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

HIGH INTENSITY FOCUSED ULTRASOUND AND OXYGEN LOAD NANOBUBBLES: TWO DIFFERENT APPROCHES FOR CANCER TREATMENT

The study of applications based on the use of ultrasound in medicine and biology for therapeutic purposes is under strong development at international level and joins the notoriously well-established and widespread use of diagnostic applications [1]. In the past few years, High Intensity Focused Ultrasound (HIFU) has developed from a scientific curiosity to an accepted therapeutic modality. HIFU is a non invasive technique for the treatment of various types of cancer, as well as non-malignant pathologies, by inducing localized hyperthermia that causes necrosis of the tissue. Beside HIFU technology, other innovative therapeutic modalities to treat cancer are emerging. Among them, an extremely innovative technique is represented by oxygen loaded nanobubbles (OLNs): gas cavities confined by an appropriately functionalized coating. This is an oxygenating drugs aimed at re-oxygenation of cancerous tissue. Oxygen deficiency, in fact, is the main hallmark of cancerous solid tumors and a major factor limiting the effectiveness of radiotherapy. In this work, these two approaches to treat tumours are under study from a metrological point of view. In particular, a complete characterization of an HIFU fields regarding power, pressure and temperature is provided while oxygen load nanobubbles are synthesized, characterized and applied in in vitro and in vivo experiments

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

Development of a medical imaging-based technology for cancer treatment

The Electrical Impedance Mammography (EIM) device is an imaging system developed at the University of Sussex for the detection of breast lesions in vivo using quadrature detection of impedance. The work describes a novel technique to integrate Ultrasound-guided Focused Ultrasound Surgery (USgFUS) with the existing EIM system. The benefits that such a system could provide include the possibility of non-invasive detection, diagnosis and treatment of breast cancer all within a single device and involving no radiation. Furthermore the timescales involved would allow the process to be considered an outpatient procedure such that a patient can be diagnosed and treated on the same day using the same device. Various geometries of transducer were investigated for physical compatibility as well as the ability to target the entire specified volume, based on the dimensions of the existing system. Simulations were performed using a custom written code based on Huygen’s principle, allowing minimum surface area and power requirements to be determined and feasibility of designs to be evaluated. The use of phase differences in the excitation signals applied to individual elements was also investigated, thus the effect of steering the simulated focus could be observed, an important factor to consider when attempting to incorporate a transducer into a device with restricted dimensions. Resulting simulated pressure fields were used to obtain acoustic intensity fields, which could then be used as inputs in the Pennes Bio-Heat Transfer Equation (BHTE) allowing temperature distributions to be observed. Preliminary studies proved the feasibility of using the suggested transducer design in conjunction with the existing EIM system. Pressure fields and heating patterns were all within acceptable limits, confirming the ability of the device to effectively ablate cancerous tissue. Additionally the capability to steer the resultant focal point was validated, and a thermal dose model was implemented allowing different heating patterns to be quantitatively compared

INVESTIGATION OF THERAPY IMPROVEMENT USING REAL-TIME PHOTOACOUSTIC IMAGING GUIDED HIGH INTENSITY FOCUSED ULTRASOUND

There are a lot of risks in cancer treatment by invasive surgery, such as bleeding, wound infection, and long recovery time, etc. Therefore, there is great need for minimally- or non-invasive treatment. High intensity focused ultrasound (HIFU) is a rapidly growing and truly non-invasive technology. It has been widely used in therapeutic applications, such as rapid tissue heating and tissue ablation. With proper imaging guidance, HIFU treatment can be performed totally noninvasively. Currently, ultrasound imaging-guided HIFU has been extensively studied. However, ultrasound imaging guidance is less precise because of the relatively low imaging contrast, sensitivity, and specificity for noninvasive detection. In this study, we employed photoacoustic imaging (PAI) technique, which has been developed a novel promising imaging technique for early cancer detection, to guide HIFU treatment. The goal of this study is to investigate the feasibility of PAI to guide, monitor in real time and enhance the HIFU therapy. In this dissertation, as the first step, the integrated PAI and HIFU system had been shown to have the feasibility to guide HIFU both ex vivo and in vivo. Then, the system was improved and developed to a real-time PAI-guided HIFU system. It is demonstrated that the sensitivity of PA detection for HIFU lesion is very high and the saturation of PA signals can be used as the indicator for tissue coagulation. During the temperature measurement using this system, laser-enhanced HIFU heating was found. Thus, we further investigated the laser enhanced technique in both HIFU heating and pulsed HIFU thrombolysis. In the HIFU therapy, laser light was employed to illuminate the sample concurrently with HIFU radiation. The resulting cavitation was detected with a passive cavitation detector. We demonstrated that concurrent light illumination during HIFU has the potential to significantly enhance HIFU by reducing cavitation threshold

