Pharmaceutical Biotechnology, Center for NanoScience (CeNS), Ludwig-Maximilians-University (LMU) Munich, Butenandtstrasse 5-13, D-81377 Munich, Germany; School of Chemistry and Chemical Engineering, South China University of Technology, 510640 Guangzhou, People's Republic of China.
Pharmaceutical Biotechnology, Center for NanoScience (CeNS), Ludwig-Maximilians-University (LMU) Munich, Butenandtstrasse 5-13, D-81377 Munich, Germany; Nanosystems Initiative Munich, Schellingstraße 4, D-80799 Munich, Germany.
J Control Release. 2014 Apr 28;180:42-50. doi: 10.1016/j.jconrel.2014.02.015. Epub 2014 Feb 22.
Native chemical ligation (NCL) was established for the conversion of sequence-defined oligomers of different topologies into targeted and PEG shielded pDNA and siRNA carriers. From an existing library of non-targeted oligoethanamino amides, six oligomers containing N-terminal cysteines were selected as cationic cores, to which monodisperse polyethylene glycol (PEG) containing terminal folic acid as targeting ligand (or terminal alanine as targeting negative control ligand) were attached by NCL. Ligated conjugates plus controls (in sum 18 oligomers) were evaluated for pDNA or siRNA gene delivery. Biophysical characteristics including nucleic acid binding in the absence or presence of serum, as well as biological activities in cellular uptake and gene transfer (or gene silencing, respectively) were determined. In most cases, the folic acid-PEG-ligated oligomers displayed a strongly improved cellular binding, uptake and gene transfer into receptor-positive KB cells as compared to the alanine-PEG controls. Changing the topological structures by increasing the number of cationic arms, adding tyrosine trimers as polyplex stabilizing domains, or histidines facilitating endosomal escape resulted in beneficial gene transfer characteristics. The screen revealed different requirements for pDNA and siRNA delivery. A folate-PEG ligated histidinylated four-arm oligomer was most effective for pDNA delivery but inactive for siRNA, whereas a folate-PEG-ligated three-arm oligomer with tyrosine trimer modifications was most effective in siRNA mediated gene silencing. The results demonstrate the site-selective NCL reaction as powerful method to modify existing oligomers. Thus multifunctional targeted carriers can be obtained with ease and used to identify lead structures for subsequent in vivo delivery.
天然化学连接（NCL）被建立用于将不同拓扑结构的序列定义的寡聚物转化为靶向和聚乙二醇屏蔽的 pDNA 和 siRNA 载体。从现有的非靶向寡乙酰胺文库中，选择了六个含有 N 端半胱氨酸的寡聚物作为阳离子核心，通过 NCL 将含有末端叶酸的单分散聚乙二醇（作为靶向配体或末端丙氨酸作为靶向阴性对照配体）连接到寡聚物上。连接的缀合物加对照物（共 18 个寡聚物）用于 pDNA 或 siRNA 基因传递的评估。评估了包括在有无血清存在下与核酸结合的生物物理特性，以及细胞摄取和基因转导（或基因沉默）的生物学活性。在大多数情况下，与丙氨酸-PEG 对照物相比，叶酸-PEG 连接的寡聚物在受体阳性 KB 细胞中的细胞结合、摄取和基因转移方面表现出显著改善。通过增加阳离子臂的数量、添加酪氨酸三聚体作为多聚物稳定结构域或组氨酸促进内涵体逃逸来改变拓扑结构，导致有益的基因转移特性。筛选揭示了 pDNA 和 siRNA 传递的不同要求。对于 pDNA 传递，带有组氨酸的叶酸-PEG 连接的四臂寡聚物最有效，但对 siRNA 无效，而带有酪氨酸三聚体修饰的叶酸-PEG 连接的三臂寡聚物在 siRNA 介导的基因沉默中最有效。结果表明，NCL 反应作为一种强大的方法，可以对现有寡聚物进行修饰。因此，可以轻松获得多功能靶向载体，并用于鉴定后续体内递送的先导结构。
