Extracellular vesicles as a next-generation drug delivery platform.

1 Review Manuscript 1 2 Extracellular vesicles as a next-generation drug delivery platform 3 4 Inge Katrin Herrmann1,2*, Matthew John Andrew Wood3 and Gregor Fuhrmann4,5,6* 5 1Nanoparticle Systems Engineering Laboratory, Institute of Energy and Process Engineering, 6 Department of Mechanical and Process Engineering, ETH Zurich, Sonneggstrasse 3, CH-8092 Zurich, 7 Switzerland. 8 2Particles Biology Interactions, Department Materials Meet Life, Swiss Federal Laboratories for 9 Materials Science and Technology (Empa), Lerchenfeldstrasse 5, CH-9014 St. Gallen, Switzerland. 10 3Department of Paediatrics and Oxford Harrington Rare Disease Centre, University of Oxford, OX1 11 3QX, United Kingdom. 12 4Helmholtz Centre for Infection Research (HZI), Biogenic Nanotherapeutics Group (BION), 13 Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus E8.1, Saarbrücken 66123, 14 Germany 15 5Department of Pharmacy, Saarland University, Campus E8.1, Saarbrücken 66123, Germany 16 6Present address: Chair for Pharmaceutical Biology, Department of Biology, Friedrich-Alexander-17 University Erlangen Nuremberg, 91058 Erlangen, Germany 18 19 *Corresponding authors: 20 IKH: phone: +41 58 765 7153, email: [email protected] 21 GF: phone: +49 68198806 1500, email: [email protected] 22 23 24 ORCID iDs: Inge K. Herrmann: 0000-0002-3018-6796; Matthew Wood: 0000-0002-5436-6011; 25 Gregor Fuhrmann: 0000-0002-6688-5126 26 27 28 29 2 Abstract 30 Extracellular vesicle (EV)-based cell-to-cell communication is conserved across all kingdoms of life. 31 There is compelling evidence that EVs are involved in major (patho)physiological processes, 32 including cellular homeostasis, infection propagation, cancer development, and cardiovascular 33 diseases. Various studies suggest that EVs have several advantages over conventional synthetic 34 carriers, opening new frontiers for modern drug delivery. Despite extensive research, clinical 35 translation of EV-based therapies remains challenging. Here, we discuss the uniqueness of EVs along 36 with critical design and development steps required to exploit their full potential as drug carriers, 37 including loading methods, in-depth characterisation, and large-scale manufacturing. We compare the 38 prospects of EVs to those of well-established liposomes and provide guidelines to direct the process of 39 developing vesicle-based drug delivery systems. 40 41 3 As the field of targeted drug delivery has expanded, nanotechnology has contributed substantially to 42 the development of smart carriers in recent decades.1 In particular, lipid-based nanocarriers offer a 43 versatile platform for drug encapsulation, which has led to clinical translation of several formulations. 44 In addition to synthetic nanocarriers, cell-derived extracellular vesicle (EV)-based carrier systems 45 have attracted considerable interest.2 46 EVs are a heterogeneous group of small, lipid-bound nanoparticles acting as key mediators of many 47 (patho)physiological processes.3 They are also being explored for the delivery of therapeutic payloads 48 to specific cells or tissues, harnessing their intrinsic tissue homing capabilities.4 From a drug delivery 49 perspective, EVs are comparable to liposomes, given that both are phospholipid-based. However, EVs 50 are assembled from a complex mixture of various lipids and surface and membrane proteins; some of 51 these components aid tissue targeting, while others ensure minimal non-specific interactions.5,6 These 52 unique protein-decorated phospholipid vesicles have been postulated to contain the specific barcodes 53 needed to find their target both locally and at distant sites. Despite extensive research, the superiority 54 of EV-based drug delivery over delivery via engineered nanocarriers, such as liposomes, and the 55 associated risk-benefit ratio remain matter of debate.7 56 Here, we critically discuss the prospects of EVs as drug delivery vehicles and as next-generation 57 therapeutics. We outline the advantages of EVs over standard delivery methods, discuss current 58 obstacles related to their clinical and industrial translation and highlight synergies with other emerging 59 fields, such as cell therapeutics (EVs are sometimes considered ‘cell-free cell therapeutics’). We also 60 propose a colour code guideline regarding experimental requirements and scientific needs to facilitate 61 the development of EVs as drug carriers to evaluate their delivery efficacy and allow benchmarking 62 against alternatives. 