Abstract:
Disclosed is a new class of lipid vesicles, liposoils, which have high oil content, low water content, and protein. The liposoils are made using a combination of a surfactant and either dried egg yolk or dried whole egg as the wall material, oil, and an aqueous diluent. Unlike most lipid vesicles, the liposoils can be made with an aqueous diluent having high salinity; in fact, sea water is a preferred aqueous diluent. Liposoils have particular applicability as a food for marine environments, such as a food source for filter feeders such as oysters. Methods of making the liposoils is also disclosed.

Description:
BACKGROUND OF THE INVENTION 
     The present invention is concerned with the materials and methods for constructing a new type of lipid vesicle, called a &#34;liposoil&#34;. Liposoils are micron-sized vesicles containing oil, dried egg, surfactant and a diluent. The diluent can vary in salinity from water suitable for injection to solutions with the salinity of seawater. Liposoils are produced using high shear force without the use of organic solvents and are freely suspendable in water. The diluent or water content of these structures is low, normally 20-40% on a volume per volume basis. Since all materials utilized in the construction of liposoils are food grade, USP or NF grade materials, liposoils are suitable for human and animal enteral use applications. Liposoils are stable at temperatures from 4° to 37° C. and are not degraded by exposure to strong acids and base. Due to the ability to use high salinity diluents in formation of these structures these materials have potential marine applications, particularly as a marine food. The size and the fact that they are constructed of edible materials allows filtration and metabolism of these structures by marine filter feeders. The liposoils have a high protein content from the dried egg used in their formation, which is also adventageous in their use aqs a marine food. 
     Liposoils differ from classic liposomes in several ways. Classic liposomes were constructed of phospholipids such as phosphatidylcholine or lecithin extracted from a variety of sources (including eggs) but dried whole egg or egg yolk was not used as a wall material. Both egg yolk and whole egg have a high protein content in addition to the lipid content. Classic liposomes do not have this high protein content, and, in fact, the protein which can be encapsulated may be limited. This limitation is not found in liposoils. In addition, only small amounts of oil, compared to large amounts of oil used in liposoils, could be incorporated before the liposome broke down. Still another difference between the classic liposomes and the liposoils is the ability to use high saline solutions in the manufacture of liposoils. High saline solutions prevented the formation of the classic liposomes. 
     The method of manufacturing liposoils is also different than that used in classical liposome formation. Classical liposomes are normally formed utilizing the Bangham method, or a variant thereof. In the Bangham method, the lipids are dissolved in organic solvent, the solvent is removed to form a film, and the film is rehydrated with an aqueous solution to form liposomes. Organic solvents are not needed to make liposoils and, in fact, may impede the formation process. 
     Although some workers in the lipid vesicle field have used procedures other than Bangham method, and materials other than classic phospholipids, there is little work on vesicles made using both phospholipids and surfactants in the vesicle walls. For example, although U.S. Pat. No. 5,234,767, entitled &#34;Hybrid Paucilamellar Lipid Vesicles&#34;, the disclosure of which is incorporated herein by reference, discusses vesicles which may have a phospholipid and nonphospholipid in the vesicle wall, the phospholipids discussed are purified phosphatidylcholine and the like, not crude dried egg yolk or dried egg. These materials do not have the high protein content of the liposoils of the present invention. In addition, there is no discussion in this patent of using high salinity diluents nor that less water could be used than lipid. 
