Intelligent biosynthetic nanobiomaterials (IBNs) for hyperthermic gene delivery
Intelligent biosynthetic nanobiomaterials (IBNs) for hyperthermic gene delivery
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DOI：
10.1007/s11095-007-9382-5
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发表时间：
2008-03-01
影响因子：
3.7
通讯作者：
Furgeson, Darin Y.
中科院分区：
文献类型：
--
作者：
Chen, Tze-Haw Howard;Bae, Younsoo;Furgeson, Darin Y.
目的.智能生物合成纳米生物材料（IBN）是由重组二嵌段共聚物K-8-ELP（1-60）组成，其中含有一个阳离子寡赖氨酸（VGK（8）G）和一个具有60个重复五肽单元的热敏弹性蛋白样多肽（ELP）嵌段[（VPGXG）（60）; X为瓦尔、Ala和Gly，比例为5：2：3]。K-8-ELP（1-60）通过递归定向连接DNA寡聚化来合成。通过SDS-PAGE和质谱法确认K-8-ELP（1-60）的纯度和分子量。从K-8-ELP（1-60）和pGL 3-Control（pGL 3-C）质粒DNA（pDNA）制备DNA聚合复合物，并通过凝胶阻滞、DLS和肝素置换DNA评价稳定性。通过在350 nm处测量浊度变化来研究聚合物的热转变曲线，并使用聚合物来抑制MCF-7细胞，同时进行细胞毒性试验。SDS-PAGE和MALDI-TOF研究显示了所需分子量的高纯度共聚物。K-8-ELP（1-60）以大于0.25的阳离子与阴离子（N/P）比浓缩pDNA，随着N/P比的增加，颗粒尺寸在115.5- 32.4nm范围内紧密分布。K-8-ELP（1-60）/pDNA和单独的K-8-ELP（1-60）的热转变温度分别为44.9和71.5 ℃。与支化聚乙烯亚胺对照相比，K-8-ELP（1-60）/pDNA复合物成功地转导了MCF-7细胞，其定性表达增强的绿色荧光蛋白（EGFP）和最小的细胞毒性。K-8-ELP（1-60）经重组方法成功地设计和纯化，其pDNA在N/P比>0.25和聚合物颗粒大小下有效和稳定地浓缩
Purpose. Intelligent biosynthetic nanobiomaterials (IBNs) were constructed as recombinant diblock copolymers, notated as K-8-ELP(1-60), containing a cationic oligolysine (VGK(8)G) and a thermosensitive elastin-like polypeptide (ELP) block with 60 repetitive pentapeptide units [(VPGXG)(60); X is Val, Ala and Gly in a 5:2:3 ratio].Methods. K-8-ELP(1-60) was synthesized by recursive directional ligation for DNA oligomerization. Purity and molecular weight of K-8-ELP(1-60) were confirmed by SDS-PAGE and mass spectrometry. DNA polyplexes were prepared from K-8-ELP(1-60) and pGL3-Control (pGL3-C) plasmid DNA (pDNA) and stability was evaluated by gel retardation, DLS, and DNA displacement with heparin. Thermal transition profiles were studied by measuring the turbidity change at 350 nm and the polyplexes were used to transfect MCF-7 cells with a concomitant cytotoxicity assay.Results. SDS-PAGE and MALDI-TOF studies showed highly pure copolymers at the desired molecular weight. K-8-ELP(1-60) condensed pDNA at a cation to anion (N/P) ratio above 0.25 with a tight distribution of particle size ranging from 115.5-32.4 nm with increasing N/P ratio. Thermal transition temperatures of K-8-ELP(1-60)/pDNA and K-8-ELP(1-60) alone were 44.9 and 71.5 degrees C, respectively. K-8-ELP(1-60)/pDNA complexes successfully transduced MCF-7 cells with qualitative expression of enhanced green fluorescent protein (EGFP) and minimal cytotoxicity compared to branched poly(ethyleneimine) controls.Conclusions. K-8-ELP(1-60) was successfully designed and purified through recombinant means with efficient and stable condensation of pDNA at N/P ratios >0.25 and polyplex particle size