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.

Induction of cell self-organization on weakly positively charged surfaces prepared by the deposition of polyion complex nanoparticles of thermoresponsive, zwitterionic copolymers.

We have developed inducible cell self-organization through weakly positively charged culture surfaces. In this study, a thermoresponsive and zwitterionic copolymer comprised of N,N-dimethylaminoethyl methacrylate (DMAEMA) and methacrylic acid (MA) (PDMAEMA-co-PMA; Mn: ∼9.7 × 104 g/mol; PDMAEMA/PMA ratio: 10) was designed for inducing cell self-organization. The copolymer formed single polymer-derived polyion complex (sPIC) nanoparticles following dissolution in an aqueous solution. The sPIC nanoparticles had a positive charge (ca. 25 mV). Self-organization occurred in adipose-derived vascular stromal cell monolayers cultivated on sPIC-deposited surfaces. There were dramatic morphological changes of these cells with the formation of capillary-like networks and single-cell aggregates with little cytotoxicity. This was a significant improvement compared with cells grown on previously developed surfaces deposited with PIC, a mixture of PDMAEMA and plasmid DNA. Thus, sPICs of PDMAEMA-co-PMA may allow for the accurate evaluation of a variety of cell behaviors with less cytotoxicity, and may facilitate additional potential medical applications such as cell-based therapy and drug discovery. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1009-1015, 2017.