Fabrication Of Chitosan Micro/Nano Functional Materials And Biomedical Application Research

With the rapid development of biomedical engineering,functional materials based on biological macromolecules such as natural polysaccharides are receiving more and more attention.Chitosan is the only natural alkaline polysaccharide in nature.It has excellent biocompatibility,good biodegradability,and various biological activities.It has great application potential in biomedical fields,especially tissue engineering and drug delivery systems.This paper focuses on the fabrication of chitosan micro/nano functional materials and their applications in biomedical field.The following aspects of work are included:(1)Preparation of porous chitosan microspheres and their application as microcarriers for 3D cell culture.Porous chitosan microspheres(CSM)were prepared through emulsification coupled with thermally induced phase separation.The morphology and microstructure of the microspheres were characterized by scanning electron microscopy,mercury injection and liquid displacement.The contact angle measurement showed that CSM had high hydrophilicity,and atomic force microscope showed that CSM had high elastic modulus.Cytotoxicity analysis and hemolysis rate test showed that CSM had excellent cytocompatibility and blood compatibility.Hepatocyte culture experiments showed that CSM not only enabled cells to adhere on the surface of microspheres,but also enabled them to enter the internal pores for growth,and the cells maintained good morphology and formed multidirectional cell connections.The function of hepatocytes was further analyzed by gene expression,protein microarray technology and Elisa.The above studies indicated that the as-prepared chitosan porous microspheres could be used as microcarriers to achieve 3D cell culture with normal cellular morphology and functions,which has great application potential in the fields of cell therapy and tissue engineering.(2)Fabrication of smart chitosan microcarriers for responsive cell detachment.Smart and responsive microcarriers based on chitosan microspheres were prepared via the host-guest interaction between ?-cyclodextrin(?-CD)and aromatic residues in gelatin.Firstly,chitosan microspheres(CSM)were prepared by emulsion crosslinking approach,the surface of which was then modified with ?-CD by chemical grafting.Further the microspheres were modified with gelatin through the host-guest interaction between ?-CD and aromatic residues in gelatin.The resultant microspheres were denoted as CSM-g-CD-Gel.Cytotoxicity evaluation showed that CSM-g-CD-Gel had good biocompatibility.High-performance cellular adhesion and growth of hepatocytes were achieved using CSM-g-CD-Gel as microcarriers.More importantly,responsive cell detachment was achieved by adding an appropriate amount of amantadine(AD)in the culture.This is attributed to the higher affinity between AD and ?-CD.The competitive binding of AD to ?-CD implied gelatin detachment,which led to cell detachment.This trypsin-free cell harvest by the responsive microcarriers could avoid the possible cellular damage and may have bright prospects in cell culture and relevant areas.(3)Preparation of chitosan nanoparticles and their nanocomposite hydrogels with gelatin for p H-responsive drug delivery systems.?-CD-modified chitosan(CS-g-CD)was synthesized by amide reaction between carboxylmethyl-?-cyclodextrin and chitosan.Nanoparticles(CS-g-CD NP)of about 150 nm were then prepared by TPP ionic cross-linking induced assembly.CS-g-CD NP exhibited p H responsiveness,which are stable under neutral conditions,but rapidly disassembled in weakly acidic environment.Supramolecular hydrogels(Gel/ CS-g-CD NP)were prepared by the host-guest interaction between ?-CD and aromatic residues in gelatin.The effect of the content of CS-g-CD NP on the hydrogels was analyzed by rheological studies.The results showed that the gelation temperature of hydrogels increased significantly with the increase of the CS-g-CD NP content while the gelation time was shortened rapidly.The elastic modulus of the hydrogels was also increased gradually with the the CS-g-CD NP content.Because of the reversibility of the interaction between CS-g-CD NP and gelatin,Gel/CS-g-CD NP hydrogels exhibited typical shear-thinning and self-healing properties.DOX-loaded CS-g-CD NP(DOX-CSCD NP)and drug-loaded Gel/CS-g-CD NP hydrogels(Gel/ DOX-CSCD NP)were prepared using DOX as the model drug.DOX-CSCD NP and Gel/ DOX-CSCD NP showed p H-responsive drug release behaviors.Drug release was relatively slow under neutral conditions,while it was faster in weakly acidic environment,and the drug release rate increased.In vitro cell evaluation experiments showed that drug-loaded nanoparticles and hydrogels had good inhibition effect against tumor cells,especially under weakly acidic conditions.These p H-responsive drug carriers are expected to be applied in targeting the tumor acidic microenvironment and drug delivery.In summary,this thesis deals with the preparation,characterization and application of three chitosan functional materials including porous chitosan microspheres,smart-responsive chitosan microspheres and chitosan nanocomposite hydrogels,which can be used as cell carriers and drug carriers for three-dimensional cell culture,detachment,and targeted controlled release of drugs.These chitosan-based micro/nano functional materials have potential applications in tissue engineering and tumor-targeted therapy,respectively.