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Influence of encapsulated enzyme on the surface properties of freeze-dried liposomes in trehalose
Colloids and Surfaces B: Biointerfaces 17 (2000) 103 – 109 www.elsevier.nl/locate/colsurfb
Influence of encapsulated enzyme on the surface properties of freeze-dried liposomes in trehalose Laura Baka´s * Instituto de In6estigaciones Bioquı´micas de La Plata (INIBIOLP), Facultad de Ciencias Me´dicas-CONICET, 60 y 120 (1900), Dto de Cs. Biolo´gicas, Facultad de Cs. Exactas UNLP, La Plata, Argentina Received 26 March 1999; accepted 29 June 1999
Abstract The interfacial properties of liposomes in which a soluble protease from Mucor meihie was encapsulated and subjected to freeze-drying procedure in the presence of trehalose, have been studied. Enzyme encapsulation in liposomes subjected to freeze-drying produces changes in the membrane interfacial properties as a consequence of penetration of some of the protein hydrophobic portion into the lipid bilayer. This membrane perturbation can be explained when considering that the presence of trehalose during the freeze-drying process brings about membrane hydrophobic defects where the protein can be accommodated without inducing any additional disorder. These interfacial changes in the membrane were detected by a decreased partition of Merocyanine 540, loss in the lytic effect of lysoderivatives and an increase in ANS binding sites in comparison to freeze-dried liposomes in the absence of protease. This information can be useful to modify the enzyme release of liposomes in a controlled way as a function of the interacting molecules present in the outside environment. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Freeze-drying; Liposomes; Enzyme encapsulation; Membrane surface properties
1. Introduction The structure of liposomes, which are concentric lipid membranes enclosing aqueous compartments, enables them to carry both lipids and water soluble components. Then, liposomes have Abbre6iations: ANS, 1,8-anilino-naphatalene sulphonate; FDT-liposomes, freeze-dried liposomes in trehalose; lysoPC, lysophosphatidylcholine; MC540, Merocyanine 540; MLV, multilamellar liposomes; PCegg, egg phosphatidylcholine; PE,phosphatidylethanolamine; SA, estearylamine. * Tel.: +54-221-4834833; fax: + 54-221-4258988. E-mail address: [email protected] (L. Baka´s)
been studied extensively for the last 15 years to elucidate their ability to transport various drugs. Up to now, it is known that hundreds of different kinds of substances such as enzymes, antitumor drugs, antibiotics and many others are entrapped in liposomes [1–3]. From this point of view, the liposome quality must be guaranteed over an acceptable time period since this drug delivery system should be successfully proved for biotechnology. Either, freezing or in particular freeze-drying of liposomes may, indeed, be a practical solution for preservation of the physical structure of liposome
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[30], but after rehydration, the associated membrane-rupturing and resealing processes may produce some loss of the encapsulated content. However, liposomes can be reduced to dry powders when dried in the presence of certain cryoprotectant such as lactose, sucrose or trehalose [4,5]. Their role is to minimize the loss of liposome-associated molecules and to prevent the fusion and irreversible aggregation of liposomes during the freeze-drying/rehydration cycles. In this regard, trehalose is particularly effective; previous studies suggest that there is a direct interaction between this sugar and the polar head groups of phospholipids [6,7]. An additional aspect to be considered is the presence of interacting macromolecules e.g. proteins during the freeze-drying process in presence of trehalose which would induce changes in the bilayer structure that modifies subsequently the interaction with the molecules present in the outside environment. Systematic studies on the membrane-protein interaction in relation to the surface properties have been reported by many authors. These investigations have shown the effects of surface active agents [8], salts [9] and lipid composition [10] on the extent of protein adsorption. However, these studies do not imply that the eventual changes in the enzyme activity are due solely to electrostatic interactions. Other surface physical properties such as hydration and the lipid phase state, can affect not only the association but also affect the protein activity. Previous results on the effect of Rennet from Mucor meihie, a soluble protease, show that this protein is adsorbed to the lipid membrane interfaces. The protease effect is related to phospholipid species, and it decreases in the order stearyamine \ phosphatidylcholine \ phosphatidylethanolamine. In the case of neutral phospholipid the effect of protease takes place only in the fluid state. However, the area change in monolayers in the fluid state is not so drastic to infer a protein penetration. [11]. On the other hand, studies dealing with the effect of liposome-encapsulated proteases on their membrane properties, are lacking. This protein which belongs to the group of the aspartyl proteases has a similar structure to the bovine chymosine [12] and can be used for the
acceleration of cheese ripening. Law and King [13] reported that enzyme encapsulation in liposomes can be a useful tool to accelerate cheese ripening; though it is necessary to select carefully the liposome interfacial properties for a better control of enzyme release into cheese under controlled conditions. Enzyme entrapment in liposomes by no means implies that the enzyme exists entirely within the aqueous phase. It is possible that during lipid bilayer build up, hydrophobic regions of the enzyme penetrate the lipid phase. Adsorption of some enzyme portion onto the liposomal surface may be detrimental to liposome formation. On the other hand, this situation can become worse when employing usual procedures in liposome technology such as freeze-drying of liposomes in the presence of cryoprotectants i.e trehalose. For these reasons, the interfacial properties of liposomes loaded with protease and freeze-dried in trehalose, were determined using Merocyanine 540 partition, lysoderivative lytic effect and ANS binding. We found, in short, that these interfacial properties are,indeed, advantageous to get enzyme release in a controlled fashion.
