Publications

Cigarette smoke (CS) represents one of the most relevant environmental risk factors for several chronic pathologies. Tissue damage caused by CS exposure is mediated, at least in part, by oxidative stress induced by its toxic and pro-oxidant components. Evidence demonstrates that extracellular vesicles (EVs) released by various cell types exposed to CS extract (CSE) are characterized by altered biochemical cargo and gained pathological properties. In the present study, we evaluated the content of oxidized proteins and phospholipid fatty acid profiles of EVs released by human bronchial epithelial BEAS-2B cells treated with CSE. This specific molecular characterization has hitherto not been performed. After confirmation that CSE reduces viability of BEAS-2B cells and elevates intracellular ROS levels, in a dose-dependent manner, we demonstrated that 24 h exposure at 1% CSE, a concentration that only slight modifies cell viability but increases ROS levels, was able to increase carbonylated protein levels in cells and released EVs. The release of oxidatively modified proteins via EVs might represent a mechanism used by cells to remove toxic proteins in order to avoid their intracellular overloading. Moreover, 1% CSE induced only few changes in the fatty acid asset in BEAS-2B cell membrane phospholipids, whereas several rearrangements were observed in EVs released by CSE-treated cells. The impact of changes in acyl chain composition of CSE-EVs accounted for the increased saturation levels of phospholipids, a membrane parameter that might influence EV stability, uptake and, at least in part, EV-mediated biological effects. The present in vitro study adds new information concerning the biochemical composition of CSE-related EVs, useful to predict their biological effects on target cells. Furthermore, the information regarding the presence of oxidized proteins and the specific membrane features of CSE-related EVs can be useful to define the utilization of circulating EVs as marker for diagnosing of CS-induced lung damage and/or CS-related diseases.

Extracellular vesicles hold great promise as a drug delivery platform for RNA-based therapeutics. However, there is a lack of experimental evidence for the intracellular trafficking of nucleic acid cargos, specifically, whether they are capable of escaping from the endolysosomal confinement in the recipient cells to be released into the cytosol and hence, interact with their cytoplasmic targets. Here, we demonstrated how red blood cell-derived extracellular vesicles (RBCEVs) release their therapeutic RNA/DNA cargos at specific intracellular compartments characteristic of late endosomes and lysosomes. The released cargos were functional and capable of knocking down genes of interest in recipient cells, resulting in tumor suppression in vitro and in an acute myeloid leukemia murine model without causing significant toxicity. Notably, surface functionalization of RBCEVs with an anti-human CXCR4 antibody facilitated their specific uptake by CXCR4+ leukemic cells, leading to enhanced gene silencing efficiency. Our results provide insights into the cellular uptake mechanisms and endosomal escape routes of nucleic acid cargos delivered by RBCEVs which have important implications for further improvements of the RBCEV-based delivery system.

Extracellular vesicles (EVs) are nowadays a target of interest in cancer therapy as a successful drug delivering tool. Based on their many beneficial biocompatible properties are designed to transport nucleic acids, proteins, various nanomaterials or chemotherapeutics. Extracellular vesicles derived from mesenchymal stem/stromal cells (MSCs) possess their tumor-homing abilities. This inspired us to engineer the MSC's EVs to be packed with chemotherapeutic agents and deliver it as a Trojan horse directly into tumor cells. In our study, human dental pulp MSCs (DP-MSCs) were cultivated with gemcitabine (GCB), which led to its absorption by the cells and subsequent secretion of the drug out into conditioned media in EVs. Concentrated conditioned media containing small EVs (potentially exosomes) significantly inhibited the cell growth of pancreatic carcinoma cell lines in vitro. DP-MSCs were simultaneously engineered to express a suicide gene fused yeast cytosinedeaminase:uracilphosphoribosyltransferase (yCD::UPRT). The product of the suicide gene converts non-toxic prodrug 5-fluorocytosine (5-FC) to highly cytotoxic chemotherapeutic drug 5-fluorouracil (5-FU) in the recipient cancer cells. Conversion of 5-FC to 5-FU had an additional effect on cancer cell's growth inhibition. Our results showed a therapeutic potential for DP-MSC-EVs to be designed for successful delivering of chemotherapeutic drugs, together with prodrug suicide gene therapy system.

