METHODS OF IMMOBILIZING LIPOSOMES IN GEL BEADS AND SIMILAR MATERIALS, GEL BEADS AND SIMILAR MATERIALS WITH IMMOBILIZED LIPOSOMES PREPARED BY SUCH METHODS AND USES THEREOF

Abstract: This invention relates to methods of immobilizing liposomes or proteoliposomes in cross-linked or non-cross-linked materials with a three-dimensional network, like gel beads or gel particles, or beads or particles of similar materials. The liposomes are trapped within the matrix or pores of the gel or material by formation of sufficiently large liposomes from a liposome suspension or an emulsion of lipids. The methods comprise preparing a suspension of liposomes and/or proteoliposomes, or an emulsion of lipids, or lipids and protein(s), and water in organic solvent; mixing the material with the suspension or the emulsion; increasing the average size of the liposomes and/or proteoliposomes which have entered the material, or effect uptake and association of emulsified lipids [and protein(s)] in the material by evaporating the organic solvent, whereby liposomes and/or proteoliposomes are formed; and removing non-immobilized liposomes and/or proteoliposomes. The used gel beads show an immobilization capacity corresponding to at least 20 mu mol of phospholipids per ml packed gel, or a total internal liposome volume of at least 50 mu mol per ml gel. The invention comprises gel beads or similar materials with immobilized liposomes, proteoliposomes and/or liposomes with included or inserted drugs, substances or macromolecules, and their use for chromatographic, medical, veterinary, biotechnological, synthetic, analytical and/or cosmetic purposes.

Description: Methods of immobilizing liposomes in gel beads and similar materials, gel beads and similar materials with immobilized liposomes prepared by such methods, and uses thereof The present invention relates to methods of immobilizing liposomes or proteoliposomes in materials like gel beads, materials with immobilized liposomes or proteoliposomes prepared by such methods, and chromatographic, medical and other uses of such gel beads or similar materials with immobilized liposomes. ipid bilayers prepared in the form of spherical vesicles, called liposomes, are commonly used as models of membranes in experimen¬ tal studies. Liposomes with membrane protein(s) in their lipid bilayers (proteoliposomes) are used for studies of protein- mediated transport across the bilayer. Liposomes can be designed to have ion-exchange, affinity and transpor properties as well as catalytic activities. Affinity ligands or catalytic macromolecules including transmembrane and peripheral membrane proteins can be hydrophobically anchored or inserted into the lipid bilayer. To simplify studies, analyses and separations by use of such properties, it is desired to immobilize the liposomes in or on a carrier, eg a gel, to be able to perform chromatographic experiments. Methods of ' immobilizing liposomes in gel beads have been described earlier by the present inventors together with M.Belew, see for example Biochimica et Biophysica Acta 924 (1987) pp 185- 19-2, Elsevier and Biochimica et Biophysica Acta 938 (1988) pp 243-256, Elsevier. These methods involve covalent coupling of ligands to the gel bead material, using specific coupling agents, to enable immobilization of the liposomes in and on the gel beads by hydrophobic interaction. To avoid the presence of free hydrophobic ligands, another method of immobilizing liposomes was developed by the present inventors, see Biochimica et Biophysica Acta, 982 (1989), p 47-52, Elsevier. This method does not involve hydrophobic ligands or covalent coupling. Here, the liposomes are entrapped (sterically immobili¬ zed) in the gel beads by dialysing solubilized lipids together with the gel beads. The amount of entrapped liposomes increases non-linearly with the initial lipid concentration and is dependent on the relative sizes of liposomes and gel pores. The known procedures of immobilizing liposomes have several drawbacks among which the most significative are, in the case of immobilization with hydrophobic ligands, the risk of undesired hydrophobic interactions and, in the case of steric immobiliza¬ tion by a dialysis procedure, the relatively low.immobilization capacity. Substantial costs are inherent in these methods and they are also time-consuming, elaborate or difficult to scale up. Therefore, it was an object of the present invention to provide a novel method of immobilizing liposomes in gel beads without said disadvantages and to provide gel beads produced by said method. These objects are achieved