A low molecular weight fraction of polyethylenimine (PEI) displays increased transfection efficiency of DNA and siRNA in fresh or lyophilized complexes

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Abstract

RNA interference (RNAi) represents a powerful method for specific gene silencing. It is mediated through small double-stranded RNA molecules (small interfering RNAs, siRNAs) which sequence-specifically trigger the cleavage and subsequent degradation of their target mRNA. One critical factor that determines the success of RNAi is the ability to deliver intact siRNAs into target cells. Polyethylenimines (PEIs) are synthetic polymers with a high cationic charge density which function as transfection reagents based on their ability to compact DNA or RNA into complexes. This paper describes the application of lyophilized PEI/siRNA complexes based on a novel polyethylenimine. By fractionation of a commercially available 25-kDa PEI using gel permeation chromatography, a low molecular weight polyethylenimine (PEI F25-LMW) with superior transfection efficacy and low toxicity in various cell lines is obtained. Complexes formed in 5% glucose, but not in 150 mM NaCl, can be lyophilized and reconstituted without loss of transfection efficacy. Furthermore, PEI F25-LMW is able to complex and fully protect siRNAs against nucleolytic degradation, and delivers siRNAs into cells where they display bioactivity. Upon lyophilization and reconstitution of PEI F25-LMW-based siRNA complexes, siRNAs are still able to efficiently induce RNAi. To further demonstrate their applicability, lyophilized PEI/siRNA complexes are employed for targeting of the growth factor VEGF. Treatment of PC-3 prostate carcinoma cells with fresh or with lyophilized complexes results in decreased cell proliferation in different assays due to the siRNA-mediated downregulation of VEGF. In conclusion, siRNAs can be applied in lyophilized formulations, and lyophilized PEI F25-LMW-based siRNA complexes represent a powerful, inexpensive, non-toxic and simple ready-to-use platform for the specific and efficient targeting of genes in vitro.

Introduction

RNA interference (RNAi) was originally recognized as an evolutionarily conserved defence mechanism in response to double-stranded RNA and represents a powerful, naturally occurring biological strategy for specific gene silencing [1]. It is mediated through 21–25 nt double-stranded RNA molecules (small interfering RNAs, siRNAs) which are intracellularly generated from long endogenous or exogenous double-stranded RNAs [2], [3], [4], [5] or directly transfected into the cells. Upon the incorporation of an siRNA into a silencing complex called RISC (RNA-induced silencing complex) and activation [6], the siRNA sequence-specifically guides the RISC complex to the target RNA and induces endonucleolytic cleavage of the mRNA strand within the target site. Due to the generation of unprotected RNA ends, this cleavage leads to the rapid degradation of the entire mRNA molecule. Since RNAi is able to provide the relatively easy ablation of the expression of a target gene, it is now commonly used as a powerful tool in biological and biomedical research. This includes the strategy of siRNA-mediated targeting for functional studies of various genes whose expression is known to be upregulated in a disease. Perhaps more important is the possibility of using genome-wide collections of siRNAs as screening tools.

Since siRNAs play a pivotal role in this process, the critical factors that determine the success of RNAi approaches are (i) the functionality of siRNAs and (ii) the ability to deliver intact siRNAs efficiently into the cells. The latter aspect requires broadly applicable and easy-to-use methods for the efficient and reproducible delivery of siRNAs in vitro. However, chemically unmodified siRNAs are rapidly degraded, and mammalian cells do not readily take up naked nucleic acids. So far, besides retroviruses, adenoviruses and lentiviruses as vehicles for RNA interference constructs, strategies to deliver siRNAs to target cells in cell culture include transduction by physical or chemical transfection, e.g. based on cationic lipids.

Polyethylenimines (PEIs) are synthetic polymers with a high cationic charge density and a protonable amino group in every third position [7], [8] and are able to deliver large DNA molecules such as 2.3 Mb yeast artificial chromosomes (YACs) [9] as well as plasmids or small oligonucleotides [7], [10], [11], [12] into mammalian cells in vitro and in vivo. This function as a potent transfection reagent relies on the PEI's ability to condense and compact the carried DNA into complexes, which form small colloidal particles allowing efficient cellular uptake through endocytosis. Additionally, this tight condensation of the DNA molecules as well as the buffering capacity of PEI in certain cellular compartments like endosomes and lysosomes also protects DNA from degradation (“proton sponge hypothesis”) [7], [8], [13], [14].

