KR20200017625A - Use of modified hyaluronic acids for delivering biomolecules or drugs in vivo - Google Patents

Abstract

The present invention provides a hydrogel platform using hyaluronic acid (HA) conjugated with a pyrogallol (PG) moiety as a substrate. The HA-PG conjugate of the present invention can be rapidly crosslinked by two different methods that either use an antioxidant or regulate the pH. The hydrogel of the present invention exhibits excellent biocompatibility and can efficiently adjust physical properties, such as crosslinking rate, elasticity, adhesive strength, etc. depending on the crosslinking method. The hydrogel of the present invention, based on such excellent stability and functionality, can be utilized in various fields including biomedicinal materials, such as drug carriers, wound treating agents, or anti-adhesion agents, medicine, cosmetics, etc.

Description

Use of modified hyaluronic acids for delivering biomolecules or drugs in vivo}

The present invention relates to the use of modified hyaluronic acid derivatives and hydrogels thereof. Specifically, the present invention relates to the use of modified hyaluronic acid derivatives and their hydrogels for the delivery of biomolecules or drugs.

As the markets of the medical, bio- and cosmetics industries expand rapidly, interest in functional biomaterials is increasing. In particular, the development of biocompatible materials using natural polymers that are more stable than chemically synthesized polymers that may cause toxicity or side effects are receiving more attention. Various studies and developments on hyaluronic acid have been performed as a biocompatible material. Hyaluronic acid is a bio-derived polymer with little side effects when applied in vivo and is hydrophilic due to the chemical structure of the sugars contained. In addition, it contains a lot of moisture, has a physical buffering effect and friction lubricating effect in the joint, and is known to be involved in the flexibility of the skin. In addition, there is a protective property against bacterial invasion from the outside, it is biodegraded at the time of transplantation in vivo by hyaluronic acid enzyme (hyaluronidase) in vivo, and is used as an important material of the drug delivery system by combining various drugs with hyaluronic acid. In particular, since hyaluronic acid has been approved by the US Food and Drug Administration, it has been widely used as a material for medical biomaterials, tissue engineering scaffolds and drug delivery polymers. In addition, hyaluronic acid is abundantly present in several different layers of the skin and, for example, is involved in hydrating functions, assisting tissues of the extracellular matrix, functions as fillers, and tissue regeneration mechanisms. Has the same complex function. However, with aging, the amount of hyaluronic acid, collagen, elastin, and other matrix polymers present in the skin decreases. For example, repeated exposure to ultraviolet light from the sun causes dermal cells to reduce their hyaluronic acid production as well as increase their rate of degradation. Loss of these materials causes wrinkles, holes, water loss, and / or other undesirable conditions that contribute to aging. Therefore, as one of methods for improving the skin condition, filler compositions containing hyaluronic acid as a main component have been widely used.

As a related art of hyaluronic acid, examples of synthesizing cross-linked insoluble hyaluronic acid derivatives using compounds having two functional groups such as bisepoxide, bishalide, formaldehyde and the like have been reported in various documents. In particular, US Pat. No. 4,582,865 discloses an example of using divinyl sulfone for crosslinking hyaluronic acid, and US Pat. No. 4,713,448 uses a crosslinking reaction with formaldehyde, US Pat. No. 5,356,883 uses various carbodiimides. A synthesis example of a hyaluronic acid derivative gel in which a carboxyl group is modified with O-acylurea or N-acylurea is disclosed. However, the hyaluronic acid crosslinked products prepared by the methods in these patents have a low stability to hyaluronic acid degrading enzymes and have a high content of unreacted chemicals, which may cause biotoxicity. In addition, there is a limit to the application of a variety of medical materials because it is not easy to control the cross-linking or physical properties to suit the intended use. Therefore, there is still a need for development of a technology that can easily control the physical properties of hyaluronic acid hydrogel while maintaining excellent biocompatibility.

Accordingly, the present inventors have endeavored to develop a technology capable of improving the functionality of hyaluronic acid, a biocompatible material, to solve such problems. As a result, a hyaluronic acid-based hydrogel platform technology modified with a pyrogallol group was developed, and thus, the present invention was completed.

Thus, it is an object of the present invention to provide a hyaluronic acid derivative prepared by modifying hyaluronic acid with a pyrogallol group and a method for producing the same.

Another object of the present invention is to provide a method for preparing a hyaluronic acid derivative hydrogel comprising crosslinking the hyaluronic acid derivative.

Another object of the present invention relates to a hyaluronic acid derivative hydrogel having a structure in which the hyaluronic acid derivative is crosslinked.

Another object of the present invention relates to a method for delivering a biomolecule or a drug using the hyaluronic acid derivative or a hydrogel thereof.

Another object of the invention is for the delivery of biomolecules or drugs. It relates to the use of modified hyaluronic acid derivatives or hydrogels thereof.

Another object of the present invention is to provide a method for delivering a biomolecule or a drug. It is to provide a carrier or drug delivery system (DDS) for drug delivery.

Another object of the present invention is to provide a medical material such as a support for tissue engineering using the hyaluronic acid derivative or a hydrogel thereof.

Another object of the present invention is to provide a wound dressing or an adhesion barrier based on the hyaluronic acid derivative.

Another object of the present invention is to provide a filler composition comprising the hyaluronic acid derivative or a hydrogel thereof.

Another object of the present invention is to provide a method for improving skin wrinkles, comprising the step of injecting the filler composition into the subcutaneous or subcutaneous of the individual.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention includes the step of crosslinking a hyaluronic acid derivative modified with gallo group, the hyaluronic acid derivative is a gallo group by a reaction between hyaluronic acid and 5'-hydroxydopamine Provided is a method of preparing a hyaluronic acid hydrogel, which has been modified.

In one embodiment of the present invention, the hyaluronic acid derivative may be represented by Formula 1, wherein the molecular weight of the hyaluronic acid derivative may be 10,000 Da to 2,000,000 Da, the galol substitution rate of the hyaluronic acid derivative is 0.1% to May be 50%.

[Formula 1]

이고, n은 1 내지 1000의 정수이다.) (In Chemical Formula 1, R ₁ is a hydroxy group or

N is an integer from 1 to 1000.

In another embodiment of the present invention, the step of crosslinking may be crosslinked by adding an oxidizing agent or a pH adjusting agent, and the oxidizing agent may be sodium periodate, hydrogen peroxide, mustard peroxidase or tyrosinase, and the pH The modulating agent may be, but is not limited to, sodium hydroxide, lithium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, magnesium hydroxide, calcium hydroxide, strontium hydroxide or barium hydroxide.

As another embodiment of the present invention, the step of crosslinking by adding the oxidizing agent, may form a crosslinking bond of the following formula (2) between the hyaluronic acid derivative.

[Formula 2]

(In Formula 2, HA 'represents hyaluronic acid in which the carboxyl group is substituted with an amide group.)

As another embodiment of the present invention, in the step of crosslinking by adding the pH adjusting agent, it is possible to form a crosslinking bond of the following formula (3) between the hyaluronic acid derivatives.

[Formula 3]

(In Formula 3, HA 'represents hyaluronic acid in which the carboxyl group is substituted with an amide group.)

In addition, the present invention provides a carrier for delivery of a biological (active) material comprising the hyaluronic acid derivative of Chemical Formula 1. In this aspect, in one embodiment of the present invention, the drug delivery carrier is an antibody, antibody fragment, protein, peptide, polypeptide, small molecule chemical compound, DNA and / or RNA, siRNA, gene, and Stem cells, including, but not limited to, adult stem cells, mesenchymal stem cells, or induced pluripotent stem cells (iPSCs). In this regard, another embodiment of the present invention, the carrier for delivery of the bioactive material provides a sustained release of the bioactive material in vivo and ex vivo.

In addition, the present invention is a hyaluronic acid hydrogel prepared by crosslinking the hyaluronic acid derivative of the formula (1), for example, hyaluronic acid hydrogel in which the crosslinking of the formula (2) or formula (3) between the hyaluronic acid derivative is formed; And it provides a carrier for delivery of a biological (active) material comprising the hyaluronic acid hydrogel. In this aspect, in one embodiment of the present invention, the drug delivery carrier is an antibody, antibody fragment, protein, peptide, polypeptide, small molecule chemical compound, DNA and / or RNA, siRNA, gene, and Stem cells, including, but not limited to, adult stem cells, mesenchymal stem cells, or induced pluripotent stem cells (iPSCs). In this regard, another embodiment of the present invention, the carrier for delivery of the bioactive material provides a sustained release of the bioactive material in vivo and ex vivo.

In addition, the present invention is a hyaluronic acid hydrogel prepared by crosslinking the hyaluronic acid derivative of the formula (1), for example, hyaluronic acid hydrogel in which the crosslinking of the formula (2) or formula (3) between the hyaluronic acid derivative is formed; And it provides a support for tissue engineering comprising the hyaluronic acid hydrogel.

The present invention also provides a filler composition comprising a hyaluronic acid derivative modified by a gallolic group or a hyaluronic acid derivative hydrogel prepared by crosslinking the same.

In one embodiment of the present invention according to this object, the molecular weight of the hyaluronic acid derivative may be 10,000 Da to 2,000,000 Da, the pyrogallol group substitution rate of the hyaluronic acid derivative is 0.1% to 50%, preferably 1% to 30%, more preferably 2% to 20%. In another embodiment of the present invention, the hyaluronic acid derivative may be contained in 0.1% (w / v) to 15% (w / v) with respect to the total filler composition.

