In the 3 months of the project duration, several advances have been made on various fronts in the MSCA project NanoNAD. These include not only the scientific goals, but also the public dissemination goals and career development goals in terms of the development of professional network and grant-writing skills for the fellow. These advances evidence the fact that the project has been on track with the progress outlined by the goals set out for the first 3 months in the project proposal.
Nanoparticle loading and characterization
The fellow synthesized new silicon dioxide (silica, SiO2) nanoparticles using Flame Spray Pyrolysis (FSP). The morphological and physicochemical properties of the nanoparticles were studied using various techniques such as transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR) and nitrogen sorption. Then, the silica nanoparticles surface was further functionalized with the silane APTES enriching their surface with amine groups. The antioxidant compounds epigallocatechin gallate (EGCG) and its derivative, gallic acid (GA) were covalently immobilized on the surface of the functionalized silica nanoparticles by implementing EDC-NHS chemistry. The resulting nano-formulations were characterized using transmission electron microscopy (TEM) where the aggregated nature of the silica nanoparticles is revealed. Fourier-transform infrared (FTIR) spectra show bands attributed to organic groups at 1400-1700 cm-1 (inserts) due to the presence of the antioxidant molecules on the surface of silica nanoparticles. Moreover, the loading efficiency of EGCG and GA were measured using thermogravimetric analysis (TGA) and their antioxidant capacity through the DPPH assay. These characteristics of our novel formulations made using home-made particles were compared with the characteristics of those made using standard commercially available A200 (SiO2) nanoparticles. The amount of the loaded GA was calculated at about 10% and 15% w/w for the in-house and commercial silica nanoparticles, respectively. For the EGCG, the loading percentage is higher for both of the nanoformulations with the TGA revealing loading percentages at approximately 15% w/w on the in-house synthesized and 22% w/w on the commercial silica nanoparticles. DPPH assay showed the retention of the antioxidant capacity of EGCG and GA even after loading into the SiO2 nanoparticles.
NAD generation in retinas treated with novel compounds
Upon start of the project, NAD assays were performed on primary cortical neurons (PCNs) to screen 6 new compounds to identify the most effective NAD-generating candidates and their lowest effective concentrations. All the compounds showed significant NAD generating capacity in these cells with a general lowest effective concentration found to be about 50 nM.
Among these compounds, BC3 and BC10 were selected to test their NAD-generating capacity within the retina by injecting them intravitreally into mouse eyes at 1 mM concentration, and measuring NAD levels in the retinas. Unexpectedly, this revealed no changes in retinal NAD levels even in EGCG injected retinas, possibly because the RGCs were not stressed like the PCNs were upon isolation. When a similar stress of axotomy was induced to RGCs by culturing retinal explants, a model of retinal axotomy, with the compounds, there was a significant increase in NAD levels in the retinas treated with EGCG, but not in those treated with BC3 or BC10 indicating that although these compounds were effective in generating NAD in PCNs, this may not be the case in RGCs, under the tested conditions. This calls for further refinement of our compounds and/or employment of different testing conditions.
Already during the three months of the fellowship, the fellow acquired interdisciplinary knowledge, technical and soft skills in the fields of materials science, neuronal and retinal metabolism, and drug discovery and development, by working side by side with highly skilled researchers and scientists, and participating in seminars and symposia. The fellow was trained in several techniques novel to him including FSP, TEM, FTIR, TGA, DPPH assays, intravitreal injections, NAD assays and drug screening. In addition, he participated in seminars organized by the beneficiary institution and in (inter)national conferences expanding his network and attracting new collaborations. The fellow will also be a first author in a high impact publication arising out of the data from the progress in the project so far.
