Small Extracellular vesicles (sEVs) hold great promise as therapeutic delivery vehicles due to their inherent biocompatibility. However, their clinical translation is limited by donor cell source dependency and inadequate targeting capabilities. To overcome these challenges, we introduce a universal surface engineering strategy that integrates lipid membrane anchoring with targeted ligand conjugation. At the core of this approach is the sEV Surface-Engineering microfluidic device (ExoSE), a dual-functional platform combining nanofluidic and microfluidic architectures. ExoSE consists of two interconnected modules: (1) a loading module that employs mechanoporation via nanochannels to transiently generate pores in sEV membranes, enabling highly efficient insertion of functionalized lipids and (2) a mixing module with specialized structures that facilitate rapid, covalent attachment of targeting ligands via optimized chemical reactions. This approach achieved lipid incorporation efficiencies of 97.93% for HEK293T-dervied sEVs and 98.47% for milk-derived sEVs, surpassing conventional co-incubation techniques. NanoFCM analysis revealed a 3- to 6-fold increase in ligand binding per sEV. Functionally, RGE peptide-modified sEVs exhibited a 54.13% increase in transmembrane transport efficiency in the in vitro model and enhanced infiltration into glioma spheroid, while AS1411 aptamer-conjugated sEVs showed 77.8% targeting specificity towards breast cancer cells, compared to 32.5% for normal breast cells. In vivo tracking in BALB/c-nude mice confirmed significantly improved brain accumulation of engineered sEVs, with no detectable hepatic or renal toxicity. Unlike traditional donor-cell-dependent genetic modification approaches, ExoSE enables universal, scalable modification of sEVs from diverse sources, including highly abundant milk-derived sEVs, and accommodates diverse ligand types such as peptides, aptamers and proteins. This device represents a transformative advancement in sEV engineering, establishing a standardized and scalable framework for precision-targeted sEV therapeutics with enhanced clinical potential.
Bacterial outer membrane vesicles (OMVs) are emerging as promising platforms for drug delivery and immunotherapy. However, bacteria only secrete a small amount of OMVs during the growth process, which seriously restricts their large-scale application. Here, a series of high-yield OMVs mutants is developed based on probiotic Escherichia coli Nissle 1917 (EcN). The mutant strain (EcNΔtolRΔmlaE) with the highest OMVs yield reported so far is identified and characterized, and its OMVs yield is 180.8 times that of the wild-type strain. More importantly, a high-yield OMVs mutant (EcNΔtolAΔnlpI) that derived OMVs can significantly improve the secretion efficiency of exogenous proteins is screened and engineered for enhanced scalability and versatility. Leveraging this platform, the prepared TOB-PslG-mOMVs nanoantibiotics, co-delivering glycosyl hydrolase (PslG) and tobramycin (TOB), synergistically disrupt biofilms and demonstrate potent antibacterial effects against Pseudomonas aeruginosa. Additionally, the prepared FI-mOMVs nanovaccines displaying the OprF190-342-OprI21-83 antigenic epitope fusion protein (FI) of P. aeruginosa can effectively induce robust humoral immune and cellular immune responses and significantly enhance protection against bacterial infection. Therefore, the OMVs nanoplatform thus represents a transformative approach, opening new avenues for combating multi-drug-resistant bacteria through innovative nanoantibiotic and nanovaccine technologies.
Extracellular vesicles (EVs) are key mediators of intercellular communication, carrying diverse molecular cargo that reflects the dynamic physiological and pathological state of their source cell. While analyses of the entire vesicular population (bulk EV) have advanced our understanding of their roles in health and disease, these approaches often obscure the heterogeneity inherent in EV populations. Emerging single-vesicle analysis technologies offer unprecedented resolution, enabling the identification of individual EV subpopulations and their distinct molecular signatures. Such approaches, combined with digital platforms, can now analyze individual molecules from single EVs, including single-molecule features such as protein, mRNA, double-stranded DNA and single-stranded DNA. This perspective explores the transformative potential of single EV technologies in clinical diagnostics and therapeutic applications. We highlight key advancements including microfluidic platforms, super-resolution microscopy and AI-driven data analyses, that are shaping and advancing the field and its applications. With the development and advancement of clinically viable single EV technologies, we are beginning to appreciate the complexity and abundance of cell type and specific EVs. We further discussed the challenges of sensitivity, specificity, standardization and scalability hindering these technologies' broad acceptance and feasibility in clinical translation. This perspective paper originates from discussions at the Chinese Society of Extracellular Vesicles (CSEV) annual meeting, held in Guangzhou, China, on 16 November 2024. At this meeting, researchers from various fields of EV research, with a particular emphasis on single EV digital, analytical and quantitative technological platforms, discussed the opportunities and challenges of this emerging single-EV-focused technology. The paper aims to provide a roadmap for integrating single EV technologies into routine EV-research and even clinical practice, paving the way for novel scientific and diagnostic tools, personalized therapies, and a deeper understanding of EV heterogeneity and EV biology.
Reliable non-invasive biomarkers for early detection of diabetic nephropathy (DN), a leading cause of chronic kidney disease, remain limited. In this study, we isolate urinary extracellular vesicles (uEVs) using wheat germ agglutinin (WGA)-conjugated magnetic beads and identify cytoskeleton-associated protein 4 (CKAP4) as a potential diagnostic biomarker for DN. Proteomic profiling and flow cytometry show that CKAP4 levels are significantly higher in uEVs from DN patients than in those from diabetic, non-diabetic renal disease (NDRD) and healthy control groups. Receiver operating characteristic (ROC) analysis demonstrates excellent diagnostic performance, with area under the curve (AUC) values of 0.9998 (sensitivity = 98.77%, specificity = 100%) for DN versus controls, and 0.9859 (sensitivity = 95.72%, specificity = 99.24%) for DN versus diabetes mellitus. CKAP4 levels, elevated even at early-stage DN, positively correlate with glomerulosclerosis, increasing with the severity of interstitial fibrosis and tubular atrophy (IFTA). Mechanistically, CKAP4-containing EVs derived from high glucose-treated podocytes promote vascular calcification in vascular smooth muscle cells via YAP signalling. These findings identify CKAP4 in podocyte-derived uEVs as a robust non-invasive biomarker for early DN detection and provide new insights into the vascular pathology associated with the disease.
Subunit vaccines are promising for disease prevention because of their safety and cost-effectiveness. However, their efficacy is limited by low immunogenicity and gastrointestinal degradation after oral administration. To address this issue, low-endotoxin Salmonella choleraesuis strain SC-L3 was engineered via lipid A modification to generate bacterial biomimetic vesicles (BBVs) with reduced endotoxin activity. BBVs were functionalized using ClyA-embedded SpyCatcher and Streptococcus protein G for dual antigen coupling, and further coated with chitosan oligosaccharides (COS) to enhance mucosal penetration and gastrointestinal stability. Using mCherry as a model antigen, we obtained optimized mCherry-CSS-BBV@COS that showed high antigen protection rates (83% and 63% in simulated gastric and intestinal fluids, respectively), capacity for lysosomal escape and effective stimulation of M1 macrophage polarization in vitro. Oral administration of mCherry-CSS-BBV@COS elicited robust systemic IgG and mucosal sIgA responses in mice. Furthermore, dual-antigen BBV conjugates (GDH-gD-Fc-CSS-BBV@COS) co-delivering Streptococcus suis glutamate dehydrogenase and pseudorabies virus gD-Fc induced antigen-specific humoral, mucosal and cellular immunity, conferring complete protection against lethal challenges with the respective pathogens. In summary, we generated a versatile, low-endotoxin BBV platform for oral combination subunit vaccines, offering a novel strategy for protection against viral and bacterial infections.
Cannabis sativa is a medicinal plant that produces a diverse array of pharmacologically active metabolites, making it a valuable resource for pharmaceutical applications. In this study, an adventitious root (AR) culture system was established from C. sativa using two representative plant growth regulators—naphthaleneacetic acid (NAA; hereafter referred to as N-ARs) and indole-3-butyric acid (IBA; hereafter referred to as I-ARs) —from which plant-derived nanovesicles (PDNVs) were subsequently isolated (hereafter N-PDNVs and I-PDNVs, respectively). The resulting N-PDNVs and I-PDNVs exhibited average diameters of 128 ± 2 and 124 ± 4 nm, respectively, with zeta potentials of −12.9 and −15.7 mV. Both PDNV types maintained structural integrity and colloidal stability under diverse external stress conditions, underscoring their physicochemical robustness. Metabolite profiling of PDNVs revealed 25 distinct metabolites. Functionally, I-PDNVs markedly enhanced dendritic cell maturation through Toll-like receptor 2 (TLR2)- and TLR4-dependent pathways, promoted T cell proliferation and activation (notably IFN-γ- and IL-17A-producing subsets), and increased natural killer (NK) cell activity compared with N-PDNVs. In immunosuppressed and tumour-bearing mouse models, I-PDNVs further augmented NK cell, Th1 and cytotoxic T lymphocyte (CTL) responses, thereby confirming their superior potential as immunotherapeutic agents. Moreover, in immunized mouse models, OVA257-264-encapsulated I-PDNVs demonstrated a clear advantage as a vaccine delivery platform by eliciting a potent OVA257-264-specific CTL response. When applied as a prophylactic cancer vaccine, they not only delayed tumour growth but also reshaped the antitumour immune landscape, characterized by enhanced CTL responses, reduced regulatory T cell frequencies and diminished exhausted CD8⁺ T cell populations. Collectively, these findings highlight the potential of I-PDNVs as dual-function PDNVs, serving both as immunotherapeutic agents and as vaccine delivery platforms for applications requiring reinforced Th1, CTL and NK cell responses.
Metastasis is the leading cause of death related to breast cancer. Premetastatic niches (PMNs), which are remodelled by the primary tumours in distant organs, are essential for the colonisation of disseminated cancer cells. The vascular niche is among the most pivotal PMNs in breast cancer lung metastasis, and the underlying mechanism remains unclear. Here, we report that breast cancer cells secrete dipeptidyl peptidase 3 (DPP3) via small extracellular vesicles (sEVs) to promote lung metastasis. Mechanistically, circulating DPP3 upregulates RAPGEF4 to activate the Rap1 signalling pathway in the lung endothelial cells through the DPP3–PFKP–YBX1 axis and promotes angiogenesis to remodel the vascular niche, thereby increasing lung metastasis. In addition, ARF4 recognises ISGylated DPP3, which facilitates its packaging into sEVs in breast cancer cells. Finally, treatment with losartan pharmacologically inhibits the ISGylation of DPP3, preventing its secretion via sEVs. In summary, our findings demonstrate that DPP3, which is encapsulated in sEVs and secreted by breast cancer cells, regulates angiogenesis in the lung and remodels vascular niches to promote breast cancer lung metastasis, making it a potential target for the diagnosis and treatment of breast cancer metastasis.
During fibrogenesis, certain negative feedback loops are elicited to restrain persistent and hyperactive fibrotic responses. Activated fibroblasts have been found to acquire anti-fibrotic phenotypes. However, the specific inhibitory modulators remain largely enigmatic. Thus, the present study aimed to examine the intrinsic autoregulatory mechanisms of fibroblasts. Here, we demonstrated that angiotensin II (AngII)-primed cardiac myofibroblast moderated subsequent profibrotic activation. More importantly, this suppressive action was dependent on small extracellular vesicles (sEVs). Strikingly, small RNA sequencing identified an abundant presence of Piwi-interacting RNAs (piRNAs) in sEVs. In cultured primary cardiac fibroblasts, piRNA-62788 was induced by AngII receptor type 2 (AT2R) stimulation and encapsulated into sEVs. Furthermore, fibrogenic responses were attenuated by piRNA-62788 overexpression, whereas aggravated by piRNA-62788 knockdown. In a mouse model of transverse aortic constriction, either piRNA-62788 agomir or circulating sEVs of patients with heart failure (HF) mitigated adverse cardiac remodelling, while piRNA-62788 inhibitor-containing sEVs accentuated myocardial fibrosis. Mechanistically, piRNA-62788 formed a functional complex with PIWI-like protein 2 (PIWIL2) and bound to the 3’ untranslated region (UTR) region of serum response factor (Srf) mRNA transcripts, leading to inhibition of the SRF signalling. Additionally, plasma sEV-derived piRNA-62788 was significantly upregulated in HF patients and negatively correlated with left ventricular ejection fraction. Collectively, we uncovered a protective negative feedback circuit controlled by AngII/AT2R/sEVs axis. Understanding this endogenous anti-fibrotic pathway may hold therapeutic promise in HF.
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