WO2008067613A1 - Liposome production - Google Patents
Liposome production Download PDFInfo
- Publication number
- WO2008067613A1 WO2008067613A1 PCT/AU2007/001891 AU2007001891W WO2008067613A1 WO 2008067613 A1 WO2008067613 A1 WO 2008067613A1 AU 2007001891 W AU2007001891 W AU 2007001891W WO 2008067613 A1 WO2008067613 A1 WO 2008067613A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- liposome
- solution
- lipid
- entrapped
- lecithin
- Prior art date
Links
- 239000002502 liposome Substances 0.000 title claims abstract description 137
- 238000004519 manufacturing process Methods 0.000 title abstract description 16
- 150000003904 phospholipids Chemical class 0.000 claims abstract description 45
- 239000000787 lecithin Substances 0.000 claims abstract description 40
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 claims abstract description 39
- 235000010445 lecithin Nutrition 0.000 claims abstract description 39
- 229940067606 lecithin Drugs 0.000 claims abstract description 39
- 238000000034 method Methods 0.000 claims abstract description 39
- 230000008569 process Effects 0.000 claims abstract description 39
- 150000001875 compounds Chemical class 0.000 claims abstract description 36
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 26
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- 150000002433 hydrophilic molecules Chemical class 0.000 claims abstract description 23
- 239000000416 hydrocolloid Substances 0.000 claims abstract description 18
- 239000000243 solution Substances 0.000 claims description 89
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 claims description 76
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- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 claims description 14
- 150000008104 phosphatidylethanolamines Chemical class 0.000 claims description 14
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- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 7
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- PZNPLUBHRSSFHT-RRHRGVEJSA-N 1-hexadecanoyl-2-octadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)O[C@@H](COP([O-])(=O)OCC[N+](C)(C)C)COC(=O)CCCCCCCCCCCCCCC PZNPLUBHRSSFHT-RRHRGVEJSA-N 0.000 description 5
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- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 4
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/10—Foods or foodstuffs containing additives; Preparation or treatment thereof containing emulsifiers
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
- A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
- A23L29/212—Starch; Modified starch; Starch derivatives, e.g. esters or ethers
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
- A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
- A23L29/238—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin from seeds, e.g. locust bean gum or guar gum
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
- A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
- A23L29/256—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin from seaweeds, e.g. alginates, agar or carrageenan
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
- A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
- A23L29/262—Cellulose; Derivatives thereof, e.g. ethers
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
- A23P10/00—Shaping or working of foodstuffs characterised by the products
- A23P10/30—Encapsulation of particles, e.g. foodstuff additives
- A23P10/35—Encapsulation of particles, e.g. foodstuff additives with oils, lipids, monoglycerides or diglycerides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
- A61K8/14—Liposomes; Vesicles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Definitions
- the invention relates to producing liposomes.
- Liposomes are structures comprising one or more concentric spheres of lipid bilayer that enclose an aqueous compartment. They have a diameter in the range of 50 nm to 100 ⁇ m and are typically prepared by the process of dispersing phospholipids in an organic solvent followed by a hydration of the lipids in an aqueous medium to form a lipid vesicle, with post size reduction and purification from the aqueous medium.
- liposomes are inherently impermeable to large, mostly polar molecules, and can therefore be used to contain or encapsulate said molecules.
- molecules include enzymes, nucleic acids and compounds such as pharmaceuticals. Accordingly, liposomes find particular application in the delivery of molecules to a biological system where they are useful for protecting molecules from degradation and for targeting molecules to particular cells or tissues.
- liposomes are conventionally made by mixing highly purified phospholipids such as phosphatidylcholine and phosphatidylethanolamine with sterols such as cholesterol or ergosterol together and an admixture of synthetic lipids which when combined are capable of producing a liposome having desired membrane characteristics.
- sterols such as cholesterol or ergosterol
- liposomes with pharmaceuticals.
- the approach has generally been to use cationic lipids such as DOTAP and stearylamine to produce these liposomes.
- cationic lipids such as DOTAP and stearylamine to produce these liposomes.
