Extracellular Vesicles as Novel Nanocarriers for Therapeutic Delivery

C H A P T E R

12 Extracellular Vesicles as Novel Nanocarriers for Therapeutic Delivery Yusuke Yoshioka*, Takahiro Ochiya*,† *Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan † Institute of Medical Science, Tokyo Medical University, Tokyo, Japan

1 INTRODUCTION The Drug Delivery System (DDS) is one of the key technologies for achieving safe treatment, as without DDS, drug molecules can easily diffuse throughout the body and affect nondiseased sites.1, 2 In recent years, nanomaterial-based DDSs have attained considerable prominence. Many kinds of nanobased drug formulations have been used to improve the therapeutic efficacy of chemical and biomolecular drugs.3–5 However, clinical translation of these systems faces two distinct issues: cytotoxicity of the materials and rapid clearance by the reticuloendothelial system (RES) or the mononuclear phagocyte system (MPS).6–8 To address these issues, methods such as coating drug-loaded nanocarriers with a polyethylene glycol (PEG) to promote stealth delivery and decrease clearance by the MPS have been introduced. Although PEGylation decreases clearance by the MPS, it also reduces the interaction of the nanoformulation with target and barrier cells, thereby decreasing the drug biodistribution in diseased tissues.9–11 In addition, development of an immune response to the PEG

Nucleic Acid Nanotheranostics https://doi.org/10.1016/B978-0-12-814470-1.00012-5

corona significantly increases the clearance of PEGylated drug nanocarriers. In extreme cases, severe allergic reactions to PEG due to immediate exposure to reactive antibodies in the blood have been reported.12 Furthermore, in the case of a DDS for tumor tissue, the EPR effect of nanoparticles is also an effective strategy. The EPR effect enables some selective tumor uptake and retention of nanoparticles due to the leaky tumor vasculature and poor lymphatic drainage in tumors, respectively.13–15 However, the delivery of nanoparticles to tumors through the EPR effect has been reported to be inefficient and provides only approximately a 25% increase in delivery compared with normal organs.16 Therefore, for the clinical translation of new nanomedicines, more continuous improvements are required. Compared with synthetic nanoformulations, endogenous DDSs have shown promising results in enhancing drug delivery and reducing side effects because of their native biocompatibility in vivo.17 In this respect, because extracellular vesicles (EVs) have the intrinsic ability to traverse biological barriers and to naturally

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Copyright # 2019 Elsevier Inc. All rights reserved.

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transport functional biomolecules between cells, they represent a novel and exciting delivery vehicle for the field of nucleic acids and proteins. In this review, we highlight the recent developments in EV-based drug delivery systems, followed by a discussion of current challenges and future perspectives.

2 EVs AS NATURAL DELIVERY VEHICLES In this article, we use the term EVs as an umbrella term for all types of vesicles present in the extracellular space. Some confusion exists in the literature regarding the terms “microvesicles” and “exosomes.” The difference between these two terms is generally based on size: exosomes are in the range of approximately 100 nm, and microvesicles are in the range of 100–1000 nm. Only a few years ago, the International Society for Extracellular Vesicles (ISEV) recommended that researchers use the term “EVs” as an umbrella term for all types of vesicles present in the extracellular space, including exosomes, shedding vesicles, melanosomes, prostasomes, and apoptotic bodies. According to this recommendation, we use the term EVs throughout the paper. First reported by Pan and Johnstone18 in 1983, EVs were disregarded as cell debris and considered part of a disposal mechanism. However, EVs have gained increasing attention because of accumulating knowledge that EVs are natural nanosized vesicles secreted by endogenous cells (Fig. 1A) and that their intrinsic roles are to transport biomolecules, including messenger RNAs (mRNAs), microRNAs (miRNAs), proteins, and lipids (Fig. 1B), between cells.19 Many reports have introduced the concept that EVs can be considered not only a waste disposal mechanism but also important mediators of intercellular communication using EV-associated biomolecules.20 A seminal paper by Valadi et al.21 in 2007 led the way in the development of the EV research area. They identified miRNA as well as mRNA inside EVs and showed the potential functionality of mRNA

