A sugar-responsive protamine insulin, its preparation method and application

Abstract

This invention relates to a method for preparing glucose-responsive protamine insulin and its application. The method involves modifying chemical groups on the cysteine ​​residues of protamine and linking them with phenylboronic acid compounds to form a complex, which is then mixed with insulin to prepare glucose-responsive protamine insulin. This glucose-responsive insulin formulation achieves an intelligent glucose response regulation mechanism. Specifically, under normal blood glucose levels, the insulin is released slowly, while under hyperglycemic conditions, its release is accelerated, thereby achieving effective blood glucose control and preventing hypoglycemia.

Description

[0001] This application claims priority to a prior Chinese application, application number 202310987884.3, filed on August 7, 2023; the description, claims, abstract and drawings of that application are incorporated herein by reference in their entirety. Technical Field

[0002] This invention belongs to the field of drug delivery technology, and more specifically, relates to a sugar-responsive protamine insulin, its preparation method, and its application. Background Technology

[0003] Currently, diabetes affects more than 537 million people worldwide, and this number is projected to exceed 783 million by 2045. Diabetes is a common chronic disease characterized by abnormal glucose metabolism due to absolute or relative insulin deficiency, with hyperglycemia as the primary clinical manifestation. Diabetes is mainly divided into two categories: type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). T1DM is primarily caused by damage to the insulin-secreting pancreatic beta cells, and it typically occurs at a younger age, characterized by absolute insulin deficiency. T2DM is mainly caused by insulin resistance, and it is more common in the elderly, characterized by relative insulin deficiency. Both T1DM and advanced T2DM require daily injections or continuous infusions of exogenous insulin to maintain blood glucose levels within the normal range.

[0004] To reduce the frequency of daily insulin injections, protamine zinc insulin (PZI) was developed in 1936. The insulin in PZI can form a complex with excess protamine and zinc, increasing the stability of the formulation and delaying the release of insulin in the body, thus prolonging the duration of insulin's action. In 1946, the more stable neutral protamine zinc insulin (NPH) was successfully developed. NPH is mainly used to provide basal insulin, controlling blood glucose levels before meals and at night to maintain stability. Because exogenous insulin is not regulated by endogenous feedback mechanisms, even with carefully designed dosing regimens and even when patients strictly adhere to their doctor's prescribed diet and lifestyle, diabetic patients will inevitably experience symptoms of hyperglycemia or hypoglycemia.

[0005] Therefore, a glucose-responsive insulin system that mimics the mechanism of action of endogenous insulin is crucial. Such a system could provide a treatment method that adjusts the insulin release rate in real time to adapt to the patient's blood glucose level, thereby improving therapeutic efficacy and reducing adverse reactions. Currently, glucose-responsive elements used to modify insulin mainly include glucose oxidase (GO). X ), phenylboronic acid (PBA) compounds, and glucose-binding molecules.

[0006] In existing technologies, insulin delivery carriers are typically prepared by combining phenylboronic acid or its derivatives with polymers. For example, patent CN115671050A uses polyacrylamide carbonate-polycaprolactone as a raw material, with phenylboronic acid derivatives forming a phenylboronic acid ester complex with dihydroxyl groups. Liver-targeted glucose-responsive nanoparticles are then prepared via W / O / W emulsification and UV crosslinking. Other researchers have also prepared PBA-modified poly(2-aminoethyl)acrylamide based on this strategy, using this material to load insulin and verifying its glucose-responsive properties and blood glucose control effects in animals. However, the above studies all use polymer materials, which involve cumbersome preparation processes, and the biocompatibility and safety of the polymers lack clinical validation.

[0007] Compared to the polymers mentioned above, protamine has better biocompatibility and safety, and there are currently no reports on protamine insulin with glucose responsiveness. Summary of the Invention

[0008] To address the unavoidable hypoglycemia issue in existing protamine insulin systems, this invention provides a glucose-responsive protamine insulin, its preparation method, and its applications. By modifying protamine cysteine ​​with chemical groups and linking them to phenylboronic acid compounds to form a complex, and then mixing this complex with insulin, glucose-responsive protamine insulin is prepared. This results in a faster glucose response rate, enabling intelligent glucose response regulation. It releases insulin slowly at normal blood glucose levels and accelerates insulin release at high blood glucose levels, thus achieving effective blood glucose control and preventing hypoglycemia.

[0009] On one hand, the present invention provides a sugar-responsive insulin carrier comprising protamine and phenylboronic acid compounds, wherein the cysteine ​​in the protamine is directly or indirectly linked to the phenylboronic acid compounds, or the phenylboronic acid compounds directly or indirectly modify the cysteine.

[0010] Protamine is a mixture of strongly basic peptides that was originally isolated from the sperm of salmon and other fish species, but is now primarily produced via biotechnology in a recombinant form; it contains more than two-thirds L-arginine. Because protamine contains amino acids with free basic side chains, it has some buffering capacity and is therefore considered a buffer.

[0011] In some cases, protamine includes natural protamine derived from salmon, herring, or trout, or protamine synthesized in a solid phase.

[0012] This invention demonstrates that by modifying cysteine ​​in protamine, the modified protamine can be linked to phenylboronic acid compounds, thereby enabling the preparation of a glucose-responsive insulin carrier. It can bind with negatively charged insulin to prepare glucose-responsive protamine insulin. When glucose levels are high, the carrier can bind to insulin, slowly releasing insulin at normal blood glucose levels and accelerating insulin release at hyperglycemia levels.

