PH-responsive drug-loaded micelle composite hydrogel for oral delivery of insulin and preparation method of pH-responsive drug-loaded micelle composite hydrogel

Abstract

The invention relates to pH-responsive drug-loaded micelle composite hydrogel and a preparation method thereof. The preparation method comprises the following steps: (1) synthesizing CB-tBu; (2) synthesis of NHS-PCB-tBu (N-hydroxysuccinimide (3) synthesis of the ABAPE-PCB; (4) preparing an amphoteric polymer micelle; (5) preparation of an insulin-loaded polymer micelle ABAPE-PCB (at) INS; and (6) preparing the pH response type drug-loaded micelle composite hydrogel ABAPE-PCB (at) INS (at) SC. The pH response type drug-loaded micelle composite hydrogel ABAPE-PCB (at) INS (at) SC disclosed by the invention has a good mucus penetrating effect and can efficiently cross an intestinal mucosa barrier; the critical micelle concentration is relatively low, and the problem of unstable micelle drug loading at present can be effectively improved; pH responsiveness is achieved, and protein drugs such as insulin can be protected to break through the physiological barrier of the gastrointestinal tract; the micro-nano structure can slowly release insulin, so that the occurrence of hypoglycemia is greatly reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a pH-responsive drug-loaded micelle composite hydrogel for oral delivery of insulin and a preparation method thereof. Background Art

[0002] Diabetes is a chronic metabolic disease, and the number of patients worldwide is increasing year by year. For patients with type 1 diabetes and some patients with type 2 diabetes who cannot control their blood sugar with oral hypoglycemic drugs, insulin injection is currently the most effective treatment because of its rapid effect and high bioavailability. However, problems such as limited accuracy in blood sugar control, severe reaction scars at the injection site, local allergic reactions and skin infections have also prompted the continuous development of new exogenous insulin delivery routes. Oral administration is considered to be the simplest, most convenient and most efficient method of administration because of its high patient compliance, low cost, non-invasiveness and ease of use. In addition, oral insulin absorption from the intestine to the portal vein and liver can better simulate the natural physiological pathway of pancreatic insulin secretion than subcutaneous injection, and is expected to reduce peripheral hyperinsulinemia. Currently, surfactants such as sodium octanoate / sodium decanoate and their derivatives are commonly used to open tight junctions to improve the efficiency of oral insulin delivery, but their increased permeability of the intestinal epithelium in vivo may cause more pathogens and toxins to penetrate the intestinal wall and other immune responses.

[0003] Among them, oral insulin using micro-nano drug delivery systems has unique advantages, such as improving oral insulin bioavailability, prolonging blood circulation time, and releasing insulin on demand. The zwitterionic material betaine has been shown to have good biocompatibility and good resistance to nonspecific protein adsorption. In addition, the neutral surface of polycarboxy betaine makes it easy to pass through the mucus barrier, and it is a substrate for proton-assisted amino acid transporter 1, which can be efficiently taken up by epithelial cells. Polymer micelles are microparticle drug delivery systems with a core-shell structure formed by self-assembly of a polymer material with one end hydrophilic and the other end hydrophobic in water. Its hydrophobic core can be used as a reservoir for poorly soluble drugs to improve the solubility of fat-soluble drugs. Compared with small molecule surfactants, polymer micelle materials have the characteristics of low critical micelle concentration, good dilution resistance, strong solubilization, and good thermodynamic stability, and are considered to be one of the most promising drug delivery systems. Sodium alginate gel has the characteristics of rapid forming and pH responsiveness. The added carboxymethyl chitosan can improve its swelling in the intestine and release drugs to a greater extent. Among them, oral insulin using micro-nano drug delivery systems has unique advantages, such as improving the bioavailability of oral insulin, prolonging blood circulation time, and releasing insulin on demand. Summary of the invention

[0004] In order to solve the problems existing in the above-mentioned prior art, the technical problem to be solved by the present invention is to provide a preparation method of a pH-responsive drug-loaded micelle composite hydrogel for oral delivery of insulin in order to overcome the defects of the prior art. The composite hydrogel of the present invention can effectively solve the problems that the oral delivery of insulin is difficult to penetrate the mucus barrier and has low bioavailability.

[0005] To achieve the above object, the technical solution of the present invention is:

[0006] A pH-responsive drug-loaded micelle composite hydrogel, wherein the drug-loaded micelle ABAPE-PCB is a zwitterionic micelle, wherein PCB, namely polycarboxybetaine, as the hydrophilic end of the nanoparticle micelle can quickly pass through the mucus layer, and can penetrate the epithelial cell layer through the proton-assisted amino acid transporter 1 to increase the absorption of intestinal drugs; ABAPE is a hydrophobic block, namely 4-aminophenylboronic acid pinacol ester with a glucose-responsive phenylboronic acid group, which is glucose-sensitive and can achieve on-demand release of insulin.

