Bioconjugate Chem. 2007, 18, 2029–2036
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Dexamethasone-Conjugated Low Molecular Weight Polyethylenimine as a Nucleus-Targeting Lipopolymer Gene Carrier Yun Mi Bae,† Hye Choi,† Seungah Lee,‡ Seong Ho Kang,‡ Young Tae Kim,§ Kihoon Nam,⊥ Jong Sang Park,⊥ Minhyung Lee,| and Joon Sig Choi*,† Department of Biochemistry, Chungnam National University, Daejeon, 305-764, Korea, Department of Chemistry and Research Institute of Physics and Chemistry (RINPAC), Chonbuk National University, Jeonju, 561-756, Korea, Department of Thoracic and Cardiovascular Surgery, Xenotransplantation Research Center, Seoul National University Hospital, Cancer Research Institute, Seoul National University College of Medicine, Seoul, 110-744, Korea, Department of Bioengineering, Hanyang University, Seoul, 133-791, Korea, and School of Chemistry, Seoul National University, 151-742, Korea. Received January 12, 2007; Revised Manuscript Received July 9, 2007
Dexamethasone, a glucocorticoid steroid, can dilate the nuclear pore complexes and translocate into the nucleus when it is bound to its glucocorticoid receptor, suggesting that the transport of DNA into the nucleus may be facilitated by the reagent. In this research, dexamethasone was conjugated to low molecular weight polyethylenimine (2kDa) for efficient translocation of the polymer/DNA complex into the nucleus. Polyethylenimine (PEI)– dexamethasone (PEI–Dexa) was synthesized by one-step reaction using the Traut’s reagent. In gel retardation assay, the PEI–Dexa/DNA complex was completely retarded at or above 0.3/1 weight ratio (polymer/DNA). The average size distributions and ζ-potential values of the complexes were measured at various weight ratios. In vitro transfection assay showed that the PEI–Dexa/DNA complex had higher gene delivery efficiency compared to PEI 2kDa/DNA complex. The localization of PEI–Dexa/plasmid DNA complexes in the nucleus was confirmed by using total internal reflection fluorescence and Nomarski differential interference contrast microscope as well as confocal microscope. Therefore, with efficient nuclear translocation and low cytotoxicity, PEI–Dexa may be useful for nonviral gene therapy.
INTRODUCTION Gene therapy is an interdisciplinary field, including basic sciences, engineering, pharmacology, and medicines. For more than two decades, it has been gaining interests and prospects as one of the most promising custom-made platform therapeutic technologies in the post-genomic era. The vehicle for encapsulation and delivery of therapeutic genes is either viral vectors or nonviral synthetic vectors, such as cationic liposomes and cationic polymers or the combination of both vectors (1). In the view of targeting and transfection efficiency, viral vectors are better than nonviral vectors, but their inevitable problems, such as immune response and possible tumorigenesis, are still difficult barriers to overcome. As an alternative, nonviral synthetic vectors have been actively studied and developed over the past decade, and some recent studies showed promising results for future clinical applications with DNA and RNA delivery (2, 3). For the synthetic vectors, the liposome system is one of the most promising carriers for delivering therapeutic genes as well as biologically active molecules into mammalian cells (4). The application of such synthetic nonviral vectors is dependent on several factors, such as the solubility in water, the size and surface charge of the complexes, and the lipid composition of liposomes. Therefore, it is necessary to control such conditions and to check every preparation step to get optimal transfection efficiency in vitro and/or in vivo. * Towhomcorrespondenceshouldbeaddressed.E-mail:
[email protected]. Tel: 82-42-821-5489. Fax: 82-42-822-7548. † Chungnam National University. ‡ Chonbuk National University. § Seoul National University College of Medicine. | Hanyang University. ⊥ Seoul National University.
