Silk protein hydrogel loaded with chlorella as well as dual-crosslinking preparation method and application of silk protein hydrogel

Abstract

The invention discloses silk protein hydrogel loaded with chlorella as well as a dual-crosslinking preparation method and application of the silk protein hydrogel, and belongs to the field of biomedical materials. A silk protein solution with excellent biocompatibility is used as a material, the hydrogel is prepared through a double crosslinking process of an ultrasonic crosslinking method and an HRP enzyme crosslinking method, and the hydrogel has the advantage of high stability of an ultrasonic method hydrogel and also has the characteristics of high elasticity, high light transmittance and the like of an HRP enzyme crosslinking method hydrogel. When the silk protein hydrogel loaded with the chlorella is applied to diabetic wounds, oxygen can be generated by means of efficient photosynthesis of the chlorella, local dissolved oxygen is continuously conveyed to the wounds, tissue regeneration and vascular repair are further promoted, and the problem that the diabetic chronic wounds are difficult to heal is effectively solved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and particularly relates to an oxygen-producing silk fibroin hydrogel loaded with active Chlorella vulgaris, a double-crosslinking preparation method and its application in promoting diabetic wound healing. Technical Background

[0002] As a prevalent epidemic disease, chronic wounds affect more than 1% of the population. Diabetes is one of the most common diseases that induce chronic wounds. For example, diabetic foot ulcers increase the probability and risk of amputation in patients, and at least 68% of these patients will die within 5 years. The formation and difficult healing of wounds in diabetic patients are closely related to multiple factors. The main reason is that diabetic patients are in a hyperglycemic state for a long time, and the hyperglycemic environment will affect the stability of hypoxia-inducible factor. The rapid hydroxylation and degradation after translation of hypoxia-inducible factor result in the inability to up-regulate vascular endothelial growth factor, the process of generating new blood vessels at the wound site is relatively slow, and the newly formed blood vessels are damaged, leading to insufficient oxygen supply, thus affecting wound healing. Therefore, hyperbaric oxygen and local gaseous oxygen are common strategies to provide sufficient oxygen to relieve the hypoxic environment of chronic wounds. However, hyperbaric oxygen therapy cannot prevent local ischemia of the wound; when using local gaseous oxygen therapy, the penetration ability of external gas to tissues is limited, and its effect of penetrating the skin is poor compared with local dissolved oxygen. Research shows that local gaseous oxygen can only penetrate through 300 μm of the dermis, which limits its role in wound healing.

[0003] In recent years, people have utilized microbial photosynthesis to produce local dissolved oxygen as a novel strategy for the healing of chronic diabetic wounds. In this invention, the natural oxygen-producing ability of Chlorella vulgaris is used to increase the oxygen level and balance the oxygen gradient within a larger wound structure. Chlorella vulgaris has various advantages such as good biocompatibility and biodegradability. Under irradiation at 650 nm, the chlorophyll released by Chlorella vulgaris is a natural photosensitizer that can generate reactive oxygen species and is an ideal oxygen producer. In addition, Chlorella vulgaris also has the functions of solidifying glucose at the wound site, antibacterial, and antioxidant, and can regulate metabolism. The wound delivery of local dissolved oxygen is achieved by encapsulating Chlorella vulgaris with silk fibroin hydrogel. Silk fibroin, as a currently known natural polymer material, has excellent processability, biocompatibility, adjustable biodegradability, low extraction cost, high safety, etc., and is usually widely used as an ideal biomaterial for skin repair. Currently, there are mainly three methods for preparing silk fibroin hydrogel: physical cross-linking method, chemical cross-linking method, and enzyme-catalyzed cross-linking method. Among them, the chemical cross-linking method has the disadvantages that the added chemical cross-linking reagents are difficult to remove, there is a certain degree of toxicity to organisms, the biocompatibility of the silk fibroin hydrogel is low, and the elasticity is poor. In this invention, a double cross-linking gelation method of ultrasonic physical cross-linking and enzyme cross-linking is selected to prepare a silk fibroin hydrogel with excellent properties. The prepared hydrogel not only has the advantages of high stability of the hydrogel prepared by the ultrasonic method but also has the characteristics of high elasticity and high light transmittance of the hydrogel prepared by the HRP enzyme cross-linking method, significantly shortening the gelation time and improving the cell compatibility.

