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

摘要 直径范围为0.5至8微米的脂质壳全氟化碳（PFC）微泡已被用于制备。 经FDA批准，用于增强血液的对比度，以进行超声诊断成像。微观尺度 超声造影剂将它们限制在血流中，消除了增强的渗透性和滞留性 (EPR)在许多肿瘤中观察到。从现有的脂质壳微泡中产生纳米颗粒的能力 造影剂使我们能够利用这些药物的无毒配方， 使用任何已建立的用于配制靶向微泡的策略来制备纳米颗粒。微泡 靶向分泌型卷曲相关蛋白2（SFRP 2）的细胞在肿瘤和 SFRP 2在正常血管系统中高度表达，并且SFRP 2在多种乳腺癌中高度表达。我们和我们的同事 发现寒冷和压力增加会导致传统的气相超声造影剂凝结成 有足够的能量，这些非回声剂恢复到 常规超声造影剂。纳米液滴具有纳米颗粒固有的优点 癌症治疗的策略：通过EPR效应增强肿瘤中的被动积累，靶向 通过靶向部分辅助的肿瘤内的累积，以及减少的全身给药。激活纳米液滴 超声提供了高度的空间选择性，防止微尺度气泡重新进入 从而保持造影剂的高局部浓度。分子成像， 询问肿瘤内的间隙空间，扩大了成像的物理范围， 整个肿瘤体积中结合造影剂的密度，以及靶向肿瘤分子的额外机会 血管内皮细胞中没有我们认为，血管外分子成像与SFRP 2- 与微泡相比，靶向纳米液滴将增加检测乳腺癌的灵敏度 分子成像，同时保持减少错误诊断所需的分子特异性。一个关键 利用分子靶向的脂质壳全氟化碳纳米液滴的障碍是根本缺乏 了解它们在正常和肿瘤组织中的生物分布。我们建议将现有的、已建立的联合收割机 产生携带生物素和DNA的SFRP 2靶向和非靶向纳米液滴制剂的技术 标记，测定其循环半衰期，并使用生物素免疫组织化学检测或定量 我们独特的DNA有效载荷的聚合酶链反应测定，以表征这些液体的生物分布 在正常和肿瘤组织中的全氟化碳液滴。建立靶向纳米液滴交叉的能力 内皮细胞层利用现有的微泡造影剂， 药物，并通过开发一种新的，高度敏感的 灵敏的超声方法，该方法利用靶向分子成像的鉴别能力， 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.