KR101201473B1 - Preparation method of self assembling drug and cell delivery system, and self assembling drug and cell delivery system prepraed therefrom - Google Patents

Abstract

The present invention relates to a method for producing a self-assembled drug and cell carrier, and a self-assembled drug and cell carrier produced by the present invention, as well as to easily and simply synthesize a polymer complex in which a host molecule or a guest molecule is introduced, The interaction between the host molecule and the guest molecule may be applied to the polymer composite to carry a useful material. Therefore, it can be usefully used as a drug-sustaining drug delivery and tissue engineering system capable of effectively delivering useful substances to a target individual or site without denaturation.

Drugs and cell carriers, magic blit

Description

Method for producing self-assembled drug and cell carrier, and self-assembled drug and cell carrier produced by the present invention TECHNICAL FIELD

The present invention relates to a method for the preparation of self-assembled drugs and cell carriers and to self-assembled drugs and cell carriers produced thereby. More specifically, the present invention relates to a method for producing self-assembled drugs and cell carriers which can carry useful substances by applying the interaction between the host molecule and the guest molecule to the polymer complex into which the host molecule or the guest molecule is introduced.

The magic bullet is a concept proposed by ringsdorf 30 years ago that combines water-soluble polymers with various functions such as bio-imaging materials, targeting materials, drugs, and materials that control the physicochemical properties of the carrier. Multifunctional drug carrier. Magiclets conjugated with various functional groups are prepared through various chemical reactions by selecting biomaterials to be conjugated. There is a risk that the activity of various biomaterials is reduced during this complex manufacturing process, and there is a problem in that the manufacturing process and materials must be newly designed according to the functional group introduced according to the purpose.

This application to the bonding system currently commercially available protein drug delivery system is a biocompatible polymer ^{Poly (ethylene glycol) PEGASYS ®,} ® PEG-INTRON ®, Macugen; Nutropin Depot ^® with PLGA microparticles as a sustained release formulation system of human growth hormone (hGH) (completely withdrawn from market in 2004 due to denaturation of proteins by degradation products of PLGA); There is Declase ^® (LG Life Sciences), a sustained release formulation system of hGH with high molecular weight hyaluronic acid.

In addition, as a water-soluble polymer having a network structure, the research on the hydrogel (hydrogel) that has a high moisture content in the water and does not dissolve in the structure similar to the tissue is being actively conducted in the bio field. Hydrogels introduce active functional groups into the polymer and induce covalent bonds between functional groups through additional chemical or external stimulation treatments in aqueous solutions to form hydrogels and support drugs or cells inside the hydrogels. This may be used as a drug carrier or a tissue engineering material. However, functional groups introduced into the polymer that influence the formation of the hydrogel may inhibit the activity of drugs or cells carried therein.

In order to solve this problem, attempts have been made to form hydrogels by physical interaction without introducing functional groups into polymers. In particular, in recent years, drugs or cells can be filled without denaturation and minimal wounds are generated. Attention has been focused on the implantable hydrogel that can be injected into the human body. Injectable hydrogels are commercially available from ReGel®, a temperature sensitive system using a triple block copolymer of PLGA-PEG-PLGA, and a variety of low critical solution temperature (LCST) phenomena. Temperature sensitive hydrogels using polymers have also been actively studied. However, since the temperature-sensitive injection hydrogel has a net structure formed by the physical bond forming micelles, it has a high initial release rate and a high rate of degradation in vivo, so that it can be applied to a drug delivery system of an anticancer drug or a protein drug for long-term proper efficacy. There is a limit to this.

Meanwhile, cucuribit [n] uril (CB [n]) is an amber hollow molecule formed by gathering n glycourils, and varies according to the number of glycourils (n = 4 to 12). Cooker bituril of size is made. In 2000, Kim and Kim and co-workers improved the method of synthesizing existing Cucurbitu [6] reels to synthesize not only the Cucurbitu [6] reel but also the cognate Cucurbitu [n] reels (n = 5, 7, 8). Each structure was identified by X-ray diffraction (J. Am. Chem. Soc. 2000, 122, 540), Korean Patent Application No. 2000-0033026 and the article (Acc. Chem. Res., 2003, 36, 621-630) to disclose the chemical formula of CB [n].

Various cucurbiturils utilize the hydrophobic space inside to form complexes with strong interactions with various guest molecules. Ionic materials and materials with high polarity, for example, various organic materials such as gas compounds, aliphatic compounds, aromatic compounds, various kinds of compounds such as insecticides, herbicides, amino acids, nucleic acids, ionic compounds, metal ions, organometal ions, etc. (J. Am. Chem. Soc. 2001, 123, 11316; European Patent 1094065; J. Org. Chem. 1986, 51, 1440). In particular, it forms a complex through a very non-covalent bond with a compound having a functional group, such as amino group, carboxylic acid, etc., the research to apply these characteristics to the development of a variety of drug delivery systems are continuously made.

An example of the use of cucurbituril in drug delivery systems has been the formation of complexes through stable non-covalent binding of oxaliplatin (Oxaliplatin), which has been recently approved by the FDA, to cucurbituril (PCT / KR02 / 01755). In addition, cucurbituril may introduce various functional groups therein, thereby linking compounds having various characteristics to cucurbituril (Korean Patent Application No. 2002-0068362, J. Am. Chem. Soc., 2003, 125, 10186). . For example, there is a patent (Korean Patent Application No. 2006-0018434) that uses the interaction of a cucurbituril host molecule and its guest molecules.

The inventors of the present invention, while studying a technique for overcoming the above-mentioned problems of the prior art, by using mutual recognition and self-assembly through the non-covalent interaction between the host molecule and the guest molecule introduced in each polymer, self-assembly carrier The present invention was completed by confirming that not only the useful substance can be loaded in the inside but also applied to the living body to effectively deliver the useful substance.

Accordingly, an object of the present invention is to provide a method for producing a self-assembled drug and cell carrier and a self-assembled drug and cell carrier produced thereby.

In addition, an object of the present invention is to provide a sustained release implantable hydrogel that is simple in manufacturing method and capable of supporting various useful materials without denaturation.

Accordingly, the present invention provides a compound represented by the following formula (I).

Formula I

[B] _m- [H] _n

In Formula I,

H is a cucurbituryl derivative, cyclodextrin derivative or calyx arene derivative, to which at least one functional group selected from amine group, carboxyl group, hydroxy group, aldehyde group, allyloxy, vinyl, acryl group, thiol group and combinations thereof is linked Or a combination thereof;

B is a monomer of a polymer compound into which a functional group selected from the group consisting of carboxyl group, aldehyde group, amine group, hydroxy group, thiol group, vinyl, allyloxy and acryl is introduced;

m is an integer from 1 to 10,000;

n is an integer from 1 to 10,000.

In the compounds of the above formula (I), H is preferably a cooker linked to at least one functional group selected from amine groups, carboxyl groups, hydroxy groups, aldehyde groups, allyloxy, vinyl groups, acrylic groups, thiol groups and combinations thereof Bitu [n] yl (n = 6-12) derivatives.

The present invention also provides a compound represented by the following formula (II).

