"Peripheral nerve injury" refers to damage or trauma affecting nerves outside the brain and spinal cord. Peripheral nerve injury results in movements or sensation impairments, and represents a serious public health problem. Although severed peripheral nerves have been effectively joined and various therapies have been offered, recovery of sensory or motor functions remains limited, and efficacious therapies for complete repair of a nerve injury remain elusive. The emerging field of mesenchymal stem cells and their exosome-based therapies hold promise for enhancing nerve regeneration and function. Mesenchymal stem cells, as large living cells responsive to the environment, secrete various factors and exosomes. The latter are nano-sized extracellular vesicles containing bioactive molecules such as proteins, microRNA, and messenger RNA derived from parent mesenchymal stem cells. Exosomes have pivotal roles in cell-to-cell communication and nervous tissue function, offering solutions to changes associated with cell-based therapies. Despite ongoing investigations, mesenchymal stem cells and mesenchymal stem cell-derived exosome-based therapies are in the exploratory stage. A comprehensive review of the latest preclinical experiments and clinical trials is essential for deep understanding of therapeutic strategies and for facilitating clinical translation. This review initially explores current investigations of mesenchymal stem cells and mesenchymal stem cell-derived exosomes in peripheral nerve injury, exploring the underlying mechanisms. Subsequently, it provides an overview of the current status of mesenchymal stem cell and exosome-based therapies in clinical trials, followed by a comparative analysis of therapies utilizing mesenchymal stem cells and exosomes. Finally, the review addresses the limitations and challenges associated with use of mesenchymal stem cell-derived exosomes, offering potential solutions and guiding future directions.

In recent years, exosomes versatility has prompted their study in the biomedical field for diagnostic, prognostic, and therapeutic applications. Exosomes are bi-lipid small extracellular vesicles (30-150 nm) secreted by various cell types, containing proteins, lipids, and DNA/RNA. They mediate intercellular communication and can influence multiple human physiological and pathological processes. So far, exosome analysis has revealed their role as promising diagnostic tools for human pathologies. Concurrently, artificial intelligence (AI) has revolutionised multiple sectors, including medicine, owing to its ability to analyse large datasets and identify complex patterns. The combination of exosome analysis with AI processing has displayed a novel diagnostic approach for cancer and other diseases. This review explores the current applications and prospects of the combined use of exosomes and AI in medicine. Firstly, we provide a biological overview of exosomes and their relevance in cancer biology. Then we explored exosome isolation techniques and Raman spectroscopy/SERS analysis. Finally, we present a summarised essential guide of AI methods for non-experts, emphasising the advancements made in AI applications for exosome characterisation and profiling in oncology research, as well as in other human diseases.

Successful implantation relies on high-quality blastocysts, uterine receptivity, and effective embryo-endometrium communication. This study investigated the effects of umbilical cord-derived mesenchymal stem cells (UC-MSC) secretomes on embryo development and implantation.

Trophoblastic spheroids and murine embryos were used to evaluate the impact of UC-MSC secretomes. Embryos obtained through superovulation were cultured in vitro and divided into five groups: a control group and four experimental groups treated with varying concentrations of UC-MSC secretomes (2.5, 5, 10, and 50 μg/mL). Embryo development competence and implantation potential were assessed in each group, and the expression levels of related genes were analyzed.

Supplementation with UC-MSC secretomes significantly enhanced trophoblast cell migration. It also stimulated endometrial cell proliferation and upregulated key implantation-related genes (LIF, LIFR, VEGFA, ITGB3, and ITGAV), improving endometrial receptivity and adhesion in trophoblastic spheroid co-cultures. While morulation rates of murine embryos remained unchanged, UC-MSC secretomes supplement significantly increased blastulation, pluripotency gene expression, and hatching rates. Supplementation with 10 and 50 μg/mL significantly increased blastocyst diameter and blastomere number, as well as embryo adhesion, outgrowth areas, and implantation rates. Additionally, growth factor analysis showed elevated VEGF-A and PDGF-AA levels in the culture media.

This study demonstrates that UC-MSC secretomes enhance both embryo development and endometrial cell function, facilitating implantation potential. These findings suggest their potential utility in supporting preimplantation embryos and improving maternal endometrial receptivity in ART.

Cancer is one of the main causes of death and a major obstacle to increasing life expectancy in all countries of the world. Lymph node metastasis (LNM) of in cancer patients indicates poor prognosis and it is an important indication to determine the therapeutic regime. Therefore, more attention should be given to the molecular mechanics of tumor lymphangiogenesis and LNM. Extracellular vesicles (EVs) are nanoscale cargo-bearing membrane vesicles that can serve as key mediators for the intercellular communication. Like Hermes, the messenger of the Greek gods, EVs can be secreted by tumor cells to regulate the LNM process. Many evidence has proved the clinical correlation between EVs and LNM in various cancer types. EVs plays an active role in the process of metastasis by expressing its connotative molecules, including proteins, nucleic acids, and metabolites. However, the clear role of EVs in the process of cancer LNM has not been thoroughly studied yet. In this review, we will summarize the clinical and mechanical findings of EVs regulating role on cancer LNM, and discuss the advanced modification of the research proposal. We propose the "PUMP" principle of EVs in LNM, including Preparation, Unleash, Migration, and Planting.

Sleep-disordered breathing (SDB) is characterized by recurrent reductions or interruptions in airflow during sleep, termed hypopneas and apneas, respectively. SDB impairs sleep quality and is linked to substantive health issues including cardiovascular and metabolic disorders, as well as cognitive decline. Recent evidence suggests a link between gut microbiota (GM) composition and sleep apnea. Indeed, GM, a community of microorganisms residing in the gut, has emerged as a potential player in various diseases, and several studies have identified associations between sleep apnea and GM diversity along with shifts in bacterial populations. Additionally, the concept of "leaky gut," a compromised intestinal barrier with potentially increased inflammation, has emerged as another key player in the potential bidirectional relationship between GM and sleep apnea. One of the potential effectors could be extracellular vesicles (EVs) underlying gut-brain communication pathways that are relevant to sleep regulation and function. Thus, therapeutic implications afforded by targeting the GM or exosomes for sleep apnea management have surfaced as promising areas of research. This review explores current understanding of the relationship between GM, exosomes and sleep apnea, highlighting key research dynamics and potential mechanisms. A comprehensive review of the literature was conducted, focusing on studies investigating GM composition, intestinal barrier function and gut-brain communication in relation to sleep apnea.

