Associate Professor University of Texas at Dallas, United States
Introduction:: The use of microbubbles for targeted drug delivery has increased significantly. Microbubbles have an advantage in creating effective unloading strategies. Under ultrasound exposure, microbubbles produce shear stress and heat that release their cargo quickly, resulting in high drug concentrations outside the carrier. In our previous work, we created hemoglobin microbubbles (HbMBs) using a unique method and observed that they exhibit different responses in oxygenated and deoxygenated media when exposed to ultrasound. In this study, we aim to explore the potential of using HbMBs for delivering drugs in an oxygen-sensitive manner. We first modified the HbMBs by pegylation and then examined how they react to ultrasound at varying frequencies in both oxygenated and deoxygenated environments. We aim to cover HbMBs with liposomes containing drugs and examine how the release of the drugs is influenced by changes in oxygen concentration.
Materials and Methods:: Hemoglobin microbubbles (HbMBs) were prepared by mixing 8 ml of hemoglobin solution (10 mg/ml) and 2 ml of tryptophan solution (10 mg/ml) and heating them to 50℃. The resulting solution was then subjected to tip sonication for 5 seconds to form HbMBs. Afterwards, the HbMBs were immediately cooled in an ice bath for 3 minutes.Then, 108 HbMBs were treated with 3 mg of Maleimide-PEG for 1 hour, followed by 3 rounds of centrifugation at 200 RCF for 2 minutes to remove the extra Maleimide-PEG. After that, sodium dithionite and sodium sulfide were introduced to the pegylated MBs, and oxygen gas was flushed into the solution for 3 minutes. Either oxygenated (DO >100%) or deoxygenated (DO< 10%) PBS was mixed with 100 ul of the solution. Then, the mixture was insonified at 3 and 1 MHz and 0.6 MPa for 1 minute. The stability of the final solutions in both environments after sonication was examined using bright field imaging.
Results, Conclusions, and Discussions:: The images in Fig. 1 show fluorescence of pegylated HbMBs. To obtain these images, FITC-PEG-Maleimide was used for incubating the HbMBs instead of PEG-Maleimide. The bright shell of the HbMBs visible in Fig. 1 indicates successful pegylation. Based on the data in Figure 2, there was no noticeable difference in the stability of HbMBs before and after sonication at 1 MHz frequency in both oxygenated and deoxygenated media, with a 78.1 ± 3.1% decrease in concentration observed for the former and a 72.4 ± 4.8% decrease for the latter. Interetingly, at 3 MHz, the breakdown of deoxygenated HbMBs was 76.9 ± 1.3%, while that of oxygenated microbubbles was only 21.1 ± 3.3%. Our results suggest that HbMBs can be preferentially destroyed at 3 MHz in a deoxygenated environment, which could have clinical value in treating hypoxic tumors or drug-delivery to metabolically active tissue.
In conclusion, we have efficiently taken the first and most important step toward creating an oxygen-sensitive drug delivery system that is capable of releasing the drug inside the tumor microenvironment rather than any off-target site. We believe that this strategy could be a prominent asset for cancer therapy, especially, for patients with metastasized cancer.