Formula II

[B] _m- [G] _n

In the formula II,

G is a C ₁ -C ₂₀ alkyl group having one or more amine groups; C ₂ -C ₂₀ alkenyl group; C ₂ -C ₂₀ alkynyl group; C ₁ -C ₂₀ alkoxy group; C ₁ -C ₂₀ aminoalkyl group; C ₄ -C ₂₀ cycloalkyl group; C ₄ -C ₇ heteroarylcyclo group; C ₆ -C ₂₀ aryl group; C ₅ -C ₂₀ heteroaryl group; C ₁ -C ₂₀ alkylsilyl group; Adamantane with a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group or C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Ferrocene or metallocene having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Carborane having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Fullerene having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Cyclam or crownether having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ An oxygen protected amino acid having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Peptides having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl, C ₆ -C ₂₀ aryl group or a C ₅ - C ₂₀ groups having a heteroaryl alkaloid; Cisplatin having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Oligonucleotides having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Rhodamine having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; And a C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Selected from the group consisting of nanoparticles having an aryl group or a C ₅ -C ₂₀ heteroaryl group or a combination thereof;

B is a monomer of a high molecular compound to which a functional group selected from the group consisting of carboxyl group, aldehyde group, amine group, hydroxy group, thiol group, vinyl, allyloxy and acryl is connected;

m is an integer from 1 to 10,000;

n is an integer from 1 to 10,000.

In addition,

In the solvent,

(i) a compound represented by formula (I),

(ii) a drug, fluorescent substance, radioisotope, target directional substance or imaging substance that interacts with a compound represented by Formula (I); And

At least one drug, fluorescent substance, radioisotope, target directional substance or imaging substance to which a functional group interacting with the compound represented by formula (I) is linked

1: mixing at 0.1 to 10 equivalents,

The functional group that interacts with the compound represented by the formula (I), C ₁ -C ₂₀ alkyl group having at least one amine group; C ₂ -C ₂₀ alkenyl group; C ₂ -C ₂₀ alkynyl group; C ₁ -C ₂₀ alkoxy group; C ₁ -C ₂₀ aminoalkyl group; C ₄ -C ₂₀ cycloalkyl group; C ₄ -C ₇ heteroarylcyclo group; C ₆ -C ₂₀ aryl group; C ₅ -C ₂₀ heteroaryl group; C ₁ -C ₂₀ alkylsilyl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Adamantane having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Ferrocene or metallocene having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group or C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Carborane having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Fullerene having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Cyclam or crownether having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ An oxygen protected amino acid having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Peptides having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group or C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Alkaloids having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Cisplatin having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Oligonucleotides having an aryl group or a C ₅ -C ₂₀ hetero aryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Rhodamine having an aryl group or a C ₅ -C ₂₀ heteroaryl group; And a C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Provided are selected from the group consisting of nanoparticles having an aryl group or a C ₅ -C ₂₀ heteroaryl group, or a combination thereof, a method for producing a self-assembling drug and cell carrier.

In addition,

In the solvent,

(i) a compound represented by formula (II),

(ii) at least one of drugs, fluorescent materials, radioisotopes, target-directed materials or imaging materials to which one or more functional groups which interact with a compound represented by formula (II) are linked

1: mixing at 0.1 to 10 equivalents,

The functional group that interacts with the compound represented by the formula (II) is selected from the group consisting of cucurbituril, cyclodextrin, calix arene and derivatives thereof, it provides a method for producing self-assembled drug and cell carrier.

In addition,

In the solvent,

(i) a compound represented by formula (I),

(ii) a compound represented by formula (II)

1: Provides a method for producing a self-assembled drug and cell carrier, characterized in that it comprises the step of mixing in 0.1 to 10 equivalents. In addition, the method further comprises mixing a useful material selected from the group consisting of drugs, fluorescent materials, radioisotopes, target directional materials, imaging materials and cells. Wherein the useful material is a) (i) mixing the compound according to claim 1 with the useful material and then (ii) mixing the compound according to claim 3 with the mixture obtained; b) mixing (i) a compound represented by formula (I) with (ii) a compound represented by formula (II), followed by mixing a useful substance; Or c) (i) a compound according to claim 1, (ii) a compound according to claim 3, and a useful substance, by mixing at the same time by adding and mixing.

The present invention also provides self-assembling drugs and cell carriers prepared by any one of the above-described manufacturing methods.

In addition,

In the solvent,

(i) mixing the compound represented by formula I with a cell,

(ii) a method for producing a self-assembled drug and a cell carrier carrying a cell comprising the step of mixing the compound represented by formula II with the resulting mixture.

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

The present invention relates to a self-assembly drug and cell carrier using a host-guest chemistry and a method for producing the same.

Guest chemistry is a branch of chemistry that studies the interaction between host and guest molecules using non-covalent interactions such as hydrogen bonding, hydrophobic-hydrophobic interactions, electrostatic interactions, and vandelwald interactions. In other words, the host molecule is a molecule capable of capturing the guest molecule by non-covalent binding method, and the guest molecule is a molecule that is collected in the host molecule or a molecule having the trapped portion. Representative main molecules include crown ether, carlix arene, cyclodextrin, cucurbituril, polymer of benzene oxide, bicyclic amine, and guest molecules vary depending on the type and chemical properties of each main molecule.

These properties of subjectivity were applied to in vivo delivery systems of useful materials to prepare self-assembled drugs and cell carriers of the present invention. In other words, the present invention is based on the characteristics of the main subject chemistry, the useful material of the present invention alone or in the form of connecting to the host molecule or a guest molecule, the host molecule or a guest molecule is coupled and delivered in a non-covalent interaction with the linked polymer Provide a system.

The main molecule can use any substance previously known as the main molecule, and is selected from the group consisting of amine group, carboxyl group, hydroxy group, aldehyde group, allyloxy, vinyl, acrylic group, thiol group and combinations thereof A substance to which the above functional groups are linked may be used. Examples include cucurbituryl derivatives, cyclodextrin derivatives and cals linked with at least one functional group selected from the group consisting of amine groups, carboxyl groups, hydroxy groups, aldehyde groups, allyloxy, vinyl, acrylic groups, thiol groups and combinations thereof. And a rixarene derivative, but are not limited thereto. The cucurbituril derivatives, cyclodextrin derivatives, and kalixarene derivatives may be used alone or in combination thereof as a main molecule, preferably a cucurbitur [n] ryl (n = 6-12) derivative or a combination thereof, more preferably Preferably a cucurbitur [6] yl derivative and / or a cucurbitur [7] yl derivative. Cookerbituryl derivatives are known in the art, and may be used by purchasing a compound which is prepared by a known method or commercially available.

