Tyr) across phospholipid membranes

BBRC Biochemical and Biophysical Research Communications 339 (2006) 761–768 www.elsevier.com/locate/ybbrc

Translocation of positively charged copoly(Lys/Tyr) across phospholipid membranes Shaoqian Liu a,*, Akira Shibata b, Satoru Ueno b, Ying Huang c, Yifan Wang a, Yuanjian Li d a

College of Chemistry and chemical Engineering, Central South University, Changsha 410083, China b Faculty of Pharmaceutical Sciences, The University of Tokushima, Tokushima 770-8505, Japan c Hunan College of Tradition Chinese Medicine, Changsha 410007, China d School of Pharmaceutical Sciences, Central South University, Changsha 410083, China Received 30 October 2005

Abstract Much attention has recently been paid to the study of positively charged polypeptides as a possible carrier for therapeutic protein or DNA delivery to cells. In this study, we have investigated the translocation of positively charged copoly(Lys/Tyr) (MW = 72000, DP = 385) across lipid membranes constituted from egg-phosphatidylcholine (EPC), dioleoyl-phosphatidylethanolamine (DOPE), as well as soybean phospholipids (SBPL) using f potential method, circular dichroism spectroscopy (CD), electrophysiology technique, fluorescence spectroscopy, and confocal laser scanning microscopy. Results of f potentials show that copoly(Lys/Tyr) associate with lipid membranes and become gradually saturated on the membranes either hydrophobically or electrostatically or both. CD studies demonstrate that the copoly(Lys/Tyr) takes and remains b-sheet conformation during its interaction with liposome membranes, indicating that the translocation process should be carpet-mode like. Data from the electrophysiology technique reveal that positively charged copoly(Lys/Tyr) can cause transmembrane currents under an applied voltage, confirming its transfer across lipid membranes. Fluorescence spectroscopy results display a three-step mechanism of translocation across membrane: adsorption, transportation, and desorption, which has been verified by results from confocal laser scanning microscopy. We provided the first direct observation that the positively charged polypeptides, copoly(Lys/Tyr), can translocate through SBPL and EPC/DOPE lipid bilayer membranes. In addition, we found that the translocation efficiency of copoly(Lys/Tyr) was higher on the EPC/DOPE lipid membrane than on the SBPL lipid membrane.
2005 Elsevier Inc. All rights reserved. Keywords: Copoly(Lys/Tyr); Lipid membrane; Translocation; Electrostatic attraction; Conformation

It has been shown that some peptides, especially positively charged ones, are able to translocate across cell plasma membranes by an unknown mechanism [1–4]. In the process of membrane translocation, basic amino acid residues have been found to play a very important role [5,6]. For instance, both human immunodeficiency virus (HIV)1 Tat (13-mer peptide), a basic peptide containing six Arg and two Lys, and Drosophila antennapedia (Antp)

*

Corresponding author. E-mail address: [email protected] (S. Liu).

0006-291X/$ - see front matter
2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.11.079

(16-mer peptide) [2], a highly basic peptide including three Arg and four Lys, have frequently been reported to possess the membrane transporting capability [4]. Futaki et al. [5] demonstrated that arginine-substituted Tat could translocate through the cell membrane. Study shows that not only can these membrane transporting peptides (MTPs) translocate through cellullar and nuclear membranes themselves, but they can also carry enzymes [7], oligonucleotides [8], and even such larger structures as liposomes [9] or nanoparticles [10] into the cells, making it possible, henceforth, for us to use these MTPs as a carrier (or vector) to efficiently deliver therapeutic DNA into target cells [11].

