- Email: [email protected]
Arginine-conjugated polypropylenimine dendrimer as a non-toxic and efficient gene delivery carrier
ARTICLE IN PRESS
Biomaterials 28 (2007) 2061–2067 www.elsevier.com/locate/biomaterials
Arginine-conjugated polypropylenimine dendrimer as a non-toxic and efficient gene delivery carrier Tae-il Kim, Jung-un Baek, Cheng Zhe Bai, Jong-sang Park School of Chemistry & Molecular Engineering, Seoul National University, San 56-1, Shillim-dong, Gwanak-gu, Seoul 151-742, Republic of Korea Received 1 November 2006; accepted 11 December 2006 Available online 29 December 2006
Abstract We synthesized arginine-conjugated polypropylenimine dendrimer G2 (DAB-8), PPI2-R for gene delivery systems. Synthesized PPI2R could retard plasmid DNA at a weight ratio of 4 completely and PPI2-R polyplexes showed a fluorescence of less than 10% over a charge ratio of 2 by PicoGreen reagent assay, suggesting its good DNA condensing ability. The size of PPI2-R polyplex was measured to about 200 nm at a charge ratio of 150. PPI2-R displayed 80–90% cell viability at even a 150 mg/mL concentration. Transfection efficiency of PPI2-R was found to be high comparable to that of PEI25kD and to be 8–214 times higher than that of unmodified PPI2 on HeLa and 293 cells. Moreover, PPI2-R showed 4 times higher transfection efficiency than PEI25kD, treating with 10 mg pDNA because of its low cytotoxicity on HeLa cells. Finally, PPI2-R showed a transfection efficiency 2–3 times higher than PEI25kD on HUVECs, showing its potency as a gene delivery carrier for primary cells. These results demonstrate that arginine-conjugation of PPI2 is successful in developing a low toxic and highly transfection efficient gene delivery carrier. r 2006 Elsevier Ltd. All rights reserved. Keywords: Gene delivery; Polypropylenimine dendrimer; Arginine-conjugation; Low cytotoxicity; High transfection
1. Introduction A number of non-viral gene delivery carriers including cationic polymers and lipids have been developed as alternatives to viral gene delivery carriers due to their advantages such as non-immunogenicity, unlimited capacity of genes delivered, and convenient handling and easy introduction of functionalities in spite of relatively low transfection efficiency [1–4]. Among them, dendrimers aroused researchers’ interest for drug and gene delivery systems because they have unique and interesting characteristics such as defined structures, inner cavities able to encapsulate guest molecules, and controllable multi-valent functionalities in their inner or outer part [5–9]. However, difficulty of synthesizing dendrimers and fitness of dendrimers as drug delivery carriers have led mainly to modification of existing dendrimers, not develCorresponding author. Tel.: +82 2 880 6660; fax: +82 2 877 5110.
E-mail address: [email protected] (J.-s. Park). 0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.12.013
opment of novel dendrimers for gene delivery systems. Among them, poly(amido amine (PAMAM) dendrimers and polypropylenimine (PPI) dendrimers are representative existing dendrimers. PAMAM dendrimers have been utilized and examined for gene delivery systems in vitro and in vivo, extensively [10,11]. PAMAM dendrimers have been also modified with PEG, amino acids, or ligands in order to enhance the gene delivery potency [12–16]. In contrast, applications of PPI dendrimers for gene delivery have been limited to a small number of works. The gene delivery results of PPI dendrimers and quaternized PPI dendrimers were mainly reported by Uchegbu and colleagues [17,18]. Also, self-assembled ternary complexes of PPI dendrimer, cucurbituril, and DNA were examined as a novel non-covalent strategy for gene delivery systems by Lim et al. [19]. PAMAM dendrimer conjugated with arginine residues was previously reported to show enhanced transfection efficiency [13]. Also, replacement of terminal lysine with arginine was reported to enhance the transfection efficiency
ARTICLE IN PRESS 2062
T.-i. Kim et al. / Biomaterials 28 (2007) 2061–2067
in dendritic poly(L-lysine) analogs by Okuda et al. [20]. This may be due to the membrane permeability and nuclear localization ability of arginine residues conjugated to the periphery of the dendrimers. So, we tried to conjugate arginine residues to PPI dendrimer in order to study the arginine conjugation effect of PPI dendrimer for the first time. Here, we report the synthesis of arginine-conjugated PPI dendrimer, PPI2-R and its characterization including cytotoxicity and transfection efficiency for gene delivery systems. 2. Materials and methods
the pbf groups of coupled arginine residues at room temperature for 1 h. After the reaction, the final product, PPI2-R was dialyzed by dialysis membrane (Spectrum Laboratories, Inc., Rancho Dominguez, CA, MWCO ¼ 1000) against ultra-pure water for 4 h and lyophilized before use for analysis and assay.
2.3. 1H NMR spectroscopy 1 H NMR spectra of the polymers were obtained using a Bruker DPX300 NMR spectrometer (300 MHz). For analysis, the polymer samples were dissolved in D2O containing 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt as an internal reference (0 ppm).
2.4. Gel retardation assays
2.1. Materials Poly(ethylenimine) (25 kDa), PPI octaamine dendrimer G2.0 (DABAm-8), N,N-diisopropylethylamine (DIPEA), piperidine, and 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO). N-hydroxybenzotriazole (HOBt), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Anaspec, Inc. (San Jose, CA). Fmoc-L-Arg(pbf)-OH was purchased from Novabiochem (San Diego, CA). Luciferase 1000 Assay System and Reporter Lysis Buffer were purchased from Promega (Madison, WI). The luciferase expression plasmid, pCN-Luci was constructed by subcloning cDNA of Photinus pyralis luciferase with 21-amino acid nuclear localization signal from SV40 large T antigen to pCN [21]. PicoGreen reagent was purchased from Molecular Probes (Eugene, OR). Fetal bovine serum (FBS), and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from GIBCO (Gaithersburg, MD). EGM and EGM-2 MV SingleQuots medium were purchased from Cambrex Bio Science (Walkersville, MD). All chemicals were used without any further purification.
PPI2-R/plasmid complexes at various weight ratios ranging from 1 to 10, were prepared in Hepes buffered saline (10 mM Hepes, 1 mM NaCl, pH 7.4). After 30 min incubation at room temperature for the complex formation, the samples were electrophoresed on a 0.7% (w/v) agarose gel and stained in an ethidium bromide solution (0.5 mg/mL), and analyzed on a UV illuminator to show the location of the DNA.
2.5. Polymer/DNA self-assembly analysis by PicoGreen assay PicoGreen reagent (200 ) was diluted 200-fold in TE buffer before the experiment. About 200 mL of the diluted PicoGreen stock solution was mixed with the same volume of blank solution or polyplex solution (1 mg DNA, 1 HBS) prepared at various charge ratios ranging from 1 to 10. After 2 min incubation, each solution was added to 1.6 mL of TE buffer in a test tube. Fluorescence was measured with a FP-750 spectrofluorometer (Jasco) at room temperature. Excitation and emission wavelengths were set at 480 and 520 nm, respectively. Results were represented as relative fluorescence (%) to DNA control.
