KR20240019910A - Hydrogel patches for wound healing or reducing scar - Google Patents

Abstract

The present invention relates to a hydrogel patch for promoting wound healing or minimizing scarring, and is a hydrogel patch made of a biocompatible polymer modified with pyrogallol loaded with PTD-DMB, or a mixed drug of PTD-DMB and valproic acid. By using , the content of collagen Ⅲ, which is important for regenerative wound healing and alleviating scarring, increases and the dermal stem cell marker CD105 and angiogenesis marker CD31 increase in the wound area, thereby promoting wound healing and minimizing scarring.

Description

Hydrogel patches for promoting wound healing or minimizing scars {Hydrogel patches for wound healing or reducing scar}

The present invention relates to a therapeutic hydrogel patch that can promote wound healing and minimize scarring, and a cosmetic pack containing the same.

The skin performs various functions in our body, and the most important function among them is that it acts as a barrier that protects our body from the outside. A state in which the normal structure of the skin, which acts as a barrier, is destroyed is called a wound or wound.

When the skin is injured in this way, the wound area is exposed and can be invaded by external bacteria, so prompt wound treatment is required to prevent further infection and excessive inflammation. Excessive inflammatory response not only prolongs the wound healing period but also causes scarring.

Wound healing clinically means complete suturing of the skin, and normal wounds, not chronic wounds, are completely healed in approximately 3-14 days. Wound healing is a complex process in which various types of cells such as keratinocytes, fibroblasts, endothelial cells, macrophages, and platelets interact and regulate.

Currently, the wound treatments mainly used in Korea include Dongkuk Pharmaceutical's 'Combined Madecassol' (launched in 1985) and Dongwha Pharmaceutical's 'Fucidin' (launched in 1980). The efficacy of the two agents is generally similar, but Fucidin has an antibacterial effect and Madecassol has a slightly stronger strength in scar relief.

Existing steroid ointments are effective for anti-inflammatory, immunosuppressive, and allergic diseases, but they cause side effects such as pimples, skin wrinkles, and folliculitis, and are not effective for bacterial diseases such as athlete's foot. Additionally, ointments containing antibiotics can develop resistance and cause various side effects when used on delicate skin wounds such as those on children.

Therefore, in order to overcome the problems of steroid ointments or antibiotic ointments, there is a need for wound treatments based on wound healing mechanisms.

In addition, accidental injuries that are more serious than everyday skin wounds, skin diseases such as melanoma, large wounds after surgery, and wounds linked to other diseases such as diabetes not only take time to heal, but often cause scars. It is becoming a big problem. Daewoong Pharmaceutical Company has launched Korea's first over-the-counter medicine, 'EZF Saesal Ointment', a wound treatment product containing Epidermal Growth Factor (EGF). Epidermal cell growth factors help wound healing by covering the skin at the wound site (re-epithelialization) and promoting the creation of new skin (granulation tissue proliferation). However, since epithelial cell growth factor is a protein, the cost and effort required to produce and maintain safety is very high, and it has the disadvantage of not having a significant effect on scars.

Meanwhile, in order to effectively restore tissues and organs damaged by disease or accident, it is necessary to efficiently deliver drugs or cells in and out of the living body. Accordingly, much research has been conducted to deliver drugs or cells, but a system that is used in actual clinical practice and shows a satisfactory level of therapeutic effect is still lacking.

Among drug delivery methods, existing simple administration methods such as oral administration have the problem of short drug residence time due to rapid decomposition and diffusion of the drug, and for effective treatment, it is necessary to inject a large amount of drug or increase the frequency of drug injection. There wasn't.

Research has been conducted on delivery methods using hydrogels to solve problems with existing drug or cell delivery methods, but most of the research has been on methods of loading drugs or cells into a hydrogel solution and cross-linking them to induce gel formation. However, since the above method is applied by injecting hydrogel into the living body, the results are likely to vary depending on the user's skill in handling the hydrogel, and user convenience is greatly reduced. In addition, because it is injected into the body through injection, there is still a risk of tissue damage and bleeding, and the physical properties and adhesive strength of existing hydrogels are often insufficient to be maintained for a long period of time in the body.

