Transfection efficiency of chitosan and thiolated chitosan in retinal pigment epithelium cells: A comparative study - PubMed (original) (raw)

Transfection efficiency of chitosan and thiolated chitosan in retinal pigment epithelium cells: A comparative study

Ana V Oliveira et al. J Pharm Bioallied Sci. 2013 Apr.

Abstract

Objective: Gene therapy relies on efficient vector for a therapeutic effect. Efficient non-viral vectors are sought as an alternative to viral vectors. Chitosan, a cationic polymer, has been studied for its gene delivery potential. In this work, disulfide bond containing groups were covalently added to chitosan to improve the transfection efficiency. These bonds can be cleaved by cytoplasmic glutathione, thus, releasing the DNA load more efficiently.

Materials and methods: Chitosan and thiolated chitosan nanoparticles (NPs) were prepared in order to obtain a NH3(+):PO(4) (-) ratio of 5:1 and characterized for plasmid DNA complexation and release efficiency. Cytotoxicity and gene delivery studies were carried out on retinal pigment epithelial cells.

Results: In this work, we show that chitosan was effectively modified to incorporate a disulfide bond. The transfection efficiency of chitosan and thiolated chitosan varied according to the cell line used, however, thiolation did not seem to significantly improve transfection efficiency.

Conclusion: The apparent lack of improvement in transfection efficiency of the thiolated chitosan NPs is most likely due to its size increase and charge inversion relatively to chitosan. Therefore, for retinal cells, thiolated chitosan does not seem to constitute an efficient strategy for gene delivery.

Keywords: Chitosan; gene therapy; non-viral vectors; thiolation; transfection efficiency.

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Conflict of interest statement

Conflict of Interest: None declared.

Figures

Figure 1

Figure 1

Reaction mechanism of N-succinimidyl-3-(2-pyridyldithio)- propionate (2) with chitosan, (1) resulting in chitosan-3-(2-pyridyldithio) propionyl (CS-PDP) (3) and hydroxysuccinimide. CS-PDP reacts with mercaptoetilamine salt, (4) producing chitosan-3-(2-Aminoethyldithio) propionyl-chitosan-3-(2-aminoethyldithio) propionyl (5) and pyridine- 2-thione (6)

Figure 2

Figure 2

Both chitosan (CS) and chitosan-3-(2-aminoethyldithio) propionyl (CS-(AEDTP)) nanoparticles (NPs) effectively protect DNA from DNAse degradation, as analyzed in a 1% agarose gel electrophoresis, with DNA visualized by GreenSafe Premium. Lanes: (a) DNA marker, (b) Plasmid DNA (pDNA), (c) pDNA + DNAse I, (d) CS-pDNA NPs, (e) CS-pDNA NPs + DNAse I, (f) CS-(AEDTP)- pDNA NPs, (g) CS-(AEDTP)-pDNA + DNAse I

Figure 3

Figure 3

DNA retention by chitosan-3-(2-aminoethyldithio) propionyl (CS-(AEDTP)) nanoparticles (NPs) after 24h of incubation with: A) Increasing concentrations of dithiothreitol; Lanes: (a) DNA marker, (b) Plasmid DNA (pDNA), (c) CS-(AEDTP) NPs, (d) to i) range from 10 mM to 100 mM according to the image. B) 0.4 M gluthatione reduced-form; Lanes: (a) DNA marker, (b) pDNA, (c) CS-(AEDTP) NPs, (d) CS-(AEDTP) NPs + NADPH + phosphate buffer, (e) CS-(AEDTP) NPs + glutathione reductase + NADPH + phosphate buffer, (f) CS-(AEDTP) NPs + PB + NADPH + phosphate buffer, (g) CS-(AEDTP) NPs + PB + GR+ NADPH + phosphate buffer. This was analyzed in a 1% agarose gel electrophoresis, with DNA visualized by GreenSafe Premium

Figure 4

Figure 4

Transmission electron microscopy microphotographs of chitosan (CS) nanoparticles and DNA loaded nanoparticles (CSDNA 5:1)

Figure 5

Figure 5

Cell survival (%) as a function of CS and chitosan-3- (2-aminoethyldithio) propionyl nanoparticles amount (μg of polymer); D407 and ARPE-19 cells were incubated for 72 h with the various concentrations of nanoparticles; (C+) untreated cells, (C−) cells treated with latex extract. Vertical bars = S.D. The number of *indicates significantly different sets of data

Figure 6

Figure 6

Transfection efficiency represented as GFP positive cell percentage as a function of polymer

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