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Molecular Recognition of Complementary Liposomes: The Enhancing Role of Cholesterol Zili Sideratou, Dimitris Tsiourvas, and Constantinos M. Paleos* Institute of Physical Chemistry, NCSR “Demokritos”, 15310 Aghia Paraskevi, Attiki, Greece
Achilleas Tsortos and George Nounesis Institute of Radioisotopes and Radiodiagnostic Products, NCSR “Demokritos”, 15310 Aghia Paraskevi, Attiki, Greece Received February 7, 2000. In Final Form: August 7, 2000 Liposomes based on hydrogenated phosphatidylcholine (PC) incorporating dihexadecyl phosphate (DHP) and 1-(4-(dihexadecylcarbamoyl) butyl)guanidinium p-toluene sulfonate (DBG), respectively, were mixed in aqueous dispersion at room temperature, and their molecular recognition effectiveness was investigated by turbidimetric measurements, microscopy, DSC, and isothermal titration calorimetry. Because of the high binding efficiency of the incorporated recognizable lipids, accurate results could be obtained for low DHP/PC and DBG/PC molar ratios (19:1), especially for enthalpy change measurements. The liposomeliposome interaction was further investigated as a function of the cholesterol content in mixed (PCcholesterol) liposomes. At appropriate cholesterol concentrations, the organization of the recognizable moieties within the liquid-ordered phase of the bilayer membrane enhances molecular recognition at the liposomal interface.
Introduction Recent reports have established that the recognition of complementary molecules in aqueous media is favored in organized molecular assemblies. In fact, binding constants differ by some orders of magnitude for the various types of molecular organizations.1 According to recent reviews,2,3 monolayer organization at air-water macroscopic interfaces constitutes the most effective medium for recognition experiments. Less effective recognition occurs at microscopic liposomal interfaces, and even less in micellar aggregates. As the closest analogues to living cells, liposomes are considered the most appropriate systems for simulating cell-cell4 as well as cell-liposome interactions, which are of importance when the latter are employed as drug-delivery systems.5,6 In molecular recognition studies involving liposomal assemblies, moieties located at the external lipid surface were reacted either with complementary molecules dissolved in the aqueous phase or with molecules incorporated in other liposomes. In either case, molecular recognition leads to a variety of interesting results. When biotin was incorporated in unilamellar liposomes, the addition of streptavidin in the aqueous phase led to tethered liposomes through the formation of biotin-streptavidin bridges.7 Analogous results were obtained for the iron (II)-induced association of lecithin vesicles incorporating a terpyfunctionalized phospholipid, a reaction that can be * To whom correspondence should be addressed. (1) Onda, M.; Yoshihara, K.; Koyano, H.; Ariga, K.; Kunitake, T. J. Am. Chem. Soc. 1996, 118, 8524. (2) Ariga, K.; Kunitake, T. Acc. Chem. Res. 1998, 31, 371, and references therein. (3) Paleos, C. M.; Tsiourvas, D. Adv. Mater. 1997, 9, 695, and references therein. (4) Cooper, G. M. The Cell, A Molecular Approach; ASM Press: Washington, DC, 1997; p 467. (5) Gregoriadis, G. Trends Biotechnol. 1995, 13, 527, and references therein. (6) New, R. R. C. Liposomes, A Practical Approach; Rickwood, D., Hames, B. D., Eds.; IRL Press: Oxford, U.K., 1990. (7) Chiruvolou, S.; Walker, S.; Israelachvili, J.; Schmitt, F.-J.; Leckband, D.; Zasadzinski, J. A. Science 1994, 264, 1753.
