Utjecaj žučnih soli na permeabilnost liposoma i mehanizam oslobađanja kalceina

master's thesis

• Title (english): The effect of bile salts on liposomal permeability and calcein's leakage mechanism
• Author: Ivan Cvetković
• Mentor: Željka Vanić (mentor)
• Committee member: Željka Vanić (predsjednik povjerenstva)
• Committee member: Ivan Pepić (član povjerenstva)
• Committee member: Renata Jurišić Grubešić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of pharmaceutical technology); Zagreb
• Defense date and country: 2017-09-28, 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: fosfatidilkolin liposomi; žučne soli
• Keywords (english): EPC liposomes; bile salts
• Language: croatian
• URN:NBN: urn:nbn:hr:163:236391
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2017-11-29 10:30:23