The guest molecule may be any compound, functional group, etc. that can interact, preferably non-covalently, with the host molecule, for example, a C ₁ -C ₂₀ alkyl group having one or more amine groups; C ₂ -C ₂₀ alkenyl group; C ₂ -C ₂₀ alkynyl group; C ₁ -C ₂₀ alkoxy group; C ₁ -C ₂₀ aminoalkyl group; C ₄ -C ₂₀ cycloalkyl group; C ₄ -C ₇ heteroarylcyclo group; C ₆ -C ₂₀ aryl group; C ₅ -C ₂₀ heteroaryl group; C ₁ -C ₂₀ alkylsilyl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Adamantane having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Ferrocene or metallocene having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Carborane having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Fullerene having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Cyclam or crownether having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ An oxygen protected amino acid having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Peptides having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group or C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Alkaloids having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Cisplatin having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Oligonucleotides having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Rhodamine having an aryl group or a C ₅ -C ₂₀ heteroaryl group; And a C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ And nanoparticles having an aryl group or a C ₅ -C ₂₀ heteroaryl group. These may be used alone or in combination of two or more thereof. The guest molecule is preferably selected and used according to the host molecule used together, more preferably a C ₁ -C ₂₀ aminoalkyl group having at least one amine group, a C ₅ -C ₂₀ heteroaryl group having at least one amine group , C ₁ -C ₂₀ aminoalkyl group containing metallocene and C ₅ -C ₂₀ heteroaryl group containing metallocene.

In addition, the present invention provides a polymer compound connected to the host molecule or guest molecule. The polymer compound may be any material used as a biomaterial, and may be a monomer or a multimer of the polymer compound. Examples include polyethylene glycol (PEO), poly lactic acid (PLA), poly glycolic acid (PGA), poly lactic-co-glycolic acid (PLGA), hyaluronic acid, chitosan, dextran or cellulose, or derivatives thereof. It may be a salt. These polymeric materials can be used individually or in combination of 2 or more types. The polymeric material can be a monomer or a multimer that can be readily prepared or commercially purchased.

In particular, hyaluronic acid (HA) may be natural or synthetic hyaluronic acid, or a modification thereof, and refers to a linear polymer polysaccharide in which β-DN-acetylglucosamine and β-D-glucuronic acid are alternately bonded. . Although there is no restriction on the molecular weight of the hyaluronic acid, preferably 0.5 to 1,000 kDa may be used, but more preferably, 5 to 200 kD in molecular weight.

In addition, the present invention provides a compound in which a main molecule or a guest molecule is linked to a polymer.

The compound in which the main molecule is linked to the polymer of the present invention is represented by the following formula (I).

Formula I

[B] _m- [H] _n

In Formula I,

H is a cucurbituryl derivative, cyclodextrin derivative or calyx arene derivative, to which at least one functional group selected from amine group, carboxyl group, hydroxy group, aldehyde group, allyloxy, vinyl, acryl group, thiol group and combinations thereof is linked Or a combination thereof;

B is a monomer of a polymer compound into which a functional group selected from the group consisting of carboxyl group, aldehyde group, amine group, hydroxy group, thiol group, vinyl, allyloxy and acryl is introduced;

m is an integer from 1 to 10,000;

n is an integer from 1 to 10,000.

In the above formula (I), compounds wherein H is a cucurbituryl derivative wherein one or more functional groups selected from amine groups, carboxyl groups, hydroxy groups, aldehyde groups, allyloxy, vinyl, acrylic groups, thiol groups and combinations thereof are linked .

In Formula I, m and n may each independently be different or the same, preferably n may be lower than m, more preferably n and m correspond to the number of moles of the monomer and the main molecule of the polymer compound, respectively N may be 0.2 times lower than or equal to the value of m.

In addition, the compound in which the guest molecule is linked to the polymer of the present invention is represented by the following general formula (II).

Formula II

[B] _m- [G] _n

In the formula II,

G is a C ₁ -C ₂₀ alkyl group having one or more amine groups; C ₂ -C ₂₀ alkenyl group; C ₂ -C ₂₀ alkynyl group; C ₁ -C ₂₀ alkoxy group; C ₁ -C ₂₀ aminoalkyl group; C ₄ -C ₂₀ cycloalkyl group; C ₄ -C ₇ heteroarylcyclo group; C ₆ -C ₂₀ aryl group; C ₅ -C ₂₀ heteroaryl group; C ₁ -C ₂₀ alkylsilyl group; A substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Adamantane having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Ferrocene or metallocene having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Carborane having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Fullerene having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Cyclam or crownether having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ An oxygen protected amino acid having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Peptides having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group or C ₅ -C ₂₀ heteroaryl group; Alkaloids having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Cisplatin having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Oligonucleotides having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Rhodamine having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; And a C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Selected from the group consisting of nanoparticles having an aryl group or a C ₅ -C ₂₀ heteroaryl group or a combination thereof;

B is a monomer of a high molecular compound having a functional group selected from the group consisting of carboxyl group, aldehyde group, amine group, hydroxy group, thiol group, vinyl, allyloxy and acryl;

m is an integer from 1 to 10,000;

n is an integer from 1 to 10,000.

The compound of formula II is a C ₁ -C ₂₀ aminoalkyl group in which G has at least one amine group, C ₅ -C ₂₀ heteroaryl group having at least one amine group, C ₁ -C ₂₀ aminoalkyl group containing a metallocene and Preferred are compounds selected from the group consisting of C ₅ -C ₂₀ heteroaryl groups containing metallocenes. In addition, the metallocene is preferably ferrocene.

In Formula II, m and n may each independently be different or the same, preferably n may be lower than m, more preferably n and m correspond to the moles of monomers and guest molecules of the polymer compound, respectively. N may be from 20% of the value of m to the same value of m.

In addition, in formula (I) and / or formula (II), B is a PEO, with or without a functional group selected from the group consisting of carboxyl group, aldehyde group, amine group, hydroxy group, thiol group, vinyl, allyloxy and acryl. (Polyethylene glycol), Polylactic acid (PLA), Poly glycolic acid (PGA), Poly Lactic-co-Glycolic acid (PLGA), hyaluronic acid, chitosan, dextran and cellulose It may be a multimer, preferably a hyaluronic acid monomer or a multimer, with or without introduction of a functional group selected from the group consisting of carboxyl group, aldehyde group, amine group, hydroxy group, thiol group, vinyl, allyloxy and acryl. .

The compound of the formula (I) or (II) of the present invention can be synthesized by an organic chemical method. For example, the compound of the formula (I) is composed of H having a carboxyl group or a hydroxyl group, B having an amine group, and 1-ethyl-3- [3- (dimethylamino) -propyl] carbodiimine (1-ethyl-3- [3- (dimethylamino) -propyl] carbodiimide (EDC), N, N'-dicyclohexylcarbodiimide (DCC), or N, N'-diisopropylcarbodiimide (DIC) By carrying out the bonding method, it can be easily produced in high yield, the compound of formula (II) is a carboxyl or hydroxy group B having an amine group G and EDC (1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide) , DCC (N, N'-Dicyclohexylcarbodiimide), or DIC (N, N'-diisopropylcarbodiimide) by performing the binding method, it can be prepared simply in high yield.

In addition, H having a thiol group may be prepared by B having a double bond, or B having a thiol group, G having a double bond, by a radical thiol-ene reaction using a photoreaction or AIBN.

In addition, H having an aldehyde group can be connected to B having an amine group, or B having an aldehyde group by imine bonding with G having an amine group, followed by treatment with a reducing agent such as NaBH ₄ to form a secondary amine.

The above-described method for preparing the compound of Formula (I) or Formula (II) is just one example, and may be prepared by any method known to those skilled in the art.