[0013] Furthermore, the cysteine ​​in the protamine is modified with chemical groups, and these chemical groups are linked to NHS-esterified phenylboronic acid compounds via chemical bonds.

[0014] Furthermore, the chemical group is linked to the protamine via a thioether bond; the chemical bond between the chemical group and the NHS-esterified phenylboronic acid compound is an amide bond.

[0015] In some methods, the thioether bond is formed by the covalent addition reaction of the cysteine ​​in protamine with the chemical group maleimide.

[0016] In some cases, the chemical groups are linked to NHS-esterified phenylboronic acid compounds via amide bonds.

[0017] Furthermore, the chemical groups include any one or more of straight-chain or branched alkyl, hydroxy, carboxyl, amino, or mercapto groups, or any one or more of alkyl, hydroxy, carboxyl, amino, or mercapto groups substituted at any position in these groups.

[0018] Furthermore, the structural formula of the chemical group includes any one or more of Formulas I to IV:

[0019]

[0020] In some embodiments, the chemical group comprises a compound of formula I, II, III or IV that modifies cysteine ​​in protamine.

[0021] Furthermore, the phenylboronic acid compounds include phenylboronic acids modified with different groups; the modifying groups include one or more of alkyl, carboxyl, halogen, nitro and amide groups.

[0022] Furthermore, the phenylboronic acid compounds are modified with carboxyl groups.

[0023] Furthermore, the phenylboronic acid compound is a p-carboxyphenylboronic acid or an o-carboxyphenylboronic acid, which may be substituted or unsubstituted.

[0024] Furthermore, the substituent is one or more of halogen and nitro.

[0025] Furthermore, the phenylboronic acid compound is selected from one or more of the following compounds:

[0026]

[0027] Furthermore, the phenylboronic acid compound is 4-carboxy-3-fluorophenylboronic acid.

[0028] Furthermore, the phenylboronic acid compound reacts with N-hydroxysuccinimide to generate NHS-esterified phenylboronic acid compound.

[0029] Furthermore, N-hydroxysuccinimide reacts with 4-carboxy-3-fluorophenylboronic acid to activate the carboxyl group of 4-carboxy-3-fluorophenylboronic acid, enabling it to further react with the amino group on the chemical group to form an amide bond.

[0030] In some methods, the link between protamine and N-hydroxysuccinimide (NHS) esterified phenylboronic acid compounds can be a direct or indirect link between a single cysteine ​​and the phenylboronic acid compound, or a direct or indirect link between each cysteine ​​of multiple cysteine ​​groups and the phenylboronic acid compound.

[0031] In some embodiments, the protamine contains nine cysteine ​​residues, which may be 1 to 9 cysteine ​​residues directly or indirectly linked to phenylboronic acid compounds.

[0032] In some embodiments, the chemical group modifies the sulfhydryl group of cysteine ​​in protamine as shown in the following reaction formula:

[0033]

[0034] In some embodiments, the reaction formula for the chemically modified protamine with NHS-esterified 4-carboxy-3-fluorophenylboronic acid is shown below (chemical group is formula II):

[0035]

[0036] Furthermore, the insulin includes any one or more of recombinant human insulin, lispro insulin, aspart insulin, detemir insulin, glargine insulin, degludec insulin, and icodec insulin, including ultra-long-acting, long-acting, short-acting, and rapid-acting insulin.

[0037] On the other hand, the present invention provides a method for preparing a sugar-responsive insulin carrier, the method comprising the following steps:

[0038] (1) The compound solution reacts with the protamine solution to obtain solution A;

[0039] (2) Solution A was reacted with NHS-esterified phenylboronic acid compound solution to prepare a sugar-responsive insulin carrier.

[0040] The structural formula of the compound includes any one or more of Formulas I to IV:

[0041]

[0042] Furthermore, the preparation method includes the following steps:

[0043] (1) React protamine with a compound of formula I, formula II, formula III or formula IV or other chemical groups that modify cysteine ​​in protamine; thereby attaching chemical groups to cysteine ​​in the protein.

[0044] (2) The modified protamine is chemically linked to NHS-esterified phenylboronic acid compounds, and the phenylboronic acid compounds are directly or indirectly linked to cysteine ​​in protamine through the modified groups to prepare a sugar-responsive insulin carrier.

[0045] In some methods, step (1) involves reacting the compound solution as shown in Formula I, Formula II, Formula III or Formula IV with a protamine solution, wherein the solvent of the compound solution or protamine solution is one or more of the following that can dissolve protamine and the compound solution: PBS (0.01M, pH=7.4), PBS (0.1M, pH=7.4), ultrapure water, deionized water, double-distilled water, etc.

[0046] In some methods, the preferred solvent is PBS (0.01M, pH=7.4) solution.

[0047] Further, the molar ratio of the compound shown in formula I, II, III or IV in step (1) to protamine is (1-50):1.

[0048] Preferably, the molar ratio is 18:1.

[0049] In some methods, the molar ratio of the compound to protamine is 18:1.

[0050] In some methods, the temperature of the reaction in step (1) is 4 to 40°C.

[0051] In some methods, the reaction described in step (1) is preferably carried out at room temperature.

[0052] In some methods, step (1) further includes purifying the chemically modified protamine solution, the purification process preferably including dialysis and drying.

