Insulin cross-linked hydrogel as well as preparation method and application thereof

Abstract

The invention provides insulin cross-linked hydrogel as well as a preparation method and application thereof. The hydrogel is prepared from insulin, oxidized dextran and a solvent medium. The insulin cross-linked hydrogel is constructed by performing Schiff base bond cross-linking on an aldehyde group of oxidized dextran and an amino group of insulin, taking a hypoglycemic drug insulin as a gel network cross-linking point and taking an oxidized dextran chain as a gel skeleton, so that the form of physical encapsulation of insulin in the past is changed. The drug hydrogel has good pH responsiveness, gel degradation can be accelerated in an acid environment, and cross-linked and fixed insulin is released. The hydrogel can respond to glucose under the action of glucose oxidase and an enzyme-like catalyst thereof, so that glucose-responsive release of insulin is realized. The insulin cross-linked hydrogel is composed of drugs as gel and only contains two components, the preparation method is simple and easy to operate, and the insulin cross-linked hydrogel has good application potential in the aspect of blood glucose regulation and control.

Description

Technical Field

[0001] The present invention relates to the field of polymer bonded drugs, and in particular to an insulin cross-linked hydrogel and a preparation method and application thereof. Background Art

[0002] Insulin is a specific polypeptide hormone for the treatment of diabetes, and there is no more effective alternative drug so far. However, since insulin is a protein that is easily inactivated in the gastrointestinal microenvironment, subcutaneous injection has always been its main route of administration. Clinically, patients with insulin-dependent diabetes (also known as type 1 diabetes) must use insulin every day to lower blood sugar and maintain normal metabolism. However, frequent injections of medication bring great pain and inconvenience to patients who need lifelong medication. Therefore, the development of safer, more convenient and effective non-injection preparations of insulin or reducing the number of medications has become an important direction for the development of insulin preparations.

[0003] Hydrogel is an important carrier for sustained release of protein drugs and shows many advantages in insulin delivery. However, at present, most research results are to load insulin in hydrogel in the form of physical coating. For example, patent CN202110591295.4 mentions a pH and α-amylase dual-responsive hydrogel system loaded with insulin, in which insulin is distributed in sodium alginate hydrogel and in the pores and cavities of porous starch. Although it can be released responsively, its release controllability still needs to be improved, and the release controllability will directly affect the efficacy of blood sugar regulation. How to simply and effectively achieve the on-demand release of insulin has become a bottleneck problem that needs to be solved in this research field. Summary of the invention

[0004] In view of this, the present invention aims to provide a Schiff base cross-linked drug hydrogel with hypoglycemic drug insulin as a cross-linking agent and oxidized dextran as a hydrogel skeleton, and its preparation method and application, for the controlled release of insulin and intelligent regulation of blood sugar. The drug hydrogel has good pH responsiveness. In an acidic environment, the Schiff base bond undergoes acid-sensitive rupture, thereby accelerating the degradation of the gel and releasing the cross-linked fixed insulin; while in a physiological environment, the degradation of the hydrogel slows down, causing the insulin release rate to decrease, which can avoid hypoglycemia caused by rapid insulin release. Therefore, it can be applied to diabetes treatment by loading a glucose response unit (glucose oxidase, gold nanoparticles, etc.). The glucose response unit can convert the surrounding glucose into gluconic acid through catalysis, and convert the sugar signal into a pH signal, thereby promoting the degradation of the gel and releasing the cross-linked fixed insulin.

[0005] In the first aspect, the present invention provides an insulin cross-linked hydrogel, whose main components include the following components in mass content: 1.0 wt% to 10.0 wt% of insulin, 5.0 wt% to 50.0 wt% of oxidized dextran, and the rest is a solvent medium; wherein the amino group on the surface of insulin forms a Schiff base bond with the aldehyde group of the oxidized dextran.

