A preparation method of a pH-responsive drug-loaded polymer vesicle

Abstract

The application discloses a preparation method of a pH-responsive drug-loaded polymer vesicle, and adopts a reversible addition-fragmentation chain transfer dispersion polymerization method to prepare a pH-responsive polymer vesicle containing a phosphorylcholine group, and after polymerization, the vesicle membrane is crosslinked, the polymer vesicle membrane hole is opened under an acidic condition to enable the drug to enter the vesicle interior, then the vesicle membrane hole is closed by adjusting to an alkaline condition, and the drug is encapsulated in the vesicle interior. The phosphorylcholine group on the vesicle membrane has good biocompatibility and interaction with the cell membrane, and the pH response of the vesicle enables the drug to be released at a tumor tissue in a low pH environment, and the crosslinked vesicle membrane structure is not easily damaged by external environment changes, and is more beneficial to the drug delivery to a target site.

Description

TECHNICAL FIELD

[0001] The present application relates to a preparation method of a pH-responsive polymersome containing a choline phosphate group and its application as a drug carrier, and belongs to the technical field of biomedical materials. BACKGROUND

[0002] Malignant tumors have become a major disease that threatens human life and health, and chemotherapy is one of the important means in clinical treatment. Chemotherapeutic drugs kill tumor cells, but also cause great damage to normal tissues and cells, such as liver damage and kidney toxicity. Therefore, it is of great significance to study drug delivery systems that can target tumors.

[0003] Polymer vesicles are a new type of drug carrier, which are spherical or spherical ordered aggregates with a double-layer closed hollow structure formed by amphiphilic block copolymers, with a hydrophilic inner cavity and a hydrophobic double-layer molecular membrane. They can load both hydrophobic and hydrophilic drugs. The designability and stability of polymer vesicles are superior to other drug carriers, and they have great application potential.

[0004] Currently reported drug-loaded polymer vesicles are mainly prepared by traditional self-assembly methods. Generally, drugs are loaded in the process of self-assembly of amphiphilic block copolymers to form particles, as described in patents CN104997755A and CN113476404A. Due to the limitations of traditional self-assembly methods, the formation steps of vesicles are relatively complicated, and generally carried out at low polymer concentrations (generally <1wt%). It is not easy to scale up production, in addition, the vesicles formed by self-assembly are prone to molecular chain disentanglement under the conditions of strong shear, high dilution or the presence of good solvents, which destroys the vesicle structure and is not conducive to the safe delivery of drugs. SUMMARY

[0005] To overcome the drawbacks of the prior art, the present application provides a preparation method of a pH-responsive drug-loaded polymer vesicle containing a choline phosphate group. The pH-responsive polymer vesicle containing a choline phosphate group is prepared by a reversible addition-fragmentation chain transfer (RAFT) dispersion polymerization induced self-assembly method. Then the vesicle membrane is crosslinked to adjust the opening and closing state of the vesicle membrane pore by its pH responsiveness to realize the encapsulation and release of drugs. The crosslinked vesicle membrane structure is not easily dissociated by external environmental changes, which is more conducive to the delivery of drugs to the target site.

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

[0007] The application discloses a preparation method of a pH-responsive drug-loaded polymeric vesicle, and the pH-responsive drug-loaded polymeric vesicle is prepared by using a reversible addition-fragmentation chain transfer dispersion polymerization method and containing a phosphocholine group; under an acidic condition, the membrane hole of the polymeric vesicle is opened to enable the drug to enter the interior of the vesicle, then the vesicle is adjusted to be alkaline to enable the membrane hole of the vesicle to be closed, and the drug is encapsulated in the interior of the vesicle, and the preparation method comprises the following steps.

