Thermally and electrically conductive silk fibroin composite, its preparation method, and applications

Abstract

The present invention provides a silk fibroin composite with thermal and electrical conductivity, along with its method of preparation. Experimental results confirm that it can enhance cellular activity, stimulate cell regeneration, and accelerate wound healing.

Description

[Technical Field]

[0001] This invention belongs to the field of hydrogels, and in particular to a hydrogel containing silk protein, which has electrical conductivity and photothermal properties, and can promote cell activity and accelerate wound healing. [Previous Technology]

[0002] Silk is composed of protein and has been used as clothing since ancient times. It was also used in early surgical sutures, demonstrating the safety, affinity, and non-allergenic properties of silk protein. Recently, the characteristics of silk protein—fibroin—have been increasingly studied and understood, leading to the development of new biomaterials to enhance its added value. Fibroin exists in a concentrated liquid form within the silkworm, which is spun into fibers and possesses a highly oriented crystalline structure. When liquid fibroin is subjected to heating, freezing, pH changes, and organic solvents, its structure and properties undergo alterations. This characteristic determines its biological activity, and the main amino acid components of fibroin also have special functions for the human body. Therefore, silk protein has great potential for use as a new high-tech material, or in nutritional, health food, and cosmetic additives.

[0003] The edible value of silk mainly depends on the digestibility and nutritional function of the organism. According to digestion and absorption experiments on mice, the digestibility of regenerated fibroin aqueous solution can reach 47.2%, and the digestibility of regenerated fibroin powder is 26%. If fibroin is decomposed to the peptide and amino acid levels, the digestibility and absorption rate can reach over 90%. Therefore, the edible value of fibroin is optimal when its molecular weight is degraded to a lower level.

[0004] When silk fibroin is hydrolyzed with strong acid, water-soluble peptides with large molecular weights can be obtained under controlled hydrolysis conditions, especially peptides with molecular weights of 1000-2000. These peptides not only maintain a stable solution state for a longer period of time but also retain certain characteristics of silk fibroin. This silk fibroin peptide solution can form a silk fibroin film after drying. The powder precipitated by adding ethanol, when analyzed by X-ray diffraction, shows the presence of crystals. Due to its moisturizing properties, it is an ideal cosmetic substrate.

[0005] Silk fibroin peptides can be used in the development and utilization of shampoos and conditioners. Due to the strong adhesion of hair to silk fibroin, a crystalline film can be formed on the surface of the hair, thus having a conditioning effect. For example, during hair dyeing, bleaching, perming, and other hair styling operations, it can protect the hair from chemical and mechanical damage. Silk fibroin peptides can also be used as a substitute in cosmetics, abrasives, composite biodegradable materials, and hard materials.

[0006] The nutritional value of fibroin mainly lies in its amino acid composition and oligopeptides. In terms of the types and amounts of amino acids, its functional value is quite high. For example, glycine (accounting for 45% of total amino acids) and serine (accounting for 12% of total amino acids) can lower blood cholesterol; regular consumption of fibroin foods can reduce vascular diseases and prevent hypertension; it can also promote insulin secretion, lower blood sugar levels, and has a preventive and therapeutic effect on diabetes. Alanine (accounting for 30% of total amino acids) can promote alcohol metabolism, enhance liver function, and has a liver-protective effect; tyrosine (accounting for 6% of total amino acids) has a function in preventing dementia, etc. Fibroin protein can be hydrolyzed into aqueous amino acids or oligopeptides using acid, making it easier for the human body to absorb. Using this method, the recovery rate of fibroin can reach over 90%, and spray drying can be used for mass production. Depending on the product needs, fibroin powder can be processed into candies, snacks, biscuits, and noodles; it can also be added to foods such as tofu and jam, or directly made into capsules, tablets, and small-packaged powders.

[0007] If a silk fibroin solution is injected onto a specific template to form a uniform thin sheet, and then dried in an ventilated environment at a specific temperature, a colorless and transparent silk fibroin film can be obtained after removal. Since this type of silk fibroin film dissolves in water, it can be briefly impregnated with 70-90% alcohol to allow water to seep out from between the silk fibroin molecules, shortening the distance between molecules and creating a compact aggregate structure, thus becoming a silk fibroin film that is insoluble or sparingly soluble in water. Medically, it is known that this insoluble silk fibroin film can be used with antibacterial drugs to become a medicated silk fibroin film, which can be used as a surface covering for external wounds and as artificial skin. Because human tissue and silk fibroin have good compatibility, it will be an ideal biomedical material. [Summary of the Invention]

[0008] This invention provides a silk protein complex comprising a silk protein hydrogel (SF hydrogel), a silk / polydopamine hydrogel (SD hydrogel), a silk / polypyrrole hydrogel (SP hydrogel), or a silk / polypyrrole / polydopamine hydrogel (SDP hydrogel). The SF hydrogel comprises ddH2O, a silk solution, horseradish peroxidase (HRP), and H2O2. The SD hydrogel comprises polydopamine (PDA), ddH2O, a silk solution, HRP, and H2O2. The SP hydrogel comprises ddH2O, a silk solution, HRP, H2O2, and polypyrrole. The SDP hydrogel comprises polydopamine (PDA), HCl, ddH2O, a silk solution, HRP, H2O2, and polypyrrole.

