SonoVue® vs. Sonazoid™ vs. Optison™: Which Bubble Is Best for Low-Intensity Sonoporation of Pancreatic Ductal Adenocarcinoma?

Abstract

The use of ultrasound and microbubbles to enhance therapeutic efficacy (sonoporation) has shown great promise in cancer therapy from in vitro to ongoing clinical studies. The fastest bench-to-bedside translation involves the use of ultrasound contrast agents (microbubbles) and clinical diagnostic scanners. Despite substantial research in this field, it is currently not known which of these microbubbles result in the greatest enhancement of therapy within the applied conditions. Three microbubble formulations-SonoVue^®, Sonazoid™, and Optison™-were physiochemically and acoustically characterized. The microbubble response to the ultrasound pulses used in vivo was simulated via a Rayleigh-Plesset type equation. The three formulations were compared in vitro for permeabilization efficacy in three different pancreatic cancer cell lines, and in vivo, using an orthotopic pancreatic cancer (PDAC) murine model. The mice were treated using one of the three formulations exposed to ultrasound from a GE Logiq E9 and C1-5 ultrasound transducer. Characterisation of the microbubbles showed a rapid degradation in concentration, shape, and/or size for both SonoVue^® and Optison™ within 30 min of reconstitution/opening. Sonazoid™ showed no degradation after 1 h. Attenuation measurements indicated that SonoVue^® was the softest bubble followed by Sonazoid™ then Optison™. Sonazoid™ emitted nonlinear ultrasound at the lowest MIs followed by Optison™, then SonoVue^®. Simulations indicated that SonoVue^® would be the most effective bubble using the evaluated ultrasound conditions. This was verified in the pre-clinical PDAC model demonstrated by improved survival and largest tumor growth inhibition. In vitro results indicated that the best microbubble formulation depends on the ultrasound parameters and concentration used, with SonoVue^® being best at lower intensities and Sonazoid™ at higher intensities.

Keywords: microbubbles; pancreatic cancer; sonoporation; targeted drug delivery; ultrasound; ultrasound-enhanced therapy.

Figure 1

Graphical rendering of the experimental setup showing how the microbubble attenuation and cavitation thresholds were measured. (A) shows an overview of the ultrasound transducers with their corresponding focal sizes and alignment. (B) shows how the attenuation was measured using only the PCD and a stainless-steel reflector. (C) shows how the cavitation thresholds were measured using the confocally arranged transducers.

Figure 2

Timeline and setup for treating a mouse using the diagnostic ultrasound probe. (A) A typical timeline where the entire imaging, treatment, and recovery procedure lasted 71 min. (B) The treatment configuration where bubbles are infused via the tail vein and ultrasound is applied via the C1-5 probe.

Figure 3

Size distribution histograms of SonoVue^®, Sonazoid™, and Optison™ measured optically. (A) The histogram normalized to the number of microbubbles counted. (B) Normalizes each bin to the total volume occupied by all the microbubble population. The cumulative sum of each histogram is 100%.

Figure 4

Concentration and mean size as a function of time for SonoVue^®, Sonazoid™, and Optison™ measured optically. SonoVue^® had the largest loss in microbubbles after 30 min where the median concentration was halved and the largest change in microbubble size and variance. Both Sonazoid™ and Optison™ showed minimal loss in concentration and change in size.

Figure 5

Acoustic measurements of the three ultrasound contrast agents. (A) The attenuation as a function of frequency for all agents at an identical particle concentration of 75 × 10³ ppmL. (B) Shows the same data, but the concentration has been adjusted that all agents have the same cross-sectional area. (C) Shows the subharmonic magnitude between 400–600 kHz, emitted from the microbubbles when excited by a 1 MHz, 25 cycle burst pulse, while (D) shows the same results but evaluating the spectral integral.