2.2. Synthesis of PPI2-R 2.6. Polyplex size measurements PPI2-R was synthesized by conjugating arginine residues to the periphery of PPI2 dendrimer as shown in Fig. 1. Firstly, PPI2 dendrimer was weighed and dissolved in anhydrous DMF. Arginine conjugation reaction was performed in anhydrous DMF for 2 days at room temperature with 32 equivalents of HOBt, HBTU, Fmoc-Arg(pbf)-OH, and 64 equivalents of DIPEA. After completion of the reaction, the reaction mixture was precipitated twice with an excess of diethyl ether. Residual precipitate was dissolved in DMF and mixed with an equal volume of piperidine (30% in DMF, v/v) at room temperature for 20 min to remove the Fmoc groups of coupled Fmoc-Arg(pbf)-OH and precipitated again with diethyl ether. Then, the reagent (95:2.5:2.5, trifluoroacetic acid/triisopropylsilane/water, v/v) was used to deprotect
The hydrodynamic diameters of the PPI2-R/plasmid DNA complexes were determined by light scattering. About 2 mL of polyplex solutions containing 5 mg of DNA were prepared at various charge ratios ranging from 10 to 200. After 30 min incubation, polyplex sizes were measured using a Zetasizer 3000HS (Malvern Instruments, UK). The laser used was a nominal 5 mW HeNe laser having a 633 nm wavelength. Scattered light was detected at a 90 1 angle. The refractive index (1.33) and the viscosity (0.89) of ultrapure water were used at 25 1C for measurements. Zetasizer 3000 (Advanced) Size mode v1.61 software was used for data acquisition. Data analysis was performed in automatic mode. Measured sizes were represented as the average values of 5 runs.
Fig. 1. The synthetic scheme of PPI2-R.
ARTICLE IN PRESS T.-i. Kim et al. / Biomaterials 28 (2007) 2061–2067
2.7. Cell lines Human cervical carcinoma cells (HeLa) and 293 human kidney transformed cells were maintained in DMEM medium with 10% FBS at 37 1C in 5% CO2. Human umbilical vein endothelial cells (HUVECs) were purchased from Cambrex (Walkersville, MD). HUVECs were also maintained in an EBM-2 MV SingleQuots medium containing 2% FBS at 37 1C in 5% CO2.
2.8. Cytotoxicity assay The cytotoxicity of the polymers was measured by MTT assay. HeLa cells were seeded in a 96-well tissue culture plate at 104 cells/well in 90 mL DMEM medium containing 10% FBS. Cells achieving 70–80% confluence after 24 h were exposed to 10 mL of the polymer solutions having various concentrations for 48 h. Then, 26 mL of stock solution of MTT (2 mg/mL in PBS) was added to each well. After 4 h of incubation at 37 1C, each medium was removed and 150 mL of DMSO was added to each well to dissolve the formazan crystals formed by proliferating cells. Absorbance was measured at 570 nm using a microplate reader (Molecular Devices Co., Menlo Park, CA) and recorded as a percentage relative to untreated control cells.
2.9. In vitro transfection 2.9.1. Transfection on HeLa cells and 293 cells HeLa cells and 293 cells were seeded at a density of 5 104 cells/well in a 24-well plate in a DMEM medium containing 10% FBS and grown to reach 70–80% confluence prior to transfection. Before transfection, the medium was exchanged with fresh serum-free medium. The cells were treated with polyplex solution containing 2 mg of plasmid DNA at different charge ratios for 4 h at 37 1C. After exchange with fresh serumcontaining medium, cells were further incubated for 2 days after transfection. Then the growth medium was removed, and the cells were rinsed with PBS and shaken for 30 min at room temperature in 120 mL of Reporter Lysis Buffer. Luciferase activity was measured by a luminescence assay. Ten microliters of the lysate was dispensed into a luminometer tube and luciferase activity was integrated over 10 s with a 2 s measurement delay in a Lumat LB 9507 luminometer (Berthold, Germany) with an automatic injection of 50 mL of Luciferase Assay Reagent. The final results were reported in terms of RLU/well. 2.9.2. Transfection with varied pDNA amount on HeLa cells HeLa cells were seeded at a density of 5 104 cells/well in a 24-well plate in a DMEM medium containing 10% FBS and grown to reach 70–80% confluence prior to transfection. PEI25kD (charge ratio ¼ 8) and PPI2-R polyplexes (charge ratio ¼ 150) containing various pDNA amounts ranging from 2 to 10 mg were prepared, respectively. The cells were treated with the polyplex solutions for 4 h at 37 1C. After exchange with fresh serum-containing medium, cells were further incubated for 2 days after transfection. Further experimental procedures were performed identically to the above transfection and assay methods. 2.9.3. Transfection on HUVECs HUVECs were seeded in a density of 2.5 104 cells/well in a specific 24well plate with a Corning CellBIND Surface (Corning Incorporated, Corning, NY) in an EBM-2 MV SingleQuots medium containing 2% FBS and grown to reach 70–80% confluence prior to transfection. Before transfection, the medium was exchanged with fresh growth factor, cytokine, and supplement-free EGM medium. Transfection experiment was also performed in a growth factor-containing EBM-2 MV SingleQuots medium and this did not have to exchange the medium with EGM medium. The cells were treated with polyplex solutions containing 2 mg of plasmid DNA at different charge ratios for 4 h at 37 1C. After exchange with fresh EBM-2 MV SingleQuots medium, cells were further incubated
2063
for 2 days after transfection. Further experimental procedures were performed identically to the above transfection and assay methods.
3. Results and discussion 3.1. Synthesis and characterization of PPI2-R PPI2-R dendrimer was synthesized via conjugation of arginine residues to the periphery of PPI G2 dendrimers with a HOBt/HBTU coupling method in DMF. PPI G2 dendrimer was chosen as a backbone dendrimer because PPI G2 dendrimer was reported to show the lowest cytotoxicity and the highest transfection efficiency among PPI dendrimers by previous study [18]. Fmoc groups and pbf groups of arginines were removed by 30% piperidine/ DMF solution and the reagent (95:2.5:2.5, trifluoroacetic acid/triisopropylsilane/water, v/v), respectively. The synthesis of PPI2-R was analyzed by 1H NMR spectroscopy as follows. 1 H NMR(D2O): d arginine(–COCHCH2CH2CH2NH–) ¼ 1.70; d arginine(–COCHCH2CH2CH2NH–) ¼ 1.94; d PPI(–NCH2CH2CH2CH2N–, –NCH2CH2CH2N–) ¼ 1.79– 2.18; d PPI(–NCH2CH2CH2CH2N–, –NCH2CH2CH2N–) ¼ 3.20–3.31; d PPI(–NCH2CH2CH2NHCO–) ¼ 3.40; d arginine(–COCHCH2CH2CH2NH–) ¼ 3.26; d arginine(– COCHCH2CH2CH2NH–) ¼ 4.00. The number of arginine residues conjugated to PPI2 dendrimer was calculated by comparing NMR peak intensities between the protons of arginine and of the PPI2 dendrimer backbone. Approximately, 7 molecules of arginine were found to be conjugated to the dendrimer possessing 8 primary amines. The molecular weight of PPI2-R dendrimer was calculated to be 1866.71 Da from the NMR result. 3.2. Characterization of polyplex formation The polyplex formation of PPI2-R was examined by agarose gel retardation assay and PicoGreen reagent assay, respectively. PicoGreen reagent is a DNA intercalating dye more sensitive than ethidium bromide [22]. So, the fluorescence of PicoGreen reagent is quenched when the DNA is condensed into particles by self-assembly with cationic polymers. PPI2 dendrimer was used as a control. Fig. 2 shows the gel retardation assay results. PPI2-R could retard plasmid DNA at a weight ratio of 4 completely, although PPI2 dendrimer could retard plasmid DNA even at a ratio of 2. This tendency was also identified in PicoGreen reagent assay (Fig. 3). PPI2-R polyplexes showed about 66% fluorescence in contrast with PPI2 polyplexes showing only 15% at a charge ratio of 1 but the value of PPI2-R decreased abruptly to less than 10% as the charge ratio increased to 2. The profiles of fluorescence quenching by polyplex formation were similar at higher charge ratios. These results show that PPI2-R can condense pDNA efficiently by self-assembly and arginine-conjugation reduces the pDNA condensing ability of the dendrimer
ARTICLE IN PRESS 2064
T.-i. Kim et al. / Biomaterials 28 (2007) 2061–2067
higher ratios. The sizes of PPI polyplexes also showed a similar tendency. Both polyplexes were found to have a narrow size distribution at all the charge ratios prepared. These results represent that the size of PPI-R polyplex is proper to gene delivery and arginine conjugation to PPI dendrimer does not affect the size of the polyplex significantly.
3.4. Cytotoxicity
Fig. 2. The agarose gel retardation assay results: (a) PPI2-R polyplex; (b) PPI2 polyplex; C ¼ plasmid DNA only.
The cytotoxicity of PPI-R was examined on HeLa cell lines by MTT assay. PEI25 kD and PPI2 dendrimer were used as controls. It is well known that PEI25 kD has high transfection efficiency but also serious cytotoxicity. As shown in Fig. 4, HeLa cell viability after treating PEI25 kD was 58% at 20 g/mL and decreased to 17% at 150 g/mL of concentration while PPI2-R displayed 80–90% cell viability in the same concentration range. Cell viability of PPI2 was also found to be similar to that of PPI2-R. In general, cytotoxicity of gene delivery carriers is known to arise from the accumulation of non-degraded and nondischarged polymers with large molecular weight and charges. This result shows that the cytotoxicity of PPP5R is minimal and the arginine conjugation to PPI2 dendrimer G2 with low molecular weight does not bring a negative effect in its cytotoxicity in spite of highly positive charges of arginine residues.
3.5. Transfection experiments
Fig. 3. The PicoGreen reagent assay results of PPI2-R polyplex and PPI2 polyplex.
somewhat at a low charge ratio. It is explained by the facts that the pKa value of a-amine of arginine in PPI2-R dendrimer is lower than that of primary amine in PPI2 dendrimer [13] and that at the same charge ratio, the number of moles of PPI2-R is 1.5 times less than that of PPI2 in polyplex solution so the self-assembly of PPI2-R with pDNA can proceed less vigorously at the charge ratio. 3.3. Size measurements of the polyplexes The proper size of polyplexes is thought to be essential for efficient gene delivery because it influences the internalization into cells of the polyplexes [1,23]. We examined the size of polyplexes by Zeta-sizer at various charge ratios ranging from 10 to 200. PPI2 dendrimer was used as a control. Table 1 shows the results. The average size of PPI2-R polyplex was 189.6 nm at a charge ratio of 10 and increased to values ranging from 200 to 250 nm at
3.5.1. Transfection on HeLa and 293 cells We performed transfection experiments of newly synthesized PPI2-R on HeLa and 293 cells in the absence of serum, respectively. PEI25 kD and PPI2 were also used as controls. All values were represented as RLU/well, not RLU/mg protein because the total cellular protein of PEI25kD-treated cells was lower than others owing to its cytotoxicity. So, this would increase RLU/mg protein values of PE125KD, not reflecting the exact transfection efficiency. PEI25 kD polyplexes were prepared at a charge ratio of 8 (optimal ratio). The transfection efficiencies of PPI2-R were increased according to the charge ratio ranging from 50 to 200 on both cell lines and the maximum efficiencies reached 27–54% of that of PEI25 kD, as shown in Fig. 5. It is a notable result that PPI2-R showed a transfection efficiency 8–214 times higher than that of unmodified PPI2 at all charge ratios examined. The result shows that arginine conjugation to PPI dendrimer leads to a highly enhanced transfection efficiency and suggests that arginine-conjugated dendritic structure also possesses a high cell permeability and transfection potency like linear oligoarginine peptides or Tat sequences, as reported by previous studies [24–26].
ARTICLE IN PRESS T.-i. Kim et al. / Biomaterials 28 (2007) 2061–2067
2065
Table 1 The average size measurements of polyplexes by Zeta-sizer Charge ratio
10
50
100
150
200
PPI2-R PPI2
189.6725.1 170.3713.0
288.1722.9 247.5714.3
207.977.0 239.176.0
203.877.2 249.5710.0
257.3711.3 244.273.7
Values are represented as nm-scale.
Fig. 4. MTT assay results of PPI2-R.
3.5.2. Transfection with varied pDNA amount Transfection experiments using varied pDNA amount ranging from 2 to 10 mg were performed in order to examine the relationship between cytotoxicity and transfection efficiency on HeLa cells. Only 2 mg of pDNA was used in the previous experiment. PEI25 kD and PPI2-R polyplexes were prepared at the fixed optimal charge ratios of 8 and 150, respectively, to avoid other factors. As shown in Fig. 6, as the pDNA amount used was increased, transfection efficiency of PEI25kD became lower. The transfection value of PEI25 kD with 10 mg pDNA was decreased to even a 20% value with 2 mg pDNA and this is thought to be due to cell death induced by significant cytotoxicity of PEI25 kD because the greater the amount of pDNA amount, the greater is the amount of PEI25 kD at a fixed charge ratio. However, conversely, the transfection efficiency of PPI2-R was increased gradually in proportion to the amount of pDNA and it reached a value 4.3 times higher than that of PEI25kD when treating 10 mg pDNA. This result shows clearly that the minimal cytotoxicity of PPI2-R enabled us to use a large amount of pDNA and the polymer in contrast to the case of PEI25kD for improved gene delivery. 3.5.3. Transfection on HUVECs HUVECs are primary cells used for examining the function and metabolic mechanism of endothelial cells in the endothelium. They are also known to be hardly transfected by non-viral gene delivery carriers and only electroporation has showed relatively high transfection efficiency [27]. We performed transfection experiments in order to investigate the potency of PPI2-R as gene delivery carriers
Fig. 5. Transfection experiment results: (a) HeLa cells; (b) 293 cells. Values were represented as RLU/well.
on HUVECs. As shown in Fig. 7, the transfection efficiency of PPI2-R was 2–3 times higher at a charge ratio of 150 than that of PEI25 kD in the presence or absence of growth factor. This high transfection efficiency showed that PPI2-R possessed great potency as a gene delivery carrier for HUVECs. Interestingly, the efficiencies of both polymers in the presence of growth factor were found to be about 3-fold lower than in its absence. It is presumed that the growth factor may interact with polyplexes and inhibit the
ARTICLE IN PRESS T.-i. Kim et al. / Biomaterials 28 (2007) 2061–2067
2066
4 times higher than that of PEI25 kD because of its low cytotoxicity. Finally, PPI2-R was found to possess a transfection efficiency 2–3 times higher than PEI25kD on HUVECs that is known to be hardly transfected primary cells, showing the potency of PPI2-R as an efficient gene delivery carrier. In conclusion, arginine-conjugation of PPI2 was proven to be a successful trial to develop a low toxic and highly transfection-efficient gene delivery carrier. Acknowledgments
Fig. 6. Transfection experiment results with varied pDNA quantity on HeLa cells.
