Ultrasound application in the presence of microbubbles is a promising strategy for intracellular delivery drug and gene, but it may also trigger other cellular responses. This study investigates the relationship between the change of cell membrane permeability generated by ultrasound-driven microbubbles and the changes in intracellular calcium concentration ([Ca2+]i). Cultured rat cardiomyoblast (H9c2) cells were exposed to a single ultrasound pulse (1 MHz, 10–15 cycles, 0.27 MPa) in the presence of a Definity™ microbubble. Intracellular transport via sonoporation was assessed in real time using propidium iodide (PI), while [Ca2+]i and dye loss from the cells were measured with preloaded fura-2. The ultrasound exposure generated fragmentation or shrinking of the microbubble. Only cells adjacent to the ultrasound-driven microbubble exhibited propidium iodide (PI) uptake with simultaneous [Ca2+]i increase and fura-2 dye loss. The amount of PI uptake was correlated with the amount of fura-2 dye loss. Cells with delayed [Ca2+]i transients from the time of ultrasound application had no uptake of PI. These results indicate the formation of non-specific pores in the cell membrane by ultrasound-stimulated microbubbles and the generation of calcium waves in surrounding cells without pores.
To investigate the effects of sonoporation, spatiotemporal evolution of ultrasound-induced changes in intracellular calcium ion concentration ([Ca 2+ ] i ) was determined using real time fura-2AM fluorescence imaging. Monolayers of Chinese hamster ovary (CHO) cells were exposed to 1-MHz ultrasound tone burst (0.2 s, 0.45 MPa) in the presence of Optison ™ microbubbles. At extracellular [Ca 2+ ] o of 0.9 mM, ultrasound application generated both non-oscillating and oscillating (periods 12-30 s) transients (changes of [Ca 2+ ] i in time) with durations of 100-180 s. Immediate [Ca 2+ ] i transients after ultrasound application were induced by ultrasound-mediated microbubble-cell interactions. In some cases, the immediately-affected cells did not return to pre-ultrasound equilibrium [Ca 2+ ] i levels, thereby indicating irreversible membrane damage. Spatial evolution of [Ca 2+ ] i in different cells formed a calcium wave and was observed to propagate outward from the immediately-affected cells at 7-20 μm/s over a distance greater than 200 μm, causing delayed transients in cells to occur sometimes 60 s or more after ultrasound application. In calcium-free solution, ultrasound-affected cells did not recover, consistent with the requirement of extracellular Ca 2+ for cell membrane recovery subsequent to sonoporation. In summary, ultrasound application in the presence of Optison ™ microbubbles can generate transient [Ca 2+ ] i changes and oscillations at a focal site and in surrounding cells via calcium waves that last longer than the ultrasound duration and spread beyond the focal site. These results demonstrate the complexity of downstream effects of sonoporation beyond the initial pore formation and subsequent diffusion-related transport through the cellular membrane.
Photoacoustic imaging is an emerging technique for anatomical and functional sub-surface imaging, but previous studies have predominantly focused on time-domain analysis. In this study, frequency-domain analysis of the radio-frequency signals from photoacoustic imaging was performed to generate quantitative parameters for tissue characterization. To account for the response of the imaging system, the photoacoustic spectra were calibrated by dividing the photoacoustic spectra (radio-frequency ultrasound spectra resulting from laser excitation) from tissue by the photoacoustic spectrum of a point absorber excited under the same conditions. The resulting quasi-linear photoacoustic spectra were fit by linear regression, and midband fit, slope, and intercept were computed from the best-fit line. These photoacoustic spectral parameters were compared between the region-of-interests (ROIs) representing prostate adenocarcinoma tumors and adjacent normal flank tissue in a murine model. The mean midband fit and intercept in the ROIs showed significant differences between cancerous and non-cancerous regions. These initial results suggest that such frequency-domain analysis can provide a quantitative method for tumor tissue characterization using photoacoustic imaging in vivo.
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