四种聚阳离子载体材料的体外细胞学及体内性质研究

摘要

Objective: To study the charicteristics of cationic polymers polyethylenimine-β -cyclodextrin ( PEI-CyD), polyethylenimine- poly-( 3-hydroxypropyl)-aspartamide ( PEI-PHPA), N, N-rnDimethyldipropylenetriamine- Bis(3-aminopropyl) amine-aspartamide (PEE-PHPA) in vitro and in vivo. Methods: PEI-PHPA, PEI-CyD and PEE-PHPA were synthesized and the chemistry structure of PEI -PHPA,PEI-CyD and PEE-PHPA was confirmed by 'H-NMR. The particle size and zeta potential of these polymers were measured, and capacity of plasmid DNA condensation was tested. The inhibition of COS-7 , A549, HEK293 and C6 cells was measured by MTT assay. The transfection efficiency was determined in HEK293 cell lines. The toxicity, tissue distribution and transfection efficiency of cationic polymers were tested in vivo. Results: When the N/P of polymers/DNA at 30,the particle sizes were close 250 nm and the zeta-potential were near 35 mv. They were able to condense DNA at N/P ratio < 5. The MTT assay showed that the IC50 of PEE-PHPA was 21. 5,20. 2,7. 30 and 37. 1 μg/ml,and that of PEI25kD was 15. 8,18.3,11.4 and 36. 7 |xg/ml in C6, COS-7, A549 and HEK293 cell lines, respectively. The cell viability of PEI-CyD and PEI-PHPA in above cell lines was over 60% . They had high transfection efficiency in HEK293 cell lines. The LD50 of PEI25Kd, PEI-CyD, PEI-PHPA and PEE-PHPA in vivo was 19.50, 100.4,521.2 and 630.0,respectively by intraperitoneal (ip) injection. The contractions of these polymers were higher in kidney than in other organs and tissues. PEE-PHPA had slight effect on kidney and liver function. Conclusion; PEE and PEI25kD have higher transfection efficiency and higher toxicity; while PC and PHPA-PEI have lower toxicity and higher transfection efficiency to be used as non-viral gene vector.%目的:对四种非病毒型聚阳离子载体材料的理化性质、体外细胞学及体内性质进行研究.方法:合成聚乙烯亚胺-环糊精(PEI-CyD)、聚乙烯亚胺-聚天冬酰胺(PEI-PHPA)和烷基胺-聚天冬酰胺(PEE-PHPA)等三种聚阳离子材料,用1H核磁共振对载体材料的结构进行确定；凝胶电泳实验研究了载体材料对质粒DNA的浓缩能力；粒径分析仪测定了载体材料结合DNA后的粒径及表面电荷.用MTT法在COS-7、A549、HEK-293和C6等细胞株上测定了四种载体材料的细胞毒性;在HEK-293细胞株上进行了体外细胞转染实验.进行了四种载体材料体内毒性、组织分布和体内携带报告基因转染实验.结果:1H核磁共振证实了它们的结构.在N/P小于40时,载体材料的平均粒径在100 ～250 nm,表面电荷在10 ～ 35 mV,适合体外细胞的吞噬.体外细胞毒性表明,PEE-PHPA在C6、COS-7、A549和HEK293细胞上的IC50值分别为21.5、20.2、7.30和37.1μg/ml,PEI 25 kD的IC50值分别为15.8、18.3、11.4和36.7μg/ml,PEI-CyD和PEI-PHPA在实验所测定的浓度范围中,细胞的存活率高于60％.四种聚阳离子材料都具有较强的DNA缩合能力,且有较好的体外基因转染能力.体内急性毒性实验表明,PEI-PHPA、PEE-PHPA载体材料为低毒性载体材料,其LD50值大于500 mg/kg,血项指标表明PEE-PHPA载体材料对肝、肾功能有轻微影响,体内分布显示,四种聚阳离子载体材料在肾富集较高.结论:PEI-CyD、PEE-PHPA和PEI-PHPA聚阳离子载体材料均有较好的体外基因转染能力,能够携带报告基因在体内表达,其中PEI-CyD和PEE-PHPA是具有应用前景的非病毒性基因药物载体.