Extracellular vesicles for drug delivery

Advanced Drug Delivery Reviews 106 (2016) 148–156

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Advanced Drug Delivery Reviews journal homepage: www.elsevier.com/locate/addr

Extracellular vesicles for drug delivery☆ Pieter Vader a,⁎, Emma A. Mol b, Gerard Pasterkamp a,b, Raymond M. Schiffelers a,⁎ a b

Department of Clinical Chemistry and Hematology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands Department of Experimental Cardiology, University Medical Center Utrecht, the Netherlands, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands

a r t i c l e

i n f o

Article history: Received 7 January 2016 Received in revised form 11 February 2016 Accepted 17 February 2016 Available online 27 February 2016 Keywords: Extracellular vesicles exosomes microvesicles drug delivery isolation biodistribution targeting nanomedicine

a b s t r a c t Extracellular vesicles (EVs) are cell-derived membrane vesicles, and represent an endogenous mechanism for intercellular communication. Since the discovery that EVs are capable of functionally transferring biological information, the potential use of EVs as drug delivery vehicles has gained considerable scientific interest. EVs may have multiple advantages over currently available drug delivery vehicles, such as their ability to overcome natural barriers, their intrinsic cell targeting properties, and stability in the circulation. However, therapeutic applications of EVs as drug delivery systems have been limited due to a lack of methods for scalable EV isolation and efficient drug loading. Furthermore, in order to achieve targeted drug delivery, their intrinsic cell targeting properties should be tuned through EV engineering. Here, we review and discuss recent progress and remaining challenges in the development of EVs as drug delivery vehicles. © 2016 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Isolation of extracellular vesicles . . . . . . . . . . . . . . . . . Loading of extracellular vesicles . . . . . . . . . . . . . . . . . 3.1. Loading of cells before extracellular vesicle isolation . . . . . 3.2. Loading of extracellular vesicles after isolation . . . . . . . 4. Biodistribution and targeting of extracellular vesicles . . . . . . . 4.1. Circulation time and biodistribution of extracellular vesicles . 4.2. Interactions of extracellular vesicles with the immune system 4.3. Specific targeting of extracellular vesicles . . . . . . . . . 5. Therapeutic effects . . . . . . . . . . . . . . . . . . . . . . . 6. Extracellular vesicle-mimetic nanovesicles . . . . . . . . . . . . 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction ☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Biologicallyinspired drug delivery systems”. ⁎ Corresponding authors at: Department of Clinical Chemistry and Hematology , University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail addresses: [email protected] (P. Vader), [email protected] (R.M. Schiffelers).

http://dx.doi.org/10.1016/j.addr.2016.02.006 0169-409X/© 2016 Elsevier B.V. All rights reserved.

Extracellular vesicles (EVs) are nano-sized membrane vesicles, released by many, if not all, cell types. EV release has been found to occur in many unicellular- as well as multicellular organisms, suggesting that it represents an evolutionary-conserved process. Mammalian

P. Vader et al. / Advanced Drug Delivery Reviews 106 (2016) 148–156

cells can release distinct types of EVs, including exosomes, microvesicles, and apoptotic bodies. This classification is based on their intracellular origin. Exosomes are thought to be the smallest vesicle type, with sizes ranging from 40 to 100 nm. They originate from intraluminal budding of multivesicular endosomes (MVE) and are released upon fusion of MVEs with the plasma membrane. In contrast, microvesicles (also referred to as ectosomes) are more heterogeneous in size and can be larger (50 nm-1 μm), and are formed through direct budding from the plasma membrane. When cells are compelled to undergo apoptosis, a heterogeneous population of vesicles (50 nm-5 μm) is released, which are named apoptotic bodies [1]. Despite these differences in origin, no uniform EV nomenclature exists as of yet, due to the overlap in vesicle sizes and the absence of subtype-specific markers. As a result, it remains difficult (if not impossible) to purify and thereby distinguish between vesicle types [2]. In this review, we therefore refer to all vesicle subtypes as extracellular vesicles (EVs). The cargo of EVs comprises small and long, coding and non-coding RNAs (e.g. mRNA, miRNA, lncRNA), lipids and proteins [3,4]. Initially, EVs were thought to act as ‘garbage bags’, with a main function in discard of cellular waste. Over the last decade, however, scientific interest in EVs has rapidly increased, after it was shown that biological information packaged in EVs could be transferred between cells, and alter the recipient cells’ phenotype. Cells can package a distinct set of biomolecules into EVs via endogenous sorting mechanisms, and release EVs constitutively or after stimulation [5,6]. EVs may subsequently be internalized by target recipient cells, resulting in transfer of mRNAs and miRNAs, which can result in production or silencing of target proteins, respectively [7–9], and of proteins, including membrane proteins [10,11]. EVs can be isolated from bodily fluids, including blood, urine, cerebrospinal fluid and saliva [12–14]. As their content reflects the status of the donor cell, EVs may be applied in diagnostics, either as pathological biomarkers or to follow treatment efficacy (reviewed in [15,16]). In addition, it has become clear that, through their important role in intercellular communication, EVs affect various processes involved in health and disease. The discovery that EVs make up a natural mechanism for information transfer between cells has stimulated interest into their potential use as a new drug delivery platform. Despite considerable research in the last 50 years, the clinical translation of conventional drug delivery platforms has been limited. The efficiency of these platforms to overcome barriers in macromolecule drug transport, such as reaching the target tissue and engaging intracellular targets, is still unsatisfactory [17]. In addition, concerns related to immunogenicity and toxicity of non-natural delivery systems remain. EVs on the other hand seem to have many features of an ideal carrier system. The EV cargo is naturally protected from degradation in the circulation [18]. EVs seem to possess intrinsic cell targeting properties, and are able to overcome natural barriers such as the blood-brain barrier [19,20]. Furthermore, it is likely that EVs utilize endogenous mechanisms for uptake, intracellular trafficking and subsequent delivery of their content in recipient cells [21]. Importantly, EVs may be nearly non-immunogenic when used autologously. Moreover, several clinical trials using EVs for immunotherapy have already demonstrated the safety of EV administration in humans [22–24]. Although EVs hold immense promise for therapeutic drug delivery, clinical applications may critically depend on the development of scalable EV isolation techniques and approaches for efficient drug loading. Furthermore, improved methods to modify their in vivo biodistribution, which is an important determinant of their therapeutic effect as it enables more specific drug delivery to target tissues, are required. In this review, we discuss new findings and recent improvements on these issues, and summarize recent successes in the use of EVs as drug delivery vehicles in animal disease models.

