Single Vesicles Research Articles

Size-based separation of extracellular vesicles investigating the relationship between Tetraspanins and RNA

BackgroundExtracellular vesicles (EVs), nano-sized particles released by cells, exhibit inherent heterogeneity, posing challenges for precise classification. This study introduces a method utilizing the coffee ring effect for size-based separation analysis, driven by the outward flow during droplet evaporation with Marangoni flow resulting from a surface tension gradient. ResultsThe controlled separation of nanoparticles based on size was applied to characterize EV and virus-like particle (VLP) samples. Tetraspanin markers exhibited distinct distribution patterns in dried droplets, with CD63-single-positive particles being smaller than those positive for CD9, CD81, or gag. Additionally, using previously developed fluorescence nanoparticle tracking analysis and transmission electron microscopy, single vesicle analysis validated the size disparities in CD-positive particles. RNA analysis through the coffee ring effect and fluorescent NTA revealed differential patterns in EVs and VLPs, providing different insights into RNA packaging. SignificanceThis multifaceted approach enhances understanding EV heterogeneity, emphasizing the potential influence of cellular origin and biogenesis pathways on particle characteristics.

Phenotypic Biomarkers of Aqueous Extracellular Vesicles from Retinoblastoma Eyes.

Recent advancements in aqueous humor (AH) cell-free DNA (cfDNA) genomics have opened new avenues for ex vivo molecular profiling of retinoblastoma (RB), the most common pediatric intraocular malignancy, where biopsy is typically prohibited. While these insights offer a genetic blueprint of the tumor, they lack multi-omic molecular phenotyping, which is essential for understanding the functional state. Extracellular vesicles (EVs), naturally present in AH, are promising by offering time-resolved phenotypic information. We employed multiplex bead-based flow cytometry and Single Extracellular Vesicle Nanoscopy (SEVEN) to analyze EV phenotypes in AH from a cohort of five RB, with three uveal melanoma (UM) and two age-matched glaucoma (GLC) samples serving as controls. The studies identified CD133-enriched EVs uniquely in RB AH, absent in both GLC and UM AH. This was corroborated by further analysis of five RB cell lines, including two commercial (Y79, Weri) and three in-house developed lines, confirming CD133 enrichment and supporting its role as an RB-specific EV marker. Single-vesicle analysis demonstrated a strong association of CD133 with CD81 and CD63, with minimal CD9 presence. These results, validated through complementary techniques, position CD133 as a critical marker in RB-derived EVs, paving the way for enhanced multi-omic RB characterization and potential advancements in clinical diagnostics.

Single Vesicle Surface Protein Profiling and Machine Learning-Based Dual Image Analysis for Breast Cancer Detection.

Single-vesicle molecular profiling of cancer-associated extracellular vesicles (EVs) is increasingly being recognized as a powerful tool for cancer detection and monitoring. Mask and target dual imaging is a facile method to quantify the fraction of the molecularly targeted population of EVs in biofluids at the single-vesicle level. However, accurate and efficient dual imaging vesicle analysis has been challenging due to the interference of false signals on the mask images and the need to analyze a large number of images in clinical samples. In this work, we report a fully automatic dual imaging analysis method based on machine learning and use it with dual imaging single-vesicle technology (DISVT) to detect breast cancer at different stages. The convolutional neural network Resnet34 was used along with transfer learning to produce a suitable machine learning model that could accurately identify areas of interest in experimental data. A combination of experimental and synthetic data were used to train the model. Using DISVT and our machine learning-assisted image analysis platform, we determined the fractions of EpCAM-positive EVs and CD24-positive EVs over captured plasma EVs with CD81 marker in the blood plasma of pilot HER2-positive breast cancer patients and compared to those from healthy donors. The amount of both EpCAM-positive and CD24-positive EVs was found negligible for both healthy donors and Stage I patients. The amount of EpCAM-positive EVs (also CD81-positive) increased from 18% to 29% as the cancer progressed from Stage II to III. No significant increase was found with further progression to Stage IV. A similar trend was found for the CD24-positive EVs. Statistical analysis showed that both EpCAM and CD24 markers can detect HER2-positive breast cancer at Stages II, III, or IV. They can also differentiate individual cancer stages except those between Stage III and Stage IV. Due to the simplicity, high sensitivity, and high efficiency, the DISVT with the AI-assisted dual imaging analysis can be widely used for both basic research and clinical applications to quantitatively characterize molecularly targeted EV subtypes in biofluids.

Pancreatic β-cells package double C2-like domain beta protein into extracellular vesicles via tandem C2 domains.

Double C2-like domain beta (DOC2B) is a vesicle priming protein critical for glucose-stimulated insulin secretion in β-cells. Individuals with type 1 diabetes (T1D) have lower levels of DOC2B in their residual functional β-cell mass and platelets, a phenotype also observed in a mouse model of T1D. Thus, DOC2B levels could provide important information on β-cell dys(function). Our objective was to evaluate the DOC2B secretome of β-cells. In addition to soluble extracellular protein, we assessed DOC2B localized within membrane-delimited nanoparticles - extracellular vesicles (EVs). Moreover, in rat clonal β-cells, we probed domains required for DOC2B sorting into EVs. Using Single Extracellular VEsicle Nanoscopy, we quantified EVs derived from clonal β-cells (human EndoC-βH1, rat INS-1 832/13, and mouse MIN6); two other cell types known to regulate glucose homeostasis and functionally utilize DOC2B (skeletal muscle rat myotube L6-GLUT4myc and human neuronal-like SH-SY5Y cells); and human islets sourced from individuals with no diabetes (ND). EVs derived from ND human plasma, ND human islets, and cell lines were isolated with either size exclusion chromatography or differential centrifugation. Isolated EVs were comprehensively characterized using dotblots, transmission electron microscopy, nanoparticle tracking analysis, and immunoblotting. DOC2B was present within EVs derived from ND human plasma, ND human islets, and INS-1 832/13 β-cells. Compared to neuronal-like SH-SY5Y cells and L6-GLUT4myc myotubes, clonal β-cells (EndoC-βH1, INS-1 832/13, and MIN6) produced significantly more EVs. DOC2B levels in EVs (over whole cell lysates) were higher in INS-1 832/13 β-cells compared to L6-GLUT4myc myotubes; SH-SY5Y neuronal-like cells did not release appreciable DOC2B. Mechanistically, we show that DOC2B was localized to the EV lumen; the tandem C2 domains were sufficient to confer sorting to INS-1 832/13 β-cell EVs. Clonal β-cells and ND human islets produce abundant EVs. In cell culture, appreciable DOC2B can be packaged into EVs, and a small fraction is excreted as a soluble protein. While DOC2B-laden EVs and soluble protein are present in ND plasma, further studies will be necessary to determine if DOC2B originating from β-cells significantly contributes to the plasma secretome.

Genetically-encoded markers for confocal visualization of single dense core vesicles.

Neuronal dense core vesicles (DCVs) store and release a diverse array of neuromodulators, trophic factors and bioamines. The analysis of single DCVs has largely been possible only using electron microscopy, which makes understanding cargo segregation and DCV heterogeneity difficult. To address these limitations, we developed genetically-encoded markers for DCVs that can be used in combination with standard immunohistochemistry and expansion microscopy, to enable single-vesicle resolution with confocal microscopy.

Genetically-encoded markers for confocal visualization of single dense core vesicles.

Neuronal dense core vesicles (DCVs) store and release a diverse array of neuromodulators, trophic factors and bioamines. The analysis of single DCVs has largely been possible only using electron microscopy, which makes understanding cargo segregation and DCV heterogeneity difficult. To address these limitations, we developed genetically-encoded markers for DCVs that can be used in combination with standard immunohistochemistry and expansion microscopy, to enable single-vesicle resolution with confocal microscopy.

Rapid Assessment of Biomarkers on Single Extracellular Vesicles Using "Catch and Display" on Ultrathin Nanoporous Silicon Nitride Membranes.