[0007] Furthermore, the pH-responsive drug-loaded micelle composite hydrogel is made of drug-loaded micelles ABAPE-PCB@INS and microsphere gel matrices sodium alginate and carboxymethyl chitosan; sodium alginate, as a natural anionic polysaccharide, will quickly gel when divalent cations (such as calcium ions) are added; in addition, sodium alginate is also a pH-sensitive polymer. Due to its rapid dissolution and stability in acidic environments, but instability in alkaline environments, it is an ideal choice for designing intestinal targeted drug delivery systems; carboxymethyl chitosan can improve the drug release of sodium alginate gel in the intestinal environment, and has the characteristics of good biocompatibility and biodegradability, low price, and non-toxicity.

[0008] A method for preparing a pH-responsive drug-loaded micelle composite hydrogel for oral delivery of insulin, comprising:

[0009] Step 1: preparing zwitterionic polymer ABAPE-PCB, specifically comprising:

[0010] Step 101, at a temperature of 45 to 50° C., dissolve ethyl 2-(dimethylamino)methacrylate and tert-butyl bromoacetate in anhydrous acetonitrile at a molar ratio of 1:1 to 2, stir for 12 to 24 hours under nitrogen protection, then add 50 to 200 mL of cold ether to precipitate a white solid, separate by suction filtration and vacuum dry for 12 to 24 hours to obtain a CB-tBu monomer;

[0011] Step 102, dissolving CB-tBu, 4-cyano-4-(phenylthiothiothio)pentanoic acid N-succinimidyl ester and 2,2′-azobisisobutyronitrile in a certain proportion in 5-20 mL of anhydrous dimethylformamide, deoxygenating, placing in an ice bath for 30-120 min under N2 protection, heating under reflux with a condenser at 60-70° C., stirring for reaction for 12-24 h, then adding 50-200 mL of ether to the reaction mixture, placing in a 4° C. refrigerator and letting stand, discarding the upper mixed solution, then adding 5-10 mL of acetone at 0° C., ultrasonically precipitating for 2-3 times, removing the supernatant, collecting the precipitate and vacuum drying for 12-24 h to obtain the intermediate NHS-PCB-tBu;

[0012] Step 103, dissolving the intermediate NHS-PCB-tBu in anhydrous N,N-dimethylformamide, and dissolving a certain amount of 4-aminophenylboronic acid pinacol ester in chloroform, mixing the two, adding a certain amount of triethylamine dropwise, adjusting the pH to 7-9, stirring at room temperature for 5-10 days, and then rotary distilling at 50°C for 30-60 minutes, and precipitating in ether, filtering the filtrate and resuspending and filtering with acetonitrile, rotary distilling at 60°C for 30-60 minutes, adding 4-8 mL TFA and stirring at room temperature for 4-8 hours, precipitating with ether and filtering, vacuum drying overnight for 12-24 hours, and then neutralizing with hydroxylamine in PBS with a pH of 7-9 until the uncoupled PCB is dissolved, and then ultrafiltration in PBS, repeating ultrafiltration in water for 2-5 times to obtain ABAPE-PCB solution, and then freeze-drying in pure water for 12-24 hours to obtain the dried polymer ABAPE-PCB;

[0013] Step 2: Prepare drug-loaded micelles ABAPE-PCB@INS by dialysis method, specifically:

[0014] A certain amount of polymer ABAPE-PCB was dissolved in phosphate buffer, and a certain amount of dimethyl sulfoxide was added after stirring for 20 to 90 minutes, and the stirring was continued for 0.5 to 3 hours; an appropriate amount of insulin solution was slowly dripped in, and the stirring was continued for 30 to 60 minutes; the ABAPE-PCB@INS solution loaded with insulin was obtained by dialyzing in deionized water at 4°C for 24 to 48 hours, and the insulin-loaded micelle freeze-dried powder was obtained after vacuum freeze drying;

[0015] Step 3: Preparation of pH-responsive drug-loaded micelle composite hydrogel ABAPE-PCB@INS@SC, specifically:

[0016] Step 301, weighing a certain mass of sodium alginate and carboxymethyl chitosan and adding them into deionized water to obtain a mixed solution of sodium alginate and carboxymethyl chitosan, which is named as SC solution; the mass of sodium alginate is 0.5 to 5 times the mass of carboxymethyl chitosan; the mass percentage of sodium alginate in the SC solution is 1% to 5%;

[0017] Step 302, adding the polymer drug-loaded micelle ABAPE-PCB@INS solution to the SC solution and stirring and mixing to obtain a pH-responsive drug-loaded micelle composite hydrogel ABAPE-PCB@INS@SC solution; the mass percentage of the drug-loaded micelle ABAPE-PCB@INS in the solution is 0.5-5 mg / mL;

[0018] Step 303, prepare pH-responsive drug-loaded micelle composite hydrogel ABAPE-PCB@INS@SC under the conditions of voltage 5-20 kV, flow rate 1-3 mL / h, rotation speed 150-400 rpm, and collection liquid of 0.2 mmol / L calcium chloride solution.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] The invention provides a method for preparing a pH-responsive drug-loaded micelle composite hydrogel for oral delivery of insulin. The method uses a sodium alginate microsphere gel system, a nanosystem containing insulin-loaded zwitterionic polymer micelles ABAPE-PCB@INS, and carboxymethyl chitosan as main raw materials, and prepares the pH-responsive drug-loaded micelle composite hydrogel for oral delivery of insulin through an electrostatic spray method. The hydrogel can respond to the physiological pH environment of the gastrointestinal tract, can efficiently penetrate the mucus barrier, and has the ability of sustained drug release. The hydrogel can be effectively used for oral delivery of protein drugs such as insulin.

[0021] The preparation method of the pH-responsive drug-loaded micelle composite hydrogel for oral delivery of insulin comprises the steps of: mixing sodium alginate and carboxymethyl chitosan in a Ca 2+ Sodium alginate microspheres are obtained by cross-linking under the presence of iodine, and the drug-loaded micelles are introduced into the sodium alginate microsphere system by physical adsorption, which can effectively protect insulin from passing through the gastric environment.

[0022] The invention discloses a method for preparing a pH-responsive drug-loaded micelle composite hydrogel for oral delivery of insulin, comprising zwitterionic polymer micelles ABAPE-PCB, which have a lower critical micelle concentration, can effectively improve the stability of micelle drug loading, and can be efficiently taken up by epithelial cells, thereby improving the bioavailability of the drug.

[0023] The preparation method of the pH-responsive drug-loaded micelle composite hydrogel for oral delivery of insulin is simple and has good reproducibility, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The synthetic route of polymer micelles in Example 1;

[0025] Figure 2is the infrared spectrum of the polymer micelle ABAPE-PCB in Example 1;

[0026] Figure 3 is a scanning electron microscope image of the polymer micelle ABAPE-PCB in Example 1;

[0027] Figure 4 Schematic diagram of mucus penetration of polymer micelle ABAPE-PCB in Example 1;

[0028] Figure 5 This is a scanning electron micrograph of the pH-responsive drug-loaded micelle composite hydrogel in Example 1;

[0029] Figure 6 The cytotoxicity test results of the pH-responsive drug-loaded micelle composite hydrogel in Example 1;

[0030] Figure 7 Schematic diagram of the hypoglycemic effect of ABAPE-PCB@INS@SC gel in the type 1 diabetic rat model in Example 1;

[0031] Figure 8 This is the blood glucose value 8 hours after administration of the ABAPE-PCB@INS@SC gel in Example 1 in the type 1 diabetic rat model. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0033] like Figure 1-8 As shown,

[0034] Example 1

[0035] This embodiment provides a method for preparing a pH-responsive drug-loaded micelle composite hydrogel for oral delivery of insulin, comprising:

[0036] Step 1: Preparation of zwitterionic polymer ABAPE-PCB, the process is as follows Figure 1 As shown, specifically including:

[0037] Step 101, at 50° C., dissolve 5 g of ethyl 2-(dimethylamino)methacrylate and 8.68 g of tert-butyl bromoacetate in anhydrous acetonitrile, stir for 24 h under nitrogen protection, then add 200 ml of cold ether to precipitate a white solid, separate by suction filtration and vacuum dry for 24 h to obtain a CB-tBu monomer;

[0038] Step 102, 1.3 g CB-tBu, 78 mg 4-cyano-4-(phenylthiothiothio)pentanoic acid N-succinimidyl ester and 31.2 mg 2,2′-azobisisobutyronitrile were dissolved in 10 mL anhydrous dimethylformamide, deoxygenated, ice-bathed for 60 min under N2 protection, heated to reflux under 70° C. condenser, stirred for reaction for 24 h, then 200 mL ether was added to the reaction mixture, placed in a 4° C. refrigerator, the upper mixed solution was discarded, then 10 mL acetone was added at 0° C., ultrasonic precipitation was performed 3 times, the supernatant was removed, the precipitate was collected and vacuum dried for 24 h, to obtain the intermediate NHS-PCB-tBu;

