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Aggregation State of the Glycoprotein Excreted by Pseudoalteromonas antarctica NF3 on a Support of Phosphatidylcholine Liposomes A. de la Maza,*,† O. Lopez,† J. L. Parra,† M. Sabe´s,‡ and J. Guinea§ Departamento de Tensioactivos, Centro de Investigacio´ n y Desarrollo (CID), Consejo Superior de Investigaciones Cientı´ficas (CSIC), C/. Jorge Girona, 18-26, 08034 Barcelona, Spain, Unitat de Biofisica, Departament de Bioquı´mica i Biologia Molecular, Facultat de Medicina, Universitat Auto` noma de Barcelona, Edifici M, 08193 Bellaterra (Barcelona), Spain, and Departamento de Microbiologı´a, Facultad de Farmacia, Universidad de Barcelona. Av. Joan XXIII, s/n 08028 Barcelona, Spain Received April 29, 1998. In Final Form: July 13, 1998 The aggregation state of the microfilm produced by the glycoprotein (GP) excreted by a new Gramnegative species, Pseudoalteromonas antarctica NF3, on a support of phosphatidylcholine (PC) liposomes was examined by transmission electron microscopy (TEM). Image analysis profiles of digitized freezefracture TEM micrographs show that the outer covering film (PC:GP weight ratio 8:2) consisted in a multilayer structure formed by 9-10 layers. The periods of the average distance of the pattern ordering in layers were of about 2.3 nm, and the thickness of the complete film was of about 25 nm. The fact that the protective effect against sodium dodecyl sulfate of PC liposomes prepared in the presence of GP sharply increased with respect to that obtained when liposomes were only outwardly coated by this compound indicates that in the first case the GP multilayer structure also affected the inner monolayer of liposomes. Image analysis of freeze-fracture TEM showed that the coatings present in these two types of liposomes had the same structure despite that the number of external layers in the first case was almost the half than that in the second.
Introduction The use of liposomes as vehicles for drug delivery is limited because of their short survival time in blood. The effect of poly(ethylene glycol) in the fusion of phospholipid vesicles and in prolonging their circulation time in blood has been recently studied.1,2 Liposomes have been also used as membrane models to study the solubilizing effect of surfactants.3-6 Prokaryotic organisms have developed in the course of evolution a broad spectrum of cell envelope structures. Despite this diversity, two separate surface enveloping structures can be distinguished, the plasma membrane and the associated cell wall proper.7-9 In earlier papers we investigated the ability of an exopolymer of glycoproteic character excreted by a new Gram-negative species, Pseudoalteromonas antarctica NF3, to coat phosphatidylcholine liposomes and to protect * To whom correspondence should be addressed. Telph. (34-93) 400.61.61. Telefax (34-93) 204.59.04. † Consejo Superior de Investigaciones Cientı´ficas. ‡ Universitat Auto ` noma de Barcelona. § Universidad de Barcelona. (1) Yang Q.-L.; Guo, Y.; Li, L.; Hui, S. W. Biophys. J. 1997, 73, 277282. (2) Edwards, K.; Johnsson, M.; Karlsson, G.; Silvander, M. Biophys. J. 1997, 73, 258-266. (3) Paternostre, M.; Meyer, O.; G.-Madelmont, C.; Lesieur, S.; Ghanam, M.; Ollivon, M. Biophys. J. 1995, 69, 2476-2488. (4) Polozava, A. I.; Dubachev, G. E.; Simonova, T. N.; Barsukov, L. I. FEBS Lett. 1995, 358, 17-22. (5) Inoue, T. Vesicles; Rosoff, M., Ed.; Marcel Dekker, Inc.: New York, 1996; pp 151-195. (6) Cladera, J.; Rigaud, J. L.; Villaverde, J.; Dun˜ach, M. Eur. J. Biochem. 1997, 243, 798-804. (7) Beveridge, T. J. Bacteria in Nature, Structure, Physiology, and Genetic Adaptability; Poindexter, J. S., Leadbetter, E. R., Eds.; Plenum: New York, 1989; Vol. 3, pp 1-65. (8) Beveridge, T. J.; Graham, L. L. Microbiol. Rev. 1991, 55, 684705. (9) Sleytr, U. B.; Messner, P. Encyclopedia of Microbiology; Lederberg, J., Ed.; Academic Press: San Diego, CA, 1992; Vol. 1, pp 605-614.
these bilayers against the action of the anionic surfactant sodium dodecyl sulfate.10 In the present work we seek to extend these investigations by studying the aggregation state of the microfilm produced by this glycoprotein on a support of phosphatidylcholine (PC) liposomes by means of transmission electron microscopy. The elucidation of this fine structure may shed light on the ability of this compound to coat PC bilayers and to protect these vesicles against the surfactant action. Materials and Methods Phosphatidylcholine (PC) was purified from egg lecithin (Merck, Darmstadt, Germany) according to the method of Singleton11 and was shown to be pure by thin layer chromatography. Sodium dodecyl sulfate was purchased from Merck and further purified by a column chromatography.12 Piperazine1,4-bis(ethanesulfonic acid) (PIPES) buffer obtained from Merck was prepared as 20 mM PIPES adjusted to pH 7.20 with NaOH, containing 110 mM Na2SO4. The glycoprotein material of glycoproteic character (GP) produced by a new Gram negative species, Pseudoalteromonas antarctica NF3, was excreted by this microorganism into the culture medium and, consequently, did not form part of the bacterial cell wall. The original isolate was obtained from a sludge sample collected at the bottom of a glacier in the region of Inlet Admiralty Bay (King George Island, South Shetland Islands),13,14 and the purified glycoprotein is at present available at laboratory scale.15 Preparation of Liposome/GP Aggregates and Interaction with Sodium Decyl Sulfate (SDS). Pure PC liposomes (10) de la Maza, A.; Parra, J. L. Langmuir 1998, 14, 42-48. (11) Singleton, W. S.; Gray, M. S.; Brown, M. L.; White, J. L. J. Am. Oil Chem. Soc. 1965, 42, 53-57. (12) Rosen, M. J. J. Colloid Interface Sci. 1981, 79, 587-593. (13) Bozal, N.; Manresa, A.; Castellvi, J.; Guinea, J. J. Polar Biol. 1994, 14, 561-567. (14) Bozal, N.; Tudela, E.; Rosello-Mora, R.; Lalucat, L.; Guinea, J. Int. J. Syst. Bacteriol. 1997, 47, 345-351. (15) Bozal, N.; Guinea, J.; Tudela, E.; Congregado, F.; Parra, J. L.; de la Maza, A.; Mercade´, M. E.; Reque, M. Spanish patent 1996, 9.700.784.
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of a defined size (about 200 nm, PC concentrated 5.0 mM) were prepared by mechanical dispersion of PC in PIPES buffer and subsequent extrusion through 800-200 nm polycarbonate membranes at 25 °C using a thermobarrel extruder equipped with a thermoregulated cell compartment (Lipex Biomembranes, Inc., Vancouver, Canada). To prepare liposomes in the presence of GP, PC was mechanically dispersed in a GP aqueous dispersion in PIPES buffer (PC: GP weight ratio 8:2). The resulting coated liposomes were then extruded through 800-200 nm polycarbonate membranes at 25 °C. No GP was detected in these membranes after extrusion,13 indicating that this compound was not adsorbed onto the surface membranes. Hence, the extrusion process did not affect the lipid/ protein ratio of liposomes. To coat outwardly liposomes, PC vesicles were combined with the same volume of GP aqueous dispersions in PIPES buffer to obtain a PC/GP mixture for weight ratio 8:2. This mixture was incubated at 25 °C in a test tube rotator, type 3025 (GFL Burgwedel, Germany) with a rotation speed of 10 min-1 for 30 min. The liposome/GP aggregates resulting from these two processes were freed of the GP compound nonassembled with liposomes. To this end, the aggregates were sedimented for 30 min at 40000g at 10 °C and then resuspended in PIPES buffer. No PC was detected by a thin-layer chromatography/flame ionization detection system (TLC-FID, Iatroscan MK-5, Iatron Lab., Inc., Tokyo Japan)16 in any supernatant despite its opalescent aspect due to the presence of free GP compound. These suspensions were used to determine the structure of these aggregates by means of freezefracture transmission electron microscopy (TEM) as well as their solubilization by SDS. The solubilization of pure and coated PC vesicles was determined quantitatively by monitoring the static light-scattering (SLS) changes of the system during the process.10,17 Aliquots of liposome/GP aggregates (freed of the nonassembled glycoprotein) were mixed with appropriate SDS solutions, and the resulting mixtures were left to equilibrate for 24 h. SLS measurements were made using the spectrofluorophotometer Shimadzu RF-540 at 25 °C with both monochromators adjusted to 500 nm.18 The assays were carried out in triplicate, and the results given are the average of those obtained. Surface tension measurements of the investigated systems were performed using the ring method19 at 25 °C with a Kru¨ss (Hamburg, Germany) tensiometer (processor tensiometer K-2), which determines directly the real surface tension values at equilibrium. TEM Applied to Freeze Fractured Liposome/GP Aggregates and Image Analysis. Liposome/GP aggregates were mounted on gold stubs and quickly frozen by dipping them into nitrogen-cooled liquid propane. Freeze-fracturing, etching, and coating were carried out at -110 °C under a vacuum better than 5 × 10-7 mbar using a BAL-TEC instrument, type BAF-060 (Balzers AG, Balzers, Lichtenstein). The platinum/carbon-coated replicas were cleaned overnight in sodium hypochlorite. After being rinsed in distilled water, they were collected on Formvarcoated copper grids. The cleaned replicas were examined with a Hitachi H-600AB transmission electron microscope operating at 75 kV. To study by image analysis the ordered regions observed in freeze fractured TEM, the images were digitalized with a Epsom GT 800 scanner, using 400 dpi (400 pixels ) 2.54 cm in the micrographs). The one-dimensional optical density profiles corresponding to the intensity variations versus the average size of lamellar periods (nanometers) corresponding to selected TEM micrographs were processed.