[0004] Before this invention, the Chinese invention patent with the application number CN202110660594.9 in the prior art disclosed a chemically cross-linked silk fibroin hydrogel. The introduction of a diglycidyl ether-based chemical cross-linking agent will have a greater impact on the biocompatibility of the material, thus causing a certain degree of toxicity to active microorganisms and being unfavorable for the storage of active microorganisms.

[0005] Another example is the Chinese invention patent with the application number CN201911234756.1, which disclosed a nano-hybrid silk fibroin hydrogel prepared by the ultrasonic method. It is prepared by blending an aqueous solution of silk fibroin with nano-hydroxyapatite and subjecting it to ultrasonic treatment. This hydrogel has poor light transmittance, and the light transmittance is close to 0% after adding hydroxyapatite to silk fibroin, and it cannot load photosynthetic microorganisms such as Chlorella vulgaris. Summary of the Invention

[0006] The object of the present invention is to provide a fibroin hydrogel loaded with Chlorella vulgaris, which uses the efficient photosynthesis of Chlorella vulgaris to produce oxygen, continuously delivers oxygen to the local wound of diabetic patients, improves the dissolved oxygen level, and oxygen can promote tissue regeneration and blood vessel repair. The present invention uses ultrasonic method and HRP enzyme crosslinking method to double crosslink and gelate to prepare fibroin hydrogel as a material carrier for embedding Chlorella vulgaris. This fibroin hydrogel has good biocompatibility, and has both the advantages of high stability of the ultrasonic hydrogel and the characteristics of high elasticity and high light transmittance of the HRP enzyme crosslinked hydrogel. Finally, the fibroin hydrogel loaded with Chlorella vulgaris is used as a diabetic wound dressing, achieving the therapeutic effect of promoting wound healing.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A fibroin hydrogel loaded with Chlorella vulgaris, characterized in that the hydrogel is formed by wrapping active Chlorella vulgaris with fibroin having excellent biocompatibility. The fibroin has light transmittance and maintains the activity of Chlorella vulgaris at the same time.

[0009] The hydrogel is prepared by double crosslinking based on ultrasonic physical crosslinking and horseradish peroxidase (HRP) crosslinking method.

[0010] A preparation method of a fibroin hydrogel loaded with Chlorella vulgaris, characterized by comprising the following steps:

[0011] Step 1) Prepare BG-11 Chlorella vulgaris medium, sterilize it, inoculate Chlorella vulgaris strain, and culture it in a light incubator; centrifuge and wash Chlorella vulgaris with PBS for standby.

[0012] Step 2) Cut open the cocoon to remove the pupa, put the cocoon into boiling sodium carbonate solution, dissolve and remove the sericin on the surface of the raw silk of the cocoon, rinse the cooked silk with running water, and dry it at room temperature for standby.

[0013] Step 3) Dissolve the dried degummed silk in preheated lithium bromide solution, and then perform dialysis; after dialysis is completed, centrifuge to remove impurities to obtain a pure fibroin solution.

[0014] Step 4) After determining the concentration of the fibroin solution, perform ultrasonic treatment in a cell ultrasonic crusher. Immediately after the ultrasonic treatment, add horseradish peroxidase (HRP), H2O2 and Chlorella vulgaris with different concentrations and mix them. Let the obtained mixed system stand at 37°C to form a gel.

[0015] According to the above preparation method, the specific parameter settings in the preparation are as follows:

[0016] In the above step 1), it is preferred that Chlorella vulgaris is cultured in a light incubator with a light intensity of 2000 Lux, a light-dark ratio of 12 h / 12 h, and a temperature of 25°C.

[0017] In step 2) above, the concentration of the sodium carbonate solution is preferably 2.12 g / L, and the cooking time of the silkworm cocoons is 30 min.

[0018] In step 3) above, it is preferred that 4 mL of 9.3 M lithium bromide solution is used per 1 g of silk, dissolved at 60 °C, and the silk is stirred every 1 h for 4 h of continuous dissolution;

[0019] In step 3) above, the molecular weight cut-off of the dialysis bag is 3000 Da, the dialysis time is 40 h - 60 h, preferably 60 h. After dialysis, the centrifugation conditions are a rotation speed of 8000 rpm, a centrifugation temperature of 4 °C, and a centrifugation time of 10 min.

