* * * * * 10 25 50 100 250 PLA-PEG nanoparticles concentration (µg/ml) Lip Fig. 11 Cell viability of DNA-PLA-PEG-NPs against Hela cell line by MTT assay (n = 3). *p \ 0.05 compared with Lip )150 % (y120 t i ilb 90 a i llv 60 e ac 30 l e H 0 * 5 NPs Lip Naked DNA A C E B D F Fig. 12 Gene transfection of pEGFP in Hela cells (9400) (a, b) The transfection results of DNA-PLA-PEG-NPs in absence and presence of serum; (c, d): The transfection results of Lipofectamine 2000 in absence and presence of serum; (e, f): The transfection results of naked DNA in absence and presence of serum indicated that the DNA-PLA-PEG-NPs were much safer than Lipofectamine 2000 to Hela cell. In Vitro Transfection Studies An efﬁcient gene delivery system is required to transport the gene into cells and see to its eventual release, leading to gene expression and subsequent protein synthesis. The current study aimed to determine the ability of DNA-PLA- PEG-NPs to transfer the reporter gene EGFP to Hela cell. Transfection ability of our established vectors DNA-PLA- PEG-NPs was investigated in comparison with the com- mercial cationic liposome based reagent, Lipofectamine 2000 which was well known to provide high transfection efﬁciency and high level of transgene expression in a range of mammalian cell types in vitro. However, it was found Nanoscale Res Lett (2009) 4:982–992 991 that cationic liposomes tended to bind with some serum protein such as albumin in vivo, forming aggregation and eliminated quickly, which led to relative lower transfection efﬁciency. Thus it was meaningful to investigate the effects of serum in transfection medium on the in vitro transfection activity of gene vectors. In the present study, transfection activities of them were compared in the system with or without serum because serum was known to inﬂuence the transfection efﬁciency of gene vectors sometimes [35]. The results observed by inversion ﬂuorescence microscope after transfection (Fig. 12) showed that DNA-PLA-PEG-NPs may successfully transfer plasmid DNA into Hela cells, and the gene can encode the green ﬂuorescent protein. It was noticeable that DNA-PLA-PEG-NPs was a much more efﬁcient carrier than the EGFP plasmid alone irrespective of serum in the medium, which could barely transfect Hela cell. In the absence of serum in the transfection medium, DNA-PLA-PEG-NPs produced slightly lower transfection efﬁciency than Lipofectamine 2000 during 24 h which might be due to the sustained release property of DNA- PLA-PEG-NPs. However, Lipofectamine 2000 showed apparently diminished transfection activities in the pres- ence of serum whereas DNA-PLA-PEG-NPs still main- tained high transfection activity in the presence of serum, which was visibly higher than that of Lipofectamine 2000. These results indicated that the serum in the transfection medium hardly inﬂuenced the transfection activity of DNA-PLA-PEG-NPs but weakened the transfection activ- ity of Lipofectamine 2000 signiﬁcantly. It might be explained that cationic liposomes tended to adsorb anionic serum protein and form a larger aggregate which could not get across the cell membrane and deliver the DNA payload to the nucleus. The DNA-PLA-PEG-NPs with PEGylation could prevent the non-speciﬁc interactions with serum protein and minimize particle aggregation in buffers especially in the transfection medium and accordingly improve the transfection efﬁciency of nanoparticles [36]. Conclusion In the present work, cationic biodegradable nanoparticles based on PEGylated PLA copolymers was formulated using the mild and simple performed nanoprecipitation technique. Report gene, pEGFP was adsorbed onto the surface of the cationic PLA-PEG-NPs through electrostatic interactions, leading to the gene loaded nanoparticles (DNA-PLA-PEG-NPs). The obtained DNA-PLA-PEG-NPs with high binding efﬁciency (95.36%), positive surface charge (?16.8 mV) and small particle size (128.9 nm) showed sustained-release of DNA in vitro within 20 days. Adsorption and release from DNA-PLA-PEG-NPs did not alter structural and functional integrity of plasmid DNA. 123