The activities performed to achieve the main objectives of the first year of DAFNE are divided in two main work packages (WPs) with a secondment period of five months carried out at the Instituto de Nanociencia y Materiales de Aragón (INMA) hosted by Bionanosurf group for the synthesis of the NMs. All the activities foreseen are listed below:
1) WP1: a) Design of siRNA for the plant species Arabidopsis thaliana (At) and Nicotiana benthamiana (Nb) to target common reporter genes, including phytoene desaturase (PDS), whose silencing induces leaf bleaching, and green fluorescent protein (GFP)); b) synthesis and characterisation of alginate nanoemulsions in a core/shell structure water in oil in water (W/O/W) using homogenisation, prilling and vortexing, c) synthesis and characterisation of Au nanoparticles (AuNPs) of about 14 nm functionalised with 25% PEG coverage (50% of SH-EG(8)-(CH2)2-COOH and 50% of SH-(CH2)3-CONH-EG(6)-(CH2)2-N3); d) synthesis and characterisation of chitosan nanovesicles and chitosan polyplexes (CHPx) using vortexing methods; e) conjugation of siRNA on AuNPs and CHPx. The conjugation efficiency was evaluated using Ultraviolet-visible spectroscopy (UV-vis), dynamic light scattering (DLS) and gel electrophoresis.
2) WP2: a) phytotoxicity tests performed using the materials synthesized in WP1 upon different administration routes (e.g. root uptake, seed priming and leaf spray/infiltration); b) RNA extraction, RT-PCR and qRT-PCR to evaluate at the molecular level the effect of the silencing; c) optical microscopy to measure the stomata density, foliar and root uptake of the NMs; d) fluorescent microscopy and confocal microscopy to study the root uptake of chitosan nanovesicles; e) computed tomography (CT) used to study the interaction of NMs with the plant cell tissue in vivo using Zeiss Xradia 510 system on At leaves. This work was supported by the National Research Facility for Lab X-ray CT (NXCT) at the µ-VIS X-ray Imaging Centre, University of Southampton, through EPSRC grant EP/T02593X.
