Anti-tumor effect of PEG-coated PLGA nanoparticles of febuxostat on A549 non-small cell lung cancer cells

Abstract

In the present study, febuxostat (FBX)-loaded PEG-coated PLGA nanoparticles (FBX-PLGA-PEG) were developed and its anticancer activity on lung cancer cells was evaluated. FBX-PLGA-PEG were prepared by nanoprecipitation technique and characterized for particle size, size distribution, entrapment efficiency, and in vitro drug release study. The optimized formulations were used to evaluate cell viability, apoptosis, cell cycle, and caspase activity in A549 lung cancer cells. The optimized formulation showed spherical particle with average particle size of 180 ± 4.72 nm, particle-size distribution 0.223 ± 0.003, entrapment efficiency (70 ± 2.56%), and drug release (99.1 ± 2.33%) at 12 h. MTT cytotoxicity assay showed better cytotoxic potential of FBX-NPs than FBX solution against NSCLC A549 cells. The lower IC₅₀ of FBX-NP (52.62 ± 2.52 µg/mL) compared to FBX (68.0 ± 4.12 µg/mL) are suggestive of a potential cytotoxic effect of nano-formulation compared to the drug itself. Furthermore, flow cytometry analysis showed significantly higher percentage of total apoptotic cells in FBX-NPs (10.38 ± 1.57%) as compared to FBX solution (2.76 ± 0.17%) showed strong proapoptotic potential of FBX nano-formulation. The increased caspase activity and percent of cells at G2/M phase of cell cycle increased for FBX nanoparticles were more effective than FBX solution in increasing caspase activity and percent of cells at G2/M phase of cell cycle. Our studies with FBX nanoparticles exhibited promising outcome which could be used as a strategies to combat lung cancer.

Keywords: Apoptosis; Febuxostat; Lung cancer; Nanoparticles; PLGA.

Fig. 1

Particle size, size distribution (a) and TEM image (b) of FBX-loaded PEG-PLGA nanoparticles

Fig. 2

In vitro drug release profile of free FBX and FBX NPs. Data are expressed as mean ± SD (n = 3)

Fig. 3

Cell viability of lung cancer A549 cells treated with different concentrations of free FBX, FBX-loaded nanoparticles (FBX NPs), and placebo NPs. Data are expressed as mean ± SD (n = 3)

Fig. 4

Dot plots of FACS analysis using an annexin V-FITC probe showed induction of apoptotic cell death: a control group, b free FBX, and c FBX NPs. d Bar graphs of FACS analysis showing greater apoptotic cell death induced by FBX NPs treatment compared to free FBX. The data were analyzed using one-way ANOVA followed by Tukey's post hoc test. Percentage of cells in each group was compared with control; p < 0.05 indicates a significant difference

Fig. 5

Flow cytometric analysis showing the effect of a control group (untreated A549 cells), b free FBX, and c FBX NPs on A549 lung cancer cell lines at 24 h on cell cycle. d Bar graph represent each cell cycle phase, Sub G1 phase, G0/G1 phase, and G2/M and S phase, and results are represented as the mean ± SD (n = 3). Y-axis represents number of cell population (%) and X-axis represents FL2H flow cytometric analysis stained with propidium iodide (PI) histograms. The data were analyzed using one-way ANOVA followed by Tukey's post hoc test. Percentage of cells in each group was compared with control; p < 0.05 indicates a significant difference. The orange peak represents G1 phase in the cell cycle and the red peak represents G2/M phase in the cell cycle

Fig. 6

Caspase activity of free FBX and FBX NPs