Delivery of short interfering RNA using endosomolytic cell-penetrating peptides. FASEB J

Department of Neurochemistry, Stockholm University, Svante Arrhenius väg 21A, S-10691 Stockholm, Sweden.
The FASEB Journal (Impact Factor: 5.04). 10/2007; 21(11):2664-71. DOI: 10.1096/fj.06-6502com
Source: PubMed


Cell-penetrating peptides (CPPs) are peptides able to promote uptake of various cargos, including proteins and plasmids. Advances in recent years imply the uptake to be endocytic, where the current hurdle for efficient intracellular delivery is material being retained in the endosomes. In this study we wanted to compare the ability of various established CPPs to deliver siRNA and induce gene silencing of luciferase, with a novel designed penetratin analog having endosomolytic properties, using a noncovalent strategy. In principal, the penetratin analog EB1 will, upon protonation in the early-late endosomes, be able to form an amphipathic alpha helix resulting in permeabilization of the endosomal membrane. We demonstrate that even though all CPPs evaluated in this study can form complexes with siRNA, there is not a direct relationship between the complex formation ability and delivery efficacy. More important, although all CPPs significantly promote siRNA uptake, in some cases no gene silencing effect can be observed unless endosomal escape is induced. We find the designed endosomolytic peptide EB1 to be far more effective both in forming complexes and transporting biologically active siRNA than its parent peptide penetratin. We believe that developing CPPs with increased endosomolytical properties is a necessary step toward achieving biological effects at low concentrations for future in vivo applications.

Download full-text


Available from: Tolga Sutlu,
  • Source
    • "However, chitosan has some drawbacks in gene delivery systems, in particular inefficient endosomal escape, failure of DNA dissociation, and low transfection efficiency,6 and many researchers have tried to overcome these problems.7–12 One approach taken to improving transport from the endosomal compartment to the cytoplasm has been to incorporate fusogenic viral peptides and polyacrylic acid in which the protonated carboxyl group plays an important role.13,14 Some researchers have also demonstrated that addition of negatively charged polyanions can improve the in vitro transfection efficiency of chitosan and other polycations by loosening the polymer/DNA complexes.15–17 "
    [Show abstract] [Hide abstract]
    ABSTRACT: Cis-aconitate-modified chitosan-g-stearic acid (CA-CSO-SA) micelles were synthesized in this study to improve the gene transfection efficiency of chitosan-g-stearic acid (CSO-SA). The CA-CSO-SA micelles had a similar size, critical micelle concentration, and morphology, but their zeta potential and cytotoxicity were reduced compared with CSO-SA micelles. After modification with cis-aconitate, the CA-CSO-SA micelles could also compact plasmid DNA (pDNA) to form nanocomplexes. However, the DNA binding ability of CA-CSO-SA was slightly reduced compared with that of CSO-SA. The transfection efficiency mediated by CA-CSO-SA/pDNA against HEK-293 cells reached up to 37%, and was much higher than that of CSO-SA/pDNA (16%). Although the cis-aconitate modification reduced cellular uptake kinetics in the initial stages, the total amount of cellular uptake tended to be the same after 24 hours of incubation. An endocytosis inhibition experiment showed that the internalization mechanism of CA-CSO-SA/pDNA in HEK-293 cells was mainly via clathrin-mediated endocytosis, as well as caveolae-mediated endocytosis and macropinocytosis. Observation of intracellular trafficking indicated that the CSO-SA/pDNA complexes were trapped in endolysosomes, but CA-CSO-SA/pDNA was more widely distributed in the cytosol. This study suggests that modification with cis-aconitate improves the transfection efficiency of CSO-SA/pDNA.
    International Journal of Nanomedicine 06/2014; 9(1):2993-3003. DOI:10.2147/IJN.S61103 · 4.38 Impact Factor
  • Source
    • "Previous studies in the DNA delivery field have demonstrated that there are a range of reagents that can be incorporated (either physically or chemically) into delivery systems to help burst endosomes: e.g., chloroquine (Wagner, 1998; Wolfert and Seymour, 1998), and various fusogenic peptides (Cho et al., 2003; Lo and Wang, 2008; Wagner, 1999). Similar strategies in general also work for siRNA delivery (Lundberg et al., 2007; Rozema et al., 2003; Rozema et al., 2007). There are examples of agents that have been demonstrated specifically to enhance endosomal release of co-delivery micelleplexes. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Tumor cells exhibit drug resistant phenotypes that decrease the efficacy of chemotherapeutic treatments. The drug resistance has a genetic basis that is caused by an abnormal gene expression. There are several types of drug resistance: efflux pumps reducing the cellular concentration of the drug, alterations in membrane lipids that reduce cellular uptake, increased or altered drug targets, metabolic alteration of the drug, inhibition of apoptosis, repair of the damaged DNA, and alteration of the cell cycle checkpoints (Gottesman et al. , 2002, Holohan et al. , 2013). siRNA is used to silence the drug resistant phenotype and prevent this drug resistance response. Of the listed types of drug resistance, pump-type resistance (e.g., high expression of ATP-binding cassette transporter proteins such as P-glycoproteins (Pgp; also known as multi-drug resistance protein 1 or MDR1)) and apoptosis inhibition (e.g., expression of anti-apoptotic proteins such as Bcl-2) are the most frequently targeted for gene silencing. The co-delivery of siRNA and chemotherapeutic drugs has a synergistic effect, but many of the current projects do not control the drug release from the nanocarrier. This means the drug payload is released before the drug resistance proteins have degraded and the drug resistance phenotype has been silenced. Current research focuses on cross-linking the carrier's polymers to prevent premature drug release, but these carriers still rely on environmental cues to release the drug payload, and the drug may be released too early. In this review, we studied the release kinetics of siRNA and chemotherapeutic drugs from a broad range of carriers. We also give examples of carriers used to co-deliver siRNA and drugs to drug-resistant tumor cells, and we examine how modifications to the carrier affect the delivery. Lastly, we give our recommendations for the future directions of the co-delivery of siRNA and chemotherapeutic drug treatments.
    Biotechnology Advances 06/2014; 32(5). DOI:10.1016/j.biotechadv.2014.05.006 · 9.02 Impact Factor
  • Source
    • "siRNA uptake does not guarantee target gene knock-down (Lundberg et al., 2007; Metwally et al., 2012) and therefore the ability of the five transfection reagents to promote target gene knock-down using siRNA was investigated. bMDM were treated with optimal concentrations of transfection reagent and siRNA against the inflammasome-associated molecule MEFV, the function of which we are currently investigating. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The manipulation of the RNA interference pathway using small interfering RNA (siRNA) has become the most frequently used gene silencing method. However, siRNA delivery into primary cells, especially primary macrophages, is often considered challenging. Here we report the investigation of the suitability of two methodologies: transient transfection and electroporation, to deliver siRNA targeted against the putative immunomodulatory gene Mediterranean Fever (MEFV) into primary bovine monocyte-derived macrophages (bMDM). Eleven commerical transfection reagents were investigated with variable results with respect to siRNA uptake, target gene knock-down, cell toxicity and type I interferon (IFN) response induction. Three transfection reagents; Lipofectamine 2000, Lipofectamine RNAiMAX and DharmaFECT 3, were found to consistently give the best results. However, all the transfection reagents tested induced an IFN response in the absence of siRNA, which could be minimized by reducing the transfection reagent incubation period. In addition, optimized siRNA delivery into bMDM by electroporation achieved comparable levels of target gene knock-down as transient transfection, without a detectable IFN response, but with higher levels of cell toxicity. The optimized transient transfection and electroporation methodologies may provide a starting point for optimizing siRNA delivery into macrophages derived from other species or other cells considered difficult to investigate with siRNA.
    Veterinary Immunology and Immunopathology 04/2014; 158(3-4). DOI:10.1016/j.vetimm.2014.02.002 · 1.54 Impact Factor
Show more