Increased intracellular diffusivity of macromolecules within a mammalian cell by low-intensity pulsed ultrasound

Whilst a number of studies have demonstrated that low-intensity pulsed ultrasound (LIPUS) is a promising therapeutic ultrasound technique that can be used for delivering mild mechanical stimuli to target tissue non-invasively, the underlying biophysical mechanisms still remain unclear. Most mechanism studies have focused explicitly on the effects of LIPUS on the cell membrane and mechanosensitive receptors. In the present study, we propose an additional mechanism by which LIPUS propagation through living cells may directly impact intracellular dynamics, particularly the diffusion transport of biomolecules. To support our hypothesis, human epithelial-like cells (SaOS-2 and HeLa) seeded on a confocal dish placed on a microscope stage were exposed to LIPUS with various exposure conditions (ultrasound frequencies of 0.5, 1 and 3 MHz, peak acoustic pressure of 200 and 400 kPa, a pulse repetition frequency of 1 kHz and a 20 % duty cycle), and the diffusivities of various sizes of biomolecules in the cytoplasm area were measured using fluorescence recovery after photobleaching (FRAP). Furthermore, giant unilamellar vesicles (GUVs) filled with macromolecules were used to examine the physical causal relationship between LIPUS and molecular diffusion changes. Nucleocytoplasmic transport coefficients were also measured by modified FRAP that bleaches the whole cell nuclear region. Extracellular signal-regulated kinases (ERK) activity (the phosphorylation dynamics) was monitored using fluorescence resonance energy transfer (FRET) microscopy. All the measurements were taken during, before and after the LIPUS exposure. Our experimental results clearly showed that the diffusion coefficients of macromolecules within the cell increased with acoustic pressure by 12.1 to 33.5 % during the sonication, and the increments were proportional to their molecular sizes regardless of the ultrasound frequency used. This observation in living cells was consistent with the GUVs exposed to the LIPUS, which indicated that the diffusivity increase was a passive physical response to the acoustic energy of LIPUS. Under the 1 MHz LIPUS exposure with 400 kPa, the passive nucleocytoplasmic transport of enhanced green fluorescent protein (EGFP) was accelerated by 21.4 %. With the same LIPUS exposure condition, both the diffusivity and phosphorylation of ERK induced by EGF treatment were significantly elevated simultaneously, which implied that LIPUS could also modify the kinase kinetics in the signal transduction process. Taken together, this study is the first attempt to uncover the physical link between LIPUS and the dynamics of intracellular macromolecules and related biological processes that LIPUS can possibly increase the diffusivity of intracellular macromolecules, leading to the changes in the basic cellular processes: passive nucleocytoplasmic transport and ERK. Our findings can provide a novel perspective that the mechanotransduction process that the intracellular region, in addition to the cell membrane, can convert the acoustic stimuli of LIPUS to biochemical signals.