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Extracellular vesicles from a natural source for tailor-made nanomaterials

Periodic Reporting for period 1 - VES4US (Extracellular vesicles from a natural source for tailor-made nanomaterials)

Reporting period: 2018-09-01 to 2019-08-31

Extracellular vesicles (EVs) have emerged as important minute entities made by cells to facilitate cell communication, mediate physiological processes or affect various pathological conditions such as the activation of immune response or the spread of cancer. Much research is currently devoted to exploiting the biotechnological potential of EVs as active carriers for different theranostic applications. Safe, efficient and specific nano-delivery systems are essential to the current therapeutic medicine, cosmetic and nutraceutics sectors. The ability to optimise the bioavailability, stability and targeted cellular uptake of a bioactive molecule while mitigating its toxicity, immunogenicity and off-target/side effects is of the utmost priority. Based on our foundation data and successful preliminary isolation and characterisation of EVs derived from an already identified natural source, VES4US aims at creating a fundamentally new bioprocessing approach to generate and functionalise EVs from a renewable biological source. VES4US will impact upon beneficiaries with short, medium and long-term timelines through the generation of tailor-made natural source-derived bionanovehicles for the targeted delivery of drugs and biocompounds in the context of future therapeutic, cosmetic or nutraceutical formulations.
The development of a new bioprocessing stream to use natural source-derived EVs as new generation nanomaterials is an inherently interdisciplinary activity that requires the combination of a variety of research fields and specialised skills, including the biology of the natural source under consideration, the bioengineering aspects of the process, the chemistry of the functionalisation reactions and the biomedical context of the applications. VES4US is highly interdisciplinary and involves team members coming from different geographical and cultural areas (Mediterranean and Balkan regions, northern and central Europe); this diversity is a very valuable feature that is positively influencing the development of the project. This interdisciplinary work is supported by an integrated research approach with partners from different disciplines and backgrounds jointly collaborating and learning from one another in the process of carrying out specific tasks. As such, in this first year of the project, the high interaction among work packages and participants has ensured the creation of a powerful and well-integrated new research team; this is one of the main results achieved so far.
In this first year of the project, the cultivation and subsequent screening of the selected strains in terms of bioactivity, content in high-value metabolites and capacity to release into their growth medium EVs have been performed and reported upon as deliverables, and the related results have established the foundations for the high-risk high-reward research activities planned in VES4US. Indeed, extracellular nanoparticles were successfully purified from the selected species and characterised using a range of biophysical and biochemical methods. The analyses permitted the selection of the strains best suited for subsequent isolation and purification of EV-like objects in later parts of the VES4US work programme. Further results allowed us to choose, optimise and scale-up the purification method for EVs (i.e. Tangential Flow Fractionation, TFF) to allow laboratory-scale production of the natural source-derived EVs. To overcome a potential pitfall in the application of the TFF method, the VES4US consortium also invested in the development of an innovative technology for EV purification, and within the first year of the project, the design strategy of this technology has been described. Similarly, VES4US has described the most promising approaches to be applied in the engineering of natural source-derived EVs. This functionalisation strategy can be directly applied to the EVs obtained from the selected natural source, without the need for parent cell engineering (metabolic labelling).
In parallel with the explosive growth of available studies and data on EVs derived from different types of cells, biofluids and tissues, the scientific community faces the urgent need for a standard and transparent way to report data, to refer to reproducible methodologies, or to deposit and search for common knowledge. Indeed, EV characterisation is needed as a starting point for the bioengineered exploitation of the natural source-derived EVs. In this context, VES4US has provided a list of Minimal Information required for Studies of Extracellular Vesicles (MISEV), specifically in the context of VES4US (VES4US-MISEV), for the not-yet-described natural source-derived EVs. VES4US-MISEV is based on the guidelines provided by the International Society for Extracellular Vesicles (MISEV2018) list which was further adapted for natural source-derived EVs on the basis of our considerations and experiments during the first year of the VES4US project.
The discovery of EVs as natural carriers of small functional molecules and proteins has raised great interest in the drug delivery field as it may be possible to harness these vesicles for the therapeutic delivery of miRNA, siRNA, mRNA, lncRNA, peptides and synthetic drugs. However, systemically delivered EVs accumulate in the liver, kidneys and spleen, and some mammalian-derived secreted EVs have shown, to date, limited pharmaceutical acceptability because of their origin (e.g. bovine milk-derived EVs). VES4US aims to overcome these limitations by developing a biocompatible and cost-effective delivery system based on natural source-derived EVs which would enhance bioavailability and improve the efficacy and safety of loaded bioactive compounds. Crucial elements of innovation can be identified at several levels. First, the choice of the natural source for the EV production system is a fully innovative approach which guarantees the production of materials from a fully renewable bio-source. Secondly, a production strategy is required which should integrate a series of cutting-edge technological innovations at each stage of the development process: selection of the strain(s), EV purification techniques, EV characterisation using novel biophysical tools (e.g. microfluidics), bioactivity tests on different model systems and functionalisation chemistry. Each of these aspects has been established individually in different contexts and the consortium partners have been selected to secure expertise in each distinct area of research and innovation. Building on this solid basis and our proof-of-concept and first year-results, VES4US proposes a high-risk high-gain programme to further develop the technological processes inherent to isolating EVs from the identified natural source and their subsequent functionalisation in the view to amalgamate them in a highly cooperative frontier research strategy. We propose an ambitious collaborative project that spans an array of biotechnology-oriented activities. VES4US will pave the way to a new generation of tailor-made natural source-derived bionanovehicles for the targeted delivery of drugs and biocompounds in the context of future therapeutics, cosmetics or nutraceutical formulations.
Graphical overview of the scientific objectives, WPs and integration of the partners