PH / blood sugar double-response insulin controlled release system as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines and nano materials, and particularly relates to a pH / blood sugar double-response insulin controlled release system as well as a preparation method and application thereof. The preparation method comprises the following steps: dissolving and mixing insulin and glucose oxidase to obtain a mixed solution; adding the mixed solution and manganese dioxide nanosheets into the acetalated dextran / dichloromethane solution, carrying out ultrasonic treatment until the mixed solution becomes milk white, then immediately adding the mixed solution into a polyvinyl alcohol solution to obtain a mixed solution, and carrying out stirring reaction at room temperature; then centrifuging and precipitating to obtain white powder, and freeze-drying the white powder to obtain the pH / blood sugar double-response insulin controlled release system. The controlled release system is constructed on the basis of acetalated dextran (Ac-Dex) and manganese dioxide (MnO2) nanosheets, has pH and glucose dual response characteristics, can realize accurate long-period controlled release of insulin, and can effectively solve the problem of insulin inactivation caused by H2O2 in the existing delivery system.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine and nanomaterials, and particularly relates to a pH / glucose dual-response insulin controlled-release system and a preparation method and application thereof. BACKGROUND

[0002] Diabetes is a global chronic metabolic disease, and insulin replacement therapy is the main means of treatment. However, it is difficult to achieve physiological blood glucose regulation by traditional subcutaneous injection of insulin, which is prone to cause the risk of hypoglycemia or hyperglycemia. In recent years, intelligent insulin delivery systems have become a research hotspot. For example, the Chinese patent application with the publication number CN108567993A loads insulin and glucose oxidase into acetalized dextran to achieve glucose-responsive release; and the Chinese patent application with the publication number CN104558236A discloses preparation of acetalized dextran and describes the feasibility of the acetalized dextran as a drug carrier. However, the existing glucose-responsive controlled-release systems do not consider or solve the following key problems: 1) insulin inactivation caused by H2O2: H2O2 produced by glucose oxidase (GOx) catalysis of glucose can oxidize insulin, reducing its biological activity; and 2) low loading capacity of microparticle insulin and short release period.

[0003] Therefore, how to maximize the activity of insulin while ensuring that the microparticles have a response release capability is a problem that needs to be solved for a new type of response insulin controlled-release system. SUMMARY

[0004] To solve the shortcomings and deficiencies of the prior art, the primary purpose of the present application is to provide a preparation method of a pH / glucose dual-response insulin controlled-release system.

[0005] Another purpose of the present application is to provide a pH / glucose dual-response insulin controlled-release system prepared by the above method. The controlled-release system is constructed based on acetalized dextran (Ac-Dex) and manganese dioxide nanosheets, has pH and glucose dual-response characteristics, can achieve precise long-period controlled release of insulin, and can effectively solve the problem of insulin inactivation caused by H2O2 in the existing delivery system.

[0006] Still another purpose of the present application is to provide the application of the above-mentioned pH / glucose dual-response insulin controlled-release system.

[0007] The purposes of the present application are achieved by the following technical solutions.

[0008] A preparation method of a pH / glucose dual-response insulin controlled-release system, comprising the following steps:

[0009] (1) insulin is weighed and dissolved in a carbonate buffer solution, then glucose oxidase is added, and ultrasonic is used to completely dissolve the mixture to obtain a mixed solution;

[0010] (2) adding the mixed solution in step (1) and manganese dioxide nanosheets (δ-MnO2) into an acetalized dextran / dichloromethane solution, ultrasonicating until the mixed solution becomes milky white, and then immediately adding the mixed solution into a polyvinyl alcohol (PVA) solution to obtain a reaction solution and stirring the reaction solution at room temperature; then performing centrifugation and precipitation to obtain white powder, and freeze-drying the white powder to obtain acetalized dextran insulin microspheres (AcDEX@INS / GOD), which are the pH / blood glucose dual-responsive insulin controlled-release system.

[0011] Preferably, the concentration of insulin in the mixed solution in step (1) is 5-20 wt%, more preferably 10 wt%.

[0012] Preferably, the concentration of glucose oxidase in the mixed solution in step (1) is 1-5 wt%, more preferably 2.8 wt%.

