archives@tulane.edu / Acoustic droplet vaporization (ADV) is an attractive alternative to traditional hepatocellular carcinoma (HCC) treatments. ADV involves injecting microdroplets into the bloodstream which then accumulate in and around the tumor’s vasculature. Once accumulated, high-power ultrasound is used to vaporize the microdroplets into larger perfluoropentane gas microbubbles which occlude blood flow and induce necrosis of the tumor without harming healthy tissue like traditional HCC treatments. This study aims to optimize ADV treatment by improving the shell composition and surface architecture of microdroplets while ensuring the treatment remains safe. In order to ensure the treatment is as effective as possible, the microdroplets must have powerful binding capabilities, guaranteeing maximum microdroplet accumulation and treatment efficacy. The binding capabilities of three microdroplet shell compositions, created by adjusting the molar percentages of the three lipids found in the shell, were investigated and found to all have equal binding abilities. The surface architecture of these microdroplets were also altered to maximise binding capabilities. Microdroplets can have either an exposed-ligand or buried-ligand surface architecture. In microdroplets with a buried-ligand surface architecture, the attached tumor-targeting ligands are hidden within a layer of longer lipid chains which allow the microdroplets to evade the immune system and circulate within the bloodstream longer, increasing treatment efficacy. It was found that microdroplets with a buried-ligand surface architecture do not have comparable binding capabilities to microdroplets with an exposed-ligand surface architecture and are therefore not a viable alternative for use in ADV. Finally, the velocity required to dislodge perfluoropentane gas microbubbles was explored to determine if the gas microbubbles can remain adhered to the tumor’s vasculature to create a strong occlusion. Since perfluoropentane gas microbubbles occlude blood flow it is imperative that the microbubbles remain in the tumor’s vasculature and do not dislodge and accumulate in other parts of the body’s vasculature. By measuring the velocity and calculating the force necessary for dislodgement and comparing those values to those found in capillaries it was concluded that the perfluoropentane gas microbubbles can withstand the force of blood flow and remain lodged in capillaries. / 1 / Chloe Celingant-Copie

Acoustic droplet vaporization (ADV) is a process in which a phase shift of a liquified droplet into a gaseous microbubble, is triggered using an ultrasonic wave. In contrast to utilizing conventional contrast agents in ultrasound, the phase change contrast agents used in ADV can extravasate into tumor tissue, and they offer a greater circulatory lifespan, thereby increasing the potential applications in which they can be utilized. In this project, the impact of different ultrasound parameters on the efficiency of ADV was investigated, using a programmable ultrasound system. Two different ultrasound sequences were designed, for imaging and vaporization of droplets. Furthermore, three different sets of experiments were performed. Firstly, the vaporization effect of different imaging voltages was investigated, whereby a setting of 15V was identified as an able voltage for the remaining experiments. Secondly, experiments concerning the effect of vaporizing frequency on the ADV efficiency were performed, including the use of single and dual frequencies. Lastly, different frequency settings were combined with varying the number of cycles, to assess how the choice of pulse length influences the vaporization. The results from the project indicate that no substantial difference in ADV efficiency is achieved when using different frequency settings for perfluoropentane droplets encapsulated by cellulose nanofibers. However, the results provide clear indications of the benefit of using longer pulse durations on the vaporization efficiency. In conclusion, further studies are required before ADV can be translated into a clinical setting.

Ultrasound-mediated drug delivery has been proposed as a safe and non-invasive method to achieve localized drug release. Drug-loaded microbubbles are injected in the vascular system and ultrasound waves are then used to localize and burst the microbubbles at a specific targeted area. The relatively large size of microbubbles however limits both their lifetime and their reach in the human body. Phase-change liquid droplets can extend the use of ultrasound contrast agents for localized drug delivery. Their smaller size provides several advantages. The droplets can reach smaller capillaries, such as those in tumors vasculature. Their lifetime is also considerably prolonged. Through the phenomenon of Acoustic Droplet Vaporization (ADV), triggered by ultrasound stimulation, the liquid-filled droplets experience a phase change and are converted into gas-filled microbubbles. The newly created microbubbles can then be disrupted by further stimulation and release their drug load in the tumor tissue. In this project, a protocol to image and burst perfluoropentane-based micro-sized droplets using a single transducer is developed using the Verasonics Ultrasound System. The pulse sequences are developed to allow close monitoring of the drug delivery by capturing a series of images before and after the vaporization or destruction of the droplets. The droplets response was assessed for different pulse voltages and durations. Mean pixel value was calculated for the regions of interest, using the images captured before and after delivery of the ultrasound pulse. Vaporization of the droplets can be achieved with low voltage (10V), whereas high voltage (50V) triggers their destruction. Combined with high voltage, pulse duration affects the rate at which droplets can be destructed.

