Interaction of Phosphatidyl Choline Based Liposomes Functionalized

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Langmuir 2002, 18, 829-835

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Interaction of Phosphatidyl Choline Based Liposomes Functionalized at the Interface with Adenine and Barbituric Acid Moieties Zili Sideratou, Dimitris Tsiourvas, and Constantinos M. Paleos* Institute of Physical Chemistry, NCSR “Demokritos”, 15310 Aghia Paraskevi, Attiki, Greece

Achilleas Tsortos, Serapion Pyrpassopoulos, and George Nounesis Institute of Radioisotopes & Radiodiagnostic Products, NCSR “Demokritos”, 15310 Aghia Paraskevi, Attiki, Greece Received August 1, 2001. In Final Form: October 21, 2001 The interaction of PC-based unilamellar liposomes incorporating the complementary 5,5 di-ndodecylbarbituric acid (DBA) or 9-hexadecyladenine (HA) in various molar ratios has been investigated using imaging, spectroscopic and calorimetric techniques. Liposomal interaction was established by turbidimetry, AFM, and optical microscopy and also by isothermal titration calorimetry. For temperatures below the main lipid phase transition, the results provide evidence for hydrogen-bonding between the adenine and the barbituric acid moieties at the lipid-water-lipid interfaces with a stoichiometry ratio of 1:1. The interaction does not lead to disruption of the liposomes but instead it affords larger aggregates. The role of cholesterol in the DBA-HA binding, at high concentrations in the PC:DBA and PC:HA bilayers has also been investigated. Evidence is found that the presence of cholesterol (at molar concentration 2:1 relative to PC) alters the DBA/HA binding mechanism. For all the systems studied, no liposomal binding could be calorimetrically detected above the main lipid phase transition temperature.

Introduction Amphiphilic-type barbituric acid derivatives have been extensively employed in studies of molecular recognition, especially at air-water interfaces.1 On the other hand alkylated adenine derivatives were able to interact with n-alkyl thymine derivatives in the hydrophobic interior of sodium dodecyl sulfate micelles.2 It has been established that the high degree of molecular organization achieved at air-water interfaces has an enhancing effect upon interaction effectiveness. On the other hand, due to the lower organization obtained at liposomal interfaces, recognition in these systems is less effective. It is, however, substantially more effective for the same interaction occurring in isotropic media.3 Furthermore, enhancement of liposomal binding may be attributed to multivalent interactions4 resulting from the simultaneous associa* To whom correspondence should be addressed. (1) (a) Honda, Y.; Kurihara, K.; Kunitake, T. Chem. Lett. 1991, 681684. (b) Ahuga, R.; Caruso, P. L.; Mo¨bius, D.; Paulus, W.; Ringsdorf, H.; Wildburg, G. Angew. Chem., Int. Ed. Engl. 1993, 32, 1033-1036. (c) Bohanon, T. M.; Denziger, S.; Fink, R.; Paulus, W.; Ringsdorf, H.; Weck, M. Angew. Chem., Int. Ed. Engl. 1995, 34, 58-60. (d) Kimizuka, N.; Kawasaki, T.; Kunitake, T. J. Am. Chem. Soc. 1993, 115, 4387-4388. (e) Motesharei, K.; Myles, D. C. J. Am. Chem. Soc. 1994, 116, 74137414. (f) Kimizuka, N.; Kawasaki, T.; Kunitake, T. Chem. Lett. 1994, 1399-1402. (g) Weck, M.; Fink, R.; Ringsdorf, H. Langmuir 1997, 13, 3515-3522. (h) Marchi-Artzner, V.; Artzner, F.; Karthaus, M.; Shimomura, K.; Ariga, K.; Kunitake, T.; Lehn, J.-M. Langmuir 1998, 14, 5164-5171. (i) Bohanon, T. M.; Caruso, P. L.; Denzigner, S.; Fink, R.; Mo¨bius, D.; Paulus, W.; Preece, J. A.; Ringsdorf, H.; Schollmeyer D. Langmuir 1999, 15, 174-184. (2) Nowick, J. S.; Cao, T.; Noronha, G. J. Am. Chem. Soc. 1994, 116, 3285-3289. (3) (a) Onda, M.; Yoshihara, K.; Koyano, H.; Ariga, K.; Kunitake, T. J. Am. Chem. Soc. 1996, 118, 8524-8530. (b) Ariga, K.; Kunitake, T. Acc. Chem. Res. 1998, 31, 371-378, and references therein. (c) Paleos, C. M.; Tsiourvas, D. Adv. Mater. 1997, 9, 695-710, and references therein. (4) Mammen, M.; Choi, S. K.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 2754-2794.

