Dual-Receptor Targeted Nanoparticles for Photodynamic Therapy of Brain Cancer

描述（申请人提供）：脑癌是一种危及生命的疾病，其特点是生存率低。选择性靶向纳米颗粒与药物结合的发展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）是一种局部治疗脑肿瘤的方法。尽管有报道称PDT可提高生存率，但PDT在脑肿瘤治疗中的广泛应用在一定程度上受到对健康组织的非靶向光毒性的阻碍。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。pc4是一种非常有前途的PDT药物，已被批准用于临床试验，其特点是黑暗中几乎无毒，光毒性高，化学结构和纯度明确。这种药物在近红外（NIR）光谱范围内起作用，它可以最有效地穿透脑组织，用于诊断光学成像和光疗。该研究的目的是开发装载pc4的金纳米颗粒（Au np - pc4）与硫代聚乙二醇涂层结合，用于靶向成像引导治疗胶质瘤脑癌。我们将采用一种新的跨学科方法，合成与pc4和栓系肽配体结合的peg包被金纳米颗粒，用于靶向脑癌细胞中过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，我们将开发具有人类EGF和Tf受体的模型细胞系和动物系统，分别或联合表达为大鼠9L胶质瘤细胞上可行的癌症生物标志物。我们将在体外和体内进行最先进的荧光成像，以表征缀合物的递送和靶向，以及确定其治疗（PDT）功效。中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的功效，以实现以下目标：特定目标1：开发和表征含有EGFR和TfR结合肽配体的聚乙二醇化Au np - pc4偶联物。将合成装载PDT药物pc4的靶向NPs，充分表征（结构，配体密度和药物负载），并优化其选择性靶向和药物释放。为了支持优化过程，我们将对每个NP共轭设计进行Au NP：受体相互作用的研究，不仅包括平衡数据，还包括使用表面等离子体共振（SPR） Biacore技术进行相互作用的动力学参数。特异性目的2：纳米颗粒靶向结合给药及PDT在携带Tf和EGF受体的9L胶质瘤细胞系中的体外疗效检测。靶向过表达的人受体的能力将通过单独或联合使用过表达人EGFR和TFR的工程9L胶质瘤细胞系进行研究。我们将使用各种实验技术，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，来检查靶向NPs的摄取和定位。然后将细胞进行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（如细胞凋亡）的前兆，我们还将使用线粒体膜电位测定、TUNEL和DNA片段测定以及细胞渗透性测定（包括台泮蓝染色和膜联蛋白V/碘化丙啶流式细胞术）来评估pc4介导的程序性细胞死亡的机制。特异性目的3:PDT治疗在胶质瘤小鼠体内的翻译和治疗后监测。我们将利用三维荧光分子断层扫描（FMT）技术研究NP偶联物在受体过表达的9L荷瘤小鼠体内的靶向性和PDT效果。我们将使用ICP/AAS元素分析和银增强免疫组织化学，以及荧光成像来评估pc4和Au NPs的相对浓度，确定靶向Au NPs和药物pc4的循环、生物分布和清除。我们还将在7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一种高度靶向的、几乎无毒的PDT增敏剂，在脑癌治疗和监测方面取得巨大进步，这种增敏剂可以局部激活，并以高时空分辨率实时检测。

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.