Department of Chemistry and Environmental Sciences, IBILCE, São Paulo State University-UNESP, São José do Rio Preto, São Paulo, Brazil.
Nanotechnology. 2013 Feb 8;24(5):055101. doi: 10.1088/0957-4484/24/5/055101. Epub 2013 Jan 11.
Chitosan has been indicated as a safe and promising polycation vector for gene delivery. However its low transfection efficiency has been a challenging obstacle for its application. To address this limitation, we synthesized chitosan derivatives which had increasing amounts of diethylethylamine groups (DEAE) attached to the chitosan main chain. The plasmid DNA VR1412 (pDNA), encoding the ß-galactosidase (ß-gal) reporter gene was used to prepare nanoparticles with the chitosan derivatives, and the transfection studies were performed with HeLa cells. By means of dynamic light scattering and zeta potential measurements, it was shown that diethylethylamine-chitosan derivatives (DEAE(x)-CH) were able to condense DNA into small particles having a surface charge depending on the polymer/DNA ratio (N/P ratio). Nanoparticles prepared with derivatives containing 15 and 25% of DEAE groups (DEAE(15)-CH and DEAE(25)-CH) exhibited transfection efficiencies ten times higher than that observed with deacetylated chitosan (CH). For derivatives with higher degrees of substitution (DS), transfection efficiency decreased. The most effective carriers showed low cytotoxicity and good transfection activities at low charge ratios (N/P). Vectors with low DS were easily degraded in the presence of lysozyme at physiological conditions in vitro and the nontoxicity displayed by these vectors opens up new opportunities in the design of DEAE-chitosan-based nanoparticles for gene delivery.
壳聚糖已被证明是一种安全且有前途的阳离子载体，可用于基因传递。然而，其低转染效率一直是其应用的一个挑战性障碍。为了解决这一限制，我们合成了壳聚糖衍生物，其主链上附着了越来越多的二乙胺乙基（DEAE）基团。将携带β-半乳糖苷酶（β-gal）报告基因的质粒 DNA VR1412（pDNA）用于制备壳聚糖衍生物的纳米颗粒，并在 HeLa 细胞中进行转染研究。通过动态光散射和zeta 电位测量表明，二乙胺乙基壳聚糖衍生物（DEAE(x)-CH）能够将 DNA 凝聚成具有表面电荷的小颗粒，这取决于聚合物/DNA 的比例（N/P 比）。含有 15%和 25% DEAE 基团的衍生物（DEAE(15)-CH 和 DEAE(25)-CH）制备的纳米颗粒的转染效率比脱乙酰壳聚糖（CH）高 10 倍。对于取代度更高的衍生物，转染效率降低。最有效的载体在低电荷比（N/P）下表现出低细胞毒性和良好的转染活性。在体外生理条件下，低取代度的载体很容易被溶菌酶降解，这些载体的无毒性质为基于 DEAE 壳聚糖的纳米载体的基因传递设计开辟了新的机会。
