Controllable drug uptake and nongenomic response through estrogen-anchored cyclodextrin drug complex

Breast cancer is a leading killer of women worldwide. Cyclodextrin-based estrogen receptor-targeting drug-delivery systems represent a promising direction in cancer therapy but have rarely been investigated. To seek new targeting therapies for membrane estrogen receptor-positive breast cancer, an estrogen-anchored cyclodextrin encapsulating a doxorubicin derivative Ada-DOX (CDE(1)-Ada-DOX) has been synthesized and evaluated in human breast cancer MCF-7 cells. First, we synthesized estrone-conjugated cyclodextrin (CDE(1)), which formed the complex CDE(1)-Ada-DOX via molecular recognition with the derivative adamantane-doxorubicin (Ada-DOX) (K(d) =1,617 M(−1)). The structure of the targeting vector CDE(1) was fully characterized using (1)H- and (13)C-nuclear magnetic resonance, mass spectrometry, and electron microscopy. CDE(1)-Ada-DOX showed two-phase drug-release kinetics with much slower release than Ada-DOX. The fluorescence polarization analysis reveals that CDE(1)-Ada-DOX binds to recombinant human estrogen receptor α fragments with a K(d) of 0.027 µM. Competition assay of the drug complex with estrogen ligands demonstrated that estrone and tamoxifen competed with CDE(1)-Ada-DOX for membrane estrogen receptor binding in MCF-7 cells. Intermolecular self-assembly of CDE(1) molecules were observed, showing tail-in-bucket and wire-like structures confirmed by transmission electronic microscopy. CDE(1)-Ada-DOX had an unexpected lower drug uptake (when the host–guest ratio was >1) than non-targeting drugs in MCF-7 cells due to ensconced ligands in cyclodextrins cavities resulting from the intermolecular self-assembly. The uptake of CDE(1)-Ada-DOX was significantly increased when the host–guest ratio was adjusted to be less than half at the concentration of CDE(1) over 5 µM due to the release of the estrone residues. CDE(1) elicited rapid activation of mitogen-activated protein kinases (p44/42 MAPK, Erk1/2) in minutes through phosphorylation of Thr202/Tyr204 in MCF-7 cells. These results demonstrate a targeted therapeutics delivery of CDE(1)-Ada-DOX to breast cancer cells in a controlled manner and that the drug vector CDE(1) can potentially be employed as a molecular tool to differentiate nongenomic from genomic mechanism.