% for GNPs respectively). vs. 71.7% indomethacin). When compared to control condition the inhibition was significantly reduced (p < 0.001). Discussion Quantitative determination of the biodistribution of GNPs Many studies have shown that nanoparticles may induce Fig. 9 presents the evolution of the distribution of GNPs with genotoxicity and cytotoxicity in cancer and normal cell lines time. We found that 30 min after administering 1500 mg/mL gold [28,29]. In the present study, GNPs were also found to be cytotoxic nanoparticles 19.3% of them accumulated in the liver, 45.2% in the to some extent to HT-29 cancer cells with an IC50 viability value of stomach, 19.6% in the intestine and 15.9% in the spleen. After 1 h, 40 mM. GNPs were found in liver (21.1%), heart (7.9%), lungs (6.5%), kidneys According to the literature [30], GNPs with a mean diameter of (25.2%) and spleen (23.6%). With time, the GNPs migrated to heart 5 nm are highly efficient in inhibiting proliferation, promoting and lungs, reaching 9.3% and 16.8%, respectively, after 24 h. apoptosis, and arresting the cell cycle at the G0/G1 phase in two lung cancer cell lines. In contrast, no obvious cytotoxicity was Histopathological examination observed for 10 nm, 20 nm, and 40 nm GNPs–treated cells. In the Liver and spleen of both control and GNP-treated groups current study, we observed that GNPs decreased the cell viability presented no alterations at any time (Figs. 10 A and 11 B , and caused moderate apoptosis of HT-29 cancer cells. This result is respectively). Concerning the heart, results showed benign blunt- in consonance with the histogram in the inset of Fig. 1B in which is 64 R.F. de Araújo et al. / Pharmacological Reports 69 (2017) 119–129 127 Fig. 12. Microscopic images of heart (A), stomach (B) and intestine (C) cells after administration of 1500 mg/kg GNPs. Magnification: 40. observed that 66% of the nanoparticles have sizes smaller that inhibition of intracellular antioxidants or accumulation of reactive 8.0 nm. In this case it may be argued the GNPs smaller than 8.0 nm oxygen, as demonstrated by Gao et al. [36] caused apoptosis, while bigger ones (44%) did not significantly Concerning the anti-inflammatory tests, the highest GNPs dose impact on both growth and apoptosis. (1500 mg/kg) generated a 49% reduction in leukocyte migration, The highest concentrations of GNPs (100 mM, 200 mM, and which attested the activation of a cellular anti-inflammatory 400 mM) did not cause statistically significant growth inhibition, a response. These findings were also confirmed by the reduction of behavior that may be a consequence of GNPs aggregation [31]. The proinflammatory cytokines IL-1 beta and TNF-alpha. A study by culture media typically comprise several components (such as Dohnert et al. [37] on traumatic tendinitis in rats revealed that the electrolytes, metal ions, and proteins) that can interact with the control group treated with gold nanoparticles showed a significant nanoparticles causing destabilization of surface charges leading to reduction of IL-1 b when compared to the untreated group (injured nanoparticle aggregation [32], especially when they are present at rats without treatment). GNPs also promoted a significant decrease high concentration. In this case the GNPs cytotoxicity is influenced in TNF-a and IL-1b levels in injured muscle [38]. because it is more difficult for the cell to internalize aggregated Another important point was to verify the anti-inflammatory, nanoparticles. This is in consonance with the caspase-3 results peripheral, and analgesic activities of GNPs since these signaling shown in Fig. 5, in which it is possible to observe strong labeling pathways may or may not be related. Acetic acid induces capillary (indicated by red narrows) corresponding to aggregated caspase-3 permeability and liberates endogenous substances that sensitize (green) in cytoplasm. An inverse correspondence between pain nerve ending, leading to inflammatory pain with an increase nanoparticle concentration and toxicity has also been observed of PGE1 and PGE2 peripherally [39]. The mechanism of analgesic by others [33,34]. activity of 1500 mg/kg GNPs could probably be the blockage of the Another point worth mentioning is the time-dependent effect or the release of endogenous substances that excite pain moderate rate of apoptosis in colorectal carcinoma cells (HT-29). nerve endings similar to that of indomethacin and NSAIDs. Thus, For the GNPs dose of 20 mM, the total apoptosis levels measured the reduction in number of writhing indicates that GNPs at the after 24 h of incubation were not statistically significant. On the dose of 1500 mg/kg might exert anti-nociceptive activity by other hand, statistically significant apoptosis levels were observed inhibition of prostaglandin synthesis or action. after 48 h of incubation. Some studies have reported that the We have observed systemic distribution of orally-administered incubation time is an essential factor for the GNPs to accumulate GNPs, which were found to be accumulated in liver, lungs and within the cells [31,35] and promote damage by oxidative stress, spleen after 24 h. Such a distribution in peripheral tissues is 65 128 R.F. de Araújo et al. / Pharmacological Reports 69 (2017) 119–129 probably due to the size of the GNPs. De Jong et al. [40] found that [10] Chen H, Dorrigan A, Saad S, Hare DJ, Cortie MB, Valenzuela SM. In vivo study of smaller GNPs spread more easily to peripheral organs than to spherical gold nanoparticles: inflammatory effects and distribution in mice. PLoS One 2013;8:e58208. larger ones. The group studied the distribution of 10 nm, 50 nm, [11] Nosrati N, Hassanpour-Ezzati M, Mousavi SZ, safi Rahmanifar M, Rezagholiyan 100 nm and 250 nm spherical gold nanoparticles administered S. Comparison of MnO2 nanoparticles and microparticles distribution in CNS intravenously, revealing that 10-nm nanoparticles were quite and muscle and effect on acute pain threshold in rats. Nanomed J 2014;1:180– widespread while the larger particles were found primarily in 90.[12] Murawala P, Tirmale A, Shiras A, Prasad BLV. In situ synthesized BSA capped spleen and liver. In other study, Bednarski et al. investigated the gold nanoparticles: effective carrier of anticancer drug Methotrexate to MCF-7 distribution of GNPs in different tissues as a function of the breast cancer cells. Mater Sci Eng C Mater Biol Appl 2014;34:158–67. administration route (iv or po) [41], nding out that themajority of [13] Ferreira EB, Gomes JF, Tremiliosi-Filho G, Gasparotto LHS. One-pot eco-friendly fi synthesis of gold nanoparticles by glycerol in alkaline medium: role of orally-administrated GNPs are excreted within 4 days. A question synthesis parameters on the nanoparticles characteristics. Mater Res Bull still open in that 2014;55:131–6. study is the biodistribution is of the GNPs remaining in the organism. In order to ll that gap, histopatholog- [14] Gasparotto LHS, Garcia AC, Gomes JF, Tremiliosi-Filho G. Electrocatalytic fi performance of environmentally friendly synthesized gold nanoparticles ical analysis was performed on a variety of tissues from mice that towards the borohydride electro-oxidation reaction. J Power Sources received 7.4-nm sized GNPs orally. GNPs were found in different 2012;218:73–8. tissues and caused extravasation of red blood cells in lungs in a [15] Lima KMG, Junior RFA, Araujo AA, Oliveira ALCSL, Gasparotto LHS. Environmentally compatible bioconjugated gold nanoparticles as efficient time-dependent manner. According to Abdelhalim [42], gold contrast agents for colorectal cancer cell imaging. Sens Actuators B-Chem nanoparticles may produce reactive oxygen species that lead to 2014;196:306–13. oxidative stress in cells and organs. The alterations in lung tissue [16] de Araújo Júnio RF, Leitão Oliveira ALCS, de Melo Silveira RF, de Oliveira Rocha HA, de França C, Pedro de Araújo AA. Telmisartan induces apoptosis and suggest that GNPs might have affected its enzimes and proteins, regulates Bcl-2 in human renal cancer cells. Exp Biol Med 2014;240:34–44. which in turn, compromissed the antioxidant defense mechanism [17] Safieh-Garabedian B, Poole S, Allchorne A, Winter J, Woolf CJ. Contribution of and led to stress in lung tissue. interleukin-1 beta to the inflammation-induced increase in nerve growth GNPs themselves provoke reactions in living systems. We have factor levels and inflammatory hyperalgesia. Br J Pharmacol 1995;115:1265– 75. shown that drug-free GNPs cause apoptosis in colorectal cancer [18] Kendall C, Ionescu-Matiu I, Dreesman GR. Utilization of the biotin/avidin cells and promote analgesic and anti-inflamatory effects in mice. system to amplify the sensitivity of the enzyme-linked immunosorbent assay GNPs were found to spread to liver, spleen, kidney and lungs, and to (ELISA). J Immunol Methods 1983;56:329–39. [19] Koster R, Anderson M, Debeer EJ. Acetic acid for analgesic screening. Fed Proc cause extravasation of red blood cells in lungs. It is well known that 1959;18:412–6. nanomaterials serve as drug carriers and countless works in [20] Bingöl B, Durrell AC, Keller GE, Palmer JH, Grubbs RH, Gray HB. Electron literature are devoted to study their interaction with organisms. transfer triggered by optical excitation of phenothiazine-tris(meta- phenylene-ethynylene)-(tricarbonyl)(bpy)(py)rhenium(I). J Phys Chem B However, as shown in this work, studies with non-functionalized 2013;117(16):4177–82. nanomaterials are also important to evaluate fully the impact of [21] Abdelhalim MAK, Jarrar BM. Histological alterations in the liver of rats induced nanomaterials on living systems. by different gold nanoparticle sizes, doses and exposure duration. J Nanobiotechnol 2012;10:5–10. [22] Garcia AC, Gasparotto LHS, Gomes JF, Tremiliosi-Filho G. Straightforward Con ict of interest synthesis of carbon-supported Ag nanoparticles and their application for thefl oxygen reduction reaction. Electrocatalysis 2012;3:147–52. [23] Astruc D, Daniel MC, Ruiz J. Dendrimers and gold nanoparticles as exo- None. receptors sensing biologically important anions. Chem Commun (Camb) 2004;23:2637–49. [24] Daniel MC, Astruc D. Gold nanoparticles: assembly, supramolecular chemistry, Funding source declaration quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology. Chem Rev 2004;104:293–346. The Brazilian agency Conselho Nacional de Desenvolvimento [25] Moskovits M, Srnova-Sloufova I, Vlckova B. Bimetallic Ag–Au nanoparticles: extracting meaningful optical constants from the surface-plasmon extinction Científico e Tecnológico (CNPq) has supported the current research spectrum. J Chem Phys 2002;116:10435–46. under the grant 442087/2014-4. [26] Foster SJ, McCormick ME, Howarth A, Aked D. Leukocyte recruitment in the subcutaneous sponge implant model of acute inflammation in the rat is not mediated by leukotriene B1. Biochem Pharmacol 1986;35:1709–17. Acknowledgments [27] Alhaider AA, Lei SZ, Wilcox GL. Spinal 5-HT3 receptor-mediated antinociception: possible release of GABA. J Neurosci 1991;11:1881–8. The authors also thank the Brain Institute at UFRN for allowing [28] Choi YJ, Kim YJ, Lee JW, Lee Y, Lim Y-B, Chung HW. Cyto-/genotoxic effect of CdSe/ZnS quantum dots in human lung adenocarcinoma cells for potential the use of the confocal microscope.The BIOPOL at the Department photodynamic UV therapy applications. J Nanosci Nanotechnol 12 2012;2160– of Biochemistry/UFRN is also gratefully acknowledged. 8. [29] Andón FT, Fadeel B. Programmed cell death: molecular mechanisms and implications for safety assessment of nanomaterials. Acc Chem Res References 2013;46:733–42. [30] Liu JKH. The history of monoclonal antibody development – progress, [1] Faulk WP, Taylor GM. An immunocolloid method for the electron microscope. remaining challenges and future innovations. Ann Med Surg 2014;3:113–6. Immunochemistry 1971;8:1081–3. [31] Trono JD, Mizuno K, Yusa N, Matsukawa T, Yokoyama K, Uesaka M. Size, [2] Huang T, Liu J, Li R, Cai W, Yu A. A novel route for preparation of PtRuMe concentration and incubation time dependence of gold nanoparticle uptake (Me = Fe, Co, Ni) and their catalytic performance for methanol into pancreas cancer cells and its future application to X-ray drug delivery electrooxidation. Electrochem commun 2009;11:643–6. system. J Radiat Res 2011;52:103–9. [3] Tang B, Sun L, Kaur J, Yu Y, Wang X. In-situ synthesis of gold nanoparticles for [32] Alkilany A, Murphy C. Toxicity and cellular uptake of gold nanoparticles: what multifunctionalization of silk fabrics. Dyes Pigm 2014;103:183–90. we have learned so far? J Nanopart Res 2016;12:2313–33. [4] Fu PP, Xia Q, Hwang H-M, Ray PC, Yu H. Mechanisms of nanotoxicity: [33] Gatoo MA, Naseem S, Arfat MY, Mahmood Dar A, Qasim K, Zubair S. generation of reactive oxygen species. J Food Drug Anal 2014;22:64–75. Physicochemical properties of nanomaterials: implication in associated toxic [5] Kim S, Ryu D-Y. Silver nanoparticle-induced oxidative stress, genotoxicity and manifestations. Biomed Res Int 2014;2014:8. apoptosis in cultured cells and animal tissues. J Appl Toxicol 2012;33:78–89. [34] AshaRani PV, Low Kah Mun G, Hande MP, Valiyaveettil S. Cytotoxicity and [6] Sahoo SK, Labhasetwar V. Nanotech approaches to drug delivery and imaging. genotoxicity of silver nanoparticles in human cells. ACS Nano 2009;3:279–90. Drug Discov Today 2003;8:1112–20. [35] Boyoglu C, He Q, Willing G, Boyoglu-Barnum S, Dennis VA, Pillai S, et al. [7] Zolnik BS, Gonzalez-Fernandez A, Sadrieh N, Dobrovolskaia MA. Minireview: Microscopic studies of various sizes of gold nanoparticles and their cellular nanoparticles and the immune system. Endocrinology 2010;151:458–65. localizations. ISRN Nanotechnol 2013;2013:13. [8] Connor EE, Mwamuka J, Gole A, Murphy CJ, Wyatt MD. Gold nanoparticles are [36] Gao W, Xu K, Ji L, Tang B. Effect of gold nanoparticles on glutathione depletion- taken up by human cells but do not cause acute cytotoxicity. Small induced hydrogen peroxide generation and apoptosis in HL7702 cells. Toxicol 2005;1:325–7. Lett 2011;205:86–95. [9] Nadler M.R., Kempter C.P., Crystallographic Data .186. Lithium. Anal Chem. [37] Dohnert MB, Venâncio M, Possato JC, Zeferino RC, Dohnert LH, Zugno AI, et al. 1959;31:2109–2109. Gold nanoparticles and diclofenac diethylammonium administered by 66 R.F. de Araújo et al. / Pharmacological Reports 69 (2017) 119–129 129 iontophoresis reduce inflammatory cytokines expression in Achilles [41] Bednarski M, Dudek M, Knutelska J, Nowin ski L, Sapa J, Zygmunt M, et al. The tendinitis. Int J Nanomed 2012;7:1651. influence of the route of administration of gold nanoparticles on their tissue [38] Samuels Y, Wang Z, Bardelli A, Silliman N, Ptak J, Szabo S, et al. High frequency distribution and basic biochemical parameters: in vivo studies. Pharmacol Rep of mutations of the PIK3CA gene in human cancers. Science 2004;304:554. 2015;67:405–9. [39] Kumar V, Singh PN, Bhattacharya SK. Anti-inflammatory and analgesic activity [42] Abdelhalim MAK. Exposure to gold nanoparticles produces pneumonia, of Indian Hypericum perforatum L. Indian J Exp Biol 2001;39:339–43. fibrosis, chronic inflammatory cell infiltrates, congested and dilated blood [40] Valiev M, Bylaska EJ, Govind N, Kowalski K, Straatsma TP, Van Dam HJJ, et al. vessels, and hemosiderin granule and emphysema. J Cancer Sci Ther NWChem: a comprehensive and scalable open-source solution for large scale 2012;4:46. molecular simulations. Comput Phys Commun 2016;181. 67 Capítulo 3 On the synergy between silver nanoparticles and doxycycline towardsthe inhibition of Staphylococcus aureus growth. Heloiza F. O. Silva, Rayane P. de Lima, Fernanda S. L. da Costa, Edgar P. Moraes, Maria C. N. Melo, Celso Sant’Anna, Mateus Eugenio, Luiz H. S. Gasparotto. RSC Advances, 2018, Vol. 8, 23578–23584 Contribuição:  Participei da proposição da ideia do estudo em questão.  Acompanhei, orientei e realizei a síntese das NanoAg, bem como a conjugação das NanoAg à DO;  Acompanhei e orientei as caracterizações dos sistemas envolvidos  Cultivei e repliquei a cepa de S. aureus  Desenvolvi a metodologia para exposição de S. aureus aos antimicrobianos.  Realizei o processamento dos dados e construção dos modelos multivariados;  Escrevi a primeira versão do manuscrito. ________________________ __________________________ Heloiza Fernanda O. da Silva Athayde Prof. Luiz Henrique da S. Gasparotto 68 TERMO DE AUTORIZAÇÃO AUTORAL Declaro para os devidos fins, que eu Heloiza Fernanda Oliveira da Silva Athayde, matrícula n SIGAA sob o nº 20151020790, primeira autora do artigo intitulado “On the synergy between silver nanoparticles and doxycycline towards the inhibition of Staphylococcus aureus growth” o utilizarei como parte integrante em minha tese para conclusão do curso de Doutorado em Química pelo Programa de Pós-Graduação em Química da Universidade Federal do Rio Grande do Norte. Comprometendo-me, mediante a apresentação do presente termo, não autorizar outros e nem utilizar o referido artigo em outros trabalhos de conclusões. Natal, 28 de agosto de 2019. ________________________________ Heloiza Fernanda Oliveira da Silva Athayde 69 RSC Advances PAPER View Article OnlineView Journal | View Issue On the synergy between silver nanoparticles and doxycycline towards the inhibition of Cite this: RSC Adv., 2018, 8, 23578 Staphylococcus aureus growth Heloiza F. O. Silva,a Rayane P. de Lima,a Fernanda S. L. da Costa,a Edgar P. Moraes,a Maria C. N. Melo,b Celso Sant’Anna,c Mateus Eugênioc and Luiz H. S. Gasparotto*a In a previous paper (RSC Adv., 2015, 5, 66886–66893), we showed that the combination of silver nanoparticles (NanoAg) with doxycycline (DO) culminated in an increased bactericidal activity towards E. coli. Herein we further investigated themetabolic changes that occurred on Staphylococcus aureus upon exposure to NanoAgwith the Received 12th March 2018 help of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) coupledwithmultivariate Accepted 21st June 2018 data analysis. It has been discovered that the combination of DOwith NanoAg producedmetabolic changes in S. DOI: 10.1039/c8ra02176g aureus that were not simply the overlap of the treatments with DO and NanoAg separately. Our results suggest rsc.li/rsc-advances that DO and NanoAg act synergistically to impede protein synthesis by the bacteria. 1 Introduction Silver nanoparticles may act via four main routes: 9 (1) adhesion to the microbial cell membrane causing damage and It is widely known that the indiscriminate administration of altering transport activity; (2) penetration inside the cell leading antibiotics has rendered pathogens resistant to a variety of broad- to organelle (ribosomes, DNA, RNA) dysfunction; (3) oxidation spectrum antibiotics.1 In order to circumvent this issue, nano- of proteins, lipids and DNA bases through oxidative stress; (4) science has worked in conjunction with biology and medicine to alteration of cell signaling. Thus, as multiple factors are altered develop more efficient bactericidal agents.2 As examples, silver simultaneously, it is logical to measure the metabolism directly nanoparticles have been used against E. coli3,4 and gold nano- instead of selecting a single marker at a time. To that end, particles for killing S. aureus.5 Some researchers have attempted infrared spectroscopy (FT-IR) emerges as an interesting tech- to combine nanoparticles with antibiotics to generate more nique for metabolic ngerprinting,10 owing to its capability to potent antimicrobial agents.6 Due to their large surface-area-to- examine proteins, carbohydrates, lipids, amino acids and fatty volume ratio and biocompatibility, inorganic nanoparticles are acids concurrently. Coupled with multivariate data analysis,10 considered ideal candidates for carrying large amounts of anti- FT-IR renders metabolic ngerprinting an excellent tool to biotics without compromising their activity. Li et al.7 demon- discriminate between groups of related biological samples, in strated that the combination of silver nanoparticles with addition to being rapid and non-destructive. amoxicillin produced stronger bactericidal effect towards In the present study, we exposed S. aureus to silver nano- Escherichia coli in comparison to the administration of the particles modied with doxycycline (DO, a member of the tetra- components separately. Our group8 showed that the conjugation cycline group) and employed FT-IR coupled with multivariate of polyvinylpyrrolidone (PVP)-capped silver nanoparticles data analysis to access variations of the S. aureus metabolism. (NanoAg) with doxycycline (DO) yielded quite a potent agent for DO-functionalized NanoAg caused the greatest alteration in the the inhibition of E. coli. An interesting question that follows is metabolism of S. aureus in comparison to that of bacteria treated what biological changes occur upon contacting bacteria with with DO and NanoAg separately, which made possible the NanoAg and DO-modied NanoAg. With that information in discrimination of bacteria subjected to those different treat- hand, it would be possible to fashion NanoAg with superior ments. These results corroborate nicely our previous work8 in biocidal activities against a broader range of pathogens. which we showed that the combination of DO with NanoAg delivered an increased antimicrobial activity towards E. coli. aBiological Chemistry and Chemometrics Research Group, Institute of Chemistry, 2 Experimental section Federal University of Rio Grande do Norte, Natal 59072-970, RN, Brasil. E-mail: lhgasparotto@ufrnet.br; Tel: +55 84 33422323 2.1 Chemicals and reagents bLaboratory of Medical Bacteriology, Center of Biosciences, Federal University of Rio Grande do Norte, Natal 59078-970, RN, Brasil Sodium hydroxide, glycerol, silver nitrate, polyvinylpyrrolidone cLaboratory of Biotechnology – Labio, National Institute of Metrology, Quality and (PVP; molecular weight ¼ 10 000), and doxycycline hyclate Technology – Inmetro, Duque de Caxias 25250-020, RJ, Brazil (>98%) were obtained from Sigma-Aldrich Chemical Co (MO, 23578 | RSC Adv., 2018, 8, 23578–23584 This journal is © The Royal Society of Chemistry 2018 70 Open Access Article. Published on 28 June 2018. Downloaded on 8/16/2019 11:06:58 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Paper RSC Advances USA). Staphylococcus aureus (ATCC® 25923™) was cultivated in 0.5 McFarland which is equivalent to 1.0 108 CFU ml1 in 0.9% laboratory. sterile saline medium. The suspension was then swabbed onto another Petri dish containing sterile Müller–Hinton agar medium 2.2 Production and characterization of NanoAg and allowed to grow at 37 C for 12 h. Aerwards, 1.5 ml of the 11 antimicrobial agents (NanoAg, DO or the DO–NanoAg conjugate)NanoAg were produced according to a reported method. was applied on the bacterial colony with the aid of a micropipette. Briey, all glassware was cleaned thoroughly with a KMnO4 + The plates were incubated for further 12 h at 37 C. NaOH solution and piranha solution. The following aqueous stock solutions were then produced: 50 mmol L1 AgNO3, 100 g L1 PVP and a solution containing 1.0 mol L1 NaOH + 2.5 ATR-FTIR analysis 1.0 mol L1 glycerol. In a beaker, determined volumes of the Bacteria were gently scraped off the Petri dish with a sterile PVP and AgNO3 solutions were dissolved in water to yield a 5 ml metal handle, placed on the sample holder of the ATR-FTIR solution. In a separate beaker, a known volume of the NaOH + equipment, and covered with a piece of aluminum foil. The glycerol solution was mixed with water to generate another 5 ml latter enhances the FTIR signal without interference due to its solution. The glycerol–NaOH solution was poured into the featureless background signal.12 FTIR spectra were acquired in AgNO3–PVP one to yield the following nal concentrations: quintuplicate from each sample of the following groups (24 0.10 mol L1 glycerol and NaOH, 10.0 g L1 PVP and 1.0 mmol samples per group): control (S. aureus without any treatment), L1 AgNO3. The NanoAg colloidal solutions had then their pH DO (S. aureus treated with doxycycline), NanoAg (S. aureus adjusted to 7 by addition of diluted HCl. treated with silver nanoparticles), and DO–NanoAg (S. aureus UV-VIS was performed with an Ocean Optics USB-650 Tide treated with the conjugate), adding up to a total of 480 spectra. spectrophotometer. FTIR in ATR mode was carried out with Measurements were conducted on a Bruker VERTEX 70 FTIR a Bruker Vertex 70 spectrophotometer and Transmission Elec- spectrometer (Bruker Optics Ltd., Coventry, UK) with a Helios tron Microscopy (TEM) images were acquired with a FEI Tecnai ATR attachment containing a diamond crystal internal reec- G2 Spirit BioTWIN microscope operating at 120 kV. tive element and a 45 incidence angle of the IR beam. Each spectrum was a result of 16 scans at a spectral resolution of 2.3 Conjugation of DO with NanoAg 4 cm1. Aer each acquisition the sample holder was cleaned Conjugation of NanoAg with DO was achieved by simple incu- with 70% alcohol (v/v). bation according to a previous protocol.8 Five milliliters of a 200 mg ml1 doxycycline stock solution were added to the same 2.6 Chemometric procedure volume of the NanoAg colloidal solution, generating a 10 ml Data import, pre-treatment and chemometric procedures were DO–NanoAg solution. The nal concentrations of NanoAg and carried out with MATLAB R2014a soware (MathWorks, USA) DO in the conjugate were 0.2  109 mol L1 and 0.2  103 1 with the PLS-toolbox version 7.5.2 (Eigenvector Research, Inc., mol L , respectively. All the above-mentioned techniques Wenatchee, WA). Raw spectra were pre-processed by selecting were employed to characterize the NanoAg–antibiotic complex. the range of 1800 cm1 to 900 cm1 (468 wavenumbers at 4 cm1 spectral resolution) and mean-centering. PCA model 2.4 Exposition of S. aureus to DO, NanoAg and DO–NanoAg was constructed with 96 samples (24 samples of each class: Staphylococcus aureus (strain ATCC® 25923™) was cultured in control, DO, DO + NanoAg, NanoAg), using 4 PCs, that explained Brain-Heart-Infusion (BHI) agar medium on a Petri dish at 37 C 97.6% of total variance. PLS-DA models were made for each two for 24 h. A microbiological strain suspension was standardized at classes of treatments (control, DO, DO + NanoAg, NanoAg). Fig. 1 (A) UV-VIS spectra of DO mixed with AgNPs (red curve) and the mathematical combination of the DO and NanoAg pure spectra. (B) Chemical structure of doxycycline. This journal is © The Royal Society of Chemistry 2018 RSC Adv., 2018, 8, 23578–23584 | 23579 71 Open Access Article. Published on 28 June 2018. Downloaded on 8/16/2019 11:06:58 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online RSC Advances Paper Fig. 2 TEM images of (A) NanoAg and (B) NanoAg mixed with doxycycline. Using the algorithm Kennard–Stone (KS), separately to each spectroscopy in conjunction with PCA and PLS-DA to evaluate class, the samples were divided into training/validation (70%) the metabolic response of S. aureus aer treatment with and prediction sets (30%). Themodel performance was evaluated NanoAg, DO and DO + NanoAg. As mentioned earlier, in by gures of merit: sensitivity, specicity and confusion matrix. a previous study we discovered that the combination of DO with NanoAg delivered a conjugate with enhanced growth inhibition 3 Results and discussion properties against E. coli compared to the constituents admin-istered separately.8 This result prompted us to investigate the 3.1 Synthesis and characterization of NanoAg metabolic impact of NanoAg, DO and DO + NanoAg on S. aureus, In this study, NanoAg were produced with glycerol in alkaline a simpler microorganism in terms of cell wall complexity. 15 medium as reducing agent at room temperature. Since glycerol is Fig. 3 presents average pre-treated spectra for each class1 produced nowadays as a byproduct of the biodiesel fabrication, its acquired in the “bio-ngerprint” range of 900–1800 cm . As supply has surpassed the current demand making glycerol a rela- noticed, it is not straightforward to distinguish the spectra tively inexpensive chemical.8 Due to its biodegradability under visually, probably because the metabolic alterations upon aerobic conditions, non-toxicity, and low price, glycerol has treatment are minute. become more attractive for generating nanoparticles than estab- The spectra were then subjected to the unsupervised Prin- lished reducing chemicals such as formamide, sodium borohy- cipal Components Analysis (PCA) classication model, followed dride and hydrazine. Fig. 1A suggests that NanoAg andDO interact by the supervised classication of Partial Least Squares to some extent. The UV-VIS spectrum of the mathematical Discriminant Analysis (PLS-DA) for the binary classication, as combination of pure DO and NanoAg spectra (blue curve) shows shown in Fig. 4. a maximum at 410 nm corresponding to the characteristic Surface The plot of the PCA discrimination function with the mean Plasmon Resonance (SPR) of PVP-stabilized spherical NanoAg, FTIR-ATR spectra (Fig. 4A) revealed a degree of segregation a peak at 365 nm due to the p-electron system located in the BCD between the classes, meaning that the methodology allowed for chromophore (see Fig. 1B), and absorptions below 300 nm due to the detection of variables that differentiate the groups which a combined contribution of the BCD system with the tricarbonyl- were then compared in pairs via PLS-DA: control vs. DO, control methane keto–enol system comprised in ring A.13 The mixing of vs. NanoAg, and control vs. DO + NanoAg. This method indi- DO with NanoAg (red curve) causes all DO absorptions to shi, cated the wavenumbers whose changes were statistically implying an interaction of that system with the NanoAg. In a previous study,8 we deeply investigated the interaction between DO and NanoAg via FTIR, showing that the capping agent, the PVP, was of paramount importance in augmenting the DO concentration around the nanoparticles. In addition to the chemical interaction, DO is kept in the vicinity of the particle due to the PVP-shell structure that encapsulates the DO.14 TEM images of NanoAg (Fig. 2B and C) showed that the conjugation with DO had practically no impact on both shape and size distribution of NanoAg, which is an important result since it can rule out size and shape effects on bacteriological experiments. 3.2 Exposition of S. aureus to DO, NanoAg and DO–NanoAg 3.2.1 Sample analysis and calibration/training dataset. The Fig. 3 Average spectrum for each original class control, DO, DO + objective of the present study was to apply the ATR-FTIR NanoAg and NanoAg. 23580 | RSC Adv., 2018, 8, 23578–23584 This journal is © The Royal Society of Chemistry 2018 72 Open Access Article. Published on 28 June 2018. Downloaded on 8/16/2019 11:06:58 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Paper RSC Advances Fig. 4 Multivariate data analysis of selected variables in the samples. (A) Principal Component Analysis (PCA) of variables by the four classes and (B–D) PLS-DA by pairs. signicant for each group. Fig. 4B–D shows the PLS-DA plots for Table 3 was constructed with the aid of the most signicant the three comparisons. For the determination of the best variables present in the loadings generated in each of the three models, a confusion matrix (Table 1) was compiled with the models. From these variables, the wave numbers responsible values of true positive, true negative and type I and II errors. for the discrimination were recovered. Literature data were used It is possible to observe in Table 1 that of the three models to assign the characteristic group to each wave number the treatments with DO and DO + NanoAg presented the retrieved. smallest errors, being classied 100% in their classes. The Control vs. DO. In order to investigate the control and DO NanoAg model presented a 50% accuracy rate, corroborating samples, as observed in Table 3, six variables were selected for the PCA discrimination function, showing that it is di 1 1fficult to PLS-DA (1647 cm , 1631 cm , 1547 cm1, 1543 cm1, nd a standard for differentiation because it presents a variance 1400 cm1 and 1080 cm1). It is interesting to note that 83.3% within the very high class. The values of the parameters of of the wave numbers responsible for the discrimination quality: sensitivity, specicity, RMSEC, RMSECV and RMSEP between the two classes are related to protein: amide I in 1647 were also taken into account for the three best models. All the best models used 2 latent variables (see Table 2). These values Table 1 Confusion table for actual and predicted groups show that there was good classication, especially for the control versus DO (sensibility: control 75% and DO 100%  Actual (%) specicity control 100% and DO 75%) and control versus DO + Predicted (%) Control DO NanoAg (sensibility: control 75% and DO + NanoAg 100%  Control 75.0 0 specicity control 100% and DO + NanoAg 75%) pairs that DO 25.0 100 presented the highest values of sensitivity and specicity. These Control DO + NanoAg results conrm the potential of FITR-ATR spectroscopy to detect Control 75.0 0 and identify groups with di DO + NanoAg 25.0 100fferent metabolic responses of S. Control NanoAg aureus aer exposure to the three antimicrobials DO, NanoAg Control 100 50.0 and DO + NanoAg. NanoAg 0 50.0 This journal is © The Royal Society of Chemistry 2018 RSC Adv., 2018, 8, 23578–23584 | 23581 73 Open Access Article. Published on 28 June 2018. Downloaded on 8/16/2019 11:06:58 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online RSC Advances Paper Table 2 Quality performance values from PLS-DA method (2 latent complex; in other words, DO interacts with the 30S portion of variables) by ATR-FTIR spectroscopy for each category of the three the ribosome thereby impairing protein synthesis. It should models therefore be noted that tetracycline are classied as bacterio- Models PLS-DA (2 LVs) static, i.e. their interaction occurs reversibly. 16,17 To this we can attribute the absence of signicant change (p < 0.05) in the Control vs. Control vs. Control vs. absorbances of the other wave numbers related to the proteins. Accuracy (%) DO NanoAg DO + NanoAg An interesting result was the presence of the band at 1080 cm1, Calibration characteristic of polysaccharides. Zmantar et al. 18 showed that Sensibility (%) 93.8 87.5 93.8 S. aureus ATCC 25923, the same strain used in the present study, Specicity (%) 100 62.5 100 produces biolm aer stress and Cerca et al.19 stated that the proteins encoded by intercellular adhesin genes (icaADBC) Prediction synthesize polysaccharide, which contributes to the formation Sensibility (%) 75.0 100 75.0 Specicity (%) 100 50.0 100 of this biolm in S. aureus. In contrast to the results obtained in RMSEC 0.284 0.388 0.201 Table 3, S. aureus showed a signicant increase in mean RMSECV 0.339 0.443 0.222 absorbance (p < 0.05) when the DO band was treated with RMSEP 0.388 0.412 0.318 1080 cm1, a characteristic of polysaccharides. Control vs. NanoAg. From the investigation between the control and NanoAg samples, ve variables were selected for and 1631 cm1, amide II in 1547 and 1543 cm1 and amino acid SPA-LDA (1641 cm1, 1635 cm1, 1547 cm1, 1543 cm1 and in 1400 cm1. Note that only the wavelength 1647 cm1 showed 1086 cm1). Of these wave numbers 80% are related to proteins a signi 1 1cant change (p < 0.05) in absorbance upon exposure to as well, namely 1641 cm and 1635 cm for amide I, while the DO. This corroborates with the literature, since this antibiotic absorptions at 1547 and 1543 cm1 are related to amide II. belongs to the class of tetracyclines, whose mechanism of Although NanoAg has an extensive list of studies involving its action is the interference in the binding of the tRNA by blocking effect against bacteria, the mechanism of action is not clearly the adhesion of aminoacyl-t-RNA, to the mRNA-ribosome known.9,20 Some authors attribute such difficulty to the fact that Table 3 Infrared band assignments of the Gram-positive S. aureus and average absorbances of the control, DO, NanoAg and DO + NanoAg classes presented in the regions (variables) used in the discrimination by PLS-DAa Model Wavelength Abscontrol Abstreated Assignment Literature Control versus DO 1647 cm1 0.51 (0.02) 0.51 (0.01) Stretching of C]O in amide (amide I) of 28–30 structural proteins. 1631 cm1 0.53 (0.02) 0.52 (0.01) Stretching of C]O in amide (amide I) of 28–30 structural proteins. 1547 cm1 0.31 (0.02)* 0.30 (0.01)* N–H bending and C–N stretching in 27–30 amide (amide II) of structural proteins. 1543 cm1 0.30 (0.02) 0.30 (0.01) N–H bending and C–N stretching in 27–30 amide (amide II) of structural proteins. 1400 cm1 0.19 (0.02) 0.19 (0.01) –COO symmetric stretching of amino 27–29 and 31 acid side chains and fatty acids 1080 cm1 0.21 (0.01)* 0.23 (0.01)* C–O–C. C–O of various polysaccharides 27–29 and 31 Control versus DO + NanoAg 1635 cm1 0.54 (0.02)* 0.50 (0.01)* b-pleated sheet structures (amide I) of 27 and 29 structural proteins. 1630 cm1 0.53 (0.02)* 0.49 (0.01)* Stretching of C]O in amide (amide I) of 28–30 structural proteins. 1543 cm1 0.30 (0.02)* 0.27 (0.01)* N–H bending and C–N stretching in 27–30 amide (amide II) of structural proteins. 1539 cm1 0.29 (0.02)* 0.25 (0.01)* N–H bending and C–N stretching in 27–30 amide (amide II) of structural proteins. 1398 cm1 0.19 (0.02)* 0.17 (0.01)* –COO symmetric stretching of amino 27–29 and 31 acid side chains and fatty acids 1078 cm1 0.21 (0.01)* 0.22 (0.01)* C–O–C. C–O of various polysaccharides 27–29 and 31 Control versus NanoAg 1641 cm1 0.53 (0.02)* 0.50 (0.05)* Stretching of C]O in amide (amide I) 28–30 1635 cm1 0.54 (0.02)* 0.50 (0.05)* b-pleated sheet structures (amide I) 27 and 29 1547 cm1 0.31 (0.02)* 0.28 (0.03)* N–H bending and C–N stretching in 27–30 amide (amide II) 1543 cm1 0.30 (0.02)* 0.28 (0.03)* N–H bending and C–N stretching in 27–30 amide (amide II) 1086 cm1 0.21 (0.01)* 0.19 (0.02)* C–O–C. C–O of various polysaccharides 27–29 and 31 a *Different averages (p < 0.05). 23582 | RSC Adv., 2018, 8, 23578–23584 This journal is © The Royal Society of Chemistry 2018 74 Open Access Article. Published on 28 June 2018. Downloaded on 8/16/2019 11:06:58 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Paper RSC Advances the antibacterial action is strongly dependent on the physical– attributed to polysaccharides. Thus, the defense by biolm chemical parameters such as size, shape, surface charge, expression was presented by S. aureus aer treatment with the concentration and colloidal state.21–26 Despite many factors, DO + NanoAg conjugate. Another important point was the Dakal et al.9 found that the antimicrobial action of NanoAg in signicant decrease in mean absorbances (p < 0.05) attributed general is linked to at least four distinct mechanisms. Accord- to protein expression exhibited aer treatment with this system, ing to them, the NanoAg act (A) inducing cellular toxicity which did not occur aer treatment with DO. Based on what has through the oxidative stress caused by the generation of reactive been presented so far, we can conclude that the conjugate oxygen species (ROS) and free radicals, (B) adhering to the caused expressive metabolic responses, even though the two surface of the wall and cell membrane, (C) interfering in the starting constituents, DO and NanoAg, were at half their orig- modulation or (D) damaging intracellular structures (mito- inal concentrations. The combination of DO and NanoAg boosts chondria, vacuoles, ribosomes) and biomolecules (proteins, the inhibition of protein synthesis. lipids and DNA) aer the endocytosis of NanoAg. Based on the selected variables (most related to proteins), we can suggest that 4 Conclusions the path of action of NanoAg synthesized and administered in our work was D, since aer the treatment of S. aureus with Herein we have shown that FT-IR coupled with multivariate NanoAg the mean absorbances suffered a signicant decrease analysis is an excellent tool to discriminate bacteria that have (p < 0.05), as shown in Fig. 5. The pathway suggested is that it been treated with DO, NanoAg and DO + NanoAg. From PCA acts more expressively in the inhibition of protein synthesis. In analysis it is clear that, although both DO and NanoAg affect agreement with the expression in Table 3, the variable related to protein synthesis, their combination promotes biological the wave number 1086 cm1 was selected which is characteristic changes in S. aureus sufficient for discrimination among the of polysaccharides that predominate in biolm expressed by S. classes. aureus aer stress.17,18 However, it was unusual to note that this treatment with free NanoAg of DO showed a signicant Conflicts of interest decrease (p < 0.05) in mean absorbance suggesting that action of the nanoparticles did not allow S. aureus to effectively express There are no conicts to declare. its biolm or even that it expressed, but the damage to DNA and polysaccharides was more expressive. Acknowledgements Control vs. DO + NanoAg. Finally, the comparison between the Control and DO + NanoAg samples allowed for the selection of The authors are grateful to CNPq (grant 442087/2014-4). six variables for SPA-LDA (1635, 1630, 1543, 1539, 1398 and 1078 cm1). Of these, 80% wave numbers are protein related, as References observed in Table 3. The wavenumber at 1635 and 1630 cm1 are assigned to amide I, 1543 and 1539 cm1 assigned to amide 1 A. J. Alanis, Resistance to Antibiotics: Are We in the Post- II and 1398 cm1 attributed to amino acid. At rst, it is possible Antibiotic Era?, Arch. Med. Res., 2005, 36(6), 697–705. to observe that the treatment with the DO + NanoAg conjugate 2 A. J. Huh and Y. J. Kwon, “Nanoantibiotics”: A new paradigm allowed for the selection of the variables observed both in the for treating infectious diseases using nanomaterials in the DO treatment and in the treatment with NanoAg. Interestingly, antibiotics resistant era, J. Controlled Release, 2011, 156(2), the DO + NanoAg conjugate caused a signicant increase in the 128–145. mean absorbance (p < 0.001) at the 1078 cm1 wavenumber 3 I. Sondi and B. Salopek-Sondi, Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria, J. Colloid Interface Sci., 2004, 275(1), 177–182. 4 S. K. Rastogi, V. J. Rutledge, C. Gibson, D. A. Newcombe, J. R. Branen and A. L. Branen, Ag colloids and Ag clusters over EDAPTMS-coated silica nanoparticles: synthesis, characterization, and antibacterial activity against Escherichia coli, Nanomedicine, 2011, 7, 305–314. 5 V. P. Zharov, K. E. Mercer, E. N. Galitovskaya and M. S. Smeltzer, Photothermal Nanotherapeutics and Nanodiagnostics for Selective Killing of Bacteria Targeted with Gold Nanoparticles, Biophys. J., 2006, 90(2), 619–627. 6 A. N. Brown, K. Smith, T. A. Samuels, J. Lu, S. O. Obare and M. E. Scott, Nanoparticles Functionalized with Ampicillin Destroy Multiple-Antibiotic-Resistant Isolates of Fig. 5 Mean average absorbances of the control, DO, NanoAg and DO Pseudomonas aeruginosa and Enterobacter aerogenes and + NanoAg classes presented in the six regions used in the discrimi- Methicillin-Resistant Staphylococcus aureus, Appl. Environ. nation by PLS-DA. Microbiol., 2012, 78(8), 2768–2774. This journal is © The Royal Society of Chemistry 2018 RSC Adv., 2018, 8, 23578–23584 | 23583 75 Open Access Article. Published on 28 June 2018. Downloaded on 8/16/2019 11:06:58 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online RSC Advances Paper 7 P. Li, J. Li, C. Wu, Q. Wu and J. Li, Synergistic antibacterial and IcaR in Staphylococcus aureus, J. Bacteriol., 2008, effects of b-lactam antibiotic combined with silver 190(19), 6530–6533. nanoparticles, Nanotechnology, 2005, 16(9), 1912. 20 S. Prabhu and E. K. Poulose, Silver nanoparticles: 8 H. F. O. Silva, K. M. G. Lima, M. B. Cardoso, J. F. A. Oliveira, mechanism of antimicrobial action, synthesis, medical M. C. N. Melo, C. Sant'Anna, M. Eugenio and applications, and toxicity effects, Int. Nano Lett., 2012, L. H. S. Gasparotto, Doxycycline conjugated with 2(32), 2–10. polyvinylpyrrolidone-encapsulated silver nanoparticles: 21 S. Pal, Y. K. Tak and J. M. Song, Does the Antibacterial a polymer's malevolent touch against Escherichia coli, RSC Activity of Silver Nanoparticles Depend on the Shape of the Adv., 2015, 5(82), 66886–66893. Nanoparticle? A Study of the Gram-Negative Bacterium 9 T. C. Dakal, A. Kumar, R. S. Majumdar and V. Yadav, Escherichia coli, Appl. Environ. Microbiol., 2007, 73(6), Mechanistic Basis of Antimicrobial Actions of Silver 1712–1720. Nanoparticles, Front Microbiol, 2016, 7, 1831. 22 R. Bhattacharya and P. Mukherjee, Biological properties of 10 D. I. Ellis and R. Goodacre, Metabolic ngerprinting in “naked” metal nanoparticles, Adv. Drug Delivery Rev., 2008, disease diagnosis: biomedical applications of infrared and 60(1), 1289–1306. Raman spectroscopy, Analyst, 2006, 131(8), 875–885. 23 M. K. Rai, S. D. Deshmukh, A. P. Ingle and A. K. Gade, Silver 11 J. F. Gomes, A. C. Garcia, E. B. Ferreira, C. Pires, nanoparticles: the powerful nanoweapon against multidrug- V. L. Oliveira, G. Tremiliosi-Filho and L. H. S. Gasparotto, resistant bacteria, J. Appl. Microbiol., 2012, 112(1), 841–852. New insights into the formation mechanism of Ag, Au and 24 M. R. Nateghi and H. Hajimirzababa, Effect of silver AgAu nanoparticles in aqueous alkaline media: alkoxides nanoparticles morphologies on antimicrobial properties of from alcohols, aldehydes and ketones as universal cotton fabrics, J. Text. Inst., 2014, 105(1), 806–813. reducing agents, Phys. Chem. Chem. Phys., 2015, 17(33), 25 A. Abbaszadegan, Y. Ghahramani, A. Gholami, 21683–21693. B. Hemmateenejad, S. Dorostkar, M. Nabavizadeh and 12 L. Cui, H. J. Butler, P. L. Martin-Hirsch and F. L. Martin, H. Sharghi, The Effect of Charge at the Surface of Silver Aluminium foil as a potential substrate for ATR-FTIR, Nanoparticles on Antimicrobial Activity against Gram- transection FTIR or Raman spectrochemical analysis of Positive and Gram-Negative Bacteria: A Preliminary Study, biological specimens, Anal. Methods, 2016, 8(3), 481–487. J. Nanomater., 2015, 2015(1), 1–8. 13 S. Schneider, M. O. Schmitt, G. Brehm, M. Reiher, 26 F. Zhang, J. A. Smolen, S. Zhang, R. Li, P. N. Shah, S. Cho, P. Matousek and M. Towrie, Fluorescence kinetics of H. Wang, J. E. Raymond, C. L. Cannon and K. L. Wooley, aqueous solutions of tetracycline and its complexes with Degradable polyphosphoester-based silver-loaded Mg+2 and Ca+2, Photochem. Photobiol. Sci., 2003, 2(1), 1107– nanoparticles as therapeutics for bacterial lung infections, 1117. Nanoscale, 2015, 7, 2265–2270. 14 M. Behera and S. Ram, Inquiring the mechanism of 27 K. Maquelin, C. Kirschner, L. P. Choo-Smith, N. V. D. Braak, formation, encapsulation, and stabilization of gold H. Ph Endtz, D. Naumann and G. J. Puppels, Identication of nanoparticles by poly(vinyl pyrrolidone) molecules in 1- medically relevant microorganisms by vibrational butanol, Appl. Nanosci., 2014, 4(1), 247–254. spectroscopy, J. Microbiol. Methods, 2002, 51(1), 255–271. 15 K. D. Young, The Selective Value of Bacterial Shape, 28 W. Jiang, A. Saxena, B. Song, B. B. Ward, T. J. Beveridge and Microbiol. Mol. Biol. Rev., 2006, 70(3), 660–703. S. C. B. Myneni, Elucidation of Functional Groups on Gram- 16 E. C. Pereira-Maia, P. P. Silva, W. B. Almeida, H. F. Santos, Positive and Gram-Negative Bacterial Surfaces Using B. L. Marcial, R. Ruggiero and W. Guerra, Tetraciclinas e Infrared Spectroscopy, Langmuir, 2004, 20, 11433–11442. Glicilciclinas: Uma Visão Geral, Quim. Nova, 2010, 33(3), 29 D. Naumann, Infrared Spectroscopy in Microbiology, 700–706. Encyclopedia of Analytical Chemistry, ed. R. A. Meyers, John 17 I. Chopra and M. Roberts, Tetracycline Antibiotics: Mode of Wiley & Sons Ltd, Chichester, 2000, pp. 102–131. Action, Applications, Molecular Biology, and Epidemiology 30 Y. Burgula, D. Khali, S. Kim, S. S. Krishnan, M. A. Cousin, of Bacterial Resistance, Microbiol. Mol. Biol. Rev., 2001, J. P. Gore, B. L. Reuhs and L. J. Mauer, Review Of Mid- 65(2), 232–260. Infrared Fourier Transform-Infrared Spectroscopy 18 T. Zmantar, B. Kouidhi, H. Miladi, K. Mahdouani and Applications For Bacterial Detection, J. Rapid Methods A. Bakhrouf, A Microtiter plate assay for Staphylococcus Autom. Microbiol., 2007, 15(1), 146–175. aureus biolm quantication at various pH levels and 31 B. Buszewski, E. Dziubakiewicz, P. Pomastowski, hydrogen peroxide supplementation, New Microbiol., 2010, K. Hrynkiewicz, J. Ploszaj-Pyrek, E. Talik, M. Kramer and 33(1), 137–145. K. Albert, Assignment of functional groups in Gram- 19 N. Cerca, J. L. Brooks and K. K. Jefferson, Regulation of the positive bacteria. Analytical Method Development and Intercellular Adhesin Locus Regulator (icaR) by SarA, B, Validation: A Concise Review, J. Anal. Bioanal. Tech., 2015, 6(1), 1–8. 23584 | RSC Adv., 2018, 8, 23578–23584 This journal is © The Royal Society of Chemistry 2018 76 Open Access Article. Published on 28 June 2018. Downloaded on 8/16/2019 11:06:58 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Capítulo 4 Conclusões e Recomendações Os resultados obtidos nos dois estudos confirmam a potencialidade das nanopartículas sintetizadas em nosso grupo de pesquisa como possíveis nanocarreadores ou mesmo agindo isoladamente, sendo assim mais um recurso para a inovação tecnológica nos campos de ação anti-inflamatória, analgésica, antitumoral e no combate a resistência bacteriana. As recomendações para futuros trabalhos seguem:  Determinar a rota metabólica envolvida na ação das NanoAu e NanoAg nos respectivos sistemas dos estudos apresentados aqui, via ensaios com cromatográficos líquidos de alta eficiência equipado com detector de massas associados à quimiometria.  Validar o método que utiliza ATR-FTIR na triagem do efeito de candidatos a antimicrobianos para bactérias gram-positivas e gram-negativas.  Avaliar o efeito das NanoAg sintetizadas em nosso grupo quando combinadas a outros antibióticos. Outras sugestões já têm sido seguidas por meio das parcerias e colaborações. 77 Apêndice A Environmentally compatible bioconjugated gold nanoparticles as efficient contrast agents for inflammation-induced cancer imaging Vinícius Barreto Garcia, Thaís Gomes de Carvalho, Luiz Henrique da Silva Gasparotto, Heloiza Fernanda Oliveira da Silva, Aurigena Antunes de Araújo, Gerlane Coelho Bernardo Guerra, Timo Schomann, Luis J. Cruz, Alan B. Chan, Raimundo Fernandes de Araújo Júnior Nanoscale Research Letters, v. 14, p.166-175, 2019 Contribuição:  Realizei a síntese e purificação das NanoAu.  Realizei a caracterização das NanoAu.  Coletei os espectros de Fluorescência.  Plotei e interpretei os espectros de fluorescência  Discuti alguns resultados com o autor principal antes da escrita da primeira versão do manuscrito.  