Nanoparticle loaded with oral protein drug for treatment of diabetes and carrier

Abstract

A nanoparticle loaded with an oral protein drug for the treatment of diabetes and a carrier. Raw materials of said nanoparticle comprise: a lipid molecule-modified zwitterionic polymer, a cationic lipid, a phospholipid, and cholesterol. The lipid molecule-modified zwitterionic polymer is composed of a lipid molecule and a polymer, wherein the lipid molecule is distearoyl phosphoethanolamine or dimyristoylglycerol, and the polymer is a poly(carboxybetaine) polymer, a poly(phosphobetaine) polymer, a poly(sulfobetaine) polymer, or poly-2-(N-oxide-N,N-diethylamino)ethyl methacrylate. Said nanoparticle significantly improves the oral bioavailability of the protein drug.

Description

A nanoparticle loaded with a protein drug for oral treatment of diabetes and a carrier TECHNICAL FIELD

[0001] The present application relates to the field of synthetic biology, in particular to a nanoparticle loaded with a protein drug for oral treatment of diabetes and a carrier. BACKGROUND

[0002] Insulin and its analogs, GLP-1, GIP and GCG receptor agonists have good application in the field of diabetes treatment. However, subcutaneous injection is currently used for clinical administration. The patient compliance and safety of subcutaneous injection are poor. In contrast, oral administration is simple, convenient, has good patient compliance and high safety. However, insulin and its analogs, GLP-1, GIP and GCG receptor agonists are usually hydrophilic molecules with large molecular weight, which cannot enter the systemic circulation well after oral administration due to the existence of multiple physiological tissue barriers, and have low bioavailability.

[0003] The main reasons for this phenomenon include: (1) the acidic environment of gastric juice and a large amount of protein / polypeptide enzymes in the digestive tract make the oral protein drugs easy to decompose and inactivate; (2) the oral protein drugs are mostly hydrophilic structures and have large molecular weight, which are difficult to pass through the intestinal mucus layer and small intestinal villus epithelial cell layer to enter the blood circulation, and then play a role in the target organs.

[0004] SUMMARY

[0005] The purpose of the present application is to provide a nanoparticle loaded with a protein drug for oral treatment of diabetes and a carrier, so as to enhance the anti-enzymatic activity and penetration ability of the loaded protein drug in the stomach, intestinal mucus layer and small intestinal villus epithelial cells, improve the absorption, and significantly improve the oral bioavailability of the protein drug.

[0006] The technical scheme adopted by the present application to solve its technical problems is:

[0007] A nanoparticle carrier loaded with a protein drug for oral treatment of diabetes, the raw materials of which include: a lipid molecule modified zwitterionic polymer, a cationic lipid, a phospholipid and cholesterol.

[0008] The lipid molecule modified zwitterionic polymer is composed of a lipid molecule and a polymer, wherein the lipid molecule is distearoyl phosphatidyl ethanolamine or dimyristoyl glycerol, and the polymer is polycarboxybetaine polymer, polyphosphobetaine polymer, polysulfobetaine polymer or poly(2-(N-oxyl-N,N-dimethylamino)ethyl methacrylate).

[0009] As a preference, the lipid molecule-modified zwitterionic polymer is selected from one or more of distearoylphosphatidylethanolamine-polycarboxybetaine polymer, distearoylphosphatidylethanolamine- polyphosphobetaine polymer, distearoylphosphatidylethanolamine-polysulfobetaine polymer, distearoylphosphatidylethanolamine-poly(2-(N-oxyl-N,N-dimethylammonium)ethyl methacrylate), dimyristoylglycerophosphatidylethanolamine-polycarboxybetaine polymer, dimyristoylglycerophosphatidylethanolamine-polyphosphobetaine polymer, dimyristoylglycerophosphatidylethanolamine-polysulfobetaine polymer, dimyristoylglycerophosphatidylethanolamine-poly(2-(N-oxyl-N,N-dimethylammonium)ethyl methacrylate).

[0010] The cationic lipid is selected from one or more of dimethyl-2-hydroxyethyl-2,3- dioleyloxypropylammonium bromide (DORIE), dimethyl-2,3-dioleyloxypropyl-2-(2- spermidinecarboxamido)ethylammonium trifluoroacetate (DOSPA), trimethyl-2,3- dioleyloxypropylammonium bromide (DOTAP), trimethyl-2,3-dioleyloxypropylammonium chloride (DOTMA), dimethyl-dioctadecylammonium bromide (DDAB), dimethyl-2- hydroxyethyl-2,3-dihexadecyloxypropylammonium bromide (DPRIE), ALC-0315, SM-102, Dlin-MC3-DMA, 1,2-dioleoyloxy-3-(dimethylamino)propane (DODAP), 1,2- dioleoyl-3-dimethylamino-propane (DODMA), preferably the cationic lipid is selected from trimethyl-2,3-dioleyloxypropylammonium bromide (DOTAP), trimethyl-2,3- dioleyloxypropylammonium chloride (DOTMA), 1,2-dioleoyl-3-(dimethylamino)propane (DODAP), 1,2-dioleoyl-3-dimethylamino-propane (DODMA).

[0011] As a preference, the phospholipid is selected from one or more of distearoylphosphatidic acid, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidic acid, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine. Preferably, the phospholipid is selected from dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC).

[0012] As a preference, the molar ratio of lipid molecule-modified zwitterionic polymer: cationic lipid: phospholipid: cholesterol = 1: 2.5-10: 1.5-9: 2-9.5.

[0013] The lipid molecule and polymer are synthesized into the lipid molecule-modified zwitterionic polymer by atom transfer radical polymerization.

[0014] A nanoparticle loaded with a protein drug for oral treatment of diabetes, which comprises a nanoparticle carrier and a protein drug for oral treatment of diabetes, wherein the nanoparticle carrier is loaded with the protein drug for oral treatment of diabetes.

[0015] Further, an oral preparation loaded with a protein drug for oral treatment of diabetes, which comprises a nanoparticle loaded with a protein drug for oral treatment of diabetes and a buffer.

[0016] The mass ratio of the nanoparticle carrier to the protein drug for oral treatment of diabetes is 20:1-5, preferably 20:1.5-3, and more preferably 20:2-2.5.

[0017] The protein drug for oral treatment of diabetes includes insulin and its analogs, G protein coupled receptor (GCPR) agonists, and the G protein coupled receptor (GCPR) agonists include GLP-1 receptor agonists, GLP-1 / GIP dual receptor agonists, and GLP-1 / GIP / GCGs triple receptor agonists.

[0018] The insulin and its analogs are selected from one or more of recombinant human insulin, insulin glargine, insulin detemir, and insulin degludec; and the G protein coupled receptor (GCPR) agonists are selected from one or more of exenatide, liraglutide, albiglutide, dulaglutide, taspoglutide, and semaglutide.

