[Description] [Invention Title]

POLYMER-LIPOSOME NANO-COMPLEXES AND THE PREPARATION METHOD THEREOF, AND THE COMPOSITION OF SKIN EXTERNAL APPLICATION CONTAINING THE SAME

[Technical Field]

The present invention relates to a polymer-liposome complex using pH-sensitive polymer and the preparation method thereof, and the composition of skin external application containing the same. More particularly, the polymer-liposome complex comprising the pH-sensitive polymer comprised of methacrylic acid and n-alkyl methacrylate and the lipid bilayer has a sensitive response to pH change, so it improves the effect as a carrier by effectively releasing the hydrophobic bioactive compounds embedded inside the lipid layer or the water-soluble bioactive compounds embedded inside the liposome structure or the hydrophilic bioactive compounds solubilized through hydroxypropyl-β-cyclodextrin into an organism. Also, the present invention relates to a polymer- lipid based drug carrier and the composition of skin external application containing the same, which can exist structurally stably in the multi-component system such as an emulsion comprised of an aqueous solution, oil, wax and various surfactant.

[Background Art]

Recently, the studies which prepare more effective lipid based nanocarrier by mixing various lipid components have been getting attention as the rise of usefulness of drug delivery system using liposome. Liposome is one of components being in the spotlight of the way of delivering gene or drug in these days. It is thermodynamically stable in the water-soluble inner nucleus and the lipid vesicle, and it has the lipid bilayer surrounding at least one inner nucleus. In the liposome, the water-soluble drug is included in the continuous lipid bilayer, but the water-insoluble drug is associated with the lipid bilayer itself.

Specially, the studies which diversify the components of the liposome in order to maximize the efficacy of the active component by delivering the bioactive compounds embedded inside the liposome to cell with a high efficiency are performing actively. For example, the preparation of the liposome comprising the lipid introduced the biocompatible polymer in order to increase the bioavailability of active components by extending the circulation time inside the organism, or the studies about i) the liposome comprising the lipid introduced the cell recognition molecule or the cell adhesion molecule in order to deliver high molecular weight drug such as peptide, protein or gene to target cell or ii) the liposome designed for releasing the bioactive compounds

embedded inside the liposome inside the cell due to the decrease in the stability of the liposome by specific condition of small organ inside the cell are proceeding.

Early liposome developed as a drug carrier could not be used as an effective system because of its colloidal and biological instability, but the recent improvement of liposome stability enables the development of anti-bacteria and anticancer liposomal systems. The liposome is also useful in decreasing the toxicity and delivering bioactive compounds for the long run by capturing the compound having the usefulness in itself or having the toxicity not permitted in the therapeutical dosage.

Further, the system releasing the bioactive compounds embedded inside the liposome in response to the change of pH of small organ in the cell has been tried in various ways. For example, dioleoylphosphatidylethanolamine (DOPE) vesicle by introducing the pH-sensitive co-surfactant was widely studied in acidic solution of pH 4.5 ~ 6.5 (Chu et al . , J. Liposome Res., 1994, 4, 361), and it was actually identified that a drug was delivered to cytoplasm of various cells more effectively than other liposomal systems.

As mentioned above, the research about the polymer-liposome complex having the shape that the liposome is surrounded by the polymer was conducted in order to make the various

functions of liposome. That is, the research had an intention to control the release the drug according to the environment of solution by changing the structure of the liposome using the pH-sensitive polymer, and it was started from the early 1980. Early research related to a liposome complex with poly (ethyl acrylic acid) and phospatidylcholine by Tirrell et al . , and they developed a system in which polymer chains are adsorbed on the outer wall of liposome, and some parts of structure of this complex change according to pH of solution (Tirrell et al . , Macromolecules, 1984, 17, 1692). In order to maintain the stability of the liposome, the latest research was designed using flexible chain-structured polymers such as poly(oxy ethylene) among water-soluble polymers. Then the aliphatic chain was embedded into the liposome when the polymer, where long alkyl chain components were slightly added

(less than 10 mol%) , was synthesized and dispersed in water along with the liposome. Among the above systems, in case the main component was the polymer including acrylic acid and other flexible chains, it was suggested that the bilayer structure of the liposome changes depending on the pH of the solution (Francis et al . , Biomacromolecules 2001, 2, 741). However, it was observed that the particle size of the liposome prepared by using said polymer increased with time. It was reported that the particle size of it increased more than 30% after three months (pH 7) .

Until now, in case of the polymer which uses acrylic acid as a single component and comprises some parts of the aliphatic chain, it was disclosed that the liposome cannot be stabilized because of the high rigidity of acrylic acid in aqueous solution (Hwang et al, Langmuir, 2001, 17, 7713) . And, when the liposome is prepared by using the polymer which consists of acrylic acid and the monomer comprising aliphatic chain as a main component, it was disclosed that the stability of such system was not good (Vial et al., Langmuir, 2005, 21, 853) .

Practically, for its maximum efficacy when the efficacy components are brought into the cell, it is more advantageous to release a large amount of the drug into the cell after its structure is totally collapsed than to release slowly after its structure is partially opened at pH 4.5 ~ 5.5, pH of ribosome inside the cell, while maintaining long-term stability under the storage conditions or until the materials reaches the target sites.

Therefore, to increase the delivery efficacy of bioactive compounds and to use the polymer-liposome complex for the composition of cosmetics, it is important to possess the system having the characteristics of : i) releasing the drug by collapsing the structure which is same function with the pH sensitive lipid-cholesterol based liposome and ii) having the long-term structural stability of the polymer-liposome

complex , not alone having an excellent stability than a current liposome.

In the preparation of liposome, a certain ratio of the cholesterol is generally added to lipid for stabilizing the lipid bilayer structure of liposome. It was known by numerous studies that structurally and thertnodynamically more stable spherical lipid bilayer structure of liposome can be formed when the cholesterol was embedded in the lipid bilayer structure. Specially, it was confirmed, by analyzing the release rate of fluorescent dye which was captured in the liposome, that the lipid bilayer of liposome embedded with cholesterol was more stable than the lipid bilayer of liposome without cholesterol (Diana Velluto, et al . , Colloids and Surfaces B: Biointerfaces, 40, 2005, 11-18) . However, it is known that the lipid-cholesterol based liposome also does not have the long-term stability in the water, and it is very unstable in the various salts used in bio systems. Further, the stability inside the composition has to be secured in order to use said compositions for the cosmetics and the skin external applications, but its structure is easy to collapse because of the various surfactants inside the composition.

Generally, drugs or bioactive compounds delivered by using liposome are mostly water-soluble materials hence the drug exists in the lipid vesicle. In delivering the water-

insoluble drugs using liposome, it is reported that the water- insoluble drugs have the similar structures with cholesterol derivatives or cholesterol, so they can be embedded in the lipid bilayer of liposome. Triterpenoids, naturally driven components having similar structure with cholesterol, are a generic name of pentacyclic compound such as ursolic acid, oleanolic acid, betulin etc. These have been known to have an effect for anti-cancer, liver protection, anti-inflammatory, anti-ulcer, anti-bacterial, anti-hyperlipemia, anti-virus, collagen synthesis in skin, promoting the lipid synthesis in skin, injury healing, etc.

The triterpenoid embedded liposome system, like a lipid- cholesterol based liposome, is stable in aqueous solution like a lipid-based liposome or a lipid-cholesterol based liposome. However, the lipid-cholesterol based liposome, in which triterpenoid is embedded, is limited in its use because the vesicle structure becomes unstable in aqueous solution containing various salts used in bio systems or in the cosmetics composition having various surfactants. Further, in the case of the other water-insoluble bioactive compounds, beside triterpenoid, such as flavonoid, the bioactive compounds can not be effectively embedded in the lipid- cholesterol based liposome. Even though it can be embedded, the stability of the lipid-cholesterol based liposome, in which the bioactive compounds is embedded, was usually not

good in aqueous solution.

However, there is a limit in delivering most water- insoluble drugs by associating with lipid bilayers, and as mentioned above, there is no report about the liposome being able to deliver the water-insoluble material except triterpenoid. About 40 percents of candidate drugs of big potential values by having a high bioactivity are not able to get into a development stage because of their low solubility. In consideration of those situations, the skills for using the liposome to various water-insoluble active compounds are seriously needed.

A recent research reported the skill for preparing the liposome after solubilizing the water-insoluble active compounds using cyclodextrin (B. McCormack, G. Gregoriadis, International Journal of Pharmaceutics, 162, 1998, 59-69) . Specially, cyclodextrin has a structure that 5-7 glucose molecules form a cyclic ring and has a hydrophobic cavity inside the molecule. Therefore, it has been widely used for solubilizing various water-insoluble drugs. Moreover, there was a research for the effect of promoting the potential transdermal absorption of cyclodextrin. These results have not clearly proved the capability of cyclodextrin as a carrier through the skin, but it can be supported as a hypothesis that cyclodextrin may improve the absorption of drugs to the inner skin by activating thermodynamic movements of the drug.

However, there are still some problems in the system using cyclodextrin together with liposome. In the case of the lipid-cholesterol based liposome, there are some reports that cyclodextrin, which is used to solubilize the water-insoluble bioactive compounds and to comprise more water-insoluble bioactive compounds in liposome, can destabilize the structure of liposome by affecting the cholesterol or lipid component of the liposome structure (D. G. Fatouros et al . , European Journal of Pharmaceutical Sciences, 13, 2001, 287-296) . Furthermore, the instability of the carrier system using cyclodextrin and liposome was indirectly shown by the following research result, which had no improvement in skin absorption at all even after introducing lipid-cholesterol based liposome to promote the absorption of ketopropen solubilized by cyclodextrin (F. Maestrelli et al . , International Journal of Pharmaceutics, 298, 2005, 55-67) . Therefore, in order to use the liposome in various fields, in which the water-soluble or water-insoluble bioactive compounds are included, the long-term stability of the liposome should be ensured while maintaining the original liposome structure within the formulation including cyclodextrin or various surfactants as well as within the aqueous solution containing diverse salts, without crystallization of water-soluble or water-insoluble bioactive compounds embedded in the liposome. In addition, there is a desperate need to develop a new liposome system that can

improve the carrier role by effectively releasing bioactive compounds within the organism through the liposome carrier having the long-term stability, and that can stably exist within various biosystems, cosmetics and compositions of skin external application.

[Disclosure] [Technical Problem]

To solve the current problems, the present inventors have been performing researches with the target of development of liposome system having the characteristics of : i) having an efficient delivery of various water-soluble and water- insoluble bioactive compounds and ii) being stable without crystallization of the water-insoluble bioactive compounds under various salts, and in cosmetics and skin external application formulation.

In result, the polymer- liposome complex prepared by using the pH-sensitive polymer having the specific composition, and components has the properties of : i) same structure with the lipid or lipid-cholesterol based liposome, ii) increasing the drug delivery efficiency in the body due to the sensitivity to the change in pH, iii) improving the absorption of bioactive compounds into skin, iv) maintaining the long-term stability without crystallization of the water- insoluble bioactive compound in aqueous solution or aqueous solution containing

various salts and v) maintaining the structure of liposome stably without crystallization of the bioactive compound in cosmetics and skin external application formulation.

Moreover, the inventors tried the solublization method of the water-insoluble bioactive compounds using hydroxypropyl-β- cyclodextrin, so that the polymer-liposome complex could deliver the water-insoluble bioactive compounds that were not able to be directly embedded in the lipid bilayer of liposome. The polymer-liposome complex including the water-insoluble bioactive compounds solubilized through hydroxypropyl-β- cyclodextrin maintained the long-term stability without precipitation/crystallization of the water-insoluble bioactive compounds in aqueous solution, prevented the decrease in the concentration of the water-insoluble compounds due to denaturation and showed an improved skin-absorption in several cases, so the present invention was complete.

