Gold nanoparticle based double-labeling of melanoma extracellular vesicles to determine the specificity of uptake by cells and preferential accumulation in small metastatic lung tumors

Abstract

Background: Extracellular vesicles (EVs) have shown great potential for targeted therapy, as they have a natural ability to pass through biological barriers and, depending on their origin, can preferentially accumulate at defined sites, including tumors. Analyzing the potential of EVs to target specific cells remains challenging, considering the unspecific binding of lipophilic tracers to other proteins, the limitations of fluorescence for deep tissue imaging and the effect of external labeling strategies on their natural tropism. In this work, we determined the cell-type specific tropism of B16F10-EVs towards cancer cell and metastatic tumors by using fluorescence analysis and quantitative gold labeling measurements. Surface functionalization of plasmonic gold nanoparticles was used to promote indirect labeling of EVs without affecting size distribution, polydispersity, surface charge, protein markers, cell uptake or in vivo biodistribution. Double-labeled EVs with gold and fluorescent dyes were injected into animals developing metastatic lung nodules and analyzed by fluorescence/computer tomography imaging, quantitative neutron activation analysis and gold-enhanced optical microscopy. Results: We determined that B16F10 cells preferentially take up their own EVs, when compared with colon adenocarcinoma, macrophage and kidney cell-derived EVs. In addition, we were able to detect the preferential accumulation of B16F10 EVs in small metastatic tumors located in lungs when compared with the rest of the organs, as well as their precise distribution between tumor vessels, alveolus and tumor nodules by histological analysis. Finally, we observed that tumor EVs can be used as effective vectors to increase gold nanoparticle delivery towards metastatic nodules.

Exploiting the Natural Properties of Extracellular Vesicles in Targeted Delivery towards Specific Cells and Tissues

“…Some EVs can also increase their residence time in circulation by displaying antiphagocytic surface markers to evade clearance by the mononuclear phagocytic system [42]. Different compounds can be incorporated into such vesicles, such as drugs, nanoparticles, lipids, proteins, peptides, RNA, siRNA and fluorescent markers [13][14][15]. Lipophilic drugs or compounds will preferentially intercalate into the membrane bilayer, while hydrophilic compounds will prefer the lumen.…”

“…For example the antineoplastic drug Paclitaxel has been incorporated into stromal mesenchymal cells, which were resistant to the drug and incorporated it into EVs, which displayed cytotoxic effects against cancer cells [50]. Another recent example is the incorporation of gold nanoparticles (AuNPs), which were incubated and taken up by cells to promote their passage through the MVB and inclusion in EVs [13]. The pre-cargo-loading strategies permit obtaining EVs containing therapeutic compounds without the need for membrane-disruptive techniques.…”

“…There is still no consensus on which of the uptake mechanisms is more important; however, available evidence shows that internalization is an active process that is significantly decreased at 4 • C or after blocking protein interactions using specific antibodies. The high degree of EV heterogenicity depending on their cellular origin contributes significantly to their uptake and available evidence suggests that this can be selectively enhanced by choosing specific cell models [13,58].…”

“…For instance, EVs from mesenchymal stem cells are taken up more efficiently by their own cells when compared to other immune cells [62]; neuroblastoma EVs are endocytosed preferentially by glial cells [63]; ovarian cancer EV cells also showed increased uptake by tumor cells when compared to epithelial cell-derived EVs [64]. A recent study compared the uptake of multiple-source EVs with their corresponding cells and showed that melanoma EVs were taken up more efficiently by melanoma cells rather than immune cells, fibroblast and endothelial cells [13]. Interestingly, the uptake of colon adenocarcinoma EVs (by melanoma cells) was also higher than non-tumor EVs, suggesting that cancer-specific molecules may play an important role in promoting the uptake of these vesicles by tumor cells.…”

“…Interestingly, some EVs show tropism towards organs related to their cellular origin (Table 1). Endothelial EVs from the brain can accumulate in the cerebral tissue [71], while melanoma EVs target preferentially melanoma metastases [13]. MSC-EVs showed increased accumulation in kidneys of mice with acute kidney injury, suggesting that the presence of a particular disease also affects their biodistribution [72].…”