Cellular Vesicles: New insights in engineering methods, interaction with cells and potential for brain targeting

Cellular vesicles (CVs) have been proposed as alternatives to exosomes for targeted drug delivery. CVs, prepared from HEK-293, B16F10 and hCMEC/D3 cells, by liposome technology methods were characterized for properties, morphology, cytotoxicity and cell uptake. CV brain-targeting potential was evaluated in vitro on the hCMEC/D3 BBB-model, and in vivo/ ex vivo. CV sizes were between 135-285 nm; zeta-potential was negative. The Dehydration Rehydration method conferred highest calcein loading and latency to CVs, compared to other methods. Increased calcein-leakage from CVs compared to liposomes, indicated their poor integrity, which was increased by pegylation. In vivo results confirmed lower liver uptake by PEG-CVs (compared to non-pegylated) proving that calcein integrity test is useful for prediction of CV-biodistribution, as used for liposomes. Cell-uptake of homologous-origin CVs was not always higher compared to that of non-homologous. Nevertheless, CVs from hCMEC/D3 demonstrated highest BBB permeability (in vitro), compared to OX-26 targeted liposomes, and brain localization (in vivo). CVs from hCMEC/D3 cells grown in different media demonstrated decreased interaction with brain cells and brain localization; significant differences in proteome of the two latter CV types were identified by proteomics, suggesting a potential methodology for identification of organotropism-determining CV-components SIGNIFICANCE STATEMENT N/A