Methods for loading therapeutics into extracellular vesicles and generating extracellular vesicles mimetic-nanovesicles.

Extracellular vesicles (EVs) are membrane bound vesicles released into the extracellular environment by eukaryotic and prokaryotic cells. EVs are enriched in active biomolecules and they can horizontally transfer cargo to recipient cells. In recent years EVs have demonstrated promising clinical applications due to their theragnostic potential. Although EVs have promising therapeutic potential, there are several challenges associated with using EVs before transition from the laboratory to clinical use. Some of these challenges include issues around low yield, isolation and purification methodologies, and efficient engineering (loading) of EVs with therapeutic cargo. Also, to achieve higher therapeutic efficiency, EV architecture and cargo may need to be manipulated prior to clinical application. Some of these issues have been addressed by developing biomimetic EVs. EV mimetic-nanovesicles (M-NVs) are a type of artificial EVs which can be generated from all cell types with comparable characteristics as EVs for an alternative therapeutic modality. In this review, we will discuss current techniques for modifying EVs and methodology used to generate and customize EV mimetic-nanovesicles.

Proteomic and Post-Translational Modification Profiling of Exosome-Mimetic Nanovesicles Compared to Exosomes.

Issues associated with upscaling exosome production for therapeutic use may be overcome through utilizing artificial exosomes. Cell-derived mimetic nanovesicles (M-NVs) are a potentially promising alternative to exosomes for clinical applicability, demonstrating higher yield without incumbent production and isolation issues. Although several studies have shown that M-NVs have similar morphology, size and therapeutic potential compared to exosomes, comprehensive characterization and to what extent M-NVs components mimic exosomes remain elusive. M-NVs were generated through the extrusion of cells and proteomic profiling demonstrated an enrichment of proteins associated with membrane and cytosolic components. The proteomic data herein reveal a subset of proteins that are highly abundant in M-NVs in comparison to exosomes. M-NVs contain proteins that largely represent the parental cell proteome, whereas the profile of exosomal proteins highlight their endosomally derived origin. This advantage of M-NVs alleviates the necessity of endosomal sorting of endogenous therapeutic proteins or RNA into exosomes. This study also highlights differences in protein post-translational modifications among M-NVs, as distinct from exosomes. Overall this study provides key insights into defining the proteome composition of M-NVs as a distinct from exosomes, and the potential advantage of M-NVs as an alternative nanocarrier when spontaneous endosomal sorting of therapeutics are limited.

An intact membrane is essential for small extracellular vesicle-induced modulation of α-synuclein fibrillization.

The misfolding and fibrillization of the protein, α-synuclein (αsyn), is associated with neurodegenerative disorders referred to as the synucleinopathies. Understanding the mechanisms of αsyn misfolding is an important area of interest given that αsyn misfolding contributes to disease pathogenesis. While many studies report the ability of synthetic lipid membranes to modulate αsyn folding, there is little data pertaining to the mechanism(s) of this interaction. αSyn has previously been shown to associate with small lipid vesicles released by cells called extracellular vesicles (EVs) and it is postulated these interactions may assist in the spreading of pathological forms of this protein. Together, this presents the need for robust characterisation studies on αsyn fibrillization using biologically-derived vesicles. In this study, we comprehensively characterised the ability of lipid-rich small extracellular vesicles (sEVs) to alter the misfolding of αsyn induced using the Protein Misfolding Cyclic Amplification (PMCA) assay. The biochemical and biophysical properties of misfolded αsyn were examined using a range of techniques including: Thioflavin T fluorescence, transmission electron microscopy, analytical centrifugation and western immunoblot coupled with protease resistance assays and soluble/insoluble fractionation. We show that sEVs cause an acceleration in αsyn fibrillization and provide comprehensive evidence that this results in an increase in the abundance of mature insoluble fibrillar species. In order to elucidate the relevance of the lipid membrane to this interaction, sEV lipid membranes were modified by treatment with methanol, or a combination of methanol and sarkosyl. These treatments altered the ultrastructure of the sEVs without changing the protein cargo. Critically, these modified sEVs had a reduced ability to influence αsyn fibrillization compared to untreated counterparts. This study reports the first comprehensive examination of αsyn:EV interactions and demonstrates that sEVs are powerful modulators of αsyn fibrillization, which is mediated by the sEV membrane. In doing so, this work provides strong evidence for a role of sEVs in contributing directly to αsyn misfolding in the synucleinopathy disorders.