International Journal of Pharmaceutics 459 (2014) 10–18
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical Nanotechnology
Gene delivery of PAMAM dendrimer conjugated with the nuclear localization signal peptide originated from fibroblast growth factor 3 Jeil Lee a , Jinwoo Jung a , Youn-Joong Kim a,c , Eunji Lee a , Joon Sig Choi a,b,∗ a b c
Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305-764, Republic of Korea Department of Biochemistry, Chungnam National University, Daejeon 305-764, Republic of Korea Korea Basic Science Institute (KBSI), 169-148 Gwahangno (52 Yeoeun-Dong), Yuseong-Gu, Daejeon 305-333, Republic of Korea
a r t i c l e
i n f o
Article history: Received 26 July 2013 Received in revised form 25 October 2013 Accepted 14 November 2013 Available online 23 November 2013 Keywords: Polyamidoamine dendrimer Mouse fibroblast growth factor 3 Nuclear localization signal Transfection Polyplex Cytotoxicity
a b s t r a c t Polyamidoamine (PAMAM) is one of the widely employed non-viral vectors in gene therapy research, and shows excellent biocompatibility and relatively low cytotoxicity. However, it has poor transfection efficiency compared with that of polyethylenimine (PEI, 25 kDa). To enhance the gene expression efficiency, we introduced the RRRK peptide from mouse fibroblast growth factor 3 (FGF3) to PAMAM, which is a known nuclear localization signal (NLS). We synthesized PAMAM-KRRR and PAMAM-RRRK to verify the difference of the induced functional status from reversal of the N-terminus. PAMAM containing the FGF3 peptide showed a transfection efficiency corresponding to that of PEI in HEK293, and HeLa cells, and showed much higher gene expression capacity than that of PEI in NIH3T3 cells with relatively decreased cytotoxicity. These results imply that introduction of the FGF 3 peptide has the potential to provide a novel PAMAM-based vector by enhancing its gene expression efficiency. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Gene therapy is a technique which can either replace deficient genes or inhibit unwanted genes expression by introduction of external therapuetic genes aiming at fundamental treatment of disease at the genetic level. There are two types of gene therapies, which use a ‘vector system’ or physical methods (Mancheno-Corvo and Martin-Duque, 2006; Niidome and Huang, 2002). At the initial stage of gene therapies, viral vectors were used and showed therapeutic efficacy for treating congenital diseases. Despite the excellent efficacy, introduction of a viral vector has safety issues including insertional mutations by the virus and potential fatality caused by excessive inflammatory responses to the viral vector. Therefore, nonviral vector systems have attracted attention as an alternative to overcome the deficiencies of viral vectors. In nonviral vector systems, a cationic polymer and lipid are majorly used for gene transfer by forming a DNA-vector complex via electrostatic interactions between the DNA and cationic vector (Jeong et al., 2007). Nonviral vector systems have the advantage of comparatively low immune responses and cytotoxicity as well as ease of quality control and mass production. However,
∗ Corresponding author at: Department of Biochemistry, Chungnam National University, Daejeon 305-764, Republic of Korea. Tel.: +82 42 821 7528; fax: +82 42 822 7548. E-mail addresses: [email protected], [email protected] (J.S. Choi). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.11.027
the poor transfection efficiency of nonviral vectors remains to be resolved. Polyethyleneimine (PEI) is a cationic polymer with excellent gene expression efficiency in transfections of numerous cell lines. As the molecular weight increases, the number of primary amines provided by PEI is also increased and shows an enhanced transgene expression capacity resulted from strong DNA condensation and a proton buffering effect by enrichment of primary amines (De Smedt et al., 2000; Jeong et al., 2007). Although an increase of molecular weight denotes improved transfection efficiency, poor biodegradability of PEI causes high cytotoxicity. To improve the biodegradability, research has focused on degradable polymer linkers. One such polymer is polyamidoamine (PAMAM) dendrimer consisting of a peptide bond between methylacylate and ethylenediamine, which possesses excellent biodegradability and biocompatibility but the transfection efficiency is still poor (Braun et al., 2005; Dufes et al., 2005). It has been reported that the complexes of dendrimers form polymer/lipid vesicles by electrostatic interaction with cellular membranes and are transferred inside the cell by formation of nano-sized hole whereas complexes of cationic lipids are fused with cellular membranes (Mecke et al., 2004, 2005; Smith et al., 2010). Many groups have studied surface modification using cell penetrating peptides, nuclear localization signal (NLS) peptides, and peptide ligands for receptor-mediated delivery to improve the transfection efficiency (Choi et al., 2004, 2006; Martin and Rice, 2007; Ramamoorthy et al., 2007). In this study, to enhance the gene expression efficiency, we synthesized novel functional PAMAM dendrimer conjugated with
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multiple NLS peptides derived from mouse fibroblast growth factor 3 (FGF3) (Kiefer et al., 1994; Martin and Rice, 2007). In FGF3, the NLS sequence is RLRRDAGGRGGVYEHLGGAPRRRK with RLRR and RRRK sequences that induce nuclear localization. We hypothesized that introduction of the RRRK sequence on the PAMAM surface would improve DNA condensation by enrichment of basic amino acids and provide active transport of relatively large transgenes by the NLS peptide leading to increased gene transfection especially in fibroblast cell lines.
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2.3. Agarose gel retardation assay
2. Experiment details
Polyplexes of PAMAM-KRRR and PAMAM-RRRK with pCN-Luci were prepared at various weight ratios (polymer/pCN-Luci) ranging from 1 to 16 in HEPES buffer (25 mM, pH 7.4). The polymers of various weight ratios in HEPES buffer with pCN-Luci at a fixed volume were mixed and incubated at room temperature for 30 min. Then, electrophoresis of polyplex-formed samples was performed in a 0.7% agarose gel with 0.5 g/mL ethidium bromide. Agarose gel electrophoresis was progressed for 30 min at 100 V and agarose gel was analyzed under a UV illuminator to confirm the location of plasmid DNA bands.
2.1. Materials
2.4. Dynamic light scattering and -potential measurements
Ethidium bromide (EtBr), dimethysulfoxide (DMSO), N,Ndiisopropylethylamine (DIPEA), N,N-dimethylformamide (DMF), PAMAM generation 4, polyethylenimine (branched, 25 kDa), trifluoroacetic acid (TFA) and triisopropylsilane (TIS) were purchased from Sigma–Aldrich (Seoul, South Korea). N-hydroxybenzotriazole (HOBt), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetra-methyluronium (HBTU) were purchased from Anaspec (San Jose, CA, USA), and Fmoc-Arg(pbf)-OH and Fmoc-Lys(Boc)-OH was obtained from Novabiochem (San Diego, CA, USA). Luciferase 1000 assay kit and reporter lysis buffer were purchased from Promega (Madison, WI, USA). The luciferase expression plasmid DNA (pCN-Luci) was prepared as reported previously. 293, HeLa, and NIH3T3 cell lines were obtained from Korean Cell Line Bank. Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM) and 100× antibiotic-antimycotic reagent were purchased from GIBCO (Gaithersburg, MD, USA). Micro BCA Protein Assay Kit was purchased from Pierce (Rockford, IL, USA). ULYSIS® Nucleric Acid Labeling Kit was purchased from Molecular Probes (Invitrogen).