The present invention also provides a method for producing a self-assembling drug and cell carrier using the compound of Formula I or Formula II.

<Method for Producing Self-Assembly Drug and Cell Carrier I>

Manufacturing Method I is characterized by the following steps:

In the solvent,

(i) with respect to the compound represented by the formula (I),

(ii) a useful substance that interacts with a compound represented by formula (I) (e.g., a drug, a fluorescent substance, a radioisotope, a target directional substance or an imaging substance), and a useful group to which a functional group interacts with the compound represented by formula (I) Mixing at least one of the materials in a range of from 0.1 to 10 equivalents.

The preparation method can be carried out under conventional laboratory conditions, such as water or buffer, and the mixing can be carried out for a period of several minutes to about 24 hours.

In the above production method, by simply mixing the component (i) and the component of (ii), the compound represented by the formula (I) of (i) in the aqueous solution is a useful molecule that interacts with the compound of (ii), or The useful substance to which the interacting functional group is linked acts as a guest molecule, and spontaneous interaction occurs to form a self-assembled polymer composite. In this case, when the component of (ii) is mixed in an amount of 1: 1 equivalent or less with respect to the component (i), the component of (ii) may be used for the component (i) without further purification. When mixing at 1: 1 equivalent or more, dialysis, filtration, chromatography, or a combination of these purification methods can be performed.

In the dialysis, water, a buffer solution, methanol, ethanol, acetonitrile or a combination thereof may be used as the dialysis solution, and the pore size of the dialysis membrane is appropriately adjusted according to the size of the customer molecule, and may be 1000 to 10000. have. In the case of chromatography, gel permeable chromatograpy (GPC), high performance chromatograpy (HPLC) can be performed, the stationary phase uses Sephadex, C18, and the mobile phase is water, buffer, methanol, ethanol, acetonitrile or Combinations of these can be used.

In the above, the functional group that interacts with the compound represented by the formula (I), C ₁ -C ₂₀ alkyl group having one or more amine groups; C ₂ -C ₂₀ alkenyl group; C ₂ -C ₂₀ alkynyl group; C ₁ -C ₂₀ alkoxy group; C ₁ -C ₂₀ aminoalkyl group; C ₄ -C ₂₀ cycloalkyl group; C ₄ -C ₇ heteroarylcyclo group; C ₆ -C ₂₀ aryl group; C ₅ -C ₂₀ heteroaryl group; C ₁ -C ₂₀ alkylsilyl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Adamantane having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Ferrocene or metallocene having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group or C ₅ -C ₂₀ heteroaryl group; Carborane having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Fullerene having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Cyclam or crownether having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ An oxygen protected amino acid having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Peptides having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Alkaloids having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ Cisplatin having an aryl group or a C ₅ -C ₂₀ heteroaryl group; Oligonucleotides having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Rhodamine having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; And a C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ It may be selected from the group consisting of nanoparticles having an aryl group or a C ₅ -C ₂₀ heteroaryl group or a combination thereof.

Preferably, the functional group interacting with the compound represented by Formula I includes a C ₁ -C ₂₀ aminoalkyl group having at least one amine group, a C ₅ -C ₂₀ heteroaryl group having at least one amine group, and metallocene It can be selected from the group consisting of C ₁ -C ₂₀ aminoalkyl group and C ₅ -C ₂₀ heteroaryl group containing a metallocene. The metallocene is preferably ferrocene.

<Method for producing self-assembled drug and cell carrier II>

Preparation Method II is characterized by the following steps:

In the solvent,

(i) a compound represented by formula (II),

(ii) at least one of useful substances (eg, drugs, fluorescent materials, radioisotopes, target directional materials or imaging materials) to which at least one functional group that interacts with a compound represented by Formula II is 1: 0.1 Mixing in 10 equivalents.

The preparation method can be carried out under conventional laboratory conditions, such as water or buffer, and the mixing can be carried out for a period of several minutes to about 24 hours.

The functional group interacting with the compound represented by Formula II may be selected from the group consisting of cucurbituril, cyclodextrin, calyx arene, derivatives thereof, and salts thereof.

Useful materials to which at least one functional group that interacts with the compound represented by Formula II are linked are described in the literature (Org. Biomol. Chem., 2005, 3, 2122, Chem. Soc. Rev., 2007, 36, 267, Acc. Chem. Res., 2003, 36, 621, J. Inorg. Biochem. 2008, 102, 2060).

In the above production method, by simply mixing the component (i) and the component of (ii), the compound represented by the formula (I) of (i) in the aqueous solution is a guest molecule, and a functional group interacting with the compound of (ii) is connected. The useful substance acts as the main molecule, spontaneously interacting to form a self-assembled polymer carrier. In this case, when the component of (ii) is mixed in an amount of 1: 1 equivalent or less with respect to the component (i), the component of (ii) may be used for the component (i) without further purification. If 1: 1 equivalent or more is mixed, dialysis, filtration, chromatography, or a combined purification method thereof may be performed.

In the dialysis, water, a buffer solution, methanol, ethanol, acetonitrile or a combination thereof may be used as the dialysis solution, and the pore size of the dialysis membrane is appropriately adjusted according to the size of the customer molecule, and may be 1000 to 10000. have. In the case of chromatography, gel permeable chromatograpy (GPC), high performance chromatograpy (HPLC) can be performed, and the stationary phase uses Sephadex, C18, and the mobile phase is water, buffer, methanol, ethanol, acetonitrile or Combinations of these can be used.

<Method for producing self-assembled drug and cell carrier III>

Manufacturing Method III is characterized by the following steps:

In the solvent,

(i) a compound represented by formula (I),

(ii) a compound represented by formula (II)

1: mixing at 0.1 to 10 equivalents, preferably at most 1 equivalent.

The preparation method can be carried out under conventional laboratory conditions, such as water or buffer, and the mixing can be carried out for a period of several minutes to about 24 hours. In addition, the equivalent of the two or more mixtures is preferably not more than 1 equivalent in total.

The preparation method III may further comprise mixing a useful substance selected from the group consisting of drugs, fluorescent materials, radioisotopes, target directional materials, imaging materials and cells. The useful substance may be mixed with any one or both of these compounds and then combined with each mixture before mixing the compound represented by the formula (I) with the compound represented by the formula (II), or the compound represented by the formula (I) Useful substances can be added after mixing the compound represented by II. The order of addition of the useful substance may be determined according to the general substance such as the useful substance, the compound represented by the formula (I) and / or the compound represented by the formula (II), the nature, the content, the route of administration, the purpose of administration, the frequency of administration, and the like. have. The addition ratio of useful substance can be suitably selected according to the kind of useful substance.

The cell may be a cell derived from prokaryotic, eukaryotic, preferably mammalian, more preferably human derived cell. In particular, cancer cells, tissue cells of organs (eg, cancer cells, bone cells, gastric cells, enterocytes, lung cells, hepatocytes, brain cells, vascular cells), immune cells, red blood cells, white blood cells, lymphocytes, and the like can be exemplified.

