English   español  
Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/177537
Share/Impact:
Statistics
logo share SHARE   Add this article to your Mendeley library MendeleyBASE
Visualizar otros formatos: MARC | Dublin Core | RDF | ORE | MODS | METS | DIDL
Exportar a otros formatos:
Title

Impact of physicochemical properties of DNA/PEI complexes on transient transfection of mammalian cells

AuthorsGonzález Domínguez, Irene; Grimaldi, Natascia ; Cervera, Laura; Ventosa, Nora ; Gòdia, Francesc
KeywordsTransient transfection
DNA/PEI complexes
Mammalian cells
Particle tracking analysis
Electron microscopy
Issue Date25-Mar-2019
PublisherElsevier
CitationNew Biotechnology 49: 88-97 (2019)
AbstractPolyethyleneimine (PEI) has been used extensively for transient gene expression (TGE) in mammalian cell cultures. However, the relationship between DNA/PEI complex preparation and their biological activity has not been fully established. Here, a systematic study of DNA/PEI complexes, their physicochemical properties during formation and their transfection efficiency was performed on a virus-like particle (VLP) production platform. The same chemically defined cell culture medium for DNA/PEI complex formation was used as an alternative to simple ionic solutions to minimize changes in complex properties during transfection. Upon formation, an initial concentration of 1E + 10 DNA/PEI complexes/mL underwent partial aggregation with an average size of 300 nm. The participation of NaCl ions in the evolution of complexes was analyzed by X-ray spectroscopy, stressing the relevance of complexing media composition in TGE strategies. After 15 min incubation, 250 complexes plus aggregates per cell were estimated at the time of transfection. Such heterogeneous preparations cannot be easily characterized; subsequently, nanoparticle tracking analysis (NTA) and cryo-electron microscopy were combined to achieve a complete picture of the preparation. Finally, the contribution of each DNA/PEI complex subpopulation was tested by drug inhibition endocytosis. Interestingly, all complexes delivered DNA efficiently and high size aggregates, which enter through macropinocytosis, when inhibited presented a major contribution to transfection efficiency. There is a need to understand the physicochemical factors that participate in DNA delivery protocols. Hence, this study provides new insights into the characterization of DNA/PEI complexes that will assist in more productive and reproducible TGE strategies.
Publisher version (URL)http://dx.doi.org/10.1016/j.nbt.2018.09.005
URIhttp://hdl.handle.net/10261/177537
ISSN1871-6784
Appears in Collections:(ICMAB) Artículos
Files in This Item:
File Description SizeFormat 
GonzalezDominguez__NewBiotech_2019_postprint.pdf Embargoed until March 25, 20202,09 MBAdobe PDFThumbnail
View/Open    Request a copy
Show full item record
 


WARNING: Items in Digital.CSIC are protected by copyright, with all rights reserved, unless otherwise indicated.