Extracellular Vesicle-Associated IL4 Displays Enhanced Anti-Inflammatory Properties in Microglial Cells Giulia Marostica, Chiara Frigè, Annamaria Finardi, Alessandra Mandelli, Susanna Manenti, Stella Siciliani, Paola Podini, Ivan De Curtis, Luca Muzio, Elena Rossi, Paola de Candia, Roberto Furlan Journal of Extracellular Biology, 2026 Neuroinflammation and neurodegeneration are strictly related phenomena, characterized by dysregulation of microglia, central nervous system (CNS) resident immune cells. Interleukin‐4 (IL4) has shown beneficial abilities to re‐establish microglial homeostasis in experimental models of CNS traumatic injury, stroke and multiple sclerosis, but its optimal administration system remains uncertain. Here, we show that extracellular vesicles (EVs) released by engineered murine microglia BV2 cells constitutively expressing IL4 induced a faster and enhanced anti‐inflammatory phenotype in wild type BV2 cells (as assessed by IL4R downstream signalling), compared with soluble IL4. This effect was blunted by an anti‐IL4 antibody, while it was not dampened by knocking out the IL4 receptor α subunit in EV‐releasing BV2 cells, suggesting that IL4 was localized on the EV surface and did not necessitate to be co‐conveyed with the receptor to exert its function. BV2 cells treated with EV‐associated IL4, compared with soluble IL4, demonstrated delayed permanence of IL4R in the early endosome, suggesting amplified signalling effects. In conclusion, we here show that the association of IL4 with microglia‐derived EVs improve its anti‐inflammatory effect in an in vitro murine microglial model, which may inform on novel therapeutic opportunities to restore microglia in human disorders marked by neuroinflammation.
Intranasal Administration of Extracellular Vesicles Derived from Adipose Mesenchymal Stem Cells Has Therapeutic Effect in Experimental Autoimmune Encephalomyelitis Barbara Rossi, Federica Virla, Gabriele Angelini, Ilaria Scambi, Alessandro Bani, Giulia Marostica, Mauro Caprioli, Daniela Anni, Roberto Furlan, Pasquina Marzola, Raffaella Mariotti, Gabriela Constantin, Bruno Bonetti, Ermanna Turano Cells, 2025 Adipose stem cells (ASCs) are a subset of mesenchymal stem cells with validated immunomodulatory and regenerative capabilities that make them attractive tools for treating neurodegenerative disorders, such as multiple sclerosis (MS). Several studies conducted on experimental autoimmune encephalomyelitis (EAE), the animal model of MS, have clearly shown a therapeutic effect of ASCs. However, controversial data on their efficacy were obtained from I- and II-phase clinical trials in MS patients, highlighting standardization issues and limited data on long-term safety. In this context, ASC-derived extracellular vesicles from (ASC-EVs) represent a safer, more reproducible alternative for EAE and MS treatment. Moreover, their physical characteristics lend themselves to a non-invasive, efficient, and easy handling of intranasal delivery. Using an in vitro setting, we first verified ASC-EVs’ ability to cross the human nasal epithelium under an inflammatory milieu. Magnetic resonance corroborated these data in vivo in intranasally treated MOG35-55-induced EAE mice, showing a preferential accumulation of ASC-EVs in brain-inflamed lesions compared to a stochastic distribution in healthy control mice. Moreover, intranasal treatment of ASC-EVs at the EAE onset led to a long-term therapeutic effect using two different experimental protocols. A marked reduction in T cell infiltration, demyelination, axonal damage, and cytokine production were correlated to EAE amelioration in ASC-EV-treated mice compared to control mice, highlighting the immunomodulatory and neuroprotective roles exerted by ASC-EVs during EAE progression. Overall, our study paves the way for promising clinical applications of self-administered ASC-EV intranasal treatment in CNS disorders, including MS.
Extracellular vesicles from adipose mesenchymal stem cells target inflamed lymph nodes in experimental autoimmune encephalomyelitis Ermanna Turano, Ilaria Scambi, Roberta Bonafede, Silvia Dusi, Gabriele Angelini, Nicola Lopez, Giulia Marostica, Barbara Rossi, Roberto Furlan, Gabriela Constantin, Raffaella Mariotti, Bruno Bonetti Cytotherapy, 2024 Background aimsAdipose mesenchymal stem cells (ASCs) represent a promising therapeutic approach in inflammatory neurological disorders, including multiple sclerosis (MS). Recent lines of evidence indicate that most biological activities of ASCs are mediated by the delivery of soluble factors enclosed in extracellular vesicles (EVs). Indeed, we have previously demonstrated that small EVs derived from ASCs (ASC-EVs) ameliorate experimental autoimmune encephalomyelitis (EAE), a murine model of MS. The precise mechanisms and molecular/cellular target of EVs during EAE are still unknown.MethodsTo investigate the homing of ASC-EVs, we intravenously injected small EVs loaded with ultra-small superparamagnetic iron oxide nanoparticles (USPIO) at disease onset in EAE-induced C57Bl/6J mice. Histochemical analysis and transmission electron microscopy were carried out 48 h after EV treatment. Moreover, to assess the cellular target of EVs, flow cytometry on cells extracted ex vivo from EAE mouse lymph nodes was performed.ResultsHistochemical and ultrastructural analysis showed the presence of labeled EVs in lymph nodes but not in lungs and spinal cord of EAE injected mice. Moreover, we identified the cellular target of EVs in EAE lymph nodes by flow cytometry: ASC-EVs were preferentially located in macrophages, with a consistent amount also noted in dendritic cells and CD4+ T lymphocytes.ConclusionsThis represents the first direct evidence of the privileged localization of ASC-EVs in draining lymph nodes of EAE after systemic injection. These data provide prominent information on the distribution, uptake and retention of ASC-EVs, which may help in the development of EV-based therapy in MS.
