Amphiphilic polymer, polymer vesicle as well as preparation method and application of amphiphilic polymer and polymer vesicle

Abstract

The invention discloses an amphiphilic polymer, a polymer vesicle as well as a preparation method and application of the polymer vesicle, and belongs to the technical field of biological medicines. The amphiphilic polymer provided by the invention has at least one hydrophobic end and at least one hydrophilic end, can spontaneously form polymer vesicles, and the polymer vesicles have better stability, biocompatibility and nanoparticle size, and have better loading rate on polypeptide hypoglycemic drugs such as insulin, GLP-1 and derivatives thereof, and the like. The drug-loaded polymer vesicles can resist digestion of protease, break through in-vivo complex physiological barriers and realize oral delivery of drugs, so that the amphiphilic polymer has a better application prospect in drug delivery.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an amphiphilic polymer, a polymer vesicle, and a preparation method and application thereof. Background Art

[0002] Diabetes has become a major health threat to humans, which is mainly caused by insufficient insulin secretion or insulin dysfunction. Exogenous insulin and glucagon-like peptide-1 (GLP-1) analogs can be used as drugs for the treatment of diabetes, but due to their large molecular weight and unstable properties, they are limited by the harsh absorption environment of the gastrointestinal tract and are difficult to enter the intestinal epithelial cells to exert their effects. The adjacent cells at the apical membrane of intestinal epithelial cells are tightly connected, which is a barrier to the intercellular transport pathway. A series of intracellular pathways will promote the entry of exogenous proteins into the lysosomal pathway, resulting in drug degradation, or the exogenous proteins are sent back to the mucosal surface for luminal secretion rather than basolateral secretion, resulting in the inability of the drug to enter the circulatory system to exert its efficacy. Usually, biological drugs are used in clinical practice by subcutaneous injection. Long-term and frequent use of drugs results in poor patient compliance and poor results. Therefore, in order to alleviate the pain of patients and the inconvenience of frequent injections, it has been a hot topic for researchers to develop oral polypeptide hypoglycemic drug compositions with good therapeutic effects, high absorption efficiency, high bioavailability, simple preparation methods, and stable properties to achieve in vivo delivery of biological macromolecular drugs. The research and development of non-injection routes of administration of protein peptide hypoglycemic drugs has long been a hot topic among pharmaceutical researchers at home and abroad. Oral administration has always been the main research direction because of its economy, convenience, and accurate dosage. Relatively speaking, oral administration is a more painless, convenient, relatively safe, and low-cost way of administration.

[0003] Chinese patent application document CN103260608A discloses a solid composition comprising a GLP-1 agonist and N-(8-(2-hydroxybenzoyl)amino) caprylate (SNAC), wherein SNAC is a small molecule delivery agent. The combination of SNAC and semaglutide enables semaglutide to be partially absorbed in the stomach. SNAC can neutralize the low pH value of the microenvironment around the tablet, prevent gastric degradation, and cross the intestinal barrier through a transcellular pathway. When semaglutide enters the blood, semaglutide and SNAC are easily dissociated. The dissolution of SNAC in the stomach can increase the pH of the local environment, which can not only improve the solubility of semaglutide, but also buffer the acidic environment in the stomach to resist degradation by gastric peptidases. It can exert a hypoglycemic effect in a manner comparable to the efficacy of subcutaneous injection of semaglutide. SNAC technology effectively improves the stability of semaglutide in the stomach and promotes intestinal absorption, allowing polypeptide drugs to enter the systemic circulation smoothly. However, the oral bioavailability of oral semaglutide is only about 1% in this literature, which is far less than that of subcutaneous injection. It needs to be taken once a day, with a dose of 7 or 14 mg per tablet. Food should not be taken 6 hours before or 0.5 hours after taking it, and the presence of nonspecific absorption enhancers may lead to potential adverse reactions.

[0004] Existing studies have shown that a TGR5 receptor is expressed on the basolateral membrane of human enteroendocrine L cells. Bile acids can stimulate the secretion of GLP-1 after binding to it. GLP-1 can stimulate pancreatic β cells to secrete insulin and inhibit α cells from secreting glucagon, which has a good effect on lowering postprandial blood sugar. However, as a macromolecular polymer connected by ester bonds, polymerized bile acids are complex to prepare and have unstable chemical properties. In addition, due to the large negative surface potential of the nanoparticles, they are not effective when crossing the mucus layer of intestinal epithelial cells, so they have certain limitations in intestinal absorption.

[0005] Therefore, it is necessary to provide a new drug delivery system that can effectively deliver polypeptide glucose-lowering drugs such as insulin and GLP-1 analogs to address the deficiencies in the prior art. Summary of the invention

[0006] The purpose of the present invention is to provide an amphiphilic polymer, a polymer vesicle, and a preparation method and application thereof. The amphiphilic polymer in the present invention has at least one hydrophobic end and at least one hydrophilic end, and can spontaneously form a polymer vesicle, which has good stability and biocompatibility, and has a good loading rate for polypeptide hypoglycemic drugs such as insulin, GLP-1 and its derivatives. The drug-loaded polymer vesicle can resist protease digestion, break through the complex physiological barriers in the body, and achieve oral delivery of drugs.

[0007] In the first aspect, the present invention provides an amphiphilic polymer having a structure as shown in the following formula (I): ; Wherein, the R group is selected from at least one of the following formulas (A1)-(A5): , , , , ; And m and n are positive integers ≥ 1, Represents the connection position; the molecular weight of the amphiphilic polymer is 1000-20000Da.

