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Effective polyethylenimine-mediated gene transfer into human endothelial cells
Violeta Zaric1, Denis Weltin, Patrick Erbacher
1Laboratoire de Recherche sur l'Angiogenèse, Faculté de Médecine, Strasbourg, France.
The Journal of Gene Medicine
|February 24, 2004
Summary
Polyethylenimines (PEIs) show promise for gene delivery to endothelial cells. Optimal conditions using linear PEI (L-PEI) and its glucose-grafted derivative (L-PEI-Glc(4)) were determined for efficient gene transfer in HUVEC cells.
Area of Science:
- Biomaterials Science
- Molecular Biology
- Cardiovascular Research
Background:
- Non-viral vectors, like cationic polymers, offer higher biosafety for gene delivery compared to viral vectors.
- Polyethylenimines (PEIs) are effective cationic polymers for gene delivery across various cell types.
- Targeting primary endothelial cells with PEIs presents a potential therapeutic strategy for ischemic cardiovascular diseases.
Purpose of the Study:
- To compare the gene transfer efficacies of linear PEI (L-PEI) and a glucose-grafted derivative (L-PEI-Glc(4)) in human umbilical vein endothelial cells (HUVEC).
- To determine optimal conditions, including incubation time and serum concentration, for PEI-mediated gene delivery.
- To investigate the intracellular trafficking of PEI/DNA complexes.
Main Methods:
- Efficacy comparison of L-PEI and L-PEI-Glc(4) using a luciferase reporter gene in HUVEC.
- PEI/DNA complexes were prepared in sodium chloride (NaCl) or glucose solutions.
- Assessment of transfection efficiency under varying incubation times (2, 4, 24 h) and serum concentrations (2%, 30%).
- Intracellular fate analysis using YOYO-1 labeled complexes via flow cytometry (FACS) and confocal microscopy.
Main Results:
- PEI/DNA complexes formed in NaCl demonstrated higher transfection efficiency than those formed in glucose.
- Optimal transfection was achieved with L-PEI-Glc(4) complexed DNA incubated for 4 hours, resulting in 50% GFP-positive cells.
- High serum concentration reduced L-PEI-Glc(4) efficiency but also decreased L-PEI toxicity.
- FACS indicated ~90% cellular internalization of DNA complexes, with confocal microscopy revealing vesicular transport to the perinuclear region.
Conclusions:
- Optimal gene delivery conditions in HUVEC involve using L-PEI or L-PEI-Glc(4) complexed with DNA in 150 mM NaCl, with incubation times of 2 and 4 hours, respectively.
- Cell entry is not a rate-limiting step for PEI-mediated gene delivery.
- PEIs are effective vectors for gene transfer into human endothelial cells.