according to the characterizing parts of claims 1 and 14, respectively. The novel method of the invention utilizes the effect or effects that when liposomes are fused or lipid material (for example fragments or micelles) are associated into liposomes, the newly formed liposomes are either sterically entrapped or entangled due to their relatively large size or they become otherwise entrapped or entangled in the three-dimensional network of the material used. Preferred materials are gel beads or particles made of polysaccharides, such as agarose, dextran and glucomannan; polyacrylamide; allylpolysaccharides cross-linked with N,N r - methylene-bis-acrylamide; and silica. The gel bead material can be cross-linked or non-cross-linked and be hydrophilic or hydrophobic. The preferred embodiments of the method according to the invention will now be described further with reference to the following Examples and accompanying drawings. In the Examples, the following solutions were used: Solution A: 150 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl (pH 7.4) Solution B: 140 mM NaCl, 10 mM calcein (pH 7.4) 100 mM EYP (=egg yolk phospholipid) solution : 100 mM EYP, 150 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl (pH 7,4), 125 mM cholate 290 mM EYP solution: 290 mM EYP, 150 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl (pH 7,4), 580 mM cholate EXAMPLE 1 Steric immobilization of liposomes by freeze-thawing of a mixture of liposomes and suction-dried moist σel beads Gel beads for use as carriers of immobilized liposomes were rinsed three times with solution A or B. The gel was then dried on a column by suction with a water- et pump (aspirator vacuum pump) just until no more liquid was sucked out from the column. This is here termed suction-drying. 0.7 g of suction-dried gel of Sephacryl S-1000 or 0.6 g of Sepharose 2B (in both cases corresponding to 0.5 ml of packed gel) was placed in a 10-ml plastic conical-bottom tube and 0.55 ml of liposome suspension was added to the gel. The liposome suspension had been prepared by applying 6-8.5 ml of 100 mM EYP solution on a Sephadex G-50 column and eluting with solution A at room temperature. Thereaf¬ ter, the liposome suspension had been concentrated, for example, by using a sample concentrator such as Minicon B (from Amicon). Suspensions of different phospholipid concentrations were used. In some cases, calcein was encapsulated in the liposomes. The tube was filled with nitrogen and sealed. The gel was mixed with the liposomes by vortexing and kept under nitrogen for at least 30 min to equilibrate the gel beads with the liposomes. For mixtures of high liposome concentrations longer times were used. The mixture was frozen by immersing the tube into a dry ice/etha- nol bath (about -75°C) or into liquid nitrogen for 10 min and thawed in a 25°C water-bath. The tube was kept in the water-bath for 10 minutes. Non-immobilized liposomes were removed with three portions of solution A by centrifugations at 150 x g for 3 x 5 min. The gel with immobilized liposomes was packed into a column and further washed with at least 10 column volumes of solution A. The immobilized liposomes were finally dissolved and eluted from the column with 50 mM cholate solution for determination of the immobilization capacity (the amount of immobilized phospholipids per gel volume) by a phosphorus assay according to the method of G.R. Bartlett, J. Biol. Chem. 234 (1959) 466. EXAMPLE 2 Steric immobilization of liposomes by freeze-thawinσ of a mixture of liposomes and freeze-dried σel beads In this Example, the immobilization of liposomes was achieved by mixing a concentrated liposome suspension with freeze-dried gel beads instead of suction-dried beads. The gel used was Sephacryl S-1000 in an amount corresponding to 0.5 ml packed volume. The gel contained in a 10-ml roun -bottomed flask was immersed into dry-ice/ethanol and the gel-bead suspension was frozen to a thin shell while whirling the flask. Thereafter, the gel was freeze-dried at room temperature over¬ night. Then 1.1-1.2 ml of concentrated liposome suspension was introduced to the dried gel bead shell (76 mg). The materials were mixed by vortexing and were kept under nitrogen at room temperature for about 3 h to swell the gel beads and to equilib¬ rate the gel beads with the liposomes. The mixture was freeze- thawed as described in Example 1. The washing procedures were also done as described in Example 1. EXAMPLE 3 Steric immobilization of liposomes by freeze-dryinσ and rehydra- tion of a mixture of liposomes and suction-dried σel beads The procedures described