Recent advances in research on extracellular vesicles have significantly enhanced their potential as therapeutic agents for neurological diseases. Owing to their therapeutic properties and ability to cross the blood-brain barrier, extracellular vesicles are recognized as promising drug delivery vehicles for various neurological conditions, including ischemic stroke, traumatic brain injury, neurodegenerative diseases, glioma, and psychosis. However, the clinical application of natural extracellular vesicles is hindered by their limited targeting ability and short clearance from the body. To address these limitations, multiple engineering strategies have been developed to enhance the targeting capabilities of extracellular vesicles, thereby enabling the delivery of therapeutic contents to specific tissues or cells. Therefore, this review aims to highlight the latest advancements in natural and targeting-engineered extracellular vesicles, exploring their applications in treating traumatic brain injury, ischemic stroke, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, glioma, and psychosis. Additionally, we summarized recent clinical trials involving extracellular vesicles and discussed the challenges and future prospects of using targeting-engineered extracellular vesicles for drug delivery in treating neurological diseases. This review offers new insights for developing highly targeted therapies in this field.

Different cancers remain major global health challenges due to their diverse biological behaviors and significant treatment hurdles. The aging of populations and lifestyle factors increase cancer occurrence and place increasing pressure on healthcare systems. Despite continuous advancements, many cancers remain fatal due to late-stage diagnosis, tumor heterogeneity, and drug resistance, thus necessitating urgent development of innovative treatment solutions. Therapeutic nucleic acids, a new class of biological drugs, offer a promising approach to overcoming these challenges. The recent Nucleic Acids and Nanobiosystems in Cancer Theranostics (NANCT) conference brought together internationally recognized experts from 15 countries to discuss cutting-edge research, spanning from oncolytic viruses to anticancer RNA nanoparticles and other emerging nanotechnologies. This review captures key insights and developments, emphasizing the need for interdisciplinary translation of scientific advancements into clinical practice and shaping the future of personalized cancer treatments for improved therapeutic outcomes.

Small extracellular vesicles (sEVs) derived from mesenchymal stromal cells (MSCs) hold substantial promise for therapeutic applications, including immune modulation and tissue regeneration. However, challenges such as batch-to-batch variability, donor material diversity, and the lack of standardized potency testing remain significant barriers to clinical translation. The recent U.S. Food and Drug Administration (FDA) approval of Ryoncil (remestemcel-L) for steroid-refractory acute graft-versus-host disease (aGvHD) in pediatric patients represents a crucial milestone for MSC-based therapies, offering also valuable insights for the development of MSC-EV therapies. This approval highlights the critical need to address variability and standardization issues in MSC products. Strategies like immortalizing MSCs and expanding them clonally can improve scalability, consistency, and overcome limitations inherent to cellular MSC therapies. With the FDA's decision signaling significant progress, optimizing MSC expansion protocols and refining potency testing methods will be crucial for advancing MSC-EVs as a viable therapeutic option, overcoming current challenges, and expanding clinical applications.

Presbycusis, also referred to as age-related hearing loss, poses a substantial burden on both individuals and society. The hallmark of presbycusis is a progressive decrease in auditory sensitivity. Irreversible hearing loss occurs due to the limited regenerative capacity of spiral neurons and peripheral cochlear hair cells (HCs). Although hearing aids and cochlear implantations (CIs) are established approaches for alleviating symptoms of presbycusis, there are currently no preventive or curative measures available. This article provides a comprehensive discussion on the research progress pertaining to the classification, molecular mechanism, genetic susceptibility, as well as the applications and prospects of diverse therapeutic interventions of presbycusis. Building upon these discussions, promising interventions like gene therapy and stem cell (SC) therapy are proposed for their potential value in restoring cochlear function; thus aiming to pave new avenues for prevention and cure of presbycusis.

Osteoarthritis (OA) is a joint disease characterized by articular cartilage degradation. Persistent low-grade inflammation defines OA pathogenesis, with crucial involvement of pro-inflammatory M1-like macrophages. While mesenchymal stromal cells (MSC) and their small extracellular vesicles (sEV) hold promise for OA treatment, achieving consistent clinical-grade sEV products remains a significant challenge. This study aims to develop fully characterized, reproducible, clinical-grade batches of sEV derived from umbilical cord (UC)-MSC for the treatment of OA while assessing its efficacy and safety. Initially, a standardized, research-grade manufacturing protocol was established to ensure consistent sEV production. UC-MSC-sEV characterization under non-cGMP conditions showed consistent miRNA and protein profiles, suggesting their potential for standardized manufacturing. In vitro studies evaluated the efficacy, safety, and potency of sEV; animal studies confirmed their effectiveness and safety. In vitro, UC-MSC-sEV polarized macrophages to an anti-inflammatory M2b-like phenotype, through STAT1 modulation, indicating their potential to create an anti-inflammatory environment in the affected joints. In silico studies confirmed sEV's immunosuppressive signature through miRNA and proteome analysis. In an OA mouse model, sEV injected intra-articularly (IA) induced hyaline cartilage regeneration, validated by histological and μCT analyses. The unique detection of sEV signals within the knee joint over time highlights its safety profile by confirming the retention of sEV in the joint. The product development of UC-MSC-sEV involved refining, standardizing, and validating processes in compliance with GMP standards. The initial assessment of the safety of the clinical-grade product via IA administration in a first-in-human study showed no adverse effects after a 12 month follow-up period. These results support the progress of this sEV-based therapy in an early-phase clinical trial, the details of which are presented and discussed in this work. This study provides data on using UC-MSC-sEV as local therapy for OA, highlighting their regenerative and anti-inflammatory properties and safety in preclinical and a proof-of-principle clinical application.

