Polymer, insulin sustained release preparation containing polymer and application

Abstract

The invention discloses a polymer, the polymer comprises a diphenylboronic acid structural unit capable of being combined with glucose and a hydrophilic polymerizable structural unit, and the diphenylboronic acid structural unit can improve the combining capacity and selectivity of glucose. The invention further discloses an insulin sustained release preparation containing the polymer, the preparation has different insulin release rates under different glucose concentrations, insulin release is accelerated in a hyperglycemia environment to reduce blood sugar, and the preparation can reduce insulin wrong release caused by fructose and other diol substances. The invention also discloses a preparation method of the insulin sustained-release preparation. The insulin sustained-release preparation is prepared by self-assembling negatively charged insulin and positively charged bisphenylboronic acid modified polymer through electrostatic interaction, and the insulin sustained-release preparation has the characteristic of high load rate. The prepared insulin sustained-release preparation can release insulin, and the blood glucose level can be reduced after the released insulin is combined with an insulin receptor in a body.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a polymer, an insulin sustained-release preparation containing the polymer, and applications thereof. Background Art

[0002] Diabetes, as a chronic disease, affects more than 537 million people worldwide. At present, the main method of insulin delivery is still subcutaneous direct injection of insulin solution, which has problems in insulin storage, administration comfort, and hypoglycemia side effects. There is an urgent need for a stable, safe, and long-acting closed-loop insulin delivery system to improve patient compliance. However, commercially available insulin pumps and the insulin microneedle patches and insulin complexes under development still have defects and challenges in terms of insulin stability, drug loading rate, release controllability, and response selectivity.

[0003] Phenylboronic acid (PBA) that responds to glucose is widely used to construct insulin complexes. However, the commonly used phenylboronic acid structure has a lower binding ability to glucose than fructose, salicylic acid, tetracycline and other common diol interferents in the body. Taking fructose as an example, the concentration of fructose in the human body is usually less than 1 mg / dL. However, after consuming a large amount of fructose in a short period of time (such as drinking beverages with high fructose content and consuming a lot of fruit), the fructose level in the body can rise to 18 mg / dL, which may lead to competitive binding between fructose and insulin complexes with PBA as the response unit, causing the erroneous release of insulin and thus leading to hypoglycemia.

[0004] Publication No. CN118255997A discloses a polymer, an insulin composition containing the polymer, and its application. The polymer is composed of three different structural units, wherein structural unit III contains a phenylboronic acid structure. However, in the polymer constructed by the present invention, one monomer contains only one phenylboronic acid, and this structure may cause the insulin compound to bind to fructose, etc., causing the wrong release of insulin.

[0005] Studies have shown that diphenylboronic acid (DBA) with a suitable structure can improve its binding ability and selectivity to glucose. Therefore, the strategy based on the DBA glucose response unit can improve the specific release of insulin while reducing the erroneous release of insulin.

[0006] Reference 1 (Zuo Minzan, Wang Leyong. Multiresponsive Supramolecular Theranostic Nanoplatform Based on Pillar[5]arene and Diphenylboronic Acid Derivatives for Integrated Glucose Sensing and Insulin Delivery. Small, 14, e1801942, 2018, doi: 10.1002 / smll.201801942) discloses a diphenylboronic acid derivative with a specific structure, which optimizes its selective recognition of glucose, and then uses water-soluble pillar arene and diphenylboronic acid derivatives to self-assemble through host-guest interaction to form water-soluble supramolecular vesicles, and encapsulate insulin and glucose oxidase to construct a multi-responsive supramolecular integrated nanoplatform for glucose recognition and insulin delivery. In a hyperglycemic environment, the vesicles that synergistically encapsulate insulin and glucose oxidase can selectively recognize glucose, thereby causing the destruction of the vesicle structure and releasing insulin to regulate blood sugar levels. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a polymer, which comprises a biphenylboronic acid structural unit capable of binding to glucose, and the structural unit can improve the binding ability and selectivity to glucose.

[0008] A polymer, the structure of which is shown in formula (1),

[0009]

[0010] A is One of For connection location

[0011] R 1 , R 2 are independently selected from hydrogen atoms, C 1 ~C 6 At least one of an alkyl group or a halogen group; R 3 , R 4 Each independently selected from C 1 ~C 6 Alkyl, n=5-800, m=1-100.

[0012] Glucose in the form of α-furanose is a special sugar compound that can bind two boronic acid moieties through its two pairs of binding sites at the 1,2- and 3,5,6-positions. In the present invention, the polymer comprises a structural unit having two phenylboronic acid structures at the same time. This double phenylboronic acid structure can bind to two pairs of cis-diols (1,2- and 5,6-cis-diols of glucose) in a synergistic manner, thereby achieving selective recognition of glucose.

[0013] In the present invention, when an electron-withdrawing group (halogen) is introduced into the benzene ring of phenylboronic acid in formula (1), the electron cloud density is reduced due to the inductive effect, the Lewis acidity of the boronic acid group is stronger, the pKa is reduced, and the boronic acid group is more inclined to be converted into a tetrahedral form, which can generate a stable and hydrophilic phenylboronic acid ester with glucose. The synthesis of this type of monomer only requires replacing the phenylboronic acid raw material with a phenylboronic acid raw material containing an alkyl or halogen substitution.

[0014] Preferably, the structure of the polymer is as follows,

[0015]

[0016] Wherein, n=5~800, m=1~100, For the connection location.

[0017] The present invention also provides a method for preparing the polymer, which is simple and convenient to perform addition polymerization on carbon-carbon double bonds on different monomers through free radical copolymerization.

[0018] A method for preparing a polymer comprises the following steps: subjecting a monomer containing A, a monomer containing bisphenylboronic acid and an initiator to a free radical copolymerization reaction in a solvent, dialyzing and freeze-drying to obtain a polymer, wherein the structures of the monomer containing A and the monomer containing bisphenylboronic acid are respectively shown in formula (2) and formula (3),

[0019]

[0020] A is One of

[0021] R 1 , R 2 are independently selected from hydrogen atoms, C 1 ~C 6 At least one of an alkyl group or a halogen group; R 3 , R 4 Each independently selected from C 1 ~C 6 of alkyl.

[0022] Preferably, the molar ratio of the monomer containing biphenylboronic acid to the monomer containing A is 1-2:3-19.

[0023] In the present invention, by adjusting the molar ratio of the monomer containing A to the monomer containing bisphenylboronic acid, a polymer having a bisphenylboronic acid content of 5% to 40% can be obtained.

[0024] Preferably, the initiator is an azo initiator or an inorganic peroxide initiator.

[0025] In the present invention, the azo initiator may be azobisisobutyronitrile (AIBN), azobisisoheptanenitrile (ABVN), etc., and the inorganic peroxide initiator may be hydrogen peroxide, ammonium persulfate, potassium persulfate, etc.

[0026] Preferably, the solvent is one or more of alcohol or water.

[0027] Preferably, the volume ratio of the total mass of the monomer containing A and the monomer containing biphenylboronic acid to the solvent is 100-400 mg / mL.

[0028] Preferably, the reaction temperature of the free radical copolymerization reaction is 35-75° C., and the reaction time is 3-10 h.

[0029] Preferably, the dialysis step is to dialyze the solution after the reaction using a 3500-15000 kDa dialysis membrane for at least 5 times, and each dialysis time is 6-8 hours.

[0030] The present invention also provides an insulin sustained-release preparation comprising the above polymer, which has different insulin release rates under different glucose concentrations, accelerates insulin release to lower blood sugar under high blood sugar conditions, and can reduce erroneous insulin release caused by other diol substances such as fructose.

[0031] An insulin sustained-release preparation comprises the above polymer and insulin.

[0032] In the present invention, the polymer with a positively charged quaternary ammonium salt structure can better adsorb negatively charged insulin and improve the loading rate of insulin in the insulin sustained-release preparation. Under normal blood sugar environment, the combination of glucose and phenylboronic acid groups will reduce the positive charge density on the polymer chain to release a small amount of insulin, thereby providing slow and continuous insulin and allowing high-dose injection. Under high blood sugar environment, the combination of a large number of phenylboronic acid groups with glucose will accelerate the release of insulin and thus reduce blood sugar.

[0033] Preferably, the insulin is porcine insulin, human insulin, recombinant human insulin or FITC-porcine insulin.

[0034] In the present invention, the FITC-insulin is obtained by labeling porcine insulin with fluorescein isothiocyanate.

[0035] Preferably, the mass ratio of the polymer to insulin is 1-4:1-4.

