Department of Pharmaceutical and Pharmacological Sciences , University of Padova , Via F. Marzolo 5 35131 Padova , Italy.
Department of Chemistry and Department of Physics , University of Illinois at Chicago , Chicago , Illinois 60607 , United States.
Mol Pharm. 2019 Apr 1;16(4):1678-1693. doi: 10.1021/acs.molpharmaceut.9b00014. Epub 2019 Mar 21.
A novel unconventional supramolecular oligo-cationic structure (Agm-M-PEG-OCH) has been synthesized to yield high efficiency therapeutic oligonucleotide (ON) delivery. Agm-M-PEG-OCH was obtained by a multistep protocol that included the conjugation of agmatine (Agm) moieties to maltotriose (M), which was further derivatized with one poly(ethylene glycol) (PEG) chain. Gel electrophoresis analysis showed that the 19 base pairs dsDNA model ON completely associates with Agm-M-PEG-OCH at 3 N/P molar ratio, which is in agreement with the in silico molecular predictions. Isothermal titration calorimetry (ITC) analyses showed that the Agm-M-PEG-OCH/ON association occurs through a combination of mechanisms depending on the N/P ratios resulting in different nanostructures. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that the Agm-M-PEG-OCH/ON polyplexes have rod-shape structure with a mean diameter of 50-75 nm and aspect ratio depending on the N/P ratio. The polyplexes were stable over time in buffer, while a slight size increase was observed in the presence of serum proteins. Cell culture studies showed that neither Agm-M-PEG-OCH nor polyplexes displayed cytotoxic effects. Cellular uptake depended on the cell line and polyplex composition: cellular internalization was higher in the case of MCF-7 and KB cells compared to MC3T3-E1 cells and polyplexes with smaller aspect ratio were taken-up by cells more efficiently than polyplexes with higher aspect ratio. Finally, preliminary studies showed that our novel carrier efficiently delivered ONs into cells providing gene silencing.
一种新型非常规超分子寡阳离子结构（Agm-M-PEG-OCH）已被合成，以实现高效治疗寡核苷酸（ON）传递。Agm-M-PEG-OCH 通过多步方案获得，包括将胍丁胺（Agm）部分连接到麦芽三糖（M）上，然后进一步用一个聚乙二醇（PEG）链衍生。凝胶电泳分析表明，19 个碱基对的双链 DNA 模型 ON 完全与 3 N/P 摩尔比的 Agm-M-PEG-OCH 结合，这与计算机分子预测一致。等温热力学滴定（ITC）分析表明，Agm-M-PEG-OCH/ON 结合是通过取决于 N/P 比的多种机制发生的，导致不同的纳米结构。动态光散射（DLS）和透射电子显微镜（TEM）显示，Agm-M-PEG-OCH/ON 超分子复合物具有棒状结构，平均直径为 50-75nm，取决于 N/P 比的纵横比。在缓冲液中，超分子复合物随时间稳定，而在存在血清蛋白时观察到轻微的尺寸增加。细胞培养研究表明，Agm-M-PEG-OCH 或超分子复合物均无细胞毒性作用。细胞摄取取决于细胞系和超分子复合物的组成：MCF-7 和 KB 细胞的细胞内摄取高于 MC3T3-E1 细胞，并且具有较小纵横比的超分子复合物比具有较高纵横比的超分子复合物更有效地被细胞摄取。最后，初步研究表明，我们的新型载体能够有效地将 ON 递送到细胞中，从而实现基因沉默。
