"Extracellular vesicles (EVs) have emerged as key mediators of intercellular communication and valuable biomarkers for various diseases. However, traditional EV isolation and detection methods often struggle with efficiency, scalability, and purity, limiting their clinical utility. Recent advances in microfluidic and paper-based technologies offer innovative solutions that enhance EV isolation and detection by reducing sample volume, accelerating processing times, and integrating multiple analytical steps into compact platforms. These technologies hold significant promise for advancing point-of-care diagnostics, enabling rapid disease detection, personalized treatment monitoring, and better patient outcomes. For example, early detection of cancer biomarkers through EVs can facilitate timely intervention, potentially improving survival rates, while rapid infectious disease diagnostics can support prompt treatment. Despite their potential, challenges such as standardization, scalability, and regulatory hurdles remain. This review discusses recent advancements in microfluidic and paper-based EV diagnostic technologies, their comparative advantages over traditional methods, and their transformative potential in clinical practice."

Parkinson disease (PD) is the second most common neurodegenerative disease. While most cases are sporadic, in ~ 5%-10% of PD patients the disease is caused by mutations in several genes, among them GBA1 (glucocerebrosidase beta 1) and LRRK2 (leucine-rich repeat kinase 2), both prevalent among the Ashkenazi Jewish population. LRRK2-associated PD tends to be milder than GBA1-associated PD. Several recent clinical studies have suggested that carriers of both GBA1 and LRRK2 mutations develop milder PD compared to that observed among GBA1 carriers. These findings strongly suggested an interplay between the two genes in the development and progression of PD. In the present study Drosophila was employed as a model to investigate the impact of mutations in the LRRK2 gene on mutant GBA1-associated PD. Our results strongly indicated that flies expressing both mutant genes exhibited milder parkinsonian signs compared to the disease developed in flies expressing only a GBA1 mutation. This was corroborated by a decrease in the ER stress response, increase in the number of dopaminergic cells, elevated levels of tyrosine hydroxylase, reduced neuroinflammation, improved locomotion and extended survival. Furthermore, a significant decrease in the steady-state levels of mutant GBA1-encoded GCase was observed in the presence of mutant LRRK2, strongly implying a role for mutant LRRK2 in degradation of mutant GCase.

The tumor microenvironment (TME) plays a crucial role in the development and progression of gastric cancer (GC). The TME comprises a network of cancer cells, immune cells, fibroblasts, endothelial cells, and extracellular matrix components, which provide a supportive niche for cancer cells. This study investigates the role of TME-derived exosomal competitive endogenous RNAs (ceRNAs), particularly long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), as major regulating agents in GC development. Exosomal ceRNAs control gene expression across several TME components, amplifying cancer hallmarks like cell proliferation, invasion, metastases, and chemoresistance. They promote dynamic interplay between cancer cells and adjacent stromal cells, enabling tumor development through immune suppression, angiogenesis, and epithelial-mesenchymal transition (EMT). Exosomal ceRNAs can modify the TME, creating a pro-tumorigenic milieu and preparing cancer cells to avoid immunological responses, defy death, and adapt to therapeutic pressures. This review highlights the understudied interactions between the TME and exosomal ceRNAs in gastric cancer and emphasizes their potential utility as diagnostic and therapeutic tools.

The COVID-19 pandemic, caused by SARS-CoV-2, has significantly affected global health, with severe inflammatory responses leading to tissue damage and persistent symptoms. Macrophage-derived extracellular vesicles (EVs) are involved in the modulation of immune responses, but their involvement in SARS-CoV-2-induced inflammation and senescence remains unclear. Triggering receptors expressed on myeloid cell-1 (TREM-1) are myeloid cell receptors that amplify inflammation, described as a biomarker of the severity and mortality of COVID-19. This study investigated the composition and effects of macrophage-derived EVs stimulated by SARS-CoV-2 (MφV-EVs) on the recipient cell response. Our results, for the first time, show that SARS-CoV-2 stimulation modifies the cargo profile of MφV-EVs, enriching them with TREM-1 and miRNA-155 association, along with MMP-9 and IL-8/CXCL8. These EVs carry senescence-associated secretory phenotype (SASP) components, promote cellular senescence, and compromise antibacterial defenses upon internalization. Our findings provide evidence that MφV-EVs are key drivers of inflammation and immune dysfunction, underscoring their potential as therapeutic targets in COVID-19.

While mesangial IgA deposition is the pathognomonic feature of IgA nephropathy (IgAN), the extent of mesangial IgA accumulation does not correlate with the future risk of kidney failure. This has led to the search for other serum factors that may influence clinical outcome. The emergence of microRNAs (miRs) as negative regulators of gene expression and the increasingly recognized role of extracellular miRs in intercellular communication has prompted study of the influence of miRs on inflammatory and scarring pathways in the kidneys.

Here, next generation sequencing and subsequent qPCR validation identified a significant increase in the serum levels of miR-483-5p, largely packaged within exosomes.

Levels of miR-483-5p in serum exosomes were greatest in those IgAN patients with higher levels of proteinuria who subsequently developed kidney failure. Exosomal miR-483-5p content significantly correlated with numerous soluble isoforms of the tumor necrosis factor (TNF) receptor super family suggesting lymphocytes as a source of the miR-enriched exosomes. In PBMC miR-483-5p expression was almost exclusively seen in CD19+ lymphocytes. Activation of a human IgA secreting B-cell line with soluble TNFR1 induced miR-483-5p synthesis and enrichment within exosomes. Exposure to miR-483-5p-enriched B cell exosomes resulted in a proinflammatory phenotypic change in cultured human collecting duct epithelial cells, likely mediated through suppression of the transcription factor SOCS3. miR-483-5p-enriched exosomes were also present in the urine of patients with IgAN.

