Liposomes containing particulate materials

{
  "publication_number": "US2002041895A1",
  "country": "US",
  "kind": "A1",
  "title": "Liposomes containing particulate materials",
  "abstract": "A method is provided for the formation of liposomes of 0.1 μm to 50 μm in diameter having unilamella or multilamella structure and containing water insoluble or undissolved particulate materials comprising (a) forming liposomes and removing substantially all of any organic solvent used in their preparation, (b) freeze drying the liposomes so formed and then (c) rehydrating them in intimate admixture with the particulate material. Preferred encapsulated materials are particulate materials, most preferably microorganisms, plant or animal cells or water insoluble structures having organic solvent labile biochemical or immunological activity, but any water insoluble particulate may be encapsulated using the method. For example catalysts or drugs that are sparingly soluble may also be so incorporated such that slow release into the a patients body may be provided while release of detergents included in the many liposome preparation protocols may be avoided.",
  "claims": [
    "1. A method for forming liposomes of greater than 0.1 μm diameter containing undissolved or water insoluble particulate biologically, chemically or physically active material comprising (a) forming liposomes (b) freeze drying the liposomes so formed and then (c) rehydrating them in intimate admixture with the material to be contained therein. 2. A method for the formation of unilamella liposomes containing undissolved or water insoluble particulate material as claimed in claim 1 comprising (a) forming unilamella liposomes of size sufficiently large to accommodate the particulate material to be included therein, (b) freeze drying the liposomes so formed and then (c) rehydrating them in intimate admixture with the particulate material. 3. A method for the formation of multilamella liposomes containing undissolved or water insoluble particulate material as claimed in claim 1 comprising (a) forming unilamella liposomes of diameter less than that of the multilamella liposome to be produced (b) freeze drying the liposomes so formed in the presence of material to be contained therein and then (c) rehydrating the freeze dried liposomes and material. 4. A method as claimed in any one of the preceding claims wherein substantially all of any organic solvent used in the preparation of the liposomes in step (a) is removed before the rehydration. 5. A method as claimed in any one of the preceding claims wherein substantially all of any organic solvent used in the preparation of the liposomes in step (a) is removed prior to step of freeze drying (b). 6. A method as claimed in any one of the preceding claims wherein the substantially water insoluble or undissolved particulate material comprises a microorganism, a plant or animal cell or a water insoluble structure having organic solvent labile biochemical or immunological activity. 7. A method as claimed in any one of claims 1, 2 or 4 to 6 wherein the step (a) of forming liposomes comprises the formation of liposomes of diameter 0.1 μm to 50 μm. 8. A method as claimed in claim 7 wherein the liposomes are giant liposomes of diameter of 1 μm to 30 μm. 9. A method as claimed in any one of the preceding claims wherein step (b) is carried out with the material to be encapsulated already intimately mixed with the liposomes. 10. A method as claimed in any one of the preceding claims wherein a protectant against the effects of water loss is added to the liposome product after rehydration. 11. A method as claimed in claim 10 wherein the protectant is trehalose. 12. A method as claimed in any one of the preceding claims wherein step (c) is carried out by controlled addition of water in a small quantity just sufficient to produce a suspension, followed after a first period of several minutes by a similar amount of a suitable buffer biologically acceptable to the material to be encapsulated such as to retain its desired activity; and the suspension so obtained mixed with a larger volume of buffer after a second period of several minutes. 13. A method as claimed in claim 12 wherein the first and second periods are of from 20 to 40 minutes each. 14. A method as claimed in claim 12 or 13 wherein the buffer added is phosphate buffered saline of about pH 7.4. 