2. Materials and methods
2.1. Lipids and enzymes Egg yolk phosphatidylcholine (ePC) and monomyristoylphosphatidylcholine (LysoPC) were obtained from Avanti Polar Lipids (Birmingham, AL). Thin layer chromatography using a chloroform: water: methanol (65:25:25) mixture gave a single spot under iodine vapor. Therefore, lipids were used without further purification. Trehalose and protease (Rennet from Mucor meihie MW 34000) were obtained from Sigma (Saint Louis, MO). The enzyme purity was checked by SDS polyacrylamide gel electrophoresis. A solution of the protein was dialyzed against MilliQ water in order to eliminate the salt. Merocyanine 540 (MC540) and Anilinonaphtalene sulphonic acid (ANS) were obtained from Molecular Probes, Eugene (OR).
L. Baka´s / Colloids and Surfaces B: Biointerfaces 17 (2000) 103–109
2.2. Liposome preparation and enzyme encapsulation The experiments were performed on freeze-driedrehydrated liposomes prepared as follows: egg phosphatidylcholine (PCegg) from a chloroform solution was dried in a round botton flask under vacuum. An enzyme solution (2% w/v) in 0.1 M trehalose was entrapped in MLV by dispersing 100 mg of a PCegg film, and shaking vigorously for 2 h. Disruption of lipid film can be facilitated by the addition of a few clean glass beads. The milky liposomal suspension was frozen in liquid N2 and thawed in water at 25°C, at least four times to increase the encapsulated volume as well as the trapping efficiency, and to reach an equilibrated interlamellar solute concentration [14]. One volume of liposomal suspension was freeze-dried. The powder obtained was rehydrated at 25°C by adding an equivalent volume of water. Separation of the enzyme-containing liposomes from the non-entrapped enzyme was carried out by centrifugation at 10 000×g; pellets were washed twice and finally resuspended in 20 ml distilled water. The enzyme entrapment was measured by detecting the entrapped proteolytic activity: Prior to this assay, liposome membranes were disrupted with Triton X100 (2% v/v), and 20% resulting protease inhibition was taken into account. For this whole procedure, around 30% encapsulation efficiency was obtained. Liposomes prepared in 0.1 M trehalose (without protease) and freeze-dried were used as control for the different assays.
2.3. Clotting acti6ity Milk clotting activity determinations were done following the procedure of Arima et al. [15]. Briefly, this method is useful to determine the enzymatic activity of proteases using casein or milk as a substrate. The clotting time was determined by the visual detection of the appearance of clots. For this purpose, 100 ml of milk dispersion were mixed with 10 ml of the assayed sample and incubated at 35°C. A 10% solution of skim milk powder in 10 mM CaCl2 at pH 6.5 was used as substrate. Activity measurements were performed at pH 6.5.
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2.4. Merocyanine 540 absorption spectra MC540 has been used to investigate membrane properties of a number of different cells and phospholipid vesicles. The dye can easily be incorporated into bilayers of phospholipid model membranes and partitions into the lipid phase when it is added externally to an aqueous liposome suspension. MC 540 resides slightly above the domain of the glycerol backbone of neutral and charged phospholipids, and it is here where the dye is very sensitive to structural alterations in the lipids. The dye binds preferentially to membrane crystalline phase. In fact, it can be used to detect phase transition of bilayers [16]. MC540 shows one peak at 500 nm and another at 530 nm when dissolved in water. On the other hand, when dissolved in a non-polar solvent, it shows one peak at 570 nm In the presence of liposomes in the gel state in the aqueous suspension, the MC540 spectrum is comparable to that corresponding to water. When liposomes are in the fluid state the spectra show a small shoulder at 530 nm and a defined peak at 567 nm [17]. For this purpose, MC540 spectra in the presence of liposomes under different conditions using 1:200 dye:lipid ratio, after rehydration of dehydrated lipsomes were obtained in a Hitachi 100-60 double beam spectrophotometer. Dye-free liposome suspension served as reference to compensate for turbidity effects. Stock solutions of MC540 were prepared in ethanol to facilitate solubilization and they were stored at − 20°C
2.5. Determination of the lytic effect of lysoderi6ati6es Lysis was measured by the decrease of turbidity at 450 nm upon titration of a liposome dispersion with a lysoPC solution at 25°C. After the addition of each aliquot of lysoPC solution, the mixture was maintained at constant temperature until a constant absorbance value was obtained. Turbidity was monitored in a Hitachi double beam spectrophotometer at 450 nm. The decrease in turbidity occurring when a liposome dispersion is titrated with a lysocompound would denote the disappearance of particles larger than the wavelength [18].
