WO2008123685A1 - Liposome sensitive to ph or reductive condition and method of preparing the same - Google Patents

Abstract

Provided are a liposome formed by self-assembling a cucurbituril derivative of formula 1 in the specification, such as a liposome, in which a drug is encapsulated and/or a targeting material is embedded, and a method of preparing such a liposome.

Description

LIPOSOME SENSITIVE TO PH OR REDUCTIVE CONDITION AND METHOD OF PREPARING THE SAME

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims the benefit of Korean Patent Application No. 10-2007- 0033349, filed on April 4, 2007, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a liposome and a method of preparing the same, and more particularly, to a liposome composed of a cucurbtturi! derivative, which does not decompose in blood when administered to the body, but decomposes in low pH conditions inside an endosome or in a reductive condition inside a cell when absorbed by the cell through endocytosis, so that the liposome does not affect systemically but can affect specifically in a tissue, and a method of preparing the same.

2. Description of the Related Art

Much time and money has been invested into the development of pharmacologically active substances. The effective application of pharmacologically active substances requires efficient drug delivery systems. Thus, much research has been conducted on the development of drug delivery systems in many countries, and gene delivery substances relating to these drug delivery systems now form a very large market.

Conventional methods of effectively delivering pharmacologically active substances, for example, drugs and genes, use a retroviral vector, nanoparticles, a liposome, etc. The method using a retrovirus has a high transfection efficiency, but is limited in use because it induces an in vivo immune reaction. Artificially synthesized nanoparticles do not induce an in vivo immune reaction and are relatively stable, and has lower production costs. However, the nanoparticles cannot effectively encapsulate drugs or genes. A liposome designed as a drug delivery system refers to a vesicle that has the structure of a bimolecular layer and is obtained by suspending an amphiphile in water. A liposome can encapsulate a large amount of pharmacologically active substance. A liposome having a modified surface can be specifically transported to a target site, and thus can be used as a targeting liposome which can increase the concentration of a pharmacologically active substance only around the target organ or target tissue.

Cucurbituril was first reported by R. Behrend, E. Meyer, and F. Rusche in 1905. In 1981 , this substance was rediscovered by W. Mock and his coworkers. W. Mock and his coworkers correctly characterized cucurbituril as a hexameric macrocyclic compound with the chemical formula C₃₆H₃₆N₂₄Oi₂, which was confirmed by X-ray diffraction (J. Am. Chem. Soc. 1981 , 103, 7367). They named it cucurbituril[6]. Since then, an improved method of synthesizing cucurbituril[6] has been disclosed (DE 196 03 377 A1 ). In 2000, Kimoon Kim and his coworkers reported improved preparation and separation of cucurbituril[6] and its homologues, cucurbiturils[n] (n = 5, 7, 8), and identified their structures by X-ray diffraction (J. Am. Chem. Soc. 2000, 122, 540).

Meanwhile, WO 00/68232 discloses cucurbituril[n] represented by Reference Diagram 1 below: [Reference Diagram 1]

where n is an integer from 4 to 12.

The above-described cucurbituril derivatives are compounds including unsubstituted glycoluril monomer units. Cucurbituril is a macrocyclic compound and has a lipophilic cavity and two hydrophilic entrances at upper and lower ends. Lipophilic interactions occur in the lipophilic cavity of the cucurbituril, and hydrogen bonding, polar-polar interactions, and positive charge-polar interactions occur in the two hydrophilic entrances, each of which has six carbonyl groups. Therefore, cucurbituril can include various compounds by forming very stable non-covalent bonds with these compounds. Cucurbituril forms a complex, particularly with a compound having an amino group or a functional group such as a carboxyl acid, ferrocene, and adamamtane, by forming a very stable un-covalent linkage.

In addition, Kimoon Kim and his coworkers synthesized a liposome by using amphiphilic cucurbituril[n] derivatives. In the liposome synthesized using the amphiphilic cucurbiturilfn] derivatives, various kinds of compounds can be embedded by un-covalent bonds in the cavity of the cucurbituril which forms the liposome.

Therefore, the liposome can be used as a targeting drug delivery system (J. Am.

Chem. Soc. 2005, 127, 5006-5007; Korea Patent Publication No. 2005-0102295).

In general, a liposome enters a cell by endocytosis. Then, the liposome is surrounded by an endosome inside the cell. The pH inside the endosome is lower than outside the cell, meaning that the endosome is acidic. The pH inside the endosome is about 6.5 or lower. Since the inside of the endosome is acidic, a substance having a functional group that easily decomposes in acidic conditions, such as ester, orthoester, phosphoester, acetal, or imine etc. may easily decompose. In addition, the inside of the cell has a reductive condition (about 5 mM glutathione) compared to the outside of the cell, so that a disulfide functional group decomposes into thiols (Angew. Chem. Int. Ed. 45, 8. 1198., Biomacromolecules 2005, 6, 24.)

In the discovery of the present invention, the inventors performed research into a liposome composed of amphiphilic cucurbituril deliveries having excellent drug delivery properties. As a result, they found that if a functional group that easily decomposes in acidic conditions inside an endosome, such as ester, orthoester, phosphoester, acetal, imine etc, or a functional group that decomposes in reductive conditions inside a cell, such as a disulfide, is introduced to a liposome system formed of a cucurbituril derivative, then when the liposome enters a cell by endocytosis, the liposome can act as an excellent drug delivery system which decomposes in tissue but not in blood, so that drugs can be delivered to the tissue, without causing any systemic side effects.

SUMMARY OF THE INVENTION The present invention provides a liposome composed of a cucurbituril derivative, which does not decompose in blood but decomposes after being absorbed by tissue.

The present invention also provides a liposome composed of a cucurbituril derivative, modified with a targeting compound.

The present invention also provides a liposome composed of a cucurbituril derivative, encapsulating a pharmacologically active substance.

The present invention also provides a method of preparing the liposome composed of a cucurbituril derivative, which does not decompose in blood but decomposes after being absorbed by tissue.

According to an aspect of the present invention, there is provided a liposome formed by self-assembling a cucurbituril derivative of formula 1 :

where X is O, S, or NH; Ai and A₂ are respectively OR¹ and OR², SR¹ and SR², or NHR¹ and NHR² wherein each of R¹ and R² is independently a hydrophilic functional group so that the compound of formula 1 has an amphiphilic property to form a liposome, and R¹ or R² additionally includes ester, orthoester, acetal, imine, or disulfide in the middle thereof; and n is an integer from 4 to 20.

A surface of the liposome may be modified by embedding a targeting compound in a cavity of the cucurbituril derivative composing the liposome such that a targeting moiety of the targeting compound is exposed to the outside of the liposome. A pharmacologically active substance may be encapsulated as a guest molecule in the liposome or the liposome having its surface modified by the targeting compound.

According to another aspect of the present invention, there is provided a method of preparing a liposome formed by self-assembling the cucurbituril derivative of formula 1 , the method comprising: dissolving a cucurbituril derivative of formula 1 in an organic solvent and drying the resultant solution; and adding water to the dried compound and dispersing the compound.

According to another aspect of the present invention, there is provided a method of preparing a liposome formed by self-assembling the cucurbituril derivative of formula 1 , in which a targeting compound is embedded in a cavity of the cucurbituril derivative of formula 1 composing the liposome, the method comprising: dissolving the cucurbituril derivative of formula 1 in an organic solvent and drying the resultant solution; adding water to the dried compound and dispersing the compound; adding a targeting compound or a solution of the targeting compound to the dispersion solution and stirring the resultant mixture; and removing residual un- embedded targeting compound by dialysis.

According to another aspect of the present invention, there is provided a method of preparing a liposome formed by self-assembling the cucurbituril derivative of formula 1 , in which a pharmacologically active substance is encapsulated, the method comprising: dissolving the cucurbituril derivative of formula 1 in an organic solvent and drying the resultant solution; adding an aqueous solution of the pharmacologically active substance to the dried compound and dispersing the compound; and removing residual un-encapsulated pharmacologically active substance in the dispersion by dialysis. According to another aspect of the present invention, there is provided a method of preparing a liposome formed by self-assembling the cucurbituril derivative of formula 1 , in which a pharmacologically active substance is encapsulated and a targeting compound is embedded in a surface of the liposome, the method comprising: dissolving a cucurbituril derivative of formula 1 in an organic solvent and drying the resultant solution; adding an aqueous solution of the pharmacologically active substance to the dried compound and dispersing the compound; adding a targeting compound or a solution of the targeting compound to the dispersion solution and stirring the resultant mixture; and removing residual un-encapsulated pharmacologically active substance and residual un-embedded targeting compounds by dialysis.

BRIEF DESCRIPTION OF THE DRAWINGS The above and other features and advantages of the present invention will become more apparent by describing in detail exemplary embodiments thereof with reference to the attached drawings in which:

FIG. 1 is a schematic view of a liposome having a surface modified with a targeting compound, which encapsulates a pharmacologically active substance;

FIGS. 2 through 9 are transmission electron microscope (TEM) images of a liposome prepared by self-assembling a cucurbituril[6] derivative represented by formula 1 obtained according to an embodiment of the present invention;

FIG. 10 is a TEM image of a liposome prepared by self-assembling a cucurbituril[7] derivative represented by formula 1 obtained according to an embodiment of the present invention;

FIG. 11 is a TEM image of a liposome prepared by self-assembling a cucurbituril[8] derivative represented by formula 1 obtained according to an embodiment of the present invention; FIGS. 12 and 13 are TEM images of an albumin-encapsulating liposome obtained according to an embodiment of the present invention;

FIG. 14 is a UV absorption graph of an albumin-encapsulating liposome obtained according to an embodiment of the present invention;

FIGS. 15 and 16 are TEM images of a hydrocortisone-encapsulating liposome obtained according to an embodiment of the present invention;

FIG. 17 shows an ESI-Mass analysis of a hydrocortisone-encapsulating liposome obtained according to an embodiment of the present invention;

FIGS. 18 and 19 are TEM images of an insulin-encapsulating liposome obtained according to an embodiment of the present invention; FIG. 20 is a TEM image of a calcitonin-encapsulating liposome obtained according to an embodiment of the present invention;

FIGS. 21 and 22 are TEM images of an albumin-encapsulating liposome having a surface modified with mannose according to an embodiment of the present invention; FIGS. 23 and 24 are TEM images of a doxorubicin-encapsulating liposome having a surface modified with folate according to an embodiment of the present invention; FIG. 25 is a UV absorption graph of a doxorubicin-encapsulating liposome having a surface modified with folate according to an embodiment of the present invention;

FIGS. 26-30 are TEM images of liposomes obtained according to an embodiment of the present invention after being exposed to a pH of 5.5;

FIG. 31 shows confocal laser microscopy images of a liposome prepared according to an embodiment of the present invention and then modified with FITC- spermidine on the surface thereof, before and after the liposome is exposed to pH 5.5 or in a 5 mM glutathione solution; FIG. 32 is a TEM image of a sulforodamin G-encapsulating liposome prepared according to an embodiment of the present invention and then modified with FITC- spermidine on the surface thereof;

FIG. 33 shows confocal laser microscopy images of a sulforodamin G- encapsulating liposome prepared according to an embodiment of the present invention and then modified with FITC-spermidine on the surface thereof;

FIG. 34 shows the results of a Bradford assay of an insulin-encapsulating liposome obtained according to an embodiment of the present invention, before and after the liposome is exposed to glutathione reductive conditions;

FIG. 35 is a TEM image of a liposome prepared according to an embodiment of the present invention and then modified with FITC-spermine and folate-spermidine on the surface thereof;

FIG. 36 shows confocal laser microscopy images of KB cells which are treated with a liposome obtained according to an embodiment of the present invention in which a surface is modified with both folate-spermidine and FITC-spermine or FITC- spermine only,; and

FIG. 37 and FIG. 38 are graphs of the concentration of doxorubicin with respect to cell survivability rate of a doxorubicin-encapsulating liposome obtained according to an embodiment of the present invention, after the doxorubicin-encapsulating liposome is used to treat KB cells at various concentrations.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. In an embodiment of the present invention, a liposome is formed by self- assembling a cucurbituril derivative. The liposome includes a space filled with an aqueous solution and has a diameter of several tens to several hundreds of nanometers. The cucurbituril derivative composing the liposome includes ester, orthoester, acetal, imine, or disulfide group, so that when the liposome enters a cell by endocytosis, the cucurbituril derivative can be decomposed in the acidic conditions of pH 6.5 or less inside an endosome, or in specific reductive conditions inside the cell. Such a liposome reliably retains its shape in neutral blood when it is injected into the body, and decomposes when it is absorbed by a cell. The cucurbituril derivative composing the liposome according to the present invention may be represented by formula 1 :

where X is O, S, or NH;