[0053] The dialysis can be performed in deionized water using a dialysis bag that is conventional in the art, and the molecular weight cutoff of the dialysis bag is preferably 1k to 3.5k.

[0054] The drying process is preferably freeze-drying.

[0055] In some embodiments, the concentration of the protamine solution is preferably 0.2 to 100 mg / mL, for example 10 mg / mL.

[0056] In some embodiments, the protamine sulfate coupled with phenylboronic acid compounds in step (2) is obtained by grafting protamine sulfate with NHS-esterified phenylboronic acid compounds. Specifically, in the protamine sulfate coupled with phenylboronic acid compounds, the phenylboronic acid compounds are linked to chemical groups via amide bonds, and then the chemical groups modifying the phenylboronic acid compounds are grafted onto the cysteine ​​side chains of the protamine sulfate via thioether bonds.

[0057] The molar ratio of the protamine sulfate to the NHS-esterified phenylboronic acid compound is preferably 1:(1-54).

[0058] In some embodiments, the solvent in the protamine solution can be a conventional aqueous solvent in the art, which can dissolve protamine, including PBS (0.01M, pH=7.4), PBS (0.1M, pH=7.4), ultrapure water, deionized water, double-distilled water, etc., preferably PBS (0.01M, pH=7.4).

[0059] In some methods, the concentration of the protamine solution is preferably 1 to 200 mg / mL, for example 10 mg / mL.

[0060] In some embodiments, the solvent in the solution of the NHS-esterified phenylboronic acid compound can be a conventional water-miscible organic solvent that can dissolve the phenylboronic acid compound, preferably dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF), such as DMSO.

[0061] In some embodiments, the carboxyl-modified phenylboronic acid compound is selected from one or more of the following compounds:

[0062]

[0063] In some embodiments, the phenylboronic acid compound is 4-carboxy-3-fluorophenylboronic acid.

[0064] In some embodiments, the carboxyl-modified phenylboronic acid compound reacts with N-hydroxysuccinimide (NHS) to generate NHS-esterified phenylboronic acid compounds.

[0065] In some methods, the mixing is preferably performed by adding a solution of NHS-esterified phenylboronic acid compounds dropwise to a solution of protamine sulfate modified with chemical groups.

[0066] In some methods, the mixing process further includes one or more of the following: adjusting the pH to alkaline, stirring, adjusting the pH to acidic, dialysis, or lyophilization.

[0067] In some embodiments, the alkalinity is preferably in the pH range of 7.5 to 13.5, for example, pH 13.

[0068] In some embodiments, the solution used to adjust the pH to alkalinity is preferably a sodium hydroxide solution, a sodium carbonate solution, or a sodium bicarbonate solution, such as a sodium hydroxide solution.

[0069] In some methods, the stirring time is preferably 2 min to 24 h, for example 30 min.

[0070] In some embodiments, the acidity is preferably in the pH range of 2.0 to 6.5, for example, pH 2.

[0071] In some embodiments, the solution used to adjust the pH to acidity is preferably a hydrochloric acid solution, an acetic acid solution, or trifluoroacetic acid, such as a hydrochloric acid solution.

[0072] In some methods, the dialysis can be performed in deionized water using a dialysis bag conventional in the art. The molecular weight cutoff of the dialysis bag is preferably 1k to 10k. The purpose of the dialysis is to remove free NHS-esterified phenylboronic acid compounds.

[0073] In some embodiments, when the grafting reaction is followed by a dialysis step, the lyophilization is performed after the dialysis. The lyophilized product yields a white solid, which may be in powder or flocculent form.

[0074] In another aspect, the present invention provides a sugar-responsive protamine insulin complex, which is prepared by mixing and reacting a sugar-responsive insulin carrier and insulin as described above.

[0075] The glucose-responsive protamine insulin complex provided by this invention is an amorphous flocculent precipitate that can form an insulin reservoir upon subcutaneous injection. Under hyperglycemic conditions, the high concentration of glucose binding to phenylboronic acid groups immediately reduces the positive charge density of phenylboronic acid-modified protamine, breaking the phenylboronic acid ester bonds, thereby reducing the electrostatic attraction between insulin and the polymer and promoting insulin release. Under normal blood glucose conditions, the strong electrostatic force causes the protamine insulin complex to release a very small amount of insulin, providing a slow and sustained insulin release and reducing the occurrence of hypoglycemia.

[0076] In another aspect, the present invention provides a method for preparing a sugar-responsive protamine insulin complex, which includes the following steps: mixing the sugar-responsive insulin carrier solution as described above with an insulin solution in water, adjusting the pH to 6.5-8.0, and obtaining the sugar-responsive protamine insulin complex.

[0077] In some embodiments, the pH is preferably 7.4.

[0078] In some embodiments, the mass ratio of the sugar-responsive insulin carrier solution to the insulin solution is (0.5-10):1, preferably (1-2):1, for example 1:1, 2:1 or 4:1.

[0079] In some embodiments, the concentration of the sugar-responsive insulin carrier solution is 1–200 mg / mL, for example, 10 mg / mL.

[0080] In some embodiments, the preparation method of the sugar-responsive insulin carrier solution includes the following steps: mixing the sugar-responsive insulin carrier with weakly acidic water to obtain the sugar-responsive insulin carrier solution; wherein the pH range of the weakly acidic water is 2.0 to 7.0, preferably 2.0 to 3.0; the weakly acidic water can be hydrochloric acid solution, phosphate buffer, deionized water or pure water, such as hydrochloric acid solution.

