Journal of Extracellular Biology

BackgroundExtracellular vesicles (EVs) are nano and macro-sized, lipid-bound particles, involved in cellular communication. Interestingly, cancer-derived EVs show a heterologous and cross-species tumour tropism which makes them a potential tool for efficient delivery of therapeutic small interfering RNA (siRNA) to the tumour cells. MethodsEVs derived from glioblastoma cells (U373P and U373vIII) were loaded with EGFRvIII siRNA to develop a targeted therapeutic strategy against glioblastoma. EV biodistribution was evaluated using fluorescent indocyanine green (ICG) staining followed by ex vivo imaging. Different loading strategies, including passive loading, sonication, saponin-mediated membrane permeabilization, electroporation, and transfection were assessed for their efficiency in loading siRNA into EVs. The efficiency of each method was evaluated by nano flowcytometry, in vitro uptake assay followed by immunoblot (western blot) analysis. Eventually, the most effective formulation was tested for the systemic siRNA administration and selective tumour delivery in vivo, followed by evaluation of tumour size and EGFRvIII expression. ResultsHere, we showed that siRNA transfection into EVs was the most effective loading strategy, as confirmed by nano-flow cytometry, uptake assays, and western blot analysis, achieving over 90% knockdown efficiency in vitro for EVs carrying EGFRvIII siRNA. In vivo, EGFRvIII siRNA-loaded EVs homed to the tumour site and downregulated EGFRvIII expression compared with the PBS-siRNA control group; however, no significant tumour shrinkage was observed. ConclusionEGFRvIII-targeting, glioblastoma cell-derived EVs can be used as siRNA delivery carriers for targeted gene therapy in glioblastoma. However, further optimization of siRNA delivery and treatment duration is required.

BackgroundP-glycoprotein (P-gp/ABCB1) is a key efflux transporter that maintains barrier integrity by clearing xenobiotics and toxic metabolites. At the feto-maternal interface, trophoblast-derived extracellular vesicles (CTC-EVs) naturally and transiently transfer functional P-gp to maternal decidual cells, restoring lost and or reduced P-gp function (exofection) to sustain pregnancy homeostasis. A similar loss of P-gp at the blood brain barrier (BBB) contributes to impaired amyloid-{beta} (A{beta}) clearance and neuroinflammation in Alzheimers disease. We investigated whether CTC-EV-mediated exofection could restore P-gp function in human brain endothelial cells (hBECs) and enhance A{beta} clearance under inflammatory and neurodegenerative conditions. MethodsCTC-EVs were isolated and characterized by nanoparticle tracking analysis and western blotting for P-gp and EV markers. Transcriptomic profiling of CTC-EVs identified enrichment of transporter-related genes, including solute carriers and ABC transporters, along with inflammatory mediators. Network analysis revealed coordinated modules linking EV cargo to transporter regulation, endocytosis/trafficking pathways, and inflammatory remodeling processes converging on BBB efflux activity. hBECs were exposed to LPS (500 ng/mL, 48 h) with or without CTC-EVs. P-gp expression was assessed by immunofluorescence (mean fluorescence intensity, MFI) and western blotting, while functional efflux was measured using Calcein-AM assays. A{beta} oligomer transport was evaluated using a transwell hBEC model. In vivo, 3xTg-AD mice received intravenous CTC-EVs (1x10L/day for 5 days), followed by assessment of P-gp expression, A{beta} burden, and neuroinflammatory markers. Pharmacokinetic studies in P-gp knockout mice were conducted to confirm functional transporter recovery. ResultsLPS exposure significantly reduced P-gp expression in hBECs (41.3% decrease in MFI, p=0.0084), which was restored by CTC-EVs (46.7% increase vs. LPS, p=0.0121). Exofection increased P-gp by a 2.1-fold following EV treatment as determined by western blot. Functional assays demonstrated enhanced efflux, with a 38.5% reduction in intracellular Calcein fluorescence (p<0.001). Network-informed mechanisms supported coordinated regulation of transporter and trafficking pathways. CTC-EVs improved A{beta} transport across inflamed hBEC monolayers. In vivo, EV-treated 3xTg-AD mice exhibited increased P-gp expression in the frontal cortex (38.6%) and hippocampus (42.1%), reduced A{beta} plaque burden (27.9%), and decreased inflammatory markers (IL-1{beta} and TNF-, p<0.05). In P-gp knockout mice, EVs reduced brain drug accumulation by 22.4% (p=0.032), confirming restoration of transporter function. ConclusionCTC derived EVs are natural carriers of functional transporter proteins and restore efflux capacity in compromised endothelial barriers. Integration of transcriptomic and network analyses highlights coordinated regulation of transporter, trafficking, and inflammatory pathways underlying exofection. This reproductive biology inspired strategy offers a promising therapeutic approach for enhancing A{beta} clearance and mitigating neuroinflammation in Alzheimers disease.