[0019] The present application has the following advantages:

[0020] The present application first uses dimyristoyl glycerol / distearyl phosphatidyl ethanolamine to modify polycarboxybetaine polymer, polyphosphobetaine polymer, polysulfobetaine polymer and polymethacrylic acid-2-(N-oxidized-N,N-diethylamino)ethyl ester to synthesize a new type of lipid molecule modified zwitterionic polymer. The new zwitterionic polymer of the present application can participate in self-assembly to form lipid nanoparticles with more stable structure, smaller particle size and more uniform dispersion, and has super strong drug loading capacity, which provides safety guarantee for subsequent long-term treatment; at the same time, the new zwitterionic polymer can form a very stable hydration layer with water molecules through ionic bond in a physiological environment, resist non-specific protein adsorption, significantly prolong the circulation time of drugs in blood, effectively reduce the risk of enzyme inactivation of protein and polypeptide drugs, and further enhance the diffusion and penetration of the system in intestinal mucus.

[0021] The nanoparticles constructed in the application are applied to the field of protein and polypeptide drug delivery, and provide a safe and efficient candidate delivery platform for the further development of the field. The nanoparticles are administered to model mice for treatment, and through the detection of blood glucose, drug metabolism kinetics and oral glucose tolerance and other pharmacodynamic indexes, a new and different composition ratio is screened out. The application of the ratio significantly reduces the blood glucose level of the model mice and rats, improves the blood drug peak value of the model mice and rats after administration, significantly improves the insulin resistance and other symptoms of the model mice and rats, and realizes safe and efficient treatment of diseases. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a nanoparticle particle size diagram prepared in Examples 1-3;

[0023] Figure 2 is a nanoparticle drug loading rate diagram prepared in Examples 1-3;

[0024] Figure 3 is a nanoparticle cytotoxicity diagram prepared in Examples 1-3;

[0025] Figure 4 is a model mouse 24h blood glucose change diagram administered with the nanoparticles prepared in Examples 1-3;

[0026] Figure 5 is a nanoparticle particle size diagram prepared in Examples 4-6;

[0027] Figure 6 is a nanoparticle drug loading rate diagram prepared in Examples 4-6;

[0028] Figure 7 is a nanoparticle cytotoxicity diagram prepared in Examples 4-6;

[0029] Figure 8 is a model mouse 24h blood glucose change diagram administered with the nanoparticles prepared in Examples 4-6;

[0030] Figure 9 is a model mouse hypoglycemic curve area under the curve diagram administered with the nanoparticles prepared in Examples 7-18. DETAILED DESCRIPTION

[0031] The technical solutions of the application will be further specifically described below through specific examples.

[0032] In the application, unless specified, the raw materials and equipment used can be purchased from the market or commonly used in the art. The methods in the following examples are conventional methods in the art, unless otherwise specified.

[0033] In the following specific implementation: the mass ratio of the lipid molecule modified zwitterionic polymer, the cationic lipid, the phospholipid and the cholesterol to the protein drug is 20mg:(1-5)mg, preferably 20mg:(1.5-3)mg, and further preferably 20mg:(2-2.5)mg.

[0034] As a specific embodiment 1, the present application provides a preparation method of nanoparticles loaded with GLP-1 receptor agonist, comprising the following steps:

[0035] (1) Dissolve dimyristyl glycerol-polycarboxybetaine polymer, phospholipid, cholesterol and cationic lipid in a mixed solution of ethanol and chloroform; the total mass concentration of the mixed solution is 1-50 mg / mL, preferably 10-40 mg / mL, and further preferably 15-25 mg / mL; the volume ratio of ethanol to chloroform in the mixed solution of ethanol and chloroform is 0.05-5:1, preferably 0.1-3:1, and further preferably 0.3-2:1.

[0036] (2) Spin the above mixed solution and uniformly disperse it into a thin film in a dry Schlenk flask; the spin temperature is 20-60°C, preferably 25-55°C, and further preferably 30-50°C. The spin speed is 30-200 rpm, preferably 50-180 rpm, and further preferably 70-160 rpm. The spin time is 1-30 min, preferably 5-25 min, and further preferably 10-20 min.

[0037] (3) Add an aqueous semaglutide solution and a buffer solution to the above Schlenk flask and ultrasonicate to obtain a mixed solution; the mass concentration of semaglutide is 0.1-50 mg / mL, preferably 0.5-30 mg / mL, and further preferably 1-20 mg / mL. The buffer solution is a phosphate buffer solution, a borate buffer solution, an acetate buffer solution, a carbonate buffer solution, a citrate buffer solution, a Tris-HCl buffer solution, and HEPES, preferably a borate buffer solution, an acetate buffer solution, a carbonate buffer solution, a citrate buffer solution, and a Tris-HCl buffer solution, and further preferably an acetate buffer solution, a carbonate buffer solution, and a citrate buffer solution. The pH of the buffer solution is 0.5-7.4, preferably 1-6.8, and further preferably 2-5.8.

[0038] (4) Dialyze the above mixed solution with water as the external water phase to obtain GLP-1 analogue nanoparticles based on dimyristyl glycerol-polycarboxybetaine polymer. The dialysis time is 2-24 h, preferably 3-12 h, and further preferably 6-8 h.

[0039] Preferably, the polycarboxybetaine polymer comprises polycarboxybetaine methacrylate.

[0040] Preferably, the polycarboxybetaine methacrylate has a degree of polymerization of any integer from 1 to 500, including but not limited to 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 38, 40, 44, 48, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 480, or 490, preferably any integer from 20 to 300, and further preferably any integer from 20 to 100.

[0041] Preferably, the method for preparing the dimyristylglycerol-polycarboxybetaine methacrylate (with a theoretical degree of polymerization of 50) comprises:

[0042] Into a dry Schlenk flask was added dimethylaminoethyl methacrylate (0.6245 g, 3.97 mmol) and 20 mL of anhydrous dichloromethane, stirred at 10 °C for 10 min, and β-propiolactone (0.3437 g, 4.77 mmol) was quickly added. The reaction was allowed to proceed for 5 h, dichloromethane was removed using a rotary evaporator, and an appropriate amount of acetone was added to filter. The white solid was then washed twice with dichloromethane and twice with diethyl ether, and dried under vacuum to obtain the carboxybetaine methacrylate.

[0043] DMG (0.15 g, 0.29 mmol) was weighed into 20 mL of anhydrous dichloromethane, and triethylamine (0.0887 g, 0.879 mmol) was added. The solution was stirred at room temperature for 1 h, and 2-bromo-2-methylpropionyl bromide (0.10087 g, 0.439 mmol) was added. The solution was heated to 45 °C and condensed to reflux for 10 h. At the end of the reaction, the reaction solution was transferred to a separatory funnel, washed once with acid and three times with deionized water. The dichloromethane phase was then dehydrated with anhydrous Na2SO4, and the solid Na2SO4 was removed by filtration. The solvent dichloromethane was removed using a rotary evaporator to obtain white solid DMG-Br.

