Bioconjugate Chem. 1999, 10, 773−780
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Chain Length of Cationic r-Helical Peptide Sufficient for Gene Delivery into Cells Takuro Niidome,*,† Keiko Takaji,† Mamiko Urakawa,† Naoya Ohmori,† Akihiro Wada,‡ Toshiya Hirayama,‡ and Haruhiko Aoyagi† Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, Nagasaki 852-8521, Japan, and Department of Bacteriology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan. Received February 4, 1999; Revised Manuscript Received May 19, 1999
To define the minimal peptide length needed for gene delivery into mammalian cells, we synthesized several peptides with shortened chain lengths from the amino-termini of the original amphiphilic peptides (46, Ac-LARL-LARL-LARL-LRAL-LRAL-LRAL-NH2, and Hel 11-7, KLLK-LLLK-LWKKLLKL-LK), which have been known to have gene transfer abilities into cells. Each synthetic peptide was studied for its ability to bind and aggregate with plasmid DNA and the structural change of the peptide caused by binding with the DNA to establish a relative in vitro gene transfection efficiency in COS-7 cells. As a result, the deletion of eight amino acid residues of 46 had little influence on their ability, whereas that of 12 amino acid residues remarkably reduced the abilities to make aggregates and transfer the DNA into the cell. In the case of the Hel 11-7 series peptides, deletion of amino acid residues caused a considerable reduction in abilities to bind and form aggregates with DNA and to transfer the DNA into cell in due order. In summary, 16 and 17 amino acid residues were sufficient to form aggregates with the DNA and transfer the DNA into the cells in the deletion series of 46 and Hel 11-7, respectively. Furthermore, it was indicated that reduction of membrane perturbation activity of the peptide-DNA complex due to deletion of the peptide chain length caused suppression of the transfection efficiency even if the complex was incorporated into the cells. Transfer of the complex to cytosol mediated by membrane perturbation activity of the peptide is an important step for efficient protein expression from its cDNA. The results of this study will make it easy to design and synthesize a functional gene carrier molecule such as a carbohydrate-modified peptide used in targeted gene delivery.
INTRODUCTION
An efficient gene-transfer technique is necessary for the development of gene therapy. Various methods are being developed to transfect mammalian cells with foreign genes. In these situations, nonviral gene delivery systems are expected to introduce genes into cells without several problems, which are pointed out in transfection methods using viral vectors, such as endogenous virus recombinations, oncogenic effects and immunogenic reactions (1, 2). Nonviral delivery systems generally consist of a cationic polymer such as polylysine delivartives (35), polyethyleneimines (6) and polyamidoamine dendrimers (7), or cationic lipids (8) that electrostatically form a complex with the negatively charged DNA that can be taken up by the cells. Furthermore, Gottschalk et al. and Wadhwa et al. reported that synthetic peptides containing several cationic amino acids are useful as a gene carrier molecule (9, 10). The low molecular and homogeneous peptides are expected to easily construct a functional carrier molecule by attaching to a receptor ligand such as a carbohydride or peptide. Recently, novel gene transfer techniques, in which an amphiphilic R-helical peptide containing cationic amino acids is used as a gene carrier into cells, have been reported. Wyman et al. employed a peptide, KALA * To whom correspondence should be addressed. Phone and Fax: 81-95-843-7306. E-mail:
[email protected]. † Department of Applied Chemistry. ‡ Department of Bacteriology.
(WEAK-LAKA-LAKA-LAKH-LAKA-LAKA-LKAC-EA), which is derived from the sequence of the amino-terminal segment of the HA-2 subunit of the influenza virus hemagglutinin involved in the fusion of the viral envelope with the endosomal membrane (11). This peptide showed several functions in the transfection process, e.g., condensing DNA and causing an endosome-membrane perturbation, which enables it to deliver the incorporated DNA to the cytosol, which is essential for efficient transfection. Similarly, we have also reported the transfection technique, which is mediated by some amphiphilic R-helical peptides consisting of 24 amino acids, including six arginines (e.g., Ac-LARL-LARL-LARL-LRAL-LRALLRAL-NHCH3, 46) (12, 13). Furthermore, to clarify in detail the importance of the hydrophobic region in the amphiphilic peptide in relation to its transfection efficiency, we have employed the Hel series peptides (e.g., KLLK-LLLK-LWLK-LLKL-LL, Hel 13-5), which consist of 18 residues and have systematically varied the hydrophobic-hydrophilic balance in the amphiphilic structures (14, 15). As a result, the peptide with a large hydrophobic region could strongly bind to the plasmid DNA and form large aggregates with the DNA. In addition, it could be said that the hydrophobic region of the peptides plays an important role in the disruption of the endosomal membrane in the targeted cell, which can transfer the incorporated DNA to cytosol and prevent the degradation of the DNA in the lysosomal vesicles. As is well-known, a peptide is readily synthesized due to the development of an automatic peptide synthesis
10.1021/bc990012d CCC: $18.00 © 1999 American Chemical Society Published on Web 07/30/1999
774 Bioconjugate Chem., Vol. 10, No. 5, 1999
apparatus and reagents for synthesis. From this point of view, it is expected that the transfection method mediated by the peptide is easily accepted by many researchers taking part in a gene therapy study. Development of an efficient gene transfer system using peptide with simple structure will cause many applications to gene therapy. In this paper, for the purpose of refining of the peptide structure, we investigated the influence of the peptide chain length on gene transfer ability. We synthesized several peptides with shortened chain lengths from the amino-termini of amphiphilic peptides 46 and Hel 11-7, which have transfection abilities (13, 15), and then evaluated the DNA-binding, aggregate-forming, and transfection abilities into cultivated cells. MATERIALS AND METHODS
Chemicals and Instruments. Reagents used for the synthesis and analysis were of reagent grade. All amino acid derivatives and reagents for peptide synthesis were purchased from Watanabe Chemical (Hiroshima, Japan). Ethidium bromide and alamer Blue were purchased from Wako chemicals (Osaka, Japan). Egg yolk phosphatidylcholine, bovine brain phosphatidylserine, and bovine brain sphingomyeline were purchased from Sigma (St. Louis, MO). Peptide purification was carried out on a Hitachi L7100 system (Tokyo, Japan) with a semipreparative RP-HPLC1 column, YMC-Pack ODS column (10 × 250 mm, Kyoto, Japan) using the following solvent system: 0.1% trifluoroacetic acid (TFA)-0.08% TFA in acetonitrile. Flow rate was 3.0 mL/min, and elution was monitored at 220 nm. Mass spectra were obtained by fast-atom bombardment mass spectrometry (FAB-MS) using a JEOL JMS-DX303 mass spectrometer (Tokyo, Japan) or by matrix-assisted time-of-flight mass spectrometry (MALDI TOF-MS) using a Shimadzu Kratos Kompact MALDI III apparatus (Kyoto, Japan). Plasmid DNA which contains a luciferase gene and SV40 promoter (PicaGene control vector, PGVC) was purchased from Toyo Ink (Tokyo, Japan). Closed circular plasmid DNA was purified by ultracentrifugation in CsCl gradients. The plasmid preparations showed a major band of closed circular DNA and minor amount (