Glucocorticoid receptor is an intracellular receptor, which regulates gene expression in the presence of specific ligands. In the presence of glucocorticoid, the receptor binds to the ligand and the receptor–ligand complex is translocated into the nucleus (5). It is also reported that the nuclear pore complexes were dilated by the glucocorticoid receptor in the presence of dexamethasone, a potent glucocorticoid (6, 7). This dilation effect is considered to be helpful for nonviral gene delivery, which is favorable for translocation of polymer/DNA complex into the nucleus. Also, it was suggested that the transport of the polymer/DNA complex into the nucleus may be facilitated by the ligand–receptor-mediated transport when glucocorticoid is conjugated to the polymer. It was successfully verified in some reports using dexamethasone-mediated gene delivery (8–10), and we recently reported that the conjugation of dexamethasone to polyamidoamine dendrimer (PAMAM) increased the transfection efficiency of the polymer (11). The goal of this study is to synthesize and characterize a lipopolymer composed of nontoxic small molecular weight polyethylenimine (PEI) (2kDa) and dexamethasone. We synthesized dexamethasone conjugated PEI with a Traut’s coupling reagent. PEI–Dexa might have improved transfection efficiency due to efficient translocation of PEI–Dexa/DNA complex into the nucleus. To prove the hypothesis, we characterized physicochemical properties, cytotoxicity, and in vitro transfection efficiency of PEI–Dexa. Also, the localization of the PEI–Dexa/ DNA complex in the nucleus was verified by confocal, total internal reflection fluorescence (TIRF), and Nomarski differential interference contrast (DIC) microscopy.
EXPERIMENTAL METHODS Materials. Polyethylenimine (branched, 2kDa, and 25kDa), polyamidoamine dendrimer (PAMAM, generation 4), ethidium
10.1021/bc070012a CCC: $37.00 2007 American Chemical Society Published on Web 09/13/2007
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bromide, and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Korea. Dexamethasone-21-mesylate was purchased from Steraloids Inc. Luciferase 1000 assay kit and reporter lysis buffer were purchased from Promega (Madison, WI). The luciferase expression plasmid (pCN-Luci) was prepared as reported previously (12). HepG2 and 293 cell lines were obtained from Korean Cell Line Bank. PicoGreen reagent and Alexa Fluor 488 protein labeling kit were purchased from Invitrogen. Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), and 100× antibiotic-antimycotic reagent were purchased from GIBCO (Gaithersburg, MD). Micro BCA protein assay kit and 2-iminothiolane (Traut’s reagent) were purchased from Pierce (Rockford, IL). Synthesis of PEI–Dexa. The conjugation reaction was performed as reported previously with some modification (8, 11). Low molecular weight PEI (2 kDa) was dissolved in 1.8 mL of anhydrous dimethyl sulfoxide (DMSO) with 2-fold molar excess of Traut’s reagent and dexamethasone-21-mesylate. To minimize the crosslinking side reaction, we used anhydrous DMSO and tried to keep away humidity during the reaction. The reaction was allowed to proceed for 4 h at room temperature and was quenched by the addition of an excess amount of cold ethyl acetate. The precipitated product was solubilized in water and dialyzed for 1 day against pure water using dialysis membrane (MWCO 1000). It was further freeze-dried and a white product was obtained (60% yield). The product was solubilized in DMSO-d6/D2O for 1H NMR analysis (300 MHz, Korea Basic Science Institute). Gel Retardation Assay and PicoGreen Assay. Polymer/ plasmid complexes were prepared at various weight ratios ranging from 0.1 to 0.5 in Hepes-buffered saline (20 mM Hepes, 150 mM NaCl, pH 7.4). After 30 min incubation at room temperature for complex formation, the samples were subject to electrophoresis on a 0.7% (w/v) agarose gel containing ethidium bromide (0.5 µg/mL of the gel). After electrophoresis, the gel was analyzed on a UV illuminator to show the location of the DNA. PicoGreen assay for complex formation was also performed as reported previously (12). Dynamic Light Scattering and ζ-Potential Measurements. The hydrodynamic diameters and ζ-potential values of polyplexes at various weight ratios were determined by the Zetasizer Nano ZS system (Malvern Instruments, UK) (12). After 30 min incubation, the complex solution was diluted to a final volume of 2 mL prior to measurements. The complex size was measured using Dispersion Technology Software 4.30, and data analysis was performed in automatic mode. The refractive index (n ) 1.33) and the viscosity (0.89) of ultrapure water were used at 25 °C for