Background/Objectives: Age-related macular degeneration (AMD) is the third leading cause of irreversible blindness in elderly individuals aged over 50 years old. Oxidative stress plays a crucial role in the etiopathogenesis of multifactorial AMD disease. The phospholipid bilayer EVs derived from the culture-conditioned medium of human induced pluripotent stem cell (hiPSC) differentiated retinal organoids aid in cell-to-cell communication, signaling, and extracellular matrix remodeling. The goal of the current study is to establish and evaluate the encapsulation of a hydrophobic compound, cannabidiol (CBD), into retinal organoid-derived extracellular vesicles (EVs) for potential therapeutic use in AMD. Methods: hiPSC-derived retinal organoid EVs were encapsulated with CBD via sonication (CBD-EVs), and structural features were elucidated using atomic force microscopy, nanoparticle tracking analysis, and small/microRNA (miRNA) sequencing. ARPE-19 cells and oxidative-stressed (H2O2) ARPE-19 cells treated with CBD-EVs were assessed for cytotoxicity, apoptosis (MTT assay), reactive oxygen species (DCFDA), and antioxidant proteins (immunohistochemistry and Western blot). Results: Distinct miRNA cargo were identified in early and late retinal organoid-derived EVs, implicating their roles in retinal development, differentiation, and functionality. The therapeutic effects of CBD-loaded EVs on oxidative-stressed ARPE-19 cells showed greater viability, decreased ROS production, downregulated expression of inflammation- and apoptosis-related proteins, and upregulated expression of antioxidants by Western blot and immunocytochemistry. Conclusions: miRNAs are both prognostic and predictive biomarkers and can be a target for developing therapy since they regulate RPE physiology and diseases. Our findings indicate that CBD-EVs could potentially alleviate the course of AMD by activating the targeted proteins linked to the adenosine monophosphate kinase (AMPK) pathway. Implicating the use of CBD-EVs represents a novel frontline to promote long-term abstinence from drugs and pharmacotherapy development in treating AMD.

This review deals with the application of extracellular vesicles (EVs) in the treatment of various neuropsychiatric disorders, including mood disorders, neurodegeneration, psychosis, neurological insults and injuries, epilepsy and substance use disorders. The main challenges of most neuropsychiatric pharmaceuticals nowadays are how to reach the central nervous system at therapeutic concentration and maintain it long enough and how to avoid undesirable side effects caused by unsatisfying toxicity. Extracellular vesicles, as very important mediators of intercellular communication, can have a variety of therapeutic qualities. They can act neuroprotective, regenerative and anti-inflammatory, but they also have characteristics of a good drug delivery system, including their nano- scale size, biological safety and abilities to cross BBB, to pack drugs within the lipid bilayer, and not to trigger an immunological response. Besides, due to their presence in readily accessible biofluids, they are good candidates for biomarkers of the disease, its progression and therapy response monitoring. Alternations in EVs' cargo profiles can reflect the pathogenesis of neuropsychiatric disorders, but they could also affect the disease outcomes. In the future, EVs could help physicians to tailor treatment strategies for individual patients, however, more extensive studies are needed to standardize isolation, purification and production procedures, increase efficacy of drug loading and limit unwanted effects of innate EVs' content.

Size exclusion chromatography (SEC) is a well-established method for the isolation of extracellular vesicles (EVs), but the large elution volumes necessitate a concentration step prior to proteomics analysis. This concentration step can lead to a significant EV loss. Here we report an EV proteomics approach that enables the isolation of EVs into just 80 μL, which is directly compatible with proteomics analysis without the need for a prior concentration. EVs were characterized by transmission electron microscopy, Western blot, and nanoparticle tracking analysis, all of which confirmed the presence of small EVs. Proteomics analysis of the EVs was performed and benchmarked against those isolated by using an automated UHPLC-SEC platform. The novel workflow identified more proteins and more EV markers, including 96 of the 100 top exosomal proteins from the ExoCarta database, compared to 91 identified using EV samples isolated by UHPLC-SEC. When applied to EVs isolated from pancreatic cancer cell lines, the workflow demonstrated higher sensitivity for previously reported EV markers of pancreatic cancer.

Blood transfusion is an irreplaceable clinical treatment. Blood components are differentiated and stored according to specific guidelines. Storage temperatures and times vary depending on the blood component, but they all release extracellular vesicles (EVs) during storage. Although blood transfusions can be life-saving, they can also cause many adverse transfusion reactions, among which the effects of EVs are of increasing interest to researchers. EVs are submicron particles that vary in size, composition, and surface biomarkers, are encapsulated by a lipid bilayer, and are not capable of self-replication. EVs released by blood cells are important contributors to pathophysiologic states through proinflammatory, coagulant, and immunosuppressive effects, which in turn promote or inhibit the associated disease phenotype. Therefore, this review explores the potential mechanisms of hematopoietic-derived EVs in transfusion-associated adverse reactions and discusses the potential of the latest proteomics tools to be applied to the analysis of EVs in the field of transfusion medicine with a view to reducing the risk of blood transfusion.

