Preparation method of quick-response hydrogel sensitive to pH (potential of hydrogen) and ROS (reactive oxygen species)

Abstract

The invention relates to a preparation method of quick response hydrogel sensitive to pH and ROS, and belongs to the technical field of hydrogel. In order to solve the problems of easy infection, difficult healing, lack of a specific drug sustained release system for a wound and the like in an emergency, the multi-stimulation response hydrogel can better adapt to the change of the environment in a human body, and can accelerate the wound healing process. And the sustained release of specific drugs can be realized, and the common infection problem and the difficult problem of slow healing in the wound healing process can be solved. The rapid response hydrogel sensitive to pH and ROS can perform specific drug release aiming at wounds, is beneficial to improvement of treatment efficiency and reduction of pain of patients and treatment cost, and is a novel material with a potential wide application prospect. By means of the continuous drug release function of the intelligent hydrogel, rapid healing of wounds can be promoted, the infection risk is reduced, and the treatment success rate is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrogels, and in particular relates to a method for preparing a fast-responsive hydrogel that is sensitive to pH and ROS. Background Art

[0002] In daily life, wounds are formed due to skin damage caused by various external factors such as impact, cutting, friction, etc. These wounds usually destroy the integrity of the skin and cause the loss of some normal tissues. In the process of wound healing, this process can be divided into four stages: hemostasis, inflammation, proliferation and reconstruction. First of all, hemostasis is the body's rapid response to the wound. Through the aggregation of platelets and the formation of fibrin, blood loss can be stopped within a few minutes, thereby achieving initial hemostasis of the wound. However, in wound treatment in emergency situations, wounds are extremely susceptible to bacterial invasion and infection, which increases the difficulty of emergency treatment of wounds. In addition, there is currently a lack of effective specific drug sustained release systems for emergency treatment of susceptible wounds.

[0003] pH value is one of the important physical and chemical indicators of the human body. Under normal circumstances, the surface of human skin is weakly acidic, with a pH value of approximately between 4.0 and 6.0. However, once the skin is damaged, the pH value at the wound will undergo a series of complex changes due to the leakage of microvessels. When the pH value is close to the physiological pH value for bacterial growth, bacterial infection may occur, which in turn triggers an inflammatory response, resulting in a prolonged wound healing time. Reactive oxygen species (ROS) are free radicals with high oxidative activity produced by mitochondria in cells, which can hinder wound healing. ROS produced at the wound or during bacterial infection also have this property. Therefore, by preparing hydrogels with reactive oxygen scavenging function, the level of ROS on the wound surface can be effectively reduced, thereby accelerating the wound healing process. In addition, many diseases cannot achieve significant therapeutic effects by simply adjusting the pH value and reactive oxygen (ROS) level at the wound, and corresponding specific drugs must be used for treatment. Due to the complexity of the in vivo environment and the uncertainty of disease development, single stimulus-responsive hydrogels often find it difficult to achieve the expected therapeutic effect. Summary of the invention

[0004] The purpose of the present invention is to solve the problems of easy infection, difficult healing, and lack of a specific sustained drug release system for wounds in emergency situations. A method for preparing a fast-responsive hydrogel that is sensitive to pH and ROS is provided, and a multi-stimulus responsive hydrogel is obtained, which can better adapt to changes in the human body's internal environment, and can accelerate the healing process of both acute and chronic wounds. The prepared responsive hydrogel that is sensitive to pH and ROS can achieve sustained release of specific drugs, and can solve the common infection problems and slow healing problems in the wound healing process; the fast-responsive hydrogel that is sensitive to pH and ROS of the present invention can release specific drugs for wounds, which helps to improve treatment efficiency, reduce the patient's pain and treatment costs, and is a new material with potential broad application prospects. Through the sustained drug release function of this smart hydrogel, it can promote rapid healing of wounds, reduce the risk of infection, and improve the success rate of treatment.

[0005] The preparation method of the pH and ROS sensitive fast responsive hydrogel of the present invention is carried out according to the following steps:

[0006] Step 1: Preparation of aminated silk fibroin (CSF)

[0007] (1) Cut the cocoons and remove impurities from the surface;

[0008] (2) Degumming: The degumming process comprises: preparing a sodium carbonate aqueous solution with a mass fraction of 0.001% to 0.5%, wherein the mass ratio of the silkworm cocoons to the sodium carbonate aqueous solution is 1:50; placing the silkworm cocoons in the sodium carbonate aqueous solution and boiling for 25 to 35 minutes, taking out the silkworm cocoons, and washing them 2 to 4 times with deionized water at 55 to 65° C.;

[0009] Repeat the degumming process 3 to 5 times; place the degummed silk in an oven at 55 to 65° C. for 10 to 14 hours to obtain silk fibroin cotton;

[0010] (3) Weighing silk cotton, mixing calcium chloride, water and silk cotton, the mass ratio of silk cotton, calcium chloride and water is 1:15.85:20; boiling for 8 to 12 minutes, cooling and filtering, and then dialyzing for 2 to 4 days, replacing the water with deionized water every 6 to 9 hours, putting the dialyzed solution into a centrifuge tube, centrifuging it at 2 to 6°C for 15 to 25 minutes in a low-temperature centrifuge, and the centrifuge speed is 7500 to 8500 rpm; then filtering to remove solid insolubles and taking the supernatant, freezing the supernatant at -15 to -25°C, and finally freeze-drying to obtain silk fibroin (SF);

