Assessing the oxygen scavenging capacity and myocardial gas embolization risk of ultrasonically activated phase shift perfluorobutane droplets

Abstract

This study investigated oxygen scavenging efficiency and the risk of embolization of the cardiac vasculature using ultrasound-triggered phase-shift perfluorobutane (PFB) droplets in vitro and ex vivo. The emulsion comprised lipid-shelled perfluorobutane core droplets with a modal diameter of 0.98 ± 0.03 μm. The droplets were prepared using a high-pressure microfluidizer. The embolization risk was assessed using a modified ex vivo rat Langendorff preparation to accommodate an EkoSonic™ Endovascular Device. The EkoSonic™ Device was composed of an infusion catheter and an ultrasonic core to generate ultrasound at 2.35 MHz and nucleate acoustic droplet vaporization of the droplets. The oxygen scavenging efficiency was studied in an isolated beating heart and an in vitro flow phantom setup with target concentrations ranging from 0.05 × 10^-4 to 5.0 × 10^-4 mL mL^-1. Gas embolization from the acoustic droplet vaporization (ADV)-nucleated microbubbles was assessed based on cardiac perfusion and cardiac functional parameters. No change in cardiac perfusion was observed when using droplets with target concentrations below 1.5 × 10^-4 mL mL^-1, either with or without ultrasound insonation of the droplets. Oxygen scavenging increased with increasing droplet target concentration. The ADV transition efficiency increased with increasing droplet concentration between 0.05 × 10^-4 and 0.5 × 10^-4 mL mL^-1 and decreased for higher concentrations. The conclusion of this study was that ultrasound-triggered phase-shift perfluorobutane droplets effectively scavenge oxygen without causing significant embolization at concentrations below 1.5 × 10^-4 mL mL^-1. Oxygen scavenging increased with higher droplet concentrations, whereas the transition efficiency of ADV reached the largest value at 0.5 × 10^-4 mL mL^-1, indicating an optimal performance balancing safety and efficacy exists.

Fig. 1. (a) Timeline of Langendorff perfusion for isolated rat hearts. (b) Schematic diagram of the Langendorff apparatus. The Langendorff apparatus featured a reservoir of oxygenated KHB (partial pressure of oxygen (P_O2) of 523 ± 34 mmHg). KHB was pumped through the system in tubing (cyan) within water-jacketing (blue) to ensure a temperature of 37 °C. Cardiac function assessment utilized a balloon placed in the left ventricle, while perfusion flow rate was monitored through an in-line flow probe. Droplets were introduced into the Langendorff system via a syringe pump through the coolant lumen of the EkoSonic™ infusion catheter. The water jacketing tubing was filled with KHB. The EkoSonic™ ultrasonic core (yellow) was also inserted through the coolant lumen. A 2.35 MHz pulsed ultrasound tone burst was applied to the droplets using a 6 cm treatment zone EkoSonic™ ultrasound core. The P_O2 in the buffer entering the heart was measured using a flow-through oxygen sensor. (Created with https://BioRender.com.)

Fig. 2. Schematic diagram of the in vitro flow phantom. The oxygenated deionized water reservoir and tubing were warmed. Droplets were infused through the flow system using a syringe pump through an EkoSonic™ infusion catheter, which was maintained at 37 °C. The oxygenated water had an initial partial pressure of oxygen (P_O2) of 553 ± 8 mmHg. A 2.35 MHz pulsed ultrasound tone burst (40 cycles) insonified the droplets using a 6 cm treatment zone EkoSonic™ ultrasonic core. The P_O2 in the fluid downstream of the catheter was measured using a flow-through oxygen sensor. The figure was reproduced from Benton et al., which used an identical setup.

Fig. 3. Volume-weighted distribution of (a) the coarse emulsion droplet solution and (b) droplets emulsion solution after one passage of high shear pressure homogenization at 10 000 PSI. The droplet emulsion had a nominal modal diameter of 0.98 ± 0.03 μm. The lines and shading are the mean and standard error mean, respectively (N = 4 for both measurements).

Fig. 4. Size distribution of droplet emulsion solutions stored at 4 °C during 23-day storage. The days post manufacturing are indicated in the legends where D0 is the day of droplet manufacturing. Each line is the average of n = 3 measurements. The lines and shading are the mean and standard error mean of the triplicate measurements, respectively.

Fig. 5. PFB droplet concentration as a function of time at room temperature (22.6 °C ± 0.1 °C) up to 5 h. Each line is the average of n = 3 measurements.

