METHOD OF ENCAPSULATING ACTIVE INGREDIENTS IN LIPOSOMES

Abstract

A method for encapsulating active ingredients in liposomes having an active ingredient solution encapsulated with a bilayer composed of two monomolecular layers of amphiphilic compounds comprises: (a) providing the active ingredient solution; (b) providing an emulsion by emulsifying the active ingredient solution in a first liquid in the presence of the amphiphilic compound; (c) providing a liquid phase; (d) contacting the emulsion with the liquid phase to form a phase boundary; and (e) centrifuging the emulsion and the liquid phase that are in contact with one another via the phase boundary, wherein, on passage of the phase boundary, the amphiphilic compound enriched there is added onto the monomolecular inner layer to form a monomolecular outer layer, in order to create the bilayer.
The first liquid of the emulsion is chosen such that the solubility of the amphiphilic compound in the first liquid is not more than 1×10.sup.−4 mol/l.

Claims

1. A method of encapsulating active ingredients in liposomes, comprising: a solution, especially a hydrophilic solution, of the active ingredient and at least one bilayer composed of two at least monomolecular layers of at least one first amphiphilic compound, especially from the group of the lipids, wherein the active ingredient solution is encapsulated by the at least one bilayer, comprising the following steps: (a) providing the active ingredient solution, especially a hydrophilic active ingredient solution, of the active ingredient to be encapsulated by dissolving the active ingredient in at least one solvent, especially a hydrophilic solvent; (b) providing a first emulsion by emulsifying the active ingredient solution from step (a) in at least one first liquid, especially a hydrophobic first liquid, having zero or sparing miscibility with the at least one solvent of the active ingredient solution, in the presence of the at least one first amphiphilic compound, in order to accumulate an at least monomolecular inner layer of the at least one first amphiphilic compound onto droplets of the active ingredient solution emulsified in the at least one first liquid; (c) providing a liquid phase, especially a hydrophilic liquid phase, having zero or sparing miscibility with the at least one first liquid of the first emulsion from step (b); (d) contacting the first emulsion composed of the at least one first liquid and having the droplets of the active ingredient solution that are emulsified therein and have the at least monomolecular inner layer of the at least one first amphiphilic compound that has been accumulated thereon from step (b) with the liquid phase from step (c) to form a phase boundary between the first emulsion from step (b) and the liquid phase from step (c), wherein the at least one first amphiphilic compound is enriched at the phase boundary; and (e) centrifuging the first emulsion from step (b) and the liquid phase from step (c) that are in contact with one another via the phase boundary, in order to transfer the droplets of the active ingredient solution present in the first emulsion and having the at least monomolecular inner layer of the at least one first amphiphilic compound that has been accumulated thereon from the at least one first liquid of the first emulsion from step (b) into the liquid phase from step (c), wherein, when the droplets of the active ingredient solution having that at least monomolecular inner layer of the at least one first amphiphilic compound that has been accumulated thereon passes the phase boundary, the at least one first amphiphilic compound enriched there is accumulated on the at least monomolecular inner layer of the at least one first amphiphilic compound of the droplets of the active ingredient solution to form an at least monomolecular outer layer thereof, in order to produce the at least one bilayer composed of the two at least monomolecular layers of the at least one first amphiphilic compound, wherein the at least one first liquid of the first emulsion from step (b) is chosen such that the solubility of the at least one first amphiphilic compound in the at least one first liquid is not more than 1×10.sup.−4 mol/l.

2. The method as claimed in claim 1, wherein the at least one first liquid of the first emulsion from step (b) is chosen such that the solubility of the at least one first amphiphilic compound in the first liquid is not more than 0.5×10.sup.−4 mol/l, especially not more than 1×10.sup.−3 mol/l, preferably not more than 1×10.sup.−6 mol/l.

3. A method of encapsulating active ingredients in liposomes, comprising: a solution, especially a hydrophilic solution, of the active ingredient and at least one bilayer composed of two at least monomolecular layers of at least one first amphiphilic compound and at least one second amphiphilic compound, especially each from the group of the lipids, wherein the active ingredient solution is encapsulated by the at least one bilayer, comprising the following steps: (a) providing an active ingredient solution, especially a hydrophilic active ingredient solution, of the active ingredient to be encapsulated by dissolving the active ingredient in at least one solvent, especially a hydrophilic solvent; (b) providing a first emulsion by emulsifying the active ingredient solution from step (a) in at least one first liquid, especially a hydrophobic first liquid, having zero or sparing miscibility with the at least one solvent of the active ingredient solution, in the presence of the at least one first amphiphilic compound, in order to accumulate an at least monomolecular inner layer of the at least one first amphiphilic compound on droplets of the active ingredient solution emulsified in the at least one first liquid; (c) providing a mixture of a liquid phase, especially a hydrophilic liquid phase, having zero or sparing miscibility with the at least one first liquid of the first emulsion from step (b) with the at least one second amphiphilic compound; (d) contacting the first emulsion composed of the at least one first liquid and having the droplets of the active ingredient solution that are emulsified therein and have the at least monomolecular inner layer of the at least one first amphiphilic compound that has been accumulated thereon from step (b) with the mixture of the liquid phase with the at least one second amphiphilic compound from step (c) to form a phase boundary between the first emulsion from step (b) and the mixture from step (c), wherein at least the at least one second amphiphilic compound is enriched at this phase boundary; and (e) centrifuging the first emulsion from step (b) and the mixture from step (c) that are in contact with one another via the phase boundary, in order to transfer the droplets of the active ingredient solution present in the first emulsion and having the at least monomolecular inner layer of the at least one amphiphilic compound that has been accumulated thereon from the at least one first liquid of the first emulsion from step (b) into the liquid phase of the mixture from step (c), wherein, when the droplets of the active ingredient solution having that at least monomolecular inner layer of the at least one first amphiphilic compound that has been accumulated thereon passes the phase boundary, the at least one second amphiphilic compound enriched there is accumulated on the at least monomolecular inner layer of the at least one first amphiphilic compound of the droplets of the active ingredient solution to form an at least monomolecular outer layer thereof, in order to produce the at least one bilayer composed of the two at least monomolecular layers of the at least one first amphiphilic compound and the at least one second amphiphilic compound, wherein the at least one first liquid of the first emulsion from step (b) is chosen such that the solubility of the at least one first amphiphilic compound in the at least one first liquid is not more than 1×10.sup.−4 mol/l.

4. The method as claimed in claim 3, wherein the at least one first liquid of the first emulsion from step (b) is chosen such that the solubility both of the at least one first amphiphilic compound and of the at least one second amphiphilic compound in the at least one first liquid is not more than 1×10.sup.−4 mol/l.

5. The method as claimed in claim 3, wherein the at least one first liquid of the first emulsion from step (b) is chosen such that the solubility at least of the at least one first amphiphilic compound, especially both of the at least one first amphiphilic compound and of the at least one second amphiphilic compound, in the first liquid is not more than 0.5×10.sup.−4 mol/l, especially not more than 1×10.sup.−5 mol/l, preferably not more than 1×10.sup.−6 mol/l.

6. The method as claimed in claim 3, wherein the mixture of the liquid phase, especially the hydrophilic liquid phase, with the at least one second amphiphilic compound used in step (c) is a second emulsion of the liquid phase and at least one second liquid, especially a hydrophobic second liquid, having zero or sparing miscibility therewith with the at least one second amphiphilic compound, in that the at least one second liquid is emulsified in the liquid phase in the presence of the at least one second amphiphilic compound in order to accumulate an at least monomolecular layer of the at least one second amphiphilic compound on droplets of the second liquid emulsified in the liquid phase and to immobilize the at least one second amphiphilic compound on these droplets in this way; and on centrifugation of the first emulsion from step (b) and the mixture in the form of the second emulsion from step (c) that are in contact with one another via the phase boundary the droplets of the at least one second liquid with the at least monomolecular layer of the at least one second amphiphilic compound accumulated thereon are constantly transferred from the liquid phase of the second emulsion to the phase boundary between the first emulsion and the second emulsion, in order to constantly enrich the at least one second amphiphilic compound at this phase boundary.

7. The method as claimed in claim 6, wherein the at least one second liquid, especially the hydrophobic second liquid, having zero or sparing miscibility with the liquid phase, especially the hydrophilic liquid phase, of the second emulsion is chosen to correspond to the at least one first liquid, especially hydrophobic first liquid, of the first emulsion from step (b).

8. The method as claimed in claim 6, wherein the mixture in the form of the second emulsion from step (c) is created by first providing a mixture of the liquid phase from step (c) and the at least one second amphiphilic compound; and then emulsifying the second liquid in this mixture to form the second emulsion, by dispersing the at least one second liquid into this mixture.

9. The method as claimed in claim 1, wherein the first emulsion from step (b) is created by first providing a mixture of the active ingredient solution of the active ingredient to be encapsulated from step (a) and the at least one first amphiphilic compound; and then emulsifying this mixture to form the first emulsion from step (b) in the at least one first liquid by dispersing the mixture into the at least one first liquid.

