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bioRxiv · 10.1101/2024.08.28.610030

Investigation of Lipid-PEG Anchorage of Proteins onto Mesenchymal Stem Cell Derived Extracellular Vesicles for Intracellular Delivery: A Quantitative Assessment

Marquant, A.·Berthelot, J.·Bich, C.·Ibn Elfekih, Z.·Simon, L.·Robin, B.·Chopineau, J.·Tianpei Wang, D.·Jay Emerson, S.·Wang, A.·Benedetti, C.·Langlois, S.·Guglielmi, L.·Martineau, P.·Aubert-Pouessel, A.·Morille, M.
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Abstract

Despite biomolecule delivery is a natural function of Extracellular Vesicles (EVs), low loading of exogenous macromolecules such as proteins into EVs limits 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 critical parameters governing the association of a lipid-anchored cargo protein to EVs stills needed to confirm the relevance of this anchorage strategy. Therefore, we sought to identify these parameters in a precise and quantitative manner, using bulk and single nanoparticle analysis methods to identify protein loading capacity and subsequent intracellular delivery. We identified incubation temperature, cargo concentration, Lipid Anchor (LA) structure (lipid nature, linker) and EV origin 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. Structural properties of LA influenced not only cargo protein/EV association, but also 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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=58 SRC="FIGDIR/small/610030v3_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1fed681org.highwire.dtl.DTLVardef@1b4d728org.highwire.dtl.DTLVardef@1998e3aorg.highwire.dtl.DTLVardef@488be3_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Marquant, A., Berthelot, J., Bich, C., Ibn Elfekih, Z., Simon, L., Robin, B., Chopineau, J., Tianpei Wang, D., Jay Emerson, S., Wang, A., Benedetti, C., Langlois, S., Guglielmi, L., Martineau, P., Aubert-Pouessel, A., Morille, M.. 2024-08-29. Investigation of Lipid-PEG Anchorage of Proteins onto Mesenchymal Stem Cell Derived Extracellular Vesicles for Intracellular Delivery: A Quantitative Assessment. https://doi.org/10.1101/2024.08.28.610030

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