阅读说明：本技术 一种具有ros响应的两亲性聚合物自组装胶束的制备方法 (Preparation method of amphiphilic polymer self-assembled micelle with ROS response ) 是由 代凤英 李新坡 胡浩东 张博 王少腾 于 2021-10-09 设计创作，主要内容包括：本发明提供了一种具有ROS响应的两亲性聚合物自组装胶束的制备方法。本发明提供的聚合物首先通过丙烯酰氯对分子量为2000的葡聚糖进行改性得到AC-Dex,再通过开环反应(ROP)制备一定分子量的聚乳酸大分子链转移剂(PLLA-CTA),并将其与AC-Dex进行可逆加成-断裂链转移聚合(RAFT)形成两亲性聚合物。该聚合物在水中可分别自组装成包载柠檬酸、维生素c、没食子酸的胶束,除具有良好的生物相容性和生物可降解性外,聚乳酸还具有良好的氧化应激敏感性,遇到H-(2)O-(2)会发生断裂,聚合物裂解后释放出柠檬酸、维生素c、没食子酸。所以该纳米载体具有很好的氧化应激响应药物释放的特性及潜在的阿尔茨海默症治疗应用前景。(The inventionProvides a preparation method of an amphiphilic polymer self-assembled micelle with ROS response. The polymer provided by the invention is prepared by modifying glucan with the molecular weight of 2000 by acryloyl chloride to obtain AC-Dex, preparing a polylactic acid macromolecular chain transfer agent (PLLA-CTA) with a certain molecular weight by a ring opening Reaction (ROP), and performing reversible addition-fragmentation chain transfer polymerization (RAFT) on the polylactic acid macromolecular chain transfer agent and the AC-Dex to form an amphiphilic polymer. The polymer can be respectively self-assembled into micelles for encapsulating citric acid, vitamin c and gallic acid in water, besides good biocompatibility and biodegradability, the polylactic acid also has good oxidative stress sensitivity, and when encountering H 2 O 2 Can be broken, and the polymer is cracked to release citric acid, vitamin c and gallic acid. Therefore, the nano-carrier has good oxidative stress response drug release characteristics and potential application prospects in Alzheimer disease treatment.)

1. The invention discloses a preparation method of an amphiphilic polymer self-assembly micelle with ROS response, which comprises the steps of firstly modifying glucan with the molecular weight of 2000 by acryloyl chloride to obtain AC-Dex, then preparing a polylactic acid macromolecular chain transfer agent (PLLA-CTA) with a certain molecular weight by a ring opening Reaction (ROP), and carrying out reversible addition-fragmentation chain transfer polymerization (RAFT) on the polylactic acid macromolecular chain transfer agent and the AC-Dex to form the PLLA-g-Dex amphiphilic polymer. The polymer can be respectively self-assembled into micelles for loading citric acid, vitamin c and gallic acid in water.

2. The method of preparing the ROS-responsive amphiphilic polymer self-assembled micelle of claim 1, comprising the steps of:

(1) l-lactide is polymerized into a poly-L-lactic acid macromolecular chain transfer agent (PLLA-CTA) by a Ring Opening Polymerization (ROP) method.

(2) Dissolving PLLA-CTA obtained in step (1) in anhydrous DMSO, adding acryloyl chloride modified dextran (AC-Dex) and initiator (AIBN), heating to 70 ℃ for reaction for 12h after three freeze-thaw cycles under nitrogen, and forming polymer by reversible addition-fragmentation chain transfer polymerization

(3) Dialyzing the reaction mixture obtained in the step (2) with deionized water for 24-48 hours, and then freeze-drying to obtain the amphiphilic polymer of polylactic acid grafted glucan.

(4) Preparing the amphiphilic polymer self-assembled drug-loaded micelle of polylactic acid grafted glucan, dissolving the amphiphilic polymer of polylactic acid grafted glucan with citric acid, vitamin c and gallic acid in DMSO respectively, then dropwise adding 5mL of water into the mixed solution stirred at high speed for self-assembling for 10-40 minutes, then dialyzing for 24-48 hours with deionized water, filtering with a filter membrane to obtain a micelle-containing mixed solution, and freeze-drying to obtain the amphiphilic polymer self-assembled drug-loaded micelle of polylactic acid grafted glucan (PLLA-g-Dex).

