Hepatic Gene Delivery System Electrostatically Assembled with

Mar 27, 2014 - In this study, a novel liver-targeted gene delivery vector was developed by electrostatically coating the cationic complex of pDNA and ...
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Hepatic Gene Delivery System Electrostatically Assembled with Glycyrrhizin Tomoaki Kurosaki,† Saki Kawanabe,† Yukinobu Kodama,† Shintaro Fumoto,‡ Koyo Nishida,‡ Hiroo Nakagawa,† Norihide Higuchi,† Tadahiro Nakamura,† Takashi Kitahara,† and Hitoshi Sasaki*,† †

Department of Hospital Pharmacy, Nagasaki University Hospital, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan Department of Pharmaceutics, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan

‡

ABSTRACT: In this study, a novel liver-targeted gene delivery vector was developed by electrostatically coating the cationic complex of pDNA and polyethylenimine (PEI) with glycyrrhizin (GL). The ternary complex, pDNA/PEI/GL, had approximately 100 nm stable particles with a negative charge surface. pDNA/PEI/GL showed high gene expression comparable to that of the complex of pDNA and PEI (pDNA/PEI) in human hepatoma cell line HepG2 without cytotoxicity and agglutination. After intravenous injection of pDNA/PEI/GL into mice, the highest gene expression was observed in the liver. pDNA/PEI/GL showed significantly higher gene expression in parenchymal cells than in nonparenchymal cells. On the basis of these results, we evaluated the pharmacological activity of the ternary complex including the pDNA encoding insulin (pCMV-Ins). The pCMVIns/PEI/GL decreased blood glucose concentrations 24 h after its intravenous administration to mice. The ternary complex of pDNA, PEI, and GL may be a promising liver-targeted gene vector. KEYWORDS: DNA, self-assembly, drug delivery, surface modification, glycyrrhizin

1. INTRODUCTION The liver, the largest solid organ in the body, has a wide range of functions, including detoxification, protein synthesis, and the production of biogenic substances necessary for digestion. Liver disease is a broad term that includes many fatal diseases, such as chronic hepatitis, liver cirrhosis, fibrosis, and liver failure.1,2 The definitive treatment for chronic liver disease at present is a liver transplant. However, the liver is an exceptional organ because of its unlimited regenerative capacity, and many liver disorders that are either inherited (e.g., hemophilia and hypercholesterolemia) or acquired (e.g., hepatitis and cancer) ultimately will require gene therapy for cure.3 The liver is also anticipated to be able to produce the necessary proteins by gene therapy to cure enzyme-deficiency diseases and type I diabetes. Furthermore, particulate complexes can reach hepatocytes because liver fenestra is relatively large. Therefore, the liver is considered to be a potential target for gene therapy. Glycyrrhizin (GL) is one of the main compounds extracted from the root of Glycyrrhiza glabra (licorice). In Japan, GL has been used as a drug for allergic inflammation since 1948 and for chronic hepatitis since 1979; Stronger Neo-Minophagen C, an injectable solution including GL as the main element, has been widely used for the treatment of chronic active hepatitis.4−8 It has been proven that there are specific binding sites of GL on the cellular membrane of in vitro rat hepatocytes.9,10 Following intravenous administration, GL is rapidly cleared from the circulation by saturable uptake into the rat liver.11 These results © 2014 American Chemical Society

imply that GL may be able to be used as a novel ligand for hepatocyte-specific gene delivery. In fact, some new drug carriers surface modified with glycyrrhizin (such as liposomes and albumin nanoparticles) were prepared and proved to be more efficient on hepatocyte-targeted delivery compared with conventional carriers.12−14 We hypothesized that GL is able to coat pDNA/cationic polymer complexes electrostatically without covalent binding because of its anionic character, and the coated complexes will be taken up by cells via the GL-mediated pathway. This pharmaceutical modification would have several benefits, such as easy manufacturing, simple application to various types of cationic gene delivery vectors, improving targeting efficiency, and decreased toxicity by neutralizing cationic charges. We therefore developed a ternary complex of pDNA, polyethylenimine (PEI), and anionic GL by electrostatic interaction. The ternary complex was stable and showed high gene expression in the human hepatoma cell line HepG2. After intravenous administration of the ternary complex to mice, high gene expression was observed, specifically in the liver. Furthermore, the ternary complex including pDNA encoding Received: Revised: Accepted: Published: 1369

July 11, 2013 March 13, 2014 March 27, 2014 March 27, 2014 dx.doi.org/10.1021/mp400398f | Mol. Pharmaceutics 2014, 11, 1369−1377

