Publications

Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. While −80◦C storage is a widespread approach, some authors proposed improved storage strategies with conflicting results. Here, we designed a systematic study to assess the impact of −80◦C storage and freeze-thaw cycles on EVs. We tested the differences among eight storage strategies and investigated the possible fusion phenomena occurring during storage. EVs were collected from human plasma and murine microglia culture by size exclusion chromatography and ultracentrifugation, respectively. The analysis included: concentration, size and zeta potential (tunable resistive pulse sensing), contaminant protein assessment; flow cytometry for the analysis of two single fluorescent-tagged EVs populations (GFP and mCherry), mixed before preservation. We found that −80◦C storage reduces EVs concentration and sample purity in a time-dependent manner. Furthermore, it increases the particle size and size variability and modifies EVs zeta potential, with a shift of EVs in sizecharge plots. None of the tested conditions prevented the observed effects. Freezethaw cycles lead to an EVs reduction after the first cycle and to a cycle-dependent increase in particle size. With flow cytometry, after storage, we observed a significant population of double-positive EVs (GFP+-mCherry+). This observation may suggest the occurrence of fusion phenomena during storage. Our findings show a significant impact of storage on EVs samples in terms of particle loss, purity reduction and fusion phenomena leading to artefactual particles. Depending on downstream analyses and experimental settings, EVs should probably be processed from fresh, non-archival, samples in majority of cases.

Hypothesis The implementation of the proposal from the European Chemical Agency (ECHA) to restrict the use of nanoplastics (NP) and microplastics (MP) in consumer products will require reliable methods to perform size and mass-based concentration measurements. Analytical challenges arise at the nanometre to micrometre interface, e.g., 800 nm–10 µm, where techniques applicable at the nanometre scale reach their upper limit of applicability and approaches applicable at the micrometre scale must be pushed to their lower limits of detection. Experiments Herein, we compared the performances of nine analytical techniques by measuring the particle size distribution and mass-based concentration of polystyrene mixtures containing both nano and microparticles, with the educational aim to underline applicability and limitations of each technique. Findings Light scattering-based measurements do not have the resolution to distinguish multiple populations in polydisperse samples. Nanoparticle tracking analysis (NTA), nano-flowcytometry (nFCM) and asymmetric flow field flow fractionation hyphenated with multiangle light scattering (AF4-MALS) cannot measure particles in the micrometre range. Static light scattering (SLS) is not able to accurately detect particles below 200 nm, and similarly to transmission electron microscopy (TEM) and flow cytometry (FCM), is not suitable for accurate mass-based concentration measurements. Alternatives for high-resolution sizing and concentration measurements in the size range between 60 nm and 5 µm are tunable resistive pulse sensing (TRPS) and centrifugal liquid sedimentation (CLS), that can bridge the gap between the nanometre and micrometre range.

The measurement of physicochemical properties of polydisperse complex biological samples, for example, extracellular vesicles, is critical to assess their quality, for example, resulting from their production and isolation methods. The community is gradually becoming aware of the need to combine multiple orthogonal techniques to perform a robust characterization of complex biological samples. Three pillars of critical quality attribute characterization of EVs are sizing, concentration measurement and phenotyping. The repeatable measurement of vesicle concentration is one of the key‐challenges that requires further efforts, in order to obtain comparable results by using different techniques and assure reproducibility. In this study, the performance of measuring the concentration of particles in the size range of 50–300 nm with complementary techniques is thoroughly investigated in a step‐by step approach of incremental complexity. The six applied techniques include multi‐angle dynamic light scattering (MADLS), asymmetric flow field flow fractionation coupled with multi‐angle light scattering (AF4‐MALS), centrifugal liquid sedimentation (CLS), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), and high‐sensitivity nano flow cytometry (nFCM). To achieve comparability, monomodal samples and complex polystyrene mixtures were used as particles of metrological interest, in order to check the suitability of each technique in the size and concentration range of interest, and to develop reliable post‐processing data protocols for the analysis. Subsequent complexity was introduced by testing liposomes as validation of the developed approaches with a known sample of physicochemical properties closer to EVs. Finally, the vesicles in EV containing plasma samples were analysed with all the tested techniques. The results presented here aim to shed some light into the requirements for the complex characterization of biological samples, as this is a critical need for quality assurance by the EV and regulatory community. Such efforts go with the view to contribute to both, set‐up reproducible and reliable characterization protocols, and comply with the Minimal Information for Studies of Extracellular Vesicles (MISEV) requirements.

Within this study, the performance and limitations of tunable resistive pulse sensing (TRPS) was evaluated to characterize submicron particles in unstressed and heat stressed monoclonal antibody (mAb) solutions. These were compared with microfluidic resistive pulse sensing (MRPS), resonant mass measurement (RMM), and nanoparticle tracking analysis (NTA). For TRPS and MRPS measurements, an adjustment of ionic strength was required to achieve suitable measurement conditions. The addition of electrolytes is potentially critical for protein formulations and therefore the effect of salt concentration and pH on submicron particle levels was further investigated. Heat stress caused a sharp increase in particle levels between 250-900 nm, observable by all four techniques. Due to reduced colloidal stability, indicated by increased attractive forces and reduced aggregation onset temperatures in the presence of sodium chloride, protein aggregation was observed in heat stressed mAb only after the addition of sodium chloride. Achieving adequate ionic strength by replacing sodium chloride with other electrolytes similarly resulted in reduced colloidal stability and protein aggregation. It is recommended that protein samples prone for aggregation in the presence of high ionic strength should not be analyzed by RPS measurements after the addition of electrolytes. However, protein samples containing already required ionic strength can be analyzed by any of the four techniques.

Virus-like particles (VLPs) are excellent platforms for the development of influenza vaccine candidates. Nonetheless, their characterization is challenging due to VLPs’ unique biophysical and biochemical properties. To cope with such complexity, multiple analytical techniques have been developed to date (e.g., single-particle analysis, thermal stability, or quantification assays), most of which are rarely used or have been successfully demonstrated for being applicable for virus particle characterization. In this study, several biophysical and biochemical methods have been evaluated for thorough characterization of monovalent and pentavalent influenza VLPs from diverse groups (A and B) and subtypes (H1 and H3) produced in insect cells using the baculovirus expression vector system (IC-BEVS). Particle size distribution and purity profiles were monitored during the purification process using two complementary technologies — nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS). VLP surface charge at the selected process pH was also assessed by this last technique. The morphology of the VLP (size, shape, and presence of hemagglutinin spikes) was evaluated using transmission electron microscopy. Circular dichroism was used to assess VLPs’ thermal stability. Total protein, DNA, and baculovirus content were also assessed. All VLPs analyzed exhibited similar size ranges (90–115 nm for NTA and 129–141 nm for TRPS), surface charges (average of −20.4 mV), and morphology (pleomorphic particles resembling influenza virus) exhibiting the presence of HA molecules (spikes) uniformly displayed on M1 protein scaffold. Our data shows that HA titers and purification efficiency in terms of impurity removal and thermal stability were observed to be particle dependent. This study shows robustness and generic applicability of the tools and methods evaluated, independent of VLP valency and group/subtype. Thus, they are most valuable to assist process development and enhance product characterization.

With the approval of the first siRNA-based drugs, non-viral siRNA delivery has gained special interest in industry and academia in the last two years. For non-viral delivery, positively charged lipid and polymer formulations play a central role in research and development. However, nanoparticle size characterization, particularly of polydisperse formulations, can be very challenging. Tunable resistive pulse sensing for particle by particle measurements of size, polydispersity, zeta potential and a direct concentration promises better assessment of nanoparticle formulations. However, the current application is not optimized for positively charged particles. A supplier-provided coating solution for difficult-to-measure samples does not allow for successful measurements of positively charged nanoparticles. This article describes a new coating solution based on choline-chloride. Coating is verified by current–voltage (I-V) recordings and ultimately tested on a positively charged nanoparticle formulation comprising of siRNA and PEG-PCL-PEI polymer. This coating allows successful size, polydispersity index (PDI) and concentration measurement by tunable resistive pulse sensing of positively charged PEI-based polyplexes. This article provides the foundation for further characterization of polyplexes as well as other positively charged nanoparticle formulations based on particle by particle measurements.

Tunable resistive pulse sensing (TRPS, qNano Gold, IZON Ltd.) was investigated as a method to quantify submicron particles (SMPs) between 0.1 and 1 µm in solutions of biopharmaceuticals. To reduce sample dilution, a spiking-in approach was used to add the appropriate amount of electrolytes required for the measurement. For correct particle quantification, an electrolyte concentration of at least 50 mM sodium chloride was needed. Intra- and inter-nanopore variability were below 5% for size and below 10% for concentration measurements when analyzing polystyrene standard beads. Submicron particle counts in a stir stressed IgG1 monoclonal antibody formulation resulted in a non-symmetrical, almost bell-shaped size distribution with a maximum at 250 nm when using a NP300 nanopore (IZON Ltd.). It was shown that particle counts are heavily underestimated below 250 nm, and therefore it is recommended to quantify particle counts by TRPS in samples with heterogeneous particle size distributions (e.g., biopharmaceuticals) only starting from the maximum of the histogram towards the upper limit of detection.

Proteomic analysis of small extracellular vesicles (sEVs) poses a significant challenge. A ‘gold-standard’ method for plasma sEV enrichment for downstream proteomic analysis is yet to be established. Methods were evaluated for their capacity to successfully isolate and enrich sEVs from plasma, minimise the presence of highly abundant plasma proteins, and result in the optimum representation of sEV proteins by liquid chromatography tandem mass spectrometry. Plasma from four cattle (Bos taurus) of similar physical attributes and genetics were used. Three methods of sEV enrichment were utilised: ultracentrifugation (UC), size-exclusion chromatography (SEC), and ultrafiltration (UF). These methods were combined to create four groups for methodological evaluation: UC + SEC, UC + SEC + UF, SEC + UC and SEC + UF. The UC + SEC method yielded the highest number of protein identifications (IDs). The SEC + UC method reduced plasma protein IDs compared to the other methods, but also resulted in the lowest number of protein IDs overall. The UC + SEC + UF method decreased sEV protein ID, particle number, mean and mode particle size, particle yield, and did not improve purity compared to the UC + SEC method. In this study, the UC + SEC method was the best method for sEV protein ID, purity, and overall particle yield. Our data suggest that the method and sequence of sEV enrichment strategy impacts protein ID, which may influence the outcome of biomarker discovery studies.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. Despite several emerging vaccines, there remains no verifiable therapeutic targeted specifically to the virus. Here we present a highly effective small interfering RNA (siRNA) therapeutic against SARS-CoV-2 infection using a novel lipid nanoparticle (LNP) delivery system. Multiple siRNAs targeting highly conserved regions of the SARS-CoV2 virus were screened, and three candidate siRNAs emerged that effectively inhibit the virus by greater than 90% either alone or in combination with one another. We simultaneously developed and screened two novel LNP formulations for the delivery of these candidate siRNA therapeutics to the lungs, an organ that incurs immense damage during SARS-CoV-2 infection. Encapsulation of siRNAs in these LNPs followed by in vivo injection demonstrated robust repression of virus in the lungs and a pronounced survival advantage to the treated mice. Our LNP-siRNA approaches are scalable and can be administered upon the first sign of SARS-CoV-2 infection in humans. We suggest that an siRNA-LNP therapeutic approach could prove highly useful in treating COVID-19 disease as an adjunctive therapy to current vaccine strategies.

Metastatic spread of a cancer to secondary sites is a coordinated, non-random process. Cancer cell-secreted vesicles, especially exosomes, have recently been implicated in the guidance of metastatic dissemination, with specific surface composition determining some aspects of organ-specific localization. Nevertheless, whether the tumor microenvironment influences exosome biodistribution has yet to be investigated. Here, we show that microenvironmental cytokines, particularly CCL2, decorate cancer exosomes via binding to surface glycosaminoglycan side chains of proteoglycans, causing exosome accumulation in specific cell subsets and organs. Exosome retention results in changes in the immune landscape within these organs, coupled with a higher metastatic burden. Strikingly, CCL2-decorated exosomes are directed to a subset of cells that express the CCL2 receptor CCR2, demonstrating that exosome-bound cytokines are a crucial determinant of exosome-cell interactions. In addition to the finding that cytokine-conjugated exosomes are detected in the blood of cancer patients, we discovered that healthy subjects derived exosomes are also associated with cytokines. Although displaying a different profile from exosomes isolated from cancer patients, it further indicates that specific combinations of cytokines bound to exosomes could likewise affect other physiological and disease settings.

Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells.

Ionizing radiation (IR)-induced bystander effects contribute to biological responses to radiation, and extracellular vesicles (EVs) play important roles in mediating these effects. In this study we investigated the role of bone marrow (BM)-derived EVs in the bystander transfer of radiation damage. Mice were irradiated with 0.1Gy, 0.25Gy and 2Gy, EVs were extracted from the BM supernatant 24 h or 3 months after irradiation and injected into bystander mice. Acute effects on directly irradiated or EV-treated mice were investigated after 4 and 24 h, while late effects were investigated 3 months after treatment. The acute effects of EVs on the hematopoietic stem and progenitor cell pools were similar to direct irradiation effects and persisted for up to 3 months, with the hematopoietic stem cells showing the strongest bystander responses. EVs isolated 3 months after irradiation elicited no bystander responses. The level of seven microRNAs (miR-33a-3p, miR-140-3p, miR-152-3p, miR-199a-5p, miR-200c-5p, miR-375-3p and miR-669o-5p) was altered in the EVs isolated 24 hour but not 3 months after irradiation. They regulated pathways highly relevant for the cellular response to IR, indicating their role in EV-mediated bystander responses. In conclusion, we showed that only EVs from an early stage of radiation damage could transmit IR-induced bystander effects.

Background Subclinical mastitis, the inflammation of the mammary gland lacking clinical symptoms, is one of the most prevalent and costly diseases in dairy farming worldwide. Milk microRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) have been proposed as potential biomarkers of different mammary gland conditions, including subclinical mastitis. However, little is known about the robustness of EVs analysis regarding sampling time-point and natural infections. To estimate the reliability of EVs measurements in raw bovine milk, we first evaluated changes in EVs size and concentration using Tunable Resistive Pulse Sensing (TRPS) during three consecutive days of sampling. Then, we analysed daily differences in miRNA cargo using small RNA-seq. Finally, we compared milk EVs differences from naturally infected udder quarters with their healthy adjacent quarters and quarters from uninfected udders, respectively. Results We found that the milk EV miRNA cargo was very stable over the course of three days regardless of the health status of the quarter, and that infected quarters did not induce relevant changes in milk EVs of adjacent healthy quarters. Chronic subclinical mastitis induced changes in milk EV miRNA cargo, but neither in EVs size nor concentration. We observed that the changes in immunoregulatory miRNAs in quarters with chronic subclinical mastitis were cow-individual, however, the most upregulated miRNA was bta-miR-223-3p across all individuals. Conclusions Our results showed that the miRNA profile and particle size characteristics remained constant throughout consecutive days, suggesting that miRNAs packed in EVs are physiological state-specific. In addition, infected quarters were solely affected while adjacent healthy quarters remained unaffected. Finally, the cow-individual miRNA changes pointed towards infection-specific alterations.

Background: Exosomes derived from cancer cells encapsulate various kinds of tumor-specific molecules and thus can interact with adjacent or distant cells to mediate information exchange. Long non-coding RNAs (lncRNAs) in exosomes have the potential as diagnostic and prognostic biomarkers in different types of cancers. The current study was aimed to identify circulating exosomal lncRNAs for the diagnosis of colorectal cancer (CRC). Methods: Exosomes were isolated from the serum by ultracentrifugation and verified by transmission electron microscope (TEM), qNano, and immunoblotting. Exosomal lncRNAs FOXD2-AS1, NRIR, and XLOC_009459 were selected by lncRNA microarray and validated by qPCR in 203 CRC patients and 201 healthy donors. The receiver operating characteristic curve (ROC) was used to assess the diagnostic efficiency of serum exosomal lncRNAs. Results: Exosomal FOXD2-AS1, NRIR, and XLOC_009459 (TCONS_00020073) levels were significantly upregulated in 203 CRC patients and 80 early-stage CRC patients compared to 201 healthy donors, possessing the area under the curve (AUC) of 0.728, 0.660, and 0.682 for CRC, as well as 0.743, 0.660, and 0.689 for early-stage CRC, respectively. Notably, their combination demonstrated the markedly elevated AUC of 0.736 for CRC and 0.758 for early-stage CRC, indicating their potential as diagnostic biomarkers for CRC. Conclusions: Our data suggested that exosomal lncRNAs FOXD2-AS1, NRIR, and XLOC_009459 act as the promising biomarkers for the diagnostics of CRC and early-stage CRC.

