How sonoporation disrupts cellular structural integrity: morphological and cytoskeletal observations

Posters: no. 1Control ID: 1672429OBJECTIVES: In considering sonoporation for drug delivery applications, it is essential to understand how living cells respond to this puncturing force. Here we seek to investigate the effects of sonoporation on cellular structural integrity. We hypothesize that the membrane morphology and cytoskeletal behavior of sonoporated cells under recovery would inherently differ from that of normal viable cells. METHODS: A customized and calibrated exposure platform was developed for this work, and the ZR-75-30 breast carcinoma cells were used as the cell model. The cells were exposed to either single or multiple pulses of 1 MHz ultrasound (pulse length: 30 or 100 cycles; PRF: 1kHz; duration: up to 60s) with 0.45 MPa spatial-averaged peak negative pressure and in the presence of lipid-shelled microbubbles. Confocal microscopy was used to examine insitu the structural integrity of sonoporated cells (identified as ones with exogenous fluorescent marker internalization). For investigations on membrane morphology, FM 4-64 was used as the membrane dye (red), and calcein was used as the sonoporation marker (green); for studies on cytoskeletal behavior, CellLight (green) and propidium iodide (red) were used to respectively label actin filaments and sonoporated cells. Observation started from before exposure to up to 2 h after exposure, and confocal images were acquired at real-time frame rates. Cellular structural features and their temporal kinetics were quantitatively analyzed to assess the consistency of trends amongst a group of cells. RESULTS: Sonoporated cells exhibited membrane shrinkage (decreased by 61% in a cell’s cross-sectional area) and intracellular lipid accumulation (381% increase compared to control) over a 2 h period. The morphological repression of sonoporated cells was also found to correspond with post-sonoporation cytoskeletal processes: actin depolymerization was observed as soon as pores were induced on the membrane. These results show that cellular structural integrity is indeed disrupted over the course of sonoporation. CONCLUSIONS: Our investigation shows that the biophysical impact of sonoporation is by no means limited to the induction of membrane pores: e.g. structural integrity is concomitantly affected in the process. This prompts the need for further fundamental studies to unravel the complex sequence of biological events involved in sonoporation.postprin

Developmental delays and subcellular stress as downstream effects of sonoporation

Posters: no. 2Control ID: 1672434OBJECTIVES: The biological impact of sonoporation has often been overlooked. Here we seek to obtain insight into the cytotoxic impact of sonoporation by gaining new perspectives on anti-proliferative characteristics that may emerge within sonoporated cells. We particularly focused on investigating the cell-cycle progression kinetics of sonoporated cells and identifying organelles that may be stressed in the recovery process. METHODS: In line with recommendations on exposure hardware design, an immersion-based ultrasound platform has been developed. It delivers 1 MHz ultrasound pulses (100 cycles; 1 kHz PRF; 60 s total duration) with 0.45 MPa peak negative pressure to a cell chamber that housed HL-60 leukemia cells and lipid-shelled microbubbles at a 10:1 cell-tobubble ratio (for 1e6/ml cell density). Calcein was used to facilitate tracking of sonoporated cells with enhanced uptake of exogenous molecules. The developmental trend of sonoporated cells was quantitatively analyzed using BrdU/DNA flow cytometry that monitors the cell population’s DNA synthesis kinetics. This allowed us to measure the temporal progression of DNA synthesis of sonoporated cells. To investigate whether sonoporation would upset subcellular homeostasis, post-exposure cell samples were also assayed for various proteins using Western blot analysis. Analysis focus was placed on the endoplasmic reticulum (ER): an important organelle with multi-faceted role in cellular functioning. The post-exposure observation time spanned between 0-24 h. RESULTS: Despite maintaining viability, sonoporated cells were found to exhibit delays in cell-cycle progression. Specifically, their DNA synthesis time was lengthened substantially (for HL-60 cells: 8.7 h for control vs 13.4 h for the sonoporated group). This indicates that sonoporated cells were under stress: a phenomenon that is supported by our Western blot assays showing upregulation of ER-resident enzymes (PDI, Ero1), ER stress sensors (PERK, IRE1), and ER-triggered pro-apoptotic signals (CHOP, JNK). CONCLUSIONS: Sonoporation, whilst being able to facilitate internalization of exogenous molecules, may inadvertently elicit a cellular stress response. These findings seem to echo recent calls for reconsideration of efficiency issues in sonoporation-mediated drug delivery. Further efforts would be necessary to improve the efficiency of sonoporation-based biomedical applications where cell death is not desirable.postprin

