US 2010.0086573A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0086573 A1 Anderson (43) Pub. Date: Apr. 8, 2010

(54) COMPOSITION AND METHOD FOR (52) U.S. Cl...... 424/401; 424/450; 514/458 PREPARING STABLE UNILAMELLAR LPOSOMAL SUSPENSION (57) ABSTRACT (76) Inventor: Penelope M. Anderson, Ada, MI Compositions and methods for producing and using stable (US) transparent to translucent unilamellar liposomal Suspensions are described. The Suspensions include a liposome prepara Correspondence Address: tion having a uniform plurality of unilamellar liposomal par IN RE: 28533 ticles with a mean particle size between about 50 nm to about BRINKS, HOFER, GILSON & LIONE 290 nm. The particles are suspended in an external phase P.O. BOX 10395 composition that has a density between about 0.95 g/cc and CHICAGO, IL 60610 (US) about 1.25 g/cc, and that is present in an amount between about 30% to about 75% of the weight of the liposomal (21) Appl. No.: 12/245,528 Suspension. The liposome preparation is formed from an aqueous liposomal Solution that includes an oil-soluble com (22) Filed: Oct. 3, 2008 position and a water-soluble composition. The oil-soluble composition is present at a concentration between about 5% Publication Classification to about 33% by weight of the liposomal solution and the (51) Int. Cl. water-soluble composition is present at a concentration A 6LX 9/27 (2006.01) between about 67% to about 95% by weight of the liposomal A 6LX 3L/355 (2006.01) Solution.

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FG. US 2010/0O86573 A1 Apr. 8, 2010

COMPOSITION AND METHOD FOR sec' at 21°C. (centigrade). The external phase composition PREPARING STABLE UNILAMELLAR is present in an amount between about 30% to about 75% of LPOSOMAL SUSPENSION the weight of the liposomal Suspension. The liposomal Sus pension has a refractive index between about 1.30 and about 1.45 and is stable with a uniform distribution of particles in 0001. This invention relates generally to compositions and neatformata temperature of between about 4°C. to about 50° methods for producing stable, transparent to translucent C. for a period of at least 30 days, or at 21°C. for a period of unilamellar liposomal Suspensions with or without loaded at least 180 days, or both. The liposomal suspension includes active agents for cosmetic, therapeutic, or diagnostic appli a plurality of unilamellar liposomal particles having a mean cations. particle size between about 50 nm to about 290 nm. The liposome preparation is formed from an aqueous liposomal BACKGROUND Solution that includes an oil-soluble composition and a water soluble composition. The oil-soluble composition is present 0002. A liposome is a closed generally spherical vesicle at a concentration between about 5% to about 33% by weight with a membrane composed of a phospholipid bilayer that is of the liposomal solution, the water-soluble composition at a capable of encapsulating water-soluble, hydrophilic mol concentration between about 67% to about 95% by weight of ecules in their aqueous core and embedding oil-soluble, the liposomal solution. The oil-soluble composition includes hydrophobic molecules in the hydrophobic region of the a coupling agent, at least one phospholipid, at least one rigid bilayer. Liposomes typically are Small particles, between ity enhancer, and an antioxidant. about 25 nm to 1000 nm and composed of a variety of phos 0006. In another embodiment, the liposome preparation is pholipids, including naturally-derived phospholipids with formed using an oil-soluble composition that includes one or mixed lipid chains, such as egg phosphatidylethanolamine, or more of a lecithin preparation, butylene glycol, ceramide of pure components like DOPE (dioleolylphosphatidyletha IIIB, B-sitosterol, and tocopherol. In this embodiment, the nolamine). Liposomes are widely used in topical drug prepa liposomal Suspension includes glycerin at a concentration rations, by providing a carrier mechanism for deposition of between about 35% to about 75% of the liposomal suspen active compounds into the intrinsic layers of the skin. Benefits S1O. of employing this technology include controlled release, 0007. In another aspect, a method for making a stable selective delivery, enhanced bioavailability and stability, and unilamellar liposomal Suspension includes: (a) preparing an increased absorption of desired actives. In addition, lipo oil-soluble composition comprising a coupling agent, at least somes have a great affinity and compatibility with the skin one phospholipid, a rigidity enhancer, and an antioxidant; (b) due to their chemical resemblance to the extracellular lipids preparing a water-soluble composition; (c) combining the surrounding internal skin cells. The lipid bilayer of a lipo water-soluble composition with the oil-soluble composition some can fuse with other bilayers, for example cellular mem to form a multilamellar, multivesicular liposome preparation; branes, thus delivering the liposome contents. (d) converting the multilamellar, multivesicular liposome 0003. The commercial development of liposomes has preparation to a unilamellar liposome preparation; and (e) been plagued by poor stability, clarity, and short shelf life. adding the unilamellar liposome preparation to an external Liposome vesicles tend to fuse together or agglomerate, par phase composition having a density within a range of about ticularly when they are exposed to Surfactants, solvents, 0.95 g/cc to about 1.25 g/cc and a viscosity between about 2.5 adverse pH conditions, elevated temperatures, or even water, cP and about 40,000 cp at a shear rate of 10 sec' at 21°C. to for long periods of time. form the liposomal Suspension comprising a plurality of unilamellar liposomal particles each having a mean particle SUMMARY size between about 50 nm to about 290 nm, wherein the 0004 Compositions and methods for producing stable liposomal suspension has a refractive index between 1.30 and transparent to translucent liposomal Suspensions are 1.45 and retains its stability with a uniform distribution of described herein. The present invention is premised on the particles in neat form at about 4°C. to about 50° C. for a discovery that particular combinations of liposomal compo period of at least 30 days, or at 21°C. for a period of at least nents can be dispersed in an external phase composition, Such 180 days, or both. that the liposomal Suspension retains a unimodal distribution 0008. In a further aspect, a method for preparing a cos of uniformly sized particles exhibiting stability at various metic formulation includes combining a liposomal Suspen elevated or reduced temperatures with or without active sion of the present invention with a cosmetically suitable agents for up to a year or more. Specifically, by reducing the matrix to form a cosmetic formulation in the form of a cream, mean diameter of the distribution of dispersed liposomal lotion, gel, serum, tonic, emulsion, paste, or spray. particles, minimizing the density differences between dis 0009. The liposomal suspensions of the present invention persed liposomal particles and the external phase composi are characterized by an enhanced stability and biological tion, increasing the viscosity of the external phase composi activity profile and may be useful in any one of a variety of tion, and building rigidity into the bilayer membrane of cosmetic, therapeutic, or diagnostic applications where lipo vesicles, the stability, clarity and shelf life of the liposomal Somes are used. In addition, the translucent nature of the Suspension can be enhanced without sacrificing the structural disclosed liposomal Suspensions provides an aesthetically integrity or bioactivity of the liposomal particles. advantageous appearance over more opaque formulations 0005. In one aspect, a stable unilamellar liposomal sus used in the prior art. pension includes a liposome preparation Suspended in an external phase composition having a density between about BRIEF DESCRIPTION OF THE DRAWINGS 0.95 g/cc and about 1.25 g/cc and a viscosity between about 0010 FIG. 1A is an electron micrograph of a liposomal 2.5 cB (centipoise) and about 40,000 cp at a shear rate of 10 Suspension according to an embodiment of Table 2 taken US 2010/0O86573 A1 Apr. 8, 2010

using a Zeiss-902 transmission electron microscope (TEM) at contents of all references, patents, and published applications 30,000x magnification: FIG. 1B and 1C are TEM micro cited throughout this patent are hereby incorporated by ref graphs of the liposomal suspension in FIG. 1A at 180,000x erence herein. magnification. Exemplary lipid bilayers are identified by 0026. In one aspect, the present invention provides a stable arrows in FIGS 1B and 1C. translucent unilamellar liposomal suspension that includes a 0011 FIG. 2 is a particle size analysis of another embodi liposome preparation, which comprises a plurality of unila ment according to the liposomal formulation depicted in mellar liposomal particles having a mean particle size FIGS. 1A-1C after 19 months of shelf life. between about 50 to about 290 nm, where the liposome prepa 0012 FIG. 3 is a particle size analysis of a liposomal ration is Suspended in an external phase composition com Suspension according to an embodiment of Table 4 after 2.5 prising between about 30% to about 75% of the weight of the months of shelf life in ambient temperature conditions by liposomal Suspension. The external phase composition has a dynamic laser light scattering using a NanotracR N150 Sub density between about 0.95 g/cc and about 1.25 g/cc and a micron Particle Size Analyzer. viscosity between about 2.5 cFandabout 40,000 cp at a shear 0013 FIG. 4 is a particle size analysis of a liposomal rate of 10 sec' at 21°C. The liposomal suspension has a Suspension according to an embodiment of Table 4 after 2.5 refractive index between about 1.30 and about 1.45 and is months of shelf life under ambient temperature conditions. stable in neat form at a temperature of between about 4°C. to 0014 FIG. 5 is a particle size analysis of a liposomal about 50° C. for a period of at least 30 days, or at 21°C. for a formulation according to an embodiment of Table 4 after 19 period of at least 180 days, or both. months of shelf life under ambient temperature conditions. 0027 Liposome preparations and Suspensions 0015 FIG. 6 is a particle size analysis of a liposomal 0028. A liposome preparation includes a plurality of lipo suspension according to Table 5 after 2.5 months of shelf life Somal particles or vesicles in an aqueous liposomal Solution under ambient temperature conditions. or a plurality of lyophilized (freeze-dried) liposomal particles 0016 FIG. 7 is a particle size analysis of a liposomal originating from the liposomal solution. The liposomal par suspension according to Table 6 after 2.5 months of shelf life ticles or vesicles in the liposome preparation may be in a under ambient temperature conditions. multilamellar, multivesicular form or they may be in a sub 0017 FIG. 8 is a TEM micrograph of a liposomal cream stantially unilamellar form. A liposome preparation is added formulation taken using a Zeiss-902 electron microscope at to the external phase composition to form a liposomal Sus 11,430x magnification. Liposomes are identified by arrows. pension in accordance with the present invention as further 0018 FIG. 9 is a TEM micrograph of a liposomal cream described below. The liposomal particles in the liposome formulation taken using a Zeiss-902 electron microscope at preparation are formed from an aqueous liposomal Solution 30,000x magnification. Liposomes are identified by arrows. comprised of an oil-soluble composition and a water-soluble composition. DETAILED DESCRIPTION (0029 Oil-Soluble Composition 0019. In order to provide a clear and consistent under 0030 The oil-soluble composition includes a coupling standing of the specification and claims, the following defi agent, a phospholi"d or phospholipid mixture, a rigidity nitions are provided. The term “liposomal solution” refers to enhancer, and an antioxidant. The oil-soluble composition in a preparation of liposomal particles or vesicles in a concen the liposomal Suspension of the present invention will gener trated, aqueous Solution prior to suspension in an external ally comprise a plurality of lipids normally present in native phase composition. cellular membranes, including phospholipids, ceramides, 0020. The term “liposome preparation” refers to a prepa sphingolipids, cholesterol, and triglycerides, or other lipids ration of liposomal particles or vesicles. The liposome prepa Such as phytosterols from plants. The oil-soluble composition ration may be a liposomal Solution containing liposomal par is present at a concentration between about 5% to about 33% ticles in an aqueous solution (for example, prior to Suspension by weight of the aqueous liposomal solution and the water in an external phase composition) or it may be a preparation soluble composition is present in a concentration between of lyophilized (freeze-dried) liposomal particles or vesicles about 67% to about 95% by weight of the aqueous liposomal prepared from a liposomal solution. Solution. 0021. The term “liposomal solution” refers to a liposome 0.031 Coupling Agents preparation comprising liposomal particles or vesicles in an 0032. The oil-soluble composition in the liposomal sus aqueous solution prior to lyophilization or Suspension in an pension of the present invention will include at least one external phase composition. coupling agent. A coupling agent includes a connective 0022. The term “liposomal suspension” refers to a lipo medium Suitable for Solubilizing (for example, transiently or Some preparation Suspended in an external phase composi preferentially) immiscible phases of differing polarity suit tion. able for solubilizing immiscible phases of differing polarity 0023 The term “coupling agent” refers to a connective and for maintaining stability of the liposome preparation or medium suitable for solubilizing immiscible phases of differ liposomal Suspension. The coupling agent may be character ing polarity and for effectuating and preserving stability of ized by a dielectric constant of about 10.5 or greater at 23°C., the liposome preparation or liposomal Suspension. which is added to the liposomal preparations or Suspensions 0024. The term “rigidity enhancer refers to a sphin of the present invention to facilitate preferential or “transient’ golipid, ceramide, or sterol functioning to reduce the move solubilization and to effectuate and preserve liposomal sta ment or fluidity offatty acid chains when embedded in a lipid bility. A coupling agent, such as a glycol, for example, may bilayer and promoting the packing thereof. include both hydrocarbon and hydroxyl moieties which can 0025. Unless indicated otherwise, all proportions and per be utilized, for example, to simultaneously solubilize hydro centages cited in this disclosure are by weight. Further, the phobic or hydrophilic compounds, respectively. US 2010/0O86573 A1 Apr. 8, 2010