Oligohydramnios (decreased amniotic fluid volume for gestational age) is a severe condition associated with high morbidity and mortality mainly due to fetal pulmonary hypoplasia. Currently, there are limited treatment options to promote fetal lung development. Administration of stem cells and their derivates have shown promising regenerative properties for several fetal and neonatal diseases related to arrested lung development. Herein, we first characterized pulmonary hypoplasia secondary to oligohydramnios in a surgical rat model. Experimental induction of oligohydramnios led to impaired lung growth, branching morphogenesis (fewer airspaces with decreased Fgf10, Nrp1, Ctnnb1 expression), proximal/distal progenitor cell patterning (decreased Sox2 and Sox9 expression), and TGF-β signaling. We then tested antenatal administration of extracellular vesicles derived from amniotic fluid stem cells (AFSC-EVs). In oligohydramnios lungs, AFSC-EV administration improved lung branching morphogenesis and airway progenitor cell patterning at least in part through the release of miR-93-5p. Our experiments suggest that AFSC-EV miR-93-5p blocked SMAD 7, resulting in upregulation of pSMAD2/3 and restoration of TGF-β signaling. Conversely, oligohydramnios lungs treated with antagomir 93-5p transfected AFSC-EVs had decreased branching morphogenesis and TGF-β signaling. This is the first study reporting that antenatal administration of stem cell derivatives could be a potential therapy to rescue lung development in fetuses with oligohydramnios.

Extracellular vesicles (EVs) are heterogeneous entities secreted by cells into their microenvironment and systemic circulation. Circulating EVs carry functional small RNAs and other molecular footprints from their cell of origin, and thus have evident applications in liquid biopsy, therapeutics, and intercellular communication. Yet, the complete transcriptomic landscape of EVs is poorly characterized due to critical limitations including variable protocols used for EV-RNA extraction, quality control, cDNA library preparation, sequencing technologies, and bioinformatic analyses. Consequently, there is a gap in knowledge and the need for a standardized approach in delineating EV-RNAs. Here, we address these gaps by describing the following points by (1) focusing on the large canopy of the EVs and particles (EVPs), which includes, but not limited to - exosomes and other large and small EVs, lipoproteins, exomeres/supermeres, mitochondrial-derived vesicles, RNA binding proteins, and cell-free DNA/RNA/proteins; (2) examining the potential functional roles and biogenesis of EVPs; (3) discussing various transcriptomic methods and technologies used in uncovering the cargoes of EVPs; (4) presenting a comprehensive list of RNA subtypes reported in EVPs; (5) describing different EV-RNA databases and resources specific to EV-RNA species; (6) reviewing established bioinformatics pipelines and novel strategies for reproducible EV transcriptomics analyses; (7) emphasizing the significant need for a gold standard approach in identifying EV-RNAs across studies; (8) and finally, we highlight current challenges, discuss possible solutions, and present recommendations for robust and reproducible analyses of EVP-associated small RNAs. Overall, we seek to provide clarity on the transcriptomics landscape, sequencing technologies, and bioinformatic analyses of EVP-RNAs. Detailed portrayal of the current state of EVP transcriptomics will lead to a better understanding of how the RNA cargo of EVPs can be used in modern and targeted diagnostics and therapeutics. For the inclusion of different particles discussed in this article, we use the terms large/small EVs, non-vesicular extracellular particles (NVEPs), EPs and EVPs as defined in MISEV guidelines by the International Society of Extracellular Vesicles (ISEV).

The emergence of extracellular vesicles (EVs), which are natural lipid bilayer membrane structures facilitating intercellular substance and information exchange, has sparked innovative approaches in drug development and carrier enhancement. Plant-derived EVs notably offer advantages including low preparation cost, low immunogenicity, flexible drug delivery, high stability, good tissue permeability, and high inherent medicinal value compared to their animal-derived counterparts. Despite these promising attributes, the research on plant-derived EVs remains fragmented and lacks comprehensive synthesis. This review aims to address this gap by summarizing the isolation methods, biological characteristics, and storage techniques of plant-derived EVs. Additionally, we explore the potential of plant-derived EVs as therapeutic agents and drug carriers for treating various diseases. Finally, we delineate the current impediments to plant-derived EV development and highlight future research directions. By providing a detailed overview, we hope to facilitate further research and application in this emerging field.