[0020] In step 4) above, the mass percentage concentration of the silk fibroin solution is 5%, the ultrasonic power of the silk fibroin solution in the cell ultrasonic crusher is 120 W - 210 W, preferably 180 W, and the ultrasonic time is 30 s. The ultrasonic process is carried out with the centrifuge tube placed in an ice box.

[0021] In step 4) above, the final concentration of HRP in the mixed system is 0.192 mg / mL, and the final concentration of H2O2 is 0.036 wt%.

[0022] In step 4) above, the final concentration of Chlorella vulgaris in the mixed system is 10 6 cells / mL - 5×10 8 cells / mL, preferably 5×10 8 cells / mL.

[0023] Use of the obtained silk fibroin hydrogel loaded with Chlorella vulgaris as an external patch for promoting diabetic wound healing.

[0024] The technical solution proposed by the present invention has the following advantages:

[0025] (1) A silk fibroin hydrogel loaded with Chlorella vulgaris provided by the present invention. Chlorella vulgaris has various advantages such as being inexpensive and easily available, having good biocompatibility, antibacterial and antioxidant properties, and can efficiently produce oxygen through photosynthesis, and is an ideal oxygen producer.

[0026] (2) A silk fibroin hydrogel loaded with Chlorella vulgaris provided by the present invention. The silk fibroin hydrogel is prepared by double cross-linking of the ultrasonic method and the HRP enzyme cross-linking method, combining the advantages of the two preparation methods: ultrasound can improve the stability of the hydrogel, and the enzyme cross-linking induced by HRP makes the hydrogel have high elasticity, high light transmittance, can fully apply to and conform to the activity of Chlorella vulgaris and the drug effect, and can significantly shorten the gel time, and the gel can be formed in about 15 min.

[0027] (3) The silk fibroin hydrogel loaded with Chlorella vulgaris provided by the present invention has good biocompatibility and antibacterial properties, can effectively prevent bacterial infection and absorb wound exudate, and has excellent light transmittance and adhesiveness as a wound dressing. Description of the Drawings

[0028] Figure 1 It is a physical picture of free Chlorella vulgaris in BG-11 medium.

[0029] Figure 2 It is a flowchart for the preparation of silk fibroin hydrogel loaded with Chlorella vulgaris.

[0030] Figure 3 It is a physical picture of silk fibroin hydrogel loaded with Chlorella vulgaris.

[0031] Figure 4 It is a scanning electron micrograph of pure silk fibroin hydrogel.

[0032] Figure 5 It is a scanning electron micrograph of silk fibroin hydrogel loaded with Chlorella vulgaris.

[0033] Figure 6 It is the XRD curve of silk fibroin hydrogel prepared by different gelation methods.

[0034] Figure 7 It is the light transmittance curve of silk fibroin hydrogel prepared by different gelation methods.

[0035] Figure 8 It is the fluorescein release curve of silk fibroin hydrogel prepared by different gelation methods.

[0036] Figure 9 It is the mass change curve and silk fibroin release curve of silk fibroin hydrogel prepared by different gelation methods.

[0037] Figure 10 It is the oxygen production data graph of silk fibroin hydrogel loaded with different concentrations of Chlorella vulgaris.

[0038] Figure 11 It is the wound healing diagram and wound area histogram of different groups in the mouse diabetes model.

[0039] Figure 12 It is the H&E and Masson staining diagrams of the skin at the wound of different groups in the mouse diabetes model. Detailed Embodiments

[0040] The following will be described in detail through specific implementation cases and the accompanying drawings of the specification, but the present invention is not limited to the following embodiments.

[0041] Culture of Chlorella vulgaris:

[0042] Prepare 1500 mL of BG-11 medium and dispense it into 10 conical flasks, 150 mL per flask. Sterilize at 121 °C for 20 min. As Figure 1 shown, Chlorella vulgaris FACHB-482 (10 6 cells / mL) was purchased from the Freshwater Algae Culture Collection of the Chinese Academy of Sciences and inoculated into BG-11 medium at an inoculation amount of 20%. The inoculated Chlorella vulgaris was cultured in a light incubator at a light intensity of 2000 Lux, a light-dark ratio of 12 h / 12 h, and 25 °C.

