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Background: Extracellular vesicles (EVs) are involved in intercellular communication and various biological processes. They hold clinical promise for the diagnosis and management of a wide range of pathologies, including cancer, cardiovascular diseases and degenerative diseases, and are of interest as regenerative therapies. Understanding the complex structure of these EVs is essential to perceive the current challenges associated with their analysis and characterization. Today, challenges remain in terms of access to high-yield, high-purity isolation methods, as well as analytical methods for characterizing and controlling the quality of these products for clinical use., Results: We isolated EVs from the same immortalized human cell culture supernatant using two commonly used approaches, namely differential ultracentrifugation and membrane affinity. Then we evaluated EV morphology, size, zeta potential, particle and protein content, as well as protein identity using cryogenic electron microscopy, nanoparticle tracking analysis, asymmetric field flow fractionation (AF4) and size exclusion chromatography (SEC) coupled to multi angle light scattering, bicinchoninic acid assay, electrophoretic light scattering, western blotting and high-resolution mass spectrometry. Compared to membrane affinity isolation, dUC is a more efficient isolation process for obtaining particles with the characteristics expected for EVs and more specifically for exosomes. To validate an isolation process, cryogenic electron microscopy is essential to confirm vesicles with membranes. High resolution mass spectrometry is powerful for understanding the mechanism of action of vesicles. Separative methods, such as AF4 and SEC, are interesting for separating vesicle subpopulations and contaminants., Significance: This study provides a critical assessment of eight different techniques for analyzing EVs, some of which are mandatory for in-depth characterization and deciphering, while others are more appropriate for routine analysis, once the production and isolation process has been validated. The strengths and limitations of the different approaches used are highlighted., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Tumor-derived extracellular vesicles detection has emerged as an important clinical liquid biopsy approach for cancer diagnosis. In this work, we developed a novel hybrid plasmonic nanocavity consisting of hexagonal Au nanoplates nanoarray, SnS 2 /Au nanosheet layer and biomimetic lipid bilayer. Firstly, the hybrid plasmonic nanocavity combined the optical confinement for the ECL regulation and the biological recognition for the detection of extracellular vesicles. Secondly, MXene-derived Ti 2 N QDs have been prepared as ECL nanoprobe to label extracellular vesicles. Moreover, biomimetic lipid bilayer with specific aptamer was used to identify extracellular vesicles and integrate Ti 2 N QDs into the nanocavity with membrane fusion strategy. Due to the significant electromagnetic field enhancement at the cavity region, the hybrid plasmonic nanocavity provided strong field confinement to concentrate and redistribute the ECL emission of QDs with a 9.3-fold enhancement. The hybrid plasmonic nanocavity-based ECL sensing system improved the spatial controllability of EVs analysis and the accurate resolution of specific protein. It achieved the sensitive detection of extracellular vesicles in ascites and successfully distinguished the peritoneal metastasis of gastric cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Anthocyanin (ACN) has attracted considerable attention due to its wide range of physiological effects. However, challenges such as poor stability and limited bioavailability have hindered its utilization in functional foods. To address these issues, this research utilized milk-derived extracellular vesicles (MEV) as carriers for encapsulating and binding ACN through various techniques, including ultrasonic, electroporation, saponin treatment, incubation, and freeze-thaw cycles. The objective of these approaches was to enhance the stability of ACN and improve its oral delivery. Notably, the ACN-loaded MEV (MEV-ACN) prepared through ultrasonic exhibited small particle sizes and good stability under processing, storage, and simulated digestion conditions. Cellular studies revealed that MEV-ACN exhibited pro-oxidant properties and induced oxidative stress, leading to cell apoptosis with greater efficacy compared to free ACN. These findings suggest that encapsulating ACN within MEV can significantly enhance its processing and oral stability, as well as strengthening its dietary defense capabilities in anti-tumor applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

The cerebrospinal fluid (CSF) is a key matrix for discovery of biomarkers relevant for prognosis and the development of therapeutic targets in pediatric central nervous system malignancies. However, the wide range of protein concentrations and age-related differences in children makes such discoveries challenging. In addition, pediatric CSF samples are often sparse and first prioritized for clinical purposes. The present work focused on optimizing each step of the proteome analysis workflow to extract the most detailed proteome information possible from the limited CSF resources available for research purposes. The strategy included applying sequential ultracentrifugation to enrich for extracellular vesicles (EV) in addition to analysis of a small volume of raw CSF, which allowed quantification of 1351 proteins (+55% relative to raw CSF) from 400 μL CSF. When including a spectral library, a total of 2103 proteins (+240%) could be quantified. The workflow was optimized for CSF input volume, tryptic digestion method, gradient length, mass spectrometry data acquisition method and database search strategy to quantify as many proteins a possible. The fully optimized workflow included protein aggregation capture (PAC) digestion, paired with data-independent acquisition (DIA, 21 min gradient) and allowed 2989 unique proteins to be quantified from only 400 μL CSF, which is a 340% increase in proteins compared to analysis of a tryptic digest of raw CSF.

