Versatile Nuclear Localization Signal-Based Oligopeptide as a Gene Vector

Abstract

To develop a versatile nuclear-targeted gene vector, nuclear localization signal (NLS) oligopeptides combining cysteine (C), histidine (H), and stearic acid (STR) were investigated in this study. The original SV40 sequence (SV40: Pro-Lys-Lys-Lys-Arg-Lys-Val) was selected as the NLS sequence, and physical characterizations of various NLS-based oligopeptides (CSV40C, STR-CSV40C, and STR-CH₂SV40H₂C), including mean diameter, zeta-potential, complex condensation, and decondensation, were evaluated. In addition, cellular and nuclear uptake of plasmid DNA (pDNA) and gene expression in COS7 and dendritic cells (JAWS II) were determined. As a result, C and STR enhanced formation of a smaller and more stable nano-complex with pDNA based on ionic interactions, the disulfide linkage and hydrophobic interactions. STR-CSV40C and STR-CH₂SV40H₂C had significantly higher cellular uptake ability and transfection efficiency than SV40 and CSV40C. In particular, STR-CH₂SV40H₂C had higher nuclear uptake and gene expression efficiency than STR-CSV40C. Furthermore, STR-CH₂SV40H₂C could deliver pDNA to the nuclei and had high gene expression efficiency in dendritic cells. Our results indicate that STR-CH₂SV40H₂C is a promising gene delivery system in non- or slow-dividing cells.

Barriers in gene delivery include DNA packaging-unpacking, cell binding, internalization, endosomal escape, and nuclear localization,¹⁾ and these barriers are common in post-mitotic, non- or slow-dividing, and quiescent cells,²⁾ such as dendritic and neuronal cells. An early microinjection study revealed that less than 1% of cells expressed transgenes when a plasmid was injected into the cytoplasm, while up to 50% of cells expressed transgenes when a plasmid was directly injected into the nucleus.³⁾ The nuclear localization signal (NLS), which is recognized by nuclear transport proteins of the importin family,⁴⁾ could overcome the nuclear membrane barrier and promote nuclear translocation. Among NLS sequences, the most extensively studied is the SV40 large-T antigen, especially in its shortest form, Pro-Lys-Lys-Lys-Arg-Lys-Val.⁵⁾ However, the NLS hardly enhances gene expression efficiency, because NLS cannot promote DNA packaging-unpacking, cell binding, internalization, and endosomal escape.

To improve nuclear import efficiency, the direct or indirect attachment of cell-penetrating peptides (CPPs) with NLS to DNA or gene carriers, which could facilitate nuclear localization, attracted much attention.⁶⁾ For example, researchers attempted to chemically conjugate NLS to DNA. However, the NLS peptides chemically coupled to DNA did not result in significant increases in transfection,⁷⁾ because the NLS was involved in nuclear transfer rather than cellular uptake. Therefore, NLS-mediated gene expression is insufficient because of poor cellular uptake and endosomal escape. Another study observed a weak 4-fold increase in transgene expression only when 5 NLS peptides were covalently coupled to plasmid DNA (pDNA) by diazo coupling through a polyethylene glycol (PEG) chain, and no increase existed when the NLS peptides were directly coupled to DNA.⁸⁾ Peptide nucleic acids (PNAs) were also employed to attach the NLS to DNA in a sequence-specific, non-covalent manner, but it required individual PNA synthesis for each targeted DNA sequence.⁹⁾ Therefore, efficient gene delivery using NLS peptides has several requirements, including cellular uptake, endosomal escape, stable pDNA condensation in an extracellular environment, and decondensation in a nuclear environment.

Thiol groups are susceptible to cross-linking through oxidation when forming intramolecular and intermolecular disulfide bridges.¹⁰⁾ The disulfide bonds are very stable,¹¹⁾ are often present in biological systems, and provide structural stability.¹²⁾ However, their stabilizing effect is reversible under reducing conditions, which are found in the cytoplasm or intranuclear environment. The intracellular reduction of disulfide bonds is most likely mediated by small redox molecules such as glutathione (GSH), either alone or with the help of redox enzymes.¹³⁾ GSH is the most abundant intracellular sulfhydryl inside the cell and is present in millimolar concentrations; however, it is only present in micromolar concentrations in the blood plasma.¹⁴⁾ The concentration of GSH has been reported to be approximately 4 mM in the cytoplasm and approximately 20 mM in the nucleus, where it is required for DNA synthesis and DNA repair as well as to maintain a number of transcription factors in the reduced state.^13,15,16) The subcellular distribution of GSH indicates that intracellular reduction of disulfide bonds will preferentially proceed in the cytoplasm and nucleus.¹³⁾

