Aim: Liquid biopsies hold significant potential for the minimally invasive diagnosis of tumors and other diseases. While the clinical application of cell-free DNA (cfDNA) methodologies is emerging, the implementation of tumor-derived extracellular vesicles (EVs) as validated biomarkers is hindered by substantial preanalytical variations. In this work, we standardized the preanalytical procedures of blood collection for subsequent serial isolation of plasma cfDNA and EVs from a single blood collection tube.

Methods: We compared the impact of blood preservation tubes and storage to enable proteomic profiling of resulting EVs in addition to cfDNA extraction and sequencing. Following a stringent method of large EV (lEV) and small EV (sEV) isolation, consisting of differential ultracentrifugation and size exclusion chromatography, we evaluated the protein concentration, particle number, quality and integrity of the isolated EVs. Subsequent proteomic analyses of EV isolates revealed the complexity of the respective tube-biased proteomes, allowing the interpretation of EV origins as well as contamination sources.

Results: While ACD-A and Citrate tubes yield satisfactory results in the preservation of EV proteomes, only Streck RNA, Norgen, and PAX tubes can maintain high cfDNA purity for up to 7 days. When aiming for multiomics analyses, Streck RNA tubes showed the most stable performance across the tested parameters for both bioanalytes. Furthermore, we detected greater variability in protein composition in sEVs than in lEVs after 7 days of storage; thus, sEVs might be more susceptible to storage effects.

Conclusion: Our clinically applicable workflow provides the basis for informed choice of liquid biopsy tubes along with a ready-to-use protocol to retrieve both genomic and EV proteomic biomarker information for multiomics biomarker-based liquid biopsy studies.

In the rapidly developing field of skin care, non-surgical facial aesthetics are becoming increasingly favored by consumers. Plant-derived extracellular vesicles (PDEVs) have attracted much attention due to their low toxicity, cellular communication function, and ability to carry bioactive molecules, including proteins, lipids, nucleic acids, and small molecules with pharmacological activities. Recent in vitro studies have shown that PDEVs enhance the transdermal delivery of drugs and improve skin condition, suggesting promising applications in facial aesthetics. In this review, we provide a comprehensive overview of the application of PDEVs in anti-scarring, anti-aging, and anti-pigmentation therapies. We also discuss current limitations in their application and potential solutions to address these challenges. In conclusion, this review analyzes the roles and mechanisms of PDEVs in facial aesthetics and aims to support their future clinical application.

Extracellular vesicles (EVs) have significant potential as therapeutic agents and as sources of diagnostic, predictive, and prognostic nucleic acid biomarkers. However, variability in EV workflows and inadequate standardization of downstream analysis pose major obstacles to reproducibility. The MISEV (Minimal Information for Studies of Extracellular Vesicles) guidelines provide essential domain-specific recommendations for EV isolation, characterization, analysis, nomenclature, and reporting, but deliberately refrain from prescribing methods for the molecular quantification of EV cargo. Among the analytical platforms used in EV studies, quantitative reverse transcription PCR (RT-qPCR) is the most critical method for validating and quantifying EV-associated RNA. The recently revised MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) 2.0 guidelines offer a detailed foundation for ensuring analytical validity in RT-qPCR-based quantitative applications. The proposed model of harmonizing general and domain-specific guidelines provides a scalable blueprint for improving reproducibility across complex biomarker development workflows in molecular diagnostics.

Aim: Extracellular vesicles (EVs) play a pivotal role in tumor progression, influencing the tumor microenvironment. Despite significant research, the targeted analysis of EVs directly derived from primary tumors remains limited, particularly in ovarian cancer. The majority of existing studies have focused on EVs derived from peritoneal fluid (ascites), which encompasses contributions from different cell types. This study aims to isolate and characterize EVs secreted specifically by ovarian cancer spheroids derived from primary patient ascites.

Methods: A three-dimensional cell culture model was employed to cultivate tumor spheroids in a defined medium, with EVs purified via differential ultracentrifugation and size-exclusion chromatography. Purified EVs were characterized by nanoparticle tracking analysis, nanoflow cytometry, and electron microscopy prior to performing high-resolution mass spectrometry.

