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. 2010 Dec 7;19(2):355–361. doi: 10.1038/mt.2010.262

Antifibrotic Effect of MMP13-encoding Plasmid DNA Delivered Using Polyethylenimine Shielded With Hyaluronic Acid

Eun-Joong Kim 1,2, Hee-Jeong Cho 2, Daeui Park 3, Ji Yeon Kim 3, Young Bong Kim 4, Tae Gwan Park 5, Chang-Koo Shim 1, Yu-Kyoung Oh 2
PMCID: PMC3034855  PMID: 21139571

Abstract

The imbalanced expression of matrix metalloproteinases (MMPs) is associated with liver fibrosis, one of the most common chronic liver diseases. Enhanced expression of MMPs by gene therapy is emerging as a promising antifibrotic strategy, but the effectiveness of this approach depends on reliable systems for delivering MMP genes. Here, we evaluated a newly designed hyaluronic acid (HA)-shielded delivery system for systemic administration of plasmid DNA encoding MMP13 (pMMP13), and tested whether the enhanced expression of MMP13 ameliorates liver fibrosis in mice. In the CCl4-induced liver fibrosis model, systemic administration of pMMP13 using HA and polyethylenimine (PEI) significantly increased the expression of MMP13 and reduced collagen deposition. Moreover, following delivery of pMMP13 in a HA-shielded PEI complex, the serum levels of aspartate transaminase were reduced to levels approaching those in untreated normal mice. These results indicate that the delivery of pMMP13 using HA-shielded PEI enhances the efficiency of MMP13 expression in the liver, and highlight the potential of pMMP13 gene therapy as an antifibrotic strategy.

Introduction

Liver fibrosis is characterized by the excess accumulation and alteration of extracellular matrix (ECM) molecules, including collagen, in the tissue. Liver fibrosis can progress to liver cirrhosis, liver failure, and portal hypertension.1 Moreover, fibrosis may accelerate experimental hepatocarcinogenesis.2 Given the clinical significance of liver fibrosis and advances in our understanding of the molecular mechanisms underlying liver fibrosis, it is not surprising that antifibrotic therapies have been widely investigated as a treatment strategy.3 However, these approaches have achieved only limited success,4 and there are currently no drugs approved for antifibrotic purposes in humans.5 Thus, there is a strong unmet need for effective antifibrotic therapies.

One of the molecular targets of antifibrotic approaches is matrix metalloproteinase 13 (MMP13). In liver tissue, various MMPs are known to play pivotal roles in fibrolysis.6 MMP13 (collagenase 3), in particular, plays a crucial role in the cleavage and remodeling of ECM components,7 and its expression levels are decreased after induction of liver fibrosis.8 These previous findings indicate that increased expression of MMPs might prevent the deposition of fibrillar collagens and promote the resolution of fibrosis. Indeed, increased MMP expression or activity has been suggested as a promising strategy for the treatment of hepatic fibrosis.9,10

One strategy for enhancing the expression of MMPs is gene therapy. MMP1 gene therapy has been reported to attenuate liver fibrosis in rats,10,11 and MMP8 gene therapy has been shown to ameliorate liver cirrhosis in an experimental rat model.12 In both of these studies, adenoviral vectors were used to deliver genes encoding MMPs to the liver tissues. However, the complexity of production, limited packaging capacity, lack of targeting ability, and safety concerns, such as mutagenesis and immunogenicity, crucially limit the utility of viral gene delivery systems for clinical applications.13 Therefore, nonviral delivery systems that are safe, convenient, and capable of highly efficient liver-targeted delivery of genetic cargo may be needed to effectively treat liver fibrosis using a gene therapy approach.

