Use of silk fibroin fusion protein in preparation of topical product for skin damage

Abstract

Provided is a use of a silk fibroin fusion protein in preparation of a topical product for skin damage. The silk fibroin fusion protein is formed by combining a neurotrophic factor NT-3 and a silk fibroin light chain by means of a flexible linker chain, and the silk fibroin fusion protein can be self-assembled with a silk fibroin protein to form a novel material capable of playing a stable role for a long period of time in repairing skin damage. The material can inhibit inflammation, promote type III collagen deposition and hair follicle regeneration, and allow for scar-free wound healing.

Description

Application of silk fibroin fusion protein in the preparation of topical products for skin damage Technical Field

[0001] The invention belongs to the field of biomedical materials, and particularly relates to a silk fibroin fusion protein with NT3 activity and application thereof in repairing skin damage. Background Art

[0002] With the development of emerging disciplines such as tissue engineering and biomaterials, an increasing number of biomaterials are being used to treat skin injuries. Suitable biomaterials for repairing skin injuries should promote wound healing and reduce scar formation. Currently, many wound dressings, including hydrogels, inorganic composite microspheres, and functional nanofiber membranes, have been designed for wound healing. However, wound healing is a time-consuming process that can cause pain and further damage. To reduce wound pain and accelerate wound healing, the design of innovative bioactive materials is essential. Natural biomaterials, such as chitosan, collagen, gelatin, and silk fibroin, all have structures similar to those of the extracellular matrix. They not only have low cytotoxicity and good cytocompatibility, but also low antigenicity. Natural biomaterials are beneficial in inducing cell proliferation, migration, and differentiation. Furthermore, because they have specific recognition sites for cell surface receptors, they are less likely to induce immune rejection. Silk fibroin is an insoluble bioprotein with excellent biocompatibility in biomedical applications and has been approved by the US Food and Drug Administration (FDA) as a biomaterial for human medical use. Currently, silk fibroin is widely used in tissue engineering scaffolds due to its biocompatibility and slow biodegradability. In addition, silk fibroin can accelerate wound healing by promoting the reorganization of collagen.

[0003] The nerve growth factor family, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), has been shown to play a key role in cardiovascular development. Literature reports that intramuscular injection of NT-3 significantly promotes angiogenesis in ischemic limb muscles. Angiogenesis is a key step in promoting wound healing.

[0004] However, NT-3 diffuses rapidly at the site of injury, making it crucial to find an effective method to maintain an appropriate NT-3 concentration. Furthermore, the instability of NT-3 in the body and its limited availability significantly limit its use. Furthermore, research on NT-3 has largely focused on its effects on axonal regeneration and promoting recovery from spinal cord injuries or sciatic nerve injuries, with few reports on its effects on skin injuries.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a silk fibroin fusion protein, which combines neurotrophic factor NT-3 with silk fibroin light chain through a flexible linker chain to form a recombinant protein. This recombinant protein can self-assemble with silk fibroin to form a new material that can repair skin damage with a long-term stable effect. This material can inhibit inflammation, promote type III collagen deposition, promote hair follicle regeneration, and heal wounds without scars.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A silk fibroin fusion protein, whose amino acid sequence is:

[0009] In the present invention, the preparation method of the above protein is:

[0010] (1) Construction of recombinant expression vector: A gene fragment containing silk fibroin light chain and NT-3 was synthesized and connected to the pET-30 expression vector to complete the construction of the recombinant expression vector.

[0011] (2) Expression and purification of recombinant protein: The recombinant expression vector obtained in step (1) was transferred into BL21 Escherichia coli, induced to express in a small test tube at 37°C and identified, then amplified and collected, and ultrasonically disrupted. The recombinant protein was purified using Ni-NTA for identification and detection.

[0012] Application of the above silk fibroin fusion protein in the preparation of external use products.

[0013] Furthermore, the external use product also includes regenerated silk fibroin.

