Influence of DPPC Liposome Concentration on the Fluorescence Properties of PRODAN and LAURDAN K. A. Kozyra, J. R. Heldt, G. Gondek, P. Kwiek, and J. Heldt a Institute of Experimental Physics, University of Gda´nsk, ul. Wita Stwosza 57, 80-952 Gda´nsk, Poland a Institute of Physics, Pomeranian Pedagogical Academy, ul. Arciszewskiego 22B, 76-200 Słupsk, Poland Reprint requests to Dr. K. A. K.; E-mail: [email protected] Z. Naturforsch. 59a, 809 – 818 (2004); received July 21, 2004 Fluorescence spectral features of PRODAN and LAURDAN in phospholipid vesicles of different phase states were investigated. The results indicate that in the liquid crystalline phase the dominant emission results from the charge transfer (CT) excited state, whereas in the gel state of the mem- brane the emission from the locally excited (LE) state dominates. The fluorescence time studies point out that there are two radiation modes, one starting from only vibrationally relaxed excited states S1(LE)ν ((S1(CT)ν ) and the other from a totally thermally equilibrated state S1(LE)EQ (S1(CT)EQ). In accordance with the obtained decay time dependencies, the fluorescence emission from total non- equilibrated excited states consists of a dominant or minor radiation process in the LE or CT band emission. Key words: PRODAN, LAURDAN; Locally Excited and Charge Transfer States; Fluorescence Decay Time. 1. Introduction PRODAN (6-propionyl-2-dimethylaminonaphtal- ene) and LAURDAN (6-dodecanoyl-2-dimethylami- nonaphtalene) have been widely used to study the structure and dynamics of biological systems, e. g. membrane constituents, surfaces, large biological molecules, cells, etc. [1 – 5]. Particular attention has been paid to these organic molecules since they are capable of simultaneously creating locally excited (LE) and charge (CT) transfer excited states [5 – 9]. Therefore their luminescence properties are very Fig. 1. Chemical structure of PRODAN (n = 1) and LAUR- sensitive to changes of the surrounding microenviron- DAN (n = 10). ment, its heterogeneity, polarity, viscosity and ability to raise specific intermolecular interactions between tions, LAURDAN, due to the aliphatic tail of -(CH2) the solute and solvent molecules [8 – 11]. groups, forms micelles, a particular type of aggregates, It has been shown in a series of papers [8 – 12], in which the twisting rotations of the –N(CH3)2 group that LAURDAN in glycerol solution is an inhomoge- are damped [9]. The number of micelle aggregates and neous spectroscopic medium in which multi-channel types of conformational forms of LAURDAN in glyc- luminescence phenomena take place. The spectro- erol solutions depends on temperature, and for a given scopic inhomogenity is caused by a distribution of temperature an equilibrium distribution is achieved space conformational forms possessing different twist- [8, 9]. ing angles ϕ between the mutual orientation planes of In biological membranes, which are complex as- the dimethyl group and the naphthalene moiety (see semblies of lipids and proteins, the fluorescence probes Fig. 1), and by accompanying intra- and intermolec- are located in defined positions of the bilayer depth. ular charge transfer phenomena. At higher concentra- This location depends on the phase state of the bilayer, 0932–0784 / 04 / 1100–0809 $ 06.00 c 2004 Verlag der Zeitschrift f¨ur Naturforschung, T¨ubingen · http://znaturforsch.com 810 K. A. Kozyra et al. · DPPC Liposome Concentration and Fluorescence Properties of PRODAN and LAURDAN which can be changed by variation of the temperature Instr., Hannover, Germany) at 42 ◦C. The lipid film was or pressure [12, 13]. In practical studies, the biologi- resuspended in an appropriate amount of Tris buffer cal membranes are often replaced by model systems, (0.1 M, pH 7.4), and vigorously vortexed at a temper- such as phospholipid vesicles forming single or multi- ature above the phase transition, giving a lipid concen- ple bilayers, which are dispersed in aqueous medium. tration of approximately 2mg·ml−1. For the prepara- In such solvent mixtures, the radiation of fluorescent tion of small unilamellar vesicles (SUV), the resulting probe molecules often differs from that in neat solvents multilamellar vesicle (MLV) dispersion was sonicated [15, 16]. Thus the absorption and emission spectra re- with a Bandelin sonoplus HD70 (Bandelin Electronics, sult from ensembles in different environments. Lumi- Berlin, Germany) for 15 min at maximal power (cycle nescence studies allow to determine quantitatively the 30%) under nitrogen and transferred to a thermostat- phase state of such phospholipid solutions [4, 12]. ted membrane extruder system (Lipex Biomembranes In the literature concerning the photoluminescence Inc., Vancouver, Canada), which allowed the extrusion properties of PRODAN and LAURDAN in neat sol- of unilamellar vesicles of 25 nm final diameter. The vents and phospholipid vesicles, the distinguishable final lipid concentration of the SUV suspension was fluorescence states are defined differently, e.g., as non- determined for each preparation [17] and stored under relaxed (blue fluorescence) and