Vesicles of variable sizes produced by a rapid extrusion procedure

Mayer, Lawrence D.; Hope, Michael J.; Cullis, Pieter R.

doi:10.1016/0005-2736(86)90302-0

Cited by 1,688 publications

(1,126 citation statements)

References 19 publications

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“…28 The desired mixtures of lipids were dried in glass tubes first under nitrogen and then lyophilized overnight to obtain lipid films. The films were suspended for 1 h on a water-bath at 40-508C in Tris-HCl buffer, pH 5 7.0 (10 mM Tris, 10 mM NaCl), sealed with parafilm under nitrogen, and then freeze-thawed five times under nitrogen to produce large multilamellar vesicles.…”

Section: Preparation Of Large Unilamellar Vesiclesmentioning

confidence: 99%

Membrane interactions of designed cationic antimicrobial peptides: The two thresholds

2008

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ABSTRACT:Novel cationic antimicrobial peptides (CAPs) designed in our lab-typified by sequences such as KKKKKKAAX-AAXAAXAA-NH 2 , where X 5 Phe/Trp-display high antibacterial activity but exhibit little or no hemolytic activity towards human red blood cells even at high doses.To clarify the mechanism of their selectivity for bacterial versus mammalian membranes and to increase our understanding of the relationships between primary sequence and bioactivity, a library of derivatives was prepared by increasing segmental hydrophobicity, in which systematic substitutions of Ala for two, three, or four Leu residues were made. Conformationally constrained dimeric and cyclic derivatives were also synthesized. The peptides were examined for activity against pathogenic bacteria (Pseudomonas aeruginosa),

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Section: Preparation Of Large Unilamellar Vesiclesmentioning

confidence: 99%

Membrane interactions of designed cationic antimicrobial peptides: The two thresholds

2008

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“…Unilamellar liposomes were prepared using an extrusion procedure as described previously. 19, 20 The final lipid concentration of the stock solution was 7.4 × 10 Ϫ3 M. The water-soluble ADPA singlet oxygen quencher was then added to solutions of DMPC unilamellar liposomes at the desired concentration. Finally, the preparation was incubated for 1 h at 25 ЊC with BCA in methanol.…”

Section: Preparation Of Unilamellar Liposomesmentioning

confidence: 99%

Determination of the singlet oxygen quantum yield of bacteriochlorin a: a comparative study in phosphate buffer and aqueous dispersion of dimiristoyl-l-α-phosphatidylcholine liposomes

Hoebeke

Damoiseau

2002

Photochem Photobiol Sci

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The production of singlet oxygen ( 1 O 2 ) by bacteriochlorin a (BCA) was studied in phosphate buffer and in dimyristoyl--α-phosphatidylcholine (DMPC) unilamellar liposomes. The comparative method used to measure 1 O 2 production was a quantitative analysis of photooxidation reactions leading to the loss of absorbance of the water-soluble specific probe: anthracene-9,10-dipropionic acid. Rose Bengal, whose 1 O 2 quantum yield (Φ RB ) is well known in alcohols and phosphate buffer, was used as the standard for the quantification of the BCA singlet oxygen production. Our results confirm quantitatively that solubilization of BCA in liposomes leads to an increase in 1 O 2 production. Indeed, the quantum yield of 1 O 2 production by BCA (Φ BCA ) is 0.05 in phosphate buffer and 0.33 in DMPC liposomes.Furthermore, the diffusion characteristics of 1 O 2 produced by BCA bound to liposome were also examined using the isotopic lifetime enhancement effect of D 2 O. It was shown that 1 O 2 spent at least 70% of its lifetime in the vesicular environment.

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“…LUV of approximately 0.1 pm diameter were formed by extrusion under N2 pressure through Nucleopore polycarbonate membranes (Mayer et al, 1986). POPC and POPG were mixed in chloroform and the solution dried under a stream of NZ.…”

Section: Vesicle Preparationmentioning

confidence: 99%

Interactions between human defensins and lipid bilayers: Evidence for formation of multimeric pores

1994

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Defensins comprise a family of broad-spectrum antimicrobial peptides that are stored in the cytoplasmic granules of mammalian neutrophils and Paneth cells of the small intestine. Neutrophil defensins are known to permeabilize cell membranes of susceptible microorganisms, but the mechanism of permeabilization is uncertain. We report here the results of an investigation of the mechanism by which HNP-2, one of 4 human neutrophil defensins, permeabilizes large unilamellar vesicles formed from the anionic lipid palmitoyloleoylphosphatidylglycerol (POPG). As observed by others, we find that HNP-2 (net charge = +3) cannot bind to vesicles formed from neutral lipids. The binding of HNP-2 to vesicles containing varying amounts of POPG and neutral (zwitterionic) palmitoyloleoylphosphatidylcholine (POPC) demonstrates that binding is initiated through electrostatic interactions.Because vesicle aggregation and fusion can confound studies of the interaction of HNP-2 with vesicles, those processes were explored systematically by varying the concentrations of vesicles and HNP-2, and the P0PG:POPC ratio. Vesicles (300 pM POPG) readily aggregated at HNP-2 concentrations above 1 pM, but no mixing of vesicle contents could be detected for concentrations as high as 2 pM despite the fact that intervesicular lipid mixing could be demonstrated. This indicates that if fusion of vesicles occurs, it is hemi-fusion, in which only the outer monolayers mix at bilayer contact sites. Under conditions of limited aggregation and intervesicular lipid mixing, the fractional leakage of small solutes is a sigmoidal function of peptide concentration. For 300 pM POPG vesicles, 50% of entrapped solute is released by 0.7 p M HNP-2. We introduce a simple method for determining whether leakage from vesicles is graded or all-or-none. We show by means of this fluorescence "requenching" method that native HNP-2 induces vesicle leakage in an all-or-none manner, whereas reduced HNP-2 induces partial, or graded, leakage of vesicle contents. At HNP-2 concentrations that release 100% of small (-400 Da) markers, a fluorescent dextran of 4,400 Da is partially retained in the vesicles, and a 18,900-Da dextran is mostly retained. These results suggest that HNP-2 can form pores that have a maximum diameter of approximately 25 A. A speculative multimeric model of the pore is presented based on these results and on the crystal structure of a human defensin.

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Vesicles of variable sizes produced by a rapid extrusion procedure

Cited by 1,688 publications

References 19 publications

Membrane interactions of designed cationic antimicrobial peptides: The two thresholds

Membrane interactions of designed cationic antimicrobial peptides: The two thresholds

Determination of the singlet oxygen quantum yield of bacteriochlorin a: a comparative study in phosphate buffer and aqueous dispersion of dimiristoyl-l-α-phosphatidylcholine liposomes

Interactions between human defensins and lipid bilayers: Evidence for formation of multimeric pores

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