Water-Solubility of Partially N-Acetylated Chitosans PDF

Carbohydrate Polymers 25 (1994) 65 70 ' 1994 Elsevier Science Limited. Printed in Great Britain. Al...

Carbohydrate Polymers 25 (1994) 65 70 ' 1994 Elsevier Science Limited. Printed in Great Britain. All rights reserved ELSEVIER 0144-861 7(94)0003 ! -X 0144-8617/94/$07.00 Water-solubility of partially N-acetylated chitosans as a function of pH: effect of chemical composition and depolymerisation Kjell M.Vfirum, Mette H. Ottoy & Olav Smidsrod Norwegian Biopolymer Laboratory (NOBIPOL), Division of Biotechnology, The Norwegian Institute of Technology (NTH), University of Trondheim, N-7034 Trondheim-NTH, Norway (Received 25 March 1994; revised version received and accepted 9 May 1994) The solubility of four partially N-acetylated chitosans with fraction of acetylated units (FA) of 0.01, 0.17, 0.37 and 0.60 as a function of pH was investigated. The chitosan with FA = 0.60 was soluble at all pH-values between 4 and 9. The solu- bility versus pH curve of the other chitosans, showed that all chitosans precipi- tated between pH 6 and 7.5, but with increasing solubility at higher pH-values with increasing FA. Such solubility differences may have profound effects on enzyme accessability and biological effects of chitosans. The three chitosans with the lowest FA values were depolymerised by treatment with nitrous acid, and the fraction of water-soluble material at pH 7.5 was determined. The almost fully deacetylated chitosan was completely insoluble at pH 7.5 in the depolymerisation range investigated, while the most acetylated chitosan (FA = 0.60) was fully soluble at all pH-values. However, the two chit- osans with FA--0.17 and 0.37 could be fractionated into a neutral-soluble and a neutral-insoluble fraction. The amount of neutral-soluble material increased with decreasing depolymerisation. The neutral-soluble and the neutral-insoluble frac- tion differed in both chemical composition and degree of polymerisation. Generally, the neutral-soluble fraction had a higher fraction of acetylated units and a lower degree of depolymerisation than the neutral-insoluble fraction. This compositional heterogeneity of the degraded chitosans was shown to be consis- tent with what is expected from the theoretical random degradation of chitosans with a Bernoullian (random) distribution of acetylated and deacetylated units. INTRODUCTION may complicate the use e f chitosans in other applica- tions (i.e. biological effects at physiological pH-values). Chitosan is commercially prepared by N-deacetylation Considerable efforts have been made in synthesising of chitin (1--~4-1inked 2-acetamido-2-deoxy-]3-D-gluco- derivatives of chitin and chitosan (Roberts, 1992), and pyranose---GlcNAc) and may be considered as a family some of these derivatives, i.e. N-carboxymethylchitosan of unbranched binary hetero-polysaccharides of and chitosan phosphates, have been prepared in order fl(1-+4)-linked GIcNAc (A-unit) and 2-amino-2-deoxy- to extend the water-solubility of chitosans to neutral /LD-glucopyranose- GlcN (D-unit) of varying compo- pH. sition. The A- and D-units have been shown to be It has, however, been shown that chitosans which are randomly distributed in water-soluble partially N- soluble at neutral pH-values can be made by controlling acetylated chitosans (V~rum et al., 1991a, b). the fraction of acetylated units (FA). Chitosans with full It is well known that chitosan is insoluble in water at neutral-solubility were obtained with relatively high neutral and basic pH-values. Commercial chitosans molecular weight chitosans with FA between 0.4 and 0.6 usually contain a fraction of acetylated units (FA) (Sannan et al., 1976). Both the physical-chemical and between 0 and 0.2, and are only soluble in aqueous biological properties of chitosans are dependent on the solutions at acidic pH-values. This lack of water-solu- chemistry of the polymer (FA and the distribution of the bility of commercial chitosans at neutral pH-values may monomers along the chain), the molecular weight, the be an advantage for certain applications (i.e. easy to pH, and the ionic strength of the solution. This study remove from solutions by adjusting the pH) while it was aimed at comparing the solubilities of chitosans as 65 66 K.M. Vdrum, M.H. Ottoy, O. Smidsrod a function of pH, and at preparing well-defined chit- stopped by adjusting the pH to 4.5 with NaOH. The osans with full neutral-solubility at pH 7.5. Some of the chitosan was converted to the chitosan-HCI salt, filtra- neutral-soluble fractions have been applied in a study ted and lyophilised. where pH is restricted to neutrality (i.e. soluble chit- osans' ability to induce TNF-~ production from human Fractionation of degraded chitosans monocytes (Otterlei et al., 1994)). Solutions of the degraded chitosans were made by dissolving the chitosan in distilled water overnight. MATERIALS AND M E T H O D S NaOH was added dropwise to the chitosan solution, while the pH was continually monitored until it became Chitosans stable at pH 7.5 for 30 min. The precipitates were easily separated from the supernatant, with little or no gel-like Four chitosans with different fractions of acetylated precipitates, while the sodiumhydroxide was added. The units were used in this study. All chitosans, except for solutions were centrifuged, and the supernatant and Chit4, were prepared by heterogeneous deacetylation, precipitate separated by decanting. The supernatant was and the acid-soluble fraction prepared as previously converted to the chitosan-HCl salt, lyophilised and described (V~rum et al., 1992). Chitl (FA=0.01) was weighted (neutral-soluble fraction). The precipitate was prepared by further heterogeneous deacetylation of a solubilised by adding 2% acetic acid, converted to the commercial chitosan, while Chit4 was prepared by chitosan-HCl salt, lyophilised and weighted (neutral- homogeneous deacetylation. The chitosans were conver- insoluble fraction). ted into the chloride salt (chitosan-HCl) by dissolution in acetic acid, dialysis against excess 0.2 M NaCI and Chemical composition further dialysis against distilled water. The chitosan- hydrochlorides were finally filtrated and lyophilised. No The fraction of acetylated units and the diad frequencies detectable amount of acetate could be determined in the were determined by high-field proton NMR-spectro- 500 MHz N M R of the chitosans, where the acetate scopy as previously described (V~rum et al., 1991a). methylprotons are well separated from the other protons on chitosan (including the acetyl protons) Physical properties Fractionation of undegraded chitosans Intrinsic viscosities of chitosans were determined as previously described (Draget et al., 1992). The number- Equal volumes of a I% chitosan solution in 0.1 M NaCI average molecular weights were estimated from the and a solution containing a certain amount of NaOH Mark Houwink-Sakurada equation as reported by were mixed. The pH-value obtained was determined Anthonsen et al. (1993). The degree of scission was after leaving the solution for 30min, and thereafter calculated as I/DP,, where DPn is the number-average centrifuged. The distribution of the chitosan between degree of polymerisation. the centrifugate and the precipitate was determined by weighing the fractions (after dialysis and lyophilisation). Calculation of compositional distribution of oligomers The procedure is based on pH-solubility experiments with alginates described by Haug and Larsen (1963). The compositional distributions of oligomers of differ- ent lengths when a binary copolymer is randomly Nitrous acid degradation of chitosans degraded and the distribution of monomer units along the chain is random, are binomial (Frensdorff and Solutions of chitosan hydrochloride (chitosan-HC1) Pariser, 1963). Thus, the equation for the calculation of were made by dissolving the chitosan in distilled water the mole fractions ( f ) of an n-mer containing x A-units overnight, which was then diluted with an equal volume and (n-x) D-units is: of 4% acetic acid, and the desired amount of NaNO2 added. The next day the solution was reduced conven- f ( A = x) = ( ~ ) F ~ ( I -..... , tionally with sodiumborohydride. The chitosan was converted to the hydrochloride salt, as described above, where FA is the fraction of acetylated units in the unde- filtrated and lyophilised. graded chitosan molecule. Hydrochloric acid degradation of chitosans RESULTS AND DISCUSSION To a solution of chitosan-HC1 (Chit3) in distilled water was added HC1 to pH 2.5. The solution was heated to The insolubility in water at neutral and basic pH-values 80°C for approximately 100h, cooled and the reaction is a well-known property of commercial chitosans.  Effect o f chemical composition and depolymerisation on water solubility O/" chitosans 67 Table 1. Chemical composition and physical properties of chitosans m _ _ Sample FA FAA FAD = FDA FDD NA ND (ml/g) Mo Chitl