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2. Isolation of extracellular vesicles One of the prerequisites for clinical application of EVs is standardization of the isolation process with regards to yield, reproducibility, and purity [25]. Furthermore, such an isolation method should be scalable and robust. For large scale EV production, the manufacturing process sequentially involves expansion of the donor cell line, collection of the conditioned medium, and EV purification. Thus far, several methods for the isolation of EVs have been described. The most commonly used method is differential ultracentrifugation (UC). EVs are isolated based on sedimentation at high g-forces. Generally, this method comprises low speed spins to remove cell debris, followed by high speed spins to pellet EVs. Sucrose density gradients may subsequently be utilized to separate vesicle types based on density, and to further purify vesicles from protein aggregates [26,27]. Disadvantages of UC and sucrose gradients include the time-consuming protocol, operator-dependent yield, and possible aggregation and rupture of EVs due to high shear forces [28]. Isolation of EVs using UC may therefore not be useful for clinical practice, and novel isolation techniques are topic of intensive investigation. Two distinct approaches for isolation can be discriminated. The first approach, immunoaffinity isolation, is based on selective capture of EVs that bear specific marker proteins on their surface. This could be important when separation of EV subtypes is required, although it is currently unknown whether specific subtypes are more or less feasible for drug delivery purposes. Clayton et al. developed an isolation method to capture EVs derived from antigen-presenting cells (APCs) using antibody-coated magnetic beads. Using antibodies specific for major histocompatibility complex (MHC) class II, a specific EV subtype (i.e. exosomes) could be isolated [29]. A different antibody-based method to isolate EVs was described by Ashcroft et al., who used a microfluidic circuit to isolate CD41-positive platelet-derived EVs in plasma. EVs were captured with an anti-CD41 antibody-coated mica surface [30]. This standardized and quick method requires a very low amount of plasma (10 μl) and could be adjusted for other sources of EVs in the future. However, the absence of well-defined EV markers may thus lead to isolation of only specific EV subsets or EVs derived from specific cell types, and successful elution of intact EVs from the beads might prove challenging. Furthermore, immunoaffinity isolation protocols are not very attractive for clinical applications, since EVs are isolated at a very small scale. The second approach comprises methods that isolate EVs based on their size. With these methods, considerable efforts have already been made to improve scalability of EV isolation. Lamparski et al. showed increased recovery of MHC class II-expressing EVs using a combination of ultrafiltration and ultracentrifugation of EVs in a 30% sucrose/deuterium oxide cushion, for the first time showing that it is possible to isolate EVs for clinical application [31]. However, a drawback of this method is the low EV recovery, hindering application in large clinical studies. In an attempt to isolate EVs using filtration techniques only, Heinemann et al. developed an easy three-step protocol [32]. First, a 0.1 μm pore size polyethersulfone (PES) membrane was used to remove dead cells and cell debris. The sample was then passed through a 500 kDa molecular weight cut-off modified PES filter to remove free proteins and reduce large volumes, followed by isolation of EVs using a 0.1 um Track Etch filter. A comparison of sequential filtration versus UC showed that although filtration resulted in a slight reduction of EV yield compared to UC, it resulted in isolation of a more specific subset of EVs. The major advantage of this method is the fast and fully automatable protocol, although non-specific EV protein binding to the membranes leading to lower recovery may present a limitation. The use of size-exclusion chromatography (SEC) for EV isolation from plasma was first described by Boing et al. [33]. Fractionation of plasma using a sepharose CL-2B column resulted in fast and specific separation of proteins, HDL, and EVs. SEC isolation also resulted in a higher recovery of EVs compared to UC. Increased standardization of EV isolation was reported by Welton et al., who employed a

