Light-scattering measurements.

<p>Light-scattering measurements.</p

Stoichiometry of the cationic lipopolymer/DNA complex.

<p>Percentage of plasmid DNA association as a function of cationic lipopolymer/pCH110 (a and b) or pDsRed (c and d) plasmid DNA ratios (mol of lipids: mol of base pairs) for the DMPC:DC_8,9PC:DOTAP (1:1:0.2), DMPC:DC_8,9PC:SA (1:1:0.2), and DMPC:DC_8,9PC:MCL (1:1:0.2) formulations.</p

Z-potential measurements.

<p>Z-potential measurements.</p

Study of cationic lipopolymer/DNA interaction by flow cytometry.

<p>Flow cytometry analysis results for (a) SSC-H versus FSC-H and (b) FL2 versus FL1 values for polymerized DMPC:DC_8,9PC:DOTAP (1:1:0.2)/SYBR^® Green I-labeled pDsRed plasmid DNA complexes and (c) SSC-H versus FSC-H and (d) FL2 versus FL1 values for polymerized DMPC:DC_8,9PC:MCL (1:1:0.2)/SYBR^® Green I-labeled pDsRed plasmid DNA complexes.</p

Effect of different incubation media on the cationic lipopolymer/DNA interaction.

<p>The different cationic lipopolymers were incubated with the pDsRed plasmid DNA in a 16:1 (mol of lipids: mol of base pairs) ratio in the medium (water, PBS or MEM) indicated on the right. Each lane was loaded with 1 μg of plasmid DNA. Lanes correspond to: (1) pDsRed alone with an additional 10-min incubation in the indicated medium, (2) pDsRed alone with an additional 10-min incubation in the presence of 10% v/v FBS, (3) cationic lipopolymer (stated above the gel picture)/pDsRed plasmid DNA complex formed in the indicated medium with an additional 10-min incubation without FBS, (4) cationic lipopolymer (stated above the gel picture)/pDsRed plasmid DNA complex formed in the indicated medium with an additional 10-min incubation in the presence of 10% v/v FBS, (5) cationic lipopolymer (stated above the gel picture)/pDsRed plasmid DNA complex formed in the indicated medium with an additional 10-min incubation in the presence of 50% v/v FBS, (6) cationic lipopolymer (stated above the gel picture)/pDsRed plasmid DNA complex formed in the indicated medium with 10% v/v FBS and with an additional 10-min incubation in the presence of 50% v/v FBS. The two main topological plasmid conformations, relaxed and negatively supercoiled, are indicated with arrows on the left of the figure noted as relax and -supercoiled, respectively.</p

Polymerization confirmation.

<p>Absorbance as a function of wavelength (nm) for the DMPC:DC_8,9PC:DOTAP (1:1:0.2), DMPC:DC_8,9PC:MCL (1:1:0.2) and DMPC:DC_8,9PC:SA (1:1:0.2) mixtures, prepared with the copolymerization methodology, after 20 UV irradiation cycles. Peaks observed around 480 and 520 nm are indicative of polymer formation.</p

Optimization of the study of cationic lipopolymer/DNA interaction by flow cytometry.

<p>Flow cytometry analysis results for non-polymerized DMPC:DC_8,9PC:MCL (1:1:0.2) liposomes, used to set control values of (a) SSC-H values (related to particle complexity) versus FSC-H values (related to particle size) and (b) FL1 values (where SYBR^® Green I-labeled plasmid DNA fluorescence is detected) and FL2 values (where cationic lipopolymer fluorescence is detected). (c) and (d) show the results for polymerized DMPC:DC_8,9PC:MCL (1:1:0.2) for SSC-H versus FSC-H and FL2 versus FL1 values respectively. (e) and (f) show the results for non-polymerized DMPC:DC_8,9PC:MCL (1:1:0.2)/SYBR^® Green I-labeled pDsRed plasmid DNA complexes for SSC-H and FSC-H values and FL2 versus FL1 values respectively.</p

Cytotoxicity determination.

<p>Cell viability percentage as a function of total lipid concentration (mM) for L929 cells incubated with cationic lipopolymers alone (a) or complexed with pDsRed plasmid DNA in a 16:1 (mol of lipids: mol of base pairs) ratio (b); and for Vero cells incubated with cationic lipopolymers alone (c) or complexed with pDsRed plasmid DNA in a 16:1 (mol of lipids: mol of base pairs) ratio (d). Statistical analysis was performed by Two-Way ANOVA with Tukey´s multiple comparisons post-test. *<0.05; **<0.01; ***<0.001; ****<0.0001.</p

Serum nucleases digestion assay.

<p>The different cationic lipopolymers DMPC:DC_8,9PC:DOTAP (1:1:0.2) and DMPC:DC_8,9PC:MCL (1:1:0.2)/pDsRed plasmid DNA complexes (16:1 mol of lipids: mol of base pairs ratio) were formed in water, PBS or MEM (indicated on the right). Each lane was loaded with 1 μg of plasmid DNA. Lanes correspond to: (1) pDsRed alone with a 24-h incubation in the indicated medium, (2) pDsRed alone with a 24-h incubation in the presence of 50% v/v FBS, (3) cationic lipopolymer (stated above the gel picture)/pDsRed plasmid DNA complex formed in the indicated medium with a 24-h incubation in the indicated medium without FBS, and (4) cationic lipopolymer (stated above the gel picture)/pDsRed plasmid DNA complex formed in the indicated medium with a 24-h incubation in the presence of 50% v/v FBS. The two main topological plasmid conformations, relaxed and negatively supercoiled, are indicated with arrows on the left of the figure noted as relax and -supercoiled, respectively. Degraded DNA is also indicated with an arrow on the left of the figure.</p

Hemolysis percentage (% H).

<p>Hemolysis percentage (% H).</p