Emerging Significance and Therapeutic Potential of Extracellular vesicles

Extracellular vesicles (EVs), are membrane-bound vesicles that have many advantages over traditional nanocarriers for drug and gene delivery. Evidence from recent studies indicate that EVs have therapeutic capability with chemical or biological modification. Tumor-derived exosomes (TEXs) were used as a new type of antigens or tumor vaccines in anti-tumor immunotherapy. With superior characteristics, modified EVs were applied to loaded and delivered synthetic drugs, silencing RNA, and microRNA for treatment. Different surface functionalization strategies have been proposed to improve the therapeutic functions of EVs. Appropriately modified EVs for disease intervention provide new avenues for effective clinical treatment strategies. Therefore, this review aimed at elucidating the therapeutic functions of EVs to generate new ideas for treatment and to unlock their hidden potential in translational medicine.


Introduction
Cancer is a global human health problem that is associated with severe pain and heavy economic burdens to patients and their families [1]. Studies on EVs have elucidated on new strategies for cancer diagnosis and treatment. In recent years, nanomaterials for drug delivery have played an important role in the treatment of cancer [2]. However, they are associated with certain limitations. Therefore, studies are evaluating the value of EVs in the development of a comprehensive drug delivery system and as a circulating biomarker in cancer diagnosis [3]. The International Society for Extracellular Vesicles (ISEV) recognized the potential of EVs as treatments for cancer. EVs or exosome nanovesicles (NVs), which combine the characteristics of cells and nanocarriers, are clinically used in tumor diagnosis [4], and targeted anticancer therapies, such as breast [5], pancreatic [6], lung [7], and liver cancers [8].
In guidelines released by the ISEV, depending on their biogenesis and generation pathways, EVs can be broadly classified into three subpopulations: exosomes (EXOs, 30 to 200 nm in size), microvesicles (MVs, 200-1000 nm), and apoptotic bodies (ABs, larger than 1000 nm) [9]. EVs are extracellular nanovesicles secreted by various kinds of cells including dendritic cells (DC) [10], mesenchymal stem cells (MSCs) [11], neural cells [12], epithelial cells [13], and a variety of tumor cells [14]. Interestingly, EVs are also distributed in serum, urine, saliva, or any other body fluids [15,16]. They contain and tranfer diversified bioactive molecules include lipids, nucleic acids, and various proteins inside or on the surface of EVs, such as receptors, enzymes, transcription factors, and extracellular matrix proteins into adjacent or distant cells through the systemic circulation, participating in intracellular and intercellular communication, and regulate host tumor cell interactions [17]. As a general rule, of the targeting and fusion proteins present on the surface of EVs, the most abundant are integrins and Tetraspanins. The

Ivyspring
International Publisher members of the tetraspanin family, such as CD9, CD63, CD81 and CD82, regulate intercellular signal transduction [18,19]. As EVs contain biomolecules active from maternal cells, they can regulate function, fate, and shape of target cells, participating in different pathological and physiological conditions [20,21]. Conserved proteins participate in cytoskeleton formation (β-actin, myosin and tubulins), metabolism (glyceraldehyde 3-phosphate dehydrogenase) and protein folding (Hsp70) [22,23]. Cell-type-specific proteins such as notch ligands, β-Catenin, Wnt as well as intercellular cell signaling mediators mainly involved in cell signaling pathways such as TNF-α, TGF-β, and IL-1β [24]. Of course, EVs also contain nucleic acids, including mRNA, miRNA, DNA, and mitochondrial DNA (mtDNA). These nucleic acids are mainly involved in inflammation and as diagnostic biomarkers in tumors [25,26]. In recent years, scientists have paid more attention to EVs due to their key roles in biological systems.
Exosomal liposome structures allow the loading of various drugs, which enhances drug delivery to specific targets. EVs, as carriers, can transport their cargos to particularly intracellular locations in a target-specific manner across the plasma membrane. As natural nucleic acid and protein carriers, they have been used as vectors for targeted delivery of these molecules [27,28]. Based on their endogenous performances and multifunctional properties, EVs have a clinical potential for the development of efficient therapeutic options for cancer.
Evidence from recent studies indicate that EVs have therapeutic potential in tumors [29], neurological diseases [30] and immune diseases [31]. For instance, exosomes are not only used as carriers for tumor treatment drugs [32], but also as tumor immunotherapy, especially for those extracted from tumor cells robustly eliciting anti-tumor immune responses [33,34]. Given their low immunogenicity and high biocompatibility, exosomes can stably stay in the circulatory system for longer periods. Targeted administration of exosomes carrying drugs to tumor lesions can double the anti-tumor effect of such drugs. When used as carriers, EVs not only provide intrinsic immunomodulatory activity, but also have many advantages such extended circulation half-life, high biocompatibility, transfection efficiency, low immunogenicity and minimal reversion to virulence, over traditional nanocarriers for drug and gene delivery [34]. Hence, EVs may provide opportunities to enhance or broaden the innate therapeutic capability with chemical or biological modification.

