Factors Influencing the Repeated Transient Optical Droplet Vaporization Threshold and Lifetimes of Phase Change, Perfluorocarbon Nanodroplets.

Perfluorocarbon nanodroplets (PFCnDs) are sub-micrometer emulsions composed of a surfactant-encased perfluorocarbon (PFC) liquid and can be formulated to transiently vaporize through optical stimulation. However, the factors governing repeated optical droplet vaporization (ODV) have not been investigated. In this study, we employ high-frame-rate ultrasound (US) to characterize the ODV thresholds of various formulations and imaging parameters and identify those that exhibit low vaporization thresholds and repeatable vaporization. We observe a phenomenon termed "preconditioning", where initial laser pulses generate reduced US contrast that appears linked with an increase in nanodroplet size. Variation in laser pulse repetition frequency is found not to change the vaporization threshold, suggesting that "preconditioning" is not related to residual heat. Surfactants (bovine serum albumin, lipids, and zonyl) impact the vaporization threshold and imaging lifetime, with lipid shells demonstrating the best performance with relatively low thresholds (21.6 ± 3.7 mJ/cm2) and long lifetimes (t1/2 = 104 ± 21.5 pulses at 75 mJ/cm2). Physiological stiffness does not affect the ODV threshold and may enhance nanodroplet stability. Furthermore, PFC critical temperatures are found to correlate with vaporization thresholds. These observations enhance our understanding of ODV behavior and pave the way for improved nanodroplet performance in biomedical applications.

Leveraging the Imaging Transmit Pulse to Manipulate Phase-Change Nanodroplets for Contrast-Enhanced Ultrasound.

Phase-change perfluorohexane nanodroplets (PFHnDs) are a new class of recondensable submicrometer-sized contrast agents that have potential for contrast-enhanced and super-resolution ultrasound imaging with an ability to reach extravascular targets. The PFHnDs can be optically triggered to undergo vaporization, resulting in spatially stationary, temporally transient microbubbles. The vaporized PFHnDs are hyperechoic in ultrasound imaging for several to hundreds of milliseconds before recondensing to their native, hypoechoic, liquid nanodroplet state. The decay of echogenicity, i.e., the dynamic behavior of the ultrasound signal from optically triggered PFHnDs in ultrasound imaging, can be captured using high-frame-rate ultrasound imaging. We explore the possibility to manipulate the echogenicity dynamics of optically triggered PFHnDs in ultrasound imaging by changing the phase of the ultrasound imaging pulse. Specifically, the ultrasound imaging system was programmed to transmit two imaging pulses with inverse polarities. We show that the imaging pulse phase can affect the amplitude and the temporal behavior of PFHnD echogenicity in ultrasound imaging. The results of this study demonstrate that the ultrasound echogenicity is significantly increased (about 78% improvement) and the hyperechoic timespan of optically triggered PFHnDs is significantly longer (about four times) if the nanodroplets are imaged by an ultrasound pulse starting with rarefactional pressure versus a pulse starting with compressional pressure. Our finding has direct and significant implications for contrast-enhanced ultrasound imaging of droplets in applications such as super-resolution imaging and molecular imaging where detection of individual or low-concentration PFHnDs is required.

Design and Demonstration of a Configurable Imaging Platform for Combined Laser, Ultrasound, and Elasticity Imaging.

This paper introduces a configurable combined laser, ultrasound, and elasticity (CLUE) imaging platform. The CLUE platform enables imaging sequences capable of simultaneously providing quantitative acoustic, optical, and mechanical contrast for comprehensive diagnosis and monitoring of complex diseases, such as cancer. The CLUE imaging platform was developed on a Verasonics ultrasound scanner integrated with a pulsed laser, and it was designed to be modular and scalable to allow researchers to create their own specific imaging sequences efficiently. The CLUE imaging platform and sequence were demonstrated in a tissue-mimicking phantom containing a stiff inclusion labeled with optically-activated nanodroplets and in an ex vivo mouse spleen. We have shown that CLUE imaging can simultaneously capture multi-functional imaging signals providing quantitative information on tissue.

Ultrasonic Cavitation-Enabled Treatment for Therapy of Hypertrophic Cardiomyopathy: Proof of Principle.