From the pioneering days of cell therapy to the achievement of bioprinting organs, tissue engineering, and regenerative medicine have seen tremendous technological advancements, offering solutions for restoring damaged tissues and organs. However, only a few products and technologies have received United States Food and Drug Administration approval. This review highlights significant progress in cell therapy, extracellular vesicle-based therapy, and tissue engineering. Hematopoietic stem cell transplantation is a powerful tool for treating many diseases, especially hematological malignancies. Mesenchymal stem cells have been extensively studied. The discovery of induced pluripotent stem cells has revolutionized disease modeling and regenerative applications, paving the way for personalized medicine. Gene therapy represents an innovative approach to the treatment of genetic disorders. Additionally, extracellular vesicle-based therapies have emerged as rising stars, offering promising solutions in diagnostics, cell-free therapeutics, drug delivery, and targeted therapy. Advances in tissue engineering enable complex tissue constructs, further transforming the field. Despite these advancements, many technical, ethical, and regulatory challenges remain. This review addresses the current bottlenecks, emphasizing novel technologies and interdisciplinary research to overcome these hurdles. Standardizing practices and conducting clinical trials will balance innovation and regulation, improving patient outcomes and quality of life.

In this study, we developed a biosensor using a gold screen-printed electrode (Au-SPE) functionalized with mercaptoundecanoic acid (MUA) and an antibody for detecting the vascular-endothelial cadherin (CD144) as a endothelial biomarker protein on extracellular vesicles (EVs) isolated from saliva. The MUA functionalization provides a stable platform for immobilizing the CD144 antibody, ensuring the detection of the target protein. This biosensor combines Au-SPE technology with an immunoassay, offering a rapid, sensitive, and non-invasive method for detection of CD144 carried by EVs. Characterization of saliva-derived EVs using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) confirmed their morphology and size, which fell within the expected range of 80-180 nm. NTA indicated a lower concentration of particles in saliva-EVs than in serum-EVs (controls), highlighting the need for sensitive detection of EV cargos in this type of EV. Immunodetection confirmed the presence of CD144 in both saliva and serum-derived EVs, with higher concentrations in serum. Functionalization of Au-SPEs with MUA and CD144 antibodies was confirmed by significant resistance changes, and atomic force microscopy (AFM) was used to verify the preservation of EV morphology and their capturing post-immune adsorption. A calibration curve demonstrated the high sensitivity of the biosensor prototype for detecting CD144-positive EVs, with a limit of detection (LOD) of 0.111 ng mL-1 and a limit of quantification (LOQ) of 0.37 ng mL-1, requiring only 3 μL of EV-sample. This biosensor shows potential as a novel method for detecting and studying endothelial biomarkers associated with cardiovascular disease in EVs isolated from saliva, a capability not currently available with existing tools. Furthermore, it provides a key platform for expanding research to other biomarkers and diseases by monitoring protein cargos in the EVs, enhancing its utility across diverse clinical applications.

In this study, YIGSR-functionalized exosomes (Exo) are engineered and hybridized with lipid polymeric nanoparticles (LPNPs) followed by loading of chemotherapy Dasatinib (DST) to spatially target laminin receptors on tumors. Exo derived from differentiated macrophages are engineered with YIGSR targeting peptides.These YIGSR-Exo are subsequently fused with LPNPs membranes using the freeze-thaw method, resulting in fused hybrid YIGSR-Exo, which are then loaded with DST, creating DST-FuNP@YIGSR-Exo and targeted breast cancer (BC), leading to enhanced mitochondrial membrane potential (54.50 ±5.0%), increased reactive oxygen species (59.50 ± 6.0%), and apoptosis (63 ± 6.5%), ultimately inducing cell death. Further, cellular uptake and receptor blocking studies confirm the binding affinity and interaction of DST-FuNP@YIGSR-Exo with laminin receptors, Intravenous pharmacokinetic analysis of DST-FuNP@YIGSR-Exo reveals a significant improvement in AUC0-∞, with a 20.84-fold increase compared to free DST and a 1.61-fold enhancement over DST-FuNP@Exo. This is further supported by in vivo imaging and demonstrated improved tumor localization. A tumor regression study shows a 6.8-fold reduction in tumors. Tumor tissue-specific IHC for the Ki67 proliferative marker is significantly reduced in the targeted formulation. The potential of DST-FuNP@YIGSR-Exo as an effective carrier for delivering chemotherapeutic drugs, paving the path for the advancement of biologically obtained nanocarriers for targeted breast cancer.

Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases and remains one of the leading causes of cancer-related mortality worldwide. The high mortality rate is primarily driven by delayed diagnosis, rapid metastasis, and frequent recurrence. Tumor-derived exosomes (TEXs) have emerged as critical mediators in NSCLC progression, offering valuable insights into the tumor microenvironment. Exosomes are small membrane vesicles that facilitate intercellular communication and transport bioactive molecules, including proteins, RNAs, and DNAs, thereby reflecting the genetic complexity of tumors. These exosomes play a key role in promoting tumor metastasis, epithelial-mesenchymal transition (EMT), neovascularization, drug resistance, and immune evasion, all of which are pivotal in the development of NSCLC. This review explores the diverse roles of TEXs in NSCLC progression, focusing on their involvement in pre-metastatic niche formation, tissue metastasis, and immune modulation. Specifically, we discuss the roles of exosome-associated RNAs and proteins in NSCLC, and their contribute to tumor growth and metastasis. Furthermore, we explore the potential of TEXs as biomarkers for NSCLC, emphasizing their application in diagnosis, prognosis, and prediction of resistance to targeted therapies and immunotherapies.

Pregnancy involves extracellular vesicles (EVs) through mechanisms that are poorly understood to date. Furthermore, it is not surprising that EVs may also be involved in the pathophysiology of pre-eclampsia (PE) and gestational hypertension, two clinical conditions with high morbidity and mortality, given their capacity to mediate intracellular communications and regulate inflammation and angiogenesis. We searched major online scientific search engines (PubMed, Google Scholar, Scopus, WES, Embase, etc.) using the terms "Preeclampsia", "Pregnancy", "Hypertension", "Pregnancy-related hypertension", "Extracellular vesicles", "Biomarkers", "Gestation" AND "Obstetrics". Finding potential early biomarkers of risk or illness progression would be essential for the optimum care of expectant mothers with the aforementioned conditions. Nevertheless, none of the various screening assays that have been discovered recently have shown high predictive values. The analysis of EVs in the peripheral blood starting from the first trimester of pregnancy may hold great promise for the possible correlation with gestational hypertension problems and represent a marker of the early stages of the disease. EVs use may be a novel therapeutic approach for the management of various illnesses, as well. In order to define EVs' function in the physiopathology of pregnancy-associated hypertension and PE, as well as their potential as early biomarkers and therapeutic tools, we have compiled the most recent data in this review.

Extracellular vesicles (EV) are membranous vesicles considered as significant players in cell-to-cell communication. Milk provides adequate nutrition, transfers immunity, and promotes neonatal development, and milk EV are suggested to play a crucial role in these processes. Milk samples were obtained on days 0, 7, and 14 after parturition from sows receiving either a standard diet (ω-6:ω-3 = 13:1) or a test diet enriched in ω-3 (ω-6:ω-3 = 4:1). EV were isolated using ultracentrifugation coupled with size exclusion chromatography, and characterized by nanoparticle tracking analysis, transmission electron microscopy, and assessment of EV markers via Western blotting. The lipidome was determined following a liquid chromatography-quadrupole time-of-flight mass spectrometry approach. Here, we show that different stages of lactation (colostrum vs mature milk) have a distinct extracellular vesicle lipidomic profile. The distinct lipid content can be further explored to understand and regulate milk EV functionalities and primordial for enabling their diagnostic and therapeutic potential.

Extracellular vesicles (EVs) are emerging as viable tools in cancer treatment due to their ability to carry a wide range of theranostic activities. This review summarizes different forms of EVs such as exosomes, microvesicles, apoptotic bodies, and oncosomes. It also sheds the light onto isolation methodologies, characterization techniques and therapeutic applications of all discussed EVs. Evidence indicates that EVs are particularly effective in delivering chemotherapeutic medications, and immunomodulatory agents. However, the advancement of EV-based therapies into clinical practice is hindered by challenges including EVs heterogeneity, cargo loading efficiency, and in vivo stability. Overall, EVs have the potential to change cancer therapeutic paradigms. Continued research and development activities are critical for improving EV-based medications and increasing their therapeutic impact.

In the domain of respiratory illnesses, asthma remains a critical obstacle. The heterogeneous nature of this chronic inflammatory disease poses challenges during its treatment. Glucocorticoid-based combination drug therapy now dominates clinical treatments for asthma; however, glucocorticoid resistance, numerous adverse effects, the incidence of inadequate drug delivery, and other factors need the development of more effective therapies. In recent years, there has been extensive research on nanotechnology in medicine. It has been shown in studies that these drug delivery systems can greatly enhance targeting and bioavailability and decrease the toxicity of medication. Nanoparticle drug delivery systems offer improved therapeutic efficacy compared to conventional administration techniques. Nanotechnology enables advancements in precision medicine, offering benefits for heterogeneous conditions such as asthma. This review will examine the critical factors of asthma to consider when formulating medications, as well as the role of nanomaterials and their mechanisms of action in pulmonary medicine for asthma treatment.

The study aims to evaluate and compare two advanced proteomic techniques, nanoLC-MALDI-MS/MS and nanoLC-TIMS-MS/MS, in characterizing extracellular vesicles (EVs) from the bronchoalveolar lavage fluid (BALF) of patients with asthma and idiopathic pulmonary fibrosis (IPF). Pulmonary diseases, driven by pollutants and infections, often necessitate detailed analysis of BALF to identify diagnostic biomarkers and therapeutic targets. EVs, which include exosomes, microvesicles, and apoptotic bodies, are isolated using filtration and ultracentrifugation, and their morphology, concentration, and size distribution are assessed through transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The proteomic profiles of these EVs are then analyzed using the aforementioned techniques, highlighting their unique and common proteins. The study found that nanoLC-TIMS-MS/MS identified significantly more proteins compared to nanoLC-MALDI-MS/MS. Functional analysis via Gene Ontology revealed pathways related to inflammation and cell signaling, underscoring the role of EVs in disease pathophysiology. The findings suggest that EVs in BALF can serve as valuable biomarkers and therapeutic targets in respiratory diseases, providing a foundation for future research and clinical applications.

Stochiometric issues, encompassing both the quantity and heterogeneity of extracellular vesicles (EVs) derived from tumour or other tissues in blood, pose important challenges across various stages of biomarker discovery and detection, affecting the integrity of data, introducing losses and artifacts during blood processing, EV purification and analysis. These challenges shape the diagnostic utility of EVs especially within the framework of established and emerging methodologies. By addressing these challenges, we aim to delineate crucial parameters and requirements for tumour-specific EV detection, or more precisely, for tumour identification via EV based assays. Our endeavour involves a comprehensive examination of the layers that mask or confound the traceability of EV markers such as nucleic acids and proteins, and focus on 'low prevalence-low concentration' scenario. Finally, we evaluate the advantages versus limitations of single-particle analysers over more conventional bulk assays, suggesting that the combined use of both to capture and interpret the EV signals, in particular the EV surface displayed proteins, may ultimately provide quantitative information on their absolute abundance and distribution.