[0014] In the present invention, the preparation method of the regenerated silk fibroin is:

[0015] Step 1: Degumming mulberry silk by boiling in 0.5% NaHCO3 solution;

[0016] Step 2: After washing the degummed silk fibers, the fibers were dissolved in a mixture of CaCl2, H2O, and C2H5OH (molar ratio 1:8:2), and then dialyzed with distilled water. The molecular weight cutoff of the dialysis bag was approximately 12,000-14,000 Da. The regenerated silk fibers were freeze-dried to obtain the obtained fibers.

[0017] An external preparation comprises the silk fibroin fusion protein and regenerated silk fibroin.

[0018] Furthermore, in the external preparation, the silk fibroin fusion protein is fixed on the regenerated silk fibroin by self-assembly with the regenerated silk fibroin.

[0019] Furthermore, in the external preparation, the usage ratio of regenerated silk fibroin and silk fibroin fusion protein is: 2 μL of 100 μg / mL silk fibroin fusion protein solution is added to every 1 mL of 0.2 g / mL regenerated silk fibroin solution.

[0020] The application of the external preparation in the preparation of products for promoting the healing of skin wounds to scar-free state.

[0021] Application of the external preparation in preparing products for inhibiting skin wound inflammation.

[0022] Application of the above external preparation in the preparation of products for promoting hair follicle regeneration.

[0023] The silk fibroin fusion protein and regenerated silk fibroin described in the present invention can be made into a solution and applied directly or mixed with a dressing commonly used for skin injuries to treat skin injuries. The mixed solution can also be dried in a refrigerator at four degrees Celsius to form a film to obtain a silk fibroin film with NT-3 activity, which can then be made into gauze or a bandage for wound treatment. Beneficial effects:

[0024] The main materials used in the present invention are silk fibroin and silk fibroin light chain fused with NT-3. No toxic or side-effect substances such as cross-linking agents and surfactants are added during the preparation process, so the invention has good biocompatibility.

[0025] The skin damage repair material provided by this invention incorporates a silk fibroin light chain fused with NT-3, retaining the disulfide bonds of the light chain and adding a flexible linker chain between the NT3 and the light chain. During self-assembly of the silk fibroin in a silk solution, NT-3 is anchored to the silk fibroin along with the disulfide-bonded silk fibroin light chain. The flexible chain of the FIBL-Linker-NT3 facilitates the binding of NT3 to other NT3 chains in different directions.

[0026] In the process of repairing skin damage, through the observation of wound recovery, silk and silk fibroin light chain fused with NT-3 showed obvious effects in promoting wound recovery, inhibiting inflammation at the wound, promoting type III collagen deposition, increasing blood vessel area, epithelial length and the number of new hair follicles, and promoting skin damage to heal without scars. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 shows a schematic diagram of the recombinant protein FIBL-Linker-NT3 and the Western blot verification results.

[0028] The left image in Figure 2 shows the silver staining results of silk fibroin (SF) and silk fibroin heavy chain (SFH), and the right image shows the Western blot results of SFH-FIBL-Linker-NT3.

[0029] FIG3 shows the experimental results of Example 2 in which the recombinant protein promotes the recovery of mouse skin damage.

[0030] FIG4 shows the experimental results of mouse skin wound inflammation in Example 3.

[0031] FIG5 shows the experimental results of type III collagen deposition in mouse skin wounds in Example 4.

[0032] FIG6 shows the experimental results of angiogenesis, length of new epithelium, and number of new hair follicles in mouse skin wounds in Example 5. DETAILED DESCRIPTION

[0033] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0035] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0036] Example 1

[0037] 1. FIBL-Linker-NT3 recombinant protein expression and verification

[0038] (1) A recombinant expression vector was constructed, the codons of the silk fibroin light chain and the NT3 active peptide were optimized, and a flexible linker chain was added in the middle. Then, the genes were synthesized and the synthesized genes were constructed into the vector pET-30a by enzyme ligation. Sequencing verified the successful construction of the recombinant expression vector pET-FIBL-Linker-NT3.

