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Glycolipid and Monoclonal Immunoglobulin-Glycolipidic Liposomes Spread onto High Ionic Strength Buffers: Evidence for a True Monolayer Formation Agne`s P. Girard-Egrot,*,† Jean-Paul Chauvet,‡ Paul Boullanger,§ and Pierre R. Coulet† Laboratoire de Ge´ nie Enzymatique, UMR-CNRS 5013, Universite´ Claude Bernard Lyon 1, 43 Bvd du 11 novembre 1918, F-69622 Villeurbanne cedex, France, IFoS, UMR-CNRS 5621, Equipe Bioinge´ nierie des Interfaces et Reconnaissance Ge´ ne´ tique, Ecole Centrale de Lyon, 36 avenue Guy de Collongue, F-69131 Ecully cedex, France, and Laboratoire de Chimie Organique 2, UMR-CNRS 5622, Universite´ Claude Bernard Lyon 1, Ecole Supe´ rieure de Chimie Physique Electronique de Lyon, 43 Bvd du 11 novembre 1918, F-69622 Villeurbanne cedex, France Received July 14, 2000. In Final Form: November 13, 2000 The kinetics of the formation of an interfacial film obtained after spreading synthetic glycolipid and monoclonal immunoglobulin-glycolipidic liposomes onto high ionic strength buffered subphases were studied by measuring the evolution of the surface pressure with time, and the experimental data were analyzed theoretically according to a model previously proposed.1 The spreading kinetics of the multilamellar glycolipidic vesicles can be explained by the mechanistic scheme described for small unilamellar phospholipidic vesicles2-4 and based on a double control process (irreversible diffusion into the subphase and interfacial transformation of the closed vesicles in the surface film). In addition, the specific behavior of the glycolipidic liposomes, observed within the first minute after spreading, reveals that these liposomes exhibit surface-active properties related to their efficient interfacial disintegration. The theoretical analysis applied to the experimental data suggests that the multilamellar liposomes disintegrate at the interface into a true monomolecular film with a high rate constant of transformation. The insertion of a monoclonal immunoglobulin (IgG1) into these liposomes does not fundamentally modify their interfacial behavior and simply accelerates their disintegration. The formation of a true monolayer has been evidenced also in the presence of the immunoglobulin. The antibody insertion, characterized through transmission electron microscopy, leads to a structural modification of the liposomal membranes. This modification, weakening the membranous system, induces an additional retrodiffusion of the proteo-glycolipidic liposomes toward the interface and enhances their disintegration. The presence of the immunoglobulin in the glycolipidic film has been characterized by front face fluorescence emission spectroscopy with a mixed fluorescently labeled IgG-glycolipid film transferred onto a quartz plate. The occurrence of the interactions between the immunoglobulin and the glycolipid in the liposomal membrane prior to the monolayer formation appeared as a key step enabling the protein to stay included in the lipidic matrix during both monolayer formation and compression.
Introduction The spreading of phospholipidic vesicles at the air/water interface constitutes an alternative to produce interfacial films.3,4 We have recently proposed to use the spreading of liposomes associated with enzymatic proteins5 to form an active proteo-lipidic interfacial film as an artificial membrane model. The formation of an interfacial film based on liposome spreading, first observed by Verger and co-workers6,7 and * Corresponding author. E-mail:
[email protected]. † Laboratoire de Ge ´ nie Enzymatique, UMR-CNRS 5013, Universite´ Claude Bernard Lyon 1. ‡ IFoS, UMR-CNRS 5621, Equipe Bioinge ´ nierie des Interfaces et Reconnaissance Ge´ne´tique, Ecole Centrale de Lyon. § Laboratoire de Chimie Organique 2, UMR-CNRS 5622, Universite´ Claude Bernard Lyon 1, Ecole Supe´rieure de Chimie Physique Electronique de Lyon. (1) Panaı¨otov, I.; Ivanova, Tz.; Balashev, K.; Proust, J. Colloid Surf., A 1995, 102, 159. (2) Ivanova, T.; Georgiev, G.; Panaı¨otov, I.; Ivanova, M.; LaunoisSurpas, M. A.; Proust, J. E.; Puisieux, F. Prog. Colloid Polym. Sci. 1989, 79, 24. (3) Launois-Surpas, M. A.; Ivanova, Tz.; Panaı¨otov, I.; Proust, J. E.; Puisieux, F.; Georgiev, G. Colloid Polym. Sci. 1992, 270, 901. (4) Ivanova, Tz.; Raneva, V.; Panaı¨otov, I.; Verger, R. Colloid Polym. Sci. 1993, 271, 290. (5) Marron-Brignone, L.; More´lis, R. M.; Chauvet, J.-P.; Coulet, P. R. Langmuir 2000, 16, 498.
further analyzed on a theoretical basis by Schindler,8 has been explained by a simple kinetic mechanism1-3 wherein two independent processes occur, either an irreversible liposome diffusion into the subphase or a liposomal disintegration at the interface. With the aim of obtaining a glycolipidic film including an immunoglobulin, we have previously tested the possibility of forming an immunological proteo-lipidic film through the spreading at the air-water interface, of synthetic glycolipid liposomes including immunological proteins of an ascitic fluid. In this first study,9 the proteoglycolipidic vesicles appeared as efficient carriers to transport a large amount of proteinaceous material retained at the air/water interface in the lipidic film. Moreover, the presence of the immunological proteins slightly modified the kinetics of the spreading of glycolipidic vesicles and an additional retrodiffusion process of the proteo-glycolipidic liposomes from a subsurface layer toward the interface has been postulated to explain the kinetics patterns. However, in this previous study, the (6) Verger, R.; Pattus, F. Chem. Phys. Lipids 1976, 16, 285. (7) Pattus, F.; Desnuelle, P.; Verger, R. Biochim. Biophys. Acta 1978, 507, 62. (8) Schindler, H. Biochim. Biophys. Acta 1979, 555, 316. (9) Girard-Egrot, A. P.; More´lis, R. M.; Boullanger, P.; Coulet, P. R. Colloids Surf., B 2000, 18, 125.
