1. E-mail: beta@fisica.uminho.pt
ENERGY TRANSFER STUDIES IN CATIONIC MIXED LIPOSOMES: C12E8 AND TEMPERATURE EFFECTS
M.E.C.D.Real Oliveira, A.L.F.Baptista, P.J.G.Coutinho, E.M.S. Castanheira
Universidade do Minho - Departamento de Física - Campus de Gualtar Braga-Portugal
INTRODUCTION
The aim of this work is to study the influence of temperature and non-ionic surfactant C12E8 (polyoxyethylene 8 lauryl ether) in different kinds of LUVs (large unilamellar vesicles), namely from soybean lecithin, cationic double chain surfactant DODAB (dioctadecyldimethylammonium bromide) and lecithin/DODAB (1:1) mixture. We used UV-Visible absorption and fluorescence measurements of the lipid probes NBD-PE (N-7-nitro-1,2,3-benzodiazoyl phosphatidylethanolamine) and N-Rh-PE (N-lissamine rhodamine B sulfonyl phosphatidylethanolamine), and followed the direct energy transfer from donor (NBD-PE) to acceptor (N-Rh-PE). The controlled variables were the temperature and the surfactant-to-lipid molar ratio.
ENERGY TRANSFER
STRUCTURES
LIPIDS FLUORESCENCE PROBES
NBD-PE
N-Rh-PE
Efficiency of Energy Transfer
In gel phase, fluorescence probes tend to aggregate.
As we can see from variations in absorption spectra, the phase transition temperature (Tm) of DODAB is nearly 45 ºC.
The emission spectra were decomposed using a sum of Gaussian functions (1 or 3) for NBD-PE and 3 log-normal functions for N-Rh-PE. The various NBD-PE components result either from different locations or from NBD-PE aggregate emission.
Energy transfer for N-Rh-PE results in alterations of NBD-PE spectral shape, which can be explained by different efficiencies for the various components.
The energy transfer efficiency for each NBD-PE component was determined using a similar decomposition of NBD-PE emission in absence of N-Rh-PE.
Energy transfer can distinguish three regions in DODAB system (gel phase, intermediate phase and a liquid-crystalline phase). In gel phase, kDET/kF decreases due to the energy transfer to N-Rh-PE aggregates, which are non-emissive. Above 25 ºC, kDET/kF increases due to higher mobility of the molecules as the temperature rises.
In lecithin/DODAB system, we can detect two regions (gel phase below Tm and liquid-crystalline phase above it). Lecithin decrease the Tm of DODAB to 35 ºC. In gel phase and in liquid- -crystalline phase, this system has the same behaviour as DODAB.
In liquid-crystalline phase the process of energy transfer from NBD-PE to N-Rh-PE is more efficient, due to the fluidization caused by the presence of lecithin.
TEMPERATURE EFFECT
DODAB
LECITHIN
LECITHIN /DODAB (1:1)
SURFACTANT EFFECT
With addition of surfactant, the average distance from donor to acceptor molecules increases, as a result of incorporation of surfactant molecules in the vesicle bilayer. Upon formation of mixed micelles, which should be smaller, the probability of having a micelle with both NBD-PE and N-Rh-PE is low, although the average distance is smaller than in vesicles.
Using energy transfer, we can detect three regions (mixed vesicles, mixed vesicles+mixed vesicles and mixed micelles) in lecithin and lecithin/DODAB systems.
With the addition of surfactant in liquid-crystalline phase (lecithin), the kDET/kF decreases more than in the gel phase (DODAB).
In lecithin/DODAB system, where two phases coexist (gel and liquid-crystalline), the trend is similar as in lecithin, but the slope is smoother as in DODAB.
In general, the component at ~540 nm is the main responsible for the observed energy transfer. The differences observed when all NBD-PE emission spectra is taken into account should probably arise from variations of NBD-PE localization and/or its aggregation.