Nanoparticles can acquire a plasma protein corona defining their biological identity. Corona functions were previously considered for cell-derived extracellular vesicles (EVs). Here we demonstrate that nano-sized EVs from therapy-grade human placental-expanded (PLX) stromal cells are surrounded by an imageable and functional protein corona when enriched with permissive technology. Scalable EV separation from cell-secreted soluble factors via tangential flow-filtration (TFF) and subtractive tandem mass-tag (TMT) proteomics revealed significant enrichment of predominantly immunomodulatory and proangiogenic proteins. Western blot, calceinbased flow cytometry, super-resolution and electron microscopy verified EV identity. PLX-EVs partly protected corona proteins from protease digestion. EVs significantly ameliorated human skin regeneration and angiogenesis in vivo, induced differential signalling in immune cells, and dose-dependently inhibited T cell proliferation in vitro. Corona removal by size-exclusion or ultracentrifugation abrogated angiogenesis. Re-establishing an artificial corona by cloaking EVs with fluorescent albumin as a model protein or defined proangiogenic factors was depicted by superresolution microscopy, electron microscopy and zeta-potential shift, and served as a proof-of-concept. Understanding EV corona formation will improve rational EVinspired nano-therapy design.

Extracellular vesicles (EVs) are membranous structures in biofluids with enormous diagnostic/prognostic potential for application in liquid biopsies. Any such downstream application requires a detailed characterization of EV concentration, size and morphology. This study aimed to observe the native morphology of EVs in human cerebrospinal fluid after traumatic brain injury. Therefore, they were separated by gravity-driven size-exclusion chromatography (SEC) and investigated by atomic force microscopy (AFM) in liquid and cryogenic transmission electron microscopy (cryo-TEM). The enrichment of EVs in early SEC fractions was confirmed by immunoblot for transmembrane proteins CD9 and CD81. These fractions were then pooled, and the concentration and particle size distribution were determined by Tunable Resistive Pulse Sensing (around 1010 particles/mL, mode 100 nm) and Nanoparticle Tracking Analysis (around 109 particles/mL, mode 150 nm). Liquid AFM and cryo-TEM investigations showed mode sizes of about 60 and 90 nm, respectively, and various morphology features. AFM revealed round, concave, multilobed EV structures; and cryo-TEM identified single, double and multi-membrane EVs. By combining AFM for the surface morphology investigation and cryo-TEM for internal structure differentiation, EV morphological subpopulations in cerebrospinal fluid could be identified. These subpopulations should be further investigated because they could have different biological functions.

Mesenchymal stem cells (MSCs) have been proven to promote tissue repair. However, concerns related to their clinical application and regulatory hurdles remain. Recent data has demonstrated the proregenerative secretome of MSCs can result in similar effects in the absence of the cells themselves. Within the secretome, exosomes have emerged as a promising regenerative component. Exosomes, which are nanosized lipid vesicles secreted by cells, encapsulate micro-RNA (miRNA), RNA, and proteins that drive MSCs regenerative potential with cell specific content. As such, there is an opportunity to optimize the regenerative potential of MSCs, and thus their secreted exosome fraction, to improve clinical efficacy. Exercise is one factor that has been shown to improve muscle progenitor cell function and regenerative potential. However, the effect of exercise on MSC exosome content and function is still unclear. To address this, we used an in vitro culture system to evaluate the effects of mechanical strain, an exercise mimetic, on C2C12 (muscle progenitor cell) exosome production and proregenerative function. Our results indicate that the total exosome production is increased by mechanical strain and can be regulated with different tensile loading regimens. Furthermore, we found that exosomes from mechanically stimulated cells increase proliferation and myogenic differentiation of naïve C2C12 cells. Lastly, we show that exosomal miRNA cargo is differentially expressed following strain. Gene ontology mapping suggests positive regulation of bone morphogenetic protein signaling, regulation of actin-filament-based processes, and muscle cell apoptosis may be at least partially responsible for the proregenerative effects of exosomes from mechanically stimulated C2C12 muscle progenitor cells.

Synaptic dysfunction is an early mechanism in Alzheimer's disease that involves progressively larger areas of the brain over time. However, how it starts and propagates is unknown. Here we show that amyloid-β released by microglia in association with large extracellular vesicles (Aβ-EVs) alters dendritic spine morphology in vitro, at the site of neuron interaction, and impairs synaptic plasticity both in vitro and in vivo in the entorhinal cortex-dentate gyrus circuitry. One hour after Aβ-EV injection into the mouse entorhinal cortex, long-term potentiation was impaired in the entorhinal cortex but not in the dentate gyrus, its main target region, while 24 h later it was also impaired in the dentate gyrus, revealing a spreading of long-term potentiation deficit between the two regions. Similar results were obtained upon injection of extracellular vesicles carrying Aβ naturally secreted by CHO7PA2 cells, while neither Aβ42 alone nor inflammatory extracellular vesicles devoid of Aβ were able to propagate long-term potentiation impairment. Using optical tweezers combined to time-lapse imaging to study Aβ-EV-neuron interaction, we show that Aβ-EVs move anterogradely at the axon surface and that their motion can be blocked through annexin-V coating. Importantly, when Aβ-EV motility was inhibited, no propagation of long-term potentiation deficit occurred along the entorhinal-hippocampal circuit, implicating large extracellular vesicle motion at the neuron surface in the spreading of long-term potentiation impairment. Our data indicate the involvement of large microglial extracellular vesicles in the rise and propagation of early synaptic dysfunction in Alzheimer's disease and suggest a new mechanism controlling the diffusion of large extracellular vesicles and their pathogenic signals in the brain parenchyma, paving the way for novel therapeutic strategies to delay the disease.

Myotonic dystrophy type 1 (DM1) is the most common adult-onset muscular dystrophy, primarily characterized by muscle wasting and weakness. Many biomarkers already exist in the rapidly developing biomarker research field that aim to improve patients’ care. Limited work, however, has been performed on rare diseases, including DM1. We have previously shown that specific microRNAs (miRNAs) can be used as potential biomarkers for DM1 progression. In this report, we aimed to identify novel serum-based biomarkers for DM1 through high-throughput next-generation sequencing. A number of miRNAs were identified that are able to distinguish DM1 patients from healthy individuals. Two miRNAs were selected, and their association with the disease was validated in a larger panel of patients. Further investigation of miR-223-3p, miR-24-3p, and the four previously identified miRNAs, miR-1-3p, miR-133a-3p, miR-133b-3p, and miR-206-3p, showed elevated levels in a DM1 mouse model for all six miRNAs circulating in the serum compared to healthy controls. Importantly, the levels of miR-223-3p, but not the other five miRNAs, were found to be significantly downregulated in five skeletal muscles and heart tissues of DM1 mice compared to controls. This result provides significant evidence for its involvement in disease manifestation.

Background Extracellular vesicles (EVs) contain thousands of proteins and nucleic acids, playing an important role in cell–cell communications. Sertoli cells have been essential in the testis as a “nurse cell”. However, EVs derived from human Sertoli cells (HSerCs) have not been well investigated. Methods EVs were isolated from HSerCs via ultracentrifugation and characterized by transmission electron microscopy, tunable resistive pulse sensing, and Western blotting. The cargo carried by HSerCs-EVs was measured via liquid chromatography-mass spectrometry and GeneChip miRNA Arrays. Bioinformatic analysis was performed to reveal potential functions of HSerCs-EVs. Results A total of 860 proteins with no less than 2 unique peptides and 88 microRNAs with high signal values were identified in HSerCs-EVs. Biological processes related to molecular binding, enzyme activity, and regulation of cell cycle were significantly enriched. Specifically, many proteins in HSerCs-EVs were associated with spermatogenesis and regulation of immune system, including Septins, Large proline-rich protein BAG6, Clusterin, and Galectin-1. Moreover, abundant microRNAs within HSerCs-EVs (miR-638, miR-149-3p, miR-1246, etc.) had a possible impact on male reproductive disorders such as asthenozoospermia and oligozoospermia. Conclusions Our study has shown that HSerCs-EVs contain diverse components such as proteins and microRNAs. Further research is required to evaluate HSerCs-EVs in spermatogenesis, which are underutilized but highly potent resources with particular promise for male infertility.

Extracellular vesicles (EV) are an emerging technology as immune therapeutics and drug delivery vehicles. However, EVs are usually stored at −80 °C which limits potential clinical applicability. Freeze-drying of EVs striving for long-term stable formulations is therefore studied. The most appropriate formulation parameters are identified in freeze-thawing studies with two different EV types. After a freeze-drying feasibility study, four lyophilized EV formulations are tested for storage stability for up to 6 months. Freeze-thawing studies revealed improved colloidal EV stability in presence of sucrose or potassium phosphate buffer instead of sodium phosphate buffer or phosphate-buffered saline. Less aggregation and/or vesicle fusion occurred at neutral pH compared to slightly acidic or alkaline pH. EVs colloidal stability can be most effectively preserved by addition of low amounts of poloxamer 188. Polyvinyl pyrrolidone failed to preserve EVs upon freeze-drying. Particle size and concentration of EVs are retained over 6 months at 40 °C in lyophilizates containing 10 mm K- or Na-phosphate buffer, 0.02% poloxamer 188, and 5% sucrose. The biological activity of associated beta-glucuronidase is maintained for 1 month, but decreased after 6 months. Here optimized parameters for lyophilization of EVs that contribute to generate long-term stable EV formulations are presented.

Immune-inflammatory activation impacts extracellular vesicles (EVs), including their miRNA cargo. There is evidence for changes in the EV miRNome in inflammation-associated neuropsychiatric disorders. This mouse study investigated: (1) effects of systemic lipopolysaccharide (LPS) and chronic social stress (CSS) on plasma EV miRNome; and (2) physiological, transcriptional, and behavioural effects of peripheral or central delivered LPS-activated EVs in recipient mice. LPS or CSS effects on the plasma EV miRNome were assessed by using microRNA sequencing. Recipient mice received plasma EVs isolated from LPS-treated or SAL-treated donor mice or vehicle only, either intravenously or into the nucleus accumbens (NAc), on three consecutive days. Bodyweight, spleen or NAc transcriptome and reward (sucrose) motivation were assessed. LPS and CSS increased the expression of 122 and decreased expression of 20 plasma EV miRNAs, respectively. Peripheral LPS-EVs reduced bodyweight, and both LPS-EVs and SAL-EVs increased spleen expression of immune-relevant genes. NAc-infused LPS-EVs increased the expression of 10 immune-inflammatory genes. Whereas motivation increased similarly across test days in all groups, the effect of test days was more pronounced in mice that received peripheral or central LPS-EVs compared with other groups. This study provides causal evidence that increased EV levels impact physiological and behavioural processes and are of potential relevance to neuropsychiatric disorders.

Background Extracellular vesicles (EVs) are produced by all cell types and serve as biological packets delivering a wide variety of molecules for cell-to-cell communication. However, the biology of the EV extravesicular surface domain that we have termed EV ‘biocorona’ remains underexplored. Upon cell secretion, EVs possess an innate biocorona containing membrane integral and peripheral constituents that is modified by acquired constituents post secretion. This distinguishes EVs from synthetic nanoparticulate biomaterials that are limited to an adsorption-based, acquired biocorona. Methods The EV biocorona molecular constituents were radiolabeled with 125I to study biocorona constituents and its surface dynamics. As example toolset applications, 125I-EVs were utilized to study EV cell trafficking and the stability of the EV biocorona during storage. Results The biocorona of EVs consisted of proteins, lipids, DNA and RNA. The cellular uptake of 125I-EVs was temperature dependent and internalized 125I-EVs were rapidly recycled by cells. When 125I-EVs were stored in a purified state, they exhibited time and temperature dependent biocorona shedding and proteolytic degradation that was partially inhibited in the presence of serum. Conclusion The EV biocorona is complex and dynamic. Radiolabeling of the EV biocorona enables a unique platform methodology to study the biocorona and will facilitate unlocking EV's full clinical translation potential. General significance The EV biocorona affects EV mediated biological processes in health and disease. Acquiring knowledge of the EV biocorona composition, dynamics, stability and structure not only informs the diagnostic and therapeutic translation of EVs but also aids in designing biomimetic nanomaterials for drug delivery.

Background Physical exercise is an essential factor in preventing and treating metabolic diseases by promoting systemic benefits throughout the body. The molecular factors involved in this process are poorly understood. Micro RNAs (miRNAs) are small non‐coding RNAs that inhibit mRNA transcription. MiRNAs, which can participate in the benefits of exercise to health, circulate in plasma in extracellular particles (EP). Horses that undergo endurance racing are an excellent model to study the impact of long‐duration/low intensity exercise in plasma EP miRNAs. Objectives To evaluate the effects of 160 km endurance racing on horse plasma extracellular particles and their miRNA population. Study design Cohort study. Methods We collected plasma from 5 Arabian horses during five time‐points of an endurance ride. Extracellular particles were purified from plasma and characterised by electron microscopy, resistive pulse sensing (qNano), and western blotting. Small RNAs were purified from horse plasma EP, and sequencing was performed. Results Endurance racing increased EP concentration and average diameter compared to before the race. Western blotting showed a high concentration of extracellular vesicles proteins 2 h after the race, which returned to baseline 15 h after the race. MicroRNA differential expression analysis revealed increasing levels of eca‐miR‐486‐5p during and after the race, and decreasing levels of eca‐miR‐9083 after the end. Conclusions This study adds new data about the variation in plasma EP concentrations after long‐distance exercise and brings new insights about the roles of exercise‐derived EP miRNAs during low‐intensity endurance exercise.

Nanosized extracellular vesicles (EVs) have been found to play a key role in intercellular communication, offering opportunities for both disease diagnostics and therapeutics. However, lying below the diffraction limit and also being highly heterogeneous in their size, morphology and abundance, these vesicles pose significant challenges for physical characterization. Here, we present a direct visual approach for their accurate morphological and size-based profiling by using scanning electron microscopy (SEM). To achieve that, we methodically examined various process steps and developed a protocol to improve the throughput, conformity and image quality while preserving the shape of EVs. The study was performed with small EVs (sEVs) isolated from a non-small-cell lung cancer (NSCLC) cell line as well as from human serum, and the results were compared with those obtained from nanoparticle tracking analysis (NTA). While the comparison of the sEV size distributions showed good agreement between the two methods for large sEVs (diameter > 70 nm), the microscopy based approach showed a better capacity for analyses of smaller vesicles, with higher sEV counts compared to NTA. In addition, we demonstrated the possibility of identifying non-EV particles based on size and morphological features. The study also showed process steps that can generate artifacts bearing resemblance with sEVs. The results therefore present a simple way to use a widely available microscopy tool for accurate and high throughput physical characterization of EVs.