10.1021/la001015d CCC: $20.00 © 2001 American Chemical Society Published on Web 01/25/2001
An Interfacial Film Based on Liposome Spreading
proteo-glycoliposomes were prepared using directly mouse ascitic fluid which constitutes a rich medium of monoclonal antibodies but which contains also many contaminants such as host proteins, lipids, and cell debris. Then, in the present study, we propose to investigate carefully the interfacial spreading behavior of both glycolipid and immunoglobulin-glycolipid liposomes prepared with purified monoclonal antibodies. To provide evidence for the retrodiffusion process of the proteo-glycolipid liposomes, very high ionic strength buffers were used as subphases. Moreover, the interfacial film obtained after transformation of closed liposomes at the air/liquid interface has been reported to have a more or less complex structure, either formed by partly destructed liposomes1,3 or assumed to be a true monomolecular film.6,7 In the theoretical analysis previously proposed by Panaı¨otov and Ivanova’s group,1 the molecular area deduced from a monolayer isotherm of the pure lipid monomer is used to analyze the kinetics of the surface coverage by the lipid molecules provided by the interfacial liposome disintegration. The application of this model to the experimental curves allows some information about the nature of the interfacial film formed by liposome disintegration. The kinetics of the interfacial film formation after the spreading of the glycolipid and IgG-glycolipid liposomes has been followed through the evolution of surface pressure with time. Then, the Panaı¨otov and Ivanova’s group theoretical analysis has been applied to the π-t isotherm curves using the monomolecular monolayer of the glycolipid monomer as reference. The elastic properties of the interfacial film obtained in the absence or in the presence of the immunoglobulin have been analyzed through π-A compression isotherms and monolayer compressibility (Cs). The presence of the immunoglobulin in the glycolipidic film has been evidenced after IgG labeling through fluorescence emission spectroscopy. Experimental Section 1. Materials. The synthesis10 and the ability11 to form vesicles in an aqueous medium of the glycolipid (3,6,9,12-tetraoxa-10undecyloxymethyl) tricosyl 2-acetamido-2-deoxy-β-D-glucopyranoside (1) have been previously described.
Ultrapure water (resistivity ) 18.2 MΩ‚cm) obtained from a milli-Q four-cartridge purification system (Millipore, France) was used to prepare chromatographic buffers, buffered-subphases, and the different solutions used. Glycine-NaOH (1.5 M), NaCl (3 M, pH 8.9) used as loading buffer, and sodium citrate (0.1 M, pH 5.0) used as elution buffer for affinity chromatography were filtered through a Minisart membrane of 0.20 µm (Sartorius, Germany) prior to use. All other reagents were of analytical grade. Quartz substrates, 35 mm × 9.5 mm × 1.25 mm, purchased from Thuet-Biechelin (France) were cleaned with an ionic detergent (Hellmanex II, Eurolabo, France), thoroughly rinsed with ultrapure water, and immediately dried and stored under a filtered dried-air flow. Just prior to use, they were ultimately soaked for a few minutes in chloroform (analytical-reagent grade, Rectapur, Prolabo, France). 2. Mouse Monoclonal IgG1 Purification. Mouse ascitic fluid generated from hybridoma producing monoclonal antibodies (10) Boullanger, P.; Sancho-Camborieux, M.-R.; Bouchu, M.-N.; Marron-Brignone, L.; More´lis, R. M.; Coulet, P. R. Chem. Phys. Lipids 1997, 90, 63. (11) Sancho, M.-R., Ph.D. Thesis, University of Lyon, Lyon, France, 1994.
