AU2007329193A1

AU2007329193A1 - Liposome production

Info

Publication number: AU2007329193A1
Application number: AU2007329193A
Authority: AU
Inventors: Yan Er; Yuanita Listiohadi; Desheng Liu; Michael Patane
Original assignee: Protech Research Pty Ltd
Current assignee: Protech Research Pty Ltd
Priority date: 2006-12-08
Filing date: 2007-12-07
Publication date: 2008-06-12
Also published as: WO2008067613A1

Description

WO 2008/067613 PCT/AU2007/001891 1 Liposome production Field of the invention The invention relates to producing liposomes. Background of the invention 5 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 ptm 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. 10 Previously described as artificial cells, liposomes are inherently impermeable to large, mostly polar molecules, and can therefore be used to contain or encapsulate said molecules. Examples of such 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 15 molecules from degradation and for targeting molecules to particular cells or tissues. There are a number of factors that limit the commercial application of liposomes. One factor is cost. More specifically, 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 20 which when combined are capable of producing a liposome having desired membrane characteristics. These purified phospholipids are very expensive and are generally in the order of 1 to 10 dollars per gram. Further, as noted above, some commercial applications involve the use of liposomes with pharmaceuticals. In these applications, it is desirable to produce a liposome that 25 has a net cationic charge as these liposomes tend to more readily fuse with a target membrane. The approach has generally been to use cationic lipids such as DOTAP and WO 2008/067613 PCT/AU2007/001891 2 stearylamine to produce these liposomes. However, apart from their expense, some of these cationic lipids are cytotoxic. Another factor is that to date it has generally been very difficult to encapsulate multiple components into a single liposome. This becomes an issue where the commercial 5 application requires a liposome to deliver or provide more than one payload compound. Examples are in the snack food or cosmetic industries where it is desirable to use a liposome to provide a flavour profile to a food or deliver a complex mixture of compounds to the skin, some which may be hydrophobic, others of which may be hydrophilic. 10 Further, it is becoming clearer that compounds that are entrapped or otherwise encapsulated in a liposome may over time leach from the liposome. This is of obvious concern where the leakage of the compound reduces the efficacy of a product, leads to spoilage of a product, or deleteriously effects health, for example where a cytotoxic drug leaks from a liposome. 15 Summary of the invention In certain embodiments there is provided a process for producing a liposome including: - forming a solution from lecithin wherein the solution is enriched for phosphatidylcholine; - contacting the solution that is enriched for phosphatidylcholine with an aqueous 20 solution to form a liposome; wherein the solution that is enriched for phosphatidylcholine includes phosphatidylcholine in an amount that constitutes about 80 mole % of lipids in the solution. In another embodiment there is provided a process for entrapping a hydrophobic 25 compound in a liposome including: WO 2008/067613 PCT/AU2007/001891 3 - providing an 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. 5 In another embodiment there is provided a process for entrapping a hydrophilic compound in a liposome including: - providing an aqueous solution including a hydrophilic compound to be entrapped in a liposome and an ionic polymer for binding to the hydrophilic compound; and - contacting the aqueous solution with a lipid solution to form a liposome having the 10 hydrophilic compound entrapped therein. In other embodiments, there are provided liposomes produced by the above described process and compositions containing same. In a further embodiment, there is provided a liposome having one or more components entrapped therein including: 15 - a lipid bilayer including phospholipids extracted from lecithin, the lipid bilayer having one or more hydrophobic compounds entrapped within; - a 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. 20 Detailed description of the embodiments 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 WO 2008/067613 PCT/AU2007/001891 4 polar molecules or components in the liposome and hence inhibit leakage of components. In more detail, in certain embodiments, the inventor has sought to reduce production costs of liposome manufacture. He has done this by determining a process by which an 5 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. Thus in certain embodiments there is provided a process for producing a liposome. The process includes: - forming a solution from lecithin wherein the solution is enriched for 10 phosphatidylcholine; - contacting the solution that is enriched for phosphatidylcholine with an aqueous solution to form a liposome; wherein the solution that is enriched for phosphatidylcholine includes phosphatidyicholine in an amount that constitutes about 80 mole % of lipids in the 15 solution. In more detail, 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. 20 In the former, 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. Prior to the invention, it was not known how much phosphatidylcholine would be required to make a stable liposome using a relatively impure source, such as a solution enriched for 25 phosphatidylcholine as obtained from lecithin. Further, it was not known how much phosphatidylcholine would be required in these circumstances to be able to encapsulate multiple compounds.