[0006] The structural formula of the oxidized dextran is shown in Formula I, wherein the degree of polymerization of the first repeating unit is x, the degree of polymerization of the second repeating unit is y, and x and y satisfy 1≤x / y≤19; the oxidized dextran is prepared by mixing dextran and an oxidant, wherein the molecular weight of the dextran is 1 kDa to 2000 kDa, and the degree of oxidation of the oxidized dextran is 5.0% to 50%; Formula I The solvent medium used was ultrapure water, PBS buffer or physiological saline.

[0007] Preferably, the concentration of the insulin is 1.0 wt% to 10.0 wt%, more preferably 4.0 wt% to 6.0 wt%; Preferably, the molecular weight of dextran is 50 kDa to 500 kDa, and the degree of oxidation is 10% to 30%; the degree of oxidation refers to the degree to which hydroxyl groups (-OH) are oxidized to aldehyde groups (-CHO) in the oxidized dextran molecules, and is usually used to describe the molar percentage of aldehyde groups in the oxidized dextran, in units of percentage (%).

[0008] Preferably, the insulin is one of recombinant human insulin, low-protamine zinc insulin, aspart insulin and the like.

[0009] In a second aspect, the present invention provides a method for preparing a cross-linked insulin hydrogel, specifically: The oxidized dextran and the solvent medium are mixed, insulin is added, and after being fully mixed, the mixture is placed in a constant temperature shaker to react at a rotation speed of 10 to 200 r / min to obtain the insulin cross-linked hydrogel.

[0010] The reaction temperature is preferably 4 to 50°C; more preferably 25 to 40°C.

[0011] The reaction is carried out at a pH value of 6 to 8; the reaction time is preferably 0.1 to 72 h, more preferably 0.5 to 6 h.

[0012] In a third aspect, the present invention provides an application of an insulin cross-linked hydrogel in intelligent blood sugar regulation, specifically for preparing an intelligent blood sugar regulation carrier.

[0013] Compared with the prior art, the beneficial effects of the present invention are at least: The present invention does not need to use a variety of complex polymer materials as the gel skeleton, but uses the drug insulin itself as a cross-linking agent and oxidized dextran as a cross-linking molecule to form a binary system. The prepared hydrogel contains only two components, which simplifies the raw materials while maintaining good biocompatibility and pH responsiveness; wherein, the cross-linked drug insulin, in addition to being a hyperglycemia treatment drug, can also be used as a cross-linking point to promote the formation of hydrogels, and forms a cross-linked hydrogel network with oxidized dextran through Schiff base bonds, thereby avoiding the free diffusion of drugs due to swelling or other effects of hydrogels, and facilitating the precise release and regulation of drugs. In addition, the hydrogel system can make the hydrogel have glucose responsiveness through loaded glucose oxidase or gold nanoparticles, realize the glucose responsive release of insulin, and achieve the purpose of intelligent blood sugar control. This insulin cross-linked hydrogel has positive significance and application prospects for reducing the uncontrollable burst release of insulin and improving drug efficacy.

[0014] The preparation method of the present invention is simple and easy to use. During the preparation process, the therapeutic drug insulin is directly converted into a component of the hydrogel network, which not only simplifies the material design but also is more conducive to regulating the linkage relationship and release behavior between the drug and the carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the gelation mechanism of the insulin cross-linked hydrogel of the present invention.

[0016] Figure 2 This is a gelation photograph of the insulin cross-linked hydrogel obtained in Example 1 of the present invention.

[0017] Figure 3 These are scanning electron microscope morphology photographs of the hydrogel prepared in Example 1 of the present invention, wherein A is a scanning electron microscope photograph at 10 μm, and B is a scanning electron microscope photograph at 2 μm.

[0018] Figure 4 This is a rheological property test diagram of the hydrogel prepared in Example 1 of the present invention.

[0019] Figure 5 This is the degradation curve of the hydrogel prepared in Example 2 of the present invention under physiological environment.

[0020] Figure 6 This is the degradation curve of the hydrogel prepared in Example 2 of the present invention in an acidic environment. DETAILED DESCRIPTION

[0021] In order to more clearly illustrate the case of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described below; it is obvious that the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the protection scope of the present invention.