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bromophenol blue) and loaded onto a 0.8% agarose gel. Two micrograms of heparin sulfate was added to pDNA/PEI to assess the stability of the complex. Electrophoresis (i-Mupid J; Cosmo Bio, Tokyo, Japan) was carried out at 50 V in running buffer solution (40 mM Tris/HCl, 40 mM acetic acid, and 1 mM EDTA). The retardation of DNA was visualized with ethidium bromide staining using a FluorChem Imaging System (Alpha Innotech, San Leandro, CA, USA). 2.5. In Vitro Gene Expression Experiment. The human hepatoma cell line HepG2 was obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer, Tohoku University, Japan. HepG2 cells were maintained in DMEM supplemented with 10% FBS and antibiotics (culture medium) in a humidified atmosphere of 5% CO2 in air at 37 °C. The cells were plated on 24-well collagencontaining plates (Becton-Dickinson, Franklin Lakes, NL, USA) at a density of 2.5 × 104 cells/well and cultivated in 500 μL of culture medium. In the transfection experiment for complexes, after 24 h of preincubation, the medium was replaced with 500 μL of Opti-MEM I medium or culture medium, and each complex containing 1 μg of pCMV-Luc was added to the cells and incubated for 2 h. After transfection, the medium was replaced with culture medium, and cells were cultured for a further 22 h at 37 °C. After 22 h of incubation, the cells were washed with PBS and then lysed in 100 μL of lysis buffer (pH 7.8 and 0.1 M Tris/HCl buffer containing 0.05% Triton X-100 and 2 mM EDTA). Ten microliters of lysate samples were mixed with 50 μL of luciferase assay buffer (Picagene, Toyo Ink, Tokyo, Japan), and the light produced was immediately measured using a luminometer (Lumat LB 9507; EG & G Berthold, Bad Wildbad, Germany). The protein content of the lysate was determined by a Bradford assay using BSA as a standard. Absorbance was measured using a microplate reader (Multiskan Spectrum; Thermo Fisher Scientific Inc., Waltham, MA, USA) at 595 nm. Luciferase activity was recorded as relative light units (RLU) per mg protein. To visualize the uptake of the complexes and gene expressions, HepG2 cells were transfected by each complex constructed with pEGFP-C1, Rh-PEI, and GL as described above. After 22 h of incubation, the relative levels of Rh-PEI and GFP expression were characterized using fluorescence microscopy (200× magnification). 2.6. Inhibition Study. For the inhibition study, the cells were transfected as described above with pDNA/PEI or pDNA/PEI/GL in transfection medium containing various concentrations of GL. After transfection, the medium was replaced with culture medium, cells were cultured for a further 22 h at 37 °C, and then the luciferase activities were determined. Also, for the determination of the endocytotic pathway, after 23 h of preincubation, the cells were treated with 0.014 mM chlorpromazine (CPZ) as an inhibitor of clathrin-mediated endocytosis, 0.2 mM genistein as an inhibitor of caveolaemediated endocytosis, or 1 mM amiloride as an inhibitor of macropinocytosis for 1 h. After treatment, the pDNA/PEI/GL complexes were added to the medium containing each inhibitor and incubated for 2 h. After 2 h of transfection, the medium was replaced with culture medium, cells were cultured for a further 22 h at 37 °C, and then the luciferase activities were determined. 2.7. WST-1 Assay. Cytotoxicity tests of complexes on HepG2 cells were carried out using a WST-1 commercially

insulin was able to decrease blood glucose concentrations after its intravenous administration to mice.

