Preparation And Properties Of Chitosan-based PCA And PEDOT Hydrogels

Oxidative stress injury characterized by excessive accumulation of free radicals is closely related to ageing and a variety of neurodegenerative diseases.The development of nerve tissue engineering scaffolds with antioxidant ability,to deal with a continuous oxidative stress injury in the process of nerve repair and regeneration,is one of the potential methods for the treatment of related diseases.At the same time,given the critical regulatory role of bioelectrical signals in the development,repair,and regeneration of the nervous system,the combination of organic conductive polymers and natural/synthetic polymer materials,the construction of novel conductive scaffolds with excellent biocompatibility by chemical or physical methods has become one research focus in nerve tissue engineering in recent years.Phenolic acid grafted chitosan(CS)has the good antioxidant ability and can significantly reduce the oxidative stress damage of cells and bodies in a variety of in vivo and in vitro models.Therefore,on the one hand,based on the proper therapeutic effect of natural plant polyphenol protocatechuic acid(PCA)on a variety of in vitro and in vivo models of neurodegenerative diseases,antioxidant water-soluble CS derivative,carboxymethyl chitosan(CMCS)derivative and porous hydrogel scaffolds modified by PCA were developed.On the other hand,taking advantage of the excellent chemical stability and high electrical conductivity of poly(3-ethylenedioxythiophene)(PEDOT),PEDOT/CMCS porous conductive hydrogel scaffolds with high water absorption were prepared by in-situ interfacial polymerization.This paper provides a feasible way for the development of new nerve tissue engineering scaffolds with the antioxidant ability and electrical conductivity through the research on these two kinds of hydrogel scaffolds.In this paper,water-soluble PCA-g-CS polymer was synthesized by green free radical grafting polymerization(hydrogen peroxide(H2O2)/ascorbic acid(Vc)redox pair).The chemical structure of the polymer was characterized by FTIR,1H NMR,XRD,and UV-Vis.The antioxidant ability was tested.Compared to the control CS group,the scavenging ability of PCA-g-CS to DPPH and hydroxyl radical was increased to 5.5 times and 3.5 times,respectively.The cytotoxicity of the material was verified by mouse nerve cell PC 12 cells.PCA-g-CS aqueous solution(0.05,0.10,0.2,0.2,0.8 mg/mL)had no apparent cytotoxicity,and with the extension of culture time,the number of cells increased in a dose-dependent manner,which might have a specific ability to promote cell proliferation.The modified materials had protective effects on PC 12 cells injured by H2O2 and glutamate(Glu),and could antagonize the injury of PC 12 cells induced by H2O2 for 24 h or Glu for 48 h in a dose-dependent manner.Compared with H2O2-injured and Glu-injured control group,the cell viability of the two groups treated with 0.8 mg/mL PCA-g-CS increased by about 1.3 times.The above results confirmed that the water-soluble CS grafted with PCA retained the antioxidant and neuroprotective abilities from PCA,which had a specific significance for the application of CS-based antioxidant materials in the repair of nerve injury.The H2O2 used in free radical polymerization can oxidize and destroy the glycosidic bonds on the molecular chain of CS,and the modified CS with good solubility but low molecular weight can be obtained,which is challenging to prepare the scaffold alone.For this reason,by using EDC/NHS chemical cross-linking method and using CMCS as the reaction substrate,the PCA-g-CMCS polymer with the highest grafting amount of 64.6 mg PCA equivalent/g was obtained by adjusting the pH value of the reaction system and the molar ratio of raw materials.The chemical structure of the polymer was characterized by FTIR,'H NMR,XRD,and UV-Vis.Compared to the raw CMCS,the water solubility of PCA-g-CMCS decreased,and the pH sensitivity from CMCS was retained.When pH ?5.75 and pH?7.25,the optical transmittance was close to 100%,and it was flocculated when pH was 5.75-7.25.The in vitro antioxidant activity test(DPPH and ABTS free radical scavenging abilities,total antioxidant ability,and reducing power)and ferrous ion chelating ability showed that the grafting of PCA significantly improved the antioxidant ability and ferrous ion chelating ability of PCA-g-CMCS in vitro.Subsequently,PCA-g-CMCS porous hydrogel scaffolds were prepared by freeze-drying and EDC/NHS cross-linking curing.Compared to CMCS scaffolds,the grafting of PCA reduced the water absorption and in vitro degradation rate of the modified scaffolds,increased the compression modulus,and increased DPPH and ABTS free radical scavenging ability by 7.5 times and 5.9 times,respectively,and maintained the in vitro release activity of PCA within 15 days.The cytotoxicity of the scaffolds was verified by human nerve cell SH-SY5Y cells.It was found that the PCA-g-CMCS scaffolds had no cytotoxicity and could antagonize the oxidative damage induced by H2O2.Compared to the injury control group,the cell viability of the modified scaffold increased by about 1.39 times,which might have a specific neuroprotective effect.This antioxidant scaffold provided a new idea for potential applications such as antioxidant drug release and nerve tissue engineering.Finally,to endow CMCS scaffolds with electrical conductivity,PEDOT/CMCS conductive scaffolds were prepared by in-situ interfacial polymerization,and their physicochemical properties and cytotoxicity were evaluated.In this paper,a layer of PEDOT conductive nano-coating was chemically polymerized on the pore wall of CMCS scaffold by in-situ interfacial polymerization method based on the difference of solubility of the superabsorbent CMCS scaffold and EDOT monomer in the aqueous phase and organic phase(n-hexane),and the PEDOT/CMCS composite conductive scaffolds with different PEDOT contents were obtained.The introduction of PEDOT coating reduced the water absorption and in vitro degradation rate,improved the surface roughness,compression modulus,and electrical conductivity of the conductive scaffold.The conductive scaffold had a high water absorption and could absorb at least 17 times its weight of water,and the highest conductivity could reach 4.68×10-3 S.cm-1.Conductive scaffolds did not show obvious cytotoxicity and was beneficial to the adhesion,proliferation and long-term culture of PC 12 cells(more than 9 days).It could be applied in nerve tissue engineering.To sum up,this paper expands the application range of CS materials in neural tissue engineering and successfully prepared new CS-based PCA modified water-soluble antioxidant derivatives,antioxidant scaffolds,and conductive scaffolds.These two scaffolds had good antioxidant activity in vitro and electrical conductivity,respectively,and did not show obvious cytotoxicity.Through mouse and human nerve cell experiments,it was proved that PCA modified materials could reduce the oxidative damage of the two kinds of cells.Conductive hydrogel scaffolds were beneficial to the adhesion,proliferation,and long-term culture of mouse PC 12 cells.Therefore,these two kinds of scaffolds might play a positive role in the repair of nerve injury.In the future,it is expected to combine these two materials to develop CS-based composite hydrogel scaffolds with dual functions of antioxidation and conductivity,which can be used for nervous system repair,regeneration,and functional recovery.