[0008] a. Preparation of the cross-linked polymeric vesicle: by using a polymerization-induced self-assembly method, 2-(diisopropylamino)ethyl methacrylate (DIPEMA), 2-methacryloyloxyethyl phosphocholine (MPC), glycidyl methacrylate (GlyMA), a self-prepared macromolecular chain transfer agent, an initiator and a solvent are added into a reaction bottle, the reaction bottle is sealed after oxygen is removed, and the polymeric vesicle emulsion is obtained by reacting at 30-80 DEG C for 5-8 hours;

[0009] b. The polymeric vesicle emulsion prepared in step a is diluted by 10-20 times, diamine is added to react with the epoxy groups on the polymeric vesicle, and the cross-linked polymeric vesicle is obtained after stirring at room temperature for 24 hours;

[0010] c. The prepared cross-linked polymeric vesicle is dispersed in an acidic buffer solution, the drug is added, and the solution is stirred at room temperature for 12-24 hours; then the solution is adjusted to be alkaline, the unloaded drug is removed by dialysis, and the drug-loaded polymeric vesicle is obtained.

[0011] The preparation method of the pH-responsive drug-loaded polymeric vesicle, in step a, according to the molar ratio, n DIPEMA +MPC+GlyMA : n 大分子链转移剂 : n 引发剂 =(80-300):1:(1 / 5-1 / 3), the solid content of the polymeric vesicle emulsion is 5-50%, and the molar ratio of the DIPEMA, the MPC and the GlyMA is (1-19):(0.01-5):1.

[0012] The preparation method of the pH-responsive drug-loaded polymeric vesicle, the preparation method of the self-prepared macromolecular chain transfer agent is as follows: polyoxyethylene, a small-molecule chain transfer agent and a catalyst are added into dichloromethane, a dehydrating agent is added into the reaction system after being dissolved, the macromolecular chain transfer agent solution is obtained by stirring at room temperature for 48-72 hours, the macromolecular chain transfer agent is obtained by filtering and precipitating in diethyl ether.

[0013] The preparation method of the pH-responsive drug-loaded polymer vesicle, the polyoxyethylene is polyoxyethylene monomethyl ether, the molecular weight is 300-10000; the small molecule chain transfer agent is a dithioester or a trithioester containing a carboxyl group at one end; the catalyst is 4-dimethylaminopyridine DMAP; the dehydrating agent is N,N'-dicyclohexyl carbodiimide DCC; the molar ratio of the small molecule chain transfer agent to the polyoxyethylene is (1-5):1, the molar ratio of the dehydrating agent to the polyoxyethylene is (1-5):1, the molar ratio of the catalyst to the small molecule chain transfer agent is (0.05-0.2):1, and the solid content of the reaction system is 5-20%.

[0014] The preparation method of the pH-responsive drug-loaded polymer vesicle, the solvent is a mixed solvent of alcohol and water, the alcohol is ethanol or methanol, and the weight ratio of the alcohol to water added is (1-9):1.

[0015] The preparation method of the pH-responsive drug-loaded polymer vesicle, the initiator is a thermal initiator or a redox initiator.

[0016] The preparation method of the pH-responsive drug-loaded polymer vesicle, the thermal initiator is an azo initiator, and the redox initiator is potassium persulfate / sodium bisulfite.

[0017] The preparation method of the pH-responsive drug-loaded polymer vesicle, in step c, the acidic buffer solution is a buffer solution with a pH of 1.0-6.5; and the solution is adjusted to be alkaline with a pH of 7.0-9.0.

[0018] The preparation method of the pH-responsive drug-loaded polymer vesicle, in step c, the drug loaded is a water-soluble drug, and the water-soluble drug includes but is not limited to one of doxorubicin hydrochloride, nucleic acids, proteins, and polypeptide drugs.

[0019] The present application has the following beneficial effects:

[0020] The present application selects a pH-responsive polymer vesicle containing a phosphocholine group and having a crosslinked vesicle membrane as a drug carrier, utilizes the pH responsiveness of the polymer vesicle, the pH response range is 5.0-7.0, the polymer vesicle membrane pores are opened under acidic conditions to make the drug enter the inside of the vesicle, then the vesicle membrane pores are closed by adjusting the solution to be alkaline, the drug is encapsulated in the inside of the vesicle, and the pH responsiveness is utilized to realize the release of the drug at the tumor tissue in a low-pH environment; the phosphocholine group increases the biocompatibility of the polymer vesicle and the interaction between the polymer vesicle and the cell membrane; in addition, the crosslinked vesicle membrane structure is not easily destroyed by external environmental changes, and is more beneficial to the delivery of the drug to the target site. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a transmission electron microscope photo of the polymer vesicle prepared in Example 1 of the present application.