The early detection of neurodegenerative diseases necessitates the identification of specific brain-derived biomolecules in peripheral blood. In this context, our investigation delineates the role of amyloid precursor-like protein 1 (APLP1)-a protein predominantly localized in oligodendrocytes and neurons-as a previously unidentified biomarker in extracellular vesicles (EVs). Through rigorous analysis, APLP1+ EVs from human sera were unequivocally determined to be of cerebral origin. This assertion was corroborated by distinctive small RNA expression patterns of APLP1+ EVs. The miRNAs' putative targets within these EVs manifested pronounced expression in the brain, fortifying their neurospecific provenance. We subjected our findings to stringent validation using Thy-1 GFP M line mice, transgenic models wherein GFP expression is confined to hippocampal neurons. An amalgamation of these results with an exhaustive data analysis accentuates the potential of APLP1+ EVs as cerebrally originated biomarkers. Synthesizing our findings, APLP1+ EVs are postulated not merely as diagnostic markers but as seminal entities shaping the future trajectory of neurodegenerative disease diagnostics.

Small extracellular vesicles (sEVs) are an important part of intercellular communication. They are phospholipid bilayer particles that carry active biomolecules such as proteins, various nucleic acids, and lipids. In recipient cells, sEVs can alter cellular functions, including cancer development and premetastatic niche formation in distant organs. Moreover, sEVs can carry cancer-specific features, which makes them promising biomarker candidates. However, the interactions of sEVs with biological barriers and consequences thereof, are not clarified yet. The blood-saliva barrier is crucial for preventing the entry of pathogens and (in)organic substances into the bloodstream, as well as molecule filtration from blood to saliva. The effects of brain derived DU145 prostate cancer (PCa) sEVs on a human submandibular salivary gland barrier (SSGB) in vitro were investigated. Small EVs were harvested from normoxic (N, atmospheric O2) or hypoxic (H, 1% O2) conditions, fluorescently labeled with CellTrackerTM Orange and thoroughly characterized. HTB-41 B2 cells were used as SSGB model cultured on 24-well ThinCert® inserts. After model optimization indicating effects of serum and serum-sEVs on barrier properties, PCa sEVs were applied to the basolateral (blood) side in either 10% serum, or serum-free conditions, and barrier integrity was continuously monitored for 40 hours. This study found that H and N PCa sEVs were uptaken by the SSGB in vitro model in similar quantities regardless of the media composition in the basolateral compartment. Permeation of fluorescent PCa sEVs into the apical compartment was not detectable with the applied methods. However, treatment with H and N sEVs under different serum conditions revealed distinct molecular clusters after hierarchical analysis of mRNA data measured by high-throughput qPCR, which were partly reflected at the protein level. For example, serum-reduction dependent decrease of barrier properties was accompanied with the decrease of CDH1 or Claudin-7 expression. Interestingly, the presence of H sEVs significantly increased the number of sEV-sized particles in the apical compartment of the SSGB model compared to basolaterally added N sEVs. This functional effect on the number of particles in the saliva (apical) compartment induced by different sEVs applied in the blood (basolateral) compartment might be a new approach to understand one possible mechanism how differences of salivary EVs might occur which then could be used as biomarker.

Loss of surface sialic acid by neuraminidases is known as 'desialylation'. Platelets are desialylated in bacterial or viral infections, during storage, senescence, various mutations, platelet auto antibodies, hemostasis and shear stress. In this review the recent literature on the different sialic acid capped glycan structures will be covered as well as platelet desialylation in inherited glycan disorders and induced by external neuraminidases.

Neuraminidases are released from platelet intracellular stores and translocated to the platelet surface. Apart from clearance, loss of surface sialic acid by neuraminidases ('desialylation') affects platelet signaling including ligand binding and their procoagulant function. Platelets are also desialylated in infections, various mutations, presence of platelet auto antibodies.

Since platelet desialylation occurs in various healthy and pathological conditions, measuring desialylation might be a new diagnostic tool.

Background: Extracellular vesicles (EVs) can carry pathological cargo, contributing to disease progression. The enzyme neutral sphingomyelinase 2 (nSMase2) plays a critical role in EV biogenesis, making it a promising therapeutic target. Our lab previously identified a potent and selective inhibitor of nSMase2, named DPTIP (IC50 = 30 nM). Although promising, DPTIP exhibits poor pharmacokinetics (PKs) with a low oral bioavailability (%F < 5), and a short half-life (t1/2 ≤ 0.5 h). To address these limitations, we previously developed DPTIP prodrugs by masking its phenolic hydroxyl group, demonstrating improved plasma exposure in mice. Recognizing that species-specific metabolic differences can influence prodrug PK, we expanded our studies to evaluate selected prodrugs in both mice and dogs. Methods: The scaleup of selected prodrugs was completed and two additional valine- ester based prodrugs were synthesized. Mice were dosed prodrugs via peroral route (10 mg/kg equivalent). For dog studies DPTIP was dosed via intravenous (1 mg/kg) or peroral route (2 mg/kg) and prodrugs were given peroral at a dose 2 mg/kg DPTIP equivalent. Plasma samples were collected at predetermined points and analyzed using developed LC/MS-MS methods. Results: In mice, several of the tested prodrugs showed similar or improved plasma exposures compared to DPTIP. However, in dog studies, the double valine ester prodrug 9, showed significant improvement with an almost two-fold increase in DPTIP plasma exposure (AUC0-t = 1352 vs. 701 pmol·h/mL), enhancing oral bioavailability from 8.9% to 17.3%. Conclusions: These findings identify prodrug 9 as a promising candidate for further evaluation and underscore the critical role of species-specific differences in prodrug PKs.

