Preparation, Properties And In Vitro Cytotoxicity Of Chitosan Films As Drug Sustained-release System

[Objective] To identify the best chitosan film preparation to be used for drug sustained-release system through the evaluation of the sustained-release property, degradation property, and cytotoxicity to osteoblast of various chitosin film preparations.[Materials and Methods] 1. Using the 24 hour's swelling ratio as the evaluation index , single factor tests and orthogonal experiments were designed to determine the best combination of chitosan film preparations. The main variables which effect the preparation are: acetic acid concentration (0.5%, 1%, 1.5%, 2%), chitosan concentration (1mg/ml,2mg/ml,4mg/ml,8mg/ml), STPP concentration (4%, 6%, 8%, 10%), and cross-linking time (0.5h, 1h, 1.5h, 2h). 2. BSA was added into the chitosan films prepared with the various parameters as a protein drug model. Drug release rate was determined at the 1st, 2nd, 4th, 6th and 8th day with Coomassie brilliant blue G250. In a separate study, chitosan films were placed into the test tubes with 30ml buffer solution and 10⁷U/L lysozyme. These test tubes were incubated at 37℃in a shaking culture box. The degradation rate at the 1st, 2nd , 3rd and 4th week were calculated. 3. Osteoblasts derived from fetal rat calvarial were cultured on chitosan films. Cell proliferation was tested by MTT Assay by measuring OD at 0.5, 1, 2, 3, and 5 days, respectively. The relative growth rate was calculated and the cytotoxicity was graded.[Results] Single Factor Tests: Through the swelling ratio evaluation, chitosan film prepared with 1% acetic acid, chitosan concentration of 2 mg/ml; 8% STPP solution and 1 hour of cross- linking time, exhibits the best drug sustained-release property. Orthogonal Experiment: Due to existence of interaction among various factors, L₁₆ (4⁴) orthogonal experiment was selected to evaluate the parameters. The best combination is 1% acetic acid, chitosan concentration of 2mg/ml, 6% STPP concentration, and cross-linking time of one hour. The swelling ratio of the best parameter combination was compared with the minimum swelling ratio group in the orthogonal experiment. The difference is statistically significant (P<0.05). Drug Release Rate: The sustained-release rates of chitosan films prepared with the best parameter combination are 20.1% , 23.77% , 26.44% , 30.97% , 33.13% at 1,2,4,6, and 8 days. During the first day, the release of the BSA is quick, with the prolonged time, the release rates of BSA stablized at a lower level than the first day. Degradation Rate: The degradation rates of the films are 13.67% , 21.67%, 31.75%, 38.73% at the 1w, 2w, 3w, and 4w, respectively . With the prolonged time, the weight of chitosan films decrease gradually in the buffer solution. Cytotoxicity Experiment: The relative growth rates of the osteoblast exposed directly to chitosan films are above 100% at the 0.5 d, 2 d, 3 d, and 5 d; cytotoxicity grade is 0. For day 1, cell relative growth rate is over 90%, and the cytotoxicity grade is 1. Through the LSD statistic analysis, there is no statistical difference between the absorptance of chitosan film group and the negative control group (P > 0.05), however, compared with the positive control group and the blank group, the chitosan film group showed significant difference in absorptance (P < 0.01).[Conclusions] (1)Through the single factor tests and orthogonal experiments, the optimal conditions for preparation of the best chitosan film drug sustained release system are: 1% acetic acid; chitosan concentration of 2mg/ml, 6% STPP solution, with cross-linking time of 1h. (2)The resulting chitosan film releases 33.13% of BSA within 8 days. The drug release results demonstrate that this chitosan film possessed good sustained release ability. (3)Under the effect of lysozyme, 36.73% of this chitosan film was degraded within four weeks. It shows a good rate of degradation. (4)Cytotoxicity of this chitosan film is grade 0 or 1, which meets the safety standard. This chitosan film has good biocompatibility.