Extracellular vesicles (EVs) are increasingly explored as diagnostic and therapeutic platforms. Translation is constrained by protocols that must balance efficacy and safety, especially for EVs from tumor or immortalized cells that may carry oncogenic or immunostimulatory cargo. This study presents a rapid and reproducible engineering of EVs using bidistilled water as the sole reagent. The hypotonic environment induces osmotic lysis of EVs isolated from B lymphocytes, generating a remodeled vesicle population. The process markedly reduces a broad spectrum of intraluminal components with potential immunogenic or tumor-promoting activity, while preserving the vesicular membrane scaffold. Osmotic remodeling is associated with reorganization of membrane-associated lipids and proteins and with a preferential uptake of the remodeled vesicles by tumor cells over non-malignant counterparts in our in vitro model. The engineered EVs retain structural integrity and are amenable, at a proof-of-concept level, to reloading with defined bioactive compounds, suggesting their potential as customizable delivery systems. Overall, this osmotic remodeling strategy provides a versatile technological platform for the generation of membrane-derived vesicles with tunable properties and promising applicability in drug delivery, nanodiagnostics, and immune modulation, whose translational performance and safety will need to be established in source- and indication-specific preclinical studies.

Helminths modulate host immunity, and in this respect, the porcine whipworm Trichuris suis has been explored clinically as an immunotherapy for inflammatory diseases. T. suis secretes a complex mixture of excretory/secretory products (ESP), including extracellular vesicles (EVs), nanosized membranous particles with bioactive cargoes of lipids, proteins and nucleic acids that are implicated in intercellular communication within organisms. Here, we characterise the EV protein and miRNA cargo of T. suis to identify potential roles in host-parasite communication. In contrast to EVs from clade III/V helminth species characterised to date, we show that T. suis EVs did not suppress LPS-induced TNF production by human macrophages under the conditions tested, in contrast to non-vesicular ESP fractions which did. By generating the first extracellular miRNA complement for T. suis and quantifying EV-associated versus free miRNAs across larval and adult stages, we identify selective enrichment of two miRNAs in adult EVs with homology to human miR-22-3p and miR-29-3p, canonical anti-fibrotic and tumour-suppressor miRNAs. Functional assays in human colon cancer epithelial cells reveal that these helminth-derived orthologues downregulate conserved oncogenic targets, indicating evolutionary conservation of their regulatory capacity. Our findings uncover a previously unrecognised axis of whipworm biology in which selective EV-mediated export of anti-fibrotic miRNAs has the potential to modulate host tissue remodeling independently of classical immunomodulation, with implications for understanding helminth-driven epithelial homeostasis and cancer risk.

Plant-derived extracellular vesicles (EVs) have shown numerous health benefits, including modulation of the human gut microbiota. Herein, we employed isothermal microcalorimetry (IMC) to explore the effects of ginger-derived nanovesicles (G-NVs) on the growth and metabolic activity of Bacteroides thetaiotaomicron (Bt), a dominant saccharolytic commensal with promising next-generation probiotic potential. Bt was exposed to either G-NVs or the ginger extract separated from G-NVs (G-CTL) in defined media under anaerobic conditions. Both ginger samples enhanced bacterial specific growth rate and maximum metabolic activity, inducing the latter earlier. However, higher biomass production and greater secretion of acetic, succinic and propionic acids occurred only in response to the G-CTL. Complete sugar depletion and unchanged free amino acid levels indicated preferential carbohydrate utilisation by Bt. Overall, these findings revealed that Bt's metabolic state is shaped by both G-NVs and G-CTL, yet through distinct mechanisms, with G-NVs inducing rapid stimulation without increasing total metabolic output and G-CTL providing a sustained, dose-dependent effect. To our knowledge, this is the first study applying IMC to monitor in real-time the impact of EVs on microbial growth and metabolism, underscoring IMC's utility for mechanistic studies of EV-microbe interactions. Furthermore, this research sets the ground for innovative strategies in nutraceutical and microbiome-targeted therapy development.

Assembly of plasmonic nanoparticles (NPs) generates unique optical properties through coupling of the localized surface plasmon resonance (LSPR) of individual NPs. However, precisely controlling and monitoring how mesoscale assembly dictates final optical properties remain key challenges in designing advanced plasmonic materials. Here, we introduce "nanoplasmonic isosbestics" as optical descriptors of the mesoscale organization of gold nanoparticles (AuNPs) on soft templates. Unlike isosbestic points in molecular spectroscopy, which describe chemical equilibria, our numerical simulations demonstrate that nanoplasmonic isosbestics emerge from the coexistence of individual AuNPs and AuNP clusters, where the interparticle spacing determines the isosbestic wavelength. By templating AuNP assembly onto synthetic free-standing lipid bilayers with tunable membrane rigidity, we experimentally achieve precise control over interparticle spacing and prove that it is mirrored by univocal modulation of the isosbestic wavelength. This provides a fundamental understanding of the structure-function relationship in plasmonic systems, linking, for the first time, nanoplasmonic isosbestics to interparticle spacing and equilibrium structure in plasmonic assemblies. On the analytical perspective, nanoplasmonic isosbestics provide noninvasive optical fingerprints of the templates, opening to appealing applications. As a proof of concept, we apply this approach to profile the stiffness of two extracellular vesicle (EVs) classes─mesenchymal stem cell (MSC)-derived and red blood cell-derived EVs─both recognized for their biological and translational potential.

In certain cell systems, small extracellular vesicles bearing PD-L1 (PD-L1+ sEVs) have been shown to suppress T-cell immunity. We investigated whether a distinct profile of PD-L1+ sEVs exists in human follicular fluid (FF), a microenvironment where immune tolerance is crucial for proper follicular development. We characterized the expression and colocalization of CD63, CD81, CD9, and PD-L1 in sEVs derived from FF of women undergoing fertility treatments (n = 10), utilizing single-particle interferometric reflectance imaging sensing combined with single-particle antibody capture and immunofluorescence labeling. Additionally, sEV size distribution was analysed via atomic force microscopy. These integrated techniques revealed that the majority of tetraspanin-expressing EVs in human FF are smaller than 50 nm. Statistical analysis revealed a significant difference in PD-L1 co-expression across CD63, CD81, and CD9, confirming a preferential association of PD-L1 with CD9+ sEVs. Coefficients of variation across the cohort further indicated that PD-L1/CD9 co-expression was the most consistent among patients, suggesting a stable and distinct sEV subpopulation. These findings underscore the potential of PD-L1+ sEVs as biomarkers for immune regulation in reproductive treatments. The discovery of distinct PD-L1+ sEV subpopulations suggests a role in modulating immune responses within the follicular microenvironment. Further studies are warranted to investigate the functional relevance of these vesicles in predicting fertility outcome, promoting local immune tolerance, and facilitating follicular development.

Abstract Emerging evidence highlights the key role of microRNA (miR)-21 in cell-to-cell communication and tumorigenesis. However, limited knowledge exists on the levels and clinical meaning of miR-21 in extracellular vesicles (EVs) of patients with breast cancer (BC). We assessed EV-derived miR-21 levels in one hundred women: 30 with early BC (EBC), 30 with metastatic BC on treatment progression (MBC), 30 cancer survivors on follow-up (FU) and 10 healthy donors (HD) as age- and body mass index (BMI)-matched controls. EVs isolated from serum samples were characterized using nanoparticle tracking analysis, scanning electron microscopy and atomic force microscopy to detect their concentration, size, morphology and mechanical properties. The levels of miR-21 in EVs was evaluated using real time PCR and compared between groups (EBC, MBC and FU vs. HD) by calculating the fold change and ΔΔCt statistic. EVs size and concentration did not differ significantly among patient groups. In the EBC group, the clinical stage at diagnosis and tumor subtype did not influence miR-21 levels. The levels of miR-21 were higher in the MBC group than in the HD group (p = 0.029), mainly in those who were human epidermal growth factor receptor 2 (HER2)+ (p = 0.0005) and hormone receptor-positive (p = 0.036). In particular, in the HER2 + subgroup, the miR-21 levels were significantly higher in those with active BC (both EBC and MBC) than in HDs (p = 0.002). Our findings suggest that miR-21 may be a promising biomarker for diagnosis and tumor activity, mainly in HER2 + BC.

Parasitic helminths secrete extracellular vesicles (EVs) into their host tissues to modulate immune responses, but the underlying mechanisms are poorly understood. We demonstrate that Ascaris EVs are efficiently internalised by monocytes in human peripheral blood mononuclear cells and increase the percentage of classical monocytes. Furthermore, EV treatment of monocytes induced a novel anti-inflammatory phenotype characterised by CD14+, CD16-, CC chemokine receptor 2 (CCR2-) and programmed death-ligand 1 (PD-L1)+ cells. In addition, Ascaris EVs induced T cell anergy in a monocyte-dependent mechanism. Targeting professional phagocytes to induce both direct and indirect pathways of immune modulation presents a highly novel and efficient mechanism of EV-mediated host-parasite communication. Intra-peritoneal administration of EVs induced protection against gut inflammation in the dextran sodium sulphate model of colitis in mice. Ascaris EVs were shown to affect circulating immune cells and protect against gut inflammation; this highlights their potential as a subject for further investigation in inflammatory conditions driven by dysregulated immune responses. However, their clinical translation would require further studies and careful consideration of ethical implications.