Pancreatic cancer is a highly malignant tumor with poor prognosis, emphasizing the need for accurate early diagnosis. EVs, as mediators of intercellular communication, carry DNA, RNA, and proteins that show differential but not tumor-specific expression patterns in pancreatic cancer. Studies have shown that combining RNA markers in EVs (such as miRNA, circRNA, and lncRNA) with serum CA 19-9 testing can significantly enhance diagnostic accuracy for pancreatic cancer. EV-associated proteins have exhibited favorable diagnostic performance in early-stage pancreatic cancer in preliminary studies, though their clinical applicability remains to be further validated. Furthermore, mutations in KRAS, TP53, and SMAD4 genes within EVs offer a promising avenue for non-invasive liquid biopsy. However, challenges such as standardization, low sensitivity, and specificity still hinder the clinical application of EVs. Future research should focus on strategies including multi-omics integration, AI-assisted analysis, multi-marker combined detection, and large-scale clinical validation to further improve the diagnostic capability for pancreatic cancer. Overcoming these obstacles may position EVs as a vital tool in the diagnosis of pancreatic cancer.

Extracellular vesicles, exosomes, have garnered significant attention in the field of cancer therapy, one of the world's deadliest diseases. Exosomes from cancer cells participate in the development of cancer. Inhibition of exosome biogenesis may be a promising way to combat cancer. Numerous drugs and agents have been assessed to inhibit exosome biogenesis, release, and uptake, which are the main factors contributing to cancer progression. Different drugs target several intracellular mechanisms to stop the exosome signalling pathway. They affect various intracellular pathways; for example, they can disrupt the endosomal sorting complex or interfere with the intracellular trafficking of exosomes Furthermore, some of them suppress or modulate genes and proteins involved in exosome generation and release. Exosome inhibition may also be associated with different side and non-targeting effects. Pre-clinical studies show promising outcomes; however, some challenges need to be addressed in future studies. This review describes the properties of exosome inhibitor agents, focusing on the specific pathways involved in exosome biogenesis, release, and uptake.

In the past decade, with the rise of research on plant-derived extracellular vesicles (PDEVs), scientists have been continuously exploring the bioactivity of PDEVs. Many PDEVs have been shown to possess a variety of biological activities. Given that the specific characteristics of EVs are believed to be related to their source cells, PDEVs from traditional Chinese medicinal herbs hold significant potential for development. In this study, lotus (Nelumbo nucifera Gaertn.) leaves were selected as the source of PDEVs, and the impact of different isolation methods on their characteristics was evaluated, while their potential biological activities were also assessed. Lotus-derived EVs (LDEVs) were isolated by using tangential flow filtration (TFF), ultracentrifugation (UC), density gradient ultracentrifugation (DGU), and size-exclusion chromatography (SEC), respectively. The mean sizes of LDEVs isolated by various methods were in the range of 130-160 nm. Although the LDEVs isolated by the TFF method had a lower zeta potential, it exhibited the highest purity, with a yield of 3.69 ± 0.43 × 109 particles/g lotus leaves. Notably, LDEVs isolated by different methods all demonstrated the ability to attenuate LPS-induced inflammation in RAW264.7 cells, significantly decreasing the nitrite concentration in the culture medium. Furthermore, LDEVs also showed potential for wound healing, promoting the migration of HaCaT cells in vitro. LDEVs also demonstrated internalization by RAW264.7 and HaCaT cells. These results support the potential of LDEVs for biomedical applications while also suggesting that TFF is a promising and viable strategy for large-scale PDEV isolation.

Exosomal microRNAs (ex-miRs), encapsulated in extracellular vesicles (EVs), play a vital role in facilitating paracrine communication among granulosa cells (GCs), cumulus cells (CCs), and the oocyte inside follicular fluid (FF). These small non-coding RNAs are crucial for regulating folliculogenesis, oocyte maturation, and early embryonic development via modulating intracellular signaling networks. Dysregulation o has been associated with reproductive disorders such as polycystic ovarian syndrome (PCOS), diminished ovarian reserve (DOR), and inadequate ovarian response (POR), impacting oocyte quality and fertility outcomes. This narrative review consolidates molecular data from current human and animal studies regarding ex-miR expression patterns, functional targets, and pathway involvement within the context of assisted reproductive technologies (ARTs). A literature-based analysis was undertaken, focusing on signaling pathways, pathogenic processes, and clinical implications. Specifically, ex-miRs-such as miR-21, miR-34c, miR-143-3p, miR-155-5p, miR-339-5p, and miR-424-5p-were identified as regulators of critical pathways including phosphoinositide 3-kinase (PI3K)-AKT, ERK1/2, TGF-β/SMAD, and Rb-E2F1. These ex-miRs regulate apoptosis, glycolysis, mitochondrial function, and cell cycle expansion to influence oocyte competence. Pathological patterns in PCOS and POR are associated with altered ex-miR expression that disrupts metabolic and developmental signaling. Research utilizing animal models confirmed that modifications in EV-associated miRNA influence in vitro maturation (IVM) efficiency and blastocyst quality. Ex-miRs serve as intriguing non-invasive biomarkers and potential therapeutic targets for ARTs. Their mechanical involvement in oocyte and follicular physiology positions them for integration into forthcoming precision-based infertility therapies. For its implementation in reproductive medicine, EV profiling requires standardization and further functional validation in clinical environments.

