Enhanced Nuclear Import and Transfection Efficiency of TAT Peptide

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Enhanced Nuclear Import and Transfection Efficiency of TAT PeptideBased Gene Delivery Systems Modified by Additional Nuclear Localization Signals Wen-Jie Yi, Juan Yang, Cao Li, Hui-Yuan Wang, Chen-Wei Liu, Li Tao, Si-Xue Cheng, Ren-Xi Zhuo, and Xian-Zheng Zhang* Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China ABSTRACT: Cellular uptake and nuclear localization are two major barriers in gene delivery. In order to evaluate whether additional nuclear localization signals (NLSs) can improve gene transfection efficiency, we introduced different kinds of NLSs to TAT-based gene delivery systems to form three kinds of complexes, including TATPV/DNA, TAT/DNA/PV, and TAT/DNA/HMGB1. The DNA binding ability of different vectors was evaluated by agarose gel electrophoresis. The in vitro transfections mediated by different complexes under different conditions were carried out. The cells treated by different complexes were observed by confocal microscopy. The MTT assay showed that all complexes did not exhibit apparent cytotoxicity in both HeLa and Cos7 cell lines even at high N/P ratios. The luciferase reporter gene expression mediated by TAT-PV/DNA complexes exhibited about 200-fold enhancement as compared with TAT/DNA complexes. Confocal study showed that, except TAT/DNA/PV, all other complexes exhibited enhanced nuclear accumulation and cellular uptake in both HeLa and Cos7 cell lines. These results indicated that the introduction of nuclear localization signals could enhance the transfection efficacy of TATbased peptides, implying that the TAT peptide-based vectors demonstrated here have promising potential in gene delivery.

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INTRODUCTION Gene therapy has attracted a great deal of attention for the treatment for both acquired and inherited diseases since the first clinical trial in 1990.1,2 The success of gene therapy is mainly dependent on the gene delivery vectors since naked nucleic acids are easily degraded either in vitro or in vivo. Generally, the gene delivery vectors can be divided into two types, viral gene vectors and nonviral ones.3,4 Although viruses are capable of delivering nucleic acids into cells effectively, safety problems such as endogenous virus recombination, oncogenic effects and unexpected immune response do limit their applications.5 After several medical accidents caused by viral vectors,6−8 nonviral vectors such as cationic liposomes9 and polymers10 have gained much attention as attractive alternatives due to their safety, multifunction, low cost, and easy preparation. Cellular internalization of therapeutic agents is a critical issue in gene delivery because the plasma membranes are nearly impermeable barriers. To across these barriers, cell-penetrating peptides (CPPs) have been developed as delivery agents for various therapeutic agents.11,12 A typical example of CPPs is HIV-1 TAT, the first found and most widely used CPP, which is derived from the transcriptional activator protein encoded by human immunodeficiency virus type 1 (HIV-1).13 One main characteristic of TAT protein is the ability to cross the plasma membrane of neighboring cells.14 The minimal sequence used © 2011 American Chemical Society

as transduction domain of TAT is the amino acid residues of 48 to 57 (Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg);15−17 after being covalently or noncovalently linked to cargo molecules, TAT peptide can mediate efficient intracellular delivery. As nuclear transport is a major barrier in gene delivery, several attempts to target DNA to nucleus have been reported, including the use of electrostatic binding of DNA to cationic nuclear localization signal (NLS) -containing peptides or proteins.18,19 One of the most frequently used NLS peptide sequences is PKKKRKV, presented in the Simian Virus 40 large T antigen. It was reported that a combination of the NLS peptide to vectors could improve the nuclear import of plasmid DNA, and thereby benefit the transfection efficiency.20−22 HMGB1 is a nuclear protein which has been used to carry DNA to pass through the nuclear pore complexes.23−26 TCHD is an amphiphilic molecule which has been studied to enhance the nuclear uptake by nonselective gating of nuclear pores.27 To improve nuclear accumulation, as well as cellular uptake, the combination of nuclear localization signals and cellpenetrating peptides has attracted much research interest.28,29 Recently, we constructed a new gene vector made up of cellpenetrating peptide R8 with N-terminal stearylated nuclear Received: October 9, 2011 Revised: December 9, 2011 Published: December 11, 2011 125

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localization signal.30 As for TAT peptide, some literatures reported that TAT peptide possessed the ability of nuclear localization.31 However, other literature certified that TAT was localized primarily in the cytosol of cells, with very limited nuclear accumulation.32 To improve the transfection efficacy of TAT, in this study, we introduced different kinds of nuclear localization signals, including NLS sequence PKKKRKV, nuclear protein high mobility group box protein 1 (HMGB1) to the TAT-based gene vector. In addition, trans-cyclohexane1,2-diol (TCHD) was also used in the DMEM medium to improve the nuclear accumulation of the TAT peptide.

solution) was mixed with TAT solution at a particular N/P ratio (N/P = 20). Then, NaCl solution was added to a total volume of 50 μL. After incubation at 37 °C for 15 min, PV solution with certain volume was added to the solution containing TAT/DNA complexes. Then, NaCl solution was added to a total volume of 100 μL, and incubated at 37 °C for 15 min. TAT/DNA/HMGB1 ternary complexes were prepared as follows. 5 μL DNA solution (200 ng/μL in 40 mM Tris−HCl buffer solution) was mixed with TAT solution at a particular N/P ratio (N/P = 20). After incubation at 37 °C for 15 min, HMGB1 solution with a particular concentration was then added to the TAT/DNA complex-containing solution due to a certain HMGB1/DNA weight ratio (w/w = 2) and incubated at 37 °C for 15 min. Agarose Gel Electrophoresis. The stabilities of all complexes with different compositions were evaluated by agarose gel electrophoresis assay. In brief, for TAT/DNA and TAT-PV/DNA binary complexes, 0.5 μL DNA (200 ng/μL in 40 mM Tris−HCl buffer solution) was mixed with the vector solutions at different N/P ratios. Then, 150 mM NaCl solution was added to a total volume of 8 μL and the complexes were incubated at 37 °C for 30 min. For TAT/DNA/PV ternary complexes, 0.5 μL DNA (200 ng/μL) was mixed with TAT solution at a particular N/P ratio (N/P = 20) and incubated at 37 °C for 15 min. Then, different volumes of PV solution were added to the solution containing TAT/DNA complexes. Then, NaCl solution was added to a total volume of 8 μL, and the system incubated at 37 °C for another 15 min. After complexes were electrophoresed on the 0.7% (w/v) agarose gel containing GelRed with TAE running buffer at 80 V for 60 min, a Vilber Lourmat UV-transiluminator was used for the visualization of DNA. Naked DNA was used as control. Particle Size and Zeta Potential Measurements. The particle size and zeta potential of all complexes were measured by a Nano-ZEN3600 zetasizer (Malvern Instruments) at room temperature. All complexes with different compositions were prepared as mentioned above, and deionized water was added to a total volume of 1 mL for dilution. Cell Culture and Amplifications of Plasmid DNA. Human cervix carcinoma (HeLa) cells and African green monkey kidney (Cos7) cells were acquired from China Center for Typical Culture Collection (Wuhan, China) and were incubated in DMEM medium containing 10% FBS and 1% antibiotics at 37 °C in humidified atmosphere with 5% CO2. pGL-3 plasmid was used as a reporter gene. pGL-3 plasmid was first transformed in Escherichia coli JM 109 and then was amplified in terrific broth media at 37 °C overnight. After that, an EndoFree QiAFilter Plasmid Giga Kit was used for purification of the plasmid. The purified plasmid DNA was dissolved in TE buffer solution and stored at −20 °C before use. In Vitro Cytotoxicity Assay. The cytotoxicity assay in vitro was examined in HeLa and Cos7 cells by MTT assay. Cells were seeded in 96-well plates at a density of 6 × 103 cells/well in 200 μL DMEM medium containing 10% FBS. After 24 h of incubation, the medium was replaced with 200 μL serum-free DMEM. And then for TAT/DNA, TAT-PV/DNA, TAT/ DNA/PV, and TAT/DNA/HMGB1 complexes, the vector/ DNA complex-containing solutions (20 μL containing 0.2 μg DNA) with different compositions were added into each well, and cultured at 37 °C for 4 h. For transfection mediated by addition of TCHD in DMEM medium, the TAT/DNA