Extracellular vesicles (EVs) are routinely released from nearly all cell types as transport vehicles and for cell communication. Crucially, they contain biomolecular content for the identification of health and disease states that can be detected from readily accessible physiological fluids, including urine, plasma, or saliva. Despite their clinical utility within noninvasive diagnostic platforms such as liquid biopsies, the currently available portfolio of analytical approaches are challenged by EV heterogeneity in size and composition, as well as the complexity of native biofluids. Quartz crystal microbalance with dissipation monitoring (QCM-D) has recently emerged as a powerful alternative for the phenotypic detection of EVs, offering multiple modes of analyte discrimination by frequency and dissipation. While providing rich data for sensor development, further progress is required to reduce detection limits and fully exploit the technique’s potential within biosensing. Herein, we investigate the impact of nanostructuring the sensor electrode surface for enhancing its detection capabilities. We employ self-assembly of the block copolymer polystyrene-block-poly(4-vinylpyridine) to create well defined 2D gold islands via selective impregnation of the pyridine domain with gold precursors and subsequent removal of the template. When matched to the EV length scale, we find a 4-fold improvement in sensitivity despite a 4-fold reduction in area for analyte and ligand anchoring in comparison to a flat sensor surface. Creation of tailored and confined sensing regions interspersed by non-binding silica provides optimal spatial orientation for EV capture with reduced steric effects and negative cooperativity of grafted antibodies, offering a promising route for enhanced binding efficiency and performance of sensor platforms.

Despite significant therapeutic advances, lung cancer remains the leading cause of cancer-related death worldwide1. Non-small cell lung cancer (NSCLC) patients have a very poor overall five-year survival rate of only 10–20%. Currently, TNM staging is the gold standard for predicting overall survival and selecting optimal initial treatment options for NSCLC patients, including those with curable stages of disease. However, many patients with locoregionally-confined NSCLC relapse and die despite curative-intent interventions, indicating a need for intensified, individualised therapies. Epithelial-to-mesenchymal transition (EMT), the phenotypic depolarisation of epithelial cells to elongated, mesenchymal cells, is associated with metastatic and treatment-refractive cancer. We demonstrate here that EMT-induced protein changes in small extracellular vesicles are detectable in NSCLC patients and have prognostic significance. Overall, this work describes a novel prognostic biomarker signature that identifies potentially-curable NSCLC patients at risk of developing metastatic NSCLC, thereby enabling implementation of personalised treatment decisions.

A mechanistic understanding of cell-autonomous skeletal muscle changes after injuries can lead to novel interventions to improve functional recovery in an aged population. However, major gaps in our understanding persist owing to limitations of commonly used biological aging models. Two-dimensional cell culture represents an artificial environment, while aging mammalian models are contaminated by influences from non-muscle cells and other organs. We created a three-dimensional muscle aging system to overcome the limitations of these traditional platforms. Here, we first show that old muscle constructs (OMC) manifest a sarcopenic phenotype, as evidenced by hypotrophic myotubes, reduced contractile function, and decreased regenerative capacity compared to young muscle constructs (YMC). OMC also phenocopy the regenerative responses of aged muscle to two interventions, pharmacological and biological. Next, interrogation of muscle cell-specific mechanisms that contribute to impaired regeneration over time reveals that an aging-induced increase of complement component 4b (C4b) delays muscle progenitor cell amplification and impairs functional recovery. However, administration of complement factor I, a C4b inactivator, improves muscle regeneration in vitro and in vivo, indicating C4b inhibition may be a novel approach to enhance aged muscle repair. Collectively, our model exhibits capabilities to study cell-autonomous changes in skeletal muscle during aging, regeneration, and intervention.

Aberrant inflammation in the endometrium impairs reproduction and leads to poor fertility. Small extracellular vesicles (sEV) are nanoparticles 30-200nm in-size and contain transferable bioactive molecules that reflect the parent cell. Holstein-Friesian dairy cows with divergent genetic merit, high- (n = 10) and low-fertile (n = 10), were identified based on fertility breeding value (BV), cow ovulation synchronization and postpartum anovulatory intervals (PPAI). In this study, we evaluated the effects of sEVs enriched from plasma of high-fertile (HF-EXO) and low-fertile (LF-EXO) dairy cows on inflammatory mediator expression by bovine endometrial epithelial (bEEL) and stromal (bCSC) cells. Exposure to HF-EXO in bCSC and bEEL cells yielded higher expression of PTGS1 and PTGS2 compared to the control. Pro-inflammatory cytokine IL1-α, IL-8/CXCL8 and IL-12α genes were downregulated in bCSC cells exposed to HF-EXO. In contrast, sEV exposure significantly lowered anti-inflammatory cytokine levels (CX3CL1 and IL-4) regardless high or low fertile states. Further, exposure to HF-EXO downregulated DES gene expression level in bCSC compared to the control. Our findings demonstrate that sEVs influence differential gene expression in endometrial cells, specifically genes relate to inflammation. Further, sEV from high-fertile animals acts in a unique direction to de-activate prostaglandin synthases in both bCSC and bEEL cells, and de-activate pro-inflammatory cytokines in the endometrial stroma. The results indicate identifying circulating sEV as a potential biomarker of fertility.