Cationic polymer-based nonviral delivery systems provide a number of advantages over viral vectors, including simplicity of production, the ability to package plasmids of any size, and structural modifiability, which allows in vivo distribution to be modulated by introducing targeting moieties.14 Among cationic polymers, polyethylenimine (PEI) has been most widely used as a gene delivery system owing to its relatively high transfection efficiencies,15 and prolonged gene expression.16 Despite these advantages, PEI has limited liver-targeting ability and a less-than-ideal cytotoxicity profile.17

Hyaluronic acid (HA), a natural biocompatible polymer, has been shown to enhance liver distribution and protect against the cytotoxicity of carriers. Several studies have reported high expression levels of the HA receptor for endocytosis in liver tissue, indicating that HA may facilitate receptor-mediated endocytosis in the liver.18,19,20 Moreover, most endogenous HA is generally transported and metabolized in liver cells, suggesting that the HA component might confer liver-targeting potential on carrier systems.21,22

In this study, we hypothesized that enhanced expression of MMP13 in liver tissue might ameliorate liver fibrosis. To test this hypothesis, we developed a system for delivering an MMP13 expression plasmid using PEI shielded with HA. Here, we report that HA shielding significantly increased the viability of transfected cells in vitro, and show that systemic administration of an MMP13-encoding gene using PEI shielded with HA greatly increased the expression of MMP13 in liver tissue and reduced collagen I deposition in an experimental mouse model of liver fibrosis.

Results

Optimization of HA/PEI/pMMP13 complexes

The MMP13 complementary DNA was inserted into pIRES2-EGFP vector containing an internal ribosome entry site (IRES) and enriched green fluorescent protein (EGFP) sequences (Figure 1a). The resulting recombinant MMP13-encoding pIRES2-EGFP was named as plasmid DNA encoding MMP13 (pMMP13). Since IRES permits the translation of both MMP13 and EGFP from a single mRNA transcript, EGFP can be used as a surrogate marker of exogenously administered MMP13 gene. In this study, pIRES2-EGFP vector without MMP13 complementary DNA (pVector) was used as an expression vector control.

Figure 1.

Figure 1

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Schematic representation for the construction of pMMP13 and formation of HA-shielded PEI and plasmid DNA ternary complex for gene delivery. (a) MMP13 cDNA was inserted into the BglII/SalI sites of the pIRES2-EGFP expression vector encoding EGFP. The recombinant plasmid DNA encoding both MMP13 and EGFP was abbreviated as pMMP13. (b) Plasmid DNA was electrostatically complexed to cationic PEI, resulting in binary complexes. The binary complexes with surplus cationic charges were then coated with negatively charged HA, providing ternary complexes. cDNA, complementary DNA; EGFP, enriched green fluorescent protein; HA, hyaluronic acid; IRES, internal ribosome entry site; PEI, polyethylenimine; pMMP13, plasmid DNA encoding matrix metalloproteinases 13.

To enhance the organ and cell level delivery efficiency, negatively charged pMMP13 was electrostatically complexed with cationic PEI and shielded with HA (HA/PEI/pMMP13 complexes) (Figure 1b). The formation of HA/PEI/pMMP13 complexes was physicochemically confirmed by measuring the change in zeta-potentials, which were different for HA/PEI/pMMP13 ternary complexes with different HA and PEI composition. At a HA:PEI molar ratio of 0:1, the zeta-potential value of binary complexes was 23.1 ± 4.4 mV (Figure 2a). As the HA:PEI molar ratio increased, the zeta-potential values decreased due to the added negative charges of HA. At a HA:PEI molar ratio in the ternary complexes of 0.1:1, the zeta-potential was −10.9 ± 1.5 mV.

Figure 2.

Figure 2

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Zeta-potential and cell viability of HA/PEI/pMMP13 complexes. (a) Zeta-potentials of HA/PEI/pMMP13 were evaluated by varying the ratios of HA to PEI. (b) Following treatment of Hepa 1-6 cells with HA/PEI/pMMP13, the cytotoxicity was measured by an MTT assay. *Significantly higher than other groups (P < 0.05, ANOVA and Student–Newman–Keuls test). HA, hyaluronic acid; PEI, polyethylenimine; pMMP13, plasmid DNA encoding matrix metalloproteinases 13.

After confirming complex formation of the various HA/PEI/pMMP13 formulations, we determined the optimal composition of HA by assessing the viability of cells treated with each of the complexes. HA content significantly affected cell viability. As the amount of HA relative to PEI increased, cell viability significantly increased, reaching a maximum at a HA:PEI molar ratio of 0.1:1 (Figure 2b). This molar ratio of 0.1:1 was selected as the optimal ratio, and was used in all subsequent experiments.