[0008] In the present invention, the amphiphilic polymer is organically combined with two hydrophilic monomers and a hydrophobic long-chain dibasic acid, and has a hydrophobic end and a hydrophilic end, and can spontaneously form a vesicle, which effectively improves the stability of the polymer. When the amphiphilic polymer is used as a drug carrier, the methoxy polyethylene glycol amine at its hydrophilic end exhibits high mucus permeability, promotes mucosal adhesion in the small intestine, increases the chance of drug release in the absorptive intestinal wall villi, and also increases the stability and biocompatibility of the drug carrier nanoparticles; the polyamine compound at its hydrophobic end modifies the hydrophobic long-chain dibasic acid, so that the hydrophobic end is positively charged, which is conducive to combining negatively charged proteins, polypeptides, small molecule organic drugs, etc., especially drugs such as insulin or polypeptide substances that can be efficiently loaded. In addition, the polyamine compound (especially arginine) in the drug carrier can penetrate cells, and it promotes the trans-intestinal epithelial cell transport of drugs through binding to glycosaminoglycans on the cell surface and energy-dependent endocytosis, and enhances the cell uptake ability of the drug carrier nanoparticles, especially insulin. Therefore, the above-mentioned amphiphilic polymers can make the loaded drugs (especially insulin) resist digestion by proteases, break through the complex physiological barriers in the body, and achieve oral delivery of drugs.

[0009] In a second aspect, the present invention provides a method for preparing the above-mentioned amphiphilic polymer, comprising the following steps: S1, mixing a first hydrophilic monomer with a hydrophobic long-chain dibasic acid, performing an amide reaction, and obtaining an intermediate compound after separation and purification; S2, mixing the intermediate compound with a second hydrophilic monomer, performing an amide reaction, and obtaining an amphiphilic polymer after separation and purification; Wherein, in step S1, the first hydrophilic monomer has a structure as shown in the following formula (B): ; The hydrophobic long-chain dibasic acid has a structure as shown in the following formula (C): ; In step S2, the second hydrophilic monomer is selected from at least one of metformin, arginine, histidine, lysine, and octanediamine; And m and n are positive integers ≥1.

[0010] In the present invention, two hydrophilic monomers and a hydrophobic long-chain dibasic acid are connected by an amide bond to obtain an amphiphilic polymer. The entire preparation process is simple, the reaction conditions are mild, and it is suitable for large-scale industrial production applications.

[0011] In some embodiments, in step S1, the molar ratio of the first hydrophilic monomer to the hydrophobic long-chain dibasic acid is 1:(1.5-2), for example, it can be 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2 or other ratios within this range; the temperature of the amide reaction is 25-60°C, for example, it can be 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C or other values ​​within this range; the time is 12h-72h, for example, it can be 12h, 24h, 36h, 48h, 60h, 72h or other values ​​within this range.

[0012] It can be understood that the amide reaction is carried out in an organic solvent, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) can be added to promote the amide reaction; wherein, the amount of EDC and NHS added can be conventionally adjusted according to actual use needs, and the organic solvent can also be selected according to actual use needs. Conventional organic solvents in the prior art, for example, the organic solvent in the present invention is preferably anhydrous ethanol.

[0013] In some embodiments, in step S1, the hydrophobic long-chain dibasic acid comprises a C12-C18 dibasic acid.

[0014] In some embodiments, in step S1, separation and purification specifically comprises: performing dialysis treatment using a dialysis bag with a molecular weight cutoff of 400-600Da.

[0015] In some embodiments, in step S2, the molar ratio of the intermediate compound to the second hydrophilic monomer is 1:(1.5-2), for example, it can be 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2 or other ratios within this range; the temperature of the amide reaction is 25-60°C, for example, it can be 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C or other values ​​within this range; the time is 12h-72h, for example, it can be 12h, 24h, 36h, 48h, 60h, 72h or other values ​​within this range.

[0016] It can be understood that the amide reaction is carried out in an organic solvent, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) can be added to promote the amide reaction; wherein, the amount of EDC and NHS added can be conventionally adjusted according to actual use needs, and the organic solvent can also be selected according to actual use needs. Conventional organic solvents in the prior art, for example, the organic solvent in the present invention is preferably anhydrous ethanol.

[0017] In some embodiments, in step S2, separation and purification specifically comprises: performing dialysis treatment using a dialysis bag with a molecular weight cutoff of 400-600Da.

[0018] In a third aspect, the present invention provides polymer vesicles, which are obtained by assembling the above-mentioned amphiphilic polymer or the amphiphilic polymer prepared by any of the above-mentioned preparation methods with phospholipids.

[0019] The polymer vesicles provided by the present invention have a nanometer-scale particle size, can realize efficient loading of insulin or polypeptide substances, and have a stable structure, can improve the stability of insulin or polypeptide active ingredients in the gastrointestinal tract, and the bioavailability.

[0020] In some embodiments, the particle size of the polymer vesicle is 10 to 250 nm, for example, 10 nm, 50 nm, 100 nm, 150 nm, 200 nm, 250 nm or other values ​​within this range.

[0021] In the present invention, by controlling the particle size of the polymer vesicles within a specific range, the possibility of passing through the transport protein can be increased, and the polymer vesicles can also interact with the luminal layer through mucus binding, showing a longer retention time in the mucus, which is beneficial to controlling drug release, thereby improving bioavailability.

