KR101503492B1 - Liposomes capsule containing lipid and lysosomal enzymes, and manufacturing method thereof - Google Patents

Abstract

More particularly, the present invention relates to a liposome having an antibacterial function and a liposome surface which is firm and does not easily release a lysosomal enzyme in liposomes, and a method for producing the same. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a liposome comprising lipids and a lysosomal enzyme, .

Description

TECHNICAL FIELD [0001] The present invention relates to a liposome capsule containing lipid and a lysosomal enzyme, and a method of manufacturing the liposome capsule containing lipid and lysosomal enzymes,

TECHNICAL FIELD The present invention relates to a liposome capsule containing lipids and a lysosomal enzyme and a preparation method thereof, and more particularly to a liposome capsule having an antibacterial function and a liposome surface which is firm and does not easily release a lysosomal enzyme in liposomes, .

Lysosome is a small organ of cells with a size of 0.25 ~ 0.5 ㎛ which contains a large amount of hydrolytic enzymes and extinguishes. It is smaller than mitochondria and plays a role of extinguishing foreign substances such as bacteria and aging cells ). In addition to being involved in the metabolism of intracellular components, it plays an important role in the intracellular digestion of extrinsic foreign matter, especially by phagocytosis.

Lysosomes are fractionated midway between mitochondria and microsomes by the centrifugal fractionation method of the cell lysis solution. The lysosomes are wrapped in a membrane structure that is different from the mitochondria. This lysosome is hydrolyzed The presence of enzymes is known. For example, acid phosphatase, acid phosphatase, ribonucleic acid hydrolase, protease, β-glucuronase, and arylsulfatase are typical enzymes. However, only a few enzymes in these lysosomes are known, and the functional properties of many enzymes have not yet been confirmed.

On the other hand, liposomes, also known as lipid vesicles, are fully closed lipid bilayer membranes containing the trapped volume containing the aqueous medium. The lipid bilayers often consist of phospholipids such as lecithin and related materials such as glycolipids. The liposome may be a monolayer having a single membrane bilayer, or a multilayer having one or more membrane bilayers, with an aqueous space between the different membrane bilayers. The bilayer consists of two lipid monolayers, each having a hydrophobic portion oriented toward each other facing outward so that the hydrophilic portion faces the aqueous phase.

Liposomes are formed when phospholipids or other suitable bipolar molecules are allowed to expand in water or aqueous solutions. If a water-soluble substance is contained in the aqueous phase during the expansion process, the water-soluble substance can be entrapped in the aqueous phase between the lipid bilayers. In addition, if bipolar molecules having polar groups are used, the polar groups can be stretched into an internal or external aqueous phase, dissolved in lipids, and incorporated into the bilayer themselves.

The lipid bilayer membrane often functions in a manner similar to a cell membrane. In addition, liposomes can bind living cells as if they were organelles themselves. As a result, there is a growing interest in using liposomes as carriers for delivery of compounds having specific biological or pharmaceutical properties to patients.

However, some difficulties have arisen with regard to the use of liposomes as a means of delivery and targeting to drugs and other compounds, one of which is that liposomes prepared by conventional techniques using conventional formulations exhibit phase unstability over time Which can lead to liposome leaks, breakdown, deposition and phase separation, fusion, agglomeration or gelation upon storage of the medicament. Nevertheless, the reason that liposomes are widely studied and utilized as mobile materials for specific materials is that liposomes are easy to manufacture, are freely adjustable to the desired size, are biocompatible carriers, and have no limitations on encapsulating materials.

When encapsulating a particular substance in a liposome to achieve a particular purpose associated with the function or effect of the particular substance, the following are considered: The extent to which a particular substance is encapsulated in liposomes, the amount of liposomes introduced into the liposomes, the extent to which the encapsulated liposomes are not degraded and the liposomes reach the target point in a stable state, and whether the desired function is achieved after reaching the target point. Many studies have been conducted to solve the problems of encapsulating a specific substance in a liposome or a method of allowing a liposome reaching a target point to exert a desired function efficiently, Much research is needed on how to improve the extent to which a substance is encapsulated in liposomes.

