摘要
分别制备了以支化小分子量聚乙烯亚胺(PEI-1.8k)为引发剂,引发苯丙氨酸-NCA开环聚合得到聚乙烯亚胺-聚苯丙氨酸(PEI1.8k-g-PPhe)以及聚乙烯亚胺接枝苯丙氨酸单体(PEI1.8k-g-Phe)的系列基因载体材料.利用核磁、粒度、zeta电位仪、荧光光度计、流式细胞仪以及激光共聚焦显微镜对PEI1.8k-g-PPhe,PEI1.8k-g-Phe以及PEI1.8k-g-PPhe/DNA和PEI1.8k-g-Phe/DNA复合物颗粒进行了系统的表征.研究结果表明,最佳转染条件下,PEI1.8k-g-PPhe10/DNA复合物颗粒的粒径约为150 nm,表面电位约为16 m V.在人源宫颈癌(He La)和人源乳腺癌(MCF-7)2种细胞系中均具有较高的基因转染效率,且最佳转染效率可达到PEI-25k的12倍.MTT细胞毒性实验分别比较了PEI1.8k-g-PPhe和PEI1.8k-g-Phe对He La细胞毒性的大小.从实验结果可见,苯丙氨酸引入的方式及数量决定着其细胞毒性的大小.PEI1.8k-g-PPhe和PEI1.8k-g-Phe都具有较低的细胞毒性(材料在较高浓度1 mg/m L时的细胞存活率大于70%).内吞实验结果表明,PEI1.8k-g-PPhe由于接入了具有规则聚合链的聚苯丙氨酸,而易于被He La细胞内吞.PEI1.8k-g-PPhe10/DNA复合物颗粒相比于PEI-25k/DNA,PEI-1.8k/DNA和PEI1.8k-g-PPhe/DNA具有更高的细胞内吞效率.
To search safe and evaluate non-viral nucleic acids carrier, a series of polyphenylalanine and phenylalanine grafted low molecular weight PEI with a molecular weight of 1.8 × 10^3（PEI1.8k-g-PPhe and PEI1.8k-g-Phe） were prepared by NCA ring opening polymerization initiated by PEI-1.8k and phenylalanines conjugation to PEI-1.8k, respectively. The polymers and the complexes of PEI1.8k-g-PPhe/DNA and PEI1.8k-g-Phe/DNA were characterized by nuclear magnetic resonance（NMR） analysis, particle size analysis, zeta potential analysis, luciferase analysis, flow cytometry（FCM） analysis and confocal laser scan microscopy（CLSM）. In order to compare the property for compacting DNA into nanoparticles, the particle size and zeta potential analysis were carried out. Both PEI1.8k-g-PPhe and PEI1.8k-g-Phe showed suitable particle size and zeta potential for gene delivery. The particle size of PEI1.8k-g-PPhe10/DNA complexes were about 150 nm and the zeta potentials were about ＋16 m V, which were suitable for the in vitro experiments. Moreover, cell viability, after treating with different copolymers at various concentrations, was studied by an MTT assay. The reduced cytotoxicity of PEI1.8k-g-PPhe and PEI1.8k-g-Phe may be because of the introduction of neutral hydrophobic phenylalanine moieties. And both of the PEI1.8k-g-PPhe and PEI1.8k-g-Phe had lower cytotoxicity（above 70% viability at a higher concentration 1 mg/m L） than that of PEI with a molecular weight of 2.5 × 10^4（PEI-25k） in He La cells. The in vitro gene transfection of PEI1.8k-g-PPhe10 and PEI1.8k-g-Phe10 was conducted in human cervical cancer（He La） and breast cancer（MCF-7） cells. In both of the cells, PEI1.8k-g-PPhe10 exhibited much higher gene transfection efficiency. PEI1.8k-g-PPhe10/DNA complexes showed remarkable gene transfection efficiency, which was about twelve times higher than that of PEI-25 k. The endocytosis efficiency of PEI1.8k-g-PPhe10/DNA and PEI1.8k-g-Phe10/DNA were quantified using flow cytometry. Due to their regular polymer chain, PEI1.8k-g-PPhe10/Cy5-DNA showed better internalization efficiency than PEI1.8k-g-Phe10/ Cy5-DNA, PEI-25k/Cy5-DNA and PEI-1.8k/Cy5-DNA. The CLSM assay was carried out to verify the internalization efficiency together which also indicated that PEI1.8k-g-PPhe10/Cy5-DNA induced higher intracellular uptake efficiency than the others.
作者
林琳
郭兆培
陈杰
田华雨
陈学思
Lin Lin Zhao-pei Guo Jie Chen Hua-yu Tian Xue-si Chen(Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun130022)
出处
《高分子学报》
SCIE
CAS
CSCD
北大核心
2017年第2期321-328,共8页
Acta Polymerica Sinica
基金
国家自然科学基金(基金号21474104
51233004
51520105004
51390484)
吉林省科技发展计划(项目号20160204032GX)
中共中央组织部"万人计划"青年拔尖人才项目资助