BackgroundSmall extracellular vesicles (EVs) are nanosized, endosome-derived particles which transfer RNA, proteins, and bioactive molecules to mediate intercellular communication. While EV signaling has been observed in many organ systems, it remains unclear whether glomerular endothelial cell (GEC)-derived small EVs directly interact with podocytes in vivo or how they traverse the glomerular basement membrane (GBM). MethodsGEC-derived small EVs were characterized by nanoparticle tracking analysis, electron microscopy, RAMAN spectroscopy and flow cytometry. Cargo composition was analyzed by proteomics, and microRNA (miR) profiling. Functional and structural features were examined using protease, collagenase, adhesion, and multimodal imaging assays. GEC-derived small EV uptake and downstream transcriptional effects were studied in cultured podocytes, while in vivo trafficking was assessed by injection of labeled small EVs into transgenic zebrafish larvae under baseline conditions, puromycin-induced damage, and cd2ap-knockdown. ResultsGECs released bona fide exosome-like small EVs carrying a highly cell type-specific miR cargo. Small EV transfer to podocytes induced a defined transcriptional response consistent with miR-mediated repression of target genes involved in extracellular matrix organization, cell cycle regulation, and cellular stress responses. Proteomic analyses revealed enrichment of surface proteases and integrin-associated proteins that conferred sustained proteolytic activity and enabled GEC-derived small EV migration through extracellular matrix surrogates. In vivo, circulating small EVs traversed the GBM and localized selectively to podocytes in healthy glomeruli, whereas glomerular injury permitted small EV entry into the tubular compartment. ConclusionThese findings provide first in vivo evidence that GEC-derived small EVs can cross the GBM and impact on podocytes. By identifying integrin- and protease-dependent mechanisms which facilitate vesicle passage, this study redefines the GBM as a dynamic interface of heterocellular, vesicle-mediated communication.

AbstractInflammation and oxidative stress are key drivers in the pathogenesis of chronic lung diseases, including asthma, pulmonary fibrosis, and chronic obstructive pulmonary disease. Extracellular vesicles derived from the marine microalga Tetraselmis chuii, referred to as nanoalgosomes, have recently gained attention as natural nanocarriers that possess inherent antioxidant and anti-inflammatory properties. In this study, we investigated the biocompatibility and protective effects of aerosolized nanoalgosomes in a bronchial epithelial-macrophage co-culture model at the air-liquid interface. Co-cultures of CALU-3 epithelial cells and differentiated THP-1 macrophages were primed with aerosolised nanoalgosomes and subsequently exposed to either oxidative stress (tert-butyl hydroperoxide) or an inflammatory stimulus (lipopolysaccharide; LPS). Epithelial barrier integrity and cytotoxicity were evaluated using transepithelial electrical resistance and lactate dehydrogenase release assays, respectively, while intracellular reactive oxygen species levels and cytokine secretion were measured to assess antioxidant and immunomodulatory responses. Nanoalgosomes were non-cytotoxic, preserved epithelial barrier integrity, and significantly reduced oxidative stress. In addition, nanoalgosomes priming attenuated LPS-induced secretion of pro-inflammatory cytokines (IL-1{beta}, IL-6, IL-8, IL-18, TNF-) as well as the anti-inflammatory cytokine IL-10, suggesting a balanced immunomodulatory response. Overall, aerosolized nanoalgosomes maintained epithelial homeostasis and mitigated both oxidative and inflammatory stress, underscoring their potential as a safe, sustainable, and effective therapeutic strategy for chronic inflammatory lung diseases. Given their natural origin, excellent biocompatibility, and suitability for aerosol delivery, nanoalgosomes represent a promising class of inhalable biotherapeutics.