Conventional studies have found that a synthetic agent such as a nonionic surfactant, an emulsifying stabilizer or a polymer emulsifier is added to the liposome. However, there still exists a problem that the liposome is stabilized and the release of the enzyme inside the liposome is not suppressed until the desired site is reached. Furthermore, the problem of difficulty in improving the degree of encapsulation of liposomal enzymes with liposomes has not been solved.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in order to solve the above-mentioned problems, and it is an object of the present invention to provide a liposome which can stabilize the liposome and control the release of the enzyme in the liposome until a desired site is reached. It is also intended to provide a method for enhancing the degree of encapsulation of a specific substance into a liposome and a liposome produced by the method.

The present invention provides a liposome capsule comprising 2.5 to 20 parts by weight of lysosomal enzyme per 100 parts by weight of lipid.

According to a preferred embodiment of the present invention, the lysosomal enzyme is selected from the group consisting of lysosomelanzyme, cathepsin B, cathepsin D, cathepsin A, acid carboxypeptide hydrolase, collagen hydrolase, neutral protease, One selected from the group consisting of starase, hyaluronidase, lysozyme, neuraminidase, fucosidase, triacylglycerol, phosphatidic acid, sphingomyelin, ribonucleic acid hydrolase and deoxyribonucleic acid hydrolase Or more.

According to another preferred embodiment of the present invention, the lipid is selected from the group consisting of L-alpha-phosphatidylcholine, L-alpha-phosphatidylserine, phosphatidylethanolamine, Phosphatidylcholine, phosphatidylcholine, phosphatidic acid, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, diureoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dilinoleoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine and 1,2-dipitanoyl-3-sn-phosphatidylcholine (1 , 2-diphytanoyl-3-sn-phosphatidylcholine).

According to another preferred embodiment of the present invention, the liposome may further comprise cholesterol.

According to another preferred embodiment of the present invention, the liposome may contain 10 to 100 parts by weight of cholesterol and 2.5 to 20 parts by weight of lysosomal enzyme per 100 parts by weight of lipid.

According to another preferred embodiment of the present invention, the lysosomal enzyme may be extracted from at least one selected from the group consisting of yeast, insects, plants and animals.

According to another preferred embodiment of the present invention, the average diameter size of the liposome may be 50 to 500 nm.

The present invention also relates to a method for preparing a pharmaceutical composition, which comprises mixing a lipid, a cholesterol and a solvent to prepare a mixture; Removing the solvent from the mixture to produce a liposome film; And adding a lysosomal enzyme to the liposome film to prepare a liposome encapsulated with the lysosomal enzyme; The present invention provides a method for producing a liposome capsule.

According to a preferred embodiment of the present invention, the lysosomal enzyme may be extracted from at least one selected from the group consisting of yeast, insects, plants and animals.

According to another preferred embodiment of the present invention, the mixture contains 0.1 to 1 part by weight of lipid and 0.01 to 1 part by weight of cholesterol per 100 parts by weight of the solvent, and the lysosomal enzyme added to the liposome film is 100 parts by weight 5 to 10 parts by weight of lysosomal enzyme may be added.

According to another preferred embodiment of the present invention, the lysosomal enzyme is selected from the group consisting of lysosomelanzyme, cathepsin B, cathepsin D, cathepsin A, acid carboxypeptide hydrolase, collagen hydrolase, , Losartan, hyaluronidase, lysozyme, neuraminidase, fucosidase, triacylglycerol, phosphatidic acid, sphingomyelin, ribonucleic acid hydrolase and deoxyribonucleic acid hydrolase And may include at least one selected.

According to another preferred embodiment of the present invention, the lipid is selected from the group consisting of L-alpha-phosphatidylcholine, L- alpha -phosphatidylserine, phosphatidylethanolamine, Phosphatidylcholine, phosphatidylcholine, phosphatidic acid, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioloylphosphatidylcholine dioleoyl phosphatidylcholine, dilinoleoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine and 1,2-dipitanoyl-3-sn-phosphatidylcholine 1,2-diphytanoyl-3-sn-phosphatidylcholine).

According to another preferred embodiment of the present invention, the solvent may be at least one selected from the group consisting of chloroform and methanol.

