986 Structural Integrity Determination Structural integrity of plasmid DNA released from nanopar- ticles at selective time points was evaluated by agarose gel electrophoresis. To determine plasmid stability during release, the samples released at several time points (i.e. 2 h, 8 h, 24 h, 4 days, 10 days, 15 days and 20 days) along with control untreated plasmid DNA were applied to 0.8% agarose gel in TAE buffer. Band separation for topological structure of plasmid DNA was observed after gel electrophoresis. Functional Integrity Determination The in vitro transfection experiment was performed to investigate the functional integrity of released DNA. Hela cells (ATCC) were seeded into 24-well plates at a density of 1 9 105 cells/well 24 h prior to transfection and 1 lg of DNA of was used per well (n = 6 wells per formulation). An aliquot of untreated DNA solution and supernatants of nanoparticles in vitro release at different time points after moderate concentration (1 day, 10 days, 20 days, equiva- lent to 1 lg DNA) were complex with Lipofectamine 2000 according to the manufacturer’s instructions [28]. After incubation for 4 h at 37 C in 5% CO2 incubator, the ori- ginal incubation media was replaced with 1 mL of com- plete medium and cells were incubated sequentially until 24 h post transfection. The percentage of ﬂuorescent cells (deﬁned as cells having ﬂuorescence levels above that of untreated control cells) presented in every 10,000 cells counted by the ﬂow cytometry was calculated. Transfection experiments were performed in triplicates. Cell Viability Test of DNA-PLA-PEG-NPs The cytotoxicity of DNA-PLA-PEG-NPs was evaluated by MTT method in Hela cell line [19]. Brieﬂy, the cells were seeded into a 96-well microtiter plates at a density of 1 9 104 cells per well in 0.2 mL of RPMI 1640 culture medium supplemented with 10% fetal bovine serum (FBS) and antibiotics in 5% CO2 incubator at 37 C overnight. After that, the culture medium was replaced by 200 lL fresh serum-free RPMI 1640 medium with different con- centrations of the nanoparticles (expressed as PLA-PEG concentration, 5, 10, 25, 50, 100 and 250 lg/mL) and Lipofectamine 2000 in comparison. The applied dosage of gene vectors (Lipofectamine 2000/DNA complex and DNA-PLA-PEG-NPs) were proportional to that used in transfection experiment. After incubation for 24 h, 20 lL of MTT stock solution in PBS (5 mg/mL, pH 7.4) was added into each well at a ﬁnal concentration of 0.5 mg/mL MTT. The plate was then incubated at 37 C in 5% CO2 for 4 h. The medium was removed and 150 lL DMSO was added to dissolve the formazan crystals. The plate was read 123 Nanoscale Res Lett (2009) 4:982–992 spectrophotometrically at 570 nm by microplate reader (Model 680, BIO-RAD, USA). The cell viability (%) was calculated and compared with the untreated control (100%) according to the following equation: Cell viability ð%Þ ¼ OD570ðsamplesÞ=OD570ðcontrolÞ 100 OD570(samples) represented measurements from the cells treated with samples and OD570(control) from the untreated cells. Statistical Analysis All measurements were collected in triplicates and exper- iments were repeated three times. Values were expressed as mean ± standard deviation. Unpaired Student’s t-test was used to assess statistical signiﬁcant differences (p \ 0.05) between the group means. In Vitro Transfection Assays of DNA-PLA-PEG-NPs The transfection activity of DNA-PLA-PEG-NPs was evaluated in Hela cell lines using plasmid DNA, encoding enhanced green ﬂuorescence protein (EGFP) as reporter gene in the transfection studies. The cells were seeded into 24-well plates at a density of about 1 9 105 cells per well in 1 mL of RPMI 1640 culture medium with 10% FBS, 24 h prior to transfection. At a conﬂuence level of 70–80%, cells were washed twice with PBS, and, respectively, incubated with 500 lL of media (with or without 10% FBS) containing 1 lg of DNA in transfection vectors at 37 C. Lipofectamine 2000 (Invitrogen) was used as positive control, and the formulation of Lipofectamine/ DNA complex was carried out according to the manufac- turer’s protocols. 1 lg of plasmid DNA was used as neg- ative control. The cells were incubated with the vectors for 4 h, in the presence or absence of serum medium. The transfection media was then replaced with 1 mL of fresh complete culture media, and the cells were incubated sequentially until 24 h post transfection. Detection of expression of EGFP was carried out using an inverted ﬂuorescent microscope with an attachment for ﬂuorescent observation (OLYMPUS, ZX71, Japan) and the picture was captured using a 4009 objective. Transfection exper- iments were performed in triplicates. Results and Discussions Formation of PLA-PEG-NPs In this study we have designed a novel cationic PLA-PEG nanoparticles modiﬁed by CTAB as a nonviral vector for