In recent years, the significant role of anticancer drugs in cancer treatment has garnered considerable attention. However, the application of these drugs is largely limited by their short half-life in blood circulation, low cellular uptake efficiency, and off-target effects. Exosomes, which serve as crucial messengers in intercellular communication, exhibit unique advantages in molecular delivery compared to traditional synthetic carriers, thereby offering new possibilities for modern drug delivery systems. Exosomes possess organotropic functions and are naturally produced by cells, making them promising candidates for natural drug delivery systems with organotropic properties and minimal side effects. These naturally derived carriers can achieve stable, efficient, and selective delivery of anticancer drugs, thereby enhancing the efficacy and potential of anticancer agents in cancer immunotherapy. This review provides a concise overview of the unique characteristics of exosomes related to anticancer drug delivery, strategies for utilizing exosomes as carriers in cancer therapy, and the latest advancements in the field.

Spinal cord injury (SCI) is a devastating event for the central nervous system (CNS), often resulting in the loss of sensory and motor functions. It profoundly affects both the physiological and psychological well-being of patients, reducing their quality of life while also imposing significant economic pressure on families and the healthcare system. Due to the complex pathophysiology of SCI, effective treatments for promoting recovery remain scarce. Mesenchymal stem cell-derived exosomes (MSC-Exos) offer advantages such as low immunogenicity, good biocompatibility, and the ability to cross the blood-spinal cord barrier (BSCB). In preclinical studies, they have progressively shown efficacy in promoting SCI repair and functional recovery. However, the low yield and insufficient targeting of MSC-Exos limit their therapeutic efficacy. Currently, genetic engineering and other preprocessing techniques are being employed to optimize both the yield and functional properties of exosomes, thereby enhancing their therapeutic potential. Therefore, this paper provides an overview of the pathophysiology of SCI and the biogenesis of exosomes. It also summarizes current approaches to optimizing exosome performance. Additionally, it details the mechanisms through which optimized exosomes provide neuroprotection and explores the potential of combined treatments involving MSC-Exos and hydrogels.

Although employing exosomes (EXOs) for promoting tissue repair is already in the pipeline as a new cell-free wound treatment, the rapid clearance of EXOs is still a challenge. This study assesses the effectiveness of a hybrid design of nanofibers and hydrogel in the controlled delivery of EXOs to wounds for an enhanced healing process. EXOs are isolated from the human placenta-derived stem cells and characterized by a novel nano-fluorescent dot blot assay. They are incorporated into an alginate hydrogel composited with a nanofibrous layer of poly(ε-caprolactone) to mimic the bilayer structure of the dermis and epidermis. The scaffold characteristics, including morphology, mechanobiological properties, physical properties, anti-inflammatory activity, and cytocompatibility are comprehensively evaluated. The tailored hydrophilic/hydrophobic design of the scaffolds presents controlled degradability, controlled EXOs release, enhanced cell proliferation, hemostatic activity with insignificant hemolysis, and a balance of strength and conformability suitable for full-thickness wound milieu. The repair of full-thickness wounds is further investigated in a rat model. Animal study results indicate that the EXO-loaded scaffolds accelerate wound closure, inflammation reduction, re-epithelialization, and collagen synthesis. For the latter, a collagen content of 22 % and 33 % higher than that for the unloaded scaffold and the control was observed, respectively.

Neutrophil-derived extracellular vesicles (NEVs) are critically involved in disease progression and are considered potential biomarkers. However, the tedious processes of NEV separation and detection restrain their use. Herein, we presented an integrated microfluidic chip for NEV (IMCN) analysis, which achieved immune-separation of CD66b+ NEVs and multiplexed detection of their contained miRNAs (termed NEV signatures) by using 10 μL serum samples. The optimized microchannel and flow rate of the IMCN chip enabled efficient capture of NEVs (>90%). After recognition of the captured NEVs by a specific CD63 aptamer, on-chip rolling circle amplification (RCA) reaction was triggered by the released aptamers and miRNAs from heat-lysed NEVs. Then, the RCA products bound to molecular beacons (MBs), initiating allosteric hairpin structures and amplified "turn on" fluorescence signals (RCA-MB assay). Clinical sample analysis showed that NEV signatures had a high area under curve (AUC) in distinguishing between healthy control (HC) and gastric cancer (GC) (0.891), benign gastric diseases (BGD) and GC (0.857). Notably, the AUC reached 0.912 with a combination of five biomarkers (NEV signatures, CEA, and CA199) to differentiate GC from HC, and the diagnostic accuracy was further increased by using a machine learning (ML)-based ensemble classification system. Therefore, the developed IMCN chip is a valuable platform for NEV analysis and may have potential use in GC diagnosis.

Exosomes are critical mediators of cell-to-cell communication in physiological and pathological processes, due to their ability to deliver a variety of bioactive molecules. Tumor-derived exosomes (TDEs), in particular, carry carcinogenic molecules that contribute to tumor progression, metastasis, immune escape, and drug resistance. Thus, TDE inhibition has emerged as a promising strategy to combat cancer. In this review, we discuss the key mechanisms of TDE biogenesis and secretion, emphasizing their implications in tumorigenesis and cancer progression. Moreover, we provide an overview of small-molecule TDE inhibitors that target specific biogenesis and/or secretion pathways, highlighting their potential use in cancer treatment. Lastly, we present the existing obstacles and propose corresponding remedies for the future development of TDE inhibitors.

Background: Identification of targetable biomarkers to improve early disease detection and overall patient outcomes is becoming an urgent need in clinical oncology. Ovarian cancer (OC) has one of the highest mortality rates among gynecological cancers. It is asymptomatic and almost always diagnosed at an advanced stage (III or IV), leading to a 5-year survival rate of approximately 35%. Methods: Current therapeutic approaches for OC are very limited and mainly consist of cytoreductive surgery and cisplatin plus taxane-based chemotherapy. No gender and tumor specific biomarkers are known. Exosomes, lipid bilayer vesicles of endocytic origin secreted by most cell types, represent sources of information for their involvement in the onset and progression of many diseases. Hence, research on exosome contents as tools and targets in precise oncology therapy provides knowledge essential to improving diagnosis and prognosis of the disease. Results: This review attempts to give an overview of how exosomes are implicated in ovarian carcinoma pathogenesis to trigger further cancer exosome-based investigations aimed at developing ovarian cancer fine-tuning diagnostic methodologies. Conclusions: It is essential to investigate exosome-based cancer drugs to advance understanding, improve treatment plans, create personalized strategies, ensure safety, and speed up clinical translation to increase patients' overall survival and quality of life. Papers published in PubMed and Web of Science databases in the last five years (2020-2024) were used as a bibliographic source.

