Intracellular DNA release and elimination correlate poorly with transgene expression after non-viral transfection

被引：34

作者：

Ruponen, Marika ^{[1
]}

Arkko, Satu ^{[1
]}

Urtti, Arto ^{[2
]}

Reinisalo, Mika ^{[1
]}

Ranta, Veli-Pekka ^{[1
]}

机构：

[1] Univ Kuopio, Dept Pharmaceut, Fac Pharm, FIN-70211 Kuopio, Finland

[2] Univ Helsinki, Ctr Drug Res, FIN-00014 Helsinki, Finland

来源：

JOURNAL OF CONTROLLED RELEASE | 2009年 / 136卷 / 03期

基金：

芬兰科学院;

关键词：

Intracellular; Elimination; DNA release; Non-viral transfection; Quantitative PCR; CALCIUM-PHOSPHATE TRANSFECTION; RESONANCE ENERGY-TRANSFER; MAMMALIAN-CELLS OCCURS; MEDIATED GENE-TRANSFER; CATIONIC LIPOSOMES; PLASMID DNA; POTENTIAL BARRIER; DELIVERY-SYSTEM; ENDOCYTOSIS; GLYCOSAMINOGLYCANS;

D O I：

10.1016/j.jconrel.2009.02.012

中图分类号：

O6 [化学];

学科分类号：

070301 [无机化学];

摘要：

The intracellular limiting steps in non-viral gene delivery are still unclear. The purpose of this study was to quantize intracellular DNA release and elimination kinetics after transfection with various non-viral carriers (calcium phosphate precipitates, branched poly(ethyleneimine), poly-L-lysine, DOTAP, DOTAP/DOPE) and to correlate these factors with transgene expression. Intracellular kinetics of DNA was determined by novel quantitative method based on qRT-PCR and DNase treatment. Intracellular elimination of DNA after calcium phosphate transfection was rapid (half-life of 0.37 h) whereas the amount of DNA in the cells was stable for at least 136 h after poly(ethyleneimine) mediated transfection. Intracellular elimination half-lives for DNA delivered by other carrier systems ranged from 9 to 27 h. Calcium phosphate precipitates are not able to protect DNA, which explains the short elimination half-life. In the case of other carriers DNA is after complex removal mostly carrier bound but after 24 h the major fraction of DNA is in the released or loosened state. Overall, neither total nor released amount of intracellular DNA correlates with the transgene expression. (C) 2009 Elsevier B.V. All rights reserved.

引用

页码：226 / 231

页数：6

共 31 条

[1]

An endogenous calcium-dependent, caspase-independent intranuclear degradation pathway in thymocyte nuclei:: Antagonism by physiological concentrations of K+ ions [J].

Ajiro, Kozo ;

Bortner, Carl D. ;

Westmoreland, Jim ;

Cidlowski, John A. .

EXPERIMENTAL CELL RESEARCH, 2008, 314 (06) :1237-1249

[2]

Quantitative three-dimensional analysis of the intracellular trafficking of plasmid DNA transfected by a nonviral gene delivery system using confocal laser scanning microscopy [J].

Akita, H ;

Ito, R ;

Khalil, IA ;

Futaki, S ;

Harashima, H .

MOLECULAR THERAPY, 2004, 9 (03) :443-451

[3]

Transfer of high copy number plasmid into mammalian cells by calcium phosphate transfection [J].

Batard, P ;

Jordan, M ;

Wurm, F .

GENE, 2001, 270 (1-2) :61-68

[4]

A VERSATILE VECTOR FOR GENE AND OLIGONUCLEOTIDE TRANSFER INTO CELLS IN CULTURE AND IN-VIVO - POLYETHYLENIMINE [J].

BOUSSIF, O ;

LEZOUALCH, F ;

ZANTA, MA ;

MERGNY, MD ;

SCHERMAN, D ;

DEMENEIX, B ;

BEHR, JP .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1995, 92 (16) :7297-7301

[5]

BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3

[6]

Fluorescence resonance energy transfer: Evaluation of the intracellular stability of polyplexes [J].

Breunig, Miriam ;

Lungwitz, Uta ;

Liebl, Renate ;

Goepferich, Achim .

EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS, 2006, 63 (02) :156-165

[7]

Quantitative comparison of intracellular unpacking kinetics of polyplexes by a model constructed from quantum Dot-FRET [J].

Chen, Hunter H. ;

Ho, Yi-Ping ;

Jiang, Xuan ;

Mao, Hai-Quan ;

Wang, Tza-Huei ;

Leong, Kam W. .

MOLECULAR THERAPY, 2008, 16 (02) :324-332

[8]

Cellular dynamics of EGF receptor-targeted synthetic viruses [J].

de Bruin, Karla ;

Ruthardt, Nadia ;

von Gersdorff, Katharina ;

Bausinger, Ralf ;

Wagner, Ernst ;

Ogris, Manfred ;

Braeuchle, Christoph .

MOLECULAR THERAPY, 2007, 15 (07) :1297-1305

[9]

Endocytosis and intracellular processing accompanying transfection mediated by cationic liposomes [J].

Friend, DS ;

Papahadjopoulos, D ;

Debs, RJ .

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES, 1996, 1278 (01) :41-50

[10]

Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery [J].

Godbey, WT ;

Wu, KK ;

Mikos, AG .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1999, 96 (09) :5177-5181

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