Langmuir, Vol. 17, No. 4, 2001 1201 directed against acetylcholinesterase from Bungarus fasciatus venom was generously supplied by Dr. J. Grassi (SPI, CEA Saclay, Gif sur Yvette, France). Monoclonal antibodies were purified from ascites by protein A chromatography. A 1.5-mL portion of ascitic fluid was previously diluted 4 times with the adsorption buffer and filtered on 0.45 µm cellulose acetate membrane (Nalge`ne, France). The filtrate was loaded onto a 1 mL protein A-Hyper D F (BioSepra, France) column (1.6 cm × 0.8 cm) equilibrated in an adsorption buffer. Chromatography was carried out at room temperature with a flow rate of 15 cm/h (7.5 mL/min) obtained by gravity. After washes with 4 column volumes, stepwise elution was performed with 0.75 ()fraction 0) and 2 × 1.5 ()fractions 1 and 2) column volumes of elution buffer. Elution fractions 1 and 2 were neutralized at pH 7.0 by 275 µL of glycineNaOH buffer, 3 M NaCl, pH 8.9. Protein A column was regenerated with 4 volumes of 0.1 M NaOH, neutralized by 1 M phosphate buffer pH 7.0 and stored at 4 °C in 1 M NaCl-20% ethanol. The protein A gel was renewed after three purifications. The profile of protein elution was monitored by absorbance at 278 nm. The immunoglobulin was totally eluted in fraction 1; no absorbance was obtained for fractions 0 and 2. Immunoglobulin concentration was calculated using the specific extinction coefficient for IgG, E1% ) 13.5 at 278 nm12 and the mean concentration of fraction 1 after several runs was 4.5-5 mg/mL, corresponding to a 8 mg total amount of purified IgG. The actual A278/A251 ratio,12 as purity degree indicator of the fraction which contains IgG, was comprised in the range 2.2-2.4. The purity of fraction 1 was assessed by polyacrylamide gel electrophoresis under reducing and denaturing conditions, according to the method of Laemmli.13 SDS-PAGE was carried out in a mini-protean II apparatus (Bio-Rad, France) and gels were stained either with Coomassie brilliant blue R or silver nitrate. After protein A purification, only the two expected bands at ca. 50 and 25 kDa, corresponding to heavy and light chains of IgG, were obtained (data not shown). 3. FITC Labeling of Purified IgG. The purified antibody was labeled with fluorescein isothiocyanate (FITC) according to the FluoroTag FITC Conjugation Kit procedure (SIGMA, St Quentin Fallavier, France). Fraction 1 was previously dialyzed overnight at 4 °C against a 10 mM phosphate-buffered saline (PBS) solution at pH 7.4. The final fluorescein/protein (F/P) molar ratio, equal to 0.7, was determined by absorption spectroscopy using the ratio between the intrinsic protein adsorption at 280 nm and the fluorescein absorption at 495 nm. 4. Glycolipid and IgG1-Glycolipid Liposome Formation. The glycolipid and proteo-glycolipidic liposomes were prepared by mechanical dispersion above the main transition temperature (Tm < 8 °C) as previously described.8 To form the IgG1-glycolipidic liposomes, the dry glycolipid film was directly dispersed in 1 mL of IgG fraction 1 used without further dialysis since the 1.5 M glycine-NaOH buffer, 3 M NaCl, pH 8.9, favors hydrophobic interactions.14 The final glycolipid concentration and the lipidprotein ratio were 10 mg/mL and 2:1, respectively. To form mixed liposomes with the fluorescent-labeled antibody, the dry film was dispersed in 1 mL of the antibody solution with a percent molar fraction of fluorescent antibody (FITC-IgG) in the total protein amount equal to 5.1%. Then, all the liposomal suspensions were stored at 4 °C for 1 week without apparent modification of their spreading kinetics. 5. Liposome Characterization by Electron Microscopy. Electron micrographs were obtained from Bright Fields Transmission Electron Microscopy performed with a Phillips CM 120 microscope at the Electronic Microscopy Center of University Claude Bernard in Lyon (France). Negative staining was performed using the classical drop method. After vesicle adsorption onto carbon-coated grids, samples were stained with 2% phosphotungstic acid and observed at a magnification of 100 000. The glycolipid liposomes are multilamellar vesicles (MLV) with a mean external diameter of 150 nm and four to six closed lamellae closely apposed and readily distinguishable.9 (12) Tijssen, P. Laboratory techniques in biochemistry and molecular biology: practice and theory of enzyme immunoassays; Burdon, R. H., van Knippenberg, P. H., Eds.; Elsevier: Amsterdam, 1985; Vol. 15, p 117. (13) Laemmli, U. K. Nature 1970, 227, 680. (14) Egrot, C. BioSepra France, personal communication.
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Figure 1. Time-dependent increase of surface pressure after spreading of various volumes of the glycolipid liposomal suspension onto two different subphases, a 1.5 M phosphate buffer pH 7.4 (A and C) and a 1.5 M glycine-NaOH buffer, 3 M NaCl, pH 8.9 (B and D), at two constant trough areas, 382.5 cm2 (A and B) and 772.5 cm2 (C and D): a, 2 µL; b, 5 µL; c, 10 µL. Insets C and D: time-dependent variation of surface pressure within the first minutes after spreading under the same conditions as in Figure 1C,D. 6. Spreading Kinetics of Glycolipid and IgG1-Glycolipidic Liposomes. The measurement of interfacial parameters was performed as previously described9 with a computerized KSV 3000 Langmuir-Blodgett trough (KSV, Finland). Briefly, various volumes of liposomal suspensions were spread over a 1.5 M phosphate buffer, pH 7.4 (127 mS/cm),15 or a 1.5 M glycineNaOH buffer, 3 M NaCl, pH 8.9 (163 mS/cm),15 used as a subphase thermostated at 20 ( 0.5 °C for two constant trough areas, 382.5 or 772.5 cm2. At this temperature, above the main phase transition temperature of the glycolipid, the liposome membranes were in the liquid crystalline state. The kinetics of the surface film formation were followed by recording the variation of surface pressure (π) with time (t) for 35 min. Zero on the time scale corresponded to the beginning of the spreading procedure. 7. π-A Isotherm Diagrams of the Interfacial Films. The interfacial film formed after liposome spreading was symmetrically compressed at a rate of 7.5 cm2/min by two barriers moving at a speed of 5 mm/min. The interfacial parameters, collapse surface pressure (πc), and two-dimensional compressibility (Cs) of the monolayer, were determined from the isotherm. Cs was directly calculated from the slope of π-A isotherm by KSV software according to the following equation:
Cs ) -1/A(∂A/∂π)T It is expressed in m/mN and can be related to the elasticity (reciprocal of compressibility) of the interfacial film. 8. π-A Isotherm Diagrams of Glycolipid Monomer Monolayer. Glycolipid monomolecular films were formed by spreading 25 or 70 nmol of monomer in a chloroform-methanol solution (2:1, by volume). The same subphase with a high ionic strength buffer was used. After a 10 min lag time for solvent (15) Conductivity was used as indicator of the ionic strength.
evaporation, the monolayer was compressed in a continuous mode at a rate of 7.5 cm2/min. 9. Front-Face Fluorescence Emission Spectroscopy. The fluorescence experiments were performed on one LangmuirBlodgett (LB) monolayer transferred onto a quartz substrate with a SPEX Fluorolog 2 spectrofluorimeter. The excitation wavelength (λexc) was set at 490 nm. Stationary emission fluorescence spectra were recorded in front face mode within the range 505-605 nm and corrected from the background arising from the scattered light of a control monomer glycolipid monolayer transferred under the same conditions. The analysis directional angle θ equaled 22°.