Results: This approach allowed the identification of known cancer-associated proteins, including danger molecules, which are linked to poor prognosis. Moreover, enzyme-linked immunosorbent assay (ELISA) analysis demonstrated that the ascites abundance levels of novel candidates [RAB14 (Ras-related protein Rab-14), SCAMP3 (secretory carrier membrane protein 3), and FAM3C (FAM3 metabolism regulating signaling molecule C)] correlated with patients’ progression-free survival, further validating their clinical relevance. Finally, we used the Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database to compare our dataset with The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) data. Thereby, we revealed a signature of three TOP genes encoding proteins within our dataset (CORO1B, LAMP2, MSLN), which were differentially expressed in ovarian cancer patients compared to healthy individuals.

Conclusion: This study provides the first proteomic profile of EVs derived directly from primary tumor spheroids, and paves the way for a better mechanistic understanding of EV-associated proteins and for the development of biomarkers or therapeutic strategies.

As global aging intensifies, the issue of bone aging has become increasingly prominent. Osteoporosis and osteoarthritis, which are common complications of bone aging, significantly impair patients’ quality of life due to their high prevalence and disability rates, thereby presenting a major public health challenge. Extracellular vesicles (EVs), nanoscale particles released by cells, are regarded as an ideal platform for bone aging due to their high biocompatibility, ease of modification, and significant therapeutic efficacy. This review provides a comprehensive summary of the latest advancements in mammalian-, bacterial-, and plant-derived EVs, particularly in the context of bone aging. Furthermore, organoids, as lab-grown models replicating organ physiology, produce organoid-derived EVs that represent an especially promising avenue for therapeutic application. This review focuses on exploring potential therapeutic strategies that capitalize on the unique advantages of each EV type for treating bone aging. It is anticipated that a thorough comprehension of these EV types will unveil new avenues for bone aging treatment.

The crosstalk between the skeletal muscles and the liver is receiving growing attention, as patients with chronic liver disease often develop a loss of skeletal muscle mass. In these patients, particularly those with metabolic dysfunction-associated steatotic liver disease, physical exercise improves insulin sensitivity and hepatic steatosis. However, excessive exercise may impair mitochondrial function, inflammation, and liver health. The study by Liu et al. demonstrates that overtraining promotes liver fibrosis through myocyte-derived small extracellular vesicles. Here, we comment on the novelty of these findings and areas to be developed in the future.

Aim: This study aimed to evaluate the reproducibility of the isolation and characterization of feces-derived bacterial membrane vesicles.

Methods: Human fecal samples (n = 12) stored at -80 °C were thawed, sampled, and then refrozen. From these samples, bacterial membrane vesicles were isolated through ultrafiltration, ultracentrifugation and size exclusion chromatography. Vesicle-associated DNA was characterized by marker [16 ribosomal DNA (rDNA)] sequencing to determine composition. The same fecal samples were thawed again after > 6 months of storage at -80 °C to repeat this procedure. Compositions and other vesicle characteristics were compared to investigate effects of storage and freeze/thawing on sample stability. In addition, for four of the fecal aliquots, the bacteria were subjected to marker gene sequencing alongside their derived membrane vesicles.

Results: No significant differences were observed in the pre- and post freeze/thawing composition of feces-derived bacterial membrane vesicles [permutational multivariate analysis of variance (PERMANOVA) P = 0.356] or bacteria (PERMANOVA P = 0.721) as determined by 16S rDNA sequencing. Additionally, no significant differences were observed in vesicle size, concentration, and associated protein or DNA content. These results indicate that, long-term storage of feces at -80 °C and an additional freeze/thawing cycle does not induce compositional or qualitative changes to vesicle repertoires.

Conclusion: These reproducibility findings hold great relevance for research on (gut)bacteria derived membrane vesicles. Our results indicate that fecal samples can be stably preserved at -80 °C for bacterial and vesicle isolations as their characteristics remain stable over time.

Aim: This study aims to investigate how the gut microbiota communicates with the host via bacterial extracellular vesicles (BEVs), given that direct contact between microbes and the healthy intestinal epithelium is prevented by a sterile mucin gel layer. Understanding these indirect interactions is critical because the specific pathways and mediators of microbiota-host interactions are incompletely understood. Tracking BEVs in vivo however is particularly challenging due to their nanoscale size and complex molecular composition.

