Comparison of Polymeric siRNA Nanocarriers in a Murine LPS-Activated Macrophage Cell Line: Gene Silencing, Toxicity and Off-Target Gene Expression
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Tumor necrosis factor α (TNF-α) plays a key role in the progression of rheumatoid arthritis and is an important target for anti-rheumatic therapies. TNF-α expression can be silenced with small interfering RNA (siRNA), but efficacy is dependent on efficient and safe siRNA delivery vehicles. We aimed to identify polymeric nanocarriers for anti-TNF-α siRNA with optimal efficacy and minimal off-target effects in vitro.
TNF-α silencing with polymeric siRNA nanocarriers was compared in lipopolysaccharide-activated RAW 264.7 macrophages by real-time reverse transcription (RT)-PCR. Expression of non-target genes involved in inflammation, apoptosis, and cell cycle progression was determined by RT-PCR, toxicity evaluated by propidium iodide and annexin V staining.
PAMAM dendrimers (G4 and G7) and dextran nanogels mediated remarkably high concentration-dependent gene silencing and low toxicity; dioleoyltrimethylammoniumpropane-modified poly(DL-lactide-co-glycolide acid) nanoparticles, thiolated, trimethylated chitosan and poly[(2-hydroxypropyl)methacrylamide 1-methyl-2-piperidine methanol] polyplexes were less efficient transfectants. There were minor changes in the regulation of off-target genes, mainly dependent on nanocarrier and siRNA concentration.
Dextran nanogels and PAMAM dendrimers mediated high gene silencing with minor toxicity and off-target transcriptional changes and are therefore expected to be suitable siRNA delivery systems in vivo.
KEY WORDSdelivery macrophages polymer siRNA TNF-α
2-aminoethyl methacrylate hydrochloride
analysis of variance
cyclin-dependent kinase 7
cellular apoptosis susceptibility protein 1L
dextran hydroxyethyl methacrylate
Dulbecco´s Modified Eagle´s Medium
enhanced green fluorescent protein
fetal bovine serum
N-hydroxysuccinimidyl-activated methoxypolyethylene glycol 5000 propionic acid
oligoadenylate Synthetase-Like Protein 1d
polyfect (based on PAMAM dendrimers)
poly((2-hydroxypropyl)methacrylamide 1-methyl-2-piperidine methanol)
small interfering RNA
thiolated N,N,N-trimethylated chitosan
tumor necrosis factor α
ACKNOWLEDGMENTS & DISCLOSURES
We gratefully thank Kirsten Vikkelsø Madsen for valuable scientific discussions concerning real time RT-PCR, Lasse Bengtson for preparation of the DOTAP/PLGA nanoparticles, and Dr. Michael Timm for testing pyrogen levels of the delivery systems (all from the Faculty of Pharmaceutical Sciences, University of Copenhagen). This study has been carried out with financial support from the Commission of the European Communities, Priority 3 “Nanotechnologies and Nanosciences, Knowledge Based Multifunctional Materials, New Production Processes and Devices” of the Sixth Framework Programme for Research and Technological Development (Targeted Delivery of Nanomedicine: NMP4-CT-2006-026668). We are grateful to the Danish Agency for Science, Technology and Innovation, the Drug Research Academy and the Carlsberg Foundation for financial support for the Zetasizer Nano ZS, Nanodrop 2000 C Spectrophotometer and the LightCycler® 480 system, respectively. The funding sources had no involvement in the study design, in the collection, analysis and interpretation of data, just as they had no involvement in the writing of the report and the decision to submit the paper for publication.
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