EVs in cancer immunotherapy
Immunotherapy has shown promising prospects in the treatment of cancers, and EVs are currently applied in tumor immunotherapy. EVs have several advantages and appear to be a highlight of new pattern for cancer immunotherapy at present [35]. The use of EVs as a new type of antigens in anti-tumor immunotherapy has raise some concerns. By reactivating the patient's immune system, EVs exploit autoimmune cells, especially CD8+ T cells, to generate anti-tumor responses [36]. Although the potential of these therapies is widely known, there is still significant room for improvement. Associated immune checkpoint therapies tend to be ineffective and severe autoimmunity could also occur. Many tumors, due to genetic, biological and other factors make it unlikely that some patients will respond to these therapies. To overcome some of these obstacles, innovative approaches with lower toxicity and providing more frequent and durable response are needed. One such treatment is the use of nanoparticles, particularly EVs. Research has shown that TEXs potentiate PD-L1 function and suppress immune response [37]. In addition, in patients with melanoma receiving PD-1 blockade, PD-L1 levels in exosomes are correlated with tumor burden and treatment response. Although it is unclear whether the exosomal PD-L1 is directly related the failure of anti-PD-1 therapies, PD-L1-containing exosomes may be regulators and biomarkers of resistance to treatment.
Surface engineered EVs can actively participate in tumor immunotherapy. CD47, a "don't eat me" signal, limits the ability of macrophages to engulf tumor cells by binding to SIRPα [38]. Therefore, the exosomes carrying SIRPα variants may serve as immune checkpoint blockers, thereby antagonize the interaction between CD47 and SIRP. This will enhance tumor phagocytosis and exert effective anti-tumor T cell response [39]. Although the immunotherapeutic effects of dendritic cell vaccine have been reported, it is still unclear how tumor-associated exosome-based dendritic cell (DC) vaccine-based anti-tumor immunity can be induced to achieve antitumor effects [40]. Studies have shown that dendritic cell-derived exosomes (DEX) can regulate the immune responses to cancer [41]. DEXs stabilizes vesicles, and are not easily degraded or inactivated. Research on DEXs has shown that DEX is rich in membranous proteins such as major histocompatibility complexes class I (MHC class I), MHC class II molecules, CD63, CD81, and integrin, and has a strong immune activation effect [42]. The use of EVs as tumor vaccines has shown promising anticancer effects in vivo, and results from clinical trials on EVs have been feasible. A novel DEX vaccine with antigens and matured with either the TLR-3 ligand induced robust activation of melanoma-specific CD8(+) T cells and the recruitment of cytotoxic CD8(+) T cells, NK and NK-T cells to the tumor site, resulted in significantly reduced tumor growth and enhanced survival [43]. A phase I clinical trials to test DEXs loaded MAGE tumor antigens in patients with non-small cell lung cancer (NSCLC) showed that some patients experienced long term stability of disease and activation of immune effectors [44]. A phase II clinical trial testing the clinical benefit of DEX loaded with MHC class I-and class II-restricted cancer antigens as maintenance immunotherapy confirmed the capacity of DEX to boost the NK cell arm of antitumor immunity in patients with advanced NSCLC [45]. DEX maintain the key functions of DCs in their ability to present tumor-associated antigens (TAAs) and to activate TAA-specific immune responses [46]. To improve immunogenicity, exosomal antigen-adjuvant co-delivery systems have been engineered for cancer immunotherapy. Exosomes derived from genetically engineered tumor cells containing endogenous tumor antigens and CpG DNA with immunostimulatory have been found to confer anti-tumor effects [47]. Therefore, blocking the biogenesis and release of TEX seems to be a potential anti-tumor strategy. GW4869, an inhibitor of nSMase2, has been found to block ceramide synthesis. Treatment with GW4869 reduced lung metastasis in tumor-bearing mice. A combination of GW4869 with cisplatin and gefitinib provided strong antitumor effect [48].

