Stabilization of oligonucleotide-polyethylenimine complexes by freeze-drying: physicochemical and biological characterization

被引:53
作者
Brus, C
Kleemann, E
Aigner, A
Czubayko, F
Kissel, T
机构
[1] Univ Marburg, Dept Pharmaceut & Biopharm, D-35037 Marburg, Germany
[2] Univ Marburg, Sch Med, Dept Pharmacol & Toxicol, D-35033 Marburg, Germany
关键词
oligonucleotides; polyethylenimine; ribozymes; lyophilization; atomic force microscopy;
D O I
10.1016/j.jconrel.2003.10.021
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
In the present study the lyophilization of oligodeoxynucleotide-polyethylenimine (ODN-PEI) complexes was investigated regarding the maintenance of physicochemical properties and influence on biological activity. To achieve this, we used PEI of different molecular weights, in the range of 800-0.8 kDa, as complexing agents for unmodified ODN and ribozymes. The hydrodynamic diameter was measured by photon correlation spectroscopy (PCS) and the zeta potential was determined using laser Doppler anemometry (LDA) of ODN complexes with PEI derivatives of different molecular weights both before and after lyophilization. Atomic force microscopy (AFM) was used to visualize freshly prepared, stored and lyophilized complexes in solution. The biological activity of the ODN, as well as of plasmid DNA, in lyophilized PEI complexes was examined and compared to freshly prepared complexes using standard transfection assays. All PEI derivatives formed very small complexes with ODN displaying hydrodynamic diameters ranging from 15 to 30 nm. Marginal changes in size after lyophilization were observed for ODN-PEI complexes. In contrast, plasmid complexed with PEI was found to aggregate. In either cases minimal or no influence of the added amount of Iyoprotectant was observed. The shape of the very small and highly condensed ODN complexes was not altered by lyophilization as seen in the AFM images. The transfection efficiency of lyophilized ribozyme-PEI complexes relative to freshly prepared complexes was approximately 100%, whereas a decrease was seen for lyophilized plasmid-PEI complexes. An additive of the lyoprotectants trehalose, mannitol or sucrose preserved biological activity. This study demonstrates the particular suitability of ODN-PEI complexes to be formulated as lyophilized systems with no loss in physical stability or biological activity. (C) 2004 Elsevier B.V. All rights reserved.
引用
收藏
页码:119 / 131
页数:13
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