Obesity increases the risk of depression. Evidence shows that peripheral inflammation, glycemic dysregulation, and hyperactivity within the hypothalamic-pituitary-adrenal axis are implicated in both obesity and depression. In this study we investigated the impact of visceral adipose tissue (VAT), a crucial characteristic of obesity, on stress susceptibility in obese mice. Age-matched mice were fed with chow diet (CD) or high-fat diet (HFD), respectively, for 12 weeks. CD mice were deprived of VAT and received transplantation of VAT from HFD mice (TransHFD) or CD mice (TransCD). Extracellular vesicles (EVs) were prepared from VAT of CD or HFD mice, and intravenously injected (100 μg, 4 times in 2 weeks) in naïve mice or injected into hippocampus (5 μg, 4 times in 2 weeks) through implanted bilateral cannula. Depression-like behaviors were assessed 14 days after transplantation. We showed that HFD mice exhibited significantly higher body weight gain and impaired insulin and glucose tolerance, accompanied by increased stress susceptibility. Transplantation of VAT or VAT-derived EVs from HFD mice caused synaptic damage and promoted stress susceptibility in recipient mice. Through inhibiting miRNA biogenesis in the VAT and miRNA sequencing analysis, we demonstrated that miR-140-5p was significantly upregulated in both VAT-EVs and hippocampus of HFD mice. Overexpression of hippocampal miR-140-5p in naïve mice not only facilitated acute stress-induced depression-like behaviors, but also decreased hippocampal CREB-BDNF signaling cascade and synaptic plasticity. Conversely, knockdown of miR-140-5p in the VAT, VAT-EVs or hippocampus of HFD mice protected against acute stress, reducing stress susceptibility that were mediated via CREB-BDNF pathway. In summary, VAT-EVs or the cargo miRNAs in obese mice promote synaptic damage and stress susceptibility, providing potential therapeutic targets for metabolism-related affective disorders.

In the recent decade, extracellular vesicles (EVs) have been released from nearly all the kingdoms, modulating intercellular communication and maintaining the human body's homeostasis by regulating different cellular processes. Among EVs, exosomes are the emerging field in biopharmaceuticals. They have lipid bilayer ranging from 30 to 150 nm in size and encompass DNA, RNA, protein lipids, etc. Their sources are widespread, easy to acquire, and cost-effective in manufacturing. This review focuses on the detailed classification of exosomes existing in nature, knowledge and application of omics, therapeutic, diagnostic and theranostic application of exosomes. It covers diseases such as cancer, infectious diseases (viral, bacterial, fungal infections), neurodegenerative diseases, metabolic diseases, lifestyle diseases (diabetes, cardiovascular, gastric disorder (IBD)), autoimmune disorders and their biodistribution. This article unfolds the recent progress in the exosomes arena and covers all the regulatory considerations (FDA, EMA, and other nations) involved with it. Moreover, a detailed discussion about clinical trials and its manifestation with exosomes and challenges associated with their isolation procedures, reproducibility, and safety concerns.

Although osteoarthritis (OA) is the most prevalent form of arthritis, there is still no effective treatment capable of combining immunomodulatory effects with cartilage repair. Extracellular vesicles (EVs) represent a promising new generation of cell-free therapies for OA. Blood-derived products, including plasma, are an easily available and abundant source of EVs with anti-inflammatory and regenerative properties. In this study, our objective was to analyze the effect of platelet poor plasma-derived extracellular vesicles (PPP-EVs) on stimulated OA chondrocytes in vitro. We hypothesize that PPP from healthy donors could be a suitable source of EVs that can modulate the inflammatory environment of OA chondrocytes.

Cartilage and synovial membrane (SM) were obtained from patients with OA and whole blood from healthy donors. Synovial membrane-conditioned media (CM / SM) was analyzed using multiplex immunoassays. EVs were isolated from PPP using size exclusion chromatography (SEC) and characterized by nanoparticle tracking analysis (NTA), Western blot, and flow cytometry (FC). The phenotype of the chondrocytes was analyzed using fluorescence microscopy and RT-qPCR. Chondrocytes were stimulated with IL-1β or CM/SM for 24 h. The impact of PPP-EVs on stimulated chondrocyte gene expression was evaluated using RT-qPCR.

The PPP-EVs isolated by SEC were positive for the tetraspanins CD9, CD63, and CD81. The chondrocyte phenotype was confirmed by positive expression of Collagen II and Aggrecane. CM/SM and IL-1β caused inflammatory changes in chondrocytes, which was observed by increased expression of the genes MMP-1, MMP-3 and MMP-13, RANTES, TSG-6, and YKL-40 compared to the control. PPP-EVs added to stimulated chondrocytes for 24 h significantly decreased the expression of the chondrocyte gene YKL-40, TSG-6 and MMP-1.

