Silk fibroin hydrogel wound dressing as well as preparation method and application thereof

Abstract

The invention relates to the technical field of wound dressings, and discloses a silk fibroin hydrogel wound dressing as well as a preparation method and application thereof. The preparation method of the silk fibroin hydrogel wound dressing comprises the following steps: S1, degumming silk fibroin to obtain silk fibroin fibers; s2, carrying out allyl grafting modification on the silk fibroin fibers; s3, carrying out phenylboronic acid grafting modification on the silk fibroin subjected to grafting modification in the step S2; and S4, adding a photoinitiator into the product obtained in the step S3, and gelling through ultraviolet irradiation to obtain the silk fibroin hydrogel wound dressing. The preparation method of the hydrogel wound dressing is simple and easy to operate. The prepared hydrogel wound dressing not only has a porous three-dimensional structure and good biocompatibility, but also is natural, non-toxic and good in adhesion. Due to the introduced dynamic boric acid bonds, the hydrogel can carry polyhydroxy drugs, quantitative drugs can be released according to the environmental requirements of wounds, and the hydrogel has relatively high glucose sensitivity and responsiveness.

Description

Technical Field

[0001] The present invention relates to the technical field of wound dressings, and particularly relates to a silk fibroin hydrogel wound dressing, a preparation method thereof, and an application thereof. Background Art

[0002] Diabetic skin ulcers are a serious diabetic complication. The mechanism of diabetic ulcers is relatively complex and there is currently no clear mechanism. Approximately 20% of moderate to severe ulcer wounds with infection will lead to a certain degree of amputation. Therefore, diabetic skin ulcers are also considered to be a highly destructive complication for diabetic patients. During the healing process of diabetic skin ulcers, multiple factors interact, including an unbalanced inflammatory response caused by the excessive recruitment of neutrophils, phagocytes, and pro-inflammatory factors; an imbalance between oxidation and antioxidant effects caused by the excessive accumulation of reactive oxygen species; persistent hyperglycemia; wound hypoxia and nutritional deficiency caused by lack of angiogenesis; and wound infection caused by invading pathogens. All these factors prolong the course of diabetic skin ulcers and the wounds do not heal for a long time.

[0003] Currently, the standard treatments applied to improve diabetic wounds include debridement, improved blood glucose regulation, infection control, and moist dressings to help the wounds heal and reconstruct blood circulation. In order to accelerate the wound healing process, a variety of biocompatible modified bioactive materials have been developed as moist dressings to carry wound-healing drugs. However, simple bioactive hydrogel dressings cannot specifically solve the environmental problems that occur during the wound healing process. Summary of the Invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides a silk fibroin hydrogel wound dressing, a preparation method thereof, and an application thereof. The preparation method of the hydrogel wound dressing of the present invention is simple and easy to operate. The prepared hydrogel wound dressing not only has a three-dimensional porous three-dimensional structure and good biocompatibility, is naturally non-toxic and has good adhesiveness. Moreover, the introduced dynamic borate bond enables the hydrogel dressing to continuously release drugs according to the blood glucose environment of the wound after carrying the drugs, has high glucose sensitivity and responsiveness, and can more specifically relieve problems such as inflammation, bacterial infection, and oxidative stress in the wound environment, and promote the healing of ulcer wounds.

[0005] A first object of the present invention is to provide a preparation method of a silk fibroin hydrogel wound dressing, comprising the following steps:

[0006] S1. Degumming the silk fibroin to obtain silk fibroin fibers;

[0007] S2. Performing allyl graft modification on the silk fibroin fibers;

[0008] S3. Then performing phenylboronic acid graft modification on the silk fibroin graft-modified in step S2;

[0009] S4. Add a photoinitiator to the product obtained in step S3 and carry out ultraviolet light curing to form a gel, and then a silk fibroin hydrogel wound dressing can be obtained.

[0010] Silk fibroin is a natural bioactive compound extracted from silkworm cocoons. Due to its good mechanical properties, biocompatibility, biodegradability and other characteristics, it can be processed into various forms such as hydrogels, electrospinning, and porous scaffolds. Silk fibroin hydrogels can activate fibroblast activity and proliferation, activate immune response during the wound healing process, and have strong antibacterial, antioxidant and anti-inflammatory potentials. Since about 1 / 4 of the polar amino acids are contained in the peptide chain of silk fibroin, bioactive groups are introduced by chemical or physical methods to endow it with specific functions.

[0011] Different from the hydrogel microneedle dressing which is a composite system formed by repeatedly drying and stacking different gel solutions, the Silkma-PBA hydrogel wound dressing of the present invention starts from the chemical synthesis path. First, silk fibroin is modified with glycidyl methacrylate (GMA) to make it have the property of in-situ gelation under ultraviolet light. On this basis, phenylboronic acid is grafted onto silk fibroin to obtain a hydrogel system with silk fibroin as the substrate and carrying dynamic borate bonds to achieve glucose response. The hydrogel wound dressing prepared by the present invention can release the carried drug according to the blood glucose environment requirements of the wound, promoting rapid wound healing.