Control of cell penetration enhancer shielding and endosomal escape-kinetics crucial for efficient and biocompatible siRNA delivery Alessio Malfanti, Haider Sami, Anna Balasso, Giulia Marostica, Busra Arpac, Francesca Mastrotto, Giuseppe Mantovani, Elisa Cola, Martina Anton, Paolo Caliceti, Manfred Ogris, Stefano Salmaso Journal of Controlled Release, 2023 Although cationic liposomes are efficient carriers for nucleic acid delivery, their toxicity often hampers the clinical translation. Polyethylene glycol (PEG) coating has been largely used to improve their stability and reduce toxicity. Nevertheless, it has been found to decrease the transfection process. In order to exploit the advantages of cationic liposomes and PEG decoration for nucleic acid delivery, liposomes decorated with tetraArg-[G-1]-distearoyl glycerol (Arg4-DAG) dendronic oligo-cationic lipid enhancer (OCE) and PEG-lipid have been investigated. Non decorated or OCE-decorated lipoplexes (OCEfree-LPX and OCE-LPX, respectively) were obtained by lipid film hydration using oligonucleotide (ON) solutions. PEG and OCE/PEG decorated lipoplexes (PEG-OCEfree-LPX and PEG-OCE-LPX, respectively) were obtained by post-insertion of 2 or 5 kDa PEG-DSPE on preformed lipoplexes. The OCE decoration yielded lipoplexes with size of about 240 nm, 84% loading efficiency at 10 N/P ratio, ten times higher than OCEfree-LPX, and prevented the ON release when incubated with physiological heparin concentration or with plasma. The PEG decoration reduced the zeta potential, enhanced the lipoplex stability in serum and decreased both hemolysis and cytotoxicity, while it did not affect the lipoplex size and ON loading. With respect to OCEfree-LPX, the OCE-LPX remarkably associated with cells and were taken up by different cancer cell lines (HeLa and MDA-MB-231). Interestingly, 2 or 5 kDa PEG decoration did not reduce either the cell interaction or the cell up-take of the cationic lipoplexes. With siRNA as a payload, OCE enabled efficient internalization, but endosomal release was hampered. Post-transfection treatment with the lysosomotropic drug chloroquine allowed to identify the optimal time point for endosomal escape. Chloroquine treatment after 12 to 20 h of LPX pre-incubation enabled siRNA mediated target knockdown indicating that this is the time window of endo-lysosomal processing. This indicates that OCE can protect siRNA from lysosomal degradation for up to 20 h, as shown by these rescue experiments.
The impact of storage on extracellular vesicles: A systematic study Stefano Gelibter, Giulia Marostica, Alessandra Mandelli, Stella Siciliani, Paola Podini, Annamaria Finardi, Roberto Furlan Journal of Extracellular Vesicles, 2022 Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. While −80°C storage is a widespread approach, some authors proposed improved storage strategies with conflicting results. Here, we designed a systematic study to assess the impact of −80°C storage and freeze‐thaw cycles on EVs. We tested the differences among eight storage strategies and investigated the possible fusion phenomena occurring during storage. EVs were collected from human plasma and murine microglia culture by size exclusion chromatography and ultracentrifugation, respectively. The analysis included: concentration, size and zeta potential (tunable resistive pulse sensing), contaminant protein assessment; flow cytometry for the analysis of two single fluorescent‐tagged EVs populations (GFP and mCherry), mixed before preservation. We found that −80°C storage reduces EVs concentration and sample purity in a time‐dependent manner. Furthermore, it increases the particle size and size variability and modifies EVs zeta potential, with a shift of EVs in size‐charge plots. None of the tested conditions prevented the observed effects. Freeze‐thaw cycles lead to an EVs reduction after the first cycle and to a cycle‐dependent increase in particle size. With flow cytometry, after storage, we observed a significant population of double‐positive EVs (GFP+‐mCherry+). This observation may suggest the occurrence of fusion phenomena during storage. Our findings show a significant impact of storage on EVs samples in terms of particle loss, purity reduction and fusion phenomena leading to artefactual particles. Depending on downstream analyses and experimental settings, EVs should probably be processed from fresh, non‐archival, samples in majority of cases.
Extracellular Vesicles in Neuroinflammation Giulia Marostica, Stefano Gelibter, Maira Gironi, Annamaria Nigro, Roberto Furlan Frontiers in Cell and Developmental Biology, 2021 Extracellular vesicles (EVs) are a heterogenous group of membrane-bound particles that play a pivotal role in cell–cell communication, not only participating in many physiological processes, but also contributing to the pathogenesis of several diseases. The term EVs defines many and different vesicles based on their biogenesis and release pathway, including exosomes, microvesicles (MVs), and apoptotic bodies. However, their classification, biological function as well as protocols for isolation and detection are still under investigation. Recent evidences suggest the existence of novel subpopulations of EVs, increasing the degree of heterogeneity between EV types and subtypes. EVs have been shown to have roles in the CNS as biomarkers and vehicles of drugs and other therapeutic molecules. They are known to cross the blood brain barrier, allowing CNS EVs to be detectable in peripheral fluids, and their cargo may give information on parental cells and the pathological process they are involved in. In this review, we summarize the knowledge on the function of EVs in the pathogenesis of multiple sclerosis (MS) and discuss recent evidences for their potential applications as diagnostic biomarkers and therapeutic targets.
