Injectable In Situ-Forming and Self-Healing Hydrogels and Micelles for Sustained Ocular Drug Delivery: From Design to In Vivo Preclinical Evaluation

Abstract

The work presented in this thesis aimed to develop and evaluate drug delivery systems suitable for the treatment of chronic posterior ocular diseases. To this end, hydrogel- and micelle-based delivery systems were designed and evaluated using in vitro and in vivo models to determine their physicochemical properties, drug release characteristics, cytocompatibility, ocular distribution, and safety. A thermosensitive hydrogel for sustained intraocular protein delivery was successfully developed using oxo-ester mediated native chemical ligation of two complementary ABA triblock polymers. The hydrogel formed under mild physiological conditions, allowing encapsulation of proteins while helping preserve their biological activity. Sustained release was demonstrated for different protein molecules, with release profiles dependent on the protein and polymer composition. Quantitative release of an anti-VEGF Fab fragment was achieved over 17 days. In vitro studies with retinal pigment epithelium cells showed no significant toxicity of either the polymer building blocks or the crosslinked hydrogel, demonstrating the suitability of this platform for ocular drug delivery. Diels–Alder core-crosslinked flower-like micelles were also investigated as a potential intraocular drug delivery system. The micelles exhibited favourable physicochemical properties, efficient cellular uptake, and good cytocompatibility in vitro. Following intravitreal injection in rats, the micelles were rapidly cleared from the vitreous, although some accumulation at the inner limiting membrane and limited transport towards the optic nerve were observed. No adverse effects or retinal abnormalities were detected in vivo. These findings indicate that the micelles have potential as intraocular drug carriers, but further optimisation is required to improve vitreous retention and cell-specific targeting. The most promising results were obtained with a self-healing thermosensitive hydrogel for sustained dexamethasone delivery. The hydrogel combined thermogelation with covalent crosslinking to produce an injectable formulation capable of recovering its structure after injection. In vitro, dexamethasone was released according to first-order kinetics through ester hydrolysis for more than 430 days without burst release. LC-MS analysis confirmed that dexamethasone was released predominantly in its native form, and cytocompatibility studies demonstrated no toxicity of the delivery system at clinically relevant concentrations. In vivo evaluation further confirmed the potential of this hydrogel. In rats, the hydrogel gradually degraded following intravitreal injection and was completely cleared within five weeks, with no adverse histological findings despite minor injection-related complications. In rabbits, dexamethasone was detected in aqueous humour for 57 days after a single intravitreal injection, with first-order release kinetics and therapeutic drug concentrations maintained throughout the study. The formulation was well tolerated, and no treatment-related adverse ocular effects were observed. Overall, the results demonstrate that injectable thermosensitive hydrogels are a promising platform for sustained intraocular drug delivery. The discrepancy between the in vitro and in vivo performance of the investigated delivery systems highlights the need for more versatile in vitro models that better mimic the vitreous environment during preclinical development. Collectively, these findings showed the potential of injectable hydrogels for sustained local release of small molecules and proteins.