The present invention is significant in the point that it improved the efficiency of the water-soluble and water- insoluble bioactive compounds by the rapid structural collapse at the specific pH and at the same time it satisfied the requirements of maintaining particle stability without precipitation/crystallization and decrease in the concentration of bioactive compounds under any storing conditions before use, which had not achieved by the precedent

technology. It also solves the problem that various water- insoluble bioactive compounds cannot be effectively embedded in the liposome in the lipid or lipid-cholesterol based liposome system. Also, the present invention has usefulness and meanings to supply the polymer- liposome complex having improved stability in various salts and cosmetics composition relative to the lipid-cholesterol based liposome system and enhanced skin-absorption of the water-soluble and the water- insoluble bioactive compounds .

An object of the present invention is to provide a polymer-liposome complex comprised of the pH-sensitive random copolymer comprising two monomers of methacrylic acid and n- alkyl methacrylate and the lipid bilayer and the preparation method thereof .

Another object of the present invention is to provide a polymer-liposome complex more comprising cholesterol and the preparation method thereof.

Another object of the present invention is to provide a polymer-liposome complex which the water-soluble bioactive compound is included inside the lipid bilayer of the liposome of said polymer- liposome complex.

Another object of the present invention is to provide a polymer-liposome complex which the water-insoluble bioactive compound is embedded inside the lipid bilayer of the liposome

of said polymer-liposome complex.

Another object of the present invention is to provide a polymer-liposome complex which the water-soluble bioactive compound or the water-insoluble bioactive compound solubilized through hydroxypropyl-β-cyclodextrin is included inside the liposome of said polymer-liposome complex.

Another object of the present invention is to provide a polymer-liposome complex having the characteristics of : i) having 50 ~ 400 nm of particle size depending on the concentration of the polymer and the lipid, the water- soluble/insoluble bioactive compound included/embedded in the lipid bilayer or the water-soluble bioactive compound or the water-insoluble bioactive compound, solubilized through hydroxypropyl-β-cyclodextrin, included inside the liposome, ii) maintaining the vesicle shape which is same with the lipid comprising no polymer or the lipid-cholesterol based liposome and iii) forming the structure which the hydrophobic part of the polymer is assembled between lipid, lipid-cholesterol or lipid-water-insoluble bioactive compound based lipid bilayer. Another object of the present invention is to provide a polymer-liposome complex comprising the pH-sensitive polymer which maximizes the efficacy of the active component by improving the effect of the carrier caused by responsing to the change in pH of the target organelle in the cell, collapsing its lipid bilayer and releasing the bioactive

compound after the bioactive compound stably embedded inside the polymer-liposome complex or inside the lipid bilayer of the polymer-liposome complex is moved to the target site, and improves the skin permeability of the bioactive compound. Another object of the present invention is to provide a polymer-liposome complex comprising the polymer which can maintain stable vesicle structure from cosmetics and the skin external application formulation by keeping the vesicle structure stable from salts or surfactants in aqueous system which cause instability of liposome structure caused by assembling with the lipid or the lipid/cholesterol bilayer, tying the bilayer strongly and protecting the outer wall at the same time.

[Technical Solution]

The present invention relates to a polymer- liposome complex using pH-sensitive polymer and the preparation method thereof, and the composition of skin external application containing the same. More particularly, the polymer-liposome complex comprises the pH-sensitive polymer comprising two monomers of methacrylic acid and n-alkyl methacrylate as shown in formula 1 and the liposome structure comprising lipid bilayer, and the water-soluble/water-insoluble bioactive compounds can be included/embedded in the lipid bilayer and the water-soluble bioactive compounds or the water-insoluble

bioactive compounds solubilized through hydroxypropyl-β- cyclodextrin can be embedded inside the liposome structure .

(Formula 1)

n=7~22, mole ratio for χ:y = 90:10 ~ 50:50

In the present invention, the polymer comprising two monomers of methacrylic acid and stearyl methacrylate as shown in formula 2 can be more comprised in poly (methacrylic acid- co-n-alkyl methacrylate) random copolymer of formula 1. In the formula 2, more preferably, n = 0.85 ~ 0.6, m = 0.15 ~ 0.4.

(Formula 2 )

n = 0.99 ~ 0.5,m = 0.01 ~ 0.5

Hereinafter, the present invention is described in detail.

The polymer used in the polymer-liposome complex of the present invention is the pH-sensitive polymer comprising methacrylic acid monomer introduced for the pH-sensitivity and n-alkyl methacrylate monomer introduced for the assembly with

a lipid bilayer of liposome.

Therefore, the polymer-liposome complex comprising said polymer shows a response to pH, so the structural stability is decreased in the range below pH 5.0, and the embedded bioactive compound is released by the collapse of the structure in the lower pH. In the result, said polymer- liposome complex can be used for skin external application. In the case of delivering the bioactive materials using the polymer-liposome complex, it is possible to prepare skin external application maximizing the efficacy of the bioactive compound because it has excellent skin permeability and the embedded bioactive compound is released inside the cell according to the pH change if it is absorbed to the skin.

Generally, it has been known that the polymer prepared by using methacrylic acid changes its structure according to the pH change in aqueous solution, and the range and the degree of the pH-sensitivity depend on the number of alkyl chain present in monomer, the molecular weight of the polymer and the polydispersity index showing the molecular weight distribution of the polymer .

In n-alkyl methacrylate monomer introduced for the assembly with a lipid bilayer of liposome, the length of alkyl chain is decided according to the kind of lipid assembled. If n > 7, that is, alkyl chain is more than octyl, it can assemble with lipid or lipid-cholesterol bilayer, preferably,

n = 11 ~ 21. According to the purposes, more cholesterol can be used in lipid bilayer or the monomer containing cholesterol component in place of n-alkyl chain can be introduced.

The polymer comprising said methacrylic acid monomer and n-alkyl methacrylate monomer is polymerized by the general free radical thermal initiation method, and anionic or cationic polymerization can be used to control the molecular weight distribution. The mixing ratio of methacrylic acid (or acrylic acid, x of formula 1) and n-alkyl methacrylate (or methacrylate substituted with cholesterol, y of formula 1) used in said polymerization is 90 : 10 ~ 50 : 50 in mole ratio, preferably, 85 : 15 ~ 60 : 40. In the complex comprising said methacrylic acid monomer and n-alkyl methacrylate monomer, if n-alkyl methacrylate is less than 10% in mole ratio, there is a possibility that a part of polymer is present independently in aqueous solution and it acts as a surfactant. If n-alkyl methacrylate exceeds 50%, a hydrophobic part of polymer increases, so it can lead to instability in the structure of liposome in the preparation of the polymer-liposome complex. The molecular weight of the polymer comprising said methacrylic acid monomer and n-alkyl methacrylate monomer affects the vesicle size of the polymer-liposome complex and the stability of the polymer-liposome complex, specially, the long-term structural stability. The range of the molecular weight can be used in the polymer-liposome complex is 5,000 ~

200,000 in the number average molecular weight, preferably, 10,000 ~ 50,000.

Stearyl methacrylate introduced for the assembly with a lipid bilayer of liposome in the present invention can be assembled with the lipid bilayer according to the kind of lipid assembled. According to the purposes, the monomer containing cholesterol component in place of stearyl chain can be introduced.

Further, phospholipids or nitrolipids, which have a fatty acid chain of 12 ~ 24 carbons, can be used as the component of lipid bilayer in the preparation of the polymer-liposome complex of the present invention. Generally, the phospholipids are preferable. For example, one or more than two mixture selected from the group consisting of derivatives of the synthetic lipids and fatty acid mixtures obtained by hydrolysis of the synthetic lipids such as dicetylphosphate, distearoylphosphatidylcholine, dioleoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine , dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylserine , eleostearoylphosphatidylcholine , eleostearoylphosphatidylethanolamine and eleostearoylphosphatidylserine, which are the hydrogenation products of natural phospholipids selected from egg yolk

lecithin (phosphatidylcholine) , soybean lecithin, lysolecithin, sphingomyelin, phosphatidic acid, phosphatidylserine, phosphatidylglycerol , phosphatidylinositol , phosphatidylethanolamine, diphosphatidylglycerol, cardiolipin and plastnalogen, is used.

In the case of mixing two phospholipids, the composition such as phosphatidylcholine : phosphatidylethanolamine, phosphatidylcholine : phosphatidylglycerol, phosphatidylcholine : phosphatidylinositol, phosphatidylcholine : phosphatidic acid or phosphatidylcholine : dioleoylphosphatidylethanolamine can be used. The mixing ratio changes according to the components, but the mixing ratio of maximum component compared to minimum component is preferably less than 1: 5. For example, in the case of phosphatidylcholine : dioleoylphosphatidylethanolamine, the range of mole ratio is 5 : 1 - 1 : 5. Therefore, various ratios such as 1 : 1, 2 : 1, 3 : 1, 4 : 1, 5 : 1, 1 : 5, 1 : 4, 1 : 3 or 1 : 2 can be used. Also, in the case of mixing three phospholipids, phosphatidylcholine: dioleoylphosphatidylethanolamine : phosphatidylserine can be used in various ratios such as 1:1:1, 2:1:1, 3:1:2, 3:2:1, 3:2:2, 4:1:1 or 4:2:1 within the range of 1 : 5 mole ratio.

Cholesterol can be more mixed according to need of the polymer-liposome complex of the present invention. The addition of cholesterol disturbs self-assembly of lipid-

polymer bilayer, but it helps the formation of a stable spherical liposome by improving the curvature of liposome.

The ratio of poly (methacrylic acid-co-n-alkyl methacrylate) random copolymer, which is pH-sensitive polymer used for having the pH-sensitivity and maintaining the structural stability in the polymer-liposome complex of the present invention, and lipid is as follows. By weight basis, 1 ~ 50 wt%, preferably 5 ~ 30 wt% of poly (methacrylic acid-co-n- alkyl methacrylate) random copolymer is used, and 50 ~ 99 wt%, preferably 50 ~ 90 wt% of lipid is used of total weight of the vesicle components. Also, by weight basis, 1 - 50 wt% of cholesterol of total weight of the vesicle components can be more comprised.

The content of pH-sensitive polymer and lipid used in the polymer-liposome complex is 0.001 ~ 15 wt%, preferably 1 - 10 wt% compared to polymer-liposome complex aqueous solution.

The water-soluble bioactive compounds or the water- insoluble bioactive compounds solubilized through hydroxypropyl-β-cyclodextrin are embedded inside the polymer- liposome complex. Said water-soluble bioactive compounds are one or more than two whitening, anti-oxidation and anti- wrinkle materials selected from the group consisting of N- butyldeoxynoj irimycin which is known as whitening materials, 1-deoxynoj irimycin, castanospermin, streptomyces culture

extract (SCE) , calcium pentatheine sulfonate, arbutin, vitamin C (ascorbic acid) , ethylascorbyl ether, vitamin C derivative such as sodium ascorbyl phosphate, a-ketoglutaric acid which is known as anti-wrinkle materials and epigallocatechin gallate (EGCG) . The amount of said water-soluble bioactive compounds is 0.001 ~ 10 wt%, preferably 0.01 ~ 5 wt% compared to polymer-liposome complex aqueous solution.

Also, said water-insoluble bioactive compounds solubilized through hydroxypropyl-β-cyclodextrin are selected from the group consisting of rhubarb extract which has an anti-oxidation, skin-stabilizing and whitening effect, rhaponticin which is an indicator ingredient of rhubarb extract and undecylenoyl phenylalanine which is known as whitening materials. The amount of said water-insoluble bioactive compounds is 0.001 ~ 10 wt%, preferably 0.1 - 2 wt% compared to polymer-liposome complex aqueous solution.