[0039] Step 103, 0.73g of the intermediate NHS-PCB-tBu was dissolved in 8.6mL of anhydrous N,N-dimethylformamide, and 0.1g of 4-aminophenylboronic acid pinacol ester was dissolved in 60mL of chloroform. After the two were mixed, 200μL of triethylamine was added dropwise, the pH was adjusted to 8.5, and the mixture was stirred at room temperature for 5d, then rotary evaporated at 50°C for 30min, and precipitated in 100mL of ether. After filtering the filtrate, it was resuspended and filtered with 10mL of acetonitrile, rotary evaporated at 60°C for 30min, and then 4mL of TFA was added and stirred at room temperature for 4h, 100mL of ether was added to precipitate and filter, vacuum dried overnight for 24h, and then neutralized with 20mM hydroxylamine in PBS with a pH of 8 until the uncoupled PCB was dissolved, and then ultrafiltered in PBS, and ultrafiltration was repeated 5 times in water to obtain ABAPE-PCB solution, and then freeze-dried in pure water for 24h to obtain the dried polymer ABAPE-PCB.

[0040] Step 2: Prepare drug-loaded micelles ABAPE-PCB@INS by dialysis method, specifically:

[0041] Dissolve 50 mg of polymer ABAPE-PCB in phosphate buffer, stir for 90 min, add 10 mL of dimethyl sulfoxide, and continue stirring for 3 h. Slowly drip 10 mL of insulin solution and continue stirring for 60 min. Dialyze in 4 ° C deionized water for 48 h to obtain insulin-loaded ABAPE-PCB@INS solution, and vacuum freeze-dry to obtain insulin-loaded micelle freeze-dried powder.

[0042] Step 3: Preparation of pH-responsive drug-loaded micelle composite hydrogel ABAPE-PCB@INS@SC, specifically:

[0043] Step 301, weighing 0.64 g of sodium alginate and 0.16 g of carboxymethyl chitosan and adding them into 40 mL of deionized water to obtain a mixed solution of sodium alginate and carboxymethyl chitosan, which is named as SC solution; the mass of sodium alginate is 4 times the mass of carboxymethyl chitosan; the mass percentage of sodium alginate in the SC solution is 1.6%;

[0044] Step 302, adding 40 mg of polymer drug-loaded micelle ABAPE-PCB@INS solution to 40 mL of SC solution and stirring to obtain a pH-responsive drug-loaded micelle composite hydrogel ABAPE-PCB@INS@SC solution; the mass percentage of drug-loaded micelle ABAPE-PCB@INS in the solution is 1 mg / mL;

[0045] Step 303, prepare pH-responsive drug-loaded micelle composite hydrogel ABAPE-PCB@INS@SC under the conditions of voltage 10 kV, flow rate 1 mL / h, rotation speed 150 rpm, and collection liquid being 0.2 mmol / L calcium chloride solution.

[0046] Example 2

[0047] This embodiment provides a method for preparing a pH-responsive drug-loaded micelle composite hydrogel for oral delivery of insulin, comprising:

[0048] Step 1: preparing zwitterionic polymer ABAPE-PCB, specifically comprising:

[0049] Step 101, at 45° C., dissolve 5 g of ethyl 2-(dimethylamino)methacrylate and 8.68 g of tert-butyl bromoacetate in anhydrous acetonitrile, stir for 12 h under nitrogen protection, then add 200 mL of cold ether to precipitate a white solid, separate by suction filtration and vacuum dry for 24 h to obtain a CB-tBu monomer;

[0050] Step 102, 1.3 g CB-tBu, 78 mg 4-cyano-4-(phenylthiothiothio)pentanoic acid N-succinimidyl ester and 31.2 mg 2,2′-azobisisobutyronitrile were co-dissolved in 10 mL anhydrous dimethylformamide, deoxygenated, ice-bathed for 60 min under N2 protection, heated to reflux under 60° C. condenser, stirred for reaction for 12 h, then 200 mL ether was added to the reaction mixture, placed in a 4° C. refrigerator and allowed to stand, the upper mixed solution was discarded, then 10 mL acetone was added at 0° C., ultrasonic precipitation was performed 3 times, the supernatant was removed, and the precipitate was collected and vacuum dried for 24 h to obtain the intermediate NHS-PCB-tBu;