Cardiovascular (CV) disease remains the leading cause of morbidity and mortality worldwide, highlighting the necessity of understanding its underlying molecular and pathophysiological pathways. Conversely, physical activity (PA) and exercise are key strategies in reducing CV event risks. Detecting latent CV conditions in apparently healthy individuals, such as athletes, presents a unique challenge. The early identification and treatment of CV disorders are vital for long-term health and patient survival. Cardiac troponin is currently the most commonly used biomarker for assessing CV changes in both athletes and the general population. However, there remains considerable debate surrounding the mechanisms underlying exercise-induced troponin elevations and its release in non-ischemic contexts. Thus, there is a pressing need to identify and implement more sensitive and specific biomarkers for CV disorders in clinical practice. Indeed, research continues to explore reliable biomarkers for evaluating the health of athletes and the effectiveness of physical exercise. It is essential to analyze current evidence on troponin release in non-ischemic conditions, post-strenuous exercise, and the complex biological pathways that influence its detection. Furthermore, this study summarizes current research on cytokines and exosomes, including their physiological roles and their relevance in various CV conditions, especially in athletes. In addition, this paper gives special attention to underlying mechanisms, potential biomarkers, and future perspectives.

Extracellular Vesicles (EVs) became a focus of clinical research when experimental and pre-clinical studies showed that they mimic their parent cells' regenerative and therapeutic effects and their cargo carries disease-specific diagnostic and prognostic biomarkers. Since the publication of data forms an endpoint of the study, this review specifically focused on the published clinical trials done with EVs. For brevity, this review was restricted to the last 10 years. Unexpectedly, the literature search showed that very few clinical trials assessing the therapeutic applications of EVs were published in this period indicating that they have not reached their desired endpoint. Conversely, most studies showed the potential of EVs present in various biofluids as a promising source of diagnostic and prognostic biomarkers for various diseases, and predictive markers to assess the effectiveness of therapy. This stark difference in the numbers could perhaps be due to the time-consuming regulatory processes involved in the clinical-grade preparation and characterization of EVs, and the determination of their safety and effective dose regimens. One wonders whether fast-tracking regulatory affairs could help accelerate the therapeutic use of EVs. This aspect needs urgent attention.

With the rapid development of drug delivery systems, extracellular vesicles (EVs) have emerged as promising stars for improving targeting abilities and realizing effective delivery. Numerous studies have shown when compared to conventional strategies in targeted drug delivery (TDD), EVs-based strategies have several distinguished advantages besides targeting, such as participating in cell-to-cell communications and immune response, showing high biocompatibility and stability, penetrating through biological barriers, etc. In this review, we mainly focus on the mass production of EVs including the challenges and strategies for scaling up EVs production in a cost-effective and reproducible manner, the loading and active targeting methods, and examples of EVs as vehicles for TDD in consideration of potential safety and regulatory issues associated. We also conclude and discuss the rigor and reproducibility of EVs production, the current research status of the application of EVs-based strategies to targeted drug delivery, clinical conversion prospects, and existing chances and challenges.

Extracellular vesicles (EVs) are cell-derived small membrane structures that transport various molecules. They have emerged as potential circulating biomarkers for monitoring responses to cancer therapies. This study aimed to comprehensively characterize plasma-carried EVs in hormone receptor-positive (HR+) metastatic breast cancer (MBC) patients treated with first-line CDK4/6 inhibitors (iCDK4/6) combined with endocrine therapy. MBC patients were classified into three groups based on their response to therapy: resistant, intermediate or sensitive. In a prospective cohort, we monitored the concentration of circulating EVs, analyzed their lipid signature and correlated these factors with treatment response. To facilitate the translation of EV research to clinical practice, we established a three-step procedure: (1) EVs were isolated from plasma using semi-automatized size exclusion chromatography (SEC); (2) EV concentration, termed vesiclemia, was determined by drop counting via interferometric light microscopy (ILM); and (3) EV lipid composition was analyzed by mass spectrometry. ILM-based vesiclemia values were highly fluctuating upon iCDK4/6 treatment, while early increase associated with accelerated progression. Of note, vesiclemia remained a steady parameter over a 1-year period in age-matched healthy women. Additionally, analysis of the EV cargo unveiled a distinct sphingolipid profile, characterized by increased levels of ceramides and sphingomyelins in resistant patients within the first 2 months of treatment. Based on 16 sphingolipid species, sensitive and resistant patients were correctly classified with an overall accuracy of 82%. This specific sphingolipid pattern was exclusively discernible within EVs, and not in plasma, highlighting the significance of EVs in the early prediction of individual responses to iCDK4/6 and disease progression. Overall, this study provides insights of the longitudinal characterization of plasma-borne EVs in both a healthy group and HR+ MBC patients under iCDK4/6 therapies. Combined vesiclemia and EV sphingolipid profile emphasize the promising potential of EVs as non-invasive biomarkers for monitoring early treatment response.

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.

Extracellular vesicles (EVs) are lipid nanoparticles and play an important role in cell-cell communications, making them potential therapeutic agents and allowing to engineer for targeted drug delivery. The expanding applications of EVs in next generation medicine is still limited by existing tools for scaling standardized EV production, single EV tracing and analytics, and thus provide only a snapshot of tissue-specific EV cargo information. Here, we present the Snorkel-tag, for which we have genetically fused the EV surface marker protein CD81, to a series of tags with an additional transmembrane domain to be displayed on the EV surface, resembling a snorkel. This system enables the affinity purification of EVs from complex matrices in a non-destructive form while maintaining EV characteristics in terms of surface protein profiles, associated miRNA patterns and uptake into a model cell line. Therefore, we consider the Snorkel-tag to be a widely applicable tool in EV research, allowing for efficient preparation of EV standards and reference materials, or dissecting EVs with different surface markers when fusing to other tetraspanins in vitro or in vivo.