In order to overcome the limitations of the prior art, the present inventors used a hyaluronic acid derivative modified with pyrogallol to create a hydrogel that not only has excellent drug delivery efficiency and user convenience, but is also advantageous for clinical applications because it does not require oxidizing agents, such as wound healing or A patch containing a drug with excellent efficacy in alleviating scars was produced.

Republic of Korea Patent No. 1932668 Republic of Korea Patent No. 1721028

The purpose of the present invention is to provide a therapeutic hydrogel patch that can promote wound healing and minimize scarring.

In addition, another object of the present invention is to provide a cosmetic mask pack that can promote wound healing and minimize scars.

The hydrogel patch for promoting wound healing or minimizing scars of the present invention to achieve the above object is made of a biocompatible polymer modified with drug-loaded pyrogallol,

The drug is a single drug consisting of a polypeptide fused with a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a polypeptide consisting of an amino acid sequence encoding PTD (protein transduction domain), or a mixture of the fused polypeptide and valproic acid. It may be a mixed drug.

The mixed drug is a polypeptide fused with a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a polypeptide consisting of an amino acid sequence encoding PTD (protein transduction domain), and valproic acid are mixed at a molar ratio of 1:100-1000. You can.

The single drug or mixed drug may be contained at a concentration of 0.3 to 10 mg/ml.

The hydrogel patch increases the content of collagen III in the wound area, so that the ratio of collagen III/collagen I may be 2 to 70.

The hydrogel patch can increase CD105, a dermal stem cell marker, and CD31, a marker of angiogenesis, at the wound site.

The biocompatible polymer may be selected from the group consisting of hyaluronic acid, heparin, cellulose, dextran, alginate, chitosan, chitin, collagen, gelatin, chondroitin sulfate, and pectin.

The biocompatible polymer may be hyaluronic acid.

The PTD (protein transduction domain) may include poly R8, HIV-Tat, HSV VP22, Antp, or Transportan.

The polypeptide may additionally have a linker inserted between the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence encoding the PTD.

The hydrogel patch may be a medical device patch.

In addition, the cosmetic mask pack for promoting wound healing or minimizing scars of the present invention for achieving the other purposes described above includes a hydrogel patch made of a biocompatible polymer modified with drug-loaded pyrogallol,

The drug is a single drug consisting of a polypeptide fused with a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a polypeptide consisting of an amino acid sequence encoding PTD (protein transduction domain), or a mixture of the fused polypeptide and valproic acid. It may be a mixed drug.

Wound healing and scar formation are alleviated by PTD-DBM (single drug) or a mixed drug combining PTD-DBM and VPA, but the hydrogel of the present invention in which the single drug or mixed drug is supported on HA-PG, respectively When a patch is used, it shows a better effect of promoting wound healing and alleviating scar formation than when using only the above drugs or mixed drugs.

In addition, when the hydrogel patch containing the single drug or mixed drug of the present invention is attached to the wound area, the content of collagen III increases, which is a pattern observed in wound healing without scars in fetal wound healing. Based on this, it can be seen that scars can be minimized.

In addition, when the hydrogel patch containing the single drug or mixed drug of the present invention is attached to the wound area, CD105, a dermal stem cell marker, and CD31, an angiogenesis marker, increase, which not only promotes wound healing but also minimizes scarring. .

The hydrogel patch containing a single drug or a mixed drug of the present invention can not only be used as a medical device, but can also be used as a cosmetic mask pack containing a hydrogel patch containing a single drug or a mixed drug.

