Use of SjP40 protein, composition for promoting skin tissue repair and preparation method thereof, and pharmaceutical preparation

Abstract

The application relates to the field of biological medicine, and specifically provides application of SjP40 protein, a composition for promoting skin tissue repair, a preparation method of the composition, and a pharmaceutical preparation. The SjP40 protein from a Japanese blood fluke is applied to the field of skin damage repair, the SjP40 can significantly accelerate wound closure, reduce the level of inflammatory cell infiltration, and enhance capillary and appendage regeneration, and the overall repair quality is superior to that of an existing treatment method. Meanwhile, the SjP40 protein is combined with a hydrogel to realize local controlled release and stable release of the SjP40 protein at a wound surface, the problem of rapid degradation of protein activity in vitro is overcome, and the bioavailability and treatment efficiency are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the application of SjP40 protein, and further relates to a composition for promoting skin tissue repair and a preparation method thereof and a pharmaceutical preparation. BACKGROUND

[0002] Skin, as the largest physical barrier organ of the human body, plays a key role in resisting external damage and pathogen invasion. The repair process of skin defects caused by common clinical trauma, surgical incision, chronic diseases (such as diabetes, pressure sores) or infection is often accompanied by persistent inflammatory response, new vascularization disorder and abnormal extracellular matrix remodeling. For full-thickness injury or wound related to metabolic diseases, the existing treatment methods have clear limitations: conventional dressing change is difficult to effectively regulate the wound microenvironment; growth factor preparations (such as EGF, bFGF) are easily diluted by exudate, resulting in reduced activity, and are also costly; physical therapy such as negative pressure sealing drainage is complex to operate and has insufficient applicability for superficial wounds.

[0003] Research has found that SjP40 protein, as an immune regulatory molecule derived from Schistosoma japonicum, can inhibit excessive inflammatory response by inducing macrophages to polarize to M2 anti-inflammatory phenotype, can regulate TGF-β / Smad signaling pathway to reduce tissue fibrosis, and can promote the migration of vascular endothelial cells and angiogenesis. However, existing research mainly focuses on the application of SjP40 in the field of liver fibrosis and immune diseases, and there is no report on the application of skin repair. SUMMARY

[0004] In view of the above problems, the present application provides the application of SjP40 protein in the preparation of a drug or skin care product for skin tissue repair, and further provides a SjP40 protein hydrogel composition capable of promoting skin tissue repair, which can accelerate wound closure, reduce inflammatory cell infiltration, and enhance capillary and adnexal regeneration.

[0005] In a first aspect, the present application provides the application of SjP40 protein in the preparation of a drug or skin care product for skin tissue repair.

[0006] Preferably, the skin tissue repair includes inflammatory wound repair, knife wound repair, bed sore repair, diabetic complication ulcer repair, burn repair, scald repair, frostbite repair, radiation-induced skin damage repair, postoperative infectious incision healing, or promotion of growth after skin transplantation.

[0007] Preferably, the dosage form of the drug or skin care product includes gel, water, cream, ointment, cream, spray, injection, needle or dressing.

[0008] In a second aspect, the present application provides a composition for promoting skin tissue repair, comprising SjP40 protein and Pluronic F127 hydrogel.

[0009] Preferably, the hydrogel can be at least one of gelatin-sodium alginate composite hydrogel, chitosan hydrogel, sodium hyaluronate hydrogel, carbomer gel, chitosan / gelatin / β-glycerophosphate composite hydrogel, in addition to Pluronic F127 hydrogel.

[0010] Preferably, the composition further comprises at least one of growth factors, antibacterial peptides, silver ions, antioxidants, stem cell-derived exosomes or hemostatic agents.

[0011] The SjP40 protein in the present application can also be used in combination with the following bioactive substances to construct a multifunctional synergistic repair system: (1) combined with growth factors (such as bFGF, EGF), which can improve the rate of epithelial reconstruction; (2) combined with antibacterial peptides or silver ions, which can enhance the anti-infection ability; (3) combined with antioxidants (such as vitamin C, glutathione), which are suitable for high-stress or chronic wounds; (4) combined with stem cell-derived exosomes, which can be used for severe tissue defect repair; (5) combined with hemostatic agents, which can be used for postoperative or bleeding wounds.

[0012] Preferably, the mass fraction of Pluronic F127 hydrogel is 20% to 30%. More preferably, the mass fraction of Pluronic F127 is 26%.

[0013] Preferably, in the composition, the concentration of SjP40 protein is 0.3 to 0.7 mg / mL. More preferably, the concentration of SjP40 protein is 0.5 mg / mL.

[0014] The present application combines SjP40 protein with Pluronic F127 (PF127) hydrogel to achieve local controlled release and stable release of SjP40 protein at the wound site, overcoming the problem of rapid degradation of protein activity in vitro, improving its bioavailability and treatment efficiency; PF127 hydrogel can quickly gel at body temperature to form a soft protective layer, effectively isolating external pollution, while providing a humid environment to further promote wound healing. Through animal model experiments, it has been confirmed that the SjP40+PF127 composition constructed by the present application is significantly better than the SjP40 protein alone or the hydrogel alone in terms of wound healing speed, tissue reconstruction integrity, and inflammatory cell infiltration, and shows obvious synergistic effect. The composite design not only has anti-inflammatory and repair functions, but also significantly improves the stability and actual efficiency of protein drugs in vitro application.

[0015] Preferably, in addition to the PF127 hydrogel, the SjP40 protein can also be compounded with the following medical hydrogel systems to form external gel compositions with different physicochemical properties and sustained-release characteristics, specifically: compounded with gelatin-sodium alginate complex hydrogel, which can promote cell adhesion and tissue growth; compounded with chitosan hydrogel, which has natural antibacterial properties and hemostatic effect; compounded with sodium hyaluronate hydrogel, which has good moisturizing and biocompatibility; compounded with carbomer gel, which is a classic drug carrier and is easy to control the rheological properties; compounded with chitosan / gelatin / β-glycerophosphate complex hydrogel, which can regulate the gel temperature and degradation rate. In practical applications, the hydrogel system can be flexibly selected according to the skin condition of different patients, the depth of the wound and the healing speed requirements.

[0016] In a third aspect, the application provides a preparation method of the above-mentioned composition, comprising the following steps: mixing Pluronic F127 hydrogel with a mass fraction of 20% to 30% with SjP40 protein to obtain the composition; the concentration of SjP40 protein in the composition is 0.3 to 0.7 mg / mL.

[0017] In a fourth aspect, the application provides a pharmaceutical preparation comprising the above-mentioned composition, which is in the form of an external gel, a patch or a spray.

[0018] Preferably, the composition can be prepared into the following different forms of delivery systems according to different clinical application scenarios: (1) a microneedle hydrogel system: SjP40 is directly delivered to the dermis through a polymer microneedle structure, enhancing the penetration and improving the bioavailability of the protein; (2) a patch-type hydrogel dressing: the gel is loaded on a support film material to make a flexible dressing suitable for large-area wound management; (3) a spray-type thermosensitive gel: the gel is quickly covered on the wound by low-temperature spraying, and the gel is formed into a sterile protective film when the body temperature triggers the gelation; (4) an injection-type in-vivo gel system: applied to deep skin tissue or subcutaneous postoperative wound cavity, the gel is formed into a film by in-vivo temperature triggering to reduce exudation and scarring.

[0019] Preferably, the composition can be designed into the following dosage forms to adapt to different use scenarios according to different clinical application scenarios: (1) a flowable gel for irregular wounds; (2) a gel patch for large-area chronic wounds; (3) a hydrogel bead, microsphere system for precise management; (4) a composite nanogel for transdermal systems; (5) a lyophilized patch / film for transportation and storage.

[0020] Compared with the prior art, the application has the following beneficial effects:

[0021] (1) The SjP40 protein derived from Schistosoma japonicum is applied to the field of skin injury repair for the first time, breaking through the original application limitation in the direction of anti-inflammatory and anti-fibrosis, and widening the biomedical application boundary of the SjP40 protein. The experimental results show that the SjP40 protein can significantly accelerate wound closure, reduce the level of inflammatory cell infiltration, and enhance capillary and appendage regeneration, and has unique advantages in promoting wound healing, inhibiting inflammation, promoting angiogenesis and tissue reconstruction, and realizes the repair quality superior to existing treatment methods.

