Advanced Technology Education, Research and Application Center, Mersin University, 33343 Mersin, Turkey.
J Biomater Sci Polym Ed. 2012;23(5):577-92. doi: 10.1163/092050611X555272. Epub 2011 Feb 10.
Primary smooth muscle cells (SMC) isolated from the aorta of fetal calf were transfected with a green fluorescent protein (GFP)-encoding plasmid DNA, which was carried by a water-soluble and temperature-sensitive N-isopropylacrylamide-based (NIPAAm-based)-co-polymer, either poly(N-isopropylacrylamide-co-2-methacryloamidohistidine) (poly(NIPAAm-co-MAH)) or monosized PEGylated nanoparticle poly(styrene/poly(ethylene glycol) ethyl ether methacrylate/N-(3-(dimethylamino)propyl) methacrylamide) (poly(St/PEG-EEM/DMAPM)). Poly(NIPAAm-co-MAH) co-polymer was synthesized by solution polymerization of n-isopropylacrylamide (NIPAAm) and 2-methacrylamidohistidine (MAH). Monosized cationic nanoparticles were produced by emulsifier-free emulsion polymerization of styrene, PEG ethyl ether methacrylate and N-[3-(dimethyl-amino) propyl] methacrylamide, in the presence of a cationic initiator, 2,2-azobis (2-methylpropionamidine) dihydrochloride. The structure of poly(St/PEG-EEM/DMAPM) and poly(NIPAAm-co-MAH) was confirmed by(1) H-NMR and FT-IR spectroscopy. Particle size/size distribution and surface charges of both carriers were measured by Zeta Sizer. The LCST behavior of poly(NIPAAm-co-MAH) co-polymer was followed spectrophotometrically. Poly(St/PEG-EEM/DMAPM) nanoparticles, with an average size of 78 nm and zeta potential of 54.4 mV, and an average size of 200 nm with a zeta potential of 54.2 mV, and poly(NIPAAm-co-MAH) were used in the transfection studies. The cytotoxicity of the vectors was tested using the MTT method. According to conditions for the transfection study (polymer/cell ratio and polymer-cell incubation period), cell loss was only 4 and 15% with poly(St/PEG-EEM/DMAPM) sized 78 and 200 nm, respectively. Poly(NIPAAm-co-MAH) cytotoxicity was insignificant. Poly(NIPAAm-co-MAH) uptake efficiency in SMCs was around 85%, but gene expression efficiency were low compared to poly(St/PEG-EEM/DMAPM)/pEGFP-N2 conjugates because of the low zeta potential of the co-polymer. Polymer uptake efficiencies of the nanoparticles were 90-95%. GFP expression efficiency was 68 and 64% after transfection with pEGFP-N2 conjugate with 78 and 200 nm sized poly(St/PEG-EEM/DMAPM) nanoparticles.
从胎牛主动脉分离的原代平滑肌细胞（SMC）用携带水溶性和温敏 N-异丙基丙烯酰胺基（NIPAAm 基）共聚物的绿色荧光蛋白（GFP）编码质粒 DNA 转染，该共聚物由聚（N-异丙基丙烯酰胺-co-2-丙烯酰基组氨酸）（poly（NIPAAm-co-MAH））或单分散聚乙二醇化纳米颗粒聚（苯乙烯/聚乙二醇乙醚甲基丙烯酸酯/N-（3-（二甲氨基）丙基）甲基丙烯酰胺）（poly（St/PEG-EEM/DMAPM））组成。通过 N-异丙基丙烯酰胺（NIPAAm）和 2-丙烯酰基组氨酸（MAH）的溶液聚合合成了 poly（NIPAAm-co-MAH）共聚物。通过在阳离子引发剂 2,2-偶氮双（2-甲基丙脒）二盐酸盐存在下，无乳化剂乳液聚合苯乙烯、PEG 乙基醚甲基丙烯酸酯和 N-[3-（二甲氨基）丙基]甲基丙烯酰胺，制备了单分散的阳离子纳米颗粒。通过（1）H-NMR 和 FT-IR 光谱证实了 poly（St/PEG-EEM/DMAPM）和 poly（NIPAAm-co-MAH）的结构。通过 Zeta Sizer 测量了两种载体的粒径/粒径分布和表面电荷。通过分光光度法跟踪了 poly（NIPAAm-co-MAH）共聚物的 LCST 行为。聚（St/PEG-EEM/DMAPM）纳米粒子的平均粒径为 78nm，表面电位为 54.4mV，平均粒径为 200nm，表面电位为 54.2mV，用于转染研究。使用 MTT 法测试了载体的细胞毒性。根据转染研究的条件（聚合物/细胞比和聚合物-细胞孵育时间），聚（St/PEG-EEM/DMAPM）粒径为 78nm 和 200nm 的细胞损失分别仅为 4%和 15%。聚（NIPAAm-co-MAH）的细胞毒性可以忽略不计。SMC 中 poly（NIPAAm-co-MAH）的摄取效率约为 85%，但与聚（St/PEG-EEM/DMAPM）/pEGFP-N2 缀合物相比，基因表达效率较低，因为共聚物的表面电位较低。纳米颗粒的聚合物摄取效率为 90-95%。用 78nm 和 200nm 聚（St/PEG-EEM/DMAPM）纳米颗粒转染 pEGFP-N2 缀合物后，GFP 表达效率分别为 68%和 64%。
