Permeability Enhancement of Lipid Vesicles to Nucleotides by Use of

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Permeability Enhancement of Lipid Vesicles to Nucleotides by Use of Sodium Cholate: Basic Studies and Application to an Enzyme-Catalyzed Reaction Occurring inside the Vesicles Mike Treyer, Peter Walde,* and Thomas Oberholzer Institut fu¨ r Polymere, Eidgeno¨ ssische Technische Hochschule Zentrum, Universita¨ tstrasse 6, CH-8092 Zu¨ rich, Switzerland Received July 17, 2001. In Final Form: October 18, 2001 The effect of the bile salt sodium cholate on the permeability properties of lipid vesicles (liposomes) to mononucleotides at pH 8.0 (0.1 M Tris-HCl) in the presence of 5 mM MgCl2 was investigated. The vesicles mainly used were unilamellar with diameters of about 100 nm, prepared from POPC (1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine). Depending on the total amount of cholate and POPC, the vesicles could be loaded with ADP (adenosine 5′-diphosphate), UMP (uridine 5′-monophosphate), UDP (uridine 5′-diphosphate), or UTP (uridine 5′-triphosphate), under conditions where bilayer solubilization did not occur, Re , Resat, with Re being the molar ratio of cholate in the vesicle bilayer to POPC under conditions of the uptake experiments (Re) or under conditions of bilayer saturation with cholate (Resat), as determined by light scattering (turbidity) measurements. The uptake experiments were preceded by a detailed study of the interaction between cholate and the POPC vesicles. Most importantly, freeze-fracture electron microscopy showed that vesicle solubilization begun at Re values lower than Resat estimated turbidimetrically. This finding indicates that care has to be taken if vesicle solubilization studies are based only on the measurements of changes in turbidity. Although a number of investigations have been devoted in the past to the cholate-induced release of water-soluble molecules from vesicles, the present work is probably the first detailed study on the reverse process. Nucleotide uptake by the vesicles at subsolubilizing cholate concentration was confirmed in the case of ADP by using vesicles containing entrapped Micrococcus luteus polynucleotide phosphorylase, which catalyzed the endovesicular polymerization of exovesicularly added ADP to poly-A. These latter experiments show that enzyme-containing vesicles can be used as nanoreactor systems in which the permeability of the bilayers is selectively altered by a surfactant, allowing the uptake of substrate molecules but not the release of the entrapped enzyme or reaction product.

Introduction Lipid vesicles (liposomes)smultilamellar or unilamellarsare polymolecular aggregates that are, under equilibrated, osmotically balanced conditions, spherical structures with diameters ranging from about 30 nm (small vesicles) to 100-200 nm (large vesicles) to dozens of micrometers (giant vesicles).1,2 In addition to their use in drug delivery3,4 or in nanoparticle synthesis,5,6 lipid vesicles are currently also considered as reasonable simple models for protocells in studies on the origins of life.7 Quite generally, in all those cases where lipid vesicles are investigated as nano- or micrometer-sized reaction compartments, the permeability properties of the lipid * To whom correspondence should be addressed: Tel +41-1-632 04 73; fax +41-1-632 10 73; e-mail [email protected]. (1) Lasic, D. D. Liposomes: from Physics to Applications; Elsevier: Amsterdam, 1993. (2) Luisi, P. L., Walde, P., Eds. Giant Vesicles; Perspectives in Supramolecular Chemistry, Vol. 6; John Wiley and Sons: Chichester, England, 2000. (3) Gregoriadis, G., Ed. Liposomes as Drug Carriers: Recent Trends and Progress; John Wiley and Sons: Chichester, England, 1988. (4) Lasic, D. D., Papahadjopoulos, D., Eds. Medical Applications of Liposomes; Elsevier: Amsterdam, 1998. (5) Heywood, B. R.; Fendler, J. H.; Mann, S. J. J. Colloid Interface Sci. 1990, 138, 295-298. (6) Kennedy, M. T.; Korgel, B. A.; Monbouquette, H. G.; Zasadzinski, J. Chem. Mater. 1998, 10, 2116-2119. (7) (a) Deamer, D. W. In The Molecular Origins of Life: Assembling Pieces of the Puzzle; Brack, A., Ed.; Cambridge University Press: New York, 1998; pp 189-205. (b) Monnard, P.-A.; Deamer, D. W. Origins Life Evol. Biosphere 2001, 31, 147-155. (c) Chakrabarti, A. C.; Breaker, R. R.; Joyce, G. F.; Deamer, D. W. J. Mol. Evol. 1994, 39, 555-559.