EVs as drug carriers in cancer
Exploitation of EVs in anti-tumor research has gained momentum in recent years, and many studies have evaluated EVs -related antitumor effects. Compared with EVs, delivery of drug formulations such as liposomes [49], micelles [50], and microcapsules [51] using traditional carriers is limited in clinical application, due to immune rejection, low drug loading, and poor targeting. EVs, as carriers, do not elicit immune responses in the bloodstream like other nanoparticle formulations [52]. Given their superior characteristics to natural or synthetic polymers and liposomes, many studies ( Table 1) have reported that synthetic drugs, silencing RNA, and microRNA can be loaded into modified EVs and delivered into tumors yielding good results.
The most common treatments for malignant tumors are chemotherapy, radiation surgery, or combination therapy. Cancer targeted therapy is an emerging treatment approach in anti-tumor therapy. However, the success of targeted therapy requires a drug delivery carrier with low immunogenicity and low toxicity. Doxorubicin (DOX) was loaded into exosomes through electroporation, and delivered to breast cancer tumor cells by engineered exosomes leading to effective targeting [53]. The main advantage of using exosomes to carry biological molecules over other nanoparticles is that they possess natural ability to carry bio-related molecules such as nucleic acid drugs and can activate the immune system. Ohno et al. modified exosomes with a GE11 peptide that specifically binds to epidermal growth factor receptor (EGFR) and was loaded with let-7a miRNA, a regulator that reduced cell division and altered the cell cycle [54]. A synthetic multivalent antibodies retargeted exosome (SMART-Exosome) was designed to cross-link tumor cells with T cells and induce a strong immune response which effectively killed tumor cells in vivo and in vitro [55]. Although numerous drugs have been designed for prevention of cancer progression and suppress tumor development, of the efficacy of such drugs is limited by the low bioavailability and high toxicity. A highly biocompatible tumor cell-targeted delivery system has been designed to deliver imperialine (a less toxic anti-cancer agent) into NSCLC cells using exosome-like vesicles (ELVs) [56]. Given the high expression of alpha 3 beta 1 on NSCLC cells, they modified integrin alpha 3 beta 1-binding octapeptide cNGQGEQc to create an ELV platform for targeting tumor. This platform not only significantly improved accumulation and retention of imperialine in the tumor, but also exhibited extremely low systemic toxicity in vitro and in vivo. Natural compounds such as anthocyanins (Anthos) found in berries are limited by low permeability and oral bioavailability. Munagala et al. delivered exosomes loaded with anthocyanins from raw milk to mice with lung cancer [52]. They reported that exosomes provided an effective alternative for oral delivery of Anthos for efficient systemic delivery and robust bioavailability. For nervous system tumors, the blood-brain barrier (BBB) limits the entry of therapeutic drugs into the brain. Yang et al. tried to further their zebrafish studies pass through loading siRNA in the exosomes [57].

