ImmuBubbles: Antibody-Shelled Gas-Core Nanobubbles as Ultrasound Contrast Agents

High Resolution Image Download MS PowerPoint Slide Ultrasound (US)-responsive nanobubbles (NBs) enable image-guided, localized delivery of biologics; however, the surface area of conventional antibody-loaded NBs limits their loading capacity. Although antibodies such as trastuzumab (Herceptin) offer high target specificity and prolonged half-lives, their penetration into solid tumors is impeded by abnormal vasculature and a dense extracellular matrix. To address these challenges, we developed ImmuBubbles (IBs), self-assembled antibody-shelled gas-core NBs produced by high-speed agitation of trastuzumab solutions in perfluoropropane. Hydrophobic residues within the IgG Fc region promoted self-assembly at the gas–liquid interface, forming shells without lipid incorporation. Herceptin-shelled IBs (Her-IBs) were uniform (∼95 nm) and positively charged (ζ ≈ + 10 mV) and generated US contrast both in vitro and in vivo. Moreover, cryo-transmission electron microscopy revealed spherical shells enclosing electron-lucent cores, consistent with a gas-filled morphology. To improve in vivo persistence, Pluronic L61 (L61) was coincorporated, yielding L61-Her-IBs with increased particle yield and enhanced echogenicity in cardiac imaging, with detectable contrast in tumor imaging. Antibody-dependent cellular cytotoxicity activity was preserved for L61-Her-IBs after high-speed mixing and therapeutic US (TUS; 1 MHz) exposure, consistent with maintained bioactivity. In SKOV3 xenografts, systemic L61-Her-IBs + TUS showed a trend toward tumor growth suppression compared with Herceptin monotherapy, without evident systemic toxicity. Collectively, these findings demonstrate that antibody-shelled NBs can function as US-responsive contrast agents while retaining antibody-mediated biological activity. To the best of our knowledge, this is the first report of self-assembled antibody-shelled NBs as ultrasound contrast agents that retain antibody function. With additional shell stabilization, such as covalent cross-linking using antibody-drug conjugate-style linkers, IBs may provide a platform for improving the precision of therapeutic ultrasound–guided interventions in solid tumors and tissues protected by restrictive biological barriers, including the central nervous system.