Anna-Laurence Schachner-Nedherer1, Oliver Werzer1, Karin Kornmueller2, Ruth Prassl2, Andreas Zimmer1. 1. Department of Pharmaceutical Technology and Biopharmacy, Institute of Pharmaceutical Sciences, University of Graz, Graz 8010, Austria. 2. Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Biophysics, Medical University of Graz, Graz 8010, Austria.
Abstract
BACKGROUND: Endogenously expressed microRNAs (miRNAs) have attracted attention as important regulators in post-transcriptionally controlling gene expression of various physiological processes. As miRNA dysregulation is often associated with various disease patterns, such as obesity, miRNA-27a might therefore be a promising candidate for miRNA mimic replacement therapy by inhibiting adipogenic marker genes. However, application of naked nucleic acids faces some limitations concerning poor enzymatic stability, bio-membrane permeation and cellular uptake. To overcome these obstacles, the development of appropriate drug delivery systems (DDS) for miRNAs is of paramount importance. METHODS: In this work, a triple combination of atomic force microscopy (AFM), brightfield (BF) and fluorescence microscopy was used to trace the cellular adhesion of N-TER peptide-nucleic acid complexes followed by time-dependent uptake studies using confocal laser scanning microscopy (cLSM). To reveal the biological effect of miRNA-27a on adipocyte development after transfection treatment, Oil-Red-O (ORO)- staining was performed to estimate the degree of in lipid droplets accumulated ORO in mature adipocytes by using light microscopy images as well as absorbance measurements. RESULTS: The present findings demonstrated that amphipathic N-TER peptides represent a suitable DDS for miRNAs by promoting non-covalent complexation through electrostatic interactions between both components as well as cellular adhesion of the N-TER peptide - nucleic acid complexes followed by uptake across cell membranes and intracellular release of miRNAs. The anti-adipogenic effect of miRNA-27a in 3T3-L1 cells could be detected in mature adipocytes by reduced lipid droplet formation. CONCLUSION: The present DDS assembled from amphipathic N-TER peptides and miRNAs is capable of inducing the anti-adipogenic effect of miRNA-27a by reducing lipid droplet accumulation in mature adipocytes. With respect to miRNA mimic replacement therapies, this approach might provide new therapeutic strategies to prevent or treat obesity and obesity-related disorders.
BACKGROUND: Endogenously expressed microRNAs (miRNAs) have attracted attention as important regulators in post-transcriptionally controlling gene expression of various physiological processes. As miRNA dysregulation is often associated with various disease patterns, such as obesity, miRNA-27a might therefore be a promising candidate for miRNA mimic replacement therapy by inhibiting adipogenic marker genes. However, application of naked nucleic acids faces some limitations concerning poor enzymatic stability, bio-membrane permeation and cellular uptake. To overcome these obstacles, the development of appropriate drug delivery systems (DDS) for miRNAs is of paramount importance. METHODS: In this work, a triple combination of atomic force microscopy (AFM), brightfield (BF) and fluorescence microscopy was used to trace the cellular adhesion of N-TER peptide-nucleic acid complexes followed by time-dependent uptake studies using confocal laser scanning microscopy (cLSM). To reveal the biological effect of miRNA-27a on adipocyte development after transfection treatment, Oil-Red-O (ORO)- staining was performed to estimate the degree of in lipid droplets accumulated ORO in mature adipocytes by using light microscopy images as well as absorbance measurements. RESULTS: The present findings demonstrated that amphipathic N-TER peptides represent a suitable DDS for miRNAs by promoting non-covalent complexation through electrostatic interactions between both components as well as cellular adhesion of the N-TER peptide - nucleic acid complexes followed by uptake across cell membranes and intracellular release of miRNAs. The anti-adipogenic effect of miRNA-27a in 3T3-L1 cells could be detected in mature adipocytes by reduced lipid droplet formation. CONCLUSION: The present DDS assembled from amphipathic N-TER peptides and miRNAs is capable of inducing the anti-adipogenic effect of miRNA-27a by reducing lipid droplet accumulation in mature adipocytes. With respect to miRNA mimic replacement therapies, this approach might provide new therapeutic strategies to prevent or treat obesity and obesity-related disorders.
Authors: Sang Yun Kim; A Young Kim; Hyun Woo Lee; You Hwa Son; Gha Young Lee; Joo-Won Lee; Yun Sok Lee; Jae Bum Kim Journal: Biochem Biophys Res Commun Date: 2010-01-07 Impact factor: 3.575