[0011] (4) dissolving 580-620 mg of 1,6-hexanediamine in 2-4 mL of deionized water, and adjusting the pH to 6.3-6.7 to obtain a 1,6-hexanediamine solution;

[0012] (5) dissolving 190-210 mg of silk fibroin in 8-12 mL of deionized water to obtain a silk fibroin aqueous solution;

[0013] (6) adding the prepared 1,6-hexanediamine solution dropwise to the stirred silk fibroin aqueous solution, and then adding 190-210 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and reacting for 5-7 hours;

[0014] (7) 190-210 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, and the reaction solution was stirred at room temperature overnight;

[0015] (8) filtering with filter paper, dialyzing, and freeze-drying to obtain the aminated silk fibroin;

[0016] Step 2: Preparation of aminated silk fibroin grafted with dihydrocaffeic acid (CSF-DA)

[0017] (1) taking the prepared aminated silk fibroin obtained in step 1 and dissolving it in deionized water to obtain an aminated silk fibroin aqueous solution with a concentration of 95 to 105 mg / mL;

[0018] (2) adding dihydrocaffeic acid (DA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (NHS) to the aqueous solution of aminated silk fibroin to obtain a mixed solution, adjusting the pH to 5.0-6.0, stirring and reacting for 11-13 hours to obtain aminated silk fibroin grafted with dihydrocaffeic acid (CSF-DA);

[0019] The molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1:1;

[0020] The molar ratio of dihydrocaffeic acid to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:2;

[0021] (3) placing the aminated silk fibroin grafted with dihydrocaffeic acid in deionized water for 2 to 4 days and freeze-drying to obtain CSF-DA;

[0022] Step 3: Preparation of aldehyde-deoxyglucan (DC)

[0023] (1) 0.011-0.012 g of dextran, 0.995-1.005 g of p-carboxybenzaldehyde and 0.49-0.51 g of 4-dimethylaminopyridine were dissolved in 39-41 mL of dimethyl sulfoxide to obtain a solution;

[0024] (2) adding 1.195-1.205 g of dicyclohexylcarbodiimide to the solution and reacting at room temperature overnight;

[0025] (3) removing insoluble impurities in the reaction solution by filtration, and then gradually adding the reaction solution dropwise to a mixed solution of ethyl acetate and petroleum ether, wherein the volume ratio of ethyl acetate to petroleum ether is 10:1; diluting and dissolving the obtained white product with 90-110 mL of deionized water to obtain an aqueous solution; finally, freeze-drying the aqueous solution to obtain aldehyde-modified dextran;

[0026] Step 4: Preparation of N 1 -(4-Bromobenzyl)-N 3 -(4-Bromophenyl)-N 1 ,N 1 ,N 3 ,N 3 -Tetramethylpropane-1,3-diamine (TSPBA)

[0027] (1) Weigh 0.48-0.52 g of 4-(bromomethyl)phenylboronic acid and 0.95-1.05 g of tetramethylpropylenediamine into a round-bottom flask, add 8-12 mL of anhydrous N,N-dimethylformamide, adjust the temperature of the reaction system to 55-65° C., and react overnight;

[0028] (2) After the reaction is completed, wait for the reaction system to cool to room temperature, add 90-110 mL of tetrahydrofuran to the reaction system, and wait for a white solid to precipitate; pour out the supernatant to obtain a white solid, wash it with tetrahydrofuran, collect the white solid, and place the white solid in a vacuum drying oven to dry to obtain N 1 -(4-Bromobenzyl)-N 3 -(4-Bromophenyl)-N 1 ,N 1 ,N 3 ,N 3 -Tetramethylpropane-1,3-diamine (TSPBA);

[0029] Step 5: Preparation of pH- and ROS-sensitive responsive hydrogels (CSF / DC / CSF-DA / TSPBA hydrogels)

[0030] The aminated silk fibroin obtained in step 1, the aminated silk fibroin grafted with dihydrocaffeic acid obtained in step 2, the aldehyde-modified glucan obtained in step 3, and the N 1 -(4-Bromobenzyl)-N 3 -(4-Bromophenyl)-N 1 ,N 1 ,N 3 ,N 3-Tetramethylpropane-1,3-diamine was dissolved in PBS buffer solution to obtain CSF solution, DC solution, CSF-DA solution and TSPBA solution, and after equal volumes were vortex mixed evenly, the solution was allowed to stand at room temperature to obtain CSF / DC / CSF-DA / TSPBA hydrogel.

[0031] The beneficial effects of the present invention compared to the prior art are:

[0032] 1. The silk fibroin (SF) used in the present invention is a natural high molecular weight fibrous protein extracted from silk cocoons, which accounts for 75% of silk. Silk fibroin also has the advantages of good biocompatibility and controllable degradability. SF has been applied to food, textile, cosmetics, biomedicine and other fields in various forms such as microspheres and gels. Dextran, as one of the polysaccharide compounds, has a large number of hydroxyl groups on its molecular chain, which can be used to prepare hydrogels with different functions through modification and other steps. The present invention aminates silk fibroin and cross-links it with aldehyde-modified dextran by Schiff base reaction to obtain a hydrogel that is rapidly responsive to pH stimulation and porous and capable of loading drugs.