Auxiliei nos novos ensaios e respostas solicitadas pelos revisores. ________________________ __________________________ Heloiz Fernanda O. da Silva Athayde Prof. Luiz Henrique da S. Gasparotto 78 Garcia et al. Nanoscale Research Letters (2019) 14:166 https://doi.org/10.1186/s11671-019-2986-y NANO EXPRESS Open Access Environmentally compatible bioconjugated gold nanoparticles as efficient contrast agents for inflammation-induced cancer imaging Vinícius Barreto Garcia1,3, Thaís Gomes de Carvalho1,3, Luiz Henrique da Silva Gasparotto5, Heloiza Fernanda Oliveira da Silva5, Aurigena Antunes de Araújo4, Gerlane Coelho Bernardo Guerra4, Timo Schomann6,7, Luis J. Cruz6, Alan B. Chan7 and Raimundo Fernandes de Araújo Júnior1,2,3,6* Abstract For many cancers, early detection is the key to improve survival and reduce the morbidity, which is associated with radical resections due to late diagnosis. Here, we describe the efficiency of primary antibody-conjugated gold nanoparticles (AuNPs) to specifically target chronic inflammatory processes, specially M2 macrophages, in tissue sections of ulcerative colitis (UC) and steatohepatitis in rats which may lead to colorectal cancer and liver carcinoma, respectively. In this study, we demonstrate that AuNPs synthesized by a simple, inexpensive, and environmentally compatible method can be easily conjugated with the antibodies anti-COX-2, anti-MIF, and Alexa Fluor® 488 (ALEXA) to perform immunofluorescence staining in inflamed tissues. Moreover, we showed that primary antibody-conjugated gold nanoparticles (AuNPs) can be used to target M2 macrophages by flow cytometry. We designed three immunofluorescence staining protocols of tissue section with AuNPs for 30 min and overnight incubation, as well as one flow cytometry protocol of M2 macrophage labeling with AuNPs for 30min. Immunofluorescence and flow cytometry results suggest that conjugation was achieved by direct adsorption of antibodies on the AuNPs surface. When compared to the standard ALEXA protocol in immunofluorescence (IF) and flow cytometry (FC), our 30-min incubation protocol using AuNPs instead of ALEXA decreased from approximately 23 h to 5 h for IF and from 4 h to 1 h for FC, proving to be less laborious, which makes the method eligible for inflammation-induced cancer diagnostic. Keywords: Golden nanoparticles, M2 macrophages, Immunofluorescence, Inflammatory processes, Cancer diagnostics, Ulcerative colitis Introduction The physicochemical properties of AuNPs allow them In medical and biological research, the interest in gold to be used in many medical studies, such as genomics, nanoparticles (AuNPs) for optical microscopy studies and biosensitivity, immunoassay, clinical chemistry, detection, diagnostic procedures, especially confocal laser micros- and photothermolysis of microorganisms and cancer copy, is increasing. The use of antibody/AuNP conjugates cells [1]. Faulk and Taylor [4] described the first method allows real-time detection of gold uptake into living cells of antibody conjugation with colloidal gold for direct elec- (e.g., cancer cells) at the level of single particles, enabling tron microscopical visualization of the surface antigens of the estimation of intracellular amounts of NPs [1–3]. salmonellae. Since then, many studies were developed aiming at the application of nanoparticles conjugated with * Correspondence: araujojr@cb.ufrn.br biomacromolecules, such as antibodies, lectins, and en- 1Department of Morphology, Federal University of Rio Grande do Norte, zymes, in different fields, e.g., biochemistry, microbiology, Natal, RN 59072-970, Brazil immunology, and morphology [1, 4]. In cancer studies, 2Post-Graduation Programme in Structural and Functional Biology, Federal University of Rio Grande do Norte, Natal, RN 59072-970, Brazil highly specific and sensitive AuNP-based contrast agents Full list of author information is available at the end of the article are used for both X-ray and optical imaging modalities, © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 79 Garcia et al. Nanoscale Research Letters (2019) 14:166 Page 2 of 12 since AuNPs are able to enhance fluorescence intensity of USA). Anti-COX-2 (cyclooxygenase-2) and anti-MIF conjugated molecules [5, 6]. (macrophage migration inhibitory factor) primary anti- In 2014, we demonstrated that AuNPs can be applied to bodies were acquired from Santa Cruz Biotechnology the indirect immunofluorescence (IF) staining method in (São Paulo, Brazil), whereas Goat Anti-Rat IgG H&L cell cultures [6]. In the present study, we describe three Alexa Fluor® 488 secondary antibody was purchased new methods for immunofluorescence (IF) staining using from ABCAM® (Cambridge, UK). Fluoroshield Mounting AuNPs. Here, we use tissue sections and compare the Medium with DAPI (20 ml) from ABCAM® (Cambridge, fluorescence intensity from each of these methods to the UK) was used for counterstaining of DNA. standard staining protocol with Alexa Fluor® 488 (ALEXA) antibody (A1) with the goal of optimizing a fluorescent Production and Characterization of AuNPs and technique for clinical application [7–9]. Fluorescence Spectroscopy Fluorescence imaging (FI) for cancer cell targeting uti- Spherical AuNPs (7.1-nm) were produced and character- lizes a variety of optical imaging technologies in order to ized according to a study by Gasparotto et al. [12]. Briefly, improve the detection of early neoplasia based on molecu- all glassware was kept in KMnO4 +NaOH solution over- lar signatures specific for cancer [10]. Since 2013, there night, rinsed with deionized water, kept in H2O2 +H2SO4 has been a rapid increase in the number of clinical trials solution (1:1 v/v) for 10min, again rinsed with deionized using FI. Screening is generally considered for high-risk water, and dried prior to use. PVP (0.20 g) and gold chloride patients, which is based on a combination of lifestyle fac- (6.80mg) were dissolved in 10ml of water. In a separate tors, genetics, or a personal history of disease, while sur- beaker, glycerol (0.18 g) and NaOH (0.080 g) were dissolved veillance is reserved for patients with a diagnosis of in 10ml of water. The glycerol-NaOH solution was then dysplasia/chronic inflammation or those with suspected added to the AuCl3-PVP solution to yield the following malignancy. FI may aid in identifying malignant lesions final concentrations: 1.0mmol/L−1 Au3+, 0.10M NaOH, with improved specificity and sensitivity compared to cur- 0.10M glycerol, and 10 g/L−1 PVP. The final mixture had a rently available techniques. Furthermore, FI-based screen- deep-red color due to the formation of AuNPs. The col- ing may provide a less invasive, more cost-effective way to loidal ultraviolet-visible absorption spectra of the AuNPs detect cancerous or pre-cancerous lesions. Specifically, the were acquired with an Evolution 60S UV–visible spectro- ability of FI to detect lesions earlier than conventional photometer (Thermo Scientific, MA, USA). Fluorescence screening methods will not only result in improved treat- spectroscopy was carried out with a RF-5301 PC spectro- ment outcomes but also in reduced treatment costs as it fluorophotometer (Shimadzu, Kyoto, Japan). will prevent the need for multimodality care, which is re- quired for those diagnosed at later diagnosis [11]. In this Experimental Design study, we demonstrate that antibody/AuNPs conjugates All three protocols were tested on control groups of two can successfully be applied for imaging of chronic inflam- chronic inflammatory models, acetic acid-induced ul- mation by means of fluorescence microscopy. cerative colitis (UC) [7], and alcohol-induced steatohe- patitis [8, 9] in rats. Methods/Experimental Acetic acid-induced UC was performed on female Wis- Aim of the Study tar rats (220 ± 20 g of BW), obtained from the Biotechnol- The aim of this study is to demonstrate how fluorescence ogy Center/Universidade Federal da Paraiba. The animals properties of gold nanoparticles can be used in IF and flow were divided into two groups (n = 5 per group): acetic acid cytometry techniques. In addition, we compared the pro- control and non-colitic rats. The animals were fasted over- tocols tested with AuNPs to the standard protocol using night and anesthetized with ketamine (70mg/kg, 10%) ALEXA and proved that it is possible to use the AuNPs in and xylazine (10mg/kg, 2%). UC was induced according a protocol, which is faster, but as reliable as the standard to methods originally described by MacPherson and Pfeif- protocol. fer [13] and modified by Millar et al. [14]. A catheter was carefully inserted into the colon. Then, 10% acetic acid Chemicals and Reagents (0.5ml) in 0.9% saline was instilled into the lumen of the Gold trichloride (30 wt% in HCl), polyvinylpyrrolidone colon. The non-colitic group received 0.5ml of 0.9% saline (PVP, MW= 10.000), sodium hydroxide, dialysis tubing intracolonically. The rats were maintained in a supine cellulose membrane, and glycerol were products of Trendelenburg position for 30 s to prevent leakage of the Sigma-Aldrich Chemical Co (Saint Louis, USA). Sulfuric intracolonic instillation. acid and hydrogen peroxide were purchased from Vetec The animals were fasted overnight and euthanized with (Rio de Janeiro, Brazil). Phosphate-buffered saline (PBS) an overdose of thiopental, 48 h after induction. Next, the solution and bovine serum albumin (BSA, 5%) were pur- colon was removed aseptically, rinsed with PBS, and chased from Life Technologies Corporation© (California, placed on an ice-cold plate. The colon was cleaned of fat 80 Garcia et al. Nanoscale Research Letters (2019) 14:166 Page 3 of 12 and mesentery. Then, each specimen was weighed and its following the standard protocol [8]. Prior to the prepar- length measured under a constant load (2 g). Afterward, ation of the sections for indirect IF staining, the slides the colon was opened longitudinal and scored for macro- were kept in an oven at 60 °C for 24 h. scopically visible damage on a scale of 0 to 10 according to the criteria described by Bell et al. [15]. Immunofluorescence and Protocols Designs Alcohol steatohepatitis was induced in male Wistar rats Three tissue sections (4 μm) from each animal were (290 ± 10 g of BW) obtained from the Department of Bio- deparaffinized in xylene and washed in a series of de- physical and Pharmacology – Federal University of Rio creasing concentrations of ethanol, from 99% ethanol to Grande do Norte (UFRN). The animals were divided into 50% ethanol. Finally, tissue sections were washed twice two groups (n = 5 per group): alcoholic and non-alcoholic in dH2O and one wash with PBS. Antigen retrieval was rats. Ethanol solution (7 g/kg body weight of 30% v/v) was performed by placing the sections in a 10mM sodium used as a chronic dose for the alcoholic group. Animals in citrate with 0.05% Tween 20 at 95 °C for 30 min and the non-alcoholic group orally received an equivalent vol- subsequently at room temperature (RT) for 20 min. ume of saline solution (0.9% NaCl) by gavage. Gavage proce- Background noise/signal was reduced by incubating the dures were performed once a day in both groups for 28 sections in 0.1% Sudan black in 70% alcohol at RT for days. 20 min, followed by three washes in 0.02% PBS-Tween On day 29, euthanasia was performed by intraperito- 20. The samples were permeabilized in 0.2% neal injection of 7.5 ml/kg ketamine (50 mg/ml) and 2.5 Triton-X-100 in PBS (three washes, 5 min each), washed ml/kg Xylazine (20 mg/ml). Before euthanasia, all animal in PBS, and then blocked with PBS, 5% BSA, dH2O, and groups were fasted for 12 h. Once unconscious, the ani- Triton-X-100. Slides were incubated with block solution mals underwent cardiac puncture followed by removal in a humidity chamber for 2 h. As shown in Table 1 and of the liver. Liver fragments were immersed in 10% buff- Fig. 1, the incubation of the anti-COX-2 and anti-MIF ered formaldehyde for histopathological analysis. primary antibodies was performed following three differ- Animals from both acetic acid-induced UC and ent protocols (NP1, NP2, and NP3). These protocols alcohol-induced steatohepatitis were housed under were compared to two ALEXA-based protocols that standard conditions (12-h light/dark cycle, 22 ± 0.1 °C, differ in the time of incubation of the primary anti- and 50–55% humidity) with ad libitum access to food body—the ALEXA standard protocol (overnight incu- and water. Animals were treated according to the ethical bation) adopted by our research group as standard principles for animal experimentation. protocol for most procedures (A1) and ALEXA modi- fied protocol (A2; 30 min incubation). NP1 is a Histology nanoparticle-dependent immunofluorescence staining Five paraffin blocks from the positive controls (acetic method with 30 min for primary incubation. Primary acid control and alcoholic control) of each inflammation antibodies were diluted in 1% BSA at ratios of 1:500 model were used for indirect IF staining. The primary (anti-COX-2) and 1:400 (anti-MIF), and each sample antibodies anti-COX-2 and anti-MIF were found to be was incubated in a humidity chamber at RT (20 °C) good inflammation markers in acetic acid-induced UC for 30 min. Next, 100–150 μl of AuNPs were added to and alcohol-induced steatohepatitis, respectively, by the samples, which were left incubated at RT (20 °C) providing a reliable staining in terms of fluorescence for another 30 min. In the second nanoparticle- intensity that can be used to compare different dependent protocol (NP2), primary antibodies were diluted protocols. in 1% BSA and applied directly to the slides, which were left The tissues were fixed in 10% buffered formaldehyde, in the refrigerator overnight. Therefore, A2 and NP1 are dehydrated with ethyl alcohol (70%, 80%, 90%, 95%, and both 30-min incubation protocols, but A2 is performed P.A.), clarified in Xylol and impregnated in paraffin with a secondary fluorescent antibody (ALEXA), whereas Table 1 Comparison of immunofluorescence protocols based on ALEXA and AuNPs Deparaffinization, rehydration Antigen Retrieval - Primary antibodies ALEXA/ AuNPs incubation PBS washes and slides and PBS washes. block solution incubation assembling with DAPI Immunofluorescence Protocols A1 1h 3,8h (230 min) 18h ALEXA (1h) 20 min A2 1h 3,8h (230 min) 30 min ALEXA (30 min) 20 min NP1 1h 3,8h (230 min) 30 min AuNPs (30 min) 20 min NP2 1h 3,8h (230 min) 18h AuNPs (1h) 20 min NP3 1h 3,8h (230 min) 18h ALEXA + AuNPs (1h) 20 min 81 Garcia et al. Nanoscale Research Letters (2019) 14:166 Page 4 of 12 Fig. 1 Images of hematoxylin and eosin staining of acetic acid-induced UC and alcohol-induced steatohepatitis control groups. a Negative control of UC Model (× 100; scale bar = 100 μm). b Acetic acid-induced UC. The red arrow indicates leukocyte infiltration of the mucosa area (× 100; scale bar = 100 μm). c Negative control of steatohepatitis (× 200; scale bar = 50 μm). d Alcohol-induced steatohepatitis. The black arrow indicates leukocyte infiltration (× 200; scale bar = 50 μm) NP1 has AuNPs as fluorophores. This also applies for the software (Carl Zeiss) was utilized to quantitatively evalu- overnight-incubation protocols A1 and NP2. After the re- ate the intensity of fluorescence pixel by pixel for each spective incubation times, the slides of each protocol were picture of the ALEXA channel. At least three samples of washed three times with PBS to remove the excess primary each animal (five animals per group) were analyzed and antibody. Then, 100–150 μl of nanoparticles were added to five pictures were taken from each fragment with the × each slide in NP2, whereas ALEXA (1:400) was added in 10 objective. A1. After 1 h of incubation with AuNPs (NP2) and ALEXA (A1), all slides were washed three times with PBS. The third protocol (NP3) was design to explore the Induction of M2-polarized TAMs In Vitro amplifying properties of AuNPs in fluorescent systems. For evaluation of primary antibody-conjugated gold In NP3, AuNPs were applied during dilution of ALEXA nanoparticles (AuNPs) in M2 macrophages, RAW 264.75 in 1% BSA. Three dilutions of secondary antibody cells (5 × 10 cells/well in a 12-well plate) were cultured (1:200, 1:400, and 1:800) in BSA with AuNPs were in complete medium with 10% fetal bovine serum sup- tested in order to compare the fluorescence intensities plemented with 20 ng/ml IL-4 for 24 h. After treatment of each dilution to A1. The dilutions were prepared in with IL-4, cells were washed three times with serum-free such a way that the volume of AuNPs was proportional DMEM, followed by culture in the same medium for 48 to the volume of BSA, e.g., for a 1:200 dilution, 2 μl of h. Cells were analyzed using a Telaval 31 light micro- ALEXA was diluted in 199 μl of AuNPs + 199 μl of scope (ZEISS, Oberkochen, Germany). BSA. The incubation time for the primary antibodies in A1 and NP3 was approximately 18 h (overnight), Flow Cytometry whereas the incubation time for ALEXA + AuNPs sus- After incubation with IL-4 for 48 h, RAW 264.7 cells pension was 1 h. and M2 macrophages (1.5 × 104 cells/well in a 12-well Finally, samples were mounted using Fluoroshield plate) were collected with a scraper and blocked with Mounting Medium with DAPI. The slides were stored in 0.5 g BSA in 100 ml PBS (PBA) for 45 min, followed by a light-protected box and kept at 4 °C until microscop- incubation with PerCP-conjugated anti-mouse CD163 ical analysis. Fluorescent images were obtained with a (1:1000) and FITC-conjugated anti-mouse CD86 Zeiss Observer z.1 upright microscope for fluorescence (1:1000) at 4 °C for 60 min. Besides, M2 macrophages and bright field imaging (× 20 and × 10 objectives, Carl were labeled with COX-2 and MIF primary antibodies Zeiss, Jena, Germany) with AxioCam MRc. The arith- and so conjugated to AuNPs. M2-polarized TAMs were metic mean parameter of the Zeiss ZEN lite blue edition collected and fixed with 2% paraformaldehyde in PBS for 82 Garcia et al. Nanoscale Research Letters (2019) 14:166 Page 5 of 12 Table 2 Comparison of flow cytometry protocols based on ALEXA and AuNPs Overral time 2% Formaldehyde Primary antibody Incubation with Washes Resuspension fixation (10 min, 37°C) incubation fluorochromeconjugated substances and analysis Flow Cytometry Protocols Standard 4h Yes 1h ALEXA 488 diluted in PBA - 1 h 2x Yes AuNPs 1h No 30 min AuNPs - 30 min None Yes 10min, followed by incubation with the anti-COX-2 and 520 nm). Following the final washing step, labeled cells anti-MIF primary (1:100) for 60min at 4 °C and; after- were analyzed in a BD FACSCanto II (BD Biosciences, the ward, the cells were labeled with goat anti-rabbit Alexa® Netherlands) and FlowJo software (version 10.1; Tree Star Fluor 488-conjugated secondary antibody (Thermo Fisher Inc., UK). All flow cytometry analyses were performed Scientific) in incubation solution (1:100) at room with samples in triplicate and repeated three times. Com- temperature for 60min as described in the Alexa standard parison between standard ALEXA and AuNPs protocols protocol [16]. Following the protocol (N1) where AuNPs are shown in Table 2. replace the goat anti-rabbit Alexa® Fluor 488-conjugated secondary antibody, M2 macrophages were harvested, Statistical Analysis washed with PBS, and incubated with the anti-COX-2 and Analysis of variance (ANOVA) and Bonferroni’s post anti-MIF primary (1:100) in incubation solution at room hoc test were performed for immunofluorescence ana- temperature for 30min. Then, the cells were incubated lysis. For flow cytometry analysis, the significant dif- with AuNPs (1:5) at 4 °C for 30min (AuNPs excitation at ferences between the groups were calculated using Fig. 2 Comparison of fluorescence intensity among groups. a Fluorescence intensity of AuNPs in A2, NP1, and NP2 in comparison to A1-only acetic acid-induced UC (anti-COX-2 antibody). b All dilutions of NP3 (1:200, 1:400, and 1:800) in comparison to the fluorescence intensity of A1 (1:400)-only acetic acid-induced UC (anti-COX-2 antibody). c Comparison of the fluorescence intensity among acetic acid-induced animals and negative controls after short-time (A2 and NP1) and overnight incubation (A1 and NP2). d Fluorescence intensity of AuNPs in A2, NP1, and NP2 in comparison to A1-only alcohol-induced steatohepatitis (anti-MIF antibody). e All dilutions of NP3 (1:200, 1:400, and 1:800) in comparison to the fluorescence intensity of A1 (1:400)-only alcohol-induced of steatohepatitis (anti-MIF antibody). f Comparison of the fluorescence intensity among alcohol-induced steatohepatitis animals and negative controls after 30-min incubation (A2 and NP1) and overnight incubation (A1 and NP2). Statistics: ANOVA and Bonferroni’s post hoc test, #p ≥ 0.05, **p < 0.01,***p < 0.001, and ****p < 0.0001 83 Garcia et al. Nanoscale Research Letters (2019) 14:166 Page 6 of 12 ANOVA and Dunn’s test, as indicated. A p < 0.05 was and d demonstrate that the fluorescence intensities of considered statistically significant for all analysis A2, NP1, and NP2 are not significantly different in performed. comparison to A1 protocol (#p > 0.05), suggesting that 30-min incubation protocols (either using ALEXA or Results AuNPs) may be applicable in a diagnostic context. Microscopy Analysis In addition, the NP3 protocol (Fig. 2b, e) demon- The section in Fig. 1a shows a tissue section stained with strated that it is possible to combine the AuNPs to hematoxylin and eosin (negative control). The chronic ALEXA so that the detected fluorescence intensity was inflammatory process induced by acetic acid is depicted increased (***p < 0.001), allowing even double of the di- in Fig. 1b, which reveals loss of tissue architecture with lution of ALEXA without the fluorescence decreasing consequent destruction of the epithelium, a reduction in relative to the 1:400 dilution originally tested on A1 goblet cells, the presence of hemorrhages and leukocytes (#p > 0.05). near the injured sites. In figure fatty droplet accumula- Finally, we compared the diseased groups to the healthy tion and inflammatory infiltrate (neutrophils and lym- groups (Fig. 2f) to demonstrate that all protocols tested, phocytes) were seen in the livers from rats subjected to either with ALEXA or with AuNPs, are effective in detect- chronic alcohol exposure. ing the onset of inflammatory processes characterized by In terms of fluorescence intensity, AuNPs looked antigens COX-2 and MIF. Figures 3 and 4 support the similar to ALEXA, which is a conventional fluoro- data shown graphically in Fig. 2. Both ALEXA and AuNPs phore for IF staining. In both liver and colon are represented in green staining, whose distribution cor- samples, the differences in the fluorescence intensity responds to sites of increased leukocyte infiltration and of NP1 and NP2 were minimal or non-existent when tissue injury. compared to A1. In Fig. 2, multiple comparisons In terms of clinical application, the NP1 protocol proved among the IF protocols tested using anti-COX-2 and to be the most promising, since, when compared to the anti-MIF primary antibodies are shown. Figures 2a standard A1 protocol, it allows a saving of 18 h. Fig. 3 Anti-COX-2 IF images of colon samples from the saline (negative control) and the acetic acid-induced UC groups stained with green fluorescent compounds (ALEXA, AuNPs, or both) and DAPI (blue) for nuclei. a Negative control stained with ALEXA standard protocol (A1) with anti-COX-2 and ALEXA (green). b Negative control stained with modified ALEXA protocol (A2) with anti-COX-2 and ALEXA. c Negative control stained with nanoparticle protocol 1 (NP1) with anti-COX-2 and AuNPs (green) instead of ALEXA. d Negative control stained with nanoparticle protocol 2 (NP2) with anti-COX-2 and AuNPs instead of ALEXA. e Positive control stained with A1. f Positive control stained with A2. g Positive control stained with NP1. h Positive control stained with NP2. i Positive control stained with nanoparticle protocol (NP3) with anti-COX-2 +AuNPs with 1:200 ALEXA. j Positive control stained with NP3 (with 1:400 dilution of ALEXA). k UC positive control stained with NP3 (with 1:800 dilution of ALEXA). Magnifications, × 200. Scale bar = 50 μm 84 Garcia et al. Nanoscale Research Letters (2019) 14:166 Page 7 of 12 Fig. 4 Anti-MIF IF images of liver samples from the saline (negative control) and alcohol-induced steatohepatitis groups stained with green fluorescent compounds (ALEXA, AuNPs, or both) and DAPI (blue) for nuclei. a Negative control stained with ALEXA standard protocol (A1) with anti-MIF and ALEXA (green). b Negative control stained with ALEXA modified protocol (A2) with anti-MIF and ALEXA. c Negative control stained with nanoparticle protocol 1 (NP1) with anti-MIF and AuNPs (green) in place of ALEXA. d Negative control stained with nanoparticle protocol 2 (NP2) with anti-MIF and AuNPs in place of ALEXA. e Positive control stained with A1. f Positive control stained with A2. g Positive control stained with NP1. h Positive control stained with NP2. i Positive control stained with nanoparticle protocol 3 (NP3) with anti-MIF + AuNPs with 1:200 ALEXA. j Positive control stained with NP3 with a 1:400 dilution of ALEXA. k Positive control stained with NP3 with a 1:800 dilution of ALEXA. Magnifications, × 200. Scale bar = 50 μm Flow Cytometry: Primary Antibody-Conjugated Gold Fluorescence Spectroscopy Nanoparticle (AuNP)-Labelled M2 Macrophages In order to measure the fluorescence emission spectra To further characterize M2 macrophages, the expres- for purified AuNPs in the absence and presence of sion of M1 (CD86)- and M2 (CD163)-related makers anti-COX-2 (Fig. 6a), anti-MIF (Fig. 6b), and ALEXA in IL-4-stimulated RAW 264.7 cells were analyzed in (Fig. 6c), the excitation wavelength was fixed at 320 flow cytometry. Flow cytometry results showed that nm and the emission was recorded in the range of the M2-related markers such as CD163 receptor 650 to 900 nm. An increase of the excitation wave- were significantly higher than the native cell (Fig. 5a, length did not change the emission range of the par- c, p < 0.001) while CD86 receptor expression did not ticles, thus excluding the possibility of a scattering show any difference between M2 macrophages and process. Fluorescence emissions were slightly in- naive cells (Fig. 5b, c, p > 0.05). The results suggested creased with anti-COX-2 uptake (increased 11.93 that IL-4 (20 ng/ml) successfully induced the alter- absorbance units (a.u.) for the highest antibody con- ation of classical macrophages to M2 macrophages. centration), anti-MIF (increased of 12.15 a.u. for the After this step, M2 macrophages were incubated with highest antibody concentration), and ALEXA (in- COX-2 and MIF primary antibodies and then labeled creased of 3.18 a.u. for the highest antibody concen- with AuNPs to compare the efficiency of primary tration) in combination with AuNPs. antibody-conjugated gold nanoparticles (AuNPs) to the standard protocol (ALEXA). The results showed Discussion that either COX-2 and MIF antibody-conjugated gold It has been shown that AuNPs have fluorescence proper- nanoparticles (Fig. 5d, f, p < 0.001) or ALEXA (Fig. 5e, ties, due to their chemical structure and size, and can f, p < 0.001) showed higher fluorescence intensity in act as amplifying molecules of fluorescent signals. More M2 macrophages when compared to unstained specific, AuNP fluorescence itself originates from auro- samples. philic interactions on the surface of gold atoms [17, 18]. 85 Garcia et al. Nanoscale Research Letters (2019) 14:166 Page 8 of 12 Fig. 5 COX-2 and MIF antibody-conjugated gold nanoparticles (AuNPs) have high affinity on the surface of M2 macrophages. a–c RAW 264.7 cells were stimulated with IL-4 (20 ng/ml) for 24 h, followed by flow cytometry analysis to quantify the amount of CD163, an M2 macrophage marker, and CD86, an M1 marker. d, f Either COX-2 and MIF antibody-conjugated gold nanoparticles or e, f ALEXA 488 showed higher fluorescence intensity in M2 macrophages when compared to unstained samples. Data are expressed as mean ± SD, #p > 0.05, ***p < 0.001, p < 0.0001. Representative flow data shown are from experiments independently performed at least three times The data from Fig. 6 is similar to the findings of studies the sites of injury caused by acetic acid or ethanol in the on silver nanoparticles, which owe the increased fluores- respective model. Inflammation is also characterized by cence emission to the competition between adsorbent the leukocyte-dependent release of several cytokines and species and the molecular oxygen dissolved in solution. mediators in response to a pathogen or a stressful condi- Therefore, the increased fluorescence emission of gold tion. Among these cytokines, COX-2 and MIF are nanoparticles could be attributed to the fact that BSA is known to be markers for several inflammation processes, adsorbed on their surface [19]. since they are pro-inflammatory cytokines. The green The fluorescence signal is a function of the surface plas- staining observed in Fig. 3 and Fig. 4 is due to the re- mon resonance phenomenon (SPR). Hence, the increase in lease of COX-2 and MIF cytokines, respectively. the amount of free electrons leads to a more intense SPR In chronic UC and liver steatohepatitis, macro- signal, since SPR is the collective oscillation of free elec- phages are the main cells found which can be classi- trons. The adsorbed BSA prevents the surface free electrons fied into two major types: M1 macrophages and M2 from binding to the oxygen molecules, leading to an in- macrophages. The classically activated macrophages crease of the fluorescence signal [20, 21]. Anti-COX-2, (M1 macrophages) are proinflammatory and play a anti-MIF, and ALEXA may have been adsorbed on the pivotal role in host defense against infection which is AuNPs, thereby preventing O2 from reaching the surface of associated with iNOS and IL-23 production and their the nanoparticles. This is supported by Fig. 6, where the in- cell surface-expressed CD86 or HLA-DR that attract crease of the fluorescence signal occurred at very low con- killer cells like neutrophils and/or direct Th1 (cyto- centrations of the different antibodies. It is worth to notice, toxic) responses and stimulate further M1-type re- however, that ALEXA required a higher concentration to sponses [22]. Meanwhile, the alternatively activated cause O2 isolation of the AuNPs surface. macrophages (M2 macrophages) are associated with The chronic inflammation process is characterized by the responses to anti-inflammatory reactions as well the infiltration of immune cells, mainly macrophages, at as tissue remodeling [23]. In the tumor context, it has 86 Garcia et al. Nanoscale Research Letters (2019) 14:166 Page 9 of 12 Fig. 6 Fluorescence emission spectra of AuNPs excited at 320 nm in the presence of anti-COX-2 (a), anti-MIF (b), and ALEXA (c). The TEM image of the spherical nanoparticles used in this study, showing its size and distribution (d). Excitation and emission slits were 10 nm. The concentration of AuNPs was held constant at 29.6 ng/ml−1. The concentrations of antibody are 100 ng/ml−1 (blue curve), 150 ng/ml−1 (red curve), and 200 ng/ml−1 (green curve) been documented that M2-polarized macrophages Studies show MIF presence in the sera after hepatic promote pro-tumor functions by production of a large resection or expression in the course of liver cancer array of growth factors such as COX-2 and MIF for progression [30]. MIF is involved in COX-2 and PGE2 tumor cells, which are essential for tumor proliferation upregulation and directly promotes tumorigenesis by [24]. inhibition of p53 accumulation, which is a classic tumor It is well established that UC is an important risk suppressor gene that can promote cell cycle arrest and factor for colonic epithelial dysplasia and adenocarcin- apoptosis in response to DNA damage [31]. oma [25, 26]. According to Agoff et al. [25], increased In a previous study [6], we have demonstrated that malondialdehyde (MDA) levels and upregulation of AuNPs can be easily conjugated with the antibodies Bcl-2 during UC are potential mechanisms to explain anti-β-catenin and anti-E-cadherin to specifically target the relationship between COX-2 overexpression and colorectal carcinoma cells, whose clinical value can be neoplastic progression. In UC, the inflammation found in an early diagnosis of cancer through boosts COX-2 activity, leading to genetic damage non-invasive methods in body fluids such as saliva and through increased production of MDA. MDA is a urine. In addition, we developed a new protocol to de- by-product of COX-mediated prostaglandin synthesis crease the 27 h that are usually needed for the standard and lipid peroxidation, and it is also constitutively protocol to about 1 h needed for our improved protocol, produced by COX-1. Since COX-2 upregulates Bcl-2 which makes this method eligible for a clinical colorectal expression, it leads to resistance to apoptosis in cancer diagnostic. UC-associated neoplasia [27]. In this study, we took advantage of the properties of MIF is a multipotent cytokine in the innate immune AuNPs conjugated with primary antibodies and applied responses that contributes to hepatic injury driven by them for the indirect IF staining method of tissue sec- alcohol-induced steatohepatitis [28]. When steatohe- tions. At this point, we cannot affirm whether the inter- patitis has developed, the liver morphology rarely action of the antibody with the nanoparticle is purely goes back to normal, even after cessation. In addition, physical or if there is a chemical bond, since the amount there is a higher risk of the development of cirrhosis, of antibodies used in this method are far too small to which is the last stage of alcoholic liver disease produce discernible signals in Fourier-transform infrared (ALD) before hepatocellular carcinoma (HCC) [29]. spectroscopy. Thus, we rely only on fluorescence data 87 Garcia et al. Nanoscale Research Letters (2019) 14:166 Page 10 of 12 that suggest that conjugation was achieved by direct ad- arranged on the surface of M2 macrophage as a fast sorption of antibodies on the AuNPs surface. Such en- alternative to analyze the immune profile of hancement has been rationalized in terms of inflammation-induced cancer tissues by flow cytometry. competition between adsorbing species and molecular Although the standard protocol is laborious, the inten- oxygen dissolved in solution, as explained before by sity of marking to both the primary antibodies was Lima et al. [6]. We found that the replacement of higher than primary antibody-conjugated gold nanopar- ALEXA by the AuNPs in NP1 saves about 18 h (similar ticles. However, the primary antibody-conjugated gold to A2), when compared to standard protocol and NP2 nanoparticles needed less reagents and the time saving that require about 24 h, each, from the antigenic re- was higher as seen in the standard protocol (4 h) and in trieval to the assembling of the slides for microscopic primary antibody-conjugated gold nanoparticles N1 analysis (Fig. 7 and Table 1). The time for incubation protocol (1 h) (Fig. 7b). These results suggest that M2 with the primary antibody was 30min for A2 and NP1, macrophages can be targeted with primary but overnight (18 h) for A1, NP2, and NP3 (Fig. 7a and antibody-conjugated gold nanoparticles either to diag- Table 1). nosis or therapy. Moreover, we demonstrated MIF and COX-2 primary The time saving, the specificity, and the low cost pro- antibody-conjugated gold nanoparticles can be vided by NP1 are especially important in cancer Fig. 7 Comparative schemes illustrating the immunofluorescence (a) and flow cytometry protocols used in this study (b). In immunofluorescence, AuNPs may be applied in 30-min incubation protocols as fluorescence-enhancing agents, providing faster results with comparable fluorescence levels to the traditional ALEXA protocol, which makes NP1 a suitable protocol for cancer diagnose. Time was counted from the antigen retrieval to the end of the second incubation. In flow cytometry, although the ALEXA marking was higher than that of the AuNPs, the N1 protocol with AuNPs allowed a greater saving of reagents as well as reduced the time required for the technique from 4 to 1 h 88 Garcia et al. Nanoscale Research Letters (2019) 14:166 Page 11 of 12 diagnosis, when fast and accurate results are highly re- Ethics Approval quired. It is important to highlight that, although the Acetic acid-induced UC study was approved by the Ethics Committee on Animal Use (protocol number: 0608/2013) from Federal University of Paraiba. models adopted for this work were based on inflamma- Alcohol steatohepatitis experiments were approved by the UFRN Ethics tion diseases, the inflammation markers used in this Committee (approval number: 018/2015). study are highly cancer-correlated and AuNPs provide multiple possibilities for application in clinical research Competing Interests and diagnosis. The authors declare that they have no competing interests. Conclusions Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in All nanoparticle protocols tested showed similar fluorescent published maps and institutional affiliations. intensities to those observed in standard IF, extending the application of AuNPs, not only in research, but also in clin- Author details 1Department of Morphology, Federal University of Rio Grande do Norte, ical diagnostics. When diluted with ALEXA, AuNPs allow Natal, RN 59072-970, Brazil. 2Post-Graduation Programme in Structural and greater dilutions with acceptable fluorescence intensity. Functional Biology, Federal University of Rio Grande do Norte, Natal, RN More importantly, AuNPs can be used in faster protocols 59072-970, Brazil. 3Post-Graduation Programme in Health Science, Federal University of Rio Grande do Norte, Natal, RN 59072-970, Brazil. 4Department (e.g., 30-min incubation protocols), completely substituting of Biophysics and Pharmacology, Post-Graduation Programme in Public ALEXA and providing a way to develop further technolo- Health, Post-Graduation Programme in Pharmaceutical Science, Federal gies that will improve cancer diagnose and other diseases. University of Rio Grande do Norte, Natal, RN 59072-970, Brazil. 5Group of Biological Chemistry and Chemometrics, Institute of Chemistry, Federal We believe that these findings will contribute to advance University of Rio Grande do Norte, Natal, RN 59072-970, Brazil. 6Translational research and diagnostic procedures that utilize IF methods Nanobiomaterials and Imaging, Department of Radiology, Leiden University as well as widen the applications of AuNPs in Medical Center, 2333, ZA, Leiden, the Netherlands. 7Percuros B.V, 2333, CL, Leiden, the Netherlands. biotechnology. Received: 17 January 2019 Accepted: 22 April 2019 Abbreviations A1: ALEXA standard protocol; A2: ALEXA modified protocol; ALD: Alcoholic liver disease; ALEXA: Alexa Fluor® 488®; AuNP: Gold nanoparticles; References BSA: Bovine serum albumin; COX-2: Cyclooxygenase-2; FC: Flow cytometry; 1. Dykman LA, Khlebtsov NG (2011) Gold nanoparticles in biology and FI: Fluorescence imaging; IF: Immunofluorescence; MDA: Malondialdehyde; medicine: recent advances and prospects. Acta Naturae. 3(2):34–55 MIF: Macrophage migration inhibitory factor; NP1: Nanoparticles protocol 2. Wang G, Stender AS, Sun W, Fang N (2010) Optical imaging of non- 1NP2Nanoparticles protocol 2; NP3: Nanoparticles protocol 3; SPR: Surface fluorescent nanoparticle probes in live cells. Analyst. 135(2):215–221 plasmon resonance; UC: Ulcerative colitis 3. Klein S, Petersen S, Taylor U, Rath D, Barcikowski S (2010) Quantitative visualization of colloidal and intracellular gold nanoparticles by confocal Acknowledgements microscopy. J Biomed Opt. 15(3):036015 The authors are grateful to the Brain Institute (Federal University of Rio 4. Faulk WP, Taylor GM (1971) An immunocolloid method for the electron Grande do Norte) and the Leiden University Medical Center for their microscope. Immunochemistry. 8(11):1081–1083 contributions to the present study. We acknowledge support by 5. Kang KA, Nguyen MD (2017) Gold nanoparticle-based fluorescent contrast postdoctoral fellowship from Raimundo Fernandes de Araujo Junior by agent with enhanced sensitivity. Adv Exp Med Biol 977:399–407 CAPES 88881.119850/2016-01 and the European Commission where RFde A, 6. Lima KMG, Junior RFA, Araujo AA, Oliveira ALCSL, Gasparotto LHS (2014) ABC, and LJC have received funding from a MSCA-ITN-2015-ETN Action grant Environmentally compatible bioconjugated gold nanoparticles as efficient contrast (proposal number: 675743; project: ISPIC). agents for colorectal cancer cell imaging. Sens Actuator B-Chem. 196:306–313 7. Araújo DFS, Guerra GCB, Júnior RFA, Antunes de Araújo A, Antonino de Assis PO, Nunes de Medeiros A et al (2016) Goat whey ameliorates intestinal inflammation Funding on acetic acid-induced colitis in rats. J Dairy Sci. 99(12):9383–9394 This research was funded by CNPq/ Universal 2016 and number 425786/ 8. Araujo Junior RF, Garcia VB, Leitao RF, Brito GA, Miguel Ede C, Guedes PM et 2016-1. The APC was funded by MSCA-ITN-2015-ETN/European Commission al (2016) Carvedilol improves inflammatory response, oxidative stress and Action grant 675743, project: ISPIC. Check carefully that the details given are fibrosis in the alcohol-induced liver injury in rats by regulating Kuppfer cells accurate and use the standard spelling of funding agency names at https:// and hepatic stellate cells. PLoS One. 11(2):e0148868 search.crossref.org/funding, any errors may affect your future funding. 9. de Carvalho TG, Garcia VB, de Araujo AA, da Silva Gasparotto LH, Silva H, Guerra GCB et al. (2018) Spherical neutral gold nanoparticles improve anti- Availability of Data and Materials inflammatory response, oxidative stress and fibrosis in alcohol- The datasets used and analyzed during the current study are available from methamphetamine-induced liver injury in rats. Int J Pharmaceutics. 548:1–14. the corresponding author on reasonable request. 10. Joshi BP, Duan X, Kwon RS, Piraka C, Elmunzer BJ, Lu S et al (2016) Multimodal endoscope can quantify wide-field fluorescence detection of Barrett's neoplasia. Endoscopy. 48(2):A1–a13 Authors’ Contributions 11. Tipirneni KE, Rosenthal EL, Moore LS, Haskins AD, Udayakumar N, Jani AH RFAJ and VBG contributed to the experimental design. VBG, TGC, and GCBG et al (2017) Fluorescence imaging for cancer screening and surveillance. conducted the experiments in the animal models. HFOS, LHSG, and LJC Mol Imaging Biol 19(5):645–655 conducted the formulation analysis and manipulation of AuNPs. RFAJ and 12. Gasparotto LHS, Garcia AC, Gomes JF, Tremiliosi-Filho G (2012) AAA conducted the formal analysis. RFAJ, VBG, and TS contributed to the Electrocatalytic performance of environmentally friendly synthesized gold writing and preparation of the original draft. RFAJ, VBG, TGC, and TS nanoparticles towards the borohydride electro-oxidation reaction. J Power contributed to the writing and editing of the review. Supervision was done Sources. 218:73–78 by RFAJ. Project administration was done by RFAJ and ABC. All authors read 13. MacPherson BR, Pfeiffer CJ (1978) Experimental production of diffuse colitis and approved the final manuscript. in rats. Digestion. 17(2):135–150 89 Garcia et al. Nanoscale Research Letters (2019) 14:166 Page 12 of 12 14. Millar AD, Rampton DS, Chander CL, Claxson AW, Blades S, Coumbe A et al (1996) Evaluating the antioxidant potential of new treatments for inflammatory bowel disease using a rat model of colitis. Gut. 39(3):407–415 15. Bell CJ, Gall DG, Wallace JL (1995) Disruption of colonic electrolyte transport in experimental colitis. Am J Physiol. 268(4 Pt 1):G622–G630 16. Huang H, Koelle P, Fendler M, Schroettle A, Czihal M, Hoffmann U et al (2014) Niacin reverses migratory macrophage foam cell arrest mediated by oxLDL in vitro. PLoS One. 9(12):e114643 17. Z-j Z, C-x W, Wang Y, Niu S-h, C-g L, D-g F (2007) Fluorescent property of gold nanoparticles with different surface structures. Chinese J Chem Phys. 20(6):796–800 18. Zuber A, Purdey M, Schartner E, Forbes C, van der Hoek B, Giles D et al (2016) Detection of gold nanoparticles with different sizes using absorption and fluorescence based method. Sens Actuator B-Chem. 227:117–127 19. Alqudami A, Annapoorni S (2007) Fluorescence from metallic silver and iron nanoparticles prepared by exploding wire technique. Plasmonics. 2(1):5–13 20. Xiang H, Zhang X, Neuhauser D, Lu G (2014) Size-dependent plasmonic resonances from large-scale quantum simulations. J Phys Chem Lett. 5(7): 1163–1169 21. Ringe E, Langille MR, Sohn K, Zhang J, Huang J, Mirkin CA et al (2012) Plasmon length: a universal parameter to describe size effects in gold nanoparticles. J Phys Chem Lett. 3(11):1479–1483 22. Tarique AA, Logan J, Thomas E, Holt PG, Sly PD, Fantino E (2015) Phenotypic, functional, and plasticity features of classical and alternatively activated human macrophages. Am J Respir Cell Mol Biol. 53(5):676–688 23. Atri C, Guerfali FZ, Laouini D (2018) Role of Human Macrophage Polarization in Inflammation during Infectious Diseases. Int J Mol Sci. 19(6):1–15. 24. Erreni M, Mantovani A, Allavena P (2011) Tumor-associated macrophages (TAM) and inflammation in colorectal cancer. Cancer Microenviron. 4(2):141–154 25. Agoff SN, Brentnall TA, Crispin DA, Taylor SL, Raaka S, Haggitt RC et al (2000) The role of cyclooxygenase 2 in ulcerative colitis-associated neoplasia. Am J Pathol. 157(3):737–745 26. Morson BC (1985) Precancer and cancer in inflammatory bowel disease. Pathology. 17(2):173–180 27. Bronner MP, Culin C, Reed JC, Furth EE (1995) The bcl-2 proto-oncogene and the gastrointestinal epithelial tumor progression model. Am J Pathol. 146(1):20–26 28. Barnes MA, McMullen MR, Roychowdhury S, Pisano SG, Liu X, Stavitsky AB et al (2013) Macrophage migration inhibitory factor contributes to ethanol- induced liver injury by mediating cell injury, steatohepatitis, and steatosis. Hepatology. 57(5):1980–1991 29. Testino G, Leone S, Borro P (2014) Alcohol and hepatocellular carcinoma: a review and a point of view. World J Gastroenterol 20(43):15943–15954 30. Han Y, Zhang C (2010) Macrophage migration inhibitory factor plays a pivotal role in hepatocellular carcinoma and may be a noninvasive imaging target. Med Hypotheses. 75(6):530–532 31. Conroy H, Mawhinney L, Donnelly SC (2010) Inflammation and cancer: macrophage migration inhibitory factor (MIF)--the potential missing link. QJM. 103(11):831–836 90 Apêndice B Functionalization of gold nanoparticles with two aminoalcohol-based quinoxaline derivatives for targeting phosphoinositide 3-kinases (PI3K). Janine Araújo, Fabrício G. Menezes, Heloiza F. O. Silva, Davi S. Vieira, Sergio R. B. Silva, Adailton J. Bortoluzzi, Celso Sant’Anna, Mateus Eugenio, Jannyely M. Neri, Luiz H. S. Gasparotto. New Journal of Chemistry, v. 43, p. 1803-1811, 2019 Contribuição:  Realizei a síntese e funcionalização das NanoAu  Realizei a caracterização das NanoAu funcionalizadas com as quinoxalinas.  Fiz o tratamento e interpretação dos dados de caracterização  Auxiliei na escrita da primeira versão do manuscrito  Realizei os ajustes sugeridos pelos revisores nas figuras. ________________________ __________________________ Heloiza Fernanda O. da Silva Athayde Prof. Luiz Henrique da S. Gasparotto 91 NJC PAPER View Article OnlineView Journal Functionalization of gold nanoparticles with two aminoalcohol-based quinoxaline derivatives for Cite this:DOI: 10.1039/c8nj04314k targeting phosphoinositide 3-kinases (PI3Ka)† Janine Araújo,a Fabrı́cio G. Menezes,a Heloiza F. O. Silva,a Davi S. Vieira,a Sergio R. B. Silva, b Adailton J. Bortoluzzi,c Celso Sant’Anna,d Mateus Eugenio,d Jannyely M. Neria and Luiz H. S. Gasparotto *a Quinoxaline derivatives have attracted considerable attention due to their vast range of applications that includes electroluminescence and biomedicine. Concerning the latter, the literature has shown that compounds with a quinoxaline motif bind quite efficiently to phosphatidylinositol-4,5-bisphosphate 3-kinases (PI3Ks), which are enzymes found to be overexpressed in some types of neoplasms. In the present study, gold nanoparticles (AuNPs) were easily functionalized with 2,3-diethanolminoquinoxaline (DEQX) and 2-(2,3-dihydro-[1,4]oxazino[2,3-b]quinoxalin-4-yl)ethanol (OAQX). We made use of glycerol in alkaline media as reducing agent and the quinoxalines served as capping ligands to stabilize the AuNPs. This is the first Received 23rd August 2018, report on the modification of a nanostructure with quinoxalines. Functionalization confers nanoparticles Accepted 16th December 2018 the required specificity to target only cancer cells, which opens possibilities for phototherapy since the DOI: 10.1039/c8nj04314k modified AuNPs would concentrate in the tumor tissue as a consequence of PI3Ka overexpression. Molecular dynamics simulations have shown that DEQX and OAQX are potential inhibitors of PI3Ka rsc.li/njc since they bind to the active site of the enzyme in a way similar to other known inhibitors. 1 Introduction has been implicated in that severe type of neoplasm. 