Within this project, the consortium has greatly advanced the current state of the art knowledges from different perspectives. From a biological point of view, proteins that are essential for EVs tropism to specific bone tissues have been discovered. The novel methodology which enables to identify them is per se a discovery and has been fully set within the project course. High resolution microscopy and flow cytometry techniques have been combined together in an unprecedent hgh-resolution technique, allowing the innovative discovery of the proteins ansd lipid features at EVs surface. The same techniques are also available for the characterization of the novel EV mimics, to evaluate their physical chemical properties, as well as their targeting and cargo transport functionalities.
The consortium has also explored the dynamics of phospholipids once self-assembled in a lipidic bilayer as a vesicle or on a top of hard surface with and without sharp edges or high curvatures, like on small nanoparticles. These results have greatly advanced the knowledge of which physical and chemical parameters are affecting the lipid behavior, fluidity, dynamics and rearrangements of lipid in the shell and from the inner and the outer leaflets.
Concerning the formulation of EV-mimics, different solutions were studied and set, allowing to produce smart porous and breakable nanoparticles being highly reproducible even in high scale production, colloidally stable in aqueous and biological media up to in vivo environment, and reacting to specific endogenous stimuli. Lipids formulations fairly mimicking natural EVs composition were developed at increasing levels of complexity, enabling to achieve high tropism towards bone tissues. As a whole, the developed EV-mimics are core-shell nanoparticles, able to transport proteins or other biomolecules within the porous breakable core, while the shell is made by peculiar lipids and proteins or protein fractions fully mimicking the natural EV attitude to target bone tissue. These results have been demonstrated up to in vivo setting, resulting in a stimuli-responsive nanosized platform with all the key and essential components to mimic the natural EVs.
Ad-hoc in vitro and in vivo models have been established and specifically set to test the efficacy of the proposed approach towards bone metabolic diseases.
It is therefore clear the importance of the proposed research in different fields, from academic and scientific to industrial and socio-economic ones. First of all, the impact on healthcare is highly important, being the proof of concept of the present project designated to solve a rare disease and try to cover the gap over un unmet clinical need. Furthermore, the discovery on the key features enabling metastatic potential in EVs will advance enormously the knowledge on cancer metastasis and related diagnostic and therapeutic approaches. From a scientific point of view, the consortium is confident to have developed a new class of advanced smart and biomimicking nanotools, opening new horizons in nanomedicine and in many adjacent fields.
From an industrial point of view, the production of standardized, purified, clinical grade artificial EV-mimics will positively impact on the pharmaceutical and biomedical industries. In a future perspective, the impact on the nano- and personalized medicine and the clinical translation is highlighted, but also the benefit for the society with the generation of new trends in pharmacology. The test-bed EV-mimics can also enable the extension of the design principles, as well as the imaging and analysis techniques to other nano-objects (e.g. proteins assembly, virus-like particles, polymeric or surfactant micelles), to develop a diverse library of materials and transfer concepts to corresponding disciplines, such as biophysics, polymer physics, synthetic chemistry, and nonequilibrium statistical mechanics.
The newly enabled technology can impact beyond clinical applications in the markets of cosmetics, paints and composite formulations.
In general, the proposed research can in future bring to vast new job opportunities for highly skilled specialists in data analytics, modelling, synthetic chemistry, biology and translational medicine.
