Development and in vivo evaluation of novel bioresponsive nanocubosomal in-situ gelling system for ophthalmic delivery of Amphotericin B.

Amphotericin B (AmB), the drug of choice for fungal keratitis, displays poor aqueous solubility, physiological instability, and pH-dependent aggregation causing ocular irritation. This study aimed to develop novel reconstitutable bioresponsive Nanocubosomal in situ gelling system (NCG) of AmB to address the above-mentioned limitations. Initially, 13 batches of Nanocubosomal Dispersion (NCD) were formulated using Quality by Design-driven central composite design with glyceryl monooleate as lipid, poloxamer 407 as surfactant, and dimethyl sulfoxide as solvent, employing high-shear homogenization and ultrasonication. The optimised NCD exhibited nanoscale size (158.9 ± 2.6 nm), low polydispersity (0.162 ± 0.007), high entrapment efficiency (91.2 ± 1.1%), and colloidal stability (zeta potential -32.8 ± 1.3 mV). Due to low pH-solubility (0.2 ± 0.03 µg/mL) at pH 7.4 and moderate lipophilicity (log P: 0.46 ± 0.01), AmB would get preferentially localised in lipid domain. The optimised batch of NCD was finally lyophilised with Carbopol 934 (0.3%w/v) and mannitol (5%w/v) before gamma sterilisation to obtain NCG. NCG demonstrated bioresponsive behaviour, maintaining low pre-instillation viscosity (12 ± 0.9 mPa.s) while instantly transforming into resilient pseudoplastic gel (22,230 ± 310 mPa.s) at ocular pH that is likely to prolong ocular retention. The in-vitro drug release from NCG at 24 h was found to be significantly lower compared to marketed lyophilised Amfocare® (p < 0.0001), indicating sustained release behaviour. NCG displayed significantly enhanced (p < 0.01) antifungal activity against Candida albicans compared to Amfocare®. Draize testing in rabbits ruled out possibility of ocular irritation, demonstrating improved tolerability of NCG. Overall, NCG, by its ability to maintain AmB in monomeric form, represents a promising, stable, effective, irritation-free in situ gelling lipid-based platform for treatment of fungal keratitis.