Endres Thomas, Zheng Mengyao, Kılıç Ayşe, Turowska Agnieszka, Beck-Broichsitter Moritz, Renz Harald, Merkel Olivia M, Kissel Thomas
Department of Pharmaceutics and Biopharmacy, Philipps-Universität Marburg , Ketzerbach 63, 35037 Marburg, Germany.
Mol Pharm. 2014 Apr 7;11(4):1273-81. doi: 10.1021/mp400744a. Epub 2014 Mar 12.
Amphiphilic triblock copolymers represent a versatile delivery platform capable of co-delivery of nucleic acids, drugs, and/or dyes. Multifunctional cationic triblock copolymers based on poly(ethylene glycol), poly-ε-caprolactone, and polyethylene imine, designed for the delivery of siRNA, were evaluated in vitro and in vivo. Moreover, a nucleic acid-unpacking-sensitive imaging technique based on quantum dot-mediated fluorescence resonance energy transfer (QD-FRET) was established. Cell uptake in vitro was measured by flow cytometry, whereas transfection efficiencies of nanocarriers with different hydrophilic block lengths were determined in vitro and in vivo by quantitative real-time PCR. Furthermore, after the proof of concept was demonstrated by fluorescence spectroscopy/microscopy, a prototype FRET pair was established by co-loading QDs and fluorescently labeled siRNA. The hydrophobic copolymer mediated a 5-fold higher cellular uptake and good knockdown efficiency (61 ± 5% in vitro, 55 ± 18% in vivo) compared to its hydrophilic counterpart (13 ± 6% in vitro, 30 ± 17% in vivo), which exhibited poor performance. FRET was demonstrated by UV-induced emission of the acceptor dye. Upon complex dissociation, which was simulated by the addition of heparin, a dose-dependent decrease in FRET efficiency was observed. We believe that in vitro/in vivo correlation of the structure and function of polymeric nanocarriers as well as sensitive imaging functionality for mechanistic investigations are prerequisites for a more rational design of amphiphilic gene carriers.
两亲性三嵌段共聚物是一种多功能递送平台，能够共同递送核酸、药物和/或染料。对基于聚乙二醇、聚-ε-己内酯和聚乙烯亚胺设计用于递送小干扰RNA（siRNA）的多功能阳离子三嵌段共聚物进行了体外和体内评估。此外，还建立了一种基于量子点介导的荧光共振能量转移（QD-FRET）的对核酸解包敏感的成像技术。体外细胞摄取通过流式细胞术测量，而不同亲水嵌段长度的纳米载体的转染效率则通过定量实时聚合酶链反应在体外和体内进行测定。此外，在通过荧光光谱/显微镜证明概念验证后，通过共负载量子点和荧光标记的siRNA建立了一个原型FRET对。与性能较差的亲水性对应物（体外为13±6%，体内为30±17%）相比，疏水性共聚物介导的细胞摄取量高5倍，且具有良好的敲低效率（体外为61±5%，体内为55±18%）。通过受体染料的紫外线诱导发射证明了FRET。通过添加肝素模拟复合物解离后，观察到FRET效率呈剂量依赖性降低。我们认为，聚合物纳米载体的结构与功能的体外/体内相关性以及用于机理研究的灵敏成像功能是更合理设计两亲性基因载体的先决条件。