Results and Discussion 1. Kinetics of the Interfacial Film Formation from the Spreading of Glycolipid Liposomes. The kinetics of film formation after the liposome spreading at the air/ liquid interface were followed by recording the variation of the surface pressure with time over 35 min at two constant trough areas (382.5 and 772.5 cm2). Figure 1 displays the surface pressure variation for various volumes of the liposomal suspension spread over a phosphate buffer, pH 7.4 (127 mS/cm), or a glycine-NaOH buffer containing NaCl, pH 8.9 (163 mS/cm), as a subphase. Depending on the spread volume and the trough area, the kinetics of the surface film formation were different. For the smallest area (382.5 cm2) and whatever the subphase was (Figure 1A,B), the spreading of a 5 or 10 µL volume (curves b and c, respectively) led to an instantaneous surface pressure increase which carried on with time until reaching a plateau. Spreading volumes larger than 10 µL leads to kinetic curves practically the same as that obtained with a volume of 10 µL indicating an interfacial
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Langmuir, Vol. 17, No. 4, 2001 1203
saturation at this level. For the spreading of a 2 µL volume (curve a), no evolution with time was observed; the surface pressure reached quasi-instantaneously an equilibrium with no more variation with time. This modification of the kinetics behavior depending on the spread volume was also observed for the largest trough area (Figure 1C,D). In this case, a real kinetic effect was only obtained for the 10 µL spread volume (curve c). For the two smallest volumes (curves a and b), a very low equilibrium π value was reached immediately after spreading. However, if the surface pressure variation was more precisely analyzed within the first minute for these two latter cases (insets in panels C and D of Figure 1), it appeared that the surface pressure increased instantaneously after spreading but rapidly decreased to reach the equilibrium value. It is noteworthy that this minute variation was reproducible and did not correspond to an artifact related to the spreading procedure. This variation also exists within the first minutes for the spreading of a 2 µL volume on the smallest trough area (curve a, Figure 1A,B). Moreover, for a same spread volume of 5 µL (curves b in the different figures), the spreading kinetics were dependent on the trough area. They shift from a continuous surface pressure variation for the smallest area (Figure 1A,B) to a small amplitude increase reaching almost instantaneously the π equilibrium value for the largest one (Figure 1C,D). Consequently, the kinetics pattern was straight related to the ratio between the trough area and the initial spread amount, that is to say the liposome surface density. Finally, even if the general spreading behavior of the glycolipid liposomes was the same on both subphases, i.e., glycine and phosphate buffers, it must be underlined that in the case where the interface was saturated, the π value reached within 35 min was higher with the NaCl-glycine buffer pH 8.9 than that obtained with the phosphate buffer pH 7.4 (curve c, Figure 1A,B). The surface pressure increase versus time, after spreading liposomal suspensions, has been intensively studied with different sizes of phospholipidic liposomes (small unilamellar liposomes (SUV),1,3,4,16 LUV,17 oligolamellar,2 and MLV17) and, in each instance, is attributed to an interfacial transformation process of liposomes into a surface film with a more or less complex structure. In the case of the multilamellar glycolipidic liposomes studied here, it is assumed that the surface pressure increase corresponds also to a disintegration of the liposomes into a glycolipidic superficial film, as previously shown with a lower ionic strength phosphate-buffered subphase.5,8 The higher the ionic strength, the greater is the opening of the liposomes. The kinetics mechanism of the interfacial film formation after liposome spreading at the air/liquid interface has been carefully investigated.1-4 It can be described by a simple scheme wherein two simultaneous competitive processes occur, corresponding to both (i) an irreversible diffusion process of closed liposomes into the subphase and (ii) an irreversible interfacial disintegration process of closed liposomes into an interfacial film. This surface layer is either formed by partly destructed liposomes which act as a source of lipid monomers1,3 or assumed to be a true monomolecular film.6,7 According to Launois-Surpas et al.,3 diffusion-controlled kinetics are obtained for the spreading of small amounts of liposomes and transformation-controlled kinetics for large ones. The interpretation of the kinetics patterns obtained with the glycolipid
liposomes (Figure 1) can be based on this mechanistic scheme. Regardless of the subphase used, when the surface pressure increases with time, the kinetics must be governed by the disintegration process (ii) and when the π equilibrium is reached quasi-instantaneously, the diffusion process (i) must be preponderant, limiting the disintegration of liposomes at the air/liquid interface. However, the predominance of one or both processes was not related in the present study to the initial spread amount but to the liposomal surface density, depending itself on both the spread amount and the trough area. Considering the first minute of surface film formation, it was noticeable that whatever the initial liposome surface density was, the surface pressure increased immediately after the spreading procedure. If the surface density was initially high, the liposome disintegration persisted and the phenomenon was principally controlled by the transformation process. The main mechanical contributions