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 http://www.selleckchem.com/products/Gefitinib.html 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 ��l) containing 2 ��g of pMMP13 in naked form or complexes were added with 2 unit of DNase I (Invitrogen, Carlsbad, CA), and incubated at 37 ��C Dacomitinib 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.