Injectable insulin hydrogel with glucose responsiveness as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological materials, and particularly relates to an injectable insulin hydrogel with glucose responsiveness and a preparation method and application thereof. The invention develops an injectable hydrogel which has glucose responsiveness and is easy to degrade, and the injectable hydrogel is used for precisely controlled release of insulin. The hydrogel is prepared by combining silk fibroin and phenylboronic acid, based on the self-adjusting response of phenylboronic acid to glucose, the hydrogel shows specific response to the concentration of glucose in blood, and long-acting release can be achieved. Moreover, the hydrogel can be completely metabolized in vivo within a short time, has no adverse reaction in animal experiments, and shows good in-vivo applicability. Besides, the silk fibroin-phenylboronic acid-based hydrogel is simple to prepare and low in production cost, and a new method is provided for developing long-acting intelligent insulin preparations and using injectable hydrogel for glucose metabolism or / and clinical treatment of insulin dysfunction diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and more specifically, relates to an injectable insulin hydrogel with glucose responsiveness, and a preparation method and application thereof. Background Art

[0002] Diabetes is a common chronic disease worldwide, and its main feature is the increase in physiological blood glucose levels, which in turn leads to serious complications. At present, the main treatment method is still subcutaneous injection of exogenous insulin. Diabetic patients need to undergo one or more subcutaneous injections every day. Frequent injections lead to low patient compliance, and are also prone to cause local tissue necrosis, infection, nerve damage and insulin amyloidosis, and physiological glucose homeostasis cannot be achieved. In addition, due to the low solubility of insulin in fat and its easy inactivation, its bioavailability is low. In order to avoid the above problems, a variety of insulin delivery systems have been developed in recent years, including oral administration, pulmonary inhalation, nasal administration and transdermal microneedle administration. However, these methods include subcutaneous injections, and most of them are given after the blood sugar rises after a meal, which cannot respond to blood sugar changes in time to reduce blood sugar to the normal range. In addition, the dosage is not accurate, which may lead to hyperglycemia or hypoglycemia. Therefore, the development of a new type of insulin delivery system to reduce the injection frequency and achieve precise release of insulin that responds quickly to changes in blood sugar concentration is an urgent problem to be solved.

[0003] Glucose-responsive self-regulating insulin delivery system can automatically release the corresponding amount of insulin according to the blood sugar level of the human body, which is expected to overcome the drawbacks of traditional diabetes injection therapy. The reported glucose-responsive controlled-release system is mainly based on three response units, including glucose oxidase (GOx), concanavalin A (Con A), and phenylboronic acid (PBA). GOx and Con A are both protein substances, which are easily affected by environmental changes and are unstable during storage, use, and sterilization. The PBA system based on the formation of reversible covalent bonds with glucose molecules has good stability and shows a broader application prospect in the self-regulating insulin delivery system.

[0004] Injectable hydrogels have attracted extensive attention in the field of drug delivery and tissue engineering due to their advantages such as convenient administration, minimally invasive treatment, matching irregular defects and good biocompatibility. The porous morphology of hydrogels can encapsulate various drug molecules in their matrix, and then controllably release the drugs according to the diffusion coefficient of the encapsulated drugs and the pore size of the gel network. At the same time, injectable hydrogels have the advantages of minimally invasive delivery, without surgical implantation and infection risks. The development of glucose-responsive injectable hydrogels for painless and continuous delivery of insulin can further enhance blood sugar control and reduce the burden of patient compliance, which is of great significance for the treatment of diabetes. At the same time, based on the glucose responsiveness of PBA, injectable hydrogels have shear-thinning and self-healing properties, and can restore the gel state after subcutaneous injection.