Ultrasound myocardial cavitation-enabled treatment was applied to the SS-16BN rat model of hypertrophic cardiomyopathy for proof of the principle underlying myocardial reduction therapy. A focused ultrasound transducer was targeted using 10-MHz imaging (10 S, GE Vivid 7) to the left ventricular wall of anesthetized rats in a warmed water bath. Pulse bursts of 4-MPa peak rarefactional pressure amplitude were intermittently triggered 1:8 heartbeats during a 10-min infusion of a microbubble suspension. Methylprednisolone was given to reduce initial inflammation, and Losartan was given to reduce fibrosis in the healing tissue. At 28 d post therapy, myocardial cavitation-enabled treatment significantly reduced the targeted wall thickness by 16.2% (p <0.01) relative to shams, with myocardial strain rate and endocardial displacement reduced by 34% and 29%, respectively, which are sufficient for therapeutic treatment. Premature electrocardiogram complexes and plasma troponin measurements were found to identify optimal and suboptimal treatment cohorts and would aid in achieving the desired impact. With clinical translation, myocardial cavitation-enabled treatment should fill the need for a new non-invasive hypertrophic cardiomyopathy therapy option.

Multiple ultrasound cavitation-enabled treatments for myocardial reduction.

BACKGROUND: Ultrasound myocardial cavitation enabled treatment (MCET) is an image-guided method for tissue reduction. In this study, a strategy of fractionated (multiple) treatments was tested for efficacy. METHODS: Dahl SS rats were anesthetized and prepared for treatment with a focused ultrasound transducer in a warm water bath. Aiming at the anterior left ventricular wall was facilitated by imaging with a 10 MHz phased array (10S, GE Vivid 7, GE Vingmed Ultrasound, Horten, Norway). MCET was accomplished at 1.5 MHz by pulse bursts of 4 MPa peak rarefactional pressure amplitude, which were intermittently triggered 1:8 from the ECG during infusion of a microbubble suspension for cavitation nucleation. Test groups were sham, a 200 s treatment, three 200 s treatments a week apart, and a 600 s treatment. Treatment outcome was observed by plasma troponin after 4 h, echocardiographic monitoring and histology at 6 wk. RESULTS: The impacts of the fractionated treatments summed to approximately the same as the long treatment; e. g. the troponin result was 10.5 ± 3.2 for 200 s, 22.7 ± 5.4 (p < 0.001) for the summed fractionated treatments and 29.9 ± 6.4 for 600 s (p = 0.06 relative to the summed fractionated). While wall thickness was not reduced for the fractionated treatment, tissue strain was reduced by 35% in the target area relative sham (p < 0.001). CONCLUSION: The ability to fractionate treatment may be advantageous for optimizing patient outcome relative to all-or nothing therapy by surgical myectomy or alcohol ablation.

Passive Microlesion Detection and Mapping for Treatment of Hypertrophic Cardiomyopathy.

Intermittent high intensity ultrasound pulses with circulating contrast agent microbubbles can induce scattered microlesions of potential value for myocardial reduction therapy. This paper presents an in vitro setup imitating the treatment for monitoring development. A preclinical imaging system with a single element transducer, synchronization and receive-only imaging transducer array has been implemented on a research platform. Contrast agent microbubbles pumped in a dialysis tubing setup were exposed to high intensity focused ultrasound at 1.0/3.5 MHz center frequencies. Polystyrene spheres were employed as linear scatterers compared to contrast agents for system transfer function equalization. A cavitation mapping technique was employed to spatially locate and depict microbubble activity during treatment. For high acoustic pressure amplitudes a 5 dB difference between contrast agent and solid spheres was observed and spatially mapped. The in-plane resolution was 4.5 mm for axial and 1.5 mm laterally. In the future, this cavitation detection scheme will be applied to monitor in vivo microlesioning in real-time.

Maturation of Lesions Induced by Myocardial Cavitation-Enabled Therapy.

Myocardial contrast echocardiography at enhanced therapeutic parameters may be a novel means of tissue reduction therapy, as for hypertrophic cardiomyopathy. Dahl/SS rats were anesthetized and treated with high-amplitude pulsed ultrasound guided by 10-MHz ultrasound images. Contrast microbubbles were infused via the tail vein during intermittent pulse-burst exposure at 4 MPa. A sham group, a low-impact group (group A, 5 cycle pulses with Gaussian modulation and 1:4 trigger for 5 min) and a high-impact group (group B, 10 cycle pulses with 4-ms square modulation and 1:8 trigger for 10 min) were tested. The higher exposure used in group B yielded more substantial injury than the lower exposure in group A. Treated rats in both groups A and B had significant increases in wall thickness measured by echocardiography the next day, which returned to normal by the end of 6 wk. Six weeks after ultrasound exposure, heart tissue samples exhibited tissue fibrosis in Masson's trichrome stained histology. Maturation of lesions involved fibrosis replacement, preserving structural tissue integrity. This study indicates that myocardial injury noted previously progresses into permanent loss of myocardial tissue that may be sufficient for possible hypertrophic cardiomyopathy therapy. More research is needed to define the treatment parameters required for symptomatic relief for hypertrophic cardiomyopathy.