[0022] Figure 2 Transmission electron micrograph of the crosslinked polymer vesicles prepared in Example 1 of the present application;

[0023] Figure 3 Particle size diagram of the crosslinked polymer vesicles prepared in Example 3 of the present application;

[0024] Figure 4 Transmission electron micrograph of the crosslinked polymer vesicles prepared in Example 3 of the present application in pH 4.0 buffer solution;

[0025] Figure 5 Transmission electron micrograph of the polymer vesicles loaded with the drug doxorubicin hydrochloride (DOX·HCl) prepared in Example 3 of the present application;

[0026] Figure 6 UV absorption spectrum of the vesicles loaded with DOX and the drug doxorubicin hydrochloride (DOX·HCl) prepared in Example 4 of the present application. DETAILED DESCRIPTION

[0027] The 2-methacryloyloxyethyl phosphorylcholine MPC is selected in the copolymer monomer of the polymer vesicles of the present application, which is a monomer suitable for simulating the polar group of cell membrane phospholipid. The structure of the monomer contains a double bond at one end, which can be copolymerized with DIPEMA and GlyMA to connect the polymer segment, and contains a phosphorylcholine group at the other end, which can imitate the structure of the extracellular phospholipid bilayer, thereby increasing the biocompatibility of the prepared polymer vesicles and the interaction with the cell membrane (the cell membrane is mainly composed of a bilayer membrane of phospholipids, proteins, cholesterol and sugars, in which phospholipids are the most basic and most abundant substances, and the phosphorylcholine group is the hydrophilic end group of phospholipids), so that the prepared polymer vesicles are more suitable for use as a drug carrier.

[0028] The preparation of the pH-responsive drug-loaded polymer vesicles containing the phosphorylcholine group of the present application adopts a simple and efficient RAFT dispersion polymerization induced self-assembly method, and the functional monomer is introduced during polymerization to prepare the pH-responsive polymer vesicles containing the phosphorylcholine group in one step. The vesicle membrane is crosslinked after polymerization, the structure is stable, the morphology can be maintained in an acidic buffer solution, and the vesicle structure is also stable after drug loading.

[0029] The particle size range of the polymer vesicles prepared in the present application is 100-1000 nm; the pH response range of the crosslinked polymer vesicles is 5.0-7.0; and the loaded drug is a water-soluble drug.

[0030] The present application will be further described below in conjunction with examples. Example 1

[0031] Step a, 5.70 g of mPEG with molecular weight of 1900, 1.674 g of CPADB, 0.072 g of DMAP and 25 mL of anhydrous CH2Cl2 were weighed into a 100 mL round bottom flask and stirred, 0.9285 g of DCC was dissolved in 5 mL of anhydrous CH2Cl2 and added dropwise into the above system, the reaction was carried out at room temperature for 48 h, after filtration, it was precipitated in ethyl ether for three times and vacuum dried to obtain a macromolecular chain transfer agent.

[0032] Step b, the product in step a, 1.0343 g of DIPEMA, 0.2045 g of MPC, 0.1969 g of GlyMA, 0.0036 g of potassium persulfate, 0.0014 g of sodium bisulfite, 6.1757 g of ethanol and 2.6467 g of deionized water were added into a 25 mL reaction bottle, argon was bubbled for 30 min under ice water bath, then the reaction bottle was sealed after three times of pumping and exhaust operation, and the reaction was carried out in a 30℃ oven for 7 h, the reaction bottle was taken out and placed in an ice water bath to stop the reaction, to obtain mPEG-b-P(DIPEMA-MPC-GlyMA) polymer vesicles, TEM test was carried out on the polymer vesicles, and the test results are shown in Figure 1 It can be seen that the product obtained by the method of the present application is a polymer vesicle structure. Figure 1

[0033] Step c, 1.0 g of the product in step b was diluted 20 times with ethanol and water mixed solvent, then ethylenediamine was added, and the mixture was stirred at room temperature overnight to obtain crosslinked polymer vesicles, and the TEM results are shown in Figure 2 It can be seen that the product obtained by the method of the present application is a polymer vesicle structure. Example 2

[0034] Step a, the preparation process of the macromolecular chain transfer agent is the same as that in step a of Example 1.