Distant metastasis is a primary cause of mortality and contributes to poor surgical outcomes in cancer patients. Before the development of organ-specific metastasis, the formation of a pre-metastatic niche is pivotal in promoting the spread of cancer cells. This review delves into the intricate landscape of the pre-metastatic niche, focusing on the roles of tumor-derived secreted factors, extracellular vesicles, and circulating tumor cells in shaping the metastatic niche. The discussion encompasses cellular elements such as macrophages, neutrophils, bone marrow-derived suppressive cells, and T/B cells, in addition to molecular factors like secreted substances from tumors and extracellular vesicles, within the framework of pre-metastatic niche formation. Insights into the temporal mechanisms of pre-metastatic niche formation such as epithelial-mesenchymal transition, immunosuppression, extracellular matrix remodeling, metabolic reprogramming, vascular permeability and angiogenesis are provided. Furthermore, the landscape of pre-metastatic niche in different metastatic organs like lymph nodes, lungs, liver, brain, and bones is elucidated. Therapeutic approaches targeting the cellular and molecular components of pre-metastatic niche, as well as interventions targeting signaling pathways such as the TGF-β, VEGF, and MET pathways, are highlighted. This review aims to enhance our understanding of pre-metastatic niche dynamics and provide insights for developing effective therapeutic strategies to combat tumor metastasis.

The oviduct and uterus provide an optimal environment for early embryo development, where effective communication between the embryo and the maternal reproductive tract is crucial for establishing and maintaining pregnancy. Oviductal and uterine-derived EVs play pivotal roles in this maternal-embryonic communication and in facilitating early embryo development. However, despite the ability of in vitro culture methods to produce viable embryos, the lack of exchange between the embryo and the mother often results in lower-quality embryos than those derived in vivo. Therefore, there is a pressing need to increase our understanding of the physiological mechanisms underlying embryo interaction with the oviduct and endometrium through EVs and to develop models capable of mimicking the in vivo environment. This review aims to provide up-to-date insights into the communication between the mother and pre-implantation bovine embryo, exploring their applications and perspectives in the field.

Small extracellular vesicles (sEV) are small membrane-bound nanovesicles with a size range below 200 nm that are released by all types of cells. sEV carry a diverse cargo of proteins, lipids, glycans, and nucleic acids that mimic the content of producer cells. sEV mediate intercellular communication and play a key role in a broad variety of physiological and pathological conditions. Recently, numerous reports have emerged examining the role of sEV in viral infections. A significant number of similarities in the sEV biogenesis pathways and the replication cycles of viruses suggest that sEV might influence the course of viral infections in diverse ways. Besides directly modulating virus propagation by transporting the viral cargo (complete virions, proteins, RNA, and DNA), sEV can also modify the host antiviral response and increase the susceptibility of cells to infection. The network of mutual interactions is particularly complex in the case of oncogenic viruses, deserving special consideration because of its significance in cancer progression. This review summarizes the current knowledge of interactions between sEV and oncogenic viruses, focusing on sEV abilities to modulate the carcinogenic properties of oncoviruses.

Lymphatic vessels are essential for preventing the accumulation of harmful components within peripheral tissues, including the artery wall. Various endogenous mechanisms maintain adequate lymphatic function throughout life, with platelets being essential for preserving lymphatic vessel integrity. However, since lymph lacks platelets, their impact on the lymphatic system has long been viewed as restricted to areas where lymphatics intersect with blood vessels. Nevertheless, platelets can also exert long range effects through the release of extracellular vesicles (EVs) upon activation. We observed that platelet EVs (PEVs) are present in lymph, a compartment to which they could transfer regulatory effects of platelets. Here, we report that PEVs in lymph exhibit a distinct signature enabling them to interact with lymphatic endothelial cells (LECs). In vitro experiments show that the internalization of PEVs by LECs maintains their functional integrity. Treatment with PEVs improves lymphatic contraction capacity in atherosclerosis-prone mice. We suggest that boosting lymphatic pumping with exogenous PEVs offers a novel therapeutic approach for chronic inflammatory diseases characterized by defective lymphatics.

Recent advancements in nanomedicine and biotechnology have unveiled the remarkable potential of plant-derived extracellular vesicles (PDEVs) as a novel and promising approach for cancer treatment. These naturally occurring nanoscale particles exhibit exceptional biocompatibility, targeted delivery capabilities, and the capacity to load therapeutic agents, positioning them at the forefront of innovative cancer therapy strategies. PDEVs are distinguished by their unique properties that facilitate tumor targeting and penetration, thereby enhancing the efficacy of drug delivery systems. Their intrinsic biological composition allows for the evasion of the immune response, enabling the efficient transport of loaded therapeutic molecules directly to tumor sites. Moreover, PDEVs possess inherent anti-cancer properties, including the ability to induce cell cycle arrest and promote apoptotic pathways within tumor cells. These vesicles have also demonstrated antimetastatic effects, inhibiting the spread and growth of cancer cells. The multifunctional nature of PDEVs allows for the simultaneous delivery of multiple therapeutic agents, further enhancing their therapeutic potential. Engineering and modification techniques, such as encapsulation, and the loading of therapeutic agents via electroporation, sonication, and incubation, have enabled the customization of PDEVs to improve their targeting efficiency and therapeutic load capacity. This includes surface modifications to increase affinity for specific tumor markers and the encapsulation of various types of therapeutic agents, such as small molecule drugs, nucleic acids, and proteins. Their plant-derived origin offers an abundant and renewable source to produce therapeutic vesicles, reducing costs and facilitating scalability for clinical applications. This review provides an in-depth analysis of the latest research on PDEVs as emerging anti-cancer agents in cancer therapy.

