Methods for loading therapeutics into extracellular vesicles and generating extracellular vesicles mimetic-nanovesicles

Journal Pre-proofs Methods for loading therapeutics into extracellular vesicles and generating extracellular vesicles mimetic-nanovesicles Amirmohammad Nasiri Kenari, Lesley Cheng, Andrew F. Hill PII: DOI: Reference:

S1046-2023(19)30266-X https://doi.org/10.1016/j.ymeth.2020.01.001 YMETH 4843

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Methods

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1 October 2019 5 December 2019 2 January 2020

Please cite this article as: A.N. Kenari, L. Cheng, A.F. Hill, Methods for loading therapeutics into extracellular vesicles and generating extracellular vesicles mimetic-nanovesicles, Methods (2020), doi: https://doi.org/10.1016/ j.ymeth.2020.01.001

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Methods for loading therapeutics into extracellular vesicles and generating extracellular vesicles mimetic-nanovesicles Amirmohammad Nasiri Kenari1, Lesley Cheng1 and Andrew F. Hill*1 1Department

of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Australia *Authors to whom correspondence should be addressed: [email protected] Keywords: Extracellular vesicles, exosome mimetic-nanovesicles, engineering, therapeutic loading, cargo modification Running title: Extracellular vesicles and mimetic engineering

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ABSTRACT Extracellular vesicles (EVs) are membrane bound vesicles released into the extracellular environment by eukaryotic and prokaryotic cells. EVs are enriched in active biomolecules and they can horizontally transfer the cargo to distant recipient cells. In recent years EVs have demonstrated promising clinical applications due to their theragnostic potential. Although EVs have promising therapeutic potential, there are several challenges associated with using EVs before transition from the laboratory to clinical use. Some of these challenges include issues around low yield, isolation and purification methodologies, and poor engineering (loading) of EVs with therapeutic cargo. Also, to achieve higher therapeutic efficiency, EVs architecture and cargo may need to be manipulated prior to clinical application. Some of these issues have been addressed by developing biomimetic EVs. EV mimetic-nanovesicles (MNVs) are a type of artificial EVs which can be generated from all cell type with comparable characteristics as EVs for an alternative therapeutic modality. In this review, we will discuss current techniques for modifying EVs and methodology used for generation and customizing of EVs mimeticnanovesicles.

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INTRODUCTION Nano drug delivery systems (NDDs) have several advantages over other therapeutic modalities. NDDs permit the specific loading of therapeutics and promote protection from degradation, in addition to enhancing drug delivery efficiency and the ability to pass biological barriers. Moreover, surface engineering with ligands of interest can increase targeted delivery of therapeutics, thus minimizing offtarget effects and enhancing overall efficiency [1]. In this context, extracellular vesicles (EVs) have shown several advantages over other NDDs such as liposomes (self-assembled phospholipid membranous bound vesicles) [2]. EVs is a collective term used for lipid bilayer membrane bound vesicles secreted by eukaryotic and prokaryotic cells. In recent years there have been attempts to update nomenclature and further sub-characterization of EVs to different groups. Based on the most widely accepted nomenclature, EVs range from small (<200nm) to medium/large (>200nm) vesicles [3]. Based on the biogenesis pathway and molecular composition, EVs can be broadly classified into three subtypes. Exosomes (40-150 nm) are endosomally derived vesicles with higher expression of tetraspanin proteins such as CD63, CD81, and CD9. Exosomes play a fundamental role in many physiological and pathological conditions due to the composition of their cargo and the presence of specific proteins within their membrane. Microvesicles (150-1000 nm) are formed through shedding of the plasma membrane and are enriched in annexin A1. Apoptotic bodies (1-5 μm) are released to the extracellular environment by cells undergoing apoptosis and display high expression of Annexin V [4]. Among EVs subtype, the term exosomes has been widely used in literature as the potent class of EVs for therapeutic application and will be the subtype mostly described in this review.

Recent research has demonstrated the therapeutic potential of exosomes in the treatments of several diseases as they display numerous advantages in overcoming the challenges associated with conventional nanomedicine therapeutics [2, 5-18]. Tumor cell derived exosomes exhibit anti cancer

effect by inhibiting tumor growth [19, 20], while those from liver nonparenchymal cells and macrophages have shown therapeutic potential against viral infection [21, 22]. Mesenchymal stem cell

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derived exosomes have demonstrated they could be used as a mediator of tissue regeneration by maintaining the function of stem cells [23, 24].

Collectively, the innate ability of EVs to cross biological barriers and protect cargo from proteases and nucleases in combination with nanomedicine characteristics make them an ideal tool for clinical application. Of particular interest is the common obstacle in delivering drugs or biological therapeutic molecules into the brain for the treatment of central nervous system diseases. Regardless of the route of administration, EVs have been shown to cross the blood-brain barrier (BBB) and reach the olfactory region of the brain where they are taken up by neurons, microglia and oligodendrocytes [5, 25, 26].

Although exosomes have displayed some promising therapeutic applications, the therapeutic field still requires extensive multidisciplinary efforts to improve research grade exosomes to clinical grade exosomes. To some extent, this can be achieved by overcoming the current challenges such as heterogeneity, low yield EV secretion and isolation procedure. In recent years some of the challenges associated with using EVs for therapeutic application have been addressed by developing mimeticnanovesicles (M-NVs). Generation of M-NVs demonstrated high yield nanovesicles formation similar to exosomes biophysical characteristics with the advantage of simple isolation procedures.