In another embodiment of the present invention, the filler composition is in a liquid state in vitro and can form a gelled state in the body without a crosslinking agent. In yet another embodiment of the present invention, the filler composition is any one selected from the group consisting of tear bone area, glacial fold area, eye area, forehead area, dorsal area, nasal wrinkle area, lateral wrinkle area, and neck wrinkle area. It can be injected at one site. In another embodiment of the present invention, the filler composition, fibroblast growth factor (FGF), epidermal growth factor (EGF), keratinocyte growth factor (KGF), transforming growth factor alpha (TGF-α), Transformation growth factor beta (TGF-β), granulocyte formation stimulating factor (GCSF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet derived growth factor-BB (PDGF) -BB), brain-derived neurotrophic factor (BDNF), and glial-derived neurotrophic factor (GDNF) may further comprise any one cell growth factor selected from the group. As another embodiment of the present invention, the filler composition may further include any one component selected from the group consisting of local anesthetics, antioxidants, vitamins, and combinations thereof.

In addition, the present invention comprises the steps of preparing a hyaluronic acid derivative by introducing a pyrogallol group into the skeleton of glucuronic acid of hyaluronic acid; And adding the hyaluronic acid derivative to an aqueous medium to mix the same.

In addition, the present invention provides a method for improving skin wrinkles, comprising the step of injecting the filler composition into the subcutaneous or subcutaneous of the individual.

The present invention also provides a wound dressing or an adhesion barrier, including a hyaluronic acid derivative modified with a gallo group or a hyaluronic acid derivative hydrogel prepared by crosslinking the same.

Techniques for preparing hyaluronic acid hydrogels according to the present invention include the steps of crosslinking them under appropriate oxidation or specific pH conditions, based on galulol-modified hyaluronic acid derivatives. The present invention can effectively control the physical properties such as the crosslinking rate, elasticity, adhesive strength according to each crosslinking method with excellent biocompatibility of the hydrogel, it can be utilized in various fields such as medical and cosmetics.

1 is a diagram illustrating a synthesis process of a hyaluronic acid derivative according to the present invention.
Figure 2 is a result of the analysis of the structure of the hyaluronic acid derivatives according to the present invention (a) FT-IR or (b) H-NMR.
Figure 3 is a schematic diagram showing the change in appearance and crosslinking of the hyaluronic acid hydrogel by different crosslinking methods.
Figure 4 is a diagram showing the cross-linking mechanism based on the results of (a) analysis of the change in the chemical structure by UV-vis in the manufacturing process of the hyaluronic acid hydrogel using NaIO ₄ , and (b).
FIG. 5 is a diagram illustrating a crosslinking mechanism based on UV-vis analysis of (a) the chemical structure change and (b) the hyaluronic acid hydrogel using NaOH.
Figure 6 confirms the formation rate of the hyaluronic acid hydrogel according to the present invention, (a) as a result of visually confirming the formation of the hydrogel over time, and (b) the change in the elastic modulus over time This is the confirmed result.
Figure 7 confirms the strength and elasticity of the hyaluronic acid hydrogel according to the present invention, (a) as a result of confirming the change in the elastic modulus at 0.1 to 1Hz, and (b) the elastic modulus and tan δ (G '' / G ' ) Is the result of comparison.
8 shows the change in strength and elasticity of the hyaluronic acid hydrogel crosslinked with NaIO ₄ according to the molecular weight and concentration of the hyaluronic acid derivative, (a) the change in the elastic modulus at 0.1 to 1Hz, and (b) It is the result of comparing elastic modulus and tan δ (G '' / G ').
Figure 9 confirms the adhesive force of the hyaluronic acid hydrogel according to the present invention, (a) measuring the change in the force applied to the rheological device according to the distance between the plates, and (b) the result of quantifying the adhesive force .
Figure 10 confirms the swelling and decomposition of the hyaluronic acid hydrogel according to the present invention, hyaluronic acid synthesized by the addition of 5-hydroxydopamine of (a) 0.5X or (b) 1.0X compared to the molar concentration of hyaluronic acid It is the result of quantifying and evaluating the swelling rate and decomposition rate of the hyaluronic acid hydrogel manufactured using the derivative.
Figure 11 confirms whether the hyaluronic acid hydrogel according to the present invention whether the cytotoxic and inflammatory response induced, (a) and (b) after encapsulation of the cells to evaluate the toxicity of HAPG hydrogel 1, 3, And Live / Dead staining at 7 days (scale bar = 100 μm) (a) and hADSCs viability (b). (c) is the result of an enzyme linked immunosorbent assay for the quantification of TNF-α secreted from macrophages (RAW 264.7) when co-cultured with HA-PG hydrogels crosslinked with NaIO ₄ or NaOH solution ( n = 4, ** p <0.01 vs LPS group).
Figure 12 confirms the in vivo suitability of the hyaluronic acid hydrogel according to the present invention, (a) is HA-PG hydro recovered at 0, 7, 28, and 84 days after implantation into the subcutaneous region from the subcutaneous region of the mouse The form of the gel (DS 8%) is shown. (b) shows the in vivo biodegradation profile of HA-PG. (c) and (d) show H & E staining (c and toluidine blue staining (d) of hydrogel recovered with adjacent tissues on day 28 after transplantation, respectively (scale bar = 300 μm).
Figure 13 confirms the in vivo degradation of the hyaluronic acid hydrogel according to the present invention, after transplanting the mouse subcutaneous (a) visually confirmed the volume change of the hydrogel, and (b) the weight of the remaining hydrogel The result is confirmed.
Figure 14 confirms the availability of the hydrogel using NaIO ₄ as a drug delivery formulation, (a) before or after lyophilization, as a result of confirming the formation and presence of microparticles, and (b) to the microparticles This is the result of confirming the discharge amount of the encapsulated BSA.
15 shows the possibility of using a hydrogel using NaIO ₄ as a drug delivery agent, (a) shows a crosslinking reaction using NaIO ₄ , and (b) formation of HA-PG ultrafine particles (scale bar-50 μm). (C) shows the cumulative amount of VEGF released from bulk HA-PG hydrogels or HA-PG microparticles (PBS, 37 ° C.) (n = 4).
Figure 16 shows the effect of VEGF delivery and treatment of hydrogel with NaIO ₄ in the hindlimb ischemia mouse model, (a) muscle injection of HA-PG microparticles containing VEGF on day 0 (drug injection day) ) And the results observed on day 28, (b) the results are shown numerically (n = 4-5), (c) H & E staining and MT staining results of normal mouse tissue as a control (Scale bar-100 μm). (d) Quantitative fibrosis in the ischemic leg ( n = 12, ** p <0.01 vs PBS group, ## p <0.01 vs HA.PG group, and @ p <0.05 vs VEGF group), ( e) Immune staining of muscles of the ischemic leg against α-SMA (arteriole formation) and vWF (capillary formation). Black arrows indicate the formation of small arteries (α-SMA positive) and capillary (vWF positive) in ischemic tissues (scale bar = 100 μm), respectively. (f) shows the number of lumens and vWF positive capillaries stained α-SMA positive in ischemic muscle ( n = 18.20, * p <0.05, and ** p <0.01 vs PBS group, ## p <0.01 vs HA.PG group, and @ p <0.01 vs VEGF group).
17 shows the preparation of tissue adherent HA-PG hydrogels using NaOH-mediated crosslinking. (a) Hyaluronic acid derivative hydrogel formation by NaOH-mediated crosslinking is shown. (b) The adhesion of hyaluronic acid derivative hydrogels crosslinked with NaOH or NaIO ₄ is compared. (c) compares the average separation strength of each HA-PG hydrogel ( n = 3, ** p <0.01 vs NaIO4 group).
In FIG. 18, (a) briefly illustrates the adhesion chemistry of NaOH-derived HA-PG hydrogels, and (b) shows direct adhesion to various mouse organs (heart, kidney and liver). It is a photograph.
In FIG. 19, (a) shows NaOH-derived HA-PG hydrogels on the mouse liver surface and H & E staining of the hydrogels attached on the liver tissue surface after 7 days (scale bar = 500 μm). (b) implants hADSCs (human adipose tissue-derived stem cells) on mouse liver using HA-PG crosslinked by NaOH, and then immunostaining of hADSCs in the original liver tissue and the hydrogel construct of the present invention together. It is shown. Dil-labeled cells were detected before transplantation (arms). Immunostained for CD44 and cell nuclei counterstained with DAPI (right) (scale bar = 500 μm)
Figure 20 shows the gelation by the oxidative power in the body of the HA-PG solution according to the present invention, Figure 20a is a diagram showing the gelation by injection and oxidative power of the HA-PG solution; 20B is a result of visually confirming the skin of a mouse including a hydrogel formed in the body.
FIG. 21 is a view showing whether the hydrogel formed in the body is maintained in the body after injecting HA-PG solution according to the present invention into the skin, and FIG. 5A is a result of visually confirming the hydrogel formed in the body (Week 0, 10, 24); 5b is a result of measuring the weight change of the hydrogel formed in the body for about 6 months.
Figure 22 confirms the conditions for the formation and injection of HA-PG hydrogel of the HA-PG solution according to the present invention, Figure 22a is a schematic diagram showing the formation process of the HA-PG hydrogel; And FIG. 22B is a result of comparing the size of the injectable injection needle with a conventional filler product (Megafill, Perlane).
Figure 23, after the HA-PG solution according to the present invention was injected into the skin, confirming the wrinkle improvement effect before and after the injection, Figure 23a is a result of comparing the wrinkle-induced skin with a replica template; And FIG. 23B is a result of quantifying and comparing the area, length, and depth of wrinkles.
Figure 24 is a comparison of the body filler performance with conventional filler products (Megafill, Perlane), Figure 24a is a HA-PG solution (200KDa, 1MDa), Megafill product, Perlane product after injection into the skin, Hydrogels formed in the body were visually confirmed (D0, 14, 28, 56, 84, 168, 252); 24B is a result of measuring the weight change of the hydrogel formed in the body for about 9 months.
25 is a comparison of the body filler performance with conventional filler products (Megafill, Perlane), Figure 25a is a HA-PG solution (200KDa, 1MDa), Megafill product, Perlane product after injection into the skin according to the present invention , The result of visual confirmation of the hydrogel formed in the body (D0, 14, 28, 56, 84, 168); 25B is a result of measuring the volume change of the hydrogel formed in the body for about 9 months.
Figure 26 confirms the adhesion in the body of the hydrogel formed in the body, Figure 26a is a result of the Perlane product; And FIG. 26B is the result of HA-PG solution (200 KDa, 1 MDa).
27 is a comparison of the possibility of injecting with conventional filler products (Megafill, Perlane), using a needle of various sizes needles (21, 25, 29, 30G) HA-PG solution (200 KDa, 1 MDa), Megafill products, Perlane products, the result of the extrusion force change was confirmed.
FIG. 28 is a comparison of the possibility of injection with a conventional filler product (Megafill, Perlane), Figure 28a is a HA-PG solution (200 KDa, 1M Da), Megafill product, using a 30G needle Injecting the Perlane product, the result of confirming the extrusion force change; And FIG. 28B is a result of confirming the extrusion force change when injecting HA-PG solution (200 KDa, 1MDa), Megafill product, and Perlane product according to the present invention using a 29G needle.
29 is a comparison of the possibility of injecting with conventional filler products (Megafill, Perlane), Figure 29a is a HA-PG solution according to the present invention using the needles (21, 25, 29, 30G of various sizes) 200 KDa, 1M Da), Break Loose Force, compared with the Megafill, Perlane product; And FIG. 29B shows a dynamic glide force when injecting HA-PG solution (200 KDa, 1 MDa), Megafill product, and Perlane product according to the present invention using needles 21, 25, 29, and 30G of various sizes. Is the result of comparing.
Figure 30 shows the effect of improving the wrinkles before and after injection, after injection into the skin HA-PG solution containing the epidermal growth factor (20 ng / ml, 1 μg / ml, 20 μg / ml) according to the present invention 30A shows the comparison of the wrinkle-induced skin with a replica template; And FIG. 30B is a result of quantifying and comparing the area, length, and depth of wrinkles.
31 shows the pathology of HCT by injecting HA-PG solution containing epidermal growth factor (20 ng / ml, 1 μg / ml, 20 μg / ml) in accordance with the present invention into the skin and staining the OCT frozen sections thereof. The result is a biopsy.
32 is a skin tissue regeneration effect between the conventional filler product (Perlane) and the HA-PG solution containing the epidermal growth factor and the HA-PG solution containing the epidermal growth factor (10 μg / ml) according to the present invention Was compared over time (1 month), and the results of pathological examination were performed by H & E staining of OCT frozen sections.
33 is a skin tissue regeneration effect between the conventional filler product (Perlane) and the HA-PG solution containing the epidermal growth factor and the HA-PG solution containing the epidermal growth factor (10 μg / ml) according to the present invention Was compared over time (1 month). OCT frozen sections were stained with MT (Masson's Trichrome) for histopathologic examination.
Figure 34 is a result of applying the HA-PG solution according to the present invention to the wound site, visually confirming the hydrogel formation and adhesion according to the wound site.
35 is a view briefly illustrating a process of formulating a HA-PG solution in powder form according to the present invention.
36 is a result of visually confirming the hydrogel formed in the body after injecting a re-solubilized HA-PG solution into the skin in the form of a lyophilized powder according to the present invention.
FIG. 37 shows the storage stability of the HA-PG solution according to the present invention. The HAPG solution is stored at room temperature (25 ° C.) or refrigerated (4 ° C.) while gelling in a storage container.
38 is to confirm the storage stability of the HA-PG solution according to the present invention, injecting nitrogen gas into the storage container to block the contact between the HA-PG solution and oxygen, and then stored for 3 days in a refrigerated (4 ℃) state , On the other hand, the HA-PG solution was stored for 10 days in a frozen state (-80 ℃), after which the solution was injected into the skin to confirm the hydrogel formed in the body with the naked eye.

