A pH-responsive core-shell structured insulin-encapsulated nanomicelle and a preparation method thereof

Abstract

The application discloses a pH response core-shell structure encapsulated insulin nanomicelle and a preparation method thereof. The encapsulated insulin mass accounts for 91% of the total mass of the insulin dissolved in an acidic aqueous solution, and the encapsulated insulin mass accounts for 13% of the mass of the encapsulated insulin nanomicelle. The average particle size of the micelle is 190 nm, and the Zeta potential is -19 mV. The nanomicelle is stable, the encapsulation efficiency and the drug loading capacity of the encapsulated insulin are 91% and 13% respectively, the cumulative release of the insulin mass percentage in a simulated human gastric juice environment with pH=1.2 for 2 h, 4 h and 10 h is 12%, 14% and 20% respectively, and the release of the insulin mass percentage in a simulated human intestinal juice environment with pH=7.4 for 2 h and 10 h is 55% and 77% respectively. Compared with the pH response block located in the core of the encapsulated insulin nanomicelle, the pH response block located in the shell of the encapsulated insulin nanomicelle has high stability in the simulated human gastric juice and has a slow-release behavior in the simulated human intestinal juice, and is more suitable for the bioavailability of the oral insulin of the human body.

Description

Technical Field

[0001] This invention relates to biomedical polymer materials and drug-loaded nanomicelles, specifically a pH-responsive core-shell structured encapsulated insulin nanomicelle, its preparation method, and its application. It is also applicable to other orally administered hydrophobic drug nanomicelles. Background Technology

[0002] Diabetes has become a major killer threatening human health. For type 1 diabetes, insulin is the only drug to lower blood sugar. However, long-term and frequent insulin injections not only cause great pain and inconvenience to patients, but also often lead to side effects such as local skin edema, infection, and subcutaneous fat atrophy. Oral insulin has advantages such as convenient administration, conformity to the physiological metabolic pattern of insulin, and good treatment compliance. The drug can reach the intestine smoothly for targeted release, improving drug utilization and efficacy.

[0003] In recent years, pH-responsive amphiphilic nanomicelles encapsulating insulin drugs have been developed. These nanomicelles encapsulate hydrophobic insulin within pH-responsive polymer nanomicelles, releasing the drug in response to different pH values ​​in the human stomach and intestines. Zhang et al. (Zhang CY, Yang YQ, Huang TX, et al. Self-assembled pH-responsive MPEG-b-(PLA-co-PAE)block copolymer micelles for anticancer drug delivery[J]. Biomaterials,2012,33(26):6273-6283) synthesized a pH-responsive amphiphilic polymer MPEG-b-(PLA-co-PAE) for the controlled release of anticancer drugs. This pH-responsive amphiphilic copolymer can self-assemble into core-shell micelles in low-concentration aqueous solutions. Its pH-responsive hydrophobic block PAE is located in the polymer core, encapsulating the hydrophobic drug. It is insoluble in water at an ambient pH of 7.4, but releases the drug through amino protonation at an ambient pH below 6.5. Patent CN102432783A discloses a pH-responsive random copolymer with hydrophobic groups. It self-assembles into polymer micelles with a core formed by hydrophobic groups and pH-responsive groups and a shell formed by hydrophilic groups. Hydrophobic drugs are encapsulated in the micelle core, but the pH-responsive groups in the core affect its sensitivity to environmental pH values. Li Dan's patent CN 108379225A discloses an amphiphilic oral insulin-carrying nanomicelle. The nanomicelle has a particle size of 150-750 nm and a zeta potential of -42 to -20 mV. The amphiphilic block copolymer in the nanomicelle is poly(polylactide methacrylate-co-methacrylic acid)-b-poly(polymethacrylate monomethoxy polyethylene glycol) ester. The micelle core is formed by hydrophobic pH-sensitive block poly(polylactide methacrylate-co-methacrylic acid) [P(PLAMA-co-MAA)], while the shell is formed by hydrophilic poly(polymethacrylate monomethoxy polyethylene glycol) ester. The drug loading is 13.6% and the encapsulation efficiency is 80.2%. In a simulated human gastric juice environment with pH = 0.9-1.8, the insulin release rate is 15% after 2 hours. In a simulated human intestinal juice environment with pH = 5.1-7.8, the insulin release rates are 53% and 90% after 2 hours and 10 hours, respectively.Yang Qianqian's patent CN107519484A discloses a method for preparing insulin-loaded nanomicelles with pH sensitivity and membrane adhesion. The polymer is a poly(methyl methacrylate-co-methacrylic acid)-b-poly(aminoethyl methacrylate) copolymer, consisting of a hydrophobic pH-sensitive block poly(methyl methacrylate-co-methacrylic acid) micelle core and a hydrophilic poly(aminoethyl methacrylate) shell. The insulin loading is 8.8%, and the encapsulation efficiency is 75%. In a simulated human gastric fluid environment with pH = 0.9-1.8, the insulin release rate is 20% after 2 hours. In a simulated human intestinal fluid environment with pH = 5.1-7.8, the insulin release rates are 23% and 60% after 2 hours and 10 hours, respectively. The encapsulation efficiency refers to the percentage of insulin mass encapsulated in the pH-responsive core-shell structure relative to the total insulin mass dissolved in pharmaceutical hydrochloric acid. The insulin loading rate refers to the percentage of the encapsulated insulin mass relative to the total mass of the pH-responsive core-shell structure insulin-loaded nanomicelles. Summary of the Invention

