Journal
JOURNAL OF CONTROLLED RELEASE
Volume 133, Issue 3, Pages 178-184Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jconrel.2008.10.006
Keywords
Transferrin; Diphtheria toxin; Site-directed mutagenesis; Intracellular trafficking; Targeted cancer therapy
Funding
- Wallace H. Coulter Foundation Early Career Award
- Cancer Research Coordinating Committee
- USPHS [R01 (DK 21739)]
- Canadian institutes of Health Research-Canadian Blood Services Blood Utilization and Conservation Initiative [MOP 81286]
- Michael Smith Foundation for Health Research
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We previously demonstrated that decreasing the iron release rate of transferrin (Tf), by replacing the synergistic anion carbonate with oxalate, increases its in vitro drug carrier efficacy in HeLa cells. In the current work, the utility of this strategy has been further explored by generating two Tf mutants, K206E/R632A Tf and K206E/K534A Tf, exhibiting different degrees of iron release inhibition. The intracellular trafficking behavior of these Tf mutants has been assessed by measuring their association with HeLa cells. Compared to native Tf, the cellular association of K206E/R632A Tf and K206E/K534A Tf increased by 126 and 250%, respectively. Surface plasmon resonance studies clearly indicate that this increase in cellular association is due to a decrease in the iron release rate and not to differences in binding affinity of the mutants to the Tf receptor (TfR). Diphtheria toxin (DT) conjugates of K206E/R632A Tf and K206E/K534A Tf showed significantly increased cytotoxicity against HeLa cells with IC50 values of 1.00 pM and 0.93 pM, respectively, compared to a value of 1.73 pM for the native Tf conjugate. Besides further validating our strategy of inhibiting iron release, these Tf mutants provide proof-of-principle that site-directed mutagenesis offers an alternative method for improving the drug carrier efficacy of Tf. (C) 2008 Elsevier B.V. All rights reserved.
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