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2.6. 1,8 -Anilinonaphthalene binding ANS binding experiments were performed by incubating the probe with an increasing liposome concentration for 30 min. In all cases the ANS concentration was 10 − 5 M. Fluorometric assays were done in a Perkin Elmer 2000 spectrofluorometer at 470 and 372 nm for the emission and the excitation wavelength, respectively. The fluorescence efficiency of this dye in water is very low, but it is significantly increased in the presence of a hydrophobic interface provided by both membranes and proteins [19].
3. Results and discussion The effect of encapsulated protease on the external surface properties of the lipid bilayers was determined by adding MC 540. Dye can be easily partitioned into lipid phase when added to an aqueous liposome suspension [16]. It was studied following the intensity variation of the peak at 567 nm which denotes the ability of the dye monomer to partition into the fluid membranes. It has been proposed that in membranes, MC 540 resides slightly above the domain of the glycerol backbone of phospholipids, being here where the dye is very sensitive to structural alterations in the lipid bilayers. The MC 540 spectrum, obtained by adding the probe to egg PC liposomes prepared in trehalose 0.1 M, after freeze-drying and rehydrating procedure, is shown in Fig. 1. As seen in this figure, the 567 nm absorbance peak, generated by MC monomer, increased with respect to the control without trehalose. No changes were observed when protease was added externally to a dispersion of liposomes freeze-dried in trehalose (FDTliposomes). The increase in the peak at 567 nm of MC540 indicates that the surface membranes properties of the FDT liposomes are significantly affected. Trehalose is likely to penetrate the phospholipid headgroups during the dehydration step. After rehydration, trehalose would be able to promote bilayer expansion; then fractures may be exposed along with an increase in the MC partition. The existence of these defects implies a separation of the phospholipid headgroups and a
loose packing of the fatty acid region. Indeed, liposomes freeze-dried in trehalose exhibit a higher dye binding capacity. These hydrophobic defects can not be observed when the process is carried out simultaneously in the presence of trehalose and protease. In this case, the MC partition is less favoured than that in PCegg liposomes (see Fig. 1). The freeze-drying process in the presence of sugar promotes defects which may induce lipid–protein interactions. These hydrophobic fractures can arrange a protein molecule without inducing any additional general disorder which is energetically unfavorable [20].The lipid– protein interaction is promoted only when the freeze-drying process takes place. The MC 540 spectra obtained from liposomes loaded with protease before the freeze-drying process overlapped those of the PC liposome control. In these experiments, the external non-encapsulated protein was separated by centrifugation.
Fig. 1. Visible spectra of Merocyanine 540 (10 − 5 M) in the presence of liposomes: ( ) eggPC liposomes dispersed in 10 mM Tris – HCl; () eggPC liposomes freeze – dried –rehydrated (F– D – R) in 0.1 M trehalose with or without protease addition after rehydration; ( ) eggPC liposomes F– D –R in 0.1 M trehalose and 0.5 mM protease. All spectra were obtained at 25°C. MC540 was added from a stock solution to the cuvette after F– D – R process to yield a final concentration of 10 − 5 M of the dye.
L. Baka´s / Colloids and Surfaces B: Biointerfaces 17 (2000) 103–109
Fig. 2. Action of lysoPC on egg PC liposomes prepared in 0.1 M trehalose and F–D –R in the ( ) absence or ( ) presence of protease. The decrease in absorbance value at 450 nm was used as index of lytic action. The liposome dispersions were titrated with lysoPC solution of 2 mg ml − 1 at 25°C and vigorously stirred. Samples were allowed to equilibrate to achieve a constant value for each addition.
Changes in the surface properties of liposomes induced by the presence of protein during the freeze-drying process were also studied by lysophospholipid sensibility and ANS binding. Fig. 2 shows changes induced by lysoPC on the 450-nm absorbance of PC liposome dispersion freeze-dried in trehalose either in the presence or absence of protease. The lytic effect denotes a decrease in the turbidity of liposome dispersion due to a reorganization of the lipid bilayer in micelles [21]. It is known that liposome response to lysoderivatives is closely related to defects such as the gel–liquid crystalline coexistence domains or those ones generated by the dehydration process in the presence of trehalose and other facts that promote an increase in the surface hydrophobicity [22,23]. The freeze-drying process in presence of trehalose gives rise to membrane surface defects in which the lysoPC can penetrate and destabilize the bilayer structure. In the concentration range studied, the addition of lysoPC to trehalose freeze-dried liposome dispersion pro-
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duces a gradual decrease in the absorbance value. A response to lysocompounds similar to that found in liposome freeze-dried in trehalose was obtained with liposomes in the fluid state in the presence of protease [23]. Conversely, lysoPC did not produce any detectable change in the turbidity when the protein was present during the freezedrying process as shown in Fig. 2. The binding of ANS after the addition of 2.5× 10 − 5 M ANS to the dispersion of liposomes freeze-dried in trehalose either in the absence or presence of protein, is shown in Fig. 3. In all cases the external protein had been previously separated by centrifugation). ANS is a sensitive probe to both membranes and proteins [19]. The model proposed by Haynes [24] suggests that ANS molecules bind to preexisting sites, and the changes produced in the ANS binding to membranes as well as to proteins could be related to a change in the number of binding sites. The presence of proteins during the freeze-drying step generated those binding sites by the intercalation of any protein domain into lipid bilayer. This domain is exposed on the external surface of
Fig. 3. ANS binding to egg PC liposomes prepared in 0.1 M trehalose and F– D – R in the absence ( ) or presence ( ) of protease. Solutions containing 2.5 × 10 − 5 M ANS were titrated with aliquots of 2 mg ml − 1 of liposomes.
