Cytochalasin-B-Inducible Nanovesicle Mimics of Natural Extracellular Vesicles That Are Capable of Nucleic Acid Transfer

Abstract

Extracellular vesicles provide cell-to-cell communication and have great potential for use as therapeutic carriers. This study was aimed at the development of an extracellular vesicle-based system for nucleic acid delivery. Three types of nanovesicles were assayed as oligonucleotide carriers: Mesenchymal stem cell-derived extracellular vesicles and mimics prepared either by cell treatment with cytochalasin B or by vesicle generation from plasma membrane. Nanovesicles were loaded with a DNA oligonucleotide by freezing/thawing, sonication, or permeabilization with saponin. Oligonucleotide delivery was assayed using HEK293 cells. Extracellular vesicles and mimics were characterized by a similar oligonucleotide loading level but different efficiency of oligonucleotide delivery. Cytochalasin-B-inducible nanovesicles exhibited the highest level of oligonucleotide accumulation in HEK293 cells and a loading capacity of 0.44 ± 0.05 pmol/µg. The loaded oligonucleotide was mostly protected from nuclease action.

Cytochalasin B-Induced Membrane Vesicles from Human Mesenchymal Stem Cells Overexpressing IL2 Are Able to Stimulate CD8+ T-Killers to Kill Human Triple Negative Breast Cancer Cells

“…Large-scale production of EVs for clinical use is not without its challenges, the most prominent of which is achieving a sufficient yield of EVs when endogenously produced [ 26 ]. Alternative approaches are being applied to increase EV yields, one of which is the use of cytochalasin B. Cytochalasin B causes actin filament dissociation and the shaking of such cytochalasin B-treated cells causes cell disintegration and the formation of multiple vesicles that are built from cell plasma membrane [ 27 ]. Use of cytochalasin B-induced membrane vesicle (CIMV) production offers a new approach to simplify the isolation of vesicles on a large scale to enable their use in clinical practice.…”

Antigen-specific stimulation and expansion of CAR-T cells using membrane vesicles as target cell surrogates

“…1A ). TEM images demonstrate that the diameter of the AVs ranges from 200 to 2 000 nm.The AVs looked creasy, which is typical for the relatively large artificial vesicles 11 .…”

“…Membrane blebbing was induced by treatment of cells with cytochalasin B. Cytochalasin B rapidly disrupts the actin cytoskeleton, resulting in extrusion of the nucleus, detachment of the plasma membrane, and formation of long tubular extensions from the cell 12,16 . These extensions can be sheared off by shaking to form vesicles which carry a replica of surface proteins expressed by the source cell 11 ( Figure 1 B and C ). We envisioned that AVs could serve as a mimic of tumor cells, since they would possess the same set of surface molecules.…”

“…We felt that a promising strategy to obtain high numbers of functional antigen-specific CAR-T cells would be to stimulate CAR-T cells using antigen exposed on cell membrane fragments organized on relatively uniform microcytoshperes that lack a nucleus. It is known that the blebbing of cell membranes caused by cytochalasin B treatment results in the generation of microvesicles surrounded by plasma membrane [11][12][13] . Importantly, it has been shown that surface molecules present on the original plasma membrane are also displayed on the membrane surface of these vesicles, and in their native membrane-anchored form, with both proper glycosylation and folding 14,15 .…”

Perspectives in Manipulating EVs for Therapeutic Applications: Focus on Cancer Treatment

“…So far, several studies employed saponin-based loading technique, showing its high efficacy [ 31 , 53 , 56 , 66 , 67 , 68 ]. This strategy likely preserves the integrity of EV membranes [ 45 ], and can be employed for encapsulating of molecules that are insensitive to detergents.…”