A study on the change in plasma membrane potential during sonoporation

Posters: no. 4Control ID: 1680329OBJECTIVES: There has been validated that the correlation of sonoporation with calcium transients is generated by ultrasound-mediated microbubbles activity. Besides calcium, other ionic flows are likely involved in sonoporation. Our hypothesis is the cell electrophysiological properties are related to the intracellular delivery by ultrasound and microbubbles. In this study, a real-time live cell imaging platform is used to determine whether plasma membrane potential change is related to the sonoporation process at the cellular level. METHODS: Hela cells were cultured in DMEM supplemented with 10% FBS in Opticell Chamber at 37 °C and 5% CO2, and reached 80% confluency before experiments. The Calcein Blue-AM, DiBAC4(3) loaded cells in the Opticell chamber filled with PI solution and Sonovue microbubbles were immerged in a water tank on a inverted fluorescence microscope. Pulsed ultrasound (1MHz freq., 20 cycles, 20Hz PRF, 0.2-0.5MPa PNP) was irradiated at the angle of 45° to the region of interest for 1s.The real-time fluorescence imaging for different probes was acquired by a cooled CCD camera every 20s for 10min. The time-lapse fluorescence images were quantitatively analyzed to evaluate the correlation of cell viability, intracellular delivery with plasma membrane potential change. RESULTS: Our preliminary data showed that the PI fluorescence, which indicated intracellular delivery, was immediately accumulated in cells adjacent to microbubbles after exposure, suggesting that their membranes were damaged by ultrasound-activated microbubbles. However, the fluorescence reached its highest level within 4 to 6 minutes and was unchanged thereafter, indicating the membrane was gradually repaired within this period. Furthermore, using DIBAC4(3), which detected the change in the cell membrane potential, we found that the loss of membrane potential might be associated with intracellular delivery, because the PI fluorescence accumulation was usually accompanied with the change in DIBAC4 (3) fluorescence. CONCLUSIONS: Our study suggests that there may be a linkage between the cell membrane potential change and intracellular delivery mediated by ultrasound and microbubbles. We also suggest that other ionic flows or ion channels may be involved in the cell membrane potential change in sonoporation. Further efforts to explore the cellular mechanism of this phenomenon will improve our understanding of sonoporation.postprin

Gallium Nitride Based High-Power Switched HIFU Pulser with Real-Time Current/Voltage Monitoring

High-Intensity Focussed Ultrasound (HIFU) techniques make use of ultrasound transducers capable of delivering high powers to be delivered at high frequencies. Real-time monitoring of power delivered can avoid damage to the transducer and injury to patients due to overexposure. This paper demonstrates the real-time current and voltage monitoring capabilities of a new Gallium-Nitride (GaN) based switched mode transmit pulser developed for the University of Leeds High-Intensity Focussed Ultrasound Array Research Platform (HIFUARP) system, which uses a novel approach of using an Analog Front End (AFE) floating on the transmitter output to provide high bandwidth current measurement

Real-time imaging of cellular dynamics during low-intensity pulsed ultrasound exposure