Environmental degradation and accumulation of plastics results in micro- and nanoplastics (MNPLs) that are small enough to cross biological barriers, including the blood-brain barrier. Microglia, resident immune cells of brain, are critical regulators of neuroimmune homeostasis and represent a cellular target of nanoplastic exposure. In this study, we assessed the neurotoxic effects of two sizes of polystyrene nanoplastics (PS-NPs; 100 nm and 500 nm) using integrated in vivo and in vitro exposure and washout paradigms. In vivo exposure in mice (60 days; 0.15 or 1.5 mg/day) showed the accumulation of both PS-NP sizes in the cerebral cortex without histopathological damage. However, cortical microglia showed pronounced morphological remodeling, observed as increased expression of Iba1 and GFAP. Transcriptomic profiling of cortical tissue revealed a strong size-dependent response. The 100 nm PS-NP group revealed 18 DEGs (|log2FC| [&ge;] 2, padj < 0.05), whereas the 500 nm PS-NPs showed more than 4,000 DEGs, including upregulation of immune- and microglia-associated genes (CCL5, CXCL10, LCN2, LYZ2) and downregulation of synaptic and neuronal signaling genes (GRIN2B, SYN1, STX1B, MAP1B, ITPR1/2). In vitro assessment, using BV2 microglia cells, showed internalization of PS-NPs via the endolysosomal pathway, with strong co-localization to Rab7- and LAMP2-positive compartments and prolonged intracellular retention following exposure washout. Also, microglial activation markers (Iba1, CD68) exhibited a transient, size- and concentration-dependent increase, correlated with intracellular particle burden rather than cumulative exposure. Overall, these findings demonstrate that PS-NPs accumulate in brain, driving size-dependent microglia activation and transcriptomic reprogramming, even after cessation of exposure to PS-NPs. HighlightsO_LIPS-NPs (100 nm and 500 nm) reach mouse cerebral cortex following 60-day oral exposure. C_LIO_LIPS-NPs were internalized by microglia; accumulated in endolysosomal compartments. C_LIO_LIPS-NP exposure induced transient microglial activation without sustained cytotoxicity. C_LIO_LIMicroglial activation was correlated with intracellular PS-NPs burden. C_LIO_LITranscriptomics revealed disruption of neuroimmune and microglial regulatory pathways. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/712727v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1aba3eaorg.highwire.dtl.DTLVardef@1967641org.highwire.dtl.DTLVardef@12da637org.highwire.dtl.DTLVardef@1fb8441_HPS_FORMAT_FIGEXP M_FIG C_FIG

Extracellular vesicles (EVs) are small lipid structures secreted by cells that originate from the cell surface (typically enriched in the tetraspanin (tspan) CD9) or from multivesicular bodies (typically enriched in the tspan CD63). Current methods for studying EVs involve concentrating and purifying EVs, without providing information about the distance or amount of EVs that may transfer from one cell to another. Here, we developed a coculture assay of human mammary MCF-7 cells to study the transfer of mCherry-CD81 or mCherry-CD9 from "donor" cells to a lawn of "acceptor" cells stained with cell tracker blue or green (CTB/CTG), non-transferrable fluorescent dyes. Using confocal fluorescence microscopy, we observed the presence of spots containing mCherry-CD81 or mCherry-CD9 outside donor cells, concentrated at short distance from donor cells and that overlapped with CTB signal, suggestive of their internalization in acceptor cells. Endogenous CD63, CD81 and CD9 also transferred more efficiently at short distances, even in the presence of a flow, as shown by immunostaining cocultures of wild type and KO CD-63, or -9, or -81 cells with antibodies directed against these tspans. Computation of the (x,y,z) coordinates of tspans-containing spots revealed a double polarized transfer: in (x,y), it distributed along a gradient that started from donor cells and decreased with the distance, and in (z), it was stronger in basal compared to upper planes, a (z) polarization that was affected by syntenin-1 depletion in donor cells. Simultaneous monitoring of CD9/CD81 transfer from into double CD81/CD9 KO cells showed that cells transferred more CD81 spots than of CD9. At the basal level, CD63 and CD81 spots were plasma membrane derived as they almost always contained CD9+, and resembled membranous remnants of migration. However, live cell imaging showed migration independent secretion of EVs in the extracellular space, in upper planes. Altogether, not only is our coculture assay suitable for the direct qualitative and quantitative study of EV-transfer, but it highlighted shared three-dimensional features of EV markers transfer between cells.

