988 120 100 ) % (y 80 c n iice 60 f f gE 40 n i d inB 20 0 10/1 50/1 100/1 150/1 NPs/DNA (w/w) 200/1 500/1 Fig. 3 Binding efﬁciency of PLA-PEG-NPs with DNA at different mass ratio (n = 3) Fig. 4 TEM imaging of PLA-PEG-NPs (a) and DNA-PLA-PEG-NPs (b) Physicochemical Characterization of PLA-PEG-NPs and DNA-PLA-PEG-NPs The transmission electron micrograph of PLA-PEG-NPs and DNA-PLA-PEG-NPs was illustrated in Fig. 4. It was shown that the obtained nanoparticles appeared similar spherical in shape and separated from each other. Figure 5 showed a representative size distribution proﬁle of the nanoparticles. The polydispersity index (PDI) of PLA- PEG-NPs and DNA-PLA-PEG-NPs were 0.185 and 0.161, respectively, demonstrating a relatively narrow particle size distribution. The resultant PLA-PEG-NPs and DNA- PLA-PEG-NPs were small in size with mean particle size of 89.7 and 128.9 nm, respectively, and positive surface charge with zeta potential of ?28.9 and ?16.8 mV, respectively. It could be explained that while DNA com- bined with PLA-PEG-NPs, the particles size increased and the absolute value of surface charge decreased due to the 123 Nanoscale Res Lett (2009) 4:982–992 neutralization effect between the anionic DNA and the cationic nanoparticles. Protection of DNA-PLA-PEG-NPs Against DNase I Degradation of DNA by nuclease, such as DNase I, is a major barrier for gene delivery both in vitro and in vivo. To test whether the resultant nanoparticles can protect loaded plasmid DNA from nucleases digestion, the nanoparticles were exposed to DNase I. Figure 6 showed that naked plasmid DNA (lane 4) was completely digested by DNase I at 0.1 U/lg DNA within 30 min of incubation, conﬁrming the activity of nucleases. While DNA extracted from DNA- PLA-PEG-NPs remained intact supercoiled form in the investigated concentrations of DNase I within 30 min of incubation (lane 2–4), similar to the DNA control in lane 5. These results demonstrated that DNA-PLA-PEG-NPs indeed could protect the loaded DNA from degradation by DNase I at determinate concentrations for 30 min, and maintained the structural integrity of gene. Stability of DNA-PLA-PEG-NPs in Plasma Figure 7 showed the stabilization of DNA-PLA-PEG-NPs in plasma. It could be observed that the original plasmid DNA bands in lane 1 disappeared at the exposure of 10% human plasma for 1 h in lane 2, suggesting that the plasmid DNA had been degraded completely by nucleases in plasma. However, DNA-PLA-PEG-NPs remained rela- tively stable in the presence of plasma, with most loaded DNA detained in the well. This conﬁrmed that the DNA- PLA-PEG-NPs could not only bind DNA efﬁciently onto its surface but also partially protect the condensed DNA against degradation by plasma protein, which was prom- ising to improve the in vivo stability of DNA and favored the in vivo delivery of theoretical gene. In Vitro Release of DNA-PLA-PEG-NPs The stability of plasmid DNA in the condition identical to in vitro release of DNA-PLA-PEG-NPs was investigated in the present study. As shown in Fig. 8, the DNA percent content at the predetermined time point maintained above 95% during 20 days. It was clearly indicated that the plasmid DNA could remain extremely stable in the adoptive condition during the monitoring time without substantial degradation, that was, the release medium has potential to improve the stability of plasmid DNA and prevent its degradation. This result could be explained that dsDNA has higher stability at high salt concentrations and physiological pH which was in agreement with other report [33]. Therefore, the content of DNA released from