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Abstract Due to the fast-changing global climate, conventional agricultural systems have to deal with more unpredictable and harsh environmental conditions leading to compromise food production. The application of phytonanotechnology can ensure safer and more sustainable crop production, allowing the target-specific delivery of bioactive molecules with great and partially explored positive effects for agriculture, such as an increase in crop production and plant pathogen reduction. In this study, the effect of free pterostilbene (PTB) and poly(lactic-co-glycolic) acid (PLGA) nanoparticles (NPs) loaded with pterostilbene was investigated on Solanum lycopersicum L. metabolism. An untargeted NMR-based metabolomics approach was used to examine primary and secondary metabolism whereas a targeted HPLC–MS/MS-based approach was used to explore the impact on defense response subjected to anti-oxidant effect of PTB, such as free fatty acids, oxylipins and them impact on hormone biosynthesis, in particular salicylic and jasmonic acid. In tomato leaves after treatment with PTB and PLGA NPs loaded with PTB (NPs + PTB), both NPs + PTB and free PTB treatments increased GABA levels in tomato leaves. In addition, a decrease of quercetin-3-glucoside associated with the increase in caffeic acid was observed suggesting a shift in secondary metabolism towards the biosynthesis of phenylpropanoids and other phenolic compounds. An increase of behenic acid (C22:0) and a remodulation of oxylipin metabolism deriving from the linoleic acid (i.e. 9-HpODE, 13-HpODE and 9-oxo-ODE) and linolenic acid (9-HOTrE and 9-oxoOTrE) after treatment with PLGA NPs and PLGA NPs + PTB were also found as a part of mechanisms of plant redox modulation. To the best of our knowledge, this is the first study showing the role of PLGA nanoparticles loaded with pterostilbene in modulating leaf metabolome and physiology in terms of secondary metabolites, fatty acids, oxylipins and hormones. In perspective, PLGA NPs loaded with PTB could be used to reshape the metabolic profile to allow plant to react more quickly to stresses.

Abstract Botrytis cinerea, responsible for grey mold diseases, is a pathogen with a broad host range, affecting many important agricultural crops, in pre and post harvesting of fruits and vegetables. Commercial fungicides used to control this pathogen are often subjected to photolysis, volatilization, degradation, leaching, and runoff during application. In this context, the use of a delivery system, based on poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) represents an innovative approach to develop new pesticide formulations to successfully fight B. cinerea infections. In order to study NPs uptake, B. cinerea conidia and mycelium were treated with PLGA NPs loaded with the high fluorescent probe coumarin 6 (Cu6-PLGA NPs) and analyzed under ApoTome fluorescence microscopy. The observations revealed that 50 nm Cu6-PLGA NPs penetrated into B. cinerea conidia and hyphae, as early as 10 min after administration. Pterostilbene, a natural compound, and fluopyram, a synthetic antifungal, were entrapped in PLGA NPs, added to B. cinerea conidia and mycelium, and their antifungal activity was tested. The results revealed that the compounds loaded in NPs exhibited a higher activity against B. cinerea. These results lay the foundations for the use of PLGA NPs as a new strategy in plant pest management.

Abstract Keratinocytes, the main cell type of the skin, are one of the most exposed cells to environmental factors, providing a first defence barrier for the host and actively participating in immune response. In fact, keratinocytes express pattern recognition receptors that interact with pathogen associated molecular patterns and damage associated molecular patterns, leading to the production of cytokines and chemokines, including interleukin (IL)-6. Herein, we investigated whether mechanical energy transported by low intensity ultrasound (US) could generate a mechanical stress able to induce the release of inflammatory cytokine such IL-6 in the human keratinocyte cell line, HaCaT. The extensive clinical application of US in both diagnosis and therapy suggests the need to better understand the related biological effects. Our results point out that US promotes the overexpression and secretion of IL-6, associated with the activation of nuclear factor-κB (NF-κB). Furthermore, we observed a reduced cell viability dependent on exposure parameters together with alterations in membrane permeability, paving the way for further investigating the molecular mechanisms related to US exposure.

Abstract Ultrasound (US) induced transient membrane permeabilisation has emerged as a hugely promising tool for the delivery of exogenous vectors through the cytoplasmic membrane, paving the way to the design of novel anticancer strategies by targeting functional nanomaterials to specific biological sites. An essential step towards this end is the detailed recognition of suitably marked nanoparticles in sonoporated cells and the investigation of the potential related biological effects. By taking advantage of Synchrotron Radiation Fourier Transform Infrared micro-spectroscopy (SR-microFTIR) in providing highly sensitive analysis at the single cell level, we studied the internalisation of a nanoprobe within fibroblasts (NIH-3T3) promoted by low-intensity US. To this aim we employed 20 nm gold nanoparticles conjugated with the IR marker 4-aminothiophenol. The significant Surface Enhanced Infrared Absorption provided by the nanoprobes, with an absorbance increase up to two orders of magnitude, allowed us to efficiently recognise their inclusion within cells. Notably, the selective and stable SR-microFTIR detection from single cells that have internalised the nanoprobe exhibited clear changes in both shape and intensity of the spectral profile, highlighting the occurrence of biological effects. Flow cytometry, immunofluorescence and murine cytokinesis-block micronucleus assays confirmed the presence of slight but significant cytotoxic and genotoxic events associated with the US-nanoprobe combined treatments. Our results can provide novel hints towards US and nanomedicine combined strategies for cell spectral imaging as well as drug delivery-based therapies.

Abstract Among different therapeutic applications of Ultrasound (US), transient membrane sonoporation (SP) - a temporary, non-lethal porosity, mechanically induced in cell membranes through US exposure - represents a compelling opportunity towards an efficient and safe drug delivery. Nevertheless, progresses in this field have been limited by an insufficient understanding of the potential cytotoxic effects of US related to the failure of the cellular repair and to the possible activation of inflammatory pathway. In this framework we studied the in vitro effects of very low-intensity US on a human keratinocyte cell line, which represents an ideal model system of skin protective barrier cells which are the first to be involved during medical US treatments. Bioeffects linked to US application at 1 MHz varying the exposure parameters were investigated by fluorescence microscopy and fluorescence activated cell sorting. Our results indicate that keratinocytes undergoing low US doses can uptake drug model molecules with size and efficiency which depend on exposure parameters. According to sub-cavitation SP models, we have identified the range of doses triggering transient membrane SP, actually with negligible biological damage. By increasing US doses we observed a reduced cells viability and an inflammatory gene overexpression enlightening novel healthy relevant strategies.