Impact of physicochemical properties of DNA/PEI complexes on transient transfection of mammalian cells.
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| Title: | Impact of physicochemical properties of DNA/PEI complexes on transient transfection of mammalian cells. |
|---|---|
| Authors: | González-Domínguez, I.1 Irene.Gonzalez@uab.cat, Grimaldi, N.2,3, Cervera, L.1, Ventosa, N.2,4, Gòdia, F.1 |
| Source: | New Biotechnology. Mar2019, Vol. 49, p88-97. 10p. |
| Subjects: | Mammalian cell cycle, Gene transfection, Gene expression, Cell culture, X-ray spectroscopy, Nanoparticles |
| Abstract: | Highlights • DNA/PEI complexes formed immediately and underwent aggregation. • 1E+10 complexes/mL with 300-nm sizes were found after DNA/PEI complex formation. • 250 complexes per cell and aggregates were found at the time of transfection. • Morphology and composition of complexes were analyzed by EM. • Complex aggregates were required to enhance HEK 293 transient transfection. Abstract Polyethyleneimine (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. [ABSTRACT FROM AUTHOR] |
| Copyright of New Biotechnology is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.) | |
| Database: | Engineering Source |
| FullText | Text: Availability: 0 |
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| Header | DbId: egs DbLabel: Engineering Source An: 133766258 AccessLevel: 6 PubType: Academic Journal PubTypeId: academicJournal PreciseRelevancyScore: 0 |
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| Items | – Name: Title Label: Title Group: Ti Data: Impact of physicochemical properties of DNA/PEI complexes on transient transfection of mammalian cells. – Name: Author Label: Authors Group: Au Data: <searchLink fieldCode="AR" term="%22González-Domínguez%2C+I%2E%22">González-Domínguez, I.</searchLink><relatesTo>1</relatesTo><i> Irene.Gonzalez@uab.cat</i><br /><searchLink fieldCode="AR" term="%22Grimaldi%2C+N%2E%22">Grimaldi, N.</searchLink><relatesTo>2,3</relatesTo><br /><searchLink fieldCode="AR" term="%22Cervera%2C+L%2E%22">Cervera, L.</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Ventosa%2C+N%2E%22">Ventosa, N.</searchLink><relatesTo>2,4</relatesTo><br /><searchLink fieldCode="AR" term="%22Gòdia%2C+F%2E%22">Gòdia, F.</searchLink><relatesTo>1</relatesTo> – Name: TitleSource Label: Source Group: Src Data: <searchLink fieldCode="JN" term="%22New+Biotechnology%22">New Biotechnology</searchLink>. Mar2019, Vol. 49, p88-97. 10p. – Name: Subject Label: Subjects Group: Su Data: <searchLink fieldCode="DE" term="%22Mammalian+cell+cycle%22">Mammalian cell cycle</searchLink><br /><searchLink fieldCode="DE" term="%22Gene+transfection%22">Gene transfection</searchLink><br /><searchLink fieldCode="DE" term="%22Gene+expression%22">Gene expression</searchLink><br /><searchLink fieldCode="DE" term="%22Cell+culture%22">Cell culture</searchLink><br /><searchLink fieldCode="DE" term="%22X-ray+spectroscopy%22">X-ray spectroscopy</searchLink><br /><searchLink fieldCode="DE" term="%22Nanoparticles%22">Nanoparticles</searchLink> – Name: Abstract Label: Abstract Group: Ab Data: Highlights • DNA/PEI complexes formed immediately and underwent aggregation. • 1E+10 complexes/mL with 300-nm sizes were found after DNA/PEI complex formation. • 250 complexes per cell and aggregates were found at the time of transfection. • Morphology and composition of complexes were analyzed by EM. • Complex aggregates were required to enhance HEK 293 transient transfection. Abstract Polyethyleneimine (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. [ABSTRACT FROM AUTHOR] – Name: AbstractSuppliedCopyright Label: Group: Ab Data: <i>Copyright of New Biotechnology is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.) |
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| RecordInfo | BibRecord: BibEntity: Identifiers: – Type: doi Value: 10.1016/j.nbt.2018.09.005 Languages: – Code: eng Text: English PhysicalDescription: Pagination: PageCount: 10 StartPage: 88 Subjects: – SubjectFull: Mammalian cell cycle Type: general – SubjectFull: Gene transfection Type: general – SubjectFull: Gene expression Type: general – SubjectFull: Cell culture Type: general – SubjectFull: X-ray spectroscopy Type: general – SubjectFull: Nanoparticles Type: general Titles: – TitleFull: Impact of physicochemical properties of DNA/PEI complexes on transient transfection of mammalian cells. Type: main BibRelationships: HasContributorRelationships: – PersonEntity: Name: NameFull: González-Domínguez, I. – PersonEntity: Name: NameFull: Grimaldi, N. – PersonEntity: Name: NameFull: Cervera, L. – PersonEntity: Name: NameFull: Ventosa, N. – PersonEntity: Name: NameFull: Gòdia, F. IsPartOfRelationships: – BibEntity: Dates: – D: 25 M: 03 Text: Mar2019 Type: published Y: 2019 Identifiers: – Type: issn-print Value: 18716784 Numbering: – Type: volume Value: 49 Titles: – TitleFull: New Biotechnology Type: main |
| ResultId | 1 |
