title: Transferrin-oligomers as potential carriers in anticancer drug delivery.
abstract: OBJECTIVE
  To investigate if the cross-linking of transferrin receptor (TfR) induced by Tf-oligomers alters the endocytosis of receptor-ligand complexes in cultured tumor cells and hence increases intracellular drug release.
  
  
  METHODS
  An average of 3.5 Tf molecules per aggregate were cross-linked either by using homobifunctional linker (1, 11-bis-maleimidotetraethyleneglycol) [Tf(3.5-BM(PEO)4)] or heterobifunction linker [succinimidyl 4-(-p-maleimidophenyl)-butyrate] (Tf(3.5-SMPB)). Cell surface binding and competition experiments with 125I-Tf for TfR binding were studied to demonstrate that Tf-oligomers maintain specificity of the TfR-binding. To determine the degradation of Tf-oligomers in TfR-mediated endocytosis, cultured tumor cells were pulsed for 15 min with 125I-Tf-oligomers and chased for 2 h at 37 degrees C in the presence of excess unlabeled Tf. The chase medium was subjected to TCA precipitation to separate the intact and degraded Tf. To investigate if the alteration of TfR-trafficking facilitates the intracellular release of the drug from the Tf-conjugated form, methotrexate (MTX) was conjugated to Tf-oligomer (Agg-Tf-MTX) and its antiproliferative activity was compared with monomeric-Tf-MTX (Mono-Tf-MTX) in human colon carcinoma (Caco-2) cells, human breast adenocarcinoma (MCF-7) cells, wild-type Chinese hamster ovary (CHO) cells, and MTX-resistant CHO (CHO-MTX-RII) cells.
  
  
  RESULTS
  TfR-mediated degradation of Tf-oligomers was higher than that of monomeric Tf in both Caco-2 and MCF-7 cells. The IC50 of Agg-Tf-MTX was lower than that of Mono-Tf-MTX in both tumor cell lines. The IC50 of MTX and Mono-Tf-MTX in CHO-MTX-RII cells was higher than that in wild-type CHO cells, whereas the Agg-Tf-MTX was almost identical in both the resistant and wild-type cells.
  
  
  CONCLUSIONS
  Cross-linking of TfR induced by oligomeric Tf binding alters the intracellular trafficking of Tf-TfR complexes, redirects them out of the recycling pathway, and targets them to intracellular degradation in cultured tumor cells. The alteration of TfR-trafficking facilitates the intracellular release of the drug from the Tf-conjugated form. Consequently, Agg-Tf-MTX is more effective than Mono-Tf-MTX as a TfR-mediated antiproliferative agent in tumor cells, as well as in MTX-resistant transport deficient cells. Therefore, Tf-oligomers are potentially effective TfR-targeting carriers for intracellular delivery of anticancer drugs.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angles, California 90089, USA.
authors.forename: Ching-Jou
authors.initials: CJ
authors.lastname: Lim
authors.forename: Wei-Chiang
authors.initials: WC
authors.lastname: Shen
mesh_terms: D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000493:pharmacokinetics; D000970:Antineoplastic Agents / Q000008:administration & dosage*; D016466:CHO Cells; D018938:Caco-2 Cells; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D006224:Cricetinae; D004337:Drug Carriers; D016503:Drug Delivery Systems; D004705:Endocytosis / Q000187:drug effects; D006801:Humans; D007202:Indicators and Reagents; D007976:Leupeptins / Q000378:metabolism; D008727:Methotrexate / Q000493:pharmacokinetics; D011108:Polymers; D011956:Receptors, Cell Surface / Q000378:metabolism; D011990:Receptors, Transferrin / Q000187:drug effects; D014168:Transferrin / Q000737:chemistry*
chemical_list: D000964:Antimetabolites, Antineoplastic; D000970:Antineoplastic Agents; D004337:Drug Carriers; D007202:Indicators and Reagents; D007976:Leupeptins; D011108:Polymers; D011956:Receptors, Cell Surface; D011990:Receptors, Transferrin; D014168:Transferrin; C032854:leupeptin; D008727:Methotrexate