in recipient cells. After this report, many researchers attempted to identify the functions of EV-associated miRNAs because the report from Valadi et al. did not clarify the function of EVassociated miRNAs in recipient cells. In 2010, three groups independently reported that EVs contain miRNA transferred between cells and subsequently suppress the target genes in recipient cells.22–24 Thus, EVs have potential as natural delivery vehicles for proteins and nucleic acids. Furthermore, cells constantly release EVs into the extracellular space and systemic circulation, such as blood, urine, and saliva.19 The presence of the lipid bilayer protects the EV cargo from enzymatic degradation as the EVs move from donor to recipient cells. EVs are nanosized vesicles that are biocompatible, stable, nonmutagenic, biological barrier permeable, and that have low immunogenicity, rationalizing their potential use as an ideal vehicle for therapeutic delivery.25–28 EVs potentially can function as biomolecule carriers for therapeutic purposes. EVs derived from engineered cells may realize the goal of delivering small RNA to a specific cellular environment with high efficiency and safety. Moreover, EVs are expected to have tropism for specific cells or organs. For instance, Hoshino et al.29 described a distinct integrin expression pattern in EVs that correlates with the metastatic organotropism of cancer cells. The EV-associated integrins α6β4 and α6β1 are associated with lung metastasis, while αVβ5 is associated with liver metastasis. In other words, EVs have their own protein “zip codes” (or “shipping tag”) that address them to specific target organs or cells. Indeed, we have an opportunity to understand natural targeting delivery systems with EVs.

3 EXOSOME AND MICROVESICLE BIOGENESIS EVs are heterogeneous organelles in nature and can also be broadly classified as exosomes and microvesicles based on the biogenesis pathway through which they arise (Fig. 1A).

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FIG. 1 Schematic representation of EV biogenesis and molecular composition of EVs. (A) Exosomes are released by cells when intracellular organelles called multivesicular bodies (MVBs) fuse with the plasma membrane. Microvesicles are shed directly from the plasma membrane. (B) EVs pack a variety of cellular components, including nucleic acids (e.g., DNA, mRNA, and miRNA), lipids (e.g., cholesterol), and various types of proteins, such as membrane proteins, tetraspanins, adhesion molecules, enzymes chaperones, and various tissue-specific proteins.

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In general, exosome biogenesis consists of two steps: the inward budding of the membranous vesicles of endosomes and their release into a structure known as a multivesicular body (MVB). Early endosomes mature into late endosomes.19 During this maturation, contents fated to be degraded or exported outside the cell are enriched in vesicles that bud inward to the lumen of late endosomes. The accumulation of these vesicles inside the late endosomes is termed an MVB. MVBs then fuse with the plasma membrane to release exosomes through the process of exocytosis or alternatively can fuse to the lysosomes for degradation. Compared with exosomes, MVs are larger (100–1000 nm in diameter) and are secreted by shedding or budding from the plasma membrane.19 In addition to size and the process through which they are formed, microvesicles differ from exosomes not only in lipid content but also in protein content.

4 POTENTIAL APPLICATIONS OF EVs IN THERAPEUTIC DELIVERY EVs are naturally secreted lipid bilayer membrane-enclosed nanosized vesicles, enabling them to cross biological membrane/ barriers and deliver their payloads to recipient cells with virus-like efficiency. Moreover, EVs are less immunogenic, noncytotoxic, and nonmutagenic compared with other existing viralor liposome-based gene delivery vehicles.26 These characteristics suggest that EVs can be developed as an ideal vehicle for therapeutic delivery.28 Therefore, in recent years, several studies have highlighted situations in which an EV-based drug delivery system has improved disease conditions. For example, the first demonstration of this type of delivery was in 2010 when Sun et al.30 showed that mouse lymphoma cell EL4-derived EVs complexed with curcumin exhibited improved circulation and targeting in comparison with the free drug.

Curcumin is a natural polyphenol derived from turmeric. Curcumin is relatively unstable and highly hydrophobic, making it poorly soluble in aqueous buffers31; thus, it has been difficult to harness its therapeutic potential for clinical purposes. To overcome the stability and hydrophobic limitations of curcumin, the authors used EVs as a vehicle. Their loading method consisted of incubating EL4-derived EVs with curcumin in phosphate-buffered saline (PBS) at 22°C for 5 min before purification by gradient centrifugation, as curcumin is known to cause lipid rearrangement and changes in lipid fluidity of the cell membrane and the lipid membrane bilayer of EVs can be used for passively loading hydrophobic cargo. After this report, several studies showed that hydrophobic molecules, such as anticancer drugs, could be spontaneously packaged into EVs under ambient conditions.32–35 Doxorubicin has been loaded into unmodified EVs isolated from mouse breast cancer 4T1, human breast cancer MCF-7, and human prostate cancer PC3 cell lines through incubation at 37°C for 2 h.35 In this study, EV-associated doxorubicin was reported to inhibit tumor growth in vivo to a significantly higher extent than liposomal doxorubicin. Another study tested the intranasal administration of EVs loaded with curcumin or another hydrophobic compound, JSI-124 (cucurbitacin I), for the treatment of brain inflammatory diseases and brain tumor.32 JSI-124 is an inhibitor of signal transducer and activator of transcription 3 (STAT3). Both curcumin and JSI-124 were also loaded via incubation and gradient centrifugation. Administration of both EV variants resulted in protection against Lipopolysaccharide (LPS)-induced brain inflammation as well as reduced encephalomyelitis progression and GL26 tumor growth. These results demonstrated that this strategy could provide a noninvasive therapeutic approach for treating brain inflammatoryrelated diseases and brain tumors. Regarding the therapeutic cargo, small RNAs, such as small interfering RNA (siRNA)