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EXPERIMENTAL PROCEDURES Materials. 2-Chlorotrityl chloride resin (100−200 mesh, loading: 1.26 mmol/g), N-fluorenyl-9-methoxycarbonyl (FMOC) protected L-amino acids (FMOC-Gly-OH, FMOCArg(Pbf)-OH, FMOC-Lys(Boc)-OH, FMOC-Gln(Trt)-OH, FMOC-Pro-OH, FMOC-Val-OH), o-benzotriazol-N,N,N′,N′tetramethyluronium hexafluorophosphate (HBTU) were purchased from GL Biochem Ltd. and used as received. Diisopropylethylamine (DiEA) was acquired from GL Biochem Ltd. and distilled prior to use. N-Hydroxybenzotriazole (HOBt), piperidine, trifluoroacetic acid (TFA), and phenol were purchased from Shanghai Reagent Chemical Co. and used directly. N,N-Dimethylformamide (DMF) and dichloromethane (DCM) were obtained from Shanghai Reagent Chemical Co. and distilled before use. Ethanedithiol (EDT), triisopropylsilane (TIS), thioanisole, and polyethylenimine (branched PEI, Mw 25 kDa) were obtained from ACROS and used as received. QIAfilter plasmid purification Giga Kit was purchased from Qiagen. GelRed was purchased from Biotium. Fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin−streptomycin, 0.25% Trypsin-EDTA solution A, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and Dulbecco’s phosphate buffered saline (PBS) were obtained from Invitrogen. Dimethyl sulfoxide (DMSO), high mobility group box 1 (HMGB1), and trans-cyclohexane1,2-diol (TCHD) were obtained from Sigma-Aldrich. Molecular probes (YOYO-1 and Hoechst 33258) were purchased from Invitrogen. The Micro BCA protein assay kit was acquired from Pierce. All other reagents were analytically pure and used directly. Synthesis of Peptides and Preparation of Different Complexes. GRKKRRQRRR-NH2 (TAT), PKKKRKV-NH2 (PV), and GRKKRRQRRRPKKKRKV-NH2 (TAT-PV) were synthesized manually using standard solid-phase synthesis method based on Fmoc (1-(9H-fluoren-9-yl)-methoxycarbonyl)/tert-butyl chemistry.33,34 The molecular weights of peptides were detected by electrode spray ionization mass spectrometry (ESI-MS). Both TAT/DNA and TAT-PV/DNA binary complexes were prepared as follows. The peptide was dissolved in 150 mM NaCl buffer with a certain concentration and stored at −20 °C until use. The peptide/DNA complexes were prepared by mixing particular volume of peptide solution with 5 μL DNA solution (200 ng/μL in 40 mM Tris−HCl buffer solution) with different N/P ratios. Then, 150 mM NaCl solution was added into the complexes to a total volume of 100 μL and vortexed for 10 s. The system was incubated at 37 °C for 30 min to form stable complexes. TAT/DNA/PV ternary complexes were prepared as follows. 5 μL DNA solution (200 ng/μL in 40 mM Tris−HCl buffer 126

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Scheme 1. Gene Delivery Mediated by Different TAT Peptide-Based Gene Vectors, Including TAT/DNA, TAT-PV/DNA, TAT/DNA/PV, TAT/DNA/HMGB1, and TAT/DNA with TCHD in DMEM Medium

complex-containing solutions (20 μL containing 0.2 μg DNA) at N/P ratio of 20 were added into each well. After transfection for 4 h, the medium was removed and serum-free DMEM medium with TCHD was added and cultured for another 1 h. Thereafter, 20 μL of MTT (5 mg/mL) solution was added into each well and further incubated for 4 h. Then, the medium was removed and 200 μL DMSO was added to dissolve the formazan crystals. The absorption was evaluated at 570 nm using a microplate reader (BIO-RAD, Model 550, USA). The cell viability was calculated according to the equation: Cell viability = [OD 570 (treated) − OD 570 (background)/ OD570(untreated) − OD570(background)] × 100%. In Vitro Transfection. The transfecion efficiencies of all complexes including TAT-PV/DNA, TAT/DNA/PV, and TAT/DNA/HMGB1 in the absence of TCHD and TAT/ DNA with the presence of TCHD were evaluated in HeLa and Cos7 cell lines. TAT/DNA complexes were used as control. Cells were seeded in 24-well plates at a density of 6 × 104 cells/well and cultured in 1 mL DMEM medium containing 10% FBS at 37 °C. After incubation for 24 h, the medium was replaced by 1 mL serum-free DMEM containing complexes with different compositions and incubated for 4 h. For transfection in the presence of TCHD, after cultured for 4 h, the medium was replaced and serum-free DMEM with different contents of TCHD was added into each well and cultured for another 1 h. Thereafter, the medium was replaced by fresh DMEM containing 10% FBS and incubated for another 44 h. In order to assess the luciferase expression of plasmid DNA, the medium was removed and 200 μL phosphate buffered saline (PBS, 0.1 M, pH 7.4) was added into each well to wash out the medium remained. Then, cells were lysed with 200 μL reporter lysis buffer each well. The luciferase activity was assessed with chemiluminometer (Lumat LB9507, EG&G Berthold, Germany). The total protein was measured with a BCA protein assay kit. Luciferase activity was determined as RLU/mg protein. Cellular Uptake Study. The cellular uptake study was measured by a confocal laser scanning microscope. Cells were seeded into 6-well plates at a density of 1 × 105 per well and