63 64 Uniqueness of extracellular vesicles biology and function 65 Composition of EVs. EV secretion appears to be an evolutionarily conserved process present 66 throughout all kingdoms of life.8 Regarding fundamental biology, EV research focuses on 67 understanding the biogenesis and release of these natural carriers and their fate upon interaction with 68 4 target cells. This also comprise the genotypic and phenotypic responses that EVs induce and the 69 mechanisms by which EVs mediate cell-to-cell communication.5,9 70 Several subtypes of EVs, including exosomes, ectosomes, microvesicles, membrane vesicles, and 71 apoptotic bodies, have been identified.10 These EVs have been isolated from various sources, 72 including mammalian and prokaryotic cell cultures, blood plasma, bovine milk, and plants.8 Each EV 73 subpopulation may be derived via distinct biogenesis pathways, and because their precise biogenic 74 origin is impossible to ascertain in most cases, a comprehensive characterisation of the vesicles is 75 crucial. In addition, different EV formulations may have substantially different size distributions; thus, 76 standardised characterisation is challenging.11 The general recommendation in the field is to use ‘EVs’ 77 as a general term. Importantly, the general concept of ‘the EV’ does not exist—currently, the term 78 ‘EV’ comprises a heterogeneous population, as indicated in the Minimal Information for Studies of 79 Extracellular Vesicles (MISEV) guidelines.12 Proteomic evidence suggests that an EV core protein 80 signature (e.g., CD63, CD9, or CD81) of highly expressed vesicular proteins is commonly shared 81 between EVs of diverse parent cell origins.13 Various tetraspanins are commonly used as molecular 82 markers of EVs. In contrast to the previous MISEV guidelines, there are no typical EV markers that 83 need to be identified on EVs, but careful discrimination of EVs from contaminants, such as protein 84 aggregates and viruses, is important. To add an additional layer of complexity, vesicles still carry 85 parent cell-specific signatures, which are crucial components permitting target cell interactions in 86 distinctly different manners.14 In addition to the core signature of highly expressed and highly enriched 87 vesicular proteins, other typically low-abundance and less-enriched protein components are present; 88 these proteins reflect the specific parent cell origin of the EVs and may also vary depending on the 89 nature and biogenesis of different EV subpopulations.15 From a drug delivery perspective, this 90 complexity needs to be understood via comprehensive (multi)omics studies16 and addressed in all 91 characterisation and production processes (Fig. 1). Here, we elucidate the assembly of EVs on the 92 cellular/molecular level and their mediation of selective intercellular signalling activities to extend 93 biomedical research.17 94 Uptake and biological role of EVs Under physiological conditions, EVs are signal carriers involved in 95 the homeostasis of several processes and of events during cell development, e.g., cell differentiation.18 96 5 EV-mediated cross-talk may occur unidirectionally or reciprocally, i.e., one cell sends information to 97 another with or without reciprocal signal transmission from the recipient cell, respectively, or even via 98 systemic communication, during which EVs traffic to various tissues and organs. This interaction may 99 involve not only the release and delivery of EV cargo but also cell surface interactions and target cell 100 modulation, such as immune cell activation by major histocompatibility complex-peptide interactions. 101 The mechanisms by which EVs are taken up by their target cells are still poorly understood, and 102 examples from the literature are often specific for a certain type of vesicle.5 Currently known cellular 103 entry routes of EVs range from receptor-mediated endocytosis, lipid raft interactions, clathrin 104 interactions, phagocytosis, macropinocytosis, and possibly direct fusion.9 Like many other 105 nanocarriers, EVs are taken up into endosomes need to escape the endosome to release their cargo into 106 the cytosol. Endosomal escape is associated with degradation in acidic compartments of the lysosomal 107 pathway, which could impair the integrity of EV cargoes.19 Although EVs were initially postulated to 108 be an unprecedented route for direct cell membrane fusion and cytosolic delivery,20 vesicle uptake has 109 been confirmed to be a very complex mechanism, which requires more in-depth evaluation exploring 110 subcellular analyses based on high-resolution microscopy or novel live-cell reporters.21 On the other 111 hand, the biological effects induced by EVs are currently well known. During oncogenesis, tumour 112 cells increase their yield of EVs, allowing not only the modulation of surrounding healthy cells, 113 immune cell dysregulation, and tumour proliferation but also communication with distant tissues, e.g., 114 during angiogenesis.22 Glioblastoma cells were shown to secrete EVs capable of immunosuppression 115 by blocking T cell activation and receptor stimulation.23 Moreover, widely used cytotoxic drugs, such 116 as taxanes, may also induce shedding of EVs with pro-metastatic properties.24 Although the role of 117 EVs in tumour biology has been investigated extensively, the development of new tools for treatment 118 and diagnostics is still hampered by the absence of tumour-specific EV markers. 119 A comparable modulatory role of EVs has been observed in the progression of resistance to infections. 120 In the context of viral infections, some EVs may carry viral proteins from infected cells and follow 121 comparable biogenesis pathways.25 Furthermore, bacteria utilise EVs for the transmission of resistance 122 genes and virulence factors,26 which has sparked interest in the development of bacterial vesicles for 123 vaccination applications.27 Bacterial EVs from non-pathogenic or probiotic bacterial sources may also 124 6 be harnessed as potential EV-based delivery carriers, and their production may be readily scalable by 125 cultivation of EV-producing bacteria in small fermenters.28,29 This is a promising avenue for the 126 manufacturing of EVs with novel functionalities and in conjunction with biomaterials.30,31 However, 127 immunogenicity requires more detailed evaluation for bacterial vesicles than for mammalian EVs 128 owing to the potential presence of lipopolysaccharides, as recently discussed in detail.32 129 130 Extracellular vesicle-based drug carriers 131 Development. With the development of new analytical tool, it has been found that many previously 132 applied isolation techniques are not specific for EVs and lead to the inclusion of contaminants. 133 Methods are constantly refined, but they often expose the limitations in the field, making it difficult 134 for new researchers to follow progress in the state-of-the-art methods. For every drug nanocarrier, a 135 comprehensive physico-chemical characterisation and its interactions in biological environments must 136 be investigated for therapeutic development. While liposomes have been extensively evaluated for 137 efficacy and biocompatibility both in vitro and in vivo, methodologies well adapted to the considerably 138 more complex EVs are lacking. These natural vesicles are assembled and packaged in a cell-specific 139 manner, e.g., cancer-derived EVs, carry molecular information distinct from that carried by stem cell140 or blood cell-derived EVs. While challenging from the perspective of drug carrier development, these 141 properties make EVs a promising biomarker for liquid biopsies in several applications.33 142 In regenerative medicine, EVs derived from mesenchymal stem cells (MSCs) are already under 143 clinical assessment34 for future use in nanodelivery (Table 1). Stem cell-derived EVs can induce 144 immune cells to undergo modulation from an activated inflammatory state to a tolerant regulatory 145 state. Some of the strategies used to stimulate EV shedding and enhance yield can also be used for 146 MSCs. N-methyldopamine and norepinephrine induced an increase in MSC-derived EV production 147 without altering their modulatory capacity.35 Other approaches apply physical stimuli such as pH 148 variations or low oxygen conditions, but their long-term effect on the physiological properties of EVs 149 needs to be evaluated. In a murine wound healing model, MSC-EVs were associated with secretion of 150 an interleukin-1 receptor antagonist and induced rapid gingival healing.36 Comparable effects have 151 been shown for systemic application of MSC-EVs in patient trials, which has unfortunately led to the 152 7 use of ‘exosome’ products in unapproved applications. The Food and Drug Administration recently 153 stated that serious adverse effects were experienced by patients in Nebraska treated with unapproved 154 products marketed as containing exosomes37. The agency emphasised that there are currently no 155 regulatory approved EV products and that some clinics “deceive patients with unsubstantiated claims 156 about the potential for these products to prevent, treat or cure various diseases or conditions”. 157 Importantly, any therapeutic application of EVs requires transparent reporting of data on vesicle 158 manufacturing and characterisation, suitable quality control provisions, preclinical safety, and 159 efficacy.38 Moreover, a rational clinical trial design and regulatory monitoring are important to ensure 160 patient safety, as recently indicated by the international societies on stem cells and EVs.39 To support 161 the use of MSC-EVs, functional assays that allow in vitro-in vivo correlation of the therapeutic 162 potency of different stem cell preparations must be developed.40 Despite these caveats, ongoing efforts 163 to produce EVs from MSCs under good manufacturing process-like conditions41,42 and to design 164 upscaling approaches43 will be instrumental for their development as drug carriers. 165 8 Table 1. Ongoing clinical trials with extracellular vesicles 166 # Name* Status Condition Type of extracellular vesicles§ Location NCT number Stem cell-derived extracellular vesicles 1 A Clinical Study of Mesenchymal Progenitor Cell Exosomes Nebulizer for the Treatment of Pulmonary Infections Recruiting; Phase 1/2 Drug-resistant infections Mesenchymal stem/progenitor cell-derived exosomes Shanghai, China NCT04544215 2 Effect of Microvesicles and Exosomes Therapy on β-Cell Mass in Type I Diabetes Mellitus (T1DM) Unknown status Diabetes mellitus type 1 Mesenchymal stem cellderived exosomes Sahel, Egypt NCT04213248 3 Evaluation of Safety and Efficiency of Exosome Inhalation in SARSCoV-2 Associated Pneumonia. Enrolling by invitation; Phase 1/2 SARS-CoV-2 pneumonia Mesenchymal stem cellderived exosomes Samara, Russia NCT04213248 4 A Pilot Clinical Study on Inhalation of Mesenchymal Stem Cells Exosomes for the Treatment of Severe Novel Coronavirus Pneumonia Completed; Phase 1 SARS-CoV-2 pneumonia Mesenchymal stem cellderived exosomes Shanghai, China NCT04213248 5 Safety and Efficiency of Method of Exosome Inhalation in COVID-19 Associated Pneumonia Enrolling by invitation; Phase 2 SARS-CoV-2 pneumonia Mesenchymal stem cellderived exosomes Samara, Russia NCT04602442 6 Effect of UMSCs Derived Exosomes on Dry Eye in Patients With cGVHD Recruiting; Phase 1/2 Dry eye Umbilical mesenchymal stem cellderived exosomes Guangzhou, China NCT04213248 7 MSC-Exos Promote Healing of MHs Recruiting; early Phase 1 Macular holes Mesenchymal stem cellderived exosomes Tianjin, China NCT04213248 8 A Tolerance Clinical Study on Aerosol Inhalation of Mesenchymal Stem Cells Exosomes in Healthy Volunteers Recruiting; Phase 1 Safety and tolerance studies Mesenchymal stem cellderived exosomes Shanghai, China NCT04213248 9 Allogenic Mesenchymal Stem Cell Derived Exosome in Patients with Acute Ischemic Stroke Completed; Phase 1/2 Cerebrovascul ar disorders Mesenchymal stromal cellderived exosomes Tehran, Iran NCT03384433 10 Evaluation of Adipose Derived Stem Cells Exosomes in Treatment of Periodontitis Recruiting; early Phase 1 Periodontitis Adipose-derived stem cellderived exosomes Cairo, Egypt NCT04213248 Allogenic and autologous extracellular vesicles 9 Table 1. Continued 167 # Name* Status Condition Type of extracellular vesicles§ Location NCT number 11 Safety and Efficacy Evaluation of Allogenic Adipose MSC-Exosomes in Patients with Alzheimer's Disease Recruiting; Phase 1/2 Alzheimer’s disease Allogenic adipose mesenchymal stem cellderived exosomes Shanghai, China NCT04213248 12 A Clinical Study of Mesenchymal Stem Cell Exosomes Nebulizer for the Treatment of ARDS Not yet recruiting; Phase 1/2 