Control ID: 1671584Oral Session 5 - Bioeffects of therapeutic ultrasoundOBJECTIVE: Although the therapeutic potential of low-intensity pulsed ultrasound is unquestionable, the wave-matter interactions involved in the process remain to be vaguely characterized. Here we seek to undertake a series of in-situ cellular imaging studies that aim to analyze the mechanical impact of low-intensity pulsed ultrasound on attached fibroblasts from three different aspects: membrane, cytoskeleton, and nucleus. METHODS: Our experimental platform comprised an in-house ultrasound exposure hardware that was coupled to a confocal microscopy system. The waveguided ultrasound beam was geometrically aligned to the microscope’s fieldof-view that corresponds to the center of a polystyrene dish containing fibroblasts. Short ultrasound pulses (5 cycles; 2 kHz PRF) with 0.8 MPa peak acoustic pressure (0.21 W/cm2 SPTA intensity) were delivered over a 10 min period. Live imaging was performed on both membrane (CellMask) and cytoskeleton (actin-GFP, tubulin-RFP) over the entire observation period (up to 30 min after end of exposure). Also, pre- and post-exposure fixed-cell imaging was conducted on the nucleus (Hoechst 33342) and two cytoskeleton components related to stress fibers: F-actin (phalloidin-FITC) and vincullin (Alexa Fluor 647 conjugated). To study whether mechanotransduction was responsible in mediating ultrasound-cell interactions, some experiments were conducted with the addition of gadolinium that blocks stretch-sensitive ion channels. RESULTS: Cell shrinkage was evident over the course of low-intensity pulsed ultrasound exposure. This was accompanied with contraction of actin and tubulin. Also, an increase in central stress fibers was observed at the end of exposure, while the nucleus was found to have decreased in size. Interestingly, after the exposure, a significant rebound in cell volume was observed over a 30 min. period. These effects were not observed in cases with gadolinium blockage of mechanosensitive ion channels. CONCLUSIONS: Our results suggest that low-intensity pulsed ultrasound would transiently induce remodeling of a cell’s membrane and cytoskeleton, and it will lead to repression of nucleus. This indicates that ultrasound after all represents a mechanical stress on cellular membrane. The post-exposure outgrowth phenomenon is also of practical relevance as it may be linked to the stimulatory effects that have been already observed in low-intensity pulsed ultrasound treatments.postprin

Study on the effect of the sonication power and intersonication cooling time on the near field temperature in magnetic resonance high intensity focused ultrasound

Background: MR-HIFU is a technological breakthrough for non-surgical approach for treating tumour. MR-HIFU is safe with many advantages however complications may occur, at target and the near-field region. Specific ultrasound setup at MR-HIFU console must be made prior to ablation process, which can affect the near-field temperature. The purpose of this study was to investigate how intersonication cooling time and sonication power influence the near-field temperature. Methodology: Philips QA phantom was used for ablation. A total of 100 ablations were made with different intersonication cooling time, which are 30 s, 60 s, 90 s and 120 s and different sonication power, which are 40 W, 80 W, 120 W, 160 W and 200 W. The near-field region was taken at 2cm from the base of phantom. The data were evaluated using statistical analysis. Level of significance was determined (p < 0.05). Results: There was no significant correlation between intersonication cooling time and the near-field temperature, with p = 0.089. However, there is a significant correlation between sonication power and the near-field temperature with p = 0.001. Regression analysis indicates that the sonication power significantly predicted the temperature changes [R2 = .416, F(1,18) = 14.533, p = .001] Conclusion: There is no relationship between intersonication cooling time with near-field temperature. However, there is relationship between sonication power and near-field temperature

Treatment of brain cancer and ischaemic stroke utilising High Intensity Focus Ultrasound (HIFU) guide with MRI

In this thesis high intensity focused ultrasound (HIFU) is utilized for cancer treatment (thermal mode) and treatment of ischaemic stroke (mechanical mode). These two applications were investigated in vitro and in vivo models. MRI was utilized to monitor the lesions created by HIFU either in thermal or cavitation mode in freshly excised lamb brain tissue in vitro, and in rabbit brain in vivo. Additionally, MRI was used to monitor lesions deep in tissue for both in vitro and in vivo exposures. All three MRI sequences used (T1-W FSE, T2-W FSE and FLAIR) were able to detect lesions. Both thermal and bubbly lesions were best monitored using T1-W FSE with excellent contrast, proving the potential of HIFU to treat reliably tumours in the brain. A HIFU system was also used to assist thrombolysis in cooperation with a thrombolytic drug such as recombinant tissue plasminogen activator (rt-PA) in vitro and in vivo. It was shown that higher intensity results to higher volume of dissolved clot, but there is a limit of the intensity to be used in order to avoid heating of the clot and the surrounding tissue. The goal in this study was to achieve temperature elevation not exceeding 1ºC (called safe temperature). It was found that the larger the beam area the larger the dissolved clot volume. Also, the lower the frequency, the larger the volume of the dissolved clot. The results reported herein point to the use of frequency around 0.5 MHz and pulsing to optimize thrombolysis and skull penetration and at the same time avoiding unwanted heating. Finally, an Acrylonitrile Butadiene Styrene (ABS) phantom skull model was developed in order to evaluate the propagation of ultrasound using a single element transducer. The skull model was appropriately designed so that it has the same attenuation as a human skull. It was demonstrated that using a frequency of 0.5 MHz versus 1 MHz, ultrasound propagation through the phantom skull was higher. Therefore, higher frequency has poor skull penetration and a small beam size at the focus, while low frequencies have better skull penetration but with the risk of reaching the unpredictable effect of cavitation. The developed system has proven to successfully create large lesions in the brain and at the same time, these lesions are successfully monitored with excellent contrast using MRI (T1-W FSE) enabling the accurate determination of the margins of these lesions. The results reported in this study point to the use of frequency around 0.5 MHz and pulsing to optimize thrombolysis and skull penetration and at the same time avoiding unwanted heating. For treating tumours located deep in the brain and for dissolving thrombus causing an acute ischaemic stroke, further extensive clinical studies will be needed before this technology is applied to humans