Breast cancer (BC) remains a significant global health challenge characterized by its heterogeneity and treatment complexities. Extracellular vesicles (EVs) are small membranous particles released by cells, facilitating intercellular communication by transporting bioactive molecules such as proteins, lipids, and nucleic acids. Tumor-derived EVs have emerged as pivotal regulators in the tumor microenvironment (TME) and drivers of BC progression. These EVs carry diverse cargoes of bioactive molecules, influencing critical processes such as immune modulation, angiogenesis, and metastasis. By altering the behaviors of immune cells including macrophages, dendritic cells, and T cells, tumor-derived EVs contribute to immune evasion and tumor growth. Furthermore, Tumor-derived EVs play a role in mediating drug resistance, impacting the effectiveness of therapeutic interventions. Understanding the multifaceted roles of BC tumor-derived EVs is essential for the development of innovative therapeutic strategies. Targeting pathways mediated by EVs holds promise for enhancing the efficacy of cancer treatments and improving patient outcomes. This comprehensive review provides insights into the intricate interactions of tumor-derived EVs in immune modulation and BC progression, highlighting potential therapeutic targets and avenues for novel cancer therapies.

Diabetes is an extremely costly disease, one-third of which are attributed to the management of diabetic foot disease including chronic, non-healing, diabetic foot ulcers (DFUs). Therefore, much effort is needed to understand the pathogenesis of DFUs and novel therapeutics. We utilized exosome staining to confirm the interaction between fibroblast-derived exosomes and macrophages. Subsequently, we employed public data and qPCR to screen for upregulated miRNAs in fibroblast-derived exosomes in DFUs. The relationship between was validate miR-93-5 and ATG16L1 through data prediction and dual-luciferase reporter assays. A variety of molecular biology experiments were used for subsequent pathway validation. Additionally, we established Atg16l1MKI and Nlrp3MKO mice for further validation. We identified that miR-93-5p derived from fibroblasts played an important role in M1 macrophages polarization. Predicted by database, we found that miR-93-5p can bind to ATG16L1 mRNA, thereby influencing macrophage autophagy mediated by ATG16L1 in the clearance of ROS, thus activating the NLRP3 signaling pathway. In vivo, miR-93-5p antagomir treatment accelerated diabetic wound healing and induced M2 macrophage polarization. Fibroblasts and macrophages show cell crosstalk during the development of DFUs by miR-93-5p, and that antagomir treatment may be a promising and technically advantageous alternative to DFUs therapies.

As life quality improves and the life pressure increases, people's awareness of maintaining healthy skin and hair grows. However, the use of bioactive peptides in regenerative medical aesthetics is often constrained by the high molecular weight, which impedes skin penetration. In contrast, extracellular vesicles not only possess regenerative properties but also serve as effective carriers for bioactive peptides. Given their anti-inflammatory and bactericidal properties, capacity to promote angiogenesis, optimize collagen alignment, facilitate re-epithelialization and stimulate hair growth, extracellular vesicles become an emerging and promising solution for skin regeneration treatments. The combination of peptides and extracellular vesicles enhances therapeutic efficacy and improves the bioavailability of bioactive peptides. In this review, we summarize the functions of bioactive peptides and plant- and animal-derived extracellular vesicles in regenerative medicine with cosmetology, along with examples of their combined applications. Additionally, we provide an overview of peptides and extracellular vesicles currently available on the market and in clinical practice, discussing the challenges and solutions associated with their use.

Skeletal muscle atrophy (sarcopenia) is a serious complication of liver cirrhosis, and chronic muscle inflammation plays a pivotal role in its pathologenesis. However, the detailed mechanism through which injured liver tissues mediate skeletal muscle inflammatory injury remains elusive. Here, it is reported that injured hepatocytes might secrete mtDNA-enriched extracellular vesicles (EVs) to trigger skeletal muscle inflammation by activating the cGAS-STING pathway. Briefly, injured liver secreted increased amounts of EVs into circulation, which are then engulfed primarily by macrophages in skeletal muscle and subsequently induce cGAS-STING signaling and its-mediated inflammatory response in muscles. In contrast, suppression of hepatic EV secretion or STING signaling significantly alleviated cirrhosis-induced skeletal muscle inflammation and muscle atrophy in vivo. Circulating EVs from cirrhotic patients showed higher levels of mtDNA, and the levels of EV-mtDNA positively correlated with the severity of liver injury. In injured hepatocytes, mitochondrial damage promoted the release of cytosolic mtDNA and the subsequent secretion of mtDNA-enriched EVs. This study reveals that injured hepatocyte-derived EVs induce skeletal muscle inflammation via the mtDNA‒STING axis, while targeted blockade of liver EV secretion or STING signaling represents a potential therapeutic approach for preventing cirrhosis-associated skeletal muscle atrophy.

Extracellular vesicles (EVs) are heterogeneous vesicles released by donor cells that can be taken up by recipient cells, thus inducing cellular phenotype changes. Since their discovery decades ago, roles of EVs in modulating initiation, growth, survival and metastasis of cancer have been revealed. Recent studies from multifaceted perspectives have further detailed the contribution of EVs to cancer drug resistance; however, the role of EV uptake in conferring drug resistance seems to be overlooked. In this comprehensive review, we update the EV subtypes and approaches for determining EV uptake. The biological basis of EV uptake is systematically summarized. Moreover, we focus on the diverse uptake mechanisms by which EVs carry out the intracellular delivery of functional molecules and drug resistance signaling. Furthermore, we highlight how EV uptake confers drug resistance and identify potential strategies for targeting EV uptake to overcome drug resistance. Finally, we discuss the research gap on the role of EV uptake in promoting drug resistance. This updated knowledge provides a new avenue to overcome cancer drug resistance by targeting EV uptake.

Working memory (WM) is a dynamic process linked to whole-brain functional connectome time-varying re-configuration. The neural dynamics underlying WM deficits in adolescents with early-onset schizophrenia (EOS), who have higher genetic loads and immature WM neural substrates, still remain unclear.

