Intracellular compartmentalization of DNA fragments in cultured airway epithelial cells mediated by cationic lipids

The amount and intracellular distribution of DNA fragments (491-bp) was characterized after transfection in vitro with a commercially available cationic lipid. Localization of fragment to the nucleus, its subcellular distribution, and integrity within the cells was determined for various times after...

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Bibliographic Details
Published in:Pharmaceutical research 1999-07, Vol.16 (7), p.1020-1025
Main Authors: HOLMES, A. R, DOHRMAN, A. F, ELLISON, A. R, GONCZ, K. K, GRUENERT, D. C
Format: Article
Language:English
Subjects:
Biological and medical sciences
Bronchi - metabolism
Bronchi - pathology
Cation Exchange Resins - administration & dosage
Cation Exchange Resins - pharmacokinetics
Cations
Cell Nucleus - metabolism
Cells, Cultured
Cystic fibrosis
Cystic Fibrosis - metabolism
Cystic Fibrosis - pathology
Cytoplasm - metabolism
DNA - administration & dosage
DNA - pharmacokinetics
Drug Carriers
Drug Stability
Epithelial Cells - metabolism
Epithelial Cells - physiology
Gene therapy
General pharmacology
Humans
Intracellular Fluid - metabolism
Labeling
Lipids
Lipids - administration & dosage
Lipids - pharmacokinetics
Medical sciences
Oligonucleotides - administration & dosage
Oligonucleotides - genetics
Oligonucleotides - pharmacokinetics
Pharmaceutical technology. Pharmaceutical industry
Pharmacology. Drug treatments
Phosphorus Radioisotopes
Ratios
Subcellular Fractions - metabolism
Transfection
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Description
Summary:The amount and intracellular distribution of DNA fragments (491-bp) was characterized after transfection in vitro with a commercially available cationic lipid. Localization of fragment to the nucleus, its subcellular distribution, and integrity within the cells was determined for various times after transfection. Cystic fibrosis (CF) airway epithelial cells were transfected with 32P and FITC labeled single-stranded (ss) or double-stranded (ds) DNA fragments complexed with Lipofectamine at various charge ratios. A 511 (+/-) charge ratio was found to be the optimal ratio for transfection of both ss-and dsDNA. After a 5 h exposure, 7.51 +/- 0.89% of the radioactivity was associated with the nuclear fraction whereas only 1.07 +/- 0.23%, was found in the nuclear fraction when dsDNA was used. The nuclear radioactivity detected after a 24 h exposure was only 1/3 of that after 5 h. Analysis of fragment stability in the cytosolic and nuclear fractions showed the presence of intact fragment in each subcellular compartment. No intranuclear/intracellular fragment could be detected in control experiments with naked DNA. Conclusions. The results from these experiments indicate that small fragments of DNA can be efficiently and rapidly transferred intact to the cell nucleus using cationic lipids and that ssDNA fragments are more effective than dsDNA fragments for nuclear delivery.
ISSN:0724-8741
1573-904X
DOI:10.1023/A:1018927531003