Time-point-based analysis of gold nanoparticles in MCF-7 cells following ultrasound irradiation: quantitative and label-free intracellular characterization.

Ultrasound-mediated nanoparticle delivery has garnered increasing attention in recent years; however, the time-lapse intracellular distribution of gold nanoparticles (GNPs) following ultrasound exposure has not been adequately explored yet, particularly with respect to quantitative uptake and optimized ultrasound parameters. Here, we provide analysis addressing these gaps by employing quantitative and label-free intracellular tracking to elucidate the interactions between ultrasound and GNPs in MCF-7 breast cancer cells. MCF-7 cells treated with GNPs were exposed to 40 kHz ultrasound for 5, 10, and 20 min. Following irradiation, the cells were incubated for 0, 3, or 24 h to assess post-ultrasound intracellular distribution. Confocal imaging was performed to visualize label-free intracellular GNP clusters, enabling spatial analysis of nanoparticle dispersion. Ultrasound irradiation reduced the maximum cluster size of intracellular GNPs with increasing exposure time, reaching up to a 22% decrease compared to the control group. Optical diffraction tomography further revealed sonoporation upon ultrasound exposure, indicating ultrasound-induced membrane permeabilization. GNP concentrations measured by inductively coupled plasma atomic emission spectroscopy demonstrated that GNP uptake increased immediately post-irradiation, showing a 3.5-fold rise in the 20 min group. However, this enhancement diminished over time, with intracellular GNP levels across all groups converging around 10 h post-treatment. Nonetheless, ultrasound promoted a more uniform cytoplasmic distribution of GNPs. These findings highlight ultrasound as a rapid yet transient enhancer of intracellular GNP delivery and dispersion. Our label-free and quantitative approach enabled the spatiotemporal assessment of GNP dynamics, revealing time-dependent uptake and sonoporation.