We used dynamic voxel-wise degree centrality (dDC) to explore the dynamic profile of whole-brain functional connectome in 51 adolescents with EOS and 45 healthy controls (HCs) during an n-back task. We assessed the group-related dDC time-varying variability and clustered meta-states differences between EOS and HCs. Correlation analysis also applied between the detected areas with clinical symptoms and WM performances, and detected areas further allowed for image transcription analyses.

We did not observe any group-related differences in the dDC time-varying instability. In the clustered dominant state 1, when facing with increased WM loads, EOS showed decreased dDC compared with HCs in the left insula, anterior and posterior lobe of the cerebellum, bilateral inferior parietal lobule, left pons, bilateral superior temporal gyrus, rectus gyrus, precuneus, bilateral inferior frontal gyrus (IFG), etc. Enrichment analysis reveals these detected areas related to synaptic function, neuronal communication, and metabolic processes.

This is the first study to investigate the abnormal time-varying pattern of the whole-brain connectome in EOS during the WM task and its molecular foundation. It demonstrated impaired neural resource allocation between frontoparietal, default-mode, and salience networks and the associated metabolic processes may underlie WM deficits in EOS, which can provide knowledge for targeted interventions and future research.

Skin trauma often results from pain, swelling, and scarring and can significantly interfere with daily activities. Extracts from the American cockroach, a rapidly reproducing insect, have been recognized for therapeutic properties in wound management. Traditional extraction methods use solvents such as ethanol to obtain the active compounds, but these methods may compromise the intrinsic biological properties of American cockroach extracts. In this study, we investigated the use of nanovesicles isolated from fresh American cockroaches in skin wound treatment and focused on their biological characteristics and therapeutic efficacy. Fresh and dried American cockroach nanovesicles (F-ACNVs and D-ACNVs, respectively) were procured via ultrahigh-speed centrifugation. We found that F-ACNVs exhibited superior cell proliferation-promoting activity. By employing metabolomics, proteomics, and long noncoding RNA (lncRNA) omics, we identified a rich repertoire of metabolites, proteins, and lncRNAs within F-ACNVs. In vitro and in vivo experiments demonstrated that F-ACNVs significantly enhanced the proliferation and migration of human umbilical vein endothelial cells (HUVECs) and human skin keratinocytes (HACATs) as well as the repair of skin mechanical trauma. These effects may be mediated through the activation of angiogenic signaling pathways. Our research introduces a novel therapeutic strategy for treating skin trauma and offers insight into the medicinal potential of insects such as the American cockroach while emphasizing the importance of preserving the intrinsic biological properties of insects for optimal therapeutic outcomes.

Cancer immunotherapy has revolutionized oncology, offering new hope for patients with previously incurable cancers. However, solid tumors remain a significant challenge due to immune evasion, therapeutic resistance, and the immunosuppressive tumor microenvironment. Exosomes, a specialized subset of extracellular vesicles, have emerged as promising tools in cancer therapy owing to their unique role in intercellular communication and immune modulation. These vesicles transport antigens, major histocompatibility complex (MHC) molecules, and immune-modulatory cargo, positioning them as potential platforms for cancer vaccines, drug delivery systems, and combinatorial therapies. Advances in engineered exosomes have improved drug bioavailability, tumor targeting, and immune stimulation, showcasing their potential in personalized medicine. This review highlights their multifaceted role in the tumor microenvironment, and their mechanisms of action in solid cancer therapy. Additionally, we discuss emerging strategies to overcome clinical and technical hurdles, paving the way for novel and effective cancer treatments.

This study is designed to explore the prognostic significance of exosome-related genes (ERGs) and their impact on the the tumor microenvironment (TME) of pancreatic cancer.

Transcriptomic data alongside clinical details of patients with PC were retrieved from both The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) repository. A collection of 121 exosome-associated genes were obtained from the ExoBCD database. For constructing a risk scoring model, the absolute shrinkage and selection operator (LASSO) regression method was employed. Gene set enrichment and variance analyses were facilitated by the clusterProfiler and GSVA R software tools. Additionally, CIBERSORT was used to estimate immune cell infiltration levels. Lastly, the TIDE algorithm was leveraged to evaluate the connection between gene expression and drug sensitivity. A series of experiments were used to verify the role of DLGAP5 in PC.

Two unique molecular clusters were uncovered, and our analysis revealed a connection between ERG dysregulation across multiple layers and patient demographic, histopathological attributes, prognosis, as well as immune cell infiltration patterns within the TME. An ERG_score was developed for forecasting overall survival and its predictive capacity was confirmed in PC cases. A precise nomogram was established to enhance the clinical utility of the ERG_score. Patients in the low-risk group exhibited higher immune and ESTIMATE scores than that in the high-risk group, displaying an improved overall survival (OS). The ERG_score was associated with cancer stem cell (CSC) index and drug sensitivity. Crucial evaluations of ERGs illuminated the significance of DLGAP5, emphasizing its expression in PC and its contributory role in tumor growth stimulation.

Our investigation reveals a correlation between the exosome-related risk assessment signature and the survival outcome as well as immune cell infiltration in patients with PC. This finding potentially paves the way for enhanced therapeutic strategies for PC.

Exosomes, cell-derived vesicles produced by cells, are fascinating and drawing growing interest in biomedical exploration due to their exceptional properties. There is intriguing evidence that exosomes are involved in major biological processes, including diseases and regeneration. Exosomes from mesenchymal stem cells (MSCs) have shown promising outcomes in regenerative medicine. Numerous studies suggest that exosomes have several advantages over conventional synthetic nanocarriers, opening novel frontiers for innovative drug delivery. Regenerative medicine has demonstrated the profound therapeutic outcomes of engineered or loaded exosomes from MSCs. Different methods are being used to modify or/load exosomes. These exosomes can improve cell signaling pathways for bone and cartilage diseases, liver diseases, nerve tissues, kidney diseases, skin tissue, and cardiovascular diseases. Despite extensive research, clinical translation of these exosomes remains a challenge. The optimization of cargo loading methods, efficiency, physiological stability, and the isolation and characterization of exosomes present some challenges. The upcoming examination should include the development of large-scale, quality-controllable production approaches, the modification of drug loading approaches, and numerous in vivo investigations and clinical trials. Here, we provided an informative overview of the extracellular vesicles and modification/loading methods of exosomes. We discuss the last exosome research on regeneration disorders, highlighting the therapeutic applications of MSCs-derived exosomes. We also highlight future directions and challenges, underscoring the significance of addressing the main questions in the field.

