Combinatorial Therapy of Letrozole- and Quercetin-Loaded Spanlastics for Enhanced Cytotoxicity against MCF-7 Breast Cancer Cells

Abstract

Breast cancer is the most widespread cancer in women with rising incidence, prevalence, and mortality in developed regions. Most breast cancers (80%) are estrogen receptor-positive, indicating that disease progression could be controlled by estrogen inhibition in the breast tissue. However, drug resistance limits the benefits of this approach. Combinatorial treatment could overcome the resistance and improve the outcome of breast cancer treatment. In the current study, we prepared letrozole-(LTZSPs) and quercetin-loaded spanlastics (QuSPs) using different edge activators-Tween 80, Brij 35, and Cremophor RH40-with different concentrations. The spanlastics were evaluated for their average particles size, surface charge, and percent encapsulation efficiency. The optimized formulations were further examined using transmission electron microscopy, Fourier transform infrared spectroscopy, in vitro drug release and ex vivo skin permeation studies. The prepared spherical LTZSPs and QuSPs had average particle sizes ranged between 129-310 nm and 240-560 nm, respectively, with negative surface charge and high LTZ and Qu encapsulation (94.3-97.2% and 97.9-99.6%, respectively). The in vitro release study of LTZ and Qu from the selected formulations showed a sustained drug release for 24 h with reasonable flux and permeation through the rat skin. Further, we evaluated the in vitro cytotoxicity, cell cycle analysis, and intracellular reactive oxygen species (ROS) of the combination therapy of letrozole and quercetin either in soluble form or loaded in spanlastics against MCF-7 breast cancer cells. The LTZSPs and QuSPs combination was superior to the individual treatments and the soluble free drugs in terms of in vitro cytotoxicity, cell cycle analysis, and ROS studies. These results confirm the potential of LTZSPs and QuSPs combination for transdermal delivery of drugs for enhanced breast cancer management.

Keywords: breast cancer; combination therapy; letrozole; quercetin; spanlastics.

Figure 1

Representative transmission electron microscope images of the selected formulations (A) LTZSPs (formulation L8) and (B) QuSPs (formulation Q5).

Figure 2

FTIR spectra of (A) letrozole (LTZ), Span 60, Brij 35, blank spanlastics (Blank SPs), and letrozole loaded spanlastics (LTZSPs, formulation L8). (B) Quercetin (Qu), Span 60, Tween 80, blank spanlastics (Blank SPs), and quercetin-loaded spanlastics (QuSPs, formulation Q5).

Figure 3

Cumulative in vitro release profiles of (A) The optimized LTZSPs compared to free LTZ dispersion in hydroalcoholic PBS solution pH 7.4 at 37 °C. (B) The optimized QuSPs compared to free Qu dispersion in PBS pH 7.4 plus 1% Tween 80 at 37 °C. Data are expressed as mean ± SD (n = 3). Abbreviations: LTZ; letrozole, Qu; quercetin, LTZSPs; letrozole-loaded spanlastics, QuSPs; quercetin-loaded spanlastics.

Figure 4

Ex vivo permeation profile of (A) The optimized LTZSPs (formulation L8), compared to free LTZ dispersion in hydroalcoholic PBS solution pH 7.4 at 37 °C. (B) The optimized QuSPs (formulation Q5), compared to free Qu dispersion in PBS pH 7.4 plus 1% Tween 80 at 37 °C. Data are presented as mean ± SD, n = 3. Abbreviations: LTZ; letrozole, Qu; quercetin, LTZSPs; letrozole-loaded spanlastics, QuSPs; quercetin-loaded spanlastics.

Figure 5

Cytotoxicity assessment using MTT assay against MCF-7 cell line. (A) Dose–response curves of MCF-7 cells to treatment with LTZ, Qu, and their combination and also LTZSPs, QuSPs, and their combination using indicated concentration range using an MTT assay after 48 h of treatment. (B) The mean IC₅₀ of LTZ, Qu, and their combination and also LTZ-Qu, LTZSPs, and their combination against MCF-7 cells. Data are expressed as mean ± SD (n = 3). *** p < 0.001, ** p < 0.01, * p < 0.05. Abbreviations: LTZ; letrozole, Qu; quercetin, LTZSPs; letrozole-loaded spanlastics, QuSPs; quercetin-loaded spanlastics, ns; not significant.

Figure 6

Cell cycle studies of MCF-7 cells treated with soluble LTZ, Qu, and their combination and also LTZSPs, QuSPs, and their combination using indicated concentrations. (A) Percentage of apoptotic cells. (B) Representative histograms indicating percentage of MCF-7 cells in each cell cycle. Abbreviations: LTZ; letrozole, Qu; quercetin, LTZSPs; letrozole-loaded spanlastics, QuSPs; quercetin-loaded spanlastics.

Figure 7

ROS levels using ELISA assay against MCF-7 cell line. MCF-7 cells were treated with selected concentrations of LTZ and Qu and their combination and also LTZ–Qu, LTZSPs, QuSPs and their combination and measured using an ELISA after 24 h of treatment. All samples show significance difference compared to control. Data are expressed as mean ± SD (n = 3). *** p < 0.001, * p < 0.05. Abbreviations: LTZ; letrozole, Qu; quercetin, LTZSPs; letrozole-loaded spanlastics, QuSPs; quercetin-loaded spanlastics.