Nanosized extracellular vesicles (EVs) are released by all cells to convey cell-to-cell communication. EVs, including exosomes and microvesicles, carry an array of bioactive molecules, such as proteins and RNAs, encapsulated by a membrane lipid bilayer. Epithelial cells, endothelial cells, and various immune cells in the lung contribute to the pool of EVs in the lung microenvironment and carry molecules reflecting their cellular origin. EVs can maintain lung health by regulating immune responses, inducing tissue repair, and maintaining lung homeostasis. They can be detected in lung tissues and biofluids such as bronchoalveolar lavage fluid and blood, offering information about disease processes, and can function as disease biomarkers. Here, we discuss the role of EVs in lung homeostasis and pulmonary diseases such as asthma, chronic obstructive pulmonary disease, cystic fibrosis, pulmonary fibrosis, and lung injury. The mechanistic involvement of EVs in pathogenesis and their potential as disease biomarkers are discussed. Finally, the pulmonary field benefits from EVs as clinical therapeutics in severe pulmonary inflammatory disease, as EVs from mesenchymal stem cells attenuate severe respiratory inflammation in multiple clinical trials. Further, EVs can be engineered to carry therapeutic molecules for enhanced and broadened therapeutic opportunities, such as the anti-inflammatory molecule CD24. Finally, we discuss the emerging opportunity of using different types of EVs for treating severe respiratory conditions.

​​Exosomes, as natural intercellular messengers, are gaining prominence as delivery vehicles in nanomedicine, offering a superior alternative to conventional synthetic nanoparticles for cancer therapeutics. Unlike lipid, polymer, or metallic nanoparticles, which often face challenges related to immunogenicity, targeting precision, and off-tumor toxicity, exosomes can effectively encapsulate a diverse range of therapeutic agents while exhibiting low toxicity, favorable pharmacokinetics, and organotropic properties. This review examines recent advancements in exosome bioengineering over the past decade. Innovations such as microfluidics-based platforms, nanoporation, fusogenic hybrids, and genetic engineering have significantly improved loading efficiencies, production scalability, and pharmacokinetics of exosomes. These advancements facilitate tumor-specific cargo delivery, resulting in substantial improvements in retention and efficacy essential for clinical success. Moreover, enhanced biodistribution, targeting, and bioavailability-through strategies such as cell selection, surface modifications, membrane composition alterations, and biomaterial integration-suggests a promising future for exosomes as an ideal nanomedicine delivery platform. We also highlight the translational impact of these strategies through emerging clinical trials. Additionally, we outline a framework for clinical translation that focuses on: cargo selection, organotropic cell sourcing, precision loading methodologies, and route-specific delivery optimization. In summary, this review emphasizes the potential of exosomes to overcome the pharmacokinetic and safety challenges that have long impeded oncology drug development, thus enabling safer and more effective cancer treatments.

Studies based on extracellular vesicles (EVs) have been multiplying exponentially for almost two decades, since they were first identified as vectors of cell-cell communication. However, several of these studies display a lack of rigor in EVs characterization and isolation, without discriminating between the different EV populations, thus generating conflicting and unreproducible results. There is therefore a strong need for standardization and guidelines to conduct studies that are rigorous, transparent, reproducible and comply with certain nomenclatures concerning the type of EVs used. The International Society for Extracellular Vesicles (ISEV) published the Minimum Information for Studies of Extracellular Vesicles (MISEV) in 2014, updating it in 2018 and 2023 to reflect different study contexts and technical advancements. The primary objective of this review is to inform future authors about EVs, including their history, nomenclature, and technical recommendations for the for isolation and functionality analysis for conducing EV-based studies according to current standards. Additionally, it aims to inform reviewers about the key parameters required for characterizing EV preparations.

Exosomes are a type of membrane vesicle secreted into the extracellular medium by most cell types. They have a great potential for clinical practice as noninvasive biomarkers for diagnosis of various diseases, prognosis, and monitoring of therapy, which stimulates the development of simple methods for isolating exosomes from biological fluids. A novel affine material based on aminosilanized superparamagnetic core‒shell nanoparticles for fast isolation of urinary exosomes is reported. Iron oxide nanoparticles coated with amino organosilane have been synthesized. The structural and magnetic characteristics of the resulting nanoparticles have been studied by transmission electron microscopy and ferromagnetic resonance. The surface of the synthesized nanoparticles has been chemically functionalized with lectin (concanavalin A), and the efficiency of the obtained material as a sorbent for affine exosome isolation from human urine has been demonstrated. A highly purified fraction of exosomes 90-200 nm in size has been obtained. The exosomal nature of the isolated vesicles has been confirmed by bioluminescent solid-phase microassay of tetrasporine receptor markers. The presence of exosomal miR-21 in the isolated human urine exosome samples has been established.

Mesenchymal stem cell (MSC) therapy, with its unique properties, has garnered interest in cancer treatment. Exosomes (EXOs)-derived from MSC retain the paracrine components of MSCs and demonstrate increased stability, minimal immunogenicity, and low risk of unintended tumorigenesis. Enhanced endocytosis methods make them versatile delivery vehicles for therapeutic cargo. MSC-EXOs can either promote or inhibit carcinogenesis, mediated by paracrine factors and various RNA molecules, particularly microRNAs (miRNAs). The prospect of using MSC-EXOs as a delivery tool for antitumor miRNAs in solid tumor therapy is promising. Exosomes' intrinsic tumor-targeting abilities and low immunogenicity make them ideal for delivering miRNAs, which have shown potential as cancer therapeutics. miRNAs within MSC-EXOs molecules can stimulate tumor growth or induce non-apoptotic cell death pathways, such as ferroptosis, depending on context. Ferroptosis is a kind of controlled cell death that is associated with the pathophysiology of several illnesses and includes iron metabolism. There is growing evidence that miRNAs carried by exosomes derived from MSCs may control ferroptosis in tumor cells by altering key genes related to antioxidant defense, lipid peroxidation, and iron metabolism. Understanding their complex mechanisms in the tumor microenvironment and optimizing their cargo are critical steps toward harnessing their full therapeutic potential. This review provides a comprehensive overview of MSC-EXOs and their role in cancer treatment. We also discuss the potential of MSC-EXOs as delivery vehicles for miRNAs to enhance therapeutic efficacy, as well as the role of exosomal miRNAs in the induction of ferroptosis.