Small extracellular vesicles (sEV) are a class of extracellular vesicles (30–150 nm), delivering molecules including proteins, metabolites, and microRNAs (miRNAs), involved in physiological intercellular crosstalk and disease pathogenesis. The present pilot study aims are (I) to develop an easy and fast protocol for the isolation of sEV from plasma of mast cell tumor (MCT)-affected dogs; (II) to evaluate if miR-21-5p (sEV-miR-21-5p), a miRNA overexpressed by MCT, is associated with sEV. Seventeen dogs have been enrolled in the study: 4 healthy and 13 (6 with and 7 without nodal metastasis) MCT-affected dogs. sEV were isolated using size exclusion chromatography (SEC) (IZON column 35nm) and were characterized by Western blot, Nanoparticle tracking analysis, and transmission electron microscopy. sEV-miR-21-5p was quantified using digital PCR. sEV expressed the specific markers CD9 and TSG101, and a marker of mast cell tryptase. The sEV mean concentration and size were 2.68E + 10 particles/ml, and 99.6 nm, 2.89E + 10 particles/ml and 101.7 nm, and 3.21E + 10 particles/ml and 124 nm in non-metastatic, nodal metastatic, and healthy samples, respectively. The comparative analysis demonstrated that the level of sEV-miR-21-5p was significantly higher in dogs with nodal metastasis compared to healthy (P = 0.038) and without nodal metastasis samples (P = 0.007). In conclusion, the present work demonstrated that a pure population of sEV can be isolated from the plasma of MCT-affected dogs using the SEC approach and that the level of sEV-miR-21-5p is higher in nodal metastatic MCT-affected dogs compared with healthy and MCT-affected dogs without nodal involvement.

Endocytosis is an essential biological process for nutrient absorption and intercellular communication; it can also be used to accelerate the cellular internalization of drug delivery carriers. Clarifying the cellular uptake mechanisms of unidentified endogenous and exogenous molecules and designing new effective drug delivery systems require an accurate, specific endocytosis analysis methodology. Therefore, we developed a method to specifically evaluate cellular internalization via three main endocytic pathways: clathrin- and caveolae-mediated endocytosis, and macropinocytosis. We first revealed that most known endocytosis inhibitors had no specific inhibitory effect or were cytotoxic. Second, we successfully established an alternative method using small interfering RNA to knock down dynamin-2 and caveolin-1, which are necessary for clathrin- and caveolae-mediated endocytosis, in HeLa cells. Third, we established another method to specifically analyze macropinocytosis using rottlerin on A431 cells. Finally, we validated the proposed methods by testing the cellular internalization of a biological molecule (insulin) and carriers (nanoparticles and cell-penetrating peptides). Through this study, we established versatile methods to precisely and specifically evaluate endocytosis of newly developed biopharmaceuticals or drug delivery systems.

The secretion of extracellular vesicles and particles (EVPs) is an important mechanism of cellular communication. In this work, we demonstrate a functional role of EVPs in mechanisms regulating gastric acid secretion. HGT-1 cells were used as a model system to assess proton secretion. First, in order to prove EVP secretion by HGT-1 cells, EVPs were isolated by size exclusion chromatography and characterized by nanoparticle tracking analysis, Western blot, and cryo transmission electron microscopy. For examination of the potential role of EVPs in proton secretion, HGT-1 cells were treated with pharmacological EV-inhibitors, resulting in a reduction of histamine-induced proton secretion. To demonstrate the functional role of EVPs in the mechanism of proton secretion, EVP-conditioned supernatant was collected after stimulation of HGT-1 cells with histamine, fractionated, and subjected to an activity screening. The results revealed constituents of the HGT-1-derived secretome with an MW of >100 kDa (including EVPs) to modulate proton secretion, while smaller constituents had no effect. Finally, a dose-dependent modulatory effect on proton secretion of HGT-1 cells was demonstrated by isolated HGT-1-derived EVPs. Hence, this study presents first results on the potential function of EVPs as a previously undiscovered mechanism of regulation of gastric acid secretion by parietal cells.

