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Conversion of natural plant nanovesicles into nutraceutical delivery system

Periodic Reporting for period 1 - nanoTOM (Conversion of natural plant nanovesicles into nutraceutical delivery system)

Reporting period: 2018-10-01 to 2020-09-30

Nano-scale encapsulation is an innovative strategy that is emerging for the bioavailability enhancement of poorly absorbed high-value bio-actives. In this context, the overall aim of nanoTOM is to exploits edible plant-derived nanovesicles as vehicles for the encapsulation, protection, release and bioavailability enhancement of selected nutraceuticals. NanoTOM focuses on nanovesicles from tomato (Solarium lycopersicum), a high commercial and nutritional value agricultural product.
Plants contain numerous types of membrane-surrounded nanometer sized vesicles that are morphologically similar to mammalian extracellular vesicles (EVs). EVs can transfer proteins, oligonucleotides, lipids and other metabolites to recipient cells, and have been shown to play important roles in cell-to-cell, interspecies and inter-kingdom communication. Edible plant-derived vesicles are efficiently up-taken by intestinal cells, non-toxic, naturally contains an array of bioactives, and thus represents a valid resource for nanovectors.
NanoTOM project has realized the following specific research objectives:
1. set-up an integrated analytical pipeline for the isolation, purification, characterization, encapsulation, uptake and toxicological profiling of plant-derived nanovesicles;
2. determined the physicochemical characteristics and biocargo composition of tomato-fruit and root exudate-derived nanovesicles by using the integrated analytical pipeline;
3. evaluated the toxicity and the bioactivity and encapsulation properties of the vesicles.
None of these objectives have been addressed before. The results achieved in nanoTOM expanded the current knowhow in the edible plant –derived nanovesicles research field. Moreover, the results obtained in nanoTOM shows encouraging perspective towards industrialization.
Work performed
An integrated work-plan has been used to guide the research and training towards the final goals:
Chemical, biological, age and gender dependent nutritional characteristics of several phytochemicals have been studied at the beginning of nanoTOM project. D1.1. describes a set of high-end value substances that we selected for encapsulation in future functional foods.
In nanoTOM we have studied three different sources for the production of S. lycopersicum vesicles: root, leaf and fruit. At the beginning of project, hydroponic culture was set up at IBBR-CNR in Portici to produce starting material for root EVs and leaf apoplastic vesicles (AVs). Two genotypes, Red Setter and MicroTom were studied. Apoplastic fluid (AF) was obtained by vacuum infiltration and centrifugation from leaf tissues. Tomato fruit (Piccadilly variant) was obtained from a local shop. (D1.2)
Production of micro and nanovesicles from S. lycopersicum was one of the main tasks all over the project. We set up different methods and optimized buffer conditions to maximize the production yield. Gradient ultracentrifugation (gUC) in continuous and stepwise modes and size exclusion chromatography (SEC) were used to purify nanovesicles from tomato fruit. The yield we obtained was considerably higher than the actual state-of-the-art using mammalian cell culture or other novel resources, like milk or microalgae. We have produced more than 10 mg MVs and nanovesicles from tomato fruit during project lifetime (100 times more than expected during the submission). (D1.3)
Established, TEM, AFM, SEM, NTA, DLS and innovative MRPS, BioSAXS physiochemical methods, were used to characterize morphology (TEM, SEM, AFM), particle size distribution (DLS, NTA, MRPS) and concentration (NTA and MRPS). (D2.1)
Molecular characterization: Proteins were identified and quantified using in-gel and in-solution digestions and label free shot-gun proteomics. A thin layer chromatography (TLC)-based lipid profiling was established at EVs-MS laboratory and lipid profiles of nano and microvesicles were generated. (D2.2)
Biological, uptake nutritional and toxicological characterizations was performed in collaboration with E. Di Schiavi (IBBR-CNR, Napoli) and M. Guescini (University of Urbino, Urbino). Off note is the set-up of C. elegans model system for the bioassay (motility, life span, toxicity and effect on the neurodegenerative disorders) of plant-derived vesicles at Dr. Di Schiavi lab. Antiproliferative activity, apoptosis, toxicity and anti-inflammatory effects of vesicles were assayed using THP-1 cells-based assays. D2.3-D2.5)
Development of a novel purification method: in collaboration with H2020-FETOpen VES4US project, we have worked on the setup of a novel scalable and renewable isolation method for the isolation/purification of nanovesicles (sensitive information) (D3.1-D3.3) moreover, exploratory experiments were performed to load nanovesicles isolated in Task 1.2 with curcumin using extrusion method. (D3.5-D3.7)
We have successfully integrated the newly developed isolation/purification method (T1.3) with a wide array of physiochemical (T2.1) molecular (T2.2) and T2.3 biological characterization methods. This comprehensive platform was used to produce and characterize tomato fruit derived nanovesciles. (D4.1)

Overview of the results and their exploitation and dissemination
nanoTOM produced exploitable results, and these are 1.) development of a scalable method for the isolation of tomato fruit nanovesicles, 2.) the biological and nutritional properties of natural and loaded nanovesicles (under exploitation) could be a real breakthroughs for nutra-, pharma- and cosmeceutical applications, 3.) development of a complex platform and expertise for the characterization of plant derived nanomaterials.
At project startup a logo was created and used during and over project lifetime. Moreover, short description of nanoTOM project objectives was published at IBBR-CNR webpage (https://ibbr.cnr.it//ibbr/info/people/gabriella-pocsfalvi).
Dissemination of the research results: Conferences where results of nanoTOM were presented: ISEV-2019 conference, 1st EVIta symposium-2019, FSciT-2019, World Summit on Advancement in Food Science and Technology, 1st and 2nd VES4US-NA seminar series, 2nd VES4US-NA seminar series, organized together with VES4US H2020- FetOpen project, 26-February 2020, Napoli, Italy
Workshops we participated: Super resolution microscopy: Live demonstration; Nanobiomedicine in Naples: The next future of theranostics, Road show 2019 organized by Beckmann Coulter
One open access paper has been published in peer reviewed high impact journals that describes the intriguing interplay between SARS-19 and extracellular vesicles (https://doi.org/10.1159/000511402) and we two other manuscripts on tomato root exudate and tomato fruit-derived nanovesicles are under review. To reach wider public, nanoTOM has participated FutureTechWeek, 2019, Global European Biotech week 2019 and Futuro Remoto 4.0.
Tomatoes are the major dietary source of the antioxidant lycopene and have been largely consumed and studied because of their health benefits. In nanoTOM for the first time have we have produced nanovesicles from three different parts of S. lycopersicum plant: root, leaf and fruit. An integrated platform was set up for the reliable production and characterization of nanovesicles from tomato fruit. The obtained yield is very promising. Tomato-derived nanovesicles could be used as future delivery vector for the formulation of next generation functional foods.