[0004] To address the problems of low encapsulation efficiency, low drug loading, slow drug release, and insufficient pH sensitivity in existing pH-responsive core-shell structures for encapsulating insulin nanomicelles, this invention features the following: (1) A poly(methacrylic acid-co-methacrylic acid-polyethylene glycol) block (P(MAA-co-PEGMA)) formed by the random copolymerization of pH-responsive methacrylic acid (MAA) and hydrophilic methacrylic acid monomethoxy polyethylene glycol ester (PEGMA) serves as the outer shell of the insulin nanomicelle core-shell structure of this invention, enabling rapid and sensitive response to changes in environmental pH to release the encapsulated insulin; (2) To address the problem that the absence of pH-responsive methacrylic acid (MAA) in the core of the insulin nanomicelle core-shell structure would reduce the core space and decrease the insulin loading capacity, the degree of polymerization of the hydrophobic polymethacrylic acid polylactide in the core is increased. (2) Increase its chain length to encapsulate more hydrophobic insulin drugs, while also being able to self-assemble into a white pH-responsive core-shell structure of encapsulated insulin nanomicelles (if the degree of polymerization of hydrophobic polymethyl methacrylate polylactide is too high and the chain length is too long, it will affect the self-assembly formation of pH-responsive core-shell structure of encapsulated insulin nanomicelles); (3) The average particle size of the pH-responsive core-shell structure of encapsulated insulin nanomicelles obtained based on (1) and (2) is 190 nm, the Zeta potential is -19 mV, the nanomicelles are stable, and the encapsulation efficiency and drug loading of encapsulated insulin are 91% and 13%, respectively. Compared with encapsulated insulin nanomicelles with pH-responsive blocks located in the core, the encapsulated insulin nanomicelles with pH-responsive blocks located in the shell have high stability in simulated human gastric juice and have sustained-release behavior in simulated human intestinal juice, which is more suitable for the bioavailability of human oral insulin.

[0005] Technical solution of the present invention

[0006] The first step of the technical route of this invention is a one-pot two-step method to synthesize brush-shaped hydrophobic poly(lactic acid lactide) (PPLAMA) by first performing ring-opening polymerization of lactide and then performing RAFT polymerization (Reversible Addition-Fragmentation Chain Transfer Polymerization). Specifically, first, ring-opening polymerization of lactide is performed to graft cyclic lactide onto hydroxyethyl methacrylate monomers containing terminal hydroxyl groups to obtain a brush-shaped polylactic acid monomer (PLA-HEMA) with hydroxyethyl methacrylate end caps. Then, a free radical initiator azobisisobutyronitrile and a chain transfer agent 4-cyano-4-(phenylcarbonylthio)valerate are added to perform RAFT polymerization to obtain brush-shaped hydrophobic poly(lactic acid lactide) (PPLAMA). The second step involves feeding the pH-responsive monomer methacrylic acid (MAA) and the hydrophilic monomer poly(methacrylic acid monomethoxy polyethylene glycol) (PEGMA) together, and then randomly copolymerizing them onto poly(methacrylic acid polylactide) via RAFT polymerization to obtain a pH-responsive amphiphilic copolymer: poly(methacrylic acid polylactide)-b-poly(methacrylic acid-co-methacrylic acid monomethoxy polyethylene glycol) ([PPLAMA-bP(MAA-co-PEGMA)]). The third step involves dialysis self-assembling the hydrophobic insulin drug with the pH-responsive amphiphilic copolymer to form pH-responsive drug-loaded nanomicelles with a core-shell structure. The core is a brush-like structure of hydrophobic poly(methacrylic acid polylactide) blocks encapsulating the hydrophobic insulin drug, and the outer shell is a pH-responsive hydrophilic block poly(methacrylic acid-co-methacrylic acid monomethoxy polyethylene glycol) ester.

[0007] The technical features of this invention are as follows:

[0008] 1. A pH-responsive hydrophilic block, characterized in that:

[0009] (1) The pH-responsive hydrophilic block is a poly(methacrylic acid-co-methacrylic acid monomethoxy polyethylene glycol ester) (P(MAA-co-PEGMA)) formed by random copolymerization of pH-responsive methacrylic acid (MAA) and hydrophilic methacrylic acid monomethoxy polyethylene glycol ester (PEGMA), and its chemical molecular structure is as follows:

[0010]

[0011] In the above formula, m = 5, which is the degree of polymerization of polyethylene glycol ester (PEG); z = 10, which is the degree of polymerization of polyethylene glycol monomethoxy ester (PEGMA); and y = 60, which is the degree of polymerization of methacrylic acid (MAA).