2. MATERIALS AND METHODS 2.1. Chemicals. PEI (branched form, average molecular weight (MW) of 25,000) was purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Glycyrrhizic acid dipotassium salt (MW 899.12) was obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Fetal bovine serum (FBS) was purchased from Cosmo Bio (Tokyo, Japan). Bovine serum albumin (BSA, minimum 98%, lyophilized powder and low endotoxin) was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), antibiotics (penicillin 100 U/mL; streptomycin 100 μg/mL), and other culture reagents were obtained from Invitrogen Corp. (Carlsbad, CA, USA). WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt) and 1-methoxy PMS (1-methoxy-5-methylphenazinium methylsulfate) were obtained from Dojindo Laboratories (Kumamoto, Japan). Rhodamine-PEI (Rh-PEI) was prepared in our laboratory as described in our previous report.15 Also, PEI was labeled with XenoLight CF750 (CF750-PEI) using a Fluorescent Rapid Antibody Labeling Kit (Caliper Life Science, Hopkinton, MA, USA). All other chemicals were of reagent grade. 2.2. Construction of pDNA. pCMV-Luc was constructed by subcloning the Hind III/Xba I firefly luciferase cDNA fragment from the pGL3-control vector (Promega, Madison, WI, USA) into the polylinker of the pcDNA3 vector (Invitrogen). Enhanced green fluorescent protein (GFP) encoding the plasmid (pEGFP-C1) and pCMV6-XL5 plasmid containing human insulin cDNA (pCMV-Ins) were purchased from Clontech (Palo Alto, CA, USA) and OriGene Technologies (Rockville, MD, USA), respectively. The pDNA was amplified using an EndoFree Plasmid Giga Kit (Qiagen GmbH, Hilden, Germany). The pDNA was dissolved in 5% dextrose solution and stored at −80 °C until use. 2.3. Preparation of Complexes. The theoretical charge ratio of pDNA/PEI was calculated as the molar ratio of PEI nitrogen to pDNA phosphate. An appropriate amount of stock PEI solution (1 mg/mL, pH 7.4) was mixed with a diluted solution of pDNA (1 mg/mL) by pipetting thoroughly to prepare pDNA/PEI at a charge ratio of 8. To prepare the complex containing GL, the theoretical charge ratio of GL to pDNA was calculated as the molar ratio of GL carboxylate to pDNA phosphate. GL glucose solution (20 mg/mL) was added to pDNA/PEI at charge ratios of 4, 8, 12, 16, and 20 (pDNA/ PEI/GL4, 8, 12, 16, and 20, respectively). The total volume of the complex solution containing 10 μg of pDNA was 100 μL, for example, in the case of pDNA/PEI/GL12, 10 μL of pDNA solution (1 mg/mL), 10 μL of PEI solution (1 mg/mL), 16 μL of GL solution (20 mg/mL), and 64 μL of 5% dextrose solution were mixed. 2.4. Physicochemical Properties of Complexes. The particle size and ζ-potential of complexes were measured with Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, United Kingdom). The number-fractioned mean diameter and Zaverage diameter are shown. Transmission electron microscopy (JEM-1230; JEOL, Tokyo, Japan) was used to observe the configuration of pDNA/PEI/GL12. To evaluate the formative ability of the complex, 10 μL aliquots of complex solution containing 1 μg of pDNA were mixed with 2 μL of loading buffer (30% glycerol and 0.2% 1370

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Table 1. Particle Size and ζ-Potential of the Complexesa particle size (nm)

a

complexes

number-fraction

Z-average

ζ-potential (mV)

pDNA/PEI pDNA/PEI/GL4 pDNA/PEI/GL8 pDNA/PEI/GL12 pDNA/PEI/GL16 pDNA/PEI/GL20

60.7 ± 15.1 1319 ± 65.7** 135.2 ± 14.1 100.4 ± 0.5 95.5 ± 27.8 97.7 ± 14.0

217.4 ± 12.0 2352 ± 206** 316.7 ± 19.1 293.7 ± 7.9 217.6 ± 1.8 212.7 ± 2.6

+52.2 ± 1.4 −1.3 ± 0.1** −11.6 ± 0.5** −21.2 ± 0.3** −26.6 ± 0.8** −30.0 ± 0.1**

Each data represents the mean ± SD (n = 3). **: P < 0.01 vs pDNA/PEI.

2.11. In Vitro Gene Expression Using Primary Culture of Hepatocytes. Primary parenchymal cells and nonparenchymal cells were isolated from male ddY mice as reported previously.16,17 The liver was perfused with perfusion buffer (Ca2+ and Mg2+-free HEPES solution, pH 7.2) for 10 min and then with HEPES solution containing 5 mM CaCl2 and 0.05% (w/v) collagenase (type I) (pH 7.5) for 10 min. After the liver was excised, the cells were dispersed in ice-cold DMEM by gentle stirring and filtered through cotton mesh sieves. The dispersed cells were centrifuged and washed three times at 50g for 1 min to separate the pellets containing parenchymal cells and the supernatant containing nonparenchymal cells. The supernatant was centrifuged at 200g for 2 min to yield pellets containing nonparenchymal cells. Parenchymal and nonparenchymal cells were resuspended separately in ice-cold DMEM. The cell numbers and viability were determined by the trypan blue exclusion method. The cells were cultured on 24-well collagen-coated plates with DMEM containing FBS, 10−8 M insulin, and 10−6 M dexamethasone at a density of 2.5 × 104 (parenchymal cell) and 5.0 × 105 (nonparenchymal cells). The in vitro transfection experiment using primary cultured cells was carried out according to the method described above. 2.12. Liver Toxicological Experiments. The ddY mice were injected intravenously with pDNA/PEI/GL12. At 6 h after injection, the mice were sacrificed, and blood samples were obtained. The activities of aspartate transaminase (AST) and alanine transaminase (ALT) in the serum were determined with biochemical test kits (Wako Pure Chemical Industries, Ltd., Osaka, Japan). 2.13. Pharmacological Experiment. pCMV-Ins was used to evaluate the pharmacological effect by gene expression in the liver instead of model pDNA. Male ddY mice were fasted for 24 h and were assigned randomly to one of five groups: control, naked pCMV-Ins group, pCMV-Ins/PEI group, pCMV-Luc/ PEI/GL12 group, and pCMV-Ins/PEI/GL12 group. Glucose concentrations were measured in blood 24 h after intravenous administration of 5% glucose solution, naked pCMV-Ins, pCMV-Ins/PEI, pCMV-Luc/PEI/GL12 group, and pCMVIns/PEI/GL12. Blood glucose was determined with a blood glucose test kit (glucose CII-test; Wako). 2.14. Statistical Analysis. Statistical significance between two groups was identified by Student’s t test. Multiple comparisons among groups were made by Dunnett’s pairwise multiple comparison t test. P < 0.05 indicated significance.

