Preparation And Properties Of Modified Chitosan/Dextran Composite Hydrogel Based On Schiff Base Reaction

Background:Hydrogels may be defined as hydrophilic three-dimensional network structures which can absorb huge amounts of water or other physiological liquids.Different types of biodegradable hydrogels have been used as drug delivery carriers,cell carriers and tissue engineering scaffolds.Chitosan（CS）has been widely used in the preparation of hydrogels because of its great biocompatibility,biodegradability and cell-bounding capability.However,poor solubility of chitosan in water and most organic solvents has greatly limited its application.Thiolated chitosan can be prepared by modifying hydroxyl and amino groups in CS or both at the same time.The introduction of highly hydrophilic sulfhydryl groups could destroy the crystal structure of chitosan and improve its solubility.In addition,thiolated chitosan can form disulfide bonds with mucous glycoproteins on the mucous membrane and display pronounced adhesion.The disulfide bonds of thiolated chitosan formed within or between molecular chains also endow it with characteristics of in-situ gelation,which makes it has been widely used in the fields of drug delivery,tissue engineering and wound healing.The Schiff reaction is a family of reactions which can form dynamic Schiff base linkages,and it is one of the best methods to prepare biocompatible hydrogels.Chen et al.developed a hydrogel network by a Schiff-base reaction between thiolated chitosan（CS-NAC）and oxidized dextran（ODEX）and mainly explored the effects of different thiol group content on the physicochemical and biological properties of the hydrogel and evaluated its potential in the field of wound heaing.In this study,the same hydrogel composed of CS-NAC and ODEX was prepared.We didn’t change the thiol group content but focused on the effects of different oxidized degrees of ODEX on several physicochemical properties of the hydrogel in this experiment,in addition,drug release characteristics and biocompatibility of the hydrogel were also tested to evaluate the potential applications of the series of hydrogels in drug delivery and tissue engineering.Objective:Thiolated chitosan and oxidized dextran with different degrees of oxidation were prepared and characterized.A series of hydrogels were prepared by Schiff base reaction between amine groups of the thiolated chitosan and aldehyde groups of the oxidized dextran.The chemical structures of hydrogels were identified.The effects of ODEX with different oxidation degrees on microstructure,swelling behavior,rheological behavior,degradability and biocompatibility of hydrogels were also investigated.The drug release behavior of bovine serum albumin（BSA）in the environment with reducing agent or not were also tested to evaluate the reduction responsiveness of hydrogels.Methods:1.Hydroxybenzotriazole（HOBt）and EDC·HCl（N-（3-Dimethylaminopropyl）-N′-ethylcarbodiimide hydrochloride）were used as catalysts to mediate the amidation reaction between CS and NAC.2.The chemical structure of CS-NAC was identified by ¹H nuclear magnetic resonance spectroscopy（¹H NMR）and fourier transform infrared spectroscopy（FT-IR）,and the amount of thiol groups of CS-NAC was determined spectrophotometrically via Ellman’s reagent.3.Sodium periodate was used to oxidize dextran.Three kinds of ODEXs with different oxidation degrees were prepared by changing the molar ratio of sodium periodate to dextran monomer.Chemical structures of ODEXs were identified by FT-IR,and the oxidation degrees of ODEXs were determined by hydroxylamine hydrochloride titration.4.Three groups of hydrogels made of ODEX with different oxidation degrees were prepared by Schiff base reaction.The Schiff base linkages and disulfide linkages in hydrogels were identified by FT-IR and Raman spectroscopy.5.The microstructure of hydrogels was tested by scanning electron microscope（SEM）,the rheological behavior of hydrogels were detected by rheometer,and the swelling behavior and degradability of hydrogels were also measured by weighing method,so as to explore the effects of different oxidation degrees of ODEX on the physicochemical properties of the hydrogels.6.The release characteristics of BSA from hydrogels were measured and the effects of different concentrations of dithiothreitol（DTT）on the release rate of hydrogels were also explored to evaluate the reduction responsiveness of them.7.The extract liquid of the series of hydrogels were prepared,and the toxicity of each group on L929 cells was determined by CCK-8 method to evaluate the biocompatibility of the series of hydrogels.Results:1.The successful grafting of NAC onto CS was confirmed by FT-IR and ¹H NMR.The free thiol group content of CS-NAC was（351.67±38.23）μmol/g,and the total thiol group content was（388.45±41.56）μmol/g.More than 90%of the thiol groups in the final products were still free.2.The characteristic peak of FT-IR at 1733 cm^-1 proved that dextran had been successfully oxidized.The actual oxidation degrees of ODEXs in three groups determined by hydroxylamine hydrochloride titration were all lower than their theoretical oxidation degree,and the difference decreased with the increase of dosage of oxidizing agent.3.The characteristic peak of FT-IR at 1640 cm^-1 proved that the formation of Schiff base bond in hydrogels,and the characteristic peak of Raman spectrum at 500cm^-1 proved the formation of disulfide bond in hydrogels.4.The increase of the oxidation degree of ODEX enhanced the internal cross-linking of hydrogels,resulting in smaller pore size,declined swelling behavior,slower degradation rate,higher mechanical strength and lower thixotropy,and the range of variation of these properties decreased with the increase of oxidation degree.5.The series of hydrogels could release BSA in a sustainable way.The release rate increased in the presence of reducing agent and could be affected by the concentration of reducing agent,demonstrating the reduction responsiveness of the series of hydrogels.6.The viability of L929 cells could maintain more than 90%after co-cultured with extracts of hydrogels in three groups for 1-3 days,which proved that the series of hydrogels had good biocompatibility.Conclusions:The swelling behavior、rheological property、degradability and microstructure of the series of hydrogels prepared by Schiff base reaction could be affected by the oxidation degree of ODEX.Within a certain range,the change degree of physicochemical properties of the series of hydrogels decreased with the increase of oxidation degree of ODEX.In addition,the series of hydrogels,which had good biocompatibility,could release BSA in a sustainable way and showed a certain reduction responsiveness.