Natural killer cells are innate lymphocytes equipped with the ability to rapidly identify and eliminate cancer cells. However, cancer cells release nanosized extracellular vesicles that can induce an immunosuppressive tumor microenvironment, subsequently hindering natural killer cell immunosurveillance. Studies have reported that extracellular vesicles derived from different cancers, such as acute myeloid leukemia, melanoma, mesothelioma, head and neck squamous carcinoma, lung carcinoma, breast cancer, hepatocellular carcinoma, and pancreatic ductal adenocarcinoma, can induce natural killer cell dysfunction by suppressing cytolytic proteins and downregulating expression of receptors involved in the recognition of oncogenic cells. Additionally, cancer-derived extracellular vesicles can interfere with natural killer cell survival, proliferation, cell migration, and metabolic functions. Therefore, extracellular vesicle-induced natural killer cell suppression has emerged as a key target for research and new therapeutic approaches to recover and enhance the tumoricidal potential of these immune cells. Here, we summarize the current knowledge regarding cancer-derived extracellular vesicles and natural killer cell interactions, their role in immunosuppression, implications for developing efficient cellular immunotherapies and outstanding questions in this field.

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by low-grade inflammation, cartilage breakdown, and persistent pain. Despite its prevalence, current therapeutic strategies primarily focus on symptom management rather than modifying disease progression. Monoclonal antibodies and cytokine inhibitors targeting inflammatory pathways, including TNF-α and IL-1, have shown promise but remain limited by inconsistent efficacy and safety concerns. Long-acting biologic therapies-ranging from extended-release formulations, such as monoclonal antibodies and cytokine inhibitors, to gene therapy approaches-have emerged as promising strategies to enhance treatment durability and improve patient outcomes. Extracellular vesicles (EVs) have gained particular attention as a novel delivery platform due to their inherent stability, biocompatibility, and ability to transport therapeutic cargo, including biologics and immunomodulatory agents, directly to joint tissues. This review explores the evolving role of EVs in OA treatment, highlighting their ability to extend drug half-life, improve targeting, and modulate inflammatory responses. Additionally, strategies for EV engineering, including endogenous and exogenous cargo loading, genetic modifications, and biomaterial-based delivery systems, are discussed.

Unlike traditional small-molecule agents, biopharmaceuticals, like synthetic RNAs, enzymes, and monoclonal antibodies, are highly vulnerable to environmental conditions. Preservation of their functional integrity necessitates advanced delivery methods. Being biocompatible, extracellular vesicles (EVs) gained attention as a promising system for delivering biopharmaceuticals, addressing challenges related to the stability and efficacy of sensitive therapeutic molecules. Indeed, EVs can cross biological barriers like the blood-brain barrier, delivering therapeutic cargo to tissues that are traditionally difficult to reach. Recent innovations in surface modification technologies, including ligand and antibody attachment, have further enhanced EVs' targeting capabilities, making them particularly effective in personalized medicine. Here, we review the versatile suitability of EVs for being next-generation delivery vehicles of biopharmaceuticals, including current standings, practical challenges, and possible future directions of the technology.

Osteosarcoma (OS) calls for early diagnosis to significantly improve patient survival rates. Exosomes hold significant potential as noninvasive biomarkers for the early diagnosis of cancer. Here, we design a microfluidic device to purify and analyze plasma-derived exosomes by label-free surface-enhanced Raman spectroscopy (SERS) profiling for OS diagnosis. Exosomes were isolated, purified, and enriched using a size-dependent microfluidic chip with tangential flow filtration, achieving a high recovery rate of 82 %. The isolated exosomes were then analyzed by label-free SERS using a nanoarray chip with self-assembly monolayers of gold nanoparticles (GNPs). Exosomes originating from different OS cell types were differentiated based on the intrinsic SERS signals. Our approach was further employed to analyze the plasma-derived exosomes from healthy donors and OS patients without the need for specific biomarker labeling. A machine learning-based diagnostic model for OS was constructed, achieving an accuracy of 93 %. The findings indicate that our method is valuable for noninvasive and precise diagnosis of OS and could be generalized to other diseases in the future.

Vesicles, are categorized as artificial (i.e., liposomes) or natural (i.e., extracellular vesicles (EVs)) and play significant roles in drug-delivery and biomarker-screening applications. Liposomes, as a representative form of artificial vesicle, are spherical lipid structures composed of one or more artificially synthesized phospholipid bilayers. Liposomes are highly biocompatible and bioavailable, very stable, and easily synthesized; hence, they are among the most commonly used and frequently applied nanocarriers in targeted drug-delivery systems (DDS). EVs are natural small membrane-bound vesicles actively secreted by cells and contain a variety of components, including nucleic acids, proteins, and lipids. They also serve as important mediators of intercellular communication. As the smallest EV subtype, with diameters of only 30-100 nm, exosomes contain unique biomolecules that are considered to be the fingerprints of the parent cells. In the pathological state, the content of exosomes will change; consequently, exosomes are potential disease-diagnosis biomarkers. Recent clinical trials have shown that exosomes are ideal nanocarriers in targeted drug-delivery therapies for a variety of diseases. Compared with traditional artificial liposomal carriers, exosomes display unique advantages and provide the DDS field with new possibilities. Liposomes and exosomes are receiving increasing levels of attention in the drug-delivery and biomarker-screening fields. This article introduces techniques for the preparation of liposomes, and the enrichment and separation of exosomes, and delves into research progress on their use in drug-delivery and biomarker-screening applications. Finally, challenges facing the use of liposomes and exosomes in clinical applications are discussed.