Background: Tyrosine kinase inhibitors (TKI) were initially demonstrated as an efficacious treatment for renal cell carcinoma (RCC). However, after a median treatment length of 14 months, a vast majority of patients develop resistance. This study analyzed a combination therapy of tipifarnib (Tipi) + sunitinib that targeted exosome-conferred drug resistance. Methods: 786-O, 786-O-SR (sunitinib resistant), A498, A498-SR, Caki-2, Caki-2-SR, and 293T cells were cultured. Exosomes were collected using differential ultracentrifugation. Cell proliferation, Jurkat T cell immune assay, and immunoblot analysis were used for downstream analysis. Results: SR exosomes treatment displayed a cytotoxic effect on immune cells. This cytotoxic effect was associated with increased expression of PD-L1 on SR exosomes when compared to sunitinib-sensitive (SS) exosomes. Additionally, Tipi treatment downregulated PD-L1 expression on exosomes derived from SR cell lines. Tipi’s ability to downregulate PD-L1 in exosomes has a significant application within patients. Exosomes collected from patients with RCC showed increased PD-L1 expression over subjects without RCC. Next, exosome concentrations were then compared after Tipi treatment, with all SS cell lines displaying an even greater reduction. On immunoblot assay, 293T cells showed a dose-dependent increase in Alix with no change in either nSMase or Rab27a. Conversely, all the SS and SR cell lines displayed a decrease in all three markers. After a cell proliferation employed a 48-h treatment on all SS and SR cell lines, the drug combination displayed synergistic ability to decrease tumor growth. Conclusions: Tipifarnib attenuates both the exosome endosomal sorting complex required for endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways, thereby blocking exosome biogenesis and secretion as well as downregulating PD-L1 on SS and SR cells.

Background Breast cancer is the most common cancer, and the leading cause of cancer-related deaths, among females world-wide. Recent research suggests that extracellular vesicles (EVs) play a major role in the development of breast cancer metastasis. Axillary lymph node dissection (ALND) is a procedure in patients with known lymph node metastases, and after surgery large amounts of serous fluid are produced from the axilla. The overall aim was to isolate and characterize EVs from axillary serous fluid, and more specifically to determine if potential breast cancer biomarkers could be identified. Methods Lymphatic drain fluid was collected from 7 patients with breast cancer the day after ALND. EVs were isolated using size exclusion chromatography, quantified and detected by nanoparticle tracking analysis, electron microscopy, nano flow cytometry and western blot. The expression of 37 EV surface proteins was evaluated by flow cytometry using the MACSPlex Exosome kit. Results Lymphatic drainage exudate retrieved after surgery from all 7 patients contained EVs. The isolated EVs were positive for the typical EV markers CD9, CD63, CD81 and Flotillin-1 while albumin was absent, indicating low contamination from blood proteins. In total, 24 different EV surface proteins were detected. Eleven of those proteins were detected in all patients, including the common EV markers CD9, CD63 and CD81, cancer-related markers CD24, CD29, CD44 and CD146, platelet markers CD41b, CD42a and CD62p as well as HLA-DR/DP/DQ. Furthermore, CD29 and CD146 were enriched in Her2+ patients compared to patients with Her2- tumors. Conclusions Lymphatic drainage exudate retrieved from breast cancer patients after surgery contains EVs that can be isolated using SEC isolation. The EVs have several cancer-related markers including CD24, CD29, CD44 and CD146, proteins of potential interest as biomarkers as well as to increase the understanding of the mechanisms of cancer biology.

Extracellular vesicles (EVs) play important roles in diabetes mellitus (DM) via connecting the immune cell response to tissue injury, besides stimulation to muscle insulin resistance, while DM is associated with increased risks for major cardiovascular complications. Under DM, chronic hyperglycemia, and subsequent increase in the production of reactive oxygen species (ROS) further lead to cardiac growth remodeling and dysfunction. The purinergic drug ticagrelor is a P2Y12 receptor antagonist. Although it is widely used in cardioprotection, the underlying molecular mechanism of its inhibitory effect on diabetic cardiomyopathy is poorly elucidated. Here, we aimed to understand how ticagrelor exerts its cardio-regulatory effects. For this purpose, we investigated the anti-oxidative and cardioprotective effect of EVs derived from ticagrelor-pretreated cardiomyocytes under DM conditions. To mimic DM in cardiomyocytes, we used high glucose incubated H9c2-cells (HG). HG cells were treated with EVs, which were derived from either ticagrelor-pretreated or untreated H9c2-cells. Our results demonstrated that ticagrelor-pretreated H9c2-derived EVs significantly decreased the hyperglycemia-induced aberrant ROS production, prevented the development of apoptosis and ER stress, and alleviated oxidative stress associated miRNA-expression profile. Importantly, EVs derived from ticagrelor-pretreated H9c2-cells enhanced endothelial cell migration and tube formation, suggesting a modulation of the EV profile in cardiomyocytes. Our data, for the first time, indicate that ticagrelor can exert an important regulatory effect on diabetic cardiomyopathy through extracellular vesicular modulation behind its receptor-inhibition-related effects.

The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function.

Development of nanotechnology and corresponding industries during the last decade resulted in a new challenge for analytical science. This includes an ultrasensitive detection and characterization of nanoparticles of different origin and other nanomaterials in various media, including so complex ones as food, biological or environmental samples. The goal of this review is a systematic analysis of possible approaches and description of physical principles behind these methods. The main attention is paid to optical methods which are considered by authors to be mostly effective for the formulated task. Different approaches for detection and analysis of nanoparticles in a volume as well as of those adsorbed on a surface are discussed. While the technologies based on direct analysis of nanoparticle suspensions belong to the established approaches whose development potential has been in large extent exhausted, the novel technologies based on the surface sensing of adsorbed nanoparticles demonstrate intensive development. Therefore, the final part of the review is focused on the wide-field surface plasmon resonance microscopy. It allows one an ultrasensitive detection and characterization of individual nanoparticles of different origin in complex media and provides numerous possibilities for subsequent chemical identification of the detected particles using a hyphenation with other analytical technologies.

Ischemic conditioning and exercise have been suggested for protecting against brain ischemia-reperfusion injury. However, the endogenous protective mechanisms stimulated by these interventions remain unclear. Here, in a comprehensive translational study, we investigated the protective role of extracellular vesicles (EVs) released after remote ischemic conditioning (RIC), blood flow restricted resistance exercise (BFRRE), or high-load resistance exercise (HLRE). Blood samples were collected from human participants before and at serial time points after intervention. RIC and BFRRE plasma EVs released early after stimulation improved viability of endothelial cells subjected to oxygen-glucose deprivation. Furthermore, post-RIC EVs accumulated in the ischemic area of a stroke mouse model, and a mean decrease in infarct volume was observed for post-RIC EVs, although not reaching statistical significance. Thus, circulating EVs induced by RIC and BFRRE can mediate protection, but the in vivo and translational effects of conditioned EVs require further experimental verification.

Renal Cell Carcinoma (RCC) is the most common form of kidney cancer, with clear cell RCC (ccRCC) representing about 85% of all RCC tumors. There are limited curable treatments available for metastatic ccRCC because this disease is unresponsive to conventional targeted systemic pharmacotherapy. Exosomes (Exo) are small extracellular vesicles (EVs) secreted from cancer cells with marked roles in tumoral signaling and pharmacological resistance. Ketoconazole (KTZ) is an FDA approved anti-fungal medication which has been shown to suppress exosome biogenesis and secretion, yet its role in ccRCC has not been identified. A time-course, dose-dependent analysis revealed that KTZ selectively decreased secreted Exo in tumoral cell lines. Augmented Exo secretion was further evident by decreased expression of Exo biogenesis (Alix and nSMase) and secretion (Rab27a) markers. Interestingly, KTZ-mediated inhibition of Exo biogenesis was coupled with inhibition of ERK1/2 activation. Next, selective inhibitors were employed and showed ERK signaling had a direct role in mediating KTZ’s inhibition of exosomes. In sunitinib resistant 786-O cells lines, the addition of KTZ potentiates the efficacy of sunitinib by causing Exo inhibition, decreased tumor proliferation, and diminished clonogenic ability of RCC cells. Our findings suggest that KTZ should be explored as an adjunct to current RCC therapies.

Background Small extracellular vesicles (sEV) play a crucial role in immune responses to viral infection. However, the composition of sEV derived from children with viral pneumonia remains ill defined. Methods First, we performed mass spectrometry-based label-free proteomic analysis of urinary sEV in 7 children with viral pneumonia, 4 children with Mycoplasma pneumoniae pneumonia and 20 healthy children. Then a total of 33 proteins were selected to validate by multiple reaction monitoring analysis in an independent cohort of 20 healthy children and 29 children with pneumonia. Results In the discovery phase, a total of 1621 proteins were identified, while 260 proteins have differential expression in children with viral pneumonia compared to healthy children. Biological pathways primarily associated with neutrophil degranulation, carbohydrate metabolism and endocytosis were enriched in children with viral pneumonia. Finally, the abundance of eight proteins was verified to be significantly higher in children with viral pneumonia than in healthy children. Conclusions This pilot study with proteomic profiles of urinary sEV provided insights to the host response to viral pathogen exposure and potential diagnostic biomarkers for children with viral pneumonia, and served as the basis for understanding the fundamental biology of infection.

Clear evidence shows that tumor could secrete microRNAs (miRNAs) via exosomes to modulate tumor microenvironment (TME). However, the mechanisms sorting specific miRNAs into exosomes are still unclear. In order to study the biological function and characterization of exosomal miRNAs, we performed whole-transcriptome sequencing in 59 patients’ whole course cerebrospinal fluid (CSF) small extracellular vesicles (sEV) and matched glioma tissue samples. The results demonstrate that miRNAs could be divided into exosome-enriched miRNAs (ExomiRNAs) and intracellular-retained miRNAs (CLmiRNAs), and exosome-enriched miRNAs generally play a dual role. Among them, miR-1298-5p was enriched in CSF exosomes and suppressed glioma progression in vitro and vivo experiments. Interestingly, exosomal miR-1298-5p could promote Immunosuppressive effects of myeloid-derived suppressor cells (MDSCs) to facilitate glioma. Therefore, we found miR-1298-5p had different effects on glioma cells and MDSCs. Mechanically, downstream signaling pathway analyses showed that miR-1298-5p plays distinct roles in glioma cells and MDSCs via targeting SETD7 and MSH2, respectively. Moreover, reverse verification was performed on the intracellular-retained miRNA miR-9-5p. Thus, we confirmed that tumor-suppressive miRNAs in glioma cells could be eliminated through exosomes and target tumor-associated immune cells to induce tumor-promoting phenotypes. Glioma could get double benefit from it. These findings uncover the mechanisms that glioma selectively sorts miRNAs into exosomes and modulates tumor immunity.

Epstein-Barr virus (EBV) causes malignant carcinomas including B cell lymphomas accompanied by the systemic inflammation. Previously, we observed that phosphatidylserine (PS)-exposing subset of extracellular vesicles (EVs) secreted from an EBV strain Akata-transformed lymphoma (Akata EVs) convert surrounding phagocytes into tumor-associated macrophages (TAMs) via induction of inflammatory response, which is in part mediated by EBV-derived micro RNAs. However, it is still unclear about EV-carried other potential inflammatory factors associated with TAM formation in EBV lymphomas. To this end, we sought to explore proteomic and phospholipidomic profiles of PS-exposing EVs derived from EBV-transformed lymphomas. Mass spectrometric analysis revealed that several immunomodulatory proteins including integrin αLβ2 and fibroblast growth factor 2 (FGF2) were highly expressed in PS-exposing Akata EVs compared with another EBV strain B95-8-transformed lymphoma-derived counterparts which significantly lack TAM-inducing ability. Pharmacological inhibition of either integrin αLβ2 or FGF2 hampered cytokine induction in monocytic cultured cells elicited by PS-exposing Akata EVs, suggesting the involvement of these proteins in EV-mediated TAM induction in EBV lymphomas. In addition, phospholipids containing precursors of immunomodulatory lipid mediators were also enriched in PS-exposing Akata EVs compared with B95-8 counterparts. Phospholipidomic analysis of fractionated Akata EVs by density gradient centrifugation further demonstrated that PS-exposing Akata EVs might be identical to certain Akata EVs in low density fractions containing exosomes. Therefore, we concluded that a variety of immunomodulatory cargo molecules in a certain EV subtype are presumably conducive to the development of EBV lymphomas.

Therapeutic benefits of curcumin for inflammatory diseases have been demonstrated. However, curcumin's potential as a clinical therapeutic has been hindered due to its low solubility and stability in vivo. We hypothesized that a hybrid curcumin carrier that incorporates albumin-binding and extracellular vesicle (EV) encapsulation could effectively address the current challenges of curcumin delivery. We further postulated that using dissolvable microneedle arrays (dMNAs) for local delivery of curcumin-albumin-EVs (CA-EVs) could effectively control skin inflammation in vivo. Mild sonication was used to encapsulate curcumin and albumin into EVs, and the resulting CA-EVs were integrated into tip-loaded dMNAs. In vitro and in vivo studies were performed to assess the stability, cellular uptake, and anti-inflammatory bioactivity of dMNA-delivered CA-EVs. Curcumin in CA-EVs exhibited at least five-fold higher stability in vitro than naïve curcumin or curcumin-EVs without albumin. Incorporating CA-EVs into dMNAs did not alter their cellular uptake or anti-inflammatory bioactivity. The dMNA embedded CA-EVs retained their bioactivity when stored at room temperature for at least 12 months. In rat and mice models, dMNA delivered CA-EVs suppressed and significantly reduced lipopolysaccharide and Imiquimod-triggered inflammation. We conclude that dMNA delivery of CA-EVs has the potential to become an effective local-delivery strategy for inflammatory skin diseases. STATEMENT OF SIGNIFICANCE: We introduce and evaluate a skin-targeted delivery system for curcumin that synergistically combines albumin association, extracellular-vesicle encapsulation, and dissolvable microneedle arrays (dMNAs) . In vitro, curcumin-albumin encapsulated extracellular vesicles (CA-EVs) inhibit and reverse the LPS-triggered expression of inflammatory transcription factor NF-κB. The integration of CA-EVs into dMNAs does not affect them physically or functionally. Importantly, dMNAs extend EV storage stability for at least 12 months at room temperature with minimal loss in their bioactivity. We demonstrate that dMNA delivered CA-EVs effectively block and reverse skin inflammation in vivo in mouse and rat models.

Background MicroRNAs (miRNAs) have been reported to play important roles in regulating natural killer (NK) cell cytotoxicity to cancer cells. Objective This study aimed to investigate the effects and potential mechanism of miR-30c in regulating NK cell cytotoxicity to lung cancer cells. Methods Primary NK cells were derived from the peripheral blood of lung cancer and normal participants. Exosomes were isolated and validated via transmission electron microscopy and nanoparticle tracking analysis. The levels of miR-30c, polypeptide N-acetylgalactosaminyltransferase 7 (GALNT7) and proteins in PI3K/AKT pathway were determined using quantitative real-time polymerase chain reaction or western blot. Tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) levels and the cytotoxicity of effector NK cells to target lung cancer cells were measured via enzyme linked immunosorbent assay, cell apoptosis or xenograft experiments. The relationship between miR-30c and GALNT7 was analyzed by luciferase activity, RNA pull-down and RNA immunoprecipitation assays. And a xenograft mice model was established to verify the effect of miR-30c in regulating NK cell cytotoxicity to lung cancer cells in vivo. Results NK cell-derived exosomes carrying miR-30c, and miR-30c level was significantly downregulated in primary NK cells of lung cancer patients. MiR-30c overexpression promoted TNF-α and IFN-γ secretion and enhanced the cytotoxicity of interleukin 2 (IL-2)-treated NK cells to lung cancer cells, while knockdown of miR-30c played an opposite effect in regulating the cytotoxicity of NK cells to lung cancer cells. GALNT7 was a target of miR-30c and was negatively regulated by miR-30c. Besides, miR-30c targeted GALNT7 to exert its function in regulating NK cell cytotoxicity. Furthermore, GALNT7 prompted the activation of PI3K/AKT pathway in NK cells. Additionally, miR-30c overexpression enhanced NK cell cytotoxicity to lung cancer cells and inhibited tumor growth in vivo.ConclusionmiR-30c enhanced NK cell cytotoxicity to lung cancer cells via decreasing GALNT7 and inactivating the PI3K/AKT pathway, suggesting that regulating miR-30c expression maybe a promising approach for enhancing NK cell-based antitumor therapies.

