Functional hydrogel for promoting hair follicle regeneration as well as preparation method and application of functional hydrogel

Abstract

The invention relates to the technical field of biological medicines, and provides functional hydrogel for promoting hair follicle regeneration as well as a preparation method and application of the functional hydrogel. The functional hydrogel comprises a hydrogel matrix and an SHP2 inhibitor SHP099 carried on the hydrogel matrix. The invention further provides a corresponding preparation method and application in preparation of dressing for treating scar wound surfaces. The functional hydrogel constructed by the invention is used as a powerful tool for researching the formation of a dermis shell, not only plays a huge role in the fundamental research of the formation of skin appendages in the future, but also has a wide market application prospect in the aspects of wound healing promotion and hair follicle regeneration promotion dressings.

Description

Technical Field

[0001] The invention relates to the technical field of skin scar repair, and in particular to a functional hydrogel for promoting hair follicle regeneration, and a preparation method and application thereof. Background Art

[0002] As the main external barrier of the human body, the skin has important functions such as resisting external damage, regulating body temperature, and excreting metabolic products. After the skin is damaged and a wound is formed, a compensatory regeneration process will be initiated. When the wound is too large and too deep, the skin regeneration process is blocked, and scar tissue without skin appendages replaces normal skin tissue to form scars. The formation of scars not only affects the appearance, but more importantly, there are no hair follicles and the sebaceous glands and sweat glands formed around the hair follicles in the scar tissue, which impairs the skin function and affects the patient's quality of life. [1-3] However, there is still a lack of effective clinical treatment methods to inhibit scar formation. Therefore, developing new technologies to help wound skin appendages to quickly rebuild is crucial to treating scars and improving patients' quality of life.

[0003] Biocompatible hydrogel three-dimensional scaffold systems can mimic extracellular matrix and promote cell migration and proliferation. Therefore, they are widely used in various tissue damage repair. [4-6] UV-cured methacryloyl gelatin (GelMA) has the potential to be used as a wound dressing for the clinical treatment of scars due to its good biocompatibility, degradability and low immunogenicity. [7] However, the existing methacryloyl gelatin has a single function and is difficult to promote the regeneration of skin hair follicles. SHP099 has the potential to treat cancer as a SHP2 inhibitor [8] At the same time, SHP2 inhibitors can also treat various diseases caused by tissue fibrosis by inhibiting inflammatory responses. [9-11] . However, there has never been a report that SHP099 can promote the regeneration of skin hair follicles. In the field of hair follicle regeneration research, most of the existing studies have focused on hair follicle stem cells. Existing technologies mostly promote hair follicle regeneration by increasing the activity of hair follicle stem cells. However, as a skin appendage with a multi-level complex structure, the regeneration and maintenance of hair follicles involve the synergistic effects of multiple tissues. Among them, the dermal shell composed of smooth muscle plays a key role in the formation and maintenance of hair follicles. However, there has been no report of bioactive molecules that can effectively regulate dermal shell formation. Therefore, there is no method to promote hair follicle regeneration by promoting dermal shell regeneration. Summary of the invention

[0004] The present invention is based on the above problems. In view of the shortcoming of the single function of the currently used methacryloyl gelatin, the present invention discloses a functional hydrogel carrying SHP099, which releases SHP099 in situ and promotes the regeneration of hair follicles on the wound surface through the hydrogel scaffold system. The present invention discloses for the first time that SHP099 has the function of promoting wound healing and promoting hair follicle regeneration; at the same time, the present invention discloses for the first time that SHP099 has the ability to promote the formation of the dermal shell of hair follicles, and is the first small molecule inhibitor that has been proven to regulate the dermal shell. The function of the currently used methacryloyl gelatin is further improved to give it the function of promoting the formation of the dermal shell of hair follicles, thereby promoting the regeneration of hair follicles, and realizing skin reconstruction.

[0005] The research process of the present invention is as follows: first, a functional hydrogel carrying SHP099 with specific mechanical properties is prepared, and the swelling coefficient, morphology and drug release efficiency of the hydrogel are characterized; then, a mouse full skin defect model is used to verify the effect of the functional hydrogel on wound repair, and it is clear that the functional hydrogel carrying SHP099 has the function of quickly rebuilding the skin structure; further experimental results clarify the regulatory effect of SHP099 on the dermal shell of hair follicles.