[0035] Step b, the product in step a, 1.0343 g of DIPEMA, 0.2045 g of MPC, 0.1969 g of GlyMA, 0.0022 g of azobisisobutyronitrile AIBN, 8.3921 g of ethanol and 5.5948 g of deionized water were added into a 25 mL reaction bottle, argon was bubbled for 30 min under ice water bath, then the reaction bottle was sealed after three times of pumping and exhaust operation, and the reaction was carried out in a 65℃ oven for 6 h, the reaction bottle was taken out and placed in an ice water bath to stop the reaction, to obtain mPEG-b-P(DIPEMA-MPC-GlyMA) polymer vesicles, and the vesicle membrane was crosslinked according to step c in Example 1. Example 3

[0036] Step a, the preparation process of the macromolecular chain transfer agent is the same as that in step a of Example 1.​

[0037] Step b, the product in step a 0.1394 g, 1.6367 g DIPEMA, 0.7553 g MPC, 0.3636 g GlyMA, 0.0043 g potassium persulfate, 0.0017 g sodium bisulfite, 15.6654 g ethanol, 10.4436 g deionized water were added into a 50 mL reaction bottle, argon was bubbled for 30 min under ice water bath condition, then the reaction bottle was sealed after three times of pumping operation, and was reacted in a 30 °C oven for 7 h. The reaction bottle was taken out and put into an ice water bath to stop the reaction. The mPEG-b-P(DIPEMA-MPC-GlyMA) polymer vesicle was obtained.

[0038] Step c, the preparation of cross-linked polymer vesicle was the same as step c in Example 1. The results were shown in Figure 3 The average particle size of the obtained product was about 220 nm.

[0039] Step d, 4.0 g of the cross-linked polymer vesicle emulsion in step c was centrifuged and dispersed in a pH 4.0 buffer solution, and 0.01 g of DOX·HCl was added. After stirring at room temperature for 24 h, the pH value was adjusted to 8.0, and then it was dialyzed in a PBS buffer solution for 3 days to remove the unloaded DOX. Figure 4 is a transmission electron micrograph of the cross-linked polymer vesicle in a pH 4.0 buffer solution. It can be seen from the figure that the cross-linked polymer vesicle can maintain a good morphology in a pH 4.0 buffer solution; Figure 5 is a transmission electron micrograph of the vesicle loaded with DOX. It can be seen that the morphology of the vesicle loaded with DOX can still be maintained. Example 4

[0040] The preparation process of the cross-linked polymer vesicle was the same as steps a-c in Example 1.

[0041] Step d, 2.0 g of the cross-linked vesicle emulsion prepared in step c was centrifuged and dispersed in a pH 5.0 buffer solution, and 0.002 g of DOX·HCl was added. After stirring at room temperature for 24 h, the pH value was adjusted to 8.0, and then it was dialyzed in a PBS for 3 days to remove the unloaded DOX. Figure 6 is the ultraviolet absorption spectrum of the prepared polymer vesicle loaded with DOX, polymer vesicle and DOX·HCl. It can be seen that the vesicle loaded with drug has a clear DOX absorption peak at 480 nm, indicating that DOX is loaded into the vesicle. Example 5

[0042] The preparation process of the cross-linked polymer vesicle was the same as steps a-c in Example 1.

[0043] Step d, 4.0 g of the crosslinked niosomal emulsion prepared in step c was dispersed in pH 4.0 buffer solution after centrifugation, and 0.005 g of DOX-HCl was added. After stirring at room temperature for 24 h, the pH value was adjusted to 8.0, and then it was dialyzed in PBS for 3 days to remove the unencapsulated DOX. The drug loading was 19.08% and the encapsulation efficiency was 38.56% by UV quantitative method.