0033. The coupling agent allows for preferential solubili hydronium ion shielding. Alternatively, coupling agents may Zation of oil-phase components, including phospholipids and be used at concentrations exceeding the phospholipid con rigidity enhancers at higher temperatures, whereby the cou centration where there is a need to solubilize not only the oil pling agent migrates into the bilayer membrane components phase components, but actives in the water-soluble composi during formation of the liposomal structures in the liposomal tion as well. Solution and then, upon cooling, migrates back into the water 0036 Phospholipids phase due to its hydrophilic properties under ambient tem 0037. The oil-soluble composition in the liposomal sus perature conditions. Thus, the coupling agent may be charac pension of the present invention includes at least one phos terized as a “transient solubilizer capable of solubilizing pholipid or phospholipid mixture. Exemplary phospholipids oil-soluble components, including lipids and nonpolar Sub include, glycerophospholipids comprising saturated and/or stances, at high temperatures, and water-soluble components, unsaturated fatty acyl moieties, including phosphatidylcho including polar Substances at lower, ambient temperatures line, phosphatidylethanolamine, phosphatidylinositol, phos (and an inability or reduced ability to solubilize lipids under phatidic acid, N-acylphosphatidylethanolamine, lysophos these low temperature conditions). Accordingly, the coupling pholipids, and cardiolipins; sphingomyelin; plasmalogens; agent has a “changing or transient functionality that is tem hydrogenated, partially hydrogenated, or unhydrogenated perature dependent so as to facilitate bilayer membrane for derivatives thereof; analogs thereof; and mixtures thereof. mation, including temperature phase combinations produc 0038 Lysophospholipids are obtained when an acyl radi ing liquidified forms of cursory multilamellar, multivesicular cal is cleaved off by a phospholipase A from phospholipids liposomal structures that can be effectively processed into (e.g. lysolecithins). Cardiolipins, such as 1,3-bisphosphatidyl unilamellar structures under high shear conditions. The lower glycerol, are phospholipids comprised of two phosphatidic temperatures avoid compromising the biological activity of acids linked via glycerol. Sphingomyelins are a type of sph active Substances and reduce the effects of overheating asso ingolipid typically comprised of a phosphorylcholine and a ciated with, for example, high shear/high pressure equipment. ceramide. Plasmalogens are ether lipids having in which the 0034. In contrast to coupling agents, other oil-soluble first carbon position of glycerol has an ether-linked alkene, solubilizers that can dissolve bilipid membranes, such as the second carbon has a typical ester-linked fatty acid, the ethanol, may compromise liposomal stability and can present third carbon typically having a phospholipid head group like an explosion hazard when processing a liposomal Solution choline or ethanolamine. through high pressure devices, including microfluidic 0039 Exemplary phospholipid mixtures include lecithins, devices. Moreover, at concentrations above about 3%, etha including crude lecithins, which have been deoiled, fraction nol can disrupt the density balance of the colloidal system and ated, spray-dried, acetylated, hydrolyzed, hydroxylated, reduce the viscosity of the external phase so as to negatively hydrogenated, and/or enriched with phosphatidylcholine, impact upon colloidal stability and/or cause the collapse of phosphatidylinositol, or combinations thereof. Lecithins and gellant structures. lecithin mixtures available commercially. In some embodi 0035 Applicants have unexpectedly discovered that ments, natural sources of lecithin (soy/egg) may be used. Such incorporation of a coupling agent not only serves to facilitate as phosphatidylcholine-enriched preparations derived from solubilization of oil-soluble components, but functions as a Soy. stability enhancer, independent of whether oil-soluble actives 0040 Commercially available soy/egg lecithin prepara are included. In particular, Applicants have found that lipo tions and phospholipid concentrates, include those sold under Somal Suspensions lacking a coupling agent, but otherwise the trade names Phospholipon R 90G. Phospholipon R 90H, identical in composition, did not achieve Suitable stability. As Phospholipon R 85G, Phospholipon R 80H, Phospholipon(R) Such, Applicants believe that the use of coupling agents in the 80, Phosal(R) 50 PG, Phosal(R) 50 SA, Phosal(R) 53 MCT, and liposomal Suspensions of the present invention does not con Phosal(R) 75SA, which are available from American Lecithin stitute an art-recognized, result-effective variable with Company, Oxford Conn.; Lipoid R, S75, Lipoid R. S100, respect to stability. Suitable coupling agents include butylene Lipoid R. SPC, and Lipoid R SL80, which are available from glycol, propylene glycol, hexylene glycol, polyethylene gly Lipoid GmbH, Ludwigshafen Germany: Epikurone 125F, cols, silicone glycols, such as PEG-12 dimethicone, as well as Epikuron(R) 135F, Epikurone 130P, Epikuron(R) 130G, Epiku analogs, derivatives, and mixtures thereof. The coupling ron(R) 100H. Epikuron(R) 145V, Epikuron(R) 170, Epikuron(R) agents for use in the present invention may be present in a 200 SH, Epikuron(R) 200, Emulmetik R950, Emulmetik R900 collective amount ranging from about 1% to about 10% by and Emulmetik R. 300, Emulfluide F30 (Emulfluid, Lipotin weight of the liposomal Suspension, more desirably from Nebr.), Lipotin(R) 100, Lipotin(R) SB, Lipotine 100J, Lipotin(R) about 3% to about 8% by weight of the liposomal suspension. H. LipotinRNA, LipotinR AH, and Lipopur,R), which are Generally, the concentration of coupling agent(s) should available from Degussa Texturant Systems UK Ltd.; Terra equal or exceed the total concentration of phospholipids in the drille R V 408 and TerradrillR V 1075, which are commer liposomal solution. The use of coupling agent concentrations cially available from Cognis:Yellow thin(R) 100, Yellowthin(R) between 10-15% may allow for slightly greater solubilization 200, Lecistar Sune 100, and Yellow thin SunR 200, which are of active ingredients, particularly when using higher phos available from Sternchemie; Lecinol S-10 (hydrogenated pholipid concentrations (e.g., 1.5x). However, it is believed lecithin), which is available from Barnett Industries; Lipo H that coupling agent concentrations above 10% may cause a (hydrogenated soybean lecithin comprised of 20% phosphati phase inversion where the oil-phase exists in an external dylcholine), which is available from Lucas Meyer Cosmetics, continuum, thereby preventing liposomal microencapsula South Plainfield, N.J.; and LanchemR PE-130K, which is tion. Under these conditions, water is repelled from the lipid available from Lambent Technologies, Gurnee, Ill. Solution in a manner consistent with water-in-oil emulsions. 0041. The phospholipids for use in the present invention This can be observed visually under a microscope or it can may be present in a collective amount ranging from about 1% deduced by an inability to accurately measure pH due to to about 10% by weight of the aqueous liposomal solution US 2010/0O86573 A1 Apr. 8, 2010 prior to lyophilization or addition to the external phase com N-acyl group is other than those found in naturally-occurring position, more desirably from about 3% to about 8% by ceramides. Ceramides may be differentiated from one weight of the liposomal Solution. In addition, the aqueous another, for example, by degree of Saturation and chain liposomal solution will desirably contain a water to lipid ratio length. Ceramide derivatives include, for example, those between about 3:1 to 9:1. derivatized with one or more saturated or unsaturated fatty 0042. In one embodiment, the oil-soluble composition acids, or those modified by phosphorylation or Sulfation. includes a phospholipid preparation or mixture comprising at 0048 Type 3 ceramides constitute a mixture of different least 60% phosphatidylcholine by weight of the mixture, at molecules having a phytosphingosinebackbone that are char least 70% by weight, at least 80% by weight, at least 85% by acterized by the general name N-acylphytosphingosine, weight, or at least 90% by weight of the mixture. In another wherein the acyl group is saturated and has a chain length of embodiment, the oil-soluble composition includes a phos 14 to 30 carbons. In one embodiment, the ceramide may be a pholipid mixture comprising phosphatidylcholine at a con ceramide or phytosphingosphine derivatized with a monoun centration between about 80% to about 95% by weight of the saturated fatty acid, Such as N-oleoylphytosphingosine (cera phospholipid mixture. Additionally, the above described mide 3B). Additional ceramides are disclosed in U.S. Pat. phospholipid mixtures may comprise between about 1% to Nos. 5,693,677 and 6,001,375 to Lambers et al., the disclo about 10% by weight of the liposomal suspension, desirably sures of which are incorporated by reference herein. between about 3% to about 8% by weight of the liposomal 0049 Ceramides for use in the present invention may be suspension. It is believed that the use of phospholipid con present in a collective amount ranging from about 0.01% to centrations above 15%.will cause phase inversion resulting in about 1.00% by weight of the liposomal suspension, more water-in-oil emulsions instead of encapsulated liposomes. desirably from about 0.025% to about 0.20% by weight of the 0043. Rigidity Enhancers liposomal Suspension. 0044) The oil-soluble composition in the liposomal sus 0050 Sterols for use in the present invention represent an pension of the present invention will include at least one additional class of rigidity enhancers, which includes choles rigidity enhancer. Rigidity enhancer(s) include at least one terols, phytosterols, and organic acid derivatives, and/or salt sphingolipid, ceramide, Sterol, or combination thereof, which forms thereof. Phytosterols are plant-derived lipids bearing a function to reduce the movement or fluidity of fatty acid sterol skeleton. Phytosterols can synchronize metabolic chains when embedded in a lipid bilayer and to promote cycles of epidermal cells in skin and can enhance the strength packing of the lipid bilayers. The increased rigidity can of a septum having a bilayer membrane structure in a lipo reduce rupture and leakage of vesicles and can shift the Zeta Some or other membranous structure. potential or charge in the membrances so as to suppress 0051. The present invention may utilize a member of any attraction between particles. It is believed that the rigidity of the phytosterol categories, including the 4-desmethyl enhancer(s) help to stabilize the charge or Zeta potential in the sterols, 4-monomethylsterols, and 4.4-dimethylsterols. liposomal particles. Exemplary phytosterols include sitosterol, beta-sitosterol, 0045 Sphingolipids represent key epidermal lipids Stigmasterol, campesterol, chalinosterol, clionasterol, brassi involved in maintaining the skin's barrier function. Sphin casterol, alpha-spinasterol, A5-avennasterol, lupenol, dan golipids, Such as Sphingosine, Sphinganine, Sphingomyelin, costerol, desmoSterol, and poriferasterol. and phytosphingosine, generally comprise a long sphingoid 0052. Non-limiting examples of sterols additionally base as the central group or “backbone', including an amide include cholesterol, organic acid derivatized cholesterols, linked long-chain fatty acid and a head group. There are Such as cholesterol hemisuccinate, phytocholesterol, bisab hundreds of known classes of sphingolipids with different olol, Steroid hormones, such as hydrocortisone, as well as head groups (e.g. cholinephosphate, glucose, galactose, sterol aliphatic acid esters, such as cholesterol Sulfate, cho polysaccharides) and with different fatty acids and sphingoid lesterol butyrate, cholesterol isobutyrate, cholesterol palmi bases. Phytosphingosine is a ceramide precursor, concen tate, cholesterol Stearate, lanosterol acetate, ergosterol palmi trated in healthy stratum corneum that is converted to sphin tate, and phytosterol n-butyrate; and sterol esters of Sugar golipids by enzymes in the skin. esters, such as cholesterol glucuronide, lanosterol glucoron 0046 Ceramides constitute a family of sphingolipids, ide, 7- dehydrocholesterol glucuronide, ergosterol glucu which are extremely insoluble and difficult to formulate. ronide, and cholesterol gluconate. Exogenously administered ceramides must be able to pen 0053 Sterols for use in the present invention may be etrate the stratum corneum in order to reach the lipidlamellae present in a collective amount ranging from about 0.01% to of the permeability barrier. It is believed that one of the causes about 1.00% by weight of the liposomal suspension, more of dry skin is a reduction in the amount of ceramides within desirably from about 0.025% to about 0.20% by weight of the the intercellular lipid lamellae. It is therefore desirable to be liposomal Suspension. able successfully to replace these depleted lipids by topical 0054. In one embodiment, the liposomal suspension administration using liposomal Suspensions of the present includes at least two rigidity enhancers, including at least one invention. The liposomal Suspensions of the present invention phytosterol and either a ceramide or a sphingosine. In a provide a useful means for delivery of ceramides. desired embodiment, the liposomal suspension includes cera 0047. The ceramide may be a naturally-occurring ceram mide IIIB and B-sitosterol. ide, synthetic ceramide, glycosylceramide, or derivatives 0055. The rigidity enhancers for use in the present inven therefrom. Naturally-occurring ceramides may be derived tion may be presentina collective amount ranging from about from animals, plants, or microorganisms. Ceramides in skin 0.02% to about 1.00% by weight of the liposomal suspension, are primarily composed of 6 chromatographically distinct more desirably from about 0.05% to about 0.40% by weight fractions or families categorized as types I, II, III, IV, V, VIa, of the liposomal Suspension. and VIb (or 1, 2, 3, 4, 5, 6a, and 6b). Synthetic (or hybrid) 0056. Applicants have unexpectedly discovered a rela ceramides are ceramide-like sphingolipids, wherein the tionship between the use of rigidity enhancers and liposomal US 2010/0O86573 A1 Apr. 8, 2010

