KR102593915B1 - Mechanical Strength Control of Supramolecular Self-assembled Hyaluronic Acid Hydrogel - Google Patents

Abstract

The present invention provides a supramolecular self-assembled hyaluronic acid hydrogel.
The supramolecular self-assembled hyaluronic acid hydrogel according to the present invention can control the mechanical strength depending on the type of hyaluronic acid-cyclodextrin derivative, so it is possible to provide optimal mechanical strength required for the application field of hyaluronic acid hydrogel.

Description

{Mechanical Strength Control of Supramolecular Self-assembled Hyaluronic Acid Hydrogel}

The present invention relates to the control of mechanical strength of supramolecular self-assembled hyaluronic acid hydrogel.

Hydrogel is composed of hydrophilic polymers with a three-dimensional structure and has a high moisture content while being insoluble in water, providing an environment similar to living tissue. The polymers that make up the hydrogel can be composed of synthetic polymers or bio-derived polymers. Bio-derived polymers include gelatin, collagen, agarose, alginic acid, chitosan, starch including amylose, pullulan, dextran, and hyaluronic acid. You can use it. These bio-derived polymers can form the three-dimensional structure of a hydrogel.

Polymers such as alginic acid, chitosan, flurane, dextran, and hyaluronic acid lack self-assembly properties, and a metal or chemical crosslinking agent is required to form a three-dimensional structure of a hydrogel. When such a chemical cross-linking agent is used, the mechanical strength of the hydrogel can be controlled depending on the amount of the cross-linking agent or polymer when forming the hydrogel. However, since cross-linking agents can react with drugs, enzymes, cells, etc., there is a disadvantage that it is difficult to stably support drugs, enzymes, cells, etc. in the hydrogel.

On the other hand, gelatin, agarose, etc. dissolve in water at high temperatures, and at low temperatures, hydrophobic residues in the polymer become physically entangled, forming a supramolecular self-assembled hydrogel. In the case of such temperature-dependent supramolecular self-assembling hydrogels, there is a disadvantage in that it is difficult to support drugs, enzymes, cells, etc. with low stability against heat because a high temperature is required to initially melt the polymer.

Meanwhile, the formation of a host-guest capture complex means that a hydrophobic guest compound is trapped in a hydrophobic cavity within a hydrophilic host compound and forms a complex through non-covalent bonds. A representative bio-derived host compound is cyclodextrin, a cyclic oligosaccharide. Cyclodextrins can be divided into alpha (6)-, beta (7)-, and gamma (8)- depending on the number of glucose they consist of. In the case of beta-cyclodextrin, it is known that it is possible to form a strong Host-Guest supramolecular entrapment complex with adamantane. Therefore, hyaluronic acid-cyclodextrin (HA-CD) is substituted by covalently bonding beta-cyclodextrin to the glycoskeleton of hyaluronic acid, which has excellent biocompatibility, and adamantane is covalently bonded to the hyaluronic acid glycoskeleton. A supramolecular self-assembled hydrogel was developed using substituted (HA-Ad). Hyaluronic acid derivatives substituted with cyclodextrin and adamantane are suitable for carrying drugs, enzymes, and cells because they are based on the formation of a non-covalent Host-Guest trapping complex, and can also be injected into the body. However, because the optimal mechanical strength of the hydrogel required varies depending on the supported material, mechanical strength control is required. For example, strong mechanical strength is required for sustained release of drugs, and in the case of cells, differentiation is controlled depending on the mechanical strength of the matrix or scaffold in which they are cultured.

Since the supramolecular self-assembled hyaluronic acid hydrogel is due to the ability to form a trapping complex of cyclodextrin and adamantane, the mechanical strength of the hydrogel can be controlled by adjusting the ability to form the complex. Therefore, in the present invention, by controlling the type of cyclodextrin substituted for the hyaluronic acid skeleton and the length of the linker between hyaluronic acid and cyclodextrin, the degree of freedom of the cyclodextrin is changed to control the formation of a trapping complex with adamantane, thereby improving mechanical strength. It is possible to raise it.

The purpose of the present invention is to provide a supramolecular self-assembled hyaluronic acid hydrogel prepared by supramolecular self-assembly of cyclodextrin and adamantane.

Additionally, the present invention aims to provide a method for controlling the mechanical strength of supramolecular self-assembled hyaluronic acid hydrogel.

The present invention relates to hyaluronic acid-cyclodextrin derivatives; and hyaluronic acid-adamantane derivatives,

The hyaluronic acid-cyclodextrin derivative is a supramolecular self-assembled hyaluronic acid hydrocarbon with controlled mechanical strength in which the carboxyl group of D-glucuronic acid of hyaluronic acid and the hydroxy group at the 6th carbon position of cyclodextrin are bonded through a linker. Provides gel.

Additionally, the present invention provides a method for producing the above-described supramolecular self-assembled hyaluronic acid hydrogel, comprising mixing a hyaluronic acid-cyclodextrin derivative and a hyaluronic acid-adamantane derivative.

The present invention provides a supramolecular self-assembled hyaluronic acid hydrogel prepared by supramolecular self-assembly of cyclodextrin and adamantane.

The mechanical strength of the hydrogel can be controlled depending on the type of hyaluronic acid-cyclodextrin derivative, making it possible to provide optimal mechanical strength required for applications of supramolecular self-assembled hyaluronic acid hydrogel. Additionally, it is possible to control the ability to form a trapping complex.

Figure 1 shows the synthesis diagram of a hyaluronic acid-cyclodextrin derivative.
Figure 2 is an NMR spectrum of a hyaluronic acid-cyclodextrin derivative, (a) is a 3' derivative with a cyclodextrin degree of substitution of 17%, (b) is a 3' derivative with a cyclodextrin degree of substitution of 49%, and (c) is a 3' derivative with a cyclodextrin degree of substitution of 49%. The NMR spectrum of the 4' derivative with cyclodextrin substitution degree of 28% is shown.
Figure 3 shows the synthesis diagram of hyaluronic acid-adamantane derivative.
Figure 4 shows the rheological properties of the supramolecular self-assembled hyaluronic acid hydrogel.
Figure 5 shows a comparative graph of Tan δ (loss factor) of supramolecular self-assembled hyaluronic acid hydrogel.
Figure 6 shows film formation results and skin application examples.

The present invention relates to hyaluronic acid-cyclodextrin derivatives; and hydrogels prepared from hyaluronic acid-adamantane derivatives.

In the present invention, it is possible to provide a supramolecular self-assembled hyaluronic acid hydrogel with controlled mechanical strength by changing the length of the linker between cyclodextrin and D-glucuronic acid of hyaluronic acid.

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

In the present invention, hyaluronic acid not only has biocompatibility and biodegradability properties, but also has transdermal delivery properties, so it can be safely applied to the human body and has the advantage of being applicable to transdermal drug delivery systems for various protein drugs and chemical drugs, including antigen proteins. has

In the present invention, unless explicitly stated otherwise, 'Hyaluronic acid (HA)' is expressed by the general formula 1 below and refers to a polymer having repeating units of N-acetyl-D-glucosamine and D-glucuronic acid. It is used to include all salts or derivatives of hyaluronic acid.

[General Formula 1]

In General Formula 1, n may be an integer of 25 to 8,000 or 25 to 1,000.

In the present invention, tetrabutylammonium salt of hyaluronic acid (HA-TBA) can be used as the salt of hyaluronic acid.

In the present invention, the 'hyaluronic acid derivative' is based on the basic structure of hyaluronic acid of General Formula 1 and contains an amine group, an aldehyde group, a vinyl group, a thiol group, an allyloxy group, and N-succinimidyl-3-(2-pyridyl group). All variants of hyaluronic acid into which functional groups such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and N-hydroxysuccinimide (NHS) are introduced. refers to For example, the hyaluronic acid derivatives include HA-diaminobutane, HA-hexamethylenediamine, HA-aldehyde, and HA-adipic acid dihydrazide (HA). -Adipic Acid Dihydrazide, HA-ADH), HA-2-Aminoethyl methacrylate hydrochloride, HA-Spermine, HA-spermidine (HA- spermidine), HA-SPDP, HA-NHS, etc. can be used.

The hyaluronic acid is present in most animals and can be safely applied to the human body as a linear polysaccharide polymer that is biodegradable, biocompatible, and has no immune response. Hyaluronic acid can be used for many purposes because it performs several different roles in the body depending on its molecular weight.

The composition of hyaluronic acid, a salt of hyaluronic acid, or a derivative of hyaluronic acid used in the present invention is not limited, but preferably has a molecular weight of 10,000 to 3,000,000 daltons (Da).

Additionally, in the present invention, unless explicitly stated otherwise, “hydrogel” refers to a gel using water as a dispersion medium. The hydrogel may be formed when a hydrosol loses its fluidity due to cooling or when a hydrophilic polymer with a three-dimensional network structure and a microcrystalline structure expands by containing water. Hydrogels made of electrolyte polymers often exhibit high absorbency and are used in many fields as absorbent polymers. Among hydrogels, there are some whose expansion ratio changes discontinuously due to phase transition due to temperature, pH, etc.

In the present invention, “supramolecule” refers to a molecular complex (or trapping complex) formed by gathering molecules or ions through non-covalent bonds such as hydrogen bonding, electrostatic interaction, or van der Waals attraction. Since the representative non-covalent bonds that form the structure of supramolecular structures are very weak compared to covalent bonds, the structure of supramolecular materials can easily change depending on the surrounding environment, and using this feature, the shape of the material can be arbitrarily controlled. A representative principle for forming supramolecular structures is self-assembly. Self-assembly refers to the phenomenon in which molecules are assembled through spontaneous interactions.

In the present invention, “supramolecular self-assembled hyaluronic acid hydrogel” is a hydrogel manufactured by the manufacturing method according to the present invention, and is manufactured from a hyaluronic acid-cyclodextrin derivative and a hyaluronic acid-adamantane derivative, cyclodextrin and adamantine. It refers to a hydrogel manufactured by the supramolecular reaction of thein.

Hydrogels according to the invention can be prepared from hyaluronic acid-cyclodextrin derivatives and hyaluronic acid-adamantane derivatives.

In the present invention, hyaluronic acid-cyclodextrin derivative (HA-CD derivative) refers to a derivative in which hyaluronic acid and cyclodextrin are combined. The carboxyl group of D-glucuronic acid of the hyaluronic acid and the hydroxyl group at the 6th carbon position of cyclodextrin can form a bond through a linker, and specifically, a bond can be formed through an amide bond.

In one embodiment, the linker may have the structure -NH-(CH ₂ )m-NH-, and the hyaluronic acid-cyclodextrin derivative may be a compound represented by Formula 1 below.

[Formula 1]

In Formula 1, n may be an integer of 25 to 8,000 or 25 to 1,000. Additionally, m may be an integer of 2 to 8 or an integer of 4 to 6. In the above range of m, excellent mechanical properties can be imparted, and in particular, high mechanical strength can be imparted even with a low degree of substitution of cyclodextrin.

In one embodiment, the degree of substitution of the cyclodextrin in the hyaluronic acid-cyclodextrin derivative may be 10 to 50%, 10 to 40%, or 25 to 35%. In general, if the degree of substitution of cyclodextrin is low, the mechanical properties of the manufactured supramolecular hydrogel are poor. In addition, when the degree of substitution is large, the internal hydrophobic properties of the cyclodextrin increase, lowering the solubility of the polymer in water and inhibiting the hydrogen bonding and anionic properties that the carboxy group of hyaluronic acid may have, thereby inhibiting the hyaluronic acid skeleton itself. There is a risk that structural characteristics may change. The hyaluronic acid-cyclodextrin derivative according to the present invention has the advantage of having a low degree of substitution and high solubility in aqueous solvents. At this time, the degree of substitution can be calculated as follows.

Degree of substitution = (molar amount of hyaluronic acid repeating unit with cyclodextrin bound) * (molar amount of hyaluronic acid repeating unit with and without cyclodextrin bound) * 100

In the present invention, hyaluronic acid-adamantane (CD-Ad) derivative refers to a derivative in which hyaluronic acid and adamantane are combined. Specifically, the hydroxyl group of hyaluronic acid and the carboxyl group of adamantane may form a bond through an ester bond or amide bond.

In one embodiment, adamantane may be used to include all salt or derivative forms of adamantane. For example, Adamantaneacetic acid (Ada-acetic acid) or (1-adamantyl)phenol, in which the carboxyl group is substituted with adamantane, can be used.

The hyaluronic acid-adamantane derivative may be represented by the following formula (2).

[Formula 2]

In Formula 2, n may be an integer of 25 to 8,000 or 25 to 1,000.

In one embodiment, the degree of substitution of adamantane in the hyaluronic acid-adamantane derivative may be 10 to 50% or 10 to 35%. Within the above range, a hydrogel with excellent mechanical strength can be manufactured.

In the present invention, the hydrogel includes the above-described hyaluronic acid-cyclodextrin derivative; and hyaluronic acid-adamantane derivatives. Specifically, the hydrogel can be manufactured by supramolecular reaction of cyclodextrin of hyaluronic acid-cyclodextrin derivative and adamantane of hyaluronic acid-adamantane derivative.

In the present invention, a supramolecular sieve, that is, a hydrogel, can be produced using cyclodextrin. Cyclodextrin is a cyclic oligosaccharide with a hydrophobic cavity formed through the α-1,4 bond of 6 to 8 glucose molecules. α-cyclodextrin has 6 glucose molecules, and β-cyclodextrin has 7 glucose molecules. Dextrins, and eight γ-cyclodextrins. At this time, the molecular weight, size of the hydrophobic cavity, solubility, etc. of the cyclodextrin may vary depending on the number of glucose molecules forming the cyclodextrin.

According to X-ray analysis, the structure of cyclodextrin shows that the hydroxyl groups bonded to C2 and C3 are spread outward, and the hydroxyl group bonded to C6 is also spread in the opposite direction, so the outer ring is overall hydrophilic. . On the other hand, the hydrogen ions of C3 and C5 and the oxygen of ether are located inside the cyclodextrin structure, so the internal cavity is hydrophobic. Therefore, the hydrophilic outer shell of the entire structure allows it to dissolve well in polar solvents such as water, while forming hydrophobic pores inside the structure that are opposite to the outer shell. This enables complex formation through host-guest interaction, which is the greatest characteristic of cyclodextrin.

The guest material enters the pores of cyclodextrin with a certain size and forms a complex through structural fitting. Depending on the type of cyclodextrin, the height of the pores is the same, but the diameter and volume are different. In the present invention, adamantane is used as a guest material. The adamantane is a highly symmetric and stable compound that has a structure in which four cyclohexane rings are condensed into a basket shape, and can form a bond with cyclodextrin through host-guest interaction.

That is, in the present invention, a hydrogel can be produced through host-guest interaction of cyclodextrin of a hyaluronic acid-cyclodextrin derivative and adamantane of a hyaluronic acid-adamantane derivative.

In one embodiment, the content ratio of the hyaluronic acid-cyclodextrin derivative and the hyaluronic acid-adamantane derivative may be 1:0.1 to 1:10, 1:0.5 to 1:2, or 1:1.

In one embodiment, the supramolecular self-assembled hyaluronic acid hydrogel may be an aqueous solution-based buffer solution. The content of hydrogel in the buffer solution may be 1 to 50% by weight, 5 to 20% by weight, or 5 to 15% by weight.

In one embodiment, the hydrogel of the present invention may contain useful substances. The type of the useful material is not particularly limited, and may be, for example, one or more selected from the group consisting of drugs, useful materials for functional cosmetics, fluorescent materials, radioactive isotopes, targeting materials, imaging materials, and culture media required for cell culture. there is.

A hydrogel containing a useful substance can function as a drug carrier for delivering the useful substance.

The drug is a substance that can inhibit, suppress, alleviate, relieve, delay, prevent or treat a disease or symptom in animals, including humans. Examples include paclitaxel, doxorubicin, docetaxel, 5- 5-fluoreuracil, oxaliplatin, cisplatin, carboplatin, berberine, epirubicin, doxycycline, gemcitabine, rapa rapamycin, tamoxifen, herceptin, avastin, tysabri, erbitux, campath, zevalin, humira , mylotarg, reopro, rituxan, zenapax, simulect, orthoclone, synagis, erythropoietin, epidermal growth factor (EGF), human growth hormone ( hGH), thioredoxin, Fel d1, Api m1, myelin basic protein, Hsp60, and dnaJ can be used.

The useful substance of the functional cosmetic is one selected from the group consisting of anti-wrinkle functional ingredients, whitening functional ingredients, UV-blocking functional ingredients, ingredients derived from natural products, skin microbiome, functional strains, and fermented products of skin microbiome and functional strains. You can use the above. In addition, adhesives, thickeners, pH adjusters, flavoring agents, wetting agents, and preservatives included in cosmetic formulations can be additionally used.

The fluorescent material may be a fluorescent material commonly used in the industry to which the present invention pertains, examples of which include fluorescein, rodamine, Dansyl, Cy, and anthracene. You can use it.

The radioactive isotopes may be ³ H, ¹⁴ C, ²² Na, ³⁵ S, ³³ P, ³² P and ¹²⁵ I.

The target-directing material refers to any material that can selectively recognize, bind, or deliver a specific target material, examples of which include RGD (arginine-leucine-aspartic acid), TAT (threonine-alanine-threonine), and MVm ( Peptides such as methionine-valine-Dmethionine), peptides that recognize specific cells, antigens, and antibodies. Folic acid, nucleic acids, aptamers, or carbohydrates (e.g., glucose, fructose, mannose, galactose, ribose, etc.) can be used.

