Journal of Extracellular Vesicles最新文献

Proteolysis targeting chimeras (PROTACs) represent an emerging targeted cancer therapy approach. However, their poor cell penetration and instability in vivo pose daunting challenges for wide-spread clinical usage. To enhance the in vivo therapeutic efficacy of PROTACs, we introduced extracellular vesicles (EVs) for in vivo PROTAC delivery, which is leveraged by a novel microfluidic droplet-based EV electro-transfection system (μDES). We previously developed YX968 PROTAC, which can selectively degrade both HDAC3 and HDAC8 in triple negative breast cancer (TNBC) cells and effectively suppress the tumour cell growth without provoking global hyperacetylation. In this manuscript, we demonstrated that YX968 loaded EVs via the μDES system can retain the optimal integrity of drug loaded EVs with improved loading efficiency compared to other transfection approaches, which, in turn, significantly enhances the therapeutic function of PROTAC in vivo in TNBC mouse models. Intraperitoneal injections of YX968 loaded EVs led to significantly enhanced intratumoral degradation of HDAC3 and HDAC8 than YX986 alone, which resulted in advanced TNBC tumour inhibition without noticeable tissue toxicity. Such EV-based delivery strategy, with a scalable EV loading approach, enhanced the in vivo PROTAC drug stability and bioavailability and improved tissue penetration and targeting, filling an important gap in the clinical translation of PROTAC-based cancer therapy.

In neurones, like in any other cell, their function often relies on the fine-tuning of their protein levels, which is achieved by the balance between protein synthesis and turnover. Defects in protein homeostasis frequently lead to neuronal dysfunction and neurological disorders. Given their extreme morphological complexity and high compartmentalization, neurones highly depend on the asymmetrical distribution of their proteome. The common belief is that proteins that sustain axonal, dendritic and synaptic functions are synthesized in the soma and then transported to distal neuronal compartments. However, there is a complementary mechanism by which the mRNAs, and not the proteins, are transported to distal subneuronal domains, and once they reach their destination, they are locally translated. Although once considered heretical, local translation (or local protein synthesis) is now widely accepted by the scientific community. Nonetheless, there is one question that remains largely unexplored in the field, and that is whether local translation in dendrites, axons and synapses is fully regulated by the neurone itself or if non-neuronal cells (e.g., glia) can modulate this mechanism in a non-cell-autonomous manner. Here, we combined primary neuronal cultures, astrocyte-derived extracellular vesicle (EVs) isolation, and proteomics to investigate whether astroglial EVs modulate local translation in axons. We show that EVs released by astrocytes exposed to amyloid-β peptide (Aβ) enhance protein synthesis specifically in distal axons and increase synaptic integrity. Proteomics analysis and western blotting identified the ribosomal protein Rps6 as an astroglial Aβ-EV cargo delivered to axons. Interestingly, genetic downregulation revealed the contribution of vesicular Rps6 to translation regulation in axons and synaptic integrity. To our knowledge, this is the first report that directly demonstrates glial control of local translation in neurones through EVs, revealing a novel glia-to-neurone communication mechanism in an experimental model of Alzheimer's disease (AD).

C1q is released by microglia, localizes on weak synapses and acts as a tag for microglial synaptic pruning. However, how C1q tags synapses during the pruning period remains to be fully elucidated. Here, we report that C1q is delivered via extracellular vesicles by microglia to pre-synaptic sites that externalize phosphatidylserine. Using approaches to increase or reduce vesicles production in microglia, by C9orf72 knock out or pharmacological inhibition, respectively, we provided mechanistic evidence linking extracellular vesicle release to pre-synaptic remodelling in neuron-microglia cultures. In C9orf72 knockout mice, we confirmed larger production of microglial extracellular vesicles and showed augmented C1q presynaptic deposition associated with enhanced engulfment by microglia in the early postnatal hippocampus. Finally, we provide evidence that microglia physiologically release more vesicles during the period of postnatal circuit refinement. These findings implicate abnormal release of microglial extracellular vesicles in both neurodevelopmental and age-related disorders characterized by dysregulated microglia-mediated synaptic pruning.

Extracellular vesicles (EVs) are an attractive delivery vehicle with biological activity, intrinsic homing, low immunogenicity, and engineerability; however, challenges remain regarding loading and functional delivery of mRNA. Here, we developed a novel approach to load mRNA through low pH-induced fusion of EVs with lipid nanoparticles (LNPs) to generate hybrid EVs (HEVs). Conventional characterization showed that HEVs preserved classical features of EVs. Single particle analysis revealed successful loading of mRNA and incorporation of LNP components into HEVs. The combined properties from EV and LNP contributed to the excellent cell tolerability of HEV, overcoming dose-limit toxicity, and functional delivery of mRNA by HEV. We further elucidated the mechanism of HEV-mediated intracellular delivery of mRNA. Our results showed that in contrast to source EVs, HEVs were capable of inducing endosomal escape, facilitating intracellular delivery of mRNA. Furthermore, HEVs functionally delivered mRNA in vivo and displayed extrahepatic delivery capacity with predominant functional distribution in spleen. Our results suggest HEVs as a promising EV-based delivery platform for mRNA delivery.

The concentration of cells is a key component of modern blood tests. Given the biomarker potential of extracellular vesicles (EVs) in blood, we aimed to establish reference ranges for blood cell-derived EVs using flow cytometry. To address the orders-of-magnitude variability in reported EV concentrations between different flow cytometers (FCMs), we first validated a calibration methodology to enable reproducible EV concentration measurements. The methodology was evaluated in an interlaboratory comparison study and shows that calibration reduces the median absolute deviation of EV concentrations measured on 25 different FCMs from 67 % to 25 %–31 %. The calibration methodology was then used to determine reference ranges of erythrocyte-, leukocyte-, and platelet-derived EVs in human blood plasma in a cohort of healthy individuals (n = 224). This study demonstrates that calibration enables comparable concentration measurements of blood cell-derived EVs, thereby bringing EVs one step closer to clinical applications.

Efficient and cell-specific delivery remains a major barrier to realising the full therapeutic potential of modalities such as mRNA and CRISPR-based gene editors. Here, we report a versatile delivery platform based on engineered ARRDC1-mediated microvesicles (ARMMs) capable of delivering cargo to defined cell populations. By decorating ARMMs with engineered Nipah virus (NiV)-derived fusion and attachment proteins conjugated to cell-specific ligands, we enable selective binding and membrane fusion-mediated cargo release. ARMMs functionalized with anti-CD8 single-chain variable fragment (scFv) delivered protein, mRNA, or CRISPR-Cas9 base editor selectively to CD8⁺ T cells. Similarly, ARMMs displaying a designed ankyrin repeat protein (DARPin) targeting the GluA4 receptor enabled delivery to parvalbumin-positive (PV⁺) neurons. In vivo, administration of targeted ARMMs resulted in functional delivery to CD8⁺ splenocytes and PV⁺ cortical neurons in mice. These findings establish surface-engineered ARMMs as a programmable and modular system for precision delivery of therapeutic macromolecules, with broad applicability in gene and RNA-based medicine.

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 OprF_190-342-OprI_21-83 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.