Takako Nishiya

Mechanistic study on toxicity of positively charged liposomes containing stearylamine to blood: use of carboxymethyl chitin to reduce toxicity

Abstract Toxic effects have been reported for positively charged liposomes containing stearylamine (SA). In the present work, we have explored (i) fusion between SA liposomes and erythrocyte ghost (EG) membranes by resonance energy transfer assay, (ii) hemolysis induced by SA liposomes, and (iii) turbidity changes in native plasma on contact with SA liposomes, in the presence or absence of carboxymethyl chitin (CM-chitin). In the absence of CM-chitin, EG-SA liposome fusion, extents of hemolysis and turbidity changes in native plasma depended upon the concentration of SA. In the presence of CM-chitin, at a concentration of 10−3 or 10−2% ( w v ), EG-SA liposome fusion was inhibited. In EG-SA liposome fusion experiments, EG-SA liposome aggregation was observed in the absence of CM-chitin. However, in the presence of CM-chitin, SA liposome aggregation was observed. Extents of hemolysis and turbidity changes in native plasma induced by SA liposomes were reduced depending upon the CM-chitin concentration. These results indicate that association of SA liposome to CM-chitin reduces the propensity of SA liposomes to interact with blood components.

Quantitative analysis of carboxymethyl chitin adsorbed on a liposome surface

Abstract A convenient and precise method for the quantitative determination of carboxymethyl chitin (CM-chitin) on a liposome surface has been developed. CM-chi

Circular dichroism study of bacteriorhodopsin-lipid interaction

Delipidated bacteriorhodopsin purified from purple membrane of H. halobium was reconstituted with the circular dichroism active phospholipid. The observed circular dichroism spectra in the 450-700 nm region characteristic of bacteriorhodopsin showed the temperature dependence characterized by a midpoint at ca. 45 degrees C and this spectral change showed the disaggregation of bacteriorhodopsin trimer to monomer. The circular dichroism spectra in the 250-400 nm region characteristic of the azo chromophore of phospholipid exhibited a remarkable temperature dependence synchronized with the disaggregation of bacteriorhodopsin, suggesting that a large proportion of the phospholipid is present as boundary lipid.

Interaction of stearylamine-liposomes with erythrocyte ghosts: analysis of membrane lipid mixing and aqueous contents mixing, and the effect of carboxymethyl chitin on the interaction

Abstract We have investigated the interaction of stearylamine (SA)-containing liposomes (SA-liposomes) with erythrocyte ghosts (EGs), utilizing the Tb-dipicolinate (Tb-DPA) assay for the mixing of the aqueous contents and a resonance energy transfer (RET) assay for the mixing of lipid. The results demonstrate that SA-liposomes and EGs, after aggregation, have a great tendency to mix their lipid before the mixing of the internal contents. The mixing of their contents takes place inside the vesicles due to the fusion of SA-liposomes and EGs, followed by the leakage of the contents from the vesicles. We have also explored the properties of the interaction of SA-liposomes with carboxymethyl chitin (CM-chitin), an inhibitor of the lipid mixing between SA-liposomes and EGs, using fluorescein-labeled CM-chitin (Flu-CMC). The polarization value of Flu-CMC increased upon SA-liposome binding, the amount of Flu-CMC on the liposome surfaces increased with the increase in the concentration of phospholipid and SA, and it decreased by 20–40% in the presence of 10 mM Ca 2+ ions, which have a high affinity for CM-chitin. These results show that the association of CM-chitin with SA-liposomes could be due to both electrostatic and hydrophobic interactions between SA-liposomes and CM-chitin.

Circular dichroism active artificial phospholipids for the study of molecular membrane dynamics focused on lipid—lipid interaction

Abstract Artificial phospholipid with an azobenzene chromophore in a close proximity to the asymmetric carbon of the choline skeleton was prepared. The artificial phospholipid was miscible with the natural phospholipid and provides CD information sensitive to the membrane motion.

The use of circular dichroism active phospholipid to study lipid transfer between liposomes: effects of cholesterol and melittin on spontaneous lipid transfer

Abstract The circular dichroism (CD) active phospholipid bis (4′-n- octanoxyazobenzene-4-carboxyl)- l -α-phosphatidylcholine (CDPC) was used to study the effects of cholesterol and melittin on the spontaneous lipid transfer between liposomes. For this purpose, CD spectral changes with time were observed at 20°C and 50°C for a mixture of liposomes composed of CDPC and egg lecithin (EPC) (50%CDPC-EPC-liposomes) and pure EPC-liposomes, and for a mixture of liposomes composed of CDPC and cholesterol (50%CDPC-CHO-liposomes) and pure EPC-liposomes. In both cases, CDPC molecules were found to be diluted by the EPC matrix without significant changes in the liposome size. The spontaneous transfer of CDPC was dramatically accelerated with the rise of temperature in the 50% CDPC-EPC-liposomes/EPC-liposomes system, and it was less temperature dependent in the 50%CDPC-CHO-liposomes/EPC-liposomes system. In the presence of melittin, the acceleration of CDPC transfer accompanied by CD enhancement was observed only in the 50%CDPC-EPC-liposomes/EPC-liposomes system at 20°C.

