Preparation of human epidermal growth factor/low-molecular-weight chitosan conjugates and their effect on the proliferation of human dermal fibroblasts in vitro

Abstract

Low-molecular-weight chitosan (LMC) having molecular weight of 10535 was obtained through acid hydrolysis of crude chitosan (CS-0) from shrimp shells and subsequent ultra-filtration. LMC was characterized by FT-IR, H-1 NMR spectroscopy and gel permeation chromatography (GPC). To stabilize the three-dimensional structure of the EGF moiety, EGF was attached to LMC using a water-soluble carbodiimide (EGF-LMC). The amount of EGF in EGF-LMC, after purification using dialysis tubing (molecular weight cut-off 12000 Da), was measured by indirect enzyme-linked immunosorbent assay (ELISA). Cytotoxicity and relative cell proliferation tests of with added EGF-LMC were performed on human dermal fibroblasts. No inhibitory effects of EGF-LMC were found during cell growth. EC2000 (EGF-LMC added with 0.338 nmol of EGF) appeared to have a stronger stimulatory effect than did E200 on fibroblast proliferation over a 96 h incubation period. Therefore, we believe that EGF-LMC is a potent wound-healing agent because of its mitogenicity and potent efficiency.