Further, the amount of hydroxypropyl-β-cyclodextrin used for solubilizing the water-insoluble bioactive compounds embedded inside the polymer-liposome complex of the present invention is 3 ~ 15 times, preferably 5 - 10 times of the content of water-insoluble bioactive compounds.

If the bioactive compounds embedded in the lipid bilayer of the polymer-liposome complex of the present invention are water-insoluble, it is one selected from the group consisting of triterpenoid of ursolic acid, oleanolic acid, betulinic

acid, betulin and b-boswellic acid having a similar structure with cholesterol, flavonoid of diosmetin, quercetin and genestein, phloretin and drabae semen extract. The amount of said water-insoluble bioactive compounds is 0.001 ~ 5 wt%, preferably 0.1 ~ 2 wt% compared to total polymer-liposome complex.

Liposome is generally prepared by using ultrasonic or high pressure homogenizer. The polymer-liposome complex according to the present invention is prepared using high pressure homogenizer by the steps of : a) mixing poly (methacrylic acid-co-n-alkyl methacrylate) copolymer of formula 1 and lipidto organic solvent which is miscible with water, and heating and dissolving the mixture at 50 ~ 70 ^° C; b) mixing said a) mixture and water heated to 50 ~ 70 °Q and first dispersing the mixture using homogenizer ; and c) obtaining the vesicles from said b) mixture using high pressure homogenizer.

(Formula 1)

In said a) step, cholesterol can be π=7~22, mole ratio for χ:y = 90:10 ~ 50:50 more mixed. The organic

solvent which is miscible with water is not limited, but it is preferable to be selected from ethanol, methanol, isopropyl alcohol, acetone and tetrahydrofuran . Also, the water- insoluble bioactive compound can be mixed in a) step. After a) step, a-1) mixing hydroxy-propyl-β-cyclodextrin and water-insoluble bioactive compound to water, and heating and solubilizing water-insoluble bioactive compound using homogenizer at 60 ~ 70 ^° C ; and a-2) controlling pH of said b) solution more than 7 using acid or base ; can be more comprised.

Further, in b) step, the water-soluble bioactive compound can be comprised in heated water. To obtain the nanoparticles, high pressure homogenizer is used in c) step. Its pressure can be controlled to 100 ~ 1000 bar depending on particle size and dispersity of product, and 500 ~ 1000 bar is preferable.

Also, after c) step, d) step to control the concentration of polymer-liposome complex by removing the remaining organic solvent and water using rotary evaporator can be more comprised depending on the used organic solvent.

The polymer-liposome complex of the present invention obtained by said preparation method has the particle size of 50 ~ 400 nm depending on the concentration of polymer and lipid, the concentration of water-insoluble bioactive compound embedded in lipid bilayer of liposome or the concentration of

water-soluble bioactive compound embedded inside the liposome or the concentration of water-insoluble bioactive compound solubilized through hydroxypropyl-β-cyclodextrin, and maintain same structure and particle form with lipid not comprising polymer or lipid-cholesterol based liposome.

As shown in FIG. 1, the polymer-liposome complex can improve the effect as a carrier by effectively releasing the bioactive compound embedded inside the lipid layer within the cells because it not only has structure which hydrophobic part of polymer is assembled between lipid or lipid/cholesterol based bilayer but also is sensitive response to pH change. The polymer-liposome complex in which the water-insoluble bioactive compound is embedded, as shown in FIG. 2, can improve the effect as a carrier by effectively releasing the water-insoluble bioactive compound embedded inside the lipid bilayer within the cells because it not only has structure which hydrophobic part of polymer is assembled between lipid- water- insoluble bioactive compound based lipid bilayer but also is sensitive response to pH change. Also, as shown in FIG. 2, polymer is assembled with lipid-water- insoluble bioactive compound based lipid bilayer, and keeps the liposome structure stable, by tying the lipid bilayer strongly and protecting the outer wall of liposome at the same time, from salts or surfactants in aqueous solution which cause instability of

liposome structure. In addition, it helps so that the water- insoluble bioactive compounds embedded in the lipid bilayer do not form the crystal in aqueous solution.

Further, as shown in FIG. 3, the water-insoluble bioactive compound solubilized through hydroxypropyl-β- cyclodextrin is embedded in the polymer-liposome complex. The polymer is assembled with lipid-cholesterol based lipid bilayer, and keeps the liposome structure stable from salts or surfactants in aqueous solution which cause instability of liposome structure by tying the lipid bilayer strongly and protecting the outer wall of liposome.

The composition of skin external application comprising polymer-liposome complex of the present invention is not limited in its formulation, and it can be formulated to skin softener, astringent, astringent lotion, nutritious cream, massage cream, eye cream, eye essence, essence, cleansing cream, cleansing lotion, cleansing foam, cleansing water, pack, powder, makeup base, foundation, body lotion, body cream, body oil, body essence, body cleanser, hair dye, shampoo, rinse, toothpaste, mouth wash solution, hair setting agent, hair tonic, lotion, ointment, gel, cream, patch and spray etc.

[Advantageous Effects] As described above, polymer-liposome complex using pH-

sensitive polymer according to the present invention is designed to be capable of controlled release of drugs depending on the change in pH of solution by using the characteristic of changing its structure depending on pH of aqueous solution.

Moreover, polymer-liposome complex of the present invention has a much improved delivery of active components to the body with better efficacy, by effectively releasing bioactive compounds, which are embedded in polymer-liposome complex, within cells since its stability deteriorates rapidly at a particular pH, and shows excellent skin absorption. Additionally, polymer-liposome complex using pH-sensitive polymer according to the present invention has much improved stability in regard to various salts and cosmetics formulation compared to the well-known lipid-cholesterol based liposome system. It has the effect not only to prevent titer decrease of bioactive compounds that are embedded in it but also to improve great skin absorption.

Further, by introducing pH-sensitive polymer, polymer- liposome complex of the present invention could stably embed water-insoluble bioactive compounds, which could be embedded in lipid-cholesterol based liposome, as well as the other water-insoluble bioactive compounds, which could not be embedded, in the lipid bilayer of polymer-liposome complex. It could embed water-insoluble bioactive compounds in the lipid

bilayer, excluding triterpenoid, which could not be embedded in lipid-cholesterol based liposome.

Also, polymer-liposome complex of the present invention could include various water- insoluble bioactive compounds, which were impossible to be embedded in the lipid bilayer of lipid-cholesterol based liposome, by being solubilized through hydroxypropyl-β-cyclodextrin. Regarding various cyclodextrins, it was verified that its structure was relatively stable compared to that of the lipid-cholesterol based liposome, which indicates its high value of practical use.

[Description of Drawings]

FIG. 1 shows the schematic diagram of polymer-liposome complex . FIG. 2 shows the schematic diagram of polymer-liposome complex, in which the water-insoluble bioactive compounds are embedded .

FIG. 3 shows the schematic diagram of polymer-liposome complex, in which the water-insoluble bioactive compounds solubilized through hydroxypropyl-β-cyclodextrin are comprised. FIG. 4 shows the photograph of crystal of betulin in aqueous solution using polarizing microscope.

FIG. 5 shows the photograph of polymer- liposome complex (Example 24), in which the betulin in aqueous solution is embedded, using polarizing microscope.

FIG. 6 shows the photograph of crystal of oleanolic acid in aqueous solution using polarizing microscope.

FIG. 7 shows the photograph of polymer-liposome complex (Example 28) , in which the oleanolic acid in aqueous solution is embedded, using polarizing microscope.

FIG. 8 shows the photograph of crystal of diosmetin in aqueous solution using polarizing microscope.

FIG. 9 shows the photograph of lipid-cholesterol based liposome (Comparative Example 9) , in which the diosmetin in aqueous solution is not embedded effectively, using polarizing microscope.

FIG. 10 shows the photograph of polymer-liposome complex (Example 30) , in which the diosmetin in aqueous solution is embedded, using polarizing microscope. FIG. 11 shows the initial particle size distributions of lipid-cholesterol based liposome comprising the cyclodextrin ( (a) Comparative Example 12 : vesicle-only, (b) Comparative Example 13 : HP-β-CD, (c) Comparative Example 14 : β-CD, (d) Comparative Example 15 : M-β-CD, (e) Comparative Example 16 : DM-β-CD, (f) Comparative Example 17 : TM-β-CD, (g) Comparative Example 18 : γ-CD) .

FIG. 12 shows the initial particle size distributions of polymer- liposome complex comprising the cyclodextrin ( (a)

Example 34: vesicle-only, (b) Example 35 : HP-β-CD, (c) Example 36 : β-CD, (d) Example 37 : M-β-CD, (e) Example 38 : DM-β-CD,

(f) Example 39 : TM-β-CD, (g) Example 40 : γ-CD) .

FIG. 13 shows the particle size distributions at 4 weeks after preparation of lipid-cholesterol based liposome comprising the cyclodextrin ( (a) Comparative Example 12 : vesicle-only, (b) Comparative Example 13 : HP-β-CD, (c) Comparative Example 14 : β-CD, (d) Comparative Example 15 : M- β-CD, (e) Comparative Example 16 : DM-β-CD, (f) Comparative Example 17 : TM-β-CD, (g) Comparative Example 18 : γ-CD) .

FIG. 14 shows the particle size distributions at 4 weeks after preparation of polymer-liposome complex comprising the cyclodextrin ((a) Example 34: vesicle-only, (b) Example 35 : HP-β-CD, (c) Example 36 : β-CD, (d) Example 37 : M-β-CD, (e) Example 38 : DM-β-CD, (f) Example 39 : TM-β-CD, (g) Example 40 : γ-CD) . FIG. 15 shows the heat capacity vs temperature of polymer-liposome complex (Example 5) according to the present invention in aqueous solution.

FIG. 16 shows the heat flow vs temperature when the former lipid-cholesterol based liposome (Comparative Example 2) dispersion is mixed to nanoemulsion composition.

FIG. 17 shows the heat flow vs temperature when the polymer-liposome complex (Example 4) dispersion according to the present invention is mixed to nanoemulsion composition.

FIG. 18 shows the particle size distribution of nanoemulsion of Experimental Example 4.

FIG. 19 shows the particle size distribution of polymer- liposome complex (Example 25) , in which the betulin is embedded, of Experimental Example 4.

FIG. 20 shows the particle size distribution of lipid- cholesterol based liposome (Comparative Example 6) , in which the betulin is embedded, of Experimental Example 4.

FIG. 21 shows the particle size distribution of nanoemulsion composition comprising polymer-liposome complex, in which the betulin is embedded, of Experimental Example 4. FIG. 22 shows the particle size distribution of nanoemulsion composition comprising lipid-cholesterol based liposome of Experimental Example 4.

FIG. 23 shows the photo of structure change vs pH of the former lipid-cholesterol based liposome (Comparative Example 2) and polymer-liposome complex (Example 1 and 4) according to the present invention in aqueous solution of Experimental

Example 5.

FIG. 24 shows the photo of structure change vs pH of the former lipid-cholesterol based liposome (Comparative Example 6) and polymer- liposome complex (Example 25) , in which the betulin is embedded, according to the present invention in aqueous solution of Experimental Example 5.

FIG. 25 shows the skin permeability of arbutin using

Franz-Cell of Experimental Example 7 ( (a) lipid-cholesterol based liposome comprising arbutin, (b) polymer-liposome

complex comprising arbutin, (c) aqueous solution comprising only arbutin) .

FIG. 26 shows the skin permeability of arbutin using

Franz-Cell after mixing with the emulsion of Experimental Example 7 ( (a) lipid-cholesterol based liposome comprising arbutin, (b) polymer-liposome complex comprising arbutin, (c) aqueous solution comprising only arbutin) .