[0036] The present invention also provides a method for preparing the above-mentioned insulin sustained-release preparation, in which negatively charged insulin and positively charged diphenylboronic acid-modified polymer are self-assembled into the insulin sustained-release preparation through electrostatic interaction, and the preparation has the characteristic of high loading rate.

[0037] A method for preparing an insulin sustained-release preparation comprises the following steps: dissolving the above-mentioned polymer and insulin in acidified water, adjusting the pH to neutral with alkaline solution to form an insulin complex, adding PBS solution to adjust the insulin concentration, and resuspending after centrifugation to obtain the insulin sustained-release preparation.

[0038] Preferably, the acidified water is a hydrochloric acid aqueous solution, and the concentration of the acidified water is 0.01-1M.

[0039] Preferably, the mass volume ratio of the total mass of the polymer and insulin to the acidified water is 5 to 20 mg / mL.

[0040] Preferably, the alkali solution is a sodium hydroxide aqueous solution, and the concentration of the alkali solution is 0.01-1M.

[0041] Preferably, the centrifugal speed is 10000-20000 rpm, and the centrifugal time is 5-10 min.

[0042] The present invention also provides the use of the above-mentioned insulin sustained-release preparation in the preparation of medicines for treating diabetes. The insulin sustained-release preparation can release insulin, and the released insulin can reduce blood sugar levels after binding to insulin receptors in the body.

[0043] In the present invention, the diabetes mellitus may refer to a disease with a blood sugar level greater than normal, specifically a fasting blood sugar ≥ 7.0 mmol / L or a 2-hour blood sugar ≥ 11.1 mmol / L or a glycosylated hemoglobin ≥ 6.5%.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] (1) The polymer of the present invention comprises a biphenylboronic acid structural unit capable of binding to glucose, and the structural unit can improve the binding ability and selectivity to glucose.

[0046] (2) The insulin sustained-release preparation of the present invention has different insulin release rates under different glucose concentrations, which accelerates the release of insulin to lower blood sugar in a hyperglycemic environment, and the preparation can reduce the erroneous release of insulin caused by other diol substances such as fructose.

[0047] (3) The preparation method of the insulin sustained-release preparation of the present invention self-assembles negatively charged insulin and positively charged diphenylboronic acid-modified polymer into an insulin sustained-release preparation through electrostatic interaction, and has the characteristic of high loading rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is the specific synthesis route of the monomer containing biphenylboronic acid in Example 1.

[0049] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of compound 8 (AADBA) in Example 1.

[0050] Figure 3 It is the carbon NMR spectrum of compound 8 (AADBA) in Example 1.

[0051] Figure 4 The following is the hydrogen nuclear magnetic resonance spectrum of the polymer prepared in Example 1.

[0052] Figure 5 This is the gel permeation chromatography (GPC) spectrum of the polymer prepared in Example 1.

[0053] Figure 6 The following is the hydrogen nuclear magnetic resonance spectrum of the polymer prepared in Example 2.

[0054] Figure 7 The following is the hydrogen nuclear magnetic resonance spectrum of the polymer prepared in Example 3.

[0055] Figure 8 This is the hydrogen nuclear magnetic resonance spectrum of the polymer prepared in Example 4.

[0056] Fig. 9 This is a schematic diagram of the principle of the insulin sustained-release preparation prepared in Example 5.

[0057] Fig.10 This is the hydrogen nuclear magnetic resonance spectrum of the polymer prepared in Comparative Example 1.

[0058] Fig.11 This is a test graph of the responsiveness of the 1 mg / mL insulin sustained-release preparation obtained in Example 5 to glucose.

[0059] Fig.12 The single release curves of the insulin sustained-release preparations obtained in Examples 5, 6, 7, 9, 10 and 13 in glucose and fructose solutions, wherein A to F are the single release curves of Examples 5, 6, 7, 9, 10 and 13, respectively.

[0060] Fig.13The single release curves of the insulin sustained-release preparation obtained in Example 5 and the insulin sustained-release preparation containing monophenylboronic acid polymer obtained in Comparative Example 1 in glucose and fructose solutions, wherein A is the single release curve of Example 5, and B is the single release curve of Comparative Example 1.

[0061] Fig.14 The figures are the in vitro multiple-cycle cumulative release curves of the insulin sustained-release preparation obtained in Example 5 and the insulin sustained-release preparation containing monophenylboronic acid polymer obtained in Comparative Example 1 in glucose and fructose solutions, respectively, wherein A is the in vitro multiple-cycle cumulative release curve of Example 5, and B is the in vitro multiple-cycle cumulative release curve of Comparative Example 1. DETAILED DESCRIPTION

[0062] The present invention will be further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited to the following examples.

