Lipid Nanoparticle-Mediated Amplified Detection of Active Extracellular Triple Helicases

脂质纳米颗粒介导的活性细胞外三重螺旋酶的放大检测

• 批准号: 1306154
• 负责人: Sanku Mallik
• 金额: $ 34万
• 依托单位: North Dakota State University Fargo
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-15 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1306154&HistoricalAwards=false
• 关键词: Lipid; Nanoparticle; Mediated; Amplified; Detection

This award by the Biomaterials program in the Division of Materials Research to South Dakota State University is to study peptide/substrate-decorated liposomes for detection of peptide-cleaving active enzymes [matrix metalloproteinases (MMPs) and the disintegrin class of metalloproteinases (ADAMs)] that are associated with cancer tumor growth and metastasis. This award is cofunded by the Particulate and Multiphase Processes program in the Division of Chemical, Bioengineering, Environmental, and Transport Systems. This award would develop a complementary strategy to determine the catalytically active forms of these enzymes that hydrolyze triple helical peptides. The commercially available test kits using enzyme-linked immunosorbent assay (ELISA) measures the total concentrations of these enzymes (i.e., both catalytically active and inactive forms). To determine the active enzyme concentration, this research will use liposomes to harbor highly selective, triple helical lipo-peptide substrates on their surface, and the horseradish peroxidase (HRP) enzyme will be encapsulated in the aqueous liposome lumen. Hydrolysis of the lipo-peptides by the cognate enzymes will destabilize the liposomes lipid bilayer, releasing HRP, and bringing it in contact with a chemiluminescent substrate in the external buffer. Subsequent oxidation of the substrate catalyzed by HRP will generate and amplify the luminescent signal. Hence, the proposed methodology will not only amplify the signal like ELISA, but also will detect and quantify the catalytically active forms of the enzymes. Mechanistic studies to optimize the lipo-peptide substrates, the destabilization of the liposomes and the amplification of the signal generated are parts of this research project. Graduate and undergraduate students will work in this project and will be trained on peptide synthesis, molecular biology, biophysics and analytical chemistry. Visits to the Native American tribal community colleges in North Dakota to recruit and train undergraduate students are part of this project. Additionally, during the summer months, high school students will be trained in this interdisciplinary research activity.Many enzymes contribute to the invasion of the cancer cells and their metastasis. Several kits are commercially available to determine the amounts of these enzymes. However, these kits measure the total amounts of these enzymes and not the concentrations contributing to the metastasis. This research project will develop new strategies to determine the concentrations of active forms of of these two enzymes. As part of this project, lipid vesicles will be prepared that will 'recognize' the active metastasis-causing enzymes that in turn will release another enzyme from the interior of the vesicles and this in turn will result in luminescent signal. This sequence of events will generate and amplify the signal as a function of time, and would quantify the active form of the circulating enzymes. Hence, the method will be very selective, sensitive and quantitative. The proposed research is collaboration between a Chemist and a Biochemist, and the team will train graduate students in various techniques of molecular biology, peptide synthesis, and biophysical and analytical chemistry. The investigators plan to visit the tribal community colleges in the nearby Native American reservations of North Dakota to recruit undergraduate students to be trained in the PIs' research activities, and will be mentored to pursue graduate education. As part of the North Dakota Governor's School program, the research team will train high school students in this project during the summer months. The results from this research will be disseminated through publications and presentations in scientific meetings as well as in the Native American community colleges.

该奖项由南达科他州州立大学材料研究部生物材料项目授予，旨在研究肽/底物修饰的脂质体，用于检测与癌症肿瘤生长和转移相关的肽裂解活性酶[基质金属蛋白酶（MMP）和金属蛋白酶的去整合素类（亚当斯）]。该奖项由化学，生物工程，环境和运输系统部门的颗粒和多相过程计划共同资助。该奖项将开发一个互补的策略，以确定这些酶的催化活性形式，水解三螺旋肽。使用酶联免疫吸附测定（ELISA）的市售测试试剂盒测量这些酶的总浓度（即，催化活性和非活性形式）。为了确定活性酶的浓度，本研究将使用脂质体来在其表面上具有高度选择性的三螺旋脂肽底物，并且辣根过氧化物酶（HRP）酶将被封装在水性脂质体腔中。 通过同源酶水解脂肽将使脂质体脂质双层不稳定，释放HRP，并使其与外部缓冲液中的荧光底物接触。 随后由HRP催化的底物的氧化将产生并放大发光信号。 因此，所提出的方法不仅可以像ELISA那样放大信号，而且还可以检测和量化酶的催化活性形式。优化脂肽底物、脂质体不稳定和所产生信号放大的机制研究是该研究项目的一部分。研究生和本科生将在这个项目中工作，并将接受肽合成，分子生物学，生物物理学和分析化学的培训。访问北达科他州的美洲土著部落社区学院以招募和培训本科生是该项目的一部分。 此外，在夏季的几个月里，高中生将接受跨学科研究活动的培训。许多酶有助于癌细胞的侵袭和转移。 几种试剂盒可商购获得以测定这些酶的量。 然而，这些试剂盒测量这些酶的总量，而不是有助于转移的浓度。 该研究项目将开发新的策略来确定这两种酶的活性形式的浓度。作为该项目的一部分，将制备脂质囊泡，其将“识别”引起转移的活性酶，这些酶反过来将从囊泡内部释放另一种酶，这反过来将导致发光信号。 这一系列事件将产生和放大作为时间函数的信号，并将量化循环酶的活性形式。 因此，该方法将是非常有选择性，灵敏度和定量。 拟议的研究是化学家和生物化学家之间的合作，该团队将在分子生物学，肽合成，生物物理和分析化学的各种技术方面培训研究生。 调查人员计划访问附近北达科他州美洲原住民保留地的部落社区学院，招募本科生接受PI研究活动的培训，并将接受指导，接受研究生教育。作为北达科他州州长学校项目的一部分，研究小组将在夏季的几个月里对高中生进行这一项目的培训。这项研究的结果将通过出版物和在科学会议上以及在美洲土著社区学院中的介绍来传播。