5,6 The clarification of the molecular mechanism of cancer signaling Cancer is a multiple-factor malady caused by accumulation of allowed the development of pharmacological inhibitors that genetic mutations that lead to abnormal proliferation of tissues. An target specific proteins to halt or cease tumor cell progression.7 important consequence of decoding the human genome sequence As an example, monoclonal antibodies have been considerably was the facilitation of the analysis of cancer genomes.1 This advance successful for cancer therapy in recent years,8 as they can be was of paramount importance for locating the most frequently precisely engineered to fit a specific protein’s active site in order mutated genes that are directly correlated to overexpressed proteins to inhibit its action. However, the production of monoclonal upon tumor growth.2 As examples, there are E-cadherin and antibodies is a laborious and time-consuming technology.9 b-catenin, which are proteins that play important roles in cell–cell An interesting approach to circumvent these features is the adhesion and whose overexpression is associated with colorectal design of molecules that have high affinity to the desired carcinomas.3,4 In the case of brain tumor, atypical augmenta- protein’s site and are easily synthesized through simple tion of phosphatidylinositol-4,5-bisphosphate 3-kinases (PI3Ks) routes.10 Quinoxaline derivatives have attracted considerable attention due to their vast range of applications that includes luminescence11 and biomedicine.12 Concerning the latter, com- a Biological Chemistry and Chemometrics Research Group, Institute of Chemistry, pounds with a quinoxaline motif are quite efficient antitumor13 Federal University of Rio Grande do Norte, Lagoa Nova, 59072-970, Natal, RN, and anticancer agents14,15 as they inhibit enzymes involved in Brazil. E-mail: lhgasparotto@ufrnet.br; Tel: +55 84 3342 2323 b cancer proliferation, motility and differentiation. Mielcke et al.16Brain Institute, Federal University of Rio Grande do Norte, Morro Branco, 59056-450, Natal, RN, Brazil found that quinoxaline-derived calchones were able to inhibit c Laboratório de Bioinorgânica e Cristalografia, Departamento de Quı́mica, glioma cell proliferation through binding with the PI3K enzyme. Universidade Federal de Santa Catarina, 88040-900 Florianópolis-SC, Brazil The PI3Ka enzyme, which is a subclass of PI3Ks, is regarded as d Laboratory of Microscopy Applied to Life Science – Lamav, National Instituto of potential therapeutic target due its role in several types of Metrology, Quality and Tecnology – Inmetro, Duque de Caxias, 25250-020, cancer.17,18 In this context, Wu et al.19,20 reported the design Rio de Janeiro, RJ, Brazil † Electronic supplementary information (ESI) available. See DOI: 10.1039/ and synthesis of a series of biologically active 2,3-dissubstituted c8nj04314k quinoxaline derivatives and also made use of computational This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019 New J. Chem. 92 Published on 17 December 2018. Downloaded by University of New England on 1/21/2019 3:00:15 AM. View Article Online Paper NJC chemistry to suggest binding modes between the PI3Ka active Covalent bonds involving hydrogen were restrained by LINCS site and the quinoxaline derivatives. algorithm35 while SPC water molecules geometries were con- Nowadays, targeted therapies may be substantially enhanced strained by the SETTLE36 algorithm. Motion equations were by bringing nanotechnology to the field, as it has been employed integrated by a leap-frog37 algorithm with time steps of 2.0 fs. successfully in imaging,21,22 diagnosing23 and cancer treatment.24 The long-range interactions were computed using particle- However, nanomaterials usually lack specificity and must be mesh Ewald sum, PME,38 with a cutoff of 10 Å also used for previously functionalized in order to become a task-specific van der Waals interactions. An energy minimization step using hybrid.25 In particular, the surface of gold nanoparticles (AuNPs) steepest descent39 was employed before the equilibration may be modified with a variety of compounds such as proteins, phase. The properties related with structural dynamics were DNA, and antibodies,26 to confer them the specificity required monitored by root-mean-square deviations (RMSD) using Ca for targeted therapies. atoms and the initial structure of the simulation (t = 0 ns) as Herein, the functionalization and stabilization of AuNPs reference structure. The hydrogen bonds (HB) between amino with two synthetic quinoxaline derivatives, 2,3-diethanolmino- acids residues and ligands were identified based on geometric quinoxaline (DEQX) and 2-(2,3-dihydro-[1,4]oxazino[2,3-b]quinoxalin- criteria, the distance between acceptor and donor o 3.5 Å, and 4-yl)ethanol (OAQX) were described. To the best of our knowledge, the angle donor-hydrogen-acceptor o 301.40 The interaction this is the first study on the functionalization of AuNPs with potential energy (IPE) for ligand-residue and ligand-water quinoxaline-based molecules. Another point worth mentioning PNi PNj is that, despite the low solubility of heterocyclic units in water, molecules can be computed according to Eij , where Eij i j we were able to functionalize the gold nanoparticles with both is the interaction energy between a group of atoms from ligand quinoxaline-based molecules in aqueous solution, which is quite (i) and a group of atoms from residue j, and Ni and Nj are an important aspect for medicinal purposes.27 Computational the total number of atoms on molecules/residues i and j, studies suggested good affinity to the active site of the PI3Ka. In respectively. The cutoff for this calculation was set to 6 Å from this way, based on these preliminary results, the quinoxaline- each ligand. AuNPs conjugates are envisaged as dual-function hybrid units Surface electrostatic potentials were generated using the that (i) may prevent the proliferation of tumor cells that are USCF Chimera software with Adaptative Poisson–Boltzmann dependent on the PI3Ka enzyme and (ii) could be suitable for Solver (APBS) interface41 and the images were produced by cancer phototherapy since the modified AuNPs concentrates in VMD visualization software.42 the tumor tissue as a consequence of the PI3Ka overexpression. 2.2 Materials and instrumentation All chemicals were purchased from commercial sources (Sigma- 2 Experimental section Aldrich and Tedia) and used without further purification. Melting 2.1 Computational methodology points weremeasured in aMicroquı́micaMQAPF-301 and the values The association of DEQX and OAQX with the PI3Ka binding site were not corrected. Elemental analysis of carbon, hydrogen and was first studied via docking using the Autodock Vina software.28 oxygen was performed in a Carlo Erba E-110 instrument. UV-vis The tridimensional structure of PI3Ka enzyme was obtained analyses were carried out with an Evolution 60S UV-visible spectro- from a 2.8 Å resolution X-ray structure (PDB entry 3HHM).29 photometer (Thermo Scientific). Infrared spectra were acquired in a The structure of PI3Ka enzyme was prepared for docking 283 Perkin-Elmer instrument. Nuclear magnetic resonance (NMR) with AutoDockTools 1.5.6 (ADT).30 The three-dimensional struc- analyses were conducted in a Varian Mercury Plus-400 MHz. Trans- tures of DEQX and OAQX were built with Avogadro software.31 mission electronmicroscopy (TEM) images were acquired with a FEI 2 A 25 30 30 Å3 grid was centered near the center of the binding Tecnai G Spirit BioTWIN microscope operating at 120 kV. pocket, and the exhaustiveness parameter was set to 32. Molecular dynamics (MD) simulations were then performed 2.3 Synthesis and spectroscopic characterization of DEQX with the structures from docking procedures. The minimum and OAQX distance of the quinoxaline-PI3Ka complex to the box wall was 2.3.1 2,3-Dichloroquinoxaline (DCQX). This starting material at least 12 Å, hence the dimensions of the simulation box was prepared by a two-step protocol according to.43 The protocol were chosen accordingly. The systems were neutralized with involves the acid catalyzed cyclization of o-phenylenediamine and sodium and chloride ions until reaching the concentration of oxalic acid to generate 1,4-dihydroquinoxaline-2,3-dione, which 0.15 mol L1. The AMBER ff03 force field32 was employed with reacts with thionyl chloride to afford the starting material DCQX the with the GROMACS 5.0 software package. The topology and at 79% yield. parameters for the ligands were generated with the General 2.3.2 2,3-Diethanolminoquinoxaline (DEQX). A solution of Amber Force Fields with structure and charges calculated using DCQX (0.50 g, 2.5 mmol) in ethanolamine (5 ml) was kept at HF/6-31G(d) as the level of theory and RESP fit.33 All quantum 100 1C for three hours. Afterwards, 25 ml of cold water were mechanics calculations were performed using GAMESS. MD simula- added and the final aqueous solution was maintained in ice tions were carried out at neutral pH in NVT ensemble at a tempera- bath for 8 h. The resulting solid was filtered and recrystallized ture of 310 K controlled by the V-rescale thermostat algorithm.34 from water to give the pure compound. New J. Chem. This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019 93 Published on 17 December 2018. Downloaded by University of New England on 1/21/2019 3:00:15 AM. View Article Online NJC Paper Mp 179 1C (Lit. 175–177 1C44). IR (KBr) nmax (cm 1): 3413, comprised thirty two neighboring residues, the results revealed 3199, 2974, 2921, 1555, 1514, 1465, 1047, 765. 1H NMR that the following twelve interacted with both quinoxalines only (400 MHz, CDCl3) d (ppm) 7.38 (m, 2H), 7.15 (m, 2H), 7.03 slightly, with energies no lower than 0.1 kcal mol1: Ile771, (t, J = 5.0 Hz), 4.83 (t, J = 5.4 Hz), 3.66 (m, 4H), 3.56 (m, 4H). Leu779, Glu798, Leu807, Asp810, Leu814, Gly837, Cys838, Ser919, 13C NMR (100 MHz, DMSO-D6) d (ppm) 143.8, 136.4, 124.5, Lys924, His931, and Asp933. The other twenty residues are listed 123.2, 59.4, 43.6. Elemental analysis for C12H16N4O2: C, 58.05; in Fig. 1A with their respective IPE for both quinoxalines. Con- H, 6.50; N, 22.57; found: C, 58.12; H, 6.63; N, 22.68. Yield: 85%. sidering an arbitrary cutoff of 1.0 kcal mol1, only six residues 2.3.3 2-(2,3-Dihydro-[1,4]oxazino[2,3-b]quinoxalin-4- (Ile932, Met922, Ser854, Val851, Ile800, Met772) are hotspots yl)ethanol (OAQX). Prepared similarly to DETQ, except by the for both quinoxalines. Gln859, Val850, Ser773 are hotspots for use of diethanolamine instead ethanolamine. DEQX, while Asn853 and Trp780 are hotspots for OAQX. On the Mp 160 1C (Lit. 159–161 1C45). IR (KBr) n 1max (cm ): 3284, 2992, other hand, nine residues (Phe930, Thr856, His855, Arg852, 2862, 1658, 1566, 1456, 1342, 1058, 771. 1H NMR (400 MHz, Glu849, Ile848, Tyr836, Ser774 and Pro778) interact with neither CDCl3) d (ppm) 7.54–7.51 (m, 2H), 7.40–7.37 (m, 1H), 7.29–2.26 quinoxalines. Fig. 1B and C present a visualization of the local (m, 1H), 4.83 (t, J = 5.2 Hz, 1H), 4.47–4.45 (m, 2H), 3.77–3.71 structure of the hotspots for (A) DEQX and (B) OAQX. (m, 6H). 13C NMR (100 MHz, DMSO-D6) d (ppm) 147.6, 142.8, Val851 and Ser854 deserve further attention due to their high 139.2, 134.9, 127.7, 126.1, 124.7, 123.8, 64.3, 60.0, 49.9, 46.1. quinoxaline-P3Kia IPE (Fig. 1A). In the case of Val851, the more Elemental analysis for C12H13N3O2: C, 62.33; H, 5.67; N, 18.17; attractive IPE is due to the hydrogen bond between its amide found: C, 62.40; H, 5.80; N, 18.26. Yield: 81%. backbonemoiety and the hydroxyl group of the quinoxalines. This type of interaction is reported to be crucial to kinase-inhibitor 2.4 Preparation of quinoxaline-functionalyzed AuNPs complexes.48,49 Similarly, the Ser854 residue has an amide back- AuNPs were prepared through a previously reported method46,47 bone and a side-chain hydroxyl conferring it high potential to that was slightly modified in this study. In the method, glycerol in form hydrogen bonds with ligands. 19 The short distance of these alkaline medium is used as reducing agent and the modification hydrogen bonds, (around 2.22 Å and 2.05 Å for DEQX and OAQX, consisted in replacing polyvinylpirrolidole (the stabilizing agent) respectively) and the high occurrence frequency (100% and with the quinoxalines synthesized in this work. The concentra- around 89.5% for DEQX and OAQX, respectively) strongly suggests tions of gold ions, glycerol and NaOH were kept at 0.5 mmol L1, that the Ser854 is also a key residue for the specific inhibition 0.10 mol L1 and 0.10 mol L1, respectively. The amounts process. In both cases, the intermolecular hydrogen bonds of DEQX and OAQX varied as such to generate the following are also favored by the flexibility of 2-hydroxyethyl group Au3+/quinoxaline molar ratios: 1 : 1, 1 : 0.5, 1 : 0.2, 1 : 0.1, 1 : 0.05, (–CH2CH2OH) of DEQX and OAQX, respectively. 1 : 0.01, and 1 : 0.005. The reaction was carried out by adding Although Ile932 is not as energetic as Val851 and Ser854, it a 5 mL solution of glycerol + NaOH into another 5 mL solution qualifies as a hotspot due to its non polar nature that allows for that contained Au3+ and a quinoxaline. The final mixtures had hydrophobic interactions with the aromatic ring portion of the red-to-violet colors characteristic of gold nanoparticles. quinoxalines. Furthermore, the absence of a Ile932 residue would lead to total loss of enzyme activity.49 Val850, Ile848, Ile800, Met922, Phe930, and Ile932 residues are constituents of ATP 3 Results and discussion binding site interacting as hotspots via hydrophobic contacts with DEQX and OAQX. The other residues presented in the Fig. 1A play 3.1 Molecular modeling a collaborative role as secondary hotspots. Concerning electrostatic PI3Ka are well established enzymes as promising targets for interactions, it is known that they may guide binding processes in cancer therapy in humans. Small molecules based on quinoxaline biological environments.41 In Fig. 1D and E, electrostatic surfaces moiety have their anti-tumor activity hypothesized in terms of of the P3KIa in the presence of DEQX and OAQX clearly show that binding to P3Kia.19 It is suggested that hydrogen bonds between the hydroxyl group is highly adaptive in biological environment oxygen atoms of amine moieties and Val851 and Ser854 residues due to its amphoteric functionality. are crucial to the activity, with some other possible interactions MD simulations have shown that DEQX and OAQX are involving arylsulfonyl units. The present investigation provides an potential inhibitors of PI3Ka since they bind to the active site analysis initially based on molecular docking models between of the enzyme in a way similar to other known inhibitors.48–50 PI3Ka enzyme and DEQX and OAQX molecules and then the The correct protein–ligand binding is designed by long range inter- best structures from docking were used for MD simulations. actions optimizing hydrogen bonds and charge complementarity. Since host–guest intermolecular interactions could be a primary Approximately 67% of the protein–ligand (small organic mole- condition for a successfully structure-based drug design, we have cules) complexes have hydrogen bonds mediating recognition analyzed the binding site of the receptor by interaction potential through NH (backbone) groups of a residue and oxygen atoms energy (IPE) from MD simulations to obtain the individual con- of the ligand.51 tribution of each amino acid residue, ranking the most relevant interactions among the molecules. 3.2 Synthesis and characterization of the target quinoxalines The 6 Å sphere centered on each quinoxaline molecule Classically, quinoxaline cores and their derivatives are was used for the calculation of IPE. Although such a sphere synthesized through reactions of aromatic o-diamines and This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019 New J. Chem. 94 Published on 17 December 2018. Downloaded by University of New England on 1/21/2019 3:00:15 AM. View Article Online Paper NJC Fig. 1 (A) Interaction potential energy (IPE) for each residue. The values are shown for DEQX (dashed bars) and OAQX (filled bars). The dashed red line indicates the arbitrary limit for the binding process. Structural representation of (B) DEQX and (C) OAQX inside the binding pocket of PI3Ka. Water molecules not shown. Electrostatic surface representation of the binding pocket of PI3Ka containing (D) DEQX and (E) OAQX. a-dialdehydes/a-diketones in the presence of a catalyst.52 Other The DEQX and OAQXmolecules were fully characterized through synthetic routes that involve reaction of DCQX with nucleophilic elemental analysis, infrared spectroscopy, 1NMR and 13C NMR species are also available in literature. The structural features spectroscopies. Also, OAQX had its structure confirmed by X-ray of this substrate allow the generation of innumerable 2- and/or crystallography (Fig. 2 – inset), as shown in ESI.† Both synthetic 2,3-substituted quinoxalines, including biologically relevant quinoxaline derivatives were soluble enough in water to allow compounds such as those that present anti-cancer activity due the synthesis of AuNPs in aqueous medium. to interactions with PI3Ka.19,20 In this study, DEQX and OAQX were obtained from reactions of DCQX43 with ethanolamine and 3.3 Production of DEQX-AuNPs and OAQX-AuNPs diethanolamine, respectively (Fig. 2). DEQX originated from In this study, Au3+ ions were reduced by glycerol in alkaline the reaction of DCQX with two equivalents of ethanolamine medium at room temperature. As it is biodegradable under via double N-nucleophilic attack, while OAQX was generated aerobic conditions, non-toxic, and relatively inexpensive, by reacting one equivalent of diethanolamine via cyclization glycerol is a ‘‘greener’’ option for generating nanoparticles when processes involving simultaneous N- and O-nucleophilic attacks. compared to common reducing chemicals such as formamide, Fig. 2 Synthetic protocol of DEQX and OAQX from DCQX – conditions: ethanolamine or diethanolamine, 100 1C, 3 h. Inset: ORTEP illustration of OAQX determined by X-ray crystallography. New J. Chem. This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019 95 Published on 17 December 2018. Downloaded by University of New England on 1/21/2019 3:00:15 AM. View Article Online NJC Paper sodium borohydride and hydrazine.53 It is well known that a decreased with the augmentation of DEQX concentration. In suitable stabilizing agent is also required in order to control contrast, the concentration of OAQX impacted only slightly on size and aggregation of nanoparticles in solution.54 To this end, the amount of AuNPs freshly produced, as evidenced by similar we investigated the ability of DEQX and OAQX to stabilize gold level of absorbances regardless of the Au3+/OAQX molar ratio nanoparticles. In this way, not only would the AuNPs be (Fig. 3B). This may be explained by the formation of a relatively appropriately available in solution, but they would also be stable complex between gold ions and DEQX that competes unquestionably functionalized with the quinoxalines employed with the reduction of Au3+. In an experiment devised to check in this study. Fig. 3 presents UV-vis spectra of AuNPs capped on this hypothesis, a Au3+ solution was mixed with another of with DEQX (A) and OAQX (B) acquired at different Au3+/qui- DEQX in absence of glycerol and NaOH and UV-vis spectra were noxaline molar ratios. The Au3+ concentration was fixed at recorded at different times as presented in Fig. 4A. The shoulder at 0.50 mmol L1. Upon increasing the amount of quinoxalines, 410 nm that arises with time is attributed to a gradual formation of a the UV-vis spectra tended to acquire the shape typically related yellow-to-orange complex between gold ions andDEQX. Khan et al.56 to spherical AuNPs,46 as seen in Fig. 3A and B. The shape of reported the formation of a stable yellow-orange complex from AuNPs will be confirmed later by TEM. The signal observed in the the reaction between cetyltrimethylammonium bromide (CTAB) UV-vis experiments reflects the surface plasmon resonance (SPB) and gold ions. In their study the same shoulder at 410 nm evolved of oscillating electrons on the surface of the nanoparticles.55 An over time uponmixing CTAB with gold ions. Furthermore, a yellow important difference between DEQX and OAQX is that, compara- coordination complex has been recently isolated from the reaction tively, a much lower concentration of the former is sufficient to of KAuCl4 and quinoxaline, 57 which is a further evidence that Au3+ generate AuNPs. A DEQX concentration of 25 mmol L1 (which is prone to complexation with at least some types of quinoxalines. corresponds to the molar ratio of 1 : 0.05) is sufficient for the On the other hand, similar behaviour is not observed for OAQX at generation of AuNPs, while at least 100 mmol L1 of OAQX is the same interval. Upon mixing Au3+ with OAQX, a slight intensi- required for the same purpose. Based on these results, DEQX fication of the pale yellow is observed only after 30 min, which was more efficient than OAQX for the formation of AuNPs. might suggest weak complexation to some degree. Another important feature worth mentioning is the relative It is important to note that after 24 h the absorbance of the amount of AuNPs generated with DEQX and OAQX. In Fig. 3A, DEQX-stabilized AuNPs solutions increased for Au3+/DEQX the lower absorbance over the Au3+/DEQX molar ratio range molar ratios between 1 : 0.05 and 1 : 0.5 (Fig. 5A), meaning that of 1 : 0.005–1 : 0.5 indicates that the amount of nanoparticles in the 24 hour interval the Au3+-DEQX complex slowly released Fig. 3 UV-vis spectra of AuNPs stabilized with (A) DEQX and (B) OAQX at different Au3+/quinoxalines molar ratios. Fig. 4 UV-vis spectra of solutions containing (A) Au3+ + DEQX and (B) Au3+ + OAQX acquired at different times. Glycerol and NaOH are absent. In both cases the concentrations of Au3+ and quinoxalines were 0.50 mmol L1 and 0.25 mmol L1, respectively. This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019 New J. Chem. 96 Published on 17 December 2018. Downloaded by University of New England on 1/21/2019 3:00:15 AM. View Article Online Paper NJC Fig. 5 UV-vis spectra of AuNPs stabilized with (A) DEQX and (B) OAQX at different Au3+/quinoxalinesmolar ratios. Spectra were acquired 24 h after the synthesis. gold ions for further formation of AuNPs. Fig. 5A also shows that AuNPs. A closer inspection of Fig. 5B reveals that there was a Au3+/DEQX molar ratios of 1 : 0.01 and 1 : 0.005 were not suffi- decrease in absorbance for Au3+/OAQX molar ratios other than cient for stabilization of AuNPs. On the other hand, absorbances 1 : 1, which implies that only in the latter condition stable AuNPs from OAQX-capped AuNPs (Fig. 5B) varied only marginally, could be obtained with OAQX as capping agent. which corroborates the hypothesis that OAQX binds weakly to Fig. 6 presents TEM images of AuNPs capped with DEQX (A) Au3+ and, therefore, gold ions were promptly available to form and OAQX (B) at Au3+/quinoxaline molar ratios of 1 : 0.2 and Fig. 6 TEM images of AuNPs capped with (A) DEQX and (B) OAQX synthesized at Au3+/quinoxaline molar fractions of 1 : 0.2 and 1 : 1, respectively. Circularity analysis of (C) DEQX-capped AuNPs and (D) OAQX-capped AuNPs. New J. Chem. This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019 97 Published on 17 December 2018. Downloaded by University of New England on 1/21/2019 3:00:15 AM. View Article Online NJC Paper Fig. 7 FTIR in ATR mode of (A) DEQX-adsorbed AuNPs and (B) OAQX-adsorbed AuNPS. The spectra of the pure quinoxalines are also shown for comparison. 1 : 1, respectively. It is important to note that while the histo- quinoxaline were spherical in shape, slight differences in the gram of DEQX-stabilized AuNPs is centered at around 8.0 nm, proportion of non-spherical AuNPs were observed between the one related to OAQX-stabilized AuNPs is shifted to 11.5 nm, samples. In the range 0.70–0.79, OAQX and DEQX delivered meaning that even at a lower concentration DEQX is more 20% and 8.7% of non-spherical nanoparticles, respectively. efficient than OAQX in delivering smaller nanoparticles. Fig. 6C Therefore, not only is DEQXmore efficient in delivering smaller and D depict circularity analysis of the AuNPs produced in this nanoparticles, it provides more homogeneous ones in terms of study. A circularity value of 1 means a perfectly symmetrical morphology as well. circle and values equal or below 0.90 denote non-spherical Fig. 7 presents FTIR spectra of DEQX, OAQX the respective morphologies. While over 60% of AuNPs produced by either quinoxaline-adsorbed AuNPs in ATR mode. Differences in the Fig. 8 Proposed mechanism for the formation of AuNPs with (A) DEQX and (B) OAQX. This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019 New J. Chem. 98 Published on 17 December 2018. Downloaded by University of New England on 1/21/2019 3:00:15 AM. View Article Online Paper NJC range of 1450–1600 cm1, which is related to CQN, CQC and the nanoparticle surface. In addition, there is no need of N–H bonds, suggest that DEQX (Fig. 7A) binds to the AuNPs employing a further stabilizing agent (e.g.: polymer) since both surface through nitrogen atoms. The bands at 1039 cm1 (C–O quinoxalines may act as stabilizers at appropriate concentrations. from alcohol) and 1053 cm1 (C–N from amine) for pure DEQX MD simulations indicated that DEQX and OAQX bind to PI3Ka become broader when the latter is adsorbed onto the AuNPs, through a variety of amino-acid residues, a feature that makes suggesting that those functional groups are involved in the those quinoxalines eligible to inhibit PI3Ka enzymes. The combi- interaction with gold. In the case of OAQX (Fig. 7B), the nation of synthetic organic chemistry with nanoscience and MD spectrum for functionalized AuNPs is far less defined than that simulations is an interesting approach to conceiving new for DEQX-AuNPs. This might be an indicative that OAQX weakly potential platforms for cancer diagnostic and treatment. binds to AuNPs and, therefore, is removed from the metallic surface during the centrifugation process. UV-vis and TEM results already showed that OAQX is less efficient than DEQX Conflicts of interest in stabilizing AuNPs. The general features for the spectrum are similar to the DEQX, including the region comprising CQN There are no conflicts to declare. and CQC that becomes broader when OAQX is adsorbed onto AuNPs. Considerable alterations in bands associated to the ether group at 1056 cm1 and 1203 cm1 indicated that the Acknowledgements oxygen of the saturated cycle also contributes to the interaction The authors gratefully acknowledge CNPq, FAPERN, CAPES with the gold surface. For both quinoxaline-adsorbed AuNPs, and FINEP for the financial support of this work. This research the band at 1039 cm1 and 1022 cm1 for DEQX and OAQX, was supported by High Performance Computing Center at respectively, related to the alcoholic group remains at the same NPAD/UFRN. frequency, suggesting that it is not involved in the ligand- metal coordination. In both spectra, absorptions due to water bending at approximately 1635 cm1 denote that AuNPs are References hydrated to some extent.58,59 1 D. A. Wheeler and L. Wang, Genome Res., 2013, 23, 3.4 Mechanism of AuNPs formation 1054–1062. In light of the results from UV-Vis and IR-ATR, we propose the 2 R. T. Dorsam and J. S. Gutkind, Nat. Rev. Cancer, 2007, 7, following mechanism for the formation of AuNPs stabilized 79–94. by the quinoxalines produced in the current study (Fig. 8): 3 E. Tsanou, D. Peschos, A. Batistatou, A. Charalabopoulos and firstly, the quinoxaline derivatives bind to Au3+ generating a K. Charalabopoulos, Anticancer Res., 2008, 28, 3815–3826. coordination complex that releases gold ions (quickly or slowly 4 S. Kapitanović, T. Čačev, M. Antica, M. Kralj, G. Cavrić, depending on the quinoxaline) which are then reduced by K. Pavelić and R. Spaventi, Exp. Mol. Pathol., 2006, 80, glycerol to form AuNPs. At this point, the strong alkaline 91–96. medium induces deprotonation of DEQX, which, in turn, binds 5 F. E. Bleeker, S. Lamba, C. Zanon, R. J. Molenaar, T. J. M. to the gold surface more strongly than OAQX since the anionic Hulsebos, D. Troost, A. A. van Tilborg, W. P. Vandertop, NQC–N atomic arrangement of the former is more basic than S. Leenstra, C. J. F. van Noorden and A. Bardelli, BMC the NQC–O system of the latter. Cancer, 2014, 14, 1–12. Molecular simulation studies have investigated the molecular 6 F. E. Bleeker, R. J. Molenaar and S. Leenstra, J. Neurooncol., interactions between peptides and Au-Pd nanoparticles60 and the 2012, 108, 11–27. stabilization effect of organic molecules on Au nanoparticles.61 7 P. J. Roberts and C. J. Der, Oncogene, 2007, 26, 3291–3310. Heinz and coworkres60 have demonstrated that short peptides (up 8 L. M. Weiner, R. Surana and S. Wang, Nat. Rev. Immunol., to 12 residues) prefer to bind in vacant sites on fcc lattice of the Au 2010, 10, 317–327. surface molecules present structural/chemical requirements to bind 9 J. K. H. Liu, Ann. Med. Surg., 2014, 3, 113–116. preferably in vacant sites on Au fcc lattice at the {111} surface. 10 D.-F. Shi, T. D. Bradshaw, S. Wrigley, C. J. McCall, Concerning the orientation of the molecule on the gold surface, for P. Lelieveld, I. Fichtner and M. F. G. Stevens, J. Med. Chem., both systems, the binding of quinoxalines to the gold surface is 1996, 39, 3375–3384. more likely to take place perpendicularly, which is consistent with 11 A. J. Hallett, B. M. Kariuki and S. J. A. Pope, Dalton Trans., previously reports involving pyridine derivatives where vertical 2011, 40, 9474–9481. adsorption is more energetically favorable than flat adsorption62,63 12 P. Marques-Gallego, M. A. Gamiz-Gonzalez, F. R. Fortea-Perez, M. Lutz, A. L. Spek, A. Pevec, B. Kozlevcar and J. Reedijk, Dalton Trans., 2010, 39, 5152–5158. 4 Conclusion 13 M. N. Noolvi, H. M. Patel, V. Bhardwaj and A. Chauhan, Eur. J. Med. Chem., 2011, 46, 2327–2346. We have shown that AuNPs can be easily functionalized with 14 S.-H. Lee, N. Kim, S.-J. Kim, J. Song, Y.-D. Gong and OAQX and DEQX, with the latter adsorbing more strongly onto S.-Y. Kim, J. Cancer Res. Clin. Oncol., 2013, 139, 1279–1294. New J. Chem. This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019 99 Published on 17 December 2018. Downloaded by University of New England on 1/21/2019 3:00:15 AM. View Article Online NJC Paper 15 V. Desplat, M. Vincenzi, R. Lucas, S. Moreau, 38 T. Darden, D. York and L. Pedersen, J. Chem. Phys., 1993, 98, S. Savrimoutou, N. Pinaud, J. Lesbordes, E. Peyrilles, 10089–10092. M. Marchivie, S. Routier, P. Sonnet, F. Rossi, L. Ronga 39 G. Arfken, Mathematical Methods for Physicists, Academic and J. Guillon, Eur. J. Med. Chem., 2016, 113, 214–227. Press, Orlando, FL, 3rd edn, 1985. 16 T. R. Mielcke, A. Mascarello, E. Filippi-Chiela, R. F. Zanin, 40 D. van der Spoel, P. J. van Maaren, P. Larsson and G. Lenz, P. C. Leal, L. D. Chirardia, R. A. Yunes, R. J. Nunes, N. Tı̂mneanu, J. Phys. Chem. B, 2006, 110, 4393–4398. A. M. O. Battastini, F. B. Morrone and M. M. Campos, Eur. 41 N. A. Baker, D. Sept, S. Joseph, M. J. Holst and J. A. J. Med. Chem., 2012, 48, 255–264. McCammon, Proc. Natl. Acad. Sci. U. S. A., 2001, 98, 17 S. I. Chiosea, J. R. Grandis, V. W. Y. Lui, B. Diergaarde, 10037–10041. J. H. Maxwell, R. L. Ferris, S. W. Kim, A. Luvison, M. Miller 42 W. Humphrey, A. Dalke and K. Schulten, J. Mol. Graphics, and M. N. Nikiforova, BMC Cancer, 2013, 13, 1–7. 1996, 14(33–38), 27–38. 18 Y. Samuels, Z. Wang, A. Bardelli, N. Silliman, J. Ptak, 43 F. da Silva Miranda, A. M. Signori, J. Vicente, B. de Souza, S. Szabo, H. Yan, A. Gazdar, S. M. Powell, G. J. Riggins, J. P. Priebe, B. Szpoganicz, N. S. Gonçalves and A. Neves, J. K. V. Willson, S. Markowitz, K. W. Kinzler, B. Vogelstein Tetrahedron, 2008, 64, 5410–5415. and V. E. Velculescu, Science, 2004, 304, 554. 44 D. E. Ames and M. I. Brohi, J. Chem. Soc., Perkin Trans. 1, 19 P. Wu, Y. Su, X. Liu, B. Yang, Q. He and Y. Hu, Bioorg. Med. 1980, 1384–1389. Chem., 2012, 20, 2837–2844. 45 C. A. Obafemi, W. Pfleiderer and F. Taiwo, Molbank, 2006, 20 P. Wu, Y. Su, X. Liu, L. Zhang, Y. Ye, J. Xu, S. Weng, Y. Li, 2006, M508. T. Liu, S. Huang, B. Yang, Q. He and Y. Hu, Eur. J. Med. 46 L. H. S. Gasparotto, A. C. Garcia, J. F. Gomes and Chem., 2011, 46, 5540–5548. G. Tremiliosi-Filho, J. Power Sources, 2012, 218, 73–78. 21 A. M. Maley, Y. Terada, S. Onogi, K. J. Shea, Y. Miura and 47 A. G. Garcia, P. P. Lopes, J. F. Gomes, C. Pires, E. B. Ferreira, R. M. Corn, J. Phys. Chem. C, 2016, 120, 16843–16849. R. G. M. Lucena, L. H. S. Gasparotto and G. Tremiliosi-Filho, 22 K. M. G. Lima, R. F. A. Junior, A. A. Araujo, A. L. C. S. New J. Chem., 2014, 38, 2865–2873. L. Oliveira and L. H. S. Gasparotto, Sens. Actuators, B, 2014, 48 Z. A. Knight, B. Gonzalez, M. E. Feldman, E. R. Zunder, 196, 306–313. D. D. Goldenberg, O. Williams, R. Loewith, D. Stokoe, 23 I. N. Brigger, C. Dubernet and P. Couvreur, Adv. Drug A. Balla, B. Toth, T. Balla, W. A. Weiss, R. L. Williams and Delivery Rev., 2012, 64(supplement), 24–36. K. M. Shokat, Cell, 2006, 125, 733–747. 24 X. Huang, I. H. El-Sayed, W. Qian and M. A. El-Sayed, J. Am. 49 E. H. Walker, M. E. Pacold, O. Perisic, L. Stephens, Chem. Soc., 2006, 128, 2115–2120. P. T. Hawkins, M. P. Wymann and R. L. Williams, Mol. Cell, 25 R. A. Sperling and W. J. Parak, Philos. Trans. R. Soc., A, 2010, 2000, 6, 909–919. 368, 1333–1383. 50 R. Marone, V. Cmiljanovic, B. Giese and M. P. Wymann, 26 M. C. Daniel and D. Astruc, Chem. Rev., 2004, 104, 293–346. Biochim. Biophys. Acta, 2008, 1784, 159–185. 27 K. T. Savjani, A. K. Gajjar and J. K. Savjani, ISRN Pharm., 51 K. Chen and L. Kurgan, PLoS One, 2009, 4, e4473. 2012, 2012, 195727. 52 M. M. Heravi, S. Taheri, K. Bakhtiari and H. A. Oskooie, 28 O. Trott and A. J. Olson, J. Comput. Chem., 2010, 31, 455–461. Catal. Commun., 2007, 8, 211–214. 29 D. Mandelker, S. B. Gabelli, O. Schmidt-Kittler, J. Zhu, I. Cheong, 53 J. F. Gomes, L. H. S. Gasparotto and G. Tremiliosi-Filho, C.-H. Huang, K. W. Kinzler, B. Vogelstein and L. M. Amzel, Proc. Phys. Chem. Chem. Phys., 2013, 15, 10339–10349. Natl. Acad. Sci. U. S. A., 2009, 106, 16996–17001. 54 E. B. Ferreira, J. F. Gomes, G. Tremiliosi-Filho and 30 G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner, L. H. S. Gasparotto, Mater. Res. Bull., 2014, 55, 131–136. R. K. Belew, D. S. Goodsell and A. J. Olson, J. Comput. Chem., 55 I. H. El-Sayed, X. Huang and M. A. El-Sayed, Nano Lett., 2009, 30, 2785–2791. 2005, 5, 829–834. 31 M. D. Hanwell, D. E. Curtis, D. C. Lonie, T. Vandermeersch, 56 M. N. Khan, T. A. Khan, S. A. Al-Thabaiti and Z. Khan, E. Zurek and G. R. Hutchison, J. Cheminf., 2012, 4, 1–17. Spectrochim. Acta, Part A, 2015, 149, 889–897. 32 Y. Duan, C. Wu, S. Chowdhury, M. C. Lee, G. Xiong, W. Zhang, 57 B. Ð. Glišić, B. Warżajtis, N. S. Radulović, U. Rychlewska and R. Yang, P. Cieplak, R. Luo, T. Lee, J. Caldwell, J. Wang and M. I. Djuran, Polyhedron, 2015, 87, 208–214. P. Kollman, J. Comput. Chem., 2003, 24, 1999–2012. 58 Á. I. López-Lorente, M. Sieger, M. Valcárcel and B. Mizaikoff, 33 C. I. Bayly, P. Cieplak, W. Cornell and P. A. Kollman, J. Phys. Anal. Chem., 2014, 86, 783–789. Chem., 1993, 97, 10269–10280. 59 J.-W. Park and J. S. Shumaker-Parry, J. Am. Chem. Soc., 2014, 34 G. Bussi, D. Donadio and M. Parrinello, J. Chem. Phys., 2007, 136, 1907–1921. 126, 014101. 60 H. H. Heinz, B. L. Farmer, R. B. Pandey, J. M. Slocik, 35 B. Hess, H. Bekker, H. J. C. Berendsen and J. G. E. M. Fraaije, S. S. Patnaik, R. Pachter and R. R. Naik, J. Am. Chem. Soc., J. Comput. Chem., 1997, 18, 1463–1472. 2009, 131, 9704–9714. 36 S. Miyamoto and P. A. Kollman, J. Comput. Chem., 1992, 13, 61 E. L. da Rocha, G. F. Caramori and C. R. Rambo, Phys. Chem. 952–962. Chem. Phys., 2013, 15, 2282–2290. 37 W. F. Van Gunsteren and H. J. C. Berendsen, Mol. Simul., 62 K. Tonigold and A. Groß, J. Chem. Phys., 2015, 132, 224701. 1988, 1, 173–185. 63 V. J. Gandubert and B. Lennox, Langmuir, 2005, 21, 6532. This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019 New J. Chem. 100 Published on 17 December 2018. Downloaded by University of New England on 1/21/2019 3:00:15 AM. Apêndice C Spherical neutral Gold nanoparticles improve anti- inflammatory response, oxidative stress and fibrosis in alcohol-methamphetamine-induced liver injury in rats. Thaís Gomes de Carvalho, Vinícius Barreto Garcia, Aurigena Antunes de Araújo, Luiz Henrique da Silva Gasparotto, Heloiza F. O. Silva, Gerlane Coelho Bernardo Guerra, Emilio de Castro Miguel, Renata Ferreira de Carvalho Leitão, Deiziane Viana da Silva Costa, Luis J Cruz, Alan B. Chan, Raimundo Fernandes de Araújo Júnior. International Journal of Pharmaceutics, 2018, Vol. 548, 1–14 Contribuição:  Realizei a síntese e purificação das NanoAu.  Realizei a caracterização das NanoAu.  Fiz o tratamento e interpretação dos dados de caracterização  Participei das discussões antes da escrita do manuscrito.  Auxiliei no feedback e outros ensaios sugeridos pelos revisores. ________________________ __________________________ Heloiza Fernanda O. da Silva Athayde Prof. Luiz Henrique da S. Gasparotto 101 International Journal of Pharmaceutics 548 (2018) 1–14 Contents lists available at ScienceDirect International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm Spherical neutral gold nanoparticles improve anti-inflammatory response, T oxidative stress and fibrosis in alcohol-methamphetamine-induced liver injury in rats Thaís Gomes de Carvalhoa,c, Vinícius Barreto Garciaa,c, Aurigena Antunes de Araújod, Luiz Henrique da Silva Gasparottoe, Heloiza Silvae, Gerlane Coelho Bernardo Guerrad, Emilio de Castro Miguelf, Renata Ferreira de Carvalho Leitãog, Deiziane Viana da Silva Costag, Luis J Cruzh, Alan B. Chani, Raimundo Fernandes de Araújo Júniora,b,c,⁎ a Department of Morphology, Federal University of Rio Grande do Norte, Natal 59072-970, RN, Brazil b Post-Graduation Programme in Structural and Functional Biology, Federal University of Rio Grande do Norte, Natal 59072-970, RN, Brazil c Post-Graduation Programme in Health Science, Federal University of Rio Grande do Norte, Natal 59072-970, RN, Brazil d Department of Biophysics and Pharmacology, Post-Graduation Programme in Public Health, Post-graduation Programme in Pharmaceutical Science, Federal University of Rio Grande do Norte, Natal 59072-970, RN, Brazil eGroup of Biological Chemistry and Chemometrics, Institute of Chemistry, Federal University of Rio Grande do Norte, Natal 59072-970, RN, Brazil fDepartment of Physical/Analytical Center/UFC, Fortaleza, CE, Brazil g Department of Morphology/Post-graduate Program in Morphology/UFC, Fortaleza, CE, Brazil h TranslationalNanobiomaterials and Imaging, Department of Radiology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands i Percuros B.V, 2333 CL Leiden, The Netherlands A R T I C L E I N F O A B S T R A C T Keywords: This study aimed to elucidate the anti-inflammatory, anti-oxidant and antifibrotic effects of gold nanoparticles Gold nanoparticles (GNPs) in rats subjected to liver injury with ethanol and Methamphetamine (METH). The liver injury was in- Ethanol duced by gavage administrations of 30% alcoholic solution (7 g/kg) once a day during 28 days, followed by Methamphetamine METH (10mg/kg) on the 20th and 28th days of treatment. GNPs treatment (724.96 µg/kg) during the ethanol Toxicity and Kupffer cells and METH exposure was associated with reduced steatosis, hepatic cord degeneration, fibrosis and necrosis. Furthermore, there was a reduction in biochemical markers of liver damage and oxidative stress, and pro- inflammatory cytokines IL-1β and TNF-α, compared to ethanol+METH group alone. A decrease of FGF, SOD-1 and GPx-1 expression was also observed. GNPs down-regulated the activity of Kupffer cells and hepatic stellate cells affecting the profile of their pro-inflammatory cytokines, oxidative stress and fibrosis through modulation of signaling pathways AKT/PI3K and MAPK in ethanol+METH-induced liver injury in a rat model. 1. Introduction psychostimulant drugs such as methamphetamine (METH) or cocaine (Kedia et al., 2007) in order to enhance and prolong the drug effects. Many drugs, including alcohol and stimulants, are used in social Evidence suggests that the co-administration of ethanol and cocaine contexts, perhaps because they enhance prosocial behaviors such as produces amplified and extended subjective effect (Ikegami et al., social bonding, talking, and empathy (Kirkpatrick and De Wit, 2013; 2002), fewer studies have examined the somatic impact of the METH- Sayette et al., 2012). Poly-substance use is a term coined to denote the ethanol combination in brain (Zendulka et al., 2012; Almalki et al., concomitant or sequential consumption of more than one psychoactive 2018; Wells et al., 2016). Concerning toxic effects on liver, (Koriem and drug over an interval of at least 12months for either therapeutic or Soliman, 2014) found that a hoop of edema in the periportal area recreative purposes. Amongst the substances that are co-administered compresses the surrounding hepatocytes, leading to formation of hy- with illicit drugs, ethanol is undoubtedly the most prevalent chemical peremic vessels. The association of ethanol and METH produces a responsible for the increasing number of hospital admissions and deaths variety of histological abnormalities in liver such as abundant cyto- (Winkler et al., 2016). Ethanol is frequently co-used with plasmic lipid droplets diffusely distributed along the lobules. Besides, a ⁎ Corresponding author: Federal University of Rio Grande do Norte, Lagoa Nova, SN, 1524 Natal, Brazil. E-mail address: araujojr@cb.ufrn.br (R.F. de Araújo Júnior). https://doi.org/10.1016/j.ijpharm.2018.06.008 Received 16 February 2018; Received in revised form 24 May 2018; Accepted 4 June 2018 Available online 07 June 2018 0378-5173/ © 2018 Elsevier B.V. All rights reserved. 102 T.G. de Carvalho et al. International Journal of Pharmaceutics 548 (2018) 1–14 pronounced deposition of collagen fibers between the hepatocytes and Scientific, according to the law 10.357 of December 27, 2001, which the endothelial cells of the sinusoids results in an apparent reduction of establishes rules for production, control and inspection of chemical the size and density of the hepatocytes in the sinusoids and in the bile substances. canaliculi (Pontes et al., 2008). Murine macrophage cells (RAW 264.7; cat. no. TIB-71) were ob- Three mechanisms have been proposed for alcoholic liver injury: (i) tained from American Type Culture Collection (Manassas, VA, USA). acetaldehyde toxicity (Guo et al., 2009); (ii) metabolic generation of Dulbecco’s modified Eagle’s medium (DMEM) was purchased from reactive oxygen species (ROS) or exposure to oxidative stress (Tang Invitrogen Corporation (Carlsbad, CA, USA); fetal bovine serum from et al., 2014); and (iii) oxidative stress in hepatocytes caused by immune Hyclone Company (Logan, UT, USA). response (Chen et al., 2011; Park et al., 2013). The latter has been Antibodies anti-TGF- β, FGF, SOD-1, GPX, IL-1β, TNF-β and MIF found in alcoholic-liver-injury patients (Chen et al., 2011), which helps were obtained from Santa Cruz Biotechnology Enterprise, Brazil. prevent ethanol-induced liver disease. Streptavidin-HRP-conjugated secondary antibody (Biocare Medical, In liver injury, activated Kupffer cells release a number of soluble Concord, CA, USA), TrekAvidin-HRP Label+ Kit (Biocare Medical, agents, including cytokines, such as TGF-β, and TNF-α, ROS (Kang Dako, USA), IL-1β, IL-10, TNF-α and ELISA kit (R&D Systems, et al., 2008) and factor nuclear kappa B (NF-κB) translocates to the Minneapolis, MN, USA) were also utilized in this study. nucleus, in which it binds to the promoter of target genes such as TNF-α and other pro-inflammatory cytokines (Son et al., 2011). These factors 2.2. Cell culture act on hepatic stellate cell (HSC), which are localized in the para- sinusoidal space (Wen et al., 2013). The activated phosphatidylinositol- RAW 264.7 macrophage cells were grown in DMEM medium with 4,5-bisphosphate 3-kinase (PI3-K) participate in regulation of HSC mi- 10% fetal bovine serum. Cells were seeded at a density of 5× 104 cells gration, proliferation, collagen secretion and adhesion(Friedman, 2000) per well in 6-well plates and reached 50–60% confluence 24-h after besides being involved in regulating a number of cellular responses, seeding, just before exposure to the non-cytotoxic concentration of such as cell growth, survival and migration. The protein kinase B (Akt GNPs in 10 μg/mL for treatment (Zhang et al. 2011). The cells were or PKB) is downstream of PI3-K and activation of Akt is associated with maintained in a 5% CO2 incubator at 37 °C and 95% humidified air. HSC proliferation and α1 (I) collagen transcription and translation (Reif Cells were subsequently analysed by a phase-contrast microscope. et al., 2003). Functionalized spherical gold nanoparticles (GNPs) with controlled 2.3. Immunofluorescence geometrical and optical properties are the subject of intensive studies and biomedical applications, including genomics, biosensorics, im- RAW 264.7 macrophage cells were plated onto glass coverslips in 24 munoassay (Bartneck et al., 2014; He et al. 2008), clinical chemistry well plates (5×104 cells/well) and allowed to grow for 24 h. The cells (Bartneck et al., 2014), laser phototherapy of cancer cells (Gunes et al., were then washed, fixed with 1% paraformaldehyde, permeabilized 2010), the targeted delivery of drugs (Aslan et al., 2004), DNA and with Triton-X and incubated with 100 μL of GNPs and 4′,6-diamidino-2- antigens (Dykman and Bogatyrev, 2000), optical bioimaging and the phenylindole was used for nuclear stainingfor 10min in a humid at- monitoring of cells (Hirsch et al., 2003) and tissues with the use of mosphere at room temperature. Control experiments were performed state-of-the-art detection systems. under the same conditions but without GNPs addition. The glass cov- Investigation of GNPs cytotoxicity focuses on their size, shape, doses erslips were then directly observed with the Leica DM5500 B fluores- and surrounding ligands being those chemically positive and negative cence microscope (filter settings: TXR, Cy7, FITC and DAPI), equipped charged responsible for causing cell damages through oxidative al- with a condenser using laser excitation from 512 to 542 nm. terations in mitochondrial membrane potential (De and Vincent, 2008; Schaeublin et al., 2011). Spherical neutral GNPs are more suitable for biomedical application due to the their inability in removing electrons 2.4. Animal from molecules, such as membrane lipids and mitochondria, as well as generating free radicals (Loumaigne et al., 2010; Fröhlich, 2012). Local Thirty six Wistar rats male weighing between 270 g and 300 g, ob- macrophages like kupffer cells ingest foreign materials such as patho- tained from the animal house of Department of Biophysical and gens and nanoparticles, and recruit additional macrophages to the in- Pharmacology – Federal University of Rio Grande do Norte (UFRN), amed microenvironment, producing cytokines including TNF-α, IL-6 Natal, Brazil, were randomly divided into six groups of six animals eachfl and TGF-β that up-regulate the in ammatory process (Tilg and and used for experiments. Animals were housed in cages with free ac-fl Alexander, 2008). Positively charged nanoparticles are preferentially cess to food and water at temperature and humidity controlled en- taken up by monocyte-derived macrophages and Kup er cells that have vironment under a 12 h light/dark cycle. Animals were treated ac-ff an M2-like phenotype which produce high level of TGF-β (MacParland cording to the ethical principles for animal experimentation. All et al., 2017). experiments were approved by UFRN Ethics Committee (approval In this context, macrophages like Kupffer cells therefore not only number: 018/2015). represent attractive targets for nanomedicine in liver disease, but they also need to be considered as potential particle scavenging cells in any 2.5. Preparation and administration of ethanol kind of parenteral nanoparticle administration. As such, changing local Kupffer cell behavior may affect the progress of inflammation-related 7 g per kg body weight of 30% v/v ethanol solution was used as disorders. Therefore, the aim of the present work was to evaluate the chronic dose in this experiment and 30 g of absolute ethanol was dis- anti-inflammatory, antioxidant and antifibrotic effect of GNP in an solved in distilled water and made up to 100ml, then 6.2 ml of the animal model of ethanol and METH-induced liver injury through an solution was daily administered for 28 days to each rat treated with analysis of several markers. ethanol (de Araújo et al., 2016). 2. Methods 2.6. Preparation and administration of methamphetamine 2.1. Chemicals We used the dose of 10mg/kg for METH, in which 25mg of METH were diluted in 10ml of distilled water. Each animal received 0.1 ml of The absolute ethyl alcohol (PA 99.8%) was obtained from Vetec this solution, which corresponds to 2.5 mg in each dose (Halpin et al., Quimica, Brazil. Methamphetamine (METH) was acquired from Fisher 2013). 2 103 T.G. de Carvalho et al. International Journal of Pharmaceutics 548 (2018) 1–14 2.7. Production of gold nanoparticles stress analysis, Western blot quantification, qPCR and ultra-structural evaluation using the Transmission Electron Microscopy technique. Gold nanoparticles (GNPs) were obtained from a partnership with Other liver fragments were immersed in 10% buffered formalin for the Department of Chemistry of the Federal University of Rio Grande do histopathological Analysis. Norte-UFRN, Brazil. These GNPs are produced as described by (Gasparotto et al., 2012). 3. Biodistribution of gold nanoparticles First, Au3+ was reduced by glycerol in alkaline medium and poly- vinylpyrrolidone was used to stabilize the gold nanoparticles. Diluted 3.1. Conjugation of near-infrared (NIR) fluorescence to the GNPs HCl was then added to bring the solution pH to 7 and generate neutral GNPs. Considering the quantitative transformation of gold ions into Firstly, GNPs were conjugated to the thiol group of the alpha-amino- nanoparticles, the concentration of GNPs was estimated to be 197 μg/ omega-mercapto poly(ethylene glycol) hydrochloride (SH-PEG-NH2, mL−1. The final mixture has a dark-red color due to the GNPs formation MW 3.000 g/mol) during 3 h at room temperature. The excess of non- of 7.4 ± 1.6 nm in size. conjugated SH-PEG-NH2 was removed by dialysis (for three days in a A Zeta-Meter 3.0+ system (Zeta-Meter Inc., USA) at a temperature membrane Spectra/MWCO: 6-8000) against sodium citrate 2.2mM. of 25 ± 2 °C was used to determine the electrophoretic mobility of the The GNPs-PEG-NH2 were coupled to the IRDye® 680RD NHS ester in GNPs colloidal solution (20ml). The zeta potential was calculated using bicarbonate buffer (pH 8.1) during 12 h at room temperature. The the Smoluchowski equation: GNPs-PEG-IRDye complex was then purified by dialysis (for four days μ η0 in a membrane Spectra/MWCO: 6-8000) against sodium citrate 2.2 mM ζ E= ε ε and the solution was changed two times a day.0 r where µE is the electrophoretic mobility, ƞ0 is the continuous phase 3.2. Ex vivo fluorescence imaging of major organs and quantification viscosity, Ɛ0 is the permittivity of a vacuum, and r is the relative per- meability of the continuous phase. 