Application of Gold Nanoparticles to Increase the Efficacy of Radiation Therapy

纳米颗粒（小于100纳米的颗粒）作为疾病检测和药物输送的工具在医学领域引起了越来越多的兴趣。纳米医学领域虽然仍处于起步阶段，但随着我们开始了解与纳米材料的益处、毒性和环境影响相关的问题，它拥有巨大的希望。胶体金是氯化金与柠檬酸钠结合合成的中性金颗粒。几十年来，它一直被安全地用于关节炎患者的治疗。这种颗粒的直径为5-100纳米，可以与蛋白质、多肽、合成药物和核苷酸不可逆地联系在一起。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。理论上，在高z元素的k边吸收能量处辐照会导致俄歇电子和光电子的发射，在它们附近释放出大量的能量。在相同剂量的外照射下，这种二次辐射会对肿瘤细胞造成额外的损伤。如果肿瘤靶向金纳米颗粒的系统管理导致颗粒优先运输到肿瘤组织，我们将能够利用这一特性通过增加沉积在肿瘤中的辐射剂量来增强外部光束照射的功效，同时最大限度地减少对正常组织的毒性。由于俄歇电子在短距离内沉积的大量能量会破坏附着在金纳米粒子上的分子的化学结合，因此金纳米粒子也可以用作辐射触发释放治疗剂的运载工具。此外，金可以很容易地被热中子激活，释放出411kev的伽马射线，为SPECT监测其生物分布提供了一种手段。因此，除了俄格电子对肿瘤细胞可能的直接作用外，我们的纳米颗粒被激活用于成像它们的分布，包含肿瘤特异性靶向剂进行选择性递送，并且仅在最佳时间（由实时成像定义）在目标体积内释放活性药物，可能为肿瘤特异性递送各种肿瘤药物提供手段，包括那些目前因其疏水性和限制应用的药物。因此，补充当前的治疗策略，特别是论证放射治疗的有效性。该项目是一个包括核物理、分析化学、细胞表面受体识别、生长因子信号通路、靶向治疗、与单克隆抗体和粘附体相关的免疫学、蛋白质化学、药物化学、放射化学、放射学和分子成像等一系列不同学科的联合努力。我们结合了CCR放射肿瘤学分支、分子成像和纳米生物学项目、NIH成像探针开发中心和NIST分析化学部门的优势，将该项目合并为靶向药物输送的综合方法。随着我们将项目推向临床领域，我们将与CCR临床放射和医学肿瘤学家合作制定适当的治疗策略。研究设计目标1：辐射输运计算将使用ESG4蒙特卡罗模拟包或ROB辐射物理部门开发的治疗计划软件进行。组织中沉积的辐射剂量随金浓度和外部照射质量的变化将使用标准方法计算。目标2：该目标将与NIH成像探针开发中心的Garry Griffith博士及其团队合作进行。金纳米颗粒将被聚乙二醇化以防止其聚集，并与HER2或egfr特异性的附着体分子结合。为了成像，近红外荧光分子（如AlexaFluor）也将被附着。如果生物分布研究给出了有希望的结果，纳米粒子将被装载治疗药物。目标3：通过不同类型的连接剂与荧光染料结合的金纳米颗粒将暴露在越来越多剂量的x射线下。辐照后，纳米粒子将从含有由x射线触发的俄歇电子从金中释放出来的分子的溶液中分离出来。这些分子的荧光将被测量，并与暴露于x射线有关。目的4：肿瘤细胞对聚乙二醇化胶体金纳米颗粒与不同类型辐射的反应将通过克隆生存试验来表征。人类乳腺癌细胞系将在含金纳米粒子或不含金纳米粒子的情况下进行培养，并暴露在不同剂量的辐射下。所施加的x射线的能量在5kev到60mev的范围内变化。辐射对细胞存活的影响将被量化和评估。这一系列实验将有助于验证Aim 1的预测剂量，并允许为体内研究选择适当的细胞系、辐射类型和剂量。目标5：这项工作将与国家标准与技术研究所分析化学部门的Dr. Gregory Downing及其团队合作进行。利用NIST核反应堆的热中子端口，金纳米粒子将被不同通量的中子照射，并通过测量活化金发出的伽马射线来评估其活化程度。对先前附着在金纳米粒子上的分子的附带损伤将通过这些纳米粒子在辐照后的物理化学特性的变化来评估。目的6：利用光学成像和单光子发射计算机断层扫描（SPECT）分别对近红外荧光信标标记的纳米粒子和中子激活的纳米粒子进行体外和体内的生物分布研究。目的7：在荷瘤小鼠体内研究x射线金纳米颗粒的协同作用。成组的荷瘤动物将接受：1)仅放射治疗；2)只有黄金；3)辐射加金的组合。未治疗的动物将作为对照。肿瘤大小和生存率将用于评估每种治疗的疗效。目的8：为了评估金纳米颗粒递送治疗剂的可行性和拟议的辐射触发药物递送系统的有效性，我们将在荷瘤小鼠中比较拟议的辐射触发药物递送系统与标准治疗干预的治疗效果。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。

Background and Significance Nanoparticles (particles less than 100 nm) have generated increasing interest in the field of medicine as tools for disease detection, and drug delivery. The field of nanomedicine, while still in its infancy, holds tremendous promise as we begin to understand issues related to the benefits, toxicity and environmental impact of nanoscale materials. Colloidal gold is a neutral gold particle synthesized through the combination of gold chloride and sodium citrate. It has been used safely for decades as a therapeutic for patients with arthritis. The particle measures 5-100 nm in diameter and can be linked irreversibly to proteins, peptides, synthetic drugs and nucleotides. 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. Theoretically, the irradiation of high-Z elements at their K-edge absorption energy leads to emission of Auger electrons and photoelectrons, releasing a large amount of energy at their immediate vicinity. This secondary radiation will result in extra damage to tumor cells at the same dose of the applied external radiation. If systemic administration of tumor-targeted gold nanoparticles results in the preferential trafficking of the particles to tumor tissue, we will be able to utilize this property to enhance the efficacy of external beam irradiation by increasing the radiation dose deposited into the tumor while minimizing toxicity to normal tissues. Since the high amount of energy deposited by Auger electrons at short distances will break chemical binding of molecules attached to the gold nanoparticles, gold nanoparticles could be also used as delivery vehicles for radiation-triggered release of therapeutic agents. In addition, gold can be easily activated with thermal neutrons to emit 411 keV gamma rays, to provide a means for monitoring their biodistribution by SPECT. Therefore, in addition to a possible direct effect of Auger electrons on tumor cells, our nanoparticles activated for imaging of their distribution, containing tumor-specific targeting agents for selective delivery, and releasing the active drug within a target volume only at optimal time (defined by real-time imaging), may provide means for tumor-specific delivery of a variety of tumorocidal agents, including those whose application is currently limited due to their hydrophobicity and, thereby, complement current therapeutic strategies