[0081] In some methods, the concentration of the insulin solution may be 1 to 100 mg / mL, for example 10 mg / mL.

[0082] In some methods, the preparation of the insulin solution includes the following steps: mixing insulin with weakly acidic water to obtain an insulin solution; wherein the pH range of the weakly acidic water is 2.0 to 7.0, preferably 2.0 to 3.0; the weakly acidic water can be hydrochloric acid solution, phosphate buffer, deionized water or pure water, such as hydrochloric acid solution.

[0083] In some methods, the glucose-responsive insulin carrier solution is mixed with the insulin solution, and the pH is adjusted to 6.5–7.4, for example, to 7.4.

[0084] In some methods, the formation of a white flocculent precipitate after pH adjustment is the glucose-responsive protamine-insulin complex. This glucose-responsive protamine-insulin complex is soluble or readily soluble in weakly acidic water, and insoluble or slightly soluble in weakly alkaline water.

[0085] In some methods, the pH adjustment may further include a centrifugation step, the purpose of which is to collect a white flocculent precipitate and remove unreconstituted insulin from the supernatant.

[0086] In some embodiments, the diabetes may be type 1 diabetes or type 2 diabetes; preferably type 1 diabetes or late-stage type 2 diabetes.

[0087] In some methods, the sugar-responsive protamine insulin complex can be resuspended in phosphate buffer or saline and administered via subcutaneous injection.

[0088] Without violating common sense in the field, the conditions of the above-described embodiments can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0089] In this invention, "room temperature" refers to 18℃~25℃.

[0090] The reagents and raw materials used in this invention are all commercially available.

[0091] The beneficial effects of this invention are as follows:

[0092] 1. The protamine analog modified with phenylboronic acid designed in this invention is a biodegradable positively charged polymer capable of charge reversal, which binds to insulin to achieve stable and efficient glucose-responsive insulin release.

[0093] 2. The use of protamine sulfate, which is already in clinical use, has solved the biocompatibility and safety issues of insulin-carrying systems, and has improved insulin stability and prolonged blood glucose control time.

[0094] 3. By selecting appropriate compounds to modify protamine, the constructed glucose-responsive insulin carrier exhibits a faster and more sensitive glucose response rate after being combined with insulin. Attached Figure Description

[0095] Figure 1 The 1H NMR spectrum of 4-carboxy-3-fluorophenylboronic acid-N-hydroxysuccinimide (FPBA-NHS);

[0096] Figure 2 The 1H NMR spectrum of the reaction product (compound 2) of protamine modified with chemical group formula I and FPBA-NHS;

[0097] Figure 3 The 1H NMR spectrum of compound 4 in Example 2;

[0098] Figure 4 The 1H NMR spectrum of compound 6 in Example 3;

[0099] Figure 5 The 1H NMR spectrum of compound 8 in Example 4;

[0100] Figure 6 This is a transmission electron microscope image of glucose-responsive protamine insulin complex I in Example 5;

[0101] Figure 7 This is a transmission electron microscope image of the sugar-responsive protamine-insulin complex III in Example 5;

[0102] Figure 8 The glucose-responsive insulin release curve of glucose-responsive protamine insulin complex I in Example 5;

[0103] Figure 9 The glucose-responsive insulin release curve of glucose-responsive protamine insulin complex III in Example 5;

[0104] Figure 10 The results show the blood glucose levels in diabetic mice after administration of commercially available protamine insulin (NPH) in Example 6.

[0105] Figure 11 The results show the blood glucose levels in diabetic mice after administration of glucose-responsive protamine insulin complex I in Example 6.

[0106] Figure 12 The results show the blood glucose levels in diabetic mice after administration of glucose-responsive protamine insulin complex III in Example 6. Detailed Implementation

[0107] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0108] Example 1: Preparation of a sugar-responsive insulin carrier by modifying protamine with chemical formula I and 4-carboxy-3-fluorophenylboronic acid, thereby preparing a sugar-responsive protamine insulin complex.

[0109] The preparation process of the sugar-responsive protamine insulin complex provided in this embodiment is as follows:

[0110] 1. Preparation of 4-carboxy-3-fluorophenylboronic acid-N-hydroxysuccinimide (FPBA-NHS)

[0111] Chemical reaction formula for the synthesis of FPBA-NHS:

[0112]

[0113] Weigh 10 g of 4-carboxy-3-fluorophenylboronic acid (FPBA) and 10 g of N-hydroxysuccinimide (NHS) into a 200 mL round-bottom flask, add 150 mL of N,N-dimethylformamide (DMF), then add 15 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), 2.2 g of 4-dimethylaminopyridine (DMAP), and 12.5 mL of triethylamine to the system. React in an ice-water bath for 2 days. After 2 days, transfer the pale pink mixture to a 250 mL separatory funnel, add 150 mL of dichloromethane, and extract three times with 50 mL of 1 mol / L HCl. Separate the layers, collect the lower dichloromethane layer, and add an appropriate amount of anhydrous MgSO4 to remove excess water. After removing anhydrous MgSO4 by filtration, dichloromethane was removed by rotary evaporation, yielding a white powdery product with the following structural formula: Using deuterated dimethyl sulfoxide as a solvent, through 1 Characterized by H-NMR (see [reference]). Figure 1 ).