The use of peruorocarbon lled droplets for use as Phase Changing Contrast Agents (PCCAs) is a promosing eld. These capsules also have potential to be used for mediated drug delivery. The phase change, which has given the capsules their name, is the process when the capsule transforms from a droplet into a bubble. This process is referred to as Acoustic Droplet Vaporization (ADV) and can be induced with the use of ultrasonic waves. In this study a new type of Perfluorpentane (PFC5) capsules which are stabilized with Cellulose Nano Fibers (CNF) have been evaluated for its potential as a PCCA. To investigate this potential a setup was designed in which the capsules could be exposed to ultrasound waves. Following the ultrasound exposure the capsules were visualized under a light transmission microscope. The experiments were conducted for dierent combinations of ultrasound parameters. For each combination eight volume distributions were created, in which two of them as reference cases were not exposed to ultrasound waves. Six cases with the ultrasound ring with different levels of acoustic power, resulting in peak negative pressures ranging from 0.144 to 0.291 MPa. The results showedfthat ADV could be observed when the frequency of the acoustic wave is 3.5 MHz, the pulse repetition frequency is 500 and the burst width is set to 12 cycles. The Peak Negative Pressure (PNP)-threshold for ADV is about 0.200 MPa. When the burst width is set to 8, ADV is also observed however to a lesser extent then when it is set to 12. These results indicate that the CNF-stabilized PFC5 capsules are promising droplets with a potential future as an alternative to currently used PCCAs.

The use of ultrasound contrast agents increases the information available for reconstruction during ultrasound imaging. Previously studied microbubbles, consisting of a gas core, are subject to limitations such as a short lifetime and a large size. Droplets with a liquid perfluorocarbon core that is stabilized by cellulose nanofibers eliminate these drawbacks, but require further investigation. By studying the frequency-dependent attenuation profile of the cellulose nanofiber coated perfluorocarbon droplets within an ultrasound field, information about the droplets as oscillators can be retrieved, enabling characterization of their physical properties. In this study, the frequency-dependent attenuation profile was experimentally acquired and compared between two concentrations, using flat transducers covering the frequency range of 1-15 MHz. The data collected in the time domain was processed and transformed into the frequency domain and the attenuation was calculated across the entire frequency range. Among the frequencies studied, the attenuation increases with frequency and covers the range of approximately 0.25-8.3 dB/cm and 0.01-3.3 dB/cm at the concentrations of 50 million droplets/ml and 10 million droplets/ml respectively. The attenuation reaches a minimum at 3 MHz within the highest concentration, compared to 4.43 MHz within the lowest. The increase of the attenuation with frequency is explained by the droplets not exhibiting large oscillations within the range covered. It is probable that the droplets do exhibit oscillations, due to a viscosity lower than that of water, but a resonance frequency is not found within the spectrum studied. This could be explained by a shell elasticity or a small droplet radius placing the resonance frequency outside of the spectrum studied, or high levels of damping broadening the resonance peak. Localizing the resonance frequency would enable characterization of these physical properties of the cellulose nanofiber shell as well as the perfluorocarbon liquid core of the droplets. The increase of the attenuation with frequency demonstrates that the droplets do not produce a maximized amount of scattering at a specific frequency within the range studied, which is observed among other oscillating particles implemented as ultrasound contrast agents. The attenuation is, however, larger than that of blood across all frequencies except for those among which the attenuation reaches its minimum. Potential errors that are affecting the results include droplet vaporization, the formation of flocs after the mechanical agitation has ceased, the experimental setup, the settings on the pulse generator, the sensitivity of the transducers and the processing code.