FIG. 27 shows the skin permeability of rhaponticin using Franz-Cell of Example 42 and Comparative Example 28 of the present invention.

FIG. 28 shows the skin permeability of rhaponticin of polymer-liposome complex and nanoemulsion, in which the rhubarb extract solubilized through hydroxypropy1-β- cyclodextrin is comprised, prepared in Example 44 and Comparative Example 31 respectively.

[Mode for Invention]

The present invention is described in more detail based on the following examples and experimental examples. But, these examples are not intended to limit the scope of the present invention.

(Example 1 ~ 17) Preparation of polymer-liposome complex using the pH-sensitive polymer.

The polymer-liposome complex using the pH-sensitive polymer was prepared by the steps of: i) heating and dissolving poly (methacrylic acid-co-n-alkyl methacrylate) copolymer, 100% hydrated oleoyl-palmitoyl/oleoyl-stearyl phosphatidylcholine mixture (Lipoid S100-3) and cholesterol to ethanol (60 ^° C), ii) adding said mixture to water (60 ^° C), iii) preparing the first dispersed polymer-liposome complex by agitating said mixture using homogenizer at 5000 rpm for 5 minutes, iv) obtaining the nanoparticles from said mixture using high pressure homogenizer (1000 bar, 3 cycle) and v) removing the remaining ethanol solution using rotary evaporator. Table 1 shows the compositions (weight basis) of said components .

(Table 1)

(Example 18 ~ 23) Preparation of polymer-liposome complex comprising the water-soluble bioactive compound.

The polymer-liposome complex comprising the water-soluble bioactive compound was prepared by the steps of: i) heating and dissolving poly (methacrylic acid-co-n-alkyl methacrylate) copolymer (mole ratio = 70 : 30, number average molecular

weight = 26,000), 100% hydrated oleoyl-palmitoyl/oleoyl- stearyl phosphatidylcholine mixture (Lipoid S100-3) , cholesterol to ethanol, ii) dissolving water-soluble bioactive compound to 300 g of water and heating it to 60 ^° C, iii) adding said mixture to bioactive compound aqueous solution heated to 60 ^° C, iv) preparing the first dispersed polymer-liposome complex by agitating said mixture using homogenizer at 5000 rpm for 5 minutes, v) obtaining the nanoparticles from said mixture using high pressure homogenizer (1000 bar, 3 cycle) and vi) removing the remaining ethanol solution using rotary evaporator. Table 2 shows the weight compositions of said components .

(Table 2) The components and the compositions used in polymer-liposome complex comprising the water-soluble bioactive compound.

(Example 24 ~ 33) Preparation of pH-sensitive polymer- liposome complex, in which the water-insoluble bioactive compound is embedded.

The pH-sensitive polymer-liposome complex, in which the water-insoluble bioactive compound is embedded, was prepared by the steps of : i) heating and dissolving poly (methacrylic acid-co-stearyl methacrylate) copolymer (mole ratio = 70 : 30, number average molecular weight = 26,000), 100% hydrated oleoyl-palmitoyl/oleoyl-stearyl phosphatidylcholine mixture (Lipoid S100-3) and water-insoluble bioactive compound to organic solvent (60 ^° C), ii) adding said mixture to water (60 ^° C), iii) preparing the first dispersed polymer-liposome complex by

agitating said mixture using homogenizer at 5000 rpm for 5 minutes, iv) obtaining the nanoparticles from said mixture using high pressure homogenizer (1000 bar, 3 cycle) , v) removing the remaining organic solvent using rotary evaporator and vi) removing some water by distillation to control the concentration of the water-insoluble bioactive compound and the polymer-liposome complex.

In Example 33, the pH-sensitive polymer-liposome complex, in which the water-insoluble bioactive compound is embedded, was prepared by using the same method of Example 24 ~ 32 except using poly (methacrylic acid-co-stearyl methacrylate) copolymer (mole ratio = 85 : 15, number average molecular weight = 20,000) . Table 3 shows the weight compositions of said components .

(Table 3) The components and the compositions used in polymer-liposome complex, in which the water-insoluble bioactive compound is embedded.

(Example 34 ~ 40) Preparation of polymer-liposome complex comprising various cyclodextrins .

The polymer-liposome complex comprising the various cyclodextrins was prepared by the steps of : i) dissolving

cyclodextrin to purified water and heating it to 60 ^° C, ii) heating and dissolving poly (methacrylic acid-co-stearyl methacrylate) copolymer (mole ratio = 70 : 30, number average molecular weight = 26,000), 100% hydrated oleoyl- palmitoyl/oleoyl-stearyl phosphatidylcholine mixture (Lipoid S100-3) and cholesterol to organic solvent (60 ^° C), iii) adding said mixture to cyclodextrin aqueous solution and heating it to 60 ^° C, iv) preparing the polymer-liposome complex comprising the cyclodextrin by agitating said mixture using homogenizer at 5000 rpm for 5 minutes, v) obtaining the nanoparticles from said mixture using high pressure homogenizer (1000 bar, 3 cycle) , vi) removing the remaining organic solvent using rotary evaporator and vii) removing some water by distillation to control the concentration of the polymer- liposome complex. Table 4 shows the weight compositions of said components.

(Table 4) The components and the compositions of polymer- liposome complex comprising the various cyclodextrins .

1) hydroxypropyl-β-cyclodextrin

2) β-cyclodextrin

3) methyl-β-cyclodextrin

4 ) ditnethyl-β-cyclodextrin 5) trimethyl-β-cyclodextrin

6) γ-cyclodextrin

(Example 41 ~ 45) Preparation of polymer-liposome complex, in which the water-insoluble bioactive compound solubilized through hydroxypropyl-β-cyclodextrin is comprised.

The polymer- liposome complex, in which the water- insoluble bioactive compound solubilized through

hydroxypropyl-β-cyclodextrin is comprised, was prepared by the steps of: i) adding hydroxypropyl-β-cyclodextrin, water- insoluble bioactive compound and acidic or basic compound to purified water and heating it to 60 ^° C, ii) solubilizing the water-insoluble bioactive compound by agitating said mixture using homogenizer at 7000 rpm for 10 minutes, iii) heating and dissolving poly (methacrylic acid-co-stearyl methacrylate) copolymer (mole ratio = 70 : 30, number average molecular weight = 26,000), 100% hydrated oleoyl-palmitoyl/oleoyl- stearyl phosphatidylcholine mixture (Lipoid S100-3) and cholesterol to organic solvent (60 ^° C), iv) adding said mixture to aqueous solution containing water-insoluble bioactive compound and heating it to 60 ^° C , v) preparing the polymer- liposome complex comprising the water- insoluble bioactive compound solubilized through hydroxypropyl-β-cyclodextrin by agitating said mixture using homogenizer at 5000 rpm for 5 minutes, vi) obtaining the nanoparticles from said mixture using high pressure homogenizer (1000 bar, 3 cycle) , vii) removing the remaining organic solvent using rotary evaporator and viii) removing some water by distillation to control the concentration of the water-insoluble bioactive compound and the polymer-liposome complex. Table 5 shows the weight compositions of said components.

(Table 5) The components and the compositions of polymer-

liposome complex, in which the water-insoluble bioactive compound solubilized through hydroxypropyl-β-cyclodextrin is comprised.

Active Composition (g) Tota compone 1 nt weig ht afte r dist ilia tion

(g) pol chol Ii acti hyd eth acid wat yme este Pi ve rox ano or er r rol d comp ypr 1 base onen opy t 1- β- eye lod ext rin

Ex. rhapont 0.3 0.07 0. 0.5 5 30 0 300 50

41 icin 8 62

(Comparative Example 1 ~ 5) Preparation of liposome comprised of lipid-cholesterol.

The liposome comprised of lipid-cholesterol was prepared by the steps of: i) heating and dissolving 100% hydrated oleoyl-palmitoyl/oleoyl-stearyl phosphatidylcholine mixture

(Lipoid S100-3) , cholesterol, ii) adding said mixture to water (60 ^° C) or bioactive compound aqueous solution (60 ^° C), iii) obtaining the nanoparticles from said mixture using high pressure homogenizer (1000 bar, 3 cycle) after agitating said mixture using homogenizer at 5000 rpm for 5 minutes and iv) removing the remaining ethanol solution using rotary evaporator. Table 6 shows the weight compositions of said components .

(Table 6)

(Comparative Example 6 ~ 11) Preparation of lipid-cholesterol based liposome, in which the water-insoluble bioactive compound is embedded.

The lipid-cholesterol based liposome, in which the water- insoluble bioactive compound is embedded, was prepared by the steps of: i) heating and dissolving 100% hydrated oleoyl- palmitoyl/oleoyl-stearyl phosphatidylcholine mixture (Lipoid S100-3) , water-insoluble bioactive compound and cholesterol to organic solvent (60°C), ii) adding said mixture to water (60 ^° C), iii) preparing the first dispersed lipid-cholesterol based liposome, in which the water-insoluble bioactive compound is embedded, by agitating said mixture using homogenizer at 5000 rpm for 5 minutes, iv) obtaining the nanoparticles from said mixture using high pressure homogenizer (1000 bar, 3 cycle) , v) removing the remaining organic solvent using rotary evaporator and vi) removing some water by distillation to control the concentration of the water-insoluble bioactive compound and the lipid-cholesterol based liposome. Table 7 shows the weight compositions of said components.

(Table 7) The components and the compositions used in lipid-cholesterol based liposome, in which the water-insoluble bioactive compound is embedded.

(Comparative Example 12 ~ 18) Preparation of lipid-cholesterol based liposome comprising various cyclodextrins .

The lipid-cholesterol based liposome comprising the various cyclodextrins was prepared by the steps of: i) dissolving cyclodextrin to purified water and heating it to 60 ^° C, ii) heating and dissolving cholesterol and 100% hydrated oleoyl-palmitoyl/oleoyl-stearyl phosphatidylcholine mixture (Lipoid S100-3) to organic solvent (60 ^° C), iii) adding said mixture to cyclodextrin aqueous solution and heating it to 60 ^° C, iv) preparing the lipid-cholesterol based liposome comprising the cyclodextrin by agitating said mixture using homogenizer at 5000 rpm for 5 minutes, v) obtaining the nanoparticles from said mixture using high pressure homogenizer (1000 bar, 3 cycle) , vi) removing the remaining organic solvent using rotary evaporator and vii) removing some water by distillation to control the concentration of the lipid-cholesterol based liposome. Table 8 shows the weight compositions of said components .

(Table 8) The components and the compositions of lipid- cholesterol based liposome comprising the various cyclodextrins .

1) hydroxypropyl-β-cyclodextrin

2) β-cyclodextrin

3) methyl-β-cyclodextrin

4) dimethyl-β-cyclodextrin

5) trimethyl-β-cyclodextrin

6) γ-cyclodextrin

(Comparative Example 19 ~ 21) Preparation of polymer-liposome complex, in which the water-insoluble bioactive compound is directly embedded without using hydroxypropyl-β-cyclodextrin.

In Comparative Example 20 ~ 21, the polymer- liposome complex, in which the water-insoluble bioactive compound is directly embedded without using hydroxypropyl-β-cyclodextrin, was prepared by the steps of: i) heating and dissolving poly (methacrylic acid-co-stearyl methacrylate) copolymer (mole ratio = 70 : 30, number average molecular weight = 26,000), cholesterol, 100% hydrated oleoyl-palmitoyl/oleoyl-stearyl phosphatidylcholine mixture (Lipoid S100-3) and water- insoluble bioactive compound to organic solvent (60 ^° C), ii) adding said mixture to water comprising acid or base (60 ^° C), iii) preparing the first dispersed polymer-liposome complex, in which the water-insoluble bioactive compound is embedded, by agitating said mixture using homogenizer at 5000 rpm for 5 minutes, iv) obtaining the nanoparticles from said mixture using high pressure homogenizer (1000 bar, 3 cycle) , v) removing the remaining organic solvent using rotary evaporator and vi) removing some water by distillation to control the concentration of the water-insoluble bioactive compound and the polymer-liposome complex.

In Comparative Example 19, the pH-sensitive polymer-

liposome complex, in which the water-insoluble bioactive compound is embedded, was prepared by using the same method of Comparative Example 20 ~ 21 except using poly (methacrylic acid-co-stearyl methacrylate) copolymer (mole ratio = 85 : 15, number average molecular weight = 20,000). Table 9 shows the weight compositions of said components.

(Table 9) The components and the compositions used in polymer-liposome complex, in which the water-insoluble bioactive compound is directly embedded without using hydroxypropy1-β-cyclodextrin.

(Comparative Example 22 ~ 26) Preparation of lipid-cholesterol based liposome, in which the water-insoluble bioactive compound solubilized through hydroxypropyl-β-cyclodextrin is comprised.

The lipid-cholesterol based liposome, in which the water- insoluble bioactive compound solubilized through hydroxypropyl-β-cyclodextrin is comprised, was prepared by the steps of: i) adding hydroxypropyl-β-cyclodextrin, water- insoluble bioactive compound and acidic or basic compound to purified water and heating it to 60 ^° C, ii) solubilizing the water-insoluble bioactive compound by agitating said mixture using homogenizer at 7000 rpm for 10 minutes, iii) heating and dissolving cholesterol and 100% hydrated oleoyl-

palmitoyl/oleoyl-stearyl phosphatidylcholine mixture (Lipoid S100-3) to organic solvent (60 ^° C), iv) adding said mixture to water-insoluble bioactive compound aqueous solution and heating it to 60 ^° C, v) preparing the lipid-cholesterol based liposome comprising the water-insoluble bioactive compound solubilized through hydroxypropyl-β-cyclodextrin by agitating said mixture using homogenizer at 5000 rpm for 5 minutes, vi) obtaining the nanoparticles from said mixture using high pressure homogenizer (1000 bar, 3 cycle) , vii) removing the remaining organic solvent using rotary evaporator and viii) removing some water by distillation to control the concentration of the water-insoluble bioactive compound and the lipid-cholesterol based liposome. Table 10 shows the weight compositions of said components.

(Table 10) The components and the compositions of lipid- cholesterol based liposome, in which the water-insoluble bioactive compound solubilized through hydroxypropyl-β- cyclodextrin is comprised.

(Comparative Example 27 ~ 30) Preparation of water-insoluble bioactive compound aqueous solution solubilized through hydroxypropy1-β-eyelodextrin .

The water-insoluble bioactive compound aqueous solution was prepared by the steps of : i) adding hydroxypropyl-β- cyclodextrin, water-insoluble bioactive compound and acidic or basic compound to purified water and heating it to 60 ^° C and ii) solubilizing the water-insoluble bioactive compound by agitating said mixture using homogenizer at 7000 rpm for 10 minutes. Table 11 shows the weight compositions of said components .

(Table 11) The components and the compositions of water- insoluble bioactive compound solubilized through hydroxypropyl-β-eyelodextrin .

(Comparative Example 31) Preparation of nanoemulsion, in which 1 wt% of the rhubarb extract is comprised.

The nanoemulsion, in which 1 wt% of the rhubarb extract is comprised, was prepared as shown in Table 12 in order to compare the titer decrease and the skin permeability of polymer- liposome complex and lipid-cholesterol based liposome comprising 1 wt% of the rhubarb extract prepared according to Example 44 and Comparative Example 29.

(Table 12) The components and the contents of

nanoemulsion, in which the rhubarb extract is comprised.

(Experimental Example 1) The measurement of the particle size and analysis on polymer-liposome complex and liposome.

The particle size of the followings were measured through the dynamic light scattering, Zetasizer 3000HSa Malvern Instruments : polymer-liposome complex using the pH-sensitive polymer (Example 1 ~ 17) , polymer-liposome complex comprising the water-soluble bioactive compound (Example 18 ~ 23), polymer-liposome complex comprising the water-insoluble bioactive compound (Example 24 ~ 33) , polymer-liposome complex comprising various cyclodextrins (Example 34 ~ 40) , polymer- liposome complex, in which the water-insoluble bioactive

compound solubilized through hydroxypropyl-β-cyclodextrin is comprised (Example 41 ~ 45) , lipid and lipid-cholesterol based liposome (Comparative Example 1 ~ 5) , lipid-cholesterol based liposome comprising the water-insoluble bioactive compound (Comparative Example 6 ~ 11) , lipid-cholesterol based liposome comprising various cyclodextrins (Comparative Example 12 ~ 18) , polymer-liposome complex, in which the water-insoluble bioactive compound is directly embedded without using hydroxypropyl-β-cyclodextrin (Comparative Example 19 ~ 21) and lipid-cholesterol based liposome, in which the water-insoluble bioactive compound solubilized through hydroxypropyl-β- cyclodextrin is comprised (Comparative Example 22 ~ 26) . The scattering angle was fixed to 90°, and the temperature was kept at 25 ^° C during the measurement. The average particle diameter was calculated by the Stokes-Einstein equation. The results are shown in Table 13.

(Table 13) The measurement of the particle size of polymer-liposome complex and lipid-cholesterol based liposome dispersed in aqueous solution.

ion

Ex. 2 200 Ex. 28 254 Com Ex. 2 150

Ex. 3 240 Ex. 29 249 Com Ex. 3 175

Ex. 4 225 Ex. 30 312 Com Ex. 4 170

Ex. 5 250 Ex. 31 243 Com Ex. 5 112

Ex. 6 150 Ex. 32 273 Com Ex. 6 109

Ex. 7 80 Ex. 33 420 Com Ex. 7 127

Ex. 8 230 Ex. 34 197 Com Ex. 8 128

Ex. 9 300 Ex. 35 193 Com Ex. 9 precipitat ion

Ex. 270 Ex. 36 189 Com Ex. 10 precipitat 10 ion

Ex. 420 Ex. 37 185 Com Ex. 11 precipitat 11 ion

Ex. precipit Ex. 38 197 Com Ex. 12 121 12 ation

Ex. 245 Ex. 39 191 Com Ex. 13 105 13

Ex. 230 Ex. 40 197 Com Ex. 14 122 14

Ex. precipit Ex. 41 132 Com Ex. 15 110 15 ation

Ex. precipit Ex. 42 115 Com Ex. 16 138 16 ation

Ex. precipit Ex. 43 195 Com Ex. 17 109

As shown in Table 13, it was found that the particle size polymer-liposome complex comprising the pH-sensitive

polymer (Example 1 ~ 23) was larger than that of liposome not comprising the pH-sensitive polymer (Comparative Example 1 ~ 5) . Also, in polymer-liposome complex of Example 1 - 23, when the amount of polymer was increased, its particle size was increased. From the particle sizes (150 nm & 80 nm) of polymer-liposome complex (Example 6 and 7) , it was found that the particle size depended on the concentration of lipid, cholesterol and polymer contained in ethanol .

Moreover, the particle size was not dramatically changed depending on the increase in the molecular weight of poly (methacrylic acid-co-n-alkyl methacrylate) , but it was observed that in case of polymer-liposome complex (number average molecular weight = 68,000) of Example 9, its particle size (300 nm) slightly increased compared to those of Example 4 and 8 using polymer having the molecular weight of 17,000 and 26,000.

Particularly, Comparative Example 1 failed to prepare the liposome due to the precipitation while trying to prepare the liposome only with phosphatidylcholine. In contrast, it was found that the particles were successfully formed with the aid of polymer such as Example 3 even when vesicle was prepared with only phosphatidylcholine . It is presumed that said result was because the polymer played a good role in forming spherical bilayer of lipid. Also, in regard to the particle size of pH-sensitive

polymers used in Example 1 - 17 depending on the length (n) of the alkyl chain of n-alkyl methacrylate, the particle sizes were similar to the case of lauryl (n = 11) in Exmaple 10 and stearyl (n = 17) in Example 1 - 9 and 14 - 17. On the contrary, octyl (n = 7) in Example 11 had great increase in the particle size and butyl (n = 3) in Example 12 had a precipitation phenomenon without forming particles. Also, Example 13, which used cholesterol instead of n-alkyl methacrylate, had a similar particle size to lauryl (n = 11) in Example 10 and stearyl (n = 17) in Example 1 - 9 and 14 - 17.

In addition, regarding the suitable mole ratio of methacrylic acid and stearyl methacrylate, since precipitation was made in Example 15 - 17which formed particles from polymer-liposome complex comprising different mole ratio of methacrylic acid and stearyl methacrylate, it was verified that among complexes consisted of methacrylic acid and stearyl methacrylate, the mole ratio of stearyl methacrylate was between 10% and 50%. It was also found that the particle formation of polymer-liposome complex was greatly affected by the mole ratio of methacrylic acid and stearyl methacrylate, which composed the above poly (methacrylic acid-co-n-alkyl methacrylate) copolymer, and the length (n) of the alkyl chain of n-alkyl methacrylate.

In the polymer-liposome complexes comprising the water- soluble active compounds (Example 18 - 23) , the change in

particle sizes depending on the kind of active compounds was not nearly observed in the same composition and they showed almost the same particle sizes with the polymer-liposome complex of Example 1 ~ 17, which did not comprise active compounds .

As shown in Table 13 above, the polymer-liposome complex

(Example) where the water-insoluble bioactive compounds are embedded had more stable form of liposome with a fixed particle size compared to the lipid-cholesterol based liposome (Comparative Example) where the water-insoluble bioactive compounds are embedded.

The polymer-liposome complexes comprising triterpenoid such as betulin, ursolic acid, oleanolic acid, etc. (Example 24 ~ 29) and lipid-cholesterol based liposome (Comparative Example 6 - 8) had formed stable liposome with particle sizes, because triterpenoid, different from other water-insoluble bioactive compounds, has similar molecular shape to cholesterol and is embedded in the lipid bilayer of liposome as shown in FIG. 2, so that it helps the formation of structurally and thermodynamically more stable spherical lipid bilayer structure of liposome .

On the other hand, the particle size of polymer-liposome complex comprising pH-sensitive polymer (Example) was larger than that of lipid-cholesterol based liposome not comprising the pH-sensitive polymer (Comparative Example) .

Also, the polymer-liposome complexes of Example 30 ~ 33, in which the water-insoluble bioactive compounds such as diosmetin, phloretin, drabae semen extract except triterpenoid are embedded, had fixed particle sizes and made the stable form of liposome. However, the lipid-cholesterol based liposome of Comparative Example 9 - 11 had a precipitation phenomenon and the liposome comprising the water-insoluble bioactive compound was not formed.

Therefore, it was figured that the pH-sensitive polymer comprised in polymer-liposome complex of the present invention played a supporting role in keeping the lipid bilayer stable, not alone with a pH-sensitivity to the lipid bilayer of liposome as shown in FIG. 2.

FIG. 4 - 10 shows the photographs of betulin crystal in aqueous solution, Example 24, oleanolic acid crystal in aqueous solution, Example 28, diosmetin crystal in aqueous solution, Comparative Example 9 and Example 30 using polarizing microscope. From FIG. 4 - 10, it was observed that betulin, oleanolic acid and diosmetin remained as crystals in aqueous solution and the polymer-liposome complex where betulin, oleanolic acid, and diosmetin were embedded (Example 24, 28 and 30) did not form the crystals of the above betulin, oleanolic acid and diosmetin in aqueous solution. Also, diosmetin was not effectively embedded in the lipid- cholesterol based liposome, so its crystal was formed.

