Article

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

Department of Neurochemistry, Stockholm University, Svante Arrhenius väg 21A, S-10691 Stockholm, Sweden.
The FASEB Journal (Impact Factor: 5.48). 10/2007; 21(11):2664-71. DOI: 10.1096/fj.06-6502com
Source: PubMed

ABSTRACT 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

Full-text

Available from: Tolga Sutlu, Jun 29, 2015
0 Followers
 · 
127 Views
  • Source
    [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; DOI:10.1016/j.biotechadv.2014.05.006 · 8.91 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The PepFect family of cell-penetrating peptides (CPPs) was designed to improve the delivery of nucleic acids across plasma membranes. We present here a comparative study of two members of the family, PepFect3 (PF3) and PepFect6 (PF6), together with their parental CPP transportan-10 (TP10), and their interactions with lipid membranes. We show that the addition of a stearyl moiety to TP10 increases the amphipathicity of these molecules and their ability to insert into a lipid monolayer composed of zwitterionic phospholipids. The addition of negatively charged phospholipids into the monolayer results in decreased binding and insertion of the stearylated peptides, indicating modification in the balance of hydrophobic versus electrostatic interactions of peptides with lipid bilayer, thus revealing some clues for the selective interaction of these CPPs with different lipids. The trifluoromethylquinoline moieties, in PF6 make no significant contribution to membrane binding and insertion. TP10 actively introduces pores into the bilayers of large and giant unilamellar vesicles, while PF3 and PF6 do so only at higher concentrations. This is consistent with the lower toxicity of PF3 and PF6 observed in previous studies.
    Biochimica et Biophysica Acta 03/2012; 1818(3):915-24. DOI:10.1016/j.bbamem.2011.12.028 · 4.66 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides that can be used to deliver a variety of cargos into cells. However, it is still debated which routes CPPs employ to gain access to intracellular compartments. To assess this, most previously conducted studies have relied on information which is gained by using fluorescently labeled CPPs. More relevant information whether the internalized conjugates are biologically available has been gathered using end-point assays with biological readouts. Uptake kinetic studies have shed even more light on the matter because the arbitrary choice of end-point might have profound effect how the results could be interpreted. To elucidate uptake mechanisms of CPPs, here we have used a bioluminescence based assay to measure cytosolic delivery kinetics of luciferin-CPP conjugates in the presence of endocytosis inhibitors. The results suggest that these conjugates are delivered into cytosol mainly via macropinocytosis; clathrin-mediated endocytosis and caveolae/lipid raft dependent endocytosis are involved in a smaller extent. Furthermore, we demonstrate how the involved endocytic routes and internalization kinetic profiles can depend on conjugate concentration in case of certain peptides, but not in case of others. The employed internalization route, however, likely dictates the intracellular fate and subsequent trafficking of internalized ligands, therefore emphasizing the importance of our novel findings for delivery vector development.
    Biochimica et Biophysica Acta 12/2011; 1818(3):502-11. DOI:10.1016/j.bbamem.2011.11.020 · 4.66 Impact Factor