Furthermore, many current challenges involve methodology for customizing cell derived EVs and MNVs to further enhance cargo loading with therapeutics of interest or surface modification for tissue specific therapeutic delivery. Modification of the surface of EVs and M-NVs has shown to increase the therapeutic delivery efficiency [5, 27, 28]. Several methods have been developed for M-NV generation and modification with an overall goal in boosting therapeutic applications of nano drug delivery modalities. Both exosomes and M-NVs are derived from cells, therefore cargo can be endogenously manipulated by modifying parental cells or exogenously loading with therapeutics following vesicle isolation or generation. The emerging field of using M-NVs as an alternative nanocarrier has mainly raised due to promising results obtained by using EVs for therapeutic application as they can potentially mimic exosomes. Treatment of various diseases could be improved by replacing exosomes with M-

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NVs that are engineered to load desired cargo, improve uptake by targeting specific cells, together with the ability to produce large quantities of M-NVs compared to exosome production. This review highlights the current techniques for manipulating exosomes and focuses in depth on the methodology to generate M-NVs by cell extrusion, in addition to the current techniques used for customizing MNVs.

Biogenesis of exosomes and its therapeutic application The biogenesis of exosomes involves the endosomal system and is initiated by the maturation of endosomes, followed by the formation of intraluminal vesicles within the MVB [29]. The MVB protects these vesicles from degradation pathways and ultimately fuses with the plasma membrane where the exosomes are released into the extracellular environment (Figure 1). Once EVs are secreted into the extracellular environment they can horizontally transfer their intact genetic, protein and lipid material and reprogram the behaviour of recipient cells [30, 31].

For some time, the therapeutic potential of mesenchymal stem cells (MSC) was thought to be due to the homing/direct seeding of the cells in the host tissue. It has recently been shown that after infusion, only a small amount of MSC remained in the host tissue [32] highlighting that the therapeutic benefits of MSC could potentially come from other indirect sources. In this regard, several studies have shown that the therapeutic function of MSC is through paracrine factors [33-36]. These findings extended the investigation into MSC conditioned medium (CM), where the acellular (independent of cells) therapeutic potential of MSC was detected [37-39]. At the time this observation lead the researchers to investigate the role of MSC EVs. Since EVs are released into the extracellular environment it has been shown, to some extent, that this therapeutic potential is derived from EVs present in the secretome [14, 38]. This finding opens up the avenue for substitution of MSC to EVs for safer, potent and non-cellular therapeutic application. Proregenerative and immunomodulatory function of MSCs are preserved in many disease models, with EVs derived from MSCs directly used for therapeutic application without being modified. However, for treatment of some diseases, EVs need to be tailored to carry the therapeutic of interest, achieve higher tissue specificity, less off-target effects, enhance circulation

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(stability) and ultimately escape host immune effects. EVs can be broadly engineered by two approaches, either endogenously at the cellular level where the therapeutics are introduced to parental cell or exogenously following isolation by incorporating therapeutics through physical or chemical modification. In the next section, we will highlight the methods for manipulating EVs and how the customized EVs improve the therapeutic application.

Cell engineering as a method for manipulating extracellular vesicles C1C2 domain of Lactadherin The most studied approach for endogenously loading protein into EVs is by protein fusion whereby, genes for EV enriched proteins can be fused with protein of interest genes. This method does, however, rely on the existing cell machinery for sorting therapeutic proteins into EVs. Lactadherin is localized into the membrane of exosomes by specifically binding through C1C2 domain with phosphatidylserine enriched in exosome membrane [40, 41]. To load proteins on to EVs by this method, the gene of interest is fused (as a chimeric fusion) with the C1C2 domain of Lactadherin and subsequently transfected to donor cells. Modified exosomes harbour the C1C2 fragment and mediate anchoring on surface of the vesicle. This method is also known as exosome display technology. In this context, genes for interleukin 2 and granulocyte macrophage colony-stimulating factor were ligated between leader sequence and upstream of C1C2 terminal of Lactadherin by replacing EGF domain. The recombinant exosomes were functionally active and produced antibodies against human leukocyte antigen [42]. Furthermore, this method has been utilised for presenting chicken egg ovalbumin (OVA) antigen in association with exosomes through fusion of OVA to C1C2 domain of Lactadherin [43]. Similarly, human epidermal growth factor receptor 2 antigen (HER2) fused to the C1C2 domain of Lactadherin resulted in successful exosome antigen targeting [44].

Lamp2b Another protein utilised by the gene fusion method is the lysosome-associated membrane glycoprotein 2b (Lamp2b) which is present on the membrane of exosomes [45]. Using this technique, various therapeutics such as siRNA, miRNA, doxorubicin (DOX) and imatinib were successfully delivered to

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target the site of interest. The first study involved the fusion of Lamp2b with the central nervous systemspecific rabies viral glycoprotein (RVG) peptide. RVG mediates the specific binding to acetylcholine receptors expressed on neuroendothelial and neuronal cells. In this method, the RVG sequence was cloned between the signal peptide and N terminus of Lamp2b coding sequences and transfected into immature dendritic cells. The engineered exosomes were further loaded with siRNA against the BACE1 gene and systematically injected to mice. The modified exosomes passed the BBB and successfully mediated BACE1 knockdown [5]. Similar protein fusion strategies have been used for engineering bone marrow mesenchymal stem cells (BM-MSCs) for the production of RVG-Lamp2b exosomes. In this study engineered exosomes mediated miR-124 delivery to the ischemic region of the brain and successfully promoted neurogenesis following systematic administration [46]. In another example, iRGD, a targeting peptide for αv integrin, was fused with Lamp2b for specific targeting to tumor tissue. This suggests the Lamp2b fusion strategy is not only limited to brain but it can also be fused with organ specific peptides for targeted tissue delivery. Modified exosomes were further loaded with Dox and demonstrated tumor growth inhibition upon administration [47]. Human interleukin-3 gene fused with Lamp2b loaded with imatinib caused tumor reduction by specifically targeting tumor cells [48]. Fusion of cardiomyocyte specific peptide (CMP) to N terminus of Lamp2b increased the uptake of cardiosphere-derived (CDCs) exosomes in cardiac in comparison to unmodified exosomes [49]. Furthermore, in a separate study using bone marrow stromal cells (BMSCs), the ischemic myocardiumtargeting peptide (IMTP) was fused to N terminus of Lamp2b and isolated exosomes were used in the treatment of myocardial infarction through tissue specific targeting [50]. Collectively, these studies demonstrate the potential of peptide fusion to exosomal Lamp2b for specific tissue targeting despite any hindrance with a biological barrier.

