Lipid Nanoparticle-Mediated Amplified Detection of Active Extracellular Triple Helicases

该奖项由南达科他州立大学材料研究部的生物材料计划颁发，旨在研究多肽/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多肽裂解活性酶[基质金属蛋白酶(MMPs)和去整合素类金属蛋白酶(ADAMS)]。该奖项由化学、生物工程、环境和运输系统分部的颗粒和多相工艺计划共同资助。该奖项将开发一种补充策略，以确定这些酶的催化活性形式，这些酶水解三螺旋多肽。市面上可买到的检测试剂盒使用酶联免疫吸附试验(ELISA)来测量这些酶的总浓度(即催化活性和非活性形式)。为了确定酶的活性浓度，本研究将使用脂质体在其表面包裹高选择性的三螺旋脂肽底物，并将辣根过氧化物酶(HRP)包裹在脂质体的水腔中。同源酶对脂肽的水解会破坏脂体脂双层的稳定性，释放HRP，并使其与外部缓冲液中的化学发光底物接触。HRP催化底物的后续氧化将产生并放大发光信号。因此，所提出的方法不仅可以放大像酶联免疫吸附试验那样的信号，还可以检测和定量酶的催化活性形式。优化脂肽底物的机理研究、脂质体的失稳以及产生的信号的放大都是这一研究项目的一部分。研究生和本科生将在这个项目中工作，并将接受关于肽合成、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原住民部落社区学院招收和培训本科生是该项目的一部分。此外，在夏季的几个月里，高中生将接受这一跨学科研究活动的培训。许多酶有助于癌细胞的入侵和转移。市面上有几种试剂盒可以用来测定这些酶的含量。然而，这些试剂盒测量的是这些酶的总量，而不是导致转移的浓度。这项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，将准备脂泡，它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并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，该方法将是非常选择性、灵敏和定量的。这项拟议的研究是一名化学家和一名生物化学家之间的合作，该团队将培训研究生分子生物学、肽合成以及生物物理和分析化学的各种技术。调查人员计划访问附近北达科他州印第安人保留地的部落社区大学，招募本科生接受PI的研究活动培训，并将在指导下继续研究生教育。作为北达科他州州长学校计划的一部分，研究小组将在夏季的几个月里培训高中生进行这一项目。这项研究的结果将通过出版物和在科学会议上的陈述以及在美洲原住民社区大学中传播。

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.