governing the opening process, mainly due to short-range forces,18 are reinforced by the high liposome surface density. On the contrary, if the surface density was initially low, the first surface pressure increase was of smaller amplitude and immediately followed by an unexpected drop before reaching the π equilibrium value. This specific behavior, not yet reported to our knowledge, can be explained as follows: as this effect is instantaneous, the presence of closed liposomes in the initial interfacial film cannot be excluded. However, the increase of the surface pressure implies a cohesive effect between the components of this initial film and consequently suggests the presence of glycolipid moleculessor partly disrupted liposomess provided by the disintegration of some liposomes, immediately after spreading. At this stage, the drop in surface pressure can be ascribed to an irreversible diffusion toward the subphase, of the major part of the liposomes which remained closed in this initial surface film. Therefore, this initial surface pressure variation suggests that some closed glycolipid liposomes can stay momentary intact at the air/liquid interface in a transient mixed surface film, before diffusing into the subphase. Moreover, it is noteworthy that this particular behavior was also observed with lower ionic strength buffered subphases and appeared systematically for a low surface density. 2. Theoretical Analysis. In the theoretical analysis previously proposed by Panaı¨otov and Ivanova’s group,1,4,19 the limiting case for the interfacial reorganizationcontrolled process is realized under interfacial saturation conditions; the liposomal concentration in the first subsurface layer formed by the closed liposomes spread and the surface transformation processes are not affected by the liposome diffusion into the bulk phase. Then, the irreversible transformation of closed liposomes into molecular surface structures can be described by a Langmuirlike adsorption kinetics equation, neglecting the desorption term. In this model, the rate of transformation of closed liposomes into open ones is proportional to both the number of closed liposomes able to be transformed in the first subsurface layer and the available free surface area.4 After integration of the adsorption kinetics equation, these authors obtained
(16) Vikholm, I.; Peltonen, J.; Teleman, O. Biochim. Biophys. Acta 1995, 1233, 111. (17) Obladen, M.; Popp, D.; Scho¨ll, C.; Schwarz, H.; Ja¨hnig, F. Biochim. Biophys. Acta 1983, 735, 215.
(18) Panaı¨otov, I.; Ivanova, Tz.; Sanfeld, A. Adv. Colloid Interface Sci. 1992, 40, 147. (19) Panaı¨otov, I.; Proust, J. E.; Raneva, V.; Ivanova, Tz. Thin Solid Films 1994, 244, 845.
(
ln 1 -
)
C0d n* t ) -k n∞* n∞*
(1)
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Figure 2. Plot ln(1 - θ) versus time obtained by applying eq 1 using the monomolecular isotherm as reference and the experimental data of line c of Figure 1A (a) or of Figure 3A (b), according to the theoretical model proposed by Panaı¨otov et al.1
where n* is the number of destructed liposomes adsorbed onto 1 cm2 area at time t and n∞* is the maximum number of these structures in a close-packed layer; n*/n∞* represents the degree of coverage of the surface at time t and 1 - n*/n∞* is the available free surface area at time t; k is the surface transformation rate constant of the process (in time-1) and d is the liposome diameter; C0 is the concentration of closed liposomes in the first subsurface layer corresponding to the initial liposomal concentration of the spreading solution under saturating conditions4 and C0d corresponds to the initial number per surface unit of closed spread liposomes present in the first subsurface layer and able to be transformed. If a complete transformation of all closed liposomes from the first subsurface layer into open structure is achieved, these authors have assumed, as a working hypothesis, that the limit value of the unknown surface density of the open structures, n∞*, equals C0d. Then, eq 1 becomes
(
ln 1 -
)
n* ) -kt C0d
(2)
If the interfacial transformation of liposomes leads to the formation of a true monomolecular layer, the time dependence of the degree of surface coverage θ ) n*/n∞*(t) can be deduced from the comparison between the experimental kinetic curves π(t) and a π-A isotherm diagram of pure glycolipid, where A ) 1/Γ (Γ is the surface concentration expressed in number of molecules per cm2) and θ ) Γ/Γ∞. By comparing the kinetics curves π(t) of Figure 1A (curve c, under interfacial saturation conditions) with a reference isotherm π(1/Γ) obtained on the same high ionic buffered subphase, we deduce Γ(t) and, finally, θ ) Γ/Γ∞ (with Γ∞ ) 1.6 × 1014 molecules/cm2 for the closepacked monolayer). As for the proposed model,1 this analysis was applied during the surface pressure increase, just before reaching the equilibrium π (i.e., within a period of 1 or 2 min for the glycolipidic liposomes). Figure 2 (curve a) shows the experimental data of ln(1 - θ) as a function of time for the glycolipidic liposomes spread on the high ionic strength phosphate-buffered subphase. The fact that the linear regression line nearly passed through the origin (at -0.064), with a correlation coefficient r ) 0.977, indicates that the experimental data are adequately supported by eq 1. The monomolecular monolayer is then a good reference to analyze the interfacial film formation kinetics from glycolipid liposome spreading. This result suggests that the glycolipid liposomes disintegrate at the interface into a true monomolecular film. The good
