Ronald E. Kumon

Intracellular delivery and calcium transients generated in sonoporation facilitated by microbubbles.

Ultrasound application in the presence of microbubbles is a promising strategy for intracellular drug and gene delivery, 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 ([Ca(2+)](i)). Cultured rat cardiomyoblast (H9c2) cells were exposed to a single ultrasound pulse (1MHz, 10-15cycles, 0.27MPa) in the presence of a Definity(TM) microbubble. Intracellular transport via sonoporation was assessed in real time using propidium iodide (PI), while [Ca(2+)](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 uptake with simultaneous [Ca(2+)](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 [Ca(2+)](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.

The size of sonoporation pores on the cell membrane

Sonoporation generates transient pores on the cell membrane and has been exploited as a promising intracellular drug and gene delivery strategy. The size of the pores in the membrane resulting from sonoporation determines the size of molecules or agents that can be delivered using the technique. However, size information has not been readily available due to the challenges in measuring dynamic, submicron-sized pores. Post ultrasound assays such as AFM or SEM, often time consuming and labor intensive, have been used to gauge pore size but are intrinsically limited to static measurements that may not accurately represent the relevant size. We have previously demonstrated the utility of voltage clamp techniques for monitoring sonoporation in real time via the trans-membrane current (TMC) change of a single cell under voltage clamp. Using Xenopus oocytes as the model system in this study, the TMC change during sonoporation (0.2 s, 0.3 MPa, 1 MHz) of single cells in solution with Definity microbubbles was recorded in a whole cell voltage clamp configuration. As the changes of the TMC are related to the diffusion of ions through the pores on the membrane, they can potentially provide relevant information of the pore size generated in sonoporation. By controlling the microbubble concentration, experiments were designed to allow measurement of the TMC corresponding to a single pore on the membrane. An electro-diffusion model was developed to relate the TMC with pore size from the ion flow through the pores on the membrane.

Frequency-domain analysis of photoacoustic imaging data from prostate adenocarcinoma tumors in a murine model

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 noncancerous regions. These initial results suggest that such frequency-domain analysis can provide a quantitative method for tumor tissue characterization using photoacoustic imaging in vivo.

SPATIOTEMPORAL EFFECTS OF SONOPORATION MEASURED BY REAL-TIME CALCIUM IMAGING

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 a 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 nonoscillating and oscillating (periods 12 to 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 that was observed to propagate outward from the immediately affected cells at 7-20 microm/s over a distance >200 microm, 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.

Modulation of Intracellular Ca2+ Concentration in Brain Microvascular Endothelial Cells in vitro by Acoustic Cavitation

Localized delivery of therapeutic agents through the blood-brain barrier (BBB) is a clinically significant task that remains challenging. Ultrasound (US) application after intravenous administration of microbubbles has been shown to generate localized BBB opening in animal models but the detailed mechanisms are not yet fully described. The current study investigates the effects of US-stimulated microbubbles on in vitro murine brain microvascular endothelial (bEnd.3) cells by monitoring sonoporation and changes in intracellular calcium concentration ([Ca(2+)](i)) using real-time fluorescence and high-speed brightfield microscopy. Cells seeded in microchannels were exposed to a single US pulse (1.25 MHz, 10 cycles, 0.24 MPa peak negative pressure) in the presence of Definity microbubbles and extracellular calcium concentration [Ca(2+)](o) = 0.9 mM. Disruption of the cell membrane was assessed using propidium iodide (PI) and change in the [Ca(2+)](i) was measured using fura-2. Cells adjacent to a microbubble exhibited immediate [Ca(2+)](i) changes after US pulse with and without PI uptake and the [Ca(2+)](i) changes were twice as large in cells with PI uptake. Cell viability assays showed that sonoporated cells could survive with modulation of [Ca(2+)](i) and uptake of PI. Cells located near sonoporated cells were observed to exhibit changes in [Ca(2+)](i) that were delayed from the time of US application and without PI uptake. These results demonstrate that US-stimulated microbubbles not only directly cause changes in [Ca(2+)](i) in brain endothelial cells in addition to sonoporation but also generate [Ca(2+)](i) transients in cells not directly interacting with microbubbles, thereby affecting cells in larger regions beyond the cells in contact with microbubbles.

Mechanisms of microbubble-facilitated sonoporation for drug and gene delivery

Ultrasound-mediated delivery facilitated by microbubbles provides a novel means for intracellular drug and gene delivery and particularly a noninvasive strategy uniquely suitable for clinical applications. Spatiotemporally controllable application of ultrasound energy combined with microbubbles make it possible for site-specific delivery of therapeutic agents to the region-of-interest with minimal undesirable systemic side effects. By inducing rapid expansion/contraction and/or collapse of microbubbles, ultrasound application can temporarily increase the cell membrane permeability (sonoporation) to create a physical route for impermeable agents to enter the cells. Sonoporation is transient and dynamic, involving complex processes of bubble physics, bubble–cell interactions, and subsequent cellular effects that all affect the ultimate delivery outcome. This review summarizes the studies on the important aspects of the mechanisms of ultrasound-mediated delivery, provides illustrative examples of applications, and discusses the challenges and limitations of the technique. Microbubbles have been used for several decades as a contrast agent for ultrasound imaging [1]. The small size of microbubbles allows them access to well-perfused organs when injected into the vasculature, and their gas core efficiently reflects and scatters the incident ultrasound field, thereby increasing image contrast between the vasculature and the surrounding tissue. Recent developments in microbubble technology have enabled molecular imaging via targeting of the microbubbles to molecular markers of disease expressed on the surface of cells [2]. In addition to imaging, innovation in microbubbles has opened new opportunities for targeted drug and gene delivery. Ultrasound excitation of microbubbles has been exploited to increase vascular and cell membrane permeability and facilitate the passage of therapeutic agents across the vascular barrier and cell membrane into the cytoplasm for drug and gene transfection [3–7]. The ultrasound delivery technique, with the advantage of noninvasive, spatiotemporaly controllable ultrasound application combined with functionalized micro-bubbles, holds great promise to provide new therapeutic strategies.

