CN110358063B - Preparation method of block copolymer mPEG-b-PLA - Google Patents

Preparation method of block copolymer mPEG-b-PLA Download PDF

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CN110358063B
CN110358063B CN201910319500.4A CN201910319500A CN110358063B CN 110358063 B CN110358063 B CN 110358063B CN 201910319500 A CN201910319500 A CN 201910319500A CN 110358063 B CN110358063 B CN 110358063B
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lactide
pla
block copolymer
polyethylene glycol
monomethyl ether
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CN110358063A (en
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唐键
胡霏
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Shenzhen Puluo Meikang Materials Co ltd
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    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/66—Polyesters containing oxygen in the form of ether groups
    • C08G63/664—Polyesters containing oxygen in the form of ether groups derived from hydroxy carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
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    • C08G63/82—Preparation processes characterised by the catalyst used
    • C08G63/823—Preparation processes characterised by the catalyst used for the preparation of polylactones or polylactides
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
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    • C08G63/90—Purification; Drying

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Abstract

The invention discloses a preparation method of a block copolymer mPEG-b-PLA, which comprises the following steps: pretreating raw materials of polyethylene glycol monomethyl ether and lactide; respectively dissolving polyethylene glycol monomethyl ether and lactide, adding amidine catalyst into the polyethylene glycol monomethyl ether, adding lactide under stirring, polymerizing for 10-180min under an anaerobic condition, and terminating the reaction by adopting a terminator to obtain a crude product; the crude product is dissolved by eluent and then separated by a chromatographic column to obtain a purified product. The invention provides a preparation method of an amphiphilic block copolymer with high yield, high purity and narrow molecular weight distribution. The preparation method is solution polymerization and organic catalysis, and can accurately control reaction conditions; purification can be performed using chromatography and centrifugation, respectively. The molecular weight dispersity of the obtained block copolymer can be controlled within 1.1.

Description

Preparation method of block copolymer mPEG-b-PLA
Technical Field
The invention belongs to the technical field of pharmaceutical chemistry, relates to a synthesis and purification method of a block copolymer, and particularly relates to a preparation method of the block copolymer mPEG-b-PLA.
Background
The block copolymer mPEG-b-PLA is a good carrier of therapeutic drugs, and can increase the solubility of insoluble drugs such as anticarcinogens, reduce the side effects of the drugs and the like. As the synthesis reaction of the block polymer is ring-opening polymerization, the prior art commonly uses bulk polymerization, but the bulk polymerization needs high temperature, the reaction time usually needs more than 12h, the polymerization condition is difficult to be stably controlled, the production efficiency is low, and the molecular weight of the product and the distribution of the molecular weight are difficult to be controlled.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of a diblock copolymer mPEG-b-PLA which has the advantages of high efficiency, reaction at normal temperature, short reaction time and capability of obtaining a product with narrow molecular weight distribution aiming at the defects of the prior art.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a preparation method of a block copolymer mPEG-b-PLA comprises the following steps:
(1) pretreatment of raw materials: dissolving polyethylene glycol monomethyl ether and lactide as raw materials in an organic solvent, removing water, and filtering for later use; recrystallizing the lactide, and drying for later use;
(2) synthesis of mPEG-b-PLA: weighing polyethylene glycol monomethyl ether and lactide, wherein the weight ratio of polyethylene glycol monomethyl ether: the weight ratio of lactide is (0.1-30) to 1; dissolving polyethylene glycol monomethyl ether and lactide with volatile organic solvents respectively to obtain a polyethylene glycol monomethyl ether solution and a lactide solution; then, adding amidine catalyst into polyethylene glycol monomethyl ether, adding lactide under stirring, polymerizing for 10-180min under an anaerobic condition, and terminating the reaction by adopting a terminator to obtain a crude product; wherein, the adding amount of the amidine catalyst is 0.1 to 3 percent of the total mole number of the reaction raw materials; the addition amount of the terminator is 0.1 to 3 percent of the total mole number of the reaction raw materials;
(3) and (3) column chromatography purification: dissolving the crude product with eluent, and separating by a chromatographic column to obtain a purified product, wherein the eluent is a volatile organic solvent. Preferably, the volatile organic solvent in step (3) is a lower chain alcohol or a substituted alkane or a mixture of the lower chain alcohol and the substituted alkane.