agreement with the theoretical model confirms that at high liposomal surface density (high spread volume, small trough area), the phenomenon is well controlled by the transformation process; then, we supposed that it becomes controlled by the diffusion process for the lowest liposomal surface density. The kinetic constant of the liposome interfacial transformation, k, has been deduced from the slope using the theoretical eq 1. Taking into account that the initial glycolipid concentration is 10 mg/mL and that the liposomes are made of four to six bilayers with a mean diameter of 150 nm, we calculated that C0d ) 1.47 × 108 liposomes/cm2. According to Ivanova et al.,4 the limiting value of n∞*, corresponding to closely packed lipid molecules in a strict monomolecular film, equals 1/(2πd2). For the glycolipid liposome disintegration, we obtained a transformation rate constant, k, equal to 6.8 × 10-2 s-1, which differs by 1 order of magnitude from the values reported for phospholipid small unilamellar liposomes (SUV) in liquid crystalline state (for example, k ) 1.5 × 10-3 s-1 for DMPC1 or DOPC9 and k ) 2.3 × 10-3 s-1 for DLPC1). At the air/liquid interface, the liposome disintegration for the glycolipidic MLV being complete and faster than that for the phospholipidic SUV, we obtained a true monolayer with a lower amount of liposomal suspension (the spread volume was 100 to 1000 times higher for phospholipids) and the equilibrium surface pressure was reached within a shorter period (5 min for glycolipids compared to 50-60 min for phospholipids). Consequently, the presence of a measurable surface pressure, soon after spreading of the liposomal suspension, is due to the rapidity of the disintegration process and gives the opportunity to identify the liposome diffusion through the initial drop of the surface pressure observed (within the first minute period) at low liposome surface density. The high efficient disintegration of the multilamellar structure into a true monolayer can be related to the intrinsic properties of the glycolipid molecule: a strong amphiphatic balance20 and a very low main transition phase temperature (above 8 °C). In the present work, the high efficient disintegration has also been reinforced by the high subphase ionic strength which limited the liposome diffusion in the bulk phase and favored their interfacial adsorption. However, it must be underlined that the same rapid and efficient interfacial disruption of the glycolipid vesicles has been obtained for lower ionic strength buffered subphases (equilibrium surface pressure reached in a few minutes) (data not shown). 3. Kinetics of the Interfacial Film Formation from Spreading of IgG1-Glycolipid Liposomes. Figure 3 shows the surface pressure variation after spreading various volumes of IgG-glycolipid liposomes over both subphases, i.e., glycine and phosphate buffers. Whatever the subphase was, for the largest trough area (Figure 3C,D), again the particular kinetics behavior of the glycolipid liposomes within the first minutes was obtained for both 2 and 5 µL spread volumes (curves a and b). However, in the presence of immunoglobulin, the surface pressure did not reach an equilibrium value and continued to increase with time. For the spreading of a 10 µL volume (curve c), the kinetics behavior at the beginning of the process was similar to that obtained with the glycolipid vesicles alone, but with the antibody, the surface pressure did not reach a plateau within 35 min and continued to slightly increase. This late modification of the spreading (20) Marron-Brignone, L.; More´lis, R. M.; Coulet, P. R. J. Colloid Interface Sci. 1997, 191, 349.
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Figure 3. Time-dependent increase of surface pressure after spreading of various volumes of the IgG-glycolipid liposomes onto two different subphases: a 1.5 M phosphate buffer pH 7.4 (A and C) and a 1.5 M glycine-NaOH buffer, 3 M NaCl, pH 8.9 (B and D), at two constant trough areas, 382.5 cm2 (A and B) and 772.5 cm2 (C and D): a, 2 µL; b, 5 µL; c, 10 µL.
kinetics in the presence of the protein was also observed with the small trough area (Figure 3A,B). In this case, whatever the spread volume was (curves a, b, and c), the surface pressure linearly increased after the initial jump, which reflected a continuous adsorption phenomenon of the liposomes at the interface. As for the glycolipid liposomes alone, the spreading kinetics of a 5 µL volume (curves b) was also dependent on the trough area; the kinetics was predominantly controlled by a diffusion process for the large one and became predominantly controlled by a transformation process for the small one. The maximal surface pressure value obtained with the proteo-glycolipidic liposomes within 35 min was markedly higher than the value obtained with the glycolipidic ones, except for the spreading of 5 and 10 µL volumes on the phosphate buffer subphase (curves b and c, Figure 3A) or for the spreading of a 10 µL volume on the glycine buffer subphase (curve c, Figure 3B). In these latter cases, the interfacial saturation was reached. The theoretical analysis previously described has been also applied for IgG-glycolipid liposomes spread under interfacial saturation conditions. The plot of ln(1 - θ) as a function of time using experimental data of π(t) kinetics of Figure 3A (curve c) and the glycolipid monomolecular isotherm as reference, is presented in Figure 2 (curve b). The presence of the immunoglobulin does not affect the surface coverage by the glycolipid. The linear regression line intercepts the ordinate near the origin, at -0.012, with r ) 0.981, suggesting that, theoretically, a true glycolipidic monolayer is formed even in the presence of the immunoglobulin. The transformation rate constant calculated from the slope taking into account the modification of the mean external diameter of the liposomes in the presence of the IgG (see below), was equal to 9.2 × 10-2 s-1.