[0009] The present invention also provides a method for preparing a silk protein complex, which includes a step of washing silk: silkworm cocoons are added to a solution of Na2CO3 and ddH2O at 100-120 °C, covered with aluminum foil, and rotated at 250 rpm for 30 minutes. After boiling, the cocoons are rubbed with ddH2O three times. The above process is repeated three times, and the last rubbing is rubbed with ddH2O for a total of six times. The washed silk is then dried for 24 hours to obtain clean silk. A step of dissolving silk: ddH2O is slowly added to LiBr and a stir bar is added to stir at 200 rpm to dissolve it evenly, preparing a 9.3 M LiBr solution. The LiBr solution is then placed in a water bath at 85 °C. The dried silk is then added to the LiBr solution in batches and stirred at 200 rpm. After all the silk has been added, the solution is stirred at 85 °C and 200 rpm for 1 hour to form a silk solution. The first impurity removal step involves cooling the stirred silk solution to room temperature and then centrifuging it for the first time at 4700g for 10 minutes. Following this, a second centrifugation is performed at 8000g for 5 minutes. After centrifugation, the supernatant of the centrifuged silk solution is collected and filtered under vacuum using 4μm, 90mm filter paper, taking care to avoid bubble formation during filtration to obtain a clean silk solution free of impurities. The second step involves dialysis, where the silk solution is placed in a dialysis bag with MWCO: 6-8 kD and approximately 16cm in length. The dialysis bag is then immersed in 2.5 L of ddH2O for dialysis. A stir bar is added at room temperature, and the mixture is stirred at 150 rpm, ensuring the water temperature does not exceed 30°C. The ddH2O is replaced with clean ddH2O, and dialysis continues until the pH of the silk solution in the dialysis bag reaches approximately 7.0. The third step involves concentrating the silk solution, where the dialysis bag is immersed in approximately 800 mL of 15% PEG solution. In a (Polyethylene glycol) solution, a stir bar is added and the mixture is stirred at 150 rpm to concentrate the silk solution. After concentration at room temperature for 20 hours, the concentration of the silk solution is measured to be higher than 15%. The silk solution is then removed, dispensed, and stored at -20°C to obtain the desired silk solution. A silk solution post-processing step is then performed to obtain the silk protein complex of the present invention.

[0010] In one embodiment, if the silk fibroin complex is a silk fibroin hydrogel (SF hydrogel), the post-treatment step of the silk solution is to take 560 μL of ddH2O and mix it with 320 μL of the prepared 20% silk solution, 50 μL of HPR with a concentration of 1 U / μL (to be protected from light), and 70 μL of H2O2 with a concentration of 0.64% (to be protected from light), and react it in an incubator at 37°C for 1 hour in the dark to form a gel, thereby obtaining the silk fibroin hydrogel.

[0011] In one embodiment, if the silk fibroin complex is a silk / polydopamine hydrogel (SD hydrogel), the post-treatment step of the silk solution is to mix 250 μL of ddH2O with 2 mg of 2% Dopamine and 30 μL of 1N NaOH, and polymerize them in a water bath at 37°C for 1 hour in the dark to form polydopamine (PDA); then, mix PDA with 25 μL of 1N HCl (to adjust the pH to pH 7), 205 μL of ddH2O, 320 μL of 20% silk solution, 100 μL of 1U / μL HPR, and 70 μL of 1.29% H2O2 (to be kept out of the light), and react them in an incubator at 37°C for 1 hour in the dark to form a gel, thereby obtaining the silk / polydopamine hydrogel.

[0012] In one embodiment, if the silk fibroin complex is a silk / polypyrrole hydrogel (SP hydrogel), the post-treatment step of the silk solution is as follows: prepare 75 mL of phosphate buffered saline (PBS) solution with pH 4.5; add 0.2 g (207 μL) of pyrrole and stir at 320 rpm at 4°C to prepare a pyrrole solution; add silk fibroin hydrogel to the pyrrole solution and stir continuously; add 0.816 g of Ammonium persulphate (APS) to 25 mL of ddH2O to prepare an APS solution as an oxidant (mole ratio pyrrole:APS = 1:1.2); slowly add the APS solution to the pyrrole solution containing the hydrogel to induce a polymerization reaction to form polypyrrole; and continue stirring at 320 rpm. Stir at 4°C for 2 hours; after completion, remove the hydrogel and rinse with ddH2O to remove unfixed polypyrrole to obtain silk / polypyrrole hydrogel.

[0013] In one embodiment, if the silk fibroin complex is a silk / polypyrrole / polydopamine hydrogel (SDP hydrogel), the post-treatment step of the silk solution is as follows: prepare 75 mL of phosphate (PBS) solution with a pH of 4.5; add 0.2 g (207 μL) of pyrrole and stir at 320 rpm at 4°C to prepare a pyrrole solution; add the silk / polydopamine hydrogel to the pyrrole solution and stir continuously; add 0.816 g of Ammonium persulphate (APS) to 25 mL of ddH2O to prepare an APS solution as an oxidant (mole ratio pyrrole:APS = 1:1.2); slowly add the APS solution to the pyrrole solution containing the hydrogel to induce a polymerization reaction to form polypyrrole; and continue stirring at 320 rpm. Stir at 4°C for 2 hours; after completion, remove the hydrogel and rinse with ddH2O to remove unfixed polypyrrole, thus obtaining the silk / polypyrrole / polydopamine hydrogel.

[0014] The present invention also provides the use of a silk fibroin complex for promoting wound healing, wherein the silk fibroin complex can promote cell activity and stimulate cell regeneration.

