Nanoparticle-Mediated Histotripsy Using Dual-Frequency Pulsing Methods.

OBJECTIVE: Nanoparticle-mediated histotripsy (NMH) is a novel ablation method that combines nanoparticles as artificial cavitation nuclei with focused ultrasound pulsing to achieve targeted, non-invasive, and cell-selective tumor ablation. The study described here examined the effect of dual-frequency histotripsy pulsing on the cavitation threshold, bubble cloud characteristics, and ablative efficiency in NMH. High-speed optical imaging was used to analyze bubble cloud characteristics and to measure ablation efficiency for NMH inside agarose tissue phantoms containing perfluorohexane-filled nanocone clusters, which were previously developed to reduce the histotripsy cavitation threshold for NMH. METHODS: Dual-frequency histotripsy pulsing was applied at a 1:1 pressure ratio using a modular 500 kHz and 3 MHz dual-frequency array transducer. Optical imaging results revealed predictable, well-defined bubble clouds generated for all tested cases with similar reductions in the cavitation thresholds observed for single-frequency and dual-frequency pulsing. RESULTS: Dual-frequency pulsing was seen to nucleate small, dense clouds in agarose phantoms, intermediate in size of their component frequencies but closer in area to that of the higher component frequency. Red blood cell experiments revealed complete ablations were generated by dual-frequency NMH in all phantoms in <1500 pulses. This result was a significant increase in ablation efficiency compared with the ∼4000 pulses required in prior single-frequency NMH studies. CONCLUSION: Overall, this study indicates the potential for using dual-frequency histotripsy methods to increase the ablation efficacy of NMH.

Investigation of Optimum Production Conditions and the Stability of ß-Cyclodextrin-Perfluorocarbon Nanocone Clusters for Histotripsy Applications.

Nanocone clusters (NCCs) have been developed as clusters with inclusion complexes of FDA-approved ß-cyclodextrin (ßCD) and perfluorocarbons (PFC) (i.e., perfluoropentane (PFP) and perfluorohexane (PFH)) and have shown promise in nanoparticle-mediated histotripsy (NMH) applications owing to their lowered cavitation threshold, ease of production, and fluorocarbon quantification. However, there is still a lack of information on the best conditions of the synthesis of NCCs as a product that can have a maximum determinable fluorocarbon content and maintain the stability of the NCC during synthesis and when used as histotripsy agents or exposed to physiological conditions. These concerns about the stability of the clusters and the best possible formulation are investigated in the current work. The cluster formation potential was tested taking into consideration the nature of both PFCs and ßCD by employing different synthesis conditions in terms of solution and environmental parameters such as concentration of solvent, stoichiometry between ßCD and PFCs, temperature, pH, solvent type, etc. The best route of synthesis was then translated into various batch sizes and investigated in terms of the PFC loading and yield. These studies revealed that preparing NCCs in double-distilled water in an ice bath at the optimized solution concentration gave the highest yields and optimal PFC loading, as determined from gas chromatography. Furthermore, the stability of the clusters with different stoichiometries was scrutinized in varying concentrations, mechanical disruption times, pH levels, and temperature conditions, showing effects on each cluster's particle size in dynamic light scattering, visualized in transmission electron microscopy, and cavitation behavior in agarose gel tissue phantoms. These studies revealed stable clusters for all formulations, with PFH-containing NCCs emerging to be the most stable in terms of their cluster size and bubble formation potential in histotripsy. Finally, the shelf life of these clusters was investigated using DLS, which revealed a stable cluster. In conclusion, NCCs have shown high stability in terms of both synthesis, which can be replicated in gram-level production, and the cluster itself, which can be exposed to harsher conditions and still form stable bubbles in histotripsy.

Bubble cloud characteristics and ablation efficiency in dual-frequency intrinsic threshold histotripsy.

Histotripsy is a non-thermal focused ultrasound ablation method that destroys tissue through the generation and activity of acoustic cavitation bubble clouds. Intrinsic threshold histotripsy uses single-cycle pulses to generate bubble clouds when the dominant negative pressure phase exceeds an intrinsic threshold of ∼25-30 MPa. The ablation efficiency is dependent upon the size and density of bubbles within the bubble cloud. This work investigates the effects of dual-frequency pulsing schemes on the bubble cloud behavior and ablation efficiency in intrinsic threshold histotripsy. A modular 500 kHz:3 MHz histotripsy transducer treated agarose phantoms using dual-frequency histotripsy pulses with a 1:1 pressure ratio from 500 kHz and 3 MHz frequency elements and varying arrival times for the 3 MHz pulse relative to the arrival of the 500 kHz pulse (-100 ns, 0 ns, and +100 ns). High-speed optical imaging captured cavitation effects to characterize bubble cloud and individual bubble dynamics. The effects of dual-frequency pulsing on lesion formation and ablation efficiency were also investigated in red blood cell (RBC) phantoms. Results showed that the single bubble and bubble cloud size for dual-frequency cases were intermediate to published results for the component single-frequencies of 500 kHz and 3 MHz. Additionally, bubble cloud size and dynamics were shown to be altered by the arrival time of the 3 MHz pulse with respect to the 500 kHz pulse, with more uniform cloud expansion and collapse observed for early (-100 ns) arrival. Finally, RBC phantom experiments showed that dual-frequency exposures were capable of generating precise lesions with smaller areas and higher ablation efficiencies than previously published results for 500 kHz or 3 MHz. Overall, results demonstrate dual-frequency histotripsy's ability to modulate bubble cloud size and dynamics can be leveraged to produce precise lesions at higher ablation efficiencies than previously observed for single-frequency pulsing.

Bubble Cloud Characteristics and Ablation Efficiency in Dual-Frequency Intrinsic Threshold Histotripsy.

Histotripsy is a non-thermal focused ultrasound ablation method that destroys tissue through the generation and activity of acoustic cavitation bubble clouds. Intrinsic threshold histotripsy uses single-cycle pulses to generate bubble clouds when the dominant negative pressure phase exceeds an intrinsic threshold of ~25-30 MPa. The ablation efficiency is dependent upon the size and density of bubbles within the bubble cloud. This work investigates the effects of dual-frequency pulsing schemes on the bubble cloud behavior and ablation efficiency in intrinsic threshold histotripsy. A modular 500 kHz:3 MHz histotripsy transducer treated agarose phantoms using dual-frequency histotripsy pulses with a 1:1 pressure ratio from 500 kHz and 3 MHz frequency elements and varying arrival times for the 3 MHz pulse relative to the arrival of the 500 kHz pulse (-100 ns, 0 ns, and +100 ns). High-speed optical imaging captured cavitation effects to characterize bubble cloud and individual bubble dynamics. The effects of dual-frequency pulsing on lesion formation and ablation efficiency were also investigated in red blood cell (RBC) phantoms. Results showed that the single bubble and bubble cloud size for dual-frequency cases were intermediate to published results for the component single frequencies of 500 kHz and 3 MHz. Additionally, bubble cloud size and dynamics were shown to be altered by the arrival time of the 3 MHz pulse with respect to the 500 kHz pulse, with more uniform cloud expansion and collapse observed for early (-100 ns) arrival. Finally, RBC phantom experiments showed that dual-frequency exposures were capable of generating precise lesions with smaller areas and higher ablation efficiencies than previously published results for 500 kHz or 3 MHz. Overall, results demonstrate dual-frequency histotripsy's ability to modulate bubble cloud size and dynamics can be leveraged to produce precise lesions at higher ablation efficiencies than previously observed for single-frequency pulsing.