[0005] In recent years, silk fibroin hydrogels have been widely used in drug delivery, tissue engineering, and regenerative medicine engineering. Silk fibroin has good biocompatibility, biodegradability, low immunogenicity, excellent physical and mechanical properties, and has been found to have great application prospects. However, silk fibroin requires a relatively long gel time (4 days) in aqueous media and is not suitable for preparing injectable hydrogels. In order to accelerate its gelation process, currently commonly used methods include vortexing, ultrasound, electric field, or with the help of additives such as surfactants, acids, salts and polymers. As in the prior art (Maity, Biswanath, Hariharan Moorthy, and Thimmaiah Govindaraju. "Glucose-Responsive Self-Regulated Injectable Silk Fibroin Hydrogel for Controlled Insulin Delivery." ACS Applied Materials & Interfaces 15.43 (2023): 49953-49963.), a glucose-responsive hydrogel based on modified phenylboronic acid and silk fibroin was prepared. The hydrogel firmly fixes the glucose sensor element and insulin in its pores, and can release insulin in a controllable manner. After injecting the above hydrogel, the blood sugar level can be maintained at a normal blood sugar level for 36 hours and then slowly increases. However, the hydrogel takes a long time (28 days) to be completely degraded, and its blood sugar level control still needs to be further improved. In addition, the above hydrogel needs to react for a long time (up to 6 hours) at room temperature, and it is also necessary to perform dialysis, and the preparation steps are complicated. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing insulin-loaded hydrogels, such as poor degradation performance (complete degradation time ≥ 28 days) and the need to further improve the blood sugar control level, and to provide an injectable insulin hydrogel with glucose responsiveness.

[0007] The purpose of the present invention is to provide a method for preparing the injectable insulin hydrogel.

[0008] Another object of the present invention is to provide applications of the injectable insulin hydrogel.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention protects an injectable insulin hydrogel with glucose responsiveness, wherein the injectable insulin hydrogel comprises a hydrogel matrix and insulin, wherein the insulin is encapsulated in the hydrogel matrix;

[0011] The hydrogel matrix is ​​constructed by phenylboronic acid and silk fibroin; the mass ratio of phenylboronic acid to silk fibroin is (2-12):1.

[0012] In order to avoid the side effects of frequent insulin injections in the treatment of diabetes, the present invention adopts a green chemical synthesis method to combine silk fibroin with phenylboronic acid to prepare a new type of biodegradable glucose-responsive injectable hydrogel for precise controlled release of insulin. Among them, phenylboronic acid can act as a cross-linking agent to accelerate its gelation by forming hydrogen bonds with silk fibroin, and can also be used as a glucose detector to specifically respond to glucose concentration. In addition, phenylboronic acid changes the cross-linked network structure of the hydrogel by forming phenylboronic acid esters with glucose, releasing the insulin encapsulated therein, regulating and maintaining the physiological level of glucose (normal postprandial blood sugar should be less than 199.8mg / dL); the good biocompatibility and biodegradability of silk fibroin enable the drug delivery system to be completely metabolized in the human body, without toxic byproducts, and has good in vivo applicability.

[0013] More preferably, the mass ratio of phenylboric acid to silk fibroin is (6-12):1.

[0014] More preferably, the mass ratio of phenylboric acid to silk fibroin is (6-8):1.

[0015] More preferably, the mass ratio of phenylboric acid to silk fibroin is 6:1.

[0016] Preferably, the mass concentration of insulin in the injectable insulin hydrogel is 0.15-1 mg / mL. An appropriate amount of insulin can be loaded according to actual conditions to achieve an effective and targeted therapeutic effect.

[0017] The invention also protects a preparation method of the injectable insulin hydrogel, comprising the following steps: mixing a phenylboric acid solution and a silk fibroin solution, adding insulin, fully mixing, and standing to obtain the injectable insulin hydrogel.

[0018] Furthermore, the phenylboric acid solution is obtained by dissolving phenylboric acid in an ethanol aqueous solution.

[0019] Furthermore, the concentration of the ethanol aqueous solution is 60-80 vol%, more preferably 70 vol%.

[0020] Preferably, the concentration of the phenylboric acid solution is 80 to 120 mg / mL, more preferably 90 to 110 mg / mL.

[0021] Furthermore, the silk fibroin can be purchased from the market or prepared by oneself.

[0022] Furthermore, the preparation method of the silk fibroin solution comprises the following steps: sequentially degumming, dissolving, purifying and filtering the raw material rich in silk fibroin to obtain the silk fibroin solution.

[0023] Preferably, the raw material rich in silk fibroin is silkworm cocoons.

[0024] Preferably, the degumming treatment method is alkali degumming, organic acid degumming, soap boiling degumming, enzymatic degumming or high temperature and high pressure water degumming.

[0025] Furthermore, the alkaline degumming is to place the silkworm cocoons in a certain concentration of alkaline reagent solution and boil them to dissolve the sericin in the solution, and then separate the silk fibroin by dialysis, centrifugation, filtration, etc. This method is simple and easy to implement and is one of the commonly used degumming methods.

[0026] Furthermore, the organic acid degumming method is to use an organic acid (such as tartaric acid) to remove the sericin from the cocoon. By adjusting the concentration of tartaric acid and the treatment conditions, the sericin can be effectively removed from the cocoon while maintaining the integrity and good mechanical properties of the silk fibroin.

