Application of Gold Nanoparticles to Increase the Efficacy of Radiation Therapy

背景与意义近二十年来，人们一直在探索高原子序数元素（如碘）对癌症放疗的辐射剂量增强作用。碘脱氧尿苷（IUdR）作为一种小分子辐射剂量增强剂（smRDE），由于其易于并入细胞DNA而被广泛使用。随后将高能光子外照射到含iudr的靶细胞上，可触发光电辐射二次发射（即俄歇/二次电子发射或x射线荧光）。由此产生的smRDE触发发射可切割核DNA双链，可诱导放射性致敏细胞死亡。在这种smrde介导的放射治疗中，已经证明了几个优点。smRDEs的触发辐射通常在数微米（这是细胞的典型长度尺度）内衰减，因此产生的细胞毒性高度依赖于smRDEs的细胞位置。因此，只有位于靶细胞内的smrde在辐射作用下才具有高毒性，而在靶细胞外则可降低其脱靶毒性。虽然这类smRDE可以提高放射治疗效果，减少副作用，但安全有效地将smRDE输送到靶组织仍然是临床应用的主要缺点之一。碘附着肿瘤靶向抗体用于改善其生物分布，但由于DNA的快速脱卤化机制以及靶受体在癌细胞上的异质性和表达有限，其靶向性和治疗效果不理想。此外，由于碘化合物对宿主器官有毒副作用，应避免长期使用高剂量碘化合物治疗。为了克服这些限制，我们提出了一种基于功能化聚合物修饰纳米粒子的纳米封装RDE（纳米RDE）平台的发展。NanoRDE平台可以显示出几个优势：首先，纳米orde的生物分布可以通过实体肿瘤组织中增强的渗透和保留（EPR）效应得到极大改善，这允许纳米orde在病变部位的选择性积累（被动靶向）。其次，大量的纳米orde可以很容易地通过内吞途径在靶细胞中内化，这是一种与小分子完全不同的细胞内化机制。此外，随后的酸性内体环境可以作为额外化疗药物的ph敏感释放的触发器。在常规化疗中，肿瘤细胞耐多药特性的迅速发展成为临床肿瘤治疗中的一个关键问题。因此，很明显，联合治疗比单一治疗更有利于癌症的完全缓解。为此，抗癌药物缀合的nanoRDE将在本项目中作为放疗和化疗多模式递送平台的第一个例子进行演示。金纳米颗粒已被测试用于改善化疗和放疗。tnfalpa - peg胶体金纳米颗粒CYT-6091 （cittimmune, Gaithersburg， MD）已被证明可以选择性地输送到肿瘤组织。这种药物在临床试验中被评估为有选择性地将TNFalpha输送到肿瘤的能力。金纳米颗粒的尺寸范围很广。胶体金作为一种高z元素，除了其作为纳米载体的特性外，还可能增加特异性递送到靶细胞的辐射剂量。在这个系统中，金纳米粒子（AuNPs）被用作放射治疗的无机纳米粒子，以及化疗药物的递送平台。由于金的k边在80.7 keV处，以Au k边能级的x射线辐射可以触发aunp的光电辐射二次发射。由此产生的辐射可通过电离诱导目标分子降解，并可与周围的水分子相互作用产生可破坏目标分子的活性氧。因此，在体外模型系统中已经证明了在x射线辐射下质粒DNA和人类蛋白质的aunp致敏降解。此外，当照射含有aunp的肿瘤细胞时，由于细胞质细胞器的持续应激，检测到凋亡细胞死亡增加。此外，在人类患癌小鼠模型中也观察到AuNPs对放射治疗的体内疗效增强。然而，这些aunp显示出非常差的药代动力学结果，部分原因是它们作为裸胶体颗粒的表面功能有限。近年来，AuNPs由于其生物相容性和众所周知的表面化学性质而被广泛应用于生物领域。利用已知的反应，可以很容易地用巯基端盖聚合物修饰AuNPs，这可以显著改变药代动力学。这种功能聚合物可以通过高度可控的可逆加成-破碎链转移（RAFT）自由基聚合制备，该聚合允许广泛的单体共聚。由于金k边在80.7 keV处，且在光电主导的x射线光谱中增强效果最佳，因此可以利用单能x射线优化辐照条件。最后，金可以很容易地被热中子激活，释放出411kev的伽马射线，这可以为SPECT监测其生物分布提供一种手段。假设：功能化金纳米颗粒可用于her2靶向递送和触发治疗剂的释放，包括放射增敏剂。her2靶向AuNP的开发a) Au-NP的生物分布及药代动力学表征b)中子活化Au-NP在体内生物分布的SPECT评价c) AuNP与外照射联合在体外和体内的效果测试2。her2靶向AuNP在肿瘤特异性递送治疗药物中的应用a)优化AuNP所携带分子的触发释放条件b)与现有制剂相比，AuNP递送药物的体外和体内疗效测试。成就:1。利用中子活化定量组织中金浓度的方法已经开发出来，并在2009年美国核学会和美国化学学会年会上提出。聚乙二醇化纳米颗粒的生产方法已经建立和测试。x射线触发的荧光分子从金纳米颗粒释放进行了测试，初步结果的海报展示在2009年美国化学学会年会的胶体和表面分部获得了最佳美国化学学会（ACS）海报。4. 我们的理论工作描述了纳米粒子作为基于光动力治疗的放射增敏剂的要求，表明金纳米粒子是其他人提出的纳米闪烁体的有利替代品。

Background and Significance Over the last two decades, radiation dose enhancement by high atomic number elements such as iodine has been explored for cancer radiotherapy. As a small-molecule radiation dose enhancer (smRDE), iododeoxyuridine (IUdR) has been used due to its facile incorporation into cellular DNA. The subsequent external irradiation of high energy photons on the IUdR-containing target cells can trigger the secondary emission of photoelectric radiation (i.e. Auger/secondary electron emission or X-ray fluorescence). The resulting triggered emission from smRDE can cleave the nuclear DNA double strands that can induce the radio-sensitized cell death. In this smRDE-mediated radiotherapy, several advantages have been demonstrated. The triggered emission from smRDEs generally decays in several um which is a typical length scale of a cell so that the resulting cytotoxicity is highly dependent on the cellular location of smRDEs. Therefore, only those of smRDEs located inside