10. The method as claimed in claim 1, wherein the at least one first liquid used in the first emulsion from step (b) is a hydrophobic liquid which is especially selected from the group of: liquid, partly or wholly halogenated hydrocarbons, including the fluorocarbons, silicone oils, and siloxanes, including mixtures thereof.

11. The method as claimed in claim 1, wherein the at least one solvent used in the active ingredient solution from step (a) is a hydrophilic solvent, especially a water- and/or alcohol-based solvent.

12. The method as claimed in claim 1, wherein: the at least one first liquid of the first emulsion with the droplets of the active ingredient solution emulsified therein and having the at least monomolecular inner layer of the at least one first amphiphilic compound accumulated thereon from step (b) is chosen such that it has a lower melting point than the active ingredient solution; the first emulsion from step (b) is cooled down to a temperature between the melting point of the at least one first liquid of the first emulsion and the melting point of the active ingredient solution, in order to convert the droplets of the active ingredient solution emulsified in the at least one first liquid and having the at least monomolecular inner layer of the at least one first amphiphilic compound accumulated thereon from step (b) to the solid state; and then the first emulsion from step (b) in the solid state of the active ingredient solution is contacted with the liquid phase or mixture of the liquid phase with the at least one second amphiphilic compound that has zero or sparing miscibility with the at least one first liquid of the first emulsion from step (b), to form the phase boundary according to step (d), and the first emulsion from step (b) and the liquid phase or the mixture of the liquid phase with the at least one second amphiphilic compound that are in contact with one another via the phase boundary in step (e) are centrifuged.

13. The method as claimed in claim 12, wherein the active ingredient solution having the at least monomolecular inner layer of the at least one first amphiphilic compound accumulated thereon is kept in the solid state during the centrifuging, in order to move it, on account of a resultant difference in density, from the phase boundary in the direction of the liquid phase or the mixture of the liquid phase with the at least one second amphiphilic compound.

14. The method as claimed in claim 1, wherein the at least one bilayer composed of the two at least monomolecular layers of the at least one first amphiphilic compound or the two at least monomolecular layers of the at least one first amphiphilic compound and of the at least one second amphiphilic compound, especially exclusively the outer at least monomolecular layer of the bilayer, is modified by reaction with hydrophilic polymer conjugates.

15. The method as claimed in claim 1 is performed batchwise in a batchwise centrifugation device; semicontinuously in a batchwise centrifugation device; or continuously in a flow-operated continuous centrifugation device.

16. The method as claimed in claim 3, wherein the first emulsion from step (b) is created by: first providing a mixture of the active ingredient solution of the active ingredient to be encapsulated from step (a) and the at least one first amphiphilic compound; and then emulsifying this mixture to form the first emulsion from step (b) in the at least one first liquid by dispersing the mixture into the at least one first liquid.

17. The method as claimed in claim 3, wherein the at least one first liquid used in the first emulsion from step (b) is a hydrophobic liquid which is especially selected from the group of: liquid, partly or wholly halogenated hydrocarbons, including the fluorocarbons, silicone oils, and siloxanes, including mixtures thereof.

18. The method as claimed in claim 3, wherein the at least one solvent used in the active ingredient solution from step (a) is a hydrophilic solvent, especially a water- and/or alcohol-based solvent.

19. The method as claimed in claim 3, wherein the at least one first liquid of the first emulsion with the droplets of the active ingredient solution emulsified therein and having the at least monomolecular inner layer of the at least one first amphiphilic compound accumulated thereon from step (b) is chosen such that it has a lower melting point than the active ingredient solution; the first emulsion from step (b) is cooled down to a temperature between the melting point of the at least one first liquid of the first emulsion and the melting point of the active ingredient solution, in order to convert the droplets of the active ingredient solution emulsified in the at least one first liquid and having the at least monomolecular inner layer of the at least one first amphiphilic compound accumulated thereon from step (b) to the solid state; and then the first emulsion from step (b) in the solid state of the active ingredient solution is contacted with the liquid phase or mixture of the liquid phase with the at least one second amphiphilic compound  that has zero or sparing miscibility with the at least one first liquid of the first emulsion from step (b), to form the phase boundary according to step (d), and the first emulsion from step (b) and the liquid phase or the mixture of the liquid phase with the at least one second amphiphilic compound  that are in contact with one another via the phase boundary in step (e) are centrifuged.

20. The method as claimed in claim 3, wherein the at least one bilayer composed of: the two at least monomolecular layers of the at least one first amphiphilic compound or the two at least monomolecular layers of the at least one first amphiphilic compound and of the at least one second amphiphilic compound, especially exclusively the outer at least monomolecular layer of the bilayer, is modified by reaction with hydrophilic polymer conjugates.

21. The method as claimed in claim 3 is performed: batchwise in a batchwise centrifugation device; semicontinuously in a batchwise centrifugation device; or continuously in a flow-operated continuous centrifugation device.

Description

[0091] Further features and advantages of the invention will be apparent from the description that follows of working examples with reference to the drawings. The drawings show:

[0092] FIG. 1 a highly schematized view for illustration of the production of essentially symmetric liposomes by means of a first embodiment of a method of the invention for encapsulation of active ingredients in liposomes;

[0093] FIG. 2 a highly schematized view for illustration of the production of essentially asymmetric liposomes by means of a second embodiment of a method of the invention for encapsulation of active ingredients in liposomes;

[0094] FIG. 3 a schematic cross-sectional view of half the cross section of an embodiment of a centrifuge device for continuous performance of a method of encapsulation of active ingredients in liposomes;

[0095] FIG. 4 a transmission electron micrograph of symmetric liposomes composed of an active ingredient solution of 10 μg/ml of the mistletoe extract viscumin in 15 mM aqueous phosphate buffer, encapsulated in a bilayer of the lipid dipalmitoylphosphatidylcholine (DPPC), produced according to working example 1;

[0096] FIG. 5 a transmission electron micrograph of symmetric liposomes composed of an active ingredient solution of 10 μg/ml the mistletoe extract viscumin in 15 mM aqueous phosphate buffer, encapsulated in a bilayer of the lipid distearoylphosphatidylcholine (DSPC), produced according to working example 2; and

[0097] FIG. 6 a transmission electron micrograph of symmetric liposomes composed of an active ingredient solution of 10 μg/ml of the mistletoe extract viscumin in 15 mM aqueous phosphate buffer, encapsulated in a bilayer composed of the lipids dioleoylphosphatidylcholine (DOPC) and cholesterol in a molar ratio of 60:40, prepared according to working example 3.

[0098] FIG. 1 shows a situation during the formation of primarily symmetric liposomes in a first embodiment of a method of the invention for encapsulation of active ingredients in liposomes L by an enlarged detail view in a highly schematized manner. What can be seen in the right-hand section of FIG. 1 is a droplet, for example with a diameter between about 0.1 μm and about 200 μm, of an active ingredient solution 1, which has been produced by dissolving the active ingredient beforehand according to step (a) in a solvent. The solvent in the present case is, for example, a hydrophilic water- and/or alcohol-based solvent.

[0099] As additionally apparent in the right-hand section of FIG. 1, a monomolecular (inner) layer of a first amphiphilic compound 2, for example a lipid, has accumulated on the droplets of the active ingredient solution 1 to form a pre-liposome M, wherein the polar regions of the first amphiphilic compound 2 have become aligned in the direction of a hydrophilic active ingredient solution 1, and the nonpolar regions have become aligned in the direction of a first liquid 3—hydrophobic here—that has zero or only sparing miscibility with the solvent surrounding the droplets of the active ingredient solution 1. For production of a first emulsion 4 in this form, the disperse phase of which is formed by the droplets of the hydrophilic active ingredient solution 1 and the continuous phase of which by the hydrophobic first liquid 3, a mixture of the active ingredient solution 1 and the first amphiphilic compound 2, for example, has first been provided beforehand in step (b), and then this mixture has been dispersed into the first liquid 3 to obtain the first emulsion 4. The—hydrophobic—first liquid 3, which, in the present case, is a liquid halogenated hydrocarbon, for example, in the form of one or more fluorocarbons, has been chosen such that the solubility of the first amphiphilic compound 2 is less than 10.sup.−4 mol/l—here, less than 10.sup.−3 mol/l (see further details below). In addition, the first liquid 3 has preferably also been selected in such a way that it has a lower melting point than the active ingredient solution 1 emulsified in the first emulsion 4 (likewise, see further details below). The—hydrophobic—first liquid 3 also has a different density than the active ingredient solution 1, where the fluorocarbon used in the present case has a higher density than the aqueous and/or alcoholic active ingredient solution 1. At the same time, it is of course also possible in principle to use a “lighter” first liquid 3 compared to the active ingredient solution 1, i.e. one having a lower density by comparison.

[0100] As apparent from the left-hand section of FIG. 1, it is additionally possible for a liquid phase 5—hydrophilic here—having zero or sparing miscibility with the first liquid 3 of the first emulsion 4 to have been provided in step (c), which, for example, may be chosen to correspond to the solvent of the active ingredient solution 1 and consequently may, for example, likewise be aqueous and/or alcoholic.

