Langmuir 1995,11,3742-3747
3742
Comparative Study of Fluorescence Anisotropy in Surface Monolayers of Emulsions and Bilayers of Vesicles Hiroyuki Saito,? Kenji Nishiwaki,+Tetsurou Handa,*st Shinzaburo Ita,$ and Koichiro Miyajima? Faculty of Pharmaceutical Sciences, and Department of Polymer Chemistry, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan Received April 21, 1995. In Final Form: July 5, 1995@ Surface rigidities of emulsion particles composed of triglyceride (TG)and phosphatidylcholine (PC),and 1,3,5PC vesicles were investigated using the fluorescent probe l-[4-(trimethylamino)phenyllphenylhexatriene (TMA-DPH),which was anchored at the phospholipid-water interface. Steady-state fluorescence anisotropy of TMA-DPH in surface monolayers of emulsion particles was higher than that in bilayers of vesicles. Alonger fluorescencelifetime ofTMA-DPHin emulsion surface monolayers was observed compared to bilayers. These results indicated that the surface monolayers ofemulsion particles were more rigid than bilayers, consistent with the finding that the PC molecules formed a condensed monolayer at the TGsaline interface (Handa, T.; Saito, H.; Miyajima, K. Biochemistry 1990,29,2884-2890). Distribution of cholesterol (Chol)between the emulsion surface and core was evaluated on the basis of interfacial tension measurements. The anisotropy value in the emulsion particles did not change up to 20 mol % and began to increase at about 30mol % or greater of surface Chol, in contrast to continuousincreases in the fluorescence anisotropy in bilayer vesicles. When an approximately 20 mol % of Chol was present in surface layers, the fluorescence lifetimes of TMA-DPH increased for bilayer vesicles but did not change for emulsion particles. Fluorescence lifetimes increased for both emulsion particles and bilayer vesicles with 40 mol % of surface Chol. These effects of Chol on the fluorescence properties of emulsion particles and bilayers suggested that Chol was accommodated in a different way for the surface monolayers of emulsion particles and bilayers of vesicles.
Introduction Lipid emulsions consisting of triglyceride (TG) cores and surface monolayers of phosphatidylcholine (PC) are physical models for TG-rich lipoproteins. In plasma, TGrich lipoproteins, such as chylomicrons and very low density lipoproteins, bind to capillary endothelia and undergo hydrolysis of TG by lipoprotein lipases. The hydrolysis of TG reduces the core size, resulting in the budding of excess surface monolayer components into b i l a y e r ~ . l -When ~ these remnant bilayers dissociate from the lipoprotein particles, surface lipid and apolipoproteins are transferred to the high-density lipoprotein fraction. Interactions among PC, free cholesterol (Chol), and apolipoproteins at a lipoprotein surface are considered to play important roles in these processes. The Chol content in TG-rich emulsions affects the interactions of apolipoprotein A-1 (apoA-1) and E (apoE) with the emulsion particle^.^ We have demonstrated high and low lateral affinities of apoA-1 with PC and Chol a t the triolein-saline interface, re~pectively.~ Aviram et a1.6 have shown that the increase in Chol concentration oflow density lipoproteins leads to a suppression of the lipoprotein binding to 5-774 cells and a retardation ofcellular degradations of the lipoprotein. Redgrave et aL7 have
* Author to whom correspondence should be addressed. Phone: (75)753-4565. Fax: (75)761-2698. E-mail: handatsr@ pharmsun.pharm.kyoto-u.ac.jp. Faculty of Pharmaceutical Sciences. Department of Polymer Chemistry. Abstract published i n Advance A C S Abstracts, September 15, 1995. (1)Miller, K. W.; Small, D. M. J. Biol. Chem. 1983, 258, 1377213784. (2) Miller, K. W.; Small, D. M. Biochemistry 1983,22, 443-451. (3) Tall, A. R.; Small, D. M. Adu. Lipid Res. 1980, 17, 1-51. (4) Derksen, A,; Small, D. M. Biochemistry 1989,28, 900-906. (5) Handa, T.; Saito, H.; Tanaka, I.; Kakee,A.; Tanaka, K.; Miyajima, K. Biochemistry 1992,31, 1415-1420. (6)Aviram, M.; Keidar, S.; Rosenblat, M.; Brook, G. J. J. Biol. Chem. 1991,266, 11567-11574. @
