Thiroredoxin Targeted Nanoparticles for Cancer Research

描述(申请人提供)：目前前列腺癌研究中的一个重要临床问题是预测复发的可能性。我们的假设是，在肿瘤切片上使用显示硫氧还蛋白的纳米颗粒的组织学将改进目前的标准诊断方法(例如PSA水平和Gleason评分)，以预测复发的可能性。利用这种生物纳米技术，我们构建了一个由DNA支架组成的纳米颗粒，展示了三个拷贝的甲基转移酶-硫氧还蛋白融合蛋白。DNA支架已经被修改，以便可以使用荧光素显示癌细胞。我们的初步工作表明，显示硫氧还蛋白的纳米颗粒选择性地与LNCaP和MCF-7结合，而不与PC-3、COS-7和原代前列腺上皮细胞结合，这表明该纳米颗粒选择性地针对某些类型的癌细胞。我们建议跟进这些初步发现，具体目标如下：特定目标1：(I)生成包含Y-结DNA支架的纳米颗粒，显示三个相同的甲基转移酶硫氧还蛋白融合蛋白(YRII-Trx-F)，数量达到特定目标2和3中描述的实验所需的数量。(Ii)产生并纯化直接用荧光素标记的硫氧还蛋白(Trx-F)作为对照。具体目的2：测试显示硫氧还蛋白(YRII-TRX-F)和直接用荧光素标记的硫氧还蛋白(TRX-F)的纳米颗粒在不检测原代前列腺基质细胞和原代前列腺上皮细胞的情况下区分各种可用的前列腺癌细胞系(例如LNCaP、PC-3、DU-145、MDA 2b和22Rv1)的能力。具体目的3：检测硫氧还蛋白纳米颗粒(YRII-TRX-F)和直接荧光素标记的硫氧还蛋白(TRX-F)，确定与冷冻前列腺癌切片的结合是否与Gleason评分和/或复发相关。为了进行比较，将在每个肿瘤样本的切片上进行免疫组化，以使用抗PSMA抗体和AND-AMACR抗体来评估PSMA和AMACR的表达水平。每一种诊断化验都将被分析，以确定格里森和、抗体结合强度和纳米颗粒的荧光强度之间是否存在相关性。我们的长期目标有两个：首先，我们希望我们的发现将提供一个新的参数，有助于建立预测复发的诺模图。其次，纳米颗粒的模块化设计允许我们定制设计，以针对几乎所有可能进入细胞的癌症进行靶向。如果这项研究的结果表明，这种纳米颗粒可以特异性地靶向侵袭性的前列腺癌细胞，那么我们就可以修改潜在的治疗药物进入细胞的设计。这两个目标都超出了本提案的范围。项目简介目前，我们用于确定谁有前列腺癌复发危险的工具没有应有的帮助，因此我们无法继续进行额外的治疗，直到癌症再次出现。我们已经开发出一种技术，可以创造纳米级(分子大小)的颗粒，这种颗粒可以定位在癌细胞上，并使它们发光。在这项建议中，我们将尝试确定我们在显微镜下看到的颜色是否允许我们挑选出那些可能在治疗后复发的癌症，并帮助可能再次患上癌症的患者采取额外的行动。

DESCRIPTION (provided by applicant): Abstract Currently an important clinical problem in prostate cancer research is predicting the likelihood of recurrence. Our hypothesis is that histology using nanoparticles displaying thioredoxin on tumor sections will improve on the current standard diagnostic methods (e.g. PSA level and Gleason's score) in predicting the likelihood of recurrence. Using this bionanotechnology we have constructed a nanoparticle consisting of a DNA scaffold displaying three copies of the methyltransferase-thioredoxin fusion protein. The DNA scaffold has been modified so as to permit the visualization of the cancer cells using fluorescein. Our preliminary work shows that the nanoparticle displaying thioredoxin binds selectively to LNCaP and MCF-7, but not PC-3, COS-7 and Primary Prostate Epithelial Cells, suggesting that the nanoparticle is selectively targeting certain types of cancer cells. We propose to follow up on these preliminary findings with the following Specific Aims: Specific Aim 1: (i) Generate nanoparticles comprising a Y-junction DNA scaffold displaying three identical methyltransferase thioredoxin fusion proteins (YRII-Trx-F) in quantities necessary for the experiments described in Specific Aims 2 and 3. (ii) Generate and purify thioredoxin labeled directly with fluorescein (Trx-F) as a control. Specific Aim 2: Test the nanoparticle displaying the thioredoxin (YRII-Trx-F) and thioredoxin labeled directly with fluorescein (Trx-F) for their ability to discriminate between various available prostate cancer cell lines: (e.g. LNCaP, PC-3, DU-145, MDA PCa 2b and 22Rv1) while not detecting primary prostate stromal cells, and primary prostate epithelial cells. Specific Aim 3: Test the nanoparticle displaying the thioredoxin (YRII-Trx-F) and thioredoxin labeled directly with fluorescein (Trx-F) determine whether or not the binding to frozen prostate tumor sections can be correlated with Gleason's score and/or recurrence. For comparison, immunohistochemistry will be performed on sections of each tumor sample to evaluate the level of expression of PSMA and AMACR using anti-PSMA antibodies and and-AMACR antibodies. Each diagnostic assay will be analyzed to see if there is a correlation between the Gleason's Sum, antibody binding intensity and fluorescent intensity of the nanoparticles. Our long-term goals are two-fold: First, we hope that our findings will provide a new parameter that can contribute to a nomogram for the prediction of recurrence. Second, the modular design of the nanoparticle allows us to custom design it to target almost any cancer for potential delivery into cells. If the results of this study show that this nanoparticle can specifically target aggressive prostate tumor cells, we could then modify the design for potential delivery of a therapeutic into the cells. Both goals are beyond the scope of the present proposal. Project Narrative Currently our tools for determining who is in danger of having prostate cancer return are not as helpful as they should be, so we cannot follow up with additional treatment until the cancer reappears. We have developed a technology for creating nanoscale (molecule size) particles that can home in on the cancer cells and make them glow. In this proposal we will attempt to determine whether or not the color we see under the microscope will permit us to pick out those cancers that are likely to come back after treatment and help the patients who are likely to come down with the cancer again to take additional action.