[0051] Step 103, 0.73g of the intermediate NHS-PCB-tBu was dissolved in 8.6mL of anhydrous N,N-dimethylformamide, and 0.1g of 4-aminophenylboronic acid pinacol ester was dissolved in 60mL of chloroform. After the two were mixed, 200μL of triethylamine was added dropwise, the pH was adjusted to 8, and the mixture was stirred at room temperature for 10d, then rotary evaporated at 50°C for 60min, and precipitated in 100mL of ether. After filtering the filtrate, the mixture was resuspended and filtered with 10mL of acetonitrile, rotary evaporated at 60°C for 60min, 4mL of TFA was added, stirred at room temperature for 8h, 100mL of ether was added, precipitated and filtered, dried overnight in vacuum for 24h, and then neutralized with 20mM hydroxylamine in PBS at pH 8 until the uncoupled PCB was dissolved, and then ultrafiltered in PBS, and ultrafiltration was repeated 5 times in water to obtain ABAPE-PCB solution, which was then freeze-dried in pure water for 24h to obtain the dried polymer ABAPE-PCB.

[0052] Step 2: Prepare drug-loaded micelles ABAPE-PCB@INS by dialysis method, specifically:

[0053] Dissolve 100 mg of polymer ABAPE-PCB in phosphate buffer, stir for 90 min, add 10 mL of dimethyl sulfoxide, and continue stirring for 0.5 h. Slowly drip 10 mL of insulin solution and continue stirring for 60 min. Dialyze in 4 ° C deionized water for 48 h to obtain insulin-loaded ABAPE-PCB@INS solution, and vacuum freeze-dry to obtain insulin-loaded micelle freeze-dried powder.

[0054] Step 3: Preparation of pH-responsive drug-loaded micelle composite hydrogel ABAPE-PCB@INS@SC, specifically:

[0055] Step 301, weighing 0.64 g of sodium alginate and 0.16 g of carboxymethyl chitosan and adding them into 40 mL of deionized water to obtain a mixed solution of sodium alginate and carboxymethyl chitosan, which is named as SC solution; the mass of sodium alginate is 4 times the mass of carboxymethyl chitosan; the mass percentage of sodium alginate in the SC solution is 1.6%;

[0056] Step 302, adding 40 mg of polymer drug-loaded micelle ABAPE-PCB@INS solution to 40 mL of SC solution and stirring to obtain a pH-responsive drug-loaded micelle composite hydrogel ABAPE-PCB@INS@SC solution; the mass percentage of drug-loaded micelle ABAPE-PCB@INS in the solution is 1 mg / mL;

[0057] Step 303, prepare pH-responsive drug-loaded micelle composite hydrogel ABAPE-PCB@INS@SC under the conditions of voltage 10 kV, flow rate 3 mL / h, rotation speed 400 rpm, and collection liquid being 0.2 mmol / L calcium chloride solution.

[0058] Example 3

[0059] This embodiment provides a method for preparing a pH-responsive drug-loaded micelle composite hydrogel for oral delivery of insulin, comprising:

[0060] Step 1: preparing zwitterionic polymer ABAPE-PCB, specifically comprising:

[0061] Step 101, at 45° C., dissolve 5 g of ethyl 2-(dimethylamino)methacrylate and 8.68 g of tert-butyl bromoacetate in anhydrous acetonitrile, stir for 24 h under nitrogen protection, then add 200 mL of cold ether to precipitate a white solid, separate by suction filtration and vacuum dry for 24 h to obtain a CB-tBu monomer;

[0062] Step 102, 1.3 g CB-tBu, 78 mg 4-cyano-4-(phenylthiothiothio)pentanoic acid N-succinimidyl ester and 31.2 mg 2,2′-azobisisobutyronitrile were co-dissolved in 10 mL anhydrous dimethylformamide, deoxygenated, ice-bathed for 60 min under N2 protection, heated to reflux under 60° C. condenser, stirred for reaction for 24 h, then 200 mL ether was added to the reaction mixture, placed in a 4° C. refrigerator, the upper mixed solution was discarded, then 10 mL acetone was added at 0° C., ultrasonic precipitation was performed 3 times, the supernatant was removed, and the precipitate was collected and vacuum dried for 24 h to obtain the intermediate NHS-PCB-tBu;

