CQA and DoE guided development of brimonidine tartrate loaded polymeric nanoparticles for sustained ocular delivery and in vitro neuroprotective evaluation in glaucoma

Glaucoma is a progressive neurodegenerative disorder characterized by retinal ganglion cell (RGC) loss, in which oxidative stress and neuronal damage play key roles beyond intraocular pressure (IOP)-dependent mechanisms. Conventional therapies are limited by poor ocular bioavailability, rapid precorneal elimination, and insufficient neuroprotective efficacy. In this study, brimonidine tartrate (BT)-loaded polymeric nanoparticles (NPs) were developed as a multifunctional ocular delivery system, guided by critical quality attributes (CQAs) and a supportive design of experiments (DoE)-based optimization approach to ensure optimal physicochemical performance. The optimized NP formulation (V-BT-NP) exhibited nanoscale size (∼180 nm), acceptable polydispersity, and a positively charged surface, supporting colloidal stability and potential ocular retention. The formulation provided sustained drug release over 30 h, with release kinetics best described by the Peppas–Sahlin model, indicating a diffusion-dominated multi-mechanistic release profile. Biological evaluation using R28 retinal cells demonstrated that V-BT-NP improved cellular responses under oxidative and excitotoxic stress conditions compared to free BT. The NP system improved cell viability and metabolic activity while providing comparatively improved cytoprotective effects. In addition, cell-free antioxidant studies demonstrated moderately improved radical scavenging activity following NP encapsulation. Importantly, gene expression analysis revealed partial restoration of β-III-tubulin (TUBB3) expression, a marker associated with neuronal cytoskeletal integrity, consistent with potential neuroprotective effects at the molecular level. Sterility assessment further confirmed that 0.20 μm membrane filtration ensured microbiological safety, supporting the suitability of the developed system for ocular applications. Overall, this study demonstrates that CQA- and DoE-guided NP systems integrating sustained drug release and cytoprotective cellular responses represent a promising approach for ocular delivery in glaucoma management. The findings suggest that such nano-enabled strategies may contribute to improved ocular drug delivery and cellular resilience in retinal neurodegenerative conditions.