Method of preparing lipid vesicles by ultrasonic treatment, the use of this method and apparatus for its application.

The invention relates to a method for the production of uniform, small unilamellar so-called lipid vesicles, particularly to a method for transferring lamellar arranged lipids in lipid vesicles in which the to_'disintegrating lipid structures (lamellae) are subjected in a suspension liquid inside a sonication vessel at a substantially constant temperature to an ultrasonic treatment. The invention further comprises suitable devices for performing the method.

Lipid vesicles are needed, for example for medical therapeutic and scientific purposes. Medicines that have an intracellular target must be able to the cell membrane happen. Many effectors that control the intracellular metabolism and are formed in the cell, can not leave the cell still penetrate into this from the outside. The therapeutic use of this substance class therefore makes a transport mechanism is required which allows hineinzuschleusen non-membrane-permeable substances in the cells without these substances can leave the cells. Such a transport mechanism is also intended to be as independent as possible of the active substance to be transported, so they can carry any type of effector, on the other hand be cell-specific, that allow transportation only in certain cells. A transportation system with the above-mentioned properties represent lipid vesicles, which may include a variety of substances, such as enzymes, drugs, chelating agents, hormones, cell-effectors, antigens, antibodies, interferon inducers and genes. In the lipid vesicles, the solvent and the dissolved substances in the solvent of phospholipid bilayer membranes are enclosed. The lipid membrane has a thickness of 4 nm, which vesicles can assume a diameter of 25 to 12o nm. The size of the vesicles can be determined by means of laser light scattering, by ultracentrifugation, gel filtration, or scanning electron microscopes.

An important application of lipid vesicles is the incorporation of inositol (IHP) in red blood cells by the method described by YC Nicolau and K. Gersonde (US Patent 4,192,869), to reduce the oxygen affinity of hemoglobin. We know namely, that for example in the storage of blood, the oxygen affinity of hemoglobin in the red blood cells are constantly increasing. Also observed in certain diseases increased oxygen affinity of hemoglobin. This increased affinity for oxygen results in that only a small proportion of the oxygen is bound to hemoglobin, and circulates in the blood that is actually delivered to the tissue. This high 0₂Affinity of hemoglobin can be reduced by binding of specific effectors to the hemoglobin. The strongest effector of this type is the inositol (IHP). The incorporation of IHP is achieved in that intact cells with IHP-loaded are incubated where lipid vesicles, said IHP is introduced into the cell by fusion of lipid membranes of the cell and the vesicles and there achieves its effect, namely the change the 0₂- Affinity of hemoglobin, measured as a "right shift" hemoglobin-O₂Dissociation curve. After returning these IHP-loaded red blood cells in the circulation, a significantly higher proportion of data stored in the red blood cells is O₂- Delivered quantity in the periphery. This property of the modified red blood cells is maintained throughout the life of the cell.

For the incorporation of inositol into red blood cells small, unilamellar IHP-loaded lipid vesicles with a diameter of up to 2o So nm required. It is known to produce lipid vesicles by disintegration of lipid suspensions in the ultrasonic field. The progress in the application of lipid vesicles in the therapy takes place only very slowly, since the preparation of suitable for fusion with the red blood cell lipid vesicles is connected in a sufficient amount with considerable difficulties. The suitable for this purpose lipid vesicles must namely can not be produced in large quantities, but also to be reproducible of uniform size and therefore metered in therapeutic use. The retrospective application of separation methods for separating appropriate fractions of lipid vesicles raises many problems, eg

Maintain sterility and use of complicated and time-consuming separation techniques that greatly reduce the biological effectiveness of the vesicles, which have only a half-life of about 1 day. The only method which allows the production of large quantities of vesicles in a short time, the disintegration in the ultrasound. Apart from the nature and composition of the lipid membrane of the vesicles depend the success and reproducibility of scientific study or therapeutic treatment, ie introduction of IHP in red blood cells, much of the size of lipid vesicles from. The control whether the lipid vesicles have formed in sufficient homogeneity and thus quality and in sufficient quantity at the disintegration of the lipid suspension, carried out in such a way that the produced lipid vesicles desired IHP Einschleusungsversuche in red blood cells performs intracellular IHP chemical evidence and the hemoglobin 0₂Dissociation curve of intact cells or measuring the desired biological or therapeutic effect of IHP-loaded red blood cells in animal experiments prove. The results of the tests or the success of the treatment can therefore only after extensive studies are evaluated, and the success of the ultrasound treatment, namely the production of qualified for the merger with the red blood cell vesicles, are recognized only in retrospect.

It has been found that the production of lipid vesicles - particularly in large volumes, such as are required for therapeutic methods - is difficult with constant properties with the help of known ultrasonic technique. Despite adherence apparently completely same external conditions during the disintegration of the lipid suspensions in the ultrasonic field, it has not been possible to always get the same yield of so-called small unilamellar and thus fusion effective lipid vesicles.

The invention is based on the object to further develop the above-mentioned process for preparing .Lipid vesicles that successfully inject effectors into cells that the efficiency of the process is increased, and that a high yield of the desired in each case in a reproducible manner purpose is achieved highly effective lipid vesicles.

The invention is that the most effective for the intended purpose vesicle or vesicle size, and optimal for achieving this vesicle or vesicle size ultrasonic frequency and intensity is determined, and that the thus determined optimum ultrasonic frequency and intensity at is held constant for the rest the same treatment conditions, characterized in that continuously measured during the ultrasound treatment of the actual value of the frequency and the intensity of the ultrasonic field in the reaction medium, and depending on this feedback, the output power and the frequency of the oscillation generator feeding electric generator is regulated.

The invention is based on the finding that when the vesicles for therapeutic purposes size or size distribution of the vesicles of the effectiveness thereof is of decisive importance, and on the other hand, the size and homogeneity of the vesicles produced in the ultrasonic field in sensitive way on the constancy and intensity of acting on the lipids ultrasonic field depend. While it has been customary to measure the power coupled into the sounder sound energy and to keep it constant, the invention proposes that measure in the reaction medium effective sound energy and to specifically change, and in such a way that the effective acoustic energy in the reaction medium itself is held constant at its optimum value. By measuring the effective sound energy in the reaction medium itself, and by using this actual value for regulating the coupled sound energy, all influences are compensated which influence the effective sound intensity and sound frequency on vesicle-education center, such as different and changing the absorption of sound energy with as the reaction proceeds, changing geometric conditions within the reaction medium upon the occurrence of gas bubbles during sonication, and reflection of a frequency band of sound waves, which interferes with the emitted by the sounder sound waves, and where appropriate leads in phase-shifted superimposition to extinction of the sound energy.

So if you initially find, pursuant to the inventive method using the known examination methods optimum for each purpose vesicle or the optimal distribution curve of the most effective vesicle, then determined in another series of experiments the optimal ultrasonic conditions such as frequency and sound pressure, which lead to the desired distribution curve , and then holding the thus determined optimum ultrasonic intensity and ultrasonic frequency constant within the reaction medium during the reaction period, is obtained at a very high yield for the particular purpose extremely effective lipid vesicles.

The inventive method can not only for the production of lipid vesicles, but with the same success also for the treatment of natural biological membranes and apply their conversion into vesicles, for example at the Erforschun g of the function and structure of membrane enzymes. Again, there is in fact necessary, the membrane enzymes reproducibly converted into vesicles of a certain size, which is not of sufficient security is possible by the known processes.

Further details and advantages of the method according to the invention are described below with reference to the drawings and with reference to exemplary embodiments.