- DOTAP DOTAP
- stearylamine stearylamine
- the solution that is enriched for phosphatidylcholine includes phosphatidylcholine in an amount that constitutes about 80 mole % of lipids in the solution.
- aqueous solution including a dispersion of a lipid and a hydrophobic compound to be entrapped in a liposome
- aqueous solution including a hydrophilic compound to be entrapped in a liposome and an ionic polymer for binding to the hydrophilic compound
- liposomes produced by the above described process and compositions containing same.
- a liposome having one or more components entrapped therein including:
- lipid bilayer including phospholipids extracted from lecithin, the lipid bilayer having one or more hydrophobic compounds entrapped within;
- hydrocolloid mass and or an unfractionated lecithin component forming a core, said core being encapsulated by the lipid bilayer and having one or more hydrophylic compounds entrapped within.
- the inventor has devised a low cost process for making liposomes that are capable of entrapping multiple components whether they be hydrophobic or hydrophilic.
- the liposomes are non toxic and may be provided with a net cationic charge to assist with binding target membranes. Further, they tend to more completely entrap smaller more polar molecules or components in the liposome and hence inhibit leakage of components.
- the inventor has sought to reduce production costs of liposome manufacture. He has done this by determining a process by which an impure source of phospholipid, such as lecithin, could be used in place of pure or refined phospholipid sources that are conventionally used for liposome manufacture.
- a process for producing a liposome includes:
- the solution that is enriched for phosphatidylcholine includes phosphatidylcholine in an amount that constitutes about 80 mole % of lipids in the solution.
- the inventor has determined the critical amount of phosphatidylcholine that is required in an organic solvent for production of liposomes in circumstances where the phosphatidylcholine is provided in the form of a solution that is enriched for phosphatidylcholine, instead of being provided in the form of pure phosphatidylcholine.
- the solution that is enriched for phosphatidylcholine contains other lipids and other compounds that affect the capacity for liposome formation, and in particular, for encapsulation of multi-components of both hydrophobic and hydrophilic nature.
- the solution enriched for phosphatidylcholine includes phosphatidylcholine in an amount that constitutes about 80 mole % of lipids in the solution enriched for phosphatidylcholine, preferably about 85 mole %.
- the solution enriched for phosphatidylcholine further includes phosphatidylethanolamine.
- This has a smaller head group than phosphatidylcholine and hence modulates the packing density of phosphatidylcholine. It is generally included in an amount of less than about 10 mole % of lipids.
- the solution enriched for phosphatidylcholine may also contain ancillary phospholipids or lipid / triglyceride derivatives including phosphatidyl inositol, phosphatidyl serine, phosphatidic acid or cocoa butter. These may be provided in amounts of up to about 7 mole % of lipids in the solution enriched for phosphatidylcholine.
- the amount of lipid in the solution enriched for phosphatidylcholine is generally between about from 1 g/mL to about 2 g/mL.
- lecithin is extracted into an organic solvent, such as an alcohol, to form an extract.
- the extract so formed is more or less a homogenous dispersion of lecithin in which substantially all of the phosphatidylcholine of the lecithin as been partially solubilized and about 50% of the phosphatidylethanolamine of the lecithin has been partially solubilized.
- This normally requires a solvent to lecithin ratio of about 3:1.
- solvent i.e. about 2:1
- not enough phosphatidylcholine is extracted into the solvent.
- solvent is wasted and too much energy is required to remove it in the concentration step that follows.
- the temperature of the extract is heated to form a precipitate and supernatant.
- the heating of the extract increases the solubility of the phosphatidylcholine and phosphatidylethanolamine in the extract. It also causes other phospholipids in the extract to precipitate to form a dark brown toffee like substance. This is observed within about 30 minutes heating the extract at about 40 0 C. Above this temperature, there is no significant benefit in so far as increasing solubility of phosphatidylcholine. Further, the precipitate tends to burn, and may cause spoilage. However, other temperatures, higher or lower than as stated may be used depending on the type of organic solvent used.
