Koning G A, Morselt H W, Velinova M J, Donga J, Gorter A, Allen T M, Zalipsky S, Kamps J A, Scherphof G L
Department of Physiological Chemistry, Groningen University Institute for Drug Exploration (GUIDE), Faculty of Medical Sciences, University of Groningen, A. Deusinglaan 1, 9713 AV, Groningen, The Netherlands.
Biochim Biophys Acta. 1999 Aug 20;1420(1-2):153-67. doi: 10.1016/s0005-2736(99)00091-7.
A monoclonal antibody against the rat colon carcinoma CC531 was covalently coupled to liposomes containing a dipalmitoylated derivative of the anticancer drug FUdR as a prodrug in their bilayers. We investigated the in vitro interaction of these liposomes with CC531 target cells and the mechanism by which they deliver the active drug FUdR intracellularly to the cells by monitoring the fate of the liposomal bilayer markers cholesterol-[(14)C]oleate and [(3)H]cholesteryloleylether as well as the (3)H-labeled prodrug and colloidal gold as an encapsulated liposome marker. After binding of the immunoliposomes to the cell surface, only limited amounts were internalized as demonstrated by a low level of hydrolysis of liposomal cholesterol ester and by morphological studies employing colloidal gold-labeled immunoliposomes. By contrast, already within 24 h immunoliposome-incorporated FUdR-dP was hydrolyzed virtually completely to the parent drug FUdR intracellularly. This process was inhibited by a variety of endocytosis inhibitors, indicating that the prodrug enters and is processed by the cells by a mechanism involving an endocytic process, resulting in intracellular FUdR concentrations up to 3000-fold higher than those in the medium. Immunoliposomes containing poly(ethyleneglycol) (PEG) chains on their surface, with the antibody coupled either directly to the bilayer or at the distal end of the PEG chains were able to deliver the prodrug into the tumor cells at the same rate as immunoliposomes without PEG. Based on these observations, we tentatively conclude that during the interaction of the immunoliposomes with the tumor cells the lipophilic prodrug FUdR-dP is selectively transferred to the cell surface and subsequently internalized by constitutive endocytic or pinocytic invaginations of the plasma membrane, thus ultimately delivering the prodrug to a lysosomal compartment where hydrolysis and release of parent drug takes place. This concept allows for an efficient delivery of a liposome-associated drug without the need for the liposome as such to be internalized by the cells.
一种抗大鼠结肠癌CC531的单克隆抗体与脂质体共价偶联，该脂质体在其双层膜中含有作为前药的抗癌药物氟尿苷（FUdR）的二棕榈酰化衍生物。我们通过监测脂质体双层标记物胆固醇 - [（14）C]油酸酯和[（3）H]胆固醇油酸醚以及（3）H标记的前药和作为包封脂质体标记物的胶体金的命运，研究了这些脂质体与CC531靶细胞的体外相互作用以及它们将活性药物FUdR细胞内递送至细胞的机制。免疫脂质体与细胞表面结合后，脂质体胆固醇酯的低水平水解和使用胶体金标记的免疫脂质体的形态学研究表明，只有有限量的脂质体被内化。相比之下，在24小时内，掺入免疫脂质体的FUdR - dP几乎完全在细胞内水解为母体药物FUdR。这个过程受到多种内吞作用抑制剂的抑制，表明前药通过涉及内吞过程的机制进入细胞并被细胞处理，导致细胞内FUdR浓度比培养基中的浓度高3000倍。表面含有聚乙二醇（PEG）链的免疫脂质体，抗体要么直接偶联到双层膜上，要么偶联到PEG链的远端，能够以与不含PEG的免疫脂质体相同的速率将前药递送至肿瘤细胞。基于这些观察结果，我们初步得出结论，在免疫脂质体与肿瘤细胞相互作用期间，亲脂性前药FUdR - dP被选择性地转移到细胞表面，随后通过质膜的组成型内吞或胞饮内陷作用被内化，从而最终将前药递送至溶酶体区室进行水解并释放母体药物。这个概念允许高效递送与脂质体相关联的药物，而不需要脂质体本身被细胞内化。