[0033] 2. The present invention prepares aminated silk fibroin grafted with dihydrocaffeic acid (CSF-DA) having a catechol structure, and CSF-DA and N-phenylboronic acid having a phenylboronic acid structure are 1 -(4-Bromobenzyl)-N 3 -(4-Bromophenyl)-N 1 ,N 1 ,N 3 ,N 3 -Tetramethylpropane-1,3-diamine (TSPBA) was added to the CSF / DC hydrogel. The aminated silk fibroin grafted dihydrocaffeic acid (CSF-DA) contained a certain number of amino groups, which could undergo Schiff base reaction and cross-linking with the free aldehyde groups in the aldehyded dextran. Moreover, the catechol structure in CSF-DA could react with the phenylboronic acid structure in TSPBA to generate dynamic phenylboronic acid esters. The addition of the two components enhanced the stability of the gel structure.

[0034] 3. The phenylboronic acid ester structure is sensitive to reactive oxygen species (ROS) and has ROS stimulation responsiveness; the Schiff base structure is sensitive to pH, unstable under acidic conditions, and has pH stimulation responsiveness. The CSF / DC / CSF-DA / TSPBA hydrogel with two structures has rapid responsiveness to pH and ROS stimulation, and therefore has the structural basis for loading drugs and self-responsive drug release. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the UV / visible absorbance-wavelength curve of glycine (Gly);

[0036] Figure 2 It is the linear fitting curve between the maximum absorption peak of glycine (Gly) at 348nm and the concentration of Gly;

[0037] Figure 3 is the UV / visible absorbance-wavelength curve of silk fibroin (SF);

[0038] Figure 4 is the linear fitting curve between the maximum absorption peak of silk fibroin (SF) at 348 nm and SF concentration;

[0039] Figure 5 is the UV / visible absorbance-wavelength curve of aminated silk fibroin (CSF);

[0040] Figure 6 The maximum absorption peak of aminated silk fibroin (CSF) at 348 nm and the linear fitting curve of CSF concentration;

[0041] Figure 7 This is the UV / visible absorbance-wavelength curve of aminated silk fibroin grafted with dihydrocaffeic acid (CSF-DA);

[0042] Figure 8 The maximum absorption peak of aminated silk fibroin grafted with dihydrocaffeic acid (CSF-DA) at 348 nm and the linear fitting curve of CSF-DA concentration;

[0043] Fig. 9 It is the Fourier transform infrared spectrum of aldehyde-dextran (Dextran-CHO, DC);

[0044] Fig.10 N 1 -(4-Bromobenzyl)-N 3 -(4-Bromophenyl)-N 1 ,N 1 ,N 3 ,N 3 -H NMR spectrum of tetramethylpropane-1,3-diamine (TSPBA);

[0045] Fig.11 is a scanning electron micrograph of CSF / DC / CSF-DA / TSPBA hydrogel;

[0046] Fig.12 is the pH response cumulative release curve;

[0047] Fig.13 Cumulative release curve of ROS response. DETAILED DESCRIPTION

[0048] The technical solution of the present invention is not limited to the specific implementation modes listed below, but also includes any reasonable combination of the specific implementation modes.

[0049] Specific implementation method 1: The preparation method of the pH and ROS sensitive fast responsive hydrogel in this implementation method is carried out according to the following steps:

[0050] Step 1: Preparation of aminated silk fibroin (CSF)

[0051] (1) Cut the cocoons and remove impurities from the surface;

[0052] (2) Degumming: The degumming process comprises: preparing a sodium carbonate aqueous solution with a mass fraction of 0.001% to 0.5%, wherein the mass ratio of the silkworm cocoons to the sodium carbonate aqueous solution is 1:50; placing the silkworm cocoons in the sodium carbonate aqueous solution and boiling for 25 to 35 minutes, taking out the silkworm cocoons, and washing them 2 to 4 times with deionized water at 55 to 65° C.;

[0053] Repeat the degumming process 3 to 5 times; place the degummed silk in an oven at 55 to 65° C. for 10 to 14 hours to obtain silk fibroin cotton;

[0054] (3) Weighing silk cotton, mixing calcium chloride, water and silk cotton, the mass ratio of silk cotton, calcium chloride and water is 1:15.85:20; boiling for 8 to 12 minutes, cooling and filtering, and then dialyzing for 2 to 4 days, replacing the water with deionized water every 6 to 9 hours, putting the dialyzed solution into a centrifuge tube, centrifuging it at 2 to 6°C for 15 to 25 minutes in a low-temperature centrifuge, and the centrifuge speed is 7500 to 8500 rpm; then filtering to remove solid insolubles and taking the supernatant, freezing the supernatant at -15 to -25°C, and finally freeze-drying to obtain silk fibroin (SF);

[0055] (4) dissolving 580-620 mg of 1,6-hexanediamine in 2-4 mL of deionized water, and adjusting the pH to 6.3-6.7 to obtain a 1,6-hexanediamine solution;

[0056] (5) dissolving 190-210 mg of silk fibroin in 8-12 mL of deionized water to obtain a silk fibroin aqueous solution;

[0057] (6) adding the prepared 1,6-hexanediamine solution dropwise to the stirred silk fibroin aqueous solution, and then adding 190-210 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and reacting for 5-7 hours;

[0058] (7) 190-210 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, and the reaction solution was stirred at room temperature overnight;

[0059] (8) filtering with filter paper, dialyzing, and freeze-drying to obtain the aminated silk fibroin;

[0060] Step 2: Preparation of aminated silk fibroin grafted with dihydrocaffeic acid (CSF-DA)

[0061] (1) taking the prepared aminated silk fibroin obtained in step 1 and dissolving it in deionized water to obtain an aminated silk fibroin aqueous solution with a concentration of 95 to 105 mg / mL;