Interaction of B lymphocyte-derived miR-enriched exosomes with tubular epithelial cells may provide an explanation for the progressive tubulointerstitial scarring and loss of kidney function seen in IgAN.

Analysis of tumoral RNA from bone marrow (BM) biopsy is essential for diagnosing childhood B-cell acute lymphoblastic leukemia (B-ALL), risk stratification, and monitoring, by detecting fusions and gene expression patterns. However, frequent BM biopsies are invasive and traumatic for patients. Small extracellular vesicles (sEVs) circulating in blood contain a variety of biomolecules, including RNA, that may contribute to cancer progression, offering a promising source of non-invasive biomarkers from liquid biopsies. While most EV studies have focused on small RNAs like microRNAs (miRNAs), the role of longer RNA species, including messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), remains underexplored despite their demonstrated potential for risk-based patient stratification when starting from BM biopsies. We used immuno-purification to isolate sEVs from peripheral blood at diagnosis in B-ALL patients and cell model-based conditioned culture medium (CCM) with ETV6::RUNX1 and TCF3::PBX1 fusions. Using whole-transcriptome sequencing targeting transcripts over 200 nt and a novel data analysis pipeline, we identified 102 RNA transcripts (67 mRNAs, 16 lncRNAs, 10 circRNAs, 4 pseudogenes, and 5 others) in patient-derived sEVs. These transcripts could serve as biomarkers for two distinct molecular subgroups of B-ALL, each with different risk profiles at diagnosis. This is the first study characterizing the long transcriptome in blood-derived sEVs for childhood B-ALL, highlighting the potential use of circulating RNAs for improved risk-based stratification.

Macrophages play a key role in wound healing. Dysfunction of their M0 polarization to M2 leads to disorders of the wound immune microenvironment and chronic inflammation, which affects wound healing. Regulating the polarization of M0 macrophages to M2 macrophages is an effective strategy for treating wound healing. Mesenchymal stem cells (MSCs) deliver endogenous regulatory factors via paracrine extracellular vesicles, which may play a key role in wound healing, and previous studies have shown that apoptotic bodies (ABs) are closely associated with inflammation regression and macrophage polarization. However, the specific regulatory mechanisms involved in ABs remain unknown. In the present study, we designed an MSC-AB (MSC-derived AB)-loaded polycaprolactone (PCL) scaffold, evaluated the macrophage phenotype and skin wound inflammation in vivo and in vitro, and explored the ability of MSC-AB-loaded PCL scaffolds to promote wound healing. Our data suggest that the PCL scaffold regulates the expression of the CCL-1 gene by targeting the delivery of mmu-miR-21a-5p by local sustained-release MSC-ABs, and drives M0 macrophages to program M2 macrophages to regulate inflammation and angiogenesis, thereby synergistically promoting wound healing. This study provides a promising therapeutic strategy and experimental basis for treating various diseases associated with imbalances in proinflammatory and anti-inflammatory immune responses.

Salivary gland diseases encompass a broad range of conditions, including autoimmune, inflammatory, obstructive, and neoplastic disorders, significantly impacting oral health and overall well-being. Recent research has highlighted the crucial role of exosomes, small extracellular vesicles, in these diseases. Exosomes mediate intercellular communication by transferring bioactive molecules such as proteins, microRNAs, and lipids, positioning them as potential diagnostic biomarkers and therapeutic agents. In primary Sjögren's syndrome (pSS), exosomes derived from Epstein-Barr virus-infected B cells and activated T cells transfer key microRNAs that impair calcium signaling, contributing to glandular dysfunction. Exosome-based biomarkers like Ro/SSA and La/SSB, found in saliva, serum, and tears, offer non-invasive diagnostic tools for early disease detection. Furthermore, mesenchymal stem cell-derived exosomes show promise in treating pSS by modulating immune responses and promoting tissue repair. While exosomes hold promise for the diagnosis and treatment of other salivary gland diseases, such as radiation-induced xerostomia and sialolithiasis, their application remains limited, necessitating further research to unlock their full diagnostic and therapeutic potential. This review focuses on the role of exosomes in salivary gland diseases, with an emphasis on pSS, and highlights the need for future clinical applications and large-scale trials.

Bone reconstruction in the maxillofacial region typically relies on autologous bone grafting, which presents challenges, including donor site complications and graft limitations. Recent advances in tissue engineering have identified highly pure and proliferative dedifferentiated fat cells (DFATs) as promising alternatives. Herein, we explored the capacity for osteoblast differentiation and the osteoinductive characteristics of extracellular vesicles derived from DFATs (DFAT-EVs).

DFATs were isolated from human buccal fat pads, cultured to confluency, and placed in either a standard or osteogenic induction medium. After culturing for 3 days, the conditioned medium was used to generate EVs using the size-exclusion chromatography and concentration filter method.

Characterization of DFAT-EVs revealed typical EV morphology and positive markers (CD9 and CD63), with no differences between the two groups. In vitro assays demonstrated that EVs derived from the osteogenic induction medium (OI-EVs) significantly increased alkaline phosphatase activity and osteogenesis-related genes (Runx2 and collagen type I) compared to control EVs. Next-generation sequencing identified differentially expressed miRNAs, and gene ontology analysis suggested pathways involved in osteoblast differentiation.