[0022] The present invention provides a hydrogel cross-linked by oxidizing dextran aldehyde groups and insulin amino groups through Schiff base bonds, and its gel formation mechanism is as follows: Figure 1 As shown, it has good biocompatibility and pH sensitivity.

[0023] The invention provides an insulin cross-linked hydrogel, the main components of which include the following components in mass content: 1.0 wt% to 10.0 wt% of insulin, 5.0 wt% to 50.0 wt% of oxidized dextran, and the rest being a solvent medium; wherein the amino groups on the surface of the insulin form Schiff base bonds with the aldehyde groups of the oxidized dextran, the oxidized dextran constitutes a hydrogel skeleton, and the solvent medium is one of pure water, PBS buffer or physiological saline.

[0024] The present invention has no particular limitation on the source of the oxidized dextran, and it is preferably prepared according to the following method: Dextran and sodium periodate (NaIO4) are reacted in water to obtain oxidized dextran with an oxidation degree of 10% to 50%. The molecular weight of the dextran is preferably 50 kDa to 500 kDa, and the concentration is preferably 10.0 wt% to 30.0 wt%.

[0025] In the present invention, the dextran aqueous solution is preferably prepared by dissolving a certain mass of dextran with secondary water. The molecular weight of the dextran is preferably 50 kDa to 500 kDa. The present invention has no special restrictions on the order of adding the dextran aqueous solution and the NaIO4 aqueous solution, and the specific operation is as follows: after a certain mass of dextran is fully dissolved with secondary water, a certain proportion of NaIO4 aqueous solution is added to react, and stirred for a period of time in the dark. The temperature of the reaction for preparing oxidized dextran in the present invention is preferably 25 to 40 ° C; the time is preferably 12 to 24 h. Afterwards, the obtained reaction solution is added to a dialysis bag for dialysis for a certain period of time, and the water is changed regularly; finally, the dialyzed solution is freeze-dried to obtain an oxidized dextran product with a certain aldehyde content.

[0026] The embodiment is as follows: 5.0 g of dextran (100 kDa) is accurately weighed into a round-bottom flask, and 25 mL of phosphate buffer (10 mM, pH = 4.5) is added thereto for dissolution. The solution may be heated and stirred appropriately to accelerate the dissolution rate. After the dextran is completely dissolved, 0.67 g of the newly prepared NaIO4 solution is added dropwise to the dextran solution through a constant pressure dropping funnel under ice bath, light protection and stirring conditions. After the NaIO4 solution is added dropwise, the ice bath is removed, the reaction mixture is placed at room temperature and continues to be stirred for 1.5 hours in the dark for full oxidation. Subsequently, an ethylene glycol solution in an equimolar ratio with NaIO4 is added, and stirring is continued for 15 minutes to terminate the oxidation reaction. Then, the reaction mixture is transferred to a pre-treated dialysis bag (Mw: 3kDa) and incubated with deionized water at room temperature for 3 days. The deionized water is replaced every 6 hours on the first day and every 12 hours on the next two days to remove unreacted monomers and inorganic salts as much as possible. After dialysis, oxidized dextran with an oxidation degree of 10% was obtained by freeze drying.

[0027] After obtaining the oxidized dextran, the embodiment of the present invention fully mixes the oxidized dextran solution and the insulin solution, and reacts them at a temperature of 4°C to 50°C, preferably 25°C to 40°C, to obtain a dextran / insulin cross-linked drug hydrogel.

[0028] In one embodiment of the present invention, a certain mass of oxidized dextran and insulin are fully dissolved in a solvent medium, preferably fully mixed on a shaker at a suitable temperature, and a dextran / insulin cross-linked drug hydrogel is obtained by reaction. In an acidic environment, the Schiff base bond breaks, and the hydrogel degradation and insulin release are promoted. Preferably, the reaction is carried out under a pH value of 6 to 8. The reaction time is preferably 0.1 hour to 72 hours, more preferably 0.5 hour to 6 hours.

[0029] The present invention uses a scanning electron microscope to characterize the morphology of the obtained insulin cross-linked hydrogel. The result shows that the hydrogel has a good network pore structure, uniform pore distribution, and relatively uniform pore size, which is conducive to the release of drugs.

