Introduction: Growth factor (GF) delivery has been widely investigated as a promising treatment for progressively degenerative diseases. However, GFs that act in a diffusible manner are generally unstable in their environment because of rapid inactivation prior to reaching their targets. Since tissue repair occurs over long time frames, continuous dosing at a minimum effective dose is required. Current GF delivery strategies are limited by the fragility and uncontrollable release of the proteins, and eventually result in low treatment efficacy. We have developed an affinity-based release system using transient interaction between the protein and versatile hydrogel matrix to sustain and control the release of therapeutics. This system is able to deliver a diversity of bioactive proteins with tunable rate of release under mild conditions.
Material and Methods: A polymeric matrix formed from hyaluronic acid (HA) and methylcellulose (MC) provides an injectable and fast gelling hydrogel delivery system for protein delivery. To achieve sustained release of proteins from the hydrogel, IGF-1 was expressed as fusion protein with the Src homology 3 domain (SH3) in E.coli, SH3-IGF-1. MC was immobilized with one of two SH3 peptide binding ligands, which has weaker or stronger affinity towards the SH3 domain. MC-SH3 peptide binding ligand was then combined with unmodified HA to form HAMC-ligand. Release of SH3-IGF-1 in different hydrogels was investigated in vitro under conditions that mimic the in vivo environment.
Results and Discussion:
Both HAMC-WBP and HAMC-SBP attenuated the rate of SH3-IGF-1 release in comparison to HAMC alone. After 24 hours, approximately 67% of SH3-IGF-1 was released from HAMC, 40% from HAMC-WBP and 19% from HAMC-SBP. HAMC and HAMC-WBP released SH3-IGF-1 for 4 days and 10 days, respectively. HAMC-SBP gels appeared to stop releasing SH3-IGF-1 after 24 h, with only small nanogram quantities of SH3-IGF-1 released over subsequent timepoints.
Release rates were compared using a plot of cumulative fractional protein release against the square root of time. The linear fit of the data is indicative of Fickian diffusion, and the slopes of the curves are proportional to the protein diffusivity within the gel. All k values were significantly different between HAMC (k = 2.59 x 10-3), HAMC-WBP (k = 1.35 x 10-3), and HAMC-SBP (k = 7.92 x 10-3). HAMC and HAMC-SBP had Fickian diffusion release for the first 8 h, while HAMC-WBP extended Fickian diffusion to 48 h.
Conclusions: Sustained release of therapeutics can make a dramatic improvement on the effectiveness of the delivery system. Herein, we showed that the release of the fusion protein formed by SH3 domain and the therapeutics can be controlled through physical binding interactions with a biomaterial matrix. The release profile is linear and tunable. By further investigation of the protein-ligand interaction, we are able to achieve tunable release of multiple proteins and at distinct rates of release.
Ontario Institute for Regenerative Medicine, OIRM; Canadian Institutes of Health Research, CIHR; Natural Sciences and Engineering Research Council of Canada, NSERC; Connaught Foundation Scholarship