6 weeks oldfemale BALB/c mice (n= 3–4) (Charles River, France) were sacrificed after 48 h of intravenous injection (I.V) of the GNPs at 2.8. Dosage and administration of gold nanoparticles three different concentrations (GNP1;GNP2 and GNP3). After 48 h, all major organs were excised for ex vivo fluorescence imaging. Images The doses of GNPs were chosen through a pilot project, the doses of were acquired 700 nm at a resolution of 85mm. The data were ana- 700 μg/kg, 1000 μg/kg and 1.500 μg/kg were tested. The dose 700 μg/ lyzed using Pearl Impulse software, version 3.01 (LI-COR Biosciences, kg showed best results in decreased inflammatory cytokines IL-1β and Lincoln, NE, USA). Total fluorescence intensity was determined by TNF-α (de Araújo et al., 2017). The dose of 700 μg/kg was used to drawing a region of interest (ROI). The size and shape of the ROI was calculate the doses to be used in the experiment, based on a gold na- the same. noparticle formulation with a concentration of 197 μg/mL−1. The doses were adjusted so that for all groups treated by the oral 3.3. Antioxidant activity of GNPs route by gavage the final volume used was standardized in 1ml. In order to evaluate the dose-dependent effect, the doses were fractionated The antioxidant effect of GNPs was evaluated through the GSH into 3: starting with the highest dose: 724.96 µg/kg (GNP3); an inter- consumption, MDA formation and MPO inhibition. Liver samples were mediate dose corresponding to 362.48 µg/kg (GNP2); and finally the harvested as described above and stored at −80 °C until required for third dose with the lowest concentration: 181.48 µg/kg (GNP1). assay. After homogenisation and centrifugation (2000×g for 20min), MPO activity was determined by a previously described colorimetric 2.9. Induction of ethanol and METH-induced liver injury method (De Araújo et al. 2016). Results are reported as units of MPO per gram of tissue. The protocol for hepatic injury induction by exposure to alcohol and To quantify the increase in free radicals in the liver sample, MDA METH is summarized below: content was measured via the assay described by (Esterbauer and Kevin, 1990). Liver samples were suspended in buffer Tris HCl 1:5 (w/ 1) Animals were treated with GNP1, GNP2 and GNP3 one hour before v) and minced with scissors for 15 s on an ice-cold plate. The resulting ethanol administration (30%, 7 g/kg) by oral gavage. Each treat- suspension was homogenised for 2min with an automatic Potter ment had three doses to be tested: 181.48 μg/kg, 362.48 µg/kg and homogenizer and centrifuged at 2500×g at 4 °C for 10min. The su- 724.96 µg/kg, respectively. pernatants were assayed to determine MDA content. The results are 2) Saline solution (NaCl 0.9%) was administered by oral gavage once a expressed as nanomoles of MDA per gram of tissue. week every six days during the first and second week of hepatic GSH levels in the liver tissues were measured to antioxidant. GSH injury induction. Methamphetamine was administrated by oral ga- content was measured via the assay described by (de Araújo et al., vage on the 3rd and 4th weeks in place of the saline solution. Saline/ 2017). Liver samples (5 per group) were stored at 70 °C until use. Liver Methamphetamine were administered by oral gavage 3 h after tissue homogenates (0.25 ml of a 5% tissue solution prepared in 0.02M ethanol. In the non-methamphetamine groups, saline solution was EDTA) were added to 320ml of distilled water and 80ml of 50% TCA. used by oral gavage to simulate the methamphetamine in the 3rd Samples were centrifuged at 3000 rpm for 15min at 4 °C. The super- and 4th week. natant (400ml) was added to 800ml of 0.4M Tris buffer at pH 8.9 and 3) Step 1 was repeated 7 days a week during 28 days. 20 μL of 0.01M DTNB. The absorbance of each sample was measured at 4) Step 2 was repeated once a week every 7 days until the end of 420 nm, and the results were reported as level of GSH per milligram of 28 days of injury induction. tissue. Euthanasia was performed on the 29th day by intraperitoneal in- 3.4. Cytokine analysis jection of Ketamine 7.5ml/kg (50mg/ml) and Xylazine 2.5 ml/kg (20mg/ml). All animal groups were fasted for 12 h to perform the Liver samples (three samples per group) were stored at −80 °C until subsequent biochemical analysis. Once unconscious, the animals un- use. The tissue was homogenized and processed as described by (Safieh- derwent cardiac puncture followed by removal of the liver. Garabedian et al., 1995). Levels of IL-1β (detection range: Liver fragments were frozen at −80 °C for cytokine and oxidative 62.5–4000 pg/mL; sensitivity or lower limit of detection [LLD]: 3 104 T.G. de Carvalho et al. International Journal of Pharmaceutics 548 (2018) 1–14 12.5 ng/mL of recombinant mouse IL-1β), IL-10 (detection range: material was washed with 4× sodium cacodylate (15min each bath). A 62.5–4000 pg/mL; sensitivity or LLD: 12.5 ng/mL of recombinant drop of 1.6% Potassium Ferrocyanide (FCK) and 2% Osmium Tetroxide mouse IL-10) and TNF-α (detection range: 62.5–4000 pg/mL; sensi- for 1 h in a darkroom was added, followed by 2 washes with 0.1M tivity or LLD: 50 ng/mL of recombinant mouse TNF-α) in the intestinal sodium cacodylate for 15min and two washes with distilled water. “In samples were determined with a commercial ELISA kit (R&D Systems, block” contrast with 0.5% uranyl acetate in a darkroom for 2 h under Minneapolis, MN, USA), as described previously. All samples were refrigeration, dehydrated with acetone in different concentrations, in- within the wavelength used in UV–VIS spectrophotometry (absorbance filtrated and included in resin. Ultra-thin sections (1 μm) are stained measured at 490 nm). with toluidine blue and examined under Zeiss transmission electron microscope, model EM 902 at 80 Kv. 3.5. Histological analysis from hepatic parenchyma 3.7. Immunohistochemical staining Liver samples were fixed in 10% neutral buffered formalin, dehy- drated and embedded in paraffin. Sections of 5 μm thickness were ob- Thin sections of liver (4 μm) were obtained from each group with a tained for hematoxylin–eosin staining (H&E) and examined by light microtome and transferred to gelatine-coated slides. Each tissue section microscopy (40×, Nikon E200 LED). Three sections of liver (six ani- was then deparaffinised and rehydrated. The liver tissue slices were mals per group) were analyzed by two pathologists. Liver pathology washed with 0.3% Triton X-100 in phosphate buffer (PB) and quenched was scored as follows: steatosis (the percentage of liver cells containing with endogenous peroxidase (3% hydrogen peroxide). Tissue sections fat): < 25%=1, 25–50%=2, 50–75%=3,> 75%=4.; inflamma- were incubated overnight at 4 °C with primary antibodies (Santa Cruz tion and necrosis: 1 focus per low-power field; 2 or more foci. Pathology Biotechnology, INTERPRISE, Brazil) against TGF- β, FGF, SOD-1, GPX-1 was scored in a blinded manner by one of the authors and by an outside e IL-1β. Dilution tests (3 dilutions) were performed with all antibodies expert in rodent liver pathology (Nanji et al., 1989). The main values of to identify the 1:800; 1:600; 1:800; 1:1000 and 1:600, dilutions as scores were used for statistical analysis. appropriate, respectively. Slices were washed with phosphate buffer Histological sections were stained using picrosirius red staining kit and incubated with a streptavidin/HRP-conjugated secondary antibody (1% Sirius red in saturated picric acid; EasyPath, Indaiatuba, Brazil) for (Biocare Medical, Concord, CA, USA) for 30min. Immunoreactivity to 24 h, or haematoxylin and eosin (Easypath) and examined under light the various proteins was visualized with a colorimetric-based detection microscopy (Nikon Eclipse 2000 equipped with Nikon DS-Fi2; Nikon kit following the protocol provided by the manufacturer (TrekAvidin- Corporation, Tokyo, Japan). For the purpose of quantitative analysis HRP Label+ Kit from Biocare Medical, Dako, USA). Sections were the collagen content, randomly sampled two hundred light microscope counter-stained with hematoxylin. Known positive controls and nega- images (200X) per liver specimen, including large centrilobular veins tive controls were included in each set of samples. Planimetry micro- and large portal tracts (≥150mm) were analyzed. About 20 polarized scopy (Nikon E200 LED, Morphology Department/UFRN) with a high- light microscopy images using an Olympus BX60 microscope (Olympus, power objective (40×) was utilised to score the intensity of cell im- Tokyo, Japan) (200X) per specimen were captured and analyzed using a munostaining: 1= absence of positive cells; 2= small number of po- color threshold detection system developed in ImageJ (National sitive cells or isolated cells; 3=moderate number of positive cells; and Institutes of Health). Known positive and negative controls were in- 4= large number of positive cells. Labelling intensity was evaluated by cluded in each batch of samples. Tissue reactivity in all groups (nega- two previously trained examiners in a double-blind fashion. Three tive control, alcohol group and treated group with GNPs) was assessed. tissue sections per animal (six animals per group) were evaluated. Values are expressed as percentage of positive area. Contrast index measurements were obtained from selected area×100/total area po- 3.8. Immunofluorescence microscopy sitioned across the regions of interest (three samples per animal). Moreover, hepatic fibrosis was quantified using by (Ishak et al., 1995) Three tissue sections from each animal (six animals per group) were scoring system: level 1 indicating the absence of fibrosis; level 2 in- deparaffinized in xylene and washed in a series of concentrations of dicated enlargement of portal area; level 3 was assigned to fibrous ethanol and PBS. Antigen retrieval was performed by placing the sec- expansion of most portal areas; level 4 was assigned to lobules with tions in a 10mM sodium citrate with 0.05% Tween 20 for 40min at fibrous expansion of most portal areas with occasional portal to portal 95 °C. Autofluorescence background noise was reduced by incubating bridging; level 5 was assigned to lobules with fibrous expansion of most the sections in 0.1% Sudan black in 70% alcohol for 40min at room portal areas with marked bridging (portal to portal and portal to cen- temperature (RT). The sections were incubated overnight with rabbit tral); level 6 was assigned to lobules with marked bridging (portal to anti-IL-1β, TNF- β and MIF primary antibody (1:200, 1:400 e 1:400 portal and portal to central) with occasional nodules (incomplete cir- Abcam, EUA and Santa Cruz Biotechnology, USA, respectively), in rhosis); level 7 was observed Cirrhosis in the lobules. blocking solution/1% normal goat serum; Abcam, USA and Santa Cruz Blood samples collected from the rat and were centrifuged at 3000g Biotechnology, USA, respectively) at 4 °C, washed three times in PBS/ for 10min, and resultant supernatants were used to measure the blood 0.2% triton X-100 for 5min and incubated with Alexa Fluor 488- alanine aminotransferase level (ALT), aspartate aminotransferase (AST) conjugated goat anti-rabbit secondary antibody (1:500 in BSA 1%) and to evaluate the alcohol-induced liver injury. Liver cytosolic protein DAPI nuclear counterstain (Sigma, USA). Finally, the sections were solution was used to measure the hepatic triglyceride concentration mounted with Vectashield medium. Fluorescent images were obtained (mg/g total liver protein, % of control) as a marker of the alcohol-in- as described by (Araújo et al., 2016). duced lipid surplus in the liver. The levels of ALT and hepatic trigly- ceride were measured with an automatic analyzer (FDC4000; Fuji 3.9. Western blot analyses expression Medical Systems, Tokyo, Japan). The data is presented as means with their standard errors (SEM). The liver segments were homogenized in RIPA lysis buffer (25mM Tris-HCL, pH 7.6; 150mM NaCl; 5 mM EDTA; 1% NP40; 1% triton X- 3.6. Transmission electronic microscopy 100; 1% sodium deoxycholate; 0.1% SDS) and protease inhibitor (1 µL inhibitor: 100 µL RIPA). For protein extraction, liver samples were In order to evaluate the uptake of the GNPs by hepatic cells, 0.5 cm centrifuged (17min, 4 °C, 13000 rpm) and the supernatant was col- fragments were taken from samples of each treatment and fixed in lected. Protein concentrations were determined through the acid assay Karnovisky Solution (2.5%) and paraformaldehyde (2.5%) in buffer of (Thermo Fisher Scientific) according to the manufacturer‘s protocol. 0.1 M cacodylate) for approximately 4 h at 4 °C. After fixation the SDS-polyacrylamide gel electrophoresis (10% or 8%) were performed 4 105 T.G. de Carvalho et al. International Journal of Pharmaceutics 548 (2018) 1–14 using 50 µg of protein (previously prepared with sample buffer, BioRad and denatured at 95 °C for 5min). Then, the protein was transferred to a PVDF membrane (BioRad) for 2 h, blocked with 5% BSA for 1 h, in- cubated overnight with a primary antibody (mouse anti-β actin, sc- 81178, 1:500, Santa Cruz Biotechnology; mouse anti-TGFβ- 1/2- sc80346, 1:200, Santa Cruz Biotechnology; mouse anti-ERK1/ERK2, 136200, 1:500, Invitrogen; Goat anti-Iba-1, ab107159, Abcam) and a secondary antibody (goat anti-rabbit, 656120, Invitrogen, 1:1000; goat anti-mouse IgG, 626520, Invitrogen, 1:500; or rabbit anti-goat, A16142, Invitrogen, 1:1000) for 1 h and 30min. The membranes were incubated using the ECL system according to the manufacturer’s in- structions (BioRad) and the chemiluminescence signal was detected using the ChemiDocTM XRS system (BioRad). Densitometric quantifi- cation of bands was done through the software ImageJ (NIH, Bethesda, MD, USA). 3.10. Analysis of mRNA expression Total RNA was extracted from liver tissue with trizol reagent (Invitrogen Co. USA) and the SV Total RNA Isolation System (Promega, Madison, WI). First-strand cDNA was synthesized from 1 μg of total RNA with the ImProm-IITM Reverse Transcriptase System for RT-PCR (Promega) according to the manufacturer’s protocol. Real-time quan- titative PCR analyses of GAPDH, PCI, PCIII, NF-Kβ, F4/80, AKT and PI3K mRNAs were performed with SYBR Green Mix in the Applied Biosystems1 7500 FAST system (Applied Biosystems, Foster City, CA), according to a standard protocol with the following primers of Table 1. The reference gene for normalization was selected from an analysis of 18S (GenBank sequence NM_003286.2), ubiquitin C (UBC, NM_021009.4), β-actin (ACTB, NM_001101.3) and glyceraldehyde-3- phosphate dehydrogenase (GAPDH, NM_002046.3) genes. GAPDH was chosen as the reference gene because it did not present different am- plification patterns (Zhang et al., 2014) As it is a constitutive gene for eukaryotes, the reference gene should not be modified in the disease. All analyses were performed in a 7500 fast real-time PCR instrument (Applied Biosystems, CA, USA). The standard PCR conditions were as follow: 50 °C for 2min and 95 °C for 10min, followed by forty 30-s cycles at 94 °C, a variable annealing primer temperature for 30 s, and Fig. 1. Tecidual biodistribution of GNPs. Main target organs of GNPs 48 h post 72 °C for 1min. Reactions were carried in duplicate according to the i.v. injection of 14 µg/ml (A). Quantitative representation of GNPs distribution TaqMan® fast universal PCR master mix (Applied Biosystems, Foster intensity at 700 nm in different organs (B). Quantification of GNPs biodis- City, CA, USA) protocol in a total volume of 10 μL containing ap- tribution in the main organs. proximately 20 ng cDNA. Mean Ct values were used to calculate the relative expression levels of the target genes for the experimental 4. Results groups, relative to those in the negative control group; expression data were normalized relative to the housekeeping gene GAPDH using the 4.1. Gnps biodistribution 2−ΔΔCt formula. After I.V. injection of GNPs, we can observe that the liver, kidneys 3.11. Statistical analysis and brain were the tissues where nanoparticles migration occurred most strongly, indicated in Fig. 1A, by greater fluorescence intensity. In The data is presented as means with their standard errors (SEM) or the Fig. 1B, shows the quantification of GNPs biodistribution in the as medians. Analysis of variance (ANOVA) followed by Bonferroni test main organs and that biodistribution of gold nanoparticles have a dose- was used to in parametric tests. The Kruskal-Wallis and Dunn tests was dependent effect on the concentrations tested. used to compare medians for non-parametric tests (Graph Pad Prism 5.0 Software, La Jolla, CA, USA). Table 1 Primer Sequences used for PCR. mRNA Oligonucleotideprimers Annealing primer temperature GAPDH forward: 5′ AAC TTT GGC ATC GTG GAA GG 3′ reverse: 5′ GTG GAT GCA GGG ATG ATG TTC 3′ 60 °C NF-κβ forward: 5′ TCT GCT TCC AGG TGA CAG TG 3; reverse: 5′ ATC TTG AGC TCG GCA GTG TT 3′ 55,2 °C PCI forward: 5′ CAG GGA GTA AGG GAC ACG AA 3′;reverse: 5′ TCC CAC AGC AGT TAG GAA CC 3′ 56,8 °C PCIII forward: 5′ ATG GTG GCT TTC AGT TCA GC 3′;reverse: 5′ TGG GGT TTC AGA GAG TTT GG 3′ 55,2 °C F4/80 forward: 5′ GCC CTT CCA ACT CAT GT 3′ reverse: 5′ AGG GAA TCC TTT TGC ATG TG 3′ 55,1 °C AKT forward: 5′ TCA CCT CTG AGA CCG ACA CC 3′ reverse: 5′ ACT GGC TGA GGA GAA CTG G 3′ 58,5 °C PI3K forward: 5′ AAC TTG GCA TGG AAG G 3′,reverse: GTG GAT GCA GGG ATG ATG TTC 3′ 55,5 °C 5 106 T.G. de Carvalho et al. International Journal of Pharmaceutics 548 (2018) 1–14 Fig. 2. Intracellular localization of GNPs in macrophage. (B and D.1) and in Kupffer cells Lysossomos (E-G). Negative control, cell without GNPs (A and C). DAPI in blue (C and D) and GNPs in green (D.1). Kc: Kupffer cells; N: nucleus; Triangle: GNPs; L: lysosomes; Star: hepatocyte; Two square: Disse’s spaces. Magnification: 400x. Uptake of nanoparticles was evaluated by Fluorescence microscopy (D1). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 4.2. Intracellular localization of GNPs in macrophage 4.4. Effect of GNPs treatment on inflammation In vitro assays were performed in order to investigate how GNPs The combined treatment with ethanol and METH resulted in in- interact with the cells and whether they are internalized. We observed creased levels of IL-1β (P < 0.001) and TNF-α (P < 0.001) compared that GNPs appeared to be internalized by macrophages, likely in the to saline group (Fig. 3D and E). As noted, such combined treatment may form of agglomerates/aggregates as shown by the light microscopy induce side effects which could be reduced by GNPs treatment. Speci- (Fig. 2B and B.1), and in the spectrum of bright green by immuno- fically, IL-1β and TNF-α levels in ethanol+METH+GNP3 group were fluorescence microscopy (Fig. 2D.1). The absence of GNPs in control lower than GNPs absence (P < 0.05 and P < 0.001, respectively). On cells is represented by a lack of agglomerates (Fig. 2A) and absence of the other hand, it can also be seen in Fig. 3F, that the combined the bright green spectrum (Fig. 2C). When the distribution of the GNPs treatment of ethanol+METH+GNP3 was able to increase IL-10 levels in the liver was evaluated, all mice exposed to GNPs accumulations of (P < 0.0001) compared to ethanol+METH treatment alone. nanoparticles were traced in Kupffer cells (Fig. 2E and F), and Fig. 2G. presents a Kupffer cell from the control group that did not receive 4.5. Histopathological analysis GNPs. After analyzing the cells and the liver fragments, we observed that the GNPs appear to be primarily localized in the cytoplasm close to At the end of the treatments, the animals were euthanized and their nuclei and excluded from them. livers excised for histopathological analysis. From then on, after 28 days of saline administration, no pathological changes were observed in the negative control group (Fig. 4A–C), as indexed by a semiquantitative 4.3. Effects of GNPs on MPO activity and on MDA and GSH levels scoring system. However, as seen in Fig. 4E–G, the rats liver from ethanol+METH group exhibited fat accumulation, lymphocytes and Livers from the ethanol+METH group had significantly greater neutrophils infiltrate, and necrosis (thin arrows, circle with arrow heads myeloperoxidase (MPO) activity than livers harvested from the saline and stars, respectively), resulting in a high pathology scores (Fig. 4M). group (P < 0.0001), and this increase was attenuated in the Additionally, the ethanol+METH group had significantly greater ethanol+METH+GNP3 group that received treatment with steatosis than the saline group. On the other hand, ethanol and METH- 724.96 µg/kg GNPs (Fig. 3A). This same group, in turn, significantly induced liver damage was reduced when associated with GNP3 treat- increased glutathione (GSH) levels (P < 0.0001) compared to positive ment(P < 0.001) compared to its respective control without GNPs control group (Fig. 3C). The malonyldialdehyde (MDA) formation was (Fig. 4G–K). The same effect, however, was not observed for significantly increased in the ethanol+METH group when compared ethanol+METH + GNP1/GNP2 treated groups(Fig. 4I and J). Re- to saline group (P < 0.01) and GNPs treated groups (P < 0.01, duced inflammation was most clearly observed in the P < 0.001), as seen in Fig. 3B. ethanol+METH+GNP3 treated group (Fig. 4K), which exhibited decreased areas of steatosis, inflammatory infiltrate contains neu- trophils and lymphocytes and reduced levels of necrosis relative to 6 107 T.G. de Carvalho et al. International Journal of Pharmaceutics 548 (2018) 1–14 Fig. 3. Modulation of the antioxidant activity and cytokine profile by GNPs. Myeloperoxidase activity (MPO) (A), malondialdehy deformation (MDA) (B), and reduced glutathione levels(GSH) (C). Cytokine analysis(D–F). METH, methamphetamine; GNP1, gold nanoparticles 181.48 µg/kg; GNP2, gold nanoparticles 362.48 µg/kg, GNP3, gold nanoparticles 724.96 µg/kg. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ethanol+METH group alone. (P < 0.01), as observed in the Fig. 6 and 7. These changes were con- Negative control group livers had weak staining limited to cen- sistent with the normalization toward non-ethanol and METH exposure trilobular veins as shown in the Fig. 4D. Liver sections from the positive levels. control group exhibited marked portal fibrosis and staining between the Evaluation of IL-1β, TNF-α and macrophage migration inhibitory hepatic cords (Fig. 4H). The treated group with GNP3 had a sig- factor (MIF) was carried out by labelling with antibodies conjugated nificative (P < 0.01) fibrotic response as can be seen when comparing with fluorescent agent. Cellular IL-1β, TNF-α and MIF labelling (green) Fig. 4H, L and M. Morphometric quantification of Sirius red stained were strong and diffuse in the ethanol+METH group(Fig. 8B, E and areas demonstrated an attenuation of the fibrotic process in the treated H), poorly marked in the ethanol+METH+GNP3 group (Fig. 8C, F group compared to the positive control group. Fig. 4M shows that and I), and absent in saline group (Fig. 8A, D, and G). Densitometric treatment of the GNP3 group had an attenuation of the fibrotic process analysis confirmed that there were significantly increased IL-1β, TNF-α in the liver, according to the analysis of the tissue fractions with the and MIF immunoreactivities in the ethanol+METH group, relative to application of the Ishak scores. the saline group, and lower immunoreactivity in the In addition, treatment with ethanol and METH combined with ethanol+METH+GNP3 group(Fig. 8J, K, and L). GNP3 was able to reduce the AST, ALT and hepatic triglycerides levels (Fig. 5A–C) when compared to the control groups. However, GNPs 4.7. Gnps treatment decreased NF-κB, F480, AKT, PI3K, PCI and PCIII treatment modulated this ethanol and METH-induced hepatosteatosis mRNA expression and liver injury (Fig. 5). After analyzing the data of ALT, AST, hepatic Triglycerides, pro-and anti-inflammatory cytokines, anatomopatholo- For all evaluated genes the relative mRNA expression was decreased gical analysis, we concluded that among the 3 doses tested the GNP3 when the groups were treated with ethanol+METH+GNP3 in rela- was the one that presented the best results, therefore the following tests tion to their respective control without the GNP3 (P < 0.0001 or as Immunohistochemistry, Immunofluorescence, Electronic Microscopy P < 0.001), as observed in Fig. 9A–F. RT-PCR-Real Time, Western Blot, were performed only with the nega- tive and postoperative control groups and the group treated with the 4.8. IBA-1, ERK1/ERK2 and TGF-β expression best dose of GNPs (GNP3). The final product of ionized calcium-binding adaptor molecule 1 4.6. Immunohistochemistry and immunofluorescence (IBA-1), ERK1/ERK2 and TGF-β genes expression into rats liver, ex- posed to ethanol+METH, was evaluated by western blot as show The histopathological analysis was followed with antibody labeling Fig. 9G. Increased expression of IBA-1, ERK1 and TGF-β and decreased for transforming growth factor-beta (TGF-β), fibroblast growth factor expression of ERK2in the ethanol+METH+GNP3 group were ob- (FGF), SOD-1 and GPX-1 observation. When compared to alone served. The final product of IBA, ERK1/ERK2 and TGF-β genes ex- ethanol+METH exposure, the ethanol+METH + GNP3 group was pression into rats’ liver, exposed to ethanol+METH, was evaluated by able to reduce the TGF-β, FGF, SOD-1 (P < 0.001) and GPX-1 levels western blot as show Fig. 9G. Increased expression of IBA-1, ERK1 and 7 108 T.G. de Carvalho et al. International Journal of Pharmaceutics 548 (2018) 1–14 Fig. 4. Histological analysis from hepatic parenchyma. Group treated with saline so- lution (A–D), alcohol 30% + methamphe- tamine (METH) (E–H) and groups treated with GNP1 191.24 μg/kg (I), GNP2 362.8 μg/kg (J), GNP3 724.96 µg/kg (K-L). Fibrosis analysis by Picrosirius staining (D, H and L). The area fraction of total fibrosis, including fibrosis in the portal tract area, in rats with GNP3 group induced liver injury in relation to the Ishak score. Graphical re- presentation of the mean histopathological score from each treated group (M). Arrow, fatty changes within hepatocytes; arrow head, neutrophils; star: necrosis area; circle, lymphocytes and neutrophils infiltrate. Magnification 400×. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. TGF-β and decreased expression of ERK2 in the observed intense marking intense labeling of the analyzed proteins. ethanol+METH+GNP3 group were observed. No overexpression of IBA-1, ERK1 and TGF-β, already in the positive control group we Fig. 5. Biochemical assessments of liver function. (A) Aspartate aminotransferase (AST), (B) Alanine aminotransferase(ALT). (C) Triglyceride. METH, metham- phetamine; GNP1, gold nanoparticles 181.48 µg/kg; GNP2, gold nanoparticles 362.48 µg/kg, GNP3, gold nanoparticles 724.96 µg/kg. **p < 0.01; ***p < 0.001; ****p < 0.0001). 8 109 T.G. de Carvalho et al. International Journal of Pharmaceutics 548 (2018) 1–14 Fig. 6. Immunohistochemicalanalysis from hepatic parenchyma. The saline (A, D, G, and J), alcohol+methamphetamine (B, E, H and K) and alcohol+methamphetamine+GNP 724.96 μg/kg (C, F, I and L) groups were labeled for evaluation of TGF-β(A-C) and FGF (D-F) growth factors, and for oxidative stress through SOD-1(G-I) and GPX-1 (J-L). METH, methamphetamine; GNP3, gold nanoparticles 724.96 µg/kg. Magnification 400×. 5. Discussion in the treatment with GNPs. Previously our group, observed that the highest GNPs dose (1500 μg/kg) generated 49% of reduction in leuko- This study is the first into examine the anti-inflammatory, anti- cyte migration, which attested the activation of a cellular anti-in- oxidant and anti-fibrotic activity of GNPs in a rat model after ethanol flammatory response, while the groups received GNPs showed de- and METH-induced liver injury through an analysis of markers of the creases levels in the pro-inflammatory cytokine IL-1β(700 and 1500 μg/ inflammatory, oxidant and fibrotic processes. This was perfectly evi- kg, P < 0.05) and TNF-α(700, 1000 and 1500 μg/kg, P < 0.001) denced by a significant increase in several biochemical parameters such compared to positive control group (Araújo et al., 2017). Other studies as oxidative stress, biomarkers of inflammation, severe fat accumula- also showed this relation of importance of the size of GNP and increased tion, necrosis and accumulation of collagen type I and III around the expression of proinflamatory cytokines (Khan et al., 2013; Ibrahim hepatic triad which resulting in an strong steatosis beyond elevated et al., 2018). neutrophil, MPO, and pro-inflammatory cytokine levels were also found From the biodistribution tests, we have shown that after 48 h GNPs all these changes were well evidence in the positive control groups. migrated mainly to the spleen, kidney and liver, reaching 2×105, When comparing the results obtained in this research with the re- 5× 105 and 6× 105 ID/g, respectively, corroborating with the results sults obtained previously (de Araújo et al., 2017), we have noticed that seen earlier (de Araújo et al., 2017) and evidencing that after admin- the dose used and the size of the GNPs are a key factor for effectiveness istration these GNPs migrate to the nucleus of Kupffer cells in the liver. 9 110 T.G. de Carvalho et al. International Journal of Pharmaceutics 548 (2018) 1–14 Fig. 7. Immunohistochemistry scores. Graphical representation of the mean scores from alcohol+methamphetamine+GNP 724.96 μg/kg treated group for TGF-β (A), FGF (B), SOD-1 (C) and GPX-1 (D) immunoreactivity. **p < 0.01; ***p < 0.001; ****p < 0.0001. Studies have shown that the extent of cerebral uptake of anionic 0,724 (724 µg/kg) ppm GNPs in the treatment groups in the groups that nanoparticles is higher than cationic and neutral GNPs (Noor et al., presented the best results. 2016; Goodman et al., 2004). In this way, the surface of the NPs should Some works have showed a non-immunogenic character of the be considered in the neurotoxicity profile and in the distribution, since GNPs or have even proven their anti-inflammatory effect, consisting of neutral charges present irrelevant neurotoxicity (Masserini, 2013). Not the reactive oxygen and nitrite species inhibition as well as pro-in- all types of nanoparticles are feasible for use in a strategy whose ob- flammatory cytokines in macrophages (Kurniawan et al., 2017; Zhang jective is to cross the blood-brain barrier (BBB). Surface characteristics et al., 2011). In vivo experiments also conducted on several animal vary depending on the nanomaterials used and it was found that neutral models of inflammatory conditions have confirmed the anti-in- nanoparticles and low concentrationanionic effects have no effect on flammatory and antioxidant properties of the GNPs, manifested by a this barrier (De Jong and Borm 2008). decrease in the levels of pro-inflammatory cytokines (IL-1β,TNF-α) and In previous studies, we observed that results indicated that GNPs at oxidative tissue damage markers (Araújo et al., 2017; Tsai et al., 2012; the low concentrations below to 2 ppm did not show any toxicity, but at Dohnert et al., 2012; Sumbayev et al., 2013). the higher concentrations, significant changes were observed in the Oxidative stress is known to play a crucial role in METH-induced organ (Araújo et al., 2017; Zhang et al., 2010; Mironava et al., 2010). toxicity in the brain and other tissues, as evidenced by previous studies. However, this accumulation of GNPs in the liver did not cause any However, a comparison of oxidative effects of MET in different organs morphological changes in this organ (de Araújo et al., 2017). GNPs has not been sufficiently studied previously (Tokunaga et al., 2006). It (13 nm in diameter) after 28 days existed with the amount of has been suggested that the hepatic catalase level is negatively asso- 1.5%–9.2% in the kidneys, while the amount is negligible in the urine ciated with the severity of alcoholic liver injury (de Araújo et al., 2016; (Yang et al., 2007). Extreme changes in the histopathology of lung and Powell et al., 2010) and that SODs scavenge hydroxyl peroxides gen- liver tissues caused by spherical GNPs with 5–10 nm size in 5 (5000 µg/ erated in the cytosol and mitochondria, thereby terminating autoxida- kg), 10 (10.000 µg/kg), and 100 (100.0000 µg/kg) ppm treatment tion. Gold nanoparticles can increase the anti-oxidant defense enzymes groups, been the pathological changes (lung and liver tissue) in treat- and creating a sustained such as GSH, SOD, Catalase and GPx in dia- ment group (Au 100 ppm) more intense compared to the other groups betic mice to normal, by inhibition of lipid peroxidation and ROS (Abdelhalim et al., 2011). However, in this work was used 0,724 ppm generation (Barathmanikanth et al., 2010). In this work, ethanol asso- GNPs in the treatment groups. However, in our work was used dose of ciated to METH imbalanced the hepatocellular antioxidant system, 10 111 T.G. de Carvalho et al. International Journal of Pharmaceutics 548 (2018) 1–14 Fig. 8. Modulation of expression of IL-1β, TNF-α and MIF. Representative photomicrographs of IL-1β(A-C), TNF-α(D-F) and MIF(G-I) immunoreactivity in liver specimens from each group (green) with DAPI nuclear counterstained (blue). Graphical representation of the contrast index from IL to 1β (J), TNF-α (K) and MIF (L). METH, methamphetamine; GNP3, gold nanoparticles 724.96 µg/kg. ***p < 0.001; ****p < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) inflammatory cytokines and liberated the free radicals (Khan et al., (Bautista, 2002). This hepatotoxic effect was also confirmed by the 2012), as evidenced by the decrease in GSH level and the increased decrease in liver weight when METH was administered to ethanol pre- levels of MDA and MPO in hepatic tissue. GNPs724.96 µg/kg, however, exposed rats and by histological analysis of liver sections by light and increased hepatic GSH level and SOD-1 and GPX-1 expression, as well electron microscopy, which gives further evidences that the con- as decreased the MDA and MPO levels. Other studies in model of acute comitant exposure to METH and ethanol results in a marked aggrava- peritonitis study suggest that gold NPs of 10 nm diameter produce tion of the hepatotoxic effects such as fat accumulation, lymphocytes significant lipid peroxidation in rat liver however lungs and heart do and neutrophils infiltrate, necrosis and fibrosis (Araújo et al., 2016). not show any oxidative stress (Khan et al., 2012). That probably do not GNPs724.96 µg/kg, in turn, reduced all these histopathologic features. apply to our GNPs because they are smaller in size. Hepatocyte apoptosis causes recruitment of inflammatory cells to Ethanol pre-treatment was also able to exacerbate METH-induced damaged liver and release of pro-fibrogenic cytokines (TGF-β1, IL-6, IL- hepatotoxicity, which could be ascertained by the significant increase 1β, TNF-α) (Potter and Mezey, 2007; Seki et al., 2007). IL-10 is a potent of plasma transaminases activities, (hepatic lesion biomarkers), when anti-inflammatory molecule that has been shown to inhibit TNF-α and animals were exposed to ethanol and METH. The increase in AST, ALT IL-1 cytokines production and to suppress NF-κB activation (Mandal and triglycerides levels were already described for both compounds in et al., 2010). IL-10 reduces nitric oxide and reactive oxygen inter- humans (Ellis et al., 1996; Yue et al., 2006) and rats (Beitia et al., 2000; mediates macrophage production, and also reduces adhesion molecules Montet et al., 2002). Increased prostaglandin E2 (PGE2) causes trigly- and chemokines expression (Gao, 2012). Bone marrow(BM)-derived cerides accumulation in hepatocytes, and therefore, a state of steatosis and liver resident macrophages (Kupffer cells, KC) produce TGF-β1 in 11 112 T.G. de Carvalho et al. International Journal of Pharmaceutics 548 (2018) 1–14 Fig. 9. Gene expression at mRNA and protein levels. Relative mRNA expression fromNF-κB (A), F480 (B), AKT (C), PI3K (D), Procollagen I (E) and Procollagen III (F) genes. Results are presented as fold-change of the media values, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed by mean ± SEM (**p < 0.001; ****p < 0.0001). Protein expression from IBA-1, ERK1/ERK2 and TGF-β genes (Fig. 9G) was detected in total protein extracts determined by western blot, with detection of β-actin used as a loading control. Graphical representation densitometry analysis the result of analysis western blot (Fig. 9H). METH, methamphetamine; GNP3, gold nanoparticles 724.96 µg/kg. In Fig. 9G Figs. 1–3 represent the analyzed groups 1-negative control group, 2-positive control group and 3 group treated with GNPs 724.96 μg/kg. Data were representative of at least two similar experiments. the fibrotic liver (Potter and Mezey, 2007). TGF-β1 is a critical for proliferation, collagen secretion and adhesion(Friedman, 2000) besides activation of fibrogenic myofibroblasts, which in response to injury up- being involved in regulating a number of cellular responses, such as cell regulate α-smooth muscle actin (α-SMA) and secrete extracellular ma- growth, survival and migration. The AKT is downstream of PI3-K and trix proteins, mostly collagen Type I (Col) (Seki et al., 2007). activation of AKT is associated with HSC proliferation and α1 (I) col- Hepatic stellate cells (HSCs) are the major source of fibrogenic lagen transcription and translation (Reif et al., 2003). In the liver, myofibroblast in liver injury. There is an overwhelming evidence that macrophage-associated PI3K activation promotes cytokine production activated HSCs are the major producers of the fibrotic matrix and that and subsequent hepatocyte proliferation early following partial hepa- HSC apoptosis is the primary mechanism of regression of liver fibrosis tectomy (Jackson et al., 2008). Hepatocyte-associated PI3K regulates (Kweon et al., 2003). Inflammation is the main characteristic in liver growth following a reduction in the liver volume, a process involving fibrosis and KCs are considered to be the primary source of in- AKT activation. The activated PI3K/AKT was found to participate in flammatory cytokines (Nieto, 2006). In this study, we show that IL-1β, regulation of HSC migration, proliferation, collagen secretion, and ad- TNF-α levels and PCI and PCIII mRNA expression were increased while hesion (Reif et al., 2003; Aslan et al., 2004). In the present work, GNPs IL-10 cytokine was decreased in ethanol+METH group alone. On the 724.96 µg/kg inhibited macrophage-specific adhesion (F4/80), PI3K other hand, inverted results were found in the 724.96 µg/kg GNPs. and AKT mRNA expression. Besides, our findings show that GNPs724.96 µg/kg treatment inhibited Compared with GNPs, negatively charged were retained longer in NF-κB mRNA expression significantly. liver and spleen, presumably due to internalization by Kupffer cells and The stimulatory effect of macrophage migration inhibitory factor macrophages causing the formation of oxygen reaction species (Wang (MIF) is, at least partly, dependent on IL-1β and IL-23 production, and et al., 2016). GNPs appear to a type of be one of the promising treat- involves the signaling pathways of MAP kinase (MAPK) (Gordon et al., ment for hepatic injury, when the GNPs enter in the bloodstream, they 2014). The activated PI3-K participate in regulation of HSC migration, are trapped by the Kupffer cells in the liver, or if smaller than about 12 113 T.G. de Carvalho et al. International Journal of Pharmaceutics 548 (2018) 1–14 4–6 nm partially be filtrated into the preurine (Sadauskas et al., 2007; 135–137. Sereemaspun et al., 2008) where the nanoparticle retention in the liver Ellis, A.J., Wendon, J.A., Portmann, B., Williams, R., 1996. Acute liver damage and ec- stasy ingestion. Gut 38 (3), 454–458. http://dx.doi.org/10.1136/gut.38.3.454. and spleen is low suggesting elimination through the kidneys. This Esterbauer, Hermann, Cheeseman, Kevin H., 1990. Determination of aldehydic lipid mechanism seems to be very efficient and capable of protecting the rest peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol. of the organism from the nanoparticles (Sadauskas et al., 2007). 186 (C), 407–421. http://dx.doi.org/10.1016/0076-6879(90)86134-H. In our study, we showed that 724.96 µg/kg GNPs down-regulated Friedman, S.L., 2000. Molecular regulation of hepatic fibrosis, an integrated cellular re-sponse to tissue injury. J. Biol. Chem. 275 (10), 2247–2250. http://dx.doi.org/10. the activity of Kupffer cells and hepatic stellate cells affecting the 1074/jbc.275.4.2247. profile of their pro-inflammatory cytokines, oxidative stress and fibrosis Fröhlich, Eleonore, 2012. The role of surface charge in cellular uptake and cytotoxicity of through modulation of signaling pathways AKT/PI3K and MAPK. medical nanoparticles. Int. J. Nanomed. http://dx.doi.org/10.2147/IJN.S36111. Gao, Bin, 2012. Hepatoprotective and anti-inflammatory cytokines in alcoholic liver disease. J. Gastroenterol. Hepatol. 27 (Suppl 2), 89–93. http://dx.doi.org/10.1111/j. Acknowledgments: 1440-1746.2011.07003.x. Gasparotto, Luiz H S, Garcia, Amanda C., Gomes, Janaina F., Tremiliosi-Filho, Germano, 2012. Electrocatalytic performance of environmentally friendly synthesized gold The authors are grateful to the Brain Institute (Federal University of nanoparticles towards the borohydride electro-oxidation reaction. J. Power Sources Rio Grande do Norte) and the Leiden University Medical Center for 218, 73–78. http://dx.doi.org/10.1016/j.jpowsour.2012.06.064. their contributions to the present study. We acknowledge support by Goodman, Catherine M., McCusker, Catherine D., Yilmaz, Tuna, Rotello, Vincent M., 2004. Toxicity of gold nanoparticles functionalized with cationic and anionic side postdoctoral fellowship from the Raimundo Fernandes de Araujo Junior chains. Bioconjug. Chem. 15 (4), 897–900. http://dx.doi.org/10.1021/bc049951i. by CAPES 88881.119850/2016-01 and the European Commission Gordon, Siamon, Plüddemann, Annette, Estrada, Fernando Martinez, 2014. Macrophage where RFde A, ABC and LJC have received funding from a MSCA-ITN- heterogeneity in tissues: phenotypic diversity and functions. Immunol. Rev. 262 (1), 36–55. http://dx.doi.org/10.1111/imr.12223. 2015-ETN Action grant (proposal number: 675743; project: ISPIC). Gunes, Y., Y, L.Y., Gumrukcuoglu, H.A., Tuncer, M., 2010. Role of echocardiography in the evaluation of atrial function and diseases. Minerva Cardioangiol. 58 (3), Conflict of interest statement 379–397. http://www.ncbi.nlm.nih.gov/pubmed/20485242. Guo, Rui, Zhong, Li, Ren, Jun, 2009. Overexpression of aldehyde dehydrogenase-2 at- tenuates chronic alcohol exposure-induced apoptosis, change in akt and pim signal- The authors declare no conflicts of interest. ling in liver. Clin. Exp. Pharmacol. Physiol. 36 (5–6), 463–468. http://dx.doi.org/10. 1111/j.1440-1681.2009.05152.x. References Halpin, Laura E., Gunning, William T., Yamamoto, Bryan K., 2013. Methamphetamine causes acute hyperthermia-dependent liver damage. Pharmacol. Res. Perspect. 1 (1). http://dx.doi.org/10.1002/prp2.8. Abdelhalim, K. Mohamed Anwar, Jarrar, Bashir M., 2011. Gold nanoparticles induced He, Wei, Cheng, Zhi Huang, Yuan, Fang Li, Jian, Ping Xie, Rong, Ge Yang, Pei, Fu Zhou, cloudy swelling to hydropic degeneration, cytoplasmic hyaline vacuolation, poly- Wang, Jian, 2008. One-step label-free optical genosensing system for sequence-spe- morphism, binucleation, karyopyknosis, karyolysis, karyorrhexis and necrosis in the cific dna related to the human immunodeficiency virus based on the measurements of liver. Lipids Health Dis. 10 (1), 166. http://dx.doi.org/10.1186/1476-511X-10-166. light scattering signals of gold nanorods. Anal. Chem. 80 (22), 8424–8430. http://dx. Almalki, Atiah H., Das, Sujan C., Alshehri, Fahad S., Althobaiti, Yusuf S., Sari, Youssef, doi.org/10.1021/ac801005d. 2018. Effects of sequential ethanol exposure and repeated high-dose methampheta- Hirsch, L.R., Stafford, R.J., Bankson, J.A., Sershen, S.R., Rivera, B., Price, R.E., Hazle, mine on striatal and hippocampal dopamine, serotonin and glutamate tissue content J.D., Halas, N.J., West, J.L., 2003. Nanoshell-mediated near-infrared thermal therapy in wistar rats. Neurosci. Lett. 665 (Supplement C), 61–66. http://dx.doi.org/10. of tumors under magnetic resonance guidance. Proc. Natl. Acad. Sci. U.S.A. 100 (23), 1016/j.neulet.2017.11.043. 13549–13554. http://dx.doi.org/10.1073/pnas.2232479100. Araújo, Raimundo Fernandes, De, Vinícius Barreto, Garcia, Renata Ferreira, Leitão, De Ibrahim, Khalid Elfaki, Bakhiet, Amel Omer, Awadalla, Maaweya Elaeed, Khan, Haseeb Carvalho, Brito, Gerly Anne De Castro, De Castro, Emilio, Miguel, Paulo Marcos, Ahmad, 2018. A priming dose protects against gold nanoparticles-induced proin- Guedes, Matta, Araújo, Aurigena Antunes De, 2016. Carvedilol improves in- flammatory cytokines mrna expression in mice. Nanomedicine 13 (3), 313–323. flammatory response, oxidative stress and fibrosis in the alcohol-induced liver injury http://dx.doi.org/10.2217/nnm-2017-0332. in rats by regulating kuppfer cells and hepatic stellate cells. PLoS One 11 (2). http:// Ikegami, Aiko, Olsen, Christopher M., Fleming, Sheila M., Guerra, Erik E., Bittner, dx.doi.org/10.1371/journal.pone.0148868. Michael A., Wagner, Jeremy, Duvauchelle, Christine L., 2002. Intravenous ethanol/ Araújo, Raimundo Fernandes, de, de Araújo, Aurigena Antunes, Pessoa, Jonas Bispo, cocaine self-administration initiates high intake of intravenous ethanol alone. Freire Neto, Franscisco Paulo, da Silva, Gisele Ribeiro, C.S. Leitão Oliveira, Ana Pharmacol. Biochem. Behav. 72 (4), 787–794. http://dx.doi.org/10.1016/S0091- Luiza, de Carvalho, Thaís Gomes, et al., 2017. Anti-inflammatory, analgesic and anti- 3057(02)00738-4. tumor properties of gold nanoparticles. Pharmacol. Rep. 69 (1), 119–129. http://dx. Ishak, Kamal, Baptista, Amelia, Bianchi, Leonardo, Callea, Francesco, De Groote, Jan, doi.org/10.1016/j.pharep.2016.09.017. Gudat, Fred, Denk, Helmut, et al., 1995. Histological grading and staging of chronic Aslan, Kadir, Lakowicz, Joseph R., Geddes, Chris D., 2004. Nanogold-plasmon-resonance- hepatitis. J. Hepatol. 22 (6), 696–699. http://dx.doi.org/10.1016/0168-8278(95) based glucose sensing. Anal. Biochem. 330 (1), 145–155. http://dx.doi.org/10.1016/ 80226-6. j.ab.2004.03.032. Jackson, Lindsey N, Shawn D Larson, Scott R Silva, Piotr G Rychahou, L Andy Chen, Barathmanikanth, Selvaraj, Kalishwaralal, Kalimuthu, Sriram, Muthuirulappan, Pandian, Suimin Qiu, Srinivasan Rajaraman, et al. 2008. “PI3K/Akt Activation Is Critical for Sureshbabu Ram Kumar, Youn, Hyung-Seop, Eom, Soohyun, Gurunathan, Early Hepatic Regeneration after Partial Hepatectomy” 536: 1401–10. https://doi. Sangiliyandi, 2010. Anti-oxidant effect of gold nanoparticles restrains hyperglycemic org/10.1152/ajpgi.00062.2008. conditions in diabetic mice. J. Nanobiotechnol. 8, 16. http://dx.doi.org/10.1186/ Jong, Wim H, De, Borm, Paul JA., 2008. Drug delivery and nanoparticles:applications and 1477-3155-8-16. hazards. Int. J. Nanomed. 3 (2), 133–149. http://dx.doi.org/10.2147/IJN.S596. Bartneck, M., Warzecha, K.T., Tacke, F., 2014. Therapeutic targeting of liver inflamma- Kang, N.J., Lee, K.M., Kim, J.H., Lee, B.K., Kwon, J.Y., Lee, K.W., Lee, H.J., 2008. tion and fibrosis by nanomedicine. Hepatobiliary Surg. Nutr. 3 (6), 364–376. http:// Inhibition of gap junctional intercellular communication by the green tea polyphenol dx.doi.org/10.3978/j.issn.2304-3881.2014.11.02. (-)-epigallocatechin gallate in normal rat liver epithelial cells. J Agric Food Chem 56 Bautista, A.P., 2002. Acute ethanol binge followed by withdrawal regulates production of (21), 10422–10427. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve reactive oxygen species and cytokine-induced neutrophil chemoattractant and liver &db=PubMed&dopt=Citation&list_uids=18828601. injury during reperfusion after hepatic ischemia. Antioxid. Redox Signalling 4 (5), Kedia, Satish, Sell, Marie A, Relyea, George, 2007. Mono- versus polydrug abuse patterns 721–731. http://dx.doi.org/10.1089/152308602760598864. among publicly funded clients. Subst. Abuse Treat. Prev. Policy 2, 33. http://dx.doi. Beitia, G., Cobreros, A., Sainz, L., Cenarruzabeitia, E., 2000. Ecstasy-induced toxicity in org/10.1186/1747-597X-2-33. rat liver. Liver 20 (1), 8–15. http://dx.doi.org/10.1034/j.1600-0676.2000. Khan, Haseeb A., Mohamed Anwar Abdelhalim, K., Al-Ayed, Mohammed S., Alhomida., 020001008.x. Abdullah S., 2012. Effect of gold nanoparticles on glutathione and malondialdehyde Chen, Ya-Ling, Chen, Li-Ju, Bair, Ming-Jong, Yao, Mei-Lan, Peng, Hsiang-Chi, Yang, Sien- levels in liver, lung and heart of rats. Saudi J. Biol. Sci. 19 (4), 461–464. http://dx. Sing, Yang, Suh-Ching, 2011. Antioxidative status of patients with alcoholic liver doi.org/10.1016/j.sjbs.2012.06.005. disease in Southeastern Taiwan. World J. Gastroenterol.: WJG 17 (8), 1063–1070. Khan, Haseeb A., Mohamed Anwar Abdelhalim, K., Alhomida, Abdullah S., Al-Ayed, http://dx.doi.org/10.3748/wjg.v17.i8.1063. Mohammed S., 2013. Effects of naked gold nanoparticles on proinflammatory cyto- De, Mrinmoy, Rotello, Vincent M., 2008. Synthetic ‘chaperones’: nanoparticle-mediated kines mRNA expression in rat liver and kidney. BioMed. Res. Int. 2013. http://dx.doi. refolding of thermally denatured proteins. Chem. Commun. (Cambridge, England) org/10.1155/2013/590730. 30, 3504–3506. http://dx.doi.org/10.1039/b805242e. Kirkpatrick, Matthew G., De Wit, Harriet, 2013. In the company of others: social factors Dohnert, Marcelo B., Venâncio, Mirelli, Possato, Jonathann C., Zeferino, Rodrigo C., alter acute alcohol effects. Psychopharmacology 230 (2), 215–226. http://dx.doi. Dohnert, Luciana H., Zugno, Alexandra I., De Souza, Cláudio T., Paula, Marcos M S, org/10.1007/s00213-013-3147-0. Luciano, Thais F., 2012. Gold nanoparticles and diclofenac diethylammonium ad- Koriem, Khaled M M, Soliman, Rowan E., 2014. Chlorogenic and caftaric acids in liver ministered by iontophoresis reduce inflammatory cytokines expression in achilles toxicity and oxidative stress induced by methamphetamine. J. Toxicol. 2014. http:// tendinitis. Int. J. Nanomed. 7, 1651–1657. http://dx.doi.org/10.2147/IJN.S25164. dx.doi.org/10.1155/2014/583494. Dykman, L.A., Bogatyrev, V.A., 2000. Use of the dot-immunogold assay for the rapid Kurniawan, Alfin, Gunawan, Farrel, Nugraha, Adi Tama, Ismadji, Suryadi, Wang, Meng diagnosis of acute enteric infections. FEMS Immunol. Med. Microbiol. 27 (2), Jiy, 2017. Biocompatibility and drug release behavior of curcumin conjugated gold 13 114 T.G. de Carvalho et al. International Journal of Pharmaceutics 548 (2018) 1–14 nanoparticles from aminosilane-functionalized electrospun poly(N-Vinyl-2- Hutchison, Jim, Schlager, John J, Hussain, Saber M, 2011. Surface charge of gold Pyrrolidone) fibers. Int. J. Pharm. 516 (1–2), 158–169. http://dx.doi.org/10.1016/j. nanoparticles mediates mechanism of toxicity. Nanoscale 3, 410–420. http://dx.doi. ijpharm.2016.10.067. org/10.1039/c0nr00478b. Kweon, Young-Oh, Paik, Yong-Han, Schnabl, Bernd, Qian, Ting, Lemasters, John J, Seki, Ekihiro, De Minicis, Samuele, Osterreicher, Christoph H, Kluwe, Johannes, Osawa, Brenner, David A, 2003. Gliotoxin-mediated apoptosis of activated human hepatic Yosuke, Brenner, David A., Schwabe, Robert F., 2007. TLR4 enhances TGF-beta sig- stellate cells. J. Hepatol. 39 (1), 38–46. http://dx.doi.org/10.1016/S0168-8278(03) naling and hepatic fibrosis. Nat. Med. 13 (11), 1324–1332. http://dx.doi.org/10. 00178-8. 1038/nm1663. Loumaigne, Matthieu, Richard, Alain, Laverdant, Julien, Nutarelli, Daniele, Débarre, Sereemaspun, Amornpun, Rojanathanes, Rojrit, Wiwanitkit, Viroj, 2008. Effect of gold Anne, 2010. Ligand-induced anisotropy of the two-photon luminescence of spherical nanoparticle on renal cell: an implication for exposure risk. Ren. Fail. 30 (3), gold particles in solution unraveled at the single particle level. Nano Lett. 10 (8), 323–325. http://dx.doi.org/10.1080/08860220701860914. 