Prehod porotvornega sredstva skozi fosfolipidno membrano

Tinev, Megi (author), Ziherl, Primož (mentor), Božič, Bojan (comentor)

Ciljno delovanje in toksičnost porotvornih antibiotikov, kot je nistatin, sta neposredno povezana z njihovo sposobnostjo prehajanja skozi fosfolipidne membrane. Da bi raziskali, ali in na kakšen način ta sredstva prehajajo skozi fosfolipidno membrano, smo razvili eksperimentalno metodo, ki temelji na merjenju časa od prenosa velikanskih unilamelarnih vesiklov (GUV) in multivesikularnih vesiklov (MVV) v raztopino porotvornega sredstva do njihovega poka. MVV-ji so sestavljeni iz notranjega (inGUV) in zunanjega (outGUV) vesikla, pri čemer čas poka vsakega določimo ločeno. Do pokov vesiklov pride zaradi hitre in številčne tvorbe por v membrani, kar je mogoče le pri visokih koncentracijah porotvornega sredstva. Vesikle smo opazovali s faznokontrastno mikroskopijo. Merilo, ki pove, ali sredstvo lahko preide skozi membrano ali ne, je razlika v času, ki ga inGUV preživi v raztopini po poku outGUV-a, v primerjavi s časom poka GUV-a enake velikosti. Če je ta čas krajši, to kaže, da je inGUV prišel v stik s porotvornim sredstvom že pred pokom outGUV-a, kar pomeni, da sredstvo prehaja skozi membrano. Na podlagi izmerjenih časov pokov smo lahko ocenili tudi spodnjo mejo membranske prepustnosti za dano porotvorno sredstvo. Z razvito metodo smo pokazali, da porotvorno sredstvo nistatin lahko prehaja skozi membrano iz POPC, ki vsebuje ergosterol (15 ali 45 mol\%), kadar je ta izpostavljena raztopini z visoko koncentracijo nistatina (250 ali 500 $\mu$M). V teh pogojih namreč pride do pokov vesiklov. Na podlagi rezultatov smo predlagali mehanizem prehoda nistatina, po katerem je prehod mogoč le, kadar nistatin v membrani tvori transmembransko poro.

• Language: Slovenian
• Keywords: fosfolipidni dvosloj, porotvorno sredstvo, transmembranska pora, nistatin
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FMF - Faculty of Mathematics and Physics
• Year: 2025
• PID: 20.500.12556/RUL-173408
• COBISS.SI-ID: 249621251
• Publication date in RUL: 17.09.2025

Secondary language

• Language: English
• Title: Passage of the pore-forming agent through phospholipid membrane

Abstract

The targeted activity and toxicity of pore-forming antibiotics such as nystatin are directly linked to their ability to cross phospholipid membranes. To investigate whether such agents can pass through the lipid bilayer and to what extent, we developed an experimental method based on measuring the time between the transfer of giant unilamellar vesicles (GUVs) and multivesicular vesicles (MVVs) into a solution of the pore-forming agent and the time of their rupture. MVVs are composed of an inner (inGUV) and an outer (outGUV) vesicle, and the rupture time of each is recorded separately. Vesicle rupture occurs due to rapid and extensive pore formation in the membrane, which happens only at high concentrations of the pore-forming agent. Vesicles were observed using phase-contrast microscopy. The key indicator of membrane permeability is the difference between the time that inGUV remains intact after the rupture of the outGUV and the rupture time of control GUV of the same size. If the inGUV ruptures sooner, this indicates that it came into contact with the pore-forming agent before the outGUV ruptured, implying that the agent permeated the membrane of the outGUV. Based on the measured rupture times the lower limit of membrane permeability for a given agent could also be estimated. Using this method, we demonstrated that the pore-forming agent nystatin can pass through a POPC membrane containing ergosterol (15 or 45 mol\%) when exposed to high nystatin concentrations (250 or 500~$\mu$M). Under these conditions, vesicle rupture occurs due to pore formation in the membrane. Based on our results, we proposed a mechanism in which nystatin passes through the membrane only when it forms a transmembrane pore.

• Keywords: phospholipid bilayer, pore-forming agent, transmembrane pore, nystatin