Hypoxia induces changes in the secretion of extracellular vesicles (EVs) in several non-neuronal cells and pathological conditions. EVs are packed with biomolecules, such as microRNA(miR)-21-5p, which respond to hypoxia. However, the true EV association of miR-21-5p, and its functional or biomarker relevance, are inadequately characterised. Neurons are extremely sensitive cells, and it is not known whether the secretion of neuronal EVs and miR-21-5p are altered upon hypoxia. Here, we characterised the temporal EV secretion profile and cell viability of neurons under hypoxia. Hypoxia induced a rapid increase of miR-21a-5p secretion in the EVs, which preceded the elevation of hypoxia-induced tissue or cellular miR-21a-5p. Prolonged hypoxia induced cell death and the release of morphologically distinct EVs. The EVs protected miR-21a-5p from enzymatic degradation but a remarkable fraction of miR-21a-5p remained fragile and non-EV associated. The increase in miR-21a-5p secretion may have biomarker potential, as high blood levels of miR-21-5p in stroke patients were associated with significant disability at hospital discharge. Our data provides an understanding of the dynamic regulation of EV secretion from neurons under hypoxia and provides a candidate for the prediction of recovery from ischemic stroke.

Extracellular vesicles (EVs), nanovesicles released by cells to effectively exchange biological information, are gaining interest as drug delivery system. Yet, analogously to liposomes, they show short blood circulation times and accumulation in the liver and the spleen. For tissue specific delivery, EV surfaces will thus have to be functionalized. We present a novel platform for flexible modification of EVs with target-specific ligands based on the avidin-biotin system. Genetic engineering of donor cells with a glycosylphosphatidylinositol-anchored avidin (GPI-Av) construct allows the isolation of EVs displaying avidin on their surface, functionalized with any biotinylated ligand. For proof of concept, GPI-Av EVs were modified with i) a biotinylated antibody or ii) de novo designed and synthesized biotinylated ligands binding carbonic anhydrase IX (CAIX), a membrane associated enzyme overexpressed in cancer. Functionalized EVs showed specific binding and uptake by CAIX-expressing cells, demonstrating the power of the system to prepare EVs for cell-specific drug delivery.

In this work, we present a microfluidic amperometric immunosensor for cancer biomarker claudin7 (CLD7) determination in circulating extracellular vesicles (EVs) as well as its validation in colorectal cancer (CC) patients. The device is based on synthetized nanosized MIL-125-NH2 particles, covalently anchored to the central channel of the microfluidic immunosensor. This nanomaterial was employed as efficient platform for anti-CLD7 monoclonal antibodies immobilization for specifically recognize and capture CLD7 in EVs samples. Afterwards, the amount of this trapped CLD7 was quantified by HRP-conjugated anti-CLD7-antibody. HRP reacted with its enzymatic substrate in a redox process which resulted in the appearance of a current whose magnitude was directly proportional to the level of CLD7 in the sample. This immunosensor, under optimum conditions, gave the limit of detection for CLD7 of 0.1 pg mL−1, with a wide linear range from 2 to 1000 pg mL−1. The results reported herein open up the use of porous open framework platforms for sensing applications for biomedicine and diagnosis.

Extracellular vesicles (EVs) released from cells into the blood facilitate intercellular communication and serve as new biomarkers to understand the pathophysiology of several conditions. Although the importance of the cargo inside EVs has been extensively studied, the sizes of EVs that vary with different types of cancers are relatively poorly explored. Here, we show that pancreatic cancer cell-derived EVs are significantly smaller than non-cancer cell-derived EVs. The smaller size distribution of these EVs was confirmed by specifically isolating and examining tumor-derived EVs from the heterogeneous EV population isolated from the sera of patients with pancreatic ductal adenocarcinoma. In vitro analyses mimicking tumor microenvironment conditions revealed that low glucose conditions reduced the size distribution and increased the level of unsaturated fatty acids in the tumor-derived EVs. Because the lipid composition defines the fluidity of the membrane, the results suggest that the alterations in the size of EVs could be due to the alteration of the fluidity and stability of the membrane covering the EVs. Furthermore, the uptake of smaller EVs by recipient cells was increased, which may lead to enhanced functional results. These results provide fundamental insights into the factors defining the size of EVs, which may be important for developing cancer screening methods and understanding cancer-related pathophysiology.