In this study, we confirmed that PPP is a suitable source of EVs, which can be efficiently isolated by SEC. We found that PPP-EVs were capable of decreasing the expression of inflammatory genes in OA chondrocytes stimulated with IL-1β or CM/SM. This study provides preliminary results on PPP-EVs as an affordable and promising option to modulate the catabolic microenvironment of OA chondrocytes in vitro.

Extracellular vesicles (EVs) are promising therapeutic biomaterials capable of transferring their cargo molecules and external drugs to other cells in vivo and contain various biomarkers that can be used in liquid biopsies. The clinical application of EVs requires an efficient EV enrichment system for the large-scale production or high-throughput isolation of EVs from liquid samples, such as culture media, plant juices, and body fluids. However, current EV enrichment methods, such as ultrafiltration and ultracentrifugation, have limited applicability owing to their associated costs, inefficiency, scalability, and centrifugation time. Herein, we describe the development of a nanosieve based on a superabsorbent polymer for selective EV enrichment. The nanosieve absorbs small molecules while expelling large molecules, such as EVs, through the nanosized channels. We successfully concentrated EVs from clinical samples, such as serum and plasma, with superior cost and time efficiencies. The nanosieves did not interact with the EVs during enrichment, allowing the retention of their therapeutic functions. In addition, the nanosieve surface was specifically engineered to provide multifunctionality to effectively promote EV capture from bulk solutions. Overall, our nanosieve-based EV enrichment method is effective, time- and cost-saving, versatile, scalable, and modulable, and is an excellent option for EV production.

Topology and bioactive molecules are crucial for stimulating cellular and tissue functions. To regulate the chronic wound microenvironment, mono-assembly technology is employed to fabricate a radial egg white hydrogel loaded with lyophilized adipose tissue-extracellular vesicles (radial EWH@L-EVs). The radial architecture not only significantly modified the gene expression of functional cells, but also achieved directional and controlled release kinetics of L-EVs. Through the synergy of topographical and inherent bioactive cues, radial EWH@L-EVs effectively reduced intracellular oxidative stress and promoted the polarization of macrophages toward an anti-inflammatory phenotype during the inflammatory phase. Afterward, radial EWH@L-EVs facilitated the centripetal migration and proliferation of fibroblasts and endothelial cells as the wound transitioned to the proliferative phase. During the latter remodeling phase, radial EWH@L-EVs accelerated the regeneration of granulation tissue, angiogenesis, and collagen deposition, thereby promoting the reorganization chronic wound. Compared with the gold standard collagen scaffold, radial EWH@L-EVs actively accommodated the microenvironment via various functions throughout all stages of diabetic wound healing. This can be attributed to the orientation of topological structures and bioactive molecules, which should be considered of utmost importance in tissue engineering.

Diabetic foot ulcers (DFUs) represent a serious complication of diabetes with high incidence, requiring intensive treatment, prolonged hospitalization, and high costs. It poses a severe threat to the patient's life, resulting in substantial burdens on patient and healthcare system. However, the therapy of DFUs remains challenging. Therefore, exploring cell-free therapies for DFUs is both critical and urgent. Exosomes, as crucial mediators of intercellular communication, have been demonstrated potentially effective in anti-inflammation, angiogenesis, cell proliferation and migration, and collagen deposition. These functions have been proven beneficial in all stages of diabetic wound healing. This review aims to summarize the role and mechanisms of exosomes from diverse cellular sources in diabetic wound healing research. In addition, we elaborate on the challenges for clinical application, discuss the advantages of membrane vesicles as exosome mimics in wound healing, and present the therapeutic potential of exosomes and their mimetic vesicles for future clinical applications.

Rescuing or compensating mitochondrial function represents a promising therapeutic avenue for radiation-induced chronic wounds. Adult stem cell efficacies are primarily dependent on the paracrine secretion of mitochondria-containing extracellular vesicles (EVs). However, effective therapeutic strategies addressing the quantity of mitochondria and mitochondria-delivery system are lacking. Thus, in this study, we aimed to design an effective hydrogel microneedle patch (MNP) loaded with stem cell-derived mitochondria-rich EVs to gradually release and deliver mitochondria into the wound tissues and boost wound healing. We, first, used metformin to enhance mitochondrial biogenesis and thereby increasing the secretion of mitochondria-containing EVs (termed "Met-EVs") in adipose-derived stem cells. To verify the therapeutic effects of Met-EVs, we established an in vitro and an in vivo model of X-ray-induced mitochondrial dysfunction. The Met-EVs ameliorated the mitochondrial dysfunction by rescuing mitochondrial membrane potential, increasing adenosine 5'-triphosphate levels, and decreasing reactive oxygen species production by transferring active mitochondria. To sustain the release of EVs into damaged tissues, we constructed a Met-EVs@Decellularized Adipose Matrix (DAM)/Hyaluronic Acid Methacrylic Acid (HAMA)-MNP. Met-EVs@DAM/HAMA-MNP can load and gradually release Met-EVs and their contained mitochondria into wound tissues to alleviate mitochondrial dysfunction. Moreover, we found Met-EVs@DAM/HAMA-MNP can markedly promote macrophage polarization toward the M2 subtype with anti-inflammatory and regenerative functions, which can, in turn, enhance the healing process in mice with skin wounds combined radiation injuries. Collectively, we successfully fabricated a delivery system for EVs, Met-EVs@DAM/HAMA-MNP, to effectively deliver stem cell-derived mitochondria-rich EVs. The effectiveness of this system has been demonstrated, holding great potential for chronic wound treatments in clinic.

