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

项目编号：cbet1403564首席研究员：Efrosini kokkoli研究机构：明尼苏达大学-双子市分校：设计和表征用于特定癌症治疗的多靶向基因递送纳米颗粒到2030年，全球将有2700万癌症病例，每年1700万癌症死亡，7500万人在确诊后的五年内存活。主动靶向在肿瘤细胞表面表达的粘附分子是一个很有前途的研究领域，靶向纳米颗粒递送到癌症是一个有趣的领域，可能为改善治疗耐药癌症的治疗提供希望。不幸的是，迄今为止，通过选择性识别单个细胞受体的癌症特异性很少得到充分实现，由于一些目标疾病标志物也在肿瘤以外的区域表达，因此难以意外地传递到健康器官。这项工作的重点是非病毒基因传递载体对癌细胞和健康细胞的特异性传递问题。建议开发一种多靶点治疗系统，这将使癌细胞的识别更加特异性。这项工作的假设是，通过设计一个模块化的多靶向非病毒系统，可以实现对癌细胞的更高程度的特异性：i)引入了同时靶向过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级靶向，ii）靶向NF-kB的转录活性上调，NF-kB在健康细胞中不活跃，而在包括癌症在内的各种疾病中高度上调，从而引入了一种新的转录靶向形式作为特异性靶向的第二级。为了验证这一假设，我们设置了以下任务：研究任务1：评估肽浓度对肽-两亲分子和聚乙二醇（PEG）分子混溶性的影响。Langmuir-Blodgett膜将通过混合脂质、脂化PEG和特异性结合α (5) β(1)和α (6) β(4)整合素的肽-两亲体来模拟双配体隐身脂质体的界面。多肽浓度对多肽与聚乙二醇混合行为的影响将通过原子力显微镜进行研究。研究任务2：设计多靶向隐形脂质体，用肽两亲体功能化，特异性结合α (5) β(1)和α (6) β(4)整合素，并将其递送到不同整合素表达水平的细胞中。用两种多肽的不同浓度制备多肽功能化隐身脂质体。脂质体将被递送到整合素表达水平不同的不同癌细胞中。荧光染料最初将被封装，脂质体的结合和内化将通过平板测定和共聚焦显微镜研究进行评估。脂质体将在NF-kB启动子控制下递送白喉毒素片段A （DTA）基因，因此将进一步评估其促进特异性转录靶向的作用。整合素表达水平不同的细胞的毒性将通过平板试验进行评估。研究任务3：对多靶点隐身脂质体进行体内评价。研究任务2中的双配体配方将被证明与DLD-1结肠癌细胞结合最好，该任务将通过微pet /CT成像和生物分布研究来评估其在体内到达结肠癌肿瘤部位的能力。非靶向和单配体隐身脂质体将作为对照。单配体和双配体的最佳配方将进一步评估其功效和毒性。在这些研究中，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.​