[0101] As additionally apparent from the two sections of FIG. 1, the first emulsion 4 composed of the first hydrophobic liquid 3 comprising the pre-liposomes M composed of the droplets of the active ingredient solution 1 emulsified therein and having the monomolecular (inner) layer of the first amphiphilic compound 2 accumulated thereon from step (b) has been contacted with the liquid hydrophilic phase 5, followed by formation of a phase boundary 6 between the first liquid 3, i.e. the continuous phase of the first emulsion 4, and the liquid phase 5 on account of their sparing miscibility with one another. The first amphiphilic compound 2 which has typically been added in excess to the first emulsion 4 for this purpose has been enriched here at this phase boundary 6, and, on account of the low solubility thereof in accordance with the invention in the first liquid 3 of the first emulsion 4, there has nevertheless been no formation of an organogel as a result of excessive accumulation of the first amphiphilic compound 2 at the phase boundary 6; instead, it takes up only one or a few molecular layers.

[0102] Step (e) of the method, finally, envisages centrifugation of the first emulsion 4 and the liquid phase 5 that are in contact with one another via the phase boundary 6, in order to transfer the droplets of the active ingredient solution 1 that are present in the first emulsion 4 and comprise the monomolecular inner layer of the first amphiphilic compound 2 accumulated thereon in the direction of the arrow P.sub.1 from the first liquid 3 of the first emulsion 4 through the phase boundary 6 with the molecules of the first amphiphilic compound 2 enriched thereon into the liquid phase 5, wherein, when it passes through the phase boundary 6, the first amphiphilic compound 2 is accumulated at the monomolecular inner layer of the first amphiphilic compound 2 of the droplets of the active ingredient solution 1 to form a further monomolecular—outer—layer thereof, in order to produce the bilayer composed of two monomolecular layers of the first amphiphilic compound 2, i.e. in order to form finished liposomes L from the pre-liposomes M. The outer layer of the bilayer of the first amphiphilic compound 2 has an opposite orientation from the inner layer, meaning that the nonpolar regions of the first amphiphilic compound 2 of the outer layer point in the direction of the polar regions of the inner layer, i.e. in the direction of the encapsulated hydrophilic active ingredient solution 1 now encapsulated in a liposome L, whereas the polar regions of the outer layer of the bilayer point in the direction of the—hydrophilic—liquid phase 5 surrounding the liposome L.

[0103] Since the pre-liposomes M formed from the droplets of the (hydrophilic) active ingredient solution 1 with the inner layer of the first amphiphilic compound 2 accumulated thereon have a lower density than the surrounding (hydrophobic) first liquid 3 of the first emulsion 4, they experience a force acting in the direction of the arrows P.sub.1 in the centrifugal field, which accelerates them in centripetal direction and brings them to the phase boundary 6 covered with a very substantially monomolecular layer of the first amphiphilic compound 2. The pre-liposome M is pushed against this layer of the first amphiphilic compound 2 enriched at this phase boundary 6 such that the monomolecular layer of the first amphiphilic compound 2 enriched at the phase boundary 6 adjoins the inner layer of the first amphiphilic compound 2 accumulated on the droplets of the active ingredient solution 1, and the bilayer of the finished liposome L is formed through hydrophobic interaction of two monomolecular layers, which is in dispersed form in the (hydrophilic) liquid phase 5 in the centrifugal field after further movement in the direction of the arrows P.sub.1. In the case of a primarily ball-shaped or spherical envelope, the droplet of the active ingredient solution 1 or the pre-liposome M consequently gives rise to a symmetric liposome L having both an inner and an outer layer of the first amphiphilic compound 2 in the bilayer that forms its membrane. At the same time, it should be pointed out here that, rather than a (single) first amphiphilic compound 2, it is of course also possible to use a mixture of such compounds, for example a mixture of multiple lipids, which then respectively form the inner and outer layers of the bilayer of the liposome L.

[0104] After more or less complete formation of liposomes L, it is finally possible to separate the liquid phase 5 in which the liposomes L are dispersed from the first liquid 3, which is possible in a simple manner because the miscibility of the hydrophilic liquid phase 5 with the hydrophobic first liquid 3 is very sparing at most, and because of the different density.

[0105] For the reasons mentioned above, it is additionally possible that the first emulsion 4 is cooled to a temperature between the melting point of the (hydrophobic) first liquid 3 and the melting point of the (hydrophilic) active ingredient solution 1, in order to convert the active ingredient solution 1 of the droplets emulsified in the first liquid 3 with the monomolecular inner layer of the first amphiphilic compound 2 accumulated thereon to the solid state, after which the first emulsion 4 in the solid state of the droplets of the active ingredient solution 1 is contacted with the (hydrophilic) liquid phase 5 to form the phase boundary 6, and the first emulsion 4 and the liquid phase 5 that are in contact with one another via the phase boundary 6 are centrifuged, especially keeping the droplets of the active ingredient solution 1 constantly in the solid state of matter.

[0106] In addition, it is possible, for example, where required to modify the finished liposomes L with polymer conjugates, for example those based on polyethylene glycol (PEG), by attaching them by electrostatic means, for example, to the first amphiphilic compound 2 of the outer layer of the bilayer (not shown).

[0107] FIG. 2, in which components corresponding to FIG. 1 have been given the same reference numerals, shows a situation during the formation of primarily asymmetric liposomes in a second embodiment of a method of the invention for encapsulating active ingredients in liposomes L by an enlarged detail view in a highly schematized manner. What can be seen here in the top right-hand section of FIG. 2—insofar as analogously to the right-hand section of FIG. 1—is a droplet, for example having a diameter between about 0.1 μm and about 200 μm, of an active ingredient solution 1, which has been produced by dissolving the active ingredient in a solvent beforehand according to step (a). The solvent in the present case is, for example, a hydrophilic water- and/or alcohol-based solvent.

[0108] As additionally apparent in the top right-hand section of FIG. 2, a monomolecular (inner) layer of a first amphiphilic compound 2, for example a lipid, has been accumulated on the droplets of the active ingredient solution 1 to form a pre-liposome M, and the polar regions of the first amphiphilic compound 2 have become aligned in the direction of the hydrophilic active ingredient solution 1, and the nonpolar regions in the direction of a first liquid 3—hydrophobic here—having zero or only sparing miscibility with the solvent surrounding the droplets of the active ingredient solution 1. For production of a first emulsion 4 in such a form, the disperse phase of which is formed by the droplets of the hydrophilic active ingredient solution 1 and the continuous phase of which is formed by the hydrophobic first liquid 3, a mixture of the active ingredient solution 1 and the first amphiphilic compound 2, for example, has first been produced beforehand in step (b), and then this mixture has been dispersed into the first liquid 3 to obtain the first emulsion 4. The—hydrophobic—first liquid 3, which, in the present case, is a liquid halogenated hydrocarbon, for example, in the form of one or more fluorocarbons has been selected here such that the solubility of the first amphiphilic compound 2 is less than 10.sup.−4 mol/l— here less than 10.sup.−3 mol/l (see further details below). In addition, the first liquid 3 has preferably also been selected such that it has a lower melting point than the active ingredient solution 1 emulsified in the first emulsion 4 (likewise see further details below). The—hydrophobic—first liquid 3 also has a different density than the active ingredient solution 1, where the fluorocarbon used in the present case has a higher density than the aqueous and/or alcoholic active ingredient solution 1. At the same time, it is of course also possible in principle to use a “lighter” first liquid 3 compared to the active ingredient solution 1, i.e. one having comparatively lower density.

[0109] As apparent from the bottom left-hand section of FIG. 2, in addition, a mixture 7 of a liquid phase—hydrophilic here—having zero or sparing miscibility with the first liquid 3 of the first emulsion 4 with a second amphiphilic compound 8, for example again in the form of a lipid, has been provided in step (c), wherein the liquid phase of this mixture 7, for example, may again be chosen to correspond to the solvent of the active ingredient solution 1 and consequently, for example, may likewise be aqueous and/or alcoholic. The mixture 7 in the present case is a second emulsion 9 composed of the liquid phase of the mixture 7 and a second liquid 10—hydrophobic here—of zero or sparing miscibility therewith and the second amphiphilic compound 8, which has been produced by emulsifying the second (hydrophobic) liquid 10 in the (hydrophilic) liquid phase of the mixture 7 in the presence of the second amphiphilic compound 8, such that the continuous phase of the second emulsion 9 is formed by the (hydrophilic) liquid phase, and the disperse phase of the second emulsion 9 by the (hydrophobic) second liquid 10, which may especially be chosen to correspond to the (hydrophobic) first liquid 3 of the first emulsion 4. In this way, a monomolecular layer of the second amphiphilic compound 8 has been accumulated on the droplets of the (hydrophobic) second liquid 10 emulsified in the (hydrophilic) liquid phase of the mixture 7, and the second amphiphilic compound 8 has consequently been immobilized on these droplets to form amphiphile carriers M′.