Hereinafter, the present invention will be described in detail.

The present invention includes a step of crosslinking a hyaluronic acid derivative modified with a gallo group, wherein the hyaluronic acid derivative is a method for preparing a hyaluronic acid hydrogel, wherein the hyaluronic acid is modified with a gallo group by a reaction between hyaluronic acid and 5'-hydroxydopamine. to provide.

As used herein, the term "hyaluronic acid (HA)" refers to D-glucuronic acid (D-glucuronic acid, GlcA) and N-acetyl-D-glucosamine (GlcNAc) group β 1,3-glyco A high molecular weight linear polysaccharide comprising repeating units of disaccharides linked by a seed bond (β 1,3-glycosidic bond), which refers to both hyaluronic acid and salts thereof, but is not limited thereto. Examples of the salts include sodium salts, potassium salts, magnesium salts, calcium salts and aluminum salts.

The disaccharide repeating units of hyaluronic acid are represented by the following Chemical Formula 4, and the repeating units may be 1 to 1,000, but is not limited thereto.

[Formula 4]

Hyaluronic acid has been found in ocular free fluids, synovial fluid in joints, chicken chives and the like and is known as a highly biocompatible biomaterial. Hyaluronic acid hydrogels, despite their high applicability to biomaterials, suffer from difficulties in application as biomaterials (eg drug carriers, tissue engineering supports) due to the limitations of mechanical properties resulting from the natural polymer itself. . Accordingly, the present inventors prepared a hyaluronic acid derivative by introducing a galol group having a high oxidizing ability into hyaluronic acid (Preparation Example 1), and the hyaluronic acid derivative was crosslinked under appropriate oxidation or specific pH conditions (pH 8-9) to hydrogel By manufacturing (Production Example 2), there is a technical significance that the physical properties such as the crosslinking speed, elasticity, adhesive strength and the like can be efficiently controlled (Examples 1 and 3).

As used herein, the term "hyaluronic acid derivative" or "hyaluronic acid-pyrogallol conjugate" is interpreted to include all of hyaluronic acid or a salt thereof in which a galol group is introduced into the skeleton of glucuronic acid of hyaluronic acid or a salt thereof. do.

In one embodiment, the terminal of the disaccharide repeat unit of Formula 4, specifically, may be prepared by the reaction between the carboxy group and 5'- hydroxy dopamine. The hyaluronic acid derivative prepared by the above reaction includes one or more repeating units represented by the following Chemical Formula 5, and may be represented by the following Chemical Formula 1.

[Formula 5]

[Formula 1]

이고, n은 1 내지 1,000의 정수이다.) (In Chemical Formula 1, R ₁ is a hydroxy group or

N is an integer from 1 to 1,000.

In addition, the molecular weight of the hyaluronic acid derivative may be 10,000 Da to 2,000,000 Da, the galol substitution rate of the hyaluronic acid derivative may be 0.1% to 50%, but is not limited thereto.

The "substitution rate" means that a specific functional group of hyaluronic acid or a salt thereof is replaced or modified with a galolo group. Substitution rate to galol group is defined as the ratio of the repeating unit in which galol group was introduce | transduced among all the hyaluronic acid repeating units, By definition, it is more than 0 and 1 or less, or more than 0% and 100% or less, or more than 0 mol% and 100 mol% or less It can be expressed as a numerical value of.

As used herein, the term "hydrogel" refers to a three-dimensional structure of a hydrophilic polymer having a sufficient amount of water, and for the purposes of the present invention, the hydrogel is formed between gallon-modified hyaluronic acid derivatives. Hydrogel is shown.

The process of preparing the hyaluronic acid hydrogel may be carried out by a crosslinking reaction of the hyaluronic acid derivative, and further comprising preparing a hyaluronic acid hydrogel precursor solution by mixing the hyaluronic acid derivative with PBS for the crosslinking reaction. It may include. Such crosslinking may be formed into hydrogels by chemical crosslinking, physical crosslinking or biological crosslinking by UV irradiation. Here, the chemical crosslinking by UV irradiation may include crosslinking using photo-crosslinking or a reactive crosslinker, and the biological crosslinking may include crosslinking or DNA using a binding force between heparin and growth factors. Crosslinking using a complementary bond, and the like, and physical crosslinking may include crosslinking by hydrogen bonding, crosslinking by hydrophobic (hydrophobic) interaction, or crosslinking using electrostatic interaction, but is preferably an oxidizing agent or a pH adjusting agent. It can be added and crosslinked. The oxidant may be sodium periodate, hydrogen peroxide, mustard peroxidase or tyrosinase, and the pH adjusting agent may be sodium hydroxide, lithium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, magnesium hydroxide, calcium hydroxide, strontium hydroxide or It may be barium hydroxide, but is not limited thereto.

In one embodiment, when the cross-linking by adding the oxidizing agent, a cross-linked bond of the following formula (2) is formed between hyaluronic acid derivatives. In formula (2), HA 'represents hyaluronic acid in which the carboxyl group is substituted with an amide group.

[Formula 2]

As another embodiment, when crosslinking by adding the pH adjusting agent, crosslinking of the hyaluronic acid derivative to form a crosslink of the formula (3). In formula (3), HA 'represents hyaluronic acid in which the carboxyl group is substituted with an amide group.

[Formula 3]

In one embodiment of the present invention, the hyaluronic acid hydrogel was prepared using the sodium iodate as the oxidizing agent or the sodium hydroxide as the pH adjusting agent in the crosslinking step of the hyaluronic acid derivative modified with gallo groups (see Preparation Example 2). As a result, the hyaluronic acid hydrogel prepared according to each crosslinking method was able to confirm a marked difference in physical properties such as crosslinking rate, strength, elasticity, adhesiveness, and decomposition mode with excellent biocompatibility (see Examples 1 to 3). .

In one embodiment of the present invention, a filler composition comprising a hyaluronic acid derivative or hyaluronic acid hydrogel of the present invention is provided. The filler composition of the present invention is provided in a liquid or solution state in which a hyaluronic acid derivative having a galol group introduced thereon is mixed on an aqueous medium. In particular, when injected into the body, even if such a solution does not contain a cross-linking component, it can form a hydrogel with the body's oxidative power without supplementation between individuals to replenish skin tissue and play a role in maintaining the moisture of the skin. In one embodiment, the aqueous medium is Phosphate buffered saline (PBS), and the hyaluronic acid derivative is preferably 0.1% (w / v) to 20.0% (w / v), based on the total filler composition, More preferably, it may contain 0.3% (w / v) to 10.0% (w / v), but may be prepared by various modifications or variations depending on the content ratios in the aqueous medium and the filler composition which are well known in the art.