The critical role of neutrophils in pathological inflammation, notably in various autoimmune disorders, is currently the focus of renewed interest. Here, we demonstrate for the first time that activation of neutrophils with various inflammatory stimuli induces the release of extracellular vesicles (EVs) that are internalized by endothelial cells (ECs), thus leading to the transfer of miR-223, miR-142-3p and miR-451 and subsequent endothelial damage. Indeed, while miR-223 has little effect on EC responses, we show that the induced expression of miR-142-3p and miR-451 in ECs results in profound cell damage, especially in inflammatory conditions, characterized by a dramatic increase in cell apoptosis, impaired angiogenic repair responses, and the induction of IL-6, IL-8, CXCL10 and CXCL11 expression. We show that the strong deleterious effect of miR-142-3p may be due in part to its ability to block the activation of ERK1/2 and eNOS-mediated signals in ECs. miR-142-3p also inhibits the expression of RAC1, ROCK2 and CLIC4, three genes that are critical for EC migration and angiogenic responses. Importantly, miR-223, miR-142-3p and miR-451 are markedly increased in kidney biopsies from patients with active ANCA-associated vasculitis, a severe autoimmune disease that is prototypical of a neutrophil-induced microvascular damage. Taken together, our results suggest that miR-142-3p and miR-451 released in EVs by activated neutrophils can target EC to trigger an inflammatory cascade and induce direct vascular damage, and that therapeutic strategies based on the inhibition of these miRNAs in ECs will have implications for neutrophil-mediated inflammatory diseases.

Stem cells undergo cytokine-driven differentiation, but this process often takes longer than several weeks to complete. A novel mechanism for somatic stem cell differentiation via phagocytosing ‘model cells’ (apoptotic differentiated cells) was found to require only a short time frame. Pluripotent-like Muse cells, multipotent mesenchymal stem cells (MSCs), and neural stem cells (NSCs) phagocytosed apoptotic differentiated cells via different phagocytic receptor subsets than macrophages. The phagocytosed-differentiated cell-derived contents (e.g., transcription factors) were quickly released into the cytoplasm, translocated into the nucleus, and bound to promoter regions of the stem cell genomes. Within 24 ~ 36 h, the cells expressed lineage-specific markers corresponding to the phagocytosed-differentiated cells, both in vitro and in vivo. At 1 week, the gene expression profiles were similar to those of the authentic differentiated cells and expressed functional markers. Differentiation was limited to the inherent potential of each cell line: triploblastic-, adipogenic-/chondrogenic-, and neural-lineages in Muse cells, MSCs, and NSCs, respectively. Disruption of phagocytosis, either by phagocytic receptor inhibition via small interfering RNA or annexin V treatment, impeded differentiation in vitro and in vivo. Together, our findings uncovered a simple mechanism by which differentiation-directing factors are directly transferred to somatic stem cells by phagocytosing apoptotic differentiated cells to trigger their rapid differentiation into the target cell lineage.

Biological scaffolds derived from decellularized tissues are being investigated as a promising approach to repair volumetric muscle losses (VML). Indeed, extracellular matrix (ECM) from decellularized tissues is highly biocompatible and mimics the original tissue. However, the development of fibrosis and the muscle stiffness still represents a major problem. Intercellular signals mediating tissue repair are conveyed via extracellular vesicles (EVs), biologically active nanoparticles secreted by the cells. This work aimed at using muscle ECM and human EVs derived from Wharton Jelly mesenchymal stromal cells (MSC EVs) to boost tissue regeneration in a VML murine model. Mice transplanted with muscle ECM and treated with PBS or MSC EVs were analyzed after 7 and 30 days. Flow cytometry, tissue analysis, qRT-PCR and physiology test were performed. We demonstrated that angiogenesis and myogenesis were enhanced while fibrosis was reduced after EV treatment. Moreover, the inflammation was directed toward tissue repair. M2-like, pro-regenerative macrophages were significantly increased in the MSC EVs treated group compared to control. Strikingly, the histological improvements were associated with enhanced functional recovery. These results suggest that human MSC EVs can be a naturally-derived boost able to ameliorate the efficacy of tissue-specific ECM in muscle regeneration up to the restored tissue function.

Background: MSC-derived extracellular vehicles (EVs) exhibit a protective functional role in renal ischemia/reperfusion injury (RIRI). Recent studies have revealed that mitophagy could be a potential target process in the treatment of RIRI. However, whether MSC-derived EVs are involved in the regulation of mitophagy in RIRI remains largely unknown to date. Methods: RIRI model was established in vivo in mice by subjecting them to renal ischemia/reperfusion. TCMK-1 cells were subjected to hypoxia/reoxygenation (H/R) stimulation to mimic RIRI in vitro. BMSCs and BMSC-derived EVs were isolated and identified. Renal injury was assessed using H&E staining. The qPCR and western blot analyses were conducted to detect the mRNA and protein levels. Apoptosis was evaluated using the TUNEL assay and flow cytometry analysis. The EVs, autophagosomes, and mitochondria were observed using TEM. The colocalization of autophagosomes with mitochondria was confirmed through the confocal assay. The direct binding of miR-223-3p to NLRP3 was validated through the dual-luciferase assay. Results: BMSCs and BMSC-derived EVs were successfully isolated from mice and identified. The protective effect of BMSC-derived EVs against RIRI was validated both in vitro and in vivo, which was indicated by a decrease in apoptosis and inflammasome activation and an increase in mitophagy. However, this protective effect was impaired in the miR-223-3p-depleted EVs, suggesting that miR-223-3p mediated this protective effect. Further mechanistic investigation revealed that miR-223-3p suppressed inflammasome activation to enhance mitophagy by directly targeting NLRP3. Conclusion: In conclusion, the protective role of BMSC-derived EVs and exosome-delivered miR-223-3p in RIRI was validated. Exogenous miR-223-3p directly targeted NLRP3 to attenuate inflammasome activation, thereby promoting mitophagy.

Non-small-cell lung cancer (NSCLC) is defined as the most universally diagnosed class of lung cancer. Cisplatin (DDP) is an effective drug for NSCLC, but tumors are prone to drug resistance. The current study set out to evaluate the regulatory effect of long non-coding RNA (lncRNA) small nucleolar RNA host gene 12 (SNHG12) in extracellular vesicles (EVs) derived from carcinoma-associated fibroblasts (CAFs) on DDP resistance in NSCLC cells. Firstly, NSCLC cells were treated with EVs, followed by detection of cell activity, IC50 values, cell proliferation and apoptosis, and Cy3-SNHG12. We observed that CAFs-EVs promoted IC50 values and cell proliferation and inhibited apoptosis. In addition, we learned that lncRNA SNHG12 carried by CAFs-EVs into NSCLC facilitated DDP resistance of NSCLC cells. Furthermore, ELAV like RNA binding protein 1 (HuR/ELAVL1) binding to lncRNA SNHG12 and X-linked inhibitor of apoptosis (XIAP) was verified and RNA stability of XIAP was also verified CAFs-EVs promoted RNA stability and transcription of XIAP, while silencing HuR could partially-reverse this promoting effect. Further joint experimentation showed that silencing XIAP partially inhibited DDP resistance in NSCLC cells. Additionally, the tumor growth and the positive rate of Ki67 and HuR were detected, which showed that CAFs-oe-EVs promoted the tumor and the positive rate of Ki67, as well as the levels of lncRNA SNHG12, HuR, and XIAP in vivo. Collectively, our findings indicated that lncRNA SNHG12 carried by CAFs-EVs into NSCLC cells promoted RNA stability and XIAP transcription by binding to HuR, thus augmenting DDP resistance in NSCLC cells.

Adipose tissue represents an abundant source of mesenchymal stem cells (MSC) for therapeutic purposes. Previous studies have demonstrated the anti-inflammatory potential of adipose tissue-derived MSC (ASC). Extracellular vesicles (EV) present in the conditioned medium (CM) have been shown to mediate the cytoprotective effects of human ASC secretome. Nevertheless, the role of EV in the anti-inflammatory effects of mouse-derived ASC is not known. The current study has investigated the influence of mouse-derived ASC CM and its fractions on the response of mouse-derived peritoneal macrophages against lipopolysaccharide (LPS). CM and its soluble fraction reduced the release of pro-inflammatory cytokines, adenosine triphosphate and nitric oxide in stimulated cells. They also enhanced the migration of neutrophils or monocytes, in the absence or presence of LPS, respectively, which is likely related to the presence of chemokines, and reduced the phagocytic response. The anti-inflammatory effect of CM may be dependent on the regulation of toll-like receptor 4 expression and nuclear factor-κB activation. Our results demonstrate the anti-inflammatory effects of mouse-derived ASC secretome in mouse-derived peritoneal macrophages stimulated with LPS and show that they are not mediated by EV.

Growth plate cartilage has limited self-repair ability, leading to poor bone bridge formation post-injury and ultimately limb growth defects in children. The current corrective surgeries are highly invasive, and outcomes can be unpredictable. Following growth plate injury, the direct loss of extracellular matrix (ECM) coupled with further ECM depletion due to the inhibitory effects of inflammation on the cartilage matrix protein greatly hinder chondrocyte regeneration. We designed an exosome (Exo) derived from bone marrow mesenchymal stem cells (BMSCs) loaded ECM-mimic hydrogel to promote cartilage repair by directly supplementing ECM and anti-inflammatory properties. Aldehyde-functionalized chondroitin sulfate (OCS) was introduced into gelatin methacryloyl (GM) to form GMOCS hydrogel. Our results uncovered that GMOCS hydrogel could significantly promote the synthesis of ECM due to the doping of OCS. In addition, the GMOCS-Exos hydrogel could further promote the anabolism of chondrocytes by inhibiting inflammation and ultimately promote growth plate injury repair through ECM remodeling.

Several reports have described a beneficial effect of Mesenchymal Stromal Cells (MSCs) and of their secreted extracellular vesicles (EVs) in mice with experimental colitis. However, the effects of the two treatments have not been thoroughly compared in this model. Here, we compared the effects of MSCs and of MSC-EV administration in mice with colitis induced by dextran sulfate sodium (DSS). Since cytokine conditioning was reported to enhance the immune modulatory activity of MSCs, the cells were kept either under standard culture conditions (naïve, nMSCs) or primed with a cocktail of pro-inflammatory cytokines, including IL1β, IL6 and TNFα (induced, iMSCs). In our experimental conditions, nMSCs and iMSCs administration resulted in both clinical and histological worsening and was associated with pro-inflammatory polarization of intestinal macrophages. However, mice treated with iEVs showed clinico-pathological improvement, decreased intestinal fibrosis and angiogenesis and a striking increase in intestinal expression of Mucin 5ac, suggesting improved epithelial function. Moreover, treatment with iEVs resulted in the polarization of intestinal macrophages towards and anti-inflammatory phenotype and in an increased Treg/Teff ratio at the level of the intestinal lymph node. Collectively, these data confirm that MSCs can behave either as anti- or as pro-inflammatory agents depending on the host environment. In contrast, EVs showed a beneficial effect, suggesting a more predictable behavior, a safer therapeutic profile and a higher therapeutic efficacy with respect to their cells of origin.

Abstract Proteins are found both outside and inside of extracellular vesicles (EVs) and govern the properties and functions of EVs, while also constituting a signature of the cell of origin and of biological function and disease. Outer proteins on EVs can be directly bound by antibodies to either enrich EVs, or probe the expression of a protein on EVs, including in a combinatorial manner. However, co-profiling of inner proteins remains challenging. Here, we present the high-throughput, multiplexed analysis of extracellular vesicle inner and outer proteins (EVPio). We describe the optimization of fixation and heat-induced protein epitope retrieval for EVs, along with oligo-barcoded antibodies and branched DNA signal amplification for sensitive, multiplexed and high-throughput assays. We captured 4 subpopulations of EVs from colorectal cancer cell lines HT29 and SW403 based on EpCAM, CD9, CD63 and CD81 expression, and quantified the co-expression of 8 outer (integrins and tetraspanins) and 4 inner (heat shock, endosomal and inner leaflet) proteins. The differences in co-expression patterns were consistent with the literature and known biological function. In conclusion, EVPio analysis can simultaneously detect multiple inner and outer proteins in EVs immobilized on a surface, opening the way to extensive combinatorial protein profiles for both discovery and clinical translation.

IL-33 is a key mediator of chronic airway disease driven by type 2 immune pathways, yet the nonclassical secretory mechanism for this cytokine remains undefined. We performed a comprehensive analysis in human airway epithelial cells, which revealed that tonic IL-33 secretion is dependent on the ceramide biosynthetic enzyme neutral sphingomyelinase 2 (nSMase2). IL-33 is cosecreted with exosomes by the nSMase2-regulated multivesicular endosome (MVE) pathway as surface-bound cargo. In support of these findings, human chronic obstructive pulmonary disease (COPD) specimens exhibited increased epithelial expression of the abundantly secreted IL33Δ34 isoform and augmented nSMase2 expression compared with non-COPD specimens. Using an Alternaria-induced airway disease model, we found that the nSMase2 inhibitor GW4869 abrogated both IL-33 and exosome secretion as well as downstream inflammatory pathways. This work elucidates a potentially novel aspect of IL-33 biology that may be targeted for therapeutic benefit in chronic airway diseases driven by type 2 inflammation.

Acute liver injury (ALI) induced by chemicals or viruses can progress rapidly to acute liver failure (ALF), often resulting in death of patients without liver transplantation. Since liver transplantation is limited due to a paucity of donors, expensive surgical costs, and severe immune rejection, novel therapies are required to treat liver injury. Extracellular vesicles (EVs) are used for cellular communication, carrying RNAs, proteins, and lipids and delivering them intercellularly after being endocytosed by target cells. Recently, it was reported that EVs secreted from human hepatocytes have an ability to modulate the immune responses; however, these roles of EVs secreted from human hepatocytes were studied only with in vitro experiments. In the present study, we evidenced that EVs secreted from human hepatocytes attenuated the CCL4-induced ALI by inhibiting the recruitment of monocytes through downregulation of chemokine receptor in the bone marrow and recruitment of neutrophils through the reduction of C-X-C motif chemokine ligand 1 (CXCL1) and CXCL2 expression levels in the liver.

Extracellular vesicles (EVs) bearing biomolecules from parental cells can represent a novel source of disease biomarkers and are under intensive study for their clinical potential. Tunable resistive pulse sensing (TRPS) quantifies the magnitude of a small ionic resistive pulse current to determine the size, concentration, and zeta potential of EVs. Environmental noise is a common limiting factor that affects the precision of sensing devices. TRPS is particularly vulnerable to environmental noise, including both mechanical and electrical. The upper detection limit of the TRPS relies on the physical size of the elastomeric tunable nanopore. The lower limit relies on the electrical signal-to-noise ratio. Guided by simulation, we designed an external device to suppress environmental noise for TRPS measurement. Both mechanical and electrical environmental noise reductions were observed after using the shield. The study also validated the noise reduction function of the shield by quantifying EVs from different cell origins. Detection of EVs smaller than 200 nm was improved by using the shield; which was reported challenging for conventional quantification methods. The study highlighted a feasible approach to solve environmental noise challenges for TRPS based EV quantification.

Extracellular vesicles (EVs) are increasingly studied as vectors for drug delivery because they can transfer a variety of molecules across biological barriers. SerpinB3 is a serine protease inhibitor that has shown a protective anti-apoptotic function in a variety of stressful conditions. The aim of this study was to evaluate protection from oxidative stress-induced damage, using extracellular vesicles that overexpress SerpinB3 (EVs-SB3) in order to enhance the effect of extracellular vesicles on cellular homeostasis. EVs-SB3s were obtained from HepG2 cells engineered to overexpress SerpinB3 and they revealed significant proteomic changes, mostly characterized by a reduced expression of other proteins compared with EVs from non-engineered cells. These EV preparations showed a significantly higher protection from H2O2 induced oxidative stress in both the hepatoma cell line and in primary cardiomyocytes, compared to cells treated with naïve EVs or SerpinB3 alone, used at the same concentration. In conclusion, the induction of SerpinB3 transgene expression results in the secretion of EVs enriched with the protein product that exhibits enhanced cytoprotective activity, compared with naïve EVs or the nude SerpinB3 protein.

