Extracellular Vesicles — A Versatile Biomaterial
Authors: Juliane Nguyen, Gregor Fuhrmann
Journal: Advanced Healthcare Materials (2022)

In recent years, extracellular vesicles (EVs) have attracted a lot of interdisciplinary interest from biomedical, bioengineering, and biomaterials researchers. EVs are nanoscale, membrane-bound particles produced by all kingdoms of life including eukaryotes, bacteria, and fungi.[1-4] According to the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines, EV is the generic term for cell-derived particles enclosed by a lipid bilayer, regardless of the organism producing them. Due to their important physiological and pathophysiological roles, EVs have been studied as therapeutics, in drug delivery applications, and as novel diagnostics.[5-7] There are two subclasses of mammalian cell-derived EVs: microvesicles, which are produced by plasma membrane blebbing, and exosomes, which are produced by budding of the late endosomal membrane, which then fuses with the plasma membrane to release multiple small EVs at once. These two types of EV overlap in size, buoyant density, and surface marker expression, so their individual isolation has proven difficult. Bacteria form EVs by blebbing from the (outer) membrane, a mechanism found in all bacterial species studied to date.[8] Bacterial vesicles serve important functions including the delivery of virulence factors,[9] nutrient acquisition,[2] host cell modulation, and biofilm architecture.[10] To apply these natural carrier systems to biomedical applications, a few challenges need to be addressed, not least reproducible loading and large-scale characterization of heterogeneous EV preparations. In addition, a deeper understanding of EV-mediated cellular crosstalk is needed to inform robust in vitro models for mechanistic studies. The present themed issue aims to link recent developments in biomaterials, tissue culture, and model development with biological evaluations of EVs. We have compiled an exciting collection of articles that shine light on some of the ongoing challenges in using EVs as smart biomaterials. The composition and biological function of EVs are highly dependent on the conditions in which parent cells are cultured. Christopher Millan, Daniel Eberli, and co-workers (manuscript 2002067) assessed the impact of 3D engineered microtissues on EVs secreted by benign and malignant prostate cells and found that 3D-cultured cells produced EVs significantly different in function and composition to EVs isolated from 2D cultures, highlighting the value of engineered 3D microtissue cultures for EV studies. Similarly, in their systematic analysis, Jeremy P. Bost, Osama Saher, and colleagues showed how exogenous factors such as serum and signaling factors in the cell culture medium can affect EV production, secretion, and composition (manuscript 2101658). Both studies highlight the importance of external factors on EV composition and function. While the potential for EVs as delivery vehicles is widely recognized, their immunomodulatory functions have attracted particular attention over recent years. In their article, Mei He and colleagues discuss EV composition and surface receptor expression with an emphasis on how these parameters affect EV immunogenicity and their potential as cancer immunotherapeutics (manuscript 2100650). Looking into enhanced characterization methods, Paolo Arosio and co-workers (manuscript 2100021) focused on the problem of reproducible and efficient EV sizing and classification by introducing a microfluidic device that accurately determines vesicle concentration and composition. Their device was shown to simultaneously detect and distinguish EV subtypes and any non-vesicular impurities, factors which are extremely important to quantify when studying the biological effects of EVs. Jean-Christophe Leroux and co-workers present a highly relevant comparison of various loading methods for EVs—including saponin treatment, sonication, fusion, freeze-thawing, and osmotic shock—and assessed their influence on the biological activity (manuscript 2100047). They found that the loading efficacy of hydrophilic drugs varied between different loading methods and that not all techniques preserved the biological functionality of vesicles. Pieter Vader and co-workers (manuscript 2101202) engineered EVs as RNA drug carriers by hybridizing them with liposomes. Using this simple approach, the loading capacity of EVs was increased without compromising their biological functionality. Mesenchymal stem cell (MSC) EVs are known for their angiogenic effects. Steven M. Jay and colleagues increased the therapeutic efficacy of MSC EVs by loading them with long non-coding RNA HOX transcript antisense RNA (manuscript 2002070). They found that MSCs overexpressing HOTAIR produced HOTAIR-enriched EVs that promoted angiogenesis and wound healing in diabetic mice. Ramasamy Paulmurugan and colleagues created a nanococktail composed of polymeric nanocarriers coated in EVs for systemic miRNA delivery (manuscript 2101387). They showed that the nanococktail displayed tumor tropism and inhibited tumor growth. To combine vesicles with biomaterials, Junnan Tang, Xiaolin Cui, Jinying Zhang, and colleagues (manuscript 2100312) developed an injection-free