The interest in extracellular vesicles (EVs) has been increased in recent years due to their potential application in diagnosis and therapy of severe diseases. The versatile fields of application due to the numerous possible cargos and the targeted delivery system make them a promising biopharmaceutical product. However, their broad size range as well as varied surface protein content result in challenges for the purification, characterization, and quantification. In this study a novel method, based on high-resolution flow cytometry, was examined for the enumeration of EVs in purified as well as crude process samples. In addition to quantification, samples were characterized by dynamic light scattering, zeta potential measurement, and analytical size exclusion chromatography. It has been demonstrated that EVs were successfully enumerated with the novel method, offering great benefits for development and monitoring of EV processes.

Fibroblast growth factor-2 (FGF2) has multiple roles in cutaneous wound healing but its natural low stability prevents the development of its use in skin repair therapies. Here we show that FGF2 binds the outer surface of dermal fibroblast (DF)-derived extracellular vesicles (EVs) and this association protects FGF2 from fast degradation. EVs isolated from DF cultured in the presence of FGF2 harbor FGF2 on their surface and FGF2 can bind purified EVs in absence of cells. Remarkably, FGF2 binding to EVs is restricted to a specific subpopulation of EVs, which do not express CD63 and CD81 markers. Treatment of DF with FGF2-EVs activated ERK and STAT signaling pathways and increased cell proliferation and migration. Local injection of FGF2-EVs improved wound healing in mice. We further demonstrated that binding to EVs protects FGF2 from both thermal and proteolytic degradation, thus maintaining FGF2 function. This suggests that EVs protect soluble factors from degradation and increase their stability and half-life. These results reveal a novel aspect of EV function and suggest EVs as a potential tool for delivering FGF2 in skin healing therapies.

Age-related macular degeneration (AMD) is a leading cause of blindness. Vision loss is caused by the retinal pigment epithelium (RPE) and photoreceptors atrophy and/or retinal and choroidal angiogenesis. Here we use AMD patient-specific RPE cells with the Complement Factor H Y402H high-risk polymorphism to perform a comprehensive analysis of extracellular vesicles (EVs), their cargo and role in disease pathology. We show that AMD RPE is characterised by enhanced polarised EV secretion. Multi-omics analyses demonstrate that AMD RPE EVs carry RNA, proteins and lipids, which mediate key AMD features including oxidative stress, cytoskeletal dysfunction, angiogenesis and drusen accumulation. Moreover, AMD RPE EVs induce amyloid fibril formation, revealing their role in drusen formation. We demonstrate that exposure of control RPE to AMD RPE apical EVs leads to the acquisition of AMD features such as stress vacuoles, cytoskeletal destabilization and abnormalities in the morphology of the nucleus. Retinal organoid treatment with apical AMD RPE EVs leads to disrupted neuroepithelium and the appearance of cytoprotective alpha B crystallin immunopositive cells, with some co-expressing retinal progenitor cell markers Pax6/Vsx2, suggesting injury-induced regenerative pathways activation. These findings indicate that AMD RPE EVs are potent inducers of AMD phenotype in the neighbouring RPE and retinal cells.

Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (<200 nm, sEVs) are sufficiently abundant and accessible for developing genotyping protocols. We investigated this by extracting total intravesicular nucleic acid content from sEVs isolated from the conditioned cell medium of the human NCI-H1975 cell line containing the epidermal growth factor (EGFR) gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (<35–100 bp) present in the sEVs served as a template. Using qEV size exclusion chromatography (SEC), significantly lower yield and higher purity of isolated sEV fractions were obtained as compared to exoEasy membrane affinity purification and ultracentrifugation. Nevertheless, we detected the EGFR T790M mutation in the sEVs’ lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance EGFR T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows.

Exosomes are a class of extracellular vesicles that play a role in intercellular signaling under diverse contexts. Here, we describe a protocol that has been optimized for the isolation and characterization of exosomes from a Drosophila melanogaster cell line using size exclusion chromatography (SEC). The specific focus of this protocol was to examine the starvation-induced exosome loading of a protein. For complete details on the use and execution of this protocol, please refer to Pandey et al. (2021).

Biopharmaceutics Classification System (BCS) class II and IV drugs exhibit low solubility and suffer a limitation in oral administration. Exosomes have attracted intensive attention in the efficient delivery of such compounds. However, low gastrointestinal stability and high production cost of exosomes hinder their development as drug carriers. Here, milk exosomes are functionalized with phosphatidylserine and are capable of improving the solubility of BCS class II and IV drugs, resulting in facilitating the oral delivery of the drugs. A natural flavonoid, α-mangostin, is loaded into exosomes (AExo) to enhance the antibacterial efficiency, demonstrated by clearing 99% of bacteria in macrophages. Furthermore, AExo exhibits high mucus penetrability and shows a significant therapeutic efficacy in two animal infection models. Collectively, this work expands the application of exosomes from bovine milk with simple operation and low cost, shedding light on the potential of milk exosomes in improving the solubility of drugs to enhance the efficacy of oral administration.