Histidine has been used to enhance endosomal escape of polyplexes. The imidazole ring of histidine is a weak base and has a pK_a around 6.0, which makes it charged at the low pH encountered in the endosomes and allows it to scavenge protons. This unique property of histidine allows histidine-bearing pDNA complexes to be released from endosomes.^17–20) In our previous study, we determined the transfection efficacies of several histidine-including peptides in the presence of chloroquine, which is a well-known endosome-disrupting agent that can promote endosomal escape and enhanced transfection efficacy,²¹⁾ compared with that in the absence of chloroquine, and reported the increasing the number of histidines enhanced endosomal escape.²²⁾

Recently, some groups have shown that stearylation of CPPs can be a successful strategy to improve delivery of oligonucleotides and plasmids.^23–25) We have previously shown that stearylation of arginine-histidine-cysteine block peptides promotes efficient oligonucleotides and plasmids delivery utilizing a non-covalent vectorization approach.^20,22,26) Although delivery of plasmids with CPPs has been observed numerous times,^27–30) the relative efficiency of unmodified CPPs in promoting plasmid delivery has been poorly studied. Stearyl modification has been one of the most efficient strategies for plasmid delivery on arginine-rich peptide carriers.^{20,22,23,31,32)}

To develop a multifunctional gene vector, NLS peptides were used in this study to enhance characteristics of intracellular trafficking performance, including condensation, cellular uptake, endosomal escape, and release of pDNA in nuclei; various SV40 based-oligopeptides modified by cysteine (C) with a thiol group, histidine (H), and stearic acid (STR) were also investigated. In addition, physicochemical characterizations of the various NLS-based vectors (CSV40C, STR-CSV40C, and STR-CH₂SV40H₂C) were evaluated, including mean diameter, zeta-potential, complex condensation, and decondensation. Furthermore, cellular and nuclear uptake and gene expression in COS7 and dendritic cells (JAWS II) were determined.

MATERIALS AND METHODS

Materials

Plasmid DNA comprising a subcloned luciferase cDNA fragment at the HindIII and BamHI sites of pcDNA3.1 (pCMV-Luc) was amplified in Escherichia coli (DH5α) and purified using an Endfree Plasmid Maxi kit (Qiagen Sciences, MD, U.S.A.), followed by ethanol precipitation and dilution in Tris/ethylenediaminetetraacetic acid (EDTA) buffer. Plasmid EGFP-N1 (pEGFP, BD Clontech Laboratories, Inc., CA, U.S.A.), which codes for green fluorescence proteins (GFP), was purchased from TaKaRa Bio Inc. (Shiga, Japan). Plasmid DNA concentration was measured based on UV absorption at 260 nm. The Luciferase Assay System (Promega Co., Ltd.; Madison, WI, U.S.A.) was used for determining luciferase activity. The Mirus Label IT® Cy5 labeling kit (TaKaRa Bio Inc.) was used for fluorescent labeling of pDNA. Lipofectamine® (Life Technologies Japan Co., Tokyo, Japan) was used as a positive control for gene transfection efficiency analysis.

COS7 and JAWS II cells were purchased from American Type Culture Collection (WA, U.S.A.). Cell culture medium, Dulbecco’s modified Eagle’s medium (DMEM), Minimum Essential Medium (MEM) alpha, certified fetal bovine serum (FBS), penicillin/streptomycin stock solutions, and 0.25% trypsin–EDTA were purchased from Gibco® (Life Technologies Japan Co.). Recombinant mouse Granulocyte Macrophage colony-stimulating Factor (GM-CSF) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was purchased for generation of immature dendritic cells.

The 3 peptides (SV40, CSV40C, and CH₂SV40H₂C) were synthesized as pDNA vectors using the F-moc-solid-phase peptide synthesis method with an ABI 433 A peptide synthesizer (Applied Biosystems Japan, Ltd., Tokyo, Japan). The sequences of peptides are shown in Table 1. All peptides were purified by reverse-phase high-performance liquid chromatography (HPLC) before use. The molecular weights of peptides were determined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. STR was conjugated to the N-terminal of CSV40C or CH₂SV40H₂C using the solid-phase peptide synthesis method.^{20,22,23,26,31)}

Table 1. Peptide Sequences and Molecular Weights

Peptides	Sequences	Mass (m/z)
		Calculated	Measured
SV40	PKKKRKV	883.15	884.67
CSV40C	CPKKKRKVC	1089.44	1090.12
STR-CSV40C	CH3(CH2)16-CONH-CPKKKRKVC	1355.92	1356.32
STR-CH2SV40H2C	CH3(CH2)16-CONH-CHHPKKKRKVHHC	1904.48	1904.46

P: Proline, K: Lysine, R: Arginine, V: Valine, C: Cysteine, H: Histidine, STR: Stearic acid.