Exosomes are small extracellular vesicles produced by most cell types and contain proteins, lipids, and nucleic acids (non-coding RNAs, mRNA, and DNA) that can be released by donor cells to influence the function of recipient cells. Skin photoaging is the premature aging of skin structures caused by prolonged exposure to ultraviolet (UV), as demonstrated by depigmentation, roughness, rhytides, elastosis, and precancerous alterations. Exosomes are associated with aging processes such as oxidative damage, inflammation, and senescence. Exosomes' anti-aging properties have been linked to various in vitro and preclinical investigations. There are still several unanswered questions about the use of MSC exosomes for skin rejuvenation, despite encouraging results. Uncertainty surrounds the precise processes by which exosomes stimulate the creation of collagen, skin tissue via a variety of mechanisms, including reduced matrix metalloproteinase (MMP) expression, increased collagen and elastin production, and modulation of intracellular signaling pathways and intercellular communication. These findings suggest the therapeutic potential of exosomes in skin aging. This review provides information on the molecular mechanisms and consequences of exosome anti-aging.

Exosomes are extracellular vesicles that received attention for their potential use in the treatment of various injuries. They communicate intercellularly by transferring genetic and bioactive molecules from parent cells. Although exosomes hold immense promise for treating neurodegenerative and oncological diseases, their actual clinical use is very limited because of their biogenesis and secretion. Recent studies have shown that small molecules can significantly enhance exosome biogenesis, thereby remarkably improving yield, functionality, and therapeutic effects. These molecules modulate critical pathways toward optimum exosome production in a mode that is either ESCRT dependent or ESCRT independent. Improved exosome biogenesis may provide new avenues for targeted cancer therapy, neuroprotection in neurodegenerative diseases, and regenerative medicine in wound healing. This review explores the role of small molecules in enhancing exosome biogenesis and secretion, highlights their underlying mechanisms, and discusses emerging clinical applications. By addressing current challenges and focusing on translational opportunities, this study provides a foundation for advancing cell-free therapies in regenerative medicine and beyond.

Despite the transformative impact of immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 pathway in cancer therapy, up to 80% of patients fail to respond, necessitating reliable predictive biomarkers to guide treatment decisions. Recent studies highlight the critical role of tumor-derived exosomal PD-L1 in immune evasion, and its potential as a diagnostic and prognostic biomarker in cancer immunotherapy. However, significant challenges remain in elucidating the functional roles of PD-L1+ exosomes in immune suppression, as current methods lack the ability to precisely and simultaneously characterize and monitor exosome secretion and the corresponding immune modulation on site. To address this, we developed an integrated microfluidic platform that combines a digital nanoplasmonic immunoassay with a cell-on-a-chip system, enabling in situ monitoring of PD-L1+ exosome secretion and exosome-mediated T cell immune responses. This nanoplasmonic immunoassay integrated cell-on-a-chip (NIIC) creates a localized co-cultured microenvironment that facilitates exosome-mediated cellular interactions without direct contact. The NIIC employs machine-learning assisted signal processing for highly sensitive detection of both exosomes and cytokines, providing spatial and quantitative analysis of immune modulation in situ. Using this system, we demonstrated that PD-L1+ exosomes from cancer cells significantly suppressed IFN-γ and IL-2 secretion in neighboring T cells, offering direct insights into exosome-mediated immune suppression. The NIIC platform represents a powerful tool for advancing the understanding of exosome-driven immune modulation and holds potential for predicting clinical responses to anti-PD-1/PD-L1 therapies, paving the way for more personalized cancer immunotherapy strategies.

Previous studies found that cigarette smoke (CS) exposure could induce NSCLC malignancy and miRNA dysregulation. Yet, the association of CS-induced miRNA dysregulation and NSCLC malignancy has not been clearly understood. This study aimed to evaluate the effect of CS exposure in smokers on the expression of miR-10b-5p and miR-320b in extracellular vesicles (EVs) from NSCLC patients.

Bioinformatic analysis was conducted to validate miRNA candidates. Blood and tissue samples were collected from NSCLC patients (n = 21) with smoking and non-smoking history. EVs were isolated from plasma and miRNAs were extracted from the isolated EVs. The miRNAs relative expression was analyzed and compared.

In silico analysis identified miR-320b and miR-10b-5p as potential biomarkers for diagnosing NSCLC in smokers. Experimental analysis revealed differential expression of EVs-associated miRNAs in NSCLC patients with smoking and non-smoking histories. EVs-associated miR-10b-5p was significantly overexpressed in smoker NSCLC patients (p = 0.000), while miR-320b expression was significantly lower in this group (p = 0.018). Additionally, smoking intensity influenced miRNA expression, with higher smoking intensity correlating with increased miR-10b-5p expression and decreased miR-320b expression. ROC analysis demonstrated that EVs were a superior source of miRNAs compared to plasma for NSCLC diagnostics. miR-10b-5p and miR-320b in EVs showed higher diagnostic performance (AUC 0.878; 0.739) compared to plasma (AUC 0.628; 0.559).

CS exposure induces different expression of miR-10b-5p and miR-320b in EVs of NSCLC patients with smoking history. EV-related miR-10b-5p and miR-320b showed potential to be utilized as prognostic biomarker for smokers NSCLC patients.

Obstetric antiphospholipid syndrome (OAPS) is a pregnancy-related complication characterized by trophoblast pyroptosis and placental injury induced by antiphospholipid antibodies (aPLs). M2-polarized macrophage-derived exosomes (M2-exos) exert anti-inflammatory, immunomodulatory, and growth-promoting effects in various autoimmune diseases and tumors. However, their role in OAPS is not yet clear. Therefore, in this study, we isolated M2-exos from M2 macrophages and investigated their effects on trophoblast proliferation, death, migration, invasion, and pyroptosis following stimulation using aPLs.

First, we established an animal model of OAPS and thereafter treated the OAPS mice with exogenous M2-exos via injection through the tail vein. Then to clarify the roles of miR-20a-5p and thioredoxin-interacting protein (TXNIP) in OAPS, we performed gain- or loss-of-function assays, and used GraphPad Prism software to analyze the collected data with statistical significance set at P < 0.05.

MicroRNAs (miRNAs) sequencing revealed the enrichment of miR-20a-5p in M2-exos, and these M2-exos significantly alleviated aPLs-induced trophoblast dysfunction. Our results also indicated that M2-exos delivered miR-20a-5p to trophoblast cells directly targeted thioredoxin-interacting protein (TXNIP), and thus suppressed the TXNIP/NLRP3 pathway, reduced pyroptosis and inflammation in trophoblast cells, and improved placental function and fetal development.