tion of multiple recognizable moieties attached at the external interface of liposomes. The interactions between liposomes may simulate cellcell and/or cell-liposome processes.5 Because of some resemblance of these aggregates to cell membranes, cellliposome interactions are encountered when liposomes are employed as drug delivery systems.6 The incorporation of amphiphilic 5,5 di-n-dodecylbarbituric acid (DBA) and its complementary molecule 9-hexadecyladenine (HA) in phosphatidyl choline (PC) liposomes may be of significant interest for inducing the interaction of liposomes. These model liposomal systems are primarily prepared for drug delivery applications. Within this context, our recognition investigations were carried out between complementary cationic liposomes,7 based on didodecyldimethylammonium bromide and bearing the same recognizable moieties as above. Analogous is the work by Lehn et al.,8 where PC-based liposomes incorporating amphiphilic-type derivatives of barbituric acid and triaminopyrimidine respectively were used. In both cases, the liposomal interaction gave rise to the formation of larger aggregates. Several other examples9 of molecular recognition studies of complementary liposomes have been reported over the (5) Paleos, C. M.; Sideratou, Z.; Tsiourvas, D. ChemBioChem. 2001, 2, 305-310. (6) (a) Gregoriadis, G. Trends in Biotech. 1995, 13, 527-537, and references therein. (b) New, R. R. C. In Liposomes, A Practical Approach; Rickwood, D., Hames, B.D., Eds.; IRL Press: Oxford, 1990. (7) Paleos, C. M.; Sideratou, Z.; Tsiourvas, D. J. Phys. Chem. 1996, 100, 13898-13900. (8) Marchi-Artzner, V.; Jullien, L.; Gulik-Krzywicki, T.; Lehn, J.-M. Chem. Commun. 1997, 117-118. (9) (a) Chiruvolou, S.; Walker, S.; Israelachvili, J.; Schmitt, F.-J.; Leckband, D.; Zasadzinski, J. A. Science 1994, 264, 1753-1756. (b) Constable, E. C.; Meijer, W.; Nardin, C.; Mundwiler, S. Chem. Commun. 1999, 1483-1484. (c) Paleos, C. M.; Tsiourvas, D.; Kardassi, D. Langmuir 1999, 15, 282-284. (d) Marchi-Artzner, V.; Jullien, L.; Belloni, L.; Raison, D.; Lacombe, L.; Lehn, J.-M. J. Phys. Chem. 1996, 100, 13844-13856.

10.1021/la011223l CCC: $22.00 © 2002 American Chemical Society Published on Web 01/10/2002

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Scheme 1. The interaction of the complementary moieties of DBA and HA at the external surfaces of liposomes.

Materials. Soybean hydrogenated phosphatidylcholine (Phospholipon 90H, Nattermann Phospholipid GMBH) was used. 5,5 di-n-dodecylbarbituric acid (DBA) was synthesized12 by the reaction of ethyl di(n-dodecyl) malonate with urea. 9-Hexadecyladenine (HA) was prepared13 by reacting adenine with 1-bromohexadecane. Liposome Preparation. Unilamellar liposomes were prepared by the extrusion method14 employing a laboratory extruder (LiposoFast-Pneumatic, Avestin Inc.).15 In a

typical experiment for preparing 4 mL dispersion of liposomes, 0.036 mmol (9 × 10-3 M) of PC, were mixed with 0.009 mmol (2.25 × 10-3 M) of 5,5 di-n-dodecylbarbituric acid (DBA) or 9-Hexadecyladenine (HA) at a molar ratio of PC: DBA(HA) ) 4:1. In addition, mixed liposomes were prepared by the addition of 0.018 mmol (4.5 × 10-3 M) of cholesterol (CHOL) to PC at a molar ratio of PC: CHOL ) 2:1. Liposomal dispersions were stable for more than 5 days, although for the characterization the dispersions were used in less than 24 h following their preparation. Recognition Experiments. The interaction between liposomes was studied by turbidimetry, microscopic techniques, and isothermal titration microcalorimetry employing liposomal dispersions as described in the respective experiments. Turbidimetric Studies. The aggregation of liposomes resulting from the recognition during the titration of the complementary liposomes was monitored by measuring 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:DBA liposomes (9 × 10-4 M PC, 2.25 × 10-4 M DBA) and PC: HA liposomes (9 × 10-4 M PC, 2.25 × 10-4 M HA) were mixed. Similar experiments were performed with the liposomes containing 50% molar content of cholesterol with respect to PC, i.e., 4.5 × 10-4 M CHOL. Microscopical Studies. AFM was employed in order to determine the size of the originally prepared liposomes and also the size of the aggregates resulting following their interaction. The images were obtained with a MultiMode Nanoscope III Microscope (Digital Instruments) employing the Tapping Mode operation. Samples were observed by placing droplets of liposome dispersions on freshly cleaved mica surface as previously described.7,9c