[0063] The raw materials used in the present invention are all commercially available.

[0064] Example 1: Preparation of polymer

[0065] The specific route of the monomer containing bisphenylboronic acid is as follows Figure 1 As shown, 2,5-dimethylbenzylamine is first protected with di-tert-butyl dicarbonate in dichloromethane to obtain compound 2, and compound 2 is further protected with di-tert-butyl dicarbonate to obtain compound 3. Compound 3 is brominated with N-bromosuccinimide to produce compound 4. Compound 4 is then reacted with dimethylamine in tetrahydrofuran to produce compound 5, which is then reacted with 2-bromomethylphenylboronic acid to produce compound 6. Compound 6 is deprotected with a 3M HBr aqueous solution to obtain compound 7, which is then reacted with acryloyl chloride in an alkaline environment to obtain compound 8. The following are the specific synthesis steps.

[0066] (1) Synthesis of Compound 2

[0067] Compound 1 (9.55 g, 71 mmol) and triethylamine (TEA, 11.3 mL, 82 mmol) were added to 100 mL of dichloromethane, di-tert-butyl dicarbonate (25.3 g, 128 mmol) was dissolved in 50 mL of dichloromethane and then slowly added to the mixture, stirred at room temperature and monitored by TLC until the reaction was complete, and the solvent was removed in vacuo after 4 hours of reaction. The crude product was recrystallized in n-hexane, washed and dried to obtain a white solid compound (13.5 g, 80%).

[0068] (2) Synthesis of Compound 3

[0069] Under nitrogen protection, compound 2 (13 g, 55 mmol), di-tert-butyl dicarbonate (14.4 g, 66 mmol) and 4-dimethylaminopyridine (DMAP, 0.67 g, 5.5 mmol) were dissolved in 100 mL of anhydrous acetonitrile, heated to 60 ° C with stirring and monitored by TLC until the reaction was complete. After reacting for 24 hours, the solvent was removed in vacuo. The product was purified by silica gel column chromatography using petroleum ether / ethyl acetate = 95:5 as the eluent to obtain a colorless oil (14.1 g, 76.0%).

[0070] (3) Synthesis of Compound 4

[0071] Compound 3 (5 g, 15 mmol), N-bromosuccinimide (NBS, 5.8 g, 33 mmol) and a trace of azobisisobutyronitrile (AIBN) were dissolved in 100 mL of carbon tetrachloride, nitrogen was bubbled for 30 minutes to remove oxygen, and heated to 65°C. During the reaction, 365 nm ultraviolet light was used to continue to initiate the reaction and the reaction was monitored by TLC. After 4 hours of reaction, the solvent was removed in vacuo. The product was purified by silica gel column chromatography using petroleum ether / ethyl acetate = 100:2 as the eluent to obtain a white solid (4.3 g, 60%).

[0072] (4) Synthesis of Compound 5: Take 6 mL of 2M tetrahydrofuran solution of dimethylamine, cool it to -78°C with a dry ice acetone bath, and cool it for 10 minutes. At the same time, dissolve compound 4 (1.08 g, 2.75 mmol) in 10 mL tetrahydrofuran, add it dropwise to the cooled dimethylamine solution under vigorous stirring, warm the reaction solution to room temperature and stir for 2 hours. After the reaction is completed, pour the reaction solution into 200 mL of ethyl acetate, wash it three times with 1 mol / L potassium carbonate aqueous solution, dry the organic phase with anhydrous sodium sulfate for 30 minutes, remove the solvent in vacuo, and obtain intermediate product 5 (0.84 g, 2.6 mmol, 95.1%).

[0073] (5) Synthesis of Compound 6

[0074] The intermediate product 5 and 2-bromomethylphenylboronic acid (2.45 g, 8.25 mmol) and potassium carbonate (1.22 g, 8.8 mmol) were dissolved in 10 mL of DMF, nitrogen was bubbled for 30 minutes to remove oxygen, and the mixture was heated to 60°C. After reacting for 12 hours, the reaction solution was added dropwise to a solution of petroleum ether / ethyl acetate = 1:2 (200 mL) for precipitation. The filtered residue was dissolved in methanol, and the sample was purified by C18 reverse phase silica gel column chromatography, eluted with 30% methanol, and the methanol was removed under reduced pressure and freeze-dried to obtain a white solid (1.08 g, 47%).

[0075] (6) Synthesis of Compound 7

[0076] Compound 6 was dissolved in 5 mL of pure water, 1 mL of hydrobromic acid was added to remove the Boc protecting group, and the mixture was stirred at room temperature overnight. The pH of the reaction solution was adjusted to 4-5 with 1 mol / L sodium hydroxide solution, and the sample was purified by C18 reverse phase silica gel column chromatography, eluted with pure water, and freeze-dried to obtain a white solid (687 mg, 98%).

[0077] (7) Synthesis of Compound 8

[0078] Compound 7 (150 mg, 0.23 mmol) was dissolved in H 2 O:THF = 2:1 mixed solvent, total solvent volume 6mL, add 146.28mg of Na 2 CO 3 (1.38mmol), the reaction flask was placed in an ice bath, 62.45mg of acryloyl chloride (0.69mmol) was dissolved in a small amount of anhydrous THF and added dropwise to the reaction flask. After the addition was completed, the reaction was continued for 3h. The pH was detected during the reaction. If the pH was acidic, sodium carbonate was added to adjust it to alkalinity. The reaction solution was decompressed to remove THF, and the pH was adjusted to 4-5 with a 0.1M HBr aqueous solution. The sample was purified by C18 reverse phase silica gel column chromatography, eluted with 30% methanol, and freeze-dried to obtain a white solid, which was compound 8 (hereinafter referred to as AADBA) (159.8mg, 98.7%), and its hydrogen spectrum and carbon spectrum were as follows Figure 2 and Figure 3 shown.

[0079] (9) Preparation of polymer

[0080] AADBA (100 mg, 0.142 mmol) was added to the reaction flask, and 1200 μL of ethanol and 60 μL of water were added as solvents. 2 Bubble to deoxygenate, add 142.03 mg of N-vinyl pyrrolidone (136.6 μL, 1.278 mmol), dissolve 2.42 mg of AIBN (1% of the total mass of AADBA and N-vinyl pyrrolidone), heat to 65°C, and react for 5 hours. Use 7000 kDa dialysis membrane for dialysis separation, change water 5 times, and freeze-dry after dialysis to obtain a polymer as a white solid (192 mg, 79.3%). The hydrogen spectrum of the polymer is as follows Figure 4 As shown, the gel permeation chromatography (GPC) spectrum of the polymer is as follows Figure 5 As shown, the weight average molecular weight (Mw) of the polymer is 25032 and the polydispersity coefficient (PD) is 1.78.

[0081] Examples 2-4: Preparation of polymers

[0082] The preparation methods of Examples 2 to 4 are the same as that of Example 1, with the differences shown in the following table.

[0083] Table 1: Differences in the preparation methods of Examples 1 to 4

[0084]

[0085] Example 5: Preparation of sustained-release insulin preparation

[0086] FITC-porcine insulin (1 mg) and the polymer obtained in Example 1 (1 mg) were dissolved in 0.2 mL of HCl aqueous solution (0.1 M), mixed and added with NaOH aqueous solution (1 M) to adjust the pH to 7.4 to obtain an insulin sustained-release preparation, the principle diagram of which is shown in FIG. Fig. 9 shown.

[0087] The volume was made up to 1 mL with PBS (10 mM, pH 7.4), and the supernatant was removed after centrifugation. The mixture was resuspended in 1 mL of PBS for washing. The washing operation was repeated three times, and the insulin concentration of the supernatant was detected each time. Finally, an insulin sustained-release preparation with an insulin concentration of 1 mg / mL was prepared for subsequent experiments.

[0088] Examples 6-13: Preparation of sustained-release insulin preparations

[0089] The preparation methods of Examples 6 to 13 are the same as that of Example 5, with the differences shown in the following table.

[0090] Table 2: Differences in the preparation methods of Examples 5 to 13

[0091]

[0092] Test method for insulin loading rate: Test the concentration of insulin in the supernatant washed three times during the preparation of the insulin sustained-release preparation in Examples 5 to 13 to obtain the total amount of insulin washed away; loading rate = (total amount of insulin added - total amount of insulin washed away) / total amount of insulin added * 100%.