The spreading kinetics of the IgG-glycolipidic vesicles can be interpreted as follows: the presence of the immunoglobulin does not fundamentally change the general spreading behavior of the glycolipid liposomes, at least at the beginning of the process; when the liposome surface density is high, the kinetics of the film formation is mainly governed by the transformation process and a true monomolecular film is formed; when the surface density is low, the diffusion process of the liposomes toward the subphase becomes preponderant and controls the kinetics. However, the quasi-linearly increase of the surface pressure obtained a few minutes after spreading can be ascribed to a small and continuous retrodiffusion of the proteo-glycolipidic liposomes from the subsurface layer to the interface. This retrodiffusion phenomenon has been already proposed to explain the kinetics patterns obtained after spreading of glycolipid liposomes including ascitic fluid containing immunological proteins onto a low ionic strength buffered subphase.9 The retrodiffusion effect is undoubtedly verified here with very high ionic strength buffered subphases. Moreover, the maximal surface pressure obtained within 35 min is higher for the proteoglycolipidic liposomes. This result indicates that the liposome disintegration not only was accelerated in the presence of the immunoglobulin (transformation rate constant k is higher) but also was enhanced. The mechanism of the film formation after liposome spreading is mainly related to short-range hydrophobic molecular interactions and consequently must be dependent on the state of the liposomal membranes.1 The structure of the membranous system of glycolipid vesicles in the presence of immunoglobulin has been characterized through transmission electron microscopy (Figure 4). Whereas the membranous system of the glycolipidic liposomes is welldefined with four to six closed and closely apposed lamellae
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Figure 4. TEM observation of glycolipid liposomes (A) or mixed IgG1-glycolipid liposomes (B). In micrograph A, the bright outside structure corresponds to a crystallization shell. In micrograph B, the bright filamentous background (arrows) corresponds to proteinaceous materials. The mean external diameter of the IgG1-glycolipid liposomes is ca. 100-110 nm (magnification of 100 000).
(micrograph 4A); the membranous lamellae stacking of the mixed IgG1-glycolipidic liposomes is difficult to observe (micrograph 4B). This modification of the membranous system can be attributed to the insertion of the protein molecules into the liposomal membranes, which weakens their structural organization. This destabilization of liposomal membranes is responsible for the retrodiffusion of the proteo-glycolipidic liposomes toward the interface where their disruption was enhanced. It is noteworthy that these two consecutive phenomena (retrodiffusion and interfacial disruption) are clearly observed only for very small spread amounts. For the highest liposome surface density, even if the presence of the immunoglobulin enhanced the retrodiffusion phenomenon, the disintegration of the liposomes is limited by the rapid saturation of the interface. This saturation was observed during the spreading procedure wherein it was visible to the naked eye that beyond a 10 µL spread volume, the proteo-glycolipidic liposomes could not disintegrate and sank into the subphase. 4. Isotherms of Glycolipidic and Proteo-Glycolipidic Interfacial Films. The interfacial films made from the interfacial disintegration of the glycolipidic or proteoglycolipidic liposomes were compressed 35 min after the spreading procedure. The isotherms obtained from a 5 µL volume spread onto both subphases with a large area trough are displayed in Figure 5. The isotherm of the glycolipid monomer monolayer formed by spreading of an organic solution has been added as a reference (dotted line b). For both subphases, the isotherm obtained after spreading of the glycolipid liposomes (a) are similar to the reference one (b); the one made from the liposomal suspension is only shifted toward the smallest areas, indicating that some liposomal materials has been lost in the subphase, since the same theoretical amount of 70 nmol of glycolipid molecules has been spread in both cases. The two-dimensional compressibility (Cs) has been calculated from the slope of isotherms a and b (insets in Figure 5A,B) and appears rigorously the same all over the surface pressure range. This result confirms that the interfacial film formed by the interfacial liposome disintegration behaves as a monomer monolayer, with the same elastic properties. Using different subphases or changing the initial liposome surface density leads to the same conclusions. The isotherms obtained from the proteo-glycolipidic liposome disintegration are noticeably different (curve c,
Figure 5. Isotherms of interfacial film formed over a 1.5 M phosphate buffer pH 7.4 (A) or a 1.5 M glycine-NaOH buffer, 3 M NaCl, pH 8.9 (B): after spreading of 5 µL of the glycolipid liposomal suspension (a); 70 µL of a glycolipid monomer organic solution (70 nmol) (b); 5 µL of IgG-glycolipid liposomal suspension (c). Insets in parts A and B correspond to the compressibility (Cs) against surface pressure of the respective isotherms (a) and (b).
Figure 5A,B). The presence of the immunoglobulin has an expanding effect upon the glycolipid monolayer, occurring all over the surface pressure range. Until the collapse, the proteo-glycolipidic isotherm was shifted toward larger areas, particularly on glycine buffer (Figure 5B). This behavior, observed for the different liposome surface densities used, suggests that a large part of the immunoglobulin remains at the interface even at high surface pressures. So, the interfacial films made from the spreading of the IgG-glycolipid liposomes should really correspond to a mixed proteo-lipidic layer. The two-dimensional compressibility curves for the proteo-glycolipidic films obtained with different volumes of liposomal suspension are presented in Figure 6. As a reference, the Cs curve of the glycolipidic interfacial film obtained after spreading of liposomal suspension has been added. The presence of the immunoglobulin in lipidic matrix was characterized by an increase in the compressibility Cs at a surface pressure of ca. 30 mN/m. Whatever the spread volume was, this Cs variation was always noticed, but its intensity depended on the initial liposome surface density. The magnitude of the Cs variation was decreased by increasing the spread volume. The increase in the compressibility observed systematically in the presence of the protein reflects a decrease in the monolayer elasticity. This elasticity modification can be related to an expulsion or a molecular reorientation of
An Interfacial Film Based on Liposome Spreading
Figure 6. Compressibility (Cs) against surface pressure of monolayers obtained after spreading of various volumes of IgGglycolipid liposomal suspension onto a 1.5 M phosphate buffer pH 7.4 (A) or a 1.5 M glycine-NaOH buffer, 3 M NaCl, pH 8.9 (B) with a large trough area of (772.5 cm2): a, 2 µL; b, 5 µL; c, 10 µL. Curve d corresponds to the compressibility curve obtained after spreading of 5 µL of glycolipid liposomal suspension (reference curve).