The size and -potential value of PEI 25 kDa, PAMAM generation 4, PAMAM-KRRR, and PAMAM-RRRK/pCN-Luci complexes were determined by a Zeta-potential & Particle Size Analyzer ELS-Z (Photal, Otsuka Electronics, Japan) and Zetasizer Nano ZS (Malvern Instruments, UK). pCN-Luci (10 g) was used for polyplex formation with polymers. Each polyplex was prepared in a total volume of 800 L with distilled water and analyzed at the weight ratios showing optimal transfection efficiency. 2.5. Transmission electron microscopy PAMAM-KRRR was prepared with pCN-Luci (5 g) in distilled water. PAMAM-KRRR/pCN-Luci was mixed at 16:1 (w/w) and incubated at room temperature for 30 min. Then, 10 L of PAMAMKRRR/pCN-Luci polyplex was placed on the surface of a 300-mech copper grid and dried at natural dry condition for 4 h. Electron micrographs were optained by an energy filtering transmission electron microscope (EM912, Carl Zeiss Inc., Germany) at 120 kV (Shcharbin et al., 2009; Tang and Szoka, 1997). 2.6. Heparin competition assay
2.2. Synthesis of PAMAM-KRRR and PAMAM-RRRK Conjugation of amino acids to the PAMAM generation 4 surface was performed in a mixture of anhydrous DMF and DMSO of 2:1 (v/v) for 18 h at room temperature with 4 equivalents of HOBt, HBTU and Fmoc-Lys(Boc)-OH and 8 equivalents of DIPEA. The product was precipitated in diethyl ether and washed with excess ether. The Fmoc protecting group of Fmoc-Lys(Boc)-conjugated PAMAM was eliminated by addition of 30% piperidine in DMF (v/v). After 3 h of the deprotection reaction, mixtures were precipitated in diethyl ether and washed in an excess of ether. To conjugate FmocArg(pbf)-OH additionally, a coupling reaction of Fmoc-Arg(pbf)-OH with the product was performed in DMF for 18 h at room temperature with 4 equivalents of HOBt, HBTU, and Fmoc-Arg(pbf)-OH and 8 equivalents of DIPEA. The subsequent deprotection reaction, precipitation process, and elongation of additional amino acids were performed as described in Fig. 1 (Kumar et al., 2010). PAMAM-RRRK was also synthesized as described above. After amino acid elongation to PAMAM generation 4 was finalized, the existing protecting group (BOC and pbf) of conjugated PAMAM generation 4 was removed by deprotection solution (95:2.5:2.5, trifluoroacetic acid/triisopropylsilane/H2 O, v/v) and the final product was precipitated in diethyl ether and washed in an excess of ether. The final product was dissolved in water, placed in a dialysis membrane (MWCO 10,000, Spectra/por) and dialyzed with distillated water overnight. Then, the product was collected as a white powder after lyophilization. The synthesis yield for final products were usually over 99% and 1 H NMR spectra (400 MHz, D2 O) of the synthesized polymers are shown in Fig. 2.
Polyplexes of PEI 25 kDa, PAMAM generation 4, PAMAM-KRRR, and PAMAM-RRRK/pCN-Luci were prepared at the 4:1 (w/w). Then, heparin solution of various concentrations was added at polyplexes and incubated at room temperature for 30 min. Each polyplexes was prepared in a total volume of 200 L with HEPES buffer (25 mM, pH 7.4) and 200 L PicoGreen reagent diluted in TE buffer (10 mM Tris, 0.5 mM EDTA, pH 7.5) was added and incubated for 2 min. Finally, the polyplexes were prepared in a total volume of 2 mL with 1.6 mL TE buffer and relative fluorescence intensity was analyzed by a spectrofluorometer (Quantech Digital Filter Fluorometer, Thermo Scientific, USA). The excitation and emission wavelengths were 490 and 520 nm, respectively. 2.7. Cell culture and transfection assay Human embryonic kidney (HEK) 293 cells, HeLa human epithelial carcinomacells and NIH3T3 mouse embryonic fibroblasts were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic mixture. Cell lines were maintained in an incubator (at 37 ◦ C with 5% CO2 , and 95% humidity). For the transfection assay, cells were cultured in a 96-well plate at a density of 1.8 × 104 cells/well for 16 h to 70–80% confluence. The polyplexes were prepared by mixing pCN-Luci (0.5 g) with PAMAM-KRRR and PAMAM-RRRK at various weight ratios for 30 min at room temperature. To compare the transfection efficiency, PEI 25 kDa and PAMAM generation 4/pCN-Luci complexes were prepared as the control group. Cultured cells were treated with polyplex solutions for 24 h at 37 ◦ C. The cells were washed with Dulbecco’s Phosphate Buffered Saline (DPBS), and
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Fig. 1. Synthesis of PAMAM-KRRR and PAMAM-RRRK.
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Fig. 2.
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H NMR spectra of PAMAM-KRRR (A) and PAMAM-RRRK (B).
100 L of reporter lysis buffer (Promega, Madison, WI) was added to each well. After 30 min of incubation at room temperature, the cell lysates were harvested to microcentrifuge tubes and centrifuged at 13,200 rpm for 10 min. The protein concentration of lysates was measured by a Micro BCATM protein assay kit. Luciferase activity was measured using a Lumat LB 9507 instrument (Berthold Technology, Bad Wildbad, Germany). Luciferase activity was indicated as RLU/g total protein.
plates for 1 day prior to addition of the polymers. The cells were treated with PEI 25 kDa, PAMAM generation 4, PAMAM-KRRR, and PAMAM-RRRK at various concentrations and were incubated for 24 h at 37 ◦ C. Then, 10 L WST-1 reagent was added at each well and incubated for 2 h at 37 ◦ C. Then, the absorbance was measured at 450 nm using a microplate reader (VersaMax, Molecular Devices, USA).
2.8. Cytotoxicity assay
2.9. Preparation of Alexa Fluor 532-labeled pCN-Luci and confocal microscopy
The cytotoxicity of polymers was evaluated by a WST-1 assay. Cells were cultured at a density of 1.8 × 104 cells/well in 96-well
pCN-Luci was mixed with 3 M sodium acetate (pH 5.2) buffer solution with ethanol. Then, the mixture was frozen at −70 ◦ C for
Fig. 3. Agarose gel retardation assay for polyplexs of PEI 25 kDa, PAMAM generation 4, PAMAM-KRRR and PAMAM-RRRK with plasmid DNA. Plasmid DNA only (lane 1), the weight ratios of PAMAM-KRRR/pDNA (lanes 2–8), PAMAM-RRRK/pDNA = 1, 2, 4, 6, 8, 12, 16 (lanes 11–17), PEI 25 kDa (lane 9), and PAMAM generation 4 (lane 10).
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30 min. After thawing at room temperature, the mixture was centrifuged at 12,000 rpm for 15 min. The precipitate was washed with 70% ethanol and was dried using nitrogen gas. For DNA denaturation, nuclease-free H2 O component H was added to the dried precipitate, followed by incubation in a water bath at 95 ◦ C for 5 min. The precipitate was cooled on ice and centrifuged again. ULS reagent (10 L) was added to the precipitate, followed by incubation at 80 ◦ C for 15 min and then cooling on ice. Excess ULS reagent was eliminated by gel filtration using a MicroSpin G-25 column (Sigma–Aldrich). NIH3T3 cells were seeded at a density 5 × 103 cells/well in a confocal dish and cultured for 16 h to 70–80% confluence. Alexa Fluor 532-labeled pCN-Luci and unlabelled pCN-Luci were mixed at 1:1 (w/w). Then, the DNA mixture was complexed with PEI 25 kDa, PAMAM generation 4, PAMAMKRRR, and PAMAM-RRRK. PEI 25 kDa and PAMAM generation 4/pCN-Luci complexes were prepared at 2:1 (w/w) and 4:1 (w/w), respectively, and both PAMAM-KRRR and PAMAM-RRRK/pCN-Luci complexes were prepared at 16:1 (w/w). Each weight ratio represented optimal transfection efficiency. Cells were treated with the complexes for 24 h. The amount of pCN-Luci mixture was fixed at 0.5 g/well. Confocal microscopy images were obtained by a Zeiss LSM 5 Live confocal laser microscope.
2.10. Statistical analysis The statistical analysis was performed via the unpaired Student’s t-test (GraphPad Prism 5). Differences between groups were considered statistically significant at P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).
