Abstract |
Generally, the major obstacles for efficient gene delivery are cellular internalization and endosomal escape of nucleic acid such as plasmid DNA (pDNA) or small interfering RNA ( siRNA). We previously developed Pluronic P123 modified polypropyleneimine (PPI)/pDNA (P123-PPI/pDNA) polyplexes as a gene delivery system. The results showed that P123-PPI/pDNA polyplexes revealed higher transfection efficiency than PPI/pDNA polyplexes in multidrug resistant breast cancer cells. As a continued effort, the present investigation on the factors influencing the transfection efficiency, cellular uptake mechanisms, and intracellular fate of P123-PPI/pDNA polyplexes is reported. The presence of P123 was the main factor influencing the transfection efficiency of P123-PPI/pDNA polyplexes in MCF-7/ADR cells, but other parameters, such as N/P ratio, FBS concentration, incubation time and temperature were important as well. The endocytic inhibitors against clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and macropinocytosis were involved in the internalization to investigate their effects on the cellular uptake and transfection efficiency of P123-PPI/pDNA polyplexes in vitro. The data showed that the internalization of P123-PPI/pDNA polyplexes was obtained from both CME and CvME. Colocalization experiments with TRITC- transferrin (CME indicator), Alexa Fluor 555-CTB (CvME indicator), monoclonal anti-α- tubulin (microtubule indicator), and LysoTracker Green (Endosome/lysosome indicator) were carried out to confirm the internalization routes. The results showed that both CME and CvME played vital roles in the effective transfection of P123-PPI/pDNA polyplexes. Endosome/lysosome system and skeleton, including actin filament and microtubule, were necessary for the transportation after internalization.
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Authors | Jijin Gu, Junguo Hao, Xiaoling Fang, Xianyi Sha |
Journal | Colloids and surfaces. B, Biointerfaces
(Colloids Surf B Biointerfaces)
Vol. 140
Pg. 83-93
(Apr 01 2016)
ISSN: 1873-4367 [Electronic] Netherlands |
PMID | 26741268
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Copyright | Copyright © 2015 Elsevier B.V. All rights reserved. |
Chemical References |
- Polypropylenes
- poly(propyleneimine)
- pluronic block copolymer P123
- Poloxalene
- DNA
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Topics |
- Actin Cytoskeleton
(metabolism)
- Breast Neoplasms
(genetics, metabolism, pathology)
- Cell Survival
(genetics)
- DNA
(chemistry, genetics, metabolism)
- Drug Resistance, Multiple
- Drug Resistance, Neoplasm
- Endocytosis
- Endosomes
(metabolism)
- Humans
- Lysosomes
(metabolism)
- MCF-7 Cells
- Microscopy, Confocal
- Plasmids
(chemistry, genetics, metabolism)
- Poloxalene
(chemistry)
- Polypropylenes
(chemistry)
- Transfection
(methods)
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