Fig. 6. The change in the perfusion flow rate relative to the last 5 min of stabilization throughout the 45 min experiments without (a) and with (b) ultrasound exposure for five different droplet concentrations (0.25 × 10⁻⁴, 0.5 × 10⁻⁴, 1.5 × 10⁻⁴, 2.5 × 10⁻⁴, and 5 × 10⁻⁴ mL mL⁻¹). The first 20 min of the experiment was the stabilization phase (before infusion). The grey shaded area on the graph demarks the 2 min of droplet infusion (during infusion) followed by a 3 min period that accounts for approximately 1 min to infuse the dead volume of the EkoSonic™ infusion catheter and the time to flow from the end of the catheter to heart. The time from 25 min to 45 min represents the heart recovery period (after infusion). Each value is mean ± SEM, 6 hearts per group, except for 2.5 × 10⁻⁴ mL mL⁻¹ where 5 hearts were used and 5 × 10⁻⁴ mL mL⁻¹ where only 4 hearts were used without ultrasound due to three of four hearts ceasing perfusion before the 45 min time point. Using reduced animal numbers was deemed ethical.

Fig. 7. The change in left ventricular developed pressure (LVDP) relative to last 5 min of stabilization throughout the 45 min experiments without (a) and with (b) ultrasound exposure for five different droplet concentrations (0.25 × 10⁻⁴, 0.5 × 10⁻⁴, 1.5 × 10⁻⁴, 2.5 × 10⁻⁴, 5 × 10⁻⁴ mL mL⁻¹). The first 20 min of the experiment was the stabilization phase (before infusion). The grey shaded area on the graph demarks the 2 min of droplet infusion (during infusion) followed by a 3 min period that accounts for approximately 1 min to infuse the dead volume of the EkoSonic™ infusion catheter and the time to flow from the end of the catheter to heart. The time from 25 min to 45 min represents the heart recovery period (after infusion). Each value is mean ± SEM, 6 hearts per group, except for 2.5 × 10⁻⁴ mL mL⁻¹ where 5 hearts were used and 5 × 10⁻⁴ mL mL⁻¹ where only 4 hearts were used without ultrasound due to three of four hearts ceasing perfusion before the 45 min time point. Using reduced animal numbers was deemed ethical.

Fig. 8. The change of partial pressure of oxygen (P_O2) relative to the last 5 min of stabilization throughout 45 min of perfusion without ADV (a) and with ADV (b), respectively using the Langendorff apparatus for 5 different droplet concentrations (0.25 × 10⁻⁴, 0.5 × 10⁻⁴, 1.5 × 10⁻⁴, 2.5 × 10⁻⁴, 5 × 10⁻⁴ mL mL⁻¹). The first 20 min of the experiment was the stabilization phase (before infusion). The grey shaded area on the graph demarks the 2 min of droplet infusion (during infusion) followed by a 3 min period that accounts for approximately 1 min to infuse the dead volume of the EkoSonic™ infusion catheter and the time to flow from the end of the catheter to heart. The time from 25 min to 45 min represents the heart recovery period (after infusion). Each value is mean ± SEM, 6 hearts per group, except for 2.5 × 10⁻⁴ mL mL⁻¹ where 5 hearts were used and 5 × 10⁻⁴ mL mL⁻¹ where only 4 hearts were used without ultrasound due to three of four hearts ceasing perfusion before the 45 min time point. Using reduced animal numbers was deemed ethical.

Fig. 9. The magnitude of oxygen scavenging before- and during-ADV using the in vitro flow phantom setup with droplet concentrations of 0.0 × 10⁻⁴ (no droplet, negative control), 0.05 × 10⁻⁴, 0.25 × 10⁻⁴, 0.5 × 10⁻⁴, 2.5 × 10⁻⁴, and 5 × 10⁻⁴ mL mL⁻¹. The hashed bars represent oxygen scavenging observed without ultrasound (before ADV). The solid-colored bars are the amount of oxygen scavenging observed with ultrasound (during ADV).

Fig. 10. The calculated ADV transition efficiency for the five different target droplet concentrations 0.05 × 10⁻⁴, 0.25 × 10⁻⁴, 0.5 × 10⁻⁴, 2.5 × 10⁻⁴, 5 × 10⁻⁴ mL mL⁻¹. The transition efficiency was calculated based on the change in the partial pressure of oxygen after droplet infusion with and without ADV using the Radhakrishnan model.