Thermally Triggered Multivalent Targeting of Tumors

描述（由申请人提供）：这项竞争性NIH R 01更新申请的总体目标是创建一种肿瘤靶向细胞穿透肽（CPP）纳米级药物递送系统，以将治疗有效载荷靶向广泛的实体瘤。该提议基于以下观察结果：寡精氨酸肽在其细胞渗透能力中显示出强阈值效应;在CPP中低于6个连续精氨酸（Arg）残基的阈值时，观察到很少的细胞摄取，而在Arg残基的该阈值数量以上时，存在显著的细胞摄取。我们假设这种阈值效应与寡聚精氨酸CPP中连续Arg残基的数量无关，而是反映了局部Arg残基密度。该假设预测，在其亲水端上呈现< 6个Arg残基的二嵌段共聚物的触发自组装成胶束应该提供表现出数字化的系统。 “关-开”细胞吸收。为了测试这一假设并开发外部触发的CPP药物递送系统，将合成弹性蛋白样多肽（ELPBC）的热响应性二嵌段共聚物，使得ELPBC亲水末端上的Arg残基的数目将低于在37 ℃下有效细胞摄取ELPBC单聚体所需的阈值。在外部加热至39-42 ℃（高于ELPBC的临界胶束化温度）的肿瘤中，ELPBC将自组装成修饰有Arg残基的胶束，ELPBC冠中的局部Arg残基密度将超过有效细胞摄取所需的阈值，从而导致肿瘤内有效的细胞内摄取。将合成具有一系列结构变量的Arg呈递ELPBC，测试其在血清中39至42 ℃之间的自组装和稳定性，并通过流式细胞术定量其温度触发的细胞摄取，并通过荧光显微镜观察。然后，从这些研究中选择的最佳Arg呈递ELPBC将被测试其药代动力学、组织分布及其使肿瘤消退的能力， 它们与螯合在ELPBC胶束核心内的吉西他滨融合。该提议的重要性在于，它将提供一种主动靶向肿瘤的受体非依赖性方法，该方法适用于广泛的癌症类型并规避受体靶向的限制;此外，该靶向方式搭载于现有的热疗技术，使得其可以容易地部署在临床中。这里提出的药物载体设计是创新的，因为据我们所知，它是第一次尝试控制细胞 通过在临床相关条件下通过外部触发的自组装来操纵纳米级支架上的Arg残基的局部密度来摄取癌症治疗剂。

DESCRIPTION (provided by applicant): The overall goal of this competing NIH R01 renewal application is to create a tumor-targeted cell-penetrating peptide (CPP) nanoscale drug delivery system to target therapeutic payloads to a wide range of solid tumors. This proposal builds upon the observation that oligoarginine peptides display a strong threshold effect in their cell penetration ability; below a threshold of 6 consecutive arginine (Arg) residues in the CPP, little cell uptake is observed, while above this threshold number of Arg residues, there is significant cell uptake. We hypothesized that this threshold effect is not related to the number of sequential Arg residues in the oligoarginine CPP, but instead is reflective of the local Arg residue density. This hypothesis predicts that the triggered self-assembly of a diblock copolymer, that presents < 6 Arg residues on its hydrophilic end, into a micelle should provide a system that exhibits digital "off-on" cell uptake. To test this hypothesis and develop an externally triggered CPP drug delivery system, thermally responsive diblock copolymers of an elastin-like polypeptide (ELPBCs) will be synthesized such that the number of Arg residues on the hydrophilic terminus of the ELPBC will be below the threshold required for efficient cellular uptake of ELPBC unimers at 37 ¿C. In tumors that are externally heated to 39-42 ¿C, which is greater than the critical micellization temperature of the ELPBC, the ELPBC will self-assemble into micelles decorated with Arg residues, and the local Arg residue density in the corona of the ELPBC will exceed the threshold required for efficient cellular uptake, thereby resulting in efficient intracellular uptae within the tumor. Arg-presenting ELPBC with a range of architectural variables will be synthesized, tested for self-assembly and stability between 39 and 42 ¿C in serum, and their temperature-triggered cellular uptake will be quantified by flow cytometry and visualized by fluorescence microscopy. Optimal Arg-presenting ELPBC selected from these studies will then be tested for their pharmacokinetics, tissue distribution and their ability to regress tumors, when they are fused to gemcitabine that is sequestered within the core of the ELPBC micelles. The significance of this proposal is that it will provide a receptor independent method of actively targeting tumors that is applicable to a broad range of cancer types and circumvents the limitations of receptor targeting; furthermore this targeting modality piggybacks on to existing hyperthermia technology, so that it can be readily deployed in the clinic. The drug carrier design presented here is innovative because it is, to our knowledge, the first attempt to control cellular uptake of cancer therapeutics by manipulating the local density of Arg residues on a nanoscale scaffold by externally triggered self-assembly under clinically relevant conditions.