OPENING THE BLOOD BRAIN BARRIER FOR MOLECULAR IMAGING

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。由于血脑屏障（BBB）保护脑组织免受外来分子的侵害，大分子药物通过血液供应进入中枢神经系统（CMS）通常是不可能的。决定物质从血液渗透到中枢神经系统的因素是脂溶性、分子大小和电荷。血脑屏障阻止分子量大于180的离子化水溶性材料的渗透。因此，大多数潜在的分子显像剂不能通过血液供应到达脑组织。一项允许这些药物到达脑组织的技术将为目前无法进行的脑部疾病的诊断和监测打开新的可能性之门。这种技术还将产生一种新的研究工具，利用分子成像技术来研究动物模型中的脑功能和疾病。为了优化超声诱导血脑屏障破坏的过程，我们将首先研究不同的声学参数，以确定血脑屏障破坏的最佳值。在兔实验中，我们将改变超声频率、爆发长度、重复频率和超声持续时间，并使用MRI造影剂或必要时使用其他示踪剂来测量血脑屏障的破坏。此外，我们将研究不同的市售超声造影剂。目标将是确定哪些参数导致最大的血脑屏障破坏，而不会对大脑造成不必要的损害。在这项工作结束时，我们预计我们将有将用于临床的参数。我们还将描述我们可以使用荧光微球（来自供应商，如卡尔斯巴德的Invitrogen， CA）将什么大小的药物输送到大脑。这些球体可以在不同的直径（以及激发和发射波长）购买，并用荧光显微镜成像和定量。可以同时注入不同波长的激发或荧光球，从而提供不同大小药剂分布程度的定量图像。我们将在超声后立即注射微球，并在稍后的时间内观察血脑屏障重新封闭的时间过程。这些时间将从第一个目标的实验中确定：我们将在血脑屏障大约关闭25%和50%的时候注入球体。从这些测量中，我们可以评估试剂的分子大小是否取决于超声后的时间。我们还将能够确定血脑屏障的关闭是否取决于示踪剂的大小，或者是否同时关闭所有代理商。最后，我们将继续研究利用声发射信号监测过程的方法。在我们的初步工作中，我们发现超声频率的谐波在超声过程中急剧增加，导致血脑屏障中断。在此基础上，我们将开发一个自动化系统，利用发射信号实时控制超声爆发，我们希望能够在线确定正确的超声强度，以最大限度地破坏血脑屏障，而不会引起惯性空化。有这样一种方法来指导手术将是很重要的，因为当聚焦到活体组织深处时很难确定声强度，尤其是当超声通过完整的头骨传递时。在这项工作中，我们将继续努力通过超声诱导血脑屏障破坏将治疗药物输送到动物大脑。在我们的初步工作中，我们已经证明，我们可以将临床相关剂量的化疗药物（脂质体阿霉素）输送到正常大鼠的大脑中，我们已经证明，我们可以将抗体（多巴胺D4受体靶向抗体）输送到小鼠的大脑中。我们将测试Doxil¿对接种于大鼠脑胶质瘤的输送。我们将通过一系列MRI研究比较这些肿瘤的生长情况，包括有血脑屏障破坏和没有血脑屏障破坏肿瘤周围组织。我们还将使用荧光测定法（激发：480 nm；发射：590 nm）量化阿霉素输送到大脑的量。此外，我们将研究Herceptin¿的递送，Herceptin¿是一种人源化抗人表皮生长因子受体2 （HER2 / c-erbB2）单克隆抗体，临床用于治疗乳腺癌患者，并在控制局部和远端乳腺癌病变方面取得了巨大成功。然而，当这些癌症转移到大脑时，由于血脑屏障的存在，这种药物的有效性受到限制。首先，我们将在老鼠的实验中证明我们可以将这种抗体输送到正常的大脑中。接下来，我们将在裸鼠大脑中接种乳腺癌肿瘤，以测试该药物联合血脑屏障破坏时的有效性。在实验中，换能器将连接到我们的mri兼容定位装置上，并将其浸入除气去离子水的水箱中。我们目前有用于临床1.5 t和3T临床扫描仪的系统。在接下来的一年里，我们也将为我们的4.7T动物磁铁构建一个系统。被麻醉的动物将被放在一个塑料托盘上。水箱和动物头部之间的声学耦合将通过装满水的塑料袋来实现。在实验之前，光束路径上的毛发将被去除。在将动物放置在系统上之前，通过对组织模拟幻象中超声产生的加热成像，将焦点位置定位在MRI坐标空间中。基于这种配准，我们可以在解剖MR图像的指导下精确定位大脑中的病灶，精度约为0.5 mm。超声后使用标准MRI造影剂（如Magnevist¿，Berlex Inc., Wayne NJ）可立即确认血脑屏障开口。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The delivery of large molecular agents into the central nervous system (CMS) via the blood supply is often impossible because the blood brain barrier (BBB) protects the brain tissue from foreign molecules. The factors that determine penetration of substances from the blood to the CNS are lipid solubility, molecular size, and charge. The BBB prevents penetration of ionized water-soluble materials with molecular weight greater than 180. Thus most of the potential molecular imaging agents cannot reach the brain tissue via the blood supply. A technique that allows these agents to reach the brain tissue will open the door to new possibilities for the diagnosis and monitoring of brain disorders that currently cannot be performed. Such a technique would also result in a new research tool to investigate brain function and disorders in animal models using molecular imaging. Optimization of ultrasound-induced BBB disruption To optimize the procedure, we will first investigate different acoustic parameters to determine the best values for BBB disruption. In experiments in rabbits, we will vary the ultrasound frequency, burst length, repetition frequency, and sonication duration, and gauge the BBB disruption using MRI contrast agents, or as needed, other tracers. Further, we will investigate different commercially-available ultrasound contrast agents. The goal will be to determine which parameters result in the largest BBB disruption without causing unwanted damage to the brain. At the end of this work, we anticipate that we will have the parameters that will be used clinically. We will also characterize what sized agents we can deliver to the brain using fluorescent microspheres (from a vendor such as Invitrogen, Carlsbad, CA). These spheres can be purchased at different diameters (and excitation and emission wavelengths) and imaged and quantified with fluorescent microscopy. Spheres that are excited or fluoresce at different wavelengths can be injected at the same time, thereby providing quantitative images of the extent of the distribution of different size