Search Results (309)

Alzheimer’s disease (AD) lacks effective disease-modifying therapies, and extracellular vesicles (EVs) derived from adipose-derived mesenchymal stromal cells (ADMSCs) have emerged as promising therapeutic candidates. In this study, we investigated the brain biodistribution and dose-dependent effects of intranasally administered ADMSC-EVs in female APP/PS1 mice, with age-matched wild-type mice and vehicle-treated transgenic mice serving as controls. EV biodistribution was assessed using PKH26 labeling, cognitive performance was evaluated using the Morris water maze, Y-maze, and novel object recognition tests, and hippocampal amyloid pathology and plasma AD-related biomarkers were analyzed. Intranasally delivered ADMSC-EVs rapidly reached multiple brain regions, including the hippocampus, improved learning and memory performance, and reduced hippocampal amyloid-β 1-42 (Aβ42) deposition and plaque burden. These effects followed a nonlinear dose–response pattern, with reduced efficacy at low doses and no additional benefits at high doses. Notably, partial behavioral and pathological benefits persisted after treatment cessation. Together, these findings show that intranasal ADMSC-EVs exert therapeutic effects in APP/PS1 mice and support the importance of dose optimization and post-treatment durability in the development of EV-based interventions for AD. Full article

Obesity is a major global health challenge characterized by chronic low-grade inflammation, oxidative stress, and an increased risk of cardiometabolic disorders. Although sex-related differences in inflammatory and redox biomarkers have been reported in obese populations, the molecular mechanisms underlying this heterogeneity remain incompletely understood. In this study, we applied a proteomics-based approach to investigate urinary extracellular vesicles from 45 obese individuals (BMI 30–40 kg/m²; age 50–70 years) in order to identify molecular signatures associated with metabolic dysregulation. Shotgun proteomics analysis performed by nanoLC–MS/MS enabled the identification of 3822 proteins. Hierarchical clustering of proteomic profiles revealed two distinct molecular groups, predominantly enriched in males (Group I) and females (Group II). Label-free quantitative analysis identified 466 differentially abundant proteins between the two clusters. Functional enrichment analysis highlighted pathways associated with immune response, metabolic regulation, and redox homeostasis, including glycolysis/gluconeogenesis, lysosome activity, leukocyte transendothelial migration, and glutathione, cysteine and methionine metabolism. Notably, proteins related to ferroptosis were enriched, suggesting the involvement of iron-dependent oxidative cell death mechanisms in the metabolic imbalance observed in a subset of subjects. Furthermore, the non-enzymatic glycosylation of urinary proteins was significantly higher in Group I compared with Group II (p = 0.0002), indicating increased formation of advanced glycation products in individuals with a more pronounced pro-oxidant state. Preliminary follow-up data suggested a higher incidence of pathological events, including cardiovascular complications, among individuals belonging to Group I. Overall, these findings demonstrate that urinary proteomic profiling can identify distinct molecular phenotypes among obese individuals and highlight oxidative stress, ferroptosis, and protein glycation as potential determinants of metabolic vulnerability, supporting the use of non-invasive proteomic approaches for improved risk stratification in obesity. Full article

Exosomes are a kind of nanoscale extracellular vesicle secreted by almost all cell types and considered promising biomarkers for disease diagnosis since they could carry abundant proteins, nucleic acids, and lipids that reflect parental cell states. However, conventional exosome detection methods suffer from several limitations including insufficient specificity, low throughput, high costs, and inadequate sensitivity for clinical applications. By contrast, field-effect transistor (FET) biosensors are a promising alternative by enabling label-free, real-time, and ultrasensitive detection of exosomes through direct transduction of biorecognition events into electrical signals. This review first introduces the fundamental principles and device structure of FET biosensors, as well as exosome isolation strategies. The recent advances in exosome analysis using FET-based biosensors are then presented, which are categorized into two primary strategies: (1) direct detection of intact exosomes based on surface markers, including tetraspanin proteins (CD9, CD63, CD81, etc.) and disease-specific biomarkers, and (2) detection of exosomal contents including microRNA and protein biomarkers following exosome lysis. Finally, we discuss current challenges of FET-based exosome detection and provide perspectives on future developments. Full article

