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

Ultrasound metrology and phantom materials for validation of photoacoustic thermometry

High intensity focused ultrasound is an emerging non-invasive cancer therapy during which a focused ultrasound beam is used to destroy cancer cells within a confined volume of tissue. In order to increase its successful implementation in practice, an imaging modality capable of accurately mapping the induced temperature rise in tissue is necessary. Photoacoustic thermometry, a rapidly emerging technique for non-invasive temperature monitoring, exploits the temperature dependence of the Grüneisen parameter of tissues, which leads to changes in the recorded photoacoustic signal amplitude with temperature. However, the implementation of photoacoustic thermometry approaches is hindered by a lack of rigorous validation. This includes both the equipment and methodology used. This work investigates the effect of temperature on ultrasound transducers used in photoacoustic thermometry imaging as well as characterisation of potential phantom materials for its validation. The variation in transducer sensitivity with temperature is investigated using two approaches. The first one utilises a reference transducer whose output power is known as a function of temperature to characterise the sensitivity of the hydrophone. As the knowledge of variability of transducer output with temperature is not readily available, two standard metrology techniques using radiation force balances and laser vibrometry are extended beyond room temperature to characterise the effect of temperature on the output of PZT tranducers. For the second approach to transducer sensitivity calibration, a novel method is developed utilising water as a laser-generated ultrasound source and validated using the self-reciprocity calibration method. The calibrated hydrophone is then used to characterise the relevant temperature-dependent properties of several phantom materials in a custom-built setup. The measurement results are used to determine the most suitable phantom for photoacoustic thermometry. Finally, the phantom is heated and imaged in a proof-of-concept photoacoustic thermometry setup using a linear array. These contributions are of vital importance for allowing the translation of photoacoustic thermometry into clinical practice

Color-Changing Reflection Hologram for Quality Assurance of Therapeutic Ultrasound Systems

The acoustic output of clinical therapeutic ultrasound equipment requires regular quality assurance (QA) testing to ensure the safety and efficacy of the treatment and that any potentially harmful deviations from the expected output power density are detected as soon as possible. A hologram, consisting of a reflection grating fabricated in an acrylate photopolymer film, has been developed to produce an immediate, visible, and permanent change in the color of the reconstructed hologram from red to green in response to incident ultrasound energy. The influence of the therapeutic ultrasound insonation parameters (exposure time, ultrasound power density, and proximity to the point of maximum acoustic pressure) on the hologram’s response has been investigated for two types of therapeutic ultrasound systems: a sonoporation system and an ultrasound physiotherapy system. Findings show that, above a switching temperature of 45 °C, the ultrasound-induced temperature rise produces a structural change in the hologram, which manifests as a visible color change. The area of the color change region correlates with the ultrasound exposure conditions. The suitability of the hologram as a simple and quick QA test tool for therapeutic ultrasound systems has been demonstrated. A prototype ultrasound testing unit which facilitates user-friendly, reproducible testing of the holograms in a clinical setting is also reported

High Power CMUTs: Design and experimental verification

Cataloged from PDF version of article.Capacitive micromachined ultrasonic transducers (CMUTs) have great potential to compete with piezoelectric transducers in high-power applications. As the output pressures increase, nonlinearity of CMUT must be reconsidered and optimization is required to reduce harmonic distortions. In this paper, we describe a design approach in which uncollapsed CMUT array elements are sized so as to operate at the maximum radiation impedance and have gap heights such that the generated electrostatic force can sustain a plate displacement with full swing at the given drive amplitude. The proposed design enables high output pressures and low harmonic distortions at the output. An equivalent circuit model of the array is used that accurately simulates the uncollapsed mode of operation. The model facilities the design of CMUT parameters for high-pressure output, without the intensive need for computationally involved FEM tools. The optimized design requires a relatively thick plate compared with a conventional CMUT plate. Thus, we used a silicon wafer as the CMUT plate. The fabrication process involves an anodic bonding process for bonding the silicon plate with the glass substrate. To eliminate the bias voltage, which may cause charging problems, the CMUT array is driven with large continuous wave signals at half of the resonant frequency. The fabricated arrays are tested in an oil tank by applying a 125-V peak 5-cycle burst sinusoidal signal at 1.44 MHz. The applied voltage is increased until the plate is about to touch the bottom electrode to get the maximum peak displacement. The observed pressure is about 1.8 MPa with −28 dBc second harmonic at the surface of the array

