Engineering Microbubbles with the Buried-Ligand Architecture for Targeted Ultrasound Molecular Imaging
Microbubbles are gaseous microspheres stabilized with phospholipid monolayer shells. Because of their compressible gas core, they are highly echogenic. Taking advantage of this property, microbubbles are used as ultrasound contrast agents for signal enhancement. In addition, they are being developed for targeted diagnostic molecular imaging applications. Previous studies have shown that targeted microbubbles could induce complement activation and reduce their circulation persistence. In order to avoid the undesired immune response, a novel stealth microbubble design that consisted a bimodal poly(ethylene glycol) (PEG) brush layer, named buried-ligand architecture (BLA), was introduced. However, in order to utilize this BLA design for targeted imaging, it is essential to characterize the kinetics of ligand conjugation to BLA microbubbles and further study their in vitro and in vivo immunogenicity and contrast persistence properties. In this project, ligand conjugation to BLA microbubbles was characterized using molecules with large molecular weight difference. Microbubbles with various PEG surface architectures were formulated, and ligands with large molecular weight difference were used to conjugate to the microbubbles in order to study the feasibility of generating targeted microbubbles using the post-labeling technique. It was shown that small ligands could be conjugated to BLA microbubbles to generate targeted contrast agents using post-labeling. A surprising result was observed during the experiment that complex surface microstructures could be induced simply through streptavidin-biotin binding. This was the first time that these wrinkled structures were generated on the surface of microbubbles using a non-mechanical method. In vitro immunogenicity studies showed that BLA microbubble indeed induced less complement activation than microbubbles with monomodal PEG brush layers, or exposed-ligand architecture (ELA) microbubbles. In vivo contrast persistence studies further demonstrated the improved circulation time of the BLA microbubbles and showed that the buried-ligand design did not compromise their ability for signal enhancement. The results presented in this project supported the previous findings that microbubbles with the buried-ligand architecture had reduced immunogenicity with prolonged circulation persistence and were more suitable to be developed for targeted molecular imaging applications.
Biomedical engineering, Chemical engineering, Ligands, Microbubbles, Ultrasonic imaging
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