功能性纳米羟基磷灰石的生物相容性及安全性评价

采用水热合成法制备的铽离子掺杂的精氨酸修饰的纳米羟基磷灰石颗粒(HAP/Arg),对HAP/Arg的生物安全性进行体内外研究,探讨其作为基因治疗药物载体的可行性。选择人正常血管内皮细胞(HAEC)以及人肿瘤细胞株(Hela细胞)作为体外细胞模型,用酶标免疫检测仪检测其在0、25、50~200 μg/mL HAP/Arg纳米颗粒影响下的细胞存活率,用LDH法检测其在不同浓度的 HAP/Arg纳米颗粒影响下的细胞膜的结构完整性。选择健康小鼠作为动物模型,将其分为低剂量组(10 μg/mL/20 g 体重)、中剂量组(100 μg/mL/20 g 体重)、高剂量组(500 μg/mL/20 g 体重)及对照组,进行死亡计数、血清生化指标检测以及倒置荧光显微镜观察主要脏器的病理学特征。根据急性毒性实验结果,将健康小鼠分为低剂量组(0.09 mg/(kg·d))、中剂量组(4.5 mg/(kg·d))、高剂量组(225 mg/(kg·d))及对照组,按照新药临床前安全性评价标准判断其生殖毒性。结果表明:0、25、50~200 μg/mL的HAP/Arg纳米颗粒在4、24、48、72 h不同时间下对所选择的HAEC细胞和Hela细胞的正常生长和细胞膜结构均无明显影响。在动物的急性毒性实验和生殖毒性实验中,HAP/Arg各剂量组及对照组小白鼠均未出现明显的毒性反应及死亡情况,病理切片也均未见主要脏器结构有炎症及损伤性变化。统计学分析表明,HAP/Arg各剂量组与对照组相比均无显著差异(P>0.05)。这些结果为下一步构建一种高效、安全、简便的新型纳米基因转导系统打下基础。

Study on Biological Safety of Functionalized Nano-Hydroxyapatite

To investigate the feasibility of nano-hydroxyapatite with arginine-functionalized and Tb³⁺doped HAP/Arg as a gene drug carrier, and to study the biological safety of HAP/Arg. Normal human vascular endothelial cells (HAEC) and human tumor cells (Hela) were selected as cell models in vitro, under the exposure of 0, 25, 50 to 200 μg/mL of HAP/Arg nanoparticles, the cell survival rate was measured by the enzyme labeled immunoassay instrument, and the structural integrity of the cell membrane of which was examined by LDH method. Healthy mice were divided into low dose group (10 μg/mL/20 g weight), middle dose group (100 μg/mL/20 g weight), high dose group (500 μg/mL/20 g weight ) and control group, to count number of deaths, to detect serum biochemical indexes and to observe pathologic features of main organs by inverted fluorescence microscope. According to the results of acute toxicity experiment, healthy mice were divided into low dose group (0.09 mg/(kg·d)), middle dose group (4.5 mg/(kg·d)), high dose group (225 mg/(kg·d) ) and control group, according to the preclinical safety evaluation standard to judge its reproductive toxicity. The results showed that HAP/Arg with concentration of 0, 25, 50 and 200 μg/mL did not affect normal growth and cell membrane structure under 4, 24, 48 and 72 h of HAEC cells and Hela cells. In experiments on animal acute toxicity and general reproductive toxicity, there were no obvious toxic reaction and death in the mice of each dose group and control group, the inflammation and injury were not found in the main organs. Statistical analysis shows that each dosage groups of HAP/Arg has no significant difference compared with control group (P>0.05). Above results are expected to be useful for developing a novel efficient, safe and convenient gene delivery system for the next research step.