Table 1. Sequences and Molecular Weights of Peptides Peptide Sequence TAT PV TAT-PV

GRKKRRQRRR-NH2 PKKKRKV-NH2 GRKKRRQRRRPKKKRKVNH2

[M+nH]n+

Molecular Weight

466.6 (n = 3) 442.6 (n = 2) 566.6 (n = 4)

1396.7 883.1 2261.8

cultured with 2 mL DMEM containing 10% FBS at 37 °C for 1 day. Green molecular probe YOYO-1 and blue molecular probe Hoechst 33258 were used to tag pGL-3 and stain nucleus, respectively. One microgram plasmid was mixed with 2.5 μL YOYO-1 solution (10 μM/L) and incubated at 37 °C for 30 min before addition of vectors. After 30 min of incubation, the complexes were added into each well and cultured for 4 h. Then, the complexes were removed and the cells were washed with serum-free DMEM 3 times and PBS 3 times. After that, the nucleus were stained with 20 μL (2 μg/μL) Hoechst 33258. After incubation for further 15 min, the cells were washed again with serum-free DMEM and PBS, and then 1 mL fresh DMEM containing 10% FBS was added. The fluorescence was observed by a confocal laser scanning microscope (Confocal-Si, Nikon, Japan).

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RESULTS AND DISCUSSION

Synthesis of Peptides. In this study, different nuclear localization signals, including PKKKRKV sequence and HMGB1 were introduced to the TAT-based gene vector to form different vector/DNA complexes (Scheme 1). All peptides including GRKKRRQRRR-NH2 (TAT), PKKKRKVNH2 (PV), and GRKKRRQRRRPKKKRKV-NH2 (TAT-PV) were synthesized manually using standard FMOC solid-phase synthesis technique. The molecular weight of peptides was determined by [M+nH]n+ founded in ESI-MS. As shown in Table 1, the molecular weights of TAT, PV, and TAT-PV detected in ESI-MS were 466.6 [M+3H]3+, 442.6 [M+2H]2+, and 566.6 [M+4H]4+, respectively, which coincided with their theoretical molecular weights. 127

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Figure 1. Agorose gel electrophoresis retardation assay of (A) TAT/DNA at N/P ratios from 0 to 30; (B) TAT-PV/DNA at N/P ratios from 0 to 30; and (C) TAT/DNA/PV, to form the ternary complexes, DNA was packed with TAT first, following by the addition of PV peptide at the indicated N/P ratio of 0 to 30. The N/P ratio between TAT and DNA was 20 for all groups, and the final N/P ratios listed as TAT/DNA/NLS were 20/1/0 to 20/1/30.

Figure 2. Particle size of (A) TAT/DNA at N/P ratios ranging from 1 to 30; (B) TAT-PV/DNA at N/P ratios ranging from 1 to 30; and (C) TAT/ DNA/PV at N/P ratios ranging from 20/1/0 to 20/1/30. Data are shown as mean ± SD (n = 3).

DNA Binding Ability of Different Complexes. DNA binding ability is generally considered to be a prerequisite for gene delivery system.35 In this study, the binding and condensing abilities of the vector/DNA complexes with different N/P ratios were evaluated by gel retardation assay. As shown in Figure 1A,B, the TAT and TAT-PV peptides were able to condense DNA at N/P ratio higher than 10 and 5, respectively, which showed that both TAT and TAT-PV peptides possessed strong DNA condensing abilities. We also evaluated the DNA condensing ability of TAT/DNA/PV complexes. As shown in Figure 1C, no significant difference was observed by addition of PV peptide into TAT/DNA complexes even at a high N/P ratio of 30. This further indicated that TAT peptide was effective in binding and condensing DNA and the binding ability was not susceptible to PV peptide. The particle size and zeta potential can reveal the DNA binding ability of vectors and influence the intracellular uptake and subsequent gene transfection efficiency. The particle sizes of different complexes were measured in 150 mM NaCl solution in order to simulate the physiological conditions. As shown in Figure 2A, the average size of the TAT/DNA