15. A method as claimed in any one of the preceding claims wherein the freeze-drying step (b) is carried out by freeze drying a suspension of liposomes and material to be encapsulated, and the total volume of water and saline added in rehydration step (c) is sufficient to provide from 1 to 10 times that of the volume of the suspension. 16. A method as claimed in any one of the preceding claims wherein the liposomes formed in step (a) comprise a lipid composition comprising phosphatidylcholine (PC) and/or distearoylphosphatidyl choline (DSPC). 17. A method as claimed in claim 16 wherein the lipid composition further comprises one or more of cholesterol, phosphatidyl glycerol (PG) and triolein (TO). 18. A method as claimed in any one of the preceding claims wherein liposome formation step (a) is carried out by mixing a chloroform solution of a lipid composition with sucrose solution to form an emulsion, and that is mixed with a similar ether water emulsion to provide a water in oil in water emulsion, whereupon substantially all of the organic solvent is removed from that, resulting in generation of liposomes. 19. A method for the separation of liposomes from non-entrapped water insoluble or undissolved particulate materials characterised in that it places a mixture of the two on a density gradient and centrifuges it, the fractions of the gradient are removed, those containing the separated liposomes collected, and the liposomes separated from these with the free materials being collected in the lower fractions. 20. A method as claimed in claim 19 wherein the density gradient is a sucrose gradient from 0.4 to 4M in strength, or a gradient including an equivalent density range and/or sugar. 21. A method as claimed in claim 1 to 20 wherein the material to be contained within the liposome comprises a living microorganism or plant or animal cell, characterised in that after the rehydration step (c) the microorganism or cell is provided with nutrients through the liposome wall such that it is cultured and multiplies. 22. A method as claimed in claim 21 wherein the nutrients are provided with an inhibitor of the microorganism or cell's ability to metabolise the lipid of the liposome. 23. A method as claimed in claim 21 or 22 wherein the cell is in the form of a spore. 24. A liposome obtained by a method as claimed in any one of claims 1 to 21. 25. A liposome characterised in that it contains one or more live or attenuated microorganisms, plant or animal cells, or water insoluble structures having organic solvent labile biochemical or immunological activity. 26. A liposome characterised in that it contains live or attenuated virus, bacteria and/or protozoa. 27. A liposome as claimed in any one of claims 23 to 26 wherein the live or attenuated microorganisms are selected from measles, polio virus, Bordetella pertussis, Bacille Calmette-Guerin and Salmonella typhi. 28. A liposome as claimed in claim 25 or 27 wherein the water insoluble non-living structures comprise cytokine, enzyme, antigen or antibody bearing support materials. 29. A liposome as claimed in any one of claims 23 to 28 characterised in that it further contain water soluble biochemically or immunologically active materials. 28. A liposome preparation comprising liposomes as claimed in any one of claims 22 to 27 being of 0.1 μm to 50 μm in diameter and containing viable bacterial spores in excess of 4 spores per vesicle per 6.2 μm mean diameter sphere of liposome. 29. A liposome preparation as claimed in claim 28 wherein the liposomes are multilamella and the number of viable bacterial spores is 6 spores per 3.2 μm mean diameter sphere of liposome or more. 30. A liposome preparation as claimed in claim 28 wherein the liposomes are unilamella and the number of viable bacterial spores is 13 spores per 7.2 μm mean diameter sphere of liposome or more. 31. A composition comprising a physiologically acceptable carrier and one or more liposomes or a liposome preparation as claimed in any one of claims 23 to 30. 32. A method as claimed in any one of claims 1 to 22 substantially as described in Example 1 or Example 2. 33. A liposome as claimed in any one of claims 23 to 32 substantially as claimed in Example 1 or Example 2."
  ],
  "cpc": [
    "A61K 9/1277",
    "A61K 9/127",
    "Y10S 977/907",
    "Y10S 977/918"
  ],
  "assignees": [
    "UNITE KINGDOM DEFENCE EVALUATI"
  ],
  "filing_date": "2001-09-27",
  "publication_date": "2002-04-11",
  "priority_date": "1993-10-07",
  "application_number": "US-96360101-A",
  "family_id": "10743157"
}