Comparison of antisense oligonucleotide drug delivery systems

Antisense oligonucleotides (AS-ONs) are specific drugs to inhibit gene expression at the transcriptional level. They possess a poor bioavailability and can be degraded by nucleases very rapidly. Therefore, a strong need for the development of oligonucleotide drug delivery systems exists. In the present study, two commercially available liposomes (DOTAP, lipofectin), one artificial virus capsoid (polyoma VP1), two cationic acrylate nanoparticles and two protamine-based nanoparticle preparations (proticles) were compared. Physical parameters of all carrier systems including z-average size, size distribution and surface charge regarding were determined. Cellular uptake was measured by a microplate fluorescence quantification method and, in addition, was visualized in mouse fibroblasts by confocal laser scan microscopy (CLSM). A comparison of cytotoxicity of the different drug delivery systems was performed in vitro using a MTT assay. Mouse fibroblasts which were stable transfected with the cDNA of a N-methyl-d-aspartate (NMDA) receptor also served as functional antisense oligonucleotide test system based on excitotoxicity (cell death). In addition, the efficiency of our oligonucleotide delivery systems was compared on the level of protein expression by Western blotting. Concluding the results, an increased uptake of the ON was found (2–18fold) for all delivery systems compared to the free ON. Protamine-based nanoparticles showed a very low cytotoxicity in contradiction to all other carrier systems. Lipofectin could be identified as the most potent delivery system in terms of antisense effect, followed by protamine nanoparticles and DOTAP. Sequence-specific antisense effects up to 80% were observed in the functional cell death assay. The highest reduction of NMDA expression was obtained from liposomal preparations with approximately 60% analyzed by Western blot. D 2004 Elsevier B.V. All rights reserved.