- the supernatant including predominantly phosphatidylcholine, some phosphatidylethanolamine and even less of other phospholipids and triglycerides, is collected.
- the supernatant generally has a light brown translucent appearance.
- the temperature of the supernatant is adjusted to form a further precipitate and further supernatant.
- the temperature of the supernatant is lowered to cause phase transition of phosphatidyl serine and phosphatidyl inositol and remaining ancillary lipids or derivatives in the solution, hence leading to precipitation of these compounds.
- This temperature is about -20 0 C in ethanol. It may be higher or lower in other organic solvents.
- the precipitate formed is a light gelatinous like mass, similar to a loose gel and having a soft, tacky consistency.
- the further supernatant has a clear straw coloured appearance. 7 001891
- the further supernatant is concentrated to form a solution enriched for phosphatidylcholine.
- concentration is achieved by evaporation of the solvent.
- the solvent is ethanol
- the solvent is heated to the boiling point of ethanol, which is about 79 0 C. Higher or lower temperatures can be used depending on the boiling temperature of the solvent.
- the inventor has found that the solution recovered from the further supernatant is enriched for phosphatidylcholine. It contains about 80 to 85 mole percent phosphatidylcholine. The inventor has found that this solution can be used for formation of liposomes as a substitute for pure phosphatidylcholine.
- the solution recovered from the further supernatant is concentrated to about 1 to 2 g/mL of lipid.
- the solution may become too sticky or otherwise too difficult to handle in subsequent steps for liposome production.
- lipids may be present in the solution enriched for phosphatidylcholine.
- Phosphatidylethanolamine is one example. This may be present in an amount of 5 to 10 mole percent.
- Other lipids including phosphatidyl serine, phosphatidyl inositol or phosphatidic acid may be present in the composition enriched for phosphatidylcholine, ideally at less than 7 mole % of total lipid of the solution enriched for phosphatidylcholine.
- further lipids may be added to the solution enriched for phosphatidylcholine.
- lipids include cholesterol, ergosterol and linoleic, oleic and palmitic fatty acids or tri-glycerides.
- Cholesterol or ergosterol may be provided in an amount of about 1 to 5 mole percent. These molecules are particularly useful for tightening or otherwise increasing the degree of crystallinity of the lipid bilayers of a liposome formed from the solution enriched for phosphatidylcholine and in some circumstances inferring endocytosis across cell membranes. This may also reduce loss of components entrapped in the liposome and provide a trigger for cell recognition.
- the linoleic and oleic fatty acid triglycerides may be derived from cocoa butter having a defined melting point of less than about 37 0 C.
- the cocoa butter may be present in an amount of from 5 to 10 mole percent. Cocoa butter may be further useful for facilitating encapsulant release at a defined melting temperature.
- the lipid concentration of the solution enriched for phosphatidylcholine is normally about 1 g/ml, although higher or lesser concentrations are possible by controlling evaporation.
- Ethanol is typically used as an organic solvent, although other like alcohols and organic solvents may be used.
- the temperatures that are used in the formation of the solution enriched for phosphatidylcholine may be higher or lower than those stated. This may in part depend on the source of the lecithin, for example, whether it has been pre-fractionated or refined, whether it is from soy, egg or other source such as dairy, and the type of organic solvent that is used to dissolve it.
- soy lecithin is particularly useful as a starting material for obtaining the solution enriched for phosphatidylcholine.
- the inventor estimates the raw material costs for phospholipid as used in production of liposomes made according to these embodiments to be in the order of 30 cents/gram. In comparison, the inventor estimates the conventional approaches which tend to use 90-95 mole % phosphatidylcholine to incur costs of the order of $1.20 /gram of phospholipid as used commercially.
- the solution enriched for phosphatidylcholine may further include one or more molecules to be entrapped into the liposome. These molecules are generally hydrophobic and hence are dissolved in the solution enriched for phosphatidylcholine. As detailed further below, apart from the cost reduction advantage of the process, the solution enriched for phosphatidylcholine contains a greater diversity of molecules. Accordingly the solution enriched for phosphatidylcholine has a much greater range of polarities. This means that the solution has a much greater capacity for entrapping a range of molecular species than can be provided by conventional approaches that use purer sources of phospholipids to form liposomes.