available cell proliferation reagent. The stock solution of WST1 (5.5 mM) and 1-methoxy PMS (2 mM) was prepared in sterilized PBS, and a mixture was prepared by mixing 4.5 mL of WST-1 solution and 0.5 mL of 1-methoxy PMS solution just before the experiments. HepG2 cells were plated at 1.0 × 104 cells/well in 96-well collagen-coated plates (Becton-Dickinson, Franklin Lakes, NJ, USA). The complexes containing 1 μg of pDNA in 100 μL of Opti-MEM I medium or culture medium were added to each well and incubated for 2 h. After incubation, the medium was replaced with 100 μL of culture medium and incubated for another 22 h. The medium was replaced with 100 μL of culture medium, and 10 μL of WST-1 mixture solution (4.95 mM WST-1 and 0.2 mM 1-methoxy PMS) was added to each well. The cells were incubated for 2 h at 37 °C, and absorbance was measured at a wavelength of 450 nm with a reference wavelength of 630 nm, using a microplate reader. The results are shown as a percentage of untreated cells (control). 2.8. Agglutination Experiment. Erythrocytes from mice were washed three times at 4 °C by centrifugation at 5000 rpm (Kubota 3700; Kubota, Tokyo, Japan) for 5 min and resuspended in PBS. A 2% (v/v) stock suspension of erythrocytes was prepared for the agglutination study. The complexes were added to the erythrocyte suspension and incubated for 15 min at room temperature. A 10 μL sample was placed on a glass plate, and agglutination was observed by microscopy (400× magnification). 2.9. Animals. Animal care and experimental procedures were performed in accordance with the Guidelines for Animal Experimentation of Nagasaki University with approval from the Institutional Animal Care and Use Committee. Male ddY mice (5−6 weeks old) were purchased from Japan SLC (Shizuoka, Japan). After shipping, mice were acclimatized to the environment for at least 1 day before the experiments. 2.10. In Vivo Gene Expression and Tissue Disutribution Experiment. Naked pDNA, pDNA/PEI, and pDNA/ PEI/GL12 including 40 μg of pDNA were prepared for in vivo gene expression experiments. A 300 μL sample of complexes was injected into mice via the tail vein. The liver, kidney, spleen, heart, and lungs of the mice were dissected 6 h after injection. The tissues were homogenized in lysis buffer and centrifuged at 15,000 rpm for 5 min, and the supernatants were used for luciferase assays. Luciferase activity is indicated as RLU per tissue. To visualize the distribution of the complexes, mice were intravenously injected by each complex constructed with pDNA, CF750-PEI, and GL as described above. The liver, kidney, spleen, heart, and lungs of the mice were dissected 6 h after injection, and the relative levels of CF750-PEI were monitored using an in vivo imaging system (IVIS imaging system, IVIS Lumina 2, Caliper Life Sciences, Cheshire, UK).

3. RESULTS 3.1. Physicochemical Properties of Complex. In the preliminary experiment, the complex of pDNA and PEI at a charge ratio 1:8 of the phosphate of pDNA nitrogen to PEI 1371