[0012] (2) The pH-responsive hydrophilic block is a pH-responsive hydrophilic block in a light red pH-responsive amphiphilic copolymer with a number-average molecular weight of 10824 g / mol and a molecular weight distribution index (Mw / Mn) of 1.30. The pH-responsive amphiphilic copolymer is poly(poly(methacrylic acid-co-methacrylic acid)-b-poly(ethylene glycol monomethoxyl)) ([PPLAMA-bP(MAA-co-PEGMA)]), and its chemical molecular structure is as follows:

[0013]

[0014] In the above formula, x = 30 represents the degree of polymerization of poly(PLAMA) methacrylate. The following block in the formula represents poly(PLAMA) methacrylate, a hydrophobic block with a brush-like structure:

[0015]

[0016] 2. A pale red, pH-responsive amphiphilic copolymer, poly(methacrylic acid polylactide-b-poly(methacrylic acid-co-methacrylic acid monomethoxy polyethylene glycol ester), with a number-average molecular weight of 10824 g / mol and a molecular weight distribution index (Mw / Mn) of 1.30, characterized in that: the poly(methacrylic acid-co-methacrylic acid monomethoxy polyethylene glycol ester) blocks in the polymer poly(methacrylic acid polylactide-b-poly(methacrylic acid-co-methacrylic acid monomethoxy polyethylene glycol ester)) have pH-responsive hydrophilicity, and the brush-like poly(methacrylic acid polylactide) blocks have hydrophobicity; when 0.001 g / mL of insulin solution is added to a 0.01 g / mL solution of the polymer, it can self-assemble to form an average particle size distribution by dialysis. White, pH-responsive core-shell structured insulin-encapsulated nanomicelles with a diameter of 190 nm, a Zeta potential of -19 mV, and encapsulation efficiency and drug loading of 91% and 13%, respectively, are described. The nanomicelles consist of a pH-responsive hydrophilic poly(methacrylate-co-methacrylate monomethoxy polyethylene glycol) shell and a brush-like hydrophobic poly(methacrylate-polylactide) core encapsulating the hydrophobic insulin. In a simulated human gastric fluid environment at pH 1.2, the cumulative insulin release percentages at 2 h, 4 h, and 10 h were 12%, 14%, and 20%, respectively. In a simulated human intestinal fluid environment at pH 7.4, the insulin release percentages at 2 h and 10 h were 55% and 77%, respectively.

[0017] 3. A method for preparing white, pH-responsive core-shell structured insulin-encapsulated nanomicelles with an average particle size of 190 nm, a Zeta potential of -19 mV, an encapsulation efficiency of 91%, and a drug loading of 13%, characterized by:

[0018] (1) One-pot two-step synthesis of brush-like hydrophobic polymethacrylate polylactide

[0019] Hydroxyethyl methacrylate, lactide, stannous isooctanoate, and solvent were added to a reactor. The first step, ring-opening polymerization of lactide, was carried out at 100°C for 15 hours under an argon atmosphere. This process grafted cyclic lactide onto hydroxyethyl methacrylate monomers containing terminal hydroxyl groups, forming a brush-like structure of hydroxyethyl methacrylate-terminated polylactic acid macromonomer (PLA-HEMA). Then, azobisisobutyronitrile (AIBN) and 4-cyano-4-(phenylcarbonylthio)valerate were added as free radical initiators, and the second step, reversible addition-fragmentation chain transfer polymerization, was carried out for 24 hours to obtain a brush-like structure of hydrophobic poly(poly(methacrylate-polylactide)). The molar ratio of hydroxyethyl methacrylate, lactide, stannous isooctanoate, azobisisobutyronitrile, 4-cyano-4-(phenylcarbonylthio)valerate, and solvent was 30:30:0.3:0.1:1:77.

[0020] (2) Synthesis of pH-responsive amphiphilic copolymer: poly(polylactic acid methacrylate)-b-poly(methacrylate-co-methacrylate monomethoxy polyethylene glycol ester)

[0021] The hydrophobic poly(poly(methacrylic acid polylactide)) with a brush-like structure obtained in (1), the free radical initiator azobisisobutyronitrile, methacrylic acid, monomethoxy polyethylene glycol methacrylic acid, and solvent were polymerized at 80°C for 24 h in an argon atmosphere to obtain the pH-responsive amphiphilic copolymer: poly(poly(methacrylic acid polylactide)-b-poly(methacrylic acid-co-methoxy polyethylene glycol methacrylic acid); the molar ratio of poly(poly(methacrylic acid polylactide), free radical initiator azobisisobutyronitrile, methacrylic acid, monomethoxy polyethylene glycol methacrylic acid, and solvent was 1:0.1:60:10:77;

[0022] Before adding the above (1) and (2) materials, argon gas is first passed through the reactor to remove oxygen. After adding the materials, the reaction liquid is frozen into a slurry and then vacuumed. It is then melted at room temperature (25°C) and argon gas is passed through again to remove oxygen from the reaction system. The solvent is any one of toluene, tetrahydrofuran, N,N-dimethylformamide, and 1,4-dioxane.

[0023] (3) The pH-responsive amphiphilic copolymer was dissolved in N,N-dimethylformamide solvent at 25°C and stirred to form a polymer solution of 0.01 g / mL; the hydrophobic drug insulin was dissolved in pharmaceutical hydrochloric acid of pH=2 and 0.01 mol / L at 25°C with stirring to prepare an insulin solution of 0.001 g / mL, and then added to the aforementioned 0.01 g / mL polymer solution at 25°C and 85 r / min with stirring, and stirred for 30 min to form a mixed drug polymer solution containing insulin, and then transferred to a dialysis bag with a molecular weight cutoff of MWCO=7000D, and dialyzed at 25°C for 24 h to complete the self-assembly process, and then filtered with a 0.45 μm filter membrane. The filtrate was freeze-dried at -45°C for 24 h to obtain a white pH-responsive core-shell structure encapsulated insulin nanomicelles;

[0024] The dialysate is an acetate-sodium acetate buffer solution with a pH of 5;

[0025] The dialysis was performed by changing the dialysate every 2 hours during the first 12 hours of dialysis, and by changing the dialysate every 6 hours during the next 12 hours of dialysis. The volume ratio of the dialysate to the mixed drug polymer solution was 10:1.