Stability of pMMP13 in PEI or HA/PEI complexes

Complexation with PEI or HA/PEI protected pMMP13 against DNase I and enhanced the stability in the blood (Figure 3). Naked pMMP13 completely degraded within 0.5 hours after incubation in DNase I (Figure 3a). Unlike naked form, pMMP13 complexed to PEI or HA/PEI did not degrade until 24 hours of incubation. Similarly, the stability of pMMP13 in the blood increased following intravenous administration in PEI or HA/PEI complexes (Figure 3b). The blood levels of pMMP13 were expressed as plasmid copy numbers per 100 ng of genomic DNA. As compared to naked pMMP13, PEI/pMMP13, and HA/PEI/pMMP13 showed 84- and 22-fold higher levels of pMMP13 plasmid copy number in the blood, respectively.

Figure 3.

Figure 3

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Stability of pMMP13 in vitro and in vivo. (a) Naked pMMP13, PEI/pMMP13, or HA/PEI/pMMP13 was incubated with DNase I. The samples were collected at various time points. pMMP13 was extracted from the samples and loaded onto a 1% agarose gel. (b) Mice were injected intravenously with pMMP13 at 1 mg/kg dose in naked form or complexes. At 4 hours after injection, DNA was isolated from blood and pMMP13 copy numbers were determined by quantitative RT-PCR. The data are the mean ± SE (n = 5). *Significantly higher than naked pMMP13 (P < 0.05, ANOVA and Student–Newman–Keuls test). ANOVA, analysis of variance; HA, hyaluronic acid; PEI, polyethylenimine; pMMP13, plasmid DNA encoding matrix metalloproteinases 13.

Liver distribution of pMMP13 in PEI or HA/PEI complexes

In vivo administration of pMMP13 in HA/PEI complexes significantly increased the liver distribution in comparison with pMMP13/PEI complexes. As an indicator of preferential liver distribution, the ratios of pMMP13 plasmid copy numbers in the liver to those in the blood were compared (Figure 4). The relative liver-to-blood distribution ratio of pMMP13 was 18.2 ± 12.0 for PEI/pMMP group. Notably, the liver-to-blood ratio of pMMP13 was 875.5 ± 248.5 for the group treated with HA/PEI/pMMP, 48-fold higher than the value observed in PEI/pMMP group.

Figure 4.

Figure 4

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Liver distribution of pMMP13 in PEI or HA/PEI complexes. Mice were intravenously injected with pMMP13 (1 mg/kg) in PEI complexes or HA-shielded PEI complexes. At 4 hours after injection, gDNA was extracted from blood and liver tissues. pMMP13 copy numbers in the liver were divided by those in the blood. The data are the mean ± SE (n = 5). gDNA, genomic DNA; HA, hyaluronic acid; PEI, polyethylenimine; pMMP13, plasmid DNA encoding matrix metalloproteinases 13.

Effect of HA shielding on the in vivo toxicity of PEI

In line with the in vitro cytotoxicity data (Figure 2b), HA shielding significantly reduced the in vivo toxicity of PEI (Figure 5). When mice were intravenously administered with various doses of pMMP13 in PEI or HA/PEI complexes, the tolerable doses differed between the two carriers. Although the administration of pMMP13 at a dose of 1.0 mg/kg provided 100% survival regardless of the carriers, the escalation of pMMP13 dose to 1.5 mg/kg using PEI alone revealed 0% survival of mice. In contrast, the administration of pMMP13 in HA/PEI complexes provided 100% survival at the same dose, and did not show lethality even at 3.0 mg/kg dose. For 1 month after injection, no change in behaviors had been observed in the mice treated with HA/PEI/pMMP13.

Figure 5.

Figure 5

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Survival rates of mice after intravenous administration of pMMP13 in PEI or HA/PEI complexes. Mice in each group (n = 5) were intravenously injected with pMMP13 in PEI or HA-shielded PEI complexes. The behavior changes of survived mice were checked for 1-month postdose. HA, hyaluronic acid; PEI, polyethylenimine; pMMP13, plasmid DNA encoding matrix metalloproteinases 13.