Isolating DFATs from buccal fat pads under osteogenic induction conditions offers a procedure confined to the oral cavity, eliminating the need for harvesting from other sites. Thus, DFAT-EVs hold promise for promoting bone regeneration in maxillofacial applications.

Exosomes are vesicles produced by the human body for carrying certain information from one cell to another. The carriers are nanosized vesicles carrying a wide variety of cargo like RNA, DNA, and proteins. Exosomes are also being used in the early diagnosis of various diseases and disorders. Current research focuses on exosomes tailoring for achieving therapeutic potential in various diseases and disorders. Besides this, their biocompatibility, stability, adjustable efficacy, and targeting properties make them attractive vehicles for formulation developers. Various preclinical studies suggested that the exosome culture cells are also modified with certain genes to achieve the desirable properties of resultant exosomes. The human body also produces some other vesicles like Ectosomes and Exomeres produced along with exosomes. Additionally, vesicles like Migrasomes are produced by migrating cells and apoptotic bodies, and Oncosomes are produced by cancer cells which can also be useful for the diagnosis of various diseases and disorders. For the separation of desired exosomes from other vesicles some latest techniques that can be useful viz differential centrifugation, density gradient centrifugation, and immunoaffinity purification have been discussed. Briefly, this review summarized various techniques of isolation of purified exosomes along with an overview of the application of exosomes in various neurodegenerative disorders and cancer along with various latest aspects of exosomes in disease progression and management which might be beneficial for the researchers.

Immunotherapies such as immune checkpoint blockade (ICB) therapy and chimeric antigen receptor T-cell (CAR-T) therapy have ushered in a new era of tumor treatment. However, most patients do not benefit from immunotherapy due to limitations such as narrow indications, low response rates, and high rates of adverse effects. Toxoplasma gondii (T. gondii), a specialized intracellular protozoan, can modulate host immune responses by inhibiting or stimulating cytokines. The ability of T. gondii to enhance an organism's immune response was found to have a direct anti-tumor effect and enhance the sensitivity of patients with tumors to ICB therapy. However, the application of T. gondii for tumor therapy faces several challenges, such as biosafety concerns. Exosomes, a subtype of extracellular vesicle that contains active components such as proteins, nucleic acids, and lipids, have become effective therapeutic tools for various diseases, including tumors. Parasites, such as T. gondii, mediate the communication of pathogens with immune cells and modulate host cellular immune responses through exosomes. Growing evidence indicates that T. gondii-derived exosomes mediate communication between pathogens and immune cells, modulate host immune responses, and have great potential as new tools for tumor therapy. In this review, we highlight recent advances in isolation and identification techniques, profiling analysis, host immunomodulatory mechanisms, and the role of T. gondii-derived exosomes in tumor immunotherapy. Additionally, we emphasize the potential of T. gondii-derived exosomes as delivery platform to enhance anti-tumor efficacy in combination with other therapies. This review proposes that T. gondii-derived exosomes may serve as a novel tool for tumor immunotherapy owing to their ability to activate host immune function and properties such as high modifiability, stability, and low toxicity. This work will assist in promoting the application of parasite exosomes in tumor therapy.

Multivesicular bodies (MVBs) are vesicles of endosomal origin containing intraluminal vesicles, which upon fusion with plasma membrane, secrete exosomes. They play a significant role in the physiology and pathology of type-2 diabetes (T2D) due to disrupted intercellular communication. The role of MVBs and their influence on insulin secretory granules (ISGs) of β-cells or their characterization is yet to be uncovered. In our study, we compared MVBs to largely well-characterized ISGs in β-cells. This study compares the density, localization, and exocytosis of CD63+ compartments (CD63+c) with NPY labelled ISGs (NISGs) in β-cells. For this, tetraspanin CD63 was exploited to majorly label MVBs in β-cells. These labels preserve the structural integrity of labelled compartments and mostly do not localize with other endo-lysosomal compartments. This study showed that the β-cells have a significantly higher density of NISGs than CD63+c. CD63+c and NISGs are spatially localized apart within β-cells. The proteins that localize with CD63+c are different from the ones that localize with NISGs. Exocytosis of NISGs occurs at the periphery of the β-cells and takes significantly less time when compared to the release of CD63+c, which is non-peripheral and takes a longer duration. Mechanistically, the availability of CD63+c for exocytosis was assessed and found that an equilibrium is maintained between docking and undocking states at the plasma membrane. Although there are a high number of short-term residing, visiting CD63+c at the plasma membrane, the availability of CD63+c for exocytosis is maintained due to docking and undocking states. Further, a significant reduction in the density of NISGs and CD63+c was observed in β-cells isolated from T2D donors compared to healthy counterparts. Studying the effect of MVBs on insulin secretion in physiological and T2D conditions has huge potential. This study provides a strong basis to open new avenues for such future studies.

The endothelial cells of the blood circulation are exposed to hemodynamic forces, such as cyclic strain, hydrostatic forces, and shear stress caused by the blood fluid's frictional force. Endothelial cells perceive mechanical forces via mechanosensors and thus elicit physiological reactions such as alterations in vessel width. The mechanosensors considered comprise ion channels, structures linked to the plasma membrane, cytoskeletal spectrin scaffold, mechanoreceptors, and junctional proteins. This review focuses on endothelial mechanosensors and how they alter the vascular functions of endothelial cells. The current state of knowledge on the dysregulation of endothelial mechanosensitivity in disease is briefly presented. The interplay in mechanical perception between endothelial cells and vascular smooth muscle cells is briefly outlined. Finally, future research avenues are highlighted, which are necessary to overcome existing limitations.