[0043] Preparation of silk fibroin solution:

[0044] Cut open the silkworm cocoon to remove the pupa. After boiling 4 L of water, add 8.48 g of anhydrous sodium carbonate. After dissolution, add 10 g of silkworm cocoon and boil for 30 min for degumming. Rinse with running water until the sericin falls off, soak in deionized water for 4 h (change water every hour), and dry.

[0045] Stir and dissolve 155 g of lithium bromide in 128 mL of water to prepare a 9.3 mol / L lithium bromide solution. Dissolve 5 g of degummed silk in 20 mL of preheated 60 °C lithium bromide solution and heat at 60 °C for 4 hours.

[0046] Put the prepared silk fibroin solution into a 3000 Da dialysis bag and place it in 4000 mL of deionized water for magnetic stirring dialysis. Change water 3 times in the first 6 h, then change water every 12 h for 2.5 days. After completion, centrifuge the dialysis solution at 8000 rpm and 4 °C for 10 min to remove impurities.

[0047] Preparation of silk fibroin hydrogel loaded with Chlorella vulgaris is as Figure 2 shown:

[0048] (1) Taking the preparation of 1 mL of the system as an example, dilute the prepared silk fibroin solution with pure water to 5% (mass percentage concentration) for standby. First, centrifuge the Chlorella vulgaris solution to obtain Chlorella vulgaris precipitate, and wash it repeatedly three times by centrifugation with PBS buffer solution. The centrifugation conditions are 5000 rpm and 7 min. Resuspend Chlorella vulgaris with pure water to a fixed concentration, and the concentration of Chlorella vulgaris is 5×10 9 cells / mL.

[0049] (2) Add 748 μL of 5% (mass percentage concentration) silk fibroin solution into a centrifuge tube, and perform ultrasonic treatment for 30 s in a cell ultrasonic crusher. During the ultrasonic process, place the centrifuge tube in an ice box. Set the ultrasonic power to 180 W. Immediately after the ultrasonic treatment, add 32 μL of 6 mg / mL HRP, 120 μL of 0.3 wt% H2O2, and 100 μL of Chlorella vulgaris into the silk fibroin solution, and mix quickly and evenly (if no Chlorella vulgaris is mixed in the pure silk fibroin hydrogel, add 100 μL of pure water to make up the volume) to obtain a mixed system. The final concentration of HRP in the mixed system is 0.192 mg / mL, the final concentration of H2O2 is 0.036 wt%, and the final concentration of Chlorella vulgaris is 5×10 8 cells / mL.

[0050] (3) Use a pipette to transfer the mixed solution into a mold, and place it in a 37 °C constant temperature oven to stand for gelation. The prepared Chlorella vulgaris hydrogel is as Figure 3 shown.

[0051] Characterize the morphology of the hydrogel by scanning electron microscopy. After freeze-drying the pure silk fibroin hydrogel for 12 h, sputter gold for SEM testing. The results are as Figure 4 shown. Observe the unique three-dimensional spatial structure of the silk fibroin hydrogel. This morphology enables the silk fibroin hydrogel to provide a basic living space for the loaded Chlorella vulgaris and a microenvironment for internal molecular diffusion and other processes. After freeze-drying the silk fibroin hydrogel loaded with Chlorella vulgaris for 12 h, sputter gold for SEM testing. The results are as Figure 5 shown. Chlorella vulgaris is embedded in the silk fibroin network scaffold, indicating the successful synthesis of the material.

[0052] Use X-ray diffraction (XRD) technology to evaluate the content of β-sheet structure in three different silk fibroin hydrogels.

[0053] Take a 1 mL system as an example:

[0054] (a) Prepare a double-crosslinked silk fibroin hydrogel: 748 μL of 5% (mass percentage concentration) silk fibroin solution is ultrasonically treated for 30 s at an ultrasonic power of 180 W, and then 32 μL of HRP (6 mg / mL), 120 μL of H2O2 (0.3 wt%), and 100 μL of pure water are added to obtain a mixed system. The final concentration of HRP in the mixed system is 0.192 mg / mL, the final concentration of H2O2 is 0.036 wt%, and gelation occurs after standing at 37 °C for 15 min.