(10) Note: A thorough discussion of the subject in Faraday Discussions of the Chemical Society, 1986, 81, pages 77, 209, 340, 341. (11) So¨derlund, T.; Jutila, A.; Kinnunen, P. K. J. Biophys. J. 1999, 76, 896-907. (12) Tsiourvas, D.; Sideratou, Z.; Haralabakopoulos, A, A.; Pistolis, G.; Paleos, C. M. J. Phys. Chem. 1996, 100, 14087-14092. (13) Browne, D. T.; Eisinger, J.; Leonard, N. J. J. Am. Chem. Soc. 1968, 90, 7302-7323.

(14) (a) Bangham, A. D.; Standish, M. M.; Watkins, J. C. J. Mol. Biol. 1965, 13, 238-252. (b) Olson, F.; Hunt, C. A.; Szoka, F. C.; Vail, W. J.; Papahadjopoulos, D. Biochim. Biophys. Acta 1979, 557, 9-23. (c) Hope, M. J.; Bally, M. B.; Webb, G.; Cullis, P. R. Biochim. Biophys. Acta 1985, 812, 55-65. (15) MacDonald, R. C.; MacDonald, R. I.; Menco, B. Ph. M.; Takeshita, K.; Subbarao, N. K.; Hu, L. Biochim. Biophys. Acta 1991, 1061, 297303.

last 10 years, while the subject has recently been reviewed.5 To investigate interaction effectiveness of liposomes exclusively resulting from hydrogen bonding, unilamellar liposomes consisting of neutral lipids i.e., hydrogenated PC and cholesterol (CHOL) have been used in the present study. Since hydrogen bonding is rather weak, a relatively high molar content of DBA and HA had to be employed for quantitatively assessing liposome recognition properties and obtaining measurable thermodynamic parameters. At these high concentrations of recognizable molecules incorporated in the PC and the PC-cholesterol based liposomes, the bilayer order may be severely affected. In fact, the effects of additives on bilayer order and other properties of liposomes have been investigated since the mid-eighties10 and are still the focus of intense research.11 It is thus necessary that structural features, the dynamic heterogeneity and the stability of the initially prepared complementary liposomes, be characterized. The interaction of the complementary moieties of DBA and HA at the external surfaces of liposomes, shown in Scheme 1., was investigated in the gel as well as in the liquid crystalline phase of the membranes employing spectroscopic, microscopic, and calorimetric techniques. Experimental Section

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Liposomal aggregates resulting after association of the original liposomes were imaged by video enhanced phase contrast optical microscopy employing an Olympus BX50 microscope coupled with a Kodak Megaplus model 1.4i camera using an IC-PCI image board (Imaging Technology Inc.) and analyzed by SigmaScan Pro v4.0 image analysis software (SPSS Inc.). Thus, to a dispersion of liposomes prepared from PC:CHOL:DBA (9 × 10-4 M PC, 4.5 × 10-4 M CHOL and 2.25 × 10-4 M DBA) increasing quantities of PC:CHOL:HA liposomes (9 × 10-3 M PC, 4.5 × 10-3 M CHOL and 2.25 × 10-3 M HA) 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 microscope table for observation. The interaction was spontaneous (see also discussion in microcalorimetric studies) and the images remained unchanged throughout the microscopic observations. Differential Scanning Calorimetry. Differential scanning calorimetric experiments were performed with a VP-DSC calorimeter (Microcal Inc., Northampton, MA). The instrument’s active cell volume is 0.514 mL. Prior to scanning, all the liposomal suspensions were degassed for ∼ 15 min under vacuum. Heating and cooling scans were carried out from 20 to 70 °C at a rate of 20 K/h while the pressure was automatically kept at ∼ 30 psi. Isothermal Titration Calorimetry. Isothermal titration microcalorimetric experiments were carried out on a MCS-ITC calorimeter (Microcal Inc., Northampton, MA).16 A liposomal suspension (9 × 10-3 M PC and 2.25 × 10-3 M DBA) was placed in a 250 µL titration syringe while an analogous suspension (1.8 × 10-3 M PC and 4.5 × 10-4 M HA) was placed in a 1.334 mL reaction cell. The PC:DBA unilamellar liposomes were titrated in the PC: HA suspension via 23-injection sequences of 10 µL per injection. The injections were pre-programmed at 800second intervals and were performed automatically at 27 °C and 58 °C under 400 rpm stirring. The exothermic heat flow (dQ/dt) data were collected every one second for the first 200 s after each injection and every five seconds for the remaining time interval and were analyzed using the inbuilt Origin software. Identical experiments were carried out for PC:CHOL:DBA liposome dispersions (9 × 10-3 M PC, 4.5 × 10-3 M CHOL, 2.25 × 10-3 M DBA) titrated with PC:CHOL:HA (9 × 10-4 M PC, 4.5 × 10-4 M CHOL, 2.25 × 10-4 M HA) at 27 °C and 58 °C. The following control experiments were also performed under identical experimental conditions at the same temperatures, i.e., PC:DBA liposomes (9 × 10-3 M PC, 2.25 × 10-3 M DBA) in PC liposomes (1.125 × 10-3 M PC), PC:DBA liposomes (9 × 10-3 M PC, 2.25 × 10-3 M DBA) in water, PC:CHOL:DBA liposomes (9 × 10-3 M PC, 4.5 × 10-3 M CHOL, 2.25 × 10-3 M DBA) in PC:CHOL liposomes (9 × 10-4 M PC, 4.5 × 10-4 M CHOL), PC:CHOL:DBA liposomes (9 × 10-3 M PC, 4.5 × 10-3 M CHOL, 2.25 × 10-3 M DBA) in water. Calcein-Entrapped Liposomes and Interaction. The assessment of the stability of the recognizable liposomes during the interaction process was performed by entrapping calcein in their interior, allowing liposomes to interact and measuring the resulting fluorescence intensity. The method that was employed for the preparation of recognizable liposomes (see Experimental Section) was modified, however, at the stage of calcein entrapment. Thus, the lipid film consisting of PC, CHOL, and HA was hydrated with 4 mL of calcein-containing buffer solution, (16) Wiseman, T.; Williston, S.; Brandts, J. F.; Lin, L. N. Anal. Biochem. 1989, 179, 131-137.

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composed of 35 mM calcein, 15 mM NaCl and 0.5 mM Tris, and adjusted to pH 7.0 using NaOH solution (0.1 M). Non-encapsulated calcein was separated from unilamellar liposomes by gel filtration. Thus 0.5 mL of liposomal solution was diluted eight times with buffer solution without calcein, and subjected to gel-filtration through a Sephadex G-50 column (1.5 cm × 28 cm). A buffer solution was used as an eluent composed of 15 mM NaCl and 0.5 mM Tris adjusted to pH 7.0 with 0.1 M HCl. Brown-colored fractions were collected. The release of calcein during interaction experiments, resulting from possible destruction of the liposomes, was investigated by the addition of PC:CHOL:DBA liposomes to calcein-entrapped PH:CHOL:HA liposomes and was monitored by measuring the fluorescence intensity change at 520 nm with excitation at 490 nm. A Perkin-Elmer LS-5B Spectrophotometer was used. Results and Discussion Considering that the interaction of the recognizable moieties of DBA and HA at the lipid-water-lipid interfaces is exercised by enthalpically weak hydrogen bonding, to facilitate the thermodynamic studies, both recognizable moieties were incorporated in the bilayer at high molar ratios relative to PC (PC:DBA)4:1), (PC:HA)4:1). At these molar ratios the molecular order and the lateral heterogeneity of the PC bilayer is considerably affected17 and even the mechanism driving the main lipid phase transition is altered.18 The enhancing role of cholesterol in liposomal recognition was established in our previous work19 for the guanidinium/phosphate complementary pair. In those experiments cholesterol was incorporated at high concentrations relative to PC (PC:CHOL)2:1), while the additives were present in significantly smaller molar quantities (19:1) relative to PC. Thus a liquid-ordered phase was induced for the liposomal membrane. Under these conditions, a more effective recognition was achieved because of the enhanced lateral mobility of the recognizable molecules and the high degree of the alkyl-chain conformational order. However, as previously discussed for the PC:CHOL liposomes of the present study, the high molar content of the recognizable lipids can modify the established phase diagrams20 and thus the structural and dynamic properties21 of the bilayers leading to fluid membranes in a solid-ordered/liquid-ordered phase coexistence.17 To explore the phases of the bilayers vs temperature, for all the unilamellar liposomes employed in the present study, DSC experiments have been performed. Differential Scanning Calorimetric Studies. Differential scanning microcalorimetric experiments were carried out in the temperature region of the gel to liquidcrystalline phase transition of the unilamellar liposomes involved in the present study. The DSC thermograms are presented in Figure 1 and the thermodynamic parameters (17) (a) Mouritsen, O. G.; Jørgensen, K. Chem. Phys. Lipids 1994, 73, 3-25. (b) Mouritsen, O. G.; Jørgensen, K. BioEssays 1992, 14, 129136. (18) Jutila, A.; Kinnunen, P. K. J. J. Phys. Chem. 1997, 101, 76357640. (19) Sideratou, Z.; Tsiourvas, D.; Paleos, C. M.; Tsortos, A.; Nounesis, G. Langmuir 2000, 16, 9186-9191. (20) (a) Ipsen, J. H.; Karlstrom, G.; Mouritsen, O. G.; Wennerstroem, H.; Zuckermann, M. J. Biochim Biophys. Acta 1987, 905, 162-172. (b) Thewalt, L.; Bloom, M. Biophys. J. 1992, 63, 1176-1181. (c) Vist, M. R.; Davis, J. H. Biochemistry 1990, 29, 451-464. (d) Trandum, C.; Westh, P.; Jørgensen, K.; Mouritsen, O. G. Biophys. J. 2000, 78, 2486-2492. (21) (a) Nielsen, M.; Miao, L.; Ipsen, J. H.; Zuckermann, M. J.; Mouritsen, O. G. Phys. Rev. E 1999, 59, 5790-5803. (b) Virtanen, J. V.; Somerharju, P. J. Phys. Chem. B 1999, 103, 10289-10293. (c) Smondyrev, A. M.; Berkowitz, M. L. Biophys. J. 1999, 77, 2075-2089.

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Table 1. Thermodynamic Parameters for the Gel - Liquid-crystalline Phase Transition of the Various Unilamellar Liposomal Dispersions PC PC:HA (4:1) PC:DBA (4:1) PC:CHOL:HA (4:2:1) PC:CHOL:DB A (4:2:1)

Tma (°C)

∆Hb (kcal mol-1)

∆T1/2c (°C)

∆HvHd (kcal mol-1)

C.U.e (number of molecules)

52.1 ( 0.1 50.7 ( 0.1 51.3 ( 0.1 51.4 ( 0.3 52.6 ( 0.3

11.9 ( 0.7 7.1 ( 0.5 7.2 ( 0.5 2.3 ( 0.3 1.3 ( 0.3

2.8 ( 0.1 3.3 ( 0.1 5.1 ( 0.1 9.7 ( 0.4 9.4 ( 0.4

261 ( 29 217 ( 24 144 ( 16 74.8 ( 11 77.5 ( 11

22 31 20 33 60

a T ) transition temperature. b ∆H ) enthalpy change. c ∆T m 1/2 ) full-width-at-half-maximum of the Cp Peak. enthalpy change (see text). e C. U. ) size of Cooperative Unit (see text).

Figure 1. DSC thermograms for the gel - liquid-crystalline phase transition of the various unilamellar dispersions used in the study: a. PC, b. PC:HA (4:1), c. PC:DBA (4:1), d. PC:CHOL (4:1), e. PC:CHOL:HA (4:2:1), f. PC:CHOL:DBA (4:2:1), g. PC: CHOL (2:1). The weak ∆Cp anomalies at temperatures higher than the main ∆Cp peak are contributions from a small percentage of multilamellar liposomes in the dispersion. The data have been shifted vertically for clarity.

are summarized in Table 1. Besides the transition temperature Tm, and the total enthalpy change ∆H, the ratio of the van’t Hoff enthalpy change (∆HvH) to the total enthalpy is also listed in Table 1.22 This ratio is an estimate of the size of the cooperative unit (C. U.) that is driving the phase transition. It is, however, an approximate parameter since it is derived by an oversimplification of the kinetic process,23 yet indicative of the cooperativity in the bilayer. For PC liposomes, the values of Tm, ∆H, and C.U. are in agreement with previous findings for unilamellar liposomes.19 From the results shown in Table 1, it may be concluded that the incorporation of DBA and HA in the PC bilayers at 25% molar concentrations relative to PC, induces a decrease in both Tm and ∆H. It also causes a gradual broadening of the Cp peaks. This effect is indicative of the disruption of the translational order within the bilayer. On the other hand, there is little effect on the number of molecules (PC and additives) in the C.U. indicating that the distribution of DBA and HA molecules within the bilayer is quite uniform and does not substantially affect the lateral heterogeneity.17 For cholesterol concentrations higher than 25% relative to PC, it is well established that a liquid-ordered phase is formed with only short-range translational order but long-range, alkyl-chain conformational order.17 However, for the PC:CHOL unilamellar liposomes of the present study, the high molar concentration of DBA and HA enforces a considerable degree of lateral heterogeneity in the bilayer and the lipids are not in the liquid-ordered phase but instead in a solid-ordered/ liquid-ordered phase coexistence. As shown in Figure 1e and 1f, the main lipid phase transition is clearly detectable and reversible and it is characterized by small values of ∆H. The size of C.U. shows an abrupt increase in the case

d

∆HvH ) van’t Hoff

Figure 2. Turbidity change during recognition experiments between PC:DBA and PC:HA liposomes with cholesterol (50% relative to PC, filled circles) or without cholesterol (open circles). Horizontal axis represents the percent molar concentration of PC:HA liposomes in the aqueous dispersion.

of the PC:CHOL:DBA (4:2:1) liposomes indicating a moreordered fluid state of the bilayer. Turbidimetric Studies. Turbidimetry was employed for measuring interaction effectiveness as extensively applied in analogous studies.19,24 Turbidity increase during titration was attributed to the appearance of a greater number of large particles in the dispersion, formed because of the interaction of the liposomes. Turbidity change for the PC:DBA liposomes as a function of the added quantity of PC:HA liposomes without cholesterol and with 50% cholesterol relative to PC, is shown in Figure 2. It is deduced from this curve that the interaction of liposomes becomes most effective at 1:1 molar ratio of the recognizable molecules and also that the incorporation of cholesterol in the bilayer of liposomes enhanced the recognition effectiveness, a result which is in agreement with the previous studies.19 Microscopy Studies. The interaction between complementary liposomes was also indirectly observed by microscopic investigations. Thus with AFM microscopy (Tapping Mode), which has been employed for visualizing sensitive liposomal systems, it was possible to observe liposomes with diameters ranging between 50 and 140 nm as shown in Figure 3. Upon the addition of 10% PC: CHOL:HA liposomes to PC:CHOL:DBA liposome aggregates ranging in size between 300 and 450 nm were observed because of the association of the complementary (22) Mason, J. T. Methods Enzymol. 1998, 295, 468-494. (23) Genz, A.; Holzwarth, J. F.; Tsong, T. Y. Biophys. J. 1986, 50, 1043-1051. (24) (a) Stamatatos, L.; Leventis, R.; Zuckermann, M. J.; Silvius, J. R. Biochemistry 1988, 27, 3917-3925. (b) Eggens, I.; Fenderson, B.; Toyokuni, T.; Dean, B.; Stroud, M.; Hakomori, S. J. Biol. Chem. 1989, 264, 9476-9484. (c) Hasegawa, M.; Kaku, T.; Kuroda, M.; Ise, N.; Kitano, H. Biotechnol. Appl. Biochem. 1992, 15, 40-47. (d) Stewart, R, J.; Boggs, J. M. Biochemistry 1993, 32, 10666-10674.

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Figure 4. Phase contrast optical microscopy images of liposome aggregates after mixing samples of the complementary liposomes PC:CHOL:DBA (9 × 10-4 M PC, 4.5 × 10-4 M CHOL, 2.25 × 10-4 M DBA) with PC:CHOL:HA liposomes (9 × 10-3 M PC, 4.5 × 10-3 M CHOL, 2.25 × 10-3 M HA) at 1:1 molar ratio for the recognizable moieties. The bar in the lower right corner indicates 5 µm.

Figure 3. AFM images (taken with the Tapping Mode) of liposomes consisting of PC:CHOL:DBA [A], PC:CHOL:HA [B], and after mixing of 10% PC:CHOL:HA with PC:DHOL:DBA [C].

particles. By increasing the quantity of the added liposomes, the bigger aggregates obtained were observed even with optical microscopy. These particles sometimes encapsulate smaller aggregates as shown in Figure 4. Stability Studies. Association of liposomes because of the interaction of their complementary groups does not lead to their disruption. This is deduced from the fact that in the experiments with entrapped calcein, fluorescence intensity is slightly increased. It has been found that calcein, remaining entrapped in the interior of the liposomes at high concentrations, fluoresces only slightly because of self-quenching.25 However if liposomes were disrupted, calcein will be diluted in the bulk aqueous phase