measurements. The measured value is presented as the average size plus or minus a standard deviation of three runs. Cell Culture and in vitro Transfection. Human embryonic kidney 293 cells and human liver carcinoma HepG2 cells were grown in DMEM with 10% FBS. The cells were routinely maintained on plastic tissue culture dishes at 37 °C in an incubator with a humidified atmosphere containing 5% CO2/ 95% air. All media routinely contained antibiotic–antimycotic agent. For the transfection studies, cells were seeded at a density of 2 × 105 cells/well in 24 well flat-bottomed microassay plates 24 h before transfection. Polyplexes were prepared at various weight ratios and added to the cells. For competition assay, dexamethasone stock solution prepared in ethanol was added to each well (final ethanol concentration was 1.6%, v/v) and incubated for 30 min before polyplexes were added. The dexamethasone and polyplexes were incubated for 24 h in the presence of 10% FBS before assay. The amount of plasmid
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DNA was fixed at 1 µg/well. The cells were then incubated for 1 day at 37 °C in a 5% CO2 incubator before assay. Luciferase Assay. After transfection, the cells were washed with phosphate-buffered saline (PBS), and 150 µL of reporter lysis buffer (Promega, Madison, WI) was added to each well. After 15 min of incubation at room temperature, the cells were harvested and transferred to microcentrifuge tubes. After 15 s of vortexing, the cells were centrifuged at 12 000 rpm for 3 min. The protein concentrations of the extracts were determined by using protein assay kit. Luciferase activity was measured in terms of relative light units (RLU) using a luminometer. The final values of luciferase were reported in terms of RLU/µg total protein. Cytotoxicity Assay. Evaluation of cytotoxicity was performed by the MTT assay (13). Cells were seeded at a density of 2 × 104 cells/well in 96 well microassay plates and were incubated for 1 day before adding the polymers. After the addition of each polymer, cells were further incubated for 24 h at 37 °C. After the incubation, 24 µL of 2 mg/mL MTT solution in DPBS was added. The cells were incubated for 4 h at 37 °C and then MTTcontaining medium was removed, and 150 µL of DMSO was added to dissolve the formazan crystal formed by live cells. Absorbance was measured at 570 nm using a microplate reader (VersaMax, Molecular Devices). Fluorescence Labeling of PEI and PEI–Dexa and Complex Formation with Plasmid DNA. PEI and PEI–Dexa (2 mg/mL) were prepared in 0.1 M sodium bicarbonate and mixed with the reactive Alexa Fluor 488 dye followed by stirring for 1 h at room temperature. Each reaction mixture was dialyzed (MWCO 1000) against pure water and freeze-dried yielding a fluorescent product. The Alexa488-labeled PEI or PEI–Dexa and 1.0 µg of plasmid DNA were mixed in 200 µL of FBSfree DMEM (GIBCO, Gaithersburg, MD) to form complexes at a charge ratio of 4. The mixture was incubated for 30 min at room temperature. DNA-linked dendrimer solution was then added to the cells and incubated for the indicated time prior to imaging. The complexes were added to the cells and incubated for 24 h before imaging experiments. Cell Culture Preparation for Microscopy. 293 cells were grown in a cell culture dish (T25, Falcon), in a DMEM medium supplemented with antibiotic–antimycotic agent (GIBCO) and 10% FBS (GIBCO). The cells were maintained in an incubator with a humidified atmosphere containing 5% CO2/95% air. Approximately 2 × 104 cells per well were seeded in bare cover glass (22 mm sq, No. 1 Dow Corning, Corning, NY) the day before the experiment, and the cells were maintained with serum-free DMEM. The luciferase expression plasmid (pCNLuci) was used to prove that the dynamics and localization of polyethyleneimine (PEI) dendrimer and PEI–dexamethasone (PEI–Dexa) conjugated fluorescent dye Alexa 488. HEK 293 cells were examined by adding the DNA-linked functional dendrimer in cover glass before 24 h. Before initiating an experiment, the medium was then removed, and the adherent cells were rinsed twice with DPBS to remove the unbound complexes. A cover glass was placed on the all-side polished dove prism (BK7, 15 mm × 63 mm × 15 mm, n ) 1.522, Korea Electro-Optics Co., Ltd., Korea). Surface and interior localization of the polymer/DNA complexes were detected using the epifluorescence and TIRF microscope. Nomarski DIC imaging was used to examine the details of the living cell structure. Confocal Microscopy Experiments. 293 cells were seeded at a density of 2 × 105 cells/well on a cover glass (18 mm sq) that was spread in each well of a 6 well plate (Falcon) and incubated at 37 °C for a day. Alexa Fluor 488-labeled PEI–Dexa/ plasmid DNA complexes were prepared at a 4/1 weight ratio (polymer/plasmid DNA). The amount of plasmid DNA was