[0022] In a fourth aspect, the present invention provides a method for preparing polymer vesicles as described above, comprising the following steps: dissolving an amphiphilic polymer and a phospholipid in an organic solvent, removing the organic solvent, adding an aqueous solution for hydration, and obtaining polymer vesicles after ultrasonic treatment and / or filtration.

[0023] In the present invention, polymer vesicles with small particle size are prepared by thin film hydration method, the reaction conditions are mild, the preparation process is simple, the raw materials are easily available, and it is suitable for large-scale industrial production and application.

[0024] It is understandable that the organic solvent can be selected from conventional organic solvents in the prior art according to actual use requirements. For example, the organic solvent in the present invention is preferably at least one of methanol and acetonitrile.

[0025] In some embodiments, the mass volume ratio of the amphiphilic polymer and phospholipid to the organic solvent is (0.9-10.8 mg):1 mL, for example, it can be 0.9 mg:1 mL, 2 mg:1 mL, 4 mg:1 mL, 6 mg:1 mL, 8 mg:1 mL, 10.8 mg:1 mL or other ratios within this range.

[0026] In some embodiments, removing the organic solvent specifically includes: removing the organic solvent in a rotary evaporator at a temperature of 30°C to 50°C (for example, 30°C, 35°C, 40°C, 45°C, 50°C or other values ​​within the range) until a uniform thin film is formed in the rotary evaporator.

[0027] In some embodiments, the pH value of the aqueous solution is 2-10, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or other values ​​within the range.

[0028] In some embodiments, the aqueous solution includes at least one of an aqueous hydrochloric acid solution, an aqueous sodium hydroxide solution, and a phosphate buffer solution.

[0029] In some embodiments, the hydration time is 2 to 10 min, for example, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min or other values ​​within the range.

[0030] In some embodiments, ultrasonic treatment specifically includes: the temperature of ultrasound is 20-30°C, for example, it can be 20°C, 22°C, 25°C, 28°C, 30°C or other values ​​within the range; the power is 20-60W, for example, it can be 20W, 25W, 30W, 35W, 40W, 45W, 50W, 55W, 60W or other values ​​within the range; the time is 2-10min, for example, it can be 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10min or other values ​​within the range.

[0031] In some embodiments, the filtration treatment specifically comprises: filtering 15-25 times using a filter with a particle size of 0.45 μm.

[0032] In a fifth aspect, the present invention provides the use of the above-mentioned amphiphilic polymer, the amphiphilic polymer prepared by any of the above-mentioned preparation methods, the above-mentioned polymer vesicles or the polymer vesicles prepared by the above-mentioned preparation methods in drug delivery.

[0033] It is understandable that the drug can be routinely selected according to actual use needs. For example, the drug in the present invention is preferably at least one of insulin, GLP-1 and its derivatives.

[0034] In a sixth aspect, the present invention provides the use of the above-mentioned amphiphilic polymer, the amphiphilic polymer prepared by any of the above-mentioned preparation methods, the above-mentioned polymer vesicles or the polymer vesicles prepared by the above-mentioned preparation methods in the preparation of drugs for preventing and / or treating diabetes.

[0035] In a seventh aspect, the present invention provides a pharmaceutical composition for preventing and / or treating diabetes, comprising any of the above-mentioned polymer vesicles or the polymer vesicles prepared by the above-mentioned preparation method and an active component.

[0036] In some embodiments, the active ingredient is preferably at least one of insulin, GLP-1 and derivatives thereof.

[0037] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0038] In the present invention, the term "pharmaceutically acceptable carrier" refers to an adjuvant widely used in the field of drug production. The adjuvant is mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method to dissolve the active ingredient at a desired rate after the subject receives the administration, or promote the effective absorption of the active ingredient after the subject receives the composition. Pharmaceutical adjuvants can be inert fillers, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. Pharmaceutical adjuvants can include one or more of the following adjuvants: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0039] In some embodiments, the dosage form of the pharmaceutical composition comprises an oral formulation.

[0040] In some preferred embodiments, the oral preparation comprises at least one of a tablet, a capsule, a granule, a suspension, an oral solution, and a syrup.

[0041] The beneficial effects of the present invention are as follows: different from the prior art, the amphiphilic polymer provided by the present invention has at least one hydrophobic end and at least one hydrophilic end, which can spontaneously form polymer vesicles. The polymer vesicles have good stability, biocompatibility and nanoparticle size, and have a good loading rate for polypeptide hypoglycemic drugs such as insulin, GLP-1 and its derivatives. The drug-loaded polymer vesicles can resist digestion by proteases, break through the complex physiological barriers in the body, and achieve oral delivery of drugs. Therefore, the amphiphilic polymer has good application prospects in drug delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the amphiphilic polymer p(mPEG-C13-MET) prepared in Example 1 of the present invention; Figure 2 This is a graph showing the cytotoxicity test results of the polymer p(mPEG-C13-MET) vesicles prepared in Example 5 of the present invention and the INS-p(mPEG-C13-MET) vesicles prepared in Example 9. DETAILED DESCRIPTION

[0043] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions and conditions described in the manual, or according to conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all available from commercial channels unless otherwise specified.

[0045] Example 1 This embodiment provides a method for preparing an amphiphilic polymer p(mPEG-C13-MET).

