Experimental Oncology Laboratory, Shaare Zedek Medical Center, Jerusalem, Israel.
J Control Release. 2009 Jun 5;136(2):155-60. doi: 10.1016/j.jconrel.2009.02.002. Epub 2009 Feb 7.
Receptor-directed targeting of ligand-bearing liposomes to tumor cells may enhance therapeutic efficacy by intracellular delivery of a concentrated payload of liposomal drug. The goal of this study was to assess whether Her2-targeted pegylated liposomal doxorubicin (PLD) retains its binding ability to Her2-expressing target cells through circulation in the blood and extravasation to tumor interstitial fluid.
PLD was grafted with a lipophilic conjugate of an anti-Her2 scFv antibody fragment at an approximate ratio of 7.5, 15, or 30 ligands per liposome. BALB/c mice were injected with J6456 lymphoma cells into the peritoneal cavity to generate malignant ascites used as a model for tumor interstitial fluid. When abdominal swelling developed, Her2-targeted (HT-) PLD and non-targeted PLD were injected into the mice i.v. at a dose of 15 mg/kg. The ascitic fluid was collected 48 h later, ascitic tumor cells were removed, and the doxorubicin levels in the cell-free ascitic fluid and plasma were determined. Binding of the cell-free ascitic fluid was tested in vitro against two Her2-expressing human tumor cell lines (N87, SKBR-3) and compared to the binding of shelf formulations (not passaged in vivo) of HT-PLD and PLD, by measuring the amount of cell-associated doxorubicin.
Plasma and ascitic fluid levels of HT-PLD were only slightly below those of PLD indicating that, the Her2 ligand did not cause any significant change in the clearance rate of PLD. The in vitro binding of HT-PLD containing ascitic fluid to Her2-expressing cells was increased 10 to 20-fold above that of PLD-containing ascitic fluid, similarly to the 20-fold difference in binding between shelf Her2-PLD and PLD. The in vitro cytotoxicity of ascitic fluid containing HT-PLD tested against Her2-expressing tumor cells was far greater than that of PLD, and similar to that of the shelf formulations, indicating that the selective pharmacological activity of HT-PLD is preserved after in vivo passage. Optimal results were obtained with HT-PLD formulated with 15 ligands per liposome.
HT-PLD retains most of its original binding capacity to Her2-expressing cells after in vivo passage indicating that the ligand is stably maintained in vivo in association with the doxorubicin liposomal carrier, and confirming the validity of the post-formulation ligand grafting approach for liposome targeting. Targeting of PLD using a Her2 antibody fragment provides an important means of in vivo selective drug delivery to tumors expressing the Her2 receptor.
配体携带的脂质体通过靶向受体向肿瘤细胞进行递药，可能会通过将脂质体药物的浓缩载药递送至细胞内来提高治疗效果。本研究的目的是评估 Her2 靶向性的聚乙二醇化多柔比星脂质体（PLD）在血液循环和向肿瘤间质液外渗过程中是否保留其与 Her2 表达靶细胞的结合能力。
将 PLD 与抗 Her2 scFv 抗体片段的亲脂性缀合物以每脂质体约 7.5、15 或 30 个配体的比例进行嫁接。将 J6456 淋巴瘤细胞注入 BALB/c 小鼠的腹腔中，生成恶性腹水，作为肿瘤间质液的模型。当腹部肿胀发展时，以 15mg/kg 的剂量将 Her2 靶向性（HT-）PLD 和非靶向性 PLD 静脉内注射入小鼠体内。48 小时后收集腹水，去除腹水肿瘤细胞，并测定细胞游离腹水和血浆中的多柔比星水平。通过测量细胞相关的多柔比星量，在体外测试细胞游离腹水针对两种表达 Her2 的人类肿瘤细胞系（N87、SKBR-3）的结合情况，并与 HT-PLD 和 PLD 的货架制剂（未在体内传代）的结合情况进行比较。
HT-PLD 的血浆和腹水水平仅略低于 PLD，表明 Her2 配体未使 PLD 的清除率发生任何显著变化。含有 HT-PLD 的腹水与表达 Her2 的细胞的体外结合增加了 10 至 20 倍，与货架上 Her2-PLD 和 PLD 之间的 20 倍差异相似。针对表达 Her2 的肿瘤细胞进行测试时，含有 HT-PLD 的腹水的体外细胞毒性远大于 PLD，与货架制剂相似，表明 HT-PLD 的选择性药理活性在体内传代后得以保留。每脂质体含有 15 个配体的 HT-PLD 制剂可获得最佳效果。
HT-PLD 在体内传代后保留了其与表达 Her2 的细胞的大部分原始结合能力，表明配体在体内与多柔比星脂质体载体稳定结合，并证实了脂质体靶向的制剂后配体嫁接方法的有效性。使用 Her2 抗体片段对 PLD 进行靶向治疗为表达 Her2 受体的肿瘤提供了一种重要的体内选择性药物递送手段。