[0022] (2) The application constructs an external gel preparation with good controlled release function and tissue adhesion performance. PF127 is a liquid at low temperature, which is convenient for protein loading; after contacting with body temperature, it quickly forms a gel to form a protective layer at the wound site and continuously release SjP40. The design not only improves the protein stability and local utilization rate, but also enhances the residence time and treatment persistence of the preparation at the wound site, and belongs to a new combination of protein-gel synergistic treatment.

[0023] (3) The hydrogel composition containing the SjP40 protein provided by the application can be simply prepared under low temperature conditions without complex process, and can be prepared into an external gel, a plaster or a spray type product, and has strong expandability and is suitable for different types of skin wounds (such as postoperative wounds, abrasions, diabetic foot, etc.), and has high clinical transformation value and market application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the application, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0025] Figure 1 The wound contour of each group of mice in the effect experiment example of the application is shown in the figure;

[0026] Figure 2 The broken line graph of the wound healing rate of each group of mice in the effect experiment example of the application with the change of injury time is shown in the figure;

[0027] Figure 3 The HE staining result graph of the wound of each group of mice in the effect experiment example of the application is shown in the figure; wherein (a) is the PBS group, (b) is the SjP40 group, (c) is the PF127 group, and (d) is the SjP40+PF127 group;

[0028] Figure 4 The Masson staining result graph of the wound of each group of mice in the effect experiment example of the application is shown in the figure; wherein (a) is the PBS group, (b) is the SjP40 group, (c) is the PF127 group, and (d) is the SjP40+PF127 group;

[0029] Figure 5 Figure 1 shows the wound collagen volume fraction of each group of mice in the effect experiment of the present application. DETAILED DESCRIPTION

[0030] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, a brief introduction to the embodiments will be given below. Obviously, the embodiments described below are some embodiments of the present application, and other embodiments can be obtained by those skilled in the art without creative effort based on these embodiments.

[0031] If the specific experimental steps or conditions are not specified in the embodiments, the operations or conditions can be performed according to the conventional experimental steps described in the literature in the art. The raw materials or instruments used are conventional products that can be obtained by purchase, including but not limited to the raw materials or instruments used in the embodiments of the present application.

[0032] Embodiment 1

[0033] The present embodiment provides a SjP40 protein hydrogel composition, and the raw materials for preparing the composition are SjP40 protein and Pluronic F127 hydrogel (abbreviated as PF127 hydrogel). The mass fraction of the PF127 hydrogel is 26%, and the concentration of the SjP40 protein in the composition is 0.5 mg / mL.

[0034] The SjP40 protein used in the present embodiment is synthesized by Beijing Huada Protein Research and Development Center Co., Ltd.

[0035] The preparation process of the SjP40 protein hydrogel composition provided in the present embodiment is as follows:

[0036] Pluronic F127 is added to deionized water, and magnetic stirring is performed until complete dissolution to prepare PF127 hydrogel with a mass fraction of 26%. Then, SjP40 protein is added to the PF127 hydrogel, and the mixture is mixed at 4°C to form a SjP40 protein hydrogel composition, and the concentration of the SjP40 protein in the composition is 0.5 mg / mL.

[0037] Embodiment 2

[0038] The present embodiment provides a SjP40 protein hydrogel composition, and the raw materials for preparing the composition are SjP40 protein and Pluronic F127 hydrogel (abbreviated as PF127 hydrogel). The mass fraction of the PF127 hydrogel is 20%, and the concentration of the SjP40 protein in the composition is 0.3 mg / mL.

[0039] The SjP40 protein used in this embodiment is synthesized by Beijing Huada Protein Research and Development Center Co., Ltd.

[0040] The preparation process of the SjP40 protein hydrogel composition provided in this embodiment is as follows:

[0041] Pluronic F127 was added to deionized water, and magnetic stirring was performed until complete dissolution to prepare a PF127 hydrogel with a mass fraction of 20%. Then, SjP40 protein was added to the PF127 hydrogel, and the mixture was mixed at 4°C to form a SjP40 protein hydrogel composition. The concentration of SjP40 protein in the composition was 0.3 mg / mL.

[0042] Example 3

[0043] The SjP40 protein hydrogel composition provided in this embodiment is prepared from SjP40 protein and Pluronic F127 hydrogel (abbreviated as PF127 hydrogel). The mass fraction of the PF127 hydrogel is 30%, and the concentration of SjP40 protein in the composition is 0.7 mg / mL.