relaxed (red fluores- nitrogen in darkness at 4 ◦C to avoid lipid peroxidation. cence) singlet states [14, 22]. Hereafter the above sin- All liposomal preparations were used within 2 weeks. glet excited states will be named locally excited S1(LE) and charge transfer S1(CT) states in accordance with 2.3. Fluorescence Measurements [6 – 9, 19 – 21]. This paper is devoted to the characterization of Fluorescence measurements of LAURDAN and the fluorescence emission properties of PRODAN and PRODAN in SUV prepared from DPPC were carried LAURDAN as functions of the phospholipid con- out in a computer-controlled Perkin Elmer LS-50 spec- centration and temperature of the solution. Different trofluorimeter equipped with a thermostatted cuvette lengths of (CH2)n residues in the molecules under (Julabo Labortechnik, Seelbach, Germany). PRODAN study allow the determination of the influence of solva- and LAURDAN were added from a 1 mM stock solu- tion processes and bilayer phase states on the fluores- tion in DMF. All samples were incubated for 1 h be- cence properties. The performed study includes steady fore the measurements. The excitation wavelength was state and time resolved spectroscopic measurements 380 nm. A 5 nm slit width was set for both excitation of both probe molecules in phospholipid solutions at and emission. 25 ◦C and 50 ◦C. The fluorescence lifetime measurements were per- formed by means of the Time-Correlated Single Pho- 2. Materials and Methods ton Counting Technique (TCSPC). We used a vio- let (λ = 403 nm) pulsed semiconductor laser LDH-C 2.1. Chemicals 400/PDL-800B (PicoQuant GmbH, Germany), emit- ting pulses of approximately 70 ps duration (FWHM). PRODAN and LAURDAN were purchased from The average laser output power was about 1 mW. The Molecular Probes, Eugene, OR. High purity DPPC laser repetition could be varied up to 40 MHz. To (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) was select the correct polarization plane for light illumi- purchased from Lipoid KG (Ludwigshafen, Germany) nating the samples we decided to employ a Babinet- and used without further purification. Ethanol and Soleil compensator (RKQ10, Bernhard Halle Nachfol- DMF (N,N-dimethylformamide ) were of spectro- ger GmbH, Germany) rather than a ”conventional” in- scopic grade and were provided by Merck (Darmstadt, put polarizer. In this way we could easily rotate the Germany). polarization plane of the laser beam, keeping at the 2.2. Preparation of Liposomes same time the highest available intensity of excita- tion. The fluorescence light emitted by the samples DPPC dissolved in ethanol was evaporated to de- was directed to a monochromator slit by a carefully posit a thin lipid film on the wall of a glass tube. selected quartz lens. We applied a SpectraPro-300i The final traces of residual solvent were removed after monochromator (Acton Research Corporation, USA), overnight storage under vacuum (Vacutherm, Heraeus equipped with three diffraction gratings (300, 600 and K. A. Kozyra et al. · DPPC Liposome Concentration and Fluorescence Properties of PRODAN and LAURDAN 811 Fig. 2. PRODAN and LAURDAN excitation and emission spectra obtained in small unilamellar phospholipid vesicles com- posed of gel (25 ◦C) and liquid crystalline (50◦) phases. 1200 grooves per mm; blaze 500 nm). The monochro- 3. Results and Discussion mator entrance and exit slits were adjustable from 10 µm to 3 mm (2.7 nm/mm spectral resolution for 3.1. Excitation and Emission Spectra 1200 g/mm grating). The selected observation wave- lengths were 440 nm and 500 nm. The measurements Figure 2 shows excitation and emission spectra were performed at 25◦ and 50 ◦C. In order to elimi- of PRODAN and LAURDAN in small unilamellar vesicles (SUV) composed of DPPC (at molar ratio nate the influence of anisotropy on the decay curves, ◦ an analyzer set at the magic angle was positioned be- 1/500) at 25 and 50 C. Analyzing the spectra of both tween the monochromator and the sample. The fluo- molecules in a phospholipid bilayer, it follows that: rescence light was registered by a H5783P photomul- • The fluorescence excitation spectra (in the spec- tiplier (Hamamatsu Photonics K.K., Japan). Next, the tral region of the long wavelength absorption band) photomultiplier signal was captured by a TimeHarp have two peaks. At room temperature (gel phase state) 100 PC-board for TCSPC (4096 channels; 36 ps reso- the long wavelength maximum (λ =∼ 380 nm) is higher lution) (PicoQuant GmbH, Germany). Finally, the flu- than that at λ =∼ 350 nm. At 50 ◦C (liquid crystalline orescence decay times were calculated (using the Mar- state) the two intensity maxima show the reverse de- quardt algorithm) by means of “FluoFit” multiexpo- pendence. They are slightly shifted to shorter wave- nential fluorescence decay fitting software (PicoQuant lengths (by about 10 nm). The fluorescence excitation GmbH, Germany). The quality of the fit was assessed spectra at 50 ◦C were measured by detecting radiation over the entire decay and tested with a plot of weighted at λ = 500 nm emitted by the ensemble of molecules χ 2 residuals and the reduced value.