Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET

<p>Abstract</p> <p>Background</p> <p>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 <it>in vivo</it>.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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 <it>in vivo </it>applications.</p

Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET-1

and in Tris-HCl, followed by incubation in PBS (C,D). Fitting curves using Eqs. 2–3 are also shown.<p><b>Copyright information:</b></p><p>Taken from "Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET"</p><p>http://www.biomedcentral.com/1472-6750/8/20</p><p>BMC Biotechnology 2008;8():20-20.</p><p>Published online 26 Feb 2008</p><p>PMCID:PMC2275333.</p><p></p

Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET-3

Tris-HCl, pH 7.4 (-○-); Lipoplexes prepared in PBS (-◆-); Lipoplexes prepared in Tris-HCl and incubated on PBS (-△-); Lipoplexes prepared in Tris-HCl, incubated on PBS, followed by incubation on FBS (-□-).<p><b>Copyright information:</b></p><p>Taken from "Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET"</p><p>http://www.biomedcentral.com/1472-6750/8/20</p><p>BMC Biotechnology 2008;8():20-20.</p><p>Published online 26 Feb 2008</p><p>PMCID:PMC2275333.</p><p></p

Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET-0

Onor probe (d) on DNA (BOBO-1).<p><b>Copyright information:</b></p><p>Taken from "Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET"</p><p>http://www.biomedcentral.com/1472-6750/8/20</p><p>BMC Biotechnology 2008;8():20-20.</p><p>Published online 26 Feb 2008</p><p>PMCID:PMC2275333.</p><p></p

Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET-2

, incubated in PBS, followed by incubation in FBS for 30 min. (open symbols) and 2 h (filled symbols). Fitting curves using Eqs. 2–3 are also shown.<p><b>Copyright information:</b></p><p>Taken from "Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET"</p><p>http://www.biomedcentral.com/1472-6750/8/20</p><p>BMC Biotechnology 2008;8():20-20.</p><p>Published online 26 Feb 2008</p><p>PMCID:PMC2275333.</p><p></p