agents. We will inject the microspheres immediately after sonication and at later times to investigate the time course of the resealing of the BBB. These times will be determined from the experiments in the first aim: we will inject the spheres at the times when the BBB is roughly 25% and 50% closed. From those measurements we can evaluate whether the molecular size of the agent that can be delivered depends on the time after sonication. We will also be able to determine whether the closing of the BBB depends on the size of the tracer or if it closes to all agents at the same time. Finally, we will continue our work investigating methods to monitor the procedure using acoustic emission signals. In our preliminary work, we found that a sharp increase in harmonics of the ultrasound frequency occurred during sonications that resulted in BBB disruption. Based on this work, we will develop an automated system that uses the emission signals in real time to control the ultrasound bursts and we hope to be able to determine online the correct ultrasound intensities to use to maximize the BBB disruption without inducing inertial cavitation. Having such a method to guide the procedure will be important because it is difficult to determine the acoustic intensity when focusing deep into living tissue ¿ especially when the sonications are delivered through the intact skull. Delivery of therapeutics in animal models In this work, we will continue our efforts in delivering therapeutics to the animal brain through the ultrasound-induced BBB disruption. In our preliminary work, we have demonstrated that we can deliver clinically relevant dosages of a chemotherapy agent (liposomal doxorubicin ¿ Doxil¿) to the normal rat brain, and we have demonstrated that we can deliver antibodies (dopamine D4 receptor-targeting antibodies) into the brains of mice. We will test the delivery of Doxil¿ into gliomas inoculated in rat brain. We will compare the growth of these tumors for cases with and without BBB disruption of the tissue surrounding the tumors through serial MRI studies. We will also quantify the amount of doxorubicin delivered to the brain using fluorometry (excitation: 480 nm; emission: 590 nm). In addition, we will investigate the delivery of Herceptin¿, a humanized anti-human epidermal growth-factor receptor 2 (HER2 / c-erbB2) monoclonal antibody that is used clinically used to treat breast cancer patients and has shown great success in controlling local and distal breast cancer lesions. When these cancers metastasize to the brain, however, this effectiveness of this agent has been limited because of the BBB. First, we will demonstrate that we can deliver this antibody into the normal brain in experiments in mice. Next, we will inoculate breast cancer tumors in the brains of nude rats to test the effectiveness of this agent when its delivery is combined with BBB disruption. Methods For the experiments, the transducer will be attached to our MRI-compatible positioning device and submerged in a tank of degassed, deionized water. We currently have systems available for our clinical 1.5 and 3T clinical scanners. In the upcoming year, we will also construct a system for our 4.7T animal magnet as well. The anesthetized animal will be placed on its back on a plastic a tray. Acoustic coupling between the water tank and the animal¿s head will be achieved with plastic bag filled with water. The hair in the beam path will be removed prior to the experiments. Before the animal is placed on the system, the focal position will be located in the MRI coordinate space by imaging the heating produced by sonications in a tissue-mimicking phantom. Based on this registration, we can accurately target the focus in the brain with an accuracy of ~0.5 mm using anatomical MR images as a guide. BBB opening can be confirmed immediately after sonication using standard MRI contrast agents (such as Magnevist¿, Berlex Inc., Wayne NJ).