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）：脑癌是一种威胁生命的疾病，其特征是生存率较低。与药物结合的选择性靶向纳米颗粒的发展对于改善这种侵略性类型的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，有望用于脑肿瘤治疗。尽管报道了生存的提高，但在脑肿瘤疗法中，PDT的广泛使用已被非针对性的光毒性对健康组织的非靶标的部分受到阻碍。提高肿瘤靶向和PDT药物的持续递送的选择性将大大增强脑癌治疗的成功。 PC 4是一种高度有希望的PDT药物，已批准用于临床试验，其特征是在黑暗，高光毒性和明确定义的化学结构和纯度中虚拟无毒。该药物在近红外（NIR）光谱范围内运行，该光谱范围最有效地穿透了脑组织，用于诊断光学成像和光疗。拟议的研究的目的是开发PC 4加载金纳米颗粒（Au NP-PC 4）与硫醇化的PEG涂层共轭，用于靶向成像引导的神经胶质瘤脑癌的疗法。我们将适应一种新型的跨学科方法，以合成与PC 4结合的PEG涂层的金纳米颗粒和链状肽配体，以靶向表皮生长因子（EGF）和克服蛋白（TF）细胞表面受体，这些因子（TF）细胞表面受体在脑癌细胞中均过表达。通过应用遗传细胞工程，我们将使用人类EGF和TF受体开发模型的细胞系和动物系统，它们在大鼠9L胶质瘤细胞上分别或共同表示为可行的癌症生物标志物。我们将在体外和体内荧光成像中执行最先进的状态，以表征结合物的递送和靶向，并确定其治疗性（PDT）功效。中心假设是，使用双靶向配体概念将显着提高PDT纳米粒子对脑癌的选择性。为了检验这一假设，我们将在体外和体内和体内纳米颗粒PDT疗效以实现以下目的：特定目标1：开发和表征含有EGFR和TFR结合肽配体的eGFR和TFR结合肽配体。将合成带有PDT药物PC 4的靶向NP，完全表征（就结构，配体密度和药物加载而言），并优化用于选择性靶向和药物释放。为了支持优化过程，我们将对每种NP共轭设计的受体相互作用研究进行AU NP：不仅包括平衡数据，还包括使用表面等离子体等离子体共振（SPR）Biacore技术的相互作用的动力学参数。具体目标2：在TF和EGF受体9L胶质瘤细胞系中，靶向纳米颗粒结合递送和PDT功效测试。将使用过表达人EGFR和TFR的工程9L神经胶质瘤细胞系研究靶向过表达人体受体的能力。我们将使用各种实验技术（包括银色增强免疫组织化学，实时共聚焦激光扫描显微镜和透射电子显微镜）检查目标NP的摄取和定位。然后将对细胞进行PDT，并使用MTT测定法评估细胞活力。 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.特定目的3：在胶质瘤肿瘤轴承小鼠中PDT治疗和疗法后监测的体内翻译。我们将使用3维荧光分子断层扫描（FMT）研究NP结合物在受体过表达的9L肿瘤轴承小鼠中的NP偶联物在体内的NP靶向和PDT疗效。我们将使用ICP/AAS元素分析和增强银免疫组织化学以及荧光成像来确定靶向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。我们还将在七天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高度靶向的，几乎无毒的PDT敏化剂对脑癌的联合治疗和监测进行了巨大改进，可以通过高时空分辨率实时局部激活和审问。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。细胞表面受体靶向的金纳米颗粒与PC 4（一种光动力疗法药物）结合时，可以是用于提高这些脑癌检测特异性的分子成像剂。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神经胶质瘤癌小鼠模型的体内研究。通过提高肿瘤靶向的选择性，我们可以潜在地维持PDT药物的局部递送，从而大大增强了脑癌治疗的成功。