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commercially available column [34]. Furthermore, collection of only the EV-containing eluate fractions reduced the isolation time to 10-15 minutes. Recently, Nordin et al. reported the isolation of EVs from large volumes of cell culture media by ultrafiltration combined with sizeexclusion chromatography (SEC) [35]. The sample volume was first reduced by ultrafiltration using 100 kDa-cutoff spin filters, followed by EV isolation using SEC. They demonstrated that this method resulted in a higher EV yield when compared to UC. Furthermore, electron microscopy data showed more biophysically intact EVs after SEC isolation, which had impact on their subsequent biodistribution in vivo. These novel methods based on filtration and/or SEC could very well be adjusted and optimized for large-scale clinical application, including for drug delivery purposes. Taken together, these studies also underline that medical application of EVs still poses significant challenges. From a pharmaceutical perspective, the reproducibility of composition and purity is particularly demanding. Especially since we know that out of the different classes of biomolecules associated with EVs, such as nucleic acids, proteins and lipids, there are hundreds of species of each class that may contribute to the overall effects. In many instances, the therapeutic molecules have only partly been identified. This is important as identification of the active agents in the composition dictates quality control. It is conceivable that, similar as for stem cell therapy, a framework of Good Practices will need to be established in which also the particular pharmacokinetic and pharmacodynamic qualities of the EV product are defined on a case-by-case basis [25]. At the same time, the first clinical studies have already been conducted demonstrating that clinical application of EVs is safe and feasible. 3. Loading of extracellular vesicles The lipid bilayer membrane serves as a natural barrier to protect EV cargo from degradation in the bloodstream. However, the presence of this membrane as well as the EV endogenous content make loading of exogenous cargo (i.e. therapeutics) into EVs challenging. Nevertheless, several methods to load EVs have already been described. Here, we provide an overview of two different loading approaches. One approach is based on loading of therapeutics into cells from which the EVs are derived, which, using the endogenous loading machinery of the cells, may result in subsequent EV loading with the drug of interest. The second approach involves loading of EVs after their isolation. An overview of the described loading techniques is schematically depicted in Fig. 1. 3.1. Loading of cells before extracellular vesicle isolation EVs are natural carriers of small RNAs, including miRNAs. Binding of miRNAs (or siRNAs) to a target mRNA can cause either mRNA degradation or repression of protein translation, resulting in target gene silencing. This process is known as RNA interference [36]. Small RNAs may also be used therapeutically, for the treatment of diseases in which specific genes are overactive, such as cancer. However, RNAs typically require delivery to their site-of-action, which is the cytosol of target cells. To achieve loading of small RNAs into EVs, transfection-based approaches have been proposed. Kosaka et al. showed that transfection of HEK293 and COS-7 cells with a vector for expression of miR-16, -21, -143, -146a, or -155 resulted in overexpression of these miRNAs in cells and, as a result of endogenous RNA secretory mechanisms, in their subsequent active release in EVs [37]. Subsequently, these miRNA-containing EVs were able to induce gene silencing in recipient COS-7 cells. Similarly, other reports have shown that using vectorinduced expression of small RNAs in cells, small RNA loading into EVs can be achieved [38,39]. Alternatively, EV donor cells may be transfected with small RNAs directly [40,41]. The main disadvantage of these approaches however is that the level of RNA that is incorporated into EVs may depend on the RNA species and/or sequence, although the