Sudden sensorineural hearing loss (SSNHL) is characterized by rapid, unexplained loss of hearing within a 72-hour period and exhibits a high incidence globally. Despite this, the outcomes of therapeutic interventions remain largely unpredictable, especially for those with profound hearing loss. Extracellular vesicles (EVs), nano-sized entities containing biological materials, are implicated in the development of numerous diseases. The specific relationship between EVs and both the severity and treatment effectiveness of SSNHL, however, is not well understood.

This study involved the analysis of medical records from the Department of Otolaryngology (September 1, 2020 - December 31, 2022) of patients diagnosed with SSNHL according to the 2015 Guidelines for Diagnosis and Treatment of Sudden Deafness in China. Peripheral blood samples from patients with various types of SSNHL before and after treatment were collected, alongside samples from healthy volunteers serving as controls. Plasma EVs were isolated using gel rejection chromatography and analyzed for concentration, marker presence, and morphology using Nanosight, Western blot, and transmission electron microscopy (TEM), respectively. Proteomics and miRNA assessments were conducted to identify differentially expressed proteins and miRNAs in the plasma EVs of SSNHL patients and healthy volunteers. Key proteins were further validated through Western blot analysis. Enzyme-linked immunosorbent assay (ELISA) was utilized to determine the levels of complement C3 in plasma EVs, and correlation analyses were performed with audiological data pre- and post-treatment.

Plasma from SSNHL patients of varying types was collected and their EVs were successfully isolated and characterized. Proteomic analysis revealed that complement C3 levels in the plasma EVs of patients with profound SSNHL were significantly higher compared to healthy controls. Differential expression of miRNAs in plasma EVs and their related functions were also identified. The study found that the level of complement C3 in plasma EVs, but not the total plasma complement C3, positively correlated with the severity of SSNHL in patients exhibiting positive therapeutic responses, particularly in those with initially lower levels of EV-associated complement C3. After treatment, complement C3 level was decreased in patients with initially higher levels of EV-associated complement C3. No significant correlation was observed between changes in plasma EV-derived complement C3 levels and the degree of hearing loss in either responders or non-responders among patients with profound SSNHL.

Differential profiles of proteins and miRNAs were identified in patients with profound SSNHL. Notably, plasma EV-derived complement C3 was linked to both the severity and early treatment effectiveness of patients with profound SSNHL.

The recently published "Minimal information for studies of extracellular vesicles - 2023 (MISEV2023)" in the Journal of Extracellular Vesicles has provided practical solutions to the numerous challenges extracellular vesicles (EVs) researchers face. These guidelines are imperative for novice and experienced researchers and promote unity within the EV community. It is strongly recommended that laboratories working with EVs make MISEV2023 an essential handbook and that researchers actively promote these guidelines during laboratory meetings, journal clubs, seminars, workshops, and conferences. A collective effort from EV researchers is crucial to steer the progress of EV science in a positive direction.

Mesenchymal stromal cells (MSCs) exert immunomodulatory effects, primarily through released extracellular vesicles (EVs). For the clinical-grade manufacturing of MSC-EV products culture conditions need to support MSC expansion and allow the manufacturing of potent MSC-EV products. Traditionally, MSCs are expanded in fetal bovine serum-supplemented media. However, according to good manufacturing practice (GMP) guidelines the use of animal sera should be avoided. To this end, human platelet lysate (hPL) has been qualified as an animal serum replacement. Although hPL outcompetes animal sera in promoting MSC expansion, hPL typically contains components of the coagulation system that need to be inhibited or removed to avoid coagulation reactions in the cell culture. Commonly, heparin is utilized as an anticoagulant; however, higher concentrations of heparin can negatively impact MSC viability, and conventional concentrations alone do not sufficiently prevent clot formation in prepared media.

To circumvent unwanted coagulation processes, this study compared various clotting prevention strategies, including different anticoagulants and calcium chloride (CaCl2)-mediated declotting methods, which in combination with heparin addition was found effective. We evaluated the influence of the differently treated hPLs on the proliferation and phenotype of primary bone marrow-derived MSCs and identified the CaCl2-mediated declotting method as the most effective option. To determine whether CaCl2 declotted hPL allows the manufacturing of immunomodulatory MSC-EV products, EVs were prepared from conditioned media of MSCs expanded with either conventional or CaCl2 declotted hPL. In addition to metric analyses, the immunomodulatory potential of resulting MSC-EV products was assessed in a recently established multi-donor mixed lymphocyte reaction assay.

Our findings conclusively show that CaCl2-declotted hPLs support the production of immunomodulatory-active MSC-EV products.

