Temperature-responsive polymeric carriers incorporating hydrophobic monomers for effective transfection in small doses.
A series of thermoresponsive ternary random copolymers, poly[N-isopropylacrylamide (PIPAAm)-co-(dimethylamino)ethylmethacrylate (DMAEMA)-co-butylmethacrylate (BMA)], was synthesized and their in vitro gene transfection efficiency in cell culture was evaluated. A control copolymer containing 20 mol% DMAEMA units, IP-20D (mole ratio of IPAAm/DMAEMA/BMA=80/20/0 in feed, no BMA units) was inert in transfection. In contrast, copolymer IP-20D-10B (IPAAm/DMAEMA/BMA=70/20/10 in feed) effectively transfected plasmid DNA into COS-1 cell cultures even under small dosing conditions of 0.1 microg of plasmid DNA per well in a 96-well plate, suggesting that incorporation of the appropriate amount of hydrophobic unit is crucial to transfection efficiency. Gene expression was much more significant when transfected by the IP-20D-10B carrier in comparison with control homopolymer poly-DMAEMA, and almost equal to that of the highly competent lipid carrier, LipofectAMINE PLUS trade mark. Furthermore, the transfection efficiency of IP-20D-10B is altered in a thermally responsive manner. By temporarily lowering the cell culture incubation temperature to 20 degrees C in the posttransfection period, gene expression doubled over that for incubation temperature at 37 degrees C. The DNA EtBr intercalation assay suggested that DNA affinity for IP-20D-10B is decreased by lowering incubation temperature, implying that the thermally regulated gene expression could provide more efficient DNA release from the polymeric carrier.
Takeda
Naoya
N
Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku, Tokyo 162-8666, Japan.
Nakamura
Emiko
E
Yokoyama
Masayuki
M
Okano
Teruo
T
D000180:Acrylic Resins / Q000138:chemical synthesis; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002522:Chlorocebus aethiops; D004247:DNA / Q000008:administration & dosage / Q000235:genetics; D004337:Drug Carriers; D004996:Ethidium; D005456:Fluorescent Dyes; D015870:Gene Expression; D007364:Intercalating Agents / Q000737:chemistry; D008027:Light; D008970:Molecular Weight; D010316:Particle Size; D010957:Plasmids; D011721:Pyrenes / Q000737:chemistry; D012542:Scattering, Radiation; D013696:Temperature; D014162:Transfection / Q000379:methods*
D000180:Acrylic Resins; D004337:Drug Carriers; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D011721:Pyrenes; C492295:poly(N-isopropylacrylamide-co-(dimethylamino)ethylmethacrylate-co-butylmethacrylate); D004247:DNA; D004996:Ethidium