As used herein, the term "filler" refers to an injectable substance that replenishes skin tissue, such as by injecting a biocompatible material into the skin or subcutaneously to improve wrinkles and restore aesthetic appearance. Currently, as a filler manufacturing material approved by the FDA or MFDS, there are collagen, hyaluronic acid, calcium hydroxylapatite, polymatic acid and the like. Among them, hyaluronic acid is a substance similar to human components, and it can be used without skin reaction test, and it has the property of attracting 214 water molecules per molecule, which can effectively maintain the moisture of the skin. Accounted for%. In the filler composition according to the present invention, by using a hyaluronic acid derivative having a galol group having a high oxidizing power, hydrogels can be formed only by oxidizing power in the body without the addition of a crosslinking agent, thereby improving biocompatibility, and forming a hydrogel formed in the body as described above. The gel can maintain its shape in a transparent state for a long time (at least 6 months or more), showing excellent anti-wrinkle effect according to the properties of the hyaluronic acid described above, as well as excellent adhesion and stability in the body compared to conventional filler products, There is a technical significance that it can stably scan the target site irrespective of the extrusion force (Examples 2 and 3).

In addition, the filler compositions of the present invention may be formulated in powder form, more specifically in lyophilized powder form, to provide ease of use and storage stability. On the other hand, the filler composition requires a pretreatment step of dissolving and solubilizing in an aqueous medium such as PBS before injection into the skin, but direct application of the solutiond filler composition is also possible depending on storage and formulation conditions.

In another embodiment, to impart an effective skin regeneration effect, the filler composition of the present invention may further comprise a cell growth factor or vitamin. The cell growth factor is a generic term for polypeptides that promote cell division, growth, and differentiation, preferably fibroblast growth factor (FGF), epithelial growth factor (EGF), keratinocyte growth factor (KGF), Transformation growth factor alpha (TGF-α), transformation growth factor beta (TGF-β), granulocyte formation stimulating factor (GCSF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor ( HGF), platelet-derived growth factor-BB (PDGF-BB), brain-derived neurotrophic factor (BDNF), and glial-derived neurotrophic factor (GDNF), the cell growth factor being from 20 ng / ml to It may be contained at a concentration of 20 μg / ml, but is not limited thereto.

As another embodiment, to relieve pain during the infusion process, the filler composition of the present invention may further comprise a local anesthetic. The topical anesthetics include ambucaine, amalanone, amolanone, amylocaine, benoxinate, benzocaine, benzocaine, beoxycaine, biphenamine, Bupivacaine, butacaine, butamben, butamben, butanilicaine, butethamine, butethamine, butoxycaine, carticaine, chloroprocaine, Cocaethylene, Cocaine, Cyclomethicain, Dibucaine, Dimethisoquinone, Dimethocaine, Diferodon, Dicyclonin, Exgonidine, Exgonin, Ethyl Chloride, Ethidocaine, Beta-Yucane, Eu Prosine, phenalcomin, formokine, hexylcaine, hydroxytetracaine, isobutyl p-aminobenzoate, rheinokine mesylate, reboxadol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, Methyl Chloride, Mirtecaine, Nephine, Octacaine, Ortho Phosphorus, oxetazaine, paretoxycaine, phenacaine, phenol, piperocaine, pyridocaine, polydocanol, pramoxin, prilocaine, procaine, propanocaine, propparacaine, propicocaine, propoxycaine , Pseudococaine, pyrrocaine, ropivacaine, bupivacaine, salicylic alcohol, tetracaine, tolylcaine, trimecaine, zolamine and salts thereof and may be selected from the group of The amount may be preferably contained in an amount of 0.1 wt% to 5.0 wt%, more preferably 0.2 wt% to 1.0 wt% based on the total filler composition weight, but is not limited thereto.

In another embodiment, the filler composition of the present invention may further include an antioxidant to prevent oxidation and decomposition of the hydrogel formed by gelling in the body. The antioxidant may be selected from the group consisting of polyols, mannitol, and sorbitol, the amount of the antioxidant is preferably from 0.1% to 5.0% by weight, more preferably from 0.2% to 1.0% by weight of the total filler composition It may be contained in weight percent, but is not limited thereto.

As another aspect of the invention, the present invention comprises the steps of preparing a hyaluronic acid derivative by introducing a galol group into the skeleton of the glucuronic acid of hyaluronic acid; And adding the hyaluronic acid derivative to an aqueous medium to mix the same.

In the present invention, the step of adding and mixing the hyaluronic acid derivative to the aqueous medium, in order to prevent gelation before the injection of the filler composition in the liquid state, preferably blocked contact with oxygen and other oxidation sources (for example , Injection of nitrogen gas) may be performed at conditions of about 4 ° C. to 28 ° C., and / or pH 4 to 8.

 In one embodiment, the aqueous medium is a phosphate buffered saline (PBS), the hyaluronic acid derivative may be of Formula 1 by the reaction between hyaluronic acid and 5'- hydroxy dopamine, the filler of the present invention The composition can be prepared by adding the above-described cell growth factor local anesthetics, antioxidants, vitamins, and combinations thereof.

In another aspect of the present invention, there is provided a method for improving skin wrinkles, the method comprising injecting the filler composition intradermal or subcutaneously. In the present invention, the filler composition target site may be any part of the body, such as the face, neck, ears, chest, hips, arms, hands, etc. of the individual, and preferably, as the wrinkle area of the skin, the tear bone area, the brow It may be any one selected from the group consisting of a wrinkle area, an eye area, a forehead area, a dorsal area, a nasal wrinkle area, a side wrinkle area, and a neck wrinkle area, but are not limited thereto. In addition, in the present invention, "individual" means a subject in need of improvement of skin wrinkles, and more specifically, includes all of human or non-human primates and the like.

In particular, the filler composition of the present invention can be easily injected or injected into the target site irrespective of the extrusion force. In the injecting step, the filler composition may be injected intradermally or subcutaneously using a needle or a cannula of various sizes, and as a filler injection method, for example, a serial punture that stings several times little by little Way; Linear threading method that advances the needle about 10mm and then scans it little by little while moving backward; After stabbing once, the Fanning method may be used to repeatedly scan in the same manner by twisting a slight angle without removing the needle injected by the linear threading method.

As another embodiment of the present invention, a hyaluronic acid hydrogel prepared by crosslinking a hyaluronic acid derivative of Chemical Formula 1, hyaluronic acid hydrogel in which a crosslinking bond of Chemical Formula 2 is formed between hyaluronic acid derivatives; And it provides a drug delivery carrier, drug delivery system or tissue engineering support comprising the hyaluronic acid hydrogel.

In still another aspect of the present invention, a hyaluronic acid hydrogel prepared by crosslinking a hyaluronic acid derivative of Chemical Formula 1 includes: a hyaluronic acid hydrogel having a crosslinking bond of Chemical Formula 3 formed between hyaluronic acid derivatives; And it provides a drug delivery carrier, drug delivery system, or tissue engineering support comprising the hyaluronic acid hydrogel.

The hyaluronic acid hydrogel of the present invention can be used as an artificial extracellular matrix as an effective skeleton for drug delivery, and can realize nano- or micro-particulate forms by the excellent oxidation ability of the hyaluronic acid derivative in which the galol group is modified. There is technical significance. The drug is not particularly limited, but preferably is a chemical, small molecule, peptide, antibody, antibody fragment, nucleic acid, including DNA, RNA or siRNA, protein, gene, virus, bacteria, antibacterial, antifungal, anticancer, anti-inflammatory, or Mixtures thereof, and the like, but are not limited thereto.

In addition, the hyaluronic acid hydrogel of the present invention can be used as a support for tissue engineering based on excellent elasticity and adhesion. Tissue engineering means that a cell-support complex is prepared by culturing cells or stem cells isolated from a patient's tissue in a scaffold, and then transplanting the prepared cell-support complex again in vivo, wherein the hyaluronic acid Hydrogels can be implemented with supports similar to living tissue, in order to optimize the regeneration of living tissue and organs. Therefore, it can be used for gene therapy or cell therapy.

In addition, the hydrogel of the present invention may be utilized as cosmetics and medical materials such as wound healing agents, wound coating materials, anti-adhesion agents, or dental matrices, but is not limited thereto.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Production Example

Preparation Example 1 Preparation of Hyaluronic Acid Derivative Modified by Gallol Group

As shown in FIG. 1, a hyaluronic acid derivative modified with a galol group of the present invention was prepared. Specifically, hyaluronic acid (MW 200K, Lifecore Biomedical, IL, USA) is completely dissolved in water (TDW), 1 equivalent of NHS (N-hydroxysuccinimide, Sigma, St. Louis, MO, USA) and 1.5 equivalents of EDC (1- (3-dimethylaminopropyl) -3-ehtylcarbodiimide hydrochloride, Thermo Scientific, Rockford, IL, USA) was added and after 30 minutes, 1 equivalent of 5'-hydroxydopamine (5-hydroxydopamine as PG moiety) , Sigma) was added to react for 24 hours at pH 4 ~ 4.5. Two different molar ratios of the reactants were used to prepare HA-PG conjugates (HA: EDC: NHS: PG = 1: 1.5: 1: 1 or 2: 1.5: 1: 1). Subsequently, EDC, NHS, 5'-hydroxydopamine was converted into PBS and water-based dialysis (Cellu / Sep ™, dialysis membrane (6.8 kDa cut-off, Membrane Filtration Products Inc., Seguin, TX, USA)). The solvent was evaporated through freeze-drying to prepare the hyaluronic acid derivative of the present invention. In order to confirm the synthesis of the hyaluronic acid derivatives, Fourier transform-infrared spectroscopy (FT-IR) (Vertex 70, Bruker, Billerica, MA, USA) and proton nuclear magnetic resonance (H-NMR) (Bruker 400 MHz, Bruker) As a result of the analysis, as shown in FIG. 2A, the newly formed amide bonds were confirmed through strong peaks in the frequency range of about 1,580 cm ⁻¹ to 1,700 cm ⁻¹ , and as shown in FIG. 2B, 6.5 ppm and 3 ppm The peaks in the vicinity confirmed the structures of the aromatic benzene rings and -CH ₂ CH ₂ -of 5'-hydroxydopamine, respectively. From the above results, it can be seen that the hyaluronic acid derivative of the present invention introduces 5'-hydroxydopamine into an amide bond formed by the carboxyl group of hyaluronic acid and the amine period of 5'-hydroxydopamine. To calculate the degree of substitution of the galol group for the HA backbone, HA-PG conjugates were dissolved in PBS (pH 5) at 1 mg / ml and the absorbance of the solution was 283 nm (UV-visible spectrophotometer (Cary 100 UV-vis). , Varian Inc., Palo Alto, CA, USA) Standard curves obtained by serial dilution of 5-hydroxydopamine solution (from 1 mg / mL concentration) of% carboxyl in HA substituted with PG Calculated using.