[0063] Step 103, 0.73g of the intermediate NHS-PCB-tBu was dissolved in 8.6mL of anhydrous N,N-dimethylformamide, and 0.1g of 4-aminophenylboronic acid pinacol ester was dissolved in 60mL of chloroform. After the two were mixed, 200μL of triethylamine was added dropwise, the pH was adjusted to 8, and the mixture was stirred at room temperature for 5d, then rotary evaporated at 50°C for 30min, and precipitated in 100mL of ether. After filtering the filtrate, it was resuspended and filtered with 10mL of acetonitrile, rotary evaporated at 60°C for 30min, and then 4mL of TFA was added and stirred at room temperature for 4h, 100mL of ether was added to precipitate and filter, vacuum dried overnight for 24h, and then neutralized with 20mM hydroxylamine in PBS with a pH of 8 until the uncoupled PCB was dissolved, and then ultrafiltered in PBS, and ultrafiltration was repeated 5 times in water to obtain ABAPE-PCB solution, and then freeze-dried in pure water for 24h to obtain the dried polymer ABAPE-PCB.

[0064] Step 2: Prepare drug-loaded micelles ABAPE-PCB@INS by dialysis method, specifically:

[0065] Dissolve 100 mg of polymer ABAPE-PCB in phosphate buffer, stir for 90 min, add 10 mL of dimethyl sulfoxide, and continue stirring for 3 h. Slowly drip 10 mL of insulin solution and continue stirring for 60 min. Dialyze in 4 ° C deionized water for 48 h to obtain insulin-loaded ABAPE-PCB@INS solution, and vacuum freeze-dry to obtain insulin-loaded micelle freeze-dried powder.

[0066] Step 3: Preparation of pH-responsive drug-loaded micelle composite hydrogel ABAPE-PCB@INS@SC, specifically:

[0067] Step 301, weighing 0.48g of sodium alginate and 0.16g of carboxymethyl chitosan and adding them into 40mL of deionized water to obtain a mixed solution of sodium alginate and carboxymethyl chitosan, which is named as SC solution; the mass of sodium alginate is 3 times the mass of carboxymethyl chitosan; the mass percentage of sodium alginate in the SC solution is 1.2%;

[0068] Step 302, adding 40 mg of polymer drug-loaded micelle ABAPE-PCB@INS solution to 40 mL of SC solution and stirring to obtain a pH-responsive drug-loaded micelle composite hydrogel ABAPE-PCB@INS@SC solution; the mass percentage of drug-loaded micelle ABAPE-PCB@INS in the solution is 2 mg / mL;

[0069] Step 303, prepare pH-responsive drug-loaded micelle composite hydrogel ABAPE-PCB@INS@SC under the conditions of voltage 20 kV, flow rate 3 mL / h, rotation speed 400 rpm, and collection liquid being 0.2 mmol / L calcium chloride solution.

[0070] Example 4

[0071] This embodiment provides a method for preparing a pH-responsive drug-loaded micelle composite hydrogel for oral delivery of insulin, comprising:

[0072] Step 1: preparing zwitterionic polymer ABAPE-PCB, specifically comprising:

[0073] Step 101, at 50° C., dissolve 5 g of ethyl 2-(dimethylamino)methacrylate and 11 g of tert-butyl bromoacetate in anhydrous acetonitrile, stir for 36 h under nitrogen protection, then add 200 mL of cold ether to precipitate a white solid, separate by suction filtration and vacuum dry for 24 h to obtain a CB-tBu monomer;

[0074] Step 102, 1.3 g CB-tBu, 78 mg 4-cyano-4-(phenylthiothiothio)pentanoic acid N-succinimidyl ester and 31.2 mg 2,2′-azobisisobutyronitrile were dissolved in 10 mL anhydrous dimethylformamide, deoxygenated, ice-bathed for 60 min under N2 protection, heated to reflux under 70° C. condenser, stirred for reaction for 24 h, then 200 mL ether was added to the reaction mixture, placed in a 4° C. refrigerator, the upper mixed solution was discarded, then 10 mL acetone was added at 0° C., ultrasonic precipitation was performed 3 times, the supernatant was removed, the precipitate was collected and vacuum dried for 24 h, to obtain the intermediate NHS-PCB-tBu;

[0075] Step 103, 0.73g of the intermediate NHS-PCB-tBu was dissolved in 8.6mL of anhydrous N,N-dimethylformamide, and 0.1g of 4-aminophenylboronic acid pinacol ester was dissolved in 60mL of chloroform. After the two were mixed, 200μL of triethylamine was added dropwise, the pH was adjusted to 8, and the mixture was stirred at room temperature for 5d, then rotary evaporated at 50°C for 30min, and precipitated in 100mL of ether. After filtering the filtrate, it was resuspended and filtered with 10mL of acetonitrile, rotary evaporated at 60°C for 30min, and then 4mL of TFA was added and stirred at room temperature for 4h, 100mL of ether was added to precipitate and filter, vacuum dried overnight for 24h, and then neutralized with 20mM hydroxylamine in PBS with a pH of 8 until the uncoupled PCB was dissolved, and then ultrafiltered in PBS, and ultrafiltration was repeated 5 times in water to obtain ABAPE-PCB solution, and then freeze-dried in pure water for 24h to obtain the dried polymer ABAPE-PCB.

