Chemical Modifications of Natural Polymers and Their Technological

Chapter 4 Chemical Modifications of Natural Polymers and Their Technological Relevance Shalaby W. Shalaby and Kishore R. Shah Downloaded by STANFORD UNIV GREEN LIBR on April 9, 2013 | http://pubs.acs.org Publication Date: July 18, 1991 | doi: 10.1021/bk-1991-0467.ch004

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Bioengineering Department, Clemson University, Clemson, SC 29634-0905 Ethicon, Inc., Somerville, NJ 08876

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Natural polymers which are water-soluble or can be rendered water-soluble by chemical modification have been the subject of extensive technical reviews and original research reports, over the past fifty years. Early interests in this area were associated with the food, leather, paper and textile industries and to a lesser extent, the cosmetics and pharmaceutical industries. Proteins, such as collagen and gelatin, as well as polysaccharides, such as cellulose derivatives and starch, were dominant among all natural polymers and their derivatives. Over the past 20 years and until recently, the polymer and allied industries have focused on synthetic polymers and limited to moderate level of efforts were directed toward the modification of natural polymers. With the new interests in the biomedical and pharmaceutical industries in these polymers over the past few years, impressive activities on the modification of natural polymers to meet the growing needs are being recognized and noted in recent reviews. (Ford, 1986; Glass, 1986; Molyneux, 1982; Pariser and Lombardi, 1989; Skjak-Braek and Sanford, 1989; Shalaby, 1988). It was, therefore, decided to focus the discussion in this chapter on the most recent advances pertinent to the chemical modification of natural polymers, which are of particular interest to those in the biomedical, pharmaceutical and allied industries. Chitosan Chitosan, a partially deacetylated product of the natural polysaccharide chitin, is based on glucosamine and acetylated glucosamine units. Depending on the free amine content of the chain, chitosan exhibits variable degrees of solubility in dilute aqueous media. In the most common grade of chitosan the mole ratio of acetylated to deacetylated amine groups is 30/70. Not only does this ratio affect the polymer solubility but also it determines its susceptibility to enzyme and hence its biodegradability. Thus, a good fraction of the research activities on chitosan modification pertained to modification of the substituted and unsubstituted amine side groups of the polysaccharide chain as well as the remaining two hydroxyl 3

Current address: ConvaTec, Bristol-Myers Squibb Co., CN9254, Princeton, NJ 08543 0097-6156/91/0467-0074$06.00/0 © 1991 American Chemical Society

In Water-Soluble Polymers; Shalaby, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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functionalities (Panser and Lombardi, 1989, Sanford 1989). Given below is an outline of major research activities on such chemical modifications of chitosan. Recognizing the ability of chitosan to adsorb cupric ions, Koyama and Taniguchi (1986) have prepared homogeneously crosslinked chitosan, using glutaraldehyde, toward enhancing its uptake of the transition metal ions. A second approach to enhance chitosan adsorption of metal ions entailed its phosphorylation (Nishi, et al. 1987). The interaction of blood with chitosan derivatives has been addressed by Kurita (1986) and Hirano, et al. 1986) who were interested in cyanoethylated and sulfated chitosan, respectively. The cyanoethyl chitosan was studied in conjunction with cellulose nitrate membranes for microfiltration, while chitosan sulfate was evaluated for its anticoagulant activity. Kurita (1986) has studied several other derivatives of chitosan including N-alkyl, N-carboxybenzyl, N-carboxymethyl, and carboxyacyl chitosan. He also studied the complete deacetylation, succinylation and reductive amination of chitosan. Properties and biological activities of chitosan derivatives having modulated solubility and ionic behavior has been treated by a few recent investigators. The amphoteric N-carboxymethyl and N-carboxybutyl chitosan have been reported by Muzzarelli (1989) to be more effective than chitosan as chelating agents of metals ions. They were also noted as being more effective bacteriostatic agents than other chitosans. Hydroxypropylation of chitosan and the effect of the ratio of 0 - / N substitution and the overall degree of substitution on the solubility and related properties of resulting derivatized chitosan was studied by Maresch, Clausen and Lang (1989), N,Ocarboxymethyl chitosan was prepared and its properties were studied by Davies, Elson and Hayes (1989) as a new water-soluble polymer with potential use in a few applications. N-carboxymethyl chitosan, on the other hand, was reported by Bioagini, et al. (1989) to induce neovascularization. The authors related their findings to the understanding of healing mechanisms for chitosantreated wounds. Cellulose Among the recent studies on the modification of cellulose toward increasing its solubility in organic solvents and not necessarily water were those dealing with its cyanoethylation and subsequent reduction by borane-tetrahydrofuran to aminopropyl cellulose followed by grafting amino acid N-carboxyanhydrides (Miyamoto, et al. 1986; Hasegawa, et al. 1988). The resulting cellulose/polypeptide graft copolymers were shown to be soluble in common organic solvents. Similar grafts can be made starting with the commercially available aminoethyl cellulose (Miyamoto, et al. 1986; Miyamoto, et al. 1987). Although the technological importance of these polymers is not fully recognized, Miyamoto, etal. (1986), have described members of this family of graft copolymers to have excellent antithrombogenic properties. Starch An extensive review of new developments and potential applications of starch