Methods: To address these challenges, we developed a highly sensitive Nanoluciferase (NanoLuc) system for luminescence-based detection of BEVs produced by the model human commensal bacterium Bacteroides thetaiotaomicron. This approach was evaluated in germ-free and specific-pathogen-free mice, with comparisons between administration routes demonstrating the advantages of this system for in vivo BEV labelling over conventional lipophilic dyes.

Results: We report, for the first time, that BEVs endogenously produced in the gastrointestinal tract (GIT) of mice can deliver bioactive NanoLuc protein to multiple organ tissues, including the central nervous system. Our findings establish that naturally occurring BEVs in the GIT are capable of traversing multiple host barriers, including the intestinal epithelium, vascular endothelium, and the blood-brain-barrier, to access tissues such as the brain and eyes.

Conclusion: These findings advance our understanding of BEV-mediated microbe-host interactions and demonstrate the potential of BEVs as vehicles for long-distance delivery of bioactive therapeutics.

Mesenchymal stromal cell-derived small extracellular vesicles (MSC-sEVs) have emerged as a promising cell-free alternative to MSC-based therapies, offering superior safety, scalability, and stability profiles. These nanosized vesicles are now widely regarded as the principal therapeutic effectors of MSCs, capable of recapitulating many of the benefits attributed to their parental cells. However, their successful clinical translation depends on overcoming key challenges, particularly those related to product variability, viral safety, and the definition of mechanistically relevant potency-associated critical quality attributes (CQAs). This review explores the sources of MSC-sEV variability, including MSC tissue origin, manufacturing parameters, and limitations associated with primary and pluripotent stem cell-derived MSCs. The use of immortalized monoclonal MSC lines is discussed as a potential solution to improve batch consistency. Regulatory frameworks such as the International Council for Harmonisation (ICH) guideline Q5A(R2) are also highlighted for ensuring viral safety in sEV manufacturing processes. A major focus is the critical evaluation of microRNAs (miRNAs) - long regarded as leading candidates for potency CQAs in MSC-sEV products. Despite their prevalence in the extracellular vesicle literature, mounting evidence challenges their functional relevance in therapeutic contexts. Studies consistently show that miRNAs are underrepresented in sEVs, occur at very low copy numbers, and lack essential components (e.g., Argonaute proteins) required for canonical RNA interference. Moreover, the efficiency of EV internalization and endosomal escape remains exceedingly low, rendering miRNA-based gene regulation mechanistically implausible at physiologically relevant doses. These findings call into question the widespread assumption that miRNAs are primary effectors of MSC-sEV activity.

Aim: Precancerous lesions of gastric cancer (PLGC) represent a critical window for prevention. Developing non-invasive tools that can reliably detect these lesions is therefore a prerequisite for lowering gastric-cancer incidence. Recent work has highlighted the diagnostic promise of plasma extracellular vesicle DNAs (evDNAs) and the 5-hydroxymethylcytosine (5hmC)-Seal epigenomic platform. Here we profiled genome-wide 5hmC patterns in circulating evDNA to discover biomarkers and build a classification model.

Methods: We performed whole-genome 5hmC-Seal on plasma evDNAs from 67 PLGC patients and 67 healthy individuals. By identifying trend-expressed differentially hydroxymethylated regions (DhMRs), we used machine learning algorithms to screen for diagnostic biomarkers of PLGC and established a corresponding diagnostic model.

Results: We ultimately constructed a diagnostic model comprising nine DhMRs. In the test set, the area under the curve (AUC) value was 0.963, with an accuracy of 0.886, sensitivity of 95.45%, and specificity of 81.82%. These results indicate that DhMRs in evDNA can serve as diagnostic biomarkers for PLGC, with good diagnostic capability and reliability. Correlation analysis showed a strong association between the DhMRs in the diagnostic model and clinical pathological indicators of PLGC.

Conclusion: We developed a non-invasive diagnostic model for PLGC by profiling 5hmC in plasma evDNA. In both accuracy and inter-batch robustness, it surpasses all previously reported assays. Our findings establish plasma-evDNA 5hmC profiling as a reliable, minimally invasive strategy for the early detection and precise diagnosis of gastric precancerous lesions, and provide a new translational and clinical framework for future work.

