KR102075476B1 - hydrogel comprising phenylboronic acid conjugated polymer - Google Patents

Abstract

The present invention relates to a hydrogel comprising a polymer in which phenylboronic acid is bound and a drug delivery system using the same. More specifically, the present invention relates to a hydrogel including a natural product having a cis-diol functional group and a phenylboronic acid-coupled polymer, which is excellent in pH, glucose and reactive oxygen species (ROS) sensitivity. Drug delivery system and drug delivery system application.

Description

Hydrogel comprising phenylboronic acid conjugated polymer

The present invention relates to a hydrogel comprising a polymer in which phenylboronic acid is bound and a drug delivery system using the same.

Biopharmaceuticals using biopolymers such as proteins and nucleic acids are more likely to be degraded by enzymes or eliminated by the immune system as compared to conventional pharmaceuticals using low molecular weight compounds. Accordingly, it is necessary to develop a drug carrier and its system that can be used as a polymer material for drug delivery, and respond to external stimuli closely related to the biological environment such as pH, glucose, temperature and reactive oxygen species (ROS). Do.

As a general drug delivery system, studies have been made to increase the enhanced permeability and retention effect into cells using polyethylene oxide-based polymers.

In addition, a drug and gene delivery system using a polysaccharide is a study using a biodegradable cellulose, typically a drug carrier by preparing a hydrogel utilizing the sol-gel variation characteristics according to the unique temperature of methyl cellulose There were many cases where they wanted to utilize. In addition, many other polysaccharides have been recognized for their excellent biocompatibility in that they are non-immunogenic and are being actively researched as drug and gene delivery systems.

In addition, the drug and gene delivery system using phenylboronic acid (PBA), a molecule having a number of diols and B (OH) ₂ which is a structural characteristic of phenylboronic acid, a kind of boronic acid Can be reversibly covalently bonded. This covalent bond is called a Boronic ester bond, and when reacted with glucose, the bond is released due to a competitive reaction. This causes the hydrogel into which phenylboronic acid is introduced to swell and release the drug or gene contained therein. Such a system is highly likely to be a smart drug treatment in the future, but there is a limitation that the biocompatibility is not excellent so far, more research is needed.

Hydrogels, on the other hand, contain more than 90% of water and are biocompatible, so they are widely used as drug carriers. There are several ways to make such a hydrogel, which is made using chemical bonds or physical bonds. Inducing chemical bonds requires chemicals such as photoinitiators or external stimuli such as light. In addition, there is a possibility that chemical impurities may remain even after synthesizing the hydrogel, so that applying the hydrogel directly to the wound may cause inflammation in our body. First of all, the actual burden on the patient is very high because the hydrogel must be implanted directly through the wound. Therefore, the technology for making an injection hydrogel that can be applied directly to the wound is not only applicable to various sites but also has great applicability, so research and development are required.

Therefore, it can be used as a polymer material for drug delivery and has high responsiveness to external stimuli such as pH, glucose and reactive oxygen species (ROS), and can be used in clinical practice without adding an initiator or chemical substance. New drug carriers need to be developed.

The present inventors can be used as a polymer material for drug delivery to solve the above problems, high responsiveness to external stimuli such as pH, glucose (glucose) and reactive oxygen species (ROS), initiator or chemical While researching new drug carriers that do not contain phosphorus, polymaleic anhydrides are often found in the natural environment with cis-diol functional groups and phenylboronic acid with cancer-specific properties. By conjugation with anhydride (PolyMALEIC), hydrogels are formed easily enough for clinical use, and the hydrogels are highly sensitive to pH, glucose and reactive oxygen species (ROS). It was confirmed that the present invention was completed.

Accordingly, an object of the present invention is to provide a hydrogel comprising a natural product having a cis-diol functional group and a phenylboronic acid-coupled polymer and a drug delivery system using the same.

