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

Figures & data

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.

Table 1 Molecular weights of various polymers detected by gel permeation chromatography in the absence or presence of reduction reagents

Polymers	Mw/10^3 Da	Mn/10^3 Da	PDI
OEI800-SSx	12	3.9	3.1
OEI800-SSx + GSH	1.6	0.78	2.1
OEI800-SSx + DTT	0.99	0.75	1.3
OEI800-SeSex	18	5.3	3.4
OEI800-SeSex + GSH	8.1	2.5	3.2
OEI800-SeSex + DTT	3.9	2.6	1.5
OEI800-SeSex + NaBH4	1.4	0.79	1.8
OEI800-Sex	15	5.7	2.8
OEI800-Sex + NaBH4	15	5.8	2.7
PEI25k	16	4.5	3.6
OEI800	0.76	0.52	1.5

Abbreviations: DTT, 1,4-dithiothreitol; M_w, weight-average molecular weight; M_n, number-average molecular weight; PDI, polydispersity index; OEI, oligoethylenimine; PEI, polyethylenimine; GSH, glutathione.

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.

Figure S1 NMR spectrum of DSeDPA. (A) ⁷⁷Se NMR spectrum; (B) ¹H NMR spectrum; (C) ¹³C NMR spectrum.

Abbreviation: DSeDPA, 3, 3’-Diselanediyldipropanoic acid; NMR, nuclear magnetic resonance.

Figure S2 NMR spectrum of SeDPA. (A) ¹H NMR spectrum; (B) ¹³C NMR spectrum.

Abbreviation: SeDPA, 3, 3’-selenodipropanoic acid; NMR, nuclear magnetic resonance.

Figure S3 GPC traces of OEI₈₀₀-SS_x, OEI₈₀₀-SeSe_x, OEI₈₀₀-Se_x and the OEI₈₀₀ derivatives in the absence or presence of reduction treatment.

Abbreviations: GPC, gel permeation chromatography; OEI, oligoethylenimine; DTT, 1,4-dithiothreitol; GSH, glutathione.

Figure S4 The percentage of transfected cell for the complexes of pEGFP with OEI₈₀₀-SeSe_x, OEI₈₀₀-Se_x, OEI₈₀₀-SS_x, PEI_25k and OEI₈₀₀ was quantified by flow cytometry analysis in B16F10 cell lines at 48 hours.

Note: n = 3.

Abbreviations: pEGFP, enhanced green fluorescent protein encoding plasmid; PEI, polyethylenimine; OEI, oligoethylenimine.