Glucose and ketone body dual-response long-acting insulin delivery system as well as preparation method and application thereof

Abstract

The invention belongs to the field of drug delivery, and particularly relates to a glucose and ketone body dual-response long-acting insulin delivery system as well as a preparation method and application thereof. The glucose and ketone body dual-response long-acting insulin delivery system provided by the invention comprises a drug crystal, a polymer coating wrapping the drug crystal and a phenylboronic acid based cationic polymer modified in the polymer coating. The long-acting insulin can prolong the treatment time of the glucose-responsive insulin delivery carrier, meanwhile, the glucose-responsive insulin delivery carrier has a ketone body response behavior, ketoacidosis is inhibited, and the problems that an existing glucose-responsive insulin delivery carrier is short in treatment time, a ketone body-responsive insulin delivery carrier is difficult to construct and the like are solved.

Description

Technical Field

[0001] The present invention belongs to the field of drug delivery, and in particular relates to a glucose and ketone body dual-responsive long-acting insulin delivery system and a preparation method thereof. Background Art

[0002] Diabetes is a metabolic disease characterized by elevated blood sugar caused by defective insulin secretion and / or impaired biological action. Currently, exogenous insulin replacement is the main treatment for type 1 and late-stage type 2 diabetes. This therapy requires extremely high insulin doses and frequent blood sugar testing and insulin injections. Despite this, poor blood sugar control and the risk of hypoglycemia are still very common. Poor blood sugar control is a high-risk factor for diabetic complications, including amputation, blindness, and kidney failure. In addition, hypoglycemia can lead to behavioral and cognitive disorders, epilepsy, loss of consciousness, coma, brain damage, or death. Therefore, it is of great significance to develop a safer and more effective method for blood sugar control.

[0003] In a healthy human body, pancreatic beta cells can sense blood sugar fluctuations and use this as a response source to speed up or slow down insulin release. Therefore, researchers have developed a glucose-responsive insulin delivery system that can simulate the behavior of pancreatic beta cells releasing insulin in response to blood sugar levels in the body, greatly improving the effectiveness and safety of insulin replacement therapy. Glucose-responsive insulin delivery carriers can respond to changes in glucose concentration in the body by changing their ability to bind to glucose and undergoing structural changes such as swelling / contraction, dissolution, pore size, and degradation. Through the above-mentioned response behavior, the sugar-responsive carrier will accelerate the release of insulin when the glucose concentration in the body increases, and inhibit insulin release after the glucose concentration decreases. At present, the duration of the efficacy of glucose-responsive insulin carriers is poor, and the general treatment time is 1 to 2 days. Therefore, there is an urgent need to construct a long-acting glucose-responsive insulin delivery system.

[0004] Diabetic ketoacidosis is one of the acute complications of diabetes. Specifically, under the influence of various triggers, diabetic patients experience a significant insulin deficiency and an inappropriate increase in glucose-producing hormones. This leads to the breakdown of fatty acids to produce excessive ketone bodies, resulting in pathological changes such as hyperglycemia, hyperketosis, ketonuria, dehydration, electrolyte imbalance, and metabolic acidosis. Insulin inhibits the breakdown of fatty acids, preventing the production of excessive ketone bodies. Therefore, developing ketone-responsive insulin delivery vehicles can effectively inhibit the occurrence of ketoacidosis. However, constructing a ketone-responsive insulin delivery system remains a huge challenge, and there are currently no reports on ketone-responsive insulin delivery vehicles. Summary of the Invention

[0005] The present invention provides a long-acting insulin delivery system that is both glucose- and ketone-responsive. This system addresses the short treatment duration of existing glucose-responsive insulin delivery vehicles and the difficulty in constructing ketone-responsive insulin delivery vehicles. The drug carrier of the present invention boasts an ultra-high drug loading capacity and a long drug release time, extending the treatment duration of glucose-responsive insulin delivery vehicles while also enabling ketone-responsive behavior to inhibit the occurrence of ketoacidosis.

[0006] In one aspect, the present invention provides a method for preparing a glucose and ketone body dual-responsive long-acting insulin delivery system, characterized in that it comprises:

[0007] Monomer A undergoes in situ polymerization on the surface of drug crystals to form a polymer coating;

[0008] B covalently couples a phenylboronic acid-based cationic polymer in the polymer coating.

