阅读说明：本技术 mPEG-b-PLC两亲性嵌段共聚物的制备方法和多西紫杉醇纳米制剂及制备方法 (Preparation method of mPEG-b-PLC amphiphilic block copolymer, docetaxel nano-preparation and preparation method ) 是由 顾全荣 袁守军 李琳娜 王东海 于 2020-04-20 设计创作，主要内容包括：本发明公开了一种mPEG-b-PLC两亲性嵌段共聚物的制备方法、多西紫杉醇纳米制剂及制备方法。该多西紫杉醇纳米制剂包括多西紫杉醇、脂肪酸和mPEG-b-PLC两亲性嵌段共聚物,制备包括步骤：将多西紫杉醇溶解于第一有机溶剂,得到第一溶液；将mPEG-b-PLC两亲性嵌段共聚物溶解于第二有机溶剂,得到第二溶液；将脂肪酸溶解于第三有机溶剂,得到第三溶液；混合第一溶液、第二溶液和第三溶液,去除有机溶剂,获取混合物；水化所述混合物,用纳米过滤器过滤,获取滤液；冷冻所述滤液,获取多西紫杉醇纳米冰块；冷冻干燥所述多西紫杉醇纳米冰块,获取多西紫杉醇纳米制剂。本发明多西紫杉醇纳米制剂抗肿瘤、降低肿瘤转化率疗效明显,毒副作用低。(The invention discloses a preparation method of mPEG-b-PLC amphiphilic block copolymer, a docetaxel nanometer preparation and a preparation method. The docetaxel nanometer preparation comprises docetaxel, fatty acid and mPEG-b-PLC amphiphilic block copolymer, and the preparation comprises the following steps: dissolving docetaxel in a first organic solvent to obtain a first solution; dissolving the amphiphilic block copolymer of mPEG-b-PLC in a second organic solvent to obtain a second solution; dissolving fatty acid in a third organic solvent to obtain a third solution; mixing the first solution, the second solution and the third solution, and removing the organic solvent to obtain a mixture; hydrating the mixture, and filtering with a nanofilter to obtain a filtrate; freezing the filtrate to obtain docetaxel nanometer ice blocks; and freeze-drying the docetaxel nanometer ice blocks to obtain the docetaxel nanometer preparation. The docetaxel nanometer preparation has obvious curative effects of resisting tumor and reducing tumor transformation rate and has low toxic and side effects.)

1. The preparation method of the amphiphilic block copolymer of mPEG-b-PLC is characterized by comprising the following steps:

(1) drying mPEG in vacuum;

(2) mixing mPEG with stannous isooctanoate, adding DL-lactide in N₂Reacting for 3-5 hours at 160-180 ℃ in the atmosphere to obtain a reactant;

(3) cooling to room temperature under N₂Under the atmosphere, adding reactants into a mixed solvent of ethyl acetate and n-heptane, after completely dissolving, adding anhydrous ether to remove unreacted lactide and lactide homopolymer generated by reaction, and filtering to obtain a crude product of the amphiphilic block copolymer of mPEG-b-PLC;

(4) vacuum drying the crude product of the mPEG-b-PLC amphiphilic block copolymer;

(5) dissolving the mPEG-b-PLC amphiphilic block copolymer crude product in water, adding EDTA disodium to complex with tin ions in the EDTA disodium to form a complex, dialyzing for 20-50 hours by using a dialysis bag to remove the complex, and drying to obtain the mPEG-b-PLC amphiphilic block copolymer; or dissolving the mPEG-b-PLC amphiphilic block copolymer crude product in water, adding the solution into a dialysis bag, adding EDTA disodium into a dialysis medium to be complexed with tin ions in the solution to form a complex, dialyzing for 20-50 hours to remove the complex, and drying to obtain the mPEG-b-PLC amphiphilic block copolymer;

wherein the content of tin element in the mPEG-b-PLC amphiphilic block copolymer is 5-40 ppm.

