Deposition transfection technology using a DNA complex with a thermoresponsive cationic star polymer.
A novel non-viral gene transfection method in which DNA complexes were kept in contact with a deposition surface (deposition transfection) was developed. We designed a novel aqueous thermoresponsive adsorbent material for DNA deposition, which was a star-shaped copolymer with 4-branched chains. Each chain is comprised of a cationic poly(N,N-dimethylaminopropyl acrylamide) (PDMAPAAm) block (Mn: ca. 3000 g x mol(-1)), which formed an inner domain for DNA binding and a thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) block (Mn: ca. 6000 g x mol(-1)), which formed an outer domain for surface adsorption. Complex formation between the copolymer and the luciferase-encoding plasmid DNA occurred immediately upon simple mixing in an aqueous medium; polyplexes approximately 100 nm in size were formed. Because the lower critical solution temperature of the polyplexes was approximately 35 degrees C, they could deposit on the substrate by precipitation from an aqueous solution upon warming, which was confirmed by quartz crystal microbalance (QCM) method and water contact angle measurement. When COS-1 cells were cultured on the polyplex-deposited substrate in a culture medium, the luciferase activity observed was higher than that observed on a DNA-coated substrate with or without the cationic polymer before and after complete adhesion and by conventional solution transfection using the polyplexes. The activity was enhanced with an increase in the charge ratio (surfactant/pDNA) with permissible cellular cytotoxicity.
Zhou
Yue-Min
YM
Department of Bioengineering, Advanced Medical Engineering Center, National Cardiovascular Center Research Institute, Japan.
Ishikawa
Ayaka
A
Okahashi
Ryohei
R
Uchida
Kingo
K
Nemoto
Yasushi
Y
Nakayama
Mitsuko
M
Nakayama
Yasuhide
Y
D000178:Acrylamides / Q000138:chemical synthesis* / Q000633:toxicity; D000180:Acrylic Resins / Q000138:chemical synthesis* / Q000633:toxicity; D000818:Animals; D019556:COS Cells; D002412:Cations; D002470:Cell Survival / Q000187:drug effects; D011232:Chemical Precipitation; D002522:Chlorocebus aethiops; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D017930:Genes, Reporter; D008156:Luciferases / Q000378:metabolism; D015394:Molecular Structure; D010316:Particle Size; D013696:Temperature; D014162:Transfection / Q000379:methods*
D000178:Acrylamides; D000180:Acrylic Resins; D002412:Cations; C493602:N,N-dimethylaminopropylacrylamide; C052970:poly-N-isopropylacrylamide; D004247:DNA; D008156:Luciferases