Authors: James J. Armstrong, Richard Zhang, Liu Hong, Cindy M. L. Hutnik

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

Abstract Body:

Purpose: The subconjunctival injection of anti-metabolites prior to glaucoma microstent insertion has become a popular technique. Essentially, this “pre-forms” the filtration bleb allowing the insertion of the microstent into the preformed bleb, ab interno. Currently, aqueous preparations of Mitomycin C are used. However, aqueous solvents cause rapid dispersion of drugs and provide no lasting physical support to the bleb walls - contributing to inconsistent results. Thermosensitive chitosan hydrogels transform from an injectable liquid state at room temperature, into a gelled state at temperatures approximating the subconjunctival space. The purpose of this study was to engineer a thermosensitive hydrogel for injection into the subconjunctival physiological environment to serve as a depot for sustained drug delivery after glaucoma surgery.

Study Design: Experimentalmaterials synthesis, in vitro biocompatibility and in vitro efficacy experiments using glaucoma patient derived Tenon’s capsule fibroblasts.

Methods: High molecular weight chitosan was solubilized at various concentrations in 1% acetic acid. Next, the solution was dialyzed against 300 volumes of double-distilled H₂O to remove the remaining acetic acid. Beta-glycerophosphate (β-GP) was then added dropwise, on ice, until a pH of 7, 7.1 and 7.2 was reached and samples collected. Tube inversion tests within a heated water bath were used to assess gelation time vs temperature. Acetylsalicylic acid (ASA) was loaded into gel samples as a test compound. A perfusion system was used to perfuse PBS through ASA-loaded chitosan gels at the rate of aqueous humor production (2.6ul/min) in order to estimate the release of ASA over time under physiological conditions. Human Tenon’s capsule fibroblasts were cultured on chitosan gels at 37°C to assess cytotoxicity, as well as in collagen matrices to evaluate cell-mediated collagen contraction as a measure of in vitro scarring activity.

Results: Chitosan and β-GP solutions of pH 7, 7.1 and 7.2 were prepared successfully as described. Tube inversion tests revealed that time to gelation and the temperature of gelation were adjustable by pH. Samples of pH 7.2 underwent gelation the quickest, in under two minutes at temperatures ranging from 34 to 40°C. ASA was successfully loaded into the chitosan gels and exhibited a delayed release profile upon perfusion. Cytotoxic effects of the gel on human Tenon’s capsule fibroblasts were minimal and ASA loaded gels inhibited cell-mediated collagen contraction compared to vehicle control.

Conclusions: Chitosan and beta-glycerophosphate hydrogels possess thermosensitive characteristics and can deliver small molecule therapeutics such as ASA in a delayed release manner. These properties would make chitosan-based drug delivery systems highly desirable for glaucoma surgery, specifically for bleb formation and stabilization.