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doctoral thesis
The effect of using wet lipid films, obtained by vesicle fusion, on electroformation of giant unilamellar vesicles with respect to different lipid compositions and charge, cholesterol concentration, and solution ionic strength
2025. urn:nbn:hr:166:489348
Mardešić, Ivan
University of Split
Faculty of Science
Department of Physics

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Mardešić, I. (2025). The effect of using wet lipid films, obtained by vesicle fusion, on electroformation of giant unilamellar vesicles with respect to different lipid compositions and charge, cholesterol concentration, and solution ionic strength (Doctoral thesis). Split: University of Split, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:166:489348

Mardešić, Ivan. "The effect of using wet lipid films, obtained by vesicle fusion, on electroformation of giant unilamellar vesicles with respect to different lipid compositions and charge, cholesterol concentration, and solution ionic strength." Doctoral thesis, University of Split, Faculty of Science, 2025. https://urn.nsk.hr/urn:nbn:hr:166:489348

Mardešić, Ivan. "The effect of using wet lipid films, obtained by vesicle fusion, on electroformation of giant unilamellar vesicles with respect to different lipid compositions and charge, cholesterol concentration, and solution ionic strength." Doctoral thesis, University of Split, Faculty of Science, 2025. https://urn.nsk.hr/urn:nbn:hr:166:489348

Mardešić, I. (2025). 'The effect of using wet lipid films, obtained by vesicle fusion, on electroformation of giant unilamellar vesicles with respect to different lipid compositions and charge, cholesterol concentration, and solution ionic strength', Doctoral thesis, University of Split, Faculty of Science, accessed 06 April 2025, https://urn.nsk.hr/urn:nbn:hr:166:489348

Mardešić I. The effect of using wet lipid films, obtained by vesicle fusion, on electroformation of giant unilamellar vesicles with respect to different lipid compositions and charge, cholesterol concentration, and solution ionic strength [Doctoral thesis]. Split: University of Split, Faculty of Science; 2025 [cited 2025 April 06] Available at: https://urn.nsk.hr/urn:nbn:hr:166:489348

I. Mardešić, "The effect of using wet lipid films, obtained by vesicle fusion, on electroformation of giant unilamellar vesicles with respect to different lipid compositions and charge, cholesterol concentration, and solution ionic strength", Doctoral thesis, University of Split, Faculty of Science, Split, 2025. Available at: https://urn.nsk.hr/urn:nbn:hr:166:489348

