(452b) Poly(N-Isopropylacrylamide):Collagen Hydrogels for Tunable Syneresis and Drug Delivery

Poly(N-isopropylacrylamide; PNIPAM)-based hydrogels are used extensively in biomedical applications due to their thermoresponsive properties. Above 32°C, PNIPAM-based hydrogels exhibit a hydrophilic-to-hydrophobic switch that is associated with a contraction in volume (i.e. syneresis) as water is expelled from the hydrophobic polymer structures. Their utility may be enhanced by the integration of a natural polymer, such as collagen, that improves biocompatibility and enables modulation of hydrogel physico-chemical properties by variation of the copolymer ratio. The objective of this study was to fabricate hybrid PNIPAM:collagen hydrogels and investigate the effects of variable composition on temperature-dependent syneresis and protein release kinetics. Hydrogels (5% total mass/vol) were polymerized at 20°C with varying NIPAM monomer:collagen mass ratios (100:0-50:50). SEM microscopy was used to qualitatively examine the effects of composition on hydrogel microstructure. To evaluate syneresis, hydrogel volumes were measured at 37°C with digital calipers over four hours and related to initial volumes at 20°C. Pure PNIPAM hydrogels exhibited the most rapid syneresis upon exposure to increased temperature at 37°C, with the largest changes in volume occurring within in the first hour. The equilibrium swelling ratio, defined as the swollen volume to collapsed volume, varied from 16-1.5 for hydrogels with 100% to 50% PNIPAM content. Fluorescent bovine serum albumin (FITC-BSA) was used as a model protein to investigate release kinetics. BSA-loaded hydrogels were incubated in PBS at 20°C or 37°C, and BSA concentrations in solution were measured with fluorescence spectroscopy. Incremental increases in collagen content decreased both the rate of syneresis and the equilibrium volume change observed after 4 hours. Commensurate with decreased syneresis, the integration of collagen attenuated the initial release kinetics of BSA over one hour at 37°C. The extent of BSA release from PNIPAM hydrogels after 1 hour is significantly greater at 37°C relative to 20°C, owing to the rapid collapse of polymer conformation above 32°C. In contrast, incremental increased collagen content diminishes the temperature dependence of BSA release, which suggests that even small amounts of collagen moderate the thermosensitivity of PNIPAM. Hydrogels formulated with increased collagen (25-50%) further demonstrate a decrease in the extent of syneresis and a commensurate increase in the duration of BSA release. Specifically, 50:50 hydrogels exhibited sustained BSA delivery up to 30 days at 37°C, while pure PNIPAM hydrogels released the majority of the BSA load after several days. To evaluate biocompatibility, L929 fibroblasts were used as a model cell line and seeded onto collapsed hydrogels. Cytotoxicity was quantified after 24 hours at 37°C with a fluorescence-based assay that probes cell membrane integrity. Pure PNIPAM hydrogels showed 48% cell viability relative to controls plated on tissue culture plastic. Cell viability was improved significantly with 5% collagen, and both 25% and 50% collagen hydrogels showed similar levels of cell viability as controls. These results suggest that PNIPAM:collagen hydrogels support cell adhesion and culture, and may be useful for delivering bioactive macromolecules for directing specific cellular functions. Moreover, the rate and duration of drug delivery may be tuned by manipulation of the hydrogel copolymer ratio.