3. Results 3.1. Synthesis of PAMAM-KRRR and PAMAM-RRRK In previous studies, peptides enriched with cationic amino acids have shown an efficient translocation capacity via the cell membrane as well as a superior DNA condensation capacity and transfection efficiency. In this study, we used RRRK peptide from the bipartite NLS of FGF3. The RRRK sequence was conjugated on the surface of PAMAM dendrimer generation 4 (Fig. 1). The synthesized product was dissolved in D2 O for 1 H NMR (400 MHz). As shown in Fig. 2, the 1 H NMR spectra of the synthesized polymers are like as followings. PAMAM-KRRR: ı 1.41 ( CHCH2 CH2 CH2 CH2 NH2 of the lysine unit), 1.65 ( CHCH2 CH2 CH2 NH of the arginine unit and CHCH2 CH2 CH2 CH2 NH2 of the lysine unit), 1.80 ( CHCH2 CH2 CH2 NH of the arginine unit and CHCH2 CH2 CH2 CH2 NH2 of the lysine unit), 2.46 ( NCH2 CH2 CO of the PAMAM unit), 2.72 ( CONHCH2 CH2 N of PAMAM unit and NCH2 CH2 N of PAMAM unit), 2.90 ( NCH2 CH2 CO of PAMAM unit), 2.98 ( CHCH2 CH2 CH2 NH of the arginine unit and CHCH2 CH2 CH2 CH2 NH2 of the lysine unit) and 3.27 ( CONHCH2 CH2 N of PAMAM unit and CONHCH2 CH2 NHCO of PAMAM unit) PAMAM-RRRK: ı 1.43 ( CHCH2 CH2 CH2 CH2 NH2 of the argiof the lysine unit), 1.65 ( CHCH2 CH2 CH2 NH nine unit and CHCH2 CH2 CH2 CH2 NH2 of the lysine unit), 1.80 ( CHCH2 CH2 CH2 NH of the arginine unit and CHCH2 CH2 CH2 CH2 NH2 of the lysine unit), 2.45 ( NCH2 CH2 CO of the PAMAM unit), 2.70 ( CONHCH2 CH2 N of PAMAM unit and NCH2 CH2 N of PAMAM unit), 2.88 ( NCH2 CH2 CO of PAMAM unit), 2.99 ( CHCH2 CH2 CH2 NH of the arginine unit CHCH2 CH2 CH2 CH2 NH2 of the lysine unit) and 3.25 and ( CONHCH2 CH2 N of PAMAM unit and CONHCH2 CH2 NHCO of PAMAM unit). To study the polymer function by
Table 1 Size measurement using dynamic light scattering and zeta potential analysis of polymer/pDNA complexes.
Weight ratio (polymer:DNA) Mean size (nm) Zeta potential (mV) Polydispersity index
PEI
PAMAM
PAMAMKRRR
PAMAMRRRK
2:1
4:1
16:1
16:1
225.4 ± 13 36.8 ± 4 0.246
188.5 ± 11 38.4 ± 5 0.277
139.5 ± 13 22.7 ± 5 0.277
153.8 ± 15 22.2 ± 5 0.292
changing the peptide terminal, we synthesized PAMAM-KRRR and PAMAM-RRRK. 3.2. Agarose gel retardation assay To verify formation point of PAMAM-KRRR and PAMAMRRRK/pCN-Luci polyplexes, we performed agarose gel retardation assays (Fig. 3). The polyplexes were prepared at various weight ratios and analyzed in 0.7% (w/v) agrose gel. Polyplexes of PEI and PAMAM generation 4/pCN-Luci were used as the control and prepared at 2:1 (w/w) and 4:1 (w/w), respectively, which are ratios for optimal transfection effiecieny. PAMAM-KRRR and PAMAMRRRK/pCN-Luci polyplexes were retarded at weight ratios between 2:1 and 4:1. Complete retardation of pCN-Luci was observed at 4:1 (w/w). 3.3. Size and -potential measurements of PAMAM-KRRR and PAMAM-RRRK/pCN-Luci complexes We prepared polymer/pCN-Luci complexes as described in Table 1. Each polymer was efficiently condensed with pCN-Luci and formed nanometer-sized polyplexes of sizes ranging from 140 to 225 under conditions for optimal transfection. The introduction of KRRR and RRRK peptides on the PAMAM surface resulted in more compact polyplexes than those of PEI 25 kDa and unmodified PAMAM generation 4. Under the same conditions, we also measured -potential of the polyplexes. Althought cationic amino acids were introduced to PAMAM surface, the surface charge density of PAMAM-KRRR and PAMAM-RRRK was around 22 mV, and the value was approximately 15 mV lesser than of PAMAM generation 4 and PEI 25 kDa. 3.4. Transmission electron microscopy of PAMAM-KRRR/plasmid DNA polyplexes Dendriplexes of PAMAM-KRRR/pCN-Luci were observed by transmission electron microcscopy (TEM). Each dendriplexs exhibited a tightly condensed spherical shape (Fig. 4). Although aggregates of dendriplexes were also observed, the complexes showed a similar size (between 120 and 150 nm) compared with the measured diameter using dynamic light scattering (DLS). 3.5. Evaluation of polyplex stability by polyanion competition assay To verify the stability of polymer/pCN-Luci complexes, we performed polyanion competition assay using heparin (Fig. 5). The stability of polyplexes was analyzed by relative fluorescence of picogreen agent. The PAMAM generation 4/pCN-Luci polyplexes have begun to dissociate in the presence of heparin solution at concentrations between 20 and 30 g/mL and were completely disintegrated over 30 g/mL. Polyplexes of PAMAM-KRRR and PAMAM-RRRK showed increased stability than that of native PAMAM but these polyplexes were completely disintegrated at
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concentrations over 40 g/mL while PEI 25 kDa/pCN-Luci polyplexes maintained intact complexes.
3.6. Transfection efficiency of different cell lines The transgene expression efficiency of PAMAM generation 4 containing the RRRK peptide fragment originated from FGF3 was evaluated in a transfection assay. PAMAM-KRRR/pCN-Luci polyplexes were prepared at various weight ratios. The transfection assays using these polyplexes were performed with HEK293, HeLa, and NIH3T3 cell lines in the presence of 10% FBS. The PEI 25 kDa and PAMAM generation 4 were used as controls (polyplexes of polymer/pCN-Luci were prepared at 2:1 and 4:1 weight ratios, respectively). The transfection efficiency depended on the cell line and showed predominant gene expression efficiency for NIH3T3 cells compared with that using PEI 25 kDa. The transfection efficiency of PAMAM-KRRR for HEK293 and HeLa cell lines also showed similar expression efficiencies in comparison with that of PEI 25 kDa. The weight ratio for optimal expression efficiency was 6:1 (w/w) for HEK293 and HeLa cell lines and 16:1 for the NIH3T3 cell line (Fig. 6). The introduction of the RRRK sequence on the surface of PAMAM generation 4 enhanced the transfection efficiency by 4–2300 fold compared with that of the original PAMAM generation 4. PAMAM-RRRK was also analyzed at the same weight ratio as that of PAMAM-KRRR, and showed increased transfection efficiency.
3.7. Cytotoxicity evaluation
Fig. 4. TEM images of Dendriplexes of PAMAM (A) and PAMAM-KRRR (B).
As shown in Fig. 7, we performed cytotoxicity assays using HEK293, HeLa, and NIH3T3 cell lines. In the presence of 10% FBS, the cells were cultured for 16 h to 70–80% confluence. Next, the cells were treated with the polymers at various concentrations for 24 h. The PEI 25 kDa as a control showed strong cytotoxicity whereas PAMAM generation 4 showed much less cytotoxicity in all cell lines. PAMAM-KRRR and PAMAM-RRRK indicated contrary cytotoxic tendency. Although the RRRK sequences were introduced on the surface of PAMAM generation 4, the cytotoxicity of PAMAMKRRR in all cell lines showed a similar level compared with that of native PAMAM generation 4 while PAMAM-RRRK showed a rapid increase of cytotoxicity at concentrations over 90 g/mL.
Fig. 5. Polyanion competition assay of polyplexes using heparin. PEI 25 kDa (䊉), PAMAM generation 4 (), PAMAM-KRRR (), and PAMAM-RRRK ().
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Fig. 6. Transfection efficiency of PAMAM-KRRR and PAMAM-RRRK in various cell lines. (A) HEK293, (B) HeLa, and (C) NIH3T3 cells. Data are shown as mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001.