reversed by the addition of Na2EDTA.8 Different results were obtained, however, when dihexadecyl phosphate liposomes interacted with the guanidinium cation (C(NH2)3+) and its derivatives.9 In this case liposomes decrease in size and are finally transformed into micelles. In cases where liposome-liposome interactions are encountered, Lehn et al.10 found that for oppositely charged phosphatidylcholine liposomes (incorporating dihexadecyl phosphate and octadecylamine, respectively), a progressive charge neutralization occurred without fusion. Molecular recognition often leads to the formation of larger liposomes,11 as is the case with mixed liposomes of didodecyldimethylammonium bromide (incorporating 5,5didodecylbarbituric acid and 9-hexadecyladenine, respectively) and also with complementary lecithin liposomes containing amphiphilic-type derivatives of barbituric acid and triaminopyrimidine, respectively.12 In the present study, the role of cholesterol in affecting molecular recognition and binding of certain complementary liposomes (with formulations to be described below) has been investigated at various concentrations of incorporated cholesterol. Cholesterol is a basic constituent of cell membranes, and it is known to affect their structure and properties. Therefore, experiments with liposomes incorporating varying amounts of cholesterol can be considered as a useful model for simulating cholesterol’s function in cell-cell recognition processes. Systematic work has already been carried out on cholesterol’s role in the stability,13 permeability,14 and phase transitions of (8) Constable, E. C.; Meier, W.; Nardin, C.; Mundwiler, S. Chem. Commun. 1999, 1483. (9) Paleos, C. M.; Tsiourvas, D.; Kardassi, D. Langmuir 1999, 15, 282. (10) Marchi-Artzner, V.; Jullien, L.; Belloni, L.; Raison, D.; Lacombe, L.; Lehn, J.-M. J. Phys. Chem. 1996, 100, 13844. (11) Paleos, C. M.; Sideratou, Z.; Tsiourvas, D. J. Phys. Chem. 1996, 100, 13898. (12) Marchi-Artzner, V.; Jullien, L.; Gulik-Krzywicki, T.; Lehn, J.M. Chem. Commun. 1997, 117. (13) Kirby, C.; Clarke, J.; Gregoriadis, G. Biochem. J. 1980, 186, 591. (14) Raffy, S.; Teissie´, J. Biophys. J. 1999, 76, 2072.
10.1021/la000166d CCC: $19.00 © 2000 American Chemical Society Published on Web 10/31/2000
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Scheme 1. Schematic Interaction of Complementary Liposomes
unilamellar liposomes.15 In this connection, phospholipidcholesterol phase diagrams16-19 have been established, and structural and dynamic properties of mixed phosphatidlylcholine-cholesterol bilayers20-23 are well-understood. Additionally, recent reports address the association and effects of certain alcohols on lipid bilayer with and without cholesterol.24-28 For the present investigation, lipids functionalized with guanidinium and phosphate groups were employed as the recognizable moieties. These groups were chosen because they interact strongly both by electrostatic and hydrogenbonding forces, promoting by their combined action the noncovalent binding.1 Thus, in our experiments, liposomes based on hydrogenated phosphatidylcholine (PC) and (15) Genz, A.; Holzwarth, J. F.; Tsong, T. Y. Biophys. J. 1986, 50, 1043. (16) Ipsen, J. H.; Karlstro¨m, G.; Mouritsen, O. G.; Wennerstro¨em, H.; Zuckermann, M. J. Biochim. Biophys. Acta 1987, 905, 162. (17) Thewalt, L. J.; Bloom, M. Biophys. J. 1992, 63, 1176. (18) Vist, M. R.; Davis, J. H. Biochemistry 1990, 29, 451. (19) Trandum, C.; Westh, P.; Jørgensen, K.; Mouritsen, O. G. Biophys. J. 2000, 78, 2486. (20) Mouritsen, O. G.; Jørgensen, K. Chem. Phys. Lipids 1994, 73, 3. (21) Nielsen, M.; Miao, L.; Ipsen, J. H.; Zuckermann, M. J.; Mouritsen, O. G. Phys. Rev. E 1999, 59, 5790. (22) Virtanen, J. A.; Somerharju, P. J. Phys. Chem. B 1999, 103, 10289. (23) Smondyrev, A. M.; Berkowitz, M. L. Biophys. J. 1999, 77, 2075. (24) Zhang, F.; Rowe, E. S. Biochemistry 1992, 31, 2005. (25) Rosser, M. F. N.; Lu, H. M.; Dea, P. Biophys. Chem. 1999, 81, 33. (26) Trandum, C.; Westh, P.; Jørgensen, K.; Mouritsen, O. G. J. Phys. Chem. B 1999, 103, 4751. (27) Trandum, C.; Westh, P.; Jørgensen, K.; Mouritsen, O. G. Biochim. Biophys. Acta 1999, 1420, 179. (28) Westh, P.; Trandum, C. Biochim. Biophys. Acta 1999, 1421, 261.