[0012] Contact angle and swelling tests were carried out on both Silkma grafted and modified in step S2 and Silkma-PBA hydrogel grafted and modified in step S3. The results showed that the addition of phenylboronic acid greatly improved the hydrophilicity of the hydrogel, making Silkma-PBA a high-moisture-retaining type of hydrogel dressing. In clinical applications, the hydrophilicity of the wound dressing can keep the wound moist, and at the same time can absorb wound exudate, reducing the risk of bacterial infection.

[0013] According to a preferred embodiment of the present invention, in step S1, the degumming treatment is carried out with sodium carbonate for degumming;

[0014] And / or, the silk fibroin needs to be cut into pieces before degumming treatment.

[0015] According to a preferred embodiment of the present invention, in step S2, the steps of acryloyl graft modification are: graft-modify the silk fibroin fibers treated in step S1 with glycidyl methacrylate (GMA), and then carry out dialysis and freeze-drying to obtain Silkma.

[0016] According to a preferred embodiment of the present invention, in step S3, the step of grafting and modifying with phenylboronic acid is as follows: reacting Silkma with phenylboronic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS), and then performing dialysis and freeze-drying to obtain Silkma-PBA.

[0017] Specifically, the reaction mechanism of step S3 is to activate the carboxyl group through EDC and NHS, making it easier for the carboxyl group of Silkma to undergo a coupling reaction with the amino group of 3-aminophenylboronic acid. The specific role is that EDC reacts with the carboxyl group to generate a reactive intermediate. This intermediate has high reactivity and can undergo a coupling reaction with the amino group to form an amide bond. NHS reacts with the reactive intermediate generated by EDC to form an NHS ester intermediate. This intermediate is more stable than the original carboxyl group and can improve the yield and efficiency of the reaction.

[0018] According to a preferred embodiment of the present invention, the specific steps of the degumming treatment are as follows: boiling and degumming the shredded silk with a sodium carbonate solution, and drying to obtain the silk fibroin fiber.

[0019] According to a preferred embodiment of the present invention, in step S2, before the grafting and modification with propenyl, a drying and dissolving step is also performed; the drying and dissolving step is as follows: dissolving the dried silk fibroin fiber in a 9.5M - 11M lithium bromide solution at 60 - 80°C for 1 - 2 hours until the silk fibroin fiber is dissolved.

[0020] And / or, the mass ratio of the silk fibroin fiber to the volume of glycidyl methacrylate (GMA) is 10:(1 - 5), preferably 10:(2 - 3);

[0021] And / or, the reaction temperature for the grafting and modification with propenyl is 60 - 80°C, and the reaction time is 2 - 5 hours;

[0022] And / or, after the grafting and modification with propenyl, dialysis with deionized water is performed for 3 - 5 days, changing the water every 3 - 5 hours, and after dialysis, freeze-drying is performed with a freeze dryer.

[0023] The mass ratio of the silk fibroin fiber to the volume of glycidyl methacrylate (GMA) is 10:

[0024] (1 - 5), that is, 10 g of silk fibroin fiber requires 1 ml - 5 ml of glycidyl methacrylate; 20 g of silk requires 2 ml - 10 ml of glycidyl methacrylate.

[0025] According to a preferred embodiment of the present invention, in step S3, the mass ratio of Silkma to phenylboronic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) is 1:0.96:0.115:0.4;

[0026] And / or, before the graft modification of phenylboronic acid, Silkma is mixed with deionized water to prepare a Silkma solution; the mass ratio of Silkma to deionized water is 1:200;

[0027] And / or, the graft modification reaction of phenylboronic acid is carried out at room temperature, and the reaction time is 12 - 72 h, preferably 24 h;

[0028] And / or, after the graft modification of phenylboronic acid, it is also dialyzed with deionized water for 1 - 3 days, and the water is changed every 3 - 5 hours. After dialysis, it is freeze-dried by a freeze dryer.

[0029] According to a preferred embodiment of the present invention, in step S4, the photoinitiator is lithium phenyl(2,4,6-trimethylbenzoyl)phosphate (LAP);

[0030] And / or, the addition amount of LAP is 1 - 20 wt‰, preferably, the addition amount is 2 wt‰.

[0031] The present invention selects amino-phenylboronic acid modification. The reaction occurs between the amino group of phenylboronic acid and the carboxyl group of silk fibroin. It can also be replaced with carboxyl-phenylboronic acid, that is, the carboxyl group of phenylboronic acid reacts with the amino group of silk fibroin. In theory, replacing carboxyl-phenylboronic acid can also produce Silkma-PBA hydrogel.