Extracellular vesicles (EVs) are particles released from cells that facilitate intercellular communication and have tremendous diagnostic and therapeutic potential. Bulk assays lack the sensitivity to detect rare EV subsets relevant to disease, and while single EV analysis techniques remedy this, they are often undermined by complicated detection schemes and prohibitive instrumentation. To address these issues, a microfluidic technique for EV characterization called "catch and display for liquid biopsy (CAD-LB)" is proposed. In this method, minimally processed samples are pipette-injected and fluorescently labeled EVs are captured in the nanopores of an ultrathin membrane. This enables the rapid assessment of EV number and biomarker colocalization by light microscopy. Here, nanoparticles are used to define the accuracy and dynamic range for counting and colocalization. The same assessments are then made for purified EVs and for unpurified EVs in plasma. Biomarker detection is validated using CD9 and Western blot analysis to confirm that CAD-LB accurately reports relative protein expression levels. Using unprocessed conditioned media,CAD-LB captures the known increase in EV-associated ICAM-1 following endothelial cell cytokine stimulation. Finally, to demonstrate CAD-LB's clinical potential, EV biomarkers indicative of immunotherapy responsiveness are successfully detected in the plasma of bladder cancer patients treated with immune checkpoint blockade.

Tetraspanins, GLAST and L1CAM Quantification in Single Extracellular Vesicles from Cerebrospinal Fluid and Serum of People with Multiple Sclerosis.

Objective: This study aimed to unravel the single tetraspanin pattern of extracellular vesicles (EVs), L1CAM+ and GLAST+ EV levels as diagnostic biomarkers to stratify people with multiple sclerosis (pwMS), specifically relapsing-remitting (RRMS) and primary progressive (PPMS). Methods: The ExoView platform was used to directly track single EVs using a clinically feasible volume of cerebrospinal fluid (CSF) and serum samples. This technology allowed us to examine the patterns of classical tetraspanin and quantify the levels of L1CAM and GLAST proteins, commonly used to immunoisolate putative neuron- and astrocyte-derived EVs. Results: The tetraspanin EV pattern does not allow us to differentiate RRMS, PPMS and non-MS donors neither in CSF nor serum, but this was associated with the type of biofluid. L1CAM+ and GLAST+ EVs showed a very low presence of tetraspanin proteins. Additionally, a significant decrease in the particle count of L1CAM+ EVs was detected in L1CAM-captured spots, and L1CAM+ and GLAST+ EVs decreased in GLAST-captured spots in the CSF from PPMS subjects compared to RRMS. Interestingly, only GLAST+ EVs exhibited a lower quantity in the CSF from PPMS compared to both MS and non-MS samples. Finally, GLAST+ EVs demonstrated a medium negative and significative correlation with GFAP levels-a biomarker of MS progression, astrocyte damage and neurodegenerative processes. Conclusions: ExoView technology could track neural EV biomarkers and be potentially useful in the diagnostic evaluation and follow-up of pwMS. GLAST+ EVs might provide insights into the etiology of PPMS and could offer small windows to elucidate the molecular mechanisms behind its clinical presentation.

Comparison of nanoimaging and nanoflow based detection of extracellular vesicles at a single particle resolution.

The characterization of single extracellular vesicle (EV) has been an emerging tool for the early detection of various diseases despite there being challenges regarding how to interpret data with different protocols or instruments. In this work, standard EV particles were characterized for single CD9+, single CD81+ or double CD9+/CD81+ tetraspanin molecule positivity with two single EV analytic technologies in order to optimize their EV sample preparation after antibody labelling and analysis methods: NanoImager for direct stochastic optical reconstruction microscopy (dSTORM)-based EV imaging and characterization, and Flow NanoAnalyzer for flow-based EV quantification and characterization. False positives from antibody aggregates were found during dSTORM-based NanoImager imaging. Analysis of particle radius with lognormal fittings of probability density histogram enabled the removal of antibody aggregates and corrected EV quantification. Furthermore, different machine learning models were trained to differentiate antibody aggregates from EV particles and correct EV quantification with increased double CD9+/CD81+ population. With Flow NanoAnalyzer, EV samples were prepared with different dilution or fractionation methods, which increased the detection rate of CD9+/CD81+ EV population. Comparing the EV phenotype percentages measured by two instruments, differences in double positive and single positive particles existed after percentage correction, which might be due to the different detection limit of each instrument. Our study reveals that the characterization of individual EVs for tetraspanin positivity varies between two platforms-the NanoImager and the Flow NanoAnalyzer-depending on the EV sample preparation methods used after antibody labelling. Additionally, we applied machine learning models to correct for false positive particles identified in imaging-based results by fitting size distribution data.