Further, in the preparation method of the block copolymer mPEG-b-PLA, preferably, after the step (3), the method further comprises the following steps:
(4) o/w/preparation of micelles: dissolving the purified product separated by column chromatography in a volatile organic solvent, preferably dissolving low-carbon-chain alkane, mixing with water, fully emulsifying, and stirring until the organic solvent is completely volatilized to obtain micelle;
(5) centrifuging: and centrifuging the micelle, taking supernatant and freeze-drying to obtain a final product.
Further, in the preparation method of the block copolymer mPEG-b-PLA, the volatile organic solvent is preferably selected from low carbon chain alkane, substituted alkane or alcohol, and the carbon chain length is C1-C10。
Further, in the preparation method of the block copolymer mPEG-b-PLA, preferably, in the step (2), the oxygen-free condition is under vacuum or in an inert gas.
Further, in the preparation method of the block copolymer mPEG-b-PLA, in the step (2), the terminating agent is preferably an organic acid. At least one of benzoic acid, glacial acetic acid, oxalic acid and succinic acid is preferred.
Further, in the preparation method of the block copolymer mPEG-b-PLA, the amidine catalyst is preferably TBD, DBN or DBU.
Further, in the preparation method of the block copolymer mPEG-b-PLA, in the step (3), the filler for column chromatography is sephadex with the granularity of 100-400 meshes.
Further, in the preparation method of the block copolymer mPEG-b-PLA, in the step (3), the filler for column chromatography is preferably glucose gel of G type or LH type, and preferably the filler for column chromatography is glucose gel of G25, G50, G100, LH20 or LH 60.
Further, in the preparation method of the block copolymer mPEG-b-PLA, preferably, in the step (3), the eluent is methanol, ethanol, dichloromethane, chloroform or a mixture of two or more of them.
The invention adopts a solution polymerization method, can complete the block polymerization reaction in a short time at normal temperature by the catalysis of amidine catalysts, and achieves the aim of accurately controlling the molecular weight of the product and the molecular weight distribution thereof by controlling the feeding ratio, the dosage of the catalyst and the adopted terminating agent and the reaction time and by the comprehensive effect of the above conditions.
After the reaction is finished, the two-block polymer product is purified by two methods of column chromatography and density centrifugation respectively. The molecules with different space volumes of column chromatography can separate small molecular weight compounds such as monomers, catalysts, terminating agents and the like in a system after reaction from polymers through the characteristic of different time in the filler, so that the aim of purification is fulfilled. The centrifugal method is based on the fact that particles with different particle sizes have different sedimentation rates, the amphiphilic polymer is easy to form micelles in water, the micelles are prepared by an o/w method, the amphiphilic polymer can form micelles with different particle sizes according to molecular weights, and finally the centrifugal method can separate the copolymers with different molecular weights to achieve purification.
Drawings
The invention will be further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a nuclear magnetic resonance spectrum of example 1 of the present invention;
FIG. 2 is a nuclear magnetic resonance spectrum of example 2 of the present invention;
FIG. 3 is a GPC chart of example 2 of the present invention;
FIG. 4 is a NMR spectrum of example 3 of the present invention;
FIG. 5 is a NMR spectrum of example 4 of the present invention;
FIG. 6 is a GPC chart of example 4 of the present invention;
FIG. 7 is a GPC chart after centrifugation in example 4 of the present invention;
FIG. 8 is a NMR spectrum of example 5 of the present invention;
FIG. 9 is a nuclear magnetic resonance spectrum of example 6 of the present invention;
FIG. 10 is an absorbance release curve for a sustained release experiment according to an embodiment of the present invention;
FIG. 11 is a nuclear magnetic resonance spectrum of a comparative example;
FIG. 12 is a GPC chart of the comparative example.
Detailed Description
For a more clear understanding of the technical features, objects and effects of the present invention, embodiments of the present invention will now be described in detail with reference to the accompanying drawings.
A block copolymer mPEG-b-PLA is a high molecular polymer comprising a hydrophilic segment and a hydrophobic segment, wherein the hydrophilic segment is polyethylene glycol monomethyl ether with a molecular weight of 300-20000, the hydrophobic segment is polylactic acid with a molecular weight of 200-15000, the hydrophobic segment is terminated by an organic acid, and the weight ratio of the hydrophilic segment to the hydrophobic segment is (0.1-30): 1. within the above molecular weight range, it may be arbitrarily selected, for example: mPEG1000-b-PLA9000, mPEG1000-b-PLA3000, mPEG1000-b-PLA1000, mPEG5000-b-PLA3000, mPEG5000-b-PLA5000, mPEG5000-b-PLA8500, mPEG1000-b-PLA10000, mPEG6000-b-PLA15000, mPEG6000-b-PLA200 and mPEG300-b-PLA 5000. The block copolymer mPEG-b-PLA was prepared by the following preparation method.