stability. More particularly, Applicants have discovered that bic acid where the alkyl portion has from 8 to 20 carbon the stability of liposomes depend on the incorporation of atoms. With respect to the esters, they may be selected from rigidity enhancers over a relatively narrow concentration the group consisting of fatty acid mono-, di-, tri- or tetra range. Specifically, incorporation of rigidity enhancers at lev esters of ascorbic acid. For example, Such esters include, but els exceeding 1.0% or exceeding a 1:4 ratio by weight relative are not limited to ascorbyl palmitate, ascorbyllaureate, ascor to the phospholipids, was found to shift the morphology of the byl myristate, ascorbyl Stearate, ascorbyl dipalmitate, ascor microstructure to lamellar sheets, in contrast to spherical byl dilaurate, ascorbyl dimyristate, ascorbyl distearate, ascor liposomal vesicles. For example, 100-400x magnification byl tripalmitate, ascorbyl trilaurate, ascorbyl trimyristate, microscopy has confirmed the creation of lamellar sheets ascorbyltristearate, tetrahexyldecylascorbate, ascorbyltetra when using rigidity enhancers at greater than 25% of the laurate, ascorbyl tetramyristate, ascorbyl tetrastearateL, phospholipid levels, which were further converted to large L-ascorbyl palmitate, L-ascorbyl isopalmitate, L-ascorbyl needle structures upon aging that were detectable to the naked dipalmitate, L-ascorbyl isostearate, L-ascorbyl distearate, eye. L-ascorbyl diisoStearate, L-ascorbyl myristate, L-ascorbyl 0057 Antioxidants isomyristate, L-ascorbyl 2-ethylhexanoate, L-ascorbyl di-2- 0058 According to the present invention, at least one anti ethylhexanoate, L-ascorbyl oleate and L-ascorbyl dioleate, oxidant is embedded in a liposome. The antioxidant may, for tetrahexyldecyl ascorbate; phosphates of L-ascorbic acid example, be embedded in the phospholipid bilayer of the Such as L-ascorbyl-2-phosphate and L-ascorbyl-3-phos liposome, in the aqueous center of the liposome, or both. The phate: Sulfates of L-ascorbic acid such as L-ascorbyl-2-sul antioxidant(s) may function to protect the liposome from fate and L-acorbyl-3-sulfate; their salts with alkaline earth oxidative damage or degradation resulting either from the metals such as calcium and magnesium. aqueous center of the liposome or from reactive oxygen spe 0064. With respect to the salts, they may be selected from cies (ROS) outside the liposome. Indeed, one major action of the phosphates and Sulfates, desirably phosphate. The ascor antioxidants in cells is to prevent damage due to the action of bic acid phosphate is generally selected from L-ascorbic acid ROS. Reactive oxygen species include hydrogen peroxide 3-phosphate, L-ascorbic acid 2-phosphate, L-ascorbic acid (H2O), the Superoxide anion (O), and free radicals such as 3-pyrophosphate and bis(L-ascorbic acid 3.3-) phosphate. the hydroxyl radical (OH). These molecules are unstable and Desirably, the ascorbic acid phosphate is magnesium or highly reactive, and can damage cells by chemical chain Sodium ascorbyl phosphate; more desirably, magnesium reactions such as lipid peroxidation. ascorbyl phosphate. Likewise, the ascorbic acid Sulfate is 0059 Antioxidants may be incorporated into liposomes of generally selected from L-ascorbic acid 3-sulfate, L-ascorbic the present invention in a number of different ways. In one acid 2-sulfate, L-ascorbic acid 3-pyrosulfate and bis example, a lipid soluble antioxidant may be directly embed (L-ascorbic acid 3.3-) sulfate. ded into the lipid bilayer of the liposome. In another example, 0065. Additional antioxidants include tetrahexyldecyl a water-soluble antioxidant may be encapsulated in the aque ascorbate, butylated hydroxytoluene (BHT), butylated ous center of the liposome. In a further example, a lipid hydroxyanisole, methylgentisate, L-carnosine, tert-butylhy soluble antioxidant may be embedded in the lipid bilayer and droquinone (TBHQ), glutathione, including derivatives, a water-soluble antioxidant is encapsulated in the aqueous combinations, and mixtures thereof. In one example, water center of the lipid bilayer. soluble L-carnosine may be incorporated into the aqueous 0060. In another example, the antioxidant may be a sin center of the liposome for use as an antioxidant, in view of its glet-oxygen Scavenger. In another example, the antioxidant ability to protect the liposome from oxidative damage via its may be both water-soluble and a singlet-oxygen Scavenger or aqueous center. both lipid soluble and a singlet-oxygen Scavenger. In a further 0.066 Antioxidants for use in the present invention may be example, more than one antioxidant might be embedded in present in a collective amount ranging from about 0.01% to the liposome of the present invention. about 1.00% by weight of the liposomal suspension, more 0061 Tocopherol (i.e. vitamin E) and ascorbic acid (i.e. desirably from about 0.025% to about 0.50% by weight of the Vitamin C) are examples of antioxidants that may be embed liposomal Suspension. ded in the liposomes of the present invention. Suitable toco 0067 Water-Soluble Composition pherol antioxidants include tocopherol; monomethyl, dim 0068. The water-soluble composition will be primarily ethyl, or triethyl derivatives oftocol, including but not limited comprised of water with or without loaded active (water to, alpha tocopherol, beta tocopherol, gamma tocopherol, soluble) agents, which are further described below. Gener delta tocopherol, epsilon tocopherol, Zeta tocopherol, and eta ally, the water-soluble composition will range from about tocopherol; tocopherol esters, and derivatives therefrom, 10% to about 65% by weight of the liposomal suspension or including tocopheryl acetate, tocopheryl Succinate, toco from about 67% to about 95% by weight of the liposome pheryl benzoate, tocopheryl propionate, tocopheryl Sorbate, preparation. tocopheryl oleate, tocopheryl orotate, tocopheryl linoleate, 0069. External Phase Composition tocopheryl nicotinate, and the 2-ethyl-hexanoate ester. 0070 The liposomal particles in the liposomal suspension 0062. In addition to its antioxidant function, ascorbic acid are surrounded by an external composition comprising an (Vitamin C) and its derivatives are additionally known to aqueous solution including one or more viscosity promoting promote collagen synthesis. Ascorbic acid derivatives useful agents collectively sufficient for the external phase composi in the present invention include all enantiomers whether sin tion to have a density between about 0.95 g/cc to about 1.25 gly or in combination. Desirably, the ascorbic acid is provided g/cc and/or a viscosity generally between about 2.5 cP and in the levo form. In addition, the ascorbic acid or its deriva about 40,000 cF at a shear rate of 10 sec' at 21°C. The one tives may be in a water-soluble or an oil-soluble form. or more viscosity promoting agents may include one or more 0063) Non-exclusive examples of vitamin C (ascorbic thickening agents, gellant agents, or both. In a desired acid) derivatives include, for example, alkyl esters of L-ascor embodiment the external phase composition has a viscosity US 2010/0O86573 A1 Apr. 8, 2010

