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 30 min. 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.
Highlights
In medical and biological research, the interest in gold nanoparticles (AuNPs) for optical microscopy studies and diagnostic procedures, especially confocal laser microscopy, is increasing
In a previous study [6], we have demonstrated that Gold nanoparticles (AuNP) can be conjugated with the antibodies anti-β-catenin and anti-E-cadherin to target colorectal carcinoma cells, whose clinical value can be found in an early diagnosis of cancer through non-invasive methods in body fluids such as saliva and urine
We found that the replacement of Alexa Fluor® 488 (ALEXA) by the AuNPs in Nanoparticles protocol 1NP2Nanoparticles protocol 2 (NP1) saves about 18 h, when compared to standard protocol and NP2 that require about 24 h, each, from the antigenic retrieval to the assembling of the slides for microscopic analysis (Fig. 7 and Table 1)
Summary
In medical and biological research, the interest in gold nanoparticles (AuNPs) for optical microscopy studies and diagnostic procedures, especially confocal laser microscopy, is increasing. The physicochemical properties of AuNPs allow them to be used in many medical studies, such as genomics, biosensitivity, immunoassay, clinical chemistry, detection, and photothermolysis of microorganisms and cancer cells [1]. Many studies were developed aiming at the application of nanoparticles conjugated with biomacromolecules, such as antibodies, lectins, and enzymes, in different fields, e.g., biochemistry, microbiology, immunology, and morphology [1, 4]. Highly specific and sensitive AuNP-based contrast agents are used for both X-ray and optical imaging modalities, Garcia et al Nanoscale Research Letters (2019) 14:166 since AuNPs are able to enhance fluorescence intensity of conjugated molecules [5, 6]
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