[0027] Furthermore, the soap boiling degumming method utilizes the alkaline environment of neutral soap and the action of surfactant to effectively remove sericin from the cocoons. The cocoons are placed in a neutral soap solution and boiled, and the sericin is dissolved and the fibroin is separated through the steps of stirring and rinsing.

[0028] Furthermore, the enzymatic degumming method is to place the silkworm cocoons in a solution containing alkaline protease for treatment, which can gently remove sericin while maintaining the biological activity and integrity of silk fibroin. This is to utilize the specificity and selectivity of alkaline protease to selectively decompose sericin without affecting silk fibroin.

[0029] Furthermore, the high temperature and high pressure water degumming is to place the cocoons in high temperature and high pressure water for treatment, which can quickly remove sericin because the solubility of sericin will increase significantly in a high temperature and high pressure water environment.

[0030] Furthermore, as a preferred manner, the preparation method of the silk fibroin comprises the following steps: degumming the silk cocoons under alkaline conditions to obtain silk fibroin fibers, washing and drying the obtained silk fibroin fibers, adding a solvent, and dialyzing after fully dissolving them, taking the retentate for centrifugation, collecting the supernatant, and performing microporous filtration to obtain the silk fibroin fibers.

[0031] The solvent is selected from lithium bromide solution (preferably 8-10 mol / L), tetrabutylammonium hydroxide (TBAOH) aqueous solution (preferably 35-55 wt%), sodium hydroxide-urea solution (Na(OH)2-CH4N2O, the mass ratio of sodium hydroxide to urea in the solution is 1:(4-6)), calcium chloride aqueous solution (CaCl2-H2O, preferably 40-45 wt%) or calcium chloride / ethanol / water mixed solution (CaCl2-C2H6O-H2O, the molar ratio of the three is preferably 1:(1-3):(7-9)).

[0032] Preferably, the concentration of the silk fibroin solution is 40-60 mg / mL.

[0033] Furthermore, the alkaline condition is provided by an alkaline reagent, and the alkaline reagent is selected from one or more of sodium carbonate, sodium bicarbonate, sodium hydroxide, and potassium hydroxide, more preferably sodium carbonate.

[0034] Furthermore, the alkaline reagent is used at a concentration of 0.02 to 0.2 M (mol / L).

[0035] Furthermore, the molecular weight cut-off of the dialysis bag used in the dialysis is 3500-10000 MW.

[0036] Furthermore, the pore size of the micropores is 0.2-5 μm.

[0037] Furthermore, the sufficient mixing time is 0.5 to 2 minutes.

[0038] Furthermore, the standing time is 20 to 55 minutes.

[0039] The present invention also protects the use of the injectable insulin hydrogel in preparing insulin preparations.

[0040] Compared with the prior art, the present invention has the following beneficial effects: the present invention develops a glucose-responsive and degradable injectable hydrogel for precise controlled release of insulin. The hydrogel is prepared by combining silk fibroin with phenylboronic acid. Based on the self-regulating response of phenylboronic acid to glucose, it shows a specific response to the glucose concentration in the blood and can achieve long-term release, avoiding the problems of poor compliance of patients due to repeated injections of insulin and the inability to accurately control the release of insulin in the prior art. Moreover, the hydrogel can be completely metabolized in vivo, and no adverse reactions were observed in animal experiments, showing good in vivo applicability. In addition, the silk fibroin-phenylboronic acid-based hydrogel is simple to prepare and has low production cost, which provides a new method for the development of long-acting intelligent insulin preparations and the use of injectable hydrogels for clinical treatment of glucose metabolism or / and insulin dysfunction diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The data statistical graphs of texture analysis of phenylboronic acid-silk fibroin-based hydrogels prepared in Examples 1, 3, 5, 7, 9, 11, and 13 are shown; wherein A is a data statistical graph of gel strength, B is a data statistical graph of hardness, C is a data statistical graph of cohesion, and D is a data statistical graph of adhesion.

[0042] Figure 2 Statistical graph of the swelling properties of phenylboronic acid-silk fibroin-based hydrogels prepared in Examples 1, 3, 5, 7, 9, 11, and 13 at the same glucose concentration.

[0043] Figure 3 This is a statistical graph of the swelling properties of the hydrogel SFPH6 obtained in Example 7 at different glucose concentrations.