WO 2008/067613 PCT/AU2007/001891 5 In one embodiment, 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 %. In certain embodiments the solution enriched for phosphatidylcholine further includes 5 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. In certain embodiments the solution enriched for phosphatidylcholine may also contain ancillary phospholipids or lipid / triglyceride derivatives including phosphatidyl inositol, 10 phosphatidyl seine, 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 I g/mL to about 2 g/mL. 15 In these embodiments the solution that is enriched for phosphatidylcholine may be obtained by the following steps: - extracting lecithin into an organic solvent, such as an alcohol, to form an extract; - adjusting the temperature of the extract to form a precipitate and a supernatant; - collecting the supernatant; 20 - adjusting the temperature of the supernatant to form a further precipitate and further supernatant; - concentrating the further supernatant to form a solution enriched for phosphatidylcholine.

WO 2008/067613 PCT/AU2007/001891 6 In a first step of this embodiment, 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 5 lecithin has been partially solubilized. This normally requires a solvent to lecithin ratio of about 3:1. At lower amounts of solvent, i.e. about 2:1, not enough phosphatidylcholine is extracted into the solvent. At higher amounts of solvent, i.e. greater than 3:1, the solvent is wasted and too much energy is required to remove it in the concentration step that follows. 10 In a second step of this embodiment, 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 *C. Above this 15 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. In a third step of this embodiment, the supernatant including predominantly 20 phosphatidylcholine, some phosphatidylethanolamine and even less of other phospholipids and triglycerides, is collected. The supernatant generally has a light brown translucent appearance. In a fourth step of this embodiment, the temperature of the supernatant is adjusted to form a further precipitate and further supernatant. In this step, the temperature of the 25 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 '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. 30 The further supernatant has a clear straw coloured appearance.