A preparation method of a block copolymer mPEG-b-PLA comprises the following steps:
(1) raw material treatment: weighing polyethylene glycol monomethyl ether and lactide, dissolving the polyethylene glycol monomethyl ether in a volatile organic solvent, removing water, and filtering for later use; recrystallizing the lactide, and drying for later use; wherein, polyethylene glycol monomethyl ether: the weight ratio of lactide is (0.1-30) to 1; the volatile organic solvent is low-carbon-chain alkane or substituted alkane, and the length of the carbon chain is C1-C10。
(2) Synthesis of mPEG-b-PLA: dissolving polyethylene glycol monomethyl ether and lactide with volatile organic solvent respectively to obtain polyethylene glycol monomethyl ether solution and lactide solution, wherein the volatile organic solvent in the step is preferably low-carbon-chain alkane or substituted alkane, and the length of the carbon chain is C1-C10. Adding amidine catalyst into the polyethylene glycol monomethyl ether solution, adding lactide solution under stirring, polymerizing for 10-180min under an anaerobic condition, and terminating the reaction by adopting a terminator to obtain a crude product; wherein, the adding amount of the amidine catalyst is 0.1 to 3 percent of the total mole number of the reaction raw materials; the terminator is organic acid, the addition amount of the terminator is 0.1-3% of the total mole number of the reaction raw materials, and the terminator is preferably at least one of benzoic acid, glacial acetic acid, oxalic acid and succinic acid. The amidine-based catalyst is preferably TBD, DBN or DBU. The anaerobic condition is preferably vacuum or inert gas, wherein the vacuum degree under vacuum is 0.08-0.1 MPa, and the inert gas is nitrogen or argon.
(3) And (3) column chromatography purification: dissolving the crude product with eluent, and separating with chromatographic column to obtain purified product, wherein the eluent is volatile organic solvent, preferably low carbon chain lower alcohols or substituted alkanes or mixture of low carbon chain alcohols and substituted alkanes, most preferably methanol, ethanol, dichloromethane, trichloromethane, tetrachloromethane or mixture of two or more thereof. The filler of the column chromatography is sephadex with the granularity of 100-400 meshes. The packing material of the column chromatography is glucose gel of G type or LH type. Most preferably, the packing for column chromatography is glucose gel of G25, G50, G100, LH20 or LH 60.
After purification, the following steps can be included:
(4) o/w/preparation of micelles: dissolving the purified product separated by column chromatography with volatile organic solvent, preferably low-carbon-chain alkane, mixing with water, emulsifying, and stirring until the organic solvent is completely volatilized to obtain micelle;
(5) centrifuging: and centrifuging the micelle, taking supernatant and freeze-drying to obtain a final product.
The following is a detailed description of specific examples:
example 1, a method for preparing a block copolymer mPEG-b-PLA (mPEG1000-b-PLA9000) comprising the steps of:
(1) raw material treatment: dissolving polyethylene glycol monomethyl ether (PEG1000) and lactide in dichloromethane, removing water with molecular sieve, and filtering; recrystallizing lactide with ethanol twice, and drying for later use;
(2) synthesis of mPEG-b-PLA: dissolving pretreated 1g of mPEG1000 in 50mL of dichloromethane, stirring at 500r/min to obtain an mPEG1000 solution, and adding 240 mu L of DBN; dissolving 9g of lactide in 50mL of dichloromethane to obtain a lactide solution, transferring the lactide solution into mPEG1000 solution, pumping out air, reacting for 180min under the vacuum degree of 0.1Mpa, adding 110 mu l of glacial acetic acid to stop the reaction, continuously stirring for 10min, and removing dichloromethane to obtain a crude product. Wherein, polyethylene glycol monomethyl ether: the weight ratio of lactide is 1:9, and the addition amount of amidine catalyst DBN is 3 percent of the total mole number of reaction raw materials; the amount of the terminator added was 3% of the total moles of the reaction raw materials.
(3) And (3) column chromatography purification: after 2g of the crude product have been dissolved with 10mL of eluent (dichloromethane), they are separated by column chromatography using Sephadex LH60 as filler and washed with 20 sample volumes of the above eluent. Collecting main peak fraction, drying to obtain white powder, and performing nuclear magnetic test.