[0110] As is additionally apparent from all four sections of FIG. 2, the first emulsion 4 composed of the first hydrophobic liquid 3 with the pre-liposomes M composed of the droplets of the active ingredient solution 1 emulsified therein and having the monomolecular (inner) layer of the first amphiphilic compound 2 accumulated thereon from step (b) has been contacted with the second emulsion 9 composed of the hydrophilic liquid phase, in which there are emulsified the amphiphile carriers M′ composed of the droplets of the hydrophobic second liquid 10 and having the second amphiphilic compound 8 accumulated thereon, after which a phase boundary 6 has formed between the first liquid 3, i.e. the continuous phase of the first emulsion 4, and the liquid phase, i.e. the continuous phase of the second emulsion 9, owing to the sparing mutual miscibility thereof. The second amphiphilic compound 8 that has been added in excess to the second emulsion 9 for this purpose has been enriched here at this phase boundary 6, and, on account of the low solubility thereof in accordance with the invention in the first liquid 3 of the first emulsion 4 of less than 10.sup.−4 mol/l, there has nevertheless been no formation of an organogel as a result of excessive accumulation of the first amphiphilic compound 2 at the phase boundary 6; instead, it takes up only one or a few molecular layers. The enrichment of the first amphiphilic compound 2, the solubility of which in the first liquid 3 of the first emulsion 4 is likewise less than 10.sup.−4 mol/l, at the phase boundary 6 is very small at most since the proportion of the first amphiphilic compound 2 has firstly been adjusted in such a way that it has been very substantially accumulated as a monomolecular inner layer at the droplets of the active ingredient solution 1; secondly, the very low solubility of the first amphiphilic compound 2 in the first liquid 3 of the first emulsion 4 prevents excessive accumulation thereof at the phase boundary 6 or even the formation of an organogel.

[0111] Step (e) of the method finally envisages centrifugation of the first emulsion 4 and the second emulsion 9 that are in contact with one another via the phase boundary 6, in order firstly to transfer the droplets of the active ingredient solution 1 present in the first emulsion 4 with the monomolecular inner layer of the first amphiphilic compound 2 accumulated thereon in the direction of the arrows P.sub.1 from the first liquid 3 of the first emulsion 4 through the phase boundary 6 with the molecules of the second amphiphilic compound 8 enriched thereon into the liquid continuous phase of the second emulsion 9, wherein, when it passes through the phase boundary 6, the second amphiphilic compound 8 is accumulated on the monomolecular inner layer of the first amphiphilic compound 2 of the droplets of the active ingredient solution 1 to form a further monomolecular—outer—layer thereof, in order to produce the bilayer composed of two monomolecular layers, namely firstly of the first amphiphilic compound 2 (inner layer) and secondly of the second amphiphilic compound 8 (outer layer), i.e. in order to form the finished liposomes L from the pre-liposomes M. The outer layer of the bilayer of the second amphiphilic compound 8 has an opposite orientation from the inner layer composed of the first amphiphilic compound 2, i.e. the nonpolar regions of the second amphiphilic compound 8 of the outer layer point in the direction of the polar regions of the first amphiphilic compound 2 of the inner layer, i.e. in the direction of the hydrophilic active ingredient solution 1 now encapsulated in a liposome L, while the polar regions of the second amphiphilic compound 8 of the outer layer of the bilayer point in the direction of the—hydrophilic—liquid phase of the second emulsion 9 surrounding the liposome L (in this regard see the two upper sections of FIG. 2).

[0112] Since the pre-liposomes M composed of the droplets of the (hydrophilic) active ingredient solution 1 with the inner layer of the first amphiphilic compound 2 accumulated thereon have a lower density than the surrounding (hydrophobic) first liquid 3 of the first emulsion 4, they experience a force acting in the direction of the arrows P.sub.1 in the centrifugal field, which accelerates them in centripetal direction and brings them to the phase boundary 6 covered with an at least monomolecular layer of the second amphiphilic compound 8. The pre-liposome M is pushed against this layer of the second amphiphilic compound 8 enriched at the phase boundary 6 with such a force that the monomolecular layer of the second amphiphilic compound 8 enriched at the phase boundary 6 adjoins the inner layer of the first amphiphilic compound 2 accumulated on the droplets of the active ingredient solution 1, and hydrophobic interaction of two monomolecular layers gives rise to the bilayer of the finished liposome L, which, after further movement in the direction of the arrows P.sub.1, is dispersed in the (hydrophilic) liquid phase of the second emulsion 9 in the centrifugal field. In the case of a primarily ball-shaped or spherical shell, the droplets of the active ingredient solution 1 or the pre-liposome M consequently forms a liposome L having, in the bilayer that forms its membrane, firstly an inner layer of the first amphiphilic compound 2 and secondly an outer layer of the second amphiphilic compound 8. Consequently, it is firstly possible to produce symmetric liposomes L when the first amphiphilic compound 2 is chosen to correspond to the second amphiphilic compound 8; secondly, it is especially possible to produce asymmetric liposomes L when the first amphiphilic compound 2 is chosen differently than the second amphiphilic compound 8. At the same time, it should be pointed out here that, rather than a (single) first amphiphilic compound 2 and/or second amphiphilic compound 8, it is of course also possible to use a mixture of such compounds, for example a mixture of multiple lipids, which then respectively form the inner and outer layers of the bilayer of the liposome L (in this regard likewise see the two upper sections of FIG. 2).

[0113] As also apparent from the two lower sections of FIG. 2, the centrifuging of the first emulsion 4 and the second emulsion 9 that are in contact with one another via the phase boundary 6 constantly transfers the droplets of the second liquid 10 with the monomolecular layer of the second amphiphilic compound 8 accumulated thereon, i.e. the amphiphile carriers M′, from the liquid phase of the second emulsion 9 in the direction of the arrows P.sub.2 to the phase boundary 6 between the first emulsion 4 and the second emulsion 9, in order to constantly enrich the second amphiphilic compound 8 at the phase boundary 6 in the centrifugal field, where, as a result of the accumulation elucidated in the paragraph above, it is consumed as the outer layer on the pre-liposomes M to form the liposomes L.

[0114] Since the amphiphile carriers M′ composed of the droplets of the (hydrophobic) second liquid 10 with the second amphiphilic compound 8 accumulated thereon have a higher density than the surrounding (hydrophilic) liquid phase of the second emulsion 9, they experience a force that acts in the direction of the arrows P.sub.2 in the centrifugal field, which accelerates them in centrifugal direction and brings them to the phase boundary 6. As they do so, they shed their monomolecular layer of the second monomolecular compound 8 as they pass into the first (hydrophobic) liquid 3, i.e. into the continuous phase of the first emulsion 4, at the phase boundary 6. In this way, the layer of the second amphiphilic compound 8 deposited at the phase boundary 6 is constantly renewed as a result of the phase transfer of the droplets of the second (hydrophobic) liquid, or the second amphiphilic compound 8 is continuously “replenished” at the phase boundary 6, whereas it is continuously consumed as the outer layer by the above-described accumulation to form the liposomes L.

[0115] After more or less complete formation of the liposomes L or after more or less complete consumption of the amphiphile carriers M′ composed of the second liquid 10 with the second amphiphilic compound 8 accumulated thereon that were originally provided in the second emulsion 9, it is ultimately possible to separate the liquid phase in which the liposomes L are dispersed from the first liquid 3, which is possible in a simple manner because the miscibility of the hydrophilic liquid phase with the hydrophobic first liquid 3 is very sparing at most, and because of their different density.

[0116] For the reasons mentioned above, it is additionally possible that the first emulsion 4 is cooled down to a temperature between the melting point of the (hydrophobic) first liquid 3 and the melting point of the (hydrophilic) active ingredient solution 1, in order to convert the active ingredient solution 1 of the droplets having the monomolecular inner layer of the first amphiphilic compound 2 accumulated thereon that are emulsified in the first liquid 3 into the solid state, after which the first emulsion 4 in the solid state of the droplets of the active ingredient solution 1 is contacted with the (hydrophilic) liquid phase of the second emulsion 9, i.e. with the continuous phase thereof, to form the phase boundary 6, and the first emulsion 4 and the second emulsion 9 that are in contact with one another via the phase boundary 6 are centrifuged, especially keeping the droplets of the active ingredient solution 1 constantly in the solid state of matter.

[0117] In addition, it is firstly possible if required to modify the finished liposomes L with polymer conjugates, for example those based on polyethylene glycol (PEG), for example by attaching them electrostatically to the second amphiphilic compound 8 of the outer layer of the bilayer (not shown). Secondly, the second embodiment of the method of the invention especially offers the option that the second amphiphilic compound 8 used, which forms the monomolecular outer layer of the bilayer of liposomes L, is one or more lipids, where polymer conjugates have already been attached beforehand to at least some molecules of these lipids.