2. The preparation method of the mPEG-b-PLC amphiphilic block copolymer as claimed in claim 1, wherein the step (1) is specifically: drying mPEG for 3-5 hours in vacuum at 50-60 ℃; the step (2) is specifically as follows: preparing stannous isooctanoate into toluene solution of stannous isooctanoate or dichloromethane solution of stannous isooctanoate, mixing with mPEG, sealing, and blowing high-purity N at 70 deg.C₂To form N₂Adding DL-lactide in N atmosphere₂In the atmosphere at 160-180 DEG CReacting for 3-5 hours under the condition to obtain a reactant; in the step (2), the amount of the toluene solution of stannous isooctanoate or the dichloromethane solution of stannous isooctanoate added is 0.2-1% of the mass of mPEG; the mass ratio of mPEG to DL-lactide in the step (2) is 1: (0.5 to 1).

3. The method for preparing the mPEG-b-PLC amphiphilic block copolymer according to claim 1, wherein the volume ratio of the reactants to the mixed solvent of ethyl acetate and n-heptane in the step (3) is 1: (1-2); and (4) drying at 35-45 ℃ for 25-35 hours in vacuum.

4. The preparation method of the mPEG-b-PLC amphiphilic block copolymer as claimed in claim 1, characterized in that, in the step (5), disodium EDTA is added in the form of disodium EDTA aqueous solution, the concentration of the disodium EDTA aqueous solution is 0.0075 g/L-0.15 g/L, and the addition amount is such that the mass of the disodium EDTA is 1-2% of the mass of the crude product of the mPEG-b-PLC amphiphilic block copolymer; the step of dialyzing for 20-50 hours by using a dialysis bag specifically comprises the following steps: putting the solution to be dialyzed into a dialysis bag, putting a dialysis medium into the dialysis bag for dialysis for 20-50 hours, and replacing the dialysis medium every 5-7 hours; the dialysis medium is ultrapure water.

5. A docetaxel nano-formulation comprising docetaxel and the mPEG-b-PLC amphiphilic block copolymer prepared by the preparation method as set forth in any one of claims 1 to 4; the mass ratio of the docetaxel to the mPEG-b-PLC amphiphilic block copolymer is 1: 3-100.

6. A method for preparing docetaxel nano-formulation as set forth in claim 5, which comprises the steps of:

s1, dissolving docetaxel in a first organic solvent to obtain a first solution;

s2, dissolving the mPEG-b-PLC amphiphilic block copolymer in a second organic solvent to obtain a second solution;

s3, dissolving the fatty acid in a third organic solvent to obtain a third solution;

s4, mixing the first solution, the second solution and the third solution, and removing the organic solvent to obtain a mixture;

s5, hydrating the mixture, and filtering the mixture by using a nano filter to obtain filtrate;

s6, freezing the filtrate to obtain docetaxel nano ice blocks;

s7, freeze-drying the docetaxel nanometer ice blocks to obtain docetaxel nanometer preparations;

wherein the mass ratio of the docetaxel to the mPEG-b-PLC amphiphilic block copolymer is as follows: 1: 3-100.

7. The method for preparing docetaxel nano-formulation according to claim 6, wherein the first and third organic solvents are absolute ethyl alcohol; the second organic solvent is acetonitrile; the fatty acid is selected from any one of lauric acid, palmitic acid, oleic acid and linoleic acid.

8. The method for preparing docetaxel nano-formulation according to claim 6, wherein the step S4 is specifically: and stirring and mixing the first solution, the second solution and the third solution in a water bath at 60 ℃ for 30 minutes, and removing the organic solvent at the temperature of 60 ℃ under negative pressure to obtain a mixture of paclitaxel and the amphiphilic block copolymer of mPEG-b-PLC.

9. The method for preparing docetaxel nano-formulation according to claim 6, wherein the step S5 is specifically: hydrating the mixture with distilled water, shaking and mixing for 15-30 minutes to obtain a mixed solution, and filtering the mixed solution with a 200-nanometer filter to obtain a filtrate.

10. The method of preparing docetaxel nano-formulation according to claim 6, wherein the temperature of freezing the filtrate in the step S6 is-40 ℃; step S7 specifically includes: and drying the paclitaxel nano ice blocks for 48-72 hours by using a freeze dryer to obtain the docetaxel nano preparation.

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a preparation method of an mPEG-b-PLC amphiphilic block copolymer, a docetaxel nano-preparation and a preparation method thereof.