[0032] In a certain embodiment of the present invention,

[0033] Step (1): Cut the silk into pieces and degum it with sodium carbonate to obtain silk fibroin fibers;

[0034] Step (2): Graft-modify the silk fibroin fibers with glycidyl methacrylate (GMA), and after dialysis and freeze-drying, obtain Silkma;

[0035] Step (3): Dissolve the freeze-dried Silkma powder in deionized water to form a Silkma solution, add phenylboronic acid (PBA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to react, and then carry out dialysis and freeze-drying to obtain Silkma-PBA;

[0036] Step (4): Dissolve the freeze-dried Silkma-PBA in deionized water, add the photoinitiator lithium phenyl(2,4,6-trimethylbenzoyl)phosphate (LAP), and irradiate with ultraviolet light to form a gel to obtain the glucose-responsive silk fibroin hydrogel wound dressing Silkma-PBA.

[0037] Preferably, the specific process of step (1) is as follows: Boil and degum the shredded silk cocoons with a sodium carbonate solution, and obtain silk fibroin fibers after drying.

[0038] Preferably, the specific process of step (2) is as follows: Dissolve the dried silk fibroin fibers in a 9.5 M lithium bromide solution at 80 °C, react for 2 hours, and after the silk fibroin fibers are dissolved, add glycidyl methacrylate in a ratio of 10:3 (by mass of silk fibroin fibers to volume of glycidyl methacrylate), react at a temperature of 60 °C for 3 hours.

[0039] Further, the dialysis and freeze-drying are specifically as follows: Pour the solution after the reaction into a dialysis bag, dialyze with deionized water for 4 days, change the water every 3 - 5 hours, and after dialysis, freeze-dry with a freeze-dryer for 5 days to obtain Silkma for standby.

[0040] Preferably, the specific process of step (3) is as follows: Stir and dissolve the freeze-dried Silkma powder in a ratio of 1 g of Silkma to 200 ml of deionized water, and then stir and react the Silkma, EDC, NHS, and 3-aminophenylboronic acid at a ratio of 1 g:0.96 g:0.115 g:0.4 g at room temperature for 1 day.

[0041] Further, the dialysis and freeze-drying are specifically as follows: After the reaction, pour it into a dialysis bag, dialyze with deionized water for 2 days, change the water every 3 - 5 hours, and after dialysis, freeze-dry with a freeze-dryer for 5 days to obtain Silkma-PBA for standby.

[0042] Preferably, the specific process of step (4) is as follows: Dissolve the freeze-dried Silkma-PBA powder in deionized water at a concentration of 10%, add 2 wt‰ of LAP, and irradiate with ultraviolet light for 2 minutes to form a gel.

[0043] The second object of the present invention is to provide a silk fibroin hydrogel wound dressing prepared by the above preparation method.

[0044] The third object of the present invention is to provide the silk fibroin hydrogel wound dressing, which is mixed with a drug solution and used for the treatment of diabetic skin ulcer wounds.

[0045] Different from the physical preparation method of layered drying with multifunctional hydrogel microneedles (that is, layer A is dried in a mold first, and then solution B is added for drying to obtain the product of A + B), the Silkma-PBA hydrogel in the present invention is prepared by chemical synthesis, that is, the carboxyl group is activated by EDC and NHS to react with the amino group, and phenylboronic acid and silk fibroin are chemically coupled to obtain a hydrogel product with glucose-responsive function (that is, solutions A and B are mixed to obtain product C through a chemical reaction). Moreover, the medical dressing involved in the present invention is applied to diabetic skin ulcer wounds. Considering the irregularity of the wounds, the hydrogel microneedle mold does not have the function of arbitrarily modifying the shape and size. However, the GMA added by modifying the Silkma-PBA hydrogel can utilize the characteristic of in-situ gelation to fully cover the wound surface in the solution state first, and then ultraviolet light irradiation is carried out to make the solution become an immobile gel state, achieving full coverage of the wound. As a wound dressing, it is not easy to displace and can better release the wound-healing drugs carried by the hydrogel.

[0046] The Silkma-PBA hydrogel of the present invention is designed specifically for diabetic skin ulcer wounds, and the drugs carried are mainly anti-inflammatory and antibacterial drugs that promote wound healing. The reaction between phenylboronic acid and glucose is used for individualized drug delivery, and the drug dosage is intelligently adjusted according to the actual blood glucose level of the wounds of different patients. The difference from drug delivery to diabetic patients through microneedles is that the purpose of microneedles is to carry hypoglycemic drugs such as insulin to achieve self-regulated drug delivery through the reaction between phenylboronic acid and glucose, so as to avoid risks such as hypoglycemic reactions, pain during subcutaneous injection of insulin pens, and skin damage existing in the self-administered drugs of diabetic patients.

[0047] Compared with microneedle preparations, the characteristic of in-situ gelation of Silkma-PBA under ultraviolet light irradiation can avoid the displacement of drugs, enable the drugs to completely cover the wound, and continuously release; compared with drug delivery methods such as microneedle patches, the Silkma-PBA hydrogel can be easily wiped off during the process of changing drugs and will not cause secondary damage.

