Heat-induced transfer of protons from chitosan to glycerol phosphate produces chitosan precipitation and gelation.

Recently, chitosan dissolved in solutions containing glycerol phosphate (GP) were found to undergo a sol-gel transition when heated and the proposed gelling mechanism was based on increasing hydrophobic interactions with temperature. Subsequently, an investigation of ionization and precipitation behavior of chitosan, including dependencies on temperature, added salt, and fraction of deacetylated monomers (fD) was performed. This latter study revealed important differences in the temperature dependence of pKa of chitosan versus GP and led us to propose an alternative hypothesis for the mechanism of gelation in chitosan-GP systems whereby heat induces transfer of protons from chitosan to glycerol phosphate thereby neutralizing chitosan and allowing attractive interchain forces to form a physical gel. To investigate this specific molecular thermogelling mechanism, temperature ramp experiments on dilute chitosan-GP solutions were performed. Chitosans with fD of 0.72 and 0.98 were used to prepare solutions with a range of molar ratios of GP to chitosan glucosamine monomer of 1.25 to 10 and with 0 or 150 mM added monovalent salt. Light transmittance measurements were performed simultaneously to indicate precipitation in these dilute systems as a surrogate for gelation in concentrated systems. Measured temperatures of precipitation ranged from 15 to 85 degrees C, where solutions with less GP (used in a disodium salt form) had lower precipitation temperatures. A theoretical model using acid-base equilibria with temperature dependent pKa's, including the electrostatic contribution from the polyelectrolyte nature of chitosan, was used to calculate the degree ionization of chitosan (alpha, the fraction of protonated glucosamine monomer) as a function of temperature and showed a significant decrease in alpha with increased temperature due to proton transfer from chitosan to GP. This heat-induced proton transfer from chitosan to GP was experimentally confirmed by 31P NMR measurements during temperature ramp experiments since the chemical shift of 31P of GP is an indicator of its level of protonation. By assuming average temperature independent values of alpha p that were calculated from measured T(p), the model was able to accurately predict measured temperatures of precipitation (T(p)) of all chitosan-GP mixtures. The resulting alpha(p) were temperature independent but increased with increased chitosan fD and with increased salt. Measurements and theory revealed that T(p) can be adjusted in a predictable manner by changing the chitosan-GP molar ratio and thereby systematically tailored to obtain a large range of precipitation temperatures. Finally, similar temperature ramp experiments using inorganic phosphate and MES in place of GP demonstrated that the temperature-induced precipitation of chitosan also occurs with these buffers, confirming that the key feature of the buffer used with chitosan is its ability to absorb heat-stimulated release of chitosan protons and facilitate chitosan neutralization. A theoretical expression for the variation of chitosan ionization degree with temperature in a system composed of two titratable species (chitosan and buffer) was derived and allowed us to establish the required characteristics of the buffer for efficient heat-stimulated proton transfer between a chitosan and the buffer. These results provide a useful explanation for the mechanism of heat-induced gelation of chitosan-based systems that could be exploited for numerous practical applications.

[1]  R. Muzzarelli,et al.  Biochemistry, histology and clinical uses of chitins and chitosans in wound healing. , 1999, EXS.

[2]  M. Amiji Pyrene fluorescence study of chitosan self-association in aqueous solution , 1995 .

[3]  K. Yao,et al.  Chitosan and its derivatives--a promising non-viral vector for gene transfection. , 2002 .

[4]  H. Fukada,et al.  Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride , 1998, Proteins.

[5]  H. Nalwa,et al.  Handbook of polyelectrolytes and their applications , 2002 .

[6]  M. Buschmann,et al.  Ionization and solubility of chitosan solutions related to thermosensitive chitosan/glycerol-phosphate systems. , 2007, Biomacromolecules.

[7]  R. Misra,et al.  Biomaterials , 2008 .

[8]  J. Desbrières,et al.  Characterization of chitosan by steric exclusion chromatography , 2001 .

[9]  P. Carreau,et al.  Effect of urea on solution behavior and heat-induced gelationof chitosan-β-glycerophosphate , 2006 .

[10]  M. Shive,et al.  Chitosan-glycerol phosphate/blood implants improve hyaline cartilage repair in ovine microfracture defects. , 2005, The Journal of bone and joint surgery. American volume.

[11]  D Chandler,et al.  Temperature and length scale dependence of hydrophobic effects and their possible implications for protein folding. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[12]  Dong Wang,et al.  Rheological characterisation of thermogelling chitosan/glycerol-phosphate solutions , 2001 .

[13]  T. Fujinaga,et al.  Accelerating effects of chitosan for healing at early phase of experimental open wound in dogs. , 1999, Biomaterials.

[14]  D. Chandler,et al.  Hydrophobicity at Small and Large Length Scales , 1999 .

[15]  J. Desbrières,et al.  Hydrophobic derivatives of chitosan: characterization and rheological behaviour. , 1996, International journal of biological macromolecules.

[16]  P. Carreau,et al.  Physical gelation of chitosan in the presence of beta-glycerophosphate: the effect of temperature. , 2005, Biomacromolecules.

[17]  O. Philippova,et al.  Two types of hydrophobic aggregates in aqueous solutions of chitosan and its hydrophobic derivative. , 2001, Biomacromolecules.

[18]  Rudolph A. Marcus,et al.  Calculation of Thermodynamic Properties of Polyelectrolytes , 1955 .

[19]  J. Leroux,et al.  Novel injectable neutral solutions of chitosan form biodegradable gels in situ. , 2000, Biomaterials.

[20]  宁北芳,et al.  疟原虫var基因转换速率变化导致抗原变异[英]/Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A , 2005 .

[21]  M. Buschmann,et al.  High efficiency gene transfer using chitosan/DNA nanoparticles with specific combinations of molecular weight and degree of deacetylation. , 2006, Biomaterials.

[22]  F. Hoppe-Seyler Ueber Chitin und Cellulose , 1894 .

[23]  S. Hirano,et al.  Chitosan: A Biocompatible Material for Oral and Intravenous Administrations , 1990 .

[24]  O. Smidsrod,et al.  Water-solubility of partially N-acetylated chitosans as a function of pH: effect of chemical composition and depolymerisation , 1994 .

[25]  J. A. V. BUTLER,et al.  Theory of the Stability of Lyophobic Colloids , 1948, Nature.

[26]  R. Mumper,et al.  Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery. , 1998, Journal of controlled release : official journal of the Controlled Release Society.

[27]  A. Neuberger,et al.  Dissociation constants of 2-amino-2-deoxy-D-glucopyranose , 1969 .

[28]  C. Iversen,et al.  Characterization of association phenomena in aqueous systems of chitosan of different hydrophobicity , 1999 .