Small extracellular vesicles (sEV) have emerged as a novel mode of intercellular material transport and information transmission. It has been suggested hormones may regulate the production and function of sEV. However, the specific impact of growth hormone-releasing hormone (GHRH) on pituitary sEV production and the role of sEV in the regulation of the GHRH-GH-IGF axis has not been previously reported. The results of the present study demonstrated that GHRH increased the production of pituitary sEV by promoting the expression of Rab27a. More importantly, GHRH induced alterations in protein and miRNA levels within GH3-sEV components. Notably, GH3-sEV with GHRH treatment exhibited the enhanced ability to impede BRL 3A cell proliferation and the expression of IGF-1. Conclusively, for the first time, we corroborate the influence of GHRH on pituitary sEV, thereby presenting novel evidence for how sEV participates in the balance of the GHRH-GH-IGF axis. Importantly, this study provides new insight into a novel balance mechanism mediated by sEV within the endocrine system.

Lung cancer is frequently detected in an advanced stage and has an unfavourable prognosis. Conventional therapies are ineffective for the treatment of metastatic lung cancer. While certain molecular targets have been identified as having a positive response, the absence of appropriate drug carriers prevents their effective utilization. Lung cancer cell-derived exosomes (LCCDEs) have gained attention for their involvement in the development of cancer, as well as their potential for use in diagnosing, treating, and predicting the outcome of lung cancer. This is due to their biological roles and their inherent ability to transport biomolecules from the donor cells. Lung cancer-associated cell-derived extracellular vesicles (LCCDEVs) have the ability to enhance cell proliferation and metastasis, influence angiogenesis, regulate immune responses against tumours during the development of lung cancer, control drug resistance in lung cancer treatment, and are increasingly recognised as a crucial element in liquid biopsy evaluations for the detection of lung cancer. Therapeutic exosomes, which possess inherent intercellular communication capabilities, are increasingly recognised as effective vehicles for targeted drug delivery in precision medicine for tumours. This is due to their exceptional biocompatibility, minimal immunogenicity, low toxicity, prolonged circulation in the bloodstream, biodegradability, and ability to traverse different biological barriers. Currently, multiple studies are being conducted to create new means of diagnosing and predicting outcomes using LCCDEs, as well as to develop techniques for utilizing exosomes as effective carriers for medication delivery. This paper provides an overview of the current state of lung cancer and the wide range of applications of LCCDEs. The encouraging findings and technologies suggest that the utilization of LCCDEs holds promise for the clinical treatment of lung cancer patients.

Exosomes, membrane-bound extracellular vesicles, ranging from approximately 30-200 nm in diameter, are released by almost all cell types and play critical roles in intercellular communication. In response to the prevailing stress, the exosome-bound protein signatures vary in abundance and composition. To identify the bronchoalveolar lavage fluid (BALF) exosome-bound proteins associated with mucoinflammatory lung disease and to gain insights into their functional implications, we compared BALF exosomes-derived proteins from adult Scnn1b transgenic (Scnn1b-Tg+) and wild type (WT) mice. A total of 3,144 and 3,119 proteins were identified in BALF exosomes from Scnn1b-Tg+ and WT mice, respectively. Using cutoff criteria (Log2 fold-change > 1 and adjusted p-value < 0.05), the comparison of identified proteins revealed 127 and 30 proteins that were significantly upregulated and downregulated, respectively, in Scnn1b-Tg+ versus WT mice. In addition, 52 and 27 proteins were exclusively enriched in Scnn1b-Tg+ and WT mice, respectively. The identified exosome-bound proteins from the homeostatic airspaces of WT mice were mostly relevant to the normal physiological processes. The protein signatures enriched in the BALF exosomes of Scnn1b-Tg+ mice were relevant to macrophage activation and mucoinflammatory processes. Ingenuity pathway analyses revealed that the enriched proteins in Scnn1b-Tg+ mice contributed to the inflammatory responses and antimicrobial defense pathways. Selective proteins including, RETNLA/FIZZ1, LGALS3/Galectin-3, S100A8/MRP8, and CHIL3/YM1 were immunolocalized to specific cell types. The comparative analysis between enriched BALF exosome proteins and previously identified differentially upregulated genes in Scnn1b-Tg+ versus WT mice suggested that the compartment-/cell-specific upregulation in gene expression dictates the enrichment of their respective proteins in the lung airspaces. Taken together, this study demonstrates that the BALF exosome-bound protein signatures reflect disease-relevant disturbances. Our findings suggest that the exosomes carry disease-relevant protein signatures that can be used as a diagnostic as well as predictive biomarkers for mucoinflammatory lung disease.