[0076] Step 2: Prepare drug-loaded micelles ABAPE-PCB@INS by dialysis method, specifically:

[0077] Dissolve 80 mg of polymer ABAPE-PCB in phosphate buffer, stir for 90 min, add 10 mL of dimethyl sulfoxide, and continue stirring for 1 h. Slowly drip 10 mL of insulin solution and continue stirring for 30 min. Dialyze in 4 ° C deionized water for 24 h to obtain insulin-loaded ABAPE-PCB@INS solution, and vacuum freeze-dry to obtain insulin-loaded micelle freeze-dried powder.

[0078] Step 3: Preparation of pH-responsive drug-loaded micelle composite hydrogel ABAPE-PCB@INS@SC, specifically:

[0079] Step 301, weighing 0.64 g of sodium alginate and 0.16 g of carboxymethyl chitosan and adding them into 40 mL of deionized water to obtain a mixed solution of sodium alginate and carboxymethyl chitosan, which is named as SC solution; the mass of sodium alginate is 4 times the mass of carboxymethyl chitosan; the mass percentage of sodium alginate in the SC solution is 1.6%;

[0080] Step 302, adding 80 mg of polymer drug-loaded micelle ABAPE-PCB@INS solution to 40 mL of SC solution and stirring to obtain a pH-responsive drug-loaded micelle composite hydrogel ABAPE-PCB@INS@SC solution; the mass percentage of drug-loaded micelle ABAPE-PCB@INS in the solution is 2 mg / mL;

[0081] Step 303, prepare pH-responsive drug-loaded micelle composite hydrogel ABAPE-PCB@INS@SC under the conditions of voltage 15 kV, flow rate 1 mL / h, rotation speed 300 rpm, and collection liquid being 0.2 mmol / L calcium chloride solution.

[0082] Performance Evaluation

[0083] The synthetic route of polymer micelles in Example 1;

[0084] is the infrared spectrum of the polymer micelle ABAPE-PCB in Example 1;

[0085] This is the SEM image of the polymer micelle ABAPE-PCB in Example 1, and it can be seen that the zwitterionic polymer micelle is spherical in shape;

[0086] Schematic diagram of mucus penetration of polymer micelle ABAPE-PCB in Example 1;

[0087] This is a scanning electron micrograph of the pH-responsive drug-loaded micelle composite hydrogel in Example 1;

[0088] The cytotoxicity test results of the pH-responsive drug-loaded micelle composite hydrogel in Example 1 were obtained by testing the cytotoxicity of the hydrogel using the CCK-8 method. The results showed that the hydrogel had good biocompatibility.

[0089] This is a schematic diagram of the hypoglycemic effect of ABAPE-PCB@INS@SC gel in Example 1 in a type I diabetic rat model. Compared with subcutaneous injection of insulin, the microsphere gel has a more excellent hypoglycemic effect and can greatly reduce the occurrence of hypoglycemia;

[0090] This is the blood glucose value in the type I diabetic rat model 8 hours after administration of the ABAPE-PCB@INS@SC gel in Example 1. It can be seen that there is no significant difference in the blood glucose level between diabetic rats and normal rats 8 hours after administration of the drug-loaded gel.

[0091] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A pH-responsive drug-loaded micelle composite hydrogel, characterized in that: The drug-loaded micelle ABAPE-PCB is a zwitterionic micelle, in which PCB, namely polycarboxybetaine, as the hydrophilic end of the nanoparticle micelle can quickly pass through the mucus layer and can penetrate the epithelial cell layer through the proton-assisted amino acid transporter 1 to increase the absorption of intestinal drugs; ABAPE is a hydrophobic block, namely 4-aminophenylboronic acid pinacol ester with a glucose-responsive phenylboronic acid group, which is glucose-sensitive and can achieve on-demand release of insulin.

2. A pH-responsive drug-loaded micelle composite hydrogel according to claim 1, characterized in that: The pH-responsive drug-loaded micelle composite hydrogel is made of drug-loaded micelles ABAPE-PCB@INS and microsphere gel matrices sodium alginate and carboxymethyl chitosan. As a natural anionic polysaccharide, sodium alginate will quickly gel when divalent cations are added. In addition, sodium alginate is also a pH-sensitive polymer. Due to its rapid dissolution, it is stable in acidic environments, but unstable in alkaline environments. It is an ideal choice for designing intestinal targeted drug delivery systems. Carboxymethyl chitosan can improve the drug release of sodium alginate gel in the intestinal environment, and has the characteristics of good biocompatibility and biodegradability, low price, and non-toxicity.