Extracellular vesicles are key intercellular messengers that modulate the function of target cells by carrying effectors, either at their surface or in their lumen. In the latter case, their action depends on the ability to deliver their content into the cytosol of target cells. How efficiently EVs deliver their content upon interaction with their target cell is thus a central question for understanding the functional impact of this mode of action. To address this question, signal-driven bimolecular interactions between two partners located respectively in the EV lumen and the target cell cytosol have become a widely used strategy to detect the cytosolic delivery EV content. However, the detection of cytosolic delivery with these assays was often tributary to the artificial enhancement of the fusion between EV and cell membranes, through for instance VSV-G fusogenic protein expression. Here we provide a robust and quantitative LUCiferase-based complementation assay (HiBiT/LgBiT), to quantify the Internalization and cytosolic Delivery of EV content: LUCID-EV. By optimizing the signal-to-noise ratio of the assay, the method for loading HiBiT fragment into EVs (fusion to a lipid-binding domain rather than to tetraspanins), and the intracellular position of LgBiT (associated to membranes), we could quantify cytosolic delivery from various non-VSV-G-expressing EVs into target immune dendritic cells. Importantly, this delivery did not involve the acidic late endosomes environment required for VSV-G-dependent EV cytosolic delivery. The limited efficacy of the process highlights the need for highly sensitive assays like the one described here. Further development of the LUCID-EV assay could help identifying EV/target cells pairs with enhanced cytosolic delivery properties and characterize the cellular route for delivery.

Extracellular vesicles (EVs) mediate cell-to-cell communication and are considered potential drug delivery vehicles. Nevertheless, whether EV-embedded cargo can be efficiently delivered into the cytosol of recipient cells remains debated. Here, we investigated the fate of syntenin, a well-established internal cargo of small EVs (sEVs). Using quantitative assays, we show that [~]85% of internalized sEV-embedded syntenin can be delivered to the cytosol of recipient cells within short periods of time. Yet, even at low dose, we find that the internalization of sEVs carrying syntenin is rather inefficient ([~]0.03% of the administered dose). Moreover, we observe that the capture of sEVs by recipient cells is non-saturable over time and largely more efficient than their internalization. Finally, we identify the N-terminal domain of syntenin and the phosphorylation state of a Src-targeted tyrosine residue in this domain, as key determinants for its incorporation into sEVs that support cytosolic delivery. These findings challenge, current views in the field by indicating that sEV internalization may be a marginal process (on the contrary to capture) and that cytosolic delivery can be highly efficient. Moreover, our study identifies molecular determinants governing cytosolic delivery of sEV-embedded syntenin.

Extracellular vesicles (EVs) are key mediators of intercellular communication, yet their molecular profiles across tissues and species remain poorly characterized, particularly due to currently available methods requiring a large amount of biological material (tissue or biofluids). Here, we established a workflow allowing the deep phenotyping of EV cargos starting from single samples of human and mouse origin. We took advantage of standardised EV isolation procedures and multi-omic techniques for the isolation and analysis of EVs from brain and plasma of human and mouse, integrating flow cytometric profiling, proteomics, miRNA sequencing, and fatty acid profiling. Here we report specific brain-derived EVs proteome, enriched in neuronal and glial proteins, polyunsaturated fatty acids profiles, and distinct miRNAs. At the periphery, we also report plasma-derived EVs signatures reflecting immune, metabolic, and systemic transport functions. Despite these expected material-specific differences, EVs from the same source displayed greater similarity across species than EVs from different material, supporting the translational relevance of mouse models. Importantly, using state-of-the-art miRNA profiling approach, we identified novel EV-specific miRNAs in human and mouse brain EVs, potentially allowing the exploration of new roles in neuronal signalling. Overall, we report here a method enabling deep multi-omic characterization from minimal starting material, offering a practical approach for studies with limited biological samples. These findings also demonstrate that the origin strongly shapes EV composition, highlighting conserved and species-specific molecular features, and provide a scalable framework for multi-omic investigations of EV biology. Summary StatementWe present a standardised workflow allowing multi-omic profiling of brain and plasma-derived EVs from minimal human and mouse material. Our findings reveal both tissue-specific and species specific EV molecular signatures.

