M. Ogris et al., THE SIZE OF DNA TRANSFERRIN-PEI COMPLEXES IS AN IMPORTANT FACTOR FOR GENE-EXPRESSION IN CULTURED-CELLS/, Gene therapy, 5(10), 1998, pp. 1425-1433
Under physiological salt concentration, plasmid DNA complexed with tra
nsferrin-conjugated or unmodified polyethylenimine (PEI, 800 kDa) form
s huge (up to > 1000 nm) aggregates, unless the individual components
are mixed at a highly positive nitrogen/phosphate (N/P) charge ratio.
At low ionic strengths, however, small particles with an average size
of 40 nm are formed over a broad range of N/P ratios. Interestingly, i
n transfection experiments these small particles result in a 10-fold (
B16F10 cells) to more than 100-fold (Neuro2A cells, K562 cells) reduce
d luciferase gene expression efficiency in comparison to the large com
plexes formed in physiological salt solutions. Limited transport of th
e small particles to the cell surfaces is one possible reason for this
effect. Application of the small particles in more concentrated form
and over extended periods of time improves transfection activity Reduc
ed intracellular release may be another explanation for the decreased
transfection efficiency; incubation with chloroquine or incorporation
of the endosomolytic peptide INF5 into the small complexes enhances ge
ne expression approximately 10-fold. Analysis of gene expression at th
e cellular level using a green fluorescence protein reporter gene and
flow cytometry revealed that the differences in overall gene expressio
n largely result from different intensities per expressing cell while
the difference in the percentage of expressing cells is less substanti
al.