[0044] DMG-Br (0.242 g), carboxybetaine methacrylate (5.03 g), Cu(I)Br (0.107 g), dichloromethane (5 mL) and ethanol (15 mL) were added into a clean and dry Schlenk flask, and three freeze-thaw cycles were performed. A mixture of N,N,N',N',N"-pentamethyldiethylenetriamine (PMDETA) (0.130 g) and ethanol (1 mL) was injected into the above frozen reaction system using a disposable syringe, and three freeze-thaw cycles were performed. The reaction system was stirred at 60 °C for 24 h, and then dialyzed against deionized water for 24 h using a Cellu Sep H1 membrane (MWCO 3500) as the inner phase. The sample in the dialysis bag was dried in a freeze dryer to obtain dimyristyl glycerol-poly-carboxybetaine methacrylate.

[0045] Using the above method, by changing the amount of carboxybetaine methacrylate, dimyristyl glycerol-poly-carboxybetaine methacrylate with other theoretical degrees of polymerization can be synthesized.

[0046] As a specific embodiment 2, the present application provides a preparation method of a nanoparticle loaded with a GLP-1 / GIP dual agonist, which comprises the following steps:

[0047] (1) Dissolve dimyristyl glycerol-poly-carboxybetaine polymer (same as embodiment 1), phospholipid, cholesterol and cationic lipid in dichloromethane to form a mixed solution; the total mass concentration of the mixed solution is 1-50 mg / mL, preferably 10-40 mg / mL, and further preferably 15-25 mg / mL.

[0048] (2) Mix the above mixed solution with a tarpide aqueous solution at a certain volume ratio, and perform ultrasonic treatment for a certain time until a stable water-in-oil emulsion is formed; the mass concentration of the tarpide aqueous solution is 0.1-50 mg / mL, preferably 0.5-30 mg / mL, and further preferably 1-20 mg / mL. The volume ratio of the mixed solution to the tarpide aqueous solution is 1-10:1, preferably 1.5-8:1, and further preferably 2-5:1. The ultrasonic power is 3%-70%, preferably 5%-60%, and further preferably 8%-45%. Preferably, the ultrasonic time is 0.5-10 min, preferably 1-8 min, and further preferably 2-6 min.

[0049] (3) Evaporate the above water-in-oil emulsion in a dry Schlenk flask under reduced pressure to obtain a nanoparticle suspension;

[0050] (4) The above suspension is dialyzed with water as the external water phase (same as Embodiment 1) to obtain a GLP-1 / GIP dual agonist nanoparticle preparation based on dimyristyl glycerol-polycarboxybetaine polymer.

[0051] As a specific embodiment 3, the present application provides a preparation method of a GLP-1 / GIP / GCG triple receptor agonist-loaded nanoparticle, which comprises the following steps:

[0052] (1) Dimyristyl glycerol-polycarboxybetaine polymer (same as Embodiment 1), phospholipid, cholesterol and cationic lipid are dissolved in an organic solvent to form a mixed solution (concentration same as Embodiment 1); the organic solvent includes dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF) and acetonitrile, preferably N,N-dimethylformamide (DMF), acetonitrile, and further preferably N,N-dimethylformamide (DMF).

[0053] (2) Retalipotide is dissolved in deionized water to form a mixed solution with a certain mass concentration; the mass concentration of the retalipotide aqueous solution is 0.1-50 mg / mL, preferably 0.5-30 mg / mL, and further preferably 1-20 mg / mL.

[0054] (3) The above two solutions are placed in an LNP synthesizer at a certain volume ratio (same as Embodiment 2), and a nanoparticle suspension is prepared at a certain flow rate; the flow rate is 10-200 ml / min, preferably 20-180 ml / min, and further preferably 30-160 ml / min.

[0055] (4) The above suspension is dialyzed with water as the external water phase (same as Embodiment 1) to obtain a GLP-1 / GIP / GCG receptor agonist nanoparticle preparation based on dimyristyl glycerol-polycarboxybetaine polymer.

[0056] As a specific embodiment 4, the present application provides a preparation method of an insulin-loaded nanoparticle, which comprises the following steps:

[0057] (1) Dissolve dimyristylglycero-polyphosphobetaine polymer: dimyristylglycero-poly(2-methacryloyloxyethyl phosphorylcholine) (monomer is replaced by 2-methacryloyloxyethyl phosphorylcholine, the molar ratio of reactants and other steps are the same as the preparation method of dimyristylglycero-polycarboxybetaine methacrylate (as an example of theoretical degree of polymerization 50) of embodiment 1), phospholipid, cholesterol and one of cationic lipids (ALC-0315, SM-102, Dlin-MC3-DMA, 1,2-dioleoyl-3-(dimethylamino) propane (DODAP), 1,2-dioleoyl-3-dimethylamino-propane (DODMA)) in a mixed solution of ethanol and chloroform (the same as embodiment 1);

[0058] (2) The above mixed solution is rotary evaporated (the same as embodiment 1) and uniformly dispersed into a thin film in a dry Schlenk flask.

[0059] (3) Add an aqueous insulin solution (concentration the same as embodiment 1) and a buffer solution (the same as embodiment 1) to the above Schlenk flask and ultrasonicate to obtain a mixed solution.

[0060] (4) Dialyze the above mixed solution with water as the external water phase (the same as embodiment 1) to obtain insulin nanoparticles based on dimyristylglycero-polyphosphobetaine polymer.

[0061] As a specific embodiment 5, the present application provides a preparation method of insulin-loaded nanoparticles, which comprises the following steps:

[0062] (1) Dissolve dimyristylglycero-poly(2-methacryloyloxyethyl phosphorylcholine) (monomer is replaced by 2-methacryloyloxyethyl phosphorylcholine, the molar ratio of reactants and other steps are the same as the preparation method of dimyristylglycero-polycarboxybetaine methacrylate (as an example of theoretical degree of polymerization 50) of embodiment 1), phospholipid, cholesterol and one of cationic lipids (ALC-0315, SM-102, Dlin-MC3-DMA, 1,2-dioleoyl-3-(dimethylamino) propane (DODAP), 1,2-dioleoyl-3-dimethylamino-propane (DODMA)) in dichloromethane to form a mixed solution (concentration the same as embodiment 1);

[0063] (2) Mix the above mixed solution with an aqueous insulin solution (concentration the same as embodiment 1) at a certain volume ratio (volume ratio the same as embodiment 2) and ultrasonicate for a certain time (the same as embodiment 2) until a stable water-in-oil emulsion is formed;

[0064] (3) Evaporate the above water-in-oil emulsion in a dry Schlenk flask under reduced pressure to obtain a nanoparticle suspension;

[0065] (4) The above suspension is dialyzed against water as the external water phase (as in embodiment 1) to obtain a insulin nanoparticle formulation based on dimyristylglycerol-poly(2-methacryloyloxyethyl phosphorylcholine).