     The ability of the liposoils to have high oil content and be made using high salinity diluents such as sea water leads to an optimum usage in the marine environment, particularly in the marine food environment. Little or no work has been done in this field using liposomes because of the stability problems in this high salinity environment. The liposoils of the present invention are much better suited to this environment than classic liposomes or even most lipid vesicles using nonphospholipid materials. In addition, most nonphospholipid materials used to make lipid vesicles are not food grade or safe for incorporation into the food chain. 
     Accordingly, an object of the invention is to provide a new type of lipid vesicle, the liposoil, which has high oil content, utilizes dried egg or egg yolk, and can be made in a high salinity environment. 
     A further object of the invention is to provide a method of making the liposoils of the invention. 
     A still further object of the invention is to provide a marine food and/or pharmaceutical which is useful in a high salinity environment. 
     These and other objects and features of the invention will be apparent from the following description and the claims. 
     SUMMARY OF THE INVENTION 
     The present invention features a method of forming a composition containing a new type of structure, designated herein as the liposoil, and the composition itself. This composition, which contains oil encapsulated in the liposoil lipid structure, is made by blending a surfactant selected from the group consisting of polyoxyethylene sorbitan fatty acid esters, sorbitan fatty acid esters, polyoxyethylene nonylphenyl ethers, octylphenoxy-polyethoxyethanols, and mixtures thereof with a wall-forming material and an excess of oil to form a lipid preblend. The wall-forming material is dried egg yolk solids or dried whole egg solids and the oil is preferably an edible oil, most preferably an oil selected from the group consisting of almond oil, apricot seed oil, canola oil, castor oil, coconut oil, cod liver oil, corn oil, cottonseed oil, menhaden oil, jojoba bean oil, linseed oil, macadamia nut oil, mineral oil, mink oil, olive oil, palm oil, peanut oil, safflower oil, sardine oil, sesame oil, squalene, sunflower seed oil, and wheat germ oil. This preblend is then blended with an aqueous diluent to form the liposoil composition, the lipid preblend being greater in volume than the aqueous diluent. While a variety of diluents can be used, preferred diluents have greater than physiologically normal saline content and may include natural or synthetic sea water. Preferred liposoil compositions are lipid structures less than 1.2 microns in diameter. These compositions may also include an oil or aqueous soluble therapeutic active agent. If an aqueous soluble agent is used, it is added to the aqueous diluent before blending with the lipid preblend, while if an oil soluble agent is used, it is added to the oil prior to the forming of the lipid preblend. 
     The compositions of the invention have particular utility as foods for marine animals, particularly marine filter feeders such as oysters and clams. Both egg yolk and dried whole egg have a high protein content, so the marine food made using liposoils provides needed dietary protein as well as lipid. These compositions can also contain a therapeutically active agent, e.g., an antibiotic, which could prevent some of the common illnesses of the filter feeders. By using the compositions of the invention as a food for these marine animals, more careful control may be had over their diet. 
     The following detailed description of the invention will further amplify particular uses and methods of manufacture for the composition of the invention. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The lipid structures of the present invention (designated herein liposoils) provide inherently different properties than classic liposomes or other lipid structures. These structures combine the stability of lipid vesicles with high oil content and high salinity from the aqueous diluent which allows them to be used in environments which are detrimental to the stability of classic lipid vesicles. In addition, these vesicles should not be as susceptible to problems such as the donnan effect as are other vesicles. Accordingly, they may be used in harsher environmental conditions. 
     The following examples more clearly illustrate how the liposoils are made and their properties. These examples are purely illustrative and are not intended to limit the invention. 
    
    
     EXAMPLE 1 
     In this example, the preferred materials for making the liposoils, and their methods of preparation, are described. For preparation of the liposoils, at least one oil from Table 1 is combined with a surfactant from Table 2 and a wall-forming material from Table 3. After premixing these materials, water or a suitable diluent from Table 4 is injected into this mixture. The preferred ratio of oil:surfactant:wall material in the pre-mixed materials is 25:3:1 on a volume/volume/weight basis. The preferred ratios of the pre-mixed materials to water is 4:1 (20% diluent) or 3:2 (40% diluent). Liposoils can be produced with reciprocating syringe instrumentation, continuous flow instrumentation, or high speed mixing equipment. The mixers described in U.S. Pat. No. 4,895,452, entitled &#34;Method and Apparatus for Producing Lipid Vesicles&#34;, the disclosure of which is incorporated herein by reference, can all be used. Other mixers, such as French presses or microfluidizers such as are described in U.S. Pat. No. 4,911,928, entitled &#34;Paucilamellar Lipid Vesicles&#34;, the disclosure of which is incorporated herein by reference, can also be used. Particles created at this 3:2 ratio range in diameters from 44 to 1,197 nanometers. 
     
                       TABLE 1______________________________________List of Oils Utilized in Preparation of Liposoils______________________________________     Almond oil, sweet     Apricot seed oil     Canola oil     Castor oil     Coconut oil     Cod Liver oil     Corn oil     Cotton seed oil     Jojoba bean oil     Linseed oil, boiled     Macadamia nut oil     Medhaden Oil     Mineral oil     Mink oil     Olive oil     Palm oil     Peanut oil     Sardine Oil     Safflower oil     Sesame oil     Squalane     Sunflower seed oil     Wheat germ oil______________________________________ 
    
     
                       TABLE 2______________________________________List of Surfactants Utilized in Preparation of Liposoils______________________________________Polyoxythylene Sorbitan Festers and Sorbitan EstersTween 20Tween 40Tween 60Tween 80Tween 85Span 85Nonylphenol Polyethylene Glycol Ethers(alkylphenol-hydroxypolyoxyethylene)Poly(oxy-1,2-ethaneolyl),alpha-(4-nonylphenal)-omega-hydroy-, branched(i.e., Tergitol NP-6 Surfactant)Poly(oxy-1,2-ethaneolyl),alpha-(4-nonylphenal)-omega-hydroy-, branched(i.e., Tergitol NP-7 Surfactant)Poly(oxy-1,2-ethaneolyl),alpha-(4-nonylphenal)-omega-hydroy-, branched(i.e., Tergitol NP-8 Surfactant)Poly(oxy-1,2-ethaneolyl),alpha-(4-nonylphenal)-omega-hydroy-, branched(i.e., Tergitol NP-9 Surfactant)Poly(oxy-1,2-ethaneolyl),alpha-(4-nonylphenal)-omega-hydroy-, branched(i.e., Tergitol NP-12 Surfactant)Nonylphenol Polyethylene Glycol Ether mixtures(ie. Tergital NP-70 (70% AQ) Surfactant)OctylphenoxypolyethosyethanolsTriton  X-15Triton  X-100Triton  X-102Triton  X-114______________________________________ 
    
     
                       TABLE 3______________________________________List of Wall Materials Utilized in Preparation of Liposoils.______________________________________Dried whole eggDried egg yolk______________________________________ 
    
     
                       TABLE 4______________________________________List of Diluents Utilized in Preparation of Liposoils.______________________________________Water for injectionPhosphate buffered salineSeawater______________________________________ 
    
     By varying these components, custom liposoils can be formed. 
     EXAMPLE 2 
     In this Example, various specific liposoil formulations are described. Table 5 lists the materials utilized to produce one formulation of liposoils utilizing water as the diluent and their sizing parameters on Coulter LS230 laser sizing apparatus (Table 6). A brief description of the method of production of the liposoils is also given. 
     
                       TABLE 5______________________________________Preparation of Liposoils Utilizing Water as the Diluent.Chemical Component     Amount______________________________________Soybean oil (Oil)      25       mLPolysorbate 80 (Tween 80) (Surfactant)                  3        mLDried egg (Wall material)                  1        g______________________________________ 
    
     The oil-surfactant-wall material components are mixed for 60 seconds. Two mL of water is injected into three mL of the mixture using reciprocating syringe instrumentation. 
     
                       TABLE 6______________________________________             LS-230      LS-230             Mean Diameter                         RangePreparation       (nanometers)                         (nanometers)______________________________________Liposoils         583         326-945(SBO/Tw8O/Dried whole egg/WFI)______________________________________ 
    
     Table 7 lists the materials utilized to produce a different formulation of liposoils utilizing phosphate buffered saline as the diluent. Sizing data on this preparation from a Coulter LS230 laser sizing apparatus follows in Table 8. 
     
