Lipid Nanoparticle-Mediated Amplified Detection of Active Extracellular Triple Helicases

North Dakota State University Fargo, Fargo ND

Investigators

Abstract

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.