The present invention provides a method for effectively producing liposomes by enhancing the encapsulation of lysosomal enzymes using lipid and lysosomal enzymes, and a liposome produced by the above method, wherein the liposomes have improved stability and do not release drug in the liposome during storage There is an effect of providing a liposome capable of safely moving to a target site to exhibit activity.

FIG. 1 is an image of a liposome prepared by adding 10 mg of L-.alpha.-phosphatidylcholine only to 500 .mu.g of lysosomal enzyme in Example 1. FIG.
Fig. 2 is an image of a liposome prepared by using 9 mg of lipid (L-a-phosphatidylcholine) and 1 mg of cholesterol in 500 ㎍ of lysosomal enzyme in Example 2. Fig.
3 is an image of a liposome prepared by using 8 mg of lipid (L-alpha-phosphatidylcholine) and 2 mg of cholesterol in 500 ㎍ of lysosome enzyme in Example 2. Fig.
4 is an image of a liposome prepared by using 7 mg of lipid (L-a-phosphatidylcholine) and 6 mg of cholesterol in 500 ㎍ of lysosome enzyme in Example 2. Fig.
5 is an image of a liposome prepared by using 5 mg of lipid (L-alpha-phosphatidylcholine) and 5 mg of cholesterol in 500 ㎍ of lysosome enzyme in Example 2. Fig.
Fig. 6 is a graph showing the amount of lysosomal enzyme released for 6 days measured in the liposome performed in Experimental Example 3 to evaluate the stability of the liposome prepared in Examples 1 and 2. Fig.

Hereinafter, the present invention will be described in more detail.

As described above, many studies have been conducted so far to solve the problems of methods of encapsulating a specific substance in a liposome and methods of allowing a liposome reaching a target point to efficiently exhibit a desired function. However, Much research is needed on how to stabilize liposomes and how to enhance the extent to which certain substances are encapsulated in liposomes.

Accordingly, the present invention sought to solve the above-mentioned problems by providing liposomes containing lipid and lysosomal enzymes and encapsulating the lysosomal enzymes. Thus, it is possible to provide a liposome in which the degree of encapsulation of the lysosomal enzyme is improved, the stability of the liposome produced is improved, and the problem of destruction of the liposome before reaching the target point is solved.

The liposomal capsules of the present invention include lipids and lysosomal enzymes and can provide that the lysosomal enzymes are encapsulated. In addition, the liposome capsules may preferably contain 2.5 to 20 parts by weight of lysosomal enzyme per 100 parts by weight of lipid, and more preferably 6 to 15 parts by weight of lysosomal enzyme per 100 parts by weight of lipid.

If the liposome enzyme is contained in an amount of less than 2.5 parts by weight or more than 20 parts by weight based on 100 parts by weight of the lipid, there may arise a problem that the efficiency of liposome formation is lowered.

The lysosome of the present invention has a large amount of hydrolytic enzymes in eukaryotic cells, and besides its metabolism, plays an important role in the intracellular digestion of extraneous foreign matter by phagocyte function. Unlike the mitochondria of the host organism, it is enclosed in a single-layer membrane structure, and its interior is composed of a hydrolytic enzyme having an optimum pH in a weakly acidic state, for example, acidic dephosphate hydrolase, ribonucleic acid hydrolase, Chloritease or arylsulfatease, and the like.

The lysosomal enzyme is not particularly limited as long as it is a substance exhibiting an antimicrobial activity isolated or purified in a lysosome. Preferably, lysosomal enzyme, cathepsin B, cathepsin D, cathepsin A, acid carboxypeptide hydrolase, The enzyme is selected from the group consisting of protease, neutral protease, elastase, hyaronidase, lysozyme, neuraminidase, fucosidase, triacylglycerol, phosphatidic acid, sphingomyelin, ribonucleic acid hydrolase and deoxyribose And a nucleic acid hydrolase, and more preferably, it may contain lysosomal enzyme.

In addition, the lysosomal enzyme is not particularly limited as long as it is a microorganism such as a eukaryote, in one embodiment, a yeast, or an eukaryotic cell of an animal. Preferably, the lysosome enzyme is isolated from one or more selected from the group consisting of yeast, insect, And more preferably, those isolated from Saccharomyces cerevisiae or bread yeast can be used, and most preferably, those isolated from brewer's yeast can be used.

