Periodic Reporting for period 1 - greenEV (Development of a novel continuous Extracellular Vesicle production system)
Período documentado: 2020-12-01 hasta 2022-11-30
Plants have shaped human history and society since the beginning of our joint evolution. With the emerging recognition of world population feeding, global climate change and limited non-renewable energy resources, the relevance of plant biology and biotechnology is becoming increasingly important.
In this context, MSCA greenEV project aimed to set the bases for the production, characterization and use of a novel type of nanocarrier that can be isolated from plant resources. A nanocarrier is nanomaterial being used as a transport system for another substance, such as a drug. Commonly used nanocarriers include liposomes, micelles, carbon-based materials, polymers and other synthetic substances. Bio-based materials with novel characteristics however are emerging. Amongst these, there are extracellular vesicles (EVs), biomembrane-enclosed tiny structures that are secreted by living cells. They are natural transporters of proteins, lipids, RNAs and other small molecules. EVs have recently been recognized as an important communication system between close and distant cells and even different organisms. Mammalian cell-derived EVs are under intense exploitation by pharmaceutical industries as high versatility nano-based delivery system. Upscaling of mammalian cell cultures for the EV production is however economically and technologically challenging. Moreover, green resources would better fit to the ethical and sustainable production of EVs with interesting market opportunities. Here comes the main idea of greenEV project to use plant cell suspension cultures (CSCs) for the production of EVs. We have built our project on the following important pillars:
1. Establishment of CSCs of two selected plant species in the beneficiary laboratory for the production of CSC-derived EVs;
2. Setup methods and protocols for the isolation of CSC-derived EVs and perform an in-depth biophysical and biocargo characterization;
3. Load the plant EVs with a selected high-end value natural biomolecule to demonstrate their applicability in pharmaceutical and cosmeceutical sectors.
GreenEV has selected two plant species for its research activities: Ginkgo biloba L. (ginkgo) tree and Solanium lycopersicum (tomato) plant. Ginkgo was chosen because of its importance in our human history and culture as well as in plant evolution. Ginkgo tree is living fossil often used in streets and parks for its attractive golden leaves in autumns, but it is also rich in substances with elevated healing properties. Tomato, on the other hand, is one of the most important vegetables for human nutrition and the most accessible natural source of lycopene.
GreenEV project has successfully achieved the above main and specific objectives through its 11 deliverables. The results achieved in greenEV have expanded the current knowhow in the plant-derived EV research field. The results obtained in greenEV show translational challenges and opportunities as well as encouraging perspectives towards biotechnological applications.
In this context, MSCA greenEV project aimed to set the bases for the production, characterization and use of a novel type of nanocarrier that can be isolated from plant resources. A nanocarrier is nanomaterial being used as a transport system for another substance, such as a drug. Commonly used nanocarriers include liposomes, micelles, carbon-based materials, polymers and other synthetic substances. Bio-based materials with novel characteristics however are emerging. Amongst these, there are extracellular vesicles (EVs), biomembrane-enclosed tiny structures that are secreted by living cells. They are natural transporters of proteins, lipids, RNAs and other small molecules. EVs have recently been recognized as an important communication system between close and distant cells and even different organisms. Mammalian cell-derived EVs are under intense exploitation by pharmaceutical industries as high versatility nano-based delivery system. Upscaling of mammalian cell cultures for the EV production is however economically and technologically challenging. Moreover, green resources would better fit to the ethical and sustainable production of EVs with interesting market opportunities. Here comes the main idea of greenEV project to use plant cell suspension cultures (CSCs) for the production of EVs. We have built our project on the following important pillars:
1. Establishment of CSCs of two selected plant species in the beneficiary laboratory for the production of CSC-derived EVs;
2. Setup methods and protocols for the isolation of CSC-derived EVs and perform an in-depth biophysical and biocargo characterization;
3. Load the plant EVs with a selected high-end value natural biomolecule to demonstrate their applicability in pharmaceutical and cosmeceutical sectors.
GreenEV has selected two plant species for its research activities: Ginkgo biloba L. (ginkgo) tree and Solanium lycopersicum (tomato) plant. Ginkgo was chosen because of its importance in our human history and culture as well as in plant evolution. Ginkgo tree is living fossil often used in streets and parks for its attractive golden leaves in autumns, but it is also rich in substances with elevated healing properties. Tomato, on the other hand, is one of the most important vegetables for human nutrition and the most accessible natural source of lycopene.
GreenEV project has successfully achieved the above main and specific objectives through its 11 deliverables. The results achieved in greenEV have expanded the current knowhow in the plant-derived EV research field. The results obtained in greenEV show translational challenges and opportunities as well as encouraging perspectives towards biotechnological applications.
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.
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.
In greenEV we have established callus and batch cell suspension cultures of ginkgo biloba and tomato cell lines for the continuous and reliable biomanufacturing of the ginkgo and tomato extracellular vesicles. We have characterized the ginkgo and tomato EVs and proved their vesicular characters. We have loaded PDNVs from ginkgo seed with astaxanthin. Plant CSC-derived EVs are promising alternatives to mammalian EVs for biotechnological exploitation.