[0013] Preferably, the acetalized dextran in step (2) is prepared by the following steps: completely dissolving dextran in DMSO (dimethyl sulfoxide), then adding pyridine p-toluenesulfonate and 2-methoxypropene to react, adding triethylamine to the reaction solution after a period of time to quench the reaction, discarding the supernatant after centrifugation, washing the precipitate with water, and freeze-drying the product to obtain acetalized dextran (AcDEX).

[0014] More preferably, the mass ratio of pyridine p-toluenesulfonate to 2-methoxypropene is 1:100-250, more preferably 1:160, and the mass ratio of pyridine p-toluenesulfonate to glucose is 1:1-5.

[0015] More preferably, the molecular weight of the glucose is 10-70 kDa, most preferably 70 kDa.

[0016] More preferably, the reaction time is 5-7 h, more preferably 6 h; the reaction is preferably performed at room temperature.

[0017] More preferably, the mass ratio of 2-methoxypropene to triethylamine is 1:3-7.0, more preferably 1:4.4.

[0018] Preferably, the manganese dioxide nanosheets in step (2) can be directly purchased from the market, or are prepared by the following steps: adding tetramethylammonium hydroxide pentahydrate (TMAOH·5H2O) dissolved in H2O2 solution into an aqueous solution of MnCl2·4H2O, stirring, then centrifuging, washing, and drying, and then adding the product into water, ultrasonicating, and then centrifuging to obtain δ-MnO2, which are the manganese dioxide nanosheets.

[0019] More preferably, the molar ratio of tetramethylammonium hydroxide pentahydrate to MnCl2·4H2O is 4:1.

[0020] More preferably, the stirring time is 24-36h, preferably at room temperature; the drying time is 12-24h, and the ultrasonic time is 10-15h.

[0021] Preferably, the mass concentration of the acetylated dextran in the acetylated dextran / dichloromethane solution in step (2) is 1.5%.

[0022] Preferably, in step (2), the volume ratio of the dichloromethane solution to the polyvinyl alcohol solution is 1:4-6, more preferably 1:4.17; the concentration of the polyvinyl alcohol solution is 2-5%, more preferably 5%.

[0023] Preferably, in step (2), the mass ratio of insulin, glucose oxidase, manganese dioxide nanosheet, acetylated dextran, and polyvinyl alcohol is 5:4:(0-6):9:75.

[0024] Preferably, the ultrasonic time in step (2) is 80-100s.

[0025] Preferably, the mass fraction of the manganese dioxide nanosheet in the reaction solution in step (2) is 0-1wt%.

[0026] Preferably, the stirring time of the reaction solution in step (2) at room temperature is 1-3h.

[0027] Preferably, the centrifugation time in step (2) is 10-20min.

[0028] The pH / blood glucose dual-responsive insulin controlled-release system can be used for preparing a drug for treating diabetes.

[0029] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0030] 1) The novel insulin controlled-release system designed in the present application can respond to the release system, has a high insulin loading rate and a long release period.

[0031] 2) The novel insulin controlled-release system designed in the present application can respond to release insulin while ensuring the biological activity of insulin. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is a synthetic route diagram of AcDEX in Example 1.

[0033] Figure 2 is a SEM diagram and particle size analysis diagram of AcDEX10 prepared in Example 1.

[0034] Figure 3is the SEM image and particle size analysis chart of AcDEX10 prepared in Example 1.

[0035] Figure 4 is the insulin-responsive release curve of AcDEX10@INS and AcDEX70@INS loaded with insulin only in different pH solutions.

[0036] Figure 5 is the insulin-responsive release curve of AcDEX10@INS / GOD and AcDEX70@INS / GOD loaded with insulin and glucose oxidase in different glucose concentrations.

[0037] Figure 6 is the test chart of the protection of δ-MnO2 on insulin activity. DETAILED DESCRIPTION

[0038] The present application will be further described in conjunction with the examples and the accompanying drawings, but the embodiments of the present application are not limited thereto. The raw materials involved in the present application can be directly purchased from the market. For the process parameters not specifically mentioned, the conventional techniques can be referred to.

[0039] The molecular weight of dextran used in the examples is 10 kDa and 70 kDa; the enzyme activity unit of glucose oxidase is 100 U / mg.