3. The method for preparing the ROS-responsive amphiphilic polymer self-assembled micelle according to claim 2, wherein the mass ratio of the ROS-responsive amphiphilic polymer self-assembled micelle to the ROS-responsive amphiphilic polymer self-assembled micelle is PLLA-CTA: AC-Dex ═ 1: 2-1: 5.

4. The method for preparing the ROS-responsive amphiphilic polymer self-assembled micelle according to claim 3, wherein the mass ratio of PLLA-g-Dex to citric acid is 2: 1-10: 1; PLLA-g-Dex, vitamin c 2: 1-10: 1; PLLA-g-Dex and gallic acid are 2: 1-10: 1.

Technical Field

The invention relates to an ROS-responsive polylactic acid grafted glucan polymer drug-loaded micelle and a preparation method thereof, relates to the field of pharmaceutical preparations, targeted therapy and the like, and particularly relates to a drug carrier, the micelle and a preparation method and application thereof.

Background

Alzheimer's Disease (AD) is a neurodegenerative disease in which patients have symptoms such as memory impairment, dysfunction, agnosia, loss of visual spatial ability, and neuronal loss. To date, the causative pathogenic mechanism of AD has not been found, and it can only be temporarily slowed down by clinical drugs. As society develops, more and more people become infested with AD, and as life expectancy increases, AD will become more prevalent. According to survey data, the total population of people with AD and other forms of dementia worldwide reaches about 5000 million people, most of which are elderly people aged 65 years and older, and it is expected that the number of AD diseases will double by 2030. Therefore, how to relieve and treat AD becomes a great problem in clinical treatment.

The interaction of oxidative stress with inflammation plays an important role in the pathogenesis of a variety of chronic diseases. Chronic inflammatory reactions persist, severely hampering tissue repair and promoting degenerative changes (e.g. AD). The AD drugs currently available on the market are mainly divided into two categories: N-methyl-D-aspartate (NMDA) receptor blockers and acetylcholinesterase inhibitors (AchE I), but have significant drawbacks for the treatment of severe AD patients. Through research, oxidative stress molecules are found to be related to the early pathogenic cause of AD, and although any AD antioxidant therapy which is not approved by the FDA at present, the antioxidant experimental therapy obtains remarkable scientific research results in animal models of AD, which provides a new idea for slowing or treating AD, and the method has low side effect which is expected.

According to the relevant literature, the nano-drug compounds currently used in clinical treatment mainly include: polymer-based nano-carriers, lipid-based nano-carriers, drug conjugate nano-carriers and the like. Therefore, the development of safer and more effective nanoparticles for treating AD diseases has become the focus of research of researchers. Most of the nano-drug carriers developed have some defects or side effects. For example, the drug carrier does not have a specific targeting and site-specific function, and can not be released to a lesion part to reduce the treatment effect; poor biocompatibility, poor in vivo circulation properties, and the like. Therefore, the design of intelligent drug controlled release drug carriers is at hand.

On the basis, the research designs an amphiphilic polymer self-assembly micelle with ROS responsiveness, the micelle is sensitive to oxidative stress, the polylactic acid grafted glucan amphiphilic polymer self-assembly micelle with oxidative stress Response (ROS) is prepared, and meanwhile, citric acid, vitamin c and gallic acid are entrapped. Dextran is one of a few macromonomers with good biocompatibility, has excellent biocompatibility and has no side effect on human bodies. Polylactic acid and its derivatives are sensitive to functional groups and are sensitive to oxidative stress (H)₂O₂) The polylactic acid can be hydrolyzed under the environment to achieve the purpose of separating hydrophilic and hydrophobic components, so that the entrapped citric acid, vitamin c and gallic acid are released.