Tetraspanin modification Another group of proteins which are highly abundant in exosomes are members of the tetraspanin family such as CD63, CD81 and CD9. Tetraspanins are classically used for characterization of exosomes and recently has been widely used as a skeleton for fusion with green fluorescent protein (GFP) for visualization of EVs. In particular, CD63 has been used as a candidate for fusion with

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fluorescent protein for in vitro and in vivo visualisation of exosomes. For instance, in one example, Immune cell Raji B and J77 T cells were transfected with CD63 fused with GFP construct. This method led to successful visualization and uptake in recipient cells which was validated by flow cytometry, western blot and confocal microscopy [51]. Although, using CD63-GFP does not have a direct point to a therapeutic angle, it can help our understanding of the biogenesis of exosomes or vesicles enriched with CD63 and monitor their trafficking and fate of the vesicles upon uptake by recipient cells [52, 53]. In addition, CD9 and CD81 were successfully used for chimeric fusion like CD63-GFP. Collectively, this highlights the potential of this method for fusing the protein of interest into a diverse family of tetraspanins [54]. CD63 has also been used as a scaffold for engineering two fluorescent proteins simultaneously; GFP fused to the inner loop of CD63 and ruby red fluorescent protein (RFP) to the larger outside loop of CD63 resulting in a dual fluorescent reporter. This study demonstrates the potential of CD63 for incorporating proteins to both extravesicular and intravesicular regions of EVs [54]. This method could be applicable when the protein of interest need to be loaded in cargo or exposure on the surface of EVs for downstream applications. Recently, using in situ labelling release of vesicles from neuron to brain observed by using the CD63 fusion with copGFP (variant of GFP) [55]. This method relies on Cre-dependent exosome reporter (CD63-GFPf/f) mice characterised by a loxP-floxed stop codon upstream of CD63-copGFP with His-tag in C-terminal. Collectively, the fusion of GFP or similar fluorescent protein with tetraspanin protein could overcome artefact and issues reported to be associated with lipophilic EVs labelling dye [56].

The cargo of exosomes can be modified using optogenetically encoded proteins such as engineered EXPLORs [57]. Using this system cells must be cultured under the incubator with the light on to allow for protein-protein interactions to occur. There are two main components in EXPLORs which need to interact for cargo loading. In the first component, the protein of interest is fused to the photoreceptor cryptochrome 2 (CRY2) and in the second component, a truncated version of CRY-interacting basichelix-loop-helix 1 (CIBN) is fused to EGFP-tagged CD9. Since this technique relies on optogenetic system, upon blue light illumination, the protein of interest migrates towards CIBN located in inner

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cell membrane and is ultimately incorporated to the exosome during the biogenesis process. Once the light is switched off the cargo protein will be released from CIBN-EGFP-CD9 into intravesicular of exosomes [57]. This method can be used for loading protein to vesicles enriched with CD9.

GPI anchor It has been shown that certain proteins such as PrP (cellular prion protein) and CD59 (decay accelerating factor (DAF) are anchored on the surface of exosomes through the addition of a glycosylphosphatidylinositol (GPI) moiety [30, 58]. Inspired by GPI anchoring, proteins can be engineered to be sorted on the surface of exosomes. For example, the signal peptide from DAF-derived GPI was used to fuse with EGa1 nanobody. GPI anchor mediated the sorting of EGa1 nanobody on the surface of exosomes. This modification on exosomal surfaces enhanced the exosomes binding to tumor cells through EGa1 targeting epidermal growth factor receptor EGFR [59]. This approach can be beneficial for targeted delivery of EVs to specific tissue with minimal off-target effects.

Protein-protein interaction for manipulating extracellular vesicles Proteins can be specifically loaded into exosomes using classical late-domain (L-domain) trafficking pathways. Ndfip1 is a protein with L-domain which have been shown to interact with target proteins through its three L-domain (PPxY) and subsequently sort the protein of interest into exosomes. For example, Nedd4 is a Ndfip1 binding protein which binds to Ndfip1 through its WW domain. Based on this mechanism the WW tag can be added to a protein of interest (Cre) for specific loading into exosomes. Using nasal delivery the modified exosomes delivered functional WW-Cre to the brain and demonstrated Cre-mediated recombination [26]. Another method used to engineer exosomes includes using the pDisplay vector (commercially available) which can express the protein of interest on the surface of cells through the transmembrane domain of platelet-derived growth factor receptor. It has been shown that GE11 peptide can specifically bind to EGFR [60]. To generate modified exosomes which can harbor the GE11 to cancer cells, GE11 was fused to pDisplay vector and transfected to HEK293 cells. GE11 modified exosomes were further loaded with Let-7a miRNA and following administration delivered the functional miRNA to recipient cells expressing EGFR and inhibited tumor

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development [61]. Taken together, these studies demonstrated a diverse range of EVs engineering using either vesicles innate trafficking system or commercially available vector.

Using RNA binding proteins to customize extracellular vesicles cargo Nucleic acids such as RNA can bind to proteins enriched in EVs and are actively involved in packaging proteins into the vesicles. Targeted and modular EV loading (TAMEL) methodology has been used for active loading of RNA to exosomal cargo. This technique has two main components involving EV enriched protein (EEP) fused with RNA binding protein (RBP). An example of this is the MS2 bacteriophage coat protein (RBP) fused to Lamp2b (EEP). Using this system cellular RNA interacts with the RNA binding domain of MS2 through motif recognition and subsequently sorted into intravesicular region of exosomes due to presence of exosomes enriched protein Lamp2b [62].