the protein during the compression. In the case of the IgG1-glycolipidic interfacial film, even if it cannot be excluded that some parts of the protein molecules are expelled from the air/liquid interface during the compression, the modification of the monolayer elasticity is more probably due to a reorientation of the immunoglobulin in the glycolipidic matrix, since the protein molecules are always present even at the highest surface pressures. Moreover, the very high ionic strength of the subphase is likely unfavorable for the expulsion of molecules which have surface active properties. The reorientation of the immunoglobulin in the glycolipidic interfacial film has been extensively studied under various ionic strength buffered subphase conditions and it will be the aim of further developments. 5. Presence of the Immunoglobulin in the Interfacial Film. To provide experimental evidence of the presence of the immunoglobulin in the interfacial film, the IgG-glycolipid interfacial film was transferred onto a germanium plate in order to apply attenuated total reflectance Fourier transform infrared spectroscopy. However, for the high ionic strength buffered subphase, the high buffer viscosity favored the deposition of buffer components and the absorption bands of both glycolipid and protein molecules were masked by the corresponding bands of subphase additives. This drawback was observed even for subphases with low ionic strengths, down to a concentration of 100 mM phosphate or 100 mM glycine buffer. Then, a mixed interfacial film has been formed with fluorescently labeled IgG (FITC-IgG) by spreading a FITC-IgG-glycolipidic liposomal suspension onto 0.1
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Figure 7. Front-face fluorescence emission spectra of the mixed FITC-labeled IgG-glycolipid monolayer formed over a 0.1 M phosphate buffer pH 7.4 (A) or a 0.1 M glycine-NaOH buffer, 0.2 M NaCl, pH 8.9, (B) and transferred onto quartz substrate. (Spreading volume ) 5 µL; area trough ) 772.5 cm2.) λexc ) 490 nm; θ ) 22°.
M phosphate buffer, pH 7.4, or 0.1 M glycine-NaOH buffer, 0.2 M NaCl, pH 8.9, as a subphase. After a 35 min lag-time, the film was compressed and transferred onto a quartz substrate. It must be mentioned that the labeling of the IgG did not modify the spreading behavior of the IgG-glycolipid liposomes; the same spreading kinetics and monolayer elastic properties were obtained. The transfer surface pressure of the interfacial film formed over lower ionic strength buffered subphases has been chosen to give a monolayer compression state analogous to that obtained at a surface pressure of 34 mN/m on the highest ionic strength buffered subphases. The front-face fluorescence emission spectra of one FITC-IgG-glycolipid layer is shown Figure 7. For both subphases, an emission fluorescence band was observed with a maximum at 524 nm corresponding to the maximum emission wavelength of FITC label. The fluorescence obtained for the transferred FITC-IgG-glycolipid attests to the presence of the immunoglobulin in the glycolipidic interfacial film. Then, the interactions which are created in the liposomal membrane between the glycolipid and the immunoglobulin appears to be strong enough both to allow the antibody to stay included in the glycolipidic matrix at the time where the film is formedsi.e., liposome disintegrationsand to keep the immunoglobulin inserted in the lipidic film during the compression. Conclusion This study focuses on the interfacial behavior of glycolipid liposomes spread onto high ionic strength
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buffered subphases. These vesicles appear to present, like the phospholipidic liposomes, a double control process (diffusion and transformation) of their spreading kinetics. However, the glycolipid vesicles exhibit a high efficient disintegration at the interface enabling the glycolipid multilamellar vesicles to be transformed into a true monomolecular interfacial film, as it has been shown applying the theoretical analysis previously described.1,4,19 This fast and complete interfacial transformation, due to the strong amphipathic balance of glycolipid molecules,20 allows both the formation of the monolayer with a very small amount of material and the rapid surface saturation. The extreme rapidity of such a process has given us the opportunity to follow the liposome diffusion at the beginning of the process. The insertion of an immunoglobulin into these liposomes does not fundamentally modify their general interfacial behavior at least at the beginning of the process, but it accelerates their interfacial transformation process. Applying the same theoretical analysis, the formation of a true glycolipidic monolayer has been evidenced even in the presence of the immunoglobulin. The immunoglobulin insertion into the liposomal membranes leads to a modification of the membranous system. This insertion, likely intramembranous, destabilizes the structural organization of the membrane of proteo-liposomes, which induces a retrodiffusion of the proteo-glycolipidic vesicles from the subsurface layer toward the interface and
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enhances their disintegration. This retrodiffusion phenomenon appears to be specific of the IgG-glycolipidic vesicles. Moreover, the interactions created between the glycoprotein and the glycolipid in the liposomal membranes before the monolayer film formation, allow the immunoglobulin to stay inserted in the lipidic matrix during the interfacial disintegration and the strength of these interactions prevents the immunoglobulin from being totally ejected during the compression. The presence of the immunoglobulin in the lipidic matrix has been evidenced even after the transfer of the mixed interfacial film. Then, the spreading of preformed IgG-glycolipid liposomes appears to be an efficient method for obtaining a true monomolecular interfacial film including protein, with stable proteo-lipidic interactions (compared to protein adsorption onto preformed monolayer). The immunoaffinity of such a film is now under investigation. Acknowledgment. Thanks are due to Dr. J. Grassi (CEA, Saclay, France) for his generous gift of the ascitic fluid, to Dr. C. Bon (Institut Pasteur, Paris, France) for his interest in this work, and to BioSepra (France) for their technical assistance in affinity chromatography. This work was partially supported by the CNRS (De´partements SDV et SPI/Interface BiologiesAction incitative Biocapteurs). LA001015D