suggested that the presence of Chol in TG-rich emulsions is a critical determinant of the catabolism rate in rat plasma. On the other hand, it has been shown that phospholipid vesicles coexist with emulsion particles in commercially produced lipid emulsions and sonicated emulsions composed of TG and PC.s-12 We have shown the coexistence of vesicles and emulsion particles on the basis of monolayerhilayer equilibrium. l3 Phospholipids of the coexistingvesicles are accumulated in plasma after the injection of emulsions in animals, leading to main precursors of abnormal lipoproteins, L p - x ' ~ .Hydrolysis ~ of TG proceeded much faster in emulsion particles than in the TGPC mixed 1ip0somes.l~In addition, Chol increases the binding capacity of apoA-1to small unilamellar vesicles,15 while the binding capacities of apoA-1 and apoE to TGPC emulsion particles either do not change or decrease with an increase in Chol ~ o n t e n t .For ~ a full understanding of these phenomena, a detailed knowledge of differences in the surface properties of lipid emulsions and vesicles is required. In the current study, we investigated the surface properties of lipid emulsions and bilayer vesicles and specifically examined the effect of Chol on these properties. (7) Redgrave, T. G.;Vassiliou, G. G.; Callow, M. J. Biochim. Biophys. Acta 1987; 921, 154-157. (8) FBrBzou, J.;Lai, N.-T.; Leray, C.; Hajri, C.; Frey, A,; Cabaret, Y.; Courtieu. J.:Lutton, C.: Bach. A. C. Biochim. Biovhvs. . _ Acta 1994,1213, 149-158. (9) Hajri, T.; FBrBzou, J.; Lutton, C. Biochim. Biophys. Acta 1990, 1047, 121-130. (10)Komatsu, H.; Handa, T.; Miyajima, K. Chem. Pharm. Bull. 1994, 42, 1715-1719. (11) Rotenberg, M.; Rubin, M.; Bor, A,; Meyuhas, D.; Talmon,Y.; Lichtenberg, D. Biochem. Biophys. Acta 1991, 1086, 265-272. (12) Westesen, K.; Wehler, T. J. Pharm. Sci. 1992, 81, 777-786. (13) Handa, T.; Saito, H.; Miyajima, K. Biochemistry 1990,29,2884C.; Olivecrona, T.; Bengtsson-Olivecrona, G. Eur. J.
0743-746319512411-3742$09.00/0 0 1995 American Chemical Society
Fluorescence Anisotropy in Emulsions and Bilayers Emulsion particles were carefully isolated from coexisting vesicles, and their surface properties were compared with those of bilayer vesicles of the identical particle size with the emulsion particles. To assess differences in these properties, we utilized the fluorescent probe 1-[4-(trimethylamino)phenyllphenylhexa-1,3,Btriene(TMA-DPH), which is anchored at the phospholipid-water interface with the DPH moiety intercalated between the fatty acyl chains. We found that the steady-state anisotropy and the fluorescence lifetime of TMA-DPH in the emulsion surface monolayers were quite different from those in bilayers of vesicles.
Langmuir, Vol. 11, No. 10, 1995 3743 emulsion suspensions (5 mL). The TMA-DPH to phospholipid molar ratios were 1:50-200 for emulsions and 1:lOO-400 for vesicles, respectively. The suspension was then incubated for 1 h in the dark. Steady-state fluorescence anisotropy, r8, was measured in a SHIMADZU RF-5000 spectrofluorometer at 25 "C. Excitation was at 360 nm through HOYA U360 and TOSHIBA UV-31filters. Fluorescence emission was measured through a cutoff filter HOYA L42 at 434 nm. Samples were serially diluted to give a constant value of r6in order to eliminate the influence of the light scattering. The r8 was determined according to the following equations.
