Dados do Resumo

Título

ENHANCING RAMAN SPECTROSCOPY (SERS) FOR IMPROVED AND PRECISE CHARACTERIZATION OF EXTRACELLULAR VESICLES (EVs) IN CANCER CELL BIOLOGY

Introdução

Studies on Extracellular Vesicles (EVs) highlight their significant therapeutic potential and contributions to the knowledge of cell biology, whose impacts on cancer biology have demonstrated a crucial role in modulating tumour processes. Therefore, effective analytical techniques are imperative to comprehensively characterise EVs, ranging from their surface chemical composition to their internal content.

Objetivo

To guarantee precision and reproducibility, Raman spectroscopy technique using Au and Ag nanoparticles (NPs), are incorporated together with EVs of size from 10 to 15 times larger, they can create a plasmonic effect of amplification of the Raman signal, called SERS-Raman spectroscopy.

Métodos

To use data already published, a protocol was developed and validated in our laboratory to increase the reproducibility of the results with the use of AuNP+EVs and compare its results with AgNP+EVs, as already validated in the literature and with unsatisfactory results. Dark Field Hyperspectral Microscopy (Cytoviva) was used to monitor the real-time interaction of EVs with Au and AgNPs, confirming the vesicular integrity and chemical interactions on the surface of the EVs. In addition, Confocal Raman Microscopy (WItec) characterized Au and AgNPs+EVs at 532 nm and 633 nm lasers, using the drip technique on a glass slide, under drying at room temperature of the EVs+NPs, EVs and NPs solutions, to obtain the comparison spectra. Due to the fluorescence phenomenon, common in biomolecules, all spectra were normalized with a reference baseline and in long accumulation periods with low laser power to obtain the Raman signal.

Resultados

The spectrum obtained allowed the evaluation of the surface chemical compositions of the EVs, with a cut-off resolution for the EVs+AuNPs compared to the EVs+AgNPs. Although the literature already uses AgNPs as an analysis method, the AuNPs showed great promise and a reproducibility similar to that observed for the Ag. The spectra obtained, after treatment with the reference baseline to eliminate the fluorescence effect, allowed the visualization of the characteristic peaks of organic groups of relevance for a liposome, however with little reproducibility and clarity between what could be noise or Raman signal, whose better resolution for the organic groups linked to protein and glycidic structures was obtained with the use of Au than with AgNPs. The analyses performed using the AgNP spectra were based on the comparison of the spectra obtained with only the AgNPs and with the EVs+AgNPs, to verify the displacements and changes after the interaction of the EVs with the NPs.

Conclusões

Thus, from the implementation of AuNPs we were able to propose a new study platform for EVs, in addition to that already used in the literature with Ag, creating new alternatives to characterize their surfaces with more precise spectroscopic techniques, such as SERS. In addition, it is worth highlighting the excellent reproducibility achieved with the protocol, as well as the quality of the data obtained, despite the large amount of fluorescence, the use of both Au and Ag NPs allowed us to evaluate the differences and similarities in the results obtained, which are complementary.

Financiador do resumo

FAPESP, CAPES, CNPq, BNDS, Ministério da Saúde, EMBRAPII

Palavras Chave

EVs; Spectroscopy; NPs