2817–2824. http://dx.doi.org/10.1021/nl100737y. Son, Yong, Cheong, Yong-Kwan, Kim, Nam-Ho, Chung, Hun-Taeg, Kang, Dae Gill, Pae, MacParland, Sonya A., Tsoi, Kim M., Ouyang, Ben, Ma, Xue Zhong, Manuel, Justin, Hyun-Ock, 2011. Mitogen-activated protein kinases and reactive oxygen species: how Fawaz, Ali, Ostrowski, Mario A., et al., 2017. Phenotype determines nanoparticle can ROS activate MAPK pathways? J. Signal Trans. 2011, 792639. http://dx.doi.org/ uptake by human macrophages from liver and blood. ACS Nano 11 (3), 2428–2443. 10.1155/2011/792639. http://dx.doi.org/10.1021/acsnano.6b06245. Sumbayev, Vadim V., Yasinska, Inna M., Garcia, Cesar Pascual, Gilliland, Douglas, Lall, Mandal, Palash, Pritchard, Michele T., Nagy, Laura E., 2010. Anti-inflammatory pathways Gurprit S., Gibbs, Bernhard F., Bonsall, David R., Varani, Luca, Rossi, François, and alcoholic liver disease: role of an adiponectin/interleukin-10/heme oxygenase-1 Calzolai, Luigi, 2013. Gold nanoparticles downregulate interleukin-1β-induced pro- Pathway. World J. Gastroenterol. 16 (11), 1330–1336. http://dx.doi.org/10.3748/ inflammatory responses. Small 9 (3), 472–477. http://dx.doi.org/10.1002/smll. wjg.v16.i11.1330. 201201528. Masserini, Massimo, 2013. Nanoparticles for brain drug delivery. ISRN Biochem. 2013, Tang, Yuhan, Li, Yanyan, Haiyan, Yu., Gao, Chao, Liu, Liang, Xing, Mingyou, Liu, 1–18. http://dx.doi.org/10.1155/2013/238428. Liegang, Yao, Ping, 2014. Quercetin attenuates chronic ethanol hepatotoxicity: im- Mironava, Tatsiana, Hadjiargyrou, Michael, Simon, Marcia, Jurukovski, Vladimir, plication of ‘free’ iron uptake and release. Food Chem. Toxicol. 67, 131–138. http:// Rafailovich, Miriam H, 2010. Gold nanoparticles cellular toxicity and recovery: effect dx.doi.org/10.1016/j.fct.2014.02.022. of size, concentration and exposure time. Nanotoxicology 4 (1), 120–137. http://dx. Tilg, Herbert, Moschen, Alexander R., 2008. Inflammatory mechanisms in the regulation doi.org/10.3109/17435390903471463. of insulin resistance. Mol. Med. (Cambridge, Mass.) 14 (3–4), 222–231. http://dx.doi. Montet, Anne-Marie, Oliva, Laurence, Beaugé, Françoise, Montet, Jean-Claude, 2002. Bile org/10.2119/2007-00119.Tilg. salts modulate chronic ethanol-induced hepatotoxicity. Alcohol Alcohol. (Oxford, Tokunaga, Itsuo, Kubo, Shin-ichi, Ishigami, Akiko, Gotohda, Takako, Kitamura, Osamu, Oxfordshire) 37 (1), 25–29. 2006. Changes in renal function and oxidative damage in methamphetamine-treated Nanji, Amin A., Mendenhall, Charles L., French, Samuel W., 1989. Beef fat prevents al- rat. Legal Med. (Tokyo, Japan) 8 (1), 16–21. http://dx.doi.org/10.1016/j.legalmed. coholic liver disease in the rat. Alcohol.: Clin. Exp. Res. 13 (1), 15–19. http://dx.doi. 2005.07.003. org/10.1111/j.1530-0277.1989.tb00276.x. Tsai, Chiau-Yuang, Shiou-Ling Lu, Chia-Wen Hu, Chen-Sheng Yeh, Gwo-Bin Lee, and Nieto, Natalia, 2006. Oxidative-stress and IL-6 mediate the fibrogenic effects of rodent Huan-Yao Lei. 2012. “Size-Dependent Attenuation of TLR9 Signaling by Gold kupffer cells on stellate cells. Hepatology 44 (6), 1487–1501. http://dx.doi.org/10. Nanoparticles in Macrophages.” Journal of Immunology (Baltimore, Md. : 1950) 188 1002/hep.21427. (1): 68–76. https://doi.org/10.4049/jimmunol.1100344. Noor, Neveen A, Heba M Fahmy, and Iman M Mourad. 2016. “Evaluation of the Potential Wang, Jun Ying, Chen, Jie, Yang, Jiang, Wang, Hao, Shen, Xiu, Sun, Yuan Ming, Guo, Neurotoxicity of Gold Nanoparticles in the Different Rat Brain Regions” 4531 Meili, Zhang, Xiao Dong, 2016. Effects of surface charges of gold nanoclusters on (December): 114–29. long-term in vivo biodistribution, toxicity, and cancer radiation therapy. Int. J. Park, Ho Young, Choi, Hee Don, Eom, Hyojin, Choi, Inwook, 2013. Enzymatic mod- Nanomed. 11, 3475–3485. http://dx.doi.org/10.2147/IJN.S106073. ification enhances the protective activity of citrus flavonoids against alcohol-induced Wells, Peter G., Shama Bhatia, Danielle M. Drake, and Lutfiya Miller-Pinsler. 2016. “Fetal liver disease. Food Chem. 139 (1–4), 231–240. http://dx.doi.org/10.1016/j. Oxidative Stress Mechanisms of Neurodevelopmental Deficits and Exacerbation by foodchem.2013.01.044. Ethanol and Methamphetamine.” Birth Defects Research Part C - Embryo Today: Pontes, Helena, Duarte, Jos Alberto, de Pinho, Paula Guedes, Soares, Maria Elisa, Reviews. https://doi.org/10.1002/bdrc.21134. Fernandes, Eduarda, Dinis-Oliveira, Ricardo Jorge, Sousa, Carla, et al., 2008. Chronic Wen, Donghai, Huang, Xinzhong, Zhang, Min, Zhang, Liying, Chen, Jing, Yong, Gu., Hao, exposure to ethanol exacerbates MDMA-induced hyperthermia and exposes liver to Chuan Ming, 2013. Resveratrol attenuates diabetic nephropathy via modulating an- severe MDMA-induced toxicity in CD1 mice. Toxicology 252 (1–3), 64–71. http://dx. giogenesis. PLoS One 8 (12). http://dx.doi.org/10.1371/journal.pone.0082336. doi.org/10.1016/j.tox.2008.07.064. Winkler, Madeline C., Greager, Emilee M., Stafford, Jacob, Bachtell, Ryan K., 2016. Potter, James J., Mezey, Esteban, 2007. Acetaldehyde increases endogenous adiponectin Methamphetamine self-administration reduces alcohol consumption and preference and fibrogenesis in hepatic stellate cells but exogenous adiponectin inhibits fi- in alcohol-preferring P rats. Addict. Biol. http://dx.doi.org/10.1111/adb.12476. brogenesis. Alcohol. Clin. Exp. Res. 31 (12), 2092–2100. http://dx.doi.org/10.1111/ Yang, Raymond S H, Chang, Louis W., Jui Pin, Wu., Tsai, Ming Hsien, Wang, Hsiu Jen, j.1530-0277.2007.00529.x. Kuo, Yu Chun, Yeh, Teng Kuang, Yang, Chung Shi, Lin, Pinpin, 2007. Persistent tissue Powell, Christine L., Bradford, Blair U., Craig, Christopher Patrick, Tsuchiya, Masato, kinetics and redistribution of nanoparticles, quantum dot 705, in mice: ICP-MS Uehara, Takeki, O’Connell, Thomas M., Pogribny, Igor P., et al., 2010. Mechanism for quantitative assessment. Environ. Health Perspect. 115 (9), 1339–1343. http://dx. prevention of alcohol-induced liver injury by dietary methyl donors. Toxicol. Sci. 115 doi.org/10.1289/ehp.10290. (1), 131–139. http://dx.doi.org/10.1093/toxsci/kfq031. Yue, Min, Ni, Qun, Chao Hui, Yu., Ren, Ke Ming, Chen, Wei Xing, Li, You Ming, 2006. Reif, Shimon, Lang, Alon, Lindquist, Jeffery N., Yata, Yutaka, Gäbele, Erwin, Scanga, Transient elevation of hepatic enzymes in volunteers after intake of alcohol. Andrew, Brenner, David A., Rippe, Richard A., 2003. The role of focal adhesion ki- Hepatobiliary Pancreatic Dis. Int. 5 (1), 52–55. nase-phosphatidylinositol 3-kinase-Akt signaling in hepatic stellate cell proliferation Zendulka, O., Sabová, M., Juřica, J., MacHalíček, M., Švéda, P., Farková, M., Šulcová, A., and type I collagen expression. J. Biol. Chem. 278 (10), 8083–8090. http://dx.doi. 2012. The effect of methamphetamine on biotransformation of ethanol: pilot study. org/10.1074/jbc.M212927200. Acta Facultatis Pharm. Univ. Comenianae 59 (2), 63–71. http://dx.doi.org/10.2478/ Sadauskas, Evaldas, Wallin, Håkan, Stoltenberg, Meredin, Vogel, Ulla, Doering, Peter, v10219-012-0026-4. Larsen, Agnete, Danscher, Gorm, 2007. Kupffer cells are central in the removal of Zhang, Qin, Hitchins, Victoria M, Schrand, Amanda M, Hussain, Saber M, Goering, Peter nanoparticles from the organism. Part Fibre Toxicol. 4 (3), 10. http://dx.doi.org/10. L, 2011. Uptake of gold nanoparticles in murine macrophage cells without cyto- 1186/1743-8977-4-10. toxicity or production of pro-inflammatory mediators. Nanotoxicology 5 Safieh-Garabedian, B., Poole, S., Allchorne, A., Winter, J., Woolf, C.J., 1995. Contribution (September), 284–295. http://dx.doi.org/10.3109/17435390.2010.512401. of interleukin-1 beta to the inflammation-induced increase in nerve growth factor Zhang, Xiao Dong, Hong Ying, Wu., Di, Wu., Wang, Yue Ying, Chang, Jian Hui, Zhai, Zhi levels and inflammatory hyperalgesia. Br. J. Pharmacol. 115 (7), 1265–1275. https:// Bin, Meng, Ai Min, Liu, Pei Xun, Zhang, Liang An, Fan, Fei Yue, 2010. Toxicologic doi.org/0007-1188/95. effects of gold nanoparticles in vivo by different administration routes. Int. J. Sayette, Michael A., Creswell, Kasey G., Dimoff, John D., Fairbairn, Catharine E., Cohn, Nanomed. 5 (1), 771–781. http://dx.doi.org/10.2147/IJN.S8428. Jeffrey F., Heckman, Bryan W., Kirchner, Thomas R., Levine, John M., Moreland, Zhang, Juan, Tang, Hongju, Zhang, Yuqing, Deng, Ruyuan, Shao, Li, Liu, Yun, Li, Richard L., 2012. Alcohol and group formation: a multimodal investigation of the Fengying, Wang, Xiao, Zhou, Libin, 2014. Identification of suitable reference genes effects of alcohol on emotion and social bonding. Psychol. Sci. 23 (8), 869–878. for quantitative RT-PCR during 3T3-L1 adipocyte differentiation. Int. J. Mol. Med. 33 http://dx.doi.org/10.1177/0956797611435134. (5), 1209–1218. http://dx.doi.org/10.3892/ijmm.2014.1695. Schaeublin, Nicole M, Braydich-Stolle, Laura K, Schrand, Amanda M, Miller, John M, 14 115 Apêndice D Lipopolysaccharides and peptidoglycans modulating the interaction of Au nanoparticles with cell membranes models at the air-water interface. da Silva, R. L. C. G.;da Silva, H. F. O.; da Silva Gasparotto, L. H., Caseli, L. BiophysicalChemistry, 2018,Vol.238, 22-29 Contribuição:  Realizei a síntese e purificação das NanoAu.  Realizei a caracterização das NanoAu.  Tratei os dados e preparei as figuras relacionadas às etapas que realizei ativamente que foram incluídas no manuscrito. ________________________ __________________________ Heloiza Fernanda O. da Silva Athayde Prof. Luiz Henrique da S. Gasparotto 116 Biophysical Chemistry 238 (2018) 22–29 Contents lists available at ScienceDirect Biophysical Chemistry journal homepage: www.elsevier.com/locate/biophyschem Lipopolysaccharides and peptidoglycans modulating the interaction of Au T naparticles with cell membranes models at the air-water interface Rafael Leonardo Cruz Gomes da Silvaa, Heloiza Fernanda Oliveira da Silvab, Luiz Henrique da Silva Gasparottob, Luciano Caselia,⁎ a Department of Chemistry, Federal University of São Paulo, Diadema, SP, Brazil bGroup of Biological Chemistry and Chemometrics, Institute of Chemistry, Federal University of Rio Grande do Norte, Natal 59072970, RN, Brazil H I G H L I G H T S G R A P H I C A L A B S T R A C T • Au nanoparticles were stabilized with PVP. • They were incorporated in membrane models. • Surface properties of the films was affected. • Monolayer composition modulates in- teraction. A R T I C L E I N F O A B S T R A C T Keywords: Understanding the interactions between nanoparticles and biological surfaces is of great importance for many Gold nanoparticles areas of nanomedicine and calls for detailed studies at the molecular level using simplified models of cellular Airwater interface membranes. In this paper, water-dispersed polyvinylpyrrolidonestabilized gold nanoparticles (AuNPs) were Cell membrane models incorporated in floating monolayers of selected lipids at the air-water interface as cell membrane models. Monolayers Surface pressure-area isotherms showed the condensation of glycoside-free lipid monolayers, suggesting their adsorption on the nanoparticle surface through the hydrophilic head groups. On the other hand, monolayers containing glycoside derivatives expanded upon AuNPs incorporation, pointing that the supramolecular struc- ture formed should facilitate the incorporation of these nanoparticles in cellular membranes. These findings can be therefore correlated with the possible toxicity, microbicide and antitumorigenic effects of these nanoparticles in lipidic surfaces of erythrocyte and microbial membranes. 1. Introduction development of metallic nanoparticles applied to nanomedicine since these materials have presented antimicrobial activity [2], acting in the Among the properties of interest presented by nanoscale particles, inhibition of pathogens such as fungi and bacteria, or even helping in one can mention their capacity to interact with biological interfaces the necrosis of tumor tissues by hyperthermia, as in the case of nano- [1]. In this context, it is possible to mention the growing study and particles with magnetic properties [3]. Understanding the mechanism ⁎ Corresponding author. E-mail address: lcaseli@unifesp.br (L. Caseli). https://doi.org/10.1016/j.bpc.2018.04.007 Received 3 April 2018; Received in revised form 18 April 2018; Accepted 24 April 2018 Available online 25 April 2018 0301-4622/ © 2018 Elsevier B.V. All rights reserved. 117 R.L.C.G. da Silva et al. Biophysical Chemistry 238 (2018) 22–29 of interaction between nanoparticles and cell membranes is of parti- with these compounds. As a result, by using now AuNPs instead of cular importance in the development of novel therapeutic agents since silver, we also intend in this paper to understand the role of the che- many of these compounds are potentially toxic and can sometimes be mical nature of the metallic nanoparticle in the thermodynamic, harmful to humans [4]. Thus, the use of Langmuir films as experimental rheological, and structural aspects of metallic colloidal dispersions in- models for the study of interactions taking place in nanobiotype in- teracting with floating monolayers at the air-water interface. terfaces has gained attention because of the simplicity of these systems, whose dynamics of interfacial processes is less laborious to be taken 2. Materials and methods into account [5]. Numerous studies have used nanoparticles with modified surfaces as The lipids dipalmitoylphosphatidylcholine (DPPC) and dipalmi- a way to understand better the mode of action and the types of inter- toylphosphatidylglycerol (DPPG) were purchased from SigmaAldrich action occurring between these materials and phospholipid monolayers. and each one was dissolved in chloroform (Synth, PA) to result in a As examples one can cite silica nanoparticles (SiO2) [6–8], graphene concentration of 0.5mg/mL. The water employed in this work was oxide, fullerene, carbon nanotubes, and nanodiamonds [9–12], which purified using a MilliQ®Plus system (resistivity 18.2 MΩ cm, pH 5.5). had their effects studied in relation to surface films mimicking cell Peptidoglycan from Bacillus subtilis (PG) and lipopolysaccharide form E. membranes. In addition to the compounds mentioned above, it is also coli (LPS) were obtained from SigmaAldrich and dissolved in water to possible to highlight the importance of colloidal gold and silver in render a 0.5 mg/mL solution. biological systems. The inhibitory and bactericidal properties of silver AuNPs were synthesized according to a method reported elsewhere ions have been known for many years [13–16], and some forms of silver [34]. Briefly, all glassware was cleaned with a KMnO4+ NaOH solution have been shown to be effective in curing different types of infections and piranha solution. The following stock solutions were then produced [17]. Another important aspect related to silver that should be taken in water: 50 mmol L1 HAuCl4, 100 g L1 PVP and a solution containing into account is its lower toxicity to human cells when compared to 1.0 mol L−1 NaOH +1.0 mol L1 glycerol. In a beaker, determined vo- other metallic nanoparticles [18], which contributed to the incorpora- lumes of the PVP and HAuCl4 solutions were dissolved in water to yield tion of this compound in the production of clothing and materials for a 5mL solution. In a separate beaker, a known volume of the NaOH + surgical purposes [19,20]. glycerol solution was mixed with water to generate another 5mL so- Particularly, gold nanoparticles (AuNPs) have also been applied in lution. The glycerolNaOH solution was poured into the HAuCl −4 PVP several kinds of nanotools since it has been found novel abilities in one to yield the following final concentrations: 0.10 mol L−1 glycerol various fields of science [21], such as coating glasses to change their and NaOH, 10.0 g L−1 PVP and 1.0 mmol L−1 HAuCl4. The AuNPs properties and multicolor optical coding for biological assays. They are colloidal dispertions had then their pH adjusted to 7 by addition of also employed to enhance electroluminescence and quantum efficiency diluted HCl. in organic light emitting diodes. General speaking, the use of Au na- Lipid Langmuir monolayers were formed spreading pre-determined noparticles materials has enabled new types of new sensors and devices, aliquots of the lipid solutions on the airwater interface. For that, a mini- as well as an antimicrobial potential agent [22]. Also it has been shown KSV Langmuir trough equipped with a surface pressure sensor (the exceptional properties as a binder, which makes gold an important ally Wilhelmy method) was filled with water. The nanoparticles were in- in controlled drug delivery processes [23], tumor detection [24] and serted after lipid spreading in the aqueous subphase in the vicinities of gene therapy [25]. Furthermore, AuNPs, due to their biocompatibility the air-water interface. After at least 10 min allowed for solvent eva- and unique plasmonic properties [26–29], have been advantageously poration, mobile barriers were actioned compressing the airwater in- used in many types of therapeutic and diagnostic methods [30,31]. terface at a rate of 5 Å2 molecule−1 min−1. For mixed lipidAuNPs Their importance in medical research has prompted studies to un- monolayers, 30 min were waited for homogenization. Surface pressure scramble the interaction of this material with monomolecular films, as (π) were measured as long as the film area (A) decreased, obtaining π-A already found in the literature [32–34]. Since little is known about the isotherms. For polarization modulation infrared reflection-absorption mechanism involved in the interaction of nanoparticle systems with cell spectroscopy (PMIRRAS) studies, the airwater interface was com- membranes, and it is not possible to accurately describe all the inter- pressed up to 30mN/m and a KSV PMI 550 instrument (KSV actions that occur in a nanobiotype interface [18], colloidal dispersions Instruments, Ltd., Helsinki, Finland), operating with a modulation fre- of metallic nanoparticles can be inserted into Langmuir films, which act quency of 84 kHz, and an incidence angle to the normal of 75° obtained as cell membrane models, and different characterization techniques can a minimum of 6000 scans for each spectrum, with a resolution of be applied in order to better investigate the interactions that occur at 8 cm−1. Control experiments using only the capping polymer (without the molecular level. AuNPs) were carried out in order to check the influence of PVP on the With these ideas in mind, in this paper we employed lipid Langmuir surface activity of the supramolecular systems. monolayers to mimic the first barrier encountered by AuNPs, which All experiments were carried out at a controlled room temperature were dispersed in water and stabilized with polyvinylpyrrolidone (PVP) (25 ± 1 °C). Each π-A isotherm and spectrum was obtained at least as shown in a previous studies [35,36]. To that end, we employed the three times to ensure the reproducibility of the experiments so that only lipids dipalmitoylphosphatidylcholine (DPPC), which have been em- isotherms and spectra that were highly reproducible are shown. ployed as a model lipid for erythrocyte membranes [37], dipalmitoyl- phosphatidylglycerol (DPPG), which have been used as a model lipid in 3. Results and discussion bacterial cytoplasm membrane [38] and is also superexpressed lipid in tumorigenic cells [39]. We also investigated the role of peptidoglycans In this work, AuNPs were produced via the reduction of Au3+ with (PG) and lipopolysaccharides (LPS) considering that these substances glycerol in alkaline medium. Glycerol is nowadays quite an inexpensive are encountered at the peptidoglycan cell wall and in the periplasmic chemical readily biodegradable under aerobic conditions, therefore a space of grampositive and gram-negative bacteria as well as on the more environmentally correct option compared to common reducing surface of the other lipidic membrane of gram-negative bacteria. It is chemicals such as formamide, sodium borohydride and hydrazine. important to emphasize that similar study was carried out with AgNPs Fig. 1A shows a UV–vis spectrum of asprepared AuNPs produced by [40], also stabilized with PVP. In that paper, it was shown that AgNPs simple addition of NaOH-glycerol to AuCl3-PVP at room temperature. condensed all monolayers of the pure lipids essayed, with distinction The colloidal AuNPs spectrum had a maximum absorbance (λMax) at observed only in the infrared spectra and morphological images of the 520 nm, a value typical for spherical gold nanoparticles [41–43]. The surface. However, mixtures with glycosidic compounds presented a symmetry of the band implies a fair similarity in the shape of the na- divergent expansion of the monolayer, pointing to a specific interaction noparticles and low degree of aggregation in the solution [44]. The 23 118 R.L.C.G. da Silva et al. Biophysical Chemistry 238 (2018) 22–29 0.5 A B 0.4 0.3 0.2 0.1 400 500 600 700 Fig. 1. (A) UV–vis spectrum of the colloidal AuNPs with (B) their respective TEM image. Inset: size distribution of the AuNPs. suggests the interaction of the nanoparticles with the polar heads of the lipid, stabilizing the lateral repulsion between the hydrophilic moieties of the monolayer. Similar effect is observed for AgNPs stabilized with the same compound (PVP) [40], indicating the low ability of penetra- tion of both kind of nanoparticles into the alkyl chains of DPPC, which would lead to an expansion of the monolayer (shift to higher areas). PVP alone was also essayed in the concentrations found in the nano- particles and it does not present enough surface activity owing to its low relative concentration [40]. However, although the effect observed for AgNPs was also to con- dense DPPC monolayers [40], the effects for that case were significantly more pronounced than those observed for AuNPs (Fig. 2A). For silver, at 30 mN/m the isotherm shifted about 10 Å2/DPPC molecule, while for gold, this shift was about 4 Å2. Therefore, the effect of both kinds of nanoparticles seems analogous, which is the condensation of the monolayer. Such effect is explained by the interaction of the nano- particle with the polar heads of the lipids, minimizing lateral repul- sions. However, comparing the extension of the lipid monolayer con- densation, we note that this effect is much more effective for Ag nanoparticles. It is clear therefore that such effect is obviously depen- dent on the chemical nature of the metallic particle that constitutes the core of the colloidal dispersion. Ag and Au also diverge in terms of average size and polydispersity. Also, while for silver, the particles have sizes in the range of 16–30 nm, gold presents sizes around 7 nm. Then, it is likely that the size of the particles may also influence the their ability for condensing the monolayer. Panel B shows the quasi-equilibrium rheological properties of the monolayer by means of the compressibility modulus, which is at a given temperature T defined by –A(∂π/∂A)T. Therefore, the values of this parameter at a given molecular area can be calculated directly by the derivative of the π-A isotherm and furnishes information on the phy- sical states of the monolayer as well as possible phase transitions. The depression seen in the curve between 85 and 70 Å2 for pure DPPC is related to the firstorder transition between the states liquid-expanded and liquid-condensed. For the monolayer with AuNPs incorporated, this Fig. 2. Surface pressure (A) and Compressibility modulus (B)area isotherms for depression is not so well defined anymore, which indicates that the monolayers of pure DPPC without and with AuNPs in the aqueous subphase (as packing of the DPPC molecules when compressed at the air-water in- indicated on the charts). terface is affected by the presence of the nanoparticles. This behavior is similar to that observed for AgNPs [40], and, in fact, this is expected for TEM image illustrated that the as-prepared AuNPs were spherical in any mixture involving lipid Langmuir monolayers that present first- shape, thus corroborating the UV–vis results. The mean particle size order transitions. Usually mixtures provide a more flexible mechanism calculated from the distribution (inset of Fig. 1B) was 6.8 nm ± 1.6 of packing with more possibilities of rearrangements. As a result, the nm. energy given by the compressing barriers must be employed not only to Fig. 2 shows the tensiometric properties of DPPC monolayers com- phase transitions, but also to intermolecular interactions changes re- pressed from the gaseous state to their collapse. Panel A shows a typical lated to molecular rearrangements, providing the increase of the sur- π-A isotherm [45], presenting the following 2D states: gaseous, liquid- face pressure. expanded, and liquid-condensed. Collapse of the monolayer occurred at Fig. 3 shows the PMIRRAS spectra for DPPC monolayers at 30 mN/ 50 Å2/molecule. With the insertion of AuNPs, the isotherms are shifted m. This surface pressure was chosen because it represents the lateral to lower areas indicating the condensation of the monolayer. This fact pressure of natural cellular membrane [46]. The spectra are divided in 24 119 R.L.C.G. da Silva et al. Biophysical Chemistry 238 (2018) 22–29 the interactions between adjacent methylene groups present in the lipid alkyl chains. The condensation of the monolayer must force the chains to approach each other affecting the viscoelastic properties of the film. Fig. 3B shows the PMIRRAS for DPPC in the region where some of the bands related to its hydrophilic region appear. Although the low resolution in the region, the main bands of DPPC usually appear at approximately 1730 cm−1 (C]O stretches), 1225 cm−1 and 1100 cm−1 (PO −2 asymmetric and symmetric stretches, respectively). Some bands related to hydration of the polar heads usually appear at 1500–1700 cm−1, related to water bending modes. With AuNPs, the C]O stretching mode is shifted to higher energies, and the phosphate bands seem to increase their relative maximum intensity of their peaks, as a consequence of the nanoparticle interaction with the vicinities of the monolayer polar heads. For AgNPs, phosphate and carbonyl bands are also affected [40], evidencing the interaction of the nanoparticles with the vicinities of the polar head of the lipid. We also employed DPPG because it is a lipid that is frequently en- countered in several cells, including in bacteria [38] and in tumorigenic cells [39]. The lipid signature of induced lymphoma is reported by elevated phosphatidylglycerol levels when compared with normal tis- sues [48]. The increased-phosphatidylglycerol is also found in renal cell and hepatocellular carcinomas [49,50]. Phosphatidylglycerol also serves as a precursor of cardiolipin, found in mitochondrial membranes as well a common lipid encountered in several bacteria [51]. Fig. 4 shows the effect of AuNPs on DPPG monolayer. General speaking, the effect is very similar to that encountered for DPPC, showing the shift of the isotherms to lower molecular areas with the introduction of the nanoparticle. DPPG presents a typical curve [52], Fig. 3. PMIRRAS spectra for monolayers of pure DPPC without and with AuNPs in the aqueous subphase (as indicated on the charts). two panels in order to better analyze the bands related to the alkyl chains stretching mode (2800–3000 cm−1) and other modes related to the hydrophilic region of the phospholipid (1000–1800 cm−1). Panel A shows two mains bands, one centered at 2850 cm−1, attributed to the symmetric stretching mode of CH2, and the other one centered at 2920 cm−1, attributed to the asymmetric stretching mode of CH2. With AuNPs these bands change significantly: the symmetric band peak is shifted to higher wavelengths while the asymmetric one is shifted to lower energies. Also, the intensities between the maximum of the peaks is altered: both peaks present approximately the same intensity with AuNPs, while the peak for the symmetric stretches is significantly higher than that related to the asymmetric stretches for the pure DPPC monolayer. As the asymmetric band is more sensitive to the lateral order than the symmetric one [47], these changes in the relative in- tensities upon nanoparticle incorporation is an indicative of a more disordered monolayer for DPPC-AuNP system. Other fact that corro- borates this hypothesis is the presence of a wide band centered at ap- proximately 2960 cm−1, attributed to the CeH stretches in CH3. Al- though the monolayer is condensed with the adsorption of AuNPs and also these nanoparticles probably interact with the polar heads of the phospholipid, the changes caused in the rheological properties of the monolayer, as observed in Fig. 2B, may impact in the viscoelasticity of film, promoting the disorder of the packed monolayer. Disordering of the monolayer was also observed for AgNPs adsorbed on DPPC mono- layers [40]. Although differences in the profile of the spectra when Fig. 4. Surface pressure (A) and Compressibility modulus (B)area isotherms for comparing AgNP-DPPC and AuNP-DPPC can be found, the interaction monolayers of pure DPPG without and with AuNPs in the aqueous subphase (as of the nanoparticle with the polar heads of the lipid affects indirectly indicated on the charts). 25 120 R.L.C.G. da Silva et al. Biophysical Chemistry 238 (2018) 22–29 displaying a sudden increase of the surface pressure at about 60–70 Å2 they are more defined with the nanoparticles. For the phosphate bands, when it goes from the gaseous state to the liquid-condensed (LC) state, the asymmetric band is shifted to higher energies with AuNPs and the collapsing at about 50 Å2. With the AuNPs, the shift of the curves to symmetric band disappears. While the effect on the hydrophilic region lower molecular areas is more evident in the LC state, where for a given reflects the interaction of the nanoparticles with the lipid heads, the surface pressure the variation of lipid molecular area is about 2–3 Å2. effect on the CH2 region reflects the condensation of the monolayer and This indicates again a stabilization of the lateral repulsions of the a more compacted state of the monomolecular film. General speaking, monolayer caused by interactions of the nanoparticles probably with for DPPG, PMIRRAS also indicates that the interaction of the nano- the polar heads of the lipid. Panel B also evidences the condensation of particle with the polar heads of the lipid affects the organization of the the monolayer since the compressional modulus curve is shifted to alkyl chains, altering the mechanism of molecular packing. However lower molecular areas with the presence of AuNPs, which is consistent the effects are also distinctive on the apolar groups since the peaks in with the surface pressurearea isotherms that originated these curves. It PMIRRAS for the methylene stretches are shifted to higher energies for can be also noted that the values of compressional modulus attained at DPPG and not for DPPC. a given surface pressure are similar for both curves, showing neglecting As the bacteria cell envelope is a multilayered structure enclosed by alterations in the rheological properties of the film with or without the peptidoglycan cell wall and for gramnegative bacteria, an outer mem- nanoparticles. Similar effects were also observed for AgNPs [40], with brane containing lipopolysaccharides additionally encases the cellular condensation occurring with comparable degrees. This differentiates membrane, we decided to prepare monolayers composed by DPPG and from the effect observed for DPPC, whose condensation depended on glycosidic compounds that surround the plasmatic membrane of bac- the chemical nature of the nanoparticle. This is expected since DPPC teria since some reports on Au nanoparticles consider their possible presents a LE phase and an LE-LC transition region. These states are bactericide effect [22]. For that, we employed mixtures of peptidogly- more susceptible to external stimulus (such as adsorption of substances cans from Bacilus subtilis I (PG) and lipopolysaccharides from E. Colli coming from subphase) as they are in a less compacted phase. (LPS) representing the major components of bacteria's cell wall and Fig. 5 shows the PMIRRAS spectra for DPPG. For the pure lipid outer membrane, respectively. monolayer, the symmetric and asymmetric stretches are centered at Results for PG are presented in Fig. 6. The adsorption of PG on the 2840 and 2920 cm−1 respectively, and with AuNPs both peaks are DPPG monolayer shifts the isotherms to higher areas as a consequence shifted to higher energies. In the hydrophilic region, the C]O of its incorporation into the lipid chains. With the presence of AuNPs in stretching band appears split in two bands (1720 and 1750 cm−1) and the aqueous subphase, we observe a shift of the mixed PGDPPG monolayer isotherm to higher areas at low surface pressures, also Fig. 6. Surface pressure (A) and Compressibility modulus (B)area isotherms for Fig. 5. PMIRRAS spectra for monolayers of pure DPPG without and with AuNPs monolayers of DPPG –PG without and with AuNPs in the aqueous subphase (as in the aqueous subphase (as indicated on the charts). indicated on the charts). 26 121 R.L.C.G. da Silva et al. Biophysical Chemistry 238 (2018) 22–29 Fig. 8. Surface pressure (A) and Compressibility modulus (B)area isotherms for Fig. 7. PMIRRAS spectra for monolayers of pure DPPG-PG without and with monolayers of DPPG–LPS without and with AuNPs in the aqueous subphase (as AuNPs in the aqueous subphase (as indicated on the charts). indicated on the charts). indicating the expansion of the monolayer. Such expansion is less sig- overlapping of bands related to glycosidic structure is noted, being also nificant for surface pressures higher than 5mN/m, where a condensa- changed upon AuNPs incorporation. tion of the monolayer is further observed as a consequence of the With LPS (Fig. 8), the behavior is similar to that observed for PG: the AuNPs expelling towards the aqueous subphase interacting externally lipopolysaccharides expands the DPPG monolayer, and the adsorption to the polar heads of the mixed monolayer. This effect can be also of AuNPs expands even more the monolayer at lower surface pressure observed in the compressional modulusarea isotherms (Panel B) and values, but condenses it at higher values of surface pressure. However, was the first result indicating an expansion of the monolayer provoked PMIRRAS spectra (Fig. 9) show not only changes in the hydrophilic by the adsorption of AuNPs instead of a merely film condensation for all region (Panel B), but also in the hydrophobic region (Panel A), in- surface pressures attained in the isotherm, as observed for the films dicating changes in the organization of the lipid matrix caused by the composed only by lipids (DPPC or DPPG). This suggests a specific in- new supramolecular system formed at the interface. teraction of the nanoparticle with the peptidoglycosidic groups of PG. It Therefore, the effect of AuNPs on the monolayers herein studied is likely that the supramolecular structure formed by AuNPs/GP may depends strongly on the nature of the lipids and glycosides. It is im- facilitate the access to the monolayer expanding them at low surface portant to emphasize that the literature shows some few examples of pressures. AuNPs interacting with lipid monolayers at the air-water interface PMIRRAS spectra (Fig. 7) shows that the CH2 stretching region [53–57]. For instance, citrate-coated gold nanoparticles are reported (Panel A) is little affected by the presence of AuNPs, in contrast to that [53] to expand DPPC and DPPG monolayers in a greater extent only in a observed for the hydrophilic region (Panel B), visually more affected. form of nanorods. It is clear therefore the inability of these nano- For the DPPGPG spectrum, the bands centered at 1541 cm−1 are at- particles to penetrate into the hydrophobic moieties of the lipids, whose tributed to the amide II vibration (NeH bends coupled with CeN activity is restricted on the hydrophilic surface. Cysteine-capped gold stretches) and those at 1656 and 1681 cm−1 are attributed to amide I nanoparticles expanded octadecylamine (ODA) monolayers, but this bands (C]O stretching mode). These bands confirm the adsorption of activity occurred only at basic pHs and after a long time of waiting PG at the lipid monolayer, since they are related to peptide bonds, between the lipid spreading [54]. This was attributed to the ionized encountered in the PG structure. These bands are altered with AuNPs carboxylic acid groups exposed on the surface of the particles, leading indicating the interaction of the nanoparticles inside the backbone to strong electrostatic attractions with the protonated ODA monolayer. structure of the peptidoglycan as the position of these bands is de- Hexadecanethiolatecapped Au NPs with an average core diameter of 2 termined by the backbone conformation and the hydrogen bonding nm have been incorporated into DPPC monolayers and they do not pattern. Also between the bands related to phosphate stretches, 27 122 R.L.C.G. da Silva et al. Biophysical Chemistry 238 (2018) 22–29 evidences that the condensation of the lipid monolayer caused by the nanoparticles is independent of the chemical nature of the lipid since we employed not only zwitterionic and negative charged lipids (DPPC and DPPG respectively), but also positively charged (DODAB), fluidizer (DOPC) and condenser (cholesterol) lipids. This noticeably indicates that the driving action of these PVPcaped AuNPs on the lipid monolayer is stabilizing the film through interactions with its polar head by means of electrostatic interactions (coulombic or van der Waals interactions). However, when the lipid monolayer has glycosidic compounds in- corporated (LPS or GP), the effect alters, since expansion at low surface pressures is found. The capping agent also must play an important role in such interactions that, as demonstrated in the literature with AgNPs with similar in size but coated with different molecules interacting in a different way with lipid Langmuir monolayers taken as model bio- membrane [58]. It is evident therefore that that the correlation of the interaction of AuNPs with real cellular membranes may not be straightforward and the dependence on different parameters such as capping of nano- particles, type of monolayer, degree of hydrophilicity, size of the par- ticles, and effects on the viscoelastic properties may be taken into ac- count. These results highlight therefore the importance of understanding the interactions between metallic nanoparticles and components of bacterial and mammalian membranes at the molecular level, which may be important to assess potential longterm tox- icological and antitumorigenic effects. 4. Conclusions Our results showed that AuNPs stabilized with PVP affect the phy- sicochemical properties of Langmuir monolayers of selected lipids. They condensed pure lipid monolayers at the airwater interface due to electrostatic interactions with the polar heads groups of the lipids, but distinctive effects on the apolar groups were also observed since the peaks in PMIRRAS for the methylene stretches are shifted to higher energies for DPPG and not for DPPC. In contrast, monolayers composed with mixture of lipids and glycosidic compounds expanded upon AuNPs Fig. 9. PMIRRAS spectra for monolayers of pure DPPG-LPS without and with incorporation. Comparing with AgNPs prepared in similar conditions AuNPs in the aqueous subphase (as indicated on the charts). [40], similarities and differences can be highlighted. Whereas both kinds of nanoparticles may condense pure lipid monolayers, suggesting in uenced the surface pressurearea isotherms, but altered the nuclea- the inability of the nanoparticles to penetrate them, mixtures of lipidsfl tion, growth, and morphology of the condensed domains in monolayers with glycosidic compounds present a divergent expansion. However, of DPPC [54]. Torrano et al. incorporated AuNPs in DPPC and DPPG clear differences when the effects from each nanoparticle are compared monolayers using both negatively and positively charged nanoparticles could be observed. These differences include degree of condensation of either synthesized in aqueous solution by reduction of gold (III) in the zwitterionic monolayers, and different alteration patterns in the vi- presence of citrate anions (Cit), or prepared by surface modi cation of brational spectra, evidencing alteration not only in the viscoelasticfi Au(Cit)NPs with a cationic polyelectrolyte [56]. They showed that properties of the film, but also in the structural organization for the these nanoparticles a ects more signi cantly DPPG monolayers than lipid monolayers. Such differences can be attributed not only to theff fi DPPC ones, with the e ects being larger for the positively charged chemical nature of Ag and AuNPs, but also to their different averageff nanoparticles. They claim that while the more signi cant e ect for sizes.fi ff positively charged NPs is consistent with the higher toxicity in cellular We therefore believe that these finds may help understand how membranes (which generally have negatively charged membranes), AuPNs nanoparticles affect lipid interfaces that mimic cellular mem- and the presence of smaller e ects from the negative ones indicates that branes, being therefore correlated with their bioactivity in live systemsff the correlation may not be straightforward and the e ect of counter and also as nanotools for other biological applications.ff ions may have to be taken into account. Also, cysteine or glutathione coated spherical and rod shaped Au NPs [33] as well dodeca- Acknowledgements nethiolcapped gold nanoparticles (AuNPs) with an average core dia- meter of 6 nm [57] are shown to expand DPPC monolayers and alters its We thank FAPESP (2015/102530) and CNPq (projects 400896/ compressional properties. Herein, we employed a hydrophilic system 20168 and 442087/20144) for the research grants. Rafael Cruz receives formed by PVPAuNPs, and oppositely to what was observed by most a scholarship from FAPESP (2015/095863). part of the other AuNPs, the nanoparticles did not expand any pure lipid monolayers. This present work shows results related do DPPC and References DPPG monolayers, but we obtained similar results for several other li- pids chosen (results not shown), namely DODAB (dimethyldioctadecy- [1] A.E. Nel, D. Velegol Mädler, T. Xia, E.M.V. Hoek, P. Somasundaran, F. Klaessig,F. Castranova, T. Mike, Understanding biophysicochemical interactions at the na- lammonium bromide), cholesterol, DOPC (dioleoylglyceropho- nobio interface, Nat. Mater. 8 (2009) 543–547. sphocholine), and mixtures of DPPC/cholesterol and DPPC/DOPC. This [2] M. Sureshkumar, Y.D. Siswanto, K.C. ee, magnetic antimicrobial nanocomposite 28 123 R.L.C.G. da Silva et al. Biophysical Chemistry 238 (2018) 22–29 based on bacterial cellulose and silver nanoparticles, J. Mater. Chem. 20 (2010) [31] D. Pissuwan, S.M. Valenzuela, M.B. Cortie, Trends Biotechnol. 24 (2006) 62. 6948–6955. [32] A.A. Torrano, A.S. Pereira, O.N. Oliveira Jr., A. BarrosTimmons, Probing the in- [3] P. Chagas, A.C. da Silva, E.C. Passamani, J.D. Ardisson, L.C.A. de Oliveira, teraction of oppositely charged gold nanoparticles with DPPG and DPPC Langmuir J.D. Fabris, R.M. Paniago, D.S. Monteiro, M.C. Pereira, δFeOOH: a super- monolayers as cell membrane models, Colloid Surface B. 108 (2013) 120–126. paramagnetic material for controlled heat release under AC magnetic field, J. [33] N. Ábrahám, E. Csapó, G. Bohus, I. Dékány, Interaction of biofunctionalized gold Nanopart. Res. 15 (2013) 1544. nanoparticles with model phospholipid membranes, Colloid Polym. Sci. 292 (2014) [4] A. Nel, T. Xia, L. Madler, N. Li, Toxic potential of materials at the nanolevel, Science 2715–2725. 311 (2006) 622–627. [34] A. Mogilevsky, R. Jelinek, Gold nanoparticle selfassembly in twocomponent lipid [5] T.M. Nobre, F.J. Pavinatto, L. Caseli, A.B. Timmons, P. DynarowiczŁatka, Langmuir monolayers, Langmuir 27 (2011) 1260–1268. O.N. Oliveira Jr., Interactions of bioactive molecules & nanomaterials with [35] J.F. Gomes, A.C. Garcia, E.D.B. Ferreira, C. Pires, V.L. Oliveira, G. TremiliosiFilho, Langmuir monolayers as cell membrane models, Thin Solid Films 593 (2015) L.H.S. Gasparotto, New insight into the formation mechanism of the ag, Au and 158–188. AgAu nanoparticles in aqueous alkaline media: alkoxides from alcohols, aldehydes [6] R.K. Harishchandra, M. Saleem, H.J. Galla, Nanoparticle interaction with model and ketones as the universal reducing agent, Phys Chem Chem Phys 17 (2015) lung surfactant monolayers, J. R. Soc. Interface. 7 (2010) S15–S16. 21683–21693. [7] A.K. Sachan, H.J. Galla, Understanding the mutual impact of interaction between [36] R.F.de Araújo, A.A. de Araújo, J.P. Pessoa, F.P. Freire Neto, G.R. da Silva, hydrophobic nanoparticles and pulmonary surfactant monolayer, Small 10 (2014) A.L.C.S. Leitão Oliveira, T.G. de Carvalho, H.F.O. Silva, M. Eugênio, C. Sant'anna, 1069–1075. L.H.S. Gasparotto, Antiinflammatory, analgesic and antitumor properties of gold [8] E. Santini, E. Guzmán, F. Ravera, M. Ferrari, L. Liggieri, Properties and structure of nanoparticles, Pharmacol. Rep. 69 (2017) 119–129. interfacial layers formed by hydrophilic silica dispersions and palmitic acid, Phys. [37] R.N.M. Weijer, Lipid composition of cell membranes and its relevance in type 2 Chem. Chem. Phys. 14 (2012) 607. diabetes mellitus, Curr. Diabetes Rev. 8 (2012) 390–400. [9] J.Y. Wang, R. Liu, Y.M. Su, W. Li, Embedded arbon anotubes nanoparticles in [38] P.R. Beining, E. Huff, B. Prescott, T.S. Theodore, Characterization of the lipids of plasma membrane induce cellular calcium outflow imbalancing, J. Nanosci. mesosomal vesicles and plasma membranes from Staphylococcus aureus, J. Nanotecnol. 14 (2014) 4058–4065. Bacteriol. 121 (1975) 137–143. [10] T.J. Matshaya, A.E. Lanterna, A.M. Granados, R.W.M. Krause, B. Maggio, R.V. Vico, [39] S. BeloribiDjefaflia, S. Vasseur, F. Guillaumond, Lipid metabolic reprogramming in Distinctive interactions of oleic acid covered magnetic nanoparticles with saturated cancer cells citation, Oncogenesis 5 (2016) e189. and unsaturated phospholipids in Langmuir monolayers, Langmuir 30 (2014) 5888. [40] R.L.C.G. da Silva, H.F.O. da Silva, L.H.S. Gasparotto, L. Caseli, How the interaction [11] J. Cancino, T.M. Nobre, O.N. Oliveira Jr., S.A.S. Machado, V. Zucolotto, A new of PVPstabilized ag nanoparticles with models of cellular membranes at the air- strategy to investigate the toxicity of nanomaterials using Langmuir monolayers as water interface is modulated by the monolayer composition, J. Colloid Interf. Sci. membrane models, Nanotoxicology 7 (2013) 61–70. 512 (2018) 792–800. [12] A. Chakraborty, N.J. Mucci, M.L. Tan, A. Steckley, T. Zhang, M.L. Forrest, P. Dhar, [41] D. Astruc, M.C. Daniel, J. Ruiz, Dendrimers and gold nanoparticles as exoreceptors Phospholipid composition modulates carbon nanodiamondinduced alterations in sensing biologically important anions, Chem. Commun. (Camb) 23 (2004) phospholipid domain formation, Langmuir 31 (2015) 5093–5104. 2637–2649. [13] Y. Matsumura, K. Yoshikata, S.I. Kunisaki, T. Tsuchido, Mode of bactericidal action [42] M.C. Daniel, D. Astruc, Gold nanoparticles: assembly, supramolecular chemistry, of silver zeolite and its comparison with that of silver nitrate, Appl. Environ. quantumsizerelated properties, and applications toward biology, catalysis, and Microbiol. 69 (2003) 4278–4281. nanotechnology, Chem. Rev. 104 (2004) 293–346. [14] V. Alt, T. Bechert, P. Steinrucke, M. Wagener, P. Seidel, E. Dingeldein, R. Schnettler, [43] M. Moskovits, I. SrnovaSloufova, B. Vlckova, Bimetallic agau nanoparticles: ex- An in vitro assessment of the antibacterial properties and cytotoxicity of nano- tracting meaningful optical constants from the surfaceplasmon extinction spectrum, particulate silver bone cement, Biomaterials 25 (2004) 4383–4391. J. Chem. Phys. 116 (2002) 10435–10446. [15] C. Baker, A. Pradhan, L. Pakstis, D.J. Pochan, S.I. Shah, Synthesis and antibacterial [44] H. DvorySobol, E. Sagiv, D. Kazanov, A. BenZe'ev, N. Arber, Targeting the active properties of silver nanoparticles, J. Nanosci. Nanotechnol. 5 (2005) 244–249. betacatenin pathway to treat cancer cells, Mol. Cancer Ther. 5 (2006) 2861–2871. [16] J.R. Morones, J.L. Elechiguerra, A. Camacho, K. Holt, J.B. Kouri, J.T. Ramirez, [45] K.J. Klopfer, T.K. Vanderlick, Isotherms of dipalmitoylphosphatidylcholine (DPPC) M.J. Yacaman, The bactericidal effect of silver nanoparticles, Nanotechnology 16 monolayers:features revealed and features obscured, J. Colloid Interf. Sci. 182 (2005) 2346–2353. (1996) 220–229. [17] R.J. White, S. Fumarola, J. Denyer, Interim advice on silver dressings in paediatric [46] Blume, A comparative study of the phase transitions of phospholipid bilayers and wound and skin care, Br. J. Nurs. 20 (2011) S11. monolayers, Biochim. Biophys. Acta 557 (1979) 32–34. [18] N. Duran, P.D. Marcato, R. Conti, L.O. Alves, F.T.M. Costa, M. Brocchi, Potential use [47] R.G. Snyder, S.L. Hsu, S. Krim, Vibrational specta in the CH stretching region and of silver nanoparticles on pathogenic bacteria, their toxicity and possible mechan- the structure of the polymethylene chain, Spectrochim. Acta 34 (1978) 395–406. isms of action, J. Braz. Chem. Soc. 21 (2010) 949–959. [48] L.S. Eberlin, M. Gabay, A.C. Fan, A.M. Gouw, R.J. Tibshirani, D.W. Felsher, [19] P. Velmurugan, S.M. Lee, M. Cho, J.H. Park, S.K. Seo, H. Myung, K.S. Bang, B.T. Oh, R.N. Zare, Alteration of the lipid profile in lymphomas induced by MYC over- Antibacterial activity of silver nanoparticlecoated fabric and leather against odor expression, Proc. Natl. Acad. Sci. U. S. A. 111 (2014) 10450–10455. and skin infection causing bacteria, Appl. Microbiol. Biotechnol. 98 (2014) [49] R.H. Perry, D.I. Bellovin, E.H. Shroff, A.I. Ismail, T. Zabuawala, D.W. Felsher, 8179–8189. R.N. Zare, Characterization of MYCinduced tumorigenesis by in situ lipid profiling, [20] S. Sivolella, E. Stellini, G. Brunello, C. Gardin, L. Ferroni, E. Bressan, B. Zavan, Anal. Chem. 85 (2013) 4259–4262. Silver nanoparticles in alveolar bone surgery devices, J. Nanomat. 