Figure 1A shows the control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group of the Topical group; Patch group control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group on day 0 (0 d), day 3 (3 d), day 6 (6 d), and day 9 (9 d) ) and photos showing the scar area on day 11 (11 d); Figure 1B shows the quantification of the scar area on the 11th day of wound treatment in each group using the Image J program.
Figure 2A shows the control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group of the Topical group; This is an H&E staining image on the 11th day of wound treatment in the control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group in the Patch group; Figure 1B shows scar width (au) measured in wound tissue on the 11th day of wound treatment in each group.
Figure 3A shows the control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group of the Topical group; Immunohistochemical staining images for collagen I and collagen III on the 11th day of wound treatment in the control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group in the Patch group; Figure 3B is a graph in which the average intensity of collagen I and collagen III of each group was measured and quantified by the ratio of collagen III/collagen I.
Figure 4A shows the Topical group's control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group; This is an immunohistochemical staining image for CD105 in the wound dermis on the 11th day of wound treatment in the control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group in the Patch group; Figure 4B is a graph quantifying the number of CD105 positive cells in the wound dermis of each group.
Figure 5A shows the control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group of the Topical group; This is an immunohistochemical staining image for CD31, an angiogenesis marker, on the 11th day of wound treatment in the control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group in the Patch group; Figure 5B is a graph quantifying the number of CD31 positive cells in each group.

The present invention relates to a therapeutic hydrogel patch that can promote wound healing and minimize scarring, and a cosmetic mask pack containing the same.

The hydrogel patch refers to a thin membrane-shaped structure with a certain thickness made of biocompatible polymer, and can be cut into a desired shape and used.

Hereinafter, the present invention will be described in detail.

The hydrogel patch for promoting wound healing or minimizing scars of the present invention is made of a biocompatible polymer modified with drug-loaded pyrogallol, and the drug is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and PTD (protein transduction) A single drug consisting of a polypeptide fused with a polypeptide consisting of an amino acid sequence encoding a domain, or a polypeptide consisting of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and an amino acid sequence encoding a PTD (protein transduction domain) It may be a mixed drug containing a mixture of fused polypeptide and valproic acid.

Biocompatible polymer modified with pyrogallol (HA-PG)

The pyrogallol is a 1,2,3-trihydroxybenzene with three hydroxy groups (-OH) adjacent to each other, and forms covalent crosslinks with various functional groups through an oxidation reaction. do. In particular, due to its rapid oxidation properties, natural oxidation can occur within a few minutes without treatment with an oxidizing agent in an environment with high oxygen concentration, such as within the human body. Therefore, when applying the hydrogel patch in actual clinical practice, it has the advantage of being able to be applied directly without additional treatment with an oxidizing agent. there is.

The biocompatible polymer may be selected from the group consisting of hyaluronic acid, heparin, cellulose, dextran, alginate, chitosan, chitin, collagen, gelatin, chondroitin sulfate, and pectin, and is preferably hyaluronic acid.

The hydrogel patch made of a biocompatible polymer modified with pyrogallol (hereinafter referred to as a hydrogel patch) is a solution-based hydrogel made of a biocompatible polymer modified with pyrogallol (hereinafter referred to as bulk hydrogel). ), it has a nanofiber-based porous structure and has excellent swelling properties, so it can effectively carry cells or drugs within the patch even in an in vivo environment. In addition, hydrogel patches have better elasticity, mechanical properties, and tissue adhesion than bulk hydrogels, so they can effectively deliver drugs to tissues in vivo.

The hydrogel patch of the present invention may have a thickness of 0.05 to 10.0 mm, and preferably may have a thickness of 0.1 to 5.0 mm. If the thickness of the hydrogel patch is less than the lower limit, durability and adhesion after drug loading may decrease, and if it exceeds the upper limit, the drug release time frame may be prolonged.

Single drug (PTD-DBM) or combination drug (PTD-DBM and valproic acid combined)

The drug of the present invention is a single drug consisting of a polypeptide (PTD-DBM) fused with a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a polypeptide consisting of an amino acid sequence encoding a PTD (protein transduction domain), or the PTD -A mixed drug containing DBM and valproic acid (VPA) is included.

The PTD-DBM (protein transduction domain-fused Dvl-binding motif) is a peptide that interferes with CXXC5-Dishevelled (Dvl) interaction.