the target cells can deliver high toxicity upon radiation but their off-target toxicity can be reduced out of the cells. Although such smRDEs can improve the radiotherapeutic efficacy with reduced side effects, safe and effective delivery of smRDE to target tissue remains one of the major drawbacks to clinical applications. As iodine-attached tumor-targeting antibodies were used to improve their biodistribution, their targeting and therapeutic efficacies were not satisfied due to the rapid dehalogenation mechanism in DNA as well as the heterogeneity and limited expression of target receptors on cancer cells. Furthermore, the prolonged treatments with high dosages of iodine compounds should be avoided due to their toxic side effects to the host organs. To overcome these limitations, we propose the development of a nano-encased RDE (nano-RDE) platform, based on functionalizable polymer-modified nanoparticles. NanoRDE platforms can demonstrate several advantages: First, the biodistribution of nanoRDE can be highly improved by the enhanced permeation and retention (EPR) effect in solid tumor tissue, that allows for the selective accumulation of nanoRDE at diseased sites (passive targeting). Second, high amount of nanoRDE can be readily internalized in target cells by endocytic pathway which is known as a completely different cellular internalization mechanism from that of small molecules. In addition, the subsequent acidic endosomal environments can be used as a trigger for the pH-sensitive release of additional chemotherapeutic agents. In conventional chemotherapy, the rapid developments of multidrug-resistant characteristics in cancer cells cause a critical problem in clinical cancer treatments. As such, it is obvious that a combinational therapy is highly favorable for the complete remission of cancers rather than a single-type treatment. To this end, anti-cancer drug-conjugated nanoRDE as a first example of multimodal delivery platform for both radio- and chemotherapy will be demonstrated in this project. Gold nanoparticles have been tested for improvement of both chemotherapy and radiotherapy . The TNFalpa-PEG-colloidal gold nanoparticle, CYT-6091 (Citimmune, Gaithersburg, MD), has been shown to selectively traffic to tumor tissue. This agent has been evaluated for its ability to selectively deliver TNFalpha to tumors in clinical trials. Gold nanoparticles are available in a broad range of sizes. In addition to its properties as a nano-carrier, colloidal gold, being a high-Z element, may also increase the radiation dose delivered specifically to the target cells. In this system, gold nanoparticles (AuNPs) are used as inorganic nanoRDEs for radiotherapy as well as a delivery platform for chemotherapeutic agents. Due to the K-edge of gold at 80.7 keV, X-ray radiation with the energy level of Au K-edge can trigger the secondary emission of photoelectric radiation from AuNPs. The resulting radiation can induce the degradation of target molecules by ionization and can interact with surrounding water molecules to produce