[0062] (2) adding dihydrocaffeic acid (DA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (NHS) to the aqueous solution of aminated silk fibroin to obtain a mixed solution, adjusting the pH to 5.0-6.0, stirring and reacting for 11-13 hours to obtain aminated silk fibroin grafted with dihydrocaffeic acid (CSF-DA);

[0063] The molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1:1;

[0064] The molar ratio of dihydrocaffeic acid to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:2;

[0065] (3) placing the aminated silk fibroin grafted with dihydrocaffeic acid in deionized water for 2 to 4 days and freeze-drying to obtain CSF-DA;

[0066] Step 3: Preparation of aldehyde-deoxyglucan (DC)

[0067] (1) 0.011-0.012 g of dextran, 0.995-1.005 g of p-carboxybenzaldehyde and 0.49-0.51 g of 4-dimethylaminopyridine were dissolved in 39-41 mL of dimethyl sulfoxide to obtain a solution;

[0068] (2) adding 1.195-1.205 g of dicyclohexylcarbodiimide to the solution and reacting at room temperature overnight;

[0069] (3) removing insoluble impurities in the reaction solution by filtration, and then gradually adding the reaction solution dropwise to a mixed solution of ethyl acetate and petroleum ether, wherein the volume ratio of ethyl acetate to petroleum ether is 10:1; diluting and dissolving the obtained white product with 90-110 mL of deionized water to obtain an aqueous solution; finally, freeze-drying the aqueous solution to obtain aldehyde-modified dextran;

[0070] Step 4: Preparation of N 1 -(4-Bromobenzyl)-N 3 -(4-Bromophenyl)-N 1 ,N 1 ,N3 ,N 3 -Tetramethylpropane-1,3-diamine (TSPBA)

[0071] (1) Weigh 0.48-0.52 g of 4-(bromomethyl)phenylboronic acid and 0.95-1.05 g of tetramethylpropylenediamine into a round-bottom flask, add 8-12 mL of anhydrous N,N-dimethylformamide, adjust the temperature of the reaction system to 55-65° C., and react overnight;

[0072] (2) After the reaction is completed, wait for the reaction system to cool to room temperature, add 90-110 mL of tetrahydrofuran to the reaction system, and wait for a white solid to precipitate; pour out the supernatant to obtain a white solid, wash it with tetrahydrofuran, collect the white solid, and place the white solid in a vacuum drying oven to dry to obtain N 1 -(4-Bromobenzyl)-N 3 -(4-Bromophenyl)-N 1 ,N 1 ,N 3 ,N 3 -Tetramethylpropane-1,3-diamine (TSPBA);

[0073] Step 5: Preparation of pH- and ROS-sensitive responsive hydrogels (CSF / DC / CSF-DA / TSPBA hydrogels)

[0074] The aminated silk fibroin obtained in step 1, the aminated silk fibroin grafted with dihydrocaffeic acid obtained in step 2, the aldehyde-modified glucan obtained in step 3, and the N 1 -(4-Bromobenzyl)-N 3 -(4-Bromophenyl)-N 1 ,N 1 ,N 3 ,N 3 -Tetramethylpropane-1,3-diamine was dissolved in PBS buffer solution to obtain CSF solution, DC solution, CSF-DA solution and TSPBA solution, and after equal volumes were vortex mixed evenly, the solution was allowed to stand at room temperature to obtain CSF / DC / CSF-DA / TSPBA hydrogel.

[0075] This embodiment has the following beneficial effects:

[0076] 1. The silk fibroin (SF) used in this embodiment is a natural high molecular weight fibrous protein extracted from silk cocoons, which accounts for 75% of silk. Silk fibroin also has the advantages of good biocompatibility and controllable degradability. SF has been used in food, textile, cosmetics, biomedicine and other fields in various forms such as microspheres and gels. Dextran, as one of the polysaccharide compounds, has a large number of hydroxyl groups on its molecular chain, which can be used to prepare hydrogels with different functions through modification and other steps. In this embodiment, silk fibroin is aminated and cross-linked with formaldehyde dextran by Schiff base reaction to obtain a hydrogel that is rapidly responsive to pH stimulation and porous and capable of loading drugs.

[0077] 2. In this embodiment, aminated silk fibroin grafted with dihydrocaffeic acid (CSF-DA) having a catechol structure is prepared, and CSF-DA and N- 1 -(4-Bromobenzyl)-N 3 -(4-Bromophenyl)-N 1 ,N 1 ,N 3 ,N 3 -Tetramethylpropane-1,3-diamine (TSPBA) was added to the CSF / DC hydrogel. The aminated silk fibroin grafted dihydrocaffeic acid (CSF-DA) contained a certain number of amino groups, which could undergo Schiff base reaction and cross-linking with the free aldehyde groups in the aldehyded dextran. Moreover, the catechol structure in CSF-DA could react with the phenylboronic acid structure in TSPBA to generate dynamic phenylboronic acid esters. The addition of the two components enhanced the stability of the gel structure.

[0078] 3. The phenylboronic acid ester structure is sensitive to reactive oxygen species (ROS) and has ROS stimulation responsiveness; the Schiff base structure is sensitive to pH, unstable under acidic conditions, and has pH stimulation responsiveness. The CSF / DC / CSF-DA / TSPBA hydrogel with two structures has rapid responsiveness to pH and ROS stimulation, and therefore has the structural basis for loading drugs and self-responsive drug release.