[0046] Specifically, the preparation method comprises the following steps: S1. First, weigh 97.732 mg (0.4 mmol) of tridecanedioic acid and add it into a 50 mL round-bottom flask. Add 5 mL of anhydrous ethanol and stir to dissolve it to obtain a tridecanedioic acid solution. Weigh methoxypolyethylene glycol amine (mPEG-NH 2 ) 400 mg (0.2 mmol) was added to a 50 mL beaker, and 20 mL of anhydrous ethanol was added and heated to dissolve to obtain mPEG-NH 2 Solution; weigh 230.2 mg (1.2 mmol) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and add it to a 10 mL centrifuge tube, add 2 mL of anhydrous ethanol to dissolve it, and obtain an EDC solution; weigh 138 mg (1.2 mmol) of N-hydroxysuccinimide (NHS) and add it to a 5 mL centrifuge tube, add 2 mL of anhydrous ethanol to dissolve it, and obtain an NHS solution; Then, the EDC solution and NHS solution were slowly added to the tridecanedioic acid solution, and the reaction was stirred at room temperature (25°C) for 0.5 h. Then, mPEG-NH 2The solution was stirred and reacted for 24 h at room temperature (25 °C). The obtained product was added to a dialysis bag (MWCO was 500 Da), dialyzed in anhydrous ethanol for 24 h, then dialyzed in deionized water for 24 h, and finally precooled at -80 °C and dried at -49 °C for 30 h to obtain a loose white powder (mPEG-C13-COOH). S2. First, weigh 160 mg (0.0719 mmol) of mPEG-C13-COOH and add it to a 50 mL round-bottom flask, add 20 mL of anhydrous ethanol, heat and stir to dissolve it, and obtain an mPEG-C13-COOH solution; weigh 23.72 mg (0.143 mmol) of metformin hydrochloride (Met) and add it to a 10 mL centrifuge tube, add 5 mL of anhydrous ethanol and heat to dissolve it, and obtain a Met solution; weigh 20.668 mg (0.1078 mmol) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and add it to a 5 mL centrifuge tube, add 1 mL of anhydrous ethanol to dissolve it, and obtain an EDC solution; weigh 12.41 mg (0.1078 mmol) of N-hydroxysuccinimide (NHS) and add it to a 5 mL centrifuge tube, add 1 mL of anhydrous ethanol to dissolve it, and obtain an NHS solution; Then, the EDC solution and the NHS solution were slowly added to the mPEG-C13-COOH solution, and the reaction was stirred at room temperature (25°C) for 0.5 h. Subsequently, the Met solution was slowly added to the reaction solution, and the reaction was continued to be stirred at room temperature (25°C) for 24 h. The resulting product was added to a dialysis bag (MWCO is 500Da), first dialyzed in anhydrous ethanol medium for 24 h, then transferred to deionized water for dialyzation for 24 h, and finally pre-cooled at -80°C and dried at -49°C for 30 h to obtain a loose white powder, which is the amphiphilic polymer p(mPEG-C13-MET).

[0047] Example 2 This embodiment provides a method for preparing an amphiphilic polymer p(mPEG-C13-Arg).

[0048] Specifically, the preparation method comprises the following steps: First, 160 mg (0.0719 mmol) of mPEG-C13-COOH prepared in step S1 of Example 1 was weighed and added to a 50 mL round-bottom flask, and 20 mL of anhydrous ethanol was added and heated and stirred to dissolve it to obtain an mPEG-C13-COOH solution; 24.9106 mg (0.143 mmol) of arginine (Arg) was weighed and added to a 10 mL centrifuge tube, and 1 mL of ultrapure water was added to dissolve it to obtain an Arg solution; 20.668 mg (0.1078 mmol) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) was weighed and added to a 5 mL centrifuge tube, and 1 mL of anhydrous ethanol was added to dissolve it to obtain an EDC solution; 12.41 mg (0.1078 mmol) of N-hydroxysuccinimide (NHS) was weighed and added to a 5 mL centrifuge tube, and 1 mL of anhydrous ethanol was added to dissolve it to obtain an NHS solution; Then, the EDC solution and the NHS solution were slowly added to the mPEG-C13-COOH solution in sequence, and the reaction was stirred at room temperature (25°C) for 0.5 h. Subsequently, the Arg solution was slowly added to the reaction solution, and the reaction was continued to be stirred at room temperature (25°C) for 24 h. The resulting product was added to a dialysis bag (MWCO is 500Da), first dialyzed in anhydrous ethanol medium for 24 h, then dialyzed in deionized water for 24 h, and finally pre-cooled at -80°C and dried at -49°C for 30 h to obtain a loose white powder, which is the amphiphilic polymer p(mPEG-C13-Arg).

[0049] Example 3 This embodiment provides a method for preparing an amphiphilic polymer p(mPEG-C13-His).

[0050] Specifically, the preparation method comprises the following steps: First, 160 mg (0.0719 mmol) of mPEG-C13-COOH prepared in step S1 of Example 1 was weighed and added to a 50 mL round-bottom flask, and 20 mL of anhydrous ethanol was added and heated and stirred to dissolve it to obtain an mPEG-C13-COOH solution; 22.187 mg (0.143 mmol) of histidine (His) was weighed and added to a 10 mL centrifuge tube, and 1 mL of ultrapure water was added to dissolve it to obtain a His solution; 20.668 mg (0.1078 mmol) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) was weighed and added to a 5 mL centrifuge tube, and 1 mL of anhydrous ethanol was added to dissolve it to obtain an EDC solution; 12.41 mg (0.1078 mmol) of N-hydroxysuccinimide (NHS) was weighed and added to a 5 mL centrifuge tube, and 1 mL of anhydrous ethanol was added to dissolve it to obtain an NHS solution; Then, the EDC solution and the NHS solution were slowly added to the mPEG-C13-COOH solution in sequence, and the reaction was stirred at room temperature (25°C) for 0.5 h. Subsequently, the His solution was slowly added to the reaction solution, and the reaction was continued to be stirred at room temperature (25°C) for 24 h. The resulting product was added to a dialysis bag (MWCO is 500Da), first dialyzed in anhydrous ethanol medium for 24 h, then dialyzed in deionized water for 24 h, and finally pre-cooled at -80°C and dried at -49°C for 30 h to obtain a loose white powder, which is the amphiphilic polymer p(mPEG-C13-His).