3.8. Confocal microscopy To verify intranuclear transfer of plasmid DNA by PAMAMKRRR, we used confocal microscopy (Fig. 8). As shown in Table 1, polyplexes of the polymers at weight ratios for optimal transfection efficiency were prepared with pCN-Luci labeled with Alexa 532 fluorescent dye. The polyplexes of each polymer were incubated with NIH3T3 cells in the presence of 10% FBS for 24 h. The nuclei were indicated as blue and the location of polymer/pCN-Luci polyplexes was indicated as red. Compared with other polymers, polyplexes of PAMAM-KRRR/pCN-Luci were mostly distributed in the nucleus whereas polyplexes of PAMAM-RRRK were located in the cytosol. These results suggest that high gene transfection efficiency of PAMAM-KRRR might be caused by introduction of the NLS from FGF3. 4. Discussion We evaluated physical properties such as the size, potential, shape, and formation point of polyplexes and assessed their transfection efficiency and cytotoxicity. Despite excellent biodegradability, we observed a low transfection efficiency of the native PAMAM dendrimer, which was dependent on the cell line. To increase the gene transfer efficiency, we introduced the NLS
Fig. 7. Cytotoxicity assay in (A) HEK293, (B) HeLa and (C) NIH3T3 cells using WST assay. PEI 25 kDa (䊉), PAMAM generation 4 (), PAMAM-KRRR (), and PAMAMRRRK (). The data of respective point indicates average and standard deviation from quadruplicate.
from FGF3 on the surface of PAMAM generation 4 (Figs. 1 and 2). The PAMAM-KRRR with plasmid DNA formed a complete polyplex at a weight ratio of 4:1 (Fig. 3), and formed more compact polyplexes at 16:1 (w/w). Under the same condition, polyplex sizes were approximately 140 nm (Table 1 and Fig. 4) and showed a potential of 22.7 mV (Table 1). PAMAM-KRRR would exist mostly as a positively charged form at neutral pH because the pKa value of an arginine guanidine group is higher than those of amines of native PAMAM and PEI 25 kDa. Therefore, the formation of more compact polyplexes and relatively lower -potential value by PAMAM-KRRR compared with those of native PAMAM or PEI 25 kDa might be caused by tight packaging of plasmid DNA by PAMAMKRRR. PAMAM-RRRK had the N-terminal RRRK sequence in reverse and showed similar physical properties as those of PAMAM-KRRR. Even though PAMAM-KRRR still showed the cell line dependent transfection efficacy, the efficiency was increased significantly in all cell lines tested as RRRK sequences were introduced on the surface of PAMAM generation 4 (Fig. 6). Interestingly, PAMAM-KRRR showed higher transfection efficiency especially in the NIH3T3 cell line than that of PEI 25 kDa.
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Fig. 8. Confocal microscopy images of polyplexs of PEI 25 kDa (A), PAMAM generation 4 (B), PAMAM-KRRR (C) and PAMAM-RRRK (D) with plasmid DNA in NIH3T3 cells. The left row indicates DAPI-labelled nuclei (blue), middle row is Alexa Fluor532-labelled plasmid DNA (red), and right row indicates the merged images between left and middle. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
According to results of polyanion competition assay using heparin (Fig. 5), the polyplexes of PAMAM-KRRR and PAMAM-RRRK indicated a little bit more improved stability than that of native PAMAM. The polyplexes of PAMAM, PAMAM-KRRR, and PAMAMRRRK were completely dissociated in the presence of heparin at over 40 g/mL concentration levels while PEI 25 kDa polyplexes maintained stable complexes. In combination with results of heparin competition assay and cell line dependent transfection efficiency, it is considered that the remarkable transfection efficiency of PAMAM-KRRR is caused by a function of multimeric RRRK sequences as NLS as well as by the increased polyplex stability. It was reported that PAMAM dendrimer shows increased trasfection efficiency and cytotoxicity according to the increase of PAMAM generation (Braun et al., 2005; Jevprasesphant et al., 2003). Despite higher molecular weight than that of native PAMAM, PAMAM-KRRR showed lower cytotoxicity in all cell lines (Fig. 7) whereas PEI 25 kDa showed higher cytotoxicity. However, a more interesting observation was markedly distinguishable in vitro behavior between PAMAM-KRRR and PAMAM-RRRK. The transfection efficiency difference between PAMAM-KRRR and PAMAM-RRRK was observed from a minimal of 3–20 fold and cytotoxicity was significantly different when each polymer was used at concentrations over 90 g/mL. Confocal microscopy (Fig. 8) showed a sharp contrast between the two polymers. Polyplexes of PAMAM-KRRR/pCN-Luci were primarily distributed in the nucleus
whereas polyplexes of PAMAM-RRRK/plasmid DNA were located in the cytoplasm. Peptides with normal sequences are known to perform correct biological activities whereas peptides with a reverse sequence are unable to perform normal function and influence cytotoxicity (Buchet et al., 1996; Guptasarma, 1992; Rai, 2007; Verdoliva et al., 1995). According to the study of our group (Choi et al., 2004), the introduction of lysines on the surface of PAMAM showed relatively higher cytotoxicity than that of PAMAMarginine. Consequently, the difference of transfection efficiency and cytotoxicity between PAMAM-KRRR and PAMAM-RRRK could be caused by the difference of structural and biological effects of the multimeric NLS fragments.
5. Conclusion As a result, the introduction of RRRK peptides on the surface of PAMAM generation 4 has shown increased transfection efficiency in comparison with that of native PAMAM and indicated higher transfection efficiency especially in the NIH3T3 cell line. Despite higher molecular weight than that of native PAMAM, PAMAMKRRR showed lower cytotoxicity in all cell lines. These results imply that introduction of RRRK peptides would make PAMAM-based vector a promising cationic polymer for transfection of fibroblast cell lines. The introduction of NLS on the surface of cationic polymer
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might contribute to efficiency improvement of PAMAM and other nonviral vectors. Acknowledgment This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (MSIP) (2013, University-Institute cooperation program). References Braun, C.S., Vetro, J.A., Tomalia, D.A., Koe, G.S., Koe, J.G., Middaugh, C.R., 2005. Structure/function relationships of polyamidoamine/DNA dendrimers as gene delivery vehicles. J. Pharm. Sci. – US 94, 423–436. Buchet, R., Tavitian, E., Ristig, D., Swoboda, R., Stauss, U., Gremlich, H.U., de La Fournière, L., Staufenbiel, M., Frey, P., Lowe, D.A., 1996. Conformations of synthetic  peptides in solid state and in aqueous solution: relation to toxicity in PC12 cells. Biochim. Biophys. Acta 1315, 40–46. Choi, J.S., Nam, K., Park, J.-y., Kim, J.-B., Lee, J.-K., Park, J.-s., 2004. Enhanced transfection efficiency of PAMAM dendrimer by surface modification with l-arginine. J. Control. Release 99, 445–456. Choi, J.S., Ko, K.S., Park, J.S., Kim, Y.H., Kim, S.W., Lee, M., 2006. Dexamethasone conjugated poly(amidoamine) dendrimer as a gene carrier for efficient nuclear translocation. Int. J. Pharm. 320, 171–178. De Smedt, S.C., Demeester, J., Hennink, W.E., 2000. Cationic polymer based gene delivery systems. Pharm. Res. 17, 113–126. Dufes, C., Uchegbu, I.F., Schatzlein, A.G., 2005. Dendrimers in gene delivery. Adv. Drug Delivery Rev. 57, 2177–2202. Guptasarma, P., 1992. Reversal of peptide backbone direction may result in the mirroring of protein-structure. FEBS Lett. 310, 205–210. Jeong, J.H., Kim, S.W., Park, T.G., 2007. Molecular design of functional polymers for gene therapy. Prog. Polym. Sci. 32, 1239–1274. Jevprasesphant, R., Penny, J., Jalal, R., Attwood, D., McKeown, N.B., D’Emanuele, A., 2003. The influence of surface modification on the cytotoxicity of PAMAM dendrimers. Int. J. Pharm. 252, 263–266.