Oxylipins are powerful signalling compounds derived from polyunsaturated fatty acids (PUFAs) and involved in regulating the immune system response. A mass spectrometry-based method was developed and validated for the targeted profiling of 52 oxylipins (e.g., isoprostanoids, prostaglandins, epoxy- and hydroxy-fatty acids, specialized pro-resolving mediators) and 4 PUFAs in small urinary extracellular vesicles (uEVs). Ultrasound-assisted extraction using a 50:50 v/v MeOH:H 2 O mixture ensured optimal analytical performances. Limits of detection ranged between 10 and 400 pg/mL for oxylipins and 0.10-3 ng/mL for PUFAs. Satisfactory recoveries (85-116 %) and good intra- and inter-day precisions (RSD ≤15 %) were obtained for all the analytes. The reliability of the procedure was tested in a real case scenario by monitoring ultramarathon runners during the world Tor des Géants® (TDG) race. Both F 2 - and E 2 -isoprostanes were detected in small uEVs of the ultramarathon runners, suggesting the onset of an oxidant insult. 5-F 2t -IsoP exhibited significant pre- to post-race variations, thus potentially representing a non-invasive marker of in-vivo lipid peroxidation. The presence of specialized pro-resolving mediators suggests the activation of pro-resolution signalling cascade resolving inflammation. These outcomes may help manage post-exercise recovery and improve training., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Similar to mammalian cells, most bacteria can release nano-sized membrane vesicles (MVs) into the extracellular environment. MVs contain lipids, bioactive proteins, nucleic acids, and metabolites, and play important roles in microbial physiology. MVs have great potential for immunotherapeutic applications, such as bacterial vaccines and cancer immunotherapy. However, because of the diversity in content and heterogeneity in size of MVs, the clinical application of MVs has been limited. Recently, the use of MVs combined with nanoparticles (NPs) has been shown to be effective in improving the homogeneity, stability and function of MVs. In this review, we focus on studies of MVs combined with NPs (MV-NPs) and describe the use of these MV-NPs in biotechnology, especially in bacterial vaccine and cancer immunotherapy., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Plant-derived extracellular vesicles (PLEVs), as a type of naturally occurring lipid bilayer membrane structure, represent an emerging delivery vehicle with immense potential due to their ability to encapsulate hydrophobic and hydrophilic compounds, shield them from external environmental stresses, control release, exhibit biocompatibility, and demonstrate biodegradability. This comprehensive review analyzes engineering preparation strategies for natural vesicles, focusing on PLEVs and their purification and surface engineering. Furthermore, it encompasses the latest advancements in utilizing PLEVs to transport active components, serving as a nanotherapeutic system. The prospects and potential development of PLEVs are also discussed. It is anticipated that this work will not only address existing knowledge gaps concerning PLEVs but also provide valuable guidance for researchers in the fields of food science and biomedical studies, stimulating novel breakthroughs in plant-based therapeutic options., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work. There is no professional or other personal interest of any nature or kind in any product, service and/or company that can be construed as influencing the position presented in the manuscript., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Extracellular vesicles (EVs) are lipid-bound membrane vesicles released from cells, containing active compounds, which can be found in different foods. In this review, the role of food-derived vesicles (FDVs) as immunomodulatory drivers is summarized, with a focus on sources, isolation techniques and yields, as well as bioavailability and potential health implications. In addition, gaps and perspectives detected in this research field have been highlighted. FDVs have been efficiently extracted from different sources, and differential ultracentrifugation seems to be the most adequate isolation technique, with yields ranging from 10 8 to 10 14 EV particles/mL. Animal studies show promising results in how these FDVs might regulate different pathways related to inflammation. Further investigation on the production of stable components in a cost-effective way, as well as human studies demonstrating safety and health-promoting properties, since scarce information has been reported until now, in the context of modulating the immune system are needed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Metals in living organisms and environments are essential for key biological functions such as enzymatic activity, and DNA and RNA synthesis. This means that disruption of metal ion homeostasis and exchange between cells can lead to diseases. EVs are believed to play an essential role in transporting metals between cells, but the mechanism of metal packaging and exchange remains to be elucidated. Here, we established the elemental composition of EVs at the nanoscale and single-vesicle level and showed that the metal content depends on the cell type and culture microenvironment. We also demonstrated that EVs participate in the exchange of metal elements between cells. Specifically, we used two classes of EVs derived from papaya fermented fluid (PaEVs), and decidual mesenchymal stem/stromal cells (DEVs). To show that EVs transfer metal elements to cells, we treated human osteoblast-like cells (MG63) and bone marrow mesenchymal stem cells (BMMSCs) with both classes of EVs. We found that both classes of EVs contained various metal elements, such as Ca, P, Mg, Fe, Na, Zn, and K, originating from their parent cells, but their relative concentrations did not mirror the ones found in the parent cells. Single-particle analysis of P, Ca, and Fe in DEVs and PaEVs revealed varying element masses. Assuming spherical geometry, the mean mass of P was converted to a mean size of 62 nm in DEVs and 24 nm in PaEVs, while the mean sizes of Ca and Fe in DEVs were smaller, converting to 20 nm and 30 nm respectively. When EVs interacted with BMMSCs and MG63, DEVs increased Ca, P, and Fe concentrations in BMMSCs and increased Fe concentration in MG63, while PaEVs increased Ca concentrations in BMMSCs and had no effect on MG63. The EV cargo, including proteins, nucleic acids, and lipids, differs from their origin in composition, and this variation extends to the element composition of EVs in our study. This fundamental understanding of EV-mediated metal exchange between cells could offer a new way of assessing EV functionality by measuring their elemental composition. Additionally, it will contribute novel insights into the mechanisms underlying EV production and their biological activity.

Extracellular vesicles (EVs), biomimetics, and other biological nanoparticles (BNs) produced from human cells are gaining increasing attention in the fields of molecular diagnostics and nanomedicine for the delivery of therapeutic cargo. In particular, BNs are considered prospective delivery vehicles for different biologics, including protein and RNA therapeutics. Moreover, EVs are widely used in molecular diagnostics for early detection of disease-associated proteins and RNA. Technical approaches for measuring biologics mostly originated from the field of EVs and were later adopted for other BNs, such as extracellular vesicle-mimetic nanovesicles, membrane nanoparticles (nanoghosts), and hybrid nanoparticles, with minimal modifications. Here, we demonstrate that BNs are highly resistant to protocols that severely underestimate the protein and RNA content of BNs, and provide the relevance of these data both for general BNs characterization and practical applications of CRISPR/Cas-based therapies. We demonstrate that the addition of saponin leads to an ∼2- to 7-fold enhancement in protein isolation and an ∼2- to 242-fold improvement in RNA recovery rates and detection efficiency. Differences in the proteolipid contents of BNs, measured by Raman and surface-enhanced Raman spectroscopy, correlate with their susceptibility to saponin treatment for cargo extraction. Finally, we develop a unified protocol using saponin to efficiently isolate proteins and RNA from the BNs. These data demonstrate that previously utilized protocols underestimate BN cargo contents and offer gold standard protocols that can be broadly adopted into the field of nanobiologics, molecular diagnostics, and analytical chemistry.

Apoptotic bodies as plentiful extracellular vesicles generated from apoptotic cells play a central role in signal transduction and homeostasis regulation and simultaneously switch death to regeneration to a certain extent. Herein, we designed engineered apoptotic bodies derived from T cells to have the capacity of inflammation regulation and cartilage affinity. The engineered apoptotic bodies as a natural anti-inflammation factor were encapsulated into lubricating hydrogel microspheres to achieve an injectable microsphere complex for the treatment of osteoarthritis (OA). In the above therapeutic system, the engineered apoptotic bodies acted as a biochemical cue to regulate the inflammatory microenvironment and promote chondrocyte cartilage homeostasis, whereas the lubricating hydrogel microspheres served as a biophysical stimulation to effectively reduce the friction of the cartilage surface, restore the cartilage stress, and control the slow delivery of the encapsulated engineered apoptotic bodies by friction degradation. Consequently, the current work creates an injectable and multifunctional therapeutic microsphere to advance cartilage remodeling and OA therapy.

Extracellular vesicles (EVs) are a diverse population of membrane structures produced and released by cells into the extracellular space for the intercellular trafficking of cargo molecules. They are implicated in various biological processes, including angiogenesis, immunomodulation, and cancer cell signaling. While much research has focused on their biogenesis or their effects on recipient cells, less is understood about how EVs are capable of traversing diverse tissue environments and crossing biological barriers. Their interactions with extracellular matrix components are of particular interest, as such interactions govern diffusivity and mobility, providing a potential basis for organotropism. To start to untangle how EV-matrix interactions affect diffusivity, we use high speed epifluorescence microscopy, single particle tracking, and confocal reflectance microscopy to analyze particle mobility and localization in extracellular matrix-mimicking hydrogels composed of collagen I. EVs are compared with synthetic liposomes and extruded plasma membrane vesicles to better understand the importance of membrane composition on these interactions. By treating EVs with trypsin to digest surface proteins, we determine that proteins are primarily responsible for EV immobilization in collagen I hydrogels. We next use a synthetic peptide competitive inhibitor to narrow down the identity of the proteins involved to argynylglycylaspartic acid (RGD) motif-binding integrins, which interact with unincorporated or denatured nonfibrillar collagen. Moreover, the effect of integrin inhibition with RGD peptides has strong implications for the use of RGD-peptide-based drugs to treat certain cancers, as integrin inhibition appears to increase EV mobility, improving their ability to infiltrate tissue-like environments.

Last decade, extracellular vesicles (EVs) attracted a lot of attention as potent versatile drug delivery vehicles. We reported earlier the development of EV-based delivery systems for therapeutic proteins and small molecule chemotherapeutics. In this work, we first time engineered EVs with multivalent cationic lipids for the delivery of nucleic acids. Stable, small size cationized EVs were loaded with plasmid DNA (pDNA), or mRNA, or siRNA. Nucleic acid loaded EVs were efficiently taken up by target cells as demonstrated by confocal microscopy and delivered their cargo to the nuclei in triple negative breast cancer (TNBC) cells and macrophages. Efficient transfection was achieved by engineered cationized EVs formulations of pDNA- and mRNA in vitro. Furthermore, siRNA loaded into cationized EVs showed significant knockdown of the reporter gene in Luc-expressing cells. Overall, multivalent cationized EVs represent a promising strategy for gene delivery., (© 2024. The Author(s).)

