Association of poly I:C RNA and plasmid DNA onto MnO nanorods mediated by PAMAM

Brooke Parker-Esquivel
Kristin J. Flores, MSU Graduate Student
Daniel Louiselle, MSU Undergraduate
Michael Craig, Missouri State University
Lifeng Dong, Missouri State University
Richard Garrad, Missouri State University
Kartik C. Ghosh, Missouri State University
Adam Wanekaya, Missouri State University
Garry Glaspell
Robert K. DeLong, Missouri State University

Abstract

In this study, manganese oxide (MnO) nanorods and its association with polyamidoamine dendrimer (PAMAM) and macromolecular RNA were analyzed. Because manganese is found naturally in cells and tissues and binds proteins and nucleic acids, nanomaterials derived from manganese, such as first generation MnO, may have potential as a biocompatible delivery agent for therapeutic or diagnostic biomedical applications. Nucleic acids have a powerful influence over cell processes, such as gene transcription and RNA processing; however, macromolecular RNA is particularly difficult to stabilize as a nanoparticle and to transport across cell membranes while maintaining structure and function. PAMAM is a cationic, branching dendrimer known to form strong complexes with nucleic acids and to protect them from degradation and is also considered to be a cell penetrating material. There is currently much interest in polyinosinic:polycytidylic RNA (poly I:C) because of its potent and specific immunogenic properties and as a solo or combination therapy. In order to address this potential, here, as a first step, we used PAMAM to attach poly I:C onto MnO nanorods. Morphology of the MnO nanorods was examined by field emission scanning electron microscopy (FESEM) and their composition by energy dispersive X-ray microanalysis (EDX). Evidence was generated for RNA:PAMAM:MnO nanorod binding by a gel shift assay using gel electrophoresis, a sedimentation assay using UV spectroscopy, and zeta potential shifts using dynamic laser light scattering. The data suggest that RNA was successfully attached to the MnO nanorods using PAMAM, and this suggestion was supported by direct visualization of the ternary complexes with FESEM characterizations. In order to confirm that the associations were biocompatible and taken up by cells, MTT assays were carried out to assess the metabolic activity of HeLa cells after incubation with the complexes and appropriate controls. Subsequently, we performed transfection assays using PAMAM:MnO complexes with pDNA encoding a green fluorescent protein reporter gene instead of RNA. The results suggest that the complexes had minimal impact on metabolic activity and were readily taken up by cells, and the fluorescent protein was expressed. From the evidence, we conclude that complexes of PAMAM:MnO interact with nucleic acids to form associations that are well-tolerated and readily taken up by cells. © 2012 American Chemical Society.