Analysis of Single Urinary Extracellular Vesicles from Subregions of the Nephron as a Diagnostic Tool in Early Diabetic Nephropathy

Analysis of Single Urinary Extracellular Vesicles from Subregions of the Nephron as a Diagnostic Tool in Early Diabetic Nephropathy

Multiparametric profiling of HER2-enriched extracellular vesicles in breast cancer using Single Extracellular VEsicle Nanoscopy

BackgroundPatients with HER2-positive breast cancer can significantly benefit from HER2-directed therapy – such as the monoclonal antibody trastuzumab. However, some patients can develop therapy resistance or change HER2 status. Thus, we urgently need new, noninvasive strategies to monitor patients frequently. Extracellular vesicles (EVs) secreted from tumor cells are emerging as potential biomarker candidates. These membrane-delimited nanoparticles harbor molecular signatures of their origin cells; report rapidly on changes to cellular status; and can be frequently sampled from accessible biofluids.ResultsUsing Single Extracellular VEsicle Nanoscopy (SEVEN) platform that combines affinity isolation of EVs with super-resolution microscopy, here we provide multiparametric characterization of EVs with ~ 8 nm precision and molecular sensitivity. We first interrogated cell culture EVs affinity-enriched in tetraspanins CD9, CD63, and CD81; these transmembrane proteins are commonly found on EV membranes. SEVEN robustly provided critical parameters of individual, tetraspanin-enriched EVs: concentration, size, shape, molecular cargo content, and heterogeneity. Trastuzumab-resistant cells (vs. trastuzumab-sensitive) secreted more EVs. Additionally, EVs from trastuzumab-resistant cells had lower tetraspanin density and higher HER2 density. We also evaluated EVs affinity-enriched in HER2; we found that these EVs (vs. tetraspanin-enriched) were larger and more elongated. We further optimized analytical sample processing to assess a rare population of HER2-enriched EVs from patient plasma. In breast cancer patients with elevated HER2 protein expression (vs. controls), HER2-enriched EVs had distinct characteristics including typically increased number of tetraspanin molecules and larger size. Importantly, these EVs were on average 25-fold more abundant compared to no cancer controls.ConclusionsSEVEN revealed unique characteristics of HER2-enriched EVs in cultured cells and complex biological fluid. In combination with current clinical approaches, this method is well poised to support precise therapeutic decisions.Graphical abstract

Simultaneous Protein and RNA Analysis in Single Extracellular Vesicles, Including Viruses.

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

Improving Specificity for Ovarian Cancer Screening Using a Novel Extracellular Vesicle–Based Blood Test: Performance in a Training and Verification Cohort

The low incidence of ovarian cancer (OC) dictates that any screening strategy needs to be both highly sensitive and highly specific. This study explored the utility of detecting multiple colocalized proteins or glycosylation epitopes on single tumor-associated extracellular vesicles (EVs) from blood. The novel OC Test employs immunoaffinity capture of tumor-associated EVs followed by proximity-ligation qPCR to detect combinations of up to three biomarkers to maximize specificity and measures multiple combinations to maximize sensitivity. A high-grade serous carcinoma (HGSC) case-control training set of EDTA plasma samples from 397 women was used to lock down the test design, the data interpretation algorithm, and the cut-off between cancer and non-cancer. Performance was verified and compared to CA125 in an independent blinded case-control set of serum samples from 390 women (132 controls, 66 HGSC, 83 non-HGSC OC, 109 benign). In the verification study, the OC Test showed a specificity of 97.0% (128/132; 95% CI: 92.4%-99.6%), a HGSC sensitivity of 97.0% (64/66; 95% CI: 87.8%-99.2%), and an AUC of 0.97 (95% CI 0.93-0.99) and also detected 73.5% (61/83; 95% CI: 62.7%-82.6%) of the non-HGSC OC cases. This test exhibited fewer false positives in subjects with benign ovarian tumors, non-ovarian cancers, and inflammatory conditions when compared to CA125. The combined sensitivity and specificity of this new test suggests it may have potential in OC screening.