The encapsulation of the lysosomal enzyme means that the lysosomal enzyme is encapsulated in the lipid, which can prevent the lysosomal enzyme from being released to the outside during storage to lose the physiological activity of the lysosomal enzyme and, while the lysosomal enzyme is moving to the desired target site It is possible to prevent the physiological activity from being lost.

The lipid may be any one separated from animal or plant, or synthesized, but is preferably a phospholipid, more preferably L-alpha-phosphatidylcholine, L-alpha-phosphatidylcholine, Phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoylphosphatidylcholine (hereinafter referred to as " dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dilinoleoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, oleoyl phosphatidylcholine) and 1,2-dipitanoyl-3-sn-phosphatidylcholine (1,2-diphytanyl l-3-sn-phosphatidylcholine), and most preferably L-alpha-phosphatidylcholine.

Further, according to one embodiment of the present invention, the liposome may further contain cholesterol, and the cholesterol is not particularly limited as long as it is usually produced or sold. Further, the liposome further containing cholesterol may contain 10 to 100 parts by weight of cholesterol and 2.5 to 20 parts by weight of lysosomal enzyme per 100 parts by weight of lipid.

If cholesterol is contained in an amount of less than 10 parts by weight based on 100 parts by weight of lipid, the efficiency of liposome formation may be lowered. If the cholesterol is contained in an amount of more than 100 parts by weight based on 100 parts by weight of lipid, There is a problem that the degree of encapsulation of the capsule can be reduced.

The average diameter of the liposome capsules is not particularly limited as long as it is nano-sized, but it is preferable to provide liposomes of 50 nm to 500 nm. If the average diameter of the liposome exceeds 500 nm, there is a possibility that the range of application of the liposome becomes small.

In addition, the liposome capsules of the present invention may have antimicrobial activity by a lysosomal enzyme, and may be manufactured and / or used as a cosmetic composition and / or a pharmaceutical composition.

Further, the present invention relates to a method for preparing a pharmaceutical composition, which comprises mixing a lipid, a cholesterol and a solvent to prepare a mixture; Removing the solvent from the mixture to produce a liposome film; And adding a lysosomal enzyme to the liposome film to prepare a liposome encapsulated with the lysosomal enzyme; The present invention provides a method for producing a liposome capsule.

First, the steps of preparing a mixture by mixing lipid, cholesterol, and a solvent will be described.

The lipid may be any one separated from animal or plant, or synthesized, but is preferably a phospholipid, more preferably L-alpha-phosphatidylcholine, L-alpha-phosphatidylcholine, Phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoylphosphatidylcholine (hereinafter referred to as " dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dilinoleoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, oleoyl phosphatidylcholine) and 1,2-dipitanoyl-3-sn-phosphatidylcholine (1,2-diphytanyl l-3-sn-phosphatidylcholine), and most preferably L-alpha-phosphatidylcholine.

The above-mentioned cholesterol is not particularly limited as long as it is usually produced or sold.

Further, the solvent is usually used in the production of liposomes, and is not particularly limited as long as it can dissolve lipids and cholesterol. Preferably, the solvent may include at least one selected from the group consisting of chloroform and methanol, Chloroform may be used.

The mixing ratio of the mixture is not particularly limited as far as it includes lipid, cholesterol and a solvent. Preferably, the mixture contains 0.1 to 1 part by weight of lipid and 0.01 to 1 part by weight of cholesterol per 100 parts by weight of the solvent.

If less than 0.1 parts by weight of the lipid is contained relative to 100 parts by weight of the solvent, there may arise a problem that the efficiency of liposome formation is decreased. When the liposome is contained in an amount exceeding 1 part by weight based on 100 parts by weight of the solvent, There is a problem that the degree of encapsulation of the capsule can be reduced.

In addition, if less than 0.01 part by weight or more than 1 part by weight of cholesterol is contained in 100 parts by weight of the solvent, a problem that the degree of encapsulation of the lysosomal enzyme decreases may occur.

Next, the steps of preparing the liposome film by removing the solvent from the mixture will be described.

