Mechanism of alpha-cyclodextrin induced hemolysis. 2. A study of the factors controlling the association with serine-, ethanolamine-, and choline-phospholipids.

A nuclear magnetic resonance (NMR) spectroscopy and molecular modeling study of the interaction between alpha-cyclodextrin (alpha-CD) and phospholipids with serine, ethanolamine, or choline headgroups is presented. The experimental approach is based on 31P and 1H NMR measurements on small unilamellar vesicles (SUV), multilamellar systems (MLV), and aqueous suspensions of lipids using a direct complex preparation with alpha-CD. Molecular dynamics computer simulations are used to investigate the trajectory of alpha-CD in the vicinity of a membrane surface and the influence of the charge and dipole moment of the phospholipid headgroups. These factors of charge and orientation of dipole moment seem to play a key role in the interaction of phospholipids with alpha-CD and reflect very well the experimentally observed selectivity of the phospholipid -alpha-CD approach. However, with this approach, there is no evidence for the formation of a complex with the phospholipid headgroup (except for phosphatidylinositol) that results from electrostatic forces. Rather, after a possible extraction of the lipid from the membrane, a classical inclusion of the sn-2 chain in the cavity of alpha-CD occurs. This step depends on the alkyl chain length and saturation state of the lipids as well as on their organization (i.e., as vesicles or dispersions). Based on our results, chemical modifications of the alpha-CD molecule to control the hemolytic properties of alpha-CD are discussed.

Mechanism of alpha-cyclodextrin-induced hemolysis. 1. The two-step extraction of phosphatidylinositol from the membrane.

It has been suggested that the interaction of cyclodextrins with the lipid components of the erythrocyte membranes is the determining factor in the hemolysis induced by these cyclic oligosaccharides. In the case of alpha-cyclodextrin (cyclomaltohexose), phospholipids have been identified as the cell target. In our study, evidence for the interaction between alpha-cyclodextrin and different phospholipids has been obtained using synthetic membranes. Since phosphatidylinositol (PI) showed the strongest affinity for alpha-cyclodextrin, it has been selected to investigate the respective contributions of the polar head group and the aliphatic chains to the association process using 31P, 2H, and 1H NMR spectroscopy. In this work, we describe the two-step extraction of PI from the membrane following its association with alphaCD: a cyclodextrin molecule is first attracted to the membrane surface by electrostatic remote interactions and associates with the lipid head group. Then the whole PI molecule is extracted, and inclusion of its unsaturated sn-2 acyl chain into another alphaCD molecule occurs in the bulk.

Molecular dynamics study of an alpha-cyclodextrin-phosphatidylinositol inclusion complex.

Cyclodextrins are cyclic oligosaccharides known for their ability to include substrate molecules in their hydrophobic cavity. Moreover, cyclodextrins show a hemolytic activity when mM concentrations are added to blood. This hemolysis is commonly interpreted as a massive dissociation of phospholipids from the cell membrane due to the formation of complexes with the cyclodextrins. In the literature, a complexation between alpha-cyclodextrin (alpha CD) and phosphatidylinositol (PI) specific to the inositol headgroup has been proposed. But the need for the detailed interaction mechanism between the two molecules motivated the present work based on molecular dynamics simulations. Investigation of long range electrostatic interactions shows that a mutual approach of the molecules is only possible when the primary hydroxyl side of alpha CD faces the inositol headgroup of PI. This orientation is also the most favourable from adiabatic-and free-energy profiles calculated along a reaction coordinate that leads to an inclusion of PI into alpha CD. For free energy simulations, partial hydration of the model has been used. A study of glycosidic bond dihedral angles in alpha CD shows an increase in dihedral fluctuations before complexation and a dihedral "freezing" once the complex is formed.