incorporating dihexadecyl phosphate (DHP) and 1-(4(dihexadecylcarbamoyl) butyl)guanidinium p-toluene sulfonate (DBG), respectively, were allowed to interact (Scheme 1). The interaction took place at room temperature, well below the lipid main phase transition, and molecular recognition effectiveness at the liposomal interfaces was investigated by turbidimetric29-32 and microscopical methods9,11,12 as well as with isothermal titration calorimetry.26 The strong interaction between the DHP and the DBG groups allowed us to perform experiments at low concentrations of these molecules relative to PC (molar ratios of PC/DBG and PC/DHP ) 19:1). The effect of additives on bilayer order and other properties should not be disregarded, as set forth in the Discussions of the Faraday Society.33 At the low molar ratio used in our study, however it may be safely assumed that the incorporation of the recognizable molecules within the PC-cholesterol bilayer will not drastically disturb the molecular order of the lipid bilayer. This assumption has been confirmed by DSC microcalorimetric results, presented below. Experimental Section Materials. Soybean hydrogenated phosphatidylcholine (Phospholipon 90H), Nattermann Phospholipid GMBH, and dihexadecyl phosphate (Sigma) were used. 1-(4-(Dihexadecylcarbamoyl) (29) Stamatatos, L.; Leventis, R.; Zuckermann, M. J.; Silvius, J. R. Biochemistry 1988, 27, 3917. (30) Eggens, I.; Fenderson, B.; Toyokuni, T.; Dean, B.; Stroud, M.; Hakomori, S. J. Biol. Chem. 1989, 264, 9476. (31) Hasegawa, M.; Kaku, T.; Kuroda, M.; Ise, N.; Kitano, H. Biotechnol. Appl. Biochem. 1992, 15, 40. (32) Stewart, R. J.; Boggs, J. M. Biochemistry 1993, 32, 10666. (33) Note: Lipid Vesicles and Membranes. Discuss. Faraday Soc. 1986, 81, pp 77, 209, 340, 341.
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butyl)guanidinium p-toluene sulfonate was prepared as described in the literature.1 Liposome Preparation. Unilamellar liposomes were prepared by the extrusion method,34-36 employing a laboratory extruder (LiposoFast-Pneumatic, Avestin Inc.).37 In a typical experiment for preparing a 2-mL dispersion of liposomes, 0.038 mmol (1.9 × 10-2 M) of PC and 0.002 mmol (1.0 × 10-3 M) of DHP or DBG (molar ratio 19:1) were mixed with various concentrations of cholesterol, up to 50% molar with respect to PC. Liposomal dispersions were stable for more than 5 days; however, in the characterization experiments the dispersions were used in less than 24 h following their preparation. Recognition Experiments. Recognition reactions were performed and evaluated by turbidimetric studies, optical microscopy, and isothermal titration microcalorimetry employing liposomal dispersions as specified in the respective experiments (see below). Characterization. Turbidimetric Studies. The aggregation of liposomes resulting from the recognition during the titration of the complementary liposomes was monitored by measurement of the turbidity change at 400 nm using a Lamba-16 spectrophotometer (Perkin-Elmer) in a cuvette kept at 25 °C. For these experiments, dispersions of PC-DHP liposomes (1.9 × 10-4 M PC, 1.0 × 10-5 M DHP) were mixed with PC-DBG liposomes (1.9 × 10-4 M PC, 1.0 × 10-5 M DBG). Various quantities of cholesterol, up to 50% molar with respect to PC, were incorporated into these dispersions. Control experiments were performed as described below (see Results and Discussion). Optical and AFM Microscopical Studies. AFM was employed to determine the sizes of the originally prepared liposomes. The images were obtained with a Multi-Mode Nanoscope III microscope (Digital Instruments) employing the tapping mode operation. Droplets of liposome dispersions were placed on a freshly cleaved mica surface, as previously described,9,11 to allow observation of samples. Liposomal aggregates resulting after association of the original liposomes were imaged by video-enhanced phase-contrast optical microscopy employing an Olympus BX-50 microscope coupled with a Kodak Megaplus model 1.4i camera using an IC-PCI image board (Imaging Technology Inc.). The images were then analyzed by SigmaScan Pro version 4.0 image analysis software (SPSS Inc.). Thus, to a dispersion of liposomes prepared from PCcholesterol-DHP (4.8 × 10-3 M PC, 2.4 × 10-3 M cholesterol, and 2.5 × 10-4 M DHP), increasing quantities of PC-cholesterolDBG liposomes (4.8 × 10-2 M PC, 2.4 × 10-2 M cholesterol, and 2.5 × 10-3 M DBG) were gradually added up to a 1:1 final molar ratio for the recognizable moieties. After each addition, the resulting mixture was quickly agitated and subsequently transferred to the microscopic table for observation. The interaction was spontaneous (see also discussion in Microcalorimetric Studies), and images remained the same