mechanisms that are involved in RNA sorting into EVs largely remain to be elucidated [5,6,42]. Although most studies to date have investigated small RNAs as therapeutic cargo to be loaded in EVs, other types of therapeutics may also be incorporated, including mRNAs, proteins, and small molecules. Pascucci et al. recently showed successful loading of Paclitaxel (PTX) into EVs, by incubating mesenchymal stromal cells (MSCs) with a high dosage of PTX. Subsequently, MSCs produced EVs loaded with PTX, which were shown to be able to inhibit in vitro tumor growth [43]. In addition, Tang et al. showed that tumor cells incubated with chemotherapeutic drugs package these drug into EVs. To stimulate formation of drug-loaded EVs, cells were irradiated with ultraviolet light to induce apoptosis [44]. Lee et al. recently demonstrated for a variety of compounds that delivery into cells using fusogenic liposomes also leads to their loading into EVs released from these cells [45]. Although shown to be feasible for loading of hydrophobic as well as hydrophilic compounds, it is unclear whether this approach also leads to incorporation of synthetic lipids from the liposomes into EVs. 3.2. Loading of extracellular vesicles after isolation The second approach involves loading of EVs after their isolation. For some hydrophobic drugs, EV loading may be achieved through direct mixing. This approach was employed by Sun et al., who loaded EVs with curcumin, an anti-inflammatory drug, by simple incubation at 22°C for 5 minutes. Retention in EVs increased curcumin in vitro solubility and stability, and in vivo bioavailability. Intraperitoneal injection of curcumin-loaded EVs resulted in protection against lipopolysaccharide (LPS)-induced septic shock in mice, suggesting that this loading approach may also be feasible for clinical use [46]. Mixing has also been successfully employed to load PTX into EVs [47]. Others have shown that mild sonication may further improve PTX incorporation [48]. For hydrophilic compounds, including RNA, a major obstacle is formed by the lipid bilayer membrane, which restricts passive loading into EVs. One suggested method to achieve small RNA loading after EV isolation is electroporation. This approach is based on spontaneous pore formation in membranes to compensate for changes in voltage after stimulation with an electrical signal. Alvarez-Erviti et al. were the first to report apparent successful siRNA loading into EVs by electroporation. Importantly, subsequent systemic administration of siRNAloaded EVs in mice resulted in inhibition of Beta-Site APP-Cleaving Enzyme 1 mRNA and protein expression in the brain [19]. The use of electroporation for siRNA loading was also investigated by Walhgren et al. Encapsulation was confirmed by fluorescent microscopy, northern blotting, and flow cytometry. EV-mediated delivery of siRNA to monocytes and lymphocytes was observed, which resulted in inhibition of mitogen-activated protein kinase 1 in vitro [49]. These studies suggested that electroporation could lead to encapsulation of small RNAs in EVs, without affecting EV integrity and function. In contrast, when Kooijmans et al. investigated the efficiency of electroporation for siRNA loading into EVs, no significant differences in siRNA retention between samples electroporated in the presence or absence of EVs could be measured [50]. Instead, they found that electroporation of siRNA results in formation of extensive siRNA aggregates even in the absence of EVs, which could lead to possible overestimation of the siRNA encapsulation efficiency. Therefore, caution should be taken with the interpretation of studies using electroporation for loading in the absence of proper control experiments. In addition, electroporation might cause aggregation or fusion of EVs themselves, as has been shown for liposomes [51]. In an attempt to minimize EV aggregation after electroporation, Hood et al. investigated the use of membrane stabilizers. The use of trehalose pulse media (TPM) was shown to maximize EV colloidal stability, most likely due to the biologic stabilization properties of trehalose [52]. In an attempt to load EVs with the antioxidant enzyme catalase, several loading strategies were investigated by Haney et al., including simple incubation at room temperature, saponin-mediated

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Fig. 1. Animated overview of extracellular vesicle loading strategies. Left panel: Loading of EVs via transfection of the donor cell with a vector encoding small RNA or via incubation of the donor cell with small molecules. Using the endogenous sorting machinery of donor cell, EVs are subsequently loaded with the therapeutics. Right panels: Loading of EVs after their isolation. Electroporation is based on spontaneous pore formation after stimulation with an electrical signal. Other approaches include incubation at RT, repeated freeze-thaw cycles, application of ultrasonic frequencies, treatment with a detergent-like molecule (e.g. saponin), or extrusion. Abbreviations: EV = extracellular vesicle, RT = room temperature.

permeabilization, sonication, freeze-thaw cycles, and extrusion [53]. The highest loading efficiencies were obtained with sonication, extrusion or saponin treatment, although sonication and extrusion of EVs resulted in significant size increases as observed by dynamic light scattering, nanoparticle tracking analysis and atomic force microscopy. EVs loaded by sonication or by saponin treatment protected neurons against reactive oxygen species production in vitro and, after intranasal administration, decreased microglial activity compared to injection of free catalase or PBS in an acute brain inflammation mouse model. Although this study demonstrates the feasibility of these strategies for loading EV with large proteins, it remains to be determined how

possible disruption of the EV integrity during sonication or extrusion procedures or saponin treatment affects their immunogenicity. A similar comparison of strategies for loading of hydrophilic porphyrins was performed by Fuhrmann et al [54]. They observed an eleven-fold increase in loading efficacy after EV loading by saponin treatment compared to loading by passive incubation or extrusion. They also compared EV loading with hydrophobic and hydrophilic porphyrins by passive incubation at room temperature or electroporation. EV loading efficiency was higher for hydrophobic porphyrins compared to the hydrophilic compounds. Interestingly, in both cases electroporation increased the encapsulation efficiency compared to passive incubation.

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Hydrophobic porphyrin-loaded EVs showed improved cellular uptake compared to free drug or porphyrins-loaded liposomes. Reduced cell viability was observed in cancer cells after treatment with porphyrinloaded EVs followed by irradiation. Together, these studies demonstrate that various approaches for EV loading are starting to become available. Still, selection of the most suitable method for clinical application remains to be determined. 4. Biodistribution and targeting of extracellular vesicles 4.1. Circulation time and biodistribution of extracellular vesicles In vivo biodistribution and targeting of specific tissues by therapeutics are important factors for their effectiveness. Issues associated with administration of free drugs, including unfavorable pharmacokinetics and lack of selectivity, may be overcome by the use of delivery vehicles. For this reason, Wiklander et al. investigated the tissue distribution of 1,1'-dioctadecyl-3,3,3',3'tetramethylindotricarbocyanine iodide (DiR)-labeled EVs from various cell sources [55]. Twenty-four hours after intravenous (i.v.) injection in mice, they observed the highest fluorescent signal in the liver, followed by spleen, gastrointestinal tract, and lungs. Furthermore, they showed that cell source, EV dose, and route of administration affect EV distribution, which may have implications for the design and feasibility of therapeutic studies using EVs. Injection of higher EV doses resulted in relatively lower liver accumulation compared to lower doses, possibly caused by saturation of the mononuclear phagocyte system. Systematic comparison of the biodistribution of intraperitoneally (i.p), subcutaneously (s.c), and i.v. injected EVs revealed that i.p. and s.c. injections resulted in reduced EV accumulation in liver and spleen and enhanced pancreas and GI tract accumulation compared to i.v. injections. With a similar aim of visualizing EV biodistribution and clearance in vivo, Lai et al. developed an elegant EV imaging reporter system [56]. EVs were engineered to express a fusion of membrane-bound Gaussia luciferase (Gluc) reporter and a biotin domain, allowing multimodal imaging. In a time frame of 30 minutes to 6 hours after i.v. injection of Gluc-EVs in athymic nude mice, highest bioluminescent signals were observed in spleen and liver. Surprisingly, after subsequent transcardial perfusion with PBS, highest Gluc-EV signals were observed in kidneys, and perfused spleens showed only minimal signals across all time points. This difference suggests that the majority of splenic accumulation is caused by EV storage in the spleen rather than uptake by the tissue itself. Comparable Gluc-EV signals before and after perfusion were observed for liver and lungs, thus indicating active uptake of EVs in these organs. Most EVs were cleared after 6 hours, as a result of active uptake of EVs by cells or hepatic clearance. Imai et al. employed a similar approach to assess the importance of macrophages in EV clearance. After systemic administration of Gluc-lactadherin (Gluc-LA)-labeled EVs derived from melanoma cells, macrophage-depleted mice displayed higher Gluc-LA EV accumulation in liver, spleen, and lungs, and delayed EV clearance from blood compared to untreated mice [57]. This suggests an important role for macrophages in EV clearance. Instead of following EV biodistribution using labeled EVs, Bala et al. loaded miRNA-155, an inflammatory mediator, into EVs and determined organ distribution of miRNA-155-loaded EVs after i.v. administration in miRNA-155-/- mice. After perfusion, highest miRNA-155 signals were again observed in the liver, followed by adipose tissue and lungs, whereas the lowest signals were observed in muscle and kidneys [58]. Together, these reports demonstrate that systemically administered EVs (1) have a short half-life and (2) are rapidly taken up by the mononuclear phagocyte system (MPS), particularly in the liver and spleen. Seemingly, this mechanism of clearance resembles that described for synthetic nanoparticles, such as liposomes [59]. Moreover, it was recently shown that the EV clearance rate and biodistribution profile is

similar to that of phosphatidylcholine : cholesterol liposomes and liposomes made from the lipid extract of EVs [60]. Further studies should reveal whether EV clearance by the MPS is in fact due to their intrinsic properties, or whether it is a result of the exogenous characteristics of EVs obtained from in vitro cultures, presence of non-self-labels that are present on the EV surface, or the quantity of vesicles that are injected [59]. Interestingly, Kooijmans et al. recently showed that providing stealth properties to EVs via introduction of polyethylene glycol (PEGylation) onto the EV surface significantly increased their circulation time in mice. PEGylation of EVs may therefore be employed to increase accumulation in tumors or at sites of inflammation, which are characterized by localized increased vascular permeability [61]. Besides reports describing biodistribution of EVs after systemic injection, the possibility of EV administration via other routes has also been investigated. For some therapeutic applications, local drug administration may actually be preferred, e.g. when targeting the central nervous system. Zhuang et al. showed that intranasal administration of mouse lymphoma EVs loaded with the anti-inflammatory drug curcumin resulted in their localization to the brain. Curcumin levels peaked at 1 hour after administration, and a significant amount could still be detected after 12 hours. No toxicity or abnormal behavior was observed in mice upon EV treatment [20]. In addition, intranasal administration of catalase-loaded EVs resulted in higher EV accumulation in brain tissue after 4 hours in a mouse model of Parkinson’s disease, compared to intravenous injection [53]. Establishing the most efficient EV administration route for each application may therefore help to improve therapeutic efficacy. 4.2. Interactions of extracellular vesicles with the immune system EVs are predominantly submicron particles, which gives them a large surface area to volume ratio. In addition, because of their particulate nature, they can also display repeating molecular patterns on their surface which can be important in their interactions with the immune system. Together, these EV qualities constitute a reactive surface for molecules and cells, especially after injection into the blood stream. Within plasma, several protein cascades might interact with the vesicular surface. Classically, phosphatidylserine present on the extracellular vesicle surface has been described to recruit several phosphatidylserine-binding proteins of the coagulation cascade. The vesicles provide a catalytic surface by bringing the proteins in close proximity to each other, thereby increasing reaction speed. As a result, EVs are generally described to promote coagulation and thrombosis. Another important protein cascade in plasma is the complement system. Complement has been described to be activated by liposomes and contribute to their clearance [62]. For EVs, the situation seems to be more complex. For human antigen presenting cells, for example, EVs contain CD55 and CD59 membrane regulators of complement. Both regulators were shown to be important in limiting complement deposition on the vesicles as well as EV lysis [63]. These results indicate that EVs carry complement inhibitors that may promote their survival in the blood stream. At the same time, another study on tumor cell derived EVs showed that these activated both C5a and soluble S protein bound C5b-9, in a concentration dependent manner [64]. Activation was dependent on the presence of calcium indicating that activation occurred through the classical or lectin pathway. Interestingly, EVs from malignant cell lines were more potent in complement activation than EVs from a non-malignant cell line. Taken together these results indicate that the source and composition of EVs is important in their net effect on the complement cascade. The intrinsic tissue distribution profile of isolated EVs underlines that a large fraction of EVs interacts with the RES. In particular macrophages in liver and spleen are responsible for EV uptake, which could impact the cellular phenotype. This intrinsic uptake by antigenpresenting cells has been regarded as a possible route for vaccination. Because of the resemblance of EV composition to the composition of