Primary Sjögren syndrome (pSS) is a chronic autoimmune disease mainly affecting salivary and lacrimal glands. The current pSS biomarkers, serum autoantibodies, are negative in many pSS patients diagnosed with histopathology changes, indicating the need of novel biomarkers. The current therapies of pSS are merely short-term symptomatic relief and can't provide effective long-term remedy. Extracellular vehicles (EVs) are nano-sized lipid bilayer-delimited particles spontaneously released by almost all types of cells and carrying various bioactive molecules to mediate inter-cellular communications. Recent studies found that EVs from salivary gland epithelial cells and immune cells play essential roles in pSS pathogenesis. Correspondingly, EVs and their cargos in plasma and saliva are promising candidate biomarkers for pSS diagnosis. Moreover, EVs from mesenchymal stem cells have shown promises to improve pSS treatment by modulating immune responses. This review summarizes recent findings in roles of EVs in pSS pathogenesis, diagnosis, and treatment of pSS, as well as related challenges and future research directions.

Vibrio cholerae is an important foodborne pathogen. Cholix cytotoxin (Cholix), produced by V. cholerae, is a novel eukaryotic elongation factor 2 (eEF2) adenosine diphosphate ribosyltransferase that causes host cell death by inhibiting protein synthesis. However, the role of Cholix in the infectious diseases caused by V. cholerae remains unclear. Some bacterial cytotoxins are carried by host extracellular vesicles (EVs) and transferred to other cells. In this study, we investigated the effects of EV inhibitors and EV-regulating proteins on Cholix-induced hepatocyte death. We observed that Cholix-induced cell death was significantly enhanced in the presence of EV inhibitors (e.g., dimethyl amiloride, and desipramine) and Rab27a-knockdown cells, but it did not involve a sphingomyelin-dependent pathway. RNA sequencing analysis revealed that desipramine, imipramine, and EV inhibitors promoted the Cholix-activated c-Jun NH2-terminal kinase (JNK) pathway. Furthermore, JNK inhibition decreased desipramine-enhanced Cholix-induced poly (ADP-ribose) polymerase (PARP) cleavage. In addition, suppression of Apaf-1 by small interfering RNA further enhanced Cholix-induced PARP cleavage by desipramine. We identified a novel function of desipramine in which the stimulated JNK pathway promoted a mitochondria-independent cell death pathway by Cholix.

Exosomes are small vesicles containing proteins, nucleic acids, and biological lipids, which are responsible for intercellular communication. Studies have shown that exosomes can be utilized as effective drug delivery vehicles to accurately deliver therapeutic substances to target tissues, enhancing therapeutic effects and reducing side effects. Mesenchymal stem cells (MSCs) are a class of stem cells widely used for tissue engineering, regenerative medicine, and immunotherapy. Exosomes derived from MSCs have special immunomodulatory functions, low immunogenicity, the ability to penetrate tumor tissues, and high yield, which are expected to be engineered into efficient drug delivery systems. Despite the promising promise of MSC-derived exosomes, exploring their optimal preparation methods, drug-loading modalities, and therapeutic potential remains challenging. Therefore, this article reviews the related characteristics, preparation methods, application, and potential risks of MSC-derived exosomes as drug delivery systems in order to find potential therapeutic breakthroughs.

This review focusses on the significance of fluorescent, phosphorescent labelling and tracking of extracellular vesicles (EVs) for unravelling their biology, pathophysiology, and potential diagnostic and therapeutic uses. Various labeling strategies, such as lipid membrane, surface protein, luminal, nucleic acid, radionuclide, quantum dot labels, and metal complex-based stains, are evaluated for visualizing and characterizing EVs. Direct labelling with fluorescent lipophilic dyes is simple but generally lacks specificity, while surface protein labelling offers selectivity but may affect EV-cell interactions. Luminal and nucleic acid labelling strategies have their own advantages and challenges. Each labelling approach has strengths and weaknesses, which require a suitable probe and technique based on research goals, but new tetranuclear polypyridylruthenium(II) complexes as phosphorescent probes have strong phosphorescence, selective staining, and stability. Future research should prioritize the design of novel fluorescent probes and labelling platforms that can significantly enhance the efficiency, accuracy, and specificity of EV labeling, while preserving their composition and functionality. It is crucial to reduce false positive signals and explore the potential of multimodal imaging techniques to gain comprehensive insights into EVs.

Epilepsy is a common neurologic disorder. While a good clinical solution is still missing, studies have confirmed that exosomes (Exos) derived from adipose-derived stem cells (ADSCs) had a therapeutic effect on various diseases, including neurological diseases. Therefore, this study aimed to reveal whether ADSC-Exo treatment could improve kainic acid (KA)-induced seizures in epileptic mice. ADSCs and Exos were isolated. Mice were generated with KA-induced epileptic seizures. ELISA was used to detect inflammatory factor expression. Luciferase reporter analysis detection showed a relationship among miR-23b-3p, STAT1, and glyoxylate reductase 1 (GlyR1). ADSC-Exos had a protective effect on KA-induced seizures by inhibiting inflammatory factor expression and the M1 microglia phenotype. The result showed that miR-23b-3p played an important role in the Exo-mediated protective effect in KA-induced seizures in epileptic mice by regulating STAT1 and GlyR1. Luciferase reporter analysis confirmed that miR-23b-3p interacted with the 3'-UTR of STAT1 and GlyR1. The miR-23b-3p inhibited M1 microglia-mediated inflammatory factor expression in microglial cells by regulating STAT1 and GlyR1. The downregulation of miR-23b-3p decreased the protective effect of ADSC-Exos on KA-induced seizures in epileptic mice. The miR-23b-3p from ADSC-Exos alleviated inflammation in mice with KA-induced epileptic seizures.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a global pandemic, which severely endangers public health. Our and others' works have shown that the angiotensin-converting enzyme 2 (ACE2)-containing exosomes (ACE2-exos) have superior antiviral efficacies, especially in response to emerging variants. However, the mechanisms of how the virus counteracts the host and regulates ACE2-exos remain unclear. Here, we identified that SARS-CoV-2 nonstructural protein 6 (NSP6) inhibits the production of ACE2-exos by affecting the protein level of ACE2 as well as tetraspanin-CD63 which is a key factor for exosome biogenesis. We further found that the protein stability of CD63 and ACE2 is maintained by the deubiquitination of proteasome 26S subunit, non-ATPase 12 (PSMD12). NSP6 interacts with PSMD12 and counteracts its function, consequently promoting the degradation of CD63 and ACE2. As a result, NSP6 diminishes the antiviral efficacy of ACE2-exos and facilitates the virus to infect healthy bystander cells. Overall, our study provides a valuable target for the discovery of promising drugs for the treatment of coronavirus disease 2019.

