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Effect of covalent fluorescence labeling of plasmid DNA on its intracellular processing and transfection with lipid-based carriers

Koen Rombouts (UGent) , Thomas Martens (UGent) , Elisa Zagato (UGent) , Jo Demeester (UGent) , Stefaan De Smedt (UGent) , Kevin Braeckmans (UGent) and Katrien Remaut (UGent)
(2014) MOLECULAR PHARMACEUTICS. 11(5). p.1359-1368
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NB-Photonics
Abstract
The development of biotechnological pharmaceutics, like macro- and nanocarriers, can benefit greatly from studying their characteristics in situ using advanced fluorescence microscopy methods. While choosing the optimal labeling method for visualizing the carrier or its cargo is crucial, densities alter the intracellular processing of the molecule is it seldom receives attention. The possibility that high labeling considered, but how and at which point this interference happens is not yet studied. The aim of this study was to elucidate the effect of labeling density on the cellular trafficking of labeled pDNA. Due to the drastic effect on expression levels for higher labeling densities, we tried to determine at which steps in the intracellular processing labeled pDNA behaves different than its nonlabeled counterpart. Therefore, different labeling densities, up to the manufacturer's recommended density, were tested. It was found that the cellular uptake remains unaffected, while the affinity for lipids is increased, which affects dissociation from the lipid-based complex and may affect endosomal escape. Also, nuclear injections clearly demonstrated that transcription is affected. The information and methodology, included in this work, could be helpful in determining if the labeling method and density used yields biological relevant results for the intended research question.
Keywords
DELIVERY, HELA-CELLS, NANOPARTICLES, CELL TRANSFECTION, GENE-THERAPY, SINGLE-PARTICLE TRACKING, advanced microscopy techniques, gene therapy, drug delivery, intracellular trafficking, transfection pathway, lipid-based carriers, pDNA, fluorescent labeling, plasmid DNA, VECTORS, COMPLEXES, MICROSCOPY, TRAFFICKING

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Citation

Please use this url to cite or link to this publication:

Chicago
Rombouts, Koen, Thomas Martens, Elisa Zagato, Jo Demeester, Stefaan De Smedt, Kevin Braeckmans, and Katrien Remaut. 2014. “Effect of Covalent Fluorescence Labeling of Plasmid DNA on Its Intracellular Processing and Transfection with Lipid-based Carriers.” Molecular Pharmaceutics 11 (5): 1359–1368.
APA
Rombouts, K., Martens, T., Zagato, E., Demeester, J., De Smedt, S., Braeckmans, K., & Remaut, K. (2014). Effect of covalent fluorescence labeling of plasmid DNA on its intracellular processing and transfection with lipid-based carriers. MOLECULAR PHARMACEUTICS, 11(5), 1359–1368.
Vancouver
1.
Rombouts K, Martens T, Zagato E, Demeester J, De Smedt S, Braeckmans K, et al. Effect of covalent fluorescence labeling of plasmid DNA on its intracellular processing and transfection with lipid-based carriers. MOLECULAR PHARMACEUTICS. 2014;11(5):1359–68.
MLA
Rombouts, Koen, Thomas Martens, Elisa Zagato, et al. “Effect of Covalent Fluorescence Labeling of Plasmid DNA on Its Intracellular Processing and Transfection with Lipid-based Carriers.” MOLECULAR PHARMACEUTICS 11.5 (2014): 1359–1368. Print.
@article{4410554,
  abstract     = {The development of biotechnological pharmaceutics, like macro- and nanocarriers, can benefit greatly from studying their characteristics in situ using advanced fluorescence microscopy methods. While choosing the optimal labeling method for visualizing the carrier or its cargo is crucial, densities alter the intracellular processing of the molecule is it seldom receives attention. The possibility that high labeling considered, but how and at which point this interference happens is not yet studied. The aim of this study was to elucidate the effect of labeling density on the cellular trafficking of labeled pDNA. Due to the drastic effect on expression levels for higher labeling densities, we tried to determine at which steps in the intracellular processing labeled pDNA behaves different than its nonlabeled counterpart. Therefore, different labeling densities, up to the manufacturer's recommended density, were tested. It was found that the cellular uptake remains unaffected, while the affinity for lipids is increased, which affects dissociation from the lipid-based complex and may affect endosomal escape. Also, nuclear injections clearly demonstrated that transcription is affected. The information and methodology, included in this work, could be helpful in determining if the labeling method and density used yields biological relevant results for the intended research question.},
  author       = {Rombouts, Koen and Martens, Thomas and Zagato, Elisa and Demeester, Jo and De Smedt, Stefaan and Braeckmans, Kevin and Remaut, Katrien},
  issn         = {1543-8384},
  journal      = {MOLECULAR PHARMACEUTICS},
  keyword      = {DELIVERY,HELA-CELLS,NANOPARTICLES,CELL TRANSFECTION,GENE-THERAPY,SINGLE-PARTICLE TRACKING,advanced microscopy techniques,gene therapy,drug delivery,intracellular trafficking,transfection pathway,lipid-based carriers,pDNA,fluorescent labeling,plasmid DNA,VECTORS,COMPLEXES,MICROSCOPY,TRAFFICKING},
  language     = {eng},
  number       = {5},
  pages        = {1359--1368},
  title        = {Effect of covalent fluorescence labeling of plasmid DNA on its intracellular processing and transfection with lipid-based carriers},
  url          = {http://dx.doi.org/10.1021/mp4003078},
  volume       = {11},
  year         = {2014},
}

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