Oscillation And Gibbs-Donnan Equilibrium Of Na~+ And K~+ Concentrations Inside And Outside The Liposome

Because of the similarity to living cells in morphology,liposomes have been widely used as a model for simulating cells in the fields of the toxicology,pharmacology and biology which has become a important technique and investigation method in life science.The prerequisite for the liposomes acting as experimental model was stability.Vesicles were not a general thermodynamically stable system. The mean size,the lamellarity and the physical stability of the vesicles depended not only on the chemical structure and size of the amphiphiles used,but on the preparation methods of vesicle as well as the environmental ion factors.The thin film hydration method and ultrasonic method were used to optimize the liposomes' preparation through the one-way analysis and the orthogonal technique:soyebeen phosphatidylcholine and cholesterol were mixed in chloroform,giving molar ratio 1:1.Then the organic solvent was removed under vacuum by using a rotary evaporator in a water bath for 10 min.The rotating speed was 5～6 rounds/rain.The flask was then flushed with nitrogen for 1-2 min to remove remnants of the solvent.The pH7.8 phosphate buffer was added to the dry film in the flask to give a final phospholipid concentration of 1mg/mL.The film was removed by shaking the flask and homogenized by ultrasonic wave under ultrasonic frequency of 80%for 5min.The liposomes could be kept at room temperature for about a week.In order to better simulate the cell and the ion environment,the artificial cell was defined as the liposome in a certain ion environment. The most important simple inorganic ions Na⁺,K⁺,Ca²⁺ were chosen to investigate their influences on the liposomes' stability by measuring the absorbance,the Zeta potential,the size and counting the number under the microscope.An appropriate ion concentration range of the original solution was found to be 0.9-3 mM/L for Na⁺,1-7 mM/L for K⁺ and 0.3-1 mM/L,for Ca²⁺ respectively to keep the liposomes stable and the number almost unchanged.The stability of the liposomes was destroyed by a relatively lower concentration of Ca²⁺ or a relatively higher concentration of K⁺,which might be attributed to the smallest ion radius of K⁺.At the same time,the Na,K,Ca ion selective electrodes were used to measure the ion concentration outside the liposomes when adding the mixture,pH value was measured by pH electrode.The inner ion concentration was calculated so as to find the transmembrane mechanism of the ion when there was a competition between two cations in the system.We found that when Na⁺,K⁺ or Ca²⁺ added separately to the liposomes,its permeation followed the Donnan equilibrium;when Na⁺ and K⁺ were added collectively to the liposomes,their permeation behavior might be described by the Gibbs-Donnan equilibrium.Whether the ions added separately or collectively,the inner concentration was much higher than the bulk concentration.The permeation of the ions on both sides of the liposomes kept no equilibrium, which was caused by Donnan effect.Since its first report in the artificial liquid membrane by Yoshikawa, chemical oscillation had been a big attract for many researchers.In our experiment,the oscillation of Na⁺ and K⁺ concentrations across the liposome membrane was discovered and approved,which was a fluctuation around the equilibrium,a dynamic process,but not a phenomenon running to equilibrium.The one-way analysis and orthogonal technique were used to optimize the oscillation in the liposomes as follows:the material was yolk lecithin;the mol ratio of yolk lecithin to Cholesterol was 1:1;the ratio of Na⁺ to K⁺ was 2 mM:1 mM.There was the conservation of charge in the oscillation system.The anion oscillation was complementary to that of the cation.The oscillation phenomenon was based on the Gibbs-Donnan equilibrium.The sufficient condition for oscillation was deduced to be Gibbs-Donnan equilibrium. The necessary condition for oscillation was that,on the one hand,the concentration of one ion demonstrated the Marangoni effect which meant that the concentration was beyond the liposomes' stable ion concentration range and that the absorption of the ion provided the power for dissolving liposome;on the other hand,the concentration of the second ion was within the liposomes' stable ion concentration range and the diffusion of the ion provided the transmembrane power,accordingly a non-periodical and irregular oscillation of the ion appeared.Because of the great influence of temperature on ion penetration,heat changes were measured by VP-DSC by adding the same moles of ions to the liposomes.In our experiment,the ratio of the Na⁺ to K⁺ was 2 mM:1 mM when added collectively,while the concentrations of Na⁺ and K⁺ were 3 mM when added singly.The measurement lasted for 30 min in every system.The result showed that with the rise in temperature,the oscillation system kept exothermic all the time,while the systems with no oscillation phenomenon changed from exothermic to endothermic state, and compared with the other two systems,the oscillation system gave the largest absolute values of the heat under the same temperature.The experiment results not only had an important significance for the application of the liposome experiment model at different situations,but also enlightened the understanding of the ion condition of the cells.