2012 (2012) [50] E.H. Shroff, L.S. Eberlin, V.M. Dang, A.M. Gouw, M. Gabay, S.J. Adam, 975842. D.I. Bellovin, P.T. Tran, W.M. Philbrick, A. GarciaOcana, S.C. Casey, Y. Li, [21] A.G. Rada, H. Abbasib, M.H. Afzalib, Gold nanoparticles: synthesising, character- C.V. Dang, R.N. Zare, D.W. Felsher, MYC oncogene overexpression drives renal cell izing and reviewing novel application in recent years, Phys. Procedia 22 (2011) carcinoma in a mouse model through glutamine metabolism, Proc. Natl. Acad. Sci. 203–208. USA 112 (2015) 6539–6544. [22] A. Gharatape, S. Davaran, R. Salehi, H. Hamishehkar, H Engineered gold nano- [51] M.A. Kiebish, X. Han, H. Cheng, J.H. Chuang, T.N. Seyfried, Cardiolipin and elec- particles for photothermal cancer therapy and bacteria killing, RSC Adv 6 (2016) tron transport chain abnormalities in mouse brain tumor mitochondria: lipidomic 111482–111516. evidence supp Orting the Warburg theory of cancer, J. Lipid Res. 49 (2008) [23] H. Gang, P. Ghosh, V.M. Rotello, Multifunctional gold nanoparticles for drug de- 2545–2556. livery. Bioapplications of nanoparticles, Adv. Exp. Medicine Biol. 620 (2007) [52] D. Vollhardt, V.B. Fainerman, S. Siegel, Thermodynamic and textural character- 48–56. ization of DPPG phospholipid monolayers, J. Phys. Chem. B 104 (2000) 4115–4121. [24] K. Urbańska, B. Pająk, A. Orzechowski, J. Sokołowska, M. Grodzik, E. Sawosz, [53] P.M.P. Lins, V.S. Marangoni, T.M. Uehara, P.B. Miranda, V. Zucolotto, M. Szmidt, P. Sysa, The effect of silver nanoparticles (AgNPs) on proliferation and J. CancinoBernardi, Differences in the aspect ratio of old nanorods that induce apoptosis of in ovo cultured glioblastoma multiforme (GBM) cells, Nanoscale Res. defects in cell membrane models, Langmuir 33 (2017) 14286–14294. Lett. 10 (2015) 98. [54] K.M. Mayya, A. Gole, N. Jain, S. Phadtare, D. Langevin, M. Sastry, Timedependent [25] D.A. Giljohann, D.S. Seferos, A.E. Prigodich, P.C. Patel, C.A. Mirkin, Gene regula- complexation of cysteinecapped gold nanoparticles with octadecylamine Langmuir tion with polyvalent siRNA−nanoparticle conjugates, J. Am. Chem. Soc. 131 onolayers at the irater interface, Langmuir 19 (2003) 9147–9154. (2009) 2072–2073. [55] S. Tatur, A. Badia, Influence of hydrophobic alkylated gold nanoparticles on the [26] C.M. Cobley, J. Chen, E.C. Cho, L.V. Wang, Y. Xia gold nanostructures: engineering phase behavior of monolayers of DPPC and clinical lung surfactant, Langmuir 28 their plasmonic properties for biomedical applications, Chem. Soc. Rev. 35 (2006) (2012) 628–639. 1084. [56] A.A. Torranoa, Â.S. Pereira, O.N. Oliveira Jr., A. BarrosTimmons, Probing the in- [27] Q. Wei, A. Wei, V. Weissig, G.G.M. D'souza (Eds.), Organellespecific Pharmaceutical teraction of oppositely charged gold nanoparticles with DPPG and DPPC Langmuir Nanotechnology, John Wiley & Sons, New Jersey, 2010, p. 26. monolayers as cell membrane models, Colloid Surf. B 108 (2013) 120–126. [28] N.T.L. Thanh, L.A.W. Green, Functionalisation of nanoparticles for biomedical ap- [57] S.S. You, C.T.R. Heffern, Y. Dai, M. Meron, J.M. Henderson, W. Bu, W. Xie, K. Yee, plications, NanoToday 5 (2010) 213–230. C. Lee, B. Lin, Liquid surface Xray studies of gold nanoparticle−phospholipid films [29] E. Hutter, J.H. Fendler, Exploitation of localized surface plasmon resonance, Adv. at the air/water interface, J. Phys. Chem. B 120 (2016) 9132–9141. Mater. 16 (2004) 1685–1706. [58] J.V.M. Girón, R.V. Vico, B. Maggio, E. Zelaya, A. Rubert, G. Benítez, P. Carro, [30] X. Huang, P.K. Jain, I.H. ElSayed, M.A. ElSayed, Gold nanoparticles: interesting R.C. Salvarezza, M.E. Vela, Role of the capping agent in the interaction of hydro- optical properties and recent applications in cancer diagnostics and therapy, philic ag nanoparticles with DMPC as a model biomembrane, Environ. SciNano 3 NanomedicineUK 5 (2007) 681–693. (2016) 462–467. 29 124 Apêndice E Apoptosis in human liver carcinoma caused by gold nanoparticles in combination with carvedilol is mediated via modulation of MAPK/Akt/mTOR pathway and EGFR/FAAD proteins. De Araújo RF Jr, Pessoa JB, Cruz LJ, Chan AB, De Castro Miguel E, Cavalcante RS, Brito GAC, Silva HFO, Gasparotto LHS, Guedes PMM, Araújo AA. International Journal of Oncology, 2018, Vol. 52, 1, 189-200 Contribuição:  Realizei a síntese e purificação das NanoAu.  Realizei a caracterização das NanoAu.  Tratei os dados e preparei as figuras relacionadas às etapas que realizei ativamente que foram incluídas no manuscrito.  Participei de discussões antes da escrita do manuscrito.  Auxiliei no feedback aos revisores. ________________________ __________________________ Heloiza Fernanda O. da Silva Athayde Prof. Luiz Henrique da S. Gasparotto 125 INTERNATIONAL JOURNAL OF ONCOLOGY 52: 189-200, 2018 Apoptosis in human liver carcinoma caused by gold nanoparticles in combination with carvedilol is mediated via modulation of MAPK/Akt/mTOR pathway and EGFR/FAAD proteins RAIMUNDO F. DE ARAúJO Jr1-3*, JONAS B. PESSOA2*, LUIS J. CRUz4, ALAN B. CHAN5, EMíLIO DE CASTRO MIGUEL6, RôMULO S. CAvALCANTE3, GERLY ANNE C. BRITO7, HELOIzA FERNADA O. SILvA8, LUIz H.S. GASPAROTTO8, PAULO M.M. GUEDES9 and AURIGENA A. ARAúJO10 1Department of Morphology, 2Post Graduation Programme in Structural and Functional Biology, 3Post Graduation Programme in Health Science, Federal University of Rio Grande do Norte, Natal 59072-970, RN, Brazil; 4Translational Nanobiomaterials and Imaging, Department of Radiology, Leiden University Medical Center; 5Percuros B.v., 2333 CL Leiden, The Netherlands; 6Department of Physical/Analytical Center/UFC; 7Department of Morphology/Postgraduate Program in Morphology/UFC, Fortaleza, CE; 8Group of Biological Chemistry and Chemometrics, Institute of Chemistry; 9Department of Parasitology and Microbiology and Post Graduation Program in Parasitary Biology; 10Department of Biophysics and Pharmacology, Post Graduation Programme in Public Health, Post Graduation Programme in Pharmaceutical Science, Federal University of Rio Grande do Norte, Natal 59072-970, RN, Brazil Received June 29, 2017; Accepted September 21, 2017 DOI: 10.3892/ijo.2017.4179 Abstract. In cancers, apoptosis signaling pathways and cell real-time PCR; and western blot analysis for MAPK/ERK, survival and growth pathways responsible for resistance Akt and mTOR. Oxidative stress evaluation was performed to conventional treatments, such as Pi3K/Akt/mTOR and by reduced glutathione (GSH) and malondialdehyde (MDA) mitogen-activated protein kinase (MAPK) become dysregu- levels. Intracellular GNPs targets were identified by transmis- lated. Recently, alternative treatments to promote tumor cell sion electron microscopy. After exposure to a combination of death have become important. The present study reports on GNPs (6.25 µg/ml) and carvedilol (3 µM), death as promoted the antitumor and cytoprotective action of gold nanoparticles by apoptosis was detected using flow cytometry, for expres- (GNPs) and carvedilol in combination and in isolated appli- sion of pro-apoptotic proteins FADD, caspase-3, caspase-8 and cation. Apoptosis was analyzed by FITC/propidium iodide sub-regulation of anti-apoptotic MAPK/ERK, Akt, mTOR, staining flow cytometry; caspase-3, caspase-8, Bcl-2 and EGFR and MDR1 resistance. Non-tumor cell cytoprotection MAPK/ERK activity by immunofluorescence microscopy; with GSH elevation and MDA reduction levels was detected. gene expression of proteins related to cell death as Akt, GNPs were identified within the cell near to the nucleus mTOR, EGFR, MDR1, survivin, FADD and Apaf, by the when combined with carvedilol. The combination of GNP and carvedilol promoted downregulation of anti-apoptotic and drug resistance genes, over-regulation of pro-apoptotic proteins in tumor cells, as well as cytoprotection of non-tumor Correspondence to: Professor Raimundo Fernandes de Araújo Jr, cells with reduction of apoptosis and oxidative stress. Department of Morphology, Federal University of Rio Grande do Norte, Campus Universitário Lagoa Nova, CEP 59078-970, Caixa Introduction postal 1524, Natal, RN, Brasil E-mail: araujojr@cb.ufrn.br Cancer, a serious public health problem worldwide, is respon- Abbreviations: sible for countless deaths each year and is currently considered GNPs, gold nanoparticles; Carv, carvedilol; EGFR, epidermal growth factor receptor; Erk, extracellular the second leading cause of death on the planet (1-3). In addi- signal-regulated kinases; FADD, fas-associated protein with death tion, various malignant tumor types do not have effective domain; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; treatment (4-8), due to the ability of tumor cells to evade MDA, malondialdehyde; MAPK, mitogen activated protein kinases; death, by presenting changes in apoptosis pathway protein MDR1, multidrug resistance genes-1; mTOR, mechanistic target of levels (9-11). Changes in other cellular pathway proteins rapamycin; Pi3K, phosphatidylinositide 3-kinases such as Pi3k/Akt/mTOR and MAPK/ERK, which are highly dysregulated in malignant tumors, also corroborate the ability Key words: apoptosis, cancer, gold nanoparticles, carvedilol, of malignant cells to evade apoptosis death, contributing to combination chemotherapy resistance (12-14). The expression of multi- drug resistance genes, such as MDR1, has been implicated 126 190 ARAúJO Jr et al: APOPTOSIS CAUSED BY GNPs IN COMBINATION wITH Carv vIA MAPK/Akt/mTOR PATHwAY as the main cause of chemoresistance (15,16). Furthermore, determined at 24 and 48 h for HepG2 (1x105 cells) at different conventional chemotherapeutic treatments are known for their concentrations of GNPs (1-50 µg/ml, aqueous suspension) and side-effects on non-tumor cells, such as the strong oxidative carvedilol [1.5-300 µM, dissolved in dimethyl sulfoxide (DMSO) stress (17-19). 1%]. The cells were placed into 6-well plates. Briefly, cell aliquots Moreover, there is a need for alternative treatments that were mixed with the same volume 0.5% (w/v) trypan blue and promote apoptosis in tumor cells and yet do not negatively affect incubated at room temperature for 5 min. The number of viable normal cells. In this regard, GNPs have been highlighted in the cells was calculated using a hemocytometer. literature as promising agents with large pooling surfaces for various drugs (20), which concentrate on tumor tissues (21,22), Annexin V and propidium iodide staining. The apoptotic assay are resistant to corrosion and present low toxicity to the was conducted according to Araújo Jr et al (54). HepG2 and biological system (23-25). In addition, various studies reported HEK-293 were plated in 6-well plates (2x105 cells/well) with an effective antitumor action with non toxicity to normal 2 ml medium/well. After 24 h, concentrations of GNPs (3 and cells (26-29). The antihypertensive carvedilol is often used as 6.25 µg/ml), cisplatin (15 µg/ml) and carvedilol (1.5 and 3 µM) a non-selective inhibitor of adrenergic receptors (30), is known were added (24 and 48 h), respectively. In parallel, control cells for its cardiovascular and antioxidant benefits (31-34) and has were maintained in culture medium without GNPs, carvedilol recently presented good antitumor activity, such as growth or cisplatin. For observation of combined action, the cells were inhibition of neuroblastoma cell lines (35) and rat glioma cell treated with GNPs (3 and 6.25 µg/ml) and at 24 h treated with line (36), suppressing migration and invasion of malignant carvedilol (1.5 and 3 µM). After another 24 h, they were analyzed. breast cells (37), preventing carcinogenesis in rat epidermal The cells were then assayed using the Annexin v-FITC/PI lineages (38) and promoting apoptosis in tumoral hepatic and apoptosis detection kit I (BD Biosciences, San Diego, CA, USA). oral cell linages (39,40). The discovery of new substances and Annexin v-FITC and propidium iodide (PI) were added to the combinations is fundamental to the process of establishing new cellular suspension according to the manufacturer's instructions. treatments against cancer (41-43). Combining substances has A total of 1x106 cells from each sample was then analyzed by proven to be extremely effective due to the lower doses used, FACSCalibur cytometer (BD Biosciences, Franklin Lakes, NJ, the decreased adverse effects, and the possibility of acting on USA), and FlowJo software (BD Biosciences). Annexin v-FITC- different signaling pathways (44-50). In addition, the discovery positive/PI-negative cells were identified as cells in the early of substances that promote inhibition of dysregulated pathways stages of apoptosis, while Annexin v-FITC-positive/PI-positive such as Pi3k/Akt/mTOR and MAPK/Erk would positively cells were identified as cells in the late stages of apoptosis, or as modulate apoptosis in tumor cells (12,51,52). In the present cells undergoing necrosis. study, we investigated the effects of combined carvedilol and GNPs action on both tumor and non-tumor cells. Glutathione (GSH) levels. Antioxidant GSH levels in cell lines were measured [adapted from Rahman et al (55) and Materials and methods Costa et al (56)]. HepG2 and Hek-293 were plated in 6-well plates (2x105 cells/well) with 2 ml medium/well. After 24 h, Reagents. The reagents were purchased as indicated: Dulbecco's GNPs (6.25 µg/ml) was added and after 24 h, 3 µM carvedilol. modified Eagle's medium (DMEM; Life Technologies, Grand A homogenate of cells (100 µl of cell in 500 µl EDTA 0.02 M) Island, NY, USA); 10% (v/v) heat-inactivated fetal bovine were added to 320 µl of distilled water and 80 µl of 50% trichlo- serum (FBS; Cultilab Materiais para Cultura de Células Ltda, roacetic acid (TCA). Samples were centrifuged at 3,000 rpm Campinas, Brazil); trypsin/ EDTA (ethylenediaminetetraacetic for 15 min at 4˚C. The supernatant (100 µl) was added to 200 µl acid) (Gibco-BRL, Life Technologies, Grand Island, NY, USA); of 0.4 M Tris buffer at pH 8.9 and 20 µl of 0.01 M 5,5'-dithiobis cisplatin (citoplax, 50 mg; Bergamo, Taboão da Serra, Brazil); (2-nitrobenzoic acid) (DTNB). The absorbance of each sample gold nanoparticles GNPs (Institute of Chemical, UFRN, was measured at 420 nm, in a spectrophotometric/microplate Natal, Brazil); carvedilol (Farmafórmula, Natal, Brazil); gold reader Polaris and the results were reported as units of GSH (III) chloride (30% wt. in HCl), sodium hydroxide, glycerol, per milligram. and polyvinylpyrrolidone (PvP, molecular weight, 10,000 Da) were products from Sigma-Aldrich. The synthesis and charac- Malondialdehyde levels. Malondialdehyde (MDA) is an end terization of GNPs was the described by de Araújo et al (53). product of lipid peroxidation. To quantify the increase in GNPs, carvedilol and cisplatin solutions were filtered using a free radicals in non-cancer (HEK-293) and cancer (HepG2) 0.22-mm minipore membrane. cells, MDA content was measured via the assay described by Esterbauer and Cheeseman (57). Cell samples were suspended Cell culture. The human cell lines hepatocellular carcinoma in buffer; Tris HCl 1:5 (w/v), and minced with scissors for (HepG2) and human non-cancerous renal cell line (HEK-293) 15 sec on an ice-cold plate. The resulting suspension was were purchased from the Culture Collection of the Federal homogenized for 2 min with an automatic Potter homogenizer University of Rio de Janeiro (RJCB Collection, Rio de Janeiro, and centrifuged at 11,000 rpm at 4˚C for 10 min. The super- Brazil). HepG2 and Hek-293 cells were maintained in DMEM natants were assayed in order to determine the MDA content. supplemented with 10% (v/v) heat-inactivated FBS. The absorbance of each sample was measured at 586 nm. The results are expressed as nanomoles of MDA per cell. Cell viability. In order to determine GNP and carvedilol doses to promote and maintain low inhibition in cancer cells, cell viability Immunofluorescence, Bcl-2, MAPK/ERK, caspase-3 and was determined by trypan blue exclusion assay. The viability was caspase-8 activity. HepG2 cells were plated on glass 127 INTERNATIONAL JOURNAL OF ONCOLOGY 52: 189-200, 2018 191 coverslips in 24-well plates (5x104 cells/well). After 24 h, they GCT GGA AAC CT-3' and reverse, 5'-AGC CCG GAT GAT were treated with the GNPs (6.25 µg/ml), cisplatin (15 µg/ml) ACA AAC AG-3', annealing primer temperature, 55.6˚C); and carvedilol (3 µM) for 48 h. For combined action, we used MDR1 (forward, 5'-GTG TGG TGA GTC AGG AAC CTG GNPs (6.25 µg/ml) + carvedilol (3 µM). The cells were then TAT-3' and reverse, 5'-TCT CAA TCT CAT CCA TGG TGA washed, and fixed with paraformaldehyde, permeabilized by CA-3', annealing primer temperature, 57˚C); FADD (forward, Triton-X, and incubated with anti-Bcl-2 mouse polyclonal 5'-TCT CCA ATC TTT CCC CAC AT-3' and reverse, 5'-GAG antibody, rabbit polyclonal anti-caspase-3 antibody (Abcam, CTG CTC GCC TCC CT-3', annealing primer temperature, San Francisco, CA, USA), rabbit anti-caspase-8 monoclonal 58.7˚C); and Apaf-1 (forward, 5'-CCT CTC ATT TGC TGA antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), TGT CG-3' and reverse, 5'-TCA CTG CAG ATT TTC ACC and monoclonal mouse antibody anti-MAPK/ERK (Invitrogen, AGA-3', annealing primer temperature, 56.9˚C). The experi- Carlsbad, CA, USA) diluted 1:500 in phosphate-buffered ments were performed in triplicate. The standard PCR saline (PBS) containing bovine serum albumin (BSA; 5%; Life conditions were as follow: 50˚C for 2 min and 95˚C for 10 min, Technologies do Brasil Ltda, São Paulo, Brazil) for 1 h at RT followed by 40 of 30-sec cycles at 94˚C, a variable annealing in a humid atmosphere. The primary antibody was detected primer temperature for 30 sec and at 72˚C for 1 min. Mean Ct with Alexa Fluor 488 goat anti-rabbit or anti-mouse secondary values were used to calculate the relative expression levels of antibody (Abcam), and 4',6-diamidino-2-phenylindole (Life the target genes for the experimental groups as relative to Technologies do Brasil Ltda) was used for nuclear staining. those in the negative control group; expression data were The immunostained coverslips were examined under Axio normalized relative to the housekeeping gene GAPDH using Observer z.1, inverted fluorescence and brightfield. the 2-ΔΔCt formula. Fluorescent images were obtained on a Carl zeiss Laser Scanning Microscope (LSM 710, 20X objectives; Carl zeiss, Western blot analysis. Cells (HepG2) were lysed in buffer Oberkochen, Germany). Negative controls and treated groups [Tris-HCl 50 mM, NaCl 150 mM, Triton X-100 1%, EDTA were included in each batch of samples. Cell reactivity in all 1 mM, sodium pyrophosphate 20 mM, pH 7.4 containing a groups (negative, GNPs, carvedilol, GNPs + carvedilol and protease inhibitors cocktail (Roche), NaF (10 mM), DTT cisplatin) was assessed by computerized densitometric analysis (1 mM), PMSF (0.1 mM) and sodium vanadate (1 mM)] on of the captured digital images with the aforementioned immu- ice. To confirm equal loadings, total protein concentration was nofluorescence microscope. Average densitometric values determined using the Bradford method (Bio-Rad Laboratories, were calculated in ImageJ software (http://rsb.info.nih.gov/ij/). Hercules, CA, USA). Proteins were resolved using SDS-PAGE Contrast index measurements were obtained from the formula and then transferred to a polyvinylidene diflouride (PVDF) [(selected area x 100)/total area] after removal of background membrane. Non-specific binding sites on the membrane were in regions of interest (three samples per group). blocked using 5% non-fat skimmed milk and incubated with the primary antibody anti-Akt (1:500; Abcam), anti-mTOR Real-time PCR. HepG2 cells were plated in 6-well plates (1:500; Abcam), and MAPK/ERK (1:200; Abcam), overnight (2x105 cells/well) with 2 ml medium/well. After 24 h, concen- at 4˚C, followed by incubation with the appropriate secondary trations of gold nanoparticles (6.25 µg/ml), cisplatin (15 µg/ml) antibodies: Akt α-rat peroxidase 1:1,000; mTOR-rabbit and carvedilol (3 µM) were added for 48 h. For combined peroxidase 1:2,000 and MAPK/ERK α-rat peroxidase 1:1,000. action, GNPs (6.25 µg/ml) + (3 µM) carvedilol was used. The Proteins were detected using the ECL Plus kit (Perkin-Elmer, cells were collected with cell scrapers and total RNA was San Jose, CA, USA). isolated from cells using TRIzol reagent. The total RNA was extracted from cell samples using RNeasy Mini kit (Qiagen, Transmission electronic microscopy (TEM). Cells (HepG2) Tokyo, Japan) from QIAcube following the manufacturer's at a density of 3x105 were plated into (GNPs sensitized and guidelines. The total RNA extracted underwent reverse tran- treated) 6-well plates, and after 48 h were collected with scriptase activity using the High capacity RNA-to-cDNA kit trypsin, centrifuged at 1,500 rpm for 5 min, and washed (Applied Biosystems, Ltd., Tokyo, Japan). Real-time quantita- with PBS. The cell pellet was fixed with 2.5% glutaralde- tive PCR analyses of EGFR, Akt, mTOR, survivin, MDR-1, hyde + paraformaldehyde 4% + sodium cacodylate buffer FADD, Apaf-1 and GAPDH mRNAs were performed with (0.1 M) for 4 h at 4-8˚C. Afterwards, the samples were washed SYBR-Green Mix in the Applied Biosystems® 7500 FAST in 0.05 M sodium cacodylate (3x30 min), post-fixed in 1% system (Applied Biosystems, Foster City, CA, USA), according OsO4 + 1% potassium ferrocyanide (2 h), washed again (3x) to a standard protocol with the following primers: GAPDH with 0.05 M sodium cacodylate (3x30 min), and then serially (forward, 5'-AAC TTT GGC ATC GTG GAA GG-3' and dehydrated in ethanol 50, 70, 90 and 100, for 30 min each. reverse, 5'-GTG GAT GCA GGG ATG ATG TTC-3', annealing Polymerization of the resin was performed at 60˚C for 48 h. primer temperature, 60˚C); EGFR (forward, 5'-TGA TAG Finally, ultramicrotomy was performed followed by staining ACG CAG ATA GTC GCC-3' and reverse, 5'-TCA GGG CAC (uranyl acetate 1% + 1% lead citrate 1 h), and electron micro- GGT AGA AGT TG-3', annealing primer temperature, 56.6˚C); scope visualization (Tescan transmission, vega 3 model). Akt (forward, 5'-ACG GCA TGG ACT TTA CCA AG-3' and reverse, 5'-GCG GGT GAA AGA CAG GAA TA-3', annealing Statistical analysis. All experiments were performed in tripli- primer temperature, 55˚C); mTOR (forward, 5'-TTG AGG cate, and the significant differences between the groups were TTG CTA TGA CCA GAG AGA A-3' and reverse, 5'-TTA calculated using the analysis of variance and the Bonferroni's CCA GAA AGG ACA CCA GCC AAT G-3', annealing primer test, as indicated. A P<0.05 was considered statistically temperature, 58.3˚C); survivin (forward, 5'-TAC AGC TTC significant. 128 192 ARAúJO Jr et al: APOPTOSIS CAUSED BY GNPs IN COMBINATION wITH Carv vIA MAPK/Akt/mTOR PATHwAY Figure 1. Cell viability to determine the cell growth. (A) Effect of different doses of GNPs on hepatic tumor cells (HepG2) at 24 and 48 h. The 12.5 µg/ml dose caused greater cellular growth inhibition at both times. (B) Effect of different doses of carvedilol on hepatic tumor cells (HepG2) at 24 and 48 h. Doses >6.25 µM promoted strong inhibition of cell growth. *P<0.05; ***P<0.001. Figure 2. Flow cytometry to determine the cell death for apoptosis. Effect of different doses of GNPs, carvedilol, and cisplatin on early and late apoptosis in HepG2 cells. (A-F) At 24 h and (G-N) at 48 h. Control (A and G), GNPs 3 µg/ml (B and H), GNPs 6.25 µg/ml (C and I), Carv 1.5 µM (D and J), Carv 3 µM (E and K), cisplatin 15 µg/ml (F and L). GNPs 3 µg/ml + Carv 1.5 µM (M) and GNPs 6.25 µg/ml + Carv 3 µM (N). (O) Early apoptosis and (P) late apoptosis. *P<0.05, **P<0.01 and ***P<0.001. 129 INTERNATIONAL JOURNAL OF ONCOLOGY 52: 189-200, 2018 193 Figure 3. Flow cytometry to determine cell death by apoptosis. Effect of different doses of GNPs, carvedilol, and cisplatin on early and late apoptosis in HEK- 293 cells at 24 h (A-F) and 48 h (G-N). Control (A and G), GNPs 3 µg/ml (B and H), GNPs 6.25 µg/ml (C and I), Carv 1.5 µM (D and J), Carv 3 µM (E and K), cisplatin 15 µg/ml (F and L). GNPs 3 µg/ml + Carv 1.5 µM (M) and GNPs 6,25 µg/ml + Carv 3 µM (N). (O) Total apoptosis. *P<0.05. Results (P<0.001). At 48 h, the 1.5 and 3 µM carvedilol doses main- tained low inhibition (Fig. 1B). The other doses of carvedilol Cell viability. The GNPs doses 1, 3 and 6.25 µg/ml, at promoted greater cellular growth inhibition (P<0.001). DMSO 24 h (Fig. 1A) promoted low inhibition of cellular viability. at 1% was used as the carvedilol vehicle. The other GNPs doses promoted greater cellular growth inhibition, when compared to lower doses. The most signifi- Detection of apoptosis, by flow cytometer. For HepG2 cant was 12.5 µg/ml (P<0.05). At 48 h, GNPs doses of 3 cells, combined treatment (GNP 6.25 µg/ml + carvedilol and 6.25 µg/ml maintained low inhibition similar to that at 3 µM) induced early (P<0.05) and late (P<0.001) apoptosis 24 h (Fig. 1A), and for GNPs doses of 1, 25 and 50 µg/ml, at 48 h (Fig. 2O-P). The isolated dose of carvedilol (3 µM) there was cellular growth when compared to the same doses induced early apoptosis at 48 h (Fig. 2O; P<0.01). GNPs at 24 h. The carvedilol doses that promoted low inhibition of (6.25 µg/ml) induced late apoptosis at 48 h, P<0.05. Doses of cellular viability were 1.5 and 3 µM at 24 h (Fig. 1B). The other GNPs and carvedilol did not induce statistically significant carvedilol doses promoted greater cellular growth inhibition apoptosis in non-tumor cells (Fig. 3). Combined treatment 130 194 ARAúJO Jr et al: APOPTOSIS CAUSED BY GNPs IN COMBINATION wITH Carv vIA MAPK/Akt/mTOR PATHwAY Figure 4. Oxidative stress markers. (A) GSH measurement, important protein in balance of oxidative stress. The combined treatment modulated GSH levels on HEK-293 (*P<0.05; **P<0.01 and ***P=0.0003) and HepG2 (*P<0.05; **P<0.01 and ***P<0.0001). (B) MDA measurement, indirect marker of oxidative stress. After combined treatment with GNPs and Carv, there was a reduction of the statistically significant levels of malondialdehyde in tumor and non-tumoral lines. *P<0.05; **P<0.01. Figure 5. Detection of caspase-3, caspase-8, Bcl-2 and MAPK/ERK. HepG2 cells stained with DAPI (blue), anti-caspase-3, anti-caspase-8, anti-Bcl-2, and anti-MAPK/ERK antibodies (green) (A). Caspase-3 and caspase-8 were detected in all treated groups, yet MAPK/ERK was not. Bcl-2 did not alter expres- sion. Contrast index for caspase-3, *P<0.05 and **P<0.01 (B); caspase-8, ***P<0.001 and #P>0.05 (C); Bcl-2, #P>0.05 and ***P<0.001 (D) and MAPK/ERK, ****P<0.0001 (E). All groups treated with GNPs + Carv showed high immunoreactivity for caspase-3, caspase-8 and low immunoreactivity for MAPK/ERK. 131 INTERNATIONAL JOURNAL OF ONCOLOGY 52: 189-200, 2018 195 Figure 6. Gene and protein expression. Relative expression of genes related to survival and tumor resistance by real-time PCR. (A) FADD and Apaf-1, *P<0.05 and **P<0.01; (B) surviving and MDR1, *P<0.05 and **P<0.01; (C) EGFR, Akt and mTOR, *P<0.05, **P<0.01, ***P<0.001. (D) western blot analysis of proteins related to survival and tumor proliferation. After exposure to the GNPs and carvedilol combination, there was a large decrease in the protein levels of Akt. mTOR and MAPK/ERK have a good reduction. β-actin was used as internal control. Lane 1, Control; lane 2, GNP 6.25 µg/ml + Carv 3 µM. (GNP 6.25 µg/ml + carvedilol 3 µM) promoted cytoprotec- overcoming the control and cisplatin treated groups. This tion (P<0.05; Fig. 3O). when HepG2 and HEK-293 cells same result was observed for the HepG2 tumor cells (Fig. 4B). were treated with 15 µg/ml cisplatin, apoptosis was detected at 24 and 48 h after treatment (Figs. 2F and L and 3F and L, Immunofluorescence of caspase-3, caspase-8, Bcl-2 and respectively). The goal of using a non-tumor cell is to have as MAPK/ERK. After combined treatment with GNPs (6.25 µg/ml) a control a non-tumoral lineage to analyze its behavior against + carvedilol (3 µM), caspase-3 and caspase-8 marking was the treatment. HEK-293 is used to study cytotoxicity because noted in all treated groups (Fig. 5A). Densitometric analysis of its reliable growth despite its karyotype complex with altera- confirmed significant increases in caspase-3 (P<0.01; Fig. 5B), tions. This makes toxicity studies more consistent (54,58-62). and caspase-8 (P<0.001; Fig. 5C). In addition, there was no statistically significant change in Bcl-2 expression for the Oxidative stress. The reduced glutathione and lipid peroxi- group treated with GNPs and carvedilol (Fig. 5D; P>0.05). dation levels were observed from GSH and MDA assays, There was a decrease in MAPK/ERK immunoreactivity in respectively. Due to the fact that the combined treatment with the HepG2 cells treated with GNPs and carvedilol and GNP GNPs (6.25 µg/ml) + carvedilol (3 µM), at 48 h increased the (6.25 µg/ml) + carvedilol (3 µM) for 48 h (P<0.0001; Fig. 5E). protection for HEK-293, it was observed that the levels of GSH increased statistically significantly for both HEK-293 (P<0.05) Gene and protein expression by RT-PCR and western blot and HepG2 (P<0.001), as shown in Fig. 4A. Cisplatin promoted analysis. From the gene expression analysis, it was observed reduction of GSH levels in both cell lines. Regarding lipid that FADD elevation was statistically significant for all peroxidation for the non-tumoral lineage, there was a great groups, those treated separately with GNPs (6.25 µg/ml) and reduction in malondialdehyde levels using the combined treat- carvedilol (3 µM) (P<0.05), and in combined treatment GNPs ment with GNPs (6.25 µg/ml) + carvedilol (3 µM) (P<0.01), (6.25 µg/ml) + carvedilol (3 µM) (P<0.01), yet not observed 132 196 ARAúJO Jr et al: APOPTOSIS CAUSED BY GNPs IN COMBINATION wITH Carv vIA MAPK/Akt/mTOR PATHwAY GNPs (6.25 µg/ml) was concentrated in the vicinity of the plasma membrane. In the combined treatment with GNPs (6.25 µg/ml) + carvedilol (3 µM), GNPs displayed intra-nuclear and perinuclear concentration. Discussion Data from the literature demonstrate the importance of using low doses for drug combination studies that aim to reduce side-effects and increase the field of action in different signaling pathways (63-65). In the present study, low doses of GNPs (3 and 6.25 µg/ml) and carvedilol (1.5 and 3 µM) were selected using the cell viability test, in order to use them in combination. Studies in the literature demonstrate that low doses of GNPs are more effective in inhibiting cell prolifera- tion than larger doses (53,66). In relation to carvedilol, very high doses cause high inhibition of cell growth (67); a risk for a combined treatment. The pro-apoptotic activities of GNPs, carvedilol, and their combined use were analyzed by flow cytometry, both for tumor and non-tumoral lines. The combi- nation of GNPs (6.25 µg/ml) + carvedilol (3 µM) promoted significant initial and late apoptosis in hepatic tumor cells, while promoting reduction of total apoptosis in the non-tumoral lineage. Studies in the literature report the ability of GNPs to induce apoptosis in several tumor cell lines without interfering with non-tumoral lineages (27,29,68,69). Carvedilol has itself also been shown to be an inducer of apoptosis in tumor cells and to protect normal cells (36,70,71). Figure 7. Transmission electron microscopy for evaluation of intracellular The cytoprotective effect of the combined treatment for targets of treatment. (A) GNPs without carvedilol concentrated in the plasma non-cancerous cells was evidenced through GSH dosage; an membrane, indicated by black arrows. (B) For combined treatment, GNPs important balancing antioxidant system peptide (72,73). After concentrated inside and beside the nucleus, indicated by black arrows. N, treatment, GSH levels were elevated, which is important for nucleus. reducing oxidative stress, which is related to apoptosis induc- tion in several tissues (74-77). In addition, GNPs and carvedilol have the ability to promote elevation of GSH levels (78-81). for APAF-1 levels (Fig. 6A). In relation to survivin levels, no Furthermore, a decrease in MDA levels were evidenced, an group treated with GNPs and carvedilol demonstrated a reduc- indirect biomarker for oxidative stress, as related to plasma tion, meanwhile MDR-1 levels showed statistically significant membrane damage (82-84). This is a significant result, since reductions for those treated with GNPs (6.25 µg/ml), and one of the main side-effects of chemotherapy is lipid peroxida- for combined treatment GNPs (6.25 µg/ml) + carvedilol tion (85,86). (3 µM) (P<0.01; Fig. 6B). Regarding the Akt, mTOR and In the tumoral lineage, we observed GSH level elevation and EGFR levels, the groups treated with combined treatment decreased MDA levels, demonstrating the antioxidant action GNPs (6.25 µg/ml) + carvedilol (3 µM) showed statistically of combined treatment when compared to cisplatin, which is significant expression declines P<0.001, P<0.05 and P<0.05, known for its strong oxidative stress promotion (18,19). This respectively (Fig. 6C). The groups treated with carvedilol result was highly positive, since tumor cells have high rates (3 µM) showed statistically significant reductions only for of reactive oxygen species, which trigger the activation of EGFR (P<0.01); and those treated with GNP showed statisti- proteins such as Pi3K, Akt and MAPK/ERK; responsible for cally significant reductions only for Akt (P<0.05) and EGFR both cell proliferation and survival (87,88). (P<0.05). The pro-apoptotic activity of the treatment was confirmed The anti-apoptotic protein levels after combined treatment by the positive immunoreactivity of caspase-3 and caspase-8, of GNPs (6.25 µg/ml) + carvedilol (3 µM) are highlighted proteins involved in the extrinsic apoptosis pathway (89), in Fig. 6D. The results showed a large reduction of Akt and these were the most significant in the group treated with mTOR. MAPK/ERK has a good reduced protein expression the combination. In addition, after treatments with GNPs when compared to the control group. β-actin was used as (6.25 µg/ml) + carvedilol (3 µM), there was a decrease in internal control. immunoreactivity for the MAPK/ERK, anti-apoptotic protein. A similar result is observed in western blot analysis. There Transmission electron microscopy. TEM was performed were no significant changes for Bcl-2. These results indicate in order to ascertain locations where gold nanoparticles a prominent participation of the extrinsic apoptosis pathway, would concentrate, applied without carvedilol (Fig. 7A) and since Bcl-2 is an anti-apoptotic protein related to the intrinsic in combination with carvedilol (Fig. 7B). Applied alone, apoptosis pathway (mitochondrial) (54,90). GSH elevation and 133 INTERNATIONAL JOURNAL OF ONCOLOGY 52: 189-200, 2018 197 MDA decreases, promoted by the combined treatment with However, there remains the need for further chemical interac- GNPs (6.25 µg/ml) + carvedilol (3 µM), may have corrobo- tion and intracellular unfolding studies. rated with mitochondria protection, justifying non-activation The present study demonstrated the ability of the combined of the intrinsic apoptosis pathway (91-96). The statistically GNPs and carvedilol treatment to induce apoptosis in the significant elevation of FADD, mainly by combined treatment tumor cells by exclusively activating the extrinsic pathway. with GNPs (6.25 µg/ml) + carvedilol (3 µM), confirms activa- Activation of this pathway is advantageous compared to the tion of the extrinsic apoptosis pathway, since it participates intrinsic pathway, since it can be triggered independently of therein (97-99). Levels of APAF-1, a protein involved in the the p53 gene, which in many tumors is inactive or absent (127). intrinsic pathway (100), did not present a significant differences. In addition, studies have demonstrated that treatments with The combined treatment with GNPs (6.25 µg/ml) + targeting intrinsic pathway induction may also promote posi- carvedilol (3 µM) was extremely effective on gene expression tive selection of tumor cells, while evading the mitochondrial of anti-apoptotic proteins such as Akt and mTOR, matching pathway (128). In addition to these results, the combined treat- or overcoming the action of cisplatin. Similar effect was ment protected non-tumor cells, reducing oxidative stress and observed in western blot analysis. Inhibition of these proteins, consequently, apoptosis. The novel findings in these studies as well as MAPK/ERK, is critical for induction of apop- highlight a promising alternative for future cancer treatments. tosis (12,51,52,101). Furthermore, other studies indicate that treatments with targeting of the Pi3K/Akt/mTOR pathway Acknowledgements and the MAPK/ERK protein induce early and late apop- tosis (102-104), as also obtained in the present study. The authors are grateful to the Brain Institute (Federal The decrease in EGFR levels is of great relevance since it is University of Rio Grande do Norte), the BIOPOL (Department related to activation of Akt/mTOR and MAPK/ERK (105-108). of Biochemistry, Federal University of Rio Grande do Norte), the However, it is possible that the combined treatment may be Federal University of Ceará and the Leiden University Medical acting on EGFR-independent pathways that activate Akt and Center for their contributions to the present study. Universal mTOR; and causing depletion (14,109-115). Levels of survivin 476996/2013-9 CNPq 2013. CAPES 88881.119850/201601. were unchanged for treatment. However, in the same figure, This study was also supported by the European Commission there was a statistically significant reduction in MDR1 levels where R.F. De Araújo, A.B. Chan and L.J. Cruz have received for both the combined treatment and the gold nanoparticles funding from a MSCA-ITN-2015-ETN action grant (proposal alone. Studies have shown that the use of nanoscale systems has no. 675743; project: ISPIC). excellent effects (this includes inorganic nanoparticles, such as gold nanoparticles), on the expression of genes related to multi- References drug resistance, such as MDR1 (116,117). The decrease in the expression of this protein presents potential as a new strategy 1. Hales S, Chiu A, Husain A, Braun M, Rydall A, Gagliese L, to combat one of the main problems of cancer treatment, resis- zimmermann C and Rodin G: The quality of dying and death in tance to treatment (118). Carvedilol, although showing no effect cancer and its relationship to palliative care and place of death. J Pain Symptom Manage 48: 839-851, 2014. on MDR1 expression, has been reported in the literature as a 2. Siegel RL, Miller KD and Jemal A: Cancer statistics, 2016. CA potential MDR1 inhibitor (119,120). Studies in the literature Cancer J Clin 66: 7-30, 2016. have demonstrated that treatments acting on drug resistance 3. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D and Bray F: Cancer incidence genes promote both early and late apoptosis (121). and mortality worldwide: Sources, methods and major patterns TEM did reveal GNPs internalization. However, we noted in GLOBOCAN 2012. Int J Cancer 136: E359-E386, 2015. that when given alone, GNPs accumulated near the plasmatic 4. Özdemir F, Akalın G, Şen M, Önder NI, Işcan A, Kutlu HM and Incesu z: Towards novel anti-tumor strategies for hepatic cancer: membrane. Previous studies have reported that endosomal/ ε-viniferin in combination with vincristine displays pharma- lysosomal vesicles can imprison GNPs preventing complete codynamic synergy at lower doses in HepG2 cells. OMICS 18: action in the cellular interior, and are the greatest barrier that 324-334, 2014. 5. Ling CQ: Problems in cancer treatment and major research of GNPs need to overcome to reach the cellular nucleus (main integrative medicine. zhong Xi Yi Jie He Xue Bao 1: 168-170, target) (122-125). However, after administration of carvedilol, 2003 (In Chinese). concentration of GNPs was observed in the vicinity of the 6. Agrawal S: Late effects of cancer treatment in breast cancer survivors. South Asian J Cancer 3: 112-115, 2014. cell nucleus, both intra- and peripherally. The data possibly 7. Oberstein PE and Olive KP: Pancreatic cancer: why is it so hard explain the fact that the results for GNPs administered alone to treat? Therap Adv Gastroenterol 6: 321-337, 2013. show smaller indices as compared to the combined treatment, 8. Michaelson MD, Cotter SE, Gargollo PC, zietman AL, Dahl DM and Smith MR: Management of complications of prostate cancer which was more effective. But even the isolated action of the treatment. CA Cancer J Clin 58: 196-213, 2008. gold nanoparticle can reduce, although less than combined 9. Fernald K and Kurokawa M: Evading apoptosis in cancer. Trends treatment, the levels of survival and proliferation proteins Cell Biol 23: 620-633, 2013.10. Labi v and Erlacher M: How cell death shapes cancer. Cell Death such as Akt, EGFR, MDR-1 and MAPK/ERK, demonstrated Dis 6: e1675, 2015. in the present study, and suggest the way in which the GNPs 11. Ran LK, Chen Y, zhang zz, Tao NN, Ren JH, zhou L, Tang H, inhibit proliferation. Concerning the combined treatment, Chen X, Chen K, Li wY, et al: SIRT6 οverexpression Potentiates carvedilol may be acting through other signaling pathways, or apoptosis evasion in hepatocellular carcinoma via BCL2-associated X protein-dependent apoptotic pathway. Clin Cancer may be facilitating gold nanoparticle escape from endosomal/ Res 22: 3372-3382, 2016. lysosomal vesicles. Han et al (126) reported that carvedilol has 12. Cai Y, Tan X, Liu J, Shen Y, wu D, Ren M, Huang P and Yu D: a role during receptor recycling in endosomal/lysosomal tran- Inhibition of PI3K/Akt/mTOR signaling pathway enhances the sensitivity of the SKOv3/DDP ovarian cancer cell line to siting of vascular smooth muscle cell beta-adrenergic receptors. cisplatin in vitro. Chin J Cancer Res 26: 564-572, 2014. 134 198 ARAúJO Jr et al: APOPTOSIS CAUSED BY GNPs IN COMBINATION wITH Carv vIA MAPK/Akt/mTOR PATHwAY 13. Lu z and Xu S: ERK1/2 MAP kinases in cell survival and 35. Pasquier E, Street J, Pouchy C, Carre M, Gifford AJ, Murray J, apoptosis. IUBMB Life 58: 621-631, 2006. Norris MD, Trahair T, Andre N and Kavallaris M: β-blockers 14. wang C, Cigliano A, Delogu S, Armbruster J, Dombrowski F, increase response to chemotherapy via direct antitumour and Evert M, Chen X and Calvisi DF: Functional crosstalk between anti-angiogenic mechanisms in neuroblastoma. Br J Cancer 108: AKT/mTOR and Ras/MAPK pathways in hepatocarcinogen- 2485-2494, 2013. esis: Implications for the treatment of human liver cancer. Cell 36. Erguven M, Yazihan N, Aktas E, Sabanci A, Li CJ, Oktem G and Cycle 12: 1999-2010, 2013. Bilir A: Carvedilol in glioma treatment alone and with imatinib 15. Katayama K, Noguchi K and Sugimoto Y: Regulations of in vitro. Int J Oncol 36: 857-866, 2010. P-glycoprotein/ABCB1/MDR1 in human cancer cells. New J Sci 37. Dezong G, zhongbing M, Qinye F and zhigang Y: Carvedilol 2014: e476974, 2014. suppresses migration and invasion of malignant breast cells 16. Yang X, Uziely B, Groshen S, Lukas J, Israel v, Russell C, by inactivating Src involving cAMP/PKA and PKCδ signaling Dunnington G, Formenti S, Muggia F and Press MF: MDR1 gene pathway. J Cancer Res Ther 10: 998-1003, 2014. expression in primary and advanced breast cancer. Lab Invest 79: 38. Chang A, Yeung S, Thakkar A, Huang KM, Liu MM, 271-280, 1999. Kanassatega RS, Parsa C, Orlando R, Jackson EK, Andresen BT, 17. Chiara F, Gambalunga A, Sciacovell i M, Nicoll i A, et al: Prevention of skin carcinogenesis by the β-blocker Ronconi L, Fregona D, Bernardi P, Rasola A and Trevisan A: carvedilol. Cancer Prev Res (Phila) 8: 27-36, 2015. Chemotherapeutic induction of mitochondrial oxidative stress 39. Hsieh YD, Chi CC, Chou CT, Cheng JS, Kuo CC, Liang wz, activates GSK-3α/β and Bax, leading to permeability transition Lin KL, Tseng LL and Jan CR: Investigation of carvedilol- pore opening and tumor cell death. Cell Death Dis 3: e444, evoked Ca²+ movement and death in human oral cancer cells. J 2012. Recept Signal Transduct Res 31: 220-228, 2011. 18. Dasari S and Tchounwou PB: Cisplatin in cancer therapy: 40. Cheng JS, Huang CC, Chou CT and Jan CR: Mechanisms of Molecular mechanisms of action. Eur J Pharmacol 740: 364-378, carvedilol-induced [Ca2+]i rises and death in human hepatoma 2014. cells. Naunyn Schmiedebergs Arch Pharmacol 376: 185-194, 19. Marullo R, werner E, Degtyareva N, Moore B, Altavilla G, 2007. Ramalingam SS and Doetsch Pw: Cisplatin induces a mitochon- 41. Cohen DJ and Hochster HS: Rationale for combining biotherapy drial-ROS response that contributes to cytotoxicity depending in the treatment of advanced colon cancer. Gastrointest Cancer on mitochondrial redox status and bioenergetic functions. PLoS Res 2: 145-151, 2008. One 8: e81162, 2013. 20. Dreaden EC, Austin LA, Mackey MA and El-Sayed MA: Size 42. Patutina OA, Mironova NL, vlassov vv and zenkova MA: matters: Gold nanoparticles in targeted cancer drug delivery. New approaches for cancer treatment: Antitumor drugs based on Ther Deliv 3: 457-478, 2012. gene-targeted nucleic acids. Acta Naturae 1: 44-60, 2009. 21. Jain S, Hirst DG and O'Sullivan JM: Gold nanoparticles as novel 43. Siddiqui M and Rajkumar Sv: The high cost of cancer drugs and agents for cancer therapy. Br J Radiol 85: 101-113, 2012. what we can do about it. Mayo Clin Proc 87: 935-943, 2012. 22. Lee J, Chatterjee DK, Lee MH and Krishnan S: Gold nanopar- 44. Tannock IF: Combined modality treatment with radiotherapy ticles in breast cancer treatment: Promise and potential pitfalls. and chemotherapy. Radiother Oncol 16: 83-101, 1989. Cancer Lett 347: 46-53, 2014. 45. Brito AF, Ribeiro M, Abrantes AM, Pires AS, Teixo RJ, 23. Alkilany AM and Murphy CJ: Toxicity and cellular uptake of Tralhão JG and Botelho MF: Quercetin in cancer treatment, gold nanoparticles: what we have learned so far? J Nanopart alone or in combination with conventional therapeutics? Curr Res 12: 2313-2333, 2010. Med Chem 22: 3025-3039, 2015. 24. Alvarenga ÉC, Caires A, Ladeira LO, Gamero EJP, Andrade LM, 46. Mierzwa ML, Nyati MK, Morgan MA and Lawrence TS: Recent Paz MTL and Leite M de F: Potenciais alvos terapêuticos contra advances in combined modality therapy. Oncologist 15: 372-381, o câncer. Cienc Cult 66: 43-48, 2014. 2010. 25. Naha PC, Chhour P and Cormode DP: Systematic in vitro 47. Li J, wang Y, zhu Y and Oupický D: Recent advances in delivery toxicological screening of gold nanoparticles designed for nano- of drug-nucleic acid combinations for cancer treatment. J Control medicine applications. Toxicol In vitro 29: 1445-1453, 2015. Release 172: 589-600, 2013. 26. Butterworth KT, Coulter JA, Jain S, Forker J, McMahon SJ, 48. Collery P, Mohsen A, Kermagoret A, D'Angelo J, Morgant G, Schettino G, Prise KM, Currell FJ and Hirst DG: Evaluation Desmaele D, Tomas A, Collery T, wei M and Badawi A: of cytotoxicity and radiation enhancement using 1.9 nm Combination of three metals for the treatment of cancer: Gallium, gold particles: Potential application for cancer therapy. rhenium and platinum. 1. Determination of the optimal schedule Nanotechnology 21: 295101, 2010. of treatment. Anticancer Res 32: 2769-2781, 2012. 27. Coulter JA, Jain S, Butterworth KT, Taggart LE, Dickson GR, 49. Law MR, wald NJ, Morris JK and Jordan RE: value of low McMahon SJ, Hyland wB, Muir MF, Trainor C, Hounsell AR, dose combination treatment with blood pressure lowering drugs: et al: Cell type-dependent uptake, localization, and cytotoxicity Analysis of 354 randomised trials. BMJ 326: 1427, 2003. of 1.9 nm gold nanoparticles. Int J Nanomedicine 7: 2673-2685, 50. Morton CO, Chau M and Stack C: In vitro combination therapy 2012. using low dose clotrimazole and photodynamic therapy leads 28. Murawala P, Tirmale A, Shiras A and Prasad BLv: In situ to enhanced killing of the dermatophyte Trichophyton rubrum. synthesized BSA capped gold nanoparticles: Effective carrier BMC Microbiol 14: 261, 2014. of anticancer drug methotrexate to MCF-7 breast cancer cells. 51. Chang L, Graham PH, Ni J, Hao J, Bucci J, Cozzi PJ and Li Y: Mater Sci Eng C 34: 158-167, 2014. Targeting PI3K/Akt/mTOR signaling pathway in the treatment 29. Patra HK, Banerjee S, Chaudhuri U, Lahiri P and Dasgupta AK: of prostate cancer radioresistance. Crit Rev Oncol Hematol 96: Cell selective response to gold nanoparticles. Nanomedicine 507-517, 2015. (Lond) 3: 111-119, 2007. 52. Chappell wH, Steelman LS, Long JM, Kempf RC, Abrams SL, 30. Budni P, Pedrosa RC, Dalmarco EM, Dalmarco JB, Frode TS Franklin RA, Bäsecke J, Stivala F, Donia M, Fagone P, et al: and wilhelm Filho D: Carvedilol enhances the antioxidant effect Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR inhibitors: of vitamins E and C in chronic Chagas heart disease. Arq Bras Rationale and importance to inhibiting these pathways in human Cardiol 101: 304-310, 2013. health. Oncotarget 2: 135-164, 2011. 31. Arozal w, watanabe K, veeraveedu PT, Ma M, Thandavarayan RA, 53. de Araújo RF, de Araújo AA, Pessoa JB, Freire Neto FP, Sukumaran v, Suzuki K, Kodama M and Aizawa Y: Protective da Silva GR, Leitão Oliveira AL, de Carvalho TG, Silva HF, effect of carvedilol on daunorubicin-induced cardiotoxicity and Eugênio M, Sant'Anna C, et al: Anti-inflammatory, analgesic and nephrotoxicity in rats. Toxicology 274: 18-26, 2010. anti-tumor properties of gold nanoparticles. Pharmacol Rep 69: 32. Arumanayagam M, Chan S, Tong S and Sanderson JE: 119-129, 2017. Antioxidant properties of carvedilol and metoprolol in heart 54. de Araújo Júnior RF, Leitão Oliveira ALC, de Melo Silveira RF, failure: A double-blind randomized controlled trial. J Cardiovasc de Oliveira Rocha HA, de França Cavalcanti P and de Araújo AA: Pharmacol 37: 48-54, 2001. Telmisartan induces apoptosis and regulates Bcl-2 in human 33. Dandona P, Ghanim H and Brooks DP: Antioxidant activity of renal cancer cells. Exp Biol Med (Maywood) 240: 34-44, 2015. carvedilol in cardiovascular disease. J Hypertens 25: 731-741, 55. Rahman I, Kode A and Biswas SK: Assay for quantitative deter- 2007. mination of glutathione and glutathione disulfide levels using 34. Li YC, Ge LS, Yang PL, Tang JF, Lin JF, Chen P and enzymatic recycling method. Nat Protoc 1: 3159-3165, 2006. Guan XQ: Carvedilol treatment ameliorates acute coxsacki- 56. da Costa CM, dos Santos RC and Lima ES: A simple automated evirus B3-induced myocarditis associated with oxidative stress procedure for thiol measurement in human serum samples. J reduction. Eur J Pharmacol 640: 112-116, 2010. Bras Patol Med Lab 42: 345-350, 2006. 135 INTERNATIONAL JOURNAL OF ONCOLOGY 52: 189-200, 2018 199 57. Esterbauer H and Cheeseman KH: Determination of aldehydic 79. Sgobbo P, Pacelli C, Grattagliano I, villani G and Cocco T: lipid peroxidation products: Malonaldehyde and 4-hydroxynon- Carvedilol inhibits mitochondrial complex I and induces resis- enal. Methods Enzymol 186: 407-421, 1990. tance to H2O2-mediated oxidative insult in H9C2 myocardial 58. Al-Sheddi ES, Al-Oqail MM, Saquib Q, Siddiqui MA, Musarrat J, cells. Biochim Biophys Acta 1767: 222-232, 2007. Al-Khedhairy AA and Farshori NN: Novel all trans-retinoic Acid 80. Barathmanikanth S, Kalishwaralal K, Sriram M, Pandian SR, derivatives: Cytotoxicity, inhibition of cell cycle progression and Youn HS, Eom S and Gurunathan S: Anti-oxidant effect of gold induction of apoptosis in human cancer cell lines. Molecules 20: nanoparticles restrains hyperglycemic conditions in diabetic 8181-8197, 2015. mice. J Nanobiotech 8: 16, 2010. 59. Kimura H, Sakai K, Arao T, Shimoyama T, Tamura T and 81. Yakimovich NO, Ezhevskii AA, Guseinov Dv, Smirnova LA, Nishio K: Antibody-dependent cellular cytotoxicity of cetuximab Gracheva TA and Klychkov KS: Antioxidant properties of gold against tumor cells with wild-type or mutant epidermal growth nanoparticles studied by ESR spectroscopy. Russ Chem Bull 57: factor receptor. Cancer Sci 98: 1275-1280, 2007. 