Myelodysplastic syndrome (MDS) is a clonal disorder of hematopoietic stem cells (HSCs), characterized by ineffective hematopoiesis and a high risk of progression to acute myeloid leukemia. Elucidating the mechanism underlying the dysfunction of MDS-HSCs is crucial for exploring the pathogenesis of the syndrome. While previous studies have implicated mesenchymal stem cells (MSCs), a principal component of the bone marrow (BM) microenvironment, in the inhibition of normal hematopoiesis, the precise molecular mechanisms have not been fully elucidated. In this study, we investigated the effects of MSCs from MDS patients on hematopoietic functions of HSCs from a metabolic perspective.

MSCs were isolated from BM of MDS patients. The proliferation, apoptosis, differentiation and support for hematopoiesis of these cells were analyzed using CCK-8 assay, FC and induction medium and CFU (colony forming units) assay, respectively. Expression levels of metabolic molecules were used as indicators to screen MSCs with different metabolic pathways and were detected by RT-PCR and Western blotting. Exosome derived from MSCs were isolated from the culture supernatant and confirmed by Transmission Electron Microscope, Dynamic Light Scattering and Western blotting. The effects of these exosomes on HSCs were analyzed using the same methods as those used to assess MSCs function.

Our findings demonstrated that MDS-MSCs exhibited significant functional impairments, including reduced proliferation, impaired differentiation, diminished support for hematopoiesis, and increased apoptosis. Notably, we observed an upregulation of lipid metabolism in these MSCs, which appears to contribute to their dysfunction. Intriguingly, the aberrant lipid metabolic profile can be effectively reversed by the administration of etomoxir (ETO), an inhibitor of carnitine palmitoyltransferase 1A (CPT-1A). Furthermore, MSCs with enhanced lipid metabolism could transmit this dysfunction to HSCs through the secretion of exosomes that are enriched in CPT-1A.

We suggest that the MDS BM microenvironment disrupts MSCs metabolism by increasing the expression of CPT-1A, which impairs the ability to support normal HSCs. Interestingly, the suppressive effect is mediated by exosomes rich in CPT-1A, which derived from MSCs. These findings provide novel insights into MDS MSCs-metabolism-Exosome axis in ineffective hematopoiesis and offer new strategies for the treatment of MDS.

The villous trophoblast cells are of fundamental importance because they fulfill a variety of functions that are vital for the growth of the fetus and the maintenance of pregnancy. A simple in vitro villous trophoblast cell model that grows on standard tissue culture plates has been utilized for various functional studies on villous trophoblast cells. Despite the potential value of incorporating electron microscopy analysis in reports on functional analysis of primary human trophoblast cells, electron microscopy analysis is exclusively ancillary to functional analysis in previous publications. In the context of autophagy research of villous trophoblast cells using primary trophoblast cells, a detailed ultrastructural analysis of autophagy flux using electron microscopy is imperative; however, it has not been conducted to date. In this study, we isolated term villous trophoblast cells (i.e., cytotrophoblast cells, CTB cells) using the most up-to-date isolation method for isolating pure CTB cells from human term placenta and investigated the ultrastructural dynamic process of autophagy of cultured CTB cells by means of transmission electron microscopy. The initial 6 h culture resulted in CTB cell aggregation; however, the majority of CTB cells did not differentiate into syncytial cells. In contrast, after 72 h, CTB cells exhibited a promotion of differentiation into syncytial cells. The electron microscopy analysis revealed the upregulation of autophagy and visualized unique autophagic profiles during differentiation into syncytial cells, which exhibited perinuclear accumulation of extremely large autophagosomes/autolysosomes. This study provides novel insights into the reproductive biology of primary trophoblast cells, thereby demonstrating the substantial value of primary trophoblast cells as research resources.

Exosomes served as "communicators" to exchange information among different cells in the nervous system. Our previous study demonstrated that the enhanced spinal synaptic transmission contributed to chronic visceral pain in irritable bowel syndrome. However, the underlying mechanism of primary sensory neuron (PSN)-derived exosomes on spinal transmission remains unclear. In this study, an exosome visualization method was established to specifically track exosomes derived from PSNs in CD63-GFPf/+ (green fluorescent protein) mice. Neonatal maternal deprivation (NMD) was adopted to induce chronic visceral pain in CD63-GFPf/+ male mice. The exosome visualization technology demonstrated that NMD increased visible PSN-derived exosomes in the spinal dorsal horn, enhanced spinal synaptic transmission, and led to visceral pain in CD63-GFPf/+ male mice. The PSN-derived exosomal miR-1306-3p sorted from spinal dorsal horn activated P2X3R, enhanced spinal synaptic transmission, and led to visceral pain in NMD mice. Moreover, upregulation of Rab27a in dorsal root ganglia mediated the increased release of PSN-derived exosomes, and intrathecal injection of siR-Rab27a reduced visible PSN-derived exosomes in spinal cord, suppressed spinal synaptic transmission, and alleviated visceral pain in NMD mice. This and future studies would reveal the detailed mechanisms of PSN-derived exosomes and provide a potential target for clinical treatment of chronic visceral pain in patients with irritable bowel syndrome.

Glaucoma is one of the leading causes of irreversible blindness, characterized by progressive visual field loss. Several risk factors are associated with developing the disease. However, the exact mechanisms or pathological pathways involved are still unknown. There is an urgent need to find the mechanisms and biomarkers for early detection and therapy to halt progression or cure the disease. Extracellular vesicles (EVs), specifically exosomes, have emerged as a crucial player in all aspects of glaucoma, including pathogenesis to therapeutic application with their cell-cell communication properties.

We performed a literature search on PubMed, Google Scholar, and Web of Science using different keywords. Next, we reviewed the literature with studies focusing on the role of EVs as a causative factor in disease progression, biomarker discovery based on their contents, and protection from glaucoma.

Studies summarized here provide reports of differential EV miRNA and protein expression alterations when communicating with aqueous humor drainage tissues. We described how EV contents are involved in various pathways, including extracellular matrix remodeling and miRNA-mediated oxidative stress transmission between outflow tissues, thereby contributing to glaucoma. Extracellular vesicles, mainly derived from mesenchymal stem cells protecting the optic nerve from degeneration, have also been discussed as potential therapies for glaucoma.

Overall, this review provides a comprehensive discussion of the role of extracellular vesicles in glaucoma. We identified the challenges in finding major signaling molecules of glaucoma etiology. Lastly, we highlighted future directions to improve the treatment of glaucoma by extracellular vesicles.