[SEQ ID NO: 1]: RKTGHQICKFRKC

The amino acid sequence of SEQ ID NO: 1 is a DBM peptide (Dvl minimal binding peptide) corresponding to some amino acid sequences of proteins CXXC4 and CXXC5 (CXXC-Type Zinc Finger Protein 5) that exist in the body, and CXXC5 binds to Dvl (Dishevelled). This blocks signal transduction by the Wnt/β-catenin signaling system. The DBM peptide blocks the interaction between CXXC5 and Dvl and activates the Wnt/β-catenin signaling system.

In addition, in order for the peptide (DBM) of the amino acid sequence of SEQ ID NO: 1 to penetrate the cell membrane and induce intracellular infiltration, an amino acid sequence encoding a protein transduction domain (PTD) can be additionally inserted into the amino acid sequence of SEQ ID NO: 1. there is.

The PTD mainly consists of basic amino acids such as arginine or lysine, and by adding the PTD to the N-terminal or C-terminal of the amino acid sequence of SEQ ID NO: 1, it can penetrate the cell membrane and induce penetration into the cell.

The PTD may include poly R8, HIV-Tat, HSV VP22, Antp or Transportan, where poly R8 is the amino acid sequence of SEQ ID NO: 2, HIV-Tat is the amino acid sequence of SEQ ID NO: 3, and HSV VP22 is the amino acid sequence of SEQ ID NO: 2. The amino acid sequence of 4, Antp may have the amino acid sequence of SEQ ID NO: 5, or Transportan may have the amino acid sequence of SEQ ID NO: 6.

[SEQ ID NO: 2]: RRRRRRRR

[SEQ ID NO: 3]: YGRKKRRQRRR

[SEQ ID NO: 4]: DAATATRGRSAASRPTERPRAPARSASRPRRPVE

[SEQ ID NO: 5]: RQIKIWFQNRRMKWKK

[SEQ ID NO: 6]: AGYLLGKINLKALAALAKKIL

Additionally, in the polypeptide according to the present invention, a linker may be additionally inserted between the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence encoding PTD. A specific example of a linker inserted between the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence encoding PTD is a sequence of 4 to 12 amino acids, which may be 4 to 8 glycines, and the polypeptide in which the linker is additionally inserted is It may have the amino acid sequence of SEQ ID NO: 7 below.

[SEQ ID NO: 7]: RRRRRRRRGGGGRKTGHQICKFRKC

In addition, valproic acid (VPA) is used as a glycogen synthase kinase 3β (GSK3β) inhibitor, and when used together with PTD-DBM, it can further promote wound healing and alleviate scar formation.

The PTD-DBM and valproic acid may be mixed at a molar ratio of 1:100-1000, preferably 1:200-500. Based on PTD-DBM, if the content of valproic acid is less than the above lower limit, the ratio of collagen III/collagen I may be lowered, and if it is more than the above upper limit, the CD105 marker and CD31 marker may not increase.

The single drug or mixed drug of the present invention can be carried in the hydrogel patch at a concentration of 0.3 to 10 mg/ml, preferably 1 to 8 mg/ml. If the concentration of the mixed drug loaded is less than the lower limit, the effect may be minimal, and if it exceeds the upper limit, the effect of alleviating scars may be reduced.

When the hydrogel patch containing the single drug or mixed drug of the present invention is attached to the wound area, the content of collagen III, which is important for regenerative wound healing, increases at the wound area, so that the ratio of collagen III/collagen I is 2 to 70, preferably Since it increases to 15 to 65, more preferably 52 to 60, scars can be minimized.

In addition, when the hydrogel patch containing the single or mixed drug of the present invention is attached to the wound area, CD105, a dermal stem cell marker, and CD31, an angiogenesis marker, increase, which not only promotes wound healing but also minimizes scarring.

The hydrogel patch containing a single drug or a mixed drug of the present invention can not only be used as a medical device, but can also be used as a cosmetic mask pack containing a hydrogel patch containing a single drug or a mixed drug.

Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are merely illustrative of the present invention, and it is clear to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present invention. It is natural that such variations and modifications fall within the scope of the attached patent claims.