Claims

1. A method for preparing a pH-responsive drug-loaded polymersome, characterized by: The preparation method of pH-responsive polymersomes containing phosphocholine groups by reversible addition-fragmentation chain transfer dispersion polymerization, and the vesicle membrane is cross-linked after polymerization, the polymer vesicle membrane hole is opened under acidic conditions, the drug enters the inside of the vesicle, then the solution is adjusted to alkaline to close the vesicle membrane hole, and the drug is encapsulated in the inside of the vesicle, the preparation method comprises the following steps: a. Preparation of polymer vesicles: 2-(diisopropylamino) methyl acrylate DIPEMA, 2-methacryloyloxyethyl phosphocholine MPC, glycidyl methacrylate GlyMA, self-made macromolecular chain transfer agent, initiator and solvent are added to the reaction bottle, the bottle is sealed after deoxidization, and the reaction is carried out at 30-80℃ for 5-8h to obtain polymer vesicle emulsion; b. The polymer vesicle emulsion prepared in step a is diluted by 10-20 times, and diamine is added to react with the epoxy groups on the polymer vesicle, and after stirring at room temperature for 24h, cross-linked polymer vesicles are obtained; c. The prepared cross-linked polymer vesicles are dispersed in an acidic buffer solution, a drug is added, stirred at room temperature for 12-24h, then the solution is adjusted to alkaline, and the unloaded drug is removed by dialysis to obtain drug-loaded polymer vesicles; In step a, the molar ratio of n DIPEMA +MPC+GlyMA : n 大分子链转移剂 : n 引发剂 =(80~300):1:(1 / 5~1 / 3), the solid content of the polymeric vesicle emulsion is 5-50%, and the molar ratio of DIPEMA, MPC and GlyMA is (1~19):(0.01~5):

1.

2. The method for preparing pH-responsive drug-loaded polymer vesicles according to claim 1, characterized in that: The preparation method of the self-made macromolecular chain transfer agent is as follows: polyoxyethylene, small molecule chain transfer agent and catalyst are added to dichloromethane, after dissolution, a dehydrating agent is added to the reaction system, and after stirring at room temperature for 48-72h, a macromolecular chain transfer agent solution is obtained, which is filtered and precipitated in ethyl ether to obtain the macromolecular chain transfer agent.

3. The method of claim 2, wherein the pH-responsive drug-loaded polymersomes are prepared by the following steps: (1) dissolving the polymer and the drug in a solvent to form a solution; (2) adding the solution to the water phase to form the pH-responsive drug-loaded polymersomes. The polyoxyethylene is polyoxyethylene monomethyl ether, and the molecular weight is 300-10000; the small molecule chain transfer agent is a dithioester or a trithioester containing a carboxyl group at one end; the catalyst is 4-dimethylaminopyridine DMAP; the dehydrating agent is N,N'-dicyclohexyl carbodiimide DCC; the molar ratio of the small molecule chain transfer agent to polyoxyethylene is (1-5):1, the molar ratio of the dehydrating agent to polyoxyethylene is (1-5):1, the molar ratio of the catalyst to the small molecule chain transfer agent is (0.05-0.2):1, and the solid content of the reaction system is 5-20%.

4. The method for preparing pH-responsive drug-loaded polymer vesicles according to claim 1, characterized in that: The solvent is a mixed solvent of alcohol and water, the alcohol is ethanol or methanol, and the weight ratio of the alcohol to water is (1-9):

1.

5. The method of claim 1, wherein the pH-responsive drug-loaded polymersomes are prepared by: The initiator is a thermal initiator or a redox initiator.

6. The method of claim 5, wherein the pH-responsive drug-loaded polymersomes are prepared by: The thermal initiator is an azo initiator, and the redox initiator is potassium persulfate / sodium bisulfite.

7. The method of claim 1, wherein the pH-responsive drug-loaded polymersomes are prepared by: In step c, the acidic buffer solution is a buffer solution with a pH of 1.0-6.5; and the solution is adjusted to alkaline with a pH of 7.0-9.

0.

8. The method of claim 1, wherein the pH-responsive drug-loaded polymersomes are prepared by: In step c, the encapsulated drug is a water-soluble drug, and the water-soluble drug includes one of doxorubicin hydrochloride, nucleic acid, and polypeptide drugs.

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