- the aqueous solution to which the solution enriched for phosphatidylcholine is brought into contact with may also contain one or more molecules to be entrapped in a liposome. It may also contain unfractionated lecithin and one or more ionic polymers for binding to a molecule to be entrapped in a liposome. These embodiments of the process are discussed in more detail below.
- a number of approaches are available for the solution enriched for phosphatidylcholine and the aqueous solution to contact to form a liposome. Examples include homogenisation, dehydration followed by re-hydration, sonication and ethanol injection. These approaches are described in more detail below.
- the inventor has also recognised that conventional approaches for making liposomes, which use highly purified or otherwise refined phospholipids, tend to limit the range of components that may be entrapped in a liposome. He has also recognised that a diverse range of polarities of compounds in impure phospholipid preparations would be useful for entrapment of multiple compounds in a liposome production process. He recognised that this range of polarities could be provided by using lecithin as a source of phospholipid for production of liposomes.
- a process for entrapping a hydrophobic compound in a liposome includes:
- aqueous solution including a dispersion of a lipid and a hydrophobic compound to be entrapped in a liposome; and - contacting the aqueous solution with a lipid solution to form a liposome having the hydrophobic compound entrapped therein.
- the dispersion of lipid such as lecithin
- the aqueous solution with a diversity of lipids having a range of polarities, hence facilitating the binding of a range of hydrophobic compounds to be entrapped in the liposome.
- hydrophobic compounds tend to interact with the fatty acyl chains of the lecithin lipids of the aqueous solution leading to entrapment when the liposome is formed.
- the lipid dispersion in the aqueous solution also improves the miscibility of the aqueous solution with the lipid solution.
- hydrophobic compounds examples are described further herein.
- the lipid solution to which the aqueous solution is brought into contact may be a solution enriched for phosphatidylcholine as described above. This advantageously provides for the liposomes to be formed without the expense of using pure phospholipids. Further, this lipid solution enables an even greater range of molecules to be entrapped as it also contains a greater range of polarities than a solution formed from pure phospholipids.
- the lipid solution may be one produced by dissolving at least one pure phospholipid, in particular phosphatidylcholine, in an organic solvent to an amount of at least about 80 mole %. Sterols may also be added.
- the aqueous solution may be pH modified to provide the liposome formed according to the process with a net cationic charge. It may also contain an ionic polymer (with or without post additions of calcium ions) for inhibiting leakage of an entrapped compound from the liposome. These embodiments are discussed in more detail below.
- the lecithin is provided in the aqueous solution in an amount to provide a concentration of lipid therein that is less than the concentration of lipid in the lipid solution.
- concentration of lipid in the aqueous solution is generally about 0.05 g/mL although higher or lower concentrations may be used depending on the type of lipid dispersed in the aqueous solution.
- an ionic polymer such as a hydrocolloid is particularly useful for entrapping a hydrophilic molecule in a liposome and for preventing or inhibiting an entrapped hydrophilic molecule from leaking from a liposome.
- a process for entrapping one or more hydrophilic compounds in a liposome includes:
- aqueous solution including a hydrophilic compound to be entrapped in a liposome and an ionic polymer for binding to the hydrophilic compound
- the ionic polymer is a hydrocolloid.
- the hydrocolloid is generally cationic at pH of 5.0 or less.
- Suitable hydrocolloids are those that are homogenously dissolved and that form a low viscosity solution that does not gel with an anticipated higher ratio of carboxyl groups or available hydrogen ions at a pH of 4 to 5, as well as providing the lowest anionic to cationic effect when prepared as a liposomes.
- hydrocolloids examples include low molecular weight carboxymethyl cellulose, maltodextrin and sodium alginate. These are described in more detailed below. Kappa, iota and lambda carrageenan, guar gum, xanthan gum and starch may also be hydrocolloids.