Preparation Example 1. Hydrogel (HA-PG)

Hydrogel (HA-PG) is synthesized through carbodiimide chemistry.

Hyaluronic acid (HA, molecular weight 200 kDa, Lifecore Biomedical, Chaska, MN, USA) was completely dissolved in TDW (triple distillation water) at a concentration of 10 mg/ml, and then mixed with hyaluronic acid and 1-(3-dimethylaminopropyl). -3-Ethylcarbodiimide hydrochloride (EDC, Thermo Fisher Scientific, Waltham, MA, USA) was added at a molar ratio of 1:1.5 and stirred for 15 minutes. Afterwards, NHS was added so that the molar ratio of hyaluronic acid and N-hydroxysuccinimide (NHS, Sigma-Aldrich, St. Louis, MO, USA) was 1:1, and the mixed solution was incubated at pH 5.5 for another 15 minutes. After activating the carboxyl group of hyaluronic acid by stirring, 5-hydroxydopamine hydrochloride was added to the reaction solution so that the molar ratio of hyaluronic acid and 5-hydroxydopamine hydrochloride (Sigma-Aldrich) was 1:1 to adjust the pH. HA-PG was obtained by stirring for 24 hours while maintaining the temperature of 4.5 to 5.0.

Unreacted chemicals and by-products were removed through dialysis using a 6-8 kDa barrier membrane (Membrane Filtration Products Inc., Seguin, TX, USA) against acidic PBS (Biosesang, Seongnam, Gyeonggi), and the generated HA- PG was lyophilized and stored at 4°C until use. The successful synthesis of HA-PG was confirmed by 1H-NMR spectroscopy at 300 MHz (Bruker, Billerica, MA, USA).

Example 1. PTD-DBM alone

To confirm the efficacy of the polypeptide containing the amino acid sequence of SEQ ID NO: 1, poly R8 (RRRRRRRR) and a polyglycine linker (GGGG) at the amino acid terminus of SEQ ID NO: 1, lysine at the carboxyl terminus, and FITC (fluorescein), a fluorescent dye, were added. PolyR8-DBM peptide added with isothiocyanate was synthesized and used.

PolyR8-DBM: RRRRRRRRGGGGRKTGHQICKFRKCK-(FITC)

Example 2. Mixed drug (PTD-DBM+VPA)

PTD-DBM

To confirm the efficacy of the polypeptide containing the amino acid sequence of SEQ ID NO: 1, poly R8 (RRRRRRRR) and a polyglycine linker (GGGG) at the amino acid terminus of SEQ ID NO: 1, lysine at the carboxyl terminus, and FITC (fluorescein), a fluorescent dye, were added. PolyR8-DBM peptide added with isothiocyanate was synthesized.

PolyR8-DBM: RRRRRRRRGGGGRKTGHQICKFRKCK-(FITC)

mixed drugs

A mixed drug was obtained by mixing the PolyR8-DBM peptide (PTD-DBM) and valproic acid at a molar ratio of 1:500.

Comparative Example 1. VPA alone

Valproic acid was used alone.

Comparative Example 2. EGF alone

Epidermal growth factor (EGF) was used to promote wound recovery.

<Test example>

The control group is an untreated group.

In the drawing, Topical means that the drug prepared in Examples and Comparative Examples was applied topically to the mouse, and Patch is a hydrogel in which the drug prepared in Examples and Comparative Examples was supported on HA-PG prepared in Preparation Example 1. It's a patch.

Specifically, the hydrogel patch was prepared by dissolving the HA-PG prepared in Preparation Example 1 in PBS at a concentration of 1% (w/v) and then placing the drugs prepared in Examples and Comparative Examples in a pre-designed 1cm x 1cm rectangular mold. Each loaded HA-PG solution was poured and completely freeze-dried to obtain each HA-PG patch. The obtained HA-PG patch was stored at -20°C before use to avoid moisture.

animal testing

6-week-old male C3H mice weighing 21 to 26 g obtained from Coretech were adapted to the new environment for 1 week and the effects of the drugs prepared in Examples and Comparative Examples or hydrogel patches loaded with the drugs on wound healing and alleviation of scar formation were examined. It was used to measure impact. They were raised in acrylic cages (45 And constant temperature (20-24 ℃) and humidity (45-65%) were maintained. We monitored them for a week to help them adapt to the changed environment, maintained their sleep cycle, and checked for abnormal behavior.