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and miRNA, are powerful tools for therapy.36 These small RNAs inhibit the expression of specific target genes.37,38 In recent years, small RNA has become increasingly important in drug development because of its high specificity, significant efficacy, minor side effects, and ease of synthesis.39 Although small RNA has the potential to be a powerful therapeutic drug, its delivery remains a major limitation. One limitation is that naked small RNAs suffer from rapid degradation in the extracellular environment and are not efficiently internalized by cells.40 To address this problem, nanocarriers, including liposome, are the most common means of delivering unstable naked small RNA to the targeted tumor sites as they protect the small RNA from nucleases in the blood and undesirable immune responses, thus assisting in endocytosis.40 Researchers have focused on the body’s own RNA transport service, including EVs, which may serve as an attractive alternative. Alvarez-Erviti et al.41 reported the first proofof-concept study on the delivery of exogenous siRNA using EVs. In this study, primary dendritic cells from murine bone marrow were harvested and engineered to express the neuron-specific Rabies virus glycoprotein (RVG) peptide, which was fused to an EV membrane expressing the protein Lamp2b. The surface expression of RVG on EVs allows the EVs to specifically target the acetylcholine receptor. Several studies showed that RVG peptides can be used in delivering therapeutic agents across the blood brain barrier (BBB) and targeting cells.42 Subsequently, researchers purified the RVG-EVs from dendritic cell cultures and loaded EVs with siRNA by electroporation. Systemically administered RVGmodified EVs delivered glyceraldehyde 3-phosphate dehydrogenase (GAPDH) siRNA to neurons, microglia, and oligodendrocytes in the brain, resulting in specific gene knockdown, whereas nonspecific uptake of siRNA in other tissues was not observed. Notably, repeated administration of RVG-targeted EVs did not

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induce inflammation in mice. Using this approach, Beta-secretase 1 (BACE1), a target in Alzheimer’s disease, could also be knocked down by siRNA in mouse brains.41 These results suggested that EVs can cross the BBB, can be targeted by surface modification, and can be loaded with nucleic acid drugs. The same method was used to load EVs with siRNA. The EVs then successfully delivered the siRNA to HeLa cells, endothelial cells, monocytes, and lymphocytes in vitro.43–45 With regard to biodistribution and targeting strategies for EV-based drug delivery, another study used EVs to deliver let-7a miRNA in a targeted manner to epithelial growth factor receptor (EGFR)-overexpressing breast cancer cells in mice.46 In this study, the authors selected GE11 or EGF as ligand peptides on the surface of EVs. The GE11 peptide (YHWYGYTPQNVI) or EGF was cloned into a pDisplay vector and transfected into HEK293 cells. The GE11 peptide binds specifically to EGFR on the surface of EVs, and these engineered EVs have been shown to bind specifically to EGFR-expressing xenograft breast cancer tissue in RAG2KO mice. The data suggested that intravenous injection of the let7a-loaded GE11-targeting EVs could deliver miRNA to the EGFR-expressing tumor and that the EVs successfully inhibited the tumor growth in a mouse xenograft model.46

5 THE CHALLENGES AND LIMITATIONS OF EV-BASED DRUG DELIVERY After these reports, although it is thought that the advances in engineered EV-based DDSs make them a useful tool for therapy, there are number of limitations and challenges that need to be addressed. The following sections describe current progress in EVs for therapeutic delivery and challenges to be overcome for EV-based DDSs.

