N-acetylglucosamine-conjugated block copolymer consisting of poly(ethylene oxide) and cationic polyaspartamide as a gene carrier for targeting vimentin-expressing cells

Abstract

Gene therapy is not successful due to lack of safe gene delivery vector, low transfection efficiency and inability to target the particular cells. Here, we synthesized a biocompatible block copolymer (abbreviated as PASPG) which consists of cationic poly[(aspartamide)(spermine)] for complexation with DNA and enhancing transfection efficiency due to buffering ability of spermine, poly(ethylene oxide)(PEO) for stability after systemic administration of the gene and N-acetylglucosamine (GlcNAc) as a specific ligand to target vimentin-expressing cells. Primarily, PASPG showed efficient complexation with DNA. Cell viability assay demonstrated that PASPG had low toxicity compared to polyethylenimine 25K. Furthermore, PASPG showed higher transfection efficiency in vimentin-expressing cells than vimentin-deficient ones due to the recognition of GlcNAc in the polymeric gene carrier by vimentin in the cells for the receptor-mediated endocytosis of PASPG. Favorably, the serum had no effect on transfection efficiency of PASPG due to the presence of hydrophilic PEO in the block copolymer. This study reveals that GlcNAc-coupled biocompatible block copolymer can specifically deliver gene to vimentin-expressing cells.

Introduction

Gene delivery with viral vectors has emerged as an important tool for the treatment of genetic deficiencies. However, clinical use of viral vectors encounters many problems including acute toxicity, immunogenicity, and limited cargo capacity. Therefore, non-viral vectors have received much attention for gene delivery because of their biosafety, high flexibility of size of delivered gene and easy production (Mao et al., 2010, Kim et al., 2007). However, the existing non-viral vectors are far less efficient owing to their low level of gene transfection (Anderson et al., 2003), high cytotoxicity (Lynn and Langer, 2000), inability to target the particular cells and low stability after systemic administration (Osada et al., 2009). Although several studies have put forward block copolymers as gene carriers to overcome the low stability after systemic administration of these non-viral vectors (Oba et al., 2011, Itaka et al., 2010, Han et al., 2009, Pittella et al., 2011), a specific ligand is required to the outer shell of the block copolymers for active targeted gene delivery.

Linear polyethylenimine (PEI), a polyamine consists of repeating aminoethylene units, is one of the most widely used polycations for gene transfection due to its DNA binding ability. Moreover, the protonation degree of linear PEI increases when pH decreases from the extracellular pH (∼7.4) to the endosomal pH (∼5.5) which facilitates the endosomal escape of the gene carrier during DNA transfection (Boussif et al., 1995). Although PEI exhibits a relatively high transfection efficiency, it induces considerable cytotoxicity inhibiting its use for clinical applications. To overcome this problem, a number of polyamines have been introduced into the side chain of the N-substituted polyaspartamides to obtain fine-tuned polycations achieving efficient gene transfection with reduced cytotoxicity (Uchida et al., 2011). Consequently, polycationic polymers with primary amine end (e.g. spermine and tetraethlyenepentamine) in their core-forming structures were more efficient for siRNA cellular uptake compared to polymers with tertiary amine end (N,N-dimethyldipropylenetriamine) (Xiong et al., 2009). Thus, fine-tuning of the repetitive amine units in the polycation side chain, may provide a new design concept for non-viral gene delivery systems directed toward clinical applications.

Kidney fibrosis, a progressive loss of kidney function, is a life-threatening disease which can be treated only by chronic dialysis or renal transplantation (Schievenbusch et al., 2010). The main executive cells in this process are the myofibroblasts generated from various sources such as resident fibroblasts, endothelial cells via endothelial-mesenchymal transition and renal tubular epithelial cells via epithelial-mesenchymal transition (EMT) (Zeisberg et al., 2008). Recently, vimentin was recognized as a marker for the EMT which played a significant role in organ fibrosis (Zeisberg and Kalluri, 2004). Besides, the cytoskeleton of the vimentin was dramatically altered by interaction with N-acetylglucosamine (GlcNAc), which is closely related with pathological events such as fibrosis and tumorigenesis (Komura et al., 2012).

Few attempts have been made to inhibit the kidney fibrosis by gene therapy using viral vectors as gene carriers (Tu et al., 2008, Zhao et al., 2012). Yang et al. delivered hepatocyte growth factor (HGF) gene via systemic administration into mice by hydrodynamic gene transfer technique to ameliorate kidney fibrosis (Yang et al., 2001). Similarly, Yuan et al. reported that N-acetylated low molecular weight chitosan specifically delivered prednisolone to the renal tubular cells in the kidney through receptor-ligand specific interaction (Yuan et al., 2009, Yuan et al., 2011).

Receptor-mediated endocytosis via receptor–ligand interaction is a promising approach of gene delivery. Vimentins at the cell surface are recently known to bind GlcNAc residue (Ise et al., 2010), therefore, the cell surfaces of various vimentin-expressing cells could be targeted by using this GlcNAc residue as a specific ligand for receptor-mediated delivery system. In fact, we have recently reported that GlcNAc-coupled PEI specifically interacts with vimentin-expressing cells such as 293T and HeLa cells for efficient transfection of green fluorescent protein and luciferase genes (Kim et al., 2011).

In this study, we prepared a block copolymer composed of cationic polyaspartamide for complexation with DNA and enhancing transfection efficiency due to buffering ability by spermine, poly (ethylene oxide) (PEO) for stability after systemic administration of the gene and GlcNAc as a specific ligand to target vimentin-expressing cells. We examined physicochemical properties, transfection efficiency, cytotoxicity and effect of serum on the transfection efficiency of the block copolymer/DNA complexes. Besides, the cell targetability of GlcNAc-conjugated block copolymer to vimentin-expressing kidney cells was evaluated.