Mildly acidic tumor microenvironment for diagnostic and therapeutic nanomedicine

描述（由申请人提供）：在这项拟议的研究中，纳米结构将使用重组技术进行工程设计，以包含两种不同类型的细胞穿透肽（CPPs），即两亲肽，即模型两亲肽（MAP, klalklalkkaalkla）或来自HIV Tat蛋白的阳离子肽（YGRKKRRQRRR）。在我们的实验室中，这两种CPPs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。一种高度pH敏感的组氨酸-谷氨酸（HE）共聚物序列将与CPPs连接，以防止CPPs在非靶细胞中的非特异性内化，并在酸性（pH 6 - 7）肿瘤微环境表面靶向内化。根据我们的假设，CPP在肿瘤细胞的酸性表面附近积累后，其膜活性会再生，导致随后在肿瘤部位特异性地高积累。该提案将集中在两个关键的特定目标上，以评估附着在两种不同货物蛋白上的HE-CPP纳米结构，谷胱甘肽s -转移酶（32 kDa）和核糖核酸酶A (14 kDa), (i)纳米结构在ph敏感结合、内化和穿透多层细胞培养中的重组生产和体外表征，以及（ii）肿瘤靶向和穿透的体内表征。将比较两种类型的CPPs，即HE-MAP和HE-TAT，以确定哪种性质（两致病性或胍电荷）会导致更好的ph敏感激活、生物分布和肿瘤穿透。影响和意义。该提议中描述的纳米结构具有突破性技术的潜力，可以应用于几个不同的领域，包括酸性物质的开发

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.