CPE Peptide-Based Nanoparticles for the Diagnosis and Therapy of Chemotherapy Res

摘要 卵巢癌仍是妇科肿瘤中死亡率最高的癌症。虽然 许多卵巢癌患者对手术和化疗的标准组合完全有效，几乎 90%的人后来患上了复发的化疗耐药癌症，并不可避免地死于这种疾病。因此， 针对复发/化疗耐药卵巢癌的创新、有效治疗方法的开发 仍然是改善公共卫生的高度优先事项。使用高通量技术分析基因 卵巢癌的指纹，我们最近发现了编码基因的极高表达 蛋白claudin-3和claudin-4。因为Claudin-3和-4是梭状芽胞杆菌的上皮受体 产气荚膜肠毒素(CPE)，并足以介导CPE结合，我们假设使用 可生物降解的纳米粒子，如聚乳酸-乙醇酸共聚物，即PLGA-NP 与CPE结合域复合的治疗药物，靶向卵巢癌细胞，是一种新型的， 治疗化疗耐药卵巢癌的潜在高效治疗方法。 与这一观点一致，初步的体外数据清楚地显示，荧光素(FITC)标记的-C-CPE 在化疗耐药的原代OSPC细胞系中有效结合并迅速内化，表明C- CPE能结合药物，也能跨细胞肿瘤膜转运药物。更重要的是， 初步的体内实验结果表明，CPE-PLGA-NP选择性地包裹了一种荧光染料(香豆素) 化疗耐药卵巢肿瘤体内蓄积，非特异性蓄积最少 仅在其他器官的正常细胞中有背景结合。因此，这项提案 有三个相关的具体目标：1)表征荧光PLGA-NP包埋模型和治疗 药物与不同长度的CPE多肽(即30aa、17aa和9aa)络合，并评价结合活性 在多种体外试验中对原发化疗耐药卵巢的治疗效果 2)研究PLGA-NP香豆素微球的分布和药代动力学 ~(125)I标记C-CPE多肽在临床相关动物模型中的体内研究 3)检测PLGA-NP包裹DNA的治疗潜力 编码白喉毒素A自杀蛋白的基因启动子序列高度和 在卵巢癌中优先发挥活性(即，Mesothelin和HE4/WFDC2)，在体内与CPE多肽络合 原发化疗耐药卵巢癌的临床相关动物模型。在完成后 这个项目，我们将定位于将这项研究迅速转化为一种新颖、高效的治疗方法 化疗耐药疾病患者的治疗。

Abstract Ovarian carcinoma remains the cancer with the highest mortality rate among gynecological tumors. Although many ovarian cancer patients fully respond to the standard combination of surgery and chemotherapy, nearly 90% later develop recurrent chemotherapy-resistant cancer and inevitably succumb to their disease. Thus, development of innovative, effective therapies against recurrent/chemotherapy-resistant ovarian cancer remains a high priority for improving public health. Using high-throughput technologies to analyze genetic fingerprints of ovarian cancer, we have recently discovered extremely high expression of the genes encoding the proteins claudin-3 and claudin-4. Because claudin-3 and -4 are the epithelial receptors for Clostridium perfringens enterotoxin (CPE), and are sufficient to mediate CPE binding, we hypothesize that using biodegradable nanoparticles such as poly(lactic-co-glycolic acid, i.e., PLGA-NP) encapsulating therapeutic agents complexed to the binding domain of CPE, to target ovarian cancer cells, is a novel, potentially highly effective therapeutic approach to treat chemotherapy-resistant ovarian cancer. Consistent with this view, preliminary in vitro data clearly showed that fluorescein(FITC)-labeled-C-CPE effectively binds and rapidly internalizes in chemotherapy-resistant primary OSPC cell lines, suggesting that C- CPE is capable to bind and also of translocating drugs across cell tumor membrane. More importantly, preliminary in vivo results showed that CPE-PLGA-NP encapsulating a fluorescent dye (coumarin) selectively accumulate in vivo in chemotherapy resistant ovarian tumors with minimal nonspecific accumulation in RES organs and only background binding in normal cells of various other organs. Accordingly, this proposal has three related specific aims: 1) Characterize fluorescent PLGA-NP encapsulating model and therapeutic drugs, complexed to CPE peptides of different lengths (i.e., 30aa, 17aa and 9 aa), and evaluate binding activity and therapeutic efficacy of such NP in multiple in vitro assays against primary chemotherapy resistant ovarian carcinoma cell lines, 2) Examine the distribution and pharmacokinetics of PLGA-NP encapsulating coumarin complexed to 125I labeled C-CPE peptide in vivo in clinically relevant animal models of primary chemotherapy resistant ovarian carcinoma and 3) Examine the therapeutic potential of PLGA-NP encapsulating plasmid DNA encoding Diptheria Toxin A suicide protein under the control of promoter sequences of genes highly and preferentially active in ovarian cancer (i.e., mesothelin and HE4/WFDC2), complexed to CPE peptide in vivo in clinically relevant animal models of primary chemotherapy resistant ovarian carcinoma. Upon completion of this project, we will be positioned to quickly translate this research into a novel, highly effective therapeutic treatment for patients with chemotherapy-resistant disease.