This work was supported by the Korea Health 21 R&D Project of The Ministry of Health & Welfare, Republic of Korea (A04-0004), and the Gene Therapy Project of The Ministry of Science and Technology (M105340300405N3403-00410). We thank So Hee Nam for providing helpful guidance for HUVEC culture. References
Fig. 7. Transfection experiment results on HUVECs.
transfection mechanism via the formation of a biologically inert complex, leading to a reduction of transfection efficiency. 4. Conclusion Arginine-conjugated PPI dendrimer (DAB-2), PPI2-R was synthesized for gene delivery systems. PPI2-R was found to condense pDNA into particles with an average size of about 200 nm. The cell viability after treating with PPI2-R was shown to be over 80% at even a 150 mg/mL concentration, indicating that the cytotoxicity of PPI2-R was minimal. PPI2-R also showed a highly enhanced transfection efficiency compared to unmodified PPI2 dendrimer on HeLa and 293 cells. When the pDNA used was increased to 10 mg in a transfection experiment on HeLa cells, the transfection efficiency of PPI2-R was
[1] Zauner W, Ogris M, Wagner E. Polylysine-based transfection systems utilizing receptor-mediated delivery. Adv Drug Del Rev 1998;30: 97–113. [2] Luo D, Saltzman WM. Synthetic DNA delivery systems. Nat Biotechnol 2000;18:33–7. [3] Vijayanathan V, Thomas T, Thomas TJ. DNA nanoparticles and development of DNA delivery vehicles for gene therapy. Biochemistry 2002;41:14085–94. [4] Mastrobattista E, Bravo SA, van der Aa M, Crommelin DJA. Nonviral gene delivery systems: from simple transfection agents to artificial viruses. Drug Discov Today: Technol 2005;2:103–9. [5] Svenson S, Tomalia DA. Dendrimers in biomedical applications— reflections on the field. Adv Drug Del Rev 2005;57:2106–29. [6] Dufe`s C, Uchegbu IF, Scha¨tzlein AG. Dendrimers in gene delivery. Adv Drug Del Rev 2005;57:2177–202. [7] Gillies ER, Fre´chet JMJ. Dendrimers and dendritic polymers in drug delivery. Drug Discov Today 2005;10:35–43. [8] Cloninger MJ. Biological applications of dendrimers. Curr Opin Chem Biol 2002;6:742–8. [9] Patri AK, Majoros IJ, Baker Jr JR. Dendritic polymer macromolecular carriers for drug delivery. Curr Opin Chem Biol 2002; 6:466–71. [10] Eichman JD, Bielinska AU, Kukowska-Latallo JF, Baker Jr JR. The use of PAMAM dendrimers in the efficient transfer of genetic material into cells. PSTT 2000;3:232–45. [11] Esfand R, Tomalia DA. Poly(amidoamine) (PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications. Drug Discov Today 2001;6:427–36. [12] Luo D, Haverstick K, Belcheva N, Han E, Saltzman WM. Poly(ethylene glycol)-conjugated PAMAM dendrimer for biocompatible, high-efficiency DNA delivery. Macromolecules 2002;35: 3456–62. [13] Choi JS, Nam K, Park J-y, Kim J-B, Lee J-K, Park J-s. Enhanced transfection efficiency of PAMAM dendrimer by surface modification with L-arginine. J Control Rel 2004;99:445–56. [14] Kim T-i, Seo HJ, Choi JS, Jang H-S, Baek J-u, Kim K, et al. PAMAM-PEG-PAMAM: novel triblock copolymer as a biocompatible and efficient gene delivery carrier. Biomacromolecules 2004;5: 2487–92. [15] Choi JS, Ko KS, Park JS, Kim Y-H, Kim SW, Lee M. Dexamethasone conjugated poly(amidoamine) dendrimer as a gene carrier for efficient nuclear translocation. Int J Pharm 2006;320: 171–8.
ARTICLE IN PRESS T.-i. Kim et al. / Biomaterials 28 (2007) 2061–2067 [16] Wada K, Arima H, Tsutsumi T, Chihara Y, Hattori K, Hirayama F, et al. Improvement of gene delivery mediated by mannosylated dendrimer/a-cyclodextrin conjugates. J Control Rel 2005;104: 397–413. [17] Schatzlein AG, Zinselmeyer BH, Elouzi A, Dufes C, Chim YTA, Roberts CJ, et al. Preferential liver gene expression with polypropylenimine dendrimers. J Control Rel 2005;101:247–58. [18] Zinselmeyer BH, Mackay CP, Schatzlein AG, Uchegbu IF. The lower-generation polypropylenimine dendrimers are effective gene transfer agents. Pharm Res 2002;19:960–7. [19] Lim Y-b, Kim T, Lee JW, Kim S-m, Kim H-J, Kim K, et al. Selfassembled ternary complex of cationic dendrimer, cucurbituril, and DNA: noncovalent strategy in developing a gene delivery carrier. Bioconjugate Chem 2002;13:1181–5. [20] Okuda T, Sugiyama A, Niidome T, Aoyagi H. Characters of dendritic poly(l-lysine) analogues with the terminal lysines replaced with arginines and histidines as gene carriers in vitro. Biomaterials 2004;25:537–44. [21] Lee MJ, Cho SS, You JR, Lee Y, Kang BD, Choi JS, et al. Intraperitoneal gene delivery mediated by a novel cationic liposome
[22]
[23]
[24] [25] [26]
[27]
2067
in a peritoneal disseminated ovarian cancer model. Gene Ther 2002; 9:859–66. Singer VL, Jones LJ, Yue ST, Haugland RP. Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double stranded DNA quantitation. Anal Biochem 1997; 249:228–38. Mahato RI, Rolland A, Tomlinson E. Cationic lipid-based gene delivery systems: pharmaceutical perspectives. Pharm Res 1997; 14:853–9. Brooks H, Lebleu B, Vive`s E. Tat peptide-mediated cellular delivery: back to basics. Adv Drug Del Rev 2005;57:559–77. Zhao M, Weissleder R. Intracellular cargo delivery using Tat peptide and derivatives. Med Res Rev 2004;24:1–12. Wadia JS, Dowdy SF. Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer. Adv Drug Del Rev 2005;57:579–96. Ear T, Gigue`re P, Fleury A, Stankova J, Payet MD, Dupuis G. High efficiency transient transfection of genes in human umbilical vein endothelial cells by electroporation. J Immunol Methods 2001;257: 41–9.
