Pharmaceutical Research

, Volume 29, Issue 3, pp 669–682 | Cite as

Comparison of Polymeric siRNA Nanocarriers in a Murine LPS-Activated Macrophage Cell Line: Gene Silencing, Toxicity and Off-Target Gene Expression

  • Linda B. Jensen
  • Joscha Griger
  • Broes Naeye
  • Amir K. Varkouhi
  • Koen Raemdonck
  • Raymond Schiffelers
  • Twan Lammers
  • Gert Storm
  • Stefaan C. de Smedt
  • Brian S. Sproat
  • Hanne M. Nielsen
  • Camilla Foged
Research Paper



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.


delivery macrophages polymer siRNA TNF-α 





2-aminoethyl methacrylate hydrochloride


analysis of variance


cyclin a2


cyclin-dependent kinase 7


crossing point


cellular apoptosis susceptibility protein 1L


diaminobutane dendrimers


dextran hydroxyethyl methacrylate


dextran methacrylate


Dulbecco´s Modified Eagle´s Medium




encapsulation efficiency


enhanced green fluorescent protein


fetal bovine serum


fluorescein isothiocyanate






4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid










N-hydroxysuccinimidyl-activated methoxypolyethylene glycol 5000 propionic acid


amine-to-phosphate ratio


oligoadenylate Synthetase-Like Protein 1d






photochemical internalization


polydispersity index


polyethylene glycol




polyfect (based on PAMAM dendrimers)


poly((2-hydroxypropyl)methacrylamide 1-methyl-2-piperidine methanol)


propidium iodide


poly(DL-lactide-co-glycolide acid)




rheumatoid arthritis


RNA interference


reverse transcription


small interfering RNA


toll-like receptor


[2-(methacryloyloxy)ethyl]-trimethylammonium chloride


thiolated N,N,N-trimethylated chitosan


tumor necrosis factor α



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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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Linda B. Jensen
    • 1
  • Joscha Griger
    • 1
    • 6
  • Broes Naeye
    • 2
  • Amir K. Varkouhi
    • 3
  • Koen Raemdonck
    • 2
  • Raymond Schiffelers
    • 3
  • Twan Lammers
    • 3
    • 4
  • Gert Storm
    • 3
  • Stefaan C. de Smedt
    • 2
  • Brian S. Sproat
    • 5
  • Hanne M. Nielsen
    • 1
  • Camilla Foged
    • 1
  1. 1.Department of Pharmaceutics and Analytical Chemistry Faculty of Pharmaceutical SciencesUniversity of CopenhagenCopenhagen ODenmark
  2. 2.Laboratory of General Biochemistry and Physical PharmacyGhent UniversityGhentBelgium
  3. 3.Department of Pharmaceutics, Faculty of ScienceUtrecht UniversityUtrechtThe Netherlands
  4. 4.Department of Experimental Molecular ImagingRWTH Aachen UniversityAachenGermany
  5. 5.Chemconsilium GCVBooischotBelgium
  6. 6.Max Delbrueck Centre for Molecular MedicineBerlin-BuchGermany

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