A₁ and A₂ are respectively OR¹ and OR², SR¹ and SR², or NHR¹ and NHR² wherein each of R¹ and R² is independently a hydrophilic functional group, so that the compound of formula 1 has an amphiphilic property to form a liposome, and R¹ or R² additionally has a middle portion including ester, orthoester, acetal, imine, or disulfide; and n is an integer from 4 to 20. In the compound of formula 1 , the hydrophilic functional group can be any functional group that provides an amphiphilic property to the compound of formula 1 in order to form a liposome. Specifically, each of R¹ and R² is independently C₅-C_2O alkyl, C₅-C₂₀ alkenyl, C₅-C₂₀ alkynyl, C₅-C₂₀ carbonylalkyl, C₅-C₂₀, thioalkyl, C₅-C₂₀ alkylthiol, C₅-C₂₀ hydroxyalkyl, C₅-C₂₀ alkylsilyl, C₅-C₂₀ aminoalkyl, C₅-C₂₀ cycloalkylalkyl, C₅-C₂₀ heterocycloalkylalkyl, C₅-C₂₀ arylalkyl, or C₅-C₂₀ heteroarylalkyl.

Each of the alkyl, alkenyl, and alkynyl has at least one carbon atom substituted with a hetero atom selected from the group consisting of oxygen, nitrogen, and sulfur. In each of the alkyl, alkenyl, and alkynyl, the number of carbon atoms is larger than the number of substituted hetero atoms,

The hydrophilic functional group can be substituted or unsubstituted with amino, hydroxy, an amino acid, a peptide composed of two to ten amino acids, hexoses, or pentoses.

Further, the hydrophilic functional group in the compound of formula 1 can be each independently -O(C₂H₄O)_nH or -0(CaH₄O)_nCH₃ wherein n is an integer of 3 to 6, or poly ethyleneglycol or poly ethyleneglycol monomethtyl ether (M.W. = 1000 ~ 5000), wherein the hydrophilic functional group is substituted or unsubstituted with hydroxy, amino, an amino acid, a peptide composed of two to ten amino acids, hexoses, or pentoses.

Thus, each of R¹ and R² may be the hydrophilic functional group described above and additionally includes one of ester, orthoester, acetal, imine, and disulfide in the middle thereof.

For example, each of R¹ and R² may be represented by one of the structures below:

,N

— o ϊτ^^ε e)

The liposome formed by self-assembling the cucurbituril derivative of formula

1 may be provided with a targeting property by modifying its surface with a targeting compound. The cucurbituril derivative of formula 1 is an inclusion compound which has a cavity in its molecule, as illustrated in Reference Diagram 1 above, and thus a targeting compound can be embedded in the cavity.

Examples of the targeting compound that can be embedded in the cavity of the cucurbituril at a surface of the liposome include, but are not limited to, a compound of formula 2:

A B T

-(2) where A is 1 ,3-diaminopropyl, 1 ,4-diaminobutyl, 1 ,5-diaminopentyl, 1 ,6-diaminohexyl, sperminyl, spermidinyl, propylamine butylamino, pentylamino, hexylamino, biologinyl, pyridinyl, ferrocenyl, amino acid, or adamantanyl; B is selected from the group consisting of a hydrogen atom, a substituted or unsubstituted C1-C₃₀ alkyl, a substituted or unsubstituted C₂-C₃₀ alkenyl, a substituted or unsubstituted C₂-C₃₀ alkynyl, a substituted or unsubstituted C₂-C₃₀ carbonylalkyl, a substituted or unsubstituted CrC₃₀ thioalkyl, a substituted or unsubstituted CrC₃₀ alkylthiol, a substituted or unsubstituted C₁-C₃₀ alkoxy, a substituted or unsubstituted CrC₃₀ hydroxyalkyl, a substituted or unsubstituted C₁- C₃₀ alkylsilyl, a substituted or unsubstituted C₁-C₃₀ aminoalkyl, a substituted or unsubstituted C₁-C₃₀ aminoalkylthioalkyl, a substituted or unsubstituted C₅-C₃₀ cycloalkyl, a substituted or unsubstituted C₂-C₃₀ heterocycloalkyl, a substituted or unsubstituted Ce-C₃₀ aryl, a substituted or unsubstituted C₆-C₂₀ arylalkyl, a substituted or unsubstituted C₄-C₃₀ heteroaryl, and a substituted or unsubstituted C₄- C₂₀ heteroarylalkyl; and

T is a targeting moiety selected from the group consisting of a saccharide, a polypeptide, a protein, and an oligonucleotide, each of which can recognize and bond to a desired cell. Examples of the saccharide may include, but are not limited to, glucose, mannose, and galactose. Examples of the protein for the targeting moiety may include, but are not limited to, lectin, selectin, transferrin, herceptin, and antibody.

The targeting compound of formula 2 can be embedded in the cavity of the cucurbituril at the surface of the liposome as illustrated in Reference Diagram 2 below:

[Reference Diagram 2]

In the compound of formula 2, A is designed to be easily embedded in the cucurbituril derivative, specifically in the cavity of the cucurbituril derivative which is exposed at the surface of the liposome when the cucurbituril derivative forms the liposome. This strategy allows the surface of the liposome to be modified with the targeting moiety T, which is connected to A via a linkage portion B, as illustrated in Reference Diagram 2.