Aim: Liver fibrosis (LF) is a major pathological stage that may progress to end-stage chronic liver injury but currently lacks effective treatment strategies. Previous studies have shown that adipose-derived stem cell extracellular vesicles (ADSC-EVs) play crucial roles in tissue repair, immune regulation, and anti-inflammatory effects. This study aims to elucidate the therapeutic effect of ADSC-EVs in LF and reveal their regulation mechanisms in gut-liver axis dysregulation.

Methods: The LF mouse model was established by intraperitoneal injection of diethylnitrosamine/CCl₄. LF mice for ADSC-EV treatment received ADSC-EVs (200 μg per mouse) twice a week for three weeks. Then, hepatic function tests, liver and gut histopathology, and gut microbiota analyses were performed.

Results: ADSC-EVs effectively improved hepatic function, reduced collagen deposition and suppressed hepatic stellate cell activation, exhibiting potent anti-fibrotic potential in LF mice. Additionally, they significantly restored intestinal barrier integrity by reducing intestinal permeability and reinforcing the mucus barrier. Furthermore, ADSC-EV treatment regulated gut microbiota dysbiosis, increased the abundance of beneficial intestinal bacteria such as Akkermansia muciniphila. ADSC-EV intervention also elevated the level of butyric acid in cecal contents and significantly reduced systemic inflammation.

Conclusion: Our findings suggest that ADSC-EVs represent a promising novel therapeutic strategy for LF, promoting liver tissue repair, enhancing intestinal barrier function, and maintaining gut homeostasis to establish a virtuous circle within the liver-gut axis.

Urinary tract infections (UTIs) pose a significant public health challenge, affecting approximately 407 million people worldwide and causing substantial morbidity and approximately 237,000 deaths. Bacteria and fungi represent the most frequent causative microbes, leading to symptoms such as low abdominal pain, fever, frequent urination, hematuria, sepsis, inflammation of the bladder and kidney, and even death. In recent years, extracellular vesicles (EVs) have emerged as critical mediators of UTI pathogenesis. EVs are lipid bilayer nanoscale particles that carry DNA, RNA, enzymes, and other biomolecules. They can facilitate microbial colonization, immune modulation and evasion, tissue invasion, and antimicrobial agent resistance. This review summarizes current knowledge on the role of bacterial and fungal-derived EVs in UTIs, their mechanisms of action, and their potential therapeutic implications.

Aim: Autologous blood transfusions (ABT), especially those involving stored erythrocyte concentrates (ECs), are known to be misused as performance enhancers in recreational and competitive athletes. EC storage not only increases the release of extracellular vesicles (EVs), but also significantly alters the microRNA profiles. Since re-transfused EVs also appear in urine, this study was designed to evaluate whether the urinary EV-associated microRNA load could serve as a valuable indicator in the challenging detection of ABT.

Methods: Thirty healthy, recreationally active males were included and equally divided into three groups. The control group did not donate or receive any blood components. Group 1 donated about 500 mL of whole blood once, which was subsequently processed into ECs. These were stored for six weeks and then re-infused into the respective donor. Group 2 donated about 500 mL of whole blood twice, at an interval of two weeks. The obtained ECs were stored for six or four weeks, respectively, until parallel re-infusion. In all groups, urine samples were collected over three consecutive weeks before whole-blood donation to establish each individual’s baseline, as well as before re-transfusion and several hours and days afterward. Urine samples were processed and analyzed for general urinary health and creatinine levels. Urinary EVs were further isolated by immunoaffinity and characterized using transmission electron microscopy, fluorescence nanoparticle tracking analysis, and western blotting, as well as an established multiplexed bead-based flow cytometry method, followed by RNA isolation and in-depth small RNA profiling using next-generation sequencing and comprehensive data analyses.

Results: Urinary EVs presented with typical morphology of small EVs (< 200 nm) and an overall concentration of 8.79 ± 7.00 × 10¹⁰ particles/g U_Crea (urinary creatinine). Significant increases in urinary EV concentrations were detected up to three days after ABT. Apart from Alix, Syntenin, and tumor susceptibility gene 101 (TSG101), surface markers CD63, CD9, CD133/1, CD24, CD326, CD81, and CD31 were also shown to be highly abundant on urinary EVs. Impurities or contaminations were not detected. Cluster analysis based on surface markers showed a clear separation between the control and ABT group. Furthermore, microRNA profiling revealed 13 microRNAs differently regulated upon ABT with miR-155-5p, miR-320b, and miR-6869-5p being the most abundant.