Enhanced expression of MMP13 in liver tissue following administration of pMMP13 in HA/PEI complexes

The systemic administration of pMMP13 in HA/PEI complexes increased MMP13 mRNA and protein expression in liver tissue in the CCl4-induced murine liver fibrosis model. As depicted in Figure 6a, liver fibrosis in mice was established by intraperitoneal injections of CCl4, and MMP13 mRNA and protein levels were determined in liver tissue after intravenous of HA/PEI complexes containing pMMP13 or pVector. Intravenous injection of pMMP3 in HA/PEI complexes increased MMP13 mRNA in liver tissue to a level 25-fold greater than that observed in mice injected with complexes containing pVector (Figure 6b). There were no significant differences among untreated normal mice, CCl4-treated fibrotic mice injected with saline and CCl4-treated fibrotic mice administered pVector in HA/PEI complexes.

Figure 6.

Figure 6

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Levels of MMP13 mRNA in liver tissues of fibrotic mice. (a) For induction of fibrosis, mice were intraperitoneally injected with CCl4 and intravenously treated with HA/PEI/pVector or HA/PEI/pMMP13 according to the dosing schedule. The mRNA levels of MMP13 were measured from total RNA isolated from the liver tissue from normal mice, untreated fibrotic mice, or fibrotic mice treated with HA/PEI/pVector or HA/PEI/pMMP13. (b) The expression levels of MMP13 mRNA were normalized to those of GAPDH. The data are the mean ± SE (n = 4). HA, hyaluronic acid; PEI, polyethylenimine; pMMP13, plasmid DNA encoding matrix metalloproteinases 13.

In liver tissue sections, pMMP13 protein expression was indirectly assessed by monitoring EGFP being translated simultaneously from same pMMP13 vector with IRES sequences. To differentiate MMP13 expression derived from exogenously administered pMMP13 with that from endogenous MMP13 mRNA transcripts, we used a surrogate marker, EGFP. For that reason, we constructed pMMP13 by inserting DNA sequence encoding MMP13 into pIRES2-EGFP vector (Figure 1a). The in vivo expression of exogenously administered EGFP may indicate that MMP13 mRNA transcripts were produced upon intracellular delivery of pMMP13. pMMP13 protein expression was also directly observed by immunoblotting of MMP13 in liver homogenates. There was no expression of EGFP in liver tissue sections from untreated normal mice (Figure 7b). In contrast, EGFP expression was clearly evident in liver tissues of mice intravenously injected with pMMP13 in HA/PEI complexes (Figure 8d). Moreover, western-blot analyses revealed expression of MMP13 protein in the group administered HA/PEI/pMMP13, but not in other groups (Figure 7e).

Figure 7.

Figure 7

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Expression of MMP13 proteins in liver tissues of fibrotic mice. Expression of EGFP and MMP13 proteins in liver tissues was visualized by fluorescent microscopy and immunoblotting, respectively. (a,c) Representative phase-contrast and (b,d) fluorescence images were shown for tissues from (a,b) untreated liver or (c,d) pMMP13-treated liver. Bar = 100 µm. The expression of MMP13 proteins in liver tissues was analyzed by (e) immunoblotting. EGFP, enriched green fluorescent protein; HA, hyaluronic acid; PEI, polyethylenimine; pMMP13, plasmid DNA encoding matrix metalloproteinases 13.

Figure 8.

Figure 8

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Hepatic collagen deposition and serum AST after pMMP13 gene therapy. Collagen deposition was tested for the (a) liver tissues from normal mice, (b) fibrotic mice treated with saline, (c) HA/PEI/pVector, or (d) HA/PEI/pMMP13. Bar = 400 mm. The extent of collagen deposition was visualized by staining with picrosirius red. (e) The activity of AST was tested for serum samples collected from each group. *P < 0.05 as compared with the mice treated with HA/PEI/pVector (ANOVA and Student–Newman–Keuls test); **P > 0.05 as compared with untreated normal mice. ANOVA, analysis of variance; AST, aspartate aminotransferase; HA, hyaluronic acid; PEI, polyethylenimine; pMMP13, plasmid DNA encoding matrix metalloproteinases 13.