BackgroundSubclinical mastitis, the inflammation of the mammary gland lacking clinical symptoms, is one of the most prevalent and costly diseases in dairy farming worldwide. Milk microRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) have been proposed as potential biomarkers of different mammary gland conditions, including subclinical mastitis. However, little is known about the robustness of EVs analysis regarding sampling time-point and natural infections. To estimate the reliability of EVs measurements in raw bovine milk, we first evaluated changes in EVs size and concentration using Tunable Resistive Pulse Sensing (TRPS) during three consecutive days of sampling. Then, we analysed daily differences in miRNA cargo using small RNA-seq. Finally, we compared milk EVs differences from naturally infected udder quarters with their healthy adjacent quarters and quarters from uninfected udders, respectively.ResultsWe found that the milk EV miRNA cargo was very stable over the course of three days regardless of the health status of the quarter, and that infected quarters did not induce relevant changes in milk EVs of adjacent healthy quarters. Chronic subclinical mastitis induced changes in milk EV miRNA cargo, but neither in EVs size nor concentration. We observed that the changes in immunoregulatory miRNAs in quarters with chronic subclinical mastitis were cow-individual, however, the most upregulated miRNA was bta-miR-223-3p across all individuals.ConclusionsOur results showed that the miRNA profile and particle size characteristics remained constant throughout consecutive days, suggesting that miRNAs packed in EVs are physiological state-specific. In addition, infected quarters were solely affected while adjacent healthy quarters remained unaffected. Finally, the cow-individual miRNA changes pointed towards infection-specific alterations.

Drug nanocarriers (NCs) with sizes usually below 200 nm are gaining increasing interest in the treatment of severe diseases such as cancer and infections. Characterization methods to investigate the morphology and physicochemical properties of multifunctional NCs are key in their optimization and in the study of their in vitro and in vivo fate. Whereas a variety of methods has been developed to characterize “bulk” NCs in suspension, the scope of this review is to describe the different approaches for the NC characterization on an individual basis, for which fewer techniques are available. The accent is put on methods devoid of labelling, which could lead to artefacts. For each characterization method, the principles and approaches to analyze the data are presented in an accessible manner. Aspects related to sample preparation to avoid artefacts are indicated, and emphasis is put on examples of applications. NC characterization on an individual basis allows gaining invaluable information in terms of quality control, on: i) NC localization and fate in biological samples; ii) NC morphology and crystallinity; iii) distribution of the NC components (drugs, shells), and iv) quantification of NCs’ chemical composition. The individual characterization approaches are expected to gain increasing interest in the near future.

Nanoscale extracellular vesicles (EVs) represent a unique cellular derivative that reflect the therapeutic potential of mesenchymal stem cells (MSCs) toward tissue engineering and injury repair without the logistical and safety concerns of utilizing living cells. However, upon systemic administration in vivo,EVs undergo rapid clearance and typically lack controlled targeted delivery, thus reducing their effectiveness in therapeutic regenerative therapies. Here, we describe a strategy that enables long-term in vivo spatial EV retention by chemoselective immobilization of metabolically incoporated azido ligand-bearing EVs (azido-EVs) within a dibenzocyclooctyne-modified collagen hydrogel. MSC-derived azido-EVs exhibit comparable morphological and functional properties as their non-labeled EV counterparts and, when immobilized within collagen hydrogel implants via click chemistry, they elicited more robust host cell infiltration, angiogenic and immunoregulatory responses including vascular ingrowth and macrophage recruitment compared to ten times the higher dose required by non-immobilized EVs. We envision this technology will enable a wide range of applications to spatially promote vascularization and host integration relevant to tissue engineering and regenerative medicine applications.

The emergence of nanotechnology as a key enabling technology over the past years has opened avenues for new and innovative applications in nanomedicine. From the business aspect, the nanomedicine market was estimated to worth USD 293.1 billion by 2022 with a perception of market growth to USD 350.8 billion in 2025. Despite these opportunities, the underlying challenges for the future of engineered nanomaterials (ENMs) in nanomedicine research became a significant obstacle in bringing ENMs into clinical stages. These challenges include the capability to design bias-free methods in evaluating ENMs’ toxicity due to the lack of suitable detection and inconsistent characterization techniques. Therefore, in this literature review, the state-of-the-art of engineered nanomaterials in nanomedicine, their toxicology issues, the working framework in developing a toxicology benchmark and technical characterization techniques in determining the toxicity of ENMs from the reported literature are explored. Keywords: engineered nanomaterials, nanomedicine, nanotoxicology, particle tracking analysis, asymmetric flow field-flow fractionation, Taylor dispersion analysis

BackgroundAmyotrophic lateral sclerosis (ALS) is a multifactorial, multisystem motor neuron disease for which currently there is no effective treatment. There is an urgent need to identify biomarkers to tackle the disease’s complexity and help in early diagnosis, prognosis, and therapy. Extracellular vesicles (EVs) are nanostructures released by any cell type into body fluids. Their biophysical and biochemical characteristics vary with the parent cell’s physiological and pathological state and make them an attractive source of multidimensional data for patient classification and stratification.MethodsWe analyzed plasma-derived EVs of ALS patients (n = 106) and controls (n = 96), and SOD1G93A and TDP-43Q331K mouse models of ALS. We purified plasma EVs by nickel-based isolation, characterized their EV size distribution and morphology respectively by nanotracking analysis and transmission electron microscopy, and analyzed EV markers and protein cargos by Western blot and proteomics. We used machine learning techniques to predict diagnosis and prognosis.ResultsOur procedure resulted in high-yield isolation of intact and polydisperse plasma EVs, with minimal lipoprotein contamination. EVs in the plasma of ALS patients and the two mouse models of ALS had a distinctive size distribution and lower HSP90 levels compared to the controls. In terms of disease progression, the levels of cyclophilin A with the EV size distribution distinguished fast and slow disease progressors, a possibly new means for patient stratification. Immuno-electron microscopy also suggested that phosphorylated TDP-43 is not an intravesicular cargo of plasma-derived EVs.ConclusionsOur analysis unmasked features in plasma EVs of ALS patients with potential straightforward clinical application. We conceived an innovative mathematical model based on machine learning which, by integrating EV size distribution data with protein cargoes, gave very high prediction rates for disease diagnosis and prognosis.

One of the largest surfaces of the human body in contact with the environment is the respiratory epithelium, constituted by different specialized cells. Alveolar type I cells are involved in gas exchange whereas alveolar type II cells prevent the alveoli from collapsing due to the synthesis and secretion of lung surfactant (LS). This lipid-protein complex covers the alveolar surface and reduces surface tension at the air-liquid interface. LS, the first element in contact with inhaled air, is also involved in innate defense mechanisms. Surfactant protein C (SP-C) is a small hydrophobic transmembrane protein crucial for the biophysical function of LS. Different studies have revealed that the palmitoylation state of SP-C modulates important protein-lipid interactions within surfactant layers. Moreover, recent research has revealed that SP-C oligomerization, presumably through two structural motifs in SP-C sequence, could promote membrane fragmentation and enhance membrane vesicle alveolar uptake highlighting a key potential role of SP-C in LS homeostasis. In this work, we have analyzed the effect of palmitoylation on SP-C-promoted membrane fragmentation and vesicle uptake in the LS context. To do so, we have compared the behavior in different assays of the native palmitoylated protein and a recombinant SP-C version lacking palmitoyl chains, once reconstituted in two different lipid models mimicking LS membranes. Likewise, we have studied the implication of the proposed dimerization motifs in the SP-C sequence by testing synthetic peptides with selected sequence variations. Results from tunable resistive pulse sensing experiments suggest that both palmitoylation and the oligomerization state of SP-C are important to promote fission of membranes. Protein oligomerization and membrane fragmentation have been also analyzed with respect to membrane vesicle internalization by alveolar-derived cell lines, as evaluated by flow cytometry of cell cultures exposed to fluorescent lipid/protein complexes.

Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messengers, have high potential as biomarkers. EVs are usually collected from in vitro sources, such as cell culture media or biofluids, and not from tissues. Techniques enabling direct collection of EVs from tissues will extend the applications of EVs. We compared methods for separating EVs from solid liver, heart, and skeletal muscle. Compared with a precipitation method, an ultracentrifugation-based method for collection of EVs from solid tissues yielded a higher proportion of EVs positive for EV-related markers, with minimum levels of intracellular organelle-related markers. Some tissue-specific modifications, such as a sucrose cushion step, may improve the yield and purity of the collected EVs.

Purpose: In a benchwork particle counting analytical evaluation, the number and type of particles in intravitreal injection formulations of three different agents against vascular endothelial growth factor were investigated. Methods: Commercially available ready-to-use aflibercept and brolucizumab glass syringes, vials containing bevacizumab (off-label use in ophthalmology), and repackaged ready-to-use plastic syringes containing bevacizumab were tested without filtration. Total visible, subvisible, and nanoparticles numbers and size distributions were quantified using light obscuration, flow imaging, resonant mass measurement (RMM), tunable resistive pulse sensing, and dynamic light scattering. Results: Repackaged bevacizumab showed overall low particle numbers, aflibercept showed high numbers of micrometer sized particles but low nanoparticle numbers, brolucizumab showed low to moderate numbers of micrometer sized particles but high nanoparticle numbers. RMM measurements identified particles in the nanometer range as either proteinaceous or silicon oil; the nature of the other particles was not further evaluated. Conclusions: Repackaged bevacizumab shows no inferior particle quality compared to ready-to-use products. It is relevant to study nanoparticle load of the products as the micrometer-sized particle numbers do not in all cases correlate to nanoparticle counts. Particularly for the high concentration product Beovu (brolucizumab), high nanoparticle numbers were found despite low numbers of micrometer sized particles. Silicone oil droplets did not account for high particle numbers as the measured numbers were low. Translational Relevance: Different side effects are registered in different frequencies with different intravitreal anti-VEGF-drugs and syringes, which are applied by injection by small 30G needles through the sclera directly to the intravitreal cavity. The study of nanoparticles and silicone oil droplets may be able to contribute to narrowing down the causes.

Astrocytes-derived extracellular vesicles (EVs) are key players in glia-neuron communication. However, whether EVs interact with neurons at preferential sites and how EVs reach these sites on neurons remains elusive. Using optical manipulation to study single EV-neuron dynamics, we here show that large EVs scan the neuron surface and use neuronal processes as highways to move extracellularly. Large EV motion on neurites is driven by the binding of EV to a surface receptor that slides on neuronal membrane, thanks to actin cytoskeleton rearrangements. The use of prion protein (PrP)-coated synthetic beads and PrP knock out EVs/neurons points at vesicular PrP and its receptor(s) on neurons in the control of EV motion. Surprisingly, a fraction of large EVs contains actin filaments and has an independent capacity to move in an actin-mediated way, through intermittent contacts with the plasma membrane. Our results unveil, for the first time, a dual mechanism exploited by astrocytic large EVs to passively/actively reach target sites on neurons moving on the neuron surface.

Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication. They have important roles in numerous physiological and pathological processes, and show considerable promise as novel biomarkers of disease, as therapeutic agents and as drug delivery vehicles. Intriguingly, however, understanding of the cellular and molecular mechanisms that govern the many observed functions of EVs remains far from comprehensive, at least partly due to technical challenges in working with these small messengers. Here, we highlight areas of consensus as well as contentious issues in our understanding of the intracellular and intercellular journey of EVs: from biogenesis, release and dynamics in the extracellular space, to interaction with and uptake by recipient cells. We define knowledge gaps, identify key questions and challenges, and make recommendations on how to address these.

MicroRNAs (miRNAs) are cargo carried by extracellular vesicles (EVs) and are associated with cell–cell interactions. The response to the cellular environment, such as disease states, genetic/metabolic changes, or differences in cell type, highly regulates cargo sorting to EVs. However, morphological features during EV formation and secretion involving miRNA loading are unknown. This study developed a new method of EV loading using cell resealing and reconstituted the elementary miRNA-loading processes. Morphology, secretory response, and cellular uptake ability of EVs obtained from intact and resealed HeLa cells were comparable. Exogenously added soluble factors were introduced into multivesicular endosomes (MVEs) and their subsequent secretion to the extracellular region occurred in resealed HeLa cells. In addition, miRNA transport to MVEs and miRNA encapsulation to EVs followed a distinct pathway regulated by RNA-binding proteins, such as Argonaute and Y-box binding protein 1, depending on miRNA types. Our cell-resealing system can analyze disease-specific EVs derived from disease model cells, where pathological cytosol is introduced into cells. Thus, EV formation in resealed cells can be used not only to create a reconstitution system to give mechanistic insight into EV encapsulation but also for applications such as loading various molecules into EVs and identifying disease-specific EV markers.

Extracellular Vesicles and Circulating Nucleic Acids is an open access journal, focusing on extracellular vesicles and circulating nucleic acids including DNA, RNA, and miRNA and their therapeutic use.

Skeletal muscle growth and maintenance depend on two tightly regulated processes, myogenesis and muscle regeneration. Both processes involve a series of crucial regulatory molecules including muscle-specific microRNAs, known as myomiRs. We recently showed that four myomiRs, miR-1, miR-133a, miR-133b, and miR-206, are encapsulated within muscle-derived exosomes and participate in local skeletal muscle communication. Although these four myomiRs have been extensively studied for their function in muscles, no information exists regarding their endogenous and exosomal levels across age. Here we aimed to identify any age-related changes in the endogenous and muscle-derived exosomal myomiR levels during acute skeletal muscle growth. The four endogenous and muscle-derived myomiRs were investigated in five skeletal muscles (extensor digitorum longus, soleus, tibialis anterior, gastrocnemius, and quadriceps) of 2-week-1-year-old wild-type male mice. The expression of miR-1, miR-133a, and miR-133b was found to increase rapidly until adolescence in all skeletal muscles, whereas during adulthood it remained relatively stable. By contrast, endogenous miR-206 levels were observed to decrease with age in all muscles, except for soleus. Differential expression of the four myomiRs is also inversely reflected on the production of two protein targets; serum response factor and connexin 43. Muscle-derived exosomal miR-1, miR-133a, and miR-133b levels were found to increase until the early adolescence, before reaching a plateau phase. Soleus was found to be the only skeletal muscle to release exosomes enriched in miR-206. In this study, we showed for the first time an in-depth longitudinal analysis of the endogenous and exosomal levels of the four myomiRs during skeletal muscle development. We showed that the endogenous expression and extracellular secretion of the four myomiRs are associated to the function and size of skeletal muscles as the mice age. Overall, our findings provide new insights for the myomiRs' significant role in the first year of life in mice.

Extracellular vesicles (EVs) are small membrane-bound particles, which include exosomes, micro vesicles (MVs) and various-sized vesicles, released by healthy and diseased cells. EVs also include other vesicular structures, such as large apoptotic bodies (1–5 μm), as well as membrane particles (50–80 nm) originating from the plasma membrane. However, exosomes are nanosize (≈30–100 nm) extracellular vesicles of endocytic origin that are bud-off by most types of cells and circulate in bodily fluids. Extracellular nanovesicles contain a large variety of biomolecules, including miRNA, RNA, DNA, proteins, signaling peptides and lipids, that can have diagnostic and therapeutic value. The spectrum of the existing scientific interest in extracellular nanovesicles is comprehensive, which ranges from understanding their functions and pathways to their potential clinical usage. EVs can be obtained from different body fluids with minimally invasive techniques (e.g., urine, plasma, serum), so they are most useful in disease diagnosis. High yield and purity contribute to the accurate diagnosis of various diseases, but damaged EVs and impurities can cause misinterpreted results. Over the last decade, a plethora of approaches have been developed for examining EVs using optical and non-optical tools. However, EV isolation methods have different yields and purities. Moreover, the isolation method that is most appropriate to maximize EVs recovery depends on the different experimental situations. This review explores the emerging use of micro and nano-technologies to isolate and characterize exosomes and microvesicles (MVs) from different biological samples, and the application of these technologies for the monitoring and diagnosis of different pathological conditions.