Preparation Example 2 Preparation of Hyaluronic Acid Hydrogel

The hyaluronic acid hydrogel was prepared by cross-linking the hyaluronic acid derivative of Preparation Example 1, wherein the cross-linking method was a hydroxide of sodium periodate (NaIO ₄ ) or a pH regulator (pH 8-9), which is an oxidizing agent. Sodium (NaOH) was used respectively. Specifically, after dissolving the hyaluronic acid derivative in PBS (1% (w / v), 2% (w / v)), 4.5 mg / ml of NaIO ₄ Alternatively, crosslinking was performed while mixing 0.08 M NaOH at a ratio of 1.5: 1 to 4: 1 relative to the volume of the hyaluronic acid derivative solution, and as shown in FIG. Gels were prepared.

To specifically confirm the crosslinking of the hyaluronic acid hydrogel, it was analyzed by UV-vis (Ultraviolet-visible spectroscopy). In the case of using NaIO ₄ , as shown in FIG. 4, it can be seen that the wavelength region of 350 to 400 nm is changed over time (FIG. 4A), which indicates the instantaneous formation and reduction of radicals, which are intermediates in the oxidation process. Meaning, it was found that the biphenol (biphenol) is formed by the radical polymerization reaction (Fig. 4b). In addition, in the case of using NaOH, as shown in FIG. 5, it was confirmed that the wavelength region of 600 nm was changed over time (FIG. 5A), which was caused by the charge transfer complex and [5 + 2] tautomerization during oxidation. It was found that benzotropolone was formed (FIG. 5B).

Example

Example 1 Change of Physical Properties of Hyaluronic Acid Hydrogel According to Crosslinking Method

In this embodiment, the physical properties of the hyaluronic acid hydrogel according to the difference in the crosslinking method of Preparation Example 2 was compared. Meanwhile, in the preparation of the hydrogel, the molar ratio of 5'-hydroxydopamine to hyaluronic acid (0.5X (HA: EDC: NHS: 5'-hydroxydopamine = 2: 1.5: 1: 1), 1X (HA: EDC: NHS: 5'-hydroxydopamine = 1: 1.5: 1: 1)) was able to adjust the substitution rate of galol to 4-5% (0.5X), or 8-9% (1X) (not shown). The change of physical properties of hydrogel according to the degree of substitution of 5'-hydroxydopamine was also compared. Specifically, changes in hydrogel formation and modulus of elasticity over time were compared according to the crosslinking method, and the elasticity, adhesion, swelling, and disintegration of the crosslinking method and the degree of substitution of 5'-hydroxydopamine were compared. Analyzed.

1-1. Comparison of Formation Rates of Hyaluronic Acid Hydrogels

As shown in FIG. 6A, when NaIO ₄ was used, the light brownish hydrogel was instantaneously formed through an instant crosslinking reaction, whereas when NaOH was used, the blue hydrogel was formed relatively slowly. Could. Also, were measured and the storage modulus (G ') and loss modulus (G'') with the lapse of time, categories which call groups are cross-linked to the introduced hyaluronic acid derivative using NaIO ₄ hydrogels (HA-CA (NaIO ₄ )) And the above results. As a result, as shown in Figure 6b, as all the oxidation progressed, the hydrogel was stably formed (G '>G''). In particular, when confirming the formation time of the hydrogel through the point of contact between the storage modulus curve and the loss modulus curve, it is about 2 to 3 minutes when crosslinked using NaOH, about 30 seconds for HA-CA (NaIO ₄ ) On the other hand, when NaIO ₄ was used, it was found that the crosslinking proceeded rapidly.

The viscoelastic coefficient of hyaluronic acid was analyzed by measuring the storage modulus (G ′) and loss modulus (G “) in the frequency sweep mode in the frequency range of 0.1 to 1 Hz. The elasticity of hyaluronic acid was calculated by dividing the storage coefficient by the loss coefficient at 1 Hz (n = 45). Gelation kinetics of HA-PG were measured in a time sweep mode at a controlled strain and frequency of 10% and 1 Hz, respectively, with rheometers (MCR 102, Anton Paar, VA, USA). Two oxidants (NaIO 4 and NaOH) were added 30 minutes after the initial measurement of G 1 and G ”. Adhesion of the hydrogel was measured by recording the separation stress of the fully crosslinked hydrogel between the probe and the substrate plate on a rheometer (MCR 102, Anton Paar) (n = 3). The pulling speed of the probe was 5 μm / sec.

1-2. Comparison of Elasticity and Adhesion

As shown in Figure 7a, despite the difference in the degree of substitution of the cross-linking method or 5'- hydroxy dopamine, both the storage modulus (G ') was measured to a certain level higher than the loss modulus (G''), It was confirmed that the hydrogel was formed stably. In addition, when the elastic modulus (Elastic modulus) representing the strength of the hydrogel and tan δ (G '' / G ') representing the degree of elasticity was calculated, as shown in FIG. 7B, in the case of using NaIO ₄ , The strength of the gel was better, and all of them had excellent elasticity even at a low substitution rate, whereas when NaOH was used, excellent elasticity was obtained as the substitution rate was increased. In addition, in the same cross-linking method, it was found that each of these excellent physical properties also improved as the substitution rate of 5'-hydroxydopamine was increased (0.5X <1X). Based on the above results, the physical properties of the hydrogels crosslinked with NaIO ₄ according to the molecular weight (40, 200, 500 kDa) and the concentration (1% (w / v), 2% (w / v)) of the hyaluronic acid derivatives As a result of confirming the difference, as shown in Figure 8, all the storage modulus (G ') was measured to a certain level higher than the loss modulus (G''), it was confirmed that the hydrogel was formed stably (Fig. 8a ), The larger the molecular weight of hyaluronic acid, the higher the concentration, the strength and elasticity of the hydrogel was found to be improved (Fig. 8b). In addition, in order to evaluate the adhesion of the hydrogel, crosslinking the hydrogel between each plate of the rheology instrument (Bohlin Advanced Rheometer, Malvern Instruments, Worcestershire, UK) and measuring the force applied to the instrument while increasing the spacing of the plate was, and the hyaluronic acid derivative introduced catechol group and the hydrogel (HA-CA ₍₄ NaIO)) crosslinked using NaIO ₄ compares the results. As a result, as shown in FIG. 9, the hydrogel formed by NaOH and HA-CA (NaIO ₄ ) showed excellent adhesion, whereas the hydrogel formed by NaIO ₄ was hardly observed.

1-3. Comparison of Swelling and Degradation Patterns

As shown in FIG. 10, there were slightly different swelling and degradation patterns depending on the crosslinking mode and the degree of substitution of 5′-hydroxydopamine. Swelling properties were evaluated by measuring the weight of hydrogel remaining at specific time points (day 0, 1, 2, 3, 5, 7, 14 and 28). Specifically, HA-PG was incubated at PBS, 37 ° C. and the swelling rate was calculated by the following equation: (Wt-Wi) / Wi X 100, where Wt is the weight of the hydrogel at each time point and Wi is the initial time point. The weight of the hydrogel (n = 4 to 5). To examine the degradation profile, the swelled HA-PG hydrogel was treated with hyaluronidase (hyaluronidase (5 units / mL, Sigma) for 3 days and the weight of remaining hyaluronic acid was measured at each time point (n = 4 to 7, 0, 2, 5, 12, 24, 48 and 72 hours) When NaIO ₄ was used rather than NaOH, the swelling rate was lower and the decomposition proceeded more rapidly. When the substitution rate of was increased, the swelling rate and decomposition rate tended to decrease.To sum up these results, even when using hyaluronic acid derivatives of the same structure, hydrogels were crosslinked in different binding forms depending on the crosslinking method. (Production Example 2), which indicates a change in inherent physical properties such as strength, elasticity, adhesion, swelling and disintegration, and vice versa, even if the same crosslinking scheme is adopted, It can be seen that the physical properties also change depending on the structure of the ruronic acid derivative.

Example 2. Cytotoxicity and Biocompatibility Analysis

In this example, to evaluate the cytotoxicity and biocompatibility of the hyaluronic acid hydrogel of Preparation Example 2. First, in order to confirm the toxicity and inflammatory response in 3D cell culture, human adipose-derived stem cell (hADSC) (1.0 x 10 ⁶ cells per 100 μL of hydrogel) was cultured on a hydrogel, and LIVE / DEAD viability / cytotoxicity kit (Invitrogen, Carlsbad, CA, USA) was subjected to Live / Dead staining at each time point (day 1, 3 and 7) according to the manufacturer's instructions. Stained cells were observed with a confocal microscope (LSM 880, Carl Zeiss, Oberkochen, Germany), and viability was quantified by counting viable and dead cells from fluorescence images (n = 4 to 5). hADSC was obtained from ATCC (ATCC, Manassas, VA, USA) and supplemented with Growth Kit-low serum (ATCC) and 1% penicillin / streptomycin (Invitrogen) in Mesenchymal Stem Cell Basal Medium (ATCC) Incubated.