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products and other polysaccharides in biotechnology, diagnostics, and pharmaceuticals has been recently published (Yalpani, M . , 1987). The use of hydroxyethyl starch for regulation of osmotic pressure in blood substitutes based on artifical perfluorohexamethylenetetramine and synthetic phospholipids has been claimed (Dandliker, etal.. 1988). The pharmacokinetics of hydroxyethyl starch and dextran, the most commonly used plasma expanders, has been recently reviewed (Klotz and Kroemer, 1987). The complex compositions of these polysaccharides and biphasic time-dependent decline of their plasma concentrations render meaningful pharmacokinetic analysis difficult. Epichlorhydrin crosslinked starch hydrogels, conjugated to proteolytic enzymes (chymotrypsin, trypsin, etc.) and lysozyme, have been reported for use as biologically active wound shield (Zamek, et a l . . 1988). Similarly, hydrogel wound dressing, comprising of proteolytic enzymes immolized in glutaraldehyde crosslinked oxidized starch, has been described (Aliaga, et al.. 1988). Kulicke, et al. (1989) have studied dynamic mechanical properties of hydrogels based on starch, oxidized starch, and amylopectin, all crosslinked with epiehlorhydrin. Pectin The major use of pectins, which are made up primarily of *-l,4-poly(galacturonic acid) partially esterified with methanol, is in the food industry. The degree of esterification affects both the aqueous solubility and the gel-forming ability. The use of polyelectrolyte complex, formed by interaction of pectin with diethylaminoethyl dextran, as an anticholesteremic agent has been claimed by Kito, et a l . . (1986). Complexation of selected model polypeptides with potassium pectates and pectinates of various esterification degrees has been reported by Bystricky, et al. (1988). It was observed that the complex-forming efficacy continuously decreases with the increase in the degree of esterification of pectin. Alginate Alginate is a collective term for a family of copolymers containing segments of 1,4linked ρ -D-manuronic and *-L-guluronic acid residues (M-block and G-block, respectively) in varying proportions and sequential arrangements (Martinsen, Skjak and Smidsrod, 1989). Most of the recent work on alginates deals with correlating the chain composition with the properties of the alginate salts and particularly those with calcium ions, for their demonstrated importance in the biomedical, pharmaceutical and textile industries. It has been documented earlier that calcium alginate forms gels and the gel-forming properties are strongly correlated with the proportion and lengths of the blocks of contiguous L-guluronic acid residues (Gblocks) in the polysaccharide chain (Smidsrod, Haug and Wittington, 1972). In a recent study (Martinsen, Skjak and Smidsrod, 1989), it was shown that calcium alginate beads, made from alginate with 70% G-blocks having an average degree of polymerization of at least 15, exhibit the highest mechanical strength and lowest shrinkage. One of the important applications of the calcium alginate beads is the encasing of living cells and immobilization of enzymes. For this reason purity of



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the soluble alginate is considered critical to the success of the calcium alginate beads as carriers of enzymes or living cells. Polyphenolic contaminants are found in commercial alginates and their detection and removal was addressed in a recent report by Skiak-Braek, Murano and Paoletti (1989). Being a polyanion, alginate can complex with the polycationic chitosan. A recent patent has described the utility of such complex using modified alginates (Daly, Keown and Knorr, 1989). Thus, capsules, for use as carriers of enzymes or cells, with modulated properties, were claimed to have been prepared from chitosan alginate wherein the alginate component is 10 to 60% esterified with 1,2propylene glycol. This esterification was pursued to increase capsule permeability. As usual, the capsule hardening is achieved by allowing the chitosan alginate to react with calcium chloride. McKnight, et al. (1988) have reported modification of calcium alginate beads by formation of a polyelectrolyte complex membrane with chitosan. The effect of the chitosan molecular weight and the degree of deacetylation upon the membrane wall forming characteristics was studied. Hyaluronic Acid Modification of this natural, tissue lubricant to explore new applications, was made possible through its availability in sufficient quantities as a fermentation product. In two patent applications, Delia Valle (1987,1988) disclosed two key modifications of hyaluronic acid to produce biodegradable materials for use by the biomedical and pharmaceutical industries. In one instance, the carboxyl groups of the polymer were esterified with monohydric alcohols or reacted with basic drugs and the products were geared primarily to pharmaceutical applications (Delia Valle, 1987). Hunt, etal. (1990) have studied transport properties of thin films formed from alkyl esters of hyaluronic acid having varying degrees of alkyl group chain lengths. Small, neutral and positively charged molecules showed relatively high permeability through the films. Whereas, smaller permeability values were observed for negatively charged molecules and for solutes of molecular weights greater than 3000. Toward the preparation of biodegradable plastics primarily for sanitary and surgical articles Delia Valle (1988) used polydric alcohols to produce a crosslinked hyaluronic acid (1988). Maison and Debelder (1986, 1988) have described crosslinking of hyaluronic acid with polyfunctional reagents, such as diepoxides, to produce water-swellable and biodegradable materials for use as surgical implants and as adjuvants for the prevention of postsurgical adhesions between body tissues. Protçins This section focuses on modification of proteins such as enzyme and growth factors which are intended, primarily, to increase their stability without impairing their biological activities. With the new development in recombinant D N A products, growth factors can be obtained in sufficient purity to study their chemical modification. In a recent study of the human epidermal growth factor (EGF) Njieha and Shalaby (1989) have shown that acylation of the primary amino-group of this simple protein increases its stability without compromising its biological activities. This was supported by data based on receptor binding and mitotic assays.