[0044] The SjP40 protein used in this embodiment is synthesized by Beijing Huada Protein Research and Development Center Co., Ltd.

[0045] The preparation process of the SjP40 protein hydrogel composition provided in this embodiment is as follows:

[0046] Pluronic F127 was added to deionized water, and magnetic stirring was performed until complete dissolution to prepare a PF127 hydrogel with a mass fraction of 30%. Then, SjP40 protein was added to the PF127 hydrogel, and the mixture was mixed at 4°C to form a SjP40 protein hydrogel composition. The concentration of SjP40 protein in the composition was 0.7 mg / mL.

[0047] Example 3

[0048] The healing of skin damage in mice was investigated using different dressings, and the specific process is as follows:

[0049] 1. Establishment of skin damage model: C57 strain mice were selected, and their species, age, size, and other aspects were similar. The back of the mouse was depilated and iodophor was used for disinfection. A sterile punch with a diameter of 5 mm was used to make a standardized circular wound on the back to avoid damaging the muscle layer. After disinfection, Tegaderm bandage was immediately covered to fix and increase skin tension to avoid skin shrinkage around the wound to affect healing, simulating the human skin wound healing process.

[0050] 2. Grouping and drug treatment: 24 mice were randomly divided into 4 groups, and drug administration was performed once a day, continuously for 7 days:

[0051] Control group (PBS group): 20 μL of sterile saline was applied topically to each wound.

[0052] SjP40 group: Apply 20μL of SjP40 protein aqueous solution (0.5mg / mL) to each wound.

[0053] PF127 group: 20 μL of PF127 hydrogel with a mass fraction of 26% was applied topically to each wound.

[0054] SjP40+PF127 group: Apply 20 μL of the SjP40 protein hydrogel composition provided in Example 1 of this invention to each wound.

[0055] 3. Wound observation and assessment:

[0056] (1) Monitoring of wound healing rate: Wound images were taken at a fixed distance on postoperative days 0, 2, 4, 6, 8, and 10, and the healing rate was calculated.

[0057] Healing rate = (Initial area - Unhealed area) / Initial area × 100%;

[0058] The wound outlines of each group on days 0, 2, 4, 6, 8, and 10 are shown in the following figures. Figure 1 As shown in the figure, the line graphs depicting the changes in wound healing rate over time for each group are as follows: Figure 2 As shown. By Figure 1 and Figure 2 It was found that all wounds healed completely within approximately 14 days post-surgery. However, on days 2, 4, 6, and 10, the healing rates of the SjP40 group, PF127 group, and SjP40+PF127 group were significantly higher than those of the PBS group. Among these, the PF127+SjP40 group showed the most significant increase in wound healing rate, while there was no significant difference in wound healing rate between the SjP40 group and the PF127 group (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0059] Statistical analysis was performed using one-way ANOVA. Brown-Forsythe multiple comparisons were used to compare the treatment groups at different time points. Error bars represent the mean ± standard error.

[0060] (2) Histopathological examination: On the 10th day after surgery, wound tissue from each group was taken for HE staining to assess the pathological state of the skin wounds in each group. The HE staining results for each group are as follows: Figure 3 As shown, where, Figure 3 (a) is the PBS group. Figure 3 (b) is group SjP40. Figure 3 (c) is group PF127. Figure 3(d) is SjP40+PF127 group, in the figure, the dotted line represents the skin wound area, "▲" represents the epidermis layer, "↓" represents the hair follicle, and "→" represents the sebaceous gland. From Figure 3 It can be seen that the dotted line represents the skin wound area, and compared with the PBS group, the inflammatory cell infiltration in the dermis layer of the wound area of the SjP40 group, the PF127 group and the PF127+SjP40 group is obviously reduced; "▲" is the epidermis layer, and the epidermis layer of the wound area of the PBS group is obviously thicker than that of the surrounding normal skin area, while the SjP40 group, the PF127 group and the PF127+SjP40 group are not obvious; "↓" represents the hair follicle, and "→" represents the sebaceous gland. Compared with the PBS group, a small amount of skin appendages such as hair follicles and sebaceous glands can be seen in the wound area of the SjP40 group and the PF127 group, while a large amount of skin appendages have been formed in the wound area of the PF127+SjP40 group on the 10th day, and the dermis layer structure of the skin wound is similar to that of the normal skin around the wound.

