Petra Pullmannová

The ionic strength effect on the DNA complexation by DOPC — gemini surfactants liposomes

Liposome dispersions obtained from the mixture of gemini surfactants of the type alkane-α,ω-diyl-bis(alkyldimethylammonium bromide) and helper lipid DOPC create complexes with DNA showing a regular inner microstructure, identified by small angle X-ray diffraction as condensed lamellar phase (L(α)(c)). In addition to the L(α)(c) phase, a coexisting lamellar phase L(B) was also identified in the complexes formed, with periodicities in the range ~8.8-5.7nm, at ionic strengths corresponding to 50-200mM NaCl. The periodicities of L(B) phase did not correspond to those identified in liposome dispersion without DNA using small angle neutron scattering. The observed phase separation is shown to depend on the interplay between the surface charge density of cationic liposomes, ionic strength and method of complex preparation. The effect of ionic strength on complex formation was studied by isothermal titration calorimetry and zeta potential measurements. High ionic strength reduces the fraction of bound DNA in the complexes, and the isoelectric point is attained at a ratio of DNA/gemini surfactant which is lower than the one that can be estimated by calculation based on nominal charges of CLs and DNA.

The DNA-DNA spacing in gemini surfactants-DOPE-DNA complexes.

Gemini surfactants from the homologous series of alkane-α,ω-diyl-bis(dodecyldimethylammonium bromide) (CnCS12, number of spacer carbons n=2-12) and dioleoylphosphatidylethanolamine (DOPE) were used for cationic liposome (CL) preparation. CLs condense highly polymerized DNA creating complexes. Small-angle X-ray diffraction identified them as condensed lamellar phase L(α)(C) in the studied range of molar ratios CnGS12/DOPE in the temperature range 20-60°C. The DNA-DNA distance (d(DNA)) is studied in dependence to CnGS12 spacer length and membrane surface charge density. The high membrane surface charge densities (CnGS12/DOPE=0.35 and 0.4mol/mol) lead to the linear dependence of d(DNA) vs. n correlating with the interfacial area of the CnGS12 molecule.

Lipid bilayer – DNA interaction mediated by divalent metal cations: SANS and SAXD study

The structure of aggregates formed due to DNA interaction with dipalmitoylphosphatidylcholine (DPPC) in presence of Ca2+ and Zn2+ is examined using small-angle synchrotron X-ray diffraction (SAXD) and small-angle neutron scattering (SANS). SAXD detected two structures: LC - condensed lamellar phase and LX - lamellar phase with DNA strands intercalated between the adjacent lipid bilayers, without regular packing at low cation concentration (~1 mM). The high concentration of Zn2+ induces a macroscopic phase separation in mixtures. The SANS curves of DPPC+ions2+ vesicles evaluated using the strip function model have shown different modes of cations binding to the DPPC bilayers.

Stimuli responsive polymorphism of C12NO/DOPE/DNA complexes: Effect of pH, temperature and composition.

N,N-dimethyldodecylamine-N-oxide (C12NO) is a surfactant that may exist either in a neutral or cationic protonated form depending on the pH of aqueous solutions. Using small angle X-ray diffraction (SAXD) we observe the rich structural polymorphism of pH responsive complexes prepared due to DNA interaction with C12NO/dioleoylphosphatidylethanolamine (DOPE) vesicles and discuss it in view of utilizing the surfactant for the gene delivery vector of a pH sensitive system. In neutral solutions, the DNA uptake is low, and a lamellar Lα phase formed by C12NO/DOPE is prevailing in the complexes at 0.2≤C12NO/DOPE<0.6 mol/mol. A maximum of ~30% of the total DNA volume in the sample is bound in a condensed lamellar phase LαC at C12NO/DOPE=1 mol/mol and pH7.2. In acidic conditions, a condensed inverted hexagonal phase HIIC was observed at C12NO/DOPE=0.2 mol/mol. Commensurate lattice parameters, aHC≈dLC, were detected at 0.3≤C12NO/DOPE≤0.4 mol/mol and pH=4.9-6.4 suggesting that LαC and HIIC phases were epitaxially related. While at the same composition but pH~7, the mixture forms a cubic phase (Pn3m) when the complexes were heated to 80°C and cooled down to 20°C. Finally, a large portion of the surfactant (C12NO/DOPE>0.5) stabilizes the LαC phase in C12NO/DOPE/DNA complexes and the distance between DNA strands (dDNA) is modulated by the pH value. Both the composition and pH affect the DNA binding in the complexes reaching up to ~95% of the DNA total amount at acidic conditions.