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This is perhaps one of the earliest studies on the direct modification of growth factors. On the other hand, modification of enzymes has been known for many years. However, recent focus on this technology is to seek not only an increase in enzyme stability but also to transform the native enzymes to achieve new catalytic activities. Some attempts made toward this goal have been noted by Kabanov, Levashov and Martinek (1987). An interesting approach to enzyme modification was dealt with during the preparation of semisynthetic enzymes. As in the case of flavopapain (Kaiser, 1987). In general, this approach entails taking existing enzymes with appropriately reactive functional groups in their active sites and modifying these groups with appropriate coenzyme analogs in such a way that the particularly altered substrate binding sites are still accessible to a range of substrate molecules. (Kaiser, 1987). A second approach to this preparation of semisynthetic enzyme entails that conformational modification of protein (Keyes, Albert and Sarawathi, 1987). Although the success in the development of totally synthetic enzyme has been limited, recent studies on polymers containing chiral cavities for racemic resolution appear to hold some promise (Sellergren, Lepisto and Moshbach, 1988; Wulff, 1986). Most of the recent efforts were directed toward the synthesis of enzyme-analogue built polymers for the resolution of racemic «κ-amino acids and the ultimate synthesis of optically active amino acids from glycine (Wulff and Vietmeir, J. 1989). Literature Cited 1. Aliaga, I.; Monsan, P.; Fauran, F.; Couzinier, J., Fr. Demande, FR 2,600,897 (Jan. 8, 1988). 2. Biagini, G., Pugnaloni, A. Frongia, G., Gazzanelli, G., Lough, C. and Muzzarelli, R.A.A., in "Chitin, and Chitosan:Sources, Chemistry, Biochemistry Physical Properties and Applications"(Skjak-Braek, G. and Sanford, P.Α., Eds.) Elsevier Appl. Sci. New York, 1989, p.671. 3. Bystricky, S.; Malovikova, Α.; Sticzay, T.; and Blaha, K., Collect. Czech. Chem. Commun., 53,2833(1988) 4. Daly, M.M., Keown, R.W. and Knorr, D.W., U.S. Pat. (to University of Delaware) 4,808,707(1989). 5. Dandliker, W.; Watson, K.; and Drees, T., Europ. Pat. Appl. EP 261,802 (30 March, 1988). 6. Davies, D . H . , Elson, C.M. and Hayes, E.R., in "Chitin and Chitosan:Sources, Chemistry, Biochemistry, Physical Properties and Applications" (Skjak-Braek, G. and Sanford, P.Α., Eds.) Elsevier Appl. Sci. New York, 1989, p.467. 7. Debelder, A.N. and Malson, T., EP 190,215 (Aug. 13, 1986). 8. Della Valle, F., Eur. Pat. Appl. (to Fidia, SpA) 0,216,453-A (1987). 9. Della Valle, F., Eur. Pat. Appl. (to Fidia, SpA.) 0,265,116-A (1988). 10. Ford, W.T., Ed. "Polymeric Reagents and Catalysts", ACS Symp. Series #308, Am. Ch. Soc., Washington, D.C., 1986. 2

