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目的:评价不同季铵化程度的N-三甲基壳聚糖(TMC)包衣的空白脂质体对小鼠成纤维细胞L-929的毒性。方法:采用四步合成法合成不同季铵化程度的TMC,并采用IR对其结构进行确证和表征,~1H-NMR确定其季铵化程度。采用MTT比色法评价TMC作为阳离子脂质体包衣材料,在不同浓度和不同季铵化程度时对小鼠成纤维细胞L-929的毒性,计算相对增殖率,并参考《美国药典》的评价标准对其进行毒性分级。结果:IR分析TMC的特征吸收峰已明显发生了改变,表明TMC已经成功合成,~1H-NMR分析所合成的TMC,季铵化程度分别为20.2%、40.1%、64.4%,不同季铵化的TMC在浓度为0.02%,0.05%(w/w),细胞增殖率均大于80%,TMC20在浓度增大到0.2%(w/w)时,细胞增殖率仍高于80%,细胞毒性为1级,即无细胞毒性,但随着浓度和季铵化程度的提高,细胞毒性均相应增大。结论:所合成的TMC在一定的浓度和季铵化范围内,细胞毒性较低,又鉴于其具有良好的生物降解性、亲水性和生物粘附性,可望作为阳离子脂质体理想的包衣材料。
OBJECTIVE: To evaluate the toxicity of blank liposomes coated with N-trimethylchitosan (TMC) with different degrees of quaternization on mouse fibroblast L-929 cells. Methods: TMC with different degree of quaternization was synthesized by four-step synthesis method. The structure of the TMC was confirmed and characterized by IR. The degree of quaternization was determined by 1H-NMR. MTT colorimetric method was used to evaluate the toxicity of TMC as a cationic liposome coating material to mouse fibroblast L-929 cells at different concentrations and different degrees of quaternization to calculate the relative proliferation rate, and with reference to the “United States Pharmacopoeia” Evaluation criteria for its toxicity classification. Results: The characteristic absorption peak of TMC changed obviously by IR, which indicated that TMC has been successfully synthesized. The TMC synthesized by ~ 1H-NMR analysis showed that the degree of quaternization was 20.2%, 40.1% and 64.4% respectively. Different quaternization Of TMC at concentrations of 0.02%, 0.05% (w / w), cell proliferation rates were greater than 80%, TMC20 in the concentration increased to 0.2% (w / w), the cell proliferation rate was still above 80%, cytotoxicity Level 1, that is, no cytotoxicity, but with the concentration and the degree of quaternization increased, the corresponding increase in cytotoxicity. CONCLUSION: The synthesized TMC has low cytotoxicity in a certain range of concentrations and quaternization, and is expected to be ideal for cationic liposomes due to its good biodegradability, hydrophilicity and bioadhesion Coating material.