M2-exos improve pregnancy outcomes in OAPS via the miR-20a-5p/TXNIP/NLRP3 axis, and thus represent as a novel therapeutic approach for aPLs-induced OAPS.

Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) have shown considerable promise as restorative stroke treatment. In a head-to-head comparison in mice exposed to transient proximal middle cerebral artery occlusion (MCAO), sEVs obtained from MSCs cultured under hypoxic conditions particularly potently enhanced long-term brain tissue survival, microvascular integrity, and angiogenesis. These observations suggest that hypoxic preconditioning might represent the strategy of choice for harvesting MSC-sEVs for clinical stroke trials. To test the efficacy of hypoxic MSCs in a second stroke model in an additional species, we now exposed 6-8-month-old Sprague-Dawley rats to permanent distal MCAO and intravenously administered vehicle, platelet sEVs, or sEVs obtained from hypoxic MSCs (1% O2; 2 × 106 or 2 × 107 cell equivalents/kg) at 24 h, 3, 7, and 14 days post-MCAO. Over 28 days, motor-coordination recovery was evaluated by rotating pole and cylinder tests. Ischemic injury, brain inflammatory responses, and peri-infarct angiogenesis were assessed by infarct volumetry and immunohistochemistry. sEVs obtained from hypoxic MSCs did not influence infarct volume in this permanent MCAO model, but promoted motor-coordination recovery over 28 days at both sEV doses. Ischemic injury was associated with brain ED1+ macrophage infiltrates and Iba1+ microglia accumulation in the peri-infarct cortex of vehicle-treated rats. Hypoxic MSC-sEVs reduced brain macrophage infiltrates and microglia accumulation in the peri-infarct cortex. In vehicle-treated rats, CD31+/BrdU+ proliferating endothelial cells were found in the peri-infarct cortex. Hypoxic MSC-sEVs increased the number of CD31+/BrdU+ proliferating endothelial cells. Our results provide evidence that hypoxic MSC-derived sEVs potently enhance neurological recovery, reduce neuroinflammation. and increase angiogenesis in rat permanent distal MCAO.

Small extracellular vesicles (small EV) are a conserved means of communication across the domains of life and lately gained more interest in mammalian non-cancerous work as non-cellular, biological therapeutic with encouraging results in recent studies of chronic degenerative diseases. The nucleus pulposus (NP) is the avascular and aneural center of an intervertebral disc (IVD), home to unique niche conditions and affected in IVD degeneration. We investigated autologous and mesenchymal stem cell (MSC) small EVs for their potential to contribute to cell and tissue homeostasis in the NP niche via mass spectrometric proteome and functional enrichment analysis using adult and fetal donors. We compared these findings to published small EV databases and MSC small EV data. We propose several mechanisms associated with NP small EVs: Membrane receptor trafficking to modify signal responses promoting niche homeostasis; Redox and energy homeostasis via metabolic enzymes delivery; Cell homeostasis via proteasome delivery and immunomodulation beyond an association with a serum protein corona. The proteome signature of small EVs generated by NP parent cells is similar to previously published small EV data, yet with a focus on supplementing anaerobic metabolism and redox balance while contributing to the maintenance of an aneural and avascular microniche.

Investigating the mechanisms underlying the development of an immunosuppressive microenvironment within colorectal liver metastases (CRLM) is important for identifying synergistic targets for immunotherapy. The regulatory role of tumor-associated macrophage-derived extracellular vesicles (TAM-EVs) in the immune microenvironment of CRLM has not yet been fully explored. Here, we found that TAM-EVs shaped the immunosuppressive microenvironment at the invasive front in murine CRLM models, thus dampening anti-PD-1 immunotherapy. This environment is characterized by an increased tumor stemness potential and abundant neutrophil extracellular traps (NETs) formation. Mechanistically, TAM-EVs-derived fibrinogen-like 2 (FGL2) interacts with the FCGR2B receptor in tumor cells, which further activates a p-STAT3/IL-1β positive feedback loop to increase the stemness potential of cancer cells, whereas IL-1β mediates the communication between cancer cells and neutrophils. The use of an anti-IL-1β monoclonal antibody can reduce NETs production and synergize with anti-PD-1 immunotherapy, which offers clinical translational significance for CRLM therapy. The FGL2/p-STAT3/IL-1β loop correlates with an immunosuppressive microenvironment and poor prognosis in human patients with CRLM. Our results revealed the potential of enhancing the efficacy of immunotherapy via the targeted clearance of NETs using anti-IL-1β monoclonal antibodies, which have significant clinical translational value in the treatment of CRLM.

Cardiovascular diseases (CVDs) remain the leading cause of death worldwide, both in developed and developing countries. Despite the implementation of various measures in clinical practice that have shown certain curative effects, poor prognosis and irreversible pathological cardiac remodeling continue to limit the therapeutic effect of CVDs. There are still many new mechanisms worth exploring for the regulation of CVDs. Previous studies have highlighted the potential applicability of exosomes in CVDs, and significant research has been conducted in this area. In this review, we summarize the physiological mechanisms of exosomes and the basic research achievements in regulating CVDs via exosomal non-coding RNAs. We also discuss the limitations and prospects of exosome application in CVD treatment.

Extracellular vesicles (EVs) are natural carriers of biological information and play pivotal roles in intercellular communication. EVs are biocompatible, have low immunogenicity, and are capable of traversing biological barriers, making them ideal tools for disease diagnosis and therapy. Despite their promising prospects, the full realization of EVs potential faces several challenges. This article aims to comprehensively review the biological and molecular features of EVs, their applications in liver cancer and possible underlying mechanisms, and the critical challenges affecting the clinical translation of EVs-based therapies in liver cancer.