Glioma is the most common primary intracranial malignant tumor in adults, with a poor prognosis. Exosomes released by tumor cells play a crucial role in tumor development, metastasis, angiogenesis, and other biological processes. Despite this significance, the precise molecular mechanisms governing exosome secretion and their impact on tumor progression remain incompletely understood. While Choline Kinase Alpha (CHKA) has been implicated in promoting various types of tumors, its specific role in glioma pathogenesis remains unclear. Our study initially demonstrates that CHKA enhances the proliferation, migration, and invasion abilities of glioma cells. Interestingly, CHKA also stimulates the release of exosomes from glioma cells. Mechanistically, reduced CHKA expression hampers exosome secretion by elevating autophagy levels in gliomas, whereas counteracting the autophagy elevation resulting from CHKA downregulation restores the release of exosomes. Notably, exosomes derived from glioma cells with normal CHKA expression exhibit a greater capacity to promote glioma progression compared to those derived from cells with low CHKA expression. Overall, our findings suggest that CHKA modulates exosome secretion via an autophagy-dependent pathway, thereby facilitating the proliferation, migration, and invasion of glioma cells.

In the eukaryotic system, exosomes are categorized as unique extracellular vesicles with dimensions ranging from 30 to 150 nm. These vesicles contain a variety of endogenous molecules, such as proteins, DNA, mRNA, microRNA, and circular RNA. They are essential for a wide range of metabolic events and have the potential to be used as therapeutic or diagnostic targets for a number of diseases, including ovarian diseases. By inducing changes in the surrounding environment, the donor exosomes transfer their contents to the receiving cells, so demonstrating the biological implications of major interactions between cells. Mesenchymal stem cells (MSCs) have produced exosomes have shown promise as a treatment for premature organ failure (POF or POI). Furthermore, exosomal transport has many complexities, and contributes to the pathophysiology of ovarian cancer by affecting cell growth, migration, metastastsis and etc. Owing to these facts, in this paper, we present the progress developed in the understanding of exosomes as a viable therapeutic avenue and indisputable prognostic targets in ovarian disorders.

Gliomas are the most ordinary primary virulent brain tumours and commonly used clinical treatments include tumour resection, radiation therapy and chemotherapy. Although significant progress has been made in recent years in progression-free survival (PFS) and overall survival (OS) for patients with high-grade gliomas, the prognosis for patients remains poor. Chemoresistance refers to the phenomenon of decreased sensitivity of tumour cells to drugs, resulting in reduced or ineffective drug efficacy, and is an important cause of failure of tumour chemotherapy. Exosomes, a type of extracellular vesicle, are secreted by cancer cells and various stromal cells in the tumour microenvironment (TME) and transfer their inclusions to cancer cells, increasing chemoresistance. Furthermore, depletion of exosomes reverses certain detrimental effects on tumour metabolism and restores sensitivity to chemotherapeutic agents. Here, we summarised the correlation between exosomes and resistance to chemotherapeutic agents in glioma patients, the mechanisms of action of exosomes involved in resistance and their clinical value. We aimed to afford new thoughts for research, clinical diagnosis and intervention in the mechanisms of chemoresistance in glioma patients.

Chemotherapy resistance (CR) represents one of the most important barriers to effective oncological therapy and often leads to ineffective intervention and unfavorable clinical prognosis. Emerging studies have emphasized the vital significance of extracellular RNA (exRNA) in influencing CR. This thorough assessment intends to explore the multifaceted contributions of exRNA, such as exosomal RNA, microRNAs, long non-coding RNAs, and circular RNAs, to CR in cancer. We discuss the mechanisms by which exRNA facilitates drug resistance, such as modulating gene expression, influencing the tumor microenvironment, and facilitating intercellular communication. Furthermore, we examine the potential of exRNA as prognostic factor for determining oncology treatment efficacy and their emerging role as therapeutic targets. Diagnostic and prognostic applications of exRNA biomarkers are considered, alongside current methodologies for their detection and quantification. Additionally, we review recent advances in exRNA-targeted therapies, highlighting ongoing clinical trials and therapeutic strategies aimed at overcoming chemoresistance. Despite the promise of exRNA research, several challenges remain, including technical limitations and the biological complexity of exRNA networks. This review underscores the importance of continued investigation into exRNA biology and its therapeutic potential, which in the future may provide new avenues for cancer treatment and tailored medical strategies. By elucidating the role of exRNA in CR, this article aims to provide a comprehensive resource for researchers and clinicians seeking to improve the effectiveness of carcinoma management approaches.

This review highlights recent progress in exosome-based drug delivery for cancer therapy, covering exosome biogenesis, cargo selection mechanisms, and their application across multiple cancer types. As small extracellular vesicles, exosomes exhibit high biocompatibility and low immunogenicity, making them ideal drug delivery vehicles capable of efficiently targeting cancer cells, minimizing off-target damage and side effects. This review aims to explore the potential of exosomes in cancer therapy, with a focus on applications in chemotherapy, gene therapy, and immunomodulation. Additionally, challenges related to exosome production and standardization are analyzed, highlighting the importance of addressing these issues for their clinical application. In conclusion, exosome-based drug delivery systems offer promising potential for future cancer therapies. Further research should aim to enhance production efficiency and facilitate clinical translation, paving the way for innovative cancer treatment strategies.

Extracellular vesicle (EV) secretion is an important, though not fully understood, intercellular communication process. Lipid metabolism has been shown to regulate EV activity, though the impact of specific lipid classes is unclear. Through analysis of small EVs (sEVs), we observe aberrant increases in sEV release within genetic models of cholesterol biosynthesis disorders, where cellular cholesterol is diminished. Inhibition of cholesterol synthesis at multiple synthetic steps mimics genetic models in terms of cholesterol reduction and sEVs secreted. Further analyses of sEVs from cholesterol-depleted cells revealed structural deficits and altered surface marker expression, though these sEVs were also more easily internalized by recipient cells. Transmission electron microscopy of cells with impaired cholesterol biosynthesis demonstrated multivesicular and multilamellar structures potentially associated with autophagic defects. We further found autophagic vesicles being redirected toward late endosomes at the expense of autophagolysosomes. Through CRISPR-mediated inhibition of autophagosome formation, we mechanistically determined that release of sEVs after cholesterol depletion is autophagy dependent. We conclude that cholesterol imbalance initiates autophagosome-dependent secretion of sEVs, which may have pathological relevance in diseases of cholesterol disequilibrium.