Disclosure of Invention

The invention relates to a ROS-responsive polylactic acid grafted glucan amphiphilic polymer drug-loaded micelle and a preparation method thereof.

The ROS-responsive amphiphilic polymer self-assembled micelle provided by the invention is a micelle which takes macromolecular glucan as a hydrophilic segment and takes polylactic acid as a hydrophobic segment, and carries citric acid, vitamin c and gallic acid through self-assembly in water.

The amphiphilic polymer of the polylactic acid grafted glucan has a structure shown in a formula I:

the technical scheme of the invention is as follows:

synthesis of an ROS-responsive polylactic acid grafted dextran amphiphilic polymer:

the preparation method of the polylactic acid grafted glucan amphiphilic polymer comprises the following steps:

1. a method for preparing ROS responsive amphiphilic polymer self-assembled micelle. The polymer provided by the invention is prepared by firstly carrying out ring-opening reaction on L-lactide by using RAFT reagent (BSTSE) to obtain a levorotatory polylactic acid macromolecular chain transfer agent (PLLA-CTA), and then carrying out reversible addition-fragmentation chain transfer polymerization on the PLLA-CTA and acryloyl chloride modified glucan to form the polymer. The polymer is self-assembled into micelles for encapsulating citric acid, vitamin c and gallic acid in water.

2. The method of preparing the ROS-responsive amphiphilic polymer self-assembled micelle of claim 1, comprising the steps of:

(1) l-lactide is polymerized into a poly-L-lactic acid macromolecular chain transfer agent (PLLA-CTA) by a Ring Opening Polymerization (ROP) method.

(2) Dissolving PLLA-CTA obtained in step (1) in anhydrous DMSO, adding acryloyl chloride modified dextran (AC-Dex) and initiator (AIBN), heating to 70 ℃ for reaction for 12h after three freeze-thaw cycles under nitrogen, and forming polymer by reversible addition-fragmentation chain transfer polymerization

(3) Dialyzing the reaction mixture obtained in the step (2) with deionized water for 24-48 hours, and then freeze-drying to obtain the amphiphilic polymer of polylactic acid grafted glucan.

(4) Preparing the amphiphilic polymer self-assembled drug-loaded micelle of polylactic acid grafted glucan, dissolving the amphiphilic polymer of polylactic acid grafted glucan with citric acid, vitamin c and gallic acid in DMSO respectively, then dropwise adding 5mL of water into the mixed solution stirred at high speed for self-assembling for 10-40 minutes, then dialyzing for 24-48 hours with deionized water, filtering with a filter membrane to obtain a micelle-containing mixed solution, and freeze-drying to obtain the amphiphilic polymer self-assembled drug-loaded micelle of polylactic acid grafted glucan (PLLA-g-Dex).

3. The method for preparing the ROS-responsive amphiphilic polymer self-assembled micelle according to claim 2, wherein the mass ratio of the ROS-responsive amphiphilic polymer self-assembled micelle to the ROS-responsive amphiphilic polymer self-assembled micelle is PLLA-CTA: AC-Dex ═ 1: 2-1: 5.

4. The method for preparing the ROS-responsive amphiphilic polymer self-assembled micelle according to claim 3, wherein the mass ratio of PLLA-g-Dex to citric acid is 2: 1-10: 1; PLLA-g-Dex, vitamin c 2: 1-10: 1; PLLA-g-Dex and gallic acid are 2: 1-10: 1.

Description of the drawings:

FIG. 1 is a NMR chart of PLLA-g-Dex;

FIG. 2 is a transmission electron micrograph of a PLLA-g-Dex self-assembled polymer

The specific implementation method comprises the following steps:

example 1:

1. l-lactide (34.5mmol) and purified toluene (15mL) were added separately to a round-bottomed flask, the mixture was sufficiently stirred to dissolve L-lactide, and a chain transfer agent (BSTSE, 0.62mmol) and Sn (Oct) were added to the solution₂(200. mu.L) and a rotor, performing three freeze-thaw cycles under nitrogen atmosphere to remove oxygen in the reaction tube, performing oil bath reaction at 110 ℃ for 12h, finally terminating the reaction by liquid nitrogen quenching, precipitating the reacted solution with glacial methanol three times, drying in vacuum at 40 ℃ overnight to obtain a white powdery product, namely PLLA-CTA, sealing and storing at 4 ℃ for the next reaction.