IDENTIFYING AND MONITORING THE ROLES OF CAVITATION IN HEATING FROM HIGH-INTENSITY FOCUSED ULTRASOUND

For high-intensity focused ultrasound (HIFU) to continue to gain acceptance for cancer treatment it is necessary to understand how the applied ultrasound interacts with gas trapped in the tissue. The presence of bubbles in the target location have been thought to be responsible for shielding the incoming pressure and increasing local heat deposition due to the bubble dynamics. We lack adequate tools for monitoring the cavitation process, due to both limited visualization methods and understanding of the underlying physics. The goal of this project was to elucidate the role of inertial cavitation in HIFU exposures in the hope of applying noise diagnostics to monitor cavitation activity and control HIFU-induced cavitation in a beneficial manner. A number of approaches were taken to understand the relationship between inertial cavitation signals, bubble heating, and bubble shielding in agar-graphite tissue phantoms. Passive cavitation detection (PCD) techniques were employed to detect inertial bubble collapses while the temperature was monitored with an embedded thermocouple. Results indicate that the broadband noise amplitude is correlated to bubble-enhanced heating. Monitoring inertial cavitation at multiple positions throughout the focal region demonstrated that bubble activity increased prefocally as it diminished near the focus. Lowering the HIFU duty cycle had the effect of maintaining a more or less constant cavitation signal, suggesting the shielding effect diminished when the bubbles had a chance to dissolve during the HIFU off-time. Modeling the effect of increasing the ambient temperature showed that bubbles do not collapse as violently at higher temperatures due to increased vapor pressure inside the bubble. Our conclusion is that inertial cavitation heating is less effective at higher temperatures and bubble shielding is involved in shifting energy deposition at the focus. The use of a diagnostic ultrasound imaging system as a PCD array was explored. Filtering out the scattered harmonics from the received RF signals resulted in a spatially- resolved inertial cavitation signal, while the amplitude of the harmonics showed a correlation with temperatures approaching the onset of boiling. The result is a new tool for detecting a broader spectrum of bubble activity and thus enhancing HIFU treatment visualization and feedback.Gordon Center for Subsurface Sensing and Imaging Systems via NSF ERC Award Number EEC-9986821 and the U.S. Army, award number DAMD17-02-2-0014

Printed Receive Coils with High Acoustic Transparency for Magnetic Resonance Guided Focused Ultrasound.

In magnetic resonance guided focused ultrasound (MRgFUS) therapy sound waves are focused through the body to selectively ablate difficult to access lesions and tissues. A magnetic resonance imaging (MRI) scanner non-invasively tracks the temperature increase throughout the tissue to guide the therapy. In clinical MRI, tightly fitted hardware comprised of multichannel coil arrays are required to capture high quality images at high spatiotemporal resolution. Ablating tissue requires a clear path for acoustic energy to travel but current array materials scatter and attenuate acoustic energy. As a result coil arrays are placed outside of the transducer, clear of the beam path, compromising imaging speed, resolution, and temperature accuracy of the scan. Here we show that when coil arrays are fabricated by additive manufacturing (i.e., printing), they exhibit acoustic transparency as high as 89.5%. This allows the coils to be placed in the beam path increasing the image signal to noise ratio (SNR) five-fold in phantoms and volunteers. We also characterize printed coil materials properties over time when submerged in the water required for acoustic coupling. These arrays offer high SNR and acceleration capabilities, which can address current challenges in treating head and abdominal tumors allowing MRgFUS to give patients better outcomes