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

Gang Cheng1 National Engineering Research Center for Biomaterials, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan, People’s Republic of China;2 Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan, People’s Republic of China

Yiyan He1 National Engineering Research Center for Biomaterials, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan, People’s Republic of China

Li Xie1 National Engineering Research Center for Biomaterials, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan, People’s Republic of China

Yu Nie1 National Engineering Research Center for Biomaterials, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan, People’s Republic of ChinaCorrespondence[email protected] [email protected]

Bin He1 National Engineering Research Center for Biomaterials, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan, People’s Republic of China

Zhirong Zhang2 Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan, People’s Republic of China

Zhongwei Gu1 National Engineering Research Center for Biomaterials, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan, People’s Republic of ChinaCorrespondence[email protected] [email protected]

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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 (OEI₈₀₀) via an active ester containing diselenide bonds. Disulfide bonds cross-linked with OEI₈₀₀-SS_x and monoselenide bonds linked with OEI₈₀₀-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 OEI₈₀₀ derivatives and Cy5-labeled DNA.

Results

Diselenide-conjugated OEI₈₀₀ (OEI₈₀₀-SeSe_x) polymer carriers of high molecular weight were successfully synthesized. After compacting with DNA, the OEI₈₀₀-SeSe_x polymers formed nanoparticles with an average size of 140 nm at an adequate C/P ratio. OEI₈₀₀-SeSe_x showed reduction-responsive degradation properties similar to those of the OEI₈₀₀-SS_x via gel permeation chromatography, dynamic light scattering, and transmission electron microscopy. OEI₈₀₀-SeSe_x showed much lower cytotoxicity than PEI_25k, and significantly higher transfection efficiency than OEI₈₀₀ in both B16F10 and HeLa cells. Transfection of luciferase in the OEI₈₀₀-SeSe_x group was comparable with that of standard PEI_25k and traditional reduction-sensitive polymer OEI₈₀₀-SS_x groups. Furthermore, intracellular degradation of OEI₈₀₀-SeSe_x and dissociation with DNA were also confirmed by confocal laser scanning microscopy.

Conclusion

The OEI₈₀₀-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 OEI₈₀₀-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:

Acknowledgments

This study was supported by the National 973 programs, 2011CB606206, National Natural Science Foundation of China (51133004, 81000657, 50830105, 31070849), Ministry of Science and Technology (2010DFA 51550), Research Fund for the Doctoral Program of Higher Education of China (20100181120075), and International Cooperative Foundation of Sichuan Province (2009HH0001)

Disclosure

The authors report no conflicts of interest in this work.

Supplementary figures

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 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 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 S3 GPC traces of OEI800-SSx, OEI800-SeSex, OEI800-Sex 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.