between about 800 cF and 20,000 cBata shear rate of 10 sec' desired non-Newtonian embodiment, the fluid has pseudo at 21°C. Alternatively, when using higher gellant concentra plastic shear-thinning properties characteristic of Carbopol R. tions, the external phase viscosity may be raised to about polymers. 60,000 to 80,000 cp at a shear rate of 10 sec' at 21°C., albeit (0076. At a relatively low shear rate of 10 sec', the vis with reduced clarity. cosity of an external phase composition containing gellants or 0071. The external phase composition generally com thickeners conferring non-Newtonian flow characteristics prises a density closely matching that of the liposomal par may vary between about 500 cF to about 80,000 cF at 21°C., between about 800 cP and 40,000 cP, or between about 2,000 ticles dispersed therein and is incorporated into the liposomal cP and 20,000 cB. In one embodiment, when using a gellant, Suspension of the present invention in an amount Sufficient to Such as a Carbopole polymer, the Viscosity of the external promote liposomal particle stability and reduce liposomal phase composition at 21°C. is about 284,000 cp at 0.1 sec', particle migration, flocculation, or agglomeration. about 60,000 cF at 1 sec', about 11,200 cp at 10 sec', and 0072 The external phase composition includes one or about 6,852 cP at 20 sec'. In another embodiment, when more viscosity promoting agents, including thickening using a thickening agent, such as Xanthan gum, the Viscosity agents, densification agents, gellants, gums, and other viscos of the external phase composition at 21°C. is about 110,000 ity promoting agents known to those of skill in the art. Vis cPat 0.1 sec', about 18,670 cF at 1 sec', about 2.341 cPat cosity promoting agents for use in the present invention may 10 sec', and about 1,272 cF at 20 sec'. be empirically selected to increase Viscosity and enhance 0077 Viscosity measurements may be obtained using a stability of the liposomal formulations herein. Desirably, suitable viscometer, including Brookfield Syncho-Lectric these agents are chosen to preserve the translucent or trans Model RVT Viscometer, Haake Rotovisco Model RV-12 Vis parent nature of the liposomal Suspension. Further, the com cometer, or other Suitable viscometers (or rheometers such as positions may be adjusted by using agents selected to to TA Instruments AR-1000N) known to those of skill in the art provide a comparatively greater impact on viscosity or den for measuring viscosities of Newtonian fluids or apparent sity as desired. Exemplary viscosity promoting agents viscosities of Non-Newtonian materials at low shear rates at include glycerin; celluloses, including hydroxyethyl given rotation speeds according to ASTM D1084-88, ASTM hydroxypropyl-, hydroxymethyl-, hydroxypropylmethylcel D1824-87, D2196-86 and other ASTM protocols relating to measurement of viscosity. luloses, and their derivatives, including various celluloses 0078. In forming a liposomal suspension, the external those under the tradenames Natrosol (R), Methocel(R), and Ber phase composition may be added to a liposomal solution mocollf; light non-ionic emulsions; and polysorbates. containing liposomal particles or it may be added to a lyo Exemplary gellants and/or thickening agents include gum philized (freeze-dried) preparation of liposomal particles pre matrix agents, including Xanthan gum, and acrylate poly pared from a liposomal Solution. Based on theoretical entrap mers, including high molecular weight homo- and copoly ment Volume data, the weight percent of the liposomal mers of acrylic acid crosslinked with polyalkenyl polyethers particles relative to the liposomal Suspension may range or divinyl glycol under the tradename Carbopol R, including between about 0.96% and about 54% by weight. Carbopol R 934,940, 941,980,981,1342, 1382, 2020, 2050, 0079. When added to a liposomal solution, the external and various hydroxyethyl acrylate/acryloyidimethyltaurate phase composition may be present in an amount between copolymers under the tradename Simulgel(R). about 30% to about 75% by weight of the liposomal suspen 0073. The external phase composition may have Newto sion, from about 55% to about 70% by weight of the liposo nian or non-Newtonian flow characteristics. In one embodi mal suspension, from about 65% to about 70% by weight of ment, the one or more viscosity promoting agents includes an the liposomal suspension, or about 65% by weight of the aqueous solution having Newtonian flow characteristics, liposomal Suspension. Additionally, the weight ratio of the Such as glycerin. The Viscosity of Newtonian fluids, including liposomal Solution to the external phase composition may glycerol, is independent of shear rates at given rotation speeds range from about 0.002 to about 0.54. In a desired embodi imparted to the fluid. External phase compositions compris ment, the liposomal Suspension comprises between about ing Newtonian flow characteristics may have a viscosity 35% to about 75% glycerin, more desirably between about between about 500 cP to about 10,000 cP, or between about 50% to about 65% glycerin when added to a liposomal solu 800 cp to about 2,000 cP. tion. Where gellant(s), including neutralized Carbopole are 0074. In another embodiment, the one or more viscosity used in the external phase, the level of gellant(s) should not promoting agents may include one or more agents at concen exceed approximately 1.0% of the liposomal Suspension or trations between about 0.10% to about 1.0%, such as poly visual clarity/transparency/translucency may be reduced in meric gellantagents, which confer non-Newtonian flow char the liposomal Suspension. Generally, the liposome prepara acteristics. Non-Newtonian fluids including, for example, tion should be diluted with the external phase composition polymeric gellants or thickeners, exhibit reduced viscosity as within 24 hours of preparation to reduce instability. a function of shear rate. However, compared to Newtonian 0080. The refractive indices for liposomal suspensions fluids Such as glycerin, polymeric gellants can confer non may range from about 1.3 to about 1.45. By way of example, Newtonian flow characteristics at low concentrations and can the refractive indices for liposomal Suspensions comprising provide an apparent viscosity that is comparatively less Newtonian fluids in the external phase composition, Such as affected by temperature. glycerin, may typically range from about 1.40 to about 1.45 0075. The viscosity of non-Newtonian fluids may be clas and may typically range from about 1.35 to about 1.40 when sified in terms of shear and time dependency. Thus, a non using non-Newtonian fluids employing gums or gellants, Newtonian fluid may be shear-thinning and shear-thickening. including refractive indices between about 1.39 to about 1.40 Further, a reduction in apparent viscosity may be time-inde for Xanthan gum mixtures, and about 1.37 for external phase pendent (pseudoplastic) or time-dependent (thixotropic). In a compositions comprising polymeric Carbopols(R). US 2010/0O86573 A1 Apr. 8, 2010

