A novel type of self-assembled nanoparticles as targeted gene carriers: an application for plasmid DNA and antimicroRNA oligonucleotide delivery

Abstract

In this study, a new type of amphiphilic cetylated polyethyleneimine (PEI) was synthesized, and then polylactic-co-glycolic acid (PLGA)/cetylated PEI/hyaluronic acid nanoparticles (PCPH NPs) were developed by self-assembly as a novel type of gene-delivering vehicle. The PCPH NPs showed good DNA-condensation ability by forming polyplexes with small particle size and positive zeta potential. The transfection efficiency and cytotoxicity of PCPH NPs were evaluated as plasmid DNA vectors to transfect HepG2 in vitro. PCPH NPs exhibited much lower cytotoxicity and higher gene-transfection efficiency than PEI (25,000) and commercial transfection reagents. Furthermore, PCPH NPs were used as an anti-miR-221 vector for transfecting HepG2 cells, and anti-miR-221 was effectively transfected into cells and produced a greater inhibitory effect on cancer-cell growth by PCPH NPs. These results demonstrate that PCPH NPs can be a promising nonviral vector for gene-delivery systems.

Keywords: PLGA; anti-miR-221; gene delivery; hyaluronic acid; polyethyleneimine.

Figure 1

Prepared pathway of PLGA/PEI-cet/HA/plasmid DNA nanoparticles and schematic drawing of self-assembled cationic micellar nanoparticles and loading of plasmid DNA. Abbreviations: PLGA, polylactic-co-glycolic acid; PEI, polyethyleneimine; PEI-cet, cetylated PEI; HA, hyaluronic acid.

Figure 2

¹H-NMR spectrum of the PEI-cet copolymer in CDCl₃. Abbreviations: NMR, nuclear magnetic resonance; PEI, polyethyleneimine; PEI-cet, cetylated PEI; HA, hyaluronic acid.

Figure 3

Transmission electron microscopy images of free PLGA NPs (A); PCP NPs (B), and PCPH NPs (C). Abbreviations: PLGA, polylactic-co-glycolic acid; NPs, nanoparticles; PCP, PLGA/cetylated polyethyleneimine; PCPH, PCP/hyaluronic acid.

Figure 4

Gel retardation assay of pDNA binding with PCP NPs and PCPH NPs. Notes: (A) PCPH/pDNA nanocomplexes. Lane 1, pDNA alone; lanes 2–8, PCPH/pDNA nanocomplexes with N:P ratios 0.5, 1, 2, 4, 6, 8, and 10. (B) PCP/pDNA nanocomplexes. Lane 1, pDNA alone; lanes 2–6, PCP/pDNA complexes with N:P ratios 0.5, 1, 2, 4, and 10. Abbreviations: pDNA, plasmid DNA; PCP, polylactic-co-glycolic acid/cetylated polyethyleneimine; NPs, nanoparticles; PCPH, PCP/hyaluronic acid.

Figure 5

Cell-viability profile of PCPH/pDNA (N:P ratio 12), PCP/pDNA (N:P ratio 8), and PEI/pDNA (N:P ratio 6) at various concentrations against HepG2 cells. Notes: The concentration of different nanocomplexes in medium was converted to nitrogen concentration in medium. Percentage viability of cells is expressed relative to control cells. The error bar indicates the standard deviation in triplicate experiments. Abbreviations: PCPH, PCP/hyaluronic acid; pDNA, plasmid DNA; PCP, polylactic-co-glycolic acid/cetylated PEI; PEI, polyethyleneimine.

Figure 6

Transfection efficiency of different transfection reagents to HEK293 cells. Notes: (A) Transfection efficiency of different transfection reagents to HEK293 cells determined by flow-cytometry analysis at different N:P ratios (n=3, P<0.01). (B) EGFP expression in HEK293 cell line transfected with different transfection reagents. The images were obtained at magnification of 100×. Abbreviations: PEI, polyethyleneimine; PCP, polylactic-co-glycolic acid/cetylated PEI; PCPH, PCP/hyaluronic acid; Lipo, Lipofectamine; pDNA, plasmid DNA.

Figure 7

Transfection efficiency of different transfection reagents to HepG2 cells. Notes: (A) Transfection efficiency of different transfection reagents to HepG2 cells determined by flow-cytometry analysis at different N:P ratios (n=3, **P<0.01). (B) EGFP expression in HepG2 cell line transfected with different transfection reagents. The images were obtained at magnification of 100×. Abbreviations: PEI, polyethyleneimine; PCP, polylactic-co-glycolic acid/cetylated PEI; PCPH, PCP/hyaluronic acid; Lipo, Lipofectamine; pDNA, plasmid DNA.

Figure 8

Transfection efficiency of various transfection reagents on untreated HepG2 cells or HepG2 cells treated with CD44 siRNA. Notes: Data given as means ± standard deviation (n=3). **P<0.01. Abbreviations: PEI, polyethyleneimine; PCP, polylactic-co-glycolic acid/cetylated PEI; PCPH, PCP/hyaluronic acid; NC, negative control; siRNA, small interfering RNA.

Figure 9

Confocal fluorescence microscopy images of HepG2 cells treated with naked FAM oligonucleotides, PEI/FAM oligonucleotides, PCP/FAM oligonucleotides, and PCPH/FAM oligonucleotides, respectively. Notes: The cell nuclei were stained with Hoechst 33342 (blue). FAM-labeled small interfering RNA appears in green. Magnification was 400×. Abbreviations: FAM, fluorescein amidite; PEI, polyethyleneimine; PCP, polylactic-co-glycolic acid/cetylated PEI; PCPH, PCP/hyaluronic acid.

Figure 10

The results of HepG2 cells transfected with anti-miR-221 using PCPH NPs. Notes: (A) Relative expression change of miR-221 level in HepG2 cells. (B) Western blot analysis of p27^Kip1 in the HepG2 cells after treatment with different formulations. (C) Cell cycle profiles of HepG2 cells after treatment with different formations; (D) cell proliferation of HepG2 cells after treatment with different formations (images obtained 3 days after treatment); (a) untreated group; (b) pure PCPH NPs; (c) PCPH/mismatched AMO-transfected HepG2 cells; (d) PCPH/anti-miR-221-transfected HepG2 cells. Magnification of the images is 200×. Abbreviations: PCPH, polylactic-co-glycolic acid/cetylated polyethyleneimine/hyaluronic acid; NPs, nanoparticles; AMO, anti-microRNA oligonucleotide; miR, microRNA; NC, negative control.