The outbreak of coronavirus disease 2019 (COVID-19) severely endangers global public health. The efficacy of vaccines and antibodies declined with the rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutants. Angiotensin-converting enzyme 2-containing exosomes (ACE2-exos) therapy exhibits a broad neutralizing activity, which could be used against various viral mutations. Our study here revealed that SARS-CoV-2 nonstructural protein 6 inhibited the production of ACE2-exos, thereby promoting viral infection to the adjacent bystander cells. The identification of a new target for blocking SARS-CoV-2 depends on fully understanding the virus-host interaction networks. Our study sheds light on the mechanism by which the virus resists the host exosome defenses, which would facilitate the study and design of ACE2-exos-based therapeutics for COVID-19.

Extracellular vesicles (EVs) are natural lipid nanoparticles secreted by most types of cells. In malignant cancer, EVs derived from cancer cells contribute to its progression and metastasis by facilitating tumor growth and invasion, interfering with anticancer immunity, and establishing premetastasis niches in distant organs. In recent years, multiple strategies targeting cancer-derived EVs have been proposed to improve cancer patient outcomes, including inhibiting EV generation, disrupting EVs during trafficking, and blocking EV uptake by recipient cells. Developments in EV engineering also show promising results in harnessing cancer-derived EVs as anticancer agents. Here, we summarize the current understanding of the origin and functions of cancer-derived EVs and review the recent progress in anticancer therapy targeting these EVs.

Exosomes are the smallest subset of extracellular signaling vesicles secreted by most cells with a diameter in the range of 30-150 nm. Their use has gained great momentum recently due to their ability to be utilized as diagnostic tools with a vast array of therapeutic applications. Over 5000 publications are currently being published yearly on this topic, and this number is only expected to dramatically increase as novel therapeutic strategies continue to be investigated. This review article first focuses on understanding exosomes, including their cellular origin, biogenesis, function, and characterization. Thereafter, overviews of the quantification methods and isolation techniques are given with discussion over their potential use as novel therapeutics in regenerative medicine.

Due to its multiple features, including the ability to orchestrate remote communication between different tissues, the exosomes are the extracellular vesicles arousing the highest interest in the scientific community. Their size, established as an average of 30-150 nm, allows them to be easily uptaken by most cells. According to the type of cells-derived exosomes, they may carry specific biomolecular cargoes used to reprogram the cells they are interacting with. In certain circumstances, exosomes stimulate the immune response by facilitating or amplifying the release of foreign antigens-killing cells, inflammatory factors, or antibodies (immune activation). Meanwhile, in other cases, they are efficiently used by malignant elements such as cancer cells to mislead the immune recognition mechanism, carrying and transferring their cancerous cargoes to distant healthy cells, thus contributing to antigenic invasion (immune suppression). Exosome dichotomic patterns upon immune system regulation present broad advantages in immunotherapy. Its perfect comprehension, from its early biogenesis to its specific interaction with recipient cells, will promote a significant enhancement of immunotherapy employing molecular biology, nanomedicine, and nanotechnology.

This review focuses on the role of small extracellular vesicles in the pathophysiological mechanisms of retinal degenerative diseases. Many of these mechanisms are related to or modulated by the oxidative burden of retinal cells. It has been recently demonstrated that cellular communication in the retina involves extracellular vesicles and that their rate of release and cargo features might be affected by the cellular environment, and in some instances, they might also be mediated by autophagy. The fate of these vesicles is diverse: they could end up in circulation being used as markers, or target neighbor cells modulating gene and protein expression, or eventually, in angiogenesis. Neovascularization in the retina promotes vision loss in diseases such as diabetic retinopathy and age-related macular degeneration. The importance of micro RNAs, either as small extracellular vesicles' cargo or free circulating, in the regulation of retinal angiogenesis is also discussed.

As the only bionormal nanovesicle, exosomes have high potential as a nanovesicle for delivering vaccines and therapeutics. We show here that the loading of type-1 membrane proteins into the exosome membrane is induced by exosome membrane anchor domains, EMADs, that maximize protein delivery to the plasma membrane, minimize protein sorting to other compartments, and direct proteins into exosome membranes. Using SARS-CoV-2 spike as an example and EMAD13 as our most effective exosome membrane anchor, we show that cells expressing a spike-EMAD13 fusion protein produced exosomes that carry dense arrays of spike trimers on 50% of all exosomes. Moreover, we find that immunization with spike-EMAD13 exosomes induced strong neutralizing antibody responses and protected hamsters against SARS-CoV-2 disease at doses of just 0.5-5 ng of spike protein, without adjuvant, demonstrating that antigen-display exosomes are particularly immunogenic, with important implications for both structural and expression-dependent vaccines.

Chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI) are pathways for selective degradation of cytosolic proteins in lysosomes and late endosomes, respectively. These autophagic processes share as a first step the recognition of the same five-amino-acid motif in substrate proteins by the Hsc70 chaperone, raising the possibility of coordinated activity of both pathways. In this work, we show the existence of a compensatory relationship between CMA and eMI and identify a role for the chaperone protein Bag6 in triage and internalization of eMI substrates into late endosomes. Association and dynamics of Bag6 at the late endosome membrane change during starvation, a stressor that, contrary to other autophagic pathways, causes a decline in eMI activity. Collectively, these results show a coordinated function of eMI with CMA, identify the interchangeable subproteome degraded by these pathways, and start to elucidate the molecular mechanisms that facilitate the switch between them.

Extracellular vesicles (EVs) are nanoscopic packages that are heterogeneous and bona fide players in hepatic physiology and pathology as they are involved in intercellular communication. EVs carrying bioactive cargoes rich in lipids, proteins or nucleic acids are implicated in the onset and progression of liver diseases. Liver pathology using liver biopsy has been assessed for several intricate conditions such as viral hepatitis, alcoholic and non-alcoholic fatty liver disease, hepatic malignancies and drug-induced liver injury. The lacunae, however, lie in early diagnosis and timely treatment of the above conditions, underscoring the need for non-invasive, accurate diagnostic tools that could replace the gold standard method of tissue biopsy. In this regard, EVs have emerged as promising candidates that could serve as potential biomarkers. In the last two decades, EVs, owing to their multifaceted charm in bringing out cell-free therapeutic responses and the ability of their cargoes to be applied to novel biomarkers, have drawn the great attention of researchers with the advancement and clinical application of liquid biopsy. In this review, we recapitulate the role of EVs and provide insights into the promising role of these small packages as biomarkers in liver pathology.

The scourge of type-1 diabetes (T1D) is the morbidity and mortality it and its complications cause at a younger age. This propels the constant search for better diagnostic, treatment, and management strategies, with the ultimate quest being a cure for T1D. Recently, the therapeutic potential of exosomes has generated a lot of interest. Among the characteristics of exosomes of particular interest are (a) their regenerative capacity, which depends on their "origin", and (b) their "content", which determines the cell communication and crosstalk they influence. Other functional capacities, including paracrine and endocrine homeostatic regulation, pathogenic response ability resulting in insulin secretory defects or β-cell death under normal metabolic conditions, immunomodulation, and promotion of regeneration, have also garnered significant interest. Exosome "specificity" makes them suitable as biomarkers or predictors, and their "mobility" and "content" lend credence to drug delivery and therapeutic suitability. This review aims to highlight the functional capacities of exosomes and their established as well as novel contributions at various pathways in the onset and progression of T1D. The pathogenesis of T1D involves a complex crosstalk between insulin-secreting pancreatic β-cells and immune cells, which is partially mediated by exosomes. We also examine the potential implications for type 2 diabetes (T2D), as the link in T2D has guided T1D exploration. The collective landscape presented is expected to help identify how a deeper understanding of exosomes (and their cargo) can provide a framework for actionable solutions to prevent, halt, or change the very course of T1D and its complications.

Extracellular vesicles (EVs) are membrane-limited organelles mediating cell-to-cell communication in health and disease. EVs are of high medical interest, but their rational use for diagnostics or therapies is restricted by our limited understanding of the molecular mechanisms governing EV biology. Here, we tested whether PDZ proteins, molecular scaffolds that support the formation, transport, and function of signal transduction complexes and that coevolved with multicellularity, may represent important EV regulators. We reveal that the PDZ proteome (ca. 150 proteins in human) establishes a discrete number of direct interactions with the tetraspanins CD9, CD63, and CD81, well-known EV constituents. Strikingly, PDZ proteins interact more extensively with syndecans (SDCs), ubiquitous membrane proteins for which we previously demonstrated an important role in EV biogenesis, loading, and turnover. Nine PDZ proteins were tested in loss-of-function studies. We document that these PDZ proteins regulate both tetraspanins and SDCs, differentially affecting their steady-state levels, subcellular localizations, metabolism, endosomal budding, and accumulations in EVs. Importantly, we also show that PDZ proteins control the levels of heparan sulfate at the cell surface that functions in EV capture. In conclusion, our study establishes that the extensive networking of SDCs, tetraspanins, and PDZ proteins contributes to EV heterogeneity and turnover, highlighting an important piece of the molecular framework governing intracellular trafficking and intercellular communication.

MicroRNA (miRNA) profiles vary with the nutritional and pathological conditions of cattle. In this study, we aimed to investigate the effects of inulin supplement on miRNA profiles derived from serum extracellular vesicles (EVs). Our goal was to determine the differences in miRNA expressions and analyse the pathways in which they are involved. Based on the results of California mastitis test and milk somatic cell counts, ten lactating cows with subclinical mastitis were randomly divided into two groups: an inulin group and a control group (n = 5 in each group). The inulin group received a daily supplement of 300 g of inulin while the control group did not receive any supplementation. After a 5-week treatment period, serum-derived EV-miRNAs from each cow were isolated. High-throughput sequencing was conducted to identify differentially expressed miRNAs. GO and KEGG bioinformatics analysis was performed to examine the target genes of these differentially expressed miRNAs. The EV-RNA concentration and small RNA content were not affected by the inulin treatment. A total of 162 known miRNAs and 180 novel miRNAs were identified from 10 samples in the two groups. Among the known miRNAs, 23 miRNAs were found to be differentially expressed between the two groups, with 18 upregulated and five downregulated in the inulin group compared to the control group. Pathway analysis revealed the involvement of these differentially expressed miRNAs in the regulation of cell structure and function, lipid oxidation and metabolism, immunity and inflammation, as well as digestion and absorption of nutrients. Overall, our study provides a molecular-level explanation for the reported beneficial health effects of inulin supplementation in cows with subclinical mastitis.