The nuclear magnetic results are shown in FIG. 1: delta 1.8ppm as solvent peak, CH of mPEG2In the case of delta-3.6 ppm, CH of PLA3And CH at δ 1.6ppm and δ 5.1ppm, the integral ratio is 1.00: 4.07: 1.34, the copolymer structure is mPEG1000-b-PLA8848 and the polymerization degree is 98.3 percent calculated by the integral ratio of the characteristic peaks; column chromatographyAnd (3) purification: the yield was 93.5% and the purity was 99.5%.
Example 2, a method for preparing a block copolymer mPEG-b-PLA (mPEG1000-b-PLA3000) includes the steps of:
(1) raw material treatment: dissolving polyethylene glycol monomethyl ether (PEG1000) and lactide in dichloromethane, removing water with molecular sieve, and filtering; recrystallizing the lactide twice with ethanol, and drying for later use, wherein the weight ratio of polyethylene glycol monomethyl ether: the weight ratio of lactide is 1: 3.
(2) Synthesis of mPEG-b-PLA: dissolving pretreated 2g of mPEG1000 in 50mL of dichloromethane, stirring at 500r/min to obtain an mPEG1000 solution, and adding 130 mu g of TBD; dissolving 6g of lactide in 50mL of dichloromethane to obtain a lactide solution, transferring the lactide solution into the mPEG1000 solution, pumping out air, and reacting for 30min at the vacuum degree of 0.09 Mpa; the reaction was stopped by adding 89. mu.g of benzoic acid, stirring was continued for 10min and the crude product was obtained after removal of the dichloromethane. Wherein, polyethylene glycol monomethyl ether: the weight ratio of lactide is 1:3, and the addition amount of amidine catalyst TBD is 1.5 percent of the total mole number of the reaction raw materials; the amount of the terminator added was 1.5% of the total moles of the reaction raw materials.
(3) And (3) column chromatography purification: after 3g of the crude product have been dissolved with 10mL of eluent (ethanol: DCM ═ 2:1), separation is carried out by column chromatography on sephadex LH20, rinsing with 20 loading volumes of eluent. Collecting main peak fractions, drying to obtain a transparent paste, and performing nuclear magnetic test.
The nuclear magnetic results are shown in FIG. 2: copolymer Structure, CH of mPEG2In the case of delta-3.6 ppm, CH of PLA3And CH at δ 1.6ppm and δ 5.1ppm, the integral ratio is 1.00: 1.21: 0.38, the copolymer structure is mPEG1000-b-PLA2642 calculated by the integral ratio of characteristic peaks, and the polymerization degree is 88.1 percent; and (3) column chromatography purification: the yield is 94.4 percent, and the purity is 99.5 percent; the GPC results are shown in FIG. 3: the unimodal distribution, the dispersity is 1.07, which shows that the product only has one polymer with continuous molecular weight and the molecular weight distribution is narrow.
Example 3, a method for preparing a block copolymer mPEG-b-PLA (mPEG1000-b-PLA1000) comprising the steps of:
(1) raw material treatment: dissolving polyethylene glycol monomethyl ether (PEG1000) and lactide in dichloromethane, removing water with molecular sieve, and filtering; recrystallizing lactide with ethanol twice, and drying for use.
(2) Synthesis of mPEG-b-PLA: dissolving pretreated 5g of mPEG1000 in 50mL of dichloromethane to obtain mPEG1000 solution, stirring at 500r/min, and adding 60 mu l of DBU; dissolving 5g of lactide in 50mL of dichloromethane to obtain a lactide solution, transferring the obtained lactide solution into mPEG1000 solution, pumping out air, introducing nitrogen, and reacting for 40min under the protection of nitrogen; adding 36 mul oxalic acid to terminate the reaction, continuing to stir for 5min, and removing dichloromethane to obtain a crude product. Wherein, polyethylene glycol monomethyl ether: the weight ratio of lactide is 1:1, and the addition amount of amidine catalyst DBU is 1 percent of the total mole number of reaction raw materials; the amount of the terminator added was 1% of the total moles of the reaction raw materials.
(3) And (3) column chromatography purification: after 3G of the crude product have been dissolved with 10mL of eluent (methanol), they are separated by column chromatography using sephadex G50 as packing and washed with 20 sample volumes of eluent. Collecting main peak fractions, drying to obtain transparent colloidal liquid, and performing nuclear magnetic test.
The nuclear magnetic results are shown in FIG. 4: CH of mPEG2In the case of delta-3.6 ppm, CH of PLA3And CH at δ 1.6ppm and δ 5.1ppm, the integral ratio is 1.00: 0.42: 0.13, the copolymer structure is mPEG1000-b-PLA913 and the polymerization degree is 91.30 percent according to the integral ratio of the characteristic peaks; and (3) column chromatography purification: the yield is 92.5 percent, and the purity is 99.5 percent.