Of the drawings:<ul><li>Fig. 1 is a suitable for the production of smaller amounts of lipid vesicle suspensions device having the features of the invention, partly in schematic representation, and</li><li>FIG. 2 a for the production of lipid vesicle suspensions in sterile packs for clinical use in liter quantities suitable device, also in partially schematic representation.</li></ul>

The reaction medium 1 in the form of a suspension of lipid in a suitable dispersing agent is located in the inner cylindrical tube 2 of the double-walled glass reaction vessel 3. The bottom of the reaction vessel 3 has a solid to the reaction chamber 3 opening in which a sound sensor 5 is inserted. The sound sensor 5 is bonded and sealed using an epoxy resin layer 6 with the surrounding the opening wall. 7 From the top immersed in the reaction medium 1 is an ultrasonic vibrator. 8 The from the reaction vessel 3 upwardly projecting part 9 of the vibration sensor 8 is provided with a coupling 9 for the power supply.

The double wall of the reaction vessel, a cavity 1o is formed, which is traversed by cooling water. The pipe member 11 and 12 are used to feed and to discharge the cooling water. The tubular connector 13 leads into the reaction space above the reaction medium 1 and is used to supply an inert gas such as argon. Top of the reaction chamber is closed by a cover 14. The remaining between the lid 14 and the ultrasonic transducer and the vessel wall column, the inert gas is under slight overpressure can escape.

The sound sensor 5 has as actual sound pressure or sound intensity receiver at its top, which is in contact with the reaction medium 1 end a piezoelectric disc 18, which incorporated in the case shown radially in a holding tube, and glued into this holding tube for example with epoxy resin and is sealed. The 18 provided by the piezo disc electric signal is a measure of the effective sound frequency and the effective sound intensity in the reaction medium. This electrical signal represents the actual value of the control circuit and is fed via the line control amplifier 2o the 21st On the oscilloscope 22, the effective frequency and the sound intensity can optionally followed visually, and if necessary on hand any subsequent adjustments to the frequency and the output power of the RF generator 23rd

The variable gain amplifier 21 is set via a setpoint adjustment device 24 of the desired value for the ultrasonic intensity, and a target value adjusting apparatus 25 of the desired value for the ultrasonic frequency. In case of deviations of the actual values ​​of the frequency and the intensity of the ultrasonic field in the reaction medium of the predetermined nominal values ​​on the line 26 of the ultrasonic generator 23 is activated, the frequency and / or output power can be changed until the measured within the reaction medium 1 effective values ​​with the preset setpoints match. Of the high frequency generator 23 of the ultrasonic transmitter 8 is supplied via the line 28 with the necessary voltage.

The apparatus shown in Fig. 2 is suitable for the production of lipid vesicles in larger quantities. The reaction vessel is an open-topped cylindrical rectangular pan 3o of corrosion-resistant Cr-Ni steel. The cooling of the liquid 31 in the reaction vessel is effected by a cooling coil arrangement 32, which is inserted into the trough located in the 3o liquid 31st Under the bottom of the tub 3o eight electro-acoustic transducer 33 are arranged, the vibratory generator 34 are coupled to the bottom of the tub on the outside of 3o. The sound energy is transferred to the liquid 31 in pan 3o. In the liquid 31 emerges from the top of the sound sensor 35 a. The reaction medium itself is located in a sealed sterile bag 36, which is maintained through the coil assembly 32 within the fluid 31 slightly above the bottom of the tub. The sound sensor 35 is lowered so far that the acoustic Took head, ie, the piezoelectric disc 37 is pressed against the sterile bag 36, and so the ultrasonic field detected in the reaction medium.

The signal 37 delivered by the piezo disc controls via line 38 to the control amplifier 39 and provides this the actual values ​​for the control process. The target values ​​for the frequency and the intensity of the ultrasound to be determined by the setpoint - Vorgäbeeinrichtung 4o and 41. In case of deviation of the actual values ​​of the preset reference values ​​is controlled via line 42 of the controllable electrical generator 43 whose over to the electromagnetic transducer 33 line 44 given output power and / or frequency can be changed as long match to setpoints and actual values. The 37 measured by the piezo disc frequency and amplitude of the ultrasonic field can beobachtet.werden visually on the oscilloscope 45, so that if necessary a manual intervention in the control is possible.