In order to achieve the above object, according to an embodiment of the present invention provides a hydrogel including a natural product having a cis-diol group and a polymer in which phenylboronic acid is bonded.

In one embodiment of the present invention, the natural product having cis-diol may be at least one selected from the group consisting of tannic acid, ellagic acid, gallic acid, and humic acid. have.

The phenylboronic acid-bonded polymer may be prepared by the reaction of a succinic anhydride moiety of the maleic anhydride polymer with phenylboronic acid bonded to an amine.

In addition, the maleic anhydride polymer has a structure of the following [Formula 1],

Phenylboronic acid to which the amine is bonded may have a structure of the following [Formula 2].

[Formula 1]

(n is 20 to 5000)

[Formula 2]

In addition, the phenylboronic acid-bonded polymer may include a polymer having a structure represented by the following [Formula 3] generated by the bonding of [Formula 1] and [Formula 2].

[Formula 3]

(In Formula 3, x is an integer of 20 to 5000, y is an integer of 20 to 5000.)

According to another embodiment of the present invention, a drug delivery system including the hydrogel is provided.

The drug delivery system may be one in which the hydrogel is sensitive to any one or more changes selected from the group consisting of pH conditions, glucose concentrations, and reactive oxygen species (ROS) concentrations in cells.

At this time, the pH sensitivity of the hydrogel may be pH 5 or less.

Glucose-sensitive conditions of the hydrogel may be at least 5mM of glucose concentration.

The reactive oxygen sensitivity conditions of the hydrogel may be 90 to 150μM active oxygen concentration.

Hydrogel comprising a natural product having a cis-diol functional group and a phenylboronic acid-bound polymer of the present invention is excellent in pH, glucose and reactive oxygen species (ROS) sensitivity, Drug delivery systems and drug delivery systems that respond to closely related external stimuli can be provided.

In addition, the hydrogel according to the present invention can be self-healing (self-healing), it is excellent in the stability of the material.

1 shows ¹ H NMR of p PBA (poly (phenylbo ronic acid-co-maleic anhydride)) which is a maleic anhydride polymer bonded to phenylboronic acid.
2 is an image of a hydrogel prepared by combining a phenylboronic acid-bound polymer and tannic acid.
Figure 3 shows the results of the physical property evaluation through the flow system of the hydrogel prepared by combining the phenyl boronic acid and tannic acid.
Figure 4 is an image showing the results of the pH sensitivity evaluation of the hydrogel prepared by combining the phenyl boronic acid and tannic acid.
5 is an image showing the results of sensitivity evaluation on the glucose concentration of the hydrogel prepared by combining the phenyl boronic acid and tannic acid.
Figure 6 is an image showing the results of sensitivity evaluation on the active oxygen concentration of the hydrogel prepared by combining the phenyl boronic acid and tannic acid.
7 is an image showing a self-healing effect of a hydrogel prepared by combining a phenylboronic acid-bound polymer and tannic acid.

The present invention provides a hydrogel including a natural product having a cis-diol group and a polymer having phenylboronic acid bound thereto.

The natural product having the cis-diol group may be obtained from nature or obtained by separating from the natural product. Specifically, it may be at least one selected from the group consisting of tannic acid, ellagic acid, gallic acid, and humic acid (Fulvic acid). It doesn't work.

At this time, tannic acid is a kind of astringent polyphenol derived from plants, and has a weak acidity (Pka = 10) because there are many phenol groups. Because it has a large number of hydroxyl groups, it is known that it can stick with proteins or catch precipitation. Many of the fruits and foods we eat every day contain tannic acid. This tannic acid is very biocompatible and can be used as a biomaterial.

In addition, the natural product having the cis-diol group may be bonded to boronic acid, and the form bonded to boronic acid is called boronic acid-diol bond. Such binding can form a cancer specific drug delivery system by forming a covalent bond that is reversible and not noncovalent.