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Tung et al. [6] have shown that using positively charged MTPs as a DNA carrier can induce the electrostatic neutralization and consequently the condensation of DNA, and thus upgrade the delivery efficiency of DNA to target cells. Sorgi et al. [12] reported that a combination of protamine sulfate, a polycationic peptide, with cationic liposomes favors DNA delivery in vitro. The carrier complexed with a cationic polypeptide containing an integrin-targeting motif, liposome, and plasmid DNA demonstrates significant receptor-specific enhancement of transfection in porcine arterial endothelial cells, but not in porcine vascular smooth muscle cells [13]. These studies indicated that the positively charged polypeptides might be potential DNA carriers with a relatively high membrane translocation efficiency because there are a plentiful of positive charges in these polypeptide chains, which could facilitate the DNA condensation, complexation, and electrostatic interaction with lipid membranes [14]. Though there exists a great deal of evidence for the translocation of MTPs [1,2,15] or the poly(Lys)–DNA complex [12,16– 20] across cellular and nuclear membranes, no specific membrane translocating mechanism has been illustrated yet. Studies showed that during the process of membrane translocation, the electrostatic interaction between basic amino acid residues and lipid bilayer membranes is one of the crucial factors [21]. It has also been uncovered that the cell-translocating mechanism is most likely via non-specific interactions with lipid membranes [22]. The role of hydrophobic residues during the membrane translocation and the point of whether or not any particular secondary structure promotes the translocation of polypeptides over a potential barrier in the lipid membrane are not well understood. What is known in common is the existence of positively charged residues such as Lys and Arg in the amino acid sequences of MTPs. On the basis of these observations, we chose positively charged copoly(Lys/Tyr) to investigate its interaction with phospholipid bilayer membranes to understand better about the membrane translocation of polypeptide. This study is mainly focused on how copoly(Lys/Tyr) translocates across lipid membranes constituted from egg-phosphatidylcholine (EPC), dioleoyl-phosphatidylethanolamine (DOPE), and soybean phospholipids (SBPL) using f potential (ZP), circular dichroism spectroscopy (CD), electrophysiology technique (ET), fluorescence spectroscopy (FS), and confocal laser scanning microscopy (CLSM) methods. As will be shown in the following sections, according to our study, translocation of copoly(Lys/Tyr) across different lipid membranes has been experimentally demonstrated and characteristics of the membrane permeability of copoly(Lys/Tyr) have been illustrated. Materials and methods Materials and labeling of copoly(Lys/Tyr). Copoly(Lys/Tyr) (MW = 72000, DP = 385), egg-phosphatidylcholine (EPC), soybean