Acute respiratory distress syndrome Allogeneic human mesenchymal stem cellderived exosomes Ruijin, China NCT04602104 13 MSC extracellular vesicles in Dystrophic Epidermolysis Bullosa Not yet recruiting; Phase 1 Dystrophic epidermolysis bullosa Allogeneic mesenchymal stem cellderived extracellular vesicles Aegle Therapeutics NCT04173650 14 Effect of Plasma Derived Exosomes on Cutaneous Wound Healing Enrolling by invitation; early Phase 1 Ulcer Autologous exosome-rich plasma Kumamoto, Japan NCT04213248 Other cells or extracellular vesicle sources 15 COVID-19 Specific T Cell Derived Exosomes Active; Phase 1 SARS-CoV-2 pneumonia T cell-derived exosomes Kayseri, Turkey NCT04213248 16 Extracellular Vesicle Infusion Therapy for Severe COVID-19 Not yet recruiting; Phase 2 SARS-CoV-2 pneumonia, acute respiratory distress syndrome Bone marrow derived extracellular vesicles Direct Biologics NCT04493242 17 Edible Plant Exosome Ability to Prevent Oral Mucositis Associated with Chemoradiation Treatment of Head and Neck Cancer Active; Phase 1 Head and neck cancer, oral mucositis Grape exosomes and fentanyl patch Louisville, USA NCT04213248 Drug loaded extracellular vesicles 18 iExosomes in Treating Participants with Metastatic Pancreas Cancer with KrasG12D Mutation Not yet recruiting; Phase 1 Metastatic pancreatic adenocarcino ma, pancreatic ductal adenocarcino ma Mesenchymal stromal cellderived exosomes loaded with siRNA against KrasG12D Houston, USA NCT04213248 19 Study Investigating the Ability of Plant Exosomes to Deliver Curcumin to Normal and Colon Cancer Tissue Active; Phase 1 Colon cancer Plant exosomes loaded with curcumin Louisville, USA NCT04213248 20 Trial of a Vaccination with Tumor Antigen-loaded Dendritic Cellderived Exosomes Completed; Phase 2 Non-small cell lung cancer Dendritic cellderived exosomes loaded with antigen Villejuif, France NCT04213248 *Trials currently listed on clinicaltrials.gov; for consistency, trial names are indicated as listed on clinicaltrials.gov. 168 §The type and/or source of extracellular vesicles are indicated as given by the study sponsor and were not verified regarding 169 suitable characterisation (e.g., exosomes and microvesicles). 170 16 homing properties of EVs and on potential immunogenic and oncogenic considerations. Additionally, 344 genetic stability, host cell impurities (such as pathogens, and especially viruses) and EV yields play a 345 major role in the parent cell selection process.81 Once selected, these host cells are cultured to 346 efficiently yield EVs with the appropriate phenotype. Suggested methods include multi-layered culture 347 flasks, bioreactors, and hollow fibre cartridges. For small-scale manufacturing, cells can be expanded 348 in shake flasks, spinners, roller bottles, wave bags, or bioreactors. For large-scale cell culture, cells can 349 be grown in stainless steel bioreactors (up to 20,000 L scale), platform-rocker wave bags (up to 500 L 350 scale), or even in disposable bioreactors (up to 2,000 L scale).81 From a process safety perspective, 351 closed systems are preferred; however, these systems are more difficult to monitor than open 352 systems.42 Genetic drift and contamination need to be monitored closely, following protocols used in 353 the plants producing biologicals and cell therapies. Accumulating evidence indicates that bovine milk-354 derived EVs may be a valid alternative source for obtaining large amounts of biocompatible vesicles. 355 Although feasibility studies using milk EVs as drug carriers are underway,82 large-scale isolation of 356 pure vesicles from complex milk stills needs optimisation.62 357 Optional endogenous drug loading. Parent cells may be engineered to boost EV production and/or 358 yield EVs with enhanced properties. Several methods to load EVs with different molecules have been 359 experimentally evaluated.83 These methods include (i) endogenous techniques in which the EV-360 producing cells also equip vesicles with drug cargo or modified structural protein/RNA components or 361 (ii) exogenous approaches in which drugs are loaded into EVs post isolation. The endogenous loading 362 technique has a lower degree of complexity if the producer cells directly shed EVs containing the 363 desired molecule.84 Endogenous loading approaches have also been used for the