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, ¹H NMR, and ³¹P NMR, and the degree of substitution of the grafted phosphonium and ammonium and mPEG was calculated by ¹H 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.

在阳离子聚合物中，壳聚糖 (CS) 因其生物相容性、生物降解性和安全性而成为一种很有前途的非病毒载体。然而，其在生理 pH 值下的溶解度较差，阻碍了其在基因传递中的应用。在本研究中，新型水溶性壳聚糖衍生物含有三乙基氯化鏻（（N-（1-羧丁基-4-三乙基氯化磷）壳聚糖 = TEPB-CS））或三乙基氯化铵（（N-（1-羧丁基-4-三乙基氯化铵） ) 壳聚糖 = TEAB-CS)) 通过碳二亚胺介导的偶联反应合成具有将阳离子单元与壳聚糖骨架分开的 4 碳间隔基。合成了两种不同取代度的季鏻（43% 和 21%）和铵（40.6% 和 26%）。具有较高取代度的聚合物在水性介质中显示出较高的溶解度，并进一步接枝了 mPEG（TEAB-CS-g-mPEG 和 TEPB-CS-g-mPEG）并用于 DNA 复合、细胞毒性、细胞凋亡和基因转染研究. 上述聚合物的化学表征通过 FTIR 进行，¹ H NMR和³¹ P NMR，接枝的鏻和铵和mPEG的取代度由¹计算核磁共振。聚合物以 2.8:1 的聚合物与 pDNA 的重量比有效结合 pDNA。根据动态光散射实验，在水和磷酸盐缓冲液 (pH 7.4) 中，小牛胸腺 DNA 与聚合物复合物的平均直径在 140 – 330 nm 范围内。当聚合物/DNA 的重量比超过 2:1 时，这些复合物在水中的 Zeta 电位变为正值。大多数这些聚合物对 HeLa 细胞的生存能力有轻微影响，最终浓度为 10 µg/mL 或更高。在存在 100 µg/mL TEPB-CS-g-mPEG 的情况下，细胞活力仅受到适度影响，并且还发现该衍生物在所有四种聚合物中显示出最高的转染效率。通过流式细胞术测量的细胞凋亡表明，TEAB-CS-g-mPEG 和 TEPB-CS-g-mPEG 分别在 100 µg/mL 的浓度下均未诱导明显和轻度的细胞凋亡。总之，本研究提供的证据表明，这些聚乙二醇化的 TEPB-CS 和 TEAB-CS 是安全有效的载体，可用于高效的核酸递送。