Extracellular vesicles (EVs) are lipid bilayer-enclosed vesicles released by cells. EVs encapsulate proteins and nucleic acids of their parental cell and efficiently deliver the cargo to recipient cells. These vesicles act as mediators of intercellular communication and thus play a crucial role in various physiological and pathological processes. Moreover, EVs hold promise for clinical use. They have been explored as drug delivery vehicles, therapeutic agents, and targets for disease diagnosis. In the landscape of cancer research, while strides have been made in EV-focused cancer physiopathology, liquid biopsy, and drug delivery, the exploration of EVs as immunotherapeutic agents may not have seen substantial progress to date. Despite promising findings reported in cell and animal studies, the clinical translation of EV-based cancer immunotherapeutics encounters challenges. Here, we review the existing strategies used in EV-based cancer immunotherapy, aiming to propel the development of this emerging yet crucial field.

Obesity is a major public health issue that is associated with metabolic diseases including diabetes mellitus type 2 and metabolic syndrome. This pathology leads to detrimental cardiovascular health and secondary effects, such as lipotoxicity, inflammation, and oxidative stress. Recently, extracellular vesicles (EVs) have been highlighted as novel players participating in human physiology and pathophysiology. In obesity, adipose tissue is related to the active shedding of adipocyte-derived extracellular vesicles (AdEVs). The current review explores and highlights the role of AdEVs and their cargo in obesity and metabolic syndrome. AdEVs are proposed to play an important role in obesity and its comorbidities. AdEVs are biological nanoparticles mainly shed by visceral and subcutaneous adipose tissue, acting in physiological and pathophysiological conditions, and also carrying different cargo biomolecules, such as RNA, microRNA (miRNA), proteins, and lipids, among others. RNA and miRNA have local and systemic effects affecting gene expression in target cell types via paracrine and endocrine actions. State of the art analyses identified some miRNAs, such as miR-222, miR-23b, miR-4429, miR-148b, and miR-4269, that could potentially affect cell pathways involved in obesity-related comorbidities, such as chronic inflammation and fibrosis. Similarly, AdEVs-proteins (RBP4, perilipin-A, FABP, mimecan, TGFBI) and AdEVs-lipids (sphingolipids) have been linked to the obesity pathophysiology. The current knowledge about AdEVs along with further research would support and reveal novel pathways, potential biomarkers, and therapeutic options in obesity.

Extracellular vesicles (EVs) are nanosized biomolecular packages involved in intercellular communication. EVs are released by all cells, making them broadly applicable as therapeutic, diagnostic, and mechanistic components in (patho)physiology. Sample purity is critical for correctly attributing observed effects to EVs and for maximizing therapeutic and diagnostic performance. Lipoprotein contaminants represent a major challenge for sample purity. Lipoproteins are approximately six orders of magnitude more abundant in the blood circulation and overlap in size, shape, and density with EVs. This study represents the first example of an EV purification method based on the chemically-induced breakdown of lipoproteins. Specifically, a styrene-maleic acid (SMA) copolymer is used to selectively breakdown lipoproteins, enabling subsequent size-based separation of the breakdown products from plasma EVs. The use of the polymer followed by tangential flow filtration or size-exclusion chromatography results in improved EV yield, preservation of EV morphology, increased EV markers, and reduced contaminant markers. SMA-based EV purification enables improved fluorescent labeling, reduces interactions with macrophages, and enhances accuracy, sensitivity, and specificity to detect EV biomarkers, indicating benefits for various downstream applications. In conclusion, SMA is a simple and effective method to improve the purity and yield of plasma-derived EVs, which favorably impacts downstream applications.

Neonatal disorders, particularly those resulting from prematurity, pose a major challenge in health care and have a significant impact on infant mortality and long-term child health. The limitations of current therapeutic strategies emphasize the need for innovative treatments. New cell-free technologies utilizing extracellular vesicles (EVs) offer a compelling opportunity for neonatal therapy by harnessing the inherent regenerative capabilities of EVs. These nanoscale particles, secreted by a variety of organisms including animals, bacteria, fungi and plants, contain a repertoire of bioactive molecules with therapeutic potential. This review aims to provide a comprehensive assessment of the therapeutic effects of EVs and mechanistic insights into EVs from stem cells, biological fluids and non-animal sources, with a focus on common neonatal conditions such as hypoxic-ischemic encephalopathy, respiratory distress syndrome, bronchopulmonary dysplasia and necrotizing enterocolitis. This review summarizes evidence for the therapeutic potential of EVs, analyzes evidence of their mechanisms of action and discusses the challenges associated with the implementation of EV-based therapies in neonatal clinical practice.