WO 2008/067613 PCT/AU2007/001891 7 In a fifth step of this embodiment, the further supernatant is concentrated to form a solution enriched for phosphatidylcholine. In some embodiments, concentration is achieved by evaporation of the solvent. Where the solvent is ethanol, the solvent is heated to the boiling point of ethanol, which is about 79 0C. Higher or lower 5 temperatures can be used depending on the boiling temperature of the solvent. In more detail, 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. 10 Typically the solution recovered from the further supernatant is concentrated to about 1 to 2 g/mL of lipid. Depending on the solvent, at higher lipid concentrations, the solution may become too sticky or otherwise too difficult to handle in subsequent steps for liposome production. Other lipids may be present in the solution enriched for phosphatidylcholine. 15 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 phosphatidycholine. 20 In certain embodiments, further lipids may be added to the solution enriched for phosphatidylcholine. These 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 25 liposome formed from the solution enriched for phosphatidycholine 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. To further minimise production costs, it has been found that the linoleic and oleic fatty acid triglycerides may be derived from cocoa butter having a defined melting point of less WO 2008/067613 PCT/AU2007/001891 8 than about 37 '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. As noted above, the lipid concentration of the solution enriched for phosphatidyicholine 5 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 10 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. The inventor has found soy lecithin to be particularly useful as a starting material for 15 obtaining the solution enriched for phosphatidylcholine. Further, in some circumstances, it may be necessary to add pure phospholipid together with the solution enriched for phosphatidylcholine, to provide the solution with the appropriate amount of lipid. However, even in these circumstances, the cost of making the liposomes is much less than that incurred where pure phospholipid sources are 20 used for liposome production. Overall, 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 25 of phospholipid as used commercially. Where the objective of the liposome production process is to entrap molecules into the liposome, the solution enriched for phosphatidylcholine may further include one or more WO 2008/067613 PCT/AU2007/001891 9 molecules to be entrapped into the liposome. These molecules are generally hydrophobic and hence are dissolved in the solution enriched for phosphatidycholine. As detailed further below, apart from the cost reduction advantage of the process, the solution enriched for phosphatidylcholine contains a greater diversity of molecules. 5 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 10 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 phosphatidycholine 15 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. As outlined above, the inventor has also recognised that conventional approaches for making liposomes, which use highly purified or otherwise refined phospholipids, tend to 20 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. 25 In another embodiment there is provided a process for entrapping a hydrophobic compound in a liposome. The process includes: - providing an aqueous solution including a dispersion of a lipid and a hydrophobic compound to be entrapped in a liposome; and WO 2008/067613 PCT/AU2007/001891 10 - contacting the aqueous solution with a lipid solution to form a liposome having the hydrophobic compound entrapped therein. In this embodiment, the dispersion of lipid, such as lecithin, provides the aqueous solution with a diversity of lipids having a range of polarities, hence facilitating the 5 binding of a range of hydrophobic compounds to be entrapped in the liposome. These 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. 10 Examples of hydrophobic compounds 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 15 be entrapped as it also contains a greater range of polarities than a solution formed from pure phospholipids. Notwithstanding the above, in certain embodiments, 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 20 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. 25 Typically 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. The concentration of lipid in the aqueous solution is generally about 0.05 g/mL WO 2008/067613 PCT/AU2007/001891 11 although higher or lower concentrations may be used depending on the type of lipid dispersed in the aqueous solution. The inventor has also found that an ionic polymer, such as a hydrocolloid is particularly useful for entrapping a hydrophilic molecule in a liposome and for preventing or 5 inhibiting an entrapped hydrophilic molecule from leaking from a liposome. Thus, in a further embodiment there is provided a process for entrapping one or more hydrophilic compounds in a liposome. The process includes: - providing an aqueous solution including a hydrophilic compound to be entrapped in a liposome and an ionic polymer for binding to the hydrophilic compound; and 10 - contacting the aqueous solution with a lipid solution to form a liposome having the hydrophilic compound entrapped therein. Typically 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 15 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. Examples of hydrocolloids that are particularly useful in these embodiments include low molecular weight carboxymethyl cellulose, maltodextrin and sodium alginate. These are described in more detailed below. Kappa, iota and lambda carrageenan, guar gum, 20 xanthan gum and starch may also be hydrocolloids. In certain embodiments, the aqueous solution is provided with an acid pH, for example a pH in the range of 4.0 to 5.0. In this range, 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. 25 Advantageously, it has also been found that in this range, 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.

WO 2008/067613 PCT/AU2007/001891 12 In these embodiments, 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 5 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. 10 Notwithstanding the above, in certain embodiments, the lipid solution may be one produced by dissolving at least one pure phospholipid in an organic solvent. In certain embodiments, 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 15 solution containing divalent cations, preferably about 50 mM calcium ions, and a hydrophilic compound to be loaded into the liposome. According to this embodiment, 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 20 charge. In a further embodiments, there is provided a liposome having one or more components entrapped therein including: -a lipid bilayer including phospholipids extracted from lecithin, the lipid bilayer having one or more hydrophobic compounds entrapped within; 25 -a 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 comounds entrapped within.

