Periodic Reporting for period 1 - NanoNAD (Nanoparticle-mediated ocular delivery of novel NAD-boosting small molecules for ophthalmic disease.)
Periodo di rendicontazione: 2025-04-01 al 2027-03-31
Glaucoma is a common neurodegenerative disease affecting ~80 million people worldwide and is the leading cause of irreversible blindness. It is characterized by the progressive degeneration and loss of retinal ganglion cells (RGCs), the output neurons of the retina. Age, genetics, and high intraocular pressure (IOP) are major risk factors. Current treatment strategies only target IOP lowering. However, 42% of treated patients go blind in at least one eye, demonstrating the need for alternative neuroprotective therapies.
Several studies including ours show that NAD decline is one of the earliest changes occurring in glaucoma patients and animal models, both systemically and within the retina. Restoring NAD levels through supplementation have been found to be neuroprotective in both animals and humans.
In NanoNAD, we aim to develop an ocular delivery of nano-formulations containing novel neuron-specific NAD-boosting small molecules that can protect the RGCs. Such nano-formulations would also allow for a sustained release of these compounds enabling fewer doses as opposed to multiple daily doses that current treatments require. Finally, success of NanoNAD in glaucoma would have therapeutic implications for other more common neurodegenerative diseases like Alzheimer’s and Parkinson’s diseases, due to their shared disease mechanisms.
Several studies including ours show that NAD decline is one of the earliest changes occurring in glaucoma patients and animal models, both systemically and within the retina. Restoring NAD levels through supplementation have been found to be neuroprotective in both animals and humans.
In NanoNAD, we aim to develop an ocular delivery of nano-formulations containing novel neuron-specific NAD-boosting small molecules that can protect the RGCs. Such nano-formulations would also allow for a sustained release of these compounds enabling fewer doses as opposed to multiple daily doses that current treatments require. Finally, success of NanoNAD in glaucoma would have therapeutic implications for other more common neurodegenerative diseases like Alzheimer’s and Parkinson’s diseases, due to their shared disease mechanisms.
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.
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.
The activities from the project so far led to the production of SiO2 nanoparticles in large scale using Flame Spray Pyrolysis, generation and characterization of our novel nano-formulations containing EGCG and GA.
The NAD generating capacity of 6 novel small molecules was confirmed in PCNs using luminometric NAD assays. Then, the capacity to generate NAD in RGCs was tested for two of these compounds (BC3 and BC10) by injecting them intravitreally or incubating them with the retinal explant model of glaucoma. Although BC3 and BC10 have been effective in inducing NAD production in primary cortical neurons, they were not so effective in achieving this in RGCs. This is important information regarding the translation of our results from PCNs to RGCs, allowing us to employ appropriate approaches in choosing the right candidates involving the use of stressed RGC models to test the NAD-generating capacities. This understanding is relevant beyond just our goals, as it informs future translational studies involving such models.
The NAD generating capacity of 6 novel small molecules was confirmed in PCNs using luminometric NAD assays. Then, the capacity to generate NAD in RGCs was tested for two of these compounds (BC3 and BC10) by injecting them intravitreally or incubating them with the retinal explant model of glaucoma. Although BC3 and BC10 have been effective in inducing NAD production in primary cortical neurons, they were not so effective in achieving this in RGCs. This is important information regarding the translation of our results from PCNs to RGCs, allowing us to employ appropriate approaches in choosing the right candidates involving the use of stressed RGC models to test the NAD-generating capacities. This understanding is relevant beyond just our goals, as it informs future translational studies involving such models.