Extracellular vesicles (EVs) hold significant promise as biomarkers, but their clinical translation is constrained by variability in pre-analytical handling and isolation. EV isolation methods directly shape which EV populations are captured and characterized, yet systematic method comparisons across multiple analytical dimensions are limited. We comprehensively evaluated eleven EV isolation methods to define their performance and applications. EVs were quantified by NanoFCM, profiled for tetraspanins (CD9, CD63, CD81) via MSD assays, and further characterized by LC-MS/MS proteomics. We show that different EV isolation methods recover different EV populations. Our data provide guidance on method selection based on downstream application needs and serve as a look-up tool if a protein of interest is detected. EV isolation methods broadened proteome coverage but showed divergent performance and recover different EV populations. While all methods captured EVs in the 50-150nm range, centrifugation and ultracentrifugation identified the broadest proteomes (up to 1093 proteins) driven by higher plasma protein carryover. Conversely, ExoEasy and qEV 70 isolated larger EVs and achieved stronger depletion of abundant plasma proteins but showed lower proteome coverage. A total of 117 proteins were detected across all isolation methods. Pre-clearing samples removed contaminants but at the cost of protein identifications. We demonstrate that method selection must align with the specific analytical goal: centrifugation for comprehensive proteome profiling, affinity/size-exclusion methods for contaminant-sensitive assays, and precipitation for high-throughput applications. This systematic characterization provides an evidence-based framework and look-up resource for matching isolation strategies to downstream applications and research questions. Graphical Abstract for Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/710675v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@12ad967org.highwire.dtl.DTLVardef@270e4eorg.highwire.dtl.DTLVardef@1c41bcorg.highwire.dtl.DTLVardef@11fb236_HPS_FORMAT_FIGEXP M_FIG C_FIG This study evaluated 11 extracellular vesicle (EV) isolation methods which enriched distinct EV subpopulations with varying degrees of contaminants. No single approach optimized purity or proteome coverage; in this paper we present an Evidence-Based Framework to select plasma EV isolation methods based on downstream application needs.

Efficient, aggregation-free extracellular vesicles (EVs) labeling is essential for studying their dynamics in-vitro and in-vivo. However, traditional dyes introduce limitations including aggregation, membrane intercalation, fluorescence transfer and inconsistent performance across EV sources thus distorting quantification, altering surface properties and confounding uptake and biodistribution analyses. Here, we systematically evaluated CytoLight, a luminal dye traditionally used for live-cell imaging, as an alternative for EV quantification, characterization, uptake analysis and in-vivo tracking, benchmarking it against PKH26, CFSE and ExoBrite across multiple platforms. CytoLight generated stable, intravesicular fluorescence without aggregation or membrane alteration, eliminating artifacts characteristic of conventional dyes. Using fluorescence-NTA and single-EV flow cytometry, CytoLight showed more consistent labeling across EV types than CFSE or ExoBrite, while avoiding PKH-related micelle-driven artifacts and exhibited compatibility with CD81 dual-detection. In uptake assays, CytoLight produced EV-specific endocytosis-dependent internalization signals exceeding labeled-BPS/protein controls. In-vivo, CytoLight-labeled EVs enabled fluorescent biodistribution mapping showing conventional EV tropism patterns distinguishable from labeled-PBS controls. These findings establish CytoLight as an effective, aggregation-free EV-labeling strategy. Its stability, specificity, compatibility with single-EV platforms and reliable performance in both cellular uptake and biodistribution studies position CytoLight as a practical, scalable alternative to current dyes, providing a stronger foundation for standardized and reproducible EV research.

Breastfeeding provides health benefits in childhood, reducing the frequency of gastrointestinal and respiratory infections. Breastmilk (BM) is a rich source of bioactive molecules including extracellular vesicles (EVs), which exert immunomodulatory signalling in recipient cells, with cargo that is affected by maternal characteristics. Here we investigated the biophysical characteristics of BM-EVs from mothers with (asthmatic BM-EVs) or without asthma (control BM-EVs) and their effect on the release of cytokines from primary human hTERT-immortalized airway smooth muscle cells (hASMs) from asthmatic or non-asthmatic (control) donors. BM-EVs were isolated using size exclusion chromatography (N=5/group), characterized biophysically and by EV-specific protein markers. In addition, BM-EV were co-cultured (48h) with primary hASM cells from both non-asthmatic (control) and asthmatic donors to determine the effect on cytokine release. All participants were Caucasian and the BM was collected 12-15 weeks postpartum. BM-EVs showed the presence of intact and small-EVs ([~]100 nm). Asthmatic BM-EVs appeared to have a smaller average EV size (135.6 nm) vs. controls (148.3 nm, p=0.0613), but [~]5-fold higher concentration of both total (p=0.0014) and small EVs (p=0.0016). The expression of EV subtype protein expression was reduced in asthmatic BM-EVs vs. control BM-EVs: CD63 by 86% (p=0.0224), flotillin-1 by 40% (p=0.0196), CD9 by 24% (p=0.0646) and HSP70 by 69% (p=0.0873). Asthmatic BM-EVs co-cultured with hASMs from control donors decreased pro-inflammatory cytokine release: MCP-1 by 55% (p=0.0286), IL-6 by 45% (p=0.0801) and IL-2 by 32% (p=0.0970) vs. control-BM-EVs. Conversely, asthmatic BM-EVs co-cultured with hASMs from asthmatic donors increased secretion of anti-inflammatory cytokine IL-10 by 32% (p=0.0660), and IL-1Ra by 75% (p=0.0875), and pro-inflammatory IL-2 by 57% (p=0.0688) vs. control-BM-EVs. Internalization of control and asthmatic BM-EVs was confirmed by labelled EV uptake experiments. No detrimental effects on cell viability with BM-EV treatment were observed. In summary, asthmatic BM-EVs are smaller and enriched in BM, and exert differential effects on cytokine release in a BM-donor and recipient-cell specific manner. Given that BM can enter infant airways, the immunomodulatory effects of BM-EVs on hASMs warrants further investigation to delineate the under underlying mechanisms.