Extending the use of polyethylenimines to RNA complexation, we have shown previously that certain low molecular weight PEIs are able to complex small RNA molecules like 37 nt ribozymes. Upon complexation, the ribozymes were protected against enzymatic or non-enzymatic degradation and efficiently delivered into target cells in vitro and in vivo, where they were released displaying full bioactivity [15], [16], [17]. Under certain conditions, the PEI/ribozyme complexes retained their physical stability and biological activity also after lyophilization [18], as it has also been shown previously for DNA [19]. In more recent studies, we also demonstrated that the non-covalent complexation of synthetic siRNAs with a commercially available low molecular weight polyethylenimine (Jet-PEI) leads to the efficient siRNA stabilization and delivery [20].

In this paper, we describe the application of lyophilized PEI/siRNA complexes. By gel permeation chromatography of a commercially available 25 kDa PEI, we obtain a low molecular weight PEI fraction (PEI F25-LMW) with superior transfection efficacies and low toxicity in various cell lines. When the PEI F25-LMW-based DNA or siRNA complex formation is performed in 5% glucose, complexes can be lyophilized and reconstituted without loss of transfection efficacy and with full protection of the siRNAs. Treatment of PC-3 prostate carcinoma cells with fresh or with lyophilized complexes targeting the growth factor VEGF results in the siRNA-mediated downregulation of VEGF and to decreased cell proliferation. Thus, we show that lyophilized PEI F25-LMW-based siRNA complexes may provide a powerful ready-to-use platform for the specific and efficient targeting of genes.

Section snippets

Gel permeation chromatography and polyethylenimines

For the purification of the low molecular weight polyethylenimine PEI F25-LMW from 25 kDa PEI (Al 25-kDa, free base, water free, Sigma-Aldrich, Taufkirchen, Germany), gel permeation chromatography was performed based on a method described previously [21], but with several modifications. 3 g Sephadex G-50 resin (Amersham Biosciences, Freiburg, Germany) was pre-swollen in 300 ml 150 mM NaCl, and possible binding sites for PEI were blocked by adding 500 mg of the 25-kDa PEI described above to the

Results

In order to obtain a low molecular weight polyethylenimine with high transfection efficacy and low toxicity, gel permeation chromatography was employed to subfractionate commercially available 25-kDa PEI into 1 ml fractions. The elution profile (see Fig. 1a for a representative run) revealed a single large peak starting at fraction 28 with a peak maximum at fractions 32/33. While no other discrete peak was detectable, the determination of the PEI concentrations in later fractions revealed the

Discussion

In several studies, PEIs have been successfully used for non-viral gene delivery in vitro and in vivo. The strong buffer capacity, described by the ‘proton sponge hypothesis’ [7], seems to be responsible for the fact that PEI-based delivery does not require endosome disruptive agents for lysosomal escape. While initial publications showed increased transfection efficacies when using high molecular weight PEIs [27], more recent studies demonstrated the advantages of certain low molecular weight

Acknowledgements

We are grateful to Olga Bier, Robert Prinz, Helga Radler and Andrea Wüstenhagen for expert technical assistance and to Hermann Kalwa for valuable scientific input.

References (43)

  • C.M. Wiethoff et al.

    Barriers to nonviral gene delivery

    J. Pharm. Sci.

    (2003)
  • M.C. Molina et al.

    Maintenance of nonviral vector particle size during the freezing step of the lyophilization process is insufficient for preservation of activity: insight from other structural indicators

    J. Pharm. Sci.

    (2001)
  • T.J. Anchordoquy et al.

    Low molecular weight dextrans stabilize nonviral vectors during lyophilization at low osmolalities: concentrating suspensions by rehydration to reduced volumes

    J. Pharm. Sci.

    (2005)
  • M. Reinisalo et al.

    Freeze-drying of cationic polymer DNA complexes enables their long-term storage and reverse transfection of post-mitotic cells

    J. Control. Release

    (2006)
  • V. Oberle et al.

    Lipoplex formation under equilibrium conditions reveals a three-step mechanism

    Biophys. J.

    (2000)
  • A. Fire et al.

    Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

    Nature

    (1998)
  • A.J. Hamilton et al.

    A species of small antisense RNA in posttranscriptional gene silencing in plants

    Science

    (1999)
  • E. Bernstein et al.

    Role for a bidentate ribonuclease in the initiation step of RNA interference

    Nature

    (2001)
  • S.M. Elbashir et al.

    RNA interference is mediated by 21- and 22-nucleotide RNAs

    Genes Dev.

    (2001)
  • S.M. Hammond et al.

    An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells

    Nature

    (2000)
  • O. Boussif et al.

    A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine

    Proc. Natl. Acad. Sci. U. S. A.

    (1995)
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    These authors contributed equally to the work.

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