Characteristics of Pyrene Phospholipid/γ-Cyclodextrin Complex

Abstract

Recently, it was demonstrated that gamma-cyclodextrins (gamma-CDs) greatly accelerates transfer of hydrophobic pyrene-labeled and other fluorescent phospholipid derivatives from vesicles to cells in culture (). To understand better the characteristics of this process, we studied the interaction of gamma-CD with pyrene-labeled phosphatidylcholines (PyrPCs) using a variety of physical methods. Either one or both of the acyl chains of PC was labeled with a pyrene moiety (monoPyrPCs and diPyrPCs, respectively), and the length of the labeled chain(s) varied from 4 to 14 carbons. Fluorescent binding assays showed that the association constant decreases strongly with increasing acyl chain length. PyrPC/gamma-CD stoichiometry was 1:2 for the shorter chain species, but changed to 1:3 when the acyl chain length exceeded 8 (diPyrPCs) or 10 (monoPyrPCs) carbons. The activation energy for the formation of diPyr(10)PC/gamma-CD complex was high, i.e., +92 kJ/mol, indicating that the phospholipid molecule has to fully emerge from the bilayer before complex formation can take place. The free energy, enthalpy, and entropy of transfer of monoPyrPC from bilayer to gamma-CD complex were close to zero. The absorption, Fourier transform infrared, and fluorescence spectral measurements and lifetime analysis indicated that the pyrene moiety lies inside the CD cavity and is conformationally restricted, particularly when the labeled chain is short. The acyl chains of a PyrPC molecule seem to share a CD cavity rather than occupy different ones. The present data provide strong evidence that the ability of gamma-CD to enhance intermembrane transfer of pyrene-labeled phospholipids is based on the formation of stoichiometric complexes in the aqueous phase. This information should help in designing CD derivatives that are more efficient lipid carriers then those available at present.

Introduction of phospholipids to cultured cells with cyclodextrin

“…Cell viability was not signifi cantly com- decreased at higher me ␤ -CD concentrations. This is not unexpected, because the affi nity of phospholipids for cyclodextrin (relative to that for the bilayer) has been shown to increase with decreasing hydrophobicity ( 9 ). Thus, if the concentration of me ␤ -CD is too high, it binds much of aqueous media presents a major obstacle here.…”

Electrospray Ionization Mass Spectrometry and Exogenous Heavy Isotope-labeled Lipid Species Provide Detailed Information on Aminophospholipid Acyl Chain Remodeling

“…The degree of incorporation diminished nearly exponentially with lipid hydrophobicity (data not shown). This is characteristic for CD-mediated monomeric lipid transfer (44), and therefore the labeled PE and PS species were most probably incorporated into cells (probably the exoplasmic leaflet of the plasma membrane) as lipid monomers rather than as components of adhering donor vesicles cf (45). Supporting this conclusion, only negligible amounts of the donor vesicle marker 22:0/22:0-PC, which is too hydrophobic to be efficiently transferred by cyclodextrins, was detected in cells (not shown).…”

Interaction of fluorescent phospholipids with cyclodextrins

“…The extent of this binding depends on the chain length and degree of saturation of the fatty acyl chains as well as on the CD cavity size (see (Huang and London, 2013)). It has been shown that the affinity of pyrene-labeled phospholipids for CDs increases with decreasing hydrophobicity (Tanhuanpää et al, 2001). For the experiments in buffer, additionally the organization of the analogs has to be taken into account, which depends on the fatty acyl chain length.…”

“…Since fluorescence moieties are characterized by hydrophobic structures they may also bind to CDs (Kinsland and Wiechelman, 1984;Takakusa et al, 2001;Tanhuanpää et al, 2001;Fakayode et al, 2007). In this context, a carboxyethylated gCD has been shown to interact with various long chain fluorescently labeled phospholipids enhancing their translocation from lipid vesicles to cellular plasma membranes (Tanhuanpää and Somerharju, 1999;Tanhuanpää et al, 2001). The process of lipid extraction from membranes and the complexation of pyrene-labeled lipids by gCD were characterized in detail, indicating that the fluorescence moiety and both acyl chains of the analogs are bound to the same CD cavity.…”

“…The process of lipid extraction from membranes and the complexation of pyrene-labeled lipids by gCD were characterized in detail, indicating that the fluorescence moiety and both acyl chains of the analogs are bound to the same CD cavity. The high activation energy of lipid removal by CD favors a model, in which the phospholipid molecules are first completely extracted from the bilayer and then associate with several CD molecules in the aqueous phase (Tanhuanpää et al, 2001).…”