A sustained-release microsphere for loading polypeptide drugs and a preparation method thereof

Abstract

The present application provides a drug-loaded microsphere for loading polypeptide drugs, the polypeptide drug delivery drug-loaded microsphere is W1 / O / W2 structure, which is composed of polypeptide drug active substance and hydrophilic gel as the inner water phase (W1), organic solvent of water-insoluble polymer as the oil phase (O), and solution containing emulsifier as the outer water phase (W2). The sustained-release microsphere of the present application introduces hydrophilic temperature-sensitive gel and water-insoluble polymer to co-load drugs, increases the hydrophilicity of the carrier, and reduces the solubility of polypeptide drugs by preparing metal ion-polypeptide drug active substance insoluble complex, thereby controlling the drug release behavior. Compared with the conventional microsphere, the sustained-release microsphere of the present application has the advantages of reducing drug burst release, shortening the drug release platform period, making the overall drug release behavior of the drug tend to zero-order release, releasing more stably, and improving the compliance of the microsphere preparation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a kind of sustained-release microspheres for loading polypeptide drug and preparation method thereof. BACKGROUND

[0002] Polypeptide and protein drugs have shown great potential for decades due to their low toxicity and high specificity. In addition to vaccination and diagnosis, the main application of polypeptide and protein drugs is the treatment of chronic diseases, autoimmune diseases, AIDS and cancer, etc. Due to the low absorption and high enzymatic degradation of polypeptides and proteins, oral, pulmonary, nasal, ocular and transdermal administration are not commonly used in long-term treatment. In contrast, intravenous injection, implantation and subcutaneous injection, intramuscular injection, etc. are usually used for drug administration. However, due to the short half-life of most peptide and protein drugs in the body, the drug content in the plasma is very high at the beginning of administration, and the drug is rapidly cleared after entering the body, and soon falls below the effective blood drug concentration. Patients need long-term and high-frequency injection of drugs to maintain the effect of drug treatment, and long-term injection leads to poor compliance of patients. In addition, the rich proteases in the metabolic system and the fluctuation of pH can cause denaturation or degradation of proteins, and thus induce serious immune reactions.

[0003] In order to overcome these shortcomings, researchers have developed sustained-release drug delivery systems, which, by prolonging drug release time, greatly improve patient compliance while avoiding repeated administration. In particular for drugs with short half-life, the sustained-release drug delivery system changes the pharmacokinetic parameters of the drug in the body and improves the bioavailability. In the study of sustained-release drug delivery systems, multifunctional polymer drug-loaded materials are usually used as carriers. Common high molecular materials include synthetic polyesters, polyamides, polyamino acids, among which one of the most popular polymers is polylactic acid-glycolic acid copolymer (PLGA), which has been approved by the US Food and Drug Administration (FDA) for clinical use and drug delivery due to its superior biocompatibility and biodegradability.

[0004] However, polypeptide microsphere preparations using polylactic acid-glycolic acid copolymer (PLGA) as a carrier are prone to burst release and long release plateau, such as the marketed preparation There is almost no release in the body for 5-14 days. At the same time, PLGA has poor hydrophilicity, and water is not easy to enter the inside of the microspheres, its hydration degradation process is slow, and the distribution of drugs in PLGA microspheres is uneven. Therefore, in order to shorten the release plateau of polypeptide microspheres, improve hydrophilicity and improve the in vivo release behavior. It is a technical problem to be solved in the art to seek a sustained-release delivery drug-loaded microsphere that reduces drug burst release, shortens the release plateau of the microsphere and prolongs the rapid release period. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application aims to provide a polypeptide drug-loaded sustained-release microsphere, which has strong hydrophilicity, can prolong the rapid release period and shorten the release plateau of the microsphere.

[0006] To achieve the object of the present application, the present application adopts the following technical solutions:

[0007] In a first aspect, the present application provides a polypeptide drug-loaded microsphere, which is composed of a polypeptide drug active substance, a hydrophilic gel, a water-insoluble polymer and a solution containing an emulsifying agent.

[0008] The polypeptide drug delivery microsphere has a W1 / O / W2 structure, and is composed of a polypeptide drug active substance and a hydrophilic gel to form an inner water phase (W1), an organic solvent of a water-insoluble polymer as an oil phase (O), and a solution containing an emulsifying agent as an outer water phase (W2).

[0009] Preferably, the hydrophilic gel is at least one of dimeric lactic acid-glycolic acid copolymer polyethylene glycol, poloxamer, xanthan gum, sodium alginate, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, povidone, carbomer, alginate, chitosan, and copolymers and / or mixtures thereof.

[0010] Preferably, the hydrophilic gel is dimeric lactic acid-glycolic acid copolymer polyethylene glycol.

[0011] Preferably, the water-insoluble polymer is at least one of poly(lactic acid-co-glycolic acid), polylactide, polyglycolide, poly(lactide-co-glycolide), copolymers thereof with polycaprolactone or polyethylene glycol, polycaprolactone, copolymers thereof with polyethylene glycol, polyhydroxybutyric acid, polyhydroxyvaleric acid, poly(p-dioxanone), chitosan, alginic acid and its salts, polycyanoacrylate, fibrin, polyanhydride, polyorthoester, polyamide, polyphosphazene, polyphosphoester, and copolymers and / or mixtures thereof.

[0012] Preferably, the water-insoluble polymer is poly(lactic acid-co-glycolic acid).

[0013] Preferably, the emulsifier is any one or mixture of several of polyvinyl alcohol, oleic acid soap, stearic acid soap, lauric acid soap, rosin oil soap, alkyl sulfate, alkyl benzene sulfonate, alkyl sulfonate, alkyl naphthyl sulfonate, lignin sulfonate, phosphate ester, sulfate ester, quaternary ammonium salt, alkyl ammonium salt, lecithin, fatty acid glyceride, sucrose fatty acid ester, fatty acid sorbitan, polysorbate, polyoxyethylene fatty acid ester, polyoxyethylene fatty alcohol ether, polyoxyethylene-polyoxypropylene block copolymer, fluorocarbon surfactant, silicon-containing surfactant, biosurfactant, crown ether-type surfactant, gum arabic, gum tragacanth, gelatin, apricot kernel gum, egg yolk, polyvinylpyrrolidone, or solid microparticle emulsifier.

[0014] Preferably, the emulsifier is a mixed solution of polyvinyl alcohol and NaCl.

[0015] Preferably, the organic solvent of the oil phase (O) in the polypeptide drug delivery microsphere is any one or mixture of several of dichloromethane, glacial acetic acid, acetonitrile, trifluoroacetic acid, dimethyl sulfoxide, anhydrous diethyl ether, hexane, n-heptane, aliphatic hydrocarbon, halogenated hydrocarbon, fatty acid ester, aromatic hydrocarbon, ether, anhydrous diethyl ether, cyclohexane, n-hexane, n-heptane.

[0016] Preferably, the organic solvent is dichloromethane.

[0017] Preferably, the concentration of the water-insoluble polymer in the polypeptide drug delivery microsphere is 10-500 mg·mL -1 ; the concentration of polyvinyl alcohol is 0.1-10%; and the concentration of NaCl is 0-10%.

[0018] Preferably, the concentration of the water-insoluble polymer in the polypeptide drug delivery microsphere is 30-250 mg·mL -1 , preferably 100 mg·mL -1 ; the concentration of polyvinyl alcohol in the outer water phase is 0.1-2%, preferably 1%; and the concentration of NaCl in the outer water phase is 1-5%, preferably 5%.

[0019] Preferably, the mass ratio of the polypeptide drug active substance to the water-insoluble polymer (i.e., drug loading ratio, W Ex :W PLGA (%)) in the polypeptide drug delivery microsphere is 1-20%; the volume ratio of the inner water phase (W1) to the oil phase (O) is 1:2-20; and the volume ratio of the oil phase (O) to the outer water phase (W2) is 1:2-10.