in the first paragraph of Example 1 were repeated with the exception that the mixture was frozen as a thin shell and not thawed. Thereafter, it was freeze-dried at room temperature overnight. Then the mixture in the thin shell was rehydrated with 1.1-1.2 ml of deionized water or diluted solution A to a final composition corresponding to solution A. Thereafter, the mixture was flushed with nitrogen gas and left under nitrogen at room temperature for at least 3 h to complete the rehydration process. Non-immobilized liposomes were removed by washing procedures as described above in Example 1. Optionally, the hydrated mixture can be freeze-thawed to increase the immobilization capacity. EXAMPLE 4 Steric immobilization of liposomes by freeze-dryinq and rehydra¬ tion of a mixture of liposomes and freeze-dried gel beads In this Example the procedures were the same as in Example 3, except that 1-2 ml of concentrated liposome suspension was added to the freeze-dried gel, which was kept under nitrogen at room temperature for 3 h. The mixture was then freeze-dried and rehydrated by addition of 1.1-1.2 ml of deionized water, diluted solution A or solution A. Washing procedures were done as described in Example 1 above. EXAMPLE 5 Steric immobilization of liposomes in gel beads by reverse phase evaporation with freeze-dried σel beads 1. Freeze-dryinq of gel beads. 0.7 g of suction-dried Sephacryl S-1000 gel beads was transferred to a 25-ml round-bottomed flask. The gel was freeze-dried as described in Example- 2. 2. Emulsifying an aqueous solution in an organic solution of EYP by sonication. Emulsion 1. 200 mg of EYP was dissolved in 2 ml of diethyl ether in a 10-ml test tube fitted with a screw-cap lined with aluminium foil. 0.6 ml of solution A was rapidly injected into the lipid solution through a 0.4 mm x 20 mm needle from a 1 ml syringe. The solutions were mixed by use of a vortex mixer for 30 s and sonicated in a water-bath sonicator for 10 min. Emulsion 2. 200 mg of EYP was dissolved in 15 ml of diethyl ether solution in a 25-ml vial with a screw-cap. 1.5 ml of solution A was rapidly injected into the lipid solution as described above. The solutions were mixed by use of a vortex mixer for 30 s and sonicated in a water-bath sonicator for 20 min. 3. Mixing the emulsion with the freeze-dried gel beads. The emulsion 1 or 2 was immediately transferred to the 25-ml flask containing 76 mg of freeze-dried gel beads. The flask was closed with a stopper and gently shaken for 1-3 h at room temperature. 4. Removal of the organic solvent by rotary evaporation. The flask containing the mixture of the emulsion and the gel beads was connected to a rotary evaporator. For the mixture of emulsion 1 and the gel beads, evaporation was done with gentle rotation under water-jet-vacuum with maximal flow of water through the pump, at 37 β C. The drying was continued for about 10 min until the mixture was dry. For the mixture of emulsion 2 and the gel beads, evaporation was done with much lower flow of water through the water-jet pump. This drying was continued for 3-4 h until a gel-like mixture was formed. 5. Formation of a liposome suspension. The final dried material was mixed vigorously by use of a vortex mixer and then, if needed, 0.5-1.5 ml of solution A was added to the materials. Some of the liposomes formed inside the gel-bead pores and had a sufficient size in relation to the pore size and thus became immobilized. To remove the non-immobilized EYP liposomes, the same washing procedures as in Example 1 above were used. EXAMPLE 6 Steric immobilization of liposomes in σel beads by reverse phase evaporation with solvent-dried gel beads 1. Solvent-dried σel beads. The gel used was Sephacryl S-1000 in an amount corresponding to 0.5-ml packed volume. The gel was washed with 50 ml of each of 20%, 50% and 98% ethanol, followed by 50 ml of diethyl ether, to remove most of the water in the gel matrix. The solvent-dried gel beads were placed in a 50-ml round- bottomed flask. 