[0009] Specifically, in A: the mass ratio of the drug crystals to the monomers is 1:0.4-1.2;

[0010] In B: the covalent coupling is a Schiff base reaction, comprising: mixing the polymer coating with a phenylboronic acid-based cationic polymer in a buffer solution, and adding a cross-linking agent to couple the cationic polymer to the polymer coating.

[0011] Specifically, the modification ratio of phenylboronic acid groups in the phenylboronic acid-based cationic polymer is 10%-30%;

[0012] Preferably, the modification ratio of phenylboronic acid groups in the phenylboronic acid-based cationic polymer is 25%-30%.

[0013] More preferably, the modification ratio of phenylboronic acid groups in the phenylboronic acid-based cationic polymer is 30%.

[0014] Specifically, the mass ratio of the polymer coating to the phenylboronic acid-based cationic polymer is 1:0.5-1.

[0015] Specifically, the cross-linking agent is a dialdehyde small molecule with a concentration of 1-4 mM.

[0016] Specifically, the preparation method comprises:

[0017] (1) Preparation of phenylboronic acid cationic polymer;

[0018] (2) Preparation of polymer coating: dopamine, ammonium persulfate and drug were added to the buffer solution and mixed, washed and freeze-dried to obtain product 1, i.e., polymer coating;

[0019] (3) adding the product 1 to a solution of a phenylboronic acid cationic polymer to react and obtain a reaction solution;

[0020] (4) adding glutaraldehyde to the reaction solution of step (3), centrifuging the precipitate, washing, and freeze-drying;

[0021] The drug in step (2) is insulin or an insulin analog.

[0022] Specifically, in step (2), the concentration of dopamine in the buffer solution is 0.2-2 g / L, preferably 1 g / L; the concentration of ammonium persulfate is 0.2-1.2 g / L, preferably 0.6 g / L; and the concentration of insulin is 1.5-10 g / L, preferably 2.5 g / L.

[0023] Specifically, in step (3), the phenylboronic acid cationic polymer is a phenylboronic acid-based polyamino acid polymer.

[0024] Specifically, the concentration of the phenylboronic acid-based polyamino acid polymer in the reaction solution is 0.5-3.7 g / L;

[0025] Preferably, the concentration of the phenylboronic acid-based polyamino acid polymer in the reaction solution is 3.7 g / L;

[0026] Specifically, the solution concentration of product 1 in the reaction solution is 2-10 g / L;

[0027] Preferably, the solution concentration of product 1 in the reaction solution is 5 g / L.

[0028] Specifically, the mass concentration ratio of the phenylboronic acid-based polyamino acid polymer to the product 1 in the reaction solution is 1:4-1:1;

[0029] Preferably, the mass concentration ratio of the phenylboronic acid-based polyamino acid polymer to the product 1 in the reaction solution is 3:4.

[0030] Specifically, the buffer solution in step (2) is PBS buffer solution.

[0031] Specifically, the phenylboronic acid-based polyamino acid polymer described in step (3) is dissolved in PBS buffer.

[0032] Specifically, the glutaraldehyde solution is a solution with a volume concentration of 10%-50%;

[0033] Preferably, the glutaraldehyde solution is a solution with a volume concentration of 50%.

[0034] Specifically, the amount of glutaraldehyde solution added is 0.3‰-0.4‰ of the volume of the reaction solution;

[0035] Preferably, the amount of glutaraldehyde solution added is 0.36‰ of the volume of the reaction solution.

[0036] Specifically, the phenylboronic acid-based polylysine polymer is prepared from polylysine and fluorophenylboronic acid-N-hydroxysulfosuccinimide.

[0037] Specifically, the preparation method of the phenylboronic acid-based polylysine polymer includes:

[0038] 1) Dissolve polylysine in PBS buffer to obtain solution 1;

[0039] 2) dissolving fluorophenylboronic acid-N-hydroxysulfosuccinimide in dimethyl sulfoxide to obtain solution 2;

[0040] 3) Add solution 2 to solution 1 under ice bath and adjust pH to neutral;

[0041] 4) Dialysis and freeze-drying.

[0042] On the other hand, the present invention also provides a long-acting insulin delivery system prepared according to the aforementioned preparation method.

[0043] In yet another aspect, the present invention further provides a use of the aforementioned long-acting insulin delivery system in the preparation of a drug for preventing or treating diabetes.

