Liposomal Polyamine-Dialkyl Phosphate Conjugates as Effective Gene Carriers: Chemical Structure, Morphology, and Gene Transfer Activity
Liposomal Polyamine-Dialkyl Phosphate Conjugates as Effective Gene Carriers: Chemical Structure, Morphology, and Gene Transfer Activity
复制标题
DOI：
10.1021/bc900376y
复制
发表时间：
2010-05-01
影响因子：
4.7
通讯作者：
Nango, Mamoru
中科院分区：
文献类型：
--
作者：
Dewa, Takehisa;Asai, Tomohiro;Nango, Mamoru
合成阳离子脂质是有前途的基因治疗转染剂。我们在此报道，磷酸二烷基酯的多胺缀合物与天然脂质结合并以脂质体（聚阳离子脂质体：PCL）的形式组装，在COS-1细胞系中具有高转染活性。此外，我们描述了原子力显微镜（AFM）揭示的 PCL/DNA 复合物的功能形态。缀合物由磷酸二烷基酯（烷基链长度为 12、14 或 16 个碳）通过与聚胺分子、亚精胺、精胺或聚乙烯亚胺 (PEI(1800)) 反应合成。 [Dewa，T.，等人。生物共轭化学。 2004, 15, 824]。由亚精胺和C16结合物与磷脂和胆固醇（结合物/磷脂/胆固醇=1/1/1摩尔比）组成的PCL表现出比流行的商业产品高3.6倍的活性。系统测试揭示了转基因活性与多胺物理性质的明显相关性，特别是，较长的烷基链和较低分子量的多胺（亚精胺、精胺）有利于在较高的氮/磷酸比= 24（N/P，缀合物中氮与DNA中磷酸盐的化学计量比）下获得高功效。低分子量聚胺基 PCL 与质粒 DNA（脂质复合物）形成 150-400 nm 颗粒，其基因转移活性比相应缀合物的胶束聚集体（不含磷脂和胆固醇）高出 3 倍。相比之下，基于PEI的PCL形成大聚集体（类似于1μm），与胶束聚集体形式一样，其活性较低。低分子量聚胺基 PCL 的活性随着 lipoplex 的 NIP 线性增加，直至 N/P = 24。通过琼脂糖凝胶电泳、动态光散射 (DLS) 和 AFM 检查 lipoplex 的形成。在较低的 N/P = 5 时，观察到复合物 (I urn) 的大聚集体，其中 DNA 分子松散堆积。在较高的 NIP 下，脂质复合物转化为具有层状结构的较小颗粒（150-400 nin），其中 DNA 分子紧密堆积。 lipoplex 的这种形态特征与转染对 N/P 的依赖性相关，因为层状结构提供了优异的转染。 AFM 还表明脂质复合物显着分解。当 lipoplex 暴露于酸性条件 (pH 4) 时，会释放 DNA。相对于活性较低的胶束聚集体形式（对 pH 值变化无反应），讨论了双层脂质复合物变态的转染活性的重要性。
Synthetic cationic lipids are promising transfection agents for gene therapy. We report here that polyamine conjugates of dialkyl phosphates, combined with natural lipids and assembled in the form of liposomes (polycationic liposome: PCL), possess high transfection activity in the COS-1 cell line. Furthermore, we describe the functional morphology of the PCL/DNA complexes as revealed by atomic force microscopy (AFM). The conjugates were synthesized from dialkyl phosphates (with alkyl chain lengths of 12, 14, or 16 carbons) by reaction with the polyamine molecules, spermidine, spermine, or polyethylenimine (PEI(1800)). [Dewa, T., et al. Bioconjugate Chem. 2004, 15, 824]. The PCL composed of the spermidine and C16 conjugate combined with phospholipid and cholesterol (conjugate/phospholipid/cholesterol = 1/1/1 as a molar ratio) exhibited 3.6 times higher activity than that of a popular commercial product. Systematic tests revealed clear correlations of the transgene activity with physical properties of the polyamine, in particular, that longer alkyl chains and the lower molecular weight polyamines (spermidine, spermine) favor high efficacy at the higher nitrogen/phosphate ratio = 24 (N/P, stoichiometric ratio of nitrogen in the conjugate to phosphate in DNA). The low molecular weight polyamine-based PCLs, which formed 150-400 nm particles with plasmid DNA (lipoplexes), exhibited similar to 3-fold higher gene transfer activity than micellar aggregates (lacking phospholipid and cholesterol) of the corresponding conjugate. In contrast, the PEI-based PCL formed large aggregates (similar to 1 mu m), that, like the micellar aggregate form, had low activity. Activity of the low molecular weight polyamine-based PCLs increased linearly with the NIP of the lipoplex up to N/P = 24. Formation of lipoplexes was examined by agarose gel electrophoresis, dynamic light scattering (DLS), and AFM. At the lower N/P = 5, large aggregates of complex ( I urn), in which DNA molecules were loosely packed, were observed. At higher NIP, lipoplexes were converted into smaller particles (150-400 nin) having a lamellar structure, in which DNA molecules were tightly packed. Such morphological features of the lipoplex correlate with the dependence of transfection on the N/P in that the lamellar structures gave superior transfection. AFM also indicated that the lipoplexes disassembled significantly. releasing DNA, when the lipoplexes were exposed to acidic conditions (pH 4). The significance for transfection activity of the metamorphosis of bilayer lipoplexes is discussed relative to that of the less active micellar aggregate form, which is unresponsive to pH change.