Study on the purification of lipid membranes of giant unilamellar vesicles using a locally developed low cost technique

Abstract

Lipid membranes of giant unilamellar vesicles (GUVs) with diameters greater than 10 m are promising model membranes for investigating the physical and biological properties of biomembranes of cells. To study the interaction of various membranes active agents like nanoparticles and peptides, elasticity of membrane, shape change, phase change, membrane rupture, electroporation, purified and similar-sized oil-free GUVs are necessary. In the proposed technique, the gravitational energy is used to continue the flow of buffer and a locally available low cost roller clamp regulator, which is used in medical sector to give saline into human body, is used to control the flow rate of buffer with GUVs suspension. This technique has been characterized in terms of size distribution, average size, flow rate and efficiency of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC)-GUVs prepared by the natural swelling method. The GUVs were purified using three different flow rates 1.0, 1.5 and 2.0 mL/min. After purification of GUVs, the diameters of 300-400 GUVs have been measured and plotted a histogram. The histograms are fitted with a theoretical equation and the fitting is evaluated by the co-efficient of determination (R2). In each case, the values of R2 were more than 0.90. The average diameter of unpurified and purified GUVs is calculated by taking mean and variance from the histogram. Before purification, the average sizes of GUVs of two independent experiments are 10.  0.7, 11.2  3.7 and 8.8  0.4 m. Whereas the average sizes of purified GUVs of two independent experiments are 16.9  0.8, 18.7  0.3 and 20.0  0.1 m for the flow rates 1.0, 1.5 and 2.0 mL/min. respectively. After purification, the average sizes of GUVs are increased because the smaller GUVs are passed away through the filter during the purification. In well reported membrane filtering method, the average sizes of unpurified GUVs of two independent experiments are 8.1  0.8, 11.3  4.3 and 7.7  0.3 m and the average sizes of purified GUVs of two independent experiments are 17.6  2.1, 19.7  1.2 and 20.0  0.1 m for the flow rates 1.0, 1.5 and 2.0 mL/min. respectively. It illustrates that the new technique gives the similar results within the error bar to that of membrane filtering method. So, it can be declared that this technique can perform the purification of GUVs using only gravity without any electromechanical devices and the apparatuses are repeatedly usable. This technique is ecofriendly, time saving and cost effective. Therefore, it will be a low cost non-electromechanical future tool for the purification of GUVs with using gravity.