The method of removing the solvent from the mixture is not particularly limited as long as it is a method for producing the liposome film in the production of the liposome. Preferably, a method of removing the solvent using a vacuum evaporator or an oven can be used, The solvent may be removed by treating the mixture with a vacuum evaporator at 40 to 50 ° C for 5 to 20 minutes to remove the solvent and / or drying the resultant in an oven for 1 to 3 hours.

Next, a step of preparing a liposome encapsulated with a lysosomal enzyme by adding a lysosomal enzyme to the liposome film will be described.

The lysosomal enzyme is not particularly limited as long as it is a substance exhibiting an antimicrobial activity isolated or purified in a lysosome. Preferably, lysosomal enzyme, cathepsin B, cathepsin D, cathepsin A, acid carboxypeptide hydrolase, The enzyme is selected from the group consisting of protease, neutral protease, elastase, hyaronidase, lysozyme, neuraminidase, fucosidase, triacylglycerol, phosphatidic acid, sphingomyelin, ribonucleic acid hydrolase and deoxyribose And a nucleic acid hydrolase, and more preferably, it may contain lysosomal enzyme.

In addition, the lysosomal enzyme is not particularly limited as long as it is a microorganism such as a eukaryote, in one embodiment, a yeast, or an eukaryotic cell of an animal. Preferably, the lysosome enzyme is isolated from one or more selected from the group consisting of yeast, insect, And more preferably, those isolated from at least one selected from the group consisting of Saccharomyces cerevisiae or Bacillus subtilis may be used, and most preferably, they may be isolated from brewer's yeast.

Furthermore, the form of the lysosomal enzyme to be added to the liposome film is usually manufactured or sold, and is not particularly limited as long as it contains a lysosomal enzyme. Preferably, a sodium phosphate buffer solution containing a lysosomal enzyme is used But is not limited thereto.

The encapsulation of the lysosomal enzyme means that the lysosomal enzyme is encapsulated in the lipid, which can prevent the lysosomal enzyme from being released to the outside during storage to lose the physiological activity of the lysosomal enzyme and, while the lysosomal enzyme is moving to the desired target site It is possible to prevent the physiological activity from being lost.

The mixing ratio of the liposome film and the lysosomal enzyme is not particularly limited as long as it is capable of producing the liposome. Preferably, 5 to 10 parts by weight of the lysosomal enzyme may be added to 100 parts by weight of the liposome film.

If less than 5 parts by weight or more than 10 parts by weight of the lysosomal enzyme is added to 100 parts by weight of the liposomal film, the encapsulation degree of the lysosomal enzyme may decrease.

The method for producing the liposome encapsulated with the lysosomal enzyme is not particularly limited as long as it is a method for producing liposomes. Preferably, the liposome is prepared by vortexing and / or an ultrasonic processor for 1 to 10 minutes at 0 to 25 ° C For 1 to 10 seconds, may be used. The intensity of the ultrasonic waves used in the ultrasonic processor may be 30 to 45 Hz. If the ultrasonic intensity is less than 30 Hz, a problem may arise that the size of the generated lysosome is greatly increased. When the ultrasonic intensity is more than 45 Hz, the liposome generated before the ultrasonic treatment may be broken May occur.

The liposome of the present invention produced through the above-mentioned method can stably encapsulate the lysosomal enzyme to easily utilize the physiological activity of the lysosomal enzyme, and thus can be utilized as a functional cosmetic composition and a pharmaceutical composition.

Hereinafter, the structure and effect of the present invention will be described in more detail with reference to examples and comparative examples. However, this embodiment is only an example for explaining the present invention in more detail, and the scope of the present invention is not limited to these embodiments.

[ Example ]

Preparation Example  1. Production of lysosomal enzymes

Lysosomal enzyme was isolated from bacillus yeast (Saccharomyces cerevisiae). The brewer's yeast strain was purchased from Korea Research Institute of Bioscience and was added to the YPD medium. The YPD medium was incubated for 12 hours in an incubator at 30 ° C shaking at 200 rpm. The YPD medium contains 1% yeast extract, 2% peptone and 4% glucose.