A liposome formed by self-assembling the cucurbituril derivative of formula 1 and a liposome having the targeting compound embedded in its surface can function as drug carriers. Thus, a pharmacologically active substance can be encapsulated as a guest molecule in an empty space of the liposome. When the liposome according to the present invention encapsulates drugs and is injected into the body, the liposome remains stable in blood, but when it is absorbed by a cell through endocytosis, a functional group, which is included in R¹ or R² of the cucurbituril derivative of formula 1 and decomposes in a pH of 6.5 or less or in reductive conditions inside the cell, may be cut, allowing the liposome to decompose. Accordingly, the liposome encapsulating a drug according to the present invention does not release the drug into the blood, to prevent side effects due to a systemic reaction caused by the drug, but releases the drug after being absorbed by tissue, to obtain the desired drug effect. In addition, the liposome encapsulating a drug according to the present invention further includes a targeting material embedded in the liposome, so that the liposome can specifically react with a target site in the body and thus prevent side effects due to the reaction of the drug with sites other than the target site. Accordingly, the liposome according to the present invention can release a drug after the cucurbituril derivative formula 1 composing the liposome is absorbed by the tissue, and the cucurbituril derivative formula 1 composing the liposome can embed a targeting material in its cavity, and thus, the drug can specifically react with a target site. As a result, the liposome can be used as an efficient drug delivery system. FIG. 1 is a schematic view of a pharmacologically active substance encapsulating liposome having a surface modified with the targeting compound of formula 2.

Examples of the pharmacologically active substance may include an organic compound, a protein, an oligonucleotide, etc. Examples of the organic compound may include, but are not limited to, hydrocortisone, prednisolone, spironolactone, testosterone, megesterol acetate, danasole, progesterone, indomethacin, amphotericin B, and a mixture thereof.

Examples of the protein may include, but are not limited to, human growth hormone, G-CSF (granulocyte colony-stimulating factor), GM-CSF (granulocyte- macrophage colony-stimulating factor), erythropoietin, a vaccine, an antibody, insulin, glucagon, calcitonin, ACTH (adrenocorticotropic hormone), somatostatin, somatotropin, somatomedin, parathyroid hormone, thyroid hormone, a hypothalamus secretion, prolactin, endorphin, VEGF (vascular endothelial growth factor), enkephalin, vasopressin, a nerve growth factor, an opioid not naturally occurring, interferon, asparaginase, alginase, superoxide dismutase, trypsin, chymotrypsin, pepsin, and a mixture thereof.

A method of preparing a liposome formed by self-assembling the cucurbituril derivative of formula 1 includes: dissolving the cucurbituril derivative of formula 1 in an organic solvent and drying the resultant solution; and adding water to the dried compound and dispersing the compound.

A method of preparing a pharmacologically active substance encapsulating liposome formed by self-assembling the cucurbituril derivative of formula 1 includes: dissolving the cucurbituril derivative of formula 1 in an organic solvent and drying the resultant solution; adding an aqueous solution of the pharmacologically active substance to the dried compound and dispersing the compound; and removing residual un-encapsulated pharmacologically active substance from the dispersion by dialysis.

A method of preparing a liposome which is formed by self-assembling the cucurbituril derivative of formula 1 and has a surface modified with a targeting compound includes: dissolving the cucurbituril derivative of formula 1 in an organic solvent and drying the resultant solution; adding water to the dried compound and dispersing the compound; adding the targeting compound or a solution of the targeting compound to the dispersion solution and stirring the resultant mixture; and removing residual un-embedded targeting compound by dialysis.

A method of preparing a pharmacologically active substance-encapsulating liposome which is formed by self-assembling the cucurbituril derivative of formula 1 and has a surface modified with a targeting compound includes: dissolving a cucurbituril derivative of formula 1 in an organic solvent and drying the resultant solution; adding an aqueous solution of a pharmacologically active substance to the dried compound and dispersing the compound; adding the targeting compound or a solution of the targeting compound to the dispersion solution and stirring the resultant mixture; and removing residual un-encapsulated pharmacologically active substance and residual un-embedded targeting compound by dialysis. In all of the above methods of preparing a liposome, the organic solvent may be a solvent capable of solubilizing the cucurbituril derivative. Examples of the organic solvent may include, but are not limited to, chloroform, methanol, dimethylsulfoxide, dichloromethane, dimethylformamide, tetrahydrofuran, and a mixture thereof. In adding of water or the aqueous solution of the pharmacologically active substance to the dried cucurbituril derivative and dispersing the compound after the cucurbituril derivative is dissolved in an organic solvent and dried, the volume of added water or aqueous solution may be varied such that the concentration of the cucurbituril derivative lies in the range of 10^~6 to 10^~2 M. The water or aqueous solution of the pharmacologically active substance may further include a buffering agent to maintain a neutral pH of the reaction solution during the process of preparing the liposome to prevent the decomposition of the liposome. Examples of the buffering agent include, but are not limited to, PBS, HEPES, and acetate buffer solution. After the addition of the water or aqueous solution of the pharmacologically active substance, the cucurbituril derivative must be uniformly dispersed in the water, preferably, by sonication with an ultrasonic device.

To form a liposome with a modified surface property by embedding the targeting compound of Formula 2 in the surface of a liposome or a pharmacologically active substance encapsulating liposome, the solution of the targeting compound is added to the dispersion of the liposome, and then the resultant mixture is stirred. This stirring process may be performed at a temperature ranging from room temperature to 40 ^°C . If the stirring temperature is too high, the solvent evaporates, thereby resulting in a deformation or decomposition of the liposome. The stirring process can be performed for about 30 minutes to 2 hours, typically 1 hour. Alternatively, the targeting compound may be added directly to the dispersion solution of the liposome instead of dissolving the targeting compound in a solvent prior to the adding to the dispersion of the liposome.

As described above, a liposome or a pharmacologically active substance encapsulating liposome may be formed by self-assembling the cucurbituril derivative in water or an aqueous solution of the pharmacologically active substance and dispersing the same therein. Further, a liposome having an embedded targeting compound providing a modified surface property may be prepared by embedding the targeting compound of formula 2 in a surface of the liposome. The liposomes may have diameters of several tens to 1000 nm and can be identified using an optical microscope, light-scattering, a scanning electron microscope (SEM), or a transmission electron microscope (TEM).

[Advantageous Effects] As described above, a liposome formed by self-assembling a cucurbituril derivative having a functional group that is sensitively dissociated in acidic or reductive conditions according to the present invention is stable in blood, and decomposes after the liposome is absorbed by a cell. The cucurbituril derivative of the liposome can be embedded with a targeting compound, so that systemic side effects of a drug can be prevented and the drug can react only with the desired target tissue. Therefore, the liposome according to the present invention can be used as an effective drug delivery system.

