Extracellular Vesicles as Novel Drug-Delivery Systems through Intracellular Communications

Abstract

Since it has been reported that extracellular vesicles (EVs) carry cargo using cell-to-cell comminication according to various in vivo situations, they are exprected to be applied as new drug-delivery systems (DDSs). In addition, non-coding RNAs, such as microRNAs (miRNAs), have attracted much attention as potential biomarkers in the encapsulated extracellular-vesicle (EV) form. EVs are bilayer-based lipids with heterogeneous populations of varying sizes and compositions. The EV-mediated transport of contents, which includes proteins, lipids, and nucleic acids, has attracted attention as a DDS through intracellular communication. Many reports have been made on the development of methods for introducing molecules into EVs and efficient methods for introducing them into target vesicles. In this review, we outline the possible molecular mechanisms by which miRNAs in exosomes participate in the post-transcriptional regulation of signaling pathways via cell-cell communication as novel DDSs, especially small EVs.

Keywords: adeno-associated virus; extracellular vesicles; intracellular communication; membrane vesicle production; non-coding RNAs.

Figure 1

Secretions of extracellular vesicles (EVs). EVs are divided into three main populations according to their diameters: exosomes at 30 to 150 nm, microvesicles (MVs) at 100 to 1000 nm, and apoptotic bodies at 50 to 5000 nm. The exosome biogenesis involves the formation of multivesicular bodies and transfer to the plasma membrane, where the exosomal contents, proteins, and miRNAs are released via their fusion. The formation of both MVs and apoptotic bodies is accompanied by budding and blebbing of the cell membrane to pinch off new vesicles, respectively.

Figure 2

Mechanisms of exosome biogenesis and secretion. Upon the binding of ligand with exosome, the endocytosis process involves inward budding of the cell membrane, which occurs at lipid rafts containing common membrane proteins, such as tetraspanins (e.g., CD9, CD81, CD63, etc.), MHC class I and class II, and adhesion molecules (e.g., integrins, cadherins, etc.). The internalized cargoes, including proteins, nucleic acids, and lipids are sorted into early endosomes, which mature into late endosomes. The intraluminal vesicles (ILVs) of the late endosomes are formed through budding from the perimeter membrane into the endosome lumen following the encompassing the bioactive molecules by ESCRT-dependent or -independent pathways. Multivesicular bodies (MVBs) containing some ILVs are selectively led into two different pathways, i.e., lysosomal degradation or secretion of exosomes toward the extracellular space through the fusion of lysosomes, or the plasma membrane in the case of exocytosis, which are regulated by the Rab GTPase family. miRNAs transcribed from nuclei are incorporated into the ILVs via interactions with RNA-binding proteins.

Figure 3

Macrophage phagocytoses apoptotic cells via externalization of phosphatidylserine (PS) on EVs induced apoptosis as “eat me signal”. In apoptotic conditions, PS in the plasma membranes of cells externalizes, which is recognize by macrophages. Further, like the plasma membrane, the surface of the EVs has the PS, leading to uptake into macrophages.

Figure 4

The AAV2 genome structure. The genome of AVV consists of a linear single-stranded DNA spanning approximately 4.7 kilobases (kb), flanking with two 145 nucleotides of long inverted terminal repeats (ITRs) at their terminals. Within the genome, the two viral genes rep and cap code for non-structural and structural proteins, respectively. The non-structural (rep) coding region is regulated by two promoters, p5 and p19, resulting in the generation of a set of four overlapping proteins, Rep78, Rep68, and Rep52, Rep40, respectively. The cap gene encodes three structural capsids (VP1 to VP3) by promoter p40 and the assembly-activating protein (AAP), which promotes virion assembly and the membrane-associated accessory protein (MAAP) from an alternative open reading frame (ORF). X ORF at the 3′ end of the cap gene has a specific role in AAV DNA replication.