Extracellular Vesicles (EV) have become an interesting focus as novel biomarkers of disease and are increasingly reported upon in humans and other species. The Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018) guidelines were published to improve rigor and standardisation within the EV field and provide a framework for the reliable isolation and characterisation of EV populations. However, this rigor and standardisation has been challenging in the area of comparative medicine. Herein we present the successful isolation of EVs from human and canine plasma using Size Exclusion Chromatography and characterise these EVs according to best international practice. This study provides evidence for the reliable comparison of human and canine EVs isolated by this approach, and a baseline description of the EVs from healthy dogs to inform future biomarker studies. This work also demonstrates that the MISEV2018 guidelines can be successfully applied to EVs isolated from canine plasma.

Epithelial ovarian cancer (EOC) is an often-fatal malignancy marked by the development of resistance to platinum-based chemotherapy. Thus, accurate prediction of platinum drug efficacy is crucial for strategically selecting postoperative interventions to mitigate the risks associated with suboptimal therapeutic outcomes and adverse effects. Tissue-derived extracellular vesicles (tsEVs), in contrast to their plasma counterparts, have emerged as a powerful tool for examining distinctive attributes of EOC tissues. In this study, 4D data-independent acquisition (DIA) proteomic sequencing was performed on tsEVs obtained from 58 platinum-sensitive and 30 platinum-resistant patients with EOC. The analysis revealed a notable enrichment of differentially expressed proteins that were predominantly associated with immune-related pathways. Moreover, pivotal immune-related proteins (IRPs) were identified by LASSO regression. These factors, combined with clinical parameters selected through univariate logistic regression, were used for the construction of a model employing multivariate logistic regression. This model integrated three tsEV IRPs, CCR1, IGHV_35 and CD72, with one clinical parameter, the presence of postoperative residual lesions. Thus, this model could predict the efficacy of initial platinum-based chemotherapy in patients with EOC post-surgery, providing prognostic insights even before the initiation of chemotherapy.

Currently, clinical indicators for evaluating endothelial permeability in sepsis are unavailable. Endothelium-derived extracellular vesicles (EDEVs) are emerging as biomarkers of endothelial injury. Platelet endothelial cell adhesion molecule (PECAM) and vascular endothelial (VE)-cadherin are constitutively expressed endothelial intercellular adhesion molecules that regulate intercellular adhesion and permeability. Herein, we investigated the possible association between EDEVs expressing intercellular adhesion molecules (PECAM+ or VE-cadherin+ EDEVs) and endothelial permeability and sepsis severity.

Human umbilical vein endothelial cells (HUVECs) were stimulated with tumor necrosis factor alpha (TNF-α) directly or after pretreatment with permeability-modifying reagents such as angiopoietin-1, prostacyclin, or vascular endothelial growth factor (VEGF) to alter TNF-α-induced endothelial hyperpermeability. Endothelial permeability was measured using the dextran assay or transendothelial electrical resistance. Additionally, a prospective cross-sectional observational study was conducted to analyze circulating EDEV levels in patients with sepsis. EDEVs were examined in HUVEC culture supernatants or patient plasma (nonsepsis, n = 30; sepsis, n = 30; septic shock, n = 42) using flow cytometry. The Wilcoxon rank-sum test was used for comparisons between 2 groups. Comparisons among 3 or more groups were performed using the Steel-Dwass test. Spearman's test was used for correlation analysis. Statistical significance was set at P < .05.

TNF-α stimulation of HUVECs significantly increased EDEV release and endothelial permeability. Pretreatment with angiopoietin-1 or prostacyclin suppressed the TNF-α-induced increase in endothelial permeability and inhibited the release of PECAM+ and VE-cadherin+ EDEVs. In contrast, pretreatment with VEGF increased TNF-α-induced endothelial permeability and the release of PECAM+ and VE-cadherin+ EDEVs. However, pretreatment with permeability-modifying reagents did not affect the release of EDEVs expressing inflammatory stimulus-inducible endothelial adhesion molecules such as E-selectin, intracellular adhesion molecule-1, or vascular cell adhesion molecule-1. The number of PECAM+ EDEVs on admission in the septic-shock group (232 [124, 590]/μL) was significantly higher (P = .043) than that in the sepsis group (138 [77,267]/μL), with an average treatment effect of 98/μL (95% confidence interval [CI], 2-270/μL), and the number of VE-cadherin+ EDEVs in the septic-shock group (173 [76,339]/μL) was also significantly higher (P = .004) than that in the sepsis group (81 [42,159]/μL), with an average treatment effect (ATE) of 79/μL (95% CI, 19-171/μL); these EDEV levels remained elevated until day 5.

EDEVs expressing intercellular adhesion molecules (PECAM+ or VE-cadherin+ EDEVs) may reflect increased endothelial permeability and could be valuable diagnostic and prognostic markers for sepsis.

