Gene expression control by temperature with thermo-responsive polymeric gene carriers

A thermo-responsive copolymer, poly(N-isopropylacrylamide (IPAAm)-co-2-(dimethylamino)ethyl methacrylate (DMAEMA)-co-butylmethacrylate (BMA)), was synthesized and its in vitro gene transfection efficiency at different incubation temperatures was evaluated. A copolymer containing 8 mol% DMAEMA and 11 mol% BMA (P(IP-8DA-11BM)) had a lower critical solution temperature (LCST) at 21 degreesC, therefore the copolymer was insoluble above 21 degreesC and soluble below 21 degreesC. The LCST of P(IP-8DA-11BM) solution was not affected by the presence of salmon DNA. This copolymer was complexed with plasmid DNA, and the stability of the complex was analyzed by gel electrophoresis. DNA was completely retained in the complex, which was observed in the gel loading slot at 37 degreesC. At 20 degreesC, DNA was found to be partially dissociated from the complex by the appearance of the same band as DNA in the control experiment. These results clearly show that complex formation/dissociation was modulated by temperature alteration. The transfection efficiency of polymer-plasmid complexes was evaluated in COS-1 cells using pCMV-lacZ plasmid, encoding for P-galactosidase as a reporter gene. The transfection efficiency of PDMAEMA homopolymer incubated at 37 degreesC for 48 h was greater than that incubated at 20 degreesC for 3 h and 37 degreesC for 45 h. In contrast, the transfection efficiency of P(IP-8DA-11BM) incubated at 20 degreesC for 3 h and 37 degreesC for 45 h was much higher than that incubated at 37 degreesC for 48 h. Such an increased transfection efficiency on lowering the temperature is considered to be due to appropriate formation/dissociation control of P(IP-8DA-11BM)-DNA complexes. (C) 2000 Elsevier Science B.V. All rights reserved.