Characterization of vascular gene transfer using a novel cationic lipid.
BACKGROUND
Cationic liposomes are an alternative non-viral vector for gene therapy, but several factors affect transfection efficiency. A novel cationic lipid, o-ethyldioleoylphosphatidylcholinium (EDOPC), was studied for characterization of the time course and effects of lipid composition, concentration, charge ratio, mixing techniques, passage number, and stimulated state on transfection of human vascular cells, represented by human umbilical vein endothelial cells (HUVEC).


METHODS
HUVEC cultures were seeded at a density of 45,000 cells/well in 24-well plates and incubated overnight. Triplicate wells were transfected with samples of EDOPC/reporter plasmid for 2 h, followed by a 24-h expression time, which was the peak expression time point in an initial time-course experiment. Measuring luciferase in cell lysates quantitated gene expression.


RESULTS
Transfection of HUVEC with EDOPC was optimal with a concentration of 100 microgram lipid/well, ratio of 3:1 EDOPC:plasmid, fractional mixing of lipid and plasmid, centrifugation, and incubation in serum-free media. Transfections in sequential passages showed striking decreases in gene expression and regression analysis revealed the relationship: RLU = 120,000 - (10, 400 x passage number), r(2) = 0.947. HUVEC activated by cytokine stimulation remain susceptible to gene transfer specifically with EDOPC.


CONCLUSIONS
During transfection of HUVEC with cationic lipid species, an increase in passage number is associated with linear reduction in luciferase expression, and hence passage number must be controlled in comparative experiments. Characteristics of EDOPC may permit site-specific efficient transfection of activated human vascular cells that can be isolated from serum by mechanical methods.
Matsumura
J S
JS
Department of Surgery and the Feinberg Cardiovascular Research Institute, Northwestern University Medical School, Chicago, Illinois, 60611, USA.
Kim
R
R
Shively
V P
VP
MacDonald
R C
RC
Pearce
W H
WH
D001798:Blood Proteins / Q000494:pharmacology; D002411:Cation Exchange Resins / Q000378:metabolism; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004337:Drug Carriers; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D050356:Lipid Metabolism; D008055:Lipids; D008156:Luciferases / Q000235:genetics; D009829:Oleic Acids / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000494:pharmacology; D013096:Spermine / Q000031:analogs & derivatives / Q000378:metabolism; D014162:Transfection / Q000187:drug effects / Q000379:methods; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D001798:Blood Proteins; D002411:Cation Exchange Resins; D004337:Drug Carriers; D008055:Lipids; C086724:Lipofectamine; D009829:Oleic Acids; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C120779:o-ethyldioleoylphosphatidylcholinium; C061134:dioctadecylamidoglycylspermine; D013096:Spermine; D004247:DNA; D008156:Luciferases; D005998:Glycine