[0051] Example 4 This embodiment provides a method for preparing an amphiphilic polymer p(mPEG-C13-Lys).

[0052] Specifically, the preparation method comprises the following steps: First, 160 mg (0.0719 mmol) of mPEG-C13-COOH prepared in step S1 of Example 1 was weighed and added to a 50 mL round-bottom flask, and 20 mL of anhydrous ethanol was added and heated and stirred to dissolve it to obtain an mPEG-C13-COOH solution; 20.90517 mg (0.143 mmol) of lysine (Lys) was weighed and added to a 10 mL centrifuge tube, and 1 mL of ultrapure water was added to dissolve it to obtain a Lys solution; 20.668 mg (0.1078 mmol) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) was weighed and added to a 5 mL centrifuge tube, and 1 mL of anhydrous ethanol was added to dissolve it to obtain an EDC solution; 12.41 mg (0.1078 mmol) of N-hydroxysuccinimide (NHS) was weighed and added to a 5 mL centrifuge tube, and 1 mL of anhydrous ethanol was added to dissolve it to obtain an NHS solution; Then, the EDC solution and the NHS solution were slowly added to the mPEG-C13-COOH solution in sequence, and the reaction was stirred at room temperature (25°C) for 0.5 h. Subsequently, the Lys solution was slowly added to the reaction solution, and the reaction was continued to be stirred at room temperature (25°C) for 24 h. The resulting product was added to a dialysis bag (MWCO is 500Da), first dialyzed in anhydrous ethanol medium for 24 h, then dialyzed in deionized water for 24 h, and finally pre-cooled at -80°C and dried at -49°C for 30 h to obtain a loose white powder, which is the amphiphilic polymer p(mPEG-C13-Lys).

[0053] Example 5 This embodiment provides a method for preparing polymer p(mPEG-C13-MET) vesicles.

[0054] Specifically, the preparation method comprises the following steps: The amphiphilic polymer p(mPEG-C13-MET), egg yolk lecithin E80 and mPEG-DSPE prepared in Example 1 were weighed and dissolved in methanol respectively to obtain a p(mPEG-C13-MET) solution, an E80 solution and an mPEG-DSPE solution with a final concentration of 5 mg / mL. 120 μL of mPEG-DSPE solution, 600 μL of E80 solution and 360 μL of p(mPEG-C13-MET) solution were added to a 10 mL round-bottom flask, and the mixture was evaporated under reduced pressure at 45° C. for 3 min to form a uniform polymer film on the inner wall of the flask. 2 mL of PBS solution was added, and the film was shaken at room temperature (25° C.) to remove the film. The mixture was ultrasonicated at 28° C. for 2 min with an ultrasonic power of 20 W, and then filtered using a 0.45 μm filter to obtain a uniform polymer p(mPEG-C13-MET) vesicle dispersion.

[0055] Example 6 This embodiment provides a method for preparing polymer p(mPEG-C13-Arg) vesicles.

[0056] Specifically, the preparation method is basically the same as that in Example 5, except that the amphiphilic polymer p(mPEG-C13-MET) prepared in Example 1 is replaced by the amphiphilic polymer p(mPEG-C13-Arg) prepared in Example 2 to obtain a polymer p(mPEG-C13-Arg) vesicle dispersion.

[0057] Example 7 This embodiment provides a method for preparing polymer p(mPEG-C13-His) vesicles.

[0058] Specifically, the preparation method is basically the same as that in Example 5, except that the amphiphilic polymer p(mPEG-C13-MET) prepared in Example 1 is replaced by the amphiphilic polymer p(mPEG-C13-His) prepared in Example 3 to obtain a polymer p(mPEG-C13-His) vesicle dispersion.

[0059] Example 8 This embodiment provides a method for preparing polymer p(mPEG-C13-Lys) vesicles.

[0060] Specifically, the preparation method is basically the same as that in Example 5, except that the amphiphilic polymer p(mPEG-C13-MET) prepared in Example 1 is replaced by the amphiphilic polymer p(mPEG-C13-Lys) prepared in Example 4 to obtain a polymer p(mPEG-C13-Lys) vesicle dispersion.

[0061] Example 9 This example provides a method of using the polymer vesicles prepared in the above examples 5-8 to load insulin to obtain drug-loaded polymer vesicles.

[0062] Specifically, the drug-loaded polymer vesicles were prepared by ultrasonic method: the amphiphilic polymer p(mPEG-C13-MET), egg yolk phosphatidylcholine E80 and mPEG-DSPE prepared in Example 1 were weighed, and dissolved in methanol respectively to obtain a p(mPEG-C13-MET) solution, E80 solution and mPEG-DSPE solution with a final concentration of 5 mg / mL; insulin was weighed and dissolved in PBS solution to obtain an insulin solution with a final concentration of 4 mg / mL; 120 μL of mPEG-DSPE solution, 600 μL of E80 solution and 360 μL of p(mPEG-C13-MET) solution were added to a 10 mL round-bottom flask, and vacuum evaporated at 45°C for 3 min to form a uniform polymer film on the inner wall of the flask; 2 mL The PBS solution was shaken at room temperature (25°C) to remove the film, and ultrasonicated at 28°C for 2 min with an ultrasonic power of 20 W. Then, it was filtered using a 0.45 μm filter to obtain a uniform p(mPEG-C13-MET) vesicle dispersion. After ultrasonication at 20 W for 2 s, 500 μL of insulin solution was immediately added to obtain an INS-p(mPEG-C13-MET) vesicle dispersion.