Introduction: Extracellular vesicles (EVs) are nanosized membrane vesicles that are naturally secreted by almost all cells and have gained considerable attention. Many studies have applied EVs to the treatment of brain diseases and validated their effectiveness. Although only a few EVs can penetrate the blood‒brain barrier (BBB) into the brain after administration, it has been proven that EVs and their cargos exert their effects by interacting with brain cells. PKH dyes are commonly used to stain EVs for distribution studies. However, systematic investigations of imaging characteristics of the PKH-labeled EVs distributed in the brain are still scarce., Methods: We stained EVs derived from mesenchymal stem cells with PKH26 or PKH67. PKH26-labeled EVs and PKH67-labeled EVs were administered at the same time into each mouse while PKH26-labeled EVs were given through tail veins and PKH67-labeled EVs were given through intraperitoneal injection. Confocal microscopy was used to explore the distribution difference of two types of EVs given via different routes in the brain., Results: The fluorescence of PKH26 and PKH67 had nearly identical distributions in brain slices after 1 h, 6 h, 12 h and 1 day of EV administration. Under the same confocal parameters, brain slices without EVs administration demonstrated the same result. However, liver slices from mice administered with labeled EVs showed obviously different distributions of two types PKH fluorescence., Discussion: These findings raise questions about the ability of PKH dyes as labels for EVs when explore the EV brain distribution observed via confocal microscopy., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Wan et al.)

Background: Detailed characterization of extracellular vesicles (EVs) is crucial for their application in medical diagnostics. However, the complexity of their chemical composition and the heterogeneity of EV populations make their characterization challenging. Here we describe two analytical procedures that can help overcome this challenge., Methods: Small EVs were isolated from conditioned cell culture media using ultracentrifugation and characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Raman spectroscopy was used to assess the overall composition of the isolated samples and lipids extracted from them. Sulfophosphovanillin (SPV) colorimetric assay was used to quantify the contents of lipid., Results: Six samples of EVs were characterized. The lipid contents measured using SPV assay was in reasonable agreement with the quantitative estimates based on the particle size and concentration measured using NTA. The most peaks observed in the Raman spectra could be attributed to either proteins or lipids, and their origins was confirmed by lipid extraction. The protein-to-lipid ratio was estimated based on the Raman spectra., Conclusions: The experiential procedures described in this study will help to overcome the challenge of quick and highly informative characterization of the EVs., (© 2024 The Author(s). Published by IMR Press.)

Extracellular vesicles (EVs) harbor abundant glycans that mediate various functions, such as intercellular communication and disease advancement, which play significant roles in disease progression. However, the presence of EV heterogeneity in body fluids and the complex nature of the glycan structures have posed challenges for the detection of EV glycans. In this study, we provide a streamlined method integrated, me mbrane- s pecific s eparation with lectin-induced ag gr e gation strategy (MESSAGE), for multiplexed profiling of EV glycans. By leveraging a rationally designed lectin-induced aggregation strategy, the expression of EV glycans is converted to size-based signals. With the assistance learning machine algorithms, the MESSAGE strategy with high sensitivity, specificity, and simplicity can be used for early cancer diagnosis and classification, as well as monitoring cancer metastasis via 20 μL plasma sample within 2 h. Furthermore, our platform holds promise for advancing the field of EV-based liquid biopsy for clinical applications, opening new possibilities for the profiling of EV glycan signatures in various disease states.

Extracellular vesicles (EVs) are cell-derived membrane-bound particles with molecular cargo reflective of their cell of origin. Analysis of disease-related EVs and associated cargo from biofluids is a promising tool for disease management. To facilitate the analysis of intravesicular molecules, EV lysis is needed. Moreover, highly sensitive and multiplexed detection methods are required to achieve early diagnostics. While cell lysis approaches have been well studied, the analysis of EV lysis methods and their effects on downstream molecular detection is lacking. In this work, we analyzed chemical, thermal, and mechanical EV lysis methods and determined their efficiency based on EV particle concentration and immunoassay activity. We, for the first time, discovered that vortex was an efficient EV lysis method and used it for detection of surface and intravesicular markers in a highly sensitive multiplexed reverse phase immunoassay on a gold-nanoparticle-embedded membrane. In phosphate-buffered saline, detection limits up to 3 orders of magnitude lower than enzyme-linked immunosorbent assay were achieved. In spiked human plasma, detection limits as low as 7.27 × 10 4 EVs/mL were achieved, making it suitable for early diagnostics. These results demonstrated an effective pipeline for lysing and molecular analysis of EVs from complex biofluids, paving the way for their broad applications in biomedicine.

Extracellular vesicles (EVs), especially those derived from stem cells, have emerged as a novel treatment for promoting wound healing in regenerative medicine. However, the clinical application of mammalian cells-derived EVs is hindered by their high cost and low yields. Inspired by the ability of EVs to mediate interkingdom communication, we explored the therapeutic potential of EVs released by the probiotic strain Lactobacillus rhamnosus GG (LGG) in skin wound healing and elucidated the underlying mechanism involved. Using full-thickness skin wound-healing mouse models, we found that LGG-EVs accelerated wound healing procedures, including increased re-epithelialization and promoted angiogenesis. Using in vitro experiments, we further demonstrated that LGG-EVs boosted the proliferation and migration capacities of both epithelial and endothelial cells, as well as promoted endothelial tube formation. miRNA profiling analysis revealed that miR-21-5p was highly enriched in LGG-EVs and LGG-EV treatment significantly increased miR-21-5p level in recipient cells. Mechanically, LGG-EVs induced regulatory effects via miR-21-5p mediated metabolic signaling rewiring. Our results suggest that EVs derived from LGG could serve as a promising candidate for accelerating wound healing and possibly for treating chronic and impaired healing conditions., (© 2024. The Author(s).)

Anti-PD1 immune checkpoint blockade (ICB) has shown promising results for treating several aggressive cancers, enhancing patient survival rates. The variability in clinical response to anti-PD1 ICB is thought to be driven by patient-specific biology and heterogeneity within the tumor microenvironment. Tumor-derived extracellular vesicles (TDEVs), nano-sized particles released from tumor cells, can modulate the tumor microenvironment, leading to immunosuppression and tumor progression. Hence, TDEVs may contribute to the variability in treatment response and play a crucial role in the failure of anti-PD1 immunotherapy. In this study, we develop a systems biology approach to interrogate the role of TDEVs on the response dynamics for anti-PD1 blockade. Our results suggest that the detection and profiling of TDEVs can help screen patients for anti-PD-1 immunotherapy. Moreover, the results in this study suggest that TDEVs and IL-12 can potentially be liquid biopsy biomarkers to profile patient response to anti-PD1 ICB and tailor patient-specific treatment protocols. Importantly, the methodology is generalizable to other types of cancer immunotherapies. Therefore, the collection of patient-specific liquid biopsy data, and the implementation of those data into the systems biology framework, may offer the opportunity to discover new biomarkers for patient drug screening and enable the continuous monitoring of patient response to treatment and adaptation of patient-specific immunotherapy treatment protocols to overcome therapeutic resistance.