phospholipids (SBPL), and dioleoyl-phosphatidylethanolamine (DOPE) were purchased from Sigma (St. Louis, MO, USA). The NBD-F (4-Fluoro-7-nitrobenzo-2-oxa-1,3-diazole)was obtained from Dojindo Laboratories (Kumamoto, Japan). All peptides and lipids above were used without further purification. Hepes and Tris were purchased from ICN Biomedicals. Chloroform, methanol, and hexane were from Kanto Chemical, INC, Japan. Hepes buffer A (10 mMHepes/100 mMKCl/Tris, pH 7.40) and Hepes buffer B (10 mMHepes/10 mMKCl/Tris, pH 7.40) were used. Copoly(Lys/Tyr) stock solution (1.3 · 10 4 M) was prepared by dissolving an appropriate amount of peptides in Hepes buffer A. Solutions of lipids for constructing planar lipid membranes in electrophysiology technique experiments were obtained as follows: SBPL solution was 10 mg/1 ml hexane and EPC/DOPE (molar ratio 1:1) 5.24 mg/ 0.5 ml hexane. Labeling of copoly(Lys/Tyr) with NBD-F was performed according to the Kazuhiro method [23] and the label ratio was 0.7. Liposome preparation for f potential, CD, and fluorescence measurements. Chloroform solutions of SBPL and EPC/DOPE (molar ratio 1:1) in a 250 ml round-bottomed flask were evaporated under a gentle nitrogen stream to form a thin lipid film on the flask surface [24]. The film was further processed by the mild nitrogen stream for 2–3 h to remove the remaining solvents. The Hepes buffer B solution was then added in the flask and then the flask was sealed and stored at room temperature in the dark for 24 h. The lipid film was gradually stripped off the glass surface during the incubation. The mixture was sonicated by a bath-type sonicator for several minutes under ice bath. To achieve uniform size liposome distribution, the resultant liposomes were extruded through a 0.20 lm filter of disposable syringe filter unit, DISMIC-13 cp (Tokyo Roshi Kaisha, Japan) seven times. The liposomes thus prepared were stable for about 1 week in the dark condition at room temperature. Giant liposome preparation for CLSM. The lipid (5 mg) was dissolved in 1 ml of hexane in a 100 ml round-bottomed flask and the solvent was evaporated under a gentle nitrogen stream for 10 h to form a thin lipid film on the surface of the lower portion of the flask. The dried lipid film was hydrated by 10 ml Hepes buffer B solution and the flask was then sealed under argon and incubated at 20 C for 24 h. The lipid film was gradually stripped off the glass surface during the incubation and formed a giant liposome suspension [25]. Electrophysiology technique. Planar lipid bilayer membranes with different components of lipids as described above were formed on the small hole according to Montal M–Mueller P technique [26]. Membrane formation was verified by monitoring membrane capacitance and resistance. After the formation of stable bilayer membranes, an aliquot of copoly(Lys/Tyr) was added to the cis cell where Hepes buffer A had been placed earlier. The trans cell was connected to the voltage-holding electrode and the cis cell was held on the ground. A polypeptide was then added to the cis cell. The voltage clamp data were recorded by the Ag– AgCl electrodes immersed in a 3 M KCl solution with 0.2 M KCl agar bridges attached to a high-resolution voltage clamp amplifier. The signals were amplified and displayed on a digital oscilloscope. The data were subsequently digitized and analyzed using a pClamp 4.5 software (Axon Instruments, Foster City, CA). All experiments were performed with symmetrical buffer solutions at room temperature (20 C). f potential and particle sizing. f potentials and particle sizes of liposomes consisting of different lipids were measured by Nicomp 380 ZLS f potential/particle sizer (Santa Barbara, CA, USA). f potentials were measured before and after the addition of copoly(Lys/Tyr) with different concentrations. Prior to the measurement, particle sizes of the same liposome suspensions were measured. Both measurements were performed in a 1 cm · 1 cm cuvette at room temperature. Circular dichroism spectroscopy. The CD spectra of copoly(Lys/Tyr) during the process of interacting with the lipid membrane were detected from 260 to 190 nm with a computer-interfaced JASCO J-600 spectropolarimeter (Japan Spectroscopic) at 25 C. Samples were prepared by mixing the unilamellar liposomes with peptide solution, resulting in a mixture in which the concentration of copoly(Lys/Tyr) was 0.6 lM and that of the SBPL lipid was 0.5 mM, or a mixture in which the concentration of copoly(Lys/Tyr) was 0.6 lM and that of EPC/DOPE lipid was 0.48 mM. The samples were shaken before being loaded into the

S. Liu et al. / Biochemical and Biophysical Research Communications 339 (2006) 761–768 spectropolarimeter. Measurements were carried out with 1 cm · 1 cm cuvette and started after 10 min of polypeptide addition to the liposome suspensions. The spectra were obtained by subtracting the spectrum of the liposome suspension from that of the liposome suspension containing the polypeptide and averaged over nine scans with a scan speed of 20 nm/min. Fluorescence measurement. Fluorescence measurements were performed by a fluorescence spectrophotometer (Hitachi, F-4500). Excitation and emission slit widths were set at 10 nm each. Since the excitation and emission wavelengths of tyrosine fluorophore were at 280 and 305 nm, respectively, emission scan was undertaken in the range of 285–350 nm with a scan speed of 240 nm/min. Measurements were administered in a 1 cm · 1 cm quartz cell placed in a thermostatically controlled cell holder at a temperature of 20 C. The ratio of peptides to lipids in the liposome suspension was about 1:1000. When copoly(Lys/Tyr) was added to the liposome suspension, time dependence of the fluorescence intensity at 310 nm was immediately recorded and then followed for a duration of 10 h. Confocal laser scanning microscopy. Samples for CLSM experiments were prepared by mixing the giant liposomes with NBD-F labeled copoly(Lys/Tyr) in a PLL-coated glass dish (Matsunami Glass IND., Japan) using a molar ratio of peptide to lipid of 1:863 for SBPL and 1:973 for EPC/DOPE. The CLSM used was a laser scanning confocal imaging system (Zeiss, LSM-410, Germany) equipped with an argon ion laser (488 nm) and a He–Ne laser (543 nm). Because the excitation and emission wavelengths of NBD-F fluorescein were at 470 and 530 nm, respectively, its fluorescence was excited by an argon ion laser and the emission was observed by a band filter (515–565 nm). To prevent photobleaching, the confocal microscope was operated under conservative laser intensity and time exposure conditions. The temperature of the observation chamber was maintained at 20 C during the experiments. The images of liposomes were automatically recorded every 10 min.