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5.1 Appropriate EV Source for Engineered EVs Although conventional nanocarriers (e.g., liposomes) can be easily produced, EVs are composed of complex biomolecules and are completely manufactured by living cells. Indeed, reconstitution of EVs from chemically defined materials has never been accomplished. Therefore, an initial requirement before obtaining an efficient EV-based DDS is the choice of the optimal source of EVs and determination of whether a sufficient number of EVs can be generated. First, the successful clinical application of EV-based therapy requires that the safety of the patient is ensured. Many studies have shown that cancer cell-derived EVs promote cancer progression.47–49 For instance, cancer cells secrete EVs to modulate their microenvironment and induce cancer “hallmarks,” including cell proliferation, resistance to apoptosis, angiogenesis, and invasion and metastasis via their components.47–49 Thus, the intrinsic biological functions of EVs must be accounted for when designing therapeutic carriers. It has been reported that mesenchymal stem cell (MSC)– derived EVs can be used in medical applications for tissue repair and wound healing.50–52 For instance, Katsuda et al.53 reported the possibility of using MSC-derived EVs as a therapeutic for Alzheimer’s disease. One of the neuropathological hallmarks of Alzheimer’s disease is the accumulation of Aβ in the brain because of an imbalance between Aβ production and clearance.54 Furthermore, several studies described that EVs derived from MSCs have therapeutic potential for various diseases. In other words, although careful consideration is necessary for clinical application, these reports imply that the usage of MSC-EVs with therapeutic cargo can be considered safe. Moreover, MSCs are known for their low immunogenicity, making allogeneic applications possible.55,56 For example, Pascucci et al.34 observed that paclitaxeltreated MSCs mediated strong anticancer effects because of their capacity to take up the drug and

later release it in EVs. In this study, paclitaxeltreated MSC-EVs induced dose-dependent inhibition of proliferation in CFPAC-1 human pancreatic cancer cells as well as 50% inhibition of tumor growth. Another important issue is the production cost. As the field is moving closer to clinical applications, the realization of high vesicle yield with minimal production cost is of increasing importance. In this respect, the choice of EV-producing cells is critical. In the field of EV research, most of the studies have used cell culture supernatant; however, billions of cells must be cultured in vitro in order to harvest a few micrograms of EVs.57 This process of production is inefficient and limits the potential of EV technology for personalized therapeutic delivery. Therefore, some research groups have started to focus on alternative sources of EVs (Fig. 2). They chose agricultural products, such as fruits and milk, as sources of EV because agricultural products are relatively economically practical and scalable sources from which EVs can be isolated. For example, EV-like nanoparticles isolated from grapefruits were called grapefruit-derived nanovectors (GNVs), as the food ingested everyday includes edible plantderived EV-like nanoparticles and an average person’s gut is exposed to many billions of such nanoparticles.58–60 These reports showed that GNVs can deliver siRNAs, DNA expression vectors, miRNAs, proteins, and chemotherapeutic agents in different types of cells and animal models without inducing toxicity. Notably, they also reported that folic acid-binding GNVs significantly increased the targeting efficiency to cells that also express folate receptors.59,60 Another agricultural source of reliable, scalable, and safe EVs for therapeutic delivery is bovine milk. Munagala et al.61 reported that EVs from bovine milk can be applied as a DDS to deliver anticancer drugs without inducing toxicity. They then tested these drug-loaded EVs on lung tumor cells in culture and in xenograft models and found that the EV-encapsulated formulations have enhanced biological efficacy compared with the free drug, especially when the

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FIG. 2 Workflow for EV-based DDS. The left column presents the postloading methods. The first step is the choice of an adequate source of EVs. EVs are isolated and then loaded with the desired therapeutic molecules via direct manipulation of the EVs. The right column presents the preloading methods. The preloading methods involve either transfection of genes (for plasmid vector or siRNA) or preloading of chemical drugs in the EV-producing cells. Furthermore, to obtain targeting ability, conjugation of targeting ligands to the surface of isolated EVs can be achieved using chemical linkers.

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tumor-targeting ligand folic acid was added to the EVs. According to this report, the yield of bovine milk-derived EVs is at least 100-fold higher than those of cell culture-derived EVs. In another study, Somiya et al.62 showed that EVs obtained from defatted milk by combining acid treatment and ultracentrifugation are purer than those obtained from previously reported methods and that the yield of these EVs was over 10 μg/mL-whey. Based on their calculation of cost, bovine milk is at least 1000-fold more cost effective than serum-free medium for the production of the same amount of EVs. In addition, intravenously injected milk-derived EVs did not show any systemic toxicity or anaphylaxis-inducing immunoglobulin E in in vivo mice experiments. Therefore, milkderived EVs can deliver therapeutic molecules and may be a biocompatible material with nonimmunogenic properties in humans. However, milk is one of the major causes of food allergy. Up to 0.1% of adults suffer from milk allergies,63 while infants have milk allergies more frequently.64 These populations cannot receive milk-derived EV-containing formulations. On the other hand, it is often the case that increasing EV production in cultured cells can solve the cost problem. For example, culturing dendritic cells for extended times65 or at low pH can increase EV production by up to 10-fold.66 To overcome this problem, Watson et al.67 used a hollow-fiber bioreactor for efficient EV production, and compared with conventional culture, this bioreactor presented many advantages, which likely contribute to the increased EV production yield. In this study, the developed method yields 40-fold more EV particles per volume of conditioned medium compared with conventional cell culture.

