ABSTRACT Diabetic retinopathy is a leading cause of blindness in the industrialized world and has a global prevalence of an estimated 95 million people (1). Proliferative diabetic retinopathy (PDR) and diabetic macular edema (DME) originate from persistently elevated glucose levels leading to microvascular ischemia, retinal neovascularization (RNV), and vascular leakage (2, 3). Antibodies and therapeutics designed to sequester free vascular endothelial growth factor (VEGF) are the current standard of care (4-8). Due to the short half-life of anti-VEGF therapies, monthly intravitreal injections are needed to maintain remission (4-7). Repeat injections risk intraocular inflammation, infection, and ocular hemorrhage (9). An alternative approach is to use RNA interference (RNAi) to silence the expression of the pathogenic proteins. The recent advancements in siRNA modifications (10) and FDA approval of the third siRNA therapeutic in as many years demonstrate the renewed potential of RNAi, though like other anti-VEGF therapies the duration of action is a key limitation. We propose to evaluate the feasibility of intracellular RNA therapeutics delivered by intravitreal injection of a nanoparticle carrier to inhibit neovascularization and extend the duration of therapeutic efficacy substantially relative to current treatments. We recently demonstrated the effectiveness of intravitreally administered fusogenic porous silicon nanoparticles (F-pSiNPs) for VEGF-siRNA delivery in a DL-alpha-aminoadipic acid (DL-AAA) rabbit model of RNV. This project aims to rigorously test and optimize this system for extended efficacy. In Aim 1, we will evaluate two methods of siRNA loading into the nanoparticles: calcium silicate condensation and grafting of cyclic silanes. These systems will be optimized for loading capacity, encapsulation efficiency, and in vitro release kinetics. The fusogenic-lipid membrane coating of the F-pSiNP system will be optimized using extrusion and solvent exchange methods. The candidate formulations will be characterized by spectroscopic, DLS, and Cryo- EM methods. Cellular uptake and duration of action will be validated in vitro using RT-qPCR and flow cytometry. The most promising formulations will then be tested by intravitreal injection in Aim 2 using VEGF-siRNA and Ang-2-siRNA payloads in the DL-AAA model of RNV (11, 12). F-pSiNPs will be given as a single dose and monitored for 6 months for changes to vascular leakage using fluorescein angiography. These results will then be benchmarked against commercially available antibody therapeutics aflibercept and faricimab. In Aim 3, F- pSiNP formulations tested in Aim 2 will be conjugated with pendent surface peptides to test the hypothesis that selective cellular targeting may dramatically improve efficacy. The targeting and internalization peptide iRGD will be used for these studies. iRGD is currently in clinical development to improve chemotherapeutic uptake in tumors, and was selected for its...