Department of Pharmacy, and Center for Nanoscience (CeNS), Ludwig-Maximilians Universität , Butenandtstrasse 5-13, D-81377 Munich, Germany.
Biomacromolecules. 2011 Jun 13;12(6):2039-47. doi: 10.1021/bm101464n. Epub 2011 Apr 27.
Aim of this study was the site-specific conjugation of an epidermal growth factor (EGF)-polyethylene glycol (PEG) chain by click chemistry onto a poly(amido amine) (PAMAM) dendron, as a key step toward defined multifunctional carriers for targeted gene delivery. For this purpose, at first propargyl amine cored PAMAM dendrons with ester ends were synthesized. The chain terminal ester groups were then modified by oligoamines with different secondary amino densities. The oligoamine-modified PAMAM dendrons were well biocompatible, as demonstrated in cytotoxicity assays. Among the different oligoamine-modified dendrons, PAMAM-pentaethylenehexamine (PEHA) dendron polyplexes displayed the best gene transfer ability. Conjugation of PAMAM-PEHA dendron with PEG spacer was conducted via click reaction, which was performed before amidation with PEHA. The resultant PEG-PAMAM-PEHA copolymer was then coupled with EGF ligand. pDNA transfections in HuH-7 hepatocellular carcinoma cells showed a 10-fold higher efficiency with the polyplexes containing conjugated EGF as compared to the ligand-free ones, demonstrating the concept of ligand targeting. Overall gene transfer efficiencies, however, were moderate, suggesting that additional measures for overcoming subsequent intracellular bottlenecks in delivery have to be taken.
本研究的目的是通过点击化学将表皮生长因子 (EGF)-聚乙二醇 (PEG) 链特异性连接到聚酰胺-胺 (PAMAM) 树枝状分子上，作为实现靶向基因传递的定义多功能载体的关键步骤。为此，首先合成了带有末端酯基的炔丙胺核 PAMAM 树枝状分子。然后，通过不同二级氨基密度的低聚胺修饰链末端酯基。细胞毒性测定表明，修饰后的低聚胺改性 PAMAM 树枝状分子具有良好的生物相容性。在不同的低聚胺改性树枝状分子中，PAMAM-五亚乙基六胺（PEHA）树枝状聚合物的基因传递能力最佳。通过点击反应将 PAMAM-PEHA 树枝状分子与 PEG 间隔臂进行连接，该反应在与 PEHA 进行酰胺化之前进行。然后将所得的 PEG-PAMAM-PEHA 共聚物与 EGF 配体偶联。与不含配体的聚合物相比，与 PAMAM-PEHA 树枝状分子偶联的 EGF 可使 HuH-7 肝癌细胞中的 pDNA 转染效率提高 10 倍，证明了配体靶向的概念。然而，总体基因转移效率适中，这表明必须采取额外的措施来克服传递过程中的后续细胞内瓶颈。