[0066] As a specific embodiment 6, which differs from embodiment 3 in that the insulin is replaced by exenatide, the dimyristylglycerol-polycarboxybetaine polymer is replaced by dimyristylglycerol-poly(2-methacryloyloxyethyl phosphorylcholine), and otherwise is the same as embodiment 3.

[0067] Example 1

[0068] This example prepares an oral formulation based on a GLP-1 receptor agonist, which comprises nanoparticles encapsulating a GLP-1 receptor agonist and a buffer solution, the nanoparticles being based on a polycarboxybetaine polymer encapsulating a GLP-1 receptor agonist, and the buffer solution being an acetic acid-sodium acetate solution with pH = 4. The method of preparing the oral formulation comprises the following steps:

[0069] Dimyristylglycerol-polycarboxybetaine methacrylate (1.5 mg), distearoylphosphatidylcholine (1 mg), cholesterol (0.75 mg) and trimethyl-2,3-dioleoyloxypropylammonium bromide (1.675 mg) are dissolved in a mixed solution of ethanol and chloroform (volume ratio 1:2) (15 mL);

[0070] The above mixed solution is rotary evaporated in a dry Schlenk flask, the rotary evaporation temperature is 36°C, the rotation speed is 150 rpm, and the time is 20 min, so as to uniformly disperse it into a thin film;

[0071] The above Schlenk flask is added with a semaglutide aqueous solution (10 mg / mL, 0.1 mL) and an acetic acid-sodium acetate solution with pH = 4 (0.9 mL), and a mixed solution is obtained by ultrasonic treatment under an ultrasonic power of 600 w for 5 min;

[0072] A GLP-1 receptor agonist nanoparticle based on dimyristylglycerol-polycarboxybetaine polymer is obtained.

[0073] Example 2

[0074] This example describes the preparation of an oral formulation based on GLP-1 / GIP receptor agonist comprising nanoparticles encapsulating GLP-1 / GIP receptor agonist and a buffer solution, said nanoparticles being nanoparticles encapsulating GLP-1 / GIP receptor agonist based on polycarboxybetaine polymer, said buffer solution being a citric acid-sodium citrate solution at pH = 5.2. The method for preparing said oral formulation comprises the following steps:

[0075] Dipalmitoylglycerol-polycarboxybetaine methacrylate (1.6 mg), distearoylphosphatidylcholine (1.1 mg), cholesterol (0.68 mg) and trimethyl-2,3-dioleoyloxypropylammonium bromide (1.82 mg) were dissolved in chloroform (3 mL) to form a mixed solution;

[0076] The above mixed solution (3 mL), tirzepatide aqueous solution (0.35 mg / mL, 3 mL) and citric acid-sodium citrate solution at pH = 5.2 (6 mL) were mixed at a volume ratio of 1:1:2, and ultrasonicated at a power of 550 w for 12 min until a stable water-in-oil emulsion was formed;

[0077] The above water-in-oil emulsion was evaporated in a dry Schlenk flask at 42 °C and 170 rpm for 25 min under reduced pressure to obtain a nanoparticle suspension;

[0078] The above suspension was dialyzed against water as the external water phase for 18 h to obtain a GLP-1 / GIP receptor agonist nanoparticle formulation based on dipalmitoylglycerol-polycarboxybetaine polymer.

[0079] Example 3

[0080] This example describes the preparation of an oral formulation based on GLP-1 / GIP / GCG receptor agonist comprising nanoparticles encapsulating GLP-1 / GIP / GCG receptor agonist and a buffer solution, said nanoparticles being nanoparticles encapsulating GLP-1 / GIP / GCG receptor agonist based on polycarboxybetaine polymer. The method for preparing said oral formulation comprises the following steps:

[0081] Dipalmitoylglycerol-polycarboxybetaine methacrylate (1.6 mg), distearoylphosphatidylcholine (1.1 mg), cholesterol (0.68 mg) and trimethyl-2,3-dioleoyloxypropylammonium bromide (1.82 mg) were dissolved in chloroform (3 mL) to form a mixed solution;

[0082] Retevzotide was dissolved in deionized water to form a mixed solution with a concentration of 0.35 mg / mL;

[0083] The two mixed solutions above were placed in an LNP synthesizer at a volume ratio of 1:3, and a nanoparticle suspension was prepared at a flow rate of 50 mL / min and 150 mL / min;

[0084] The mixed solution above was dialyzed against water as an external aqueous phase for 12 hours to obtain a GLP-1 / GIP / GCG receptor agonist nanoparticle based on dimyristyl glycerol-polycarboxybetaine methacrylate.

[0085] Example 4

[0086] This example prepared an oral preparation based on insulin and its analogs, which included nanoparticles loaded with insulin and its analogs based on poly(2-methacryloyloxyethyl phosphorylcholine) and a buffer solution of acetic acid-sodium acetate solution with pH = 4. The preparation method of the oral preparation included the following steps:

[0087] Dimyristyl glycerol-poly(2-methacryloyloxyethyl phosphorylcholine) (1.875 mg), distearoylphosphatidylcholine (1 mg), cholesterol (0.675 mg), and 1,2-dioleoyl-3-(dimethylamino) propane (1.45 mg) were dissolved in a mixed solution of ethanol and chloroform (volume ratio 1:2) (15 mL);

[0088] The mixed solution above was rotary evaporated in a dry Schlenk flask, the rotary evaporation temperature was 36°C, the rotation speed was 150 rpm, and the time was 20 min, so that it was uniformly dispersed into a thin film;

[0089] The aqueous solution of insulin and its analogs (recombinant human insulin, 10 mg / mL, 0.1 mL), acetic acid-sodium acetate solution with pH = 4 (0.9 mL) were added to the above Schlenk flask, and a mixed solution was obtained by ultrasonic treatment at an ultrasonic power of 600 w for 5 min;

[0090] Thus, insulin and its analogs nanoparticles based on dimyristyl glycerol-poly(2-methacryloyloxyethyl phosphorylcholine) were obtained.

