Quantifying the Average Number of Nucleic Acid Therapeutics per Nanocarrier by Single Particle Tracking Microscopy
| Autor: | Kristiaan Neyts, Stefaan C. De Smedt, Kevin Braeckmans, Griet Van Imschoot, Jo Demeester, Roosmarijn E. Vandenbroucke, Elisa Zagato, Katrien Remaut, Heleen Dewitte, Lotte Vermeulen |
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| Rok vydání: | 2018 |
| Předmět: |
Pharmaceutical Science
Nanoparticle LIPOSOMES liposomal gene delivery SPT Transfection 030226 pharmacology & pharmacy 01 natural sciences DOTAP/DOPE 010309 optics MINICIRCLE DNA VECTORS Fatty Acids Monounsaturated 03 medical and health sciences DELIVERY MOLECULES 0302 clinical medicine Plasmid PLASMID DNA Nucleic Acids 0103 physical sciences Drug Discovery Microscopy Biological fluids Medicine and Health Sciences nucleic acid delivery NANOPARTICLES Liposome Biological Products Drug Carriers Chemistry Phosphatidylethanolamines GENE-THERAPY Quaternary Ammonium Compounds BIOLOGICAL-FLUIDS Single-particle tracking Liposomes Nucleic acid Biophysics Molecular Medicine nanocarrier Nanoparticles COMPLEXES Nanocarriers Plasmids |
| Zdroj: | MOLECULAR PHARMACEUTICS |
| ISSN: | 1543-8392 1543-8384 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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