Associate Professor University of Texas at Dallas, United States
Introduction:: Hemoglobin microbubbles (HbMBs) have been proposed as a potential oxygen carrier for therapeutic applications such as organ transplantation, cancer therapy, and wound healing. Recently we have proposed that HbMBs can potentially be used to detect blood oxygen levels in vivo. However, the formulation of stable hemoglobin microbubbles is a challenging task due to the uncontrolled crosslinking that affects the resulting bubble size and stability. This study focused on optimizing parameters for sonication to create HbMBs, followed by analyzing their size, shape, and stability for potential in vivo applications. The results of the study show that HbMBs can be effectively manufactured for in vivo application using simple modifications of the probe sonication technique.
Materials and Methods:: We prepared a solution by dissolving 10 mg/mL hemoglobin powder in a 10% glycerol, 10% polypropylene glycol PBS solution. The solution was mixed with tryptophan at a ratio of 4:1 (hemoglobin:tryptophan) and homogenized. Then, it was heated to 50ºC. Two methods were used to test size and concentration. The first method involved sonication with a tip sonicator for 2, 5, and 10 seconds at 100% amplitude. The second method involved sonication with a tip sonicator for 2 seconds at 50%, 75%, and 100% amplitude, followed by immediate cooling of the solution by immersing it in an ice bath. After allowing the solution to cool down fully, sodium dithionate and sodium sulfate stock solution were added to reduce it. Then, oxygen gas was passed over the surface of the solution for a duration of 3 minutes to saturate it. After preparing the HbMBs, separate oxy and deoxy solutions were created to test the stability of HbMBs over time. The solution obtained with (2 seconds and 100% amplitude) setting was used, to test the stability with two independent variables. The first one involved preparing solution with sodium dithionate and sodium sulfate. The second one involved preparing solution without sodium dithionate and sodium sulfate.
Results, Conclusions, and Discussions:: Based on our findings, we observed that hemoglobin microbubbles (HbMbs) can have different sizes and concentrations depending on the sonication conditions used. Our results indicate that increasing the sonication time (2, 5, 10 seconds) at 100% amplitude can result in larger size (µm) and lower concentration [Figure 1]. Similarly, increasing the sonication amplitude (50%, 75%, 100%) for 2 seconds can lead to larger size (µm) and lower concentration [Figure 2].
The stability testing results indicate that microbubbles are more stable at refrigerated temperatures (4ºC) for samples containing Sodium dithionite and Sodium sulfate, compared to room temperatures over a period of 48 hours (2 days) [Figure 3]. For samples without Sodium dithionite and Sodium sulfate, microbubbles are stable at both 4ºC and room temperature, but degradation is quicker at room temperature than at refrigerated temperatures (4ºC) over a period of 48 hours (2 days) [Figure 4].
Overall, by simple modification of the tip sonication protocol, we are able to minimize bubble sizes, maximize number of bubbles created, and generate stable bubbles for in vitro and in vivo studies, enabling cheaper and more rapid development of HbMBs for acoustic BOLD imaging applications.
Acknowledgements (Optional): : I would like to sincerely thank to: My supervisor Dr. Shashank Sirsi for giving me chance to work on this exciting project and supporting me for various aspects of life. My colleagues Sugandha Chaudhary, Ghazal Rastegar, Bahareh Kian Pour, and Teja Pathour for all their support and providing a good ambience for the team work.
References (Optional): : 1. Wong, M., & Suslick, K. S. (1994). Sonochemically produced hemoglobin microbubbles. MRS Proceedings, 372, 89-94. doi: 10.1557/proc-372-89
2. Chaudhary, S., Akter, N., Rajeev, A., Hwang, M., & Sirsi, S. (2021). Hemoglobin microbubbles for in vivo blood oxygen level dependent imaging: Boldly moving beyond MRI. The Journal of the Acoustical Society of America, 150(4_Supplement), A27. doi: 10.1121/10.0006335
3. Winslow, R. M. (1991). Hemoglobin-based red cell substitutes. Baltimore: John Hopkins University Press.
4. Jocelyn, P. C. (1972). Biochemistry of the SH group: The occurrence, chemical properties, metabolism and biological function of thiols and disulphides. Academic Press.