[0091] Example 5

[0092] This example prepared an oral preparation based on insulin and its analogs, which included nanoparticles loaded with insulin and its analogs based on poly(2-methacryloyloxyethyl phosphorylcholine) and a buffer solution of citric acid-sodium citrate solution with pH = 5.2. The preparation method of the oral preparation included the following steps:

[0093] Dipalmitoylglycerol-poly(2-methacryloyloxyethyl phosphoryl choline) (2.1 mg), distearoylphosphatidyl choline (1.3 mg), cholesterol (0.86 mg) and 1,2-dioleoyl-3-(dimethylamino)propane (1.56 mg) were dissolved in chloroform (3 mL) to form a mixed solution;

[0094] The above mixed solution (3 mL), an aqueous solution of insulin and its analogues (degludec, 0.5 mg / mL, 3 mL) and a citric acid-sodium citrate solution (pH = 5.2, 3 mL) were mixed at a volume ratio of 1:1:1, and an ultrasonic power of 500 w was used to ultrasonicate for 10 min until a stable water-in-oil emulsion was formed;

[0095] The above water-in-oil emulsion was evaporated in a dry Schlenk flask at 45°C and a rotation speed of 180 rpm under reduced pressure for 20 min to obtain a nanoparticle suspension;

[0096] The above suspension was dialyzed against water as an external aqueous phase for 24 h to obtain an insulin and its analogues nanoparticle preparation based on dipalmitoylglycerol-poly(2-methacryloyloxyethyl phosphoryl choline).

[0097] Example 6

[0098] This example prepared an insulin and its analogues-based oral preparation, which comprises nanoparticles loaded with insulin and its analogues and a buffer solution, and the nanoparticles are insulin and its analogues-loaded nanoparticles based on poly(2-methacryloyloxyethyl phosphoryl choline). The preparation method of the oral preparation comprises the following steps:

[0099] Dipalmitoylglycerol-poly(2-methacryloyloxyethyl phosphoryl choline) (3.6 mg), distearoylphosphatidyl choline (2.2 mg), cholesterol (1.48 mg) and 1,2-dioleoyl-3-(dimethylamino)propane (3.58 mg) were dissolved in N,N-dimethylformamide (0.3 mL) to form a mixed solution;

[0100] Insulin and its analogues (insulin aspart) were dissolved in a dilute hydrochloric acid solution (pH = 2) to form a mixed solution with a mass concentration of 0.5 mg / mL;

[0101] The above two mixed solutions were placed in an LNP synthesizer at a volume ratio of 1:3, and a nanoparticle suspension was prepared at a flow rate of 50 mL / min and 150 mL / min;

[0102] The above mixed solution was dialyzed for 12 hours with water as the external water phase to obtain insulin and its analog nanoparticles based on dimyristyl glycerol-poly(2-methacryloyloxyethyl phosphoryl choline).

[0103] Example 7

[0104] This example prepared an oral preparation based on GLP-1 receptor agonist, which comprises nanoparticles encapsulating GLP-1 receptor agonist and a buffer solution, the nanoparticles are poly-carboxybetaine polymer-based nanoparticles encapsulating GLP-1 receptor agonist, and the buffer solution is an acetic acid-sodium acetate solution with pH = 4. The preparation method of the oral preparation comprises the following steps:

[0105] Dipalmitoyl phosphatidyl ethanolamine-poly-carboxybetaine methacrylate (3 mg), distearoyl phosphatidyl choline (2 mg), cholesterol (1.5 mg), and 1,2-dioleoyl-3-dimethylamino-propane (3.4 mg) were dissolved in a mixed solution of ethanol and trichloromethane (volume ratio 1:2) (15 mL);

[0106] The above mixed solution was rotary evaporated in a dry Schlenk flask, the rotary evaporation temperature was 36°C, the rotation speed was 150 rpm, and the time was 20 min, so as to uniformly disperse it into a thin film;

[0107] The above Schlenk flask was added with a semaglutide aqueous solution (10 mg / mL, 0.2 mL) and an acetic acid-sodium acetate solution with pH = 4 (0.8 mL), and a mixed solution was obtained by ultrasonic treatment under an ultrasonic power of 600 w for 5 min;

[0108] Thus, GLP-1 receptor agonist nanoparticles based on dipalmitoyl phosphatidyl ethanolamine-poly-carboxybetaine polymer were obtained.

[0109] The preparation method of dipalmitoyl phosphatidyl ethanolamine-poly-carboxybetaine polymer:

[0110] The preparation method of the above dipalmitoyl phosphatidyl ethanolamine-poly-carboxybetaine methacrylate (taking a theoretical polymerization degree of 50 as an example) comprises:

[0111] A dry Schlenk flask was added with dimethylaminoethyl methacrylate (0.6245 g, 3.97 mmol) and 20 mL of anhydrous dichloromethane, stirred at 10°C for 10 min, quickly added with β-propiolactone (0.3437 g, 4.77 mmol), reacted for 5 h, removed the dichloromethane by using a rotary evaporator, and added with an appropriate amount of acetone for filtration, to obtain a white solid, which was then washed twice with dichloromethane and diethyl ether respectively, and vacuum dried, to obtain carboxybetaine methacrylate.

[0112] Weighed 2,3-dioleoyl-sn-glycero-3-phosphoethanolamine (DSPE) (0.22 g, 0.29 mmol) in 20 mL of anhydrous dichloromethane, added triethylamine (0.0887 g, 0.879 mmol), stirred at room temperature for 1 h. Added 2-bromo-2-methylpropionyl bromide (0.10661 g, 0.464 mmol) to the above solution, heated to 45 °C, and condensed the refluxing reaction for 10 h. At the end of the reaction, the reaction solution was transferred to a separatory funnel, washed with acid once, and deionized water three times. The dichloromethane phase was dried over anhydrous Na2SO4, and the solid Na2SO4was removed by filtration. The solvent dichloromethane was removed using a rotary evaporator to obtain white solid DSPE-Br.

[0113] DSPE-Br (0.353 g), carboxybetaine methacrylate (5.03 g), Cu(I)Br (0.107 g), dichloromethane (5 mL), and ethanol (15 mL) were added to a clean and dry Schlenk flask, and three freeze-thaw-degassing-thaw cycles were performed. A mixture of N,N,N',N',N"-pentamethyldiethylenetriamine (PMDETA) (0.130 g) and ethanol (1 mL) was injected into the above frozen reaction system using a disposable syringe, and three freeze-thaw-degassing-thaw cycles were performed. The reaction was stirred at 60 °C for 24 h, and the reaction system was taken up in a Cellu Sep H1 membrane (MWCO 3500) with deionized water as the external phase, and dialyzed for 24 h. The sample in the dialysis bag was dried in a freeze dryer to obtain 2,3-dioleoyl-sn-glycero-3-phosphoethanolamine-poly(carboxybetaine methacrylate).

[0114] Using the above method, other theoretical degree of polymerization of 2,3-dioleoyl-sn-glycero-3-phosphoethanolamine-poly(carboxybetaine methacrylate) can be synthesized by changing the amount of carboxybetaine methacrylate.