2. Emulsifying an aqueous solution in an organic solution of EYP bv sonication. Appropriate amounts (usually 40-100 mg) of EYP was dissolved in 5 ml of diethyl ether in a 100-ml flask. Solution A or B with an appropriate volume (usually 0.6-2 ml) was added into the organic phase followed by water-bath sonication in a coldroom (5 C C) under protection of nitrogen gas for 5-10 min until a clear emulsion was obtained. Such emulsion did not separate into two phases at room temperature during 30 min. 3. Rotary evaporation. The emuslion was transferred to the 50-ml flask containing the solvent-dried gel beads. The flask was then connected to a rotary evaporator with a membrane pump and a vacuum controller. Most of the ether in the mixture was removed at 650-700 mbar by vacuum suction at 25°C while rotating the flask at 200 rpm for 30-40 min, until the mixture formed a shell on- the wall in the flask. For brevity the mixture in this stage is termed the "shell mixture". Evaporation was continued for 15- 30 min under the same experimental conditions except that the vacuum pressure was 450-500 mbar. During the continued evapora¬ tion the shell mixture was converted to a viscous suspension as liposomes were formed, in case of emulsions which contained a relatively large volume (≥l.O ml) of aqueous solution. For emulsions with less than 1.0 ml of an aqueous solution the conversion from the shell mixture to the suspension occurred concomitant with formation of liposomes when the shell mixture was agitated vigorously by use of a vortex mixer, or when extra aqueous solution was added to the shell mixture. During the conversion process the solvent-dried gel beads swoll to some extent upon hydration with water released from the emulsion. Some of the liposomes which were formed inside the bead-pores had sufficient size in realtion to the pore size and thus became immobilized. 4. Removal of non-immobilized liposomes. Non-immobilized liposomes were removed by centrifugation and by column-washing as described i Example 1 above. Procedures 1-4 above could be done in about 2 h. RESULTS Steric immobilization by freeze-thawinσ Suction-dried (large-pore) gel beads In Figure 1, the effect of liposome (phospholipid) concentration on immobilization capacities upon freezing and thawing is illustrated with reference to Example 1. The curves represent immobilization in Sephacryl S-1000 (♦ and •) and in Sepharose 2B (A ). Freezing and thawing was performed by using solution B containing 10 mM calcein (♦), and by using solution A (•, o, A Δ, and *). Control experiments were performed under the same experimental conditions as for the freeze-thawing immobilization but without freezing and thawing of the mixture of liposomes and gel beads (o and Δ). (*) shows the immobilization capacity, of proteoliposomes (with red cell membrane proteins) in Sephacryl S-1000 by freezing-thawing. (!) shows the immoblization capacity in Sepharose 2B following the dialysis technique according to prior art cited above. As appears from the figure, the immobilization capacity increased almost linearly from 6.5 to 80 and from 3.5 to 40 μmol of phospholipids per milliliter of gel for Sephacryl S-1000 and for Sepharose 2B gels, respectively, with an increase in the liposome concentration up to 300 mM phospholipids (• and A in Fig. 1, respectively). The capacity of immobilizing proteoliposomes with red cell membrane proteins was slightly lower than for liposomes without proteins (*, Fig. 1). The amounts of EYP liposomes immobilized upon freezing and thawing were approximately twice as high in Sephacryl S-1000 (•, Fig. 1) as in Sepharose 2B gel ( A, Fig. 1). Sephacryl S-1000 gel beads of allyldextran cross- linked with N,N-methylene-bis-acryl-amid has large pores and may be more accessible to large liposomes formed by freeze-thawing. On the other hand, the higher immobilization capacity of Sephacryl S-1000 could be due to the fact that it was easier to drain more water from Sephacryl S-1000 than from Sepharose 2B gel by a water-jet pump. This might result in the uptake of more liposomes into the bead-pores of Sephacryl S-1000 compared to Sepharose 2B. Control experiments showed that less than 1.3 and 0.7 μmol of phospholipids were adsorbed on Sephacryl S-1000 and on Sepharose 2B gels (o and Δ, Fig. 1), respectively, when liposomes .were mixed with the beads without subsequent freeze- thawing. By using solution B, wherein the 10 mM Tris of solution A was replaced by 10 mM calcein, it was found that, upon increasing the liposome concentration from 80 to 190 mM phospholipids, the immobilization capacity increased sharply from 20 to 80 μmol phospholipids per ml gel (♦, Fig. 1), corresponding to an increase of up to 30%. compared with the capacity when using solution A (•, Fig. 1) in this concentration range. We observered that the viscosity of the concentrated liposome solution decreased significantly in the presence of the solution B. Obviously this increased the rate of diffusion of the liposome into the gel pores. Suction-dried (small-pore) gel beads Liposomes could also be immobilized in some gels with relatively small pores by freeze-thaw immobilization according to Example 1 as shown below in Table 