Small extracellular vesicle loading systems in cancer therapy: Current status and the way forward
Introduction
Cancer has long been a global burden and is a leading cause of death worldwide [1,2]. Over the past decades, several breakthrough therapies have been developed, including targeted and immune therapies [3,4]. Currently, comprehensive treatment, which consists of a combination of chemotherapy, surgery, radiotherapy as well as the burgeoning targeted therapy or immune therapy, is used for advanced cancers. Although systemic chemotherapy exists as the most conventional strategy to inhibit cancer progression [5], most chemotherapeutic agents are highly toxic to both cancerous and healthy cells, and the low specificity of chemotherapeutic agents results in unwanted toxicity and side effects [6,7]. To address these problems, targeting drug delivery systems (DDSs) have been intensively studied and developed, and the most promising DDSs to have emerged in the past two decades are nanocarriers [8].
Typically, synthesized nanoparticles (NPs) range from 100 to 1000 nm in size and have several advantages, such as high drug-loading capacity (due to their high surface area to volume ratio), adjustable physiochemical properties and modifiable flexibiliy [8,9]. NPs represent a large family, which includes the eight most representative nanocarriers and their ramifications: liposomes, micelles, dendrimers, meso-porous silica nanoparticles, gold nanoparticles, superparamagnetic iron oxide nanoparticles (SPIONs), carbon nanotubes and quantum dots [8,10]. However, a major concern regarding NP-based DDSs is that the fate of the artificial nanocarriers in a physiological environment depends on its inherent characteristics, such as chemical composition, size, shape, specific surface area, surface charge, as well as surface modifications. Consequently, while an ideal nanocarrier should have a good balance of all these factors, this is impossible to achieve in reality [8], [9], [10], [11]. For example, abundant blood proteins usually interact with NPs to form a dynamic “nanoparticle-protein corona,” which results in some unwanted and uncertain biological effects [9]. In addition, most of the conventional nanocarriers tend to accumulate in different vital organs such as the lungs, spleen, kidneys, liver and heart, and, therefore, these organs may be vulnerable to greater toxicity [8], [9], [10], [11], [12]. As a result, it is difficult to construct a widely applicable DDS based on conventional NPs in the foreseeable future, and, as such, the discovery and design of more biocompatible tools are urgently needed.
Recently, the emerging application of exosomes and exosome-mimics in targeted drug and therapeutics delivery is being widely recognized. Exosomes, with a diameter of 30–150 nm, are a specific subtype of extracellular vesicles (EVs) originating from the late endosomal compartment [13]. They are secreted by all cell types and have been found in numerous body fluids, including blood, amniotic fluid, urine, malignant ascites, cerebrospinal fluid, breast milk, saliva, lymph, bile and pancreatic juice under both healthy and disease conditions [13,14]. As shown in Figure 1A, exosomes contain lipids, proteins, messenger RNAs (mRNAs) and micro RNAs (miRNAs)[16,17]. However, the mechanisms of exosome biogenesis and cargo sorting remain to be understood. The exosomes released by donor cells may be selectively taken up by recipient cells, so that exosomes serve as mediators in intercellular communications [13,14,18]. As far as we know, receptor-ligand interactions, direct membrane fusion and endocytosis/phagocytosis are three ways by which exosomes interact with recipient cells [19]. Their nanoscale size, potential to carry biomolecules, specificity of organtropism, easy accessibility, ability to pass through the blood-brain barrier and innate biocompatibility make exosomes suitable therapeutics carriers as compared with artificial nanocarriers in cancer therapy [20,21].
Although exosomes derived from malignant cells may promote cancer by altering the function of normal cells, exosomes can also be engineered or naturalized to deliver therapeutics to treat cancers. A consensus has not been reached on specific markers of exosomes, making purification of one specific subpopulation of extracellular vesicles (e.g., exosomes) difficult to achieve. Consequently, “exosomes” used in many research studies may be potentially contaminated with other heterogeneous populations of extracellular vesicle subtypes [22,23]. According to the latest Minimal Information for Studies of Extracellular Vesicles 2018 proposed by the International Society for Extracellular Vesicles, “small extracellular vesicles (<200 nm, sEVs)” might be a more appropriate term. Therefore, we have adopted the term “sEVs” in this review to refer to nanovesicles, including exosomes and exosome-mimics. The sEV-based delivery systems comprise three basic components: vesicles, cargoes and decorations. Therefore, we review studies in the field from these aspects, and discuss the approaches of cargo loading and surface modification.