520-523, 2008. 60. Świątek Ł, Rajtar B, Pawlak K, Ludwiczuk A, Głowniak K and 82. Nielsen F, Mikkelsen BB, Nielsen JB, Andersen HR and Polz-Dacewicz M: In vitro evaluation of cytotoxicity of n-hexane Grandjean P: Plasma malondialdehyde as biomarker for extract from Alnus sieboldiana male flowers on vERO and oxidative stress: Reference interval and effects of life-style HEK293 cell lines. JPCCR 7: 110-107, 2014. factors. Clin Chem 43: 1209-1214, 1997. 61. Lapique N and Benenson Y: Digital switching in a biosensor 83. Gaweł S, Wardas M, Niedworok E and Wardas P: circuit via programmable timing of gene availability. Nat Chem Malondialdehyde (MDA) as a lipid peroxidation marker. wiad Biol 10: 1020-1027, 2014. Lek 57: 453-455, 2004 (In Polish). 62. Selvaraj v, Bodapati S, Murray E, Rice KM, winston N, 84. Ho E, Karimi Galougahi K, Liu CC, Bhindi R and Figtree GA: Shokuhfar T, zhao Y and Blough E: Cytotoxicity and genotox- Biological markers of oxidative stress: Applications to cardio- icity caused by yttrium oxide nanoparticles in HEK293 cells. Int vascular research and practice. Redox Biol 1: 483-491, 2013. J Nanomed 9: 1379-1391, 2014. 85. Sangeetha P, Das UN, Koratkar R and Suryaprabha P: Increase 63. Jia J, zhu F, Ma X, Cao z, Cao zw, Li Y, Li YX and Chen Yz: in free radical generation and lipid peroxidation following Mechanisms of drug combinations: Interaction and network chemotherapy in patients with cancer. Free Radic Biol Med 8: perspectives. Nat Rev Drug Discov 8: 111-128, 2009. 15-19, 1990. 64. Richardson PG, Siegel DS, vij R, Hofmeister CC, Baz R, 86. Esfahani A, Ghoreishi z, Nikanfar A, Sanaat z and Jagannath S, Chen C, Lonial S, Jakubowiak A, Bahlis N, et al: Ghorbanihaghjo A: Influence of chemotherapy on the lipid Pomalidomide alone or in combination with low-dose dexa- peroxidation and antioxidant status in patients with acute methasone in relapsed and refractory multiple myeloma: A myeloid leukemia. Acta Med Iran 50: 454-458, 2012. randomized phase 2 study. Blood 123: 1826-1832, 2014. 87. Cabello CM, Bair wB III and wondrak GT: Experimental 65. Nijhof IS, Franssen LE, Levin M-D, Bos GMJ, Broijl A, therapeutics: Targeting the redox Achilles heel of cancer. Curr Klein SK, Koene HR, Bloem AC, Beeker A, Faber LM, et al: Opin Investig Drugs 8: 1022-1037, 2007. Phase 1/2 study of lenalidomide combined with low-dose 88. Liou GY and Storz P: Reactive oxygen species in cancer. Free cyclophosphamide and prednisone in lenalidomide-refractory Radic Res 44: 479-496, 2010. multiple myeloma. Blood 128: 2297-2306, 2016. 89. García M and vecino E: vías de señalización intracelular que 66. Gatoo MA, Naseem S, Arfat MY, Dar AM, Qasim K and zubair S: conducen a la apoptosis de las células de la retina. Arch Soc Esp Physicochemical properties of nanomaterials: Implication in Oftalmol 78: 351-364, 2003. associated toxic manifestations. BioMed Res Int 2014: 498420, 90. Kang MH and Reynolds CP: Bcl-2 inhibitors: Targeting mito- 2014. chondrial apoptotic pathways in cancer therapy. Clin Cancer 67. Coelho M, Moz M, Correia G, Teixeira A, Medeiros R and Res 15: 1126-1132, 2009. Ribeiro L: Antiproliferative effects of β-blockers on human 91. Ahmad S, white Cw, Chang LY, Schneider BK and Allen CB: colorectal cancer cells. Oncol Rep 33: 2513-2520, 2015. Glutamine protects mitochondrial structure and function in 68. Baharara J, Ramezani T, Divsalar A, Mousavi M and oxygen toxicity. Am J Physiol Lung Cell Mol Physiol 280: Seyedarabi A: Induction of apoptosis by green synthesized gold L779-L791, 2001. nanoparticles Through activation of caspase-3 and 9 in human 92. Drake J, Sultana R, Aksenova M, Calabrese v and cervical cancer cells. Avicenna J Med Biotechnol 8: 75-83, 2016. Butterfield DA: Elevation of mitochondrial glutathione by 69. Connor EE, Mwamuka J, Gole A, Murphy CJ and wyatt MD: gamma-glutamylcysteine ethyl ester protects mitochondria Gold nanoparticles are taken up by human cells but do not cause against peroxynitrite-induced oxidative stress. J Neurosci acute cytotoxicity. Small 1: 325-327, 2005. Res 74: 917-927, 2003. 70. zhao Y, Xu Y, zhang J and Ji T: Cardioprotective effect of 93. Marí M, Morales A, Colell A, García-Ruiz C and Fernández- carvedilol: Inhibition of apoptosis in H9c2 cardiomyocytes Checa JC: Mitochondrial glutathione, a key survival antioxidant. via the TLR4/NF-κB pathway following ischemia/reperfusion Antioxid Redox Signal 11: 2685-2700, 2009. injury. Exp Ther Med 8: 1092-1096, 2014. 94. Cheng J, wang F, Yu DF, wu PF and Chen JG: The cytotoxic 71. Carvalho Rodrigues MA, Gobe G, Santos NA and Santos AC: mechanism of malondialdehyde and protective effect of Carvedilol protects against apoptotic cell death induced by carnosine via protein cross-linking/mitochondrial dysfunction/ cisplatin in renal tubular epithelial cells. J Toxicol Environ reactive oxygen species/MAPK pathway in neurons. Eur J Pharmacol 650: 184-194, 2011. Health A 75: 981-990, 2012. 95. Ayala A, Muñoz MF and Argüelles S: Lipid peroxidation: 72. Lu SC: Glutathione synthesis. Biochim Biophys Acta 1830: Production, metabolism, and signaling mechanisms of 3143-3153, 2013. malondialdehyde and 4-hydroxy-2-nonenal. Oxid Med Cell 73. Townsend DM, Tew KD and Tapiero H: The importance of gluta- Longev 2014: 360438, 2014. thione in human disease. Biomed Pharmacother 57: 145-155, 96. Qin J, Kang Y, Xu z, zang C, Fang B and Liu X: Dioscin 2003. prevents the mitochondrial apoptosis and attenuates oxidative 74. Kannan K and Jain SK: Oxidative stress and apoptosis. stress in cardiac H9c2 cells. Drug Res (Stuttg) 64: 47-52, 2014. Pathophysiology 7: 153-163, 2000. 97. Bang S, Jeong EJ, Kim IK, Jung YK and Kim KS: Fas- and 75. Takahashi A, Masuda A, Sun M, Centonze vE and Herman B: tumor necrosis factor-mediated apoptosis uses the same binding Oxidative stress-induced apoptosis is associated with alterations surface of FADD to trigger signal transduction. A typical in mitochondrial caspase activity and Bcl-2-dependent altera- model for convergent signal transduction. J Biol Chem 275: tions in mitochondrial pH (pHm). Brain Res Bull 62: 497-504, 36217-36222, 2000. 2004. 98. Osborn SL, Sohn SJ and winoto A: Constitutive phosphoryla- 76. Chang wK, Yang KD, Chuang H, Jan JT and Shaio MF: tion mutation in Fas-associated death domain (FADD) results in Glutamine protects activated human T cells from apoptosis by early cell cycle defects. J Biol Chem 282: 22786-22792, 2007. up-regulating glutathione and Bcl-2 levels. Clin Immunol 104: 99. Xerri L, Devilard E, Bouabdallah R, Stoppa AM, Hassoun J 151-160, 2002. and Birg F: FADD expression and caspase activation in 77. Estrela JM, Ortega A and Obrador E: Glutathione in cancer B-cell lymphomas resistant to Fas-mediated apoptosis. Br J biology and therapy. Crit Rev Clin Lab Sci 43: 143-181, 2006. Haematol 106: 652-661, 1999. 78. zubairi MB, Ahmed JH and Al-Haroon SS: Effect of adrenergic 100. Campioni M, Santini D, Tonini G, Murace R, Dragonetti E, blockers, carvedilol, prazosin, metoprolol and combination of Spugnini EP and Baldi A: Role of Apaf-1, a key regulator of prazosin and metoprolol on paracetamol-induced hepatotoxicity apoptosis, in melanoma progression and chemoresistance. Exp in rabbits. Indian J Pharmacol 46: 644-648, 2014. Dermatol 14: 811-818, 2005. 136 200 ARAúJO Jr et al: APOPTOSIS CAUSED BY GNPs IN COMBINATION wITH Carv vIA MAPK/Akt/mTOR PATHwAY 101. Brazil DP, Yang zz and Hemmings BA: Advances in protein 115. Farabaugh SM, Boone DN and Lee Av: Role of IGF1R in breast kinase B signalling: AKTion on multiple fronts. Trends Biochem cancer subtypes, stemness and lineage differentiation. Front Sci 29: 233-242, 2004. Endocrinol (Lausanne) 6: 59, 2015. 102. Daniele S, Costa B, zappelli E, Da Pozzo E, Sestito S, Nesi G, 116. Kapse-Mistry S, Govender T, Srivastava R and Yergeri M: Campiglia P, Marinelli L, Novellino E, Rapposelli S, et al: Nanodrug delivery in reversing multidrug resistance in cancer Combined inhibition of AKT/mTOR and MDM2 enhances glio- cells. Front Pharmacol 5: 159, 2014. blastoma multiforme cell apoptosis and differentiation of cancer 117. Salomon JJ and Ehrhardt C: Nanoparticles attenuate stem cells. Sci Rep 5: 9956, 2015. P-glycoprotein/MDR1 function in A549 human alveolar epithe- 103. Li C, Xin P, Xiao H, zheng Y, Huang Y and zhu X: The dual lial cells. Eur J Pharm Biopharm 77: 392-397, 2011. PI3K/mTOR inhibitor NvP-BEz235 inhibits proliferation and 118. Callaghan R, Luk F and Bebawy M: Inhibition of the multidrug induces apoptosis of burkitt lymphoma cells. Cancer Cell Int 15: resistance P-glycoprotein: Time for a change of strategy? Drug 65, 2015. Metab Dispos 42: 623-631, 2014. 104. Liu z, Ruan HJ, Gu PQ, Ding wY, Luo XH, Huang R, zhao w 119. Kakumoto M, Sakaeda T, Takara K, Nakamura T, Kita T, and Gao LJ: The roles of p38 MAPK and ERK1/2 in coplanar Yagami T, Kobayashi H, Okamura N and Okumura K: Effects polychlorinated biphenyls-induced apoptosis of human extravil- of carvedilol on MDR1-mediated multidrug resistance: lous cytotrophoblast-derived transformed cells. Cell Physiol Comparison with verapamil. Cancer Sci 94: 81-86, 2003. Biochem 36: 2418-2432, 2015. 120. wessler JD, Grip LT, Mendell J and Giugliano RP: The 105. Freudlsperger C, Burnett JR, Friedman JA, Kannabiran vR, P-glycoprotein transport system and cardiovascular drugs. J Am Chen z and van waes C: EGFR-PI3K-AKT-mTOR signaling in Coll Cardiol 61: 2495-2502, 2013. head and neck squamous cell carcinomas: Attractive targets for 121. Mitsiades CS, Treon SP, Mitsiades N, Shima Y, Richardson P, molecular-oriented therapy. Expert Opin Ther Targets 15: 63-74, Schlossman R, Hideshima T and Anderson KC: TRAIL/Apo2L 2011. ligand selectively induces apoptosis and overcomes drug resis- 106. Gan Y, Shi C, Inge L, Hibner M, Balducci J and Huang Y: tance in multiple myeloma: Therapeutic applications. Blood 98: Differential roles of ERK and Akt pathways in regulation of 795-804, 2001. EGFR-mediated signaling and motility in prostate cancer cells. 122. Huang X, Kang B, Qian w, Mackey MA, Chen PC, Oyelere AK, Oncogene 29: 4947-4958, 2010. El-Sayed IH and El-Sayed MA: Comparative study of 107. Seshacharyulu P, Ponnusamy MP, Haridas D, Jain M, Ganti AK photothermolysis of cancer cells with nuclear-targeted or cyto- and Batra SK: Targeting the EGFR signaling pathway in cancer plasm-targeted gold nanospheres: Continuous wave or pulsed therapy. Expert Opin Ther Targets 16: 15-31, 2012. lasers. J Biomed Opt 15: 058002, 2010. 108. Kidger AM and Keyse SM: The regulation of oncogenic 123. Yang CJ and Chithrani DB: Nuclear targeting of gold nanopar- Ras/ERK signalling by dual-specificity mitogen activated ticles for improved therapeutics. Curr Top Med Chem 16: protein kinase phosphatases (MKPs). Semin Cell Dev Biol 50: 271-280, 2016. 125-132, 2016. 124. Kodiha M, wang YM, Hutter E, Maysinger D and Stochaj U: 109. Gao Y, Moten A and Lin HK: Akt: A new activation mechanism. Off to the organelles - killing cancer cells with targeted gold Cell Res 24: 785-786, 2014. nanoparticles. Theranostics 5: 357-370, 2015. 110. Liu P, Begley M, Michowski w, Inuzuka H, Ginzberg M, Gao D, 125. Yanes RE, Tarn D, Hwang AA, Ferris DP, Sherman SP, Tsou P, Gan w, Papa A, Kim BM, et al: Cell-cycle-regulated Thomas CR, Lu J, Pyle AD, zink JI and Tamanoi F: Involvement activation of Akt kinase by phosphorylation at its carboxyl of lysosomal exocytosis in the excretion of mesoporous silica terminus. Nature 508: 541-545, 2014. nanoparticles and enhancement of the drug delivery effect by 111. Aeder SE, Martin PM, Soh Jw and Hussaini IM: PKC-eta exocytosis inhibition. Small 9: 697-704, 2013. mediates glioblastoma cell proliferation through the Akt and 126. Han SO, Xiao K, Kim J, wu JH, wisler Jw, Nakamura N, mTOR signaling pathways. Oncogene 23: 9062-9069, 2004. Freedman NJ and Shenoy SK: MARCH2 promotes endocy- 112. Fan Qw, Cheng C, Knight zA, Haas-Kogan D, Stokoe D, tosis and lysosomal sorting of carvedilol-bound β2-adrenergic James CD, McCormick F, Shokat KM and weiss wA: EGFR receptors. J Cell Biol 199: 817-830, 2012. signals to mTOR through PKC and independently of Akt in 127. El-Deiry wS: Insights into cancer therapeutic design based glioma. Sci Signal 2: ra4, 2009. on p53 and TRAIL receptor signaling. Cell Death Differ 8: 113. Mendoza MC, Er EE and Blenis J: The Ras-ERK and 1066-1075, 2001. PI3K-mTOR pathways: Cross-talk and compensation. Trends 128. Sayers TJ: Targeting the extrinsic apoptosis signaling pathway Biochem Sci 36: 320-328, 2011. for cancer therapy. Cancer Immunol Immunother 60: 1173-1180, 114. Denduluri SK, Idowu O, wang z, Liao z, Yan z, Mohammed MK, 2011. Ye J, wei Q, wang J, zhao L, et al: Insulin-like growth factor (IGF) signaling in tumorigenesis and the development of cancer drug resistance. Genes Dis 2: 13-25, 2015. 137 Apêndice F How the Interaction of PVP-stabilized Ag Nanoparticles with Models of Cellular Membranes at the Air-Water Interface is Modulated by the Monolayer Composition Rafael LeonardoCruz Gomes da Silva, Heloiza Fernanda Oliveira da Silva, Luiz Henriqueda Silva Gasparotto, LucianoCaseli. Journal of Colloid and Interface Science, 2018, Vol. 512, 792-800 Contribuição:  Realizei a síntese e purificação das NanoAg.  Realizei a caracterização das NanoAg.  Tratei os dados e preparei as figuras relacionadas às etapas que realizei ativamente que foram incluídas no manuscrito. ________________________ __________________________ Heloiza Fernanda O. da Silva Athayde Prof. Luiz Henrique da S. Gasparotto 138 Journal of Colloid and Interface Science 512 (2018) 792–800 Contents lists available at ScienceDirect Journal of Colloid and Interface Science journal homepage: www.elsevier .com/locate / jc is Regular Article How the interaction of PVP-stabilized Ag nanoparticles with models of cellular membranes at the air-water interface is modulated by the monolayer composition Rafael Leonardo Cruz Gomes da Silva a, Heloiza Fernanda Oliveira da Silva b, Luiz Henrique da Silva Gasparotto b, Luciano Caseli a,⇑ aDepartment of Chemistry, Federal University of São Paulo, Diadema, SP, Brazil bGroup of Biological Chemistry and Chemometrics, Institute of Chemistry, Federal University of Rio Grande do Norte, Natal 59072-970, RN, Brazil g r a p h i c a l a b s t r a c t a r t i c l e i n f o a b s t r a c t Article history: The antimicrobial property of silver nanoparticles (AgNPs) is believed to be associated to their interaction Received 26 September 2017 with biointerfaces such as microbial cell membranes, encouraging research on the identification of mem- Revised 23 October 2017 brane sites capable of AgNPs binding. Although significant progress has been made in that regard, the Accepted 24 October 2017 exact molecular mechanism of action is yet to be fully understood. In this study, AgNPs dispersed in Available online 26 October 2017 aqueous media and stabilized with polyvinylpyrrolidone were incorporated in Langmuir monolayers of selected lipids that served as cell membrane models. Results from pressure-area isotherms, vibrational Keywords: spectroscopy and Brewster angle microscopy revealed condensation of glycoside-free lipid monolayers, Silver nanoparticles Langmuir monolayers evidencing that the AgNPs interact mostly with the lipid hydrophilic head groups. In contrast, the Air–water interface monolayers of systems containing glycoside derivatives were found to expand upon AgNPs incorporation, indicating that the glycosidic compounds might facilitate the incorporation of these nanoparticles in cellular membranes. These data can be therefore correlated with the possible toxicity and microbicide effect of AgNPs in lipidic surfaces of mammalian and microbial membranes.  2017 Elsevier Inc. All rights reserved. ⇑ Corresponding author. E-mail address: lcaseli@unifesp.br (L. Caseli). https://doi.org/10.1016/j.jcis.2017.10.091 0021-9797/ 2017 Elsevier Inc. All rights reserved. 139 R.L. Cruz Gomes da Silva et al. / Journal of Colloid and Interface Science 512 (2018) 792–800 793 1. Introduction form (Synth, PA) to result in a concentration of 0.5 mg/mL. Laminin from Engelbreth- Holm-Swarm murine sarcoma basement mem- Silver nanoparticles (AgNPs) dispersed in water are colloid sys- brane, peptidoglycan from Bacillus subtilis, and lipopolysaccharide tems that can be employed in surgical and hygienic supplies due to form E. Coli were obtained from Sigma-Aldrich and dissolved in their antimicrobial properties [1–3]. Although their bactericidal water to render a 0.5 mg/mL solution. effect is well known, the molecular mechanism of action on bacte- AgNPs were synthesized according to a method reported else- rial membranes is yet to be completely clarified [4]. Studies on the where [22,23]. Briefly, all glassware was kept overnight in a KMnO4 biocidal effect of AgNPs against E. Coli [5] and others types of bac- + NaOH solution, rinsed with deionized water, kept for 10 min in terium [6] have indicated that AgNPs disrupt the bacterial mem- an H2O2 + H2SO4 solution (1:1 v/v), again rinsed with deionized brane and penetrate the cell wall and the plasmatic membrane, water and dried prior to use. The following stock solutions were releasing silver ions into the cytoplasm. The bactericidal effect is prepared: 50 mmol L1 AgNO3, 200 g l1 PVP, and 1.0 mol L1 proposed to be associated with the inactivation of vital enzymes NaOH -1. In a beaker, 0.50 mL of the PVP and 40 lL AgNO3 solutions [7,8], loss of ability of DNA to replicate [9], and structural changes were dissolved in water to yield a 5-mL solution. In a separate bea- in cell membranes [9,10]. Hence, the unraveling of molecular inter- ker, 1.0 mL of the NaOH was mixed in water to generate a 5-mL actions between nanoparticles and phospholipids is fundamental solution. Afterwards, the NaOH solution was added to the AgNO3- to comprehend mechanisms of action at the molecular level for PVP one to yield a 10 mL solution with the following final concen- future biological applications. The molecular impact of AgNPs on trations: 0.20 mmol L1 Ag+, 0.010 mol L1 NaOH and 10 g/L PVP. mammalian cells is also of interest since they can cause inflamma- The final solution had a deep-yellow color due to the formation tion, denaturation of proteins, oxidative stress and disruption of of AgNPs and was neutralized with diluted HCl. cell membranes [11]. For the formation of the lipid Langmuir monolayers, pre- Given that the cell membrane is quite a complex system, the determined aliquots of the lipid solutions were spread on the air– use simplified models may help unravel the underlying mecha- water interface of a water subphase that filled amini-KSV Langmuir nism of its interaction with nanoparticles [12]. Such models trough equipped with a surface pressure sensor (the Wilhelmy include black lipid membranes, bilayers [13,14], and lipid Lang- method). For preliminary tests, AgNpswere spread on the air–water muir monolayers [15], with the latter consisting of monomolecular interface to test their surface activity. Lipids and nanoparticles were films formed at the air–water interface that permit easy manipula- incorporated separately at the air–water interface, with the tion of the chemical composition and surface density [16]. nanoparticles inserted after lipid spreading in the aqueous subphase Although the relevance in using lipids organized as floating films in the vicinities of the air–water interface. After at least 10 min as cell membrane models is undebatable, studies using Langmuir allowed for solvent evaporation, mobile barriers were actioned to monolayers to investigate the action of AgNPs are still scarce compress the air–water interface at a rate of 5 Å2molecule1 min1. [17–20]. For instance, Giron et al. [18] proposed that the expansion For mixed lipid-AgNPs monolayers, at least 30 min were waited for of dimyristoylphosphatidylcholine (DMPC) lipid monolayers nanoparticle homogenization. The surface pressure (p) were caused by AgNPs depends on the nature of the molecules that measured as long as the film area (A) decreased, obtaining p-A iso- capped the AgNPs. Also, highly-concentrated AgNPs stabilized with therms. For polarization modulation infrared reflection–absorption a polyether block polymers amide and dispersed in ionic liquids spectroscopy (PM-IRRAS) studies, the monolayer was compressed are reported to affect the rheological properties of lipid monolayers until 30 mN/m. This surface pressure was maintained during the at the air–water interface [17], showing that not only is the effect obtainment of the spectra. For that, a KSV PMI 550 instrument dependent on the capping molecule, but also on the dispersing (KSV Instruments, Ltd., Helsinki, Finland), that operates with a medium. modulation frequency of 84 kHz, and an incidence angle to the nor- In this paper, we employed lipid Langmuir monolayers to mimic mal of 75was employed. A minimum of 6000 scans were obtained the first barrier encountered by silver nanoparticles dispersed in for each spectrum, with a resolution of 8 cm1, and the incoming water and stabilized with PVP. In a previous study [21], it was lightwas continuouslymodulated between the p and s polarization, shown that the PVP-capped AgNPs were quite effective in inhibit- allowing simultaneous measurements of the spectra for both ing the growth of E. Coli. Moreover, when combined with an antibi- polarizations. Control experiments using only the capping polymer otic from the group of tetracyclines, the PVP-AgNPs displayed (without Ag) were performed in order to check the role of PVP on enhanced inhibitory properties. It would be of interest to decouple the surface activity of the dispersions containing the nanoparticles. such effect by investigating the role of the AgNPs alone. To that PVP solutions, at the same concentration of the dispersions, were end, we employed a myriad of lipids such as dipalmitoylphosphati co-spreadwith the lipids in a similar procedure employed forAgNPs, dylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and tensiometry and vibrational experiments were performed in dimethyldioctadecylammonium bromide (DODAB), dioleoylglyc- order to verify their specific surface activity on lipid monolayers. erophosphocholine (DODAB), cholesterol, as well as mixtures of Brewster Angle Microscopy (BAM) images were obtained with them. We also investigated the role of peptidoglycans and a micro-BAM from KSV-Instruments at a surface pressure of lipopolysaccharides since these substances are encountered at 30 mN/m. This value of surface pressure was chosen to investigate the peptidoglycan cell wall or periplasmic space of gram-positive the monolayer by PM-IRRAS and BAM because it corresponds to and gram-negative bacteria as well as on the surface of the other the lateral pressure of cell membranes [24]. lipidic membrane of gram-negative bacteria. All experiments were carried out at a controlled room temper- ature (25 ± 1 C). Each surface pressure-area isotherm, spectrum, and BAM image was obtained at least three times to ensure the 2. Materials and methods reproducibility of the experiments. In this paper, only isotherms, spectra, and images that were highly reproducible are shown. The water employed in this work was purified using a MilliQ- Plus system (resistivity 18.2 MX cm, pH 5.5). The lipids dipalmi toylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol 3. Results and discussion (DPPG), dimethyldioctadecylammonium bromide (DODAB), dioleoylglycerophosphocholine (DOPC), and cholesterol were pur- Fig. 1A shows a UV–vis spectrum of the colloidal AgNps chased from Sigma-Aldrich and each one was dissolved in chloro- prepared using PVP, which concomitantly served as reducing and 140 794 R.L. Cruz Gomes da Silva et al. / Journal of Colloid and Interface Science 512 (2018) 792–800 A B 160 120 80 40 0 5 10 15 20 25 30 35 40 size/nm 300 350 400 450 500 550 600 λ / nm Fig. 1. (A) UV–vis spectrum of PVP-capped AgNPs. (B) TEM image of the AgNPs. Inset: size distribution obtained by measuring 500 particles. stabilizing agent in alkaline medium and at room temperature monolayers at the air–water interface seems to be so far indepen- (25 C). It has been demonstrated [21] that molecules containing dent of the chemical nature of the lipid. a carbonyl group prone to be nucleophilically attacked by hydroxyl It is important to emphasize that the isotherms progressively in high-pH media can generate alkoxides which are the actual shift to lower molecular areas with increasing amount of AgNPs, responsible for the reduction of silver ions. Since PVP contains a but no further condensation of the monolayers was observed for ketone-carbonyl group that can undergo nucleophilic addition, it concentrations higher than shown in the isotherms. This means is able to reduce silver and gold ions, as also shown elsewhere that we worked with a concentration value near the point of [22,25]. The colloidal AgNps had a single maximum absorbance saturation of the effect. It is also important to mention that no at around 410 nm, a value that has been associated with spherical hysteresis was observed in cycles of compression-expansion for silver nanoparticles. The TEM of AgNPs in Fig. 1B indicates that monolayers of all chemical compositions employed in this work most of the nanoparticles obtained from the process were spherical (not shown). This suggests that the film decompression provides in shape. The inset in Fig. 1B shows that the majority of AgNPs have a rapid return of the floating film to the less condensed states of sizes in the range of 16–30 nm. the lipid monolayer. The absence of hysteresis also indicated that a supposed desorption of lipids from the air–water interface by 3.1. Pure lipid monolayers the AgNPs is improbable given that viscoelastic effects related to desorption/re-adsorption would probably lead to a hysteresis Fig. 2A shows the effect of AgNPs on the surface pressure-area effect. Furthermore, it is important to report that the incorporation isotherms of selected lipids. Particularly, DPPC is a zwitterionic of pure PVP (the capping agent) has no significant impact on the lipid that has been employed in several kinds of cell models at monolayers, indicating that the effects observed so far must be the air–water interface, including mammalian [26] and microbial exclusively originated from the supramolecular system consisting [27] ones. The typical isotherm for pure DPPC features a plateau of Ag/PVP nanoparticles-lipid. between 65 and 45 Å2 indicating the transition between the states In order to better investigate the chemical nature of the interac- named liquid-expanded and liquid-condensed. The presence of the tion between the AgNPs and the lipid monolayers, the air–water AgNPs shifted the isotherm to lower areas denoting the condensa- interface of the systems were studied via infrared spectroscopy. tion of the monolayer, suggesting that at these conditions the To simplify the discussion, in Fig. 3, we present only the spectra nanoparticles may not penetrate between the alkyl chains of the for the systems involving DPPC, and for the other lipids we present lipid, which would otherwise lead to the expansion of the mono- their respective spectra in the Supplementary Materials Section. layer (shift to higher molecular areas). Therefore, the AgNPs should Panel A focus on the 2800–3000 cm1 region, where the main interact with the polar heads of the lipid minimizing the lateral bands for the alkyl chain of the lipids appear in the PM-IRRAS spec- repulsion between adjacent lipids. Such effect is similar for the tra. Usually, two main bands are found in this region, one centered other lipids, (Fig. 1 – Panels B-E). DPPG is a negatively charged lipid at approximately 2850 cm1, attributed to CAH symmetric largely employed as model for bacterial membranes [28], and stretches of CH2, and another centered at about 2920 cm1, attrib- DODAB is a synthetic positively charged non-naturally occurring uted to CAH antisymmetric stretches of CH2. Other bands may lipid, employed to contrast with the effect for the negative charged appear in this region and are usually assigned to CAH stretches lipids. For both cases, the result is similar in terms of degree of con- of CH3, being their presence in the PM-IRRAS spectra usually densation, showing that this effect is not, at a first sight, directly assigned to a degree of fluidity or disorder of the monolayer [30]. dependent on the charge of the lipid polar head. Although AgNPs were initially assumed not penetrate in the alkyl Another aspect worth investigating is the interaction of the chains of the lipids at the air–water interface, the CAH bands are AgNPs with a more fluid monolayer. To this end, DOPC has been remarkably affected by the presence of the nanoparticles. For selected as a representative lipid since it contains an unsaturation instance, for DPPC, the two main bands are shifted to higher in each one of its alkyl chains that gives fluidity to the monolayer. wavenumbers (to 2944 and 2867 cm1) as consequence of mono- Once more we observe (Panel D) the condensation of the film due layer disordering [30]. For the other lipids, distinctive changes to the presence of the nanoparticle, as also observed for cholesterol are noted in the profile of the spectra upon incorporation of the (Panel E), which is a lipid with a known effect of film condensing AgNPs. For DPPG, the intensity of the band centered at 2839 when in some lipid mixtures [29]. This fact then shows that the cm1 was lower compared to that at 2919 cm1 upon incorpora- condensation of the monolayer upon AgNP incorporation is neither tion of AgNPs. For DODAB, the band centered at 2916 cm1 split dependent on the fluidizing nor the condensing nature of the lipid. in two new bands. For DOPC and cholesterol the bands were Therefore, such condensing effect of the nanoparticles on lipid ill-defined, probably due to the high degree of fluidity of DOPC 141 Abs (a.u.) Frequency R.L. Cruz Gomes da Silva et al. / Journal of Colloid and Interface Science 512 (2018) 792–800 795 70 A 60 B 60 DPPC 50 DPPG 50 40 40DPPC+ Ag 30 30 DPPG+Ag 20 20 10 10 0 0 20 30 40 50 60 70 80 90 Area per molecule (Å2) 20 40 60 80 100 120 Area per molecule (Å2) C 50 D 40 40 DOPC+Ag DODAB 30 30 DODAB+Ag DOPC 20 20 10 10 0 0 40 50 60 70 80 90 100 110 120 60 80 100 120 140 160 180 Area per molecule (Å2) Area per molecule (Å2) 50 E 40 30 Cholesterol+Ag 20 Cholesterol 10 0 38 40 42 44 46 48 50 52 Area per molecule (Å2) Fig. 2. Surface pressure-area isotherms for monolayers of pure lipids (as indicated on the graphs) without and with AgNPs in the aqueous subphase (indicated as Ag on the charts). and the lack of linear alkyl chains in cholesterol (as it is a cycloalk- Fig. 3B shows the PMIRRAS for the 1000–1800 cm1 region ane). Nonetheless, general changes in the profile of the spectra are where main bands related to the hydrophilic regions of DPPC observed when the AgNPs adsorb on the monolayers. Therefore, appear. For DODAB and cholesterol there are no relevant bands even considering that the AgNPs do not penetrate the alkyl chains in that region. For DPPC, DOPC and DPPC, bands related to vibra- of the lipids, their interaction with the polar heads must affect the tions of phosphate and carbonyl groups (absent for DODAB and organization and ordering of the lipids at the air–water interface. cholesterol) are present. For DPPC, particularly (Fig. 3B), the band The condensation of the monolayer forces the alkyl chains to centered at 1723 cm1 is related to the carbonyl stretching mode approach each other and the consequent increase in the degree of the lipid and splits in two bands upon AgNP adsorption. Between of packing and decrease of the compressibility moduli alters the 1000 and 1300 cm1 various bands related to P@O appear, and for PMIRRAS spectra related to the CAH stretching mode region. pure DPPC, a band centered at 1090 cm1 is prominent. The P@O 142 Surface Pressure (mN/m ) Surface Pressure (mN/m ) Surface Pressure (mN/m ) Surface Pressure (mN/m ) Surface Pressure (mN/m ) 796 R.L. Cruz Gomes da Silva et al. / Journal of Colloid and Interface Science 512 (2018) 792–800 A 2944 50 A2912 2867 2850 40 DPPC + Cholesterol + Ag DPPC + Ag 30 DPPC + Cholesterol 20 DPPC 10 0 3000 2950 2900 2850 2800 Wavenumber (cm-1) 30 40 50 60 70 80 90 Area per molecule (Å2 ) B 1723 50 B 1190 1090 40 DPPC + Ag 30 DPPC + DOPC 20 DPPC + DOPC + Ag DPPC 10 1800 1600 1400 1200 1000 0 Wavenumber (cm-1) 30 40 50 60 70 80 90 100 110 120 Fig. 3. PMIRRAS for monolayers for DPPC monolayers without and with AgNPs in Area per molecule (Å2 ) the aqueous subphase (indicated as Ag on the charts). Fig. 4. Surface pressure-area isotherms for monolayers of mixed DPPC:Cholesterol (1:1 M proporation) (A), DPPC:DOPC (1:1 M proporation) (B) without and with AgNPs in the aqueous subphase (indicated as Ag on the charts). band disappears with nanoparticle incorporation and small bands appear in this region, however centered at other wavenumbers. Other bands appear between 1300 and 1700 cm1 and are assigned shifted to lower areas as they did for the pure lipids. For the other to CH2 and H2O bending modes. An intense band near 1600 cm1 is mixed monolayers employed in this work (Fig. 6B) the effect is attributed to water bends and it is usually related to an artefact similar. In this case, we employed a mixture of DPPC with a due to difference of reflectivity between the air–water interface fluidizing lipid: DOPC. This composition was chosen because it is covered and uncovered by the monolayer [31]. Since all spectra a representative parameter to investigate the effect of the nanopar- are subtracted from the spectrum of the pure air–water interface, ticles on a mixture between a lipid that forms a well-packed mono- bands related to interfacial water molecules are improbable in layer and another that forms a fluid monolayer. For pure DOPC the principle. However, possible reorganization of interfacial water condensation is significant, but for the mixed DPPC-DOPC mono- molecules after monolayer formation may lead to the appearance layer the shift to lower areas occurs to greater extension, revealing of water vibration bands, interfering in the spectrum. For DPPG that the lipid with a more fluid nature, such as DOPC, favors the and DOPC (Supplementary Materials Section), the bands in this condensing effect of the nanoparticles. region also suffer some sort of alteration as well, mainly for car- PM-IRRAS spectra for the mixed monolayers (Supplementary bonyl and phosphate groups, two representative regions in the Materials Section) also show alteration in bands for asymmetric hydrophilic region of these phospholipids, indicating a remarkable and symmetric CH2 stretches (2919 and 2854 cm1, respectively). interaction of the AgNPs with these groups. As for the nanoparticles in the pure lipid monolayers, the effects are somewhat similar: the AgNPs may adsorb on the polar heads 3.2. Mixed lipid monolayers of the lipid without penetrating the monolayer. This affects the polar heads of the lipid as shown the PM-IRRAS, and may indirectly In Fig. 4, we discuss a series of results obtained from selected affect the alignment of the alkyl chains, distorting the apolar tails mixed monolayers. Fig. 4A shows isotherms for mixed monolayers and also changing the spectra for the CH2 stretching mode region. of DPPC and cholesterol (1:1 M proportion). It is known that cholesterol and DPPC present attractive interactions at the air–wa- 3.3. Mixed monolayers with glycosidic compounds ter interface [29], causing the molecular areas of the isotherms to present values that are lower than those expected by the rule of The bacteria cell envelope is a multilayered structure that additivity. With AgNPs, the isotherm for the mixed monolayer protects these organisms from outside hostilities. For instance, 143 PM-IRRAS signal (arb. units) PM-IRRAS signal (arb. units) Surface Pressure (mN/m) Surface pressure (mN/m) R.L. Cruz Gomes da Silva et al. / Journal of Colloid and Interface Science 512 (2018) 792–800 797 60 A 200 A 175 50 DPPC 150 DPPC + Ag 40 125 DPPC 30 DPPC+GP 100 DPPC+GP+Ag 20 75 10 50 0 25 20 40 60 80 100 120 140 160 020 30 40 50 60 70 80 90 100 Area per molecule (Å2) Area per molecule (Å2) 200 60 B B DPPC 50 150 DPPC + PG 40 DPPC + PG + Ag 100 30 50 20 10 0 0 0 10 20 30 40 50 60 70 60 80 100 120 140 160 Surface Pressure (mN/m) Area per molecule (A2) Fig. 6. Surface Elasticity-Area (A) and –Surface Pressure (B) isotherms for DPPC and DPPC) without and with AgNPs in the aqueous subphase (indicated as Ag on the 60 charts). C 50 DPPC of peptidoglycans from Bacilus subtilis I (PG) and lipopolysaccha- 40 rides from E. Colli (LPS) representing the major components of DPPC + LPS bacteria’s cell wall and outer membrane, respectively. In addition, 30 DPPC + LPS + Ag to represent glycosidic compounds from human beings, we employed mixtures of DPPC with laminin (GP), a major glycopro- 20 tein of the extracellular matrix of basement membranes. Results for GP are presented in Fig. 5A. The adsorption of GP on the DPPC monolayer shifts the isotherms to higher areas as a 10 consequence of its incorporation into the lipid chains. At surface pressures higher than 35 mN/m, the expansion of the monolayer 0 is negligible, which suggests the expelling of GP towards the aque- ous subphase, probably interacting with the polar heads of the 40 60 80 100 120 140 160 2 lipids. With the presence of AgNPs in the aqueous subphase, weArea per molecule (Å ) observe a shift of the mixed GP-DPPC monolayer isotherm to higher areas at low surface pressures, also indicating the expansion Fig. 5. Surface pressure-area isotherms for DPPC monolayers with GP (2.5  103 mg/L) (A), DPPC monolayers with PG (2.5103 mg/L) incorporated from of the monolayer. Such expansion is less significant for surface the aqueous subphase (B), and DPPC monolayers with LSP (2,5. 103 mg/L) pressures higher than 5 mN/m. This was the first result indicating incorporated from the aqueous subphase (C) without and with AgNPs in the an expansion of the monolayer provoked by the adsorption of aqueous subphase (indicated as Ag on the charts). AgNPs instead of a film condensation, as observed for the films composed only by lipids, suggesting a specific interaction of the nanoparticle with glycosidic groups of GP. It is likely that the gram-negative bacteria are enclosed by a thin peptidoglycan cell supramolecular structure formed by AgNPs/GP may facilitate the wall which itself is encased by an outer membrane containing access to the monolayer expanding them at low surface pressures. lipopolysaccharides. Taking this into consideration, we decided to For PG, however, there is no significant alteration in the iso- prepare monolayers composed by DPPC, a major lipid in any cellu- therm for pure DPPC (Fig. 5B). On the other hand, the addition of lar membrane [32] and glycosidic compounds that surround the AgNPs at higher molecular areas leads to the expansion of the plasmatic membrane of bacteria. For that, we employed mixtures monolayer. It is noted surface pressure values higher than zero 144 Surface Pressure (mN/m) Surface Pressure (mN/m) Surface Pressure (mN/m) Surface Elasticity (mN/m) Surface Elasticity (mN/m) 798 R.L. Cruz Gomes da Silva et al. / Journal of Colloid and Interface Science 512 (2018) 792–800 Table 1 PM-IRRAS spectra were also obtained (Supplementary Materials Values of Surface Elasticity obtained from the p-A isotherms for the monolayers Section) and they show that AgNPs interact with the monolayer via studied in this work at a surface pressure of 30 mN/m. glycosidic and peptide chains of laminin, which may be related to E at 30 mN/m (mN/m) E at 30 mN/m (mN/m) the expansion of the monolayer for DPPC/GP films instead of the without AgNPs with AgNPs condensation as observed for films constituted only by lipids. DPPC 120 100 DPPG 94 125 3.4. Rheological properties of the monolayers DODAB 96 102 Cholesterol 950 705 DOPC 142 95 Fig. 6 and Table 1 show the impact of AgNPs on the rheological DPPC+Cholesterol 360 301 properties of the lipid monolayers employed in the present study. DPPC+DOPC 99 90 The surface elasticity (E), also known as compressional modulus, DPPC+GP 79 70 can be estimated by the surface pressure-area (p-A) isotherms DPPC+PG 351 332 DPPC+LPS 222 210 using the expression: ¼  @pE A @A T even for DPPC molecular areas as high as 160 Å2. Overall, this This parameter is important because the flow and shear behavior of indicates a distinctive mechanism of interaction between the the monolayer is dependent on the phase properties of monolayers nanoparticle and the mixed monolayer with a compound contain- that often present viscoelastic features [33]. In addition, the ing a glycosidic chain. With monolayer compression this expansion literature suggests [34] that the lateral compressibility instead of is suppressed, probably due to molecular accommodation. In the lateral pressure is the relevant quantity for partitioning of mole- Fig. 5C a similar behavior is observed for the lipopolysaccharide cules and their conformational changes into biological membranes. (LPS). The amount inserted in the DPPC monolayer changed only In general, biological membranes are fluid but with densely- slightly the surface pressure-area isotherm. The addition of AgNPs, aggregated surface domains that regulate the viscoelastic properties on the other hand, led to the expansion of the monolayer at higher of the interface. For DPPC (Fig. 6A), we note that it reaches high areas. This behavior is, therefore, very similar to that for PG. values of elasticity (as high as 170 mN/m) featuring the Fig. 7. BAM images obtained at 30 mN/m for the monolayers as indicated. 145 R.L. Cruz Gomes da Silva et al. / Journal of Colloid and Interface Science 512 (2018) 792–800 799 liquid-condensed state. Concerning the E-A isotherm, a region monolayers at the air–water interface. Bothun et al. examined the between 56 and 50 Å2 with neglecting elasticity corresponds to interactions between polymer coated anionic (AgCOOH) and the first order transition between the liquid-expanded and liquid- cationic (AgNH) silver nanoparticles and mixtures of anionic condensed phases. Upon addition of AgNPs, it is clear the shift to and zwitterionic lipids [17], and observed that the interaction lower areas due to the condensation of the monolayer with an evi- with the lipids monolayers depends on the charge of the dent relative increase of elasticity values at the liquid-condensed nanoparticle surface and the nature of the lipid monolayers. Par- regime between 10 and 50 mN/m (see panel B). At 30 mN/m, for ticularly, they reported that AgCOOH inserted into the monolay- instance, the elasticity increases from 120 to 100 mN/m, indicating ers caused lipid condensation at high initial surface pressures (20 the formation of a more rigid monolayer (less compressible). Only and 30 mN/m). They suggested that electrostatic and charge- close to the collapse do we note higher values of elasticity for the dipole interactions with the lipids were responsible for condensa- mixed system in relation to the pure lipid monolayer (better visual- tion. Girón et al. [18] showed that AgNPs coated with different ized in panel A). molecules such as citrate (CIT-AgNPs) and 4-mercaptobenzoic Consistently, the condensation of the DOPC and cholesterol acid (MBA-AgNPs), both negatively charged at physiological pHs, monolayers (Table 1) can be also directly related to the increase interact in a different way with DMPC Langmuir monolayers in their surface elasticity. In contrast, for DPPG and DODAB the sur- taken as model biomembrane. At low surface pressures, both face elasticity increases upon AgNPs incorporation at 30 mN/m. types of AgNPs interact in a similar way, expanding the phospho- This suggests a direct dependence on the charge of the polar lipid molecular area. However, CIT-AgNPs are excluded from the groups of the lipid given that DPPG is a negatively charged lipid interface upon compression, whereas MBA-AgNPs remain and DODAB is a positively one. For the mixed lipid monolayer adsorbed. They reported neither penetration nor changes in the (DPPC/cholesterol and DPPC/DOPC), we have also observed an reflectivity of DMPC, as observed from BAM images, which increase in the elasticity values, reinforcing the fact that AgNPs pointed out that the interaction was confined to the AgNP/DMPC incorporated in monolayers of lipids with no net charges make interface. Anaya et al. [19] employed membrane lipid extracts the monolayer more rigid and therefore less compressible. from chemostat cultures exposed to casein-coated Ag nanoparti- For the lipid monolayer containing glycosidic compounds, the cles and observed that AgNPs condensed the films, causing rigid- elasticity also increased, indicating that GP, PG and LPS may neu- ity in the floating monolayers. Although our paper is specific for tralize the charges existing in the lipid polar heads, and the incor- PVP-capped AgNPs, it is notable that the previous papers on other poration of the AgNPs into the mixed monolayer may make the AgNPs report mainly the condensation of lipid films formed at the film more rigid, as already observed for the neutral lipids. air–water interface as a main effect. It is clear therefore the inability of these nanoparticles to penetrate into the hydrophobic 3.5. Morphology of the monolayers moieties of the lipids, whose activity is restricted on the hydro- philic surface. Herein, we found similar results in terms of Fig. 7 shows BAM images of the monolayers studied in this surface-pressure-area isotherms for several types of lipids as well work. As previously reported [35], for pure DPPC, DPPG and DOPC as their mixtures. These results highlight therefore the impor- there is no phase contrast at 30 mN/m. The addition of AgNPs led tance of understanding the interactions between metallic to no alterations in the morphology, except for small bright nanoparticles and components of bacterial and mammalian mem- domains in the case of DOPC. In contrast, the heterogeneous mor- branes at the molecular level, which may be important to assess phology observed for cholesterol monolayers is due to the irregular potential long-term toxicological effects. packing of the monomolecular film during the interfacial compres- sion, which may cause superposition of layers. Although hetero- geneity remains upon addition of AgNPs, the pattern is 4. Conclusions somewhat distinct as denoted by bright domains distributed along the surface. For mixed monolayers, the films were found to be In this work, we discovered that silver nanoparticles stabilized more irregular in terms of morphology and distribution of aggre- with PVP affect the physicochemical properties of floating lipid gates. For DPPC-cholesterol monolayers, some fractal structures monolayers at the air–water interface. Tensiometry and vibrational appear due to defects from non-homogeneous mixing between spectroscopy showed that AgNPs condense DPPC, DPPG, DODAB, the lipids. Although AgNPs bring certain degree of homogeneity, cholesterol and DOPC monolayers through the interaction of the some domains appeared and the pattern of reflectivity is quite dis- hydrophilic groups in the lipids. Additionally, the condensation tinctive. For the DPPC-DOPC monolayer, the surface is also homo- affects the organization of the alkyl chains of the lipids, as observed geneous with various small domains distributed along the surface. by PM-IRRAS. While this behavior is somewhat indistinguishable The higher fluidity of the DOPC must have mitigated the formation for lipids of different natures in terms of chemical structure, net of defects upon monolayer compression. With AgNPs the surface charge and degree of fluidity, mixtures with glycosidic compounds density of these small aggregates seems to increase as a conse- presented a divergent expansion of the monolayer, pointing to a quence of the higher stabilization already observed by the surface specific interaction. The morphological in situ analysis of the air– pressure-area isotherms, where a more considerable condensation water interface showed the degree of homogeneity was highly of the Langmuir film is noted. The heterogeneous monolayer com- dependent on the chemical nature of the floating monolayer. This posed of DPPC and the glycoprotein laminin (GP) tends to become fact basically differs from other AgNPs dispersed in aqueous med- homogenized upon incorporation of AgNPs due to the GP incorpo- ium with other capping agents, that shown basically condensation ration into the DPPC alkyl chains. With PG and LPS, on the other of the lipid monolayers [18–20]. This present work therefore brings hand, the monolayer is homogeneous. Although the effects of the following new concepts and innovations: AgNPs on DPPC mixtures are similar in terms of tensiometry and molecular spectroscopy, the morphology strongly depends on the (i) The impact of PVP-AgNPs on the surface activity of lipid glycosidic derivative employed. monolayers dispersed in water depends on the chemical Overall, this paper shows that the impact of AgNPs on the composition of the film. monolayers herein studied depends strongly on the nature of (ii) PVP-AgNPs in cell membrane models at the air–water inter- the lipids and glycosides. It is important to emphasize that the lit- face affect both hydrophobic and hydrophilic regions, with a erature shows some few examples of AgNPs interacting with lipid prominent effect in the hydrophilic region of the lipid layer. 146 800 R.L. Cruz Gomes da Silva et al. / Journal of Colloid and Interface Science 512 (2018) 792–800 (iii) Disrupting of the structure of the lipid monolayer associated Langmuir monolayers as cell membrane models, Thin Solid Films 593 (2015) to the expansion of the monolayer was observed only when 158–188. [13] R.P. Carney, Y. Astier, T.M. Carney, K. Voïtchovsky, P.H. Jacob Silva, F. Stellacci, glycosidic compounds are present at the film. Electrical method to quantify nanoparticle interaction with lipid bilayers, ACS (iv) Vibrational spectroscopy coupled with BAM studies showed Nano 9 (2013) 932–942. that the morphological and structural properties of the [14] M.R. de Planque, S. Aghdaei, T. Roose, H. Morgan, Electrophysiological characterization of membrane disruption by nanoparticles, ACS Nano 5 monolayer with AgNPs adsorbed are modulated by the com- (2011) 3599–3606. ponents of the floating film. [15] E. Rascol, J.M. Devoisselle, J. Chopineau, The relevance of membrane models to (v) The interaction of AgNPs with lipids depends on the charge understand nanoparticles-cell membrane interactions, Nanoscale 8 (2016) 4780–4798. of the polar head groups which, in turn, directly affects the [16] B. Brockman, Lipid monolayers: why use half a membrane to characterize membrane elasticity. protein-membrane interactions?, Current Opin Struct. Biol. 9 (1999) 438–443. [17] G.D. Bothun, N. Ganji, I.A. Khan, A. Xi, C. Bobba, Anionic and cationic silver This work helps understand how metallic nanoparticles dis- nanoparticle binding restructures net-anionic PC/PG monolayers withsaturated or unsaturated lipids, Langmuir 33 (2017) 353–360. persed in aqueous media stabilized with polymers affect lipid [18] J.V. Maya Girón, R.V. Vico, B. Maggio, E. Zelaya, A. Rubert, G. Benítez, P. Carro, R. interfaces that mimic cellular membranes or bacteria’s cell wall. C. Salvarezza, M.E. Vela, Role of the capping agent in the interaction of These results may be used to correlate with the microbicide activ- hydrophilic Ag nanoparticles with DMPC as model biomembrane, Environ. Sci-Nano 2 (2016) 462–472. ity or long-term toxicological effects of AgNPs in live systems. [19] N.M. Anaya, F. Faghihzadeh, N. Ganji, G. Bothun, V. Oyanedel-Craver, Comparative study between chemostat and batch reactors to quantify Acknowledgements membrane permeability changes on bacteria exposed to silver nanoparticles, Sci. Total Environ. 