Increasing sensitivity and discrimination of breast cancer diagnosis using molecularly targeted, extravascular ultrasound imaging with ultrasound-activated, phase-change nanodroplet contrast agent

摘要

ABSTRACT Lipid shelled perfluorocarbon (PFC) microbubbles ranging from 0.5 to 8 microns in diameter have been approved by the FDA to enhance the contrast of blood for diagnostic ultrasound imaging. The micro scale of ultrasound contrast agents limits them to the bloodstream, eliminating the enhanced permeability and retention (EPR) observed in many tumors. The ability to create nanoparticles from existing lipid-shelled microbubble contrast agents lets us leverage the non-toxic formulations of these agents, and lets us create novel targeted nanoparticles using any of the established strategies for formulating targeted microbubbles. Microbubbles targeted to Secreted Frizzled Related Protein 2 (SFRP2) showed significant discrimination between tumor and normal vasculature, and SFRP2 is highly expressed in a variety of breast cancers. We and our colleagues found that cold and increased pressure caused conventional gas-phase ultrasound contrast to condense into lipid-shelled nanodroplets of liquid PFC. With sufficient energy, these non-echogenic agents revert back to conventional ultrasound contrast agents. Nanodroplets share the advantages inherent to nanoparticle strategies for cancer therapy: passive accumulation in tumors enhanced by the EPR effect, targeted accumulation within tumors aided by targeting moieties, and reduced systemic dosing. Activating nanodroplets with ultrasound provides a high degree of spatial selectivity, preventing re-entry of the micro scale bubbles into the vasculature, thus maintaining a high local concentration of contrast agent. Molecular imaging that interrogates the interstitial space within tumors widens the physical scope of imaging with the payoff of a higher density of bound contrast throughout the tumor volume, and additional opportunities to target tumor molecules that are not found in the vascular endothelium. We propose that extravascular molecular imaging with SFRP2- targeted nanodroplets will increase the sensitivity of detecting breast cancer compared to microbubble molecular imaging while maintaining the molecular specificity needed to decrease false diagnoses. A critical barrier to leveraging molecularly targeted, lipid shelled perfluorocarbon nanodroplets is a fundamental lack of knowledge about their biodistribution in normal and tumor tissue. We propose to combine existing, established techniques to create SFRP2-targeted, and non-targeted nanodroplet formulations carrying a biotin and DNA label, determine their circulating half-lives, and use immunohistochemical detection of biotin or quantitative polymerase chain reaction assays of our unique DNA payload to characterize the biodistribution of these liquid perfluorocarbon droplets in normal and tumor tissues. Establishing the ability of targeted nanodroplets to cross the endothelial cell layer leverages existing microbubble contrast agents, promotes development of novel nano agents, and increases the impact of ultrasound for detecting breast cancer by developing a novel, highly sensitive ultrasound method that leverages the discriminatory power of targeted molecular imaging in the vascular and extravascular space of breast lesions.