bilayer play a key role. Sometimes, it may be desired that the bilayer permeability is modified by the addition of bilayer-soluble molecules, hopefully leading to an increase in the permeability for small molecules, without significantly altering the permeability for large ones. One such case is enzyme-containing lipid vesicles,8-10 and an obvious idea to try to alter the bilayer permeability is to use micelleforming surfactants.11-13 Surfactants (detergents) show affinity for lipid bilayers without destroying them, as long as the surfactant concentration is not higher than the lowest concentration for bilayer solubilization (see below). In the following, after a short general introduction into the field of vesicle permeabilization by micelle-forming detergents, we report on the use of sodium cholate for the modification of the bilayer permeability to mononucleotides in the case of vesicles formed from POPC (1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). It will particularly be shown that in the presence of sublytic cholate concentrations nucleotides pass from the external (exovesicular) medium into the vesicle’s interior, the endovesicular space. This latter point was proven by use (8) (a) Walde, P. Curr. Opin. Colloid Interface Sci. 1996, 1, 638-644, and references therein. (b) Walde, P.; Ichikawa, S. Biomol. Eng. 2001, 18, 143-177. (9) Kaszuba, M.; Jones, M. N. Biochim. Biophys. Acta 1999, 1419, 221-228. (10) Blocher, M.; Walde, P.; Dunn, I. J. Biotechnol. Bioeng. 1999, 62, 36-43. (11) Annesini, M. C.; Bragulia, C. M.; Memoli, A.; Palermiti, L. G.; Di Sario, S. Biotechnol. Bioeng. 1997, 55, 261-266. (12) Annesini, M. C. Chem. Biochem. Eng. Q. 1998, 12, 1-17. (13) Oberholzer, T.; Meyer, E.; Amato, I.; Lustig, A.; Monnard, P.-A. Biochim. Biophys. Acta 1999, 1416, 57-68.

10.1021/la011111u CCC: $22.00 © 2002 American Chemical Society Published on Web 01/18/2002

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of vesicles containing the enzyme polynucleotide phosphorylase (PNPase). Vesicle Permeabilization by Detergents Basic Aspects of the Detergent-Induced Permeabilization of Phospholipid Vesicles. The addition of micelle-forming detergents to phospholipid vesicles leads to an increase in the permeability of the vesicle bilayer for low molecular weight, water-soluble substances if the concentration of the added detergent is below the concentration at which vesicle solubilization occurs.14-23 In a first approximation, the interaction of detergent molecules with phospholipid bilayers can be described by a simple three-stage model (stages I-III).16,19,23-30 In stage I, detergent molecules partition from the aqueous phase into the bilayer, leading often to a swelling and/or fusion14b of the vesicles (slight but significant increase in vesicle size).16 During this process, mixed phospholipid/detergent vesicles are formed. There is a maximal total detergent concentration at which the vesicles are saturated with surfactant molecules, abbreviated as DTsat. DTsat depends on the phospholipid concentration. If the detergent concentration is further increased, stage II is reached, in which detergent-saturated vesicles coexist with mixed phospholipid/detergent micelles. In stage III no vesicles exist anymore. This last stage starts at that minimal total detergent concentration at which complete vesicle solubilization is reached (DTsol), characterized by the presence of mixed phospholipid/detergent micelles which are in equilibrium with free, nonassociated detergent molecules. As proposed before,22,31 the relevant parameter for characterizing mixed vesicle-detergent systems is Re, the so-called effective (molar) ratio, defined as Re ) Db/L, where Db and L are the concentrations of bound detergent and (phospho)lipid, respectively. Furthermore, DT ) Db + Dw, where Dw is the concentration of the detergent in the aqueous medium. DTsat depends linearly on L: DTsat ) ResatL + Dwsat, where Resat is the effective ratio at (14) (a) Ueno, M. Biochemistry 1989, 28, 5631-5634. (b) Sun, C.; Ueno, M. Colloid Polym. Sci. 2000, 278, 855-863. (15) Walde, P.; Sunamoto, J.; O’Connor, C. J. Biochim. Biophys. Acta 1987, 905, 30-38. (16) (a) Paternostre, M.-T.; Roux, M.; Rigaud, J.-L. Biochemistry 1988, 27, 2668-2677. (b) Paternostre, M.; Meyer, O.; Gabrielle-Madelmont, C.; Lesieur, S.; Ghanam, M.; Ollivon, M. Biophys. J. 1995, 69, 24762488. (17) Ruiz, J.; Gon˜i, F. M.; Alonso, A. Biochim. Biophys. Acta 1988, 937, 127-134. (18) Nagawa, Y.; Regen, S. L. J. Am. Chem. Soc. 1992, 114, 16681672. (19) Edwards, K.; Almgren, M. Langmuir 1992, 8, 824-832. (20) de La Maza, A.; Parra, J. L.; Garcia, M. T.; Ribosa, I.; Sanchez Leal, J. J. Colloid Interface Sci. 1992, 148, 310-316. (21) Lasch, J. Biochim. Biophys. Acta 1995, 1241, 269-292. (22) (a) Inoue, T.; Yamahata, T.; Shimozawa, R. J. Colloid Interface Sci. 1992, 149, 345-358. (b) Inoue, T. In Vesicles; Surfactant Science Series, Vol. 62; Rosoff, M., Ed.; Marcel Dekker Inc.: New York, 1996; pp 151-195. (23) Albalak, A.; Zeidel, M. L.; Zucker, S. D.; Jackson, A. A.; Donovan, J. M. Biochemistry 1996, 35, 7936-7945. (24) Lichtenberg, D.; Robson, R. J.; Dennis, E. A. Biochim. Biophys. Acta 1983, 737, 285-304. (25) (a) Almog, S.; Kushnir, T.; Nir, S.; Lichtenberg, D. Biochemistry 1986, 25, 2597-2605. (b) Almog, S.; Litman, B. J.; Wimley, W.; Cohen, J.; Wachtel, E. J.; Barenholz, Y.; Ben-Shaul, A.; Lichtenberg, D. Biochemistry 1990, 29, 4582-4592. (26) Nomura, F.; Nagata, M.; Inaba, T.; Hiramatsu, H.; Hotani, H.; Takiguchi, K. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 2340-2345. (27) Levy, D.; Gulik, A.; Seigneuret, M.; Rigaud, J.-L. Biochemistry 1990, 29, 9480-9488. (28) Silvander, M.; Karlsson, G.; Edwards, K. J. Colloid Interface Sci. 1996, 179, 104-113. (29) Wenk, M. R.; Seelig, J. J. Phys. Chem. B 1997, 101, 5224-5231. (30) Kragh-Hansen, U.; le Maire, M.; Møller, J. V. Biophys. J. 1998, 75, 2932-2946. (31) Lichtenberg, D. Biochim. Biophys. Acta 1985, 821, 470-478.