                       TABLE 7______________________________________Preparation of Liposoils Utilizing Phosphate Buffered Salineas the Diluent.Chemical Component     Amount______________________________________Soybean oil (Oil)      25       mLPolysorbate 80 (Tween 80) (Surfactant)                  3        mLDried whole egg (Wall material)                  1        g______________________________________ 
    
     As mentioned above, the oil-surfactant-wall material components are mixed for 60 seconds to form a lipid preblend. Two mL of PBS is injected into three mL of the mixture using reciprocating syringe instrumentation. Table 8 shows the sizing parameters for the liposoils obtained with these materials. 
     
                       TABLE 8______________________________________             LS-230      LS-230             Mean Diameter                         RangePreparation       (nanometers)                         (nanometers)______________________________________Liposoils         563         313-917(SBO/Tw8O/Dried whole egg/PBS)______________________________________ 
    
     Table 9 lists the materials utilized to produce still another formulation of liposoils utilizing seawater as the diluent. Sizing data on this preparation from a Coulter LS230 laser sizing apparatus follows in Table 10. 
     
                       TABLE 9______________________________________Preparation of Liposoils Utilizing Seawater as the Diluent.Chemical Component     Amount______________________________________Soybean oil (Oil)      25       mLPolysorbate 80 (Tween 80) (Surfactant)                  3        mLDried egg (Wall material)                  1        g______________________________________ 
    
     The oil-surfactant-wall material components are mixed for 60 seconds to form the lipid preblend. Two mL of seawater is injected into three mL of the mixture using reciprocating syringe instrumentation. Table 10 shows sizing data for this formulation. 
     
                       TABLE 10______________________________________             LS-230      LS-230             Mean Diameter                         RangePreparation       (nanometers)                         (nanometers)______________________________________Liposoils         558         323-883(SBO/Tw8O/Dried whole egg/seawater)______________________________________ 
    
     EXAMPLE 3 
     In Example 2, whole dried egg was used as the wall material. In this example, dried egg yolk is used instead. Table 11 lists the materials utilized to produce liposoils utilizing dried egg yolk instead of whole dried egg and Table 12 shows their sizing parameters on Coulter LS230 Laser sizing apparatus. 
     
                       TABLE 11______________________________________Preparation of Liposoils Utilizing Water as the Diluent andDried Egg Yolk.Chemical Component     Amount______________________________________Soybean oil (Oil)      25       mLPolysorbate 80 (Tween 80) (Surfactant)                  3        mLDried egg yolk (Wall material)                  1        g______________________________________ 
    
     The oil-surfactant-wall material components are mixed for 60 seconds. Two mL of water is injected into three mL of the mixture using reciprocating syringe instrumentation. The resulting liposoils were then sized using the laser sizing device. 
     
                       TABLE 12______________________________________             LS-230      LS-230             Mean Diameter                         RangePreparation       (nanometers)                         (nanometers)______________________________________Liposoils         380         203-577(SBO/Tw8O/Dried egg yolk/WFI)______________________________________ 
    
     As in Example 2, various diluents were used in the preparation of different formulations of the liposoils. Table 13 lists the materials utilized to produce liposoils utilizing phosphate buffered saline as the diluent and dried egg yolk instead of whole dried egg. Sizing data on this preparation from a Coulter LS230 laser sizing apparatus follows in Table 14. 
     
                       TABLE 13______________________________________Preparation of Liposoils Utilizing Phosphate Buffered Salineas the Diluent and Dried Egg Yolk.Chemical Component     Amount______________________________________Soybean oil (Oil)      25       mLPolysorbate 80 (Tween 80) (Surfactant)                  3        mLDried egg yolk (Wall material)                  1        g______________________________________ 
    
     The oil-surfactant-wall material components are mixed for 60 seconds. Two mL of saline is injected into three mL of the mixture using reciprocating syringe instrumentation. Table 14 shows the sizes of the resulting liposoils. 
     