Results Fig. 1 demonstrates the effect of copoly(Lys/Tyr) on the f potential of SBPL (0.46 mM) and EPC/DOPE (0.40 mM) liposomes. Before the addition of copoly(Lys/Tyr), f potentials of SBPL and EPC/DOPE liposomes are 24.8 and 5.5 mV, respectively. The f potentials in the two cases increase sharply after the addition of copoly(Lys/ Tyr) concentration from 1.0 · 10 7 to 3.0 · 10 7 M. Then as more copoly(Lys/Tyr) is added, increase of the f poten-

tial slows down and gradually saturates within the range of copoly(Lys/Tyr) concentrations from 0.8 to 1.0 lM. Liposomal sizes have been scaled before the measurement of f potential. The size of the SBPL liposome was found to be 161.8 nm (84.2%) and 45.7 nm (15.8%), and that of the EPC/DOPE liposome 149.6 nm (79.7%) and 76.2 nm (20.3%). CD spectra of copoly(Lys/Tyr) interacting with SBPL or EPC/DOPE liposome together with that of copoly(Lys/Tyr) in buffer B are shown in Fig. 2. During CD spectra measurements, the concentration of SBPL and EPC/DOPE lipids was 0.5 and 0.48 mM, respectively, and that of copoly(Lys/Tyr) was 0.6 lM. The negative peak of copoly(Lys/Tyr) in the buffer at about 218 nm can be assigned to a typical b-sheet secondary structure [27]. Upon binding to both liposomes, copoly(Lys/Tyr) renders spectra with similar shapes that are slightly redshifted with almost no change in the conformation type. Fig. 3 displays the current intensity as a function of the elapsing time in the absence (A) or presence (B) of copoly(Lys/Tyr) at the constant voltage of 100 mV applied on the EPC/DOPE planar bilayer and 120 mV on the SBPL planar bilayers, respectively. The recording time for curves (A) and (B) in both cases is 160 s. The downward straight lines in controls represent capacitive transient in response to the application of voltage pulses (for 1 s). Without the addition of copoly(Lys/Tyr), there is no transmembrane current signal detected in the two cases, indicating that these lipid membranes are stable under the applied voltage. After an appropriate amount of copoly(Lys/Tyr) is added, the lines begin with zero current and shortly after transmembrane current signals were ensued. After the applied voltage is switched off in 160 s, the current signal rapidly decays to zero, suggesting that the lipid membranes have not been broken. However, in general, the lipid membranes become unstable either when more than 140 mV voltage is applied or when a high concentration of peptides

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is added. When the voltage polarity is switched, no transmembrane current signal will be observed. Fluorescence intensities at 310 nm from EPC/DOPE and SBPL liposome suspensions after the addition of copoly(Lys/Tyr) vs. the elapsing time are shown in Fig. 4. After the addition, with the ratio of peptide to lipid of about 1:1000, the fluorescence intensity in the SBPL liposome system decreases steeply in first 30 min, then its descending speed slows down a little bit from 30 to 300 min, and finally in the next phase it reaches a relatively flat state from 300 to 600 min. Although almost the same conclusion can be drawn for the EPC/DOPE system, its fluorescence intensity decrease in the first 30 min is not as sharp as that of SBPL.