[0118] While the embodiments of a method of the invention for encapsulation of active ingredients in symmetric or asymmetric liposomes L that has been elucidated above with reference to FIGS. 1 and 2 can in principle be performed in a batchwise or else semicontinuous manner as mentioned above, the invention especially opens up the option of a continuous performance, which is consequently particularly advantageous on the industrial scale, of the two embodiments of the method.

[0119] FIG. 3 shows a schematic cross-sectional view of one embodiment of a centrifuge device suitable for continuous performance of the method of encapsulation of active ingredients in liposomes, wherein FIG. 3, for reasons of illustration, shows only half the cross section of the essentially rotationally symmetric centrifuge device. The flow-operated continuous centrifuge device shown in FIG. 3 is rotatable about its longitudinal center axis 11, and the centrifugal field that can be generated thereby is indicated by the arrows P.sub.3. The centrifuge device comprises a centrifuge chamber 13 which is bounded by a circumferential wall 12 and extends over the majority of its axial length. On its left-hand side in FIG. 3, the centrifuge device has two separate inlets 14, 15 that are disposed, for example, essentially coaxially to one another. The first inlet 14 opens into a radially outer circumferential section of the centrifuge chamber 13, while the second inlet 15 opens into a radially inner central section of the centrifuge chamber 13. In the inlet region (on the left in FIG. 3) of the centrifuge device, for this purpose, there is an inlet weir 16 that extends essentially in radial direction thereof, which leaves clear firstly a radially outer, for example approximately annular, passage orifice between the first inlet 14 and the radially outer section of the centrifuge chamber 13, and secondly a radially inner, for example roughly circular, passage orifice between the second inlet 15 and the radially inner section of the centrifuge chamber 13, such that fluid media applied simultaneously to the first inlet 14 and the second inlet 15, by means of the inlet weir 16, are at first kept separate from one another, after which, as a result of the passage through the inlet weir 16, they are transferred into the common centrifuge chamber 13 on the radial outside on the one hand and on the radial inside on the other hand, specifically into the radially outer section thereof on the one hand and into the radially inner section thereof on the other hand.

[0120] On its right-hand side in FIG. 3, the centrifuge device has two separate outlets 17, 18, again arranged essentially coaxially to one another, for example. The first outlet 17 opens out from the radially outer circumferential section of the centrifuge chamber 13, while the second outlet 18 opens out from the radially inner central section of the centrifuge chamber 13. In the outlet region (on the right in FIG. 3) of the centrifuge device, for this purpose, there is an outlet weir 19 that extends essentially in radial direction thereof, which leaves clear firstly a radially outer, for example roughly annular, passage orifice between the first outlet 17 and the radially outer section of the centrifuge chamber 13, and secondly a radially inner, for example roughly circular, passage orifice between the second outlet 18 and the radially inner section of the centrifuge chamber 13, such that two fluid media of different density being centrifuged within the centrifuge chamber 13, which have especially zero or only sparing miscible with one another, after passing through the outlet weir 19, are separated from one another on the radial inside on the one hand and on the radial outside on the other hand, after which they are transferred into the first outlet 17 on the one hand and into the second outlet 18 on the other hand, in order to remove them from the centrifuge device.

[0121] In the centrifuge chamber 13, in the region of the section on the outlet side thereof, but upstream of the outflow weir 19, there is also a first retaining weir 20 that extends essentially in radial direction thereof, which is configured essentially in the form of a circular ring, for example, and extends from the outer circumferential wall 12 of the centrifuge device inward by a radial distance R.sub.1, where this radial distance R.sub.1, i.e. the radial width of the first retaining weir 20, appropriately corresponds at least to the radial width of the passage orifice between the radially outer end of the outlet weir 19 and the circumferential wall 12, or preferably at least slightly exceeds it. In addition, in the centrifuge chamber 13, in the region of the section on the outlet side thereof, but once again upstream of the outlet weir 19, there is a second retaining weir 21 that likewise extends essentially in radial direction thereof, which, in the present case, is in the form of a circular ring, for example, and extends between a section of the centrifuge chamber 13 which is roughly in the middle in radial terms to close to the central axis of rotation 11 of the centrifuge device, although it may instead also be of essentially circular configuration and consequently may have no central passages (not shown). The radial distance R.sub.2 of the second retaining weir from the central axis of rotation 11, i.e. the radial width of the second retaining weir 21, appropriately corresponds at least to the radial width of the passage orifice between the radially inner end of the outlet weir 19 and the central axis of rotation 11, or preferably exceeds it at least slightly.

[0122] For continuous performance of the embodiments of the method of the invention described above with reference to FIGS. 1 and 2, firstly, the first emulsion 4 from step (b), wherein the hydrophobic first liquid has a higher density than the hydrophilic liquid phase 5 (FIG. 1) or than the hydrophilic continuous phase of the second emulsion 9 (FIG. 2), is supplied continuously to the first (radially outer) inlet 14 of the centrifuge device; secondly, the hydrophilic liquid phase 5 (FIG. 1) or the second emulsion 9 (FIG. 2) is supplied continuously to the second (radially inner) inlet 15 of the centrifuge device. In this case, the first emulsion 4 on the one hand and the hydrophilic liquid phase 5 (FIG. 5) or the second emulsion 9 (FIG. 2) on the other hand are first kept separate from one another by the inlet weir 16 in the inlet region of the centrifuge device, and subsequently, as soon as they have passed through the inlet weir 16 through its radially outer passage orifice on the one hand and through its radially inner passage orifice on the other hand, enter the radially outer section on the one hand and the radially inner section on the other hand of the common centrifuge chamber 13, by way of which they are contacted with one another in step (d) and the phase boundary 6 is formed. A further function of the inlet weir 16 over or under which flow may pass is to not blanket the (radially outer) inlet region of the first emulsion 4 into the centrifuge chamber 13 with the hydrophilic liquid phase 5 (FIG. 1) or with the second emulsion 9 (FIG. 2) having comparatively lower density, in order that no uncontrolled bilayers of the active ingredient solution droplets 1 with the monomolecular layer of the first amphiphilic compound 2 accumulated thereon that are present in the first emulsion 4 (cf. FIGS. 1 and 2) are generated.

[0123] In the centrifuge chamber 13, through which the flow direction is axial, i.e. from left to right in FIG. 3, the centrifuging step (e) elucidated specifically above with reference to FIGS. 1 and 2 also takes place, wherein the droplets of the active ingredient solution 1 with the monomolecular (inner) layer of the first amphiphilic compound 2 of the first emulsion 4 accumulated thereon that are emulsified in the first liquid 3 (cf. also FIGS. 1 and 2) can be accumulated in the centrifuge chamber 13 by means of the first retaining weir 20. One function of the first retaining weir 20 is consequently also that the emulsified droplets of the active ingredient solution 1 provided with a monolayer of the first amphiphilic compound 2 in the first emulsion 4 that have not yet been converted to a liposome L provided with a bilayer are not entrained out of the centrifuge chamber 13 via the first outlet 17, but reach the phase boundary 6 beforehand by virtue of buoyancy forces, in order to be able to be very substantially converted there to the liposomes L having a bilayer.

[0124] If, in accordance with the second embodiment of the method of the invention outlined with reference to FIG. 2, in step (c), a second emulsion 9 composed of the hydrophilic liquid phase with the droplets of the hydrophobic second liquid 10 that have been provided with a monomolecular layer of the second amphiphilic compound 8 emulsified therein is used, these droplets may be accumulated in the centrifuge chamber 13 by means of the second retaining weir 21. One function of the second retaining weir 21 in this case is consequently primarily that the emulsified droplets of the second hydrophobic liquid 10 provided with a monolayer of the second amphiphilic compound 8 in the second emulsion 9 which has not yet separated out the second amphiphilic compound 8 at the phase boundary 6 are not entrained out of the centrifuge chamber 13 via the second outlet 18, but reach the phase boundary 6 beforehand by virtue of the centrifugal forces induced, in order to very substantially enrich the second amphiphilic compound 8 there.

[0125] Finally, firstly the hydrophilic liquid phase 5 comprising liposomes provided with the bilayer composed of the two monomolecular layers of the first amphiphilic compound 2 (FIG. 1) or the continuous hydrophilic liquid phase of the second emulsion 9 comprising the liposomes L provided with the bilayer composed of the first amphiphilic compound 2 (inner layer) and the second amphiphilic compound 8 (outer layer) (FIG. 2), and secondly the hydrophobic first liquid 3 are separated from one another by means of the outlet weir 19 from the common centrifuge chamber 13, and withdrawn from the centrifuge device firstly via the second outlet 18 and secondly via the first outlet 17. In FIG. 3, the liquid fill levels within the centrifuge chamber 13 that can be established by means of the weirs over and under which the flows can pass can be seen. The (lighter phase) having lower density—here the hydrophilic liquid phase 5 (FIG. 1) or the continuous hydrophilic liquid phase of the second emulsion 9 (FIG. 2)—flows over the outlet weir 19 here only when the liquid column of the two phases 5 and 4, or 9 and 4, generates the same hydrostatic pressure up to the radially inner end of the outlet weir 19 as the liquid column of the “heavy” phase having comparatively higher density—the hydrophobic first liquid 3 of the first emulsion 4 here—up to the radially inner end 17a of the first outlet 17. One function of the outlet weir 19 over or under which the flows can pass is thus primarily to ensure complete separation of the phases 5 and 4, or 9 and 4, where the distance of the phase boundary 6 from the radially outer end of the outlet weir 19 should advantageously be adjusted such that the phase boundary 6 is incapable of reaching the radially outer end of the outlet weir 19 even in the event of fluctuations.