Other studies have identified specific miRNA motif sequences where the RNA binding proteins can bind and sort miRNA into exosomes. It has been shown that sumoylated heterogenous ribonucleoprotein A2B1 (hnRNPA2B1) recognise EXOmotifs (specific short sequence present in miRNA) and then sort miRNA into exosomes [63]. Similarly, synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP) was found to bind to a hEXO motif which is a specific sequence in certain miRNA causing it to be sorted into ILVs. Together, this data suggests that using RNA binding proteins and a known sequence-specific motif we can manipulate exosome cargo for therapeutic RNA loading and delivery [64].

An alternative approach to therapeutic exosome loading has been demonstrated by overexpressing miRNA in the parental/donor cells of interest. This method results in passive packaging of the overexpressed product into exosomes [61, 65-68], however, there is some concern about the specificity and relative abundance of the overexpressed product ultimately packaged into exosomes. Furthermore, overexpression of RNA and protein in cells can impact the cells innate biology and cause unnecessary

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downstream effects prior to it being packaged into exosomes. Therefore, it is important to investigate the impact of cell modification before engineering EVs.

Exogenous loading of therapeutics to extracellular vesicles Once EVs are isolated from the extracellular environment, they can be exogenously modified with therapeutics. This approach has been extensively used for loading of small molecules and nucleic acids to EVs. In this method, therapeutic molecules have to be internalized into exosomes through the exosomal membrane. Exogenous method can be useful for loading of variety of therapeutics with simple techniques.

Incubation of therapeutics with exosomes One non-invasive method for loading small drugs into exosomes is via co-incubation. This method allows the loading of hydrophobic drugs through passive diffusion. Drugs such as withaferin A, bilberry-derived, anthocyanidins, curcumin, docetaxel, paclitaxel and celastrol have successfully been loaded into exosomes via the incubation method [25, 69-71]. Curcumin was mixed with exosomes resuspended in buffer and incubated at 22 °C for 5 minutes. The curcumin loaded exosomes were purified following sucrose density gradient. Exosomes containing curcumin exhibited enhanced solubility, stability and bioavailability of loaded curcumin in vitro and in vivo [71]. Furthermore, using an incubation strategy, JSI124 an inhibitor for signal transduction and activator of transcription 3 (Stat3) was loaded to exosomes. Modified exosomes was active and functional in the brain following intranasal delivery [25]. In addition to loading drugs using the incubation method, modified siRNAs were also successfully loaded into exosomes. In this method, hydrophobic siRNA (hsiRNA) was conjugated with cholesterol for hydrophobic interaction and with the single-stranded phosphorothioated for internalization of siRNA through exosomal membranes. This method successfully downregulated the expression of the Huntingtin mRNA and protein in a dose-dependent manner [72]. Similarly, in a separate study, cholesterol conjugated siRNA successfully loaded into exosomes and exhibited

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silencing of human antigen R (HuR) [73]. It should be noted that loading exosomes using these incubation strategies require the optimisation of incubation temperature, time, volume of buffer and ratio of therapeutic to EVs. Collectively, simple incubation of therapeutics with EVs under optimal condition can enhance the loading efficiency.

Electroporation Electroporation is a well-utilised method for loading therapeutic molecules into exosomes. This involves mixing the therapeutic with exosomes in electroporation buffer, and applying a high voltage electrical charge to create transient pores on the exosomal membrane. This temporal membrane interruption allows the permeabilization of the therapeutic into exosomes. Following electroporation, exosomes can be recovered by incubation at an appropriate temperature. To remove any extravesicular therapeutics from solution, EVs need to be washed and further purified, before use in functional assays. This methodology was first applied using siRNA targeting BACE1 in the brain and demonstrated efficient knockdown of BACE1 gene [5]. Further studies used the electroporation approach for loading siRNA, miRNA, DNA and Dox into exosomes [2, 47, 74-78]. It has since been shown that electroporation can cause siRNA precipitation and aggregation which could lead to false interpretation of siRNA loading efficiency [79].

Despite this limitation, the clinical grade production of iExosomes (exosomes loaded with siRNA targeting oncogenic KRAS) have shown successful electroporation of siRNA into exosomes [2, 80]. Also, several attempts were performed for optimization of electroporation for loading therapeutics into EVs [81, 82]. The therapeutic loading through electroporation is an active area of EV engineering and we find this is a controversial topic based on available literature. This variation on loading efficiency or artefacts associated with self-aggregation of siRNA is probably due to several factors, including electroporation devices, run settings, buffer composition, nature of EVs and therapeutics being loaded.

Sonication, extrusion, hypotonic dialysis, Freeze-thaw cycles, saponin, CaCl2 and lipofectamine reagent

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In general, molecules can be loaded into EVs either using mechanical interruption such as sonication and extrusion or chemical interruption such as hypotonic, saponin, CaCl2 and lipofectamine reagent (Figure 2).

Sonication is an alternative method for loading a variety of molecules such as siRNA, miRNA, ssDNA, paclitaxel and Dox into EVs [83, 84]. Loading EV cargo with sonication requires consideration of the potential to cause damage or lysis of the EVs. Also, other methods such as extrusion and hypotonic dialysis have been used for loading drugs into exosomes [85]. For hypotonic dialysis, EVs and therapeutics are mixed with hypotonic solution and due to osmolarity, EV membranes permeabilize allowing the therapeutics to pass through the membrane. The mixture can be further subject to an isotonic solution for remodelling EVs and encapsulation of surrounding therapeutics. EVs can be purified based on common EV purification strategies.