[0044] Specifically, the diabetes is type 1 diabetes.

[0045] In yet another aspect, the present invention further provides a medicine comprising the aforementioned long-acting insulin delivery system.

[0046] Beneficial effects of the present invention:

[0047] (1) The present invention provides a long-acting insulin delivery system that is dual-responsive to glucose and ketone bodies, wherein the drug carrier has a high drug loading capacity, a long drug release time, and can prolong the treatment time of the glucose-responsive insulin delivery carrier.

[0048] (2) The present invention provides a long-acting insulin delivery system that responds to both glucose and ketone bodies, which can inhibit the occurrence of ketoacidosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Schematic diagram of the preparation method of a long-acting insulin delivery system that responds to both glucose and ketone bodies.

[0050] Figure 2 This is the H-NMR spectrum of phenylboronic acid-based polylysine polymer.

[0051] Figure 3 This is a scanning electron micrograph of the responsive polymer conformable coating.

[0052] Figure 4 The drug loading and encapsulation efficiency of the responsive polymer conformal coating.

[0053] Figure 5 Figure 2 shows the release of glucose (A) and ketone bodies (B) in response to insulin after polymer conformal coating.

[0054] Figure 6 The therapeutic effect of a long-acting insulin carrier that responds to both glucose and ketone bodies and a glargine insulin control group on diabetic mice.

[0055] Figure 7 These are the blood glucose curves of diabetic mice in the response preparation treatment group (A), insulin treatment group (B) and untreated group (C) after intraperitoneal injection of 1.5 g / kg glucose, as well as the blood glucose curves of diabetic mice in the response preparation treatment group (D) after intraperitoneal injection of 0.83 g / kg ketone bodies, the response preparation treatment group without intraperitoneal injection of ketone bodies (E) and untreated group after intraperitoneal injection of whole body (F).

[0056] Figure 8 Figure 3 shows the changes in plasma insulin content in diabetic mice after intraperitoneal injection of glucose (A) and the changes in plasma insulin content in diabetic mice after intraperitoneal injection of ketone bodies (B). DETAILED DESCRIPTION

[0057] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0058] In the following embodiments:

[0059] Poly-L-lysine: Sigma Aldrich, P2636.

[0060] Fluorophenylboronic acid-N-hydroxysulfosuccinimide (FPBA-NHS): Science Advances, 2019, 5(7):eaaw4357.

[0061] Recombinant human insulin crystals: Solarbio, I8830.

[0062] Example 1 Preparation of Responsive Formulation (Phenylboronic Acid Modified Polydopamine Coated Recombinant Human Insulin Crystals)

[0063] 1. Preparation of phenylboronic acid-based polylysine polymer

[0064] (1) Dissolve 100 mg of poly-lysine (molecular weight 30-70 kDa) in 10 mL of PBS buffer (0.01 M, pH = 7.4) to obtain solution 1;

[0065] (2) Dissolve 39.6 mg of fluorophenylboronic acid-N-hydroxysulfosuccinimide (FPBA-NHS) in 5 mL of dimethyl sulfoxide (DMSO) to obtain solution 2;

[0066] (3) Add solution 2 described in step (2) dropwise to solution 1 described in (1) under ice bath conditions, and adjust the pH to about 7 with 1M sodium hydroxide solution during the addition process;

[0067] (4) The reaction was continued for 30 min, and the product was dialyzed using a dialysis bag with a molecular weight cut-off of 3500 Da, and finally freeze-dried to obtain a phenylboronic acid-based polylysine polymer. Figure 2 As shown, it was proved that phenylboronic acid-based polylysine was successfully synthesized.

[0068] 2. Modification of phenylboronic acid-based polylysine polymer on polymer coating

[0069] (1) 8 mg of dopamine, 2.4 mg of ammonium persulfate, and 10 mg of recombinant human insulin crystals were added to 4 mL of PBS buffer, magnetically stirred for 4 h, washed three times with PBS buffer, and freeze-dried to obtain product 1, i.e., polydopamine-coated recombinant human insulin crystals;

[0070] (2) Dissolve 7.5 mg of phenylboronic acid-based polylysine polymer in 1.5 mL of PBS buffer (0.01 M, pH = 7.4) to obtain solution 3;

[0071] (3) Dissolve 10 mg of product 1 in 0.5 mL of PBS buffer (0.01 M, pH = 7.4) to obtain solution 4;

[0072] (4) Slowly add solution 3 described in (2) dropwise to solution 4 described in (3), and stir magnetically for 15 minutes to obtain a reaction solution;

[0073] (5) Add 0.72 μL of 50% glutaraldehyde solution to the reaction solution described in (4) and stir magnetically for 1 hour;

[0074] (6) The precipitate was centrifuged to the bottom of the centrifuge tube, washed three times with PBS, and freeze-dried to obtain the responsive preparation.

[0075] The polylysine modified with phenylboronic acid in the obtained responsive preparation was fluorescently labeled with Cy5 and the fluorescence was observed under scanning electron microscopy ( Figure 3 A) and confocal images ( Figure 3 B), demonstrating the coupling of phenylboronic acid-modified polylysine into the polymer coating.

[0076] Effect Experiment Example 1 Determination of drug loading and encapsulation efficiency

[0077] 1. The drug loading capacity is determined as follows:

[0078] (1) First, a standard curve of insulin solution needs to be determined. Prepare insulin solutions of 1 mg / mL, 0.75 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.1 mg / mL, 0.05 mg / mL, and 0.025 mg / mL. Measure the light absorption intensity of insulin solutions of different concentrations using a microplate reader according to the Coomassie Brilliant Blue method (detection wavelength is 595 nm). Draw a standard curve of insulin solution with insulin concentration as the horizontal axis and absorption intensity as the vertical axis.

[0079] (2) Prepare a 1 mg / mL suspension of the test formulation, adjust the pH to 2, sonicate for 20 minutes, centrifuge at 12,000 rpm for 15 minutes, and collect the supernatant. Measure the absorbance intensity of the supernatant sample using a microplate reader (detection wavelength: 595 nm) using the Coomassie Brilliant Blue method. Calculate the recombinant human insulin concentration in the test formulation based on the standard curve, which is the drug loading.

[0080] 2. The encapsulation efficiency was determined as follows:

[0081] After the reaction in Example 1, the mass of the resulting product corresponding to the preparation was weighed, and the encapsulation efficiency was calculated according to the following formula:

[0082] Encapsulation efficiency = response preparation mass × drug loading / insulin mass added in the reaction × 100%;

[0083] The results are as follows Figure 4 As shown, the drug loading of the responsive preparation prepared in Example 1 was 92.9 wt % and the encapsulation efficiency was 82.0%.

[0084] Effect Example 2 In vitro glucose and ketone body release in response to insulin

[0085] The above responsive dosage forms were tested for in vitro glucose and ketone body responsive insulin release.

[0086] Insulin release test method:

[0087] First, determine the standard curve for insulin solution. Prepare insulin solutions of 1 mg / mL, 0.75 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.1 mg / mL, 0.05 mg / mL, and 0.025 mg / mL. Measure the absorbance of these solutions at different concentrations using a microplate reader (at a wavelength of 595 nm) using the Coomassie Brilliant Blue method. Draw a standard curve for the insulin solution using insulin concentration as the horizontal axis and absorbance intensity as the vertical axis.

[0088] 5 mg of the response preparation was weighed and dispersed in 1 mL of PBS buffer. The glucose concentration of the PBS buffer was configured to 0 mg / dL, 100 mg / dL, 200 mg / dL, and 400 mg / dL, and the ketone body concentration was configured to 0 mM, 5 mM, and 10 mM. A certain volume of the response preparation suspension was taken out at a preset time and centrifuged to obtain the supernatant. The absorption light intensity of the samples at different time points in the supernatant was measured by a microplate reader according to the Coomassie Brilliant Blue method (detection wavelength was 595 nm), thereby calculating the concentration of recombinant human insulin released by the response preparation.

[0089] Depend on Figure 5 It can be seen that the release rate of the responsive preparation of the present application is accelerated with the increase of glucose and ketone body concentrations, which proves that this responsive preparation has the characteristics of glucose and ketone body responsive insulin release.