The polymer to which the phenylboronic acid binds is not particularly limited as long as it is a polymer capable of introducing a large amount of phenylboronic acid, but preferably a maleic anhydride polymer is preferable.

Specifically, the phenylboronic acid-bonded polymer is preferably a maleic anhydride polymer containing a succinic anhydride moiety, and may be a polymer prepared by combining phenylboronic acid with a succinic anhydride moiety.

The phenylboronic acid-bound polymer may be used without limitation as long as the polymer is water-soluble after hydrolysis and includes a succinic anhydride moiety.

In the present invention, the maleic anhydride polymer may be a copolymer in which the maleic anhydride monomer is polymerized with another monomer.

The maleic anhydride monomers, for example, maleic anhydride, methyl maleic anhydride, cyclohexyl maleic anhydride, phenyl maleic anhydride and the like.

The other monomer may be, for example, a vinyl monomer, and the vinyl monomer is specifically methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, benzyl (meth) acrylate, cyclohexyl ( Meta) acrylate; Unsaturated carboxylic acids such as acrylic acid, methacrylic acid, and the like.

The anhydride of the maleic anhydride polymer is excellent in reactivity with amines or hydroxyl groups, so it is easy to introduce single molecules into the main chain of the polymer, and an anhydride ring is opened during the grafting process. By forming a carboxylic acid group it can function to increase the solubility in the aqueous solution of the polymer.

The phenylboronic acid-bonded polymer is synthesized by combining phenylboronic acid with polymethyl vinyl ether-alt-maleic anhydride (pMAnh), which is represented by the following [Formula 1]: It may have been.

[Formula 1]

(n is 20 to 5000)

As the polymer to which the phenylboronic acid is bound, aminophenyboronic acid having an amine group bonded thereto may be used to bind phenylboronic acid to the polymer including the succinic anhydride moiety. The amino phenylboronic acid is preferably amino phenylboronic acid represented by the following [Formula 2], but is not limited thereto.

[Formula 2]

At this time, the amino group of the amino phenylboronic acid maleic anhydride moiety contained in the polymer may be combined in a ring opeing through hydrolysis.

 According to an embodiment of the present invention, the phenylboronic acid-bonded polymer is produced by combining the maleic anhydride polymer of [Chemical Formula 1] and amino phenylboronic acid of [Chemical Formula 2]. Phenylboronic acid-maleic anhydride polymer (poly (phenylboronic acid-co-maleic anhydride), pPBA).

[Formula 3]

(In Formula 3, x is an integer of 20 to 5000, y is an integer of 20 to 5000.)

The polymer synthesis is very simple at room temperature because it can be achieved simply by mixing maleic anhydride polymer and amino phenylboronic acid in a solvent such as dimethyl sulfoxide (DMSO) or acetone. Is also excellent.

The phenylboronic acid-conjugated polymer can selectively target cancer because it can bind with sialic acid around cancer cells.

The hydrogel may further increase the drug delivery effect by controlling the mole ratio of a natural product having a phenylboronic acid-bonded polymer and a cis-diol group.

Preferably, when the natural product having a cis-diol group is tannic acid, there are 10 cis-diols, but five cis-diols may spatially participate in binding. Therefore, more polymers containing phenylboronic acid in the hydrogel than natural products having cis-diol groups, or molar ratios of polymers having phenylboronic acid and natural products having cis-diol groups deviate from 0.1 to 1: 1 to 5 The tumor specific drug delivery effect may be more elevated. This may be due to the fact that more polymers bound to phenylboronic acid, which do not bind to natural products with cis-diol groups, can interact with N-acetylnuraminic acid in tumor cells. However, when more phenylboronic acid-containing polymers are included, it may be difficult to form a three-dimensional network structure, thereby making it difficult to form a hydrogel.

In the hydrogel of the present invention, the molar ratio of the phenylboronic acid-bonded polymer and the natural product having the cis-diol group may be 1: 1 to 10, preferably 1: 1 to 5, and more preferably 1: 5, It is not limited to this.