BackgroundFlow virometry (FV) - the application of flow cytometry to viruses - has historically been hindered by the inability of cytometers to detect particles below [~]300 nm in size. However, advances in optics and fluidics have enabled cytometers primarily designed for cells to detect viruses and extracellular vesicles (EVs) through light scatter alone. In 2024, the CytoFLEX nano was released, marketed for the detection of particles as small as 40 nm; however, its performance has yet to be compared to a conventional instrument for FV. MethodsFV was utilized to evaluate performance of the CytoFLEX nano and a conventional flow cytometer (CytoFLEX S). Instrument scatter sensitivity was assessed using NIST beads (40-400 nm), and virus stocks [human immunodeficiency virus (HIV), human coronaviruses (HCoV)-229E and HCoV-OC43]. For fluorescence analysis, HIV virions were stained with PE- and BV421-conjugated antibodies targeting virion incorporated proteins (CD38, CD44), individually and in combination. Finally, HIV stocks were labeled with antibodies against the envelope (Env) glycoprotein and tetraspanins (CD9, CD81) to assess EVs within virus preparations. ResultsCompared to the CytoFLEX S, the CytoFLEX nano exhibited substantially greater scatter sensitivity, reflected by up to 50-fold higher signal-to-noise ratio across NIST-traceable beads and virus samples. This enabled clearer resolution of smaller populations, including bead populations < 70 nm that were undetectable on the CytoFLEX S, as well as improved resolution across all viruses. While both instruments reliably detected stained proteins on HIV virions, the CytoFLEX nano revealed a distinct population of tetraspanin-positive EVs within HIV stocks that was undetected on the CytoFLEX S. Using GFP-tagged HIV, we identified Env+ particles lacking GFP, indicating the presence of Env on EVs. ConclusionsThe CytoFLEX nano exhibited markedly improved scatter sensitivity compared to the CytoFLEX S, improving detection of viruses and enabling detection of EV populations that were undetectable on the conventional instrument. While both platforms performed similarly for surface protein labeling, additional consideration of spectral overlap was needed with the CytoFLEX nano in multicolor experiments. These findings highlight that the complementary strengths of each platform can be utilized to more comprehensively characterize virus and EV populations, providing new opportunities to investigate nanoparticle heterogeneity.

Extracellular vesicles (EVs) are lipid spheres released from cells. Research utilizing EVs has met several hurdles owing to the small size of the majority of EVs and other nanoparticles (<150 nm) and the lack of detection technologies capable of providing high-throughput single particle measurements at this scale. The use of high-throughput single particle measurements is critical for the assessment of EV heterogeneity and abundance which are features often used to assess the development of isolation protocols or particle characterization. The Coulter principle, known in the field as resistive pulse sensing (RPS), has been used for several decades to size and count cells. More recently, this technology has evolved to accommodate nanoparticle analysis. In the last decade a platform utilizing microfluidic resistive pulse sensing (MRPS) has been demonstrated for nanoparticles, offering ergonomic characterization of nanoparticles along with utilizing open format data. To date, assessment of MRPS accuracy and reporting standards have not been assessed. With the aim of increasing data accuracy, ergonomics, and reporting transparency, we developed a microfluidic resistive pulse sensing post-acquisition analysis software (RPSPASS) application for automated cohort calibration, population gating, statistical output, QC plot generation, alternative data file outputs, and standardized reporting templates.