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saturation.32 A linear dependency of DTsat on L has been verified in the case of several phospholipid-detergent systems.16,25a,27 Furthermore, for L . Db, Re ) (KDT)/(1 + LK), K being the equilibrium partition coefficient, defined as K ) Db/(LDw).33,34 Cholate-Induced Permeabilization of Phosphatidylcholine Vesicles. Detergent-induced permeabilization of phosphatidylcholine vesicles has been studied in the past for a number of detergents, in particular n-octyl β-D-glucopyranoside,14,16a n-dodecyl octaethylene glycol monoether (C12E8),14,19,35 Triton X-100,16a,18,36 Tween 80,36 and different bile salts.15,16a,22b,36 For the present work, we have selected sodium cholate, since (a) it is a wellknown “mild” biological detergent often used in membrane protein reconstitution experiments,37 (b) earlier studies have shown that the exovesicular addition of cholate increases the permeability of phospholipid bilayers to entrapped inorganic ions36,38,39 or fluorescent dye molecules,16a,40,41 and (c) using high cholate concentrations, phosphatidylcholine vesicles can be loaded with low as well as high molecular weight compounds with Mr up to 70,000.42a,13 In this last case, however, the vesicle loading with large molecules was most likely possible only due to a transient vesicle destabilization at high local cholate concentrations, followed by a vesicle re-formation process.43 In summary, despite a large number of cholate-induced permeability studies carried out in the past, mostly dyerelease experiments, nothing is known about the opposite process, namely, the effect of cholate on the permeation of molecules from the bulk medium into the vesicles interior under subsolubilizing cholate concentrations. The present paper is a contribution to this. Materials and Methods Chemicals. All chemicals were purchased and used without further purification. POPC (1-palmitoyl-2-oleoyl-sn-glycero-3(32) This linearity is a direct consequence of the model applied, which is largely a phenomenological description only valid for high lipid concentrations (>1 mM). See Roth, Y.; Opatowski, E.; Lichtenberg, D.; Kozlov, M. M. Langmuir 2000, 16, 2052-2061. (33) Schurtenberger, P.; Mazer, N.; Ka¨nzig, W. J. Phys. Chem. 1985, 89, 1042-1049. (34) If L . Db, it is more appropriate to define the partition coefficient as K* ) Db/[(Db + L)Dw], as pointed out by Lichtenberg in ref 31. Please note that the use of one single partition coefficient (K or K*) to describe surfactant partitioning over the entire surfactant concentration range is certainly a very rough approximation; it is expected that the partitioning behavior changes with increasing surfactant content within the lipid bilayer. (a) Lasch, J.; Schubert R. In Liposome Technology, 2nd ed.; Gregoriadis, G., Ed.; CRC Press: Boca Raton, FL, 1993; Vol. II, pp 233-260. (b) M. Ueno, unpublished results. (35) Ueno, M. Biochim. Biophys. Acta 1987, 904, 140-144. (36) Young, M.; Dinda, M.; Singer, M. Biochim. Biophys. Acta 1983, 735, 429-432. (37) Rigaud, J.-L.; Pitard, B.; Levy, D. Biochim. Biophys. Acta 1995, 1231, 223-246. (38) Bangham, J. A.; Lea, E. J. A. Biochim. Biophys. Acta 1978, 511, 388-396. (39) Hunt, G. R. A.; Jawaharlal, K. Biochim. Biophys. Acta 1980, 601, 678-684. (40) O’Connor, C. J.; Wallace, R. G.; Iwamoto, K.; Taguchi, T.; Sunamoto, J. Biochim. Biophys. Acta 1985, 817, 95-102. (41) de la Maza, A.; Parra, J. L. Colloids Surf. 1997, 127, 125-134. (42) (a) Schubert, R.; Wolburg, H.; Schmidt, K.-H.; Roth, H. J. Chem. Phys. Lipids 1991, 58, 121-129. (b) Schubert, R.; Beyer, K.; Wolburg, H.; Schmidt, K.-H. Biochemistry 1986, 25, 5263-5269. (43) Similarly, a cholate-induced release of entrapped dextran from phosphatidylcholine vesicles21,42b originated from high local cholate concentrations (inducing a transient partial micellization) during the release experiments.44 The influence of such kinetic effects in bile saltvesicle mixing experiments has also been discussed in another context.45 (44) Albalak, A.; Jackson, A. A.; Donovan, J. M. In Bile Acids in Gastroenterology: Basic and Clinical Advance; Hofmann, A. F., Paumgartner, G., Stiehl, A., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1997; pp 254-264. (45) Cohen, D. E.; Angelico, M.; Carey, M. C. J. Lipid Res. 1990, 31, 55-70.