The constant dialogue between the plant world and the animal world (including man among them) has been known since the time of Adam and Eve, where an apple was the origin of the evils of the world. Apart from Snow White—who might have something to object to when it comes to the use of apples—fruits, plants, and natural extracts have been known for millennia as remedies for human health-related ailments. In the light of such evidence, the aim of the present work was to investigate from a biological point of view the potential role of apple exosomes in inflammatory processes on human cells. To this end we isolated and characterized apple exosomes and treated human cells such as macrophages and NCTC L929 as cancer cells in order to evaluate the tumorigenic and anti-inflammatory effect of apple exomes. Microscopic and molecular biology analyses were conducted to characterize exosomes and to assess cell proliferation, death, and miRNA line, as well as gene expression and the uptake of exosomes by cells. The results confirm the absolute biological safety of exosomes and their anti-inflammatory effect, mediated mainly by miRNA146 production by M2 macrophages.

Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are promising candidates for regenerative medicine; however, the lack of scalable methods for high quantity EV production limits their application. In addition, signature EV-derived proteins shared in 3D environments and 2D surfaces, remain mostly unknown. Herein, we present a platform combining MSC microfiber culture with ultracentrifugation purification for high EV yield. Within this platform, a high quantity MSC solution (∼3 × 108 total cells) is encapsulated in a meter-long hollow hydrogel-microfiber via coaxial bioprinting technology. In this 3D core–shell microfiber environment, MSCs express higher levels of stemness markers (Oct4, Nanog, Sox2) than in 2D culture, and maintain their differentiation capacity. Moreover, this platform enriches particles by ∼1009-fold compared to conventional 2D culture, while preserving their pro-angiogenic properties. Liquid chromatography-mass spectrometry characterization results demonstrate that EVs derived from our platform and conventional 2D culturing have unique protein profiles with 3D-EVs having a greater variety of proteins (1023 vs 605), however, they also share certain proteins (536) and signature MSC-EV proteins (10). This platform, therefore, provides a new tool for EV production using microfibers in one culture dish, thereby reducing space, labor, time, and cost.

The mutational status of the epidermal growth factor receptor (EGFR) guides the stratification of non-small cell lung cancer (NSCLC) patients for treatment with tyrosine kinase inhibitors (TKIs). A liquid biopsy test on cell-free DNA is recommended as a clinical decision-supporting tool, although it has limited sensitivity. Here, we comparatively investigated the extracellular vesicle (EV)-RNA as an independent source for multidimensional and longitudinal EGFR profiling in a cohort of 27 NSCLC patients. We introduced and validated a new rapid, highly specific EV-RNA test with wild-type (WT) and mutant-sensitive probes (E746-A750del, L858R, and T790M). We included a cohort of 20 NSCLC patients with EGFR WT tumor tissues and systematically performed molecular EV-RNA and circulating tumor DNA analyses with clinical data statistics and biophysical profiles of EVs. At the single-patient level, we detected variegated tumor heterogeneity dynamics supported by combinations of driver EGFR mutations. EV-RNA-based mutation analysis showed an unprecedented sensitivity of over 90%. The resistance-associated mutation T790M frequently pre-existed at baseline with a gained EV-transcript copy number at progression, while the general mutational burden was mostly decreasing during the intermediate follow-up. The biophysical profile of EVs and the quantitative assessment of T790M revealed an association with tumor size determined by the sum of the longest diameters in target lesions. Vesicular RNA provides a validated tool suitable for use in clinical practice to investigate the dynamics of common driver EGFR mutations in NSCLC patients receiving TKIs.

Androgenetic alopecia (AGA) is the most common type of hair loss in men and women. Dihydrotestosterone (DHT) and androgen receptor (AR) levels are increased in patients with AGA, and DHT-AR signaling correlates strongly with AGA pathogenesis. In this study, treatment with self-assembled micelle inhibitory RNA (SAMiRNA) nanoparticle-type siRNA selectively suppressed AR expression in vitro. Clinical studies with application of SAMiRNA to the scalp and massaging to deliver it to the hair follicle confirmed its efficacy in AGA. For identification of a potent SAMiRNA for AR silencing, 547 SAMiRNA candidates were synthesized and screened. SAMiRNA-AR68 (AR68) was the most potent and could be efficiently delivered to human follicle dermal papilla cells (HFDPCs) and hair follicles, and this treatment decreased the AR mRNA and protein levels. We confirmed that 10 µM AR68 elicits no innate immune response in human PBMCs and no cytotoxicity up to 20 µM with HFDP and HaCaT cells. Clinical studies were performed in a randomized and double-blind manner with two different doses and frequencies. In the low-dose (0.5 mg/ml) clinical study, AR68 was applied three times per week for 24 weeks, and through quantitative analysis using a phototrichogram, we confirmed increases in total hair counts. In the high-dose (5 mg/ml) clinical study, AR68 was given once per week for 24 weeks and showed 83% efficacy in increasing hair counts compared with finasteride. No side effects were observed. Therefore, SAMiRNA targeting AR mRNA is a potential novel topical treatment for AGA.

In tuberous sclerosis complex (TSC), Tsc2 mutations are associated with more severe disease manifestations than Tsc1 mutations and the role of extracellular vesicles (EVs) in this context is not yet studied. We report a comparative analysis of EVs derived from isogenic renal cells except for Tsc1 or Tsc2 gene status and hypothesized that in spite of having similar physical characteristics, EVs modulate signaling pathways differently, thus leading to TSC heterogenicity. We used mouse inner medullary collecting duct (mIMCD3) cells with the Tsc1 (T1G cells) or Tsc2 (T2J cells) gene disrupted by CRISPR/CAS9. EVs were isolated from the cell culture media by size-exclusion column chromatography followed by detailed physical and chemical characterization. Physical characterization of EVs was accessed by tunable resistive pulse sensing and dynamic light scattering, revealing similar average sizes and zeta potentials (at pH 7.4) for EVs from mIMCD3 (123.5 ± 5.7 nm and -16.3 ± 2.1 mV), T1G cells (131.5 ± 8.3 nm and -19.8 ± 2.7 mV), and T2J cells (127.3 ± 4.9 nm and -20.2 ± 2.1 mV). EVs derived from parental mIMCD3 cells and both mutated cell lines were heterogeneous (>90% of EVs < 150 nm) in nature. Immunoblotting detected cilial Hedgehog signaling protein Arl13b; intercellular proteins TSG101 and Alix; and transmembrane proteins CD63, CD9, and CD81. Compared to Tsc2 deletion, Tsc1 deletion cells had reduced EV production and release rates. EVs from Tsc1 mutant cells altered mTORC1, autophagy, and β-catenin pathways differently than EVs from Tsc2-mutated cells. Quantitative PCR analysis revealed the down regulation of miR-212a-3p and miR-99a-5p in EVs from Tsc2-mutated cells compared to EVs from Tsc1-mutant cells. Thus, EV-derived miR-212-3p and mIR-99a-5p axes may represent therapeutic targets or biomarkers for TSC disease.

Background Although exosomes, as byproducts of human umbilical cord mesenchymal stem cells (hUC-MSCs), have been demonstrated to be an effective therapy for traumatic spinal cord injury (SCI), their mechanism of action remains unclear. Methods We designed and performed this study to determine whether exosomes attenuate the lesion size of SCI by ameliorating neuronal injury induced by a secondary inflammatory storm and promoting neurite outgrowth. We determined the absolute levels of all exosomal miRNAs and investigated the potential mechanisms of action of miR-199a-3p/145-5p in inducing neurite outgrowth in vivo and in vitro.ResultsmiR-199a-3p/145-5p, which are relatively highly expressed miRNAs in exosomes, promoted PC12 cell differentiation suppressed by lipopolysaccharide (LPS) in vitro through modulation of the NGF/TrkA pathway. We also demonstrated that Cblb was a direct target of miR-199a-3p and that Cbl was a direct target of miR-145-5p. Cblb and Cbl gene knockdown resulted in significantly decreased TrkA ubiquitination levels, subsequently activating the NGF/TrkA downstream pathways Akt and Erk. Conversely, overexpression of Cblb and Cbl was associated with significantly increased TrkA ubiquitination level, subsequently inactivating the NGF/TrkA downstream pathways Akt and Erk. Western blot and coimmunoprecipitation assays confirmed the direct interaction between TrkA and Cblb and TrkA and Cbl. In an in vivo experiment, exosomal miR-199a-3p/145-5p was found to upregulate TrkA expression at the lesion site and also promote locomotor function in SCI rats. ConclusionsIn summary, our study showed that exosomes transferring miR-199a-3p/145-5p into neurons in SCI rats affected TrkA ubiquitination and promoted the NGF/TrkA signaling pathway, indicating that hUC-MSC-derived exosomes may be a promising treatment strategy for SCI.

Abstract Extracellular vesicles (EVs) have been shown as key mediators of extracellular small RNA transport. However, carriers of cell-free messenger RNA (cf-mRNA) in human biofluid and their association with cancer remain poorly understood. Here, we performed a transcriptomic analysis of size-fractionated plasma from lung cancer, liver cancer, multiple myeloma, and healthy donors. Morphology and size distribution analysis showed the successful separation of medium and small EVs and non-vesicular carriers. We developed a strategy to purify and sequence ultra-low amounts of cf-mRNA from vesicular and non-vesicular subpopulations with the implementation of RNA spike-ins to control for technical variability and to normalize for intrinsic drastic differences in the amount of cf-mRNA carried in each plasma fraction. We found that the majority of cf-mRNA was enriched and protected in EVs with remarkable stability in RNase-rich environments. We observed specific enrichment patterns of cancer-associated cf-mRNA in each vesicular and non-vesicular subpopulation. The EV-enriched differentiating genes were associated with specific biological pathways, such as immune systems, liver function, and toxic substance regulation in lung cancer, liver cancer, and multiple myeloma, respectively. Our results suggest that dissecting the complexity of EVs subpopulations illuminates their biological significance and offers a promising liquid biopsy approach.

One major part of therapeutic oncolytic viruses (OV) manufacturing is a membrane-based process with a major challenge of membrane fouling and consequent product loss. A small-scale microfiltration setup was developed, allowing online transmembrane pressure (TMP) measurement through a constant flux filtration using a low volume of a representative OV solution (< 3 mL). Using this setup, the effects of different additives including various proteins (bovine serum albumin and alpha-lactalbumin) and organic polymers (polyethylene glycol and polyvinylpyrrolidone) on microfiltration of the OV solution were screened. Results demonstrated that examined proteins significantly decreased membrane fouling rates and increased the virus recoveries. An addition of 5% bovine serum albumin (BSA) to the virus solution increased the virus recovery about 6-times compared to microfiltration of the virus solution without any additive. In contrast, none of the organic polymers could imitate effects of the protein additives. In a separate set of experiment, to study effect of protein on the membrane surface, the membrane surface was pre-blocked using a BSA protein solution and then subsequently utilized to filter the virus solution. This result also demonstrated a significant increase in virus recovery through the blocked membrane, about 4-times higher virus recovery compared to a non-blocked membrane.

The treatment of central nervous system (CNS) pathologies is severely hampered by the presence of tightly regulated CNS barriers that restrict drug delivery to the brain. An increasing amount of data suggests that extracellular vesicles (EVs), i.e., membrane derived vesicles that inherently protect and transfer biological cargoes between cells, naturally cross the CNS barriers. Moreover, EVs can be engineered with targeting ligands to obtain enriched tissue targeting and delivery capacities. In this review, we provide a detailed overview of the literature describing a natural and engineered CNS targeting and therapeutic efficiency of different cell type derived EVs. Hereby, we specifically focus on peripheral administration routes in a broad range of CNS diseases. Furthermore, we underline the potential of research aimed at elucidating the vesicular transport mechanisms across the different CNS barriers. Finally, we elaborate on the practical considerations towards the application of EVs as a brain drug delivery system.

Extracellular vesicles (EVs) including exosomes act as intercellular communicators by transferring protein and microRNA cargoes, yet the role of EV lipids remains unclear. Here, we show that the pro-tumorigenic action of lymphoma-derived EVs is augmented via secreted phospholipase A2 (sPLA2)-driven lipid metabolism. Hydrolysis of EV phospholipids by group X sPLA2, which was induced in macrophages of Epstein-Barr virus (EBV) lymphoma, increased the production of fatty acids, lysophospholipids, and their metabolites. sPLA2-treated EVs were smaller and self-aggregated, showed better uptake, and increased cytokine expression and lipid mediator signaling in tumor-associated macrophages. Pharmacological inhibition of endogenous sPLA2 suppressed lymphoma growth in EBV-infected humanized mice, while treatment with sPLA2-modified EVs reversed this phenotype. Furthermore, sPLA2 expression in human large B cell lymphomas inversely correlated with patient survival. Overall, the sPLA2-mediated EV modification promotes tumor development, highlighting a non-canonical mechanistic action of EVs as an extracellular hydrolytic platform of sPLA2.

Fibrosis is a serious unintended side effect of radiation therapy. In this study, we aimed to investigate whether amphiregulin (AREG) plays a critical role in fibrosis development after total-body irradiation (TBI). We found that the expression of AREG and fibrotic markers, such as α-smooth muscle actin (α-SMA) and collagen type I alpha 1 (COL1α1), was elevated in the kidneys of 6 Gy TBI mice. Expression of AREG and α-SMA was mainly elevated in the proximal and distal tubules of the kidney in response to TBI, which was confirmed by immunofluorescence staining. Knockdown of Areg mRNA using self-assembled-micelle inhibitory RNA (SAMiRNA) significantly reduced the expression of fibrotic markers, including α-SMA and COL1α1, and inflammatory regulators. Finally, intravenous injections of SAMiRNA targeting mouse Areg mRNA (SAMiRNA-mAREG) diminished radiation-induced collagen accumulation in the renal cortex and medulla. Taken together, the results of the present study suggest that blocking of AREG signaling via SAMiRNA-mAREG treatment could be a promising therapeutic approach to alleviate radiation-induced kidney fibrosis.

Background Duchenne muscular dystrophy (DMD) is caused by mutations of the gene that encodes the protein dystrophin. A loss of dystrophin leads to severe and progressive muscle wasting in both skeletal and heart muscles. Human induced pluripotent stem cells (hiPSCs) and their derivatives offer important opportunities to treat a number of diseases. Here, we investigated whether givinostat (Givi), a histone deacetylase inhibitor, with muscle differentiation properties could reprogram hiPSCs into muscle progenitor cells (MPC) for DMD treatment. Methods MPC were generated from hiPSCs by treatment with CHIR99021 and givinostat called Givi-MPC or with CHIR99021 and fibroblast growth factor as control-MPC. The proliferation and migration capacity were investigated by CCK-8, colony, and migration assays. Engraftment, pathological changes, and restoration of dystrophin were evaluated by in vivo transplantation of MPC. Conditioned medium from cultured MPC was collected and analyzed for extracellular vesicles (EVs). Results Givi-MPC exhibited superior proliferation and migration capacity compared to control-MPC. Givi-MPC produced less reactive oxygen species (ROS) after oxidative stress and insignificant expression of IL6 after TNF-α stimulation. Upon transplantation in cardiotoxin (CTX)-injured hind limb of Mdx/SCID mice, the Givi-MPC showed robust engraftment and restored dystrophin in the treated muscle than in those treated with control-MPC or human myoblasts. Givi-MPC significantly limited infiltration of inflammatory cells and reduced muscle necrosis and fibrosis. Additionally, Givi-MPC seeded the stem cell pool in the treated muscle. Moreover, EVs released from Givi-MPC were enriched in several miRNAs related to myoangiogenesis including miR-181a, miR-17, miR-210 and miR-107, and miR-19b compared with EVs from human myoblasts. Conclusion sIt is concluded that hiPSCs reprogrammed into MPC by givinostat possessing anti-oxidative, anti-inflammatory, and muscle gene-promoting properties effectively repaired injured muscle and restored dystrophin in the injured muscle.

tRNA derived small RNAs (tRFs) have recently received extensive attention; however, the effects of tRFs in exosome as biomarkers has been less studied. The objective of this study was to validate novel diagnostic exosomal tRFs with sensitivity and specificity for non-small cell lung cancer (NSCLC). Exosomes extracted from plasma of NSCLC patients and healthy individuals were identified by transmission electron microscopy (TEM), qNano and western blots. The differentially expressed tRFs were screened by high-throughput sequencing in plasma exosomes of NSCLC patients and healthy individuals, and further verified by Quantitative Real-Time PCR (qRT-PCR). To assess the diagnostic efficacy of exosomal tRFs for NSCLC, receiver operating characteristic (ROC) curves were used next. The expression levels of exosomal tRF-Leu-TAA-005, tRF-Asn-GTT-010, tRF-Ala-AGC-036, tRF-Lys-CTT-049, and tRF-Trp-CCA-057 were significantly decreased in NSCLC patients and early-stage NSCLC patients compared to healthy individuals. Notably, the exepression of tRF-Leu-TAA-005, tRF-Asn-GTT-010, tRF-Ala-AGC-036, tRF-Lys-CTT-049, and tRF-Trp-CCA-057 in the exosomes were higher than the exosome depleted supernatant (EDS). Our results showed that the levels of exosomal tRF-Leu-TAA-005, tRF-Asn-GTT-010, tRF-Ala-AGC-036, tRF-Lys-CTT-049, and tRF-Trp-CCA-057 were significantly downregulated in NSCLC patients. This suggests that these five exosomal tRFs may be promising diagnostic biomarkers for NSCLC.

