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

ABSTRACT

Purpose

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.

Methods

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.

Results

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.

Conclusions

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 WORDS

delivery macrophages polymer siRNA TNF-α 

ABBREVIATIONS

Act

β-actin

AEMA

2-aminoethyl methacrylate hydrochloride

ANOVA

analysis of variance

Ccna2

cyclin a2

Cdk7

cyclin-dependent kinase 7

CP

crossing point

Cse1L

cellular apoptosis susceptibility protein 1L

DAB

diaminobutane dendrimers

dex-HEMA

dextran hydroxyethyl methacrylate

dex-MA

dextran methacrylate

DMEM

Dulbecco´s Modified Eagle´s Medium

DOTAP

1,2-dioleoyloxy-3-trimethylammoniumpropane

EE

encapsulation efficiency

EGFP

enhanced green fluorescent protein

FBS

fetal bovine serum

FITC

fluorescein isothiocyanate

G

generation

Gus

β-glucuronidase

HEPES

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

IL

interleukin

INF

interferon

LPS

lipopolysaccharide

LUC

luciferase

NHS-PEG

N-hydroxysuccinimidyl-activated methoxypolyethylene glycol 5000 propionic acid

N/P

amine-to-phosphate ratio

OAS1d

oligoadenylate Synthetase-Like Protein 1d

OF

oligofectamine

PAMAM

poly(amidoamine)

PCI

photochemical internalization

PDI

polydispersity index

PEG

polyethylene glycol

PEI

polyethylenimine

PF

polyfect (based on PAMAM dendrimers)

pHPMA-MPPM

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

PI

propidium iodide

PLGA

poly(DL-lactide-co-glycolide acid)

PVA

polyvinylalcohol

RA

rheumatoid arthritis

RNAi

RNA interference

RT

reverse transcription

siRNA

small interfering RNA

TLR

toll-like receptor

TMAEMA

[2-(methacryloyloxy)ethyl]-trimethylammonium chloride

TMC-SH

thiolated N,N,N-trimethylated chitosan

TNF-α

tumor necrosis factor α

Notes

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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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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