The present invention will be described in further detail with reference to the following examples. These examples are for illustrative purposes only, and are not intended to limit the scope of the present invention.

Example 1

Preparation of Liposome

3 mg of cucurbituril[6] represented by formula 1 in which X = O and each of R¹

and R² is was completely dissolved in

1 ml_ of methylalcohol, and the resultant solution was dried in air. Then, about 6 ml_ of a PBS buffer solution having a pH of 7.4 was added to the dried product, and the temperature of a water bath was controlled to 40⁰C . The product was then dispersed for about 30 minutes by sonication. Liposomes having sizes of several tens of nanometers were identified using a transmission electron microscope (TEM). The TEM image of the liposomes is shown in FIG. 2.

Examples 2-8 Preparation of Liposomes

Liposomes were prepared in the same manner as in Example 1 , except that instead of the cucurbituril[6] derivative used according to Example 1 , cucurbituril[6] derivatives, each represented by formula 1 where X = O and each of R¹ and R² has the chemical structure shown in Table 1 , were used. The liposomes were identified using TEM. As a result, the sizes of the liposomes were several tens of nanometers. The TEM images of the liposomes are shown in FIGS. 3- 9. Table 1

Examples 9-16

Preparation of Liposomes

The liposomes were prepared in the same manner as in Examples 1- 8, except that instead of the cucurbituril[6] derivatives, cucurbituril[7] derivatives having the same chemical formula as used according to Examples 1-8 except n = 7 were used. The liposomes were identified using TEM. As a result, the sizes of the liposomes were several tens of nanometers. The TEM image of the liposomes prepared according to Example 9 is shown in FIG. 10.

Examples 17-24 Preparation of Liposomes

The liposomes were prepared in the same manner as Examples 1- 8, except that instead of the cucurbituril[6] derivative, cucurbituril[8] derivatives having the same chemical formula as used according to Examples 1-8 except n = 8 were used. The liposomes were identified using TEM. As a result, the sizes of the liposomes were several tens of nanometers. The TEM image of the liposomes prepared according to Example 17 is shown in FIG. 11.

Examples 25 and 26

Albumin (protein) Encapsulating Liposome

2.3 mg of cucurbituril[6] represented by formula 1 where X=O, n=6, and each

of R¹ and R² is (Example 25) or

(Example 26) was completely dissolved in about 1 ml_ of methylalcohol, and the resultant solution was dried in air. About 6 ml_ of an aqueous solution in which 5 mg of albumin was dissolved was added to the dried product, the temperature of a water bath was controlled to 40 ⁰C , and then the product was dispersed in the aqueous solution for 30 minutes by sonication. The dispersion solution was subjected to dialysis for one day to remove albumin remaining outside the liposome. The resultant product was identified using TEM. As a result, the liposomes had sizes of 100 - 200 nm. The TEM images are shown in FIG. 12 (Example 25) and FIG. 13 (Example 26.)

In addition, the liposome prepared according to Example 25 was exposed to UV, and a strong absorption peak was identified at a wavelength of 280 nm, which is a unique absorption wavelength of albumin. Therefore, the presence of albumin in the liposome was identified. FIG. 14 is a UV absorption graph of the liposome prepared according to Example 25. Based on the results described above, it was found that a protein having a smaller size than a liposome according to the present invention is sufficiently encapsulated in the liposome.

Examples 27 and 28

Hydrocortisone (organic compound) Encapsulating Liposome

2.3 mg of cucurbituril[6] represented by formula 1 where X=O, n=6, and each

of R¹ and R² is (Example 27) or

(Example 28) was completely dissolved in about 1 ml_ of methylalcohol, and the resultant solution was dried in air. About 6 ml_ of an aqueous solution in which 1 mg of hydrocortisone was dissolved was added to the dried product, the temperature of a water bath was controlled to 40 °C , and then the product was dispersed in the aqueous solution for 30 minutes by sonication. The dispersion solution was subjected to dialysis for one day to remove hydrocortisone remaining outside the liposome. The resultant product was identified using TEM. As a result, the liposomes had sizes of 100 - 200 nm. The TEM images are shown in FIG. 15 (Example 27) and FIG. 16 (Example 28.)

In addition, ESI-Mass analysis was performed using the liposome prepared according to Example 27, and a hydrocortisone peak was identified. Therefore, the presence of hydrocortisone in the liposome was identified. FIG. 17 shows the results of the ESI-Mass analysis. Based on the results described above, it was found that an organic compound is sufficiently encapsulated in the liposome.

Examples 29 and 30 Insulin Encapsulating liposome 2.3 mg of cucurbituril[6] represented by formula 1 where X=O, n=6, and each

of R¹ and R² is (Example 29) or

(Example 30) was completely dissolved in about 1 ml_ of methylalcohol, and then the resultant solution was dried in air. About 6 ml_ of an aqueous solution in which 1 mg of insulin was dissolved was added to the dried product, the temperature of a water bath was controlled to 40 "C , and then the product was dispersed in the aqueous solution for 30 minutes by sonication. The dispersion solution was subjected to dialysis for one day to remove insulin remaining outside the liposome. The resultant product was identified using TEM. As a result, the identified liposomes had the sizes of 100-200 nm. The TEM images are shown in FIG. 18 (Example 29) and FIG. 19 (Example 30.)

Example 31

Calcitonin Encapsulating liposome

2.3 mg of cucurbituril[6] represented by formula 1 where X=O, n=6, and each

of R¹ and R² is was completely dissolved in about 1 ml_ of methylalcohol, and then the resultant solution was dried in air. About 6 ml_ of an aqueous solution in which 2 mg of calcitonin was dissolved was added to the dried product, the temperature of a water bath was controlled to 40⁰C , and then the product was dispersed in the aqueous solution for 30 minutes by sonication. The dispersion solution subjected to dialysis for one day to remove calcitonin remaining outside the liposome. The resultant product was identified using TEM. As a result, the liposomes had sizes of tens to hundreds nm. The TEM images are shown in FIG. 18 (Example 29) and FIG. 19 (Example 30.)

In addition, IR analysis was performed using the liposome prepared as described above, and an amid bond peak corresponding to a strong peptide bond was identified at a wavelength of about 1660 nm. Based on the results described above, it was found that calcitonin is sufficiently encapsulated in the liposome.

