Application of an Environmentally Sensitive Fluorophore for Rapid Analysis of the Binding and Internalization Efficiency of Gene Carriers

Nonviral gene carriers must associate with and become internalized by cells in order to mediate efficient transfection. Methods to quantitatively measure and distinguish between cell association and internalization of delivery vectors are necessary to characterize the trafficking of vector formulati...

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Bibliographic Details
Published in:Bioconjugate chemistry 2008-01, Vol.19 (1), p.377-384
Main Authors: Bergen, Jamie M, Kwon, Ester J, Shen, Tammy W, Pun, Suzie H
Format: Article
Language:English
Subjects:
Binding sites
Cells
Dithionite - chemistry
Environment
Flow Cytometry
Fluorescence
Fluorescent Dyes - analysis
Genes
Genetics
HeLa Cells
Humans
Methods
Neurons - cytology
Neurons - metabolism
Oligonucleotides - metabolism
Oxadiazoles - chemistry
Oxadiazoles - metabolism
Oxidation-Reduction
Reproducibility of Results
Sensitivity and Specificity
Time Factors
Transfection - methods
Xanthenes
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Summary:Nonviral gene carriers must associate with and become internalized by cells in order to mediate efficient transfection. Methods to quantitatively measure and distinguish between cell association and internalization of delivery vectors are necessary to characterize the trafficking of vector formulations. Here, we demonstrate the utility of nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labeled oligonucleotides for discrimination between bound and internalized gene carriers associated with cells. Dithionite quenches the fluorescence of extracellular NBD-labeled material, but is unable to penetrate the cell membrane and quench internalized material. We have verified that dithionite-mediated quenching of extracellular materials occurs in both polymer- and lipid-based gene delivery systems incorporating NBD-labeled oligonucleotides. By exploiting this property, the efficiencies of cellular binding and internalization of lipid- and polymer-based vectors were studied and correlated to their transfection efficiencies. Additionally, spatiotemporal information regarding binding and internalization of NBD-labeled gene carriers can be obtained using conventional wide-field fluorescence microscopy, since dithionite-mediated quenching of extracellular materials reveals the intracellular distribution of gene carriers without the need for optical sectioning. Hence, incorporation of environmentally sensitive NBD−oligos into gene carriers allows for facile assessment of binding and internalization efficiencies of vectors in live cells.
ISSN:1043-1802
1520-4812
DOI:10.1021/bc700315v