[0044] Figure 4 The data statistical graph of the in vitro biodegradability of the hydrogels obtained in Examples 1, 3, 5, 7, 9, 11, and 13.

[0045] Figure 5 This is a statistical graph of the biodegradability of the hydrogel SFPH6 obtained in Example 7 in Wistar rats.

[0046] Figure 6 This is a statistical graph of the injectability test data of the hydrogel SFPH6 obtained in Example 7.

[0047] Figure 7 This is a graph showing the insulin release curve of the drug-loaded hydrogel iSFPH6 obtained in Example 8 at different glucose concentrations.

[0048] Figure 8 The data statistics chart is a blood glucose controlled release experiment of the hydrogel SFPH6 obtained in Example 7, the drug-loaded hydrogel iSFPH6 obtained in Example 8, insulin (Insulin) and PBS in animal experiments.

[0049] Fig. 9 This is a statistical graph of the cytotoxicity test data of the hydrogel SFPH6 obtained in Example 7. DETAILED DESCRIPTION

[0050] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0051] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0052] Example 1 Phenylboronic acid-silk fibroin-based hydrogel (SFPH2) for glucose responsiveness and insulin delivery

[0053] Preparation of Phenylboronic Acid-Silk Fibroin-Based Hydrogels for Glucose Responsiveness and Insulin Delivery:

[0054] S1. Prepare solution A: Add 5g of silkworm cocoons to 0.02M sodium carbonate solution and boil for 30 minutes to remove sericin. Wash the obtained silk fibroin fibers with ultrapure water, dry them at room temperature overnight, then add 9.3M lithium bromide solution at a ratio of 20% (wt / vol), put them in a 60°C oven to dissolve for 4 hours, then transfer them to a 3500MW dialysis bag, dialyze with ultrapure water for three days, collect the supernatant by centrifugation, and filter them twice with a needle filter (pore size of 5μm) to obtain a purified silk fibroin solution. The purified silk fibroin solution is diluted to 50mg / mL with ultrapure water.

[0055] S2. Prepare Solution B: Dissolve 5 g of phenylboric acid in 50 mL of 70% ethanol solution to prepare a 100 mg / mL phenylboric acid solution.

[0056] S3. Preparation of glucose-responsive hydrogel: 1 mL of solution A was mixed with 1 mL of solution B, and the mixture was evenly pipetted and allowed to stand for 50 to 55 min to obtain a hydrogel (SFPH2) with a phenylboronic acid:silk fibroin mass ratio of 2:1.

[0057] Example 2 An injectable insulin hydrogel with glucose responsiveness (iSFPH2)

[0058] Preparation of hydrogels loaded with fluorescently labeled insulin (FITC-insulin):

[0059] S1. Prepare liquid A: same as step S1 of Example 1.

[0060] S2. Prepare liquid B: same as step S2 of Example 1.

[0061] S3. Preparation of FITC-insulin-loaded hydrogel: Mix 1 mL of solution A with 1 mL of solution B, add 1.25 mg of FITC-insulin, pipette and mix evenly, let stand for 50 to 55 min to obtain a drug-loaded hydrogel (iSFPH2) with a phenylboronic acid:silk fibroin mass ratio of 2:1.

[0062] Example 3 Phenylboronic acid-silk fibroin-based hydrogel (SFPH3) for glucose responsiveness and insulin delivery

[0063] Preparation of Phenylboronic Acid-Silk Fibroin-Based Hydrogels for Glucose Responsiveness and Insulin Delivery:

[0064] S1. Prepare liquid A: same as step S1 of Example 1.

[0065] S2. Prepare liquid B: same as step S2 of Example 1.

[0066] S3. Preparation of glucose-responsive hydrogel: 1 mL of solution A was mixed with 1.5 mL of solution B, and the mixture was evenly pipetted and allowed to stand for 50 to 55 min to obtain a hydrogel (SFPH3) with a phenylboronic acid:silk fibroin mass ratio of 3:1.

[0067] Example 4 An injectable insulin hydrogel with glucose responsiveness (iSFPH3)

[0068] Preparation of hydrogels loaded with fluorescently labeled insulin (FITC-insulin):

[0069] S1. Prepare liquid A: same as step S1 of Example 1.

[0070] S2. Prepare liquid B: same as step S2 of Example 1.

[0071] S3. Preparation of FITC-insulin-loaded hydrogel: 1 mL of solution A was mixed with 1.5 mL of solution B, 1.5625 mg of FITC-insulin was added, pipette-blotted evenly, and allowed to stand to obtain a drug-loaded hydrogel (iSFPH3) with a phenylboronic acid:silk fibroin mass ratio of 3:1.