1. Table 1 Immobilization capacit ' of EYP liposomes in small-pore gel beads upon freeze-thaw procedures. Gels Amount of suction- Concentration of phosp o- Immobilization capacity dried gel * 1 ipids in the suspension of added EYP liposomes ^JIDI pnosDnoiiDiαs/mi gel (ς) (mM) Sepharose 65 0.7 295 54 Sepharose CL-6B 0.66 300 61 Suπerose m 6 0.57 295 45 Sephacryl 5-400 0.56 300 56 # All the amounts of suction-dried gels correspond to 0.5 ml packed gel volume under the experimental conditions used. The immobilization capacities of these gel were higher at 300 mM lipid concentration than was the 35 μmol/ml capacity of Sepharose 2B (A. Fig. 1), but lower than the corresponding 78 μmol/ml capacity of Sephacryl S-1000 (β, Fig. 1). Freeze-dried gel beads In Example 2, still higher capacities than in Example 1 were obtained when the gel beads were freeze-dried before mixing with liposome suspensions. The concentration of phospholipids in the liposome suspensions needed for high immobilization capacity was thus lower than in the case of suction-dreid moist gel beads. The results are shown below in Table 2. Table 2 Immobilization capacity in Sephacryl S-1000 gel beads upon freezing and thawing of the mixture of freeze-dried beads and EYP liposomes. Amount of Concentration of phospho- Amount of immobilized Immobilization capacity freeze-απeα gela lipids in the suspension of phospholipids added EYP liposomes (mg) (mM) (iimol) (μmol phospholipids/ml gel) 76 93 76 200 76 252 76 270 a 76 mg of freeze-dried gel corresponds to 0.5 ml of packed gel volume under the experimental conditions used. b The specific internal volume was determined by use of en¬ capsulated 10 mM calcein. It was 0.88 μl per μmol of phospholi¬ pids. Compared to the corresponding concentrations of Example 1 (see Fig. 1, •), the capacities increased by approximately 50% with a liposome concentration of 270 mM, and by approximately 120% with concentrations of 93 and 200 mM. Thus, a higher capacity was obtained at relatively lower concentration of liposomes. Steric immobilization by freeze-dryinq and rehvdration Suction-dried gel beads The effect of the total amounts of phospholipids on immobiliza¬ tion capacities in Sephacryl S-1000 gel beads upon freeze-drying and rehydration are shown in Figure 2 with reference to Example 3. The lipid concentrations of the EYP liposome suspension that was mixed with the suction-dried gel beads prior to freeze-drying were 39, 165 and 255 mM, with the volumes of the suspensions being 2, 1 and 0.55 ml, respectively. The corresponding amounts of lipids are given on the x-axis. Figure 2 shows a relatively high immobilization capacity, 62 μmol per ml gel, following freeze-drying and rehydration of a liposome suspension comprising 165 μmol phospholipids. It was noted that the capacities were increased by increasing the amounts of phospholipids in the above mentioned liposome suspensions having varying concentrations and volumes. Compared to the freeze- thawing procedure according to Example 1, an advantage of immobilization following freeze-drying and rehydration seems to be that high capacity can be obtained by using a lower concen¬ tration of liposomes. Freeze-dried gel beads In Example 4, as Table 3 shows, high capacities were also obtained. Table 3 _ _ Immobilization capacity in Sephacryl S-1000 gel beads upon freeze-drying and rehydration of the mixture of freeze-dried beads and EYP liposomes. Amount of immobilized Immobilization capacity phospholipids (iimol) (μmol phospholipids/ml gel) 52^ 104 56.3 1 13- # The specific internal volume was determined by use of en¬ capsulated 10 mM calcein. It was 0.54 μl per μmol of phospholipids. However, the specific internal volume of the liposomes was 38% lower than that obtained by the procedure in Example 2. Steric immobilization by reverse phase evaporation I. Freeze-dried σel beads Liposomes could also be immobilized in Sephacryl S-1000 gel beads by steric immobilization according to Example 5. The resulting immobilization capacity by using Emulsion 1 in Example 5 was 128 μmol of phospholipids per μl gel. Internal volume Figure 3 shows the total internal volumes and the specific internal volumes of immobilized EYP liposomes at varying concentrations. EYP liposomes with encapsulated 10 mM calcein were immobilized in Sephacryl S-1000 by freeze-thawing according to Example 1. The symbol (β) denotes specific internal volume of the immobilized liposomes expressed in μl per μmol phospholipid. The symbol (o) denotes the total volume in μl per ml gel, which is calculated by multiplying the immobilization-capacities (data from