reactive oxygen species that can damage the target molecules. As such, AuNP-sensitized degradations of plasmid DNA and human proteins upon X-ray radiation have been demonstrated in in vitro model systems. Additionally, when AuNP-containing tumor cells were irradiated, increased apoptotic cell death was detected due to the continuous stress on cytosolic organelles. Furthermore, enhanced in vivo efficacy of AuNPs upon radiotherapy was also observed in human cancer-bearing mouse models. However, these AuNPs have shown very poor pharmacokinetic results due in part to their limited surface functionality as a bare colloidal particle. Recently, AuNPs have been used in a wide range of biological applications due to their biocompatibility and well-known surface chemistry. Using the known reactions, AuNPs can be readily modified with thiol-end-capped polymers that can significantly alter the pharmacokinetics. This functional polymer can be prepared by highly controllable reversible addition-fragmentation chain-transfer (RAFT) radical polymerization which allows for copolymerization of a wide range of monomers. As the gold K-edge is at 80.7 keV and the enhancement is optimal in the photoelectric-dominated X-ray spectrum, irradiation conditions could be optimized by using monoenergetic X-rays. Finaly, gold can be easily activated with thermal neutrons to emit 411 keV gamma rays, which could provide a means for monitoring their biodistribution by SPECT. Hypothesis: Functionalized gold nanoparticles can be used for HER2-targeted delivery and triggered release of therapeutic agents, including radiosensitizers Research Aims 1. Development of HER2-targeted AuNP a) Characterization of biodistribution and pharmacokinetics of Au-NP b) Assessment of biodistribution of neutron-activated Au-NP in vivo by SPECT c) Testing the effect of combining AuNP with external irradiation in vitro and in vivo 2. Application of HER2-targeted AuNP for tumor-specific delivery of therapeutic agents a) Optimization of conditions required for triggered release of the molecules carried by Au NP b) In vitro and in vivo testing of the efficacy of Au-NP-delivered drugs as compared with current formulations. Accomplishments: 1. Methods for quantification of gold concentration in the tissue using neutron activation have been developed and presented on 2009 Annual Meetings of the American Nuclear Society and American Chemical Society 2. Methods for production of pegylated nanoparticles have been established and tested 3. X-ray-triggered release of fluorescent molecules from gold nanoparticles was tested and a poster presentation of the preliminary results received Best American Chemical Society (ACS) poster at the ACS Division of Colloid and Surface on the 2009 Annual Meetings of ACS. 4. Our theoretical work describing the requirements of nanoparticles to be used as photodynamic therapy-based radiosensitizers indicating that gold nanoparticles present an advantageous alternative to nano-scintillatots proposed by others has been published in Radiation Research.