Renal ischemia-reperfusion injury (RIRI) is a complex disorder characterized by both intrinsic damage to renal tubular epithelial cells and extrinsic inflammation mediated by cytokines and immune cells. Unfortunately, there is no cure for this devastating condition. Extracellular vesicles (EVs) are nanosized membrane-bound vesicles secreted by various cell types that can transfer bioactive molecules to target cells and modulate their function. EVs have emerged as promising candidates for cell-free therapy of RIRI, owing to their ability to cross biological barriers and deliver protective signals to injured renal cells. In this review, we provide an overview of EVs, focusing on their functional role in RIRI and the signaling messengers responsible for EV-mediated crosstalk between various cell types in renal tissue. We also discuss the renoprotective role of EVs and their use as therapeutic agents for RIRI, highlighting the advantages and challenges encountered in the therapeutic application of EVs in renal disease.

Wound repair is a complex problem for both clinical practitioners and scientific investigators. Conventional approaches to wound repair have been associated with several limitations, including prolonged treatment duration, high treatment expenses, and significant economic and psychological strain on patients. Consequently, there is a pressing demand for more efficacious and secure treatment modalities to enhance the existing treatment landscapes. In the field of wound repair, cell-free therapy, particularly the use of mesenchymal stem cell-derived exosomes (MSC-Exos), has made notable advancements in recent years. Exosomes, which are small lipid bilayer vesicles discharged by MSCs, harbor bioactive constituents such as proteins, lipids, microRNA (miRNA), and messenger RNA (mRNA). These constituents facilitate material transfer and information exchange between the cells, thereby regulating their biological functions. This article presents a comprehensive survey of the function and mechanisms of MSC-Exos in the context of wound healing, emphasizing their beneficial impact on each phase of the process, including the regulation of the immune response, inhibition of inflammation, promotion of angiogenesis, advancement of cell proliferation and migration, and reduction of scar formation.

The increasing research and rapid developments in the field of exosomes provide insights into their role and significance in human health. Exosomes derived from various sources, such as mesenchymal stem cells, cardiac cells, and tumor cells, to name a few, can be potential therapeutic agents for the treatment of diseases and could also serve as biomarkers for the early detection of diseases. Cellular components of exosomes, several proteins, lipids, and miRNAs hold promise as novel biomarkers for the detection of various diseases. The structure of exosomes enables them as drug delivery vehicles. Since exosomes exhibit potential therapeutic applications, their efficient isolation from complex biological/clinical samples and precise real-time analysis becomes significant. With the advent of microfluidics, nano-biosensors are being designed to capture exosomes efficiently and rapidly. Herein, we have summarized the history, biogenesis, characteristics, functions, and applications of exosomes, along with the isolation, detection, and quantification techniques. The implications of surface modifications to enhance specificity have been outlined. The review also sheds light on the engineered nanoplatforms being developed for exosome detection and capture.

Valvular heart disease represents a significant burden to the healthcare system, with approximately 5 million cases diagnosed annually in the US. Among these cases, calcific aortic stenosis (CAS) stands out as the most prevalent form of valvular heart disease in the aging population.  CAS is characterized by the progressive calcification of the aortic valve leaflets, leading to valve stiffening. While aortic valve replacement is the standard of care for CAS patients, the long-term durability of prosthetic devices is poor, calling for innovative strategies to halt  or reverse disease progression. Here, we explor the potential use of novel extracellular vesicle (EV)-based nanocarriers for delivering molecular payloads to the affected valve tissue. This approach aims to reduce inflammation and potentially promote resorption of the calcified tissue.

Engineered EVs loaded with the reprogramming myeloid transcription factors, CEBPA and Spi1, known to mediate the transdifferentiation of committed endothelial cells into macrophages. We evaluated the ability of these engineered EVs to deliver DNA and transcripts encoding CEBPA and Spil into calcified aortic valve tissue obtained from patients undergoing valve replacement due to aortic stenosis. We also investigated whether these EVs could induce the transdifferentiation of endothelial cells into macrophage-like cells.

Engineered EVs loaded with CEBPA + Spi1 were successfully derived from human dermal fibroblasts. Peak EV loading was found to be at 4 h after nanotransfection of donor cells.  These CEBPA + Spi1 loaded EVs effectively transfected aortic valve cells, resulting in the successful induction of transdifferentiation, both in vitro with  endothelial cells and ex vivo with valvular endothelial cells, leading to the development of anti-inflammatory macrophage-like cells.

Our findings highlight the potential of engineered EVs as a next generation nanocarrier to target aberrant calcifications on diseased heart valves. This development holds promise as a novel therapy for high-risk patients who may not be suitable candidates for valve replacement surgery.

The online version contains supplementary material available at 10.1007/s12195-023-00783-x.