[0072] Example 5 Phenylboronic acid-silk fibroin-based hydrogel (SFPH4) for glucose responsiveness and insulin delivery

[0073] Preparation of Phenylboronic Acid-Silk Fibroin-Based Hydrogels for Glucose Responsiveness and Insulin Delivery:

[0074] S1. Prepare liquid A: same as step S1 of Example 1.

[0075] S2. Prepare liquid B: same as step S2 of Example 1.

[0076] S3. Preparation of glucose-responsive hydrogel: 1 mL of solution A was mixed with 2 mL of solution B, and the mixture was evenly pipetted and allowed to stand for 45 to 50 min to obtain a hydrogel (SFPH4) with a phenylboronic acid:silk fibroin mass ratio of 4:1.

[0077] Example 6 An injectable insulin hydrogel with glucose responsiveness (iSFPH4)

[0078] Preparation of hydrogels loaded with fluorescently labeled insulin (FITC-insulin):

[0079] S1. Prepare liquid A: same as step S1 of Example 1.

[0080] S2. Prepare liquid B: same as step S2 of Example 1.

[0081] S3. Preparation of FITC-insulin-loaded hydrogel: 1 mL of solution A was mixed with 2 mL of solution B, 1.875 mg of FITC-insulin was added, pipette-blotted evenly, and allowed to stand for 45 to 50 min to obtain a drug-loaded hydrogel (iSFPH4) with a phenylboronic acid:silk fibroin mass ratio of 4:1.

[0082] Example 7 Phenylboronic acid-silk fibroin-based hydrogel (SFPH6) for glucose responsiveness and insulin delivery

[0083] Preparation of hydrogels loaded with fluorescently labeled insulin (FITC-insulin):

[0084] S1. Prepare liquid A: same as step S1 of Example 1.

[0085] S2. Prepare liquid B: same as step S2 of Example 1.

[0086] S3. Preparation of glucose-responsive hydrogel: 1 mL of solution A was mixed with 3 mL of solution B, and the mixture was evenly pipetted and allowed to stand for 30 to 35 min to obtain a hydrogel (SFPH6) with a phenylboronic acid:silk fibroin mass ratio of 6:1.

[0087] Example 8 An injectable insulin hydrogel (iSFPH6) with glucose responsiveness

[0088] Preparation of hydrogels loaded with fluorescently labeled insulin (FITC-insulin):

[0089] S1. Prepare liquid A: same as step S1 of Example 1.

[0090] S2. Prepare liquid B: same as step S2 of Example 1.

[0091] S3. Preparation of FITC-insulin-loaded hydrogel: 1 mL of solution A was mixed with 3 mL of solution B, 2.5 mg of FITC-insulin (i.e., insulin fluorescently labeled with fluorescein isothiocyanate) was added, and the mixture was evenly mixed with a pipette, and allowed to stand for 30 to 35 min to obtain a drug-loaded hydrogel (iSFPH6) with a phenylboronic acid:silk fibroin mass ratio of 6:1.

[0092] Example 9 Phenylboronic acid-silk fibroin-based hydrogel (SFPH8) for glucose responsiveness and insulin delivery

[0093] Preparation of Phenylboronic Acid-Silk Fibroin-Based Hydrogels for Glucose Responsiveness and Insulin Delivery:

[0094] S1. Prepare liquid A: same as step S1 of Example 1.

[0095] S2. Prepare liquid B: same as step S2 of Example 1.

[0096] S3. Preparation of glucose-responsive hydrogel: 1 mL of solution A was mixed with 4 mL of solution B, and the mixture was evenly pipetted and allowed to stand for 30 to 35 min to obtain a hydrogel (SFPH8) with a phenylboronic acid:silk fibroin mass ratio of 8:1.

[0097] Example 10 An injectable insulin hydrogel (iSFPH8) with glucose responsiveness

[0098] Preparation of hydrogels loaded with fluorescently labeled insulin (FITC-insulin):

[0099] S1. Prepare liquid A: same as step S1 of Example 1.

[0100] S2. Prepare liquid B: same as step S2 of Example 1.

[0101] S3. Preparation of FITC-insulin-loaded hydrogel: 1 mL of solution A was mixed with 4 mL of solution B, 3.125 mg of FITC-insulin was added, pipette-blotted evenly, and allowed to stand for 30 to 35 min to obtain a drug-loaded hydrogel (iSFPH8) with a phenylboronic acid:silk fibroin mass ratio of 8:1.