approach for delivering EVs to treat myocardial infarction by entrapping them in a hydrogel network. They found that the EVs were slowly released from the network and improved cardiac function in mice treated with the EV-laden hydrogel. In a similar approach, Haifeng Liu, Yubo Fan, and co-workers used an injectable hydrogel to deliver EVs loaded with growth and transcription factors (manuscript 2100334). They showed that the hydrogel increased EV stability and significantly improved neovascularization and recovered limb function after critical limb ischemia. When using EVs as a delivery vehicle, effectively loading therapeutic cargo into EVs is key. Gordana Vunjak-Novakovic and colleagues (manuscript 2101557) discuss engineering approaches that utilize the endogenous cellular machinery for cargo loading into EVs as well as approaches that allow cargo loading through external manipulation of EVs. In their paper, Natalia Higuita-Castro, Daniel Gallego-Perez, and their team show how myeloid-derived suppressor cells (MDSCs) can be used to deliver EVs to the tumor niche (manuscript 2101619). After non-viral transfection, MDSCs continue to home to cancerous tissue and release EVs to mediate antitumoral responses via paracrine signaling. Following on with further physicochemical characterizations, Wolfgang Friess and co-workers comprehensively studied the influence of lyophilization (freeze-drying) and cryoprotectants on EV stability (manuscript 2100538). This evaluation included several common excipients and examined up to six months of storage stability, since long-term storage is ultimately required for the technological translation of EVs into real-world practice. To better characterize pathogen-derived vesicles for vaccination approaches, Gregor Fuhrmann and his group studied the influence of the growth conditions of streptococci on the immunomodulatory capacity of their bacterial vesicles (manuscript 2101151). Proteomic analyses showed that vesicles isolated at later bacterial growth stages were no better at modulating primary human immune cells than those isolated at earlier timepoints. Expanding on the role of EVs, Natalia Higuita-Castro and co-workers present a detailed study of the manufacturing process of neurogenic EVs for delivery to the central nervous system (manuscript 2100805). They found that designer EVs could be a promising delivery platform to modulate pro-neuronal responses and that modifying the EVs with glutamate receptors facilitated the deployment of EVs to the brain. Claus-Michael Lehr and his research team (manuscript 2101180) were interested in developing an in vitro model based on bacterial membrane vesicles to predict the diffusion of common antibiotics through the Gram-negative bacterial membrane. They showed that their vesicle-based model had a greater predictive accuracy than commonly applied liposomal models, emphasizing the importance of membrane proteins in vesicular transport. When developing EVs as drug carriers, it is pivotal to assess them against appropriate lipid-based comparator systems. In their review manuscript, Alika Nagelkerke and co-workers (manuscript 2100639) discuss the similarities and differences between lipid nanocarriers and EVs to ensure that both research areas benefit from each other in the near future. Following up to this, James C. Iatridis and co-workers reviewed the potential role of EVs in regenerative medicine, focusing on intervertebral disc degeneration (manuscript 2100596). They evaluated several preclinical studies and identified challenges associated with the clinical translation of EV therapeutics in this research area. We believe that this collection of articles covers a wide range of relevant EV research in the biomedical arena, and we are grateful for the opportunity to co-edit this special issue. The authors declare no conflict of interest. Juliane Nguyen is associate professor and Vice Chair in the Division of Pharmacoengineering and Molecular Pharmaceutics, School of Pharmacy, at the University of North Carolina at Chapel Hill. Her lab develops novel protein-, RNA-, and exosome-based delivery platforms for treating myocardial infarction and cancer. Dr. Nguyen’s work has been recognized with the NSF CAREER Award (2018), the Young Innovator Award from the Biomedical Engineering Society (2019), and the AAPS Emerging Leader Award (2019). She received her Ph.D. in pharmaceutical sciences from the Philipps-University of Marburg (Germany) with Thomas Kissel and was a Deutsche Forschungsgemeinschaft Postdoctoral Fellow at UCSF with Frank Szoka. Gregor Fuhrmann has been a professor of pharmaceutical biology at the Friedrich-Alexander-University Erlangen-Nürnberg since 2021. He obtained his doctoral degree from the Swiss Federal Institute of Technology ETH Zurich with Jean-Christophe Leroux in 2013, and was a postdoc at Imperial College London from 2013–2016 with Molly M. Stevens. From 2016–2021, Gregor led his junior research group at the Helmholtz Institute for Pharmaceutical Research Saarland, Germany. He is a former Marie-Curie and current ERC Starting Grant awardee, and received the 2019s Young Invetigator Award from the German Pharmaceutical Society. His research focusses on extracellular vesicles in infection and inflammatory dispositions.