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Figs. 5 and 6 show time profiles of EPC/DOPE and SBPL liposome images, respectively, as well as the fluorescence intensity histograms after addition of NBD-F-copoly(Lys/Tyr) (0.73 lM in SBPL and 0.75 lM in EPC/ DOPE). The binding and translocation of copoly(Lys/ Tyr) to/across lipid membranes are demonstrated by the fluorescence brightness both on the surfaces and in inner aqueous phases of liposomes. The brighter a spot of the image, the more NBD-F-copoly(Lys/Tyr) molecules accumulated in the place of the liposomes. This information can quantitatively be rendered by fluorescence intensity histograms, shown underneath each image. Obviously, the transfer efficiency of NBD-F-copoly(Lys/Tyr) in EPC/DOPE bilayer membrane, denoted by the fluorescence intensity in the inner aqueous phases of liposomes with the progression of time, is different from that in SBPL bilayer membranes.

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time/min Fig. 4. Change of fluorescence intensity with the progress of time of two systems constituted from copoly(Lys/Tyr) and lipsomes of different composition. Copoly(Lys/Tyr) = 0.5 lmol/L; full triangle: in SBPL (0.49 mmol/L); open square: in PC/DOPE (0.5 mmol/L, molar ratio = 1/1); buffer: 10 mmol/L Hepes/10 mmol/L KCl/Tris, pH 7.4.

One of the key steps in gene therapy is to efficiently deliver DNA to target cells. Studies show that positively charged MTPs [1,2] or polypeptides [17–19] are potential DNA carriers with improved efficiency and safety on gene delivery. Along this line and in regard to the fact that the interaction of peptides with phospholipid vesicles is crucial to understand the mechanism of biological activities, we have investigated translocation of positively charged copoly(Lys/Tyr) across phospholipid bilayer membranes. Although several types of intermolecular forces are involved in the interaction of peptides with lipid membranes [28], it is commonly accepted that primary contributions of interactions come from both hydrophobic and electrostatic effects [21]. In the present study, we employed the electrophysiology technique to examine translocation of peptides across lipid bilayer membranes, whose results exhibited unambiguously that copoly(Lys/Tyr) can activate transmembrane current under an appropriate range of applied voltage and peptide concentration. When the

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polarity of applied voltage is changed, no transmembrane current signal will be detected, validating that copoly(Lys/Tyr) has indeed penetrated both the SBPL and EPC/DOPE lipid bilayer membranes. More detailed information about the interaction of a peptide with lipid membranes has been obtained by the f potential method, circular dichroism spectroscopy, fluorescence spectroscopy, and confocal laser scanning microscopy. Before the f potential measurement was carried out, 0.20 lm filter had been used to make the liposome systems roughly uniform in particle size to mimic the curvature of cells. f potential data apparently show that positively charged copoly(Lys/Tyr) gradually accumulate on the liposome membrane surfaces of SBPL lipid or EPC/DOPE lipid after addition. f potentials increase more rapidly after the addition of copoly(Lys/Tyr) for the negatively charged SBPL liposome than for the EPC/DOPE liposome. In addition, the increased amount for SBPL is 19.30 mV,

whereas for EPC/DOPE it is 15.99 mV. Because the electrostatic force is stronger than hydrophobic interaction, we attribute the above difference to the different contribution of the two main kinds of interactions; That is, the interaction of copoly(Lys/Tyr) molecules with SBPL liposome is dictated by the electrostatic attraction, while the interaction between copoly(Lys/Tyr) and the uncharged EPC/DOPE liposome is governed by the hydrophobic contribution. When the concentration of the added copoly(Lys/Tyr) is larger than 5 · 10 7 M, the curve of the f potential reaches a relatively flat stage, showcasing that copoly(Lys/Tyr) molecules get slowly saturated on the lipid membranes. This process can be categorized as the first step, association, of the interaction between copoly(Lys/ Tyr) and lipid bilayer membranes. Results from circular dichroism spectroscopy study demonstrate that copoly(Lys/Tyr) took b-sheet conformation in buffer. No such conformational change has been