Accurate and transparent characterization of extracellular vesicle (EV) preparations is essential to ensure reproducibility, comparability, and adherence to MISEV reporting standards. However, data outputs from commonly used instruments for assessing EV size, concentration, and surface charge (zeta potential) vary widely in format and structure, complicating standardized analysis and integration across platforms. We present PHoNUPS (Plotting the Histogram of Non-Uniform Particles Sizes), free and open-source software (FOSS) developed in R, that enables unified processing, analysis, and visualization of EV characterization data. PHoNUPS computes statistics and generates standardized histograms and contour plots (for size against zeta potential) suitable for transparent reporting and cross-study comparison. The software produces high-quality, publication-ready figures. Third-party graphical editing tools allow users to refine and annotate visualizations for presentation or manuscript preparation. PHoNUPS supports multiple measurement file formats, thereby facilitating dataset integration from different instruments. PHoNUPS was developed with extensibility at its core, providing a basis for user-driven growth. We invite the EV community--researchers, analysts, and tool developers--to use PHoNUPS, share feedback on their experience and needs, and contribute to the platform by integrating additional input data formats, analytical routines, and visualization functionalities. Graphical abstractThe free software PHoNUPS processes the outputs of several different EV characterization instruments and it is extensible with further ones. It computes statistics of particle size and zeta potential distributions and it plots the corresponding histograms or contour plots. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/702479v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@b2b3a1org.highwire.dtl.DTLVardef@2f2907org.highwire.dtl.DTLVardef@2ec521org.highwire.dtl.DTLVardef@903624_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pathogenic bacterial extracellular vesicles (BEVs) can disrupt the blood-brain barrier (BBB), leading to neuroinflammation. Prior in vitro studies of this process were performed in simple models that may have lacked important physiological factors. We sought to determine if treatment with Escherichia coli-derived BEVs could directly compromise the integrity of a BBB lab-on-chip model or if an immune component was required. Our device featured isogenic human induced pluripotent stem cell-derived brain microvascular endothelial-like cells (BMECs) and pericytes separated by an ultrathin, porous silicon nitride membrane. BEVs and free lipopolysaccharide (LPS) were capable of causing upregulation of intercellular adhesion molecule-1 on the BMEC surfaces, which is important for immune cell recruitment. However, neither BEVs nor LPS at physiological doses caused pronounced loss of BMEC tight junction proteins, nor did they increase barrier permeability to small dye molecules. In contrast, stimulating THP-1 macrophages with BEVs led to increased production of pro-inflammatory cytokines, and conditioned media from the stimulated macrophages disrupted BMEC tight junctions and increased barrier permeability. Our work demonstrates the importance of incorporating an immune component in studies of BEV-mediated disruption of BBB models.

Messenger RNA (mRNA) vaccines using lipid nanoparticles (LNPs) are well-established and globally approved with acceptable safety profiles for preventing respiratory disease. Other mRNA-LNP product concepts are also emerging as novel treatments for broader clinical use. Here, we describe mRNA-LNP vaccine tissue distribution and kinetics after intramuscular dosing using three products formulated with same LNP matrix: mRNA-1273 (Spikevax), mRNA-1647 (a candidate cytomegalovirus [CMV] vaccine), and a reporter mRNA (nascent peptide-luciferase) drug product. Consistent biodistribution patterns were observed across studies: tissues with highest exposures were the injection site, draining lymph nodes, and spleen, with minimal distribution to non-lymphoid tissues. Vaccine components cleared rapidly from circulation and tissues, with complete elimination simulated to occur by [~]2 weeks. Following mRNA-1273 vaccination, Spike protein levels were transiently observed (elimination <5 days) and did not accumulate with repeated dosing. The ionizable lipid in the LNP matrix, Lipid H, underwent biotransformation and was excreted renally and hepatically, with no human-specific metabolites. Collectively, these results indicate that the LNP composition, not mRNA cargo, governs biodistribution. Furthermore, in a SARS-CoV-2 infection-free model, there was no evidence of Spike protein persistence. Overall, the data establish a framework that justifies leveraging biodistribution data across products and supports eliminating redundant animal studies.

The hollow fiber infection model (HFIM) is a translational in vitro model that links time-varying human pharmacokinetic profiles to the associated viral dynamic responses, from which pharmacokinetic/pharmacodynamic (PK/PD) targets can be derived. Establishing such targets is essential for antiviral dose selection and optimization. This is particularly important for cytomegalovirus (CMV) infection treatment, which primarily affects vulnerable patient populations. PK/PD targets for ganciclovir, the first-line drug for treatment, are not yet defined. The lack of an undefined PK/PD target makes dose optimization challenging and may result in suboptimal exposure, prolonged toxicity, and the emergence of resistance. For the first time, we have demonstrated the use of a low-cost hemodialyzer hollow fiber cartridge with application for CMV infection using ganciclovir. We have established a system that 1) supports CMV culture for PD analysis, 2) reproduces a clinically relevant ganciclovir PK profile, and 3) maintains consistent drug exposure in the infected cells, allowing reliable PK/PD analysis. Quantitative methods such as tissue culture infectious dose 50% (TCID50) and quantitative PCR were used to assess both active virus replication and genome copies production. Ganciclovir PK was measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This validation study serves as a fundamental step that can allow further PK/PD studies for ganciclovir and other antiviral agents that is still largely understudied. Consequently, this model could provide an affordable and practical platform for establishing clinically relevant PK/PD targets and guide treatment optimization.

