984 50,000 Da, synthesized by school of chemistry of Shan- dong University). Cetyltrimethyl ammonium bromide (CTAB) was purchased from Amresco (Amresco, China). pEGFP-N1 was provided by Zhejiang University (China). PicoGreen dsDNA reagent was obtained from Molecular Probes (Invitrogen, USA). Agarose was purchased from BIO-WEST (Spain). Goldview was obtained from Beijing Saibaisheng Biological Engineering Co. (Beijing, China). DNase I enzyme was obtained from Beijing Yinfeng Century Scientiﬁc Develop Co., Ltd (Beijing, China). MTT (3-[4, 5-dimehyl-2-thiazolyl]-2, 5-diphenyl-2H- tetrazo- lium bromide) were purchased from Sigma-Aldrich (China). Lipofectamine 2000 was from Invitrogen (USA). Hela cell line was obtained from American Type Culture Collection (ATCC, USA). All the other chemicals and reagents used were of analytical purity grade or higher, obtained commercially. Cationic PLA-PEG nanoparticles (abbreviated as PLA- PEG-NPs below) was prepared by the nanoprecipitation method [21] according to the optimized formulation. Typi- cally, accurately weighed (20 mg) PLA-PEG was dissolved into 5 mL acetone under sonication. The resulting polymer solution was slowly (30 mL/h) injected by a micro-syringe pump (KDS 100, USA) into 20 mL magnetically stirring (600 rpm, RCT basic stirrer, IKA, Germany) mixed sur- factant solution containing CTAB (0.1%, w/v) and Tween 80 (0.2%, w/v) and agitated for 8 h at room temperature until complete evaporation of the organic solvent. The entire dispersed system was then centrifuged (Beckman, Fullerton, CA) at 15,000 rpm, 4 C for 30 min. The pellet was re- suspended in Milli-Q water and washed three times to remove the redundant surfactant. Finally it was re-sus- pended in phosphate buffered saline (PBS, pH 7.4) solution followed by ﬁltered through 0.45 lm nitrocellulose mem- brane (Millipore) ﬁlter and was stored at 4 C until use. Preparation and Optimization of Gene Loaded Cationic PLA-PEG Nanoparticles Adsorption of Plasmid DNA onto PLA-PEG-NPs The gene loaded cationic PLA-PEG nanoparticles (abbre- viated as DNA-PLA-PEG-NPs below) were obtained by means of electrostatic attraction between the anionic plasmid DNA and the blank cationic nanoparticles. Brieﬂy, the report gene pEGFP solution was added into PLA-PEG-NPs at a ﬁxed weight ratio (PLA-PEG: DNA, w/w). The mixed liquor was kept for 20 min at room temperature and the resultant DNA-PLA-PEG-NPs were directly used for further study. 123 Preparation of Cationic PLA-PEG Nanoparticles Determination of DNA Binding Efﬁciency Nanoscale Res Lett (2009) 4:982–992 Agarose Gel Electrophoresis of DNA/Nanoparticles Complex Complex formation between nanoparticles and DNA was analyzed by agarose gel electrophoresis. The gels were prepared with 0.8% (w/v) agarose in 20 mL TAE buffer (40 mM Tris, 40 mM Acetic acid, 1 mM EDTA, pH 8.5) containing 2 lL goldview as stains. A ﬁxed amount (1 lg) of DNA was incubated with various amounts of PLA-PEG- NPs in 100 lL of PBS (pH = 7.4) (the weight ratio of PLA-PEG and DNA is 10:1, 50:1, 100:1, 150:1, 20:1, 500:1, respectively). The resultant DNA-PLA-PEG-NPs and control plasmid DNA were applied to gel electropho- resis at a constant 90 V for 25 min. After the electropho- resis, images were obtained using UV transilluminator and MultimageTM Light Cabinet (Alpha Imagers EC, Alpha Innotech Corporation) to show the location of DNA. To measure the DNA binding efﬁciency of the DNA-PLA- PEG-NPs, the free DNA was separated from the nanopar- ticles by ultracentrifugation at 15000 rpm, 4 C for 30 min and the free DNA in supernatants were collected and analyzed using ﬂuorospectrophotometry with PicoGre- enTM dsDNA quantitation reagent [22]. The ﬂuorescence was measured by ﬂuorescence spectrophotometer (HIT- ACHI 850, Japan) at excitation and emission wavelengths of 480 and 520 nm, respectively. The amount of DNA was calculated according to the linear calibration curve of DNA (F = 2 9 10-4 C ? 0.0437 R = 0.999 4). The binding efﬁciency was calculated from the following equation: DNA binding efficiency ð%) ¼ ðtotal DNA content free DNA contentÞ total DNA content 100 : Morphology, Particle Size and Zeta Potential of Nanoparticles The morphology of PLA-PEG-NPs and DNA-PLA-PEG- NPs was examined under transmission electron microscope (TEM, JEM-1200EX, Japan). Samples were prepared by placing a drop of nanoparticle suspension onto a copper grid and air-dried, following negative staining with one drop of 2% aqueous solution of sodium phosphotungstate for con- trast enhancement. The air-dried samples were then directly examined under the transmission electronic microscope. The average particle size, size distribution, and zeta potential of the nanoparticles were measured by photon correlation spectroscopy (PCS) using Zetasizer 3000 (Malvern Instruments, Malvern, England). All measure- ments were carried out in triplicates. The average particle