Biomaterials 28 (2007) 2061–2067 www.elsevier.com/locate/biomaterials
Arginine-conjugated polypropylenimine dendrimer as a non-toxic and efficient gene delivery carrier Tae-il Kim, Jung-un Baek, Cheng Zhe Bai, Jong-sang Park School of Chemistry & Molecular Engineering, Seoul National University, San 56-1, Shillim-dong, Gwanak-gu, Seoul 151-742, Republic of Korea Received 1 November 2006; accepted 11 December 2006 Available online 29 December 2006
Abstract We synthesized arginine-conjugated polypropylenimine dendrimer G2 (DAB-8), PPI2-R for gene delivery systems. Synthesized PPI2R could retard plasmid DNA at a weight ratio of 4 completely and PPI2-R polyplexes showed a fluorescence of less than 10% over a charge ratio of 2 by PicoGreen reagent assay, suggesting its good DNA condensing ability. The size of PPI2-R polyplex was measured to about 200 nm at a charge ratio of 150. PPI2-R displayed 80–90% cell viability at even a 150 mg/mL concentration. Transfection efficiency of PPI2-R was found to be high comparable to that of PEI25kD and to be 8–214 times higher than that of unmodified PPI2 on HeLa and 293 cells. Moreover, PPI2-R showed 4 times higher transfection efficiency than PEI25kD, treating with 10 mg pDNA because of its low cytotoxicity on HeLa cells. Finally, PPI2-R showed a transfection efficiency 2–3 times higher than PEI25kD on HUVECs, showing its potency as a gene delivery carrier for primary cells. These results demonstrate that arginine-conjugation of PPI2 is successful in developing a low toxic and highly transfection efficient gene delivery carrier. r 2006 Elsevier Ltd. All rights reserved. Keywords: Gene delivery; Polypropylenimine dendrimer; Arginine-conjugation; Low cytotoxicity; High transfection
1. Introduction A number of non-viral gene delivery carriers including cationic polymers and lipids have been developed as alternatives to viral gene delivery carriers due to their advantages such as non-immunogenicity, unlimited capacity of genes delivered, and convenient handling and easy introduction of functionalities in spite of relatively low transfection efficiency [1–4]. Among them, dendrimers aroused researchers’ interest for drug and gene delivery systems because they have unique and interesting characteristics such as defined structures, inner cavities able to encapsulate guest molecules, and controllable multi-valent functionalities in their inner or outer part [5–9]. However, difficulty of synthesizing dendrimers and fitness of dendrimers as drug delivery carriers have led mainly to modification of existing dendrimers, not develCorresponding author. Tel.: +82 2 880 6660; fax: +82 2 877 5110.
E-mail address: [email protected] (J.-s. Park). 0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.12.013
opment of novel dendrimers for gene delivery systems. Among them, poly(amido amine (PAMAM) dendrimers and polypropylenimine (PPI) dendrimers are representative existing dendrimers. PAMAM dendrimers have been utilized and examined for gene delivery systems in vitro and in vivo, extensively [10,11]. PAMAM dendrimers have been also modified with PEG, amino acids, or ligands in order to enhance the gene delivery potency [12–16]. In contrast, applications of PPI dendrimers for gene delivery have been limited to a small number of works. The gene delivery results of PPI dendrimers and quaternized PPI dendrimers were mainly reported by Uchegbu and colleagues [17,18]. Also, self-assembled ternary complexes of PPI dendrimer, cucurbituril, and DNA were examined as a novel non-covalent strategy for gene delivery systems by Lim et al. [19]. PAMAM dendrimer conjugated with arginine residues was previously reported to show enhanced transfection efficiency [13]. Also, replacement of terminal lysine with arginine was reported to enhance the transfection efficiency
ARTICLE IN PRESS 2062
T.-i. Kim et al. / Biomaterials 28 (2007) 2061–2067
in dendritic poly(L-lysine) analogs by Okuda et al. [20]. This may be due to the membrane permeability and nuclear localization ability of arginine residues conjugated to the periphery of the dendrimers. So, we tried to conjugate arginine residues to PPI dendrimer in order to study the arginine conjugation effect of PPI dendrimer for the first time. Here, we report the synthesis of arginine-conjugated PPI dendrimer, PPI2-R and its characterization including cytotoxicity and transfection efficiency for gene delivery systems. 2. Materials and methods
the pbf groups of coupled arginine residues at room temperature for 1 h. After the reaction, the final product, PPI2-R was dialyzed by dialysis membrane (Spectrum Laboratories, Inc., Rancho Dominguez, CA, MWCO ¼ 1000) against ultra-pure water for 4 h and lyophilized before use for analysis and assay.
2.3. 1H NMR spectroscopy 1 H NMR spectra of the polymers were obtained using a Bruker DPX300 NMR spectrometer (300 MHz). For analysis, the polymer samples were dissolved in D2O containing 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt as an internal reference (0 ppm).
2.4. Gel retardation assays
2.1. Materials Poly(ethylenimine) (25 kDa), PPI octaamine dendrimer G2.0 (DABAm-8), N,N-diisopropylethylamine (DIPEA), piperidine, and 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO). N-hydroxybenzotriazole (HOBt), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Anaspec, Inc. (San Jose, CA). Fmoc-L-Arg(pbf)-OH was purchased from Novabiochem (San Diego, CA). Luciferase 1000 Assay System and Reporter Lysis Buffer were purchased from Promega (Madison, WI). The luciferase expression plasmid, pCN-Luci was constructed by subcloning cDNA of Photinus pyralis luciferase with 21-amino acid nuclear localization signal from SV40 large T antigen to pCN [21]. PicoGreen reagent was purchased from Molecular Probes (Eugene, OR). Fetal bovine serum (FBS), and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from GIBCO (Gaithersburg, MD). EGM and EGM-2 MV SingleQuots medium were purchased from Cambrex Bio Science (Walkersville, MD). All chemicals were used without any further purification.
PPI2-R/plasmid complexes at various weight ratios ranging from 1 to 10, were prepared in Hepes buffered saline (10 mM Hepes, 1 mM NaCl, pH 7.4). After 30 min incubation at room temperature for the complex formation, the samples were electrophoresed on a 0.7% (w/v) agarose gel and stained in an ethidium bromide solution (0.5 mg/mL), and analyzed on a UV illuminator to show the location of the DNA.
2.5. Polymer/DNA self-assembly analysis by PicoGreen assay PicoGreen reagent (200 ) was diluted 200-fold in TE buffer before the experiment. About 200 mL of the diluted PicoGreen stock solution was mixed with the same volume of blank solution or polyplex solution (1 mg DNA, 1 HBS) prepared at various charge ratios ranging from 1 to 10. After 2 min incubation, each solution was added to 1.6 mL of TE buffer in a test tube. Fluorescence was measured with a FP-750 spectrofluorometer (Jasco) at room temperature. Excitation and emission wavelengths were set at 480 and 520 nm, respectively. Results were represented as relative fluorescence (%) to DNA control.
2.2. Synthesis of PPI2-R 2.6. Polyplex size measurements PPI2-R was synthesized by conjugating arginine residues to the periphery of PPI2 dendrimer as shown in Fig. 1. Firstly, PPI2 dendrimer was weighed and dissolved in anhydrous DMF. Arginine conjugation reaction was performed in anhydrous DMF for 2 days at room temperature with 32 equivalents of HOBt, HBTU, Fmoc-Arg(pbf)-OH, and 64 equivalents of DIPEA. After completion of the reaction, the reaction mixture was precipitated twice with an excess of diethyl ether. Residual precipitate was dissolved in DMF and mixed with an equal volume of piperidine (30% in DMF, v/v) at room temperature for 20 min to remove the Fmoc groups of coupled Fmoc-Arg(pbf)-OH and precipitated again with diethyl ether. Then, the reagent (95:2.5:2.5, trifluoroacetic acid/triisopropylsilane/water, v/v) was used to deprotect
The hydrodynamic diameters of the PPI2-R/plasmid DNA complexes were determined by light scattering. About 2 mL of polyplex solutions containing 5 mg of DNA were prepared at various charge ratios ranging from 10 to 200. After 30 min incubation, polyplex sizes were measured using a Zetasizer 3000HS (Malvern Instruments, UK). The laser used was a nominal 5 mW HeNe laser having a 633 nm wavelength. Scattered light was detected at a 90 1 angle. The refractive index (1.33) and the viscosity (0.89) of ultrapure water were used at 25 1C for measurements. Zetasizer 3000 (Advanced) Size mode v1.61 software was used for data acquisition. Data analysis was performed in automatic mode. Measured sizes were represented as the average values of 5 runs.
Fig. 1. The synthetic scheme of PPI2-R.
ARTICLE IN PRESS T.-i. Kim et al. / Biomaterials 28 (2007) 2061–2067
2.7. Cell lines Human cervical carcinoma cells (HeLa) and 293 human kidney transformed cells were maintained in DMEM medium with 10% FBS at 37 1C in 5% CO2. Human umbilical vein endothelial cells (HUVECs) were purchased from Cambrex (Walkersville, MD). HUVECs were also maintained in an EBM-2 MV SingleQuots medium containing 2% FBS at 37 1C in 5% CO2.
2.8. Cytotoxicity assay The cytotoxicity of the polymers was measured by MTT assay. HeLa cells were seeded in a 96-well tissue culture plate at 104 cells/well in 90 mL DMEM medium containing 10% FBS. Cells achieving 70–80% confluence after 24 h were exposed to 10 mL of the polymer solutions having various concentrations for 48 h. Then, 26 mL of stock solution of MTT (2 mg/mL in PBS) was added to each well. After 4 h of incubation at 37 1C, each medium was removed and 150 mL of DMSO was added to each well to dissolve the formazan crystals formed by proliferating cells. Absorbance was measured at 570 nm using a microplate reader (Molecular Devices Co., Menlo Park, CA) and recorded as a percentage relative to untreated control cells.
2.9. In vitro transfection 2.9.1. Transfection on HeLa cells and 293 cells HeLa cells and 293 cells were seeded at a density of 5 104 cells/well in a 24-well plate in a DMEM medium containing 10% FBS and grown to reach 70–80% confluence prior to transfection. Before transfection, the medium was exchanged with fresh serum-free medium. The cells were treated with polyplex solution containing 2 mg of plasmid DNA at different charge ratios for 4 h at 37 1C. After exchange with fresh serumcontaining medium, cells were further incubated for 2 days after transfection. Then the growth medium was removed, and the cells were rinsed with PBS and shaken for 30 min at room temperature in 120 mL of Reporter Lysis Buffer. Luciferase activity was measured by a luminescence assay. Ten microliters of the lysate was dispensed into a luminometer tube and luciferase activity was integrated over 10 s with a 2 s measurement delay in a Lumat LB 9507 luminometer (Berthold, Germany) with an automatic injection of 50 mL of Luciferase Assay Reagent. The final results were reported in terms of RLU/well. 2.9.2. Transfection with varied pDNA amount on HeLa cells HeLa cells were seeded at a density of 5 104 cells/well in a 24-well plate in a DMEM medium containing 10% FBS and grown to reach 70–80% confluence prior to transfection. PEI25kD (charge ratio ¼ 8) and PPI2-R polyplexes (charge ratio ¼ 150) containing various pDNA amounts ranging from 2 to 10 mg were prepared, respectively. The cells were treated with the polyplex solutions for 4 h at 37 1C. After exchange with fresh serum-containing medium, cells were further incubated for 2 days after transfection. Further experimental procedures were performed identically to the above transfection and assay methods. 2.9.3. Transfection on HUVECs HUVECs were seeded in a density of 2.5 104 cells/well in a specific 24well plate with a Corning CellBIND Surface (Corning Incorporated, Corning, NY) in an EBM-2 MV SingleQuots medium containing 2% FBS and grown to reach 70–80% confluence prior to transfection. Before transfection, the medium was exchanged with fresh growth factor, cytokine, and supplement-free EGM medium. Transfection experiment was also performed in a growth factor-containing EBM-2 MV SingleQuots medium and this did not have to exchange the medium with EGM medium. The cells were treated with polyplex solutions containing 2 mg of plasmid DNA at different charge ratios for 4 h at 37 1C. After exchange with fresh EBM-2 MV SingleQuots medium, cells were further incubated
2063
for 2 days after transfection. Further experimental procedures were performed identically to the above transfection and assay methods.
3. Results and discussion 3.1. Synthesis and characterization of PPI2-R PPI2-R dendrimer was synthesized via conjugation of arginine residues to the periphery of PPI G2 dendrimers with a HOBt/HBTU coupling method in DMF. PPI G2 dendrimer was chosen as a backbone dendrimer because PPI G2 dendrimer was reported to show the lowest cytotoxicity and the highest transfection efficiency among PPI dendrimers by previous study [18]. Fmoc groups and pbf groups of arginines were removed by 30% piperidine/ DMF solution and the reagent (95:2.5:2.5, trifluoroacetic acid/triisopropylsilane/water, v/v), respectively. The synthesis of PPI2-R was analyzed by 1H NMR spectroscopy as follows. 1 H NMR(D2O): d arginine(–COCHCH2CH2CH2NH–) ¼ 1.70; d arginine(–COCHCH2CH2CH2NH–) ¼ 1.94; d PPI(–NCH2CH2CH2CH2N–, –NCH2CH2CH2N–) ¼ 1.79– 2.18; d PPI(–NCH2CH2CH2CH2N–, –NCH2CH2CH2N–) ¼ 3.20–3.31; d PPI(–NCH2CH2CH2NHCO–) ¼ 3.40; d arginine(–COCHCH2CH2CH2NH–) ¼ 3.26; d arginine(– COCHCH2CH2CH2NH–) ¼ 4.00. The number of arginine residues conjugated to PPI2 dendrimer was calculated by comparing NMR peak intensities between the protons of arginine and of the PPI2 dendrimer backbone. Approximately, 7 molecules of arginine were found to be conjugated to the dendrimer possessing 8 primary amines. The molecular weight of PPI2-R dendrimer was calculated to be 1866.71 Da from the NMR result. 3.2. Characterization of polyplex formation The polyplex formation of PPI2-R was examined by agarose gel retardation assay and PicoGreen reagent assay, respectively. PicoGreen reagent is a DNA intercalating dye more sensitive than ethidium bromide [22]. So, the fluorescence of PicoGreen reagent is quenched when the DNA is condensed into particles by self-assembly with cationic polymers. PPI2 dendrimer was used as a control. Fig. 2 shows the gel retardation assay results. PPI2-R could retard plasmid DNA at a weight ratio of 4 completely, although PPI2 dendrimer could retard plasmid DNA even at a ratio of 2. This tendency was also identified in PicoGreen reagent assay (Fig. 3). PPI2-R polyplexes showed about 66% fluorescence in contrast with PPI2 polyplexes showing only 15% at a charge ratio of 1 but the value of PPI2-R decreased abruptly to less than 10% as the charge ratio increased to 2. The profiles of fluorescence quenching by polyplex formation were similar at higher charge ratios. These results show that PPI2-R can condense pDNA efficiently by self-assembly and arginine-conjugation reduces the pDNA condensing ability of the dendrimer
ARTICLE IN PRESS 2064
T.-i. Kim et al. / Biomaterials 28 (2007) 2061–2067
higher ratios. The sizes of PPI polyplexes also showed a similar tendency. Both polyplexes were found to have a narrow size distribution at all the charge ratios prepared. These results represent that the size of PPI-R polyplex is proper to gene delivery and arginine conjugation to PPI dendrimer does not affect the size of the polyplex significantly.
3.4. Cytotoxicity
Fig. 2. The agarose gel retardation assay results: (a) PPI2-R polyplex; (b) PPI2 polyplex; C ¼ plasmid DNA only.
The cytotoxicity of PPI-R was examined on HeLa cell lines by MTT assay. PEI25 kD and PPI2 dendrimer were used as controls. It is well known that PEI25 kD has high transfection efficiency but also serious cytotoxicity. As shown in Fig. 4, HeLa cell viability after treating PEI25 kD was 58% at 20 g/mL and decreased to 17% at 150 g/mL of concentration while PPI2-R displayed 80–90% cell viability in the same concentration range. Cell viability of PPI2 was also found to be similar to that of PPI2-R. In general, cytotoxicity of gene delivery carriers is known to arise from the accumulation of non-degraded and nondischarged polymers with large molecular weight and charges. This result shows that the cytotoxicity of PPP5R is minimal and the arginine conjugation to PPI2 dendrimer G2 with low molecular weight does not bring a negative effect in its cytotoxicity in spite of highly positive charges of arginine residues.
3.5. Transfection experiments
Fig. 3. The PicoGreen reagent assay results of PPI2-R polyplex and PPI2 polyplex.
somewhat at a low charge ratio. It is explained by the facts that the pKa value of a-amine of arginine in PPI2-R dendrimer is lower than that of primary amine in PPI2 dendrimer [13] and that at the same charge ratio, the number of moles of PPI2-R is 1.5 times less than that of PPI2 in polyplex solution so the self-assembly of PPI2-R with pDNA can proceed less vigorously at the charge ratio. 3.3. Size measurements of the polyplexes The proper size of polyplexes is thought to be essential for efficient gene delivery because it influences the internalization into cells of the polyplexes [1,23]. We examined the size of polyplexes by Zeta-sizer at various charge ratios ranging from 10 to 200. PPI2 dendrimer was used as a control. Table 1 shows the results. The average size of PPI2-R polyplex was 189.6 nm at a charge ratio of 10 and increased to values ranging from 200 to 250 nm at
3.5.1. Transfection on HeLa and 293 cells We performed transfection experiments of newly synthesized PPI2-R on HeLa and 293 cells in the absence of serum, respectively. PEI25 kD and PPI2 were also used as controls. All values were represented as RLU/well, not RLU/mg protein because the total cellular protein of PEI25kD-treated cells was lower than others owing to its cytotoxicity. So, this would increase RLU/mg protein values of PE125KD, not reflecting the exact transfection efficiency. PEI25 kD polyplexes were prepared at a charge ratio of 8 (optimal ratio). The transfection efficiencies of PPI2-R were increased according to the charge ratio ranging from 50 to 200 on both cell lines and the maximum efficiencies reached 27–54% of that of PEI25 kD, as shown in Fig. 5. It is a notable result that PPI2-R showed a transfection efficiency 8–214 times higher than that of unmodified PPI2 at all charge ratios examined. The result shows that arginine conjugation to PPI dendrimer leads to a highly enhanced transfection efficiency and suggests that arginine-conjugated dendritic structure also possesses a high cell permeability and transfection potency like linear oligoarginine peptides or Tat sequences, as reported by previous studies [24–26].
ARTICLE IN PRESS T.-i. Kim et al. / Biomaterials 28 (2007) 2061–2067
2065
Table 1 The average size measurements of polyplexes by Zeta-sizer Charge ratio
10
50
100
150
200
PPI2-R PPI2
189.6725.1 170.3713.0
288.1722.9 247.5714.3
207.977.0 239.176.0
203.877.2 249.5710.0
257.3711.3 244.273.7
Values are represented as nm-scale.
Fig. 4. MTT assay results of PPI2-R.
3.5.2. Transfection with varied pDNA amount Transfection experiments using varied pDNA amount ranging from 2 to 10 mg were performed in order to examine the relationship between cytotoxicity and transfection efficiency on HeLa cells. Only 2 mg of pDNA was used in the previous experiment. PEI25 kD and PPI2-R polyplexes were prepared at the fixed optimal charge ratios of 8 and 150, respectively, to avoid other factors. As shown in Fig. 6, as the pDNA amount used was increased, transfection efficiency of PEI25kD became lower. The transfection value of PEI25 kD with 10 mg pDNA was decreased to even a 20% value with 2 mg pDNA and this is thought to be due to cell death induced by significant cytotoxicity of PEI25 kD because the greater the amount of pDNA amount, the greater is the amount of PEI25 kD at a fixed charge ratio. However, conversely, the transfection efficiency of PPI2-R was increased gradually in proportion to the amount of pDNA and it reached a value 4.3 times higher than that of PEI25kD when treating 10 mg pDNA. This result shows clearly that the minimal cytotoxicity of PPI2-R enabled us to use a large amount of pDNA and the polymer in contrast to the case of PEI25kD for improved gene delivery. 3.5.3. Transfection on HUVECs HUVECs are primary cells used for examining the function and metabolic mechanism of endothelial cells in the endothelium. They are also known to be hardly transfected by non-viral gene delivery carriers and only electroporation has showed relatively high transfection efficiency [27]. We performed transfection experiments in order to investigate the potency of PPI2-R as gene delivery carriers
Fig. 5. Transfection experiment results: (a) HeLa cells; (b) 293 cells. Values were represented as RLU/well.
on HUVECs. As shown in Fig. 7, the transfection efficiency of PPI2-R was 2–3 times higher at a charge ratio of 150 than that of PEI25 kD in the presence or absence of growth factor. This high transfection efficiency showed that PPI2-R possessed great potency as a gene delivery carrier for HUVECs. Interestingly, the efficiencies of both polymers in the presence of growth factor were found to be about 3-fold lower than in its absence. It is presumed that the growth factor may interact with polyplexes and inhibit the
ARTICLE IN PRESS T.-i. Kim et al. / Biomaterials 28 (2007) 2061–2067
2066
4 times higher than that of PEI25 kD because of its low cytotoxicity. Finally, PPI2-R was found to possess a transfection efficiency 2–3 times higher than PEI25kD on HUVECs that is known to be hardly transfected primary cells, showing the potency of PPI2-R as an efficient gene delivery carrier. In conclusion, arginine-conjugation of PPI2 was proven to be a successful trial to develop a low toxic and highly transfection-efficient gene delivery carrier. Acknowledgments
Fig. 6. Transfection experiment results with varied pDNA quantity on HeLa cells.
