Process for preparing liposomes and/or biological cells containing ingredients, and their use.

p0001The invention relates to the mentioned in the patent claims the subject, ie, a process for the preparation of liposomes and / or biological cells, which receive external substances added during the production. In both cases the matter intake occurs under mild conditions. The loaded liposomes and / or cells are useful as targeted drug carriers.

p0002Liposomes are made of lipid molecules, preferably phospholipids constructed. These form in the aqueous medium spontaneously lipid bilayers arranged closed and lamellar. One or more slats separating the inner compartment of the external solution. The polar hydrophilic head groups of lipids (eg ethanolamine at cephalin, choline in lecithin) show towards the water phase, non-polar hydrophobic fatty acid residues are directed into the interior of Lippiddoppelschicht. The bilayers are two molecular layers or 4 to 5 nm thick. One differentiates the liposomes according to their size and number of lamellae and speaks of small, unilamellar (small unilamellar vesicles (SUV)) with radii up to 100 nm, large unilamellar (large unilameller vesicles (LUV)) with radii larger than 100 nm and multilamellar liposomes (multilamellar vesicles (MLV)). In aqueous phase, phospholipids stored together without any action to multilamellar liposomes, in which several Lippiddoppelschichten are onion-like arranged separated by water layers.

p0003The training lamellar arranged lipid bilayers is a consequence of the balance of physical forces and steric effects. The lipid bilayer also forms the block formation of the plasma membranes, encase the biological cells and at which proteins are embedded in the lipid matrix as the second block.

p0004The physical and physiological properties of liposomes are determined by their lipid composition, size, temperature, pH, type and strength of the buffer, the preparation conditions, history and age. The use of liposomes loaded with drugs, found in the diagnosis and treatment of great interest. Liposomes open as targeted carriers of drugs new therapy techniques (see for example R. Lawaczeck "liposomes as targeted drug carriers", German Apotheker Zeitung 127, 1771-1773 (1987), MJ Ostro "Liposomes", Sci. Am. 256, 90- 99 (1987)). For pharmaceutical purposes, are the permeability of the lipid membrane and the stability of the liposomes of interest; In addition, the physiologically Fusogenität and the kinetics of cellular uptake is important.

p0005Due to the broad interest There are a number of publications which deal with the physics, chemistry, biology, preparation and use of liposomes. A recent review article pharmaceutical orientation was from D. Lichtenberg and Y. Barenholz ( "Liposomes: preparation, characterization, and preservation" in Methods of Biochemical Analysis Vol 33, 337-462 (1988).) Published. The use of cells as drug carriers has been described, inter alia by C. Nicolau and employees ( "resealed red blood cells as a new blood transfusion product" Biblthca haemat. 51, 82-91 (1985)).

p0006To produce uni- and oligolamellarer and loaded liposomes various strategies have been developed (reviewed in Lichtenberg & Barenholz), which can be classified schematically as 1 physical destruction by shearing forces and 2. chemical disintegration by "helper molecules" of spontaneously forming ending multilamellar liposomes. The application of shear forces is not suitable in the inclusion of labile substances. Helper molecules are available in the form of detergents or organic solvent molecules. With the help of non-bilayer aggregates of lipid molecules (mostly micelles) formed. The removal of the helper molecules leads again to the spontaneously forming liposomal bilayers and the simultaneous intake of added substances. The complete removal of Detergenzmoleküle is often difficult, also, in proteinaceous Einschlußmolekülen undesired denaturation be obstructive. The uptake by biological cells can be achieved with subsequent adjustment of the isotonicity or electrically induced pore formation by hypo-osmolar disruption (lysis) of cells. Here externally existing substances are absorbed and encapsulated during closure of the membrane. A gentle lysis under isotonic, detergent and stress-free conditions is not known. There is therefore a need for a manufacturing method of ingredients-containing liposomes and / or biological cells, which does not have the abovementioned disadvantages. This object is achieved by the present invention.