Examples 32 and 33 Preparation of Albumin Encapsulating Liposome Having Surface Modified with

Mannose

2.3 mg of cucurbituril[6] represented by formula 1 where X=O, n=6, and each

of R¹ and R² is (Example 32) or

(Example 33) was completely dissolved in about 1 ml_ of methylalcohol, and the resultant solution was dried in air. About 6 mL of an aqueous solution in which 1 mg of albumin was dissolved was added to the dried product, the temperature of a water bath was controlled to 40 ^°C, and then the product was dispersed in the aqueous solution for 30 minutes by sonication to obtain a dispersion solution of liposomes. 0.5 mg of a mannose-spermidine compound having substitute spermidine at C1 position of mannose was added to the obtained dispersion solution and then stirred for 1 hour. The stirred product was subjected to dialysis for 1 day to remove un-encapsulated albumin and un-embedded mannose-spermidine compound. The resultant product was identified using TEM. As a result, the liposomes had sizes of 100 - 200 nm. The TEM images are shown in FIG. 21 (Example 32) and FIG. 22 (Example 33.)

Examples 34 and 35

Preparation of Doxorubicin Encapsulating liposome Having Surface Modified with Folic acid-spermidine

2.3 mg of cucurbituril[6] represented by formula 1 where X=O, n=6, and each

of R¹ and R² is (Example 34) or

(Example 35) was completely dissolved in about 1 mL of methylalcohol, and then the resultant solution was dried in air. About 6 mL of an aqueous solution in which 1 mg of doxorubicin was dissolved was added to the dried product, the temperature of a water bath was controlled to 40 ⁰C , and then the product was dispersed in the aqueous solution for 30 minutes by sonication to obtain a dispersion solution of liposomes. 0.5 mg of a folate-spermidine compound was added to the dispersion solution and stirred for 1 hour. The stirred product was subjected to dialysis for 1 day to remove un- encapsulated doxorubicin and un-embedded folate-spermidine compound. The resultant product was identified using TEM. As a result, the liposomes had sizes of 100 - 300 nm. The TEM images are shown in FIG. 23 (Example 34) and FIG. 24 (Example 35.) In addition, the liposome prepared according to Example 34 was exposed to UV, and strong absorption peaks were identified at unique absorption wavelengths of doxorubicin, folate, and spermidine. FIG. 25 is a UV absorption graph of the liposome prepared according to Example 34. Based on the results described above, it was found that doxorubicin can be encapsulated in a liposome and folate- spermidine can be embedded in the surface of the liposome.

Experimental Example 1

Identification of Collapse of Liposome in Acidic or Reductive Conditions (1 )

0.5 N HCI was slowly added to each of the liposome dispersion solutions prepared according to Examples 1 through 4 such that each liposome dispersion solution had a pH of 5.5, and then the resultant solution was left to sit at room temperature for about one hour. Also, glutathione was added to the solution of the liposome prepared according to Example 6 such that the concentration of glutathione was 5 mM, and then the resultant solution was left to sit at room temperature for about one hour.

Then, these samples were identified using TEM and it was found that the liposomes had lost their original shape and collapsed. The TEM images are shown in FIGS. 26 (Example 1 ), 27 (Example 2), 28 (Example 3), 29 (Example 4), and 30 (Example 6.) Based on the results described above, it was found that when a liposome according to the present invention is absorbed by the cell, the liposome can easily collapse in the acidic conditions of pH 6.5 inside an endosome or glutathione reductive conditions inside the cell.

Experimental Example 2

Identification of Collapse of Liposome in Acidic or Reductive Conditions (2) To identify that the liposome according to the present invention collapses in acidic or reductive conditions, 0.04 mg of FITC-spermidine was added to a dispersion solution of each of the liposome (liposome sensitive to acidic conditions) prepared according to Example 1 and the liposome (liposome sensitive to reductive conditions) prepared according to Example 6, and the resultant solution was stirred for one hour. The stirred solution was subjected to dialysis for one day to remove un-embedded FITC-spermidine, and then identified by confocal laser microscopy. Round green spots were observed, which indicates that the FITC-spermidine is well embedded in the surface of the liposome.

HCI was added to the dispersion solution of the liposome prepared according to Example 1 such that the liposome dispersion solution had a pH of 5.5, and the resultant solution was left to sit at room temperature for about one hour. Also, glutathione was added to the solution of the liposome prepared according to Example 6 such that the concentration of glutathione was 5 mM, and the resultant solution was left to sit at room temperature for about one hour.

Subsequently, each liposome dispersion solution was identified by a confocal laser microscopy. The green spots which had been observed above were not found. Based on the results described above, it was identified that the liposomes collapsed in the acidic or reductive conditions. FIG. 31 shows images of the liposomes obtained by confocal laser microscopy.

Experimental Example 3 Identification of Encapsulation and Surface Modification Capabilities of

Liposome: Preparation of Sulforodamin G encapsulating Liposome Having Surface Modified with FITC

2.3 mg of cucurbituril[6] represented by formula 1 where X=O, n=6, and each

of R¹ and R² is

was completely dissolved in about 1 mL of methylalcohol and the resultant solution was dried in air. Then, about 6 mL of an aqueous solution in which about 10^~7 M sulforodamin G(SRG) was dissolved was added to the dried product, the temperature of a water bath was controlled to 40 °C , and then the product was dispersed in an aqueous solution for about 30 minutes by sonication to obtain a dispersion solution of liposomes. 0.04 mg of FITC- spermidine having spermidine substituted to FITC was added to the dispersion solution of liposome, and stirred for one hour to embed the FITC-spermidine in liposome. A one-day dialysis was used to remove residual un-encapsulated sulforodamin G and residual un-embedded FITC-spermidine, and then the resultant liposome was identified by TEM. As a result, the liposomes had a size of 100 - 200 nm. FIG. 32 is a TEM image of the prepared liposome.

To identify that the sulforodamin G was encapsulated in the liposome and the FITC-spermidine was embedded in the liposome, confocal laser microscopy was used. The image obtained by the confocal laser microscopy is shown in FIG. 33. Referring to FIG. 33, green fluorescence is emitted from the FITC at the surface of the liposome, and red fluorescence is emitted from the sulforodamin G inside the liposome. As a result, it was found that sulforodamin G was encapsulated in a liposome and FITC-spermidine was embedded in the liposome. Based on the results described above, it was found that a liposome composed of a cucurbituril according to the present invention easily encapsulates a substance and the surface of the liposome can be easily modified.