[0079] Specific implementation method 2: This implementation method is different from specific implementation method 1 in that: the molecular weight cutoff of the ultrafiltration membrane used for dialysis in step 1 is 8000-14000Da.

[0080] Specific implementation method three: This implementation method is different from specific implementation methods one or two in that: the molecular weight cutoff of the ultrafiltration membrane used for dialysis in step two is 8000-14000Da.

[0081] Specific implementation method 4: This implementation method is different from any one of specific implementation methods 1 to 3 in that: the molecular weight of the dextran described in step 3 is 70,000 Da.

[0082] Specific embodiment 5: This embodiment differs from one of specific embodiments 1 to 4 in that: when washing the white solid with tetrahydrofuran in step 4, 15 to 25 mL of tetrahydrofuran is used each time, and the washing is performed 2 to 4 times.

[0083] Specific implementation method 6: This implementation method is different from any one of specific implementation methods 1 to 5 in that: the concentration of the CSF solution in step 5 is 350-500 mg / mL.

[0084] Specific implementation method 7: This implementation method is different from any one of specific implementation methods 1 to 6 in that: the concentration of the DC solution in step 5 is 190-210 mg / mL.

[0085] Specific embodiment eight: This embodiment differs from any one of specific embodiments one to seven in that: the concentration of the CSF-DA solution in step five is 190-210 mg / mL.

[0086] Specific embodiment 9: This embodiment is different from any one of specific embodiments 1 to 8 in that: the concentration of the TSPBA solution in step 5 is 190-210 mg / mL.

[0087] Specific embodiment ten: This embodiment differs from any one of specific embodiments one to nine in that: the concentrations of the CSF solution, DC solution, CSF-DA solution, and TSPBA solution in step five are 400 mg / mL, 200 mg / mL, 200 mg / mL, and 200 mg / mL, respectively.

[0088] Embodiment 1:

[0089] The preparation method of the pH and ROS sensitive fast responsive hydrogel in this embodiment is carried out according to the following steps:

[0090] Step 1: Preparation of aminated silk fibroin (CSF)

[0091] (1) Cut the cocoons, remove the impurities on the surface, and weigh 2.500 g of cocoons;

[0092] (2) Degumming: The degumming process is as follows: preparing a sodium carbonate aqueous solution with a mass fraction of 0.1%, with the mass ratio of silk cocoons to the sodium carbonate aqueous solution being 1:50; placing the silk cocoons in the sodium carbonate aqueous solution and boiling for 30 minutes, taking out the silk cocoons, and washing them three times with deionized water at 60°C;

[0093] Repeat the degumming process three times; place the degummed silk in an oven at 60°C for 12 hours to obtain silk fibroin cotton;

[0094] (3) Weighing silk cotton, mixing calcium chloride, water and silk cotton, the mass ratio of silk cotton, calcium chloride and water is 1:15.85:20; boiling for 10 minutes, cooling and filtering, and then dialyzing for 3 days, changing the water with deionized water every 8 hours, putting the dialyzed solution into a centrifuge tube, centrifuging it at 4°C for 20 minutes in a low-temperature centrifuge, and the centrifuge speed is 8000 rpm; then filtering to remove solid insolubles and taking the supernatant, freezing the supernatant at -20°C, and finally freeze-drying to obtain silk fibroin (SF);

[0095] (4) 600 mg of 1,6-hexanediamine was dissolved in 3 ml of deionized water and the pH was adjusted to 6.5 to obtain a 1,6-hexanediamine solution;

[0096] (5) dissolving 200 mg of silk fibroin in 10 mL of deionized water to obtain a silk fibroin aqueous solution;

[0097] (6) adding the prepared 1,6-hexanediamine solution dropwise to the stirred silk fibroin aqueous solution, and then adding 200 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and reacting for 6 hours;

[0098] (7) 200 mg of EDC was added, and the reaction solution was stirred at room temperature overnight;

[0099] (8) Filter through filter paper, dialyze, and finally freeze-dry to obtain CSF;

[0100] In order to detect the grafting degree of 1,6-hexanediamine, SF and CSF were tested by UV spectrophotometer. Since there is only one amino group on each molecule of glycine (Gly), glycine solutions of different concentrations were first selected to make UV / visible absorbance-concentration standard curve. The UV absorbance of solutions of different concentrations was measured respectively to obtain UV-visible absorption spectra, and the maximum absorption peak data at 348nm was read. The linear fitting curve of the maximum absorption peak data and glycine concentration can be obtained. Figure 1 and Figure 2 SF and CSF were tested using the same method. Figure 1 is the UV / visible absorbance-wavelength curve of glycine (Gly); Figure 2 It is the linear fitting curve between the maximum absorption peak of glycine (Gly) at 348nm and the concentration of Gly; Figure 3 is the UV / visible absorbance-wavelength curve of silk fibroin (SF); Figure 4 is the linear fitting curve between the maximum absorption peak of silk fibroin (SF) at 348 nm and SF concentration; Figure 5 is the UV / visible absorbance-wavelength curve of aminated silk fibroin (CSF); Figure 6The maximum absorption peak of aminated silk fibroin (CSF) at 348 nm and the linear fitting curve of CSF concentration; Figure 1 and Figure 2 Make a standard curve to compare with SF and CSF to determine the number of amino groups on each molecule; Figure 3 to Figure 6 They are SF, CSF and their maximum absorbance fitting curves respectively. Before calculating the number of amino groups per molecule, it can be seen from the absorbance on the ordinate and the change in the corresponding concentration that the number of amino groups per molecule on CSF is higher than that on SF, indicating that the amination is successful.