[0115] Example 8

[0116] The difference between this example and Example 7 is that the lipid molecule modified zwitterionic polymer is replaced by 2,3-dioleoyl-sn-glycero-3-phosphoethanolamine-poly(phosphobetaine) (2,3-dioleoyl-sn-glycero-3-phosphoethanolamine-poly(2-methacryloyloxyethyl phosphorylcholine), cationic lipid (trimethyl-2,3-dioleoyloxypropylammonium chloride), phospholipid (2,3-dioleoyl-sn-glycerol phosphatidic acid). The molar ratio of lipid molecule modified zwitterionic polymer: cationic lipid: phospholipid: cholesterol is the same as that of Example 7.

[0117] The preparation method of the distearoylphosphatidylethanolamine-polysulfobetaine polymer is the same as that of the distearoylphosphatidylethanolamine-polycarboxybetaine methacrylate (with a theoretical degree of polymerization of 50) described in Example 7, except that the monomer is replaced by 2-methacryloyloxyethyl phosphorylcholine, and the molar ratio of reactants is the same as that described in Example 7.

[0118] Example 9

[0119] The difference between this example and Example 7 is that the lipid molecule modified zwitterionic polymer is replaced by distearoylphosphatidylethanolamine-polysulfobetaine polymer (distearoylphosphatidylethanolamine-polymethacrylic acid sulfobetaine), cationic lipid (trifluoro-dimethyl-2,3-dioleyloxypropyl-2-(2-spermidinecarboxamide) ethylammonium), phospholipid (dimyristoyl lecithin). The molar ratio of lipid molecule modified zwitterionic polymer: cationic lipid: phospholipid: cholesterol is the same as that of Example 7.

[0120] The preparation method of the distearoylphosphatidylethanolamine-polysulfobetaine polymer is the same as that of the distearoylphosphatidylethanolamine-polycarboxybetaine methacrylate (with a theoretical degree of polymerization of 50) described in Example 7, except that the monomer is replaced by: methacrylic acid sulfobetaine, and the molar ratio of reactants is the same as that described in Example 7.

[0121] Example 10

[0122] The difference between this example and Example 7 is that the lipid molecule modified zwitterionic polymer is replaced by distearoylphosphatidylethanolamine-polymethacrylic acid-2-(N-oxidized-N,N-diethylamino) ethyl ester, cationic lipid (dimethyl dioctadecyl ammonium bromide), phospholipid (dipalmitoyl phosphatidic acid). The molar ratio of lipid molecule modified zwitterionic polymer: cationic lipid: phospholipid: cholesterol is the same as that of Example 4.

[0123] The preparation method of the distearoylphosphatidylethanolamine-polymethacrylic acid-2-(N-oxidized-N,N-diethylamino) ethyl ester is the same as that of the distearoylphosphatidylethanolamine-polycarboxybetaine methacrylate (with a theoretical degree of polymerization of 50) described in Example 7, except that the monomer is replaced by: diethylaminoethyl methacrylate, and the molar ratio of reactants is the same as that described in Example 7.

[0124] Example 11

[0125] The difference between this example and Example 4 is that the lipid-molecule-modified zwitterionic polymer is replaced by distearoylphosphatidyl ethanolamine-polysulfobetaine polymer (distearoylphosphatidyl ethanolamine-poly(methacrylic acid sulfobetaine)), cationic lipid (SM-102), and phospholipid (distearoylphosphatidyl choline). The molar ratio of the lipid-molecule-modified zwitterionic polymer : cationic lipid : phospholipid : cholesterol is the same as in Example 4.

[0126] The method for preparing the distearoylphosphatidyl ethanolamine-polysulfobetaine polymer is the same as the method for preparing the distearoylphosphatidyl ethanolamine-polycarboxybetaine methacrylate (with a theoretical degree of polymerization of 50) described in Example 7, except that the monomer is replaced by 2-methacryloyloxyethyl phosphoryl choline, and the molar ratio of the reactants is the same as described in Example 7.

[0127] Example 12

[0128] The difference between this example and Example 4 is that the lipid-molecule-modified zwitterionic polymer is replaced by distearoylphosphatidyl ethanolamine-polysulfobetaine polymer (distearoylphosphatidyl ethanolamine-poly(methacrylic acid sulfobetaine)), cationic lipid (SM-102), and phospholipid (distearoylphosphatidyl choline). The molar ratio of the lipid-molecule-modified zwitterionic polymer : cationic lipid : phospholipid : cholesterol is the same as in Example 4.

[0129] The method for preparing the distearoylphosphatidyl ethanolamine-polysulfobetaine polymer is the same as the method for preparing the distearoylphosphatidyl ethanolamine-polycarboxybetaine methacrylate (with a theoretical degree of polymerization of 50) described in Example 7, except that the monomer is replaced by 2-methacryloyloxyethyl phosphoryl choline, and the molar ratio of the reactants is the same as described in Example 7.

[0130] Example 13

[0131] The difference between this example and Example 4 is that the lipid-molecule-modified zwitterionic polymer is replaced by distearoylphosphatidyl ethanolamine-polymethacrylic acid-2-(N-oxidized-N,N-diethylamino)ethyl ester, cationic lipid (Dlin-MC3-DMA), and phospholipid (distearoylphosphatidic acid). The molar ratio of the lipid-molecule-modified zwitterionic polymer : cationic lipid : phospholipid : cholesterol is the same as in Example 4.

[0132] The method for preparing the distearoylphosphatidyl ethanolamine-polymethacrylic acid-2-(N-oxidized-N,N-diethylamino)ethyl ester is the same as the method for preparing the distearoylphosphatidyl ethanolamine-polycarboxybetaine methacrylate (with a theoretical degree of polymerization of 50) described in Example 7, except that the monomer is replaced by diethylaminoethyl methacrylate, and the molar ratio of the reactants is the same as described in Example 7.

[0133] Example 15

[0134] The difference between this embodiment and Embodiment 1 is that:

[0135] The mass ratio of nanoparticle carrier to protein drugs for oral treatment of diabetes is 20:1.

[0136] The molar ratio of the lipid-modified zwitterionic polymer (dimyristicoglycerol-polymethacrylic acid sulfobetaine): cationic lipid: phospholipid: cholesterol is 1:2.5:1.5:2.

[0137] Example 16

[0138] The difference between this embodiment and Embodiment 1 is that:

[0139] The mass ratio of nanoparticle carrier to protein drugs for oral treatment of diabetes is 20:1.

[0140] The molar ratio of the lipid-modified zwitterionic polymer (dimyristicoglycerol-polymethacrylate-2-(N-oxy-N,N-diethylamino)ethyl ester): cationic lipid: phospholipid: cholesterol is 1:10:9:9.5.