FIG. 4 - 10 shows that the water-insoluble bioactive compounds embedded in the polymer-liposome complex were well dispersed in the bilayer without crystallization. Accordingly, it was found that water-insoluble bioactive compounds were stably collected in the lipid bilayer of polymer- liposome complex .

In comparison of the size between the polymer-liposome complex comprising various cyclodextrins in Example 34 ~ 40 and the lipid-cholesterol based liposome comprising various cyclodextrins in Comparative Example 12 ~ 18, it was observed that the polymer-liposome complex comprising various cyclodextrins in Example 34 ~ 40 had larger particles than the lipid-cholesterol based liposome in Comparative Example 12 ~ 18, just as the polymer-liposome complex not comprising cyclodextrin.

FIG. 11 and FIG. 12 show the particle size distributions of the as-prepared lipid-cholesterol based liposome of Comparative Example 12 ~ 18 and the polymer-liposome complex of Example 34 ~ 40. In the initial stage of preparation, it was found that stable vesicles were formed in polymer- liposome complex (FIG. 12 (a) ~ (g) ) and all liposomes (FIG. 11 (a) ~ (d) , (f) and (g) ) except lipid-cholesterol based liposome comprising dimethyl-β -cyclodextrin (DM-β-CD) . Also, FIG. 13 and FIG. 14 show the particle size

distributions at 4 weeks after preparation of lipid- cholesterol based liposome of Comparative Example 12 ~ 18 and polymer-liposome complex of Example 34 ~ 40.

As shown in FIG. 13, it was observed that lipid-cholesterol based liposome (Comparative Example 14, 16 and 18) comprising some cyclodextrin (β-CD, DM-β-CD and γ-CD) had started to have unstable structure, and then their particle sizes became larger or their particles were precipitated. However, as shown in FIG. 14, the polymer-liposome complex had formed and kept long-term stable structure regarding all cyclodextrins . Accordingly, it was found that in case of vesicle comprising cyclodextrin, the vesicle prepared by using polymer-liposome complex had much greater structural stability than lipid- cholesterol based vesicle.

FIG. 15 shows the thermal property, which was measured by microcalorimetry, on polymer-liposome complex of Example 5 and liposome of Comparative Example 4. Liposome of Comparative Example 4 showed the transition point of gel to liquid crystal at around 50 ^° Q but polymer-liposome complex of Example 5 showed it at around 43 ^° C Having the transition point of gel to liquid crystal in polymer-liposome complex indicates that the self-assembly structure is being kept, which is the characteristics of bilayer. That means the aliphatic chain of stearyl methacrylate, a component of pH-sensitive polymer of

polymer-liposome complex prepared according to the present invention, is self-assembled with lipid components. Thus, as shown in FIG. 1, it was found that the pH-sensitive polymer used in polymer-liposome complex keeps liposome's unique property by assembling with lipid-cholesterol and covers the wall of bilayers.

Further, as shown in Table 14, it was observed that polymer-liposome complex comprising various cyclodextrins prepared through Example 35 ~ 40, showed the same transition temperature with polymer-liposome complex not comprising cyclodextrin (Example 34) . As a result, it can be thought that the existence and the kind of cyclodextrin does not influence on the physical property of polymer- liposome complex.

(Table 14) The transition temperature of polymer-liposome complex comprising various cyclodextrins.

1) hydroxypropyl-β-cyclodextrin

2) β-cyclodextrin

3) methyl-β-cyclodextrin

4) dimethyl-β-cyclodextrin 5) trimethyl-β-cyclodextrin 6) γ-cyclodextrin

As shown in Table 13, the polymer-liposome complex of Example 41 ~ 45 could obtain the stable vesicle aqueous solution with the particle sizes of 115 ~ 195 nm. On the other hand, the polymer-liposome complex of Comparative Example 19 ~ 21 shown in Table 13 could not obtain the polymer-liposome complex aqueous solution, and the polymer-liposome complex itself was not well formed along with the crystal formation of the water-insoluble bioactive compound. Also, it can be known that the particle size of polymer-liposome complex changed depending on the kind of water-insoluble bioactive compounds, which were solubilized through hydroxypropyl-β-cyclodextrin, and the concentration of the components of polymer-liposome complex. In addition, as presented in Table 13, through the particle size of lipid-cholesterol based liposome of Comparative Example 22 ~ 26, as the concentration of water- insoluble bioactive compounds, which were solubilized through hydroxypropyl-β-cyclodextrin, got higher, the stable liposome was not formed but the precipitation was made, which was

different from the case of polymer-liposome complex. On the contrary, in Table 11, the water-insoluble bioactive compounds, which were solubilized through hydroxypropyl-β-cyclodextrin in aqueous solution of Comparative Example 27 ~ 30, did not show any of crystal or flotage but rather a pure and transparent aqueous solution with a particular color. This indicates that the water-insoluble bioactive compounds are dispersed in aqueous solution as molecules by hydroxypropyl-β-cyclodextrin. From the result as shown in FIG. 3, it was found that when the water-insoluble bioactive compounds, which were solubilized through hydroxypropyl-β-cyclodextrin, was made to liposome using polymer- liposome complex, the water-insoluble bioactive compounds, which were solubilized through cyclodextrin, was embedded inside of polymer-liposome complex. Therefore, polymers kept the liposome structure stable from various elements that made the liposome structure unstable in aqueous solution by assembling with the lipid-cholesterol based lipid bilayer, tying the lipid bilayer strongly and protecting the outer wall of liposome.

(Experimental Example 2) The stability evaluation of polymer- liposome complex.

Table 15 below shows the change in particle sizes of the solutions by the time, using dynamic light scattering,

Zetasizer 3000HSa Malvern Instruments. The solutions were kept at low temperature (2 ^° C ) and at room temperature (25 ^° C ) respectively in order to observe the long-term stability of polymer-liposome complex of Example 1, 4, 10, 11 and 19 and lipid-cholesterol based liposome in Comparative Example 2 and 3.

Also, Table 15 below shows the change in particle sizes of the solutions by the time, using dynamic light scattering, Zetasizer 3000HSa Malvern Instruments. The solutions were kept at low temperature (2°C ), at room temperature (25 ^° C ) and at high temperature (40 "C ) respectively in order to observe the long-term stability of polymer- liposome complex, in which the water-insoluble bioactive compounds were embedded (Example 24, 25, 30 and 31) , polymer-liposome complex, in which the water- insoluble bioactive compounds solubilized through hydroxypropyl-β-cyclodextrin were embedded (Example 41 ~ 44) and lipid-cholesterol based liposome, in which the water- insoluble bioactive compounds were embedded (Comparative Example 6) .

(Table 15) The measurement of long-term stability of the particle size of polymer-liposome complex and lipid- cholesterol based liposome dispersed in aqueous solution.

Particle size (nm)

As shown in Table 15, it was found that in case of polymer-liposome complex using poly (methacrylic acid-co-n- alkyl methacrylate) copolymer, the stability of its particle became different depending on the length of the alkyl chain that was used in n-alkyl methacrylate. In Example 11, octyl (n = 7) formulated a large particle with the size of 420 nm, but formed precipitation after three weeks of storage at low temperature and room temperature, so it was confirmed that it had very little long-term stability of particle compared to other examples .

Also, it was found that in case of polymer-liposome complex that used stearyl (n = 17) and lauryl (n = 11) methacrylate as monomers (Example 1, 4, and 10), its particle

sizes had not much increased even when kept at low and room temperature for twelve months as well as eight weeks, as shown in Table 15 above. The above findings can indicate that polymer addition makes the liposome structure unstable when the length of the alkyl chain is short as described in the precedent records. It was also discovered that in order to prepare the polymer-liposome complex having an excellent stability, there should be the unique composition of the polymer and the special solution properties of the polymer in aqueous solution.

In comparison of stability between lipid-cholesterol based liposome comprising streptomyces extract (Comparative Example 3) and polymer-liposome complex (Example 19) , Comparative Example 3 had the gradually increased particle size at low and room temperature as the storage time got longer. It formed some precipitation after eight weeks, but Example 19 with the same amount of streptomyces extract had the nearly unchanged particle size after eight weeks. Thus, it was observed that polymer-liposome complex of the present invention, when it included active components, kept more stable structure in the long run than lipid-cholesterol based liposome .

Further, as shown in Table 15, regardless of the kind of

water-insoluble bioactive compounds, which are embedded in polymer-liposome complex and lipid-cholesterol based liposome, polymer-liposome complex in Example 24, 25, 30 and 31 and lipid-cholesterol based liposome in Comparative Example 6 both did not have much increased particle sizes compared to as- prepared samples even when they were kept at 2 ^° Cand 25 ^° C They showed long-term stability in aqueous solution at 2 ^° Cand 25 ^° C

On the other hand, polymer-liposome complex in Example 24, 25, 30 and 31 and lipid-cholesterol based liposome in Comparative Example 6, which were kept in aqueous solution at 40 ^° C had relatively increased particle sizes compared to any other temperatures. This might be resulted from the volume expansion of liposome by loosening of self-assembly of lipid bilayer of vesicles at high temperature of 40°C compared to low temperature (2 "C) and room temperature (25 "C).

On the contrary, as shown in Table 15, there was not a big change in particle sizes by the time when the polymer- liposome complex of Example 41 ~ 44, in which the water- insoluble bioactive compound solubilized through hydroxypropyl-β-cyclodextrin was embedded, were kept at 2°Cand 25 ^° C

On the other hand, it was observed that the particle size of polymer-liposome complex of Example 41 ~ 44, which was stored at 40 ^° C relatively increased compared to other

temperatures. This might be resulted from the volume expansion of liposome by loosening of self-assembly of lipid bilayer of vesicles at high temperature of 40 "C compared to low temperature (2 "C) and room temperature (25 "C). Also, the stability of polymer-liposome complex having cyclodextrin was confirmed by the fact that the particle size of polymer-liposome complex in aqueous solution along with hydroxypropyl- β-cyclodextrin (Example 41 ~ 44) stayed unchanged in the long time. It is considered that polymer in polymer-liposome complex plays a supporting role in tying the lipid bilayer of liposome strongly so that the stable and bilayer structure of liposome can be kept even with cyclodextrin compared to lipid-cholesterol based liposome.

Table 16 shows the result of the long-term stability according to salt addition to polymer-liposome complex of Example 4 and 25, and to lipid-cholesterol based liposome of Comparative Example 2 and 6 in the sodium phosphate buffer solution and HEPES (N-2-hydroxyethyl-piperazine-N ¹ -2-ethan sulfonate) buffer solution.

(Table 16) The long-term stability of polymer-liposome complex and lipid-cholesterol based liposome in the buffer solution.

Particle size (nm) (polydispersity) D

1) : polydispersity indicates the size distribution of

particles. Small value of polydispersity indicates the narrower distribution of particles.

As shown in Table 16, the polymer-liposome complex of Example 4 according to the present invention did not show big difference in particle sizes and polydispersity in the long time regardless of storing at distilled water and the buffer solution with salt. However, when compared to being kept in distilled water in case of lipid-cholesterol based liposome of Comparative Example 2, it was observed that the particle size became larger, polydispersity increased, and the structure went unstable when it was kept in the sodium phosphate buffer solution added with sodium phosphate and sodium chloride (NaCl) , because the buffer solution with salt influenced the particle size and polydispersity. Also, when it was kept in the HEPES buffer solution, the structure collapsed after two weeks and formed precipitation.