resulting in enhanced fluorescence since quenching is reduced.25 Thus, the fusion of these liposomes is a nonleaky process, as also established for large phosphatidylserine liposomes.26 Calcein can be entrapped in either of the two complementary liposomes, since in both cases the same information is always obtained. In the present experiments, calcein was entrapped in PC:CHOL:HA liposomes. Following their interaction, slight fluorescence was observed; it is therefore deduced that liposomes were not disrupted. Isothermal Titration Calorimetric Studies. The interaction of liposomes that was observed by the microscopic and turbidimetric studies was further investigated by isothermal titration microcalorimetry (ITC)16 monitoring the binding of DBA and HA molecules at the lipidwater-lipid interfaces. ITC experiments have been widely used for studies of ligand/lipid-membrane interactions and have been successful for partitioning experiments of various molecules such as alcohols, drugs, and other biomolecules.27 The ITC studies of liposome-liposome interactions present several problems the most important of which are fusion and disruption of the membranes contributing to the measured enthalpy change, as well as (25) Komatsu, H.; Okada, S. Chem. Phys. Lipids 1997, 85, 67-74. (26) Cevc, G.; Richardsen, H. Adv. Drug Delivery Rev. 1999, 38, 207232.

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Figure 6. Detail of the isothermal titration experiments displayed in Figure 5, where PC:DBA (4:1)/PC:HA (4:1) raw data (solid line) and control (dots) PC:DBA (4:1)/PC are superimposed. The numbers at the bottom of the peaks indicate the injection numbers in the sequence of the 23-injection experiments. Figure 5. Raw data for the heat flow vs time from 23-injection, isothermal titration experiments at 27 °C for a: unilamellar PC:DBA (4:1) liposomal dispersion (9 × 10-3 M PC, 2.25 × 10-3 M DBA) injected in unilamellar PC:HA (4:1) dispersion (1.8 × 10-3M PC, 4.5 × 10-4 M HA), b: unilamellar PC:CHOL:DBA (4:2:1) liposomal dispersion (9 × 10-3 M PC, 4.5 × 10-3 M CHOL, 2.25 × 10-3 M DBA) injected in unilamellar PC:CHOL:HA (4: 2:1) dispersion (9 × 10-4 M PC, 4.5 × 10-4 M CHOL, 2.25 × 10-4 M HA), c: typical control experiment, for instance, PC:DBA (4:1) unilamellar liposomes injected in unilamellar PC dispersion.

the geometric constraints resulting from the sphericallike topology and the dynamic heterogeneity of the liposomal surfaces. These constraining factors, especially when coupled with translational order limiting lateral mobility in the bilayer, may disable a considerable number of molecules located at either interacting membrane from participating in the binding process. Nevertheless, ITC experiments may be beneficial especially when compared to suitable control experiments in order to obtain the magnitude of the heat released or absorbed during an isothermal liposomal interaction, the reaction rates involved and the qualitative differences of various binding mechanisms. ITC raw data (exothermic heat flow vs time) for the interaction of unilamellar PC:DBA liposomes (4:1) with unilamellar PC:HA (4:1) ones at 27 °C are presented in Figure 5a. It is evident that although the thermal signal is generally weak, two different components are readily identifiable for each injection peak: a sharp exothermic spike and a second exothermic component characterized by slower reaction rates. This is demonstrated clearly in Figure 6. Control experiments on the PC:DBA (4:1) added to a PC unilamellar dispersion (Figure 5c) as well as experiments on unilamellar PC:DBA liposomes added to water, indicate that the observed sharp spike is associated with the dilution of the concentrated PC:DBA suspension. The calculated area under the curve for this process is (27) (a) Milhaud, J.; Lancelin, J. M.; Michels, B.; Blume, A. Biochim. Biophys. Acta 1996, 1278, 223-232. (b) Lobo, B. A.; Davis, A.; Koe, G.; Smith, J.; Middaugh, C. R. Arch. Biochem. Biophys. 2001, 386, 95-105. (c) Heerklotz, H.; Lantzsch, G.; Binder, H.; Klose, G.; Blume, A. J. Phys. Chem. 1996, 100, 6764-6774. (d) Bai, G.; Wang, Y.; Wang, J.; Han, B.; Yan, H. Langmuir 2001, 17, 3522-3525. (e) Trandum, C.; Westh, P.; Jørgensen, K.; Mouritsen, O. G. Biochim. Biophys. Acta 1999, 1420, 179-188.