WO 2008/067613 PCT/AU2007/001891 13 The liposome may be multi-lamellar vesicle or a unilamellar vesicle. The lipid bilayer typically contains phosphatidycholine 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 5 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 phosphatidic 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 10 above for formation of a solution that is enriched for phosphatidylcholine from lecithin. These lipids may be supplemented with pure phospholipids. Examples of hydrophobic and hydrophilic compounds include flavours, functional food ingredients, nutriceuticals, pharmacuetical and cosmeceutical compounds. Typically the hydrocolloid mass is formed from a maltodextrin, carboxy methyl cellulose 15 or sodium alginate. Typically the liposome is provided with an overall cationic charge for facilitating binding of the liposome to a target membrane. Examples Example 1 - Materials for encapsulation study: 20 Sample flavour components used for encapsulation into liposomes: Trans 2 Trans 4 Decadienal Furaneol Methyl Furanthiol Methyl Furanthiol Methyl Thiopropanal-3 WO 2008/067613 PCT/AU2007/001891 14 Methyl Furanthiol 2-Isobutyl-3-Methoxypyrazin Example 2 - Phospholipid fractionation study: The aim of this work was to fractionate and concentrate primarily phosphatidylcholine (PC) from food grade lecithin with the aim of determining the minimum PC content 5 required to produce cost efficient and stable liposome formulations. The study also determined the required inclusion ratios of other phospholipids and lipids to enhanced binding efficiencies. Up to 40 commercial lecithins, purified phospholipids, synthetic lipids, fats, triglycerides and sterols were examined and analysed for purity with the following process and 10 formula found to be most useful. Raw materials required: 1 Kg of soy lecithin ALCOLEC 40P or ULTRALEC P (approx. 25- 30% w/w PC content) 3 Ltrs of 100% ethanol AR grade Process for the fractionation of lecithin to selected phospholipids: 15 Add the soy lecithin slowly to a vessel containing ethanol and mix for up to 1 hour under low speed until homogenously dispersed. Heat the mixture to 40 0C, continue mixing and hold at this temperature for 30 minutes to precipitate the first series of ethanol insoluble components. Turn off the stirrer and allow the mixture to stand and cool for 15 mins. 20 Pour off and collect the supernatant.

WO 2008/067613 PCT/AU2007/001891 15 Lower the temperature of the supernatant to -18 0 C and hold for 12 hours to crystallize out the remaining ethanol insoluble fractions. Pour off the supernatant and filter the extract through a 0.45 micron filter. Solvent evaporate ethanol at 80 0C to produce a concentration of 1 g/ mL phospholipid, 5 measuring PC and PE to provide an enrichment of 80 to 85 % PC. Retain the evaporated ethanol for reuse as the lecithin dispersion solvent. Conduct proximate phospholipid and phosphate analysis to determine total phospholipid concentration, using HPLC analysis to determine phospholipid ratios, spectrophotometry to determine total phosphate and Gas Chromatography to confirm 10 fatty acid derivatives. Discussion of activity: 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 15 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 *C]. Negligible levels of Phosphatidyl serine (PS), Phosphatidyl inositol (PI) or Phosphatidic acid (PA) will be present in the fractionated 20 lecithin preparation. The use of these 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 25 encapsulating and retaining solutes based on phospholipid type, phase transition temperatures, charge potential, solubility and packing configuration of fatty acyl chains.

WO 2008/067613 PCT/AU2007/001891 16 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 5 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 pKa). The solutes being encapsulated may be included in the concentrated lipid suspension if 10 hydrophobic or in the injection buffer if hydrophilic. Example 3 - Formulation of an injection buffer to produce liposome with enhanced solute encapsulation Trials were conducted to review solute binding efficiencies into liposomes incorporating a range of hydrocolloids, dextrins, maltodextrins and starches over a pH and 15 temperature gradient to influence the encapsulation potential of flavour solutes based on a charge and solubility interaction between the injection solution, containing the fractionated phospholipids (with hydrophobic flavours) and the injection buffer containing less purified but hydrated lipids, a selected hydrocolloid at defined pH and hydrophilic flavours. 20 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 25 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.