Exosomes are nanoscale vesicles wrapped in lipid bilayers that are secreted by cells and carry a variety of proteins, lipids, nucleic acids, and metabolites. Exosomes are widely present in various bodily fluids and mediate intercellular communication. They participate in a variety of physiological and pathological processes, including immune regulation, angiogenesis, tumorigenesis, and metastasis, and have significant clinical diagnosis and treatment potential. Exosomes are source-rich, structurally stable, and reflect the states of their parental cells. Therefore, they are expected to serve as novel diagnostic markers for various diseases. In addition, stem-cell-derived exosomes show therapeutic potential and have the advantages of low immunogenicity, high safety and easy storage, and exhibit therapeutic potential for neurodegenerative disorder, cardiovascular disease, and cancer. Furthermore, exosomes are highly biocompatible, have natural homing properties, and are capable of easily penetrating biological barriers, making them excellent drug-delivery carriers. Isolation and enrichment of exosomes is a prerequisite for downstream analysis and application. High-purity, high-yield, and high-throughput exosome-isolation methods are expected to be used in clinical diagnosis and treatment applications. Based on the physicochemical properties of exosomes, including density, size, charge, and surface composition, exosome-isolation methods are mainly divided into density-based (e.g., differential ultracentrifugation, density-gradient ultracentrifugation), size-based (e.g., ultrafiltration, size-exclusion chromatography, field-flow fractionation), polymer-precipitation (e.g., polyethylene-glycol-based precipitation), and chemical affinity (e.g., antibody-based, aptamer-based, and surface-lipid-based lipid probes) methods. Currently, basic research into exosomes and their clinical applications face a number of challenges. Firstly, the complexity and heterogeneity of exosomes and the lack of standardized isolation methods has led to highly variable research results that hinder comparing and reproducing results between different laboratories and clinical settings. Current isolation methods are generally hindered by insufficient purity, low yield, low throughput, and difficulties separating specific subpopulations, which seriously restrict the development of the exosome field. Secondly, exosome-isolation methods that are easy to use in the clinic, have few technical requirements, and are highly efficient and inexpensive are lacking. Commonly used classical methods, such as ultracentrifugation, are time-consuming, labor-intensive, require large sample volumes, and are inappropriate for clinical settings. Methods such as immunoaffinity can be used to isolate exosomes from precious trace samples in clinical practice; however, high costs, low recoveries, and high operating requirements are shortcomings that restrict sample analysis in the clinic. In addition, robust large-scale methods for preparing exosomes are lacking. There is an urgent need to develop repeatable and scalable methods for preparing batches of high-quality exosomes owing to the rapid development of exosomes for the treatment of clinical diseases. Generally, exosome research progress is expected to greatly improve our understanding of the biological functions and components of exosomes, which will help transform the exosome research into effective diagnostic and therapeutic strategies and lead to new precision-medicine and personalized-treatment applications. This article summarizes the latest progress in exosome-isolation and -enrichment technologies and introduces the application of exosomes as disease diagnostic markers, therapeutic agents, and drug delivery carriers. Finally, the future developmental trends in exosome isolation and enrichment technologies for disease diagnosis and treatment are discussed.

A traditional Chinese medicine (TCM) monomer is a bioactive compound extracted from Chinese herbal medicines possessing determined biological activity and pharmacological effects, and has gained much attention for treating neuronal diseases. However, the application of TCM monomers is limited by their low solubility and poor ability to cross the blood-brain barrier (BBB). Exosomes are small extracellular vesicles (EVs) ranging in size from 30 to 150 nm in diameter and can be used as drug delivery carriers that directly target cells or tissues with unique advantages, including low toxicity, low immunogenicity, high stability in blood, and the ability to cross the BBB. This review discusses the biogenesis, components, stability, surface modification, isolation technology, advantages, and disadvantages of exosomes as drug carriers and compares exosomes and other similar drug delivery systems. Furthermore, exosome-encapsulated TCM monomers exert neuroprotective roles, such as anti-inflammation, anti-apoptosis, anti-mitophagy, and anti-oxidation, in various neuronal diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), and cerebral ischemia and reperfusion (CI/R) injury, as well as anti-drug resistance, anti-tumorigenesis, anti-angiogenesis, and promotion of apoptosis in brain tumors, providing more inspiration to promote the development of an exosome-based delivery tool in targeted therapy for neuronal diseases.

A vicious cycle between microbiota dysbiosis and hyperactivated inflammation, hardly disrupted by conventional therapies, remains a significant clinical challenge for periodontitis treatment. Herein, by cloaking a cascade catalysis system in an engineered macrophage membrane, a nanodecoy-based strategy, with targeted bacteria-killing and immunomodulatory abilities, is proposed for reshaping the hostile periodontitis microenvironment. Specifically, recombinant human antimicrobial peptide, LL-37, is anchored to a Toll-like receptor-enriched macrophage membrane via genetic engineering, which facilitates the specific bacteria elimination and efficient tissue retention of the nanodecoys. Moreover, the cascade catalysis system integrates L-amino acid oxidase (LAAO) with hollowed manganese dioxide (hMnO2) by reciprocal elevation of the catalytic efficiency of hMnO2 and LAAO, leading to accelerated O2 generation under a hypoxic microenvironment and disrupted metabolism of periodontopathogenic bacteria. Notably, the nanodecoys trigger the nuclear translocation of NF-E2-related factor-2 (NRF2) to reduce oxidative stress response and rewire the polarization of macrophages, thereby boosting the osteogenic differentiation of osteoblasts. Furthermore, the alveolar bone regeneration therapeutically benefits from the nanodecoys in vivo. Altogether, these results highlight the attractive functions of engineered macrophage membrane-cloaked nanodecoys for effective periodontitis treatment.

Outer membrane vesicles (OMVs) are nanoparticles released by Gram-negative bacteria during growth, mainly under stress conditions. OMV-based vaccines have played an important role in vaccination against Neisseria meningitidis serogroup B (MenB), stimulating research into novel approaches for developing more effective vaccines. OMVs released by the bacterium Neisseria lactamica have emerged as a promising platform for new vaccine development, especially as carriers in subunit vaccines. Despite their importance, some challenges remain in obtaining and purifying OMVs. The most commonly employed methods for OMV isolation and purification are ultracentrifugation (UC) and size exclusion chromatography (SEC). However, these techniques could present limitations for large-scale production and often result in low yields. This study investigated techniques such as tangential flow filtration (TFF), membrane chromatography, and mixed-mode (multimodal) chromatography as potential replacements for UC and SEC. Among the TFF methods evaluated, the sample obtained on the membrane with a 300-kDa cutoff showed a profile more similar to UC but with more than double the total protein recovery. Sartobind® Q membrane chromatography was ineffective for OMV purification, in the conditions evaluated, with a recovery of 8.7%. Conversely, multimodal Capto™ Adhere chromatography recovered 59.0%, while Capto™ Core 400 yielded a recovery of 72.0%, proving to be more effective for purification when analyzed by high-performance liquid chromatography (HPLC). Thus, combining TFF with a 300-kDa membrane followed by Capto™ Core 400 chromatography can be applied as strategy for large-scale applications offering high recovery and purity. KEY POINTS: • Evaluation of TFF, membrane and multimodal chromatography techniques for OMV purification. • Improved Neisseria lactamica OMV yields combining TFF and multimodal chromatography. • A process for OMV purification from a non-pathogenic organism feasible to scale up.