Extracellular vesicles (EVs) are cell-released vesicles that mediate intercellular communication by transferring bioactive cargo. Protein and RNA sorting into EVs has been extensively assessed, while selective enrichment of glycans in EVs remains less explored. In this study, a mass spectrometry-based approach, glycan node analysis (GNA), was applied to broadly assess the sorting of glycan features into EVs. Two metastatic variants (lung and bone) generated in mouse modes from the MDA-MB-231 human breast cancer cell line were assessed, as these EVs are known to contain distinct organotropic biomolecules. EVs were isolated from conditioned cell culture medium by tangential flow filtration and authenticated by standard techniques. GNA analysis revealed selective enrichment of several glycan features in EVs compared to the originating cells, particularly those associated with binding to the extracellular matrix, which was also observed in EVs from the parental MDA-MB-231 cell line (human pleural metastases). The bone-tropic variant displayed enrichment of distinct EV glycan features compared to the lung-tropic one. Additionally, the metastatic variants generated in mouse models displayed reduced EV glycan sorting compared to the parental metastatic cell line. This study represents the first comprehensive assessment of differences in glycan features between EVs and originating cells and provides evidence that the diversity of EV glycan sorting is reduced upon generation of variant cell lines in mouse models. Future research is likely to uncover novel mechanisms of EV glycan sorting, shed light on glycan features for EV authentication or biomarker purposes, and assess functional roles of the EV glycocode in (patho)physiology.

Extracellular vesicles and lipoproteins are lipid-based biological nanoparticles that play important roles in (patho)physiology. Recent evidence suggests that extracellular vesicles and lipoproteins can interact to form functional complexes. Such complexes have been observed in biofluids from healthy human donors and in various in vitro disease models such as breast cancer and hepatitis C infection. Lipoprotein components can also form part of the biomolecular corona that surrounds extracellular vesicles and contributes to biological identity. Potential mechanisms and the functional relevance of extracellular vesicle-lipoprotein complexes remain poorly understood. This Review addresses the current knowledge of the extracellular vesicle-lipoprotein interface while drawing on pre-existing knowledge of liposome interactions with biological nanoparticles. There is an urgent need for further research on the lipoprotein-extracellular vesicle interface, which could return important mechanistic, therapeutic, and diagnostic findings.

Extracellular vesicles (EVs) are biological nanoparticles that promote intercellular communication by delivering bioactive cargo over short and long distances. Short-distance communication takes place in the interstitium, whereas long-distance communication is thought to require transport through the blood circulation to reach distal sites. Extracellular vesicle therapeutics are frequently injected systemically, and diagnostic approaches often rely on the detection of organ-derived EVs in the blood. However, the mechanisms by which EVs enter and exit the circulation are poorly understood. Here, the lymphatic system and transport across the endothelial barrier through paracellular and transcellular routes are discussed as potential pathways for EV entry to and exit from the blood circulatory system.

Extracellular vesicles (EVs) and lipoproteins (LPPs) serve as important carriers of circulating miRNAs in peripheral blood, offering immense potential for disease diagnosis and therapeutic interventions. Due to their shared physicochemical attributes, EVs and LPPs are frequently coisolated, potentially leading to misunderstandings regarding their distinct functional roles in physiological and pathological processes. Here, we report a highly selective magnetic system based on the pH-mediated affinity displayed by cibacron blue (CB) toward EVs and LPPs, enabling successful separation and collection of these two nanoparticles without cross-contamination for subsequent circulating RNA analysis. First, we found that CB-modified magnetic beads (CBMBs) exhibit a strong affinity toward LPP particles while displaying little interaction with EVs in standard samples under physiological pH conditions. We further demonstrate that the affinity between CB molecules and bionanoparticles in plasma samples is highly pH-dependent. Specifically, CBMBs show affinities for both LPP and EV particles under neutral and acidic conditions. However, at basic pH levels, CB molecules selectively bind only to LPP particles. Consequently, the remaining EV particles present in plasma are subsequently isolated by using titanium dioxide-modified beads (TiMBs) through phospholipid affinity. The simultaneous analysis of the transcriptomic contents of EV and LPP reveals clear differences in their small RNA profiles, with the differentially expressed RNAs reflecting distinct biological processes. Significantly, in a proof-of-concept study, we successfully demonstrated a strong correlation between miRNAs carried by both EV and LPP particles with the occurrence of ocular neovascularization during the progression of diabetic retinopathy. The involved miRNAs may serve as potential biomarkers for DR diagnostics and severity classification. To sum up, this pH-mediated separation system is not only user-friendly but also highly compatible, rendering it a potent tool for probing the molecular compositions, biomarkers, and underlying biological mechanisms of EVs and LPPs.

Extracellular vesicles (EVs) are phospholipid bilayer nano-vesicles that were originally identified to deliver signals for intercellular communications. Based on the dynamic contents including proteins, nucleic acids and metabolites, EVs have been developed into diagnostic and therapeutic fields including cardiovascular diseases, neurological disorders and infectious diseases. A growing number of investigations revealed that EVs are also powerful carriers of loaded compounds and nucleic acids as enhanced treatments. Herein, we summarized the recent engineering advances related to three major issues when applying EVs in drug delivery systems: EVs isolation, drug loading strategies and targeting delivery approaches. Moreover, current applications of traditional Chinese medicine (TCM), in composition or compound form, are searched and listed as unique combinations with EVs. Further, we discuss emerging challenges and consider future directions of drug-loading EVs in therapeutic opportunities. This review discusses pros and cons of collecting, drug loading and delivery strategies of EVs as delivery systems, and highlights the promising combination with traditional Chinese medicine to help us advance its clinical application.

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.

Over the past decades, there has been an exponential increase in the development of preclinical and clinical nanodelivery systems, and recently, an accelerating demand to deliver RNA and protein-based therapeutics. Organ-specific vasculature provides a promising intermediary for site-specific delivery of nanoparticles and extracellular vesicles to interstitial cells. Endothelial cells express organ-specific surface marker repertoires that can be used for targeted delivery. This article highlights organ-specific vasculature properties, nanodelivery strategies that exploit vasculature organotropism, and overlooked challenges and opportunities in targeting and simultaneously overcoming the endothelial barrier. Impediments in the clinical translation of vasculature organotropism in drug delivery are also discussed.

The variable presence of contaminants in extracellular vesicle (EV) samples is one of the major contributors to a lack of inter-study reproducibility in the field. Well-known contaminants include protein aggregates, RNA-protein complexes and lipoproteins, which resemble EVs in shape, size and/or density. On the contrary, polysaccharides, such as hyaluronic acid (HA), have been overlooked as EV contaminants. Here, it is shown that low and medium molecular weight HA polymers are unexpectedly retained to some extent in EV fractions using two common isolation methods known for high purity: size-exclusion chromatography and tangential flow filtration. Although these isolation techniques are capable of efficient removal of non-EV-associated proteins, this is not the case for HA polymers, which are partially retained in a molecular weight-dependent manner, especially with size-exclusion chromatography. The supramolecular structure and hydrodynamic size of HA are likely to contribute to isolation in EV fractions of filtration-based approaches. Conversely, HA polymers were not retained with ultracentrifugation and polymer-based precipitation methods, which are known for co-isolating other types of contaminants. HA has a broad range of immunomodulatory effects, similar to those ascribed to various sources of EVs. Therefore, HA contaminants should be considered in future studies to avoid potential inaccurate attributions of functional effects to EVs.

Extracellular vesicles (EVs) are released from all cells in the body, forming an important intercellular communication network that contributes to health and disease. The contents of EVs are cell source-specific, inducing distinct signaling responses in recipient cells. The specificity of EVs and their accumulation in fluid spaces that are accessible for liquid biopsies make them highly attractive as potential biomarkers and therapies for disease. The duality of EVs as favorable (therapeutic) or unfavorable (pathological) messengers is context dependent and remains to be fully determined in homeostasis and various disease states. This review describes the use of EVs as biomarkers, drug delivery vehicles, and regenerative therapeutics, highlighting examples involving viral infections, cancer, and neurological diseases. There is growing interest to provide personalized therapy based on individual patient and disease characteristics. Increasing evidence suggests that EV biomarkers and therapeutic approaches are ideal for personalized medicine due to the diversity and multifunctionality of EVs.

Extracellular vesicles (EVs) play important roles in (patho)physiological processes by mediating cell communication. Although EVs contain glycans and glycosaminoglycans (GAGs), these biomolecules have been overlooked due to technical challenges in comprehensive glycome analysis coupled with EV isolation. Conventional mass spectrometry (MS)-based methods are restricted to the assessment of N-linked glycans. Therefore, methods to comprehensively analyze all glyco-polymer classes on EVs are urgently needed. In this study, tangential flow filtration-based EV isolation was coupled with glycan node analysis (GNA) as an innovative and robust approach to characterize most major glyco-polymer features of EVs. GNA is a molecularly bottom-up gas chromatography-MS technique that provides unique information that is unobtainable with conventional methods. The results indicate that GNA can identify EV-associated glyco-polymers that would remain undetected with conventional MS methods. Specifically, predictions based on GNA identified a GAG (hyaluronan) with varying abundance on EVs from two different melanoma cell lines. Enzyme-linked immunosorbent assays and enzymatic stripping protocols confirmed the differential abundance of EV-associated hyaluronan. These results lay the framework to explore GNA as a tool to assess major glycan classes on EVs, unveiling the EV glycocode and its biological functions.

Extracellular vesicles (EVs) are cell-released, heterogenous nanoparticles that play important roles in (patho)physiological processes through intercellular communication. EVs are often depicted as having a single lipid bilayer, but many studies have demonstrated the existence of multilayered EVs. There has been minimal inquiry into differences between unilamellar and multilamellar EVs in terms of biogenesis mechanisms and functional effects. This commentary speculates on potential causes and roles of multilamellar EVs and serves as a call to action for the research community to unravel the complex layers of EVs.

The native role of extracellular vesicles (EVs) in mediating the transfer of biomolecules between cells has raised the possibility to use them as therapeutic vehicles. The development of therapies based on EVs is now expanding rapidly; here we will describe the current knowledge on different key points regarding the use of EVs in a clinical setting. These points are related to cell sources of EVs, isolation, storage, and delivery methods, as well as modifications to the releasing cells for improved production of EVs. Finally, we will depict the application of EVs therapies in clinical trials, considering the impact of the COVID-19 pandemic on the development of these therapies, pointing out that although it is a promising therapy for human diseases, we are still in the initial phase of its application to patients.

Background: Treatment of diabetic wounds is a major challenge in clinical practice. Extracellular vesicles (EVs) from adipose-derived stem cells have shown effectiveness in diabetic wound models. However, obtaining ADSC-EVs requires culturing vast numbers of cells, which is hampered by the need for expensive equipment and reagents, extended time cost, and complicated procedures before commercialization. Therefore, methods to extract EVs from discarded tissue need to be developed, for immediate application during surgery. For this reason, mechanical, collagenase-digestive, and constant in-vitro-collective methods were designed and compared for preparing therapy-grade EVs directly from adipose tissue. Methods: Characteristics and quantities of EVs were detected by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting firstly. To investigate the biological effects of EVs on diabetic wound healing, angiogenesis, proliferation, migration, and inflammation-regulation assays were then evaluated in vitro, along with a diabetic wound healing mouse model in vivo. To further explore the potential therapeutic mechanism of EVs, miRNA expression profile of EVs were also identified and analyzed. Results: The adipose tissue derived EVs (AT-EVs) were showed to qualify ISEV identification by nanoparticle tracking analysis and Western blotting and the AT-EVs yield from three methods was equal. EVs also showed promoting effects on biological processes related to diabetic wound healing, which depend on fibroblasts, keratinocytes, endothelial cells, and macrophages both in vitro and in vivo. We also observed enrichment of overlapping or unique miRNAs originate from different types of AT-EVs associated with diabetic wound healing for further investigation. Conclusion: After comparative analyses, a mechanical method was proposed for preparing immediate clinical applicable EVs from adipose tissue that would result in reduced preparation time and lower cost, which could have promising application potential in treating diabetic wounds.

Joint diseases are one of the most common causes of morbidity and disability worldwide. The main diseases that affect joint cartilage are osteoarthritis and rheumatoid arthritis, which require chronic treatment focused on symptomatic relief. Conventional drugs administered through systemic or intra-articular routes have low accumulation and/or retention in articular cartilage, causing dose-limiting toxicities and reduced efficacy. Therefore, there is an urgent need to develop improved strategies for drug delivery, in particular, the use of micro- and nanotechnology-based methods. Encapsulation of therapeutic agents in delivery systems reduces drug efflux from the joint and protects against rapid cellular and enzymatic clearance following intra-articular injection. Consequently, the use of drug delivery systems decreases side effects and increases therapeutic efficacy due to enhanced drug retention in the intra-articular space. Additionally, the frequency of intra-articular administration is reduced, as delivery systems enable sustained drug release. This review summarizes various advanced drug delivery systems, such as nano- and microcarriers, developed for articular cartilage diseases.

The incidence of high-risk Human Papillomavirus (HR-HPV)-driven head and neck squamous cell carcinoma (HNSCC) is on the rise globally. HR-HPV-driven HNSCC displays molecular and clinical characteristics distinct from HPV-uninvolved cases. Therapeutic strategies for HR-HPV-driven HNSCC are under investigation. HR-HPVs encode the oncogenes E6 and E7, which are essential in tumorigenesis. Meanwhile, involvement of E6 and E7 provides attractive targets for developing new therapeutic regimen. Here we will review some of the recent advancements observed in preclinical studies and clinical trials on HR-HPV-driven HNSCC, focusing on nanotechnology related methods. Materials science innovation leads to great improvement for cancer therapeutics including HNSCC. This article discusses HPV-E6 or -E7- based vaccines, based on plasmid, messenger RNA or peptide, at their current stage of development and testing as well as how nanoparticles can be designed to target and access cancer cells and activate certain immunology pathways besides serving as a delivery vehicle. Nanotechnology was also used for chemotherapy and photothermal treatment. Short interference RNA targeting E6/E7 showed some potential in animal models. Gene editing by CRISPR-CAS9 combined with other treatments has also been assessed. These advancements have the potential to improve the outcome in HR-HPV-driven HNSCC, however breakthroughs are still to be awaited with nanomedicine playing an important role.