Cell Culture

COS7 cells were maintained in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 5% CO₂ at 37°C. Cells were seeded into culture flasks for a few days. The cells were harvested by trypsinization, washed with phosphate-buffered saline (PBS), and resuspended in DMEM. JAWS II cells were maintained in MEM alpha supplemented with 20% FBS, 1% penicillin/streptomycin, and 5% CO₂ at 37°C. Cells were seeded into culture flasks for 7 d. The cells were harvested by trypsinization, washed with PBS, and resuspended in MEM alpha.

Preparation of pDNA/Peptide Complexes

N/P ratios were defined as the molar ratio of amine groups (N) in lysine and arginine in peptide/DNA phosphate groups (P). The pDNA/peptide complexes were prepared by mixing components at N/P ratios ranging from 1 to 50 for 24 h at room temperature prior to the experiments

Physiochemical Characterization

The mean particle size and zeta-potential of the pDNA/various peptides were determined using a dynamic light scattering (DLS)-700 unit (Otsuka Electronics Co., Ltd., Osaka, Japan) and a Zeta Potential/Particle Sizer NICOMPTM 380 ZLS (Nicomp Particle Size Systems, Santa Barbara, CA, U.S.A.). The pDNA/peptide complexes were analyzed by agarose gel electrophoresis in Tris-borate–EDTA (TBE: 40 nM Tris-borate, 1 mM EDTA, pH 7.4) buffer. Various ratios of complexes were loaded onto a 1% agarose gel containing ethidium bromide (0.5 mg/mL) and electrophoresed in TBE buffer (0.25%) at 100 V for 40 min. The gel was visualized on a UV illuminator to determine the location of pDNA.

Cellular Uptake Assay

COS7 cells (2×10⁵ cells) were seeded onto 6-well culture plates. After 24 h incubation in DMEM containing 10% FBS, the cells were washed with PBS, and 1.9 mL of FBS(−) DMEM was added before transfection with naked Cy5-labeled pCMV-Luc (Cy5-pDNA) or Cy5-pDNA with SV40, CSV40C, STR-CSV40C, or STR-CH₂SV40H₂C (Cy5-pDNA: 1 µg) in 100 µL of FBS(−) DMEM. After 4 h incubation, the culture medium was aspirated and the cells were washed with PBS. After detachment by pipetting and resuspension in PBS, Cy5 fluorescence intensity in the cells was analyzed using flow cytometry (FACSCanto; Japan BD Co., Ltd., Tokyo, Japan). The cells were gated electronically according to non-transfected cell forward-scatter (FSC) and side-scatter (SSC) properties to induce the main population of cells and exclude dead cells. The 30000 events were analyzed, and the ratio of Cy5-detected cells among all transfected cells was depicted as the cellular uptake efficiency (%).

Luciferase Assay

COS7 cells (5×10⁵ cells) were seeded onto 6-well culture plates. After 24 h incubation in DMEM containing 10% FBS, the cells were rinsed with PBS, and then 1.9 mL of DMEM without FBS was added to each well. The pCMV-Luc complex solution (100 µL containing 1 µg of pCMV-Luc) was applied to each well. After 4 h, the medium was removed and replaced by 10% FBS-containing DMEM for further incubation. After 44 h, the cells were washed 3 times with PBS, lysed by addition of 100 µL of lysis buffer to each well, and left for 15 min at room temperature. Cell lysates were then collected and centrifuged at 15000 rpm for 3 min. After addition of 50 µL of luciferase substrate solution to 10 µL of cell lysate, luciferase activity was measured using a MicroLumat Plus LB96V chemiluminescence instrument (MicroLumat Plus LB96V, Berthold, Germany). The protein concentration of each cell lysate was determined using a standard BioRad protein assay kit (Bio-Rad, Hercules, CA, U.S.A.). Briefly, 5 µL of cell lysate was diluted by a factor of 160 with ultrapure water and then incubated with 40 µL of dye reagent for 30 min at room temperature; the absorbance was then measured at 595 nm using the microplate reader (Tecan Safire, Tecan Trading AG, Switzerland). The protein concentration of the cell lysate was calculated using a calibration curve generated with bovine serum albumin standards (2 mg/mL). The results are shown as relative light units (RLU) per mg of protein.