Colocalization of Cancer-Associated Biomarkers on Single Extracellular Vesicles for Early Detection of Cancer

Detection of cancer early, when it is most treatable, remains a significant challenge due to the lack of diagnostic methods sufficiently sensitive to detect nascent tumors. Early-stage tumors are small relative to their tissue of origin, heterogeneous, and infrequently manifest in clinical symptoms. Detection of their presence is made more difficult by a lack of abundant tumor-specific indicators (i.e., protein biomarkers, circulating tumor DNA, etc.) that would enable detection using a non-invasive diagnostic assay. To overcome these obstacles, we have developed a liquid biopsy assay that interrogates circulating extracellular vesicles (EVs) to detect tumor-specific biomarkers colocalized on the surface of individual EVs. We demonstrate the technical feasibility of this approach in human cancer cell line-derived EVs where we show strong correlations between assay signal and cell line gene/protein expression for the ovarian cancer-associated biomarkers BST2, FOLR1, and MUC1. Furthermore, we demonstrate that detecting distinct colocalized biomarkers on the surface of EVs significantly improves discrimination performance relative to single biomarker measurements. Using this approach, we observe promising discrimination of high-grade serous ovarian cancer versus benign ovarian masses and healthy women in a proof-of-concept clinical study.

Simultaneous detection of membrane protein and mRNA at single extracellular vesicle level by droplet microfluidics for cancer diagnosis

IntroductionSimultaneous detection of proteins and mRNA within a single extracellular vesicle (EV) enables comprehensive analysis of specific EVs subpopulations, significantly advancing cancer diagnostics. However, developing a sensitive and user-friendly approach for simultaneously detecting multidimensional biomarkers in single EV is still challenging. ObjectivesTo facilitate the analysis of multidimensional biomarkers in EVs and boost its clinical application, we present a versatile droplet digital system facilitating the concurrent detection of membrane proteins and mRNA at the single EV level with high sensitivity and specificity. MethodsThe antibody-DNA conjugates were firstly prepared for EVs protein biomarkers recognition and signal transformation. Coupling with the assembled triplex droplet digital PCR system, a versatile droplet digital analysis assay for simultaneous detection of membrane protein and mRNA at a single EV level was developed. ResultsOur new droplet digital system displayed high sensitivity and specificity. Additionally, its clinical application was validated in a breast cancer cohort. As expected, this assay has demonstrated superior performance in distinguishing breast cancer from healthy individuals and benign controls through combined detection of EVs protein and mRNA markers compared to any single kind marker detections, especially for patients with breast cancer at early stage (AUC=0.9229). ConclusionConsequently, this study proposes a promising strategy for accurately identifying and analyzing specific EV subgroups through the co-detection of proteins and mRNA at the single EV level, holding significant potential for future clinical applications.

Comparison of EV characterization by commercial high-sensitivity flow cytometers and a custom single-molecule flow cytometer.