In addition, tumor necrosis factor secreted by an inflammatory response after co-culture of immune cells (Raw 264.7) using a transwell system (permeable supports with 3.0 μm pores, Corning, New York, NY, USA) on a hydrogel ( The amount of TNF-α) was measured using an enzyme-linked immunosorbent assay (ELISA). Raw 264.7 cells were incubated overnight, then seeded onto a 96-well plate (2.0 × 10 ⁴ cells per well), then loaded with 50 μl of hydrogel through the top insert of the transwell and further incubated for 24 hours. The amount of TNF-α in the media collected from the coculture was quantified using a TNF-α enzyme-linked immunosorbent assay (ELISA) kit (R & D Systems, Minneapolis, MN, USA).

PG-HA hydrogel of in vivo To assess biocompatibility, 100 μl of HA-PG hydrogel (final concentration of 2% [w / v]) formed by NaIO 4 or NaOH was implanted subcutaneously in ICR mice. Mice (5-week-old male, OrientBio, Seongnam, Korea) Ketamine (100 mg / kg, Yuhan, Seoul, Korea) and xylazine (20 mg / kg, Bayer Korea, Ansan, Korea). For histological analysis, hyaluronic acid constructs were recovered with adjacent tissues at predetermined time points (0, 7, 14, 28, and 84 days). The recovered hyaluronic acid was fixed in 4% paraformaldehyde (Sigma) for 2 days, embedded in an OCT compound (Leica Biosystems, Wetzlar, Hesse, Germany), and cut into 6 μm thick. in vivo Cleaved samples from the experiments were stained with H & E to assess hydrogel retention. Toluidine blue staining was performed to investigate the immune response after HA-PG hydrogel implantation. PG-HA hydrogel of in vivo Degradation was assessed by measuring the residual weight of the hydrogel structures recovered at each time point (n = 3-5).

As a result, as shown in Figure 11 and 12, both forms of hydrogel according to the cross-linking method, did not show cytotoxicity until after 7 days of culture (Fig. 11 (a). (B)). TNF-α increased by LPS, when treated with the two forms of hydrogel, only a small amount was detected, and there was no significant difference from the control (NT) without any treatment (FIG. 11 c). In addition, as shown in FIG. 12, even when the hydrogel was transplanted into the mouse, no specific inflammation was observed, and its structure was maintained well without proliferation of inflammation-related cells such as macrophage cells near the transplanted hydrogel. It can be confirmed that (Fig. 12 c and d).

Example 3 In Vivo Digestion Aspect Analysis

In this example, hyaluronic acid hydrogels differing in the crosslinking mode (NaIO ₄ / NaOH) and the molar ratio (0.5X, 1X) of reacted 5'-hydroxydopamine were implanted subcutaneously into mice. On the day of transplantation, mice were sacrificed one week, four weeks, and twelve weeks thereafter, and each hydrogel was collected, the degree of swelling thereof was visually checked, and the remaining amount was calculated in vivo. Was analyzed.

As a result, as shown in Figure 13, the hydrogel cross-linked using NaIO _{4 It} was confirmed that the degree of swelling is less, it can be seen that the degradation in vivo faster. In addition, in the same crosslinking mode, when the substitution rate of 5'-hydroxydopamine (galol group) increased, the decomposition rate tended to decrease.

The results of Examples 2 and 3 suggest that the hyaluronic acid hydrogel according to the present invention can be used in the field of bio materials such as, for example, drug carriers, tissue engineering supports, and the like. In particular, considering the inherent physical properties of Example 1 and the like, the hydrogel formed by NaIO ₄ showing a rapid crosslinking rate and in vivo degradation aspect is a carrier for drug delivery in the form of microparticles, excellent adhesion and slow The hydrogel formed by NaOH, which exhibits degradation in vivo, may be used as a tissue for tissue engineering.

Example 4 Application as a Drug Delivery System Through Microparticulate Formation

4-1. HA- PG Hydrogel Microparticles  Of used protein Slow release  (sustained release)

In this embodiment, as an embodiment of the hydrogel formed by NaIO ₄ , to prepare a drug delivery formulation in the form of ultra-fine particles having a diameter of nano or micro units and to determine the efficacy thereof. First, the emulsion of the hyaluronic acid derivative solution (HA-PG) of Preparation Example 2 was induced by using an oil / water emulsion method, and an oxidizing agent (NaIO ₄ ) was added to the solution, followed by microparticles before and after freeze drying. Formation and presence were confirmed. In addition, HA-PG solution and protein (bovine serum albumin (BSA) were mixed, made into an emulsion form, and then crosslinked by adding an oxidizing agent (NaIO ₄ ) to prepare BSA-enclosed microparticles, The protein release pattern of the microparticles was confirmed. As a result, as shown in Figure 14, through the above manufacturing process was produced microparticles containing the component of the hyaluronic acid hydrogel, and remained unchanged even after the lyophilization process (Fig. 14a). In addition, the encapsulated protein was hardly released in the HA-PG bulk hydrogel, whereas it was confirmed that the protein was released at a constant rate in the HA-PG particle (FIG. 14B). ), The hydrogel formed by the microparticulate NaIO ₄ may be used as a drug delivery agent.

4-2. Slow Release of Antibodies Using HA-PG Hydrogel Microparticles

First, a therapeutic application is prepared for sustained and controlled delivery of growth factors, wherein HA-PG hydrogel microparticles are prepared using an NaIO ₄ -mediated crosslinking ultra-fast gel-forming and oil / water emulsion method. (FIG. 15 (a). HA-PG microparticles can be prepared by simply adding to a water-in-oil emulsion of HAPG solution. Rapid chemical crosslinking of HAPG emulsion with NaIO ₄ in oil phase results in moisture A large amount of HA particles sub-micro were produced in. The resulting HA-PG microparticles exhibited a sphere having an average diameter of 8.8 μm (± 3.9 μm) (FIG. 15 (b)). Encapsulated vascular endothelial growth factor (VEGF) was released slowly for 60 days, while HA-PG in bulk form was hardly released from the hydrogel construct (Fig. 15 (c)). Catechol-formulated alginines It was confirmed that growth factor encapsulated in the hydrogel was hardly released due to the strong binding of the protein to the oxidized catechol during the gelation process. It is possible to induce strong binding to the structure, but it appears that HA-PG formed of particulates can release VEGF due to the surface area for release of significantly increased growth factors.

HA-PG microparticles incorporated with VEGF (Peprotech, Rocky Hill, NJ, USA) were applied to promote therapeutic angiogenesis in peripheral vascular disease. Intramuscular injection of VEGF containing HA-PG microparticles (VEGF loading dose: 6 μg per mouse) showed a markedly improved therapeutic effect in a model of lower limb ischemia prepared by femoral artery excision and ligation. Mice were obtained from Orient Bio ((Balb / c, 6-week-old female, OrientBio). Four weeks after infusion, any leg amputation or tissue necrosis occurred in the group treated with HAPG microparticles containing VEGF. necrosis), whereas only 20% of the groups treated with HA-PG without PBS or VEGF showed improvement in ischemic limbs (Figure 16 (a)) Single dose of VEGF at the same dose (6 μg per mouse) (Single bolus) administration showed some improvement of the ischemic leg, but 50% of the ischemic mice still had leg loss or tissue necrosis (Figure 16 (b)), and also by H & E and Massons's trichrome (MT) staining. Confirmed histological analysis showed minimal muscle damage and fibrosis formation in the ischemic mouse model of the HAPG / VEGF group (FIG. 16 (c), (d)). Also, α-smooth muscle actin (α-SMA for arterial staining) ) And von Willebrand factor (vWF) cotton for capillary staining Inverse histochemical analysis showed that the arterial and capillaries significantly increased in the HAPG / VEGF group compared to the control ((PBS, HAPG, and VEGF) (FIG. 16 (e), (f)). Substantial improvement of NaOH-mediated crosslinking was used to prepare adhesive hydrogel scaffolds for non-invasive, injection-free cell transplantation (FIG. 17 (a)). Catechol-modified polymers have been shown to exhibit strong tissue adhesion through high binding affinity for various nucleophiles of oxidized catechol in proteins.

Example  5. Hyaluronic Acid Using Oxidative Strength in the Body filler  Of composition Gelation  And wrinkle dogs Check the line effect

5-1. Formation and maintenance of hydrogel using oxidative power in the body

The HA-PG solution prepared in Preparation Example 1 was injected subcutaneously, and it was then confirmed whether the injected HA-PG solution could proceed in the body without addition of a crosslinking agent. In addition, in the body conditions, by measuring the weight change of the formed HA-PG hydrogel for about 6 months, it was confirmed whether the formed HA-PG hydrogel can be maintained in the body for a long time.

As a result, as shown in FIG. 20 and FIG. 21, the HA-PG solution injected subcutaneously in the liquid state gelled within 5 minutes to form HA-PG hydrogel in the body, and the formed HA-PG hydrogel was in a transparent state. As a result, it was well maintained in the body for 6 months without any significant weight difference. In addition, as shown in FIG. 22, when the HA-PG solution is exposed to oxidative conditions in the body, gallon period quinones having strong self-oxidation ability are easily formed to polymerize hyaluronic acid and form HA-PG hydrogels. . Therefore, unlike filler products (Megafill, perlane) consisting of the polymerized form of the formulation, the filler composition of the present invention can be used in the form of a solution, so that it is easier to inject into the skin, and may contain various additional ingredients And it was found. That is, the HA-PG solution of the present invention was able to form a hydrogel in the body without the addition of a crosslinking agent, and was able to be stably maintained, thereby confirming the applicability to the filler composition.