WO 2008/067613 PCT/AU2007/001891 17 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. Remote loading of hydrophilic compounds into formed liposomes will further enhance 5 encapsulation efficiency under pH gradient with 50 mM calcium cation in solution to further enhance cationic charge potential. Example 4 Hydrocolloid studies 1. Protanal GP 2650 Sodium Alginate: viscosity 5,500-8,000 mPa at 10%w/w in solution (manufacturer's specifications). Trialled optimally at 0.1 % w/w in the injection 10 buffer at pH 4.0 with a zeta potential as an empty liposome recorded at -18 mV and then with added flavour (Furaneol) and the addition of a pH gradient infused 50mm calcium ions produced a cationic 10.3 mV response. 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 15 at pH 4.0 with 50mm calcium ions as calcium chloride. 2. 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 20 400 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. 3. Cellogen FSA Sodium Carboxymethyl Cellulose: viscosity 600-800 cps at 2% w/w in solution at 25 0C at neutral pH. Trialled optimally at 0.1 % w/w in the injection buffer at 25 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 WO 2008/067613 PCT/AU2007/001891 18 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: 5 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 "C, including ionic 10 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 15 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. Unit operations: 1. Prepare the fractionated phospholipid suspension from lecithin (Alcolec 40P) which is 20 enriched for phosphatidylcholine and dispersed in ethanol based on the process identified in the above. 2. Formulate the injection solution from the fractionated lecithin suspension enriched for phosphatidylcholine including ergosterol (<5 mole %), cocoa butter (<10 role %) and commercially purified phosphatidylcholine as required to a concentration of 80-85% PC 25 in solution. Add to this dispersion the hydrophobic solutes or flavours to be encapsulated. The addition ratio of the flavours or any solutes to the injection solution will be in the order of 10:90 to 20:80 to 30:70 solutes to fractionated lecithin suspension depending on the solubility coefficient of the materials being encapsulated.

WO 2008/067613 PCT/AU2007/001891 19 3. Prepare the injection buffer 0.2M citrate / phosphate solution (pH 4.0), containing 0.1% w/w of either carboxymethyl cellulose WF 10 or potato maltodextrin DE <10 or sodium alginate and a dispersion of prehydrated Alcolec 40P to a concentration of <5.0% w/w. Include the addition of any hydrophilic flavours or similar solubility 5 components to be encapsulated. 4. Assemble the ultrasonic injection unit by attaching either; a. 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 10 b. 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 401C 15 depending on the phase transition temperature of the lipids used in the formulation. Typically the recirculation solution will be <10 *C. 5. 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 20 flavours will be continually reused and blended for reinjected with fresh injection buffer after being analysed by GC to optimise continued additions. 6. Once a concentrated liposome suspension is formed within the aqueous buffer by repeated injections of a phosphatidylcholine enriched solution, the process will be stopped and the liposomes containing flavours will be removed and isolated by 25 ultrafiltration using a 100 kDa NMWC hollow fibre membrane. 7. The process is recommenced.

WO 2008/067613 PCT/AU2007/001891 20 Example 6 - Encapsulation efficiency data Compound Classification % Loading Approx. % EE Sulfurol Beef juice 40% Gamma Nonalactone Coconut 10% Citral Lemon grass 10% Linalool Floral 10% 2 Acetyl Thiazole Nutty 20% Ethyl Myristate Coconut 20% Trans 2 Dodecenal Coriander 10% Trans 2 Trans 4 Decadienal Fatty 10% 80% Furaneol Strawberry 0.2g/mL 20% Methyl furanthiole Roast meat 10% 40% Methyl Thiopropanal 3 Boiled potato 30% 15% 2 Isobutyl 3 Methoxypyrazin Pepper 10% 40% Swiss cheese extract Mix (phobic) 12.5% Cheddar cheese extract Mix (phobic) 10% Balsamic vinegar extract Mix (philic) 20% WO 2008/067613 PCT/AU2007/001891 21 Note: For swiss cheese, cheddar cheese and balsamic vinegar extracts, certain components have different encapsulation efficiency. Example 7 - Remote loading of Furaneol Phospholipid (2 g/mL EtOH) was injected into alginate solution (0.1%) in Phosphate 5 citrate buffer pH 4 to make liposomes containing alginate. The liposome containing alginate was ultracentrifuged (100,000 rpm, 15 minutes, 4 *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. 10 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, 40C) and washed with H 2 0 (washing with Buffer of the same pH washed the Furaneol out of the 15 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 0. Approximately 20% Furaneol travelled through liposome's membrane and was retained in the liposome. 20 It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Claims

