pHLIP Nanotechnology Platform for Cancer Imaging and Therapy

描述（由申请人提供）：我们的项目是基于使用一种水溶性膜肽phillip，我们已经通过全身荧光和PET成像证明，它可以选择性地靶向体内的酸性实体肿瘤，并将极性货物分子转移到培养的癌细胞的细胞质中。在轻度酸性条件下，phillip作为单体在细胞膜的脂质双分子层上单向插入，就像在肿瘤中发现的那样，并形成跨膜α螺旋，而在正常细胞外pH值的健康组织中，几乎不存在跨膜插入。迄今为止，在培养细胞或小鼠中均未观察到phillip暴露的毒性作用。在这里，我们建议开发一个纳米技术平台，用于选择性地向肿瘤输送成像和治疗药物，该平台基于philips -生物纳米注射器的使用。通过将装载分子附着在膜外的phillip末端，我们可以将成像或治疗探针固定在癌细胞表面，从而促进诊断、治疗和治疗监测。通过将货物通过可切割的链接连接到其插入的末端，phillip可以用于选择性地将极性，细胞不渗透的分子转移到癌细胞中。通过三个实验室的共同努力，这项有前途的技术将有可能得到广泛的发展。我们将使用pHLIP靶向来测试癌症模型，并确定肿瘤生长和发展如何与肿瘤酸度相关。为了改进pHLIP技术，我们将设计、合成和测试各种树突-pHLIP结构，以实现多种治疗和/或成像探针的肿瘤递送。我们将介绍一种通过可切割的S-S键将货物分子和荧光染料同时偶联到pHLIP的c端的合成方案，并建立pHLIP可以通过膜的脂质双分子层转运的货物分子的性质（极性，形状，电荷和大小），定义一种新的极性治疗分子，可以用于肿瘤治疗。我们将在体内测试pHLIP对两种功能性细胞不渗透分子的细胞内递送：一种毒素（phalloidin）和一种基因调节剂（Peptide核酸）。重要的是，我们将尝试通过标记的phillip -phalloidin同时检测和治疗肿瘤，这是我们第一个潜在的抗转移药物。此外，我们将开发一种两步递送方案，用于在体内癌细胞表面特异性地捆绑和组装纳米颗粒：1)使用带有结合域的pHLIP靶向肿瘤，该结合域将被捆绑在癌细胞表面；2)使用含有治疗和/或成像有效载荷的脂质体靶向pHLIP，并具有表面暴露的互补结合域。受当前版本的pHLIP特性的启发，我们将进一步评估pHLIP序列变化对肽插入膜的影响，从而设计出具有一系列有用特性的第二代纳米注射器。philips纳米技术为癌症的特定疾病成像和治疗提供了一种新的方法。我们的最终目标是改善癌症的诊断和治疗，在美国和其他发达国家，癌症占所有死亡人数的25%。在这个问题的几个方面，我们的技术发展可能是有用的，但主要的概念是选择性地将治疗和显像剂输送到肿瘤细胞中。该技术的另一个方面是，它允许使用一类新的治疗剂：不渗透细胞的分子，这种分子只会转移到病变组织的细胞中，而不会影响健康细胞。基于这些概念的治疗将表现出更高的疗效和/或显著减少副作用。这种改进对癌症治疗尤其重要，因为大多数抗癌药物都是破坏正常细胞的毒药。

DESCRIPTION (provided by applicant): Our project is based on the use of a water-soluble membrane peptide, pHLIP, which we have shown, by whole-body fluorescence and PET imaging, to selectively target acidic solid tumors in vivo and to translocate polar cargo molecules into the cytoplasms of cultured cancer cells. pHLIP inserts unidirectionally across the lipid bilayer of a cell membrane as a monomer under mildly acidic conditions, as are found in tumors and forms a transmembrane alpha helix, whereas there is practically no insertion across the membranes of cells with the normal extracellular pH of healthy tissue. To date, no toxic effects of pHLIP exposure have been observed either for cells in culture or for mice. Here we propose to develop a nanotechnology platform for selective delivery of imaging and therapeutic agents to tumors based on the use of the pHLIP-bionanosyringe. By attaching cargo molecules to the end of pHLIP that stays outside of the membrane, we can anchor imaging or therapeutic probes to the surfaces of cancer cells, facilitating diagnosis, treatment and therapeutic monitoring. By attaching cargo to its inserted end via cleavable links, pHLIP can be used for the selective translocation of polar, cell-impermeable molecules into cancer cells. By combining the efforts of three laboratories, a broad development of this promising technology will be possible. We will use pHLIP targeting to test cancer models and establish how tumor growth and development correlate with tumor acidity. To improve pHLIP technology, we will design, synthesize and test various dendrimeric-pHLIP constructs to enable delivery of multiple therapeutic and/or imaging probes to tumors. We will introduce a synthetic scheme of simultaneous conjugation of cargo molecules and fluorescent dyes to the C-terminus of pHLIP via a cleavable S-S bond and establish the properties (polarity, shape, charge and size) of cargo molecules that pHLIP can translocate through the lipid bilayer of a membrane, defining a new, polar class of therapeutic molecules that can be delivered for tumor treatment. We will test pHLIP for the intracellular delivery of two functional cell-impermeable molecules in vivo: a toxin (phalloidin) and a gene regulation agent (Peptide Nucleic Acid). Importantly, we will attempt the simultaneous detection and treatment of tumors by labeled pHLIP-phalloidin, which is our first lead for a potential antimetastatic drug. Further, we will develop a two-step delivery scheme for the specific tethering and assembly of nanoparticles at the surfaces of cancer cells in vivo: 1) targeting tumors using pHLIP with a binding domain, which will be tethered to the surface of cancer cells and 2) targeting the pHLIP with liposomes containing therapeutic and/or imaging payloads and having a surface-exposed complementary binding domain. Inspired by the properties of pHLIP in its current version, we will further evaluate the effect of pHLIP sequence variation on peptide insertion into a membrane, enabling the design of a second generation of the nanosyringe with a range of useful properties. pHLIP nanotechnology offers a new approach for the disease-specific imaging and treatment of cancers. Our ultimate goal is to improve the diagnosis and treatment of cancer, which is responsible for about 25% of all deaths in the USA and other developed countries. There are several aspects of the problem where our technology development could be useful, but the major concept is the selective delivery of therapeutic and imaging agents to cells in tumors. Another aspect of the technology is that it permits the use of a new class of therapeutic agents: cell-impermeable molecules that would be translocated into cells only in diseased tissue while not affecting healthy cells. A therapy based on these concepts would exhibit much higher efficacy and/or significantly reduced side effects. Such improvements are especially important for cancer treatment, since the majority of anti-cancer drugs are poisons that damage normal cells.