Periodic Reporting for period 3 - MIMIC-KeY (A KEY TO THE RATIONAL DESIGN OF EXTRACELLULAR VESICLES-MIMICKING NANOPARTICLES)
Reporting period: 2023-12-01 to 2025-11-30
Being able to target a specific tissue or cell type is the holy grail of targeted therapies such as drug delivery. This is still extremely challenging and the clinical applications of this approach are still limited. Nature offers examples of selective targeting, being cancer metastasis a particularly striking one. Specific types of cancer metastasize selectively in determined organs using extracellular vesicles (EVs) to a target tissue where these EVs can modify cellular behaviour.
In MIMIC-KEY project we have taken inspiration from the natural homing capabilities of tumor-derived EVs and we have synthesized fully artificial nanoparticles inspired to them with selective tissue targeting. Here cancer is not the disease to treat but a source of inspiration to build artificial EVs mimics able to fight other clinically unmet diseases, specifically metabolic bone disorders. These diseases comprise a subset of pathologies caused by an altered relationship between bone resorption and bone formation. It yields to altered bone structure and subsequent frequent fractures and bone malformation.
The ambitious MIMIC-KEY goal required two main steps:1) understanding the molecular features that determine selective tissue targeting of prostate cancer-derived EVs to the bone and 2) having the synthetic tool to mimic them in a clinically relevant formulation.
The breakthrough idea we explored and validated is the creation of artificial EV-mimics, using only essential building blocks (protein and lipids) responsible for tissue homing capabilities of natural EVs and amplifying their therapeutic functions. We have overcome the current limitations of natural EVs use (isolation, reproducible features, high cost of manufacturing). We have reported the preparation of EV-mimics, consisting in organosilica porous nanoparticles coated by special lipidic and proteins formulation, being colloidally stable, ease to prepare, reproducible, and able to target the bone tissue up to in-vivo settings.
In MIMIC-KEY project we have taken inspiration from the natural homing capabilities of tumor-derived EVs and we have synthesized fully artificial nanoparticles inspired to them with selective tissue targeting. Here cancer is not the disease to treat but a source of inspiration to build artificial EVs mimics able to fight other clinically unmet diseases, specifically metabolic bone disorders. These diseases comprise a subset of pathologies caused by an altered relationship between bone resorption and bone formation. It yields to altered bone structure and subsequent frequent fractures and bone malformation.
The ambitious MIMIC-KEY goal required two main steps:1) understanding the molecular features that determine selective tissue targeting of prostate cancer-derived EVs to the bone and 2) having the synthetic tool to mimic them in a clinically relevant formulation.
The breakthrough idea we explored and validated is the creation of artificial EV-mimics, using only essential building blocks (protein and lipids) responsible for tissue homing capabilities of natural EVs and amplifying their therapeutic functions. We have overcome the current limitations of natural EVs use (isolation, reproducible features, high cost of manufacturing). We have reported the preparation of EV-mimics, consisting in organosilica porous nanoparticles coated by special lipidic and proteins formulation, being colloidally stable, ease to prepare, reproducible, and able to target the bone tissue up to in-vivo settings.
The project is concluded with major advancements in both steps 1 and 2, mentioned above, thanks to a synergistic combination of groundbreaking and multidisciplinary approaches spanning from inorganic, organic and supramolecular chemistry, biophysics, optics, molecular and cell biology, proteomics, clinical translation and mathematical modelling in an excellent partnership throughout Europe.
In particular, the natural EVs from prostate cancer were isolated and deeply characterized, finding innovative approaches to understand their molecular features responsible for the targeting behaviour. Novel high-resolution biological techniques combining super-resolution microscopy and flow cytometry, proteomics analysis and multiscale simulations were developed and assessed to understand the targeting capabilities of natural EVs derived from tumor cells. Key proteins and relevant lipid compositions were found enabling to understand what are the key actors playing a role in natural EV homing capabilities, here in particular how EV from prostate cancer can target the bone, a typical site for metastasis. In MIMIC-KEY the bone is the target of a metabolic disease to be treated.
Concerning the preparation of EV mimics, we have set a full method for the rational design of these innovative nanoparticles. In our concept they consist of inorganic, breakable on demand nanocapsules, able to carry a relevant protein or enzyme, and embedded in a rationally designed lipid bilayer equipped with the key proteins or their derivatives for site selective targeting to the bone tissue. These artificial EV-mimics have smart features allowing a stimuli-responsive cargo release, a dynamic lipid bilayer shell and are decorated with targeting biomolecules, mimicking the functionalities of the natural EVs. Such EV-mimics have demonstrated to target both in vitro and in vivo the osteoclasts of the bone. These results will enable novel enzyme-based therapy against metabolic diseases.
The results of this research were disseminated to both the scientific audience, in dedicated topical conferences and scientific publications, and to the broad public, within scientific cafès, citizen visit to the labs or involved in dissemination events. The breakthrough idea of artificial EV-mimics, using only essential building blocks and amplifying their therapeutic functions, was also patented and is currently under study for its exploitation in relevant clinical setting considering different bone diseases.
In particular, the natural EVs from prostate cancer were isolated and deeply characterized, finding innovative approaches to understand their molecular features responsible for the targeting behaviour. Novel high-resolution biological techniques combining super-resolution microscopy and flow cytometry, proteomics analysis and multiscale simulations were developed and assessed to understand the targeting capabilities of natural EVs derived from tumor cells. Key proteins and relevant lipid compositions were found enabling to understand what are the key actors playing a role in natural EV homing capabilities, here in particular how EV from prostate cancer can target the bone, a typical site for metastasis. In MIMIC-KEY the bone is the target of a metabolic disease to be treated.
Concerning the preparation of EV mimics, we have set a full method for the rational design of these innovative nanoparticles. In our concept they consist of inorganic, breakable on demand nanocapsules, able to carry a relevant protein or enzyme, and embedded in a rationally designed lipid bilayer equipped with the key proteins or their derivatives for site selective targeting to the bone tissue. These artificial EV-mimics have smart features allowing a stimuli-responsive cargo release, a dynamic lipid bilayer shell and are decorated with targeting biomolecules, mimicking the functionalities of the natural EVs. Such EV-mimics have demonstrated to target both in vitro and in vivo the osteoclasts of the bone. These results will enable novel enzyme-based therapy against metabolic diseases.
The results of this research were disseminated to both the scientific audience, in dedicated topical conferences and scientific publications, and to the broad public, within scientific cafès, citizen visit to the labs or involved in dissemination events. The breakthrough idea of artificial EV-mimics, using only essential building blocks and amplifying their therapeutic functions, was also patented and is currently under study for its exploitation in relevant clinical setting considering different bone diseases.
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.
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.