PLGA NPs' cell uptake involves different endocytic pathways. Clathrin-independent endocytosis is the main internalization route. The cell wall plays a more prominent role than the plasma membrane in NPs' size selection. In the last years, many studies on absorption and cell uptake of nanoparticles by plants have been conducted, but the understanding of the internalization mechanisms is still largely unknown. In this study, polydispersed and monodispersed poly(lactic-co-glycolic) acid nanoparticles (PLGA NPs) were synthesized, and a strategy combining the use of transmission electron microscopy (TEM), confocal analysis, fluorescently labeled PLGA NPs, a probe for endocytic vesicles (FM4-64), and endocytosis inhibitors (i.e., wortmannin, ikarugamycin, and salicylic acid) was employed to shed light on PLGA NP cell uptake in grapevine cultured cells and to assess the role of the cell wall and plasma membrane in size selection of PLGA NPs. The ability of PLGA NPs to cross the cell wall and membrane was confirmed by TEM and fluorescence microscopy. A strong adhesion of PLGA NPs to the outer side of the cell wall was observed, presumably due to electrostatic interactions. Confocal microscopy and treatment with endocytosis inhibitors suggested the involvement of both clathrin-dependent and clathrin-independent endocytosis in cell uptake of PLGA NPs and the latter appeared to be the main internalization pathway. Experiments on grapevine protoplasts revealed that the cell wall plays a more prominent role than the plasma membrane in size selection of PLGA NPs. While the cell wall prevents the uptake of PLGA NPs with diameters over 50 nm, the plasma membrane can be crossed by PLGA NPs with a diameter of 500-600 nm.

Poly(lactic-co-glycolic) acid (PLGA)-\ud based NPs are currently considered among the most\ud promising drug carriers, nevertheless their use in\ud plants has never been investigated. In this work, for the\ud first time, we demonstrated the ability of PLGA NPs to\ud cross the plant cell wall and membrane of Vitis\ud vinifera cell cultures and grapevine-pathogenic fungi.\ud By means of fluorescence microscopy, we established\ud that PLGA NPs can enter in grapevine leaf tissues\ud through stomata openings and that they can be\ud absorbed by the roots and transported to the shoot\ud through vascular tissues. TEM analysis on cultured\ud cells showed that NPs B 50 nm could enter cells,\ud while bigger ones remained attached to the cell wall.\ud Viability tests demonstrated that PLGA NPs were not\ud cytotoxic for V. vinifera-cultured cells. The cellular\ud uptake of PLGA NPs by some important grapevinepathogenic\ud fungi has also been observed, thus suggesting\ud that PLGA NPs could be used to deliver\ud antifungal compounds within fungal cells. Overall the\ud results reported suggest that such NPs may play a key\ud role in future developments of agrobiotechnologies, as\ud it is currently happening in biomedicine

Polymeric nanoparticle-based carriers are promising agents to deliver drugs to cells. Vitis vinifera phenolic compounds are known for their antifungal activity against Candida albicans. The aim of the present study was to investigate the antifungal activity of pterostilbene or crude extracts from non-fermented grape pomace, entrapped in poly(lactic-co-glycolic) acid nanoparticles (NPs), with diameters of 50 and 150 nm, on Candida biofilm. The fluorescent probe coumarin 6 was used to study the uptake of poly(lactic-co-glycolic)acid (PLGA) NPs in planktonic cells and biofilm. The green fluorescent signal of coumarin 6 was observed in Candida biofilm after 24 and 48 hours. Both pterostilbene and crude pomace extract entrapped in NPs exerted a significantly higher anti-biofilm activity compared to their free forms. The entrapment efficiency of both pterostilbene and crude pomace extract in PLGA NPs was ~90%. At 16 µg/mL, pterostilbene loaded in PLGA NPs reduced biofilm formation of 63% and reduced mature biofilm of 50%. Moreover, at 50 µg/mL, the pomace extract loaded in NPs reduced mature biofilm of 37%. These results strongly suggest that PLGA NPs are promising nanodevices for the delivery of antifungal drugs as the crude grape pomace extract, a by-product of white wine making.

The objective of the present work was to synthesize biopolymeric nanoparticles (NPs) entrapping the resistance-inductor methyl jasmonate (MeJA) to be employed as a novel and alternative strategy in integrated pest management. NPs were prepared by using a continuous flow microfluidic reactor that allows to precisely control some features that are crucial for applications such as size, polydispersion, morphology and reproducibility. Poly(lactic-co-glycolic acid) (PLGA), a biopolymer largely studied for its use in biological applications, was chosen for the production of NPs entrapping MeJA, a biotic endogenous elicitor able to trigger plant’s defense responses. The effect of different fluid-dynamic conditions, PLGA molecular weight and concentration on NP properties (dimensions, polydispersion, morphology, stability) was evaluated. DLS and SEM were employed to characterize the obtained NPs. MeJA-loaded PLGA NPs ranging from 40 to 70 nm were administered to Vitis vinifera cell cultures, in order to evaluate the biological response in terms of stilbene biosynthesis. HPLC investigations showed a faster response when the elicitor was administered by PLGA NPs in comparison with free MeJA. This result demonstrates that the encapsulation in PLGA NPs significantly promotes MeJA cell uptake and the activation of MeJA-induced responses.