[0055] (b) Preparation of HRP silk fibroin hydrogel: 32 μL of HRP (6 mg / mL), 120 μL of H2O2 (0.3 wt%) and 100 μL of pure water were added to 748 μL of 5% (mass percentage concentration) silk fibroin solution to obtain a mixed system. The final concentration of HRP in the mixed system was 0.192 mg / mL, and the final concentration of H2O2 was 0.036 wt%. The mixture was allowed to stand at 37 °C for 60 min to form a gel.

[0056] (c) Preparation of ultrasonic silk fibroin hydrogel: 1 mL of 5% (mass percentage concentration) silk fibroin solution was ultrasonicated at a power of 240 W for 30 s and then allowed to stand at 37 °C for 5 min to form a gel.

[0057] The prepared HRP silk fibroin hydrogel, ultrasonic silk fibroin hydrogel and double-crosslinked silk fibroin hydrogel samples were quickly frozen in liquid nitrogen and then transferred to a freeze dryer for freeze-drying treatment. The dried samples were ground into fine powder with an agate mortar and gently flattened with a glass slide to make sample slices with a flat surface for testing. The β-sheet structure of silk fibroin showed characteristic diffraction peaks near 2θ = 20.5°. As Figure 6 shown, the β-sheet content of the double-crosslinked hydrogel was higher than that of the HRP hydrogel, indicating higher stability; at the same time, the β-sheet content of the double-crosslinked hydrogel was slightly lower than that of the ultrasonic hydrogel, indicating better light transmittance than the ultrasonic hydrogel.

[0058] The light transmittance performance of the hydrogel was evaluated. The light transmittance of the hydrogel determines the amount of light energy received by Chlorella, which in turn affects the ability of Chlorella to produce oxygen through photosynthesis. The HRP silk fibroin hydrogel, ultrasonic silk fibroin hydrogel and double-crosslinked silk fibroin hydrogel were scanned at full wavelength with an enzyme-labeled instrument, with a wavelength range of 350 nm - 800 nm, a step size of 5 nm, and a hydrogel thickness of 3 mm. There were 3 parallel samples in each group. The results were as Figure 7 shown. The light transmittance of the ultrasonic hydrogel was the lowest. The light transmittance of the HRP hydrogel was 95.156%, and the light transmittance of the double-crosslinked hydrogel was 88.379%, which was similar to that of the HRP hydrogel and about 6 times that of the ultrasonic hydrogel, indicating that the double-crosslinked silk fibroin hydrogel had excellent light transmittance.

[0059] Evaluate the mass transfer efficiency of the hydrogel. Good mass transfer efficiency can ensure cell viability and material transportation, etc. 10% (v / v) 50 μg / L fluorescein was embedded in HRP silk fibroin hydrogel, ultrasonic silk fibroin hydrogel, and double-crosslinked silk fibroin hydrogel respectively. The thickness of the hydrogel was 3 mm, and there were 3 parallel samples in each group. The hydrogel was soaked in 2 mL of PBS and cultured in the dark at 4 °C. Every other day, 100 μL of the culture medium was taken, and its fluorescence intensity was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The excitation wavelength was 493 nm, and the emission wavelength was 518 nm. After detection, 100 μL of PBS was added to the 24-well plate, and it was continued to be placed in the dark at 4 °C for culture. The results are as Figure 8 shown. The mass permeability of the ultrasonic hydrogel is the worst. On the 7th day, the fluorescein concentration of the HRP hydrogel was 10.00 μg / mL, the fluorescein concentration of the ultrasonic hydrogel was 7.73 μg / mL, and the fluorescein concentration of the double-crosslinked hydrogel was 15.60 μg / mL, indicating that the mass transfer efficiency of the double-crosslinked hydrogel is stable and the best.