0081. The external phase composition may further include ded molecules is not considered critical since there is a high one or more preservatives, pH adjusters, and/or water. Non cost associated with Such processing, and cosmetic molecules limiting examples of preservatives include phenoxyethanol, are non-toxic at the levels they are commonly used. chlorphenesin, methylisothiazolinone, propylene glycol, benzyl alcohol, ethanol, methylparaben, propylparaben, eth I0087. By contrast with the hydrophilic actives entrapped ylparaben, butylparaben, and isobutylparaben. Commer within the core of the liposome, hydrophobic compounds cially Suitable preservative preparations include those sold typically are entrapped in the hydrophobic regions of the lipid under the trade names Microcare(R) MTC (propylene glycol, bilayer. This type of entrapment involves a form of partition chlorphenesin, and methylisothiazolinone) and Phenonip(R) ing wherein the hydrophobic compounds are dissolved in XB (phenoxyethanol, methylparaben, propylparaben, eth Suitable solubilizing agent(s), along with the lipid. For ylparaben). Exemplary pH adjusters include L-arginine and example, if the compound is soluble in butylene glycol, triethanolamine. ethoxyglycol, ethanol, methanol, chloroform, toluene, ether, 0082) Applicants have unexpectedly discovered that con propylene glycol, polyethylene glycol, including polyethyl ventional preservatives, including chelating agents, such as ene glycol 200 and polyethylene glycol 300, or medium chain disodium EDTA can negatively impact on the stability of the triglycerides it can be dissolved along with the lipid in the above-described liposomal suspensions, and that sodium solvent. While other solvents, such as ethanol and methanol benzoate and various acids, including citric acid, Sorbic acid, can be used for solubilization of lipids and oil soluble actives, benzoic acid, and isopropyl methylphenol, can rupture the they should be removed by extraction prior to addition of the liposomal vesicles at concentration greater than 0.01%. The external phase composition. Applicants have discovered, for phenomenon of bilayer/vesicular disruption (and Subsequent example, that solvents, such as ethanol, methanol, chloro destabilization of the colloidal system) was confirmed by form, toluene, or ether at concentrations above 3% can cause instantaneous visual opacity upon addition of citric acid to a collapse of gellation, disruption in the density balance, or liposomal solution as described above. Moreover, Applicants reduction in the viscosity of the external phase, thereby have discovered that paraben preservatives have a tendency to reducing the Stability of the liposomal Suspension. migrate to the bilipid membrane due to partitioning into the oil phase, which negatively impacts upon stability. In addi I0088. By way of example, a variety of skin humectants, tion, parabens have been found to result in an overall increase barrier enhancers, essential fatty acids, and Vitamins may be in liposomal particle size upon aging. Applicants have further embedded into or associated with the liposomes of the present found that long term stability of liposomal suspensions of the invention. These elements help to retain moisture and further present invention may be sacrificed when eliminating L-argi support the skin's barrier function by preventing and revers nine from the water-phase composition. ing damage thereto. Exemplary skin humectants include 0083 Preferably, the preservatives for use in the liposomal glycerin, sodium hyaluronate, panthenol, and amino acids. suspensions of the present invention are free of EDTA, I0089 Barrier enhancers include various skin lipids, which Sodium benzoate, citric acid, Sorbic acid, benzoic acid, and help to Support and maintain a healthy skin barrier function. isopropyl methylphenol. In desired embodiments, the liposo The skin barrier function is characterized by a water imper mal Suspensions are additionally free of parabens. Desired meability barrier in skin, or more specifically, the water preservatives for use in the liposomal Suspensions of the retaining function of the stratum corneum. Lipid lamellar present invention include phenoxyethanol alone, methyl structures in the intercellular spaces of the stratum corneum isothiazolinone alone, chlorphenesin alone, chlorphenesin in are considered to be responsible for the water retaining prop combination with methylisothiazolinone, or chlorphenesin in erties of the stratum corneum combination with methylisothiazolinone and phenoxyetha 0090 Exemplary skin lipids contributing to maintenance nol. of barrier function include the above described lipids con 0084 Preservatives may be present in an amount between tained in the oil phase composition in the above described about 0.01% to about 5.00% by weight of the liposomal liposomes, including phospholipids, sphingolipids, choles suspension, from about 0.01% to about 2.00%, or between terols, and phytosterols, which can be utilized to replenish about 0.01% and about 0.30% by weight of the liposomal critical Stratum corneum lipids associated with maintaining Suspension. L-arginine or triethanolamine may be present in and enhancing the skin's barrier function. an amount between about 0.001% to about 0.10%, desirably 0091 Additional barrier enhancers include hydrolyzed between about 0.001% and about 0.010%. Soybean protein and soybean protein extract. Soybean protein I0085 Active Agents extract is known to inhibit the activity of an enzyme (i.e. I0086 Liposomal suspensions of the present invention elastase), which breaks down elastin fibers in the dermis and may further include one or more active agents (or “actives') is released to the skin in response to UV rays, dryness, and embedded into the liposomal particles of the present inven environmental stresses generally. Treatment of skin with tion. The active agents may include any active agents known hydrolyzed soybean protein extract helps keep skin elastic, in the art for cosmetic, therapeutic, or diagnostic applications. firm, Smooth, Soft, and moisturized. Hydrolyzed soybean pro The actives may be hydrophobic (i.e., oil-soluble) or hydro teins are known to stimulate production of collagen and elas philic (i.e., water-soluble) in nature. Water-soluble actives tin and provide firmness and elasticity to skin. may be embedded during the process of liposome preparation 0092 Additional barrier enhancer ingredients include via “passive loading.” This can beachieved by pre-dissolving essential fatty acids and vitamins. Non-limiting examples of the hydrophilic actives in a buffer used for hydration of the Such essential fatty acids useful in the present compositions dry lipid. Any non-embedded molecule can thereafter be include linoleic acid, linolenic acid, oleic acid, columbinic removed by dialyzing against the blank buffer, or by passing acid, arachidic acid, arachidonic acid, lignoceric acid, ner the dispersion through a SephadexTM gel column. For most Vonic acid, eicosapentanoic acid, palmitic acid, Stearic acid, cosmetic and personal care products, removal of non-embed and mixtures thereof. Other essential fatty acids known to US 2010/0O86573 A1 Apr. 8, 2010

those of ordinary skill in the art, such as those described in the the mice skin after irradiation with ultraviolet light, was above resources, are further contemplated as useful in the shown to decrease the number of nonmalignant and malig present compositions. nant skin tumors (Lu et al., Proc. Natl. Acad. Sci. USA 0093 Essential fatty acids can be derived from a variety of 99(19): 12455-60, 2002), diminish photodamage, and pro Sources. For example, the essential fatty acids may be pro mote the elimination of DNA-damaged keratinocytes (Koo et vided in the liposomal Suspensions from one or more oils al., Br. J. Dermatol., 156(5):957-64, 2007). In addition, meth derived from hydrophobic botanical extracts. Exemplary oils ylxanthines, like caffeine are believed to act as “sunless” useful in this regard include various vegetable or plant oils, tanning agents modulating the activity of biochemical path including but not limited to flaxseed oil, hempseed oil, cotton ways involved in melanogenesis, presumably by their ability seed oil, pumpkin seed oil, corn oil, canola oil, linseed oil, to inhibit cyclic-AMP phosphodiesterase and enhance the rosemary oil, soybean oil, wheat germ oil, olive oil, grape production of melanin in melanocytes, alone or in combina seed oil, Seabuckthorn seed oil, borage oil, macadamia inte tion with tanning stimulants like ultraviolet or Solar radiation. grifolia seed oil, evening primrose oil, meadowfoam seed oil, 0099 Exemplary methylxanthines for use in the present perilla ocymoides seed oil, black currant seed oil, chestnut invention include caffeine (1,3,7-trimethylxanthine), theo oil, palm oil, papaya see oil, liquorice root, corn oil, grapefruit phylline (1,3-dimethylxanthine), aminophylline, theobro seed oil, kiwi seed oil, ligonberry seed oil, passion fruit seed mine (3,7-dimethylxanthine), paraxanthine, isobutylmethyl oil, watermelon seed oil, rosehip seed oil, safflower oil, Sun Xanthine, butymethylxanthine, as well as analogs, deriva flower oil, sunflower seed oil, cottonseed oil, peanut oil, tives, and combinations thereof. In a particular embodiment, jojoba oil, sesame seed oil, poppy seed oil, vegetable oil, a methylxanthine, such as caffeine, will be present at a mixtures thereof, and hydrogenated derivatives thereof. weight. concentration of about 0.01% to about 0.30%. 0094. Alternatively, or in addition, the liposomes of the 0100 Other additives that may be included in the liposo present invention may be loaded with one or more plant mal Suspensions of the present invention will be apparent to extracts, plant ingredients, DNA repair enzymes, and/or those of skill in the art and are included within the scope of the methylxanthines. Exemplary extracts may be derived from present invention. various plant sources, including Rosemary, Centella, Echina 0101. In skin care or cosmetic products, the liposomal cea, Alpinia, Bearberry, Acerola Cherry Fermentate, or from Suspensions of the present invention can be incorporated into the plants corresponding to the above-identified oils. Addi cosmetically Suitable matrix to form a cosmetic formulation tional examples of plant extracts, plant ingredients and plant in the form of a cream, lotion, gel, serum, tonic, emulsion, derived oils are described in U.S. Pat. Appl. Publ. Nos. 2006/ paste, or spray for topical administration. 0257509 and 2007/0003536, the disclosures of which are 0102 Methods for Producing Liposomes expressly incorporated by reference herein. 0103) In another aspect, the present invention provides a 0095. The oils or extracts for use in the present invention method for producing a stable, transparent to translucent, may be present in a collective amount ranging from about unilamellar liposomal Suspension as described above. In one 0.0001% to 1% by weight of the liposomal suspension, more embodiment, a method for producing a stable, transparent to desirably from about 0.0003 wt % to about 0.003 wt %. translucent, unilamellar liposomal Suspension includes: (a) 0096 Exemplary DNA repair enzymes include bacte preparing an oil-soluble composition comprising a coupling riophage T4 pyrimidine dimer-specific endonuclease, Micro agent, at least one phospholipid, a rigidity enhancer, and an coccus luteus N-glycosylase/AP lyase, Saccharomyces cer antioxidant; (b) preparing a water-soluble composition; (c) evisiae N-glycosylase/apurinic-apyrimidinic lyase, combining the water-soluble composition with the oil Schizosaccharomyces pombe UV damage endonuclease soluble composition to form a multilamellar, multivesicular (UVDE), Chlorella virus isolate PBCV-1 pyrimidine dimer liposome preparation; (d) converting the multilamellar, mul specific glycosylase, and Anacystis nidulans photolyase. tivesicular liposome preparation to a unilamellar liposome Additional DNA repair enzymes are described in U.S. Pat. preparation; and (e) adding the unilamellar liposome prepa Appl. Publ. Nos. 2006/0257509, the disclosures of which are ration to an external phase composition having a density expressly incorporated by reference herein. The DNA repair within a range of about 0.95 g/cc to about 1.25 g/cc. The enzymes may be present in a collective amount ranging from method will typically result in a liposomal Suspension com about 0.01% to about 10% by weight of the liposomal sus prising a uniform plurality of unilamellar liposomal particles pension, more desirably from about 0.1% to about 3% by having a mean particle size between about 50 to about 290 weight. nm, wherein the liposomal Suspension has a refractive index 0097 Exemplary vitamins include the above-described between 1.30 and 1.45 and retains its stability in neat form antioxidants, as well as lipophilic vitamins, such as vitamin within an amberglassjar, colorless glassjar, or opaque plastic A, including free acids or derivatives thereof and precursors container at about 4°C. to about 50° C. for a period of at least thereof, including retinoids. Such as retinol, retinal, and reti 30 days, or at 21°C. for a period of at least 180 days, or longer. nyl esters, such as retinyl acetate, retinyl butyrate, retinyl 0104. In one embodiment, the oil-soluble composition propionate, retinyl octanoate, retinyl laurate, retinyl palmi may be prepared by sequentially adding at least one coupling tate, retinyl oleate, and retinyl linoleate; and carotenoids; agent, at least one phospholipid, at least one rigidity enhancer, B-complex vitamins, including B1: thiamine, B2: riboflavin, and/or at least one antioxidant to form a batch composition. B6: pyridoxin, panthenol, and pantothenic acid; vitamin B12 The batch compCsition may be agitated and heated to tem and combinations thereof, vitamin D, biotin, vitamin K, perature Sufficient to dissolve the components (for example, water-soluble derivatives thereof, and the like. 190-210°F). Upon cooling, one or more temperature-sensi 0098 Topical administration of methylxanthines, such as tive oil-soluble components, including oil-soluble actives caffeine, is believed to reduce the deleterious effects of DNA may be added. The batch composition may then be cooled and damage caused by exposure of skin to ultraviolet or Solar maintained (and agitated) at a temperature between about radiation. Specifically, topical administration of caffeine to 75-85 F. for phase combination with the water-soluble com US 2010/0O86573 A1 Apr. 8, 2010

