Preparation Of Disulfide Cross-linked Layer-by-Layer Self-Assembly Microcapsules And Hydrogels For Drug Controlled Release

Layer-by-Layer（LBL）self-assembly microcapsules formed by alternating adsorption of a polycation and a polyanion on a charged sacrificial template followed by dissolution of the template have attracted much attention,owing to the tunable properties of wall thickness,permeability,surface chemistry and responsiveness to external stimulus.The microcapsules were generally assembled by electrostatic interactions or hydrogen-bonded that can lead to undesired disassembly and cargos leakage from the microcapsules due to the weak nature of the non-covalent under diverse solution conditions such as pH,ionic strength and temperature.In order to circumvent this limitation a cross-linking was introduced interlayer of microcapsules.Chemical cross-linking and radiation cross-linking were more versatile method to synthesize stable covalent bonding microcapsules.However,these methods were inadequate for the application in bio-medical field because toxic chemical agents and high energy radiation can reduce biocompatibility of microcapsules.So,we proposed a very simple,non-toxic,biocompatible and rapid rate cross-linking method by using a Horseradish peroxidase（HRP）-mediated cross-linking of a thiolated polymer under physiological conditions.Hydrogels are physically or chemically cross-linked three-dimensional hydrophilic networks capable of absorbing large quantities of water while maintaining their structure.Natural hydrogels have been used widely in the fields of biomedicine,tissue engineering and bioengineering due to its excellent biocompatibility,nontoxicity and biodegradability properties.Nowadays,the construction of hydrogel has already become more and more intelligent.Smart hydrogel can be rapidly responsive to the environmental conditions with temperature,light,pH value or reductant etc.Smart hydrogel has been widely used in drug delivery and tissue engineering.As a smart hydrogel,redox-sensitive hydrogel is particularly appealing because it can be readily dissolved under mild conditions in the presence of small reductant such as glutathione（GSH）,cysteine（Cys）and dithiothreitol（DTT）.In the first part of this thesis,disulfide cross-linked chitosan/hyaluronic acid（CS/HA）hydrogels and CS/HA microcapsules were prepared and controlled drug release were studied.Firstly,the CS/HA hydrogels were prepared by HRP-mediated cross-linking reaction system.The gelation was simply conducted,by mixing of aqueous HRP,tyramine hydrochloride（Tyr）and equal amounts of thiol-modified chitosan（CS-SH）and thiol-modified hyaluronic acid（HA-SH）in PBS at 37℃.The gelation time,swelling ratio,degradation properties,rheological properties and drug release behavior of CS/HA hydrogels were explored.The gelation time can be controlled by altering the concentration of HRP and Tyr.The CS/HA hydrogels can be formed in 17min at 10 units/mL of HRP and 35 mM of Tyr,the equilibrium swelling ratio was 25.The drug release behavior showed drastic changes due to adding reducing agent DTT.The drug release was dramatically accelerated with increasing the concentration of DTT because of the degradation of disulfide bond.Secondly,shell cross-linked hollow polyelectrolyte microcapsules were fabricated based on the layer-by-layer self-assembly（LbL）technique.CS-SH and HA-SH were alternately adsorbed on FITC-dextran-CaCO₃ sacrificial templates with a diameter of 3-5μm,and followed by cross-linking of the thiol groups using HRP enzymatically cross-linked to form stable disulfide bonds,and then dissolution of the sacrificial CaCO₃ core by a chelating agent EDTA.The polyelectrolyte successively alternating deposition procedure on CaCO₃ templates can be monitored with Zeta-potential.The morphology and structure of the microcapsules are investigated by means of scanning electron microscopy（SEM）,transmission electron microscopy（TEM）and confocal laser scanning microscopic（CLSM）.The redox-sensitive microcapsules showed controlled release of FITC-dextran by adding reducing agent DTT.The release behavior of FITC-dextran can be manipulated by controlling the polyelectrolyte layers,cross-linking and the concentration of DTT.In the third part of this thesis,the HA-SH/HA-Tyr hydrogel was prepared by HRP-mediated cross-linking reaction system.The HA-SH/HA-Tyr hydrogels were prepared by the mixing of aqueous HRP,HA-SH and HA-Tyr in PBS at 37℃.The gelation time,swelling ratio,degradation properties and rheological properties were explored.The drug release properties can be tuned by changing the concentration of HA-Tyr and reducing agent DTT.The HA-SH/HA-Tyr hydrogels can be formed in35-40 min at 10 units/m L of HRP and 10 mM of HA-Tyr,and the equilibrium swelling ratio was 25.78.The mechanical properties of the HA-SH/HA-Tyr hydrogels were studied by oscillatory rheology at 37℃.Storage moduli of 3080-3090Pa were observed for HA-SH/HA-Tyr hydrogels at 10 units/m L of HRP,10 mM of HA-Tyr and 2%of HA-SH.The experimental results of DOX drug release showed that the increase of DTT concentration and decrease of HA-Tyr concentration can effectively increase release effect because of the degradation of disulfide bond.