Also, polymer-liposome complex comprising betulin of Exmaple 25 did not show much of a difference in the particle size and polydispersity when it was kept in the buffer solution without salt or with salt, but in case of lipid- cholesterol based liposome comprising betulin of Comparative Example 6, it was observed that the particle size became larger and polydispersity increased, and the structure went unstable in the sodium phosphate buffer solution added with

sodium phosphate and sodium chloride (NaCl) . When it was kept in the HEPES buffer solution, the structure collapsed and the precipitation is formed after one week.

Therefore, from Table 15 and Table 16 above, it was found that polymer-liposome complex of the present invention had kept more stable structure in the existence of water-soluble bioactive compounds and other salts than lipid-cholesterol based liposome. Also, the polymer-liposome complex, in which the water-insoluble bioactive compounds are embedded, had a more stable structure in aqueous solution with salt, which was used in biosystems, than lipid-cholesterol based liposome, in which the water-insoluble bioactive compounds were embedded.

(Experimental Example 3) The stability evaluation of polymer- liposome complex and lipid-cholesterol based liposome in nanoemulsion cosmetics formulation.

In order to compare the structural stability of polymer- liposome complex and lipid-cholesterol based liposome according to the present invention in nanoemulsion cosmetics formulation, the solutions prepared in Example 4 and

Comparative Example 2 were put into the nanoemulsion formulation with the average droplet size of 137 nm, which was formulated by the compositions and contents of Table 17, with

10 wt%, 30 wt% and 50 wt% of vesicle solutions at 60 ^° C Then, they were stirred for five minutes at 7200 rpm through the homogenizer, and after the cooling and degassing process, the formulation comprising vesicle was prepared. The thermal behaviors of these prepared formulation-vesicle mixture was observed by using the differential scanning calorimetry, DSC QlOOO, TA Instruments. The results are shown in FIG. 16 and FIG. 17.

(Table 17) The compositions and contents of nanoemulsion formulation to compare the stability of polymer-liposome complex with lipid-cholesterol based liposome in nanoemulsion formulation.

FIG. 16 shows the thermograms for the mixtures of nanoemulsion and lipid-cholesterol based liposome of Comparative Example 2 for various contents of vesicles. Liposome showed the transition point of gel to liquid crystal at around 51 ^° C and nanoemulsion showed the transition point of emulsion droplet at around 62 ^° C. However, in case of the liposome-nanoemulsion mixture, each transition point was disappeared, but a single transition point appeared. It was also found that the transition point above changed as the composition changed. If vesicle and nanoemulsion droplet exist independently and stably within the mixture, the transition point of vesicle solution and nanoemulsion formulation should be appeared at each transition point with different peak intensity. From FIG. 16, appearing a single transition point indicates that a new particle was reconstructed while liposome and nanoemulsion got mixed. It was found that when it was mixed with nanoemulsion, the initial structure of lipid- cholesterol based liposome could hardly exist any longer

inside the formulation.

FIG. 17 shows the thermograms of polymer-liposome complex of Example 4, which showed the transition point of gel to liquid crystal at 45°C. When polymer-liposome complex and nanoemulsion were mixed together, as also shown in FIG. 17, two transition points co-exist, which is characteristics of the transition point of gel to liquid crystal of polymer- liposome complex and droplet of nanoemulsion. Different from the case of liposome-nanoemulsion, this is because each polymer-liposome complex and nanoemulsion kept its own structure and stayed uniformly when polymer-liposome complex and nanoemulsion were mixed and stirred.

Thus, it was found that polymer-liposome complex of the present invention solved the problem of storage stability, which the current lipid-cholesterol liposome has. The present invention also has a very stable structure in cosmetics formulation comprising water-soluble active compound, salt and surfactant compared to the lipid-cholesterol based liposome.

(Experimental Example 4) The stability evaluation of polymer- liposome complex comprising betulin and lipid-cholesterol based liposome in nanoemulsion cosmetics formulation.

In order to compare the structural stability of polymer- liposome complex comprising water-insoluble bioactive compound of the present invention and lipid-cholesterol based liposome in nanoemulsion cosmetics formulation, the solutions prepared in Example 25 and Comparative Example 6 were mixed with the nanoemulsion formulation with the average particle size of 137 nm, which was formulated by the compositions and contents of Table 17, with 10 wt% ratio of vesicle solutions. Then they were stirred for five minutes at 7000 rpm at room temperature through the homogenizer, and after the degassing process, the nanoemulsion formulations comprising polymer-liposome complex comprising betulin and lipid-cholesterol based liposome respectively were produced.

FIG. 18-22 show the graph of the particle size using dynamic light scattering, Zetasizer 3000HSa Malvern Instruments, after storing the nanoemulsion above, polymer- liposome complex comprising betulin (Example 25) , lipid- cholesterol based liposome (Comparative Example 6) , nanoemulsion formulation comprising polymer- liposome complex comprising betulin and nanoemulsion formulation comprising lipid-cholesterol based liposome each for four weeks at room temperature .

FIG. 18 shows the particle size distribution of the nanoemulsion prepared from the composition of Table 17, FIG.

19 shows the particle size distribution of polymer-liposome complex comprising betulin (Example 25) , FIG. 20 shows the particle size distribution of lipid-cholesterol based liposome comprising betulin (Comparative Example 6) , FIG. 21 shows the particle size distribution of nanoemulsion formulation comprising polymer-liposome complex comprising betulin, and FIG. 22 shows the particle size distribution of nanoemulsion formulation comprising lipid-cholesterol based liposome comprising betulin. From FIG. 18 ~ 22 above, it was found that nanoemulsion formulation comprising polymer-liposome complex comprising betulin had much better structural stability in the formulation than nanoemulsion formulation comprising lipid- cholesterol based liposome comprising betulin. It was also found that lipid-cholesterol based liposome comprising betulin failed to keep the stable liposome structure and collapsed when it got mixed within nanoemulsion formulation.

(Experimental Example 5) The observation on pH-sensitivity of polymer-liposome complex, polymer-liposome complex comprising betulin and lipid-cholesterol based liposome.

In order to observe the pH-sensitivity of polymer- liposome complex according to the present invention, FIG. 23 shows the photographs of the solution after diluting the

concentrations of polymer-liposome complex in Example 1 and Example 4 and liposome in Comparative Example 2 to 0.1 wt% by- using sodium phosphate buffer solution in which the pH of the solution was adjusted to 3 , 4, 5, 6 and 7.4 with hydrochloric acid solution. Table 18 shows the change in particle sizes depending on pH of the solution.

Also, FIG. 24 shows the photographs of the solution after diluting polymer-liposome complex comprising betulin as water- insoluble bioactive compound of Example 25 and lipid- cholesterol based liposome of Comparative Example 6 to 0.1 wt% by using sodium phosphate buffer solution, in which the pH of the solution was adjusted to 3, 4, 5, 6 and 7.4 with hydrochloric acid solution. Table 18 shows the change in particle sizes depending on pH of the solution.

From FIG. 23, it was found that lipid-cholesterol based liposome of Comparative Example 2 was well dispersed in aqueous solution regardless of pH of the solution, and polymer-liposome complex of Example 1 and 4 was well dispersed in aqueous solution at pH above 5, but it failed to keep the stable liposome structure and collapsed in aqueous solution with pH 4 and 3.

Further, as shown in Table 18, when polymer- liposome complex of Example 1 and 4 was dispersed in the solution with pH 7.4 ~ 5, there was not much of a change in the particle

size and polydispersity compared to distilled water. It is considered that it has pH sensitivity, which it rapidly collapsed its structure at less than said pH range, probably because pH-sensitive polymer used in polymer-liposome complex has the property that dramatically changes in its form in a particular pH in aqueous solution.

On the other hand, in FIG. 23, it seems that liposome of Comparative Example 2 is well dispersed regardless of pH of the solution, but it was observed that the particle size and polydispersity much increased as pH of the solution changed, which was different from the case that it was dispersed in distilled water as shown in Table 18.

Thus, polymer-liposome complex of the present invention stably keeps its particle form at more than pH 5, but its stability deteriorates rapidly at less than pH 5, so that it can effectively release the bioactive compounds inside the cell and promote the delivery effect as a carrier.

From FIG. 24, it was found that lipid-cholesterol based liposome comprising betulin of Comparative Example 6 was well dispersed in aqueous solution regardless of pH of the solution, and polymer-liposome complex comprising betulin was well dispersed in aqueous solution at more than pH 5, but it failed

to keep its stability at less than pH 5 and the structure collapsed and precipitation appeared.

On the other hand, in FIG. 24, it seems that lipid- cholesterol based liposome comprising betulin of Comparative Example 6 was well dispersed regardless of pH of the solution, but it was observed that the particle size and polydispersity much increased as pH of the solution changed, which was different from the case that it was dispersed in distilled water as shown in Table 18.

Also, polymer-liposome complex comprising betulin of Example 25 kept stable particle form at more than pH 5, but failed to keep it at lower pH and showed rapid structural collapse. This is because the pH-sensitive polymer has dramatic change in its form and destructs the structure of complex at less than pH 5.

(Table 18) The measurement of particle sizes depending on solution acidity of polymer-liposome complex and lipid- cholesterol based liposome.

1) polydispersity indicates the size distribution of particles. Small value of polydispersity indicates the narrower distribution of particles.

(Experimental Example 6) The observation on the drug release behavior depending on pH of polymer-liposome complex.

In order to promote the efficiency of water-soluble bioactive compounds within cells, they should have the function to promote the delivery efficiency of bioactive compounds within cells using a carrier, which also has to release the drug at a certain environment within cells at the same time. The surroundings of ribosome within cells keep lower pH (pH 4.5 ~ 5.5) than the physiological condition. When there is a function to release the drug in response to the change in the environment within cells, the delivery

efficiency can be promoted within cells of water-soluble bioactive compounds. Therefore, the following experiment was conducted to observe whether the water-soluble drug released selectively depending on pH of aqueous solution comprising polymer-liposome complex of the present invention.

0.455 g of 8-hydroxypyrene-l, 3, 6-trisulfonic acid trisodium salt ("HPTS" hereafter) and 0.525 g of p-xylene-bis- pyridium bromide ("DPX" hereafter) were dissolved in the 300 ml of aqueous solution including 10 mM of HEPES (N-2- hydroxyethyl-piperazine-N' -2-ethan sulfonate) and 11 mM of sodium phosphate .

0.6 g of poly (methacrylic acid-co-stearyl methacrylate) (mole ratio = 70 : 30, number average molecular weight = 26,000), 1.2 g of 100% hydrated oleoyl-palmitoyl/oleoyl- stearyl phosphatidylcholine mixture (S100-3) and 0.15 g of cholesterol were dissolved with heat in 30 g of ethanol, then put into the buffer solution including HPTS-DPX that were heated to 65 "Q and mixed for five minutes at 5000 rpm using the homogenizer. Said first dispersed polymer-liposome complex was further mixed using high pressure homogenizer (1000 bar, 3 cycle) to nanoparticles . Then, polymer-liposome complex comprising HPTS-DPX was prepared and showed the particle size of 244 nm.

On the other hand, as a control group, 1.8 g of 100% hydrated oleoyl-palmitoyl/oleoyl-stearyl phosphatidylcholine

mixture (S100-3) and 0.2 g of cholesterol were dissolved with heat in 30 g of ethanol, and then with the same method of polymer-liposome complex, liposome comprising HPTS-DPX was prepared, which showed the particle size of 200 nm. 200μl of polymer-liposome complex comprising HPTS-DPX and lipid-cholesterol based liposome were respectively put in 20 ml of 20 mM HEPES buffer solution (pH 7.1, includes 144 mM sodium chloride) and 20 ml of 100 mM MES buffer solution (pH 3.45, includes 144 mM sodium chloride) and then dispersed. The 2 ml of this solution was sealed in the dialysis tube, and it was put into 30 ml of isotonic solution (pH 7.1 and 3.45 respectively) , which was controlled to have the same pH, and the amount of HPTS-DPX released was detected with time from the solution under contstant stirring and temperature . Table 19 shows the amount of HPTS-DPX in the detected solution through fluorescence spectroscopy (Hitachi 4500) . On the other hand, the total amount of HPTS-DPX in isotonic solution was detected through fluorescence spectroscopy (Hitachi 4500) after its structure was completely collapsed by putting polymer-liposome complex and liposome into 10% Triton X-100 solution.