[0141] Example 17

[0142] The difference between this embodiment and Embodiment 1 is that:

[0143] The mass ratio of nanoparticle carrier to protein drugs for oral treatment of diabetes is 4:1.

[0144] The molar ratio of the lipid-modified zwitterionic polymer, cationic lipid, phospholipid, and cholesterol is 1:2.5:1.5:2.

[0145] Example 18

[0146] The difference between this embodiment and Embodiment 1 is that:

[0147] The mass ratio of nanoparticle carrier to protein drugs for oral treatment of diabetes is 4:1.

[0148] The molar ratio of the lipid-modified zwitterionic polymer, cationic lipid, phospholipid, and cholesterol is 1:10:9:9.5.

[0149] Example 19

[0150] The difference between this embodiment and Example 1 is that, while keeping other conditions constant, the mass ratio of the zwitterionic polymer modified with lipid molecules to the cationic lipid is changed to seek the best therapeutic effect in the rat model, with the range being 0.76-1.85:1.

[0151] From the data in the table, the smaller the area under the curve of the glucose-lowering curve, the better the glucose-lowering effect of the nanoparticles. When the mass ratio of the lipid molecule modified zwitterionic polymer: cationic lipid is 1.03, the glucose-lowering effect is the best; as the mass ratio gradually increases or gradually decreases, the glucose-lowering effect will weaken.

[0152] Example 20

[0153] The difference between this example and Example 1 is that the cationic lipid is selected to be brominated dimethyl-2-hydroxyethyl-2,3-dioleyloxypropyl ammonium (the same molar amount as in Example 1).

[0154] Example 21

[0155] The difference between this example and Example 1 is that the cationic lipid is selected to be brominated dimethyl-2-hydroxyethyl-2,3-dioleyloxypropyl ammonium (the same molar amount as in Example 1).

[0156] Example 23

[0157] The difference between this example and Example 4 is that the cationic lipid is selected to be 1,2-dioleyl-3-dimethylamino-propane (the same molar amount as in Example 4).

[0158] Test Example 1: Detection of GLP-1 / GIP / GCG receptor agonist nanoparticle particle size

[0159] The particle size of the GLP-1 / GIP / GCG receptor agonist nanoparticles was measured using a Nano-ZS90 laser particle size analyzer, with a refractive index of 1.590, an absorption coefficient of 0.010, a temperature of 25°C, and a measurement mode of automatic. The results are shown in Figure 1. The average particle size of the GLP-1 / GIP / GCG receptor agonist nanoparticles prepared in Examples 1, 2, and 3 was about 125.6 nm, 145.3 nm, and 162.9 nm, respectively.

[0160] Test Example 2: Measurement of the embedding rate of the GLP-1 / GIP / GCG receptor agonist nanoparticles 100 μL of the GLP-1 / GIP / GCG receptor agonist nanoparticles were taken, 3 times the volume of acetonitrile was added, and the mixture was allowed to stand for 10 min. The mixture was centrifuged at 10,000 rpm for 10 min, and 100 μL of the supernatant was taken. The concentrations of semaglutide, tirzepatide and ritateglutide in the supernatant were determined by using an ultra-high performance liquid chromatograph (detection wavelength 214 nm). The embedding rate was calculated according to formula (1), and the test results are shown in Figure 2. The embedding rates of the GLP-1 / GIP / GCG receptor agonist nanoparticles prepared in Examples 1, 2 and 3 were 69.21%, 70.04% and 64.58%, respectively. Embedding rate (%) = m1 / m x 100% (1)

[0161] wherein m1 is the mass of the GLP-1 / GIP / GCG receptor agonist in the particles; and m is the total mass of the GLP-1 / GIP / GCG receptor agonist used in preparing the particles.

[0162] Test Example 3: Study of cytotoxicity

[0163] In this test example, the cytotoxicity of the GLP-1 / GIP / GCG receptor agonist nanoparticles was studied.

[0164] The cytotoxicity of the GLP-1 / GIP / GCG receptor agonist nanoparticles prepared in Examples on Caco-2 cells was determined by the tetrazolium blue (MTT) method. 1.0 x 104 Caco-2 cells were cultured in each well of a 96-well plate, and 100 μL of culture medium (DMEM + 20% fetal bovine serum + 1% double antibody) was added to each well. After 24 h of culture, the culture medium was replaced with 100 μL of fresh culture medium containing nanoparticles with different drug concentrations (0.0625 μg / mL, 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL and 1 μg / mL), and the culture was continued for another 24 h. Then, 20 μL of MTT (5 mg / mL dissolved in PBS) was added to each well, and the mixture was incubated in a cell incubator for 2 h. The absorbance was measured at a wavelength of 562 nm by using an enzyme-labeled instrument, and a cell growth curve was plotted. The results are shown in Figure 3. The GLP-1 / GIP / GCG receptor agonist nanoparticles prepared in Examples 1, 2 and 3 had low cytotoxicity.

[0165] Test Example 4: Study of the hypoglycemic effect at the animal level

[0166] The mice were fasted overnight (without water restriction) for 10 h, and then randomly divided into 4 groups, 6 mice in each group. The mice were subcutaneously injected with semaglutide, taspoglutide and ritateglutide solution (SC, 5 IU / kg), respectively, and orally administered with the GLP-1 / GIP / GCG receptor agonist-based nanoparticles prepared in Examples 4, 5 and 6 (oral, 0.05 mg / kg). The blood glucose values were measured at different time points (1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 20 h and 24 h) before and after administration. The experimental results are shown in FIG. 4, and the GLP-1 / GIP / GCG receptor agonist-based nanoparticles prepared in Examples 4, 5 and 6 all have good hypoglycemic effect.

[0167] Test Example 5: Detection of the particle size of insulin and insulin analog-based nanoparticles

[0168] The particle size of the insulin and insulin analog-based nanoparticles was measured using a Nano-ZS90 laser particle size analyzer, with the refractive index set to 1.590, the absorption coefficient set to 0.010, the temperature set to 25°C, and the measurement mode set to automatic. The results are shown in FIG. 4, and the average particle sizes of the insulin and insulin analog-based nanoparticles prepared in Examples 4, 5 and 6 were about 172.7 nm, 188.7 nm and 162.3 nm, respectively.

[0169] Test Example 6: Detection of the embedding rate of insulin and insulin analog-based nanoparticles

[0170] 100 μL of the insulin and insulin analog-based nanoparticles were taken, 3 times the volume of acetonitrile was added, and the mixture was allowed to stand for 10 min. The mixture was centrifuged at 10,000 rpm for 10 min, and 100 μL of the supernatant was taken. The concentration of insulin and insulin analog in the supernatant was determined using an ultra-high performance liquid chromatograph (detection wavelength: 214 nm). The embedding rate was calculated according to Formula (2), and the experimental results are shown in FIG. 5. The embedding rates of the insulin and insulin analog-based nanoparticles prepared in Examples 4, 5 and 6 were 58.89%, 62.59% and 57.71%, respectively.

