Utilização de nanopartículas teranósticas de ouro como método de contraste em imunofluorescência e citometria de fluxo para o diagnóstico de doenças inflamatórias associadas ao câncer

Abstract

Immunofluorescence (IF) and flow cytometry (FC) are methods known for their very high sensitivity and specificity, as well as their ability to detect potentially carcinogenic inflammatory changes, making them useful in early cancer screening. However, both techniques still have a high cost to perform, which discourages their use in many cases. In this work, we used gold nanoparticles (AuNPs) as substitutes for Alexa Fluor 488 in the IF of ulcerative colitis, steatohepatitis and also in the FC of RAW 264.7 cells polarized for the M2 profile, in order to quantify the expression of cyclooxygenase 2 (COX-2) and macrophage migration inhibitory factor (MIF), both highly expressed in inflammatory processes that precede cancer. In addition to the standard IF protocol, where primary and secondary antibody incubations are 18h and 1h, respectively, a rapid protocol of 30 min of incubation for each antibody was also developed. AuNPs were used in both protocols as substitutes for Alexa Fluor 488 (ALEXA), as well as in a third protocol, where the AuNPs were diluted with ALEXA itself. For the FC of polarized macrophages, in addition to the standard protocol, which includes 2h of primary antibody incubation followed by 2h of secondary antibody incubation, a rapid protocol of 30 min for each incubation was performed, which used AuNPs instead of ALEXA . When compared to the standard IF protocol with ALEXA, the AuNPs protocols exhibited the same fluorescence intensity (p>0.05). AuNPs also increased the fluorescence intensity of ALEXA when diluted with it (p<0.001), making it possible to double the dilution of ALEXA without compromising the fluorescence intensity (p>0.05). Although FC with ALEXA was more sensitive in detecting macrophages labeled with MIF and COX-2 (p<0.0001), the protocol with AuNPs was still able to detect a significant number of positive cells (p<0.001), requiring of a shorter runtime. Therefore, AuNPs make it possible to carry out these methods cheaper and faster, thus contributing to technological development and to the democratization of these techniques in diagnosis.