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

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

Simulation study on acousto-optics sensing of focused ultrasound

Abstract. The acousto-optics (AO) technique can provide a good contrast with high penetration depth (up to 5 cm) and can be potentially utilized in real time monitoring of the focused ultrasound (FUS) therapies. This work presents the AO simulation study on the interaction of light and FUS in the single-layer brain (SLB) medium and four-layer brain (FLB) medium. FUS pressure distribution at 0.5 MHz and 0.9 MHz frequency was simulated on k-Wave toolbox and the AO Monte Carlo (MC) algorithm was developed on MATLAB to simulate the AO effect in both mediums. The result for the SLB for both ultrasound (US) frequencies suggests that the modulation depth (MD) is high in the region of US focus with a magnitude of 2%-3% and <1% at 0.5 MHz and 0.9 MHz, respectively. Moreover, the MD decreases to 5 orders of magnitude at the source region. In the FLB, the MD decreased to 4–4.5 orders at the source and was present in the skull and US focus region with a magnitude of <1% at both US frequencies. These results suggest that AO can be utilized in sensing FUS effects on brain tissue and the AO signal-to-noise ratio (SNR) depends not only on the MD but also on the level of light intensity interacting with the US pressure

Controlling microbubble dynamics in ultrasound therapy

Microbubble-mediated focused ultrasound therapies such as blood-brain barrier opening, sonothrombolysis, and sonoporation can non-invasively deliver drugs to a targeted region. Despite the potential impact this technology could have in the clinic, there are currently concerns regarding its efficacy, uniformity and safety. These challenges ultimately stem from a limited ability to control the microbubble dynamics during ultrasound exposure. In the current thesis, we sought to design ultrasound exposure sequences and develop monitoring techniques that promote the desired acoustic cavitation activity and suppress unwanted stimuli that do not produce a safe therapeutic bioeffect. For example, violent cavitation activity could cause irreversible damage within the treatment area. The behaviour of microbubble populations exposed to low-power therapeutic ultrasound was first qualitatively studied using high-speed microscopy. Microbubbles were found to form large clusters within milliseconds of exposure and collectively coalesce into larger bubbles. Based on these observations and findings in the literature, new therapeutic sequences were designed and tested. Rapid short-pulse sonication consisted of μs-long pulses separated by short off-times. When compared to conventional ultrasound sequences, this pulse sequence enhanced the lifetime and mobility of cavitation nuclei and resulted in more uniform acoustic activity distributions. To better monitor ultrasound treatment as it evolves, we developed a method that passively measures microbubble velocities via the Doppler effect emerging in the microbubble acoustic emissions. Using standard passive cavitation detection techniques in one and two dimensions, we estimated microbubble velocities on the order of m s-1 during ultrasound exposure. Finally, we tested our new therapeutic design in a mouse model in order to improve the safety of blood-brain barrier opening. We achieved drug delivery with a similar magnitude but with a better uniformity compared to conventional sequences, thus demonstrating evidence of favourable microbubble dynamics within the targeted region. Taken together, our contribution in ultrasonic stimulation using new sequences and monitoring using passive acoustic detection techniques improves our control of microbubble dynamics in ultrasound therapy and has the potential to promote treatment efficacy and suppress unwanted damage.Open Acces