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Scheme 1. Synthesis Scheme of Polyethylenimine-Dexamethasone (PEI-Dexa)
fixed at 2 µg per well. The polymer/plasmid DNA complex was added to the cells and incubated for 24 h in 10% FBS-containing media. For nucleus labeling, cells were washed with PBS and further incubated for 30 min with 1.8 mL (4 µM) Hoechst 33342 dye (Molecular Probes, U.S.A.). The cells were then finally washed with PBS, and the cells were observed using confocal laser-scanning microscopy (Radiance 200 MP, Bio-Rad, U.K.). Combined System of Nomarski DIC and TIRFM. We used a combined system of DIC and TIRF microscope for the realtime detection of polymer/DNA complexes localization in living 293 cells. The combined microscope system was similar to the system reported recently (14). A 100× (Olympus UPLFL 100×/ 1.3 N.A., W.D. 0.1) oil immersion objective lens was used for the image acquisition. A charge-coupled device (CCD) camera (Cascade 512B, Photometrics, Tucson, AZ) was mounted on the top of the microscope. The CCD exposure time was 100 ms. The combined system, which we integrated, consists of an upright Olympus BX51 microscope (Olympus Optical Co., LTD, Shinjuku-ku, Tokyo, Japan) equipped with a DIC slider (UDICT, Olympus) and a Hg lamp for epifluorescence illumination. DIC imaging was used to examine the image and the details of the living cell structure, and epifluorescence imaging was used to detect the distribution of the polymer/DNA complexes in the bulk. The epifluorescence filter cube consisted of U-MWB2 (excitation filter, 460–490 nm; barrier filter, 520 nm; dichromatic mirror, 500 nm, Olympus Optical Co., Ltd., Shinjuku-ku, Tokyo, Japan). The TIRFM imaging was used to detect complexes within the HEK 293 cells. An argon ion laser at 488 nm with a 30 mW output (model 532-LAP-431–220, Melles Griot, Irvin, CA) was used as the excitation source for TIRF. The bare cover glass (No.1 Corning, 22 mm square) incubating the cells was placed on the all-side polished dove prism (BK7, 15 mm × 63 mm × 15 mm, n )1.522, Korea Electro-Optics Co., Ltd., Korea). The cover glass and the prism were index-matched with a drop of immersion oil (Immersol 518F, Zeiss, n )1.518). The laser beam was directed through the prism toward the cover glass/media interface. The angle of incidence, θi, was slightly greater than 72°. The laser beams
were transmitted through an optical pinhole to eliminate extraneous light and plasma lines and to reduce the laser diameter. A Uniblitz mechanical shutter (model LS3Z2, Vincent Associates, Rochester, NY) was used to reduce photobleaching. The shutter was controlled by a model VMM-D1 shutter driver. All the experiments were performed on the all-side polished dove prism without moving the sample. MetaMorph 6.3 software (Universal Imaging Co., Downingtown, PA) was used to collect the image of the individual single molecules and to process the data.
RESULTS Synthesis and Properties of PEI–Dexa. In this report, we conjugated low molecular weight PEI with the nucleus-targeting glucocorticoid dexamethasone and applied it to nonviral gene delivery experiments. PEI–dexamethasone, referred to as PEI– Dexa in this study, is one kind of functional water-soluble lipopolymers with nucleus receptor targeting characteristics. As described in Scheme 1, dexamethasone was efficiently conjugated by using the method recently reported (8, 11). In addition to the pH sensitiveness of the linkage, the benefit of this approach was that there was no cationic charge sacrifice effect. The 1H NMR data of the product is presented and each proton peak is identified (Figure 1). The peak intensity ratio was calculated between the ethyl protons of PEI backbone and the two methyl protons at 13′ and 16′ position of the five-numbered ring of dexamethasone (δ ) 0.7–0.9 ppm). From the 1H NMR calculations, it was observed that one mole of dexamethasone was conjugated per 1.3 mol of PEI. Analysis of Complex Formation by Agarose Gel Electrophoresis and PicoGreen Reagent Assay. The complex formation of PEI–Dexa with plasmid DNA was investigated by agarose gel electrophoresis and PicoGreen reagent assay. Polymer/DNA complexes were prepared at various weight ratios ranging from 0.1 to 0.5. As shown in Figure 2A,B, PEI–Dexa was more effective in retardation of complexes at each weight ratio, and it was further approved by PicoGreen reagent assay. We have already reported the same approach for polyplexes
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Figure 1. 1H NMR of PEI–Dexa.
Figure 2. The DNA complexation assay. (A) Agarose gel retardation assay. Plasmid DNA only (lane 1, 5), polymer/DNA weight ratio 0.1 (lane 2, 6), 0.3 (lane 3, 7), 0.5 (lane 4, 8). PEI 2kD (lane 2–4) and PEI–Dexa (lane 6–8). (B) PicoGreen reagent assay at various weight ratios. PEI 2 kDa (b) and PEI–Dexa () (n ) 3).
and proved that this application is much more effective and sensitive than using EtBr for the characterization of polyplexes (12). For PEI–Dexa, the fluorescence intensity decreased more rapidly compared to that of PEI 2kDa, which means that PEI–Dexa can construct more mature complexes inhibiting the PicoGreen reagent from binding the uncomplexed DNA. We think that there is another important factor governing the effective complex formation between PEI–Dexa and DNA compared to PEI. Even though the charge density of PEI–Dexa is lower at the same weight ratio than PEI, the conjugated dexamethasones will function as hydrophobic cores that may facilitate the complex formation in addition to the process of charge neutralization. So it is considered that the presence of hydrophobic dexamethasones may contribute to the complex formation and collapse into complexed particles more effectively with plasmid DNA than PEI.
Size Measurements of the PEI-Dexa/Plasmid DNA Complex. PEI/DNA and PEI–Dexa/DNA complexes were prepared at various weight ratios (polymer/DNA) and the mean particle sizes and the ζ-potential values of polyplexes were measured using the Zetasizer Nano ZS system. As presented in Figure 3, there was no remarkable difference in the mean diameter between PEI/DNA and PEI–Dexa/DNA complexes at the weight ratios tested. In addition, for ζ-potential experiments, PEI–Dexa/DNA complexes showed lower values compared to PEI/DNA complexes. Because the particle sizes of PEI–Dexa/ DNA complexes are very similar to those of PEI/DNA complexes and the ζ-potentials of PEI–Dexa/DNA show a few lower values at the same conditions, it is likely that other factors may contribute to the enhanced transfection efficiency of PEI–Dexa compared to PEI. Transfection Experiments. Low molecular weight PEI (2kDa) or PEI–Dexa was mixed with plasmid DNA at various weight ratios and each complex was evaluated in terms of the transfection efficiency in 293 and HepG2 cells in the presence of 10% FBS (Figure 4A,B). PEI 25kDa and PAMAM polymer was used as positive control reagents. After 24 h of transfection, cells were harvested for assaying the expressed luciferase activity. As in the previous report (15), the transfection efficiency of low molecular weight PEI was not remarkable at low weight ratios. In comparison, PEI–Dexa showed a greater increased level of gene expression compared to native PEI 2kDa at all weight ratios tested. As compared with the transfection efficiency of native PEI, the gene expression level of PEI–Dexa increased by 1 order of magnitude for HepG2 and at least 2 orders for 293 cells. Even though the transgene expression level was lower than PEI 25kDa for both cell lines, PEI–Dexa showed comparable transfection efficiency for 293 cells and one order of increased efficiency for HepG2 at weight ratio 8 (polymer/ DNA) in comparison with PAMAM. We also studied the effect of dexamethasone incubation on transfection efficiency at different concentration ranges from 10 to 50 nM. As shown in Figure 4C, we observed that the transfection efficiency of PEI–Dexa/DNA constructed at weight ratio 2 apparently decreased to 15%, whereas no competition effects were detected for PEI/DNA. Cytotoxicity Assay Results. The cytotoxicity of PEI–Dexa was examined and compared with PEI and PAMAM at various concentration ranges for 293 and HepG2 cells. As shown in Figure 5, PEI–Dexa showed no toxicity at the concentration ranges tested in this experiment, whereas PEI 25kDa displayed
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Figure 3. The average size data (A), and ζ-potential values (B) of PEI/ DNA and PEI–Dexa/DNA complexes at various weight ratios. Each value is the average plus or minus the standard deviation of three different experiments.
