1. A Versatile Linker for Nontoxic Polyamine-Mediated DNA Transfection
Edmund J Niedzinski, Scott K Fujii, Mike E Lizarzaburu, James G Hecker, Michael H Nantz 
Molecular Therapy 
Volume 6, Issue 2, Pages (August 2002)
DOI: /mthe
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
Synthesis of polyamines. Reagents and conditions: (A) H2C=CHC(O)Cl (3.0 eq), Et3N (3 eq), cat. DMAP, CH2Cl2, 0°C to room temperature, 24 hours; (B) diamine, 2-(N,N-dimethyl)ethanamine; or 3-(N,N-dimethylamino) propanamine; or 4-(N,N-dimethylamino)butanamine.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
Effect of pH on DNA condensation by polyamines 8–10. Formulations of polyamines 8–10 and cholesterol were added to 0.5 μ9 pCMV-luc DNA that was pre-incubated with ethidium bromide. The lipoplexes were then treated with either water (diamond), 10 mM Tris, pH 7, (square), 10 mM Tris, pH 8, (triangle), or 10 mM Tris, pH 9, (open square). The resulting fluorescence was measured at 250 nm excitation and 610 nm-emission. Each data point reflects the mean value from three samples, and the standard deviation from the mean. The background fluorescence of ethidium bromide in the absence of DNA was 4.52 RFU.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
Optimization of CHO cell transfection using polyamines 5–10. Polyamine formulations (compounds depicted as alternative black and white bars) containing equimolar DOPE (condition A) or cholesterol (condition B) were complexed with luciferase pDNA at the indicated N:P ratios and used to deliver 1 μg DNA/well. Luciferase activity in the cell lysates was determined 24 hours after the transfection. With the exception of polyamine 9, all of the polyamines were tested simultaneously to determine the optimal formulation. Each data point reflects the mean value from three transfections, and the standard deviation from the mean.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Analysis of transfection activity in CHO cells using Luciferase Assay. Polyamine 5–10 formulations containing equimolar cholesterol were complexed with luciferase plasmid at a 5:1 N:P ratio and used to deliver 1 μg DNA/well. LipofectAMINE PLUS (LFP) and TransFast (TF) were formulated at N:P ratios of 5:1 and 3:1, respectively, and used to deliver 1 μg DNA/well. Luciferase activity in the cell lysates was determined 24 hours after the transfection. Each data point reflects the mean value from three transfections, and the standard deviation from the mean.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
Analysis of transfection activity in NIH3T3 cells using Luciferase Assay. Polyamine 5–10 formulations containing equimolar cholesterol were complexed with luciferase plasmid at a 10:1 N:P ratio and used to deliver 1 μg DNA/well. LipofectAMINE PLUS (LFP) and TransFast (TF) were formulated at N:P ratios of 5:1 and 3:1, respectively, and used to deliver 1 μg DNA/well. Luciferase activity in the cell lysates was determined 24 hours after the transfection. Each data point reflects the mean value from three transfections, and the standard deviation from the mean.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

7. FIG. 6
Analysis of transfection activity and cytotoxicity in CHO cells as measured by flow cytometry. Polyamine 5–10 formulations containing equimolar cholesterol were complexed with GFP plasmid at a 5:1 N:P ratio and used to deliver 1 μg DNA/well. Cells were analyzed for GFP fluorescence to determine the transfection activity and for annexin V binding to determine cytotoxicity 24 hours after transfection. The black bars represent the percentage of the cell population that was GFP-positive and annexin V-negative. The gray bars represent percentage of the cell population that was GFP-positive and annexin V-positive. The white bars represent the percentage of the cell population that was GFP-negative and annexin V-positive. The remaining percentage of the cell population that was GFP-negative and annexin V-negative has not been represented.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

8. FIG. 7
Analysis of transfection activity and cytotoxicity in NIH3T3 cells as measured by flow cytometry. Polyamine 5–10 formulations containing equimolar cholesterol were complexed with GFP plasmid at a 10:1 N:P ratio and used to deliver 1 μg DNA/well. Cells were analyzed for GFP fluorescence to determine the transfection activity and for annexin V binding to determine cytotoxicity 24 hours after transfection. The black bars represent the percentage of the cell population that was GFP-positive and annexin V-negative. The gray bars represent percentage of the cell population that was GFP-positive and annexin V-positive. The white bars represent the percentage of the cell population that was GFP-negative and annexin V-positive. The remaining percentage of the cell population, which was GFP-negative and Annexin V-negative, is not represented.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

9. FIG. 8
Structural comparison of polyamine 9, DOSPA and DMDHP. Polyamines 5–10 and two commercially available transfection reagents, DOSPA and DMDHP, were simultaneously analyzed to demonstrate the effect of the chemical structure on transfection activity and cytotoxicity.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions