Modified linear polyethylenimine-cholesterol conjugates for DNA complexation
Modified linear polyethylenimine-cholesterol conjugates for DNA complexation
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DOI：
10.1021/bc0340565
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发表时间：
2003-07-01
影响因子：
4.7
通讯作者：
Kim, SW
中科院分区：
文献类型：
--
作者：
Furgeson, DY;Chan, WS;Kim, SW
线性聚乙烯亚胺（LPEI）是一种有效的非病毒基因载体，其转染水平等于或高于支化聚乙烯亚胺（BPEI），并且表现出比BPEI更低的细胞毒性。高分子量LPEI M-w 25 k用三种不同几何形状的胆固醇修饰：线性形状（L）、T形形状（T）和组合的线性/T形形状（LT），分别形成LPEI-胆固醇（LPC）缀合物LPC-L、LPC-T和LPC-LT。LPC/pDNA复合物的物理表征包括粒径、zeta电位、DNA酶保护、mIL-12 p70表达和细胞毒性。通过原子力显微镜（AFM）进一步确认颗粒尺寸。当N/P为10/1时，LPC-T/pDNA复合物的粒径最大，约为250 nm，表面电荷为+10 mV。这些复合物还在DNA酶I存在下有效地保护pDNA长达180分钟。用LPC-L和LPC-T转染的B16-F0细胞显示出比单独的LPEI更高的蛋白表达水平，是BPEI的两倍，但细胞活力没有任何显著损失。用EGFP流式细胞术和转染Renca细胞证实了这些结果。LPC/pDNA复合物的转染速率的差异部分是由于从复合物形成到与质膜相互作用的构象变化。这些构象变化通过胆固醇部分的疏水保护为LPEI骨架中的未质子化仲胺提供保护，我们称之为“未质子化储备”。最后，我们表明LPC缀合物通过LDL-R途径利用受体介导的内吞作用，在用hLDL-R-Ab使MCF-7细胞上的LDL-R位点饱和后，转基因表达水平降低近20%。
Linear polyethylenimine (LPEI) is an effective nonviral gene carrier with transfection levels equal or above branched polyethylenimine (BPEI) and exhibits a lower cytotoxicity profile than BPEI. High molecular weight LPEI M-w 25 k was modified with cholesterol in three different geometries: linear shaped (L), T-shaped (T), and a combined linear/T-shaped (LT) forming the LPEI-cholesterol (LPC) conjugates LPC-L, LPC-T, and LPC-LT, respectively. Physical characterization of LPC/pDNA complexes included particle size, zeta potential, DNase protection, mIL-12 p70 expression, and cytotoxicity. The particle size was further confirmed by atomic force microscopy (AFM). The LPC-T/pDNA complexes were optimal at N/P 10/1 that resulted in a particle size of similar to250 nm, which was confirmed by AFM, and a surface charge of +10 mV. These complexes also effectively protected the pDNA for up to 180 min in the presence of DNase I. B16-F0 cells transfected with LPC-L and LPC-T showed protein expression levels higher than LPEI alone and twice that of BPEI but without any significant loss in cell viability. These results were confirmed with EGFP flow cytometry and transfection of Renca cells. The differences in rates of transfection of the LPC/pDNA complexes is due in part to conformational changes from the point of complex formation to interaction with the plasma membrane. These conformation changes provide protection for unprotonated secondary amines in the LPEI backbone by hydrophobic protection of the cholesterol moiety that we termed "unprotonated reserves". Finally, we show that LPC conjugates exploit receptor-mediated endocytosis via the LDL-R pathway with transgene expression levels decreasing nearly 20% after saturating the LDL-R sites on MCF-7 cells with hLDL-R-Ab.