#Results for Action 1
We established a production of small extracellular vesicles (sEVs) from human endometrial decidual tissue-derived mesenchymal stromal cells, and from marine microalgae T. chuii, generating nanoalgosomes. Red blood cell-EVs and liposomes mimicking EVs were also used as testing surrogate materials in hybrid nanodevices (RBC-evMBDs and lpMBDs). TheMBD lipid coating were successfully functionalized with folate for macrophage targeting, producing a first set of engineered nanohybrids to be tested in pulmonary fibrosis.
On the synthetic side, two main MBD prototypes were developed: Single-core (S.C.) MBDs and Multi-core (M.C.) ones. Additionally, "soft" MBDs, gelatin-coated M.C. structures were created, representing a novel nanomaterial. MBD were also functionalized with fluorescent moieties for optical tracking. All the MBDs produced exhibited excellent features. However, S.C. MBDs were preferred for evMBD/lpMBD synthesis due to superior encapsulation performance.
During the production of the BOW materials, tailored procedures were developed for their production, storage and shipment across Consortium sites, ensuring batch-to-batch reproducibility. A shared minimal analytical package also ensured consistent quality control.
#Results for Action 2
Two microfluidic devices were developed to streamline MBD encapsulation within EV membranes:
•A single-phase device perturbing EVs via active mixing and sonication
•A two-phase water-in-oil emulsion device using osmotic shock and chaotic mixing.
The sonication device was chosen for its higher encapsulation yield, optimized, and used to produce lpMBDs, RBC-evMBDs, and MSC-evMBDs.
During the project, a comprehensive toolbox was developed to assess the physicochemical properties and functionality of evMBDs, including:
•FRET-based assay & Single-Molecule Localization Microscopy for evaluate MBD coverage
•Cryo-EM, AFM & colloidal nanoplasmonics-based assays for MBD structural and coverage analysis
•FCCS for quantifying hybrid synthesis yields
•FCS for detecting folate functionalization
•Inductive coil-based device for real-time diffusion rate, size, and concentration measurements of hybrids.
These characterization tools establish a strong foundation for further hybrid materials development, ensuring functionality and reproducibility for downstream applications.
#Results for Action 3
Extensive nanotoxicology studies were conducted, focused on assessing effects on cell metabolism, membrane integrity, clonogenicity, DNA damage, oxidative stress, paracrine signaling, and macrophage polarization. Advanced in vitro models were implemented, showing high biocompatibility and therapeutic potential of some of the pristine BOW products. Moreover, the lipid membrane coating improved safety of toxic MBDs. Plus, folate functionalization effectively enhances macrophage targeting.
In vivo experiments evaluated BOW material biodistribution, uptake, clearance, and biological responses. C. elegans studies confirmed good biocompatibility for all EVs, while pristine MBDs exhibited toxicity. In M. musculus models of lung inflammation, nanoalgosomes, MSC-EVs, and lpMBDs showed low toxicity and therapeutic promise, consistent with in vitro and C. elegans findings. Overall, lipid membrane coatings effectively reduced toxicity, supporting the safe therapeutic potential of these materials for pulmonary diseases.
Mathematical models to assess the toxicity and immune reactivity of BOW materials were also developed, including a toxicodynamic model, a cell growth model to assess proliferation across experiments, a macrophage polarization model for long-term immune outcomes, and a model to estimate uptake, clearance rates, and the Lowest Observed Adverse Effect Level in vitro. These models provide a starting point to build robust algorithms for predicting the long-term effects of these materials in vitro and extrapolating findings to in vivo systems.
#Results for Action 4
Below is a summary of the dissemination and communication activities carried out throughout the project:
• 8 Consortium meetings, 13 tech meetings, 3 Tech meetings managed by young researchers hired on the grant
• 6 Ph.D. students, 9 Post-doc hired
• 3 Ph.D. student exchange between labs
• 32 papers, 1 book chapter published
• 40+ presentations, 15+ posters at congresses
• 13 deliverables uploaded in public repositories
• 2 International Ph.D. schools organized
• 1 Italian patent application filed
• 8 Key Exploitation Results
• 14 News in media, 250 tweets posted, +10 linkedin posts made
• EIC cluster meeting in Brussels involving representatives from relevant EIC projects on EVs.
