Enhanced transfection efficiency of poly(N,N-dimethylaminoethyl methacrylate)-based deposition transfection by combination with tris(hydroxymethyl)aminomethane.
We have developed a substrate-mediated transfection method called "deposition transfection technology" using a poly(N,N-dimethylaminoethylmethacrylate) (PDMAEMA) homopolymer with both thermoresponsive and cationic characteristics. In this study, we enhanced deposition transfection efficiency by using tris(hydroxymethyl)aminomethane (Tris buffer) as a pH adjuster for transfection solution composed of PDMAEMA and plasmid DNA (pDNA). PDMAEMA with a molecular weight of 9.7 × 10(4) g mol(-1) was synthesized by photoinduced radical polymerization. The pH of PDMAEMA solution was increased gradually in the range from 8 to 11 by the addition of Tris, and then the solubility of PDMAEMA was significantly decreased and the dissolution time was extended from 15 to 40 min at Tris/PDMAEMA ratio of 1 and higher. On the other hand, while the polyion complexes (polyplexes) were formed by mixing PDMAEMA with luciferase-encoding plasmid DNA even under an excess amount of Tris at Tris/PDMAEMA ratio of 8, the binding affinity between PDMAEMA and pDNA was decreased with increasing Tris at Tris/PDMAEMA ratio of 2 and higher. When HeLa cells, smooth muscle cells, and cardiac fibroblasts were transfected by the deposition method using polyplex solution containing various amounts of Tris, the transgene expression dramatically increased at a Tris/PDMAEMA ratio of 2 in all cell types, which were more than 150-fold in HeLa cells, 40-fold in smooth muscle cells, and 30-fold in cardiac fibroblasts compared to those in the Tris-free condition. In addition, the enhanced transgene expression by Tris was sustained for over 10 days post-transfection as well as that observed in Tris-free condition. Thus, deposition transfection efficiency can be dramatically enhanced by using Tris buffer as a pH adjuster for polyplex solution.
Division of Medical Engineering and Materials, National Cerebral and Cardiovascular Center Research Institute, Japan.
Ryosuke
R
Iwai
Ryota
R
Haruki
Yasushi
Y
Nemoto
Yasuhide
Y
Nakayama
D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D004247:DNA / Q000008:administration & dosage*; D004285:Dogs; D006367:HeLa Cells; D006801:Humans; D008689:Methacrylates / Q000737:chemistry / Q000378:metabolism*; D009757:Nylons / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids / Q000008:administration & dosage*; D014162:Transfection / Q000379:methods*; D014325:Tromethamine / Q000737:chemistry / Q000378:metabolism*
D008689:Methacrylates; D009757:Nylons; C407037:poly(2-(dimethylamino)ethyl methacrylate); D014325:Tromethamine; D004247:DNA