[0102] Example 11 Phenylboronic acid-silk fibroin-based hydrogel (SFPH) for glucose response and insulin delivery 10 ) Preparation of phenylboronic acid-silk fibroin-based hydrogels for glucose responsiveness and insulin delivery:

[0103] S1. Prepare liquid A: same as step S1 of Example 1.

[0104] S2. Prepare liquid B: same as step S2 of Example 1.

[0105] S3. Preparation of glucose-responsive hydrogel: 1 mL of solution A was mixed with 5 mL of solution B, and the mixture was evenly mixed with a pipette and allowed to stand for 25 to 30 min to obtain a hydrogel with a phenylboronic acid:silk fibroin mass ratio of 10:1 (SFPH 10 ).

[0106] Example 12 A glucose-responsive injectable insulin hydrogel (iSFPH 10 )

[0107] Preparation of hydrogels loaded with fluorescently labeled insulin (FITC-insulin):

[0108] S1. Prepare liquid A: same as step S1 of Example 1.

[0109] S2. Prepare liquid B: same as step S2 of Example 1.

[0110] S3. Preparation of FITC-insulin-loaded hydrogel: 1 mL of solution A was mixed with 5 mL of solution B, 3.75 mg of FITC-insulin was added, pipetted evenly, and allowed to stand for 25 to 30 min to obtain a drug-loaded hydrogel (iSFPH) with a phenylboronic acid:silk fibroin mass ratio of 10:1. 10 ).

[0111] Example 13 Phenylboronic acid-silk fibroin-based hydrogel (SFPH) for glucose response and insulin delivery 12 ) Preparation of phenylboronic acid-silk fibroin-based hydrogels for glucose responsiveness and insulin delivery:

[0112] S1. Prepare liquid A: same as step S1 of Example 1.

[0113] S2. Prepare liquid B: same as step S2 of Example 1.

[0114] S3. Preparation of glucose-responsive hydrogel: 1 mL of solution A was mixed with 6 mL of solution B, and the mixture was evenly mixed with a pipette and allowed to stand for 20 to 30 min to obtain a hydrogel with a phenylboronic acid:silk fibroin mass ratio of 12:1 (SFPH 12 ).

[0115] Example 14 A glucose-responsive injectable insulin hydrogel (iSFPH 12 )

[0116] Preparation of hydrogels loaded with fluorescently labeled insulin (FITC-insulin):

[0117] S1. Prepare liquid A: same as step S1 of Example 1.

[0118] S2. Prepare liquid B: same as step S2 of Example 1.

[0119] S3. Preparation of FITC-insulin-loaded hydrogel: 1 mL of solution A was mixed with 6 mL of solution B, 4.375 mg of FITC-insulin was added, pipetted evenly, and allowed to stand for 20 to 30 min to obtain a drug-loaded hydrogel (iSFPH) with a phenylboronic acid:silk fibroin mass ratio of 12:1. 12 ).

[0120] The parameters of the phenylboronic acid-silk fibroin-based hydrogels prepared in Examples 1, 3, 5, 7, 9, 11, and 13 are shown in Table 1.

[0121] Table 1 Phenylboronic acid-silk fibroin-based hydrogels with different mass ratios

[0122]

[0123]

[0124] Experimental Example 1 Texture Test

[0125] The phenylboronic acid-silk fibroin-based hydrogels obtained in Examples 1, 3, 5, 7, 9, 11, and 13 were used as representatives for texture analysis using a texture analyzer. Specifically, the probe was pressed into the sample, the target speed was 0.5 mm / s, the probe was pressed into the target to a depth of 10 mm, the trigger force was 6.8 g, and the speed before the test was 1 mm / s.

[0126] The results are as follows Figure 1 As shown in the figure, gel strength, hardness, cohesiveness and adhesiveness are important parameters of the texture characteristics of gel materials, which can represent the mechanical properties of gel samples. From the experimental results, it can be seen that the gel strength and hardness of the phenylboronic acid-silk fibroin-based hydrogel obtained in Example 1 are both large, which is not conducive to injection; while the gel strength of the phenylboronic acid-silk fibroin-based hydrogels obtained in Examples 3, 5, 7, 9, 11, and 13 is between 140 and 485 g / cm 2 , the hardness is within 25-110g and has moderate mechanical properties. The hydrogel within these parameters is not easy to rupture before injection and release the drug in advance. After being injected into the body, the loaded drug can be smoothly released from the hydrogel.

