Intracellular FRET analysis of lipid/DNA complexes using flow cytometry and fluorescence imaging techniques.
Gene therapy is a promising therapeutic concept for a large number of incurable diseases. Lipid/DNA complexes (lipoplexes) are used to deliver genes into cells. However, while large efforts have been made to investigate the fate of lipoplexes once inside the cell, the rate of intracellular dissociation is still largely unknown. Analysis of the dissociation rates of DNA from lipid/DNA complexes is crucial for the evaluation of a gene delivery system's efficiency. This study introduces a new fluorescence resonance energy transfer (FRET) approach for the intracellular dissociation analysis of lipid/DNA complexes. Here, the labeling of both complex components, DNA as well as lipid, reveals whether DNA is still associated with the lipid or has dissociated. In this study the kinetic properties of complex dissociation were consistently measured with flow cytometry and fluorescence microscopy, and indicated that most complexes were dissociated after 24h in A-10 cells.
Schneider
Sebastian
S
Department of Pharmaceutical Technology and Biopharmacy, Albert-Ludwigs University, D-79104 Freiburg, Germany. schseb47@web.de
Lenz
Dominik
D
Holzer
Martin
M
Palme
Klaus
K
Süss
Regine
R
D000818:Animals; D002232:Carbocyanines / Q000737:chemistry; D002478:Cells, Cultured; D004247:DNA / Q000008:administration & dosage* / Q000737:chemistry; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008055:Lipids / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D009130:Muscle, Smooth / Q000166:cytology; D051381:Rats
D002232:Carbocyanines; D008055:Lipids; C089187:cyanine dye 3; C085321:cyanine dye 5; D004247:DNA