Ternary Complex of Plasmid DNA Electrostatically Assembled with Polyamidoamine Dendrimer and Chondroitin Sulfate for Effective and Secure Gene Delivery

Abstract

The purpose of this study was to develop a ternary complex of plasmid DNA (pDNA) electrostatically assembled with polyamidoamine (PAMAM) dendrimer and chondroitin sulfate (CS) for effective and secure gene delivery. PAMAM dendrimers are new cationic polymers that are expected to be used as gene delivery vectors. However, cationic non-viral gene vectors showed cytotoxicity by binding to negative cellular membranes. We therefore prepared a ternary complex by adding CS, an anionic polymer, and examined its usefulness. The pDNA/PAMAM dendrimer complex (PAMAM dendriplex) and the PAMAM dendriplex coated by CS (CS complex) showed nanoparticles with positive ζ-potential and negative ζ-potential, respectively. The CS complex had no cytotoxicity against B16-F10 cells and no agglutination activity, although severe cytotoxicity and high agglutination were observed in the PAMAM dendriplex. As a result of an in vitro gene expression study of B16-F10 cells, not only the PAMAM dendriplex but also the CS complex showed high transfection efficiency. The transfection efficiency of the CS complex was significantly inhibited by clathrinmediated endocytosis inhibitor (chlorpromazine), caveolae-mediated endocytosis inhibitor (genistein), and hypothermia. Tail-vein injection of the CS complex into mice led to significantly higher gene expression in the spleen than the PAMAM dendriplex. Thus, the ternary complex of pDNA electrostatically assembled with PAMAM denriplex and CS showed safe high gene expression in the spleen. This vector is expected to be useful for useful gene delivery.Key words ternary complex; chondroitin sulfate; polyamidoamine dendrimer; gene delivery Gene therapy is expected to be an effective method to treat cancer, infection, innate immunodeficiency and cardiovascular diseases.1-4) The success of gene therapy is largely dependent on the development of vectors capable of effectively delivering foreign genes into targeted cells. One major approach in gene therapy is based on cationic polymers, such as polyethylenimine (PEI), polylysine, polyarginine, and chitosan.5-8) When cationic polymer-encapsulated plasmid DNA (pDNA) makes association with the cell surface, it enters the cells by endocytosis.Polyamidoamine (PAMAM) dendrimers are new cationic polymers, which are highly branched radial polymers that have specific and systematically variable size, shape and chemical structure. Their radical structure contains a 2-carbon ethylenediamine core and primary amino groups on the surface. Successive generations (G) have increasing diameter and double the surface functional amino groups of the preceding generation.9,10) PAMAM dendrimers can be used as carriers for pDNA and show high transfection efficiency. 11,12)The pDNA/PAMAM dendrimer complexes (PAMAM dendriplex) are formed by electrostatic interactions and initiate cell entry through binding to anionic phospholipids on the cell membrane. With increasing generations of dendrimers, the PAMAM dendriplex showed higher transfection efficiency, which depended on the charge rati...

Progress in the development of Lipoplex and Polyplex modified with Anionic Polymer for efficient Gene Delivery

“…Iwanaga et al also reported that intravenous administration of ternary complexes of pDNA/ dendrigraft poly-L-lysine/CS ( Figure 5A) induced high gene expression levels in the reticuloendothelial system [63]. Furthermore, Imamura et al reported that a ternary complex of pDNA electrostatically assembled with PAMAM dendrimer and CS ( Figure 5A) was an effective and secure gene vector, and they led to signi icantly higher gene expression in the spleen than pDNA/PAMAM dendrimer polyplexes [64]. Therefore, CS coating of polyplexes may be expected to be useful for gene vectors to the spleen, and they may be a promising approach for DNA vaccination.…”

Cationic Polymers for the Delivery of Therapeutic Nucleotides

“…They also claim comparable or superior transfection efficacies with fluorinated G3, G4, and G5 PPI dendrimers as compared with Lipofectamine 2000, branched poly(ethyleneimine), SuperFect ® , PolyFect ® , as well as argininemodified dendrimer on both HEK293 and HeLa cells (Liu et al 2014a). The ternary complex of plasmid DNA polyamidoamine dendrimer and chondroitin sulfate results in no agglutination activity and no cytotoxicity against B16-F10 cells (Imamura et al 2014). PEGylation is the well-known method to improve biocompatibility and transfection efficiency (Sun et al 2014).…”

Characterization of a DNA Aptamer for Ovarian Cancer Clinical Tissue Recognition and in Vivo Imaging

“…To determine the pathway of R13 internalization, we preincubated cells with 80 μmol dynasore (inhibitor of clathrin-mediated endocytosis) [23], 50 μmol genistein (inhibitor of caveolae-mediated endocytosis) [24], or 50 μmol amiloride (inhibitor of macropinocytosis) for 30 min at 37°C [25]. Then, the cells were incubated with aptamers for 40 min at 37°C.…”