Extracellular vesicles (EVs) are membrane-enclosed bio-nanoparticles secreted by cells and naturally evolved to transport various bioactive molecules between cells and even organisms. These cellular objects are considered one of the most promising bio-nanovehicles for the delivery of native and exogenous molecular cargo. However, many challenges with state-of-the-art EV-based candidates as drug carriers still exist, including issues with scalability, batch-to-batch reproducibility, and cost-sustainability of the final therapeutic formulation. Microalgal extracellular vesicles, which we named nanoalgosomes, are naturally released by various microalgal species. Here, we evaluate the innate biological properties of nanoalgosomes derived from cultures of the marine microalgae Tetraselmis chuii, using an optimized manufacturing protocol. Our investigation of nanoalgosome biocompatibility in preclinical models includes toxicological analyses, using the invertebrate model organism Caenorhabditis elegans, hematological and immunological evaluations ex vivo and in mice. We evaluate nanoalgosome cellular uptake mechanisms in C. elegans at cellular and subcellular levels, and study their biodistribution in mice with accurate space-time resolution. Further examination highlights the antioxidant and anti-inflammatory bioactivities of nanoalgosomes. This holistic approach to nanoalgosome functional characterization demonstrates that they are biocompatible and innate bioactive effectors with unique bone tropism. These findings suggest that nanoalgosomes have significant potential for future therapeutic applications.

Extracellular vesicles (EVs) serve as an intrinsic system for delivering functional molecules within our body, playing significant roles in diverse physiological phenomena and diseases. Both native and engineered EVs are currently the subject of extensive research as promising therapeutics and drug delivery systems, primarily due to their remarkable attributes, such as targeting capabilities, biocompatibility, and low immunogenicity and mutagenicity. Nevertheless, their clinical application is still a long way off owing to multiple limitations. In this context, the Science Board of the Pharmaceuticals and Medical Devices Agency (PMDA) of Japan has conducted a comprehensive assessment to identify the current issues related to the quality and safety of EV-based therapeutic products. Furthermore, we have presented several examples of the state-of-the-art methodologies employed in EV manufacturing, along with guidelines for critical processes, such as production, purification, characterization, quality evaluation and control, safety assessment, and clinical development and evaluation of EV-based therapeutics. These endeavors aim to facilitate the clinical application of EVs and pave the way for their transformative impact in healthcare.

Extracellular vesicles (EVs) are naturally occurring vesicles secreted by cells that can transport cargo between cells, making them promising bioactive nanomaterials. However, due to the complex and heterogeneous biological characteristics, a method for robust EV manipulation and efficient EV delivery is still lacking. Here, we developed a novel class of extracellular vesicle spherical nucleic acid (EV-SNA) nanostructures with scalability, programmability, and efficient cellular delivery. EV-SNA was constructed through the simple hydrophobic coassembly of natural EVs with cholesterol-modified oligonucleotides and can be stable for 1 month at room temperature. Based on programmable nucleic acid shells, EV-SNA can respond to AND logic gates to achieve vesicle assembly manipulation. Importantly, EV-SNA can be constructed from a wide range of biological sources EV, enhancing cellular delivery capability by nearly 10-20 times. Compared to artificial liposomal SNA, endogenous EV-SNA exhibited better biocompatibility and more effective delivery of antisense oligonucleotides in hard-to-transfect primary stem cells. Additionally, EV-SNA can deliver functional EVs for immune regulation. As a novel material form, EV-SNA may provide a modular and programmable framework paradigm for EV-based applications in drug delivery, disease treatment, nanovaccines, and other fields.

Cellular and molecular alterations associated with hearing loss are now better understood with advances in molecular biology. These changes indicate the participation of distinct damage and stress pathways that are unlikely to be fully addressed by conventional pharmaceutical treatment. Sensorineural hearing loss is a common and debilitating condition for which comprehensive pharmacologic intervention is not available. The complex and diverse molecular pathology that underlies hearing loss currently limits our ability to intervene with small molecules. The present review focuses on the potential for the use of extracellular vesicles in otology. It examines a variety of inner ear diseases and hearing loss that may be treatable using exosomes (EXOs). The role of EXOs as carriers for the treatment of diseases related to the inner ear as well as EXOs as biomarkers for the recognition of diseases related to the ear is discussed.

The aim of this study was to evaluate potential synergistic effects of a single, local application of human umbilical cord MSC-derived sEVs in combination with a low dose of recombinant human rhBMP-2 to promote the regeneration of a metaphyseal femoral defect in an osteoporotic rat model.

6 weeks after induction of osteoporosis by bilateral ventral ovariectomy and administration of a special diet, a total of 64 rats underwent a distal femoral metaphyseal osteotomy using a manual Gigli wire saw. Defects were stabilized with an adapted Y-shaped mini-locking plate and were subsequently treated with alginate only, or alginate loaded with hUC-MSC-sEVs (2 × 109), rhBMP-2 (1.5 µg), or a combination of sEVs and rhBMP-2 (n = 16 for each group). 6 weeks post-surgery, femora were evaluated by µCT, descriptive histology, and biomechanical testing.

