Exploring the Potential of Hemoglobin Microbubbles for Oxygen Sensing in Ultrasound Imaging

Introduction:: Hemoglobin, a tetrameric protein composed of two alpha and two beta chains, plays a crucial role in oxygen transport within the human body due to its ability to bind and unbind oxygen molecules with its attached heme site. In this work, we describe a new type of gas-filled ultrasound contrast agent known as a hemoglobin microbubble (HMB) composed of multiple hemoglobin proteins crosslinked by disulfide bonds. We propose that these oxygen-binding microbubbles could be used for oxygen-sensing in vivo. To test our hypothesis, we obtained stable air-filled HMBs through ultrasonication and centrifugation with a median size of 4.5µm suitable for intravascular injections into the bloodstream. Spectroscopic testing showed that HMBs retained their ability to reversibly bind oxygen. Acoustic testing revealed that HMBs respond differently whether they are oxygenated or deoxygenated environments, making them ideal for oxygen sensing in vivo. By utilizing passive cavitation detection, we were able to measure significant differences in acoustic responses at the fundamental frequency(1.1 MHz), 1st and 2nd, and subharmonic frequencies, of oxygenated HMBs compared to deoxygenated HMBs. These results have been further tested by Verasonics imaging which showed higher mean pixel intensity of deoxygenated HMBs than their oxygenated counterparts. This breakthrough could lead to exciting new applications in ultrasound imaging, such as blood oxygen level detection, which is not currently possible using Ultrasound.

Materials and Methods:: We produced HMB microbubbles through a sonochemical process that uses high ultrasonic energy to create free radicals. The free radicals cause oxidation of the cysteine residues within the microbubbles and result in the formation of interprotein disulfide bonds. To determine the size distribution of the protein microbubbles, we used the Coulter counter method and brightfield microscopy. The addition of a reducing agent activates the microbubbles which change their absorbance properties between 300nm -700nm. We used our own acoustic setup to perform passive cavitation detection and test the acoustic property of HMBs in both oxygenated and deoxygenated environments. Labview and Matlab were used to visualize and analyze the non-linear frequency components. The differences in contrast enhancement from HMBs were also observed using a Verasonics Ultrasound scanner.

Results, Conclusions, and Discussions:: Our study successfully produced functional microbubbles that were highly concentrated, with a median size of 4.5 microns. Changes in the Soret peak and q-bands were observed in shell-incorporated hemoglobin bubbles, indicating the hemoglobin remained active. Acoustic characterization showed that the subharmonic signals were significantly higher in the deoxy environment in HbMBs but only marginally

different in non-oxygen-sensitive polymer controls. The mean pixel intensity measured using the Verasonics system showed that the deoxygenated hemoglobin microbubbles had higher intensity than the oxygenated hemoglobin microbubbles. In our project, we successfully created functional microbubbles and demonstrated the variation in their response between oxygenated and deoxygenated states. These microbubbles can have practical clinical use in differentiating between low and high-oxygen areas in a tumor's microenvironment through ultrasound imaging. This method can help reduce the need for expensive imaging modalities, such as PET scans and MRIs, which require radiolabelled contrast agents.


Acknowledgements (Optional): :

References (Optional): :