[0127] In addition, the mechanical strength of the injectable hydrogel should accurately match the target tissue. The elastic modulus of muscle and skin is usually in the range of 0.0002 to 0.018 MPa, so hydrogels with mechanical strength close to this range are suitable for subcutaneous injection. 10 (0.012Mpa), SFPH 12 The mechanical strength of (0.0098Mpa) is well matched with the elastic modulus of the skin and is more suitable for subcutaneous injection. The mechanical strength of the phenylboronic acid-silk fibroin-based hydrogel obtained in Examples 1, 3, and 5 is relatively large, but poorly matched with the elastic modulus of the skin and is not suitable for subcutaneous injection.

[0128] The texture test results of the insulin-loaded hydrogel (such as iSFPH6) and its corresponding drug-free phenylboronic acid-silk fibroin-based hydrogel (such as SFPH6) are basically the same.

[0129] Experimental Example 2 Swelling Test

[0130] (1) Swelling test of phenylboronic acid-silk fibroin-based hydrogels with different mass ratios

[0131] The swelling test was performed using the phenylboronic acid-silk fibroin-based hydrogels obtained in Examples 1, 3, 5, 7, 9, 11, and 13 as representatives. The hydrogels were freeze-dried and cut into small pieces, then immersed in a 37° C. PBS solution, and the gels were taken out at different time intervals, the surface water was absorbed by absorbent paper, and the gels were weighed, and the swelling was calculated by weighing.

[0132] The calculation formula of swelling property is as follows:

[0133] SR=(W s -W d ) / W d ×100%

[0134] Among them, SR is the swelling ratio;

[0135] W s is the mass of the hydrogel after swelling (i.e., after fully absorbing the solvent);

[0136] W d It is the mass of the hydrogel before swelling (i.e. dry state).

[0137] The results are as follows Figure 2 As shown in the figure, the proportion of phenylboronic acid in the hydrogel is negatively correlated with the swelling rate. The lower the proportion, the higher the swelling rate, and the higher the proportion, the lower the swelling rate. The experimental results show that the phenylboronic acid-silk fibroin-based hydrogel with a mass ratio of phenylboronic acid to silk fibroin of 6 to 12 has a moderate swelling rate. The swelling rate of the phenylboronic acid-silk fibroin-based hydrogel below this range is too high, the drug release is too fast, and the sustained release effect cannot be achieved.

[0138] (2) Swelling test of phenylboronic acid-silk fibroin-based hydrogel at different glucose concentrations

[0139] The phenylboronic acid-silk fibroin-based hydrogel (SFPH6) obtained in Example 7 was used as a representative to conduct a swelling test. The specific test method is as follows:

[0140] The freeze-dried hydrogel SFPH6 was immersed in PBS solution (pH 7.4) with different glucose concentrations (0 mg / mL, 1 mg / mL, 3 mg / mL, 5 mg / mL). The test results are as follows: Figure 3 , the higher the glucose concentration, the greater the swelling rate.

[0141] The swelling test results of the insulin-loaded hydrogel and its corresponding drug-free phenylboronic acid-silk fibroin-based hydrogel were basically the same.

[0142] Experimental Example 3 Biodegradability

[0143] The phenylboronic acid-silk fibroin-based hydrogels obtained in Examples 1, 3, 5, 7, 9, 11, and 13 were used as representatives to conduct biodegradability tests.

[0144] (1) In vitro degradation test

[0145] The prepared hydrogel was placed in a PBS solution containing proteinase K (pH 7.4). The test results were as follows: Figure 4 As shown, except for hydrogel SFPH2 which degraded within four days, the other hydrogels were completely degraded within three days.

[0146] (2) In vivo degradation test

[0147] The phenylboronic acid-silk fibroin-based hydrogel (SFPH6) obtained in Example 7 was used as a representative to perform an in vivo degradation test.

[0148] The prepared hydrogel SFPH6 was injected subcutaneously into the back of Wistar rats. The rats were killed and dissected to observe the degradation of the hydrogel in the rats. The results are shown in Figure 5 , the hydrogel was completely degraded in three days.

[0149] The hydrogels obtained in Examples 9, 11, and 13 have substantially the same in vivo degradation properties as the hydrogel obtained in Example 7.

[0150] The degradation test results of the insulin-loaded hydrogel and its corresponding drug-free phenylboronic acid-silk fibroin-based hydrogel were basically the same.