[0101] Three groups of identical absorbances were taken from the linear fitting curves of the maximum absorption peak at 348 nm of SF and CSF and the concentrations of SF and CSF, and they were fitted to the linear fitting curve of the maximum absorption peak of Gly at 348 nm and the concentration of Gly. The corresponding Gly concentration under the same absorbance could be obtained. The calculation showed that the average number of amino groups per molecule of SF and CSF were 26 and 152, respectively. The number of amino groups increased significantly compared with that before grafting, indicating that the amination was successful.

[0102] Step 2: Preparation of aminated silk fibroin grafted with dihydrocaffeic acid (CSF-DA)

[0103] (1) taking 0.2 g of the CSF obtained in step 1 and dissolving it in 20 mL of deionized water to obtain an aqueous solution of aminated silk fibroin with a concentration of 100 mg / mL;

[0104] (2) adding 0.226 g of dihydrocaffeic acid (DA), 0.474 g of EDC and 0.284 g of N-hydroxysuccinimide (NHS) to the aqueous solution of aminated silk fibroin to obtain a mixed solution, adjusting the pH to 5.0-6.0, stirring and reacting for 12 hours to obtain aminated silk fibroin grafted with dihydrocaffeic acid (CSF-DA);

[0105] (3) The aminated silk fibroin grafted with dihydrocaffeic acid was dialyzed in deionized water for 3 days and freeze-dried to obtain CSF-DA;

[0106] The molecular weight cut-off of the ultrafiltration membrane used for dialysis in step 1 and step 2 is 8000-14000Da;

[0107] The method for detecting the grafting degree of DA to CSF ​​is the same as the method for determining Gly, SF, and CSF. Figure 7 This is the UV / visible absorbance-wavelength curve of aminated silk fibroin grafted with dihydrocaffeic acid (CSF-DA); Figure 8 The maximum absorption peak of aminated silk fibroin grafted with dihydrocaffeic acid (CSF-DA) at 348 nm and the linear fitting curve of CSF-DA concentration; Figure 8 The vertical coordinate and Figure 7By comparing the vertical coordinates, it can be found that the absorbance of the maximum absorption peak decreases at the same concentration. By comparing with the glycine standard curve, it can be calculated that the number of amino groups on each molecule of CSF-DA is 18, which is significantly lower than that of CSF, indicating that DA is successfully grafted onto CSF.

[0108] Step 3: Preparation of aldehyde-deoxyglucan (DC)

[0109] (1) 0.0113 g of dextran, 1.001 g of p-carboxybenzaldehyde and 0.5 g of 4-dimethylaminopyridine were dissolved in 40 mL of dimethyl sulfoxide to obtain a solution; the molecular weight of the dextran was 70,000 Da;

[0110] (2) adding 1.2 g of dicyclohexylcarbodiimide to the solution and reacting at room temperature overnight;

[0111] (3) removing insoluble impurities in the reaction solution by filtration, and then gradually adding the reaction solution dropwise to a mixed solution of ethyl acetate and petroleum ether, wherein the volume ratio of ethyl acetate to petroleum ether is 10:1; diluting and dissolving the obtained white product with 100 mL of deionized water to obtain an aqueous solution; finally, freeze-drying the aqueous solution to obtain aldehyded dextran (DC);

[0112] Fig. 9 It is the Fourier transform infrared spectrum of aldehyde-dextran (Dextran-CHO, DC); Fig. 9 Medium, 3419cm -1 The absorption peak at 2928cm is the stretching vibration absorption peak of -OH. -1 The absorption peak at 1649.91 cm is the stretching vibration absorption peak of CH. -1 The absorption peak at 1701.49cm is the bending vibration absorption peak of -OH. In the infrared spectrum of aldehyde-modified dextran, an absorption peak not found on dextran can be seen, 1701.49cm -1 The absorption peak at 1726.46 cm is the stretching vibration absorption peak of C=O in aromatic aldehyde. -1 The absorption peak at is the stretching vibration absorption peak of C=O in the ester group obtained by the reaction of the hydroxyl group in dextran and the carboxyl group in p-formylbenzoic acid. Based on this characteristic absorption peak, it is fully proved that the esterification reaction is carried out and the product is successfully prepared.

[0113] Step 4: Preparation of N 1 -(4-Bromobenzyl)-N 3 -(4-Bromophenyl)-N 1 ,N 1 ,N 3 ,N 3 -Tetramethylpropane-1,3-diamine (TSPBA)

[0114] (1) Weigh 0.5 g of 4-(bromomethyl)phenylboronic acid and 0.1 g of tetramethylpropylenediamine into a round-bottom flask, add 10 mL of anhydrous N,N-dimethylformamide, adjust the temperature of the reaction system to 60°C, and react overnight;

[0115] (2) After the reaction is completed, the reaction system is cooled to room temperature, 100 mL of tetrahydrofuran is added to the reaction system, and a white solid is precipitated; the supernatant is poured out to obtain a white solid, which is washed with tetrahydrofuran and collected, and the white solid is placed in a vacuum drying oven and dried to obtain N 1 -(4-Bromobenzyl)-N 3 -(4-Bromophenyl)-N 1 ,N 1 ,N 3 ,N 3 -Tetramethylpropane-1,3-diamine (TSPBA);