The tumor microenvironment (TME) is a highly heterogeneous ecosystem that plays critical roles in the initiation, progression, invasion, and metastasis of cancers. Extracellular vesicles (EVs), as emerging components of the host-tumor communication, are lipid-bilayer membrane structures that are secreted by most cell types into TEM and increasingly recognized as critical elements that regulate the interaction between tumor cells and their surroundings. They contain a variety of bioactive molecules, such as proteins, nucleic acids, and lipids, and participate in various pathophysiological processes while regulating intercellular communication. While many studies have focused on the EVs derived from different body fluids or cell culture supernatants, the direct isolation of tissue-derived EVs (Ti-EVs) has garnered more attention due to the advantages of tissue specificity and accurate reflection of tissue microenvironment. In this review, we summarize the protocol for isolating Ti-EVs from different tissue interstitium, discuss the role of tumor-derived and adipose tissue-derived Ti-EVs in regulating TME. In addition, we sum up the latest application of Ti-EVs as potential biomarkers for cancer diseases.

Intervertebral disc degeneration (IVDD) is characterized by the senescence and declining vitality of nucleus pulposus cells (NPCs), often driven by mitochondrial dysfunction. This study elucidates that mesenchymal stem cells (MSCs) play a crucial role in attenuating NPC senescence by secreting mitochondria-containing microvesicles (mitoMVs). Moreover, it demonstrates that static magnetic fields (SMF) enhance the secretion of mitoMVs by MSCs. By distinguishing mitoMV generation from exosomes, this study shifts focus to understanding the molecular mechanisms of SMF intervention, emphasizing cargo transport and plasma membrane budding processes, with RNA sequencing indicating the potential involvement of the microtubule-based transport protein Kif5b. The study further confirms the interaction between Rab22a and Kif5b, revealing Rab22a's role in sorting mitoMVs into microvesicles (MVs) and potentially mediating subsequent plasma membrane budding. Subsequent construction of a gelatin methacrylate (GelMA) hydrogel delivery system further addresses the challenges of in vivo application and verifies the substantial potential of mitoMVs in delaying IVDD. This research not only sheds light on the molecular intricacies of SMF-enhanced mitoMV secretion but also provides innovative perspectives for future IVDD therapeutic strategies.

The global incidence of cancer is increasing, with estimates suggesting that there will be 26 million new cases and 17 million deaths per year by 2030. Cancer stem cells (CSCs) and extracellular vesicles (EVs) are key to the resistance and advancement of cancer. They play a crucial role in tumor dynamics and resistance to therapy. CSCs, initially discovered in acute myeloid leukemia, are well-known for their involvement in tumor initiation, progression, and relapse, mostly because of their distinct characteristics, such as resistance to drugs and the ability to self-renew. EVs, which include exosomes, microvesicles, and apoptotic bodies, play a vital role in facilitating communication between cells within the tumor microenvironment (TME). They have a significant impact on cellular behaviors and contribute to genetic and epigenetic changes. This paper analyzes the mutually beneficial association between CSCs and EVs, emphasizing their role in promoting tumor spread and developing resistance mechanisms. This review aims to investigate the interaction between these entities in order to discover new approaches for attacking the complex machinery of cancer cells. It highlights the significance of CSCs and EVs as crucial targets in the advancement of novel cancer treatments, which helps stimulate additional research, promote progress in ideas for cancer treatment, and provide renewed optimism in the effort to reduce the burden of cancer.

Noninvasive sample sources of exosomes, such as exhaled breath and sputum, which are in close proximity to the tumor microenvironment and may contain biomarkers indicative of lung cancer, are far more permissive than invasive sample sources for biomarker screening. Standardized exosome extraction and characterization approaches for low-volume noninvasive samples are critically needed. We isolated and characterized exhaled breath condensate (EBC) and sputum exosomes from healthy nonsmokers (n= 30), tobacco smokers (n= 30), and lung cancer patients (n= 40) and correlated the findings with invasive sample sources. EBC samples were collected by using commercially available R-Tubes. To collect sputum samples the participants were directed to take deep breaths, hold their breath, and cough in a collection container. Dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy were used to evaluate the exosome morphology. Protein isolation, western blotting, exosome quantification via EXOCET, and Fourier transform infrared spectroscopy were performed for molecular characterization. Exosomes were successfully isolated from EBC and sputum samples, and their yields were adequate and sufficiently pure for subsequent downstream processing and characterization. The exosomes were confirmed based on their size, shape, and surface marker expression. Remarkably, cancer exosomes were the largest in size not only in the plasma subgroups, but also in the EBC (p < 0.05) and sputum (p= 0.0036) subgroups, according to our findings. A significant difference in exosome concentrations were observed between the control sub-groups (p < 0.05). Our research confirmed that exosomes can be extracted from noninvasive sources, such as EBC and sputum, to investigate lung cancer diagnostic biomarkers for research, clinical, and early detection in smokers.

Oxidative stress is one of the main driving mechanisms of intervertebral disc degeneration(IDD). Oxidative stress has been associated with inflammation in the intervertebral disc, cellular senescence, autophagy, and epigenetics of intervertebral disc cells. It and the above pathological mechanisms are closely linked through the common hub reactive oxygen species(ROS), and promote each other in the process of disc degeneration and promote the development of the disease. This reveals the important role of oxidative stress in the process of IDD, and the importance and great potential of IDD therapy targeting oxidative stress. The efficacy of traditional therapy is unstable or cannot be maintained. In recent years, due to the rise of materials science, many bioactive functional materials have been applied in the treatment of IDD, and through the combination with traditional drugs, satisfactory efficacy has been achieved. At present, the research review of antioxidant bioactive materials in the treatment of IDD is not complete. Based on the existing studies, the mechanism of oxidative stress in IDD and the common antioxidant therapy were summarized in this paper, and the strategies based on emerging bioactive materials were reviewed.