[0015] In one embodiment, the silk protein complex can be applied by means of a gel, microneedle, film or nanoparticles.

Implementation Method

[0016] To make the description of this disclosure more detailed and complete, illustrative descriptions of embodiments and specific examples of the present invention are provided below; however, these are not the only forms of implementing or utilizing the specific examples of the present invention. The embodiments cover features of multiple specific embodiments and methods and uses for operating these specific embodiments; however, other specific embodiments may also be used to achieve the same or equivalent effects. It should be understood that these embodiments are for illustrating the present invention and not for limiting the scope of the present invention.

[0017] Glossary

[0018] ImageJ is a Java-based image processing software developed by the National Institutes of Health and the Optical and Computational Instrumentation Laboratory at the University of Wisconsin. ImageJ supports image stacking, allowing a series of images to share a single viewport, and because it is multi-threaded, it can perform time-consuming operations in parallel on multi-CPU hardware. ImageJ can also calculate the area and pixel statistics of user-defined selections and intensity thresholds. In addition to basic image operations (such as scaling, rotation, distortion, and smoothing), ImageJ can perform image area and pixel statistics, distance and angle calculations, histogram and profile plot creation, and Fourier transforms.

[0019] The simplified preparation flow chart of the silk composite of the present invention is shown in Figure 1.

[0020] Example 1: Preparation of Silk Solution

[0021] 1. Cleaning the silk: i. Weigh 5g of silkworm cocoons and cut them in half. ii. Add the cocoons to a solution of 0.763g of Na2CO3 and 360mL of ddH2O, at a temperature between 100 and 120℃. iii. Cover with aluminum foil and rotate the cocoons at 250rpm for 30 minutes. iv. After boiling, wash the cocoons three times with 1L of ddH2O each time. v. Repeat steps 2 to 4 three times (the third time, wash the cocoons six times with 1L of ddH2O each time). vi. After washing, tear the cocoons apart and spread them flat on paper towels. vii. Place the cocoons in a fume hood for 24 hours to obtain clean, dry silk.

[0022] 2. Dissolving the silk: i. Slowly add 16 mL of ddH2O to 12.9218 g of LiBr and stir at 200 rpm until dissolved to prepare a 9.3 M LiBr solution. ii. Place the prepared LiBr solution in a water bath at 85 °C. iii. Add 4 g of dried silk to the LiBr solution in batches and stir at 200 rpm to dissolve the silk. iv. After all the silk has been added, continue stirring at 85 °C and 200 rpm for 1 hour to form a silk solution.

[0023] 3. Removing impurities from the silk solution i. After cooling the stirred silk solution to room temperature, perform a first centrifugation at 4700g for 10 minutes. ii. After the first centrifugation, perform a second centrifugation at 8000g for 5 minutes. iii. Take out the supernatant of the centrifuged silk solution and filter it under vacuum. Use 4μm, 90mm filter paper for filtration, and try to avoid generating air bubbles during filtration.

[0024] 4. Dialysis of LiBr in the solution i. Using a dialysis bag with MWCO: 6~8 kD and a length of approximately 16 cm, fold one side twice, secure it with a clip, and tie rubber bands to both sides of the clip. Use a pipette with the tip of a dropper to draw the filtered silk solution into the dialysis bag, pushing out any bubbles. Fold the other side twice more, secure it with another clip, and tie rubber bands to both sides of the clip again. ii. Immerse the dialysis bag containing the solution in 2.5 L of ddH2O for dialysis, adding a stir bar and stirring at 150 rpm. (Dialysis must be performed at room temperature; the water temperature should not exceed 30℃.) iii. On the first day, change the clean ddH2O every hour and record the time, for a total of 4 changes over 3 hours, and continue dialysis until the next day. iv. On the second day, change the clean ddH2O every 2 hours and record the time, for a total of 5 changes over 8 hours, and continue dialysis until the next day. v. On the third day, change the clean ddH2O every 4 hours and record the time. Before each water change, test the pH value of the silk solution in the dialysis bag. If the pH is around 7.0, stop dialysis. Usually, changing the water 1-2 times a day will bring the pH to around 7.0.

[0025] 5. Concentration of Silk Solution i. After dialysis is stopped, immerse the dialysis bag containing the silk solution into approximately 800 mL of 15% PEG (Polyethylene glycol) solution, add a stir bar, and stir at 150 rpm to concentrate the silk solution. ii. After concentrating at room temperature for 20 hours, measure the concentration of the silk solution. If it is higher than 15%, remove the silk solution, aliquot it, and store it in a refrigerator at -20℃ to obtain the desired silk solution. (Method for measuring concentration: Take a clean petri dish and record its weight (W0). Add 100 μL of the concentrated silk solution to the petri dish, tilt and rotate the petri dish to spread the solution evenly. After completion, place the petri dish in a 65℃ oven to dry for 45 min, then place it in a drying oven for 15 min and record the weight of the petri dish (W1). The calculation method is (W1 - W0) / 0.1 x 100%.)

[0026] Example 2: Preparation of silk fibroin complex

[0027] 1. Preparation of silk fibroin hydrogel (SF hydrogel)

[0028] Take 560 μL of ddH2O and mix it with 320 μL of the prepared 20% silk solution, 50 μL of horseradish peroxidase (HRP) with a concentration of 1 U / μL (to be protected from light) and 70 μL of H2O2 with a concentration of 0.64% (to be protected from light). After reacting in an incubator at 37°C for 1 hour in the dark, a gel is formed, thus obtaining the silk protein hydrogel.