In the present invention, the drug is a substance capable of inhibiting, inhibiting, alleviating, alleviating, delaying, preventing or treating a disease or symptom in an animal including a human, and examples thereof include paclitaxel, doxorubicin, and docetaxel. ), 5-fluoreuracil, oxaliplatin, cisplatin, carboplatin, berberine, epirubicin, doxycycline, gemcitabine gemcitabine, rapamycin, tamoxifen, herceptin, avastin, tysabri, erbitux, campath, zevalin, Humira, mylotarg, Xolair, bexxar, raptiva, remicade, siRNA, aptamer, interferon, insulin , Reopro, rituxan, zenapax, simulect, o Orthoclone, synagis, erythropoietin, epidermal growth factor (EGF), human growth hormone (hGH), thioredoxin, Fel d1, Api m1, myelin basic protein (myelin basic protein), Hsp60 and dnaJ, but are not limited to these.

Here, the fluorescent material may be a fluorescent material generally used in the art to which the present invention belongs, and examples thereof include fluorescein, rodamine, dansyl, Cy, and antracene. ), But is not limited to these.

Herein, the radioisotope may be, but is not limited to, ³ H, ¹⁴ C, ²² Na, ³⁵ S, ³³ P, ³² P and ¹²⁵ I.

As used herein, the target directional material means any material that can selectively recognize, bind, or deliver a specific target material, such as RGD (arginine-leucine-aspartic acid), TAT (threonine-alanine-threonine) And peptides such as MVm (methionine-valine-Dmethionine), peptides that recognize specific cells, antigens, and antibodies. Folic acid, nucleic acid, aptamers, or carbohydrates (eg, glucose, fructose, mannose, galactose, ribose, etc.), but are not limited to these.

In the present application, the imaging material is NMR (Nuclear Resonance Spectrometer), MRI (Magnetic Resonance Imaging), PET (Positron Emission Tomography), spectroscopy such as CT (Nuclear Resonance Spectrometer), fluorescence microscope, confocal laser scanning microscope, etc. Examples of any material that can be detected through a microscope include, but are not limited to, Ga-complex, nanoparticles, and carbon nanomaterials. The Ga-complex includes Ga-DTPA, Ga-DTPA-BMA, Ga-DOT, and Ga-cyclam, and the nanoparticles include gold, silver, manganese, cadmium, selenium, tellurium, zinc, and the like. Preferably, the nanoparticles are 1 to 200 nm in size, and the carbon nanomaterials include single-walled nanotubes, multi-walled nanotubes, fullerenes, and graphenes.

Herein, the solvent may be a compound represented by the formula (I) and / or formula (II), a drug, a fluorescent substance, a radioisotope, a target directional substance, an imaging substance and / or any solvent compatible with the cell, preferably human It may be a solvent acceptable to the animal, including. Examples thereof include water, saline, buffer, ethanol, and the like, but are not limited thereto. Those skilled in the art will be able to select a suitable solvent depending on the type and content of the material to be mixed in the solvent.

In accordance with another embodiment, the present invention provides a self-assembling drug and cell carrier prepared by the above-described method. The self-assembled drug and cell carrier are used for the delivery of useful substances in vivo or in vitro to animals, including humans.

The self-assembling biocarriers prepared by the methods of Preparation Methods I and / or II may be applied to a target object as it is. However, the self-assembled drug and cell carrier prepared by the method of Preparation III may be applied to a target subject as it is after preparation, but may be applied to the inside or the surface of an animal including human or tissues, organs or cells derived therefrom. Carriers can be prepared directly in vivo by sequential injection and / or infusion of the components.

Specifically,

(a) treating a target site with (i) a mixed solution of the compound represented by formula (I) and a useful substance and then (ii) treating a solution of the compound represented by formula (II) at the same site;

(b) treating a mixed solution of (i) a compound of formula (I) and a useful substance at the target site with (ii) a solution of the compound of formula (II) simultaneously;

(c) adding a mixed solution of (i) a compound represented by formula (I) and a useful substance to (ii) a compound represented by formula (II), and then treating the target site before the hydrogel is formed; or

(d) a mixed solution of (i) the compound represented by the formula (I) and the useful substance is added to (ii) the compound represented by the formula (II), followed by treatment with the target site after the hydrogel is formed.

The present invention also provides a pharmaceutical composition comprising a self-assembled drug and cell carrier according to the present invention. The pharmaceutical composition may further include a pharmaceutically acceptable carrier or diluent, and the like, and the pharmaceutical composition may be administered by any method known to those skilled in the art, for example, oral, parenteral administration such as injection, infusion, or implantation. have. Examples of parenteral routes include intravascular, intratumoral, pericardium, transmucosal, transdermal, intramuscular, intranasal, intravenous, intradermal, subcutaneous, intraperitoneal, intraventricularly, intracranially, intravaginally, inhaled. , Workplace and so on.

The pharmaceutical composition may be used as it is, or as formulated in a form suitable for the route of administration, ie, a solid preparation, a liquid preparation, or a hydrogel, prepared by using the prepared self-assembled drug and cell carrier.

According to the present invention, it is possible to easily and simply synthesize the polymer complex in which the host molecule or the guest molecule is introduced, and also to apply the interaction between the host molecule and the guest molecule to the polymer composite to carry a useful substance. Therefore, it can be usefully used as a sustained drug delivery and tissue engineering system which can deliver useful substances to a target individual or site without denaturation.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

( Example  One) Cookerbitu [6] Reel  Linked hyaluronic acid ( CB [6]- HA Manufacturing

Main molecule-cucurbitu [6] yl derivative with allyloxy group

Polymer-Hyaluronic Acid with Thiol Group

(Allyloxy) ₁₂ CB [6] (83 mg), hyaluronic acid with a thiol group introduced (60 mg), TECP (tris (2-carboxyethyl) phosphine) (40 mg) were added to DMSO (10 mL) in a quartz tube. After dissolution, the reaction was carried out in a photoreactor for 2 hours. The reaction was purified by dialysis in distilled water and lyophilized to give hyaluronic acid with 10 mg of CB [6] introduced.

NMR measurement of the obtained compound showed that the CB [6] derivative was successfully introduced into about 6% of the total hyaluronic acid units (FIG. 1).

¹ H NMR (500 MHz, D ₂ O) δ 2.04 (br, 3H), 3.37-4.22 (br, 10H), 4.56 (br, 2H), 5.35-6.02 (br, 1.31H).

( Example  2) Cookerbitu [6] Reel  Preparation of Linked Hyaluronic Acid Compounds

Main molecule-cucurbitu [6] ryl derivative with hydroxyl group

Polymer-Hyaluronic Acid

(Hydroxy) ₁₂ CB [6] (30 mg), hyaluronic acid (60 mg), EDC (24 mg), Sulfo-NHS (18 mg) were dissolved in H ₂ O (50 mL) in a round bottom flask. After stirring, the mixture was stirred at room temperature for 2 days. The reaction was then purified by dialysis in distilled water and lyophilized to yield 50 mg of hyaluronic acid incorporating CB [6].

NMR measurement of the obtained compound showed that the CB [6] derivative was successfully introduced into about 9% of the total hyaluronic acid units.

¹ H NMR (500 MHz, D ₂ O) δ 2.04 (br, 3H), 3.37-4.22 (br, 10H), 4.56 (br, 2H), 5.35-6.00 (br, 2.1H).