EVs for gene therapy
Donor cells have also been engineered to isolate qualified exosomes that contain the gene or drug of interest [58]. Exosomes are secreted from engineered cells through the endosomal pathway, and this pathway have been hijacked with viruses and used for superior delivery of RNA in vivo [59]. Many studies have exploited the genetic material naturally carried by foreign bodies, and many cancer-based studies have investigated exosomes using microRNAs. Thus far, exosome-derived microRNAs, through target gene transcriptional repression, have the demonstrated ability to induce cell migration, inflammation, immune responses, angiogenesis, invasion, pre-metastatic niche formation and metastasis ( Figure 1). Masaki et al. found that miR-199a-3p-Exo suppressed c-Met expression, a direct target of miR-199a-3p, leading to the inhibition of cell proliferation and invasion [60]. The engineered exosomes were utilized to simultaneously deliver the anticancer drug 5-Fu and the Mir-21 inhibitor oligonucleotide (Mir-21I) to cancer cells expressing human epidermal growth factor receptor-2 (HER2) [61]. This effectively reversed drug resistance in 5-FU-resistant colon cancer cells and significantly enhanced cytotoxicity. These studies highlight the potential of exosomes encapsulated with tumor suppressor miRNAs in the treatment of cancer.   Some in vivo studies have also been conducted with satisfactory results. Introduction of T7-exo and antisense miRNA oligonucleotides against miR-21 (AMO-21) via tail vein effectively introduced AMO-21 into the brain and reduced Mir-21 levels in glioblastoma in rats [62]. Downregulation of miR-21 by AMO-21 induced the expression of PDCD4 and PTEN in tumors, thereby decreased the tumor size. In a study, miR-129-3p directly inhibited the expression of SUMO-activated enzyme subunit 1 (SAE1) by targeting 3'UTR and also suppressed the zoylation of XRCC4, leading to more DNA damage in gastric cancer cells and inhibition of the proliferation, migration and invasion of gastric cancer cells [63]. The effect of exosomal encapsulated miRNAs on tumor chemical sensitivity was also investigated. Intra tumoral injection of miR-122-containing exosomes combined with sorafenib significantly reduced the weight and volume of tumors, indicating that exosomes from miR-122 adipose derived MSCs (AMSC) may enhance the sensitivity of human hepatocellular carcinoma (HCC) cells to chemotherapy [64]. The enhanced efficacy of engineered exosomes (iExosomes) in targeting oncogenic KRAS compared to liposomes has also been studied [65]. A key driver of common mutations in pancreatic cancer is the mutational form of GTPase KRAS. Application of a targeting method called RNA interference (RNAi) successfully inhibited tumor growth and significantly increased overall survival of mice with pancreatic cancer.

Modification of EVs for targeted delivery
The composition of EVs can be modified at the cellular level. EVs from various biological sources can be modified after isolation when cell-derived exosomes are unable to meet the requirements. While preserving the membrane integrity of exosomes, functional fluorescent tags [102], imaging probes [103], immuneactivators [104], and targeted therapeutic agents can be added [105]. Modified EV surface structures allow in vivo imaging and tracking of EVs. Although EVs have been shown to be useful in vitro anticancer drug carriers, this is not the case in vivo given their non-specific toxicity and off-target effects similar to those observed in conventional chemical drugs [106]. EVs need to be functionalized with specially designated parts to optimize their transmission properties. To improve the therapeutic functions of EVs in cancer treatment, different surface functionalization strategies have been proposed as shown in Figure 2.

Surface engineering of EVs
To further increase the functions of EVs, different surface engineering strategies have been explored, which can be roughly divided into three main approaches: physical, chemical, and biological approaches.

