Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer

被引:373
作者
Boeckle, S [1 ]
von Gersdorff, K [1 ]
van der Piepen, S [1 ]
Culmsee, C [1 ]
Wagner, E [1 ]
Ogris, M [1 ]
机构
[1] Univ Munich, Dept Pharm, D-81377 Munich, Germany
关键词
nonviral vectors; PEI; size exclusion chromatography; lung gene transfer; erythrocyte aggregation; endosomal release;
D O I
10.1002/jgm.598
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Background Nonviral vectors based on polyethylenimine (PEI) usually contain an excess of PEI that is not complexed to DNA. Since unbound PEI contributes to cellular and systemic toxicity, purification of polyplexes from unbound PEI is desirable. Methods Size exclusion chromatography (SEC) was used to purify PEI polyplexes of free PEI. Transfection properties of purified polyplexes and the effect of free PEI on gene delivery were studied in vitro and in vivo after systemic application into mice. Results SEC did not change the size and zeta-potential of polyplexes. Independent of the amount of PEI used for complex formation, purified PEI polyplexes had the same final PEI nitrogen/DNA phosphate ratio of 2.5. Notably, purified PEI polyplexes demonstrated low cellular and systemic toxicity. High transfection efficiency was achieved with purified polyplexes at high DNA concentrations (8-15 mug/ml). At low DNA concentrations (2-4 mug/ml) gene transfer with purified particles was less efficient than with polyplexes containing free PEI both in vitro and in vivo. Mechanistic studies showed that free PEI partly blocked cellular association of DNA complexes but was essential for the following intracellular gene delivery. Adding free PEI to cells treated with purified particles with a delay of up to 4 h resulted in significantly enhanced transfection efficiency compared with non-purified particles or purified particles without free PEI. Conclusions This study presents an efficient method to remove free PEI from PEI polyplexes by SEC. Our results from transfection experiments demonstrate that free PEI substantially contributes to efficient gene expression but also mediates toxic effects in a dose-dependent manner. Purified polyplexes without free PEI have to be applied at increased concentrations to achieve high transfection levels, but exhibit a greatly improved toxicity profile. Copyright (C) 2004 John Wiley Sons, Ltd.
引用
收藏
页码:1102 / 1111
页数:10
相关论文
共 25 条
  • [21] Spectrophotometric determination of polyethylenimine in the presence of an oligonucleotide for the characterization of controlled release formulations
    Ungaro, F
    De Rosa, G
    Miro, A
    Quaglia, F
    [J]. JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS, 2003, 31 (01) : 143 - 149
  • [22] Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo
    Wightman, L
    Kircheis, R
    Rössler, V
    Carotta, S
    Ruzicka, R
    Kursa, M
    Wagner, E
    [J]. JOURNAL OF GENE MEDICINE, 2001, 3 (04) : 362 - 372
  • [23] Zou SM, 2000, J GENE MED, V2, P128, DOI 10.1002/(SICI)1521-2254(200003/04)2:2<128::AID-JGM95>3.0.CO
  • [24] 2-W
  • [25] Towards synthetic viruses
    Zuber, G
    Dauty, E
    Nothisen, M
    Belguise, P
    Behr, JP
    [J]. ADVANCED DRUG DELIVERY REVIEWS, 2001, 52 (03) : 245 - 253