Thermally Triggered Multivalent Targeting of Tumors

描述(由申请人提供)：这项竞争的NIH R01更新申请的总体目标是创建一种肿瘤靶向细胞穿透肽(CPP)纳米级药物输送系统，以靶向广泛的实体肿瘤的治疗有效载荷。这一建议建立在以下观察结果的基础上：低聚精氨酸肽在细胞穿透能力方面显示出很强的阈值效应；在CPP中连续6个精氨酸(Arg)残基的阈值以下，几乎没有观察到细胞摄取，而超过这个阈值数量，细胞摄取显著。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。这一假说预测，两嵌段共聚物的触发自组装，在其亲水的一端呈现-lt；6个精氨酸残基，形成胶束，应该提供一个显示数字的系统。 “开-关”的细胞摄取。为了检验这一假设和开发外部触发的CPP给药系统，将合成具有温度响应的弹性蛋白样多肽(ELPBCs)两嵌段共聚物，使ELPBC亲水末端的Arg残基数量低于37℃时细胞有效摄取ELPBC异构体所需的阈值。在外部加热到39-42℃，高于ELPBC的临界胶束温度时，ELPBC将自组装成装饰有Arg残基的胶束，ELPBC冠层中的局部Arg残基密度将超过细胞有效摄取所需的阈值，从而在肿瘤内产生有效的细胞内提升。将合成具有一系列结构变量的精氨酸递呈ELPBC，测试其在血清中39至42℃的自组装和稳定性，其温度触发的细胞摄取将通过流式细胞仪进行定量，并通过荧光显微镜可视化。从这些研究中挑选出最佳的Arg递呈ELPBC，然后将测试它们的药代动力学、组织分布和肿瘤消退的能力，当 它们与隔离在ELPBC胶束核心内的吉西他滨融合在一起。这项建议的意义在于，它将提供一种适用于广泛癌症类型的、不依赖于受体的主动靶向肿瘤的方法，并绕过了受体靶向的限制；此外，这种靶向模式依赖于现有的热疗技术，因此可以很容易地部署在临床上。这里介绍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 在临床相关条件下，通过外部触发自组装来操纵纳米支架上Arg残基的局部密度来摄取癌症治疗药物。 与公共健康相关：拟议的研究将开发一种可生物降解的温度敏感型聚合物，这种聚合物将在受到外部来源温和加热的肿瘤中形成纳米颗粒。这些纳米颗粒的外部将装饰有精氨酸残留物，并将含有一种抗癌药物。精氨酸将允许肿瘤细胞有效地摄取纳米颗粒，从而导致药物选择性和有效地治疗肿瘤，副作用很少。

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. PUBLIC HEALTH RELEVANCE: The proposed research will develop a biodegradable, temperature sensitive polymer that will morph into nanoparticles in tumors that are mildly heated by an external source. These nanoparticles will be decorated with arginine residues on their exterior and will contain a cancer drug. The arginine's will allow efficient uptake of the nanoparticles by cells in the tumor, thereby leading to selective and effective treatment of the tumor by the drug with few side-effects.