Controlled delivery using nanoparticle-based systems has attracted considerable attention; however, achieving cell-type specificity remains a major challenge. To address this issue, we focused on the intrinsic cell tropism of viruses. The hepatocyte tropism of hepatitis B virus (HBV) is mediated by interactions between its large envelope protein (L protein) and host factors, including the sodium taurocholate cotransporting polypeptide (NTCP). In this study, we explored viral-like secretory vesicles (VLSVs) displaying HBV spike proteins as a virus-inspired vesicle platform for hepatocyte targeting. We previously established a method for producing VLSVs from HBV L- and S-expressing HEK293T cells. In the present study, we developed an improved protocol using exosome-depleted fetal calf serum and optimized ultracentrifugation, resulting in VLSVs with comparable particle numbers and sizes but approximately tenfold higher protein content per particle. VLSVs were concentrated using a two-layer sucrose cushion, labeled with DiI, and purified by sucrose density gradient ultracentrifugation. We evaluated DiI uptake in hepatocyte-derived cells (HepG2 and Huh7), non-hepatic cells (MDA-MB231, H1299, HeLa, and Vero), and NTCP-overexpressing HepG2 cells. VLSVs showed preferential uptake in the following order: NTCP-overexpressing HepG2 > HepG2 > Huh7 > non-hepatic cells. Furthermore, removal of the N-terminal Flag tag from the L protein enhanced hepatocyte-associated uptake, suggesting the importance of preserving the native structure of the preS1 domain. While vesicle characterization and mechanistic validation remain to be further investigated, these findings provide a proof-of-concept for a virus-inspired vesicle platform exhibiting preferential uptake in hepatocyte-derived cells. Full article

The development of high-throughput technologies for the separation and labeling of extracellular vesicles (EVs) from whole blood is critical for downstream EV detection and analysis. However, conventional EV separation and labeling workflows are typically labor-intensive and inefficient, requiring multiple sequential processing steps. Here, we present a microfluidic platform that integrates negative magnetophoresis-based separation with mixing-enhanced on-chip labeling. The chip adopts a vertical flow channel architecture in combination with a Halbach-array magnetic field configuration, thereby overcoming the throughput limitations inherent to traditional horizontal microchannels. Parallel channels can be freely arranged above on the magnetic array to achieve ultra-high throughput processing, achieving a cell removal efficiency of 99.97% at a blood-to-sheath flow ratio of 1:5. Furthermore, by incorporating a narrow-wide channel design synergized with a herringbone–Tesla micromixer structure, the platform achieves a labeling efficiency of 91.8% within 2 min, approaching the performance of conventional 20 min incubation. This system offers both high-throughput and integration capabilities, providing a powerful technical platform for EV-related life science research. Full article

Graphene oxide (GO)–lipid hybrid nanostructures represent a promising class of multifunctional platforms for drug delivery and fluorescence-guided cellular imaging. In this study, we developed a graphene oxide-supported lipid bilayer system composed of rhodamine-labeled phosphatidylcholine (POPC-Rhod) for the delivery of the repurposed antispasmodic drug alverine citrate (ALV) to neuroblastoma cells. The hybrid nanostructures were assembled using two drug-loading strategies and characterized by UV–Vis spectroscopy, fluorescence analysis, dynamic light scattering, and atomic force microscopy to evaluate molecular interactions, vesicle size distribution, and nanomechanical properties. In vitro studies were performed using human neuroblastoma SH-SY5Y cells and their retinoic acid-differentiated neuronal-like counterparts. Confocal microscopy confirmed efficient cellular uptake of the fluorescent lipid–graphene hybrids, while viability and mitochondrial reactive oxygen species assays revealed differentiation-dependent cellular responses. ALV-loaded hybrids induced cytotoxic effects in proliferating neuroblastoma cells, whereas differentiated neuron-like cells exhibited greater tolerance and, at moderate concentrations, preserved viability despite increased oxidative stress. These findings demonstrate that graphene oxide–lipid hybrids can act as fluorescence-traceable drug delivery platforms and highlight the potential of alverine as a candidate for repurposing in neural cancer models. The system presented here provides a proof-of-concept framework for the development of multifunctional nanocarriers integrating therapeutic delivery with imaging capabilities. Full article

Streptococcus mutans (S. mutans), one of the main etiological pathogens of dental caries, forms dental plaque biofilms that drive tooth decay. Although bacterial membrane vesicles (MVs) are increasingly recognized as modulators of biofilm biology, little is known about MVs generated by S. mutans. The objective of this study is to investigate the role of S. mutans-derived MVs in the development of S. mutans biofilms formed under static conditions in plates or confocal dishes. Transmission electron microscopy and nanoparticle tracking analysis revealed that the MVs were cup-shaped with bilayered membranes and averaged 80.49 $\pm$ 32.24 nm in diameter. The addition of ≥5 µg/mL MVs enhanced biofilm formation during the initial adhesion stage (0 to 6 h), as demonstrated by crystal violet staining and XTT assays. Confocal laser scanning microscopy and scanning electron microscopy confirmed the incorporation of PKH26-labeled MVs into S. mutans biofilms and showed that supplemental MVs increased bacterial viability and extracellular polysaccharide biomass. Furthermore, RT-qPCR analysis revealed upregulated expression of genes related to adhesion and quorum-sensing systems in MV-treated biofilms. In conclusion, these findings indicate that S. muants MVs are integral biofilm components that promote biofilm establishment at the early stage of biofilm formation. Full article

Utilization of a blood pump to aid in circulating a patient’s blood, otherwise known as mechanical circulatory support, is an effective and often life-saving treatment for cardiac/pulmonary failure patients, yet adverse events remain a common complication often attributed to mechanical trauma inflicted on blood components. This work specifically focuses on erythrocyte-derived extracellular vesicles (ErEVs) as a marker of this mechanical trauma as they are elevated in patients with blood pumps and have been tied to adverse events. Despite this, ErEVs are typically neglected during device development which usually includes testing with animal blood, most commonly porcine and bovine. Flow cytometry was employed to monitor ErEVs generated during a 6 h perfusion of porcine or bovine red blood cells (RBCs) in a blood circulatory loop with the CentriMag blood pump. Successful measurement meant overcoming limitations in suitable stains for the RBCs and ErEVs of the two species. Between the two species, 12 different antibodies and dyes were evaluated, including multiple glycophorin A clones, the typical human erythrocyte antigen. Only CD46 and carboxyfluorescein succinimidyl ester (CFSE) were found to successfully and reliably label porcine and bovine RBCs, respectively. With these stains, statistically significant increases for both porcine and bovine ErEVs with perfusion time were observed. Bovine erythrocytes produced significantly more ErEVs than porcine, indicating they are more sensitive to mechanical trauma and could be useful in early-stage device development. The utility of CD46 and CFSE used for porcine and bovine ErEV detection was demonstrated for in vitro pump testing with implications for physiological and pathological research with these animals. Full article

This study leverages surface plasmon resonance (SPR) Biacore^TM technology to unveil the diagnostic potential of detecting CXCR4 on extracellular vesicles (EVs). Despite its recognized potential as a cancer biomarker, the presence of CXCR4 on EVs remains underexplored for diagnostic purposes. Using reference material (rEVs), a standardized label-free and real-time SPR biosensor is established to molecularly profile CXCR4-positive EVs. The binding interactions between immobilized antibodies and EVs isolated from different cancer cell lines revealed a unique SPR molecular fingerprint (SPR-MFP) consisting of varying expression levels of the CD9, CD63 and CD81 EV biomarkers, as well as CXCR4. There was a strong correlation between CXCR4 expression on the cellular membrane measured by flow cytometry (FCM) and the CXCR4 SPR signal of purified EVs, indicating that the chemokine receptor is actively transferred to the extracellular space. The Biacore^TM biosensor is able to directly detect and molecularly profile EVs in buffer and spiked in cell culture supernatant supplemented with 10% EV-depleted serum. Altogether, our findings illuminate the potential of SPR Biacore^TM technology in EV-related research as well as reveal the diagnostic potential of EV-associated CXCR4, offering valuable insights and paving the way for medical applications in diseases associated with aberrant CXCR4 expression. Full article

Membrane fusion is fundamental to intracellular transport and signal transduction, with its dysregulation implicated in various diseases. Deciphering its transient, microscale dynamics requires advanced sensing technologies. This review systematically evaluates optical and electrochemical sensing platforms for in vitro studies of membrane fusion. Optical sensing platforms provide greater intuitive readout of membrane fusion events, whereas electrochemical sensing platforms enable label-free, single-event resolution. We revisit classical fluorescence resonance energy transfer (FRET) strategies for lipid and content mixing, tracing their evolution from ensemble measurements to real-time, multiparameter, single-vesicle analysis. We further examine electrochemical platforms based on nanodisc-black lipid membranes (ND-BLMs) and solid-supported lipid bilayers (SLBs), highlighting their unique capabilities in characterizing fusion pore kinetics and virus–host membrane fusion. ND-BLM-based systems are irreplaceable for probing fusion pore kinetics, owing to their sub-millisecond temporal resolution and being essentially free from ion saturation and depletion effects. Meanwhile, SLB-based electrochemical sensing platforms excel at high-throughput detection of viral membrane fusion events by virtue of their excellent compatibility and facile integration. These sensors provide powerful tools for elucidating the molecular mechanisms underlying SNARE-mediated membrane fusion and viral fusion processes. Finally, this review outlines future directions centered on the integration of multimodal sensing and the construction of physiomimetic membranes, emphasizing the critical role of cross-scale, multiparameter sensing in bridging molecular mechanisms with biological functions and advancing the diagnosis and treatment of membrane fusion-related diseases. Full article

Intravenous delivery of recombinant adeno-associated virus serotype 9 can lead to reporter activation in cell types beyond the vasculature, but the routes enabling downstream parenchymal labeling remain unclear. Here, we provide a systematic, time-resolved map of parenchymal labeling after a single intravenous dose of rAAV9 encoding Cre recombinase under a ubiquitous promoter in healthy adult Ai9 reporter mice. Following retro-orbital administration, we quantified tdTomato-positive labeling across 25 targets at multiple time points over six months and observed durable reporter activation in several nonvascular parenchymal populations relevant to systemic gene-delivery applications. We also identify a set of parenchymal cell types that are consistently labeled in both this vascularly initiated reporter system and our prior adult VE-cadherin-driven reporter paradigm, supporting a connection to vascular exposure without asserting lineage relationships. These results nominate mechanistic routes for future disambiguation, including viral transcytosis across endothelium, endothelial cell transdifferentiation and extracellular-vesicle-mediated transfer. The dataset and methods provide a reference framework for investigators optimizing systemic delivery and interpreting downstream labeling in vivo. Full article

Continuous, label-free particle separation is essential for a broad range of biochemical and biomedical applications. Here, we present a microfluidic device that integrates inertial focusing and deterministic lateral displacement (DLD) within a compact channel architecture to achieve size-based particle sorting under laminar flow conditions. The design combines upstream curved channels for initial lateral positioning with downstream micropillar-embedded curved channels to enhance separation resolution. Theoretical analysis and numerical simulations were performed to optimize channel geometry and micropillar arrangement, predicting size-dependent lateral displacement driven by centrifugal forces and pillar-induced constraints. Experimental validation using glass beads of two distinct sizes (8 μm and 15 μm) demonstrated a separation efficiency exceeding 93% across a range of flow rates and particle concentrations. The device offers a simple, cost-effective, and scalable solution for passive particle sorting without external fields or labeling. The flexibility of the design configuration can be adapted for diverse applications, including extracellular vesicles, barcoded hydrogel particles, and engineered drug-delivery carriers. Full article