[0171] In the formula, m1 is the mass of insulin and insulin analog in the nanoparticles; and m is the total mass of insulin and insulin analog used in the preparation of the nanoparticles.

[0172] Test Example 7: Study of cytotoxicity

[0173] This test example studies the cytotoxicity of the insulin and insulin analog-based nanoparticles.

[0174] The cytotoxicity of the insulin and insulin analog-based nanoparticles prepared in Examples 4, 5 and 6 at different concentrations was detected by the tetrazolium blue (MTT) method. 1.0 × 10 4Caco-2 cells were added with 100 μL of culture medium (DMEM + 20% fetal bovine serum + 1% double antibody) respectively, and cultured for 24 h, and then replaced with 100 μL of fresh culture medium containing different concentrations of insulin and its analog nanoparticles (0.0625 μg / mL, 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL and 1 μg / mL) respectively, and cultured for another 24 h. Then 20 μL of MTT (5 mg / mL dissolved in PBS) was added to each well, and incubated in a cell incubator for 2 h. An enzyme marker was used to detect at a wavelength of 562 nm, and a cell growth curve was drawn. The experimental results are shown in Figure 7, and the insulin and its analog nanoparticles prepared in Examples 4, 5 and 6 have low toxicity.

[0175] Example 8 Animal Level Hypoglycemic Effect Study

[0176] After the mice were fasted overnight (without water) for 10 h, they were randomly divided into 4 groups, 6 mice in each group. Insulin and its analog solution (SC, 5 IU / kg) was subcutaneously injected, and the insulin and its analog nanoparticles of Example (oral, 50 IU / kg) was orally administered. The blood glucose value was measured before administration and at different time points (1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 20 h and 24 h) after administration. The experimental results are shown in Figure 8, and the insulin and its analog nanoparticles prepared in Examples 4, 5 and 6 have good hypoglycemic effect.

[0177] Example 9 Area under the Curve of Hypoglycemic Curve of Model Mice

[0178] After the mice were fasted overnight (without water) for 10 h, they were randomly divided into 12 groups, 6 mice in each group. The nanoparticles described in Example 19 (oral, 0.05 mg / kg) was orally administered. The blood glucose value was measured before administration and at different time points (1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 20 h and 24 h) after administration. The obtained experimental results were plotted into a hypoglycemic curve, and the area under the curve of the hypoglycemic curve was calculated (as shown in Figure 9), and the smaller the area, the better the effect.

[0179] The above-described examples are only a preferred scheme of the present application, and do not limit the present application in any form. Other variants and modifications can be made without exceeding the technical scheme recited in the claims.

Claims

1. A nanoparticle carrier loaded with a protein drug for oral treatment of diabetes, characterized in that, The raw materials include: lipid molecule modified zwitterionic polymer, cationic lipid, phospholipid and cholesterol; The lipid molecule modified zwitterionic polymer is composed of lipid molecule and polymer, wherein the lipid molecule is distearoyl phosphatidyl ethanolamine or dimyristyl glycerol, and the polymer is polycarboxybetaine polymer, polyphosphobetaine polymer, polysulfobetaine polymer or polymethacrylic acid-2-(N-oxidized-N,N-diethylamino)ethyl ester.

2. The nanoparticle carrier of claim 1, wherein, The lipid molecule modified zwitterionic polymer is selected from one or more of distearoyl phosphatidyl ethanolamine-polycarboxybetaine polymer, distearoyl phosphatidyl ethanolamine-polyphosphobetaine polymer, distearoyl phosphatidyl ethanolamine-polysulfobetaine polymer, distearoyl phosphatidyl ethanolamine-polymethacrylic acid-2-(N-oxidized-N,N-diethylamino)ethyl ester, dimyristyl glycerol-polycarboxybetaine polymer, dimyristyl glycerol-polyphosphobetaine polymer, dimyristyl glycerol-polysulfobetaine polymer and dimyristyl glycerol-polymethacrylic acid-2-(N-oxidized-N,N-diethylamino)ethyl ester.

3. The nanoparticle carrier of claim 1, wherein, The cationic lipid is selected from one or more of dimethyl-2-hydroxyethyl-2,3-dioleyloxypropyl ammonium bromide, dimethyl-2,3-dioleyloxypropyl-2-(2-argininecarboxamide)ethyl ammonium trifluoroacetate, trimethyl-2,3-dioleoyloxypropyl ammonium bromide, trimethyl-2,3-dioleoyloxypropyl ammonium chloride, dimethyldioctadecyl ammonium bromide, dimethyl-2-hydroxyethyl-2,3-bis-hexadecyloxypropyl ammonium bromide, ALC-0315, SM-102, Dlin-MC3-DMA, 1,2-dioleoyl-3-(dimethylamino)propane, 1,2-dioleoyl-3-dimethylamino-propane.

4. The nanoparticle carrier of claim 1, wherein, The phospholipid is selected from one or more of distearoyl phosphatidic acid, dipalmitoyl phosphatidyl choline, dipalmitoyl phosphatidic acid, dimyristoyl lecithin, dipalmitoyl phosphatidyl choline, distearoyl phosphatidyl choline.

5. The nanoparticle carrier according to any one of claims 1 to 4, wherein The molar ratio of lipid molecule modified zwitterionic polymer: cationic lipid: phospholipid: cholesterol = 1: 2.5-10: 1.5-9: 2-9.

5.

6. The nanoparticle carrier of claim 1, wherein, The lipid molecule and the polymer are synthesized into the lipid molecule modified zwitterionic polymer by atom transfer radical polymerization.

7. Nanoparticles loaded with a protein drug for oral treatment of diabetes, characterized in that, The composition comprises: the nanoparticle carrier of claim 1 and the protein drug for oral treatment of diabetes, wherein the nanoparticle carrier encapsulates the protein drug for oral treatment of diabetes.

8. The nanoparticle of claim 7, wherein, The mass ratio of the nanoparticle carrier: the protein drug for oral treatment of diabetes = 20:1-5.

9. The nanoparticle of claim 7 or 9, wherein, The protein drug for oral treatment of diabetes includes insulin, G protein coupled receptor agonist, and the G protein coupled receptor agonist includes GLP-1 receptor agonist, GLP-1 / GIP dual receptor agonist, GLP-1 / GIP / GCGs triple receptor agonist.

10. The nanoparticle of claim 9, wherein, The insulin is selected from one or more of recombinant human insulin, insulin glargine, insulin detemir, insulin degludec; the G protein coupled receptor agonist is selected from one or more of exenatide, lixisenatide, bexarotene, liraglutide, semaglutide, albiglutide, taspoglutide, raxofelast.

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