In vitro transfection of HeLa cells with temperature sensitive polycationic copolymers

Abstract

In this study, we investigated different types of polyethyleneimine (PEI) and their block copolymers with N-isopropylacrylamide (NIPA) as temperature-sensitive polycationic non-viral vectors for transfection of HeLa cells in cell culture media. First carboxyl-terminated poly(NIPA) was synthesized and then copolymerized with PEIs branched or linear and with two different molecular weights (2 and 25 kDa). Addition of PEI units to the poly(NIPA) chains increased the LCST values up to body temperature. Zeta potentials of the copolymers were significantly lower than the corresponding PEI homopolymers. A green fluorescent protein expressing plasmid was used as a model. Complexes of this plasmid both with PEIs and their copolymers were formed. The zeta potentials of these complexes were between -3.1 and +21.3. Higher values were observed for the complexes prepared with branched and higher molecular weight PEIs. Copolymerization caused a profound decrease in the positive charges. Particle sizes of the complexes were in the range of 190-1235 nm. Using high polymer/plasmid ratios caused aggregation. The smallest complexes were obtained with the copolymer prepared with branched PEI with 25-kDa molecular weight. Copolymers were able to squeeze plasmid DNA more at the body temperature. Cytotoxicity was observed with PEIs especially with the branched higher molecular weights. Copolymerization reduced the cytotoxicity. The best in vitro DNA uptake efficiency (70%) was achieved with the complex prepared with poly(NIPA)/PEI25B. However, poly(NIPA)/PEI25L was the most successful vector for an effective gene expression without any significant toxicity.

Cytotoxicity and apoptotic effects of nickel oxide nanoparticles in cultured HeLa cells.

“…HeLa cells were cultured in DMEM-F12 medium supplemented with 10% FCS and 1% antibiotic (10.000 unit of penicilin and streptomycin 10mg/ml in 100 ml of sterile distilled water) in a humidified incubator at 37°C and 5% CO 2 atmosfer. The cells were subcultered twice a week, using dissociation medium ( trypsin-EDTA, pH 7.4) as buffer system [19].…”

“…The plates were kept in the CO2 incubator (37°C in 5% CO 2 ) for 2-16 h. Following this incubation, HeLa cells were harvested with trypsin-EDTA. They were dyed with trypan blue (19). The viable cells were counted with a haemacytometer (C.A.…”

Cannabinoid activation of PPARα; a novel neuroprotective mechanism

“…The reporter contains three PPARbinding sites linked to a promoter controlling the gene for firefly luciferase. Transient transfection was carried out by the polyethyleneimine method with the ratio nitrogen (N) to DNA phosphate (P) of N/P ¼ 15 (Boussif et al, 1995;Turk et al, 2004). Immediately before transfection, cells were rinsed with phosphate-buffered saline (PBS) and supplemented with fresh serum-free culture medium.…”

Intelligent polymers as nonviral vectors

“…[29][30][31] Poly(NI-PA)PEI block copolymers were synthesized by using carboxyl-ended poly(NIPA) and PEI (both branched 'B' and linear 'L' and with different molecular weights) in the presence of an activating agent (EDAC). Copolymerization of poly(NIPA) chains with more hydrophilic PEI chains caused observable increases in the LCST of the homopolymer from 311C to around body temperature (36-391C).…”