Interleukin-12 Plasmid DNA Delivery by N-[(2-Hydroxy-3-trimethylammonium)propyl]chitosan-Based Nanoparticles

Abstract

Cationic polysaccharides are capable of forming polyplexes with nucleic acids and are considered promising polymeric gene carriers. The objective of this study was to evaluate the transfection efficiency and cytotoxicity of N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan salt (HTCS), a quaternary ammonium derivative of chitosan (CS), which benefits from non-ionizable positive charges. In this work, HTCS with a full quaternization of amino groups and a molar mass of 130,000 g·mol^-1 was synthesized to use for delivery of a plasmid encoding the interleukin-12 (IL-12) gene. Thus, a polyplex based on HTCS and the IL-12 plasmid was prepared and then was characterized in terms of particle size, zeta potential, plasmid condensation ability, and protection of the plasmid against enzymatic degradation. We showed that HTCS was able to condense the IL-12 plasmid by the formation of polyplexes in the range of 74.5 ± 0.75 nm. The level of hIL-12 production following the transfection of the cells with HTCS polyplexes at a C/P ratio of 8:1 was around 4.8- and 2.2-fold higher than with CS and polyethylenimine polyplexes, respectively. These findings highlight the role of HTCS in the formation of polyplexes for the efficient delivery of plasmid DNA.

Keywords: gene delivery; interleukin-12; nanoparticles; polyplex; quaternized chitosan.

Figure 1

(a) ¹H NMR (400 MHz) and (b) ¹³C NMR (100 MHz) spectra of CS (10 mg·mL⁻¹) and HTCS (20 mg·mL⁻¹) in D₂O/DCl at 65 °C. The chemical structures of sugar units are inserted.

Figure 2

Buffering capacity curves of NaCl, CS, and HTCS in pH ranging from 3.0 to 11.0.

Figure 3

The water solubility of CS and HTCS at pH ranging from 3 to 12. The transmittance of the polymeric solution was measured with UV-vis.

Figure 4

Plasmid DNA binding affinity of PEI, CS, and HTCS determined by gel retardation assay at C/P ratios of 0.5:1, 4:1, and 8:1.

Figure 5

DNase I protection assay for PEI, CS, and HTCS. The polyplexes were treated with DNase I or PBS (negative control).

Figure 6

(a) Cell viability of MCF-7 and HepG2 cells as a function of concentration of PEI, CS, and HTCS in the medium. ^a p < 0.05, CS and HTCS compared with PEI at the same concentration; ^b p < 0.01, CS and HTCS compared with PEI at the same concentration; ^c p < 0.001, CS and HTCS compared with PEI at the same concentration. * p < 0.05, HTCS compared with CS at the same concentration; ** p < 0.01, HTCS compared with CS at the same concentration; *** p < 0.001, HTCS compared with CS at the same concentration. (b) Gene transfer ability of PEI, CS, and HTCS. The levels of hIL-12 in MCF-7 cells and HepG2 cell following treatment with polyplexes at C/P ratios of 0.5, 4, and 8. The level of hIL-12 expression is presented as the concentration of the protein (pg·mL⁻¹) per seeded cell. ^a p < 0.05, CS and HTCS compared with PEI at the same C/P ratio; ^b p < 0.01, CS and HTCS compared with PEI at the same C/P ratio; ^c p < 0.001, CS and HTCS compared with PEI at the same C/P ratio. * p < 0.05, HTCS compared with CS at the same C/P ratio; ** p < 0.01, HTCS compared with CS at the same C/P ratio; *** p < 0.001, HTCS compared to CS at the same C/P ratio.