Development of a reduction-sensitive diselenide-conjugated oligoethylenimine nanoparticulate system as a gene carrier

Abstract

Background: The reduction-sensitive cationic polymer is a promising nonviral carrier for gene delivery. Until now, disulfide bonds have been the only golden standard for its design. The aim of this research was to develop a novel reduction-responsive cationic polymer as a gene carrier.

Methods: Polycationic carriers were synthesized by addition of branched oligoethylenimine 800 Da (OEI(800)) via an active ester containing diselenide bonds. Disulfide bonds cross-linked with OEI(800)-SS(x) and monoselenide bonds linked with OEI(800)-Se(x) were synthesized and compared. Their molecular weights and degradation properties were determined using gel permeation chromatography. Changes in particle size, morphology, and DNA binding were investigated by dynamic light scattering, transmission electron microscopy, and electrophoresis assay in a reduction environment. Cytotoxicity and transfection in vitro were evaluated in a murine melanoma cell line (B16F10) and a human cervical epithelial carcinoma cell line (HeLa), while intracellular degradation and dissociation with DNA were studied by confocal laser scanning microscopy with FITC-labeled OEI(800) derivatives and Cy5-labeled DNA.

Results: Diselenide-conjugated OEI(800) (OEI(800)-SeSe(x)) polymer carriers of high molecular weight were successfully synthesized. After compacting with DNA, the OEI(800)-SeSe(x) polymers formed nanoparticles with an average size of 140 nm at an adequate C/P ratio. OEI(800)-SeSe(x) showed reduction-responsive degradation properties similar to those of the OEI(800)-SS(x) via gel permeation chromatography, dynamic light scattering, and transmission electron microscopy. OEI(800)-SeSe(x) showed much lower cytotoxicity than PEI(25k), and significantly higher transfection efficiency than OEI(800) in both B16F10 and HeLa cells. Transfection of luciferase in the OEI(800)-SeSe(x) group was comparable with that of standard PEI(25k) and traditional reduction-sensitive polymer OEI(800)-SS(x) groups. Furthermore, intracellular degradation of OEI(800)-SeSe(x) and dissociation with DNA were also confirmed by confocal laser scanning microscopy.

Conclusion: The OEI(800)-SeSe(x) obtained was able to bind plasmid DNA efficiently to yield nanosized particles and had reduction sensitivity which is as efficient as that for OEI(800)-SS(x). In vitro experiments confirmed its low cytotoxicity and high transfection ability. Diselenide bonds can be used as effective and novel reduction-sensitive linkages for gene delivery.

Keywords: diselenide; gene carriers; oligoethylenimine; reduction-sensitive.

Figure 1

Synthetic schema for OEI₈₀₀-SeSe_x (1), OEI₈₀₀-Se_x (2), and OEI₈₀₀-SS_x (3). Abbreviations: OEI, oligoethylenimine; NHS, N-hydroxysuccinimide; EDC, 1-ethyl-3-[3-(dimethylamino)-propyl] carbodiimide.

Figure 2

Sizes and zeta potentials of various complexes determined by Zetasizer Nano ZS. (A) Sizes of complexes composed of PEI_25k, OEI₈₀₀, or OEI₈₀₀ derivatives and genes at different C/P ratios, (B) zeta potentials of complexes composed of PEI_25k, OEI₈₀₀ or OEI₈₀₀ derivatives and genes at different C/P ratios, (C) changes in size with or without the reductive reagent (C/P 6), and (D) zeta potential changes with or without the reductive reagent (C/P 6, n = 3). Abbreviations: DTT, 1,4-dithiothreitol; OEI, oligoethylenimine; PEI, polyethylenimine.

Figure 3

Transmission electron microscopy results for PEI_25k, OEI₈₀₀, and OEI₈₀₀-derived complexes dropped onto amorphous carbon-coated copper grids, dried, and dyed using phosphomolybdic acid. Note: Bar = 200 nm. Abbreviations: DTT, 1,4-dithiothreitol; OEI, oligoethylenimine; PEI, polyethylenimine.

Figure 4

Gel retardation assay of various cationic polymers complexed with plasmid DNA. (A) OEI₈₀₀-SS_x, (B) OEI₈₀₀-SeSe_x, (C) OEI₈₀₀-Se_x, (D) OEI₈₀₀, and (E) PEI_25k. Notes: Polymers and plasmid DNA were mixed at various mass ratios and incubated at 37°C for 30 minutes with or without DTT. The mixed suspension was subsequently applied to the agarose gel and electrophoresed at 85 V for 40 minutes. Abbreviations: DTT, 1,4-dithiothreitol; OEI, oligoethylenimine; PEI, polyethylenimine.

Figure 5

Cytotoxicity assays of the OEI₈₀₀ derivatives, OEI₈₀₀, and PEI_25k in (A) B16F10 and (B) HeLa cells. Note: The polymers were incubated with the cells for 24 hours (n = 4). Abbreviations: OEI, oligoethylenimine; PEI, polyethylenimine.

Figure 6

Luciferase transfection of the polycation-pGL3 complexes in (A) B16F10 cells and (B) HeLa cells. Notes: All the complexes were prepared in HEPES buffered glucose, and 200 ng of pGL3 per well was used for the 96-well tissue culture plate. The data show the average for experiments performed at least three times (n = 3, *P < 0.05). Abbreviations: OEI, oligoethylenimine; PEI, polyethylenimine; RLU, relative light units.

Figure 7

In vitro transfection efficiency of the complexes of pEGFP with OEI₈₀₀-SeSe_x, OEI₈₀₀-Se_x, OEI₈₀₀-SS_x, PEI_25k, and OEI₈₀₀ in B16F10 cell lines at 48 hours. Note: Bar = 200 μm. Abbreviations: OEI, oligoethylenimine; PEI, polyethylenimine; pEGFP, enhanced green fluorescent protein encoding plasmid.

Figure 8

Percentages of transfected cells for the complexes of pEGFP with OEI₈₀₀-SeSe_x, OEI₈₀₀-Se_x, OEI₈₀₀-SS_x, PEI_25k, and OEI₈₀₀ quantified by flow cytometry analysis in B16F10 cells at 48 hours Note: n = 3. Abbreviations: OEI, oligoethylenimine; PEI, polyethylenimine; pEGFP, enhanced green fluorescent protein encoding plasmid.

Figure 9

Labeled DNA and cationic polymer for intracellular investigation in B16F10 cells. Red dots: Cy5-labeled DNA; green dots: FITC-labeled PEI or OEI₈₀₀ derivatives. Note: Bar = 25 μm. Abbreviations: OEI, oligoethylenimine; PEI, polyethylenimine.

Figure 10

Schematic illustration of decondensation of reduction-sensitive complexes in the intracellular environment.