                       TABLE 14______________________________________             LS-230      LS-230             Mean Diameter                         RangePreparation       (nanometers)                         (nanometers)______________________________________Liposoils         608         349-974(SBO/Tw80/Dried egg yolk/PBS)______________________________________ 
    
     Table 15 lists the materials utilized to produce liposoils utilizing seawater as the diluent and dried egg yolk instead of whole dried egg. Sizing data on this preparation from a Coulter LS230 laser sizing apparatus follows in Table 16. 
     
                       TABLE 15______________________________________Preparation of Liposoils Utilizing Seawater as the Diluentand Dried Egg Yolk.Chemical Component     Amount______________________________________Soybean oil (Oil)      25       mLPolysorbate 80 (Tween 80) (Surfactant)                  3        mLDried egg yolk (Wall material)                  1        g______________________________________ 
    
     The oil-surfactant-wall material components are mixed for 60 seconds. Two mL of seawater is injected into three mL of the mixture using reciprocating syringe instrumentation. Table 16 shows the sizing data. 
     
                       TABLE 16______________________________________             LS-230      LS-230             Mean Diameter                         RangePreparation       (nanometers)                         (nanometers)______________________________________Liposoils         589         331-949(SBO/Tw8O/Dried whole egg/seawater)______________________________________ 
    
     EXAMPLE 4 
     In this Example, the type of oil used was varied to produce different liposoil formulations. Table 17 lists the materials utilized to produce liposoils where the oil component is varied utilizing seawater as the diluent. The volume of each oil utilized was 25 mL. The volume of surfactant (Tween 80) was 3 mL. The weight of the wall material utilized was 1 gram. In each preparation, the oil-surfactant-wall material components were mixed for 60 seconds. Two mL of seawater is injected into three mL of the mixture using reciprocating syringe instrumentation. Sizing information was determined on each preparation on a Coulter LS230 laser sizing apparatus and is shown on Table 17. 
     
                       TABLE 17______________________________________Preparation of Liposoils Containing Tween 80 and Dried WholeEgg, varying the Oil and Utilizing Seawater as the Diluent.          LS-230        LS-230          Mean Diameter RangeChemical Component          (nanometers)  (nanometers)______________________________________Almond oil, sweet          656           359-1,086Apricot seed oil          654           343-1,119Canola oil     465           240-747Castor oil     508           308-716Coconut oil    563           268-1,073Cod Liver oil  683           384-1,111Corn oil       647           401-972Cotton seed oil          637           336-1086Fish oil       610           381-911Jojoba bean oil          673           342-1,197Linseed oil, boiled          585           394-814Macadamia nut oil          666           378-1,082Mineral Oil    509           272-812Mink oil       645           347-1,090Olive oil      663           380-1,062Palm oil       604           421-829Peanut oil     684           366-1,164Safflower oil  665           380-1,067Sesame oil     680           361-1,161Squalane       549           281-956Squalene (batch 2)          686           413-1,060Sunflower seed oil          638           322-1,152Wheat germ oil 638           380-985______________________________________ 
    
     EXAMPLE 5 
     This Example illustrates various surfactants useful in formulating liposoils. Table 18 lists the materials utilized to produce liposoils where the surfactant component is varied utilizing seawater as the diluent, including sizing data. The volume of soybean oil utilized was 25 mL. The volume of surfactant was 3 mL. The weight of the wall material (dried whole egg) utilized was 1 gram. In each preparation, the oil-surfactant-wall material components were mixed for 60 seconds. Two mL of seawater is injected into three mL of the mixture using reciprocating syringe instrumentation. Sizing information was determined on each preparation on a Coulter LS230 laser sizing apparatus and is shown in Table 18. 
     
