(451c) Ligand Conjugation to Bimodal PEG Brush Layers On Microbubbles

Using microbubbles as a model system, we examined molecular diffusion and binding to colloidal surfaces with bimodal hydrated-polymer brush layers. A microbubble is a gas-filled colloidal particle with diameter less than 10 um, of which the surface comprises amphiphilic phospholipids self-assembled to form a lipid monolayer shell. Due to the compressible gas core, microbubbles provide a sensitive acoustic response and are currently used as ultrasound contrast agents. Similar to the design of long circulating liposomes, poly(ethylene glycol) (PEG) chains are typically incorporated into the shell of microbubbles to form a steric barrier against coalescence and adsorption of other macromolecules to the microbubble surface. We introduced a buried-ligand architecture (BLA) design where the microbubble surface was coated with a bimodal PEG brush. After microbubbles were generated, fluorescent ligands with different molecular weights were conjugated to the tethered functional groups on the shorter PEG chains, while the longer PEG chains served as a shield to protect these ligands from exposure to the surrounding environment. BLA microbubbles partially prevented the binding of macromolecules (>10 kDa) to the tethers due to the steric hindrance of the PEG overbrush, while allowing the uninhibited attachment of small molecules (<1 kDa). Some (~45%) of the model macromolecule fluorescein conjugated streptavidin (SA-FITC) did bind to BLA microbubbles compared to the exposed-ligand architecture (ELA), suggesting a possible phase separation between the lipid species on the surface leading to populations of revealed and concealed ligands. Ligand conjugation kinetics was independent of microbubble size, regardless of ligand size or microbubble architecture. We observed, for the first time, streptavidin-induced surface structure formation for ELA microbubbles, and discovered that this phenomenon may be measured quantitatively via flow cytometry. Most importantly, we demonstrated the feasibility of post-labeling for small-molecule ligands to BLA microbubbles as a platform to generate stealth targeted ultrasound contrast agents.