The gene transfection efficiency of thermoresponsive N,N,N-trimethyl chitosan chloride-g-poly(N-isopropylacrylamide) copolymer.
A thermoresponsive copolymer, trimethyl chitosan-g-poly(N-isopropylacrylamide) (TMC-g-PNIPAAm), was synthesized by coupling PNIPAAm-COOH to TMC. Their molecular structures were characterized by 1HNMR. The lower critical solution temperature (LCST) of TMC-g-PNIPAAm in PBS was measured as 32 degrees C by dynamic light scattering (DLS) and UV-vis spectroscopy, regardless of the grafting ratios. Upon mixing with DNA, TMC/DNA particles were formed, whose size and morphology were investigated by DLS and transmission electron microscopy, respectively. The particle size ranged from 200 to 900 nm depending on the N/P ratio and was less influenced by the temperature variation. The majority of the particles have spherical morphology. The zeta potentials of these particles were increased along with the N/P ratio. At a given N/P ratio, the zeta potentials were almost constant at 25 degrees C regardless of the existence of serum proteins. However, the values were significantly decreased at 37 degrees C in a solution containing serum protein. The affinity between DNA and TMC was examined by ethidium bromide competitive binding assay. TMC-g-PNIPAAm has stronger ability to combine with DNA at 40 degrees C when the PNIPAAm chain is collapsed. Gel electrophoresis results reveal that the vectors/DNA complexes can be formed regardless of the incubation temperature. HEK293 cell line was chosen as a model to study cellular uptake of the TMC-g-PNIPAAm/DNA particles, gene transfection and cytotoxicity. The grafting of PNIPAAm will not affect cellular uptake of the particles at 37 degrees C. The level of gene transfection could be thermally controlled. By using a temperature variation protocol, i.e. incubation of the cultured cells at 25 degrees C for a while, the gene transfection efficiency was significantly improved. Finally, the optimized gene transfection efficiency achieved by TMC-g-PNIPAAm is comparable to Lipofectamine 2000. No obvious cytotoxicity was detected for the TMC-g-PNIPAAm/DNA particles. These results suggest that TMC-g-PNIPAAm is an effective thermoresponsive gene carrier with minimal cytotoxicity, which has great promise for practical applications.
Mao
Zhengwei
Z
Key Laboratory of Macromolecular Synthesis and Functionalization, Ministry of Education, and Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China.
Ma
Lie
L
Yan
Jiang
J
Yan
Ming
M
Gao
Changyou
C
Shen
Jiacong
J
D000178:Acrylamides / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D048271:Chitosan / Q000737:chemistry*; D004577:Electron Probe Microanalysis; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005562:Formazans / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D009682:Magnetic Resonance Spectroscopy; D008855:Microscopy, Electron, Scanning; D015394:Molecular Structure; D010957:Plasmids; D011108:Polymers / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D013778:Tetrazolium Salts / Q000378:metabolism; D014162:Transfection
D000178:Acrylamides; D005456:Fluorescent Dyes; D005562:Formazans; D011108:Polymers; D013778:Tetrazolium Salts; C079782:MTT formazan; D048271:Chitosan; C067295:N-isopropylacrylamide; D016650:Fluorescein-5-isothiocyanate