Antifibrotic effect of pMMP13 administered in HA/PEI complexes

Intravenous administration of pMMP13 in HA/PEI complexes ameliorated liver fibrosis in mice. Sirius red staining of liver sections showed the presence of collagen deposition in the liver tissues of CCl4-treated mice administered saline (Figure 8b). Collagen was deposited to a similar extent in liver tissues of CCl4-treated mice administered pVector in HA/PEI complexes (Figure 8c). In contrast, injections of pMMP13/HA/PEI complexes significantly decreased the deposition of collagen in CCl4-treated mice (Figure 8d), reducing collagen to levels similar to those in untreated normal mouse livers (Figure 8a).

The administration of pMMP13 in HA/PEI complexes also promoted recovery from liver damage in the liver fibrosis model, significantly decreasing the plasma levels of aspartate aminotransferase (AST) compared with those in animals administered pVector in HA/PEI complexes. Consistent with the collagen-deposition histology data, there was no significant difference in serum AST levels between the untreated normal group and the liver fibrosis group treated with pMMP13 in HA/PEI complexes (Figure 8e).

Discussion

In this study, we demonstrated the antifibrotic effects of pMMP13 administered in HA-shielded complexes in a murine liver fibrosis model. Treatment of mice with liver fibrosis by administering HA/PEI/pMMP13 increased MMP13 mRNA and protein levels in liver tissue. Moreover, HA/PEI/pMMP13 treatment reduced collagen I deposition in liver tissue and restored plasma AST levels to values similar to those in untreated normal mice.

The shielding of PEI/pMMP13 with HA provided protection against PEI cytotoxicity. The mechanism underlying this cytoprotective effect remains to be elucidated. However, Moghimi et al.23 reported that PEI cytotoxicity was associated with activation of caspase-2 and changes in the mitochondrial membrane potential. Thus, it is possible that neutralization of the cationic charges of PEI by HA reduces the mitochondrial damage caused by PEI. Moreover, in vivo, the shielding of PEI/pMMP13 with HA may prevent the interaction of PEI with plasma proteins of the circulatory system. Indeed, the use of PEGylated PEI for complexation with plasmid DNA was shown to reduce the interaction with blood components, such as immunoglobulin M, fibrinogen and complement C3, and decrease the aggregation of erythrocytes compared with unpegylated PEI.24

The form of pMMP13 delivered to the liver may be speculated from the in vivo distribution patterns between PEI/pMMP13 and HA/PEI/pMMP13. If pMMP13 is liberated from the complexes in the blood stream, and delivered to the liver in naked form, there would be no significant differences in liver distribution patterns between PEI/pMMP13 and HA/PEI/pMMP13. Indeed, we observed that there was a striking difference in the liver-to-blood ratios between PEI/pMMP13 and HA/PEI/pMMP13 groups (Figure 4). The observation implies that pMMP13 may be delivered to the liver in PEI or HA/PEI complexes, rather than liberated form.

We observed that HA/PEI/pMMP13 induced a notable increase in the levels of MMP13 mRNA in liver tissue following systemic administration. It is unlikely that this increase is due to induction of endogenous MMP13 by HA/PEI alone because administration of the HA/PEI/pVector did not significantly increase the mRNA levels of MMP13 in liver tissue compared with those in the untreated control group. The increase in MMP13 expression in liver tissue might be explained in two ways. First, high-affinity binding of the HA shielding to HA receptor for endocytosis may increase the distribution of pMMP to liver tissue. Consistent with this, a recent report has shown that HA receptor for endocytosis is highly expressed in sinusoidal endothelial cells of the liver.25 Second, HA receptor for endocytosis are known to be involved in the cellular uptake of HA by the clathrin-coated pit pathway.26 The delivery of pMMP13 in HA-shielded PEI complexes may thus promote the intracellular uptake of the complex by receptor-mediated endocytosis. Our previous demonstration that HA/poly-ℓ-arginine delivers small interfering RNA more efficiently to cells that express HA receptors at a high density is consistent with this interpretation.20