Results and Discussion Freeze-Fractured TEM Images of Liposome/GP Aggregates. We previously reported freeze-fractured (16) Ackman, R. G.; McLeod, C. A., Banerjee, A. K. J. Planar Chromatogr.-Mod. TLC 1990, 3, 450-490. (17) Urbaneja, M. A.; Alonso, A.; Gonza´lez-Man˜as, J. M.; Gon˜i, F. M.; Partearroyo, M. A.; Tribout, M.; Paredes, S. Biochem. J. 1990, 270, 305-308. (18) de la Maza, A.; Parra, J. L. Langmuir 1996, 12, 6218-6223. (19) Lunkenheimer, K.; Wantke, D. Colloid Polym. Sci. 1981, 259, 354-366.
TEM images of PC liposomes outwardly coated by GP. The presence of increasing GP amounts resulted the spontaneous formation of a GP film that coated liposomes and protected these structures against the action of SDS. Furthermore, the thickness of this film increased as the GP proportion in bilayers increased.10 To elucidate how these structures coated and protected PC vesicles, the aggregation state of these structures was studied in detail by freeze-fracture TEM. Two images of outwardly coated vesicles (micrographs 1 and 2) and four for liposomes prepared in the presence of GP (micrographs 3-6) in both cases at the optimum PC:GP proportions (PC:GP weight ratio 8:2)10 are shown in Figure 1. A rupture in the enveloping structure of outwardly coated vesicles (micrograph 1, see arrows) shows the aforementioned GP film. A more enhanced magnification of this sample (micrograph 2) reveals that this covering film was formed by various thin GP layers that coated tightly the surface of the vesicles. Micrographs of samples 3 and 4 reveal that the covering structure of the liposomes prepared in the presence of GP was similar to that reported for the outwardly coated vesicles.10 More enhanced magnifications of these two samples (micrographs 5 [circle of micrograph 3] and 6) show that the covering film was also formed by GP layers that coated the vesicles (see arrows). In addition, no ordered symmetry was found in the coating material, in contrast with the conventional crystalline bacterial cell surface layers (S-layers).20,21 To study in detail the multilayer structure of this GP film, image analysis of the digitized TEM micrographs 2 and 5 were performed and representative optical density profiles of these two images are shown in Figure 2A and Figure 2B, respectively. Profile sections are indicated in each image. The profile of micrograph 2 showed that the GP multilayer structure had a thickness of about 25 nm, in agreement with the size reported for these aggregates measured by dynamic light-scattering.10 The periods of the average distance of the pattern ordering in layer structures (9-10 layers) were of about 2.3 nm. The profile of micrograph 5 showed that the periods of the multilayer structure were also of about 2.3 nm (5 layers). Comparison of these two profiles shows that the structure of the coatings for the two types of liposomes was the same despite that the number of external layers present in liposomes prepared in the presence of GP was almost the half than that present in the outwardly coated vesicles. It is noteworthy that this average distance is similar to the thickness of the outer monolayer of PC bilayers (2.1 nm).22 The similar architecture of these two structures may explain in part the ability of the GP layers to coat narrow PC vesicles and to form enveloping multilayer structures. In these two profiles 10 points corresponded to 3.80 pixels and to 3.85 nm, respectively (400 pixels ) 2.54 cm in the micrographs). Interaction of Liposome/GP Aggregates with SDS. Liposomes outwardly coated by GP and those prepared in the presence of GP (in both cases freed of the nonassembled glycoprotein) were treated with different SDS concentrations in order to compare their stability against this biological active surfactant. Figure 3 shows the solubilization curves of PC liposomes (b), liposomes outwardly coated with GP (O), and those prepared in the presence of GP (0). At low SDS concen(20) Sleytr, U. B. USA patents 4.849.109 and 4.886.604, 1989. (21) Ku¨pcu¨, S.; Sa´ra, M.; Sleytr, U. B. Biochim. Biophys. Acta 1995, 1235, 263-269. (22) Lasic, D. D. Liposomes: from Physics to Applications; Lasic, D. D., Ed.; Elsevier Science Publishers B.V.: Amsterdam, 1993; pp 554555.