The brewer's yeast was cultured up to an exponential phase (log-phase), followed by treatment with a centrifuge at 3,000 rpm for 10 minutes to remove the supernatant, followed by culture of the brewer's yeast cultured in the supernatant-free medium. The obtained beer yeast is tree -SO ₄ buffer solution (Tris-SO ₄ buffer, dithiothreitol (DTT in 1M per 100 ㎖; solution containing dithiothreitol)) and mixed with a suspension of beer yeast obtained by preparing a mixed solution (Suspending). The mixture was centrifuged at 3,000 rpm for 10 minutes, and the supernatant was removed to obtain yeast cells. The obtained yeast was re benefit by removing the yeast was resuspended and again centrifuged at 3,000 rpm using a sorbitol -K ⁺ buffer (sorbitol-K ⁺ buffer) supernatant again. The reacquired yeast was subjected to ultrasonic disruption (10: 0 sec on / off pulse) at 40 watts (W) for 5 minutes, centrifuged at 3,000 rpm for 10 minutes, and the supernatant was removed to obtain lysosomal enzyme. The supernatant was mixed with a destroying solution (solution containing 50 μl of 1 M phenylmethylsulfonyl fluoride (MSF) per 50 ml), and ultrasonically pulverized (10:10 sec on / off pulse) for 30 minutes After centrifugation at 500 rpm for 5 minutes, the supernatant was removed by centrifugation at 20,000 x g for 30 minutes, and lysosomal enzyme was further obtained. Finally, the obtained lysosomal enzyme was mixed with a lysis buffer solution (solution containing 1% NP-40, 1 mM DTT and 1 mM PMSF) at a ratio of 1: 1 (ex-L: L) Lt; RTI ID = 0.0 > 15,000 rpm. ≪ / RTI >

Example  One.

L-α-phosphatidylcholine (product of Sigma-Aldrich) was dissolved in 10 ml of chloroform, and then dried in a vacuum evaporator at 45 ° C. for 10 minutes. The chloroform was removed for a period of time to prepare a liposome film. Then, 1 ml of a sodium phosphate buffer containing 250 ㎍ of the lysosomal enzyme obtained in Preparation Example 1 was added to the liposome film. The liposome film containing the sodium phosphate buffer solution was vortexed for 1 minute and then ultrasonically disrupted at 40 Hz in an ice bath through an ultrasonic disintegrator to prepare a liposome capsule solution.

Example  2 to 4.

Liposomal capsules were prepared using the same procedure as in Example 1 except that the lysosomal enzymes were added to the liposomal membranes in the presence of 500 μg (Example 2), 1000 μg (Example 3) or 2000 μg (Example 4) Buffer solution was added.

Example  5 to 9.

Liposomal capsules were prepared using the same procedure as in Example 1 except that 9 mg and 1 mg (Example 5), 8 mg and 2 < RTI ID = 0.0 > (L-a-phosphatidylcholine and cholesterol (Sigma- A liposome solution was prepared by adding 1 mg (Example 6), 7 mg and 3 mg (Example 7), 6 mg and 4 mg (Example 8), or 5 mg and 5 mg (Example 9).

Experimental Example  1. Measurement of encapsulation%

Experimental Example  1-1: Example  One Liposomal  % Encapsulation% measurement

The liposome capsule solution of Example 1 was centrifuged at 13,000 rpm for 20 minutes to separate and remove the supernatant. The supernatant-separated liposome solution was analyzed by a bradford protein assay method (Bio-Rad, Richmond, CA, USA) Ltd.) was used to measure the amount of lysosomal enzyme encapsulated in the liposome. The measurement results are shown in Table 1 below.

division The amount of added lipid (mg) The amount of added lysosomal enzyme ([mu] g) Amount of encapsulated lysosomal enzyme ([mu] g) Encapsulation% Example 1 10 mg 250 [mu] g 98 [mu] g 19.6% Example 2 10 mg 500 [mu] g 120 [mu] g 24.0% Example 3 10 mg 1000 μg 480 [mu] g 48.0% Example 4 10 mg 2000 μg 451 [mu] g 22.5%

As shown in Table 1, when the liposome capsules were prepared using 250 의 of lysosomal enzyme in Example 1, the encapsulation% of the lysosomal enzyme was about 20%, but the addition amount of the lysosomal enzyme was changed to 500 또는 or 1000 로 It was confirmed that the liposome capsules of Examples 2 and 3 exhibited encapsulation of 24.0% or 48.0%, respectively. However, the liposome encapsulated in Example 4, which was prepared by adding 2000 μg of lysosomal enzyme, exhibited a reduction of encapsulation by 50% or more as compared with the liposome encapsulation of Example 3, which was prepared by adding 1000 μg of lysosomal enzyme to 22.5% .