A 7-week-old C3H mouse with resting hair follicles was anesthetized with 2,2,2-tribromoethanol (Sigma Aldrich), the hair on the back of the mouse was shaved with clippers, and a 1 cm ² wound was made on the back of the mouse. The topical treatment group (Topical) was It was applied to the wound every day, and the hydrogel patch group was applied to the wound once.

-10 groups of mice-

Topical
(Apply daily) control: phosphate buffered saline PTD-DBM: PTD-DBM of Example 1 100 uM applied daily VPA: VPA 50mM of Comparative Example 1 applied daily PTD-DBM+VPA: Mixed drug of 100 uM PTD-DBM and 50 mM VPA of Example 2 applied daily EGF: 100 uM EGF of Comparative Example 2 applied daily Patch
(Attach once) control: phosphate buffered saline PTD-DBM: PTD-DBM of Example 1 was loaded at a concentration of 0.3 mg/ml VPA: VPA of Comparative Example 1 contained at a concentration of 7.2 mg/ml PTD-DBM+VPA: Mixed drug loading of 0.3 mg/ml PTD-DBM and 7.2 mg/ml VPA of Example 2 EGF: EGF of Comparative Example 2 was contained at a concentration of 0.6 mg/ml

Data are expressed as mean ± SD. Statistical analysis is performed using an unpaired two-tailed Student's t-test. Statistical significance is presented as *P<0.05, **P<0.005, ***P<0.0005.

Test Example 1. Measurement of wound area in mouse

Figure 1A shows the control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group of the Topical group; Patch group control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group on day 0 (0 d), day 3 (3 d), day 6 (6 d), and day 9 (9 d) ) and photos showing the scar area on day 11 (11 d); Figure 1B shows the quantification of the scar area on the 11th day of wound treatment in each group using the Image J program.

As shown in Figures 1A and 1B, in both the Topical group and the Patch group, Example 1 consisting of PTD-DBM and Example 2 combining PTD-DBM and VPA promoted wound healing and scar formation compared to the other groups. It was confirmed that the formation was significantly alleviated.

Moreover, the HA-PG patch loaded with the drugs of Example 1, Example 2, Comparative Example 1, and Comparative Example 2 had a better overall effect of promoting wound healing and alleviating scar formation than when applied topically. Among them, it was confirmed that the PTD-DBM+VPA_HA-PG patch group loaded with the mixed drug combining PTD-DBM and VPA of Example 2 showed the best effect in promoting wound healing and alleviating scar formation.

Test Example 2. Hematoxylin and eosin (H&E) staining

The mouse wound tissue was fixed overnight at 4°C using 4% (w/v) paraformaldehyde (PFA), dehydrated, paraffinized, embedded in paraffin, and sectioned at a thickness of 4 μm. Sections were deparaffinized and rehydrated through a series of xylene and ethanol, and slides were incubated in Harris hematoxylin for 5 minutes and eosin for 1 minute. H&E-stained slides were observed using a bright-field optical microscope (ECLIPSE TE2000-U, Nikon).

Figure 2A shows the control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group of the Topical group; This is an H&E staining image on the 11th day of wound treatment in the control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group in the Patch group; Figure 1B shows scar width (a.u., arbitary unit) measured in wound tissue on the 11th day of wound treatment in each group.

As shown in Figures 2A and 2B, in both the Topical group and the Patch group, Example 1 consisting of PTD-DBM and Example 2 combining PTD-DBM and VPA promoted wound healing and scar formation compared to the other groups. It was confirmed that the formation was significantly alleviated.