Angiogenesis is essential for pannus formation and maintenance in rheumatoid arthritis (RA). Heat shock protein 70 kDa (HSP70) can induce angiogenesis by being released extracellularly through exosomes. Geniposide (GE) is the primary pharmacological component of the fruit of Gardenia jasminoides Ellis (GJ). In vivo, we have found that GE is able to reduce HSP70 levels in the synovium and serum of CIA-S and has anti-angiogenic effects. However, the mechanism by which GE inhibits HSP70 to improve angiogenesis is still unclear. This study aims to explore how GE inhibits the extracellular release of HSP70 and its impact on angiogenesis in human umbilical vein endothelial cells (HUVECs).

HUVECs' exosomes were extracted using ultracentrifugation and characterized through transmission electron microscope, nanoparticle tracer technology, nano-flow cytometry and Western blotting. The proliferative ability of HUVECs was assessed by EdU and CCK8 assay. Transwell and wound healing assays were used to measure the migration ability of HUVECs, while tube formation assay was employed to evaluate their tube-forming ability. The TNF-α-induced HSP70 release model in HUVECs was established, with extracellular HSP70 levels serving as an evaluation index. Immunofluorescence and co-immunoprecipitation assay were used to analyze the interaction between HSP70 and the lipid raft marker Caveolin-1 (Cav-1). Western blotting was employed to investigate the expression of SphK1/S1P/S1PRs/Gαi pathway-related proteins, and ELISA was utilized to detect extracellular S1P and HSP70 levels.

The exosomes of HUVECs contained HSP70. HUVECs were stimulated by extracellular HSP70, which enhanced their proliferation, migration, and tube-forming abilities. TNF-α (10 ng/mL) significantly increased the release of HSP70, which was inhibited by GE (25 µM-100 µM) in a concentration-dependent manner. GE reduced HSP70 in lipid rafts without affecting Cav-1. GE (100 µM) inhibited proteins in the SphK1/S1P/S1PRs/Gαi pathway, preventing HSP70 release and improving HUVECs' functions compared to the K6PC-5 (SphK1-specific agonist) and TNF-α groups.

This study found that GE inhibited the extracellular release of HSP70 by suppressing the SphK1/S1P/S1PRs/Gαi pathway, thereby producing anti-angiogenic effects in vitro. This provides a novel direction and strategy for anti-angiogenesis therapy for RA.

Cancer-associated fibroblasts (CAFs) constitute a critical component of the tumor microenvironment (TME). CAFs can be reprogrammed by cancer cells, leading to the production of extracellular vesicles (EVs). These EVs serve as carriers for bioactive substances, including proteins, nucleic acids, and metabolic products, thereby facilitating tumor progression. CAF-derived EVs exert substantial influence on tumor cell proliferation, invasion, and metastasis, the immunological environment, and the processes of lymphangiogenesis and angiogenesis. Despite their potential as non-invasive biomarkers and therapeutic delivery vehicles, the clinical application of CAF-derived EVs is currently limited by challenges in purification and precise targeting. This review delineates the diverse roles of CAF-derived EVs in tumor growth, metastasis, and immune evasion within the TME.

Painful diabetic neuropathy (PDN) is a challenging complication of diabetes with patients experiencing a painful and burning sensation in their extremities. Existing treatments provide limited relief without addressing the underlying mechanisms of the disease. PDN involves the gradual degeneration of nerve fibers in the skin. Keratinocytes, the most abundant epidermal cell type, are closely positioned to cutaneous nerve terminals, suggesting the possibility of bi-directional communication. Extracellular vesicles are lipid-bilayer encapsulated nanovesicles released from many cell types that mediate cell to cell communication. The role of keratinocyte-derived extracellular vesicles (KDEVs) in influencing signaling between the skin and cutaneous nerve terminals and their contribution to the genesis of PDN has not been explored. In this study, we characterized KDEVs in a well-established high-fat diet mouse model of PDN using primary adult mouse keratinocyte cultures. We obtained highly enriched KDEVs through size-exclusion chromatography and then analyzed their molecular cargo using proteomic analysis and small RNA sequencing. We found significant differences in the protein and microRNA content of high-fat diet KDEVs compared to KDEVs obtained from control mice on a regular diet, including pathways involved in axon guidance and synaptic transmission. Additionally, using an in vivo conditional extracellular vesicle reporter mouse model, we demonstrated that epidermal-originating GFP-tagged KDEVs are retrogradely trafficked into the dorsal root ganglion (DRG) neuron cell bodies. This study presents the first comprehensive isolation and molecular characterization of the KDEV protein and microRNA cargo in RD and HFD mice. Our findings suggest a potential novel communication pathway between keratinocytes and DRG neurons in the skin, which could have implications for PDN.

Extracellular vesicles (EVs) are small membranous carriers of protein, lipid, and nucleic acid cargoes and play a key role in intercellular communication. Recent work has revealed the previously under-recognized participation of endoplasmic reticulum (ER)-associated proteins (ERAPs) during EV secretion, using pathways reminiscent of viral replication and secretion. Here, we present highlights of the literature involving ER/ERAPs in EV biogenesis and propose mechanistic parallels with ERAPs exploited during viral infections. We propose that ERAPs play an active role in the release of EVs and viral particles, and we present views on whether viruses hijack or enhance pre-existing ERAP-dependent secretory machineries or whether they repurpose ERAPs to create new secretory pathways.

Extracellular vesicles (EVs) are lipid bilayer-bound particles released from cells that cannot replicate on their own, play a crucial role in intercellular communication, and are implicated in various physiological and pathological processes. Within the domain of embryo culture media research, extensive studies have been conducted to evaluate embryo viability by analyzing spent culture medium. Advanced methodologies such as metabolomic profiling, proteomic and genomic analyses, transcriptomic profiling, non-coding RNA assessments, and oxidative status measurements have been employed to further understand the molecular characteristics of embryos and improve selection criteria for successful implantation. In the field of EVs, only a limited number of studies have been conducted on embryo-conditioned medium, indicating a significant gap in knowledge regarding the potential role of EVs in embryo development and implantation. Therefore, this review aims to evaluate current research findings on EVs enriched from animal and human embryo spent medium. By unraveling the potential link between embryo-derived EVs and embryo selection in clinical settings, such research might enhance embryo-selection methods in assisted reproductive technologies, eventually increasing the success rates of fertility treatments and advancing our understanding of mechanisms underlying successful embryo development and implantation in humans.