throughout the microscopic observations. Microcalorimetric Studies. Microcalorimetric experiments were performed on an MCS-ITC isothermal titration microcalorimeter (Microcal Inc., Northampton, MA).38 For the calorimetric experiments, a dispersion of PC-DBG liposomes, the “titrant”, was injected into a dispersion of PC-DHP liposomes, the “titrand”. The rate of heat released (dQ/dt) as a result of interaction was monitored with time. The heat released for any particular injection (Q) equals the area under the trace of dQ/dt. Identical isothermal titration experiments were also performed with liposomal systems incorporating 50% molar cholesterol with respect to PC. Control experiments were carried out with PCcholesterol-DGB as titrant and PC-cholesterol as titrand. In addition, PC-cholesterol titrant was added to PC-cholesterol or PC-cholesterol-DHP employed as titrands. Furthermore, (34) Hope, M. J.; Bally, M. B.; Webb, G.; Cullis, P. R. Biochim. Biophys. Acta 1985, 812, 55. (35) Olson, F.; Hunt, C. A.; Szoka, F. C.; Vail, W. J.; Papahadjopoulos, D. Biochim. Biophys. Acta 1979, 557, 9. (36) Bangham, A. D.; Standish, M. M.; Watkins, J. C. J. Mol. Biol. 1965, 13, 238. (37) MacDonald, R. C.; MacDonald, R. I.; Menco, B. Ph. M.; Takeshita, K.; Subbarao, N. K.; Hu, L. Biochim. Biophys. Acta 1991, 1061, 297. (38) Wiseman, T.; Williston, S.; Brandts, J. F.; Lin, L. N. Anal. Biochem. 1989, 179, 131.
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Figure 1. Turbidity change during molecular recognition experiments between PC-DHP and PC-DBG liposomes without cholesterol (open circles) or with 50% cholesterol (filled circles). Horizontal axis represents the percent concentration of PC-DBG liposomes in the aqueous dispersion. simple PC liposomes of the above-mentioned concentrations were used. With these control experiments, the heat of dilution as well as the association ability of DBG with PC were assessed. A typical experiment is described in the paragraph that follows. Injections of 10 µl of the titrant (4.8 × 10-2 M PC, 2.4 × 10-2 M cholesterol, and 2.5 × 10-3 M DBG) were added to a 1.334-mL reaction cell containing the titrand (4.8 × 10-3 M PC, 2.4 × 10-3 M cholesterol, and 2.5 × 10-4 M DHP). Twenty injections at 200-s intervals were programmed and performed automatically at 28 °C; dQ/dt data points were collected every 1 s and were analyzed using the built-in Origin software. Differential scanning microcalorimetry measurements were carried out on a VP-DSC calorimeter (Microcal Inc., Northampton, MA), at heating rates of 20 K hr-1 and PC concentration of 1.9 × 10-2 M.
Results and Discussion Turbidimetry was used as an indirect method for assessing molecular recognition; it has been extensively applied in analogous studies.29-32 The increase in turbidity during titration is attributed to the appearance of a greater number of large particles in the dispersion, formed because of effective interaction of the liposomes. This change in turbidity for the PC-DHP liposomes as a function of the added quantity of PC-DBG liposomes is shown in Figure 1. It is evident from these results that recognition is most effective (curve maximum) at 1:1 molar ratios of the recognizable molecules. Analogous experiments were performed with liposomes, incorporating increasing quantities of cholesterol up to the levels usually present in liposomal drug-delivery systems as well as in biological cells, although the latter possess a much smaller curvature than the preparations reported here. With increasing cholesterol concentration, from 10% upward, recognition at 1:1 molar ratios of the recognizable molecules is enhanced, as demonstrated by the turbidity increase (Figure 2). It should be noted that a minor turbidity increase was also observed during the interaction of PCDBG liposomes with PC liposomes free of DHP. This increase is attributed to the significantly less effective interaction of DBG with the sterically hindered phosphate group of PC. None of the other control experiments performed showed any detectable change in turbidity. Recognition was also followed by optical microscopy. However, because the prepared liposomes were too small to be seen by optical microscopy, AFM (tapping mode), which has been employed for visualizing sensitive lipo-
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Figure 2. Turbidity measurements during recognition experiments of 1:1 molar ratios of complementary liposomes (1.9 × 10-4 M PC, 1.0 × 10-5 M DHP or DBG) as a function of cholesterol concentration with respect to PC.
Figure 4. Phase-contrast optical microscopy images of liposome aggregates after mixing samples of the complementary liposomes shown in Figure 3. The bar in the lower-left corner indicates 5 µm.