Figure 7. Isothermal titration plots ∆H vs DBA/HA molar ratio for [A]: the PC:DBA (4:1)/PC:HA (4:1) binding and [B]: the PC:CHOL:DBA (4:2:1)/PC:CHOL:HA (4:2:1) at 27 °C. The solid lines are the single-site, N ) 1, binding model [A] and a two-site model [B].

constant throughout the 23-injection titration sequence (∆H ) -120 ( 20 cal/mol DBA). On the contrary, upon subtracting the dilution contribution from the overall heat flow, the exothermic slow component associated with the liposomal binding exhibits the characteristic titration curve displayed in Figure 7A. We have attempted to analyze these data via a simple single-site binding model.15 The single-site model fits the data adequately (solid line in Figure 7A) and for a stoichiometry ratio N ) 1 (fixed parameter), ∆H ) -1.2 ( 0.2 kcal/mol DBA and the binding constant KB ) 850 ( 200 M-1 are derived. The obtained value for ∆H is expected for the formation of a DBA/HA hydrogen bond at 27 °C.28 When N is treated as a free fit parameter the following results are obtained: N ) 0.88 ( 0.22, ∆H ) -2.2 ( 0.8 kcal/mol DBA and KB ) 1500 ( 500 M-1. The two fits show no statistically (28) Ross, P. D.; Rekharsky, M. V. Biophys. J. 1996, 71, 2144-2154.

Phosphatidyl Choline Based Liposomes

significant difference. As stated earlier, even though the assumption that all the DBA and HA molecules in the liposomal suspension are available and accessible for binding is overly simplistic, the results indicate that a one-to-one binding between adenine and barbituric acid in the lipid-water-lipid interface is realistic. At temperature T ) 58 °C, above the main lipid phase transition, the situation is different and no liposomal binding is detected. This is mainly the result of the molecular disorder within the bilayer (liquid-disordered/liquid-ordered phase coexistence) that limits the capacity of DBA and HA to bind. It also results from the weakening of the hydrogen bond and the DBA-HA binding constants at these high temperatures. Isothermal titration calorimetric experiments have also been carried out for the system of unilamellar liposomes of PC:CHOL:DBA (4:2:1) interacting with PC:CHOL:HA (4:2:1) ones in order to study whether cholesterol affects liposomal recognition. The raw data at 27 °C are presented in Figure 5b and ∆H measurements in Figure 7B. Control experiments: PC:CHOL:DBA (4:2:1) liposomes added to PC:CHOL (2:1) liposomes and to water exhibit similar exothermic behavior as shown in Figure 5c. The enthalpy change per each injection is ∆H ) -75 ( 15 cal/mol DBA, constant throughout the 23-injection titration experiment. After subtracting the contribution determined by the control experiment, the ∆H values vs the DBA/HA molar ratio are plotted in Figure 7B. It is evident from these results that a single-site binding model is not adequate to describe the data, especially for the low values of the molar ratio DBA/HA. However, a two-site model (solid line in Figure 7B), as well as a two-site sequential binding model provide adequate but not the only method to fit the data. The data are nevertheless indicative that the singlesite, N ) 1, binding model will always fail to fit the data for the PC:CHOL system. In other words, the presence of

Langmuir, Vol. 18, No. 3, 2002 835

cholesterol directly affects the mechanism of binding of DBA and HA at the lipid-water-lipid interface. While calculating reaction rates from raw heat-flow ITC data (presented in Figure 5a and 5c) for the binding process and for each injection separately, by employing simple exponential fits another difference between the PC:DBA/ PC:HA and the PC:CHOL:DBA/PC:CHOL:HA unilamellar systems is noted. The reaction rates (k) are more or less constant in the case of PC:DBA/PC:HA throughout the 23-injection titration experiment (k ) 4.0 ( 0.3 ms-1). On the other hand, when cholesterol is present a clear trend in the reaction rates can be observed as a function of the injection number in the sequence. In the presence of cholesterol, the binding process is faster at low DBA/HA molar ratios (k ∼ 5 ms-1) and k monotonically decreases with each titration injection to k ∼ 2.3 ms-1. This may be another indication that cholesterol affects the DBA/HA liposomal interaction kinetics. Concluding Remarks Liposomal recognition mediated by the presence of hydrogen-bonded complementary moieties at the lipidwater interface provides evidence for binding between DBA and HA at temperatures below the main lipid transition of the membrane. This interaction leads to the formation of liposomal aggregates without any detectable disruption of the liposomes. In the presence of cholesterol, in the solid-ordered/liquid-ordered phase coexistence of the membrane, the binding mechanism is altered. This may result from the fluidity of the bilayer, attributed to the high concentration of cholesterol, which in turn contributes to a high lateral mobility of the incorporated molecules. Additional weak interactions of cholesterol with HA and DBA possibly occur. LA011223L