Native radiographs and µCT analysis confirmed superior bony union with callus formation after treatment with hUC-MSC-sEVs in combination with a low dose of rhBMP-2. This finding was further substantiated by histology, showing robust defect consolidation 6 weeks after treatment. Torsion testing of the explanted femora revealed increased stiffness after application of both, rhBMP-2 alone, or in combination with sEVs, whereas torque was only significantly increased after treatment with rhBMP-2 together with sEVs.

The present study demonstrates that the co-application of hUC-MSC-sEVs can improve the efficacy of rhBMP-2 to promote the regeneration of osteoporotic bone defects.

Extracellular vesicles (EVs) are released by all cells and contribute to cell-to-cell communication. The capacity of EVs to target specific cells and to efficiently deliver a composite profile of functional molecules have led researchers around the world to hypothesize their potential as therapeutics. While studies of EV treatment in animal models are numerous, their actual clinical benefit in humans has more slowly started to be tested. In this scoping review, we searched PubMed and other databases up to 31 December 2023 and, starting from 13,567 records, we selected 40 pertinent published studies testing EVs as therapeutics in humans. The analysis of those 40 studies shows that they are all small pilot trials with a large heterogeneity in terms of administration route and target disease. Moreover, the absence of a placebo control in most of the studies, the predominant local application of EV formulations and the inconsistent administration dose metric still impede comparison across studies and firm conclusions about EV safety and efficacy. On the other hand, the recording of some promising outcomes strongly calls out for well-designed larger studies to test EVs as an alternative approach to treat human diseases with no or few therapeutic options.

Substantial efforts have been made for local administration of small molecules or biologics in treating hearing loss diseases caused by either trauma, genetic mutations, or drug ototoxicity. Recently, extracellular vesicles (EVs) naturally secreted from cells have drawn increasing attention on attenuating hearing impairment from both preclinical studies and clinical studies. Highly emerging field utilizing diverse bioengineering technologies for developing EVs as the bioderived therapeutic materials, along with artificial intelligence (AI)-based targeting toolkits, shed the light on the unique properties of EVs specific to inner ear delivery. This review will illuminate such exciting research field from fundamentals of hearing protective functions of EVs to biotechnology advancement and potential clinical translation of functionalized EVs. Specifically, the advancements in assessing targeting ligands using AI algorithms are systematically discussed. The overall translational potential of EVs is reviewed in the context of auditory sensing system for developing next generation gene therapy.

The extracellular matrix (ECM) consists in a complex meshwork of collagens, glycoproteins, and proteoglycans, which serves a scaffolding function and provides viscoelastic properties to the tissues. ECM acts as a biomechanical support, and actively participates in cell signaling to induce tissular changes in response to environmental forces and soluble cues. Given the remarkable complexity of the inner ear architecture, its exquisite structure-function relationship, and the importance of vibration-induced stimulation of its sensory cells, ECM is instrumental to hearing. Many factors of the matrisome are involved in cochlea development, function and maintenance, as evidenced by the variety of ECM proteins associated with hereditary deafness. This review describes the structural and functional ECM components in the auditory organ and how they are modulated over time and following injury.

Up to 1.5 billion people worldwide suffer from various forms of hearing loss, with an additional 1.1 billion people at risk from various insults such as increased consumption of recreational noise-emitting devices and ageing. The most common type of hearing impairment is sensorineural hearing loss caused by the degeneration or malfunction of cochlear hair cells or spiral ganglion nerves in the inner ear. There is currently no cure for hearing loss. However, emerging frontier technologies such as gene, drug or cell-based therapies offer hope for an effective cure. In this review, we discuss the current therapeutic progress for the treatment of hearing loss. We describe and evaluate the major therapeutic approaches being applied to hearing loss and summarize the key trials and studies.

Clinical translation of gene therapy has been challenging, due to limitations in current delivery vehicles such as traditional viral vectors. Herein, we report the use of gRNA:Cas9 ribonucleoprotein (RNP) complexes engineered extracellular vesicles (EVs) for in vivo gene therapy. By leveraging a novel high-throughput microfluidic droplet-based electroporation system (μDES), we achieved 10-fold enhancement of loading efficiency and more than 1000-fold increase in processing throughput on loading RNP complexes into EVs (RNP-EVs), compared with conventional bulk electroporation. The flow-through droplets serve as enormous bioreactors for offering millisecond pulsed, low-voltage electroporation in a continuous-flow and scalable manner, which minimizes the Joule heating influence and surface alteration to retain natural EV stability and integrity. In the Shaker-1 mouse model of dominant progressive hearing loss, we demonstrated the effective delivery of RNP-EVs into inner ear hair cells, with a clear reduction of Myo7ash1 mRNA expression compared to RNP-loaded lipid-like nanoparticles (RNP-LNPs), leading to significant hearing recovery measured by auditory brainstem responses (ABR).