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the parental cell, EVs can contain antigens, for example oncogenes. As such, the EVs could represent a platform for tumor vaccination. Although some studies support dendritic cell- and tumor-derived EVs as anticancer vaccines (reviewed in [65]), recent literature suggests that EVs are predominantly immunosuppressive [66–68]. It is likely that the condition of the immune system and the state and composition of the parental cell dictates the outcome of the encounter of immune system and EVs. An important pathophysiological role of this interaction is illustrated in a recent paper by Costa-Silva et al. [69]. Here they showed that uptake of pancreatic carcinoma-derived exosomes by Kupffer cells in the liver caused these cells to secrete transforming growth factorbeta and hepatic stellate cells to produce fibronectin. This environment promoted influx of bone marrow-derived macrophages that secreted macrophage migration inhibitory factor that was shown to be involved in liver metastasis of the pancreatic cancer cells. This study underlines the important consequences of interaction of EVs with components of the immune system. 4.3. Specific targeting of extracellular vesicles As described above, administration of exogenous EVs seems to result mainly in accumulation in the spleen and liver. However, several studies have shown that it is possible to engineer EVs in such a way, that increased targeting to other tissues or specific cell types is achieved. A well-studied strategy to equip EVs with targeting properties is via transfection of EV-producing cells to drive expression of targeting moieties fused with EV membrane proteins. To accomplish increased targeting to the brain, Alvarez-Erviti et al. engineered dendritic cells (DCs) to express a fusion protein of an EV membrane anchor, lysosome-associated membrane glycoprotein 2b (Lamp2b), and brain-specific rabies viral glycoprotein (RVG), a peptide that binds the acetylcholine receptor. EVs isolated from these cells were shown to express RVG on their surface. After tail vein injection of siRNA-loaded EVs, knockdown of BACE1 mRNA and protein was demonstrated in the brains of mice, for the first time showing the potential of EVs as targeted drug delivery systems [19]. Wiklander et al. indeed found increased accumulation of these RVG-targeted EVs in the brain after systemic administration compared to unmodified EVs [55]. Similarly, Tian et al. showed that expression of a fusion of Lamp2b and αv integrin-specific iRGD peptide (internalizing RGD peptide CRGDKGPDC) in immature DCs resulted in expression of these peptides on the surface of EVs. Injection of modified EVs loaded with the chemotherapeutic drug doxorubicin in tumorbearing mice resulted in delivery of doxorubicin to the tumor site [70]. However, Hung & Leonard found that peptides fused to the Nterminus of Lamp2b are not detected in cells and on EVs, presumably as a result of degradation by endosomal proteases [71]. Glycosylation of these peptides protected against proteolytic degradation, which enhanced expression on EVs and increased target binding. Subsequent to the study of Alvarez-Erviti et al., they also investigated the influence of engineered glycosylation on the uptake of RVG-displaying EVs by neuroblastoma cells. EVs expressing glycosylated RVG peptides fused to Lamp2b were more efficiently internalized in vitro compared to EVs expressing non-glycosylated peptides. Therefore, glycosylation of targeting peptides could possibly enhance tissue targeting. To achieve targeted delivery to epidermal growth factor receptor (EGFR)-expressing tumors, Ohno et al. displayed EGFR-specific GE11peptides fused to transmembrane domains of platelet-derived growth factor receptor on EVs. They showed that miRNA-loaded EVs derived from the human embryonic kidney cell line HEK293 modified to express the GE11-peptide displayed increased accumulation in EGFRexpressing breast tumors in mice compared to untargeted EVs [72]. Together, these data highlight the possibilities of engineering of EVs to enhance specific tissue targeting, which could contribute to making EVs applicable as drug delivery systems. Besides ligand-mediated targeting, magnetic drug targeting, i.e. enhanced drug delivery to a chosen tissue by application of a magnetic

