Mildly acidic tumor microenvironment for diagnostic and therapeutic nanomedicine

描述(申请人提供)：在这项拟议的研究中，将使用重组技术设计纳米结构，以包含两种不同类型的细胞穿透肽(CPP)，即两亲性多肽，即模型两亲性多肽(MAP，KLALKLALKALKAALKLA)，或源自HIV Tat蛋白的阳离子多肽(YGRKKRRQRRR)。这两种CPP先前在我们的实验室中已经被证明通过两种不同的途径在哺乳动物细胞中具有高度的非特异性积累。一个对pH高度敏感的组氨酸-谷氨酸(HE)共聚物序列将与CPP相连，以防止该结构在非靶细胞中的非特异性内化，并在酸性(PH值6-7)的肿瘤微环境的表面进行靶向内化。根据我们的假设，在肿瘤细胞酸性表面积聚后，CPP的膜活性将被再生，导致随后在肿瘤部位的高积聚。这项建议将集中于两个关键的特定目标，以评估附着在两种不同的货物蛋白，谷胱甘肽S转移酶(32 KDa)和核糖核酸酶A(14 KDa)上的HE-CPP纳米结构，(I)在pH敏感的结合、内化和跨多层细胞培养中纳米结构的重组生产和体外表征，以及(Ii)肿瘤靶向性和渗透性的体内表征。将比较两种类型的CPP，即HE-MAP和HE-TAT，以确定哪种性质、两亲性或胍电荷将导致更好的pH敏感激活、生物分布和肿瘤穿透。影响和意义。这项提案中描述的纳米结构具有作为突破性技术的潜力，并可用于几个不同的领域，包括开发酸性 肿瘤微环境在肿瘤诊断和靶向方面的应用，以及CPPS在作为抗癌治疗药物的大分子药物输送中的应用。根据MAP和/或TAT-纳米结构是否在肿瘤部位显示出足够的靶向性和内在化，这些结构将作为大分子药物的载体非常有用，用于靶细胞的胞浆和/或核室。这些纳米结构也很容易与其他分子结合，包括聚乙二醇化以改善药代动力学/生物分布特性，靶向部分以进一步增强肿瘤特异性，以及抗癌治疗蛋白或多肽域。因此，这些纳米结构不仅有可能为肿瘤靶向药物提供一种新的方法，而且还可能导致生物技术衍生药物(即蛋白质和多肽)作为抗癌治疗药物领域的进步。

DESCRIPTION (provided by applicant): In this proposed study, nanoconstructs will be engineered using recombinant technology to contain two different types of cell penetrating peptides (CPPs), i.e. amphipathic peptide, i.e., Model Amphipathic Peptide (MAP, KLALKLALKALKAALKLA), or a cationic peptide derived from HIV Tat protein (YGRKKRRQRRR). These two CPPs have previously been shown in our laboratory to have high non-specific accumulation in mammalian cells via two distinct pathways. A highly pH-sensitive histidine-glutamic acid (HE) copolymer sequence will be linked to the CPPs to prevent non-specific internalization of the construct in non-target cells, and to target internalization atthe surface of the acidic (pH 6 - 7) tumor microenvironment. According to our hypothesis, following accumulation near the acidic surface of tumor cells, the membrane activity of the CPP will be regenerated, leading to subsequent high accumulation specifically at the tumor site. This proposal will focus on two critical specific aims to evaluate HE-CPP nanoconstructs attached to two different cargo proteins, glutathione S-transferase (32 kDa) and ribonuclease A (14 kDa), (i) recombinant production and in vitro characterization of nanoconstructs in pH-sensitive binding, internalization, and penetration across multilayered cell cultures, and (ii) in vivo characterizatin of the tumor targeting and penetration. The two types of CPPs, i.e. HE-MAP and HE-TAT, will be compared in order to determine which property, amphipathicity or guanidinium charge, will lead to better pH-sensitive activation, biodistribution, and tumor penetration. Impact and Significance. The nanoconstructs described in this proposal have potential as a breakthrough technology, and can be utilized in several different areas including the exploitation of the acidic tumor microenvironment in diagnosis and targeting tumors, and in the application of CPPs in delivery of macromolecular drugs as anticancer therapeutics. Depending on whether the MAP and/or TAT-nanoconstruct displays sufficient targeting and internalization at the tumor site, these constructs will be immensely useful as carriers of macromolecular drugs to cytosolic and/or nuclear compartments of target cells. These nanoconstructs are also easily amenable to attachment of other molecules, including PEGylation to improve pharmacokinetic/biodistribution properties, targeting moieties to further enhance tumor specificity, and anti-cancer therapeutic protein or peptide domains. Therefore, the nanoconstructs have the potential to not only provide a new method in tumor targeting of drugs, but could also lead to advancement of the field of biotechnology-derived drugs (i.e. proteins and peptides) as anti-cancer therapeutics.