5.2 EV Purification for Clinical Usage In addition to the source of EVs and EV productivity, the purification method is also an important factor in obtaining a substantial

quantity of EVs (Fig. 2). The development of EVs for therapeutic applications is conditional on the establishment of scalable, reproducible, and high-throughput methods for the purification of EVs. The most common strategy used for purification of EVs is ultracentrifugation, a method that includes performing a sequence of centrifugation steps. However, ultracentrifugation is time consuming, requires an ultracentrifuge, and results in a relatively low recovery of EVs.68 Moreover, the disadvantage of ultracentrifugation is that it is not suitable for EV isolation from large-volume EV sources. Another disadvantage of ultracentrifugation alone is the presence of protein aggregates and other non-EV particles in the obtained fraction. Clearly, if EVs are to be used as a DDS cargo, it is necessary to isolate a high-purity preparation and to recover the maximal quantity of vesicles. To increase the purity of the EV fraction, density-gradient ultracentrifugation is frequently used for efficient particle separation and is regarded as one of the best methods for EV isolation.69 However, this method results in a considerable loss of EVs, although it enables production of EV fractions with improved purity. Thus, all methods have both advantages and disadvantages, and there is no best purification method. To overcome the issues mentioned above, many researchers have developed EV purification methods and equipment. For example, in a laboratory setting, the method based on precipitation of EVs in PEG solutions is used to some extent in EV purification. This method enables fast, simple and efficient enrichment of EV70,71; however, a disadvantage of this method is the variable contamination of EVs with proteins, protein complexes, lipoproteins, etc. Another method, size exclusion chromatography (SEC), is a simple and feasible approach to isolate EVs from raw materials.72–74 The principle of SEC is separation based on a difference in size. Therefore, lipoproteins, which are present in EV sources and are similar to EVs because they have nanosized structures and

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are composed of lipids and protein complexes, can be a contaminant in the EV fraction. The advantage of SEC is that it is readily scalable because an increase in the length of columns enhances the peak resolution for particles that are close in their sizes, while an increase in the column diameter allows analysis of more concentrated samples with a larger volume. Affinity purification is supposed to be the most promising approach to obtain EVs with high purity. For example, immunoaffinity-based capture strategies are based on the highly specific interaction of a target with its antibody associated ligands that are on EVs, such as the tetraspanin family, including CD9, CD63, and CD81.75,76 This method requires knowledge of specific EV markers, which, despite many years of research, are still difficult to identify. Moreover, this method is difficult to use in mass-scale production due to limitations of the sample volume and the high cost of production for antibodies. Furthermore, EVs must be carefully handled during purification processes, while affinity between proteins is sometimes too strong for dissociation under mild conditions. Another type of EV isolation based on binding to the EV surface molecules is the use of Tim4 protein, which is able to bind phosphatidylserine (PS) on the membranes in the presence of calcium ions.77 Unlike immunoaffinity-based capture strategies, using a chelating agent, such as ethylenediaminetetraacetic acid (EDTA), can dissociate the Tim4 complexes from EVs in this method. However, the disadvantage of this method is the difficulty in distinguishing between different populations of EVs based on Tim4. Another interesting approach for EV isolation is based on the ability of heparin to bind EVs.78 EVs have been reported to bind to heparin, and this interaction is necessary for the cellular uptake of EVs.79 By utilizing the affinity between heparin and EVs, heparin affinity chromatography was developed. Although heparin affinity chromatography is useful for EV isolation, the crossreactivity with other components present in

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the corresponding media is a possible concern, and the purification steps take up to three days in this report.78 In summary, from a therapeutic point of view, when contemplating the use of EVs as DDS cargo, the purification method should be a simple, fast, and nondestructive procedure that is capable of mass-scale purification and that uses low-cost materials to isolate the EVs from EV sources.