Example 4, a method for preparing a block copolymer mPEG-b-PLA (mPEG5000-b-PLA3000) comprising the steps of:
(1) raw material treatment: dissolving polyethylene glycol monomethyl ether (PEG5000) and lactide in dichloromethane, removing water with molecular sieve, and filtering; recrystallizing lactide with ethanol twice, and drying for use.
(2) Synthesis of mPEG-b-PLA:
dissolving pretreated 5g of mPEG5000 in 50mL of dichloromethane, stirring at 500r/min to obtain an mPEG5000 solution, and adding 70 mu g of TBD; dissolving 3g of lactide in 50mL of dichloromethane to obtain a lactide solution, transferring the lactide solution into the mPEG5000 solution, pumping out air, and reacting for 60 min; adding 35 mul oxalic acid to terminate the reaction, continuing stirring for 10min, and removing dichloromethane to obtain a crude product. Wherein, polyethylene glycol monomethyl ether: the weight ratio of the lactide is 5:3, and the addition amount of the amidine catalyst TBD is 1.5 percent of the total mole number of the reaction raw materials; the amount of the terminator added was 1.5% of the total moles of the reaction raw materials.
(3) And (3) column chromatography purification: after 3G of the crude product have been dissolved with 10mL of eluent (ethanol), they are separated by column chromatography using sephadex G100 as packing and washed with 20 sample volumes of eluent ethanol. Collecting main peak fraction, drying to obtain white powder, and performing nuclear magnetic test.
Preparing the purified sample into micelle by o/w method, centrifuging at 3000r/min for 5min, taking supernatant, and freeze-drying to obtain the final purified product.
The nuclear magnetic results are shown in FIG. 5: CH of mPEG2In the case of delta-3.6 ppm, CH of PLA3And CH at 1.6ppm and 5.1ppm, the integral ratio is 1.00:0.28:0.09, the copolymer structure is mPEG5000-b-PLA2940 calculated by characteristic peak ratio, and the polymerization degree is 98%; and (3) column chromatography purification: the yield is 95.5 percent; the GPC results are shown in FIG. 6, showing a bimodal distribution with a main peak dispersity of 1.03; GPC results after centrifugation are shown in FIG. 7, which shows a monomodal distribution, a degree of dispersion of 1.04, a yield of 85.5%, and a purity of 99.5%. It can thus be seen that: after centrifugal treatment, the double peak is changed into a single peak, the by-product is separated, and the dispersity is 1.04, which shows that the product only remains a polymer with continuous molecular weight and has narrow dispersion after centrifugal separation, so that the product which can not be further separated by only adopting column chromatography can be prepared into micelle for centrifugal separation.
Example 5, a method for preparing a block copolymer mPEG-b-PLA (mPEG5000-b-PLA5000), comprising the steps of:
(1) raw material treatment: dissolving polyethylene glycol monomethyl ether (mPEG5000) and lactide as raw materials in dichloromethane, removing water by using a molecular sieve, and filtering for later use; recrystallizing lactide with ethanol twice, and drying for use.
(2) Synthesis of mPEG-b-PLA: dissolving pretreated 5g of mPEG5000 in 50mL of dichloromethane, stirring at 500r/min to obtain an mPEG5000 solution, and adding 140 mu g of TBD; dissolving 5g of lactide with 50mL of dichloromethane to obtain a lactide solution, transferring the lactide solution into mPEG5000 solution, pumping out air, controlling the vacuum degree to be 0.09Mpa, and reacting for 70 min; adding 70 mul oxalic acid to terminate the reaction, continuing to stir for 5min, and removing dichloromethane to obtain a crude product. Wherein, polyethylene glycol monomethyl ether: the weight ratio of lactide is 1:1, and the addition amount of amidine catalyst TBD is 2% of the total mole number of the reaction raw materials; the amount of the terminator added was 2% of the total moles of the reaction raw materials.
(3) And (3) column chromatography purification: after 3g of the crude product have been dissolved with 10mL of eluent (ethanol: DCM: 2:1 by volume), they are separated by column chromatography on sephadex LH60, and washed with 20 loading volumes of eluent. Collecting the main peak fraction, drying and performing nuclear magnetic test.
Preparing the purified sample into micelle by o/w method, centrifuging at 2500r/min for 5min, and drying the supernatant to obtain the final purified product.
The nuclear magnetic results are shown in FIG. 8: CH of mPEG2In the case of delta-3.6 ppm, CH of PLA3And CH at δ 1.6ppm and δ 5.1ppm with an integral ratio of 1.00:0.41:0.13, copolymer structure mPEG5000-b-PLA4476 calculated from the characteristic peak integral ratio, degree of polymerization 89.5%; and (3) column chromatography purification: the yield is 95.8%; GPC results show that the distribution is bimodal, and the dispersity of main peaks is 1.05; GPC results after centrifugation showed unimodal distribution, dispersion 1.05, yield 84.5%, purity 99.5%.
Example 6, a method for preparing a block copolymer mPEG-b-PLA (mPEG5000-b-PLA8500) comprising the steps of:
(1) raw material treatment: dissolving polyethylene glycol monomethyl ether (PEG5000) and lactide in dichloromethane, removing water with molecular sieve, and filtering; recrystallizing lactide with ethanol twice, and drying for use.
(2) Synthesis of mPEG-b-PLA: dissolving pretreated 5g of mPEG3000 in 50mL of dichloromethane, stirring at 500r/min to obtain an mPEG3000 solution, and adding 220 mu g of TBD; dissolving 8.5g lactide with 50mL dichloromethane to obtain lactide solution, transferring the lactide solution into mPEG3000 solution, pumping out air, controlling the vacuum degree to be 0.08Mpa, and reacting for 40 min; adding 130 mul succinic acid to terminate the reaction, continuing stirring for 5min, and removing dichloromethane to obtain a crude product. Wherein, polyethylene glycol monomethyl ether: the weight ratio of lactide is 1:1, and the addition amount of amidine catalyst is 2 percent of the total mole number of reaction raw materials; the amount of the terminator added was 2% of the total moles of the reaction raw materials.
(3) And (3) column chromatography purification: after 3g of the crude product have been dissolved in 10mL of eluent (dichloromethane), the Sephadex LH60 is separated by column chromatography, rinsing with 20 loading volumes of eluent. Collecting the main peak fraction, drying and performing nuclear magnetic test.
Preparing the purified sample into micelle by an o/w method, centrifuging for 5min at 2000r/min, and taking the supernatant to be dried to obtain the final purified product.
The nuclear magnetic results are shown in FIG. 9: CH of mPEG2In the case of delta-3.6 ppm, CH of PLA3And CH at δ 1.6ppm and δ 5.1ppm, with an integral ratio of 1.00: 0.74: 0.24, the copolymer structure calculated by the characteristic peak ratio is mPEG5000-b-PLA8043, and the polymerization degree is 94.6%; GPC results show that the distribution is bimodal, and the dispersity of main peaks is 1.04; GPC results after centrifugation showed unimodal distribution, divergence 1.05, yield 87.5%, and purity 99.9%.
Example 7, a method for preparing a block copolymer mPEG-b-PLA (mPEG1000-b-PLA10000) comprising the steps of:
(1) raw material treatment: dissolving polyethylene glycol monomethyl ether (PEG1000) and lactide in acetone, removing water with molecular sieve, and filtering; recrystallizing lactide with acetone twice, and drying for use.
(2) Synthesis of mPEG-b-PLA: dissolving pretreated 1g of mPEG1000 in 50mL of acetone, stirring at 500r/min to obtain an mPEG1000 solution, and adding 420 mu g of TBD; dissolving 10g of lactide in 50ml of acetone to obtain a lactide solution, transferring the obtained lactide solution into mPEG1000 solution, pumping out air, filling nitrogen, and reacting for 40 min; adding 250 mul of benzoic acid and oxalic acid (volume ratio is 10:1) to terminate the reaction, continuing stirring for 10min, and removing acetone to obtain a crude product. Wherein, polyethylene glycol monomethyl ether: the weight ratio of lactide is 1:10, and the addition amount of amidine catalyst is 3 percent of the total mole number of reaction raw materials; the amount of the terminator added was 3% of the total moles of the reaction raw materials.
(3) And (3) column chromatography purification: after 3g of the crude product was dissolved in 10mL of eluent (chloroform), column chromatography separation was performed using Sephadex LH60 as a filler, and 20 loading volumes of eluent were used for washing, collecting the main peak fraction, and drying to obtain a white powder for nuclear magnetic testing.
The crude nuclear magnetic results show that: CH of mPEG2In the case of delta-3.6 ppm, CH of PLA3And CH at δ 1.6ppm and δ 5.1ppm, an integral ratio of 1.00: 4.03:1.43, and the polymerization degree is 94.0 percent calculated by the integral ratio of the characteristic peaks; and (3) column chromatography purification: the yield was 93.9% and the purity was 99.5%.