complexes increased from about 400 to 800 nm before the N/P ratio reached 10. The particle size of complexes was large when N/P ratios were 5 and 10 due to their slightly negative or neutral zeta potentials, which suggested that complexes might aggregate at neutral conditions. When the N/P ratio was higher than 15, the particle size descended gradually, and the average size of the complexes was less than 400 nm, indicating the formation of compact complexes. For TAT-PV/DNA complexes, as presented in Figure 2B, the size changed in a similar trend. In detail, the size of TAT-PV/DNA ascended with the increasing N/P ratio when the ratio was lower than 5. When the N/P ratio was higher than 5, the particle size descended to a minimum of 325 nm at the N/P ratio of 30. As for the TAT/ DNA/PV complexes, as shown in Figure 2C, the average size descended with the increasing N/P ratio of PV/DNA, which demonstrated that the addition of positively charged PV could benefit the DNA condensing ability of the complexes. However, the effect of the addition of PV peptide on the complex size is limited and the complexes exhibited mean size between 350 and 450 nm. 128

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Figure 3. Zeta potential of (A) TAT/DNA at N/P ratios ranging from 1 to 30; (B) TAT-PV/DNA at N/P ratios ranging from 1 to 30; and (C) TAT/DNA/PV at N/P ratios ranging from 20/1/0 to 20/1/30. Data are shown as mean ± SD (n = 3).

Figure 4. Cell viabilities of TAT/DNA at N/P ratio from 1 to 30, TAT-PV/DNA at N/P ratio from 1 to 30, and TAT/DNA/PV complexes at ratio from 20/1/1 to 20/1/30 TAT/DNA N/P ratio was 20/1 for all groups) in (A) HeLa cells and (B) Cos7 cells. Data are shown as mean ± SD (n = 3).

Figure 5. Cell viabilities of TAT/DNA at N/P ratio of 20, TAT/DNA/HMGB1 at N/P ratio of 20 (HMGB1/DNA weight ratio was 2), and TAT/ DNA at N/P ratio of 20 (with 1.5% w/v TCHD in DMEM medium) in (A) HeLa cells and (B) Cos7 cells. Data are shown as mean ± SD (n = 3).

was 22.3 mV at N/P ratio of 30, while the maximum potential of TAT/DNA was 14.5 mV at the same N/P ratio. As for TAT/DNA/PV ternary complexes, the potential remained positive and increased with the increase of PV amount due to the slight positive charge of PV peptide. In Vitro Cytotoxicity. The biocompatibility of gene vectors is of importance for the success of gene therapy.36 To evaluate the cytotoxicity of vectors, MTT assay was conducted in both

The positive charge of vectors is crucial for the complexes to enter cells due to the negatively charged cell membrane.3 As shown in Figure 3, the zeta potential of TAT/DNA and TATPV/DNA binary complexes and TAT/DNA/PV ternary complexes displayed a similar increasing trend with the increase in N/P ratio. Positive potentials were observed when the N/P ratio was higher than 15 for TAT/DNA and higher than 10 for TAT-PV/DNA. For TAT-PV/DNA, the maximum potential 129

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Figure 6. Luciferase expression mediated by TAT/DNA at N/P ratio of 20, TAT-PV/DNA at N/P ratios from 1 to 30, and TAT/DNA/PV complexes at ratio from 20/1/1 to 20/1/30 (the N/P ratio between TAT and DNA was 20 for all groups) in (A) HeLa cells and (B) Cos7 cells. Data are shown as mean ± SD (n = 3).

Figure 7. Luciferase expression mediated by (A) TAT/DNA/HMGB1 at N/P ratio of 20 (HMGB1/DNA weight ratio was 2) and (B) TAT/DNA at N/P ratio of 20 (with different amounts of TCHD in DMEM medium). Data are shown as mean ± SD (n = 3).

Figure 8. Luciferase expression mediated by TAT-PV/DNA complexes at N/P ratios from 1 to 30 with or without serum: (A) in HeLa cells, and (B) in Cos7 cells. Data are shown as mean ± SD (n = 3).