Experimental Section Materials. Egg yolk phosphatidylcholine (PC) was kindly provided by Asahi Kasei Co. The purity (over 99.5%) was determined by thin-layer chromatography. Cholesterol (Chol) (over 99% purity) was purchased from Sigma and was used without further purification. Soy bean triglyceride(TG)obtained from Nakarai Tesque and triolein (TO) obtained from Taiyo Chemicals Co. were purified by silicate (Wakogel C-200, Wako Pure Chemicals) column chromatography to remove fatty acid, diglyceride, and monoglyceride, using chloroform as an eluent. The purity of TG and TO thus obtained were over 99%. TMADPH was purchased from Molecular Probes. Praseodymium(II1) nitrate hexahydrate obtained from Wako Pure Chemicals was recrystalized twice and was then kept in a deciccator until use. Deuterium oxide (over 99.9% purity) was obtained from Aldrich Chemical Co. Inc. All other chemicals were of special grade from Wako Pure Chemicals. Water was doubly distilledwith a quartz still. Preparation of Emulsions. PC, TG, and Chol were dissolved in chloroform. Aliquots of lipid stock solutions were mixed in a round-bottom flask. After evaporation ofthe solvent,the mixture was dried in vacuo for at least 12 h. Then the buffer solution (10 mM Tris-HCl,150 mM NaC1, pH 7.4)was added to the dried lipid mixture. The final concentration of total lipids was kept at 30 mM. The suspension was briefly vortexed, and then emulsified by the following two methods. (i) Ten milliliters of the suspension was transferred to the glass tube and sonicated using a UD-200 model from Tomy Seiko Co. Ltd. Sonication was performed for 30 min at power setting 100 W under a stream of nitrogen gas. (ii) Thirty milliliters of the suspension was emulsified using a high-pressure emulsifier (Nanomizer, Nanomizer Inc., Tokyo)under a pressure of 750-800 kg/cm2. In both methods, the sample temperature was maintained at about 60 "C for TG-PC emulsions. In emulsions containing Chol, on the other hand, the emulsifications were performed at about 30 "C because small particles could not be obtained at 60 "C. No free fatty acids resulting from the hydrolysis of the lipids during emulsifications were detected by thin-layer chromatography. Isolation of Emulsion Particles from Vesicles. The prepared emulsions were transferred into polyallomer tubes (25 x 89 mm). The tubes were placed in a SW-28 rotor (Beckman L7-65 ultracentrifuge) and were ultracentrifuged for 1 h at 30000g and 25 "C. The floating creamy layer was isolated with a tube slicer (HITACHI)and resuspended in the buffer. Then ultracentrifugation was again carried out for 1h at 20000g and the creamy layer was collected as the isolated emulsion particles. Preparation of Vesicles. Vesicles with homogenous size distribution were obtained by extrusion method. The dried lipid film was hydrated with the buffer solution and vortexed to form a suspension of multilamellar vesicles. The suspension was subjected to three freeze-thaw cycles, followed by successive extrusion (LipexBiomembranes Inc.). The extrusion was carried out through a 0.6-ym pore size polycarbonate filter (Nuclepore) five times under a nitrogen pressure of 2 kg/cm2and then through doubly stacked filters of 0.1pm pore size 15times under a 10-12 kg/cm2pressure. NMR Spectroscopy. 31PNMR spectra were obtained on a Brucker AC-300 spectrometer at 121.5 MHz by using 2-ys 45" pulses and application of a 50 kHz proton decoupling field during acquisition. Aparamagnetic shiftingreagent, praseodymium(II1) nitrate, was added into emulsion or vesicle samples (the final concentration was 10 mM). Fluorescence Measurement. TMA-DPH, previously dissolved in dimethyl sulfoxide (5 pL) was added into vesicle and
Z, and ZH are the fluorescence intensities parallel and perpendicular to the exciting polarized light. G represents the compensating factor for the anisotropic sensitivity of the instrument. Fluorescence lifetime measurements were performed with a nanosecond single-photon counting method at 25 "C using the following apparatus: photomultiplier (PRA model 1550),timeto-amplitude converter (ORTEC model 4571, discriminator (ORTEC model 436, 583), multichannel analyzer (HITACHI model 5051, and hydrogen gas filled flash lamp (PRAmodel 510B) with a full-width at half-maximum height of about 1.8 ns. To excite the probe, HOYA U360 and a glass filter were used, and a cutoff filter FUJI SC-41was used for detection. Deconvolution procedures were carried out according to the method of Wahl.lG Curve-fitting procedures were performed by assuming monoand biexponential decays. Measurement of Interfacial Tension. The interfacial tension of the TO-saline (10 mM Tris-HC1, 150 mM NaCl, pH 7.0) interface was measured as a function of Chol concentration in TO solutions of PC and Chol by the drop weight method at 25 "C. The inverted TO drops (TO was less dense than the surrounding saline) were formed by the use of an all-glass micrometer syringe in a double-walled jacket equipped with a Teflon stopper. More than 10 drops were formed for an experimental point of a certain Chol concentration, and the averaged value ofinterfacial tension (f0.4 mN/m) was obtained. The particulars in the interfacial tension measurement have been described e l ~ e w h e r e . ~ J ~ J ~ Lipid Analysis. Phospholipid concentration was determined by the phosphorus assay according to the method of Bartlett.18 The concentrations of triglyceride and cholesterol were determined using enzymatic assay kits Triglyceride G-test Wako and Free cholesterol E-test Wako, respectively, obtained from Wako Pure Chemicals. Particle Size Measurement. Quasi-elasticlight scattering measurements of emulsions and vesicles were performed at 25 "C on a Photal LPA-3000/3100 instrument (Photal, Otsuka Electronic Co.). The correlation functions were analyzed by the histogram method, and the weight-averaged particle sizes were evaluated. Results Coexistence of Bilayer Vesicles with Emulsion Particles. In both emulsions prepared by sonication and high-pressure emulsification, vesicles were observed beside emulsion particles. We estimated the content of vesicles coexisting with emulsion particles by 31PNMR spectra in the presence of Pr3+. Pr3+causes a downfield shift of the phosphorus signal of PC because of pseudocontact, dipole interactions.lg The addition of F W therefore results in a downfield shift of signals originated from the PC head groups in the outer monolayers of vesicles and emulsion particles (Pout), whereas signals from the head groups in the inner layers of vesicles (Pin) are not shifted. As shown in Figure lA, the peak near 0 ppm (Pin) (16) Wahl, Ph. Biophys. Chem. 1979, 10, 91-104. (17) Mukejee, P.; Handa, T. J. Phys. Chem. 1981,85,2298-2303. (18)Bartlett, G. R. J . Biol. Chem. 1969,234,466-468. (19)Bergelson, L. D. In Method in Membrane Biology; Plenum Press: New York, 1977; Vol. 9, p275-335.