The systemic gene therapy of pMMP13 using HA/PEI complexes ameliorated the collagen depositions in liver tissue. MMPs are known to play an important role in regulating ECM homeostasis, and have been studied as a potential tool for remodeling hepatic ECM and thereby inhibiting the progression of fibrogenesis. Among the various MMPs, MMP13 (collagenase 3) has been shown to degrade intact collagen and to participate in the remodeling of collagenous ECM.27 Moreover, MMP13 was shown to be involved in the degradation of newly formed matrix during the recovery from liver fibrosis.8 Thus, the reduction in collagen deposition induced by pMMP13 likely reflects increased MMP13 protein expression following delivery using HA/PEI complexes. Support for this interpretation is provided by recent adenoviral vector-mediated gene therapy experiments, which showed that an adenovirus carrying plasminogen activator inhibitor-1 shRNA facilitated matrix degradation in liver fibrosis through upregulation of MMP13.28

Although the present study specifically demonstrated the antifibrotic effects of HA/PEI-complexed pMMP13, HA/PEI systems might also be applied to deliver plasmid DNAs encoding other antifibrotic proteins that have shown promise in treating liver fibrosis. For example, MMP 1 and MMP 8 genes11,12 were reported to ameliorate liver cirrhosis in an experimental rat model, and overexpression of the cytoglobin gene29 was shown to promote recovery from fibrogenesis. Recently, human hepatocyte growth factor gene expression was reported to be effective for treatment of renal fibrosis.30 In these studies, viral vectors such as adenovirus and adeno-associated virus were used as delivery systems. This study describes an alternative to viral delivery methods, showing that nonviral HA/PEI-based delivery systems can be applied to deliver plasmid DNAs for gene therapy of liver fibrosis.

In conclusion, our results indicate that pMMP13 can be developed as an effective gene therapy target for the treatment of liver fibrosis. Moreover, effective systemic delivery of pMMP13 via HA/PEI ternary complexes should potentiate the therapeutic efficacy of pMMP13 against liver fibrosis and provide a safe alternative to viral-mediated gene transfer methods.

Materials and Methods

Construction of MMP13 expression plasmids. A recombinant plasmid that expresses MMP13 under the control of a cytomegalovirus promoter, pMMP13, was constructed by subcloning full-length MMP13 complementary DNA from the murine hepatoma cell line Hepa1-6 into the BglII/SalI site of the pIRES2-EGFP expression vector encoding EGFP (Clontech, Mountain View, CA). All plasmids were purified using Plasmid Midi Kits (Qiagen, Hilden, Germany).

Preparation and characterization of pMMP13 ternary complexes. Ternary complexes composed of pMMP13, branched PEI (MW 25 kd; Aldrich, Milwaukee, WI) and HA (MW 19 kd; LifeCore Biomedical, Chaska, MN) were prepared by initially forming the binary complex of branched PEI and pMMP13 at an N/P ratio of 10:1. The redundant positive charges on the binary complex were shielded by the addition of HA in various molar ratios. The formation of HA/PEI/pMMP13 ternary complexes was characterized by measuring zeta-potentials (ELS-8000; Otsuka, Osaka, Japan).

In vitro plasmid stability test. The stability of pMMP13 was tested using DNase I. pMMP13 was complexed to PEI at N/P ratio of 10:1. The redundant positive charges on the binary complex were shielded by the addition of HA at 0.1:1 molar ratio. Solution (20 µl) containing 2 µg of pMMP13 in naked form or complexes were added with 2 unit of DNase I (Invitrogen, Carlsbad, CA), and incubated at 37 °C for 24 hours. The samples were collected at various time points and mixed with 1 unit of heparin (USB/Amersham Life Science, Cleveland, OH). The samples were loaded onto a 1% agarose gel containing 0.2 mg/ml ethidium bromide. The plasmid bands were visualized under an UV transilluminator.

Quantification of pMMP13 by real-time PCR. The levels of pMMP13 plasmid DNA in blood and liver tissues were measured using quantitative real-time-PCR. Female balb/c mice were intravenously administered with 1 mg/kg of pMMP13 in various forms. Four hours after administration, the mice were sacrificed and genomic DNA was isolated from blood and liver tissue using DNeasy Blood & Tissue kit (Qiagen). Quantitative real-time PCR was performed using a LightCycler 480 (Roche, Basel, Switzerland) and SYBR green dye (Roche Diagnostics, Mannheim, Germany) under the following conditions: 45 cycles of 95 °C for 40 seconds (denaturation), 57 °C for 30 seconds (annealing), and 72 °C for 30 seconds (extension). The sequences of the primers used to amplify murine MMP13 were 5′-CCT TCT GGT CTT CTG GCA CAC-3′ (sense) and 5′-GGC TGG GTC ACA CTT CTC TGG -3′ (antisense). The primer sequences for murine glyceraldehyde-3-phosphate dehydrogenase were 5′-ATC ACC ATC TTC CAG GAG C- 3′ (sense) and 5′-AGA GGG GCC ATC CAC AGT CTT C-3′ (antisense). Plasmid copy numbers were calculated by standard curves of the samples containing known amounts of pMMP13.