Helicobacter pylori (H. pylori) is a Gram-negative bacterium that colonizes the human stomach and is associated with several gastric diseases, including gastritis, peptic ulcer disease, and gastric cancer. The bacterium's ability to thrive in the harsh gastric environment is due, to some extent, to its stress response mechanisms, with its heat shock proteins (HSPs) playing a putative, yet not fully understood, role in these adaptive processes. HSPs are a family of molecules, highly conserved throughout phylogenesis, that assist in protein folding, prevent aggregation, and ensure cellular homeostasis under stressful conditions. In H. pylori, HSPs contribute to survival in the stomach's acidic environment and oxidative stress. Furthermore, they aid in the bacterium's ability to adhere to gastric epithelial cells, modulate the host immune response, and form biofilms, all contributing to chronic infection and pathogenicity. The role of microbial HSPs in antibiotic resistance has also emerged as a critical area of research, as these proteins help stabilize efflux pumps, protect essential proteins targeted by antibiotics, and promote biofilm formation, thereby reducing the efficacy of antimicrobial treatments. Among bacterial HSPs, GroEL and DnaK are probably the major proteins that control most of the H. pylori's functioning. Indeed, both proteins possess remarkable acid resistance, high substrate affinity, and dual roles in protein homeostasis and host interaction. These features make them critical for H. pylori's adaptation, persistence, and pathogenicity in the gastric niche. In addition, recent findings have also highlighted the involvement of HSPs in the crosstalk between H. pylori and gastric epithelial cells mediated by the release of bacterial outer membrane vesicles and host-derived exosomes, both of these extracellular vesicles being part of the muco-microbiotic layer of the stomach and influencing cellular signalling and immune modulation. Considering their critical role in the survival and persistence of bacteria, microbial HSPs also represent potential therapeutic targets. Strategies aimed at inhibiting microbial HSP function, combined with conventional antibiotics or developing vaccines targeting microbial HSPs, could provide new avenues for the treatment of H. pylori infections and combat antibiotic resistance. This review explores the multifaceted roles of microbial HSPs in the pathogenesis of H. pylori, highlighting their contributions to bacterial adhesion, immune evasion, stress response, and antibiotic resistance.

Lemur Tail Kinase 3 (LMTK3) promotes cell proliferation, invasiveness and therapy resistance, and its expression correlates with poor survival in several different malignancies, including breast cancer. Crosstalk through extracellular vesicles (EVs) is an increasingly appreciated mechanism of cell communication within the tumour immune microenvironment, which contributes to different aspects of cancer progression and plays a pivotal role in shaping tumour fate.

Nanoparticle tracking analysis and transmission electron microscopy were used to study the effects of LMTK3 on EV size, while single particle interferometry allowed us to examine LMTK3-dependent effects on the subpopulation distribution of EVs. Quantitative mass spectrometry was used to profile LMTK3-dependent proteomics changes in breast cancer-derived EVs. Bioinformatics analysis of clinical data along with in vitro and cell-based assays were implemented to explore the effects of LMTK3-dependent EV protein cargo on the tumour immune microenvironment. To elucidate the mechanism through which LMTK3 impacts endosomal trafficking and regulates EV biogenesis, we used a variety of approaches, including in vitro kinase assays, confocal and electron microscopy, as well as in vivo subcutaneous and orthotopic breast cancer mouse models.

Here, we report that LMTK3 increases the average size of EVs, modulates immunoregulatory EV proteomic cargo and alters the subpopulation distribution of EVs released by breast cancer cells. Mechanistically, we provide evidence that LMTK3 phosphorylates Rab7, a key regulator of multivesicular body (MVB) trafficking, thereby reducing the fusion of MVBs with lysosomes and subsequent degradation of intralumenal vesicles, resulting in altered EV release. Moreover, LMTK3 causes increased packaging of phosphoserine aminotransferase 1 (PSAT1) in EVs, leading to a paracrine upregulation of phosphoglycerate dehydrogenase (PHGDH) in monocytes when these EVs are taken up. PSAT1 and PHGDH play key roles in the serine biosynthesis pathway, which is closely linked to cancer progression and regulation of monocyte behaviour. LMTK3 EV-induced elevated PHGDH expression in monocytes reduces their infiltration into breast cancer 3D spheroids and in vivo breast cancer mouse models. Furthermore, these infiltrating monocytes preferentially differentiate into pro-tumourigenic M2-like macrophages. Additional breast cancer mouse studies highlight the contribution of LMTK3-dependent EVs in the observed immunosuppressive macrophage phenotype. Finally, in vitro experiments show that pharmacological inhibition of LMTK3 reverses the pro-tumourigenic and immunomodulatory effects mediated by EVs derived from LMTK3 overexpressing cells.

Overall, this study advances our knowledge on the mechanisms of EV biogenesis and highlights a novel oncogenic role of LMTK3 in the breast TME, further supporting it as a target for cancer therapy.

Extracellular vesicles (EVs) are widely explored as vehicles for delivering therapeutic or experimental cargo to cardiomyocytes. Efforts to improve EV bioavailability in the heart, and reduce their off-target actions, require screening methods that can replicate the physiological and anatomical barriers present in the myocardium. Additionally, discovery pipelines must exercise control over EV dosage and timing, and provide a means of assessing cargo incorporation into cardiomyocytes specifically. These criteria are not generally met by experiments on cultured cells or animals. Here, we present a Langendorff-heart discovery pipeline that combines the strengths of in vivo and in vitro approaches. Langendorff-mode perfusion enables controlled exposure of beating hearts to re-circulated EVs. Following perfusion, cardiomyocytes can be isolated enzymatically for analysis, such as imaging. We tested this discovery pipeline by functionalizing EVs with beta-blockers (atenolol, metoprolol) using click-chemistry and incorporating the fluorescent protein NeonGreen2 to track the fate of EV cargo. Fluorescence in cardiomyocytes, including their nuclear regions, increased after Langendorff-treatment with beta-blocker decorated EVs, but only if these contained NeonGreen2, implicating the fluorescent cargo as the source of signal. Superior binding efficacy of beta-blockers was confirmed by referencing to the substantially lower signals obtained using wild-type EVs or EVs presenting myomaker or myomixer proteins, motifs that modestly enrich cardiac EV uptake in mice. Our findings demonstrate successful cardiomyocyte targeting using EVs decorated with beta-receptor binders. We propose the Langendorff-perfused heart as an intermediate step, nested between in vitro characterisation and animal testing, in discovery pipelines for seeking improved EV designs for the heart.