[0006] In a first aspect, the present invention provides a functional hydrogel and its preparation, including a hydrogel matrix and SHP099 carried thereon.

[0007] Preferably, the hydrogel matrix is ​​a hydrogel with good biocompatibility and low immunogenicity, such as methacryloyl dextran (DexMA), methacryloyl hyaluronic acid (HAMA), methacryloyl silk fibroin (SilMA), methacryloyl sodium alginate (AlgMA), methacryloyl chondroitin sulfate (ChSMA), methacryloyl polylysine (PLMA), methacryloyl carboxymethyl chitosan (CMCSMA), and all other controllable curing natural or modified polymer hydrogel scaffolds.

[0008] The concentration of SHP099 is 5-100 μm / mL, preferably 10 μm / mL.

[0009] Furthermore, the preparation method of the functional hydrogel comprises the following steps:

[0010] A. Hydrogel Precursor Configuration

[0011] After the initiator standard solution is prepared, it is mixed with the hydrogel matrix, shaken to fully soak the hydrogel matrix, and heated in a 40°C water bath away from light to dissolve, and shaken several times during the process to fully dissolve it; the prepared hydrogel matrix solution is immediately sterilized to prevent low-temperature gelation, thereby obtaining a hydrogel precursor solution.

[0012] Preferably, the method for preparing the initiator standard solution is as follows: take PBS, add it to a brown bottle containing the initiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP), heat it in a 45°C water bath for 15 minutes, shake it thoroughly to dissolve it, prepare it into a 0.25% (w / v) initiator standard solution, and store it at 4°C in the dark;

[0013] In order to take into account both biocompatibility and SHP099 loading, the molecular weight of the hydrogel matrix is ​​100-200KD, and the amino substitution rate is 90±5%. In the preferred embodiment of the present invention, GelMA is selected, and the photoinitiator and the GelMA solid are mixed at a volume mass ratio of 1:0.1, and the GelMA is fully infiltrated by oscillation, and heated and dissolved in a 40°C water bath in the dark for 30 minutes, and oscillated several times during the period to fully dissolve it; the prepared GelMA solution is immediately sterilized with a 0.22μm sterile syringe filter to prevent low-temperature gelation.

[0014] B. Equipped with SHP099

[0015] Add a predetermined concentration of SHP099 solution to the hydrogel precursor solution, shake it several times during the process to fully dissolve the solution, take the required amount of solution and irradiate it with 405nm ultraviolet light for tens of seconds to induce curing to form a hydrogel. The viscosity of the hydrogel is 0.021±0.06Pa.S, the storage modulus is 9638±960Pa, and the compression modulus is 27.58±5kPa.

[0016] The prepared functional hydrogel loaded with SHP099 was characterized: the microscopic morphology of the hydrogel surface and cross-section was observed using SEM, which showed that SHP099 did not affect the structural characteristics of the GelMA hydrogel; the drug release results showed that the hydrogel was able to continuously release SHP099.

[0017] The second aspect of the present invention shows the application of the above functional hydrogel. The effect of the functional hydrogel carrying SHP099 on wound repair was verified using a mouse full skin injury model, including but not limited to: (1) using a mouse model to verify the therapeutic effect of the functional hydrogel carrying SHP099 on wounds; (2) clarifying the mechanism by which the functional hydrogel carrying SHP099 promotes the reconstruction of wound skin structure.

[0018] The results showed that the functional hydrogel carrying SHP099 can promote faster wound healing and significantly promote wound epidermal regeneration and hair follicle regeneration.

[0019] The third aspect of the present invention clarifies that the functional hydrogel carrying SHP099 promotes the regeneration of hair follicles in wounds by regulating the dermal shell of hair follicles. This includes but is not limited to: (1) SHP099 is confirmed to promote the expression of Caldesmon-1 (Cald), a key protein in the dermal shell; (2) SHP099-carrying functional hydrogel promotes the formation and maintenance of the dermal shell of hair follicles in in vivo experiments.

