Biomanufacturing of Tomato-Derived Nanovesicles

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.

Extracellular Vesicles from Plants: Current Knowledge and Open Questions

“…HSP60 is found in PDEVs from sunflower, as well as HSP70, which has also been described in PDEVs from several citrus fruits [30] where HSP80 and HSP90 have been also found [4,32]. In a recent study of EVs isolated from tomato by size exclusion chromatography techniques, high levels of HSPs were found, together with lipoxygenase and ATPases [33]. Finally, proteins belonging to the Aquaporin family were found in PDEVs from citrus [4,30,34] and grape [3].…”

Identification of Tomato Infecting Viruses That Co-Isolate with Nanovesicles Using a Combined Proteomics and Electron-Microscopic Approach

“…NVs were isolated from tomato fruits (250–300 g) by ultracentrifugation, as previously described [ 14 ]. Briefly, tomatoes were washed with MilliQ water, and exocarp was removed by placing the tomatoes into hot boiling water for a couple of seconds.…”

“…Crude tomato NVs sample isolated by the differential centrifugation procedure was fractionated by dUC, using sucrose or OptiPrep (iodixanol) gradient solutions. Sucrose gUC was performed according to the procedure described in Reference [ 14 ], by under layering the sample with 8, 30 and 45% ( w / v ) sucrose cushions in a 38.5 mL polypropylene tube and centrifuging at 100,000× g for 2 h, at 4 °C, and by using a SW28Ti rotor (Beckman Coulter, Brea, CA, USA) in a Beckman Coulter Optima L-90K ultracentrifuge. Six fractions were collected from top to bottom.…”

“…Cell-derived submicron particles isolated from plant resources [ 1 , 2 , 3 , 4 , 5 , 6 , 7 ] are gaining attention both as complex biomaterials with health-promoting effects [ 8 , 9 ] and as delivery vectors for exogenous substances [ 10 ]. Nanometer-sized vesicles (NVs) have been isolated from many different plants, such as ginger [ 6 , 7 , 11 ], carrots [ 12 ], citrus species [ 4 , 13 ], grapes [ 1 , 2 ], tomato [ 1 , 14 ], blueberry [ 1 ], coconut [ 1 ], broccoli [ 15 ], wheat [ 16 ], etc., and even dried plant material [ 5 ]. Several of these isolates were shown to have anticancer [ 3 , 17 , 18 ], anti-inflammatory [ 7 , 11 , 15 ] or anti-senescence [ 19 ] activities in vitro or in vivo.…”

“…A recent work shows that NVs can be obtained with a high yield from tomatoes, using a combination of differential centrifugation (dUC) and size-exclusion chromatography (SEC) methods [ 14 ]. Tomato ( Solanum lycopersicum L.) is one of the leading vegetable crops worldwide.…”