The hydrogel has a natural product having a cis-diol group and a phenylboronic acid-bonded polymer to reversibly form a boronic ester bond, which is covalently bonded as water is removed by condensation of alcohol and boronic acid. Losing mechanism Here, the boron ester bond is pH sensitive and can react competitively with substances such as alcohol.

The hydrogel may be sensitive to any one or more selected from the group consisting of pH, glucose and reactive oxygen species (ROS), and thus may be used as drug delivery agents.

The hydrogel reversibly combines a natural product having a cis-diol group with a phenylboronic acid, thereby exhibiting a self-healing effect. In addition, the hydrogel has a self-healing effect, even if a small damage is applied can be quickly recovered to have the stability of the material.

The water content of the hydrogel (water content) is more than 90%, the water content is very high biocompatibility (biocompatibility), it is easy to use as a biomaterial.

Since the hydrogel has biodegradability, it is suitable to be used as a biomaterial applicable to the human body, and in the present invention, it contains a macromolecule having a phenylboronic acid and natural products having cis-diol, so that no harmful substances remain in the human body. And does not cause inflammation.

According to another embodiment of the present invention, a drug delivery system including the hydrogel may be provided.

The drug delivery system may implement a system in which a drug may be released in a specific environment by using sensitivity of pH, glucose, and reactive oxygen species (ROS) of the hydrogel.

Specifically, depending on the pH conditions, glucose (glucose) concentration and reactive oxygen species (ROS) concentration in the cells, the drug supported on the hydrogel may be released to deliver the drug to a specific cell or site.

In this case, the pH sensitivity of the hydrogel is less than pH 5, when the pH exceeds 5, the hydrogel does not have a pH sensitivity, and below pH 5 shows a pH sensitivity to release the drug pre-supported on the hydrogel release the drug Can be controlled.

The glucose sensitivity of the hydrogel is a glucose concentration of 5mM or more, when the glucose concentration is 5mM or more the hydrogel is decomposed by glucose, the higher the glucose concentration increases the degree of decomposition of the hydrogel. Accordingly, since the hydrogel has a sensitivity to glucose concentration, the hydrogel may be supported on the hydrogel to control the release of the drug.

The active oxygen sensitivity condition of the hydrogel is 90 to 150 μM of active oxygen concentration, and when the active oxygen concentration is 90 to 150 μM, the hydrogel is decomposed by active oxygen, especially at 110 μM of the active oxygen concentration in the cell. The gel is completely degraded. Accordingly, degradation occurs when the hydrogel is infiltrated into cells, but does not degrade when not infiltrated into cells. Since the hydrogel has a sensitivity to active oxygen concentration, the drug may be supported on the hydrogel to control the release of the drug.

The drug delivery system is such that the drug contained in the drug system is delivered by injection or other means (e.g., implanted, celiac or in a possible space, as appropriate for a person or other mammal having a therapeutically effective disease state or condition), By coating the tissue surface of the body or by coating the surface of the implantable device), in particular, the composition is preferably delivered parenterally. “Parenteral” includes intramuscular, intraperitoneal, abdominal, subcutaneous, intravenous and intraarterial.

Hereinafter, embodiments for carrying out the present invention will be described in detail, and the following examples correspond to preferred examples for carrying out the present invention, and the present invention is not limited thereto.