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Table 1 Proteolytic activity Coagulation time/s Total Latent Apparent Supernatant
75 18 0 52.5
membranes. Then, the ANS bound increased when compared to trehalose freeze-dried liposomes. On the other hand, the availability of enzyme may be influenced by its location in the liposomes. In this case, the apparent liposomal enzyme activity, which derived either from enzyme adsorbed on the liposomal surfaces or from enzymatically active extraliposomal portion of entrapped protein after removing the external enzyme by successive centrifugation steps, was negligible. Only after adding Triton X100, the latent liposome entrapped enzyme activitiy could be recovered (see Table 1). These results can be explained if we consider that phospholipid assemblies, such as liposomes, are stabilized by hydrophobic forces, inherent to the amphiphilic nature of these molecules, which are indispensable to maintain the phospholipid bilayer integrity. Nevertheless, it is possible to eliminate the water by drying liposomes in the presence of certain sugars. In this regard, trehalose is the most effective because it is able to preserve structural and functional properties of either natural or synthetic lipid membranes by mimicking the effect of water in such a way that trehalose could promote an expansion of the phospholipid lattice [25 – 27]. Trehalose reaches the head group regions when either total or partial amount of bound water is displaced, and it would remain there even when water is restored to the system [22]. In addition, it is well-known that several sugars can stabilize proteins in solution [28]. When a protein/sugar solution is dried, a compact conformation will be induced, and those domains with hydrophobic character such as Trp residues will appear exposed on the surface protein [29].
On the other hand, if we consider that enzyme entrapment in liposomes is a passive process, as liposomes are formed, they will entrap a volume of water containing the solutes. However, when liposomes are loaded with proteins and then freeze-dried in the presence of cryoprotectant, the hydrophobic regions of enzymes may penetrate the lipid phase. This close contact can only be achieved if protein encapsulation is followed by a dehydration step performed in the presence of sugar. The changes observed in the surface properties of liposomes subjected to the above described encapsulation procedures, are the consequence of the lipid–protein interaction in hydrophobic fractures that can host a protein molecule. These alterations in the fatty acyl region of the membrane produce, in consequence, changes in the packing at the membrane surface; they were sensed by MC 540, lysoPC and ANS binding. In conclusion, the use of liposomes as protein carriers requires the characterisation of interactions between the bilayer and the entrapped protein after freeze-drying procedure. In fact, this process affects not only the protein structure but also the structure and properties of liposomes. Yet, the above mentioned changes might give rise to different delivering mechanisms of encapsulated enzymes which are worth knowing in order to utilise them as macromolecule-carrier systems.
Acknowledgements This work was supported by funds from I.F.S. -Sweden. L.S.B is a member of the Research Career of the Comision de Investigaciones Cientificas de la Pcia de Buenos Aires (Argentina).
References [1] S. Wrigts, L. Huang, Adv. Drug Delivery Rev. 3 (1989) 343 – 389. [2] B. Hughes, S. Kennel, R, Lee, L. Huang, Cancer. Res. 49 (1988) 6214 – 6220. [3] W. Alkahalaf, M. El Soda, C. Gripon, L. Vassal, J. Dairy Res. 72 (1989) 2233 – 2238.
L. Baka´s / Colloids and Surfaces B: Biointerfaces 17 (2000) 103–109 [4] L. Crowe, C. Womersley, J. Crowe, D. Reide, L. Appel, A. Rudolph, Biochim. Biophys Acta 861 (1988) 131 – 140. [5] H. Hauser, G. Strauss, Biochim. Biophys. Acta 897 (1987) 331 – 334. [6] J. Crowe, L. Crowe, D. Chapman, Science 223 (1984) 701 – 703. [7] J. Crowe, L. Crowe, D. Chapman, Arch. Biochem. Biophys. 232 (1984) 400–407. [8] A. Helenius, K. Simons, Biochim. Biophys Acta 11 (1975) 415 – 429. [9] L. Wojtczak, M. Nalecz, Eur. J. Biochem. 94 (1979) 99 – 107. [10] M. Nalecz, K. Zborowski, L. Famulski, L. Wojtczak, Eur. J. Biochem. 112 (1980) 75–80. [11] L Baka´s, M.S.P. Chaazalet, D. Bernik, E.A. Disalvo, Colloids Surf. 12 (1998) 77–87. [12] M. Ottesen, W. Rickert, Compt. Rend. Trav. Lab. Carisberg. 37 (1970) 307. [13] B. Law. J. King, J. Dairy Res. 52 (1985) 183. [14] L. Mayer, M. Hope, P. Cullis A. and Janoff, Biochim. Biophys. Acta 817 (1985) 193–196. [15] K. Arima J. Yu, S. Ikasaki, Methods Enzymol. 19 (1970) 446 [16] A. Verkman, Biochemistry 26 (1987) 4050–4056. [17] P. Lelkes I. Miller, J. Membr. Biol. 52. (1989) 1 – 15.