[0029] 2. Preparation of silk / polydopamine hydrogel (SD hydrogel)

[0030] 250 μL of ddH2O was mixed with 2 mg of 2% Dopamine and 30 μL of 1N NaOH, and the mixture was reacted in a water bath at 37°C in the dark for 1 hour to form polydopamine (PDA).

[0031] Next, PDA is mixed with 25 μL of 1N HCl (to adjust the pH to pH 7), 205 μL of ddH2O, 320 μL of 20% silk solution, 100 μL of 1U / μL HRP, and 70 μL of 1.29% H2O2 (to be kept out of the light). The mixture is then reacted in an incubator at 37°C for 1 hour in the dark to form a gel, thus obtaining the silk / polydopamine hydrogel.

[0032] 3. Preparation of silk / polypyrrole hydrogel (SP hydrogel)

[0033] Prepare 75 mL of phosphate-powdered saline (PBS) solution with pH 4.5. Add 0.2 g (207 μL) of pyrrole and stir at 320 rpm at 4 °C to prepare a pyrrole solution. Add silk fibroin hydrogel to the pyrrole solution and stir continuously. Add 0.816 g of ammonium persulphate (APS) to 25 mL of ddH2O to prepare an APS solution as an oxidant (mole ratio pyrrole:APS = 1:1.2). Slowly add the APS solution to the pyrrole solution containing the hydrogel to induce a polymerization reaction and form polypyrrole. Stir continuously at 320 rpm at 4 °C for 2 hours. After completion, remove the hydrogel and wash with ddH2O to remove unfixed polypyrrole, thus obtaining the silk / polypyrrole hydrogel.

[0034] 4. Preparation of silk / polypyrrole / polydopamine hydrogel

[0035] The basic process is the same as the preparation method of silk / polypyrrole hydrogel, except that the silk protein hydrogel is replaced with silk / polydopamine hydrogel. Following the same steps, silk / polypyrrole / polydopamine hydrogel can be obtained.

[0036] Example 3: Performance Testing of Various Silk Protein Complexes (Hydrogels)

[0037] The following performance tests were all conducted using the various hydrogels prepared in Example 2.

[0038] 1. SEM Testing

[0039] Various hydrogels were freeze-dried in an environment of -50°C. Their cross-sections were quickly cut with surgical scissors. Carbon tape was attached to the stage, and the dried hydrogels were fixed on the carbon tape. A layer of platinum was deposited at 10mA for 90 seconds, and the cross-sections and internal structures of the hydrogels were observed with an electron beam energy of 5kV.

[0040] The results are shown in Figure 3. From the surface observation of SEM, it can be seen that the surface of the group coated with polymer polypyrrole (hereinafter referred to as PPy) has obvious wrinkles, while the surface of the group without PPy coating is relatively smooth.

[0041] 2. FTIR test

[0042] After drying various hydrogels in an oven at 65°C, the dried hydrogels were ground into powder and dried in an oven at 85°C for one day before being molded into ingots. Total reflectance Fourier transform infrared spectrometers were used to scan the material at wavelengths of 600~4000λ and a scanning resolution of 4 cm⁻¹, and the functional groups of the material were analyzed using Omnic software.

[0043] The results are shown in Figure 4. The functional groups of all four hydrogels exhibit the three characteristic peaks of silk protein hydrogel: Amide I, Amide II, and Amide III. The characteristic peaks of silk / polypyrrole hydrogel and silk / polypyrrole / polydopamine hydrogel also show the characteristic peaks of pyrrole, namely CN (stretching), C–H (wag), and C–H (ring vibration) at 1113 cm⁻¹, 929 cm⁻¹, and 617 cm⁻¹, respectively.

[0044] 3. Moisture content test

[0045] Take a specific amount of various hydrogels, measure the initial weight (W0) immediately after preparation, freeze-dry the hydrogels and weigh them (W1). Calculate the water content using the formula below. Formula: Water content (%) = (W0-W1) / W0 × 100%.

[0046] As shown in Figure 5, the water content of all hydrogels exceeded 80%, indicating that they have high water content and potential as wound dressings.

[0047] 4. Swelling Rate Test

[0048] Test Method: After measuring the initial weight (W0) of the hydrogel, it was placed in a pH 7.4 phosphate buffered saline (PBS) solution, and the weight of the swollen hydrogel (Wt) was measured at specific time points. The swelling rate of the hydrogel was calculated using the following formula: Formula: Swelling rate (%) = (Wt-W0) / W0 × 100%.

[0049] The results are shown in Figure 6. The swelling rates of the silk fibroin hydrogel, silk / polydopamine hydrogel, silk / polypyrrole hydrogel, and silk / polypyrrole / polydopamine hydrogel all reached their swelling saturation points after 24 hours, at 341±5%, 337±2%, 231±4%, and 319±16%, respectively. This indicates that all four groups of hydrogels showed good swelling rates and no disintegration occurred during the experiment.

[0050] 5. LED bulb luminescence test

[0051] Use a power supply to provide 3V, 20mA, connect the positive terminal to the hydrogel and the negative terminal to the bulb to form a circuit, and observe whether the bulb lights up and the brightness of the bulb.

[0052] The results are shown in Figure 7. By visual observation, it can be found that the brightness of the bulb increases as the conductivity of the hydrogel increases. This result is consistent with the conductivity result.