This work was supported by the Korea Health 21 R&D Project of The Ministry of Health & Welfare, Republic of Korea (A04-0004), and the Gene Therapy Project of The Ministry of Science and Technology (M105340300405N3403-00410). We thank So Hee Nam for providing helpful guidance for HUVEC culture. References
Fig. 7. Transfection experiment results on HUVECs.
transfection mechanism via the formation of a biologically inert complex, leading to a reduction of transfection efficiency. 4. Conclusion Arginine-conjugated PPI dendrimer (DAB-2), PPI2-R was synthesized for gene delivery systems. PPI2-R was found to condense pDNA into particles with an average size of about 200 nm. The cell viability after treating with PPI2-R was shown to be over 80% at even a 150 mg/mL concentration, indicating that the cytotoxicity of PPI2-R was minimal. PPI2-R also showed a highly enhanced transfection efficiency compared to unmodified PPI2 dendrimer on HeLa and 293 cells. When the pDNA used was increased to 10 mg in a transfection experiment on HeLa cells, the transfection efficiency of PPI2-R was
[1] Zauner W, Ogris M, Wagner E. Polylysine-based transfection systems utilizing receptor-mediated delivery. Adv Drug Del Rev 1998;30: 97–113. [2] Luo D, Saltzman WM. Synthetic DNA delivery systems. Nat Biotechnol 2000;18:33–7. [3] Vijayanathan V, Thomas T, Thomas TJ. DNA nanoparticles and development of DNA delivery vehicles for gene therapy. Biochemistry 2002;41:14085–94. [4] Mastrobattista E, Bravo SA, van der Aa M, Crommelin DJA. Nonviral gene delivery systems: from simple transfection agents to artificial viruses. Drug Discov Today: Technol 2005;2:103–9. [5] Svenson S, Tomalia DA. Dendrimers in biomedical applications— reflections on the field. Adv Drug Del Rev 2005;57:2106–29. [6] Dufe`s C, Uchegbu IF, Scha¨tzlein AG. Dendrimers in gene delivery. Adv Drug Del Rev 2005;57:2177–202. [7] Gillies ER, Fre´chet JMJ. Dendrimers and dendritic polymers in drug delivery. Drug Discov Today 2005;10:35–43. [8] Cloninger MJ. Biological applications of dendrimers. Curr Opin Chem Biol 2002;6:742–8. [9] Patri AK, Majoros IJ, Baker Jr JR. Dendritic polymer macromolecular carriers for drug delivery. Curr Opin Chem Biol 2002; 6:466–71. [10] Eichman JD, Bielinska AU, Kukowska-Latallo JF, Baker Jr JR. The use of PAMAM dendrimers in the efficient transfer of genetic material into cells. PSTT 2000;3:232–45. [11] Esfand R, Tomalia DA. Poly(amidoamine) (PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications. Drug Discov Today 2001;6:427–36. [12] Luo D, Haverstick K, Belcheva N, Han E, Saltzman WM. Poly(ethylene glycol)-conjugated PAMAM dendrimer for biocompatible, high-efficiency DNA delivery. Macromolecules 2002;35: 3456–62. [13] Choi JS, Nam K, Park J-y, Kim J-B, Lee J-K, Park J-s. Enhanced transfection efficiency of PAMAM dendrimer by surface modification with L-arginine. J Control Rel 2004;99:445–56. [14] Kim T-i, Seo HJ, Choi JS, Jang H-S, Baek J-u, Kim K, et al. PAMAM-PEG-PAMAM: novel triblock copolymer as a biocompatible and efficient gene delivery carrier. Biomacromolecules 2004;5: 2487–92. [15] Choi JS, Ko KS, Park JS, Kim Y-H, Kim SW, Lee M. Dexamethasone conjugated poly(amidoamine) dendrimer as a gene carrier for efficient nuclear translocation. Int J Pharm 2006;320: 171–8.
ARTICLE IN PRESS T.-i. Kim et al. / Biomaterials 28 (2007) 2061–2067 [16] Wada K, Arima H, Tsutsumi T, Chihara Y, Hattori K, Hirayama F, et al. Improvement of gene delivery mediated by mannosylated dendrimer/a-cyclodextrin conjugates. J Control Rel 2005;104: 397–413. [17] Schatzlein AG, Zinselmeyer BH, Elouzi A, Dufes C, Chim YTA, Roberts CJ, et al. Preferential liver gene expression with polypropylenimine dendrimers. J Control Rel 2005;101:247–58. [18] Zinselmeyer BH, Mackay CP, Schatzlein AG, Uchegbu IF. The lower-generation polypropylenimine dendrimers are effective gene transfer agents. Pharm Res 2002;19:960–7. [19] Lim Y-b, Kim T, Lee JW, Kim S-m, Kim H-J, Kim K, et al. Selfassembled ternary complex of cationic dendrimer, cucurbituril, and DNA: noncovalent strategy in developing a gene delivery carrier. Bioconjugate Chem 2002;13:1181–5. [20] Okuda T, Sugiyama A, Niidome T, Aoyagi H. Characters of dendritic poly(l-lysine) analogues with the terminal lysines replaced with arginines and histidines as gene carriers in vitro. Biomaterials 2004;25:537–44. [21] Lee MJ, Cho SS, You JR, Lee Y, Kang BD, Choi JS, et al. Intraperitoneal gene delivery mediated by a novel cationic liposome
[22]
[23]
[24] [25] [26]
[27]
2067
in a peritoneal disseminated ovarian cancer model. Gene Ther 2002; 9:859–66. Singer VL, Jones LJ, Yue ST, Haugland RP. Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double stranded DNA quantitation. Anal Biochem 1997; 249:228–38. Mahato RI, Rolland A, Tomlinson E. Cationic lipid-based gene delivery systems: pharmaceutical perspectives. Pharm Res 1997; 14:853–9. Brooks H, Lebleu B, Vive`s E. Tat peptide-mediated cellular delivery: back to basics. Adv Drug Del Rev 2005;57:559–77. Zhao M, Weissleder R. Intracellular cargo delivery using Tat peptide and derivatives. Med Res Rev 2004;24:1–12. Wadia JS, Dowdy SF. Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer. Adv Drug Del Rev 2005;57:579–96. Ear T, Gigue`re P, Fleury A, Stankova J, Payet MD, Dupuis G. High efficiency transient transfection of genes in human umbilical vein endothelial cells by electroporation. J Immunol Methods 2001;257: 41–9.