Mesenchymal stem cell-derived exosomes exhibit promising potential in tissue regeneration. Recent studies highlight its significant therapeutic potential in various stages of wound healing. However, the clinical translation of exosome-based therapy was hindered due to issues regarding low productivity and the lack of efficient production protocol to obtain a clinically relevant exosome quantity. Therefore, this study established a potential upscaling protocol to produce exosomes derived from canine adipose-derived mesenchymal stem cells (cAD-MSCs) and explored its potential for wound treatment. The potential upscaling protocol, termed VSCBIC-3-3D, was carried out using VSCBIC-3 in-house serum-free exosome-collecting solution in a three-dimensional (3D) culture system followed by the tangential flow filtration (TFF) isolation. Our findings suggest that culturing cAD-MSCs with VSCBIC-3 maintained cell morphology and viability. Compared to conventional two-dimensional (2D) protocols, The potential upscaling protocol increased exosome yield and concentration in conditioned medium by 2.4-fold and 3.2-fold, respectively. The quality assessment revealed enhanced purity and bioactivity of exosomes produced using the VSCBIC-3-3D protocol. In addition, the cAD-MSCs-derived exosomes were shown to significantly improve fibroblast migration, proliferation, and wound healing-related gene expression in vitro. This study collectively demonstrates that potential upscaling protocol establishment allowed robust production of exosomes from cAD-MSCs, which exhibit therapeutic potential for wound healing in vitro.

We present a novel acoustofluidic chromatography platform for high-throughput nanoparticle trapping and enrichment, with a focus on extracellular vesicles (EVs) from blood plasma. The system features a packed bed of polystyrene beads inside a rectangular glass capillary, acoustically actuated by a piezoelectric element. Using fluorescent polystyrene nanoparticles as small as 25 nm, we characterized device performance across a frequency range of 0.45-4 MHz, demonstrating particle trapping at all tested frequencies. The platform achieved recoveries of up to 42.9 ± 3.2% at input powers as low as 55 mW and operated at high flow rates of up to 200 μL/min. Trapping capacity reached 6.7 × 109 ± 2.5 × 109 particles for 25 nm polystyrene beads. For EV isolation, processing just 4 μL of blood plasma yielded 2 × 108 washed EV-sized particles eluted in 100 μL within 8 min. Micro BCA analysis confirmed a plasma protein background below 2 μg/mL, enabling downstream mass spectrometry. This platform provides an efficient, high-throughput approach for nanoparticle trapping and EV enrichment with minimal sample volumes, offering potential applications in diagnostics and therapeutic development. Future work will focus on optimizing bead properties for EV subpopulation separation and scaling the system for clinical applications.

Interest in Current Good Manufacturing Practices (cGMP)-grade extracellular vesicles (EVs) is expanding. Some obstacles in this new but rapidly growing field include a lack of standardization and scalability. This review focuses on automated biomanufacturing of EVs in conditioned media collected from cultured mesenchymal stromal cells (MSCs). Different automated cell culture systems are discussed, including factors affecting EV quantity and quality, isolating EVs manufactured in an automated system, and validations needed. The ultimate goal when manufacturing cGMP-grade EVs is to identify a specific application and characterize the EV population in detail. This is achieved by validating every step of the process, choosing appropriate release criteria, and assuring batch-to-batch consistency. Due to the lack of standards in the field, it is critical to ensure that the cGMP-grade EVs meet FDA standards pertaining to identity, reproducibility, sterility, safety, purity, and potency. A closed-system automated bioreactor can be a valuable tool to generate cGMP-EVs in a scalable, economical, and reproducible manner.

Exosomes, nanosized extracellular vesicles released by various cell types, are intensively studied for the diagnosis and treatment of cancer and neurodegenerative diseases, and they also display high usability in regenerative medicine. Emphasizing their diagnostic potential, exosomes serve as carriers of disease-specific biomarkers, enabling non-invasive early detection and personalized medicine. The cargo loading of exosomes with therapeutic agents presents an innovative strategy for targeted drug delivery, minimizing off-target effects and optimizing therapeutic interventions. In regenerative medicine, exosomes play a crucial role in intercellular communication, facilitating tissue regeneration through the transmission of bioactive molecules. While acknowledging existing challenges in standardization and scalability, ongoing research efforts aim to refine methodologies and address regulatory considerations. In summary, this review underscores the transformative potential of exosomes in reshaping the landscape of medical interventions, with a particular emphasis on cancer, neurodegenerative diseases, and regenerative medicine.

Extracellular vesicles (EVs) are nanovesicles that facilitate intercellular communication by carrying essential biomolecules under physiological and pathological conditions including microRNAs (miRNAs). They are found in various body fluids, such as blood, urine, and saliva, and their levels fluctuate with disease progression, making them valuable diagnostic tools. However, isolating EVs is challenging due to their small size and biological complexity. Here, we summarize the principles behind the most common EV isolation methods including ultracentrifugation, precipitation, immunoaffinity, sorting, ultrafiltration, size exclusion chromatography, and microfluidics while highlighting protocol strengths and weaknesses. We also review the main strategies to identify and quantify circulating miRNAs with a particular focus on EV-encapsulated miRNAs. Since these miRNAs hold special clinical interest derived from their superior stability and therapeutic potential, the information provided here should provide valuable guidance for future research initiatives in the promising field of disease diagnostic and treatment based on EV-encapsulated miRNAs.

Erectile dysfunction is a common and multifactorial condition that significantly impacts men's quality of life. Traditional treatments, such as phosphodiesterase type 5 inhibitors (PDE5i), often fail to provide lasting benefits, particularly in patients with underlying health conditions. In recent years, regenerative medicine, particularly stem cell therapies, has emerged as a promising alternative for managing erectile dysfunction. This review explores the potential of mesenchymal stromal/stem cells (MSCs) and their paracrine effects, including extracellular vesicles (EVs), in the treatment of erectile dysfunction. MSCs have shown remarkable potential in promoting tissue repair, reducing inflammation, and regenerating smooth muscle cells, offering therapeutic benefits in models of erectile dysfunction. Clinical trials have demonstrated positive outcomes in improving erectile function and other clinical parameters. This review highlights the promise of MSC therapy for erectile dysfunction, discusses existing challenges, and emphasizes the need for continued research to refine these therapies and improve long-term patient outcomes.

