A comprehensive work plan has been created to guide greenEV project research and training activities to achieve the overall aims. Chemical, biological, age and gender dependent nutritional characteristics of many phytochemicals have been studied (T1.1). D1.1 describes a set of high-end value substances that would highly benefit from encapsulation.
We have set up and characterized callus and CSCs of Ginkgo biloba L. and Solanium lycopersicum in WP1. The cell lines established within greenEV project are maintained at the host IBBR-CNR in Naples for the further implementation of greenEV actions beyond the project. These cell lines can be grown in conditions that allow the isolation of their secreted EVs in the cell cultures supernatant (T1.2). They are virus and pathogen free. EV yield have been optimized over the different stages of cell growth and at different cell densities.
Production of EVs from the two CSCs (WP2). We optimized buffer, temperature and differential ultracentrifugation conditions in order to increase EV yield and purity. We have set up protocols for the density gradient ultracentrifugation (DGUC), size exclusion chromatography (SEC) and tangential flow filtration (TFF) purification and separation of CSCs-derived EVs (T2.1). We have also used transformation genetics variety of tomato (GCR237) to maximize the production yield. The yield of CSC-derived EVs was: 0.38 µg/mL to 0.62 µg/mL EVs (expressed in protein amount) for mL of ginkgo cultures and somewhat lower in tomato CSC. This yield is comparable to the EV yield observed in mammalian cell culture. We have studied the physiochemical and morphological features of the CSC-derived EVs and PDNVs and collected CryoTEM, SEM, NTA, DLS and interferometry light microscopy data to prove the vesicle character of the samples (D2.1).
Molecular characterization: We have successfully integrated the optimized isolation and purification (T2.1) with the morphological, physical, molecular (T2.2) characterization methods. Proteins were identified and quantified using in-gel and in-solution digestions and label free shot-gun proteomics. A lipid profiles of nano and microvesicles were measured in T2.2. Biological, uptake and toxicological characterization of the CSCs-derived EVs are in progress, while we have obtained a solid data on tomato PNDVs (T2.3) (D2.2). Our actual setup is based on renewable batch suspension cell cultures (WP3). We have tested and confirmed the reproducibility of the production (D3.1). Moreover, we loaded ginkgo PNDVs isolated in task 2.1 with Astaxanthin using two different methods (D2.3 -D2.4).
Outcomes of greenEV project were maximized by our integrated exploitation and dissemination strategy during and over project lifetime. GeenEV produced promising results and these are 1.) Set-up a novel continues system for the isolation of ginkgo and tomato single cell type derived EVs; 2.) Employed an integrated analytical pipeline for the isolation, characterization of plant EVs; 3.) Exploited PDNVs as vehicles for delivery of a selected nutraceutical candidate 4.) setup nanoencapsulation of astaxanthin based on passive cargo loading and sonication.
At beginning of a greenEV project logo was created and used during and over project lifetime. Moreover, short description of geenEV project objectives was published at many websites as a press release at IBBR-CNR (host institute), Faculty of Health Sciences, University of Ljubljana, Slovenia and at Damanhour University websites. Research results were disseminated at conferences: ISEV-2021, EVIta symposiums 2021 and 2022, "Socratic lectures", 2020 and 2021, and 4th International Conference on “Plant Cell & Tissue Culture In Vitro 2022. Vienna, Austria. The MSCA researcher organized a workshop at Damanhour University where PI delivred a lecture. To reach wider public, geenEV has participated at the 9th Edition of the European Biotech Week in 2021.