[0053] 6. Conductivity Test

[0054] The conductivity of a hydrogel made with a mold was measured using a four-point probe. The measurement conditions were: five points were measured, and each point was measured three times.

[0055] The results are shown in Figure 8. The conductivity of the silk protein hydrogel, silk / polydopamine hydrogel, silk / polypyrrole hydrogel, and silk / polypyrrole / polydopamine hydrogel are (6.76±0.18)×10-4 S / cm, (3.12±0.09)×10-3 S / cm, (3.71±0.22)×10-2 S / cm, and (6.25±0.15)×10-2 S / cm, respectively. It can be clearly seen that the conductivity of the silk protein hydrogel to the silk / polypyrrole hydrogel after PPy coating differs by one order.

[0056] 7. NIR Test

[0057] Various hydrogels were placed in a small water bath and kept at a constant temperature of 37°C. An 808nm NIR light source with an energy of 2 W / cm2 and a distance of 2cm from the hydrogel was used to irradiate the hydrogel from top to bottom. The temperature of the hydrogel was detected and recorded in real time by a thermocouple.

[0058] The results are shown in Figure 9 and Table 1. After irradiation with near-infrared light for 5 minutes, the temperature of the silk / polypyrrole / polydopamine hydrogel group reached 49.5±0.2°C, while that of the silk / polydopamine hydrogel was 45.7±0.5°C, the silk / polypyrrole hydrogel was 42.2±0.2°C, the silk fibroin hydrogel was 38.3±0.3°C, and the phosphate buffered saline (PBS) group was 38.3±0.2°C. This indicates that the silk / polypyrrole / polydopamine hydrogel, silk / polypyrrole hydrogel, and silk / polydopamine hydrogel all exhibit photothermal conversion effects. The temperature rise trend of the silk fibroin hydrogel was the same as that of the control group PBS, indicating that it did not have a photothermal conversion effect. Among them, the silk / polypyrrole / polydopamine hydrogel had the best photothermal conversion effect and can be used as a good property for promoting drug release and antibacterial properties in hydrogels.

[0059] Table 1. Temperature changes of silk fibroin complex (hydrogel) under near-infrared light irradiation Silk fibroin complex (hydrogel) Temperature (°C) after 5 minutes PBS 38.3 ± 0.2 SF hydrogel 38.3 ± 0.3 SD hydrogel 45.7 ±0.5 SP hydrogel 42.2 ± 0.2 SDP hydrogel 49.5 ± 0.2

[0060] 8. Cell compatibility test

[0061] The hydrogel was first sterilized in a UV sterilizer, then replaced four times with PBS (12 hours each time). The hydrogel was then prepared with culture medium to a concentration of 0.1 g / ml and cultured at 37°C and 5% CO2 for 24 hours to obtain the hydrogel extract. Mouse fibroblasts (L929) were cultured at a concentration of 1×10⁴ cells / well in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours. Extracts of different concentrations were prepared with fresh culture medium and added to 96-well plates respectively. After co-culturing with the cells for 24 hours, 100 μL of MTT reagent was added and reacted at 37°C for 4 hours. The MTT reagent was then aspirated and 100 μL of DMSO was added. The absorbance at 570 nm and 630 nm was measured by UV-VIS. Cell compatibility was calculated using the following formula. Formula: (OD570Sample - OD630Sample) / (OD570Control - OD630Control) × 100%.

[0062] According to the ISO 10993-5 standard, in the biocompatibility test, the cell viability should be greater than 70%, and the cell viability of 50% extract should be greater than or equal to that of 100% extract group, then the material can be determined to be non-cytotoxic.

[0063] The results are shown in Figures 10-11. In this experiment, the cell viability of L929 cells co-cultured with different concentrations of extract solution from hydrogel was determined using the MTT assay. The results showed that the cell viability of all extract solution concentration groups was greater than the 70% required in the specification, thus confirming that the hydrogel has good biocompatibility.

[0064] 9. Electrical stimulation cell activity test

[0065] The hydrogel was first sterilized in a UV sterilizer, then replaced four times with PBS (12 hours / time). Mouse fibroblasts (L929) were directly cultured on the hydrogel at a concentration of 3 × 10⁴ cells / well. After 24 hours of culture, current and voltage were supplied by a power supply for three days of electrical stimulation, one hour per day. On the last day of the experiment, 100 μL of MTT reagent was added and reacted at 37°C for 4 hours. The MTT reagent was then aspirated and 100 μL of DMSO was added. The absorbance at 570 nm and 630 nm was measured using UV-VIS. Cell viability was represented by the following formula: Formula: (OD570Sample - OD630Sample) / (OD570Control - OD630Control) × 100%.

[0066] The results are shown in Figure 12. After three days of electrical stimulation, the cell viability of L929 cells on the hydrogel was measured by the MTT assay. The results showed that the SDP group hydrogel had the highest cell viability, confirming that conductive hydrogels can transfer current to increase cell proliferation. This is consistent with the results of the hydrogel conductivity experiment.

[0067] 10. Cell scratch test

[0068] The experiment was divided into two groups: a control group (no electrical stimulation) and an experimental group (electrical stimulation). Mouse fibroblasts (L929) were cultured at a concentration of 1 × 10⁵ cells / well in a specific plate. After the cells reached confluence, the electrical stimulation group underwent electrical stimulation for 1 hour at hours 0, 7, and 23, and photos were taken after each stimulation session for comparison with the control group. The scratch area was calculated using ImageJ software (please confirm for errors or provide additional explanations).

