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Characterization of cationic lipid DNA transfection complexes differing in susceptability to serum inhibition.

Nchinda G, Uberla K, Zschörnig O - BMC Biotechnol. (2002)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Virology, University of Leipzig, Leipzig, Germany. godwin.nchinda@ruhr-uni-bochum.de

ABSTRACT

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.

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Cytotoxicity of DOTAP (A) or DC (B) DNA complexes formed at different ratios of lipid to protamine sulfate to DNA. Two days after transfection in the presence of 10% serum the toxicity of the complexes was determined by the MTT assay. Values are expressed as the percent viability of untransfected cells.
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Figure 2: Cytotoxicity of DOTAP (A) or DC (B) DNA complexes formed at different ratios of lipid to protamine sulfate to DNA. Two days after transfection in the presence of 10% serum the toxicity of the complexes was determined by the MTT assay. Values are expressed as the percent viability of untransfected cells.

Mentions: Since toxicity of the lipid DNA complexes could limit their use, cell viability was analyzed after transfection with various ratios of lipid to DNA to protamine sulphate. For ratios of up to 64 nmol DOTAP/μg DNA cell viability was higher than 80% of the viability of untransfected cells independent of the amount of protamine sulfate added (Fig. 2A). At higher DOTAP to DNA ratios (> 64 nmol DOTAP/μg DNA), toxicity peaked at 8 μg protamine sulfate/μg DNA. High cell viabilities were observed for complexes containing DC at ratios ≤ 64 nmol lipid/μg DNA with limited correlation of the amount of protamine sulfate added to the complexes.


Characterization of cationic lipid DNA transfection complexes differing in susceptability to serum inhibition.

Nchinda G, Uberla K, Zschörnig O - BMC Biotechnol. (2002)

Cytotoxicity of DOTAP (A) or DC (B) DNA complexes formed at different ratios of lipid to protamine sulfate to DNA. Two days after transfection in the presence of 10% serum the toxicity of the complexes was determined by the MTT assay. Values are expressed as the percent viability of untransfected cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC117600&req=5

Figure 2: Cytotoxicity of DOTAP (A) or DC (B) DNA complexes formed at different ratios of lipid to protamine sulfate to DNA. Two days after transfection in the presence of 10% serum the toxicity of the complexes was determined by the MTT assay. Values are expressed as the percent viability of untransfected cells.
Mentions: Since toxicity of the lipid DNA complexes could limit their use, cell viability was analyzed after transfection with various ratios of lipid to DNA to protamine sulphate. For ratios of up to 64 nmol DOTAP/μg DNA cell viability was higher than 80% of the viability of untransfected cells independent of the amount of protamine sulfate added (Fig. 2A). At higher DOTAP to DNA ratios (> 64 nmol DOTAP/μg DNA), toxicity peaked at 8 μg protamine sulfate/μg DNA. High cell viabilities were observed for complexes containing DC at ratios ≤ 64 nmol lipid/μg DNA with limited correlation of the amount of protamine sulfate added to the complexes.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Virology, University of Leipzig, Leipzig, Germany. godwin.nchinda@ruhr-uni-bochum.de

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus