Elsevier

Carbohydrate Polymers

Volume 46, Issue 1, September 2001, Pages 39-47
Carbohydrate Polymers

Rheological characterisation of thermogelling chitosan/glycerol-phosphate solutions

https://doi.org/10.1016/S0144-8617(00)00281-2Get rights and content

Abstract

In this study we demonstrate that chitosan solutions can be neutralised up to physiological pH (∼7.2) using β-glycerol phosphate without creating immediate gel-like precipitation and furthermore that subsequent heating of these solutions induces hydrogel formation. The addition of the particular basic salt, glycerol phosphate, provides the correct buffering and other physicochemical conditions including control of hydrophobic interactions and hydrogen bonding which are necessary to retain chitosan in solution at neutral pH near 4°C and furthermore to allow gel formation upon heating to 37°C. Rheological investigation evidenced the endothermic gelation of chitosan/β-glycerol phosphate solutions and allowed the establishment of a sol/gel diagram. The gelation process appears to be governed by delicate interplay between the pH and the temperature. The role of β-glycerol phosphate is discussed in the light of relevant literature particularly those indicating the role of glycerol and polyols in the stabilisation of proteins and polysaccharides.

Introduction

Chitosan is an aminopolysaccharide obtained by alkaline deacetylation of chitin, a cellulose-like polymer present in fungal cell walls and exoskeletons of arthropods such as insects, crabs, shrimps, lobsters and other vertebrates (Muzzarelli, 1977). Chitosan is a biodegradable (Struszczyk, Wawro, & Niekraszewicz, 1991), biocompatible (Chandy and Sharma, 1990, Hirano et al., 1990a) and mucoadhesive (He et al., 1998, Henriksen et al., 1996, Lehr et al., 1992) biopolymer, which is emerging to play a significant role in biomedical applications (Felt et al., 1998, Illum, 1998, Madhavan, 1992, Malette et al., 1983, Sandford, 1988), due to its abundance and wide scope of use. Chitosan has been recommended as an appropriate material for many purposes in pharmaceutical, medical and food industries, where numerous international patents have claimed applications of chitosan in these areas (Nordquist, 1998).

The term chitosan is commonly used to describe a series of chitosan polymers with various weight average molecular weights (50kDaM̄w≤2000kDa) and degrees of deacetylation (40<DDA<98%). Chitosan is typically not soluble in water, but chitosan solutions can be obtained in acidic aqueous media which protonate chitosan amino groups, rendering the polymer positively charged and thereby overcoming associative forces between chains. When adding a strong base (i.e. NaOH) to such solutions, chitosan remains in solution up to a pH in the vicinity of 6.2. Further basification, to pH>6.2, systematically leads to the formation of a hydrated gel-like precipitate. This precipitation, or gel formation, is due to the neutralisation of chitosan amine groups and the consequent removal of repulsive interchain electrostatic forces which subsequently allows for extensive hydrogen bonding and hydrophobic interactions between chains. The inability to maintain chitosan in solution up to a physiological pH in the region of 7.0–7.4, has been the main obstacle to date in the development of certain biomedical applications of chitosan, for example as an encapsulating or delivery system for living cells or for pH-sensitive proteins. In the context of the current study, it is important to note a significant exception to the above general description of the solubility behaviour of chitosan — chitosans with a relatively low DDA, from 40 to 60%, remain in solution up to a pH near 9, and are therefore not the subject of investigation in the current work.

Here we report the preparation and characterisation of thermogelling chitosan solutions formulated at conditions including physiological pH. The endothermically gelling chitosan solution is prepared by supplementing an aqueous solution of chitosan with glycerophosphate salt, an additive which plays three essential roles: (1) to increase the pH into the physiological range of 7.0–7.4; (2) to prevent immediate precipitation or gelation; and (3) to allow for controlled hydrogel formation when an increase in temperature is imposed. Our results suggest that the addition of this particular basic salt provides the correct buffering and other physicochemical conditions including control of hydrophobic interactions and hydrogen bonding which are necessary to retain chitosan in solution at neutral pH and furthermore to allow gel formation upon heating to 37°C. This system is likely to receive considerable attention in the biomedical field, since such liquid polymer solutions can be loaded with therapeutic materials at low but non-freezing temperatures, and then injected into body sites to form degradable gel implants in situ (Chenite et al., 1999). An additional physicochemical characteristic of chitosan bodes well for its use as a scaffold or carrier system in tissue regeneration and repair and local drug and gene delivery. Namely the anionic nature of most human tissues due to the presence of glycosaminoglycans in the extracellular matrix, and the cationic character of chitosan, (at pH≈7.2, approximately 17% of amino groups are still protonated), allows for adherence of these thermally gelling solutions to tissue sites. Recently we have also shown that some formulations can be prepared to have physiological pH and osmotic pressure and to thereby offer a suitable microenvironment for living cells to maintain functional characteristics after injection and implant formation (Hoamann, Sun, Binette, McKee, & Buschmann, 2000).

Section snippets

Materials

Medium molecular weight chitosan (M̄w≈3.5×105) with a high degree of deacetylation (DDA∼91%), was generously provided by Maypro (Purchase, NY, USA). The weight average molecular weight and the DDA of chitosan were determined by using size exclusion chromatography (SEC) and 13C-NMR spectroscopy, respectively. For SEC we used a HP1100 chromatograph equipped with WAT011545 column connected to WAT011565 guard column in series (both from Waters Inc., Milford, MA), with an on-line detection obtained

Results and discussion

Chitosan solutions can be neutralised to pH values between 6.5 and 7.3 via β-GP addition, without inducing immediate precipitation or gelation, provided the temperature is maintained between 4 and 15°C. The pH of these chitosan solutions approached the physiological region when the molar concentration of β-GP exceeded the molar concentration of the amine groups of chitosan (0.110 M in Fig. 1). This ability to maintain chitosan in solution and prevent chain aggregation at neutral or nearly

Conclusions

A thermally gelling chitosan system was prepared by neutralising highly deacetylated chitosan solutions with β-glycerol phosphate to retain chitosan in solution at physiological pH. Upon heating to moderate temperatures, these solutions quickly transformed into a hydrogel structure as demonstrated by rheological measurements. Furthermore, the sol/gel transition temperature was pH-sensitive and gelling time was shown to be temperature-dependent. The molecular mechanism of gelation may involve

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