Construction And Properties Of Responsive Polysaccharide Hydrogel Based On Cellulose Acetoacetate

Hydrogels are cross-linked 3D networks of molecular chains or macromolecular aggregates which can entrap substantial amounts of water but do not dissolve in water.Responsive hydrogels have received widespread attention because they can change their sizes and shapes through reversible disassembly in response to environmental stimuli（pH,temperature,redox agents,electric,light,etc.）.Self-healing hydrogels offer substantial benefits to extend the life span of hydrogel material due to its excellent intrinsic healing ability to repair themselves after destruction.Recently,the environment problem is becoming more and more serious.At the same time,the research on natural polymers is rapidly emerging,biocompatible and biodegradable polysaccharide hydrogels have become popular in materials science.Nowadays,the preparation of responsive and self-healing polysaccharide hydrogels is mainly based on dynamic chemical bond.Responsive polysaccharide hydrogel based on Schiff base had been reported to be an attractive method,which could be effective formed between aldehyde groups and amino groups without external stimuli.However,most studies used the sodium periodate as oxidant;this could lead to the sharp degradation of polysaccharide polymer.Besides,complicated synthesis process makes it difficult to control.Common dynamic covalent bond such as disulfide bonds,Diels–Alder reactions,phenylboronate esters,and acylhydrazone bonds are often based on the complex synthetic procedures,biocompatibility is not optimal and high-cost.Therefore,a fast and efficient new approach for the preparation of polysaccharide hydrogel is still lacking.In this research,we designed responsive smart polysaccharide hydrogel derived from two natural polymers of cellulose and chitosan based on dynamic covalent enamine bonds under mild condition,and the hydrogel also showed self-healing and injectable properties.The stable 3D network of polysaccharide hydrogel was built through the formation of dynamic covalent enamine bonds between carbonyl groups on cellulose acetoacetate（CAA）and amino groups on chitosan.The self-healing,stability,responsive,and rheology properties of the polysaccharide hydrogel were studied,and the application of polysaccharide hydrogel in cell culture and drug release was explored.The main contents and results of the work are as following:（1）A responsive and self-healing polysaccharide hydrogel based on dynamic covalent enamine bonds was prepared.CAA was synthesized by reaction of cellulose with tert-butylacetoacetate（t-BAA）in ionic liquid 1-allyl-3-methylimidazolium chloride（AMIMCl）under homogeneous conditions without catalysts,and this controllable reaction with mild reaction condition.The structure of CAA were characterized by FTIR and NMR,the characteristic peaks of acetoacetyl groups were found in FTIR and NMR spectra.The degree of substitution（DS）can be controlled by varying the reaction time,temperature or molar ratio of t-BAA to cellulose,and the solubility of CAA in different solvents were tested.The results demonstrate that CAA possesses water solubility with a DS about 0.58-1.11,and all the products could be readily dissolved in DMSO.The polysaccharide hydrogel was obtained by mixing CAA aqueous solution with chitosan aqueous solution under mild condition.The 3D network of hydrogel was built through the formation of dynamic covalent enamine bonds between the acetoacetyl groups in CAA and the amino groups in chitosan.The structure of hydrogel was characterized by FTIR,the characteristic peaks of enamine bonds were found in FTIR spectrum.The gelation time was monitored by rheology test,indicating it could be adjusted by changing the concentration of CAA and chitosan.The storage modulus of hydrogel immediately recovered after the breaking strain was removed from the rhrological test,and macroscopic self healing test also demonstrated the four pieces hydrogel could heal autonomously into a single one after 40 min,indicating the enamine bond could dynamically decompose and reform under acidic condiction.The stability of hydrogel under physiological conditions were conducted under room temperature,it still remained the hydrogel state after immersing in the phosphate buffered saline（PBS,pH=7.4）for 72 h.Hydrochloric acid and sodium hydroxide were chosen as pH regulators to test the responsive properties of polysaccharide hydrogel,the hydrogel could quickly change into hydrosol after adding hydrochloric acid solution,and the hydrogel was regenerated by adding sodium hydroxide solution,indicating the hydrogel exhibited reversible acid–base stimulus responsive properties.