[0026] Technical features and effects of the present invention:

[0027] (1) The poly(methacrylic acid-co-methacrylic acid-polyethylene glycol) block (P(MAA-co-PEGMA)) formed by random copolymerization of pH-responsive methacrylic acid (MAA) and hydrophilic methacrylic acid monomethoxy polyethylene glycol ester (PEGMA) serves as the outer shell of the insulin nanomicelle core-shell structure of this invention, thus rapidly and sensitively responding to changes in environmental pH to release the encapsulated insulin.

[0028] (2) To address the issue that pH-responsive methacrylic acid (MAA) would reduce the core space and decrease the insulin loading capacity of the core due to its absence in the core of the insulin nanomicelle core-shell structure, the degree of polymerization of the hydrophobic polymethacrylic acid polylactide in the core is increased to improve its chain length and encapsulate more hydrophobic insulin drugs. At the same time, it can self-assemble into a white pH-responsive core-shell structure of insulin nanomicelles (if the degree of polymerization of the hydrophobic polymethacrylic acid polylactide is too high and the chain length is too long, it will affect the self-assembly of the pH-responsive core-shell structure of insulin nanomicelles).

[0029] (3) Based on (1) and (2), the pH-responsive core-shell structure of the present invention encapsulated insulin nanomicelles has an average particle size of 190 nm and a Zeta potential of -19 mV. The nanomicelles are stable, and the encapsulation efficiency and drug loading of encapsulated insulin are 91% and 13%, respectively. In a simulated human gastric fluid environment with pH=1.2, the cumulative insulin mass percentages released at 2 h, 4 h, and 10 h are 12%, 14%, and 20%, respectively. In a simulated human intestinal fluid environment with pH=7.4, the insulin mass percentages released at 2 h and 10 h are 55% and 77%, respectively. Compared with encapsulated insulin nanomicelles with pH-responsive blocks located in the core, these encapsulated insulin nanomicelles with pH-responsive blocks located in the shell have higher stability in simulated human gastric fluid and exhibit sustained-release behavior in simulated human intestinal fluid, making them more suitable for the bioavailability of oral insulin in humans.

[0030] (4) The preparation method of the present invention is simple and reliable, the reaction and dialysis conditions are mild, the experimental cycle is short, and it can be mass-produced and applied. Attached Figure Description

[0031] Figure 1 The reaction formula for the synthesis of PPLMA-bP (MAA-co-PEGMA) in Example 1 is shown.

[0032] Figure 2 The two compounds in Example 1 are (A) PPLAMA in deuterated chloroform and (B) PPLAMA-bP (MAA-co-PEGMA) in deuterated dimethyl sulfoxide. 1 H NMR spectrum.

[0033] Figure 3 The image shows an SEM image of insulin-loaded nanomicelles in Example 11. It can be seen that the insulin-loaded nanomicelles are spherical with an average particle size of 190 nm.

[0034] Figure 4 The image shows the particle size distribution of insulin-loaded nanomicelles in Example 11. The average particle size of the insulin-loaded nanomicelles is 190 nm.

[0035] Figure 5 The release curves of insulin-loaded nanomicelles in simulated gastric and intestinal fluids in Example 11 are shown. It can be seen that the release of insulin-loaded nanomicelles in simulated gastric fluid is slow. In the simulated human gastric fluid environment with pH=1.2, the cumulative insulin mass percentages released at 2h, 4h, and 10h are 12%, 14%, and 20%, respectively. In the simulated human intestinal fluid environment with pH=7.4, the insulin mass percentages released at 2h and 10h are 55% and 77%, respectively.

[0036] Figure 6The release curves of insulin nanomicelles encapsulated in the core of Example 15, which have a different structure from that of this patent, in simulated gastric juice (pH=1.2) and intestinal juice (pH=7.4) show low stability in simulated human gastric juice, with an insulin release of about 25% in 2 hours. They also show low sensitivity to environmental pH and a 55% insulin mass release in simulated human intestinal juice environment in 1 hour, indicating a significant burst release behavior. Detailed Implementation

[0037] To further illustrate the technical solution and features of the present invention, the experimental scheme of the present invention is described below in conjunction with embodiments.

[0038] An Agilent 1260 series gel permeation chromatography (GPC) system, equipped with an LC metering pump and a guard column (PL gel 5μm), was used. ) and PL gel A tandem column and an RI differential refractive index detector were used to determine the number-average molecular weight (Mn) and polydispersity index (Mw / Mn), where Mw is the weight-average molecular weight. The chromatographic system was calibrated using a set of monodisperse polyethylene standards. The mobile phase was HPLC-grade tetrahydrofuran, the flow rate was 1.0 mL / min, and the column temperature was 30 °C.

