Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET.
BACKGROUND
Serum and high ionic strength solutions constitute important barriers to cationic lipid-mediated intravenous gene transfer. Preparation or incubation of lipoplexes in these media results in alteration of their biophysical properties, generally leading to a decrease in transfection efficiency. Accurate quantification of these changes is of paramount importance for the success of lipoplex-mediated gene transfer in vivo.


RESULTS
In this work, a novel time-resolved fluorescence resonance energy transfer (FRET) methodology was used to monitor lipoplex structural changes in the presence of phosphate-buffered saline solution (PBS) and fetal bovine serum. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/pDNA lipoplexes, prepared in high and low ionic strength solutions, are compared in terms of complexation efficiency. Lipoplexes prepared in PBS show lower complexation efficiencies when compared to lipoplexes prepared in low ionic strength buffer followed by addition of PBS. Moreover, when serum is added to the referred formulation no significant effect on the complexation efficiency was observed. In physiological saline solutions and serum, a multilamellar arrangement of the lipoplexes is maintained, with reduced spacing distances between the FRET probes, relative to those in low ionic strength medium.


CONCLUSIONS
The time-resolved FRET methodology described in this work allowed us to monitor stability and characterize quantitatively the structural changes (variations in interchromophore spacing distances and complexation efficiencies) undergone by DOTAP/DNA complexes in high ionic strength solutions and in presence of serum, as well as to determine the minimum amount of potentially cytotoxic cationic lipid necessary for complete coverage of DNA. This constitutes essential information regarding thoughtful design of future in vivo applications.
Madeira
Catarina
C
1IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Av. Rovisco Pais, 1049-001,Portugal. catarina.madeira@ist.utl.pt
Loura
Luís M S
LM
Prieto
Manuel
M
Fedorov
Aleksander
A
Aires-Barros
M Raquel
MR
D005229:Fatty Acids, Monounsaturated / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008081:Liposomes / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D044967:Serum / Q000737:chemistry*
D005229:Fatty Acids, Monounsaturated; D007477:Ions; D008081:Liposomes; D000644:Quaternary Ammonium Compounds; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane