KR102557290B1 - Nitrogen monoxide releasing polymer, nitrogen monoxide releasing nano-structure comprising the same, and method for delivering nitrogen monoxide using the nitrogen monoxide releasing nano-structure - Google Patents

Abstract

It relates to a nitrogen monoxide-releasing polymer, a nitrogen monoxide-releasing nanostructure comprising the same, and a nitrogen monoxide delivery method using the same, comprising: a nitrogen monoxide-releasing material containing a diol group; And a polymer containing a boronic acid group in the residue of the repeating unit; chemically bonded, a nitrogen monoxide-releasing polymer, a nitrogen monoxide-emitting nanostructure including the same, and a nitrogen monoxide delivery method using the same can be provided. .

Description

Nitrogen monoxide-releasing polymer, nitric oxide-releasing nanostructure comprising the same, and nitrogen monoxide delivery method using the same NANO-STRUCTURE}

The present invention relates to a nitrogen monoxide delivery system, and more particularly, to a nitrogen monoxide-releasing polymer, a nitrogen monoxide-releasing nanostructure comprising the same, and a nitrogen monoxide delivery method using the same.

Nitrogen monoxide is generated in various cells of the human body and is known as a defense substance that exhibits anticancer and antimicrobial actions in the immune system, as a neurotransmitter in the nervous system, and as a vasodilator in the circulatory system.

Therefore, in order to use nitrogen monoxide for medical purposes such as anticancer treatment, research on new materials and methods that can more efficiently release nitrogen monoxide for the purpose of use is being conducted.

In this regard, since nitrogen monoxide is toxic at high concentrations, it may adversely affect the body when released in large amounts throughout the body. Therefore, development of a carrier for emitting nitrogen monoxide that emits nitrogen monoxide at an appropriate concentration at an appropriate rate according to the signal in the surrounding environment, or suppresses or delays the emission if necessary, but whose emission characteristics are not affected by unwanted noise signals in the surrounding environment The demand for is increasing day by day.

One aspect of the present invention is to provide a nitric oxide-releasing polymer capable of selectively delivering and releasing nitric oxide to specific cells in the body.

One aspect of the present invention is to provide a nanostructure comprising the nitrogen monoxide-releasing polymer.

One aspect of the present invention is to provide a nitrogen monoxide delivery method using the nanostructure.

One aspect of the present invention, a nitrogen monoxide-releasing material containing a diol (diol) group; And a polymer containing a boronic acid group in the residue of the repeating unit; provides a chemically bonded, nitrogen monoxide-releasing polymer.

The nitrogen monoxide releasing material including a diol group may include a vicinal or cis diol group.

The nitrogen monoxide-releasing polymer may include Structural Formula 1 below in a residue of a repeating unit.

[Structural Formula 1]

In Structural Formula 1, R ¹ is C1 to C10 substituted or unsubstituted alkylene, C5 to C12 substituted or unsubstituted cycloalkylene, C5 to C12 substituted or unsubstituted heterocycloalkylene, C5 to C12 substituted or unsubstituted arylene, or C5 to C12 substituted or unsubstituted heteroarylene, R ⁴ is a C1 to C10 substituted or unsubstituted alkyl group, R ⁵ is C5 to C12 substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group of C5 to C12, and L ¹ , R ² and R ³ are, (i) L ¹ , R ² and R ³ form a ring with each other and are substituted or unsubstituted C5 to C12 cycloalkylene, C5 to C12 substituted or unsubstituted arylene, or substituted or unsubstituted C5 to C12 heteroarylene, or (ii) L ¹ is a single or double bond, and R ² is C1 to C10 substituted or unsubstituted alkylene, C5 to C12 substituted or unsubstituted cycloalkylene, C5 to C12 substituted or unsubstituted heterocycloalkylene, C5 to C12 substituted or unsubstituted Arylene or C5 to C12 substituted or unsubstituted heteroarylene, R ³ is hydrogen or a C1 to C10 substituted or unsubstituted alkyl group.

The nitrogen monoxide-releasing polymer may include Structural Formula 2 below in a residue of a repeating unit.

[Structural Formula 2]

The nitric oxide-releasing polymer may release nitric oxide through a chemical reaction with intracellular glutathione.

The nitric oxide-releasing polymer may release nitric oxide in an environment in which the intracellular glutathione concentration is 0.5 to 10 mM.

One aspect of the present invention provides a nanostructure in which the nitrogen monoxide-releasing polymer is self-assembled.

The particle diameter of the nanostructure may be 10 to 500 nm.

One aspect of the present invention provides a nitrogen monoxide delivery method using the release of nitrogen monoxide through a reaction between the nanostructure and glutathione.

The nitrogen monoxide-releasing polymer of one aspect of the present invention does not release nitrogen monoxide in normal physiological environments, but selectively reacts with specific biomolecules to release nitrogen monoxide, thereby selectively delivering and releasing nitrogen monoxide to target cells can do. Thus, while minimizing toxicity in the human body, nitrogen monoxide can be more efficiently used for medical purposes such as anticancer treatment.

The nitric oxide-releasing nanostructure of one aspect of the present invention includes the nitrogen monoxide-releasing polymer and has a nano-sized structure, so that it can easily penetrate into blood vessels in target cells of areas such as cancer and inflammation, and target It can enable selective nitric oxide release in cells.

According to the nitric oxide delivery method of one aspect of the present invention, nitric oxide can be selectively delivered to target cells.

1 is a nuclear magnetic resonance analysis result of MA-diol-NO prepared in Examples.
2 is data obtained by measuring the nitrogen monoxide release pattern of MA-diol-NO prepared in Example.
3 is a nuclear magnetic resonance analysis result of Diol-NO-arene prepared in Example.
4 is data obtained by measuring nitrogen monoxide release patterns of Diol-NO-arene prepared in Examples.
5 is a result of nuclear magnetic resonance analysis of the nitrogen monoxide-releasing polymer prepared in Example.
6 is a transmission electron micrograph of the nitrogen monoxide-releasing nanostructure prepared in Example.
7 is a cytotoxicity test result for MDA-MB-231 cells.
8 is a cytotoxicity test result for 4T1 cells.
9 is a cytotoxicity test result for MCF-7 cells.
10 is a cytotoxicity test result for A549 cells.
11 is a cytotoxicity test result for HCT8-DR cells.

Unless otherwise defined, all terms (including technical and scientific terms) used in this specification may be used in a meaning commonly understood by those of ordinary skill in the art to which the present invention belongs. When it is said that a certain part "includes" a component throughout the specification, this means that it may further include other components, not excluding other components unless otherwise stated. In addition, singular forms also include plural forms unless specifically stated in the text.

One aspect of the present invention, as a material capable of selective delivery and release of nitrogen monoxide, a nitrogen monoxide-releasing material containing a diol (diol) group; And a polymer containing a boronic acid group in the residue of the repeating unit; provides a chemically bonded, nitrogen monoxide-releasing polymer.

As one binding material of the nitrogen monoxide-releasing polymer, the nitrogen monoxide-releasing material includes a diol group, and the nitrogen monoxide-releasing material including a diol group is a boronic acid group of a polymer containing a boronic acid group in a residue of a repeating unit. A nitrogen monoxide-releasing polymer may be formed through chemical bonding between diol groups of the nitrogen monoxide-releasing material.

More specifically, the nitrogen monoxide releasing material may include a vicinal or cis diol group. When it contains a vicinal or cis diol group, it can react more easily with a polymer containing a boronic acid group in the residue of the repeating unit, making it easier and more selective to form a nitrogen monoxide-releasing polymer. As such, nitrogen monoxide may be desirable because it may form a nitrogen monoxide-releasing polymer capable of delivering and releasing nitrogen monoxide.

The chemical bond may be specifically a covalent bond, and more specifically, a covalent bond through a dehydration reaction between the diol group of the nitrogen monoxide-releasing material containing the diol group and the boronic acid group of the polymer including the boronic acid group in the residue of the repeating unit. may be achieved.

Such a nitrogen monoxide-releasing polymer may selectively release nitrogen monoxide by reacting with a specific biomolecule by having the above-described structure. More specifically, the biomolecule may be glutathione. Glutathione is a substance that is contained in a particularly large amount in various cancer cells, and can target cancer cells by a selective chemical reaction with glutathione and the release of nitric oxide.

Accordingly, nitric oxide is not released in a physiological environment where glutathione is absent or the concentration is very low, such as blood, but nitric oxide can be selectively released in an environment where the concentration of glutathione is high, such as cancer cells. It can be released to prevent undesirable toxicity.

The nitric oxide-releasing polymer may be one that releases nitric oxide in an environment in which the intracellular glutathione concentration is 0.5 to 10 mM, and more specifically, as a lower limit of 1 mM or more, or 2 mM or more, and as an upper limit of 5 mM or less, or 3 mM or less It may be to release nitrogen monoxide in the environment, but is not necessarily limited thereto.

In addition, the nitric oxide-releasing polymer may exhibit cytotoxicity when present in a concentration of 10 μM or more in cells. However, the present invention is not necessarily limited thereto. More specifically, it may exhibit cytotoxicity when present at concentrations of 20 μM or more, 40 μM or more, 50 μM or more, or 60 μM or more in cells. The upper limit of the concentration may be 1 mM or less, 500 μM or less, 200 μM or less, or 100 μM or less, but is not limited thereto.

In addition, the nitrogen monoxide-releasing polymer may form a nanostructure by self-assembly in an aqueous solution environment such as blood. The particle size of these nanostructures may be illustratively 10 to 500 nm, but is not limited thereto.

When the nitrogen monoxide-releasing polymer forms a nanostructure by self-assembly, the target delivery and release effect of nitrogen monoxide can be further increased.

Specifically, nanostructures having a nano size can easily pass into blood vessels in cells of tissues such as cancer and inflammation, which have very weak blood vessels and loose structures.

Accordingly, the target delivery of the nanostructure itself including the nitric oxide-releasing polymer to target cells such as cancer and inflammatory cells and the effect of selectively releasing nitrogen monoxide in the target cells can be further improved.

Hereinafter, the structure of the nitrogen monoxide-releasing polymer of one embodiment of the present invention will be described in more detail.

The nitrogen monoxide-releasing polymer may include Structural Formula 1 below in a residue of a repeating unit.

[Structural Formula 1]

Wherein R ¹ is C1 to C10 substituted or unsubstituted alkylene, C5 to C12 substituted or unsubstituted cycloalkylene, C5 to C12 substituted or unsubstituted heterocycloalkylene, C5 to C12 substituted or unsubstituted It may be cyclic arylene or C5 to C12 substituted or unsubstituted heteroarylene.

Substitution in R ¹ includes, but is not limited to, an amino group, an amide group, a C1 to C10 alkoxy group, a C1 to C10 alkylamino group, a C1 to C10 alkylamide It may be substituted with an (alkylamide) group or an alkylester group of C1 to C10, and when these substituents are substituted with side branches, as well as those in which R ¹ is linked to * or B through these substituents is included.

R ⁴ may be a C1 to C10 substituted or unsubstituted alkyl group.

Substitution in R ⁴ , as a non-limiting example, may be substituted with one or more C1 to 10 alkyl groups or C1 to C10 cycloalkyl groups.

R ⁵ may be a C5 to C12 substituted or unsubstituted aryl group or a C5 to C12 substituted or unsubstituted heteroaryl group.

Substitution in R ⁵ may be substituted with at least one electron-withdrawing group, and the electron-withdrawing group may be, for example, a nitro group, a halogen group, or a nitrile group.

The L ¹ , R ² and R ³ may satisfy the following (i) or (ii).

(i) L ¹ , R ² and R ³ form a ring with each other and are substituted or unsubstituted C5 to C12 cycloalkylene, C5 to C12 substituted or unsubstituted arylene, or substituted or unsubstituted C5 to C12 Heteroarylene of can be formed.

Substitution here is a non-limiting example, and may be substituted with one or more C1 to 10 alkyl groups or C1 to C10 cycloalkyl groups.

(ii) L ¹ is a single or double bond, R ² is C1 to C10 substituted or unsubstituted alkylene, C5 to C12 substituted or unsubstituted cycloalkylene, C5 to C12 substituted or unsubstituted hetero Cycloalkylene, C5 to C12 substituted or unsubstituted arylene, or C5 to C12 substituted or unsubstituted heteroarylene, and R ³ may be hydrogen or a C1 to C10 substituted or unsubstituted alkyl group. .

Substitution here is a non-limiting example, and may be substituted with one or more C1 to 10 alkyl groups or C1 to C10 cycloalkyl groups.

Since the nitric oxide-releasing polymer of one aspect of the present invention has the above structure, it is possible to self-assemble in an in vivo environment such as blood to form a nanostructure, and at the same time to release nitric oxide through a selective reaction with glutathione can

More specifically, the nitrogen monoxide-releasing polymer of one aspect of the present invention may include Structural Formula 2 below in a residue of a repeating unit.

[Structural Formula 2]

The nitrogen monoxide-releasing polymer having such a residue may be prepared by reacting a polymer having a phenylboronic acid group as a residue and a nitrogen monoxide-releasing material having a vicinal diol under basic conditions of pH 7 or higher.

In more detail,

First, 3-methylamino-1,2-propanediol having a secondary amine group and a vicinal diol group is mixed with methanol or ( N -diazeniumdiolates) groups may be introduced by adding nitrogen monoxide in an organic solvent such as ether.

Then, by reacting with a solution containing a protecting group precursor material such as 1-fluoro-2,4-dinitrobenzene, N -diazenium diolates A protecting group can be introduced at the O-terminus of Selective release of nitric oxide by selective reaction with glutathione can be realized through the introduction of such a protecting group.

Thereafter, a polymer having a phenylboronic acid group as a residue may be reacted with a polymer having a pH of 7 or higher under basic conditions to prepare a nitrogen monoxide releasing polymer having a residue represented by Structural Formula 2.

The nitrogen monoxide-releasing polymer may be more specifically represented by Structural Formula 3 below. However, this structure is intended to be described in more detail to aid understanding of the nitrogen monoxide-releasing polymer of one aspect of the present invention, and is not necessarily intended to limit the present invention thereto.

[Structural Formula 3]

In Structural Formula 3, m is an integer from 20 to 5000, and n is an integer from 20 to 5000.

The material represented by Structural Formula 3 may be prepared by reacting a polymer having a phenylboronic acid group at its residue with a nitrogen monoxide-releasing material as described above. Hereinafter, the polymer having a phenylboronic acid group will be described in detail.

The polymer having a phenylboronic acid group may be obtained by combining phenylboronic acid with a maleic anhydride polymer containing a succinic anhydride moiety. Specifically, phenylboronic acid may be combined with a succinic anhydride moiety to form a binding site for the above-described nitrogen monoxide-releasing material.

The maleic anhydride polymer containing a succinic anhydride moiety may be a poly(methyl vinyl ether-alt-maleic anhydride) (pMAnh) polymer. However, any polymer that is water-soluble after hydrolysis and contains a succinic anhydride moiety may be used without limitation.

The number average molecular weight of the maleic anhydride polymer containing the succinic anhydride moiety may be 1000 to 1000000, more specifically 5000 to 500000, 10000 to 300000, or 25000 to 100000, but is limited thereto. it is not going to be

The polymer having the phenylboronic acid group may be prepared by binding phenylboronic acid to poly(methyl vinyl ether-alt-maleic anhydride) (pMAnh) represented by Structural Formula 4 below.

[Structural Formula 4]

(n is 20 to 10000.)

As the phenyl boronic acid, aminophenyboronic acid to which an amine group is bound may be used in order to bind to a polymer containing a maleic anhydride moiety. Amino phenylboronic acid may be 3-amino phenylboronic acid represented by Structural Formula 5 below.

[Structural Formula 5]

The amino group of amino phenylboronic acid and the maleic anhydride moiety included in the polymer may bond through a ring opening reaction through hydrolysis. Polymer synthesis is very simple because it can be done simply by mixing maleic anhydride polymer and aminophenylboronic acid in a solvent such as dimehyl sulfoxide (DMSO) or acetone at room temperature, and the synthesis efficiency is also high. great.

