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.

The increasing demand for antimicrobial surfaces, particularly in hygiene-sensitive environments, has driven the development of ceramic tiles functionalized with bioactive agents. This study proposes the use of multiple and customized approaches to characterize functionalized ceramic tiles for antibacterial purposes. Tiles incorporating two silver-based formulations (named W-Ag and S-Ag) within different glaze matrices were characterized using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) to evaluate surface morphology and elemental composition. Antibacterial efficacy was assessed against four opportunistic pathogens (Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, and Pseudomonas aeruginosa) through minimum inhibitory and bactericidal concentration assays, ISO 22196 standard procedures, bacterial adhesion assays, and biofilm visualization. Results revealed that post-firing S-Ag-treated tiles exhibited superior antibacterial activity and anti-adhesive properties compared to pre-firing W-Ag tiles, achieving substantial bacterial reduction (R>2) against all strains after 24 h, and early-stage inhibition after 4 h. W-Ag tiles showed limited efficacy, primarily against E. coli, likely due to a decrease in silver availability after high-temperature firing. Tile morphology and glaze composition significantly influenced antibacterial performance, with smoother surfaces promoting reduced bacterial adhesion. Reduced concentrations of S-Ag maintained efficacy against S. aureus but were less effective against other strains, highlighting species-specific and concentration-dependent activity. These findings underscore the critical role of deposition methods, surface morphology, and material composition in determining the antimicrobial performance of ceramic tiles. Comprehensive evaluation combining standardized assays, early-stage adhesion testing, and structural characterization is essential for optimizing bioactive tiles for real-world applications, including infection/contamination prevention in healthcare and public settings.

Cancer cells actively release extracellular vesicles (EVs) into the tumor microenvironment, where they interact with both malignant and non-malignant cells, activating signaling pathways and reshaping the microenvironment. In this study, we investigated EVs secreted by FGFR2 -amplified cancers of unknown primary (CUPs), which generate extrachromosomal circular DNA (ecDNA) as a mechanism of oncogene amplification. We found that FGFR2 -containing ecDNA is packaged into both small and large EVs, horizontally transferred to recipient cells, and remains functionally active. Upon exposure to CUP-derived EVs—either by direct administration or co-culture—cancer (NCI-N87, THP1) and non-cancer (HUVEC, fibroblasts) cells internalized FGFR2 ecDNA, which was subsequently transcribed and translated to some extent. Functionally, CUP-derived EVs polarized THP1 cells toward an M2-like phenotype and promoted HUVEC proliferation. In vivo , xenografts generated from CUP cell lines released circulating FGFR2 + EVs, which mediated the systemic transfer of FGFR2 ecDNA to distant organs. Collectively, these findings demonstrate that tumor-derived EVs can propagate and horizontally transfer oncogenic ecDNA both in vitro and in vivo , providing a possible mechanistic basis for the high metastatic potential of this tumor type.

Reliable methodologies for spatio-temporal controlled delivery of morphogens are of key importance in organoid research, regenerative medicine and developmental biology. To develop such a methodology, we constructed a magnetic nanocarrier composed of a supramolecular nanoassembly of amphiphilic cyclodextrin (SC6OH) entangling superparamagnetic iron oxide nanoparticles (SPIONs) within the surface. Upon encapsulation of a defined amount of retinoic acid (RA), the nanocarriers are remotely guided through microfluidic channels to a cell culture compartment by a specifically designed magnetic device based on electro-mechanically actuated permanent magnets. We demonstrate the efficiency of this innovative technology for the delivery of morphogens by applying it to induce the differentiation of human neuroblastoma SH-SY5Y cells into neurons. The magnetically controlled RA delivery resulted in the successful induction of neuronal differentiation with precise spatial and temporal control while minimizing reliance on complex microfluidic setups. Thus, the integration of magnetic actuation with supramolecular nanocarriers promotes new efficient routes and scalable protocols that go beyond state-of-the-art research in various bio-medical applications.

Carboranes are chemically and biologically stable boron‑carbon clusters with promising applications in medicinal chemistry. While their use in boron neutron capture therapy (BNCT) has been extensively explored, recent attention has shifted toward understanding their interactions with biological macromolecules, particularly proteins. Here, we characterize the interaction between closo-ortho-carborane and lysozyme (LSZ) using NMR spectroscopy, molecular docking and molecular dynamics simulations, and enzymatic assays. Experimental data demonstrate that carborane forms a stable 1:1 complex with LSZ (Carborane@LSZ), retaining the monomeric state and the protein fold, with only a limited number of amino acids involved in the interaction. In particular, NMR chemical shift perturbations revealed specific binding near the substrate-binding pocket, a result corroborated by molecular docking and molecular dynamic simulations. Carborane fits into a hydrophobic pocket near the substrate-binding site, where the recognition process is driven by hydrophobic interactions complemented by classical hydrogen and non-standard dihydrogen bonding. Carborane-@LSZ complex partially inhibits enzymatic activity (∼33 %). Extending this approach to bovine serum albumin (BSA) revealed similar binding principles, underscoring the generality of carborane-protein supramolecular interactions. These findings provide fundamental insights into pristine carboranes recognition by proteins and establish a foundation for designing carborane-based therapeutics and delivery platforms in nanomedicine.

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.

The extracellular vesicle (EV) route is essential for cell-to-cell communication. Cancer cells release EVs in the extracellular space, where they can interact with cancer and non-cancer cells, activating specific signaling pathways, modulating tumor microenvironment remodeling and inducing gene expression alterations. We investigated the functional role of EVs released by cancer of unknown primary (CUP), a rare disease (1-3% of novel cancer diagnoses) that presents with metastasis of unknown or uncertain origin and no apparent primary tumor. We derived three CUP cell lines from patient’s tumor cells, CUP#55A and S and CUP#96, both characterized by FGFR2 gene amplification, either in the form of trisomy (CUP#55A), chromosomal homogeneously staining region (CUP#55S) or double minute chromosomes (CUP#96). It has been recently recognized that tumors use extrachromosomal circular DNA (eccDNA) as a way to increase oncogenic amplification, thus conferring resistance to therapy and contributing to a worse survival. We demonstrated that FGFR2 amplification in CUP cell lines is associated with ecDNA generation and that this ecDNA is loaded as cargo inside EVs and exert a functional activity in nearby cells. The full-length FGFR2 DNA was detectable inside both small and large vesicles isolated from cell culture medium and we confirmed the circular nature of a fraction of this FGFR2 ecDNA using DNAse based strategies and visualizing DNA circles in cells and in EVs with atomic force microscopy. The protein surface profile of small and large EV populations revealed the presence of cancer related molecules that mirrored the cells of origin, including EpCAM and CD44, and variable degrees of FGFR2pos EVs. Cancer (NCI-N87) and non-cancer cell exposure (THP1 and HUVEC) to CUP EVs (by direct administration and co-culture) revealed an increase of FGFR2 DNA copies at 24 hours and its functional transcription in FGFR2 mRNA after 48 hours in all exposed cells. In addition to oncogenic delivery, CUP#96 EVs induced a polarization of THP1 cells towards M2 subtype, while CUP#55S EVs exerted a proliferative effect on HUVEC. In conclusion, we identified a model of oncogene amplification and EV-mediated delivery recurrent in cancer of unknown primary, whose DNA content was demonstrated to be functionally active in recipient cells and proportional with extrachromosomal DNA generation. This mechanism could contribute to the high metastatic potential of this cancer type. (The research leading to these results has received funding from AIRC under IG 2021 - ID. 25789 project - P.I. Ferracin Manuela) Irene Salamon, Giulia Gallerani, Gianluca Storci, Beatrice Fontana, Salvatore Serravalle, Francesco Valle, Marco Brucale, Marco Pagano Mariano, Andrea Cavazzoni, Roberta Roncarati, Spartaco Santi, Massimiliano Bonafè, Manuela Ferracin. Extracellular vesicle delivery of functional extrachromosomal DNA in FGFR2-amplified cancer of unknown primary [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6569.

Aerobic vaginitis is an infectious disease characterized by the overgrowth of abnormal vaginal microflora. Conventional local dosage forms are not always effective, due to their inadequate drug release and residence time within the vaginal cavity. Therefore, this study aimed to develop azithromycin (AZT)-loaded liposomes, coated with two mucoadhesive polymers, chitosan (CS) and sodium hyaluronate (HYA), to increase thedrug's stay at the site of infection and to control its release. Liposomes were developed through the thin film hydration method followed by extrusion and subsequently added to the polymer solution. Later, they were characterized by their size, surface charge, morphology, and encapsulation efficiency. Furthermore, mucoadhesive properties and drug release behavior were investigated at different pH values, e.g., 4.5 and 7.4, mimicking the physiological and pathological conditions, respectively. Finally, antimicrobial tests and in vitro permeation studies were carried out. Results showed size and surface charge variations of coated LP with respect to the uncoated ones, confirming the success of the coating process. LP possessed a good capacity to encapsulate the drug. Among all the formulations, CS-LP demonstrated superior control of drug release and greatest mucoadhesive properties at both tested pHs, as well as the highest drug accumulation inside the vaginal tissue, maintaining at the same time AZT antimicrobial effect. Overall, CS-LP could be proposed as a promising nanocarrier for AZT vaginal delivery, in virtue of its ability to achieve locally a sustained release of drug, helping to lower the dosage and administration frequency, and consequently improving treatment efficacy.

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.

Calcium phosphates-based (CaPs) nanocoatings on metallic prosthesis are widely studied in orthopedics and dentistry because they mimic the mineral component of native human bone and favor the osseointegration process. Despite the fact that different calcium phosphates have different properties (composition, crystallinity, and ion release), only stoichiometric hydroxyapatite (HA) films have been analyzed in deep. Here, we have realized films of different CaPs (HA, beta-tricalcium phosphate (β-TCP) and brushite (DCPD)) onto Ti6Al4V microrough substrates by Ionized Jet Deposition (IJD). We have implemented the heating of substrates at 400°C during deposition to see the effect on coating properties.Different film features are evaluated: morphology and topography (FEG-SEM, AFM), physical-chemical composition (FT-IR and EDS), dissolution profile and adhesion to substrate (scratch test), with a focus on how the different CaPs and temperature changed the coating features. After coating optimization, we have studied the in vitro BM-MSC behavior, in term of viability and early adhesion.We have obtained good transfer of fidelity in composition from target to coating for all CaPs, with nanostructured films formed by globular aggregates (~300 nm diameter), with homogeneous and uniform coverage of the substrate surface, without cracks. The heating during deposition has increased the adhesion of the films to the substrate, with higher stability in medium immersion and wettability, features that can improve the biological behavior of cells. All CaP coatings have showed excellent biocompatibility, with DCPD coating that promote higher cells viability at 14 days respect to HA and β- TCP films. About the early cell adhesion, the BM-MSC have showed switch from a globular to an elongated morphology at 6 hours in all coatings respect to the uncoated titanium, sign of better adhesion.From these results, the fabrication of different CaP nanocoatings with IJD can be a promising for applications in orthopedics and dentistry.

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.

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.