(Table 19) The change in release quantity of HPTS-DPX depending on pH change of polymer-liposome complex and lipid- cholesterol based liposome.

From Table 19, it was found that the amount of drug released was larger when the pH of aqueous solution was 3.45 than 7.1 in both polymer-liposome complex and lipid- cholesterol based liposome. As shown in Table 19, this is due to the increase in particle sizes and structure collapse in polymer-liposome complex and lipid-cholesterol based liposome as the pH of the solution got low.

Further, from Table 19 above, it was observed that the effect of increase in the released drug was bigger in polymer- liposome complex compared to lipid-cholesterol based liposome as pH of the solution got lower from 7.1 to 3.45. This is due to lipid-cholesterol based liposome had a little unstable structure and HPTS-DPX could not release completely, while the

structure of polymer-liposome complex was completely collapsed at less than pH 5 and HPTS-DPX could freely release.

Therefore, it was found that polymer-liposome complex of the present invention changes dramatically its structure depending on pH changes and is able to control and release drugs depending on pH change.

(Experimental Example 7) The observation on the skin permeability of polymer-liposome complex comprising active components

In order to evaluate the skin permeability of polymer- liposome complex according to the present invention, the skin permeability of the arbutin solution collected by polymer- liposome complex as a carrier, the arbutin solution collected by lipid-cholesterol based liposome and the arbutin aqueous solution without vesicle were measured.

The aqueous solution of polymer-liposome complex and lipid-cholesterol based liposome comprising 2 wt% of arbutin (Example 21 and Comparative Example 5) and 2 wt% of arbutin aqueous solution were prepared respectively. At the same time, the skin extracted from the Guinea pig was installed in Franz- cell (Hanson Research) , experiment equipment for skin permeability, and the upper side of the skin was filled with the aqueous solution of polymer-liposome complex comprising

arbutin, the aqueous solution of lipid-cholesterol based liposome comprising arbutin and the arbutin aqueous solution respectively. Then, the solution released through the skin was withdrawed every six hour. FIG. 25 shows quantified concentration of arbutin (Skin permeability, P (mg/cm ² ) ) regarding how much arbutin molecules from the aqueous solution of polymer- liposome complex comprising arbutin, the aqueous solution of lipid-cholesterol based liposome comprising arbutin and the arbutin aqueous solution was permeated through the skin, using high performance liquid chromatography (Hewlett Packard) .

From FIG. 25, it was found that as time goes lipid- cholesterol based liposome comprising arbutin (a) showed the best skin permeability and polymer-liposome complex (b) had better skin permeability than the arbutin aqueous solution without vesicle (c) , which indicates that polymer-liposome complex has the function to promote skin permeability, one of the functions of liposome.

The function of polymer- liposome complex for promoting skin permeability improves when polymer- liposome complex exists in emulsion formulation, compared to lipid-cholesterol based liposome and the aqueous solution comprising arbutin. Polymer- liposome complex prepared in Example 21 and Liposome in Comparative Example 5 and arbutin aqueous solution, all of

which were concentrated to 4wt%, into emulsion formation in Table 17 with the 1:1 weight ratio. FIG. 26 shows the result of skin permeability of said mixture using the same method above . From FIG. 26, different from FIG. 25, it was found that the mixture of polymer-liposome complex comprising emulsion formulation and arbutin had much better skin permeability than the mixture of lipid-cholesterol based liposome and emulsion and the emulsion mixture comprising only arbutin. Thus, it was found that polymer-liposome complex has excellent skin permeability not only in vesicle itself but also in multi- component systems including salt, oil, wax, etc.

However, lipid-cholesterol based liposome has lost the skin permeability that vesicle itself has and showed the almost same skin permeability ability with the emulsion formation without vesicle. This is considered to be quite related to structural stability inside the emulsion system of vesicle as described above.

(Experimental Example 8) The observation on the titer of rhubarb extract in polymer-liposome complex, in which rhubarb extract solubilized through hydroxypropyl-β-cyclodextrin was embedded, and nanoemulsion comprising rhubarb extract.

The titer stability of rhubarb extract was observed in

polymer-liposome complex, in which rhubarb extracts solubilized through hydroxypropyl-β-cyclodextrin was embedded, (Example 44) and nanoemulsion comprising rhubarb extract (Comparative Example 31) . Table 20 shows the measurement result of rhaponticin concentration, an indicative component of rhubarb extract, using high performance liquid chromatography, with time while the samples of Example 44 and Comparative Example 31 were kept at room temperature and 40 ^° C respectively.

(Table 20)

As shown in Table 20, no decrease as time passed at both room temperature and 40 ^° C was observed in the rhaponticin concentration in Example 44. However, the rhaponticin concentration in Comparative Example 31 had more titer decrease at 40°C than room temperature as time passed. Rhaponticin, the indicative component of rhubarb extract, is a

water- insoluble bioactive compound that is inclined to decrease its concentration by light or temperature. According to the result of Table 20 above, rhubarb extract comprised in polymer-liposome complex, after it is solubilized by hydroxypropyl-β-cyclodextrin, has better titer stability compared to rhubarb extract comprised in the nanoemulsion. It is assumed that this is due to the effect of preventing the decrease in the concentration of rhaponticin while rhubarb extracts are collected by hydroxypropyl-β-cyclodextrin that is used to solublize rhubarb extracts, rather than due to stability by polymer-liposome complex.

(Experimental Example 9) The comparative experiment on the skin absorption of rhubarb extract through Franz-cell.

The skin absorption of rhaponticin was compared between polymer-liposome complex comprising rhaponticin solubilized through hydroxypropyl-β-cyclodextrin and the aqueous solution where rhaponticin solubilized through hydroxypropy1- β- cyclodextrin was dispersed prepared in Example 42 and Comparative Example 28 respectively.

Also, the skin absorption of rhaponticin was compared between polymer-liposome complex comprising rhubarb extract solubilized through hydroxypropyl-β-cyclodextrin and the nanoemulsion comprising rhubarb extract prepared in Example 44

and Comparative Example 31 respectively.

The experiment of skin absorption was conducted by using the skin obtained from the albino guinea pig for 18 hours using Franz-cell equipment. FIG. 27 and FIG. 28 show the result of rhaponticin concentration that was permeated into the skin, using high performance liquid chromatography, in order to compare the skin absorption of rhaponticin.

FIG. 27 is the comparison result of skin absorption of rhaponticin between polymer-liposome complex comprising rhaponticin solubilized through hydroxypropyl-β-cyclodextrin (Example 42) and the aqueous solution, in which rhaponticin solubilized through hydroxypropyl-β-cyclodextrin was dispersed (Comparative Example 28). Rhaponticin solubilized through hydroxypropyl-β-cyclodextrin showed quite good skin permeation, but polymer-liposome complex of Example 42 had 162% higher skin permeation of rhaponticin compared to the aqueous solution of Comparative Example 28.

Although the enhancing effect of cyclodextrin or on the skin permeation has not fully examined yet, its effects have been revealed through lots of experiment results. In polymer- liposome complex, polymer plays a supporting role in tying the lipid bilayer of liposome strongly and keeps unique structure of liposome even with cyclodextrin, so that it can improve skin absorption. FIG. 28 is the comparison result of skin absorption of

rhaponticin, the indicative component of rhubarb extract, between polymer-liposome complex comprising rhubarb extract solubilized through hydroxypropyl-β-cyclodextrin (Example 44) and nanoemulsion comprising rhubarb extract (Comparative Example 31) . Compared to rhubarb extract that are embedded in nanoemulsion (Comparative Example 31) , rhubarb extracts in polymer-liposome complex (Example 44) had 842% higher skin absorption effect of rhaponticin. This is much higher skin absorption effect of rhaponticin than the current formulation and it can be a great help for various formulation prescription of water-insoluble bioactive compounds through hydroxypropyl-β-cyclodextrin and polymer-liposome complex with excellent stability.

(Experimental Example 10) The observation on the whitening effect of polymer- liposome complex comprising active component within cells.

In order to evaluate whether polymer-liposome complex comprising active component has excellent effect within cells compared to active component aqueous solution without carrier, streptomyces extract was used, which is known to have the ability to inhibit melanin formation. Polymer-liposome complex comprising the ^■ streptomyces extracts (Example 19) and streptomyces extract aqueous solution were inserted into cell

culture medium in order to keep the concentration of all streptomyces extracts in the medium cultivating human melanoma HM3KO cells as noted in Table 21. After the culture medium including the cells above was incubated for 24 hours, the cells were removed and the remaining solution was collected after centrifugal separation of the cells. Table 21 shows the ratio of amount of melanin from the intensity of absorbance peak that was produced at 490 nm using UV-vis spectroscopy.

(Table 21) The relative amount of melanin (%) compared to the vehicles (polymer-liposome complex or deionized water) of no streptomyces extract (100%) depending on the concentration of streptomyces extract aqueous solution and polymer-liposome complex comprising streptomyces extract.

As shown in Table 21, for the concetration of

streptomyces extract was 0.5 mM and 0.25 mM, the inhibition effect of melanin synthesis by melanocytes was much greater when treated with polymer-liposome complex. Even with 0.5 mM of streptomyces extracts, it showed excellent inhibition effect of melanin formation compared to 1 mM aqueous solution, and polymer-liposome complex with 0.25 mM of streptomyces extracts had greater inhibition effect of melanin formation than 0.5 mM aqueous solution. Therefore, it was found that the delivery efficiency to cells is better when polymer-liposome complexes comprising active components are used, compared to when the carriers are not used.

[Industrial Applicability]

Polymer-liposome complex using pH-sensitive polymer according to the present invention is designed to be capable of controlling and releasing drugs depending on the change in pH of solution by using the characteristics of changing its structure depending on pH of aqueous solution.

Further, by introducing pH-sensitive polymer, polymer- liposome complex of the present invention could stably embed water-insoluble bioactive compounds, which could be embedded in lipid-cholesterol based liposome, as well as the other water-insoluble bioactive compounds, which could not be embedded in lipid-cholesterol based liposome, in the lipid bilayer of polymer-liposome complex. It could embed water-

insoluble bioactive compounds in the lipid bilayer, excluding triterpenoid, which could not be embedded in lipid-cholesterol based liposome.

Also, polymer-liposome complex of the present invention could include/embed various water-insoluble bioactive compounds, which were impossible to be included/embedded in the lipid bilayer of general lipid-cholesterol based liposome, inside the liposome after being solubilized through hydroxypropyl-β-cyclodextrin. Regarding various cyclodextrins, it was verified that its structure was relatively stable compared to that of the general lipid-cholesterol based liposome .

Moreover, polymer-liposome complex of the present invention has a much improved delivery efficiecy of active components to the body and better efficacy, by effectively releasing bioactive components, which are embedded in polymer- liposome complex, within cells since its stability deteriorates rapidly at a particular pH, and shows excellent skin permeation. Additionally, polymer-liposome complex using pH-sensitive polymer according to the present invention has much improved stability in regard to various salts and cosmetics formulation compared to the well-known lipid- cholesterol based liposome system. It has the effect not only to prevent titer decrease of bioactive components that are embedded in it but also to improve great skin permeation,

which indicates its high value of practical use.