Nanoscale Res Lett (2009) 4:982–992 size was expressed in volume mean diameter and the reported value was represented as mean ± SD (n = 3). Stability Test of the DNA-PLA-PEG-NPs Against DNase I Protection of plasmid DNA from nucleases is one of the most important properties for efﬁcient gene delivery both in vitro and in vivo. To test whether DNA-PLA-PEG-NPs can protect the loaded plasmid DNA from nucleases digestion, the results of DNase I mediated digestion was evaluated using agarose gel electrophoresis [23]. In brief, 100 lL of DNA-PLA-PEG-NPs containing 1 lg of DNA were, respectively, incubated with different amounts of DNase I (0.1, 0.2 and 0.4 U/lg DNA) in DNase I /Mg2? digestion buffer (50 mM, Tris–HCl, pH 7.6, and 10 mM MgCl2). Naked DNA (1 lg) was treated with DNase I at 0.1 U/lg DNA as a reference. The suspension was incu- bated in shaking water bath (100 rpm) for 30 min at 37 C. After that, the enzymatic digestion reaction was terminated with 5 lL EDTA solution (0.5 M, pH 8.0) for 10 min at room temperature. To asses the integrity of DNA loaded in the nanoparticles, it was dissociated from the cationic nanoparticles by adding heparin solution [24], an anionic glycosaminoglycan, at ﬁnal concentration of 1% (w/v) and the suspension was then incubated in shaking water bath (100 rpm) for 3 h at 37 C. The conﬁguration of plasmid DNA in the nanoparticles after extraction was analyzed by gel electrophoresis with untreated naked DNA as a refer- ence. The samples were applied to a 0.8% (w/v) agarose gel in TAE buffer as described above. The Plasma Stability Investigation of DNA-PLA-PEG- NPs To examine resistance of DNA-PLA-PEG-NPs to DNA degradation in plasma, 25 lL of plasmid DNA solution and DNA-PLA-PEG-NPs were, respectively, incubated with 25 lL of 20% human plasma for 1 h in a 37 C incubator. Immediately following the incubation, nucleases were inactivated with 3 lL of EDTA solution (0.5 M, pH 8.0) for 10 min at room temperature. Thereafter, the plasma treated samples together with untreated DNA-PLA-PEG- NPs and DNA were all applied to 0.8% (w/v) agarose gel as described above. The In Vitro Release of DNA-PLA-PEG-NPs The Stability of Plasmid DNA in the Release Medium During the prolonged in vitro drug release, the biomacro- molecule drug may be degraded, and this may affect in vitro drug release studies [13, 25]. Therefore, it is essential 985 to select a suitable drug release medium to protect the plasmid DNA from degradation during in vitro drug release period. Based on physiological environment and the result of preliminary tests, the mixed solution containing phos- phate buffered saline solution at pH 7.4 (100 mM) and 150 mM NaCl solution was selected as the release medium for the DNA-PLA-PEG-NPs. The stability of plasmid DNA in the release medium in the duration of in vitro release was investigated at ﬁrst. Brieﬂy, 200 lL DNA solution (10 lg/mL) were diluted with isovolumic release medium mentioned above in Eppendorf tubes and then shaken horizontally in water bath at 37 C and 100 rpm. Separate tubes were used for each data point. At predetermined time intervals (2 h, 5 h, 8 h, 12 h, 24 h, 2 days, 4 days, 7 days, 10 days, 15 days, 20 days), the samples were withdrawn and the concentration of DNA was determined by the PicoGreen ﬂuorimetric assay mentioned above. The experiments were repeated three times and all measure- ments were collected in triplicates. The proﬁle of percent content of DNA versus time in release medium was drawn. Measurement of In Vitro DNA Release The proﬁles of in vitro DNA release from DNA-PLA-PEG- NPs were measured over 20 days using separate samples for each time point according to the following operations. Typically, 200 lL of DNA-PLA-PEG-NPs (DNA concen- tration: 10 lg/mL) were diluted with isovolumic release medium in Eppendorf tubes and then shaken horizontally in water bath at 37 C and 100 rpm. At predetermined time intervals, the tubes were withdrawn and centrifugated at 15,000 rpm for 30 min, the supernatants were collected for analysis. The amount of the released DNA was evaluated by the PicoGreen ﬂuorimetric assay. Background readings were corrected using the centrifugation supernatants from blank nanoparticles. Integrity Determination of Plasmid DNA After Release The molecular form of DNA (often referred to as ‘‘topol- ogy’’) has been demonstrated to affect the transfection efﬁ- ciency in vitro and in vivo [26]. Supercoiled plasmid was believed to be the most efﬁcient form for cell transfection and it was established that the order of transfection efﬁciency was supercoiled [ relaxed [ linear [27]. Fabrication conditions such as sonication, lyophilization, and change of pH were reported previously to decrease the supercoiled content. It is well known that the hydrolysis of PLA may substantially decrease the pH in PLA nanoparticles, which potentially change the topology structure of DNA and decrease the biological activity of DNA accordingly. Therefore, it was crucial to examine the plasmid structural and functional integrity following release from nanoparticles. 123