encapsulation of 364 nanoparticle-based drugs into EVs.85 Because the loading efficiency is typically limited, cells may be 365 genetically engineered to produce EVs containing the desired active components. However, this 366 approach is limited to biologically accessible drugs, such as nucleic acid and protein drugs. Recently, 367 an optogenetically engineered exosome system that integrates a blue-light responsive membrane 368 module for controllable protein–protein interactions to encapsulate large quantities of anti-369 inflammatory proteins into EVs was presented.86 While highly interesting, genetic engineering of EV-370 producing cells results in more complex production upscaling than isolation of vesicles from naïve 371 cells and is less elaborate than post-isolation engineering. 372 17 373 EV Harvesting and Engineering 374 Product separation and characterisation of prepared EVs. For EV isolation, the primary separation of 375 products from cells can be accomplished by well-established procedures for the isolation of biologics, 376 including centrifugation, depth filtration (mechanical sieving and adsorption) or tangential flow (cross 377 flow) filtration . While several strategies for EV isolation and purification, such as differential 378 ultracentrifugation (dUC), precipitation, size exclusion chromatography , affinity chromatography, and 379 tangential flow filtration, have been evaluated, there is no consensus on an appropriate EV isolation 380 technique for large-scale manufacturing.87 This lack of consensus is mostly because some procedures 381 may negatively affect the integrity and quality of EVs. Additionally, yield and product purity vary 382 among methods and reports, and the overall EV purity generally appears to be low.88 Moreover, for 383 heterogeneous EV populations, the isolation method may lead to selective isolation of one specific 384 subpopulation with higher or lower biological activity than the total population. Density gradient 385 centrifugation and dUC offer a decent compromise in terms of yield and purity. dUC is broadly 386 accepted as a common method to obtain ‘pure’ EVs, especially when using chemically defined media 387 without foetal bovine serum. However, careful evaluation of artefacts introduced by the culture 388 medium is pivotal, as it was recently shown that co-precipitation of nucleic acid impurities is possible 389 even under these conditions.89 In fact, recent studies suggest that ultrafiltration and size exclusion 390 chromatography (alone or in combination) outperform dUC in terms of both yield and purity.90,91 391 However, depending on the required EV purity, orthogonal methods that target different EV 392 characteristics may need to be integrated at the expense of yield.89 Another intrinsic problem arises 393 because most of the currently used purification techniques were originally developed for the 394 purification of viruses,42,92 which are amongst the most critical potential contaminants of EVs. 395 Recently, Barone et al. comprehensively analysed the risks, costs, and implications of viral 396 contamination in biological manufacturing.93 Virus risk mitigation strategies for general 397 biopharmaceutical manufacturing are based on three tenets: (i) prevention of viral entry by selecting 398 low-risk starting and raw materials and using manufacturing controls; (ii) testing of in-process 399 materials to ensure they are free of virus and enable lot rejection; and (iii) clearance of viral 400 contaminants (via inactivation and/or removal) from the product. While both careful selection of cells 401 18 and raw materials and testing (based on polymerase chain reaction or in vitro virus assays) can reduce 402 risk, the unique size of EVs makes the inactivation and/or removal of viral contaminants even more 403 challenging for EVs than for other biopharmaceutical products. Current strategies are based on affinity 404 rather than size exclusion. Extensive multi-step downstream processing may, however, critically force 405 manufacturing costs to increase. In addition to the challenges associated with the complete 406 characterisation of contents (including contaminants), the spatial and conformational organisation of 407 the constituents is extremely challenging to assess with existing analytical techniques. Even small 408 changes in composition or architecture can significantly affect efficacy and safety. The use of 409 functional activity assays is of utmost importance because isolation methods and the presence of other 410 EV populations or contaminants may decisively influence product activity and off-target effects. 