Conclusion: This proof-of-concept study suggests an impact of ABT on the urinary EV-microRNA cargo and yields comprehensive findings into surface markers of urinary EVs. While the adoption of urinary EV-associated microRNAs in routine doping tests requires further exploration, these data serve as a valuable basis for a variety of subsequent investigations.

Aim: Human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) persist in effectively treated HIV-infected individuals, in part due to HIV reservoirs in brain microglia, which express low levels of viral proteins such as Nef. This study aimed to elucidate how microglia release Nef into the extracellular space, where it exerts its biological functions.

Methods: Here, we systematically characterized extracellular particles released from immortalized human microglia (h-microglia) expressing Nef alone or after HIV infection. Importantly, we established a novel h-microglia model harboring a stably integrated Nef tagged with green fluorescent protein (Nef.GFP) transgene under an inducible promoter. Extracellular vesicles (EVs) were enriched from culture media and analyzed for morphology, size, concentration and molecular composition, including Nef content, by (super-resolution) fluorescence microscopy, (immunogold) transmission electron microscopy, asymmetric flow field-flow fractionation coupled to a multi-angle light-scattering detector, nanoparticle tracking analysis, and nano-flow cytometry and immunoblotting.

Results: Nef.GFP expression increased particle release up to 11.7-fold compared with controls or known stimulants adenosine triphosphate (ATP) and ionomycin. Compared to the latter, the particles were also significantly smaller (root mean square radius, Rrms = 172 nm) and displayed unique protein and density profiles. All data support the EV nature of the released particles. Approximately half of the Nef.GFP-induced EVs contained Nef (45.5% ± 15.8%), with immunogold labeling confirming its intraluminal localization. Notably, infection with HIV isolates NL4-3 and YU-2 likewise produced Nef-positive EVs distinct from virions.

Conclusion: Our findings importantly contribute to understanding the source and characteristics of extracellular Nef in the central nervous system of HIV infected individuals and offer new tools to study HIV Nef biology. Nef-laden EVs should be further investigated as potential therapeutic targets in HAND.

Although anatomically separate, the gut and lungs are interconnected through intricate pathways involving their respective microbiota, supporting the concept of a gut-lung axis. In the pediatric field, devastating intestinal pathologies such as necrotizing enterocolitis and inflammatory bowel diseases mostly affect preterm infants. In parallel, in the lung, bronchopulmonary dysplasia and chronic obstructive pulmonary disease represent pediatric unmet clinical needs. In this review, we discuss how the extracellular vesicles (EVs), nanoparticles secreted by all cell types, represent a common element in the gut-lung axis. Specifically, EVs play a dual role, serving both as novel disease biomarkers and as promising therapeutic agents.

Extracellular vesicles (EVs) are central mediators of intercellular communication in both healthy and malignant states. In normal B lymphocyte (cell) biology, EVs derived from B cells, mast cells, T cells, and mesenchymal stromal cells regulate maturation, antigen presentation, and activation. B cell-derived EVs can either suppress excessive activation to maintain immune homeostasis or amplify responses during an active immune response. Modulation of these responses often occurs via phosphoinositide 3-kinase signaling pathways in recipient cells. In B cell malignancies, such as leukemias, lymphomas, and multiple myeloma, EVs play pivotal roles in disease progression and therapy resistance. Tumor- and stromal-derived EVs can transfer pro-survival proteins, regulatory RNAs, and drug-resistance factors to directly promote tumor progression. In addition, EVs can shape the tumor microenvironment to indirectly promote tumor progression through macrophage polarization, stromal cell reprogramming, and suppression of anti-tumor immunity. Conversely, under certain conditions, B cell EVs can enhance immune surveillance by stimulating T cells and presenting antigen, highlighting their dual potential in cancer biology. Clinically, B cell-derived EVs represent promising liquid biopsy biomarkers: increases in EV abundance, expression of surface antigens, altered protein cargo, and distinct RNA signatures have been associated with disease stage, treatment response, and patient outcomes. Despite this potential, variability in EV isolation and analysis methods remains a barrier to clinical translation. Moving forward, identifying robust biomarker signatures across platforms and clarifying mechanisms of cargo selection and EV uptake will be critical for advancing diagnostic and therapeutic applications. Overall, B cell-derived EVs act as contextual regulators of immune function and malignancy, positioning them as both modulators of disease progression and promising clinical tools.