Physical methods
In this approach, physical approaches such as ultrasonic treatment, extrusion, and freeze-thaw are used to temporarily destroy lipid structures. Once the structures are removed, the vesicles spontaneously re-assemble into their natural structure. Sagar et al. hybridized macrophage-derived exosomes with liposomes through freeze-thaw method which allowed membrane fusion to form hybrid immune exosomes for doxorubicin treatment of breast cancer [107]. Since freezing and thawing can lead to denaturation of proteins, to avoid this, they applied extrusion method to promote membrane fusion.
Natural killer cell exosomes (NKsome) were prepared by simple liposome membrane extrusion technology [108]. The engineered NKsome successfully retained the targeted proteins associated with the NK cell membrane on its surface and showed higher affinity for cancer cells. Moreover, doxorubicin-loaded NKsome showed good homing efficiency in vivo, and its plasma retention time was extended by 18 h providing strong therapeutic effects. A biomimetic nanostructure (BNS) with a multimodal imaging system was designed to coat polymer nanoparticles with natural killer cell membrane (NKM), near-infrared ray (NIR) fluorescent dye and Gadolinium (Gd) conjugated magnetic resonance imaging (MRI) contrast agent [109], which allowed in vitro and in vivo tracking [110]. The physical approach of surface functionalization allows for the simple reagent free functionalization of exosomes. Compared to biological and chemical methods, physical method does not require additional reagents or cell-based systems.

Chemical methods
Chemical approaches for surface functionalization involve direct use of chemical reagents to anchor functional parts to the surface of exosomes. The transmembrane protein portion of phospholipids or amine/carboxylate on surface of exosomes can be directly functionalized with various functional groups. Alternatively, functional phospholipids obtained from exosomes can be added to exosomes by simple incubation using a hydrophobic insertion strategy. Firstly, lipid functionalization of exosomes sealed with maleimide is achieved via the hydrophobic insertion strategy. A hydrophobic insertion strategy in which maleimide-terminated DSPE-PEG-Mal is used as a labeling probe, which is labeled with fluorescent dye containing maleimide for monitoring of cell communication [111]. Secondly, azide can be linked with cargo-conjugated dibenzobicyclooctyne (DBCO) through azide-alkyne cycloaddition. Wang et al. combined metabolic markers of newly synthesized proteins or polysaccharides/glycoproteins from secreting exosomal cells with chemically active azido groups, modified and functionalized exosomes through bioorthogonal click conjugation [112]. Lastly, copper-catalyzed azide-alkyne cycloaddition is another click chemistry strategy that can be used for surface functionalization of azide-functioning exosomes. Exosomes were chemically crosslinked with alkyne groups by carbodiimide and coupled to a model azide, fluoroazide 545. The coupling had no by-effect on the size of exosomes, nor did it alter the degree of adhesion/internalization of exosomes to recipient cells. This technique is superior to other exosome-labeling methods and is likely to find widespread application in exosome research [113]. These chemical methods are widely used to functionalize biomolecules because they are easy, fast and compatible with biomolecules.

