Characterizing vesicle leakage by fluorescence lifetime measurements

Patel, Hiren; Tscheka, Clemens; Heerklotz, Heiko

doi:10.1039/b908524f

Cited by 85 publications

(131 citation statements)

References 11 publications

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“…This is similar to what was observed for the dyeinux experiments, discussed above and points towards a heterogeneous graded permeabilization. 52 In the BODIPY channel (Fig. 10h) we see an almost equal distribution of membrane staining dye, indicating that the GUV stays intact and does not disintegrate upon pore formation and copolymer translocation.…”

Section: Clsm: Membrane Integrity and Pore Formationmentioning

confidence: 99%

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Binding of amphiphilic and triphilic block copolymers to lipid model membranes: the role of perfluorinated moieties

et al. 2014

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A novel class of symmetric amphi-and triphilic (hydrophilic, lipophilic, fluorophilic) block copolymers has been investigated with respect to their interactions with lipid membranes. The amphiphilic triblock copolymer has the structure PGMA 20 -PPO 34 -PGMA 20 (GP) and it becomes triphilic after attaching perfluoroalkyl moieties (F9) to either end which leads to F 9 -PGMA 20 -PPO 34 -PGMA 20 -F 9 (F-GP). The hydrophobic poly(propylene oxide) (PPO) block is sufficiently long to span a lipid bilayer. The poly(glycerol monomethacrylate) (PGMA) blocks have a high propensity for hydrogen bonding. The hydrophobic and lipophobic perfluoroalkyl moieties have the tendency to phase segregate in aqueous as well as in hydrocarbon environments. We performed differential scanning calorimetry (DSC) measurements on polymer bound lipid vesicles under systematic variation of the bilayer thickness, the nature of the lipid headgroup, and the polymer concentration. The vesicles were composed of phosphatidylcholines (DMPC, DPPC, DAPC, DSPC) or phosphatidylethanolamines (DMPE, DPPE, POPE). We showed that GP as well as F-GP binding have membrane stabilizing and destabilizing components.PPO and F9 blocks insert into the hydrophobic part of the membrane concomitantly with PGMA block adsorption to the lipid headgroup layer. The F9 chains act as additional membrane anchors. The insertion of the PPO blocks of both GP and F-GP could be proven by 2D-NOESY NMR spectroscopy. By fluorescence microscopy we show that F-GP binding increases the porosity of POPC giant unilamellar vesicles (GUVs), allowing the influx of water soluble dyes as well as the translocation of the complete triphilic polymer and its accumulation at the GUV surface. These results open a new route for the rational design of membrane systems with specific properties.

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Section: Clsm: Membrane Integrity and Pore Formationmentioning

confidence: 99%

“…This is very similar to what was observed for some pore forming peptides (PFPs) interacting with GUVs 51 and indicates that the leakage mechanism is "heterogeneous graded". 52 Some of the vesicles might also be permeabilized by the "all or none" mechanism (GUV 4).…”

Section: Clsm: Membrane Integrity and Pore Formationmentioning

confidence: 99%

Binding of amphiphilic and triphilic block copolymers to lipid model membranes: the role of perfluorinated moieties

et al. 2014

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“…This classification was taken further by Wimley et al’s re-quenching protocol 48 . Then, the additional information provided by time-resolved fluorescence spectroscopy 49 was utilized to detect the leakage mechanism by monitoring the amount and local concentration of free and entrapped dye 50–52 . An alternative, statistical evaluation of the influx of a dye into giant vesicles 53, 54 is much more demanding with respect to time and instrumentation but can provide a histogram of leakage of a vesicle population directly.…”

