Abstract
Despite biomolecule delivery is a natural function of Extracellular Vesicles (EVs), loading of exogenous macromolecules such as proteins into EVs remains a major challenge limiting their interest as convincing protein delivery systems for health applications. In this context, lipid-anchorage of exogenous cargo into EV membrane recently emerged as a promising option to enable their vectorization into cells. Nevertheless, this option was not explored for protein intracellular delivery, and further characterization of the critical parameters governing the association of a lipid-anchored cargo protein to EVs is still needed to confirm the relevance of this anchorage strategy. Therefore, in this work, we have sought to identify these parameters in a precise and quantitative manner, working with bulk and single nanoparticle analysis methods to identify protein loading capacity and subsequent intracellular delivery. Incubation temperature, cargo concentration and Lipid Anchor (LA) structure (lipid nature and PEG size) appeared as critical factors influencing maximal EV loading capacity. Precise control of these parameters enabled to load cargo protein close to EV saturation without hindering cellular delivery. Interestingly, we evidenced that EV isolation process influenced this association. Structural properties of LA influenced not only cargo protein/EV association, but also its intracellular delivery into different carcinoma cell lines. By thoroughly characterizing Lipid-PEG-protein anchorage, this study evidences the interest of this tunable and controllable approach for efficient EV protein delivery.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Article has been shortened and clarified, and additional data moved as supplementary data. Discussion has been modified to highlight new insights. Few typos has been corrected.