5-2. Check wrinkle improvement

Based on the results of Example 5-1, it was intended to confirm the wrinkle improvement effect of HA-PG solution. Specifically, calcitriol was administered to hairless mice five times a week at 0.2 μg / day for a total of six weeks to cause wrinkles on the skin. The HA-PG solution was then injected subcutaneously into the skin, and the wrinkle-induced skin before and after injection was templated with a replica and the area, length, and depth of the wrinkles were measured using a wrinkle analysis machine and compared. As a result, as shown in FIG. 23, the area, length, and depth of the wrinkles before injection of the HA-PG solution were about 6 mm 2, about 36 mm, and about 90 μm, respectively, while after HA-PG solution injection, respectively, about As 2 mm 2, about 19 mm, and about 68 μm, it can be seen that the area, length, and depth of wrinkles are all significantly reduced. These results indicate that the HA-PG solution of the present invention can be utilized as a composition for fillers for improving skin wrinkles.

5-3. Comparison with conventional filler products

(1) Comparison of adhesion and fixation in the body

In order to more specifically confirm the applicability of the HA-PG solution as a filler, HA-PG hydrogels based on hyaluronic acid derivatives having various molecular weights (200 KDa, 1 MDa) and filler products commercially available (Megafill, Perlane) ) Body maintenance and degradation patterns were measured for about 9 months and compared. In addition, the adhesion of the HA-PG solution of the present invention and the hydrogel produced by the Perlane product was compared with the adhesion or fixability of the body. As a result, as shown in Figure 24 and 25, Megafill, Perlane products showed a tendency to decrease the weight and volume of the hydrogel in the body from about one month from the day of injection, while HA-PG hydrogel 9 It was confirmed that the shape is maintained without significant change in weight and volume for months, and it can be seen that HA-PG hydrogel has excellent retention in the body. In addition, as a result of confirming the adhesion of the hydrogel in the body, as shown in FIG. 26, while Perlane product was not fixed to the skin and showed a phenomenon of being driven to a specific site, the hydrogel with HA-PG solution adhered to and fixed to the injection site. It was confirmed that it is well maintained in vivo, this excellent body adhesion is a result derived from the reaction between the functional groups present in the skin, such as amine groups, thiol groups (thiol group) and the hyaluronic acid derivative of the present invention (Fig. 22a). That is, the HA-PG solution of the present invention indicates that it has better body stability and adhesion than the conventional filler product.

(2) Injectability test

In order to specifically confirm the excellent injection potential of HA-PG solution, injection between HA-PG hydrogel based on hyaluronic acid derivatives (200 KDa, 1 MDa) having various molecular weights and fillers commercially available (Megafill, Perlane) Extrusion force variation according to the needle size (21G, 25G, 29G, 30G) was measured using the universal testing machine (UTM), as well as the break loose force, the force required to move the syringe for the first time, and the mobility of the moving syringe. Dynamic Glide Force, the force required to quantify and compare it. As a result, as shown in Figure 27 and 28, Megafill was not extruded in the needle of the small size due to the large size of the particle, it was possible to extrude only under 21G, Perlane also 29G and 30G due to the particle size While showing an irregular extrusion force in the form, the HA-PG solution (200 KDa, 1 MDa) of the present invention was confirmed to be effectively extruded with a small force in the needle of any size including 29G and 30G. In addition, as a result of comparing the break loose force and the dynamic glide force, as shown in FIG. 29, the HA-PG solution of the present invention (200K), as compared to the Perfill which showed the high force value and the unfilled Megafill, which is not easily measured. , 1M) showed a remarkably low force value, indicating that the injection force can be easily injected without affecting the extrusion force, that is, the size of the needle. These results indicate that the HA-PG solution of the present invention can be directly injected into the target site and can be injected more stably.

5-4. Functional Filler Composition Including Cell Growth Factors

In order to confirm the application as a functional filler, epidermal growth factor (EGF; 20 ng / ml, 1 µg / ml, 20 µg / ml) was added to the skin of the hairless mouse in which the wrinkles were induced by the method of Example 5-2. The sealed HA-PG solution was injected, and the wrinkle-induced skin before and after injection was templated with a replica, and the area, length, and depth of the wrinkles were measured using a wrinkle analysis machine, and the EGF-encapsulated One month after injection of HA-PG solution, the skin tissue was taken to prepare OCT frozen sections, which were then subjected to pathological examination through H & E (Hematoxylin & eosin) staining. In addition, the hairless mouse was injected with HA-PG solution, HA-PG solution, and the conventional product Perlane, in which EGF (10 μg / ml) was enclosed, and H & E and MT (Masson's trichrome) in the same manner as described above. The comparison of skin tissue regeneration was compared.

As a result, as shown in Figure 30, compared to before injection, when the injection of the EGF-encapsulated HA-PG solution, it can be seen that the area, length, and depth of the wrinkles are all significantly reduced regardless of the size of the wrinkles formed there was. In addition, as shown in FIG. 31, the collagen fibers in the dermal layer of the skin were destroyed and had a dense density and irregular arrangement. However, as the concentration of the enclosed epidermal growth factor increased, the collagen fibers tended to be regularly arranged. , Significant skin regeneration and wrinkle improvement were found. In addition, as shown in FIGS. 32 and 33, when the HA-PG solution injected with EGF is injected, the epidermal layer thickened due to wrinkles is more prominent compared to other comparative groups (No filler, Perlane, HA-PG). Thinner, it was confirmed that the collagen density of the dermal layer is significantly increased. These results indicate that by applying the cell growth factor for skin wrinkle improvement to the HA-PG solution of the present invention, it can be used as a functional filler.

Example  6. Wound dressings, wound healing agents and anti-adhesion preparations

In order to confirm the application as a dressing preparation for wound treatment, HA-PG solution was applied to a wound-induced animal model in which the back skin of the mouse was cut to a size of 1 cm x 1 cm, and then crosslinked using the surrounding active oxygen. , Hydrogel formation of the wound site and adhesion was confirmed accordingly. As a result, as shown in Figure 34, the hydrogel film is formed within 10 minutes of applying the HA-PG solution to the wound site, it was confirmed that the stable adhesion through this. These results indicate that the HA-PG solution can be utilized as a new type of wound dressing material that has good oxidation ability and can be evenly applied to the wound without any additives.

Example 7. Powder Formulation and Storage Stability Analysis

7-1. Powder Formulation

In order to confirm the powder (powder) formulation possibility of the filler composition, as shown in FIG. 35, the HA-PG solution of Preparation Example 1 was lyophilized to prepare a filler composition in powder form. Thereafter, the filler composition in powder form was dissolved in PBS (pH7) and resolubilized, and then injected into the mouse subcutaneously to observe whether hydrogel was formed. As a result, as shown in Figure 36, re-solubilized in the freeze-dried powder state, the filler composition of the present invention was confirmed that the cross-linked by the body's oxidative power as in the past to form a hydrogel. These results indicate that the filler composition of the present invention, which is powder formulated, can provide the user with ease of use and ease of storage.

7-2. Storage stability analysis

In order to confirm the specific storage stability of the filler composition, first, the HA-PG solution of Preparation Example 1 was stored at room temperature (25 ° C.) or refrigerated (4 ° C.) to confirm gelation in a storage container. In addition, nitrogen gas is injected into the storage container to block the contact between the HA-PG solution and oxygen, and then stored in a refrigerated (4 ° C) state for 3 days, and on the other hand, the HA-PG solution is frozen (-80 ° C). After storage for 10 days, the solution was injected subcutaneously in mice to observe the formation of hydrogels. As a result, as shown in FIG. 37, the filler composition of this invention gelatinized within 1 day at room temperature and 3 days in a refrigerated state by high oxidizing power. On the other hand, as shown in FIG. 38, in the refrigerated state, gelation did not proceed even after 3 days in the refrigerated state, and the HA-PG solution was stored in the frozen state and then thawed after 10 days. In addition to maintaining the properties of the solution, it was confirmed that even when injected into the skin of the mouse, as in the past, crosslinked by the body's oxidative power to form a hydrogel. These results indicate that the filler composition of the present invention can be stored for a longer period of time under oxygen-blocked conditions or in a frozen state.

Example  8. Exenatide  ( Exenatide )of In vivo pK analysis

In vivo pharmacokinetics (pK, pharmacokinetics) of the exenatide in order to determine whether the hyaluronic acid derivatives or hydrogels thereof of the present invention can increase the in vivo activity of the low molecular compound drug exenatide It was. Exenatide and Exenatide ELISA kits were purchased from MYBio. Hyaluronic acid derivatives and hydrogels were prepared according to the preparation examples. Sprague-Dawley Rat (SD-Rat) was purchased from Daehan Biolink. The experimental group was divided into 6 groups. The first group is the control group with PBS added to SC, the second group is 4, 8, 12% with HA, and the control group with HA-PG modified PG. The third group is 4, 8, 12% with HA. PG-modified HA-PG after the first cross-linking with NaOH was added to the control group, the fourth group was the experimental group was added once the amount of Exenatide 200μg / kg, the fifth group was 4, 8, 12% in HA Experimental group mixed 200 μg / kg Exenatide with PG-modified HA-PG and put it into SC. The sixth group mixes 200 μg / kg Exenatide with HA-PG-modified PG-modified HA-PG with HA, 4, 8, and 12%. After the cross-linking, the experiment was divided into experimental groups put in SC. SD-Rat was a male of 215 ~ 245g at 7 weeks of age and was put into 270 ~ 300g of rats at 8 weeks of age. Observation and serum were obtained for 10 days after administration of each group of substances to determine the amount of Exenatide. In order to check the amount of Exenatide present in the serum (serum), a commercially available ELISA kit purchased from MyBio was tested according to the instructions and confirmed the amount.