Figure 5. Isothermal titration calorimetric results for molecular recognition of the PC/DHP with PC/DBG system without cholesterol (dashed curve) or with 50% cholesterol (solid curve). Control dilution experiment of PC with PC liposomes is shown as a dotted curve. First and ninth (inset) 10-µL injections are shown.
Figure 3. AFM images (taken with the Tapping Mode) of liposomes consisting of PC/DHP/cholesterol (A) and PC/DBG/ cholesterol (B).
somal systems,9,11 was used. The liposomes’ size was found to range between 60 and 130 nm (Figure 3). Upon mixing, interactions occur spontaneously; as a result of fusion, large aggregates are formed, which are then visible with phase-contrast optical microscopy. These particles interact further, resulting in even larger aggregates, which in
certain cases encapsulate smaller aggregates, as shown in Figure 4. The photographs depict various structures obtained at the same time immediately after the mixing of the complementary liposomes. The enhancing role of cholesterol in liposomal association, observed by the microscopic and turbidimetric studies, was further investigated and quantified by isothermal titration microcalorimetry.38 The heat released by the liposomal interaction is maximum for the (PCcholesterol-DBG)/(PC-cholesterol-DHP) system and negligible for all the control experiments. In Figure 5, results are presented for the DBG/DHP interaction in liposomes with (50% mole) or without cholesterol, as well as for one of our control experiments. The results from all our exothermic experiments are summarized in Table 1.
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Table 1. Binding Parameters for PC-based Complementary Liposomes titrant/titrand
∆H (kJ mol-1)
k(s-1)
PC-DBG/PC-DHP PC-chol-DBG/PC-chol-DHP
0.576 ( 0.035 1.113 ( 0.052
0.068 ( 0.005 0.273 ( 0.017
Control Experiments PC/PC 0.028 ( 0.008 PC-chol/PC-chol 0.034 ( 0.008 PC-DBG/PC 0.123 ( 0.012 PC-chol-DBG/PC-chol 0.206 ( 0.019 PC/PC-DHP 0.113 ( 0.012 PC-chol/PC-chol-DHP 0.165 ( 0.013
The enthalpy change for the binding (∆H) is found to be 1.113 kJ mol-1 when cholesterol is present and 0.576 kJ mol-1 in the absence of cholesterol. For the control titration experiments between PC-based liposomes with or without cholesterol, only the dilution heat for the concentrated lipid is measured (see Table 1), because in this case no interaction is taking place. Small ∆H values were measured for the other control experiments as shown in Table 1. The most notable is the one observed during the titration of PC-DBG with PC liposomes with (∆H ) 0.206 kJ mol-1) or without cholesterol (∆H ) 0.123 kJ mol-1), indicating that some interaction may be taking place between the phosphate group of PC and the guanidinium group of DBG. These calorimetric results are consistent with the findings of the turbidimetric experiments. The substantial ∆H increase recorded in the presence of cholesterol can be understood in the context of the difference in molecular order within the lipid bilayer. In the absence of cholesterol, the PC molecules are in the solid-ordered phase,16-19 characterized by a well-established translational and chain-conformational order. Only a portion of the DBG molecules will bind to DHP as a result of geometrical constraints, because of the spherical topology of the liposomes. However, the enhanced lateral mobility of the interacting molecules in the liquid-ordered phase16-19 of the PC-cholesterol mixed bilayers, where no long-range translational order exists, will most likely give rise to the formation of wide liposomal contact zones. The effect of geometrical constraints should be greatly reduced, leading to a more efficient association of the liposomal interfaces. The role of cholesterol in liposomal recognition is also evident from the reaction rates that are measured in isothermal titration experiments. Assuming singleexponential kinetics, the reaction rates (k) measured39 from the data presented in Figure 5, corrected for the instrument relaxation time,40 become approximately 4 times faster in the presence of cholesterol. The formation of large aggregates, observed microscopically, was also detected by the microcalorimetry experiments. Excess heat is observed, not following the monoexponential time decay of dQ/dt, for consecutive injections of DBG liposomes. The effect is much more pronounced in the case of the PC-cholesterol liposomes and increases with the number of injections. The kinetics involved in the binding process of the large aggregates is certainly much slower than in the case of simple liposomal binding. The data presented in the inset of Figure 5 are for the ninth 10-µL injection of DBG-PC-cholesterol liposomes. The detection of the excess heat began at the fifth and increased for additional injections. This process is currently under investigation. The enhanced effectiveness of molecular recognition observed when cholesterol is incorporated into mixed (39) Williams, A. B.; Toone, E. J. J. Org. Chem. 1993, 58, 3507. (40) Morin, P. E.; Freire, E. Biochemistry 1991, 30, 8494.
