RESUMO
A ß-cyclodextrin (ß-CD) nanosponge (NS) was synthesized using diphenyl carbonate (DPC) as a cross-linker to encapsulate the antitumor drug cyclophosphamide (CYC), thus obtaining the NSs-CYC system. The formulation was then associated with magnetite nanoparticles (MNPs) to develop the MNPs-NSs-CYC ternary system. The formulations mentioned above were characterized to confirm the deposition of the MNPs onto the organic matrix and that the superparamagnetic nature of the MNPs was preserved upon association. The association of the MNPs with the NSs-drug complex was confirmed through field emission scanning electron microscopy, energy dispersive spectroscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, dynamic light scattering, ζ-potential, atomic absorption spectroscopy, X-ray powder diffraction, selected area electron diffraction, and vibrating-sample magnetometer. The superparamagnetic properties of the ternary system allowed the release of CYC by utilizing magnetic hyperthermia upon the exposure of an alternating magnetic field (AMF). The drug release experiments were carried out at different frequencies and intensities of the magnetic field, complying with the "Atkinson-Brezovich criterion". The assays in AMF showed the feasibility of release by controlling hyperthermia of the drug, finding that the most efficient conditions were F = 280 kHz, H = 15 mT, and a concentration of MNPs of 5 mg/mL. CYC release was temperature-dependent, facilitated by local heat generation through magnetic hyperthermia. This phenomenon was confirmed by DFT calculations. Furthermore, the ternary systems outperformed the formulations without MNPs regarding the amount of released drug. The MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays demonstrated that including CYC within the magnetic NS cavities reduced the effects on mitochondrial activity compared to those observed with the free drug. Finally, the magnetic hyperthermia assays showed that the tertiary system allows the generation of apoptosis in HeLa cells, demonstrating that the MNPs embedded maintain their properties to generate hyperthermia. These results suggest that using NSs associated with MNPs could be a potential tool for a controlled drug delivery in tumor therapy since the materials are efficient and potentially nontoxic.
RESUMO
The metabolite 2-methoxyestradiol (2ME) is an endogenous estrogen metabolite with potential therapeutic properties in reproductive cancers. However, the molecular mechanisms by which 2ME exerts its anticancer activity are not well elucidated. The purpose of this study was to determine the molecular signals associated with the apoptotic effects of 2ME in a human endometrial cancer cell line. Ishikawa cells were treated with non-apoptotic (0.1 µM) or apoptotic concentrations (5 µM) of 2ME, and 12 hours later mRNA levels for Scd2, Snx6, and Spon1 were determined by real-time PCR. We then investigated by immunofluorescence and Western blot the expression and distribution of F-spondin, encoded by Spon1, in Ishikawa cells treated with 2ME 5 µM at 6, 12, or 24 h after treatment. The role of estrogen receptors (ER) in the effect of 2ME on the Spon1 level was also investigated. Finally, we examined whether 2ME 5 µM induces cell death in Ishikawa cells pre-incubated with a neutralizing F-spondin antibody. Non-apoptotic or apoptotic concentrations of 2ME decreased Scd2 and increased Snx6. However, Spon1 was only increased with the 2ME apoptotic concentration. F-spondin protein was also increased at 12 and 24 h after 2ME treatment, while 2ME-induced Spon1 increase was independent of ER. Neutralization of F-spondin blocked the effect of 2ME on the cell viability. These results show that F-spondin signaling is one of the components in the apoptotic effects of 2ME on Ishikawa cells and provide experimental evidence underlying the mechanism of action of this estrogen metabolite on cancer cells.