[0040] Example 1:

[0041] (1) Synthesis of acetalized dextran (AcDEX)

[0042] The acetalization reaction of 2-methoxypropene on the hydroxyl group of dextran is carried out under the catalysis of pyridine p-toluenesulfonate. The whole reaction system needs to be carried out in a dry and anaerobic reaction bottle. The reaction steps are as shown in Figure 1

[0043] In a 25 mL two-port bottle, 1.00 g of dextran (Mw = 10 kDa) and 10 mL of DMSO were added and stirred until the dextran was completely dissolved. Then 15.6 mg of pyridine p-toluenesulfonate was added, 3.4 mL of 2-methoxypropene was injected into the reaction solution, the two-port bottle was sealed and kept under positive N2 pressure. After 6 h of reaction at room temperature, 15 mL of triethylamine was added to the reaction solution to quench the reaction, then the dextran solution after reaction was precipitated in deionized water, the supernatant was discarded by centrifugation, the precipitate was washed with deionized water for 3 times, the product was frozen and freeze-dried, and finally the acetalized dextran (AcDEX10) was obtained.

[0044] Only the molecular weight of dextran is changed to 70 kDa, and the rest of the reaction steps remain unchanged, then the acetalized dextran AcDEX70 can be prepared.

[0045] ​(2) Preparation of acetalized dextran microspheres loaded with insulin and glucose oxidase

[0046] Weigh 50 mg of insulin and dissolve it in 0.5 mL of carbonate buffer, then add 14 mg of glucose oxidase and 5 mg of δ-MnO2, and ultrasonicate to completely dissolve. Dissolve 180 mg of acetalized dextran (AcDEX) in 6 mL of dichloromethane. Ultrasonicate the two-phase mixture with an ultrasonic probe for 90 s, pause for a few seconds, and then continue ultrasonication until the mixture turns milky white, and then immediately add it to 25 mL of a 3% PVA solution to obtain a reaction solution. Stir the reaction solution at room temperature for 2 h, centrifuge at 8000 rpm for 15 min, and wash the precipitate with deionized water 3 times, and then freeze-dry the white powder obtained to obtain the acetalized dextran insulin microspheres. When the acetalized dextran used is AcDEX10, the acetalized dextran insulin microspheres prepared are denoted as AcDEX10@INS / GOD; when the acetalized dextran used is AcDEX70, the acetalized dextran insulin microspheres prepared are denoted as AcDEX70@INS / GOD.

[0047] The SEM images of AcDEX10 and AcDEX70 and the particle size distribution obtained by analyzing the SEM images are shown in Figure 2 and Figure 3 The SEM image of AcDEX10 shows that there are defects and irregular protrusions on the surface of some microspheres, and agglomeration occurs, and the average particle size is 9.96 ± 3.04 μm. Compared with AcDEX10, AcDEX70 exhibits a more regular spherical shape, a stable particle structure, and a smooth surface, and the particle size is more uniform and normally distributed, and the average particle size is 15.57 ± 8.24 μm.

[0048] Application Experiment 2: Determination of insulin loading, entrapment efficiency, and drug loading

[0049] In the construction of acetalized dextran microparticles loaded with insulin and glucose oxidase, the final mixture (the reaction solution after stirring for two hours in step (2)) was centrifuged at 8000 rpm for 15 min, and the supernatant was collected. The concentration of free insulin in the supernatant was determined at 595 nm using a microplate reader, and the mass of free insulin in the supernatant was calculated, and the entrapment efficiency (EE) and drug loading (DL) were calculated according to the following formulas.

[0050]

[0051] The results of the determination of the encapsulation efficiency and drug loading of insulin by AcDEX10 microspheres and AcDEX70 microspheres are shown in Table 1. Among them, the acetalized dextran prepared by 10 kDa and 70 kDa molecular weight dextran is represented by AcDEX10 and AcDEX70 respectively, insulin is represented by INS, and glucose oxidase is represented by GOD.

[0052] Table 1 Determination of the loading and encapsulation efficiency and drug loading of insulin

[0053]

[0054] Note: The preparation method of AcDEX 10@INS and AcDEX 70@INS described in Table 1 is carried out according to Example 1, but no glucose oxidase is added.

