Amphiphilic Biodegradable PEG-PCL-PEI Triblock Copolymers for FRET-Capable in Vitro and in Vivo Delivery of siRNA and Quantum Dots
Amphiphilic Biodegradable PEG-PCL-PEI Triblock Copolymers for FRET-Capable in Vitro and in Vivo Delivery of siRNA and Quantum Dots
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DOI：
10.1021/mp400744a
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发表时间：
2014-04-01
影响因子：
4.9
通讯作者：
Kissel, Thomas
中科院分区：
文献类型：
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作者：
Endres, Thomas;Zheng, Mengyao;Kissel, Thomas
两亲性三嵌段共聚物代表了能够共同递送核酸、药物和/或染料的多功能递送平台。设计用于递送 siRNA 的基于聚乙二醇、聚ε-己内酯和聚乙烯亚胺的多功能阳离子三嵌段共聚物在体外和体内进行了评估。此外，建立了基于量子点介导的荧光共振能量转移（QD-FRET）的核酸解包敏感成像技术。通过流式细胞术测量体外细胞摄取，而通过定量实时PCR测定具有不同亲水块长度的纳米载体的体外和体内转染效率。此外，在通过荧光光谱/显微镜证明概念验证后，通过共同加载量子点和荧光标记的 siRNA 建立了原型 FRET 对。与亲水性共聚物（体外13+/-6％，体内30+/-17％）相比，疏水性共聚物介导了5倍高的细胞摄取和良好的击倒效率（体外61+/-5％，体内55+/-18％），而亲水性共聚物表现出较差的性能。 FRET 通过受体染料的紫外线诱导发射得到证实。通过添加肝素模拟复合物解离后，观察到 FRET 效率呈剂量依赖性下降。我们相信，聚合物纳米载体结构和功能的体外/体内相关性以及用于机理研究的敏感成像功能是更合理设计两亲性基因载体的先决条件。
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-epsilon-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.