HeLa and Cos7 cell lines. As shown in Figure 4, no significant cell toxicity was observed in both cell lines for TAT/DNA, TAT-PV/DNA, and TAT/DNA/PV complexes. Moreover, we found that the cytotoxicity of different complexes primarily depended on the N/P ratio of vector/DNA. With an increase in N/P ratio, the cell viability slightly decreases. Nevertheless, all complexes exhibited very low cytotoxicities with cell viabilities higher than 90%. We also evaluated the cell toxicity of TAT/DNA/HMGB1 complexes and TAT/DNA complexes with the presence of 1.5% w/v TCHD in DMEM medium. According to the results in Figure 5, for both cell lines, the cytotoxicity values of TAT/ DNA (with 1.5% w/v TCHD in DMEM medium) and TAT/ DNA/HMGB1 complexes were slight higher than that of TAT/DNA complexes alone, which was due to the additional toxicity of TCHD and HMGB1. Nevertheless, both complexes showed more than 85% cell viability. In Vitro Transfection. In order to observe the influence of additional nuclear localization signals on the TAT/DNA complexes, the transfection efficiencies of different complexes

were assessed in different cell lines. As shown in Figure 6, TATPV/DNA complexes exhibited significantly improved transfection efficiency. In detail, the gene expression of TAT-PV/ DNA increased with the increasing N/P ratio from 1 to 15, but further increase in N/P ratio did not result in the improvement in transfection. At N/P ratio of 15, the complexes showed the highest luciferase expression in both cell lines (HeLa: 6.29 × 107 RLU/mg Protein; Cos7: 8.12 × 107 RLU/mg Protein), which exhibited about 200-fold enhancement as compared with TAT/DNA complexes (HeLa: 3.41 × 105 RLU/mg Protein; Cos7: 4.01 × 105 RLU/mg Protein). One reason for enhanced gene efficiency was the addition of cationic PV, which increases the positive charge density of the complexes. Another reason was the addition of PV peptide causing a decrease in particle size of TAT/DNA complexes to some extent. As we know, an appropriate particle size is a prerequisite for gene transfection. Small particles may exhibit higher transfection efficiencies compared with larger ones, as small particles are able to penetrate throughout the submucosal layers while the larger particles are predominantly localized in the epithelial lining.37 130

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Figure 9. Confocal microscopy images of intracellular trafficking in HeLa cells: (A) TAT/DNA at N/P ratio of 20; (B) TAT/DNA/PV at ratio of 20/1/10; (C) TAT/DNA at N/P ratio of 20 (with 1.5% w/v TCHD in DMEM medium); (D) TAT/DNA/HMGB1 at N/P ratio of 20 (HMGB1/ DNA weight ratio was 2); and (E) TAT-PV/DNA at N/P ratio of 15.

mitosis.40 So, introduction of NLS sequence may provide a large benefit for nuclear import and subsequent transfection of TAT/DNA complexes. However, introduction of PV sequence to the gene delivery system through electrostatic complexation did not show obvious enhancement in gene expression. This is due to the fact that TAT/DNA/PV ternary complexes were unstable and the contribution of noncovalent bound NLS peptide is limited for the enhancement of gene expression. We also introduced HMGB1 and TCHD to the gene delivery systems. As shown in Figure 7, for TAT/DNA/ HMGB1 complexes with HMGB1/DNA weight ratio of 2,30 the luciferase expression increased by about 3-fold in HeLa cells and Cos7 cells (HeLa: 1.02 × 106 RLU/mg Protein; Cos7: 1.24

However, larger particles have slightly higher DNA release than small ones,38 so a medium-sized complex may be important in gene delivery. Furthermore, the introduction of NLS sequence could also benefit the gene transfection. As we know, nuclear transport of vector/DNA complexes is one major barrier in nonviral gene delivery systems. Nuclear pore complexes (NPCs) are large proteinaceous assemblies spanning the nuclear envelope, functioning as mediators of a two-way alternative between the nucleoplasmic and cytoplasmic compartments.39 Generally, macromolecules such as DNA and proteins have difficulty passing through the NPCs due to their large size, and the most possible way for DNA complexes to enter the nucleus is through nuclear envelope fracture during 131

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Figure 10. Confocal microscopy images of intracellular trafficking in Cos7 cells. (A) TAT/DNA at N/P ratio of 20; (B) TAT/DNA/PV at ratio of 20/1/10; (C) TAT/DNA at N/P ratio of 20 (with 1.5% w/v TCHD in DMEM medium); (D) TAT/DNA/HMGB1 at N/P ratio of 20 (HMGB1/ DNA weight ratio was 2); and (E) TAT-PV/DNA at N/P ratio of 15.