Enhanced Lipid Vesicle Permeability to Nucleotides Table 1. Dynamic Light Scattering Data of POPC Vesiclesa [POPC] during extrusion extrusion (mM) method 20 20 100

Ab Ac Bb

scattering angle dependency of Rh (nm) 60° 90° 120°

P at 90°

57.0 ( 1.0 56.8 ( 0.2 55.6 ( 0.6 58.4 ( 0.4 57.3 ( 0.3 56.3 ( 0.2 48.6 ( 0.6 49.5 ( 0.2 49.5 ( 0.2

0.024 0.033 0.040

a Prepared by the extrusion technique, with either “The Extruder” (method A) or the LiposoFast basic device (method B). Final extrusions were performed through polycarbonate membranes with a nominal mean pore diameter of 100 nm. Rh represents the hydrodynamic radius; P is the polydispersity index. See Materials and Methods for details. b Analyzed 1 day after extrusion and storage at room temperature. c Analyzed 1 week after extrusion and storage at room temperature.

phosphocholine) was from Avanti Polar Lipids, Inc., Pelham, AL. Sodium cholate (g99%), ADP-K‚2H2O (adenosine 5′-diphosphate monopotassium salt dihydrate, g99%), and MgCl2‚6H2O (g99%) were from Fluka, Buchs, Switzerland. UDP-K2‚3H2O (uridine 5′-diphosphate dipotassium salt trihydrate, g98.5%) and proteinase K were from Merck, Darmstadt, Germany. Trizma base (g 99.9%), Trizma hydrochloride (g 99%), UMP-Na2 (uridine 5′-monophosphate disodium salt, g98%) and UTP-Na3 (uridine 5′-triphosphate trisodium salt, 90-95%) were from Sigma, Buchs, Switzerland. Polynucleotide phosphorylase (PNPase) from Micrococcus luteus was from The Midland Certified Reagent Co., Midland, TX. POPC Vesicle Preparation. All vesicles were prepared from POPC by the extrusion technique46 with either “The Extruder” (method A, volumes between 5 and 10 mL, [POPC] ) 20 mM) from Lipex Biomembranes, Vancouver, Canada,47 or the “Liposo Fast-Basic” device (method B, volumes between 0.25 and 0.5 mL, [POPC] ) 100 mM) from Avestin, Ottawa, Canada,48 with Nucleopore track-etch polycarbonate membranes from Corning Separations Division, Acton, MA (method A) or polycarbonate membranes from Avestin (method B). In both methods, 10 freezing/thawing cycles (liquid nitrogen/room temperature) preceded the extrusions. In the case of method A, multiple extrusions were first performed by using two stacked polycarbonate membranes with a mean nominal pore diameter of 400 nm (10 times), followed by 200 nm (10 times), and finally 100 nm (10 times). In the case of method B, the vesicle suspension was extruded 21 times directly through two 100 nm membranes. The hydrodynamic radius (Rh) of the vesicles prepared were determined by dynamic light scattering and the corresponding values are listed in Table 1. For the preparation of POPC vesicles containing PNPase, three freezing/thawing cycles and method B were used with final extrusions through membranes with 100 or 400 nm pores. The buffer (0.1 M Tris-HCl and 5 mM MgCl2, pH 8.0) contained 29 units of M. luteus PNPase/mL (1 unit of PNPase catalyzes the liberation of 1 µmol of inorganic phosphate from ADP in 15 min at 37 °C in a Tris buffer, pH 9, with no template/primer added). The POPC concentration was 100 mM. This extruded vesicle suspension was treated with 50 µg of proteinase K/mL in order to degrade externally present PNPase. Nonentrapped, degraded enzyme molecules were separated from the vesicles after 2.5 h of incubation at room temperature by gel-permeation chromatography (Sepharose 4B, column length 38 cm, column diameter 1.5 cm). The concentration of POPC was determined by the Stewart assay and a corresponding calibration.49 Turbidity, Dynamic Light Scattering, and FreezeFracture Electron Microscopy Measurements. Experimental details were given in a previous paper.50 (46) Mayer, L. D.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1986, 858, 161-168. (47) Hope, M. J.; Nayar, R.; Mayer, L. D.; Cullis, P. R. In Liposome Technology, 2nd ed.; Gregoriadis, G., Ed.; CRC Press: Boca Raton, FL, 1993; Vol. I, pp 123-139. (48) MacDonald, R. C.; MacDonald, R. I.; Menco, B. Ph. M.; Takeshita, K.; Subbarao, N. K.; Hu, L. Biochim. Biophys. Acta 1991, 1061, 297303.