Extracellular vesicles (EVs) encompass a diverse range of membranous structures derived from cells, including exosomes and microvesicles. These vesicles are present in biological fluids and play vital roles in various physiological and pathological processes. They facilitate intercellular communication by enabling the exchange of proteins, lipids, and genetic material between cells. Understanding the cellular processes that govern EV biology is essential for unraveling their physiological and pathological functions and their potential clinical applications. Despite significant advancements in EV research in recent years, there is still much to learn about these vesicles. The advent of improved mass spectrometry (MS)-based techniques has allowed for a deeper characterization of EV protein composition, providing valuable insights into their roles in different physiological and pathological conditions. In this chapter, we provide an overview of proteomics studies conducted to identify the protein contents of EVs, which contribute to their therapeutic and pathological features. We also provided evidence on the potential of EV proteome contents as biomarkers for early disease diagnosis, progression, and treatment response, as well as factors that influence their composition. Additionally, we discuss the available databases containing information on EV proteome contents, and finally, we highlight the need for further research to pave the way toward their utilization in clinical settings.

Extracellular vesicles (EVs) occur in a variety of bodily fluids and have gained recent attraction as natural materials due to their bioactive surfaces, internal cargo, and role in intercellular communication. EVs contain various biomolecules, including surface and cytoplasmic proteins; and nucleic acids that are often representative of the originating cells. EVs can transfer content to other cells, a process that is thought to be important for several biological processes, including immune responses, oncogenesis, and angiogenesis. An increased understanding of the underlying mechanisms of EV biogenesis, composition, and function has led to an exponential increase in preclinical and clinical assessment of EVs for biomedical applications, such as diagnostics and drug delivery. Bacterium-derived EV vaccines have been in clinical use for decades and a few EV-based diagnostic assays regulated under Clinical Laboratory Improvement Amendments have been approved for use in single laboratories. Though, EV-based products are yet to receive widespread clinical approval from national regulatory agencies such as the United States Food and Drug Administration (USFDA) and European Medicine Agency (EMA), many are in late-stage clinical trials. This perspective sheds light on the unique characteristics of EVs, highlighting current clinical trends, emerging applications, challenges and future perspectives of EVs in clinical use.

Mesenchymal stromal cells (MSCs) have demonstrated therapeutic potential in diverse clinical settings, largely due to their ability to produce extracellular vesicles (EVs). These EVs play a pivotal role in modulating immune responses, transforming pro-inflammatory cues into regulatory signals that foster a pro-regenerative milieu. Our previous studies identified the variability in the immunomodulatory effects of EVs sourced from primary human bone marrow MSCs as a consistent challenge. Given the limited proliferation of primary MSCs, protocols were advanced to derive MSCs from GMP-compliant induced pluripotent stem cells (iPSCs), producing iPSC-derived MSCs (iMSCs) that satisfied rigorous MSC criteria and exhibited enhanced expansion potential. Intriguingly, even though obtained iMSCs contained the potential to release immunomodulatory active EVs, the iMSC-EV products displayed batch-to-batch functional inconsistencies, mirroring those from bone marrow counterparts. We also discerned variances in EV-specific protein profiles among independent iMSC-EV preparations. Our results underscore that while iMSCs present an expansive growth advantage, they do not overcome the persistent challenge of functional variability of resulting MSC-EV products. Once more, our findings accentuate the crucial need for batch-to-batch functional testing, ensuring discrimination of effective and ineffective MSC-EV products for considered downstream applications.

Despite significant advances in the development of therapeutics for hearing loss, drug delivery to the middle and inner ear remains a challenge. As conventional oral or intravascular administration are ineffective due to poor bioavailability and impermeability of the blood-labyrinth-barrier, localized delivery is becoming a preferable approach for certain drugs. Even then, localized delivery to the ear precludes continual drug delivery due to the invasive and potentially traumatic procedures required to access the middle and inner ear. To address this, the preclinical development of controlled release therapeutics and drug delivery devices have greatly advanced, with some now showing promise clinically. This review will discuss the existing challenges in drug development for treating the most prevalent and damaging hearing disorders, in particular otitis media, perforation of the tympanic membrane, cholesteatoma and sensorineural hearing loss. We will then address novel developments in drug delivery that address these including novel controlled release therapeutics such as hydrogel and nanotechnology and finally, novel device delivery approaches such as microfluidic systems and cochlear prosthesis-mediated delivery. The aim of this review is to investigate how drugs can reach the middle and inner ear more efficiently and how recent innovations could be applied in aiding drug delivery in certain pathologic contexts.