[0114] 2. Preparation of protamine sulfate modified with chemical group formula I

[0115] Chemical reaction formula for protamine modified with chemical group formula I:

[0116]

[0117] Weigh 50 mg of protamine (1.0 eq, 5 ml PBS) and 120 mg of MAL-KK-NH2 (18.0 eq, 5 ml PBS) into a 25 mL round-bottom flask and react overnight at room temperature. The resulting mixture was dialyzed four times with 4 L of deionized water. The product was then freeze-dried to obtain a white powder, compound 1.

[0118] 3. Preparation of glucose-responsive insulin carriers

[0119] The chemical reaction formula for protamine modified with chemical group formula I and FPBA-NHS is as follows:

[0120]

[0121] The preparation process is as follows:

[0122] Compound 1 (1.0 eq) was dissolved in 5 mL of deionized water to obtain an aqueous solution of compound 1. Separately, 4-carboxy-3-fluorophenylboronic acid-N-hydroxysuccinimide (FPBA-NHS, 2.0 eq) was dissolved in 5 mL of dimethyl sulfoxide (DMSO) to obtain an FPBA-NHS solution. The aqueous solution of compound 1 was cooled in an ice bath, and the FPBA-NHS solution was added dropwise to it. 1 mol / L sodium hydroxide solution was added dropwise to the mixture to maintain the pH at approximately 13.0, and the mixture was stirred for about 30 minutes. Then, 1 mol / L hydrochloric acid solution was added dropwise to the clear solution, causing a white precipitate to form first, followed by clarification, with a pH of approximately 2.0. The reaction mixture was dialyzed four times using 4 L of deionized water. The dialysis product was freeze-dried to obtain a white powder or flocculent compound 2. Using 550 μL of deuterated water containing 1 μL of trifluoroacetic acid as a solvent, the mixture was... 1 Characterized by H-NMR (see [reference]). Figure 2 ).

[0123] 4. Preparation of glucose-responsive protamine-insulin complex I

[0124] Weigh 10 mg of insulin and dissolve it in 1 mL of water. Separately weigh 10 mg of compound 2 and dissolve it in 1 mL of water. Add 15 μL of 1 mol / L hydrochloric acid solution to each solution to adjust the pH to approximately 2.0–3.0. Mix 100 μL of each solution in an EP tube and add 3.0 μL of 1 mol / L sodium hydroxide solution to adjust the pH to 7.4. Then, centrifuge to remove insulin that has not formed a complex, obtaining a white precipitate. Add 1 mL of PBS (0.01 M, pH = 7.4) to the precipitate to obtain 1 mg / mL (based on the mass of insulin) insulin-protamine complex I dispersed in PBS.

[0125] Example 2: Preparation of a sugar-responsive insulin carrier by modifying protamine with chemical group formula II and 4-carboxy-3-fluorophenylboronic acid, thereby preparing a sugar-responsive protamine insulin complex.

[0126] The preparation process of the sugar-responsive protamine insulin complex provided in this embodiment is as follows:

[0127] 1. Preparation of 4-carboxy-3-fluorophenylboronic acid-N-hydroxysuccinimide (FPBA-NHS): The preparation method is as shown in Example 1.

[0128] 2. Preparation of protamine sulfate modified with chemical group formula II

[0129] Chemical reaction formula for protamine modified with chemical group formula II:

[0130]

[0131] Weigh 50 mg of protamine (1.0 eq, 5 ml PBS) and MAL-KKK-NH2 (18.0 eq, 5 ml PBS) into a 25 mL round-bottom flask and react overnight at room temperature. The resulting mixture was dialyzed four times with 4 L of deionized water. The product after dialyzing was freeze-dried to obtain a white powder, compound 3.

[0132] 3. Preparation of glucose-responsive insulin carriers

[0133] The glucose-responsive insulin carrier prepared in this embodiment is KKK-protamine-4-carboxy-3-fluorophenylboronic acid (KKK-protamine-FPBA), and its chemical reaction formula is as follows:

[0134]

[0135] Its preparation process is as follows:

[0136] Compound 3 (1.0 eq) was dissolved in 5 mL of deionized water to obtain an aqueous solution of compound 3. Separately, 4-carboxy-3-fluorophenylboronic acid-N-hydroxysuccinimide (FPBA-NHS, 3.0 eq) was dissolved in 5 mL of dimethyl sulfoxide (DMSO) to obtain an FPBA-NHS solution. The aqueous solution of compound 3 was cooled in an ice bath, and the FPBA-NHS solution was added dropwise to it. 1 mol / L sodium hydroxide solution was added dropwise to the mixture to maintain the pH at approximately 13.0, and the mixture was stirred for about 30 minutes. Then, 1 mol / L hydrochloric acid solution was added dropwise to the clear solution, causing a white precipitate to form first, followed by clarification, with a pH of approximately 2.0. The reaction mixture was dialyzed four times using 4 L of deionized water. The dialysis product was freeze-dried to obtain a white powder or flocculent compound 4. Using 550 μL of deuterated water containing 1 μL of trifluoroacetic acid as a solvent, the mixture was... 1 Characterized by H-NMR (see [reference]). Figure 3 ).