The poly(phenylboronic acid-co-maleic anhydride) (pPBA) prepared by combining the maleic anhydride polymer of structural formula 4 and aminophenylboronic acid of structural formula 5 was prepared by combining the nitrogen monoxide A nitrogen monoxide-releasing polymer of structural formula 3 may be synthesized by reacting the emissive material with a basic condition of pH 7 or higher.

Preferred examples and comparative examples of the present invention are described below. However, the following example is only a preferred embodiment of the present invention, but the present invention is not limited to the following example.

1. Preparation of nitrogen monoxide releasing material

(One) vic - diol secondary while having amine group Synthesis of nitrogen monoxide-releasing substances using existing substances

Mixing 3-methylamino-1,2-propanediol (MA-diol) containing vic-diol and having secondary amine with methanol and Ether Sodium 1-( N -methyl-N with N -diazeniumdiolates introduced into the secondary amine of MA-diol by dissolving in a solvent, adding sodium methoxide, and then adding high-pressure nitrogen monoxide -2,3-dihydroxypropyl)-diazen-1-ium-1,2 dioleate (1-(N-methyl-N-2,3-dihydroxypropyl)-diazen-1-ium-1,2- diolate, MA-diol-NO) was synthesized.

As a result of confirmation through Nuclear Magnetic Resonance analysis (NMR), it was confirmed that the hydrogen atom environment around the part where N -diazeniumdiolates were introduced changed, and that MA-diol-NO was synthesized normally (Fig. 1).

It was confirmed that the prepared material emitted nitrogen monoxide using a Nitric oxide Analyzer Sievers 280i (GE Analytical instruments). (FIG. 2)

(2) N -Introduction of protection groups to diazeniumdiolates

A protective group was introduced into N -diazeniumdioates so that nitrogen monoxide can be selectively released by reacting with glutathione among biomolecules present in cells, rather than immediately releasing nitrogen monoxide when put in water.

After dissolving MA-diol-NO with N -diazeniumdioates in a low-temperature saturated sodium bicarbonate solution, 1-fluoro-2,4-dinitrobenzene ) O ² -(2,4-dinitrophenyl)-1-(N-methyl-N-2,3-dihydroxypropyl)-diazene with an arene group introduced at the O-terminus of N -diazeniumdioates by mixing with the solution. -1-ium-1,2 diolate (O ² -(2,4-dinitrophenyl)-1-(N-methyl-N-2,3-dihydroxypropyl)-diazen-1-ium-1,2-diolate, Diol-NO-arene) was synthesized.

As a result of Nuclear Magnetic Resonance analysis (NMR), the existence of hydrogen atoms attached to 2,4-dinitrophenyl group showed that N -diazeniumdioates It was confirmed that the arene group was bound to the O end of (Fig. 3)

In addition, the material manufactured using a nitrogen monoxide analyzer (Nitric oxide Analyzer Sievers 280i, GE Analytical instruments) does not release nitrogen monoxide when Glutathione is not present (left picture in FIG. 4), but when Glutathione is present, nitrogen monoxide It was confirmed that the emission started. (Right picture in FIG. 4)

2. Preparation of nitrogen monoxide-releasing polymers and nanostructures

(1) Poly(methyl vinyl ether-alt-maleic anhydride (pMAnh), a maleic anhydride polymer with a number average molecular weight of 80,000, and phenyl bonded with an amine group As a boronic acid, 3-aminophenylboronic acid monohydrate (PBA-NH ₂ ) was used.

First, an aqueous solution was prepared by dissolving 500 mg of pMAnh (including 3.2 mmol succinic anhydride) in acetone. Thereafter, 160 mg of PBA-NH ₂ (1 mmol) was added to the aqueous solution in which pMAnh was dissolved, and stirred at room temperature for 24 hours. After 24 hours, 10 ml of 0.1N NaOH was added to terminate the reaction by replacing unreacted succinic anhydride moiety with carboxylic acid through hydrolysis of the maleic anhydride polymer. After completion of the reaction, dialysis was carried out for two days to remove suspended solids, and lyophilization was performed to obtain poly(phenylboronic acid-co-maleic anhydride), a maleic anhydride polymer with phenylboronic acid bound thereto. maleic anhydride), pPBA) was obtained.

The synthesis of pPBA was confirmed through nuclear magnetic resonance analysis (NMR), and the amount of phenylboronic acid contained in one polymer strand was confirmed. (FIG. 5)

(2) After dissolving the prepared pPBA polymer in water, the pH was adjusted to 8 by adding NaOH. Here, in a solution of 500 mM concentration in which Diol-NO-arene prepared above was dissolved in dimethyl sulfoxide (DMSO), the amount of phenylboronic acid and the amount of Diol-NO-arene in the pPBA aqueous solution were 1 on a molar basis: It was added so as to have an equivalent of 1.

The thus prepared nitrogen monoxide emitting nanostructure was observed through a transmission electron microscope (TEM) (Fig. 6), and it was confirmed that a nanostructure having a particle diameter of about 50 nm was formed.

3. Confirmation of cancer growth inhibition potential of the prepared nitrogen monoxide-releasing polymer and nanostructure

Through a cell viability test, the possibility of suppressing cancer growth of the prepared nitric oxide-releasing nanostructure was confirmed.

MDA-MB-231, 4T1, MCF-7, A549, and HCT8-DR cells, which are cancer cells, were each dispensed at 6000 cells/well in a 96-well culture plate and cultured overnight. These cancer cells contain about 2 to 10 mM of intracellular glutathione. After incubation, each medium is replaced with a new medium, and the nanostructure prepared by the reaction of Diol-NO-arene and pPBA phenylboronic acid prepared above is treated at a concentration of 0 to 100 μM, respectively, and further incubated for 48 hours, followed by MTT assay Cytotoxicity was confirmed through an assay. (FIGS. 7 to 11)

Each index of FIGS. 7 to 11 means the following.

MA-diol: 3-methylamino-1,2,-propandiol, a precursor before the introduction of N -diazeniumdiolates

Arene: 2,4-dinitrophenol containing the protecting group Dinitrobenzene

MA-diol-NO: Substance with N-diazeniumdiolates introduced but no protecting group introduced

pPBA: poly(phenylboronic acid-co-maleic anhydride)

Diol-NO-arene: A substance in which a protecting group is introduced into MA-diol-NO

pPBA:Diol-NO-arene 1:1 complex: A nanostructure in which the molar ratio between the content of phenylboronic acid in pPBA and Diol-NO-arene is 1:1

pPBA:Diol-NO-arene 2:1 complex A nanostructure in which the molar ratio between the content of phenylboronic acid in pPBA and Diol-No-arene is 2:1

As a result, it was confirmed that MA-diol, Arene, MA-diol-NO, and pPBA did not show toxicity, but Diol-NO-arene and pPBA:Diol-NO-arene complex effectively inhibited the growth of cancer cells under conditions above a certain concentration. did In particular, looking at the marked difference in the cancer cell growth inhibitory effect before and after the introduction of the protecting group (MA-diol-NO, Diol-NO-arene), it is necessary to use specific biomolecules (glutathione) present in the cell to inhibit the growth of cancer cells. It was confirmed that it is very important to react and release nitrogen monoxide.

In addition, as pPBA and Diol-NO-arene react to form nanostructures, it is confirmed that cancer cell targets are better and selective delivery and release of nitric oxide is possible through reaction with glutathione, a biomolecule in cancer cells. .

Claims (9)

a nitrogen monoxide-releasing material containing a diol group; And a polymer containing a boronic acid group in the residue of the repeating unit; is chemically bonded to a nitrogen monoxide-releasing polymer,
The nitrogen monoxide-releasing polymer is a nitrogen monoxide-releasing polymer comprising structural formula 2 in the residue of the repeating unit.
[Structural Formula 2]
delete delete delete In claim 1,
Nitric oxide is released through a chemical reaction with intracellular glutathione.
Nitric oxide-releasing polymers. In paragraph 5,
To release nitric oxide in an environment with an intracellular glutathione concentration of 0.5 to 10 mM,
Nitric oxide-releasing polymers. The nitrogen monoxide-releasing polymer of claim 1 is self-assembled, nanostructure. In paragraph 7,
A nanostructure having a particle size of 10 to 500 nm. delete