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observed upon binding to both SBPL and EPC/DOPE lipid membranes, showing that only one kind of peptide– membrane interaction mechanism mode, called the Carpet mode, can be used to explain the whole process because the Carpet mode polypeptides can translocate across a lipid membrane without changing its conformation [29]. Comparing fluorescence spectroscopy results in Fig. 4 we can further confirm that the electrostatic attraction is the dominant driving force for the initial adsorption of positively charged peptide molecules to negatively charged lipid membranes. It is believed that the fluorescence intensity of a fluorophore is strongly affected by the characteristics of its environment, such as dielectric constant (e). Generally, a fluorophore demonstrates a weaker fluorescence intensity in a less polar environment, which has a smaller dielectric constant. Accordingly, changes of fluorescence

intensities in Fig. 4 can be classified into three distinct stages to categorize the different extent of the interaction between peptides and lipid membranes since the dielectric constants in the aqueous region, region from the lipid headgroup to glycerol, and the center of hydrophobic region are about 78, 34, and 2, respectively [30]. Like Step one described in the f potential measurement, Stage (1) here suggests that copoly(Lys/Tyr) are gradually accumulated on the outer surfaces of the liposome membranes from the outer aqueous phase (e = 78). In Stage (2), most of copoly(Lys/Tyr) molecules pass primarily via hydrophobic force through the hydrophobic region (e = 2). Finally in Stage (3), an equilibrium is likely established between the peptidie molecules desorbed from the inner surface of the membrane and those re-adsorbed onto the inner surface of the liposome membrane from the inner aqueous phase

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of liposome. This three-step mode of interaction between copoly(Lys/Tyr) and lipid membranes is subsequently verified by the change of fluorescence intensity profiles of NBD-F-copoly(Lys/Tyr) in confocal laser scanning microscopy experiments. According to the Figs. 5 and 6, the interaction of copoly(Lys/Tyr) with lipid membranes can clearly be divided into three steps: Step 1, accumulation of copoly(Lys/Tyr) molecules onto the outer surface of lipid membranes; Step 2, translocation of copoly(Lys/ Tyr) molecules across the hydrophobic region of the lipid membranes; Step 3, desorption of the copoly(Lys/Tyr) molecules from the inner lipid membrane surface to the inner aqueous phase of the liposomes. The results of electrophysiology technique measurements show that the transmembrane current caused by copoly(Lys/Tyr) (0.2 lM) in the SBPL lipid membrane under 120 mV is much smaller than that in the EPC/ DOPE membrane under 100 mV, indicating that it is easier for copoly(Lys/Tyr) to penetrate the EPC/DOPE lipid membrane under the applied voltage. In the case of CLSM, brightness from liposome images represents the fluorescence intensity and is proportional to the concentration of NBD-F-copoly(Lys/Tyr) in the liposome. Fifty minutes after NBD-F-copoly(Lys/Tyr) is added, the fluorescence intensity in the center of SBPL liposome is about 1000, while that in EPC/DOPE is more than 1800, suggesting that NBD-F- copoly(Lys/Tyr) has had more difficulty to pass through the SBPL lipid membrane than the EPC/ DOPE lipid membrane. This result can be reassured by comparing the fluorescence intensity in the center of the EPC/DOPE liposome at 60 min (more than 4000) to that of SBPL at 90 min (less than 3000). The difference in translocation efficiency of copoly(Lys/Tyr) on SBPL and EPC/ DOPE is most likely from different lipid compositions in bilayer membranes. In the case of SBPL, several natural lipid components have been used to make the liposome, in which 25% are negatively charged. Its low transfer efficiency can thus be accounted for by its anionic content since addition of an anionic composition to the membrane will decrease permeability of positively charged peptides [31]. Based on what we have found in the present work, it can be concluded that (1) copoly(Lys/Tyr) can transport across EPC/DOPE and SBPL lipid bilayer membranes, and consequently induce transmembrane currents; (2) apparently three steps are involved in the translocation of copoly(Lys/Tyr) across lipid bilayer membranes, which are adsorption, passage, and desorption; (3) on peptide interaction with membranes, although both the electrostatic and hydrophobic interactions are major contributors for the initial adsorption, the electrostatic attraction has been found to be the dominant factor if a peptide is positively charged and the lipid membrane is negatively charged; (4) by using CLSM, we have provided the first direct evidence that copoly(Lys/Tyr) binds to and translocates across a liposomal membrane with almost no conformational change; (5) finally, the translocation efficiency of