[0039] (2) The recombinant vector was transformed into BL21 Escherichia coli to construct an expression strain. A single clone was selected and transferred to 1 mL of LB liquid medium. The culture was cultured overnight at 37°C and 150 rpm. Expression was identified and the expression strain was preserved. The next day, the culture was expanded to 50 mL and shaken at 37°C until the OD600 of the culture solution was approximately 1.0. IPTG was then added to a final concentration of 1 mM and induced for 16 h at 37°C.

[0040] (3) The bacterial pellet was collected by centrifugation and ultrasonically disrupted. The target protein with the His tag was separated and purified using Ni-NTA. The expression of the target protein FIBL-Linker-NT3 was identified by SDS-PAGE and Western blot.

[0041] The results are shown in Figure 1 , indicating that the fusion protein FIBL-Linker-NT3 was successfully constructed and expressed.

[0042] 2. Silk fibroin can self-assemble with FIBL-Linker-NT3

[0043] (1) Dissolve 0.1 g of silk fibroin in 2 mL of PBS (0.1 M) at pH 7.4, add 100 μL of DTT (1 M), seal the bottle, and react at 4°C for 6 h. Place the solution in a cellulose tube (molecular cutoff value 100 kDa) and soak in distilled water for 3 days. Change the deionized water approximately every 8-10 hours. Collect the silk fibroin heavy chain (SFH) in the cellulose tube and silver stain it together with the original silk fibroin to verify the successful separation of the silk fibroin heavy chain (SFH).

[0044] (2) 100 μL of silk fibroin heavy chain SFH (7.5 μg / μL) was added to 9.9 mL of PBS (0.1 M) at pH 7.4, and then 180 μL of recombinant protein FIBL-Linker-NT3 (100 μg / mL) was added for self-assembly. The cells were then dialyzed for 3 days in a 100 kDa cellulose tube. The SFH-FIBL-Linker-NT3 in the cellulose tube was retained for further Western blot analysis. Detection with NT3 antibody revealed that the molecular band of the self-assembled SFH-FIBL-Linker-NT3 was above 100 kDa, indicating successful self-assembly. The results are shown in Figure 2.

[0045] Example 2

[0046] SF+FIBL-Linker-NT3 recombinant protein solution promotes the recovery of mouse skin injuries

[0047] (1) Blank group: 8-week-old male adult mice were purchased from the Experimental Animal Center of Nantong University and anesthetized by intraperitoneal injection of 3% sodium pentobarbital solution. A 10 mm diameter circular wound was made on the back of each mouse using a sterile punch. 100 μL of pH 7.4 PBS (0.1 M) was added daily.

[0048] (2) Place 50 g of raw silk in 2 L of boiling 0.5% NaHCO₃ solution. Boil for 30 minutes, then discard the water to remove the sericin. Rinse the silk three times with triple-distilled water. Repeat this process three times. Dry the silk in a laminar flow hood to obtain silk fibroin fibers free of sericin.

[0049] (3) Experimental Group 1: 20 g of the obtained silk fibroin fibers were dissolved in a terpolymer solvent system of CaCl2 / H2O / C2H5OH (molar ratio 1:8:2). The obtained silk fibroin solution was then dialyzed with triple-distilled water. The molecular weight cut-off of the dialysis bag was approximately 12,000-14,000 Da. After dialysis at 4°C for 3 days, the solution was freeze-dried in a freeze dryer to obtain regenerated silk fibroin. 0.2 g of regenerated silk fibroin was weighed and dissolved in 1 mL of PBS (0.1 M) at pH 7.4 to obtain a 20% silk fibroin solution. A mouse skin injury model was obtained by referring to the method of the blank group, and 100 μL of silk fibroin solution (SF) was added dropwise every day.

[0050] (4) Experimental group 2: 20 μL of recombinant protein FIBL-Linker-NT3 (100 μg / mL) was added to 1 mL of PBS (0.1 M) at pH 7.4. The mouse skin injury model was obtained by referring to the method of the blank group, and 100 μL of FIBL-Linker-NT3 recombinant protein solution was added every day.