Moreover, the HA-PG patch containing the materials of Example 1, Example 2, Comparative Example 1, and Comparative Example 2, respectively, had a better overall effect of promoting wound healing and alleviating scar formation than when applied topically. Among them, it was confirmed that the PTD-DBM+VPA_HA-PG patch group loaded with the mixed drug combining PTD-DBM and VPA of Example 2 showed a significantly excellent effect of promoting wound healing and alleviating scar formation.

Test Example 3. Immunohistochemical staining

After deparaffinizing and rehydrating 4 μm paraffin sections, slides were autoclaved in 10 mM sodium citrate buffer for antigen retrieval. After cooling, the slides were incubated in PBS and then blocked with 5% bovine serum albumin (BSA) in PBS for 30 min at room temperature, and then the slides were incubated overnight at 4°C with the following dilutions of primary antibodies.

anti-collagen I (1:200, Abcam), anti-collagen III (1:200, Novus Biologicals), anti-CD105 (1:50, R&D systems), and anti-CD31 (1:100, Abcam) in PBS. After washing, the slides were incubated with Alexa Fluor 488- or Alexa Fluor 555-conjugated IgG secondary antibodies (Invitrogen, 1:500) for 1 h at room temperature and blotted with DAPI (Boehringer Mannheim, 1:5000) for 10 min. dyed. Stained images were examined using a Nikon Eclipse Ti microscope (Nikon).

Quantification was performed using NIS Elements V3.2 software (Nikon).

3-1. Immunohistochemical staining for collagen I and collagen III.

Figure 3A shows the control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group of the Topical group; Immunohistochemical staining images for collagen I and collagen III on the 11th day of wound treatment in the control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group in the Patch group; Figure 3B is a graph in which the average intensity of collagen I and collagen III of each group was measured and quantified by the ratio of collagen III/collagen I.

As shown in Figure 3A, collagen I increased in the control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group in the Topical group, but in the control group, PTD-DBM group, and VPA in the Patch group. group, PTD-DBM+VPA group, and EGF group were confirmed to have increased collagen III compared to each group in the Topical group.

Also, as shown in Figure 3B, the ratio of collagen III/collagen I was very low at less than 10 in the control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group in the Topical group, but the Patch group It was confirmed that the control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group had a higher collagen Ⅲ/collagen I ratio compared to each group in the Topical group.

In particular, among the Patch groups, the PTD-DBM+VPA_HA-PG patch group loaded with the mixed drug combining PTD-DBM and VPA of Example 2 was confirmed to have a significantly high collagen III/collagen I ratio of 57.2.

3-2. Immunohistochemical staining for CD105 in wound dermis.

Figure 4A shows the control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group of the Topical group; This is an immunohistochemical staining image for CD105 in the wound dermis on the 11th day of wound treatment in the control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group in the Patch group; Figure 4B is a graph quantifying the number of CD105 positive cells in the wound dermis of each group.

As shown in Figures 4A and 4B, in both the Topical group and the Patch group, Example 1 consisting of PTD-DBM and Example 2 combining PTD-DBM and VPA were stem cell markers in the wound dermis compared to the other groups. It was confirmed that CD105 was significantly increased.

Moreover, the HA-PG patch loaded with the materials of Example 1, Example 2, Comparative Example 1, and Comparative Example 2, respectively, had an overall increase in CD105 more than when applied topically, and among them, PTD- of Example 2 It was confirmed that the PTD-DBM+VPA_HA-PG patch group loaded with the mixed drug combining DBM and VPA showed a significantly increased number of CD105 positive cells.

3-3. Immunohistochemical staining for CD31

Figure 5A shows the control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group of the Topical group; This is an immunohistochemical staining image for CD31, an angiogenesis marker, on the 11th day of wound treatment in the control group, PTD-DBM group, VPA group, PTD-DBM+VPA group, and EGF group in the Patch group; Figure 5B is a graph quantifying the number of CD31 positive cells in each group.

As shown in Figures 5A and 5B, in both the Topical group and the Patch group, CD31, an angiogenesis marker, was significant in Example 1 consisting of PTD-DBM and Example 2 combining PTD-DBM and VPA compared to the other groups. It was confirmed that there was a significant increase.

