Abstract
Extracellular vesicles (EVs) mediate intercellular transport of biomolecular cargo in the body, making them promising delivery vehicles for bioactive compounds. Genetic engineering of producer cells has enabled encapsulation of therapeutic proteins in EVs. However, genetic engineering approaches can be expensive, time-consuming, and incompatible with certain EV sources, such as human plasma and bovine milk. The goal of this study was to develop a quick, versatile, and simple method for loading proteins in EVs post-isolation. Proteins, including CRISPR associated protein 9 (Cas9), were bound to cationic lipids that were further complexed with MDA-MB-231 cell-derived EVs through passive incubation. Size-exclusion chromatography was used to remove components that were not complexed with EVs. The ability of EVs to mediate intracellular delivery of proteins was compared to conventional methods, such as electroporation and commercial protein transfection reagents. The results indicate that EVs retain native features following protein-loading and obtain similar levels of intracellular protein delivery as conventional methods, but display less toxicity. This method opens up opportunities for rapid exploration of EVs for protein delivery.
Highlights
In recent decades, extracellular vesicles (EVs) have generated considerable interest due to their involvement in physiological and pathological intercellular communication [1,2,3,4].EVs are composed of an external bilayer formed by lipids [5], proteins [6], and glycans [7]that enclose an aqueous core with additional bimolecular cargo, such as nucleic acids [8].EVs are released by all cells and are classified based on biogenesis, but subtypes differ in terms of size and composition
Consistent with previously published studies [45,46,65], tangential flow filtration resulted in separation and concentration of nanosized EVs (Figure 1b, Supplementary Figure S1)
Various synthetic nanoparticles and EV loading methods have been used for protein delivery
Summary
Extracellular vesicles (EVs) have generated considerable interest due to their involvement in physiological and pathological intercellular communication [1,2,3,4].EVs are composed of an external bilayer formed by lipids [5], proteins [6], and glycans [7]that enclose an aqueous core with additional bimolecular cargo, such as nucleic acids [8].EVs are released by all cells and are classified based on biogenesis, but subtypes differ in terms of size and composition. Extracellular vesicles (EVs) have generated considerable interest due to their involvement in physiological and pathological intercellular communication [1,2,3,4]. EVs are composed of an external bilayer formed by lipids [5], proteins [6], and glycans [7]. EVs are released by all cells and are classified based on biogenesis, but subtypes differ in terms of size and composition. Exosomes are small EVs (approximately 30–100 nm) that are derived from multivesicular bodies that fuse with the cell membrane [2,9]. Microvesicles are larger EVs (approximately 100–1000 nm) that are formed through cell membrane budding [2]. Apoptotic bodies are the largest EV subtype
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