[0030] The present invention has carried out rheological property test on the obtained hydrogel, and the result shows that the hydrogel has certain mechanical strength and good injectability. At the same time, the degradation behavior of the hydrogel under different pH conditions is also studied on the hydrogel of the present invention, and the result shows that the drug cross-linked hydrogel has good pH responsiveness.

[0031] In order to further understand the present application, the insulin cross-linked hydrogel and the preparation method thereof provided by the present application are specifically described below in conjunction with examples.

[0032] Example 1 Add 10 mg of 10% oxidized dextran (100 kDa) into 100 μL ultrapure water and heat in a 45 °C water bath to completely dissolve it to obtain a clear solution. Add 5.0 mg of insulin to the solution and place it in a shaker. Set the temperature to 37 °C and the speed to 150 r / min, or use mechanical stirring to evenly disperse it in the solution. After about 4-8 h of reaction, an insulin cross-linked hydrogel is obtained.

[0033] The hydrogel prepared in Example 1 of the present invention was quickly frozen by liquid nitrogen quick freezing method, and freeze-dried to obtain a freeze-dried gel sample; the sample was tested by scanning electron microscope to obtain a microscopic morphology of the gel, such as Figure 2 As shown. Figure 2 It can be seen that the complex hydrogel material has an interconnected macroporous structure, which is conducive to the transmission and release of drugs in the gel material.

[0034] Figure 3 This is a scanning electron microscope morphology photograph of the insulin cross-linked hydrogel obtained in Example 1; Figure 4 The rheological study results of the insulin cross-linked hydrogel obtained in Example 1.

[0035] Example 2 Add 10 mg of 10% oxidized dextran (100 kDa) into 100 μL ultrapure water and heat in a 60 °C water bath to completely dissolve it to obtain a clear solution. Add 3.0 mg of insulin to the solution and place it in a shaker with the temperature set to 37 °C and the speed set to 200 r / min, or use mechanical stirring to evenly disperse the insulin in the solution. After about 4-8 h of reaction in the shaker, the insulin cross-linked hydrogel is obtained.

[0036] The product obtained in this example was placed in different pH environments to study the hydrogel degradation behavior. The results showed that the insulin cross-linked hydrogel had good pH responsiveness. Figure 5 and Figure 6 The results show that the hydrogel has good pH-responsive degradation behavior, degrading slowly under physiological conditions and rapidly under acidic conditions. Since insulin is directly involved in the construction of the hydrogel, the speed of degradation represents the speed of insulin release, which further indicates that the hydrogel of the present invention can quickly release drugs under acidic pH conditions.

[0037] Example 3 Add 50 mg of 50% oxidized dextran (1 kDa) into 100 μL ultrapure water and heat in a 50°C water bath to completely dissolve it to obtain a clear solution. Add 10 mg of insulin to the solution and place it in a shaker with the temperature set to 37°C and the speed set to 200 r / min, or use mechanical stirring to evenly disperse the insulin in the solution. After about 24-48 hours of reaction in the shaker, an insulin cross-linked hydrogel is obtained.

[0038] Example 4 15 mg of 10% oxidized dextran (500 kDa) was added to 100 μL PBS buffer and heated in a 45°C water bath to completely dissolve it to obtain a clear solution. 2 mg of oligosaccharide zinc insulin was added to the solution and then placed in a shaker with the temperature set to 37°C and the speed set to 200 r / min, or mechanical stirring was used to evenly disperse the insulin in the solution. After about 12-24 h of reaction in the shaker, oligosaccharide zinc insulin cross-linked hydrogel was obtained.

[0039] Example 5 Add 50 mg of 10% oxidized dextran (100 kDa) into 100 μL ultrapure water and heat in a 45°C water bath to completely dissolve it to obtain a clear solution. Add 1 mg of aspart insulin to the solution and place it in a shaker with the temperature set to 37°C and the speed set to 10 r / min. Alternatively, use mechanical stirring to evenly disperse the insulin in the solution and react in a shaker to obtain aspart insulin cross-linked hydrogel.

