Lipidic Nano-Sized Emulsomes Potentiates the Cytotoxic and Apoptotic Effects of Raloxifene Hydrochloride in MCF-7 Human Breast Cancer Cells: Factorial Analysis and In Vitro Anti-Tumor Activity Assessment

Abstract

Raloxifene hydrochloride (RLX), an antiosteoporotic agent, has been utilized for guarding against breast cancer and recently, for the disease management owing to its estrogen antagonist activity. Nevertheless, RLX exhibits poor bioavailability that could be attributed to reduced water solubility and first pass metabolism. To overcome these challenges, this study aimed at formulating and optimizing RLX emulsomes (RLX-EMLs) to enhance the drug antitumor activity. A 4¹3¹ factorial design was employed for assessing the effect of lipoid: solid lipid ratio and solid lipid type on the emulsomes characteristics. The anticancer potential of the optimized formulation and apoptotic parameters were assessed. Vesicle size, entrapment, and release efficiency were significantly influenced by both variables, while zeta potential was influenced by lipoid: solid lipid at p < 0.05. The optimal formulation exhibited vesicle size of 236 ± 8.6 nm, zeta potential of -18.6 ± 0.7 mV, drug entrapment of 98.9 ± 4.9%, and release efficiency of 42.7 ± 1.8%. MTT assay showed concentration-dependent inhibition of MCF-7 cells viability. In addition, cells treated with RLX-EMLs showed significant arrest at G2/M phase associated with significant increase in apoptotic and necrotic cells. The enhanced cytotoxic and anti-proliferative effect of RLX-EMLs relative to raw drug was authenticated through increased Bax/Bcl-2 ratio, caspase-9 activation and depletion of mitochondrial membrane potential.

Keywords: apoptosis; cell cycle analysis; emulsomes; factorial design; mitochondrial membrane potential; raloxifene hydrochloride.

Figure 1

Response 3D plot for PL:SL weight ratio effect; X₁ and SL type; X₂ on (A) vesicle size; Y₁, (B) zeta potential; Y₂, (C) entrapment efficiency; Y₃, and (D) release efficiency after 6 h; Y₄ of RLX-EMLs. Abbreviations: RLX-EMLs, raloxifene hydrochloride emulsomes; PL:SL, lipoid: solid lipid weight ratio; SL type: solid lipid type.

Figure 2

Effect of RLX-EMLs or RLX on MCF-7 cell viability using MTT assay. The cells were treated with RLX-EMLs or RLX for 48 h. (A) dose-response curve with IC₅₀ values; (B) comparison of cell viability results using 0.1 µM of RLX. $ p < 0.0001 considered significantly different from RLX, # p < 0.0001 considered significantly different from blank EMLs.

Figure 3

Effect of RLX-EMLs or RLX on MCF-7 cell cycle changes of MCF-7 cells using flow cytometry analysis. The cells were treated with RLX-EMLs or RLX for 48 h. (A) control, (B) blank EMLs, (C) RLX, and (D) RLX-EMLs. (E) graphical presentation of each phase. All data are presented as the mean ± SE of three independent experiments. * p < 0.0001 considered significantly different from the control. $ p < 0.0001 considered significantly different from RLX, # p < 0.0001 considered significantly different from blank EMLs.

Figure 4

Effect of RLX-EMLs or RLX on MCF-7 apoptosis profile using annexin V/PI staining. (A) control, (B) blank EMLs, (C) RLX, and (D) RLX-EMLs. (E) graphical presentation of each phase (early apoptotic, late apoptotic, total apoptotic, and dead cells). All data are expressed as the mean ±SE of three independent experiments. * p < 0.05 considered significantly different from the control. $ p < 0.0001 considered significantly different from RLX, # p < 0.0001 considered significantly different from blank EMLs. ^ p < 0.05 considered significantly different from the control, RLX, or blank EMLs.

Figure 5

Effect of RLX-EMLs or RLX on Bcl-2 proteins, caspase-9 activation and MMP. (A,B) protein levels for BAX and Bcl-2 consequently. (C) Bax/Bcl-2 ratio. (D) activation level of caspase-9. (E) percentage of MMP. All data are expressed as the mean ± SE of three independent experiments. * p < 0.0001 considered significantly different from the control. $ p < 0.0001 considered significantly different from RLX.