Kiefer, P., Acland, P., Pappin, D., Peters, G., Dickson, C., 1994. Competition between nuclear-localization and secretory signals determines the subcellular fate of a single CUG-initiated form of FGF3. EMBO J. 13, 4126–4136. Kumar, A., Yellepeddi, V.K., Davies, G.E., Strychar, K.B., Palakurthi, S., 2010. Enhanced gene transfection efficiency by polyamidoamine (PAMAM) dendrimers modified with ornithine residues. Int. J. Pharm. 392, 294–303. Mancheno-Corvo, P., Martin-Duque, P., 2006. Viral gene therapy. Clin. Transl. Oncol. 8, 858–867. Martin, M.E., Rice, K.G., 2007. Peptide-guided gene delivery. AAPS J. 9, E18– E29. Mecke, A., Uppuluri, S., Sassanella, T.M., Lee, D.-K., Ramamoorthy, A., Baker Jr., J.R., Orr, B.G., Banaszak Holl, M.M., 2004. Direct observation of lipid bilayer disruption by poly(amidoamine) dendrimers. Chem. Phys. Lipids 132, 3–14. Mecke, A., Lee, D.-K., Ramamoorthy, A., Orr, B.G., Banaszak Holl, M.M., 2005. Synthetic and natural polycationic polymer nanoparticles interact selectively with fluid-phase domains of DMPC lipid bilayers. Langmuir 21, 8588–8590. Niidome, T., Huang, L., 2002. Gene therapy progress and prospects: nonviral vectors. Gene Ther. 9, 1647–1652. Rai, J., 2007. Retroinverso mimetics of S peptide. Chem. Biol. Drug Des. 70, 552–556. Ramamoorthy, A., Kandasamy, S.K., Lee, D.-K., Kidambi, S., Larson, R.G., 2007. Structure, topology, and tilt of cell-signaling peptides containing nuclear localization sequences in membrane bilayers determined by solid-state NMR and molecular dynamics simulation studies. Biochemistry 46, 965–975. Shcharbin, D., Pedziwiatr, E., Bryszewska, M., 2009. How to study dendriplexes I: Characterization. J. Control. Release 135, 186–197. Smith, P.E., Brender, J.R., Dürr, U.H., Xu, J., Mullen, D.G., Banaszak Holl, M.M., Ramamoorthy, A., 2010. Solid-state NMR reveals the hydrophobic-core location of poly (amidoamine) dendrimers in biomembranes. J. Am. Chem. Soc. 132, 8087–8097. Tang, M.X., Szoka, F.C., 1997. The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes. Gene Ther. 4, 823–832. Verdoliva, A., Ruvo, M., Villain, M., Cassani, G., Fassina, G., 1995. Antigenicity of topochemically related peptides. Biochim. Biophys. Acta 1253, 57–62.
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical Nanotechnology
Gene delivery of PAMAM dendrimer conjugated with the nuclear localization signal peptide originated from fibroblast growth factor 3 Jeil Lee a , Jinwoo Jung a , Youn-Joong Kim a,c , Eunji Lee a , Joon Sig Choi a,b,∗ a b c
Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305-764, Republic of Korea Department of Biochemistry, Chungnam National University, Daejeon 305-764, Republic of Korea Korea Basic Science Institute (KBSI), 169-148 Gwahangno (52 Yeoeun-Dong), Yuseong-Gu, Daejeon 305-333, Republic of Korea
a r t i c l e
i n f o
Article history: Received 26 July 2013 Received in revised form 25 October 2013 Accepted 14 November 2013 Available online 23 November 2013 Keywords: Polyamidoamine dendrimer Mouse fibroblast growth factor 3 Nuclear localization signal Transfection Polyplex Cytotoxicity
a b s t r a c t Polyamidoamine (PAMAM) is one of the widely employed non-viral vectors in gene therapy research, and shows excellent biocompatibility and relatively low cytotoxicity. However, it has poor transfection efficiency compared with that of polyethylenimine (PEI, 25 kDa). To enhance the gene expression efficiency, we introduced the RRRK peptide from mouse fibroblast growth factor 3 (FGF3) to PAMAM, which is a known nuclear localization signal (NLS). We synthesized PAMAM-KRRR and PAMAM-RRRK to verify the difference of the induced functional status from reversal of the N-terminus. PAMAM containing the FGF3 peptide showed a transfection efficiency corresponding to that of PEI in HEK293, and HeLa cells, and showed much higher gene expression capacity than that of PEI in NIH3T3 cells with relatively decreased cytotoxicity. These results imply that introduction of the FGF 3 peptide has the potential to provide a novel PAMAM-based vector by enhancing its gene expression efficiency. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Gene therapy is a technique which can either replace deficient genes or inhibit unwanted genes expression by introduction of external therapuetic genes aiming at fundamental treatment of disease at the genetic level. There are two types of gene therapies, which use a ‘vector system’ or physical methods (Mancheno-Corvo and Martin-Duque, 2006; Niidome and Huang, 2002). At the initial stage of gene therapies, viral vectors were used and showed therapeutic efficacy for treating congenital diseases. Despite the excellent efficacy, introduction of a viral vector has safety issues including insertional mutations by the virus and potential fatality caused by excessive inflammatory responses to the viral vector. Therefore, nonviral vector systems have attracted attention as an alternative to overcome the deficiencies of viral vectors. In nonviral vector systems, a cationic polymer and lipid are majorly used for gene transfer by forming a DNA-vector complex via electrostatic interactions between the DNA and cationic vector (Jeong et al., 2007). Nonviral vector systems have the advantage of comparatively low immune responses and cytotoxicity as well as ease of quality control and mass production. However,
∗ Corresponding author at: Department of Biochemistry, Chungnam National University, Daejeon 305-764, Republic of Korea. Tel.: +82 42 821 7528; fax: +82 42 822 7548. E-mail addresses: [email protected], [email protected] (J.S. Choi). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.11.027
the poor transfection efficiency of nonviral vectors remains to be resolved. Polyethyleneimine (PEI) is a cationic polymer with excellent gene expression efficiency in transfections of numerous cell lines. As the molecular weight increases, the number of primary amines provided by PEI is also increased and shows an enhanced transgene expression capacity resulted from strong DNA condensation and a proton buffering effect by enrichment of primary amines (De Smedt et al., 2000; Jeong et al., 2007). Although an increase of molecular weight denotes improved transfection efficiency, poor biodegradability of PEI causes high cytotoxicity. To improve the biodegradability, research has focused on degradable polymer linkers. One such polymer is polyamidoamine (PAMAM) dendrimer consisting of a peptide bond between methylacylate and ethylenediamine, which possesses excellent biodegradability and biocompatibility but the transfection efficiency is still poor (Braun et al., 2005; Dufes et al., 2005). It has been reported that the complexes of dendrimers form polymer/lipid vesicles by electrostatic interaction with cellular membranes and are transferred inside the cell by formation of nano-sized hole whereas complexes of cationic lipids are fused with cellular membranes (Mecke et al., 2004, 2005; Smith et al., 2010). Many groups have studied surface modification using cell penetrating peptides, nuclear localization signal (NLS) peptides, and peptide ligands for receptor-mediated delivery to improve the transfection efficiency (Choi et al., 2004, 2006; Martin and Rice, 2007; Ramamoorthy et al., 2007). In this study, to enhance the gene expression efficiency, we synthesized novel functional PAMAM dendrimer conjugated with
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multiple NLS peptides derived from mouse fibroblast growth factor 3 (FGF3) (Kiefer et al., 1994; Martin and Rice, 2007). In FGF3, the NLS sequence is RLRRDAGGRGGVYEHLGGAPRRRK with RLRR and RRRK sequences that induce nuclear localization. We hypothesized that introduction of the RRRK sequence on the PAMAM surface would improve DNA condensation by enrichment of basic amino acids and provide active transport of relatively large transgenes by the NLS peptide leading to increased gene transfection especially in fibroblast cell lines.
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2.3. Agarose gel retardation assay
2. Experiment details
Polyplexes of PAMAM-KRRR and PAMAM-RRRK with pCN-Luci were prepared at various weight ratios (polymer/pCN-Luci) ranging from 1 to 16 in HEPES buffer (25 mM, pH 7.4). The polymers of various weight ratios in HEPES buffer with pCN-Luci at a fixed volume were mixed and incubated at room temperature for 30 min. Then, electrophoresis of polyplex-formed samples was performed in a 0.7% agarose gel with 0.5 g/mL ethidium bromide. Agarose gel electrophoresis was progressed for 30 min at 100 V and agarose gel was analyzed under a UV illuminator to confirm the location of plasmid DNA bands.