[0048] The beneficial effects of the present invention are as follows: The preparation process of the hydrogel wound dressing of the present invention is mature, the operation is convenient, and the yield is relatively high. The prepared hydrogel wound dressing has good biocompatibility, flexibility, compressibility and other properties, and is an excellent wound dressing. Moreover, the three-dimensional porous structure of the hydrogel and the introduced dynamic borate bond enable the hydrogel to carry drugs and release glucose-sensitive drugs in response to the wound blood glucose environment in actual applications. Description of the Drawings

[0049] Figure 1 is the reaction formula of Example 1 of the present invention ( Figure 1a is the reaction formula of step S2; Figure 1b is the reaction formula of step S3);

[0050] Figure 2 Morphological feature diagram of the hydrogel dressing prepared in Example 1;

[0051] Figure 3 SEM scanning electron micrograph of Example 3;

[0052] Figure 4 Nuclear magnetic resonance spectra of the raw materials and products of Example 3;

[0053] Figure 5 Infrared spectra of Silkma and Silkma-PBA prepared in Example 3;

[0054] Figure 6 Contact angle test diagrams of Silkma and Silkma-PBA prepared in Example 3;

[0055] Figure 7 Swelling property test diagrams of Silkma and Silkma-PBA prepared in Example 3 at 15 wt% and 20 wt%;

[0056] Figure 8 shows the rheological property test diagrams of Silkma and Silkma-PBA prepared in Example 4 at 15 wt% and 20 wt%;

[0057] Figure 9 Diagrams for testing the sustained-release performance and glucose-responsive performance of DHM-loaded Silkma and Silkma-PBA;

[0058] Figure 10 shows the rheological test diagrams of the hydrogels prepared in Examples 3, 4, and 5. Detailed implementation mode

[0059] The present invention will be further described below in conjunction with specific embodiments, but it does not constitute any limitation to the present invention.

[0060] In the following examples, the raw materials are all commercially available.

[0061]

Example 1

[0062] S1. Put 20 g of shredded silk into 500 ml of sodium carbonate solution with a concentration of 0.05 M, heat it in a water bath to 100 °C, boil for 30 minutes, wash the sericin on the surface of the silk and the remaining sodium carbonate with deionized water to obtain fibroin fibers, and place them in a drying oven at 30 - 40 °C for drying for later use.

[0063] S2. Put 10 g of dried silk fibroin fibers into 50 ml of lithium bromide solution with a concentration of 11 M, heat in a water bath to 60 °C, stir with a magnetic stirrer for 1 hour until the silk fibroin fibers are completely dissolved, then add 2 ml of glycidyl methacrylate, react at 60 °C, and stir for 3 hours. After the reaction, pour the solution into a dialysis bag, dialyze with deionized water for 4 days, change the water every 3 - 5 hours. After dialysis, centrifuge at 4000 rpm / min for 15 minutes with a low-speed centrifuge, retain the supernatant, freeze it at -20 °C for 1 day, and finally freeze-dry it with a freeze dryer for 5 days to obtain Silkma for standby (the reaction formula is as shown in Figure 1a shown).

[0064] S3. Dissolve 0.4 g of freeze-dried Silkma powder in 200 ml of deionized water, remove the undissolved Silkma solid, then add 0.38 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 0.05 g of N-hydroxysuccinimide, and 0.16 g of 3-aminophenylboronic acid, and stir at room temperature for 1 day. After the reaction, pour the solution into a dialysis bag, dialyze with deionized water for 2 days, change the water every 3 - 5 hours. After dialysis, centrifuge at 4000 rpm / min for 15 minutes with a low-speed centrifuge, retain the supernatant, freeze it at -20 °C for 1 day, and finally freeze-dry it with a freeze dryer for 5 days to obtain Silkma-PBA for standby (the reaction formula is as shown in Figure 1b shown).

[0065] S4. To prepare a 10% concentration hydrogel, weigh 200 mg of freeze-dried Silkma-PBA, dissolve it in 2 ml of deionized water, then add 2 wt‰ of photoinitiator LAP, and form a gel by ultraviolet light irradiation for 2 minutes to obtain a hydrogel dressing.

[0066]

Example 2

[0067] S1. Put 20 g of shredded silk into 500 ml of sodium carbonate solution with a concentration of 0.05 M, heat in a water bath to 100 °C, boil for 30 minutes, wash the sericin on the silk surface and the residual sodium carbonate with deionized water to obtain silk fibroin fibers, and place them in a drying oven at 30 - 40 °C for drying and standby.

[0068] S2. Put 10 g of dried silk fibroin fibers into 50 ml of lithium bromide solution with a concentration of 10 M, heat it in a water bath to 60 °C, stir with a magnetic stirrer for 2 hours until the silk fibroin fibers are completely dissolved, then add 2 ml of glycidyl methacrylate, react at 60 °C, and stir for 3 hours. After the reaction, pour the solution into a dialysis bag, dialyze with deionized water for 4 days, change the water every 3 - 5 hours. After dialysis, centrifuge at 4000 rpm / min for 15 minutes with a low-speed centrifuge, retain the supernatant, freeze it at -20 °C for 1 day, and finally freeze-dry it with a freeze dryer for 5 days to obtain Silkma for standby.

