1. Introduction

Cancer is one of the most significant burdens a person can bear. Cancer is responsible for one out of every six deaths worldwide, according to the World Health Organization [1]. Most traditional antitumor small molecule chemotherapeutics and antibody drugs work by binding to target proteins, but the druggability of the target proteins limits their development. Only 3000 of the roughly 20,000 proteins encoded by the human genome are druggable, and only 700 have corresponding drugs in development [2,3]. Nucleic acid drugs can modulate extracellular and cell membrane proteins, whereas antibody drugs only act on cell membranes and extracellular proteins.

Cancer is caused by changes in genetic material, such as genetic mutations and chromosomal aberrations, which eventually lead to continued proliferation and metastasis. Nucleic acid drug therapy can begin at the source of the disease by exogenously introducing the therapeutic genes into diseased cells, correcting the disease caused by gene defect and abnormality, and achieving a therapeutic effect on the tumor [4]. Gene drugs can achieve breakthroughs in difficult-to-make protein targets and have a high potential for developing drugs for “untargetable” and “undruggable” diseases [5].

Extracellular vesicles (EVs), which are cell-derived, phospholipid-based bilayer membrane particles, are considered potential bioderived nanocarriers. Compared with synthetic lipid nanoparticles, EVs have natural biological advantages: low toxicity, low immunogenicity, exudation in tissues, ability to cross biological barriers, targeting of specific cell types, easy fusion with cell membranes, and ability to achieve endo/lysosomal escape. Moreover, nucleic acid drugs loaded in EVs are naturally protected from circulation degradation, which is a major advantage of EVs as drug delivery systems (DDS) [6-8].

2. EVs as Nucleic Acid Drug Delivery Vehicles

The International Society for Extracellular Vesicles (ISEV) is a professional social group composed of researchers and scientists in the field of EVs. It is committed to promoting global EV research and is one of the most authoritative societies in the field of EVs. As defined by the ISEV, EVs are the general term for particles that are naturally released from cells, which are separated by lipid bilayers and cannot replicate, i.e., do not contain a functional nucleus [9]. They can be endosome-derived (termed exosomes, diameter 30~150 nm) or are generated by membrane outward budding (termed ectosomes, diameter 50~1000 nm) [10]. Based on this, EVs can be applied in targeted therapy, cell-free therapy, and drug delivery systems [11,12].

Many types of cells are suitable for producing EVs based on their natural properties. Stem cells are favored for their high safety and high EV secretion and have been used in clinical studies [13]. However, as a drug delivery system, the following aspects still need to be considered: (1) EV secretion varies widely among different cell types and subpopulations [14] and may be further influenced by cell state and growth conditions [15]. Since various types of cells can generate EVs in response to endogenous or exogenous stimuli, how to improve the production of EVs is a key step for the widespread application and industrialization of EVs as DDS [16]. (2) Improving the encapsulation rate of nucleic acid drugs is also a consideration for realizing industrialization. (3) Although EVs themselves can circumvent the clearance of the mononuclear phagocytic system to a certain extent (clearing circulating particles larger than 100 nm), engineering modifications are required to maximize their circulation time and emphasize their advantages in intercellular communication [17]. (4) The different characteristics of EV producers and target cells may lead to significant differences in the efficiency of cell-to-cell communication. The efficiency of cellular uptake may be affected by surface-specific proteins, lipopolysaccharide decoration, and the overall potential (usually negative charge) of EVs. Therefore, targeting modifications for EVs have been extensively studied [18,19]. Given the foregoing, it is critical to design EVs to improve the efficiency and quality of nucleic acid drug delivery vehicles.

3. Improvements in EV Drug Delivery Systems

EVs are nano-scale vesicles with surfaces composed of a heterogeneous mixture of lipids and proteins, and naturally have the advantages of stealth, biocompatibility, and intrinsic homing ability. Although natural EVs already have certain targeting, long-term circulation, and cell entry capabilities, researchers are not limited to using natural EVs but intend to engineer them for better effects [5,20,21]. Here, we discuss improvement strategies according to the following four purposes (Figure 1): (A) to increase EV production; (B) to improve nucleic acid drug loading efficiency; (C) to extend circulation time; (D) to improve targeting capability and introduce corresponding modification methods (Table 1). At the end of this section, we compare the advantages and disadvantages of various strategies for modification purposes (Figure 2).

Figure 1. Summary of different modification strategies for EVs. (Created with BioRender.com, accessed on 23 August 2022).

4. Conclusions and Research Prospects

Currently, the application of EVs is facing the following questions: (1) Quality control is a problem. (2) The drug loading rate is still not ideal. (3) Stability is difficult to guarantee. (4) Metabolism and dynamics tracking are difficult to achieve. 

In the future, commercial EV production requires stricter GMP specifications, including the selection of EV sources, standardized cell culture techniques, downstream EV isolation, purification, and quality assessment protocols, EV detection and tracking, and the stability of EV-based preparations [22].

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