Dual-Receptor Targeted Nanoparticles for Photodynamic Therapy of Brain Cancer

描述(申请人提供)：脑癌是一种以低存活率为特征的危及生命的疾病。与药物结合的选择性靶向纳米粒子的开发对于改善这种侵袭性类型癌症的治疗和监测至关重要。光动力疗法(PDT)是一种局部治疗方法，在脑肿瘤的治疗中具有广阔的应用前景。虽然已有报道可提高存活率，但PDT在脑肿瘤治疗中的广泛应用已受到对健康组织的非靶向光毒性的部分阻碍。提高肿瘤靶向的选择性和PDT药物的持续输送将极大地提高脑癌治疗的成功率。PC4是一种非常有前途的PDT药物，已被批准用于临床试验，其特点是在黑暗中几乎无毒，高光毒性，以及明确的化学结构和纯度。这种药物在近红外(NIR)光谱范围内工作，这种光谱最有效地穿透脑组织进行诊断光学成像和光疗。这项研究的目的是开发Pc4负载的金纳米颗粒(Au-NP-Pc4)与硫代聚乙二醇化的聚乙二醇包被物，用于脑胶质瘤的靶向影像导向治疗。我们将采用一种新的跨学科方法来合成聚乙二醇包覆的金纳米颗粒，该纳米颗粒连接到Pc4和系链多肽配体，用于靶向在脑癌细胞中过度表达的表皮生长因子(EGF)和转铁蛋白(TF)细胞表面受体。通过应用基因细胞工程，我们将建立含有人EGF和TF受体的模型细胞系和动物系统，在大鼠9L胶质瘤细胞上单独或联合表达作为活的肿瘤生物标记物。我们将在体外和体内进行最先进的荧光成像，以表征偶联物的传递和靶向，以及确定它们的治疗(PDT)效果。中心假设是，使用双靶向配体概念将极大地提高PDT纳米颗粒对脑癌的选择性。为了验证这一假设，我们将在体外和体内开发和测试纳米粒子PDT的有效性，以实现以下目标：特定目标1：含有EGFR和TFR结合肽配体的聚乙二醇化Au NP-Pc 4结合物的开发和表征。将合成负载PDT药物Pc4的靶向纳米粒，充分表征(在结构、配体密度和载药量方面)，并优化选择性靶向和药物释放。为了支持优化过程，我们将使用表面等离子体共振(SPR)Biacore技术对每个NP共轭设计进行Au NP：受体相互作用研究，不仅包括平衡数据，还包括相互作用的动力学参数。特定目的2：靶向纳米粒子结合物传递及体外对携带Tf和EGF受体的9L胶质瘤细胞系的PDT疗效检测。针对过表达的人受体的能力将使用高表达人EGFR和TFR的工程化9L胶质瘤细胞系单独和联合进行研究。我们将使用各种实验技术来检测靶向NPs的摄取和定位，包括银增强免疫组织化学、实时共聚焦激光扫描显微镜和透射电子显微镜。然后对细胞进行光动力治疗，并用四甲基偶氮唑盐比色法评估细胞活性。由于PDT药物的细胞内定位是下游细胞事件(如细胞凋亡)的先兆，我们还将使用线粒体膜电位分析、TUNEL和DNA碎片分析以及细胞通透性分析(包括台盼蓝染色和Annexin V/碘化丙啶流式细胞术)来评估Pc4介导的细胞程序性死亡的机制。具体目的3：PDT疗法在荷脑胶质瘤小鼠体内的翻译及治疗后监测。我们将利用三维荧光分子断层扫描(FMT)技术，在荷瘤小鼠体内研究NP的靶向性和对受体高表达9L肿瘤小鼠的PDT效应。我们将利用电感耦合等离子体原子吸收光谱元素分析、银增强免疫组织化学和荧光成像来确定靶向Au纳米粒和药物Pc4的循环、生物分布和清除，以评估Pc4和Au纳米粒的相对浓度。我们还将在七天的时间内使用动态荧光成像检查PDT后肿瘤的病理。该项目的最终目标是使用一种高度靶向的、几乎无毒的PDT敏感剂，以高时空分辨率实时激活和询问，大幅改进脑癌的联合治疗和监测。 公共卫生相关性：恶性胶质瘤是最常见的原发脑瘤，也是人类最致命的癌症之一。当细胞表面受体靶向的金纳米颗粒与光动力治疗药物Pc4偶联时，可以作为分子显像剂用于提高对这些脑癌的检测特异性。我们的多学科研究计划包括靶向纳米颗粒的化学合成和表征、体外细胞培养研究以及人类胶质瘤小鼠模型的体内研究。通过提高肿瘤靶向的选择性，我们有可能维持PDT药物的局部输送，从而极大地提高脑癌治疗的成功。

DESCRIPTION (provided by applicant): Brain cancer is a life threatening disease characterized by low survival rates. The development of selectively targeted nanoparticles conjugated with drugs is critical for improving the treatment and monitoring of this aggressive type of cancer. Photodynamic therapy (PDT) is a localized treatment modality that is promising for brain tumor treatment. Although improvements in survival were reported, the widespread use of PDT in brain tumor therapy has been partially hampered by non-targeted phototoxicity towards healthy tissue. Improving the selectivity of tumor targeting and sustained delivery of PDT drugs will dramatically enhance the success of brain cancer therapy. Pc 4 is a highly promising PDT drug, approved for clinical trials, characterized by virtual non-toxicity in the dark, high phototoxicity, and well defined chemical structure and purity. This drug operates in the near infrared (NIR) spectral range, which penetrates brain tissue most efficiently for both diagnostic optical imaging and phototherapy. The objective of the proposed research is to develop Pc 4 loaded gold nanoparticle (Au NP-Pc 4) conjugates with a thiolated PEG coating for targeted imaging-guided therapy of glioma brain cancers. We will adapt a novel cross-disciplinary approach to synthesize PEG-coated gold nanoparticles conjugated to Pc 4 and tethered peptide ligands for targeting epidermal growth factor (EGF) and transferrin (Tf) cell surface receptors, which are overexpressed in brain cancer cells. By applying genetic cell engineering, we will develop model cell lines and animal systems with human EGF and Tf receptors, expressed separately or jointly as viable cancer biomarkers on rat 9L glioma cells. We will perform state of the art in vitro and in vivo fluorescence imaging to characterize the delivery and targeting of the conjugates, as well as determining their therapeutic (PDT) efficacy. The central hypothesis is that using a dual-targeting ligand concept will dramatically improve PDT nanoparticle selectivity to brain cancers. To test this hypothesis, we will develop and test both in vitro and in vivo nanoparticle PDT efficacy to fulfill the following aims: Specific Aim 1: Development and characterization of PEGylated Au NP-Pc 4 conjugates containing EGFR and TfR binding peptide ligands. Targeted NPs loaded with the PDT drug Pc 4 will be synthesized, fully characterized (in terms of structure, ligand density, and drug loading), and optimized for selective targeting and drug release. To support the optimization process, we will perform Au NP: receptor interaction studies of each NP conjugate design, including not only equilibrium data, but also kinetic parameters of the interactions using surface plasmon resonance (SPR) Biacore technology. Specific Aim 2: Targeted nanoparticle conjugate delivery and PDT efficacy testing in vitro in Tf and EGF receptor-bearing 9L glioma cell lines. The ability to target overexpressed human receptors will be studied using engineered 9L glioma cell lines overexpressing human EGFR and TFR, separately and in combination. We will examine the uptake and localization of targeted NPs using various experimental techniques, including silver enhancement immunohistochemistry, real time confocal laser scanning microscopy, and transmission electron microscopy. The cells will then be subjected to PDT, and cellular viability will be assessed using the MTT assay. Since intracellular localization of the PDT drug is a precursor to downstream cellular events, such as apoptosis, we will also assess the mechanism of Pc 4-mediated programmed cell death using a mitochondrial membrane potential assay, TUNEL and DNA fragmentation assays, and cell permeability assays, including trypan blue staining and Annexin V/ propidium iodide flow cytometry. Specific Aim 3: In vivo translation of PDT therapy and post-therapy monitoring in glioma tumor bearing mice. We will investigate the NP targeting and the PDT efficacy of the NP conjugates in vivo in receptor overexpressing 9L tumor bearing mice using 3-dimensional fluorescence molecular tomography (FMT). We will determine circulation, biodistribution, and clearance of the targeted Au NPs and the drug Pc 4 using ICP/AAS elemental analysis and silver enhancement immunohistochemistry, and fluorescence imaging to evaluate relative concentrations of Pc 4 and the Au NPs. We will also examine the pathology of the tumors after PDT using dynamic fluorescent imaging over a seven day period. The ultimate goal of this project is a drastic improvement of combined brain cancer treatment and monitoring using a highly targeted, virtually non-toxic PDT sensitizer that can be locally activated and interrogated in real time with high spatio-temporal resolution. PUBLIC HEALTH RELEVANCE: Malignant gliomas are the most common primary brain tumors and among the most lethal cancers in man. Cell surface receptor-targeted gold nanoparticles when conjugated with Pc 4, a photodynamic therapy drug, can be molecular imaging agents used to improve the specificity of detection of these brain cancers. Our multidisciplinary research plan involves chemical synthesis and characterization of targeted nanoparticles, in vitro cell culture studies, and in vivo studies of mouse models of human glioma carcinomas. By improving the selectivity of tumor targeting, we can potentially sustain local delivery of PDT drugs, thus dramatically enhancing the success of brain cancer therapy.