[0151] Experimental Example 4 Injectability Test

[0152] The phenylboronic acid-silk fibroin-based hydrogel (SFPH6) obtained in Example 7 was used as a representative to perform an injectability test. The specific test method is as follows:

[0153] The prepared hydrogel preparation solution was added to the syringe, and the injectability of the hydrogel was tested using 16G, 20G, and 26G needles, respectively. The results are shown in Figure 6 , the hydrogel can be injected smoothly with needles of three specifications.

[0154] The hydrogels obtained in Examples 8 to 14 have substantially the same injectability properties as the hydrogel obtained in Example 7.

[0155] Experimental Example 5 Glucose responsiveness test

[0156] The glucose-responsive injectable insulin hydrogel (iSFPH6) obtained in Example 8 was used as a representative insulin-loaded hydrogel to conduct a glucose-responsive drug release test.

[0157] (1) In vitro glucose-responsive drug release

[0158] iSFPH6 was placed in a centrifuge tube (pH 7.4) containing PBS solution with different glucose concentrations (0 mg / mL, 1 mg / mL, 5 mg / mL), in a 37°C constant temperature water bath, and its drug release percentage was detected by UV. The test results are shown in Figure 7 As shown, the greater the glucose concentration, the greater the percentage of insulin released.

[0159] (2) In vivo glucose responsiveness test (animal experiment)

[0160] The prepared hydrogel SFPH6, drug-loaded hydrogel iSFPH6, insulin and PBS were injected subcutaneously into the backs of four groups of Wistar rats, with 6 rats in each group, and the blood glucose levels of the rats were tested regularly. Figure 8 ,Compared with the insulin group, the drug-loaded hydrogel group showed excellent glucose responsiveness and controlled release behavior, and could maintain blood glucose homeostasis for two days.

[0161] The drug-loaded hydrogels obtained in Examples 10, 12, and 14 have glucose responsiveness in vivo and in vitro that is substantially similar to that of the drug-loaded hydrogel obtained in Example 8.

[0162] Experimental Example 6 Biocompatibility

[0163] The biocompatibility test was performed using the phenylboronic acid-silk fibroin-based hydrogel (SFPH6) obtained in Example 7 as a representative. The specific test method is as follows:

[0164] The prepared hydrogel was sterilized and co-cultured with cells, and the cell activity was detected at 24h and 48h. Fig. 9 , the hydrogel has no obvious toxicity to cell activity and has good biocompatibility.

[0165] The hydrogels obtained in other examples have substantially the same biocompatibility as the hydrogel obtained in Example 7.

[0166] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An injectable insulin hydrogel with glucose responsiveness, characterized in that: The injectable insulin hydrogel comprises a hydrogel matrix and insulin, wherein the insulin is encapsulated in the hydrogel matrix; The hydrogel matrix is ​​constructed by phenylboronic acid and silk fibroin; the mass ratio of phenylboronic acid to silk fibroin is (2-12):

1.

2. The injectable insulin hydrogel according to claim 1, characterized in that: The mass ratio of phenylboric acid to silk fibroin is (6-12):

1.

3. The injectable insulin hydrogel according to claim 2, characterized in that: The mass ratio of phenylboric acid to silk fibroin is (6-8):

1.

4. The injectable insulin hydrogel according to claim 1, characterized in that: The mass concentration of insulin in the injectable insulin hydrogel is 0.15-1 mg / mL.

5. The method for preparing the injectable insulin hydrogel according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: mixing phenylboronic acid solution and silk fibroin solution, adding insulin, fully mixing, and standing to obtain injectable insulin hydrogel.

6. The preparation method according to claim 5, characterized in that: The phenylboric acid solution is obtained by dissolving phenylboric acid in an ethanol aqueous solution.

7. The preparation method according to claim 6, characterized in that: The concentration of the ethanol aqueous solution is 60-80 vol%.

8. The preparation method according to claim 5, characterized in that: The concentration of the phenylboric acid solution is 80-120 mg / mL.

9. The preparation method according to claim 5, characterized in that: The preparation method of the silk fibroin solution comprises the following steps: sequentially performing degumming treatment, dissolution, purification and filtration on a raw material rich in silk fibroin to obtain the silk fibroin solution.

10. The preparation method according to claim 5, characterized in that: The concentration of the silk fibroin solution is 40-60 mg / mL.

11. Use of the injectable insulin hydrogel according to any one of claims 1 to 3 in the preparation of insulin preparations.

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