[0090] Effect Example 3 Therapeutic Effect on Type 1 Diabetic Mice

[0091] The therapeutic effect of the responsive formulation prepared in the example was evaluated on type 1 diabetic mice. The specific procedures were as follows:

[0092] 7.7 mg of the above-mentioned response preparation was weighed and dispersed in 0.7 mL of PBS buffer as an experimental sample. C57BL / 6 mice were purchased from Hangzhou Medical Institute and induced into a type 1 diabetic mouse model by intraperitoneal injection of 120 mg / kg streptozotocin. After anesthesia, 5 type 1 diabetic mice were anesthetized, and 0.1 mL of the experimental sample was injected subcutaneously into each diabetic mouse. 40 U / kg of insulin glargine was injected subcutaneously as a control. A blood glucose meter (Aviva, ACCU-CHEK) was used to measure blood glucose levels at the set time points.

[0093] like Figure 6 As shown in A, subcutaneous injection of commercial long-acting insulin glargine can only maintain normal blood sugar for 7 hours. Figure 6 As shown in B, its blood sugar can be maintained at normal levels (about 200 mg / dL) for up to 15 days. This indicates that the carrier can significantly slow down the release of insulin in the body.

[0094] Effect Example 4: Glucose and ketone body release in response to insulin in vivo

[0095] The responsive formulation of the present invention was used to evaluate the release of glucose and ketone bodies in response to insulin in vivo. The specific procedures were as follows:

[0096] Weigh 7.7 mg of the above-mentioned response preparation and disperse it in 0.7 mL of PBS buffer. C57BL / 6 mice were purchased from Hangzhou Medical Academy and induced into a type 1 diabetic mouse model by intraperitoneal injection of 120 mg / kg streptozotocin. After anesthetizing 5 type 1 diabetic mice, 0.1 mL of PBS buffer of the response formulation was injected subcutaneously into each diabetic mouse. Diabetic mice were intraperitoneally injected with 1.5 g / kg of glucose solution or 0.83 g / kg of 3-hydroxybutyric acid solution. A blood glucose meter was used to measure the blood glucose value at the set time point. In order to verify the dual-response characteristics of the response preparation, in addition to the response preparation treatment group, we also set up an insulin treatment group and an untreated group as controls.

[0097] Five type 1 diabetic mice were anesthetized and 0.1 mL of the corresponding formulation of PBS buffer was injected subcutaneously into each diabetic mouse. The diabetic mice were intraperitoneally injected with either 3 g / kg of glucose solution or 0.83 g / kg of 3-hydroxybutyrate solution. At designated time points, blood was collected from the mice's orbitals and centrifuged to obtain plasma. Plasma insulin content was measured using a human recombinant insulin ELISA kit.

[0098] like Figure 7 As shown in A, after intraperitoneal injection of 1.5g / kg glucose solution, the blood glucose of diabetic mice in the response preparation treatment group first increased slightly, then slowly decreased, and finally remained stable. Figure 7 As shown in B, the blood sugar of diabetic mice in the insulin treatment group first increased significantly and then increased slowly. Figure 7 As shown in Figure C, the blood glucose levels of the untreated diabetic mice initially rose sharply and then remained stable. This suggests that the responsive agent effectively regulates the blood glucose levels of diabetic mice, maintaining normal blood glucose levels. This is because intraperitoneal injection of glucose causes elevated blood glucose levels in the mice. Simultaneously, the responsive agent accelerates insulin release under hyperglycemic conditions, effectively controlling the elevated blood glucose levels and returning them to normal levels.

[0099] like Figure 7 As shown in D, after intraperitoneal injection of 0.83 g / kg 3-hydroxybutyric acid solution, the blood glucose level of diabetic mice in the response preparation treatment group first slowly decreased and then remained stable. Figure 7 As shown in E, without intraperitoneal injection of 3-hydroxybutyric acid solution, the blood glucose of diabetic mice in the response preparation treatment group did not decrease but remained stable. Figure 7 As shown in Figure 5, blood glucose levels in untreated diabetic mice did not decrease after intraperitoneal injection of 3-hydroxybutyrate solution, but remained stable. This suggests that 3-hydroxybutyrate accelerates insulin release in response to the formulation.

[0100] like Figure 8As shown in A, after intraperitoneal injection of 3g / kg glucose solution, the insulin content in the serum of diabetic mice in the response preparation treatment group increased with the increase of blood sugar and decreased with the decrease of blood sugar, which proves that the response preparation of the present application has the characteristics of glucose response insulin release in mice. Figure 8 As shown in Figure B, after intraperitoneal injection of 0.83 g / kg of 3-hydroxybutyric acid solution, the insulin content in the serum of diabetic mice first slowly increased and then slowly decreased, proving that the responsive preparation of the present application also has ketone body responsive insulin release characteristics in mice.

