2.11. Increased intracellular uptake of DOX by SP94-DOX-NDs under ultrasound irradiation

Because SP94-DOX-NDs are desired to deliver more DOX to tumor cells, the intracellular uptake of DOX must be determined, and this is accomplished using ultrasound irradiation. Considering cytotoxicity, the 50% inhibition concentration (IC50) of DOX was calculated. Ultrasound stimulation was performed using a therapeutic ultrasound device (WED-100, Honda Hi-Tech, Shenzhen, China), and the parameters were set as follows: fixed-frequency = 1 MHz, duty cycle = 70%, pulse repetition rate = 100 Hz, sound intensity = 0.5 W/cm², and time = 60 s (Meng et al., 2019). Cells were exposed for 2 h to different treatments (PBS, DOX, DOX-NDs, SP94-DOX-NDs) with and without ultrasound irradiation while maintaining the DOX concentration of the different groups at the same level. After fixation with 4% paraformaldehyde, cells were examined via fluorescence microscopy.