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Glass, J.E., Ed. "Water-Soluble Polymers-Beauty with Performance" Adv. Chem. Series Vol. #213, Amer. Chem. Soc. Washington, D.C., 1986. Hasegawa, O., Takahashi, S., Zuzuki, H. and Miyamoto, T., Bull. Inst. Chem. Res., Kyoto Univ. 66,93(1988). Hirano, S., Kinugawa, J. andNishioka, Α., in "Chitin in Nature and Technology" (Muzzarelli, R., Jeuniaux C., and Gooday, C. W., Eds.) Plenum Press, New York, 1986, p-461. Hunt, J.A., Joshi, H.N., Stella, V.J., and Τοpp, E . M . , J. Control. Rel., 12, 159(1990). Kabanov, A.V., Leashov, A.V. and Martinek, K., Ann. N.Y. Acad. Sci., 501, 63(1987). Kaiser, E.T., Ann. N.Y. Acad. Sci., 501, 14,(1987). Keyes, M.H., Albert, D.E. and Saraswathi, S. Ann. N.Y. Acad. Sci., 501. 63(1987). Kito, K.; Ogawa, T.; Vemida, H; Tanahaski, E . ; Ito, Y.; and Fujii, M . , Jpn. Kokgi Tokkyo Koho JP 61,106,602 (24 May, 1986). Klotz, U. and Kroemer, H., Clin. Pharmacokinetic. 12, 123(1987). Koyama, Y. and Taniguchi, Α., J. Appl. Polym. Sci. 31, 1951(1986). Kulicke, W. M . ; Aggour, Υ. Α.; Nottelmann, H . ; and Elaabee, M . , Starch/Staerke, 41, 140(1989). Kurita, K. in "Chitin in Nature and Technology" (R. Muzzarelli, C. Jeuniaux and G. W. Gooday, Eds.), Plenum Press, New York, 1986, p-287. McKnight, C.A., Ku, Α., Goosen, M.F.A., Sun, D. and Penney, C., J. Bioact. and Comp. Polym. 3, 334(1988). Malson, T., EP 272,300 (June 29, 1988). Maresch, G., Claussen, T. and Lang, G. in "Chitin and Chitosan:Sources, Chemistry, Biochemistry, Physical Properties and Applications" (Skjak, Braek, G. and Sanford, P.Α., Eds.) Elsevier Appl. Sci. New York, 1989, p-389. Martinsen, Α., Skjak-Braek, G. and Smidsrod, O., Biotech. Bioeng., 33, 79(1989). Miyamoto, Τ., Takahashi, H., Tsuji, S., Inagaki, H. and Noishiki, J. Appl. Polym. Sci., 31, 2303(1986). Miyamoto, T., Ito, H., Takahashi, S., Inagaki, H. and Noishiki, Y., in "Wood and Cellulosics" (Kennedy, J.F., Phillips, G.O. and Williams, P.Α., Eds.) E. Horwood Ltd., Chichester, U.K. 1987, Chap. 53. Molyneux, P., "Water-Soluble Polymers:Properties and Behavior" Volumes I and II., CRC Press, Boca Raton, Fl. 1982. Muzzarelli, R.A.A. in "Chitin and Chitosan:Sources, Chemistry, Biochemistry, Physical Properties and Applications" (Skjak-Braek, G. and Sanford, P.Α., Eds.) Elsevier Appl. Sci., New York 1989, p-87. Nishi, N . , Mekita, Y., Nishimura, S., Int. J. Biol. Macromol., 9, 109(1987). Njieha, F.K. and Shalaby, S.W., U.S. Pat. Applic (to Ethicon, Inc.) Doc.S-761, Ser.#383-518, filed 7/24/89.


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WATER-SOLUBLE POLYMERS Panser, E.R. and Lombardi, D.P. "Chitin Sourcebooks Guide to the Research Literature", John Wiley, New York, 1989. Sellergren, B., Lepisto, M. and Mosbach, K., J. Amer. Chem. Soc., 110, 5853(1988). Shalaby, S.W. in "Encyclopedia of Pharmaceutical Technology" Vol. 1, Marcel Dekker, New York, 1988, p-465. Skjak-Braek, G., Murano, E., and Paoletti, S., Biotech. Bioeng., 33, 90(1989). Skjak-Braek, G. and Sanford, P.Α., Eds. "Chitin and Chitosan:Sources, Chemistry, Biochemistry, Physical Properties and Applications, Elsevier Appl. Sci., New York, 1989. Smidsrod, O., Haug, A. and Wittington, S., Acta. Chem. Scand., 26, 2563(1972). Wulff, G. in "Polymeric Reagents and Catalysts" (W.T. Ford, Ed.) ACS Symposium Series #308, Washington, 1986, p.186. Wulff, G. and Vietmeir, J., Makromol. Chem., 190, 1717(1989). Yalpani, M., Prog. Biotechnol., 2, 311(1987). Zamek, J.; Jurkstovic, T.; and Kumiak, L. Czech. Patent CS 250,018 (May 15, 1988).

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Chemical Modifications of Natural Polymers and Their Technological

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