High-sensitivity flow cytometers have been developed for multi-parameter characterization of single extracellular vesicles (EVs), but performance varies among instruments and calibration methods. Here we compare the characterization of identical (split) EV samples derived from human colorectal cancer (DiFi) cells by three high-sensitivity flow cytometers, two commercial instruments, CytoFLEX/CellStream, and a custom single-molecule flow cytometer (SMFC). DiFi EVs were stained with the membrane dye di-8-ANEPPS and with PE-conjugated anti-EGFR or anti-tetraspanin (CD9/CD63/CD81) antibodies for estimation of EV size and surface protein copy numbers. The limits of detection (LODs) for immunofluorescence and vesicle size based on calibration using cross-calibrated, hard-dyed beads were ∼10 PE/∼80nm EV diameter for CytoFLEX and ∼10 PEs/∼67nm for CellStream. For the SMFC, the LOD for immunofluorescence was 1 PE and ≤ 35nm for size. The population of EVs detected by each system (di-8-ANEPPS+/PE+ particles) differed widely depending on the LOD of the system; for example, CellStream/CytoFLEX detected only 5.7% and 1.5% of the tetraspanin-labelled EVs detected by SMFC, respectively, and median EV diameter and antibody copy numbers were much larger for CellStream/CytoFLEX than for SMFC as measured and validated using super-resolution/single-molecule TIRF microscopy. To obtain a dataset representing a common EV population analysed by all three platforms, we filtered out SMFC and CellStream measurements for EVs below the CytoFLEX LODs as determined by bead calibration (10 PE/80nm). The inter-platform agreement using this filtered dataset was significantly better than for the unfiltered dataset, but even better concordance between results was obtained by applying higher cutoffs (21 PE/120nm) determined by threshold analysis using the SMFC data. The results demonstrate the impact of specifying LODs to define the EV population analysed on inter-instrument reproducibility in EV flow cytometry studies, and the utility of threshold analysis of SMFC data for providing semi-quantitative LOD values for other flow cytometers.

Expression of the αVβ3 integrin affects prostate cancer sEV cargo and density and promotes sEV pro-tumorigenic activity in vivo through a GPI-anchored receptor, NgR2.

It is known that small extracellular vesicles (sEVs) are released from cancer cells and contribute to cancer progression via crosstalk with recipient cells. We have previously reported that sEVs expressing the αVβ3 integrin, a protein upregulated in aggressive neuroendocrine prostate cancer (NEPrCa), contribute to neuroendocrine differentiation (NED) in recipient cells. Here, we examine the impact of αVβ3 expression on sEV protein content, density and function. sEVs used in this study were isolated by iodixanol density gradients and characterized by nanoparticle tracking analysis, immunoblotting and single vesicle analysis. Our proteomic profile of sEVs containing αVβ3 shows downregulation of typical effectors involved in apoptosis and necrosis and an upregulation of tumour cell survival factors compared to control sEVs. We also show that the expression of αVβ3 in sEVs causes a distinct reposition of EV markers (Alix, CD81, CD9) to a low-density sEV subpopulation. This low-density reposition is independent of extracellular matrix (ECM) protein interactions with sEVs. This sEV subset contains αVβ3 and an αVβ3 downstream effector, NgR2, a novel marker for NEPrCa. We show that sEVs containing αVβ3 are loaded with higher amounts of NgR2 as compared to sEVs that do not express αVβ3. Mechanistically, we demonstrate that sEVs containing NgR2 do not affect the sEV marker profile, but when injected in vivo intratumorally, they promote tumour growth and induce NED. We show that sEVs expressing NgR2 increase the activation of focal adhesion kinase (FAK), a known promoter of cancer cell proliferation, in recipient cells. We also show that NgR2 mimics the effect of sEVs containing αVβ3 since it displays increased growth of NgR2 transfectants in vivo, as compared to control cells. Overall, our results describe the changes that occur in cargo, density and functions of cancer cell-derived sEVs containing the αVβ3 integrin and its effector, NgR2, without affecting the sEV tetraspanin profiles.

Orthogonal analysis reveals inconsistencies in cargo loading of extracellular vesicles.

Since extracellular vesicles (EVs) have emerged as a promising drug delivery system, diverse methods have been used to load them with active pharmaceutical ingredients (API) in preclinical and clinical studies. However, there is yet to be an engineered EV formulation approved for human use, a barrier driven in part by the intrinsic heterogeneity of EVs. API loading is rarely assessed in the context of single vesicle measurements of physicochemical properties but is likely administered in a heterogeneous fashion to the detriment of a consistent product. Here, we applied a suite of single-particle resolution methods to determine the loading of rhodamine 6G (R6G) surrogate cargo mimicking hydrophilic small molecule drugs across four common API loading methods: sonication, electroporation, freeze-thaw cycling and passive incubation. Loading efficiencies and alterations in the physical properties of EVs were assessed, as well as co-localization with common EV-associated tetraspanins (i.e., CD63, CD81 and CD9) for insight into EV subpopulations. Sonication had the highest loading efficiency, yet significantly decreased particle yield, while electroporation led to the greatest number of loaded API particles, albeit at a lower efficiency. Moreover, results were often inconsistent between repeated runs within a given method, demonstrating the difficulty in developing a rigorous loading method that consistently loaded EVs across their heterogeneous subpopulations. This work highlights the significance of how chosen quantification metrics can impact apparent conclusions and the importance of single-particle characterization of EV loading.