Dermatomyositis and polymyositis (DM/PM) are systemic autoimmune diseases characterized by proximal muscle weakness. The underlying pathogenetic mechanism of this disease remains under-researched. Here, using proteomics analysis, a great overlap of differentially expressed plasma exosomal proteins involved in the complement and coagulation cascade pathway, including FGA, FGB, FGG, C1QB, C1QC, and VWF, was identified in DM/PM patients versus healthy controls. Correlation analysis showed that the expression levels of complement-associated proteins (C1QB and C1QC) correlated positively with CRP, ESR, and platelet count. ROC curve analysis demonstrated that complement and coagulation cascade-associated proteins could be strong predictors for DM/PM. In addition, we also identified several other proteins that were differentially expressed in DM and PM. The selected candidate proteins were further validated by parallel reaction monitoring (PRM) and enzyme-linked immunosorbent assay (ELISA). Together, our findings indicate that these exosome-derived proteins might participate in microvascular damage in DM/PM through the activation of the complement and coagulation cascade pathway and function as biomarkers for the clinical diagnosis of DM/PM.

The interaction of SARS-CoV-2 infection with extracellular vesicles (EVs) is of particular interest at the moment. Studying SARS-CoV-2 contaminated-EV isolates in instruments located outside of the biosafety level-3 (BSL-3) environment requires knowing how viral inactivation methods affect the structure and function of extracellular vesicles (EVs). Therefore, three common viral inactivation methods, ultraviolet-C (UVC; 1350 mJ/cm2), β-propiolactone (BPL; 0.005%), heat (56°C, 45 min) were performed on defined EV particles and their proteins, RNAs, and function. Small EVs were isolated from the supernatant of SARS-CoV-2-infected human lung epithelial Calu-3 cells by stepwise centrifugation, ultrafiltration and qEV size-exclusion chromatography. The EV isolates contained SARS-CoV-2. UVC, BPL and heat completely abolished SARS-CoV-2 infectivity of the contaminated EVs. Particle detection by electron microscopy and nanoparticle tracking was less affected by UVC and BPL than heat treatment. Western blot analysis of EV markers was not affected by any of these three methods. UVC reduced SARS-CoV-2 spike detectability by quantitative RT-PCR and slightly altered EV-derived β-actin detection. Fibroblast migration-wound healing activity of the SARS-CoV-2 contaminated-EV isolate was only retained after UVC treatment. In conclusion, specific viral inactivation methods are compatible with specific measures in SARS-CoV-2 contaminated-EV isolates. UVC treatment seems preferable for studying functions of EVs released from SARS-CoV-2 infected cells.

The oviduct provides optimum physiological and biochemical milieu essential for successful fertilization, early embryo development and facilitates functional maturation of spermatozoa. A study has revealed that spermatozoa alters the gene expression in bovine oviductal epithelial cells (BOECs) remotely via bio-active particles, thus acting as a cue to the oviduct prior to their arrival. However, very little attention has been paid to the question of whether spermatozoa could alter the cargo of extracellular vesicles (EVs) derived from BOECs. Therefore, the aim of this study was to investigate the alterations in small non-coding RNAs in EVs cargo derived from BOECs when incubated with spermatozoa in contact and non-contact co-culture models. After 4 h of incubation the EVs were isolated from the conditioned media, followed by small non-coding sequencing of the BOEC derived EVs. Our results revealed that EVs from both co-culture models contained distinct cargo in form of miRNA, fragmented mRNA versus control. The pathway enrichment analysis revealed that EV miRNA from direct co-culture were involved in the biological processes associated with phagocytosis, macroautophagy, placenta development, cellular responses to TNF and FGF. The mRNA fragments also varied within the different groups and mapped to the exonic regions of the transcriptome providing vital insights regarding the changes in cellular transcriptome on the arrival of spermatozoa. The findings of this study suggest that spermatozoa, in contact as well as remotely, alter the EV cargo of female reproductive tract epithelial cells which might be playing an essential role in pre and post-fertilization events.