Fig. 1, ♦) with the specific internal volumes (•). The specific internal volumes of the immobilized liposomes expressed in μl per μmol of phospholipids decreased gradually with an increase in the liposome concentration (•, Fig. 3). The liposome concentration thus affects the freeze-thaw fusion process. The average size of the immobilized liposomes may decrease with increasing liposome concentration, but it is also possible that, the liposomes become multilamellar or multi- vesicular to a larger extent. It is known that small unilamellar vesicles prepared by sonication increase markedly in average size and in trapped volume upon freezing and thawing and upon freeze- drying and rehydration. It has also been reported that the trap¬ ping volume decreases in the presence of divalent metal ions like Ca 2* . Similarly, the trapping volume (• in Fig. 3) might have been lowered because Solution B contains 10 mM calcein, which has a net negative charge of -3. The total internal volume expressed in μl per ml gel increased (o. Fig. 3) since the increase of the immobilization capacity (see ♦, Fig. 1) was larger than the decrease in specific internal volume. The total internal volumes of 144 and 150 μl per ml gel obtained for liposome concentrations of 190 and 293 mM, respectively, were 50% higher than the value, 74 μl per ml gel, estimated for the medium vesicles (100 nm in average diameter) immobilized by hydrophobic interaction on octyl sulfide-Sephacryl S-1000 according to prior art. II. Solvent-dried σel beads with modification of the procedure. Liposomes could also be immobilized in Sephacryl S-1000 gel beads according to Example 6 as illustrated in Fig. 4, for the case of emulsification of 100 mg of EYP in an organic phase with the aqueous solution B, which contains calcein for determination of the internal volume. The specific internal volume increased from 1.1 to 6.1 μl water per μmol phospholipid with a decrease of capacity from 60 to 12 μmol phospholipids per ml packed gel, when the volume of aqueous solution added for emulsification was increasd from 0.8 to 2.0 mi ' , as shown in Fig. 4A. Large liposomes. with a specific internal volume of 4.9-6.1 μl were immobilized and relatively high capacities were maintained. The total internal volume of the immobilized liposomes increased gradually from 55 to 88 μl water per ml packed gel upon an increase of the -addition of the aqueous solution from 0.8 to 1.6 ml (Fig. 4B). However, further increase of this volume caused the total internal volume to decrease slightly (Fig. 4B). Proteoliposomes immobilized at approximatively 10 μmol phospholipids per ml gel could be prepared by addition of integral membrane proteins from human red cells, solubilized in octyl glucoside to the immobili¬ zed liposomes pre-equilibrated with a low concentration of octyl glucoside. Applications of the invention The gel beads or similar materials produced according to the methods of the invention can be used as chromatographic media intended for a variety of applications as is apparent for a person skilled in the art. Recent review articles are: Protein, Nuclic Acid and Enzyme 35 (1990) p 1983-1998, Kyoritsu Shuppan, Tokyo; and J. Chromatogr., Liposome chromatography: Liposomes immobilized in gel beads as a stationary phase for aqueous column chromatography, Per Lundahl and Qing Yang, in press. Application can include biotechnical purposes, chemical synthesis and chemical analysis. For chemical synthesis catalytically active substances or macromolecules are incorporated into the liposomes or into their bilayers. Beads or particles with or without included, inserted or adsorbed substances or macromolecules are used for chemical analysis. It is also contemplated to incorporate one or more pharmacologi¬ cally active drug(s) into the liposomes prior to or upon immobilizing thereof in gel beads or similar materials. This is readily accomplished by mixing the drug(s) with the liposomes or the lipid material upon or prior to immobilization. The obtained gel beads or similar materials can then be implanted or injected in the human or animal body to serve as a drug delivery system having prolonged release of the drug(s) in vivo. Alternatively, they can be applicated, in any convenient form, onto the skin or other parts of the body to perform their intended action by diffusion of the drug(s). Similarly, immobilized liposomes with or without incorporated or encapsulated agents can be used for cosmetic purposes. Liposomes are well known as components in several cosmetic preparations and their action can be controlled by immobilization.