Observation of pDNA Unpacking from Complexes Treated in a Reducing Environment Using Electrophoresis

To evaluate the pDNA release function of the STR-CH₂SV40H₂C vector with a reducing agent (GSH) in similar conditions to cytosol (ca. 4 mM) or nuclei (ca. 20 mM) (Manickam and Oupický,¹³⁾ Hwang et al.^15,16)), agarose gel electrophoresis of STR-CH₂SV40H₂C/pDNA complexes treated with GSH was carried out. STR-CH₂SV40H₂C/pDNA complexes were incubated with 4 mM or 20 mM GSH and 0.01% heparin for 48 h. After incubation, STR-CH₂SV40H₂C/pDNA complexes were loaded onto a 1% agarose gel containing ethidium bromide (0.5 mg/mL) and electrophoresed in TBE buffer (0.25%) at 100 V for 40 min. The gel was visualized on a UV illuminator to assess the location of the pDNA.

Intranuclear Amount of pDNA

The Cy5-pDNA complexes were transfected to the COS7 or JAWS II cells in a manner similar to the procedure for the cellular uptake assay. After transfection, the culture medium was aspirated, and cells were washed with PBS. To collect nuclei, 500 µL of lysis buffer (pH 7.4, 10 mM tris(hydroxyl) aminomethane–HCl, 10 mM NaCl, 3 mM MgCl₂, and 1% Nonidet P-40) was added to the cells. The cells were resuspended in PBS; the fluorescence intensity of Cy5-pDNA in the nuclei was analyzed using a microplate reader (Safire Microplate Reader, Tecan) after centrifugation at 3000 rpm for 5 min. The amount of Cy5-pDNA in the nuclei was calculated using a calibration curve generated with Cy5-pDNA standards. The results are shown as the amount of pDNA per nucleus.

GFP Expression Assay

JAWS II cells (1×10⁶ cells) were seeded onto 6-well culture plates. After 24 h incubation in MEM alpha containing 20% FBS, the cells were rinsed with PBS, and then 1.9 mL of MEM alpha without FBS was added to each well. The pEGFP complex solution (100 µL containing 1 µg of pEGFP) was applied to each well. After 4 h, the medium was removed and replaced by 20% FBS-containing MEM alpha for further incubation. After 44 h, the cells were washed with PBS. After detachment by pipetting and resuspension in PBS, the GFP fluorescence intensity in the cells was analyzed using flow cytometry (FACSCanto, Japan BD Co., Ltd., Tokyo, Japan). The cells were gated electronically according to non-transfected cell FSC and SSC properties to induce the main population of cells and exclude dead cells. The 30000 events were analyzed, and the ratios of GFP-expressing cells among all transfected cells are shown as the gene transfection efficiency (%).

Statistical Analysis

Data are expressed as the mean±S.D. Statistical analysis of the data was performed using one-way ANOVA followed by Dunnett’s test. Statistical significance was defined as * p<0.05, ** p<0.01.

RESULTS

Physicochemical Characterization

We first determined the pDNA condensation of SV40, CSV40C, STR-CSV40C, and STR-CH₂SV40H₂C using electrophoresis. Figure 1 depicts the electrophoresis of pDNA complexes with SV40, CSV40C, STR-CSV40C, or STR-CH₂SV40H₂C. As shown in Fig. 1, at any N/P ratio of SV40/pDNA as well as in naked pDNA, a band of pDNA was observed, indicating that a stable complex was not formed by SV40. By contrast, the bands of pDNA complexed with CSV40C, STR-CSV40C, or STR-CH₂SV40H₂C were delayed, indicating that stable complexes were formed. We also determined the particle size and zeta-potential of pDNA complexed with SV40, CSV40C, STR-CSV40C, or STR-CH₂SV40H₂C. Table 2 depicts the particle size and zeta-potential of pDNA complexes with SV40, CSV40C, STR-CSV40C, or STR-CH₂SV40H₂C. As shown in Table 2, the particle size and zeta-potential of SV40/pDNA were up to 500 nm and there was a negative charge. By contrast, the particle size and zeta-potential of the pDNA complex with CSV40C was smaller than that of the pDNA complex with SV40 and had a positive charge. In addition, pDNA complexed with STR-CSV40C or STR-CH₂SV40H₂C showed markedly smaller sizes (<90 nm) and higher positive charges than CSV40C. These results indicate that conjugations of C and STR enhance the pDNA condensation of NLS peptides, because of disulfide linkage by thiol groups in C and hydrophobic interactions associated with STR. In addition, the result of zeta potential suggest that the basic amino acids, including lysine and arginine, in the both peptides appeared on the surface of complexes with complexes, and these amino acids were expected to mediate high cellular uptake and gene expression. Furthermore, there is no physicochemical characterization difference between STR-CSV40C, suggesting that and H did not affect the complexation with pDNA.