In addition, imaging materials include spectroscopy such as NMR (Nuclear Resonance Spectrometer), MRI (Magnetic Resonance Imaging), PET (Positron Emission Tomography), CT (Nuclear Resonance Spectrometer), fluorescence microscope, confocal laser scanning microscope, etc. Any material that can be detected through a microscope, examples of which include, but are not limited to, Ga-complexes, nanoparticles, and carbon nanomaterials. The Ga-complexes include Ga-DTPA, Ga-DTPA-BMA, Ga-DOT, and Ga-cyclam, and the nanoparticles include gold, silver, manganese, cadmium, selenium, tellurium, and zinc. Preferably, the nanoparticles are 1 to 200 nm in size, and the carbon nanomaterials include single-walled nanotubes, multi-walled nanotubes, fullerene, and graphene.

In addition, ingredients used in the industry can be used as a culture medium for cell culture without limitation.

Additionally, the present invention relates to a method for producing the above-described supramolecular self-assembled hyaluronic acid hydrogel.

The supramolecular self-assembled hyaluronic acid hydrogel according to the present invention may include the step of mixing a hyaluronic acid-cyclodextrin derivative and a hyaluronic acid-adamantane derivative.

In the present invention, the hyaluronic acid-cyclodextrin derivative can be produced by reacting hyaluronic acid and cyclodextrin.

In one embodiment, the hyaluronic acid-cyclodextrin derivative includes the steps of (a) reacting the hydroxy group at the 6th carbon position of the cyclodextrin with a diamine compound to prepare a cyclodextrin having an amine group; and

(b) It can be prepared through the step of reacting hyaluronic acid with the cyclodextrin having the amine group to produce a hyaluronic acid-cyclodextrin derivative.

In step (a), cyclodextrin having an amine group can be produced by forming a reactive group at the hydroxyl group at the 6th carbon position of cyclodextrin and then reacting it with a diamine compound.

The diamine compounds include 1,2-ethylenediamine, 1,3-propanediamine, 1,4-butanediamine, 1,5-pentanediamine, 1,6-hexanediamine, 1,7-heptanediamine, and 1,8-octanediamine. You can use more than one of your selections.

In step (b), cyclodextrin having an amine group can be used in an amount of 0.5 to 1.5 equivalents, 0.5 to 1.0 equivalents, or 0.6 to 0.8 equivalents based on 1 equivalent of hyaluronic acid. The content of the cyclodextrin used affects the degree of substitution, and the degree of substitution can be adjusted to 10 to 50% within the above content range.

Step (b) may be performed in the presence of a solvent and a coupling reagent. Water, DMSO (Dimethyl sulfoxide), or PBS (Phosphate-buffered saline) can be used as the solvent, and DMTMM (4-(4,6-Dimethoxy-1,3,5-triazin-2-yl) as the coupling reagent. -4-methylmorpholinium chloride), EDC(N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, NHS(N-Hydroxysuccinimide), Pyridine, HBTU(N′-Tetramethyl-O-(1H-benzotriazol-1-yl) uronium hexafluorophosphate) or BOP ((Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate) can be used.

During the reaction, PBS or MES (2-(N-morpholino)ethanesulfonic acid) can be used as a buffer.

Step (b) can be performed at 25 to 50°C and room temperature for 20 to 48 hours or 20 to 30 hours.

In the present invention, the degree of substitution of the hyaluronic acid-cyclodextrin derivative produced can be adjusted by adjusting the equivalent weight and reaction time of the cyclodextrin having an amine group.

The degree of substitution of these hyaluronic acid-cyclodextrin derivatives may be 10 to 50%, 10 to 40%, or 25 to 35%.

Additionally, in the present invention, the hyaluronic acid-adamantane derivative can be produced by reacting hyaluronic acid and adamantane.

Specifically, it can be prepared by dissolving hyaluronic acid and adamantane in a solvent and then reacting in the presence of a reaction reagent.

The solvent may be water, dimethyl sulfoxide (DMSO), or phosphate-buffered saline (PBS). The reaction reagent is a reagent that causes an ester reaction, such as 4-Dimethylaminopyridine (DMAP), Divinyl acetate (DVA), N,N'-dicyclohexylcarbodiimide (DCC), Adamantane anhydride, or Di-tert-butyl. dicarbonate can be used. Additionally, during the reaction, PBS or MES (2-(N-morpholino)ethanesulfonic acid) can be used as a buffer. The reaction can be performed in vacuum.

The degree of substitution of adamantane in the prepared hyaluronic acid-adamantane derivative may be 10 to 50%.

In the present invention, a hydrogel can be produced through supramolecular reaction, that is, host-guest interaction, of cyclodextrin of a hyaluronic acid-cyclodextrin derivative and adamantane of a hyaluronic acid-adamantane derivative.

In one embodiment, the content ratio of the hyaluronic acid-cyclodextrin derivative and the hyaluronic acid-adamantane derivative may be 1:0.1 to 1:10, 1:0.5 to 1:2, or 1:1.

Additionally, in one embodiment, the hydrogel can be prepared by physical mixing of a hyaluronic acid-cyclodextrin derivative and a hyaluronic acid-adamantane derivative.

In addition, the present invention relates to the above-described hyaluronic acid-cyclodextrin derivative; and a drug delivery system containing a hyaluronic acid-adamantane derivative.

Since the hydrogel according to the present invention is manufactured through self-assembly of the above-described derivatives, useful substances, specifically drugs, can be loaded inside the hydrogel. These drugs can be of the types described above.

In one embodiment, the useful substance can be easily supported in the hydrogel by mixing the hyaluronic acid-cyclodextrin derivative and the hyaluronic acid-adamantane derivative with the useful substance during the production process of the hydrogel. .