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[18] A. Carmona Ribeiro, H. Chaimovich, Biophys. J. 50 (1986) 621 [19] J. Slavik, Biochim. Biophys. Acta. (1982) 1 – 25 [20] M. Jain, D. Zakim, Biochim. Biophys. Acta 906 (1987) 33 – 68 [21] G. Senisterra, E. Disalvo, J.J. Gagliardino, Biochim. Biophys. Acta 941 (1988) 264 – 270. [22] L. Viera, S. Alonso, M. Borovyagin, M. Feliz, E. Disalvo, Biochim. Biophys. Acta. 1145 (1993) 157 – 167. [23] E. Disalvo, L. Viera, L. Baka´s G. Senisterra, J. Colloid Int. Sci. 178 (1996) 417 – 425 [24] D. Haynes, J. Membr. Biol., 17 (1974) 313 – 340. [25] J. Crowe, L. Crowe, D. Chapman, Science 223 (1984) 701 – 703. [26] R. Goodrich, J. Crowe, L. Crowe, J. Baldeschwieler, Biochemistry 23 (1991) 6912 – 6919. [27] L. Crowe, J. Crowe, D. Chapman, Arch. Biochem. Biophys 206 (1988) 289 – 296. [28] T. Arakama, S. Timasheff, Biochemistry 21 (1982) 6536– 6544. [29] N. Khanna, T. Masaki, D. Marton, J. Biol. Chem. 261 (1986) 883 – 8897. [30] Y. Ozer, A. Talsma, D.J. Crommelin, A.A. Hincal, Acta Pharmacol. Technol. 34 (1988) 129 – 137.
Influence of encapsulated enzyme on the surface properties of freeze-dried liposomes in trehalose Laura Baka´s * Instituto de In6estigaciones Bioquı´micas de La Plata (INIBIOLP), Facultad de Ciencias Me´dicas-CONICET, 60 y 120 (1900), Dto de Cs. Biolo´gicas, Facultad de Cs. Exactas UNLP, La Plata, Argentina Received 26 March 1999; accepted 29 June 1999
Abstract The interfacial properties of liposomes in which a soluble protease from Mucor meihie was encapsulated and subjected to freeze-drying procedure in the presence of trehalose, have been studied. Enzyme encapsulation in liposomes subjected to freeze-drying produces changes in the membrane interfacial properties as a consequence of penetration of some of the protein hydrophobic portion into the lipid bilayer. This membrane perturbation can be explained when considering that the presence of trehalose during the freeze-drying process brings about membrane hydrophobic defects where the protein can be accommodated without inducing any additional disorder. These interfacial changes in the membrane were detected by a decreased partition of Merocyanine 540, loss in the lytic effect of lysoderivatives and an increase in ANS binding sites in comparison to freeze-dried liposomes in the absence of protease. This information can be useful to modify the enzyme release of liposomes in a controlled way as a function of the interacting molecules present in the outside environment. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Freeze-drying; Liposomes; Enzyme encapsulation; Membrane surface properties
1. Introduction The structure of liposomes, which are concentric lipid membranes enclosing aqueous compartments, enables them to carry both lipids and water soluble components. Then, liposomes have Abbre6iations: ANS, 1,8-anilino-naphatalene sulphonate; FDT-liposomes, freeze-dried liposomes in trehalose; lysoPC, lysophosphatidylcholine; MC540, Merocyanine 540; MLV, multilamellar liposomes; PCegg, egg phosphatidylcholine; PE,phosphatidylethanolamine; SA, estearylamine. * Tel.: +54-221-4834833; fax: + 54-221-4258988. E-mail address: [email protected] (L. Baka´s)
been studied extensively for the last 15 years to elucidate their ability to transport various drugs. Up to now, it is known that hundreds of different kinds of substances such as enzymes, antitumor drugs, antibiotics and many others are entrapped in liposomes [1–3]. From this point of view, the liposome quality must be guaranteed over an acceptable time period since this drug delivery system should be successfully proved for biotechnology. Either, freezing or in particular freeze-drying of liposomes may, indeed, be a practical solution for preservation of the physical structure of liposome
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[30], but after rehydration, the associated membrane-rupturing and resealing processes may produce some loss of the encapsulated content. However, liposomes can be reduced to dry powders when dried in the presence of certain cryoprotectant such as lactose, sucrose or trehalose [4,5]. Their role is to minimize the loss of liposome-associated molecules and to prevent the fusion and irreversible aggregation of liposomes during the freeze-drying/rehydration cycles. In this regard, trehalose is particularly effective; previous studies suggest that there is a direct interaction between this sugar and the polar head groups of phospholipids [6,7]. An additional aspect to be considered is the presence of interacting macromolecules e.g. proteins during the freeze-drying process in presence of trehalose which would induce changes in the bilayer structure that modifies subsequently the interaction with the molecules present in the outside environment. Systematic studies on the membrane-protein interaction in relation to the surface properties have been reported by many authors. These investigations have shown the effects of surface active agents [8], salts [9] and lipid composition [10] on the extent of protein