Acute liver failure (ALF) is a life-threatening condition caused by rapid hepatocyte death and impaired liver regeneration. Here we show that extracellular vesicles engineered to express Signal Regulatory Protein Alpha (SIRP-EVs), produced via a scalable 3D bioreactor process with high yield and purity, exhibit significant therapeutic potential by targeting damaged cells and promoting tissue repair. SIRP-EVs block CD47, a crucial inhibitory signal on necroptotic cells, to enhance macrophage-mediated clearance of dying hepatocytes. They also deliver regenerative cargo from mesenchymal stem cells, reprogramming macrophages to support liver regeneration. In male animal models, SIRP-EVs significantly reduce liver injury markers and improve survival, demonstrating their dual-function therapeutic efficacy. By integrating the resolution of necroptosis with regenerative macrophage reprogramming, SIRP-EVs represent a promising platform for restoring liver function. These findings support the development of EV-based in vivo macrophage reprogramming therapies for ALF and other inflammation-driven diseases, paving the way for the clinical application of engineered EV therapeutics.

Lactobacillus derived extracellular vesicles (LAB-EVs) are nanosized particles secreted from Lactobacillus during fermentation, and therefore exist universally in fermented foods such as yogurt, pickles, and fermented beverages. In this study, three LAB-EVs were prepared using a simple scalable method, and then their structures, compositions, and biosafety properties were characterized. The protective properties and potential mechanisms of action of the LAB-EVs against alcoholic liver disease were studied. All three LAB-EVs alleviated alcohol-induced liver injury. It was shown by reduction of liver index, histological damage, liver function impairment, inflammation, and liver oxidative status. The results showed that three LAB-EVs positively promoted the diversity of intestinal flora in mice. Additionally, the relative hepatic protein level of Nrf-2, HO-1, and CYP2E1 was also regulated by LAB-EVs. In summary, these facts suggest that the three LAB-EVs can alleviate alcohol-induced liver damage, by positively modulating the intestinal flora and activation of the Nrf-2 signaling pathway. These results may facilitate the understanding of the composition and function of Lactobacillus fermented food and also the development of Lactobacillus fermented functional food.

Extracellular vesicles (EVs) have emerged as promising biomarkers in liquid biopsy, owing to their ubiquitous presence in bodily fluids and their ability to carry disease-related cargo. Recognizing their significance in disease diagnosis and treatment, substantial efforts have been dedicated to developing efficient methods for EV isolation, detection, and analysis. EVs, heterogeneous membrane-encapsulated vesicles secreted by all cells, contain bioactive substances capable of modulating recipient cell biology upon internalization, including proteins, lipids, DNA, and various RNAs. Their prevalence across bodily fluids has positioned them as pivotal mediators in physiological and pathological processes, notably in cancer, where they hold potential as straightforward tumor biomarkers. This review offers a comprehensive examination of advanced nanotechnology-based techniques for detecting lung cancer through EV analysis. It begins by providing a brief overview of exosomes and their role in lung cancer progression. Furthermore, this review explores the evolving landscape of EV isolation and cargo analysis, highlighting the importance of characterizing specific biomolecular signatures within EVs for improved diagnostic accuracy in lung cancer patients. Innovative strategies for enhancing the sensitivity and specificity of EV isolation and detection, including the integration of microfluidic platforms and multiplexed biosensing technologies are summarized. The discussion then extends to key challenges associated with EV-based liquid biopsies, such as the standardization of isolation and detection protocols and the establishment of robust analytical platforms for clinical translation. This review highlights the transformative impact of EV-based liquid biopsy in lung cancer diagnosis, heralding a new era of personalized medicine and improved patient care.

As functional derivatives of mesenchymal stem cells (MSCs), small extracellular vesicles (sEVs) have garnered significant attention and application in regenerative medicine. However, the technical limitations for large-scale isolation of sEVs and their heterogeneous nature have added complexity to their applications. It remains unclear if the heterogeneous sEVs represent different aspects of MSCs functions. Here, we provide a method for the large-scale production of sEVs subpopulations derived from human umbilical cord mesenchymal stem cells (HucMSCs), utilizing tangential flow filtration combined with size exclusion chromatography. The resulting subpopulations, S1-sEVs and S2-sEVs, exhibited stable variations in size, membrane-marked proteins, and carrying cargos, thereby displaying distinct functions both in vitro and in animal disease models. S1-sEVs, that highly expressed CD9, HRS and GPC1, demonstrated a greater immunomodulatory impact, while S2-sEVs with enriched expression of CD63 and FLOT1/2 possessed enhanced capacities in promoting cell proliferation and angiogenesis. These discrepancies are attributed to the specific proteins and miRNAs they contain. Further investigation revealed that the two distinct sEVs subpopulations corresponded to different biological processes: the ESCRT pathway (S1-sEVs) and the ESCRT-independent pathway represented by lipid rafts (S2-sEVs). Therefore, we propose the potential for large-scale isolation and purification of sEVs subpopulations from HucMSCs with distinct functions. This approach may provide advantages for targeted therapeutic interventions in various MSC indications.

Current approaches to oral cancer diagnosis primarily involve physical examination, tissue biopsy, and advanced computer-aided imaging techniques. However, despite these advances, patient survival rates have not significantly improved. Hence, there is a critical need to develop minimally invasive tools with high sensitivity and specificity to improve patient survival and quality of life. Liquid biopsy is a non-invasive, real-time method for predicting cancer status and potentially serves as a biomarker source for treatment response. Liquid biopsy includes rich biologically relevant components, such as circulating tumor cells, circulating tumor DNA, and extracellular vesicles (EVs). EVs are particularly intriguing due to their relatively high abundance in most biofluids, with the potential to identify specific cargo derived from circulating tumor EVs. Moreover, normal cells in lymph nodes can uptake EVs, fostering a pre-metastatic microenvironment that facilitates lymph node metastases - a common occurrence in oral cancers. This review encompasses English language publications over the last twenty years, focusing on methods for isolating EVs from saliva, blood, and lymphatic fluids, as well as the collection methods employed. Seventeen cases met the inclusion criteria according to ISEV guidelines, including 10 saliva cases, 6 blood cases, and 1 lymphatic fluid case. This review also highlighted research gaps in oral squamous cell carcinoma (OSCC) EVs, including a lack of multi-omics studies and the exploration of potential EV markers for drug resistance, as well as a notable underutilization of microfluidic technologies to translate liquid biopsy EV findings into clinical applications.

