Small diamines as modifiers for phosphatidylcholine/phosphatidylserine coatings in capillary electrochromatography

Greater stability of liposome coatings and improved resolution of model steroids in capillary electrochromatography (CEC) were sought by adding small diamines (ethylenediamine, diaminopropane, bis-tris-propane, or N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid, HEPES)) to the liposome solution before coating of fused silica capillaries. The phospholipid coatings consisted of 1 mM of 8:2 mol% phosphatidylcholine (PC)/phosphatidylserine (PS) and 5 mM of modifier in buffer solutions (acetate, phosphate, or Tris) at pH 4.0-7.4. The coating was based on a published procedure, and five steroids were used as neutral model analytes in evaluation of the coating. The results showed that under optimal conditions, the small linear diamines increased the packing density of anionic phospholipids, leading to improved separations. In addition, the choice of buffer for the liposome coating and separation appeared to influence the performance of the coatings. While buffers with amino groups take part in the phospholipid bilayer formation, buffers like phosphate may even have negative effect on coating formation. The factors affecting phospholipid coatings with diamines as modifiers are clarified.     

Adsorption of DNA onto anionic lipid surfaces

Currently self-assembled DNA delivery systems composed of DNA multivalent cations and anionic lipids are considered to be promising tools for gene therapy. These systems become an alternative to traditional cationic lipid–DNA complexes because of their low cytotoxicity lipids. However, currently these nonviral gene delivery methods exhibit low transfection efficiencies. This feature is in large part due to the poorly understood DNA complexation mechanisms at the molecular level. It is well-known that the adsorption of DNA onto like charged lipid surfaces requires the presence of multivalent cations that act as bridges between DNA and anionic lipids. Unfortunately, the molecular mechanisms behind such adsorption phenomenon still remain unclear. Accordingly a historical background of experimental evidence related to adsorption and complexation of DNA onto anionic lipid surfaces mediated by different multivalent cations is firstly reviewed. Next, recent experiments aimed to characterise the interfacial adsorption of DNA onto a model anionic phospholipid monolayer mediated by Ca^2 + (including AFM images) are discussed. Afterwards, modelling studies of DNA adsorption onto charged surfaces are summarised before presenting preliminary results obtained from both CG and all-atomic MD computer simulations. Our results allow us to establish the optimal conditions for cation-mediated adsorption of DNA onto negatively charged surfaces. Moreover, atomistic simulations provide an excellent framework to understand the interaction between DNA and anionic lipids in the presence of divalent cations. Accordingly,our simulation results in conjunction go beyond the macroscopic picture in which DNA is stuck to anionic membranes by using multivalent cations that form glue layers between them. Structural aspects of the DNA adsorption and molecular binding between the different charged groups from DNA and lipids in the presenceof divalent cations are reported in the last part of the study. Although this research work is far from biomedical applications, we truly believe that scientific advances in this line will assist, at least in part, in the rationaldesign and development of optimal carrier systems for genes and applicable to other drugs.     

Pseudo Real-Time Method for Monitoring of the Limiting Anisotropy in Membranes

Data acquisition and analysis of the time-resolved fluorescence anisotropy is typically a time consuming process preventing usage of this experimental method for monitoring of time-dependent phenomena. We describe a method for pseudo real-time monitoring of the limiting fluorescence anisotropy r(infinity) allowing to track changes of the membrane order occurring on the time scale of minutes. Principle and performance of the method is demonstrated in the time domain with the time-correlated single photon counting detection. DMPC liposomes stained with 1,6-diphenyl-1,3,5-hexatriene (DPH) have been used to test influence of the diffusion membrane potential on the membrane order during the temperature-induced phase transition in DMPC membranes. It has been found that the transmembrane field of the order of -70 mV increases the phase transition temperature by about 1.5 degrees C-2 degrees C. It is proposed that the full advantage of the method can be utilized with a gated detection, which besides a faster data acquisition brings additional advantage of excitation light suppression. The method can be also used for imaging.