- the aqueous solution is provided with an acid pH, for example a pH in the range of 4.0 to 5.0.
- a hydrocolloid such as carboxymethyl cellulose or maltodextrin becomes cationic, (in the presence of added calcium ions) facilitating binding with hydrophylic components to be entrapped in the liposome.
- the liposome produced according to the process tends to have an overall cationic charge, facilitating in some cases a fusion of the liposome with a target membrane.
- the aqueous solution may also be provided with lecithin. When hydrated, the lecithin may interact with certain hydrophylic components leading to entrapment of hydrophilic compounds in a liposome.
- the lipid solution to which the aqueous solution is brought into contact may be one formed by dissolving a composition enriched for phosphatidylcholine described above in an organic solvent. This advantageously provides for the liposomes to be formed without the expense of using pure phospholipids. Further, this lipid solution enables an even greater range of molecules to be entrapped as it also contains a greater range of polarities than a solution formed from pure phospholipids.
- the lipid solution may be one produced by dissolving at least one pure phospholipid in an organic solvent.
- the process includes the further step of remote loading a hydrophilic compound into the liposome. This is achieved by contacting a liposome produced according to the embodiment with a solution having a pH of about 7, the solution containing divalent cations, preferably about 50 mM calcium ions, and a hydrophilic compound to be loaded into the liposome.
- the hydrophylic compound may be loaded into the liposome under the pH gradient with calcium cations to activate the gel forming properties of the hydrocolloid and to provide the liposome with an overall cationic charge.
- a liposome having one or more components entrapped therein including:
- lipid bilayer including phospholipids extracted from lecithin, the lipid bilayer having one or more hydrophobic compounds entrapped within;
- the liposome may be multi-lamellar vesicle or a unilamellar vesicle.
- the lipid bilayer typically contains phosphatidylcholine in an amount of 80 to 85 mole % of the total lipids of the lipid bilayer. It may further comprise phosphatidylethanolamine in an amount of about 10 mole % of total lipids of the lipid bilayer. It may further comprise a sterol such as cholesterol or ergosterol in an amount of about 2 to 5 mole % of the total lipids of the lipid bilayer. Other phospholipids such as phosphatidyl serine, phosphatidyl inositol and phosphatide acid are generally provide in an amount of less than about 7 mole % total lipids of the bilayer.
- the lipids for formation of the lipid bi-layer may be provided by the process described above for formation of a solution that is enriched for phosphatidylcholine from lecithin. These lipids may be supplemented with pure phospholipids.
- hydrophobic and hydrophilic compounds include flavours, functional food ingredients, nutriceuticals, pharmacuetical and cosmeceutical compounds.
- the hydrocolloid mass is formed from a maltodextrin, carboxy methyl cellulose or sodium alginate.
- the liposome is provided with an overall cationic charge for facilitating binding of the liposome to a target membrane.
- PC phosphatidylcholine
- the study also determined the required inclusion ratios of other phospholipids and lipids to enhanced binding efficiencies.
- Solvent evaporate ethanol at 80 0 C to produce a concentration of 1 g/ mL phospholipid, measuring PC and PE to provide an enrichment of 80 to 85 % PC.
- a suitable formulation to produce cost efficient liposomes will include 80-85 mole percent phosphatidylcholine (PC), 5-10 mole percent Phosphatidylethanolamine (PE), 2-5 mole percent ergosterol (to tighten or increase the degree of crystallinity of the lipid bilayers thereby reducing solute losses) and 5-10 mole percent cocoa butter, [a triglyceride containing predominately linoleic acid (C18:2) and Oleic acid (C18:1) fatty acids (similar to the structure contained with the derived phospholipids) with a defined melting point between 34-37 0 C].
- Negligible levels of Phosphatidyl serine (PS), Phosphatidyl inositol (Pl) or Phosphatidic acid (PA) will be present in the fractionated lecithin preparation.