5.3 Loading Strategy of Therapeutic Molecules Into EVs Although EVs have potential as naturally equipped drug delivery vehicles, EVs should be effectively loaded with drug molecules in order to be used as a DDS. There are two general processes for loading therapeutic cargo within EVs: exogenous (postloading method) and endogenous (preloading method) (Fig. 2). Exogenous strategies mean that EVs are isolated and then loaded with the desired therapeutic molecules via direct manipulation of EVs, while endogenous strategies mean that the desired therapeutic molecules are incorporated into EVs during their biogenesis in the EV-producing cells. In the following section, we summarize some of the published exogenous and endogenous EV loading methods. 5.3.1 Postloading Methods The most frequently reported method, especially for hydrophilic membraneimpermeable components, is electroporation. Electroporation, as established by AlvarezErviti and coworkers,41 is a process by which transient pores are made in the membrane of EVs to facilitate cargo loading and is generally used to load EVs. As mentioned above, they modified electroporation protocols to load siRNAs into EVs derived from dendritic cells with an overall loading efficiency of 35%. In this method, purified EVs and therapeutic molecules are mixed together in an electroporation buffer.

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The mixture is then electroporated to disrupt the EV structure, leading to spontaneous pore formation and allowing the therapeutic molecules to incorporate into the EVs. After this report, several other groups also used electroporation for the incorporation of cargo into EVs. For instance, Shtam et al.43 packaged siRNAs against RAD51 and RAD52 into EVs derived from HeLa cells and confirmed that the EVs loaded with the siRNA against RAD51 were functional and caused massive reproductive cell death of recipient cancer cells. Recently, Kalluri’s group80,81 loaded EVs derived from normal fibroblast-like mesenchymal cells to carry siRNA or shRNA specific to oncogenic KRASG12D using electroporation and reported that treatment with these modified EVs suppressed cancer in multiple mouse models of pancreatic cancer and significantly increased their overall survival. Similar to RNA, the incorporation of exogenous DNA into EVs by electroporation has also been reported.82,83 Interestingly, the loading efficiency and capacity of DNA in EVs were dependent on the size of the DNA molecules, as linear DNA molecules less than 1000 bp in length were more efficiently transferred into EVs than larger linear DNAs and plasmid DNAs.83 This method does not apply only to exogenous nucleic acids. Electroporation can also be used to encapsulate chemotherapeutic agents into EVs. Tian et al.33 used electroporation to encapsulate doxorubicin into EVs. Intravenously injected EVs containing doxorubicin were delivered to tumor tissues, leading to inhibition of tumor growth without overt toxicity. Many studies have used electroporation for loading therapeutic molecules into EVs, whereas some researchers have posed questions about this method. Kooijmans et al.84 presented evidence that electroporation caused RNA aggregation and EV instability, thereby resulting in a low loading capacity. In fact, the real efficiency of siRNA retention in EVs after electroporation was below 0.05%. Thus, careful consideration

needs to be taken when interpreting the loading using the electroporation method. Other postloading methods are also based on transiently destabilizing the EV membrane, including repeated freeze-thaw cycles, sonication, extrusion, or saponin treatment.85,86 In other words, if the lipid bilayer of EVs is destabilized, therapeutic molecules can be loaded into EVs. As an example, Fuhrmann et al.85 destabilized the EV membrane by using saponin as a detergent and showed that incubation of small hydrophilic porphyrins with saponin increases drug loading into EVs by 11-fold compared with passive loading without saponin. In addition, Haney et al.86 compared different loading methods, such as direct incubation, sonication, freeze-thaw cycles, permeabilization with saponin, and size extrusion, to encapsulate catalase. In terms of catalase loading, EVs loaded using saponin had a higher loading efficiency that those loaded by incubation or freeze-thaw cycles but a lower efficiency than those loaded by sonication and extrusion. Furthermore, catalase-loaded EVs obtained by permeabilization with saponin had better therapeutic effects in a mouse model of Parkinson’s disease than those obtained by sonication. However, this protein loading method based on physical treatment of EVs as well as protein denaturation or destabilization may significantly limit the applicability of EVs to clinical usages for unstable proteins. Chemical-based transfection using commercial transfection reagents, such as cationic lipids, has been used in only a few studies to encapsulate molecules that naturally carry a negative charge, such as siRNA, into EVs.43,45 However, it is nearly impossible to avoid the possibility that the chemical transfection reagent is solely responsible for the delivery of nucleic acids into cells, as evidenced by the fact that a complex of nucleic acids and chemical transfection agents (without EVs) can functionally deliver the nucleic acids into cells.43 From this point of view, the chemical transfection method is not suitable for loading therapeutic molecules into EVs.

