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A comparative study on ganglioside micelles using electronic energy transfer, fluorescence correlation spectroscopy and light scattering techniques
Umeå University, Faculty of Science and Technology, Department of Chemistry. (J. Heyrovsky´ Institute of Physical Chemistry of the Academy of Sciences of the Czech Republic, Dolejsˇkova 3 182 23 Prague 8,Czech Republic)
Shemyakin & Ovchinnikov Institute, Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia. (Department of Chemistry, Biophysical Chemistry, Umeå University, Umeå, Sweden)
J. Heyrovsky´ Institute of Physical Chemistry of the Academy of Sciences of the Czech Republic, Dolejsˇkova 3 182 23 Prague 8,Czech Republic.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
2009 (English)In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 11, no 21, 4335-4343 p.Article in journal (Refereed) Published
Abstract [en]

Ganglioside (GM1) micelles have been studied by means of three different techniques: fluorescence correlation spectroscopy (FCS), electronic energy transfer, as monitored by time-resolved fluorescence spectroscopy, as well as static and dynamic light scattering. The aggregation numbers obtained, 168 ± 4, remain constant over a wide range of GM1 concentrations (0.764-156 M), are very consistent when using different donor-acceptor energy transfer pairs and have served as reference values in tests of the FCS method. It is recommended to calibrate the focal volume by using known dye concentrations. For this the rhodamine dye, 5-TAMRA, turns out to be most suitable. It is also shown that FCS provides correct values of the aggregation numbers, provided that the focal volume is calibrated by using updated values of the diffusion constant of Rhodamine 6G. These results also support recent methodological advances in FCS.

Place, publisher, year, edition, pages
Royal Society of Chemistry , 2009. Vol. 11, no 21, 4335-4343 p.
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:umu:diva-20816DOI: 10.1039/b821658dOAI: oai:DiVA.org:umu-20816DiVA: diva2:209624
Available from: 2009-03-26 Created: 2009-03-26 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Localisation of Fluorescent Probes and the estimation of Lipid Nanodomain sizes by modern fluorescence techniques
Open this publication in new window or tab >>Localisation of Fluorescent Probes and the estimation of Lipid Nanodomain sizes by modern fluorescence techniques
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[cs]
Lokalizace fluorescenčních značek a určování velikostí lipidových nanodomén pomocí moderních fluorescenčních metod
Abstract [en]

The thesis is divided into two major parts. The first part focuses on the localisation of probes in lipid/polymeric bilayers and in GM1 micelles. Included in this thesis is a new approach based on electronic energy transfer/migration (FRET/DDEM), which efficiently determines transversal positions of fluorescent molecules in lipid bilayers. This approach has been used to locate newly synthesized lipid probes in DOPC bilayers. The label was introduced at the end of sn-2 acyl chains of variable length.

Analytical models accounting for FRET exist for a limited number of basic geometries. Here, a combination of FRET and Monte Carlo simulations enables the localisation of probes in bicelles and in bilayers containing pores, i.e. in lipid systems with variable curvature, or in non-homogenous lipid systems. This approach has been used to test whether conical-like fluorescence probes have an increased affinity to highly curved regions, which would enable preferential labelling of membrane pores.

A simplified FRET model has been applied to localize 2-pyridones, a class of potential drugs, in GM1 micelles. Since the localisation of drugs within nanoparticles might influence the release kinetics and loading efficiency, knowledge about the drug location is highly relevant. It turned out that all derivatives were localised at the core-shell interface of GM1 micelles.

The second part of the thesis focuses mainly on the estimation of lipid nanodomain size by means of FRET, which still remains the most powerful method in this field. Limitations of FRET in the determination of domain size have been explored. We showed that the limitations of FRET are mainly caused by a low probes affinity to either the liquid-ordered or liquid-disordered phase. In the continuing work we provided a detailed dynamic and structural study of crosslinker-triggered formation of nanodomains. Here, two different domains have been revealed, i.e. i) domains whose size grows with increasing amount of added cholera toxin (CTxB), and to which CTxB binds tightly; ii) domains formed in membranes containing a slightly increased amount of sphingomyelin (as compared to i) whose size does not change during titration by additional CTxB and to which CTxB binds less tightly.

Abstract [cs]

Disertace je rozdělena do dvou hlavníchčástí. Prvníčást se zabývá lokalizací značek v lipidových/polymerních dvojvrstvách a v GM1micelách. V práci prezentujeme nový přístup založený na přenosu/migraci elektronické energie (FRET/DDEM), jež umožňuje efektivně určovat vertikální pozici fluorescenčních molekul uvnitř lipidové dvojvrstvy. Tato metoda byla použita k lokalizaci nově syntetizovaných lipidových značek značených na konci sn-2 acylového řetězce s různou délkou v DOPC dvojvrstvách.

Analytické modely popisující FRET existují pouze pro limitovaný počet základních geometrií. Kombinace FRETu s Monte Carlo simulacemi nicméně umožňuje lokalizaci značek v bicelách a v dvojvrstvách obsahujících póry, tj. v lipidových systémech s proměnlivým zakřivením a v nehomogenních lipidových útvarech. Tento přístup umožnil např. zjistit, zda kuželovitětvarované značky mají zvýšenou afinitu k vysoce zakřiveným oblastem dvojvrstvy, což by umožnilo preferenční značení pórů.

Lokalizovány byly rovněž tři deriváty 2-pyridonů(potencionálních léčiv) v GM1micelách za použití jednoduchého modelu zohledňujícího FRET mezi donory a akceptory nacházejícími se v micelách. Lokalizace léčiv v nanočásticích ovlivňuje kinetiku uvolňování (release kinetics) a množství látky solubilizované v micelách (loading efficiency).

Druhá část se především zabývá určováním velikostí lipidových nanodomén pomocí FRETu, který stále zůstává nejvíce výkonnou metodou v této oblasti. Zkoumány byly limitace FRETu v určování lipidových nanodomén. Ukázalo se, že tato omezení jsou především způsobena nízkou afinitou značek buď k Lonebo k Ldfázi. V navazující studii jsme poskytnuli detailní dynamickou a strukturní studii formace nanodomén indukované crosslinkerem. Objevili jsme dva typy domén: a) domény, jejichž velikost se zvětšuje s rostoucím množstvím přidaného cholera toxinu (CTxB) a k nimž se CTxB váže pevně a b) domény vzniklé v membránách se zvýšeným množstvím sfingomyelinu (ve srovnání s a)), jejichž velikost se nemění během titrace dodatečným CTxB a k nimž se CTxB váže méně pevně.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2012. 46 p.
Keyword
Electronic energy transfer, fluorescence, rafts, lipid bilayer, bicelles, micelles
National Category
Physical Chemistry
Research subject
Physical Chemistry
Identifiers
urn:nbn:se:umu:diva-52619 (URN)978-91-7459-390-7 (ISBN)
Public defence
2012-03-27, KBC-huset, KB3A9, Umeå Universitet, Umeå, 10:00 (English)
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Supervisors
Note
This thesis has been elaborated within the framework of the Agreement on Joint Supervision (co-tutelle) of an International Doctoral Degree Programme between Charles University in Prague, Czech Republic and the Department of Chemistry at Umeå University, Sweden.Available from: 2012-03-06 Created: 2012-02-28 Last updated: 2012-02-28Bibliographically approved

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