AbstractEscherichia coli A0 34/86 (EcO83) is a probiotic strain used in newborns to prevent nosocomial infections and diarrhoea. This bacterium stimulates both pro‐ and anti‐inflammatory cytokine production and its intranasal administration reduces allergic airway inflammation in mice. Despite its benefits, there are concerns about the use of live probiotic bacteria due to potential systemic infections and gene transfer. Extracellular vesicles (EVs) derived from EcO83 (EcO83‐EVs) might offer a safer alternative to live bacteria. This study characterizes EcO83‐EVs and investigates their interaction with host cells, highlighting their potential as postbiotic therapeutics. EcO83‐EVs were isolated, purified, and characterised following the Minimal Information of Studies of Extracellular Vesicles (MISEV) guidelines. Ex vivo studies conducted in human nasal epithelial cells showed that EcO83‐EVs increased the expression of proteins linked to oxidative stress and inflammation, indicating an effective interaction between EVs and the host cells. Further in vivo studies in mice demonstrated that EcO83‐EVs interact with nasal‐associated lymphoid tissue, are internalised by airway macrophages, and stimulate neutrophil recruitment in the lung. Mechanistically, EcO83‐EVs activate the NF‐κΒ signalling pathway, resulting in the nitric oxide production. EcO83‐EVs demonstrate significant potential as a postbiotic alternative to live bacteria, offering a safer option for therapeutic applications. Further research is required to explore their clinical use, particularly in mucosal vaccination and targeted immunotherapy strategies.

Since unmodified polyester fibres have no reactive groups like those in cellulose and protein fibres, they do not show an affinity for water-soluble acid, basic and direct dyestuffs. Only disperse dyestuff, a non-ionic dyestuff class with low molar mass molecules, proved to be useful for dyeing this man-made fibre following a solid-solid interaction; disperse dyestuffs do not form primary chemical bonds with polymer chains rather the dye colour is retained by H-bonds and Van der Waals forces. Herein, a new strategy for dyeing polyester fabrics with a direct dyestuff in a two-step strategy was designed and realized, using an organic-inorganic composite coating based on methyl red-loaded sol-gel modified halloysite nanotubes. In the first step two distinct reaction methods were compared to functionalize halloysite nanotubes with (3-Glycidyloxypropyl)trimethoxysilane, as a covalent crosslinker between the halloysite nanotubes and fibres, (i) in water and (ii) in ethanol, using BF3O(C2H5) and chloridric acid (HCl) as catalysts, respectively. In the second step, methyl red loaded GPTMS modified halloysite sols were applied onto polyester fabrics by impregnation. The amount of methyl red dyestuff was evaluated to be superior for the complex realized in ethanol than in water, thus promoting homogeneous nanocomposite coatings on treated polyester samples. Methyl red loaded sol-gel modified halloysite complex, as well as treated and untreated samples, were investigated to characterize their properties and morphology. NMR investigation confirmed the structure of the new complex, validating the successful dyestuff coordination reaction at GPTMS. The influence of treatments on the morphology of fibres surfaces was demonstrated by Scanning Electron Microscopy (SEM), Energy Dispersive X-ray spectroscopy (EDX), and Atomic Force Microscopy (AFM) analyses, highlighting the influence of GPTMS-based composites on the microstructure of functionalized polyester fibres. To further confirm if the suggested approach offers a stable dyestuff loading on PE, diffuse reflectance spectroscopic studies, X-ray Photoelectron Spectroscopy (XPS) and colour fastness to rubbing and washing tests were carried out on the coated polyester. All findings make sol-gel based modification of halloysite a reliable and promising method for eco-friendly dyeing processes of polyester fabrics.

Correction for ‘Photo-gain optimization in multilayer organic phototransistors by study of space-charge limited current’ by Giulia Baroni et al. , J. Mater. Chem. C , 2024, https://doi.org/10.1039/D4TC01925C.