Vitis rupestris is used as rootstock or to obtain hybrids with Vitis vinifera, due to its resistance to certain pathogens. Its resistance mechanisms are poorly understood, while it is known that stilbene neo-synthesis is a central defense strategy in V. vinifera. In the present study, the response to methyl jasmonate (MeJa) and light treatment in terms of stilbene biosynthesis and the expression of genes involved in polyphenol biosynthesis was investigated in V. vinifera and V. rupestris cells. The two species exhibited a similar constitutive stilbene content [2.50-2.80 mg g dry weight (DW)], which greatly increased in response to elicitation (8.97-11.90 mg g DW). In V. vinifera, continuous light treatment amplified the effect of MeJa, with a stilbene production that had never previously been obtained (26.49 mg g DW). By contrast, it suppressed the effect of MeJa in V. rupestris. Gene expression was consistent with stilbene production in V. vinifera, whereas discrepancies were recorded in V. rupestris that could be explained by the synthesis of stilbenes that had never before been analyzed in this species.

Several human skin diseases are associated with fungi as dermatophytes and Malassezia. Skin mycoses are increasing and new alternatives to conventional treatments with improved efficacy and/or safety profiles are desirable. For the first time, the anti-dermatophytes and the anti-Malassezia activities of Vitis vinifera seed extracts obtained from different table and wine cultivars have been evaluated. Geometric minimal inhibitory concentration ranged from 20 to 97 µg/mL for dermatophytes and from 32 to 161 µg/mL for Malassezia furfur. Dried grape seed extracts analyzed by HPLC/DAD/ESI/MS showed different quali-quantitative compositions in terms of monomeric and polymeric flavan-3-ols. The minimal inhibitory concentrations for Trichophyton mentagrophytes and for M. furfur were inversely correlated with the amount of the polymeric fraction (r = -0.7639 and r = -0.7228, respectively). Differently, the antifungal activity against T. mentagrophytes was not correlated to the content of flavan-3-ol monomers (r = 0.2920) and only weakly correlated for M. furfur (r = -0.53604). These results suggest that extracts rich in polymeric flavan-3-ols, recovered from V.  vinifera seeds, could be used for the treatment of skin fungal infections. Copyright © 2016 John Wiley & Sons, Ltd.

In the last decades, the interest in biological activity of natural compounds has been growing. In plant protection, essential oils have been reported to exhibit antiviral, antimycotic, and antiparasitic activities, and are regarded as promising for the formulation of safe antimicrobial agents. Attention has also been focused on hydrosols, the by-products of hydro-distillation of essential oils. Their production is easy, fast, and cheap, and they seem to arise less concern for human health than essential oils. Plant viruses represent a major concern for agricultural crops since no treatment compound is available for virus control. This work was aimed at evaluating the antiphytoviral effectiveness of treatments with three essential oils and corresponding hydrosols extracted from Origanum vulgare, Thymus vulgaris, and Rosmarinus officinalis on Cucurbita pepo plants infected by zucchini yellow mosaic virus or tomato leaf curl New Delhi virus. Treatments were applied either concurrently or after virus inoculation to ascertain an inhibition or curative activity, respectively. Symptoms were observed and samplings were performed weekly. Virus titer and expression levels of phenylalanine ammonia lyase gene (PAL) were measured on treated and untreated infected plants by real-time PCR. PAL gene plays an important role in plant defense response as it is involved in tolerance/resistance to phytopathogens. Results indicated that treatments were effective against tomato leaf curl New Delhi virus whether applied simultaneously with the inoculation or after. A major inhibition was observed with O. vulgare essential oil and hydrosol, resulting in 10–4-fold decrease of virus titer 3 weeks after treatment. Curative activity gave maximum results with all three essential oils and T. vulgaris and R. officinalis hydrosols, recording from 10–2-fold decrease to virus not detected 4 weeks after treatment. An induction of PAL gene expression was recorded at 12 d.p.i. and then was restored to the levels of untreated control. This allows to hypothesize an early plant defense response to virus infection, possibly boosted by treatments. Plant extracts’ composition was characterized by gas chromatography-mass spectrometry. Phenols were largely main components of O. vulgare and T. vulgaris extracts (carvacrol and thymol, respectively), while extracts from R. officinalis were based on monoterpene hydrocarbons (essential oil) and oxygenated monoterpenes (hydrosol).

After the delineation of this methodology, the compliance to a routine practice allowed us to reduce time, stress, and cost; quality and quantity of possible research increased.