and, specifically, argument the efficacy of radiation therapy. This project is a joint effort encompassing a range of different disciplines which include: nuclear physics, analytical chemistry, cell surface receptor recognition, growth factor signaling pathways, targeted therapy, immunology pertaining to mAbs and affibody, protein chemistry, medicinal chemistry, radiochemistry, radiology, and molecular imaging. We have combined the strengths of the CCR Radiation Oncology Branch, Molecular Imaging and Nanobiology Programs, with NIH Imaging Probe Development Center, and NIST Analytical Chemistry Division to merge the project into an integrated approach for targeted drug delivery. As we move the project towards the clinical arena we will partner with CCR clinical radiation and medical oncologists for appropriate therapeutic strategies. Research Design Aim 1: Radiation transport calculations will be carried out using the ESG4 Monte Carlo simulation package or the treatment planning software developed within the ROB Radiation Physics Section. The changes of the radiation dose deposited in tissues as a function of the concentration of gold and the quality of the applied external irradiation will be calculated using standard methodology . Aim 2: This aim will be carried out in collaboration with Dr. Garry Griffith and his team at NIH Imaging Probe Development Center. Gold nanoparticles will be pegylated to prevent their aggregation and conjugated with HER2- or EGFR-specific affibody molecules. For imaging, near-infrared fluorescent molecules (e.g. AlexaFluor) will be attached, too. If the biodistribution studies give promising results, the nanoparticles will be loaded with therapeutic agents. Aim 3: Gold nanoparticles, conjugated with fluorescent dyes via different types of linkers, will be exposed to increasing doses of x-rays. After irradiation, the nanoparticles will be separated from solution containing molecules released form gold by x-ray-triggered Auger electrons. The florescence of these molecules will be measured and correlated with the exposure to x-rays. Aim 4: Response of tumor cells to combination of pegylated colloidal gold nanoparticles with various types of radiation will be characterized by the clonogenic survival assay. Human breast cancer cell lines will be incubated with or without gold nanoparticles, and exposed to different doses of radiation. The energy of applied x-rays will vary in the range from 5 keV to 60 MeV. The effects of radiation on cell survival will be quantified and evaluated. This series of experiments will help in the validation of the predicted doses from Aim 1 and will allow for the selection of the appropriate cell line, and radiation type and dose for in vivo studies. Aim 5: This work will be carried out in collaboration with Dr. R. Gregory Downing and his team at Analytical Chemistry Division of the National Institute of Standards and Technology. Using the thermal neutron port at NIST nuclear reactor gold nanoparticles will be irradiated with different fluencies of neutrons and their activation assess by measurement of the gamma rays emitted from the activated gold. The collateral damage to the molecules previously attached to the gold nanoparticles will be assessed by changes in physico-chemical characteristics of theses nanoparticles post-irradiation. Aim 6: Biodistribution of nanoparticles in tumor bearing mice will be studied ex vivo and in vivo using optical imaging and single-photon emission computer tomography (SPECT) for nanoparticles labeled with near-infrared fluorescent beacons and activated with neutrons, respectively. Aim 7: The possible synergistic effect of combining x-rays gold nanoparticles will be studied in vivo using tumor bearing mice. Groups of tumor-bearing animals will be treated with: 1) radiation only; 2) gold only; and 3) combination radiation plus gold. Non-treated animals will be used as controls. Tumor size and survival will be used to assess the efficacy of each treatment. Aim 8: To assess the feasibility of gold nanoparticles for delivery of therapeutic agents and efficacy of the proposed radiation-triggered drug delivery system we will compare the therapeutic efficacy of the proposed radiation-triggered drug delivery system with standard therapeutic interventions in tumor bearing mice. 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.