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

摘要 脂壳全氟碳(PFC)微泡的直径从0.5微米到8微米不等 FDA批准增强血液对比度，用于诊断超声成像。中国的微观尺度 超声造影剂将它们限制在血液中，消除了增强的渗透性和滞留。 (EPR)在许多肿瘤中观察到。从现有的脂壳微泡中产生纳米颗粒的能力 造影剂让我们利用这些试剂的无毒配方，并让我们创建新的靶向 使用任何既定的策略形成靶向微泡的纳米颗粒。微泡 靶向分泌Frizzled2相关蛋白2(SFRP2)在肿瘤和肿瘤之间有明显的区别 血管系统正常，SFRP2在多种乳腺癌中高表达。我们和我们的同事 发现寒冷和压力增加会导致常规气相超声造影剂凝结成 液体PFC的脂壳纳米液滴。有了足够的能量，这些非回声物质就会恢复到 常规超声造影剂。纳米液滴具有纳米颗粒固有的优点 癌症治疗策略：通过EPR效应增强肿瘤中的被动蓄积，有针对性 通过靶向部分帮助肿瘤内的蓄积，并减少全身剂量。激活纳米液滴 与超声波提供了高度的空间选择性，防止微小气泡再次进入 血管系统，因此保持较高的局部造影剂浓度。分子成像 询问肿瘤内的间隙扩大了成像的物理范围，其回报是更高的 整个肿瘤体积的结合对比度密度，以及靶向肿瘤分子的更多机会 在血管内皮细胞中没有发现。我们建议用SFRP2-进行血管外分子成像。 与微泡相比，靶向纳米液滴将提高检测乳腺癌的敏感性 分子成像，同时保持减少误诊所需的分子特异性。一位批评者 利用分子靶向、脂壳全氟碳纳米液滴的障碍是根本缺乏 了解它们在正常组织和肿瘤组织中的生物分布。我们建议将现有的、已建立的 制备携带生物素和DNA的SFRP2靶向和非靶向纳米液滴制剂的技术 标记，测定它们的循环半衰期，并用免疫组织化学方法检测生物素或定量 聚合酶链式反应分析我们独特的DNA有效载荷，以表征这些液体的生物分布 正常组织和肿瘤组织中的全氟碳滴。确定靶向纳米液滴的交叉能力 内皮细胞层利用现有微泡造影剂，促进新型纳米材料的开发 试剂，并通过开发一种新的、高度有效的方法来增加超声波对检测乳腺癌的影响 灵敏的超声方法，利用靶向分子成像的分辨能力 乳腺病变的血管和血管外间隙。

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.