Synthesis of laboratory Ultrasound Contrast Agents

Abstract

Ultrasound Contrast Agents (UCAs) were developed to maximize reflection contrast so that organs can be seen clearly in ultrasound imaging. UCAs increase the signal to noise ratio (SNR) by linear and non-linear mechanisms and thus help more accurately visualize the internal organs and blood vessels. However, the UCAs on the market are not only expensive, but are also not optimized for use in various therapeutic research applications such as ultrasound-aided drug delivery. The UCAs fabricated in this study utilize conventional lipid and albumin for shell formation and perfluorobutane as the internal gas. The shape and density of the UCA bubbles were verified by optical microscopy and Cryo SEM, and compared to those of the commercially available UCAs, Definity® and Sonovue®. The size distribution and characteristics of the reflected signal were also analyzed using a particle size analyzer and ultrasound imaging equipment. Our experiments indicate that UCAs composed of spherical microbubbles, the majority of which were smaller than 1 um, were successfully synthesized. Microbubbles 10 um or larger were also identified when different shell characteristics and filters were used. These laboratory UCAs can be used for research in both diagnoses and therapies.

Figure 1

Cryo-SEM images of UCAs. Mirco-size circular bubbles can be observed in all four cases.

Figure 2

Optical images of the UCAs. The shape of the UCAs was observed under optical microscope at 1400× magnification.

Figure 3

3D tomographic images of the UCAs. In order to confirm round shape microbubbles, 3D optical images were obtained at 400× magnification.

Figure 4

Size distributions of UCAs. The size distribution of UCAs was measured with a particle size analyzer (Mastersizer 2000, Malvern Instruments Ltd., UK) and plotted against volume. In this figure ‘NF’ indicated non-filtered data, and ‘F’ indicated filtered data, respectively. The results indicate that the synthesized UCAs have approximately five times more volume percent of microbubbles in the range of 10–100 μm compared to that of commercial UCAs. If the synsthesized UCAs was filtered once, the volume percent of the larger bubble was reduced approximately four times.

Figure 5

Size distributions of UCAs by the number of microbubbles. The majority of synthesized microbubbles are adequate to use as UCAs. The use of a 5 μm membrane filter seems to over-filter, resulting in small-sized microbubbles ranging from 1–3 μm.

Figure 6

Cell toxicity test of the UCAs. The results indicate that all of the tested UCAs are not toxic to 293A human embryo kidney cell.

Figure 7

Ultrasound imaging comparison of two commercial UCAs and two synthesized UCAs over time (0, 1, 3, 5, 7, 9 min). (a) 3 days after synthesis. (b) 6 days after synthesis. (c) 9 days after synthesis. (d) 12 days after synthesis. (e) 15 days after synthesis. Ultrasound imaging results show that the synthesized microbubbles can be successfully used for imaging purposes. In particular, the synthesized lipid shell UCA shows almost equivalent imaging capabilities as Definity^®. On the other hand, the synthesized albumin shell UCA seems to less effective in increasing ultrasound imaging contrast.

Figure 8

UCA lifespan. All the UCAs seem to have limited lifespan in the case of bolus injection. Additionally, the preservation period of all of the UCAs seems to be less than six days. SonoVue^® and albumin shelled UCAs seem to decay faster after opening the vial or synthesis.

Figure 9

Staining lipid shell microbubble and the shell structure. Negative-stained TEM images with uranyl acetate and phosphotungstic acid was used to visualize the shell layer. The electron acceleration voltage was increased up to 120 kV to puncture the shell and to stain inside if possible. A fragment of the shell shown in Figure 9a was relocated to the shell surface to create Figure 9b. The fragment seems to fit the missing surface portion and form a complete shell surface. In other words, fragments of the shell do not seem to reform after being separated from the microbubbles. Figure 9c shows the bilayer-like structure of a fragment with a shell thickness of approximately 7 nm.

Figure 10

Low temperature workspace and liquefaction of perfluorobutane.

Figure 11

Toxicity evaluation setup. The 293A human embryo kidney cell line was used for toxicity testing.

Figure 12

Experiment setup for UCA response to an ultrasound imaging system. All the prepared UCAs were imaged with a commercial ultrasound imaging system (Sonoace Pico^®, Medison, Seoul, Korea).