Exosomes are small, round vesicles in the 30 and 120 nm diameter range released by all living cell types. Exosomes play many essential functions in intercellular communication and tissue crosstalk in the human body. They can potentially be used as strong biomarkers and therapeutic agents for early diagnosis, therapy response, and prognosis of different diseases. The main requirements for exosomal large-scale clinical practice application are rapid, easy, high-yield, high purity, characterization, safety, low cost, and therapeutic efficacy. Depending on the sample types, environmental insults, and exosome quantity, exosomes can be isolated from various sources, including body fluids, solid tissues, and cell culture medium using different procedures. This study comprehensively analyzed the current research progress in exosome isolation and characterization strategies along with their advantages and disadvantages. The provided information will make it easier to select exosome separation methods based on the types of biological samples available, and it will facilitate the use of exosomes in translational and clinical research, particularly in cancer. Lay abstract Exosomes have recently received much attention due to their potential to function as biomarkers and novel therapeutic agents for early diagnosis, therapeutic response, and prognosis in various diseases. This review summarizes many approaches for isolating and characterizing exosomes, focusing on developing technologies, and provides an in-depth comparison and analysis of each method, including its principles, advantages, and limitations.

Extracellular vesicles (EVs) are increasingly investigated for delivering nucleic acid (NA) therapeutics, leveraging their natural role in transporting NA and protein-based cargo in cell-to-cell signaling. Their synthetic counterparts, lipid nanoparticles (LNPs), have been developed over the past decades as NA carriers, culminating in the approval of several marketed formulations such as patisiran/Onpattro® and the mRNA-1273/BNT162 COVID-19 vaccines. The success of LNPs has sparked efforts to develop innovative technologies to target extrahepatic organs, and to deliver novel therapeutic modalities, such as tools for in vivo gene editing. Fueled by the recent advancements in both fields, this review aims to provide a comprehensive overview of the basic characteristics of EV and LNP-based NA delivery systems, from EV biogenesis to structural properties of LNPs. It addresses the primary challenges encountered in utilizing these nanocarriers from a drug formulation and delivery perspective. Additionally, biodistribution profiles, in vitro and in vivo transfection outcomes, as well as their status in clinical trials are compared. Overall, this review provides insights into promising research avenues and potential dead ends for EV and LNP-based NA delivery systems.

Plant-derived extracellular vesicles (PDEVs) are nanoscale vesicles released from plant cells into the extracellular space. While similar in structure and function to mammalian-derived EVs, PDEVs are unique due to their origin and the specific metabolites they carry. PDEVs have gained significant attention in recent years, with numerous reports isolating different PDEVs from various plants, each exhibiting diverse biological functions. However, the field is still in its early stages, and many issues need further exploration. To better develop and utilize PDEVs, it is essential to have a comprehensive understanding of their characteristics. This review provides an overview of recent advances in PDEV research. It focuses on the methods and techniques for isolating and purifying PDEVs, comparing their respective advantages, limitations, and application scenarios. Furthermore, we discuss the latest discoveries regarding the composition of PDEVs, including lipids, proteins, nucleic acids, and various plant metabolites. Additionally, we detail advanced studies on the multiple biological functions of PDEVs. Our goal is to advance our understanding of PDEVs and encourage further exploration in PDEV-based science and technology, offering insights into their potential applications for human health.

Milk-derived extracellular vesicles (mEVs) are emerging as promising therapeutic candidates due to their unique properties and versatile functions. These vesicles play a crucial role in immunomodulation by influencing macrophage differentiation and cytokine production, potentially aiding in the treatment of conditions such as bone loss, fibrosis, and cancer. mEVs also have the capacity to modulate gut microbiota composition, which may alleviate the symptoms of inflammatory bowel diseases and promote intestinal barrier integrity. Their potential as drug delivery vehicles is significant, enhancing the stability, solubility, and bioavailability of anticancer agents while supporting wound healing and reducing inflammation. Additionally, bovine mEVs exhibit anti-aging properties and protect skin cells from UV damage. As vaccine platforms, mEVs offer advantages including biocompatibility, antigen protection, and the ability to elicit robust immune responses through targeted delivery to specific immune cells. Despite these promising applications, challenges persist, including their complex roles in cancer, effective antigen loading, regulatory hurdles, and the need for standardized production methods. Achieving high targeting specificity and understanding the long-term effects of mEV-based therapies are essential for clinical translation. Ongoing research aims to optimize mEV production methods, enhance targeting capabilities, and conduct rigorous preclinical and clinical studies. By addressing these challenges, mEVs hold the potential to revolutionize vaccine development and targeted drug delivery, ultimately improving therapeutic outcomes across various medical fields.

Since chimeric antigen receptor T (CAR-T) cells were introduced three decades ago, the treatment using these cells has led to outstanding outcomes, and at the moment, CAR-T cell therapy is a well-established mainstay for treating CD19 + malignancies and multiple myeloma. Despite the astonishing results of CAR-T cell therapy in B-cell-derived malignancies, several bottlenecks must be overcome to promote its safety and efficacy and broaden its applicability. These bottlenecks include cumbersome production process, safety concerns of viral vectors, poor efficacy in treating solid tumors, life-threatening side effects, and dysfunctionality of infused CAR-T cells over time. Exosomes are nano-sized vesicles that are secreted by all living cells and play an essential role in cellular crosstalk by bridging between cells. In this review, we discuss how the existing bottlenecks of CAR-T cell therapy can be overcome by focusing on exosomes. First, we delve into the effect of tumor-derived exosomes on the CAR-T cell function and discuss how inhibiting their secretion can enhance the efficacy of CAR-T cell therapy. Afterward, the application of exosomes to the manufacturing of CAR-T cells in a non-viral approach is discussed. We also review the latest advancements in ex vivo activation and cultivation of CAR-T cells using exosomes, as well as the potential of engineered exosomes to in vivo induction or boost the in vivo proliferation of CAR-T cells. Finally, we discuss how CAR-engineered exosomes can be used as a versatile tool for the direct killing of tumor cells or delivering intended therapeutic payloads in a targeted manner.

Exosomes, nano-sized extracellular vesicles, have emerged as a promising tool for the diagnosis and treatment of various intractable diseases, including chronic wounds and cancers. As our understanding of exosomes continues to grow, their potential as a powerful therapeutic modality in medicine is also expanding. This systematic review aims to examine the progress of exosome-based clinical trials and provide a comprehensive overview of the therapeutic perspectives of exosomes.

