MATERIALS AND METHODS

Materials

1,4-Butandiol diacrylate was obtained from Merck, Germany. Butyl amine and 3-(dimethylamino)-1-propylamine were obtained from Fluka, Switzerland.

1-(3-Aminopropyl)imidazole and Polyethylenimine (Branched PEI，Mw=2500) were obtained from Sigma-Aldrich, Germany. The solvent of N,N-dimethylformamide (DMF, Tedia co., USA) was dried over calcium hydride and distilled just before use. N-[2-hydroxyethyl]

piperazine-N’-[2-ethanesulfonic acid] (HEPES) were obtained from Sigma Co. (USA).

N-methyldibenzopyrazine methyl sulfate (electron-coupling reagent) and sodium (2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide) (XTT) were purchased from Roche Co. (USA). The plasmid pCMV-LacZ (pCMV-βgal)contained a CMV promoter to drive theβ-galactosidase (LacZ) gene expression. The plasmid DNA was amplified in Escherichia coli (DH5αstrain) and purified using column chromatography (Qiagen^® Plasmid Mega kit, Germany).

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The purified plasmid DNA was dissolved in a tris(hydroxymethyl) methylamine-ethyldiaminetetraacetic acid (Tris-EDTA) buffer (pH 8.0) and determined using the ratio of UV absorbance at 260nm/280nm. Monkey SV40 transformed kidney fibroblast COS-7 cells were obtained from American Type Culture Collection (ATCC, CRL-1651). The cells were cultured in Dulbecco’smodified Eagle’smedium (DMEM,GibcoBRL Co., Ltd.) supplemented with 10%

FBS, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, and 4 mM L-glutamine, and maintained at 37 C in a humidified 5% CO₂-containing atmosphere.

Synthesis of Poly(β-amino ester) (PEDP)

The 1,4-Butandiol diacrylate with amino monomers molar ratio of 1/1 were mixed in anhydrous CH2Cl2solvent within the double-necked reaction flask under dry nitrogen purge. Then heated ranged were from room temperature to 80 °C and reacted for 0.5 to 6.5 h. The polymer was

precipitated in ethyl ether and then dried at 40 °C under vacuum. Yield was 86%. The polymer was characterized by FT-IR,¹H NMR, and¹³C NMR.

Poly(β-amino ester) Characterization

The poly(β-amino ester)s were characterized by nuclear magnetic resonance

(NMR, BrukerAVANCETM DPX-200spectrometer) and Fourier transform infrared spectroscopy (FT-IR,MATTSON GALAXY 5000 SERIESspectrophotometer). All of the chemical shifts in¹H NMR and¹³C NMR spectra were reported in parts per million (ppm). A 99.8% pure DMSO-d6was used as the solvent to characterize polymers. IR spectra were recorded on a spectrometer as KBr pellets. The molecular weights of polymers were determined by gel permeation chromatography (GPC) analysis (VISCOTEK,REFRACTIVE INDEX DETECTOR, MODEL: 504). THF was used as the eluent and

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polystyrene as the reference. The weight- and number-average molecular weights (Mw and Mn, respectively) were calibrated with standard polystyrene samples. The sample concentration in the THF was 2.5 mg/mL, and the flow rate was 1.0 mL/min.

Buffering Capacity of Polymers

Acid-base titration was used to evaluate the buffering capacity of PEI25k, PEDP, and PEDP-PEI25k . In this assay, 10 mg of polymer was dissolved in 10 mL of 150 mM NaCl, and then 100 μL of 1 N NaOH was added to the solution to adjust the pH to the alkaline range at 11.6. HCl (0.1 N) was used as the titratant to lower the pH to acidic conditions at around 2.0-2.5. Titration increment size =

100μL.

Hydrolytic degradation of polymers

PEDP was dissolved in a buffer solution (pH 7.4) with a concentration of 10mg/mL, and then incubated in a water bath at 37C for various durations. After hydrolysis for various durations, the solution was dried in a vacuum for several hours to remove water. The molecular weight of the polymer was determined using gel permeation chromatography (GPC).

Preparation and Characterization of Polymer/DNA Complexes

5.0 mg/mL of the polymer was dissolved in 20 mM HEPES buffer solution (pH 7.4), and its serial dilutions were made. The polymer serial dilutions were rapidly added into the DNA solutions to obtain polymer/DNA complexes. Then, the complexes were allowed to self-assemble in the HEPES buffer solution and incubated at room temperature for 30 min before measuring. The hydrodynamic sizes of the polymer/DNA complexes were determined by dynamic light scatter (Nicomp 380 system,

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USA).

Gel Electrophoresis of Polymer/DNA Complexes

The electrophoretic mobility of polymer/DNA complexes were measured with 0.7% agarose gel in Tris-Acetate-EDTA (TAE) buffer containing ethidium bromide (0.6 ug/mL). After electrophoresis at

100 volts for 90 min, the DNA band was visualized by UV irradiation and photographed.

XTT assay

The influence of the polymer concentration on the cell viability was evaluated in a cell culture for the various polymers. The cytotoxicities of PEDP-PEI25k/DNA and PEDP/DNA for comparison with that of PEI25k/DNA were evaluated using the XTT assay. In a 96-well plate, COS-7 cells were cultured in complete DMEM and then seeded at a density of 1.0×10⁴ cells/well. The cells were incubated at 37C and 5% CO2in a humidified atmosphere for 24 hours. Subsequently, the cells were incubated foronehourin 200 μL FBS-free DMEM containing polymer with various concentrations.

The cells were incubated in DMEM as a negative control. After 1 hour, the cells were washed with 200 L PBS solution and replaced by complete DMEM for a further 48 hours of incubation. Then, 50L of XTT labeling mixture was added to each well and the cells were further incubated at 37C for 1 hour. Results are expressed as the relative cell viability (%) with respect to control wells containing culture medium.

Transfection Protocol and ONPG Assay

COS-7 cells were used to evaluate the transfection efficiency of polymer/DNA complexes. The cells were seed in a 96-well plate (1.0×10⁴ cells per well) in complete DMEM and incubated for 24 h before transfection trials. The DNA concentration was kept constant at 5μg/mL(1.0 μg/well) and the amounts of polymers were varied. 200 μL solutions of polymer/DNA complexes was taken and incubated with cells for 1 h at 37℃. The medium was replaced afterwards with complete DMEM and the cells were incubated for another 48 h. For evaluating transfection efficiency, the cells were

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washed with 0.3 mL PBS and then permeabilized with 20μL cell lysis buffer at 4℃ for 20 min. An ONPG solution (180 μg/well) was added after lysis treatment and the cells were incubated at 37℃

for 1h. The expression of pCMV-βgalgene was measured spectrometrically using an ELISA reader at a wavelength of 405 nm.