Preparation, Proerties And Biological Evaluation Of The Mitomycin C Released Membrane Based On Modified Chitosan

Objective:The loss of the intraocular pressure (IOP) lowering effect after the glaucoma filtering surgery may be caused by collapsed of filtering bleb. Its formation is referable to scar formation between the conjunctiva and sclera. Scarring of the filtering bleb is associated with cellular proliferation, particularly fibroblast proliferation. Antimetabolite is used for inhibition of fibroblast proliferation during an operation or after, which increases the success rate of filtering surgery, but the chemical reaction might cause unexpected complications and tissue damage. Implantation can be used in glaucoma filtering surgery and create a physical barrier between the conjunctiva and sclera, but its application is limited by immunological rejection, source and other factors. Thus, one focus of the glaucoma filtering surgery is the combination of antimetabolite and implantation. An agent used for the glaucoma filtering surgery should be safe, biodegradable and easy to apply, and it should have an IOP-lowering effect. Chitosan is been widely researched and applied in medical and biomaterial fields due to its excellent characteristics. Mitomycin C (MMC) in particular is widely used as an antifibrotic agent to inhibit fibroblast proliferation in trabeculectomy. The purpose of this experiment is to prepare a drug release membrane of Mitomycin C with N-Succinyl-hydroxyethyl chitosan for glaucoma filtering surgery, study its characteristics and evaluate the effect of inhibiting fibroblast proliferation in vitro, biodegradability and biocompatibility. The experiment will evaluate the feasibility of Suc-HECTS-MMC membrane as an implant for glaucoma filtering surgery.Methods: 1.Prepare and purify hydroxyethyl chitosan (HECTS). The acquired products were characterized by FTIR. The deacetylation degree and substitution degree of hydroxyethyl are measured by 1H NMR and Elemental analysis. Cytotoxicity of HECTS is evaluated.2.N-Succinyl-hydroxyethyl chitosan (Suc-HECTS) is prepared by HECTS and succinic anhydride via ring-opening reaction. The best reaction condition is selected by analyzing the experiment conclusions in different conditions to obtain the supreme binding rate of succinic anhydride. The acquired product is characterized and the binding rate of succinic anhydride is measured by 1H NMR. Cytotoxicity of Suc-HECTS is evaluated.3. A drug loaded membrane is made of Suc-HECTS and mitomycin C (MMC). The conjugate prepared by the direct coupling of MMC with Suc-HECTS using EDC-HC1 is obtained due to cross-linking among the polymer supports. Under dark light condition, drug-loaded membranes with three different cross-linking degrees are synthesized by using BDDGE as a polymer cross-linker. Experiments for the drug release in vitro from the drug loaded membranes are performed. The characteristics of the membranes, such as FTIR, equilibrium water content, swelling ratio, permeability, anti-fibrosis in vitro, subcutaneous irritant reaction, biodegradability and biocompatibility are determined.Results:1.It shows that the substituent at the 6-position of HECTS, and the substitution degree of hydroxyethyl is 3.14. The cytotoxicity experiment in vitro shows that the material has noncytotoxicity.2. The best reaction condition of Suc-HECTS is selected and the supreme binding rate of succinic anhydride is about 40%. The cytotoxicity experiment in vitro shows that the material has noncytotoxicity. 3. The MMC loaded membranes are prepared through the covalent cross-linking between Suc-HECTS and MMC. The MMC-Suc-HECTS membranes could delay the release of MMC in vitro. The experimental results indicate the drug loaded membrane is effective on the swelling property and permeability. In the L929 fibroblast cell adherent culture, experimental results demonstrate that the destination membranes can inhibit fibroblast proliferation. In vivo, the drug loaded membranes show excellent biodegradability and biocompatibility in the experiment of implanting membranes into the subcutaneous tissue and muscle of rats. Conclusions:In this experiment, the MMC-Suc-HECTS membranes have a certain degree of flexibility, suitable permeability and hydrophilic property, drug delayed releasing ability, inhibited the fibroblast ability in vitro, good biocompatibility and biodegradability. The experimental results provide a theoretical basis for the future development of Suc-HECTS-MMC membrane as an implant for glaucoma filtering surgery.