[0020] Therefore, the present invention provides the use of the functional hydrogel in preparing a wound dressing for treating scars. Preferably, the wound dressing is a dressing that promotes hair follicle regeneration, and further, the dressing that promotes hair follicle regeneration is a dressing that improves the ability of hair follicle dermal shell formation.

[0021] Furthermore, the present invention also provides the use of SHP099 in the preparation of a hair follicle dermal shell regulator.

[0022] Beneficial protection and effects of the present invention:

[0023] The present invention uses photocurable hydrogels to carry SHP2 inhibitor SHP099 to prepare functional hydrogels, which can not only promote wound healing, but more importantly, SHP099 effectively inhibits scar formation by promoting hair follicle regeneration. The present invention discloses for the first time the therapeutic effect of SHP099 on scars. Further analysis shows that SHP099 promotes hair follicle regeneration by promoting the regeneration of the dermal shell of hair follicles. This is the first report of a small molecule active substance that promotes the regeneration of the dermal shell of hair follicles. The functional hydrogel constructed by the present invention is a powerful tool for studying the formation of dermal shells. It will not only play a huge role in the basic research on the formation of skin appendages in the future, but also have broad market application prospects in promoting wound healing and promoting hair follicle regeneration dressings. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present disclosure will be further described below in conjunction with the accompanying drawings, wherein these drawings are only for illustrating the embodiments of the present disclosure rather than for limiting the scope of the present disclosure.

[0025] Figure 1 The scanning electron micrograph of the functional hydrogel loaded with SHP099 (SHP) is shown (scale bar: 100 μm);

[0026] Figure 2 The cumulative release profile of SHP099 from the hydrogel is shown;

[0027] Figure 3 It shows that SHP099-carrying functional hydrogel promotes wound repair and hair follicle regeneration;

[0028] Figure 4 It was shown that SHP099 promoted the expression of Cald, a protein associated with the dermal shell of fibroblasts, and that functional hydrogels carrying SHP099 promoted the expression of Cald in the dermal shell of hair follicles on the wound surface. DETAILED DESCRIPTION

[0029] The following examples and experimental examples are provided to further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include a detailed description of conventional methods, which are well known to those skilled in the art and are described in many publications.

[0030] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention, and the preferred implementation methods and materials described in the specific implementation methods are for demonstration purposes only.

[0031] Example 1: Preparation and characterization of functional hydrogels loaded with SHP099

[0032] 1. Preparation of functional hydrogels carrying SHP099

[0033] Take 20mL PBS and add it to the brown bottle containing the initiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) (containing 0.05g LAP), and heat it in a 45℃ water bath for 15 minutes, shaking it thoroughly to dissolve it. The prepared 0.25% (w / v) initiator standard solution is stored at 4℃ in the dark.

[0034] Take 1mL of photoinitiator and 0.1g of GelMA solid (molecular weight 100-200KD, amino substitution rate 90±5%) and place them in a centrifuge tube. Shake to fully soak the GelMA, and heat and dissolve in a 40℃ water bath in the dark for 30 minutes. Shake several times during the process to fully dissolve it. The prepared GelMA solution is immediately sterilized with a 0.22μm sterile syringe filter to prevent low-temperature gelation.

[0035] Take 1 mL of GelMA hydrogel precursor solution, add 10 μM SHP099 solution, shake several times during the process to make the solution fully dissolved, take the required amount of solution and irradiate it with 405 nm UV for 30 seconds to induce curing to form a hydrogel (viscosity: 0.021±0.06 Pa.S; storage modulus: 9638±960 Pa; compression modulus: 27.58±5 kPa).

[0036] 2. Characterization of functional hydrogels loaded with SHP099

[0037] Microstructure: The functional hydrogel samples loaded with SHP099 and the GelMA hydrogel samples were frozen in a -80°C refrigerator overnight, and then transferred to a vacuum freeze dryer for 24 hours to obtain freeze-dried hydrogel samples. The samples were brittle after being treated with liquid nitrogen. The samples were directly glued to the conductive glue and gold was sprayed on the samples for 45 seconds using a sputtering coater to improve their conductivity. Finally, the microscopic morphology of the hydrogel surface and cross-section was observed using SEM, and photos were taken ( Figure 1 ). The results showed that SHP099 did not affect the structural characteristics of GelMA hydrogel.

[0038] Drug release: Pipette 1 ml of the functional hydrogel precursor solution carrying SHP099 into a test tube, irradiate it under 405 nm ultraviolet light for 30 seconds to solidify it into a gel; then add 10 mL of PBS (pH = 7.4) to the test tube, place it in a constant temperature oscillator at 37 ° C and an oscillation speed of 100 rpm / min; Pipette 1 ml of the drug solution, add 1 ml of PBS solution and put it back into the test tube. Use a UV spectrophotometer to measure the OD value, and then use the standard curve to correspond to the concentration value. Take 6 time points of 1, 2, 3, 4, 5, and 6 days, calculate the drug concentration according to the cumulative release formula, and draw the drug release curve based on the relationship between the cumulative drug release and time ( Figure 2 ), the results showed that the hydrogel continuously released SHP099.

[0039] Example 2: Functional hydrogel carrying SHP099 promotes the reconstruction of mouse wound skin structure

[0040] 1. Establishment of mouse skin injury model

[0041] All experimental mice were weighed and recorded using an electronic balance. The experimental animals were anesthetized with 4% chloral hydrate, which was injected intraperitoneally at 0.1 ml / 10 g of mouse body weight. After the mice were fully anesthetized, the hair on the back of the mice was shaved with a razor and then a depilatory cream was applied. After 3 minutes, the remaining hair on the back was gently wiped with gauze and clean water to fully expose the back skin. The back skin was disinfected with alcohol gauze, and a full-thickness wound was made on the back skin using a 7 mm biopsy punch and surgical scissors.

[0042] 2. Evaluate the therapeutic effect of functional hydrogel carrying SHP099 on wounds.

[0043] 30 μl of SHP099-loaded functional hydrogel precursor solution or 30 μl of GelMA hydrogel precursor solution was dripped onto the wound area. The wound was cured by 405 nm UV irradiation for 30 seconds. The day of modeling was designated as day 0, and the wound surface was photographed on days 0, 3, and 7 ( Figure 3 a), and the wound surface was obtained for histological evaluation on days 3, 7, and 14.

[0044] Wound healing rate: The wound area was calculated using Image J software. Wound healing rate = (S0-St) / S0×100%, where S0 represents the wound area on the 0th day of modeling, and St represents the wound area at time t of modeling. The results showed that the functional hydrogel carrying SHP099 promoted faster wound healing ( Figure 3 a, 3b)

[0045] H&E staining: After paraffin embedding, wound tissue sections were sliced ​​and H&E stained. The results were analyzed using Image J software. The results showed that the functional hydrogel carrying SHP099 significantly promoted wound epidermal regeneration ( Figure 3 c) and hair follicle regeneration ( Figure 3 d).

[0046] Example 3: Functional hydrogel loaded with SHP099 promotes hair follicle regeneration by regulating the dermal shell of hair follicles

[0047] 1. Clarify that SHP099 promotes the expression of key proteins in the dermis

[0048] Mouse fibroblast L929 cells were cultured in DMEM containing 10% (v / v) fetal bovine serum, 100U / ml penicillin and 100μg / ml streptomycin in a 5% CO2 and 37°C incubator. Then 5μM SHP099 was added to the culture medium to stimulate the cells. After 12 or 72 hours of stimulation, the cells were collected and the expression of the dermis shell-related protein Caldesmon-1 was characterized by Western blot. The results showed that SHP099 significantly increased the expression of Caldesmon-1 (Cald) in L929 cells ( Figure 4 a).

[0049] 2. It is clear that the functional hydrogel carrying SHP099 promotes the formation and maintenance of the dermal shell of hair follicles in in vivo experiments.

[0050] Next, immunohistochemical staining was performed on the 7-day skin tissue sections to characterize the expression of Caldesmon-1 in the wound surface. The results showed that the functional hydrogel carrying SHP099 significantly promoted the expression of Cald in the dermis around the hair follicles on the wound surface ( Figure 4 b, 4c).

[0051] The references used in the background technology of the present invention are as follows:

[0052] [1]Martin Paul.Wound Healing--Aiming for Perfect Skin Regeneration[J].Science,1997,276(5309):75-81.

