Piceatannol-Loaded Bilosome-Stabilized Zein Protein Exhibits Enhanced Cytostatic and Apoptotic Activities in Lung Cancer Cells

Abstract

Piceatannol (PIC) is a naturally occurring polyphenolic stilbene, and it has pleiotropic pharmacological properties. Moreover, PIC has cytotoxic actions among various cancer cells. In this work, preparations of PIC-loaded bilosome-zein (PIC-BZ) were designed, formulated, and characterized, and the optimized PIC-BZ cytotoxic activities, measured as half maximal inhibitory concentration (IC₅₀), against lung cancer cell line was investigated. Box-Behnken design was utilized in order to examine the effect of preparation factors on drug entrapment and particle size. PIC-BZ showed a spherical shape after optimization, and its particle size was determined as 157.45 ± 1.62 nm. Moreover, the efficiency of drug entrapment was found as 93.14 ± 2.15%. The cytotoxic activity evaluation revealed that the adjusted formulation, which is PIC-BZ formula, showed a substantially smaller IC₅₀ versus A549 cells. Cell cycle analysis showed accumulation of cells in the G2-M phase. Moreover, it showed in the sub-G1 phase, a rise of cell fraction suggestion apoptotic improving activity. Increased early and late phases of apoptosis were demonstrated by staining of cells with annexin V. Furthermore, the cellular caspase-3 protein expression was significantly raised by PIC-BZ. In addition, the wound healing experiment confirmed the results. To conclude, compared to pure PIC, PIC-BZ demonstrated a higher cell death-inducing activity against A549 cells.

Keywords: A549cells; apoptosis; bilosomes; piceatannol; zein.

Figure 1

Diagnostic plots for globule size of PIC-BZ: (A) Box–Cox plot for power transforms, (B) externally studentized residuals vs. predicted values plot, (C) externally studentized residuals vs. run number plot, and (D) predicted vs. actual values plot.

Figure 2

2D contour plot for the influence of formulation variables on the particle size of PIC-BZ.

Figure 3

Particle size distribution of the optimized PIC-BZ.

Figure 4

In vitro release profile of PIC-BZ in PBS (pH 7.4, 0.1 M) comprising 0.1% of Tween 80 at 37 ± 0.5 °C.

Figure 5

Cytotoxicity of PIC-BZ in A549. Cells were incubated with Blank-BZ, PIC-raw, or PIC-BZ for 48 h, and IC₅₀ values were determined using MTT assay. Data are the mean of 4 independent experiments ± SD. * Significantly different from Blank-PZ, p < 0.05; # significantly different from PIC-raw, p < 0.05.

Figure 6

Cellular uptake of PIC from PIC raw and optimized PIC-BZ at 2 and 4 h. Data represent mean of six independent replicates SD. * Significantly different (p < 0.05) determined by Student’s t-test.

Figure 7

Impact of Blank-BZ, PIC-raw, or PIC-BZ treatments on A549 cell cycle phases with data table (inset). Data are the mean of 4 independent experiments ± SD. * Significantly different from control, p < 0.05; # significantly different from Blank-BZ, p < 0.05; $ significantly different from PIC-raw, p < 0.05.

Figure 8

Impact of Blank-BZ, PIC-raw, or PIC-BZ treatments on the percentage of apoptotic or necrotic A549 cells with data table (inset). Total = apoptosis + necrosis; early = early apoptotic phase; late = late apoptotic phase. Data are the mean of 4 independent experiments ± SD. * Significantly different from control, p < 0.05; # significantly different from Blank-BZ, p < 0.05; $ significantly different from PIC-raw, p < 0.05.

Figure 9

Effect of Blank-BZ, PIC-raw, or PIC-BZ treatments on caspase-3 enzyme contents in A549 cells. Values are expressed as pg/mg of protein. Data are the mean of 4 independent experiments ± SD. * Significantly different from control, p < 0.05; # significantly different from Blank-BZ, p < 0.05; $ significantly different from PIC-raw, p < 0.05.

Figure 10

Wound coverage results of (A) untreated cells, (B) cells treated with blank-BZ, (C) cells treated with PIC-raw, and (D) cells treated with optimized PIC-BZ with 40× magnification.