The results showed that the mixing ratio of lipid and enzyme to maximize encapsulation of lysosomal enzyme was 0.2 to 2 mg of lysosomal enzyme per 10 mg of lipid, which is the mixing ratio for preparing liposome capsules having the highest lysosome enzyme encapsulation effect.

Experimental Example  1-2. Example  2 Liposomal  Encapsulation%

The% encapsulation of the liposome solution prepared in Examples 1 and 2 was measured in the same manner as in the measurement of the encapsulation% of liposome in Experimental Example 1-1. The measurement results are shown in Table 2 below.

division The amount of added lysosomal enzyme ([mu] g) The amount of added lipid (mg) Amount of added cholesterol (mg) Amount of encapsulated lysosomal enzyme ([mu] g) Encapsulation% Example 2  500 [mu] g 10 mg 0 mg 120 [mu] g 24% Example 5  500 [mu] g 9 mg 1 mg 370 [mu] g 74% Example 6  500 [mu] g 8 mg 2 mg 320 [mu] g 64% Example 7  500 [mu] g 7 mg 3 mg 300 [mu] g 60% Example 8  500 [mu] g 6 mg 4 mg 220 [mu] g 44% Example 9  500 [mu] g 5 mg 5 mg 200 [mu] g 40%

As can be seen from the above Table 2, it can be seen that the liposome capsules prepared by adding cholesterol encapsulate a larger amount of lysosomal enzyme. It was also found that when the ratio of cholesterol and lipid added was 1: 9 by weight to 3: 7 by weight, encapsulation of the lysosomal enzyme was increased.

Experimental Example  2. Liposome  image

20 μl of each of the liposome capsule solutions prepared in Example 2, Examples 5 to 7 and Example 9 was dissolved in 400 mesh of a copper-carbon material using an aqueous solution containing 2% acetic acid uranyl acetate And then the mesh was washed with purified water to obtain a liposome capsule. Thereafter, the liposome capsules obtained liposome images with a transmission electron microscope (manufactured by Hi-Tech Co., Ltd., Japan). The liposome images of Example 2, Examples 5 to 7 and Example 9 are shown in FIGS. 1 to 5, respectively.

As shown in Figs. 1 to 5, it was confirmed that liposomes were produced in each of the liposome solutions. However, as a result of comparing the images of FIG. 1 and FIGS. 2 to 5, it was confirmed that liposome formation was good in FIGS. 2 to 5 in which cholesterol was added than in FIG.

2 to 5, it was found that the mixing ratio of lipid and cholesterol was 9 (weight ratio), 1 (weight ratio), 7 weight ratio and 3 weight ratio (FIG. 4) It was confirmed that the liposome was formed well in the liposome solution prepared using the weight ratio and the ratio by weight of 2 (FIG. 3).

Further, as shown in the image of FIG. 3, it was confirmed that liposome capsules prepared by adding lipid and cholesterol at an amount of 8 weight ratio and 2 weight ratio produced liposome capsules of various diameters ranging from 100 to 500 nm.

Experimental Example  3. Measurement of lysosomal enzyme release

To evaluate the stability of the liposome capsules prepared in Examples 2 and 6, the amount of lysosomal enzyme released in the liposome capsules for 6 days was measured.

The liposome capsules of Example 2 and the liposome capsules of Example 6 were washed with sodium phosphate buffer (pH 6.0, 0.1 M), and the liposome capsules were added to a new sodium phosphate buffer solution to measure the lysosomal enzyme concentration Respectively. The concentration of the measured lysosomal enzyme is shown in Fig.

6, it was confirmed that the lysosomal enzyme was released from both the liposome of Example 2 and the liposome of Example 6 after one day after adding the liposome capsules to the new sodium phosphate buffer solution. However, (Using only lipids) released more lysosomal enzymes than the liposomes of Example 6 (using lipid and cholesterol).