Esophageal cancer is a common and lethal digestive system malignancy, and both treatment efficacy and patient survival rates face significant challenges. In recent years, exosomes have emerged as crucial mediators of intercellular communication, demonstrating tremendous clinical potential, particularly in the diagnosis, treatment, and prognostic evaluation of esophageal cancer. These exosomes not only serve as biomarkers for early diagnosis and prognosis but also modulate tumor growth, metastasis, and drug resistance by delivering bioactive molecules. Importantly, exosomes can act as carriers for esophageal cancer-related therapeutic agents, optimizing gene therapy strategies to enhance efficacy while reducing toxicity and side effects. Despite facing challenges in clinical applications such as purification, enrichment, and standardization of analytical methods, exosomes maintain broad prospects for application in esophageal cancer treatment, with the potential to significantly improve patient outcomes and quality of life. This review focuses on the innovative role of exosomes in the early diagnosis of esophageal cancer, exploring their application value and safety in disease monitoring and assessment of treatment response. Furthermore, this study outlines the challenges and limitations of transitioning exosome research from basic studies to clinical applications, as well as potential solutions and future research directions to address these obstacles.

Extracellular vesicles (EVs) are important mediators of intercellular communication in the tumour microenvironment. The cytokine transforming growth factor-β (TGF-β) facilitates cancer progression via EVs secreted by cancer cells, which act on recipient cells in the tumour microenvironment. However, the mechanisms of how TGF-β affects cancer cell EV release and composition are incompletely understood. Here, we systematically investigate the effects of TGF-β on the release and protein composition of EVs from breast cancer cells. TGF-β suppresses the transcription of RAB27B mediated by SMAD3 and thereby hampers EV release. Using click chemistry and quantitative proteomics, we found that TGF-β increases the quantity of protein cargo and changes the composition of EVs by downregulating RAB27B expression. The recomposed EVs, induced by TGF-β or RAB27B depletion, inhibit CD8+ T cell-mediated breast cancer killing. Our findings reveal the critical roles of TGF-β and RAB27B in cancer development by regulating EV release and composition and thus provide potential targets to improve cancer immunotherapy.

Colorectal cancer (CRC) is one of the most common cancers worldwide. The mechanisms underlying metastasis, which contributes to poor outcomes, remain elusive.

We used the Cancer Genome Atlas dataset to compare mRNA expression patterns of integrin α6 (ITGA6) and integrin β4 (ITGB4) in patients with CRC. We measured ITGA6 and ITGB4 expression levels in highly metastatic (i.e., HCT116 and SW620) and lowly metastatic (i.e., SW480 and Caco2) CRC cell lines. Exosomes were isolated from cell culture media and characterized using western blotting and nanoparticle analyses. The role of exosomes in lung metastasis was investigated using xenograft experiments in mice models, which received CRC cell injection and were treated with exosomes.

ITGA6 and ITGB4 were significantly overexpressed in CRC tissues, and ITGA6 was associated with the American Joint Committee on Cancer (AJCC) stage and outcome. ITGA6 and ITGB4, as well as exosomal ITGA6 and ITGB4, were significantly more highly expressed in HCT116 and SW620 cells than in SW480 and Caco2 cells. The proliferation and tubulogenesis of vascular endothelial cells were markedly decreased by disruption of ITGA6 and ITGB4 but were markedly increased by ectopic expression of ITGA6 and ITGB4. Exosomal ITGA6 and ITGB4 promoted CRC metastasis to the lung in vivo.

Taken together, our findings suggested that exosomal ITGA6 and ITGB4 displayed organotropism to the lung and upregulated proliferation and tubulogenic capacities, which might help reduce lung metastasis from CRC. These findings provided new insights into the mechanisms of CRC metastasis and provided novel potential therapeutic targets.

M2-polarized tumor-associated macrophages (M2 TAMs) are known to promote cancer progression, and exosomes are crucial mediators of communication within the tumor microenvironment (TME). However, the specific role of exosomes derived from M2 TAMs in pancreatic cancer (PC) progression remains poorly understood. Tyrosine kinase binding protein (TYROBP, also known as DAP12 for DNAX activating protein-12) is a transmembrane signal transduction polypeptide that interacts with immune cell receptors, influencing cellular functions via signal transduction pathways. TYROBP is prominently found in M2 TAMs exosomes, facilitating its transfer to PC cells and suggesting a potential role in PC pathogenesis.

This study initially confirmed the presence of TYROBP in M2 TAMs exosomes and its transfer to PC cells via exosomes. The impact of TYROBP on PC proliferation, apoptosis, migration, and invasion was investigated. Special attention was given to TYROBP's influence on PC metastasis and its underlying mechanisms, focusing particularly on the CD44/AKT/ERK signaling pathway.

TYROBP expression in PC cells did not significantly affect tumor cell proliferation or apoptosis but demonstrated a notable inhibitory effect on migration and invasion, which was mediated through the CD44/AKT/ERK pathway. Both in vivo and in vitro experiments consistently showed that TYROBP enhanced PC metastasis.

This study elucidates that TYROBP plays a direct role in promoting PC metastasis through its association with M2 TAMs polarization. Therefore, TYROBP represents a potential novel therapeutic target for interventions aimed at combatting PC progression.

Extracellular vesicles (EVs) are a heterogenous group of lipid bilayer bound particles naturally released by cells. These vesicles are classified based on their biogenesis pathway and diameter. The overlap in size of exosomes generated from the exosomal pathway and macrovesicles that are pinched off from the surface of the plasma membrane makes it challenging to isolate pure populations. Hence, isolated vesicles that are less than 200 nm are called small extracellular vesicles (sEVs). Extracellular vesicles transport a variety of cargo molecules, and multiple mechanisms govern the packaging of cargo into sEVs. Here, we discuss the current understanding of how miRNAs are targeted into sEVs, including the role of RNA binding proteins and EXOmotif sequences present in miRNAs in sEV loading. Several studies in human pain disorders and rodent models of pain have reported alterations in sEV cargo, including miRNAs. The sorting mechanisms and target regulation of miR-939, a miRNA altered in individuals with complex regional pain syndrome, is discussed in the context of inflammation. We also provide a broad overview of the therapeutic strategies being pursued to utilize sEVs in the clinic and the work needed to further our understanding of EVs to successfully deploy sEVs as a pain therapeutic.

