Bioactive hydrogels demonstrate mediated release of a chromophore by chymotrypsin

A model system, α-chymotrypsin (Cht) (a protease) and a cleavable peptide-chromogen (pro-drug) covalently incorporated into a hydrogel, was investigated to understand the mechanisms of covalent loading and release by enzymatic cleavage in bio-responsive delivery systems. Using EDC and Sulfo-NHS, terminal carboxyl groups of N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide, a cleavable chromogen, were conjugated to primary amines of a hydrated poly(HEMA)-based hydrogel. Hydrogel disks were incubated in buffered Cht causing enzyme-mediated cleavage of the peptide and concomitant release of the chromophore for monitoring. To investigate substrate loading and the effects of hydrogel morphology on the system, the concentration of the amino groups (5, 10, 20, and 30 mol%) and the cross-linked density (1, 5, 7, 9 and 12 mol%) were independently varied. Loading-Release Efficiency of the chromogen was shown to exhibit a positive relation to increasing amino groups (AEMA). The release rates demonstrated a negative relation to increasing cross-linked density attributed to decreasing void fractions and increasing tortuosities. The diffusion coefficient of Cht, D _0,Cht, was determined to be 6.9 ± 0.5 × 10 ^{- 7} cm ² s ^{- 1}, and the range of D _eff of Cht for 1 to 12 mol% TEGDA was determined to be 6.9 × 10 ^{- 8} to 0.1 × 10 ^{- 8} cm ² s ^{- 1}. We show how these parameters may be optimized and used to achieve programmed release rates in engineered bio-responsive systems.