Advancements in extracellular vesicle (EV) studies necessitate the development of optimized storage conditions to ensure preservation of physical and biochemical characteristics. In this study, the most common buffer for EV storage (phosphate-buffered saline/PBS) was compared to a cryoprotective 5% sucrose solution. The size distribution and concentration of EVs from two different sources changed to a greater extent after -80 °C storage in PBS compared to the sucrose solution. Additionally, molecular surface protrusions and transmembrane proteins were more prevalent in EVs stored in the sucrose solution compared to those stored in PBS. This study demonstrates, for the first time, that distinct ring-like molecular complexes and cristae-like folded membranous structures are visible upon EV degradation. Taken together, the size, concentration, molecular surface extensions, and transmembrane proteins of EVs varied substantially based on the buffer used for -80 °C storage, suggesting that biocompatible cryoprotectants, such as sucrose, should be considered for EV studies.

Neurological diseases remain some of the major causes of death and disability in the world. Few types of drugs and insufficient delivery across the blood-brain barrier limit the treatment of neurological disorders. The past two decades have seen the rapid development of extracellular vesicle-based therapeutics in many fields. As the physiological and pathophysiological roles of extracellular vesicles are recognized in neurological diseases, they have become promising therapeutics and targets for therapeutic interventions. Moreover, advanced nanomedicine technologies have explored the potential of extracellular vesicles as drug delivery systems in neurological diseases. In this review, we discussed the preclinical strategies for extracellular vesicle-based therapeutics in neurological disorders and the struggles involved in their clinical application.

Research on bacterial membrane vesicles (BMVs) is an emerging topic, and the goal is to address whether BMVs can bring translational tools to improve current therapies. In this review, we provided the updated studies on BMVs including their production, their types, and therapeutic regimens for treating infectious diseases and cancers. We described several platforms of BMVs, such as outer membrane vesicles (OMVs), inner membrane vesicles (IMVs) and double membrane vesicles (DMVs), and those structures were produced from Gram-negative or Gram-positive bacteria. We also discussed how to engineer and formulate new and novel BMVs using chemical, physical, and genetic methods. For therapies, we analyzed current methods for loading drugs in BMVs and discussed their limitations. Finally, we reviewed several therapeutic platforms of BMVs that have been exploited in improving the treatments of infectious diseases and cancers. Although BMVs offer the promising biomedical applications, it is needed to develop rigorous approaches and methods to generate reproducible and scalable drug delivery systems for translation.

Despite the multiple ongoing and novel initiatives for developing brain-targeted drug delivery systems, insurmountable obstacles remain. A perfect drug delivery device that can bypass the brain-blood barrier and boost therapeutic efficacy is urgently needed for clinical applications. Exosomes hold unrivaled benefits as a drug delivery vehicle for treating brain diseases due to their endogenous and innate attributes. Unique properties, such as the ability to penetrate physical barriers, biocompatibility, innate targeting features, ability to leverage natural intracellular trafficking pathways, favored tumor homing, and stability, make exosomes suitable for brain-targeted drug delivery. Herein, we provide an overview of recent exosome-based drug delivery nanoplatforms and discuss how these inherent vesicles can be used to deliver therapeutic agents to the brain to cure neurodegenerative diseases, brain tumors, and other brain disorders. Moreover, we review the current roadblocks associated with exosomes and other brain-targeted drug delivery systems and discuss future directions for achieving successful therapy outcomes.

Monocyte-induced endothelial cell inflammation is associated with multiple pathological conditions, and extracellular vesicles (EVs) are essential nanosized components of intercellular communication. EVs derived from endotoxin-stimulated monocytes were previously shown to carry pro-inflammatory proteins and RNAs. The role of glucose transporter-1 (GLUT-1) and glycan features in monocyte-derived EV-induced endothelial cell inflammation remains largely unexplored. This study demonstrates that EVs derived from endotoxin-stimulated monocytes activate inflammatory pathways in endothelial cells, which are partially attributed to GLUT-1. Alterations in glycan features and increased levels of GLUT-1 were observed in EVs derived from endotoxin-stimulated monocytes. Notably, inhibition of EV-associated GLUT-1, through the use of fasentin, suppressed EV-induced inflammatory cytokines in recipient endothelial cells.

The scale of the COVID-19 pandemic forced urgent measures for the development of new therapeutics. One of these strategies is the use of convalescent plasma (CP) as a conventional source for passive immunity. Recently, there has been interest in CP-derived exosomes. In this report, we present a structural, biochemical, and biological characterization of our proprietary product, convalescent human immune plasma-derived exosome (ChipEXO), following the guidelines set forth by the Turkish Ministry of Health and the Turkish Red Crescent, the Good Manufacturing Practice, the International Society for Extracellular Vesicles, and the Gene Ontology Consortium. The data support the safety and efficacy of this product against SARS-CoV-2 infections in preclinical models.

