Fluorescence properties of Laurdan in cochleate phases

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Abstract

Cochleates are lipid-based delivery system that have found application in drug and gene delivery. They are precipitates, formed as a result of interaction between cations (e.g. Ca2+) and negatively charged phospholipids such as phosphatidylserine (PS). In the present study, we investigated the utility of fluorescent probe Laurdan (6-dodecanoyl-2-dimethylamino naphthalene) to monitor cochleate phase formation. Following addition of Ca2+ to Laurdan labeled lipid vesicles comprised of brain phosphatidylserine (BPS), a significant blue shift in the emission peak maximum of Laurdan was observed and the spectral features were distinct from those observed for the gel and liquid-crystalline (LC) phases. This is consistent with the formation of anhydrous cochleate cylinders that was further confirmed by electron microscopy studies. Due to dipolar relaxation, excitation and emission generalized polarization (GPEx and GPEm) indicate transition from a LC to a rigid and dehydrated (RD) cochleate phase. These spectral changes were utilized to monitor the influence of lipid composition, ionic strength and lamellarity on the formation of cochleate phase. The results indicated that the presence of phosphatidylcholine (PC) and bulk Na+ concentration influenced the formation of cochleate structures from small unilamellar vesicles (SUV) and multilamellar vesicles (MLV) composed of PS. The presence of PC and higher bulk Na+ concentration stabilized the PS vesicles against collapse and total loss of contents, intermediate molecular events in the formation of cochleate structures. From these studies, we conclude that Laurdan fluorescence is a sensitive and a rapid method to detect cochleate phase formation.

Keywords

Ca2+–PS interaction
Cochleate
Laurdan fluorescence
Generalized polarization
Phosphatidylcholine–phosphatidylserine mixture

Abbreviations

BPS
brain phosphatidylserine
DMPC
dimyristoyl phosphatidylcholine
DLS
dynamic light scattering
EM
electron microscopy
EDTA
ethylenediaminetetraacetic acid
GP
generalized polarization
IR
infrared spectroscopy
Laurdan
6-dodecanoyl-2-dimethylamino naphthalene
LS
light scattering
LUV
large unilamellar vesicles
MLV
multilamellar vesicles
PC
phosphatidylcholine
PS
phosphatidylserine
SUV
small unilamellar vesicles

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