Biophysical Characterization of Copolymer-Protected Gene Vectors

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• 作者：Daniel Hnig ; Jason DeRouchey ; Ralf Jungmann ; Christian Koch ; Christian Plank ; Joachim O. Rdler
• 刊名：Biomacromolecules
• 出版年：2010
• 出版时间：July 12, 2010
• 年：2010
• 卷：11
• 期：7
• 页码：1802-1809
• 全文大小：301K
• 年卷期：v.11,no.7(July 12, 2010)
• ISSN：1526-4602

文摘

A copolymer-protected gene vector (COPROG) is a three-component gene delivery system consisting of a preformed DNA and branched polyethylenimine (bPEI) complex subsequently modified by the addition of a copolymer (P6YE5C) incorporating both poly(ethylene glycol) (PEG) and anionic peptides. Using fluorescence correlation spectroscopy (FCS) and atomic force microscopy (AFM), we characterized and compared the self-assembly of bPEI/DNA particles and COPROG complexes. In low salt buffer, both bPEI/DNA and COPROG formulations form stable nanoparticles with hydrodynamic radii between 60−120 nm. COPROG particles, as compared to bPEI/DNA, show greatly improved particle stability to both physiological salt as well as low pH conditions. Binding stoichiometry of the three-component COPROG system was investigated by dual-color fluorescence cross-correlation spectroscopy (FCCS). It was found that a significant fraction of P6YE5C copolymer aggregates with excess bPEI forming bPEI/P6YE5C “ghost complexes” with no DNA inside. The ratio of ghost particles to COPROG complexes is about 4:1. In addition, we find a large fraction of excess P6YE5C copolymer, which remains unbound in solution. We observe a 2−4-fold enhanced reporter gene expression with COPROG formulations at various equivalents as compared to bPEI-DNA alone. We believe that both complex stabilization as well as the capture of excess bPEI into ghost particles induced by the copolymer is responsible for the improvement in gene expression.