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Metadata
TitleThe effect of using wet lipid films, obtained by vesicle fusion, on electroformation of giant unilamellar vesicles with respect to different lipid compositions and charge, cholesterol concentration, and solution ionic strength
Title (croatian)Učinak korištenja vlažnih lipidnih filmova, dobivenih fuzijom vezikula, na elektroformaciju divovskih unilamelarnih vezikula s obzirom na različite lipidne sastave i naboj, koncentraciju kolesterola i ionsku snagu otopine
AuthorIvan Mardešić
MentorMarija Raguž (mentor)
Committee memberLucija Krce (predsjednik povjerenstva)
Committee memberŽeljka Sanader Maršić (član povjerenstva)
Committee memberMartina Perić Bakulić (član povjerenstva)
GranterUniversity of Split
Faculty of Science
(Department of Physics)
Split
Defense date and country2025-03-10, Croatia
Scientific / art field,
discipline and subdiscipline		NATURAL SCIENCES
Physics
Universal decimal classification
(UDC)		577 - Biochemistry. Molecular biology. Biophysics
Abstract
The long-term aim of this research is to investigate the properties of cholesterol bilayer domains (CBDs). Before starting experimental work, we conducted a detailed literature search and wrote a review article about all membrane models and experimental methods suitable for studying properties of CBD domains. We also described in details their known properties and functions in biological membranes. Our motivation stemmed from the beneficial role of CBDs in protecting the eye lens from cataract... More development and maintaining its homeostasis. Based on this knowledge we chosen giant unilamellar vesicles (GUVs) as an appropriate model for studying lipid portion of the fiber cell plasma membranes of the eye lens. GUVs are widely used as membrane models in studies of membrane properties, with the electroformation method being the most common approach for their production. However, several factors can affect the success of GUV electroformation. Common challenges include high cholesterol (Chol) concentrations, the use of lipid mixtures containing charged lipids, and solutions with elevated ionic strength. High Chol concentrations pose a particular problem for the traditional electroformation protocol, which involves drying the lipid film by fully evaporating the organic solvent. During this process, anhydrous Chol crystals can form, which do not integrate into the lipid bilayer, resulting in a lower Chol concentration in the vesicle bilayer compared to the original lipid mixture. To address the issue of artifactual Chol demixing, we have modified the electroformation protocol by introducing rapid solvent exchange, ultrasonication, plasma cleaning, and spin-coating techniques for the reproducible production of GUVs from damp lipid films. A high yield of GUVs was achieved for Chol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) samples with mixing ratios ranging from 0 to 2.5. At Chol/POPC mixing ratios greater than 1,5, where the GUVs may contain Chol bilayer domains (CBDs), a significant decrease in the average GUV diameter is measured. It is about 40% lower compared to that of the pure POPC bilayer. This modified approach not only minimizes Chol demixing but also successfully produces GUVs from lipid mixtures containing charged lipids and ionic solutions as the internal medium. We prepared GUVs from mixtures with up to 60 mol% of the charged lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and formed GUVs in low-concentration NaCl solutions. Less
Abstract (croatian)
Dugoročni cilj ovog rada je istraživanje svojstava čiste kolesterolove domene. Prije početka eksperimentalnog rada, istražili smo literaturu te napisali detaljni pregledni članak o različitim membranskim modelima i eksperimentalnim tehnikama prikladnima za istraživanje tih domena. Sva poznata svojstva i funkcije čiste kolesterolove domene su opisane. Glavna motivacija za istraživanje su istraživanje funkcija domene u leći oka, gdje ta domena omogućuje homeostazu stanice i ima zaštitnu ... More funkciju tijekom razvoja katarakte. Odlučili smo koristiti divovske unilamelarne vezikule (DUV) kao prikladan model za stanične membrane vlaknastih stanica leće oka. DUV-ovi se koriste kao membranski modeli za istraživanje osobina bioloških membrana, pri čemu je elektroformacija najčešće primjenjivana metoda za njihovo formiranje. Međutim, mnogo čimbenika može utjecati na uspjeh elektroformacije DUV-ova. Neki od izazova uključuju upotrebu visoke koncentracije kolesterola, nabijenih lipida i korištenje otopine s ionskom snagom. Visoke koncentracije kolesterola predstavljaju poseban problem za tradicionalni protokol, koji uključuje sušenje lipidnog filma. Tijekom ovog procesa mogu se formirati kolesterolovi kristali koji se ne ugrađuju u vezikulu, što rezultira nižom koncentracijom kolesterola u vezikuli nego u izvornoj smjesi lipida. Kako bismo se riješili problemom izlučivanja kolesterola, modificirali smo protokol elektroformacije korištenjem različitih metoda kao što su: brza izmjene otapala, sonikacija, čišćenje elektrode plazmom i brzo razmazivanje. Modificirani elektroformacijski protokol omogućuje reproducibilnu proizvodnju DUV-ova za različite omjere kolesterola i 1-palmitnska-2- oleoinska-sn-glicero-3-fosfokolina (POPC) od 0 do 2.5, uz minimiziranje izlučivanja kolesterola. Za omjere kolesterol/POPC veće od 1.5 izmjeren je pad drastični promjera DUV-ova. Promjer se smanjio oko 40% u usporedbi s čistom POPC membranom. Osim formiranja DUV-ova s visokom koncentracijom kolesterola, uspješno smo formirali DUV-ove koristeći nabijene lipide (1-palmitoil-2-oleoil-sn-glicero-3- fosfo-L-serin, POPS) i ionske otopine s niskom koncentracijom NaCl-a. Less
Keywords
Keywords (croatian)
Languageenglish
URN:NBNurn:nbn:hr:166:489348
Study programmeTitle: Doctoral Study of Biophysics
Study programme type: university
Study level: postgraduate
Academic / professional title: doktor/doktorica znanosti u području prirodnih znanosti (doktor/doktorica znanosti u području prirodnih znanosti)
Type of resourceText
Extent74 str. ; 30 cm
File originBorn digital
Access conditionsOpen access
Terms of useLicence In copyright icon
Created on2025-03-14 13:46:30
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