High-frequency rapid B-mode ultrasound imaging for real-time monitoring of lesion formation and gas body activity during high-intensity focused ultrasound ablation

The goal of this study was to examine the ability of high-frame-rate, high-resolution imaging to monitor tissue necrosis and gas-body activities formed during high-intensity focused ultrasound (HIFU) application. Ex vivo porcine cardiac tissue specimens (n = 24) were treated with HIFU exposure (4.33 MHz, 77 to 130 Hz pulse repetition frequency (PRF), 25 to 50% duty cycle, 0.2 to 1 s, 2600 W/cm2). RF data from Bmode ultrasound imaging were obtained before, during, and after HIFU exposure at a frame rate ranging from 77 to 130 Hz using an ultrasound imaging system with a center frequency of 55 MHz. The time history of changes in the integrated backscatter (IBS), calibrated spectral parameters, and echo-decorrelation parameters of the RF data were assessed for lesion identification by comparison against gross sections. Temporal maximum IBS with +12 dB threshold achieved the best identification with a receiver-operating characteristic (ROC) curve area of 0.96. Frame-to-frame echo decorrelation identified and tracked transient gas-body activities. Macroscopic (millimetersized) cavities formed when the estimated initial expansion rate of gas bodies (rate of expansion in lateral-to-beam direction) crossed 0.8 mm/s. Together, these assessments provide a method for monitoring spatiotemporal evolution of lesion and gas-body activity and for predicting macroscopic cavity formation.

High-Frequency Ultrasound M-Mode Imaging for Identifying Lesion and Bubble Activity During High-Intensity Focused Ultrasound Ablation

Effective real-time monitoring of high-intensity focused ultrasound (HIFU) ablation is important for application of HIFU technology in interventional electrophysiology. This study investigated rapid, high-frequency M-mode ultrasound imaging for monitoring spatiotemporal changes during HIFU application. HIFU (4.33 MHz, 1 kHz PRF, 50% duty cycle, 1 s, 2600‒6100 W/cm²) was applied to ex vivo porcine cardiac tissue specimens with a confocally and perpendicularly aligned high-frequency imaging system (Visualsonics Vevo 770, 55 MHz center frequency). Radio-frequency (RF) data from M-mode imaging (1 kHz PRF, 2 s × 7 mm) was acquired before, during and after HIFU treatment (n = 12). Among several strategies, the temporal maximum integrated backscatter with a threshold of +12 dB change showed the best results for identifying final lesion width (receiver-operating characteristic curve area 0.91 ± 0.04, accuracy 85 ± 8%, compared with macroscopic images of lesions). A criterion based on a line-to-line decorrelation coefficient is proposed for identification of transient gas bodies.

Characterization of the pancreas in vivo using EUS spectrum analysis with electronic array echoendoscopes

BACKGROUND Spectral analysis of the radiofrequency (RF) signals that underlie grayscale EUS images has been used to provide quantitative, objective information about tissue histology. OBJECTIVE Our purpose was to validate RF spectral analysis as a method to distinguish between chronic pancreatitis (CP) and pancreatic cancer (PC). DESIGN AND SETTING A prospective study of eligible patients was conducted to analyze the RF data obtained by using electronic array echoendoscopes. PATIENTS Pancreatic images were obtained by using electronic array echoendoscopes from 41 patients in a prospective study, including 15 patients with PC, 15 with CP, and 11 with a normal pancreas. MAIN OUTCOME MEASUREMENTS Midband fit, slope, intercept, correlation coefficient, and root mean square deviation from a linear regression of the calibrated power spectra were determined and compared among the groups. RESULTS Statistical analysis showed that significant differences were observable between groups for mean midband fit, intercept, and root mean square deviation (t test, P < .05). Discriminant analysis of these parameters was then performed to classify the data. For CP (n = 15) versus PC (n = 15), the same parameters provided 83% accuracy and an area under the curve of 0.83. LIMITATIONS Moderate sample size and spatial averaging inherent in the technique. CONCLUSIONS This study shows that mean spectral parameters of the backscattered signals obtained by using electronic array echoendoscopes can provide a noninvasive method to quantitatively discriminate between CP and PC.

TOMOGRAPHIC RECONSTRUCTION OF TISSUE PROPERTIES AND TEMPERATURE INCREASE FOR HIGH-INTENSITY FOCUSED ULTRASOUND APPLICATIONS

The acoustic and thermal properties as well as the temperature change within a tissue volume during high-intensity focused ultrasound ablation are critically important for treatment planning and monitoring. Described in this article is a tomographic reconstruction method used to determine the tissue properties and increase in temperature in a 3-D volume. On the basis of the iterative finite-element solution to the bioheat equation coupled with Tikhonov regularization techniques, our reconstruction algorithm solves the inverse problem of bioheat transfer and uses the time-dependent temperature measured on a tissue surface to obtain the acoustic absorption coefficient, thermal diffusivity and temperature increase within the subsurface volume. Numerical simulations were performed to validate the reconstruction algorithm. The method was initially conducted in ex vivo experiments in which time-dependent temperature on a tissue surface was measured using high-resolution, non-invasive infrared thermography.