In one embodiment, when preparing a drug delivery system, that is, when carrying a useful substance in a hydrogel, the useful substance is used in the form of an HA-useful substance conjugate in which the useful substance is bound to hyaluronic acid, or hyaluronic acid-A. It can be used in the form of an HA-Ad-useful substance conjugate in which a useful substance is combined with a damantane derivative. Alternatively, it can be used as a useful substance itself. The HA-useful substance conjugate can be prepared by introducing an aldehyde group into hyaluronic acid and binding a useful substance (the useful substance may contain an amine group or be modified with an amine group) to hyaluronic acid through an amine-aldehyde reaction. there is. In addition, the HA-Ad-useful substance conjugate introduces an aldehyde group into a hyaluronic acid-adamantane (HA-Ad) derivative and adds a useful substance to the HA-Ad derivative through an amine-aldehyde reaction (the useful substance contains an amine group or , can be modified with an amine group) can be manufactured by combining.

The drug delivery system can be used to deliver useful substances in vivo or in vitro to animals, including humans.

Additionally, the present invention provides a pharmaceutical composition containing the drug delivery system according to the present invention. The pharmaceutical composition may further include a pharmaceutically acceptable carrier or diluent, and the pharmaceutical composition may be administered by any method known to those skilled in the art, for example, oral or parenteral administration, such as injection, infusion, or transplantation. It can be. Examples of parenteral routes include intravascular, intratumoral, peritumoral, transmucosal, transdermal, intramuscular, intranasal, intravenous, intradermal, subcutaneous, intraperitoneal, intraventricularly, intracranially, intravaginally, and inhalation. , workplace, etc.

The pharmaceutical composition can be used as a prepared hydrogel, or can be formulated into a form suitable for the administration route, that is, a solid preparation, a liquid preparation, or a hydrogel.

Additionally, the present invention relates to a composition for transdermal delivery containing the above-described supramolecular self-assembled hyaluronic acid hydrogel.

Hyaluronic acid-cyclodextrin derivative according to the present invention; And when the supramolecular self-assembled hyaluronic acid hydrogel prepared from a hyaluronic acid-adamantane derivative is applied to the skin, a film is formed, which enables maintenance of the active substance in the formulation and effective transdermal delivery.

In one embodiment, the content of the hyaluronic acid-cyclodextrin derivative and the hyaluronic acid-adamantane derivative included in the composition for transdermal delivery may be 0.5 to 10 wt%, 1 to 5 wt%, or 1 to 2 wt%, respectively. . In the above content range, a film is formed when applied to the skin, etc., and useful substances can be effectively maintained and delivered to the dermis. If the content of the derivative is too low, problems may arise in the storage of useful substances, and a film is not formed and exists only as a fluid gel. Therefore, it is better to adjust the content range.

In one embodiment, the composition for transdermal delivery may include the above-described useful substances, specifically, anti-wrinkle functional ingredients, whitening functional ingredients, UV protection functional ingredients, natural product-derived ingredients, skin microbiome, functional strains, and skin. It may include one or more selected from the group consisting of microbiome and fermented products of functional strains.

Additionally, the present invention relates to hyaluronic acid-cyclodextrin derivatives; and a cosmetic composition containing a hyaluronic acid-adamantane derivative. The cosmetic composition according to the present invention can have the effect of improving wrinkles and can be used as a cosmetic composition for improving wrinkles.

Hereinafter, the present invention will be explained in detail by the following examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

Example.

Example 1. Hydrogel preparation

(1) Preparation of hyaluronic acid-cyclodextrin (HA-CD) derivatives

① Cyclodextrin (CD-NH) in which the hydroxy group at carbon position 6 is substituted with an alkyldiamine ₂ ) manufacturing

40 g of cyclodextrin was added to 450 ml of distilled water, heated to 60°C, and dissolved using a magnetic stirrer. The reaction solution was cooled to room temperature, 31.3 g of 1-(p-Toluenesulfonyl)imidazole was added, and stirred at room temperature for 4 hours. A NaOH solution was prepared by dissolving 50 ml of 18 g of NaOH in distilled water, which was slowly added to the above reaction solution for 20 minutes and then stirred for 10 minutes. 1-(p-Toluenesulfonyl)imidazole, which did not participate in the reaction, was removed through a filter. After adding 48.2 g of NH ₄ Cl to the filtered solution and completing the reaction, air was blown to concentrate the reaction solution to 1/2 the volume for 12 hours. The concentrated reaction solution was washed twice with 100 ml of cold water, and finally, 200 ml of acetone was poured to remove the water and dried using a vacuum oven to produce Mono-6-O-(p-toluenesulfonyl)-cyclodextrin. got it

Dissolve 10 g of Mono-6-O-(p-toluenesulfonyl)-cyclodextrin in 30 mL of DMF under oxygen-blocked argon gas, add 20 to 30 ml of 1,6-hexanediamine, and stir at 80°C for 12 hours. reacted. Acetone equivalent to 10 times the reaction solution was poured to precipitate the cyclodextrin derivative, which was dried in a vacuum oven. Using a cation exchange column resin, cyclodextrin (mono-(6-(1,6-hexamethylenediamine)-6-deoxy)-beta-cyclodextrin, 4, in which the hydroxy group at carbon 6 is replaced with an alkyldiamine group. ) was separated and lyophilized to obtain the final product.

② Preparation of hyaluronic acid-cyclodextrin (HA-CD) derivatives

The synthesis process of the hyaluronic acid-cyclodextyrin derivative is schematically shown in Figure 1.

Specifically, 100 mg of hyaluronic acid was dissolved in 20 ml of 0.1M MES buffer (pH 5.5) by stirring. Afterwards, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)4-methoxymorpholinium chloride (DMTMM) corresponding to 4 times the number of moles of hyaluronic acid was added to the hyaluronic acid solution. and stirred for 1 to 2 hours.

The cyclodextrin prepared in ① was added to the hyaluronic acid reaction solution in an amount 0.75 times the equivalent weight of hyaluronic acid, and reacted with stirring at room temperature for 24 hours to synthesize a hyaluronic acid-cyclodextrin derivative. The prepared derivative (4') was purified through dialysis and obtained through freeze-drying.