[0137] 4. Preparation of glucose-responsive protamine-insulin complex II

[0138] The reaction formula is as follows:

[0139] Weigh 10 mg of insulin and dissolve it in 1 mL of water. Separately weigh 10 mg of compound 4 and dissolve it in 1 mL of water. Add 15 μL of 1 mol / L hydrochloric acid solution to each solution to adjust the pH to approximately 2.0–3.0. Mix 100 μL of each solution in an EP tube and add 3.0 μL of 1 mol / L sodium hydroxide solution to adjust the pH to 7.4. Centrifuge to remove insulin that has not formed a complex, resulting in a white precipitate. Add 1 mL of PBS (0.01 M, pH = 7.4) to the precipitate to obtain 1 mg / mL (based on the mass of insulin) insulin-protamine complex II dispersed in PBS.

[0140] Example 3: Preparation of a sugar-responsive insulin carrier by modifying protamine with chemical group formula III and 4-carboxy-3-fluorophenylboronic acid, thereby preparing a sugar-responsive protamine insulin complex.

[0141] The preparation process of the sugar-responsive protamine insulin complex provided in this embodiment is as follows:

[0142] 1. Preparation of 4-carboxy-3-fluorophenylboronic acid-N-hydroxysuccinimide (FPBA-NHS): The preparation method is as shown in Example 1.

[0143] 2. Preparation of protamine sulfate modified with chemical group formula III

[0144] Chemical reaction formula for protamine modified with chemical group formula III:

[0145]

[0146] Weigh 50 mg of protamine (1.0 eq, 5 ml PBS) and MAL-KKKK-NH2 (18.0 eq, 5 ml PBS) into a 25 mL round-bottom flask and react overnight at room temperature. The resulting mixture was dialyzed four times with 4 L of deionized water. The product after dialyzing was freeze-dried to obtain a white powder, compound 5.

[0147] 3. Preparation of glucose-responsive insulin carriers

[0148] The glucose-responsive insulin carrier prepared in this embodiment is KKKK-protamine-4-carboxy-3-fluorophenylboronic acid (KKKK-protamine-FPBA), and its chemical reaction formula is as follows:

[0149]

[0150] Its preparation process is as follows:

[0151] Compound 5 (1.0 eq) was dissolved in 5 mL of deionized water to obtain an aqueous solution of compound 5. Separately, 4-carboxy-3-fluorophenylboronic acid-N-hydroxysuccinimide (FPBA-NHS, 4.0 eq) was dissolved in 5 mL of dimethyl sulfoxide (DMSO) to obtain an FPBA-NHS solution. The aqueous solution of compound 5 was cooled in an ice bath, and the FPBA-NHS solution was added dropwise to it. 1 mol / L sodium hydroxide solution was added dropwise to the mixture to maintain the pH at approximately 13.0, and the mixture was stirred for about 30 minutes. Then, 1 mol / L hydrochloric acid solution was added dropwise to the clear solution, causing a white precipitate to form first, followed by clarification, with a pH of approximately 2.0. The reaction mixture was dialyzed four times using 4 L of deionized water. The dialyzed product was freeze-dried to obtain a white powder or flocculent compound 6. Using 550 μL of deuterated water containing 1 μL of trifluoroacetic acid as a solvent, the mixture was... 1 Characterized by H-NMR (see [reference]). Figure 4 ).

[0152] 4. Preparation of glucose-responsive protamine-insulin complex III

[0153] Weigh 10 mg of insulin and dissolve it in 1 mL of water. Separately weigh 10 mg of compound 6 and dissolve it in 1 mL of water. Add 15 μL of 1 mol / L hydrochloric acid solution to each solution to adjust the pH to approximately 2.0–3.0. Mix 100 μL of each solution in an EP tube and add 3.0 μL of 1 mol / L sodium hydroxide solution to adjust the pH to 7.4. Centrifuge to remove any insulin that has not formed a complex, resulting in a white precipitate. Add 1 mL of PBS (0.01 M, pH = 7.4) to the precipitate to obtain 1 mg / mL (based on the mass of insulin) of insulin-protamine complex III dispersed in PBS.

[0154] Example 4: Preparation of a sugar-responsive insulin carrier by modifying protamine with chemical group formula IV and 4-carboxy-3-fluorophenylboronic acid, thereby preparing a sugar-responsive protamine insulin complex.

[0155] The preparation process of the sugar-responsive protamine insulin complex provided in this embodiment is as follows:

[0156] 1. Preparation of 4-carboxy-3-fluorophenylboronic acid-N-hydroxysuccinimide (FPBA-NHS): The preparation method is as shown in Example 1.

[0157] 2. Preparation of protamine sulfate modified with chemical group formula IV

[0158] Chemical reaction formula for protamine modified with chemical group formula IV:

[0159]

[0160] Weigh 50 mg of protamine (1.0 eq, 5 ml PBS) and MAL-KKKKKK-NH2 (18.0 eq, 5 ml PBS) into a 25 mL round-bottom flask and react overnight at room temperature. The resulting mixture was dialyzed four times with 4 L of deionized water. The product was then freeze-dried to obtain a white powder, compound 7.