[0069] S3. Dissolve 1 g of freeze-dried Silkma powder in 200 ml of deionized water, remove the undissolved Silkma solid, then add 0.96 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 0.115 g of N-hydroxysuccinimide, and 0.4 g of 3-aminophenylboronic acid, and stir at room temperature for 1 day. After the reaction, pour the solution into a dialysis bag, dialyze with deionized water for 2 days, change the water every 3 - 5 hours. After dialysis, centrifuge at 4000 rpm / min for 15 minutes with a low-speed centrifuge, retain the supernatant, freeze it at -20 °C for 1 day, and finally freeze-dry it with a freeze dryer for 5 days to obtain Silkma-PBA for standby.

[0070] S4. To prepare a 10% concentration hydrogel, weigh 200 mg of freeze-dried Silkma-PBA, dissolve it in 2 ml of deionized water, then add 2 wt‰ of photoinitiator LAP, and form a gel by irradiating with ultraviolet light for 2 minutes to obtain a hydrogel dressing.

[0071]

Example 3

[0072] S1. Put 20 g of shredded silk into 500 ml of sodium carbonate solution with a concentration of 0.05 M, heat it in a water bath to 100 °C, boil for 30 minutes, wash the sericin on the surface of the silk and the remaining sodium carbonate with deionized water to obtain silk fibroin fibers, and place them in a drying oven at 30 - 40 °C for drying for standby.

[0073] S2. Put 10 g of dried silk fibroin fibers into 50 ml of lithium bromide solution with a concentration of 10 M, heat it in a water bath to 80 °C, stir with a magnetic stirrer for 2 hours until the silk fibroin fibers are completely dissolved, then add 2 ml of glycidyl methacrylate, react at 60 °C, and stir for 3 hours. After the reaction, pour the solution into a dialysis bag, dialyze with deionized water for 4 days, change the water every 3 - 5 hours. After dialysis, centrifuge at 4000 rpm / min for 15 minutes with a low-speed centrifuge, retain the supernatant, freeze it at -20 °C for 1 day, and finally freeze-dry it with a freeze dryer for 5 days to obtain Silkma for standby.

[0074] S3. Dissolve 1 g of freeze-dried Silkma powder in 200 ml of deionized water, remove the undissolved Silkma solid, then add 0.96 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 0.115 g of N-hydroxysuccinimide, and 0.4 g of 3-aminophenylboronic acid, and stir at room temperature for 1 day. After the reaction, pour the solution into a dialysis bag and dialyze with deionized water for 2 days, changing the water every 3 - 5 hours. After dialysis, centrifuge at 4000 rpm / min for 15 minutes using a low-speed centrifuge, retain the supernatant, freeze it at -20°C for 1 day, and finally freeze-dry it using a freeze dryer for 5 days to obtain Silkma-PBA for standby.

[0075] S4. To prepare a 10% concentration hydrogel, weigh 200 mg of freeze-dried Silkma-PBA, dissolve it in 2 ml of deionized water, then add 2 wt‰ photoinitiator LAP, and form a gel by irradiating with ultraviolet light for 2 minutes to obtain a hydrogel dressing.

[0076]

Example 4

[0077] The difference from Example 3 is only that in step 2, "2 ml of glycidyl methacrylate" is replaced by "3 ml of glycidyl methacrylate".

[0078]

Example 5

[0079] The difference from Example 3 is only that in step 2, "2 ml of glycidyl methacrylate" is replaced by "1 ml of glycidyl methacrylate".

[0080]

Comparative Example 1

[0081] The difference from Example 2 is only that step S3 is not included.

[0082] That is, the steps are as follows:

[0083] S1. Put 20 g of shredded silk into 500 ml of sodium carbonate solution with a concentration of 0.05 M, heat it in a water bath at 100°C for 30 minutes, wash the sericin on the silk surface and the remaining sodium carbonate with deionized water to obtain silk fibroin fibers, and place them in a drying oven at 30 - 40°C for drying and standby.

[0084] S2. Put 10 g of dried silk fibroin fibers into 50 ml of lithium bromide solution with a concentration of 10 M, heat it in a water bath to 60 °C, and stir with a magnetic stirrer for 2 hours until the silk fibroin fibers are completely dissolved. Then add 2 ml of glycidyl methacrylate, react at 60 °C, and stir for 3 hours. After the reaction, pour the solution into a dialysis bag, dialyze with deionized water for 4 days, change the water every 3 - 5 hours. After dialysis, centrifuge at 4000 rpm / min for 15 minutes with a low-speed centrifuge, retain the supernatant, freeze it at -20 °C for 1 day, and finally freeze-dry it with a freeze dryer for 5 days to obtain Silkma for standby.

[0085] S4. To prepare a 10% concentration hydrogel, 200 mg of freeze-dried Silkma needs to be weighed, dissolved in 2 ml of deionized water, and then 2 wt‰ photoinitiator LAP is added. After being irradiated with ultraviolet light for 2 minutes, a hydrogel dressing is formed.

