Preparation and Characterization of Cationic PLA-PEG Nanoparticles for Delivery of Plasmid DNA

Abstract

The purpose of the present work was to formulate and evaluate cationic poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) nanoparticles as novel non-viral gene delivery nano-device. Cationic PLA-PEG nanoparticles were prepared by nanoprecipitation method. The gene loaded nanoparticles were obtained by incubating the report gene pEGFP with cationic PLA-PEG nanoparticles. The physicochemical properties (e.g., morphology, particle size, surface charge, DNA binding efficiency) and biological properties (e.g., integrity of the released DNA, protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in Hela cells) of the gene loaded PLA-PEG nanoparticles were evaluated, respectively. The obtained cationic PLA-PEG nanoparticles and gene loaded nanoparticles were both spherical in shape with average particle size of 89.7 and 128.9 nm, polydispersity index of 0.185 and 0.161, zeta potentials of +28.9 and +16.8 mV, respectively. The obtained cationic PLA-PEG nanoparticles with high binding efficiency (>95%) could protect the loaded DNA from the degradation by nuclease and plasma. The nanoparticles displayed sustained-release properties in vitro and the released DNA maintained its structural and functional integrity. It also showed lower cytotoxicity than Lipofectamine 2000 and could successfully transfect gene into Hela cells even in presence of serum. It could be concluded that the established gene loaded cationic PLA-PEG nanoparticles with excellent properties were promising non-viral nano-device, which had potential to make cancer gene therapy achievable.

Figure1

TEM imaging of CTAB-NPs (a) and Tween 80-NPs (b)

Figure 2

Agarose gel electrophoresis analysis of PLA-PEG-NPs combining with DNA at different mass ratio.Lane1,8: DNA Control;Lane2–7: the mass ratio of NPs/DNA was 10:1, 50:1, 100:1, 150:1, 200:1, 500:1, respectively

Figure 3

Binding efficiency of PLA-PEG-NPs with DNA at different mass ratio (n = 3)

Figure 4

TEM imaging of PLA-PEG-NPs (a) and DNA-PLA-PEG-NPs (b)

Figure 5

Representative size distribution profile for PLA-PEG-NPs (a) and DNA-PLA-PEG-NPs (b)

Figure 6

Agarose gel electrophoresis of DNA-PLA-PEG-NPs after being incubated with DNase I at different concentrations.Lane1–3: DNA-PLA-PEG-NPs incubated with different amount of DNase I at 0.1, 0.2, 0.4 U/μg DNA, respectively, for 30 min;Lane4: Naked DNA incubated with DNase I at 0.1 U/μg DNA for 30 min;Lane5: DNA control

Figure 7

Plasma stability of DNA-PLA-PEG-NPs.1: DNA control;2: Naked DNA incubated in 10% plasma for 1 h;3: DNA-PLA-PEG-NPs Control;4: DNA-PLA-PEG-NPs incubated in 10% plasma for 1 h

Figure 8

The profile of content of DNA in release medium with time (n = 3)

Figure 9

Accumulative release of DNA from DNA-PLA-PEG-NPs (n = 3)

Figure 10

Agarose gel electrophoresis of DNA released from DNA-PLA-PEG-NPs

Figure 11

Cell viability of DNA-PLA-PEG-NPs against Hela cell line by MTT assay (n = 3). *p < 0.05 compared with Lip

Figure 12

Gene transfection of pEGFP in Hela cells (×400) (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