[0039] Example 1: Preparation of a pH-responsive amphiphilic copolymer

[0040] (1) One-pot two-step synthesis of brush-like hydrophobic polymethacrylate polylactide

[0041] A magnetic stir bar was placed in a dry eggplant-shaped flask, and hydroxyethyl methacrylate, lactide, stannous isooctanoate, and toluene solvent were added. The flask was sealed, and the reaction solution was frozen into a slurry. After evacuation, the mixture was melted at 25°C, and argon gas was introduced to remove oxygen from the flask, including the reaction solution. The mixture was then placed in an oil bath at 100°C for ring-opening polymerization for 15 hours. This process resulted in the hydroxyethyl methacrylate monomer containing terminal hydroxyl groups being grafted with cyclic lactide to form a brush-like structure of hydroxyethyl methacrylate-terminated polylactic acid monomer (PLA-HEMA). Then, azobisisobutyronitrile (AIB) and 4-cyano-4-(phenylcarbonylthio)pentanoic acid (CPO4) were added as initiators. The second step of reversible addition-fragmentation chain transfer (RAFT) polymerization was carried out at 100°C for 24 hours. Tetrahydrofuran was then added to dilute and dissolve the product. The solution was uniformly added dropwise to an excess of 10 times its volume of -20°C petroleum ether to precipitate the product. The precipitate was then vacuum-dried at 40°C for 24 hours to obtain a pale red solid, which is the brush-like hydrophobic poly(methacrylate) polylactide, abbreviated as PPLAM. 30Its number-average molecular weight is 3101 g / mol, and its molecular weight distribution index Mw / Mn is 1.20. The molar ratio of the added hydroxyethyl methacrylate, lactide, stannous isooctanoate, free radical initiator azobisisobutyronitrile, chain transfer agent 4-cyano-4-(phenylcarbonylthio)valerate, and toluene is 30:30:0.3:0.1:1:77.

[0042] (2) Synthesis of pH-responsive amphiphilic copolymers

[0043] A magnetic stir bar was placed in a dried eggplant-shaped flask. Argon gas was first passed through to remove oxygen. Then, a brush-shaped hydrophobic poly(poly(methacrylic acid)-polylactide), azobisisobutyronitrile (AIB) initiator, methacrylic acid, monomethoxy polyethylene glycol methacrylate (MMY) solvent, and N,N-dimethylformamide were added. The flask was sealed, and the reaction solution was frozen into a slurry. Vacuum was applied, and the mixture was thawed at 25°C. Argon gas was then passed through to remove oxygen from the reaction flask, including the reaction solution. The mixture was then placed in an oil bath at 80°C for 24 hours for polymerization. N,N-dimethylformamide was added to dilute and dissolve the slurry. The resulting solution was then uniformly added dropwise to an excess of 10 times its volume of anhydrous diethyl ether at -20°C to precipitate the product. The precipitate was dried under vacuum at 40°C for 24 hours. The resulting pale pink solid product is a pH-responsive amphiphilic copolymer: poly(methacrylic acid)-polylactide-b-poly(methacrylic acid-co-methacrylic acid)-MMY), abbreviated as PPLAMA. 30 -bP(MAA 60 -co-PEGMA 10 The number-average molecular weight is 10824 g / mol, the molecular weight distribution index Mw / Mn is 1.30, and the added poly(poly(lactic acid) methacrylate, poly(lactic acid) lactide, free radical initiator azobisisobutyronitrile, methacrylic acid, monomethoxy polyethylene glycol methacrylate, and solvent molar ratio is 1:0.1:60:10:77.

[0044] Example 2: Preparation of a pH-responsive amphiphilic block copolymer

[0045] (1) One-pot two-step synthesis of brush-like hydrophobic polymethacrylate polylactide

[0046] The rest is the same as described in Example 1 (1), but the lactide ring-opening polymerization is carried out for 10 h to obtain a hydrophobic polymethyl methacrylate polylactide with a brush-like structure of 3065 g / mol and a molecular weight distribution index of 1.32.

[0047] (2) Synthesis of pH-responsive amphiphilic copolymers

[0048] The rest is the same as described in Example 1 (2), but the molar ratio of each reactant component is 1:0.1:60:5:77, resulting in a pH-responsive amphiphilic copolymer PPLAMA. 30 -bP(MAA60 The number-average molecular weight of PEGMA5 was 9860 g / mol, and the molecular weight distribution index was 1.35.

[0049] Example 3: Preparation of a pH-responsive amphiphilic block copolymer

[0050] (1) One-pot two-step synthesis of brush-like hydrophobic polymethacrylate polylactide

[0051] The other conditions are the same as described in Example 1 (1), but the lactide is subjected to ring-opening polymerization for 5 hours to obtain a brush-like hydrophobic polymethyl methacrylate polylactide with a number average molecular weight of 2956 g / mol and a molecular weight distribution index of 1.51.

[0052] (2) Synthesis of pH-responsive amphiphilic copolymers

[0053] The operation is the same as described in Example 1 (2), but the molar ratio of each reactant component is 1:0.1:60:15:77, to obtain the pH-responsive amphiphilic copolymer PPLAMA. 30 -bP(MAA 60 -co-PEGMA 15 The number-average molecular weight is 9273 g / mol, and the molecular weight distribution index is 1.38.

[0054] Example 4: Preparation of a pH-responsive amphiphilic block copolymer

[0055] (1) One-pot two-step synthesis of brush-like hydrophobic macromolecular poly(methacrylic acid) polylactide

[0056] The rest is the same as described in Example 1 (1), but the lactide ring-opening polymerization and RAFT polymerization are carried out simultaneously to obtain a brush-shaped hydrophobic polymethyl methacrylate polylactide with a number average molecular weight of 2813 g / mol and a molecular weight distribution index of 1.73.