Edible plant-derived nanovesicles (PDNVs) have emerged as promising nanotherapeutic strategies for various diseases, including cancer. The biochemical composition and functional properties of PDNVs vary considerably on the basis of their botanical source. Bacopa monnieri (L.) Wettst is a medicinal plant renowned for its rich phytochemical profile, yet the isolation and biological activities of B. monnieri -derived nanovesicles (BMNVs) remain unexplored. We report, for the first time, the isolation, molecular cargo profiling, and in vitro functional evaluation of BMNVs against neuroblastoma cells. The isolated BMNVs displayed a characteristic bilayer morphology with an average particle size of [~]112 nm. Mass spectrometry-based metabolite analysis revealed an enrichment of triterpenoids and triterpene saponins, whereas protein cargo analysis revealed superoxide dismutase, which is correlated with their intrinsic free radical scavenging activity. In vitro assays demonstrated that BMNVs significantly suppress neuroblastoma cell growth and induce morphological alterations. Confocal three-dimensional reconstruction confirmed the cellular internalization of the BMNVs, revealing a distinct perinuclear distribution. This study provides the first evidence of the use of BMNVs as bioactive carriers, highlighting their potential as novel nanotherapeutic agents and establishing B. monnieri as a valuable natural resource for the development of bioactive plant-derived nanovesicles for nanomedicine. HighlightsO_LIFirst isolation and biophysical characterization of Bacopa monnieri-derived nanovesicles (BMNVs). C_LIO_LIBMNVs have diverse metabolite profiles and are notably enriched in triterpenoids and triterpene saponins. C_LIO_LIThe superoxide dismutase (SOD) identified within BMNVs confers intrinsic free radical-scavenging activity. C_LIO_LIBMNVs exhibit therapeutic potential as anti-neuroblastoma agents. C_LIO_LIThese edible plant-derived nanovesicles offer a versatile, biogenic platform to explore for further development in diverse therapeutic and nutraceutical applications. C_LI

Post-acute sequelae of SARS-CoV-2 infection (PASC), commonly referred to as Long COVID, comprise a constellation of persistent, recurrent, or newly emerging symptoms that may endure for months or years following acute infection. Beyond respiratory impairment, PASC is characterized by a wide spectrum of extrapulmonary manifestations, among which neurological and neuropsychiatric symptoms are highly prevalent. Reported features include olfactory dysfunction with loss of smell and taste, fatigue, neuroinflammation, cognitive and memory impairment, depression, and anxiety, with some symptoms persisting up to one year post-infection. Despite increasing recognition of these complications, the molecular mechanisms underlying post-COVID neurological sequelae remain poorly defined. In this study, we employed a label-free quantitative (LFQ) proteomics approach to investigate protein alterations in olfactory neuroepithelium-derived stem cells (ONEs), a unique population of neural progenitors located in the olfactory mucosa at the interface between the respiratory system and both the peripheral and central nervous systems. Due to their anatomical exposure and susceptibility to SARS-CoV-2, ONEs represent a highly relevant translational model for exploring virus-associated neurobiological processes. ONEs derived from healthy donors were incubated with serum from either asymptomatic PCR-positive individuals (AS; n=4) or critically ill hospitalized patients (CR; n=6). Proteomic profiling revealed a distinct differential protein expression pattern in ONEs exposed to CR serum compared with AS serum. Altered pathways were associated with viral infection responses, respiratory and cardiovascular dysfunction, and notably, cerebrovascular and nervous system disorders. These findings highlight the vulnerability of ONEs to systemic factors associated with severe COVID-19 and provide molecular insight into mechanisms potentially contributing to persistent neurological sequelae in PASC. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/710460v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@12cfda5org.highwire.dtl.DTLVardef@c0636borg.highwire.dtl.DTLVardef@bf303eorg.highwire.dtl.DTLVardef@1f861e9_HPS_FORMAT_FIGEXP M_FIG C_FIG