Moreover, the HA-PG patch carrying the materials of Example 1, Example 2, Comparative Example 1, and Comparative Example 2, respectively, showed a greater overall increase in CD31 than when applied topically, and among them, PTD- of Example 2 It was confirmed that the PTD-DBM+VPA_HA-PG patch group loaded with the mixed drug combining DBM and VPA showed a significantly increased number of CD31 positive cells.

Claims (15)

A hydrogel patch made of a biocompatible polymer modified with drug-loaded pyrogallol,
The drug is a single drug consisting of a polypeptide fused with a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a polypeptide consisting of an amino acid sequence encoding PTD (protein transduction domain), or a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 A hydrogel patch for promoting wound healing or minimizing scarring, characterized in that it is a mixed drug consisting of a polypeptide fused with a polypeptide consisting of an amino acid sequence encoding a PTD (protein transduction domain) and valproic acid. The method of claim 1, wherein the mixed drug is a polypeptide fused with a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a polypeptide consisting of an amino acid sequence encoding PTD (protein transduction domain) and valproic acid at a ratio of 1:100-1000. A hydrogel patch for promoting wound healing or minimizing scars, characterized in that it is mixed at a molar ratio of. The hydrogel patch for promoting wound healing or minimizing scars according to claim 1, wherein the drug is contained at a concentration of 0.3 to 10 mg/ml. The hydrogel patch for promoting wound healing or minimizing scars according to claim 1, wherein the hydrogel patch increases the content of collagen III in the wound area so that the ratio of collagen III/collagen I is 2 to 70. The hydrogel patch for promoting wound healing or minimizing scars according to claim 1, wherein CD105, a dermal stem cell marker, and CD31, an angiogenesis marker, increase in the wound area by the hydrogel patch. The method of claim 1, wherein the biocompatible polymer is selected from the group consisting of hyaluronic acid, heparin, cellulose, dextran, alginate, chitosan, chitin, collagen, gelatin, chondroitin sulfate, and pectin. Hydrogel patch for minimizing scars. The hydrogel patch for promoting wound healing or minimizing scars according to claim 1, wherein the biocompatible polymer is hyaluronic acid. The hydrogel patch for promoting wound healing or minimizing scars according to claim 1, wherein the PTD (protein transduction domain) includes poly R8, HIV-Tat, HSV VP22, Antp, or Transportan. The hydrogel patch for promoting wound healing or minimizing scars according to claim 1, wherein the polypeptide further has a linker inserted between the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence encoding the PTD. The hydrogel patch for promoting wound healing or minimizing scars according to claim 1, wherein the hydrogel patch is a patch for a medical device. A cosmetic mask pack comprising a hydrogel patch made of a biocompatible polymer modified with drug-loaded pyrogallol,
The drug is a single drug consisting of a polypeptide fused with a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a polypeptide consisting of an amino acid sequence encoding PTD (protein transduction domain), or a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 A cosmetic mask pack for promoting wound healing or minimizing scars, characterized in that it is a mixed drug consisting of a polypeptide fused with a polypeptide consisting of an amino acid sequence encoding a PTD (protein transduction domain) and valproic acid. The method of claim 11, wherein the mixed drug containing the fused polypeptide and valproic acid is a polypeptide fused with a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a polypeptide consisting of an amino acid sequence encoding a PTD (protein transduction domain). A cosmetic mask pack for promoting wound healing or minimizing scars, characterized by mixing peptide and valproic acid at a molar ratio of 1:100-1000. The cosmetic mask pack for promoting wound healing or minimizing scars according to claim 11, wherein the mixed drug is contained at a concentration of 0.3 to 10 mg/ml. The cosmetic mask pack for promoting wound healing or minimizing scars according to claim 11, wherein the hydrogel patch increases the content of collagen Ⅲ in the wound area and the ratio of collagen Ⅲ/collagen Ⅰ is 2 to 70. . The cosmetic mask pack for promoting wound healing or minimizing scars according to claim 11, wherein the hydrogel patch increases CD105, a dermal stem cell marker, and CD31, an angiogenesis marker, at the wound site.