Periodontitis is a chronic inflammatory disease characterized by the progressive destruction of both soft (gingiva and periodontal ligament) and hard (cementum and alveolar bone) supporting tissues. The complex periodontal microenvironment often limits the effectiveness of current clinical treatments in achieving functional tissue regeneration. Although mesenchymal and immune cell-based therapies hold promise, concerns related to cell viability and immune compatibility limit their clinical translation. As a natural secretome, small extracellular vesicles (sEVs) are cell-secreted nanoparticles that deliver bioactive molecules for cell-to-cell communication to modulate immune response and promote tissue regeneration. To assess the translational readiness of sEVs therapy, this scoping review first outlines the current clinical trials of mesenchymal stem cells (MSCs)-sEVs in periodontitis, followed by a transition to preclinical application of integrating sEVs with biomaterial scaffolds to enhance localized regenerative outcomes. We then analyzed eight preclinical studies utilizing 3D bioprinted MSCs-sEVs/human umbilical vein endothelial cells-sEVs (or immune cell-derived sEVs) constructs in bone and vasculature regeneration models, and one study related to in vitro periodontal regeneration. These constructs exhibited improved outcomes in osteogenesis, angiogenesis, and immunomodulation, supporting their potential for future translational applications in periodontal therapy. Given the early stage of bioprinted sEVs constructs in periodontitis, we outline critical research gaps and potential future directions to overcome current technical and biological challenges. Together, this review demonstrated the translational trajectory of sEV-based strategies for periodontal regeneration. It offers a potential roadmap for utilizing sEV-based periodontal regeneration across clinical, preclinical, and biofabrication applications, highlighting their potential as next-generation, cell-free therapeutics in regenerative periodontics.

Aim: Small extracellular vesicles (sEVs) are promising noninvasive biomarkers for several malignancies, including thyroid carcinoma (TC). However, their heterogeneity is frequently overlooked in bulk-level analyses.

Methods: Plasma samples from TC and healthy controls (HC) were collected for a proximity-dependent barcoding assay (PBA) to identify plasma sEV biomarkers at the single-sEV level. We screened the potential biomarkers using the Panel260 (the panel that detects 260 proteins) of PBA in Cohort 1, and validated them using Panel550 (the panel that detects 550 proteins) in Cohort 2.

Results: Plasma exosome counts were significantly elevated in TC compared with those in HC in both Cohort 1 and Cohort 2. Receiver operating characteristic curve analysis showed that sEV counts exhibited an area under the curve > 0.75 in both cohorts. The sEV proteomic analysis revealed that sEV epithelial cell adhesion molecule (EPCAM) levels were significantly increased, whereas claudin-11, integrin alpha X, and lymphocyte-activating 3 were significantly decreased in TC compared with HC. The increase in EPCAM in the plasma and tumor tissues was confirmed by enzyme-linked immunosorbent assay and immunohistochemistry analyses, respectively. The sEV subpopulation analysis further demonstrated that EPCAM⁺ sEVs were significantly elevated in TC compared with HC in both cohorts. The reduction in sEV counts was observed in 18 out of 20 patients after the operation. The decrease in EPCAM⁺ sEVs was observed in 20 patients with TC post-operatively, whereas the reduction in the conventional biomarker serum thyroglobulin (Tg) was observed in 14 patients. TC-derived plasma sEVs promoted TC cell proliferation, migration, invasion, and TC xenograft growth.

Conclusion: EPCAM⁺ sEVs could serve as a promising biomarker for the early diagnosis of TC and perform better in monitoring post-operative remission of TC than serum Tg.