[0069] As shown in Figure 13, under electrical stimulation, the scratches disappeared after 24 hours, confirming that various hydrogels can accelerate cell migration through microcurrents.

[0070] 11. Drug release test

[0071] Test Procedure: i. Check Lines: Prepare sodium sulfate (Na2SO4) solutions with concentrations of 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, and 0.625 mg / mL, and plot check lines using the sodium sulfate quantification method. ii. Incorporate fucoidan (FC) into three sample hydrogels: silk fibroin hydrogel, silk / polydopamine hydrogel, and silk / polypyrrole / polydopamine hydrogel, to prepare hydrogels containing 2% silk fibroin, silk / polydopamine, and silk / polypyrrole / polydopamine. Maintain all hydrogels at 37°C, and quantify the FC content using the FC quantification method at time points of 0.5, 1, 2, 6, 12, 18, 24, 36, 48, and 49 hours. (Electrical stimulation for 1 hour is given from hour 48 to hour 49) iii. Quantification of FC: Dissolve 100 μL of sample in 1 mL of 1 N HCl and heat at 100°C for 1 hour. After cooling, take out 200 μL of sample HCl solution and add 1 mL of BaCl2-Gelatin reagent (0.5 g of Gelatin is dissolved in 100 mL of deionized water in a 60°C water bath, cooled, and then 0.5 g of BaCl2 is added, resulting in a white turbid state). After mixing, let stand for 15 minutes and measure the absorbance at OD360 nm.

[0072] The results are shown in Figure 14. Comparing the drug release rates before and after electrical stimulation, the drug release rate increased significantly after electrical stimulation. The increase was 10.1% in the silk / polypyrrole / polydopamine hydrogel group, 8.9% in the silk / polydopamine hydrogel group, and 8.5% in the silk protein hydrogel group. The silk / polypyrrole / polydopamine hydrogel group showed the largest increase, suggesting that electrical stimulation may also help with drug release and thus aid wound healing.

[0073] Example 4: Comparison with the inventor's previous research

[0074] 1. Hyaluronic Acid / Dopamine / Silk Hydrogel (HDS hydrogel)

[0075] In this embodiment, the first comparison is with the silk fibroin hydrogel researched by the inventors in the past, which is "Dopamine-dependent functions of hyaluronic acid / dopamine / silk fibroin hydrogels that highly enhance N-acetyl-L-cysteine ​​(NAC) delivered from nasal cavity to brain tissue through a near-infrared photothermal effect on the NAC-loaded hydrogels." The contents of this document are incorporated herein by reference in their entirety.

[0076] While previous materials in this literature also contained silk fibroin (SF), this invention further incorporates a polypyrrole coating / inclusion, which additionally increases conductivity and photothermal conversion efficiency. The improved photothermal conversion efficiency enhances probiotic effects and promotes collagen production. Furthermore, the improved biocompatibility has revealed its ability to enhance cell activity under electrical stimulation, thereby promoting wound healing. Therefore, the efficacy of the silk fibroin complex is extended from drug release to promoting cell repair and wound healing. Detailed comparisons and differences are shown in Table 2 below.

[0077] Table 2. Comparison of differences between ordinary silk protein composition and the silk protein complex (hydrogel) of this name Comparison items The inventor's past research Silk protein composition The present invention relates to a silk fibroin complex (hydrogel). Improvements and Differences Material composition Hyaluronic acid (HA), silk fibroin (SF), dopamine (DA) / polydopamine (PDA) Silk fibroin (SF), polydopamine (PDA), and polypyrrole (PPy) Adding a polypyrrole coating / inclusion increases conductivity and photothermal conversion efficiency. Application directions Drug delivery from the nasal cavity to the brain Wound dressings, promoting wound healing under electrical stimulation Applications have expanded from drug delivery to wound healing, and also include electrical conductivity and photothermal conversion applications. Photothermal conversion effect Near-Infrared (NIR) illumination increases temperature for drug release. The polypyrrole coating / inner layer significantly improves photothermal conversion efficiency, raising the temperature by up to 10°C, effectively inhibiting bacteria and promoting collagen production. The photothermal conversion efficiency has been significantly improved, and the application scope has been expanded from drug release to antibacterial and wound healing. electrical conductivity No relevant tests The conductivity is significantly improved, enabling it to drive LED bulbs. The material incorporates a polypyrrole coating / embedded component, and its electrical conductivity is verified for the first time. Biocompatibility High biocompatibility can effectively open tight junctions in nasal cavity cells. Improved biocompatibility and promotion of cell proliferation and migration under electrical stimulation help accelerate wound healing. Increase the application of electrical stimulation to enhance cell activity and wound healing ability. Antibacterial and antioxidant effects Polydopamine provides antioxidant effects Polypyrrole coating / Contains antibacterial and antioxidant properties The added antibacterial function inhibits bacterial growth and promotes wound healing. Drug release control The photothermal effect promoted the release of acetylcysteine ​​(NAC) from the hydrogel, increasing the cumulative release to 65.4%. Polypyrrole coating / inner layer can further enhance the effect of photothermal activity on drug release and improve cell repair under electrical stimulation. Expanding drug release capabilities to electrically assisted cell repair Mechanical strength and stability The combination of dopamine and hyaluronic acid enhances the mechanical strength of hydrogels, but its effects are still limited. Polypyrrole coating / Contains components that enhance overall stability and material durability The polypyrrole coating / contains elements that enhance the material's mechanical strength and stability. Application of research findings Primarily used in drug delivery research for brain diseases. Further development of wound dressings in the biomedical field Expanding applications to promote wound healing and cell repair

[0078] 2. Silk Fibroin

[0079] In this embodiment, the effects of the inventors’ previous research on pure silk fibroin (SF) are then compared, namely “Biomaterial-induced conversion of quiescent cardiomyocytes into pacemaker cells in rats.” The contents of that literature are incorporated herein by reference in their entirety.

