Andrographolide nanophytosomes exhibit enhanced cellular delivery and pro-apoptotic activities in HepG2 liver cancer cells

Abstract

Andrographolide (AG), a major active constituent of Andrographis paniculata, is known to hinder proliferation of several types of cancer cells. However, its poor solubility and cellular permeability restrict its use in clinical applications. In this study, AG-loaded phytosomes (AG-PTMs) were formulated and optimized with respect to particle size using l-α-phosphatidylcholine (PC):AG ratio and sonication time (ST) as independent variables. The optimized formula was prepared at 1:2.7 for AG:PC molar ratio and 4.9 min for ST and exhibited a particle size of 243.7 ± 7.3 nm, polydispersity index (PDI) of 0.310 and entrapment efficiency of 72.20 ± 4.53. Also, the prepared formula showed a slow release of AG over 24-h period. The antiproliferative activity of AG-PTMs was investigated against the liver cancer cell line HepG2. AG-PTMs significantly repressed the growth of HepG2 cells with an IC₅₀ value of 4.02 ± 0.14 µM. AG uptake by HepG2 cells was significantly enhanced in incubations containing the optimized formula. AG-PTMs also caused G2-M cell cycle phase arrest and increased the fraction of apoptotic cells in pre-G1 phase. These effects were associated with induction of oxidative stress and mitochondrial dysfunction. In addition, AG-PTMs significantly upregulated mRNA expression of BAX and downregulated that of BCL2. Furthermore, AG-PTMs significantly enhanced the concentration of caspase-3 in comparison to raw AG. These data indicate that the phytosomal delivery of AG significantly inhibited HepG2 cell proliferation through enhanced cellular uptake, arresting cell cycle at the G2-M phase and inducing mitochondrial-dependent apoptosis.

Keywords: Andrographolide; HepG2 cells; apoptosis; liver cancer; phytosomes.

Figure 1.

Diagnostic plots for particle size of AG-PTMs. (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.

(A) Perturbation, (B) 2D-contour and (C) 3D-surface plots for the effects and interactions between AG: PC molar ratio (X₁) and sonication time (X₂) on the AG-PTM size.

Figure 3.

Fourier-transform infrared (FTIR) of AG (A), PC (B), AG-PTMs (C) and a simple mixture of AG and PC (D).

Figure 4.

In vitro release pattern of optimized AG-PTMs after 24 h.

Figure 5.

Antiproliferative effects of AG, AG-PTMs and SORA in HepG2 cells. Data are represented as mean ± SD of six independent experiments.

Figure 6.

Cellular uptake of AG by HepG2 cells. *Significantly different from corresponding AG at p < .05 as determined by independent Student t test.

Figure 7.

Cell cycle analysis of HepG2 cells, (A) Control cells or pretreated with (B) AG, (C) AG-PTMs and (D) SORA. (E) Representative bar chart demonstrating the proportions in the cell cycle phases. Data are represented as mean ± SD of six independent experiments. *A statistically significant difference from control at p < .05, $statistically significant difference from AG at p < .05, †statistically significant difference from AG-PTMs at p < .05.

Figure 8.

Detection of apoptosis by flow cytometry in HepG2 cells pretreated with AG, AG-PTMs and SORA. Representative flow cytometric dot plots of control (A), AG (B), AG-PTMs (C) and SORA (D). Graphic presentation of early, late and total apoptosis (E). Data are represented as mean of six independent experiments ± SD. *Statistically significant differences from control at p < .05, $statistically significant differences from AG at p < .05, †statistically significant differences from AG-PTMs at p < .05.

Figure 9.

Graphic presentation of changes in MMP (A), ROS production (B) and mRNA expression of CYSC (C) in HepG2 cells pretreated with AG, AG-PTMs, and SORA. Data are represented as mean of six independent experiments ± SD. *Statistically significant differences from control at p < .05, $statistically significant differences from AG at p < .05, †statistically significant differences from AG-PTMs at p < .05.

Figure 10.

Graphic presentation of BAX (A) and BCL2 (B) mRNA expressions, and active caspase-3 concentrations (C) in HepG2 cells pretreated with AG, AG-PTMs, and SORA. Data are represented as mean of six independent experiments ± SD. *Statistically significant differences from control at p < .05, $statistically significant differences from AG at p < .05, †statistically significant differences from AG-PTMs at p < .05.