Background

Docetaxel is a derivative of paclitaxel, has anti-tumor activity like paclitaxel, has an action target in plasma coated microtubules, and has a wide anti-tumor spectrum. Docetaxel is poorly water-soluble, and thus it is required to develop a docetaxel water-soluble injection preparation for easy administration.

Taxotere (Taxotere), a docetaxel injection anticancer drug developed by Sanofi-Aventis, contains 40mg docetaxel, 1040mg polysorbate 80 and 13% ethanol per ml, and is commonly used for the treatment of breast cancer, ovarian cancer, etc. However, Taxotere is prone to toxic side effects such as bone marrow suppression, allergy, gastrointestinal reactions, fluid retention, arthralgia and the like. These toxic side effects are associated with pharmaceutical adjuvants, and therefore, modifying an adjuvant would hopefully reduce the toxic side effects of the formulation. The mPEG-b-PLC amphiphilic block copolymer can be used as a pharmaceutical auxiliary, but the mPEG-b-PLC amphiphilic block copolymer prepared by the prior art has the characteristics of wide molecular weight distribution (the molecular weight distribution is about 1.7) and high tin element content (400-600 ppm), tin element belongs to heavy metal, stannous ions are easily oxidized, and the health of human bodies can be harmed once the stannous ions enter the bodies if the amphiphilic block copolymer is not treated.

Disclosure of Invention

The invention aims to provide a novel preparation method of mPEG-b-PLC amphiphilic block copolymer, a novel docetaxel nano-preparation with low toxic and side effects and a preparation method thereof, aiming at the problems of the existing mPEG-b-PLC amphiphilic block copolymer and docetaxel preparation. In order to achieve the above purpose, the invention provides the following technical scheme:

a preparation method of an amphiphilic block copolymer of mPEG-b-PLC comprises the following steps:

(1) drying mPEG in vacuum;

(2) mixing mPEG with stannous isooctanoate, adding DL-lactide in N₂Reacting for 3-5 hours at 160-180 ℃ in the atmosphere to obtain a reactant;

(3) cooling to room temperature under N₂Under the atmosphere, adding reactants into a mixed solvent of ethyl acetate and n-heptane, after completely dissolving, adding anhydrous ether to remove unreacted lactide and lactide homopolymer generated by reaction, and filtering to obtain a crude product of the amphiphilic block copolymer of mPEG-b-PLC;

(4) vacuum drying the crude product of the mPEG-b-PLC amphiphilic block copolymer;

(5) dissolving the mPEG-b-PLC amphiphilic block copolymer crude product in water, adding EDTA disodium to complex with tin ions in the EDTA disodium to form a complex, dialyzing for 20-50 hours by using a dialysis bag to remove the complex, and drying to obtain the mPEG-b-PLC amphiphilic block copolymer; or dissolving the mPEG-b-PLC amphiphilic block copolymer crude product in water, adding the solution into a dialysis bag, adding EDTA disodium into a dialysis medium to be complexed with tin ions in the solution to form a complex, dialyzing for 20-50 hours to remove the complex, and drying to obtain the mPEG-b-PLC amphiphilic block copolymer;

wherein the content of tin element in the mPEG-b-PLC amphiphilic block copolymer is 5-40 ppm.

Preferably, the step (1) is specifically: drying mPEG for 3-5 hours in vacuum at 50-60 ℃; the step (2) is specifically as follows: mixing isooctanePreparing stannous isooctanoate toluene solution or stannous isooctanoate methylene dichloride solution from stannous acid, mixing with mPEG, sealing, and blowing high-purity N at 70 deg.C₂To form N₂Adding DL-lactide in N atmosphere₂Reacting for 3-5 hours at 160-180 ℃ in the atmosphere to obtain a reactant; in the step (2), the amount of the toluene solution of stannous isooctanoate or the dichloromethane solution of stannous isooctanoate added is 0.2-1% of the mass of mPEG; the mass ratio of mPEG to DL-lactide in the step (2) is 1: (0.5 to 1).

Preferably, the volume ratio of the reactants to the mixed solvent of ethyl acetate and n-heptane in the step (3) is 1: (1-2); and (4) drying at 35-45 ℃ for 25-35 hours in vacuum.

