Functional characterization of mammalian and plant extracellular vesicles as systems for cancer treatment

In the last years, the field of nanomedicine and drug delivery has grown exponentially, providing new platforms to carry therapeutic agents into the target sites. Extracellular vesicles (EVs) are ready-to-use, biocompatible, and non-toxic nanoparticles that are revolutionizing the field of drug delivery. EVs are involved in cell-cell communication and mediate many physiological and pathological processes by transferring their bioactive cargo to target cells. Recently, nanovesicles from plants (PDNVs) have raised the interest of the scientific community due to their high yield and biocompatibility. This study aims to first evaluate whether PDNVs may be used as drug delivery systems. We isolated and characterized nanovesicles from tangerine juice (TNVs) that were comparable to mammalian EVs in size and morphology. TNVs carry the traditional EV marker HSP70 and, as demonstrated by metabolomic analysis, contain flavonoids, organic acids, and limonoids. TNVs were loaded with DDHD1-siRNA through electroporation, obtaining a loading efficiency of 13%. The DDHD1 gene encodes the phospholipase DDHD1 protein, which plays a vital role in lipid metabolism by hydrolyzing phospholipids into fatty acids and other lipophilic substances. Overexpression of DDHD1 has been linked to increased proliferation in colorectal cancer cells, suggesting its potential as a target for cancer therapies. We found that the DDHD1-siRNA TNV complexes were able to deliver DDHD1-siRNA to human colorectal cancer cells, inhibiting the target expression by about 60%. This study represents a proof of concept for the use of PDNVs as vehicles of RNA interference (RNAi) toward mammalian cells. More recently we tested whether the approach used with PDNVs can be applied also to mammalian vesicles, in particular to EVs purified from human embryonic kidney cells (HEK293T). We focused on loading onco-suppressor microRNAs, miR-146a and miR-199a, in EVs and to test the functionality of the complex on thyroid cancer cell lines. To increase the cell-specific targeting of the EV-miRNA complex, we decorated exosomes with Hyaluronic acid (HA), since cancer cells overexpress its receptor, CD44. Preliminary results suggest that the developed electroporation approach can be also used to load exogenous RNA in mammalian EVs; also, our findings suggest that the decoration of EV with HA may enhance the delivery efficiency of miRNAs to target cells.