Claims: CLAIMS 1. Method of immobilizing liposomes and/or proteoliposomes in cross-linked or non-cross-linked material having a three- dimensional network that forms internal pores or cavities, comprising the following steps: a) preparing a suspension of liposomes and/or proteoliposomes, or an emulsion of lipids, or lipids and protein(s), and water in organic solvent; b) mixing the material with the suspension or the emulsion; c) increasing the average size of the liposomes and/or pro¬ teoliposomes which have entered the material, or effect uptake and association of emulsified lipids [and protein(s)] in the material by evaporating the organic solvent, whereby. liposomes and/or proteoliposomes are formed; thereby trapping or enclosing liposomes and/or proteoliposomes within said three-dimensional network; and d) removing non-immobilized liposomes and/or proteoliposomes. 2. Method according to claim 1, wherein the average liposome (proteoliposome) size is increased by fusion of liposomes (proteoliposomes), and/or by association of liposome (proteolipo¬ some) fragments to larger liposomes (proteoliposomes) . 3. Method according to one or both of claims 1 and 2, wherein the liposome suspension is prepared to have a 50-350 mM con¬ centration of lipids. 4. Method according to one or more of claims 1-3, wherein, the material with the three-dimensional network consists of gel beads or gel particles for example of types that have been pre- manufactured for use as packing material for chromatography or as cell carriers in cell cultures, or of similar beads or particles, and that the liposomes, or the lipids from the emulsion, enter the material by diffusion and/or aspiration and/or upon swelling of the material. 5. Method according to claim 4, wherein the material is freeze- dried prior to step b). 6. Method according to claim 4, wherein the material is suction-dried prior to step b), and wherein step c) is performed by freezing and thawing of a mixture between the material and liposomes and/or proteoliposomes. 7. Method according to claim 4, wherein the material is depleted cf water by washing with organic solvent prior to step b). 8. Method according to claim 5, wherein step c) is performed by freezing and thawing of a mixture between the material and liposomes and/or proteoliposomes. 9. Method according to one or more of claims 1-4, wherein step c) is performed by freeze-drying of a mixture between the material and liposomes and/or proteoliposomes, and subsequent rehydration. 10. Method according to claim 9, wherein freezing and subsequent thawing of the mixture of the liposomes (proteoliposomes) and the material is performed prior to step d). 11. Method according to claim 9 or 10, wherein the material in step-b) is suction-dried. 12. Method according to claim 9 or 10, wherein the material in step b) is freeze-dried. 13. Method according to one ore more of the claims 1-12, wherein the liposomes are provided with one or more pharmacologically active drug(s) prior to or upon immobilization. 14. Beads or particles of non-cross-linked .or cross-linked gel, cr other similar beads or particles, comprising immobilized liposomes, wherein the liposomes ar immobilized within the pores or cavities of the three-dimensional network of tne material according to one or more of the claims 1-13. 15. Beads or particles accordning to claim 14, wherein the amount of immobilized liposomes and/or proteoliposomes corre¬ sponds to at least 20 μmol of lipids per ml gel. 16. Gel beads according to claims 14 or 15, wherein the total internal volume of the immobilized liposomes and/or proteoliposo¬ mes is at least 50 μl per ml gel. 17. Gel beads or gel particles according to claims 14, 15 or 16, wherein the gel consists of one or more of the following components or derivatives of these: agarose; allylpolysaccharides cross-linked with N,N'-methylene-bis-acrylamide; cellulose; dextran; gelatin; glucomannan; polyacrylamide; and silica. 18. Beads or particles according to claims 14, 15 or 16, wherein the material is a ceramic material or glass. 19. Beads or particles according to one or more of the claims 14-18, wherein one or more natural or artificial membrane protein(s) is (are) incorporated into the lipid bilayers. 20. Beads or particles according to one or more of the claims 14-19, wherein catalytically active substances or macromolecules are incorporated into the liposomes or into their bilayers. 21. Beads or particles according to one or more of the claims 14-19, wherein one or more pharmacologically active drug(s) is (are) incorporated in the liposomes. 22. Use cf beads or particles according to one or more of the claims 14-20 for biotechnological purposes. 23. Use cf beads or particles according to claim 22 for chromatography. 24. Use of beads or particles according to claim 22 with or without included, inserted or adsorbed substances or macromolecu¬ les for chemical analysis. 25. Use of beads or particles according to claim 21, for medical or veterinary purposes. 26. Use of beads or particles according to one or more of the claims 14-21 for cosmetic purposes. SUBSTITUTE

Inventors: GREIJER EVA, LUNDAHL PER, SANDBERG MARIA, YANG QING, GREIJER, EVA, LUNDAHL, PER, SANDBERG, MARIA, YANG, QING
Applicants: GREIJER EVA, LUNDAHL PER, SANDBERG MARIA, YANG QING, GREIJER, EVA, LUNDAHL, PER, SANDBERG, MARIA, YANG, QING
Application year: 1991
Main classification: B01J20
IPC codes: A61K8, A61K9, A61K47, A61Q19, B01J13, B01J20, C12M1, G01N1, G01N30