( Example  3) Cooker bituril  Connected Dextran  Preparation of compounds

Main molecule-cucurbitu [6] ryl derivatives with amine groups

Polymers-Dextran derivatives with aldehyde groups

(H ₂ NCH ₂ CH ₂ SCH ₂ CH ₂ CH ₂ O-) ₁₂ CB [6] (640 mg), dextran (60 mg), EDC (24 mg), Sulfo-NHS (18 mg) in a round bottom flask After dissolving in H ₂ O (50 mL), the mixture was stirred at room temperature for 2 days. The reaction was purified by dialysis in distilled water and lyophilized to give dextran with 50 mg of CB [6] introduced. NMR measurement of the obtained compound confirmed that the CB [6] derivative was successfully introduced in about 4% of the total dextran unit units.

¹ H NMR (500 MHz, D ₂ O) δ 2.12 (br, 2H), 2.81 (br, 2H), 2.93 (br, 2H), 3.31 (br, 2H) 3.25-4.52 (br, 11H), 5.32 ( br, 1 H), 5.72 (br, 1 H).

( Example  4) Cookerbitu [7] Reilly  Preparation of Linked Hyaluronic Acid

Master Molecule-Derivatives of Cucurbituryl [7] with hydroxyl group

Polymer-Hyaluronic Acid

(Hydroxy) ₆ CB [7] (15 mg), hyaluronic acid (60 mg), EDC (24 mg), Sulfo-NHS (18 mg) were dissolved in H ₂ O (50 mL) in a round bottom flask. After that, the mixture was stirred at room temperature for 2 hours. The resulting reaction was purified by dialysis in distilled water and lyophilized to give hyaluronic acid incorporating 35 mg of CB [7]. NMR measurement of the obtained compound showed that the CB [6] derivative was successfully introduced into about 7% of the total hyaluronic acid units.

¹ H NMR (500 MHz, D ₂ O) δ 2.04 (br, 3H), 3.37-4.22 (br, 10H), 4.56 (br, 2H), 5.60-6.40 (br, 2H)

( Example  5) Spurmin  Preparation of Linked Hyaluronic Acid

Guest molecule-Spurmin

Polymer-Hyaluronic Acid

Spermine (300 mg), hyaluronic acid (60 mg), EDC (24 mg), Sulfo-NHS (36 mg) are dissolved in H ₂ O (50 mL) in a round bottom flask and then stirred at room temperature for 2 days. It was. The resulting reaction was purified by dialysis in distilled water and lyophilized to yield 65 mg of spermine introduced hyaluronic acid. The NMR measurement of the obtained compound confirmed that CB [6] derivatives were successfully introduced into about 50% of the total hyaluronic acid units.

¹ H NMR (500 MHz, D ₂ O) δ 1.78 (br, 2H), 2.04 (br, 4H), 3.14 (br, 4H), 3.37-4.22 (br, 10H), 4.52 (br, 2H).

Example 6 Preparation of Hyaluronic Acid with HMDA Linked

Guest molecule-HMDA (Hexamethylene Diamine)

Polymer-Hyaluronic Acid

HMDA (180 mg), hyaluronic acid (60 mg), EDC (24 mg), Sulfo-NHS (36 mg) were dissolved in H ₂ O (50 mL) in a round bottom flask and then stirred at room temperature for 2 days. It was. The resulting reaction was purified by dialysis in distilled water and lyophilized to yield 50 mg of spermine introduced hyaluronic acid. The NMR measurement of the obtained compound confirmed that CB [6] derivatives were successfully introduced into about 50% of the total hyaluronic acid units.

¹ H NMR (500 MHz, D ₂ O) δ 1.24 (br, 2H), 1.41 (br, 1H), 1.48 (br, 1H), 2.03 (br, 3H), 2.80 (br, 1H), 3.02 (br , 1H) 3.37-4.22 (br, 10H), 4.51 (br, 2H).

( Example  7) Ferrocenemethylamine  Preparation of Linked Hyaluronic Acid

Guest molecule-ferrocenemethylamine

Polymer-Hyaluronic Acid Derivatives with Aldehyde Group

Ferrocenemethylaminochloride (180 mg) and hyaluronic acid (60 mg) linked with an aldehyde group were dissolved in H ₂ O (50 mL) in a round bottom flask and allowed to react at room temperature for 4 hours, followed by NaBH (10 mg). It stirred for 2 hours again. The resulting reaction was purified by dialysis in distilled water and lyophilized to yield 70 mg of ferrocenemethylamine introduced hyaluronic acid. NMR measurement of the obtained compound confirmed that the CB [6] derivative was successfully introduced into about 25% of the total hyaluronic acid units.

¹ H NMR (500 MHz, D ₂ O) δ 2.03 (br, 3H), 2.80 (br, 1H), 3.02 (br, 1H), 3.37-4.22 (br, 10H), 4.32 (s, 1.3H), 4. 48 (s, 0.5H), 4.52 (s, 0.5H), 4.58 (s, 0.5H), 4.51 (br, 2H).

( Example  8) Cooker bituril  Linked hyaluronic acid and FITC ( fluorescein  isothiocyanate)-Preparation of Self-Assembled Drug Carrier of Spermidine

First ingredient-hyaluronic acid with CB [6] introduced

Second Component-FITC-spermidine Conjugate

FITC-spermidine conjugates are described in Chem. Commun. Prepared directly to the laboratory according to the method described in 2009, 71.

Dissolve hyaluronic acid (10 mg) in which CB [6] prepared in Example 1 was dissolved in 1 mL of an aqueous PBS solution, and then FITC-spermidine was added in an equivalent amount to CB [6] introduced in the first component. The mixture was stirred at room temperature for 1 hour. This was purified by dialysis in PBS aqueous solution was confirmed by fluorescence emission analysis. As a result, a carrier of hyaluronic acid (FITC-spmd @ CB [6] HA) linked with FITC-spermidine and cucurbituril at a position shifted about 9 nm from the fluorescence emission peak (FITC-spmd) corresponding to FITC-spermidine The emission peak of was observed, indicating that the self-assembled carrier of the first component and the second component was successfully formed (FIG. 2).

( Example  9) Cooker bituril  Introduced hyaluronic acid FITC - Spermidine  Of self-assembled drug carriers In vivo  administration

0.2 mL of the carrier (1 × 10 ⁻⁴ M) prepared in Example 8 was incubated for 4 hours in 1.8 mL with B16F1 cells having a receptor for hyaluronic acid. After incubation, intracellular FITC fluorescence was measured by confocal laser microscopy and flow cytometry to confirm that the carrier was well introduced into the cells.

In addition, the control group without any treatment and the experimental group treated with excess hyaluronic acid (HA) to block the HA receptor of the cells before treatment, the experimental group treated only with SPMD-FTIC were also investigated.

The results are shown in FIGS. 3 and 4.

FIG. 3 is a confocal laser micrograph of an untreated control group, SPMD-FITC alone group, HA treated group, and the self-assembled delivery vehicle treated group of Example 9 for B16f1 cells, the bottom right picture is a confocal laser micrograph This is an enlarged picture, the bottom left picture is a picture of the right and overlaps the cell image. In FIG. 3, it was confirmed that the FITC-spmd / HA-CB prepared in this experiment was successfully delivered into cells.

