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Quantitative and functional characterisation of extracellular vesicle loading with small molecules by passive adsorption versus cholesterol mediated membrane anchoring

Treton, Gwenola, Sayer, Claudia, Schürz, Melanie, Jaritsch, Maria, Mueller, Anna, Matea, Cristian-Tudor, Stajnolovic, Vesna, Melo-Benirschke, Heloisa, Be, Celine, Krembel, Caroline, Rodde, Stephane, Haffke, Matthias, Hintermann, Samuel, Marzinzik, Andreas, Ripoche, Sebastien, Bloechl, Constanin, Hollerweger, Julia, Auer, Daniela, Cabrele, Chiara, Huber, Christian, Hintersteiner, Martin, Wagner, Beatrix, Lingel, Andreas and Meisner-Kober, Nicole (2023) Quantitative and functional characterisation of extracellular vesicle loading with small molecules by passive adsorption versus cholesterol mediated membrane anchoring. Journal of controlled release, 361. pp. 694-716. ISSN 1873-4995; 0168-3659


Extracellular vesicles (EVs) are nanosized intercellular messengers that bear enormous application
potential as biological drug delivery vehicles. Much progress has been made for loading or decorating
EVs with proteins, peptides or RNA by genetic engineering of the donor cells, but post-isolation loading
with synthetic drugs and/or using EVs from natural sources remains challenging. In particular,
quantitative and unambiguous data of whether and how small molecules associate with EVs versus
other components in the samples are still lacking. Here we describe the systematic and quantitative
characterisation of EV loading with small molecules by passive adsorption based on hydrophobic
interactions versus membrane anchoring of hydrophilic ligands via cholesterol tags. As revealed by
single vesicle imaging, both ligand types bind to CD63 positive EVs, however also unspecifically to other
vesicles, particles and serum proteins. With increasing concentrations, insoluble compounds such as
Curcumin and Terbinafine agglomerate on EVs with no apparent saturation up to 106-107 molecules
per vesicle as quantified by liquid chromatography – high resolution mass spectrometry (LC-HRMS),
forming a population of large, electron-dense vesicles detected by cryo-transmission electron
microscopy (TEM). High density EV loading resulted in reduced EV cell uptake for both compounds,
and toxic gain of function for Curcumin-EVs. In contrast, Cholesterol tagging of a hydrophilic mdm2-derived cyclic peptide saturated at densities of ca 104 – 105 molecules per vesicle, whereas lipidomics showed an addition rather than replacement of endogenous Cholesterol. Cholesterol anchored ligands did not change the EV size or morphology and retained their cell uptake activity without inducing cell toxicity, however were shed from the vesicles in presence of serum. Based on these data we conclude that (1) both methods allow loading of EVs with small molecules but are prone to unspecific compound binding to other components if present in the sample, (2) Cholesterol anchoring needs substantial
optimization of formulation stability for in vivo applications, whereas (3) careful titration of loading densities is warranted when relying on hydrophobic interactions of EVs with insoluble compounds to mitigate changes in physicochemical properties and loss of EV function and potential cell toxicity.

Item Type: Article
Date Deposited: 25 Oct 2023 12:15
Last Modified: 25 Oct 2023 12:15