[0161] 3. Preparation of glucose-responsive insulin carriers

[0162] The glucose-responsive insulin carrier prepared in this embodiment is KKKKKK-protamine-4-carboxy-3-fluorophenylboronic acid (KKKKKK-protamine-FPBA), and its chemical reaction formula is as follows:

[0163]

[0164] Its preparation process is as follows:

[0165] Compound 7 (1.0 eq) was dissolved in 5 mL of deionized water to obtain an aqueous solution of compound 7. Separately, 4-carboxy-3-fluorophenylboronic acid-N-hydroxysuccinimide (FPBA-NHS, 6.0 eq) was dissolved in 5 mL of dimethyl sulfoxide (DMSO) to obtain an FPBA-NHS solution. The aqueous solution of compound 7 was cooled in an ice bath, and the FPBA-NHS solution was added dropwise to it. 1 mol / L sodium hydroxide solution was added dropwise to the mixture to maintain the pH at approximately 13.0, and the mixture was stirred for about 30 minutes. Then, 1 mol / L hydrochloric acid solution was added dropwise to the clear solution, causing a white precipitate to form first, followed by clarification, with a pH of approximately 2.0. The reaction mixture was dialyzed four times using 4 L of deionized water. The dialyzed product was freeze-dried to obtain a white powder or flocculent compound 8. Using 550 μL of deuterated water containing 1 μL of trifluoroacetic acid as a solvent, the mixture was... 1 Characterized by H-NMR (see [reference]). Figure 5 )

[0166] 4. Preparation of glucose-responsive protamine insulin complex IV

[0167] 10 mg of insulin was dissolved in 1 mL of water, and 10 mg of compound 8 was dissolved in 1 mL of water. 15 μL of 1 mol / L hydrochloric acid solution was added to each solution to adjust the pH to approximately 2.0–3.0. 100 μL of each solution was mixed in an EP tube, and 3.0 μL of 1 mol / L sodium hydroxide solution was added to adjust the pH to 7.4. The mixture was then centrifuged to remove any insulin that did not form a complex, resulting in a white precipitate. 1 mL of PBS (0.01 M, pH = 7.4) was added to the precipitate to obtain insulin-protamine complex IV dispersed in PBS at a concentration of 1 mg / mL (based on the mass of insulin). Among all the complexes, complex IV had the highest phenylboronic acid grafting ratio, leading to a higher level of free insulin in the complex IV solution and consequently, a low insulin loading rate. Therefore, complex IV will not be discussed further, and the complexes from Examples 1 and 3 are preferred for discussion.

[0168] Example 5: Validation of the efficacy of the glucose-responsive protamine insulin complex

[0169] 1. Morphological study of sugar-responsive protamine complex

[0170] In this embodiment, the morphology of the sugar-responsive protamine insulin complexes prepared in Examples 1 and 3 was observed using transmission electron microscopy:

[0171] The glucose-responsive protamine insulin complexes I and III prepared in Examples 1 and 3 were weighed separately, and water was added to make an insulin equivalent concentration of 1 mg / mL. The complexes were then sonicated for 1 min at 100 W using an ultrasonic cell disruptor. After dispersing and suspending in water, the mixture was dropped onto a copper grid. 10 μL of 5% uranyl acetate solution was added to the copper grid, and the mixture was allowed to stand for 10 min. The solution was then removed with filter paper. The samples were observed using a transmission electron microscope. The glucose-responsive protamine insulin complexes prepared in Examples 1 and 3 had similar morphologies. The transmission electron microscope images of the glucose-responsive protamine insulin complexes in Examples 1 and 3 are shown below. Figure 6 and Figure 7 As shown.

[0172] according to Figure 6 and Figure 7 It can be seen that the glucose-responsive protamine-insulin complex has a porous and loose microstructure. The formed protamine-insulin complex has an irregular shape, a size at the micromolar level, and insulin and protamine are uniformly distributed in the complex.

[0173] 2. In vitro validation of the glucose response of the glucose-responsive protamine-insulin complex

[0174] 0.1 mg of water-soluble Cy5-NHS ester was dissolved in 0.02 mL of DMSO, and 10 mg of insulin was added to 5 mL of sodium bicarbonate solution (0.1 M). The two solutions were mixed and stirred overnight at room temperature. The mixture was dialyzed three times in 4 L of deionized water in the dark, and then freeze-dried to obtain Cy5-labeled insulin.

[0175] Cy5-labeled insulin was used to prepare 1 mg / mL glucose-responsive protamine insulin complexes with the glucose-responsive insulin carriers prepared in Examples 1 and 3, respectively (steps as shown in Examples 1 and 3, respectively). Different amounts of 0.4 g / mL glucose solution were added to the solutions to obtain different initial glucose concentrations (0, 100, 200, 400 mg / dL). 50 μL of the complex was aspirated and centrifuged at 0, 15, 30, 60, 120, and 180 minutes, respectively. Then, 20 μL of the supernatant was mixed with 180 μL of PBS (0.01 M, pH = 7.4). The absorbance of the mixture at 640 nm excitation and 670 nm emission was measured using a microplate reader, and the insulin concentration in the supernatant was calculated using a pre-defined standard curve. The results are shown in [reference needed]. Figure 8 and Figure 9 In phosphate buffer at pH 7.4, the concentration of insulin in the supernatant was determined using Cy5-labeled insulin, and the glucose-responsive insulin release performance of the complex was evaluated at different glucose concentrations (0, 100, 200, and 400 mg / dL).

[0176] according to Figure 8 and Figure 9 It can be seen that the addition of glucose to the PBS solution significantly increased the insulin release rate and equilibrium insulin concentration. High glucose concentration may lead to more glucose binding to FPBA residues on the complex, thus reducing the positive charge density and decreasing the attraction between negatively charged insulin and positively charged polymers under physiological conditions, thereby promoting insulin release. Simultaneously, comparing the glucose response rates of glucose-responsive protamine insulin complexes I and III, it can be seen that with an increasing proportion of phenylboronic acid modification on protamine, the insulin release rate from the complex accelerates.