2.1. Materials
2.4. Dynamic light scattering and -potential measurements
Ethidium bromide (EtBr), dimethysulfoxide (DMSO), N,Ndiisopropylethylamine (DIPEA), N,N-dimethylformamide (DMF), PAMAM generation 4, polyethylenimine (branched, 25 kDa), trifluoroacetic acid (TFA) and triisopropylsilane (TIS) were purchased from Sigma–Aldrich (Seoul, South Korea). N-hydroxybenzotriazole (HOBt), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetra-methyluronium (HBTU) were purchased from Anaspec (San Jose, CA, USA), and Fmoc-Arg(pbf)-OH and Fmoc-Lys(Boc)-OH was obtained from Novabiochem (San Diego, CA, USA). Luciferase 1000 assay kit and reporter lysis buffer were purchased from Promega (Madison, WI, USA). The luciferase expression plasmid DNA (pCN-Luci) was prepared as reported previously. 293, HeLa, and NIH3T3 cell lines were obtained from Korean Cell Line Bank. Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM) and 100× antibiotic-antimycotic reagent were purchased from GIBCO (Gaithersburg, MD, USA). Micro BCA Protein Assay Kit was purchased from Pierce (Rockford, IL, USA). ULYSIS® Nucleric Acid Labeling Kit was purchased from Molecular Probes (Invitrogen).
The size and -potential value of PEI 25 kDa, PAMAM generation 4, PAMAM-KRRR, and PAMAM-RRRK/pCN-Luci complexes were determined by a Zeta-potential & Particle Size Analyzer ELS-Z (Photal, Otsuka Electronics, Japan) and Zetasizer Nano ZS (Malvern Instruments, UK). pCN-Luci (10 g) was used for polyplex formation with polymers. Each polyplex was prepared in a total volume of 800 L with distilled water and analyzed at the weight ratios showing optimal transfection efficiency. 2.5. Transmission electron microscopy PAMAM-KRRR was prepared with pCN-Luci (5 g) in distilled water. PAMAM-KRRR/pCN-Luci was mixed at 16:1 (w/w) and incubated at room temperature for 30 min. Then, 10 L of PAMAMKRRR/pCN-Luci polyplex was placed on the surface of a 300-mech copper grid and dried at natural dry condition for 4 h. Electron micrographs were optained by an energy filtering transmission electron microscope (EM912, Carl Zeiss Inc., Germany) at 120 kV (Shcharbin et al., 2009; Tang and Szoka, 1997). 2.6. Heparin competition assay
2.2. Synthesis of PAMAM-KRRR and PAMAM-RRRK Conjugation of amino acids to the PAMAM generation 4 surface was performed in a mixture of anhydrous DMF and DMSO of 2:1 (v/v) for 18 h at room temperature with 4 equivalents of HOBt, HBTU and Fmoc-Lys(Boc)-OH and 8 equivalents of DIPEA. The product was precipitated in diethyl ether and washed with excess ether. The Fmoc protecting group of Fmoc-Lys(Boc)-conjugated PAMAM was eliminated by addition of 30% piperidine in DMF (v/v). After 3 h of the deprotection reaction, mixtures were precipitated in diethyl ether and washed in an excess of ether. To conjugate FmocArg(pbf)-OH additionally, a coupling reaction of Fmoc-Arg(pbf)-OH with the product was performed in DMF for 18 h at room temperature with 4 equivalents of HOBt, HBTU, and Fmoc-Arg(pbf)-OH and 8 equivalents of DIPEA. The subsequent deprotection reaction, precipitation process, and elongation of additional amino acids were performed as described in Fig. 1 (Kumar et al., 2010). PAMAM-RRRK was also synthesized as described above. After amino acid elongation to PAMAM generation 4 was finalized, the existing protecting group (BOC and pbf) of conjugated PAMAM generation 4 was removed by deprotection solution (95:2.5:2.5, trifluoroacetic acid/triisopropylsilane/H2 O, v/v) and the final product was precipitated in diethyl ether and washed in an excess of ether. The final product was dissolved in water, placed in a dialysis membrane (MWCO 10,000, Spectra/por) and dialyzed with distillated water overnight. Then, the product was collected as a white powder after lyophilization. The synthesis yield for final products were usually over 99% and 1 H NMR spectra (400 MHz, D2 O) of the synthesized polymers are shown in Fig. 2.
Polyplexes of PEI 25 kDa, PAMAM generation 4, PAMAM-KRRR, and PAMAM-RRRK/pCN-Luci were prepared at the 4:1 (w/w). Then, heparin solution of various concentrations was added at polyplexes and incubated at room temperature for 30 min. Each polyplexes was prepared in a total volume of 200 L with HEPES buffer (25 mM, pH 7.4) and 200 L PicoGreen reagent diluted in TE buffer (10 mM Tris, 0.5 mM EDTA, pH 7.5) was added and incubated for 2 min. Finally, the polyplexes were prepared in a total volume of 2 mL with 1.6 mL TE buffer and relative fluorescence intensity was analyzed by a spectrofluorometer (Quantech Digital Filter Fluorometer, Thermo Scientific, USA). The excitation and emission wavelengths were 490 and 520 nm, respectively. 2.7. Cell culture and transfection assay Human embryonic kidney (HEK) 293 cells, HeLa human epithelial carcinomacells and NIH3T3 mouse embryonic fibroblasts were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic mixture. Cell lines were maintained in an incubator (at 37 ◦ C with 5% CO2 , and 95% humidity). For the transfection assay, cells were cultured in a 96-well plate at a density of 1.8 × 104 cells/well for 16 h to 70–80% confluence. The polyplexes were prepared by mixing pCN-Luci (0.5 g) with PAMAM-KRRR and PAMAM-RRRK at various weight ratios for 30 min at room temperature. To compare the transfection efficiency, PEI 25 kDa and PAMAM generation 4/pCN-Luci complexes were prepared as the control group. Cultured cells were treated with polyplex solutions for 24 h at 37 ◦ C. The cells were washed with Dulbecco’s Phosphate Buffered Saline (DPBS), and
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Fig. 1. Synthesis of PAMAM-KRRR and PAMAM-RRRK.
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Fig. 2.
1
13
H NMR spectra of PAMAM-KRRR (A) and PAMAM-RRRK (B).
100 L of reporter lysis buffer (Promega, Madison, WI) was added to each well. After 30 min of incubation at room temperature, the cell lysates were harvested to microcentrifuge tubes and centrifuged at 13,200 rpm for 10 min. The protein concentration of lysates was measured by a Micro BCATM protein assay kit. Luciferase activity was measured using a Lumat LB 9507 instrument (Berthold Technology, Bad Wildbad, Germany). Luciferase activity was indicated as RLU/g total protein.
plates for 1 day prior to addition of the polymers. The cells were treated with PEI 25 kDa, PAMAM generation 4, PAMAM-KRRR, and PAMAM-RRRK at various concentrations and were incubated for 24 h at 37 ◦ C. Then, 10 L WST-1 reagent was added at each well and incubated for 2 h at 37 ◦ C. Then, the absorbance was measured at 450 nm using a microplate reader (VersaMax, Molecular Devices, USA).
2.8. Cytotoxicity assay
2.9. Preparation of Alexa Fluor 532-labeled pCN-Luci and confocal microscopy
The cytotoxicity of polymers was evaluated by a WST-1 assay. Cells were cultured at a density of 1.8 × 104 cells/well in 96-well
pCN-Luci was mixed with 3 M sodium acetate (pH 5.2) buffer solution with ethanol. Then, the mixture was frozen at −70 ◦ C for
Fig. 3. Agarose gel retardation assay for polyplexs of PEI 25 kDa, PAMAM generation 4, PAMAM-KRRR and PAMAM-RRRK with plasmid DNA. Plasmid DNA only (lane 1), the weight ratios of PAMAM-KRRR/pDNA (lanes 2–8), PAMAM-RRRK/pDNA = 1, 2, 4, 6, 8, 12, 16 (lanes 11–17), PEI 25 kDa (lane 9), and PAMAM generation 4 (lane 10).