Preferably, in the step (5), disodium EDTA is added in the form of disodium EDTA aqueous solution, the concentration of the disodium EDTA aqueous solution is 0.0075-0.15 g/L, and the addition amount is 1-2% of the weight of disodium EDTA based on the weight of the crude product of the mPEG-b-PLC amphiphilic block copolymer; the step of dialyzing for 20-50 hours by using a dialysis bag specifically comprises the following steps: putting the solution to be dialyzed into a dialysis bag, putting a dialysis medium into the dialysis bag for dialysis for 20-50 hours, and replacing the dialysis medium every 5-7 hours; the dialysis medium is ultrapure water.

A docetaxel nano-formulation comprising docetaxel and the mPEG-b-PLC amphiphilic block copolymer prepared by the preparation method as set forth in any one of claims 1 to 4; the mass ratio of the docetaxel to the mPEG-b-PLC amphiphilic block copolymer is 1: 3-100.

In particular embodiments of the invention, the mass ratio of docetaxel to mPEG-b-PLC amphiphilic block copolymer may be, for example, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100, and the like.

A preparation method of the docetaxel nanometer preparation comprises the following steps:

s1, dissolving docetaxel in a first organic solvent to obtain a first solution;

s2, dissolving the mPEG-b-PLC amphiphilic block copolymer in a second organic solvent to obtain a second solution;

s3, dissolving the fatty acid in a third organic solvent to obtain a third solution;

s4, mixing the first solution, the second solution and the third solution, and removing the organic solvent to obtain a mixture;

s5, hydrating the mixture, and filtering the mixture by using a nano filter to obtain filtrate;

s6, freezing the filtrate to obtain docetaxel nano ice blocks;

s7, freeze-drying the docetaxel nanometer ice blocks to obtain docetaxel nanometer preparations;

wherein the mass ratio of the docetaxel to the mPEG-b-PLC amphiphilic block copolymer is as follows: 1: 3-100.

Preferably, the first organic solvent and the third organic solvent are absolute ethyl alcohol; the second organic solvent is acetonitrile; the fatty acid is selected from any one of lauric acid, palmitic acid, oleic acid and linoleic acid.

Preferably, step S4 is specifically: and stirring and mixing the first solution, the second solution and the third solution in a water bath at 60 ℃ for 30 minutes, and removing the organic solvent at the temperature of 60 ℃ under negative pressure to obtain a mixture of paclitaxel and the amphiphilic block copolymer of mPEG-b-PLC.

Preferably, step S5 is specifically: hydrating the mixture with distilled water, shaking and mixing for 15-30 minutes to obtain a mixed solution, and filtering the mixed solution with a 200-nanometer filter to obtain a filtrate.

Preferably, the temperature of freezing the filtrate in step S6 is-40 ℃; step S7 specifically includes: and drying the paclitaxel nano ice blocks for 48-72 hours by using a freeze dryer to obtain the docetaxel nano preparation.

The mPEG-b-PLC amphiphilic block copolymer prepared by the technical scheme of the invention has the advantage of low tin element content, and the docetaxel nano preparation has the advantages of low toxic and side effects, excellent anti-tumor effect, simple preparation process and low production cost.

Drawings

FIG. 1 shows the bioluminescence intensity of the animal model Day1 in the experimental examples of the present invention.

FIG. 2, Experimental example of the invention, bioluminescence 2D imaging of various animal models Day 1.

FIG. 3 shows the bioluminescence intensity of the animal model Day8 in each group according to the experimental example of the present invention.

FIG. 4, an experimental example of the present invention, groups of animal models, Day8, bioluminescence 2D imaging.

FIG. 5 shows the bioluminescence intensity of the animal models of the present invention, Day 15.

FIG. 6, an experimental example of the present invention, bioluminescence 2D imaging of various animal models Day 15.

FIG. 7 shows the bioluminescence intensity of the animal models of the present invention, Day 22.

FIG. 8 shows an experimental example of the present invention, and various animal models Day22 bioluminescence 2D imaging.

FIG. 9 shows comparison of increase times of bioluminescence intensity of each animal model group in the experimental example of the present invention.

FIG. 10 is a graph showing the comparison of the fold increase in body weight of animal models of each group in the experimental examples of the present invention.

Detailed Description

In order to make the skilled person better understand the present invention, the following examples of the present invention are clearly and specifically described, but not intended to limit the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.