As a result, as shown in Figure 39, it was shown that the SD rats of all groups used in the experiment were normal without any particular weight loss. FIG. 40 is an analysis of exenatide pk of each experimental group. In the 4 group injected with exenatide alone, the amount of exenatide in blood was continuously decreased at 0.5 hours, but it was mixed with the hyaluronic acid derivative of the present invention. In groups 5 and 6, it was confirmed that the amount of exenatide in blood was maintained for up to 168 hours.

Example  9. Leuprolide  ( Leuprolide )of In vivo pK analysis

In vivo pharmacokinetics (pK, pharmacokinetics) of the leuprolides are analyzed to determine whether the hyaluronic acid derivatives or hydrogels thereof of the present invention can increase the in vivo activity of the low molecular compound drug leuprolide. It was. Leproprolide and Leuprolide ELISA kits were purchased from MYBio. Hyaluronic acid derivatives and hydrogels thereof prepared according to the above preparation examples of the present application were used in the experiment. SD rats were purchased from Daehan Biolink. The experimental group was divided into a total of six groups. The first group was the control group injected with PBS into the subcutaneoulsly injection (SC), the second group was the control group with HA-PG modified with PG (pyrrogalol) at 4, 8, and 12% in HA. 4, 8, 12% in the HA, PG-modified HA-PG after the primary cross-linking with NaOH, the control group, the fourth group was injected once the amount of leuprolide 100 μg / kg once, The fifth group was 4, 8, 12% in HA, and the experimental group was mixed with 100 μg / kg of leuprolide in HA-PG modified PG. PG-modified HA-PG was mixed with 100 μg / kg of leuprolide and subjected to primary crosslinking with NaOH and then divided into experimental groups put into SC. SD rats were male at 210 to 235 g at 7 weeks of age and were tested at 273 to 310 g of rats at 8 weeks of age at 8 weeks of age. Observation and plasma were obtained for 10 days after administration of each group of substances to check the amount of leuprolide. In order to confirm the amount of leuprolide present in the plasma, a commercially available ELISA kit was purchased from MyBio and tested according to the manufacturer's instructions.

As a result

As a result, as shown in Figure 41, the SD rats of all the groups used in the experiment appeared to be normal without any particular weight loss. FIG. 42 shows the analysis of leuprolide pk of each experimental group. In the 4 group injected with leuprolide alone, the amount of leuprolide in blood was continuously decreased at 0.25 hours, but when mixed with the hyaluronic acid derivative of the present invention. As seen in groups 6 and 6, it was confirmed that the amount of leuprolide in the blood was maintained for up to 168 hours.

Example  10. Naltrexon  ( Naltrexone In vivo pK analysis

In vivo pharmacokinetics (pK, pharmacokinetics) of the naltrexone were analyzed to determine whether the hyaluronic acid derivatives or hydrogels thereof of the present invention can increase the in vivo activity of the low molecular compound drug naltrexone. Naltrexone was purchased from sigma. The experimental group was divided into a total of six groups. The first group was the control group injected with PBS into SC, the second group was the control group injected with HA-PG modified with PG at 4, 8, and 12% in HA, and the third group was 4, 8, And 12% PG-modified HA-PG, which was injected after primary in vitro crosslinking with NaOH, the fourth group was an experimental group injected with 100 μg / kg of naltrexone once, and the fourth group was HA 4 The experimental group injected with 100 μg / kg of naltrexone mixed with HA-PG modified with PG at 8, 8, and 12% into SC, and the sixth group with HA-PG modified with 4, 8, and 12% PG in HA The naltrexone was mixed with 100 μg / kg of NaOH and crosslinked with NaOH to the experimental group injected with SC. SD rats were males at 7 weeks of age of 213 to 239g, and the experiments were conducted at 8 weeks of age at 273 to 310 g of rats. Substances in each group were observed for 10 days after administration and naltrexone was detected and analyzed from the plasma. LC-MS / MS analyzer was used to detect naltrexone present in the plasma. Perkin-Elmer Sciex API-3000 was used as detector and the system was used for Shimadzu series 10AD VP LC system. Methanol: water (1: 1, v / v) was used for stabilization. 10 ng / ml of plasma was mixed with acetonitrile in 1% and separated by Betasil silica column. . At 0 minutes, acetonitrile: water: naltrexone was flowed at a ratio of (90: 10: 1, v / v / v) and after 1 minute acetonitrile: water: naltrexone (50: 50: 1, v / v / v) The ratio was changed. Subsequently, after 2 minutes acetonitrile: water: naltrexone (50: 50: 1, v / v / v); In 3 minutes, flow acetonitrile: water: naltrexone (90: 10: 1, v / v / v). The flow rate was 0.5 ml / min and the injection volume was 10 μl.

As a result, as shown in Figure 43, it was shown that the SD rats of all groups used in the experiment were normal without any particular weight loss. 44 shows the pk of naltrexone in each experimental group. In the 4 group injected with naltrexone alone, the amount of naltrexone in the blood was continuously decreased at 0.25 hours, but in the 5 and 6 groups mixed with the hyaluronic acid derivative of the present invention, It was confirmed that the amount of naltrexone was maintained for up to 168 hours.

Example  11. Apple Recept  ( Aflibercept In vivo pK analysis

In vivo pharmacokinetics (pK, pharmacokinetics) of the applerceptor to determine whether the hyaluronic acid derivatives or hydrogels thereof of the present invention can increase the in vivo activity of the anti-VEGF antibody drug applevercept ) Was analyzed. Apple Recept and ELISA kits for it were purchased from MYBio. The experimental group was divided into 6 groups. The first group is the control group injected with PBS into SC, the second group is the control group injected with HA-PG modified with PG at 4, 8, and 12% in HA, and the third group is 4, 8, And 12% PG-modified HA-PG, which was injected with NaOH after primary crosslinking in vitro, and the fourth group was the experimental group that once added 1 mg / kg of applericept, and the fifth group HA-PG formulated with 4, 8 and 12% in HA mixed with 1 mg / kg of applivercept in SC, 6th group modified with 4, 8 and 12% in HA The experiment was carried out by dividing 1 mg / kg of appliverscept into HA-PG, first crosslinking in vitro with NaOH, and then injecting into SC. SD rats were males at 221-239 g at 7 weeks of age and subjected to experiments at 283-304 g of rats at 8 weeks of age. Eight animals per group were made. Substances from each experimental group were observed for 10 days after administration and the amount of applericept was quantified from the plasma. To detect appliverscept present in the plasma, an ELISA kit commercially available from MyBio was used according to the manufacturer's instructions.

As a result, as shown in Figure 45, SD rats of all groups used in the experiment appeared to be normal without any particular weight loss. FIG. 46 is an analysis of the pk of the applericept of each experimental group. In the 4 groups injecting only appleburcept, the amount of appleliceptin in blood was continuously decreased at 0.5 hours, but was mixed with the hyaluronic acid derivative of the present invention. In groups 5 and 6, it was confirmed that the amount of applivercept in blood was maintained for up to 168 hours.

Example  12. Somatropin  ( Somatropin In vivo pK analysis

In order to determine whether the hyaluronic acid derivative or hydrogel thereof of the present invention can increase the in vivo activity of somatropin, the in vivo pharmacokinetics of the somatropin was analyzed. Somatropin and an ELISA kit for it were purchased from MYBio. The experimental groups were divided into a total of six groups and included 8 mice in each group. The first group is a control group injected with PBS into SC, the second group is a control group injected with HA-PG (hyaluronic acid derivative modified with pyrogallol group) modified with PG at 4, 8, and 12% in HA, respectively. The first group was HA, PG modified with POH-modified HA-PG in HAOH, and the first group was injected in vitro with NaOH in the first group, and the fourth group was 150 mU / kg of sosome somatropin. Experimental group injected once, the fifth group HA, PG-modified HA-PG at the 4%, 8, and 12%, respectively, the experimental group injected with SC mixed with 150 mU / kg of somatropin, the sixth group HA Experiments were performed by mixing 150-MU / kg of somatropin with HA-PG modified with PG at 4, 8, and 12%, respectively, followed by primary crosslinking in vitro with NaOH and then injected into SC. SD rats were males at 217-244 g at 7 weeks of age and were tested at 290-311 g of rats at 8 weeks of age. Substances in each group were observed for 10 days after administration and detected and analyzed somatropin from the plasma. An ELISA kit commercially available from MyBio was used according to the manufacturer's instructions to confirm the amount of somatropin present in the plasma.

As a result, as shown in Figure 47, the SD rats of all groups used in the experiment appeared to be normal without any particular weight loss. FIG. 48 is an analysis of the somatropin pk of each experimental group. In the 4 group injected with only somatropin, the amount of somatropin in the blood was continuously decreased at 0.5 hours, but was mixed with the hyaluronic acid derivative of the present invention. In groups 6 and 6, it was confirmed that blood somatropin was maintained for up to 168 hours.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (5)

A method for delivering a biomolecule or a drug in vivo using a hyaluronic acid derivative having the formula (1) modified with a pyrogallol group and a crosslinked hydrogel thereof:
[Formula 1]

(In Chemical Formula 1, R ₁ is a hydroxy group or

And n is an integer from 1 to 1,000. The method of claim 1, wherein the hyaluronic acid derivative has a molecular weight of 10,000 Da to 2,000,000 Da. The method of claim 1, wherein the biomolecule or drug is a nucleic acid, peptide, gene, protein, stem cell or chemical compound comprising an antibody, antibody fragment, DNA, RNA or siRNA. . Formula 1 below is modified with a pharmacologically active substance that is an antibody, an antibody fragment, a nucleic acid, a peptide, a gene, a protein, a stem cell or a compound including a DNA, RNA or siRNA, and a pyrogallol group. A pharmaceutical composition having increased pharmacological activity in vivo, comprising a hyaluronic acid derivative having and a crosslinked hydrogel thereof:
[Formula 1]

(In Chemical Formula 1, R ₁ is a hydroxy group or

And n is an integer from 1 to 1,000. The pharmaceutical composition of claim 4, wherein the hyaluronic acid derivative has a molecular weight of 10,000 Da to 2,000,000 Da.

Images