[0126] The invention is elucidated in detail hereinafter by working examples.

EXAMPLE 1

[0127] Production of symmetric liposomes from 10 μg/ml viscumin (mistletoe extract) in 15 mM phosphate buffer in a bilayer composed of the lipid dipalmitoylphosphatidylcholine (DPPC). [0128] Active ingredient solution: 10 μg/ml viscumin (active ingredient) in 15 mM aqueous phosphate buffer (PP) (hydrophilic solvent), [0129] First amphiphilic compound: dipalmitoylphosphatidylcholine (DPPC), [0130] First liquid (hydrophobic): perfluoroperhydrophenanthrene (C.sub.14F.sub.24, CAS number 306-91-2), [0131] Liquid phase (hydrophilic): 15 mM aqueous phosphate buffer (PP), [0132] Solubility of DPPC in C.sub.14F.sub.24: <10.sup.−5 mol/l (detection limit).

(a) Providing the Active Ingredient Solution:

[0133] The active ingredient viscumin is dissolved in a proportion of 10 μg/ml in 15 mM aqueous phosphate buffer (PP) as hydrophilic solvent, in order to provide the hydrophilic active ingredient solution.

(b) Providing the First Emulsion:

[0134] 150 mM of dipalmitoylphosphatidylcholine (DPPC) as the first amphiphilic compound is dissolved in ethanol in a round-bottom flask, and then the ethanol is fully evaporated by means of a rotary evaporator and subsequent placing of the round-bottom flask in a desiccator, such that the DPPC remains in the round-bottom flask as a dry film.

[0135] The hydrophilic active ingredient solution composed of 10 μg/ml in 15 mM PP from step (a) is then added to the round-bottom flask containing the DPPC, in order to obtain a mixture of 150 mM DPPC in the active ingredient solution. The DPPC (first amphiphilic compound) is leached away here from the glass wall of the round-bottom flask as a result of swelling in the hydrophilic active ingredient solution, and forms polydisperse amphiphilic aggregates that are comminuted by mechanical processing. For this purpose, 2 alternative methods are employed: [0136] extrusion through 800 nm and 400 nm pores; and [0137] use of ultrasound (Hielscher UP 200S with maximum power 200 W and an ultrasound frequency of 26 kHz): 10 seconds with a proportion of 100% of the sound treatment cycle (cycle time: about 1 second) and amplitude of 50%; the initial sound treatment for the first 10 seconds is followed by sound treatment over 10 minutes with a cycle proportion of 50% and an amplitude of 40% without cooling for comminution of the amphiphilic aggregates to an average particle size of 276 nm±50 nm.

[0138] The mixture of the active ingredient solution with the suspended aggregates of DPPC (first amphiphilic compound) produced in this way is introduced into perfluoroperhydrophenanthrene (first liquid, hydrophobic) with a proportion by volume of 1%. For emulsification, the active ingredient solution comprising the DPPC as disperse phase is suspended by means of ultrasound (Hielscher UP 200S) for 10 seconds with a cycle proportion of 100% and an amplitude of 50%, followed by 10 minutes with a cycle proportion of 50% and an amplitude of 40%, without cooling. In this way, pre-liposomes are produced from the active ingredient solution droplets having a monomolecular layer of DPPC (disperse phase of the first emulsion) in perfluoroperhydrophenanthrene (first liquid, hydrophobic as continuous phase of the first emulsion) having an average particle size of 424 nm±33 nm, a particle count of 643 kcounts/s±64 kcounts/s (derived count rate) and a polydispersity index (PDI) of 0.179. The size stability of the pre-liposomes was tested over 138 minutes and changed by less than 10% within this time.

[0139] Immediately thereafter, the first emulsion comprising the pre-liposomes is aliquoted, by dispensing 0.55 ml of the first emulsion in each case in centrifuge tubes for the subsequent batchwise centrifugation (see step (e) further down).

(c) Providing the Liquid Phase (Hydrophilic) of Zero or Sparing Miscibility with the First Liquid (Perfluoroperhydrophenanthrene, Hydrophobic) of the First Emulsion:

[0140] The hydrophilic liquid phase used is 15 mM aqueous phosphate buffer (PP), corresponding to the solvent of the active ingredient solution.

(d) Contacting the First Emulsion (Continuous Phase of the First Liquid, Perfluoroperhydrophenanthrene, Hydrophobic; Disperse Phase Composed of Pre-Liposomes of the Hydrophilic Active Ingredient Solution with Monomolecular Layer of DPPC) with the Liquid Phase (15 mM PP in Water, Hydrophilic):

[0141] In order to ensure more effective centrifugation (see step (e) further down) as a result of a higher difference in density, it is preferably possible first to freeze the aliquoted fractions of the first emulsion comprising the pre-liposomes in a freezer at −24° C. for at least 4 hours. Then the hydrophobic first liquid (perfluoroperhydrophenanthrene) of the first emulsion in which the pre-liposomes are suspended is adjusted to a controlled temperature of −3° C.±0.5° C. in an ice bath with addition of salt for about 30 minutes; the hydrophilic liquid phase in step (c) (16 mM PP in water) is set to a controlled temperature of −1° C.±0.5° C. in a thermostated bath for about 30 minutes.

[0142] The first emulsion comprising the hydrophobic first liquid as continuous phase is then contacted with the hydrophilic liquid phase by pipetting about 0.55 ml of the first emulsion comprising the pre-liposomes and 0.7 ml of the hydrophilic liquid phase into a centrifuge tube, by first layering on the first emulsion, which forms the heavier phase in the present working example, and very cautiously layering the hydrophilic liquid phase thereon, which forms the lighter phase in the present working example, so as to form a phase boundary on which the DPPC can accumulate without forming an organogel. The filling of the centrifuge tube appropriately takes place in the precooled centrifuge.

(e) Centrifuging of the First Emulsion (Continuous Phase Composed of the First Liquid, Perfluoroperhydro-phenanthrene, Hydrophobic; Disperse Phase Composed of Pre-Liposomes from the Hydrophilic Active Ingredient Solution with Monomolecular Layer of DPPC) and the Liquid Phase in Contact Therewith Via the Phase Boundary (15 mM PP in Water, Hydrophilic):

[0143] The centrifuge tube filled according to the above step (d) is finally centrifuged at 4000 g (corresponding to about 39 000 m/s.sup.2) at a centrifuge temperature of −3° C. for 30 to 60 minutes, in the course of which the pre-liposomes composed of the first emulsion or the first liquid thereof (perfluoroperhydrophenanthrene) are transferred through the phase boundary into the liquid phase (15 mM PP in water), and the DPPC enriched at the phase boundary accumulates as a second (outer) monomolecular layer on the first (inner) monomolecular layer of DPPC, in order to form the finished liposomes.

[0144] The hydrophilic liquid phase (15 mM PP in water) with the finished liposomes suspended therein is analyzed in order to determine the size of the liposomes produced in this way. For the visualization by transmission electron microscopy (TEM, Philips CM 12 with GATAN Multiscan 400 HP camera), ammonium molybdate as hydrophilic dye is added to the hydrophilic liquid phase with the liposomes produced therein, and the sample to be examined is applied to a copper grid and dried under air and in a desiccator. The dried samples are analyzed by TEM, with the dye producing a negative stain, meaning that the ammonium molybdate reduces transmission in the purely hydrophilic areas; as a result, the liposomes remain light-colored. FIG. 4 shows a TEM image of the liposomes produced in this way, which are spherical in shape and, according to an analysis by means of photon correlation spectroscopy (PCS, Malvern Zetasizer Nano ZS90), have a particle size of 305 nm±46 nm, an average particle count of 257±17 kcounts/s (derived count rate) and a polydispersity index (PDI) of 0.915.

EXAMPLE 2

[0145] Production of symmetric liposomes from 10 μg/ml viscumin (mistletoe extract) in 15 mM phosphate buffer in a bilayer composed of the lipid distearoylphosphatidylcholine (DSPC). [0146] Active ingredient solution: 10 μg/ml viscumin (active ingredient) in 15 mM aqueous phosphate buffer (PP) (hydrophilic solvent), [0147] First amphiphilic compound: distearoylphosphatidylcholine (DSPC), [0148] First liquid (hydrophobic): perfluoroperhydrophenanthrene (C.sub.14F.sub.24, CAS number 306-91-2), [0149] Liquid phase (hydrophilic): 15 mM aqueous phosphate buffer (PP), [0150] Solubility of DSPC in C.sub.14F.sub.24: <10.sup.−5 mol/1 (detection limit).