[0086]

Application Example 1

[0087] In step S4 of Example 2, add 15.63 μmol / l dihydromyricetin solution (DHM).

[0088] That is, step S4 is: To prepare a 10% concentration hydrogel, 200 mg of freeze-dried Silkma-PBA needs to be weighed, dissolved in 2 ml of deionized water, and then 2 wt‰ photoinitiator LAP and 15.63 μmol / l dihydromyricetin (DHM) solution are added. After being irradiated with ultraviolet light for 2 minutes, a hydrogel dressing is formed.

[0089]

Comparative Application Example 1

[0090] In step S4 of Comparative Example 1, add 15.63 μmol / l dihydromyricetin solution (DHM).

[0091] That is, step S4 is: To prepare a 10% concentration hydrogel, 200 mg of freeze-dried Silkma needs to be weighed, dissolved in 2 ml of deionized water, and then 2 wt‰ photoinitiator LAP and 15.63 μmol / l dihydromyricetin (DHM) solution are added. After being irradiated with ultraviolet light for 2 minutes, a hydrogel dressing is formed.

[0092] As Figure 2 shown (morphology diagram of Silkma-PBA, Figure 2 a is in the form of freeze-dried powder, Figure 2 b is in the form of aqueous solution, Figure 2 c is in the form of gelled state), the freeze-dried powder state is the general storage state of the hydrogel material (as Figure 2 a). When using the hydrogel material, first dissolve the freeze-dried powder in water to obtain an aqueous solution (as Figure 2b). Adding a photoinitiator LAP to an aqueous solution can turn the solution into a gel state by irradiating it with ultraviolet light (such as Figure 2 c).

[0093] Such as Figure 3 is the scanning electron micrograph of Silkma-PBA prepared in Example 3, Figure 3 a is at a magnification of 500 times, Figure 3 b is at a magnification of 100 times, Figure 3 c is at a magnification of 50 times). At different magnifications, a clear three-dimensional porous structure can be seen in Silkma-PBA.

[0094]

Test Example 1 - Nuclear Magnetic Resonance Spectroscopy Test

[0095] The nuclear magnetic resonance spectra of phenylboronic acid (PBA), silk fibroin fiber (Silk), and Silkma and Silkma-PBA prepared in Example 3 were tested respectively, and the obtained spectra are as Figure 4 shown.

[0096] Such as Figure 4 shown, new peak signals appeared in the Silkma and Silkma-PBA groups at 5.5 ppm and 6.2 ppm, while no similar peaks appeared in the Silk group. These peaks indicate that the epoxy group of GMA has successfully combined with the amino group in Silk, and the two new peaks represent the vinyl signals in GMA, proving that GMA has been successfully grafted onto silk fibroin, which is the basis for achieving cross-linking and gelation under ultraviolet light irradiation. Similar multiple peak signals appeared between 7.3 ppm - 7.6 ppm in the Silkma-PBA group and the PBA group, indicating that the amino group carried by phenylboronic acid has combined with the carboxyl group in silk fibroin, proving the successful grafting of PBA, which is the basis for achieving glucose responsiveness.

[0097]

Test Example 2 - Infrared Spectroscopy Test

[0098] The infrared spectra of Silkma and Silkma-PBA prepared in Example 3 were measured respectively, and the obtained spectra are as Figure 5 shown.

[0099] The characteristic absorption peaks of the Silkma and Silkma-PBA groups were compared to judge the chemical structure differences in the synthesis. Characteristic peaks of amide groups can be found at 1641 cm -1 , 1544 cm -1 and 1241 cm -1 (the Silkma-PBA group is located at 1648 cm -1 , 1544 cm -1 and 1242 cm -1)。The characteristic B-O peak unique to PBA appears at 1386 cm of Silkma-PBA -1 ; the C=O characteristic peak is visible at 1665 cm -1 .

[0100]

Test Example 3 - Contact Angle Test

[0101] The contact angles of Silkma prepared in step S2 of Example 3 and Silkma-PBA prepared in step S3 were respectively tested (using a contact angle tester). The test results are as Figure 6 shown ( Figure 6 a is the test diagram of Silkma, Figure 6 b is the test diagram of Silkma-PBA).

[0102] As Figure 6 shown, the contact angle of the Silkma-PBA hydrogel is 47.171, and the contact angle of Silkma is 5.618. The results show that the addition of phenylboronic acid improves the hydrophilicity of the Silkma-PBA hydrogel. According to clinical observations, the hydrophilicity of wound dressings can keep the wound moist, absorb wound exudate, and reduce the risk of bacterial infection.

[0103]

Test Example 4 - Swelling Performance Test

[0104] Aqueous solutions with concentrations of 15 wt% and 20 wt% were prepared using Silkma prepared in step S2 of Example 3, and aqueous solutions with concentrations of 15 wt% and 20 wt% were prepared using Silkma-PBA prepared in step S3. The swelling performance tests were then carried out on the four solutions respectively, and the test results are as Figure 7 shown.