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agarose gel. Bands of naked pDNA were not detected in pDNA/PEI or any pDNA/PEI/GL complexes, indicating the formation of stable complexes; however, the addition of heparin sulfate to pDNA/PEI was detected as naked pDNA bands. Furthermore, the bands of pDNA were not detected after the incubation of pDNA/PEI or pDNA/PEI/GL12 in the serum for 2 h (Figure 2b). 3.2. In Vitro Gene Expression Experiment. Each complex was incubated with HepG2 cells for 2 h, and luciferase activity in the cells was determined with a luminometer. The in vitro gene expressions of the complexes at various charge ratios of GL are shown in Figure 3. pDNA/PEI showed extremely high gene expression, although no gene expression was observed in naked pDNA. After transfection in transfection medium, pDNA/PEI/GL4 and pDNA/PEI/GL8 showed lower gene expressions than pDNA/PEI. pDNA/PEI/GL complexes above charge ratio 12, however, showed high gene expressions comparable to that of pDNA/PEI (Figure 3a). However, after transfection in culture medium, pDNA/PEI and pDNA/PEI/ GL showed lower luciferase activity. pDNA/PEI/GL12 showed a high gene expression compared to that of pDNA/PEI (Figure 3b). HepG2 cells were transfected with pDNA/PEI and pDNA/ PEI/GL12 containing Rh-PEI and pEFGP-C1 to visualize the uptake of complexes and gene expression (Figure 4). In pDNA/PEI and pDNA/PEI/GL12, the red dots of Rh-PEI and bright green fluorescence of GFP were highly observed in most cells, although naked pDNA showed no red dots or green fluorescence. 3.3. Inhibition Study. Inhibition studies were performed with various inhibitory agents. The gene expression of pDNA/ PEI/GL12 was assessed in medium containing 0.1 mM or 1 mM GL (Figure 5A). One millimole of GL significantly inhibited the transgene efficiency of pDNA/PEI/GL12. However, GL did not influence the transgene efficiency of pDNA/PEI (Figure 5B). Figure 4C shows the influence of endocytotic inhibitors on the transgene efficiency of pDNA/ PEI/GL12. The inhibition of caveolae-mediated endocytosis with genistein significantly decreased the transgene efficiency of pDNA/PEI/GL12 (P < 0.01), which was lower than 20%. However, chlorpromazine and amiloride had little effect on the transgene efficiency of pDNA/PEI/GL12. 3.4. Cytotoxicity and Erythrocyte Agglutination. pDNA/PEI and pDNA/PEI/GL12 were added to HepG2 cells, and cell viability was evaluated by the WST-1 assay to

(pDNA/PEI) showed the highest gene expression in HepG2 cells. GL was added to pDNA/PEI at various charge ratios of its carboxylate. The particle size and ζ-potential of the complexes were measured and are shown in Table 1. The pDNA/PEI showed 60.7 ± 15.1 nm number-fractioned mean particle size and 52.2 ± 1.4 mV ζ-potential. The pDNA/PEI/GL4 increased the particle size (1319 ± 65.7 nm) with an approximately neutral surface charge (−1.3 ± 0.1). Further addition of GL to pDNA/PEI decreased its ζ-potential and particle size, reaching a plateau at a charge ratio of 12. pDNA/PEI/GL12 was observed as clumped nanoparticles in the TEM (Figure 1).

Figure 1. Transmission electron microscopy (TEM) image of pDNA/ PEI/GL12.

The gel retardation assay was performed to examine complex formations (Figure 2a). Naked pDNA was detected as bands on

Figure 2. Gel retardation assay of each complex without serum (a) or with serum (b). (a) Naked pDNA (A), pDNA/PEI (B), pDNA/PEI/ GL4 (C), pDNA/PEI/GL8 (D), pDNA/PEI/GL12 (E), pDNA/PEI/ GL16 (F), pDNA/PEI/GL20 (G), and pDNA/PEI/heparin20 (H). Retardation of pDNA was visualized using ethidium bromide.

Figure 3. In vitro gene expression of each complex. HepG2 cells were transfected with each complex in transfection medium (a) or culture medium (b). Luciferase activity was determined 24 h after transfection. Each bar represents the mean ± SE (n = 3). *, P < 0.05; **, P < 0.01 vs pDNA/PEI. 1372

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PEI (P < 0.05). However, the distribution of pDNA/PEI to the liver was comparable to that of pDNA/PEI/GL12 (Figure 7B). Primary parenchymal and nonparenchymal cells were separately isolated from perfused livers of male ddY mice. An in vitro transfection experiment using primary cultured cells was carried out to examine the distribution of pDNA/PEI/GL12 (Figure 7C). The gene expression of parenchymal cells of the complex was significantly higher than that of nonparenchymal cells (P < 0.01). 3.6. Liver Toxicity of pDNA/PEI/GL12. pDNA/PEI/ GL12 was administrated to mice, and the in vivo liver toxicity was evaluated. pDNA/PEI/GL12 did not markedly increase AST and ALT, as shown in Figure 8A and B. 3.7. Pharmacological Experiment. To confirm the utility of pDNA/PEI/GL12 for in vivo gene therapy, pDNA/PEI/ GL12 containing pCMV-Ins were administered to mice, and their pharmacological activities were evaluated. Naked pCMVIns, pCMV-Ins/PEI, pCMV-luc/PEI/GL12, and pCMV-Ins/ PEI/GL12 were injected intravenously into mice, and glucose concentrations were measured in the serum after 24 h (Figure 9). Naked pCMV-Ins, pCMV-Ins/PEI, and pCMV-luc/PEI/ GL12 showed no significant decrease of glucose concentration compared with that of the control group. However, a significantly lower blood glucose concentration was observed in pCMV-Ins/PEI/GL12-treated mice (P < 0.01).