[0151] The production of liposomes takes place analogously to steps (a) to (e) according to example 1 and with corresponding proportions, but with the proviso that, rather than dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DPPC) is used as the first amphiphilic compound. The same applies to the transmission electron microscope analysis.

[0152] FIG. 5 shows a TEM image of the liposomes produced in this manner, which are spherical in shape and, according to PCS analysis, have an average particle size of 183 nm±51 nm, a particle count of 549±140 kcounts/s (derived count rate) and a polydispersity index (PDI) of 0.9.

EXAMPLE 3

[0153] Production of symmetric liposomes from 10 μg/ml viscumin (mistletoe extract) in 15 mM phosphate buffer in a bilayer composed of a mixture of lipids dioleoylphosphatidylcholine (DOPC) and cholesterol in a molar ratio of 60:40. [0154] Active ingredient solution: 10 μg/ml viscumin (active ingredient) in 15 mM aqueous phosphate buffer (PP) (hydrophilic solvent), [0155] First amphiphilic compound: dioleoylphosphatidylcholine (DOPC) and cholesterol in a ratio of 60:40 mol %, [0156] First liquid (hydrophobic): perfluoroperhydrophenanthrene (C.sub.14F.sub.24, CAS number 306-91-2), [0157] Liquid phase (hydrophilic): 15 mM aqueous phosphate buffer (PP), [0158] Solubility of DOPC in C.sub.14F.sub.24: <10.sup.−5 mol/1 (detection limit), [0159] Solubility of cholesterol in C.sub.14F.sub.24: <10.sup.−5 mol/1 (detection limit).

[0160] The production of liposomes takes place analogously to steps (a) to (e) according to example 1 and with corresponding proportions, but with the proviso that, rather than dipalmitoylphosphatidylcholine (DPPC), a mixture of dioleoylphosphatidylcholine (DOPC) and cholesterol with a molar ratio of 60:40 is used as the first amphiphilic compound. The same applies to the transmission electron microscope analysis.

[0161] FIG. 6 shows a TEM image of the liposomes produced in this manner, which are spherical in shape and, according to PCS analysis, have an average particle size of 108 nm±31 nm, a particle count of 1116±32 kcounts/s (derived count rate) and a polydispersity index (PDI) of 0.962.

EXAMPLE 4

[0162] Production of symmetric liposomes from pyranin-(8-hydroxy-1,3,5-pyrenetrisulfonic acid trisodium salt (HPTS) in 15 mM phosphate buffer in a bilayer of a mixture of egg lecithin having a phosphatidylcholine content of 80% (E80) and cholesterol in a molar ratio of 60:40. [0163] Active ingredient marker solution: Pyranin-8-hydroxy-1,3,5-pyrenetrisulfonic acid trisodium salt (HPTS) (surrogate) in 15 mM aqueous phosphate buffer (PP) (hydrophilic solvent), [0164] First amphiphilic compound: egg lecithin with a phosphatidylcholine content of 80% (E80) and cholesterol in a ratio of 60:40 mol %, [0165] First liquid (hydrophobic): perfluoroperhydrophenanthrene (C.sub.14F.sub.24, CAS number 306-91-2), [0166] Liquid phase (hydrophilic): 15 mM aqueous phosphate buffer (PP), [0167] Solubility of E80 in C.sub.14F.sub.24: <10.sup.−5 mol/l (detection limit), [0168] Stability of cholesterol in C.sub.14F.sub.24: <10.sup.−5 mol/l (detection limit).

(a) Providing the Active Ingredient Marker Solution:

[0169] The active ingredient marker HPTS (surrogate) is dissolved in 15 mM of aqueous phosphate buffer (PP) as hydrophilic solvent, in order to provide the hydrophilic active ingredient marker solution.

(b) Providing the First Emulsion:

[0170] The providing of the first emulsion takes place analogously to step (b) of example 1, except that the first amphiphilic compound used, rather than 150 mM DPPC, is 150 mM of a mixture of E80 and cholesterol in a molar ratio of 60:40 mol %.

[0171] In addition, in a departure from step (b) of example 1, the first emulsion comprising the pre-liposomes is aliquoted to volumes of 30 ml.

(c) Providing the Liquid Phase of Zero or Sparing Miscibility with the First Liquid (Perfluoroperhydrophenanthrene, Hydrophobic) of the First Emulsion:

[0172] The hydrophilic liquid phase provided, analogously to step (c) of example 1, is 15 mM aqueous phosphate buffer (PP), corresponding to the solvent of the active ingredient marker solution.

(d) Contacting the First Emulsion (Continuous Phase) of the First Liquid, Perfluoroperhydrophenanthrene, Hydrophobic; Disperse Phase Composed of Pre-Liposomes of the Hydrophilic Active Ingredient Marker Solution with a Monomolecular Layer of E80/Cholesterol) with the Liquid Phase (15 mM PP in Water, Hydrophilic):

[0173] The volumes of 30 ml of the first emulsion comprising the pre-liposomes that have been aliquoted as per step (b) above are used uncooled at room temperature and contacted with the hydrophilic liquid phase according to step (e) below in a flow-operated, continuous centrifuge device, as specifically elucidated above with reference to FIG. 3, forming a phase boundary at which the mixture of E80 and cholesterol can be enriched without forming an organogel.

(e) Centrifuging the First Emulsion (Continuous Phase Composed of the First Liquid, Perfluoroperhydro-phenanthrene, Hydrophobic; Disperse Phase Composed of Pre-Liposomes of the Hydrophilic Active Ingredient Marker Solution with Monomolecular Layer of E80/Cholesterol) and the Liquid Phase that has been Contacted Therewith Via the Phase Boundary (15 mM PP in Water, Hydrophilic):

[0174] First of all, the dead volume of the continuous centrifuge at 333 g (corresponding to about 3300 m/s.sup.2) is filled by continuous injection of firstly about 13 ml of the first emulsion (hydrophobic first liquid, perfluoroperhydrophenanthrene, with the pre-liposomes suspended therein) at a flow rate of about 1 ml/min, and secondly of the hydrophilic liquid phase (15 mM PP in water). Once the centrifuge chambers overflow, the two phases (hydrophilic/hydrophobic) are supplied continuously at a flow rate of about 0.3 ml/min, and centrifugation is continued at 333 g, with transfer of the pre-liposomes from the first emulsion or the first liquid thereof (perfluoroperhydrophenanthrene) through the phase boundary into the liquid phase (15 mM PP in water) and accumulation of the E80/cholesterol enriched at the phase boundary as the second (outer) monomolecular layer on the first (inner) monomolecular layer of E80/cholesterol, in order to form the finished liposomes. The total volumes of the two phases (hydrophilic/hydrophobic) are about 30 ml. On conclusion of the continuous supply of the two phases (hydrophilic/hydrophobic) after about 70 min., the two phases are centrifuged at 333 g for a further 20 minutes. The phases that flow out via the outlet weirs (cf. FIG. 3) are collected.

[0175] The hydrophilic liquid phase (15 mM PP in water) with the finished liposomes suspended therein is determined analogously to example 1 by means of photon correlation spectroscopy (PCS) in order to determine the size of the liposomes produced in this way. The liposomes produced in this way are of spherical shape and have an average particle size of 254 nm±3 nm, a particle count of 645 kcounts/s±6 kcounts/s (derived count rate) and a polydispersity index (PDI) of 0.380.

EXAMPLE 5

[0176] Production of asymmetric liposomes from 10 μg/ml viscumin (mistletoe extract) in 15 mM of phosphate buffer in a bilayer composed firstly of a mixture of egg lecithin having a phosphatidylcholine content of 80% (E80) and cholesterol in a molar ratio of 60:40 (inner molecular layer) and secondly a mixture of egg lecithin having a phosphatidylcholine content of 80% (E80) and cholesterol in a molar ratio of 80:20 (outer monomolecular layer). [0177] Active ingredient solution: 10 μg/ml viscumin (active ingredient) in 15 mM aqueous phosphate buffer (PP) (hydrophilic solvent), [0178] First amphiphilic compound: egg lecithin having a phosphatidylcholine content of 80% (E80) and cholesterol in a ratio of 60:40 mol %, [0179] Second amphiphilic compound: egg lecithin having a phosphatidylcholine content of 80% (E80) and cholesterol in a ratio of 80:20 mol %, [0180] First liquid (hydrophobic): perfluoroperhydrophenanthrene (C.sub.14F.sub.24, CAS number 306-91-2), [0181] Second liquid (hydrophobic): perfluoroperhydrophenanthrene (C.sub.14F.sub.24, CAS number 306-91-2), [0182] Liquid phase (hydrophilic): 15 mM aqueous phosphate buffer (PP), [0183] Solubility of E80 in C.sub.14F.sub.24: <10.sup.−5 mol/l (detection limit), [0184] Solubility of cholesterol in C.sub.14F.sub.24: <10.sup.−5 mol/l (detection limit).

