(190bd) Sucrose Concentration Determines Giant Unilamellar Vesicle Size during Electroformation

Unilamellar
vesicles are spherical structures consisting of a lipid bilayer with an aqueous
core. Vesicles can be used to study the effects of different solutions and
proteins on physical properties of biomembranes. For example, hydrophobic
effects influence membrane properties such as bending rigidity and compression
density (Evans and Rawicz, PRL, 1990). The relative entropy of water in bulk
and at the phospholipid surface can be calculated using a two-phase
thermodynamic model to predict how sugar molecules in solution interact with
phospholipid membranes (Tian et al., Biophys J, 2013). Disaccharide sucrose
molecules laterally expand phospholipid monolayers by increasing the area
between head groups (Crowe et al., Biochim Biophys Acta, 1984); reduce the
thickness of individual bilayers (Anderson et al., PNAS, 2011); and increase
the spacing between adjacently stacked bilayers (LeNeveu et al., Biophys J,
1977). Herein, the relationship between sucrose concentration and the size
distributions of giant unilamellar vesicles made by electroformation is
investigated.

In
this study, giant unilamellar vesicles (GUVs) are made using readily available
materials.  An electroformation chamber is constructed from indium-tin
oxide (ITO)-coated glass slides sandwiching an o-ring and is powered using a
waveform generator (Fig. 1A). Lipids are extracted from chicken egg yolks
diluted in water, methanol and chloroform. After centrifugation, the chloroform
layer containing lipids is isolated and dried with a gentle flow of air. Nile
Red in ethanol is added to the lipids, which are again dried and reconstituted
in chloroform. A drop of lipid solution is then placed in the electroformation
chamber, dried, and hydrated with the sugar solution. Electroformation proceeds
with application of 2.6 V_p-p at 440 Hz, sinusoidal, for 24 h. Multiple
preparations are run simultaneously by connecting five chambers in parallel.
 Following electroformation, GUVs are released from the ITO surface with
gentle pipetting and diluted with an equimolar glucose solution at a 1:3
volumetric ratio. Multiple images are captured from each sample using a Thorlabs
confocal fluorescence microscope (Fig. 1B). GUV size distributions are acquired
using the Analyze Particles procedure embedded in Fiji.  

Results
indicate that increasing sucrose concentration creates a greater number of
vesicles and increased vesicle area. A logarithmic relationship exists between
sucrose concentration and mean vesicle area (Fig. 1C). The relationship is
likely caused by enhanced hydration of dried bilayers with more sucrose, which
up to a threshold concentration increases interbilayer distance and swelling to
form both more and larger GUVs.

Figure
1. (A)
Diagram of electroformation chamber. (B) A representative confocal fluorescence
image of a GUV formed in 100 mM sucrose. (C) Plot of mean vesicle area as a
function of sucrose concentration. Error bars represent standard error of the
mean (SEM).