Figure 4. Phase contrast image (a) and fluorescence microscopy images (b and c) of HepG2 cells transfected with each complex. Cells were transfected with each complex containing pEGFP-C1 and RhPEI. Twenty-four hours after transfection, the uptake of Rh-PEI (b) and the expression of GFP (c) were monitored (200× magnification).

determine cytotoxicity (Figure 6A,B). For the transfection medium, pDNA/PEI showed significantly higher cytotoxicity than the control (P < 0.05). However, no cytotoxicity was observed in pDNA/PEI/GL12. Also, pDNA/PEI showed high cytotoxicity compared to that of pDNA/PEI/GL12 in the culture medium. To evaluate agglutination, pDNA/PEI and pDNA/PEI/GL12 were added to mouse erythrocytes (Figure 6C). pDNA/PEI showed severe agglutination on microscopy, but no agglutination was observed with pDNA/PEI/GL12. 3.5. Gene Expression Experiment in Mice and Hepatic Primary Culture Cells. In vivo gene expression of pDNA/PEI and pDNA/PEI/GL12 was examined in male ddY mice (Figure 7A). No gene expression was observed in any tissues after intravenous administration of naked pDNA. However, 6 h after intravenous administration of pDNA/PEI and pDNA/PEI/ GL12, luciferase activities were evaluated in several tissues. High gene expressions were observed in the liver, spleen, and lung after the administration of pDNA/PEI. In contrast, pDNA/PEI/GL12 showed significantly higher gene expression in the liver and lower gene expression in the lung than pDNA/

4. DISCUSSION GL is one of the main compounds extracted from the root of Glycyrrhiza glabra (licorice). Licorice root has been used orally as a sweetener and as a traditional medicine mainly for the treatment of peptic ulcers, hepatitis C, pulmonary, and skin diseases.18−22 Clinical and experimental studies suggest that it has several other useful pharmacological properties, such as anti-inflammatory, antiviral, antimicrobial, antioxidative, anticancer, hepatoprotective, and cardioprotective effects.23 We successfully constructed a stable complex with a negative charge surface by combining pDNA, PEI, and GL. Several advantages of PEI in the process of gene transfection have been reported;24,25 it combines with pDNA by electrostatic interaction, binding to the cell surface, and is taken up by the endocytotic pathway and releases pDNA into the cytoplasm via the so-called “proton-sponge mechanism.”26−28 We therefore selected pDNA/PEI as a reference from several commercial vectors, such as cationic phospholipids and polymers. The

Figure 5. Influence of GL on the transgene efficiency of pDNA/PEI/GL12 (A) and pDNA/PEI (B). Influence of endocytotic inhibitors on the transgene efficiency of pDNA/PEI/GL12 (C). (A) pDNA/PEI/GL12 was transfected in a medium containing various concentrations of GL. (B) pDNA/PEI2 was transfected in a medium containing 1 mM GL. (C) pDNA/PEI/GL12 was transfected in a medium with various endocytotic inhibitors. After 22 h of transfection, luciferase activity was evaluated. Each bar is the mean ± SE of three or six experiments. **: P < 0.01 vs control. 1373

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Figure 6. Cytotoxicity tests of each complex on HepG2 cells in transfection medium (A) or culture medium (B) and interaction of each complex with erythrocytes (C). (A) Cells were incubated with each complex for 2 h in transfection medium, and cell viability was measured by WST-1 assay at 24 h after treatment. (B) Cells were incubated with each complex for 2 h in culture medium, and cell viability was measured by WST-1 assay 24 h after treatment. Data are the percentage of untreated cells. Each bar represents the mean ± SE (n = 8). *: P < 0.05 vs the control. (C) PBS (a), pDNA/PEI (b), and pDNA/PEI/GL12 (c) were added to erythrocytes, and agglutination was assessed. Agglutination was observed by phase microscopy (400× magnification).

