Co-Delivery of Docetaxel and Salinomycin to Target Both Breast Cancer Cells and Stem Cells by PLGA/TPGS Nanoparticles

Abstract

Purpose: Conventional chemotherapy is hampered by the presence of breast cancer stem cells (BCSCs). It is crucial to eradicating both the bulky breast cancer cells and BCSCs, using a combination of conventional chemotherapy and anti-CSCs drugs. However, the synergistic ratio of drug combinations cannot be easily maintained in vivo. In our previous studies, we demonstrated that the simultaneous delivery of two drugs via nanoliposomes could maintain the synergistic drug ratio for 12 h in vivo. However, nanoliposomes have the disadvantage of quick drug release, which makes it difficult to maintain the synergistic drug ratio for a long time. Herein, we developed a co-delivery system for docetaxel (DTX)-a first-line chemotherapy drug for breast cancer-and salinomycin (SAL)-an anti-BCSCs drug-in rigid nanoparticles constituted of polylactide-co-glycolide/D-alpha-tocopherol polyethylene glycol 1000 succinate (PLGA/TPGS).

Methods: Nanoparticles loaded with SAL and DTX at the optimized ratio (NSD) were prepared by the nanoprecipitation method. The characterization, cellular uptake, and cytotoxicity of nanoparticles were investigated in vitro, and the pharmacokinetics, tissue distribution, antitumor and anti-CSCs activity of nanoparticles were evaluated in vivo.

Results: We demonstrated that a SAL/DTX molar ratio of 1:1 was synergistic in MCF-7 cells and MCF-7-MS. Moreover, the enhanced internalization of nanoparticles was observed in MCF-7 cells and MCF-7-MS. Furthermore, the cytotoxicity of NSD against both MCF-7 cells and MCF-7-MS was stronger than the cytotoxicity of any single treatment in vitro. Significantly, NSD could prolong the circulation time and maintain the synergistic ratio of SAL to DTX in vivo for 24 h, thus exhibiting superior tumor targeting and anti-tumor activity compared to other treatments.

Conclusion: Co-encapsulation of SAL and DTX in PLGA/TPGS nanoparticles could maintain the synergistic ratio of drugs in vivo in a better manner; thus, providing a promising strategy for synergistic inhibition of breast cancer.

Keywords: breast cancer stem cells; combined therapy; docetaxel; nanoparticles; salinomycin.

Figure 1

The cytotoxicity of SAL and DTX in MCF-7 cells and MCF-7-MS. The concentration-dependent cytotoxicity induced by SAL (A) and DTX (B) in MCF-7 cells and MCF-7-MS at 48 h. In vitro screening of SAL and DTX for synergy in MCF-7 cells (C) and MCF-7-MS (D) as a function of the SAL/DTX ratio and drug concentrations. Fraction affected means the fraction of the cell that was killed. CI values of < 1, = 1, and > 1 indicate synergy, additivity, and antagonism, respectively. Data are presented as means ± standard deviations (n = 3).

Figure 2

Characterization of nanoparticles. Size distribution (A) and zeta potential of nanoparticles (B), as determined by dynamic light scattering. The TEM image of nanoparticles (C). Bars represent 100 nm. One representative image is shown. The cumulative release of SAL or DTX from nanoparticles at pH 7.4 (D) and 5.0 (E). Data are presented as means ± standard deviations (n = 3).

Figure 3

In vitro cellular uptake of nanoparticles. MCF-7 cells (A) and MCF-7-MS (B) were treated with coumarin-6 and NC after 1 h, followed by staining with DAPI for nuclei. The green fluorescence of coumarin-6 and blue fluorescence of DAPI were analyzed by a confocal laser scanning microscopy. Bars represent 75 μm.

Figure 4

The concentration-dependent cytotoxicity induced by nanoparticles in MCF-7 cells (A, C) or MCF-7-MS (B, D). The cells were incubated for 48 h with varying concentrations of nanoparticles or free SAL or DTX, and the cell viability was evaluated by CCK-8 assays. Data are presented as means ± standard deviations (n = 3).

Figure 5

The pharmacokinetic studies after i.v. injection of SAL + DTX, NS + ND and NSD (2 mg/kg SAL and 2.1 mg/kg DTX) to SD rats via tail vein. Mean plasma concentration versus time of SAL (A) or DTX (B). Data are presented as means ± standard deviations (n = 5).

Figure 6

The mole ratio of SAL and DTX in plasma after i.v. injections of SAL + DTX, NS + ND and NSD (2 mg/kg SAL and 2.1 mg/kg DTX) to SD rats. Data are presented as means ± standard deviations (n = 5).

Figure 7

Tissue distribution in vivo. BALB/c nude mice bearing MCF-7 breast cancer-derived tumors were given tail vein injections of saline, free DiR, or NDiR. Time-dependent in vivo images of mice after treatment with formulations (A). Ex vivo images of tumors and other organs at 24 h post-injection of the formulations (B).

Figure 8

Therapeutic effects of nanoparticles in mice bearing subcutaneous MCF-7 tumors. Mice were treated with intravenous injections of the nanoparticles or free drugs (SAL 2 mg/kg; DTX 2.1 mg/kg) via the tail vein. Tumor growth curves (A). The enlarged profiles of DTX, SAL + DTX, ND, NS + ND and NSD on the growth of tumors (B). Images of excised tumors in each group at the endpoint (C). The excised tumors were weighed at the endpoint (D). *p < 0.05; **p < 0.01. Body weight change rate and tumor inhibitory rate after treatment of different formulations (E). Weight changes in mice during the treatment (F). Data are presented as means ± standard deviations (n = 6).

Figure 9

In vivo anti-CSCs activity. Image of tumorspheres of excised tumors (A). Tumorsphere-forming efficiency of excised tumor cells (B). Data are presented as means ± standard deviations (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.