Claims

1. A method for preparing a glucose and ketone body dual-responsive long-acting insulin delivery system, characterized in that: include: Monomer A undergoes in situ polymerization on the surface of drug crystals to form a polymer coating; B covalently couples a phenylboronic acid-based cationic polymer in the polymer coating.

2. The preparation method according to claim 1, characterized in that In A: The mass ratio of the drug crystal to the monomer is 1:0.4-1.2; In B: The covalent coupling is a Schiff base reaction, comprising: mixing the polymer coating with a phenylboronic acid-based cationic polymer in a buffer solution, and adding a cross-linking agent to couple the cationic polymer to the polymer coating; The modification ratio of phenylboronic acid groups in the phenylboronic acid-based cationic polymer is 10%-30%, preferably 25%-30%, and more preferably 30%. The mass ratio of the polymer coating to the phenylboronic acid-based cationic polymer is 1:0.5-1.

3. The preparation method according to claim 2, characterized in that The cross-linking agent is a dialdehyde small molecule with a concentration of 1-4 mM.

4. The preparation method according to claim 1, characterized in that include: (1) Preparation of phenylboronic acid cationic polymer; (2) Preparation of polymer coating: dopamine, ammonium persulfate and drug were added to the buffer solution and mixed, washed and freeze-dried to obtain product 1, i.e., polymer coating; (3) adding the product 1 to a solution of a phenylboronic acid cationic polymer to react and obtain a reaction solution; (4) adding glutaraldehyde to the reaction solution of step (3), centrifuging the precipitate, washing, and freeze-drying; The drug in step (2) is insulin or an insulin analog.

5. The preparation method according to claim 4, characterized in that In step (2), the concentration of dopamine in the buffer solution is 0.2-2 g / L, preferably 1 g / L; the concentration of ammonium persulfate is 0.2-1.2 g / L, preferably 0.6 g / L; and the concentration of insulin is 1.5-10 g / L, preferably 2.5 g / L.

6. The preparation method according to claim 4, characterized in that In step (3), the phenylboronic acid cationic polymer is a phenylboronic acid-based polyamino acid polymer; the solution concentration of the phenylboronic acid-based polyamino acid polymer is 0.5-3.7 g / L, preferably 3.7 g / L; the solution concentration of the product 1 is 2-10 g / L, preferably 5 g / L.

7. The preparation method according to claim 6, characterized in that The mass concentration ratio of the solution of the phenylboronic acid-based polyamino acid polymer in step (3) to the solution of the product 1 is 1:4-1:1, preferably 3:

4.

8. The preparation method according to claim 4, characterized in that The buffer solution in step (2) is PBS buffer solution, and the phenylboronic acid-based polyamino acid polymer in step (3) is dissolved in PBS buffer solution.

9. The preparation method according to claim 4, characterized in that The glutaraldehyde solution has a volume concentration of 10%-50%, preferably 50%; the amount of the glutaraldehyde solution added is 0.3‰-0.4‰ of the volume of the reaction solution, preferably 0.36‰.

10. The preparation method according to claim 4, characterized in that The phenylboronic acid-based polylysine polymer is prepared from polylysine and fluorophenylboronic acid-N-hydroxysulfosuccinimide.

11. The preparation method according to claim 10, characterized in that: The preparation method of the phenylboronic acid-based polylysine polymer comprises: 1) Dissolve polylysine in PBS buffer to obtain solution 1; 2) dissolving fluorophenylboronic acid-N-hydroxysulfosuccinimide in dimethyl sulfoxide to obtain solution 2; 3) Add solution 2 to solution 1 under ice bath and adjust pH to neutral; 4) Dialysis and freeze-drying.

12. A long-acting insulin delivery system prepared according to the preparation method according to any one of claims 1 to 11.

13. Use of the long-acting insulin delivery system according to claim 12 in the preparation of a medicament for preventing or treating diabetes.

14. The use according to claim 13, characterized in that The diabetes is type 1 diabetes.

15. A medicine comprising the long-acting insulin delivery system according to claim 12.

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