1. A liposome having one or more components entrapped therein, the liposome including: - a lipid bilayer having one or more hydrophobic compounds entrapped within; 5 - a hydrocolloid mass and/ or an unfractionated lecithin component forming a core, said core being encapsulated by the lipid bilayer and having one or more hydrophilic compounds entrapped within.

2. The liposome of claim 1 wherein the lipid bilayer includes phospholipids extracted from lecithin. 10

3. The liposome of claim 1 wherein the hydrocolloid mass is formed from a compound selected from the group consisting of maltodextrin, carboxy methyl cellulose and sodium alginate.

4. The liposome of any one of the preceding claims wherein the lipid bilayer includes phosphatidylcholine extracted from lecithin in an amount that constitutes about 15 80 mole % of lipids in the lipid bilayer.

5. The liposome of claim 4 wherein the lipid bilayer further includes phosphatidylethanolamine in an amount of less than about 10 mole % of lipids in the lipid bilayer.

6. The liposome of any one of the preceding claims wherein the hydrophobic and 20 hydrophilic compounds are selected from the group consisting of flavours, functional food ingredients, nutraceuticals, pharmaceuticals and cosmeceutical compounds.

7. The liposome of any one of the preceding claims, said liposome being provided with a cationic charge for facilitating binding of the liposome to a target membrane.

8. A process for entrapping a hydrophobic compound in a liposome including: WO 2008/067613 PCT/AU2007/001891 23 - providing an 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. 5

9. The process of claim 8 wherein the lipid dispersed in the aqueous solution is lecithin.

10. The process of claim 8 wherein the lipid solution is a solution that includes phosphatidylcholine in an amount that constitutes about 80 mole % of lipids in the solution. 10

11. The process of any one of the preceding claims wherein the lipid solution is obtained from lecithin.

12. The process of any one of the preceding claims wherein the lipid solution further includes phosphatidylethanolamine in an amount of less than about 10 mole % of lipids in the solution. 15

13. The process of any one of the preceding claims wherein the hydrophobic compound is selected from the group consisting of flavours, functional food ingredients, nutraceuticals, pharmaceutical and cosmeceutical compounds.

14. The process of any one of the preceding claims wherein the aqueous solution is pH modified to provide the liposome with a cationic charge. 20

15. The process of any one of the preceding claims wherein the concentration of lipid in the aqueous solution is generally about 0.05mg/mL.

16. A process for entrapping a hydrophilic compound in a liposome including: - providing an aqueous solution including a hydrophilic compound to be entrapped in a liposome and an ionic polymer for binding to the hydrophilic compound; and WO 2008/067613 PCT/AU2007/001891 24 - contacting the aqueous solution with a lipid solution to form a liposome having the hydrophilic compound entrapped therein.

17. The process of claim 16 wherein the ionic polymer is a hydrocolloid.

18. The process of claim 17 wherein the hydrocolloid is formed from a compound 5 selected from the group consisting of low molecular weight carboxymethyl cellulose, maltodextrin, sodium alginate, Kappa carrageenan, iota carrageenan, lambda carrageenan, guar gum, xanthan gum and starch.

19. The process of any one of claims 16 to 18 wherein the aqueous solution is provided with a pH from about 4.0 to about 5.0. 10

20. The process of claim 19 wherein the hydrophilic compound is selected from the group consisting of flavours, functional food ingredients, nutraceuticals, pharmaceuticals and cosmeceutical compounds.