Section: Introductionmentioning

confidence: 99%

Correlating antimicrobial activity and model membrane leakage induced by nylon-3 polymers and detergents

et al. 2015

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Most antimicrobial peptides act upon target microorganisms by permeabilizing their membranes. The mode of action is often assessed by vesicle leakage experiments that use model membranes, with the assumption that biological activity arises from permeabilization of the lipid bilayer. The current work aims to extend the interpretation of vesicle leakage results and examine the correlation between vesicle leakage and antimicrobial activity. To this end, we used a lifetime-based leakage assay with calcein-loaded vesicles to study the membrane permeabilizing properties of a novel antifungal polymer poly-NM, two of its analogs, and a series of detergents. In conjunction, the biological activities of these compounds against Candida albicans were assessed and correlated with data from vesicle leakage. Poly-NM induces all-or-none leakage in polar yeast lipid vesicles at the polymer’s MIC, 3 μg/mL. At this and higher concentrations, complete leakage after an initial lag time was observed. Concerted activity tests imply that this polymer acts independently of the detergent octyl glucoside (OG) for both vesicle leakage and activity against C. albicans spheroplasts. In addition, Poly-NM was found to have negligible activity against zwitterionic vesicles and red blood cells. Our results provide a consistent, detailed picture of the mode of action of Poly-NM: this polymer induces membrane leakage by electrostatic lipid clustering. In contrast, Poly-MM:CO, a nylon-3 polymer comprised of both cationic and hydrophobic segments, seems to act by a different mechanism that involves membrane asymmetry stress. Vesicle leakage for this polymer is transient (limited to <100%) and graded, non-specific among zwitterionic and polar yeast lipid vesicles, additive with detergent action, and correlates poorly with biological activity. Based on these results, we conclude that comprehensive leakage experiments can provide a detailed description of the mode of action of membrane permeabilizing compounds. Without this thorough approach, it would have been logical to assume that the two nylon-3 polymers we examined act via similar mechanisms; it is surprising that their mechanisms are so distinct. Some, but not all mechanisms of vesicle permeabilization allow for antimicrobial activity.

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“…With this alternative experiment, it is possible to estimate whether the leakage process is graded or all-or-none (Parente et al 1990;Pokorny et al 2002). In addition, Heerklotz and coauthors (Patel et al 2009) developed a method using time-resolved fluorescence approach to monitor dye release (calcein) from LUVs. This method provided evidence about the heterogeneity of graded leakage and also allowed distinguishing from allor-none leakage process.…”

Section: Model Membranes and Strategies For Investigating Lipid-packimentioning

confidence: 99%

Lipid-packing perturbation of model membranes by pH-responsive antimicrobial peptides

2017

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The indiscriminate use of conventional antibiotics is leading to an increase in the number of resistant bacterial strains, motivating the search for new compounds to overcome this challenging problem. Antimicrobial peptides, acting only in the lipid phase of membranes without requiring specific membrane receptors as do conventional antibiotics, have shown great potential as possible substituents of these drugs. These peptides are in general rich in basic and hydrophobic residues forming an amphipathic structure when in contact with membranes. The outer leaflet of the prokaryotic cell membrane is rich in anionic lipids, while the surface of the eukaryotic cell is zwitterionic. Due to their positive net charge, many of these peptides are selective to the prokaryotic membrane. Notwithstanding this preference for anionic membranes, some of them can also act on neutral ones, hampering their therapeutic use. In addition to the electrostatic interaction driving peptide adsorption by the membrane, the ability of the peptide to perturb lipid packing is of paramount importance in their capacity to induce cell lysis, which is strongly dependent on electrostatic and hydrophobic interactions. In the present research, we revised the adsorption of antimicrobial peptides by model membranes as well as the perturbation that they induce in lipid packing. In particular, we focused on some peptides that have simultaneously acidic and basic residues. The net charges of these peptides are modulated by pH changes and the lipid composition of model membranes. We discuss the experimental approaches used to explore these aspects of lipid membranes using lipid vesicles and lipid monolayer as model membranes.

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Characterizing vesicle leakage by fluorescence lifetime measurements

Cited by 85 publications

References 11 publications

Binding of amphiphilic and triphilic block copolymers to lipid model membranes: the role of perfluorinated moieties

Binding of amphiphilic and triphilic block copolymers to lipid model membranes: the role of perfluorinated moieties

Correlating antimicrobial activity and model membrane leakage induced by nylon-3 polymers and detergents

Lipid-packing perturbation of model membranes by pH-responsive antimicrobial peptides

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