A freeze-thaw cycle strategy can be used for loading of drugs into exosomes. Catalase solution was incubated with exosomes for 30 minutes and then by performing three cycles of rapid freeze/thawing (-80°C/RT), efficient loading of antioxidant catalase into exosomes was achieved [86].

Saponin is a detergent which can permeabilise the plasma membrane and it has been shown that therapeutics can be loaded into exosomes through the simple incubation of saponin, EVs and therapeutics at room temperature [85-87]. CaCl2 can be used for loading therapeutics into EVs. This protocol involves mixing exosome and therapeutics with 0.1 M final concentration of CaCl2 following incubation on ice and heat shock. When compared with electroporation, a similar loading efficiency is obtained [88]. Lipofectamine is commonly used as a classical method for cell transfection, and this method has been utilised to load siRNA into exosomes by incubation siRNA with the lipofectamine transfection reagent with exosomes. Despite the loading efficiency, it is difficult to distinguish between the lipofectamine (lipid micelles) loaded siRNA and exosome loaded siRNA due to the heterogeneous mix of micelle

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packaged siRNA versus exosomes [74, 89]. This is important to consider when using lipofectamine based exogenous loading.

Exogenous cargo loading and membrane functionalization Exosome membrane modification has traditionally been customized by manipulating parental cells, however, recent studies demonstrate methods that are effective for exosome surface engineering post isolation [27, 28] . Using exogenous method hydrophobic molecules such as aptamer, a class of short oligonucleotides (DNA or RNA) can specifically bind to EV membranes for functionalization and specific targeting. LZH8 is a DNA aptamer shown to specifically bind to HepG2 (cancer liver cell) and incorporate into the exosome membrane, resulting in functional exosomes [90]. Similarly, diacyl lipidsgc8 DNA aptamer which specifically recognizes membrane protein tyrosine kinase 7 (PTK7) incorporates into exosomes through hydrophobic interaction using the simple incubation method. The surface engineered exosomes were further loaded with Dox by electroporation [27]. In addition to DNA aptamer, three-way junction (3WJ) can be incorporated into the exosomal membrane by conjugating to cholesterol. To further achieve specific cancer cell targeting, the folate, prostate-specific membrane antigen and EGFR RNA aptamer conjugated to 3WJ. The surface decorated exosomes was loaded with siRNA against survivin and demonstrated efficient siRNA delivery and mediated the anti tumor property [91]. Similarly, the exosomal surface can be modified by sonication for dual engineering by incorporating aminoethylanisamide-polyethylene glycol (AA-PEG) for specific targeting lung cancer cell through sigma receptor and loading cargo with PTX [92].

In addition to lipid mediating exosome functionalization, peptides can also be incorporated into exosomes. Using phage display technology, a CP05 peptide demonstrated selective binding to exosomal enriched protein CD63. Using this method, authors anchored both the targeting moiety for muscle M12 and antisense oligonucleotide drugs through CP05 binding to CD63 using the simple incubation strategy [93]. Collectively, using the advanced molecular biology techniques we can now functionalize the

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exosome surface post isolation. Once the EVs membrane is decorated it can be further loaded with therapeutics of interest. Taken together, this method could be more specific and reliable on engineering EVs compare to modifying parental cell.

Application of mini-extruder in generating a nanodrug delivery system Mini-extruder technology has been in use for nearly 30 years in the design of five generations of NDD systems [94] (Figure 3). This technique was traditionally used for making liposome from lipid molecules or making homogenous small-sized liposome complexes which can be ultimately loaded with therapeutics of interest (Figure 3.A and B). The third generation of NDD system produced by mini-extruder used cells as precursors for generation of drug delivery vectors, a protocol now defined as the top down method for generation of nanovesicles [95]. This top down method for generation of cell derived nanovesicles evolved from using RBC membrane vesicles followed by co-extrusion of RBC isolated membrane with PLGA (poly lactic-co-glycolic acid). To make the RBCs NDD platform, authors first attempted to remove the haemoglobin by hypotonically lysing RBCs and then isolating the membrane fraction, termed ghost erythrocytes which are devoid from intracellular (mainly haemoglobin) content [96]. The extrusion method was subsequently used to remodel the RBC membrane to nanovesicles by sequentially extruding through 400 and 100 nm polycarbonate membranes. Also, the RBC nanovesicle were further engineered by incubation with PLGA before coextrusion through a polycarbonate membrane with 100 nm size. Collectively this study highlighted the use of the top down method for generating cell membrane-derived nanovesicles (Figure 3.C) [95]. Similarly, cancer cell membrane isolated by hypotonic lysis and extruded through 400 nm to form cancer cell membrane derived nanovesicles. To generate cancer membrane-coated nanoparticles, PLGA were mixed with cancer cell membrane and extruded through 200 nm polycarbonate membrane [97]. Recently, a similar approach was used for loading camptothecin into RBCs membrane vesicles [98]. Collectively, these studies demonstrated the potential of the mini-extruder as a device for generating nanovesicles.

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Using an extruder has evolved into a new method to generate nanovesicles for drug delivery, however extruders were initially used to produce liposomes, ‘empty’ membranous RBCs (ghost erythrocytes) and recently using whole cells as the precursor material to generate nanovesicles (fourth generation) (Figure 3.D ). Furthermore, application of mini-extruder has extended in loading of therapeutics to exosomes. In this context, exosome can be mixed with therapeutics of interest and resuspended samples subjected to the extrusion for loading vesicles into exosomes (fifth generation) (Figure 3.E ).

A growing number of studies now use the 4th generation method by utilizing whole cells as a precursor for generating cell derived nanovesicles which referred to as ‘exosome mimetic-nanovesicles’ due to their comparable biophysical characteristics to exosomes. In the next part of this review, we will discuss the methodology for generating EVs/exosomes mimetic-nanovesicles. Generating EV/exosome mimetic-nanovesicles Unlike exosomes which are produced by a distinct cellular process, M-NVs are generated in vitro and are not naturally involved in any physiological mechanism [99, 100]. M-NVs is an umbrella term used for nanovesicles generated from precursor material such as cells, exosomes or liposomes to either enhance EV therapeutic potency or potentially assemble new vesicles. These new vesicles mimic biophysical and biochemical characteristics of EVs to overcome current challenges faced using exosomes for therapeutic applications. Recently, a review published in the Journal of Extracellular Vesicles suggested a systematic classification for artificial EVs [101]. Based on this nomenclature, artificial EVs are broadly classified into semi synthetic and synthetic groups. Semi synthetic M-NVs include engineering the EVs either by manipulation of parental cells or post isolation. Synthetic methods are further divided into two classes, bottom top, or top down, which involve making vesicles either from selected individual components (proteins, nucleic acids, lipids), or fragmenting cells for generation of nanoparticles respectively [101].

Although the term exosome mimetic-nano vesicles has been widely used in publications referring to vesicles generated by any of the above-mentioned methods, in this review we define M-NVs as vesicles

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generated by the top down method using cells as the initial material for the generation of nanovesicles. Several studies using the top down method, demonstrate functional M-NVs generated from different parental cells with more than 100 fold higher particle yield compared to exosomes [99, 102-105]. The principal of M-NVs generation using the top down method is based on the advantage of fragmented lipids from cell membranes which can reassemble to spherical structures and simultaneously encapsulate surrounding molecules [105]. There are three major steps involved to produce M-NVs: 1) harvesting cells and resuspension in suitable buffer; 2) breaking the cells by mechanical force and; 3) purification/enrichment of the vesicles produced [99, 103, 104] (Figure 4). To generate M-NVs by the top down method, cells must undergo some force and shearing pressure to fragment the plasma membrane. Several methods have been developed to fragment the plasma membrane, such as through extrusion using an Avanti mini-extruder and centrifugal force, passing cells through a microchannel by microfluidic device or cell slicing using microfluidic device.

To generate M-NVs using a microfluidic device, cells are extruded through microchannels and M-NVs generated due to pressure force and extension of the cell membrane [106]. Another approach based on microfluidic devices employs cell slicing using microfabricated silicone nitride blades [107]. In both studies, crude M-NVs were further purified and collected following an Opti-prep density gradient purification.

However, in all M-NV publications to date, the most common method utilized for generation of MNVs involves extrusion using an Avanti mini extruder [78, 103, 104] (Table 1). Since M-NVs are generated from whole live cells, care must be taken to not introduce any damage specifically regarding the harvesting of adherent cell lines, as maintaining cell membrane proteome could directly reflect the M-NVs target delivery [104]. The constant factor in cell extrusion is that the cell must sequentially pass through the polycarbonate membrane with diminishing pore size as cells fragment once passed through each membrane. Currently, the most commonly used filtering combination is sequential extrusion through 10, 5, and 1 μm polycarbonate membrane [78, 100, 103, 104, 108-112]. We have shown the successful generation of M-NVs using this method [99]. In addition to the use of an extruder, M-NVs

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can be generated based on extrusion through a membrane by centrifugal force. In this method, M-NVs generated by extrusion through 10 and 5μm or 10 and 8μm do not require the use of 1 μm membrane [11, 28, 105, 113]. Once M-NVs have been generated by any of the above methods, cell debris and extravesicular biomolecules have to be removed before downstream use. M-NVs can be further purified and enriched using OptiPrep purification [78, 99, 103, 104] or size exclusion chromatography [105]. It has been shown that M-NV uptake is mediated by clathrin and caveolae mediated endocytosis. Inhibition of these pathways interfered with the functionality and uptake of M-NVs in recipient cells suggesting a similar potential route of uptake as exosomes [78].

Extracellular vesicles mimetic nano vesicles as therapeutic modalities As mentioned previously, the idea of generating M-NVs is to overcome some of the challenges associated with exosome therapeutic applications. The choice of the cell line is important for therapeutic application. EVs derived from MSCs have been shown to maintain pro-regenerative and immunomodulatory functions.

M-NVs derived from different cell types can also preserve the

therapeutic function of parental cells and therefore naïve M-NVs can be directly used for therapeutic application. For example, M-NVs generated from pancreatic β-cell (MIN6) can induce therapeutic function without any cargo modification [103]. MIN6 cells were extruded five times through 10, 5 and 1 μm and M-NVs further purified using Optiprep gradient fractionation. Following M-NV injection, the BM cells implanted in mice differentiated into insulin producing cells, demonstrating the regenerative potential of M-NVs. This finding indicated that the M-NVs contain functional cargo necessary for inducing therapeutic function upon uptake [103]. Furthermore, a similar methodology was used for the generation of M-NVs from primary hepatocyte cells [112]. M-NVs intravenously injected into the mice model of partial hepatectomy (PH), showed promising hepatocyte proliferation and liver regeneration. Furthermore, to investigate whether the M-NVs are capable of transferring active biomolecules from parental cells the Sphk2 level was checked. Sphk2 has previously been shown to be enriched in exosomes and is capable of liver regeneration [114]. Similarly, M-NVs was enriched in Sphk2 and following administration, the Sphk2 level increased in both normal and PH, indicating that M-NVs cargo can be functional upon intake by recipient cells [112].

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M-NVs were generated from Natural Killer (NK) cells by extrusion through 5 and 1 μm polycarbonate membrane and were shown to exhibit potent antitumor activity in a xenograft glioblastoma mouse model. Furthermore, the conserved and active anti-tumor function of the NK cells was demonstrated by the presence of high levels of perforin and FasL in M-NVs demonstrating innate packaging of acellular therapeutic factor [115]. Anti-inflammatory effects of M-NVs was demonstrated using intraperitoneal injection into a mouse model of sepsis (induced by bacterial outer membrane vesicles). The M-NVs therapeutic function was delivered through the upregulation of an anti-inflammatory IL-10 [111]. Therefore, similar to exosomes, the anti-inflammatory effect of MSCs can be preserved in M-NVs. Regenerative capabilities of M-NVs directly generated from MSCs have been demonstrated recently for mediating skin wound closure. In this study, M-NVs were generated by extrusion through 10 and 5 μm membranes and injections of 50 µg of M-NVs into the mouse resulted in skin regeneration seven days post treatment [116].

Collectively, these findings highlight that although M-NVs are artificially made from cells, they can preserve the contents of the parental cells. M-NVs can deliver these potentially therapeutic contents in the form of nanovesicles without the need for cargo modification. There are three possible routes for modifying M-NV cargo: 1) Engineering parental cells for passive cargo loading into M-NVs; 2) exogenous loading following M-NVs generation or; 3) loading during the extrusion process.

Modification of parental cells to load cargo into mimetic-nanovesicles Since M-NVs are artificially generated from cells, any modification to the parental cell can directly reflect the M-NVs cargo composition. This advantage can be useful when a high load of therapeutics are required to be packaged into cell derived nanovesicles. Endogenous loading of M-NVs using parental cell engineering was explored using NIH3T3 cells transduced with shRNA against human MYC and generated by extrusion. To assess the endogenous loading and M-NV uptake, M-NVs were further labelled with PKH26 and added to λ820 cells expressing human c-Myc. Following treatment, c-Myc expression was reduced at both the mRNA and protein level suggesting efficient endogenous

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loading of siRNA into M-NVs which were functional upon uptake by the recipient cell [78]. Furthermore, using the parental cell modification method, long non-coding RNA (LncRNA) was also successfully packaged into M-NVs. LncRNA-H19 was overexpressed in HEK293 and M-NVs generated by extrusion through 10, 5 and 1 μm membranes. LncRNA-H19 loaded M-NVs demonstrated successful wound healing in the treatment of diabetic wounds [100]. M-NVs generated from cells manipulated with iron oxide nanoparticles (IONP) showed successful parental cell to M-NVs passive cargo engineering. In this study, M-NVs were generated by extrusion through 10, 5, 1 μm and 400 nm pore size membranes, five times each. EM revealed the IONP was incorporated into M-NVs and using external magnetic force (guidance), M-NVs accumulated at the site of injury and enhanced spinal cord function by angiogenic, anti-apoptotic and anti-inflammatory properties. [110].

Exogenous loading of siRNA into mimetic-nanovesicles M-NVs were exogenously loaded with siRNA with 2% loading efficiency of siRNA [78]. In this study, electroporation caused no significant changes in M-NVs structure and siRNA aggregates were removed further by Optiprep density gradient fractionation. Exogenously siRNA loaded M-NVs were able to efficiently downregulate GFP mRNA and protein expression which highlights the M-NV uptake and functionality in recipient cells [78].

Loading therapeutic into mimetic-nanovesicles during the extrusion process Unlike exosomes, M-NVs can be loaded with therapeutics during their generation process. Once cells are harvested, they can be mixed with chemical drugs or biological molecules without any incubation and subjected to extrusion. As cells are fragmented by extrusion, the therapeutic molecules in the buffer can be encapsulated by lipid membranes during nanovesicle formation. This system can potentially increase the packaging of therapeutics into vesicles without any changes in parental cell biology or MNVs cargo interruption post isolation. Furthermore, this method can save time and effort required for endogenously or exogenously loading of cargo. The first study on M-NVs utilised loading via the extrusion process where cells were mixed with Dox and the resuspension was extruded. Crude M-NVs were further purified from Optiprep density gradients and Dox packaged into M-NVs was correlated to

20

Dox concentration in suspension. Intravenous Injection of 10 μg of Dox loaded M-NVs into mice demonstrated a significant decrease in tumor growth [104]. Paclitaxel, another chemotherapeutic drug, was also packaged into M-NVs during extrusion. Researchers found that by increasing drug concentration, the size of generated vesicles increased and the best loading efficiency was achieved using 50 μg of paclitaxel [109]. Taken together, the extrusion method has great potential in packaging therapeutics into M-NVs.

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Conclusion Although the EV field is new and growing, based on the current literature, EVs have shown promising therapeutic potential for many diseases. EVs can be directly isolated and used as a therapeutic system or they can be modified endogenously or exogenously to improve downstream applications. There are challenges associated with therapeutic use of EVs. These include the limiting number of EVs released by cells, heterogeneity and isolation/purification difficulty. In recent years, artificial EVs have been developed to overcome some of these challenges. Among the artificial EVs, M-NVs demonstrated comparable biophysical characteristic and therapeutic potential to exosomes. To maximise the therapeutic efficiency and specific tissue targeting, both EVs and artificial EVs can be engineered. Furthermore, the reproducibility of EV engineering is crucial for safer drug/gene delivery, especially if the therapeutically loaded EVs need to be modified before clinical application. Recent developments in the field have overcome some of the misinterpretation regarding the EV cargo loading efficiency (ratio of therapeutic in EVs versus in solution). However, further studies are required to understand cargo loading, exact mechanism of uptake, cargo release and fate of vesicles in recipient tissue. Using EVs as disease therapeutics requires a series of essential standardization steps in the protocol from large scale isolation, purification, modification, storage, method for administration and assessing long-time safety in the host.

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Table 1. List of studies used top-down method for generation of mimetic-nanovesicles

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Precursor cell U937 and Raw264.7

Application/disease

Generation method

Outcome

Methodology and Cancer

NIH/3T3 and MIN6

Diabetes mellitus

Extrusion by mini-extruder (Three times through 10, 5, and 1 μm) Extrusion by mini-extruder (Five times through 10, 5, and 1 μm)

U937 and NIH/3T3

Cancer

HEK293

Diabetic wounds

MSCs

Cancer

Raw264.7

Noninvasive imaging and biodistribution

Extrusion by mini-extruder (Three times through 10, 5, and 1 μm)

Raw264.7

Cancer

Mice primary hepatocytes cell MSCs

Liver regeneration

Extrusion by mini-extruder (Eleven times through 1 μm, 400 and 200 nm) Extrusion by mini-extruder (Five times through 10, 5, and 1 μm)

Target delivery of chemotherapeutics to malignant tumors [104] Differentiation of therapeutic insulinproducing cells from bone marrow for diabetes mellitus [103] RNAi delivery for targeting c-Myc in cancer [78] Transport LncRNA-H19 as competing endogenous RNA [100] Anti-cancer effect against breast cancer [109] Noninvasive imaging 99m using Tc-HMPAO [108] Antitumor efficacy [117]

NK92-MI

Immunotherapy

MSCs

Sepsis

MSCs

Wound healing

SH-SY5Y

BM-DCs

Proteomic characterization and comparison with exosomes Methodology

ESD3

Focus on methodology

ESCs

Regenerative medicine

Spinal cord injury

Extrusion by mini-extruder (Three times through 10, 5, and 1 μm) Extrusion by mini-extruder (Three times through 10, 5, and 1 μm) Extrusion by mini-extruder (10, 5, and 1 μm)

Extrusion by mini-extruder (Five times through 10, 5, and 1 μm and 400 nm) Extrusion by mini-extruder (Eight times through 5 and 1 μm) Extrusion by mini-extruder (Five times through 10, 5, and 1 μm) Extrusion by mini-extruder (Five times through 10 and 5 μm) Extrusion by mini-extruder (Thirteen times through 10, 5, and 1 μm) Extrusion through centrifugal force (Through 10 and 5 μm) Extrusion through centrifugal force (Three times through 10 and 5μm) Extrusion through

Promote hepatocytes proliferation in vitro and liver regeneration in vivo [112] Repairing injured spinal cord [110] Immunotherapeutic agent for treatment of cancer [115] May be clinically applicable to septic patient [111] Skin regenerative treatment [116] Composition of vesicles and potential as delivery system [99] Cancer treatment [28] Drug delivery [113]

Regenerative medicine

24

U937

Characterization and in vivo biodistribution

ESD3

Focus on methodology

ESD3

Focus on methodology

centrifugal force (Through 10 and 5 μm) Extrusion through centrifugal force (Through 10 and 8 μm) Microfluidic fabrication (force cells through hydrophilic microchannels) Microfluidic (cell slicing by silicon Nitride blades)

[11] Colocalization of vesicles at the tumor site indicate the anti-cancer strategy [105] Drug and RNA delivery system [106] Exogenous material delivery [107]

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FIGURE LEGENDS Figure 1. Schematic representation of exosomes biogenesis and uptake mechanism in intercellular communication. Exosome biogenesis is initiated upon endocytosis which forms the early endosome (EE) by inward budding of the plasma membrane. Depending on the machinery involved, the early endosome is either recycled to the plasma membrane or forms the late endosome/multivesicular bodies (MVB). Invagination of the late endosome membrane leads to the formation of ILVs within MVB. At this stage in addition to membrane proteins on the surface of ILVs originating from the plasma membrane and EE, other cargo can be specifically sorted via the TGN into EE or MVBs. The fate of the MVB is either to undergo degradation by autophagosome and lysosomes or to dock and fuse to the plasma membrane via several proteins such as actin, RAB and SNARE. The latter results in the release of exosomes into the extracellular environment. The important mechanisms involved in target and interaction of exosomes with recipient cells is found to be via exosomal cargo and the presence of distinct protein within surface membrane. This ultimately mediates intercellular communication and subsequently reprograms the behavior of the recipient cell. There are several possibilities whereby exosomes can interact with a recipient cell. Exosomes can fuse with the membrane of a recipient cell and release cargo into the cytoplasm or taken up by the assistance of surface binding signaling such as integrin. Alternatively, exosomes can undergo phagocytosis, micropinocytosis or endocytosis mediated mechanisms such as lipid raft and caveolae. Also, clathrin dependent mechanism could be associated with exosome internalization once the exosomes interacts with the cell membrane surface. Thus, the exact mechanism by which exosomes interact with recipient cells depends on the cascade of cellular signaling in donor and recipient cells. Figure 2. Simplified illustration of methods for exogenous loading of therapeutics into extracellular vesicles.Various method including incubation, electroporation, sonication, extrusion, hypotonic dialysis, Freeze-thaw cycles, saponin, CaCl2 and lipofectamine reagent Figure 3. Schematic representation of mini-extruder application for generating nano drug delivery system. (A-E) represent different precursors used for generating nanovesicles by extruding through polycarbonate membrane using mini-extruder Figure 4. Schematic representation of mimetic-nanovesicles generation. For M-NVs generation, cells have to be harvested and diluted in suitable buffer. Cells in suspension are extruded sequentially through polycarbonate membrane with diminishing pore size using a miniextruder to produce <200 nm nanovesicles. Crude nanovesicles can be further purified by Optiprep density gradient or size exclusion chromatography [99, 105, 108].

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Highlights:   

Description of methods for generating mimetic extracellular vesicles for potential therapeutic uses Methods described for loading extracellular vesicles with functional cargo Discussion of current uses of mimetic extracellular vesicles

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