**Background:** Glaucoma is a group of eye disorders characterized by progressive optic nerve neuropathy, often associated with elevated intraocular pressure (IOP). Rho-kinase (ROCK) inhibitors, such as Fasudil hydrochloride, represent a novel class of anti-glaucoma agents that not only lower IOP by increasing aqueous humor outflow through the trabecular meshwork but also improve ocular blood flow and protect optic nerve cells from oxidative stress. However, Fasudil's hydrophilic nature (log P 0.16) and low molecular weight (327.83 g/mol) pose challenges for ocular delivery, leading to low bioavailability when formulated as simple eye drops. Chitosan (Cs) nanoparticles offer mucoadhesive and permeation-enhancing properties that could improve ocular drug delivery. This study aimed to investigate the ocular absorption pathway of Fasudil, develop an optimized Cs nanoparticulate formulation, and evaluate its performance and biocompatibility.
**Methods:** Ex vivo permeation of Fasudil solution (1 mg/mL) through excised bovine cornea and sclera was studied using Franz diffusion cells with simulated tear fluid (STF, pH 7.4) at 35 ± 0.5 °C over 6 hours. A two-level full factorial design (3 factors, 8 runs plus center point) was employed to optimize Cs nanoparticles prepared by ionic gelation, varying Cs concentration (1.2, 3.7, 6.2 mg/mL), Cs:TPP mass ratio (1:1, 3:1, 5:1), and sonication time (45, 67.5, 90 s). Nanoparticles were characterized for particle size, polydispersity index (PDI), zeta potential, and entrapment efficiency (EE%). In vitro release was conducted in STF at 35 °C for 24 h using dialysis membrane. Ex vivo trans-corneal permeation of optimized Cs NPs was compared to Fasudil solution. Ocular tolerability was assessed via HET-CAM test (conjunctival irritation) and bovine corneal opacity and permeability (BCOP) assay. Cytotoxicity was evaluated on human lens epithelial cells (HLEC B-3) using neutral red uptake assay at concentrations from 1×10⁻⁵ to 1 mg/mL.
**Key Results:** Ex vivo permeation showed significantly higher Fasudil permeation through sclera than cornea: at 2 h, 109.1 µg/cm² (sclera) vs. 14.4 µg/cm² (cornea); at 6 h, 370.0 µg/cm² vs. 96.8 µg/cm². Flux and Papp were 70.4 ± 3.93 µg/cm²·h and 11.5 ± 0.64 ×10⁻⁶ cm/s for sclera vs. 20.6 ± 2.24 µg/cm²·h and 3.4 ± 0.37 ×10⁻⁶ cm/s for cornea (p < 0.05). The optimized Cs NPs (Cs concentration 1.2 mg/mL, Cs:TPP ratio 1:1, sonication 45 s) had particle size 297.0 ± 13.4 nm, PDI 0.21 ± 0.034, zeta potential 17.6 ± 2.3 mV, and EE 32.0 ± 3.2%. In vitro release showed initial burst (35.51% at 2 h) followed by sustained release (55.6% at 6 h, ~60% at 24 h), fitting Higuchi (R²=0.9460) and Korsmeyer–Peppas (R²=0.9650, n=0.35) models, indicating Fickian diffusion. Ex vivo trans-corneal permeation of Cs NPs showed 2.1-fold higher flux (43.2 ± 1.75 vs. 20.6 ± 2.24 µg/cm²·h) and Papp (7.1 ± 0.31 vs. 3.46 ± 0.37 ×10⁻⁶ cm/s) compared to Fasudil solution (p < 0.05). HET-CAM test yielded cumulative score < 0.9 (non-irritant) for Cs NPs, similar to saline. BCOP assay cumulative score was ≤ 0.5 (non-irritant). Neutral red assay showed >94% cell viability at Cs NP concentrations up to 1×10⁻³ mg/mL; at 1 mg/mL, viability decreased to 75% (p < 0.05).
**Clinical Implications:** The study demonstrates that Fasudil permeates predominantly via the scleral route, but the corneal barrier limits its absorption. Chitosan nanoparticles significantly enhance corneal permeation (2.1-fold) and provide sustained drug release, potentially reducing dosing frequency and improving patient compliance in glaucoma therapy. The excellent ocular tolerability (non-irritant to conjunctiva and cornea) and low cytotoxicity at therapeutic concentrations support the safety of this formulation. These findings suggest that Fasudil-loaded chitosan nanoparticles could be a promising alternative to conventional eye drops for glaucoma management, though further in vivo studies are needed to confirm efficacy and safety.