METHODS: Dextrin was modified by succinoylation and conjugated to recombinant human EGF (rhEGF) using 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) coupling agents. The dextrin-EGF conjugate was purified by FPLC and characterised by SDS electrophoresis (free protein) and BCA protein assay (total protein). The ability of the polymer-EGF conjugate to induce cellular proliferation was examined in rhEGF-responsive human epidermoid carcinoma cells (Hep2) in vitro in both the presence and absence of alpha-amylase.
RESULTS: The conjugates prepared contained < 0.1% free rhEGF and had a total protein content of ~ 11 wt%. It was demonstrated that dextrin-rhEGF conjugates had reduced activity in the Hep2 model (compared to rhEGF alone), but upon polymer degradation by alpha-amylase, the biological activity of rhEGF was restored to 97% (p<0.0001). At physiological concentrations of alpha-amylase, activity was restored to 40-60% (p<0.02). A sustained release phenomenon is exhibited by the conjugate, as is an increase in the potency of the conjugated EGF.
CONCLUSION: These results demonstrate the potential of bioresponsive polymer therapeutics as a novel treatment for chronic periodontal disease and wound healing. Further investigations will utilise in vitro cellular wound healing assays (chronic wound fibroblasts and keratinocytes) to assess the potential of these conjugates to direct wound healing responses.
ACKNOWLEDGEMENTS: We would like to thank the Healing Foundation (UK) and the Welsh Office for Research and Development for their support.