Associate Professor University of Texas at Dallas, United States
Introduction Hemoglobin Microbubbles (HbMBs) are a promising tool for acoustic blood oxygen level dependent (BOLD) imaging. Our preliminary studies suggest that HbMBs, due to their protein shell stabilization, exhibit improved visibility of blood arteries and other tissues. Thus, evaluation of HbMBs is crucial to advance acoustic BOLD imaging as a potential diagnostic technique. Microbubbles are safe to inject and have been approved by the FDA for ultrasound imaging of the heart for over a decade. These gas-filled bubbles are stabilized by a polymer, protein or lipid shell. HbMBs have demonstrated their unique oxygen binding properties in in-vitro, but have not been tested in in vivo applications using clinical ultrasound scanners. The ultimate goal of this study is to assess the response and stability of HbMBs using a commonly available clinical ultrasound scanning system in vitro, then test the circulation profile of hemoglobin microbubbles in immunocompetent mice, thus further demonstrating their clinical potential Materials and methods To create hemoglobin microbubbles, 10 mg/ml hemoglobin solution and 10mg/ml tryptophan solution were made using a solution containing phosphate buffer saline, 10%glycerol, and 10%propylene glycol. The solution was preheated and then subjected to sonication. Afterwards, the size and concentration of the HbMBs were measured with a Multisizer 4e Coulter Counter. Oxygenated and deoxygenated HbMBs were prepared by diluting them in a PBS solution flushed with oxygen and nitrogen, respectively. Firstly, HbMB solutions were created in varying concentrations and placed under an ultrasound clinical scanner (Siemens Acuson Sequoia C512 with 15-L cardiac transducer probe). The mechanical index was varied (0-0.6) to determine the threshold of activation, intensity, and stability of HbMBs over time. Images obtained were later analyzed through ImageJ software. Secondly, an in-vivo experiment was performed on CD1 mice mouse after removing the hair in the kidney region. The mouse was placed on a preheated plate and injected with HbMB via the tail. Subsequently, ultrasound images and videos were captured and analyzed using custom Labview software. Result Hemoglobin microbubbles improved contrast of ultrasound images on a clinical ultrasound scanner, similar to other types of microbubbles. At the concentration of 2e6 MB/ml and above, HbMB showed signal enhancement. This signal intensity increased with HbMB concentration. Moreover, the signal contrast improved with increasing Mechanical Index (MI) with significant changes for MIs below 0.2 (fig. 1 and 2). Furthermore, the acoustic intensity scattered from oxygenated HbMBs was higher at lower mechanical indices than 0.3. However, at higher mechanical indices, there were no statistically significant difference between signal intensities detected from oxygenated and deoxygenated HbMB (Fig. 3). Time-intensity curves demonstrated that these microbubbles were stable about 50 seconds under the maximum mechanical index. Discussion The results of the study indicate that contrast signal increases with MI using both B-mode and non-linear CPS mode imaging, which could be useful for monitoring HbMBs in vivo. More research is necessary to validate the use of HbMBs in circulation. However, these findings demonstrate that the use of HbMBs has the potential to significantly improve acoustic BOLD imaging. Conclusion In this study, hemoglobin microbubbles were shown to be acoustically active and their signal enhancement was evaluated using a clinical ultrasound scanner. We demonstrated that the contrast improvement depends mainly on the concentration of microbubbles and the pressure exerted on them, as well as oxygen saturation of the HbMB’s environment. These microbubbles also showed a significant contrast enhancement in the blood circulation of mice. Our ongoing studies include pegylation of the HbMBs to reduce complement activation while still maintaining their oxygen binding capabilities.