[0063] Alternatively, the drug-loaded polymer vesicles were prepared by filtration: the amphiphilic polymer p(mPEG-C13-MET), egg yolk lecithin E80 and mPEG-DSPE prepared in Example 1 were weighed, and dissolved in methanol respectively to obtain a p(mPEG-C13-MET) solution, E80 solution and mPEG-DSPE solution with a final concentration of 5 mg / mL; insulin was weighed and dissolved in PBS solution to obtain an insulin solution with a final concentration of 4 mg / mL; 120 μL of mPEG-DSPE solution, 600 μL of E80 solution and 360 μL of p(mPEG-C13-MET) solution were added to a 10 mL round-bottom flask, and vacuum evaporated at 45°C for 3 min to form a uniform polymer film on the inner wall of the flask, 500 μL of insulin solution was added, and the film was shaken at room temperature (25°C) to fall off, 2 mL of PBS solution was added, and then filtered 20 times using a 0.45 μm filter to obtain an INS-p(mPEG-C13-MET) vesicle dispersion.

[0064] Similarly, according to the above method, the amphiphilic polymer p(mPEG-C13-MET) prepared in Example 1 was replaced by the amphiphilic polymer p(mPEG-C13-Arg) prepared in Example 2, the amphiphilic polymer p(mPEG-C13-His) prepared in Example 3, and the amphiphilic polymer p(mPEG-C13-Lys) prepared in Example 4, respectively, to obtain INS-p(mPEG-C13-Arg), INS-p(mPEG-C13-His), and INS-p(mPEG-C13-Lys) vesicle dispersions.

[0065] Example 10 This example provides a method of using the polymer vesicles prepared in the above examples 5-8 to load GLP-1 and its derivatives to obtain drug-loaded polymer vesicles.

[0066] According to the method in Example 9, insulin was replaced with GLP-1 and its derivatives (GLP-1, GLP-1-ALB8, GLP-1-ABD, GLP-1-sfGFP, GLP-1-sfGFP-ALB8, GLP-1-sfGFP-ABD), respectively, to obtain GLP-1-p(mPEG-C13-MET), GLP-1-ALB8-p(mPEG-C13-MET), GLP-1-ABD-p(mPEG-C13-MET), GLP-1-sfGFP-p(mPEG-C13-MET), GLP-1-sfGFP-ALB8-p(mPEG-C13-MET), and GLP-1-sfGFP-ABD-p(mPEG-C13-MET) vesicle dispersions.

[0067] Performance Testing The amphiphilic polymers p(mPEG-C13-MET), p(mPEG-C13-Arg), p(mPEG-C13-His), and p(mPEG-C13-Lys) prepared in Examples 1-4 were subjected to nuclear magnetic resonance testing.

[0068] For example, the H NMR spectrum of the amphiphilic polymer p(mPEG-C13-MET) is as follows: Figure 1 shown.

[0069] from Figure 1 It can be seen that δ3.53ppm is mPEG-NH 2 The characteristic peak of δ1.30ppm is the characteristic peak of tridecanedioic acid, and δ2.67ppm is the characteristic absorption peak of metformin hydrochloride. The results confirm the successful synthesis of the amphiphilic polymer p(mPEG-C13-MET).

[0070] The particle sizes of the polymer p(mPEG-C13-MET), p(mPEG-C13-Arg), p(mPEG-C13-His), and p(mPEG-C13-Lys) vesicles prepared in Examples 5-8 were tested using a Malvern particle size analyzer.

[0071] For example, the polymer p(mPEG-C13-MET) vesicles have a smaller particle size of about 220 nm, a PDI of less than 0.3, a better particle size distribution, and a negatively charged surface.

[0072] The drug-loaded polymer vesicles prepared in Examples 9 and 10 (INS-p(mPEG-C13-MET), INS-p(mPEG-C13-Arg), INS-p(mPEG-C13-His), INS-p(mPEG-C13-Lys), GLP-1-p(mPEG-C13-MET), GLP-1-ALB8-p(mPEG-C13-MET), GLP-1-ABD-p(mPEG-C13-MET), GLP-1-sfGFP-p(mPEG-C13-MET), GLP-1-sfGFP-ALB8-p(mPEG-C13-MET), GLP-1-sfGFP-ABD-p(mPEG-C13-MET) vesicles) were tested for drug loading capacity (LD%) and encapsulation efficiency (EE%).

[0073] Exemplarily, the INS-p (mPEG-C13-MET) vesicle dispersion prepared in Example 9 was placed in a 100 kD ultrafiltration tube and centrifuged at 4000 rpm and 10° C. for 30 min. The ultrafiltrate was collected and the protein concentration was determined by the BCA method. The absorbance value was determined at a wavelength of 562 nm, and the drug loading capacity (LD%) and encapsulation efficiency (EE%) of the drug-loaded vesicles were calculated by the external standard method.

[0074] The results showed that the drug loading capacity and encapsulation efficiency of INS-p(mPEG-C13-MET) vesicles prepared by ultrasound method were both 44%, and the drug loading capacity and encapsulation efficiency of INS-p(mPEG-C13-MET) vesicles prepared by filtration method were both 26%.

