Delivery of Apoptosis-inducing Piperine to Triple-negative Breast Cancer Cells via Co-polymeric Nanoparticles

Materials and Methods

Cell lines and tissue culture. MDA-MB-468 breast adenocarcinoma cells were obtained from Dr. P. Lee (Dalhousie University, Halifax, NS, Canada). BT-549 breast ductal carcinoma cells were from Dr. P. Marcato (Dalhousie University, Halifax, NS, Canada). Cell lines were authenticated using the short tandem repeat method. Human dermal fibroblasts (HDF) from Lonza Inc. (Walkersville, MD, USA) were cultured in Fibroblast Basal Medium supplemented with fetal bovine serum, insulin, gentamicin sulfate-amphotericin, and fibroblast growth factor (all from Lonza) and maintained at 37°C in a humidified incubator with 5% CO₂. MDA-MB-468 cells and BT-549 cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 5 mM HEPES (all from Invitrogen Canada, Burlington, ON, Canada), and maintained at 37°C in a humidified incubator with 10% CO₂.

NP formulation. NPs were formulated by single emulsion solvent extraction, using a modification of the method developed by Fessi et al. (10). Briefly, 8.5 mg piperine (Sigma-Aldrich, Oakville, ON, Canada) and 100 mg mPEG-PLGA (5-10 kDa; Akina Inc., West Lafayette, IN, USA) were dissolved in 10 ml dichloromethane to generate the organic phase. The aqueous phase consisted of 150 ml distilled water. Lutrol® F68 (0.1%; Sigma-Aldrich) was used as a surfactant to stabilize the emulsion droplets. The organic phase was added drop-wise to the aqueous phase under constant high-speed stirring for 2 h. The emulsion was then heated to ~40°C under vacuum using a rotary evaporator to remove the dichloromethane. NPs were pelleted by ultracentrifugation at 27,000 × g for 15 min, then dissolved in water containing 5% sucrose as a cryoprotectant, flash-frozen, lyophilized and stored at room temperature. Prior to use, NPs were reconstituted in sterile water.

To formulate NPs by thin-film hydration (11), 5 mg piperine and 45 mg mPEG-PLGA were dissolved in 5 ml dichloromethane. The solution was transferred to a round-bottom flask for evaporation under vacuum at 60°C using a rotary evaporator. The resulting thin film was dissolved in saline solution and stirred at 60°C to allow for the self-assembly of polymers into piperine-containing micelles. Dialysis across a membrane with a 14 kDa cut-off against saline solution at room temperature removed any unencapsulated piperine. NPs were then flash-frozen, lyophilized, and stored at room temperature. Prior to use, NPs were reconstituted in sterile water and sonicated for 5 min with an ultrasonic probe to generate NPs of the desired size. All NP preparations were passed through a 0.2 μm syringe filter to remove polymer aggregates and any crystals of piperine.

NP characterization. NPs were placed on a grid, stained with 0.2% uranyl acetate for 30 s, and dried. The grid was examined with a JEM 1230 transmission electron microscope (JEOL USA Inc., Peabody, MA, USA). Images were captured with an ORCA-HR digital camera (Hamamatsu Photonics, Hamamatsu, Japan). Particle size was measured using AMT Image Capture Engine (version 6.02; AMT Corp., Woburn, MA, USA). Measurements of 90 randomly selected NPs yielded an average NP size.

Encapsulation efficiency was determined by spectrophotometric analysis. The absorbance of piperine at 346 nm is proportional to its concentration over the range used in this study. Absorbance of piperine dissolved in dimethyl sulfoxide (DMSO) at a range of concentrations (10-80 μg/ml) was measured using an UVM 340 microplate reader (Biochrom, Cambridge, UK) to generate a standard curve. Absorbance of piperine-loaded NPs (5 mg/ml in DMSO) was also determined. Absorbance of empty NPs was subtracted from that of piperine-containing NPs to correct for NP polymer absorbance. The standard curve was used to determine the amount of piperine in the NP preparation. Encapsulation efficiency was calculated by comparing the amount of piperine contained within NPs with the amount of piperine used in NP preparation.

Cell growth and viability assays. All experiments used low passage cultures in which the passage number at 80-90% confluency did not exceed 25 in the case of MDA-MB-488 and BT-549 cell lines, or 12 in the case of HDFs.

TNBC growth in the absence and presence of piperine-loaded NPs or free piperine was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) assay. TNBC cells were seeded in quadruplicate wells of a 96-well plate at a density of 5×10³ cells/well and cultured overnight to allow for adherence. Cultures then received medium, vehicle or 50-150 μM of piperine (free or loaded into NPs), followed by culture for 48 h. The concentrations of piperine used were previously determined to be cytotoxic for TNBC cells (4).

Cell death was assessed by flow cytometric analysis of cells stained with Annexin-V-FLUOS (Roche Diagnostics, Laval, QC, Canada) and propidium iodide (PI; Sigma-Aldrich). TNBC cells were seeded in a 6-well plate at a density of 5×10⁴ cells/well and cultured overnight to allow for adherence. Cultures then received medium, vehicle, or 150 μM piperine (free or loaded into NPs), followed by culture for 48 h. The MTT and the Annexin-V-FLUOS/PI staining assays were performed as described in detail by Greenshields et al. (4).

Statistical analysis. Microsoft Excel (Redmond, WA, USA) and GraphPad Prism (GraphPad Software, San Diego, CA, USA) were used for analysis of data from three independent experiments. Statistical significance was determined by two-tailed Student's t-test or analysis of variance (ANOVA) with the Tukey multiple comparisons post-test; differences were considered statistically significant at p<0.05.