This systematic review strictly follows PRISMA guidelines and has been registered in PROSPERO, the International Prospective Register of Systematic Reviews. It encompasses articles from January 2000 to January 2023, sourced from bibliographic databases, with targeted search terms targeting exosome applications in clinical trials. During the screening process, strict inclusion and exclusion criteria were applied, including a focus on clinical trials utilizing different cell-derived exosomes for therapeutic purposes.

Among the 522 publications initially identified, only 10 studies met the stringent eligibility criteria after meticulous screening. The selection process involved systematically excluding duplicates and irrelevant articles to provide a transparent overview.

According to our systematic review, exosomes have promising applications in a variety of medical fields, including cell-free therapies and drug delivery systems for treating a variety of diseases, especially cancers and chronic wounds. To ensure safety, potency, and broader clinical applications, further optimization of exosome extraction, loading, targeting, and administration is necessary. While cell-based therapeutics are increasingly utilizing exosomes, this field is still in its infancy, and ongoing clinical trials will provide valuable insights into the clinical utility of exosomes.

Extracellular vesicles (EVs) are heterogeneous membrane-like vesicles secreted by living cells that are involved in many physiological and pathological processes and act as intermediaries of intercellular communication and molecular transfer. Recent studies have shown that EVs from specific sources regulate tissue repair and regeneration by delivering proteins, lipids, and nucleic acids to target cells as signaling molecules. Nanotechnology breakthroughs have facilitated the development and exploration of engineered EVs for tissue repair. Enhancements through gene editing, surface modification, and content modification have further improved their therapeutic efficacy. This review summarizes the potential of EVs in tissue repair and regeneration, their mechanisms of action, and their research progress in regenerative medicine. This review highlights their design logic through typical examples and explores the development prospects of EVs in tissue repair. The aim of this review is to provide new insights into the design of EVs for tissue repair and regeneration applications, thereby expanding their use in regenerative medicine.

We have recently made the strikingly discovery that upon a muscle injury, Wnt7a is upregulated and secreted from new regenerating myofibers on the surface of exosomes to elicit its myogenerative response distally. Despite recent advances in extracellular vesicle (EVs) isolation from diverse tissues, there is still a lack of specific methodology to purify EVs from muscle tissue. To eliminate contamination with non-EV secreted proteins and cytoplasmic fragments, which are typically found when using classical methodology, such as ultracentrifugation, we adapted a protocol combining Tangential Flow Filtration (TFF) and Size Exclusion Chromatography (SEC). We found that this approach allows simultaneous purification of Wnt7a, bound to EVs (retentate fraction) and free non-EV Wnt7a (permeate fraction). Here we described this optimized protocol designed to specifically isolate EVs from hind limb muscle explants, without cross-contamination with other sources of non-EV bounded proteins. The first step of the protocol is to remove large EVs with sequential centrifugation. Extracellular vesicles are then concentrated and washed in exchange buffer by TFF. Lastly, SEC is performed to remove any soluble protein traces remaining after TFF. Overall, this procedure can be used to isolate EVs from conditioned media or biofluid that contains EVs derived from any cell type or tissue, improving reproducibility, efficiency, and purity of EVs preparations. Our purification protocol results in high purity EVs that maintain structural integrity and thus fully compatible with in vitro and in vivo bioactivity and analytic assays.

Biomimetic nanoparticles (BMNPs) are innovative nanovehicles that replicate the properties of naturally occurring extracellular vesicles, facilitating highly efficient drug delivery across biological barriers to target organs and tissues while ensuring maximal biocompatibility and minimal-to-no toxicity. BMNPs can be utilized for the delivery of therapeutic payloads and for imparting novel properties to other nanotechnologies based on organic and inorganic materials. The application of specifically modified biological membranes for coating organic and inorganic nanoparticles has the potential to enhance their therapeutic efficacy and biocompatibility, presenting a promising pathway for the advancement of drug delivery technologies. This manuscript is grounded in the fundamentals of biomimetic technologies, offering a comprehensive overview and analytical perspective on the preparation and functionalization of BMNPs, which include cell membrane-coated nanoparticles (CMCNPs), artificial cell-derived vesicles (ACDVs), and fully synthetic vesicles (fSVs). This review examines both "top-down" and "bottom-up" approaches for nanoparticle preparation, with a particular focus on techniques such as cell membrane coating, cargo loading, and microfluidic fabrication. Additionally, it addresses the technological challenges and potential solutions associated with the large-scale production and clinical application of BMNPs and related technologies.

Neurotropic viruses, characterized by their capacity to invade the central nervous system, present a considerable challenge to public health and are responsible for a diverse range of neurological disorders. This group includes a diverse array of viruses, such as herpes simplex virus, varicella zoster virus, poliovirus, enterovirus and Japanese encephalitis virus, among others. Some of these viruses exhibit high neuroinvasiveness and neurovirulence, while others demonstrate weaker neuroinvasive and neurovirulent properties. The clinical manifestations of infections caused by neurotropic viruses can vary significantly, ranging from mild symptoms to severe life-threatening conditions. Extracellular vesicles (EVs) have garnered considerable attention due to their pivotal role in intracellular communication, which modulates the biological activity of target cells via the transport of biomolecules in both health and disease. Investigating EVs in the context of virus infection is crucial for elucidating their potential role contribution to viral pathogenesis. This is because EVs derived from virus-infected cells frequently transfer viral components to uninfected cells. Importantly, EVs released by virus-infected cells have the capacity to traverse the blood-brain barrier (BBB), thereby impacting neuronal activity and inducing neuroinflammation. In this review, we explore the roles of EVs during neurotropic virus infections in either enhancing or inhibiting viral pathogenesis. We will delve into our current comprehension of the molecular mechanisms that underpin these roles, the potential implications for the infected host, and the prospective diagnostic applications that could arise from this understanding.

For the past two decades researchers have linked extracellular vesicle (EV)-mediated mechanisms to various physiological and pathological processes in the heart, such as immune response regulation, fibrosis, angiogenesis, and the survival and growth of cardiomyocytes. Although use of EVs has gathered momentum in the cardiac field, several obstacles in both upstream and downstream processes during EV manufacture need to be addressed before clinical success can be achieved. Low EV yields obtained in small-scale cultures deter clinical translation, as mass production is a prerequisite to meet therapeutic doses. Moreover, standardizing EV manufacture is critical given the inherent heterogeneity of EVs and the constraints of current isolation techniques. In this review, we discuss the critical steps for the large-scale manufacturing of high-potency EVs for cardiac therapies.