Studies
show that different surface functionalization strategies can enhance targeting of exosomes by adding targeted peptides or ligands to EV surfaces. Expression of a well-characterized exosomal membrane protein (Lamp2b) on engineered exosome DCs fused to αv integrin-specific iRGD peptide has been reported to promote tumor targeting [53]. EVs modified with GE11 peptide, a synthetic peptide that binds specifically to EGFR, could efficiently deliver let-7a miRNA to EGFR-expressing xenograft breast cancer tissues in mice, leading to a marked inhibition of tumor growth [114]. Besides, specific targeting molecules such as folic acid (FA), iron oxide, and aptamer have been used to modify EV. Aptamers with high affinity and specificity for their targets are often considered as substitutes for antibodies in targeted delivery. Targeted delivery of miR-21 to leukemia cells was achieved by modifying exosomes with the cholesterol-conjugated aptamer AS1411 [115]. Most tumors are angiogenic and produce exosomes, and novel strategies targeting exosome induced angiogenesis can reduce tumorigenesis [116]. Corrado et al. reported that carboxyamidotriazole orotate (CTO) targets tumor exosomes promoted IL-8 expression and cell adhesion of endothelial cells (ECs). Therefore, CTO inhibits the effects of these exosomes on ECs-chronic myelogenous leukaemia (CML) interaction and the migration of ECs, suppresses angiogenesis induced by exosomes [117]. This resulted in enhanced specific targeting of cancer cells and effective inhibition of tumor growth. Folate receptor (FR) is a glycosylphosphatidylinositol glycoprotein anchored on the cell surface, which is overexpressed in many epithelial malignancies, including ovarian, breast and lung cancers. Erastin-loaded exosomes labeled with FA were used to target triple negative breast cancer (TNBC) cells overexpressing FA receptors [71]. Tissue-specific delivery may also be achieved by loading exosomes with magnetic nanoparticles. It has been reported that exosomes anchored with cell-targeted peptides (CPPs) and TNF containing superparamagnetic iron oxide nanoparticles improve tumor targeting in external magnetic fields and inhibit tumor growth [118]. Surface-functionalization approach promotes expression of targeted cargo on exosomes surface. Apart from peptides, incorporating targeting proteins containing nanobodies to exosome surface has also been demonstrated as an attractive targeting strategy. Cheng et al. linked a rather sophisticated polypeptide composed of two single chain variable fragment (scFv) antibodies targeting CD3 and EGFR on top of the domain of human platelet-derived growth factor (PDGFR) [55]. Here, they integrated with EVs by the EV biogenesis process to obtain surface-functionalized EVs with antibodies. Exosomes prepared from Ovalbumin (OVA)-pulsed, activated dendritic cells were modified with anti-CTLA-4 antibody to block this inhibitory molecule and to enhance the specificity of the exosomes toward T cells. This study provides a unique strategy to endow exosomal membranes with the function of anti-CTLA-4 antibody to synergize the efficacy of cancer vaccines and checkpoint blockade on tumors [119].

Remaining concerns and future perspectives
The extraction, isolation, and identification of tumor cell-derived EVs can be used to elucidate the mechanisms underlying tumor progression and provide potential therapeutic targets for cancer patients. Secondly, EVs can be loaded in different antitumor drugs to treat cancer. Lastly, with increasing interest in tumor immunotherapy, EV immunomodulatory properties mainly include regulating antigen presentation, immune surveillance, and immune activation. It is likely that a new approach to tumor immunotherapy will be revealed through an in-depth study of the molecular mechanisms underlying the interaction between EVs and immune cells.
As intermediates of intercellular communication, EVs have heterogeneous and pleiotropic physiological and pathological roles. However, many issues still need to be addressed before its clinical application. Due to tissue and cell specificity, not all tissues and cells express these so-called EV markers. Therefore, further investigation into how to identify EVs is a significant goal for future research. Isolation of purification specific EVs is limited by technical limitations, and specific EVs by the availability of suitable biomarkers as well as expensive techniques. It is necessary that we develop standard and highly efficient methods for EV isolation, purification, characterization, and manipulation that allow these vesicles to be successfully applied in the clinic.

Conclusion
In the field of nanofabrication, EVs have been widely applied in the diagnosis and treatment of tumors, but the clinical application of exosomes still faces arduous challenges. EVs play important roles in the tumor microenvironment, tumor and non-tumor tissue. Considering their good biocompatibility, tumor targeting and low immunogenicity, EVs have been proven to be potential drug carriers. However, the complex characteristics of exosomes are not thoroughly understood, it is important to comprehensively define the many subtypes of exosomes. Although there are many challenges in the costs, technical challenges, and lack of suitable biomarkers for EVs isolation and purification, more classification and loading mechanisms of exosomes can be exploited in further research to develop efficient exosome-based drug delivery systems. Moreover, EVs have shown great promise in tumor targeting, tumor immunotherapy and inhibition of tumor metastasis. In the future, in-depth research is needed to develop effective tools for efficient drug loading, targeted modification, and studying the mechanisms that drive tumor development and response to drugs. Such efforts will also generate new ideas for the diagnosis and treatment of tumors.