(a) Providing the Active Ingredient Solution:

[0185] Viscumin, the active ingredient, is dissolved with a proportion of 10 μg/ml in 15 mM aqueous phosphate buffer (PP) as hydrophilic solvent in order to provide the hydrophilic active ingredient solution.

(b) Providing the First Emulsion:

[0186] The providing of the first emulsion is accomplished analogously to step (b) according to example 1, except that the first amphiphilic compound used, rather than 150 mM DPPC, is 150 mM of a mixture of E80 and cholesterol in a molar ratio of 60:40 mol %, such that the first emulsion consists of 1% (v/v) of hydrophilic active ingredient solution (viscumin in 15 mM aqueous PP) in the first hydrophobic liquid (perfluoroperhydrophenanthrene) with a total proportion of 1.5 mM of the first amphiphilic compound (E80 and cholesterol in a ratio of 60:40 mol %).

[0187] In this way, pre-liposomes are produced from the active ingredient solution droplets having a monomolecular layer of E80/cholesterol in a molar ratio of 60:40 mol % (disperse phase of the first emulsion) in perfluoroperhydrophenanthrene (first liquid, hydrophobic as continuous phase of the first emulsion) having an average particle size of 376 nm±22 nm, a particle count of 482 kcounts/s±35 kcounts/s (derived count rate) and a polydispersity index (PDI) of 1.0.

[0188] In addition, in a departure from step (b) according to example 1, the first emulsion comprising the pre-liposomes was aliquoted to volumes of 30 ml.

(c) Providing the Mixture—Here in the Form of the Second Emulsion—of the Liquid Phase (Hydrophilic) of Zero or Sparing Miscibility with the First Liquid (Perfluoroperhydrophenanthrene, Hydrophobic) of the First Emulsion with the Second Amphiphilic Compound with the Second Liquid Emulsified Therein (Hydrophobic):

[0189] E80/Cholesterol in a molar ratio of 80:20 mol % as the second amphiphilic compound is dissolved in ethanol in a round-bottom flask, and then the ethanol is fully evaporated by means of a rotary evaporator followed by storage of the round-bottom flask in a desiccator, so as to leave the second amphiphilic compound as a dry film in the round-bottom flask.

[0190] The hydrophilic liquid phase composed of 15 mM aqueous PP is then added to the round-bottom flask containing the E80/cholesterol in a molar ratio of 80:20 mol %, in order to obtain a mixture of 150 mM E80/cholesterol in a molar ratio of 80:20 mol % (second amphiphilic compound) in 15 mM aqueous PP (hydrophilic liquid phase). The E80/cholesterol in a molar ratio of 80:20 mol % becomes detached here from the glass wall of the round-bottom flask owing to swelling in the hydrophilic active ingredient solution, and forms polydisperse amphiphilic aggregates that can be reduced in size by mechanical processing. For this purpose, it is again possible to use 2 alternative methods: [0191] extrusion through 800 nm and 400 nm pores; and [0192] use of ultrasound (Hielscher UP 200S with maximum power 200 W and an ultrasound frequency of 26 kHz): 10 seconds with a proportion of 100% of the sound treatment cycle (cycle time: about 1 second) and an amplitude of 50%; the initial sound treatment for the first 10 seconds is followed by sound treatment over 10 minutes with a cycle proportion of 50% and an amplitude of 40% without cooling for comminution of the amphiphilic aggregates.

[0193] Then the second emulsion is produced by dispersing the hydrophobic second liquid (perfluoroperhydrophen-anthrene) into the mixture of the hydrophilic liquid phase (15 mM aqueous PP) with the suspended aggregates of E80/cholesterol in a molar ratio of 80:20 mol % (second amphiphilic compound) generated in this way with a proportion by volume of about 1%. For emulsification, the perfluoroperhydrophenanthrene as disperse phase is suspended by means of ultrasound (Hielscher UP 200S) for 10 seconds with a cycle proportion of 100% and an amplitude of 50%, followed by 30 minutes with a cycle proportion of 75% and an amplitude of 60%, without cooling, such that the second emulsion consists of 1% (v/v) hydrophobic second liquid (perfluoroperhydrophenanthrene) in the hydrophilic liquid phase (15 mM aqueous PP) with a total content of 1.5 mM of the second amphiphilic compound (E80 and cholesterol in a ratio of 80:20 mol %).

[0194] In this way, amphiphile carriers composed of the perfluoroperhydrophenanthrene droplets with a monomolecular layer of E80/cholesterol, the second amphiphilic compound, in a molar ratio of 80:20 mol % (disperse phase of the second emulsion) in 15 mM aqueous PP (liquid phase, hydrophilic as continuous phase of the second emulsion) are produced with an average particle size of 127 nm±6 nm, a particle count of 1411 kcounts/s±52 kcounts/s (derived count rate) and a polydispersity index (PDI) of 0.298.

[0195] Immediately thereafter, the second emulsion with the amphiphile carriers is also aliquoted to volumes of 30 ml.

(d) Contacting of the First Emulsion (Continuous Phase of Perfluoroperhydrophenanthrene, the First Liquid, Hydrophobic; Disperse Phase Composed of Pre-Liposomes of the Hydrophilic Active Ingredient Solution with a Monomolecular Layer of E80/Cholesterol in a Molar Ratio of 60:40 Mol %) with the Second Emulsion (Continuous Phase Composed of 15 mM PP in Water, the Liquid Phase, Hydrophilic; Disperse Phase Composed of Amphiphile Carriers Composed of Perfluoroperhydro-phenanthrene, the Second Liquid, with a Monomolecular Layer of E80/Cholesterol in a Molar Ratio of 80:20 Mol %):

[0196] The volumes of 30 ml of the first emulsion comprising the pre-liposomes that were aliquoted in step (b) above are used uncooled at room temperature and contacted in step (e) below in a flow-operated, continuous centrifugation device, as specifically elucidated above with reference to FIG. 3, with the volumes of 30 ml of the second emulsion comprising the amphiphile carriers that were aliquoted in step (c) above, for the phase boundary between the continuous phases of the first and second emulsions at which the mixture of E80 and cholesterol can accumulate without forming an organogel. [0197] (e) Centrifuging of the First Emulsion (Continuous Phase of Perfluoroperhydrophenanthrene, the First Liquid, Hydrophobic; Disperse Phase Composed of Pre-Liposomes Composed of the Hydrophilic Active Ingredient Solution with a Monomolecular Layer of E80/Cholesterol in a Molar Ratio of 60:40 Mol %) with the Second Emulsion in Contact Therewith Via the Phase Boundary (Continuous Phase Composed of 15 mM PP in Water, the Liquid Phase, Hydrophilic; Disperse Phase Composed of Amphiphile Carriers Composed of Perfluoroperhydrophenanthrene, the Second Hydrophobic Liquid, with a Monomolecular Layer of E80/Cholesterol in a Molar Ratio of 80:20 Mol %):

[0198] First of all, the dead volume of the continuous centrifuge at 1000 g (corresponding to about 9800 m/s.sup.2) is filled by continuous injection of about 13 ml firstly of the first emulsion (perfluoroperhydrophen-anthrene, hydrophobic first liquid, with the pre-liposomes suspended therein) at a flow rate of about 1 ml/min, and secondly of the second emulsion (15 mM PP in water, hydrophilic liquid phase, with the amphiphile carriers suspended therein). Once the centrifuge chambers overflow, the two phases (hydrophilic/hydrophobic) are fed in continuously at a flow rate of about 0.3 ml/min, and continuous integration continues at 1000 g. In the course of this, the amphiphile carriers from the second emulsion are first supplied continuously to the phase boundary in the centrifugal field, in order to constantly enrich the second amphiphilic compound (E80/cholesterol in a molar ratio of 80:20 mol %) at the phase boundary. Secondly, the pre-liposomes of the first emulsion or of the first liquid thereof (perfluoroperhydrophen-anthrene) are transferred through the phase boundary into the liquid phase (15 mM PP in water), with accumulation of the second amphiphilic compound enriched at the phase boundary (E80/cholesterol in a molar ratio of 80:20 mol %) as the second (outer) monomolecular layer on the first (inner) monomolecular layer of the first amphiphilic compound (E80/cholesterol in a molar ratio of 60:40), in order to form the finished liposomes. The total volumes of the two phases (hydrophilic/hydrophobic) are about 30 ml. On conclusion of the continuous supply of the two phases (hydrophilic/hydrophobic) after about 70 min., the two phases are centrifuged at 1000 g for a further 20 min. The phases that flow away via the outlet weirs (cf. FIG. 3) are collected.

[0199] The hydrophilic liquid phase (15 mM PP in water) with the finished—asymmetric—liposomes suspended therein is analyzed analogously to example 1 by means of PCS, in order to determine the average size of the liposomes produced in this way. The liposomes produced in this way, which have a different composition of the inner and outer monomolecular layer of their bilayer, are of spherical shape and have an average particle size of 367 nm±61 nm, a particle count of 3207 kcounts/s±52 kcounts/s (derived count rate) and a polydispersity index (PDI) of 0.30.