Combination of TNF-Gold Nanoparticles with Radiation

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20-30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。胶体金除了具有纳米载体的特性外，还具有高z元素的作用。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。我们相信，胶体金的这一次要性质为新的研究和临床应用开辟了一个重要的领域。具体来说，由于CYT-6091粒子对肿瘤组织的优先运输，我们可以利用这一特性，通过增加沉积在肿瘤中的辐射剂量，同时最大限度地减少对正常组织的毒性，来增强外束照射的疗效。事实上，CYT-6091目前正在CCR的I期临床试验中作为单药治疗进行评估，这使得我们的研究结果将CYT-6091与实验室的辐射结合到临床具有重要意义。研究设计目的为了确定在金颗粒中产生二次辐射的最佳辐照条件，并估计由于金纳米颗粒的存在而增加的辐射剂量，将使用ESG4蒙特卡罗模拟包或罗布辐射物理部门开发的治疗计划软件进行辐射输运计算。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。这些数据将使我们能够确定添加胶体金的相对效益，以及胶体金的最佳浓度和最佳辐射能量，以优化增强的辐射效果。目标2。肿瘤细胞对聚乙二醇化胶体金TNF纳米颗粒与各种类型辐射结合的反应将通过克隆生存测定来表征。人类乳腺癌细胞系MCF7和MDA251对TNF的敏感性不同，将在含或不含金纳米颗粒和TNF的情况下进行培养，并接受不同剂量（0 - 8 Gy）的辐射。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3。首先，对含有不同浓度金的植入物的组织等效假体进行CT扫描，以评估由于金纳米颗粒的存在而导致的组织密度变化，以及使用CT扫描监测其体内生物分布的可行性。CT扫描的结果将与通过验证的电子显微镜方法对组织中颗粒的定量进行比较。如果这些研究的结果是肯定的，将使用微型CT扫描仪比较CT扫描上的Hounsfield单位的变化与组织中测量的金浓度。具有可测量的皮下肿瘤（约0.5 cm3）的小鼠将注射CYT-6091，其剂量可能导致通过计算机模拟和体外研究确定有效的肿瘤浓度。注射后3至5小时，将进行CT扫描。每组5只小鼠，每个时间点。扫描完成后，将对小鼠实施安乐死，并收集肿瘤、肌肉、肺、肝、脾、胰腺、肾、胃、皮肤和骨骼等感兴趣的组织。所有小鼠研究将在IACUC批准的协议下进行。组织中金的浓度将用质谱技术测量。电子显微镜将用于评估金的亚细胞微分布。目标4。x射线联合CYT-6091可能的协同效应将在体内通过小鼠异种移植和同基因肿瘤模型进行研究。每组10只携带肿瘤的动物将接受以下治疗：1)仅放射治疗；2)聚乙二醇胶体金TNF；3)联合辐射加聚乙二醇胶体金TNF。未治疗的动物将作为对照。纳米粒子的应用剂量和辐射将在以前的目标中确定。肿瘤大小和生存率将用于评估每种治疗的疗效。成就:1。我们进行了体外克隆存活实验，以确定纳米颗粒与辐射联合使用对细胞存活的影响。2. 对金纳米颗粒在荷瘤小鼠体内的生物分布进行了研究。3. 使用荷瘤小鼠进行了体内研究。

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 20-30 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 also acts as a high-Z element. 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. We believe that this secondary property of colloidal gold opens a significant area of novel research and clinical application. Specifically, because of the preferential trafficking of the CYT-6091 particles to tumor tissue, we can 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. The fact that CYT-6091 is currently being evaluated as single agent therapy in the context of a Phase I clinical trial within the CCR, makes the potential for the translation of our findings combining CYT-6091 with radiation in the laboratory to the clinic significant. Research Design Aim 1. In order to identify irradiation conditions optimal for production of secondary radiation in gold particles and to estimate the increase of the radiation dose due to the presence of gold nanoparticles, 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 This data will allow us to determine the relative benefit of the addition of colloidal gold as well as the best concentrations of colloidal gold and the optimal energy of radiation to be used in order to optimize the enhanced radiation effects. Aim 2. Response of tumor cells to combination of pegylated colloidal gold TNF nanoparticles with various types of radiation will be characterized by the clonogenic survival assay. Human breast cancer cell lines, MCF7 and MDA251, with different sensitivities to TNF, will be incubated with or without gold nanoparticles and TNF, and exposed to different doses (0 - 8 Gy) 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 3. Initially, CT scans of a tissue-equivalent phantom with inserts containing different concentrations of gold will be used to assess the changes of density of tissue due to the presence of gold nanoparticles and the feasibility of using CT scans to monitor their in vivo biodistribution. Results of the CT scans will be compared to the quantification of the particles in the tissue by validated electron microscopy approaches. If the results of these studies are positive, the micro-CT scanner will be used to compare the changes of Hounsfield units on the CT scans with gold concentration measured in the tissue. Mice with measurable subcutaneous tumors (approximately 0.5 cm3) will be injected with CYT-6091 at doses that may lead to concentrations in the tumors identified as effective by the computer simulations and the in vitro studies. Three and five hours post injection, CT scans will be carried out. Groups of five mice per time point will be used. After completion of the scans, the mice will be euthanized and tissues of interest including tumor, muscle, lung, liver, spleen, pancreas, kidney, stomach, skin, and bone will be collected. All mouse studies will be performed under approved IACUC protocols. The concentration of gold in the tissues will be measured using mass spectrometry techniques. Electron microscopy will be used to assess the sub-cellular microdistribution of gold. Aim 4. The possible synergistic effect of combining x-rays with CYT-6091 will be studied in vivo using both mouse xenograft and syngeneic tumor models. Groups of 10 tumor-bearing animals will be treated with: 1) radiation only; 2) pegylated colloidal gold TNF only; and 3) combination radiation plus pegylated colloidal gold TNF. Non-treated animals will be used as controls. Applied doses of the nanoparticles and radiation will be identified in previous Aims. Tumor size and survival will be used to assess the efficacy of each treatment. Accomplishments: 1. In vitro clonogenic survival experiments have been carried out to establish the effect of nanoparticles in combination with radiation on cell survival. 2. Biodistribution studies of gold nanoparticles in tumor-bearing mice have been carried out. 3. In vivo studies using tumor bearing mice are carried out.