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. 2020 May 5;12(5):1057.
doi: 10.3390/polym12051057.

Effect of Double Substitution in Cationic Chitosan Derivatives on DNA Transfection Efficiency

Veronika D Badazhkova  1 , Sergei V Raik  1   2 , Dmitry S Polyakov  3 , Daria N Poshina  1 , Yury A Skorik  1
Affiliations

Effect of Double Substitution in Cationic Chitosan Derivatives on DNA Transfection Efficiency

Veronika D Badazhkova et al. Polymers (Basel). .

Abstract

Recently, much effort has been expended on the development of non-viral gene delivery systems based on polyplexes of nucleic acids with various cationic polymers. Natural polysaccharide derivatives are promising carriers due to their low toxicity. In this work, chitosan was chemically modified by a reaction with 4-formyl-n,n,n-trimethylanilinium iodide and pyridoxal hydrochloride and subsequent reduction of the imine bond with NaBH4. This reaction yielded three novel derivatives, n-[4-(n',n',n'-trimethylammonium)benzyl]chitosan chloride (TMAB-CS), n-[(3-hydroxy-5-(hydroxymethyl)-2-methyl-4-pyridine)methyl]chitosan chloride (Pyr-CS), and n-[4-(n',n',n''-trimethylammonium)benzyl]-n-[(3-hydroxy-5-(hydroxymethyl)-2-methyl-4-pyridine)methyl]chitosan chloride (PyrTMAB-CS). Their structures and degrees of substitution were established by 1H NMR spectroscopy as DS1 = 0.22 for TMAB-CS, DS2 = 0.28 for Pyr-CS, and DS1 = 0.21, DS2 = 0.22 for PyrTMAB-CS. Dynamic light scattering measurements revealed that the new polymers formed stable polyplexes with plasmid DNA encoding the green fluorescent protein (pEGFP-N3) and that the particles had the smallest size (110-165 nm) when the polymer:DNA mass ratio was higher than 5:1. Transfection experiments carried out in the HEK293 cell line using the polymer:DNA polyplexes demonstrated that Pyr-CS was a rather poor transfection agent at polymer:DNA mass ratios less than 10:1, but it was still more effective than the TMAB-CS and PyrTMAB-CS derivatives that contained a quaternary ammonium group. By contrast, TMAB-CS and PyrTMAB-CS were substantially more effective than Pyr-CS at higher polymer:DNA mass ratios and showed a maximum efficiency at 200:1 (50%-70% transfected cells). Overall, the results show the possibility of combining substituent effects in a single carrier, thereby increasing its efficacy.

Keywords: cell transfection; chitosan; gene delivery; polyplex.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Synthesis scheme for n-[(3-hydroxy-5-(hydroxymethyl)-2-methyl-4-pyridine)methyl]chitosan chloride (Pyr-CS) (a), n-[4-(n’,n’,n’-trimethylammonium)benzyl]chitosan chloride (TMAB-CS) (b), and n-[4-(n’,n’,n’-trimethylammonium)benzyl]-n-[(3-hydroxy-5-(hydroxymethyl)-2-methyl-4-pyridine)methyl]chitosan chloride (PyrTMAB-CS) (c).
Figure 2
Figure 2
1H NMR spectra of CS (1), TMAB-CS (2), Pyr-CS (3), and PyrTMAB-CS (4).
Figure 3
Figure 3
Ionization scheme of Pyr-CS units.
Figure 4
Figure 4
Evolution of the UV spectrum of Pyr-CS (0.125 mg/mL) with changing pH.
Figure 5
Figure 5
The hydrodynamic radii of TMAB-CS:DNA, Pyr-CS:DNA, and PyrTMAB-CS:DNA polyplexes.
Figure 6
Figure 6
DNA binding ability for Pyr-CS, PyrTMAB-CS, and TMAB-CS as determined by the relative fluorescence changes.
Figure 7
Figure 7
Atomic force microscopy (AFM) images of DNA-polyplexes with TMAB-CS (a), Pyr-CS (b), and PyrTMAB-CS (c,d).
Figure 8
Figure 8
Transfection efficiency of polyplexes at different polymer:DNA mass ratios. Individual points represent mean ± SD, n = 8.
Figure 9
Figure 9
Transfection efficiency of polyplexes at 200:1 polymer:DNA mass ratio compared to that of Lipofectin. Each column represents the so-called “five-number summary” and describes a list of five values: the minimum, the 5th percentile, the median, the 95th percentile, and the maximum. Horizontal brackets with asterisks indicate statistically significant differences: * p < 0.01; ** p < 0.001.
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