[0116] When washing the white solid with tetrahydrofuran, 20 mL of tetrahydrofuran was used each time, and the washing was performed 3 times;

[0117] Fig.10 N 1 -(4-Bromobenzyl)-N 3 -(4-Bromophenyl)-N 1 ,N 1 ,N 3 ,N 3 -H NMR spectrum of tetramethylpropane-1,3-diamine (TSPBA); TSPBA was successfully synthesized in this example;

[0118] Step 5: Preparation of pH- and ROS-sensitive responsive hydrogels (CSF / DC / CSF-DA / TSPBA hydrogels)

[0119] The CSF obtained in step 1, the CSF-DA obtained in step 2, the DC obtained in step 3, and the TSPBA obtained in step 4 were respectively dissolved in PBS buffer solution to obtain CSF solution, DC solution, CSF-DA solution, and TSPBA solution, and after equal volumes were vortex-mixed, the mixture was allowed to stand at room temperature to obtain CSF / DC / CSF-DA / TSPBA hydrogel;

[0120] The concentrations of the CSF solution, DC solution, CSF-DA solution, and TSPBA solution are 400 mg / mL, 200 mg / mL, 200 mg / mL, and 200 mg / mL, respectively;

[0121] Fig.11 This is a scanning electron microscope photo of CSF / DC / CSF-DA / TSPBA hydrogel, which shows that it has a porous structure with uniform pore distribution;

[0122] CSF / DC / CSF-DA / TSPBA hydrogel loading and response to sustained release tobramycin test:

[0123] Eight groups of freshly prepared hydrogels with equal amounts of CSF / DC / CSF-DA / TSPBA were taken. Among them, four groups were made by dissolving the raw materials in PBS buffer solution containing 200μg / mL tobramycin, which were experimental groups. Tobramycin molecules contain amino structures, which can react with free aldehyde groups in aldehyde-modified dextran to form Schiff base structures, which can achieve better drug loading effects. The other four groups were directly made with PBS buffer solution, which were blank control groups to eliminate the effect of dissolution of hydrogel elements on drug release during the response process. 200μL of PBS buffer solutions with pH=7.4 and pH=5.5 and pH=7.4 and 1mmol / L H 2 O 2 , the blank control group was added with the corresponding solutions as above. Each hydrogel was centrifuged once every hour (200rpm, 10 minutes), and a small amount of supernatant from the hydrogel was taken and stored to wait for reaction, and this operation was carried out until the release was completed. Since each molecule of tobramycin contains 5 amino groups, the method for measuring amino-grouped silk fibroin and other primitives is the same as the above. The supernatants of each group of hydrogels extracted at different times in the test were measured for the maximum absorption peak data at 348nm, and the data of the experimental group and the control group were linearly fitted with the glycine standard curve. After data analysis and processing, the cumulative release curve of tobramycin was obtained, and this test operation was repeated 3 times. The pH response cumulative release curve is shown in Figure 12, and the ROS response cumulative release curve is shown in Figure 12. Fig.13 . Fig.12 and Fig.13 It can be explained that the hydrogel prepared in this embodiment can encapsulate tobramycin, is sensitive to pH and ROS, and under weakly acidic conditions, the cleavage of its own Schiff base bond will lead to accelerated release of tobramycin, which is faster than the release rate of the hydrogel under neutral pH conditions; under conditions rich in active oxygen, tobramycin will be released due to the cleavage of its own phenylboronic ester bond.