[0061] On the 10th day after the operation, the wound tissue of each group was taken for Masson staining to evaluate the collagen fiber deposition in the skin wound area of each group. The collagen protein area of each group was quantified by image J software, and multiple images were taken along the wound bed of each sample in each group, and then the average value was compared. The Masson staining results of each group are shown in Figure 4 , where red represents muscle fibers and blue represents collagen fibers. Figure 4 (a) is the PBS group, the blue collagen fiber in the wound area is lightly colored and sparsely arranged, indicating that the collagen deposition is less; Figure 4 (b) is the SjP40 group, compared with the PBS group, the blue collagen fiber is colored darker and the density is increased, indicating that the collagen deposition is promoted; Figure 4 (c) is the PF127 group, compared with the PBS group, the blue collagen fiber is colored and the density is increased; Figure 4 (d) is the SjP40+PF127 group, the blue collagen fiber in the wound area is colored the darkest and has the highest density, and the fiber arrangement is more regular, indicating that the collagen deposition is the most significant.

[0062] The collagen volume fraction chart of each group is shown in Figure 5 Compared with the PBS group, the collagen volume fraction of the wound area of the SjP40 group, the PF127 group and the PF127+SjP40 group is significantly increased, and the collagen volume fraction of the PF127+SjP40 group is increased most obviously, but there is no obvious difference in the collagen volume fraction between the wound areas of the SjP40 group and the PF127 group (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001);

[0063] Statistical analysis was performed by one-way ANOVA and comparison was made by Brown-Forsythe multiple comparison.

[0064] 4. Results analysis: The wound healing rate of SjP40+PF127 group was significantly higher than that of other groups; HE section showed that the inflammatory infiltration of wound surface in SjP40+PF127 group was the least, the epidermal layer was not significantly thickened, and the adnexa such as hair follicle and sebaceous gland in the dermis layer recovered well. It can be seen that the composition of SjP40 protein and PF127 hydrogel can significantly accelerate the closure of skin wound, reduce inflammatory cell infiltration, promote collagen fiber deposition and skin adnexa regeneration, and help to reduce post-healing scar hyperplasia, and its repair effect is better than that of using SjP40 protein or PF127 hydrogel alone, showing a synergistic effect.

[0065] Obviously, the above examples are only examples for clearly illustrating, but not limitation to the embodiments. Other different forms of changes or variations can be made on the basis of the above description for those skilled in the art. Here, all the embodiments need not and cannot be exhausted. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. Use of SjP40 protein in preparation of a medicament or skin care product for skin tissue repair.

2. Use according to claim 1, wherein The skin tissue repair includes repair of inflammatory wounds, knife wounds, bedsores, diabetic complication ulcers, burns, scalds, frostbite, radiation-induced skin damage, postoperative infected incision healing or growth promotion after skin transplantation.

3. The use according to claim 1, wherein The dosage form of the medicament or skin care product includes gel, aqueous solution, cream, ointment, cream, spray, injection or dressing.

4. A composition for promoting repair of skin tissue, characterized by, The composition comprises SjP40 protein and Pluronic F127 hydrogel.

5. The composition of claim 4, wherein The composition further comprises at least one of growth factors, antibacterial peptides, silver ions, antioxidants, stem cell-derived exosomes or hemostatic agents.

6. The composition of claim 4, wherein The mass fraction of the Pluronic F127 hydrogel is 20% to 30%.

7. The composition of claim 4, wherein The concentration of SjP40 protein in the composition is 0.3 to 0.7 mg / mL.

8. A process for the preparation of a composition according to any one of claims 4 to 7, characterised in that, The composition comprises the following steps: Pluronic F127 hydrogel with a mass fraction of 20% to 30% is mixed with SjP40 protein to obtain the composition; the concentration of SjP40 protein in the composition is 0.3 to 0.7 mg / mL.

9. A pharmaceutical preparation comprising the composition according to any one of claims 4 to 7, characterized in that, The pharmaceutical preparation is in the form of external gel, patch or spray.

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