cytotoxicity with increasing concentration levels. This result indicates that dexamethasone conjugated to low molecular weight PEI did not cause any harmful effects at the concentration ranges for the cell lines tested in these experiments. Confocal Microscopy and TIRFM/DIC Microscopy Experiments. To identify the localization of polyplexes inside cells, confocal microscopy and TIRFM/DIC microscopy experiments were performed in parallel and compared. Each complex constructed at weight ratio 4 (polymer/DNA) was incubated with 293 cells and the difference in cell-associated fluorescence was monitored. Alexa 488 was used to label PEI and PEI–Dexa for complex-mediated fluorescence monitoring. The amount of labeled Alexa 488 for each polymer was calculated using the molar extinction coefficient of the dye at 494 nm (71 000 cm-1 M-1), and each value was 2.3 × 10-4 and 4.5 × 10-5 moles of dye per 4.0 µg of PEI and PEI–Dexa, respectively. The labeling efficiency for native PEI was about five times higher than PEI–Dexa. As shown in Figure 6, many fluorescent complexes were found inside the nucleus in the confocal microscopy image of Alexa 488-labeled PEI–Dexa/plasmid DNA complexes on 293 cells. In addition, in the TIRF/DIC microscopy images, more PEI–Dexa/DNA complexes were found in the nucleus region compared to PEI 2kD/DNA complexes (Figure 7). These results clearly indicate that PEI–Dexa/DNA complexes were more effectively translocated into the nucleus than native PEI 2kDa. This efficient translocation into the nucleus may contribute to
Figure 4. Transfection efficiency of PEI–Dexa for 293 cells (A) and HepG2 cells (B). PEI/PAMAM indicates the control data for PEI 25kDa (black) and PAMAM (gray). The numbers in the x-axis are the weight ratios of polymer/DNA complexes for PEI 2kDa (black bars) and PEI–Dexa (gray bars). The competition effects of dexamethasone incubation with each polyplex on transfection efficiency for HepG2 at different nanomolar concentrations (C). Each polyplex was constructed at the weight ratio of 2 and was introduced to cells with dexamethasone. The data are presented as average percent control values of three results of the expressed luciferase activity compared to the nontreated one.
the higher transfection efficiency of PEI–Dexa at a lower polymer concentration.
DISCUSSION The effective cell-specific targeting, endosomal escape, and nuclear localization are key factors for nonviral vectors to
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Figure 5. Cytotoxicity assay for 293 cells (A) and HepG2 cells (B) by MTT assay. Relative cell viability was calculated assuming the absorbance at 570 nm of intact control cells to be 100%. Each value is the average plus or minus the standard deviation of three different experiments.
Figure 6. Confocal microscopy images of 293 cells incubated with PEI–Dexa/DNA complexes (B). The complex was prepared as described in Experimental Methods. Hoechst 33342 was used to stain the nucleus (blue) and Alexa 488-labeled polymer was used to localize the complexes (green). The image shown in (A) is the respective transmitted light image of the same field.
improve transfection efficiency (16–18). Nuclear localization signal peptides have been employed to increase efficiency for nondividing cells as well as dividing cells (19, 20). PEI 25kDa showed the highest gene transfection efficiency among the polymer vectors that have been reported so far. However, the cytotoxicity of the high molecular weight PEI is a major obstacle in application of the polymer to clinical gene therapy approach. So, low molecular weight PEI 2kDa has been investigated as a gene carrier, because it is relatively nontoxic
compared to the higher molecular weight PEI derivatives and it showed remarkable level of transfection efficiency at high polymer/DNA ratios (15, 21–24). Many studies have been conducted to modify low molecular weight PEI for enhanced functionality and targeting activity using the polymer as a starting material. In some reports, it was attempted to construct hyperbranched PEI derivatives with high molecular weight using low molecular weight PEI as a building block and to introduce various biodegradable linkages in the backbone (25–27). In
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Figure 7. The internalization of polymer/plasmid DNA complexes in living human embryonic kidney 293 cells using various detection techniques of optical microscopes. Epifluorescence: light source, Hg lamp. TIRFM: light source, argon ion laser 488 nm; incident angle θi, 72°. DIC: light source, halogen lamp; DIC slider, U–DICT. The images are epifluorescence (A, E), TIRFM (B, F), Nomarski DIC images (C, G), and the merged images of TIRFM and DIC (D, H) of PEI 2kDa/DNA complexes at 4/1 weight ratio (top panels) and PEI–Dexa/DNA complexes at 4/1 weight ratio (bottom panels). The arrows indicate the polymer/DNA complexes.