position. The water-soluble composition and the oil-soluble cosmetically Suitable matrix to form a cosmetic formulation composition (with or without water-soluble and/or oil in the form of a cream, lotion, gel, serum, tonic, emulsion, soluble actives) may be mixed in an agitator (at for example, paste, or Spray. 50 rpm for 10-15 minutes). A multilamellar, multivesicular 0110 Transmission electron microscopy (TEM) provides liposomal particle composition may be formed and then con a means for characterizing the size, integrity, and stability of Verted to a unilamellar liposomal particle composition using the liposomes. A commonly used method of sample prepara a suitable conversion process, such as high pressure microf tion includes applying a drop of liposome Suspension diluted luidization, extrusion, high speed shearing, milling, Sonica 1:5 into a plastic-coated grid, negatively staining the material tion, selective microchannel filtration or homogenization. An with phosphotungstic acid or ammonium molybdate, thereby external phase composition may be prepared by sequentially producing a monolayer of liposomes embedded in the nega mixing together one or more and dissolving the contents tive stain. under agitation and adding the contents of the external phase 0111. More specifically a sample may be prepared for composition to a liposome preparation as described above. TEM analysis as follows: 1% aqueous agarose (w/v) is poured into plastic Petridishes and air-dried at room tempera 0105 Because the biological activity of certain active ture for 10 minutes. Formvar or collodion coated grids are ingredients may be compromised after being Subjected to made hydrophilic by glow discharge and the grids are placed temperatures in the oil-soluble batch composition of about on Whatman no. 1 filter paper. One drop of the sample sus 200' F., for example, certain active ingredients, including pension is placed on each grid. When most of the liquid is botanical Substances (such as Centella asiatica extract), may absorbed at the periphery of the grid by the filter paper, the be solubilized in the water-soluble composition under more grids are transferred to the agarose plates for 30 minutes to 1 reduced temperature conditions or they may added (and solu hour to facilitate salt and liquid diffusion into the agarose. bilized) in the oil-soluble batch composition upon further 0112 The unstained grids are viewed and photographed cooling. using a Zeiss-902 with ESI. Typically, the liposomes will 0106. A number of methodologies may be employed for appear as discrete round structures without evidence of sig converting a multilamellar, multivesicular liposome prepara nificant clumping. tion to a unilamellar liposome preparation. These include 0113 Liposomal particle sizes and stability may be evalu high pressure microfluidization, extrusion, high speed shear ated, for example, by evaluating the sizes and structures of ing, milling, Sonication, selective microchannel filtration, and liposomes in an aqueous solution or in a cream formulation homogenization. both prior to or following storage in an amber glass jar, colorless glass jar, or opaque plastic container for 1-12 0107 Microfluidization provides a desirable method for months after storage, or later. Liposomal particle sizes and producing unilamellar liposome preparations, particularly in stability may be evaluated using Suitable laser light scattering view of its capacity for producing liposome preparations in (e.g., Nanotrac(R) or Microtrac(R).multiple light scattering, bulk quantities. Microfluidization involves introduction of kinetic stability analyzer (Turbiscan RTLab Expert), and/or slurry-like concentrated lipid/water dispersions in a microf transmission electron microscopy devices and methodologies luidizer which pumps the dispersions at. a very high pressure as known to those of skill in the art. (10,000 to 20,000 psi) through microchannels of 1-5 um in 0114. In one aspect, laser light scattering may be used for diameter. The microchannels are split and impinged upon particle size quantitation. For example, the Nanotrac(R) uti each other to enhance droplet size reduction and energy input. lizes a “controlled reference' dynamic light scattering Fluid moving at a very high Velocity is split into two streams approach, which utilizes a 780 nm wavelength laser diode by forcing it through two defined microchannels. Massive shining light through a sapphire filter window into a diluted cavitation is experienced though a rapid pressure differential sample dispersion media. Laser light (in a scattered pattern of upon entry of the fluid through the tiny orifice of the microf shifted frequencies) is reflected back through the sapphire luidics chamber. A similar pressure differential is obtained window into an optical splitter that is received by a photode through conventional homogenization. The two streams are tector. The shifted frequencies of the scattered light represent then made to collide together at right-angles at very high the combination of the “controlled reference', which is light Velocity. The tremendous energy imparted by the high pres reflected by the sapphire filter (of known un-shifted fre sure differential and high velocity causes the lipids to self quency), in conjunction with the influence of the velocity of assemble into liposomes. The fluid collected at the end may Suspended sample particles experiencing Brownian motion. be re-passed through the microfluidizer one to four times until The laser is frequency shifted according to the “Doppler a homogeneous-looking dispersion, and unimodal, uniform effect”, which is proportional to the motion velocity of the particle size distribution is obtained. Suitable microfluidic distribution of particles of interest. The detector output sig devices for producing the liposome preparations in accor nals are then amplified, filtered, digitized and mathematically dance with the present invention include Micofluidics Models analyzed to quantify the particle size distribution. M110Y and M210EH (Microfluidics Corp., Newton, Mass.) 0.115. A stable liposomal system exhibits a uniform, uni 0108. Alternatively, the unilamellar liposomes of the modal distribution of particles upon aging when Subjected to present invention can be also created by Sonicating phospho reduced or elevated temperatures. A bimodal or trimodal lipids in water. Low shear rates create multilamellar lipo distribution occurs when the particles coalesce or flocculate Somes, which have many layers like an onion. Continued leading to rupture of their entrapped contents and full-scale high-shear Sonication tends to form Smaller unilamellar lipo creaming or sedimentation of the entire microstructure/col SOS. loidal system. A liposomal Suspension according to the 0109. In a further aspect, the present invention includes a present invention may retain its stability in neat form within method for preparing a cosmetic formulation, wherein a lipo an amberglassjar, colorless glass jar, or opaque plastic con Somal Suspension of the present invention is combined with a tainer at about 4°C. to about 50° C. for a period of at least 30 US 2010/0O86573 A1 Apr. 8, 2010