Claims

1. A method for preparing a fast-responsive hydrogel that is sensitive to pH and ROS, characterized in that: The preparation method of the pH and ROS sensitive fast responsive hydrogel is carried out according to the following steps: Step 1: Preparation of aminated silk fibroin (1) Cut the cocoons and remove impurities from the surface; (2) Degumming: The degumming process comprises: preparing a sodium carbonate aqueous solution with a mass fraction of 0.001% to 0.5%, wherein the mass ratio of the silkworm cocoons to the sodium carbonate aqueous solution is 1:50; placing the silkworm cocoons in the sodium carbonate aqueous solution and boiling for 25 to 35 minutes, taking out the silkworm cocoons, and washing them 2 to 4 times with deionized water at 55 to 65° C.; Repeat the degumming process 3 to 5 times; place the degummed silk in an oven at 55 to 65° C. for 10 to 14 hours to obtain silk fibroin cotton; (3) Weighing silk cotton, mixing calcium chloride, water and silk cotton, the mass ratio of silk cotton, calcium chloride and water is 1:15.85:20; boiling for 8 to 12 minutes, cooling and filtering, and then dialyzing for 2 to 4 days, replacing the water with deionized water every 6 to 9 hours, placing the dialyzed solution into a centrifuge tube, and centrifuging it at 2 to 6° C. for 15 to 25 minutes in a low-temperature centrifuge at a speed of 7500 to 8500 rpm; then filtering to remove solid insolubles and taking the supernatant, freezing the supernatant at -15 to -25° C., and finally freeze-drying to obtain silk fibroin; (4) dissolving 580-620 mg of 1,6-hexanediamine in 2-4 mL of deionized water, and adjusting the pH to 6.3-6.7 to obtain a 1,6-hexanediamine solution; (5) dissolving 190-210 mg of silk fibroin in 8-12 mL of deionized water to obtain a silk fibroin aqueous solution; (6) adding the prepared 1,6-hexanediamine solution dropwise to the stirred silk fibroin aqueous solution, and then adding 190-210 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and reacting for 5-7 hours; (7) 190-210 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, and the reaction solution was stirred at room temperature overnight; (8) filtering with filter paper, dialyzing, and freeze-drying to obtain the aminated silk fibroin; Step 2: Preparation of aminated silk fibroin grafted with dihydrocaffeic acid (1) taking the aminated silk fibroin prepared in step 1 and dissolving it in deionized water to obtain an aminated silk fibroin aqueous solution with a concentration of 95 to 105 mg / mL; (2) adding dihydrocaffeic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to the aqueous solution of aminated silk fibroin to obtain a mixed solution, adjusting the pH to 5.0-6.0, and stirring the mixture for 11-13 hours to obtain aminated silk fibroin grafted with dihydrocaffeic acid; The molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1:1; The molar ratio of dihydrocaffeic acid to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:2; (3) The aminated silk fibroin grafted with dihydrocaffeic acid was dialyzed in deionized water for 2 to 4 days and freeze-dried to obtain CSF-DA; Step 3: Preparation of Aldehyde Dextran (1) 0.011-0.012 g of dextran, 0.995-1.005 g of p-carboxybenzaldehyde and 0.49-0.51 g of 4-dimethylaminopyridine were dissolved in 39-41 mL of dimethyl sulfoxide to obtain a solution; (2) adding 1.195-1.205 g of dicyclohexylcarbodiimide to the solution and reacting at room temperature overnight; (3) removing insoluble impurities in the reaction solution by filtration, and then gradually adding the reaction solution dropwise to a mixed solution of ethyl acetate and petroleum ether, wherein the volume ratio of ethyl acetate to petroleum ether is 10:1; diluting and dissolving the obtained white product with 90-110 mL of deionized water to obtain an aqueous solution; finally, freeze-drying the aqueous solution to obtain aldehyde-modified dextran; Step 4: Preparation of N 1 -(4-Bromobenzyl)-N 3 -(4-Bromophenyl)-N 1 ,N 1 ,N 3 ,N 3 -Tetramethylpropane-1,3-diamine (1) Weigh 0.48-0.52 g of 4-(bromomethyl)phenylboronic acid and 0.95-1.05 g of tetramethylpropylenediamine into a round-bottom flask, add 8-12 mL of anhydrous N,N-dimethylformamide, adjust the temperature of the reaction system to 55-65° C., and react overnight; (2) After the reaction is completed, wait for the reaction system to cool to room temperature, add 90-110 mL of tetrahydrofuran to the reaction system, and wait for a white solid to precipitate; pour out the supernatant to obtain a white solid, wash it with tetrahydrofuran, collect the white solid, and place the white solid in a vacuum drying oven to dry to obtain N 1 -(4-Bromobenzyl)-N 3 -(4-Bromophenyl)-N 1 ,N 1 ,N 3 ,N 3 -Tetramethylpropane-1,3-diamine; Step 5: Preparation of pH and ROS-sensitive responsive hydrogels The aminated silk fibroin obtained in step 1, the aminated silk fibroin grafted with dihydrocaffeic acid obtained in step 2, the aldehyde-modified glucan obtained in step 3, and the N 1 -(4-Bromobenzyl)-N 3 -(4-Bromophenyl)-N 1 ,N 1 ,N 3 ,N 3 -Tetramethylpropane-1,3-diamine was dissolved in PBS buffer solution to obtain CSF solution, DC solution, CSF-DA solution and TSPBA solution, and after equal volumes were vortex mixed evenly, the solution was allowed to stand at room temperature to obtain CSF / DC / CSF-DA / TSPBA hydrogel.

2. The method for preparing a fast-responsive hydrogel sensitive to pH and ROS according to claim 1, characterized in that: The ultrafiltration membrane used for dialysis in step 1 has a molecular weight cutoff of 8000 to 14000 Da.

3. The method for preparing a fast-responsive hydrogel sensitive to pH and ROS according to claim 1, characterized in that: The molecular weight cut-off of the ultrafiltration membrane used for dialysis in step 2 is 8000-14000Da.

4. The method for preparing a fast-responsive hydrogel sensitive to pH and ROS according to claim 1, characterized in that: The molecular weight of the dextran in step 3 is 70,000 Da.

5. The method for preparing a fast-responsive hydrogel sensitive to pH and ROS according to claim 1, characterized in that: When washing the white solid with tetrahydrofuran as described in step 4, 15 to 25 mL of tetrahydrofuran is used each time, and washing is performed 2 to 4 times.

6. The method for preparing a fast-responsive hydrogel sensitive to pH and ROS according to claim 1, characterized in that: The concentration of the CSF solution in step 5 is 350-500 mg / mL.

7. The method for preparing a fast-responsive hydrogel sensitive to pH and ROS according to claim 1, characterized in that: The concentration of the DC solution in step 5 is 190-210 mg / mL.

8. The method for preparing a fast-responsive hydrogel sensitive to pH and ROS according to claim 1, characterized in that: The concentration of the CSF-DA solution in step 5 is 190-210 mg / mL.

9. The method for preparing a fast-responsive hydrogel sensitive to pH and ROS according to claim 1, characterized in that: The concentration of the TSPBA solution in step 5 is 190-210 mg / mL.

10. The method for preparing a fast-responsive hydrogel sensitive to pH and ROS according to claim 1, characterized in that: The concentrations of the CSF solution, DC solution, CSF-DA solution, and TSPBA solution in step 5 are 400 mg / mL, 200 mg / mL, 200 mg / mL, and 200 mg / mL, respectively.

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