The progression of liver fibrosis leads to portal hypertension and liver dysfunction. However, no antifibrotic agents have been approved for cirrhosis to date, making them an unmet medical need. Small extracellular vesicles (sEVs) of mesenchymal stem cells (MSCs) are among these candidate agents. In this study, we investigated the effects of sEVs of MSCs, analyzed their distribution in the liver post-administration, whether their effect was dose-dependent, and whether it was possible to collect a large number of sEVs.

sEVs expressing tdTomato were generated, and their uptake into constituent liver cells was observed in vitro, as well as their sites of uptake and cells in the liver using a mouse model of liver cirrhosis. The efficiency of sEV collection using tangential flow filtration (TFF) and changes in the therapeutic effects of sEVs in a volume-dependent manner were examined.

The sEVs of MSCs accumulated mostly in macrophages in damaged areas of the liver. In addition, the therapeutic effect of sEVs was not necessarily dose-dependent, and it reached a plateau when the dosage exceeded a certain level. Furthermore, although ultracentrifugation was commonly used to collect sEVs for research purposes, we verified that TFF could be used for efficient sEV collection and that their effectiveness is not reduced.

In this study, we identified some unknown aspects regarding the dynamics, collection, and capacity dependence of sEVs. Our results provide important fundamentals for the development of therapies using sEVs and hold potential implications for the therapeutic applications of sEV-based therapies for liver cirrhosis.

Extracellular vesicles (EVs) are widely recognized for their potential as drug delivery systems. EVs are membranous nanoparticles shed from cells. Among their natural features are their ability to shield cargo molecules against degradation and enable their functional internalization into target cells. Especially biological or bio-inspired large molecules (LMs), like nucleic acids, proteins, peptides, and others, may profit from encapsulation in EVs for drug delivery purposes. In the last years, a variety of loading protocols are explored for different LMs. The lack of standardization in the EV drug delivery field has impeded their comparability so far. Currently, the first reporting frameworks and workflows for EV drug loading are proposed. The aim of this review is to summarize these evolving standardization approaches and set recently developed methods into context. This will allow for enhanced comparability of future work on EV drug loading with LMs.

Cell culture-conditioned medium (CCM) is a valuable source of extracellular vesicles (EVs) for basic scientific, therapeutic and diagnostic applications. Cell culturing parameters affect the biochemical composition, release and possibly the function of CCM-derived EVs (CCM-EV). The CCM-EV task force of the Rigor and Standardization Subcommittee of the International Society for Extracellular Vesicles aims to identify relevant cell culturing parameters, describe their effects based on current knowledge, recommend reporting parameters and identify outstanding questions. While some recommendations are valid for all cell types, cell-specific recommendations may need to be established for non-mammalian sources, such as bacteria, yeast and plant cells. Current progress towards these goals is summarized in this perspective paper, along with a checklist to facilitate transparent reporting of cell culturing parameters to improve the reproducibility of CCM-EV research.

Hearing impairment is a global health problem. Stem cell therapy has become a cutting-edge approach to tissue regeneration. In this review, the recent advances in stem cell therapy for hearing loss have been discussed. Nanomaterials can modulate the stem cell microenvironment to augment the therapeutic effects further. The potential of combining nanomaterials with stem cells for repairing and regenerating damaged inner ear hair cells (HCs) and spiral ganglion neurons (SGNs) has also been discussed. Stem cell-derived exosomes can contribute to the repair and regeneration of damaged tissue, and the research progress on exosome-based hearing loss treatment has been summarized as well. Despite stem cell therapy's technical and practical limitations, the findings reported so far are promising and warrant further investigation for eventual clinical translation.

The International Society for Cell & Gene Therapy Scientific Signature Series event "Therapeutic Advances With Native and Engineered Human EVs" took place as part of the International Society for Cell & Gene Therapy 2022 Annual Meeting, held from May 4 to 7, 2022, in San Francisco, California, USA. This was the first signature series event on extracellular vesicles (EVs) and a timely reflection of the growing interest in EVs, including both native and engineered human EVs, for therapeutic applications. The event successfully gathered academic and industrial key opinion leaders to discuss the current state of the art in developing and understanding native and engineered EVs and applying our knowledge toward advancing EV therapeutics. Latest advancements in understanding the mechanisms by which native and engineered EVs exert their therapeutic effects against different diseases in animal models were presented, with some diseases such as psoriasis and osteoarthritis already reaching clinical testing of EVs. The discussion also covered various aspects relevant to advancing the clinical translation of EV therapies, including EV preparation, manufacturing, consistency, site(s) of action, route(s) of administration, and luminal cargo delivery of RNA and other compounds.

Extracellular vesicles (EVs), including exosomes and microvesicles, are released by almost all cells and found in all body fluids. Unknown proportions of EVs transmit specific information from their cells of origin to specific target cells and are key mediators in intercellular communication processes. Depending on their origin, EVs can modulate immune responses, either acting as pro- or anti-inflammatory. With the aim to analyze the immunomodulating activities of EV preparations, especially those from mesenchymal stromal cells (MSCs) in vitro, a multi-donor mixed lymphocyte reaction (mdMLR) assay was established and stressed for its reproducibility.