DNA – DOPC – gemini surfactants complexes: effect of ionic strength

Abstract The effect of ionic strength on DNA condensation by cationic liposomes prepared as a mixture of ethane-1,2-diylbis(dodecyl-dimethylammonium bromide) (C2GS12) and dioleoylphosphatidylcholine (DOPC) was studied using fluorescence spectroscopy. The DNA condensation followed by changes in emission intensity of ethidium bromide shows a strong dependence on the ionic strength of the solution. At physiologically relevant ionic strength (0.15 mol/l NaCl), the amount of DNA condensed between lipid bilayers is approximately 40% lower compared to 0.005 mol/l NaCl. The structure of formed complexes was studied using small angle X-ray diffraction (SAXD). DNA–C2GS12–DOPC complexes form a condensed lamellar phase organisation, which is partially disrupted by the increase of ionic strength. Both the lamellar repeat distance and DNA–DNA distance show dependence on C2GS12/DOPC molar ratio, temperature and also on ionic strength. We found that the method of preparation significantly affects both the quality of organisation and the structural parameters of complexes as discussed in the paper.

Study of interaction of long-chain n-alcohols with fluid DOPC bilayers by a lateral pressure sensitive fluorescence probe.

The excimer 1,2-dipyrenedecanoyl-sn-glycero-3-phosphatidylcholine (dipy10PC) fluorescence probe was used to determine effects of aliphatic alcohols (CnH2n+1OH, n = 12-18 is the even number of carbons in alkyl chain) on fluid dioleoylphosphatidylcholine (DOPC) +dioleoylphosphatidylserine (DOPS) bilayers in multilamellar vesicles at molar ratio DOPC/DOPS = 24.7. The excimer to monomer fluorescence intensity ratio increases with the increase of CnH2n+1OH/DOPC molar ratio and decreases with the CnH2n+1OH alkyl chain length n at a constant CnH2n+1OH/DOPC = 0.4 molar ratio. These effects indicate changes in the bilayer lateral pressure on the level of pyrenyl moieties location.

The microstructure of DNA-egg yolk phosphatidylcholine-gemini surfactants complexes: effect of the spacer length

Abstract Background: The length of spacer of gemini surfactants affects the DNA packing in DNA-neutral phospholipid-gemini surfactant complexes. Methods: The microstructure of complexes DNA-egg yolk phosphatidylcholine (EYPC)-alkane-α,ω-diyl-bis(dodecyl\xaddimethylammonium bromides) (CnGS, spacer n=2–12, n is even) was studied using small angle X-ray diffraction. Results: At EYPC:CnGS=1:1 mol/mol, the condensed lamellar phase was identified in complexes with CnGS, n=2–4, whereas longer spacer (n≥6) induced a hexagonal phase. The condensed lamellar phase Lαc was observed in the range 2\≤ EYPC:GnGS≤10 (mol/mol) in all complexes. The distance between adjacent DNA strands increases linearly with decreasing surface charge density of EYPC-CnGS vesicles. We determined the increase in dDNA 0.40±0.03 nm/1 mol of EYPC from the slope of dDNA=f (molEYPC/molCnGS) in the range of molar ratios 2≤EYPC:CnGS≤5. At lower surface charge density, EYPC:CnGS>5 mol/mol, the length of CnGS spacer (n=6–10) modulates the DNA-DNA distance. Conclusions: Both the short spacer of CnGS and the low molar ratio EYPC:CnGS result in the closest DNA-DNA packing. A high surface charge density of membrane was reported as a key parameter for transfection efficiency of Lαc phase-forming complexes.

Small Angle X‐ray Diffraction Study of DNA—Cationic Liposomes Aggregates

The microstructure of DNA—dioleoylphosphatidylethanolamine (DOPE)—propane‐1,3‐diyl‐bis(dodecyldimethylammonium bromide) (C3GS12) aggregates as a function of the C3GS12:DOPE molar ratio and temperature was investigated using small angle X‐ray diffraction. At 20° C, we observe a condensed lamellar phase (Lαc) with the lattice parameter d∼6.8–6.2 nm and the DNA—DNA distance dDNA∼5.8–3.2 nm decreasing with increasing content of C3GS12 in the phospholipid bilayer. Increase in temperature induces a phase transition from Lαc phase to condensed inverted hexagonal phase (HIIc). The temperature of the Lαc→HIIc phase transition increases with increasing C3GS12:DOPE molar ratio.