Diabetes mellitus (DM) is a chronic metabolic condition, characterized by hyperglycaemia, oxidative imbalance, pancreatic β-cell death, and insulin insufficiency. Angiotensin II (Ang II) increases oxidative stress, inflammation, and apoptosis, and Ang II type 1 receptor (AT1R) blockers (ARBs) can ameliorate inflammatory response and oxidative stress. However, like other small-molecule drugs, free ARBs show poor in vivo efficacy and dose-limiting toxicities. Hence, in this study, we developed nano-formulations of telmisartan (TEL), an ARB, by encapsulating it inside a murine insulinoma cell-derived extracellular vesicle (nanoTEL) and a bio-mimetic lipid nanovesicle (lipoTEL). Both nano-formulations showed spherical morphology and sustained release of TEL. In vitro , nanoTEL restored oxidative equilibrium, attenuated reactive oxygen species levels, enhanced the uptake of glucose analogue, and increased the expression of glucose transporter protein 4 better than lipoTEL. In a streptozotocin-induced murine model of diabetes, nanoTEL lowered blood glucose levels, improved glucose tolerance, and promoted insulin synthesis and secretion significantly better than lipoTEL. Moreover, nanoTEL was found superior in ameliorating the pancreatic inflammatory microenvironment by regulating NF-κBp65, HIF-1α, and PPAR-γ expression; modulating IL-1β, IL-6, tumor necrosis factor-α, IL-10, and IL-4 levels and inducing the polarization of macrophage from M1 to M2. Further, nanoTEL administration induced angiogenesis and promoted the proliferation of pancreatic cells to restore the structural integrity of the islets of Langerhans more efficiently than lipoTEL. These findings collectively suggest that nanoTEL outperforms lipoTEL in restoring the function of pancreatic β-cells by modulating the pancreatic inflammatory microenvironment and show potential for the treatment of DM.

Bovine mastitis is an infectious disease that causes substantial economic losses to the dairy industry worldwide. Current antibiotic therapy faces issues of antibiotic misuse and antimicrobial resistance, which has aroused concerns for both veterinary and human medicine. Thus, this study explored the potential of Colo EVs (bovine colostrum-derived extracellular vesicles) to address mastitis. Using LPS-induced murine mammary epithelial cells (HC11), mouse monocyte macrophages (RAW 264.7), and a murine mastitis model with BALB/C mice, we evaluated the safety and efficacy of Colo EVs, in vivo and in vitro. Colo EVs had favorable biosafety profiles, promoting cell proliferation and migration without inducing pathological changes after injection into murine mammary glands. In LPS-induced murine mastitis, Colo EVs significantly reduced inflammation, improved inflammatory scores, and preserved tight junction proteins while protecting milk production. Additionally, in vitro experiments demonstrated that Colo EVs downregulated inflammatory cytokine expression, reduced inflammatory markers, and attenuated NF-κB pathway activation. In summary, we inferred that Colo EVs have promise as a therapeutic approach for mastitis treatment, owing to their anti-inflammatory properties, potentially mediated through the NF-κB signaling pathway modulation., (© 2024. The Author(s).)

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by multiorgan involvement and complex clinical manifestations, leading to cumbersome diagnostic processes. MicroRNAs (miRNAs) in small extracellular vesicles (sEVs) have emerged as promising biomarkers for liquid biopsy. Herein, we constructed a simple multi-miRNA detection platform based on target-triggered locked hairpin DNA-functionalized Au nanoprobes (AuNP@LH) as a simple and noninvasive tool for the diagnosis and classification of SLE. The nanoprobes were prepared by modifying locked hairpin DNA designed for target miRNAs on gold nanoparticles. In the presence of target miRNAs, target-triggered hairpin assembly amplification was induced, and then fluorophore-labeled bolt DNA was released, resulting in a fluorescence signal responsive to miRNA concentration. Benefiting from the enzyme-free amplification strategy, the limits of detection (LOD) of three miRNA biomarkers for SLE were 19 pM for microRNA-146a, 66 pM for microRNA-29c, and 19 pM for microRNA-150. The proposed probes have been successfully applied to simultaneously detect multiple miRNAs in urinary sEVs from patients diagnosed with SLE and healthy controls, which exhibited good practicability in SLE diagnosis with the area under curve (AUC) of the receiver characteristic curve reaching 1.00. Furthermore, SLE patients with different disease severity can be differentiated with 81.2% accuracy. Predictably, with the advantages of low cost, rapidity, high sensitivity, and noninvasiveness, our multi-miRNA detection platform is a potential tool for multiple miRNA analysis and related clinical applications.

Extracellular vesicles (EVs) are emerging as key diagnostic biomarkers due to their widespread presence in body fluids and the proteins on their surfaces, which reflect the identity and condition of their parent cells. Research has focused on detecting EVs with biosensors that target individual transmembrane proteins (TMPs) like tetraspanins. However, due to TMP heterogeneity and the formation of tetraspanin-enriched microdomains (TEMs), cotargeting multiple TMPs is a promising strategy for enhancing EV detection. In this work, we introduce a dual-antibody surface functionalization approach using surface plasmon resonance (SPR) biosensors to cotarget tetraspanins on EVs derived from mouse macrophages. The expression of EV tetraspanin markers followed the trend of CD9 > CD63 > CD81, which was consistent with the EV detection targeting their nontetraspanin partners, exhibiting LFA-1 > ICAM-1 > VCAM-1, and suggesting a differential role of tetraspanins with their associated TMPs. Cotargeting EV tetraspanins via CD81/CD63, CD81/CD9, and CD63/CD9 dual monoclonal antibody surfaces resulted in higher EV detection compared to predictions based on binding with two monoclonal antibodies against tetraspanins without cotargeting. Furthermore, the optimization of dual monoclonal antibody surface ratios to improve cotargeting effect yielded a statistically significant enhancement in the sensitivity of EV detection. These findings underscore the importance of TEMs in designing EV-based biosensing platforms to achieve optimized sensitivity in EV detection.

Critical limb ischemia (CLI) is a peripheral arterial disease resulting from chronic inflammation of vascular systems. Recent studies have shown that inhibiting macrophage inflammation has the potential to treat CLI, and extracellular vesicles (EVs) from endothelial cells can inhibit macrophage activation. However, the limited cell-targeting capabilities and rapid clearance of EVs from the injection site limit the in vivo application of the EVs. Here, we modified endothelial EVs with platelet membranes (pM/EVs) to boost the inhibition effects on macrophage inflammation and developed an injectable alginate-based collagen composite (ACC) hydrogel for localized delivery of pM/EVs (pM/EVs@ACC) for CLI treatment. We found that pM/EVs can effectively inhibit macrophage inflammation in vitro. Furthermore, pM/EVs@ACC treatment significantly promotes the recovery of limb functions, restoring the feet' blood supply and relieving inflammation. Our findings provide compelling evidence that the pM/EVs@ACC injectable system mediating delivery of pM/EVs is a promising strategy for CLI treatment.

Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) contain a set of microRNAs with regenerative and anti-inflammatory roles. Therefore, purified MSC-EVs are envisioned as a next-generation therapeutic option for a wide array of diseases. In this protocol, we report the strategy for successfully sorting EVs from the supernatant of adipose-derived MSCs (ASCs), often used in orthopedics regenerative medicine applications. First, we described the sample preparation, focusing on EV isolation and labeling steps with carboxyfluorescein succinimidyl ester (CFSE) for fluorescence detection; subsequently, we detailed the sorting process, which constitutes the main part of the protocol. In addition to the rules defined by MISEV 2023 and MIFlowCyt EV guidelines, we applied specific experimental conditions concerning nozzle size, frequency, and sheath pressure. Morphological parameters are established using beads of diameters selected to cover the theoretical range of EV size. After ASC-EVs sorting, we performed a purity check of the sorted fraction by re-analyzing it with the sorter and verifying the EV size distribution with the nanoparticle tracking analysis technique. Due to the increasing importance of EVs, having a pure population to study and characterize is becoming crucial. Here, we demonstrate a winner strategy to set up sorting to achieve this goal.

Background: Parascaris spp. represent a significant threat to equine health worldwide, particularly in foals. The long-term survival of parasites in the host necessitates persistent modulation of the host immune response. Intercellular communication achieved through the exchange of molecules via extracellular vesicles (EVs) released from the parasite could be a crucial factor in this regard. This study aimed to isolate and characterize EVs released by adult male and female Parascaris worms and conduct a proteomic analysis to identify sex-specific proteins and potential immunomodulatory factors., Methods: Live adult Parascaris worms were collected, and EVs were isolated from spent culture media using differential ultracentrifugation. Nanoparticle tracking analysis and transmission electron microscopy confirmed the size, concentration, and morphology of the isolated EVs. Proteins within the isolated EVs were analyzed using mass spectrometry-based proteomics (LC-MS/MS)., Results: Proteomic analysis revealed a total of 113 proteins in Parascaris EVs, with several proteins showing homology to known helminth exosome proteins and exhibiting immunomodulatory functions. Sex-specific differences in EV protein composition were observed, with a distinct abundance of C-type lectins in female EVs, suggesting potential sex-specific roles or regulation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed metabolic pathways shared between male and female Parascaris EVs, as well as differences in signal transduction, and cell growth and death pathways, indicating sex-specific variations., Conclusions: These findings imply that Parascaris EVs and their protein cargo are complex. This data potentially opens avenues for discovering innovative approaches to managing and understanding helminth infection., (© 2024. The Author(s).)

Tumor necrosis factor-α (TNF-α) plays a causal role in the pathogenesis of ulcerative colitis (UC), and anti-TNF-α siRNA shows great promise in UC therapy. However, delivering siRNA with site-targeted stability and therapeutic efficacy is still challenging due to the complex and dynamic intestinal microenvironment. Here, based on the functional plant-derived ginger extracellular vesicles (EVs) and porous ZIF-8 nanoparticles, we propose a novel TNF-α siRNA delivery strategy (EVs@ZIF-8@siRNA) for UC targeted therapy. Ginger EVs show strong colon and macrophage targeting, as well as robust resistance to acidic degradation in the stomach. Moreover, 6-shogaol in ginger-derived EVs displays anti-inflammatory effects, which enhance the treatment efficiency by cooperation with TNF-α siRNA. In vitro experiments reveal that ZIF-8 nanoparticles have high TNF-α siRNA loading capacity and promote siRNA escape from cellular lysosomes. In vivo experiments show that the TNF-α level is reduced more significantly in colonic tissue than other nontargeted inflammation related factors, showing a good targeting of this composite nanoparticle. Furthermore, gut microbiota sequencing results demonstrate that the nanoparticles can promote intestinal barrier repair by regulating the intestinal microbial balance and restoring the intestinal health of UC mice. Therefore, the developed EVs@ZIF-8@siRNA nanoparticles may represent a novel colon-targeted oral drug, providing a promising therapeutic strategy for UC therapy.

Milk-derived extracellular vesicles (mEVs) are beneficial to the health of infants. However, the effect of mEVs on early intestinal inflammation is not well established. Herein, weaned colitic mice were used to explore the potential effects and underlying mechanisms of porcine mEVs (pmEVs) on intestinal inflammation during early life. We found that pmEVs administration attenuated early life intestinal inflammation and promoted colonic barrier integrity in mice. The anti-inflammatory effect of pmEVs was achieved by shifting a proinflammatory macrophage (M1) toward an anti-inflammatory macrophage (M2). Moreover, pmEVs can be absorbed by macrophages and reduce proinflammatory polarization (stimulated by LPS) in vitro. Noteworthily, ssc-let- 7 c was found to be highly expressed in pmEVs that can regulate the polarization of macrophages by targeting the tensin homologue deleted on chromosome ten (PTEN), thereby activating the PI3K/Akt pathway. Collectively, our findings revealed a crucial role of mEVs in early intestinal immunity and its underlying mechanism.

Extracellular vesicles (EVs) are a heterogeneous collection of particles that play a crucial role in cell-to-cell communication, primarily due to their ability to transport molecules, such as proteins. Thus, profiling EV-associated proteins offers insight into their biological effects. EVs can be isolated from various biological fluids, including donor blood components such as cryoprecipitate and fresh frozen plasma (FFP). In this study, we conducted a proteomic analysis of five single donor units of cryoprecipitate, FFP, and EVs derived from these blood components using a quantitative mass spectrometry approach. EVs were successfully isolated from both cryoprecipitate and FFP based on community guidelines. We identified and quantified approximately 360 proteins across all sample groups. Principal component analysis and heatmaps revealed that both cryoprecipitate and FFP are similar. Similarly, EVs derived from cryoprecipitate and FFP are comparable. However, they differ between the originating fluids and their derived EVs. Using the R-package MS-DAP, differentially expressed proteins (DEPs) were identified. The DEPs for all comparisons, when submitted for gene enrichment analysis, are involved in the complement and coagulation pathways. The protein profile generated from this study will have important clinical implications in increasing our knowledge of the proteins that are associated with EVs derived from blood components.

Extracellular vesicles (EVs) have excellent biocompatibility and long retention times in the circulation and have consequently been expected to be useful as drug-delivery systems. However, their applications have been limited because of the inability to introduce hydrophobic compounds to EVs without the use of harmful organic solvents. Herein, we developed an organic-solvent-free drug-loading technique based on the host exchange reaction. We demonstrated that the exchange reaction enabled quantitative loading of EVs with highly concentrated (0.1 mM) hydrophobic fullerene derivatives. Fullerene derivative-loaded EVs (EVs/C 60 ) could eliminate cancer cell lines more efficiently than fullerene derivative-loaded liposomes (Lip/C 60 ). Moreover, the photodynamic activity of EVs/C 60 was fivefold higher than that of the clinically available photosensitizer photofrin. EVs/C 60 could efficiently suppress tumor growth in tumor-xenograft model mice.

Extracellular vesicles (EVs) are valuable targets for liquid biopsy. However, attempts to introduce EV-based biomarkers into clinical practice have not been successful to the extent expected. One of the reasons for this failure is the lack of reliable methods for EV baseline purification from complex biofluids, such as cell-free plasma or serum. Because available one-step approaches for EV isolation are insufficient to purify EVs, the majority of studies on clinical samples were performed either on a mixture of EVs and lipoproteins, whilst the real number of EVs and their individual specific biomarker content remained elusive, or on a low number of samples of sufficient volume to allow elaborate 2-step EV separation by size and density, resulting in a high purity but utmost low recovery. Here we introduce Fast Protein Liquid Chromatography (FPLC) using Superose 6 as a matrix to obtain small EVs from biofluids that are almost free of soluble proteins and lipoproteins. Along with the estimation of a realistic number of small EVs in human samples, we show temporal resolution of the effect of the duration of postprandial phase on the proportion of lipoproteins in purified EVs, suggesting acceptable time frames additionally to the recommendation to use fasting samples for human studies. Furthermore, we assessed a potential value of pure EVs for liquid biopsy, exemplarily examining EV- and tumour-biomarkers in pure FPLC-derived fractions isolated from the serum of patients with pancreatic cancer. Consistent among different techniques, showed the presence of diseases-associated biomarkers in pure EVs, supporting the feasibility of using single-vesicle analysis for liquid biopsy., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Extracellular vesicles (EVs) are membrane-enclosed particles that have attracted much attention for their potential in disease diagnosis and therapy. However, the clinical translation is limited by the dosing consistency due to their heterogeneity. Among various characterization techniques, nanoparticle tracking analysis (NTA) offers distinct benefits for EV characterization. In this review, we will discuss the NTA technique with a focus on factors affecting the results; then, we will review the two modes of the NTA techniques along with suitable applications in specific areas of EV studies. EVs are typically characterized by their size, size distribution, concentration, protein markers, and RNA cargos. The light-scattering mode of NTA offers accurate size, size distribution, and concentration information in solution, which is useful for comparing EV isolation methods, storage conditions, and EV secretion conditions. In contrast, fluorescent mode of NTA allows differentiating EV subgroups based on specific markers. The success of fluorescence NTA heavily relies on fluorescent tags (e.g., types of dyes and labeling methods). When EVs are labeled with disease-specific markers, fluorescence NTA offers an effective tool for disease detection in biological fluids, such as saliva, blood, and serum. Finally, we will discuss the limitations and future directions of the NTA technique in EV characterization.

Immunofluorescence analysis of individual extracellular vesicles (EVs) in common fluorescence microscopes is gaining popularity due to its accessibility and high fluorescence sensitivity; however, EV number and size are only measurable using fluorescent stains requiring extensive sample manipulations. Here we introduce highly sensitive label-free EV size photometry (SP) based on interferometric scattering (iSCAT) imaging of immersed EVs immobilized on a glass coverslip. We implement SP on a common inverted epifluorescence microscope with LED illumination and a simple 50:50 beamsplitter, permitting seamless integration of SP with fluorescence imaging (SPFI). We present a high-throughput SPFI workflow recording >10,000 EVs in 7 min over ten 88 × 88 µm 2 fields of view, pre- and post-incubation imaging to suppress background, along with automated image alignment, aberration correction, spot detection and EV sizing. We achieve an EV sizing range from 37 to ∼220 nm in diameter with a dual 440 and 740 nm SP illumination scheme, and suggest that this range can be extended by more advanced image analysis or additional hardware customization. We benchmark SP to flow cytometry using calibrated silica nanoparticles and demonstrate superior, label-free sensitivity. We showcase SPFI's potential for EV analysis by experimentally distinguishing surface and volumetric EV dyes, observing the deformation of EVs adsorbed to a surface, and by uncovering distinct subpopulations in <100 nm-in-diameter EVs with fluorescently tagged membrane proteins., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

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., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Stem cell derived small extracellular vesicles (sEVs) have emerged as promising nanomaterials for the repair of bone defects. However, low retention of sEVs affects their therapeutic effects. Clinically used natural substitute inorganic bovine bone mineral (Bio-Oss) bone powder lacks high compactibility and efficient osteo-inductivity that limit its clinical application in repairing large bone defects. In this study, a poly ethylene glycol/hyaluronic acid (PEG/HA) hydrogel was used to stabilize Bio-Oss and incorporate rat bone marrow stem cell-derived sEVs (rBMSCs-sEVs) to engineer a PEG/HA-Bio-Oss (PEG/HA-Bio) composite scaffold. Encapsulation and sustained release of sEVs in hydrogel scaffold can enhance the retention of sEVs in targeted area, achieving long-lasting repair effect. Meanwhile, synergistic administration of sEVs and Bio-Oss in cranial defect can improve therapeutic effects. The PEG/HA-Bio composite scaffold showed good mechanical properties and biocompatibility, supporting the growth of rBMSCs. Furthermore, sEVs enhanced in vitro cell proliferation and osteogenic differentiation of rBMSCs. Implantation of sEVs/PEG/HA-Bio in rat cranial defect model promoted in vivo bone regeneration, suggesting the great potential of sEVs/PEG/HA-Bio composite scaffold for bone repair and regeneration. Overall, this work provides a strategy of combining hydrogel composite scaffold systems and stem cell-derived sEVs for the application of tissue engineering repair., (© 2024 IOP Publishing Ltd. All rights, including for text and data mining, AI training, and similar technologies, are reserved.)

The individual detection of human immunodeficiency virus (HIV) virions and resolution from extracellular vesicles (EVs) during analysis is a difficult challenge. Infectious enveloped virions and nonviral EVs are released simultaneously by HIV-infected host cells, in addition to hybrid viral EVs containing combinations of HIV and host components but lacking replicative ability. Complicating the issue, EVs and enveloped virions are both delimited by a lipid bilayer and share similar size and density. The feature that distinguishes infectious virions from host and hybrid EVs is the HIV genomic RNA (gRNA), which allows the virus to replicate. Single-particle analysis techniques, which provide snapshots of single biological nanoparticles, could resolve infectious virions from EVs. However, current single-particle analysis techniques focus mainly on protein detection, which fail to resolve hybrid EVs from infectious virions. A method to simultaneously detect viral protein and internal gRNA in the same particle would allow resolution of infectious HIV from EVs and noninfectious virions. Here, we introduce SPIRFISH, a high-throughput method for single-particle protein and RNA analysis, combining single particle interferometric reflectance imaging sensor with single-molecule fluorescence in situ hybridization. Using SPIRFISH, we detect HIV-1 envelope protein gp120 and genomic RNA within single infectious virions, allowing resolution against EV background and noninfectious virions. We further show that SPIRFISH can be used to detect specific RNAs within EVs. This may have major utility for EV therapeutics, which are increasingly focused on EV-mediated RNA delivery. SPIRFISH should enable single particle analysis of a broad class of RNA-containing nanoparticles.

Tumor-derived small extracellular vesicle (sEV) microRNAs (miRNAs) are emerging biomarkers for cancer diagnostics. Conventional sEV miRNA detection methods necessitate the lysis of sEVs, rendering them laborious and time-consuming and potentially leading to damage or loss of miRNAs. Membrane fusion-based in situ detection of sEV miRNAs involves the preparation of probe-loaded vesicles ( e.g. , liposomes or cellular vesicles), which are typically sophisticated and require specialist equipment. Membrane perforation methods employ chemical treatments that can induce severe miRNA degradation or leaks. Inspired by previous studies that loaded nucleic acids into EVs or cells using hydrophobic tethers for therapeutic applications, herein, we repurposed this strategy by conjugating a hydrophobic tether onto molecular beacons to aid their transportation into sEVs, allowing for in situ detection of miRNAs in a fusion-free and multiplexing manner. This method enables simultaneous detection of multiple miRNA species within serum-derived sEVs for the diagnosis of prostate cancer, breast cancer, and gastric cancer with an accuracy of 83.3%, 81.8%, and 100%, respectively, in a cohort of 66 individuals, indicating that it holds a high application potential in clinical diagnostics.

Imaging flow cytometry (IFCM) is a technique that can detect, size, and phenotype extracellular vesicles (EVs) at high throughput (thousands/minute) in complex biofluids without prior EV isolation. However, the generated signals are expressed in arbitrary units, which hinders data interpretation and comparison of measurement results between instruments and institutes. While fluorescence calibration can be readily achieved, calibration of side scatter (SSC) signals presents an ongoing challenge for IFCM. Here, we present an approach to relate the SSC signals to particle size for IFCM, and perform a comparability study between three different IFCMs using a plasma EV test sample (PEVTES). SSC signals for different sizes of polystyrene (PS) and hollow organosilica beads (HOBs) were acquired with a 405 nm 120 mW laser without a notch filter before detection. Mie theory was applied to relate scatter signals to particle size. Fluorescence calibration was accomplished with 2 μm phycoerythrin (PE) and allophycocyanin (APC) MESF beads. Size and fluorescence calibration was performed for three IFCMs in two laboratories. CD235a-PE and CD61-APC stained PEVTES were used as EV-containing samples. EV concentrations were compared between instruments within a size range of 100-1000 nm and a fluorescence intensity range of 3-10,000 MESF. 81 nm PS beads could be readily discerned from background based on their SSC signals. Fitting of the obtained PS bead SSC signals with Mie theory resulted in a coefficient of determination >0.99 between theory and data for all three IFCMs. 216 nm HOBs were detected with all instruments, and confirmed the sensitivity to detect EVs by SSC. The lower limit of detection regarding EV-size for this study was determined to be ~100 nm for all instruments. Size and fluorescence calibration of IFCM data increased cross-instrument data comparability with the coefficient of variation decreasing from 33% to 21%. Here we demonstrate - for the first time - scatter calibration of an IFCM using the 405 nm laser. The quality of the scatter-to-diameter relation and scatter sensitivity of the IFCMs are similar to the most sensitive commercially available flow cytometers. This development will support the reliability of EV research with IFCM by providing robust standardization and reproducibility, which are pre-requisites for understanding the biological significance of EVs., (© 2024 Cytek Biosciences, Inc. and The Author(s). Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

Rescuing or compensating mitochondrial function represents a promising therapeutic avenue for radiation-induced chronic wounds. Adult stem cell efficacies are primarily dependent on the paracrine secretion of mitochondria-containing extracellular vesicles (EVs). However, effective therapeutic strategies addressing the quantity of mitochondria and mitochondria-delivery system are lacking. Thus, in this study, we aimed to design an effective hydrogel microneedle patch (MNP) loaded with stem cell-derived mitochondria-rich EVs to gradually release and deliver mitochondria into the wound tissues and boost wound healing. We, first, used metformin to enhance mitochondrial biogenesis and thereby increasing the secretion of mitochondria-containing EVs (termed "Met-EVs") in adipose-derived stem cells. To verify the therapeutic effects of Met-EVs, we established an in vitro and an in vivo model of X-ray-induced mitochondrial dysfunction. The Met-EVs ameliorated the mitochondrial dysfunction by rescuing mitochondrial membrane potential, increasing adenosine 5'-triphosphate levels, and decreasing reactive oxygen species production by transferring active mitochondria. To sustain the release of EVs into damaged tissues, we constructed a Met-EVs@Decellularized Adipose Matrix (DAM)/Hyaluronic Acid Methacrylic Acid (HAMA)-MNP. Met-EVs@DAM/HAMA-MNP can load and gradually release Met-EVs and their contained mitochondria into wound tissues to alleviate mitochondrial dysfunction. Moreover, we found Met-EVs@DAM/HAMA-MNP can markedly promote macrophage polarization toward the M2 subtype with anti-inflammatory and regenerative functions, which can, in turn, enhance the healing process in mice with skin wounds combined radiation injuries. Collectively, we successfully fabricated a delivery system for EVs, Met-EVs@DAM/HAMA-MNP, to effectively deliver stem cell-derived mitochondria-rich EVs. The effectiveness of this system has been demonstrated, holding great potential for chronic wound treatments in clinic.

The biosafety concerns associated with fecal microbiota transplant (FMT) limit their clinical application in treating ulcerative colitis (UC). Gut microbiota secrete abundant extracellular vesicles (Gm-EVs), which play a critical role in bacteria-to-bacteria and bacteria-to-host communications. Herein, intestinal microbiota are trained using tea leaf lipid/pluronic F127-coated curcumin nanocrystals (CN@Lp 127 s), which can maintain stability during transit through the gastrointestinal tract. Compared with FMT, Gm-EVs derived from healthy mice significantly improve treatment outcomes against UC by reducing colonic inflammatory responses, restoring colonic barrier function, and rebalancing intestinal microbiota. Strikingly, Gm-EVs obtained from CN@Lp 127 -trained healthy mice exhibit a superior therapeutic effect on UC compared to groups receiving FMT from healthy mice, Gm-EVs from healthy mice, and FMT from CN@Lp 127 -trained healthy mice. Oral administration of Gm-EVs from CN@Lp 127 -trained healthy mice not only alleviates colonic inflammation, promotes mucosal repair, and regulates gut microbiota but also regulates purine metabolism to decrease the uric acid level, resulting in a robust improvement in the UC. This study demonstrates the UC therapeutic efficacy of Gm-EVs derived from nanomedicine-trained gut microbiota in regulating the immune microenvironment, microbiota, and purine metabolism of the colon. These EVs provide an alternative platform to replace FMT as a treatment for UC., (© 2024 Wiley‐VCH GmbH.)

Milk-derived extracellular vesicles (M-EVs) are low-cost, can be prepared in large quantities, and can cross the gastrointestinal barrier for oral administration. However, the composition of milk is complex, and M-EVs obtained by different extraction methods may affect their oral delivery. Based on this, a new method for extracting M-EVs based on cryogenic freezing treatment (Cryo-M-EVs) is proposed and compared with the previously reported acetic acid treatment (Acid-M-EVs) method and the conventional ultracentrifugation method (Ulltr-M-EVs). The new method simplifies the pretreatment step and achieves 25-fold and twofold higher yields than Acid-M-EVs and Ulltr-M-EVs. And it is interesting to note that Cryo-M-EVs and Acid-M-EVs have higher cellular uptake efficiency, and Cryo-M-EVs present the best transepithelial transport effect. After oral administration of the three M-EVs extracted by three methods in mice, Cryo-M-EVs effectively successfully cross the gastrointestinal barrier and achieve hepatic accumulation, whereas Acid-M-EVs and Ultr-M-EVs mostly reside in the intestine. The M-EVs obtained by the three extraction methods show a favorable safety profile at the cellular as well as animal level. Therefore, when M-EVs obtained by different extraction methods are used for oral drug delivery, their accumulation properties at different sites can be utilized to better deal with different diseases., (© 2024 Wiley‐VCH GmbH.)

Acoustofluidic technologies that integrate acoustic waves and microfluidic chips have been widely used in bioparticle manipulation. As a representative technology, acoustic tweezers have attracted significant attention due to their simple manufacturing, contact-free operation, and low energy consumption. Recently, acoustic tweezers have enabled the efficient and smart manipulation of biotargets with sizes covering millimeters (such as zebrafish) and nanometers (such as DNA). In addition to acoustic tweezers, other related acoustofluidic chips including acoustic separating, mixing, enriching, and transporting chips, have also emerged to be powerful platforms to manipulate micro/nano bioparticles (cells in blood, extracellular vesicles, liposomes, and so on). Accordingly, some interesting applications were also developed, such as smart sensing. In this review, we firstly introduce the principles of acoustic tweezers and various related technologies. Second, we compare and summarize recent applications of acoustofluidics in bioparticle manipulation and sensing. Finally, we outlook the future development direction from the perspectives such as device design and interdisciplinary., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2024. Published by Elsevier B.V.)

Reconstruction of a full-thickness spongy urethra is difficult because a corpus spongiosum (CS) defect cannot be repaired using self-healing or substitution urethroplasty. Small extracellular vesicles (sEVs) secreted by urine-derived stem cells (USC-sEVs) strongly promote vascular regeneration. In this study, it is aimed to explore whether USC-sEVs promote the repair of CS defects. To prolong the in vivo effects of USC-sEVs, a void-forming photoinduced imine crosslinking hydrogel (vHG) is prepared and mixed with the USC-sEV suspension. vHG encapsulated with USC-sEVs (vHG-sEVs) is used to repair a CS defect with length of 1.5 cm and width of 0.8 cm. The results show that vHG-sEVs promote the regeneration and repair of CS defects. Histological analysis reveals abundant sinusoid-like vascular structures in the vHG-sEV group. Photoacoustic microscopy indicates that blood flow and microvascular structure of the defect area in the vHG-sEV group are similar to those in the normal CS group. This study confirms that the in situ-formed vHG-sEV patch appears to be a valid and promising strategy for repairing CS defects., (© 2024 Wiley‐VCH GmbH.)

