Design and Characterization of Multi-Targeted Gene Delivery Nanoparticles for Specific Cancer Therapy

提案编号：CBET-1403564 首席研究员：Efrosini Kokkoli 机构：明尼苏达大学双城分校 标题：用于特定癌症治疗的多靶点基因传递纳米粒子的设计和表征 到 2030 年，全球可能会出现 2700 万癌症病例，每年有 1700 万人因癌症死亡，并且在诊断后五年内有 7500 万人患有癌症。主动靶向肿瘤细胞表面表达的粘附分子是一个有前途的研究领域，而靶向纳米颗粒递送至癌症是一个令人感兴趣的领域，可能为改善耐药性癌症的治疗带来希望。不幸的是，迄今为止，通过选择性识别单个细胞受体来实现癌症特异性很少能够充分实现，因为一些目标疾病标记物也在肿瘤以外的区域表达，因此会意外地递送到健康器官。 这项工作的重点是非病毒基因递送载体向癌细胞与健康细胞的特异性递送的问题。提议开发多靶点治疗系统，这将使癌细胞的识别更加特异性。这项工作的假设是，通过设计模块化多靶向非病毒系统可以实现对癌细胞的更高程度的特异性，该系统：i）引入同时靶向过度表达的癌症表面受体α（5）β（1）和α（6）β（4）整合素作为细胞外水平的第一靶向水平，以及ii）靶向上调的NF-kB转录活性，该活性在健康细胞中不活跃，而在多种细胞中高度上调。包括癌症在内的疾病，从而引入一种新形式的转录靶向作为第二级特异性靶向。为了检验这一假设，设定了以下任务： 研究任务 1：评估肽浓度对肽两亲物和聚乙二醇 (PEG) 分子混溶性的影响。 Langmuir-Blodgett 膜将被设计为通过混合脂质、脂化 PEG 和特异性结合 α(5)β(1) 和 α(6)β(4) 整联蛋白的肽两亲物来模拟双配体隐形脂质体的界面。将通过原子力显微镜研究肽浓度对肽和 PEG 之间混合行为的影响。研究任务 2：设计多靶点隐形脂质体，用特异性结合 α(5)β(1) 和 α(6)β(4) 整合素的肽两亲物进行功能化，并将其递送至具有不同整合素表达水平的细胞。将用不同浓度的两种肽来制备肽功能化的隐形脂质体。脂质体将被递送至具有不同整合素表达水平的不同癌细胞。荧光染料将首先被封装，并且脂质体的结合和内化将通过平板测定和共焦显微镜研究进行评估。脂质体将在促进特异性转录靶向方面进行进一步评估，因为它们将在 NF-kB 启动子的控制下递送白喉毒素片段 A (DTA) 基因。具有不同整合素表达水平的不同细胞的毒性将通过平板测定进行评估。研究任务3：体内评价多靶点隐形脂质体。研究任务 2 中的双配体制剂将被证明与 DLD-1 结肠癌细胞最佳结合，在此任务中将通过 microPET/CT 成像和生物分布研究评估其到达体内结肠肿瘤部位的能力。非靶向和单配体隐形脂质体将用作对照。单配体和双配体的最佳制剂将进一步评估其功效和毒性。对于这些研究，将使用 NF-kB 启动子控制下的 DTA 基因。

Proposal Number: CBET-1403564 Principal Investigator: Efrosini KokkoliInstitution: University of Minnesota - Twin CitiesTitle: Design and Characterization of Multi-Targeted Gene Delivery Nanoparticles for Specific Cancer Therapy By 2030 there could be 27 million incident cases of cancer worldwide, 17 million cancer deaths annually and 75 million persons alive with cancer within five years of diagnosis. Active targeting of an adhesion molecule that is expressed on the surface of the tumor cells, is a promising area of research and targeted nanoparticle delivery to cancer is an area of interest that may offer hope in improving the treatment of therapy-resistant cancers. Unfortunately, cancer-specificity by selective recognition of individual cellular receptors has rarely been achieved adequately to date, suffering from unintended delivery to healthy organs as some target disease markers are also expressed in areas other than tumors. The focus of this work is on the problem of specific delivery of non-viral gene delivery vehicles to cancer cells versus healthy cells. Proposed is the development of a multi-targeted therapeutic system, that will make the recognition of cancer cells more specific. The hypothesis of this work is that a higher degree of specificity for cancer cells could be achieved by designing a modular multi-targeted non-viral system that: i) introduces simultaneous targeting of the overexpressed cancer surface receptors alpha(5)beta(1) and alpha(6)beta(4) integrins as the first level of targeting at the extracellular level, and ii) targets the upregulated transcriptional activity of NF-kB that is inactive in healthy cells while it is highly upregulated in a variety of diseases including cancer, thereby introducing a new form of transcriptional targeting as the second level of specific targeting. To test this hypothesis the following tasks have been set: Research Task 1: Evaluate the effect of peptide concentration on the miscibility of peptide-amphiphiles and polyethylene glycol (PEG) molecules. Langmuir-Blodgett membranes will be designed that will mimic the interfaces of the dual-ligand stealth liposomes by mixing lipids, lipidated PEG and the peptide-amphiphiles that bind specifically to the alpha(5)beta(1) and alpha(6)beta(4) integrins. The effect of peptide concentration on the mixing behavior between the peptides and PEG will be investigated by atomic force microscopy. Research Task 2: To engineer multi-targeted stealth liposomes, functionalized with peptide-amphiphiles that bind specifically to the alpha(5)beta(1) and alpha(6)beta(4) integrins, and deliver them to cells with different integrin expression levels. Peptide-functionalized stealth liposomes will be prepared with different concentrations of the two peptides. The liposomes will be delivered to different cancer cells with varying levels of integrin expression. Fluorescent dyes will be encapsulated initially, and the binding and internalization of the liposomes will be evaluated with plate assays and confocal microscopy studies. The liposomes will be evaluated further with respect to promoting specific transcriptional targeting as they will deliver the diphtheria toxin fragment A (DTA) gene under the control of the NF-kB promoter. The toxicity of different cells with varying levels of integrin expression will be evaluated with plate assays. Research Task 3: To evaluate the multi-targeted stealth liposomes in vivo. Dual-ligand formulations from Research Task 2 that will be shown to bind best to DLD-1 colon cancer cells will be evaluated in this task for their ability to reach the colon tumor site in vivo, with microPET/CT imaging and biodistribution studies. Non-targeted and single-ligand stealth liposomes will be used as controls. The single- and dual-ligand optimal formulations will further be evaluated for their efficacy and toxicity. For these studies the DTA gene under the control of the NF-kB promoter will be employed.​