The degree of substitution of the prepared hyaluronic acid-cyclodextrin (HA-CD) derivative was 28%.

(2) Preparation of hyaluronic acid-adamantane (HA-Ad) derivatives

The synthesis process of the hyaluronic acid-adamantane derivative is schematically shown in Figure 3.

First, the TBA (Tetrabutylammonium) salt of hyaluronic acid was prepared. A hyaluronic acid solution was prepared by dissolving 1 g of hyaluronic acid in 100 ml of deionized triple distilled water. The Na (sodium) salt of hyaluronic acid was removed by passing the hyaluronic acid solution through a DOWEX 50Wx 8,400 or cation exchangeable resin column.

The pH of the hyaluronic acid eluent from which Na was removed was adjusted to pH 7-8 using 40% TBA-OH (Tetrabutylammonium hodroxide). Afterwards, the mixture was stirred for 1 hour using a magnetic stirrer and then freeze-dried to prepare the TBA salt of hyaluronic acid (6).

The TBA salt of hyaluronic acid (6), adamantane acetic acid (1-adamataneacetic acid, 7), which is 2 to 3 times the equivalent of hyaluronic acid, and 4-DMAP (4), which is 0.7 to 1 times the equivalent of hyaluronic acid. -dimethylaminopyridine) was dissolved in DMSO (Dimethyl sulfoxide). Oxygen in the reaction vessel was removed using vacuum and argon gas before and after dissolution. Afterwards, di-tert-butyl dicarbonate (an amount equivalent to ~0.5 times the standard equivalent of hyaluronic acid) was added to the reaction solution and stirred thoroughly for 24 hours using a magnetic stirrer.

After 24 hours, NaCl (sodium chloride) corresponding to the equivalent weight of hyaluronic acid was dissolved in water and added to the reaction solution. Then, 5 to 10 times more isopropyl alcohol was added to create a hyaluronic acid-adamantane derivative. precipitated. The precipitate was obtained through a filter, dissolved in water, dialyzed in a 0.1 M NaCl solution for 24 hours, and dialyzed using distilled water for an additional 48 hours.

Rather than using a immersion recovery method through the addition of isopropyl alcohol, the reaction solution was recovered, placed in a dialysis membrane, and dialyzed using a 20% (v/v) DMSO solution for 24 hours. Afterwards, additional dialysis can be performed using the above-mentioned method to obtain the product.

After dialysis, hyaluronic acid-adamantane derivatives were obtained through freeze-drying.

The degree of substitution of the prepared hyaluronic acid-adamantane derivative was 20%.

(3) Hydrogel production

A hyaluronic acid-cyclodextrin derivative solution was prepared by dissolving 4 wt% of the hyaluronic acid-cyclodextrin derivative prepared in (1) in a PBS (Phosphate buffered saline, pH 7.4) solution.

A hyaluronic acid-adamantane derivative solution was prepared by dissolving 5 wt% of the hyaluronic acid-adamantane derivative prepared in (2) in a PBS solution.

A supramolecular self-assembled hyaluronic acid hydrogel was prepared by mixing the two hyaluronic acid derivatives 1:1.

Comparative Example 1.

(1) Preparation of hyaluronic acid-cyclodextrin (HA-CD) derivatives

① Cyclodextrin (CD-NH) in which the hydroxy group at carbon position 3 is replaced with an amine group ₂ ) manufacturing

Cyclodextrin (3A-Amino-3A-deoxy-(2AS,3AS)-beta-cyclodextrin, 2), in which the hydroxy group at carbon 3 is replaced with an amine group, was manufactured by TOKYO CHEMICAL INDUSTRY CO., LTD, (Product number: A1916) ) was purchased and used.

② Preparation of hyaluronic acid-cyclodextrin (HA-CD) derivatives

100 mg of hyaluronic acid was dissolved in 10 ml of 0.1M MES buffer (pH 5.5) by stirring. Afterwards, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)4-methoxymorpholinium chloride (DMTMM) corresponding to 4 times the number of moles of hyaluronic acid was added to the hyaluronic acid solution. and stirred for 1 to 2 hours.

(1) of Example 1, except that 0.7 times the equivalent of hyaluronic acid equivalent of ① was added to the hyaluronic acid reaction solution and reacted with stirring at room temperature for 24 hours to synthesize a hyaluronic acid-cyclodextrin derivative. Hyaluronic acid-cyclodextrin derivative (2') was prepared in the same manner as above.

The degree of substitution of the prepared hyaluronic acid-adamantane derivative was 50%.

(2) Preparation of hyaluronic acid-adamantane (HA-Ad) derivatives

A hyaluronic acid-adamantane derivative was prepared in the same manner as (2) of Example 1.

(3) Hydrogel production

A hydrogel was prepared in the same manner as (3) of Example 1, except that 5 wt% of the hyaluronic acid-cyclodextrin derivative prepared in (1) was dissolved in a PBS solution to prepare a hyaluronic acid-cyclodextrin derivative solution. did.

Comparative Example 2.

(1) Preparation of hyaluronic acid-cyclodextrin (HA-CD) derivatives

① Production of cyclodextrin (CD-NH2) in which the hydroxy group at carbon position 6 is replaced with an amine group.

10 g of Mono-6-O-(p-toluenesulfonyl)-cyclodextrin was added to 100 ml of 28-30% ammonia water and stirred. 5 ml of new ammonia water was added to the reaction solution every day for 5 days. After 6 days of reaction, 7 to 10 times as much acetone as the reaction solution was added to precipitate the product, which was then vacuum dried. A cyclodextrin (6-mono-6-deoxt-beta-cyclodextrin, 3) in which the hydroxy group at carbon 6 was replaced with an amine group was prepared by using a cation column resin and freeze-drying.

②Manufacture of hyaluronic acid-cyclodextrin (HA-CD) derivatives

100 mg of hyaluronic acid was dissolved in 20 ml of 0.1M MES buffer (pH 5.5) by stirring. Afterwards, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)4-methoxymorpholinium chloride (DMTMM), which is twice the number of moles of hyaluronic acid, was added to the hyaluronic acid solution. and stirred for 1 to 2 hours.

