990 ) 120 % ( se100 a e l re 80 A DN 60 e tiav 40 l u um 20 c c A 0 0 5 20 25 10 15 Time (day) Fig. 9 Accumulative release of DNA from DNA-PLA-PEG-NPs (n = 3) model-dependent methods, and the result showed that the release behavior of DNA from DNA-PLA-PEG-NPs ﬁtted into bioexponential ﬂow equation best: 100 - Q = 78.70e - 0.9919t ? 51.37e - 0.2073t (Ra = 0.998 2, Rb = 0.993 6). It could be predicted that the established DNA-PLA-PEG-NPs with a delayed release effect could keep the loaded DNA prolonged action in vivo. Integrity Investigation of the DNA Released from DNA-PLA-PEG-NPs Plasmid DNA released from DNA-PLA-PEG-NPs at vari- ous time points was evaluated for its structural integrity on 0.8% agarose gel in comparison with control DNA (Fig. 10). It can be observed that the DNA released from DNA-PLA-PEG-NPs was predominantly supercoiled (S.C.) in form and comparable with the native DNA. Although some evidence of gradual increase in the per- centage of open circular plasmid DNA (O.C.) was visible at later time points, no linear conﬁguration was observed during the process of in vitro release. These results indi- cated that DNA-PLA-PEG-NPs could protect the loaded plasmid DNA from degradation to some extent in the release duration. The results of the functional integrity of released DNA based on in vitro transfection experiment were shown in Table 1. It was evident from the result that DNA after releasing from DNA-PLA-PEG-NPs still retained gene expression activity. In the prophase of in vitro release (1 day and 10 days), the released DNA from DNA-PLA- PEG-NPs completely maintained its bioactivity as the in Nanoscale Res Lett (2009) 4:982–992 Fig. 10 Agarose gel electrophoresis of DNA released from DNA- PLA-PEG-NPs vitro transfection efﬁciency of released DNA/lipofectamine complex was equivalent to the untreated DNA that was complexed with Lipofectamine 2000 (p [ 0.05). While in the terminal in vitro release (20 days), the transfection efﬁciency of the released DNA/Lipofectamine complex was a little lower than the untreated DNA/Lipofectamine complex (p \ 0.05). That results might be explained that more DNA had converted into open circular with release time as demonstrated by agarose gel electrophoresis described above, and the bioactivity of open circular DNA was reported to be less efﬁcient than supercoiled conﬁgu- ration [34]. Evaluation of the Cytotoxicity of DNA-PLA-PEG-NPs In vitro toxicity of DNA-PLA-PEG-NPs was evaluated by MTT assay in Hela cells. The cytotoxicity of DNA-PLA- PEG-NPs at various concentrations against Hela cell was shown in Fig. 11. Lipofectamine 2000, which is commonly used in in vitro gene transfection, was evaluated in com- parison with DNA-PLA-PEG-NPs. As illustrated in Fig. 11, the Hela cell viabilities decreased with the increased concentration of DNA-PLA-PEG-NPs while the average cell viabilities of different formulations at the studied concentrations (5–250 lg/mL) were between 80 and 120% versus control cells and were all signiﬁcantly higher than that of Lipofectamine 2000 (p \ 0.05), which Table 1 Biological activity of DNA released from DNA-PLA- PEG-NPs (n = 3) * p \ 0.05 compared with untreated DNA/lipofectamine complex 123 Transfection efﬁciency (%) Untreated DNA/lipofectamine complex 51.01 ± 2.43 Released DNA/lipofectamine complex 1 day 10 days 20 days 49.36 ± 2.83 46.95 ± 1.68 43.41 ± 2.36*