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[0057] [6]Goh MeeiChyn,Du Meng,Peng Wang Rui,Saw Phei Er,ChenZhiyi.Advancing burn wound treatment:exploring hydrogel as atransdermal drugdelivery system[J].Drug Delivery,2024,31(1):

[0058] [7] Zhu Jie, Liu Zhixiao, Wang Ling, Jin Qishu, Zhao Yunpeng, Du Antong, Ding Neng, Wang Yue, Jiang Hua, Zhu Lie. Exosome Mimetics-Loaded Hydrogel Accelerates Wound Repair by Transferring Functional Mitochondrial Proteins[J]. Front Bioeng Biotechnol, 2022, 10(

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[0061]

[10] Ye Shuhui, Zuo Bowen, Xu Lenan, Wu Yue, Luo Ruixiang, Ma Lin, Yao Wanxin, Chen Lingfeng, Liang Guang, Zhang Yanmei. Inhibition of SHP2 by the Small Molecule Drug SHP099 Prevents Lipopolysaccharide-Induced Acute Lung Injury in Mice[J]. Inflammation, 2023, 46(3): 975-86.

[0062]

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[0063] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A functional hydrogel, characterized in that: It includes a hydrogel matrix and a SHP2 inhibitor SHP099 carried thereon.

2. The functional hydrogel according to claim 1, characterized in that The hydrogel matrix is ​​selected from any one of methacryloyl gelatin GelMA, methacryloyl dextran DexMA, methacryloyl hyaluronic acid HAMA, methacryloyl silk fibroin SilMA, methacryloyl sodium alginate AlgMA, methacryloyl chondroitin sulfate ChSMA, methacryloyl polylysine PLMA, and methacryloyl carboxymethyl chitosan CMCSMA. The concentration of SHP099 was 5-100 μM / mL.

3. The method for preparing the functional hydrogel according to claim 1 or 2, characterized in that: The steps include: A. Hydrogel Precursor Configuration After preparing the initiator standard solution, mix it with the hydrogel matrix, shake it to fully infiltrate the hydrogel matrix, heat it in a 40°C water bath away from light to dissolve it, and shake it several times during the process to fully dissolve it; the prepared hydrogel matrix solution is immediately sterilized to prevent low-temperature gelation, and a hydrogel precursor solution is obtained. B. Equipped with SHP099 SHP099 solution was added to the hydrogel precursor solution, and the solution was shaken several times to fully dissolve the solution. The required amount of solution was taken and irradiated with 405nm ultraviolet light for tens of seconds to induce curing to form a hydrogel.

4. The method for preparing the functional hydrogel according to claim 3, characterized in that: The steps include: Wherein, in step A, the preparation method of the initiator standard solution is as follows: take PBS, add it to a brown bottle containing the initiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate LAP, heat it in a 45°C water bath for 15 minutes, shake it thoroughly to dissolve it, prepare it into a w / v 0.25% initiator standard solution, and store it at 4°C in the dark; When the hydrogel matrix is ​​selected from GelMA, its molecular weight is 100-200KD and the amino substitution rate is 90±5%; the photoinitiator and the GelMA solid are mixed in a volume mass ratio of 1:0.1, and the GelMA is fully infiltrated by shaking, and heated and dissolved in a 40°C water bath in the dark for 30 minutes, and shaken several times during the process to fully dissolve it; the prepared GelMA solution is immediately sterilized with a 0.22μm sterile syringe filter to prevent low-temperature gelation.

5. The method for preparing the functional hydrogel according to claim 3, characterized in that: The steps include: In step B, a predetermined concentration of SHP099 solution is added to the GelMA hydrogel precursor solution, and the solution is shaken several times to fully dissolve the solution. The required amount of solution is irradiated with 405 nm ultraviolet light for 30 seconds to induce curing to form a hydrogel.

6. Use of the functional hydrogel according to claim 1 or 2 in preparing a dressing for treating scar wounds.

7. The use according to claim 6, characterized in that: The wound dressing is a dressing that promotes hair follicle regeneration.

8. The use according to claim 7, characterized in that: The dressing for promoting hair follicle regeneration is a dressing for improving the ability of hair follicle dermal shell formation.

9. Application of SHP099 in the preparation of hair follicle dermal shell regulator.

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