Improved therapeutic efficacy of quercetin-loaded polymeric nanoparticles on triple-negative breast cancer by inhibiting uPA

Abstract

Triple negative breast cancer (TNBC) is one kind of breast cancer that demonstrates highly aggressive tumor biology. The high heterogeneity of TNBC makes its individual clinical treatment extremely blind and limited, which also introduces more challenges into the diagnosis and treatment of diseases. Urokinase-type plasminogen activator (uPA) is a high level marker for breast cancer, which mediates tumor growth and metastasis. Quercetin is a plant-derived flavonoid in many plants, which inhibits uPA and has low bioavailability and mediocre pharmaceutical efficacy. Thus, we herein developed polymeric nanoparticulate systems from PLGA-TPGS (Qu-NPs) for quercetin oral delivery and evaluated the anticancer effect of this formulation on TNBC in vitro and in vivo. Qu-NPs have a uniform spherical morphology with a mean diameter of 198.4 ± 7.8 nm and good drug loading capacity (8.1 ± 0.4%). Moreover, Qu-NPs exhibited significantly improved inhibition on the growth and metastasis in TNBC cells. Following oral gavage, a remarkable antitumor effect of Qu-NPs on 4T1-bearing mice was observed with a tumor inhibition ratio of 67.88% and fewer lung metastatic colonies. Furthermore, the inhibitory effect of quercetin on the migration of uPA knockdown MDA-MB231 cells was greatly attenuated. Together, Qu-NPs improved the significant antitumor and antimetastatic effects by inhibiting uPA, which provides a new strategy for the treatment of TNBC.

Fig. 1. Chemical structure of quercetin (3,3′,4′,5,7-pentahydroxyflavone).

Fig. 2. Morphology, particle size and zeta potential distribution of Qu-NPs, and the uniformity and stability of the nanoparticles were determined. (A) Particle size intensity distribution map of Qu-NPs. The average particle size (Z-average) of the obtained quercetin nanoparticles was 198.4 ± 7.8 nm, the dispersion coefficient (PDI) was 0.095 and the peak shape showed a single peak, and the particle size distribution was relatively uniform. (B) Zeta potential profile of Qu-NPs. The measured zeta potential result was −20 mV, and the peak shape showed a single peak, indicating that the prepared quercetin nanoparticles were also relatively stable. (C) Transmission electron microscopy (TEM) images of quercetin nanoparticles at different magnifications. (D) Scanning electron microscope (SEM) images of quercetin nanoparticles at different magnifications.

Fig. 3. The cytotoxicity of quercetin and Qu-NPs on a range of cells. (A) MDA-MB231, (B) 4T1 and (C) HELF cells were treated with quercetin and Qu-NPs at concentrations of 0, 1, 5, 10, and 50 μM for 48 hours, respectively. Cell viability was measured by MTT assay. The cytotoxic effects of Qu-NPs on TNBC cells are much stronger than that of quercetin alone, and the cytotoxicity to HELF cells was not significantly improved compared with quercetin alone.

Fig. 4. Inhibition of quercetin and Qu-NPs on the migration of triple negative breast cancer cells. In the wound healing experiment, (A) 4T1 and (B) MDA-MB231 cells were cultured in a 12-well plate until they reached a single layer fusion, and the monolayer cells were scraped with a pipette tip and then treated with different concentrations of the drugs (0, 5, 10, and 50 μM). Final images of the cells migrated into the scratches were captured at different time points (0 h, 12 h, and 24 h).

Fig. 5. Inhibition of quercetin and Qu-NPs on triple-negative breast cancer cell invasion. (A) 100 μL of matrigel (0.25 mg mL⁻¹) was coated in a Transwell chamber, and the basement membrane was hydrated with DMEM medium. 4T1 and MDA-MB231 cells were treated with different concentrations of quercetin alone (0, 5, 10, and 50 μM) and Qu-NPs (0, 5, and 10 μM) respectively for 24 hours, and stained with crystal violet to capture cell images of cells invading the subsurface. (B) and (C) are quantitative plots of 4T1 and MDA-MB231 cells invading into the lower chamber, respectively. The crystal violet on the cells was eluted with a 33% acetic acid solution and its absorbance was measured at 570 nm.

Fig. 6. The anti-tumor and anti-metastatic efficacies of Qu-NPs in vivo. The suppression of the tumor growth in Balb/c mice orthotopically implanted with 4T1 cells at the daily dosage of 30 mg kg⁻¹ Qu-NPs for 10 days. Representative graphics (A) and tumor weights (B) of resected tumors with and without treatments. (C) The tumor volume of Balb/c mice was recorded from the seventh day of tumor seeding, and the data were processed with 0.5 × length × width. The values were represented as mean ± SEM. (D) The graphics of the H&E-stained histological sections of the tumor and lungs with and without the treatments. (E) The changes of the body weights of mice orthotopically implanted with 4T1 cells with the treatment of Qu-NPs. PBS buffer as a control, the values were represented as mean ± SEM.

Fig. 7. Quercetin inhibits migration of MDA-MB231 cells by inhibiting uPA and supplemental uPA restores the migration of MDA-MB231 shuPA cells. (A) Wound healing experiments of MDA-MB231 cells knocked down of uPA (MDA-MB231 shuPA) and MDA-MB231 pLKO.1 cells transfected with empty vector as a positive control. After treating with 5 μM quercetin or 10 μM quercetin, the cell migration process was photographed at different time points (0 h, 12 h, and 24 h). (B) Statistical analysis of the wound scratch closure monitored over time in the presence of quercetin. (C) Wound healing experiments of MDA-MB231 cells knocked down by RNAi (MDA-MB231 shuPA). After treating with 5 μM quercetin, 100 nM uPA, and a combination of the two, the cell migration process was photographed at different time points (0 h, 12 h, and 24 h). (D) Statistical analysis of the wound scratch closure monitored over time in the presence of quercetin or uPA. Green areas indicate cell-free zones as determined using the MRI Wound Healing Tool of Image J. Results are representative of three independent experiments. Data are presented as the mean residual wound area at different time points as a percentage of the original wound area at 0 h ± SEM.