Extracellular vesicles (EVs) have emerged as a promising drug delivery system. Connectosomes are a specialized type of EVs that contain connexins in their membranes. Connexin is a surface transmembrane protein that forms connexin hemichannels. When a connexin hemichannel on a connectosome docks with another connexin hemichannel of a target cell, they form a gap junction that allows direct intracellular delivery of therapeutic cargos from within the connectosome to the cytoplasm of the recipient cell. In the present study, we tested the feasibility of converting connectosomes into dry powders by (thin-film) freeze-drying to enable their potential storage in temperatures higher than the recommended -80 °C, while maintaining their activity. Connectosomes were isolated from a genetically engineered HeLa cell line that overexpressing connexin-43 subunit protein tagged with red fluorescence protein. To facilitate the testing of the function of the connectosomes, they were loaded with calcein green dye. Calcein green-loaded connectosomes were thin-film freeze-dried with trehalose alone or trehalose and a polyvinylpyrrolidone polymer as lyoprotectant(s) to produce amorphous powders with high glass transition temperatures (>100 °C). Thin-film freeze-drying did not significantly change the morphology and structure of the connectosomes, nor their particle size distribution. Based on data from confocal microscopy, flow cytometry, and fluorescence spectrometry, the connexin hemichannels in the connectosomes reconstituted from the thin-film freeze-dried powder remained functional, allowing the passage of calcein green through the hemichannels and the release of the calcein green from the connectosomes when the channels were opened by chelating calcium in the reconstituted medium. The function of connectosomes was assessed after one month storage at different temperatures. The connexin hemichannels in connectosomes in liquid lost their function when stored at -19.5 ± 2.2 °C or 6.0 ± 0.5 °C for a month, while those in dry powder form remained functional under the same storage conditions. Finally, using doxorubicin-loaded connectosomes, we showed that the connectosomes reconstituted from thin-film freeze-dried powder remained pharmacologically active. These findings demonstrate that (thin-film) freeze-drying represents a viable method to prepare stable and functional powders of EVs that contain connexins in their membranes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [ROW and ZC report financial support provided by TFF Pharmaceuticals, Inc. ZC reports a relationship with TFF Pharmaceuticals, Inc., that includes equity or stocks and funding. ROW reports a relationship with TFF Pharmaceuticals, Inc., that includes consulting or advisory, equity or stocks, and funding. Conflict of interest management plans are available at UT Austin]. ZC serves as the Editor of the International Journal of Pharmaceutics for North America. This paper was subject to all the Journal’s usual procedures. The peer review was handled, and the editorial decision was made independently of ZC and his research group., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The high incidence and mortality rates associated with acute and chronic wound infections impose a significant burden on global healthcare systems. In terms of the management of wound infection, the reconstruction and regeneration of skin appendages are essential for the recovery of mechanical strength and physiological function in the regenerated skin tissue. Novel therapeutic approaches are a requisite for enhancing the healing of infected wounds and promoting the regeneration of skin appendages. Herein, a novel antimicrobial microneedle patch has been fabricated for the transdermal controlled delivery of adipose tissue-derived apoptotic vesicles (ApoEVs-AT@MNP) for the treatment of infected wounds, which is expected to achieve high-quality scarless healing of the wound skin while inhibiting the bacteria in the infected wound. The microneedle patch (MNP) system possesses adequate mechanical strength to penetrate the skin, allowing the tips to remain inside tissue for continuous active release of biomolecules, and subsequently degrades safely within the host body. In vivo transplantation demonstrates that ApoEVs-AT@MNP not only inhibits bacterial proliferation in infected wounds but also significantly promotes effective and rapid scarless wound healing. Particularly noteworthy is the ability of ApoEVs-AT@MNP to promote the rapid formation of mature, evenly arranged hair follicles in infected wounds, observed as early as 8 days following implantation, which is essential for the restoration of skin function. This rapid development of skin appendages has not been reported this early in previous studies. Therefore, ApoEVs-AT@MNP has emerged as an excellent, painless, non-invasive, and highly promising treatment for infected wounds., (© 2024. The Author(s).)

Here, we present a protocol for the direct isolation of small extracellular vesicles (sEVs) from the spleen of preclinical murine models of leukemia using ultracentrifugation. We describe steps for tissue collection, sample preparation, ultracentrifugation-based isolation, and sEV characterization. This protocol allows for efficient enrichment of both leukemia and its microenvironment-derived sEV (LME-sEV), providing a valuable tool for studying their composition and functional roles. Potential applications include investigating the role of sEV in leukemia progression and identifying biomarkers. For complete details on the use and execution of this protocol, please refer to Gargiulo et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Extracellular vesicles (EVs) are membranous nanoparticles classified based on their size and surface markers, which can be specific to various cell origins. Here, we present a protocol for the isolation of pulmonary-specific EVs in mice. We describe steps for differential centrifugation, density gradient centrifugation, and commercially available polyethylene glycol(PEG)-based precipitation, employing pulmonary-specific EV-bound chemicals and antibodies. We then detail procedures for the characterization of these EVs through nanoparticle tracking analysis, flow cytometry, scanning electron microscopy, and transmission electron microscopy. For complete details on the use and execution of this protocol, please refer to Lee et al. 1 , 2 , 3 , 4 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Small extracellular vesicles (sEVs) are lipid bilayer-enclosed particles secreted by living cells. Here, we present a protocol for the collection and isolation of sEVs derived from human umbilical cord mesenchymal stem cells (hucMSCs). We describe steps for characterizing their morphology and integrity by transmission electron microscopy (TEM) and size distribution using nanoparticle tracking analysis (NTA) and an atomic force microscope (AFM). We then detail procedures for assessing nanoparticle size analysis and molecular markers by western blotting and Flow NanoAnalyzer., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Synthetic liposomes are widely used as drug delivery vehicles in biomedical treatments, such as for mRNA-based antiviral vaccines like those recently developed against SARS-CoV-2. Extracellular vesicles (EVs), which are naturally produced by cells, have emerged as a next-generation delivery system. However, key questions regarding their origin within cells remain unresolved. In this regard, plasma membrane vesicles (PMVs), which are essentially produced from the cellular plasma membrane (PM), present a promising alternative. Unfortunately, their properties relevant to biomedical applications have not be extensively studied. Therefore, we conducted a thorough investigation of the methods used in the production of PMVs. By leveraging advanced fluorescence techniques in microscopy and flow cytometry, we demonstrated a strong dependence of the physicochemical attributes of PMVs on the chemicals used during their production. Following established protocols employing chemicals such as paraformaldehyde (PFA), N- ethylmaleimide (NEM) or dl-dithiothreitol (DTT) and by developing a modified NEM-based method that involved a hypotonic shock step, we generated PMVs from THP-1 CD1d cells. We systematically compared key parameters such as vesicle output, their size distribution, vesicular content analysis, vesicular membrane lipid organization and the mobility of a transmembrane protein. Our results revealed distinct trends: PMVs isolated using NEM-based protocols closely resembled natural vesicles, whereas PFA induced significant molecular cross-linking, leading to notable changes in the biophysical properties of the vesicles. Furthermore, our novel NEM protocol enhanced the efficiency of PMV production. In conclusion, our study highlights the unique characteristics of chemically produced PMVs and offers insights into their potentially diverse yet valuable biological functions.

Intrauterine adhesion (IUA), a prevalent etiology of female infertility, is attributed to endometrial damage. However, conventional therapeutic interventions for IUA are plagued by high recurrence rates. Human umbilical cord mesenchymal stem cell-derived extracellular vesicles (hUCMSC-EVs) demonstrate the promising therapeutic effects on IUA, but the current efficacy of extracellular vesicles (EVs) is hindered by lower retention and bioavailability. In this study, a thermosensitive hydrogel was utilized as a prolonged release carrier to improve the retention and bioavailability of hUCMSC-EVs in IUA treatment. The hydrogel-EVs complex effectively prolonged EVs retention in human endometrial stromal cells and an IUA mouse model. The complex exhibited superior protection against cellular injury, significantly alleviated endometrial damage, inhibited fibrosis, suppressed inflammation, and improved fertility compared to EVs alone. The results indicated that thermosensitive hydrogel enhanced the therapeutic capacity of EVs for IUA by prolonging their retention in the uterine environment. The hydrogel-EVs complex provides a novel strategy for the sustained release of hUCMSC-EVs in the treatment of IUA., (© 2024. The Author(s).)