The impact of vaporized nanoemulsions on ultrasound-mediated ablation

BACKGROUND: The clinical feasibility of using high-intensity focused ultrasound (HIFU) for ablation of solid tumors is limited by the high acoustic pressures and long treatment times required. The presence of microbubbles during sonication can increase the absorption of acoustic energy and accelerate heating. However, formation of microbubbles within the tumor tissue remains a challenge. Phase-shift nanoemulsions (PSNE) have been developed as a means for producing microbubbles within tumors. PSNE are emulsions of submicron-sized, lipid-coated, and liquid perfluorocarbon droplets that can be vaporized into microbubbles using short (5 MPa) acoustic pulses. In this study, the impact of vaporized phase-shift nanoemulsions on the time and acoustic power required for HIFU-mediated thermal lesion formation was investigated in vitro. METHODS: PSNE containing dodecafluoropentane were produced with narrow size distributions and mean diameters below 200 nm using a combination of sonication and extrusion. PSNE was dispersed in albumin-containing polyacrylamide gel phantoms for experimental tests. Albumin denatures and becomes opaque at temperatures above 58°C, enabling visual detection of lesions formed from denatured albumin. PSNE were vaporized using a 30-cycle, 3.2-MHz, at an acoustic power of 6.4 W (free-field intensity of 4,586 W/cm(2)) pulse from a single-element, focused high-power transducer. The vaporization pulse was immediately followed by a 15-s continuous wave, 3.2-MHz signal to induce ultrasound-mediated heating. Control experiments were conducted using an identical procedure without the vaporization pulse. Lesion formation was detected by acquiring video frames during sonication and post-processing the images for analysis. Broadband emissions from inertial cavitation (IC) were passively detected with a focused, 2-MHz transducer. Temperature measurements were acquired using a needle thermocouple. RESULTS: Bubbles formed at the HIFU focus via PSNE vaporization enhanced HIFU-mediated heating. Broadband emissions detected during HIFU exposure coincided in time with measured accelerated heating, which suggested that IC played an important role in bubble-enhanced heating. In the presence of bubbles, the acoustic power required for the formation of a 9-mm(3) lesion was reduced by 72% and the exposure time required for the onset of albumin denaturation was significantly reduced (by 4 s), provided that the PSNE volume fraction in the polyacrylamide gel was at least 0.008%. CONCLUSIONS: The time or acoustic power required for lesion formation in gel phantoms was dramatically reduced by vaporizing PSNE into bubbles. These results suggest that PSNE may improve the efficiency of HIFU-mediated thermal ablation of solid tumors; thus, further investigation is warranted to determine whether bubble-enhanced HIFU may potentially become a viable option for cancer therapy.R21 EB009493 - NIBIB NIH HH

Test materials for characterising heating from HIFU devices using photoacoustic thermometry

High intensity focused ultrasound (HIFU) is a non-invasive thermal therapy during which a focused ultrasound beam is used to destroy cells within a confined volume of tissue. Due to its increased use and advancements in treatment delivery, various numerical models are being developed for use in treatment planning software. In order to validate these models, as well as to perform routine quality checks and transducer characterisation, a temperature monitoring technique capable of accurately mapping the temperature rise induced is necessary. Photoacoustic thermometry is a rapidly emerging technique for non-invasive temperature monitoring, where the temperature dependence of the Gruneisen parameter leads to changes in the recorded photoacoustic signal amplitude with temperature. In order to use this technique to assess heating induced by HIFU in a metrology setting, a suitable test material must first be selected that exhibits an increase in the generated photoacoustic signal with temperature. In this study, the temperature dependence of the photoacoustic conversion efficiency (μaΓ) of several tissue-mimicking materials was measured for temperatures between 22 °C and 50 °C. Materials included were agar-based phantoms, copolymer-in-oil, gel wax, PVA cryogels, PVCP and silicone. This information provided a basis for the development of a volumetric phantom, which was sonicated in a proof-of-concept integrated photoacoustic thermometry system for monitoring of HIFU-induced heating. The results show the suitability of agar-based phantoms and photoacoustic thermometry to image the 3D heat distribution generated by a HIFU transducer

