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master's thesis
Utjecaj žučnih soli na permeabilnost liposoma i mehanizam oslobađanja kalceina
Zagreb: University of Zagreb, Faculty of Pharmacy and Biochemistry, 2017. urn:nbn:hr:163:236391
Cvetković, Ivan
University of Zagreb
Faculty of Pharmacy and Biochemistry
Department of pharmaceutical technology

Institutional repository: Repository of Faculty of Pharmacy and Biochemistry University of Zagreb

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Cvetković, I. (2017). Utjecaj žučnih soli na permeabilnost liposoma i mehanizam oslobađanja kalceina (Master's thesis). Zagreb: University of Zagreb, Faculty of Pharmacy and Biochemistry. Retrieved from https://urn.nsk.hr/urn:nbn:hr:163:236391

Cvetković, Ivan. "Utjecaj žučnih soli na permeabilnost liposoma i mehanizam oslobađanja kalceina." Master's thesis, University of Zagreb, Faculty of Pharmacy and Biochemistry, 2017. https://urn.nsk.hr/urn:nbn:hr:163:236391

Cvetković, Ivan. "Utjecaj žučnih soli na permeabilnost liposoma i mehanizam oslobađanja kalceina." Master's thesis, University of Zagreb, Faculty of Pharmacy and Biochemistry, 2017. https://urn.nsk.hr/urn:nbn:hr:163:236391

Cvetković, I. (2017). 'Utjecaj žučnih soli na permeabilnost liposoma i mehanizam oslobađanja kalceina', Master's thesis, University of Zagreb, Faculty of Pharmacy and Biochemistry, accessed 27 December 2024, https://urn.nsk.hr/urn:nbn:hr:163:236391

Cvetković I. Utjecaj žučnih soli na permeabilnost liposoma i mehanizam oslobađanja kalceina [Master's thesis]. Zagreb: University of Zagreb, Faculty of Pharmacy and Biochemistry; 2017 [cited 2024 December 27] Available at: https://urn.nsk.hr/urn:nbn:hr:163:236391

I. Cvetković, "Utjecaj žučnih soli na permeabilnost liposoma i mehanizam oslobađanja kalceina", Master's thesis, University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, 2017. Available at: https://urn.nsk.hr/urn:nbn:hr:163:236391

