Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
  • B01J20/291—Gel sorbents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
  • A61K8/14—Liposomes; Vesicles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Liposomes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
  • A61K9/1605—Excipients; Inactive ingredients
  • A61K9/1629—Organic macromolecular compounds
  • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
  • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
  • B01J20/28016—Particle form
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
  • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
  • B01J20/28047—Gels
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2220/00—Aspects relating to sorbent materials
  • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
  • B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography

Definitions

• 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.
• 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 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.
• Solution A 150 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl (pH 7.4)
• 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.
• 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 1 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.
• the hydrated mixture can be freeze-thawed to increase the immobilization capacity.
• Example 2 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.
• the gel was freeze-dried as described in Example- 2.
• 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.
• 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.
• 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.
• ⁇ l.O ml a relatively large volume of 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.
• the solvent-dried gel beads swoll to some extent upon hydration with water released from the emulsion.
• 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.
• the immobilization capacity increased almost linearly from 6.5 to 80 and from 3.5 to 40 ⁇ mol 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.
• 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 ⁇ mol of phospholipids were adsorbed on Sephacryl S-1000 and on Sepharose 2B gels (o and ⁇ , Fig. 1), respectively, when liposomes .were mixed with the beads without subsequent freeze- thawing.
• 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
• the immobilization capacities of these gel were higher at 300 mM lipid concentration than was the 35 ⁇ mol/ml capacity of Sepharose 2B (A. Fig. 1), but lower than the corresponding 78 ⁇ mol/ml capacity of Sephacryl S-1000 ( ⁇ , Fig. 1).
• Example 2 still higher capacities than in Example 1 were obtained when the gel beads were freeze-dried before mixing with liposome suspensions.
• Table 2 Table 2
• a 76 mg of freeze-dried gel corresponds to 0.5 ml of packed gel volume under the experimental conditions used.
• the specific internal volume was determined by use of en ⁇ capsulated 10 mM calcein. It was 0.88 ⁇ l per ⁇ mol of phospholi ⁇ pids.
• Example 1 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.
• Figure 2 shows a relatively high immobilization capacity, 62 ⁇ mol per ml gel, following freeze-drying and rehydration of a liposome suspension comprising 165 ⁇ mol 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.
• Example 4 as Table 3 shows, high capacities were also obtained.
• the specific internal volume was determined by use of en ⁇ capsulated 10 mM calcein. It was 0.54 ⁇ l per ⁇ mol of phospholipids. However, the specific internal volume of the liposomes was 38% lower than that obtained by the procedure in Example 2.
• Liposomes could also be immobilized in Sephacryl S-1000 gel beads by steric immobilization according to Example 5.
• Example 5 was 128 ⁇ mol of phospholipids per ⁇ l gel.
• 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 ( ⁇ ) denotes specific internal volume of the immobilized liposomes expressed in ⁇ l per ⁇ mol phospholipid.
• the symbol (o) denotes the total volume in ⁇ l per ml gel, which is calculated by multiplying the immobilization-capacities (data from Fig. 1, ⁇ ) with the specific internal volumes (•).
• 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.
• 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 ⁇ l water per ⁇ mol phospholipid with a decrease of capacity from 60 to 12 ⁇ mol 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.
• Proteoliposomes immobilized at approximatively 10 ⁇ mol 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.
• 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.
• 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).
• 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.

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• 1990
  • 1990-11-21 SE SE9003706A patent/SE9003706L/sv not_active Application Discontinuation
• 1991
  • 1991-11-20 WO PCT/SE1991/000788 patent/WO1992009267A1/en not_active Application Discontinuation
  • 1991-11-20 AU AU89268/91A patent/AU8926891A/en not_active Abandoned
  • 1991-11-20 EP EP19910920486 patent/EP0559698A1/en not_active Withdrawn
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