Quantitative assessment of damage during MCET: a parametric study in a rodent model.

BACKGROUND: Myocardial cavitation-enabled therapy (MCET) has been proposed as a means to achieve minimally invasive myocardial reduction using ultrasound to produce scattered microlesions by cavitating contrast agent microbubbles. METHODS: Rats were treated using burst mode focused ultrasound at 1.5 MHz center frequency and varying envelope and pressure amplitudes. Evans blue staining indicated lethal cardiomyocytic injury. A previously developed quantitative scheme, evaluating the histologic treatment results, provides an insightful analysis for MCET treatment parameters. Such include ultrasound exposure amplitude and pulse modulation, contrast agent dose, and infusion rate. RESULTS: The quantitative method overcomes the limitation of visual scoring and works for a large dynamic range of treatment impact. Macrolesions are generated as an accumulation of probability driven microlesion formations. Macrolesions grow radially with radii from 0.1 to 1.6 mm as the ultrasound exposure amplitude (peak negative) increases from 2 to 4 MPa. To shorten treatment time, a swept beam was investigated and found to generate an acceptable macrolesion volume of about 40 µL for a single beam position. CONCLUSIONS: Ultrasound parameters and administration of microbubbles directly influence lesion characteristics such as microlesion density and macrolesion dimension. For lesion generation planning, control of MCET is crucial, especially when targeting larger pre-clinical models.

Use of Theranostic Strategies in Myocardial Cavitation-Enabled Therapy.

The accumulation of microlesions induced by ultrasound interaction with contrast microbubbles in the myocardium potentially represents a new method of tissue reduction therapy. Anesthetized rats were treated in a heated water bath with 1.5-MHz focused ultrasound pulses triggered once every four heartbeats from the electrocardiogram during infusion of microbubble contrast agent. Treatment was guided by an 8-MHz B-mode imaging transducer, which also was used to provide estimates of left ventricular echogenicity as a possible predictor of efficacy during treatment. Strategies to reduce prospective clinical treatment durations were tested, including pulse modulation to simulate a theranostic scanning strategy and an increased agent infusion rate over shorter durations. Sources of variability, including ultrasound path variation and venous catheter placement, also were investigated. Electrocardiographic premature complexes were monitored, and Evans-blue stained cardiomyocyte scores were obtained from frozen sections. Left ventricular echogenicity reflected variations in the infused microbubble concentration, but failed to predict efficacy. Comparison of suspensions of varied microbubble size revealed that left ventricular echogenicity was dominated by larger bubbles, whereas efficacy appeared to be dependent on smaller sizes. Simulated scanning was as effective as the normal fixed-beam treatment, and high agent infusion allowed reduced treatment duration. The success of these theranostic strategies may increase the prospects for realistic clinical translation of myocardial cavitation-enabled therapy.

Characterization of macrolesions induced by myocardial cavitation-enabled therapy.

Intermittent high intensity ultrasound pulses with circulating contrast agent microbubbles can induce scattered cavitation caused myocardial microlesions of potential value for tissue reduction therapy. Here, computer-aided histological evaluation of the effective treated volume was implemented to optimize ultrasound pulse parameters, exposure duration, and contrast agent dose. Rats were treated with 1.5 MHz focused ultrasound bursts and Evans blue staining indicates lethal cardiomyocytic injury. Each heart was sectioned to provide samples covering the entire exposed myocardial volume. Both brightfield and fluorescence images were taken for up to 40 tissue sections. Tissue identification and microlesion detection were first done based on 2-D images to form microlesion masks containing the outline of the heart and the stained cell regions. Image registration was then performed on the microlesion masks to reconstruct a volume-based model according to the morphology of the heart. The therapeutic beam path was estimated from the 3-D stacked microlesions, and finally the total microlesion volume, here termed macrolesion, was characterized along the therapeutic beam axis. Radially symmetric fractional macrolesions were characterized via stepping disks of variable radius determined by the local distribution of microlesions. Treated groups showed significant macrolesions of a median volume of 87.3 µL, 2.7 mm radius, 4.8 mm length, and 14.0% lesion density compared to zero radius, length, and lesion density for sham. The proposed radially symmetric lesion model is a robust evaluation for myocardial cavitation-enabled therapy. Future work will include validating the proposed method with varying acoustic exposures and optimizing involved parameters to provide macrolesion characterization.