[0105] As Figure 7 shown, only Silkma-PBA (15%) decomposed within 48 h, while the swelling efficiency of Silkma-PBA was higher, and the sample reached the water absorption equilibrium within 12 h. Combining the results of the contact angle test, it shows that the addition of PBA makes Silkma-PBA a high-moisture type of hydrogel dressing.

[0106]

Test Example 5 - Rheological Property Test

[0107] Aqueous solutions with concentrations of 15 wt% and 20 wt% were prepared using Silkma prepared in step S2 of Example 4, and aqueous solutions with concentrations of 15 wt% and 20 wt% were prepared using Silkma-PBA prepared in step S3. The rheological property tests were carried out on the four samples, and the test results are shown in Figure 8 ( Figure 8a is the rheological diagram of the storage modulus (G’) under ultraviolet light illumination mode; Figure 8bIt is a graph of the loss modulus (G”) under ultraviolet light irradiation mode; Figure 8c It is a rheological diagram under viscosity mode). The shaded area is the stage of ultraviolet light irradiation, that is, ultraviolet light irradiation was carried out during 30 - 90 s in the test. The rise of the curve proves that crosslinking reaction occurred and the hydrogel changed from a solution state to a gel state.

[0108] As shown in Figure 8, through rheological tests in crosslinking mode and viscosity mode, it was observed that in the storage modulus (G’) and loss modulus (G”), Silkma has higher peaks than Silkma-PBA. This is because the addition of phenylboronic acid makes Silkma-PBA more hydrophilic while the texture becomes softer, which also indicates that Silkma has stronger mechanical properties. In the viscosity test, both Silkma and Silkma-PBA hydrogels showed a decrease in the viscosity of the hydrogel with the increase of shear rate, featuring shear thinning.

[0109]

Test Example 6 - Tests on Sustained Release Performance and Glucose Responsive Performance

[0110] The drug release test was carried out on the dihydromyricetin (DHM) carried in Application Example 1 and Comparative Application Example 1. The test results are as Figure 9 (Application Example 1 is Silkma-PBA-DHM; Comparative Application Example 1 is Silkma-DHM) shown. This test example is to verify whether the Silkma-PBA hydrogel can achieve the function of glucose response. Its response principle is achieved through the dynamic borate bond of phenylboronic acid, that is, phenylboronic acid in the Silkma-PBA hydrogel carries a certain drug (it can be Drug A, it can be Drug B, and in this experiment, it carried DHM). In a high-glucose environment, since phenylboronic acid is more likely to bind to glucose, phenylboronic acid will preferentially release the carried drug into the environment to achieve the binding with glucose. Theoretically, the Silkma-PBA hydrogel can change the rate of its own drug release according to the level of blood glucose in the environment.

[0111] As Figure 9As shown, in the 0 - 8h period, Silkma - DHM and Silkma - PBA - DHM showed a rapid release phase. From 8 - 48h, they were in a sustained release phase, and then entered a slow release phase. During the rapid release phase, due to the sensitivity of the dynamic borate bond of phenylboronic acid to glucose in the environment, a large amount of carried DHM was released by quickly binding to glucose. In the sustained release phase, it can be observed that the release curve of Silkma - PBA - DHM showed a uniform upward trend, while the release curve of Silkma - DHM was flatter without an obvious upward phase. At this stage, the hydrogel basically achieved swelling equilibrium. At this time, the slow - release performance of silk fibroin dominated, but due to the influence of the hydrophilicity of phenylboronic acid in Silkma - PBA - DHM, it could more widely open the polymer pores for drug release, so the release curve showed a uniform upward pattern. Finally, in the slow release phase, as the drug release time continued to extend, after 48h, the drug release entered a stable trend, and the corresponding cumulative drug release amounts could reach: Silkma - PBA - DHM (35.56%) and Silkma - DHM (11.42%) respectively. By comparing the drug release rates of Silkma and Silkma - PBA carrying the drug DHM in a high - glucose environment, it was verified that the Silkma - PBA hydrogel with phenylboronic acid did indeed release drugs faster than Silkma without phenylboronic acid in a high - glucose environment, that is, the glucose - responsive function was achievable.

[0112]

Comparative Experiment

[0113] The hydrogels prepared in Examples 3, 4, and 5 were subjected to rheological tests, and the test results are shown in Figure 10 ( Figure 10a is the rheological diagram of the storage modulus (G’) under ultraviolet light irradiation mode; Figure 10b is the loss modulus (G”) diagram under ultraviolet light irradiation mode).

[0114] As shown in Figure 10, the effects of different grafting ratios of GMA (10:1, 10:2, and 10:3) on the hydrogel were tested. The sample concentration was set to 20wt%. The rheological test results showed that Example 5 (the mass ratio of silk fibroin fiber to the volume of glycidyl methacrylate GMA was 10:1) could not achieve gelation under ultraviolet light, and the stability of the hydrogel decreased greatly and could not maintain the gel form.