adsorption. However, these studies do not imply that the eventual changes in the enzyme activity are due solely to electrostatic interactions. Other surface physical properties such as hydration and the lipid phase state, can affect not only the association but also affect the protein activity. Previous results on the effect of Rennet from Mucor meihie, a soluble protease, show that this protein is adsorbed to the lipid membrane interfaces. The protease effect is related to phospholipid species, and it decreases in the order stearyamine \ phosphatidylcholine \ phosphatidylethanolamine. In the case of neutral phospholipid the effect of protease takes place only in the fluid state. However, the area change in monolayers in the fluid state is not so drastic to infer a protein penetration. [11]. On the other hand, studies dealing with the effect of liposome-encapsulated proteases on their membrane properties, are lacking. This protein which belongs to the group of the aspartyl proteases has a similar structure to the bovine chymosine [12] and can be used for the
acceleration of cheese ripening. Law and King [13] reported that enzyme encapsulation in liposomes can be a useful tool to accelerate cheese ripening; though it is necessary to select carefully the liposome interfacial properties for a better control of enzyme release into cheese under controlled conditions. Enzyme entrapment in liposomes by no means implies that the enzyme exists entirely within the aqueous phase. It is possible that during lipid bilayer build up, hydrophobic regions of the enzyme penetrate the lipid phase. Adsorption of some enzyme portion onto the liposomal surface may be detrimental to liposome formation. On the other hand, this situation can become worse when employing usual procedures in liposome technology such as freeze-drying of liposomes in the presence of cryoprotectants i.e trehalose. For these reasons, the interfacial properties of liposomes loaded with protease and freeze-dried in trehalose, were determined using Merocyanine 540 partition, lysoderivative lytic effect and ANS binding. We found, in short, that these interfacial properties are,indeed, advantageous to get enzyme release in a controlled fashion.
2. Materials and methods
2.1. Lipids and enzymes Egg yolk phosphatidylcholine (ePC) and monomyristoylphosphatidylcholine (LysoPC) were obtained from Avanti Polar Lipids (Birmingham, AL). Thin layer chromatography using a chloroform: water: methanol (65:25:25) mixture gave a single spot under iodine vapor. Therefore, lipids were used without further purification. Trehalose and protease (Rennet from Mucor meihie MW 34000) were obtained from Sigma (Saint Louis, MO). The enzyme purity was checked by SDS polyacrylamide gel electrophoresis. A solution of the protein was dialyzed against MilliQ water in order to eliminate the salt. Merocyanine 540 (MC540) and Anilinonaphtalene sulphonic acid (ANS) were obtained from Molecular Probes, Eugene (OR).
L. Baka´s / Colloids and Surfaces B: Biointerfaces 17 (2000) 103–109
2.2. Liposome preparation and enzyme encapsulation The experiments were performed on freeze-driedrehydrated liposomes prepared as follows: egg phosphatidylcholine (PCegg) from a chloroform solution was dried in a round botton flask under vacuum. An enzyme solution (2% w/v) in 0.1 M trehalose was entrapped in MLV by dispersing 100 mg of a PCegg film, and shaking vigorously for 2 h. Disruption of lipid film can be facilitated by the addition of a few clean glass beads. The milky liposomal suspension was frozen in liquid N2 and thawed in water at 25°C, at least four times to increase the encapsulated volume as well as the trapping efficiency, and to reach an equilibrated interlamellar solute concentration [14]. One volume of liposomal suspension was freeze-dried. The powder obtained was rehydrated at 25°C by adding an equivalent volume of water. Separation of the enzyme-containing liposomes from the non-entrapped enzyme was carried out by centrifugation at 10 000×g; pellets were washed twice and finally resuspended in 20 ml distilled water. The enzyme entrapment was measured by detecting the entrapped proteolytic activity: Prior to this assay, liposome membranes were disrupted with Triton X100 (2% v/v), and 20% resulting protease inhibition was taken into account. For this whole procedure, around 30% encapsulation efficiency was obtained. Liposomes prepared in 0.1 M trehalose (without protease) and freeze-dried were used as control for the different assays.
2.3. Clotting acti6ity Milk clotting activity determinations were done following the procedure of Arima et al. [15]. Briefly, this method is useful to determine the enzymatic activity of proteases using casein or milk as a substrate. The clotting time was determined by the visual detection of the appearance of clots. For this purpose, 100 ml of milk dispersion were mixed with 10 ml of the assayed sample and incubated at 35°C. A 10% solution of skim milk powder in 10 mM CaCl2 at pH 6.5 was used as substrate. Activity measurements were performed at pH 6.5.