[0093] As shown in Table 2, different insulin sustained-release preparations (ie, Examples 5 to 13) can be prepared by changing the mass ratio of insulin to polymer and changing the content of AADBA in the polymer. The insulin loading rates of these insulin sustained-release preparations are all higher than 70%.

[0094] Comparative Example 1: Preparation of insulin sustained-release preparation containing monophenylboronic acid polymer

[0095] (1) Preparation of phenylboronic acid compounds

[0096] Dissolve 4-bromomethylphenylboronic acid (160 mg, 0.745 mmol) in DMF (5 mL) and drop into dimethylaminopropyl acrylamide (232.8 mg, 1.49 mmol) dissolved in DMF (5 mL), and stir at room temperature for 5 hours. After the reaction is completed, drop the reaction solution into ethyl acetate with a volume of 20 times or more to precipitate. Keep stirring during the dropwise addition. After precipitation is completed, filter and dry in a vacuum drying oven to obtain a crude product. Dissolve the crude product in a small amount of water and purify the sample by C18 reverse phase silica gel column chromatography, elute with 10% methanol, and freeze-dry to obtain a white transparent solid, which is a phenylboronic acid compound (hereinafter referred to as AAPBA) (159.8 mg, 58.0%).

[0097] (2) Preparation of polymer

[0098] AAPBA (50 mg, 0.227 mmol) obtained in step (1) was added to the reaction flask, and 400 μL of ethanol and 20 μL of water were added as solvents. 2 Bubble to deoxygenate, add 101 mg (97.1 μL, 0.909 mmol) of N-vinyl pyrrolidone, dissolve 1.51 mg of AIBN (1% of the total mass of AAPBA and N-vinyl pyrrolidone), heat to 65°C, and react for 5 hours. Use 7000 kDa dialysis membrane for separation, change water 5 times, and freeze-dry after dialysis to obtain a white solid (132 mg, 87.4%). The hydrogen spectrum of the polymer is as follows Fig.10 shown.

[0099] (3) Preparation of insulin sustained-release preparations containing monophenylboronic acid polymers

[0100] FITC-porcine insulin (1 mg) and the polymer obtained in step (2) (1 mg) were dissolved in 0.2 mL of HCl aqueous solution (0.1 M), mixed and added with NaOH aqueous solution (1 M) to adjust the pH to 7.4 to obtain an insulin sustained-release preparation containing a monophenylboronic acid polymer.

[0101] The volume was made up to 1 mL with PBS (10 mM, pH 7.4), and the supernatant was removed after centrifugation. The mixture was resuspended in 1 mL of PBS for washing. The washing operation was repeated three times, and the insulin concentration of the supernatant was detected each time. Finally, an insulin sustained-release preparation with an insulin concentration of 1 mg / mL was prepared for subsequent experiments.

[0102] Sample analysis

[0103] 1. Test on the responsiveness of insulin sustained-release preparations to glucose

[0104] Drawing of standard curve: Weigh 1 mg FITC-porcine insulin in an EP tube and add 0.1 mL of HCl solution (0.1 M), add 0.15 mL of NaOH solution (0.1 M) to completely dissolve the insulin, add PBS (10 mM, pH 7.4) to make the volume 1 mL, and obtain 1 mg / mL FITC-porcine insulin. Use 1 mg / mL FITC-porcine insulin mother solution to prepare 1, 5, 10, 20, 40, 80, 160 μg / mL insulin solutions, take 100*3 μL respectively, use a microplate reader to test the fluorescence intensity at 525 nm excited by 495 nm, and finally draw a linear standard curve of fluorescence intensity-insulin concentration.

[0105] The 1 mg / mL insulin sustained-release preparation obtained in Example 5 was cyclically exposed to glucose solutions at concentrations of 100 and 400 mg / dL (n=3). At the beginning of the experiment, the insulin sustained-release preparation (equivalent to 1 mg of insulin) was centrifuged and then mixed with 1 mL of glucose solution (400 mg / dL). After incubation on a shaker at 37°C and 400 rpm for 10 minutes, the suspension was centrifuged again and the supernatant was collected to determine the insulin concentration, and then 1 mL of glucose solution (100 mg / dL) was added. After 10 minutes, the suspension was centrifuged again and the supernatant was collected to determine the insulin concentration, and then 1 mL of glucose solution (400 mg / dL) was added. This process was repeated three times. The insulin concentration of the supernatant in the EP tube was quantitatively determined using a fluorescence method, and the insulin concentration was calculated using a standard curve.

[0106] Fig.11 The responsiveness test diagram of the 1mg / mL insulin sustained-release preparation obtained in Example 5 to glucose. As shown in the figure, in three high-sugar-normal blood sugar cycles, the amount of insulin sustained-release preparation released in high-sugar solution is greater than that in normal blood sugar solution, which fully verifies that this insulin sustained-release preparation has the ability to release insulin in response to glucose, thereby providing long-acting ultra-low-dose insulin to maintain blood sugar stability under normal blood sugar, and providing a high-dose insulin to reduce blood sugar to a normal range when blood sugar rises. The amount released each time will be slightly reduced on the one hand because the stored insulin will decline with the release of insulin, and on the other hand because centrifugation causes the insulin sustained-release preparation to aggregate.

[0107] 2. Single Kinetic Release Test of Sustained-Release Insulin Preparations

[0108] First, the insulin sustained-release preparations of Examples 5, 6, 7, 9, 10 and 13 were prepared into 160 μg / mL PBS solutions as mother solutions. Samples were added to the microplate reader according to the following groups:

[0109] PBS group: 50 μL mother solution + 125 μL PBS;

[0110] 100G group: 50 μL mother solution + 17.5 μL 10 mg / mL glucose + 107.5 μL PBS;

[0111] 400G group: 50 μL mother solution + 70 μL 100 mg / mL glucose + 55 μL PBS;

[0112] 20F group: 50 μL mother solution + 3.5 μL 10 mg / mL fructose + 121.5 μL PBS;

[0113] 100F group: 50μL mother solution + 17.5μL 10mg / mL fructose + 107.5μL PBS; after preparing the solution, put the 96-well plate into the microplate reader, set the total test time to 12h, test every 5min, shake for 10s during each test, and test the fluorescence intensity at 525nm under 495nm excitation each time. Finally, the change of fluorescence intensity over time is calculated. The results are as follows Fig.12 As shown in A to F.

[0114] The 1mg / mL insulin sustained-release preparation obtained in Example 5 was respectively configured with sugar solutions of different concentrations in a 96-well plate to form a release solution with a total amount of 100μL of insulin concentration in each well of 500μg / mL. PBS, 100mg / dL glucose (100G), 400mg / dL glucose (400G), 20mg / dL fructose (20F), 100mg / dL fructose (100F), and a mixed solution of 20mg / dL fructose and 100mg / dL glucose (100G+20F) were set up for a total of 6 release groups. The insulin sustained-release preparation (1mg / mL) containing a monophenylboronic acid polymer obtained in Comparative Example 1 and sugar solutions of different concentrations were finally configured in a 96-well plate to form a release solution with a total amount of 100μL of insulin concentration in each well of 500μg / mL. Two release groups were set up for 100mg / dL fructose (100F) and 400mg / dL glucose (400G). After preparing the solution, send the 96-well plate into the microplate reader, set the total test time to 4 hours, test every 2.5 minutes, shake for 10 seconds during each test, and test the fluorescence intensity at 525nm under 495nm excitation each time. Finally, make the change of fluorescence intensity over time. The results are as follows: Fig.13 shown.

[0115] Fig.12 A to F in the figure are the single release curves of the insulin sustained-release preparations obtained in Examples 5, 6, 7, 9, 10 and 13 in glucose and fructose solutions, respectively. Fig.13 A and B in the figure are the single release curves of the insulin sustained-release preparation obtained in Example 5 and the insulin sustained-release preparation containing monophenylboronic acid polymer obtained in Comparative Example 1 in glucose and fructose solutions, respectively. Fig.12 and Fig.13 As shown, the responsiveness of Comparative Example 1 to glucose is significantly lower than that to fructose. Compared with Example 1, the responsiveness of the insulin sustained-release preparations obtained in Examples 5, 6, 7, 9, 10 and 13 to glucose and fructose are similar, and the response to glucose is even better than that to fructose, indicating that the insulin sustained-release preparation containing bisphenylboronic acid can reduce the misidentification of fructose and the like, and avoid the misrelease of insulin as much as possible.