[0075] Embodiment 11 In this example, the polymer p(mPEG-C13-MET), p(mPEG-C13-Arg), p(mPEG-C13-His), and p(mPEG-C13-Lys) vesicles prepared in Examples 5-8, and the drug-loaded polymer vesicles (INS-p(mPEG-C13-MET), INS-p(mPEG-C13-Arg), INS-p(mPEG-C13-His), INS-p(mPEG-C13-Lys) prepared in Examples 9 and 10 were analyzed. The cytotoxicity of GLP-1-p(mPEG-C13-MET), GLP-1-ALB8-p(mPEG-C13-MET), GLP-1-ABD-p(mPEG-C13-MET), GLP-1-sfGFP-p(mPEG-C13-MET), GLP-1-sfGFP-ALB8-p(mPEG-C13-MET), and GLP-1-sfGFP-ABD-p(mPEG-C13-MET) vesicles was tested.

[0076] Specifically, the MTT method was used to measure the cytotoxicity of polymer vesicles and drug-loaded polymer vesicles at different concentrations. Caco-2 cells were plated at 1×10 4 The cells were inoculated into a 96-well plate at a density of 100 μL and cultured at 37°C and 5% carbon dioxide for 24 hours. The old culture medium was discarded, and 100 μL of culture medium containing different concentrations of polymer vesicles and drug-loaded polymer vesicles (0, 0.01, 0.1, 1, 2, 5, 10 mg / mL) was added to the cells. After incubation for another 24 hours, 20 μL of MTT (5 mg / mL) solution was added to each well. After incubation for another 4 hours, the culture medium was replaced with 125 μL of DMSO and placed on a shaker for low-speed shaking for 10 minutes. Using the blank culture medium as a control, the absorbance at 575 nm was measured with an enzyme-labeled instrument. The cell survival rate was calculated according to the following formula: Cell survival rate (%) = (absorbance of drug group - absorbance of blank group) / (absorbance of control group - absorbance of blank group) × 100%.

[0077] For example, the cytotoxicity test results of polymer p(mPEG-C13-MET) vesicles and INS-p(mPEG-C13-MET) vesicles are as follows: Figure 2 shown.

[0078] from Figure 2It can be seen that the cell survival rates of polymer p(mPEG-C13-MET) vesicles and INS-p(mPEG-C13-MET) vesicles treated with different concentrations are higher than 80%. The above results indicate that polymer p(mPEG-C13-MET) vesicles and INS-p(mPEG-C13-MET) vesicles have low toxicity to Caco-2 cells and have good safety.

[0079] Example 12 In this example, the hypoglycemic effect of the drug-loaded polymer vesicles (INS-p(mPEG-C13-MET), INS-p(mPEG-C13-Arg), INS-p(mPEG-C13-His), INS-p(mPEG-C13-Lys), GLP-1-p(mPEG-C13-MET), GLP-1-ALB8-p(mPEG-C13-MET), GLP-1-ABD-p(mPEG-C13-MET), GLP-1-sfGFP-p(mPEG-C13-MET), GLP-1-sfGFP-ALB8-p(mPEG-C13-MET), GLP-1-sfGFP-ABD-p(mPEG-C13-MET) vesicles) prepared in Examples 9 and 10 was tested in diabetic mice.

[0080] Specifically, a diabetic mouse model was first constructed: a large dose of streptozotocin (STZ) was intraperitoneally injected to induce the KM diabetic mouse model. The mice were fasted for 12 hours overnight before modeling, with free access to water during this period. According to the fasting body weight of the mice, 1% STZ solution was intraperitoneally injected at a dose of 150 mg / kg. After the injection, the mice continued to fast for 2 hours, and then resumed normal diet to avoid affecting the modeling effect. Some mice could form the model after one week, and the mice that did not form the model were fasted for 12 hours overnight, with free access to water during this period. According to the fasting body weight of the mice, 1% STZ solution was intraperitoneally injected at a dose of 15 mg / kg for 5 consecutive days to finally obtain diabetic mice.

[0081] Then, the diabetic mice were divided into five groups, 4 mice in each group. The first group was gavaged with PBS; the second group was intraperitoneally injected with insulin solution (5 IU / kg); the third group was gavaged with insulin solution (1500 IU / kg); the fourth group was gavaged with the polymer p(mPEG-C13-MET) vesicles prepared in Example 5; the fifth group was gavaged with INS-p(mPEG-C13-MET) vesicles prepared in Example 9 at a dosage of 1500 IU / kg. After measuring fasting blood glucose, gavage and injection were performed respectively. After 0.5h, 20% glucose was gavaged at 2g / Kg. Blood was collected from the tail vein at 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, and 10h according to the pre-set time, and the blood glucose of the mice was measured with a blood glucose meter and blood glucose test strips. The results are shown in Table 1 below.

[0082] Table 1 Changes in blood glucose levels in diabetic mice after administration of INS-p(mPEG-C13-MET) vesicles

[0083] Similarly, the above method was used for treatment, except that the fourth group was gavaged with the polymer p(mPEG-C13-Arg) vesicles prepared in Example 6; the fifth group was gavaged with the INS-p(mPEG-C13-Arg) vesicles prepared in Example 9 at a dose of 1500 IU / kg, and blood was collected from the tail vein at 0h, 0.5h, 1h, 1.5h, 2h, and 2.5h according to the pre-set time, and the blood glucose of the mice was measured with a blood glucose meter and blood glucose test strips. The results are shown in Table 2 below.

