Enzyme Detection with Ultrasound Contrast Agents

酶活性的检测是分子成像的主要目标之一，以帮助评估 肿瘤侵袭性、肿瘤表型选择合适的分子治疗方法，并进行监测 治疗反应。超声，最通用和最通用的成像方式，号称 高空间和时间分辨率以及对微气泡的高敏感性一直局限于 检测血管内目标。该项目旨在将检测酶的能力带入超声波。我们 已经成功地生产出乳剂和纳米气泡，其颗粒小于100 nm，能够 从血管间隙出来。我们建议使用两组粒子，每组粒子携带一种 黏附分子对，如生物素-亲和素，其相互作用的能力被阻止，直到暴露于 感兴趣的酶。这种酶激活相互作用，产生一种颗粒聚集体， 会被困在肿瘤里。因为超声后向散射与散射体的半径增加到 6次方，信号显著增加，允许聚合检测。避免使用生物亲和素 由于已知的过敏效应，我们选择了赠送的DMA链作为粘连对 因为当它们相互接触时，会迅速形成紧密的双螺旋。防止血浆降解 为了阻止相互作用，每条DMA链都被环化了一种可以被以下酶切割的肽 利息。为了实现这一点，我们提出了5个目标，这些目标的复杂性越来越高。我们将首先使用 液体氟碳乳剂，然后进入更脆弱的纳米气泡：1)我们将首先证明体外 使用由二硫键环化的DNA链，该二硫键由还原剂TCEP打开，当 链发生开放聚集，超声信号增强。2)然后我们将用一个 可被凝血酶切割的多肽连接物，如在目标1中一样，证明在体外信号在存在的情况下增加 凝血酶，然后在体内使用一种急性血栓，这提供了血管内的靶点。3)我们会重复 目的1，但多肽接头将被一种可被基质金属蛋白酶(MMPs)切割的多肽取代。我们 然后将证明信号增加在体内使用的小鼠的基质金属蛋白酶+或基质金属蛋白酶-肿瘤。4)然后我们将重复目标 1-3在对纳米气泡进行优化后使用纳米气泡，以最大化其非聚集的信号差异 和聚合态。5)对于AIMS 1至4，使用以下方法将DNA链连接到颗粒 生物素/亲和素连接物，方便使用。与AIMS 1-4并行，我们将致力于直接连接DNA链 以增加将代理人转移到诊所的可能性。

Detection of enzyme activity is one of the major goals of molecular imaging to aid in the assessment of tumor aggressiveness, tumor phenotyping to select the appropriate molecular therapeutic, and to monitor therapeutic response. Ultrasound, the most versatile and most commonly used imaging modality that boasts high spatial and temporal resolution as well as high sensitivity to microbubbles, has been limited to the detection of intravascular targets. This project aims to bring to ultrasound the ability to detect enzymes. We have successfully produced emulsions and nanobubbles with particles less than 100nm that are capable of exiting the vascular space. We propose to use two populations of particles where each carries one of the pairs of an adhesion molecule pair, such as biotin-avidin, whose ability to interact is blocked until exposed to the enzyme of interest. The enzyme activates the interaction to produce an aggregate of particles that becomes trapped in the tumor. Because ultrasound backscatter is related to the scatterer's radius raised to the 6th power, signal increases dramatically allowing aggregate detection. To avoid the use of biotion-avidin because of the known allergic effects, we selected complimentary DMA strands as the adhesion pair because they rapidly form a tight double helix when exposed to each other. To prevent plasma degradation and to block interaction, each DMA strand is cyclized with a peptide that can be cleaved by the enzyme of interest. To accomplish this we propose 5 aims that proceed with increasing complexity. We will first use liquid fluorocarbon emulsions and then proceed to the more fragile nanobubbles: 1) We will first prove invitro using DNA strands cyclized by a disulfide bond that are opened by TCEP, a reducing agent, that when the strands open aggregation occurs and ultrasound signal increases. 2) We will then cyclize the DNA with a peptide linker cleavable by thrombin and as in Aim 1, prove that signal increases in vitro in the presence of thrombin and then in vivo using an acute thrombus that provides an intravascular target. 3) We will repeat Aim 1, but the peptide linker will be replaced by a peptide cleavable by matrix metalloproteinase (MMP). We will then prove signal increase in vivo using mice with an MMP+ or MMP- tumor. 4) We will then repeat Aims 1-3 using nanobubbles after they have been optimized to maximize signal difference between their nonaggregated and aggregated state. 5) For aims 1 to 4 the DNA strands were attached to the particles using biotin/avidin linkers for convenience. In parallel with Aims 1-4 we will work to attach the DNA strands directly to the shell to increase the probability of translating the agent to the clinic.