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Another siRNA loading method involves the use of modified siRNA. Didiot et al.87 developed a robust and scalable method for loading therapeutic RNA into EVs. They used cholesterolmodified siRNA against the Huntingtin gene because the hydrophobicity imparted by the cholesterol enabled enhanced membrane association. In this study, EVs derived from U87 glioblastoma cells were simply loaded with the cholesterol-modified siRNA by incubation at 37°C for 90 min with shaking. The silencing of Huntingtin mRNA by the EVs was demonstrated in vitro using mouse primary cortical neurons as well as in vivo upon infusion into the mouse striatum. Although another study also used cholesterol-modified siRNA and showed that cholesterol-modified siRNA was loaded effectively into the EVs and that the EVs were taken up by recipient cells, the authors could not observe the EVs functionally delivering the modified siRNA to recipient cells.88 The cause of the inconsistency in efficacy between these two studies is unclear. 5.3.2 Preloading Methods The most widely used endogenous strategy for loading therapeutic molecules into EVs is transfecting the EV donor cells so that they overexpress a desired gene product that the cell will package into the lumen or membrane of the EVs for secretion. Several studies reported that miRNAs could be efficiently loaded into the EVs either by using miRNA expression vectors or by transfecting cells with precursor or miRNA-mimic oligonucleotides.23,46,89–95 In one of our previous experiments,23 pri-miRNAoverexpressing cells secreted EVs containing substantial amounts of functional miRNA. In another study, Wang et al.92 demonstrated that MSC-EVs enriched with miR-let7c using the transfection approach selectively targeted fibrotic kidneys in an in vivo model of unilateral ureteral obstruction and downregulated several profibrotic genes. However, efficient loading of EVs with an RNA of interest can be more effectively achieved by first loading the parent cell.

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In 2011, Akao et al.90 demonstrated that macrophages transfected with miR-143 mimic secrete EVs containing the miRNA at a loading efficiency of approximately 0.20%. Therefore, to efficiently load specific miRNA into EVs, it is important to understand the sorting mechanism. In this respect, Villarroya-Beltri et al.96 reported an important finding: the sumoylated form of a ubiquitously expressed RNA-binding protein, hnRNPA2B1, directs the sorting of miRNA loading into EVs through recognition of short sequence motifs overrepresented in miRNAs. Similarly, Y-box protein 1, an RNA-binding protein, was found to be necessary to sort miRNA-223 into EVs in a cell-free system and in cultured HEK293 cells.97 Furthermore, our group reported that annexin A2 can also bind to miRNAs in a sequence-independent manner and sort a broad range of miRNAs into EVs.98 These findings can provide a tool for the efficient loading of selected regulatory miRNAs into EVs. In this strategy, proteins can also be sorted into EVs by creating a fusion construct containing the protein of interest linked to a protein that is inherently associated with EVs.99–101 This approach can be used for EV-targeting purposes, as described below. Another form of endogenous loading involves incorporating therapeutic molecules, such as chemical drugs, into EVs from donor cells. For example, Pascucci et al.34 demonstrated that EVs isolated from paclitaxel-treated MSCs exhibited antiproliferative activity against cancer cells in vitro. It was shown that this anticancer drug was incorporated into EVs during their biogenesis. However, it was not clarified whether EVs became loaded with the drug in the cells or later in the growth medium after being released.

5.4 Engineering Targeting Molecules on EVs The most intriguing aspects of using EVs for therapeutic delivery are the possibilities of innate targeting. Several studies have shown that EVs have a natural targeting ability based

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on donor cells. As mentioned above, various kinds of “zip codes” (or “shipping tags”) are displayed on the surface of EVs and recognize specific molecules of recipient cells.29 When developing an EV-based DDS, we have to understand what molecules are a zip code and make good use of the zip code for targeted delivery. However, at this point, we still cannot understand specific zip codes for targeted delivery. Therefore, at the moment, many reports have been published with regard to using EVs in DDS cargo, but only a small portion of them utilize a targeting strategy for direct delivery of the DDS cargo. Among them, the RVG peptide is the most commonly used targeting ligand for brain-specific delivery. To display targeting ligands on the surface of EVs, the most commonly used technique is to insert the gene encoding the targeting proteins into the donor cells as described above. As introduced above, Alvarez-Erviti et al.41 reported that targeted in vivo delivery of siRNA with RVG-EVs through tail vein injection of mice resulted in specific siRNA delivery to the brain. In another report, Cooper et al.102 used RVG-EVs in a therapeutic model of Parkinson’s disease. Accumulation of α-synuclein aggregates in the brain is a pathological hallmark of Parkinson’s disease. The authors demonstrated that intravenous administration of the α-synuclein siRNA-loaded EVs expressing RVG protein on their surface resulted in brain-specific uptake and reversed brain α-Syn pathological conditions in mice. On the other hand, postconjugation methods are also of interest for targeting specific cells (Fig. 2). Stable conjugation of targeting ligands to the surface of isolated EVs can be achieved using chemical linkers via similar chemistry to that employed to functionalize synthetic particles and liposomes. Koojimans et al.103 showed that efficiently decorating vesicles with antiEGFR nanobodies at a high surface density via glycosylphosphatidylinositol (GPI) dramatically altered their binding and internalization by EGFR-overexpressing tumor cells.