Section snippets
sEVs
sEVs represent the core components in a sEV-based delivery system. Alternatively, NPs coated or decorated with natural cellular membranes are termed “biomimics” or “bioinspired” vesicles, and are similar to exosome-based loading systems. Li et al. and Fang et al. have given comprehensive overviews of the development of biomimic NPs [24,25]. Here, we focus on the vesicles formed by natural membranes.
Recently, the Minimal Information for Studies of Extracellular Vesicles 2018 issued guidelines on
Methods for loading cargoes into sEVs
The cargoes encapsulated in sEVs represent the therapeutic components of the sEV-based DDS. One of the key challenges is how to efficiently package cargoes into sEVs. Generally, there are three major strategies to load therapeutic agents: (i) manipulating gene expression in parent cells, so that the proteins or RNAs in exosomes or exosome-mimics are changed indirectly; (ii) incorporating therapeutic cargoes into producer cells so that a portion of the cargoes can be released into sEVs; and
Genetic modification in parent cells
Because some endogenous RNA and protein can be automatically encapsulated into exosomes, this approach incorporates cargoes into exosomes by engineering the genes of producer cells, so it is especially suitable for cytosolic and transmembrane proteins or high–molecular weight RNA that cannot be directly loaded into isolated exosomes [47]. Sterzenbach et al. reported that the evolutionarily conserved late-domain (L-domain) pathway could be used as a mechanism for loading exogenous proteins into
Incubation of parent cells with drugs
In this method, parent cells are incubated with a drug of interest and then the drug disperses into or is endocytosed by donor cells. Consequently, a fraction of the drug in the cytoplasm can be distributed into the corresponding sEVs. Pascucci et al. attempted to incubate murine SR4987 MSCs with a low dose of paclitaxel for 24 h and collected the exosomes derived from MSCs. These exosomes released by MSCs significantly inhibited proliferation of the human pancreatic cancer cell line CFPAC-1
Manipulating sEVs directly
Therapeutic agents could be directly packaged into sEVs using the following physical or chemical methods. In general, compared with manipulating the parent cells, directly manipulating sEVs is more controllable and effective in loading cargoes. With the exception of incubation, methods such as freeze/thaw cycles, electroporation, extrusion and sonication are limited by the fact that they may destroy the membrane integrity of the sEVs to variable degrees. Consequently, drugs may enter the sEVs
Cargoes
The cargo to sEVs-based delivery system is what the warhead is to a missile, and different cargoes exert different impacts on recipient cells. Applications to deliver conventional therapeutic drugs, proteins, siRNAs, miRNAs and imaging molecules in cancer treatments have been reviewed in many articles (Figure 2A). However, several novel studies have attempted to combine sEV vectors with immunotherapy, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein
Methods for modifying sEV surfaces
Similar to cell membranes, there are various functional proteins (such as adhesion molecules, ligand receptors, major histocompatibility complex (MHC) molecules and vesicle-specific markers) on the sEV membrane surfaces, which play important roles in mediating EV-to-cell recognition and interaction [90], [91], [92]. Jang et al. showed that the chemotherapeutic-agent-loaded sEVs specifically traveled to the endothelial cells in tumor tissue and suppressed tumor activity in vivo. Molecules such
Future outlook and conclusions
sEV loading systems herald the dawn of a new age of targeted therapy for cancer. However, there are several obstacles hampering the clinical application of exosomes. Several unanswered questions include the following: How to manufacture sEVs in a more standard and scalable way? How to maintain the stability and biological availability of sEVs for a long time? How to efficiently and safely load cargoes into sEVs and modify the membranes of sEVs? All these obstacles need further investigation.
A
Declaration of Competing Interest
This work was supported by grants from the National Natural Science Foundation of China (number 81700682) and the Medical and Health Technology Project of Zhejiang Province (number 2019311124).
The authors have no commercial, proprietary, or financial interest in the products or companies described in this article.
Author Contributions
All authors contributed to this paper with conception and design of the study, literature review and analysis, drafting and critical revision and editing and approval of the final version.
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