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field gradient, represents a non-invasive alternative strategy to enhance therapeutic efficacy. Delivery of drugs to tissues using magnetic iron oxide nanoparticles bound to a therapeutic agent was already introduced in the 1970s [73,74]. Recently, Silva et al. provided proof-ofconcept of using the combination of EVs and magnetic targeting for tissue-specific delivery. Therapeutics and iron oxide nanoparticles were together incubated with macrophages, which resulted in production of EVs loaded with both the therapeutic and magnetic nanoparticles. Magnetic targeting resulted in enhanced as well as spatially modulated EV and drug uptake by cancer cells in vitro [75]. However, disadvantages of this method when applied in vivo may include difficulties to target deep tissues in the body, and toxicity issues associated with the use of iron nanoparticles. 5. Therapeutic effects Despite the challenges in the isolation, loading and targeting of EVs, several reports have already demonstrated their potential for therapeutic drug delivery in various animal models of disease. The majority of these studies have focussed on cancer therapy. Due to their nanosize, EVs may achieve passive targeting to tumors via the enhanced permeation and retention (EPR) effect [76]. One of the first studies that described the use of EVs for drug delivery to tumors was performed by Mizrak et al. EVs derived from the cell line HEK293 were loaded with the suicide gene cytosine deaminase fused to uracil phosphoribosyltransferase to induce cell death in nerve sheath tumor cells upon addition of the prodrug 5-fluorocytosine. Weekly intratumoral injections of engineered EVs led to significant tumor regression in mice after 2 months [77]. Later, Ohno et al. showed that EGFR- targeted EVs loaded with let-7a miRNA delivered this miRNA to EGFR-expressing breast tumors upon systemic delivery in RAG2-/- mice, which significantly inhibited tumor development [72]. However, downregulation of let-7a target genes could not be demonstrated. In addition, Zhang et al. utilized EVs to deliver siRNA against transforming growth factor β1 (TGFβ1), a wellstudied target for cancer treatment, to sarcoma cells [78]. Using a subcutaneous tumor model in mice, they showed that i.v. injection of TGF-β1 siRNA-loaded EVs strongly suppressed TGF-β1 expression and downstream signaling in the tumors, and inhibited primary tumor growth as well as formation of lung metastases. Interestingly, siRNA doses used in this study were surprisingly low (0.4 pmol per injection). This may indicate that EVs were highly efficient in delivering their cargo to the target cells, or that other EV-mediated effects contributed to the observed inhibition of tumor growth. Besides for delivery of small RNA molecules, which critically depend on intracellular delivery to conduct their silencing function, EVs have also been employed to deliver chemotherapeutic agents, with the aim to enhance their efficacy and reduce side effects. For example, Tian et al. observed significantly improved suppression of breast tumor growth after i.v. injection of integrin-targeted, dendritic cell-derived EVs loaded with the chemotherapeutic drug doxorubicin in mice, compared to free drug. Moreover, doxorubicin was shown to cause less cardiac damage, the most important dose-limiting side effect of the drug, when packaged in EVs [70]. Furthermore, PTX-loaded EVs were shown to be more effective for inhibiting growth of Lewis lung carcinoma metastases than Taxol, a commercially available formulation of PTX [48]. Tang et al. used two different tumor models to show beneficial effects of packaging chemotherapeutic drugs into EVs [44]. Repeated i.p. injections of cisplatin-loaded EVs improved long-term survival of ovarian cancer-bearing mice, as compared to cisplatin only. Furthermore, i.v. injection of doxorubicin-loaded EVs delayed growth of established subcutaneous hepatocarcinoma. Importantly, these EV treatments did not adversely affect liver or kidney functions, which are typical side effects observed after administration of the free drugs. Based on results from this study (Fig. 2), a phase II clinical trial has been initiated which aims to evaluate the effect of chemotherapeutic drugs encapsulated in

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Fig. 2. Schematic overview of preclinical studies that formed the basis for the first clinical trials using extracellular vesicles for drug delivery. Left panel: Loading of the antiinflammatory agent curcumin into EVs was achieved via simple incubation at 22°C. Curcumin-loaded EVs were shown to attenuate autoimmune encephalitis and LPS-induced septic shock in mice. Right panel: EVs loaded with chemotherapeutic drugs inhibited hepatocarcinoma in mice after tail vein injection. EV loading was achieved by first incubating tumor cells with chemotherapeutic drugs. Then, to stimulate formation of drug-loaded EVs, cells were irradiated with ultraviolet light to induce apoptosis. Abbreviations: EV = extracellular vesicle, LPS = lipopolysaccharide, i.p. = intraperitoneal, UV = ultraviolet.

