Department of Medical Biochemistry and Global COE, University of Shizuoka Graduate School of Pharmaceutical Sciences, Shizuoka 422-8526, Japan.
Bioconjug Chem. 2011 Mar 16;22(3):429-35. doi: 10.1021/bc1004697. Epub 2011 Mar 1.
Dicetyl phosphate-tetraethylenepentamine (DCP-TEPA) conjugate was newly synthesized and formed into liposomes for efficient siRNA delivery. Formulation of DCP-TEPA-based polycation liposomes (TEPA-PCL) complexed with siRNA was examined by performing knockdown experiments using stable EGFP-transfected HT1080 human fibrosarcoma cells and siRNA for GFP. An adequate amount of DCP-TEPA in TEPA-PCL and N/P ratio of TEPA-PCL/siRNA complexes were determined based on the knockdown efficiency. Then, the biodistribution of TEPA-PCL modified with poly(ethylene glycol) (PEG) was examined in BALB/c mice. As a result, TEPA-PCL modified with PEG6000 avoided reticuloendothelial system uptake and showed long circulation in the bloodstream. On the other hand, PEGylation of TEPA-PCL/siRNA complexes caused dissociation of a portion of the siRNA from the liposomes. However, we found that the use of cholesterol-conjugated siRNA improved the interaction between TEPA-PCL and siRNA, which allowed PEGylation of TEPA-PCL/siRNA complexes without siRNA dissociation. In addition, TEPA-PCL complexed with cholesterol-conjugated siRNA showed potent knockdown efficiency in stable luciferase-transfected B16-F10 murine melanoma cells. Finally, the biodistribution of cholesterol-conjugated siRNA formulated in PEGylated TEPA-PCL was examined by performing near-infrared fluorescence imaging in Colon26 NL-17 murine carcinoma-bearing mice. Our results showed that tumor targeting with siRNA via systemic administration was achieved by using PEGylated TEPA-PCL combined with active targeting with Ala-Pro-Arg-Pro-Gly, a peptide used for targeting angiogenic endothelium.
新合成了磷酸二辛酯-四乙烯五胺（DCP-TEPA）缀合物，并将其制成脂质体以实现高效的 siRNA 递送。通过使用稳定转染 EGFP 的 HT1080 人纤维肉瘤细胞和 GFP 的 siRNA 进行敲低实验，考察了基于 DCP-TEPA 的聚阳离子脂质体（TEPA-PCL）与 siRNA 形成的复合物的形成。根据敲低效率确定了 TEPA-PCL 中适当量的 DCP-TEPA 和 TEPA-PCL/siRNA 复合物的 N/P 比。然后，在 BALB/c 小鼠中考察了用聚乙二醇（PEG）修饰的 TEPA-PCL 的体内分布。结果表明，用 PEG6000 修饰的 TEPA-PCL 避免了网状内皮系统的摄取，并在血液中表现出长循环。另一方面，PEG 化 TEPA-PCL/siRNA 复合物导致部分 siRNA 从脂质体中解离。然而，我们发现使用胆固醇偶联的 siRNA 可以改善 TEPA-PCL 与 siRNA 之间的相互作用，从而允许在不引起 siRNA 解离的情况下对 TEPA-PCL/siRNA 复合物进行 PEG 化。此外，与胆固醇偶联的 siRNA 复合的 TEPA-PCL 在稳定转染荧光素酶的 B16-F10 鼠黑色素瘤细胞中显示出强大的敲低效率。最后，通过在携带 Colon26 NL-17 鼠癌的小鼠中进行近红外荧光成像，考察了用 PEG 化的 TEPA-PCL 包封的胆固醇偶联 siRNA 的体内分布。我们的结果表明，通过使用与用于靶向血管生成内皮的肽 Ala-Pro-Arg-Pro-Gly 进行主动靶向的 PEG 化 TEPA-PCL，通过系统给药实现了 siRNA 的肿瘤靶向。