Metadata
TitleUtjecaj žučnih soli na permeabilnost liposoma i mehanizam oslobađanja kalceina
Title (english)The effect of bile salts on liposomal permeability and calcein's leakage mechanism
AuthorIvan Cvetković
MentorŽeljka Vanić (mentor)
Committee memberŽeljka Vanić (predsjednik povjerenstva)
Committee memberIvan Pepić (član povjerenstva)
Committee memberRenata Jurišić Grubešić (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Pharmacy and Biochemistry
(Department of pharmaceutical technology)
Zagreb
Defense date and country2017-09-28, Croatia
Scientific / art field,
discipline and subdiscipline		BIOMEDICINE AND HEALTHCARE
Pharmacy
Pharmacy
Abstract
Cilj ovog rada bio je ispitati utjecaj žučnih soli na permeabilnost fosfolipidnog dvosloja liposoma i oslobađanje uklopljene hidrofilne tvari. Liposomi s kalceinom (hidrofilni fluorescentni marker) pripremljeni su metodom hidratacije suhog fosfolipidnog sloja korištenjem fosfatidilkolina kao jedine lipidne komponente. Homogenizacija liposomskih disperzija provedena je ekstruzijom kroz polikarbonatne membrane veličine pora 400 nm i 100 nm. Odjeljivanje neuklopljenog kalceina provedeno je gel kromatografijom, a konačna koncentracija fosfolipida u ekstrudiranoj liposomskoj disperziji je određena Bartlett-ovom metodom. Ispitivanja utjecaja žučnih soli na mehanizam oslobađanja uklopljenog sadržaja iz liposoma provedena su tako da su liposomi s uklopljenim kalceinom inkubirani kroz dulji vremenski period u otopinama natrijevog kolata, deoksikolata ili taurokolata različitih koncentracija. U određenim vremenskim intervalima svakom je uzorku izmjereno vrijeme života slobodnog i uklopljenog kalceina korištenjem metode vremenski koreliranog pojedinačnog brojanja fotona (eng. time-correlated single photon counting, TCSPC). Dobiveni podatci su matematički obrađeni te je izračunata količina oslobođenog kalceina. Ekstruzija originalno pripravljenih liposoma film metodom je rezultirala homogenom disperzijom unilamelarnih liposoma srednjeg promjera od 141 nm i malog indeksa polidisperznosti (0,062). Sve ispitivane žučne soli imale su utjecaj na integritet liposomske mebrane što se očitovalo povećanjem permeabilnosti uklopljenog sadržaja iz liposoma (kalcein). Sve korištene otopine žučnih soli uzrokovale su postepeno otpuštanje kalceina iz liposoma (eng. graded leakage), pri čemu je za natrijev kolat i taurokolat potvrđeno da uzrokuju podvrstu heterogenog postepenog oslobađanja (heterogenous graded leakage) dok za natrijev deoksikolat to nije bilo moguće sa sigurnoću odrediti. Natrijev deoksikolat je imao najjači učinak na povećanje permeabilnosti fosfolipidnog dvosloja liposoma već u koncentraciji od 0,5 mM. Kod svih ispitivanih otopina žučnih soli dokazano je da porastom koncentracije žučne soli i duljim vremenom inkubacije raste udio oslobođenog kalceina. Međutim, pri koncentracijama žučnih soli nižim od određene kritične minimalne koncentracije nije bilo moguće postići značajnije oslobađanje uklopljenog sadržaja, bez obzira na produljenje vremena inkubacije.
Abstract (english)
The object of this research was to study the liposomal leakage in phospholipid bilayers caused by bile salts. For that purpose, calcein-loaded phosphatidylcholine liposomes were made using the thin layer method. Calcein was used, because it was the most suitable hydrophilic dye for this research. In order to get a more homogenous fraction, liposomes were extruded through 400 nm and 100 nm polycarbonate membranes. The surplus of free calcein was removed from the calcein-loaded liposomal solution by size exclusion chromatography (SEC). Because this method diluted the sample, the phospholipid concentration of the liposomal sample was determined by Bartlett method. The leakage assay of calcein-loaded liposomes was done by incubating the liposomes in solutions of sodium cholate, sodium taurocholate and sodium deoxycholate of different concentrations during a longer period of time. At predetermined time intervals, fluorescence lifetime of the free and entrapped calcein molecules was measured using the time correlated single photon counting method (TCSPC). The results were then analyzed by biexponential curve fitting and the efflux of calcein molecules was determined for each bile salt concentration and incubation time. The previously done liposomal extrusion resulted in a homogenous fraction of unilamellar liposomes (polydispersity index 0.062) that had an average diameter of 141 nm. All of the bile salts used had an effect on the permeability of liposomal membrane, causing it to leak the entrapped calcein. More precisely, all the bile salts induced graded leakage in phosphatidlycholine liposomes. While sodium cholate and taurocholate caused heterogeneously graded leakage, the exact subgroup of graded leakage could not be determined for sodium deoxycholate. However, sodium deoxycholate had the strongest effect on liposomal membranes, causing them to leak calcein at a very low concentration of 0.5 mM. It has been found that by increasing the bile salt concentration and prolonging the incubation time, one can achieve greater leakage of the entrapped dye. Although this is only true for bile salt concentrations that exceed a specific minimal concentration needed to cause distortions in liposomal bilayers.
Keywords
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:163:236391
Study programmeTitle: Pharmacy
Study programme type: university
Study level: integrated undergraduate and graduate
Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
Type of resourceText
File originBorn digital
Access conditionsOpen access
Terms of useLicence In copyright icon
Created on2017-11-29 10:30:23