[0020] Preferably, the mass ratio of the polypeptide drug active substance to the water-insoluble polymer (i.e., drug loading ratio, W Ex :W PLGAThe percentage of the water phase (W1) is 4-10%, preferably 5%; the volume ratio of the inner aqueous phase (W1) to the oil phase (O) is 1:5-10, preferably 1:10; preferably, the volume ratio of the oil phase (O) to the outer aqueous phase (W2) is 1:4-8, preferably 1:4.5.

[0021] Preferably, the polypeptide drug delivery drug-loaded microspheres further include forming an insoluble complex with a metal salt solution, wherein the insoluble complex and the hydrophilic gel constitute an inner aqueous phase (W1).

[0022] Preferably, the mass ratio of the polypeptide active substance to the metal salt solution is 1:1 to 10.

[0023] Preferably, the mass ratio of the polypeptide active pharmaceutical ingredient to the metal salt solution (i.e., M) Ex :M Zn The ratio is 1:1 to 4, preferably 1:1.

[0024] Preferably, the metal salt solution includes zinc acetate solution, zinc sulfate solution, zinc carbonate solution, zinc nitrate solution, zinc chloride solution, zinc nitrite solution, zinc sulfite solution, copper sulfate solution, copper chloride solution, copper nitrate solution, magnesium nitrate solution, magnesium chloride solution, magnesium sulfate solution, calcium chloride solution, calcium chlorate solution, calcium gluconate solution, calcium hypochlorite solution, and calcium perchlorate solution.

[0025] Preferably, the metal salt solution is a zinc acetate solution.

[0026] Preferably, the active pharmaceutical ingredient of the polypeptide is exenatide or exenatide salt.

[0027] Preferably, the active pharmaceutical ingredient of the polypeptide is semaglutide or semaglutide salt.

[0028] Secondly, the present invention provides a method for preparing drug-loaded microspheres for delivering polypeptide drugs, the method comprising the following steps:

[0029] (1) Preparation of hydrophilic thermosensitive gel;

[0030] (2) Dissolve the active substances of polypeptide drugs in the above-mentioned thermosensitive gel as the inner aqueous phase (W1);

[0031] (3) Dissolve the water-insoluble polymer in an organic solvent to form the oil phase (O);

[0032] (4) Pour the aqueous phase (W1) into the oil phase (O) and shear the solution at high speed to form W1 / O primary emulsion;

[0033] (5) Pour the W1 / O primary emulsion into the external aqueous phase (W2) containing the emulsifier and shear at high speed to form the W1 / O / W2 double emulsion;

[0034] (6) removing the organic solvent by rotary evaporation, centrifuging the above liquid, discarding the supernatant, and collecting the microspheres to obtain the polypeptide drug delivery microspheres.

[0035] Preferably, the shearing speed of the primary emulsion in the step (4) is 6000-20000 r·min -1 , and the shearing time of the primary emulsion is 0.5-10 min. -1 The shearing speed of the re-emulsion in the step (5) is 3000-12000 r·min -1 , and the shearing time of the re-emulsion is 0.5-10 min.

[0036] Preferably, the shearing speed of the primary emulsion in the step (4) is 8000-16000 r·min -1 , and preferably 12000 r·min -1 .

[0037] Preferably, the shearing time of the primary emulsion in the step (4) is 1-3 min, and preferably 3 min.

[0038] Preferably, the shearing speed of the re-emulsion in the step (5) is 8000 r·min -1 , and the shearing time of the re-emulsion is 1-3 min, and preferably 2 min.

[0039] Preferably, the preparation method of the polypeptide drug delivery microspheres further comprises the step of preparing a metal salt and polypeptide complex.

[0040] Preferably, the step of preparing a metal salt and polypeptide complex specifically comprises: uniformly mixing a metal salt solution and a polypeptide solution, incubating at low temperature, and freeze-drying to obtain a metal salt solution polypeptide complex.

[0041] Preferably, the step of incubating at low temperature specifically comprises: incubating at 4℃ for 24 hours, and the incubation solvent volume is 5-10 mL, and preferably 10 mL.

[0042] The beneficial effects of the present application are that the slow-release microspheres (Ex-gel-Ms and X-Ex-gel-Ms, "X" represents metal ions) for loading polypeptide drugs in the present application introduce hydrophilic temperature-sensitive gel (for example: PLGA-PEG-PLGA) and water-insoluble polymer (for example: PLGA) to co-load drugs, increase the hydrophilicity of the carrier, and make the polypeptide drug into X-Ex insoluble complex to reduce the solubility of the drug, thereby reducing the drug burst. The plateau of Ex-gel-Ms and X-Ex-gel-Ms is shorter than that of Ex-Ms, and the shorter the plateau, the earlier the starting point of the rapid release period, thereby improving the bioavailability of the drug. BRIEF DESCRIPTION OF DRAWINGS

[0043] The accompanying drawings are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and are meant to explain the application without limiting the scope thereof. In the drawings:

[0044] Figure 1 Figure 4 is a graph showing the effect of the ratio of the volume of the aqueous phase to the volume of the oil phase in Example 1.

[0045] Figure 2 Figure 5 is a graph showing the effect of the concentration of NaCl in the aqueous phase in Example 1; (A) 2% NaCl concentration, (B) 3% NaCl concentration, (C) 4% NaCl concentration, (D) 5% NaCl concentration.

[0046] Figure 3 Figure 6 is a graph showing the effect of the shear rate of the colostrum in Example 1; (A) 10,000 r / min shear rate of the colostrum, (B) 12,000 r / min shear rate of the colostrum, (C) 14,000 r / min shear rate of the colostrum. -1 -1 -1

[0047] Figure 4 Figure 7 is a graph showing the particle size and particle size distribution of the three kinds of microspheres; (A) Ex-Ms, (B) Ex-gel-Ms, (C) Zn-Ex-gel-Ms.

[0048] Figure 5 Figure 8 is a SEM image of the three kinds of microspheres; (A) Ex-Ms, (B) Ex-gel-Ms, (C) Zn-Ex-gel-Ms.

[0049] Figure 6 Figure 9 is a graph showing the appearance of the powder of the three kinds of microspheres.

[0050] Figure 7 Figure 10 is a graph showing the in vitro release curve of the three kinds of microspheres.

[0051] Figure 8 Figure 11 is a graph showing the blood concentration curve of the three kinds of microspheres for 24 days.

[0052] Figure 9 Figure 12 is a graph showing the blood concentration curve of the three kinds of microspheres for 24 hours. DETAILED DESCRIPTION

[0053] The scheme of the application will be explained below in conjunction with the examples. Those skilled in the art will understand that the examples below are only for the purpose of illustrating the application and should not be regarded as limiting the scope of the application. If a specific technique or condition is not specified in the examples, it is performed according to the technique or condition described in the literature in the art or according to the product manual. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained on the market. ​​​

[0054] The present embodiment provides a polypeptide drug delivery microsphere (denoted as Ex-gel-Ms), which is composed of a polypeptide drug active substance, a hydrophilic gel, a water-insoluble polymer, and a solution containing an emulsifier.

[0055] The polypeptide drug delivery microsphere has a W1 / O / W2 structure and is composed of a polypeptide drug active substance and a hydrophilic gel as an internal water phase (W1), an organic solvent of a water-insoluble polymer as an oil phase (O), and a solution containing an emulsifier as an external water phase (W2).

[0056] Exemplarily, the water-insoluble polymer can be polylactic acid-glycolic acid copolymer (PLGA), the hydrophilic gel can be dimeric lactic acid-glycolic acid copolymer polyethylene glycol (PLGA-PEG-PLGA), the organic solvent can be dichloromethane, and the solution containing an emulsifier can be a mixed solution of polyvinyl alcohol (PVA) and NaCl.

[0057] The hydrophilic gel can also be at least one of poloxamer, xanthan gum, sodium alginate, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, povidone, carbomer, alginate, chitosan, and a copolymer and / or mixture thereof.

[0058] The water-insoluble polymer can also be at least one of polylactide, polyglycolide, polylactide-co-glycolide, a copolymer thereof with polycaprolactone or polyethylene glycol, polycaprolactone, a copolymer thereof with polyethylene glycol, polyhydroxybutyric acid, polyhydroxyvaleric acid, poly-p-dioxanone, chitosan, alginic acid and its salt, polycyanoacrylate, fibrin, polyanhydride, polyorthoester, polyamide, polyphosphazene, polyphosphoester, and a copolymer and / or mixture thereof.

[0059] The organic solvent can also be any one or a mixture of several of glacial acetic acid, acetonitrile, trifluoroacetic acid, dimethyl sulfoxide, anhydrous diethyl ether, hexane, n-heptane, aliphatic hydrocarbon, halogenated hydrocarbon, fatty acid ester, aromatic hydrocarbon, ether, anhydrous diethyl ether, cyclohexane, n-hexane, n-heptane.

[0060] The emulsifier can also be any one or a mixture of several of oleic acid soap, stearic acid soap, lauric acid soap, rosin soap, alkyl sulfate, alkyl benzene sulfonate, alkyl sulfonate, alkyl naphthyl sulfonate, lignin sulfonate, phosphate ester, sulfate ester, quaternary ammonium salt, alkyl ammonium salt, lecithin, fatty acid glyceride, sucrose fatty acid ester, fatty acid sorbitan, polysorbate, polyoxyethylene fatty acid ester, polyoxyethylene fatty alcohol ether, polyoxyethylene-polyoxypropylene block copolymer, fluorocarbon surfactant, silicon-containing surfactant, biological surfactant, crown ether-type surfactant, gum arabic, gum tragacanth, gelatin, apricot kernel gum, egg yolk, polyvinylpyrrolidone, or solid microparticle emulsifier.

[0061] In the present embodiment, the mass ratio of the polypeptide drug active substance to PLGA (i.e. drug loading ratio, W Ex :W PLGA ) in the polypeptide drug delivery microspheres is 4-10%, preferably 5%; the PLGA concentration is 30-250 mg·mL -1 , preferably 100 mg·mL -1 ; the organic solvent of the oil phase (O) is dichloromethane; the volume ratio of the inner water phase (W1) to the oil phase (O) is 1:5-10, preferably 1:10; the volume ratio of the oil phase (O) to the outer water phase (W2) is 1:4-8, preferably 1:4.5; the PVA concentration in the outer water phase is 0.1-2%, preferably 1%; and the NaCl concentration in the outer water phase is 1-5%, preferably 5%.

[0062] The polypeptide drug delivery microspheres (denoted as Ex-gel-Ms) are prepared by the W1 / O / W2 multiple emulsion-solvent evaporation method, and the specific preparation process is as follows:

[0063] (1) preparing a PLGA-PEG-PLGA thermosensitive gel;

[0064] (2) dissolving the polypeptide drug active substance in the above thermosensitive gel as the inner water phase (W1);

[0065] (3) dissolving PLGA in dichloromethane as the oil phase (O);

[0066] (4) pouring the inner water phase (W1) into the oil phase (O), and shearing the solution at high speed to form a W1 / O primary emulsion;

[0067] (5) pouring the W1 / O primary emulsion into the outer water phase (W2) containing PVA and NaCl, and shearing at high speed to form a W1 / O / W2 multiple emulsion;

[0068] (6) pouring the multiple emulsion into a NaCl solution, and removing dichloromethane by rotary evaporation;

[0069] (7) centrifuging the above liquid, discarding the supernatant, and collecting the microspheres to obtain the Ex-gel-Ms microspheres.

[0070] In the above preparation method, the shearing speed of the primary emulsion in step (4) is 8000-16000 r·min -1 , preferably 12000 r·min -1 ; the shearing time of the primary emulsion is 1-3 min, preferably 3 min; the shearing speed of the multiple emulsion in step (5) is 8000 r·min -1 , and the shearing time of the multiple emulsion is 1-3 min, preferably 2 min.

[0071] The embodiment also provides a metal salt polypeptide complex gel microsphere (denoted as: X-Ex-gel-Ms), which is composed of a polypeptide drug active substance, a metal salt, a hydrophilic gel, a water-insoluble polymer, and a solution containing an emulsifier.

[0072] The polypeptide drug delivery microsphere has a W1 / O / W2 structure, and is composed of an insoluble complex of a polypeptide drug active substance and a metal salt, a hydrophilic gel as an inner water phase (W1), an organic solvent of a water-insoluble polymer as an oil phase (O), and a solution containing an emulsifier as an outer water phase (W2).

[0073] Exemplarily, the metal salt in the embodiment can be zinc acetate.

[0074] In the embodiment, the mass ratio of the polypeptide drug active substance to zinc acetate (i.e., M Ex :M Zn ) in the polypeptide drug delivery microsphere is 1:1-4, preferably 1:1; the mass ratio of the polypeptide drug active substance to PLGA (i.e., drug loading ratio, W Ex :W PLGA (%) is 4-10%, preferably 5%; the concentration of the PLGA is 30-250 mg·mL -1 , preferably 100 mg·mL -1 ; the organic solvent of the oil phase (O) is dichloromethane; the volume ratio of the inner water phase (W1) to the oil phase (O) is 1:5-10, preferably 1:10; the volume ratio of the oil phase (O) to the outer water phase (W2) is 1:4-8, preferably 1:4.5; the concentration of PVA in the outer water phase is 0.1-2%, preferably 1%; and the concentration of NaCl in the outer water phase is 1-5%, preferably 5%.

[0075] The zinc acetate polypeptide complex gel microsphere (denoted as: Zn-Ex-gel-Ms) is prepared by a W1 / O / W2 complex emulsion-solvent evaporation method, and the specific preparation process is as follows:

[0076] (1) zinc acetate solution and polypeptide solution are uniformly mixed, incubated at low temperature, and freeze-dried to obtain Zn-Ex insoluble complex;

[0077] (2) a PLGA-PEG-PLGA temperature-sensitive gel is prepared;

[0078] (3) the Zn-Ex insoluble complex is dissolved in the above-mentioned temperature-sensitive gel as an inner water phase (W1);

[0079] (4) PLGA is dissolved in dichloromethane as an oil phase (O);

[0080] (5) pour the inner water phase (W1) into the oil phase (O), high speed shearing the solution to form W1 / O primary emulsion;

[0081] (6) pour the W1 / O primary emulsion into the outer water phase (W2) containing PVA and NaCl, high speed shearing to form W1 / O / W2 multiple emulsion;

[0082] (7) pour the multiple emulsion into NaCl solution, rotary evaporation to remove dichloromethane;

[0083] (8) centrifuge the above liquid, discard the supernatant, collect the microspheres, and the Ex-gel-Ms microspheres are obtained.

[0084] In the above preparation method, the (1) low temperature incubation is specifically: 4℃, incubation for 24 hours, the incubation solvent volume is 5-10 mL, preferably 10 mL; the step (4) primary emulsion shearing speed is 8000-16000 r·min -1 , preferably 12000 r·min -1 ; the primary emulsion shearing time is 1-3 min, preferably 3 min; the step (5) multiple emulsion shearing speed is 8000 r·min -1 , the multiple emulsion shearing time is 1-3 min, preferably 2 min.

[0085] In the present embodiment, the dimeric lactic acid-glycolic acid copolymer polyethylene glycol (PLGA-PEG-PLGA) is a hydrophilic gel, and in the present application, the hydrophilic gel can also be at least one of poloxamer, xanthan gum, sodium alginate, sodium carboxymethyl cellulose, methyl cellulose, hypromellose, povidone, carbomer, alginate, chitosan, and a copolymer and / or mixture thereof.

[0086] In the present embodiment, the polylactic acid-glycolic acid copolymer (PLGA) is a water-insoluble polymer, and in the present application, the water-insoluble polymer can also be at least one of polylactic acid-glycolic acid copolymer, polylactide, polyglycolide, copolymer of lactide-glycolide and copolymer thereof with polycaprolactone or polyethylene glycol, polycaprolactone and copolymer thereof with polyethylene glycol, polyhydroxybutyric acid, polyhydroxyvaleric acid, poly-p-dioxanone, chitosan, alginic acid and its salt, polycyanoacrylate, fibrin, polyanhydride, polyorthoester, polyamide, polyphosphazene, polyphosphate, and a copolymer and / or mixture thereof.

[0087] In the present embodiment, polyvinyl alcohol (PVA) and NaCl form the outer aqueous phase to form the emulsifier. In the present application, the emulsifier can also be any one or mixture of several of the following: oleic acid soap, stearic acid soap, lauric acid soap, rosin oil soap, alkyl sulfate, alkyl benzene sulfonate, alkyl sulfonate, alkyl naphthyl sulfonate, lignin sulfonate, phosphate ester, sulfate ester, quaternary ammonium salt, alkyl ammonium salt, lecithin, fatty acid glyceride, sucrose fatty acid ester, fatty acid sorbitan, polysorbate, polyoxyethylene fatty acid ester, polyoxyethylene fatty alcohol ether, polyoxyethylene-polyoxypropylene block copolymer, fluorocarbon surfactant, silicon-containing surfactant, biological surfactant, crown ether-type surfactant, gum arabic, gum tragacanth, gelatin, apricot kernel gum, egg yolk, polyvinylpyrrolidone, or solid microparticle emulsifier.

[0088] In the present application, the organic solvent of the oil phase (O) can also be any one or mixture of several of the following: dichloromethane, glacial acetic acid, acetonitrile, trifluoroacetic acid, dimethyl sulfoxide, anhydrous diethyl ether, hexane, n-heptane, aliphatic hydrocarbon, halogenated hydrocarbon, fatty acid ester, aromatic hydrocarbon, ether, anhydrous diethyl ether, cyclohexane, n-hexane, n-heptane.

[0089] Preferably, the concentration of the water-insoluble polymer in the polypeptide drug delivery microspheres can be 10-500 mg·mL -1 ; the concentration of polyvinyl alcohol can be 0.1-10%; and the concentration of NaCl can be 0-10%.

[0090] Preferably, the mass ratio of the polypeptide drug active substance to the water-insoluble polymer (i.e., drug loading ratio, W Ex :W PLGA (%) can be 1-20%; the volume ratio of the inner aqueous phase (W1) to the oil phase (O) can be 1:2-20; and the volume ratio of the oil phase (O) to the outer aqueous phase (W2) can be 1:2-10.

[0091] Preferably, the polypeptide drug delivery microspheres further comprise forming an insoluble complex of the polypeptide drug active substance and a metal salt solution, and the insoluble complex and the hydrophilic gel form the inner aqueous phase (W1).

[0092] In the present application, the polypeptide drug active substance can also form an insoluble complex with other metal salt solutions.

[0093] In the present application, the metal salt can also be zinc sulfate, zinc carbonate, zinc nitrate, zinc chloride, zinc nitrite, zinc sulfite, copper sulfate, copper chloride, copper nitrate, magnesium nitrate, magnesium chloride, magnesium sulfate, calcium chloride, calcium chlorate, calcium gluconate, calcium hypochlorite, calcium perchlorate.

[0094] Example 1

[0095] This embodiment provides a method for preparing exenatide gel microspheres (denoted as Ex-gel-Ms), which includes the following steps:

[0096] Exenatide gel microspheres were prepared using the W1 / O / W2 double emulsion-solvent evaporation method. The specific preparation process is as follows:

[0097] (1) Accurately weigh 0.3g of PLGA-PEG-PLGA gel into a 5mL vial, add 1.5mL of distilled water, and swell in a water bath at 25℃ for 48 hours to obtain the thermosensitive gel.

[0098] (2) Accurately transfer 0.4 mL of the thermosensitive gel prepared above (containing 75 mg of PLGA-PEG-PLGA), and accurately weigh 20 mg of exenatide and dissolve it in the thermosensitive gel as the inner aqueous phase (W1).

[0099] (3) Accurately weigh 400 mg of PLGA and dissolve it in dichloromethane as the oil phase (O);

[0100] (4) Pour the aqueous phase (W1) into the oil phase (O), and use a high-speed disperser to shear the solution at a certain speed to form W1 / O primary emulsion;

[0101] (5) Pour the W1 / O primary emulsion into an external aqueous phase containing 1% PVA and 1% NaCl, and perform high-speed shearing to form a W1 / O / W2 double emulsion;

[0102] (6) Pour the double emulsion into 50 mL of NaCl solution and evaporate at 40 °C for 20 min to remove dichloromethane;

[0103] (7) The above liquid was heated to 1500 r·min -1 Centrifuge for 5 minutes, discard the supernatant, wash the collected microspheres three times with distilled water, and freeze-dry to remove water to obtain exenatide Ex-gel-Ms microspheres.

[0104] This embodiment focuses on the drug loading ratio (i.e., the ratio of peptide drug mass to PLGA mass W). Ex :W PLGA The parameters were optimized by fixing the following experimental parameters: NaCl concentration in the external aqueous phase was 1%, PVA concentration was 1%, and PLGA concentration was 100 mg·mL. -1 The volume of the inner aqueous phase was 0.4 mL, and the volume of the outer aqueous phase was 18 mL. The effects of drug loading ratios of 4%, 5%, and 6% on encapsulation efficiency and drug loading were investigated. The results are shown in Table 1.

[0105] Table 1W Ex :W PLGA The investigation

[0106]

[0107] The results show that the loading capacity of PLGA for drugs is limited, with the increase of drug loading ratio, the drug loading of microspheres first increases and then remains almost unchanged, and the encapsulation efficiency first remains unchanged and then decreases. The results show that the drug loading will not continue to increase with the continuous increase of drug dosage. When the drug concentration in the inner water phase is too large, the drug concentration gradient between the inner and outer water phases increases, the osmotic pressure of the inner water phase increases, the water in the outer water phase migrates inward, the volume of the inner water phase increases, the droplets gather in the inside, and the probability of fusion of the inner water phase through the oil phase and the outer water phase increases, and the drug leaks in this process, resulting in a decrease in the encapsulation efficiency. Therefore, under the condition that the drug loading is not much different, the prescription with higher encapsulation efficiency is selected, that is, the drug loading ratio is 5%.

[0108] In this example, the PLGA concentration is optimized. The following experimental parameters are fixed: the NaCl concentration of the outer water phase is 1%, the PVA concentration is 1%, the volume of the inner water phase is 0.4 mL, the volume of the outer water phase is 18 mL, the drug loading ratio is 5%, and the PLGA concentration is 50, 100, 200 mg·mL -1 respectively. The effect of PLGA concentration on the microsphere encapsulation efficiency is investigated, and the results are shown in Table 2:

[0109] Table 2 Effect of PLGA concentration

[0110]

[0111] The results show that when the PLGA concentration in the oil phase increases from 50 mg·mL -1 to 100 mg·mL -1 , the encapsulation efficiency and drug loading increase significantly. This is because the increase of PLGA concentration increases the viscosity of the oil phase, accelerates the precipitation of PLGA, and hinders the diffusion of drugs in the inner water phase, so the drug encapsulation efficiency increases. When the PLGA concentration continues to increase to 200 mg·mL -1 , the encapsulation efficiency no longer increases. Considering that the density of the microspheres is too large and is not conducive to the later release, the PLGA concentration of 100 mg·mL -1 is selected as the best scheme.

[0112] In this example, the volume ratio of the inner water phase to the oil phase is optimized. The volume of the inner water phase has an important influence on the initial emulsion particle size and stability during the preparation of the microspheres. The following experimental parameters are fixed: the NaCl concentration of the outer water phase is 1%, the PVA concentration is 1%, the PLGA concentration is 100 mg·mL -1 , the volume of the outer water phase is 18 mL, and the drug loading ratio is 5%. The effects of the volume ratio of the inner water phase to the oil phase of 1:5, 1:8, and 1:10 on the microsphere encapsulation efficiency, drug loading, and burst release are investigated. The results are shown in Table 3:

[0113] Table 3 Effect of the volume ratio of the inner water phase to the oil phase

[0114]

[0115] As shown in the above table, when the inner water phase: oil phase = 1:5, the encapsulation efficiency of the microspheres is only 47.63%, while when the inner water phase: oil phase is reduced to 1:10, the encapsulation efficiency of the microspheres rises to 85.17%. This is because the larger the volume of the inner water phase, the weaker the wrapping effect of the oil phase on the inner water phase, and the easier the droplets of the inner water phase to gather near the oil-water interface, and the easier the water in the inner water phase to escape to the outer water phase. When the inner water phase: oil phase = 1:5 is reduced to 1:8, the burst release decreases obviously, which is because the droplets of the inner water phase gather at the oil-water interface, leaving holes after freeze-drying, and the drug in the holes rapidly diffuses in the release medium. Therefore, the inner water phase: oil phase = 1:10 is selected as the best scheme.

[0116] In this embodiment, the volume ratio of the oil phase to the outer water phase is optimized. In the preparation of the microspheres, the volume of the outer water phase has a great influence on the particle size of the microspheres. The following experimental parameters are fixed: the NaCl concentration of the outer water phase is 1%, the PVA concentration is 1%, the PLGA concentration is 100 mg·mL -1 , the volume ratio of the inner water phase to the oil phase is 1:10, and the theoretical drug loading is 5%. The influence of the oil phase: outer water phase ratio of 1:4.5, 1:5.5 and 1:6.5 on the particle size of the microspheres is investigated, respectively. The results are shown in Table 4.

[0117] Table 4 Influence of the volume ratio of the oil phase to the outer water phase

[0118]

[0119] The results show that as the volume of the outer water phase increases, the particle size of the microspheres increases and the uniformity of the particle size decreases, which is because under the fixed shear force, the larger the volume of the water phase, the lower the overall efficiency, which is conducive to the formation of larger emulsion droplets, thereby increasing the particle size of the microspheres. According to the particle size distribution results, when the shear efficiency is high, the particle size distribution is more uniform, and therefore the oil phase: outer water phase = 1:4.5 is selected as the best scheme of the microspheres of the present application.

[0120] In this embodiment, the PVA concentration in the outer water phase is optimized. In the process of forming the double emulsion of the microspheres, an emulsifier needs to be added to the outer water phase to reduce the interfacial tension between the oil and water phases, prevent the droplets from breaking and coagulating, and improve the stability of the double emulsion. A commonly used emulsifier is polyvinyl alcohol (PVA). The following experimental parameters are fixed: the NaCl concentration of the outer water phase is 1%, the PLGA concentration is 100 mg·mL -1 , the inner water phase: oil phase = 1:10, the oil phase: outer water phase = 1:4.5, and the drug loading ratio is 5%. The influence of 0.5%, 1% and 2% PVA solution on the encapsulation efficiency and particle size is investigated, respectively. The results are shown in Table 5.

[0121] Table 5 Influence of the PVA concentration in the outer water phase

[0122]

[0123] The results show that when the PVA concentration in the external aqueous phase is from 0.5 to 1%, the encapsulation efficiency slightly increases, the particle size decreases, and the particle size uniformity improves. This is because PVA acts as a surfactant in the external aqueous phase, which enhances the stability of the multiple emulsion by reducing the surface tension, reduces the aggregation of the multiple emulsion, and thus the particle size is more uniform and the particle size decreases. The increase in the stability of the multiple emulsion increases the drug encapsulation efficiency. When the PVA concentration increases to 2%, the encapsulation efficiency of the microspheres does not significantly increase, and the particle size uniformity decreases. This may be due to the increase in PVA concentration, which increases the viscosity of the multiple emulsion, and insufficient shearing, resulting in a decrease in particle size uniformity. Therefore, 1% is selected as the optimal concentration of PVA in the external aqueous phase.

[0124] In this example, the concentration of NaCl in the external aqueous phase is optimized. In the process of preparing microspheres by multiple emulsion solvent evaporation method, the osmotic pressure of the external aqueous phase is adjusted by adding NaCl, which is an important parameter affecting the encapsulation efficiency and surface morphology of the microspheres. When the internal aqueous phase is PLGA-PEG-PLGA gel, the osmotic pressure increases, and the osmotic pressure of the internal aqueous phase has a greater impact on the encapsulation efficiency of the microspheres. The following parameters are fixed: primary emulsion shearing speed 12000 r·min -1 , primary emulsion shearing time 2 min, multiple emulsion shearing speed 8000 r·min -1 , multiple emulsion shearing time 3 min, and the effect of NaCl concentration of 2%, 3%, 4%, and 5% in the external aqueous phase on the properties of the microspheres is investigated. The results are shown in Table 6:

[0125] Table 6 Effect of NaCl concentration in the external aqueous phase

[0126]

[0127] As shown in the above table, increasing the osmotic pressure of the external aqueous phase also increases the encapsulation efficiency and drug loading. When the internal aqueous phase is replaced with PLGA-PEG-PLGA gel, the osmotic pressure of the internal aqueous phase increases significantly. Scanning electron microscopy Figure 2 shows that when the NaCl concentration in the external aqueous phase is 2%, the surface of the microspheres has many cavities, the particle size is not uniform, and there are many small particle sizes. As the NaCl concentration increases to 5%, the surface of the microspheres is smooth, the number of cavities decreases, and the particle size uniformity improves. When the internal and external osmotic pressures are balanced, the addition of PLGA-PEG-PLGA gel to the internal aqueous phase increases the viscosity of the primary emulsion, thereby improving the stability of the primary emulsion, and thus the uniformity of the prepared microspheres is improved.

[0128] In this example, the primary emulsion shearing speed is optimized. The following parameters are fixed: primary emulsion shearing time 2 min, multiple emulsion shearing speed 8000 r·min -1 , multiple emulsion shearing time 3 min, NaCl concentration 5%, and the primary emulsion shearing speed of 10000, 12000, and 14000 r·min -1The influence of the primary emulsion shearing speed on the microspheres is shown in Table 7.

[0129] Table 7 Influence of the primary emulsion shearing speed

[0130]

[0131] The results are shown in the above table: with the increase of the primary emulsion shearing speed, the encapsulation efficiency of the microspheres increases, and when the primary emulsion shearing speed is 12000 r·min -1 , the encapsulation efficiency is the highest, 91.37%, and when the primary emulsion shearing speed continues to increase, the encapsulation efficiency of the microspheres decreases. This is because when the primary emulsion shearing speed is too fast, many small droplets are formed, and the small droplets are easy to coagulate to form large droplets, resulting in drug leakage and leading to the decrease of the drug encapsulation efficiency.

[0132] Scanning electron microscope Figure 3 shows that when the primary emulsion shearing speed is 10000 r·min -1 , the microspheres have a large and uniform particle size, which is because the low shearing speed forms large primary emulsion droplets, and then the microspheres have a large particle size. When the primary emulsion shearing speed is increased to 12000 r·min -1 , the particle size of the microspheres decreases, and when the primary emulsion shearing speed continues to increase to 14000 r·min -1 , the uniformity of the particle size of the microspheres decreases obviously, and large particle size microspheres appear. This is because the small droplets in the primary emulsion coagulate to form large droplets, and then form large microspheres, and the small droplets that do not coagulate form small microspheres, leading to the uneven distribution of the overall particle size of the microspheres. Therefore, 12000 r·min -1 is selected as the optimal primary emulsion shearing speed.

[0133] In this example, the primary emulsion shearing time is optimized, and the following parameters are fixed: the primary emulsion shearing speed is 12000 r·min -1 , the re-emulsion shearing speed is 8000 r·min -1 , the re-emulsion shearing time is 3 min, and the NaCl concentration is 5%. The influence of the primary emulsion time of 1, 2 and 3 min on the microspheres is investigated, and the results are shown in Table 8.

[0134] Table 8 Investigation of the primary emulsion shearing time

[0135]

[0136] The results show that: with the increase of the initial milk shearing time, the microsphere encapsulation rate increases. Increasing the initial milk shearing time is beneficial to the full shearing of the initial milk, so that the particle size of the initial milk is more uniform, thereby increasing the encapsulation rate of the microspheres. Compared with Ex-Ms, the optimal initial milk shearing time is extended by 1 min, because the viscosity of the initial milk increases, and under the same shearing speed, the shearing efficiency decreases, and extending the shearing time is beneficial to full shearing. Therefore, 3 min is selected as the optimal shearing time of the initial milk.