Also, after 3 days, the liposome enzyme of Example 2 (lipid only) released a large amount of lysosomal enzyme of 0.4 mg, whereas the liposome of Example 6 (using lipid and cholesterol) was less than the liposome of Example 2 It was confirmed that a positive lysosomal enzyme was released.

Furthermore, after 6 days, 0.5 mg of lysosomal enzyme was released in the liposome of Example 2, and 0.2 mg of lysosomal enzyme was released in the liposome of Example 6. Thus, liposomes prepared using lipid alone were found to release liposomal enzymes about twice as much as liposomes using lipids and cholesterol.

As can be seen from the above Examples and Experimental Examples, it was confirmed that the capsules of the lysosomal enzymes were improved by the liposome capsules prepared by containing 500 to 1000 μg of lysosomal enzyme in 10 mg of lipid. In addition, it was confirmed that liposome capsules prepared by adding lipids and cholesterol to the 500 μg of lysosomal enzyme liposome capsules prepared by adding only lipids improve the degree of encapsulation of the lysosomal enzymes and stabilize the liposomal capsules to decrease the release of lysosomal enzymes I could.

Claims (13)

5 to 10 parts by weight of a lysosomal enzyme isolated from at least one selected from the group consisting of Saccharomyces cerevisiae or Bacillus subtilis, and 5 to 40 parts by weight of cholesterol relative to 100 parts by weight of lipid, wherein the lysosomal enzyme is encapsulated ). ≪ / RTI > The method according to claim 1, wherein the lysosomal enzyme is selected from the group consisting of lysosomelanzyme, cathepsin B, cathepsin D, cathepsin A, acid carboxypeptide hydrolase, collagen hydrolase, neutral protease, And at least one member selected from the group consisting of alanine, lysozyme, neuraminidase, fucosidase, triacylglycerol, phosphatidic acid, sphingomyelin, ribonucleic acid hydrolase and deoxyribonucleic acid hydrolase ≪ / RTI > The method of claim 1, wherein the lipid is selected from the group consisting of L-alpha-phosphatidylcholine, L-alpha-phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, Diaryl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, di-amyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, and 1,2-diphytanoyl-3-sn-phosphatidylcholine. 3-sn-phosphatidylcholine). ≪ / RTI > delete delete delete 4. The liposome capsule according to any one of claims 1 to 3, wherein the liposome has an average diameter of 50 to 500 nm. Lipids, cholesterol and a solvent to prepare a mixture;
Removing the solvent from the mixture to produce a liposome film; And
Preparing a liposome encapsulated liposome enzyme by adding a lysosomal enzyme isolated from at least one selected from the group consisting of Saccharomyces cerevisiae or bread enzyme to the liposome film;
Lt; / RTI >
Wherein the mixture comprises 0.1 to 1 part by weight of lipid and 0.01 to 1 part by weight of cholesterol relative to 100 parts by weight of the solvent, and the lysosomal enzyme added to the liposome film is added with 5 to 10 parts by weight of lysosomal enzyme per 100 parts by weight of the liposome film ≪ / RTI > 9. The method of claim 8, wherein the lysosomal enzyme is selected from the group consisting of lysosomelanzyme, cathepsin B, cathepsin D, cathepsin A, acid carboxypeptide hydrolase, collagen hydrolase, neutral protease, And at least one member selected from the group consisting of alanine, lysozyme, neuraminidase, fucosidase, triacylglycerol, phosphatidic acid, sphingomyelin, ribonucleic acid hydrolase and deoxyribonucleic acid hydrolase ≪ / RTI > delete 9. The method of claim 8, wherein the liposome encapsulated liposome is prepared by vortexing for 1 to 10 minutes, sonicating at 30 to 45 Hz using an ultrasonic processor at 1 to 25 < For 10 seconds. ≪ RTI ID = 0.0 > 11. < / RTI > 9. The method of claim 8, wherein the lipid is selected from the group consisting of L-alpha-phosphatidylcholine, L-alpha-phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, Diaryl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, di-amyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, and 1,2-diphytanoyl-3-sn-phosphatidylcholine. 3-sn-phosphatidylcholine). ≪ / RTI > 9. The method of claim 8, wherein the solvent comprises at least one selected from the group consisting of chloroform and methanol.

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