[0057] (2) Synthesis of pH-responsive amphiphilic block copolymers

[0058] The operation is the same as described in Example 1 (2), but the molar ratio of each reactant component is 1:0.1:60:15:77, to obtain the pH-responsive amphiphilic copolymer PPLAMA. 30 -b-(MAA 60 -co PPEGMA 15 The number-average molecular weight is 9117 g / mol, and the molecular weight distribution index is 1.45.

[0059] Example 5: Preparation of a pH-responsive amphiphilic block copolymer

[0060] (1) One-pot two-step synthesis of brush-like hydrophobic macromolecular poly(methacrylic acid) polylactide

[0061] Same as in Example 1 (1).

[0062] (2) Synthesis of pH-responsive amphiphilic block copolymers

[0063] The operation was the same as described in Example 1 (2), but methacrylic acid and methacrylate monomethoxy polyethylene glycol were fed separately. First, methacrylic acid was added for RAFT polymerization for 15 hours, and then methacrylate monomethoxy polyethylene glycol was added to continue polymerization for 24 hours. The molar ratio of each component of the reactants was 1:0.1:60:10:77, resulting in a pH-responsive amphiphilic copolymer PPLAMA with a number average molecular weight of 9842 g / mol and a molecular weight distribution index of 1.75. 30 -bP(MAA 60 -co-PEGMA 10 ).

[0064] Example 6: Preparation of a pH-responsive amphiphilic block copolymer

[0065] (1) One-pot two-step synthesis of brush-like hydrophobic macromolecular poly(methacrylic acid) polylactide

[0066] The operation is the same as described in Example 1 (1), but the molar ratio of each component of the reactants is 10:10:0.1:0.1:1, to obtain a brush-like hydrophobic polymethyl methacrylate polylactide with a number average molecular weight of 1724 g / mol and a molecular weight distribution index of 1.29.

[0067] (2) Synthesis of pH-responsive amphiphilic block copolymers

[0068] The operation is the same as described in Example 1 (2), but the molar ratio of each reactant component is 1:0.1:60:10:77, to obtain the pH-responsive amphiphilic copolymer PPLAMA. 10 -b-(MAA 60 -co PPEGMA 10 The number-average molecular weight is 9010 g / mol, and the molecular weight distribution index is 1.69.

[0069] Example 7: Preparation of a pH-responsive amphiphilic block copolymer

[0070] (1) Synthesis of macromolecular chain transfer agent poly(methacrylic acid) polylactide

[0071] The operation is the same as described in Example 1 (1), but the molar ratio of the reactant components is 20:20:0.2:0.1:1:77, resulting in a brush-like hydrophobic polymethyl methacrylate polylactide with a number average molecular weight of 2123 g / mol and a molecular weight distribution index of 1.41.

[0072] (2) Synthesis of pH-responsive amphiphilic block copolymers

[0073] The operation is the same as described in Example 1 (2), but the molar ratio of each reactant component is 1:0.1:60:10:77, to obtain the pH-responsive amphiphilic copolymer PPLAMA. 20 -b-(MAA 60 -co PPEGMA 10 The number-average molecular weight is 9730 g / mol, and the molecular weight distribution index is 1.73.

[0074] Example 8: Preparation of a pH-responsive amphiphilic block copolymer

[0075] (1) Synthesis of macromolecular chain transfer agent poly(methacrylic acid) polylactide

[0076] Same as in Example 1 (1).

[0077] (2) Synthesis of pH-responsive amphiphilic block copolymers

[0078] The operation was the same as described in Example 1 (2), but the polymerization temperature was 70℃, the solvent was 1,4-dioxane, and after the polymerization reaction was completed, tetrahydrofuran was added to dilute and dissolve the product. The resulting solution was uniformly added dropwise to petroleum ether to precipitate the product. The molar ratio of each component of the reactants was 1:0.1:60:10:77, resulting in a pH-responsive amphiphilic copolymer PPLAMA with a number average molecular weight of 10092 g / mol and a molecular weight distribution index of 1.69. 30 -bP(MAA 60 -co-PEGMA 10 ).

[0079] Example 9: Preparation of a pH-responsive amphiphilic block copolymer

[0080] (1) Synthesis of macromolecular chain transfer agent poly(methacrylic acid) polylactide

[0081] Same as in Example 1 (1).

[0082] (2) Synthesis of pH-responsive amphiphilic block copolymers

[0083] The operation was the same as described in Example 1 (2), but the polymerization temperature was 80℃, the solvent was 1,4-dioxane, and after the polymerization reaction was completed, tetrahydrofuran was added to dilute and dissolve the product. The resulting solution was uniformly added dropwise to petroleum ether to precipitate the product. The molar ratio of each component of the reactants was 1:0.1:60:10:77, resulting in a pH-responsive amphiphilic copolymer PPLAMA with a number average molecular weight of 10124 g / mol and a molecular weight distribution index of 1.72. 30 -bP(MAA 60 -co-PEGMA10 ).