Numerous studies have demonstrated that extracellular vesicles (EVs) carry a variety of noncoding RNAs (ncRNAs), which can be taken up by neighboring cells or transported to distant sites via bodily fluids, thereby facilitating intercellular communication and regulating multiple cellular functions. Within the tumor microenvironment, EV-ncRNA, on the one hand, regulate the expression of PD-L1, thereby influencing tumor immune evasion, promoting tumor cell proliferation, and enhancing tumor growth, invasion, and metastasis in vivo. On the other hand, these specific EV-ncRNAs can also modulate the functions of immune cells (such as CD8 + T cells, macrophages, and NK cells) through various molecular mechanisms, inducing an immunosuppressive microenvironment and promoting resistance to anti-PD-1 therapy. Therefore, delving into the molecular mechanisms underlying EV-ncRNA regulation of immune checkpoints presents compelling therapeutic prospects for strategies that selectively target EV-ncRNAs. In this review, we elaborate on the cutting-edge research progress related to EV-ncRNAs in the context of cancer and dissect their pivotal roles in the PD-1/PD-L1 immune checkpoint pathway. We also highlight the promising clinical applications of EV-ncRNAs in anti-PD-1/PD-L1 immunotherapy, bridging basic research with practical clinical applications.

Extracellular vesicles (EVs) are heterogeneous nanoscale membrane vesicles released by almost all cell types into the circulation. Depending on their biogenesis and cells of origin, EVs show considerable heterogeneity in their biophysical and biomolecular composition and can serve as reflective and dynamic blood biomarkers for personalized medicine. Conventional analytical technologies, however, often lack the compatibility to reveal nanoscale EV features and resolve vesicle heterogeneity. The past decade has since witnessed the development of various nanoplasmonic technologies to empower EV analysis, through bulk and single-vesicle characterization, at an unprecedented scale and resolution. These platforms achieve versatile measurements that are not only size-matched to EV dimensions but can also probe multiplexed biomolecular contents, thereby providing new insights into EV heterogeneity and enabling transformative clinical opportunities. In this review, key characteristics of EVs and their remarkable heterogeneity are introduced. The sensing principles of plasmonic platforms are also discussed, with recent technology developments highlighted to resolve EV heterogeneity, through bulk analyses of EV subpopulations as well as high-resolution single-EV measurements. An outlook is further provided on emerging opportunities, at the interface of biomarker discovery and technology innovation, to develop empowering nanoplasmonic EV platforms for personalized medicine. biosensing; bulk analysis; extracellular vesicles; nanoplasmonics; single-vesicle analysis.

Autophagy and apoptosis are two essential mechanisms regulating cell fate. Although distinct, their signaling pathways are closely interconnected through various crosstalk mechanisms. Lipid rafts are described to act as both physical and functional platforms during the early stages of autophagic and apoptotic processes. Only recently has a role for lipid raft-associated molecules in regulating EV biogenesis and release begun to emerge. In particular, lipids of EV membranes are essential components in conferring stability to these vesicles in different extracellular environments and/or to facilitate binding or uptake into recipient cells. In this review we highlight these aspects, focusing on the role of lipid molecules during apoptosis and secretory autophagy pathways. We describe the molecular machinery that connects autophagy and apoptosis with vesicular trafficking and lipid metabolism during the release of EVs, and how their alterations contribute to the development of various diseases, including autoimmune disorders and cancer. Overall, these findings emphasize the complexity of autophagy/apoptosis crosstalk and its key role in cellular dynamics, supporting the role of lipid rafts as new therapeutic targets.

Extracellular vesicles (EVs) are lipid bilayer-enclosed particles secreted by cells and ubiquitously present in various biofluids. They not only mediate intercellular communication but also serve as promising drug carriers that are capable of delivering therapeutic agents to target cells through their inherent physicochemical properties. In this review, we summarized the recent advances in EV isolation techniques and innovative drug-loading strategies. Furthermore, we emphasized the distinct advantages and therapeutic applications of EVs derived from different cellular sources in cancer treatment. Finally, we critically evaluated the ongoing clinical trials utilizing EVs for drug delivery and systematically assessed both the opportunities and challenges associated with implementing EV-based drug delivery systems in cancer therapy.

The CRISPR/Cas9 toolbox consists of modular nucleases that can be employed to efficiently modify genomic sequences with high specificity. However, delivery of the large Cas9-sgRNA ribonucleoprotein (RNP) complexes remains challenging due to their immunogenicity, size, and overall negative charge. An approach to overcome these limitations is the use of extracellular vesicles (EVs) as intracellular delivery vehicles. EVs exhibit the natural ability to carry and deliver RNA and proteins across biological barriers, and can be engineered to load and deliver a variety of biotherapeutic molecules. Previous studies have shown that efficient EV-mediated cargo delivery does not only require active loading strategies, but also benefits from strategies to release cargo from the EV membrane. Here, we load Cas9 RNP complexes into EVs by expressing sgRNAs containing MS2 aptamers (MS2-sgRNAs), alongside Cas9 and a fusion protein of CD63 and tandem MS2 coat proteins (MCPs). We demonstrate that efficient Cas9 RNP delivery can also be facilitated by modulating the binding affinity between MS2 aptamers and the MCPs. To study the effect of altering the binding affinity between the MS2 hairpin and the MCP on Cas9 RNP delivery, various mutations affecting the binding affinity were made in both the interacting MS2-hairpin and the RNA-binding domain of the MCPs. Comparing Cas9 RNP delivery of the modulated MS2-sgRNAs revealed that adapting binding affinity highly affects functional RNP delivery. Mutations resulting in high affinity did not facilitate efficient RNP delivery unless combined with a photo-inducible release strategy, showing that cargo release was a limiting factor in RNP delivery. Mutations that decreased affinity resolved this issue, resulting in Cas9 RNP delivery without the requirement of additional release strategies. However, further decreasing affinity resulted in decreased Cas9 gene-editing efficiency due to decreased levels of Cas9 RNP loading into EVs. A similar effect on functional delivery was seen after modification of the RNA-binding domain of the MCPs. Our results demonstrate that EVs are capable of functional Cas9-sgRNA complex delivery, and that modulation of binding affinity can be used to increase efficient functional delivery with non-covalent loading constructs, without the need for additional engineering strategies for cargo release.

