Exploring phosphonium and ammonium chitosan polymers and their PEGylated analogs for high performance gene delivery

Abstract

• New chitosan derivatives containing triethylammonium chloride or triethylphosphonium chloride and their PEGylated analogs have been synthesized and characterized. • The derivatives efficiently bind pDNA at low weight ratio of derivative:pDNA of 2.8:1. • Apoptosis measurements by flow cytometry indicated that the derivatives containing triethylammonium chloride or triethylphosphonium chloride and PEG induce no perceptible and mild apoptosis with their concentration at 100 µg/mL. • The present study provides evidence that these PEGylated derivatives are safe and effective vectors for efficient nucleic acid delivery. Among cationic polymers, chitosan (CS) is a promising non-viral vector due to its biocompatibility, biodegradability and safety profile. However, its application for gene delivery is hindered by its poor solubility at physiological pH. In this study, novel water-soluble chitosan derivatives containing triethylphosphonium chloride ((N-(1-carboxybutyl-4-triethylphosphonium chloride) chitosan = TEPB-CS)) or triethylammonium chloride ((N-(1-carboxybutyl-4-triethylammonium chloride) chitosan = TEAB-CS)) with 4-carbon spacers separating the cationic units from the chitosan backbone were synthesized by carbodiimide mediated coupling reactions. Two different degrees of substitution of quaternary phosphonium (43% and 21%) and ammonium (40.6% and 26%) were synthesized. The polymers with higher degrees of substitutions displayed higher solubility in aqueous media and were further grafted with mPEG (TEAB-CS-g-mPEG and TEPB-CS-g-mPEG) and used for DNA complexation, cytotoxicity, apoptosis, and gene transfection studies. Chemical characterization of the above polymers was performed by FTIR, 1 H NMR, and 31 P NMR, and the degree of substitution of the grafted phosphonium and ammonium and mPEG was calculated by 1 H NMR. The polymers efficiently bound pDNA at weight ratio of polymer:pDNA of 2.8:1. From dynamic light scattering experiments, the mean diameters of the calf thymus DNA complexes with the polymers, in water and phosphate buffer (pH 7.4), were in the range of 140 – 330 nm. Zeta potential of these complexes in water became positive as the weight ratios of the polymers/DNA exceeded 2:1. Most of these polymers mildly affect the viability of HeLa cells with a final concentration of 10 µg/mL or higher. Cell viability was only modestly compromised in the presence of 100 µg/mL of TEPB-CS-g-mPEG, and this derivative was also found to display the highest transfection efficiencies among all four polymers. Apoptosis measurements by flow cytometry indicated that both TEAB-CS-g-mPEG and TEPB-CS-g-mPEG induce no perceptible and mild apoptosis with their concentration at 100 µg/mL, respectively. Together, the present study provides evidence that these PEGylated TEPB-CS and TEAB-CS are safe and effective vectors for efficient nucleic acid delivery.