Assessment of carrier toxicity. The toxicity of PEI and HA/PEI carriers complexes was measured in vitro and in vivo. For in vitro cytotoxicity study, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay was used. Hepa 1-6 cells were seeded onto a 48-well plate at a density of 2 × 104 cells/well in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen-Gibco, Paisley, UK) and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin). After treating for 48 hours with HA/PEI/pMMP13 ternary or PEI/pMMP13 binary complexes containing 2 µg of plasmid DNA, 20 µl of MTT (5 mg/ml) was added and cells were incubated for 3 hours. After adding a 0.04 N HCl/isopropanol solution, absorbance was measured at 570 nm. Cell viability was expressed relative to untreated control cells as a percentage. For in vivo toxicity tests, female balb/c mice received single intravenous injection with various doses of pMMP13 in PEI or HA-shielded PEI complexes. In each group, five mice were allocated. The survival and abnormal behaviors of mice were checked for 1 month postdose.

Murine in vivo liver fibrosis model. Liver fibrosis was induced in mice by injecting 1.2 ml/kg of 25% (vol/vol) CCl4 in corn oil into the peritoneal cavity four times at 3-day intervals. In parallel, mice were injected intravenously with saline, or 1.5 mg/kg plasmid DNA (pMMP13 or pVector) in HA-shielded PEI complexes three times at 4-day intervals. After 18 days, the mice were sacrificed and examined.

Evaluation of MMP13 expression in liver tissue by fluorescence microscopy and immunoblotting. The levels of MMP13 protein in liver tissue were evaluated using fluorescence microscopy and immunoblotting. Liver tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline for 24 hours and stored overnight in phosphate-buffered saline containing 30% sucrose. Liver tissues were then frozen in OCT compound (Tissue-Tek; Sakura Finetek, Torrance, CA) and cryosectioned (10-µm thick sections) using a cryomicrotome (Leica CM3050 Cryostat; Leica, Wetzlar, Germany). Samples mounted on gelatin-coated slides were visualized under a fluorescence microscope (Leica). For immunoblotting of MMP13, frozen liver tissues were homogenized in ice-cold lysis buffer (10 mmol/l Tris–Cl, 150 mmol/l NaCl, 1 mmol/l ethylenediaminetetraacetic acid, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, pH 8.0). After resolving proteins by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferring to membranes, blots were probed with rabbit monoclonal anti-MMP13 (1:500 dilution; Abcam, Cambridge, MA) or β-actin antibodies (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), and immunoreactive proteins were visualized using a goat anti-rabbit alkaline phosphatase-conjugated secondary antibody.

Histological analysis of liver tissue. Type I collagen content in liver tissue was evaluated using picrosirius red staining. Extracted liver tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-µm sections. Sections were then deparaffinized, hydrated, and stained with picrosirius red as described by the manufacturer (Sigma, St Louis, MO). The images of stained liver specimens were captured by light microscopy.

Measurement of liver damage. The serum levels of AST were measured as a reliable indicator of liver damage. Blood samples were obtained from normal control groups and various groups of pMMP13-treated mice before sacrificing. AST activities in serum were determined by a UV-kinetic method using commercial kits (YD Diagnostics, Yongin, Korea).

Statistics. Statistical analyses were performed using ANOVAs with post-hoc Student–Newman–Keuls tests. SigmaStat software (version 3.5; Systat Software, Richmond, CA) was used for all analyses, and a P value <0.05 was considered statistically significant.

Acknowledgments

This work was supported by the research grants from the Korean Health Technology R&D project (A090945), Ministry for Health, Welfare, and Family Affairs, and from National Research Laboratory (ROA-2006-000-10290-0), South Korea.

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