Patients with single ventricle heart defects requires a series of staged open-heart procedures, termed Fontan palliation. However, while lifesaving, these operations are associated with significant morbidity and early mortality. The attendant complications are thought to arise in response to the abnormal hemodynamics induced by Fontan palliation, although the pathophysiology underlying these physicochemical changes in cardiovascular and other organs remain unknown. Here, we investigated the microRNA (miRNA) content in serum and serum-derived extracellular vesicles (EVs) by sequencing small RNAs from a physiologically relevant sheep model of the Fontan operation. The differential expression analysis identified the enriched miRNA clusters in (1) serum vs. serum-derived EVs and (2) pre-Fontan EVs vs. post-Fontan EVs. Metascape analysis showed that the overexpressed subset of EV miRNAs by Fontan procedure target liver-specific cells, underscoring a potentially important pathway involved in the liver dysfunction that occurs as a consequence of Fontan palliation. We also found that post-Fontan EV miRNAs were associated with senescence and cell death, whereas pre-Fontan EV miRNAs were associated with stem cell maintenance and epithelial-to-mesenchymal transition. This study shows great potential to identify novel circulating EV biomarkers from Fontan sheep serum that may be used for the diagnosis, prognosis, and therapeutics for patients that have undergone Fontan palliation.

Advancements in extracellular vesicle (EV) studies necessitate the development of optimized storage conditions to ensure preservation of physical and biochemical characteristics. In this study, the most common buffer for EV storage (phosphate-buffered saline/PBS) was compared to a cryoprotective 5% sucrose solution. The size distribution and concentration of EVs from two different sources changed to a greater extent after −80 °C storage in PBS compared to the sucrose solution. Additionally, molecular surface protrusions and transmembrane proteins were more prevalent in EVs stored in the sucrose solution compared to those stored in PBS. This study demonstrates, for the first time, that distinct ring-like molecular complexes and cristae-like folded membranous structures are visible upon EV degradation. Taken together, the size, concentration, molecular surface extensions, and transmembrane proteins of EVs varied substantially based on the buffer used for −80 °C storage, suggesting that biocompatible cryoprotectants, such as sucrose, should be considered for EV studies.

Colorectal cancer (CRC) is a leading cause of cancer-related death. There is an urgent need for new methods of early CRC detection and monitoring to improve patient outcomes. Extracellular vesicles (EVs) are secreted, lipid-bilayer bound, nanoparticles that carry biological cargo throughout the body and in turn exhibit cancer-related biomarker potential. RNA binding proteins (RBPs) are post-transcriptional regulators of gene expression that may provide a link between host cell gene expression and EV phenotypes. Insulin-like growth factor 2 RNA binding protein 1 (IGF2BP1/IMP1) is an RBP that is highly expressed in CRC with higher levels of expression correlating with poor prognosis. IMP1 binds and potently regulates tumor-associated transcripts that may impact CRC EV phenotypes. Our objective was to test whether IMP1 expression levels impact EV secretion and/or cargo. We used RNA sequencing, in vitro CRC cell lines, ex vivo colonoid models, and xenograft mice to test the hypothesis that IMP1 influences EV secretion and/or cargo in human CRC. Our data demonstrate that IMP1 modulates the RNA expression of transcripts associated with extracellular vesicle pathway regulation, but it has no effect on EV secretion levels in vitro or in vivo. Rather, IMP1 appears to affect EV regulation by directly entering EVs in a transformation-dependent manner. These findings suggest that IMP1 has the ability to shape EV cargo in human CRC, which could serve as a diagnostic/prognostic circulating tumor biomarker.

Extracellular vesicles (EVs) have therapeutic effects on osteoarthritis (OA). Some recent strategies could elevate EV's therapeutic properties including cell aggregation, co-culture, and 3D culture. It seems that a combination of these strategies could augment EV production and therapeutic potential. The current study aims to evaluate the quantity of EV yield and the therapeutic effect of EVs harvested from rabbit mesenchymal stem cells (MSCs) aggregates, chondrocyte aggregates, and their co-aggregates in a dynamic 3D culture in a rat osteoarthritis model. MSC and chondrocytes were aggregated and co-aggregated by spinner flasks, and their conditioned medium was collected. EVs were isolated by size exclusion chromatography and characterized in terms of size, morphology and surface markers. The chondrogenic potential of the MSC-ag, Cho-ag and Co-ag EVs on MSC micromass differentiation in chondrogenic media were assessed by qRT-PCR, histological and immunohistochemical analysis. 50 μg of MSC-ag-EVs, Cho-ag-EVs and Co-ag-EVs was injected intra-articularly per knee of OA models established by monoiodoacetate in rats. After 8 weeks follow up, the knee joints were harvested and analyzed by radiographic, histological and immunohistochemical features. MSC/chondrocyte co-aggregation in comparison to MSC or chondrocyte aggregation could increase EV yield during dynamic 3D culture by spinner flasks. Although MSC-ag-, Cho-ag- and Co-ag-derived EVs could induce chondrogenesis similar to transforming growth factor-beta during in vitro study, Co-ag-EV could more effectively prevent OA progression than MSC-ag- and Cho-ag-EVs. Our study demonstrated that EVs harvested from the co-aggregation of MSCs and chondrocytes could be considered as a new therapeutic potential for OA treatment.