Th2 cell extracellular vesicles promote eosinophil survival through the cytokine cargo IL-3 and prolong airway eosinophilia.

Extracellular vesicles (EVs) mediate intercellular communication during immune responses. EVs are abundant in respiratory biofluids, and the composition of EVs in the lung changes during inflammation. We aimed to quantify the contribution of T cells to airway EVs in allergic lung inflammation and ascertain their function during a type 2 inflammatory response. Genetic membrane tagging was combined with single vesicle flow cytometry to quantify T cell EVs in the airways of mice challenged with ovalbumin or house dust mite. EVs were purified from T helper type 2 (Th2) cell cultures and their functions on eosinophils assessed by flow cytometry and RNA sequencing. Th2 cell EVs were instilled into the lungs of mice to determine effects on lung eosinophilia. Finally, the function of an EV protein cargo was tested using inhibitors and blocking antibodies. T cell EVs are increased in the airways of mice with induced allergic inflammation. EVs secreted by Th2 cells inhibit apoptosis and induce activating pathways in eosinophils in vitro. This effect depends on re-stimulation through the T cell receptor. Th2 cell EVs prolong eosinophilia in vivo during allergic airway inflammation. Th2 cell EVs carry a potent form of the cytokine IL-3 on their surfaces, which inhibits apoptosis by activating Jak1/2-dependent pro-survival programs in eosinophils. Th2 cell EVs promote eosinophil survival and prolong eosinophilia during allergic airway inflammation. This function depends on the EV cargo IL-3, supporting a role for EVs as vehicles of cytokine-based communication in lung inflammation. T cells secrete extracellular vesicles in the airway during allergic lung inflammation.Th2 cell extracellular vesicles inhibit eosinophil apoptosis and prolong airway eosinophilia during allergic lung inflammation.IL-3 carried on Th2 cell EVs is a functional cargo, supporting a role for cytokine-carrying EVs as drivers of type 2 inflammation. This study supports that T cell extracellular vesicles may be important drivers of eosinophilic inflammation through the cytokine cargo IL-3, offering new insights into pro-inflammatory signaling in the allergic lung of patients with asthma.

Relationship between oligoarginine-induced membrane damage of single Escherichia coli cells and entry of the peptide into the cytoplasm

Cell-penetrating peptides (CPPs) can enter the cytosol of eukaryotic cells without killing them whereas some CPPs exhibit antimicrobial activity against bacterial cells. Here, to elucidate the mode of interaction of the CPP nona-arginine (R9) with bacterial cells, we investigated the interactions of lissamine rhodamine B red-labeled peptide (Rh-R9) with single Escherichia coli cells encapsulating calcein using confocal laser scanning microscopy. After Rh-R9 induced the leakage of a large amount of calcein, the fluorescence intensity of the cytosol due to Rh-R9 greatly increased, indicating that Rh-R9 induces cell membrane damage, thus allowing entry of a significant amount of Rh-R9 into the cytosol. To determine if the lipid bilayer region of the membrane is the main target of Rh-R9, we then investigated the interaction of Rh-R9 with single giant unilamellar vesicles (GUVs) comprising an E. coli polar lipid extract containing small GUVs and AlexaFluor 647 hydrazide (AF647) in the lumen. Rh-R9 entered the GUV lumen without inducing AF647 leakage, but leakage eventually did occur, indicating that GUV membrane damage was induced after the entry of Rh-R9 into the GUV lumen. The Rh-R9 peptide concentration dependence of the fraction of entry of Rh-R9 after a specific interaction time was similar to that of the fraction of leaking GUVs. These results indicate that Rh-R9 can damage the lipid bilayer region of a cell membrane, which may be related to its antimicrobial activity.