Figure 7. In vivo gene expression (A) and tissue distribution (B) of each complex and in vitro gene expression of pDNA/PEI/GL12 on hepatic primary culture cells (C). (A) pDNA/PEI (white bar) and pDNA/PEI/GL12 (gray bar) were injected intravenously into the mice (40 μg of pDNA per mouse). At 6 h after administration, mice were sacrificed, and each organ was dissected to quantify luciferase activity. Each bar represents the mean ± SE (n = 6). *: P < 0.05 vs pDNA/PEI. (B) pDNA/PEI and pDNA/PEI/GL12 were injected intravenously into the mice (40 μg pDNA per mouse). At 6 h after administration, mice were sacrificed, and each organ was dissected to observe the fluorescence. (C) Parenchymal and nonparenchymal cells were transfected with the complex. Luciferase activity was determined 24 h after transfection. Each bar represents the mean ± SE (n = 3). **: P < 0.01.

addition of GL decreased the ζ-potential, which reached a plateau at a charge ratio of 12 (Table 1). This result suggests the concentrated distribution of GL outside of the particles. The pDNA/PEI/GL complex above a charge ratio of 12 was approximately 100 nm. Also, we observed the complexes with TEM, as shown in Figure 1. Furthermore, the size and ζpotential of pDNA/PEI/GL12 were not significantly changed in serum (number-fraction; 175.1 ± 9.2 nm, Z-average; 215.4 ± 14.0 nm, ζ-potential; −20.8 ± 1.6 mV). The size may be sufficiently small to pass through the endothelial fenestrae in

the sinusoids, which are reported to be 150−175 nm in diameter.29 Naked pDNA was detected as a band on agarose gel, while bands of pDNA were not detected in pDNA/PEI, as shown in Figure 2. Anionic agents such as heparin sulfate are known to destroy the structure of the complex. In fact, the addition of heparin sulfate dissociated pDNA/PEI, and the band of pDNA was observed. pDNA/PEI/GL, however, did not release pDNA after electrophoresis. Also, the bands of pDNA were not detected after incubation of pDNA/PEI/GL in the serum for 2 1374

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observed even if it had an anionic charge. These results indicated that pDNA/PEI/GL12 can release DNA so that it can be transported into the nucleus, although the detailed mechanism is unclear. For clarification of the uptake mechanism, pDNA/PEI/GL12 was added to HepG2 cells in medium containing various concentrations of GL. GL concentration-dependently inhibited the transgene efficiency of pDNA/PEI/GL12 (Figure 5A). However, GL did not influence the transgene efficiency of pDNA/PEI (Figure 5B). This result suggested that anionic pDNA/PEI/GL12 was specifically taken up by the GLmediated pathway and showed high transgene efficiency, regardless of the anionic surface charge. We also performed an inhibition study with various endocytotic inhibitors, such as chlorpromazine for clathrinmediated endocytosis, genistein for caveolae-mediated endocytosis, and amiloride for macropinocytosis. The transgene efficiency of pDNA/PEI/GL12 was significantly inhibited by genistein (P < 0.01), as shown in Figure 5C. These results indicated that pDNA/PEI/GL12 was taken up by the caveolaemediated endocytotic pathway with the interposition of GL. However, pDNA/PEI complexes could be internalized by cells via both clathrin- and caveolae-mediated pathways, depending on the particle size, as shown in a previous report.33 When the particles are smaller than 200 nm, the complexes are mainly internalized by the clathrin pathway, which is not consistent with our data. The present results apparently showed a relationship with the caveolae-mediated endocytotic pathway. The dominant pathway may be influenced by coating materials, particle size distribution, cell lines, and cellular growth stage. Further experiments are necessary to reveal the detailed uptake pathway. To confirm the suitability of pDNA/PEI/GL12 for clinical use, cytotoxicity, agglutination, and in vivo transgene expression were evaluated. As cationic complexes have high cytotoxicity and cause agglutination of erythrocytes after intravenous injection, they are difficult to use in vivo. However, anionic complexes are expected to be safe because of their negative ζpotential. pDNA/PEI/GL12 showed no cytotoxicity and no agglutination, although significant cytotoxicity and severely agglutinated erythrocytes were observed with pDNA/PEI (Figure 6A−C). These results indicate that pDNA/PEI/GL12 as a vector is safer and more suitable for clinical use than pDNA/PEI. However, PEI had high cytotoxicity and was not biodegradable, so it could not be used in clinical practice. Further study is needed to develop a complex using biodegradable safe materials such as cationic polypeptides. We evaluated the in vivo gene expression of pDNA/PEI/ GL12 in mice (Figure 7A). High gene expressions were observed in the liver, spleen, and lung with pDNA/PEI and pDNA/PEI/GL12. Notably, pDNA/PEI/GL12 showed the highest gene expression in the liver. Ishida et al. reported that GL is rapidly cleared from the circulation by saturable uptake into the rat liver after intravenous administration.11 Negishi et al. investigated the tissue distribution of [3H] GL binding activity in rats.9 They found that GL binding activity was mainly located in the rat liver. In fact, pDNA/PEI/GL12 was mainly distributed to the liver (Figure 7B). We also determined the gene expression of pDNA/PEI/GL12 in primary parenchymal and nonparenchymal cells (Figure 7C). pDNA/PEI/GL12 showed significantly higher gene expression in parenchymal cells than in nonparenchymal cells (P < 0.01). This result

Figure 8. AST (a) and ALT (b) values of mice injected with pDNA/ PEI/GL12. The mice were administrated with each complex. Six hours after administration, blood was collected, and serum AST and ALT values were measured. Each value is the mean ± SE (n = 3).