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

Development of positioning devices for MRI-guided high intensity focused ultrasound (HIFU) for abdominal, thyroid and brain, tumours

High intensity focused ultrasound (HIFU) is a promising technology for a variety of therapeutic applications. This concept initiated in 1942 by Lynn Zwemer [1]. HIFU has long been known as a minimal invasive or non-invasive procedure that destroys tissue through ablation. However, it is only in recent years that clinical applications are becoming feasible, with the development of high power ultrasound transducers compatible with the MRI scanner which is used to monitor these non-invasive HIFU applications. New technologies, combined with more sophisticated treatment methods and monitoring methods allow non-invasive procedures in many areas such as the brain, eye, breast, kidney, liver, pancreas, thyroid, uterine fibroids and pancreas. Meanwhile, new investigations are underway for treading cardiac arithmia, strokes, palliative pain treatment of bone metastases and brain disorders such as Parkinson’s disease, essential tremor, and neuropathic pain. These optimistic investigations have encouraged physicians and provided them new valuable tools for medical research

Investigations of the Cavitation and Damage Thresholds of Histotripsy and Applications in Targeted Tissue Ablation.

Histotripsy is a noninvasive ultrasound therapy that controls acoustic cavitation to mechanically fractionate soft tissue. This dissertation investigates the physical thresholds to initiate cavitation and produce tissue damage in histotripsy and factors affecting these thresholds in order to develop novel strategies for targeted tissue ablation. In the first part of this dissertation, the effects of tissue properties on histotripsy cavitation thresholds and damage thresholds were investigated. Results demonstrated that the histotripsy shock scattering threshold using multi-cycle pulses increases in stiffer tissues, while the histotripsy intrinsic threshold using single-cycle pulses is independent of tissue stiffness. Further, the intrinsic threshold slightly decreases with lower frequencies and significantly decreases with increasing temperature. The effects of tissue properties on the susceptibility to histotripsy-induced tissue damage were also investigated, demonstrating that stiffer tissues are more resistant to histotripsy. In the second part of this dissertation, the feasibility of using histotripsy for targeted liver ablation was investigated in an intact in vivo porcine model, with results demonstrating that histotripsy was capable of non-invasively creating precise lesions throughout the entire liver. Additionally, a tissue selective ablation approach was developed, where histotripsy completely fractionated the liver tissue surrounding the major hepatic vessels and gallbladder while being self-limited at the boundaries of these critical structures. In the final part of this dissertation, a novel ablation method combining histotripsy with acoustically sensitive nanodroplets was developed for targeted cancer cell ablation, demonstrating the potential of using nanodroplet-mediated histotripsy (NMH) for targeted, multi-focal ablation. Studies demonstrated that lower frequency and higher boiling point perfluorocarbon droplets can improve NMH therapy. The role of positive and negative pressure on cavitation nucleation in NMH was also investigated, showing that NMH cavitation nucleation is caused directly from the peak negative pressure of the incident wave, similar to histotripsy bubbles generated above the intrinsic threshold. Overall, the results of this dissertation provide significant insight into the physical mechanisms underlying histotripsy tissue ablation and will help to guide the future development of histotripsy for clinical applications such as the treatment of liver cancer.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113591/1/evlaisav_1.pd