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30 min. After thawing at room temperature, the mixture was centrifuged at 12,000 rpm for 15 min. The precipitate was washed with 70% ethanol and was dried using nitrogen gas. For DNA denaturation, nuclease-free H2 O component H was added to the dried precipitate, followed by incubation in a water bath at 95 ◦ C for 5 min. The precipitate was cooled on ice and centrifuged again. ULS reagent (10 L) was added to the precipitate, followed by incubation at 80 ◦ C for 15 min and then cooling on ice. Excess ULS reagent was eliminated by gel filtration using a MicroSpin G-25 column (Sigma–Aldrich). NIH3T3 cells were seeded at a density 5 × 103 cells/well in a confocal dish and cultured for 16 h to 70–80% confluence. Alexa Fluor 532-labeled pCN-Luci and unlabelled pCN-Luci were mixed at 1:1 (w/w). Then, the DNA mixture was complexed with PEI 25 kDa, PAMAM generation 4, PAMAMKRRR, and PAMAM-RRRK. PEI 25 kDa and PAMAM generation 4/pCN-Luci complexes were prepared at 2:1 (w/w) and 4:1 (w/w), respectively, and both PAMAM-KRRR and PAMAM-RRRK/pCN-Luci complexes were prepared at 16:1 (w/w). Each weight ratio represented optimal transfection efficiency. Cells were treated with the complexes for 24 h. The amount of pCN-Luci mixture was fixed at 0.5 g/well. Confocal microscopy images were obtained by a Zeiss LSM 5 Live confocal laser microscope.
2.10. Statistical analysis The statistical analysis was performed via the unpaired Student’s t-test (GraphPad Prism 5). Differences between groups were considered statistically significant at P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).
3. Results 3.1. Synthesis of PAMAM-KRRR and PAMAM-RRRK In previous studies, peptides enriched with cationic amino acids have shown an efficient translocation capacity via the cell membrane as well as a superior DNA condensation capacity and transfection efficiency. In this study, we used RRRK peptide from the bipartite NLS of FGF3. The RRRK sequence was conjugated on the surface of PAMAM dendrimer generation 4 (Fig. 1). The synthesized product was dissolved in D2 O for 1 H NMR (400 MHz). As shown in Fig. 2, the 1 H NMR spectra of the synthesized polymers are like as followings. PAMAM-KRRR: ı 1.41 ( CHCH2 CH2 CH2 CH2 NH2 of the lysine unit), 1.65 ( CHCH2 CH2 CH2 NH of the arginine unit and CHCH2 CH2 CH2 CH2 NH2 of the lysine unit), 1.80 ( CHCH2 CH2 CH2 NH of the arginine unit and CHCH2 CH2 CH2 CH2 NH2 of the lysine unit), 2.46 ( NCH2 CH2 CO of the PAMAM unit), 2.72 ( CONHCH2 CH2 N of PAMAM unit and NCH2 CH2 N of PAMAM unit), 2.90 ( NCH2 CH2 CO of PAMAM unit), 2.98 ( CHCH2 CH2 CH2 NH of the arginine unit and CHCH2 CH2 CH2 CH2 NH2 of the lysine unit) and 3.27 ( CONHCH2 CH2 N of PAMAM unit and CONHCH2 CH2 NHCO of PAMAM unit) PAMAM-RRRK: ı 1.43 ( CHCH2 CH2 CH2 CH2 NH2 of the argiof the lysine unit), 1.65 ( CHCH2 CH2 CH2 NH nine unit and CHCH2 CH2 CH2 CH2 NH2 of the lysine unit), 1.80 ( CHCH2 CH2 CH2 NH of the arginine unit and CHCH2 CH2 CH2 CH2 NH2 of the lysine unit), 2.45 ( NCH2 CH2 CO of the PAMAM unit), 2.70 ( CONHCH2 CH2 N of PAMAM unit and NCH2 CH2 N of PAMAM unit), 2.88 ( NCH2 CH2 CO of PAMAM unit), 2.99 ( CHCH2 CH2 CH2 NH of the arginine unit CHCH2 CH2 CH2 CH2 NH2 of the lysine unit) and 3.25 and ( CONHCH2 CH2 N of PAMAM unit and CONHCH2 CH2 NHCO of PAMAM unit). To study the polymer function by
Table 1 Size measurement using dynamic light scattering and zeta potential analysis of polymer/pDNA complexes.
Weight ratio (polymer:DNA) Mean size (nm) Zeta potential (mV) Polydispersity index
PEI
PAMAM
PAMAMKRRR
PAMAMRRRK
2:1
4:1
16:1
16:1
225.4 ± 13 36.8 ± 4 0.246
188.5 ± 11 38.4 ± 5 0.277
139.5 ± 13 22.7 ± 5 0.277
153.8 ± 15 22.2 ± 5 0.292
changing the peptide terminal, we synthesized PAMAM-KRRR and PAMAM-RRRK. 3.2. Agarose gel retardation assay To verify formation point of PAMAM-KRRR and PAMAMRRRK/pCN-Luci polyplexes, we performed agarose gel retardation assays (Fig. 3). The polyplexes were prepared at various weight ratios and analyzed in 0.7% (w/v) agrose gel. Polyplexes of PEI and PAMAM generation 4/pCN-Luci were used as the control and prepared at 2:1 (w/w) and 4:1 (w/w), respectively, which are ratios for optimal transfection effiecieny. PAMAM-KRRR and PAMAMRRRK/pCN-Luci polyplexes were retarded at weight ratios between 2:1 and 4:1. Complete retardation of pCN-Luci was observed at 4:1 (w/w). 3.3. Size and -potential measurements of PAMAM-KRRR and PAMAM-RRRK/pCN-Luci complexes We prepared polymer/pCN-Luci complexes as described in Table 1. Each polymer was efficiently condensed with pCN-Luci and formed nanometer-sized polyplexes of sizes ranging from 140 to 225 under conditions for optimal transfection. The introduction of KRRR and RRRK peptides on the PAMAM surface resulted in more compact polyplexes than those of PEI 25 kDa and unmodified PAMAM generation 4. Under the same conditions, we also measured -potential of the polyplexes. Althought cationic amino acids were introduced to PAMAM surface, the surface charge density of PAMAM-KRRR and PAMAM-RRRK was around 22 mV, and the value was approximately 15 mV lesser than of PAMAM generation 4 and PEI 25 kDa. 3.4. Transmission electron microscopy of PAMAM-KRRR/plasmid DNA polyplexes Dendriplexes of PAMAM-KRRR/pCN-Luci were observed by transmission electron microcscopy (TEM). Each dendriplexs exhibited a tightly condensed spherical shape (Fig. 4). Although aggregates of dendriplexes were also observed, the complexes showed a similar size (between 120 and 150 nm) compared with the measured diameter using dynamic light scattering (DLS). 3.5. Evaluation of polyplex stability by polyanion competition assay To verify the stability of polymer/pCN-Luci complexes, we performed polyanion competition assay using heparin (Fig. 5). The stability of polyplexes was analyzed by relative fluorescence of picogreen agent. The PAMAM generation 4/pCN-Luci polyplexes have begun to dissociate in the presence of heparin solution at concentrations between 20 and 30 g/mL and were completely disintegrated over 30 g/mL. Polyplexes of PAMAM-KRRR and PAMAM-RRRK showed increased stability than that of native PAMAM but these polyplexes were completely disintegrated at
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concentrations over 40 g/mL while PEI 25 kDa/pCN-Luci polyplexes maintained intact complexes.