Table 2: Transition Temperatures (Tm) and Enthalpy Changes (∆H) for the Main-Phase Transition of the Various Liposomal Dispersions PC PC/DHP (19:1) PC/DBG (19:1) PC/cholesterol (10:1) PC/cholesterol/DHP (19:1.9:1) PC/cholesterol/DBG (19:1.9:1) PC/cholesterol/DHP (19:3.8:1) PC/cholesterol/DBG (19:3.8:1) PC/cholesterol/DHP (19:9.5:1) PC/cholesterol/DBG (19:9.5:1) a
Tm (°C)
∆H (kcal mol-1)
51.88 49.24 52.11 50.16 51.84 52.01 52.90 52.95 a a
11.9 10.2 12.1 7.4 8.9 9.1 6.6 6.8
No transition has been detected.
Figure 6. DSC profiles of ∆Cp vs T for the main-phase transition of liposomal dispersions: (a) PC, (b) PC/DHP (19:1), (c) PC/cholesterol/DHP (19:1.9:1), (d) PC/cholesterol/DHP (19: 3.8:1), (e) PC/cholesterol/DHP (19:9.5:1). ∆Cp traces have been shifted vertically for clarity. The concentration of PC in all the above experiments was 1.9 × 10-2 M.
liposomes at concentration levels of 10%-50% can be attributed to the structural features of lipid-cholesterol bilayers. The membrane structure contributes to a favorable exposure of the complementary groups of the recognizable lipids, and the interaction is more efficient. This finding is in agreement with recent results on cholesterol’s effect upon the molecular order of the lipid bilayer, which led to a widely accepted phase diagram.16-19 According to this phase diagram (which is relatively independent of the precise chemical structure of PC), at the temperature employed in our experiments, cholesterol present in amounts higher than 25% mol gives rise to the formation of a liquid-ordered phase. This phase is a fluid from the viewpoint of lateral disorder and diffusion, and therefore it is of importance for the mobility of membraneincorporated compounds. However, the lipid acyl-chains are characterized by a high degree of conformational order. In other words, at these high concentrations, cholesterol decouples the positional and conformational degrees of freedom of the phospholipid molecules. At concentrations exceeding 10% and up to 25%, the liquid-ordered phase coexists with the solid-ordered phase. Because the recognizable lipids incorporated are at low concentrations (1:19 molar ratio), their presence will not, as mentioned earlier, appreciably perturb the molecular order of the PC-cholesterol bilayer, and therefore the interacting moieties would most likely encounter the previously described organized environment. In fact, it has been found (DSC measurements; see Table 2) that PC-based liposomes containing the interacting components in molar ratios of PC/DHP (or DBG) 19:1 exhibit the main lipid-phase transition at temperatures very close to the Tm value of
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pure PC and at an almost identical heat capacity (Cp) versus temperature profiles. Furthermore, samples containing 10% and 20% cholesterol and incorporating the complementary lipids in the same concentration as above have Tm at ∼50 °C and gradually broadening Cp profiles, indicating that the low-temperature phase in the bilayer is a coexistence between the solid-ordered and the liquidordered phases.15,41 At 50% cholesterol concentration, no phase transition has been detected, indicating that the bilayer is in the liquid-ordered phase (see Figure 6). The enhanced association of recognizable liposomes containing cholesterol relative to those without cholesterol can be attributed to the organization of the lipids and the resulting higher lateral mobility of the recognizable molecules in the liquid-ordered phase. Apparently, molecular organization combined with fluid lateral mobility (41) Vist, M. R.; Davis, J. H. Biochemistry 1990, 29, 451.
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is required for a more efficient recognition of liposomal assemblies. On the contrary, in the solid-ordered phase, the lack of mobility does not favor such efficient interaction between liposomes. In conclusion, cholesterol incorporated into liposomes significantly modifies their molecular recognition performance. It is thus possible to tune the association ability of liposomes by changing the incorporated amounts of cholesterol in the liposomal bilayer. On the basis of this capability, model liposomal systems may be produced to explore the role of cholesterol in molecular recognition in cell membranes and drug-delivery targeting. Acknowledgment. This work was partially supported by the Brite Euram project, BRPR-CT94-0401. LA000166D