To this end, human peripheral blood-derived mononuclear cells (PBMCs) of 12 different healthy donors were pooled warranting mutual allogeneic cross-reactivity, even following an optimized freezing and thawing procedure. After thawing, mixed PBMCs were cultured for 5 days in the absence or presence of EVs to be tested. Reflecting allogeneic reactions, in the absence of EVs, pooled PBMCs form characteristic satellite colonies whose appearance can be modulated by EVs. More quantifiable, the strength of the allogenic reaction is reflected by the content of activated CD4 and CD8 T cells being recognized by means of their CD25 and CD54 expression.

Of note, connected to the use of primary cells, independent multi-donor PBMC pools differed in their capability to activate their cultured T cells. Thus, throughout the study, only pooled PBMC batches were used whose activated T-cell contents exceeded 25% of the total T-cell population at culture day 5 and whose contents were reproducibly reduced in the presence of immunomodulatory active MSC-EVs. T-cell activation-suppressing effects of the MSC-EV preparations tested were in all cases accompanied by the impact on monocytes. In the presence of immunomodulatory active MSC-EVs, more monocytes were harvested from mdMLR cultures than in their absence. Furthermore, in the absence of immunomodulatory EVs, most monocytes appeared as non-classical (CD14+CD16+) monocytes, whereas immunomodulatory active MSC-EVs promoted the appearance of classical (CD14++CD16-) and intermediate (CD14++CD16+) monocyte subpopulations.

Overall, the obtained results qualify the mdMLR assay as a robust experimental tool for the evaluation of immunomodulatory potentials of given MSC-EV samples. However, further assay development is required to develop and qualify an authority-acceptable potency assay for clinically applicable MSC-EV products.

The most clinically trialed cells, mesenchymal stromal cells (MSCs), are now known to mainly exert their therapeutic activity through paracrine secretions, which include exosomes. To mitigate potential regulatory concerns on the scalability and reproducibility in the preparations of MSC exosomes, MSC exosomes were produced using a highly characterized MYC-immortalized monoclonal cell line. These cells do not form tumors in athymic nude mice or exhibit anchorage-independent growth, and their exosomes do not carry MYC protein or promote tumor growth. Unlike intra-peritoneal injections, topical applications of MSC exosomes in a mouse model of IMQ-induced psoriasis alleviate interleukin (IL)-17, IL-23 and terminal complement complex, C5b9 in psoriatic skin. When applied on human skin explants, fluorescence from covalently labeled fluorescent MSC exosomes permeated and persisted in the stratum corneum for about 24 hours with negligible exit out of the stratum corneum into the underlying epidermis. As psoriatic stratum corneums are uniquely characterized by activated complements and Munro microabscesses, we postulated that topically applied exosomes permeate the psoriatic stratum corneum to inhibit C5b9 complement complex through CD59, and this inhibition attenuated neutrophil secretion of IL-17. Consistent with this, we demonstrated that assembly of C5b9 on purified human neutrophils induced IL-17 secretion and this induction was abrogated by MSC exosomes, which was in turn abrogated by a neutralizing anti-CD 59 antibody. We thus established the mechanism of action for the alleviation of psoriatic IL-17 by topically applied exosomes.

In a previous study, an inner ear catheter was used to deliver low- and high-dose steroids into the cochlea prior to cochlear implant electrode insertion. With this approach, more apical regions of the cochlea could be reached and a reduction of electrode impedances in the short term was achieved in cochlear implant recipients. Whether intracochlear application of drugs via the catheter is a safe method also for patients with residual hearing has not been investigated hitherto. The aim of the present study was therefore to investigate the effect of intracochlear triamcinolone application in cochlear implant recipients with residual hearing.

Patients with residual hearing were administered triamcinolone-acetonide (4 mg/ml; n = 10) via an inner ear catheter just prior to insertion of a MED-EL FLEX28 electrode. Impedances were measured at defined time points (intra-operatively, post-operatively and at first fitting) and retrospectively compared with a control group (no steroid application) and low- and high-dose group. Hearing thresholds were measured preoperatively, 3 days after surgery and at first fitting by pure tone audiometry. Pre- to postoperative hearing loss was determined at first fitting and compared to results from a previous study.

The median hearing loss after implantation (125-1,500 Hz) was 20.6 dB. Four patients (40%) showed a median hearing loss of less than 15 dB, three patients (30%) between 15 and 30 dB and three patients (30%) more than 30 dB. The median hearing loss was similar to the results obtained from our previous study showing a median hearing loss of 24 dB when using FLEX28 electrode arrays.

No difference in residual hearing loss was found when comparing application of triamcinolone-acetonide using an inner ear catheter prior to the insertion of a FLEX28 electrode array to the use of the FLEX28 electrode array without the catheter. Thus, we conclude that application of drugs to the cochlea with an inner ear catheter could be a feasible approach in patients with residual hearing.