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2.4. Merocyanine 540 absorption spectra MC540 has been used to investigate membrane properties of a number of different cells and phospholipid vesicles. The dye can easily be incorporated into bilayers of phospholipid model membranes and partitions into the lipid phase when it is added externally to an aqueous liposome suspension. MC 540 resides slightly above the domain of the glycerol backbone of neutral and charged phospholipids, and it is here where the dye is very sensitive to structural alterations in the lipids. The dye binds preferentially to membrane crystalline phase. In fact, it can be used to detect phase transition of bilayers [16]. MC540 shows one peak at 500 nm and another at 530 nm when dissolved in water. On the other hand, when dissolved in a non-polar solvent, it shows one peak at 570 nm In the presence of liposomes in the gel state in the aqueous suspension, the MC540 spectrum is comparable to that corresponding to water. When liposomes are in the fluid state the spectra show a small shoulder at 530 nm and a defined peak at 567 nm [17]. For this purpose, MC540 spectra in the presence of liposomes under different conditions using 1:200 dye:lipid ratio, after rehydration of dehydrated lipsomes were obtained in a Hitachi 100-60 double beam spectrophotometer. Dye-free liposome suspension served as reference to compensate for turbidity effects. Stock solutions of MC540 were prepared in ethanol to facilitate solubilization and they were stored at − 20°C
2.5. Determination of the lytic effect of lysoderi6ati6es Lysis was measured by the decrease of turbidity at 450 nm upon titration of a liposome dispersion with a lysoPC solution at 25°C. After the addition of each aliquot of lysoPC solution, the mixture was maintained at constant temperature until a constant absorbance value was obtained. Turbidity was monitored in a Hitachi double beam spectrophotometer at 450 nm. The decrease in turbidity occurring when a liposome dispersion is titrated with a lysocompound would denote the disappearance of particles larger than the wavelength [18].
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2.6. 1,8 -Anilinonaphthalene binding ANS binding experiments were performed by incubating the probe with an increasing liposome concentration for 30 min. In all cases the ANS concentration was 10 − 5 M. Fluorometric assays were done in a Perkin Elmer 2000 spectrofluorometer at 470 and 372 nm for the emission and the excitation wavelength, respectively. The fluorescence efficiency of this dye in water is very low, but it is significantly increased in the presence of a hydrophobic interface provided by both membranes and proteins [19].
3. Results and discussion The effect of encapsulated protease on the external surface properties of the lipid bilayers was determined by adding MC 540. Dye can be easily partitioned into lipid phase when added to an aqueous liposome suspension [16]. It was studied following the intensity variation of the peak at 567 nm which denotes the ability of the dye monomer to partition into the fluid membranes. It has been proposed that in membranes, MC 540 resides slightly above the domain of the glycerol backbone of phospholipids, being here where the dye is very sensitive to structural alterations in the lipid bilayers. The MC 540 spectrum, obtained by adding the probe to egg PC liposomes prepared in trehalose 0.1 M, after freeze-drying and rehydrating procedure, is shown in Fig. 1. As seen in this figure, the 567 nm absorbance peak, generated by MC monomer, increased with respect to the control without trehalose. No changes were observed when protease was added externally to a dispersion of liposomes freeze-dried in trehalose (FDTliposomes). The increase in the peak at 567 nm of MC540 indicates that the surface membranes properties of the FDT liposomes are significantly affected. Trehalose is likely to penetrate the phospholipid headgroups during the dehydration step. After rehydration, trehalose would be able to promote bilayer expansion; then fractures may be exposed along with an increase in the MC partition. The existence of these defects implies a separation of the phospholipid headgroups and a
loose packing of the fatty acid region. Indeed, liposomes freeze-dried in trehalose exhibit a higher dye binding capacity. These hydrophobic defects can not be observed when the process is carried out simultaneously in the presence of trehalose and protease. In this case, the MC partition is less favoured than that in PCegg liposomes (see Fig. 1). The freeze-drying process in the presence of sugar promotes defects which may induce lipid–protein interactions. These hydrophobic fractures can arrange a protein molecule without inducing any additional general disorder which is energetically unfavorable [20].The lipid– protein interaction is promoted only when the freeze-drying process takes place. The MC 540 spectra obtained from liposomes loaded with protease before the freeze-drying process overlapped those of the PC liposome control. In these experiments, the external non-encapsulated protein was separated by centrifugation.
Fig. 1. Visible spectra of Merocyanine 540 (10 − 5 M) in the presence of liposomes: ( ) eggPC liposomes dispersed in 10 mM Tris – HCl; () eggPC liposomes freeze – dried –rehydrated (F– D – R) in 0.1 M trehalose with or without protease addition after rehydration; ( ) eggPC liposomes F– D –R in 0.1 M trehalose and 0.5 mM protease. All spectra were obtained at 25°C. MC540 was added from a stock solution to the cuvette after F– D – R process to yield a final concentration of 10 − 5 M of the dye.
L. Baka´s / Colloids and Surfaces B: Biointerfaces 17 (2000) 103–109
Fig. 2. Action of lysoPC on egg PC liposomes prepared in 0.1 M trehalose and F–D –R in the ( ) absence or ( ) presence of protease. The decrease in absorbance value at 450 nm was used as index of lytic action. The liposome dispersions were titrated with lysoPC solution of 2 mg ml − 1 at 25°C and vigorously stirred. Samples were allowed to equilibrate to achieve a constant value for each addition.
Changes in the surface properties of liposomes induced by the presence of protein during the freeze-drying process were also studied by lysophospholipid sensibility and ANS binding. Fig. 2 shows changes induced by lysoPC on the 450-nm absorbance of PC liposome dispersion freeze-dried in trehalose either in the presence or absence of protease. The lytic effect denotes a decrease in the turbidity of liposome dispersion due to a reorganization of the lipid bilayer in micelles [21]. It is known that liposome response to lysoderivatives is closely related to defects such as the gel–liquid crystalline coexistence domains or those ones generated by the dehydration process in the presence of trehalose and other facts that promote an increase in the surface hydrophobicity [22,23]. The freeze-drying process in presence of trehalose gives rise to membrane surface defects in which the lysoPC can penetrate and destabilize the bilayer structure. In the concentration range studied, the addition of lysoPC to trehalose freeze-dried liposome dispersion pro-
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duces a gradual decrease in the absorbance value. A response to lysocompounds similar to that found in liposome freeze-dried in trehalose was obtained with liposomes in the fluid state in the presence of protease [23]. Conversely, lysoPC did not produce any detectable change in the turbidity when the protein was present during the freezedrying process as shown in Fig. 2. The binding of ANS after the addition of 2.5× 10 − 5 M ANS to the dispersion of liposomes freeze-dried in trehalose either in the absence or presence of protein, is shown in Fig. 3. In all cases the external protein had been previously separated by centrifugation). ANS is a sensitive probe to both membranes and proteins [19]. The model proposed by Haynes [24] suggests that ANS molecules bind to preexisting sites, and the changes produced in the ANS binding to membranes as well as to proteins could be related to a change in the number of binding sites. The presence of proteins during the freeze-drying step generated those binding sites by the intercalation of any protein domain into lipid bilayer. This domain is exposed on the external surface of
Fig. 3. ANS binding to egg PC liposomes prepared in 0.1 M trehalose and F– D – R in the absence ( ) or presence ( ) of protease. Solutions containing 2.5 × 10 − 5 M ANS were titrated with aliquots of 2 mg ml − 1 of liposomes.
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Table 1 Proteolytic activity Coagulation time/s Total Latent Apparent Supernatant
75 18 0 52.5
membranes. Then, the ANS bound increased when compared to trehalose freeze-dried liposomes. On the other hand, the availability of enzyme may be influenced by its location in the liposomes. In this case, the apparent liposomal enzyme activity, which derived either from enzyme adsorbed on the liposomal surfaces or from enzymatically active extraliposomal portion of entrapped protein after removing the external enzyme by successive centrifugation steps, was negligible. Only after adding Triton X100, the latent liposome entrapped enzyme activitiy could be recovered (see Table 1). These results can be explained if we consider that phospholipid assemblies, such as liposomes, are stabilized by hydrophobic forces, inherent to the amphiphilic nature of these molecules, which are indispensable to maintain the phospholipid bilayer integrity. Nevertheless, it is possible to eliminate the water by drying liposomes in the presence of certain sugars. In this regard, trehalose is the most effective because it is able to preserve structural and functional properties of either natural or synthetic lipid membranes by mimicking the effect of water in such a way that trehalose could promote an expansion of the phospholipid lattice [25 – 27]. Trehalose reaches the head group regions when either total or partial amount of bound water is displaced, and it would remain there even when water is restored to the system [22]. In addition, it is well-known that several sugars can stabilize proteins in solution [28]. When a protein/sugar solution is dried, a compact conformation will be induced, and those domains with hydrophobic character such as Trp residues will appear exposed on the surface protein [29].
On the other hand, if we consider that enzyme entrapment in liposomes is a passive process, as liposomes are formed, they will entrap a volume of water containing the solutes. However, when liposomes are loaded with proteins and then freeze-dried in the presence of cryoprotectant, the hydrophobic regions of enzymes may penetrate the lipid phase. This close contact can only be achieved if protein encapsulation is followed by a dehydration step performed in the presence of sugar. The changes observed in the surface properties of liposomes subjected to the above described encapsulation procedures, are the consequence of the lipid–protein interaction in hydrophobic fractures that can host a protein molecule. These alterations in the fatty acyl region of the membrane produce, in consequence, changes in the packing at the membrane surface; they were sensed by MC 540, lysoPC and ANS binding. In conclusion, the use of liposomes as protein carriers requires the characterisation of interactions between the bilayer and the entrapped protein after freeze-drying procedure. In fact, this process affects not only the protein structure but also the structure and properties of liposomes. Yet, the above mentioned changes might give rise to different delivering mechanisms of encapsulated enzymes which are worth knowing in order to utilise them as macromolecule-carrier systems.
Acknowledgements This work was supported by funds from I.F.S. -Sweden. L.S.B is a member of the Research Career of the Comision de Investigaciones Cientificas de la Pcia de Buenos Aires (Argentina).
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