Breast cancer, a leading cause of mortality among women, has been recognized as requiring improved diagnostic methods. Exosome proteins, found in small extracellular vesicles, have emerged as a promising solution, reflecting the state of their cell of origin and playing key roles in cancer progression. This review examines their potential in breast cancer diagnosis, discussing advanced isolation and characterization techniques such as ultracentrifugation and microfluidic-based approaches. Various detection methods-including electrochemical, nano-based, optical, and machine learning platforms-were evaluated for their high sensitivity, specificity, and non-invasive capabilities. Electrochemical methods were used to identify unique protein signatures for rapid, cost-effective diagnosis, while machine learning enhanced the classification of exosome proteins. Nano-based techniques leveraged nanomaterials to detect low-abundance proteins, and optical methods offered real-time, label-free monitoring. Despite their promise, challenges in standardizing protocols and integrating these diagnostics into clinical practice remain. Future directions include technological advancements, personalized medicine, and exploring the therapeutic potential of exosome proteins.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and irreversible interstitial lung disease caused by aberrant deposition of extracellular matrix in the lungs with significant morbidity and mortality. The therapeutic choices for IPF remain limited. Extracellular vesicles (EVs), as messengers for intercellular communication, are cell-secreted lipid bilayer nanoscale particles found in body fluids, and regulate the epithelial phenotype and profibrotic signaling pathways by transporting bioactive cargo to recipients in the pathogenesis of IPF. Furthermore, an increasing number of studies suggests that EVs derived from stem cells can be employed as a cell-free therapeutic approach for IPF, given their intrinsic tissue-homing capabilities and regeneration characteristics. This review highlights new sights of EVs in the pathogenesis of IPF, their potential as diagnostic and prognostic biomarkers, and prospects as novel drug delivery systems and next-generation therapeutics against IPF. Notably, bringing engineering strategies to EVs holds great promise for enhancing the therapeutic effect of anti-pulmonary fibrosis and promoting clinical transformation.

The migratory and invasive capabilities of melanoma cells contribute to metastasis. Therefore, targeting the genes driving these processes can support melanoma therapy. Rab27A and Rab27B contribute to tumor formation progression in many types of cancer through various mechanisms, including the secretion of small extracellular vesicles (sEVs). We explored the role of these GTPases in melanoma cell functioning in three RAB27A knockout (KO) cell lines (A375, DMBC12, and SkMel28) and a double RAB27A/B KO A375 cell line. The loss of RAB27A impaired the migration and invasion of DMBC12 and SkMel28 cells; however, the behavior of highly aggressive A375 cells was unaffected. The RAB27A/B double knockout moderately decreased the migratory capacity of A375 cells without disturbing their invasiveness. Additionally, the silencing of RAB27A did not affect the number and mean size of the sEVs, despite some alterations in the protein content of the vesicles. Both Rab27 isoforms can, at least partially, act independently. The potential role of Rab27A in the functioning of melanoma cells depends on the individual character of the cell line, but not on its basal expression, and seems to be unrelated to the secretion of sEVs.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of preterm infants, with its incidence rising due to improved survival rates of these infants. BPD results from a combination of prenatal and postnatal factors, such as mechanical ventilation, oxygen toxicity, and infections, all of which significantly impact the prognosis and growth of affected infants. Current treatment options for BPD are largely supportive and do not address the underlying pathology. Exosomes are cell-derived bilayer-enclosed membrane structures enclosing proteins, lipids, RNAs, growth factors, cytokines and metabolites. They have become recognized as crucial regulators of intercellular communication in various physiological and pathological processes. Previous studies have revealed the therapeutic potential of human umbilical cord mesenchymal stem cells-derived exosomes (HUCMSCs-Exos) in promoting tissue repair and regeneration. Therefore, HUCMSCs-Exos maybe a promising and effective therapeutic modality for BPD. In this review, we firstly provide a comprehensive overview of BPD, including its etiology and the mechanisms of lung injury. Then we detail the isolation, characterization, and contents of HUCMSCs-Exos, and discuss their potential mechanisms of HUCMSCs-Exos in BPD treatment. Additionally, we summarize current clinical trials and discuss the challenges in translating these findings from bench to bedside. This review aims to lay the groundwork for future clinical applications of HUCMSCs-Exos in treating BPD.

Migrasomes, vesicular structures discovered in migrating cells, arise from the junctions or tips of retraction fibers, and gradually grow to microscale vesicles. Migrasomes have garnered attention for their role in intercellular communication and potential therapeutic implications. This review presents an overview of recent advances in migrasome biology, covering the mechanisms of migrasome biogenesis, essential physiological roles, and their association with various diseases, alongside potential therapeutic applications. Furthermore, we share our perspectives on potential future directions in the study of migrasomes and highlight the challenges that remain in this developing area of research.

Significance: The study of extracellular vesicles (EVs), especially exosomes, has unlocked new avenues in understanding cellular communication and potential therapeutic applications. Recent Advances: Advancements in EV research have shown significant contributions from the International Society for Extracellular Vesicles (ISEV), in establishing methodological standards. The evolution of the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines from 2014 to 2023 reflects enhanced research rigor and reproducibility. The launch of EV-TRACK platform promotes uniformity and reproducibility by providing a centralized repository for data sharing and standardization practices. Furthermore, databases like EVpedia and ExoCarta have facilitated data sharing and collaboration within the scientific community. Concurrently, exosome-based therapies have emerged as a forefront area within regenerative medicine and targeted drug delivery, showcasing the potential of exosomes in promoting tissue regeneration. Critical Issues: Despite advancements, the field grapples with challenges such as vesicular heterogeneity, EV isolation complexity, and standardization. These issues impact research reproducibility and clinical applications. The inconsistency in exosomal preparations in clinical trials poses significant challenges to therapeutic efficacy and safety. Future Directions: The review outlines critical areas for future research, including the need for technological innovation in EV isolation and characterization, the establishment of standardized protocols, and a deeper understanding of exosome biology. The review also highlights the need to reassess guidelines, develop new EV isolation and characterization technologies, and establish standardized protocols to overcome current limitations. Emphasis is placed on interdisciplinary research and collaboration to address the complexities of EV biology, improve clinical trial design, and ultimately realize exosome's therapeutic and diagnostic potential. Continued evaluation and rigorous scientific validation are essential for successful exosome integration.