Extracellular vesicles (EVs) are cell-released nanoparticles that transfer biomolecular content between cells. Among EV-associated biomolecules, microRNAs (miRNAs/miRs) represent one of the most important modulators of signaling pathways in recipient cells. Previous studies have shown that EVs from adipose-derived mesenchymal stromal cells (MSCs) and adipose tissue modulate inflammatory pathways in macrophages. In this study, the effects of miRNAs that are abundant in adipose tissue EVs and other biogenic nanoparticles (BiNPs) were assessed in terms of altering Toll-like receptor 4 (TLR4)-induced cytokines. TLR-4 signaling in macrophages is often triggered by pathogen or damage-induced inflammation and is associated with several diseases. This study demonstrates that miR-451a, which is abundant in adipose tissue BiNPs, suppresses pro-inflammatory cytokines and increases anti-inflammatory cytokines associated with the TLR4 pathway. Therefore, miR-451a may be partially responsible for immunomodulatory effects of adipose tissue-derived BiNPs.

The regenerative promise of nanosized extracellular vesicles (EVs) secreted by cells is widely explored. Recently, the capacity of EVs purified from blood to elicit regenerative effect has begun to be evaluated. Blood might be a readily available source of EVs, avoiding need for extensive cell culturing, but there are specific issues that complicate use of the biofluid in this area. We assess the evidence for blood containing regenerative material, progress made towards delivering blood derived EVs as regenerative therapeutics, difficulties that relate to the complexity of blood and the promise of hydrogel-based delivery of EVs.

Rapidly growing interest in the study of extracellular vesicles (EVs) has led to the accumulation of evidence on their critical roles in various pathologies, as well as opportunities to design novel therapeutic EV-based applications. Efficiently exploiting the constantly expanding knowledge of the biology and function of EVs requires a deep understanding of the various possible strategies of using EVs for therapeutic purposes. Accordingly, in the present work, we have narrowed the broad therapeutic potential of EVs and consider the similarities and differences of various strategies as we articulate three major aspects (i.e., a triad) of their therapeutic uses: (i) EVs as drug targets, whereby we discuss therapeutic targeting of disease-promoting EVs; (ii) EVs as drugs, whereby we consider the natural medicinal properties of EVs and the available options for their optimization; and (iii) EVs as drug carriers, whereby we highlight the advantages of EVs as vehicles for efficacious drug delivery of natural compounds. Finally, after conducting a comprehensive review of the latest literature on each of these aspects, we outline opportunities, limitations, and potential solutions.

Exosomes are the main actors of intercellular communications and have gained great interest in the new cell-free regenerative medicine. These nanoparticles are secreted by almost all cell types and contain lipids, cytokines, growth factors, messenger RNA, and different non-coding RNA, especially micro-RNAs (mi-RNAs). Exosomes' cargo is released in the neighboring microenvironment but is also expected to act on distant tissues or organs. Different biological processes such as cell development, growth and repair, senescence, migration, immunomodulation, and aging, among others, are mediated by exosomes and principally exosome-derived mi-RNAs. Moreover, their therapeutic potential has been proved and reinforced by their use as biomarkers for disease diagnostics and progression. Evidence has increasingly shown that exosome-derived mi-RNAs are key regulators of age-related diseases, and their involvement in longevity is becoming a promising issue. For instance, mi-RNAs such as mi-RNA-21, mi-RNA-29, and mi-RNA-34 modulate tissue functionality and regeneration by targeting different tissues and involving different pathways but might also interfere with long life expectancy. Human mi-RNAs profiling is effectively related to the biological fluids that are reported differently between young and old individuals. However, their underlying mechanisms modulating cell senescence and aging are still not fully understood, and little was reported on the involvement of mi-RNAs in cell or tissue longevity. In this review, we summarize exosome biogenesis and mi-RNA synthesis and loading mechanism into exosomes' cargo. Additionally, we highlight the molecular mechanisms of exosomes and exosome-derived mi-RNA regulation in the different aging processes.

Extracellular vesicles (EVs) mediate intercellular transport of biomolecular cargo in the body, making them promising delivery vehicles for bioactive compounds. Genetic engineering of producer cells has enabled encapsulation of therapeutic proteins in EVs. However, genetic engineering approaches can be expensive, time-consuming, and incompatible with certain EV sources, such as human plasma and bovine milk. The goal of this study was to develop a quick, versatile, and simple method for loading proteins in EVs post-isolation. Proteins, including CRISPR associated protein 9 (Cas9), were bound to cationic lipids that were further complexed with MDA-MB-231 cell-derived EVs through passive incubation. Size-exclusion chromatography was used to remove components that were not complexed with EVs. The ability of EVs to mediate intracellular delivery of proteins was compared to conventional methods, such as electroporation and commercial protein transfection reagents. The results indicate that EVs retain native features following protein-loading and obtain similar levels of intracellular protein delivery as conventional methods, but display less toxicity. This method opens up opportunities for rapid exploration of EVs for protein delivery.