Development of a 1D phased ultrasonic array for intravascular sonoporation

Error on title page – year of award is 2021.Sonoporation represents a promising approach to increase targeted drug delivery efficiency by facilitating transport of therapeutic agents to the target tissue with the use of ultrasound. However, most of the current research in sonoporation is performed with external ultrasonic transducers, which hinders the applicability of the therapeutic procedure for treatment of conditions situated deeper into the patient’s body, such as liver or intestinal tumours. This Thesis presents the development process of a miniature-sized 1-3 connectivity piezocomposite 1D phased array for intracorporeal sonoporation. The device was to be incorporated into a capsule or catheter and hence the primary design constraint was the reduced size of the piezoelectric element, which was limited to 2.5 mm in width and 12 mm in length. To meet the needs of the intended application, resonance frequencies of 1.5 MHz and 3.0 MHz were considered. A simulation framework was developed for optimization of the miniature array in relation to the peak negative pressure attained at the focus to mitigate the low power output associated with the limited device dimensions. This was implemented through a multiparametric sweep of the 1-3 piezocomposite geometry-related parameters. Devices made with PZT-5H and PMN-29%PT were evaluated. The optimization algorithm was used to determine specifications for phased array designs based on the two materials and the two resonance frequencies. The 1.5 MHz devices comprised 24 elements and the 3.0 MHz ones had 32 elements. The piezocomposites were manufactured using the dice and fill technique and electroded using a novel method of electrode deposition employing spin coating of Ag ink. Subsequently, the prototype devices were driven with a commercial array controller and characterized with a calibrated needle hydrophone in a scanning tank. Two simulation profiles based on finite element analysis and time extrapolation were developed to model the acoustic beams from the arrays, which were compared and calibrated with experimental data for focal distances between 5 mm and 10 mm and beam steering angles from 0° to 40°. The results showed that modelling could be employed reliably for therapeutic planning. Both the 1.5 MHz and the 3.0 MHz, PZT-5H arrays were tested in vitro and shown to induce and control sonoporation of a human epithelial colorectal adenocarcinoma cell layer. Finally, a 24 element, 1.5 MHz, PZT-5H array was implemented in a 40 mm long by 11 mm diameter tethered, biocompatible capsule intended for in vivo operation. The device was characterized in the scanning tank for steering angles in the range 0° to 56° and focal distances between 4.0 mm and 5.7 mm, and the measured beam profiles were correlated with the simulation framework. The capsule will be tested in future ex-vivo and in-vivo experiments on insulin absorption through porcine small bowel by means of sonoporation.Sonoporation represents a promising approach to increase targeted drug delivery efficiency by facilitating transport of therapeutic agents to the target tissue with the use of ultrasound. However, most of the current research in sonoporation is performed with external ultrasonic transducers, which hinders the applicability of the therapeutic procedure for treatment of conditions situated deeper into the patient’s body, such as liver or intestinal tumours. This Thesis presents the development process of a miniature-sized 1-3 connectivity piezocomposite 1D phased array for intracorporeal sonoporation. The device was to be incorporated into a capsule or catheter and hence the primary design constraint was the reduced size of the piezoelectric element, which was limited to 2.5 mm in width and 12 mm in length. To meet the needs of the intended application, resonance frequencies of 1.5 MHz and 3.0 MHz were considered. A simulation framework was developed for optimization of the miniature array in relation to the peak negative pressure attained at the focus to mitigate the low power output associated with the limited device dimensions. This was implemented through a multiparametric sweep of the 1-3 piezocomposite geometry-related parameters. Devices made with PZT-5H and PMN-29%PT were evaluated. The optimization algorithm was used to determine specifications for phased array designs based on the two materials and the two resonance frequencies. The 1.5 MHz devices comprised 24 elements and the 3.0 MHz ones had 32 elements. The piezocomposites were manufactured using the dice and fill technique and electroded using a novel method of electrode deposition employing spin coating of Ag ink. Subsequently, the prototype devices were driven with a commercial array controller and characterized with a calibrated needle hydrophone in a scanning tank. Two simulation profiles based on finite element analysis and time extrapolation were developed to model the acoustic beams from the arrays, which were compared and calibrated with experimental data for focal distances between 5 mm and 10 mm and beam steering angles from 0° to 40°. The results showed that modelling could be employed reliably for therapeutic planning. Both the 1.5 MHz and the 3.0 MHz, PZT-5H arrays were tested in vitro and shown to induce and control sonoporation of a human epithelial colorectal adenocarcinoma cell layer. Finally, a 24 element, 1.5 MHz, PZT-5H array was implemented in a 40 mm long by 11 mm diameter tethered, biocompatible capsule intended for in vivo operation. The device was characterized in the scanning tank for steering angles in the range 0° to 56° and focal distances between 4.0 mm and 5.7 mm, and the measured beam profiles were correlated with the simulation framework. The capsule will be tested in future ex-vivo and in-vivo experiments on insulin absorption through porcine small bowel by means of sonoporation

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