HEATING IN VASCULAR TISSUE AND FLOW-THROUGH TISSUE PHANTOMS INDUCED BY FOCUSED ULTRASOUND

High intensity focused ultrasound (HIFU) can be used to control bleeding, both from individual blood vessels as well as from gross damage to the capillary bed. This process, called acoustic hemostasis, is being studied in the hope that such a method would ultimately provide a lifesaving treatment during the so-called "golden hour", a brief grace period after a severe trauma in which prompt therapy can save the life of an injured person. Thermal effects play a major role in occlusion of small vessels and also appear to contribute to the sealing of punctures in major blood vessels. However, aggressive ultrasound-induced tissue heating can also impact healthy tissue and can lead to deleterious mechanical bioeffects. Moreover, the presence of vascularity can limit one’s ability to elevate the temperature of blood vessel walls owing to convective heat transport. In an effort to better understand the heating process in tissues with vascular structure we have developed a numerical simulation that couples models for ultrasound propagation, acoustic streaming, ultrasound heating and blood cooling in Newtonian viscous media. The 3-D simulation allows for the study of complicated biological structures and insonation geometries. We have also undertaken a series of in vitro experiments, in non-uniform flow-through tissue phantoms, designed to provide a ground truth verification of the model predictions. The calculated and measured results were compared over a range of values for insonation pressure, insonation time, and flow rate; we show good agreement between predictions and measurements. We then conducted a series of simulations that address two limiting problems of interest: hemostasis in small and large vessels. We employed realistic human tissue properties and considered more complex geometries. Results show that the heating pattern in and around a blood vessel is different for different vessel sizes, flow rates and for varying beam orientations relative to the flow axis. Complete occlusion and wall- puncture sealing are both possible depending on the exposure conditions. These results concur with prior clinical observations and may prove useful for planning of a more effective procedure in HIFU treatments.Defense Advanced Research Projects Agency, the U. S. Army, and the Center for Subsurface Sensing and Imaging Systems

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

Investigation of bubble dynamics and heating during focused ultrasound insonation in tissue-mimicking materials

The deposition of ultrasonic energy in tissue can cause tissue damage due to local heating. For pressures above a critical threshold, cavitation will occur in tissue and bubbles will be created. These oscillating bubbles can induce a much larger thermal energy deposition in the local region. Traditionally, clinicians and researchers have not exploited this bubble-enhanced heating since cavitation behavior is erratic and very difficult to control. The present work is an attempt to control and utilize this bubble-enhanced heating. First, by applying appropriate bubble dynamic models, limits on the asymptotic bubble size distribution are obtained for different driving pressures at 1 MHz. The size distributions are bounded by two thresholds: the bubble shape instability threshold and the rectified diffusion threshold. The growth rate of bubbles in this region is also given, and the resulting time evolution of the heating in a given insonation scenario is modeled. In addition, some experimental results have been obtained to investigate the bubble-enhanced heating in an agar and graphite based tissue- mimicking material. Heating as a function of dissolved gas concentrations in the tissue phantom is investigated. Bubble-based contrast agents are introduced to investigate the effect on the bubble-enhanced heating, and to control the initial bubble size distribution. The mechanisms of cavitation-related bubble heating are investigated, and a heating model is established using our understanding of the bubble dynamics. By fitting appropriate bubble densities in the ultrasound field, the peak temperature changes are simulated. The results for required bubble density are given. Finally, a simple bubbly liquid model is presented to estimate the shielding effects which may be important even for low void fraction during high intensity focused ultrasound (HIFU) treatment

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

Temperature Increase Dependence on Ultrasound Attenuation Coefficient in Innovative Tissue-mimicking Materials

Although high intensity focused ultrasound beams (HIFU) have found rapid agreement in clinical environment as a tool for non invasive surgical ablation and controlled destruction of cancer cells, some aspects related to the interaction of ultrasonic waves with tissues, such as the conversion of acoustic energy into heat, are not thoroughly understood. In this work, innovative tissue- mimicking materials (TMMs), based on Agar and zinc acetate, have been used to conduct investigations in order to determine a relation between the sample attenuation coefficient and its temperature increase measured in the focus region when exposed to an HIFU beam. An empirical relation has been deduced establishing useful basis for further processes of validations of numerical models to be adopted for customizing therapeutic treatments

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