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Figure 1. Freeze-fractured TEM micrographs of PC liposome coated by GP at the PC:GP weight ratio 8:2, and PC concentration 5.0 mM. Micrographs 1 and 2 corresponded to liposomes outwardly coated by GP and micrographs 3-6 to liposomes prepared in the presence of GP.
tration an initial rise in the SLS of these systems occurred, being more pronounced for liposomes prepared in the presence of GP. This rise that took place at SDS concentrations higher than its critical micelle concentration (cmc) (0.5 mM at the working medium23) may be attributed to the incorporation of surfactant molecules into liposomes up to bilayer saturation, in agreement with
our previous work involving the interaction of SDS with PC liposomes.23 Increasing SDS amounts led to a SLS fall up to a low constant value for complete liposome solubilization, regardless of the presence and characteristics of the covering structures. It is noteworthy that the (23) de la Maza, A.; Parra, J. L. Langmuir 1995, 11, 2435-2441.
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Figure 3. Percentage changes in static light-scattering of pure PC liposomes (b), liposomes outwardly coated with GP (O), and those prepared in the presence of GP (0) against the action of sodium dodecyl sulfate (PC:GP weight ratio 8:2). PC concentration in liposomes was 5.0 mM.
Figure 2. (A) Profile of the digitalized TEM image of micrograph 2 in Figure 1. (B) Profile of the digitalized TEM image of micrograph 5 in Figure 1. The analyzed profiles are indicated in each micrograph as continuous lines.
amount of SDS needed to produce the same solubilizing effect in both types of liposomes/GP aggregates was clearly different, despite the identical proportion of GP used in both cases (PC:GP weight ratio 8:2). Thus, almost a double surfactant concentration was needed in the first case to saturate and to completely solubilize liposomes, indicating that this type of covering structure was more resistant to the surfactant action. These findings suggest that the coating structure detected on the outwardly coated liposomes also affected the inner leaflet of vesicles prepared in the presence of GP. In fact, the hydration of PC in the presence of GP (by mechanical dispersion) may favor the formation of this inner GP film. This double protection may hamper the solubilization of PC molecules, despite that the initial surfactant-PC interaction occurred in the outer vesicle leaflet. The increased difficulty in the transfer of surfactant molecules from the outer to the inner vesicle leaflet via flip-flop (complexes surfactant-PC polar heads) may explain both the increased initial SLS in liposomes prepared in the presence of GP and the increased difficulty in the formation of surfactant-PC mixed micelles. This
Figure 4. Surface tension variations of pure PC liposome suspensions (b), liposomes outwardly coated with GP (O), and those prepared in the presence of GP (0) due to the action of sodium dodecyl sulfate (PC:GP weight ratio 8:2). PC concentration in liposomes was 5.0 mM.
difficulty may be responsible for the increased resistance of these liposomes against the action of SDS.24 To confirm that the results given in Figure 3 are not a consequence of the simple adsorption of SDS onto the GP, a series of surface tension measurements were performed during the interaction of SDS with the aggregates studied. The results obtained are plotted in Figure 4. Surface tensions decreased with increasing surfactant concentration showing in all cases inflection points at SDS concentrations higher than its cmc (0.5 mM).23 Thus, the inflection point for PC liposomes (b) was reached at ∼6 mM SDS, whereas those for outwardly coated liposomes (O) and liposomes prepared in the presence of GP (0) were reached at SDS concentrations of 7.1 and 8.3 mM, respectively. These points corresponded to the surfactant concentration at which SLS starts to decrease with respect (24) Lasic, D. D. Liposomes: from Physics to Applications; Lasic, D. D., Ed.; Elsevier Science Publishers B.V.: Amsterdam, 1993; Chapter 2.
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to the initial value (see arrow Figure 3). These concentrations may be considered the cmc’s of each system, in accordance with our previous work involving the SDS/PC liposomes binary systems.25 Increasing SDS amounts did not produce appreciable changes in surface tension values. This means that a progressive formation of complex surfactant/aggregate mixed micelles took place and, consequently, the solubilization of the initial aggregates. Hence, the curves for Figure 3 are the result of solubi(25) de la Maza, A.; Parra, J. L. J. Am. Oil Chem. Soc. 1993, 70, 699-706.
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lization of the initial aggregates by SDS and not the consequence of the simple adsorption of SDS onto the GP. Acknowledgment. The freeze-fracture TEM analysis was performed at Barcelona University. We thank Dr. Carmen Lopez for his skillful work at the microscope. We are also grateful to Mr. G. von Knorring for their expert technical assistance. This work was supported by funds from DGICYT (Direccio´n General de Investigacio´n Cientı´fica y Te´cnica) (Prog. PB94-0043), Spain. LA980499Z