3.6. Transfection efficiency of different cell lines The transgene expression efficiency of PAMAM generation 4 containing the RRRK peptide fragment originated from FGF3 was evaluated in a transfection assay. PAMAM-KRRR/pCN-Luci polyplexes were prepared at various weight ratios. The transfection assays using these polyplexes were performed with HEK293, HeLa, and NIH3T3 cell lines in the presence of 10% FBS. The PEI 25 kDa and PAMAM generation 4 were used as controls (polyplexes of polymer/pCN-Luci were prepared at 2:1 and 4:1 weight ratios, respectively). The transfection efficiency depended on the cell line and showed predominant gene expression efficiency for NIH3T3 cells compared with that using PEI 25 kDa. The transfection efficiency of PAMAM-KRRR for HEK293 and HeLa cell lines also showed similar expression efficiencies in comparison with that of PEI 25 kDa. The weight ratio for optimal expression efficiency was 6:1 (w/w) for HEK293 and HeLa cell lines and 16:1 for the NIH3T3 cell line (Fig. 6). The introduction of the RRRK sequence on the surface of PAMAM generation 4 enhanced the transfection efficiency by 4–2300 fold compared with that of the original PAMAM generation 4. PAMAM-RRRK was also analyzed at the same weight ratio as that of PAMAM-KRRR, and showed increased transfection efficiency.
3.7. Cytotoxicity evaluation
Fig. 4. TEM images of Dendriplexes of PAMAM (A) and PAMAM-KRRR (B).
As shown in Fig. 7, we performed cytotoxicity assays using HEK293, HeLa, and NIH3T3 cell lines. In the presence of 10% FBS, the cells were cultured for 16 h to 70–80% confluence. Next, the cells were treated with the polymers at various concentrations for 24 h. The PEI 25 kDa as a control showed strong cytotoxicity whereas PAMAM generation 4 showed much less cytotoxicity in all cell lines. PAMAM-KRRR and PAMAM-RRRK indicated contrary cytotoxic tendency. Although the RRRK sequences were introduced on the surface of PAMAM generation 4, the cytotoxicity of PAMAMKRRR in all cell lines showed a similar level compared with that of native PAMAM generation 4 while PAMAM-RRRK showed a rapid increase of cytotoxicity at concentrations over 90 g/mL.
Fig. 5. Polyanion competition assay of polyplexes using heparin. PEI 25 kDa (䊉), PAMAM generation 4 (), PAMAM-KRRR (), and PAMAM-RRRK ().
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Fig. 6. Transfection efficiency of PAMAM-KRRR and PAMAM-RRRK in various cell lines. (A) HEK293, (B) HeLa, and (C) NIH3T3 cells. Data are shown as mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001.
3.8. Confocal microscopy To verify intranuclear transfer of plasmid DNA by PAMAMKRRR, we used confocal microscopy (Fig. 8). As shown in Table 1, polyplexes of the polymers at weight ratios for optimal transfection efficiency were prepared with pCN-Luci labeled with Alexa 532 fluorescent dye. The polyplexes of each polymer were incubated with NIH3T3 cells in the presence of 10% FBS for 24 h. The nuclei were indicated as blue and the location of polymer/pCN-Luci polyplexes was indicated as red. Compared with other polymers, polyplexes of PAMAM-KRRR/pCN-Luci were mostly distributed in the nucleus whereas polyplexes of PAMAM-RRRK were located in the cytosol. These results suggest that high gene transfection efficiency of PAMAM-KRRR might be caused by introduction of the NLS from FGF3. 4. Discussion We evaluated physical properties such as the size, potential, shape, and formation point of polyplexes and assessed their transfection efficiency and cytotoxicity. Despite excellent biodegradability, we observed a low transfection efficiency of the native PAMAM dendrimer, which was dependent on the cell line. To increase the gene transfer efficiency, we introduced the NLS
Fig. 7. Cytotoxicity assay in (A) HEK293, (B) HeLa and (C) NIH3T3 cells using WST assay. PEI 25 kDa (䊉), PAMAM generation 4 (), PAMAM-KRRR (), and PAMAMRRRK (). The data of respective point indicates average and standard deviation from quadruplicate.
from FGF3 on the surface of PAMAM generation 4 (Figs. 1 and 2). The PAMAM-KRRR with plasmid DNA formed a complete polyplex at a weight ratio of 4:1 (Fig. 3), and formed more compact polyplexes at 16:1 (w/w). Under the same condition, polyplex sizes were approximately 140 nm (Table 1 and Fig. 4) and showed a potential of 22.7 mV (Table 1). PAMAM-KRRR would exist mostly as a positively charged form at neutral pH because the pKa value of an arginine guanidine group is higher than those of amines of native PAMAM and PEI 25 kDa. Therefore, the formation of more compact polyplexes and relatively lower -potential value by PAMAM-KRRR compared with those of native PAMAM or PEI 25 kDa might be caused by tight packaging of plasmid DNA by PAMAMKRRR. PAMAM-RRRK had the N-terminal RRRK sequence in reverse and showed similar physical properties as those of PAMAM-KRRR. Even though PAMAM-KRRR still showed the cell line dependent transfection efficacy, the efficiency was increased significantly in all cell lines tested as RRRK sequences were introduced on the surface of PAMAM generation 4 (Fig. 6). Interestingly, PAMAM-KRRR showed higher transfection efficiency especially in the NIH3T3 cell line than that of PEI 25 kDa.
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Fig. 8. Confocal microscopy images of polyplexs of PEI 25 kDa (A), PAMAM generation 4 (B), PAMAM-KRRR (C) and PAMAM-RRRK (D) with plasmid DNA in NIH3T3 cells. The left row indicates DAPI-labelled nuclei (blue), middle row is Alexa Fluor532-labelled plasmid DNA (red), and right row indicates the merged images between left and middle. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
According to results of polyanion competition assay using heparin (Fig. 5), the polyplexes of PAMAM-KRRR and PAMAM-RRRK indicated a little bit more improved stability than that of native PAMAM. The polyplexes of PAMAM, PAMAM-KRRR, and PAMAMRRRK were completely dissociated in the presence of heparin at over 40 g/mL concentration levels while PEI 25 kDa polyplexes maintained stable complexes. In combination with results of heparin competition assay and cell line dependent transfection efficiency, it is considered that the remarkable transfection efficiency of PAMAM-KRRR is caused by a function of multimeric RRRK sequences as NLS as well as by the increased polyplex stability. It was reported that PAMAM dendrimer shows increased trasfection efficiency and cytotoxicity according to the increase of PAMAM generation (Braun et al., 2005; Jevprasesphant et al., 2003). Despite higher molecular weight than that of native PAMAM, PAMAM-KRRR showed lower cytotoxicity in all cell lines (Fig. 7) whereas PEI 25 kDa showed higher cytotoxicity. However, a more interesting observation was markedly distinguishable in vitro behavior between PAMAM-KRRR and PAMAM-RRRK. The transfection efficiency difference between PAMAM-KRRR and PAMAM-RRRK was observed from a minimal of 3–20 fold and cytotoxicity was significantly different when each polymer was used at concentrations over 90 g/mL. Confocal microscopy (Fig. 8) showed a sharp contrast between the two polymers. Polyplexes of PAMAM-KRRR/pCN-Luci were primarily distributed in the nucleus
whereas polyplexes of PAMAM-RRRK/plasmid DNA were located in the cytoplasm. Peptides with normal sequences are known to perform correct biological activities whereas peptides with a reverse sequence are unable to perform normal function and influence cytotoxicity (Buchet et al., 1996; Guptasarma, 1992; Rai, 2007; Verdoliva et al., 1995). According to the study of our group (Choi et al., 2004), the introduction of lysines on the surface of PAMAM showed relatively higher cytotoxicity than that of PAMAMarginine. Consequently, the difference of transfection efficiency and cytotoxicity between PAMAM-KRRR and PAMAM-RRRK could be caused by the difference of structural and biological effects of the multimeric NLS fragments.
5. Conclusion As a result, the introduction of RRRK peptides on the surface of PAMAM generation 4 has shown increased transfection efficiency in comparison with that of native PAMAM and indicated higher transfection efficiency especially in the NIH3T3 cell line. Despite higher molecular weight than that of native PAMAM, PAMAMKRRR showed lower cytotoxicity in all cell lines. These results imply that introduction of RRRK peptides would make PAMAM-based vector a promising cationic polymer for transfection of fibroblast cell lines. The introduction of NLS on the surface of cationic polymer
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