（2）An injectable polysaccharide hydrogel based on CAA,water-soluble hydroxypropyl chitosan（HPCS）,and amino-modified cellulose nanocrystals（CNC-NH₂）was prepared under physiological conditions.CNC-NH₂ acted as both physical and chemical cross-linker.The structure and properties of the polysaccharide hydrogel were characterized by FTIR,NMR,Raman,rheological,self-healing,and responsive test.The covalent enamine bonds were formed between HPCS and CAA form the FTIR,NMR and Raman spectra.The results demonstrated that the addition of CNC-NH₂ improved the gelation rate and reduced the gelation time significantly.Specifically,even at a low CNC-NH₂ loading of 0.2 wt%,the gelation time saved310 s compared to the CAA/HPCS gel.The gelation time was only 100 s with the CNC-NH₂loading of 0.8 wt%.The modulus of hydrogel could be adjusted by tuning the CNCs concentration,and the maximum storage modulus 2800 Pa was observed for a gel containing0.80 wt%of CNC-NH₂,and the CNC-NH₂ reinforced polysaccharide hydrogel exhibited significantly higher elastic modulus（150%increase at peak strength）compared to a hydrogel without the filler.The rhrological test indicating the CAA/HPCS/CNC-NH₂ polysaccharide hydrogel can endure the alternate sweep of 200%high strain and 1%low strain.The macroscopic self healing tests also demonstrated CNC-NH₂ reinforced polysaccharide hydrogel could heal autonomously into a single one,indicating the enamine bond could dynamically decompose and reform under acidic condiction.The hydrogel also exhibited pH-responsive properties,the hydrogel changed into hydrosol while pH below 2.5 and back to hydrogel again with changing pH to neutral.Stability testing demonstrated that the addition of CNCs and CNC-NH₂ showed no evident effect on the stability of polysaccharide hydrogel under physiological conditions.In addition,CCK-8 cytotoxicity study with fibroblast L929 cells demonstratedgoodbiocompatibilityofCAA/HPCS,CAA/HPCS/CNCs,and CAA/HPCS/CNC-NH₂ polysaccharide hydrogels.（3）On the basis of preparing pH-responsive hydrogel,redox sensitive disulfide moiety was introduced into the hydrogel system.A cellulose-based pH and redox multi-responsive hydrogel was prepared by simply mixing aqueous solutions of CAA and cystamine dihydrochloride（CYS）.The hydrogel 3D network was built through the formation of dynamic covalent enamine bonds and disulfide bonds.The internal morphology,structure,and mechanical properties of the cellulose hydrogel were characterized by FTIR,NMR,Raman,water retention,porosity,and rheology measurements.The covalent enamine and disulfide bonds present in hydrogel molecular structure form the FTIR and ¹³C NMR spectra.The responsive test demonstrated that cellulose hydrogel showed reversible sol–gel transitions in response to both pH and redox triggers.In addition,polysaccharide hydrogel displayed good stability under physiological conditions,without significant change after immersing in water for72 h.The results of rheology experiments demonstrated that the modulus of hydrogel can be adjusted by changing the reactant concentration and solution pH,the modulus increased with the increasing of concentration or decreasing of pH.The rheological measurement indicated the hydrogel can endure a temperature of 100℃.Gels loaded with small molecules Rhodamine B showed variable release properties in response to pH or redox stimuli,the realese rate increased with the decreasing of pH values or increasing the DTT concentration,indicating this pH/redox dual-responsive hydrogel held great potential for targeted drug delivery and smart sensors.（4）On the basis of preparing polysaccharide hydrogel based on dynamic covalent bond,a high strength double cross-linked responsive polysaccharide hydrogel films were constructed by simply soaking saturation saline solution method.First,simply mixing CAA aqueous solution with excessive chitosan solution to obtain a chemical cross-linked hydrogel,on the second step,the chemical cross-linked hydrogel was immersed into saturated sodium chloride solution to obtain a double cross-linked polysaccharide hydrogel.The internal morphology,structure of polysaccharide hydrogel were characterized by FTIR,¹³C NMR,Raman,XRD and SEM measurements.The characteristic peaks of covalent enamine bond were found in the FTIR,¹³C NMR and Raman spectra of double cross-linked hydrogel.Hydrated crystal peak appeared in the XRD spectra of double cross-linked hydrogel.The SEM tests indicated that the physical entanglement network of chitosan chains could improve the compactness of cross-linked network.The polysaccharide hydrogel held high mechanical properties because of the dynamic cross-linking of enlamine bond and the physical entanglement network of chitosan interpenetrate to each other.Besides,the mechanical properties could be adjusted by tuning the molar ratio and immering time to control the ratio of physical and chemical cross-linking.The optimal molar ratio of chitosan and CAA was 2:1,immering time was 2 h,the tensile strength of hydrogel film up to 0.23 MPa,and the compression strength was0.19 MPa.The rheological measurment indicated that the modulus of double cross-linked hydrogel was higher than that of single chemical cross-linked hydrogel,and the maximum storage modulus was up to 15000 Pa.The effect of pH value on mechanical properties of hydrogel films was discussed,demonstrating the hydrogel film showed good pH stimulus responsive properties.The mechanical properties of the double cross-linked hydrogel deceased with the decreasing of pH values.In addition,water swelling testing showed that the gel possessed higher water absorption capacity,and could retain the gel state after absorbing water.These features suggest that polysaccharide hydrogel holds great potential for appli cations in tissue engineering and drug delivery.