                       TABLE 18______________________________________Preparation of Liposoils Containing Soybean Oil and DriedWhole Egg, Varying the Surfactant and Utilizing Seawateras the Diluent.          LS-230        LS-230          Mean Diameter RangeChemical Component          (nanometers)  (nanometers)______________________________________Sorbitan DerivativesTween 20       592           329-964Tween 40       606           375-915Tween 60       571           387-798Tween 80       589           331-949Tween 85       539           339-790Span 85        363           200-544NonlyphenolPolyethyleneGlycol EthersTergitol MP-6  512           315-721SurfactantTergitol NP-7  468           290-674SurfactantTergitol NP-8  481           291-702SurfactantTergitol NP-9  376           222-552SurfactantTergitol NP-12 382           225-561SurfactantTergitol NP-70  78            45-125(70% AQ) SurfactantOctylphenoxypoly-ethoxyethanolsTriton X-15     77            44-122Triton X-100   555           386-760Triton X-102   325           168-502Triton X-114   557           349-810______________________________________ 
    
     EXAMPLE 6 
     In this Example, various physical stability tests were run on liposoils made as in Example 2 using seawater as the diluent. 
     Table 19 displays stability data on liposoils after exposure to strong acids and base. After two hour exposures of liposoils to either 1N sodium hydroxide, 1N sulfuric acid, or 1N nitric acid, no evidence of disruption of structures was noted. 
     
                       TABLE 19______________________________________pH Stability of Liposoils after a Two Hour Exposureto Strong Base or Acids              LS-230     LS-230              Mean Diameter                         RangePreparation        (nanometers)                         (nanometers)______________________________________Liposoils          589        331-949(SBO/Tw8O/dried whole egg/seawater)(Initial Preparation)Liposoils          503        286-787(SBO/Tw8O/dried whole egg/seawater)(2 hour exposure to 1N NaOH)Liposoils          512        287-819(SBO/Tw8O/dried whole egg/seawater)(2 hour exposure to 1N H.sub.2 SO.sub.4)Liposoils          533        299-851(SBO/Tw8O/dried whole egg/seawater)(2 hour exposure to IN HNO.sub.3)______________________________________ 
    
     Table 20 displays sizing information on liposoils stored at -20° C., 4° C., 25° C., 37° C. or 56° C. Liposoils were stable at 4° C., 25° C. and 37° C. but unstable at -20° C. and 56° C. 
     
                       TABLE 20______________________________________Thermal Stability of Liposoils at One Month              LS-230     LS-230              Mean Diameter                         RangePreparation        (nanometers)                         (nanometers)______________________________________Liposoils                     Unstable(SBO/Tw80/dried whole egg/seawater)STORAGE at -20° C.Liposoils          381        204-584(SBO/Tw80/dried whole egg/seawater)STORAGE at 4° C.Liposoils          275        142-427(SBO/Tw80/dried whole egg/seawater)STORAGE at 25° C.Liposoils          269        138-420(SBO/Tw80/dried whole egg/seawater)STORAGE at 37° C.Liposoils                     Unstable(SBO/TW80/dried whole egg/seawater)STORAGE at 56° C.______________________________________ 
    
     EXAMPLE 7 
     To determine whether liposoils could be used as a food for filter factors, liposoils made as in Example 2 using seawater as the diluent fed to eastern oysters free of Perkinsus marinus infection. The oysters were placed in 50 liter aerated polypropylene tanks, twelve oysters per tank and acclimated for two weeks. Water was changed every other day with estuarine water prefiltered through a series of ten micron and one micron filters. Oysters were fed daily with 0.1 g of algeal paste of Thalassiosira weisfolggi resuspended in water. After two weeks of acclamation, 50 mL of liposoils was added to each tank. This amount of material caused marked tank turbidity. All material was filtered out of estuarine water by the oysters in three hours. 
     Oysters can be grown by substitution of liposoils for standard commercial oyster food. The liposoils provide not just lipid but also high protein content from the dried egg or dried egg yolk. This protein is an important part of the feed for the marine filter feeders. In addition, a test using an antibiotic bacitracin, which was included in the lipid preblend before the formation of the liposoils, showed that the oysters tolerated the antibiotic-liposoil combination well. Liposoils have the potential for being used as both marine foods and therapeutic delivery systems. 
     The foregoing examples are non-limiting and are set forth merely to elucidate the invention. The invention is defined by the following claims.