In Vivo Cellular and Molecular Imaging Centers (ICMICs)

描述（由申请人提供）：检测酶活性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，从而选择适当的分子治疗剂，并监测治疗反应。超声是最通用和最常用的成像方式，具有高空间和时间分辨率以及对微泡的高灵敏度，但仅限于检测血管内目标。该项目旨在使超声波检测酶的能力。我们已经成功地生产了能够离开血管空间的颗粒小于100 nm的乳剂和纳米气泡。我们建议使用两个群体的颗粒，其中每个携带一对粘附分子对，如生物素-抗生物素蛋白，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，产生被困在肿瘤中的颗粒聚集体。由于超声背向散射与散射体半径的6次方相关，因此信号显著增加，从而允许聚集检测。由于已知的过敏效应，为了避免使用生物素-抗生物素蛋白，我们选择了互补DNA链作为粘附对，因为它们在相互接触时迅速形成紧密的双螺旋。为了防止血浆降解和阻断相互作用，每个DNA链都用可以被感兴趣的酶切割的肽环化。为了实现这一目标，我们提出了5个目标，这些目标的复杂性越来越大。我们将首先使用液体碳氟化合物乳液，然后进行更脆弱的纳米气泡：1）我们将首先使用通过二硫键环化的DNA链在体外证明，所述二硫键被还原剂TCEP打开，当链打开时发生聚集并且超声信号增加。2)然后，我们将用凝血酶可裂解的肽接头环化DNA，并如目的I中所述，证明在凝血酶存在下体外信号增加，然后使用提供血管内靶点的急性血栓在体内信号增加。3)我们将重复目标I，但肽接头将被基质金属蛋白酶（MMP）可裂解的肽取代。然后，我们将使用具有MMP+或MMP-肿瘤的小鼠证明体内信号增加。4)然后，在纳米气泡经过优化以最大化其非聚集状态和聚集状态之间的信号差异后，我们将使用纳米气泡重复目标1-3。5)对于目的1至4，为了方便，使用生物素/抗生物素蛋白接头将DNA链连接至颗粒。与目标1-4平行，我们将努力将DNA链直接连接到外壳上，以增加将药剂翻译到临床的可能性。

DESCRIPTION (provided by applicant): 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 DNA 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 DNA 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 in-vitro 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 I, 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 I, 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 non-aggregated 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.