Hepatocellular carcinoma cell apoptosis induced by low-intensity ultrasound with polylactic acid-polyethylene glycol nanometer microspheres loaded with sodium cantharidin: An in vitro study

Abstract

This study examines the synergistic inhibitory effect of two drugs on hepatocellular carcinoma cells using polylactic acid-polyethylene glycol (PLA-PEG) nanoparticles loaded with sodium cantharidin (SCA) combined with low-intensity ultrasound. We sought to provide an experimental basis for the cytological level of SCA combined with low-intensity ultrasound tumor treatment. The study consisted of four treatment groups: HepG2 cells treated with 2.5 g/mL SCA for 30 min then irradiated with 0.5 w/cm2 low-intensity ultrasound for 8 s (combination group), HepG2 cells treated with SCA alone (SCA group), HepG2 cells randomly irradiated with low-intensity ultrasound (ultrasound group), and HepG2 cells without intervention (control group). After each treatment, the cells were cultured for 6 h, and then the cell proliferation inhibition rate, apoptosis cycle, and intracellular calcium concentration were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry and Fluo-3 AM detection. Intracellular ultrastructural changes were observed using transmission electron microscopy. The results showed that the proliferation inhibition rate of cells in the combination group was significantly higher than that in the SCA and ultrasound groups. Annexin V-FITC and propidium iodide staining revealed that the apoptosis rate of the combination group significantly increased compared with that of the other groups. There was no significant difference in cell cycle among the different treatments after 6 h of cell culture. However, cell cycle arrest occurred in HepG2 cells at G0/G1 stage after 12 h of cell culture. Fluo-3 AM showed that the intracellular calcium concentration in the combined group was significantly higher than that in the other groups at 6 h after treatment. Transmission electron microscopy showed that apoptotic bodies and mitochondrial and endoplasmic reticulum swelling were present in the combined group after treatment. We conclude that low-intensity ultrasound (0.5 w/cm2) combined with SCA (2.5 μg/mL) synergistically induce apoptosis of hepatocellular carcinoma HepG2 cells, block cell cycle at G0/G1 phase, and disrupt intracellular calcium homeostasis. PLA-PEG can be loaded with SCA to improve drug targeting and drug concentration in the affected area.