Figure 4 is a flow cytometric graph of untreated controls, HA treated groups and FITC-spmd / HA-CB treated groups for B16f1 cells.

Through the above results, the experimental group treated with the FITC-spmd / HA-CB self-assembled transporter was significantly superior in the cell inflow rate compared to the other experimental groups (Fig. 3 and 4), which is the self-assembled type of the present invention It was found that the carrier is useful as a system for delivering a specific substance in cells. On the basis of these results, the possibility of next-generation magic block using self-assembled carrier and its effective drug delivery could be confirmed.

( Example  10) Cooker bituril  Introduced hyaluronic acid Peptides ( DWKYMVm ) - Spermidine  Preparation of Self-Assembled Drug Carriers

First ingredient-hyaluronic acid with CB [6] introduced

Second Component-Peptide (DWKYMVm) -spermidine Conjugate

Peptide (DWKYMVm) -spermidine conjugates are described in the paper Chem. Commun. It was prepared directly by the Institute according to the method described in 2009, 71.

After dissolving the hyaluronic acid (10 mg) in which CB [6] prepared in Example 1 was dissolved in 1 mL of an aqueous PBS solution, peptide (DWKYMVm) -spur was added in an equivalent amount to CB [6] introduced in the first component. The midine conjugate was added and stirred at room temperature for 1 hour. The sample purified by dialysis in PBS was confirmed by absorbance analysis of tryptophan of the peptide at 278 nm. As a result, an absorption peak was observed at 278 nm, and it was found that HA-CB / peptide (DWKYMVm) -spermidine composed of the first component and the second component was successfully formed.

( Example  11) Cooker bituril  Introduced hyaluronic acid Peptides ( DWKYMVm ) - Spermidine In vivo administration of self-assembled drug carriers

0.2 mL of the carrier (1 × 10 ⁻⁴ M) prepared in Example 10 was incubated for 4 hours in 1.8 mL of MCF7 cells overexpressing the receptor for peptide (DWKYMVm). After incubation, an increase in intracellular calcium ions generated during intracellular signaling by binding between the peptide and the receptor for the peptide was observed using confocal laser microscopy, thereby introducing cucurbituril-induced hyaluronic acid and peptide (DWKYMVm)- The efficiency of the self-assembled biocarrier of spermidine was confirmed. In order to identify intracellular calcium ions, the FLUO-3 chromophore, which binds to calcium and emits green fluorescence, was incubated with MCF7 cells for 30 minutes, and then treated with no treatment and peptide (DWKYMVm) -spermidine alone. Also investigated together (FIG. 5).

FIG. 5 is a blank of untreated control group (A), peptide (DWKYMVm) -spermidine treatment group (B), and HA-CB / peptide (DWKYMVm) -spermidine treatment group (C) of MCF7 cells. Focused laser micrographs show that spermidine effectively exerts an activity on cells.

( Example  12) Cooker bituril  Introduced hyaluronic acid, Peptides ( DWKYMVm ) - Spermidine  And FITC-S Permy Preparation of Dean's Self-Assembled Drug Carrier

First ingredient-hyaluronic acid with CB [6] introduced

Second component-DWKYMVm-spermidine conjugate

Third component-FITC-spermidine conjugate

After dissolving hyaluronic acid (10 mg) in which CB [6] prepared in Example 1 was dissolved in 1 mL of an aqueous PBS solution, peptide (DWKYMVm)-was added in 0.5 equivalents to CB [6] introduced in the first component, respectively. The spermidine conjugate and the FITC-spermidine conjugate were added and stirred at room temperature for 1 hour. The sample purified by dialysis in PBS aqueous solution was confirmed through fluorescence emission analysis and absorbance analysis.

The results are shown in FIG. FIG. 6 is a fluorescence emission peak (530 nm) corresponding to FITC-spmd (A) and tryptophan corresponding to DWKYMVm-spmd in the HA-CB / DWKYMVm-spmd / FITC-spmd carrier prepared in this experiment. This shows that all of the absorption peaks (B) are observed, and thus, it is possible to prepare a carrier in which two guest molecules (the second component and the third component) are complexed in one host molecule (first component). Confirmed.

( Example  13) Hydrogel  Produce

0.2 mL (30 mg / mL) of the hyaluronic acid compound to which the cucurbituril prepared in Example 1 was introduced and 0.2 mL (30 mg / mL) of the hyaluronic acid compound to which the HMDA was prepared in Example 6 were cut into the front part. Into a cylindrical hydrous gel was prepared. The prepared gel was pushed into a vial containing 2 mL of PBS, but did not release, and it was confirmed that a hydrogel was formed (FIG. 7). The gel was photographed by scanning electron microscopy to confirm the hydrogel state (FIG. 8).

( Example  14) the drug Supported Hydrogel  Produce

In 0.2 mL of docetaxol (10 mg / mL) aqueous solution, the hyaluronic acid compound prepared with the cucurbituril prepared in Example 1 was dissolved to a concentration of 50 mg / mL, and then the hyaluronic acid with the HMDA prepared in Example 6 was introduced. 0.2 mL of compound (50 mg / mL) was added to prepare a cylindrical hydrogel containing 0.4 mL of volume of docetaxol in a 1 mL syringe. The prepared hydrogel was placed in a vial containing 2 mL of PBS solution, and the PBS solution was taken over time to quantify the amount of drug released from the hydrogel by RP-HPLC.

9 is a graph showing the release amount of docetaxel with time of the hydrogel of Example 13. In FIG. 9, the drug was not released under the hyaluronic acid degrading enzyme-free condition, and the drug was released when the hyaluronic acid degrading enzyme was added in about 26 minutes. In other words, it can be seen that when hyaluronic acid is decomposed by the hyaluronic acid degrading enzyme present in the body, the trapped drug may be released slowly in vivo when the drug-captured hydrogel is implanted or injected in vivo.

( Example  15) In animals  Injection type Hydrogel  formation

Injecting 0.2 mL (25 mg / mL) of the cucurbituril-induced hyaluronic acid compound prepared in Example 1 into the subcutaneous rat, and then 0.2 mL of the hyaluronic acid compound incorporating the HMDA prepared in Example 6 25 mg / mL) was injected. After 10 minutes, the injected part was dissected to confirm the formation of a hydrogel.

10 is a diagram illustrating a method of directly forming a hydrogel in a mouse subcutaneously, and it was found that the hydrogel was formed directly in the body when the first and second components were continuously injected at the same position. Therefore, it can be seen that the hydrogel of the present invention can be used as an implantable hydrogel in which a hydrogel is formed in a living body.

( Example  16) cells Supported Hydrogel  Produce

The cucurbituril-induced hyaluronic acid compound prepared in Example 1 (15 mg / mL) was dissolved in 0.5 mL of PBS solution, and then mixed with preosteoblast. Thereafter, a hyaluronic acid compound (15 mg / mL) into which HMDA prepared in Example 6 was introduced was added to prepare a hydrogel gel in which cells were captured. This was observed under a microscope.