[0051] (5) Experimental group 3: 0.2 g of regenerated silk fibroin and 2 μL of recombinant protein FIBL-Linker-NT3 (100 μg / mL) were weighed and added to 1 mL of PBS (0.1 M) at pH 7.4. The mouse skin injury model was obtained by referring to the blank group, and 100 μL of SF + FIBL-Linker-NT3 recombinant protein solution was added every day.

[0052] (6) The skin injuries on the back of mice were photographed and observed on days 0, 5, and 10 after skin injury. As shown in Figure 3, SF+FIBL-Linker-NT3 was more conducive to the recovery of the skin injuries on the back of mice.

[0053] Example 3

[0054] SF+FIBL-Linker-NT3 recombinant protein film promotes the recovery of mouse skin damage

[0055] (1) Place 50 g of raw silk in 2 L of boiling 0.5% NaHCO₃ solution. Boil for 30 minutes, then discard the water to remove the sericin. Rinse the silk three times with triple-distilled water. Repeat this process three times. Place the silk in a clean hood and air dry to obtain silk fibroin fibers free of sericin.

[0056] (2) Control group: 20 g of the obtained silk fiber was dissolved in a terpolymer solvent system of CaCl2 / H2O / C2H5OH (molar ratio 1:8:2), and then the obtained silk protein solution was dialyzed with triple-distilled water. The molecular weight cut-off of the dialysis bag was about 12000-14000 Da. After dialysis at 4°C for 3 days, it was freeze-dried in a freeze dryer to obtain regenerated silk fibroin. 0.2 g of regenerated silk fibroin was weighed and dissolved in 1 mL of PBS (0.1 M) with a pH of 7.4 to obtain a 20% silk fibroin solution. A sterile small round glass slide with a diameter of 14 mm was placed in a 24-well plate, 100 μL of the silk fibroin solution was added dropwise to the small round glass slide, and the plate was placed in a refrigerator at 4°C overnight to dry and form a film. 8-week-old male adult mice were purchased from the Experimental Animal Center of Nantong University and anesthetized by intraperitoneal injection of 3% sodium pentobarbital solution. A circular wound with a diameter of 10 mm was made on the back of each mouse with a sterile punch. Apply silk film on the wound and wrap it with gauze.

[0057] (3) Experimental group: 0.2 g of regenerated silk fibroin and 2 μL of recombinant protein FIBL-Linker-NT3 (100 μg / mL) were weighed and added to 1 mL of PBS (0.1 M) at pH 7.4. After preparing a mouse skin injury model, a SF+FIBL-Linker-NT3 film was applied to the wound and wrapped with gauze.

[0058] (4) The skin injuries on the back of mice were photographed and observed on the 0th, 5th and 10th days after the skin injury. The results showed that the SF+FIBL-Linker-NT3 film was more conducive to the recovery of the skin injuries on the back of mice than the single silk film.

[0059] Example 4

[0060] SF+FIBL-Linker-NT3 reduces wound inflammation in mice

[0061] (1) Referring to Example 2, a mouse back skin injury model was prepared.

[0062] (2) Five days after skin injury, the skin tissue from the back of the mouse was collected and fixed with 4% paraformaldehyde. After 24 hours, it was dehydrated with 30% sucrose and then frozen and sectioned. The section thickness was set to 12 μm.

[0063] (3) After sectioning, immunofluorescence staining was performed, inflammatory cells were labeled with iNOS, and the inflammatory condition of the injured area was observed using a fluorescence microscope.

[0064] (4) As shown in Figure 4, the inflammation in the SF group and the SF+FIBL-Linker-NT3 group was significantly less than that in the PBS and PBS+FIBL-Linker-NT3 groups, indicating that silk fibroin can inhibit inflammation in the wound.

