During lyophilization, CaCl2 might preferentially interact with α,β-glycerophosphate (α,β-GP), inhibit transfer of protons from chitosan to α,β-GP and then inhibit aggregation of chitosan (CS). The reconstituted chitosan/α,β-GP/CaCl2 solutions could form hydrogel quickly at 36 °C, but did not form hydrogel at 20 °C and 4 °C more than two weeks.
In the present study, lyophilization was attempted to improve the long-term storage of CS/GP thermogelling systems for biopharmaceutical applications. After lyophilization, CS/α,β-GP lyophilizate could not be dissolved in water, but some metal salts, such as NaCl, CaCl2, and MgCl2 surprisingly facilitated its dissolution. X-ray powder diffraction analysis suggested that calcium ions might preferentially form salts with α,β-GP, inhibit the transfer of protons from CS to α,β-GP, and then inhibit the aggregation of CS molecules during lyophilization. Comparison of the freshly prepared CS/α,β-GP/salt solutions and the reconstituted solutions from lyophilizates showed that lyophilization clearly influenced the properties of reconstituted CS/α,β-GP/salt solutions such as gelation time, viscosity, and pH. Furthermore, the reconstituted CS/α,β-GP/CaCl2 solutions maintained thermogelling properties and formed hydrogels at 37 °C within approximately 5 min, but did not form hydrogels at 20 °C and 4 °C over 2 weeks. The model protein bovine serum albumin (BSA) was further incorporated into the CS/α,β-GP/CaCl2 system. In vitro release experiments showed the sustained release of BSA from CS/α,β-GP/CaCl2 hydrogels in a pH-sensitive manner, demonstrating that CS/α,β-GP/CaCl2 may be useful as an in situ gel-forming system.