[0177] However, it's important to note that a faster insulin release rate does not necessarily translate to better glycemic control. Insulin release rate is only one factor influencing glycemic control; other factors, such as drug metabolism and tissue sensitivity to insulin, also play crucial roles. Therefore, rigorous experimental verification of the glycemic control effects of complex I and complex III is still needed. Based on this, subsequent experiments will utilize glucose-responsive protamine insulin complex I and complex III to conduct in-depth in vivo glycemic regulation studies, comprehensively evaluating their glycemic control performance in practical applications.

[0178] Example 6: In vivo blood glucose regulation study of glucose-responsive protamine-insulin complex

[0179] Streptozotocin (STZ) was dissolved in citrate / sodium citrate buffer to prepare a 10 mg / mL solution. The resulting solution was stored on ice protected from light and used immediately after preparation. Male C57BL / 6J mice (approximately 25 g in size, clean-grade) were fasted overnight and then intraperitoneally injected with STZ solution at a dose of 120 mg / kg under light-protected conditions. Blood glucose levels were measured two weeks after injection. Successfully induced diabetic mice (blood glucose 300–500 mg / dL) were used for further research. The insulin equivalent dose of the complex was determined by equivalent conversion to the dosage of commercially available drugs. Blood glucose was monitored before and after treatment until it returned to initial levels. Blood was collected from the tail tip, and plasma glucose concentration was measured using a glucometer.

[0180] Three groups (n=5) of C57BL / 6J diabetic mice with blood glucose levels ranging from 300 to 500 mg / dL were selected. The control group received commercially available protamine insulin (NPH), while the experimental groups received glucose-responsive protamine insulin complex I and III. Both the control and experimental groups were administered insulin subcutaneously at a dose equivalent to 6.0 mg / kg. After administration, blood glucose levels were measured and recorded using an ACCU-CHEK glucometer. Figures 10-12 The results of blood glucose levels in diabetic mice after administration of the NPH group, Complex I group, and Complex III group are presented. Following subcutaneous injection of the complexes, blood glucose levels in diabetic mice returned to normal. The duration of normal blood glucose maintenance in diabetic mice in the NPH group (control group), Complex I group, and Complex III group reached 2.3±0.975 hours, 11.4±4.099 hours, and 8.8±3.174 hours, respectively. Furthermore, no hypoglycemia (<50 mg / dL) was observed in diabetic mice treated with either complex. Compared to the NPH group (control group), the duration of normal blood glucose maintenance and the incidence of hypoglycemia were prolonged in diabetic mice in the Complex I and Complex III groups.

[0181] Compared with existing insulin controlled-release systems, the glucose-responsive protamine insulin complexes I and III of the present invention exhibit significant advantages in terms of accuracy of blood glucose response, controllability of insulin release, and biocompatibility. Existing insulin controlled-release systems suffer from problems such as slow response speed, poor biocompatibility, or unstable insulin release, while complexes I and III provided by the present invention effectively solve these problems and are therefore viable options.

[0182] In the complexes I and III provided by this invention, based on the morphology study in Example 5, both exhibit a porous and loose microstructure with uniform distribution of insulin and protamine, which facilitates insulin release. The in vivo blood glucose regulation study in Example 6 showed that, compared to commercially available protamine insulin (NPH), complexes I and III significantly prolonged the maintenance time of normal blood glucose in diabetic mice and reduced the incidence of hypoglycemia. Furthermore, in summary, complex I can more precisely and rapidly regulate insulin release according to blood glucose levels, resulting in a longer blood glucose control time and thus more effectively maintaining blood glucose stability, achieving a maintenance time of normal blood glucose in diabetic mice of 11.4 ± 4.099 hours. Therefore, the structure of complex I is preferred in this invention.

[0183] The embodiments described in this invention are preferred embodiments and features. Any person skilled in the art can make changes and variations based on the spirit of the invention, and such changes and variations are also considered to fall within the scope of this invention and the limits of the independent and appended claims.

Claims

1. A sugar-responsive insulin carrier, characterized in that, The compound comprises protamine and phenylboronic acid compounds, wherein the cysteine ​​in the protamine is modified with a chemical group, which is linked to the NHS-esterified phenylboronic acid compound via a chemical bond; the chemical group is linked to the protamine via a thioether bond; the chemical bond between the chemical group and the NHS-esterified phenylboronic acid compound is an amide bond; the phenylboronic acid compound is halogen-modified phenylboronic acid; and the chemical group has the structural formula of any one or more of Formulas I to IV. 。 2. A method for preparing a glucose-responsive insulin carrier as described in claim 1, characterized in that, Includes the following steps: (1) The compound solution reacts with the protamine solution to obtain solution A; (2) Solution A reacts with NHS-esterified phenylboronic acid compound solution; The structural formula of the compound includes any one or more of Formulas I to IV: 。 3. The preparation method according to claim 2, characterized in that, The molar ratio of the compound to protamine in step (1) is (1~50):

1.

4. A sugar-responsive protamine insulin complex, characterized in that, It is prepared by mixing and reacting the carrier and insulin as described in claim 1.

5. A method for preparing a sugar-responsive protamine insulin complex, characterized in that, The sugar-responsive insulin carrier as described in claim 1 is mixed with insulin, and the pH is adjusted to 6.5-8.0 to obtain a sugar-responsive protamine insulin complex.

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