Background: Exosomal miRNA had been proved as the promising biomarkers for multiple cancers including epithelial ovarian cancer (EOC). This study aimed to validate the diagnostic accuracy of exosomal miR-320d, miR-4479, and miR-6763-5p for EOC. Materials and methods: Exosomes isolated from the plasma by ultracentrifugation were verified using TEM, qNano and western blot. MiRNAs sequencing was used to screen out the differential exosomal miRNAs and miR-320d, miR-4479, and miR-6763-5p were selected as candidates, which were further verified by RT-qPCR in 168 healthy donors and 161 primary EOC patients. Besides, the diagnostic accuracy of these three exosomal miRNAs were evaluated using the receiver operating characteristic curve (ROC). Results: MiRNAs sequencing revealed 95 differential exosomal miRNAs between EOC patients and healthy donors. Subsequently, exosomal miR-320d, miR-4479, and miR-6763-5p were significantly down regulated in EOC patients compared with healthy controls and benign patients. More importantly, these three miRNAs could serve as circulating diagnostics biomarkers for EOC, possessing areas under the curve (AUC) of 0.6549, 0.7781, and 0.6834, respectively. Moreover, these three exosomal miRNAs levels were closely associated with lymph node metastasis, meanwhile exosomal miR-320d and miR-4479 expression was related to tumor stage. Conclusion: Exosomal miR-320d, miR-4479, and miR-6763-5p might serve as potential biomarkers for EOC.

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.

OBJECTIVES: In the premature newborn, perinatal inflammation mediated by microglia contributes significantly to neurodevelopmental injuries including white matter injury (WMI). Brain inflammation alters development through neuroinflammatory processes mediated by activation of homeostatic microglia toward a pro-inflammatory and neurotoxic phenotype. Investigating immune regulators of microglial activation is crucial to find effective strategies to prevent and treat WMI. METHODS: Ex vivo microglial cultures and a mouse model of WMI induced by perinatal inflammation (interleukin-1-beta [IL-1β] and postnatal days 1-5) were used to uncover and elucidate the role of microRNA-146b-5p in microglial activation and WMI. RESULTS: A specific reduction in vivo in microglia of Dicer, a protein required for microRNAs maturation, reduces pro-inflammatory activation of microglia and prevents hypomyelination in our model of WMI. Microglial miRNome analysis in the WMI model identified miRNA-146b-5p as a candidate modulator of microglial activation. Ex vivo microglial cell culture treated with the pro-inflammatory stimulus lipopolysaccharide (LPS) led to overexpression of immunomodulatory miRNA-146b-5p but its drastic reduction in the microglial extracellular vesicles (EVs). To increase miRNA-146b-5p expression, we used a 3DNA nanocarrier to deliver synthetic miRNA-146b-5p specifically to microglia. Enhancing microglial miRNA-146b-5p overexpression significantly decreased LPS-induced activation, downregulated IRAK1, and restored miRNA-146b-5p levels in EVs. In our WMI model, 3DNA miRNA-146b-5p treatment significantly prevented microglial activation, hypomyelination, and cognitive defect induced by perinatal inflammation. INTERPRETATIONS: These findings support that miRNA-146b-5p is a major regulator of microglia phenotype and could be targeted to reduce the incidence and the severity of perinatal brain injuries and their long-term consequences. ANN NEUROL 2022;91:48-65.

Extracellular vesicles, especially the larger fraction (LEVs - large extracellular vesicles), are believed to be an important means of intercellular communication. Earlier studies on LEVs have shown their healing properties, especially in the vascular cells of diabetic patients. Uptake of LEVs by endothelial cells and internalization of their cargo have also been demonstrated. Endothelial cells change their properties under hyperglycemic conditions (HGC), which reduces their activity and is the cause of endothelial dysfunction. The aim of our study was to investigate how human umbilical vein endothelial cells (HUVECs) change their biological properties: shape, mobility, cell surface stiffness, as well as describe the activation of metabolic pathways after exposure to the harmful effects of HGC and the administration of LEVs released by endothelial cells. We obtained LEVs from HUVEC cultures in HGC and normoglycemia (NGC) using the filtration and ultracentrifugation methods. We assessed the size of LEVs and the presence of biomarkers such as phosphatidylserine, CD63, beta-actin and HSP70. We analyzed the LEVs uptake efficiency by HUVECs, HUVEC shape, actin cytoskeleton remodeling, surface stiffness and finally gene expression by mRNA analysis. Under HGC conditions, HUVECs were larger and had a stiffened surface and a strengthened actin cortex compared to cells under NGC condition. HGC also altered the activation of metabolic pathways, especially those related to intracellular transport, metabolism, and organization of cellular components. The most interesting observation in our study is that LEVs did not restore cell motility disturbed by HGC. Although, LEVs were not able to reverse this deleterious effect of HGC, they activated transcription of genes involved in protein synthesis and vesicle trafficking in HUVECs.

Virus-based biologicals are one of the most promising biopharmaceuticals of the 21st century medicine and play a significant role in the development of innovative therapeutic, prophylactic, and clinical applications. Oncolytic virus manufacturing scale can range from 5 L in research and development up to 50 L for clinical studies and reach hundreds of liters for commercial scale. The inherent productivity and high integration potential of periodic counter-current chromatography (PCC) offer a transversal solution to decrease equipment footprint and the reduction of several non-value-added unit operations. We report on the design of an efficient PCC process applied to the intermediate purification of oncolytic adenovirus. The developed ion-exchange chromatographic purification method was carried out using a four-column setup for three different scenarios: (i) variation in the feedstock, (ii) potential use of a post-load washing step to improve virus recovery, and (iii) stability during extended operation. Obtained virus recoveries (57%-86%) and impurity reductions (>80% DNA, and >70% total protein) match or overcome batch purification. Regarding process stability and automation, our results show that not only the dynamic control strategy used is able to suppress perturbations in the sample inlet but also allows for unattended operation in the case of ion exchange capture.

Proximal tubular epithelial cells (PTEC) are central players in inflammatory kidney diseases. However, the complex signalling mechanism/s via which polarized PTEC mediate disease progression are poorly understood. Small extracellular vesicles (sEV), including exosomes, are recognized as fundamental components of cellular communication and signalling courtesy of their molecular cargo (lipids, microRNA, proteins). In this study, we examined the molecular content and function of sEV secreted from the apical versus basolateral surfaces of polarized human primary PTEC under inflammatory diseased conditions. PTEC were cultured under normal and inflammatory conditions on Transwell inserts to enable separate collection and isolation of apical/basolateral sEV. Significantly increased numbers of apical and basolateral sEV were secreted under inflammatory conditions compared with equivalent normal conditions. Multi-omics analysis revealed distinct molecular profiles (lipids, microRNA, proteins) between inflammatory and normal conditions for both apical and basolateral sEV. Biological pathway analyses of significantly differentially expressed molecules associated apical inflammatory sEV with processes of cell survival and immunological disease, while basolateral inflammatory sEV were linked to pathways of immune cell trafficking and cell-to-cell signalling. In line with this mechanistic concept, functional assays demonstrated significantly increased production of chemokines (monocyte chemoattractant protein-1, interleukin-8) and immuno-regulatory cytokine interleukin-10 by peripheral blood mononuclear cells activated with basolateral sEV derived from inflammatory PTEC. We propose that the distinct molecular composition of sEV released from the apical versus basolateral membranes of human inflammatory PTEC may reflect specialized functional roles, with basolateral-derived sEV pivotal in modulating tubulointerstitial inflammatory responses observed in many immune-mediated kidney diseases. These findings provide a rationale to further evaluate these sEV-mediated inflammatory pathways as targets for biomarker and therapeutic development.

The BK polyomavirus (BKPyV) is a ubiquitous human virus that persists in the renourinary epithelium. Immunosuppression can lead to BKPyV reactivation in the first year post-transplantation in kidney transplant recipients (KTRs) and hematopoietic stem cell transplant recipients. In KTRs, persistent DNAemia has been correlated to the occurrence of polyomavirus-associated nephropathy (PVAN) that can lead to graft loss if not properly controlled. Based on recent observations that conventional dendritic cells (cDCs) specifically infiltrate PVAN lesions, we hypothesized that those cells could play a role in BKPyV infection. We first demonstrated that monocyte-derived dendritic cells (MDDCs), an in vitro model for mDCs, captured BKPyV particles through an unconventional GRAF-1 endocytic pathway. Neither BKPyV particles nor BKPyV-infected cells were shown to activate MDDCs. Endocytosed virions were efficiently transmitted to permissive cells and protected from the antibody-mediated neutralization. Finally, we demonstrated that freshly isolated CD1c+ mDCs from the blood and kidney parenchyma behaved similarly to MDDCs thus extending our results to cells of clinical relevance. This study sheds light on a potential unprecedented CD1c+ mDC involvement in the BKPyV infection as a promoter of viral spreading.

Extracellular vesicles (EVs) have been lauded as next-generation medicines, but very few EV-based therapeutics have progressed to clinical use. Limited clinical translation is largely due to technical barriers that hamper our ability to mass produce EVs, i.e., to isolate, purify, and characterize them effectively. Technical limitations in comprehensive characterization of EVs lead to unpredicted biological effects of EVs. Here, using a range of optical and non-optical techniques, we showed that the differences in molecular composition of EVs isolated using two isolation methods correlated with the differences in their biological function. Our results demonstrated that the isolation method determines the composition of isolated EVs at single and sub-population levels. Besides the composition, we measured for the first time the dry mass and predicted sedimentation of EVs. These parameters were likely to contribute to the biological and functional effects of EVs on single cell and cell cultures. We anticipate that our new multiscale characterization approach, which goes beyond traditional experimental methodology, will support fundamental understanding of EVs as well as elucidate the functional effects of EVs in in vitro and in vivo studies. Our findings and methodology will be pivotal for developing optimal isolation methods and establishing EVs as mainstream therapeutics and diagnostics. This innovative approach is applicable to a wide range of sectors including biopharma and biotechnology as well as to regulatory agencies.

Erectile dysfunction (ED) is an adverse side effect of pelvic surgery with no effective treatment. In this study, we explored whether melatonin could improve the therapeutic effects of small extracellular vesicles (sEVs), derived from mesenchymal stem cells (MSCs), on cavernous nerve injury (CNI) ED and investigated the underlying mechanisms. The sEVs from melatonin-pretreated MSCs (MT-EVs) and MSCs (NC-EVs) were isolated and applied to CNI ED. Transplantation of MT-EVs remarkably increased erectile function and reduced phenotypic modulation in CNI ED rats. MT-EVs increased Calponin 1 and SMA and decreased OPN, Vimentin, and cell migration capabilities. The therapeutic effects of MT-EVs were superior to those of NC-EVs. Sequencing implied that miR-10a-3p was enriched in MT-EVs, and directly targeted the protein kinase inhibitor α (PKIA). After the suppression of miR-10a-3p, the therapeutic actions of MT-EVs were abolished but were rescued by PKIA. Similarly, RhoA/ROCK was inhibited by MT-EVs, but this action was reversed by suppressing miR-10a-3p, accompanied by corresponding changes in PKIA. In conclusion, transplantation of MT-EVs could significantly alleviate CNI ED. MT-EVs may relieve the phenotypic modulation of the corpora cavernosum smooth muscle cells via the miR-10a-3p/PKIA/RhoA/ROCK signaling axis. These nanovesicles should be potential therapeutic vectors or bioactive materials for CNI ED.

Tumour-draining lymph nodes (LNs) undergo massive remodelling including expansion of the lymphatic sinuses, a process that has been linked to lymphatic metastasis by creation of a pre-metastatic niche. However, the signals leading to these changes have not been completely understood. Here, we found that extracellular vesicles (EVs) derived from melanoma cells are rapidly transported by lymphatic vessels to draining LNs, where they selectively interact with lymphatic endothelial cells (LECs) as well as medullary sinus macrophages. Interestingly, uptake of melanoma EVs by LN-resident LECs was partly dependent on lymphatic VCAM-1 expression, and induced transcriptional changes as well as proliferation of those cells. Furthermore, melanoma EVs shuttled tumour antigens to LN LECs for cross-presentation on MHC-I, resulting in apoptosis induction in antigen-specific CD8+ T cells. In conclusion, our data identify EV-mediated melanoma-LN LEC communication as a new pathway involved in tumour progression and tumour immune inhibition, suggesting that EV uptake or effector mechanisms in LECs might represent a new target for melanoma therapy.

In the biomedical field, there has been a requirement for developing theranostic nanomaterials with higher biosafety, leading to both diagnosis and therapy. Methylene blue (MB+) is an organic dye with both photoluminescence (PL) and photosensitization abilities to generate singlet oxygen (1O2). However, MB+ easily loses its generation ability by hydrogen reduction in vivo or by forming aggregates. In this study, MB+ immobilized on biocompatible hydroxyapatite (HA) nanoparticles was applied for the bifunctions of efficient PL and photosensitization. The MB+-immobilized HA nanoparticles (MH) formed aggregates with sizes of 80–100 nm in phosphate buffer (PB). The generation amount and efficiency of 1O2 from the nanoparticles in PB seem to depend on the immobilized MB+ amount and the percentage of the monomer, respectively. Considering the larger immobilized amount and percentage of the MB+ monomer, it was found that there was MH with the lower generation amount and efficiency of 1O2 to exhibit the highest PL intensity. The photofunctional measurement of MB+ revealed the state of MB+ molecules on the HA surface, and it was suggested that the MB+ molecules immobilized on the MH surface would form more hydrogen bonds to change their excitation states. In the cellular experiments, the Hela cancer cells reacted with the nanoparticles and showed red-color PL, indicating cellular imaging. Furthermore, the adherent cell coverage decreased by 1O2 generation, indicating the importance of the immobilization amount of the MB+ monomer. Therefore, theranostic nanomaterials with biosafety were successfully synthesized to show two photofunctions, which provide both cellular imaging and photodynamic therapy by the nanohybrid system between HA and MB+.

Non-coding RNA (ncRNA), released into circulation or packaged into exosomes, plays important roles in many biological processes in the kidney. The purpose of the present study is to identify a common ncRNA signature associated with early renal damage and its related molecular pathways. Three individual libraries (plasma and urinary exosomes, and total plasma) were prepared from each hypertensive patient (with or without albuminuria) for ncRNA sequencing analysis. Next, an RNA-based transcriptional regulatory network was constructed. The three RNA biotypes with the greatest number of differentially expressed transcripts were long-ncRNA (lncRNA), microRNA (miRNA) and piwi-interacting RNA (piRNAs). We identified a common 24 ncRNA molecular signature related to hypertension-associated urinary albumin excretion, of which lncRNAs were the most representative. In addition, the transcriptional regulatory network showed five lncRNAs (LINC02614, BAALC-AS1, FAM230B, LOC100505824 and LINC01484) and the miR-301a-3p to play a significant role in network organization and targeting critical pathways regulating filtration barrier integrity and tubule reabsorption. Our study found an ncRNA profile associated with albuminuria, independent of biofluid origin (urine or plasma, circulating or in exosomes) that identifies a handful of potential targets, which may be utilized to study mechanisms of albuminuria and cardiovascular damage.

