Institute of Biomedical Engineering, Department of Chemical Engineering, École Polytechnique, Montréal, QC, Canada.
Int J Nanomedicine. 2012;7:1399-414. doi: 10.2147/IJN.S26571. Epub 2012 Mar 13.
Chitosan, a natural polymer, is a promising system for the therapeutic delivery of both plasmid DNA and synthetic small interfering RNA. Reports attempting to identify the optimal parameters of chitosan for synthetic small interfering RNA delivery were inconclusive with high molecular weight at high amine-to-phosphate (N:P) ratios apparently required for efficient transfection. Here we show, for the first time, that low molecular weight chitosan (LMW-CS) formulations at low N:P ratios are suitable for the in vitro delivery of small interfering RNA. LMW-CS nanoparticles at low N:P ratios were positively charged (ζ-potential ~20 mV) with an average size below 100 nm as demonstrated by dynamic light scattering and environmental scanning electron microscopy, respectively. Nanoparticles were spherical, a shape promoting decreased cytotoxicity and enhanced cellular uptake. Nanoparticle stability was effective for at least 20 hours at N:P ratios above two in a slightly acidic pH of 6.5. At a higher basic pH of 8, these nanoparticles were unravelled due to chitosan neutralization, exposing their polynucleotide cargo. Cellular uptake ranged from 50% to 95% in six different cell lines as measured by cytometry. Increasing chitosan molecular weight improved nanoparticle stability as well as the ability of nanoparticles to protect the oligonucleotide cargo from nucleases at supraphysiological concentrations. The highest knockdown efficiency was obtained with the specific formulation 92-10-5 that combines sufficient nuclease protection with effective intracellular release. This system attained >70% knockdown of the messenger RNA, similar to commercially available lipoplexes, without apparent cytotoxicity. Contrary to previous reports, our data demonstrate that LMW-CS at low N:P ratios are efficient and nontoxic polynucleotide delivery systems capable of transfecting a plethora of cell lines.
壳聚糖是一种天然聚合物，是治疗性递送达质粒 DNA 和合成小干扰 RNA 的有前途的系统。试图确定壳聚糖用于合成小干扰 RNA 递送的最佳参数的报告尚无定论，高相对分子质量和高胺-磷（N：P）比显然是有效转染所必需的。在这里，我们首次表明，低相对分子质量壳聚糖（LMW-CS）制剂在低 N：P 比下适合小干扰 RNA 的体外递送。通过动态光散射和环境扫描电子显微镜分别证明，低 N：P 比的 LMW-CS 纳米粒带正电荷（ζ-电位约 20 mV），平均粒径小于 100nm。纳米粒呈球形，这种形状可降低细胞毒性并增强细胞摄取。纳米粒的稳定性在 N：P 比高于 2 时在略酸性 pH 值为 6.5 的情况下至少有效 20 小时。在较高的碱性 pH 值 8 下，由于壳聚糖中和，这些纳米粒被展开，暴露出其多核苷酸货物。通过细胞计数测量，在六种不同的细胞系中，细胞摄取率在 50％至 95％之间。增加壳聚糖的相对分子质量可提高纳米粒的稳定性，以及纳米粒保护寡核苷酸货物免受核酸酶在高于生理浓度的能力。具有足够的核酸酶保护和有效的细胞内释放的特定制剂 92-10-5 获得了最高的敲低效率。该系统获得了>70％的信使 RNA 敲低，与市售的脂质体类似，没有明显的细胞毒性。与先前的报告相反，我们的数据表明，低 N：P 比的 LMW-CS 是有效的、无毒的多核苷酸递送系统，能够转染多种细胞系。