Background Small extracellular vesicles (sEVs) are membrane vesicles released by healthy and malignant cells. sEVs are potential biomarkers for cancer diagnosis. Cervical cancer (CC) is the fourth most common cancer in females worldwide. Existing biomarkers, such as squamous cell carcinoma antigens, show low specificity. Hence, a novel biomarker for the diagnosis of CC is required. This study aimed to identify potential candidates in sEVs through proteomic analysis for the diagnosis of CC and to determine the EV protein profile to distinguish between healthy and CC serum samples. Methods The number and size distribution of sEVs in healthy controls (HC) and CC were measured using nanoparticle tracking analysis. Differential ultracentrifugation combined with size-exclusion chromatography was used to isolate and purify sEVs derived from the serum of HC and CC. The isolated sEVs were characterized using western blotting and transmission electron microscopy. Liquid chromatography-tandem mass spectrometry was used to identify and compare the protein profiles between CC and HC. EV proteins were validated using the TCGA database. Results The particle concentration in CC was marginally higher than that in HC. The mode size of the particles in CC was significantly smaller than that in the HC-derived particles. Proteomic and functional protein analyses revealed a difference in the EV protein profiles between HC and CC. We found three and 18 uniquely expressed proteins in HC and CC, respectively. Unique EV proteins in CC are involved in angiogenesis and the Ras, VEGF, and FAS signaling pathways, while EV proteins in HC are involved in cellular homeostasis. EV proteins such as C1QB, MYO3B, and NADSYN1 were significantly upregulated in CC and primary tumor tissues, whereas MAFK, OR13C9, PIK3C2, PLCB4, RAB12, and VIP were downregulated in CC sEVs and primary tumor tissues. Conclusion Our study provides useful insights into the potential of sEVs as noninvasive biomarkers for CC diagnosis. Validation with a well-designed cohort should be performed to assure the clinical diagnostic value of specific protein markers for CC sEVs.

Small extracellular vesicles (sEVs) have in recent years evolved as a source of biomarkers for disease diagnosis and therapeutic follow up. sEV samples derived from multicellular organisms exhibit a high heterogeneous repertoire of vesicles which current methods based on ensemble measurements cannot capture. In this work we present droplet barcode sequencing for protein analysis (DBS-Pro) to profile surface proteins on individual sEVs, facilitating identification of sEV-subtypes within and between samples. The method allows for analysis of multiple proteins through use of DNA barcoded affinity reagents and sequencing as readout. High throughput single vesicle profiling is enabled through compartmentalization of individual sEVs in emulsion droplets followed by droplet barcoding through PCR. In this proof-of-concept study we demonstrate that DBS-Pro allows for analysis of single sEVs, with a mixing rate below 2%. A total of over 120,000 individual sEVs obtained from a NSCLC cell line and from malignant pleural effusion (MPE) fluid of NSCLC patients have been analyzed based on their surface proteins. We also show that the method enables single vesicle surface protein profiling and by extension characterization of sEV-subtypes, which is essential to identify the cellular origin of vesicles in heterogenous samples.

The brain is protected against invading organisms and other unwanted substances by tightly regulated barriers. However, these central nervous system (CNS) barriers impede the delivery of drugs into the brain via the blood circulation and are therefore considered major hurdles in the treatment of neurological disorders. Consequently, there is a high need for efficient delivery systems that are able to cross these strict barriers. While most research focuses on the blood-brain barrier (BBB), the design of drug delivery platforms that are able to cross the blood-cerebrospinal fluid (CSF) barrier, formed by a single layer of choroid plexus epithelial cells, remains a largely unexplored domain. The discovery that extracellular vesicles (EVs) make up a natural mechanism for information transfer between cells and across cell layers, has stimulated interest in their potential use as drug delivery platform. Here, we report that choroid plexus epithelial cell-derived EVs exhibit the capacity to home to the brain after peripheral administration. Moreover, these vesicles are able to functionally deliver cargo into the brain. Our findings underline the therapeutic potential of choroid plexus-derived EVs as a brain drug delivery vehicle via targeting of the blood-CSF interface.