Experimental Example 4: Identification of Decomposition of Insulin Encapsulating Liposome prepared according to Example 30 in Glutathione Reductive Conditions

The liposome dispersion solution which had been prepared and refined by dialysis according to Example 30 was subjected to a Bradford assay, which is a typical protein quantitative test. As a result, no change occurred. Meanwhile, glutathione was added to the liposome dispersion solution which had been prepared and refined by dialysis according to Example 30 so that the concentration of the glutathione was 5 mM. The resultant solution was left to sit for one hour, and then the Bradford assay was performed. As a result, a color change occurred, indicating the presence of protein, in this case insulin (brown -> blue). The results of before and after the glutathione treatment are shown in FIG. 34.

Based on the results described above, it was found that a liposome having a disulfide group according to the present invention decomposes in reductive conditions.

Experimental Example 5

Identification of Liposome Entry into Cell by Endocvtosis 0.2 mg of FITC-spermine and 0.2 mg of folate-spermidine were added to the dispersion solution of the liposome prepared according to Example 1 , and the resultant solution was stirred for one hour. A one-day dialysis was performed to refine the dispersion solution by removing un-embedded compounds. The refined product was identified by TEM. As a result, the liposomes had a size of 100 - 300 nm. TEM images of the liposomes are shown in FIG. 35. Then, 100 ≠. of this refined liposome dispersion solution was used to treat KB cells which were sufficiently cultured in a RPMI-1640 medium (200 μi) and 5 % CO₂ at 37⁰C . Then, confocal laser microscopy was used to identify the entrance of the liposome into the cell. For a control group, the same experiment was performed as described above, except that the folate-spermidine was not added to the dispersion solution of the liposome, and the FITC-spermine alone was added to the dispersion solution of the liposome. For reference, a KB cell is a typical oral cancer cell and has a plurality of folate receptors on its surface. Accordingly, the liposome which is surface-treated with folate can enter the KB cell.

FIG. 36 shows images of the KB cells obtained by confocal laser microscopy.

Referring to FIG. 36, significantly much more folate-spermidine embedded liposome entered the KB cell than the control group. Based on the results described above, it was found that a liposome has a targeting delivery capability by being modified with a targeting material that specifically reacts with to a cell.

Experimental Example 6: Identification of Anticarcinoqen Delivery Efficiency of Liposomes prepared according to Examples 34 and 35

KB cells were loaded into 96 wells at per 4000 cells/well, sufficiently cultured under conditions including 200 μl of a RPMI-1640 medium, 5 % CO₂, and 37⁰C , and then treated with 200 μJt of each of the liposome dispersion solutions prepared according to Examples 34 and 35. At this time, the concentration of the liposome that contains doxorubicin was varied. Then, the resultant product was cultured for about 60 hours and the cell survival rate according to the treated liposome was identified by MTT experiment. As for the control group which was not treated with the liposome, the cell survival was 99% or more. Therefore, it was found that the KB cell was effectively killed in the group treated with the doxorubicin encapsulating liposomes prepared according to Examples 34 and 35. The rate of cell survival with respect to the concentration of doxorubicin is illustrated in FIG. 37 (liposome prepared according to Example 34) and FIG. 38 (liposome prepared according to Example 35.) While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.

Claims

What is claimed is:

1. A liposome formed by self-assembling a cucurbituril derivative represented by formula 1 :

where X is O, S, or NH;

A₁ and A₂ are respectively OR¹ and OR², SR¹ and SR², or NHR¹ and NHR², wherein each of R¹ and R² is independently a hydrophilic functional group so that the compound of formula 1 has an amphiphilic property to form a liposome, and R¹ or R² additionally comprises ester, orthoester, acetal, imine, or disulfide in the middle thereof; and n is an integer from 4 to 20.

2. The liposome of claim 1 , wherein the hydrophilic functional group is each independently C5-C20 alkyl, C5-C20 alkenyl, C₅-C₂O alkynyl, C₅-C₂O carbonylalkyl, C₅- C₂₀, thioalkyl, C₅-C₂₀ alkylthiol, C₅-C₂O hydroxyalkyl, C₅-C₂₀ alkylsilyl, C₅-C₂₀ aminoalkyl, C₅-C₂₀ cycloalkylalkyl, C₅-C₂₀ heterocycloalkylalkyl, C₅-C₂₀ arylalkyl, or C₅-C₂₀ heteroarylalkyl, wherein each of the alkyl, alkenyl, and alkynyl has at least one carbon atom substituted with a hetero atom selected from the group consisting of oxygen, nitrogen, and sulfur, and in each of the alkyl, alkenyl, and alkynyl , the number of carbon atoms is larger than the number of substituted hetero atoms, and the hydrophilic functional group is substituted or unsubstituted with hydroxy, amino, an amino acid, a peptide composed of two to ten amino acids, hexoses, or pentoses.

3. The liposome of claim 1 , wherein the hydrophilic functional group is each independently -0(C₂H₄O)_nH or -0(C₂H₄O)_nCH₃ wherein n is an integer of 3 to 6, or poly ethyleneglycol or poly ethyleneglycol monomethyl ether (M.W. = 1000 ~ 5000), wherein the hydrophilic functional group is substituted or unsubstituted with hydroxy, amino, an amino acid, a peptide composed of two to ten amino acids, hexoses, or pentoses.

4. The liposome of claim 1 , wherein a targeting compound is embedded in a cavity of the cucurbituril derivative composing the liposome such that a targeting moiety of the targeting compound is exposed to the outside of the liposome.

5. The liposome of claim 4, wherein the targeting compound is represented by formula 2:

A B T

^{■ ■ ■} (2) where A is 1 ,3-diaminopropyl, 1 ,4-diaminobutyl, 1 ,5-diaminopentyl, 1 ,6-

diaminohexyl, sperminyl, spermidinyl, propylamino, butylamino, pentylamino,

hexylamino, biologinyl, pyridinyl, ferrocenyl, amino acid; or adamantanyl.