days, at least about 2 months, at least about 3 months, at least I0121 The present invention also includes methods of about 6 months, at least about 19 months, or longer. As shown treating skin by topically applying the liposomal Suspensions in FIG. 5 below, liposomal formulations of the present inven of the present invention. In use, a small quantity of the com tion were found to retain their stability and unimodal particle position, for example from 1 to 100 ml, may be applied to distribution under ambient room temperature conditions for exposed areas of the skin, from a suitable container or appli at least 19 months, as reflected in a unimodal particle size cator and, if necessary, it is then spread over and/or rubbed distribution characterized by a mean particle size of about 157 into the skin using the hand or fingers or a suitable device. I0122. In one embodiment, the present invention provides a nm and a size range between about 54 nm and about 521 nm. method for preventing, arresting, reversing, ameliorating, 0116 ATurbiscan Accelerated Kinetic Stability Analyzer diminishing, reducing, or improving a sign of aging, in which can provide a traceable, quantitative method for early predic a composition of the present invention is topically applied to tion of instability of colloidal systems. The Turbiscan Ana skin in a cosmetically effective amount Sufficient to prevent, lyzer employs multiple light scattering to detect the level of arrest, reverse ameliorate, diminish, reduce, or improve a sign transmitted and backscattered light as a function of sample ofaging in skin. Exemplary signs of aging include, but are not depth for colloidal systems ranging in appearance from trans limited to, facial lines, fine lines, wrinkles, crow's feet, dark parent to opaque. The Turbiscan instrument consists of a near eye circles, blemishes, age spots, stretch marks, weakened infrared laser diode which shines photons of light throughout skin barrier repair properties, or combinations thereof. the depth of a small (1 cmx8 cm) cylindrical glass cell filled I0123. In another embodiment, the present invention pro with a sample. The photons of light emerge (1) from the vides a method for improving the aesthetic appearance of sample in a scattered pattern, which are detected simulta skin, in which a composition of the present invention is topi neously by transmission (for transparency) and backscatter cally applied to skin in a cosmetically effective amount Suf (for opacity) detectors. The ratio of backscattered to trans ficient to improve the aesthetic appearance of the skin. The mitted light is collected as a "fingerprint Scan according to improvements may relate to skin thickness, elasticity, resil user-defined time intervals. Kinetic results of the superim iency, moisturization, tone, texture, radiance, luster, bright posed data scans show the degree of homogeneity of the ness, clarity, contour, uniformity, firmness, tautness, Supple Suspension, emulsion or foam of interest. ness, softness, sensitivity, pore size, reduction in water loss, 0117 Relative to the term “stability” in the present inven or combinations thereof. tion, a “backscatter kinetic result may be defined as the 0.124. The improvements may further relate to improving relative change in backscattered light as a function of time adverse skin conditions affected by, resulting in or resulting interval defined by the user. Each scan of backscattered light from the group consisting of psoriasis, eczema, seborrhea, for the product changes with each time interval scanned by dermatitis, Sunburn, estrogen imbalance, hyperpigmentation, the laser. This change in backscattered light relates to the hypopigmentation, discoloration, yellowing, freckles, skin migration of particles within the system. In a stable liposomal atrophy, skin breakout, skin fragility, dryness, chapping, Sag Suspension, a laser Scan of backscattered light will change giness, thinning, hyperplasia, fibrosis, enlarged pores, cellu little relative to the initial scan of backscattered light. Relative lite formation, bruising, acne formation, apoptosis, cellular to the present invention, a backscatter kinetic result of less differentiation, cellular de-differentiation, prevention of than 2.0% depicts a stable liposomal Solution or Suspension tumor induction or tumor progression, viral infections, fungal under the selected temperature conditions and user-defined infections, bacterial infections, spider veins (telangectasia), time intervals. For the liposomal systems of the present inven hirsutism, rosacea, pruritis, calluses, warts, corns, or combi tion, the backscatter kinetic results were approximately 0.2 to nations thereof. 1.0% when exposed to temperatures between 25°C. to 50° C. 0.125 The signs of aging or adverse skin conditions may over a 16 hour period. Early indications of instability can be result from free radical damage, environmental agents, pol detected in a period as short as 4-8 hours for colloidal sys lutants, diet, chronological aging, premature aging, hormonal temS. aging, photo-aging, or combinations thereof. Accordingly, 0118 Modes of Administration the present compositions and methods selected for improved 0119 The liposomal suspensions of the present invention anti-aging characteristics or adverse skin conditions may are generally designed for topical administration. Generally, employ topical application of active ingredients inhibiting the compositions of the present invention are administered at enzymes or mediators that accelerate or facilitate aging, dam least on a daily basis. Administration of the compositions of age, formation of free radicals, or breakdown of skin ele the invention may continue for any suitable period of time. It ments, including, but not limited to metalloproteinases, col should be appreciated that the degree of cosmetic enhance lagenases, elastases, hyaluronidases, and proteases. The ment and degree of maintenance, improvement, restoration, active ingredients may have anti-oxidant activity, free radical or repair will vary directly with the total amount and fre Scavenging or anti-inflammatory activity, and/or they may quency of composition used. inhibit breakdown of collagen, elastin, fibronectin, hyalu 0120 Useful dosage forms can be prepared by methods ronic acid, glycosaminoglycans (GAG), or other extracellular and techniques that will be well understood by those of skill matrix elements or regulatory enzymes or mediators of the in the art and may include the use of additional ingredients in NF-kB signal transduction pathway. The active agents may producing appropriate dosage forms. In one example, a lipo also inhibit other signal transduction pathways associated Somal Suspension of the present invention is topically admin with aging, including the mediators and regulators associated istered at least once a day. In another example, a liposomal with these pathways, or combinations thereof. Suspension is administered twice daily. In a further example, I0126. In addition to improving the aesthetic or cosmetic the formulation may be administered three to five times daily. appearance of skin, the topical compositions of the present Generally, there is no limit on the amount of the formulation invention may be topically applied to enhance the general that may be administered daily. health, Vitality and appearance of the skin. For example, the US 2010/0O86573 A1 Apr. 8, 2010 11 present composition may be applied to skinto improve micro 1b/min. The water-soluble and oil-soluble components were circulation, communication among skin cells, replenishment mixed for 10-15 min. with the turbine agitator at 50 rpm. The of essential nutrients or skin constituents, or to improve the resulting multilamellar, multivesicular liposomal particle metabolism, proliferation, multiplication, turnover and/or composition was then subjected to 1-4 passes at 12,000-15, exfoliation of skin cells. 000 psi in a microfluidizer (Microfluidics M110Y and 0127 Exfoliation may be carried out with or without the M21OEH) using 80-100 psi in-feed pressure to form a unila use of alpha- or beta hydroxy acids or other exfoliants, or mellar liposomal particle composition. An external phase combinations thereof by topical application to skin. When composition was prepared by sequentially mixing together in using exfoliating agents in the compositions of the present a scrapewall turbine tank glycerin, methylparaben, arginine, invention, Sufficient anti-irritant or anti-inflammatory agents and water and dissolving the contents at 145-155°F. for about are included to neutralize the potential irritation associated 20 min. under agitation (scrapewall at 30 rpm; turbine at with exfoliating agents in the absence of Such neutralizing 300-350 rpm). Upon cooling to 140°F, additional preserva agents. tives were added to external phase composition. Then, upon 0128. It is intended that the foregoing detailed description further cooling, the microfluidized unilamellar particle com be regarded as illustrative rather than limiting. The present position was added to the external phase composition and invention is further illustrated by the following experimental mixed for 15 min. at 75-90°F. investigations and examples, which should not be construed I0131 The resulting liposomal suspension containing 60% as limiting. Further, the liposomal Suspensions in Tables 3-6 glycerin had a refractive index of 1.4 and was stable at tem are exemplary formulations, whose compositional ingredi peratures between 4°C. and 60° C. for at least 6 months. The ents and amounts may be modified in accordance with the formulation was further evaluated by Transmission Electron teachings above. Microscope (TEM) analysis (FIGS. 1A-1C), which con firmed the production of structurally intact unilamellar lipo EXAMPLE1 somes (FIGS. 1B, 1C). 0129. Preparation of a liposomal suspension. I0132 Table 1 shows a particle size analysis of a liposomal 0130. In an exemplary method for preparing a liposomal formulation according to FIGS. 1A-1C following 2 or 3 Suspension according to the present invention, an oil-soluble passes through the microfluidizer at various time intervals composition was prepared by sequentially adding to a scrape after preparation (initial. 1 month, 2 months, 3 months, and 6 wall turbine tank Phospholipon R 85, ceramide IIIB, beta months) under varying temperature storage conditions (40° sitosterol, and butylene glycol. Scrapewall agitation at 40 rpm F., ambient, and 120° F.) by dynamic laser light scattering was initiated and the batch composition was heated to a using a NanotracR N150 Submicron Particle Size Analyzer temperature (190-210°F.) sufficient to dissolve the compo (Microtrac Inc., Montgomeryville, Pa.). Each of the 25 con nents. The composition was cooled, and tocopherol was ditions listed includes particle size calculations for 75 differ added when the batch temperature dropped to 170° F. The ent tests based on 3 Scans (histograms) for each condition batch composition was then cooled to, and maintained at, a tested. As shown in Table 1, a cosmetic formulation compris temperature between 75-85 F. for phase combination with ing the liposomal suspension was characterized by a particle the water-soluble composition. While agitating the scrape size range between 56 nm to 333 nm, and a median particle wall at 50 rpm, water was slowly added to the oil-soluble size of about 140 nm following 6 months of storage under composition using an air powered gear pump at a rate of 8-10 ambient temperature conditions.

TABLE 1. Particle Size Analysis for Liposomal Formulation. Reference#PMA9330-87

2G T = 40 °F. 2G T = Ambient 2G T = 120 °F. 3G T = 40 °F. 3G T = Ambient 3G T = 120 °F. Range size nm vol% size nm vol% size nm vol% size nm vol% size nm vol% size nm vol%

Initial LOW 25.55 25.55 25.55 18.06 18.06 18.06 High 486 486 486 486 486 486 Mean 1431 100 1431 100 1431 100 125 1OO 25 1OO 125 100 1 month LOW 18.06 3O4 25.55 25.55 18.06 25.55 High 1156 2312 578 687 578 687 Mean 1812 100 1931 100 175.8 100 1514 1OO 33.9 1OO 132 100 2 months Low 18.06 25.55 36.1 18.06 18.06 25.55 High 818 578 818 687 486 1156 Mean 1542 100 153.9 100 1543 100 125.8 1OO 43.3 1OO 134 100 3 months Low 18.06 25.55 36.1 15.19 18.06 3O4 High 578 972 578 578 486 578 Mean 1692 100 156.1 100 1743 100 134.1 1OO 43.1 1OO 158 100 6 months Low 56 High 333 Mean 40 1OO

2G = 2 passes through microfluidizer 3G = 3 passes through microfluidizer **Cosmetic Formulations are only tested for physical properties under ambient conditions beyond 3 mon hs of shelflife. US 2010/0O86573 A1 Apr. 8, 2010

0.133 FIG. 2 shows the resulting unimodal, particle size nm for a paraben-free suspension employing phenoxyetha distribution of a liposomal Suspension according to Example nol, propylene glycol, chlorphenesin, and methylisothiazoli 1 after 19 months of shelf life under ambient temperature none. FIG. 4 shows a mean particle size of about 247 nm and conditions. FIG.2 shows a mean particle size of about 157 nm a size range between about 141 nm to about 518 nm for a and a size range between about 54 to about 521 nm. Suspension employing phenoxyethanol in combination with various parabens, including methylparaben, propylparaben, EXAMPLE 2 and ethylparaben. FIG. 5 shows maintenance of a unimodal 0134) Table 2 depicts a liposomal suspension made by the particle size distribution of a liposomal Suspension according process described in EXAMPLE 1. to Table 4 under ambient room temperature conditions for at least 19 months. In particular, FIG. 5 shows a unimodal TABLE 2 particle size distribution characterized by a mean particle size

Amount of about 157 nm and a size range between about 54 nm and Ingredients (percent) about 521 nm. Oil-Soluble Composition TABLE 4

Phospholipid(s) 1.0-10.O Amount Ceramide(s) O.O1-1.O Ingredients (percent) Phytosterol(s) O.O1-1.O Coupling agent(s) 1.0-10.O Oil-Soluble Composition Antioxidant(s) O.O1-1.O Water-Soluble Composition Lecithin 4.OOO Ceramide IIIB O.1SO Water 5.0-60.O Beta-sitosterol O.100 External Phase Composition Butylene glycol 4.OOO Tocopherol O.O10 Viscosity promoting agent(s) 35.0-70.O Meadowfoam Seed Oil O.1SO Preservative(s) O.O-5.O Soybean Oil O.200 Evening Primrose Oil O.OSO Perilla Ocymoides Seed Oil O.OSO Omega Plus (R) (Sunflower seed O.OSO EXAMPLE 3 oil, corn oil, Sesame seed oil, macadamia seed oil, olive oil) 0135 Table 3 depicts a liposomal suspension formed by Tocopheryl acetate O.OSO the process described in EXAMPLE 1. Water-Soluble Composition Water 27.OOO TABLE 3 External Phase Composition Amount Glycerin 58.458 Ingredients (percent) Arginine O.007 Water 2.OOO Oil-Soluble Composition Preservative(s) 3.250-3700 Lecithin 4.OOO Ceramide IIIB O.1SO Beta-sitosterol O.100 Butylene glycol 4.OOO EXAMPLE 5 Tocopherol O.O10 Water-Soluble Composition 0.137 Table 5 depicts a liposomal suspension formed by Water 27.OOO the general process described in EXAMPLE 1 in conjunction External Phase Composition with several ingredient modifications as indicated below. FIG. 6 displays the resulting unimodal, particle size distribu Glycerin 60.483 Arginine O.OO7 tion of the Table 5 suspension after 2.5 months of shelf life Water 2.OOO under ambient temperature conditions in which the liposomal Preservative(s) 2.2SO Solution was passed through a microfluidizer 2 times.

