Extracellular Vesicles for Therapeutic Nucleic Acid Delivery: Loading Strategies and Challenges

Abstract

Extracellular vesicles (EVs) are membrane vesicles released into the extracellular milieu by cells of various origins. They contain different biological cargoes, protecting them from degradation by environmental factors. There is an opinion that EVs have a number of advantages over synthetic carriers, creating new opportunities for drug delivery. In this review, we discuss the ability of EVs to function as carriers for therapeutic nucleic acids (tNAs), challenges associated with the use of such carriers in vivo, and various strategies for tNA loading into EVs.

Keywords: apoptotic bodies; drug delivery; ectosomes; exosomes; extracellular vesicles; intraluminal vesicles; microvesicles; multivesicular body; nanocarrier; nucleic acid therapeutics.

Figure 3

The ALIX–syntenin–syndecan pathway of exosome biogenesis. (A) Schematic illustration of the organization of ALIX and syntenin-1 protein domain structures [175,179]. (B) A proposed scheme for loading a tNA-conjugated ligand mimetic into exosomes. After endocytosis, syndecan is trimmered by heparanase in endosomes and subjected to proteolytic cleavage to its C-terminal fragment (I) [142,178,184]. The mimetic/receptor/HS complex can be sorted into ILVs in an ALIX- and syntenin-1- dependent manner (II–III; ALIX, syntenin-1, and ESCRTs not shown). The complex can be secreted into the extracellular environment via exosomes (IV). HS—heparan sulfate; PRD—proline-rich domain; NTD—N-terminal domain; CTD—C-terminal domain.

Figure 4

The formation of blebs and similar structures on the plasma membrane. (A) A characteristic feature of the amoeboid movement is the formation of a bleb lacking the actin cortex (I) [191]. Its size expands according to the influx of the cytoplasm (II). The bleb growth stops when the actin cytoskeleton (red lines, III) reassembles. Eventually, the bleb retracts into the plasma membrane (IV). (B) Receptor-induced biogenesis of microvesicles. Stimulation of the P2X7 receptor by ATP results in the phosphorylation of P38 MAP kinase (p38 MAPK) by src-protein tyrosine kinase (Src-K). Phosphorylation of P38 promotes the translocation of acid sphingomyelinase (A-SMase) to the outer leaflet of the plasma membrane, where it triggers ceramide-dependent microvesicle biogenesis [197]. (C) Vesicle shedding is a form of the plasma membrane repair mechanism [193]. Annexins have a high affinity for PS and play a key role in the removal of injured membrane regions. Annexin A7 (ANXA7) interacts with the ALG-2 (apoptosis linked gene-2) protein, which is a partner of ALIX. In turn, the ESCRT-III complex is assembled through ALIX and provides the damage site shedding [134,193]. In some cases, the vesicularization is preceded by the formation of filopodia-like protrusions at the damage site [195]. (D) Formation of apoptotic bodies [205]. Microtubule-rich membrane protrusions (microtubule spikes) and beaded apoptopodia can be formed in the presence or absence of membrane blebbing. Thin, string-like apoptopodia are generated between membrane blebs.

Figure 1

MVB interacts with other secretory and degradation processes in a cell. MVBs, autophagosomes, and amphisomes may undergo exocytosis or fusion with lysosomes, resulting in either degradation of their content or lysosomal exocytosis. It should be noted that some paths shown in the figure are hypothetical. The scheme was prepared on the basis of the reviews [82,84,88,89,90]. MVB—multivesicular body; PH—phagophore; CMA—chaperone-mediated autophagy; ER—endoplasmic reticulum; L—lysosome.

Figure 2

The endogenous strategies of tNA loading into EVs and schematic illustration of exosome and migrasome formation. (A) Endogenous strategies for tNA loading. The first (I) strategy is based on cell transduction, transfection, or electroporation by a tNA expressing vector. The second strategy (II) is based on the direct delivery of tNAs into EV-secreting cells. (B) Scheme of the ILV back-fusion. (C) The long tubular retraction fibers are formed by migrating cells during movement. Migrasomes localize on the tips or at the intersections of retraction fibers and contain numerous smaller (Ø 50–100 nm) vesicles. They are released into the extracellular environment after the fiber breaks. Apparently, in some cases, smaller vesicles inside them can exit into the extracellular space. ILV—intraluminal vesicle; MVB—multivesicular body; ASO—antisense oligonucleotide; siRNA—small interfering RNA.