- phospholipids derived from fractionated soy lecithin provides for a unique ratio of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) with inherent fatty acids of a defined chain length and orientation (as identified above) to assist in forming stable liposomes capable of multiple component encapsulation, for encapsulating and retaining solutes based on phospholipid type, phase transition temperatures, charge potential, solubility and packing configuration of fatty acyl chains.
- PC phosphatidylcholine
- PE phosphatidylethanolamine
- the fractionated phospholipids can be used under specified conditions to increase solute binding during an ethanol injection process to form liposomes when prepared at high lipid concentration (dispersed in ethanol and prepared as an injection solution while also incorporated [but to a less purified yet similar chemical composition] suspension of phospholipids derived from the same lecithin stock material) dispersed at a low concentration into the injection buffer together with a derived cationic charged polysaccharide at a defined fluidity (molecular weight and charge potential a pH below its p Ka).
- the solutes being encapsulated may be included in the concentrated lipid suspension if hydrophobic or in the injection buffer if hydrophilic.
- Example 3 Formulation of an injection buffer to produce liposome with enhanced solute encapsulation
- the most effective materials trialled were sodium alginate with a low viscosity and molecular weight and high sodium substitution, carboxymethyl cellulose at low fluidity [(10-100) representing a low molecular weight derivative] and a medium conversion potato based maltodextrin (containing charged phosphate side groups) with a DE ⁇ 10 and a moderate proportion of medium to low chain amylose fractions for enhanced binding of the solute without forming firm gels.
- the pH under which this injection buffer was prepared is based on a citrate and phosphate system stable at pH 4.0-5.0 providing a cationic effect to the injection buffer (contained hydrated, less purified soy lecithin with hydrophilic flavours) and an overall cationic effect to the formed liposomes formed from the anionic concentrated lipid injection solution with added calcium ions. It is believed the differential in charge has further helped to increase the overall binding of flavour solutes while producing liposomes of a defined size and stability that is suitable for flavour solute encapsulation.
- the particle size varied from 400nm to 5 micron (typically 2-5micron) and the encapsulation efficiency for Furaneol increased from 7% in water alone at pH 7.0, to 20% when injected into a 0.1% solution at pH 4.0 with 50mm calcium ions as calcium chloride.
- Paselli SA2 enzyme converted, pre-gelatinised potato starch with a DE ⁇ 10 Trialled optimally at 0.1 % w/w in the injection buffer at pH 4.0 with a zeta potential as an empty liposome recorded at -10 mV and with added flavour (Furaneol) and the addition of 50 mm calcium ions producing a cationic 7.3 mV response.
- the particle size varied from 400 nm to 5 micron (typically 2-5 micron) and the encapsulation efficiency for Furaneol increased from 7% in water alone to 15% when injected into a 0.1% solution at pH 4.0 with 50 mm calcium ions as calcium chloride.
- Cellogen FSA Sodium Carboxymethyl Cellulose: viscosity 600-800 cps at 2% w/w in solution at 25 0 C at neutral pH. Trialled optimally at 0.1 % w/w in the injection buffer at pH 4.0 with a zeta potential as an empty liposome recorded at -14 mV and with added flavour (Furaneol) and the addition of 50 mm calcium ions producing a cationic 9.6 mV response.
- the particle size varied from 400 nm to 5 micron (typically 2-5micron) and the encapsulation efficiency for Furaneol increased from 7% in water alone to 20% when injected into a 0.1% solution at pH 4.0 with 50 mm calcium ions as calcium chloride.
- Example 5 Construction of a device for producing improved liposomes for enhanced encapsulation:
- a device has been designed, constructed and trialled for the semi continuous manufacture of liposomes capable of scaled manufacture for the encapsulation of multiple components utilising fractionated lecithin enriched for phosphatidylcholine.
- the device has a circulating flow of injection buffer of varying flow rate (required to improve encapsulation efficiency) at a pH of 4-5 and temperature of 4-40 0 C, including ionic polymers and hydrated lecithin to which is injected under pressure and ultrasonic energy up to 130 kHz, a phosphatidylcholine enriched solution.
