DoE-Optimized Chitosan-Coated pH-Sensitive Liposomes for Targeted Nose-to-Brain Delivery of Temozolomide

Glioblastoma is an aggressive brain tumor with limited treatment efficacy due to poor blood-brain barrier (BBB) penetration and intrinsic resistance. Temozolomide (TMZ), the frontline chemotherapeutic, undergoes rapid hydrolysis at physiological pH, with low systemic bioavailability and efflux-mediated clearance. To address this, we developed chitosan-coated pH-sensitive liposomes (SpH-TMZ-CS-LIPO) that were optimized via Box-Behnken design, achieving 223.48 ± 1.93 particle size, 35.72 ± 0.51 zeta potential, and 78.85 ± 2.62% entrapment. The formulation exhibited pH-sensitive release, with cumulative TMZ release of 92-94% at pH 4.5-5.5, following diffusion-controlled kinetics. Ex vivo permeation across goat nasal mucosa at pH 5.5 showed a 1.39-fold increase in TMZ flux compared to Free TMZ, while mucin adsorption studies demonstrated superior mucoadhesion. In U87MG cells, SpH-TMZ-CS-LIPO showed 4.6-fold higher uptake than Free TMZ and markedly enhanced cytotoxicity, reducing viability to 3.92 ± 1.56% (IC₅₀: 0.396 ± 0.105 µg/mL; > 33-fold improvement), reflecting improved intracellular delivery and pH-responsive release. Following intranasal administration in rats, it reached a C_max of 260.82 ± 9.21 µg/mL in the brain and a brain AUC_₀₋∞ of 12,720.42 ± 252.44 µg/mL* h, > 22-fold higher than Free TMZ, with prolonged cerebral residence (MRT_₀₋∞: 36.52 h, t_₁/₂: 24.80 h) and high nose-to-brain targeting (DTI: 17.72; DTE: 177.2%; DTP: 94.3%). These effects reflect pH-sensitive CHEMS-mediated release and CS-induced mucoadhesion, ensuring optimized CNS accumulation with minimal systemic exposure. Overall, SpH-TMZ-CS-LIPO represents a rationally engineered, non-invasive nanocarrier that markedly enhances TMZ delivery, cytotoxicity, and brain-targeting efficiency, offering a promising strategy for glioblastoma therapy.