Synergistic Effect of Quercetin Magnetite Nanoparticles and Targeted Radiotherapy in Treatment of Breast Cancer

Abstract

Quercetin is a potent cancer therapeutic agent present in fruits and vegetables. The pharmaceutical uses of quercetin are limited due to many problems associated with low solubility, bioavailability, permeability, and instability. In addition, the high doses of quercetin show toxic effects in clinical and experimental studies. Therefore, a new strategy is warranted to overcome these problems without the use of toxic doses. The iron oxide nanoparticles can be used as a drug delivery system. This study aimed to prepare quercetin-conjugated magnetite nanoparticles (QMNPs) using biological simple nanoprecipitation and mediated by fungus Aspergillus oryzae. Also, we initiated in vitro and in vivo studies to determine whether QMNPs might sensitize breast cancer to radiotherapy treatment. The structural, morphological, and magnetic properties of the prepared nanoparticles were studied. The results indicated that QMNPs were spherical in shape and 40 nm in diameter. The in vitro studies showed that the incubation of MCF-7, HePG-2, and A459 cancer cells with QMNPs for 24 h effectively inhibited the growth of cancer cell lines in a concentration-dependent manner with IC50 values of 11, 77.5, and104 nmol/mL, respectively. The combination of QMNPs with irradiation (IR) potently blocked MCF-7 cancer cell proliferation and showed significant changes in the morphology of these cells as observed by bright-field inverted light microscopy. Focusing on the long-term toxicity of QMNPs (20 ml/kg), the assessment of hematological, hepatic, and renal markers indicated no toxic effect. Besides, QMNPs inhibited tumor growth and potently enhanced the lateral radiotherapy treatment in N-methyl-N-nitrosourea (MNU)-induced breast cancer in female white albino rats. These anticancer and radiosensitizing activities were ascribed to cytotoxicity, cell cycle arrest, immunomodulation, and efficiency through induction of apoptosis. In a conclusion, these observations suggest that the QMNPs combined with LRT could act as a potential targeted therapy in breast cancer.

Keywords: Nanoparticles; apoptosis; breast cancer; magnetic oxide; methyl-N-nitrosourea; quercetin; radiotherapy.

Figure 1.

(A) Diagram of animal local RT by cobalt 60 and (B) schematic illustration of the experimental design and tumor induction. MNU indicates 1-methyl-1-nitrosourea; QMNP, quercetin magnetite nanoparticles.

Figure 2.

Structural preparation and characterization of QMNPs: (A) schematic illustration of the reaction mechanisms for the overall synthesis process of the quercetin-loaded Fe3O4 nanoparticles via A. oryzae, (B) visual characterization of the synthesized QMNPs, (C) the chromatogram of UV spectrophotometry of MNPs (curve 1) and QMNPs (curve 2) mediated by A. oryzae using FeSO4 as a substrate, (D) TEM images of the synthesized MNPs (A) and QMNPs (B) mediated by A. oryzae and FeSO4 as a substrate, magnification (X = 200 000 K for A and X = 100 000 K for B). (E) XRD patterns of the synthesized MNPs (A) and QMNPs (B) mediated by A. orrzae and FeSO4 as a substrate. (F) FTIR analysis of magnetite (Fe3O4) nanoparticles (A), magnetite (Fe3O4)–quercetin with FeSO4 substrate. FTIR indicates Fourier transform infrared; MNP, magnetite nanoparticles; QMNP, quercetin magnetite nanoparticles; TEM, transmission electron microscope; UV, ultraviolet; XRD, X-ray diffraction.

Figure 3.

(A) Dose-dependent cytotoxicity and IC50 values of QMNPs on MCF-7, Hep-G2, and A-549 cell lines at 24 h treatment. (B) Estimation of the optimal radiotherapy dose on MCF-7 cell line. (C) Radiosensitization effect of QMNPs on MCF-7 cell line using; MCF-7 cells treated with 11 nmol/mL QMNPs for 24 h prior to exposure to 6 Gy γ-ray. (D) Effect of QMNPs and/or RT on cell cycle distribution. Values expressed as the mean ± SEM, n = 5. QMNP indicates quercetin magnetite nanoparticles; RT, radiotherapy; SEM, mean ± standard error. ^a1P < .001, ^a2P < .01 vs control; ^b1P < .001, ^b2P < .01, ^b3P < .05 vs RT group (4 Gy) and RT (6 Gy); ^c3P < .05 vs QMNPs group.

Figure 4.

QMNPs effect on vital indices: (A) hematological indices, (B) hepatic, renal markers, and antioxidants/oxidative markers. Values expressed as the mean ± SEM, n = 5. ALB indicates albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CAT, catalase; GSH, glutathione; MDA, malondialdehyde; PLT indicates platelets; QMNP, quercetin magnetite nanoparticles; RBC, red blood cells; SEM, mean ± standard error; SOD, superoxide dismutase; WBC, white blood cells. ^a3P < .05 vs control.

Figure 5.

QMNPs and/or RT effect on morphological and histopathological indices. (Control rat) showing normal mammary acini (arrow) embedded in the surrounding adipose tissue. (Tumor rat) showing many empty spaces (arrow) in the cribriform adenocarcinoma, the top left image is showing adenocarcinoma, notice the multiple spaces within the solid masses of the tumor some are empty and others contained pink proteinaceous secretion (arrow). (RT rat showing large eosinophilic granular areas of cancer cells’ necrosis [GN] among the tumor masses.). (QMNPs rat) showing marked necrosis of the cancer cells with its transformation into hyalinized eosinophilic material (arrow) as well as many pyknotic nuclei. QMNPs + RT rat showing necrosis and beginning of fibrous bands (arrow) among the cancer cell groups. Tumor size was measured using a standard caliber, in length and width, and the tumor was weighed upon excision it of all treated and untreated tumor-bearing rats. Number of animals (5 per group). QMNP indicates quercetin magnetite nanoparticles; RT, radiotherapy.

Figure 6.

QMNPs and/or RT effect on cell cycle, apoptosis, CD4 and CD8: (A) cell cycle analysis, (B) Bcl-2 and caspase-3 expression, (C) CD4 expression, and (D) CD8 expression. Values expressed as the mean ± SEM, n = 5. QMNP indicates quercetin magnetite nanoparticles; RT, radiotherapy; SEM, mean ± standard error. ^a1P < .001, ^a2P < .01, ^a3P < .05 vs control; ^b1P < .001, ^b2P < .01 vs tumor group; ^c1P < .001, ^c2P < .01, ^c3P < .05 vs RT group; ^d2P < .01, ^d3P < .05 vs QMNPs group.

Figure 7.

Summary of QMNPs and local radiotherapy (LRT) effect in breast cancer: in vitro and in vivo. MNU indicates 1-methyl-1-nitrosourea; QMNP, quercetin magnetite nanoparticles; RT, radiotherapy.