Increased target specificity of anti-HER2 genospheres by modification of surface charge and degree of PEGylation
Increased target specificity of anti-HER2 genospheres by modification of surface charge and degree of PEGylation
复制标题
DOI：
10.1021/mp060040v
复制
发表时间：
2006-11-01
影响因子：
4.9
通讯作者：
Kirpotin, D. B.
中科院分区：
文献类型：
--
作者：
Hayes, M. E.;Drummond, D. C.;Kirpotin, D. B.
基因组球是阳离子脂质-核酸纳米颗粒，由脂类和核酸从水/有机液体单相中组装而成，可独立溶解各组分，其中生成的颗粒尺寸均匀(70-110 nm)，并有效地结合和保护DNA。在目前的研究中，我们使用pH可滴定的脂类证明了对生殖层表面电荷的pH依赖的调节。通过将脂类与可滴定的阴离子或咪唑头基结合，在中性pH下制备具有中性或阴离子表面电荷的基因组球，并比较其在乳腺癌细胞培养中对报告基因(荧光素酶)的摄取和转染率。用荧光显微镜研究颗粒-细胞结合的程度，并用细胞荧光法进行定量。此外，还研究了在低(0.5%)和高(5%)接枝密度下用聚乙二醇(分子量2000)修饰基因球表面的效果，以及通过聚乙二醇间隔区连接的可内化的抗HER2单链抗体F5对HER2受体定向靶向的影响。PH可滴定脂质CHEM(琥珀酸甘油酯)、CHIM(1-(3-(cholesteryloxycarbonylamino)propyl)imidazole)，或DSGG(1，2-二硬脂酰甘油-3-戊二酸半酯)配方中的包合物使粒子表面电荷在中性pH时为中性或略带阴离子，在中等酸性时为阳离子，如Zeta-电位测量所示。在HER2靶向系统中，HER2过表达的SKBR-3乳腺癌细胞的转染活性和靶向性依赖于基因组球表面电荷和聚乙二醇化。最高的靶特异性与中性pH下的低阳离子电荷有关，而加入5mol%的聚乙二醇脂对基因组球与细胞的结合、内化和转染活性只有轻微的影响。讨论了这项工作对体内潜在应用的影响。
Genospheres are cationic lipid-nucleic acid nanoparticles prepared by the assembly of the lipids and nucleic acids from an aqueous/organic liquid monophase that independently dissolves the components, where the resultant particles are homogeneously sized (70-110 nm), with efficiently incorporated and protected DNA. In the present study, we demonstrate pH-dependent modulation of the Genosphere surface charge using pH-titratable lipids. By incorporation of the lipids with titratable anionic or imidazole headgroups, Genospheres with neutral or anionic surface charge at neutral pH were produced and compared for cellular uptake and transfection of a reporter gene (luciferase) in culture of breast cancer cells. The extent of particle-cell association was also studied by fluorescent microscopy and quantified by cytofluorometery. The effects of Genosphere surface modification with poly(ethylene glycol) (molecular weight 2000) at low (0.5 mol %) and high (5 mol %) grafting densities, as well as the effects of HER2-receptor-directed targeting by an internalizable anti-HER2 scFv F5, linked via PEG spacer, were also studied. Inclusion in the Genosphere formulation of pH-titratable lipids CHEMS (cholesteryl hemisuccinate), CHIM (1-(3-(cholesteryloxycarbonylamino)propyl)imidazole), or DSGG (1, 2-distearoyl-sn-glycero-3-hemiglutarate) rendered the particles surface-charge neutral or slightly anionic at neutral pH, and cationic at mildly acidic pH, as shown by zeta-potential measurements. In HER2-targeted systems, transfection activity and target specificity with HER2-overexpressing SKBR-3 breast cancer cells were dependent on Genosphere surface charge and PEGylation. The highest target specificity correlated with low cationic charge at neutral pH, while incorporation of 5 mol % PEG-lipid had only minor effects on Genosphere-cell association, internalization, and transfection activity. The implications of this work for potential in vivo applications are discussed.