Example 8, a method for preparing a block copolymer mPEG-b-PLA (mPEG6000-b-PLA200), comprising the steps of:
(1) raw material treatment: dissolving polyethylene glycol monomethyl ether (PEG6000) and lactide in chloroform, removing water with molecular sieve, and filtering; recrystallizing lactide with ethanol twice, and drying for use.
(2) Synthesis of mPEG-b-PLA: dissolving pretreated 9g of mPEG6000 in 50mL of trichloromethane, stirring at 500r/min to obtain an mPEG6000 solution, and adding 4.2 mu g of TBD; dissolving 0.3g of lactide with 50mL of trichloromethane to obtain a lactide solution, transferring the obtained lactide solution into the mPEG6000 solution, pumping out air, and reacting for 10 min; adding 2 mul oxalic acid to terminate the reaction, continuing to stir for 5min, and removing the trichloromethane to obtain a crude product. Wherein, polyethylene glycol monomethyl ether: the weight ratio of lactide is 30:1, and the addition amount of amidine catalyst is 0.1 percent of the total mole number of the reaction raw materials; the amount of the terminator added was 0.1% of the total moles of the reaction raw materials.
(3) And (3) column chromatography purification: after 3G of the crude product have been dissolved in 10mL of eluent (ethanol), the Sephadex G25 is separated by column chromatography, and 20 loading volumes of eluent are washed. Collecting the main peak fraction, drying and performing nuclear magnetic test.
The nuclear magnetic results show that: CH of mPEG2Delta is 3.6ppm,CH of PLA3And CH in δ 1.6ppm and δ 5.1ppm, base ratio 71.42: 1.00: 0.34 the copolymer structure is mPEG6000-b-PLA190 and the polymerization degree is 95 percent calculated by the characteristic peak ratio; and (3) column chromatography purification: the yield is 90.5 percent and the purity is 99.5 percent.
The block copolymer mPEG-b-PLA of the invention is used as a functional experiment of a drug sustained-release material:
paclitaxel sustained Release test
1) Standard curve
Preparing paclitaxel into 40 μ g/ml, 20 μ g/ml, 10 μ g/ml, 5 μ g/ml, 2.5 μ g/ml and 1 μ g/ml solution with absolute ethanol, testing ultraviolet absorbance, obtaining a standard curve, and obtaining a regression equation: y is 0.0232x
2) Paclitaxel micelle preparation
Dissolving 200mg mPEG5000-b-PLA3000 and 20mg paclitaxel in 2ml dichloromethane, adding into 20ml deionized water, performing ultrasonic treatment with 140w for 15min in ice-water bath, and stirring at room temperature of 200r/min until the organic solvent is completely volatilized to obtain the paclitaxel micelle.
3) Sustained release experiments
Freezing and centrifuging the micelle at a high speed of 15000rpm for 10min at 4 ℃, washing and centrifuging again after precipitation, repeatedly washing twice, combining all supernatants, measuring that the encapsulation rate is 90.1%, dispersing the precipitate obtained by centrifugation in a 3500D dialysis bag by using 10ml of PBS buffer solution, dialyzing the dialysis bag in 20ml of PBS buffer solution, placing the dialysis bag in a constant temperature oscillator at 37 ℃ and 180r/min, respectively sampling 10ml of dissolution liquid at 2h, 4h, 6h, 22h, 31h, 48h, 57h, 78h and 95, supplementing 10ml of PBS buffer solution, respectively extracting the samples by using dichloromethane, drying the dichloromethane, dissolving the dichloromethane by using 4ml of ethanol, testing absorbance to prepare a release curve, wherein the release curve is shown in figure 10. The slow release curve shows that mPEG-b-PLA can be used as a drug slow release material.
Comparative experiment: respectively weighing 5g mPEG5000 and 8.5g lactide, putting the weighed materials into a100 ml dry round-bottom flask together, heating the materials to 140 ℃ under a vacuum condition, and carrying out magnetic stirring reaction for 8 hours to obtain light yellow clear transparent viscous liquid; dissolving with dichloromethane, dripping into anhydrous cold diethyl ether for precipitation, standing overnight, filtering to obtain precipitate, and vacuum drying at room temperature to obtain the product.
The designed molecular weight structure is mPEG5000-b-PLA8500, the nuclear magnetism result is shown in figure 11, the actual molecular weight structure is mPEG5000-b-PLA6300, the polymerization degree is 74.1%, the GPC result is shown in figure 12, the bimodal distribution is formed, and the main peak dispersity is 1.15.