- Liposomes are formed by immediate contact of the phospholipids and aqueous buffer by either direct injection into the recirculating liquid or as a fine droplet aspiration into air with venturi forces drawing the phospholipids into the buffer.
- the high surface area of the injection particles ( ⁇ 10.0 micron) contributes and the recirculation or venturi effect combining shear, pressure and temperature differences is capable of producing liposomes of ⁇ 1 micron.
- the ultrasonic (liquid into air) atomising device [Sonozap] to the top of a glass venturi mixing unit and operate at an injection solution flow rate of 10ml per minute and 130 kHz energy or
- the (liquid into liquid) injection device dispersing the pre ultrasonicated injection solution at the same energy and flow rate as used with the Sonozap (liquid into air) atomising device.
- the injection buffer will recirculate through the venturi or liquid mixing unit at a flow rate of 5-10 L per minute with a temperature of recirculation maintained between 4 and 4O 0 C depending on the phase transition temperature of the lipids used in the formulation.
- the recirculation solution will be ⁇ 10 0 C.
- the liposomes will form instantly on differential phase contact and be continually concentrated in the recirculating injection buffer by ultrafiltration using a 100 kDa NMWC hollow fibre membrane.
- the excluded permeate containing any unbound flavours will be continually reused and blended for reinjected with fresh injection buffer after being analysed by GC to optimise continued additions.
- Phospholipid (2 g/mL EtOH) was injected into alginate solution (0.1%) in Phosphate citrate buffer pH 4 to make liposomes containing alginate.
- the liposome containing alginate was ultracentrifuged (100,000 rpm, 15 minutes, 4 0 C) to separate the supernatant and to concentrate the liposome.
- the concentrated liposome was resuspended with Phosphate citrate buffer pH 7 containing Furaneol (SR-1, 0.2 g/mL) and Ca at 50 mM.
- the resuspended liposome was incubated for 1-2 days in the Furaneol + Ca Phosphate citrate buffer to remote load the Furaneol and to retain Furaneol in the liposome when the alginate gelled in the presence of Ca.
- the incubated liposome was ultracentrifuged (100,000 rpm, 15 minutes, 4 0 C) and washed with H 2 O (washing with Buffer of the same pH washed the Furaneol out of the liposome) to separate the supernatant containing free (not loaded) Furaneol and concentrate the liposome containing Furaneol.
- the concentrated liposome containing Furaneol was resuspended with H 2 O.
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Abstract
Description
Claims
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AU2007329193A AU2007329193A1 (en) | 2006-12-08 | 2007-12-07 | Liposome production |
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Citations (3)
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US4089801A (en) * | 1974-07-19 | 1978-05-16 | Battelle Memorial Institute | Process for the preparation of liposomes |
US5783211A (en) * | 1996-09-18 | 1998-07-21 | Dragoco, Inc. | Liposome encapsulated active agent dry powder composition |
US6355267B1 (en) * | 1993-11-05 | 2002-03-12 | Amgen Inc. | Liposome preparation and material encapsulation method |
-
2007
- 2007-12-07 AU AU2007329193A patent/AU2007329193A1/en not_active Abandoned
- 2007-12-07 WO PCT/AU2007/001891 patent/WO2008067613A1/en active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4089801A (en) * | 1974-07-19 | 1978-05-16 | Battelle Memorial Institute | Process for the preparation of liposomes |
US6355267B1 (en) * | 1993-11-05 | 2002-03-12 | Amgen Inc. | Liposome preparation and material encapsulation method |
US5783211A (en) * | 1996-09-18 | 1998-07-21 | Dragoco, Inc. | Liposome encapsulated active agent dry powder composition |
Non-Patent Citations (1)
Title |
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NIEUWENHUYZEN W. AND SZUHAJ B.F.: "Effects of lecithins and proteins on the stability of emulsions", FETT/LIPID, vol. 100, no. 7, 1998, pages 282 - 291, XP000767809, DOI: doi:10.1002/(SICI)1521-4133(199807)100:7<282::AID-LIPI282>3.3.CO;2-N * |
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