Extracellular vesicles (EVs) derived from human bone marrow mesenchymal stem cells (BMSCs) are suggested to promote angiogenesis in a rat model of acute myocardial infarction (AMI). This study aimed to explore the underlying mechanism of BMSCs-EVs in AMI-induced heart failure (HF). BMSCs were isolated and verified, and EVs were purified and identified. After establishment of AMI-induced HF models, rats were treated with BMSCs-EVs and/or overexpressing (ov)/knocking down (kd) bone morphogenetic protein 2 (BMP2). Cardiac function, myocardial histopathological changes, angiogenesis, and vascular regeneration density were measured. Levels of pro-angiogenesis factors and cardiomyocyte apoptosis were detected. The viability and angiogenesis of hypoxic human umbilical vein endothelial cells (HUVECs) were measured. After BMSCs-EV treatment, the cardiac function of HF rats was improved, myocardial fibrosis and inflammatory cell infiltration were decreased, angiogenesis was increased, and cardiomyocyte apoptosis was inhibited. BMP2 was significantly upregulated in the myocardium. Ov-BMP2-BMSCs-EVs alleviated myocardial fibrosis and inflammatory cell infiltration, and promoted angiogenesis of HF rats, and improved the activity and angiogenesis of hypoxic HUVECs, while kd-BMP2-BMSCs-EVs showed limited protection against AMI-induced HF. BMSCs-EVs deliver BMP2 to promote angiogenesis and improve cardiac function of HF rats.

Background Acute radiation syndrome (ARS) is caused by acute exposure to ionizing radiation that damages multiple organ systems but especially the bone marrow (BM). We have previously shown that human macrophages educated with exosomes from human BM-derived mesenchymal stromal cells (MSCs) primed with lipopolysaccharide (LPS) prolonged survival in a xenogeneic lethal ARS model. The purpose of this study was to determine if exosomes from LPS-primed MSCs could directly educate human monocytes (LPS-EEMos) for the treatment of ARS. Methods Human monocytes were educated by exosomes from LPS-primed MSCs and compared to monocytes educated by unprimed MSCs (EEMos) and uneducated monocytes to assess survival and clinical improvement in a xenogeneic mouse model of ARS. Changes in surface molecule expression of exosomes and monocytes after education were determined by flow cytometry, while gene expression was determined by qPCR. Irradiated human CD34+ hematopoietic stem cells (HSCs) were co-cultured with LPS-EEMos, EEMos, or uneducated monocytes to assess effects on HSC survival and proliferation. Results LPS priming of MSCs led to the production of exosomes with increased expression of CD9, CD29, CD44, CD146, and MCSP. LPS-EEMos showed increases in gene expression of IL-6, IL-10, IL-15, IDO, and FGF-2 as compared to EEMos generated from unprimed MSCs. Generation of LPS-EEMos induced a lower percentage of CD14+ monocyte subsets that were CD16+, CD73+, CD86+, or CD206+ but a higher percentage of PD-L1+ cells. LPS-EEMos infused 4 h after lethal irradiation significantly prolonged survival, reducing clinical scores and weight loss as compared to controls. Complete blood counts from LPS-EEMo-treated mice showed enhanced hematopoietic recovery post-nadir. IL-6 receptor blockade completely abrogated the radioprotective survival benefit of LPS-EEMos in vivo in female NSG mice, but only loss of hematopoietic recovery was noted in male NSG mice. PD-1 blockade had no effect on survival. Furthermore, LPS-EEMos also showed benefits in vivo when administered 24 h, but not 48 h, after lethal irradiation. Co-culture of unprimed EEMos or LPS-EEMos with irradiated human CD34+ HSCs led to increased CD34+ proliferation and survival, suggesting hematopoietic recovery may be seen clinically. Conclusion LPS-EEMos are a potential counter-measure for hematopoietic ARS, with a reduced biomanufacturing time that facilitates hematopoiesis.

Therapeutic applications of extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have attracted considerable attention because of their immunomodulatory properties against immune-mediated, inflammatory diseases. Here, we demonstrated enhanced immunomodulatory properties of EVs secreted from endoplasmic reticulum (ER) stress inducer thapsigargin (TSG)-primed human Wharton's jelly-derived MSCs (WJ-MSCs). EVs from TSG-primed WJ-MSCs (TSG-EV) showed increased yield and expression of immunomodulatory factors, such as transforming growth factor-β1 (TGFβ), cyclooxygenase-2 (COX2), and especially indoleamine 2,3-dioxygenase (IDO), compared to control EVs. TSG-EV showed a significantly enhanced immunosuppressive effect on human peripheral blood-derived T cell proliferation and Th1 and Th17 differentiation, whereas Treg and M2-type macrophage were enriched compared to a control EV-treated group. Furthermore, TSG-EV substantially mitigated mouse experimental colitis by reducing the inflammatory response and maintaining intestinal barrier integrity. A significant increase of Tregs and M2-type macrophages in colitic colons of a TSG-EV-treated mouse suggests an anti-inflammatory effect of TSG-EV in colitis model, possibly mediated by Treg and macrophage polarization. These data indicate that TSG treatment promoted immunomodulatory properties of EVs from WJ-MSCs, and TSG-EV may provide a new therapeutic approach for treatment of colitis.

Neutrophils are classically characterized as merely reactive innate effector cells. However, the microbiome is known to shape the education and maturation process of neutrophils, improving their function and immune‐plasticity. Recent reports demonstrate that murine neutrophils possess the ability to exert adaptive responses after exposure to bacterial components such as LPS (Gram‐ negative bacteria) or LTA (Gram‐positive bacteria). We now ask whether small extracellular vesicles (EVs) from the gut may directly mediate adaptive responses in neutrophils in vitro. Murine bone marrow‐derived neutrophils were primed in vitro by small EVs of high purity collected from colon stool samples, followed by a second hit with LPS. We found that low‐dose priming with gut micro‐ biota‐derived small EVs enhanced pro‐inflammatory sensitivity as indicated by elevated levels of TNF‐α, IL‐6, ROS and MCP‐1 and increased migratory and phagocytic activity. In contrast, high‐ dose priming resulted in a tolerant phenotype, marked by increased IL‐10 and decreased transmi‐ gration and phagocytosis. Alterations in TLR2/MyD88 as well as TLR4/MyD88 signaling were cor‐ related with the induction of adaptive cues in neutrophils in vitro. Taken together, our study shows that small EVs from stools can drive adaptive responses in neutrophils in vitro and may represent a missing link in the gut–immune axis.

Ovarian cancer is highly prevalent and has high mortality rates due to metastasis and relapse. The cross communication between cancer-associated fibroblasts (CAFs) and cancer-associated macrophages (CAMs) in the ovarian tumor microenvironment leads to cancer cell invasion and metastasis. However, the role of overproduction of IL-33/ST2 in the CAFs of ovarian cancer is still unclear. The expression of IL-33, ST2, apoptosis-related proteins and epithelial-mesenchymal transition (EMT) markers was measured by western blotting. Primary normal fibroblasts and CAFs from ovarian cancerous tissue were isolated and cultured in vitro, and the medium was used to stimulate blood-derived monocytes. Flow cytometry analysis was used to detect the frequency of M2-like macrophages in blood-derived monocytes from patients with ovarian cancer. Cell invasion were evaluated using Transwell assays. A xenograft model was used to study tumor growth in ST2-knockout and wild-type NOD-SCID mice. The results demonstrated higher expression of IL-33 and ST2 in carcinoma tissues compared with in para-carcinoma tissues, and there was a survival improvement associated with elevated IL-33. IL-33 and culture supernatants from CAFs, rather than normal ovarian fibroblasts, led to a higher expression of M2 macrophage marker genes in human blood-derived monocytes. Invasion and migration were aggravated in COC1 cells co-cultured with CAF-induced CAMs, and the EMT marker genes were upregulated. It was reported that EMT marker genes were downregulated and tumor volumes were significantly reduced in ST2-deficient mice. Overall, the IL-33/ST2 axis in ovarian cancer might integrate IL-33-expressing CAFs with M2 type-like CAMs, which aggravated invasion and metastasis by promoting EMT.

The apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1), the main AP-endonuclease of the DNA base excision repair pathway, is a key molecule of interest to researchers due to its unsuspected roles in different nonrepair activities, such as: i) adaptive cell response to genotoxic stress, ii) regulation of gene expression, and iii) processing of microRNAs, which make it an excellent drug target for cancer treatment. We and others recently demonstrated that APE1 can be secreted in the extracellular environment and that serum APE1 may represent a novel prognostic biomarker in hepatocellular and non-small-cell lung cancers. However, the mechanism by which APE1 is released extracellularly was not described before. Here, using three different approaches for exosomes isolation: commercial kit, nickel-based isolation, and ultracentrifugation methods and various mammalian cell lines, we elucidated the mechanisms responsible for APE1 secretion. We demonstrated that APE1 p37 and p33 forms are actively secreted through extracellular vesicles (EVs), including exosomes from different mammalian cell lines. We then observed that APE1 p33 form is generated by proteasomal-mediated degradation and is enzymatically active in EVs. Finally, we revealed that the p33 form of APE1 accumulates in EVs upon genotoxic treatment by cisplatin and doxorubicin, compounds commonly found in chemotherapy pharmacological treatments. Taken together, these findings provide for the first time evidence that a functional Base Excision Repair protein is delivered through exosomes in response to genotoxic stresses, shedding new light into the complex noncanonical biological functions of APE1 and opening new intriguing perspectives on its role in cancer biology.

Academic and industrial groups worldwide have reported technological advances in exosome-based cancer diagnosis and prognosis. However, the potential translation of these emerging technologies for research and clinical settings remains unknown. This work overviews the role of exosomes in cancer diagnosis and prognosis, followed by a survey on emerging exosome technologies, particularly microfluidic advances for the isolation and detection of exosomes in cancer research. The advantages and drawbacks of each of the technologies used for the isolation, detection and engineering of exosomes are evaluated to address their clinical challenges for cancer diagnosis and prognosis. Furthermore, commercial platforms for exosomal detection and analysis are introduced, and their performance and impact on cancer diagnosis and prognosis are assessed. Also, the risks associated with the further development of the next generation of exosome devices are discussed. The outcome of this work could facilitate recognizing deliverable Exo-devices and technologies with unprecedented functionality and predictable manufacturability for the next-generation of cancer diagnosis and prognosis.

Metabolism, is a complex process involving the gut and the liver tissue, is difficult to be reproduced in vitro with conventional single cell culture systems. To tackle this challenge, we developed a gut-liver-axis chip consisting of the gut epithelial cell chamber and three-dimensional (3D) uniform-sized liver spheroid chamber. Two cell culture chamber compartments were separated with a porous membrane to prevent microorganisms from passing through the chamber. When the hepG2 spheroids cultured with microbiota-derived metabolites, we observed the changes in the physiological function of hepG2 spheroids, showing that the albumin and urea secretion activity of liver spheroids was significantly enhanced. Additionally, the functional validation of hepG2 spheroids treated with microbiota-derived exosome was evaluated that the treatment of the microbiota-derived exosome significantly enhanced albumin and urea in hepG2 spheroids in a gut-liver axis chip. Therefore, this gut-liver axis chip could be a potentially powerful co-culture platform to study the interaction of microbiota and host cells.

The rapid and reliable recognition of nucleic acid sequences is essential to a broad range of fields including genotyping, gene expression analysis, and pathogen screening. For viral detection in particular, the capability is critical for optimal therapeutic response and preventing disease transmission. Here, we report an approach for detecting identifying sequence motifs within genome-scale single-strand DNA and RNA based on solid-state nanopores. By designing DNA oligonucleotide probes with complementarity to target sequences within a target genome, we establish a protocol to yield affinity-tagged duplex molecules the same length as the probe only if the target is present. The product can subsequently be bound to a protein chaperone and analyzed quantitatively with a selective solid-state nanopore assay. We first use a model DNA genome (M13mp18) to validate the approach, showing the successful isolation and detection of multiple target sequences simultaneously. We then demonstrate the protocol for the detection of RNA viruses by identifying and targeting a highly conserved sequence within human immunodeficiency virus (HIV-1B).

Among many methods to mitigate the solubility limitations of drug compounds, amorphous solid dispersion (ASD) is considered to be one of the most promising strategies to enhance the dissolution and bioavailability of poorly water-soluble drugs. The enhancement of ASD in the oral absorption of drugs has been mainly attributed to the high apparent drug solubility during the dissolution. In the last decade, with the implementations of new knowledge and advanced analytical techniques, a drug-rich transient metastable phase was frequently highlighted within the supersaturation stage of the ASD dissolution. The extended drug absorption and bioavailability enhancement may be attributed to the metastability of such drug-rich phases. In this paper, we have reviewed (i) the possible theory behind the formation and stabilization of such metastable drug-rich phases, with a focus on non-classical nucleation; (ii) the additional benefits of the ASD-induced drug-rich phases for bioavailability enhancements. It is envisaged that a greater understanding of the non-classical nucleation theory and its application on the ASD design might accelerate the drug product development process in the future.

Background In endothelial cells, phospholipase C (PLC) β1-activated Ca2+ is a crucial second messenger for the signaling pathways governing angiogenesis. PLCβ1 is inactivated by complexing with an intracellular protein called translin-associated factor X (TRAX). This study demonstrates specific interactions between Globo H ceramide (GHCer) and TRAX, which highlight a new angiogenic control through PLCβ1 activation. Methods Globo-series glycosphingolipids (GSLs), including GHCer and stage-specific embryonic antigen-3 ceramide (SSEA3Cer), were analyzed using enzyme-linked immunosorbent assay (ELISA) and Biacore for their binding with TRAX. Angiogenic activities of GSLs in human umbilical vein endothelial cells (HUVECs) were evaluated. Molecular dynamics (MD) simulation was used to study conformations of GSLs and their molecular interactions with TRAX. Fluorescence resonance energy transfer (FRET) analysis of HUVECs by confocal microscopy was used to validate the release of PLCβ1 from TRAX. Furthermore, the in vivo angiogenic activity of extracellular vesicles (EVs) containing GHCer was confirmed using subcutaneous Matrigel plug assay in mice. Results The results of ELISA and Biacore analysis showed a stable complex between recombinant TRAX and synthetic GHCer with KD of 40.9 nM. In contrast, SSEA3Cer lacking a fucose residue of GHCer at the terminal showed ~ 1000-fold decrease in the binding affinity. These results were consistent with their angiogenic activities in HUVECs. The MD simulation indicated that TRAX interacted with the glycan moiety of GHCer at amino acid Q223, Q219, L142, S141, and E216. At equilibrium the stable complex maintained 4.6 ± 1.3 H-bonds. TRAX containing double mutations with Q223A and Q219A lost its ability to interact with GHCer in both MD simulation and Biacore assays. Removal of the terminal fucose from GHCer to become SSEA3Cer resulted in decreased H-bonding to 1.2 ± 1.0 by the MD simulation. Such specific H-bonding was due to the conformational alteration in the whole glycan which was affected by the presence or absence of the fucose moiety. In addition, ELISA, Biacore, and in-cell FRET assays confirmed the competition between GHCer and PLCβ1 for binding to TRAX. Furthermore, the Matrigel plug assay showed robust vessel formation in the plug containing tumor-secreted EVs or synthetic GHCer, but not in the plug with SSEA3Cer. The FRET analysis also indicated the disruption of colocalization of TRAX and PLCβ1 in cells by GHCer derived from EVs. Conclusions Overall, the fucose residue in GHCer dictated the glycan conformation for its complexing with TRAX to release TRAX-sequestered PLCβ1, leading to Ca2+ mobilization in endothelial cells and enhancing angiogenesis in tumor microenvironments.