Effect of Eudragit on In Vitro Transfection Efficiency of PEI–DNA Complexes

Materials and Methods

Materials. Polyethylenimine (PEI, branched, MW 25 kDa) was purchased from Aldrich (Milwaukee, WI, USA). Agarose was purchased from Sigma (St Louis, MO, USA). Endotoxin-free Plasmid Maxiprep Kit was purchased from Qiagen (Santa Clarita, CA, USA). Enhanced GFP plasmid (pEGFP) DNA was obtained from Clontech (Mountain View, CA, USA). RPMI-1640, fetal bovine serum (FBS), Trypsin-EDTA, and penicillin-streptomycin were purchased from Gibco BRL (Rockville, MD, USA). Luciferase assay reagent was purchased from Promega (Madison, WI, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Sigma. KB human cervical carcinoma cells were obtained from the American Type Culture Collection (Rockville, MD USA). All other chemicals were of cell culture and molecular biology quality.

Complex formation between PEI and plasmid DNA. PEI and DNA complexes were formed as described by Wanlop et al. (19). Briefly, different N/P ratios of PEI (i.e. 5, 7.5, 10, 15, 20) were prepared by dissolving PEI in HEPES buffer (20 mM HEPES, pH 7.4). pEGFP plasmid DNA solution (0.5 mg/ml) was added to different N/P ratios of PEI to form PEI–pEGFP complexes. The formed mixture was gently vortexed for 5 s before leaving it at room temperature for 15 min. A stock solution of EE100 was prepared in ethanol and added in a 0.25:1 w/w ratio to PEI solution prior to mixing with DNA solution to attain the required N/P ratio. For experiments containing EE100 alone, equivalent concentrations were used.

Gel retardation. Gel retardation analysis was used to assess the formation of electrostatic complexes between cationic polymers and plasmid DNA. It was performed with slight modification to the method outlined by Palocci et al. (20). Briefly, a 0.8% agarose gel was prepared in 1× TBE buffer with 0.5 μg/ml ethidium bromide. Polymer samples were combined with 1 μg of pEGFP plasmid at N/P ratios of 5 and 10. Samples of PEI, EE100, and PEI-EE100 were run against free GFP plasmid. Electrophoresis was conducted at 100 V, 90 mA for 45 min. Gels were photographed under UV light with a Bio-Rad 170-8126 Universal Hood with Quantity One v4.5.2 Software.

Cell culture. KB (a subline of HeLa) cells were grown in RPMI-1640 medium containing glutamine. The medium was supplemented with 10% FBS, 50 μg/ml streptomycin, and 50 μg/ml penicillin. Cells were grown in an incubator at 100% relative humidity, 37°C, and 5% CO₂.

In vitro transfection. KB cells were seeded in a 24-well plate at a density of 5×10⁵ cells/well in 500 μl of RPMI-1640 medium containing glutamine. The cells were incubated for 24 h at 37°C in a 5% CO₂ environment. After 24 h, the medium was replaced with 200 μl growth medium containing either PEI, PEI in combination with EE100, or EE100 alone with GFP, at different N/P ratios (0, 5, 7.5, 10, 15 or 20). After 4 h of incubation, the transfection medium was replaced with fresh growth medium and the cells were incubated for another 24 h at 37°C in a 5% CO₂ environment, before being analyzed for GFP expression. In all transfection protocols, the formulations used were combined with transfection vectors in serum-free medium and incubated for about 15 min at room temperature prior to mixing with cells.

GFP expression levels were analyzed by flow cytometry similar to the methods described by Zhao et al. (21). The cells were washed with PBS (pH 7.4) three times before detaching with trypsin-EDTA (0.25%) solution. The cells were then centrifuged and fixed with 500 μl of 4% formalin in phosphate-buffered saline (PBS). The cell suspension in 4% formalin was introduced into a Becton Dickinson FACS Calibur cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) to determine the level of GFP expression. For each cell sample, 1×10⁵ events were collected. The data were analyzed using BD CellQuest Pro software (Becton Dickinson). Untreated cells and cells treated with GFP alone were used as controls. All transfection experiments were performed in triplicate.

Evaluation of cytotoxicity. Cytotoxicity studies were carried out using MTT assay as described previously (15). Briefly, KB cells were seeded in a 96-well plate at a density of 10⁵ cells/well in 200 μl of growth medium. The cells were incubated for 24 h at 37°C under 5% CO₂ atmosphere. After 24 h, the cells were washed with PBS (pH 7.4), followed by addition of 15 μl of transfection vectors containing either PEI, PEI in combination with EE100, or EE100 alone. The cells were incubated with medium containing formulations for 4 h before being replaced with 200 μl of fresh medium and further incubated for 24 h at 37°C under an atmosphere with 5% CO₂. Non-treated cells and cells treated with GFP alone were used as controls and incubated for the same amount of time. After 24 h, the cells were treated with 20 μl of 5 mg/ml concentrated MTT solution and incubated for 2 h. Viable cells formed formazan crystals and these were dissolved by adding 100 μl dimethyl sulfoxide to each well. The resultant colored product was read at 550 nm using a microplate reader (340 ATTC; SLT Lab Instruments, Salzburg, Austria).

Statistical analysis. Statistical significance of differences in transfection efficiency and cell viability were examined using Student's t-test. The significance level was set at p<0.05.