[0084] Example 10: Preparation of a pH-responsive amphiphilic block copolymer

[0085] (1) Synthesis of macromolecular chain transfer agent poly(methacrylic acid) polylactide

[0086] Same as in Example 1 (1)

[0087] (2) Synthesis of pH-responsive amphiphilic block copolymers

[0088] The operation was the same as described in Example 1 (2), but the polymerization temperature was 65℃, the solvent was tetrahydrofuran, and after the polymerization reaction was completed, tetrahydrofuran was added to dilute and dissolve the product. The resulting solution was uniformly added dropwise to petroleum ether to precipitate the product. The molar ratio of each component of the reactants was 1:0.1:60:10:77, resulting in a pH-responsive amphiphilic copolymer PPLAMA with a number average molecular weight of 9873 g / mol and a molecular weight distribution index of 1.92. 30 -bP(MAA 60 -co-PEGMA 10 ).

[0089] By comparing Examples 1-10 above, pH-responsive amphiphilic block copolymers with different number-average molecular weights and molecular weight distribution indices were obtained, among which PPLAMA in Example 1... 30 -bP(MAA 60 -co-PEGMA 10 It is best to have a high number-average molecular weight of 10824 g / mol and a low molecular weight distribution index Mw / Mn of 1.30.

[0090] Example 11: Preparation of pH-responsive core-shell structured encapsulated insulin nanomicelles, measurement, and in vitro simulated release.

[0091] (1) The polymer PPLAM prepared in Example 1 was used. 30 -b-(MAA 60 -co-PPEGMA 10The polymer solution was prepared by dissolving N,N-dimethylformamide at 25°C and stirring to form a 0.01 g / mL polymer solution. Hydrophobic insulin was dissolved in a pharmaceutical-grade hydrochloric acid solution at pH 2 with stirring at 25°C to prepare a 0.001 g / mL insulin solution. This solution was then added to the aforementioned 0.01 g / mL polymer solution at 25°C and 85 r / min, and stirred for 30 min to form a mixed drug polymer solution containing insulin. This solution was then transferred to a dialysis bag with a molecular weight cutoff (MWCO) of 7000D and dialyzed at 25°C for 24 h to complete the self-assembly process of the drug-loaded micelles. After filtration through a 0.45 μm filter membrane, the filtrate was freeze-dried at -45°C for 24 h to obtain a white, pH-responsive core-shell structured encapsulated insulin nanomicelle.

[0092] (2) After dialysis, the filtrate was taken and the average particle size of the encapsulated insulin nanomicelles with a pH-responsive core-shell structure was measured to be 190 nm and the zeta potential was -19 mV using a ZS90 potential particle size analyzer.

[0093] The encapsulation efficiency (EE) and loading content (LC) of encapsulated insulin were calculated by measuring the insulin concentration in the supernatant after washing with water using high-performance liquid chromatography (HPLC). The calculation formula is as follows:

[0094]

[0095]

[0096] The drug loading percentage of the insulin-encapsulated nanomicelles was 13%, and the encapsulation efficiency of insulin was 91%.

[0097] (3) Hydrochloric acid buffer solution with pH = 1.2 (0.15M HCl - 0.05M KCl) and phosphate buffer solution with pH = 7.4 (8g NaCl - 0.2g KCl - 1.44g Na2HPO4 - 0.24g KH2PO4) were used as in vitro simulated gastric and intestinal fluids, respectively, to measure the release of insulin from the encapsulated insulin micelles. 10mg of encapsulated insulin nanomicelles were accurately weighed and dissolved in 20mL of simulated gastric and intestinal fluids, respectively. The solutions were shaken horizontally at 37℃ and 50 times / min. Samples were taken at 1h, 2h, 4h, 7h, 10h, and 12h, and corresponding simulated gastric and intestinal fluids were added. The supernatant was collected by centrifugation, and the insulin concentration was determined. The cumulative percentage of insulin released from the encapsulated insulin nanomicelles at different times was calculated using the following formula:

[0098]

[0099] In the above formula, V is the volume of simulated gastric or intestinal fluid (mL), C is the insulin concentration in the supernatant (mg / mL), W is the mass of the insulin-loaded nanomicelles (mg), and LC is the drug loading of the insulin-loaded nanomicelles (%).

[0100] The release results showed that the cumulative insulin mass percentages released at 2h, 4h, and 10h were 12%, 14%, and 20% in a simulated human gastric juice pH=1.2 environment, respectively, while the insulin mass percentages released at 2h and 10h were 55% and 77% in a simulated human intestinal juice pH=7.4 environment, respectively.

[0101] The operating steps for Example 12 are the same as those for Example 11, but the polymer used is PPLAMA, the polymer from Example 7. 20 -b-(MAA 60 -co PPEGMA 10 The obtained insulin-loaded nanomicelles had a zeta potential of -19.8 mV, an average particle size of 219 nm, a drug loading of 11%, and an encapsulation efficiency of 86%.

[0102] The operating steps for Example 13 are the same as those for Example 11, but the polymer used is PPLAMA, the polymer from Example 6. 10 -b-(MAA 60 -co PPEGMA 10 The insulin-loaded nanomicelles prepared had an average particle size of 253 nm, a zeta potential of -22 mV, a drug loading of 7%, and an encapsulation efficiency of 72%.

[0103] The operating steps for Example 14 are the same as those for Example 11, but the polymer used is PPLAMA, the polymer from Example 4. 30 -b-(MAA 60 -co PPEGMA 15 The insulin-loaded nanomicelles were prepared with an average particle size of 300.5 nm, a zeta potential of -18.7 mV, a drug loading of 11.9%, and an encapsulation efficiency of 90.2%.