[0116] III. In vitro multiple cycle cumulative release test of insulin sustained-release preparations

[0117] The insulin sustained-release preparation obtained in Example 5 was resuspended in PBS at pH 7.4 to obtain a final insulin concentration of 1 mg / dL. It was added to an EP tube to prepare a final glucose concentration of 100 mg / dL, 400 mg / dL and a fructose concentration of 20 mg / dL, 100 mg / dL, and a mixed group of glucose concentration of 100 mg / dL and fructose concentration of 20 mg / dL, and the final insulin concentration was 500 μg / mL (n=3). The above steps were repeated for the insulin sustained-release preparation containing monophenylboronic acid polymer obtained in Comparative Example 1 to obtain a release group with a final glucose concentration of 400 mg / dL and a fructose concentration of 100 mg / dL. Each release experiment was conducted for 1 hour, and 10 cycles were performed. After each cycle, 90% of the supernatant was centrifuged and added with the same volume and concentration of sugar solution without insulin. 100 μL of the released supernatant was added to a 96-well plate and the fluorescence intensity of FITC-insulin was detected by an enzyme-labeled instrument. The concentration of insulin was calculated using the standard curve. The drawing of the standard curve was the same as above. The cumulative release amount was calculated according to the following formula:

[0118] Q n =C 1 ×V 1 +C 2 ×V 2 ×0.9+C 3 ×V 3 ×0.9+......+C n ×V n ×0.9

[0119] Where Q n is the cumulative release amount in the nth cycle, C n is the insulin concentration of the supernatant detected in the nth cycle, V n is the total volume of the released liquid in the nth cycle.

[0120] Fig.14The in vitro multiple-cycle cumulative release curves of the insulin sustained-release preparation obtained in Example 5 and the insulin sustained-release preparation containing monophenylboronic acid polymer obtained in Comparative Example 1 in glucose and fructose solutions, respectively. In the long-term multiple cumulative release process, the insulin sustained-release preparation obtained in Example 5 still maintains the responsiveness to sugar solutions of different concentrations, and compared with Comparative Example 1, the insulin sustained-release preparation in Example 5 has better selectivity for glucose, and can reduce the erroneous release of insulin caused by fructose.

[0121] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polymer, characterized in that The structure of the polymer is shown in formula (1), A is One of For the connection location, R1 and R2 are each independently selected from a hydrogen atom, a C1-C6 alkyl group, and a halogen; R3 and R4 are each independently selected from a C1-C6 alkyl group, n=5-800, m=1-100.

2. The method for preparing a polymer according to claim 1, characterized in that: The following steps are involved: A monomer containing A, a monomer containing bisphenylboronic acid and an initiator are subjected to a free radical copolymerization reaction in a solvent, and a polymer is obtained after dialysis and freeze-drying; the structures of the monomer containing A and the monomer containing bisphenylboronic acid are shown in formula (2) and formula (3), respectively. A is One of R1 and R2 are each independently selected from at least one of a hydrogen atom, a C1-C6 alkyl group, and a halogen; R3 and R4 are each independently selected from a C1-C6 alkyl group.

3. The method for preparing a polymer according to claim 2, characterized in that: The molar ratio of the monomer containing biphenylboronic acid to the monomer containing A is 1-2:3-19.

4. The method for preparing a polymer according to claim 2, characterized in that: The initiator is an azo initiator or an inorganic peroxide initiator.

5. The method for preparing a polymer according to claim 2, characterized in that: The volume ratio of the total mass of the monomer containing A and the monomer containing biphenylboronic acid to the solvent is 100-400 mg / mL.

6. The method for preparing a polymer according to claim 2, characterized in that: The reaction temperature of the free radical copolymerization reaction is 35-75° C., and the reaction time is 3-10 hours.

7. An insulin sustained-release preparation, characterized in that: Comprising insulin and the polymer according to claim 1.

8. The insulin sustained-release preparation according to claim 7, characterized in that: The insulin is porcine insulin, human insulin, recombinant human insulin or FITC-porcine insulin; the mass ratio of the polymer to the insulin is 1-4:1-4.

9. The method for preparing the sustained-release insulin preparation according to claim 7 or 8, characterized in that: The method comprises the following steps: dissolving the polymer described in claim 1 and insulin in acidified water, adjusting the pH to neutral with alkaline solution to form an insulin complex, adding PBS solution to adjust the insulin concentration, resuspending after centrifugation, and obtaining an insulin sustained-release preparation.

10. Use of the sustained-release insulin preparation according to claim 7 or 8 in the preparation of a drug for treating diabetes.

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