Extracellular vesicles (EVs) are natural carriers of biomolecules that play a crucial role in cell-to-cell communication and tissue homeostasis under normal and pathological conditions, including inflammatory diseases and cancer. Since the discovery of the pro-regenerative and immune-modulating properties of EVs, EV-based therapeutics have entered clinical trials for conditions such as myocardial infarction and autoimmune diseases, among others. Due to their unique advantages-such as superior bioavailability, substantial packaging capacity, and the ability to traverse biological barriers-EVs are regarded as a promising platform for targeted drug delivery. However, achieving a sufficient accumulation of therapeutic agents at the target site necessitates a larger quantity of EVs per dose compared to using EVs as standalone drugs. This challenge can be addressed by administering larger doses of EVs, increasing the drug dosage per administration, or enhancing the selective accumulation of EVs at target cells. In this review, we will discuss methods to improve the isolation and purification of EVs, approaches to enhance cargo packaging-including proteins, RNAs, and small-molecule drugs-and technologies for displaying targeting ligands on the surface of EVs to facilitate improved targeting. Ultimately, this guide can be applied to the development of novel classes of EV-based therapeutics and to overcoming existing technological challenges.

Dental, oral, and craniofacial diseases can substantially impact the quality of human life, thereby posing a serious public health concern. Although conventional therapies such as surgery have solved these problems largely, the prognosis of patients is not always satisfactory. Cell membrane-coated nanoparticles (CMCNPs) carry nanodrugs with the help of natural cell membranes, therefore utilizing their remarkable ability to interface and interact with their surrounding environment. These nanoparticles have demonstrated substantial advantages in drug targeting, prolonging blood circulation time, penetrating biofilms, and immune escape. With the assistance of CMCNPs, the therapeutic effects of dental, oral, and craniofacial diseases can reach a higher level. CMCNPs have been applied for dental, oral, and craniofacial diseases for various conditions such as head and neck cancer, periodontal disease, and oral biosignal detection. For the therapies of head and neck cancer, CMCNPs have been widely utilized as a tool of chemotherapy, phototherapy, and immunotherapy, while yet to be exploited in imaging technique. In the end, we summarized the challenges and prospectives of CMCNPs for dental, oral, and craniofacial diseases: large-scale production with uniform standards and high quantity, extensive application directions in dental, oral, and craniofacial regions (implant, endodontics), and the promotion of its clinical application.

Extracellular vesicles (EVs) are nanovesicles containing bioactive molecules including proteins, nucleic acids and lipids that mediate intercellular and inter-organ communications, holding promise as potential therapeutics for multiple diseases. Adipose tissue (AT) serves as a dynamically distributed energy storage organ throughout the body, whose accumulation leads to obesity, a condition characterized by infiltration with abundant immune cells. Emerging evidence has illustrated that EVs secreted by AT are the novel class of adipokines that regulate the homeostasis between AT and peripheral organs. However, most of the studies focused on the investigations of EVs derived from adipocytes or adipose-derived stem cells (ADSCs), the summarization of functions in cellular and inter-organ crosstalk of EVs directly derived from adipose tissue (AT-EVs) are still limited. Here, we provide a systemic summary on the key components and functions of EVs derived from healthy adipose tissue, showing their significance on the tissue recovery and metabolic homeostasis regulation. Also, we discuss the harmful influences of EVs derived from obese adipose tissue on the distal organs. Furthermore, we elucidate the potential applications and constraints of EVs from healthy patients lipoaspirates as therapeutic agents, highlighting the potential of AT-EVs as a valuable biological material with broad prospects for future clinical use.

Small extracellular vesicles (sEVs) are proven to hold great promise for diverse therapeutic and diagnostic applications. However, batch preparation of sEVs with high purity and bioactivity is a prerequisite for their clinical translations. Herein, we present an electric field assisted tangential flow filtration system (E-TFF), which integrates size-based filtration with electrophoretic migration-based separation to synergistically achieve the isolation of high-quality sEVs from cell culture medium. Compared with the gold-standard ultracentrifugation (UC) method, E-TFF not only improved the purity of sEVs by 1.4 times but also increased the yield of sEVs by 15.8 times. Additionally, the entire isolation process of E-TFF was completed within 1 h, about one-fourth of the time taken by UC. Furthermore, the biological activity of sEVs isolated by E-TFF was verified by co-incubation of sEVs derived from human umbilical cord mesenchymal stem cells (hUCMSCs) with HT22 mouse hippocampal neuronal cells exposed to amyloid-β (Aβ). The results demonstrated that the sEVs isolated by E-TFF exhibited a significant neuroprotective effect. Overall, the E-TFF platform provides a promising and robust strategy for batch preparation of high-quality sEVs, opening up a broad range of opportunities for cell-free therapy and precision medicine.

Recent decades have witnessed substantial interest in extracellular vesicles (EVs) due to their crucial role in intercellular communication across various biological processes. Among these, plant-derived exosome-like Nanoparticles (ELNs) have rapidly gained recognition as highly promising candidates. ELNs, characterized by diverse sources, cost-effective production, and straightforward isolation, present a viable option for preventing and treating numerous diseases. Furthermore, ELNs hold significant potential as carriers for natural or engineered drugs, enhancing their attractiveness and drawing considerable attention in science and medicine. However, translating ELNs into clinical applications poses several challenges. This study explores these challenges and offers critical insights into potential research directions. Additionally, it provides a forward-looking analysis of the industrial prospects for ELNs. With their broad applications and remarkable potential, ELNs stand at the forefront of biomedical innovation, poised to revolutionize disease management and drug delivery paradigms in the coming years.

Extracellular vesicles (EVs) are promising next-generation therapeutics and drug delivery systems due to demonstrated safety and efficacy in preclinical models and early-stage clinical trials. There is an urgent need to address the immunogenicity of EVs (beyond the apparent lack of immunotoxicity) to advance clinical development. To date, few studies have assessed unintended immunological recognition of EVs. An in-depth understanding of EV-induced immunogenicity and clearance is necessary to develop effective therapeutic strategies, including approaches to mitigate immunological recognition when undesired. This article summarizes various factors involved in the potential immunogenicity of EVs and strategies to reduce immunological recognition for improved therapeutic benefit.