[0104] Example 15 operates on the same steps as Example 11, but the polymer is a pH-responsive P(PLAMA) polymer with the core located in the core. 20 -co-MAA 60 )-b-PPEGMA 10 (poly(poly(polylactic acid methacrylate-co-methacrylic acid)-b-poly(monomethoxy polyethylene glycol methacrylate)) Figure 6The results showed that the pH-responsive block encapsulated insulin nanomicelles located in the core had low stability in simulated human gastric juice at pH=1.2, with a cumulative insulin release of nearly 25% in 2 hours. However, in simulated human intestinal juice at pH=7.4, the cumulative insulin release of nearly 60% in 1 hour showed a significant burst release behavior.

[0105] Comparing Examples 11-15 above, it is evident that increasing the length of the poly(poly(lactic-co-methyl)methacrylate) hydrophobic block (PPLAMA) in the polymer, i.e., increasing the degree of polymerization x of poly(lactic-co-methyl)methacrylate from 10 to 30, increases the drug loading from 7% to 13% and the encapsulation efficiency from 86% to 91%, with Example 11 exhibiting the highest drug loading and encapsulation efficiency. Furthermore, the comparison between Examples 11 and 15 demonstrates that the pH-responsive block of the insulin nanomicelles encapsulated in the outer shell of this invention exhibits high stability in simulated human gastric fluid and sustained-release behavior in simulated human intestinal fluid, making it more suitable for the bioavailability of orally administered insulin.

Claims

1. A method for preparing white, pH-responsive core-shell structured insulin-encapsulated nanomicelles with an average particle size of 190 nm, a Zeta potential of -19 mV, an encapsulation efficiency of 91%, and a drug loading of 13%, characterized in that: (1) One-pot two-step synthesis of brush-like hydrophobic polymethacrylate polylactide Hydroxyethyl methacrylate, lactide, stannous isooctanoate, and solvent were added to a reactor. The first step, ring-opening polymerization of lactide, was carried out at 100°C for 15 hours under an argon atmosphere. This process grafted cyclic lactide onto hydroxyethyl methacrylate monomers containing terminal hydroxyl groups, forming a brush-like structure of hydroxyethyl methacrylate-terminated polylactic acid macromonomer (PLA-HEMA). Then, azobisisobutyronitrile (AIBN) and 4-cyano-4-(phenylcarbonylthio)valerate were added as free radical initiators, and the second step, reversible addition-fragmentation chain transfer polymerization, was carried out for 24 hours to obtain a brush-like structure of hydrophobic poly(poly(methacrylate-polylactide)). The molar ratio of hydroxyethyl methacrylate, lactide, stannous isooctanoate, azobisisobutyronitrile, 4-cyano-4-(phenylcarbonylthio)valerate, and solvent was 30:30:0.3:0.1:1:

77. (2) Synthesis of pH-responsive amphiphilic copolymer: poly(polylactic acid methacrylate)-b-poly(methacrylate-co-methacrylate monomethoxy polyethylene glycol ester) The hydrophobic poly(poly(methacrylic acid polylactide)) with a brush-like structure obtained in (1), the free radical initiator azobisisobutyronitrile, methacrylic acid, monomethoxy polyethylene glycol methacrylic acid, and solvent were polymerized at 80°C for 24 h in an argon atmosphere to obtain the pH-responsive amphiphilic copolymer: poly(poly(methacrylic acid polylactide)-b-poly(methacrylic acid-co-methoxy polyethylene glycol methacrylic acid); the molar ratio of poly(poly(methacrylic acid polylactide), free radical initiator azobisisobutyronitrile, methacrylic acid, monomethoxy polyethylene glycol methacrylic acid, and solvent was 1:0.1:60:10:77; Before adding the above (1) and (2) materials, argon gas is first passed through the reactor to remove oxygen. After adding the materials, the reaction liquid is frozen into a slurry and then vacuumed. It is then melted at room temperature (25°C) and argon gas is passed through again to remove oxygen from the reaction system. The solvent is any one of toluene, tetrahydrofuran, N,N-dimethylformamide, and 1,4-dioxane. (3) The pH-responsive amphiphilic copolymer was dissolved in N,N-dimethylformamide solvent at 25°C and stirred to form a polymer solution of 0.01 g / mL; the hydrophobic drug insulin was dissolved in pharmaceutical hydrochloric acid of pH=2 and 0.01 mol / L at 25°C with stirring to prepare an insulin solution of 0.001 g / mL, and then added to the aforementioned 0.01 g / mL polymer solution at 25°C and 85 r / min with stirring, and stirred for 30 min to form a mixed drug polymer solution containing insulin, and then transferred to a dialysis bag with a molecular weight cutoff of MWCO=7000D, and dialyzed at 25°C for 24 h to complete the self-assembly process, and then filtered with a 0.45 μm filter membrane, and the filtrate was freeze-dried at -45°C for 24 h to obtain a white pH-responsive core-shell structure encapsulated insulin nanomicelles; The dialysate is an acetate-sodium acetate buffer solution with a pH of 5; The dialysis was performed by changing the dialysate every 2 hours during the first 12 hours of dialysis, and by changing the dialysate every 6 hours during the next 12 hours of dialysis. The volume ratio of the dialysate to the mixed drug polymer solution was 10:1.

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