Extracellular vesicles (EVs) are known as potential biomarkers for several diseases; nevertheless, the degree of technical and biological variability is not yet adequately characterized. Because pre-analytical factors such as blood collection time and EV subpopulation could confound biomarker studies, we performed a pilot study systematically quantifying methodological and biological variability including EV-Array (surface proteins), and proteome characterization of cargo. Plasma samples from six healthy adults were collected at two time points (morning and afternoon) and plasma was analyzed with EV-Array, and isolated EVs were analyzed using nanoparticle tracking analysis (NTA), and label-free mass spectrometry (LC-MS/MS). Methodological repeatability was high for NTA particle size (3.3% CV) and LC-MS (8.2% CV), and lower for EV-Array surface markers (22.6% CV). Variations between samples were reasonable for NTA-size, EV-Array and LC-MS/MS (5–21%) and substantially lower than between-subject variation. No evidence of systemic morning–afternoon shifts in particle size and concentration or EV cargo was observed, although small effects cannot be excluded. The same was true for most surface markers, but minor but statistically significant reductions in a few specific surface markers occurred in afternoon EV-Array samples. In this pilot we therefore do not observe any major systemic diurnal bias in healthy individuals in samples collected a.m. vs. p.m. Despite the small sample size, this study underscores the importance of accounting for individual variability and methodological standardization when designing EV-based biomarker research. Full article

Background/Objectives: Neovascularization, defined as the sprouting of new blood vessels from pre-existing vasculature, is a critical pathological feature in ocular diseases such as pathological myopia and represents a leading cause of corneal vision loss. Vascular endothelial growth factor A (VEGFA) plays a pivotal role in endothelial cell proliferation, migration, survival by anti-apoptotic signaling, and vascular permeability. Dysregulation of VEGFA is closely linked to pathological neovascularization. Exosomes, nanosized phospholipid bilayer vesicles ranging from 30 to 150 nm, have emerged as promising gene delivery vehicles due to their intrinsic low immunogenicity, superior cellular uptake, and enhanced in vivo stability. This study aimed to investigate whether highly purified mesenchymal stem cell (MSC)-derived exosomes loaded with VEGFA siRNA labeled with FAM can effectively suppress pathological corneal neovascularization (CNV) via targeeted cellular transduction and VEGFA inhibition. Furthermore, we examined whether the therapeutic effect involves the modulation of the PI3K–Akt–Caspase-3 signaling axis. Methods: Exosomes purified by chromatography were characterized by electronmicroscopy, standard marker immunoblotting, and nanoparticle tracking analysis. In vitro, we assessed exosome uptake and cytoplasmic release, suppression of VEGFA mRNA/protein, cell viability, and apoptosis. In a mouse CNV model, we evaluated tissue reach and stromal retention after repeated intrastromal injections; anterior segment angiogenic indices; CD31/VEGFA immunofluorescence/immunoblotting; phosphorylated PI3K and Akt; cleaved caspase-3; histology (H&E); and systemic safety (liver, kidney, and spleen). Results: Exosomes were of high quality and showed peak efficacy at 48 h, with decreased VEGFA mRNA/protein, reduced viability, and increased apoptosis in vitro. In vivo, efficient delivery and stromal retention were observed, with accelerated inhibition of neovascularization after Day 14 and maximal effect on Days 17–19. Treatment reduced CD31 and VEGFA, decreased p-PI3K and p-Akt, and increased cleaved caspase-3. Histologically, concurrent reductions in neovascularization, inflammatory cell infiltration, and inflammatory epithelial thickening were observed, alongside a favorable systemic safety profile. Conclusions:VEGFA siRNA-loaded exosomes effectively reduce pathological CNV via a causal sequence of intracellular uptake, cytoplasmic release, targeted inhibition, and phenotypic suppression. Supported by consistent PI3K–Akt inhibition and caspase-3–mediated apoptosis induction, these exosomes represent a promising local gene therapy that can complement existing antibody-based treatments. Full article