Fig. 1. Agarose Gel Electrophoretic Analysis of Complex Formation of pDNA with Various NLS-Based Vectors

The 1% agarose gel electrophoresis was carried out in TBE buffer at 100 mV for 40 min. The gel was stained with ethidium bromide. A; pDNA ladder, B; naked pDNA, C; 1, D; 5, E; 10, F; 20, G; 30, H; 40, I; 50 N/P ratio of NLS-based vectors/pDNA.

Table 2. Mean Diameter and Zeta-Potential of pDNA/Peptide Complexes at an N/P Ratio of 10

pDNA/peptide complex	Mean diameter (nm)	Zeta-potential (mV)
SV40	563.3	−3.67
CSV40C	235.8	4.1
STR-CSV40C	85.0	33.0
STR-CH2SV40H2C	89.2	29.1

In Vitro Transfection Using COS7 Cells

We next examined cellular uptake, intranuclear amount, and gene expression efficiency of pDNA with SV40, CSV40C, STR-CSV40C, or STR-CH₂SV40H₂C in COS7 cells. Figures 2(a), (b) depict the intracellular uptake of Cy5-pDNA with SV40, CSV40C, STR-CSV40C, or STR-CH₂SV40H₂C in COS7 cells as determined by flow cytometry. There was minimal intracellular uptake of Cy5-pDNA complexes with SV40 and CSV40C, indicating that SV40 and CSV40C could not enhance the cellular uptake of pDNA because basic amino acids area, which should be the important force for attaching to the cell membrane, in SV40 and CSV40C was almost used as the force for interaction with pDNA. By contrast, STR-CSV40C or STR-CH₂SV40H₂C strikingly enhanced the cellular uptake of Cy5-pDNA, indicating that STR conjugation improved the intracellular uptake of pDNA.

Fig. 2. Cellular Uptake Ability of pDNA in COS7 Cells Transfected with Various NLS-Based Vectors

COS7 cells were transfected with naked Cy5-pDNA (1 µg) and Cy5-pDNA (1 µg) complexed with various NLS-based vectors (N/P ratio: 10). After 4 h incubation, (a) intracellular uptake efficiency (%), (b) mean fluorescence intensity, or (c) intranuclear amount (pg/nucleus) of Cy5-pDNA in COS7 cells were determined. Each bar represents the mean±S.D. (n=3). (a) ** p<0.01 vs. naked, SV40 and CSV40C group, and ^n.s. p>0.05 (not significance), (b) and (c) ** p<0.01 vs. other groups.

Figure 2(c) shows the intranuclear amount of Cy5-pDNA with SV40, CSV40C, STR-CSV40C, or STR-CH₂SV40H₂C in COS7 cells 4 h after transfection without FBS. As well as the cellular uptake, the intranuclear amounts of Cy5-pDNA complexed with SV40 and CSV40C were minimal. By contrast, STR-CSV40C or STR-CH₂SV40H₂C strikingly enhanced the intranuclear amount of Cy5-pDNA. These results suggest that the addition of STR increased cellular uptake and intranuclear transfer of pDNA because of the high affinity with the cell and endosomal membrane by increase of hydrophobicity. In addition, STR conjugation improved the pDNA condensation of SV40, and the basic amino acids area in STR-conjugated peptides might be appear on the surface of complexes, and work as a cellular and nuclear localization moiety.

Furthermore, the intranuclear amount of pDNA complexed with STR-CH₂SV40H₂C was significantly higher than that in pDNA complexed with STR-CSV40C.

We also determined the luciferase activity in COS7 cells transfected with pCMV-Luc complexed with STR-CSV40C or STR-CH₂SV40H₂C at various N/P ratios. Figure 3 depicts luciferase activity in COS7 cells transfected with pCMV-Luc complexed with STR-CSV40C or STR-CH₂SV40H₂C at various N/P ratios ranging from 5 to 50. In the case of STR-CSV40C, the luciferase activity was highest at an N/P ratio of 10. In the case of STR-CH₂SV40H₂C, luciferase activity at an N/P ratio of more than 10 produced almost the same values. In addition, luciferase activity in COS7 cells transfected with pCMV-Luc complexed with STR-CH₂SV40H₂C was significantly higher than that in pDNA complexed with STR-CSV40C.

Fig. 3. Intranuclear Amount of pDNA Luciferase Activity in COS7 Cells after Transfection with Various NLS-Based Vectors

The naked pDNA and pDNA complexed with STR-CSV40C or STR-CH₂SV40H₂C (pDNA: 1 µg, N/P ratio: 5–50) were transfected into COS7 cells (5×10⁵ cells/35-mm dish) with serum-free medium for 4 h. After transfection, the cells were washed with PBS, and incubated with FBS-containing media for 44 h. After incubation, the luciferase activity was determined. Each bar represents the mean±S.D. (n=3). * p<0.05.

These results indicate that H conjugation increase the intranucelar amount of pDNA and gene expression because the amount of pDNA which could escape from endosome/lysosome to cytoplasm was increased by endosomal escaping ability of H.

pDNA Unpacking from the STR-CH₂SV40H₂C/pDNA Complex

We next evaluated the pDNA unpacking ability from complexes in the cytoplasm or intranuclear environment after endosomal escape in the cell. Figure 4 shows the agarose gel electrophoresis of the pDNA/STR-CH₂SV40H₂C complex at an N/P ratio of 10 after treatment with various concentrations of reducing agent. GSH was used as the reducing agent, and GSH concentrations of 4 mM or 20 mM were used as mimics of the cytoplasm and intranuclear environments, respectively.^13,15,16) As a result, STR-CH₂SV40H₂C could strongly condense the pDNA in the absence of GSH or in the presence of 4 mM of GSH, whereas the most of pDNA was released from STR-CH₂SV40H₂C in the presence of 20 mM of GSH, suggesting that STR-CH₂SV40H₂C can release pDNA in the nucleus instead of the cytoplasm. This result suggested that STR-CH₂SV40H₂C can release pDNA in the nucleus and not the cytoplasm, and that this intranuclear-reducible NLS vector has the potential to efficiently release pDNA after delivering genes to the nuclei by the NLS transport pathway.

Fig. 4. In Vitro pDNA Decondensation from STR-CH₂SV40H₂C

The complexes were prepared by mixing pDNA and STR-CH₂SV40H₂C at an N/P ratio of 10. Complexes were incubated with or without GSH (4 mM or 20 mM) at 37°C for 48 h. Before electrophoresis, the complexes were treated with 0.01% heparin at 37°C for 30 min. A; pDNA ladder, B; naked pDNA, C; STR-CH₂SV40H₂C/pDNA complex, STR-CH₂SV40H₂C/pDNA complex treated with D; 4 mM or E; 20 mM GSH.

Intranuclear pDNA Amount and Gene Expression Efficiency in Mouse Dendritic Cells after Transfection with STR-CH₂SV40H₂C

Finally, to determine the transfection efficiency of STR-CH₂SV40H₂C even in non- or slow-dividing cells, both the intranuclear pDNA amount and gene expression efficiency were determined in mouse dendritic cells (JAWS II cells). Figure 5 shows the intranuclear pDNA amounts and gene expression efficiency in mouse dendritic cells (JAWS II cells) transfected with the pDNA/STR-CH₂SV40H₂C complex at an N/P ratio of 10 or 30. As shown in Fig. 5, the intranuclear pDNA amount and gene expression efficiency increased with increasing N/P ratio, which occurred with STR-CH₂SV40H₂C, and the intranuclear pDNA amount and gene expression efficiency of the STR-CH₂SV40H₂C/pDNA complex were highest at an N/P ratio of 30. Then, gene expression efficiency of the STR-CH₂SV40H₂C/pDNA complex at an N/P ratio of 30 was significantly higher than when Lipofectamine was used as a positive transfection reagent. These results indicate that the high transfection efficiency of STR-CH₂SV40H₂C, even in non-dividing dendritic cells, could be attributed to pDNA condensation by C and STR, enhancement of cellular uptake by STR and endosomal escape by H and STR, intranuclear transport by NLS, and unpacking of pDNA from complexes in high-reduction environments inside the nucleus by disulfide bonds of the thiol group in C.

Fig. 5. Intranuclear pDNA Amount and Gene Expression Efficiency in JAWS II Cells after Transfection with STR-CH₂SV40H₂C

The naked pDNA or pDNA/STR-CH₂SV40H₂C complexes (N/P ratios: 10 or 30) were transfected into JAWS II cells (1×10⁶ cells/35-mm dish) with serum-free medium for 4 h. (a) Intranuclear pDNA amount (pg/nucleus). Each bar represents the mean±S.D. (n=3). ** p<0.01, * p<0.05, and ^n.s. p>0.05 (not significance). (b) GFP expression efficiency (%). Each bar represents the mean±S.D. (n=3). ** p<0.01 vs. other groups, * p<0.05.

CONCLUSION

We demonstrated that STR-CH₂SV40H₂C, in both dividing and slow-dividing cells, enhanced transfection efficiency because of pDNA condensation, efficient cellular and nuclear uptake of pDNA, enhancement of endosomal escape, and unpacking of pDNA in the nucleus by cleavage of disulfide cross linkages in the intranuclear environment. The findings reported in this study suggest that this novel peptide vector, which contains STR, H, and C, is appropriate for the delivery of therapeutic genes.

Acknowledgments

This work was supported in part by a Grant for Private Universities provided by the Promotion and Mutual Aid Corporation for Private Schools of Japan. We thank Ms. Yumiko Suda, Ms. Yuki Hoashi, Ms. Kana Sogabe, and Ms. Asuna Shibano (School of Pharmacy, Tokyo University of Pharmacy and Life Sciences) for their technical assistance.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Wiethoff CM, Middaugh CR. Barriers to nonviral gene delivery. J. Pharm. Sci., 92, 203–217 (2003).
• 2) Brunner S, Sauer T, Carotta S, Cotten M, Saltik M, Wagner E. Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus. Gene Ther., 7, 401–407 (2000).
• 3) Capecchi MR. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell, 22, 479–488 (1980).
• 4) Pouton CW, Wagstaff KM, Roth DM, Moseley GW, Jans DA. Targeted delivery to the nucleus. Adv. Drug Deliv. Rev., 59, 698–717 (2007).
• 5) Wang HY, Chen JX, Sun YX, Deng JZ, Li C, Zhang XZ, Zhuo RX. Construction of cell penetrating peptide vectors with N-terminal stearylated nuclear localization signal for targeted delivery of DNA into the cell nuclei. J. Control. Release, 155, 26–33 (2011).
• 6) Bremner KH, Seymour LW, Logan A, Read ML. Factors influencing the ability of nuclear localization sequence peptides to enhance nonviral gene delivery. Bioconjug. Chem., 15, 152–161 (2004).
• 7) van der Aa MA, Koning GA, d’Oliveira C, Oosting RS, Wilschut KJ, Hennink WE, Crommelin DJA. An NLS peptide covalently linked to linear DNA does not enhance transfection efficiency of cationic polymer based gene delivery systems. J. Gene Med., 7, 208–217 (2005).
• 8) Nagasaki T, Myohoji T, Tachibana T, Futaki S, Tamagaki S. Can nuclear localization signals enhance nuclear localization of plasmid DNA? Bioconjug. Chem., 14, 282–286 (2003).
• 9) Brandén LJ, Mohamed AJ, Smith CI. A peptide nucleic acid nuclear localization signal fusion that mediates nuclear transport of DNA. Nat. Biotechnol., 17, 784–787 (1999).
• 10) Bravo-Osuna I, Teutonico D, Arpicco S, Vauthier C, Ponchel G. Characterization of chitosan thiolation and application to thiol quantification onto nanoparticle surface. Int. J. Pharm., 340, 173–181 (2007).
• 11) Feener EP, Shen WC, Ryser HJ. Cleavage of disulfide bonds in endocytosed macromolecules. A processing not associated with lysosomes or endosomes. J. Biol. Chem., 265, 18780–18785 (1990).
• 12) Aravindan L, Bicknell KA, Brooks G, Khutoryanskiy VV, Williams AC. A comparison of thiolated and disulfide-crosslinked polyethylenimine for nonviral gene delivery. Macromol. Biosci., 13, 1163–1173 (2013).
• 13) Manickam DS, Oupický D. Multiblock reducible copolypeptides containing histidine-rich and nuclear localization sequences for gene delivery. Bioconjug. Chem., 17, 1395–1403 (2006).
• 14) Jones DP, Carlson JL, Samiec PS, Sternberg P Jr, Mody VC Jr, Reed RL, Brown LA. Glutathione measurement in human plasma. Evaluation of sample collection, storage and derivatization conditions for analysis of dansyl derivatives by HPLC. Clin. Chim. Acta, 275, 175–184 (1998).
• 15) Hwang C, Sinskey AJ, Lodish HF. Oxidized redox state of glutathione in the endoplasmic reticulum. Science, 257, 1496–1502 (1992).
• 16) Hwang C, Lodish HF, Sinskey AJ. Measurement of glutathione redox state in cytosol and secretory pathway of cultured cells. Methods Enzymol., 251, 212–221 (1995).
• 17) Pichon C, Gonçalves C, Midoux P. Histidine-rich peptides and polymers for nucleic acids delivery. Adv. Drug Deliv. Rev., 53, 75–94 (2001).
• 18) Kumar VV, Pichon C, Refregiers M, Guerin B, Midoux P, Chaudhuri A. Single histidine residue in head-group region is sufficient to impart remarkable gene transfection properties to cationic lipids: evidence for histidine-mediated membrane fusion at acidic pH. Gene Ther., 10, 1206–1215 (2003).
• 19) Mann A, Shukla V, Khanduri R, Dabral S, Singh H, Ganguli M. Linear short histidine and cysteine modified arginine peptides constitute a potential class of DNA delivery agents. Mol. Pharm., 11, 683–696 (2014).
• 20) Tanaka K, Kanazawa T, Ogawa T, Takashima Y, Fukuda T, Okada H. Disulfide crosslinked stearoyl carrier peptides containing arginine and histidine enhance siRNA uptake and gene silencing. Int. J. Pharm., 398, 219–224 (2010).
• 21) Wolfert MA, Seymour LW. Chloroquine and amphipathic peptide helices show synergistic transfection in vitro. Gene Ther., 5, 409–414 (1998).
• 22) Tanaka K, Kanazawa T, Ogawa T, Suda Y, Takashima Y, Fukuda T, Okada H. A novel, bio-reducible gene vector containing arginine and histidine enhances gene transfection and expression of plasmid DNA. Chem. Pharm. Bull., 59, 202–207 (2011).
• 23) Futaki S, Ohashi W, Suzuki T, Niwa M, Tanaka S, Ueda K, Harashima H, Sugiura Y. Stearylated arginine-rich peptides: a new class of transfection systems. Bioconjug. Chem., 12, 1005–1011 (2001).
• 24) Nakamura Y, Kogure K, Futaki S, Harashima H. Octaarginine-modified multifunctional envelope-type nano device for siRNA. J. Control. Release, 119, 360–367 (2007).
• 25) Mäe M, El Andaloussi S, Lundin P, Oskolkov N, Johansson HJ, Guterstam P, Langel U. A stearylated CPP for delivery of splice correcting oligonucleotides using a non-covalent co-incubation strategy. J. Control. Release, 134, 221–227 (2009).
• 26) Kanazawa T, Tamura T, Yamazaki M, Takashima Y, Okada H. Needle-free intravaginal DNA vaccination using a stearoyl oligopeptide carrier promotes local gene expression and immune responses. Int. J. Pharm., 447, 70–74 (2013).
• 27) Hashida H, Miyamoto M, Cho Y, Hida Y, Kato K, Kurokawa T, Okushiba S, Kondo S, Dosaka-Akita H, Katoh H. Fusion of HIV-1 Tat protein transduction domain to poly-lysine as a new DNA delivery tool. Br. J. Cancer, 90, 1252–1258 (2004).
• 28) Ignatovich IA, Dizhe EB, Pavlotskaya AV, Akifiev BN, Burov SV, Orlov SV, Perevozchikov AP. Complexes of plasmid DNA with basic domain 47–57 of the HIV-1 Tat protein are transferred to mammalian cells by endocytosis-mediated pathways. J. Biol. Chem., 278, 42625–42636 (2003).
• 29) Liu Z, Li M, Cui D, Fei J. Macro-branched cell-penetrating peptide design for gene delivery. J. Control. Release, 102, 699–710 (2005).
• 30) Lo SL, Wang S. An endosomolytic Tat peptide produced by incorporation of histidine and cysteine residues as a nonviral vector for DNA transfection. Biomaterials, 29, 2408–2414 (2008).
• 31) Khalil IA, Futaki S, Niwa M, Baba Y, Kaji N, Kamiya H, Harashima H. Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation. Gene Ther., 11, 636–644 (2004).
• 32) Lehto T, Abes R, Oskolkov N, Suhorutšenko J, Copolovici DM, Mäger I, Viola JR, Simonson OE, Ezzat K, Guterstam P, Eriste E, Smith CIE, Lebleu B, El Andaloussi S, Langel U. Delivery of nucleic acids with a stearylated (RxR)₄ peptide using a non-covalent co-incubation strategy. J. Control. Release, 141, 42–51 (2010).