[0060] Evaluate the stability of the hydrogel. The HRP silk fibroin hydrogel, ultrasonic silk fibroin hydrogel, and double-crosslinked silk fibroin hydrogel were placed in 1 mL of BG-11 culture medium and then cultured in a light incubator with a light intensity of 2000 Lux, a light-dark ratio of 12 h / 12 h, and a temperature of 25 °C. After removing the culture medium, the mass of the hydrogel was weighed, and 200 μL of the culture medium was taken and placed in a 96-well plate to detect its OD 280 (used to detect the silk fibroin content in the culture medium), and fresh culture medium was replaced after measurement. The results are as Figure 9 shown. There was no significant large change in the mass of the double-crosslinked hydrogel and the ultrasonic hydrogel. The stability of the HRP hydrogel was poor and it had completely degraded on the 5th day. Therefore, the double-crosslinked hydrogel has the excellent characteristics of high stability of the ultrasonic hydrogel and can be stored for a long time.

[0061] Characterize the oxygen production capacity of the silk fibroin hydrogel loaded with Chlorella vulgaris through a dissolved oxygen meter. Chlorella vulgaris has the performance of photosynthetic oxygen production, and measuring the dissolved oxygen content in the solution is an important means to characterize the oxygen production capacity of the hydrogel. In a centrifuge tube, the densities of Chlorella vulgaris were prepared to be 5×10 7 cells / mL, 10 8 cells / mL, 5×10 85 mL of chlorella filament protein-loaded hydrogel at cells / mL. After adding 10 mL of PBS buffer to the surface of the hydrogel, 80 μL of 0.05 g / mL anhydrous sodium sulfite was added to prepare anaerobic water. The blank control was 10 mL of PBS buffer added with 80 μL of 0.05 g / mL anhydrous sodium sulfite. The samples were placed in an illumination incubator with an illumination intensity of 2000 Lux, a light-dark ratio of 12 h / 12 h, and a temperature of 25 °C. The dissolved oxygen content in the solution was measured every 1 h using a dissolved oxygen meter, and each group was measured in parallel three times. The results are as Figure 10 shown. During the 6-h detection, the oxygen content in the solution increased significantly. The oxygen content in the chlorella hydrogel at 5×10 8 cells / mL had a higher growth rate after 1 h. This result shows that the chlorella hydrogel at a high concentration has a high oxygen-producing ability and can produce sufficient dissolved oxygen for wound treatment.

[0062] To verify the therapeutic effect of the chlorella-loaded silk fibroin hydrogel on wounds in a diabetes model, mice were fasted and water-deprived for 12 h in advance. After weighing the mice, type I diabetes was induced by intraperitoneal injection of streptozotocin solution at a dose of 150 mg / kg. Four hours after injection, the mice were supplemented with food and glucose water. One week later, the blood glucose level of the mice was measured, and those with a blood glucose level exceeding 16.7 mM were considered to have successfully induced type I diabetes and were subjected to subsequent experiments.

[0063] The successfully constructed diabetic mice were randomly divided into four groups: the PBS group, the Gel group, the Algea group, and the Algealight group. The mice were anesthetized using isoflurane inhalation anesthesia. After the mice lost their response, the hair on the back of the mice was removed using a hair clipper. Round full-thickness skin wounds with a diameter of approximately 1 cm were cut on both the left and right sides of the back. The PBS group was the control group. The Gel group used pure silk fibroin hydrogel to treat the wounds. The Algea group used chlorella-loaded silk fibroin hydrogel to treat the wounds. The Algea light group applied the chlorella-loaded silk fibroin hydrogel to the wound and then irradiated it with 660-nm light for 2 h. Treatment was performed once every 3 days, and the wound healing condition was recorded. The results are as Figure 11 shown. The Algea light group had the best wound healing and recovery effect, and the wound area was only 6.9% on the 12th day. Compared with diabetic mice without any treatment, the Algea light group had a significant healing effect on diabetic wounds. Chlorella produces oxygen through photosynthesis and releases dissolved oxygen at the wound tissue, alleviating the hypoxic microenvironment of diabetic wounds and promoting wound healing.