[0080] This technology explores how to use silk fibroin (SF) to induce ventricular myocytes (VMs) to transform into rhythmic sinoatrial node-like cells as a biological alternative to traditional electronic pacemakers. Therefore, it not only does not produce a hydrogel, but its effects and purposes also differ from this invention. Furthermore, its effect of promoting the "conversion" of myocytes into sinoatrial node-like cells is medically significantly different from the cell function conversion effect of this invention, which promotes cell activity and cell proliferation.

[0081] Comprehensive Comparison

[0082] Compared to the inventors' prior art, the present invention adds polypyrrole to the polydopamine-silk fibroin hydrogel, thereby increasing the conductivity of the hydrogel. The SF hydrogel of the present invention itself possesses certain electrical conductivity, while the SDP hydrogel exhibits multifunctional properties, including higher conductivity and a significant photothermal conversion effect. Furthermore, in electrical stimulation experiments, the SDP hydrogel also showed the highest cell activity, further supporting its advantage in promoting cell proliferation. The increased conductivity of the present invention enables it to exhibit stronger cell activity compared to ordinary silk fibroin and common hyaluronic acid / dopamine / silk hydrogel (HDS hydrogel), thus achieving a greater effect on promoting cell activation and proliferation that is unattainable by prior art.

[0083] Conclusion

[0084] The results of the study showed that silk fibroin hydrogels possess electrical conductivity, while silk / polypyrrole / polydopamine hydrogels exhibited multifunctional properties, including high conductivity and a significant PTR effect. Furthermore, in the electrostimulation experiment, silk / polypyrrole / polydopamine hydrogels showed the highest cell viability. These findings indicate that silk / polypyrrole / polydopamine hydrogels have significant potential to accelerate wound healing. In the electrostimulation migration experiment, it was found that cell migration speed was accelerated under a simulated endogenous electric field (Figure 12), suggesting that the highly conductive silk / polypyrrole / polydopamine hydrogels can promote the conduction of the endogenous electric field. This effect is further confirmed by the high cell viability exhibited by the silk / polypyrrole / polydopamine hydrogels under electrostimulation. Therefore, these findings indicate that silk / polypyrrole / polydopamine hydrogels have the potential to accelerate wound healing.

[0085] The above experimental data are preliminary experimental results obtained under specific conditions, and are only used to facilitate understanding or reference of the technical content of the present invention. Further related experiments are required. The experimental data and results are not intended to limit the scope of the present invention.

[0086] The foregoing preferred embodiments are merely illustrative of the present invention and its technical features. The technology of these embodiments can still be implemented by various substantially equivalent modifications and / or substitutions. Therefore, the scope of the present invention shall be determined by the scope defined in the appended claims. [Simplified Explanation of the Diagram]

[0087] Figure 1 is a simplified flowchart of the preparation process of each hydrogel of the silk fibroin complex of the present invention. Figure 2 shows the visual appearance of each hydrogel of the silk fibroin complex of the present invention. Figure 3 shows the SEM results of each hydrogel of the silk fibroin complex of the present invention. Figure 4 shows the FT-IR results of each hydrogel of the silk fibroin complex of the present invention. Figure 5 shows the water content of each hydrogel of the silk fibroin complex of the present invention. Figure 6 shows the swelling rate of each hydrogel of the silk fibroin complex of the present invention. Figure 7 shows the comparison of LED brightness of the connection circuit of each hydrogel of the silk fibroin complex of the present invention. Figure 8 shows the conductivity of each hydrogel of the silk fibroin complex of the present invention. Figure 9 shows the temperature change of each hydrogel of the silk fibroin complex of the present invention under near-infrared light irradiation. Figure 10 shows the cell compatibility of extracts of different proportions of different silk fibroin complexes (hydrogels). Figure 11 shows the cell viability of L929 cells on different silk fibroin complexes (hydrogels) after 3 days under electrical stimulation. Figure 12 shows the fluorescence staining results of cells in the electrically stimulated and unstimulated groups of the various hydrogels of the silk fibroin complex of the present invention. Figure 13 shows the in vitro scratch test results of the various hydrogels of the silk fibroin complex of the present invention. Figure 14 shows the fucoidan (FC) release curves of the various hydrogels of the silk fibroin complex of the present invention. [Biomaterial Storage]

[0089] None

Claims

1. A silk fibroin complex comprising a silk fibroin hydrogel (SF hydrogel), a silk / polydopamine hydrogel (SD hydrogel), a silk / polypyrrole hydrogel (SP hydrogel), or a silk / polypyrrole / polydopamine hydrogel (SDP hydrogel), wherein the SF hydrogel comprises 93-94% ddH2O, 5.95-6.95% silk solution, and 0.01-0.1% horseradish peroxidase (HRP); wherein the SD hydrogel comprises 93-94% ddH2O, 0.15-0.25% polydopamine (PDA), 5.8-6.8% silk solution, and 0.01-0.1% HRP; wherein the SP hydrogel comprises 93-94% ddH2O, 5.8-6.8% silk solution, 0.01-0.1% HRP, and polypyrrole... 0.15~0.35%; wherein the SDP hydrogel contains 0.1~0.3% polydopamine (PDA), 92.5~93.5% ddH2O, 5.8~6.8% silk solution, 0.01~0.1% HRP and 0.15~0.35% polypyrrole.