TOWARD CLINICAL TRANSLATION OF MICROVASCULAR ULTRASOUND IMAGING: ADVANCEMENTS IN SUPERHARMONIC ULTRASOUND TECHNOLOGY

Ultrasound imaging is perhaps the safest, most affordable, and most available biomedical imaging modality. However, it suffers from poor specificity for cancer detection, particularly in breast cancer, which affects one in eight women and leads to a high incidence of unnecessary biopsies from inconclusive screening. It is well-known that malignant cancers are accompanied by abnormal angiogenesis, leading to tortuous and disorganized vasculature. Acoustic angiography, a microvascular contrast-enhanced ultrasound technique, was developed to visualize and harness this aberrant vasculature as a biomarker of malignancy. This technique applies a dual-frequency superharmonic strategy to isolate intravascular microbubble contrast from the surrounding tissue with low-frequency transmit and high-frequency receive, resulting in high-resolution microvascular maps. Preclinically, acoustic angiography has been a valuable tool for differentiating tumors from healthy tissue by quantifying vascular features like tortuosity. The preclinical success of this technique is attributed to the single-element dual-frequency transducers used, which provide contrast sensitivity and focal depth best suited for imaging small animals at high microbubble doses. In an exploratory clinical study in which these transducers were used to image the human breast, imaging depth, low sensitivity, and motion artifacts significantly degraded image quality. For acoustic angiography to be successfully translated to clinical use, the technique must be optimized for clinical imaging. In this dissertation, we explore three ways in which acoustic angiography may be improved for the clinic. First, we evaluate microbubble contrast agents to determine the composition that maximizes superharmonic generation. The results indicate that lipid-shelled microbubbles with perfluorocarbon cores, like the commercial agent, DEFINITY, produce the greatest superharmonic signal. Then, we present a novel transducer, a stacked dual-frequency array, as the next-generation device for acoustic angiography and demonstrate improvements in imaging depth and sensitivity up to 10 mm and 13 dB, respectively. We go on to apply this device in a clinical pilot study and elucidate the challenges that remain to be overcome for clinical acoustic angiography. Finally, we propose custom simulations for superharmonic imaging and identify optimal frequency combinations for imaging at depths up to 8 cm, which can be used to design dedicated clinical dual-frequency arrays in the future.Doctor of Philosoph

VISUALIZATION OF ULTRASOUND INDUCED CAVITATION BUBBLES USING SYNCHROTRON ANALYZER BASED IMAGING

Ultrasound is recognized as the fastest growing medical modality for imaging and therapy. Being noninvasive, painless, portable, X-ray radiation-free and far less expensive than magnetic resonance imaging, ultrasound is widely used in medicine today. Despite these benefits, undesirable bioeffects of high-frequency sound waves have raised concerns; particularly, because ultrasound imaging has become an integral part of prenatal care today and is increasingly used for therapeutic applications. As such, ultrasound bioeffects must be carefully considered to ensure optimal benefits-to-risk ratio. In this context, few studies have been done to explore the physics (i.e. ‘cavitation’) behind the risk factors. One reason may be associated with the challenges in visualization of ultrasound-induced cavitation bubbles in situ. To address this issue, this research aims to develop a synchrotron-based assessment technique to enable visualization and characterization of ultrasound-induced microbubbles in a physiologically relevant medium under standard ultrasound operating conditions. The first objective is to identify a suitable synchrotron X-ray imaging technique for visualization of ultrasound-induced microbubbles in water. Two synchrotron X-ray phase-sensitive imaging techniques, in-line phase contrast imaging (PCI) and analyzer-based imaging (ABI), were evaluated. Results revealed the superiority of the ABI method compared to PCI for visualization of ultrasound-induced microbubbles. The second main objective is to employ the ABI method to assess the effects of ultrasound acoustic frequency and power on visualization and mapping of ultrasound-induced microbubble patterns in water. The time-averaged probability of ultrasound-induced microbubble occurrence along the ultrasound beam propagation in water was determined using the ABI method. Results showed the utility of synchrotron ABI for visualizing cavitation bubbles formed in water by clinical ultrasound systems working at high frequency and output powers as low as used for therapeutic systems. It was demonstrated that the X-ray ABI method has great potential for mapping ultrasound-induced microbubble patterns in a fluidic environment under different ultrasound operating conditions of clinical therapeutic devices. Taken together, this research represents an advance in detection techniques for visualization and mapping of ultrasound-induced microbubble patterns using the synchrotron X-ray ABI method without usage of contrast agents. Findings from this research will pave the road toward the development of a synchrotron-based detection technique for characterization of ultrasound-induced cavitation microbubbles in soft tissues in the future