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

• 批准号: 8304224
• 负责人: James Peter Basilion
• 金额: $ 17.99万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8304224
• 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 Delivery Systems; 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

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）：脑癌是一种以低生存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颗粒的开发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）是一种局部治疗模式，是有前途的脑肿瘤治疗。尽管有报道称PDT可改善生存率，但PDT在脑肿瘤治疗中的广泛应用受到了健康组织非靶向光毒性的部分阻碍。改善肿瘤靶向的选择性和PDT药物的持续递送将显著提高脑癌治疗的成功率。Pc 4是一种非常有前途的PDT药物，已被批准用于临床试验，其特征在于在黑暗中几乎无毒，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，可以最有效地穿透脑组织进行诊断光学成像和光疗。本研究的目的是开发具有巯基化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。我们将采用一种新的跨学科的方法来合成PEG涂层的金纳米粒子共轭Pc 4和拴系肽配体靶向表皮生长因子（EGF）和转铁蛋白（Tf）细胞表面受体，这是过表达的脑癌细胞。通过应用遗传细胞工程，我们将开发具有人EGF和Tf受体的模型细胞系和动物系统，这些受体分别或共同表达为大鼠9 L胶质瘤细胞上的可行癌症生物标志物。我们将进行体外和体内荧光成像的最新技术，以表征缀合物的递送和靶向，以及确定其治疗（PDT）功效。核心假设是使用双靶向配体概念将显著提高PDT纳米颗粒对脑癌的选择性。为了验证这一假设，我们将开发和测试体外和体内纳米颗粒PDT功效，以实现以下目标：具体目标1：含有EGFR和TfR结合肽配体的PEG化Au NP-Pc 4缀合物的开发和表征。将合成装载有PDT药物Pc 4的靶向NPs，充分表征（在结构、配体密度和药物负载方面），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，还包括相互作用的动力学参数。具体目标2：在携带Tf和EGF受体的9 L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。将使用分别和组合过表达人EGFR和TFR的工程化9 L胶质瘤细胞系研究靶向过表达人受体的能力。我们将使用各种实验技术，包括银增强免疫组织化学，真实的时间共聚焦激光扫描显微镜，和透射电子显微镜检查靶向纳米颗粒的摄取和定位。然后对细胞进行PDT，并使用MTT测定法评估细胞活力。由于PDT药物的细胞内定位是下游细胞事件（如凋亡）的前体，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渗透性测定（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机制。具体目标3：PDT治疗的体内转化和荷胶质瘤小鼠的治疗后监测。我们将使用三维荧光分子断层扫描（FMT）在受体过表达的9 L荷瘤小鼠中研究NP缀合物的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靶向Au NPs和药物Pc 4的循环、生物分布和清除，以评估Pc 4和Au NPs的相对浓度。我们还将使用动态荧光成像在七天内检查PDT后肿瘤的病理学。该项目的最终目标是使用高度靶向的、几乎无毒的PDT敏化剂大幅改善脑癌联合治疗和监测，该PDT敏化剂可以以高时空分辨率在真实的时间内局部激活和询问。 公共卫生相关性：恶性胶质瘤是人类最常见的原发性脑肿瘤，也是人类最致命的癌症之一，以细胞表面受体为靶点的金纳米粒子与光动力学治疗药物Pc 4结合后，可作为分子显像剂用于提高恶性胶质瘤检测的特异性。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，从而显著提高脑癌治疗的成功率。