Grafting chitosan with polyethylenimine in an ionic liquid for efficient gene delivery.

Modifying chitosan (CS) with polyethylenimine (PEI) grafts is an effective way to improve its gene transfection performance. However, it is still a challenge to conduct the grafting with fine control and high efficiency, particularly for the modification of water-insoluble CS. Herein, a novel method to graft CS with PEI (1.8 kDa, PEI-1.8) was developed by using ionic liquid 1-butyl-3-methyl imidazolium acetate ([BMIM]Ac) as a reaction solvent, water-insoluble CS as a reaction substrate and 1,1-carbonyldiimidazole (CDI) as a linking agent. The grafting reaction was greatly accelerated and the reaction time was largely shortened to 4 h by taking advantages of the good solubility of CS, the enhanced nucleophilicity of amino groups and the preferential stability of the activated complexes in the ionic liquid. The chitosan-graft-polyethylenimine (CS-g-PEI) products were characterized by 1H NMR, FTIR and GPC. PEI-1.8 was quantitatively grafted to CS through urea linkages, and the grafting degree (GD) was conveniently tuned by varying the molar ratios of PEI-1.8 to D-glucosamine units of CS in the range of 9.0 × 10(-3) to 9.0 × 10(-2). Compared with CS, the synthesized CS-g-PEI copolymers showed higher pDNA-binding affinity, which increased with the GD as shown in Agarose gel electrophoresis. The dynamic light scattering (DLS) experiment demonstrated that the CS-g-PEI/pDNA polyplexes had suitable particle sizes and proper ζ-potentials for cell transfection. The CS-g-PEI copolymer with a medium GD of 4.5% conferred the best gene transfection, with the efficiency 44 times of CS and 38 times of PEI-1.8 in HEp-2 cells. The cytotoxicity of CS-g-PEI was tested and found nearly as low as that of CS and much lower than that of PEI.