Figure 9. Blood glucose concentration 24 h after administration of the complexes to mice. Naked pCMV-Ins, pCMV-Ins/PEI, pCMV-luc/ PEI/GL12, and pCMV-Ins/PEI/GL12 were injected intravenously into the mice (40 μg pDNA per mouse). After 24 h from administration, blood glucose concentrations were determined. Each bar is the mean ± SE (n = 4−5). **: P < 0.01 vs the control.

h (Figure 2b). These results support that pDNA/PEI/GL are stable as self-assembled nanoparticles without decomplexation, even in blood. The basic in vitro gene expression of these stable complexes was determined in HepG2 cells (Figure 3). Generally, anionic complexes cannot be taken up by cells because they electrostatically repulse the cellular membrane. However, surprisingly, pDNA/PEI/GL showed a gene expression as high as that of pDNA/PEI, despite its negative charge. Ismair et al. found that a carrier-mediated transport system participates in the uptake of GL into rat and human hepatocytes.30 The high gene expression of pDNA/PEI/GL can be explained by its specific uptake through GL into HepG2 cells. It has been proven that there are specific binding sites of GL on the cellular membrane of in vitro rat hepatocytes.9,10 However, it was reported that a very significant amount of PEI remains free when a mixing N:P ratio of 8 is used.31,32 pDNA/PEI/GL12 may include a few of the PEI/GL complex. The PEI/GL complex can inhibit the gene expression of pDNA. The lower gene expression of pDNA/PEI/GL20 may be explained by the presence of the PEI/GL complex (Figure 3). The high uptake and gene expression of pDNA/PEI/GL12 were confirmed by the observation with the complexes loading Rh-PEI and pEGFP-C1 by fluorescence microscopy (Figure 4). Cationic pDNA/PEI showed high uptake (red) or gene expression (green). However, pDNA/PEI/GL12 was also highly taken up by the cells, and high gene expression was 1375

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suggests that pDNA/PEI/GL12 is useful for gene delivery to parenchymal cells in the liver. In previous studies, we confirmed that pDNA/PEI markedly increased AST and ALT and caused liver necrosis.34 At the same time, pDNA/PEI/GL did not cause liver toxicity, such as increase of AST and ALT. These results suggested that pDNA/ PEI/GL12 could deliver the pDNA to the liver effectively and safely. The liver is a major target for gene delivery because it can produce the necessary proteins by transfection and cure enzyme-deficiency diseases and type I diabetes. We confirmed the pharmacological activity of pDNA/PEI/GL12 as a gene delivery system to hepatocytes using pCMV-Ins, which can produce insulin in cells (Figure 9). The naked pCMV-Ins, pCMV-Ins/PEI, and pCMV-Luc/PEI/GL12 groups showed no significant decrease of the glucose concentration 24 h after intravenous injection into mice compared with the control group. However, a significantly lower blood glucose concentration was observed in the pCMV-Ins/PEI/GL12 group (P < 0.05). These results indicated that pDNA/PEI/GL12 was taken up well and certainly showed insulin gene expression, followed by a decrease in blood glucose by the secretion of insulin, although further study is necessary to elucidate the detailed mechanisms. Maeda et al. also found that the mouse insulin 2 gene conjugated with tetra-(piperazino)-fullerene epoxide decreased blood glucose levels after intravenous injection.35 They found high gene expression in the liver by this gene delivery system and the same as with the pDNA/PEI/GL12based gene delivery system. The results of this study demonstrated the advantage of a complex containing GL-based transfection systems in terms of toxicity and possible therapeutic application. No cytotoxicity and hematotoxicity were found with the GL complex. In the in vivo study, the complex containing GL specifically showed high gene expressions in the liver. We also successfully decreased blood glucose concentration using pDNA encoding insulin/ PEI/GL complexes. This complex should prove useful for gene therapy, although further experiments, such as an in vivo detailed toxicity profile, are necessary before clinical use.

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5. CONCLUSIONS We developed pDNA/PEI/GL as a promising nonviral vector. GL can electrostatically coat pDNA/PEI to form stable anionic particles. The GL coating markedly decreased the toxicity of pDNA/PEI; furthermore, pDNA/PEI/GL showed high gene expressions in the liver, especially in parenchymal cells after intravenous administration.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-95-819-7245. Fax: +81-95-819-7251. E-mail: sasaki@ nagasaki-u.ac.jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. 1376

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