[0084] Table 2 Changes in blood glucose levels in diabetic mice after administration of INS-p(mPEG-C13-Arg) vesicles

[0085] Similarly, the above method was continued, except that the fourth group was gavaged with the polymer p(mPEG-C13-His) vesicles prepared in Example 7; the fifth group was gavaged with the INS-p(mPEG-C13-His) vesicles prepared in Example 9 at a dose of 1500 IU / kg, and blood was collected from the tail vein at 0 h, 0.5 h, 1 h, 1.5 h, 2 h, and 2.5 h according to the preset time, and the blood glucose of the mice was measured with a blood glucose meter and a blood glucose test strip. The results are shown in Table 3 below.

[0086] Table 3 Changes in blood glucose levels in diabetic mice after administration of INS-p(mPEG-C13-His) vesicles

[0087] Similarly, the above method was continued, except that the fourth group was gavaged with the polymer p (mPEG-C13-Lys) vesicles prepared in Example 8; the fifth group was gavaged with the INS-p (mPEG-C13-Lys) vesicles prepared in Example 9 at a dose of 1500 IU / kg, and blood was collected from the tail vein at 0h, 0.5h, 1h, 1.5h, 2h, and 2.5h according to the pre-set time, and the blood glucose of the mice was measured with a blood glucose meter and a blood glucose test strip. The results are shown in Table 4 below.

[0088] Table 4 Changes in blood glucose levels in diabetic mice after administration of INS-p(mPEG-C13-Lys) vesicles

[0089] As can be seen from Tables 1-4, after administration of INS-p (mPEG-C13-MET), INS-p (mPEG-C13-Arg), INS-p (mPEG-C13-His) and INS-p (mPEG-C13-Lys) vesicles, respectively, the blood glucose levels of diabetic mice decreased significantly. The above results indicate that the drug-loaded vesicles of the present invention can pass through the mucus barrier and intestinal epithelial cells, effectively achieve oral delivery of insulin, and can control drug release for a long time within the therapeutic window, thereby improving bioavailability.

[0090] In summary, the amphiphilic polymer provided by the present invention can spontaneously form polymer vesicles, which have good stability and biocompatibility, and have a good loading rate for polypeptide glucose-lowering drugs such as insulin, GLP-1 and its derivatives. The drug-loaded polymer vesicles can resist digestion by proteases, break through the complex physiological barriers in the body, and achieve oral delivery of drugs.

[0091] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0092] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An amphiphilic polymer, characterized in that It has the structure shown in the following formula (I): ； Wherein, the R group is selected from at least one of the following formulas (A1)-(A5): 、 、 、 、 ； And m and n are positive integers ≥ 1, represents the connection position; the molecular weight of the amphiphilic polymer is 1000-20000Da.

2. A method for preparing an amphiphilic polymer as claimed in claim 1, characterized in that: The steps include: S1, mixing a first hydrophilic monomer with a hydrophobic long-chain dibasic acid and performing an amide reaction, and obtaining an intermediate compound after separation and purification; S2, mixing the intermediate compound with a second hydrophilic monomer and performing an amide reaction, and obtaining the amphiphilic polymer after separation and purification; Wherein, in step S1, the first hydrophilic monomer has a structure as shown in the following formula (B): ； The hydrophobic long-chain dibasic acid has a structure as shown in the following formula (C): ； In step S2, the second hydrophilic monomer is selected from at least one of metformin, arginine, histidine, lysine, and octanediamine; And m and n are positive integers ≥1.

3. The method for preparing an amphiphilic polymer according to claim 2, characterized in that: In step S1, the molar ratio of the first hydrophilic monomer to the hydrophobic long-chain dibasic acid is 1:(1.5-2); The temperature of the amide reaction is 25-60°C, and the time is 12h-72h.

4. The method for preparing an amphiphilic polymer according to claim 2, characterized in that: In step S2, the molar ratio of the intermediate compound to the second hydrophilic monomer is 1:(1.5-2); The temperature of the amide reaction is 25-60°C, and the time is 12h-72h.

5. A polymer vesicle, characterized in that The polymer vesicle is obtained by assembling the amphiphilic polymer according to claim 1 or the amphiphilic polymer prepared by the preparation method of any one of claims 2 to 4 with phospholipids.

6. The polymer vesicle according to claim 5, characterized in that The particle size of the polymer vesicle is 10 to 250 nm.

7. A method for preparing a polymer vesicle according to any one of claims 5 to 6, characterized in that: The method comprises the following steps: dissolving the amphiphilic polymer and phospholipid in an organic solvent, removing the organic solvent, adding an aqueous solution for hydration, and obtaining the polymer vesicles after ultrasonic treatment and / or filtering treatment.

8. Use of the amphiphilic polymer according to claim 1, the amphiphilic polymer prepared by the preparation method according to any one of claims 2 to 4, the polymer vesicle according to any one of claims 5 to 6, or the polymer vesicle prepared by the preparation method according to claim 7 in drug delivery.

9. Use of the amphiphilic polymer according to claim 1, the amphiphilic polymer prepared by the preparation method according to any one of claims 2 to 4, the polymer vesicle according to any one of claims 5 to 6, or the polymer vesicle prepared by the preparation method according to claim 7 in the preparation of a drug for preventing and / or treating diabetes.

10. A pharmaceutical composition for preventing and / or treating diabetes, characterized in that: The invention comprises the polymer vesicle according to any one of claims 5 to 6 or the polymer vesicle prepared by the preparation method according to claim 7 and an active component.

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