[0040] Example 6 Add 10 mg of 30% oxidized dextran (1000 kDa) into 100 μL ultrapure water and heat in a 45°C water bath to completely dissolve it to obtain a clear solution. Add 3 mg of insulin to the solution and place it in a shaker with the temperature set to 37°C and the speed set to 200 r / min, or use mechanical stirring to evenly disperse the insulin in the solution. After about 36-48 hours of reaction in the shaker, the insulin cross-linked hydrogel is obtained.

[0041] Example 7 Add 10 mg of 30% oxidized dextran (60 kDa) into 150 μL ultrapure water and heat in a 45°C water bath to completely dissolve it to obtain a clear solution. Add 5 mg of insulin to the solution and place it in a shaker with the temperature set to 50°C and the speed set to 100 r / min, or use mechanical stirring to evenly disperse the insulin in the solution. After about 4-8 hours of reaction in the shaker, an insulin cross-linked hydrogel is obtained.

[0042] Example 8 8 mg of 10% oxidized dextran (1000 kDa) was added to 100 μL ultrapure water and heated in a 45 °C water bath to completely dissolve it to obtain a clear solution. 4 mg of insulin was added to the solution and placed on a shaker with the temperature set to 20 °C and the speed set to 200 r / min. After about 8-12 h of reaction, insulin cross-linked hydrogel was obtained.

[0043] Example 9 30 mg of 50% oxidized dextran (1000 kDa) was added to 100 μL ultrapure water and heated in a 60°C water bath to completely dissolve it to obtain a clear solution. 1 mg of insulin was added to the solution and mixed thoroughly. The solution was placed in a shaker and the temperature was set to 40°C and the speed was set to 150 r / min. After about 8-12 h of reaction in the shaker, insulin cross-linked hydrogel was obtained.

[0044] The above embodiments are not limitations of the present invention, and the present invention is not limited to the above embodiments. As long as the requirements of the present invention are met, they belong to the protection scope of the present invention.

Claims

1. An insulin cross-linked hydrogel, characterized in that: The invention comprises the following components by weight: 1.0wt% to 10.0wt% of insulin, 5.0wt% to 50.0wt% of oxidized dextran, and the rest being a solvent medium; wherein the amino groups on the surface of the insulin and the aldehyde groups of the oxidized dextran form Schiff base bonds; The oxidized dextran is obtained by oxidizing dextran with a molecular weight of 1 kDa to 2000 kDa, with an oxidation degree of 5.0% to 50%, and its structural formula is shown in Formula I, where x and y satisfy 1≤x / y≤19; 2. The insulin cross-linked hydrogel according to claim 1, characterized in that: The concentration of insulin is 1.0wt% to 10.0wt%.

3. The insulin cross-linked hydrogel according to claim 2, characterized in that: The concentration of insulin is 4.0wt% to 6.0wt%.

4. The insulin cross-linked hydrogel according to claim 1, characterized in that: The molecular weight of dextran is 50 kDa to 500 kDa.

5. The insulin cross-linked hydrogel according to claim 1, characterized in that: The oxidation degree of the oxidized dextran is 10% to 30%.

6. The insulin cross-linked hydrogel according to claim 1, characterized in that: The solvent medium is one of ultrapure water, PBS buffer and physiological saline.

7. The insulin cross-linked hydrogel according to claim 1, characterized in that: The insulin is one of recombinant human insulin, low-protamine zinc insulin, and aspart insulin.

8. A method for preparing the insulin cross-linked hydrogel according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: The oxidized dextran and the solvent medium are mixed, insulin is added, and after being fully mixed, the mixture is placed in a constant temperature shaker for reaction to obtain the insulin cross-linked hydrogel.

9. The method according to claim 8, characterized in that The temperature of the constant temperature shaker is 4-50°C, the rotation speed is 10-200r / min; the reaction time is 0.1-72h, and the pH value of the reaction environment is 6-8.

10. Use of the insulin cross-linked hydrogel according to any one of claims 1 to 7 in preparing a carrier for intelligent blood sugar regulation.

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