Characterization of cationic lipid DNA transfection complexes differing in susceptability to serum inhibition.
BACKGROUND
Cationic lipid DNA complexes based on DOTAP (1,2-dioleoyl-3-(trimethyammonium) propane) and mixtures of DOTAP and cholesterol (DC) have been previously optimized for transfection efficiency in the absence of serum and used as a non-viral gene delivery system. To determine whether DOTAP and DC lipid DNA complexes could be obtained with increased transfection efficiency in the presence of high serum concentrations, the composition of the complexes was varied systematically and a total of 162 different complexes were analyzed for transfection efficiency in the presence and absence of high serum concentrations.


RESULTS
Increasing the ratio of DOTAP or DC to DNA led to a dose dependent enhancement of transfection efficiency in the presence of high serum concentrations up to a ratio of approximately 128 nmol lipid/microg DNA. Transfection efficiency could be further increased for all ratios of DOTAP and DC to DNA by addition of the DNA condensing agent protamine sulfate (PS). For DOTAP DNA complexes with ratios of < or = 32 nmol/microg DNA, peak transfection efficiencies were obtained with 4 microg PS/microg DNA. In contrast, increasing the amount of PS of DC complexes above 0.5 microg PS/microg DNA did not lead to significant further increases in transfection efficiency in the presence of high serum concentrations. Four complexes, which had a similar high transfection efficiency in cell culture in the presence of low serum concentrations but which differed largely in the lipid to DNA ratio and the amount of PS were selected for further analysis. Intravenous injection of the selected complexes led to 22-fold differences in transduction efficiency, which correlated with transfection efficiency in the presence of high serum concentrations. The complex with the highest transfection efficiency in vivo consisted of 64 nmol DC/ 16 microg PS/microg DNA. Physical analysis revealed a predicted size of 440 nm and the highest zeta potential of the complexes analyzed.


CONCLUSIONS
Optimization of cationic lipid DNA complexes for transfection efficiency in the presence of high concentrations of serum led to the identification of a DC complex with high transduction efficiency in mice. This complex differs from previously described ones by higher lipid to DNA and PS to DNA ratios. The stability of this complex in the presence of high concentrations of serum and its high transduction efficiency in mice suggests that it is a promising candidate vehicle for in vivo gene delivery.
Nchinda
Godwin
G
Institute of Virology, University of Leipzig, Leipzig, Germany. godwin.nchinda@ruhr-uni-bochum.de
Uberla
Klaus
K
Zschörnig
Olaf
O
D000818:Animals; D002412:Cations; D002417:Cattle; D002460:Cell Line; D002784:Cholesterol / Q000737:chemistry; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D005260:Female; D006801:Humans; D007275:Injections, Intravenous; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D044967:Serum / Q000502:physiology*; D014162:Transfection / Q000379:methods*
D002412:Cations; D003470:Culture Media; D005229:Fatty Acids, Monounsaturated; D008055:Lipids; D008081:Liposomes; D000644:Quaternary Ammonium Compounds; D011993:Recombinant Fusion Proteins; D004247:DNA; D002784:Cholesterol; D008156:Luciferases; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane