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Effect of Electrical Parameters and Cholesterol Concentration on Giant Unilamellar Vesicles Electroformation

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Abstract

Giant unilamellar vesicles (GUVs) are used extensively as models that mimic cell membranes. The cholesterol (Chol) content in the fiber cell plasma membranes of the eye lens is extremely high, exceeding the solubility threshold in the lenses of old humans. Thus, a methodological paper pertaining to preparations of model lipid bilayer membranes with high Chol content would significantly help the study of properties of these membranes. Lipid solutions containing 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and Chol were fluorescently labeled with phospholipid analog 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiIC18(3)) and spin-coated to produce thin lipid films. GUVs were formed from these films using the electroformation method and the results were obtained using fluorescent microscopy. Electroformation outcomes were examined for different electrical parameters and different Chol concentrations. A wide range of field frequency–field strength (ff–fs) combinations was explored: 10–10,000 Hz and 0.625–9.375 V/mm peak-to-peak. Optimal values for GUVs preparation were found to be 10–100 Hz and 1.25–6.25 V/mm, with largest vesicles occurring for 10 Hz and 3.75 V/mm. Chol:POPC mixing ratios (expressed as a molar ratio) ranged from 0 to 3.5. We show that increasing the Chol concentration decreases the GUVs size, but this effect can be reduced by choosing the appropriate ff–fs combination.

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Acknowledgements

We thank Ante Bilušić, Katarina Vukojević, Damir Sapunar, Sandra Kostić, Jasna Puizina, Ivana Bočina, Ivica Aviani, and Lucija Krce for access to their lab equipment and helpful discussions and comments.

Funding

Research reported in this publication was supported by the Croatian Science Foundation (Croatia) under Grant [IP-2019-04-1958], by the National Institutes of Health (USA) under Grant R01 EY 015526, and by the Polish National Science Center under Grant [2016/22/M/NZ1/00187].

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Authors and Affiliations

  1. Department of Medical Physics and Biophysics, University of Split School of Medicine, Split, Croatia

    Zvonimir Boban, Ana Puljas & Marija Raguz

  2. University of Split, Faculty of Science, Doctoral study of Biophysics, Split, Croatia

    Zvonimir Boban

  3. Department of Physics, Faculty of Science, University of Split, Split, Croatia

    Dubravka Kovač

  4. Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI, USA

    Witold Karol Subczynski

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  1. Zvonimir Boban
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  2. Ana Puljas
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  4. Witold Karol Subczynski
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Correspondence to Marija Raguz.

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Boban, Z., Puljas, A., Kovač, D. et al. Effect of Electrical Parameters and Cholesterol Concentration on Giant Unilamellar Vesicles Electroformation. Cell Biochem Biophys 78, 157–164 (2020). https://doi.org/10.1007/s12013-020-00910-9

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