School of Chemical Biology and Pharmaceutical Sciences, Capital Medical University, Beijing, China 100069.
Mol Pharm. 2011 Aug 1;8(4):1224-32. doi: 10.1021/mp200039s. Epub 2011 Jun 20.
Arginine-glycine-aspartate (RGD) has been shown to possess a strong affinity for the integrins overexpressed in tumor cells, especially during tumor invasion, angiogenesis and metasis. Based on work from others, a novel tetrapeptide, arginine-glycine-aspartate-phenylanaline (RGDF), has been designed and studied as a homing device to direct liposomal doxorubicin (DOX) to tumor cells in this work. In order to incorporate RGDF into liposomal DOX preparations, RGDF was conjugated with three different fatty alcohols to achieve RGDF-fatty alcohol conjugates. Glycine-glycine-aspartate-phenylanaline (GGDF)-lauryl alcohol conjugate was synthesized as a negative control. RGDF-fatty alcohol conjugates (RGDFO(CH(2))(n)CH(3)) and GGDF-lauryl alcohol conjugate (L-GGDFC12-DOX) incorporated liposomal preparations were obtained by first preparing liposomes using the film dispersion method followed by loading DOX using a transmembrane pH gradient method. Because of their amphipathic nature, RGDF- or GGDF-fatty alcohol conjugates are expected to be readily incorporated into liposomes with their fatty alkanyl chains being intercalated between fatty acyl chains of liposomal bilayers and the hydrophilic peptide moiety (RGDF or GGDF) being anchored on the surface of liposomes. The particle size and zeta potential of liposomal DOX preparations containing RGDF-fatty alcohol conjugate (L-RGDF-DOXs) or L-GGDFC12-DOX were measured, and their morphology was studied using transmission electron microscopy. In vitro DOX release profile from RGDF incorporated liposomal DOX was measured. The antitumor activities of RGDF incorporated liposomal DOX preparations were evaluated in ICR mice inoculated with sarcoma S(180), which is known to express α(v)β(3) integrin. Both conventional liposomal DOX preparation (L-DOX) without RGDFO(CH(2))(n)CH(3) and L-GGDFC12-DOX were used as negative controls. Our results showed improved tumor growth inhibition with L-RGDF-DOXs over doxorubicin hydrochloride solution, L-DOX and L-GGDFC12-DOX. Pathological examination of tumor biopsy demonstrated that L-RGDF-DOXs induced enhanced tumor cell death in comparison to negative controls. Pharmacokinetic studies showed that the concentrations of DOX found in tumor sites were increased by 1.7-4.5-fold when liposomal DOX preparation containing RGDF-lauryl alcohol conjugate (L-RGDFC12-DOX) was administered in comparison to when L-GGDFC12-DOX or doxorubicin hydrochloride solution was administered. The concentrations of DOX found in the heart, which is the main site of toxic effects of DOX, were significantly reduced when L-RGDFC12-DOX was administered in comparison to when L-GGDFC12-DOX or doxorubicin hydrochloride solution was administered.
精氨酸-甘氨酸-天冬氨酸（RGD）已被证明对肿瘤细胞过度表达的整合素有很强的亲和力，特别是在肿瘤侵袭、血管生成和转移过程中。基于他人的工作，设计并研究了一种新型四肽，精氨酸-甘氨酸-天冬氨酸-苯丙氨酸（RGDF），作为一种归巢装置，将阿霉素脂质体（DOX）导向肿瘤细胞。为了将 RGDF 掺入脂质体 DOX 制剂中，将 RGDF 与三种不同的脂肪醇偶联以得到 RGDF-脂肪醇缀合物。合成甘氨酸-甘氨酸-天冬氨酸-苯丙氨酸（GGDF）-月桂醇缀合物作为阴性对照。通过首先使用薄膜分散法制备脂质体，然后使用跨膜 pH 梯度法加载 DOX，获得包含 RGDF-脂肪醇缀合物（RGDFO（CH（2））（n）CH（3））和 GGDF-月桂醇缀合物（L-GGDFC12-DOX）的脂质体制剂。由于它们的两亲性，RGDF 或 GGDF-脂肪醇缀合物有望很容易地掺入脂质体中，其脂肪烷链插入脂质体双层的脂肪酰链之间，并且亲水性肽部分（RGDF 或 GGDF）锚定在脂质体的表面上。测量了含有 RGDF-脂肪醇缀合物（L-RGDF-DOXs）或 L-GGDFC12-DOX 的脂质体 DOX 制剂的粒径和 zeta 电位，并使用透射电子显微镜研究了它们的形态。测量了 RGDF 掺入的脂质体 DOX 中 DOX 的体外释放曲线。用肉瘤 S（180）接种 ICR 小鼠评价了 RGDF 掺入的脂质体 DOX 制剂的抗肿瘤活性，肉瘤 S（180）已知表达α（v）β（3）整合素。未含有 RGDFO（CH（2））（n）CH（3）的常规脂质体 DOX 制剂（L-DOX）和 L-GGDFC12-DOX 均用作阴性对照。我们的结果表明，与盐酸多柔比星、L-DOX 和 L-GGDFC12-DOX 相比，L-RGDF-DOX 对肿瘤生长的抑制作用得到了改善。肿瘤活检的病理检查表明，与阴性对照相比，L-RGDF-DOX 诱导的肿瘤细胞死亡增强。药代动力学研究表明，与 L-GGDFC12-DOX 或盐酸多柔比星溶液给药相比，含有 RGDF-月桂醇缀合物（L-RGDFC12-DOX）的脂质体 DOX 制剂给药时，肿瘤部位 DOX 的浓度增加了 1.7-4.5 倍。与 L-GGDFC12-DOX 或盐酸多柔比星溶液给药相比，心脏（DOX 的主要毒性作用部位）中 DOX 的浓度显著降低。
