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Dual-Receptor Targeted Nanoparticles for Photodynamic Therapy of Brain Cancer

双受体靶向纳米颗粒用于脑癌光动力治疗

基本信息

批准号：
8542845
负责人：
ANN-MARIE BROOME
金额：
$ 24.43万
依托单位：
MEDICAL UNIVERSITY OF SOUTH CAROLINA
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-30 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8542845
关键词：
3-Dimensional Address Adverse effects Animals Apoptosis Applied Genetics Binding Biodistribution Biological Assay Biological Markers Blood Circulation Brain Neoplasms Cancer Patient Cancer cell line Carcinoma Cell Culture Techniques Cell Line Cell Surface Receptors Cells Chemical Structure Clinical Trials DNA Fragmentation Data Detection Development Diagnostic Disease Drug Targeting Engineering Epidermal Growth Factor Epidermal Growth Factor Receptor Equilibrium Event Flow Cytometry Fluorescence Generations Genetic Glioma Goals Gold Health Human Image Immunohistochemistry In Transferrin In Vitro Interdisciplinary Study Kinetics Laser Scanning Confocal Microscopy Lead Life Ligands Local Therapy Malignant Glioma Malignant Neoplasms Malignant neoplasm of brain Mediating Membrane Potentials Modality Modeling Molecular Monitor Mus Outcome Peptides Permeability Pharmaceutical Preparations Photochemotherapy Phototherapy Phototoxicity Primary Brain Neoplasms Propidium Diiodide Rattus Relative (related person)Reporting Research Resolution Silver Site Solubility Specificity Staining method Stains Structure Surface Plasmon Resonance Survival Rate System TdT-Mediated dUTP Nick End Labeling Assay Techniques Technology Testing Therapeutic Time Tissues Transferrin Transferrin Receptor Translating Translations Transmission Electron Microscopy Treatment Efficacy Trypan Blue Tumor Pathology Vision Water annexin A5 brain tissue cancer cell cancer therapy cancer type cellular engineering chemical synthesis density design efficacy testing fluorescence imaging glioma cell line image guided therapy improved in vivo man mitochondrial membrane molecular imaging mouse model nanoparticle nanotherapeutic novel optical imaging overexpression process optimization receptor success targeted delivery therapeutic target tomography tumor uptake virtual

项目摘要

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.

描述（由申请人提供）：脑癌是一种危及生命的疾病，其特征是存活率低。开发与药物结合的选择性靶向纳米颗粒对于改善这种侵袭性癌症的治疗和监测至关重要。光动力疗法（PDT）是一种局部治疗方式，有望用于脑肿瘤的治疗。尽管据报道生存率有所提高，但 PDT 在脑肿瘤治疗中的广泛使用受到了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药物的持续递送将极大地提高脑癌治疗的成功率。 Pc 4 是一种非常有前途的 PDT 药物，已批准用于临床试验，其特点是在黑暗中几乎无毒性、高光毒性以及明确的化学结构和纯度。该药物在近红外 (NIR) 光谱范围内发挥作用，可最有效地穿透脑组织，用于诊断光学成像和光疗。拟议研究的目的是开发带有硫醇化 PEG 涂层的 Pc 4 负载金纳米颗粒 (Au NP-Pc 4) 缀合物，用于胶质瘤脑癌的靶向成像引导治疗。我们将采用一种新颖的跨学科方法来合成与 Pc 4 和束缚肽配体缀合的 PEG 包被的金纳米颗粒，用于靶向表皮生长因子 (EGF) 和转铁蛋白 (Tf) 细胞表面受体，这些受体在脑癌细胞中过度表达。通过应用遗传细胞工程，我们将开发具有人类 EGF 和 Tf 受体的模型细胞系和动物系统，这些受体分别或联合表达为大鼠 9L 神经胶质瘤细胞上的可行癌症生物标志物。我们将进行最先进的体外和体内荧光成像，以表征缀合物的递送和靶向，并确定其治疗 (PDT) 功效。核心假设是，使用双靶向配体概念将显着提高 PDT 纳米颗粒对脑癌的选择性。为了检验这一假设，我们将开发和测试体外和体内纳米粒子 PDT 功效，以实现以下目标：具体目标 1：开发和表征含有 EGFR 和 TfR 结合肽配体的聚乙二醇化 Au NP-Pc 4 缀合物。将合成装载有 PDT 药物 Pc 4 的靶向 NP，对其进行全面表征（在结构、配体密度和药物负载方面），并针对选择性靶向和药物释放进行优化。为了支持优化过程，我们将对每个 NP 缀合物设计进行 Au NP：受体相互作用研究，不仅包括平衡数据，还包括使用表面等离子共振 (SPR) Biacore 技术的相互作用的动力学参数。具体目标 2：在携带 Tf 和 EGF 受体的 9L 神经胶质瘤细胞系中进行靶向纳米颗粒缀合物递送和体外 PDT 功效测试。将使用分别或组合过度表达人 EGFR 和 TFR 的工程化 9L 神经胶质瘤细胞系来研究靶向过度表达的人受体的能力。我们将使用各种实验技术检查靶向纳米颗粒的摄取和定位，包括银增强免疫组织化学、实时共焦激光扫描显微镜和透射电子显微镜。然后对细胞进行 PDT，并使用 MTT 测定评估细胞活力。由于 PDT 药物的细胞内定位是下游细胞事件（例如细胞凋亡）的前兆，因此我们还将使用线粒体膜电位测定、TUNEL 和 DNA 片段化测定以及细胞通透性测定（包括台盼蓝染色和膜联蛋白 V/碘化丙啶流式细胞术）评估 Pc 4 介导的程序性细胞死亡的机制。具体目标 3：PDT 疗法在荷瘤小鼠体内的转化和治疗后监测。我们将使用 3 维荧光分子断层扫描 (FMT) 研究 NP 缀合物在受体过表达 9L 荷瘤小鼠体内的 NP 靶向和 PDT 功效。我们将使用 ICP/AAS 元素分析和银增强免疫组织化学以及荧光成像来确定目标 Au NP 和药物 Pc 4 的循环、生物分布和清除率，以评估 Pc 4 和 Au NP 的相对浓度。我们还将在 7 天的时间内使用动态荧光成像检查 PDT 后肿瘤的病理学。该项目的最终目标是使用高度针对性、几乎无毒的 PDT 敏化剂来大幅改进脑癌联合治疗和监测，该敏化剂可以以高时空分辨率进行局部激活和实时询问。公共卫生相关性：恶性神经胶质瘤是最常见的原发性脑肿瘤，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 Pc 4 结合时，可以作为分子成像剂，用于提高这些脑癌检测的特异性。我们的多学科研究计划涉及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神经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 药物的局部递送，从而显着提高脑癌治疗的成功率。