Syntheses and properties of circular dichroism active phospholipids

A group of circular dichroism (CD) active phospholipids has been synthesised, in which one or both acyl chains has been replaced with a cinnamoyl or azobenzene chromophore-containing acid. Studies on the structure, CD activity and thermodynamic property of liposome membranes composed of CD active phospholipids were carried out. CD active liposomes were found to be stable, normal liposomes of approximately 550 A diameter based on the electron micrograph and dynamic light scattering, and to have thermodynamic property similar to the conventional phospholipid membranes without serious perturbation by aromatic bulk groups based on DSC. Liposomes composed of phospholipid having two trans-azobenzene chromophores showed an extremely large CD enhancement even well above Tc. This CD enhancement was drastically changed by the presence of cis-azobenzene chromophore and cis-cis isomer content after irradiation was higher than the theoretical value, suggesting the importance of interchromophore interaction in the liposome membranes.

The use of circular dichroism active phospholipid to study bacteriorhodopsin-lipid interaction. Effects of lipid to protein ratio and crosslinkings of lysine residues

Circular dichroism (CD) active phospholipid bis(4′-n-octanoxyazobenzene-4-carboxyl)-l-α-phosphatidylcholine (CDPC) was used to study bacteriorhodopsin(BR)-lipid interactions. For this purpose, delipidated BR was incorporated into the liposomes composed of CDPC and the temperature-dependent CD spectra of BR and CDPC were observed. For the liposomes in which the molar phospholipid to BR monomer ratio (L/BR) was 53, the disaggregation of BR trimer to monomer and melting of lipids were characterized by a midpoint at 35°C and 37°C, respectively. For the liposomes in which L/BR was 96, they were estimated to be 23°C and 28°C, respectively. When the liposomes in which L/BR was 96 were treated with dimethyl-3-3′-dithiobispropionimidate to crosslink the lysine residues in BR, BR was still a trimer even at 40°C, and the temperature of lipid melting was increased by 4°C compared with that of unmodified liposomes in which L/BR was 96. The temperature-dependent CD spectral changes of the liposomes incorporating BR during heating did not coincide with those during cooling. This hysteresis effect becomes smaller with the decrease in L/BR. These results confirm the idea that BR molecules restrict the motion of phospholipids and the motion of phospholipids influences the aggregation or disaggregation of BR molecules.

Interaction of melittin analogue with phospholipase A2 and/or phospholipid: importance of proline-14 to the membrane action of melittin

Abstract For peptide-membrane interactions, the secondary structure of the peptide plays an important role. Melittin, an amphipathic 26 amino acid peptide, induces a wide variety of structural perturbations of the lipid bilayer, depending on the type of lipid. These observations suggest that melittin-induced morphological changes in the lipid bilayer may be attributed to a wedge-shaped structure afforded by the Pro-14 residue. To test this hypothesis, the effects of melittin and a melittin analogue (peptide 1), in which the Pro-14 is substituted with serine, on the internal rotation of phospholipase A 2 (PLA) in the lipid bilayer, PLA-catalyzed lipid hydrolysis, and the bilayer-to-hexagonal phase transition of egg phosphatidylethanolamine (EPE) have been compared. These measurements showed that, in the presence of melittin, the mobility of PLA upon the substrate binding is reduced much more than that in the presence of peptide 1, the stimulation of PLA-catalyzed lipid hydrolysis by melittin is much higher than that by peptide 1, and the EPE bilayer is destabilized by peptide 1 whereas it is stabilized by melittin. Peptide 1 has higher hemolytic activity than melittin due to its longer amphiphilic segment, but is less able to interact with PLA and/or phospholipid, indicating that the helix flexibility afforded by the Pro-14 residue promotes the ability of melittin to form a complex with the PLA and/or phospholipid.

Mechanistic Study on Membrane Lysis by Bee Venom

Abstract Interaction of phospholipid bilayer with melittin and/or phospholipase Az (PLA) have been studied with circular dichroism (CD), fluorescence, differential scanning calorimetry (DSC), and pH-stat.