B is selected from the group consisting of a hydrogen atom, a substituted or unsubstituted CrC₃₀ alkyl, a substituted or unsubstituted C₂-C₃₀ alkenyl, a substituted or unsubstituted C₂-C₃₀ alkynyl, a substituted or unsubstituted C₂-C₃₀ carbonylalkyl, a substituted or unsubstituted C₁-C₃₀ thioalkyl, a substituted or unsubstituted CrC₃₀ alkylthiol, a substituted or unsubstituted CrC₃₀ alkoxy, a substituted or unsubstituted CrC₃₀ hydroxyalkyl, a substituted or unsubstituted C_r C₃₀ alkylsilyl, a substituted or unsubstituted CrC₃₀ aminoalkyl, a substituted or unsubstituted CrC₃₀ aminoalkylthioalkyl, a substituted or unsubstituted C₅-C₃₀ cycloalkyl, a substituted or unsubstituted 0₂-C₃₀ heterocycloalkyl, a substituted or unsubstituted C₆-C₃₀ aryl, a substituted or unsubstituted C₆-C₂₀ arylalkyl, a substituted or unsubstituted C₄-C₃₀ heteroaryl, and a substituted or unsubstituted C₄- C₂₀ heteroarylalkyl; and

T is a saccharide, a polypeptide, a protein, or an oligonucleotide, each of which recognizes and binds to a desired cell.

6. The liposome of claim 5, wherein the saccharide is glucose, mannose, or galactose.

7. The liposome of claim 5, wherein the protein is lectin, selectin, transferrin, herceptin, or antibody.

8. The liposome of claim 1 , wherein a pharmacologically active substance is encapsulated as a guest molecule in the liposome.

9. The liposome of claim 4, wherein a pharmacologically active substance is encapsulated as a guest molecule in the liposome.

10. The liposome of claim 8 or claim 9, wherein the pharmacologically active substance is an organic compound, a protein, or an oligonucleotide.

11. The liposome of claim 10, wherein the organic compound is hydrocortisone, prednisolone, spironolactone, testosterone, megesterol acetate, danasole, progesterone, indomethacin, amphotericin B, dopamine, L-DOPA, doxorubicin, paclitaxel, gleevec, oxaliplatin, cisplatin, or a mixture thereof.

12. The liposome of claim 10, wherein the protein is a human growth hormone, a G-CSF (granulocyte colony-stimulating factor), a GM-CSF (granulocyte- macrophage colony-stimulating factor), erythropoietin, a vaccine, an antibody, insulin, glucagon, calcitonin, an ACTH (adrenocorticotropic hormone), somatostatin, somatotropin, somatomedin, parathyroid hormone, thyroid hormone, a hypothalamus secretion, prolactin, endorphin, a VEGF (vascular endothelial growth factor), enkephalin, vasopressin, a nerve growth factor, an opioid not naturally occurring, interferon, asparaginase, alginase, superoxide dismutase, trypsin, chymotrypsin, pepsin, or a mixture thereof.

13. A method of preparing the liposome of claim 1 , the method comprising: dissolving a cucurbituril derivative of formula 1 in an organic solvent and drying the resultant solution; and adding water to the dried compound and dispersing the compound,

where X is O, S, or NH;

Ai and A₂ are respectively OR¹ and OR², SR¹ and SR², or NHR¹ and NHR², wherein each of R¹ and R² is independently a hydrophilic functional group so that the compound of formula 1 has an amphiphilic property to form a liposome, and R¹ or R² additionally comprises ester, orthoester, acetal, imine, or disulfide in the middle thereof; and n is an integer from 4 to 20.

14. A method of preparing the liposome of claim 4, the method comprising: dissolving a cucurbituril derivative of formula 1 in an organic solvent and drying the resultant solution; adding water to the dried compound and dispersing the compound; adding a targeting compound or a solution of the targeting compound to the dispersion solution and stirring the resultant mixture; and removing residual un-embedded targeting compound by dialysis,

where X is O, S, or NH;

A₁ and A₂ are respectively OR¹ and OR², SR¹ and SR², or NHR¹ and NHR², wherein each of R¹ and R² is independently a hydrophilic functional group so that the compound of formula 1 has an amphiphilic property to form a liposome, and R¹ or R² additionally comprises ester, orthoester, acetal, imine, or disulfide in the middle thereof; and n is an integer from 4 to 20.

15. A method of preparing the liposome of claim 8, comprising: dissolving a cucurbituril derivative of formula 1 in an organic solvent and drying the resultant solution; adding an aqueous solution of the pharmacologically active substance to the dried compound and dispersing the compound; and removing residual un-encapsulated pharmacologically active substance in the dispersion solution by dialysis,

where X is O, S, or NH;

Ai and A₂ are respectively OR¹ and OR², SR¹ and SR², or NHR¹ and NHR², wherein each of R¹ and R² is independently a hydrophilic functional group so that the compound of formula 1 has an amphiphilic property to form a liposome, and R¹ or R² additionally comprises ester, orthoester, acetal, imine, or disulfide in the middle thereof; and n is an integer from 4 to 20.

16. A method of preparing the liposome of claim 9, the method comprising: dissolving a cucurbituril derivative of formula 1 in an organic solvent and drying the resultant solution; adding an aqueous solution of the pharmacologically active substance to the dried compound and dispersing the compound; adding a targeting compound or a solution of the targeting compound to the dispersion solution and stirring the resultant mixture; and removing residual un-encapsulated pharmacologically active substance and residual un-embedded targeting compounds by dialysis,

where X is O, S, or NH;

Ai and A₂ are respectively OR¹ and OR², SR¹ and SR², or NHR¹ and NHR², wherein each of R¹ and R² is independently a hydrophilic functional group so that the compound of formula 1 has an amphiphilic property to form a liposome, and R¹ or R² additionally comprises ester, orthoester, acetal, imine, or disulfide in the middle thereof; and n is an integer from 4 to 20.

17. The method of any one of claims 13 to 16, wherein the organic solvent is chloroform, methyl alcohol, dimethylsulfoxide, dichloromethane, dimethylformamide, tetrahydrofuran, or a mixture thereof.

18. The method of any one of claims 13 to 16, wherein the water or the aqueous solution further comprises a buffer solution.

19. The method of any one of claims 13 to 16, wherein the dispersing is performed by sonication with an ultrasonic device .