Veldhoen, S, Laufer, SD, Trampe, A and Restle, T. Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: quantitative analysis of uptake and biological effect. Nucleic Acids Res 34: 6561-6573

Institut für Molekulare Medizin, Universitätsklinikum Schleswig-Holstein, Universität zu Lübeck, Ratzeburger Allee 160 23538, Lübeck, Germany.
Nucleic Acids Research (Impact Factor: 9.11). 02/2006; 34(22):6561-73. DOI: 10.1093/nar/gkl941
Source: PubMed


Cell-penetrating peptides (CPPs) have evolved as promising new tools to deliver nucleic acids into cells. So far, the majority of these delivery systems require a covalent linkage between carrier and cargo. To exploit the higher flexibility of a non-covalent strategy, we focused on the characterisation of a novel carrier peptide termed MPGalpha, which spontaneously forms complexes with nucleic acids. Using a luciferase-targeted small interfering RNA (siRNA) as cargo, we optimised the conditions for MPGalpha-mediated transfection of mammalian cells. In this system, reporter gene activity could be inhibited up to 90% with an IC50 value in the sub-nanomolar range. As a key issue, we addressed the cellular uptake mechanism of MPGalpha/siRNA complexes applying various approaches. First, transfection of HeLa cells with MPGalpha/siRNA complexes in the presence of several inhibitors of endocytosis showed a significant reduction of the RNA interference (RNAi) effect. Second, confocal laser microscopy revealed a punctual intracellular pattern rather than a diffuse distribution of fluorescently labelled RNA-cargo. These data provide strong evidence of an endocytotic pathway contributing significantly to the uptake of MPGalpha/siRNA complexes. Finally, we quantified the intracellular number of siRNA molecules after MPGalpha-mediated transfection. The amount of siRNA required to induce half maximal RNAi was 10 000 molecules per cell. Together, the combination of methods provided allows for a detailed side by side quantitative analysis of cargo internalisation and related biological effects. Thus, the overall efficiency of a given delivery technique as well as the mechanism of uptake can be assessed.