6 PHARMACOKINETICS OF EVs For therapeutic purposes, the pharmacokinetics of EVs are crucial to deliver drug molecules to a specific site in the body. Basically, upon systemic administration, nanoparticles accumulate rapidly in the liver, spleen, and lung, that is, the MPS or RES.79 Avoiding capture by the MPS is a solution to improve the pharmacokinetics of EVs. In this section, the in vivo biodistribution of EVs from various sources is summarized. Furthermore, the engineering approaches to improve the pharmacokinetics of EVs are discussed.

6.1 Biodistribution of EVs Upon Administration When EVs are administered systemically, almost all of the EVs are suddenly captured by the MPS.104–107 Macrophages in the MPS are responsible for the rapid clearance of EVs from the bloodstream.108 This phenomenon is not unique to EVs, as nanoparticles are generally taken up by the MPS. Furthermore, the negative charge on the surface of EVs, originating from the negatively charged phospholipid PS, is the main cause of uptake by macrophages because PS can be recognized by PS-binding receptor molecules on the macrophages.109 The source of EVs is also crucial to the pharmacokinetics of EVs because each EV has a different composition. EVs from different cell types showed slightly different biodistributions.110 The administration route strongly affects the pharmacokinetics of EVs. As mentioned above, intravenous injection of EVs results in rapid clearance from the blood and accumulation in MPS-related organs. Intranasal administration is a unique route to deliver drugs into the brain.32 By using food-derived EVs, oral administration is a promising approach to deliver drugs due to its minimal invasiveness,61,111 although it is still unclear whether EVs can be transferred into blood circulation via

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gastrointestinal absorption while maintaining their integrity.

6.2 Improvement in Pharmacokinetics In general, nanoparticles are taken up by the MPS upon systemic administration. PEGylation might be the most promising approach to evade capture by the MPS. PEG is a hydrophilic polymer, and when attached to nanoparticles, PEG chains cover the surface of the nanoparticle. Because of the steric hindrance effect of PEG chains, the interaction between nanoparticles and proteins/cells can be reduced, and the half-life of nanoparticles in blood circulation is prolonged.11 These strategies can be applied to EVs for improving their pharmacokinetics. In one paper that described the PEGylation of EVs, PEG chains were conjugated on the surface of EVs via a lipid anchor.112 PEGylated EVs showed a longer half-life in blood circulation than non-PEGylated EVs. However, unexpectedly, significant tumor accumulation of the EVs was not observed. This is probably due to the detachment of PEG chains during circulation. Rational and robust conjugation methods other than lipid anchoring for PEG chains is necessary for the stable PEGylation of EVs and improved pharmacokinetics.

7 CONCLUSION EVs are secreted by almost all cell types, exist in all our body fluids, and function as natural carriers of biological molecules, which makes them an attractive next-generation vehicle for DDSs. However, at the beginning of their history, about four decades ago, EVs were considered cell debris or garbage bags and were assigned little relevance. In the last decade, significant progress has been made toward harnessing EVs for therapeutic molecule delivery. Today, EVs can be an innovative DDS, as they can overcome physical and biological barriers

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and safely deliver therapeutic drugs to target tissues. However, the study of EVs is still in its early stages, and a number of challenges remain to be addressed before their successful clinical translation. The first obstacle is the question of achieving the large-scale production and purification of EVs for clinical use. In other words, large quantities of EVs should be obtained at reasonable cost. Second, there is no proven method for loading therapeutic molecules into EVs. Therefore, effective loading of therapeutic molecules into EVs is one of the major challenges associated with EV therapeutic delivery. Moreover, refinement of the methods for endowing EVs with targeting ability is required for clinical use because efficient targeting helps reduce the side effects of the drug and reduce the dose of the drug needed to achieve a therapeutic effect. Certainly, there are still many challenges, yet EV-based DDSs promise an unparalleled efficacy in the treatment of many lifethreatening conditions, including those lacking effective DDSs.

Funding This study was supported by Grant in Aid for the Japan Agency for Medical Research and Development (A-MED) through the Basic Science and Platform Technology Program for Innovative Biological Medicine (JP18am0301013) and Center of Innovation Program (COI stream) from Japan Science and Technology Agency.

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