411 Exogenous drug loading. For clinical EV translation, reproducible and technologically accessible 412 methods are needed to load them with the desired drugs.94 While loading methods for liposomes have 413 been optimised and applied in industrial production, such settings for EVs are still lacking. Exogenous 414 loading methods work passively by the association of drugs with the lipid bilayer membrane after 415 incubation,83 by attaching therapeutics to the EV surface,95 and by mechanical or chemical techniques 416 to transiently open the EV membrane to allow diffusion of compounds into the vesicle. The most 417 common approaches to temporarily permeabilize the membrane include sonication, electroporation, 418 saponin treatment, and passive incubation.96 The advantages and disadvantages of each approach 419 depend on the experimental settings, the types of drugs and source of EVs, and they can be scaled 420 (Fig. 4c). Passive incubation is a very simple method in which purified EVs are incubated with drugs 421 to allow incorporation into the vesicle membrane. Many early loading protocols followed this method 422 because it exhibits excellent performance for incorporation of hydrophobic compounds such as 423 curcumin.97 However, the stability of drugs loaded by passive incorporation across the EV membrane 424 is still unclear. For hydrophilic compounds, loading may be enhanced by the addition of saponin, 425 which has been shown to be effective for large proteins.98 Saponins are mild surfactants that induce 426 transient membrane destabilisation and may also affect biomolecules; thus, careful purification is 427 needed when saponins are used on a large scale. Mechanical methods for permeabilising the EV 428 membrane, such as electroporation or sonication, have been shown to be successful for both small 429 molecules and macromolecules.50 Even though these methods can be scaled up, their potential 430 19 influence on protein and nucleic acid drugs requires careful consideration.99 In addition to concerns 431 with maintaining the stability of biomacromolecules, the size of the drugs poses another challenge 432 during EV-loading procedures. Large enzymes >200 kDa have been successfully loaded into EVs 433 using saponin pretreatment.98,100 As the size of nucleic acids that may be encapsulated exogenously 434 into EVs is also limited, a cell nanoporation method for large-scale production of functional EVs has 435 been developed.101 EV yield and mRNA loading were enhanced by this method; however, the required 436 additional steps of transfection and electrical stimulation render its industrial adoption relatively 437 difficult. 438 Recently, an alternative method based on liposome fusion has been proposed.102 Liposomes containing 439 fusogenic lipids were incubated with EVs, and the cargo of the synthetic liposomes was merged with 440 that from the EVs. Such an approach may pave the way for efficient loading of larger molecules 441 without compromising the EV membrane.95 442 443 Downstream 444 Purification. In addition to the EV isolation and purification steps prior to drug loading, additional 445 purification steps may be necessary to remove the free drug and exclude the potential contaminants 446 introduced during post-processing (Fig. 4b and d). Magnetic immunoaffinity purification has gained 447 increasing attention owing to the high purity of the obtained products. However, low yields are 448 generally obtained, although theoretical yields may be underestimated due to contaminants. 