Figure 6

Summary of simulation results. For the used ultrasound conditions, SonoVue^® induced the largest cumulative oscillation amplitude and radiated pressure, i.e., potentially having the largest effect on the surrounding cells. The treatment pulse resulted in the largest cumulative oscillation amplitude and radiated pressure for SonoVue^® and Sonazoid™ but not for Optison™ indicating that the treatment pulse was suboptimal for the size disruption of Optison™. PNP: Peak-Negative Pressure; PPP: Peak-Positive Pressure.

Figure 7

Results of in vitro experiments evaluating the percentage of cells affected by sonoporation using SonoVue^®, Sonazoid™, and Optison™ at different acoustic conditions, different microbubble concentrations, and three difference pancreatic cancer cell lines. In general, increasing the acoustic power and microbubble concentration increased the number of calcein-positive cells. All three cell lines responded differently with BxPC-3 being the most sensitive, followed by PANC-1 and MIA PaCa-2 being the least sensitive.

Figure 8

Compiled results showing which microbubble is the best at a given ultrasound intensity and microbubble concentration for the three cell lines MIA PaCa-2, BxPC-3, and PANC-1. The color at a given point indicates which microbubble had the largest amount of calcein-positive cells. Each cell line has a different sensitivity pattern to the microbubble type and acoustic intensity. SonoVue^® or Optison™ are typically best at lower intensities (<15 mW/cm²) but Sonazoid™ was best at higher ultrasound intensities (>15 mW/cm²).

Figure 9

Quantitative metric of treatment and therapeutic response as a function of time. (A) The total bodyweight for all mice as a function of time. No mice showed a drop below the 20% body weight loss limit indicating the treatment did not have significant adverse effects. (B) The percentage increase in whole body bioluminescence as a function of time. Bioluminescence images were taken 2–3 days before treatment. An initial increase in bioluminescence is observed followed by a decreased due to necrotic cores forming. (C) The tumor volumes measured with 3D ultrasound. All microbubble groups showed significant growth inhibition. (D) The three groups treated with microbubbles to help better distinguish between groups. SonoVue^® showed significant tumor inhibition vs. Optison™ and Sonazoid™. (E) The overall survival of all groups. The number at the bottom of the groups indicates the median survival. The mice treated with SonoVue^® showed the longest survival. This survival was significant vs. drug alone and both other bubbles.

Figure 10

Qualitative photographs of bioluminescence as a function of time. Each row is a single mouse in which the bioluminescence count matched the group mean closest at week 5. At the end of treatment, the bioluminescence spread engulfs the entire abdomen of the mice treated with paclitaxel alone or paclitaxel with ultrasound. In contrast, the mice treated with sonoporation seem to have a more localized cancer spread (i.e., primary tumor and liver), potentially due to the slower growth.

Figure 11

Qualitative renderings of the tumor volumes measured using 3D ultrasound. Each row is a single mouse in which the tumor volume matched the group mean closest at week 8. All tumors have a similar size before treatment (week 2) and the difference in tumor volume development is clearly observable over the following 7 weeks.

Figure 12

Example ultrasound images and quantitative results from tumor vascularization measurements. (A) An example image of the PDAC tumor imaged with duplex mode (B-mode and power doppler). The tumor can clearly be delineated, and part of the feeding vasculature is detected. (B) A Maximum Intensity Projection (MIP) of the power doppler signal following 3D imaging. The tumor is located between the two white arrows. The feeding vessel is clearly visible in addition to microvasculature throughout the tumor. Other organs such as the spleen and kidney can also be clearly distinguished. (C) A B-Mode ultrasound image (3 MHz) of a healthy mouse with no tumor or contrast agent captured using the GE E9 scanner and C1-5 ultrasound probe to help depict the image quality and resolution. The landmarks used to locate the pancreas and tumor (such as the kidneys) can be easily delineated. (D) The same mouse but during treatment. A significant loss in image quality is observed due to the different imaging mode. (E) The quantitative results of the tumor percentage vasculature (calculated from the doppler content of the 3D tumors) as a function of time. No difference was observed between the groups. (F) The same data with every datapoint plotted. A general trend of decreasing vascular as a function of tumor volume is observed but no distinct correlations are observed.