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copoly(Lys/Tyr) is higher in the uncharged EPC/DOPE lipid membrane than in the negatively charged SBPL lipid membrane. The present study has mainly focused on model lipid membranes. However, we think the results can provide useful insights into complex mechanisms of positively charged polypeptides interacting with biomembranes. Based on our results from the current work, copoly(Lys/Tyr) may be used as a potential carrier to deliver therapeutic DNA to target cells. Acknowledgment The study was supported in part by the Japan-China Sasakawa Medical Fellowship. References [1] S. Fawell, J. Seery, Y. Daikh, C. Moore, L.L. Chen, B. Pepinsky, J. Barsoum, Tat-mediated delivery of heterologous proteins into cells, Proc. Natl. Acad. Sci. USA 91 (1994) 664. [2] D. Derossi, A.H. Joliot, G. Chassaing, A. Prochiantz, The third helix of the antennapedia homeodomain translocates through the plasma membrane and accumulates in the cell nucleus, J. Biol. Chem. 269 (1994) 10444. [3] C. Plank, B. Oberhauser, K. Mechtler, The influence of endosomedisruptive peptides on gene transfer using synthetic virus-like gene transfer system, J. Biol. Chem. 269 (1994) 12918–12924. [4] E. Vives, P. Brodin, B. Lebleu, A truncated HIV-1 Tat protein basic domain rapidly translocates through biological membranes, J. Biol. Chem. 272 (1997) 16010. [5] S. Futaki, T. Suzuki, W. Ohashi, T. Yagami, S. Tanaka, K. Ueda, Y. Sugiura, Arginine-rich peptides: an abundant source of membranepermeable peptides having potential as carriers for intracellular protein delivery, J. Biol. Chem. 276 (2001) 5836. [6] C.-H. Tung, S. Mueller, R. Weissleder, Novel branching membrane translocational peptide as gene delivery vector, Bioorg. Med. Chem. 10 (2002) 3609. [7] S.R. Schwarze, A. Ho, A. Vocero-Akbani, S.F. Dowdy, In vitro protein transduction: delivery of a biologically active protein into the mouse, Science 285 (1999) 1569. [8] A. Astriab-Fisher, D.S. Sergueev, M. Fisher, B.R. Shaw, R.L. Juliano, Antisense inhibition of P-glycoprotein expression using peptide–oligonucleotide conjugates, Biochem. Pharmacol. 60 (2000) 83. [9] V.P. Torchilin, R. Rammohan, V. Weissig, S. Levchenko, TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors, Proc. Natl. Acad. Sci. USA 98 (2001) 8786. [10] M. Lewin, N. Carlesso, C.H. Tung, X.W. Tang, D. Cory, D.H. Scadden, R. Weissleder, Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells, Nat. Biotechnol. 18 (2000) 410. [11] T. Niidome, N. Ohmori, A. Ichinose, Binding of cationic a-helical peptides to plasmid DNA and their gene transfer activities into cells, J. Biol. Chem. 272 (1997) 15307–15312. [12] F.L. Sorgi, S. Bhattacharya, L. Huang, Protamine sulfate enhances lipid-mediated gene transfer, Gene Ther. 4 (1997) 961. [13] R. Parkes, Q.-H. Meng, E. Saipati, J.R. McEwan, S.L. Hart, High efficiency transfection of porcine vascular cells in vitro with a synthetic vector system, J. Gene Med. 4 (2002) 292. [14] M. Molas, R. Bartrons, J.C. Perales, Single-stranded DNA condensed with poly-lysine result in nanometric particles that are significantly smaller, more stable in physiological ionic strength fluids and afford

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