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    • "Cell penetrating peptide (CPP) can transfer various cargos including protein (Choi et al., 2006; El- Andaloussi et al., 2007; Nakase et al., 2008), nuclear acid (Eguchi et al., 2001; Endoh & Ohtsuki, 2009), and the other drug vector (Koppelhus et al., 2008) into a range of cell types as well as the blood–brain barrier (Lindgren et al., 2000). However, the negative charge of siRNA may neutralize the positive charges of CPPs either by covalent attachment (Meade & Dowdy, 2007) or noncovalent conjugated (Veldhoen et al., 2006; Meade & Dowdy, 2008), and result to reduce efficiency of CPP. Until now, the siRNA delivery is still the key barrier for its application in clinic. "
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    ABSTRACT: Abstract The siRNA deliveries, for the siRNA's high negative charge, short serum half life, poor cellular internalization, etc, are still the key barriers for its application in clinic. In this study, several cell penetrating peptide (CPP) and dsRNA binding domain (dsRBD)-based fusion proteins have been developed and screened as the siRNA vector. The siRNA binding ability was measured by the agarose gel retardation, the cell uptaking was characterized under fluorescence microscopy, and further more RNAi effect was evaluated on the endogenous (GAPDH, western blot) and exogenous (GFP, flow cytometry analysis) genes in HeLa cell. Finally, the cytotoxicity was assessed on HeLa cells using cell counting kit-8. The efficiency of siRNA delivery by the CPP-dsRBD fusion protein was the CPP and the dsRBD dependent. Three fusion proteins showed similar efficiency of siRNA delivery when comparing to Lipofectamine RNAi Max as the siRNA carrier. These results indicated that these CPP-dsRBD-based fusion proteins were promising candidates as siRNA carriers.
    Full-text · Article · Feb 2014 · Drug Delivery
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    • "We hypothesized that minimal gene silencing resulted from limited dissociation of the siRNA from the high affinity RNA-binding domain and/or that the siRNA that did dissociate remained trapped in endocytic compartments. To test these possibilities, we treated cells with siRNA complexes in the presence or absence of chloroquine, an endosomolytic agent used to enhance gene delivery20,21,22 (Figure 6a). In contrast to treatment without chloroquine, HPRT message was reduced 92% in cells treated with either PTD-DRBD or PTD-2× DRBD (Figure 6a). "
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    ABSTRACT: Delivery of small interfering RNA (siRNA) targeted to specific cell types is a significant challenge for the development of RNA interference-based therapeutics. Recently, PTD-DRBD, a double-stranded RNA binding domain (DRBD) fused to the TAT protein transduction domain (PTD), was shown to be effective at delivering siRNA in a non-cell type-specific manner. Here, we evaluated the potential of DRBD as a general protein platform for targeted small interfering RNA (siRNA) delivery. We found that a single DRBD was insufficient to stably complex siRNA when fused to targeting peptides other than PTD, which facilitated nonspecific nucleic acid binding. In contrast to PTD-DRBD, fusion proteins containing two DRBDs (2× DRBD) yielded specific and stable siRNA binding. These proteins could mediate the cellular uptake of siRNA in vitro, though compared with PTD-DRBD gene silencing was attenuated by endosomal entrapment. Our findings suggest that unlike a single DRBD, 2× DRBD inhibits siRNA escape into the cytoplasm and/or induces an internalization pathway distinct from that of PTD-DRBD. Collectively, these data indicate that while 2× DRBD retains siRNA-binding activity when fused to different cell surface-interacting peptides, the utility of 2× DRBD for cell-specific RNA interference is limited without further protein engineering to enhance the bioavailability of the delivered siRNAs.Molecular Therapy - Nucleic Acids (2012) 1, e53; doi:10.1038/mtna.2012.43; published online 13 November 2012.
    Full-text · Article · Nov 2012 · Molecular Therapy - Nucleic Acids
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    • "Reporter genes are usually used as silencing targets to investigate the potential of siRNA delivery vectors because their expressed products are very easy to detect (Veldhoen et al. 2006; Xu et al. 2010). However, these reporter genes such as luciferase gene and GFP gene are exogenous relative to mammalian cells. "
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    ABSTRACT: Gene silencing induced by RNA interference using small interfering RNA (siRNA) provides a promising therapeutic approach for cancers. However, the lack of siRNA delivery vector has limited the development of siRNA therapy. The purpose of this study was to use the novel copolymer (mPEG5k-PCL1.2k)1.4-g-PEl10k to prepare siRNA-loaded nanoparticles for siRNA delivery. The results suggested that (mPEG5k-PCL1.2k)1.4-g-PEl10k could load siRNA to form nanoparticles with particle size less than 200 nm in a narrow distribution. Moreover, a certain density of positive charge existed onto the surfaces of nanoparticles. MTT assay results demonstrated that (mPEG5k-PCL1.2k)1.4-g-PEl10k/siRNA nanoparticles showed very low cytotoxicity. The gene silencing efficiency of (mPEG5k-PCL1.2k)1.4-g-PEl10k/siRNA nanoparticles was investigated through luciferase reporter gene assays. The expression of exogenous luciferase gene was significantly downregulated at a range of N/P ratio from 50 to 125, and was maximally inhibited at the N/P ratio of 125 with 54% and 59% reduction in MCF-7 and HepG2 cells, respectively. In the 4T1-luc cell line expressing luciferase stably, the silencing of endogenous luciferase gene also has a similar overall profile with maximal 54% reduction of luciferase expression. These results suggested that (mPEG5k-PCL1.2k)1.4-g-PEI10k/SiRNA nanoparticles could serve as a kind of highly efficient siRNA delivery system for down-regulating the expression of exogenous and endogenous target genes.
    Full-text · Article · Aug 2012 · Pharmazie
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