Article

Intracellular delivery of siRNA by cell-penetrating peptides modified with cationic oligopeptides.

DDS Institute, The Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato, Tokyo 105-8461, Japan.
Drug Delivery (Impact Factor: 2.2). 05/2009; 16(3):153-9. DOI: 10.1080/10717540902722774
Source: PubMed

ABSTRACT To achieve the effective intracellular delivery of siRNA and silence specific genes, various types of conjugates between cell-penetrating peptides (CPPs; Transportan, Penetratin, Tat) and cationic peptides were developed. Uptake, intracellular localization, cytotoxicity, and biological activity of siRNA were significantly dependent on the kind of CPP used and the length of the cationic peptides in the conjugate. Transportan-based conjugates yielded both high internalization of siRNA and strong gene silencing activity, while Penetratin- and Tat-based conjugates did not. These different properties of CPPs emphasize the importance of careful peptide selection and design when attempting the application of CPP technology.

0 Followers
 · 
93 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Interest in cell-penetrating peptides (CPPs) as delivery agents has fuelled a large number of studies conducted on cultured cells and in mice. However, only a few studies have been devoted to the behaviour of CPPs in human tissues. Therefore, we performed ex vivo tissue-dipping experiments where we studied the distribution of CPP-protein complexes in samples of freshly harvested human tissue material. We used the carcinoma or hyperplasia-containing specimens of the uterus and the cervix, obtained as surgical waste from nine hysterectomies. Our aim was to evaluate the tissue of preference (epithelial versus muscular/connective tissue, carcinoma versus adjacent histologically normal tissue) for two well-studied CPPs, the transportan and the TAT-peptide. We complexed biotinylated CPPs with avidin--galactosidase (ABG), which enabled us to apply whole-mount X-gal staining as a robust detection method. Our results demonstrate that both peptides enhanced the tissue distribution of ABG. The enhancing effect of the tested CPPs was more obvious in the normal tissue and in some specimens we detected a striking selectivity of CPP-ABG complexes for the normal tissue. This unexpected finding encourages the evaluation of CPPs as local delivery agents in non-malignant situations, for example in the intrauterine gene therapy of benign gynaecological diseases.
    Pharmaceuticals 03/2010; 3(3). DOI:10.3390/ph3030621
  • [Show abstract] [Hide abstract]
    ABSTRACT: Gene silencing by RNA interference (RNAi) is a promising therapeutic approach for a wide variety of diseases for which the biological cause is known. The main challenge remains the ineffective RNAi delivery inside the cells. Non-viral gene delivery vectors have low immunogenicity compared to viral vectors, but are constrained by their reduced transfection efficiency. Silencing of the bcr-abl gene expression by RNAi confers therapeutic potential in Chronic Myeloid Leukemia (CML), but is limited by the cytotoxicity of the existing delivery methods. Here, we present evidence that the fusion between the cell penetrating peptide (CPP) HIV-Tat (49-57) and the membrane lytic peptide (LK15), Tat-LK15, mediates high transfection efficiency in delivering short hairpin RNA (shRNA) and small interfering RNA (siRNA) targeting the BCR-ABL oncoprotein in K562 CML cells. Our results show that shRNA complexes induce a more stable gene silencing of bcr-abl when compared to silencing mediated by siRNA complexes. In addition, silencing of the BCR-ABL oncoprotein by both shRNA and siRNA delivered by Tat-LK15 is more efficient and longer lasting than that achieved using Lipofectamine and more importantly without considerable cytotoxicity. In these terms Tat-LK15 can be an alternative to DNA/siRNA delivery in difficult-to-transfect leukemic cells.
    Journal of Controlled Release 08/2010; 145(3):272-80. DOI:10.1016/j.jconrel.2010.04.011 · 7.26 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cell-penetrating peptides comprising cloned epitopes that contribute to membrane transduction, DNA-binding and cell targeting functions are known to facilitate nucleic acid delivery. Using the ITASSER software, we predicted the 3-D structure of a well characterized and efficient transfecting cell-penetrating peptide, namely TAT-Mu and its derivative TAT-Mu-AF protein that harbors a targeting ligand, the HER2-binding affibody. Our model predicts TAT-Mu-AF fusion protein as primarily comprising α-helices. The affibody in TAT-Mu-AF is predicted as a 3-helical domain that is distinct from the TAT-Mu domain. Its positioning in three-dimensional structure is oriented in a manner that possibly favors interactions with receptor and facilitates transport to the target site. The linker region between TAT-Mu and the affibody is also predicted as a helix that is likely to stabilize the overall fold of the TAT-Mu-AF complex. Further, the evaluation of secondary structure of the designed TAT-Mu-AF fusion protein by circular dichroism is in support of our predictions.
    Systems and Synthetic Biology 12/2010; 4(4):293-7. DOI:10.1007/s11693-011-9074-7