PFC microbubbles act as cavitation nuclei and lower the acoustic intensity required to achieve ablation by HIFU. Lowered energy requirements should improve HIFU safety and make it a viable option to treat a broader range of disease sites. Vaporizing liquid PFC droplets in the target area provides an attractive option for introducing these microbubbles, given the stability of PFC droplets in circulation and the fact that they can be manufactured small enough (<300 nm) to extravasate through the leaky tumor vasculature. Previous testing has confirmed that phase-shift agents using several different PFC formulations produce larger ablation lesions with lower HIFU energies [9, 17, 20]. However, clinical utility requires control over the size, shape, and location of the ablation lesion, and this control can be problematic when PFC agents are present in the acoustic field [9, 11, 20, 21], particularly if the PSNE includes a volatile PFC component.
This study explored the use of a PSNE containing highly volatile DFB (1:1 mix of DFB and DDFP) in a gel model of HIFU enhancement. Lowering the intensity needed to achieve vaporization by the addition of a volatile PFC component can minimize HIFU intensity requirements. However, since the acoustic pressure that triggers vaporization is similar to the pressure that drives cavitation, establishing a vaporization field that does not grow during lower amplitude continuous wave insonation is not possible. Therefore, these experiments focused on optimizing acoustic parameters (intensity and insonation time) to achieve controllable HIFU enhancement for a given concentration of this novel PSNE. Previous testing has defined the limits of effective acoustic intensity for this PSNE . Even when PSNE was present, no ablation was seen at intensities below 140 W/cm2, and ablation in agent-free gels started to occur above 650 W/cm2. Therefore, the current study explored the effects of varying insonation (‘on’) and separation (‘off’) times of the HIFU treatment cycle within the intensity range from 140 to 650 W/cm2. It should be noted that this intensity range corresponds well with the range (157 to 550 W/cm2) identified by Zhang P. et al. as most appropriate for PFC-enhanced HIFU . In contrast, current HIFU systems in use clinically must operate at intensities at or above 2,000 W/cm2, risking injury to tissue outside of the target site. This study demonstrated that appropriate acoustic intensities and insonation times can be selected to maintain control over ablation lesion size, shape, and location even in the presence of a PSNE containing a volatile PFC component. Additionally, since the vaporization field was not fixed, relatively large ablation lesions that propagated outward from the acoustic focus could be produced. The ablation lesions measured up to 135 mm3 in gels with PSNE, compared to lesions measuring <4 mm3 in agent-free gels, when HIFU was applied at 650 W/cm2 for 20 s.
There are interesting similarities and differences between these findings and the results of other phase-shift studies looking at HIFU enhancement by PFC nanodroplets and microdroplets in acrylamide gels. Using a DDFP-based PSNE without a more volatile component, Zhang P. et al. were also able to produce ablation lesions larger than 100 mm3. However, the required intensities (around 2,000 W/cm2) were higher . It is suspected that the differences in ablation response between these two studies resulted primarily from the presence of volatile DFB in our PSNE. Testing in solution has demonstrated that incorporating a lower boiling point PFC during the PSNE preparation decreases the vaporization threshold of the PSNE . As a result of this lower threshold, the vaporization field increased in volume throughout the continuous wave HIFU treatment cycle in our study (Figure 6). In contrast, in the presence of DDFP alone, Zhang P. et al. established a stable vaporization field using a 30-cycle high-intensity (4,586 W/cm2) pulse and followed this with 15 s of continuous wave HIFU . Additionally, it should be noted that the frequency of applied HIFU was also different in these two studies (1 vs. 3.2 MHz). PFC droplet vaporization is a frequency-dependent phenomenon, and frequency differences alter ultrasound wave attenuation, energy deposition, and microbubble cavitation .
Zhang M. et al. studied DDFP microdroplet enhanced HIFU delivered at a frequency (1.44 MHz) similar to ours . They too noted increasing ablation lesion volumes with longer insonation times (insonation duration 2 to 5 s). However, at comparable PFC agent concentrations (3 vs. 5 × 105 droplets per milliliter) and insonation duration (5 s), the ablation responses were quite different. Probably reflecting the use of larger PFC droplets (2 to 8 microns) and a higher intensity (4,000 W/cm2), Zhang M. et al. produced ablation lesions measuring 600 mm3, compared to 20 mm3 in our study .