p0007It has now been found that a liposomal and / or cellular uptake of substances, preferably pharmaceuticals for therapeutic and diagnostic purposes, surprisingly, by the application of gaseous helper molecules can be achieved. This method does not have the shortcomings of the above-outlined preparation process. In this new method, the aqueous lipid or cell suspension containing the entrapped substance in addition to the lipids, or cells according to the invention mixed with a gas. It come with gases "detergenzähnlichem effect" question, ie gases which in contrast to the hydrostatic pressure decrease the ordering of lipid molecules with increasing pressure and / or lead to a membrane solubilization. It is waited for the adjustment of the thermodynamic equilibrium with the aqueous lipid or cell suspension at the respective applied pressure. The gases used must be chemically inert with respect to the lipids and the aqueous phase and not able to interact with water as carbon dioxide or ammonia. Preferably, the use of an easily manageable, industrially available gases that do not leave a toxic residue comes in a pressure range up to 1000, preferably up to 200 bar in question. Exceeding the boiling curve in the p, T diagram in the liquid state may be advantageous. Suitable gases are preferably nitrous oxide (N₂0), halothane, argon, xenon, sulfur hexafluoride (SF₆), alkanes such as methane, ethane, propane, butane, hexane, heptane, alkenes such as ethene, propene, butene, hexene, heptene. When nitrous oxide, a pressure up to 55 bar is selected, as it is in technical pressure bottles available. For the temperature of the area between the respective freezing and boiling point is available, preferably from 0 ° to 37 ° C. The gas molecules mentioned exert a "detergenzähnlichen" effect and lead to an opening and / or solubilization of the lipid bilayer and / or diaphragm. During the relaxation phase, spontaneously develop new liposomes or close the membranes again, so that the added substances are added. This process may be repeated cyclically. Liposomes a narrowing of the liposome size on a pressure release to atmospheric pressure by size-selective separation filter is possible. The lipid composition of the liposomes can be optimized depending on the therapeutic and / or diagnostic utility, and according to the destination. In the case of biological cells in addition to the cells of the bloodstream, such as erythrocytes, leukocytes, lymphocytes and platelets are preferably cells that are maintained in culture in question.

p0008The advantages of the liposome preparation and cell loading with the "gas helper method" are obvious: The procedure is technically easy to implement and inexpensive. The gas molecules are in contrast to the conventional detergent molecules easily removed. Because of the inert nature and the remaining only in trace amounts makes the toxicity of the molecules used - if at all - only a minor role. It is advantageous to work at physiological temperatures or below. The application of inert gases in said pressure range is not harmful for most substances or cells.

p0009The method is hydro- for liposomal and cellular addition, incorporation and / or substitution and suitable lipophilic substances.

p0010The loaded liposomes may be surface-modified and used as a source or target-organ-specific drug carriers or drug store.

Examples

p0011<ul><li><b>Example 1:</b> 20 mg dimyristoyl lecithin are added to 1 ml of chloroform. The solution is introduced into the pressure vessel. Chloroform is evaporated with purified nitrogen and subsequently under vacuum. 2 ml of water containing 1 mM carboxyfluorescein (and recrystallized from ethanol), 20 mM Tris HCl adjusted to pH 8, are introduced. CO₂ and O₂ are expelled by flushing with nitrous oxide. The pressure vessel is closed and created a laughing gas pressure of 50 bar at room temperature. The solution is shaken for 0.5 hours, then slowly and isothermal to atmospheric pressure. The process is repeated 5 times. The solution is taken from the pressure vessel and, for the removal of the existing external carboxyfluorescein over an anion exchanger. The liposome fraction containing the entrapped dye and can be used for fluorescence studies.</li><li><b>Example 2:</b> as Example 1, instead of dimyristoyl lecithin 200 mg of a 4 are: 1 mixture of yolk lecithin and cholesterol taken. Carboxyfluorescein is replaced by 20 mM gadolinium-DTPA as MR contrast agents. The liposomes obtained can be used in MR diagnosis.</li><li><b>Example 3:</b> Production of erythrocytes, or red blood cell ghosts, which are loaded with carboxyfluorescein, under blutisotonen conditions. Fresh bovine blood is washed 3 times in PBS buffer pH 7.4 and freed up on the erythrocytes of other ingredients. The dark red, cloudy erythrocyte is with PBS buffer containing carboxyfluorescein, diluted so that the concentration of carboxyfluorescein 10⁻⁵ M is. The solution is placed in a pressure measuring cell. After rinsing with N₂O N₂O be 50 bar applied at 37 ° C, stirred and pursued the optical density at 675 nm. occurs between 400 and 600 min lysis of the cells, clearly visible through the typical sigmoid drop in optical density. Reference measurements under atmospheric pressure or hydrostatic pressure 50 bar up to 18 hours show no lysis of the erythrocytes. The solutions are slowly depressurized to atmospheric pressure and examined under a microscope in transmission and fluorescence. The red cells show before and after lysis, the typical discoid appearance. Non-lysed cells (controls) included unlike the meantime lysed no carboxyfluorescein. The cell fluorescence remains visible even after extensive washing. It is thus apparent that the erythrocytes was added to dye lysed state under isotonic conditions and were resealed during relaxation.</li><li><b>Example 4:</b> How Beispeil 3, only instead of sheep red Rindererythrocyten in 2 mM KH₂PO₄, 9 mM Na₂HPO₄, 3 mM KCl, 137 mM NaCl pH 7.5 is used. Lysis occurs between 55 to 70 a minute. The results are comparable to those from Example 3. FIG.</li></ul>