Aim: Pig seminal plasma (SP) contains oxytocin (OXT) at levels associated with the fertility outcomes of boars used for artificial insemination (AI). However, OXT easily volatilizes when circulating freely, making it difficult to maintain stability in AI seminal doses. The hypothesis is that OXT is stably carried in seminal extracellular vesicles (EVs). This study aimed to determine the following: (1) whether seminal EVs carry OXT and, if so, where they carry it; (2) the source of seminal EVs carrying OXT; and (3) whether the levels of OXT in seminal EVs are associated with the fertility of AI boars.

Methods: Seminal EV samples were isolated by size-exclusion chromatography from entire ejaculates and ejaculate fractions: the first 10 mL of the sperm-rich fraction (SRF; SRF-P1), the remainder of the SRF (SRF-P2), and the post-SRF fraction. The OXT concentration was measured using a direct competitive immunoassay with AlphaLISA technology and an anti-OXT monoclonal antibody.

Results: Seminal EVs carry OXT, primarily on the outer surface of the EV membrane, likely within the protein corona. The concentration of OXT in seminal EVs varied among ejaculate fractions (P < 0.0001). It was higher in SRF-P2 (3,017.68 ± 860.52 pg/mL of SP) and post-SRF (3,613.27 ± 935.08 pg/mL of SP) EVs than in SRF-P1 (1,675.15 ± 520.62 pg/mL of SP) EVs. The concentration of OXT in seminal EVs was associated with the fertility outcomes of AI boars. Higher concentrations were found in the seminal EVs of boars with a high farrowing rate and in those with a small litter size.

Conclusion: Porcine seminal EVs carry OXT outside of membranes, and those originating from the accessory sex glands are particularly enriched. The association of OXT of seminal EVs with fertility is ambivalent: it enhances farrowing rate while potentially reducing litter size, likely via its effects on myometrial contractility, which facilitates sperm transport but may hinder embryo implantation.

Aim: Representing about 15% of lung cancers, small cell lung cancer (SCLC) is an extremely aggressive disease characterized by rapid growth and early spread, leading to dismal clinical outcomes. In this study, we aimed to investigate the dual roles of exosomal long non-coding RNA (lncRNA) LYPLAL1-DT (LYPLAL1 divergent transcript) in both tumor cells and vascular endothelial cells.

Methods: The circulating levels of LYPLAL1-DT were measured using real-time polymerase chain reaction in 13 SCLC patients and 21 normal controls. Exosomes from the supernatant of cell culture medium or serum were extracted through ultracentrifugation and dyed with PKH67 green fluorescent cell linker to identify internalization. Migration and invasion assay, colony formation, Cell Counting Kit-8 (CCK-8), and tube formation assays were used to assess the malignant effects of extracellular RNAs (exRNAs) LYPLAL1-DT in exosomes.

Results: Exosomal LYPLAL1-DT is upregulated in SCLC patients and plays a dual role in promoting tumor cell aggressiveness and enhancing pro-angiogenic behavior in endothelial cells, thereby accelerating SCLC progression. Mechanistically, LYPLAL1-DT functions as a competing endogenous RNA, exerting its effects through the miR-204-5p/profilin-2, miR-204-5p/B-cell lymphoma 2 and miR-204-5p/sirtuin 1 regulatory axes. These pathways underscore the pleiotropic effects of exosomal LYPLAL1-DT and underscore its value as a promising therapeutic target.

Conclusion: In the current study, we investigated the bidirectional communication mediated by exRNA LYPLAL1-DT between SCLC and endothelial cells, while also exploring its potential regulatory targets. This research provides a potential circulating biomarker for the diagnosis, prognosis, and treatment of SCLC.

Aim: Small extracellular vesicles (sEVs) are membrane-bound nanoparticles secreted by virtually all cell types that have emerged as promising sources of protein biomarkers for a wide range of diseases, including central nervous system disorders. Blood sampling is the most informative and non-invasive biomarker source. Notably, mouse models represent essential systems for studying in vivo disease mechanisms and testing therapeutic strategies. Therefore, in this study, we investigated the suitability of two different isolation methods for sEV recovery starting from non-terminal mouse blood sampling, with the aim of identifying the most effective protocol for downstream biomarker discovery.