Finally, again testing a DDFP PSNE in acrylamide gels, Kawabata et al. applied HIFU using acoustic parameters (frequency, 1.1 MHz; intensity, 600 W/cm2; insonation duration, 5 to 20 s) similar to our study and found increasing ablation lesion size with longer insonation . However, lesion size did not increase when HIFU was applied at 2.2 MHz. The similarities and differences between these studies emphasize the fact that the ablation response during PSNE enhanced HIFU results from a complex interplay of the applied acoustic parameters as well as the type and size of the PFC agent. As a result, it is suspected that changing the ratio of DFB and DDFP in our PSNE would alter both the vaporization and ablation responses and is a potential area of future study.
The ablation lesions formed in this study were located in larger vaporization fields (microbubble clouds). The fact that the vaporization field grows throughout the entire HIFU treatment cycle when a volatile PSNE is present distinguishes this study from previous phase-shift enhancement studies. Continuous vaporization provides an opportunity to generate larger ablation lesions but also introduces unique challenges with regard to controlling the ablation response. Anticipating the extent of vaporization surrounding an ablation site is of practical importance when positioning adjacent lesions. Separating insonation by 2, 4, or 6 s did not affect the size of the vaporization field or the ablation lesion within this microbubble cloud when adjacent HIFU sites were 8 mm apart. Decreasing this separation distance could result in overlapping vaporization fields. Future study will be needed to assess the impact of overlapping vaporization fields on tissue heating and the ablation response.
In this gel model, controlled ablation could be achieved using acoustic parameters that did not result in lesion formation in agent-free regions (frequency = 1 MHz, intensity ≤ 650 W/cm2, insonation time ≤ 20 s). However, the limitations of this model should be noted. The phantoms and surrounding gels were acellular and avascular. Therefore, tissue injury that would result from mechanical cavitation effects was not captured, and heat sink effects of blood flow were not considered. Also, changes in acoustic impedance due to trapped degassed water between the acrylamide gel cap, the PFC-containing phantom, and the gellan gum mold resulted in echogenic boundaries, which may have slightly attenuated transmitted energies. Such distinct separations between agent-free and PFC-containing tissue regions would not be seen in vivo nor would the uniform distribution of PFC agent achievable in vitro.
A PSNE concentration in the range of 105 to 107 ND per milliliter was required for enhanced ablation in this gel model. Further experimentation will be needed to determine how these concentrations can be achieved in vivo. Recent testing has shown that the intravenous administration of a DDFP-based PSNE (0.5 mL/kg) resulted in the preferential deposition of nanodroplets in the vascular periphery of a rabbit tumor . These agents remained stable for hours and accumulated to a concentration sufficient to lower the energy required to reach an ablative temperature by HIFU. The DDFP PSNE used in this in vivo study was also tested in vitro by Zhang P. et al. in concentrations similar to those used in our current study . It is therefore reasonable to assume that effective tissue concentrations of our DFB-DDFP PSNE can also be achieved. However, to date, tissue concentrations of PFC nanodroplets following the administration of a PSNE have not been quantified in vivo. Magnetic resonance of fluorine or gadolinium-tagged agents may provide a tool to measure PFC tissue concentrations [20, 30]. The development of such a tool may be an important step on the path toward using PSNEs clinically for several reasons. First, there is a heterogeneous deposition of PFC in tumor tissue following the intravenous administration of a PSNE , quite different from the uniform distribution used for in vitro studies. Second, as this and other studies have now demonstrated, controlled ablation requires selecting appropriate acoustic parameters for a given PSNE concentration. Therefore, the safe application of HIFU in a field containing an unequal distribution of PFC agent may require estimating regional PFC concentrations and adjusting the acoustic parameters accordingly. We envision a magnetic resonance-guided focused ultrasound surgery system that maps the distribution of PFC tissue levels, selects appropriate acoustic parameters for the target region and desired lesion size, and monitors ablation by thermometry.