Effects of salt concentrations on the size distribution of giant unilamellar vesicles

Abstract

Lipid membranes of giant unilamellar vesicles (GUVs) of sizes 10 m or more are considered as a mimic of biomembranes of cells. In this investigation, the membranes of GUVs are prepared by a mixture of negatively charged lipid dioleoylphosphatidylglycerol (DOPG) and neutral lipid dioleoylphosphatidylcholine (DOPC) using the natural swelling method. It has been studied the effects of salt concentrations 12, 62, 112, 162, 212, 312, 362 and 412 mM containing in buffer (10 mM PIPES, pH 7.0 and 1 mM EGTA) on the size distribution and average sizes of self-assembled GUVs. The experimental results are presented in the form of set of histograms. The lognormal distribution is used to calculate the average sizes of GUVs from the mean and variance of the distribution. The values of average size of 40%DOPG/60%DOPC-GUVs (% indicates mole %) from two to three independent experiments are obtained (19.6  0.4) and (9.2  0.3) µm for 12 and 412 mM, respectively. The average size of GUVs decreases with the increase of salt concentration in buffer for fixed surface charge density of membrane. To understand the electrostatic effects due to salt concentrations, it has been also studied the effects of surface charge density in the membrane of GUVs i.e., 10%, 25%, 40%, 55%, 70% and 90% DOPG on the size distribution of GUVs in a buffer containing 162 mM. It is observed that with the rise of surface charge density the average sizes of GUVs increase. The values of average size of GUVs are obtained (11.9  0.9) and (17.9  0.9) µm for 10% and 90% DOPG, respectively at 162 mM salt. In each case of salt concentrations and surface charge density, the values of the co-efficient of determination (R2) are more than 0.90. A theory of size distribution of vesicles is developed to explain the experimental results using the Helmholtz’s free energy of the system. The size distribution histograms and average size of GUVs at various conditions are fitted with the proposed theory from which the bending modulus (Kben) is calculated for each salt concentration and surface charge density of the membrane where the values for 12 and 412 mM are obtained (35.9  0.1) and (11.1  0.4) kBT, respectively and for 10% and 90% DOPG Kben are obtained (17.0  0.2) and (30.1  0.1) kBT, respectively. To observe the effects of salt concentrations and surface charge density on the average sizes of GUVs more rigorously, it is used the purified GUVs in which purification is performed by the well known membrane filtering method. The results obtained from purified samples show a good consistency with the unpurified ones. It has shown that the variation of Kben due to the salt as well as the surface charge density is the main factor causing to change the sizes of GUVs.