TAT-PV/DNA in 10% serum was 1.63 × 107 RLU/mg protein, which is comparable to that without serum (1.86 × 107 RLU/ mg protein), which indicated that the TAT-PV vector possessed stability in serum and had potential application in vivo. Cellular Uptake Study of Vector/DNA Complexes by Confocal Laser Scanning Microscopy. In order to investigate the effects of the nuclear localization signals on the complexes in nuclear accumulation and cellular internalization, the HeLa cells and Cos7 cells treated by different complexes were observed by a confocal laser scanning

× 106 RLU/mg Protein). For TAT/DNA complexes with 1.5% w/v TCHD in DMEM medium, the luciferase expression increased by more than 2-fold in both cell lines (HeLa: 7.72 × 105 RLU/mg Protein; Cos7: 8.71 × 105 RLU/mg Protein). Those results indicated the presence of both HMGB1 and TCHD could increase nuclear transport and thus increase the luciferase expression mediated by the complexes. To assess the effect of serum, we also evaluated transfections of TAT-PV/DNA complexes in both cell lines in the presence of 10% serum. As shown in Figure 8, no significant decrease was detected in both HeLa and Cos7 cells. For example, in HeLa cells, at N/P ratio of 10, the luciferase expression of 132

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microscope. Molecular probes YOYO-1 and Hoechst 33258 were used to tag DNA and nucleus. As shown in Figure 9, after coincubating with TAT/DNA complexes with N/P ratio of 20 for 4 h, only a few green fluorescence dots could be observed in the cytoplasm and almost no green fluoroescence dots were detected in the nucleus of HeLa cells. The results were in accordance with our transfection results above, indicating the limited capacity of TAT peptide in nuclear target. As for TAT/DNA/HMGB1 with N/P of 20 and HMGB1/DNA weight ratio of 2 and TAT/ DNA complexes with 1.5% w/v TCHD in DMEM medium, the increase in the number of green fluoroescence dots detected in the cytoplasm is limited. For HeLa cells treated with TAT-PV/DNA with N/P ratio of 15, apparent increase of green fluorescence dots in the cytoplasm could be detected, and quite a few green fluorescence dots were observed in the nucleus of the cells, which indicated that the covalently bound NLS peptide contributed a lot to the cellular uptake and nuclear accumulation of TAT-based gene delivery systems. However, TAT/DNA/PV complexes did not show obvious enhancement in cellular uptake and nuclear accumulation as compared with TAT/DNA complexes. The possible reason is that the ternary complexes formed by electrostatic complexation were not stable enough and may be degraded in cytoplasm. Cellular uptake and nuclear accumulation in Cos7 cells were also detected. As shown in Figure 10, consistent with results in HeLa cells, similar trends were observed in Cos7 cells, i.e., TAT-PV/DNA complexes exhibited the highest cellular uptake and nuclear accumulation.

CONCLUSIONS In this study, we introduced different kinds of nuclear localization signals to TAT-based gene delivery systems to form three kinds of complexes, including TAT-PV/DNA, TAT/DNA/PV, and TAT/DNA/HMGB1. Trans-cyclohexane1,2-diol (TCHD) was also added in DMEM medium to improve the transfection efficiency of TAT/DNA complexes. We found that, except TAT/DNA/PV, the presence of nuclear localization signals in all other complexes benefited nuclear accumulation and resulted in enhanced cellular uptake of plasmid DNA in both HeLa and Cos7 cell lines. Moreover, the luciferase reporter gene expression mediated by TAT-PV/DNA complexes exhibited about 200-fold enhancement as compared with TAT/DNA complexes. These results indicated that additional nuclear localization signals could improve the nuclear transport and cellular uptake of TAT-based gene delivery systems. The modification strategy in this study could effectively improve the gene transfection ability of peptidebased nonviral gene vectors. AUTHOR INFORMATION

Corresponding Author

*Tel.: + 86 27 6875 5993; fax: + 86 27 6875 4509. E-mail address: [email protected] (X.Z. Zhang).

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REFERENCES

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ACKNOWLEDGMENTS

This work was financially supported by the Ministry of Science and Technology of China (2011CB606202), National Natural Science Foundation of China (20974083), and the Fundamental Research Funds for the Central Universities. 133

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