565 (2016) 841–848. [20] G.B. Soriano, R.S. Oliveira, F.F. Camilo, L. Caseli, Interaction of non-aqueous We thank FAPESP (2015/10253-0) and CNPq (projects dispersions of silver nanoparticles with cellular membrane models, J. Colloid 400896/2016-8 and 442087/2014-4) for the research grants. Rafael Interf. Sci. 496 (2017) 111–117. [21] H.F.O. Silva, K.M.G. Lima, M.B. Cardoso, J.F.A. Oliveira, M.C.N. Melo, C. Cruz receives a scholarship from FAPESP (2015/09586-3). Sant’Anna, M. Eugenio, L.H.S. Gasparotto, Doxycycline conjugated with polyvinylpyrrolidone encapsulate silver nanoparticles: a polymer’s Appendix A. Supplementary data malevolent touch against Escherichia Coli, RSC Adv. 5 (2015) 66886–66893.[22] A.C. Garcia, L.H.S. Gasparotto, J.F. Gomes, G. Tremiliosi-Filho, Straightforward synthesis of carbon supported Ag nanoparticles and their application for the Supplementary data associated with this article can be found, in oxygen reduction reaction, Electrocatalysis 3 (2012) 147–152. the online version, at https://doi.org/10.1016/j.jcis.2017.10.091. [23] J.F. Gomes, A.C. Garcia, E.B. Ferreira, C. Pires, V.L. Oliveira, G. Tremiliosi-Filho, L. H.S. Gasparotto, New insight into the formation mechanism of the Ag, Au and AgAu nanoparticles in aqueous alkaline media: alkoxides from alcohols, References aldehydes and ketones as the universal reducing agent, Phys. Chem. Chem. Phys. 17 (2015) 21683–21693. [1] S. Leon-Silva, F. Fernandez-Luqueno, F. Lopez-Valdez, Silver Nanoparticles [24] A. Blume, A comparative study of the phase transitions of phospholipid (AgNP) in the Environment: a Review of Potential Risks on Human and bilayers and monolayers, Biochim. Biophys. Acta 557 (1979) 32–34. Environmental Health, Water Air Soil Poll. 227 (2016) 306. [25] L.H.S. Gasparotto, A.C. Garcia, J.F. Gomes, G. Tremiliosi-Filho, Electrocatalytic [2] F. Furno, K.S. Morley, B. Wong, B.L. Sharp, P.L. Arnold, S.M. Howdle, R. Bayston, performance of environmentally friendly synthesized gold nanoparticles P.D. Brown, P.D. Winship, H.J. Reid, Silver nanoparticles and polymeric medical towards the borohydride electro-oxidation reaction, J. Power Sources 218 devices: a new approach to prevention of infection?, J Antimicrob. Chemoth. (2012) 73–78. 54 (2006) 1019–1024. [26] N. Hussein, C.C. Lopes, P.C.A. Pernambuco Filho, B.R. Carneiro, L. Caseli, Surface [3] R. Singh, H.S. Nalwa, Medical applications of nanoparticles in biological chemistry and spectroscopy studies on 1,4-naphthoquinone in cell membrane imaging, cell labeling, antimicrobial agents, and anticancer nanodrugs, J. models using Langmuir monolayers, J. Colloid Inter. Sci. 402 (2013) 300–306. Biomed. Nanothecnol. 7 (2011) 489–503. [27] T.E. Goto, L. Caseli, The interaction of mefloquine hydrochloride with cell [4] N. Durán, M. Durán, M.B. de Jesus, A.B. Seabra, W.J. Fávaro, G. Nakazato, Silver membrane models at the air–water interface is modulated by the monolayer nanoparticles: A new view on mechanistic aspects on antimicrobial activity, lipid composition, J. Coll. Inter. Sci. 431 (2014) 24–30. Nanomed. Nanotechnol. Biol. Med. 12 (2016) 789–799. [28] R.F. Epand, P.B. Savage, R.M. Epand, Bacterial lipid composition and the [5] I. Sondi, B. Salopek-Sondi, Silver nanoparticles as antimicrobial agent: a case antimicrobial efficacy of cationic steroid compounds (Ceragenins), Biochim study on E. coli as a model for Gram-negative bacteria, Silver nanoparticles as Biophys. Acta: Biomembr. 1768 (2007) 2500–2509. antimicrobial agent: a case study on E. coli as a model for Gram-negative [29] C. Ohe, T. Sasaki, M. Noi, Y. Goto, K. Itoh, Sum frequency generation bacteria, J. Colloid Interf. Sci. 275 (2004) 177–182. spectroscopic study of the condensation effect of cholesterol on a lipid [6] J.R. Morones, J.L. Elechiguerra, A. Camacho, K. Holt, J.B. Kouri, J.T. Ramírez, M.J. monolayer, Anal. Bioanal. Chem. 388 (2007) 73–77. Yacaman, The bactericidal effect of silver nanoparticles, Nanotechnology 16 [30] A. Dicko, H. Bourque, M. Pézolet, Study by infrared spectroscopy of the (2005) 2346–2353. conformation of dipalmitoylphosphatidylglycerol monolayers at the air–water [7] K. Nomiya, A. Yoshizawa, K. Tsukagoshi, N.C. Kasuga, S. Hirakawa, J. Watanabe, interface and transferred on solid substrates, Chem. Phys. Lipids 96 (1998) Synthesis and structural characterization of silver(I), aluminium(III) and cobalt 125–139. (II) complexes with 4-isopropyltropolone (hinokitiol) showing noteworthy [31] D. Blaudez, J.M. Turlut, J. Dufourcq, D. Bard, T. Buffeteau, B. Desbat, biological activities. Action of silver(I)-oxygen bonding complexes on the Investigations at the air/water interface using polarization modulation IR antimicrobial activities, J. Inorg. Biochem. 98 (2004) 46–60. spectroscopy, J. Chem. Soc. Faraday Trans. 92 (1996) 525–530. [8] A. Gupta, M. Maynes, S. Silver, Effects of halides on plasmid-mediated silver [32] C. Peetla, A. Stine, V. Labhasetwar, Biophysical interactions with model lipid resistance in Escherichia coli, Appl. Environ. Microbiol. 64 (1998) 5042–5045. membranes: applications in drug discovery and drug delivery, Mol Pharm. 6 [9] Q.L. Feng, J. Wu, G.Q. Chen, F.Z. Cui, T.N. Kim, J.O. Kim, A mechanistic study of (2009) 1264–1276. the antibacterial effect of silver ions on Escherichia coli and Staphylococcus [33] D. Vollhardt, V.B. Fainerman, Progress in characterization of Langmuir aureus, J. Biomed. Mater. Res. 52 (2000) 662–668. monolayers by consideration of compressibility, Adv. Colloid Interf. Sci. 127 [10] L. Nover, K.D. Scharf, D. Neumann, Formation of cytoplasmic heat shock (2006) 83–97. granules in tomato cell cultures and leaves, Mol. Cell. Biol. 9 (1983) 1648– [34] D. Marsh, Lateral pressure in membranes, Biochem. Biophys. Acta 1286 (1996) 1655. 183–223. [11] D. McShan, P.C. Ray, H. Yu, Molecular toxicity mechanism of nanosilver, J. Food [35] K.D. Souza, N. Duran, K.R. Perez, G.Z. Justo, L. Caseli, Interaction of violacein in Drug. Anal. 22 (2014) 116–127. models for cellular membranes: regulation of the interaction by the lipid [12] T.M. Nobre, F.J. Pavinatto, L. Caseli, A. Barros-Timmons, P. Dynarowicz-Latka, composition at the air-water interface, Colloid Surf. B: Biointerf. 160 (2017) O.N. Oliveira Jr., Interactions of bioactive molecules & nanomaterials with 247–253. 147 Apêndice G Dual Role of a Ricinoleic Acid Derivative in the Aqueous Synthesis of Silver Nanoparticles. Isadora Dantas Costa, Alcides de Oliveira Wanderley Neto, Heloiza Fernanda Oliveira da Silva, Edgar Perin Moraes, Eryka Thamyris Damascena Nóbrega, Celso Sant’Anna, Mateus Eugenio, Luiz Henrique da Silva Gasparotto. Journal of Nanomaterials, 2017, Vol. 2017, 1-8 Contribuição:  Acompanhei e auxilei a síntese e funcionalização das NanoAg  Coletei os espectros de UV-Visível das amostras de esgoto após contato com as NanoAg funcionalizadas.  Tratei os dados e preparei as figuras relacionadas às etapas que realizei ativamente que foram incluídas no manuscrito.  Participei das discussões pré-escrita do manuscrito. ________________________ __________________________ Heloiza Fernanda O. da Silva Athayde Prof. Luiz Henrique da S. Gasparotto 148 Hindawi Journal of Nanomaterials Volume 2017, Article ID 1230467, 8 pages https://doi.org/10.1155/2017/1230467 Research Article Dual Role of a Ricinoleic Acid Derivative in the Aqueous Synthesis of Silver Nanoparticles Isadora Dantas Costa,1 Alcides de Oliveira Wanderley Neto,2 Heloiza Fernanda Oliveira da Silva,1 Edgar Perin Moraes,1 Eryka Thamyris Damascena Nóbrega,1 Celso Sant’Anna,3 Mateus Eugenio,3 and Luiz Henrique da Silva Gasparotto1 1Biological Chemistry and Chemometrics Research Group, Institute of Chemistry, Federal University of Rio Grande do Norte, Lagoa Nova, 59072-970 Natal, RN, Brazil 2Institute of Chemistry, Federal University of Rio Grande do Norte, Lagoa Nova, 59072-970 Natal, RN, Brazil 3Laboratory of Microscopy Applied to Life Science-Lamav, National Institute of Metrology, Quality and Technology-Inmetro, Duque de Caxias, 25250-020 Rio de Janeiro, RJ, Brazil Correspondence should be addressed to Luiz Henrique da Silva Gasparotto; lhgasparotto@gmail.com Received 8 November 2016; Revised 24 January 2017; Accepted 5 February 2017; Published 23 March 2017 Academic Editor: Piersandro Pallavicini Copyright © 2017 Isadora Dantas Costa et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. We show that sodium 9,10-epoxy-12-hydroxytetradecanoate (SEAR), an epoxidized derivative of ricinoleic acid, simultaneously functioned as reducing and stabilizing agents in the synthesis of silver nanoparticles in alkaline aqueous medium.The advantage of using SEAR is its biodegradability andnontoxicity, which are important characteristics formitigation of environmental impact upon discharge of nanoparticles into terrestrial and aquatic ecosystems. The SEAR concentration was found to impact considerably the size distribution of silver nanoparticles (AgNPs). A concentration below the SEAR critical micelle concentration (CMC) generated 23 nm sized AgNPs with 10 nm standard deviation, while 50 nm sized AgNPs (𝜎 = 21 nm) were obtained at a concentration above the SEAR CMC. FTIR analysis revealed that the carboxylate that constitutes the SEAR hydrophilic head binds directly to the AgNPs surface promoting stabilization in solution. Finally, AgNPs turned into Ag S upon contact with wastewater samples 2 from Wastewater Treatment Plant at Federal University of Rio Grande do Norte (UFRN), Brazil, which is an interesting result, since Ag S is more environmentally friendly than pure AgNPs. 2 1. Introduction metallic ions, despite the fact that most reactions involving it require further safety precautions due to their extreme Silver nanoparticles (AgNPs) have found innumerable appli- exothermic nature [13, 14]. Additionally, NaBH has to be cations that comprise biomedicine [1, 2], food storage [3], 4used in large excess because it undergoes hydrolysis when sensors [4], and (electro)catalysis [5, 6]. Concerning their brought into contact with water and metallic surfaces [15]. synthesis, AgNPsmay be produced via physical [7], biological N,N-dimethylformamide [16] and hydrazine [17] have also [8], or chemical methods [9–11], being the latter more been applied as reducing agents for the production of AgNPs. adequate in terms of cost, simplicity, and quantity of formed It is important to point out, however, that those chemicals AgNPs. Chemicalmethods usually require reducing agents to must be carefully handled since they have been implicated in convert Ag+ into Ag0, followed by nucleation and growth that liver disease [18] and cancer [19], respectively. Another point lead to metallic colloidal nanoparticles. Sodium borohydride worth mentioning is that nanoparticles require some sort of [12] is the most widely used reagent for the reduction of stabilization (electrostatic [20] or steric [21]) to guarantee 149 2 Journal of Nanomaterials O (1) Peracetic acid OH O O OH 500 rpm/3 h H3C (CH2) CH CH5 2 CH CH (CH2)7 C (2) NaOH − +CH O Na3 OH Scheme 1: Epoxidation of ricinoleic acid. their proper availability in solution. Needless to say, it is 2. Experimental also desirable that a stabilizing agent be environmentally- compatible for safe handling of the final product. 2.1. Synthesis of SEAR Castor oil is a vegetable oil extracted from seeds of 2.1.1. Castor Oil Alkaline Hydrolysis and Extraction of Rici- castor bean plant (Ricinus communis L.). Originally used as noleic Acid. The procedure has been adapted from the work a laxative folk medicament, castor oil is nowadays employed by Castro Dantas et al. [32]. 30.0 g of castor oil was refluxed predominantly in cosmetic industries as a cream base in for- for 2 h with 6.0 g of KOH (Sigma-Aldrich) dissolved in a mulations of skinmoisturizers [22]. It is primarily constituted mixture of 60mL of deionized water with 60.0mL of ethanol of ricinoleic acid (approximately 90%), which can then be (Synth).The product was then washed with water and had its transformed into sodium ricinoleate via alkaline hydrolysis pH adjusted to 4.5 with 30% H SO (Sigma-Aldrich). After 2 4 [23]. Teomim et al. [24] hypothesized the use of ricinoleate- adding 20mL diethyl ether (Sigma-Aldrich), the organic based polymers as drug carriers due to their lack of toxicity phase (a mixture of fatty acids) was separated and mixed when tested in rats. Vieira et al. [25] found that topical with 3.0 g of Na SO (Sigma-Aldrich) to remove excess water. 2 4 application of ricinoleic acid exerted exceptional analgesic The mixture was then filtered, the diethyl ether (Synth) was and anti-inflammatory activity in rats. These features would removed in a rotary evaporator, and 50mLof acetone (Sigma- make castor oil derivatives eligible for nanoparticle synthesis, Aldrich) was added to the fatty acid mixture which was then provided that the 18-carbon fatty acid is capable of preventing kept at −10∘C for 48 h to promote solidification of fatty acids aggregation and excessive nanoparticle growth. So far, studies other than the ricinoleic acid. Afterwards, the two-phase have employed castor oil or ricinoleic acid as dispersive mixture was filtered to separate the liquid ricinoleic acid. media for stabilization of a variety of nanostructures that Characterization of the ricinoleic acid by FTIR and NMR include gold nanoparticles produced by sputtering [26] and can be found in Supporting Information (see Supporting wet chemistry process [27], quantum dots fabricated through Information in the Supplementary Material available online thermolysis [28], and silver and gold nanoparticles in paints at https://doi.org/10.1155/2017/1230467). [29], as well as silver nanoparticles synthesized via ablation of metallic silver [30]. A question that remains open is whether 2.1.2. Epoxidation of Ricinoleic Acid and Saponification. Epo- ricinoleic acid/ricinoleate could function concomitantly as xidized ricinoleic acid was synthesized in a two-step process. reducing and capping agents in appropriate experimental Firstly, peracetic acid was obtained by slowly mixing 12.5 g of conditions. acetic anhydride (Sigma-Aldrich) with 11.6mL of hydrogen Herein we showed, for the first time, that sodium peroxide (Sigma-Aldrich) in an ice bath. After 6 the mixture 9,10-epoxy-12-hydroxytetradecanoate (SEAR), an epoxidized was stirred magnetically at 500 rpm for 24 h, 20.0 g of rici- derivative of ricinoleic acid, simultaneously functions as noleic acid was added to it, and the chemicals were allowed reducing and stabilizing agents in the synthesis of silver to react for 3 h in order to obtain the epoxidized ricinoleic nanoparticles in alkaline aqueous medium. Recently [31], we acid. Saponification was then carried out by mechanically showed that molecules bearing hydroxyl groups can reduce stirring the epoxidized ricinoleic acid with 1.2 g of NaOH gold and silver ions in alkaline media via formation of (Sigma-Aldrich) until a white solid was obtained.The process alkoxides that are the actual reducing agents. Although SEAR is briefly presented in Scheme 1. The epoxidized ricinoleic has a hydroxyl group at the C-12 position, it is not capable acidwas then characterizedwith FTIR andNMR (Supporting of reducing Ag+. On the other hand, stable AgNPs were Information). obtained by substituting the double bond of the oil skeleton with an epoxide. The concentration of SEAR influenced the 2.2. Synthesis and Characterization of SEAR-Capped AgNPs. rate of AgNPs formation and their size distribution. Finally, The following stock solutions were prepared for the syn- we studied the behavior of SEAR-capped AgNPs spiked in thesis of AgNPs: 0.10mol l−1 SEAR, 10mM AgNO (Sigma-3 sewage from a local wastewater treatment plant. Changes in Aldrich), and 1.0mol l−1 NaOH (Sigma-Aldrich). In a typical theUV-Vis signal of the AgNPs suggested that these nanopar- experiment at room temperature, 80𝜇l of theAgNO solution 3 ticles may be interesting sensor for chemical oxygen demand is diluted in 1920𝜇l of water. In another vessel, 400𝜇l of the determination. NaOH solution is added to either 40 𝜇l or 400 𝜇l of the SEAR 150 Journal of Nanomaterials 3 solution with the final volume taken to 2.0ml through the and 1(b) show time-dependent UV-Vis spectra of solutions addition of water.The SEAR +NaOHmixture is then poured that resulted from the addition of SEAR to AgNO at room 3 into the diluted AgNO to form AgNPs. temperature and pH 13. Both solutions turned into yellow 3 UV-Vis was performed on an Ocean Optics USB-650 as a consequence of the formation of AgNPs. The yellow Tide spectrophotometer. Transmission electron microscopy color is due to the surface plasmon band (SPB) that resulted (TEM) images were acquired with a FEI Tecnai G2 Spirit from the resonant coherent dipolar oscillations of the electron BioTWIN microscope operating at 120 kV and FTIR in ATR gas (electrons of the conduction band) at the surface of mode was carried out with a Bruker Vertex 70 spectropho- nanoparticles [34].Themaximumwavelength in the colloidal tometer. AgNPs spectra after 24 h was 408 nm regardless of the SEAR concentration. Although the maximum wavelength [35] is a 2.3. Reaction of AgNPs with Local Sewage. A real effluent common parameter used to compare sizes of nanoparticles (chemical oxygen demand = 920.0mg L in a given matrix, a more detailed analysis is provided by the−1) was sampled at the Federal University of Rio Grande do Norte’s wastewater peak width at half maxima (PWHM) as studied by Brown et treatment plant (ETE-UFRN) located in the northeast of al. [36]. According to their concept, the smaller the PWHM Brazil. The wastewater is mostly composed of domestic value the narrower the size distribution of nanoparticles. effluents from the UFRN central campus. In order tomonitor Comparing the PWHM of AgNPs spectra obtained with the reaction between the wastewater sample and AgNPs with 1.0mM and 10.0mM SEAR that have roughly the same UV-Vis spectroscopy, 1.0mL of wastewater was mixed with absorbance (≈1.30), the former and the latter concentrations 1.0mL of deionized water in a 1.0 cm path-length cuvette, resulted in 90 nm and 109 nm, respectively, meaning that followed by addition of 1.0mL SEAR-capped AgNPs. UV-Vis 1.0mM SEAR provided somewhat more uniform AgNPs. spectra were then recorded at distinct times. In a separate This result will be further investigated by TEM. experiment devised to check the constitution of the AgNPs Figure 1(c) presents a plot of the absorbance at 408 nm after reacting with wastewater, 50mL of wastewater was as a function of time for AgNPs produced with 1.0mM (red mixed with 50mL of AgNPs + 50mL of deionized water solid circles) and 10.0mM (black solid squares) SEAR. Inter- and allowed to rest for 15 days. Afterwards, the solution was estingly, the formation ofAgNPs in both cases passed through centrifuged for 15min and the supernatant was discarded. an induction period (nucleation process) of approximately TheAgNPs on the bottomof the flasks were thenwashedwith 40min (Figure 1(c), inset) prior to exponential absorbance deionized water to remove soluble species and centrifuged increase related to autocatalytic growth [37] of AgNPs. for another 15min. The AgNPs pellet was dried at 40 C for Another point is that the absorbances recorded at 1.0mM∘ 4 h and subjected to energy-dispersive X-ray spectroscopy SEAR were slightly higher than those at 10.0mM SEAR up to (EDS). 40min (inset of Figure 1(c)). This is also observed in Figures1(a) and 1(b), where initial spectra from AgNPs produced with 1.0mM SEAR already present a positive curvature, 3. Results and Discussion as opposed to those from 10.0mM. These results may be reasoned by selecting the CMC as the parameter that divides 3.1. Synthesis and Characterization of SEAR-AgNPs. Gomes the two behaviors: before the SEAR CMC of 9.2mM [23], et al. [31] presented strong evidences that alkoxides from SEAR molecules are readily available in solution for the alcohols, aldehydes, and ketones are the actual reducing reduction of Ag+, leading to quick AgNPs nucleation. After species in alkaline media for the generation of silver and the CMC, on the other hand, SEAR molecules incorporated gold nanoparticles. In that study, the authors argued that into micelles must be liberated through disruption of the any molecule capable of generating an alkoxide would be micelle structure in order to make –OH groups available for eligible for reducing Au3+, Ag+, or a mixture of them, to form further reduction of Ag+. Since we are dealing with normal- their respective nanoparticles. In the present case, SEAR has phase micelles, hydrophilic “heads” contact water while the hydroxyl and epoxide groups in its skeleton. Since the exper- hydrophobic tails containing –OHpoint to themicelle center. iments were conducted in alkaline medium, we hypothesized Therefore, we concluded that the nucleation process depends the hydroxyl group on SEAR tail would be deprotonated to on free, soluble SEAR while autocatalytic growth would some extent and the epoxide ring would be open generating require disruption of micelles to liberate the –OH hidden in more hydroxyl groups to act as reducing agents.The following their core. results confirm that SEAR is indeed capable of reducing Figure 2 depicts TEM images of AgNPs produced with silver ions under the conditions employed in the present 1.0mMand 10.0mMSEARwith their respective size distribu- study. It is also important to take into consideration that tions. As seen, the AgNPs were not monodisperse; however, the concentration of the capping agent (in the present study those obtained with 10.0mM SEAR were substantially larger a role also played by SEAR) profoundly impacts on size (51 nm on average) than those from 1.0mM SEAR (21 nm). and shape of nanoparticles, especially if the surfactant forms The standard deviation also followed the same trend (23 nm, micelles [33]. Thus, we have selected SEAR concentrations twice the value of that from 1.0mM SEAR). This result of 1.0mM and 10.0mM, which correspond to concentrations corroborates the PWHM analysis (Figures 1(a) and 1(b)).The below and above the SEAR critical micelle concentration micelle structure seems to have played an important role (CMC) [23], respectively, to evaluate their impact on the in controlling size and distribution of AgNPs. As shown in reduction of Ag+ and posterior stabilization. Figures 1(a) Scheme 2, SEAR interacts with silver through hydrophilic 151 4 Journal of Nanomaterials 4 1.4 SEAR 1.0mM SEAR 10.0mM 1.2 Time 3 1.0 Time 0.8 2 0.6 0.4 1 0.2 0 350 400 450 500 350 400 450 500 Wavelength (nm) Wavelength (nm) (a) (b) 3.5 3.0 2.5 0.40 0.35 2.0 0.30 0.25 1.5 0.20 1.0 0 10 20 30 40 50 0.5 0.0 0 40 80 120 160 Time (min) SEAR 1.0mM SEAR 10.0mM (c) Figure 1: UV-Vis spectra of AgNPs produced with (a) 1.0mM SEAR (red solid circles) and (b) 10.0mM SEAR (black solid squares) recorded 5min apart. (c) Evolution of absorbance at 408 nm. Inset. Magnification of the region between 0min and 50min. Other conditions: [Ag+] = 0.20mM, [OH−] = 0.10M, 𝑇 = 25∘C. the CMC, all SEAR molecules on the AgNPs surface interact through heads with AgNPs and the hydrophobic tails prevent excessive nanoparticle growth through steric hindrance. In contrast, at concentrations higher than the CMC, not all SEAR molecules on the AgNPs surface were directly bound to it because the spherical nature of the micelle makes Ag some SEAR molecules point away from the AgNP surface.Ag The micelle structure may not be as effective as free SEAR molecules in stabilizing Ag nuclei, which then led to larger nanoparticles and broader size distribution probably through Ostwald ripening [38]. Scheme 2: On left and right sides AgNPs are stabilized by SEAR FTIR spectra in ATR mode of pure SEAR and SEAR- below and its above CMC, respectively. capped silver nanoparticles are presented in Figure 3. As the most relevant region of the SEAR spectrum comprising the range of 1200–1800 cm−1, only this region has been analyzed heads (as shown later by ATR-FTIR). It is thus safe to assume in detail. The FTIR spectrum of pure SEAR (black line) that there would be a competition between free and micelle displayed asymmetrical and symmetrical carboxylate stretch- SEAR for Ag adsorption sites. At concentrations lower than ing modes at 1564 cm−1 and 1410 cm−1, respectively [39]. 152 Absorbance Absorbance Journal of Nanomaterials 5 30 20 25 15 20 15 10 10 5 5 0 0 10 20 30 40 50 60 25 50 75 100 125 Size (nm) Size (nm) (a) (b) Figure 2: TEM images of AgNPs obtained with (a) 1.0mM and (b) 10.0mM SEAR. Other conditions: [Ag+] = 0.20mM, [OH−] = 0.10M, 𝑇 = 25∘C. 1.0 SEAR carboxylate and metallic silver. The fact that the asymmetrical stretching mode is shifted to a lower frequency 0.9 suggests that both oxygen atoms from carboxylate donate an electron pair to vacant silver orbitals, decreasing, in turn, 0.8 the double bond character due to interference with the carboxylate resonance system that probably chelated with 0.7 silver symmetrically [40]. Pure SEAR AgNPs have found amyriad of applications that comprise 0.6 c −1 SEAR-AgNPs cosmetics, textiles, and food packaging. Inevitably, waste1564 m −1 1555 cm from those products will end up in terrestrial and aquatic 0.5 ecosystems impacting biotic niche.We have therefore carried out experiments on the behavior of AgNPs in raw sewage 0.4 from a local wastewater treatment plant in order to deter- 1800 1600 1400 1200 mine the fate of the AgNPs upon contact with a real-life Wavenumber ( −1cm ) system. Similar studies conducted with AgNPs stabilized by citrate [41], polyvinylpyrrolidone [41–43], and TWEEN [41] Figure 3: FTIR in ATR mode of pure SEAR (black line) and SEAR- revealed that silver nanoparticles are prone to sulfidation that capped AgNPs (red line). leads to Ag S, which decreased silver nanoparticle toxicity 2 towards a variety of terrestrial eukaryotic organisms [42]. As we envisage the herein produced AgNPs in a variety of Upon stabilization of AgNPs (red line), the center of the applications, it is thus imperative to find out whether they asymmetrical stretching band shifts to 1555 cm−1 and the can be transformed into Ag S in order to mitigate their envi-2 shoulders of the band related to the symmetrical stretching ronmental impact. Figure 4 presents time-dependent UV-Vis are considerably suppressed. This observation suggests that spectra of AgNPs in contact with raw sewage, where AgNPs stabilization of AgNPs occurs via interaction between the produced with 1.0mM SEAR (Figure 4(a)) are consumed 153 Transmittance (%) Frequency (%) Frequency (%) 6 Journal of Nanomaterials 1.0 0–120 1.0min 0–120min Time 0.8 Time 0.8 24 h 0.6 24 h 96 h 0.6 0.4 96 h 240 h 0.4 240 h 0.2 0.2 0.0 300 400 500 600 300 400 500 600 Wavelength (nm) Wavelength (nm) (a) (b) C 1.00 0.95 Slope −3= 1 × 10 0.90 Slope −3= 2 × 10 0 20 40 60 80 100 120 Time (min) 1.0mM SEAR-AgNPS 10.0 mM SEAR-AgNPs (c) Figure 4: UV-Vis spectra of AgNPs produced with (a) 1.0mM SEAR and (b) 10.0mM SEAR in contact with raw sewage. (c) Absorbance recorded at 408 nm as a function of time for AgNPs. more quickly than those synthesized with 10.0mM SEAR conducted on AgNPs that reacted with raw sewage revealed (Figure 4(b)), as revealed by the decrease of the surface plas- the presence of sulfur confirming that sulfidation took place. mon resonance (SPB) with time. After 10 days, the intensities At this point we are not able to determine whether sulfur is of SPB from AgNPs produced with 1.0mM and 10.0mM located only on the surface of theAgNPs or in their interior as SEAR were 0.060 and 0.71, respectively, showing that the well. Although the structure of the silver sulfide nanoparticles latter is somewhat shielded from sulfidation. Figure 4(c) remains elusive (it is beyond the scope of the present study), presents the rate of absorbance decrease at 408 nm for AgNPs the fact that the AgNPs produced herein are indeed prone produced at both SEAR concentrations.The slopes calculated to sulfidation makes them adequate for a variety of real-life for both curves show that the sulfidation is faster with AgNPs applications. from 1.0mMSEAReven in the beginning of the reaction.This result is probably a consequence of a higher concentration 4. Conclusion of SEAR around the particle in the case of 10.0mM SEAR, thus hampering the access of sulfur-containing species to We have shown that SEAR, a surfactant derived from rici- the nanoparticle surface. The nanoparticle size is another noleic acid, is capable of acting as reducing and capping factor that may have played a role in sulfidation. Kaegi et agents in the synthesis of silver nanoparticles in alkaline al. [41] found that smaller silver nanoparticles are subjected medium. The fact that SEAR may form micelles had an to higher degree of sulfidation than bigger nanoparticles, a impact on size and distribution of AgNPs. Finally, in addition result that corroborates those found in the present work. EDS to SEAR being environmentally benign, the toxicological 154 Absorbance Absorbance Absorbance Journal of Nanomaterials 7 effects of AgNPs on the environment are mitigated because [13] J. Zhang, T. S. Fisher, J. P. Gore, D. Hazra, and P. V. Ramachan- AgNPs are subjected to sulfidation upon contact with raw dran, “Heat of reaction measurements of sodium borohydride sewage.These findings may propel these AgNPs into broader alcoholysis and hydrolysis,” International Journal of Hydrogen field of applications such as sensors and environmental Energy, vol. 31, no. 15, pp. 2292–2298, 2006. remediation. [14] S. W. Chaikin and W. G. Brown, “Reduction of aldehydes, ketones and acid chlorides by sodium borohydride,” Journal of the American Chemical Society, vol. 71, no. 1, pp. 122–125, 1949. Competing Interests [15] U. B. Demirci, O. Akdim, J. Andrieux, J. Hannauer, R. The authors declare that they have no competing interests. Chamoun, and P. Miele, “Sodium borohydride hydrolysis ashydrogen generator: issues, state of the art and applicability upstream from a fuel cell,” Fuel Cells, vol. 10, no. 3, pp. 335–350, References 2010. [16] I. Pastoriza-Santos and M. Liz-Marzán, “Formation and sta- [1] C.Marambio-Jones and E.M. V.Hoek, “A review of the antibac- bilization of silver nanoparticles through reduction by N,N- terial effects of silver nanomaterials and potential implications dimethylformamide,”Langmuir, vol. 15, no. 4, pp. 948–951, 1999. for human health and the environment,” Journal of Nanoparticle [17] Y. Li, Y. Wu, and B. S. Ong, “Facile synthesis of silver nanopar- Research, vol. 12, no. 5, pp. 1531–1551, 2010. ticles useful for fabrication of high-conductivity elements for [2] H. F. O. Silva, K. M. G. Lima, M. B. Cardoso et al., “Doxycy- printed electronics,” Journal of the American Chemical Society, cline conjugated with polyvinylpyrrolidone-encapsulated silver vol. 127, no. 10, pp. 3266–3267, 2005. nanoparticles: a polymer’s malevolent touch against Escherichia [18] C. A. Redlich, W. S. Beckett, J. Sparer et al., “Liver disease coli,” RSC Advances, vol. 5, no. 82, pp. 66886–66893, 2015. associated with occupational exposure to the solvent dimethyl- [3] R. Tankhiwale and S. K. Bajpai, “Graft copolymerization onto formamide,”Annals of InternalMedicine, vol. 108, no. 5, pp. 680– cellulose-based filter paper and its further development as silver 686, 1988. nanoparticles loaded antibacterial food-packaging material,” [19] B. Ritz, Y. Zhao, A. Krishnadasan, N. Kennedy, and H. Mor- Colloids and Surfaces B: Biointerfaces, vol. 69, no. 2, pp. 164–168, genstern, “Estimated effects of hydrazine exposure on cancer 2009. incidence and mortality in aerospace workers,” Epidemiology, [4] P. Vasileva, B. Donkova, I. Karadjova, and C. Dushkin, “Synthe- vol. 17, no. 2, pp. 154–161, 2006. sis of starch-stabilized silver nanoparticles and their application [20] N. Mandzy, E. Grulke, and T. Druffel, “Breakage of TiO 2 as a surface plasmon resonance-based sensor of hydrogen agglomerates in electrostatically stabilized aqueous disper- peroxide,” Colloids and Surfaces A: Physicochemical and Engi- sions,” Powder Technology, vol. 160, no. 2, pp. 121–126, 2005. neering Aspects, vol. 382, no. 1–3, pp. 203–210, 2011. [21] D. H. Napper, “Steric stabilization,” Journal of Colloid And [5] A. C. Garcia, L. H. S. Gasparotto, J. F. Gomes, andG. Tremiliosi- Interface Science, vol. 58, no. 2, pp. 390–407, 1977. Filho, “Straightforward synthesis of carbon-supported Ag [22] G. A. Burdock, I. G. Carabin, and J. C. Griffiths, “Toxicology nanoparticles and their application for the oxygen reduction and pharmacology of sodium ricinoleate,” Food and Chemical reaction,” Electrocatalysis, vol. 3, no. 2, pp. 147–152, 2012. Toxicology, vol. 44, no. 10, pp. 1689–1698, 2006. [6] Z.-J. Jiang, C.-Y. Liu, and L.-W. Sun, “Catalytic properties of [23] A. de Oliveira Wanderley Neto, T. Neuma de Castro Dantas, silver nanoparticles supported on silica spheres,” Journal of A. A. Dantas Neto, and A. Gurgel, “Recent advances on the Physical Chemistry B, vol. 109, no. 5, pp. 1730–1735, 2005. use of surfactant systems as inhibitors of corrosion on metallic [7] T. Tsuji, D.-H. Thang, Y. Okazaki, M. Nakanishi, Y. Tsuboi, and surfaces,” in The Role of Colloidal Systems in Environmental M. Tsuji, “Preparation of silver nanoparticles by laser ablation Protection, pp. 479–508, Elsevier, Amsterdam,TheNetherlands, in polyvinylpyrrolidone solutions,” Applied Surface Science, vol. 2014. 254, no. 16, pp. 5224–5230, 2008. [24] D. Teomim, A. Nyska, and A. J. Domb, “Ricinoleic acid-based [8] N. Vigneshwaran, N. M. Ashtaputre, P. V. Varadarajan, R. P. biopolymers,” Journal of Biomedical Materials Research, vol. 45, Nachane, K. M. Paralikar, and R. H. Balasubramanya, “Biologi- no. 3, pp. 258–267, 1999. cal synthesis of silver nanoparticles using the fungusAspergillus [25] C. Vieira, S. Evangelista, R. Cirillo, A. Lippi, C. A. Maggi, and flavus,”Materials Letters, vol. 61, no. 6, pp. 1413–1418, 2007. S. Manzini, “Effect of ricinoleic acid in acute and subchronic [9] H. Wang, X. Qiao, J. Chen, and S. Ding, “Preparation of silver experimental models of inflammation,”Mediators of Inflamma- nanoparticles by chemical reduction method,” Colloids and tion, vol. 9, no. 5, pp. 223–228, 2000. Surfaces A: Physicochemical and Engineering Aspects, vol. 256, [26] H. Wender, L. F. De Oliveira, A. F. Feil et al., “Synthesis of no. 2-3, pp. 111–115, 2005. gold nanoparticles in a biocompatible fluid from sputtering [10] M. M. Oliveira, D. Ugarte, D. Zanchet, and A. J. G. Zarbin, deposition onto castor oil,” Chemical Communications, vol. 46, “Influence of synthetic parameters on the size, structure, and no. 37, pp. 7019–7021, 2010. stability of dodecanethiol-stabilized silver nanoparticles,” Jour- [27] S. F. A. Morais, M. G. A. Da Silva, S. M. P. Meneghetti, and M. nal of Colloid and Interface Science, vol. 292, no. 2, pp. 429–435, R. Meneghetti, “Colloids based on gold nanoparticles dispersed 2005. in castor oil: Synthesis parameters and the effect of the free fatty [11] A. G. Garcia, P. P. Lopes, J. F. Gomes et al., “Eco-friendly acid content,” Comptes Rendus Chimie, vol. 18, no. 4, pp. 410– synthesis of bimetallic AuAg nanoparticles,” New Journal of 421, 2015. Chemistry, vol. 38, no. 7, pp. 2865–2873, 2014. [28] G. B. Shombe, E. B. Mubofu, S. Mlowe, and N. Revaprasadu, [12] T. Maneerung, S. Tokura, and R. Rujiravanit, “Impregnation “Synthesis and characterization of castor oil and ricinoleic of silver nanoparticles into bacterial cellulose for antimicrobial acid capped CdS nanoparticles using single source precursors,” wound dressing,” Carbohydrate Polymers, vol. 72, no. 1, pp. 43– Materials Science in Semiconductor Processing, vol. 43, pp. 230– 51, 2008. 237, 2016. 155 8 Journal of Nanomaterials [29] A. Kumar, P. K. Vemula, P. M. Ajayan, and G. John, “Silver- nanoparticle-embedded antimicrobial paints based on veg- etable oil,” Nature Materials, vol. 7, no. 3, pp. 236–241, 2008. [30] R. Zamiri, A. Zakaria, H. Abbastabar,M.Darroudi,M. S. Husin, and M. A. Mahdi, “Laser-fabricated castor oil-capped silver nanoparticles,” International Journal of Nanomedicine, vol. 6, no. 1, pp. 565–568, 2011. [31] J. F. Gomes, A. C. Garcia, E. B. Ferreira et al., “New insights into the formation mechanism of Ag, Au and AgAu nanoparticles in aqueous alkaline media: alkoxides from alcohols, aldehydes and ketones as universal reducing agents,” Physical Chemistry Chemical Physics, vol. 17, no. 33, pp. 21683–21693, 2015. [32] T. N. Castro Dantas, E. Ferreira Moura, H. Scatena Júnior, A. A. Dantas Neto, and A. Gurgel, “Micellization and adsorption thermodynamics of novel ionic surfactants at fluid interfaces,” Colloids and Surfaces A: Physicochemical and Engineering Aspects, vol. 207, no. 1-3, pp. 243–252, 2002. [33] Y.-Y. Yu, S.-S. Chang, C.-L. Lee, and C. R. C. Wang, “Gold nanorods: electrochemical synthesis and optical properties,” Journal of Physical Chemistry B, vol. 101, no. 34, pp. 6661–6664, 1997. [34] P.Mulvaney, “Surface plasmon spectroscopy of nanosizedmetal particles,” Langmuir, vol. 12, no. 3, pp. 788–800, 1996. [35] E. Saion, E. Gharibshahi, and K. Naghavi, “Size-controlled and optical properties of monodispersed silver nanoparticles syn- thesized by the radiolytic reduction method,” International Journal of Molecular Sciences, vol. 14, no. 4, pp. 7880–7896, 2013. [36] K. R. Brown, D. G.Walter, andM. J. Natan, “Seeding of colloidal Au nanoparticle solutions. 2. Improved control of particle size and shape,” Chemistry of Materials, vol. 12, no. 2, pp. 306–313, 2000. [37] K. Esumi, T. Hosoya, A. Suzuki, and K. Torigoe, “Formation of gold and silver nanoparticles in aqueous solution of sugar- persubstituted poly(amidoamine) dendrimers,” Journal of Col- loid and Interface Science, vol. 226, no. 2, pp. 346–352, 2000. [38] J. Polte, “Fundamental growth principles of colloidal metal nanoparticles—a new perspective,” CrystEngComm, vol. 17, no. 36, pp. 6809–6830, 2015. [39] J. Oomens and J. D. Steill, “Free carboxylate stretching modes,” The Journal of Physical Chemistry A, vol. 112, no. 15, pp. 3281– 3283, 2008. [40] G. B. Deacon, F. Huber, and R. J. Phillips, “Diagnosis of the nature of carboxylate coordination from the direction of shifts of carbonoxygen stretching frequencies,” Inorganica Chimica Acta, vol. 104, no. 1, pp. 41–45, 1985. [41] R. Kaegi, A. Voegelin, C. Ort et al., “Fate and transformation of silver nanoparticles in urban wastewater systems,” Water Research, vol. 47, no. 12, pp. 3866–3877, 2013. [42] C. Levard, E. M. Hotze, B. P. Colman et al., “Sulfidation of silver nanoparticles: natural antidote to their toxicity,” Environmental Science and Technology, vol. 47, no. 23, pp. 13440–13448, 2013. [43] C. M. Levard, B. C. Reinsch, F. M. Michel, C. Oumahi, G. V. Lowry, and G. E. Brown, “Sulfidation processes of PVP-coated silver nanoparticles in aqueous solution: impact on dissolution rate,” Environmental Science & Technology, vol. 45, no. 12, pp. 5260–5266, 2011. 156 Heloiza Fernanda O. da Silva Athayde Natal, RN - Brasil Cel.: 84 996936814 heloizafos@hotmail.com Perfil Bacharel e mestre em Química pela UFRN. Atualmente é doutoranda em Química (aprovada no exame de qualificação em 23/08/2016), pela mesma instituição. Atua principalmente nos seguintes tópicos: Síntese e caracterização de nanopartículas metálias e sua funcionalização para aplicações biomédicas (efeito anti- bacteriano/bactericida, analgésico, anti-inflamatório e anticancerígeno) e análises ambientais. Membro do Grupo de Pesquisa em Química Biológica e Quimometria (QBQ) desde 2013. Também possui experiência em: DoE, Biospectroscopy, calibração multivariada (quimiometria aplicada) e cultivo de células eucarióticas e procarióticas. Formação Acadêmica 2019 Doutorado em Química Polymers and Colloids (concluindo) Universidade Federal do Rio Grande do Norte LENA – Laboratório de Eletroquímica e Nanoprtículas Apicadas 2015 Mestrado em Química Polymers and Colloids Universidade Federal do Rio Grande do Norte LENA – Laboratório de Eletroquímica e Nanoprtículas Apicadas 2015 Licenciatura em Química Universidade Federal do Rio Grande do Norte 2010 Bacharelado em Química Universidade Federal do Rio Grande do Norte Formação Complementar 2018 Treinamento em Cultura Celular Universidade Federal do Rio Grande do Norte Departamento de Bioquímica - UFRN 2015 4th School of SAXS Data Analysis National Synchrotron Light Laboratory, LNLS, Brazil 2014 Resolução de Curva Multivariada – Teoria e Prática. Romà Tauler Universidade Federal do Rio Grande do Norte 2014 2ª Escola de Colóides e Superfície Universidade de São Paulo, USP, Brasil 2014 Aspectos Fundamentais no Controle de Tamanho Sociedade Brasileira de Química, SBQ, Brasil. 2014 Novos Desafios em Química Medicinal Sociedade Brasileira de Química, SBQ, Brasil. 2013 SAXS Workbench. National Synchrotron Light Laboratory, LNLS, Brazil 157 Honras e Prêmios 2015 Prêmio Dissertação Destaque de 2015 promovido pelo Programa de Pós-Graduação em Química da UFRN em parceria com a CRQ XV região Publicações 1. GARCIA, VINICIUS B.; DE CARVALHO, THAIS GOMES; GASPAROTTO, LUIZ HENRIQUE DA S.; SILVA, HELOIZA F. O. ; DE ARAÚJO, AURIGENA ANTUNES; GUERRA, GERLANE C. B.; SCHOMANN, TIMO; CRUZ, LUIS. J. ; CHAN, ALAN B.; DE ARAÚJO, RAIMUNDO FERNANDES. Environmentally compatible bioconjugated gold nanoparticles as efficient contrast agents for inflammationinduced cancer imaging. Nanoscale Research Letters, v. 14, p. 1-12, 2019 2. ARAUJO, JANINE; MENEZES, FABRÍCIO G.; SILVA, HELOIZA F. O. ; VIEIRA, DAVI S.; SILVA, SERGIO R. B.; BORTOLUZZI, ADAILTON J. ; SANT?ANNA, C.; EUGENIO, M.; NERI, JANNYELY M.; GASPAROTTO, LUIZ H. S. Functionalization of gold nanoparticles with two aminoalcohol-based quinoxaline derivatives for targeting phosphoinositide 3-kinases (PI3Kα). NEW JOURNAL OF CHEMISTRY, v. 43, p. 1803-1811, 2019. 3. DE CARVALHO, THAIS GOMES; GARCIA, VINICIUS B.; ARAUJO, AURIGENA A.; GASPAROTTO, LUIZ HENRIQUE DA S.; SILVA, HELOIZA F. O. ; GUERRA, GERLANE C. B.; MIGUEL, EMILIO DE CASTRO; LEITAO, RENATA FERREIRA C.; DA SILVA COSTA, DEIZIANE V.; CRUZ, LUIS. J. ; CHAN, ALAN B.; ARAUJO JUNIOR, R. F. Spherical neutral Gold nanoparticles improve anti-inflammatory response, oxidative stress and fibrosis in alcohol-methamphetamine-induced liver injury in rats. INTERNATIONAL JOURNAL OF PHARMACEUTICS, v. 548, p. 1-14, 2018. 4. SILVA, HELOIZA F. O.; LIMA, RAYANE P. ; COSTA, FERNANDA S. L. ; MORAES, EDGAR P. ; MELO, MARIA CELESTE N. ; SANT’ANNA, CELSO ; EUGENIO, M. ; GASPAROTTO, LUIZ H.S. . On the synergy between silver nanoparticles and doxycycline towards the inhibition of growth. RSC Advances, v. 8, p. 23578-23584, 2018. 5. SILVA, RAFAEL LEONARDO C. G.; SILVA, HELOIZA F. O.; GASPAROTTO, LUIZ HENRIQUE DA S.; CASELI, LUCIANO. Lipopolysaccharides and peptidoglycans modulating the interaction of Au nanoparticles with cell membranes models at the air/water interface. BIOPHYSICAL CHEMISTRY, v. 238, p. 22-29, 2018. 6. SILVA, RAFAEL LEONARDO C. G.; SILVA, HELOIZA F. O. ; GASPAROTTO, LUIZ HENRIQUE DA S.; CASELI, LUCIANO. How the interaction of PVP-stabilized Ag nanoparticles with models of cellular membranes at the air-water interface is modulated by the monolayer composition.. JOURNAL OF COLLOID AND INTERFACE SCIENCE, v. 512, p. 792-800, 2018. 7. DE ARAÚJO, RAIMUNDO FERNANDES; DE ARAÚJO, AURIGENA ANTUNES; PESSOA, JONAS BISPO; FREIRE NETO, FRANSCISCO PAULO; DA SILVA, GISELE RIBEIRO; LEITÃO OLIVEIRA, ANA LUIZA C.S.; DE CARVALHO, THAÍS GOMES; SILVA, HELOIZA F.O.; EUGÊNIO, MATEUS; SANT’ANNA, CELSO; GASPAROTTO, LUIZ H.S. Anti-inflammatory, analgesic and anti-tumor properties of gold nanoparticles. Pharmacological Reports, v. 69, p. 119-129, 2017. 8. COSTA, I. D.; WANDERLEY NETO, A. O.; MORAES, E. P.; NOBREGA, E. T. D.; SANT?ANNA, C.; EUGENIO, M.; GASPAROTTO, L. H. S.; SILVA, H. F. O. Dual Role of a Ricinoleic Acid Derivative in the Aqueous Synthesis of Silver Nanoparticles. JOURNAL OF NANOMATERIALS, v. 2017, p. 1-8, 2017. 9. DE ARAÚJO, RAIMUNDO FERNANDES; PESSOA, JONAS; CRUZ, LUIS; CHAN, ALAN ; MIGUEL, EMILIO DE CASTRO; CAVALCANTE, ROMULO; BRITO, GERLY ANNE; SILVA, HELOIZA F. O.; GASPAROTTO, LUIZ HENRIQUE DA S.; GUEDES, PAULO; ARAUJO, AURIGENA A. Apoptosis in human liver carcinoma caused by gold nanoparticles in combination with carvedilol is mediated via modulation of MAPK/Akt/mTOR pathway and EGFR/FAAD proteins. International Journal of Oncology, v. 52, p. 189, 2017. 158 10. SILVA, HELOIZA F. O.; LIMA, KÁSSIO M. G.; CARDOSO, MATEUS B.; OLIVEIRA, JESSICA F. A.; MELO, MARIA C. N.; SANT'ANNA, CELSO; EUGÊNIO, MATEUS; GASPAROTTO, LUIZ H. S. Doxycycline conjugated with polyvinylpyrrolidone-encapsulated silver nanoparticles: a polymer's malevolent touch against Escherichia coli. RSC Advances, v. 5, p. 66886-66893, 2015. Apresentações científicas 1.SILVA, H. F. O. ; DE ARAÚJO, RAIMUNDO FERNANDES; ARAUJO, AURIGENA A.; SANT'ANNA, CELSO; EUGÊNIO, MATEUS; OLIVEIRA, ANA LUIZA C.S. LEITÃO; GASPAROTTO, L.H.S. Doxycycline conjugated with polyvinylpyrrolidoneencapsulated silver nanoparticles: a polymer's malevolent touch against Escherichia coli Abstract Body. In: MRS Fall Meeting, 2016, Boston. MRS Fall Meeting, 2016. 2.SILVA, H.F.O., VALE, B.C.M., GASPAROTTO, L.H.S., MELO, M.C.N. Antibacterial potential of silver nanoparticles (AgNPs) combined with antibiotic doxycline. 4º Simpósio Internacional de Microbiologia Clínica – SIMC, 2014. 3. SILVA, H. F. O.; GASPAROTTO, L.H.S. ; LIMA, K. M. G. Síntese verde de nanopartículas de prata para aplicação antimicrobiana utilizando planejamento fatorial completo. 2014, Sociedade Brasileira de Química, SBQ, Brasil., SBQ, Brasil. 2014. Patente 1. DE ARAÚJO, RAIMUNDO FERNANDES; GARCIA, V. B.; LEITÃO OLIVEIRA, ANA LUIZA C.S.; Gasparotto, L.H.S.; SILVA, H. F. O.; ARAUJO, AURIGENA A.. SISTEMA NANOPARTICULADO DE OURO E SUA FORMA DE OBTENÇÃO APLICADOS À TÉCNICA DE IMUNOFLUORESCÊNCIA EM TECIDO PARAFINIZADO. 2016, Brasil. Patente: Privilégio de Inovação. Número do registro: BR1020160244749, título: "SISTEMA NANOPARTICULADO DE OURO E SUA FORMA DE OBTENÇÃO APLICADOS À TÉCNICA DE IMUNOFLUORESCÊNCIA EM TECIDO PARAFINIZADO", Instituição de registro: INPI - Instituto Nacional da Propriedade Industrial. Depósito: 20/10/2016 Orientações 2017 Trabalho de Conclusão de Curso. Rayane Pereira De Lima. Discriminação de respostas metabólicas de Staphylococcus aureus submetidas à Nanopartículas de prata. Química – UFRN Docência assistida 2015 Físico-química Experimental Nível: Graduação 2014 Físico-química de Superfície e Eletroquímica Nível: Graduação Referências Luiz Gasparotto José Luis Fonseca Instituto de Química, UFRN, Brasil Instituto de Química, UFRN, Brasil LENA – Laboratório de Eletroquímica e Laboratório de Membranas e Colóides Nanopartículas Aplicadas jlcfonseca.br@gmail.com lhgasparotto@gmail.com Maria Celeste Raimundo Fernandes Departamento de Microbiologi e Parasitologia, Departamento de Morfologia, UFRN, Brazil UFRN, Brazil Nanobiomateriais Translacionais e Imagem LBMED – Laboratório de Microbiologia Médica Departamento de Radiologia celmelo@gmail.com Centro Médico da Universidade de Leiden araujojr@cb.ufrn.br Kassio Lima Frank Quina Instituto de Química, UFRN, Brasil Departamento de Química-USP QBQ - Grupo de Química Biológica e Quimiometria Laboratório de Dinâmica Foto-molecular kassiolima@gmail.com quina@usp.br 159 CARTA DE CONCLUSÃO Ao iniciar esta carta me vem à mente o dia em que recebi a notícia que havia sido aprovada na seleção de doutorado. Um misto de emoções, pois ainda estava me acostumando com o fato de receber o título de mestre em Química. Permanecendo no mesmo laboratório e sendo orientada pelo mesmo orientador foi difícil assimilar que já não estava no mestrado e que a mudança na matrícula precisava refletir na minha formação que agora era destinada a não só receber o título, mas em me tornar uma profissional doutora em química. O bom foi que com o passar dos dias vieram como rito de passagem as discussões e escrita do projeto de tese. E a frase: “você será a doutora do seu doutorado, não eu!” dita pelo meu orientador ao me receber de volta para mais uma jornada permeou meus pensamentos e instigou minhas ações nesse período. O dicionário define a palavra projeto como “o desejo, intenção de fazer ou realizar (algo) no futuro”. Essa definição descreve literalmente minha intenção ao escrever meu projeto de tese. Com a visão romântica de que tudo seria realizado segundo o cronograma tão bem planejado, fez com que por vezes me frustrasse. Foram muitos nãos, quebra de equipamentos sem data para conserto, entre outros desafios. Mas hoje vejo que na realidade as vicissitudes que passei foram oportunidades para buscar sins em outros lugares e para inspirar novos trabalhos com os recursos que dispunha. Durante esses desafios pude contar com o apoio e ajuda de tantas pessoas (citadas nos agradecimentos) que possivelmente não teria tido a oportunidade de conhecê-las se tudo tivesse saído como o planejado no projeto. Ah, se pudéssemos publicar todo o aprendizado de quatro anos em periódicos! Como aprendemos com a busca pela reprodutibilidade! Sou tão grata pelas oportunidades de crescimento e aprendizado que tive durante esse processo. Se eu pudesse resumir os últimos anos, diria que vivi dez anos a cada um ano. Hoje sei que o título de doutora que pleiteio (não só com o que está escrito nessa tese) não será concedido de maneira arbitrária ou sem mérito. É sempre difícil dissertar sobre si, mas até isso o doutorado me trouxe de bom. Reconheço que não fui perfeita, mas me dediquei da melhor maneira que pude, nas condições que dispunha para chegar aqui por merecimento e ser a doutora do meu doutorado. Concluo com uma citação do professor Seixas: “Sempre poderíamos fazer melhor se tivéssemos mais um minuto. A vida exige conclusões e nos ensina a ver com bons olhos o esforço que fizemos com esse um minuto a menos”. Heloiza Athayde 160