EVs on malignant ascites and pleural effusion in advanced cancer patients. Patients are locally injected four times a week, and both therapeutic as well as side effects are recorded (clinicaltrials.gov, NCT01854866). A second ongoing clinical trial (phase I) in which EVs are being evaluated as drug delivery systems is based on two studies that showed beneficial effects of encapsulating anti-inflammatory compounds into EVs (Fig. 2). Firstly, Sun et al. showed that EVs as vehicles for curcumin increase its solubility, stability, and bioavailability. Furthermore, curcumin-loaded EVs protected mice from lipopolysaccharide (LPS)induced septic shock [46]. In a follow-up study, daily intranasal administrations of curcumin packaged in EVs delayed and attenuated experimental autoimmune encephalomyelitis, an effect not observed after administration of curcumin alone. Mechanistically, treatment success was likely caused by increased induction of apoptosis in microglial cells [20]. The clinical trial aims to study the ability of plant EVs to deliver curcumin to colon tumors. Outcome measures include curcumin concentrations in normal and cancerous tissue after oral administration and safety as well as tolerability (compared to curcumin alone) as determined by adverse events (clinicaltrials. gov, NCT01294072). Besides for cancer, EVs have been proposed as therapeutic delivery vehicle for the treatment of Parkinson’s disease (PD). PD is associated with reduced levels of several brain enzymes such as catalase, superoxide dismutase, and other antioxidants. To increase brain levels of catalase, Haney et al. intranasally administered catalase-loaded EVs in a mouse model of PD [53]. Catalase-loaded EVs were shown to reduce microglial activation and protect neurons against reactive oxygen species more efficiently compared to free catalase. Altogether, these results, although preliminary, have demonstrated that EVs are promising candidate drug carriers for the treatment of a variety of diseases.

6. Extracellular vesicle-mimetic nanovesicles Although substantial progress has been made in the development of natural EVs as drug delivery vehicles, the relatively low recovery of EVs produced by mammalian cells remains an obstacle for large-scale EV production. For this reason, the possibility of generating EV-mimetic vesicles with a substantially greater yield has attracted recent attention. Methods for preparing EV-mimetics are based on obtaining artificially generated nanovesicles from broken cells, which resemble the structural and physical features of EVs. For example, Jang et al. produced drugloaded nanovesicles by serial extrusion of monocytes through filters in the presence of chemotherapeutics, and isolated these vesicles from free drugs using an OptiPrep density gradient [79]. This production method resulted in a 100-fold increased vesicle yield compared to isolation of naturally produced EVs. Nanovesicles were shown to express lymphocyte function-associated antigen-1 (LFA-1) that can bind endothelial cell adhesion molecules (CAMs), overexpressed on activated endothelial cells, such as found in tumors. Importantly, i.v. injection of these drug-loaded nanovesicles in mice resulted in their accumulation in tumor tissue and subsequent reduction of tumor growth, without the toxic side effects present after injection of the free drug. Interestingly, EV-mimetic nanovesicles were found to be similarly effective as EVs harvested from the same cells. An alternative method to generate EVmimetic nanovesicles was proposed by Yoon et al. Living cells were sliced with microfabricated silicon nitride blades of 500 nm while flowing through a standardized microfluidic system. After slicing, nanovesicles were spontaneously formed through self-assembly of membrane fragments. Vesicles were shown to be composed of a lipid bilayer membrane and carry various membrane proteins, intracellular proteins and RNAs. The addition of polystyrene latex beads during slicing resulted in encapsulation of beads in the nanovesicles, with efficiencies up to 30% [80]. These successful examples demonstrate the

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potential of these methods to generate nanovesicles for drug delivery on a large scale. However, whether these cell-derived EV-mimetic vesicles also possess the envisioned advantages of natural EVs, including low immunogenicity and intrinsic capacity to utilize endogenous mechanisms for uptake and intracellular trafficking in recipient cells, is currently unknown. 7. Conclusions EVs are natural membrane vesicles involved in intercellular communication. The ability of EVs to transfer their content to recipient cells via endogenous uptake mechanisms makes them attractive candidates for application in drug delivery. However, current obstacles to overcome before deployment of EVs in large clinical trials are the need for both scalable EV isolation methods, and more efficient drug loading approaches for different therapeutics. Especially ultrafiltration- and SECbased EV isolation approaches seem promising for large-scale clinical application, although further optimizations remain to be implemented. An additional challenge remains shifting in vivo biodistribution of EVs from non-specific organ accumulation to accumulation in desired tissues. Although considerable efforts have been made in targeting specific cell types by engineering EVs to express cell-type specific ligands, one of the major obstacles remains inefficient drug delivery to target tissues. Gaining more insight into fundamental EV biology, in particular into how EVs target specific cell types, and whether different EV subtypes serve different physiological roles, could therefore contribute to further improvements in the development of EVs for drug delivery. Conflict of interest statement

[9]

[10]

[11]

[12] [13] [14]

[15]

[16] [17] [18] [19]

[20]

[21] [22]

RMS is the CSO of Excytex, and on the scientific advisory board of JSR Micro NV. Acknowledgements PV is supported by a VENI Fellowship (# 13667) from the Netherlands Organisation for Scientific Research (NWO). The work of EAM is part of the Project SMARTCARE-II of the BioMedical Materials institute, co-funded by the Dutch Ministry of Economic Affairs, Agriculture and Innovation and the Netherlands CardioVascular Research Initiative (CVON): the Dutch Heart Foundation, Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development, and the Royal Netherlands Academy of Sciences. The work of RMS on cell-derived membrane vesicles is supported by a European Research Council starting grant (# 260627) “MINDS” in the FP7 ideas program of the European Union.

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