In Vivo Cellular and Molecular Imaging Centers (ICMICs)

描述（由申请人提供）：酶活性检测是分子成像的主要目标之一，有助于评估肿瘤侵袭性，选择合适的分子治疗方法，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，但仅限于血管内目标的检测。这个项目旨在使超声波具有检测酶的能力。我们已经成功地制造出颗粒小于100纳米的乳剂和纳米气泡，它们能够离开血管空间。我们建议使用两种粒子，其中每个粒子携带粘附分子对中的一对，例如生物素-亲和素，其相互作用的能力被阻断，直到暴露于感兴趣的酶。这种酶激活了这种相互作用，产生了被困在肿瘤中的颗粒聚集体。由于超声后向散射与散射体半径提高到6次方有关，因此信号会急剧增加，从而允许聚合检测。为了避免因已知的过敏效应而使用生物亲和素，我们选择了互补DNA链作为粘附对，因为它们在相互暴露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，每条DNA链都被一个肽环化，这个肽可以被目标酶切割。为了实现这一目标，我们提出了5个目标，这些目标越来越复杂。我们将首先使用液态氟碳乳液，然后进行更脆弱的纳米气泡：1)我们将首先在体外证明用二硫键环化的DNA链被TCEP（一种还原剂）打开，当链打开时发生聚集，超声波信号增加。2)然后，我们将用可被凝血酶切割的肽连接物环化DNA，并像在Aim I中一样，证明在体外凝血酶存在时信号增加，然后在体内使用提供血管内靶点的急性血栓。3)我们将重复Aim I，但肽连接器将被可被基质金属蛋白酶（MMP）切割的肽所取代。然后，我们将在体内使用具有MMP+或MMP-肿瘤的小鼠来证明信号增加。4)在纳米气泡被优化后，我们将使用纳米气泡重复目标1-3，以最大化其非聚集状态和聚集状态之间的信号差异。5)为了方便起见，目的1至目的4使用生物素/亲和素连接物将DNA链连接到颗粒上。在目标1-4的同时，我们将努力将DNA链直接附着在外壳上，以增加将药物转化为临床药物的可能性。

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.