This article reports on the synthesis of an innovative smart polymer, P5-QPDMAEMA, opportunely developed with the aim of combining the responsiveness of PDMAEMA polymer and the host-guest properties of covalently linked pillar[5]arenes. Thanks to a traditional Non-Induced Phase Separation (NIPS) process performed at various coagulation pH, the blending of P5-QPDMAEMA with polyethersulfone gave rise to the formation of functional beads for the removal of organic dyes in water. Adsorption tests are carried out on all the produced blend-based beads by employing two representative dyes, the cationic methylene blue (MB), and the anionic methyl orange (MO). In particular, the P5-QPDMAEMA based beads, prepared at acidic pH, featured the best MO removal rate (i. e., 91.3 % after 150 minutes starting from a 20 mg ⋅ L-1 solution) and a high selectivity towards the removal of the selected anionic dye. Based on the adsorption kinetics and isotherm calculations, the pseudo-first order and Freundlich models were shown to be the most suitable to describe the MO adsorption behavior, achieving a maximum adsorption capacity of 21.54 mg ⋅ g-1. Furthermore, zwitterionic beads are obtained by a post-functionalization of the PDMAEMA and the P5-QPDMAEMA based beads, to test their removal capability towards both anionic and cationic dyes, as shown.

Endometriosis is a chronic inflammatory condition characterized by the presence of endometrium-like tissue outside the uterus, primarily affecting pelvic organs and tissues. In this study, we explored platelet activation in endometriosis. We utilized the STRING database to analyze the functional interactions among proteins previously identified in small extracellular vesicles (EVs) isolated from the peritoneal fluid of endometriosis patients and controls. The bioinformatic analysis indicated enriched signaling pathways related to platelet activation, hemostasis, and neutrophil degranulation. Double immunohistochemistry analysis for CD61 and MPO revealed a significant presence of neutrophils and platelets in close contact infiltrating endometriotic lesions, suggesting potential cell-cell interactions. Subsequently, we isolated small EVs from the peritoneal fluid of women diagnosed with endometriosis and from women without endometriosis who underwent surgery for non-inflammatory benign diseases. We performed single-particle phenotyping analysis based on platelet biomarkers GPIIb/IIIa and PF4 using nanoflow cytometry, as well as single-particle morphological and nanomechanical characterization through atomic force microscopy. The study demonstrated that patients with endometriosis had a notably higher proportion of particles testing positive for platelet biomarkers compared to the total number of EVs. This finding implies a potential role for platelets in the pathogenesis of endometriosis. Further research is necessary to delve into the mechanisms underlying this phenomenon and its implications for disease progression.

The aim of this study was to develop azithromycin (AZT)-loaded liposomes (LP) and niosomes (NS) useful for the treatment of bacterial skin infections and acne. LP based on phosphatidylcholine from egg yolk (EPC) or from soybean lecithin (SPC), and NS composed of sorbitan monopalmitate (Span 40) or sorbitan monostearate (Span 60) were prepared through the thin film hydration (TFH) and the ethanol injection (EI) methods. The formulations were subsequently characterized for their physico-chemical and functional properties. Vesicles prepared through TFH showed higher average sizes than the corresponding formulations obtained by EI. All the vesicles presented adequate encapsulation efficiency and a negative ζ potential, which assured good stability during the storage period (except for LP-SPC). Formulations prepared with TFH showed a more prolonged AZT release than those prepared through EI, due to their lower surface area and multilamellar structure, as confirmed by atomic force microscopy nanomechanical characterization. Finally, among all the formulations, NS-Span 40-TFH and LP-EPC-TFH allowed the highest drug accumulation in the skin, retained the antimicrobial activity and did not alter fibroblast metabolism and viability. Overall, they could ensure to minimize the dosing and the administration frequency, thus representing promising candidates for the treatment of bacterial skin infections and acne.

Hybrid lipid membranes incorporating amphiphilic copolymers have gained significant attention due to their potential applications in various fields, including drug delivery and sensing. By combining the properties of copolymers and lipid membranes, such as enhanced chemical tunability and stability, environmental responsiveness, and multidomain nature, novel membrane architectures have been proposed. In this study, we investigated the potentialities of hybrid membranes made of two distinct components: the rigid fully saturated phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the soft copolymer poly(butadiene-b-ethyleneoxide) (PBD-b-PEO). The objective was to explore the interaction of citrate-coated gold nanoparticles (AuNPs) and the hybrid membrane, aiming at constructing AuNPs-hybrid vesicles suprastructures with controlled and adjustable plasmonic properties. A series of experimental techniques were employed to investigate hybrid free-standing and supported membranes. The results revealed that the incorporation of the copolymer into the lipid membrane promotes AuNPs clustering, demonstrating a distinctive aggregative phenomenon of citrate-coated AuNPs on multidomain membranes. Importantly, we show that the size and morphology of AuNPs clusters can be precisely controlled in non-homogeneous membranes, enabling the formation of hybrid suprastructures with controlled patch properties. These results highlight the potential of lipid-copolymer hybrid membranes for designing functional materials with tailored plasmonic properties, with potential applications in nanomedicine and sensing.

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.