[0115] In summary, based on the analysis of the test results of nuclear magnetic resonance spectroscopy and infrared spectroscopy, new chemical bonds were formed in the preparation method of the present invention, which can prove the successful preparation of the Silkma - PBA hydrogel material.

[0116] Based on the results of contact angle test, swelling property test and rheological property test, the Silkma-PBA hydrogel is more hydrophilic, has a softer texture, can absorb wound exudate, and reduce the risk of bacterial infection.

[0117] Based on the sustained-release performance and glucose-responsive performance tests, it can be proved that the Silkma-PBA hydrogel prepared by the present invention can change the rate of its own drug release according to the change of blood glucose level in the environment. The Silkma-PBA hydrogel with phenylboronic acid does release drugs faster in a hyperglycemic environment than Silk without phenylboronic acid, that is, the glucose-responsive function. Due to the influence of the glucose responsiveness of phenylboronic acid and the hydrophilicity of silk fibroin in Silkma-PBA-DHM, it achieves a specific drug release function.

[0118] For any numerical value mentioned in the present invention, if there is only a two-unit interval between any minimum value and any maximum value, it includes all values increasing by one unit each time from the minimum value to the maximum value. For example, if the amount of a component, or the value of a process variable such as temperature, pressure, time, etc. is stated as 50 - 90, it means in this specification that the values 51 - 89, 52 - 88... as well as 69 - 71 and 70 - 71 etc. are specifically listed. For non-integer values, units of 0.1, 0.01, 0.001 or 0.0001 can be appropriately considered. These are only some specifically indicated examples. In this application, in a similar manner, all possible combinations of numerical values between the listed minimum value and maximum value are considered to have been disclosed.

[0119] It should be noted that the above-described embodiments are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described by referring to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than limiting words. Modifications can be made to the present invention within the scope of the claims of the present invention as specified, and the present invention can be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein. On the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A method for preparing a silk fibroin hydrogel wound dressing, characterized in that: The following steps are involved: S1, degumming the silk fibroin to obtain silk fibroin fibers; S2, performing acrylic grafting modification on silk fibroin fibers; S3, performing phenylboronic acid grafting modification on the silk fibroin grafted and modified in step S2; S4, adding a photoinitiator to the product obtained in step S3, and irradiating the product with ultraviolet light to form a gel, thereby obtaining a silk fibroin hydrogel wound dressing.

2. The preparation method according to claim 1, characterized in that: In step S1, the degumming treatment is performed using sodium carbonate; And / or, the silk fibroin needs to be shredded before degumming.

3. The preparation method according to claim 1 or 2, characterized in that: In step S2, the step of acrylic grafting modification is: the silk fibroin fiber treated in step S1 is grafted with glycidyl methacrylate, and then dialyzed and freeze-dried to obtain Silkma.

4. The preparation method according to claim 3, characterized in that: In step S3, the step of phenylboronic acid grafting modification is: reacting Silkma with phenylboronic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide, and then dialysis and freeze-drying to obtain Silkma-PBA.

5. The preparation method according to claim 2, characterized in that: In step S1, the degumming treatment specifically comprises the following steps: degumming the shredded silk by boiling it in a sodium carbonate solution, and drying it to obtain the silk fibroin fiber.

6. The preparation method according to claim 3, characterized in that: In step S2, before the acrylic grafting modification, a drying and dissolving step is performed; the drying and dissolving step is: dissolving the dried silk fibroin fiber in a 9.5M-11M lithium bromide solution at 60-80° C., reacting for 1-2 hours, until the silk fibroin fiber is dissolved; and / or, the volume ratio of the mass of the silk fibroin fiber to glycidyl methacrylate is 10:(1-5); And / or, the reaction temperature of the propylene graft modification is 60-80°C, and the reaction time is 2-5 hours; And / or, after the propylene graft modification, the product is dialyzed with deionized water for 3-5 days, with the water changed every 3-5 hours, and then freeze-dried with a freeze dryer.

7. The preparation method according to claim 4, characterized in that: In step S3, the mass ratio of Silkma to phenylboric acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:0.96:0.115:0.4; And / or, before the phenylboronic acid grafting modification, Silkma is mixed with deionized water to prepare a Silkma solution; the mass volume ratio of Silkma to deionized water is 1:200; And / or, the phenylboronic acid grafting modification reaction is carried out at room temperature for a reaction time of 12-72 hours; And / or, after the phenylboronic acid is grafted and modified, it is dialyzed with deionized water for 1-3 days, the water is changed every 3-5 hours, and after the dialysis is completed, it is freeze-dried by a freeze dryer.

8. The preparation method according to claim 1 or 2, characterized in that: In step S4, the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt; And / or, the amount of the photoinitiator added is 1-20wt‰.

9. The silk fibroin hydrogel wound dressing prepared by the preparation method according to any one of claims 1 to 8.

10. The silk fibroin hydrogel wound dressing prepared by the preparation method according to any one of claims 1 to 8, or the silk fibroin hydrogel wound dressing according to claim 9, combined with traditional Chinese medicine monomers, for use in diabetic ulcer wounds.

Images