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 neverless 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 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. Batimastat The recombinant … 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.