Preparation of Human Epidermal Growth Factor/Low-Molecular-Weight Chitosan Conjugates and Their Effect on the Proliferation of Human Dermal Fibroblasts in vitro

Tae Il Son; Se Hoon Park; Hahk Soo Kang; Young Suk Son; Chun Ho Kim; Eui-Chan Jang

Journal of Industrial and Engineering Chemistry, Vol.11, No.1, 34-41, January, 2005

Tae Il Son^†, Se Hoon Park, Hahk Soo Kang, Young Suk Son¹, Chun Ho Kim¹, and Eui-Chan Jang²

Department of Biotechnology, University of Chungang, Kyoungki-Do, Korea
¹Laboratory of Tissue Engineering Korea Institute of Radiological and Medical Sciences, Korea
²Department of Orthopaedic Surgery, College of Medicine, University of Chungang, Seoul, Korea

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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, 1H 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 hincubation period. Therefore, we believe that EGF-LMC is a potent wound-healing agent because of its mitogenicity and potent efficiency.

Keywords:Low-molecular-weight chitosan;EGF;EDC condensation;Indirect ELISA;Human dermal fibroblasts

[References]

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Carpenter G, Cohen S, J. Cell Biol., 71, 159 (1976)
Kim DC, Sugiama Y, Fuwa T, Sakamoto S, Ica T, Hanano M, Biochem. Pharmacol., 38, 241 (1989)
Jogensen PE, Regul. Pept., 23, 161 (1988)
MateoDeAcosta C, Justiz E, Skoog L, Lage A, Anticancer Res., 9, 87 (1989)
Scott-Robson S, Capala J, Carlsson S, Malmborg P, Lundqovist H, Int. J. Appl. Rad. Isotop. (1990)
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Chang K, Lee J, Fu WR, J. Food Drug Anal., 75 (2000)
Wriston JC, Febs Lett., 33, 1 (1973)
Kohn J, Wilchek M, Appl. Biochem. Biotechnol., 9, 285 (1984)
Kohn J, Lenger R, Wilchek M, Appl. Biochem. Biotechnol., 8, 227 (1983)
Blomhoff HK, Biochem. Biophys. Acta., 743, 401 (1983)
Marshall JJ, Trends Biochem. Sci., 3, 79 (1978)
Melton RG, Wiblin CN, Foster RF, Biochem. Pharmacol., 36, 105 (1987)
Qin CQ, Du YM, Xiao L, Polym. Degrad. Stabil., 76, 211 (2002)
Howling GI, Dettmar PW, Goddard PA, Hampson FC, Dornish M, Wood EJ, Biomaterials, 22, 2959 (2001)
Hirai A, Odani H, Nakajima A, Polym. Bull., 26, 87 (1991)
Varum KM, Anthonsen MW, Grasdalen H, Smidsrd O, Carbohydr. Res., 211, 17 (1991)
Varum KM, Otty MH, Smidsrd O, Carbohydr. Polym., 46, 89 (2001)
Kratz G, Anander C, Swedenborg J, Back M, Falk C, Gouda I, Larm O, Scand. J. Plast. Reconstr. Surg. Hand Surg., 31, 119 (1997)
Kratz G, Anander C, Larm O, Scand. J. Plast. Reconstr. Surg. Hand Surg., 32, 381 (1998)

[Referenced By]

[1] Gottschalk A, Glycoproteins, 2nd Edn., Elsevier, Amsterdam (1972)

[2] Lee SY, Lee SJ, Jung HT, J. Ind. Eng. Chem., 9(1), 9 (2003)

[3] Um SH, Lee JB, Choi JW, Koo KK, J. Ind. Eng. Chem., 9(3), 243 (2003)

[4] Pazur HJ, Knull HR, Simpson DL, Biochem. Biophys. Res. Commun., 40, 1 (1970)

[5] Stowell CP, Lee YC, Adv. Carbohydr. Chem. Biochem., 37, 225 (1980)

[6] Andersson A, Holmberg A, Carlsson J, Ponten J, Westermark B, Int. J. Cancer, 47, 439 (1991)

[7] Cawley DB, Herschman HR, Cell, 22, 563 (1980)

[8] Carpenter G, Cohen S, Ann. Rev. Biochem., 48, 193 (1979)

[9] Laato M, Kahari VM, Niinikoski J, Vuorio E, Biochemistry, 247, 385 (1987)

[10] Nanney LB, Clin Res., 35, 706A (1987)

[11] Carpenter G, Ann. Rev. Biochem., 56, 881 (1987)

[12] Carpenter G, Cohen S, J. Cell Biol., 71, 159 (1976)

[13] Kim DC, Sugiama Y, Fuwa T, Sakamoto S, Ica T, Hanano M, Biochem. Pharmacol., 38, 241 (1989)

[14] Jogensen PE, Regul. Pept., 23, 161 (1988)

[15] MateoDeAcosta C, Justiz E, Skoog L, Lage A, Anticancer Res., 9, 87 (1989)

[16] Scott-Robson S, Capala J, Carlsson S, Malmborg P, Lundqovist H, Int. J. Appl. Rad. Isotop. (1990)

[17] Lee MY, Var F, Shin-ya Y, Kajiuchi T, Yang JW, Process Biochem., 34(5), 493 (1999)

[18] Chang K, Lee J, Fu WR, J. Food Drug Anal., 75 (2000)

[19] Wriston JC, Febs Lett., 33, 1 (1973)

[20] Kohn J, Wilchek M, Appl. Biochem. Biotechnol., 9, 285 (1984)

[21] Kohn J, Lenger R, Wilchek M, Appl. Biochem. Biotechnol., 8, 227 (1983)

[22] Blomhoff HK, Biochem. Biophys. Acta., 743, 401 (1983)

[23] Marshall JJ, Trends Biochem. Sci., 3, 79 (1978)

[24] Melton RG, Wiblin CN, Foster RF, Biochem. Pharmacol., 36, 105 (1987)

[25] Qin CQ, Du YM, Xiao L, Polym. Degrad. Stabil., 76, 211 (2002)

[26] Howling GI, Dettmar PW, Goddard PA, Hampson FC, Dornish M, Wood EJ, Biomaterials, 22, 2959 (2001)

[27] Hirai A, Odani H, Nakajima A, Polym. Bull., 26, 87 (1991)

[28] Varum KM, Anthonsen MW, Grasdalen H, Smidsrd O, Carbohydr. Res., 211, 17 (1991)

[29] Varum KM, Otty MH, Smidsrd O, Carbohydr. Polym., 46, 89 (2001)

[30] Kratz G, Anander C, Swedenborg J, Back M, Falk C, Gouda I, Larm O, Scand. J. Plast. Reconstr. Surg. Hand Surg., 31, 119 (1997)

[31] Kratz G, Anander C, Larm O, Scand. J. Plast. Reconstr. Surg. Hand Surg., 32, 381 (1998)