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Involvement of specific mechanism in plasmid DNA uptake by mouse peritoneal macrophages
T Takagi1, M Hashiguchi, R I Mahato
1Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Japan.
Abstract:
The binding and uptake of plasmid DNA encoding luciferase reporter gene (pCMV-Luc) were studied in vitro using cultured mouse peritoneal macrophages. A significant and time-dependent cellular association of [32P]pCMV-Luc with resident macrophages was observed at 37 degrees C and this decreased at 4 degrees C. The binding at 4 degrees C was saturable and a Scatchard plot gave a maximum binding capacity of 0.81 microgram/mg-protein and a dissociation constant of 0.30 microgram/ml. The binding of [32P]-pCMV-Luc was inhibited by polyinosinic acid, dextran sulfate and salmon sperm DNA, but not by polycytidylic acid, dextran and EDTA. A confocal microscopic study demonstrated that fluorescein-labeled pCMV-Luc was internalized at 37 degrees C while only cell surface binding occurred at 4 degrees C. No significant luciferase gene expression was obtained after incubation with a high concentration (100 micrograms/ml) of pCMV-Luc. These data suggest that plasmid DNA is taken up by macrophages via a mechanism mediated by a receptor like the macrophage scavenger receptor.
Insights
Macrophages internalize plasmid DNA via a receptor-mediated pathway, likely involving scavenger receptors. This uptake mechanism is temperature-dependent and saturable, crucial for understanding gene delivery.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Macrophages play a critical role in immune responses and are targets for gene therapy.
- Understanding plasmid DNA uptake mechanisms is essential for efficient gene delivery and therapeutic applications.
Purpose of the Study:
- To investigate the in vitro binding and uptake of plasmid DNA by mouse peritoneal macrophages.
- To elucidate the characteristics and potential mechanisms of plasmid DNA internalization.
Main Methods:
- In vitro study using cultured mouse peritoneal macrophages.
- Utilized radiolabeled plasmid DNA ([32P]pCMV-Luc) for binding assays.
- Confocal microscopy to visualize cellular uptake and distribution.
- Scatchard analysis to quantify binding parameters.
Main Results:
- Significant, time-dependent binding and uptake of plasmid DNA observed at 37°C, reduced at 4°C.
- Binding was saturable, with a maximum binding capacity of 0.81 μg/mg protein and a dissociation constant of 0.30 μg/ml.
- Uptake was inhibited by polyanions like polyinosinic acid and dextran sulfate, suggesting receptor-mediated endocytosis.
- Internalization occurred at 37°C, while only surface binding was observed at 4°C.
- No significant luciferase gene expression was detected even at high plasmid DNA concentrations.
Conclusions:
- Plasmid DNA uptake by macrophages is mediated by a saturable, receptor-dependent mechanism.
- The findings suggest a role for scavenger receptors in mediating plasmid DNA internalization.
- Efficient gene expression was not achieved, indicating potential barriers in the intracellular processing or translation of the reporter gene.