Example 1 (Except that 0.75 times the equivalent of the hyaluronic acid equivalent of the cyclodextrin prepared in ① was added to the hyaluronic acid reaction solution and reacted with stirring at room temperature for 24 hours to synthesize a hyaluronic acid-cyclodextrin derivative. Hyaluronic acid-cyclodextrin derivative (3') was prepared in the same manner as 1).

The degree of substitution of the prepared hyaluronic acid-adamantane derivative was 17%.

(2) Preparation of hyaluronic acid-adamantane (HA-Ad) derivatives

A hyaluronic acid-adamantane derivative was prepared in the same manner as (2) of Example 1.

(3) Hydrogel production

A hydrogel was prepared in the same manner as (3) of Example 1, except that 5 wt% of the hyaluronic acid-cyclodextrin derivative prepared in (1) was dissolved in a PBS solution to prepare a hyaluronic acid-cyclodextrin derivative solution. did.

Comparative Example 3.

(1) Preparation of hyaluronic acid-cyclodextrin (HA-CD) derivatives

① Production of cyclodextrin (CD-NH2) in which the hydroxy group at carbon position 6 is replaced with an amine group.

It was prepared in the same manner as (1) ① of Comparative Example 2.

② Preparation of hyaluronic acid-cyclodextrin (HA-CD) derivatives

100 mg of hyaluronic acid was dissolved in 15 ml of 0.1M MES buffer (pH 5.5) by stirring. Afterwards, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)4-methoxymorpholinium chloride (DMTMM), which is twice the number of moles of hyaluronic acid, was added to the hyaluronic acid solution. and stirred for 1 to 2 hours.

Except that 1.0 times the equivalent of the hyaluronic acid equivalent of the cyclodextrin prepared in ① was added to the hyaluronic acid reaction solution and reacted with stirring at room temperature for 24 hours to synthesize the hyaluronic acid-cyclodextrin derivative (3'). Hyaluronic acid-cyclodextrin derivative (3') was prepared in the same manner as (1) in Example 1.

The degree of substitution of the prepared hyaluronic acid-adamantane derivative was 49%.

(2) Preparation of hyaluronic acid-adamantane (HA-Ad) derivatives

A hyaluronic acid-adamantane derivative was prepared in the same manner as (2) of Example 1.

(3) Hydrogel production

A hydrogel was prepared in the same manner as (3) of Example 1, except that 5 wt% of the hyaluronic acid-cyclodextrin derivative prepared in (1) was dissolved in a PBS solution to prepare a hyaluronic acid-cyclodextrin derivative solution. did.

Experimental Example 1. NMR analysis of derivatives

(1) Method

The hyaluronic acid-cyclodextrin derivatives prepared in Examples and Comparative Examples were analyzed by NMR (DPX300, Bruker, Germany).

(2) Results

The results of NMR analysis of the hyaluronic acid-cyclodextrin derivative are shown in Figure 2.

In Figure 2, (a) is a hyaluronic acid-cyclodextrin derivative prepared in Comparative Example 2 (cyclodextrin substitution degree 17%, 3'), (b) is a hyaluronic acid-cyclodextrin derivative prepared in Comparative Example 3 (cyclodextrin Degree of substitution is 49%, 3'), (c) shows the NMR spectrum of the hyaluronic acid-cyclodextrin derivative prepared in Example 1 (degree of substitution of cyclodextrin 28%, 4').

As shown in Figure 2, based on 3, which is the integral value of the acetyl group of the hyaluronic acid skeleton shown at 2.0 ppm, it can be confirmed that the hydrogen pick at carbon 1 of the glucose of cyclodextrin shown at 5.0 ppm was detected. there is. Through this, it is possible to confirm that cyclodextrin is conjugated to hyaluronic acid and the degree of substitution.

Experimental Example 2. Rheological properties of hydrogel

(1) Method

The rheological properties of the hydrogels prepared in Example 1 and Comparative Examples 1 to 3 were measured.

Specifically, the storage modulus (G') and loss modulus (G'') of the hydrogel were measured using an MCR 92 rheometer (Anton Paar, USA) at an angular frequency in the range of 0.1 to 100 rad/s and a diameter of 25 mm at 25°C. Measured with a plate.

(2) Results

The measurement results are shown in Figure 4.

In Figure 4, (a) is composed of 5 wt% HA-CD (2', degree of substitution 50%) and 5 wt% HA-Ad (6', degree of substitution 20%) prepared in Comparative Example 1, and (b) is a comparison. (c) consists of 5 wt% HA-CD (3', degree of substitution 17%) prepared in Example 2 and 5 wt% HA-Ad (6', degree of substitution 20%), and (c) is 5 wt% HA prepared in Comparative Example 3. -CD (3', degree of substitution 49%) and 5 wt% HA-Ad (6', degree of substitution 20%), (d) is 4 wt% HA-CD (4', degree of substitution) prepared in Example 1 28%) and 5 wt% HA-Ad (6', degree of substitution 20%).

As shown in FIG. 4, the hydrogel prepared in Comparative Example 1 has a higher degree of cyclodextrin substitution and thus has higher elasticity than Comparative Example 2. It can be seen that Comparative Example 3, which has a similar degree of substitution of cyclodextrin, has higher elasticity than Comparative Example 1. In other words, the hydrogel is bonded to the 6th carbon position of the cyclodextrin, and the higher the degree of substitution, the higher the elasticity.

However, it can be confirmed that Example 1 has a lower degree of cyclodextrin substitution and a lower concentration of hyaluronic acid than Comparative Examples 1 and 3, but has elasticity equal to or greater than that of Comparative Examples 1 and 3.

Experimental Example 3. Measurement of Tan δ (loss factor) of hydrogel

(1) Method

Tan δ of the hydrogels prepared in Example 1 and Comparative Examples 1 to 3 was measured.