Extracellular vesicles are nanoscale vesicles released by a diversity of cells that mediate intercellular communication by transporting an array of biomolecules. They are gaining increasing attention in cancer research due to their ability to carry specific biomarkers. This characteristic makes them potentially useful for highly sensitive, noninvasive diagnostic procedures and more precise prognostic assessments. Consequently, EVs are emerging as a transformative tool in cancer treatment, facilitating early detection and personalized medicine. Despite significant progress, clinical implementation is hindered by challenges in EV isolation, purification, and characterization. However, developing advanced biosensor technologies offers promising solutions to these obstacles. This review highlights recent progress in biosensors for EV detection and analysis, focusing on various sensing modalities including optical, electrochemical, microfluidic, nanomechanical, and biological sensors. We also explore techniques for EV isolation, characterization, and analysis, such as electron microscopy, atomic force microscopy, nanoparticle tracking analysis, and single-particle analysis. Furthermore, the review critically assesses the challenges associated with EV detection and put forward future directions, aiming to usher in a cutting-edge era of precision medicine through advanced, sensor-based, noninvasive early cancer diagnosis by detecting EV-carried biomarkers.

Gene therapy has a pivotal role in treating new diseases. In addition to the recent mRNA-based COVID-19 vaccines produced by Pfizer-BioNTech and Moderna against severe acute respiratory syndrome corona virus 2, several new gene therapies have recently been approved as effective treatments for fatal genetic disorders such as Duchenne's muscular dystrophy, familial transthyretin amyloidosis, hemophilia A, hemophilia B, spinal muscle atrophy, early cerebral autoleukodystrophy, and β-thalassemia. This review provides novel insights into RNA therapeutics focusing on endogenous RNA species, RNA structure and function, and chemical modifications that improve the stability and distribution of RNAs. Furthermore, it includes updated knowledge on clinically approved gene therapies rendering a comprehensive understanding of the biochemical basis and clinical application of gene therapies. SIGNIFICANCE STATEMENT: There have recently been significant advances in clinical translation of RNA therapeutics. This review discusses the diverse types of RNA species, RNA structure and function, backbone and chemical modifications to RNAs, and every RNA therapeutic approved for clinical use at the time of writing.

In the cardiovascular system, different types of cardiovascular cells can secrete specific exosomes and participate in the maintenance of cardiovascular function and the occurrence and development of diseases. Exosomes carry biologically active substances such as proteins and nucleic acids from cells of origin and can be used as biomarkers for disease diagnosis and prognosis assessment. In addition, exosome-mediated intercellular communication plays a key role in the occurrence and development of cardiovascular diseases and has become a potential therapeutic target. This article emphasizes the importance of understanding the mechanism of exosomes in cardiovascular diseases and systematically details the current understanding of exosomes as regulators of intercellular communication in cardiomyocytes, providing a basis for future research and therapeutic intervention.

Blood vessel formation is a necessary part of bone tissue regeneration. MSCs-sEVs play a vital role in the in vivo bone regeneration strategy. However, natural MSCs-sEVs suffer from limited blood vessel formation potency, which makes it difficult to induce vascularized bone regeneration. Here, sEVs derived from magnesium cation-activated DPSCs (Mg2+-EVs) are purified and found to have superior potential in promoting endothelial cell migration and angiogenesis, as well as BMSC proliferation and osteogenesis. The beneficial effects of Mg2+-EVs could be attributed to the enrichment of miR-451a and the subsequent regulation and activation of AKT/eNOS signaling pathways. On this basis, Mg2+-EVs are delivered on β-TCP-modified GelMA scaffolds for slow release and better bioavailability. The rat cranial defect model verifies that GelMA/β-TCP with Mg2+-EVs has enhanced potential of inducing vascularized bone regeneration. The present study provides a cation-activated strategy to modulate the cargos and contents of MSC-derived sEVs, obtaining desirable vascular promotion and bone regeneration potential. Furthermore, the developed β-TCP-modified delivery scaffolds represent a promising strategy for efficient loading and slow-release delivery of sEVs for clinical translation.

Osteoarthritis (OA) remains a leading cause of disability worldwide, with no disease-modifying therapies currently available for treatment. The infrapatellar fat pad (IFP) harbors mesenchymal stem/stromal cells (MSC) with potent immunomodulatory and regenerative properties, making them a promising candidate for OA treatment. A growing body of evidence suggests that the therapeutic effects of MSC are largely mediated by their extracellular vesicles (EVs), which carry bioactive cargo that modulates inflammation and tissue repair. However, optimizing MSC-derived EVs as a cell-free therapeutic approach requires an in-depth understanding of how culture conditions and inflammatory/hormonal priming influence their functional properties. In this study, IFP-MSC were expanded in regulatory-compliant human platelet lysate (HPL) and xeno-/serum-free (XFSF) media and primed with an inflammatory/fibrotic cocktail (TIC) with oxytocin (OXT) to assess the impact on their immunophenotypic profile and EV cargo. The immunophenotype confirmed that TIC+OXT-primed MSC retained key immunomodulatory surface markers, while EV characterization verified the successful isolation of CD63+/CD9+ vesicles. Pathway enrichment analysis of both HPL- and XFSF- TIC+OXT EVs cargo identified key miRNAs associated with immune regulation, tissue repair, and anabolic signaling. Functional assays revealed that TIC+OXT EVs promoted M2-like anti-inflammatory macrophage polarization and exhibited chondroprotective properties in chondrocytes/synoviocytes inflammatory osteoarthritic assay. These findings highlight the therapeutic potential of TIC+OXT-primed IFP-MSC-derived EVs as immunomodulatory and chondroprotective agents, offering a promising strategy for OA treatment through a clinically viable, cell-free approach.