[0055] From Table 1, it can be seen that the insulin encapsulation efficiency and drug loading of AcDEX microspheres with added glucose oxidase are higher than those of microspheres with only insulin, and the higher the molecular weight of AcDEX, the higher the drug loading. However, it is found that as the molecular weight further increases, the long polymer chain of the dextran with too high molecular weight leads to a significant increase in the viscosity of the solution, which hinders the emulsification process when preparing microparticles by emulsification method. According to the experimental results, the drug loading of AcDEX70 microspheres for insulin can reach 8.52±1.28%, which has a higher drug loading rate. Therefore, in the present application, the molecular weight of glucose is 70 kDa at most, and at this time the prepared controlled release system has a high insulin loading rate and a long release period.

[0056] Application Experiment 3: Insulin response release curve test of acetalized dextran microspheres in different pH solutions

[0057] The empty AcDEX microspheres were suspended in PBS (pH=7.4) or 0.3M acetic acid buffer (pH=5) at a concentration of 5mg / mL, and stirred at room temperature at a speed of 100rpm. The degradation of AcDEX in the reagent bottle was observed at 0, 8 and 24h, and photographed.

[0058] 75mg of AcDEX10@INS and AcDEX70@INS loaded only with insulin were placed in 5mL of PBS (pH=7.4) or acetic acid buffer (pH=5) respectively, and incubated on a 37°C shaking table. At the preset time points (0, 2, 4, 6, 8, 12, 24, 36 and 48h), the samples were taken out and centrifuged, 200μL of supernatant was aspirated, and 200μL of the original solution was added for continued incubation. The insulin content of the supernatant was determined by Coomassie Plus protein assay method, and the insulin release curve of acetalized dextran microspheres in different pH solutions was calculated and plotted.

[0059] Acetalized dextran is acid-sensitive; it maintains its original form under physiological conditions (pH=7.4), but undergoes hydrolysis under weakly acidic conditions (pH=5). For example... Figure 4 As shown in Figure A, the AcDEX70 microspheres remained an opaque suspension in PBS buffer at pH 7.4, and their state was no different from when they were first soaked after 24 hours. Conversely, the AcDEX70 microsphere suspension continued to degrade in acetate buffer at pH 5. After 8 hours of soaking, the number of suspended particles had decreased, and after 24 hours, the suspension became transparent, indicating that the microspheres had been hydrolyzed into soluble dextran.

[0060] like Figure 4 As shown in B and C, AcDEX10@INS, when incubated in a solution at pH 5, rapidly hydrolyzes and releases insulin within 12 hours, with the insulin release rate of the microspheres reaching its maximum at 24 hours. AcDEX70@INS exhibits a slower insulin release rate in a solution at pH 5 than AcDEX10@INS, but its insulin release cycle is longer, continuously releasing insulin for up to 36 hours.

[0061] The above experimental results demonstrate that acetalized dextran has excellent pH responsiveness and can be hydrolyzed and release insulin under acidic conditions. The slower the hydrolysis rate of the microspheres using AcDEX70, the longer the release cycle.

[0062] Application Experiment 4: In vitro glucose-responsive insulin release assay of acetalized dextran microspheres

[0063] 50 mg of AcDEX10@INS / GOD and AcDEX70@INS / GOD, loaded with insulin and glucose oxidase, were placed in 5 mL of PBS solution containing different glucose concentrations (0, 100, and 400 mg / dL), respectively, and incubated on a shaker at 37 °C. The samples were removed and centrifuged at preset time points (0, 2, 4, 6, 8, 12, 24, 36, and 48 h), and 200 μL of supernatant was aspirated. Then, 200 μL of the corresponding glucose solution was added and incubation continued. Insulin content was measured using the Coomassie Plus protein assay, and insulin release curves were calculated and plotted.

[0064] like Figure 5 As shown in Figure A, AcDEX10@INS / GOD microspheres, when immersed in a 400 mg / dL glucose solution, exhibited an insulin release rate of 81.57 ± 3.81% after 12 hours; its insulin release rate was slower in a 100 mg / dL glucose solution, with a maximum release rate of 62.57 ± 2.56%; and it released almost no insulin in a glucose-free solution. Figure 5As shown in Figure B, AcDEX70@INS / GOD microspheres, when immersed in a 400 mg / dL glucose solution, continuously released insulin over 48 hours, with a maximum release rate of 91.36 ± 1.28%; while the maximum insulin release rate when immersed in a 100 mg / dL glucose solution was 65.36 ± 2.94%. These results indicate that high molecular weight acetalized dextran microparticles can achieve a sustained-release therapeutic effect.