Specifically, the storage modulus and loss modulus of the hydrogel were measured using the same method as measured in Experimental Example 2, and Tan δ was calculated as the ratio of loss modulus/storage modulus.

(2) Results

The measurement results are shown in Figure 5.

In Figure 5, (a) is composed of 5 wt% HA-CD (2', degree of substitution 50%) and 5 wt% HA-Ad (6', degree of substitution 20%) prepared in Comparative Example 1, and (b) is a comparison. (c) consists of 5 wt% HA-CD (3', degree of substitution 17%) prepared in Example 2 and 5 wt% HA-Ad (6', degree of substitution 20%), and (c) is 5 wt% HA prepared in Comparative Example 3. -CD (3', degree of substitution 49%) and 5 wt% HA-Ad (6', degree of substitution 20%), (d) is 4 wt% HA-CD (4', degree of substitution) prepared in Example 1 The Tan δ measurement results of a supramolecular self-assembled hyaluronic acid hydrogel composed of 28%) and 5 wt% HA-Ad (6', degree of substitution 20%) are shown.

As shown in FIG. 5, Comparative Example 2 shows a higher Tan δ value than other examples in all angular frequency ranges, and a Tan δ value of 1 or less at an angular frequency of 30 rad/s or more. In the case of Comparative Example 1 with a high degree of substitution, a Tan δ value lower than that of Comparative Example 2 was observed at the initial angular frequency, and a Tan δ value of 1 or less was observed at an angular frequency of 10 rad/s or more, showing superior mechanical strength than Comparative Example 2. confirmed.

In the case of Comparative Example 3, which has a degree of substitution similar to Comparative Example 1 but has an amine group at the 6th carbon position, a Tan δ value lower than that of Comparative Example 1 was confirmed in all angular frequency ranges, and was less than 1 at an angular frequency of 1 rad/s or less. It was confirmed that the Tan δ value of . Example 1 showed Tan δ values similar to Comparative Example 3, but it was confirmed to have lower Tan δ values at all frequencies.

A Tan δ value lower than 1 means that the elasticity of the hydrogel is higher than the viscosity, and a lower Tan δ value means higher mechanical strength. Therefore, through this experimental example, it can be confirmed that the hydrogel prepared in Example 1 has a low degree of substitution and has excellent mechanical strength.

Experimental Example 4. Transdermal delivery cosmetic formulation based on supramolecular self-assembled hydrogel

(1) Method

A cosmetic formulation for transdermal delivery was prepared by dissolving the hyaluronic acid-cyclodextrin derivative and the hyaluronic acid-adamantane derivative prepared in Example 1 in a cosmetic composition as shown in Table 1 below at a concentration of 1 wt% or 2 wt%, respectively.

Cosmetic formulations include water, thickeners, pH adjusters, moisturizers, preservative solvents, etc., whitening functional ingredients, wrinkle improvement functional ingredients, natural product-derived functional ingredients, skin microbiome and functional strains, skin microbiome and functional strain fermentation. Water, etc. may be additionally included.

NO. INGREDIENTS LIST One WATER 2 ACRYLATES/C10-30 ALKYL ACRYLATE CROSSPOLYMER 3 TROMETHAMINE 4 HYDROXYETHYLCELLULOSE 5 HYDROLYZED HYALURONIC ACID 6 SODIUM HYALURONATE 7 DISODIUM EDTA 8 SACCHAROMYCES FERMENT FILTRATE 9 ALOE BARBADENSIS LEAF EXTRACT 10 ULMUS DAVIDIANA ROOT EXTRACT 11 METHYLPROPANEDIOL 12 1,2-HEXANEDIOL 13 GLYCERIN 14 PENTYLENE GLYCOL 15 BUTYLENE GLYCOL 16 ETHYLHEXYLGLYCERIN

(2) Results

The film formation results and skin application example of the prepared cosmetic formulation are shown in Figure 6.

The content of the hyaluronic acid-cyclodextrin derivative and the hyaluronic acid-adamantane derivative in the cosmetic formulation may be 1 to 5 wt%, and may form a film when applied to the skin together with each cosmetic ingredient.

Due to the formed film, the active ingredient is maintained and efficient transdermal delivery is possible.

Claims (13)

Contains supramolecular self-assembled hyaluronic acid hydrogel,
The supramolecular self-assembled hyaluronic acid hydrogel is a hyaluronic acid-cyclodextrin derivative; and hyaluronic acid-adamantane derivatives,
The hyaluronic acid-cyclodextrin derivative is a compound represented by the following formula (1),
A composition for transdermal delivery wherein the degree of substitution of cyclodextrin in the hyaluronic acid-cyclodextrin derivative is 25 to 35%:

[Formula 1]


In Formula 1, n is an integer from 25 to 8,000, and m is an integer from 2 to 8.
delete delete According to claim 1,
The hyaluronic acid-adamantane derivative is a composition for transdermal delivery in which the carboxyl group of hyaluronic acid and adamantane are bonded through an ester bond or amide bond.
According to claim 1,
A composition for transdermal delivery in which the degree of substitution of adamantane in a hyaluronic acid-adamantane derivative is 10 to 50%.
According to claim 1,
A composition for transdermal delivery wherein the content ratio of the hyaluronic acid-cyclodextrin derivative and the hyaluronic acid-adamantane derivative is 1:0.1 to 1:10.
According to claim 1,
The hydrogel is a composition for transdermal delivery that is formed by supramolecular reaction of cyclodextrin of a hyaluronic acid-cyclodextrin derivative and adamantane of a hyaluronic acid-adamantane derivative.
According to claim 1,
Supramolecular self-assembled hyaluronic acid hydrogel is a composition for transdermal delivery that is an aqueous solution-based buffer solution.
According to claim 1,
It additionally contains useful substances,
The useful substance is a composition for transdermal delivery, wherein the useful substance is one or more selected from the group consisting of drugs, useful substances in functional cosmetics, fluorescent substances, radioactive isotopes, target-directing substances, imaging substances, and culture media required for cell culture.
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