Long-term efficient gene delivery using polyethylenimine with modified Tat peptide Seiichi Yamano a, * , Jisen Dai a , Shigeru Hanatani a , Ken Haku a , Takuto Yamanaka a , Mika Ishioka a , Tadahiro Takayama a , Carlo Yuvienco b , Sachin Khapli c , Amr M. Moursi d , Jin K. Montclare b a Department of Prosthodontics, New York University College of Dentistry, New York, NY 10010, United States b Department of Chemical and Biomolecular Engineering, Polytechnic Institute of New York University, Brooklyn, NY 11201, United States c Division of Engineering, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates d Department of Pediatric Dentistry, New York University College of Dentistry, New York, NY 10010, United States article info Article history: Received 24 September 2013 Accepted 2 November 2013 Available online 20 November 2013 Keywords: Gene delivery Tat peptide Polyethylenimine Transfection Plasmid DNA Non-viral vector abstract Polyethylenimine (PEI), a cationic polymer, has been widely studied and shown great promise as an efficient gene delivery vehicle. Likewise, the HIV-1 Tat peptide, a cell-permeable peptide, has been successfully used for intracellular gene delivery. To improve the favorable properties of these two vectors, we combine PEI with the modified Tat peptide sequence bearing histidine and cysteine residues (mTat). In vitro mTat/PEI-mediated transfection was evaluated by luciferase expression plasmid in two cell types. mTat/PEI produced significant improvement (z5-fold) in transfection efficiency of both cell lines with little cytotoxicity when compared to mTat alone, PEI alone, or four commercial reagents. The particle size of mTat/PEI/DNA complex was significantly smaller than mTat or PEI alone, and it was correlated with higher transfection efficiency. Filipin III, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI transfection. In contrast, chlorpromazine, an inhibitor of clathrin-mediated endocy- tosis, did not. This suggested caveolae-mediated endocytosis as the transfection mechanism. Further- more, the results of in vivo studies showed that animals administered mTat/PEI/DNA intramuscularly had significantly higher and longer luciferase expression (z7 months) than those with mTat/DNA, PEI/DNA, or DNA alone, without any associated toxicity. The combination of mTat with PEI could significantly improve transfection efficiency, expanding the potential use as a non-viral gene vector both in vitro and in vivo. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Non-viral gene delivery systems are widely used in basic research and in pre-clinical gene therapy applications. Despite extensive testing of alternatives such as lipoplexes and polyplexes, transfection efficiency is generally low, except for cells transformed in vitro. The cationic polymer, polyethylenimine (PEI), is a useful delivery vehicle for oligonucleotides and ensures effective oligo- nucleotides delivery with low toxicity in spite of relatively short duration of gene expression [1]. Because PEI forms stable com- plexes with oligonucleotides, the positively charged particles are able to interact with anionic proteoglycans at the cell surface and enter cells by endocytosis [2]. PEI possesses the unique property of acting as a “proton sponge” that buffers the endosomal pH [3] and protects oligonucleotides from degradation [4]. The continuous proton influx also induces endosome osmotic swelling and rupture, which provides an escape mechanism for oligonucleotides to the cytoplasm [5]. A previous study by our group showed that, compared to other non-viral vectors, a cationic polymer-based vector is the most efficient across a wide range of cell lines [6]. The cell membrane has been recognized as a major barrier to efficient transfection [7]. In order to overcome this barrier, peptides that are capable of penetrating the plasma membrane have been employed. Cell-permeable peptides (CPPs) or protein transduction domains are derived from naturally occurring proteins such as the Tat protein of human immunodeficiency virus type 1 (HIV-1) [8,9], the structural protein VP22 of herpes simplex virus 1 [10], and the DNA binding domain (homeodomain) of Drosophila transcription * Corresponding author. Department of Prosthodontics, New York University College of Dentistry, 345 East 24th Street, 4W, New York, NY 10010, United States. Tel.: þ1 212 998 9714; fax: þ1 212 992 7100. E-mail address: sy23@nyu.edu (S. Yamano). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.11.012 Biomaterials 35 (2014) 1705e1715