Babesia microti is the primary cause of human babesiosis in North America. Despite the emergence of the disease in recent years, the pathogenesis and immune response to B. microti infection remain poorly understood. Studies in laboratory mice have shown a critical role for macrophages in the elimination of parasites and infected red blood cells (iRBCs). Importantly, the underlying mechanisms that activate macrophages are still unknown. Recent evidence identified the release of extracellular vesicles (EVs) from Babesia iRBCs. EVs are spherical particles released from cell membranes under natural or pathological conditions that have been suggested to play roles in host-pathogen interactions among diseases caused by protozoan parasites. The present study examined whether EVs released from cultured Babesia iRBCs could activate macrophages and alter cytokine secretion. An analysis of vesicle size in EV fractions from Babesia iRBCs showed diverse populations in the <100 nm size range compared to EVs from uninfected RBCs. In co-culture experiments, EVs released by B. microti iRBCs appeared to be associated with macrophage membranes and cytoplasm, indicating uptake of these vesicles in vitro. Interestingly, the incubation of macrophages with EVs isolated from Babesia iRBC culture supernatants resulted in the activation of NF-κB and modulation of pro-inflammatory cytokines. These results support a role for Babesia-derived EVs in macrophage activation and provide new insights into the mechanisms involved in the induction of the innate immune response during babesiosis.

Various cell types release extracellular vesicles (EVs) containing proteins, DNA, and RNA essential for intercellular communication. The bioactive molecules from EVs can reflect disease status and monitor progression, while their communication abilities suggest therapeutic potential. We will review various EV isolation methods, EV-enriched fluids, and studies analyzing differential mi-RNA and protein levels extracted from EVs. Specifically, tear-derived EVs, which protect their molecular content and allow for real-time monitoring of ocular conditions such as Dry Eye Disease (DED), Sjögren's disease (SJD), Ocular graft-versus-host disease (oGVHD), and Diabetic Retinopathy (DR), which all currently remain undiagnosed in patients. EVs also provide potential as carriers for gene transfer, and mesenchymal stem cell (MSCs)-derived EVs are shown to be immunomodulatory, demonstrating promise for autoimmune ocular diseases. Through the multi-omic analysis of tear-fluid content, EVs are promising biomarkers and therapeutic agents in ocular diseases.

In this study, the lack of standardized freezing protocols for sperm cryopreservation in dogs and the limited research on using exosomes in cryopreservation were considered. Additionally, unlike previous studies on sperm cryopreservation, we introduced an innovative approach using the Aqueous Two-Phase System (ATPS) method for exosome isolation. This study aimed to evaluate the sperm-protective effects of adipose tissue-derived mesenchymal stem cell exosomes (MSC-exo) and seminal plasma exosomes (SP-exo) in dog sperm cryopreservation. Ejaculates from six dogs were processed with Tris-based diluents and divided into four groups: MSC-exo, 1.5 % SP-exo, 2 % SP-exo, and control, and frozen. After thawing, sperm motility, viability, membrane integrity, chromatin integrity, morphological integrity, and gene expression levels were analyzed. The results showed that the MSC-exo group had significantly higher total motility (%60.31 ± 6.12), progressive motility (%22.09 ± 3.34), plasma membrane integrity (%66.94 ± 2.24), and viability (%70.88 ± 1.95) compared to the other groups (P < 0.05). Additionally, the normal chromatin packaging rate was highest in the MSC-exo group (%91.33 ± 0.61, P < 0.05). While some improvements were observed in the SP-exo groups, they were not as pronounced as in the MSC-exo group. No significant differences were found in gene expression levels, although an improvement trend was observed in the MSC-exo group. In conclusion, MSC-exo reduced cryopreservation-induced sperm damage and provided overall protection in sperm parameters. These findings suggest that MSC-exo could be a potential biological additive in dog sperm freezing protocols.

Small extracellular vesicles (sEVs) can provide information about the pathophysiology of the cells; therefore, sEVs have attracted considerable interest as possible diagnostic biomarkers. A key challenge lies in the necessity for simple and cost-effective sEV isolation methods to achieve high purity and yield suitable for research and clinical applications. We are introducing a comprehensive study on isolating sEVs using a novel cocktail strategy that integrates chemical precipitation and ultrafiltration with a two-step filtering process to ensure a highly pure and homogeneous population and further compared with PEG-based precipitation, ultra-centrifugation, and size-exclusion-chromatography columns. The isolated sEVs from each protocol are quantified for size and yield using nanoparticle tracking analysis, morphologically characterized through transmission electron microscopy, and validated by quantifying the expression profiles of sEV surface biomarkers. Furthermore, the study explores the applicability of our method for downstream multi-omics analyses. The results highlight the efficacy of the proposed protocol, demonstrating the ease and efficiency of isolating sEVs from different biofluids with minimal laboratory requirements and confirming the compatibility with multi-omics analyses. These findings position our method as particularly valuable for translational research, offering a promising avenue for advancing the study and application of sEVs in diagnostic and therapeutic research.

Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by chronic inflammation and progressive damage to connective tissue. It is driven by dysregulated cellular homeostasis, often leading to autoimmune destruction and permanent disability in severe cases. Over the past decade, various drug delivery systems have been developed to enable targeted therapies for disease prevention, reduction, or suppression. As an emerging therapeutic platform, extracellular vesicles (EVs) offer several advantages over conventional drug delivery systems, including biocompatibility and low immunogenicity. Consequently, an increasing number of studies have explored EV-based delivery systems in the treatment of RA, leveraging their natural ability to evade phagocytosis, prolong in vivo half-life, and minimize the immunogenicity of therapeutic agents. In this review, we first provide an in-depth overview of the pathogenesis of RA and the current treatment landscape. We then discuss the classification and biological properties of EVs, their potential therapeutic mechanisms, and the latest advancements in EVs as drug delivery platforms for RA therapy. We emphasize the significance of EVs as carriers in RA treatment and their potential to revolutionize therapeutic strategies. Furthermore, we examine key technological innovations and the future trajectory of EV research, focusing on the challenges and opportunities in translating these platforms into clinical practice. Our discussion aims to offer a comprehensive understanding of the current state and future prospects of EV-based therapeutics in RA.