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Biogenic Organotropic Wetsuits

Periodic Reporting for period 1 - BOW (Biogenic Organotropic Wetsuits)

Reporting period: 2020-11-01 to 2021-10-31

Extracellular vesicle (EV) nanoparticles are key players in inter-cellular and inter-organism communication. “Made by cells for cells”, they mediate different physiological and/or pathological processes.
The main goal of the BOW project is to explore and consolidate the technology able to impart biological surface precision, circulation and targeting abilities of EVs to superparamagnetic nanodevices (Magnetic Bead Devices, MBDs) by “dressing” them with a single- or multi-layer “wetsuit” of EV membrane “fabric”.
Specific objectives include production of high-grade extracellular vesicles with biomimetic and organotropic functions, synthesis and functionalization of MBDs, engineering of a microfluidic device for streamlined fabrication of extracellular vesicle membrane-coated MBDs (evMBDs) and evaluation of evMBD biological performances and nanotoxicity in vitro, ex vivo and in vivo.
# Action 1 “Production and characterization of the biogenic and synthetic building nanomaterials”.
- Small EVs (biogenic nanomaterials):
a) Methods and protocols to provide pure, well-characterized, stable, and reproducible sets of small EVs (< 250 nm) from the selected sources in the necessary amounts for next use (engineering, membrane wetsuit sources, and reference) were defined.
b) Two strategies for EV membrane engineering for augmented targeting have been devised and are under experimental exploration.
c) Work for setting optimal handling, storage, and shipment across the Consortium sites of the produced EVs has profitably started, providing a first necessary set of instructions for in- project logistics.

- Single- and multi-core MBDs of silica-encapsulated magnetite nanoparticles (synthetic nanomaterials) have been successfully synthesized. In parallel, a proper toolbox of state-of-the-art characterization techniques was set and implemented to analyze their key physicochemical features.

# Action 2 “Fabrication of the microfluidic dressing device”.
- Device prototypes. The microfluidic device shall sequentially open the EVs and transfer their membranes onto the MBDs (viz. encapsulate the MBDs into the EVs) in a streamlined process. Exploration of the two envisioned devices began on time and a prototype of the first was fabricated. As planned, the activity related to the second device started in parallel.
- evMBD physicochemical characterization started ahead of time (originally planned to start in month M13), driven by the need for proper analytical methods to determine the encapsulation yield of the devices (both off-line and in-line with the devices). Four orthogonal methods/techniques were developed or are under development, three of them being off-line and one in-line.

# Action 3 “Testing of the nanotoxicology and of the biological performances of the nanomaterials”.
Start of the action 3 activities, planned at month M6, was anticipated immediately after the project kick-off, in the attempt to counterbalance delays due to COVID-19 pandemic restrictions. The move turned out successful, and the first in-vivo and in-vitro testing of EVs and MBDs (which are the building blocks of evMBDs) successfully conducted. The activity was further supported by a 9 month long visit of a Ph.D. student of CSGI (Università degli Studi di Brescia research unit) with expertise on EVs at HMGU (leader partner of the action).
The application of evMBDs for therapy and diagnosis of pulmonary inflammation and fibrosis has been identified to have great potential, also in consideration of the rise of lung fibrosis cases as emergent pathology during the post-COVID era. evMBDs applicability has been preliminary explored in-vitro and therefore it will be investigated in the next using the full in vitro and in vivo Consortium capacity.

# Carried-out activities of management, exploitation, dissemination, communication, and training included:
• 3 consortium meetings;
• 3 bimonthly technical meetings;
• 1 technical meeting managed and participated by young researchers hired on the grant;
• 6 Ph.D. students hired on the grant;
• 6 post-doc students were hired on the grant;
• 1 Ph.D. student exchange between partners laboratories;
• external scientific advisory board (AB) established;
• 7 papers published in peer-reviewed international scientific journals;
• 1 book chapter;
2 public deliverables were uploaded onto open access repositories indexed in OpenAIRE (Zenodo);
• 13 news in media;
• 109 social media tweets posted.
BOW will proof and set a general, viable paradigm to recapitulate key biomimetic functions – including camouflage to the immune system and organ site/tumor targeting – to any synthetic nanodevice, while being disruptive as a first example of biogenic nanotechnology.
If successful, such a non-incremental technology will promote the progress of implantable nanodevices and nanomaterials towards sustainable production and clinical translation, contributing to strengthen and keep in the lead position European biotechnology and impacting life quality for people.