2. A method for preparing the silk protein complex as described in claim 1, comprising: a silk washing step, in which silkworm cocoons are added to a solution of Na2CO3 and ddH2O at 100-120 °C and rotated at 250 rpm for 30 minutes; after boiling, the cocoons are rubbed with ddH2O and then dried for 24 hours to obtain silk; and a silk dissolving step, in which ddH2O is added to LiBr and stirred at 200 rpm to dissolve it evenly to prepare a 9.3 M LiBr solution; the LiBr solution is then placed in a water bath at 85 °C; the dried silk is then added in batches to the LiBr solution and stirred at 200 rpm; after all the silk has been added, the mixture is stirred at 85 °C and 200 rpm for 1 hour to form a first silk solution. The process involves several steps:

1. Impurity removal: After stirring, the first silk solution is cooled to room temperature and centrifuged. The supernatant is then filtered under vacuum to obtain a second silk solution with impurities removed.

2. Dialysis: The second silk solution is placed in a dialysis bag with a MWCO of 6-8 kD. The bag is then immersed in 2.5 L of ddH2O for dialysis at room temperature with stirring at 150 rpm (water temperature not exceeding 30°C). Clean ddH2O is replaced repeatedly, and dialysis continues until the pH of the second silk solution in the dialysis bag reaches approximately 7.

0.

3. Concentration: The dialysis bag is immersed in a 15% PEG (Polyethylene glycol) solution with stirring at 150 rpm for concentration. After concentration for 20 hours at room temperature, the concentration is measured to be higher than 15%. The silk solution is then removed, dispensed, and stored at -20°C. If stored at ℃, the desired third silk solution can be obtained. The post-processing steps for the silk solution are as follows: Take 560 μL of ddH2O, 320 μL of the prepared 20% third silk solution, 50 μL of 1 U / μL HPR, and 70 μL of 0.64% H2O2, mix them in the dark, and react them in an incubator at 37℃ in the dark for 1 hour to form a gel, thus obtaining a silk protein hydrogel (SF hydrogel).

3. The method for preparing the silk protein complex as described in claim 2, wherein if the silk protein complex is a silk / polydopamine hydrogel (SD hydrogel), the post-treatment step of the silk solution is changed to mixing 250 μL of ddH2O with 2 mg of 2% Dopamine and 30 μL of 1N NaOH, and reacting in a water bath at 37°C in the dark for 1 hour to polymerize and form polydopamine (PDA); then, the PDA is mixed with 25 μL of 1N pH 7 HCl, 205 μL of ddH2O, 320 μL of the third silk solution at 20% concentration, 100 μL of 1U / μL HPR, and 70 μL of 1.29% H2O2 in the dark, and reacted in an incubator at 37°C in the dark for 1 hour to form a gel, thereby obtaining a silk / polydopamine hydrogel.

4. The method for preparing the silk fibroin complex as described in claim 2, wherein if the silk fibroin complex is a silk / polypyrrole hydrogel (SP hydrogel), the post-treatment step of the silk solution is changed to preparing 75 mL of phosphate (PBS) solution with a pH of 4.5; adding 0.2 g (207 μL) of pyrrole and stirring at 320 rpm at 4°C to obtain a pyrrole solution; adding the silk fibroin hydrogel to the pyrrole solution and stirring continuously; adding 0.816 g of Ammonium persulphate (APS) to 25 mL of ddH2O to prepare an APS solution as an oxidant (mole ratio pyrrole:APS = 1:1.2); slowly adding the APS solution to the pyrrole solution containing the hydrogel to induce a polymerization reaction to form polypyrrole; and stirring continuously at 320 rpm at 4°C for 2 hours. After hr, remove the hydrogel and rinse with ddH2O to remove unfixed polypyrrole, thus obtaining a silk / polypyrrole hydrogel.

5. The method for preparing the silk fibroin complex as described in claim 3, wherein if the silk fibroin complex is a silk / polypyrrole / polydopamine hydrogel (SDP hydrogel), the post-treatment step of the silk solution is modified as follows: 75 mL of phosphate (PBS) solution with pH 4.5 is prepared; 0.2 g (207 μL) of pyrrole is added and stirred at 320 rpm at 4°C to obtain a pyrrole solution; the silk / polydopamine hydrogel is added to the pyrrole solution and stirred continuously; 0.816 g of Ammonium persulphate (APS) is added to 25 mL of ddH2O to prepare an APS solution as an oxidant (mole ratio pyrrole:APS = 1:1.2); the APS solution is slowly added to the pyrrole solution containing the hydrogel to induce a polymerization reaction to form polypyrrole; and the mixture is stirred continuously at 320 rpm at 4°C for 2 hours. After hr; remove the hydrogel and rinse with ddH2O to remove unfixed polypyrrole, and a silk / polypyrrole / polydopamine hydrogel can be obtained.

6. Use of a silk protein complex as described in claim 1 for promoting wound healing, the silk protein complex being able to promote cell activity and stimulate cell regeneration.

7. The use of the silk protein complex as described in claim 6 for promoting wound healing, wherein the silk protein complex can be applied by means of application such as patches, ointments, gels, or microneedles.

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