Langmuir, Vol. 18, No. 4, 2002 1045 Addition of Cholate to Preformed POPC Vesicles. To POPC vesicles, which were first prepared at a concentration x mM in 0.1 M Tris-HCl and 5 mM MgCl2, pH 8.0, were added an equal volume of a sodium cholate stock solution at concentration y mM (in 0.1 M Tris-HCl and 5 mM MgCl2, pH 8) under rapid mixing, resulting in a mixture containing x/2 mM POPC and y/2 mM cholate. The turbidity of the mixture measured at 550 nm reached a constant value after equilibration for about 10 h; the reading was taken after a 15-17 h incubation time. Cholate-Induced Uptake of Nucleotides. Uptake experiments were performed in the presence of 0.1 M Tris-HCl and 5 mM MgCl2 buffer, pH 8.0, with two different final POPC concentrations: 5 mM in the experiments with ADP or 30 mM in the experiments with UMP, UDP, and UTP. If not otherwise indicated, in the case of ADP, 1 mL of ADP solution (40 mM) was first added to 1 mL of POPC vesicles (20 mM POPC). To this suspension was added 2 mL of a cholate stock solution (z mM) to obtain concentrations of 5 mM POPC, 10 mM ADP, and z/2 mM cholate. After an incubation for 15-17 h at room temperature, the taken-up nucleotides were analyzed in the following way (method I): 1 mL of the mixture was applied on a Sepharose 4B size-exclusion column (diameter 1.5 cm, length 38 cm), and 2 mL fractions were collected and analyzed at 260 nm (path length 1 cm). Sepharose 4B was from Amersham Pharmacia Biotech AB, Uppsala, Sweden. Vesicle-entrapped ADP was quantified spectrophotometrically, after the pooled vesicles were destroyed, in the presence of 40 mM cholate by using  (260 nm) ) 14 900 M-1 cm-1 (determined from a corresponding calibration curve). In all cases with UMP, UDP, and UTP, the final vesicle suspension contained 30 mM POPC, 20 mM nucleotide, and a desired amount of cholate. After incubation for 4.5 h at room temperature, the uptaken nucleotides were analyzed as follows (method II): 0.1 mL of the mixture was applied on a Bio-Gel A-15m size-exclusion spin column (1 mL plastic syringe).51 Bio-Gel A-15m (200-400 mesh size) was from Bio-Rad Laboratories Inc., Hercules, CA. A repetitive centrifugation at 1200 rpm for 2 min and 26 °C in a tabletop centrifuge, followed by addition of 50 µL of buffer solution, yielded 50 µL fractions in which the vesicles could be detected by eye due to the turbidity. The nucleotides were quantified spectrophotometrically as described above in the presence of 50 mM sodium cholate with a 60 µL quartz cell of 1 cm path length. Quantification of Poly-A. The formation of poly-A was quantified by HPLC52 with an anion-exchange column (Nucleogen DEAE 4000-7, 125/4 from Macherey-Nagel AG, Oensingen, Switzerland) and a Dionex low-pressure mixing HPLC system, connected to a photodiode array detector (detection at 260 nm). Two elution solvents A (4 M urea and 50 mM potassium phosphate, pH 7) and B (4 M urea, 50 mM potassium phosphate, and 1.5 M KCl, pH 7) were used, and a gradient from 10% B to 100% B in 30 min, followed by 5 min with 100% B, was applied.

Results and Discussion Characterization of the POPC Vesicles. All the vesicle suspensions prepared were rather monodisperse (low scattering angle dependency of Rh and low polydispersity index P, see Table 1), containing mainly unilamellar vesicles,46 as evidenced by freeze-fracture electron microscopy (data not shown). The vesicle suspensions were stable for at least 1 week (no significant change in size). While the radius of the POPC vesicles prepared with “The Extruder” was similar to what has been observed before in our group (Rh ≈ 57 nm),10,53 the vesicles prepared with the “LiposoFast-Basic” were a bit smaller (Rh ≈ 50 nm), although in both cases polycarbonate membranes (49) Stewart, J. C. M. Anal. Biochem. 1980, 104, 10-14. (50) Blo¨chliger, E.; Blocher, M.; Walde, P.; Luisi, P. L. J. Phys. Chem. B 1998, 102, 10383-10390. (51) Chonn, A.; Semple, S. C.; Cullis, P. R. Biochim. Biophys. Acta 1991, 1070, 215-222. (52) Walde, P.; Goto, A.; Monnard, P.-A.; Wessicken, M.; Lusi, P. L. J. Am. Chem. Soc. 1994, 116, 7541-7547. (53) (a) Dorovska-Taran, V.; Wick, R.; Walde, P. Anal. Biochem. 1996, 240, 37-47. (b) Lonchin, S.; Luisi, P. L.; Walde, P.; Robinson, B. H. J. Phys. Chem. B 1999, 103, 10910-10916.

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with the same nominal mean pore diameter were used for the final extrusions.54 Effect of Added Cholate on Preformed POPC Vesicles. The equilibrium partitioning of cholate between the aqueous phase and the POPC vesicles was determined by turbidimetry, similarly to what had been done before in the case of vesicles prepared from egg phosphatidylcholine (egg PC)14b,21,25a,42b,57 or from mixed egg PC/egg phosphatidic acid (9:1 molar ratio) vesicles,16a see the data for 5 and 15 mM POPC in Figure 1A. Each data point in Figure 1A represents a different sample. Following an earlier discussion,16a for each POPC concentration the cholate concentration at which the turbidity was highest was taken as an estimate of [cholate]Tsat (limit of stage I, see above). Plotting [cholate]Tsat vs [POPC] results in a linear relationship (see the experimental points, b, and the corresponding line 1 in Figure 1B). The slope of the straight line 1 in Figure 1B is the estimated Resat ()0.41 ( 0.05). The intercept point on the ordinate gives a rough estimation for [cholate]wsat (≈3.0 ( 0.8 mM). The true value of [cholate]wsat is probably lower, as recently discussed.32 Earlier it has been found in the case of similar phosphatidylcholine-cholate systems under different experimental conditions that Resat is between 0.316a,21,25a,41,43 and 0.34 ( 0.0457 and [cholate]wsat is between 2.18 mM37 and 2.8 mM.16a Our data for Resat and [cholate]wsat are therefore in good agreement with these earlier values. More recently it has been found that [cholate]wsat strongly depends on the salt concentration, ranging from ≈6.5 mM (in the presence of ≈10 mM NaCl) to ≈2 mM (in the presence of 0.3-0.5 M NaCl).57 For Resol and [cholate]wsol we obtained 0.64 ( 0.01 and 4.6 ( 0.2 mM, respectively, again in good agreement with literature data on similar vesicle-cholate systems.16a,25a,41,57 The corresponding [cholate]Tsol vs [POPC] data are given in Figure 1B (0, line 2). [Cholate]Tsol corresponds to the lowest cholate concentration at which the turbidity vanishes. Note that our own measurements indicate that [cholate]wsol * [cholate]wsat, a finding that has been discussed before58 and apparently originates from a deviation from linearity of the [detergent]T vs [lipid] plot at low [lipid].32 With the turbidimetrically determined Resat ) 0.41 ( 0.05 and [cholate]wsat ) 3.0 ( 0.8 mM, the equilibrium partition coefficient K (valid for [POPC] . [cholate]b, i.e., in stage I of the solubilization process) was calculated to be 0.14 ( 0.06 mM-1, in good agreement with literature values on similar PC-cholate systems (the reported values for K vary between 0.05 and 0.132 mM-1).16a,25a,34,59,60 The meaning of K ) 0.14 ( 0.06 mM-1 for stage I is shown in Figure 2, using the relation [cholate]w ) [cholate]T/(1 + K[POPC]), for both [POPC] ) 5 mM (line 1) and [POPC] ) 30 mM (line 2). To illustrate Figure 2, let us look at the case where the POPC concentration is (54) The reason for this difference is not completely clear. It is, however, known that the extrusion conditions, in particular the extrusion pressure,55 influence to a certain extent the resulting vesicle size. Furthermore, it is also known that the mean pore size of the membranes may vary considerably from lot to lot,56 which affects the size of the vesicles prepared. (55) Hunter, D. G.; Frisken, B. J. Biophys. J. 1998, 74, 2996-3002. (56) Information provided by Lipex Biomembranes Inc. (now Northern Lipids Inc.), Vancouver, British Columbia, Canada (http://www.northernlipids.com, accessed in October 2001). (57) Meyuhas, D.; Bor, A.; Pinchuk, I.; Kaplun, A.; Talmon, Y.; Kozlov, M. M.; Lichtenberg, D. J. Colloid Interface Sci. 1997, 188, 351-362. (58) Lichtenberg, D. In Handbook of Nonmedical Applications of Liposomes; Barenholz, Y., Lasic, D. D., Eds.; CRC Press: Boca Raton, FL, 1996; Vol. II, pp 199-218. (59) Malloy, R. C.; Binford, J. S., Jr. J. Phys. Chem. 1990, 94, 337345. (60) Ollila, F.; Slotte, J. P. Langmuir 2001, 17, 2835-2840.

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Figure 1. (A) Changes in turbidity of POPC vesicles as a function of total cholate concentration, [cholate]T, at different POPC concentrations at 25 °C. [POPC] ) 5.0 mM (b) and 15 mM (0); the buffer was 0.1 M Tris-HCl and 5 mM MgCl2, pH 8.0. The vesicles were prepared by method A (see Materials and Methods). Path length was 1 cm. (B) Relationship between [cholate]Tsat and [POPC], determined for [POPC] ) 3.75, 5.0, 9.7, 15, and 30 mM, respectively. (b) Experimental data for [cholate]Tsat and (0) experimental data for [cholate]Tsol were determined at different POPC concentrations. The vesicles were prepared by method A (except in the case of 30 mM POPC) or method B (30 mM POPC).

5 mM and [cholate]T ) 4 mM: [cholate]w is 2.4 mM (and Re ) 0.33 with K ) 0.14 mM-1). In other words, 40% of all cholate molecules are bound to the vesicle bilayer, and the molar ratio of POPC to cholate in the bilayer is about 3:1, which represents a substantial modification of the POPC bilayer. To test whether under these conditions the vesicles are indeed still intact bilayered entities, a series of freeze-fracture electron micrographs were taken; see Figure 3. From these micrographs it is clear that a transformation of mixed vesicles into mixed micelles at 5 mM POPC occurs at 4.0 mM < [cholate]T < 5.0 mM. From a statistical analysis of the electron micrographs it can be seen that the smallest vesicles tend to disappear first (data not shown), forming mixed micelles or/and possibly larger vesicles through a fusion process.14b In comparison with the turbidity measurements, from which [cholate]Tsat ) 5.1 mM, [cholate]wsat ) 3.0 mM, and

Enhanced Lipid Vesicle Permeability to Nucleotides

Langmuir, Vol. 18, No. 4, 2002 1047 Table 2. Dynamic Light Scattering Data of Equilibrated Mixed POPC/Cholate Vesicle Systems at [POPC] ) 5 mM as a Function of [Cholate]Ta [cholate]T (mM) 0 3 4 5 5.5 6 7

Figure 2. Estimation of [cholate]w as a function of the total cholate concentration ([cholate]T) for 5 mM POPC (line 1) and 30 mM POPC (line 2), with K ) 0.14 ( 0.05 mM-1. The solid lines represent the curve calculated for the mean value of K, and the dotted lines are the calculated curves for the standard deviations.

Figure 3. Freeze-fracture electron micrographs of the equilibrium structures of different POPC vesicle/cholate mixtures. [POPC] ) 5 mM; [cholate]T ) 3 mM (A), 4 mM (B), 5 mM (C), 5.5 mM (D), 6 mM (E), or 7 mM (F). The buffer was 0.1 M Tris-HCl and 5 mM MgCl2, pH 8.0. The vesicles were prepared by method A (see Materials and Methods). Length of the bar represents 200 nm. Apart from vesicles, vesicle fragments (circle), micelles (arrow), and aggregated structures (rectangle) could be detected.

K ) 0.14 mM-1, the electron microscopy data indicate that the first vesicle solubilization at [POPC] ) 5 mM starts effectively below [cholate]T ) 5 mM. The reason for this discrepancy between simple turbidity and electron

scattering angle dependency of Rh (nm) 60° 90° 120° 48.6 ( 0.6 49.5 ( 0.4 48.4 ( 0.4 48.2 ( 0.3 57.8 ( 1.0 78.3 ( 0.6 106.1 ( 5.2

49.5 ( 0.2 49.3 ( 0.4 48.9 ( 0.6 48.0 ( 0.8 58.6 ( 0.5 80.0 ( 0.8 89.4 ( 0.9

49.5 ( 0.5 49.0 ( 0.7 48.2 ( 0.5 48.4 ( 0.9 59.3 ( 0.3 80.0 ( 1.3 87.6 ( 5.4

P at 90° 0.040 0.041 0.033 0.041 0.019 0.037 0.110

a The vesicles were prepared by method B with equal volumes of the vesicle suspension (10 mM POPC), mixed with an appropriate cholate solution and analyzed without dilution with an appropriate transmission filter. The buffer was 0.1 M Tris-HCl and 5 mM MgCl2, pH 8.0. Rh represents the hydrodynamic radius; P is the polydispersity index.

microscopy data lies in the fact that turbidity measurements are not sensitive enough for detecting the disappearance of a small amount of very small vesicles. The same insensitivity was found by using dynamic light scattering measurements (Table 2). A clear change in Rh and polydispersity index only occurs above 5 mM cholate. Similarly to the present work, it has been shown, for example, in the case of egg PC and sodium dodecyl sulfate that electron microscopy is an important complementary tool to light scattering for elucidating the mechanism of vesicle solubilization.61 This section can be summarized by claiming that those experimental conditions were established under which the interaction of cholate molecules with unilamellar POPC vesicles led to the formation of mixed POPC/cholate vesicles without formation of mixed POPC/cholate micelles; in the case of 5 mM POPC, [cholate]T has to be at 4 mM or below to be on the safe side. In the case of 30 mM POPC, the electron microscopy analysis showed that [cholate]T has to be 0.27), the uptake yields were lower (see Table 4). In the case of UTP (20 mM), the importance of the order of cholate and nucleotide addition was also investigated. It was found that similar uptake yields were obtained, independently of whether the POPC vesicles were first preincubated with cholate followed by UTP addition or whether UTP and cholate were added simultaneously to the POPC vesicles ([POPC] ) 30 mM, [cholate]T ) 10 mM, data not shown).

[cholate]T (mM)

Re

uptakeb (%) [nucleotide]insidec (mM) UMP UDP UTP UMP UDP UTP

6.0 7.0 8.0 9.0 10.0 11.0 12.0 14.0

0.16 0.19 0.22 0.24 0.27 0.30 0.32 0.38

nde nd 2.3 4.1 6.0 4.0 1.9 nd

d

1.3; 1.6f nd 2.0 3.2 6.1; 7.5f 5.2 4.0 1.1; 1.4f

nd 3.3 5.2 5.4 6.6 5.0 1.0 nd

nd nd 3.5 6.2 9.1 6.1 2.9 nd

2.0; 2.4f nd 3.0 4.8 9.3; 11.4f 7.9 6.1 1.7; 2.3f

nd 5.0 7.9 8.2 10.0 7.6 1.5 nd

a [POPC] ) 30 mM; [nucleotide] ) 20 mM; the buffer was 0.1 M Tris-HCl and 5 mM MgCl2, pH 8.0. The analysis was performed after an incubation for 4.5 h at room temperature. The vesicles were prepared by method B, and the nucleotide uptake was quantified by method 2 (see Materials and Methods). b Experimentally determined; values are with respect to the total amount of nucleotide applied (20 mM). c Calculated by assuming that all nucleotide molecules taken up are in the aqueous interior of the vesicles and that all vesicles are unilamellar (with an external radius ≈ Rh ) 68 nm (as determined for this set of experiments), with a POPC mean headgroup area70,71 of 0.72 nm2 and a bilayer thickness72,73 of 3.7 nm, yielding an internal vesicle volume of 4.39 µL/µmol of POPC). The concentration of the nucleotides outside the vesicles after uptake was calculated to be between ≈22 and 23 mM. d Mean effective ratio, calculated with K ) 0.14 mM-1. e Not determined. f Incubation time 20 h.

Figure 6. Endovesicular poly-A formation catalyzed by vesicletrapped PNPase. [POPC] ) 5 mM; [cholate]T ) 4 mM (9) or 0 mM (O); the buffer was 0.1 M Tris-HCl and 5 mM MgCl2, pH 8.0. The vesicles were prepared by method B, with final extrusions through membranes with 100 nm (A) or 400 nm (B) pores. The initial exovesicularly added ADP concentration was 10 mM.

Enzyme-Catalyzed ADP Polymerization inside the Vesicles. The enzyme polynucleotide phosphorylase (PNPase) is known to catalyze the synthesis of polynucleotides from the corresponding nucleoside diphosphates; i.e., poly-A is formed from ADP.52,62 In a series of experiments, POPC vesicles were prepared containing entrapped M. luteus PNPase, and ADP (10 mM) was added

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Langmuir, Vol. 18, No. 4, 2002

Figure 7. Schematic representation of the ADP-uptake assay with vesicles containing the enzyme PNPase, which catalyzes the polymerization of ADP.

to these vesicles ([POPC] ) 5 mM) at [cholate]T ) 4 mM (Re ) 0.33). As evidenced by ion-exchange HPLC (Figure 6), endovesicular poly-A formation occurred only in the presence of 4 mM cholate, while in the absence of cholate but under otherwise identical conditions, no poly-A was formed.63 These experiments (i) confirm the nucleotide uptake studies described above and (ii) demonstrate that the enzyme-containing vesicles can be seen as a type of nanometer-sized vesicular reactor, e.g., as a protocell model,7 in which the substrate permeability is increased by the presence of cholate at a subsolubilizing concentration. A schematic drawing of this nanoreactor is shown in Figure 7. Concluding Remarks On the basis of earlier studies by Schubert et al.,42 the present investigation was focused on the uptake of nucleotides by phospholipid vesicles at low, subsolubilizing cholate concentrations in equilibrated systems. The results obtained in the present work indicate that cholate molecules permeabilize POPC vesicles to nucleotides at cholate concentrations that did not lead to vesicle solubilization. Maximal nucleotide uptake is achieved at cholate concentrations where the vesicle membrane approaches saturation with cholate molecules. Below a certain Re value (Re < 0.2) the effect of cholate on the (62) Littauer, U. Z.; Soreq, H. In The Enzymes; Boyer, P. D., Ed.; Academic Press: New York, 1982; Vol. XV, pp 517-553. (63) A possible leakage of entrapped enzyme molecules after cholate addition is unlikely since, under identical conditions and in the case of the relatively small, monomeric proteinase K (Mr ∼ 30 000), no enzyme molecules were found to cross the bilayer membrane. It can, however, not be completely excluded that some of the poly A formed exovesicularly.

Treyer et al.

uptake yields of the nucleotides tested is comparably small. It seems that a critical amount of cholate molecules need to be bound to the vesicles in order to have a permeabilizing effect. It may be that this critical value is related to the cholate concentration at which increased flip-flop of the bile salt from the outer monolayer to the inner monolayer of the vesicle membrane occurs. Indeed, despite earlier claims,65,66 it has been shown that not only un-ionized but also ionized bile salts undergo a flip-flop.64 Particularly, it has been reported before that in the case of taurineconjugated bile salts and egg phosphatidylcholine the bile salt flip-flop rate depends on the bile salt concentration within the bilayer.64 In the case of taurodeoxycholate (TDC), and in the absence of cholesterol, the flip-flop rate is more than 10 times higher at Re ) 0.15 than at Re ) 0.05,64 and it has been postulated that the bile salt flipflop is directly related to the bile salt-induced permeability increase toward Mg2+ and Ca2+. Bile salts most likely transport the divalent cations Ca2+ and Mg2+ across the phospholipid bilayers through the formation of neutral bile salt/cation complexes.44,67 In the case of our experiments, it may well be that Mg2+ complexes not only with cholate but also with the nucleotides, which would help in transporting the nucleotides across the POPC vesicle membrane. It has been reported before that, in the context of the origin of the first cells, even in the absence of cholate, adenine nucleotides show increased diffusion across phospholipid bilayers around the phase transition temperature of the lipids (e.g., 24 °C in the case of 1,2dimyristoyl-sn-glycero-3-phosphocholine)7b,7c or in the presence of divalent metal ions.68,69 In the latter case it has been argued that metal ion binding to the nucleotidess and the resulting charge neutralizationsis responsible for this increased nucleotide diffusion. Our investigation indicates that the presence of cholate seems to further increase this effect. Acknowledgment. We thank Pier Luigi Luisi for initiating this work and for critically reading the mansucript; Michaela Wessicken for taking the electron micrographs; Monica Gosh-Ray and Yasmin Ojeda for performing some of the experiments reported in the paper; Masaharu Ueno for stimulating discussions; and Jason Keiper and Fred M. Menger for supplying polynucleotide phosphorylase. LA011111U (64) Donovan, J. M.; Jackson, A. A. Biochemistry 1997, 36, 1144411451. (65) Cabral, D. J.; Small, D. M.; Lilly, H. S.; Hamilton, J. A. Biochemistry 1987, 26, 1801-1804. (66) Kamp, F.; Hamilton, J. A. Biochemistry 1993, 32, 11074-11086. (67) Donovan, J. M.; Jackson, A. A. Gastroenterology 1998, 114, A520. (68) Petkau, A.; Chelack, W. S. Can. J. Biochem. 1972, 50, 615-619. (69) Stillwell, W.; Winter, H. C. Biochem. Biophys. Res. Commun. 1974, 56, 617-622. (70) Cornell, B. A.; Middlehurst, J.; Separovic, F. Biochim. Biophys. Acta 1980, 598, 405-410. (71) Israelachvili, J. N.Intermolecular and Surface Forces; Academic Press: London, 1985; pp 246-264. (72) Huang, C.; Mason, J. T. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 308-310. (73) Tahara, Y.; Fujiyoshi, Y. Micron 1994, 25, 141-149.