聚天冬酰胺衍生物的合成及细胞毒性研究

目的 合成一种聚天冬酰胺衍生物并对其细胞水平的安全性进行评价， 为其作为药物载体应用提供研究基础。方法 通过 L-天冬氨酸热缩聚合成聚琥珀酰亚胺， 利用苯丙氨酸甲酯盐酸盐和乙醇胺对聚琥珀酰亚胺进行开环反应得到载体 PSI-Phe-EA； 利用 1H NMR 进行聚合物结构表征； 采用内标法磁氢谱计算其开环率； 通过水溶性的比较验证其亲水性变化； 采用 MTT 法对聚合物的细胞增殖抑制进行研究， 利用倒置显微镜观察聚合物对细胞微观形态的影响； 利用碘化丙啶 （PI） 染色法通过流式细胞仪研究聚合物对细胞周期的影响。结果 1H NMR 确证了开环衍生物 PSI-Phe-EA 的结构， PSI 的开环率为 40%； 乙醇胺开环后聚合物的亲水性得到了明显改善； MTT 实验表明，PSI-Phe-EA 在所检测浓度范围内 （<100 mg/L）， 对 NIH3T3 和 HepG2 两种细胞的 24 h 细胞存活率均在 80%以上； 倒置显微镜观察表明， 50 mg/L 的 PSI-Phe-EA 孵育 24 h 后以上两种细胞的形态与对照组无明显差异； 细胞周期分析表明 PSI-Phe-EA 处理与否对细胞周期分布的影响无明显差异。结论 合成的聚天冬酰胺衍生物PSI-Phe-EA 亲水性明显提高， 且对细胞的存活、 微观形态以及周期分布均无明显影响， 是一种安全的高分子材料。

Synthesis of poly asparagine derivatives and its cytotoxicity study

Objective To synthesize poly asparagine derivatives and to evaluate its safety at the cellular level, which provide research platform for its potential application as drug carrier. Methods Polysuccinimide was synthesized by thermal polymerization of L-polyaspartic acid, and the target product of PSI-Phe-EA was obtained by the ring-opening reaction of polysuccinimide using L- phenylalanine methyl ester hydrochloride and ethanol amine. The structure of PSI- Phe- EA were characterized by 1H NMR. The rate of ring-opening of PSI was calculated by internal standard method of 1H NMR. The change of hydrophilicity was studied by the comparison of solubility. The cytotoxicity and morphology modification by PSIPhe-EA at designate concentrations was investigated by MTT method and inverted microscopy respectively. The effects on cell cycles were analyzed by flow cytometry after propidium iodide (PI) staining. Results 1H NMR results confirmed the structure of PSI-Phe-EA and the ring-openning rate of PSI was 40%. The hydrophilicity of PSI-Phe-EA was greatly increased upon ring opening using ethanol amine. MTT test showed that the cell survival rates of NIH 3T3 and HepG2 cells were higher than 80% under the examined concentration (<100 mg/L). Inverted microscopy showed that 50 mg/L of PSI-PheEA treatment had no adverse effects on cell morphology. Cell cycle analysis indicated that PSI-Phe-EA treatment had no influence on cell cycles of NIH 3T3 and HepG2 cell lines. Conclusion PSI-Phe-EA showed high hydrophilicity without significant effects on the cells survival, cells morphology and cell cycles. It is a kind of safe polymer material.