[0065] Application Experiment 5: Protective Effect of δ-MnO2 on Insulin Activity

[0066] AcDEX70@INS / GOD was prepared according to the steps in Example 1, with the amounts of δ-MnO2 added being 0 mg, 2.5 mg, 5 mg, and 10 mg, respectively.

[0067] 50 mg of the prepared acetalized dextran insulin microspheres were placed in 5 mL of PBS solution with a glucose concentration of 400 mg / dL. After 48 hours, the supernatant was collected. The supernatant was then analyzed by reversed-phase high-performance liquid chromatography to determine the amount of undegraded insulin.

[0068] like Figure 6 As shown, without the addition of δ-MnO2, insulin is inactivated by H2O2, with an activity of only 52%. After the addition of δ-MnO2, the activity of insulin is greatly improved. When 5 mg of δ-MnO2 is added, the activity of insulin can reach 92%, which proves the protective function of δ-MnO2 on insulin activity.

[0069] In summary, this invention prepares acetalized dextran microparticles by using dextran of different molecular weights, identifies the relationship between different molecular weight dextrans and insulin loading rate and release cycle, optimizes the preparation strategy of acetalized dextran, and designs a composite with manganese dioxide (MnO2) nanosheets to efficiently protect insulin activity without affecting insulin response release.

[0070] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a pH / blood glucose dual-responsive insulin controlled release system, characterized in that, The method comprises the following steps: (1) Weighing insulin and dissolving it in carbonate buffer solution, then adding glucose oxidase, and ultrasonic dissolving to obtain a mixed solution; (2) Adding the mixed solution in step (1) and manganese dioxide nanosheet into an acetalized dextran / dichloromethane solution, ultrasonic dissolving until the mixed solution becomes milky white, then adding it into a polyvinyl alcohol solution to obtain a reaction solution and stirring at room temperature; then centrifuging and precipitating to obtain a white powder, and freeze-drying the white powder to obtain acetalized dextran insulin microspheres, which are the pH / blood glucose dual-responsive insulin controlled release system.

2. The production method according to claim 1, characterized by, The concentration of insulin in the mixed solution in step (1) is 5-20wt%; The concentration of glucose oxidase in the mixed solution in step (1) is 1-5wt%.

3. The production method according to claim 1, characterized by, The acetalized dextran in step (2) is prepared by the following steps: completely dissolving dextran in DMSO, then adding pyridine p-toluenesulfonate and 2-methoxy propene for reaction, adding triethylamine into the reaction solution to quench the reaction after a period of time, centrifuging and discarding the supernatant, washing the precipitate with water, and freeze-drying the product to obtain acetalized dextran.

4. The production method according to claim 3, characterized by, The mass ratio of pyridine p-toluenesulfonate to 2-methoxy propene is 1:100-250, and the mass ratio of pyridine p-toluenesulfonate to glucose is 1:1-5; The molecular weight of the glucose is 10-70kDa; The reaction time is 5-7h; The mass ratio of 2-methoxy propene to triethylamine is 1:3-7.

0.

5. The method of claim 1, wherein, The manganese dioxide nanosheet in step (2) is prepared by the following steps: adding tetramethylammonium hydroxide pentahydrate dissolved in H2O2 solution into an aqueous solution of MnCl2·4H2O, stirring, then centrifuging, washing, and drying, then adding the product into water, ultrasonic dissolving, and then centrifuging to obtain δ-MnO2, which is the manganese dioxide nanosheet.

6. The production method according to claim 5, wherein The molar ratio of tetramethylammonium hydroxide pentahydrate to MnCl2·4H2O is 4:1; The stirring time is 24-36h, the drying time is 12-24h, and the ultrasonic time is 10-15h.

7. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of insulin, glucose oxidase, manganese dioxide nanosheet, acetalized dextran, and polyvinyl alcohol is 5:4:(0-6):9:

75.

8. The method of claim 1, wherein, The ultrasonic time in step (2) is 80-100s; The mass fraction of the manganese dioxide nanosheet in the reaction solution in step (2) is 0-1wt%; The stirring time of the reaction solution at room temperature in step (2) is 1-3h.

9. A pH / blood glucose dual-responsive insulin controlled release system prepared by the preparation method of any one of claims 1-8.

10. The use of the pH / blood glucose dual-responsive insulin controlled release system of claim 9 in the preparation of a drug for treating diabetes.

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