449 Quality control—(minimal) characterisation of engineered EVs. For routine manufacturing and 450 product monitoring, critical quality attributes need to be defined. Methods to assess these attributes 451 may include evaluations of parent cell properties (e.g., evaluation of viability and surface marker 452 expression to assess the phenotype), EV characteristics (e.g., evaluation of quantity, size, and surface 453 marker expression), assessment of microbial contamination (e.g., detection of endotoxin and 454 mycoplasma) and (application-specific) functional activity.42 For assessment of batch-to-batch 455 variations, we suggest the use of a concept similar to that applicable to biosimilars—the analytical 456 characteristics of the products should be highly similar to those of the reference product. While non-457 cell culture methods (such as isolation from plasma or milk) offers interesting alternatives to cell 458 culture and access to potentially large amounts of EVs, these sources contain EVs originating from 459 20 many cell types103 that cannot be separated easily. Therefore, characterisation of the functional activity 460 of EVs is even more crucial in quantifying on-target and off-target effects. 461 462 Formulation and shelf life. Recent studies have focused on the formulation and storage conditions 463 (e.g., –80 °C vs. 4 °C) of EVs by assessing the size, charge, and number of EVs, but strong 464 correlations have not been found.95,104 Storage at 4 °C has been shown to cause aggregation and 465 damage to the EV structure.105 Moreover, even though the size and number of EVs remained 466 unchanged at −80 °C, alterations in biological activity were detected.106 Lyophilisation has been 467 investigated as an alternative for long-term storage; however, its impact on vesicle integrity during 468 reconstitution depends on the use of cryoprotectants.105 Although storage at −80 °C is recommended, it 469 is logistically most challenging and costly method (Fig. 4e). 470 Safety by design and process de-risking. While a few years ago, the mammalian cell origin of EVs 471 was a major hurdle to their clinical translation, considerable advances have been made in cell-based 472 therapeutics. Regarding safety, EVs derived from autologous cells are associated with lower risks than 473 EVs derived from heterologous cells (including cell lines). However, the time needed to produce 474 autologous EVs is often incompatible with the time available for initiating treatment. During the time 475 required for the manufacturing and quality control of patient-specific EVs, the clinical condition of the 476 patient may worsen, making it impossible to administer the personalised product. Despite the several 477 examples of autologous products developed and commercialised by pharmaceutical companies, the 478 current frameworks seem to be predominantly suited for small-scale academic production rather than 479 for large-scale pharmaceutical production, and production costs may be prohibitive. While the use of 480 allogenic EVs appears generally feasible, the selection of parent cells, assessment of immunologic and 481 oncogenic effects, and risk of viral contamination need to be minimised by continuous monitoring. 482 Selection of assays for monitoring, particularly their sensitivity, is a key challenge in determining the 483 time required for clinical translation of EV-based drug carriers. Regulators have yet to release 484 guidance on how the safety and potency of these EVs should be tested. Currently, EVs are tested batch 485 by batch, with each laboratory and company using different assays.107 486 487 21 Perspectives 488 EVs may be used as carrier systems for various drug delivery applications. Compared to standard 489 delivery methods, EVs have been shown to deliver functional cargo with decreased immune clearance 490 when administered systemically to rodents. However, more evaluation in clinically relevant systems 491 and direct, quantitative comparison with liposome-based alternatives are required to comprehensively 492 assess the risk/benefit ratio.59 Successful translation of EVs depends on the availability of cost-493 effective large-scale production, isolation, and characterisation methods with high sensitivity to assess 494 batch-to-batch variations (and their biological consequences), and the availability of widely applicable 495 methods for loading drugs (Fig. 4f). The increasing availability of new analytical techniques is 496 expected to provide new insights into the uniqueness of EVs and may inspire the engineering of next-497 generation synthetic systems. The production of artificial EVs or EV mimics can overcome challenges 498 related to sterility, mass production and regulation. 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