[0137] In this example, the emulsion shearing time is optimized, and the following parameters are fixed: initial milk shearing speed 12000r·min -1 , initial milk shearing time 3min, emulsion shearing speed 8000r·min -1 , NaCl concentration is 5%, respectively, the influence of emulsion time of 1, 2, 3min on the properties of microspheres is investigated. The results are shown in Table 9:

[0138] Table 9 Emulsion shearing time investigation

[0139]

[0140] The results show that: with the increase of the emulsion shearing time, the microsphere encapsulation rate first increases and then decreases. This is because: increasing the emulsion shearing time makes the initial milk more uniformly dispersed in the external aqueous phase, forming a more uniform emulsion, so that the drug is better wrapped in the microspheres. When the emulsion shearing time is too long, the particle size of the emulsion continues to decrease, and the drug wrapping capacity of small particle size microspheres is weaker than that of large particle size microspheres, so the encapsulation rate of the microspheres decreases, so 2min is selected as the optimal shearing time of the emulsion.

[0141] Example 2

[0142] The present example provides a preparation method of semaglutide gel microspheres (denoted as: Sem-gel-Ms), which comprises the following steps:

[0143] The semaglutide gel microspheres are prepared by W1 / O / W2 multiple emulsion-solvent evaporation method, and the specific preparation process is as follows:

[0144] (1) precisely weigh 0.3g PLGA-PEG-PLGA gel in a 5mL syringe, add 1.5mL distilled water, and swell at 25℃ water bath for 48 hours to obtain a temperature-sensitive gel;

[0145] (2) precisely take 0.4mL of the prepared temperature-sensitive gel (containing 75mg of PLGA-PEG-PLGA) and dissolve 20mg of semaglutide in the temperature-sensitive gel as the internal aqueous phase (W1);

[0146] (3) precisely weigh 400mg of PLGA dissolved in dichloromethane as the oil phase (O);

[0147] (4) Pour the inner water phase (W1) into the oil phase (O), and use a high-speed dispersion machine to shear the solution at a certain speed to form a W1 / O primary emulsion, the shearing speed of the primary emulsion is 10000 r·min -1 , and the shearing time is 3 min;

[0148] (5) Pour the W1 / O primary emulsion into an outer water phase containing 5% PVA and 1% NaCl, and high-speed shear to form a W1 / O / W2 multiple emulsion; the shearing speed of the multiple emulsion is 8000 r·min -1 , and the shearing time is 2 min;

[0149] (6) Transfer the multiple emulsion to 50 mL of an aqueous solution of NaCl with a concentration of 5%, and rotary evaporate at 40℃ for 20 min to remove dichloromethane;

[0150] (7) Centrifuge the above-mentioned liquid at 1500 r·min -1 for 5 min, discard the supernatant, wash the collected microspheres with distilled water for 3 times, and freeze-dry to remove water to obtain the semorilum peptide Sem-gel-Ms microspheres.

[0151] Example 3

[0152] The embodiment provides a preparation method of zinc acetate-exenatide compound gel microspheres (denoted as: Zn-Ex-gel-Ms), which comprises the following steps:

[0153] The zinc acetate-exenatide compound gel microspheres are prepared by using a W1 / O / W2 multiple emulsion-solvent evaporation method, and the specific preparation process is as follows:

[0154] (1) Preparation of zinc acetate-exenatide (denoted as: Zn-Ex) insoluble compound: accurately weigh 20 mg of exenatide into 5 mL of distilled water, stir and dissolve to prepare an exenatide solution, accurately weigh 20 mg of zinc acetate into 5 mL of distilled water, stir and dissolve to form a zinc acetate solution, slowly pour the zinc acetate solution into the exenatide solution, stir at room temperature for 5 min, incubate in a 4℃ refrigerator for 24 hours, and freeze-dry to obtain the Zn-Ex insoluble compound;

[0155] (2) Preparation of temperature-sensitive gel: accurately weigh 0.3 g of PLGA-PEG-PLGA gel into a 5 mL vial, add 1.5 mL of distilled water, and swell at 25℃ for 48 hours to obtain the temperature-sensitive gel.

[0156] (3) 20 mg of Zn-Ex insoluble compound is dissolved in 0.4 mL of temperature-sensitive gel (containing 75 mg of PLGA-PEG-PLGA) to serve as the inner water phase W1;

[0157] (4) 4 mL of PLGA with a concentration of 100 mg·mL-1 A dichloromethane solution was used as the oil phase O;

[0158] (5) 18 mL of aqueous solution containing 5% NaCl and 1% PVA was used as the external aqueous phase W2;

[0159] (6) Pour the internal aqueous phase W1 into the oil phase O, at 12000 r·min -1 Shear at a high speed for 3 minutes to form W1 / O primary emulsion;

[0160] (7) Pour the obtained colostrum into 18 mL of external aqueous phase W2 and incubate at 8000 r·min -1 Shear at a rotation speed for 2 minutes to form W1 / O / W2 double emulsion;

[0161] (8) Transfer the double emulsion to 50 mL of 5% NaCl aqueous solution and rotary evaporate at 40 °C for 20 min to remove dichloromethane;

[0162] (9) 1500 r·min -1 Centrifuge for 5 minutes, discard the supernatant, wash the collected microspheres three times with distilled water, and freeze-dry to remove water to obtain Zn-Ex-gel-Ms.

[0163] In this embodiment, M... Ex :M Zn The mass ratio was optimized, the reaction temperature was fixed at 4℃, and the incubation solvent was 10mL of distilled water. The reaction was carried out at M... Ex :M Zn The effect of ratios of 1:1, 1:2, and 1:4 on the properties of the Zn-Ex insoluble complex.

[0164] Table 10. Mass ratio of exenatide to zinc acetate

[0165]

[0166] The results show that: in M Ex :M Zn When the ratio changes, the change in particle size is small; when M... Ex :M Zn When the ratio is 1:1, PDI is minimized, therefore M is chosen. Ex :M Zn A ratio of 1:1 is the optimal solution.

[0167] In this embodiment, the incubation solvent volume was optimized, and the reaction temperature was kept constant at 4°C. Ex :M Zn The ratio was 1:1, and the effect of incubation solvent volumes of 5, 8, and 10 mL on the Zn-Ex insoluble complex was investigated.

[0168] Table 11 Incubation solvent volume

[0169]

[0170] The results show that with the increase of the volume of the incubation solvent, the particle size of the Zn-Ex insoluble complex slightly increases, and the PDI significantly decreases, so 10 mL is selected as the optimal volume of the incubation solvent. The prepared Zn-Ex insoluble complex is used to prepare Zn-Ex-gel-Ms according to the above method, and the microsphere encapsulation rate is 88.90%, and the drug loading is 3.47%.

[0171] The present comparative example provides a preparation method of an exenatide microsphere (denoted as: Ex-Ms) delivery drug-loaded microsphere, which comprises the following steps:

[0172] (1) 0.4 mL of an aqueous exenatide solution with a concentration of 50 mg·mL -1 as an internal aqueous phase W1;

[0173] (2) 4 mL of a dichloromethane solution of PLGA with a concentration of 100 mg·mL -1 as an oil phase O;

[0174] (3) 18 mL of an aqueous solution containing 1% NaCl and 1% PVA as an external aqueous phase W2;

[0175] (4) Pour the internal aqueous phase W1 into the oil phase O, and shear at a speed of 12000 r·min -1 for 2 min to form a W1 / O primary emulsion;

[0176] (5) Pour the obtained primary emulsion into 18 mL of the external aqueous phase W2, and shear at a speed of 8000 r·min -1 for 1 min to form a W1 / O / W2 multiple emulsion;

[0177] (6) Transfer the multiple emulsion to 50 mL of an aqueous NaCl solution with a concentration of 1%, and rotary evaporate at 40°C for 20 min to remove dichloromethane;

[0178] (7) Centrifuge at a speed of 1500 r·min -1 for 5 min, discard the supernatant, wash the collected microspheres with distilled water for 3 times, and freeze-dry to remove water to obtain the exenatide Ex-Ms microspheres.

[0179] Experimental Example Three Characterization and in Vitro Release of Three Microspheres (Ex-Ms, Ex-gel-Ms, and Zn-Ex-gel-Ms)

[0180]

[0181]

[0182] Table 12 Drug loading and encapsulation efficiency of microspheres

[0183]

[0184] 2. Particle size and size distribution of microspheres

[0185] Particle size and size distribution are key parameters affecting the properties of microspheres. The particle size of microspheres was determined by a Malvern laser particle size analyzer. The results are shown in Table 13, Figure 4

[0186] Table 13 Particle size and size distribution of microspheres

[0187]

[0188]

[0189] The results show that the uniformity of the particle size of the three kinds of microspheres is good.

[0190] 3. Appearance of microspheres

[0191] The surface of the microspheres was characterized by scanning electron microscopy SEM. The surfaces of the three kinds of microspheres were smooth and had no holes, as shown in Figure 5 The appearance of the microspheres was characterized. The microspheres were white powders with good fluidity, as shown in Figure 6

[0192] 4. In vivo release and in vitro release of the three kinds of microspheres

[0193] (1) In vitro release:

[0194] The three kinds of microspheres were subjected to in vitro release. A certain number of microspheres were accurately weighed and placed in 2 mL EP tubes. 1 mL of pH 7.4 phosphate buffer containing 0.02% Tween-80 as a microsphere aggregation inhibitor was added to each EP tube. The EP tubes were placed in a 37°C constant temperature air bath shaker and shaken at 100 r·min -1 The EP tubes were removed every day, centrifuged at 5000 r·min -1 for 5 min, the supernatant was discarded, and 1 mL of release medium was added. At 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40 days, part of the sample was removed, centrifuged and the supernatant was discarded, and the precipitate was freeze-dried. The precipitate was dissolved in 150 μL of acetonitrile, 850 μL of pure water was added, and the mixture was centrifuged at 8000 r·min -1 for 5 min. The supernatant was passed through a 0.22 μm microporous filter membrane, 20 μL of sample was injected, the drug peak area was substituted into the standard curve, the residual drug amount in the microspheres was calculated, and the cumulative drug release curve was drawn. The results of the in vitro release curve are shown in Table 14, Figure 7 .​​

[0195] Table 14 In vitro release of microspheres

[0196]

[0197] The results show that: (1) the burst release of Zn-Ex-gel-Ms is lower than that of Ex-gel-Ms, and the burst release of Ex-gel-Ms is lower than that of Ex-Ms, indicating that co-loading of PLGA-PEG-PLGA and PLGA significantly reduces the burst release, and the preparation of Zn-Ex insoluble complex can further reduce the burst release.

[0198] This is because in the preparation process of Ex-Ms microspheres, the inner water phase is unstable and easy to coagulate into larger droplets, and the macromolecular water-soluble drug is easy to distribute on the surface of the microspheres. After freeze-drying, holes are left on the surface layer, and when the microspheres are placed in the release medium, the surface layer forms a water-soluble channel, and the drug in the hole diffuses rapidly outward, resulting in the highest burst release. When the Ex-gel-Ms preparation uses gel as the inner water phase, the gel has a higher viscosity than water, thereby increasing the stability of the primary emulsion, and the primary emulsion droplets are more evenly distributed in the multiple emulsion. The gel is not easy to aggregate on the surface of the microspheres, and the drug is uniformly distributed in the microspheres. Moreover, the drug has a weak affinity with PLGA and a strong affinity with gel, and the drug dispersed in the gel will not rapidly diffuse into the release medium, and the gel plays a buffering role on the drug, thereby reducing the burst release compared with Ex-Ms. The reason why Zn-Ex-gel-Ms has the lowest burst release is that Zn-Ex has a low solubility in water, and the release medium will not immediately dissolve in water and diffuse outward after entering the microspheres, so the burst release is the least.

[0199] The platform periods of Zn-Ex-gel-Ms and Ex-gel-Ms are shorter than that of Ex-Ms, and co-loading of PLGA-PEG-PLGA and PLGA shortens the platform period. The longest platform period of Ex-Ms is due to the poor hydrophilicity of PLGA, which makes it difficult for water to enter the interior of the microspheres, and the process of PLGA hydration and degradation is slow. Moreover, the drug is not uniformly distributed in the PLGA microspheres, and a large amount of drug is distributed on the surface layer of the microspheres. When the surface layer drug is completely released, the amount of internal drug is greatly reduced, the number of hydrophilic channels composed of drug in the interior is less, the dissolution rate of the microspheres is slower, and the platform period is longer. After the inner water phase of Ex-gel-Ms is replaced by a temperature-sensitive gel, the temperature-sensitive gel has strong hydrophilicity, which attracts water in the release medium to enter the microspheres and form channels in the interior of the microspheres, accelerating the swelling process of the microspheres. After the microspheres pass the burst release period, there is still a large amount of drug in the interior, which makes the interior of the microspheres have more hydrophilic channels, so the platform period is shorter than that of Ex-Ms. The reason why Zn-Ex-gel-Ms still has a large amount of release in 1-4 days is that only a small amount of drug exists in the gel in the form of molecules during the burst release stage. During 2-4 days, the Zn-Ex insoluble complex rapidly converts into the molecular form of exenatide in the gel matrix, and this amount of drug will be rapidly released during this period.

[0200] Zn-Ex-gel-Ms release cycle is longer than Ex-gel-Ms, Ex-gel-Ms release cycle is longer than Ex-Ms, which shows that using PLGA-PEG-PLGA and PLGA to co-load drugs can prolong the release cycle, and making Exenatide into Zn-Ex insoluble complex can further prolong the release cycle. The fast release period of Ex-gel-Ms is longer than that of Ex-Ms, and the fast release period of Zn-Ex-gel-Ms is longer than that of Ex-gel-Ms, which shows that using PLGA-PEG-PLGA and PLGA to co-load drugs can prolong the fast release period, and making Exenatide into Zn-Ex insoluble complex can prolong the fast release period.

[0201] The fast release period of Ex-Ms is the shortest, because the burst release amount is too large in the early stage, the releasable drug amount is less in the later stage, and the drug has poor affinity with PLGA. When the microspheres swell, the drug rapidly migrates outward through the water-soluble pores. When the water phase in Ex-gel-Ms is replaced by gel, the microspheres swell, the drug has affinity with the gel, and the gel matrix can play a buffering role, thereby prolonging the fast release period. A part of Exenatide in Zn-Ex-gel-Ms still exists in the form of insoluble complex during the whole release period, so the fast release process is further prolonged.

[0202] (2) In vivo pharmacokinetics:

[0203] The three kinds of microspheres were injected into SD rats, and the rat in vivo pharmacokinetics experiment was carried out. The three kinds of microspheres released drugs in vivo for 24 days, and the 24d blood drug concentration curve is shown in Figure 8 , and the blood drug concentration curve within 24h is shown in Figure 9 . According to the in vivo pharmacokinetics data, the C max of Ex-Ms, Ex-gel-Ms and Zn-Ex-gel-Ms is 84.985ng·mL -1 , 42.22ng·mL -1 , 35.04ng·mL -1 respectively, and the burst release of Zn-Ex-gel-Ms is the lowest, which is better than the other two preparations; the AUC 0-24d of the three kinds of microspheres is 127.04, 151.33, 152.45ng·mL -1 ·d -1 respectively, and the relative bioavailability Fr result shows that the bioavailability AUC 0-24d of Ex-gel-Ms and Zn-Ex-gel-Ms is better than that of Ex-Ms, and the variance analysis result shows that the three kinds of preparations are bioinequivalent.

[0204] C maxComparison: Ex-Ms > Ex-gel-Ms > Zn-Ex-gel-Ms, the results show that the addition of temperature-sensitive gel PLGA-PEG-PLGA and the preparation of exenatide into Zn-Ex insoluble complex can reduce the burst release.

[0205] Fr analysis: Ex-gel-Ms > Ex-Ms, Zn-Ex-gel-Ms > Ex-Ms, the results show that the addition of temperature-sensitive gel can improve the bioavailability of the drug.

[0206] MRT comparison: Zn-Ex-gel-Ms > Ex-gel-Ms > Ex-Ms, the data show that the addition of temperature-sensitive gel can prolong the drug release time in vivo, and the preparation of exenatide into Zn-Ex insoluble complex can further prolong the release time in vivo.

[0207] When PLGA-PEG-PLGA and PLGA are combined to load drugs, PLGA-PEG-PLGA is dispersed inside the microspheres. After this temperature-sensitive material is injected into the body, it gradually swells to form a gel with the entry of water, and the gel is not diluted by water and still retains its shape. The drug is dispersed in the gel, and because the drug has a certain affinity with the gel, the gel has a buffering effect on the drug release, so the burst release is low.

[0208] After exenatide is prepared into Zn-Ex insoluble complex, the solubility of exenatide decreases, and the amount of drug dissolved in the release medium in a short time decreases, so the burst release decreases.

[0209] The release mechanism of the drug in vivo includes diffusion mechanism and erosion mechanism. The diffusion mechanism mainly exists in the burst release stage, and water enters the microsphere channel, and the microsphere transfers outward through these channels. The erosion mechanism exists in the rapid release stage, and the carrier material begins to degrade, and the drug is rapidly released. In the intermediate platform period, the drug is almost not released, and this stage is the swelling process of the carrier, and the carrier does not erode in the swelling process, and after the entire microsphere swells, a water-soluble channel is formed, and the carrier material begins to erode, so the length of the platform period depends on the swelling speed of the carrier material. PLGA-PEG-PLGA is a biodegradable temperature-sensitive gel, and the hydrophilicity of this material is higher than that of PLGA, and it is more easily to attract water into the microsphere and accelerate the swelling of the microsphere, so the platform period of Ex-gel-Ms and Zn-Ex-gel-Ms is shorter than that of Ex-Ms. The shorter the platform period, the earlier the starting point of the rapid release period, which improves the bioavailability of the drug.

[0210] The analysis of the fast release period: the amount of the later release mainly depends on the residual drug amount in the microspheres and the hydrophilicity of the carrier. The fast release period of Zn-Ex is short and the release amount is small because of the small residual drug amount and the strong hydrophobicity of the carrier. The fast release period of Ex-gel-Ms is prolonged and the release amount is increased because of the increased residual drug amount and the strong hydrophilicity of the carrier. The fast release period of Zn-Ex-gel-Ms is further prolonged because of the largest residual drug amount.

[0211] In summary: Zn-Ex-gel-Ms is superior to Ex-gel-Ms, and Ex-gel-Ms is superior to Ex-Ms.

[0212] It should be further pointed out that the polypeptide sustained-release microspheres prepared by using other hydrophilic gels, water-insoluble polymers, emulsifier-containing solutions, organic solvents and metal salt solutions in the application have the same effects as those in Examples 1, 2 and 3, and will not be described one by one here.

[0213] Although the embodiments of the application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the application, and those of ordinary skill in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the application.

Claims

1. A drug-loaded microsphere for loading polypeptide drugs, characterized in that, The microspheres are composed of a polypeptide drug active substance, a hydrophilic gel, a water-insoluble polymer, and a solution containing an emulsifier; the polypeptide drug delivery microspheres have a W1 / O / W2 structure; the inner aqueous phase (W1) is composed of the polypeptide drug active substance and the hydrophilic gel, the organic solvent of the water-insoluble polymer is the oil phase (O), and the solution containing the emulsifier is the outer aqueous phase (W2). The hydrophilic gel is a dimer lactic acid-glycolic acid copolymer polyethylene glycol; The water-insoluble polymer is a polylactic acid-glycolic acid copolymer; The emulsifier-containing solution is a mixed solution of polyvinyl alcohol with a mass concentration of 1-2% and sodium chloride with a mass concentration of 2-5%.

2. The drug-loaded microspheres as described in claim 1, characterized in that, In the drug delivery microspheres for polypeptide drugs, the oil phase (O) organic solvent is any one or a mixture of several of the following: dichloromethane, glacial acetic acid, acetonitrile, trifluoroacetic acid, dimethyl sulfoxide, anhydrous diethyl ether, n-heptane, aliphatic hydrocarbons, halogenated hydrocarbons, fatty acid esters, aromatic hydrocarbons, cyclohexane, or n-hexane.

3. The drug-loaded microspheres as described in claim 2, characterized in that, In the drug delivery microspheres for polypeptide drugs, the organic solvent is dichloromethane.

4. The drug-loaded microspheres as described in claim 1, characterized in that, The hydrophilic gel concentration in the drug-loaded microspheres for polypeptide delivery is 30-250 mg / mL. -1 .

5. The drug-loaded microspheres as described in claim 1, characterized in that, The mass ratio of the polypeptide active pharmaceutical ingredient to the water-insoluble polymer is 1-20%. The volume ratio of the internal aqueous phase (W1) to the oil phase (O) is 1:2~20; The volume ratio of the oil phase (O) to the external water phase (W2) is 1:2~10; The emulsifier-containing solution is a mixed solution of 1% polyvinyl alcohol and 5% sodium chloride by mass.

6. The drug-loaded microspheres as described in claim 5, characterized in that, The mass ratio of the polypeptide drug active substance to the water-insoluble polymer is 4-10%. The volume ratio of the internal aqueous phase (W1) to the oil phase (O) is 1:5~10; The volume ratio of the oil phase (O) to the external water phase (W2) is 1:4~8.

7. The drug-loaded microspheres as described in claim 1, characterized in that, The drug delivery microspheres for polypeptide drugs further include forming an insoluble complex with a metal salt solution, wherein the insoluble complex and the hydrophilic gel constitute an inner aqueous phase (W1), and the mass ratio of the active polypeptide drug to the metal salt solution is 1:1 to 10.

8. The drug-loaded microspheres as described in claim 7, characterized in that, The active polypeptide drug substance is exenatide or exenatide salt, or semaglutide or semaglutide salt. The metal salt solution is selected from zinc acetate solution, zinc sulfate solution, zinc carbonate solution, zinc nitrate solution, zinc chloride solution, zinc nitrite solution, zinc sulfite solution, copper sulfate solution, copper chloride solution, copper nitrate solution, magnesium nitrate solution, magnesium chloride solution, magnesium sulfate solution, calcium chloride solution, calcium chlorate solution, calcium gluconate solution, calcium hypochlorite solution, or calcium perchlorate solution.

9. A method for preparing polypeptide drug delivery microspheres as described in any one of claims 1 to 8, characterized in that, The method includes the following steps: (1) Preparation of hydrophilic thermosensitive gel; (2) Dissolve the active pharmaceutical ingredient of polypeptide in the above-mentioned thermosensitive gel as the inner aqueous phase (W1). (3) Dissolve the water-insoluble polymer in an organic solvent to form the oil phase (O); (4) Prepare a mixed solution of polyvinyl alcohol with a mass concentration of 1-2% and sodium chloride with a mass concentration of 2-5% as the external aqueous phase (W2). (5) Pour the aqueous phase (W1) into the oil phase (O) and shear the solution at high speed to form W1 / O primary emulsion; (6) Pour the W1 / O primary emulsion into the external aqueous phase (W2) containing the emulsifier and shear at high speed to form a W1 / O / W2 double emulsion; (7) Remove the organic solvent by rotary evaporation, centrifuge the above liquid, discard the supernatant, collect the microspheres, and obtain the drug-loaded microspheres for delivery of the polypeptide drug.

10. The preparation method according to claim 9, characterized in that, In step (5), the colostrum shear rate is 6000~20000 r·min. -1 Colostrum shearing time is 0.5~10 min; The re-emulsification shearing rate in step (6) is 3000~12000 r·min -1 The re-emulsification shearing time is 0.5~10 min.

11. The method for preparing drug-loaded microspheres as described in claim 9, characterized in that, The method for preparing the polypeptide drug delivery microspheres also includes the step of preparing a metal salt ion polypeptide complex.

Images