3. A method for preparing a pH-responsive drug-loaded micelle composite hydrogel for oral delivery of insulin according to claim 1 or 2, characterized in that: include: Step 1: preparing zwitterionic polymer ABAPE-PCB, specifically comprising: Step 101, at a temperature of 45 to 50° C., dissolve ethyl 2-(dimethylamino)methacrylate and tert-butyl bromoacetate in anhydrous acetonitrile at a molar ratio of 1:1 to 2, stir for 12 to 24 hours under nitrogen protection, then add 50 to 200 mL of cold ether to precipitate a white solid, separate by suction filtration and vacuum dry for 12 to 24 hours to obtain a CB-tBu monomer; Step 102, dissolving CB-tBu, 4-cyano-4-(phenylthiothiothio)pentanoic acid N-succinimidyl ester and 2,2′-azobisisobutyronitrile in a certain proportion in 5-20 mL of anhydrous dimethylformamide, deoxygenating, placing in an ice bath for 30-120 min under N2 protection, heating under reflux with a condenser at 60-70° C., stirring for reaction for 12-24 h, then adding 50-200 mL of ether to the reaction mixture, placing in a 4° C. refrigerator and letting stand, discarding the upper mixed solution, then adding 5-10 mL of acetone at 0° C., ultrasonically precipitating for 2-3 times, removing the supernatant, collecting the precipitate and vacuum drying for 12-24 h to obtain the intermediate NHS-PCB-tBu; Step 103, dissolving the intermediate NHS-PCB-tBu in anhydrous N,N-dimethylformamide, and dissolving a certain amount of 4-aminophenylboronic acid pinacol ester in chloroform, mixing the two, adding a certain amount of triethylamine dropwise, adjusting the pH to 7-9, stirring at room temperature for 5-10 days, and then rotary distilling at 50°C for 30-60 minutes, and precipitating in ether, filtering the filtrate and resuspending and filtering with acetonitrile, rotary distilling at 60°C for 30-60 minutes, adding 4-8 mL TFA and stirring at room temperature for 4-8 hours, precipitating with ether and filtering, vacuum drying overnight for 12-24 hours, and then neutralizing with hydroxylamine in PBS with a pH of 7-9 until the uncoupled PCB is dissolved, and then ultrafiltration in PBS, repeating ultrafiltration in water for 2-5 times to obtain ABAPE-PCB solution, and then freeze-drying in pure water for 12-24 hours to obtain the dried polymer ABAPE-PCB; Step 2: Prepare drug-loaded micelles ABAPE-PCB@INS by dialysis method, specifically: A certain amount of polymer ABAPE-PCB was dissolved in phosphate buffer, and a certain amount of dimethyl sulfoxide was added after stirring for 20 to 90 minutes, and the stirring was continued for 0.5 to 3 hours; an appropriate amount of insulin solution was slowly dripped in, and the stirring was continued for 30 to 60 minutes; the ABAPE-PCB@INS solution loaded with insulin was obtained by dialyzing in deionized water at 4°C for 24 to 48 hours, and the insulin-loaded micelle freeze-dried powder was obtained after vacuum freeze drying; Step 3: Preparation of pH-responsive drug-loaded micelle composite hydrogel ABAPE-PCB@INS@SC, specifically: Step 301, weighing a certain mass of sodium alginate and carboxymethyl chitosan and adding them into deionized water to obtain a mixed solution of sodium alginate and carboxymethyl chitosan, which is named as SC solution; the mass of sodium alginate is 0.5 to 5 times the mass of carboxymethyl chitosan; the mass percentage of sodium alginate in the SC solution is 1% to 5%; Step 302, adding the polymer drug-loaded micelle ABAPE-PCB@INS solution to the SC solution and stirring and mixing to obtain a pH-responsive drug-loaded micelle composite hydrogel ABAPE-PCB@INS@SC solution; the mass percentage of the drug-loaded micelle ABAPE-PCB@INS in the solution is 0.5-5 mg / mL; Step 303, prepare pH-responsive drug-loaded micelle composite hydrogel ABAPE-PCB@INS@SC under the conditions of voltage 5-20 kV, flow rate 1-3 mL / h, rotation speed 150-400 rpm, and collection liquid of 0.2 mmol / L calcium chloride solution.

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