Background: With growing age and the development of metabolic diseases, the incidence of fundus diseases, including age-related macular degeneration (AMD), pathological myopia, and diabetic retinopathy, has risen sharply, often resulting in the formation of abnormal neovascularization and damage to normal fundus tissue. Anti-vascular endothelial growth factor (VEGF) therapy has emerged as the standard treatment for neovascular fundus diseases. Although the emergence of small interfering RNA (siRNA) drugs targeting VEGF offers a new treatment option for patients with ocular vascular diseases, the requirement of frequent injection and potential safety challenges associated with their administration impose substantial burdens and risks to patients.

Objective: This study aims to develop a simple and straightforward siRNA-based formulation for intraocular injection and assess its efficacy and safety profile.

Methods: This study developed siRNA-based formulations, evaluated the VEGF inhibitory effect, and Half-maximal Inhibitory Concentration (IC₅₀) values at the mRNA level using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell viability and proliferation activity were determined using the cell counting kit-8 (CCK-8). Furthermore, the angiogenic capability of the cells was assessed using a tube formation assay. Additionally, a laser-induced mouse choroidal neovascularization (CNV) model was applied to investigate the therapeutic effect of siRNA on retinal neovascularization in vivo, and intraocular safety was evaluated using slit lamp examination and hematoxylin and eosin (H&E) staining of retinal tissue sections.

Results: siRNA formulation effectively silenced VEGF expression in human umbilical vein endothelial cells (HUVECs), indicating an mRNA inhibition rate of >85%. The treatment substantially inhibited HUVEC proliferation (compared to the control group: at 24 hours post-administration, siRNA 75 nM and 100 nM groups, p < 0.05; at 48 hours, siRNA 50 nM group, p < 0.05, and siRNA 75 nM and 100 nM groups, p < 0.005; at 72 hours, siRNA 50 nM group, p < 0.01, siRNA 75 nM and 100 nM groups, p < 0.005). Furthermore, siRNA formulations significantly suppressed angiogenesis compared to the control group (siRNA 50 nM, p < 0.05; siRNA 75 nM and 100 nM, p < 0.01). Moreover, after a single intravitreal injection, VEGF expression suppressed for up to 5 weeks, along with reduction in neovascularization area compared to the negative control group: at day 7 after administration, medium dose, p < 0.05 and high dose, p < 0.01; at day 21 after administration, low-, p < 0.05, medium dose and high-dose, p < 0.005; at day 35 post-administration, low-dose, p < 0.05, medium dose, p < 0.01, and high-dose, p < 0.005. Notably, no significant retinal toxicity was observed in normal rabbits after 3 months of vitreous injection.

Conclusion: This study confirms the therapeutic potential of VEGF-targeting siRNA formulations for retinal diseases, emphasizing their significance in promoting the clinical translation of siRNA-based therapies in ophthalmology.