another study, cholesterol was conjugated to low molecular weight PEI as a hydrophobic moiety to increase membrane perturbation effect leading to enhanced gene delivery, and they named the system as “water-soluble lipopolymer” (28). We reported that the preincubation of free dexamethasone could increase the polyplex-mediated gene transfection efficiency and that it could be attributed to the nuclear pore dilation effect of the drug (29). However, due to the reduced solubility of dexamethasone in water, the emulsification step is generally needed using organic solvents and/or other surfactants, which may cause negative effects to cell viability. So it is needed to prepare water-soluble dexamethasone derivatives to make other emulsifying additives unnecessary. The recent development of a novel functional liposomal system known as spermine–dexamethasone provided some valuable characteristics as a nonviral vector system (8). The liposome is composed of a cationic spermine headgroup and a nuclear-targeting hydrophobic ligand, dexamethasone, which showed remarkable gene transfection efficiency. Our group also recently reported the synthesis of dexamethasone-conjugated PAMAM, and that the water-soluble conjugate polymer showed higher transfection efficiency than PEI 25kDa in a specific cell line in vitro (11). In line with the previous reports, we attempted to prepare novel functional water-soluble lipopolymeric gene carriers by conjugating nontoxic low molecular weight PEI and dexamethasone. Therefore, PEI–Dexa may be a nuclear-targeting carrier with the functions of carrying the cargo attached to it into the nucleus and dilating nuclear pore complexes. These properties may contribute synergistically to the enhanced transfection efficiency of PEI–Dexa. Indeed, PEI–Dexa showed the enhanced gene transfection efficiency at lower polymer concentrations compared to native PEI, and the transfection efficiency of PEI–Dexa was greatly affected in the presence of dexamethasone for such conditions as presented in Figure 4C. So, it is presumed that there may be two possible explanations for the enhanced gene transfection efficiency. First, PEI–Dexa is one of the hydrophobic group-containing lipopolymers, which might have membrane perturbation effect like many other cationic liposomes including the water-soluble lipopolymer, PEI–cholesterol. Second, based upon the previous microscope observations that more PEI–Dexa/plasmid DNA complexes were found in the nucleus region compared to PEI 2kDa/DNA
complexes, it is possible to propose that PEI-conjugated dexamethasone might have contributed to the enhanced nucleuslocalization activity of the complexes causing greater increased transfection efficiency. Taken together, the results suggest that the conjugation of a glucocorticoid to a polymeric gene carrier may have two favorable effects. First, because dexamethasone is prescribed as a drug for the treatment of inflammation and autoimmune diseases, PEI–Dexa may be a water-soluble lipopolymeric prodrug that enhances the solubility of dexamethasone. Therefore, PEI–Dexa may have dual functions as a water-soluble steroid drug and a gene carrier for combinational approaches of drug/gene therapy. Second, in addition to the nuclear pore complex dilation effect, PEI–Dexa could act as a ligand for intracellular glucocorticoid receptors that are present in mammalian cells. So it is expected that PEI–Dexa may function as a potent nucleus-targeting signal following the ligand–receptor interaction, and the receptor may facilitate the translocation of dexamethasone-decorated complexes into the nucleus and also may function as a reagent that makes the nuclear membrane more vulnerable to the nanoparticles by enlarging the nuclear pores at the same time.
ACKNOWLEDGMENT This research was supported by the Ministry of Science and Technology (M10534030003-06N3403-00310), by grants from the KBRDG Initiative Research Program (F104AD01001106A0401-01110), and by the Korea Science and Engineering Foundation (R01-2006-000-10617-0) in Korea.
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