Using the devices described following reactions are performed, for example:

example 1

It should IHP-loaded lipid vesicles for therapeutic purposes are made, namely the introduction of IHP in red blood cells by fusion to improve 0 ₂-Freisetzungs- Properties of the red blood cells. The general method for the preparation of lipid vesicles is described in US Patent No. 4,192,869, to which reference is hereby made. The lipid vesicles of phosphatidylcholine, phosphatidylserine and cholesterol in a molar ratio of 8: 2: 7 constructed. These lipids are first dissolved in an organic solvent of 95 parts of chloroform, and 5 parts of methanol to achieve a homogeneous solution and mixture of these lipids. Then, the solvent is removed at 2O ° C in a rotary evaporator. The then in the flask remaining lipid film with an aqueous solution (here IHP) containing the biologically active substance was added and shaken, so that now form planar lipid lamellae in this suspension. This suspension contains lipids in a concentration of about 17 to 200 ug / l. The suspension is further saturated with IHP and between pH. 7₀- 8.o buffered.

In a previous series of experiments it was found that are suitable for fusion with erythrocytes and the incorporation of IHP into erythrocytes vesicles that have the composition described above and a diameter of 25 degrees - have 500A. The effectiveness is greater, the greater the amount of these vesicle formation in the respective preparation. By a further series of experiments was then determined that these desired diameter distribution can be achieved, if the lipids at an approximately constant temperature of 37 degrees Celsius with a narrow-band sound frequency of 2o kHz with an effective sound energy 3-6 W / cm²reverberate.

The above-described lipid suspension is filled under inert gas into the reaction vessel 3 (Fig. 1). The reaction vessel is flushed through the tube 13 with argon. To the desired value setting devices 24 and 25, then the optimum sound intensity and the desired sound frequency be set. The control means described ensures that the optimum sound intensity is observed as an effective, coming to the reaction liquid to the full effect sound intensity during the entire treatment time. The treatment lasts 3o to 6o minutes. During this time, the heat developed in the reaction vessel is discharged and so the temperature maintained constant by the cooling water.

The thus obtained vesicle suspension is then incubated with red blood cells at 37 degrees Celsius for 1 h. Thereafter, the red blood cells are washed in isotonic buffer pH 7.4, and the hemoglobin 0₂Dissociation curve of modified intact cells measured. The success of IHP incorporation is called a "right shift" hemoglobin 0₂- Dissociation erkannt.Der by IHP binding to hemoglobin maximum achievable 0₂-Halbsättigungsdruck Was 7.4 95 mmHg at 37 degrees Celsius and pH.

The success of controlled ultrasonic method lies in the fact that the maximum IHP Inkorporierungseffekt with volume ratios RBC (red blood cells): vesicle or lipid Konzen is reached concentrations that only 1₀ % Of the values ​​are, which had to be used in the usual uncontrolled use of ultrasound for a successful IHP incorporation. This results in a considerable economic advantage, since lipids are expensive and can not be reused. Moreover, can be achieved with the usual method reproducible results, which are an absolute prerequisite for a safe dosage in therapy.

example 2

When lipid vesicles for therapeutic purposes, there is a demand for absolute Keimfreiheit.Da sterilization of the finished vesicle suspension encounters extreme difficulties following procedure shall apply: The starting components according to Example 1 are first sterilized, and under sterile conditions in a likewise sterilized bags made of polyethylene or plasticized PVC film of about o, filled 5 mm wall thickness. The bag is sterile-sealed, and then exposed to ultrasound in the vessel according to Fig. 2 of the ultrasonic effect, the space between the vessel walls and the bag is filled with water. The measurement of the sound intensity is carried out in this case on the outside of the bag. the same results are described under the same conditions as in Example 1 obtained when the vibration sensors power supplied is increased by approximately a factor 1.65. This experimentally determined at the given test set measurements from factor represents the entering through the introduction of the bag power losses.