FIG. 11 is a photograph immediately after the preparation of the hydrogel containing the cells (A) and one week after the preparation (B), wherein the green fluorescent blue portion is labeled with the cells stained with casein AM and living cells. In FIG. 11, immediately after the hydrogel was prepared, the cells and the hydrogel were present, but after one week, the cells were supported in the hydrogel. The survival rate of the cells was more than 82%, and the number of dead cells increased with the cell culture for one week, but it was confirmed that they proliferated by forming clusters.

( Example  17) In animals  Cells Supported  Injection type Hydrogel  formation

The cucurbituril-induced hyaluronic acid compound prepared in Example 1 (25 mg / mL) was dissolved in 0.2 mL of PBS solution, then mixed with 1 × 10 ⁶ preosteoblasts and injected subcutaneously in rats. It was. Thereafter, 0.2 mL of the hyaluronic acid compound (25 mg / mL) incorporating HMDA prepared in Example 6 was injected subcutaneously at the same location. After 10 minutes, the injected part was cut and observed that a hydrogel was formed (FIG. 12).

Therefore, if the cells are mixed together and injected at the same time in the process of mixing the two components, it is thought that the cell-supported hydrogel can be injected into the living body with minimal wounds.

1 is a synthesis method (A) and a ¹ H NMR analysis result (B) of a CB [6] -hyaluronic acid (HA) conjugate prepared in Example 1. FIG.

2 is a fluorescence emission graph of FITC-spermidine / CB [6] -HA prepared in Example 8. FIG.

3 is a confocal laser micrograph of an untreated control group, SPMD-FTIC treated group, HA treated group, and FITC-spmd / HA-CB for B16f1 cells, an enlarged photograph of the lower right photograph, and the lower left is lower The photo on the right overlaps with the cell image.

4 is a flow cytometric graph of FITC-spmd / HA-CB of the untreated control, HA treated group and Example 9 for B16f1 cells.

FIG. 5 is a blank of untreated control group (A), peptide (DWKYMVm) -spermidine treatment group (B) and MC-HA / CB / peptide (DWKYMVm) -spermidine treatment group (C) of Example 10. FIG. Focus laser micrograph.

6 is a FITC-spmd fluorescence emission graph (A) and a DWKYMVm-spmd absorbance graph (B) of HA-CB / DWKYMVm-spmd / FITC-spmd of Example 12.

7 is a picture of a hydrogel gel prepared in Example 13.

8 is a scanning electron microscope (SEM) photograph of the hydrogel prepared in Example 13. FIG.

FIG. 9 is a graph showing docetaxel release over time from the drug-supported hydrogel according to Example 14. FIG.

10 is a diagram illustrating a method of directly forming a hydrogel in a mouse subcutaneous.

FIG. 11 is a photograph of a cell-supported hydrogel immediately after preparation (A) and one week after preparation (B). FIG.

12 shows the formation of a hydrogel containing cells according to Example 17.

Claims (27)

A self-assembling complex comprising 1: 0.1 to 10 equivalents of a compound represented by Formula I and a compound represented by Formula II: Formula I [B] _m- [H] _n , Formula II [B] _m- [G] _n ; In the formulas I and II, H is a cucurbitur [n] yl (n = 6 or 7) linked to a functional group selected from an amine group, a hydroxy group, allyloxy, and a combination thereof; G is selected from the group consisting of spermine, Hexamethylene Diamine (HMDA), and ferrocenemethylamine; The B is connected to a functional group selected from the group consisting of carboxyl group, aldehyde group, amine group, hydroxy group, thiol group, vinyl, allyloxy and acryl, polyethylene glycol (PEO), polylactic acid (polylactic acid, PLA), Poly glycolic acid (PGA), Poly Lactic-co-Glycolic acid (PLGA), hyaluronic acid, chitosan, dextran, or cellulose; m is an integer from 1 to 10,000; n is an integer from 1 to 10,000, n is from 20% to the same value of m. delete delete delete The self-assembling composite according to claim 1, wherein B is a hyaluronic acid to which a functional group selected from the group consisting of carboxyl group, aldehyde group, amine group, hydroxy group, thiol group, vinyl, allyloxy and acryl is linked. The self-assembled composite of claim 1, wherein the self-assembled composite is in a hydrogel state. The self-assembling composite of any one of claims 1, 5 and 6; And Useful materials selected from the group consisting of drugs, fluorescent materials, radioisotopes, target directional materials, imaging materials and cells A self-assembling useful material composition comprising a. According to claim 7, wherein the drug is paclitaxel (paclitaxel), doxorubicin (doxorubicin), docetaxel (docetaxel), 5-fluoreuracil (5-fluoreuracil), oxaliplatin, cisplatin, carboplatin (carboplatin) ), Berberine, epirubicin, doxycycline, gemcitabine, rapamycin, rapamycin, tamoxifen, herceptin, avastin, thisabri tysabri, erbitux, campath, zevalin, humira, mylotarg, Xolair, bexxar, raptiva ), Remicade, siRNA, aptamer, interferon, insulin, reopro, rituxan, zenapax, simulect, orthoclone ), Synagis, erythropoietin, epidermal growth factor (EGF), human growth hormone (hGH), thiore A composition for delivery of self-assembling useful substances, which is selected from the group consisting of thioroxin, Fel d1, Api m1, myelin basic protein, Hsp60, and dnaJ. The self-assembling useful substance delivery according to claim 7, wherein the fluorescent material is selected from the group consisting of fluorescein, rodamine, dansyl, Cy, and anthracene. Composition. 8. The composition of claim 7, wherein the radioisotope is selected from the group consisting of ³ H, ¹⁴ C, ²² Na, ³⁵ S, ³³ P, ³² P and ¹²⁵ I. 9. The method of claim 7, wherein the target directional agent recognizes a peptide, a specific cell comprising RGD (arginine-leucine-aspartic acid), TAT (threonine-alanine-threonine) or MVm (methionine-valine-Dmethionine). Peptides, antigens, antibodies. Folic acid, nucleic acid, aptamers and carbohydrates, which is selected from the group consisting of self-assembling useful substance delivery composition. The composition of claim 7, wherein the imaging material is selected from the group consisting of Ga-complexes, nanoparticles, and carbon nanomaterials. The method of claim 12, The Ga-complex is selected from the group consisting of Ga-DTPA, Ga-DTPA-BMA, and Ga-cyclam; The nanoparticles are nanoparticles having a size of 1 to 200 nm of a metal selected from the group consisting of gold, silver, manganese, cadmium, selenium, tellurium and zinc; The carbon nano material is selected from the group consisting of single-walled nanotubes, multi-walled nanotubes, fullerenes and graphene, Self-assembling useful material composition. The method of claim 7, wherein the cells are selected from the group consisting of cancer cells, bone cells, gastric cells, enterocytes, lung cells, hepatocytes, brain cells, vascular cells, immune cells, red blood cells, white blood cells, and lymphocytes, Prefabricated useful material composition. The self-assembling composite of any one of claims 1, 5 and 6; And cell Containing, tissue engineering composition. The composition of claim 15, wherein the cells are progenitor bone cells. delete delete delete delete delete delete delete delete delete delete delete

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