[0065] Example 5

[0066] The SF+FIBL-Linker-NT3 group significantly promoted the deposition of type III collagen

[0067] (1) Referring to Example 2, a mouse back skin injury model was prepared.

[0068] (2) Five days after skin injury, skin tissue from the back of the mouse was collected and fixed with 4% paraformaldehyde. After 24 hours, it was dehydrated with 30% sucrose and then frozen and sectioned. The section thickness was set to 12 μm.

[0069] (3) After sectioning, immunofluorescence staining was performed, and type III collagen was labeled with Col3. Fluorescence microscopy was used to observe the deposition of type III collagen at the lesion site.

[0070] (4) The results are shown in Figure 5. The deposition of type III collagen in the SF+FIBL-Linker-NT3 group was significantly higher than that in the other three groups, proving that the presence of silk fibroin and FIBL-Linker-NT3 can significantly promote the deposition of type III collagen and reduce scar formation.

[0071] Example 6

[0072] The SF+FIBL-Linker-NT3 group significantly promoted angiogenesis, the length of new epithelium and the number of new hair follicles

[0073] (1) Referring to Example 2, a mouse back skin injury model was prepared.

[0074] (2) The skin tissue from the back of the mouse was collected 5 days after skin injury, fixed with 4% paraformaldehyde, dehydrated with 30% sucrose after 24 hours, and then frozen and sectioned. The section thickness was set to 12 μm.

[0075] (3) After sectioning, hematoxylin-eosin (HE) staining was performed. Hematoxylin is alkaline and primarily stains the chromatin in the cell nucleus and the ribosomes in the cytoplasm purple-blue; eosin is an acidic dye that primarily stains the components of the cytoplasm and extracellular matrix red. The results are shown in Figure 6. The SF+FIBL-Linker-NT3 group effectively promoted angiogenesis, increased the length of regenerated epithelium, and increased the number of newly formed hair follicles.

Claims

1. Application of silk fibroin fusion protein in the preparation of external products for skin damage, characterized in that: The amino acid sequence of the silk fibroin fusion protein is: MHHHHHHAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDKSIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAANVINSYTDGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRIYDFEAAWDAILASSDSSFLNEEYCIVKRLYNSRN SQSNNIAAYITAHLLPPVAQVFHQSAGSITDLLRGVGNGNDATGLVANAQRYIAQAASQVHVGGGGSGGGGSYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCALSRKIGRT.

2. The use according to claim 1, characterized in that: The skin injury external product also includes regenerated silk.

3. The use according to claim 1, characterized in that: The preparation method of the regenerated silk fibroin is: Step 1, taking mulberry silk and boiling it in 0.5% NaHCO3 solution for degumming; Step 2, washing the degummed silk fiber, dissolving it in a mixture of CaCl2, H2O and C2H5OH, wherein the molar ratio of CaCl2, H2O and C2H5OH in the mixture is 1:8:2, and then dialyzing it with distilled water, the molecular weight cutoff of the dialysis bag is 12000-14000Da, and obtaining regenerated silk fibroin after freeze-drying.

4. An external preparation, characterized in that It includes silk fibroin fusion protein and regenerated silk fibroin, and the amino acid sequence of the silk fibroin fusion protein is: MHHHHHHAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDKSIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAANVINSYTDGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRIYDFEAAWDAILASSDSSFLNEEYCIVKRLYNSRN SQSNNIAAYITAHLLPPVAQVFHQSAGSITDLLRGVGNGNDATGLVANAQRYIAQAASQVHVGGGGSGGGGSYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCALSRKIGRT.

5. The external preparation according to claim 4, characterized in that The silk fibroin fusion protein is fixed on the regenerated silk fibroin by self-assembly with the regenerated silk fibroin.

6. Use of the external preparation according to claim 4 in the preparation of a product for promoting scar-free healing of skin wounds.

7. Use of the external preparation according to claim 4 in the preparation of a product for inhibiting inflammation of skin wounds.

8. Use of the external preparation according to claim 4 in the preparation of products for promoting hair follicle regeneration.

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