Enhanced Cytotoxic Activity of Docetaxel-Loaded Silk Fibroin Nanoparticles against Breast Cancer Cells

Abstract

Despite decades of research, breast cancer therapy remains a great challenge. Docetaxel is an antimicrotubule agent that is effectively used for the treatment of breast cancer. However, its clinical use is significantly hampered by its low water solubility and systemic toxicity. The current study was designed to prepare docetaxel (DXL)-loaded silk-fibroin-based nanoparticles (SF-NPs) and to screen their potential antitumor activity against breast cancer cell lines. DXL-loaded SF-NPs were prepared using a nanoprecipitation technique and were evaluated for particle size, zeta potential, entrapment efficiency, and in vitro release profile. In addition, DXL-loaded SF-NPs were screened for in vitro cytotoxicity, cellular uptake, and apoptotic potential against MCF-7 and MDA-MB-231 breast cancer cell lines. The prepared DXL-loaded SF-NPs were 178 to 198 nm in diameter with a net negative surface charge and entrapment efficiency ranging from 56% to 72%. In vitro release studies exhibited a biphasic release profile of DXL from SF-NPs with sustained drug release for 72 h. In vitro cell studies revealed that entrapment of DXL within SF-NPs significantly improved cytotoxic potential against breast cancer cell lines, compared to the free drug, and enhanced cellular uptake of DXL by breast cancer cells. Furthermore, the accumulation in the G2/M phase was significantly higher in cells treated with DXL-loaded SF-NPs than in cells treated with free DXL. Collectively, the superior antitumor activities of DXL-loaded SF-NPs against breast cancer cells, compared to free DXL, could be ascribed to improved apoptosis and cell cycle arrest. Our results highlighted the feasibility of using silk fibroin nanoparticles as a nontoxic biocompatible delivery vehicle for enhanced therapeutic outcomes in breast cancer.

Keywords: breast cancer; cytotoxicity; docetaxel; nanoparticles; silk fibroin.

Figure 1

Scanning electron microscopy (SEM) picture of DXL-loaded SF-NPs (magnification ×100,000).

Figure 2

Physicochemical characterization of DXL-loaded SF-NPs. (A) Fourier-transform infrared spectra, (B) differential scanning calorimetry thermograms, and (C) X-ray diffraction spectra.

Figure 3

In vitro release study of DXL from SF-NPs in phosphate-buffered saline (pH 5.5 and 7.4).

Figure 4

In vitro cytotoxicity of DXL-loaded SF-NPs against breast cancer cell lines. (A) MCF-7 and (B) MDAMB-231 cells were incubated with different concentrations (25–400 μg/mL) of free DXL, SF-NPs, or DXL-loaded SF-NPs for different incubation times (24, 48, and 72 h), and cell viability was then assessed by MTT assay. Data represent mean ± SD.

Figure 5

(A) Qualitative cellular uptake study of DXL-loaded SF-NPs by MCF-7 and MDA-MB-231 breast cancer cells by confocal microscopy analysis. DIC refers to differential interference contrast image. (B) Quantitative assessment of cellular uptake of DXL-loaded SF-NPs by MCF-7 and MDA-MB-231 breast cancer cells by flow cytometric analysis.

Figure 5

(A) Qualitative cellular uptake study of DXL-loaded SF-NPs by MCF-7 and MDA-MB-231 breast cancer cells by confocal microscopy analysis. DIC refers to differential interference contrast image. (B) Quantitative assessment of cellular uptake of DXL-loaded SF-NPs by MCF-7 and MDA-MB-231 breast cancer cells by flow cytometric analysis.

Figure 6

Effect of free DXL and DXL-loaded SF-NPs on cell cycle progression. Flow cytometric analysis of cell populations at different cell cycle stages of (A) untreated MCF-7 cells, (B) MCF-7 cells treated with free DXL, (C) MCF-7 cells treated with DXL-loaded SF-NPs, (D) untreated MDA-MB-231 cells, (E) MDA-MB-231 cells treated with free DXL, (F) MDA-MB-231 cells treated with DXL-loaded SF-NPs. Analysis of the cell cycle of (G) MCF-7 cells or (H) MDA-MB-231 cells treated with either free DXL or DXL-loaded SF-NP. Data represent mean ± SD.

Figure 7

Flow cytometry analysis data from annexin V-FITC staining. Dot plots representing the distribution of apoptotic cells of (A) MCF-7 cells and (B) MDA-MB-231 cells following staining with annexin V-FITC and propidium iodide. (C) Quantitative analysis of the percentage of apoptotic cells upon treatment with either free DXL or DXL-loaded SF-NPs.

Figure 7

Flow cytometry analysis data from annexin V-FITC staining. Dot plots representing the distribution of apoptotic cells of (A) MCF-7 cells and (B) MDA-MB-231 cells following staining with annexin V-FITC and propidium iodide. (C) Quantitative analysis of the percentage of apoptotic cells upon treatment with either free DXL or DXL-loaded SF-NPs.