Production Example  1-1. Maleic acid  On anhydride polymers Phenylboronic acid Combined  Synthesis of Polymer

Maleic anhydrate, pMAnh (polyvinyl vinyl ether-alt-maleic anhydride) having a molecular weight of 80,000, and phenylboronic acid combined with an amine group, 3-aminophenylboronic acid monohydrate (PBA-NH) ₂ ) A maleic anhydride polymer to which phenylboronic acid is bound was synthesized as in Fig. 1. First, an aqueous solution in which 500 mg of pMAnh (including 3.2 mmol succinic anhydride) was dissolved in DMSO (dimethyl sulfoxide) was prepared. Then, 160 mg of PBA-NH ₂ (1 mmol) was added to the aqueous solution of pMAnh and stirred for 24 hours at room temperature, after 24 hours, 10 ml of 0.1 N NaOH was added to the unreacted succinic anhydride moiety of the maleic anhydride polymer. The hydrolysis of the tee was promoted and the reaction was terminated.After completion of the reaction, dialysis for 2 days (fraction molecular weight (MWCO = 10,000)) was performed by freeze-drying, p PBA (poly ( phenylboron ic acid-co-maleic anhydride)), yield was 91%, and the conjugation ratio of PBA was calculated by ¹ H-NMR (Fig. 1) ¹ H NMR (D ₂ O, 300 MHz): 7.7- 7.0 (m, Ph, 4xH); 3.8-3.5 (m, -CH-, 1H); 3.5-3.1 (m, -OCH ₃ , 3H); 3.1-2.4 (m, CHCOO, 2H); 2.4-1.4 ( m, -CH _2- , 2H) The calculations show that the molar ratio of PBA in p PBA is 156 per 513 repeating units.

Production Example  1-2. Phenylboronic acid Combined  Polymer and Tannic acid  Containing Hydrogel  Produce

At room temperature, 20 µl of 0.848 M pPBA (poly (phenylboronic acid-co-maleic anhydride)) prepared from Preparation Example 1-1 and 20 µl of 0.16 M tannic acid, which is a natural product having cis-diol groups, were mixed in an aqueous DMSO solution at room temperature.

As shown in FIG. 2, a hydrogel was prepared through a combination of diol of tannic acid and phenylboronic acid of pPBA. In addition, after leaving the hydrogel at room temperature for 5 minutes, the diphenyl group of phenylboronic acid and tannic acid of pPBA is combined to form a hydrogel, and then, due to the hydrophobic interaction of phenylboronic acid and diol group, pPBAs are self-aggregated, It has been shown to form free-standing hydrogels.

Experimental Example  One. Phenylboronic acid Combined  On polymers Tannic acid Combined Hydrogel  Property evaluation

In order to check the physical properties of the hydrogel prepared from Preparation Example 1-2 and to determine whether the hydrogel was transferred to the hydrogel, a change in viscosity through a rheometer was observed. Specifically, the hydrogel is placed on the sample holder of the flow system, and a deformation of the material is made by applying a constant stress of 10 Pa at a frequency of 1-100 rad / sec using a 20 mm thick flat plate. The degree was confirmed by storage modulus (G ') and loss modulus (G' '). Storage modulus (G ') and loss modulus (G' ') was measured by the following equation (1). The results thereof are shown in FIG. 3.

[Equation 1]

σ = γ ₀ [G '(ω) sin (ωt)] + G''(ω) cos (ωt)]

In Equation 1,

σ is the shear stress on the material;

γ 0 is the maximum amplitude of the shear strain caused by the deformation of the material;

G 'is the storage modulus;

G '' is the loss modulus;

ω is the frequency; And

t is time.

As shown in FIG. 3, unlike the conventional hydrogel, the intersection point of storage modulus (G ′) and loss modulus (G ″) is not a rigid hydrogel, but may be used as an injection hydrogel with fluidity. Confirmed.

Experimental Example  2. Phenylboronic acid Combined  On polymers Tannic acid Combined Hydrogel  pH sensitivity evaluation

The hydrogel prepared from Preparation Example 1-2 was dispersed in an aqueous solution of pH 3, pH 7, and pH 9, and observed to show an image thereof in FIG. 4.

As shown in FIG. 4, the hydrogel was found to be stable at pH 7 and pH 9 while decaying at low pH 3. Thus, it was confirmed that the hydrogel according to the present invention has a pH sensitivity under acidic conditions.

Experimental Example  3. Phenylboronic acid Combined  On polymers Tannic acid Combined Hydrogel  According to glucose concentration Irritability  evaluation

The hydrogel prepared from Preparation Example 1-2 was dispersed in a glucose solution (5mM, 10mM, 20mM, 40mM) for each concentration, and observed to show its image in FIG. 5.

As shown in FIG. 5, the degree of decomposition of the hydrogel increases as the concentration of the glucose solution increases. Thus, it was confirmed that the hydrogel according to the present invention has sensitivity to glucose concentration.

Experimental Example  4. Phenylboronic acid Combined  On polymers Tannic acid Combined Hydrogel  For free radicals Irritability  evaluation

The hydrogel prepared from Preparation Example 1-2 was dispersed in an active oxygen solution (110 μM) and then observed to show an image thereof in FIG. 6.

As shown in FIG. 6, it was confirmed that the hydrogel was decomposed by the active oxygen solution. In addition, it was confirmed that it is applicable to the drug delivery system by treating and confirming 110 μM of an active oxygen solution having the same concentration as the cells. Thus, it was confirmed that the hydrogel according to the present invention has a sensitivity to active oxygen and is easy to apply to a drug delivery system.

Experimental Example  5. Phenylboronic acid Combined  On polymers Tannic acid Combined Hydrogel  Self-healing Evaluation of the effect

The hydrogels prepared in Preparation Example 1-2 were dyed with food colorings of red and blue, respectively, and self-resilience was evaluated. First, the hydrogel was abutted and then stretched. The photograph about this is shown in FIG.

As shown in FIG. 7, it was confirmed that the hydrogels were not separated from each other even after the hydrogels were attached, and the hydrogels had a self-healing effect. Thus, the hydrogel according to the present invention has a self-recovery effect, it was confirmed that can be quickly recovered even if a small damage is applied to have the stability of the material.

Claims (11)

natural products having a cis-diol group; And
It includes; polymer phenylboronic acid is bonded;
The natural product having the cis-diol is at least one selected from the group consisting of tannic acid, ellagic acid, gallic acid, and humic acid.
Hydrogel. delete The method of claim 1,
The phenylboronic acid-bonded polymer is a hydrogel, characterized in that prepared by the reaction of succinic anhydride moiety (maleic acid) moiety of the maleic anhydride polymer and phenylboronic acid bonded to the amine. The method of claim 3, wherein
The maleic anhydride polymer is a hydrogel, characterized in that the polymer having a structure of the following [Formula 1].
[Formula 1]

(n is 20 to 5000) The method of claim 3, wherein
The maleic anhydride polymer has a structure of the following [Formula 1], the phenyl boronic acid to which the amine is bonded, characterized in that has a structure of the following [Formula 2], hydrogel.
[Formula 1]

(n is 20 to 5000)
[Formula 2]

The method of claim 5,
The phenylboronic acid-bonded polymer is a hydrogel, characterized in that it comprises a polymer having the structure of [Formula 3], produced by the bond of the [Formula 1] and [Formula 2].
[Formula 3]

(In Formula 3, x is an integer of 20 to 5000, y is an integer of 20 to 5000.) Drug delivery system comprising a hydrogel according to claim 1. The method of claim 7, wherein
The drug delivery system is characterized in that the hydrogel is used to respond to any one or more changes selected from the group consisting of pH conditions in the cells, glucose (glucose) concentration and reactive oxygen species (ROS) concentration, Drug delivery system. The method of claim 8,
The pH-sensitive condition of the hydrogel is characterized in that less than pH 5, drug delivery system. The method of claim 8,
The glucose-sensing condition of the hydrogel is characterized in that the glucose concentration is 5mM or more, drug delivery system. The method of claim 8,
Reactive oxygen response conditions of the hydrogel is characterized in that the active oxygen concentration is 90 to 150μM, drug delivery system.

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