Saito et al.
3744 Langmuir, Vol. 11, No. 10, 1995 pout
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Figure 1. 31PNMRspectraat121.5 MHz ofTG-PC emulsions prepared by a high-pressureemulsifier in the presence of Pr3+ in the external aqueous phase, before (A, left) and after (B, right) ultracentrifugation as described under Experimental Procedures. Peaks are designated P i n for PC head groups in the inner monolayer of vesicles and Poutfor those in the outer monolayers of vesicles and emulsion particles, respectively. was observed together with the peak shifted to 20-22 ppm (Pout) in TG-PC emulsions before ultracentrifugation, indicating that a considerable amount ofvesicles coexisted with emulsion particles. On the contrary, the peak near 0 ppm almost disappeared after ultracentrifugation (Figure 1B). Similar results were observed in the emulsions containing Chol. These results indicate that the emulsion particles can be separated from coexisting vesicles effectively by the ultracentrifugation procedure described under Experimental Procedures.
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InterfacialTension and Adsorption of Chol at the TO-Saline Interface in the Presence of the PC Monolayer. We estimated the Chol adsorption behavior a t the triolein (TO)-saline interface instead of the TGsaline interface because of the better reproducibility of the interfacial tension. TG had the similar solubility in PC bilayers with TO and the distribution of Chol between the core and surface phases in the particles made from extracted !ipoprotein lipids was quite similar to that in the emulsions of T0.1,2s20Figure 2A shows the interfacial tension at the interface ( y ) as a function of Chol concentrations in the TO phase. The PC concentration in the TO phase is constant a t 4 x M (moVL), and the interface was exclusively occupied by the PC m01ecules.l~ The amounts of Chol adsorbed a t the interface ( r C h o l in mol/cm2) in the presence of the PC monolayer are correlated with the change of interfacial tension by the Gibbs adsorption equation:
where R is the gas constant. The subscript T,P,PC indicates that the measurement is performed a t constant temperature, pressure, and PC concentration. Figure 2B shows r C h o l calculated by the use of eq 2 as a function of the Chol concentration. When PC monolayer is absent, large cooperativity is observed in the Chol adsorption. The cooperativity has been ascribed to the lateral repulsive interactions between Chol and TO molecules (or the apparent attractive interaction between Chol molecules) at the i n t e r f a ~ e .The ~ PC molecules a t the interface were gradually replaced by the Chol molecules with increasing CChol. The adsorption (penetration) of Chol a t the interfacial monolayer of PC also showed a slight cooperativity. (20)Miller, K. W.; Small, D. M. J. Colloid Interface Sci. 1982,89, 466-478.
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Chol Concentration ( m mol / I ) Figure 2. (A, upper) Interfacial tension of the TO-saline (10 mM Tris-HC1, 150 mM NaCl, pH 7.0) interface as a function of Chol concentration in the TO phase. The PC concentration mom. The solubility of Chol in in the TO phase was 4 x mom. (B, 1ower)Adsorptionisotherm TO at 25 "C was 9 x of Chol at the TO-saline interface. The PC concentration in the TO phase was 4 x mom. The monolayer/TG core (liquid phase of TO) distribution coefficient, K,, is calculated by the equation:
Here r:hol and r& are the saturated adsorption amounts for Chol and PC, 4.7 x and 4.1 x mol/cm2,re~pectively.~J~ The PC and Chol concentrations in the TO phase were low (