[0064] The therapeutic effect of Chlorella-loaded silk protein hydrogel on wounds in the diabetic model was evaluated by H&E staining and Masson staining. After the wounds in the Algea light group were basically healed on the twelfth day, the mice were anesthetized with isoflurane and killed. The skin at the wound was removed, embedded, sliced, and H&E and Masson staining were performed. The results are shown in Figure 12 As shown, there were more inflammatory cell infiltrations in the wound areas of the PBS group, Gel group, and Algea group, showing a typical acute inflammatory response. The number of inflammatory cells was large and their distribution was relatively wide, indicating that the tissue was still in an active inflammatory phase and the healing process was slow. The Algea light group had fewer inflammatory factors, indicating that the skin healed faster. The collagen fibers in the Algea light group increased significantly and were arranged relatively regularly, indicating that tissue repair and reconstruction were relatively successful. The results showed that the Algea light group had the best effect in promoting wound healing in the diabetic model.

Claims

1. A fibroin hydrogel loaded with Chlorella vulgaris, characterized in that, A fibroin hydrogel loaded with Chlorella vulgaris, characterized in that the hydrogel is formed by wrapping active Chlorella vulgaris with fibroin having excellent biocompatibility, and the fibroin has light transmittance and maintains the activity of Chlorella vulgaris.

2. The silk fibroin hydrogel according to claim 1, wherein The hydrogel is prepared by double cross-linking based on ultrasonic physical cross-linking and horseradish peroxidase (HRP) cross-linking method.

3. The preparation method of the silk fibroin hydrogel according to claim 1 or 2, characterized in that, It includes the following steps: Step 1) Prepare BG-11 Chlorella vulgaris medium, sterilize it, inoculate Chlorella vulgaris strains, and culture them in a light incubator; centrifuge and wash Chlorella vulgaris with PBS for standby. Step 2) Cut open the silkworm cocoon to remove the pupa, put the silkworm cocoon into boiling sodium carbonate solution to dissolve and remove the sericin on the surface of the raw silk of the silkworm cocoon, rinse the cooked silk with running water, and air-dry it at room temperature for standby. Step 3) The degummed silk after air-drying is fully dissolved in the preheated lithium bromide solution, and then dialysis is carried out; after dialysis is completed, impurities are removed by centrifugation to obtain a pure fibroin solution. Step 4) After determining the concentration of the fibroin solution, perform ultrasonic treatment in a cell ultrasonic crusher. Immediately after the ultrasonic treatment, add horseradish peroxidase (HRP), H2O2 and Chlorella vulgaris with different concentrations and mix them. The obtained mixed system is allowed to stand at 37 °C to form a gel.

4. The preparation method according to claim 3, characterized in that, The specific parameter settings in the preparation are as follows: In the above step 1), it is preferred that Chlorella vulgaris is cultured in a light incubator with a light intensity of 2000 Lux, a light-dark ratio of 12 h / 12 h, and a temperature of 25 °C.

5. The preparation method according to claim 3, characterized in that, In the above step 2), it is preferred that the concentration of the sodium carbonate solution is 2.12 g / L and the cooking time of the silkworm cocoon is 30 min.

6. The preparation method according to claim 3, characterized in that, In the above step 3), it is preferred that 4 mL of 9.3 M lithium bromide solution is used for every 1 g of silk, dissolved at 60 °C, and the silk is stirred every 1 h for continuous dissolution for 4 h. In the above step 3), the molecular weight cut-off of the dialysis bag is 3000 Da, the dialysis time is 40 h - 60 h, preferably 60 h. After dialysis, the centrifugation conditions are a rotation speed of 8000 rpm, a centrifugation temperature of 4 °C, and a centrifugation time of 10 min.

7. The preparation method according to claim 3, characterized in that In the above step 4), the mass percentage concentration of the fibroin solution is 5%.

8. The preparation method according to claim 3, characterized in that, The ultrasonic power of the fibroin solution in the cell ultrasonic crusher is 120 W - 210 W, preferably 180 W, and the ultrasonic time is 30 S; during the ultrasonic process, the centrifuge tube is placed in an ice box. In the above step 4), the final concentration of HRP in the mixed system is 0.192 mg / mL, and the final concentration of H2O2 is 0.036 wt%.

9. The preparation method according to claim 3, characterized in that, In step 4) above, the final concentration of Chlorella in the mixed system is 10 6 cells / mL - 5×10 8 cells / mL, preferably 5×10 8 cells / mL.

10. Use of the fibroin hydrogel loaded with Chlorella vulgaris according to any one of claims 1 or 2 as an external patch for promoting diabetic wound healing.

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