Development of a thermosensitive hydrogel for sustained drug release and in situ bleb support

Authors: Richard Zhang1, James J. Armstrong1, Cindy M. L. Hutnik2. 1Western University, 2Ivey Eye Institute.

Author Disclosure Block: R. Zhang: None. J.J. Armstrong: None. C.M.L. Hutnik: None.

Abstract Title: Development of a thermosensitive hydrogel for sustained drug release and in situ bleb support.

Abstract Body:

Purpose: Anti-fibrotic agents such as mitomycin C are used to limit post-operative scarring of filtration blebs in glaucoma treatment. It has become popular to inject such anti-metabolites in the subconjunctival space in the immediate pre-operative period to modulate wound healing. However, aqueous drug preparations are rapidly dispersed and thus do not provide lasting physical support for the bleb or reproducible drug delivery, likely contributing to inconsistent and unpredictable results. Chitosan-based thermosensitive hydrogels are liquid at room temperature but gelatinous at physiological temperatures. The purpose of this study was to engineer a thermosensitive hydrogel for subconjunctival injection to serve as a sustained-release anti-fibrotic drug depot and to provide physical support for the bleb. Study Design: Experimental materials synthesis, in vitro biocompatibility and in vitro efficacy experiments using primary human Tenon's capsule fibroblasts from patients with glaucoma. Methods: Hydrogels were prepared by dissolving chitosan in 2% acetic acid, dialyzing to remove acetic acid, and adding beta-glycerophosphate (β-gp). Various recipes were tested for gelation time vs temperature using the tube inversion test. Biocompatibility was tested by culturing human Tenon's capsule fibroblasts on chitosan gels at 37°C. Resistance to flow was tested by measuring afferent pressure when perfusing a gel-filled flow chamber with saline at the rate of aqueous humor production (2.6μL/min). Drug release was tested by measuring acetylsalicylic acid (ASA) or bovine serum albumin (BSA) in the effluent when perfusing drug-loaded gels with saline at 2.6 μL/min. Results: Gelation time of chitosan/β-gp solutions is inversely related to temperature and the concentration of β-gp added to the gel. 5% and 6% β-gp mixtures gelled the quickest at 37°C. MTT and LDH assays showed that chitosan hydrogels have no significant effect on cellular metabolic activity or necrosis compared to vehicle controls. Perfusion of the hydrogel demonstrated a reproducible, transient increase in afferent pressure of 5.01 mmHg compared to an empty control that resolved by 9 hours. ASA and BSA were successfully loaded into chitosan gels and perfusion with saline elicited a delayed release of ASA/BSA. Conclusions: Chitosan/β-gp hydrogels solidify at eye-surface temperatures, are biocompatible with ocular fibroblasts, do not substantially impede flow, and can be loaded with small molecules or proteins to act as a delayed-release drug depot. These properties support the potential subconjunctival injection of chitosan to provide sustained drug-release and structural support for the bleb.