TABLE 5

EXAMPLE 4 Amount 0.136 Table 4 depicts a liposomal suspension formed by Ingredients (percent) the general process described in EXAMPLE 1 in conjunction Oil-Soluble Composition with several ingredient modifications as indicated below. Lecithin 4.OOO FIGS. 3 and 4 display the resulting unimodal, particle size Ceramide IIIB O.1SO distribution of two liposomal Suspensions according to Table Beta-sitosterol O.100 4 with different preservative combinations after 2.5 months of Butylene glycol 4.OOO Tocopherol O.O10 shelf life under ambient temperature conditions. The liposo Meadowfoam Seed Oil O.1SO mal solution in FIG. 3 was passed through a microfluidizer 4 Soybean Oil O.200 times; the liposomal solution in FIG. 4 was passed through a Evening Primrose Oil O.OSO microfluidizer 2 times. FIG. 3 shows a mean particle size of Perilla Ocymoides Seed Oil O.OSO about 87 nm and a size range between about 38 to about 187 US 2010/0O86573 A1 Apr. 8, 2010

1. A method for preparing a stable unilamellar liposomal TABLE 5-continued Suspension comprising:

Amount a. preparing an oil-soluble composition comprising a cou Ingredients (percent) pling agent, at least one phospholipid, a rigidity enhancer, and an antioxidant; Omega Plus (R) (Sunflower seed O.OSO oil, corn oil, Sesame seed oil, b. preparing a water-soluble composition; macadamia seed oil, olive oil) c. combining the water-soluble composition with the oil Tocopheryl acetate O.OSO soluble composition to form a multilamellar, multive Tetrahexyldecylascorbate O.O2S Water-Soluble Composition sicular liposome preparation; d. converting the multilamellar, multivesicular liposome Butylene glycol 4.OOO preparation to a unilamellar liposome preparation; and Centella asiatica extract O.100 Caffeine O.100 e. adding the unilamellar liposome preparation to an exter Water 22.800 nal phase composition having a density within a range of External Phase Composition about 1.05 g/cc to about 1.25 g/cc and a viscosity between about 2.5 cBandabout 40,000 cBata shear rate Glycerin 60.158 Arginine O.OO7 of 10 sec' at 21° C. to form the liposomal suspension Water 2.OOO comprising a plurality of unilamellar liposomal particles Preservative(s) 2.OOO having a mean particle size between about 50 to about 290 nm, wherein the liposomal Suspension has a refractive index between 1.30 and 1.45 and retains its stability in neat EXAMPLE 6 form at temperatures between about 4°C. to about 50° 0138 Table 6 depicts a liposomal suspension applying the C. for a period of at least30days, or at 21°C. for a period general process described in EXAMPLE 1 in conjunction of at least 180 days. 2. The method of claim 1, wherein the coupling agent is with several ingredient modifications as indicated below. selected from the group consisting of butylene glycol, propy FIG. 7 displays the resulting unimodal particle size distribu lene glycol, hexylene glycol, and polyethylene glycol, and tion of the Table 6 suspension after 2.5 months of shelf life mixtures thereof. under ambient temperature conditions. 3. The method of claim 2, wherein the coupling agent is TABLE 6 butylene glycol. 4. The method of claim 1, wherein the at least one phos Amount pholipid comprises a glycerophospholipid with varying fatty Ingredients (percent) acyl moieties or a sphingolipid. Oil-Soluble Composition 5. The method of claim 1, wherein the at least one phos pholipid comprises a phospholipid preparation comprising Lecithin 3.OOO Beta-sitosterol O.100 phosphatidylcholineata concentration of between about 80% Butylene glycol 3.OOO to about 95% by weight of the phospholipid preparation. Tetrahexyldecylascorbate O.100 Tocopheryl acetate O.200 6. The method of claim 1, wherein the rigidity enhancer is Water-Soluble Composition selected from the group consisting of sphingolipid, phy tosterol, cholesterol, and mixtures thereof. Water 27.OOO 7. The method of claim 1, comprising at least two rigidity Acerola Cherry Ferment O.100 Bearberry extract 6.690 enhancers, including at least one phytosterol and at least one External Phase Composition member selected from the group consisting of ceramide, sph ingosine, and phytosphingosine. Glycerin 37.500 Xanthan gum O.800 8. The method of claim 1, wherein the oil-soluble compo Water 26.9SO sition comprises butylene glycol, ceramide IIIB, B-sitosterol, Preservative(s) 1.OOO and tocopherol. 9. The method of claim 1, wherein the antioxidant is selected from the group consisting of tocopherol, derivatives of tocopherol, derivatives of tocopherol, alpha tocopherol, EXAMPLE 7 tocopheryl acetate, tocopheryl Succinate, ascorbic acid, deri vates of ascorbic acid, tetrahexyldecyl ascorbate, butylated 0139 Liposomal Cream Formulations hydroxyl toluene, butylated hydroxyanisole, and mixtures 0140 FIG. 8 is a TEM micrograph of a liposomal cream thereof. formulation at 11,430x magnification. FIG. 9 is a TEM 10. The method of claim 1, wherein the weight ratio of the micrograph of a liposomal cream formulation taken using a liposome solution to the external phase composition is from Zeiss-902 electron microscope at 30,000x magnification. about 0.002 to about 0.54. Liposomes are identified by arrows. Liposomes are identified 11. The method of claim 1, wherein the liposomal suspen by arrows. In FIGS. 8 and 9, a liposomal suspension was sion comprises glycerin at a concentration between about diluted 1:5 in deionized water and mixed until homogeneous. 35% to about 75% of the liposomal suspension. US 2010/0O86573 A1 Apr. 8, 2010

12. The method of claim 1, wherein the conversion com wherein the external phase composition is at a concentra prises high pressure microfluidization, extrusion, high speed tion between about 30% to about 75% by weight of the shearing, milling, Sonication, selective microchannel filtra liposomal Suspension, the external phase composition tion or homogenization. comprising a density between about 0.95 g/cc and about 13. The method of claim 1, wherein the unilamellar lipo 1.25 g/cc and a viscosity between about 2.5 cFandabout Some preparation is lyophilized prior to Suspension in the 40,000 cF at a shear rate of 10 sec' at 21°C.; and external phase composition. wherein the liposomal Suspension has a refractive index 14. A method for preparing a cosmetic formulation, com between about 1.30 and about 1.45, and retains its sta prising: bility in neat form at a temperature of between about 4 combining the liposomal Suspension of claim 1 with a C. to about 50° C. for a period of at least30days or at 21° cosmetically suitable matrix to form a cosmetic formu C. for a period of at least 180 days. lation in the form of a cream, lotion, gel, serum, tonic, 17. The liposomal suspension of claim 16, wherein the emulsion, paste, or spray. coupling agent is selected from the group consisting of buty 15. A stable unilamellar liposomal Suspension comprising: lene glycol, propylene glycol, hexylene glycol, and polyeth a liposome preparation Suspended in an external phase ylene glycol, and mixtures thereof. composition, the liposome preparation comprising a 18. The liposomal suspension of claim 16, wherein the plurality of unilamellar liposomal particles having a coupling agent is butylene glycol. mean particle size between about 50 nm to about 290 19. The liposomal suspension of claim 16, wherein the at nm, the liposome preparation formed from an aqueous least one phospholipid comprises a glycerophospholipid with liposomal solution comprised of an oil-soluble compo varying fatty acyl moieties or a sphingomyelin. sition and a water-soluble composition, the oil-soluble 20. The liposomal suspension of claim 16, wherein the at composition at a concentration between about 5% to least one phospholipid comprises a phospholipid preparation about 33% by weight of the liposomal solution, the comprising phosphatidylcholine at a concentration of water-soluble composition at a concentration between between about 80% to about 95% by weight of the phospho about 67% to about 95% by weight of the liposomal lipid preparation. Solution, wherein the oil-soluble composition comprises a lecithin 21. The liposomal suspension of claim 16, wherein the at preparation, butylene glycol, ceramide IIIB, and B-sito least one rigidity enhancer is selected from the group consist sterol; ing of sphingolipid, phytosterol, cholesterol, and mixtures wherein the external phase composition comprises glyc thereof. erinata concentration between about 35% to about 75% 22. The liposomal Suspension of claim 16, comprising at by weight of the liposomal Suspension; and least two rigidity enhancers, including at least one phytosterol wherein the liposomal Suspension has a refractive index and at least one member selected from the group consisting of between about 1.30 and about 1.45. ceramide, Sphingosine, and phytosphingosine. 16. A stable unilamellar liposomal Suspension comprising: 23. The liposomal suspension of claim 16, wherein the a liposome preparation Suspended in an external phase oil-soluble composition comprises butylene glycol, ceramide composition, the liposome preparation comprising a IIIB, B-sitosterol, and tocopherol. plurality of unilamellar liposomal particles having a 24. The liposomal suspension of claim 16, wherein the mean particle size between about 50 nm to about 290 antioxidant is selected from the group consisting of toco nm, the liposome preparation formed from an aqueous pherol, derivatives of tocopherol, alpha tocopherol, toco liposomal solution comprised of an oil-soluble compo pheryl acetate, tocopheryl Succinate, ascorbic acid, derivates sition and a water-soluble composition, the oil-soluble of ascorbic acid, tetrahexyidecyl ascorbate, butylated composition at a concentration between about 5% to hydroxyl toluene, butylated hydroxyanisole, and mixtures about 33% by weight of the liposomal solution, the thereof. water-soluble composition at a concentration between 25. The liposomal suspension of claim 16, wherein the about 67% to about 95% by weight of the liposomal liposomal suspension comprises glycerin at a concentration Solution, between about 35% to about 75% of the liposomal wherein the oil-soluble composition comprises a coupling Suspension. agent, at least one phospholipid, at least one rigidity enhancer, and an antioxidant;