After comparison of the comparative experiment with example 6, it can be seen that: the preparation method of the invention is carried out at normal temperature, the reaction time is far shorter than that of the prior art, the preparation method has the effect of accurately controlling the molecular weight of the product and the molecular weight distribution thereof, and the preparation method has higher polymerization degree which is up to more than 90 percent.
In addition, as can be seen from example 4, in the GPC results before and after the centrifugation treatment, the bimodal distribution shown in fig. 12 was changed to the mono distribution shown in fig. 7, and the by-products were completely separated. Experiments prove that: in the purification method, the chromatographic column can separate small molecules such as monomers, catalysts, terminating agents and the like; the centrifugal treatment can separate the polymer with the molecular weight close to that of the main product generated by the side reaction. Therefore, the invention can completely separate the by-products through two-step purification, and can efficiently obtain the diblock copolymer mPEG-b-PLA of the product with narrow molecular weight distribution.

Claims (6)

1. Block copolymer mPEG-b-a process for the preparation of PLA, characterized in that it comprises the following steps:
(1) pretreatment of raw materials: dissolving polyethylene glycol monomethyl ether and lactide as raw materials in an organic solvent, removing water, and filtering for later use; recrystallizing the lactide, and drying for later use;
（2）mPEG-b-synthesis of PLA: weighing polyethylene glycol monomethyl ether and lactide, wherein the weight ratio of polyethylene glycol monomethyl ether: the weight ratio of lactide is (0.1-30) to 1; dissolving polyethylene glycol monomethyl ether and lactide with volatile organic solvents respectively to obtain a polyethylene glycol monomethyl ether solution and a lactide solution; adding amidine catalyst into polyethylene glycol monomethyl ether solution, adding lactide solution under stirring, polymerizing for 10-180min under an anaerobic condition, and terminating the reaction by adopting a terminator to obtain a crude product; wherein, the amidine catalyst is added as a reaction raw material0.1% -3% of the total mole number; the addition amount of the terminating agent is 0.1-3% of the total mole number of the reaction raw materials; the volatile organic solvent is selected from low carbon chain alkane, substituted alkane or alcohol, and the carbon chain length is C1-C10(ii) a The terminator is organic acid; the amidine catalyst is TBD, DBN or DBU;
(3) and (3) column chromatography purification: dissolving the crude product with eluent, and separating with chromatographic column to obtain purified product, wherein the eluent is volatile organic solvent selected from low carbon chain alkane, substituted alkane or alcohol with carbon chain length of C1-C10。
2. The method for preparing the block copolymer mPEG-b-PLA according to claim 1, further comprising the following steps after the step (3):
(4) o/w/preparation of micelles: dissolving the purified product separated by column chromatography in a volatile organic solvent, mixing with water, fully emulsifying, and stirring until the volatile organic solvent is completely volatilized to obtain micelle;
(5) centrifuging: and centrifuging the micelle, taking supernatant and freeze-drying to obtain a final product.
3. The method for preparing the block copolymer mPEG-b-PLA as claimed in claim 1 or 2, wherein the oxygen-free condition in the step (2) is vacuum or inert gas.
4. The method for preparing the block copolymer mPEG-b-PLA according to the claim 1 or 2, wherein in the step (3), the filler for column chromatography is sephadex with the particle size of 100-400 meshes.
5. The method for preparing the block copolymer mPEG-b-PLA as claimed in claim 1 or 2, wherein the filler for column chromatography in the step (3) is glucose gel of G type or LH type.
6. The method for preparing the block copolymer mPEG-b-PLA as claimed in claim 1 or 2, wherein the eluent is methanol, ethanol, dichloromethane, chloroform or a mixture of two or more thereof in the step (3).
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CN104761710A (en) * 2014-02-14 2015-07-08 苏州海特比奥生物技术有限公司 Methoxypolyethylene glycol-lactic acid block copolymer and preparation method thereof
CN106939075A (en) * 2015-12-31 2017-07-11 银谷制药有限责任公司 A kind of hydrophilic polyester and its block copolymer

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Publication number Priority date Publication date Assignee Title
CN104761710A (en) * 2014-02-14 2015-07-08 苏州海特比奥生物技术有限公司 Methoxypolyethylene glycol-lactic acid block copolymer and preparation method thereof
CN106939075A (en) * 2015-12-31 2017-07-11 银谷制药有限责任公司 A kind of hydrophilic polyester and its block copolymer

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