Methods: We performed and compared size exclusion chromatography (SEC) and ultracentrifugation followed by iodixanol density gradient (UC-IDG). Additionally, we optimized extracellular vesicle (EV) isolation from small-volume samples of both serum and plasma, since these represent the most used sources for in vivo preclinical biomarker research. Both methods were evaluated in terms of yield, purity, and EV protein content by nanoparticle tracking analysis, electron microscopy, and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) proteomics.

Results: SEC showed a higher number of isolated vesicles and EV-associated markers, while reporting a reduced percentage of blood-abundant co-isolated proteins, compared to UC-IDG. The use of plasma as a starting material resulted in a cleaner background, showing fewer protein aggregates. The obtained results emphasize the advantage of SEC in enhancing vesicle yield and purity levels.

Conclusion: This work contributes to sEV-derived biomarker research in mouse models by confirming plasma, rather than serum, as the most reliable source of EVs and providing evidence that SEC is more suitable than UC-IDG for EV isolation.

Aim: Chemotherapy continues to be the frontline treatment for lung cancer patients. However, treatment-related toxicity and off-target effects limit the use of chemotherapy. Therefore, improvements in delivering chemotherapeutics with reduced toxicity to normal tissues are needed. In the present study, we combined nanotechnology with extracellular vesicle (EV) technology to produce tumor-targeted multifunctional EVs (tt-Mfn-EVs) as drug carriers for cancer therapy.

Methods: The tt-Mfn-EVs were formulated by exogenously loading EVs with gold nanoparticles conjugated to cisplatin (CDDP) via pH-sensitive coordination ester linkage. Attached to the outer surface of drug-loaded EVs is the transferrin ligand for targeting transferrin receptor (TfR) overexpressing lung cancer cells.

Results: The tt-Mfn-EVs were 138.2 nm in size and exhibited greater drug release kinetics at pH 5.5 compared to pH 7.2. They significantly reduced cell viability of A₅₄₉ (TfR high) lung cancer cells compared to HCC₈₂₇ (TfR low) cells and non-targeted EVs. Tt-Mfn-EVs also induced higher levels of apoptosis and DNA damage in A₅₄₉ and HCC₈₂₇ cells compared to control groups. Finally, tt-Mfn-EV-mediated cytotoxicity was minimal in normal human lung fibroblast (MRC-₉) and human embryonic kidney 293 (HEK₂₉₃) cells compared to free CDDP.

Conclusion: Our study showed that tt-Mfn-EVs exerted selective and enhanced tumor-targeted cell killing in vitro, providing an opportunity for developing EV-based drug carriers for cancer therapy.

Aim: Mesenchymal stromal cells (MSCs) exert their therapeutic effects in osteoarthritis (OA) primarily through paracrine signaling, including secreted proteins and extracellular vesicle (EV)-associated microRNAs (miRNAs). However, the contribution of tissue origin to the composition and function of these secretomes remains unclear. This study aimed to provide a comprehensive molecular and functional comparison of secretomes from adipose-derived (ASCs), bone marrow-derived MSCs (BMSCs) and human amniotic membrane-derived MSCs, with a specific focus on OA-relevant pathways.

Methods: MSCs were immunophenotyped by flow cytometry. Secretomes were profiled for 200 factors and 784 EV-miRNAs. Functional enrichment was performed using Gene Ontology and Reactome databases. In vitro, secretomes were tested on interleukin (IL)-1β-stimulated human chondrocytes to assess modulation of OA-related gene expression.

Results: All MSC secretomes shared a core of factors enriched in anti-inflammatory and matrix-regulatory functions. ASCs showed the differential expression of a few modulators, potentially shifting their chondroprotective phenotype. EV-miRNAs further distinguished the MSC types. ASCs and BMSCs clustered closely in both overall miRNA content and functional enrichment, which included pathways for extracellular matrix organization, angiogenesis and IL-6 signaling. BMSC- and ASC-EVs had a higher ratio of OA-protective to destructive miRNAs, including miR-24-3p, miR-125b-5p and miR-222-3p. Functional assays confirmed that all MSC secretomes were effective in suppressing key OA-related genes in inflamed chondrocytes, with ASCs and BMSCs having a stronger activity.

Conclusion: These findings support the development of MSC-derived cell-free therapies and emphasize the importance of molecular profiling in MSC source selection. Further studies are warranted to validate these observations and optimize MSC-based interventions for clinical translation in OA.