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Quantifying the average number of nucleic acid therapeutics per nanocarrier by single particle tracking microscopy

Elisa Zagato (UGent) , Lotte Vermeulen (UGent) , Heleen Dewitte (UGent) , Griet Van Imschoot (UGent) , Roosmarijn Vandenbroucke (UGent) , Jo Demeester (UGent) , Stefaan De Smedt (UGent) , Kristiaan Neyts (UGent) , Katrien Remaut (UGent) and Kevin Braeckmans (UGent)
(2018) MOLECULAR PHARMACEUTICS. 15(3). p.1142-1149
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Abstract
Nucleic acid biopharmaceuticals are being investigated as potential therapeutics. They need to be incorporated into a biocompatible carrier so as to overcome several biological barriers. Rational development of suitable nanocarriers requires high-quality characterization techniques. While size, concentration, and stability can be very well measured these days, even in complex biological fluids, a method to accurately quantify the number of nucleic acid therapeutics encapsulated in nanocarriers is still missing. Here we present a method, based on concentration measurements with single particle tracking microscopy, with which it is possible to directly measure the number of plasmid DNA molecules per nanoparticle, referred to as the plasmid/NP ratio. Using DOTAP/DOPE liposomes as a model carrier, we demonstrate the usefulness of the method by investigating the influence of various experimental factors on the plasmid/NP ratio. We find that the plasmid/NP ratio is inversely proportional with the size of the pDNA and that the plasmid/NP decreases when lipoplexes are prepared at lower concentrations of pDNA and nanocarrier, with values ranging from 6.5 to 3 plasmid/NP. Furthermore, the effect of pre- and post-PEGylation of lipoplexes was examined, finding that pre-PEGylation results in a decreased plasmid/NP ratio, while post-PEGylation did not alter the plasmid/NP ratio. These proof-of-concept experiments show that single particle tracking offers an extension of the nanoparticle characterization toolbox and is expected to aid in the efficient development of nanoformulations for nucleic acid-based therapies.
Keywords
SPT, nanocarrier, liposomal gene delivery, nucleic acid delivery, MINICIRCLE DNA VECTORS, BIOLOGICAL-FLUIDS, GENE-THERAPY, PLASMID DNA, NANOPARTICLES, DELIVERY, LIPOSOMES, DOTAP/DOPE, COMPLEXES, MOLECULES

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Chicago
Zagato, Elisa, Lotte Vermeulen, Heleen Dewitte, Griet Van Imschoot, Roosmarijn Vandenbroucke, Jo Demeester, Stefaan De Smedt, Kristiaan Neyts, Katrien Remaut, and Kevin Braeckmans. 2018. “Quantifying the Average Number of Nucleic Acid Therapeutics Per Nanocarrier by Single Particle Tracking Microscopy.” Molecular Pharmaceutics 15 (3): 1142–1149.
APA
Zagato, E., Vermeulen, L., Dewitte, H., Van Imschoot, G., Vandenbroucke, R., Demeester, J., De Smedt, S., et al. (2018). Quantifying the average number of nucleic acid therapeutics per nanocarrier by single particle tracking microscopy. MOLECULAR PHARMACEUTICS, 15(3), 1142–1149.
Vancouver
1.
Zagato E, Vermeulen L, Dewitte H, Van Imschoot G, Vandenbroucke R, Demeester J, et al. Quantifying the average number of nucleic acid therapeutics per nanocarrier by single particle tracking microscopy. MOLECULAR PHARMACEUTICS. 2018;15(3):1142–9.
MLA
Zagato, Elisa, Lotte Vermeulen, Heleen Dewitte, et al. “Quantifying the Average Number of Nucleic Acid Therapeutics Per Nanocarrier by Single Particle Tracking Microscopy.” MOLECULAR PHARMACEUTICS 15.3 (2018): 1142–1149. Print.
@article{8558526,
  abstract     = {Nucleic acid biopharmaceuticals are being investigated as potential therapeutics. They need to be incorporated into a biocompatible carrier so as to overcome several biological barriers. Rational development of suitable nanocarriers requires high-quality characterization techniques. While size, concentration, and stability can be very well measured these days, even in complex biological fluids, a method to accurately quantify the number of nucleic acid therapeutics encapsulated in nanocarriers is still missing. Here we present a method, based on concentration measurements with single particle tracking microscopy, with which it is possible to directly measure the number of plasmid DNA molecules per nanoparticle, referred to as the plasmid/NP ratio. Using DOTAP/DOPE liposomes as a model carrier, we demonstrate the usefulness of the method by investigating the influence of various experimental factors on the plasmid/NP ratio. We find that the plasmid/NP ratio is inversely proportional with the size of the pDNA and that the plasmid/NP decreases when lipoplexes are prepared at lower concentrations of pDNA and nanocarrier, with values ranging from 6.5 to 3 plasmid/NP. Furthermore, the effect of pre- and post-PEGylation of lipoplexes was examined, finding that pre-PEGylation results in a decreased plasmid/NP ratio, while post-PEGylation did not alter the plasmid/NP ratio. These proof-of-concept experiments show that single particle tracking offers an extension of the nanoparticle characterization toolbox and is expected to aid in the efficient development of nanoformulations for nucleic acid-based therapies.},
  author       = {Zagato, Elisa and Vermeulen, Lotte and Dewitte, Heleen and Van Imschoot, Griet and Vandenbroucke, Roosmarijn and Demeester, Jo and De Smedt, Stefaan and Neyts, Kristiaan and Remaut, Katrien and Braeckmans, Kevin},
  issn         = {1543-8384},
  journal      = {MOLECULAR PHARMACEUTICS},
  keyword      = {SPT,nanocarrier,liposomal gene delivery,nucleic acid delivery,MINICIRCLE DNA VECTORS,BIOLOGICAL-FLUIDS,GENE-THERAPY,PLASMID DNA,NANOPARTICLES,DELIVERY,LIPOSOMES,DOTAP/DOPE,COMPLEXES,MOLECULES},
  language     = {eng},
  number       = {3},
  pages        = {1142--1149},
  title        = {Quantifying the average number of nucleic acid therapeutics per nanocarrier by single particle tracking microscopy},
  url          = {http://dx.doi.org/10.1021/acs.molpharmaceut.7b00999},
  volume       = {15},
  year         = {2018},
}

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