Biomanufacturing of Tomato-Derived Nanovesicles.

Ramesh Bokka,Samuele Raccosta,Giorgia Adamo,Tamás Csizmadia,Immacolata Fiume,Gabriella Pocsfalvi,Simon Sugár,Mauro Manno,Anna Paulina Ramos,Lilla Turiák

doi:10.3390/foods9121852

Abstract

Micro- and nano-sized vesicles (MVs and NVs, respectively) from edible plant resources are gaining increasing interest as green, sustainable, and biocompatible materials for the development of next-generation delivery vectors. The isolation of vesicles from complex plant matrix is a significant challenge considering the trade-off between yield and purity. Here, we used differential ultracentrifugation (dUC) for the bulk production of MVs and NVs from tomato (Solanum lycopersicum L.) fruit and analyzed their physical and morphological characteristics and biocargo profiles. The protein and phospholipid cargo shared considerable similarities between MVs and NVs. Phosphatidic acid was the most abundant phospholipid identified in NVs and MVs. The bulk vesicle isolates were further purified using sucrose density gradient ultracentrifugation (gUC) or size-exclusion chromatography (SEC). We showed that SEC using gravity column efficiently removed co-purifying matrix components including proteins and small molecular species. dUC/SEC yielded a high yield of purified vesicles in terms of number of particles (2.6 × 1015 particles) and protein quantities (6.9 ± 1.5 mg) per kilogram of tomato. dUC/gUC method separated two vesicle populations on the basis of buoyant density. Proteomics and in silico studies of the SEC-purified MVs and NVs support the presence of different intra- and extracellular vesicles with highly abundant lipoxygenase (LOX), ATPases, and heat shock proteins (HSPs), as well as a set of proteins that overlaps with that previously reported in tomato chromoplast.

Highlights

MVs and NVs were isolated from tomato fruit (Piccadilly variety) as red color pellets obtained by differential ultracentrifugation (dUC) after the 15,000× g and 100,000× g centrifugation steps, respectively (Figure 1)
Since size-exclusion chromatography (SEC) has not yet been applied to the purification of plant-derived vesicles, we aimed to compare the performance of SEC to the more frequently used gradient ultracentrifugation (gUC) method
Differential ultracentrifugation was used to prepare samples enriched in MVs and NVs

Summary

Introduction

The study of nanometer-sized vesicles (NVs) isolated from whole plants or plant organs has opened a new branch of research in the field of extracellular vesicles (EVs). [1,2,3] Several recent reports and review articles describe the successful isolation of NVs from a great variety of edible fruits and vegetables (Table 1) [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15]. Plant-derived vesicle isolates are more complex than mammalian cell-derived EVs and contain both intra- and extracellular vesicles [7]. There is a growing interest in advanced strategies for the production of NVs from plant resources due to their numerous promising applications, especially in the nutraceutical [6], cosmeceutical [8], and therapeutic fields [9]. Due to their inherent role in intracellular trafficking, native NVs are efficiently taken up by recipient cells to which they transfer their lipids, mRNAs, microRNAs, and protein biocargo [10,11]. Ginger-derived NVs were proven to be efficient against Alzheimer’s disease in a rat model [17]

Methods

Results

Conclusion