2. Dextran with a molecular weight of 2000 (Dex, 0.1mmol) was dissolved in DMSO (250mL) in a round-bottomed flask, and a mixed solution of DMSO/DMF (5mL/2mL) and 300. mu.L of triethylamine were added thereto. The round bottom flask was then placed in an ice-water bath, and then 150. mu.L of acryloyl chloride was dissolved in 2mL of DMF in an isopiestic dropping funnel and added dropwise to the round bottom flask at a rate of one drop per three seconds with stirring until the addition was complete. After a subsequent reaction time of 12h at room temperature. The solution was removed and dialyzed against dialysis bag (Mw ═ 1,000Da) for 72h, and lyophilized to obtain a white powder sample.

3. PLLA-CTA (300mg) and AC-Dex (1.0g) were added to the reaction tube, and finally AIBN (2.8mg) was added thereto under N₂After three freeze-thaw cycles under ambient conditions, the reaction was stopped by exposure to air at 70 ℃ for 12 hours, followed by removal. Dialyzing with dialysis bag (Mw ═ 5,000Da) for 72h, taking out the dialyzate, lyophilizing to obtain white powder sample, sealing, and storing at 4 deg.C.

4. Dissolving 20mg of PLLA-g-Dex and 5mg of citric acid in 2mL of DMSO (dimethylsulfoxide), then dropwise adding 5mL of deionized water into the mixed solution stirred at a high speed for self-assembly for 12h, then dialyzing with the deionized water for 24 h to obtain a polymer mixed solution, and freeze-drying to obtain the PLLA-g-Dex polymer self-assembly drug-loaded micelle.

Example 2:

1. l-lactide (30.0mmol) and purified toluene (13mL) were added to a round-bottomed flask, and stirred sufficiently to dissolve L-lactide sufficiently, and a chain transfer agent (BSTSE, 0.58mmol) and Sn (Oct) were added to the solution₂(200. mu.L) and a rotor, performing three freeze-thaw cycles under nitrogen atmosphere to remove oxygen in the reaction tube, performing oil bath reaction at 110 ℃ for 12h, finally terminating the reaction by liquid nitrogen quenching, precipitating the reacted solution with glacial methanol three times, drying in vacuum at 40 ℃ overnight to obtain a white powdery product, namely PLLA-CTA, sealing and storing at 4 ℃ for the next reaction.

2. Dextran with a molecular weight of 2000 (Dex, 0.2mmol) was dissolved in DMSO (250mL) in a round-bottomed flask, and a mixed solution of DMSO/DMF (5mL/2mL) and 600. mu.L of triethylamine were added thereto. The round bottom flask was then placed in an ice-water bath, and then 300 μ L of acryloyl chloride was dissolved in 2mL of DMF in an isopiestic dropping funnel and added dropwise to the round bottom flask at a rate of one drop per three seconds with stirring until the addition was complete. After a subsequent reaction time of 12h at room temperature. The solution was removed and dialyzed against dialysis bag (Mw ═ 1,000Da) for 72h, and lyophilized to obtain a white powder sample.

3. PLLA-CTA (450mg) and AC-Dex (1.0g) were added to the reaction tube, and finally AIBN (3.0mg) was added thereto under N₂After three freeze-thaw cycles under ambient conditions, the reaction was stopped by exposure to air at 70 ℃ for 12 hours, followed by removal. Dialyzing with dialysis bag (Mw ═ 5,000Da) for 72h, taking out the dialyzate, lyophilizing to obtain white powder sample, sealing, and storing at 4 deg.C.

4. Dissolving 20mg of PLLA-g-Dex and 5mg of vitamin c in 2mL of DMSO, dropwise adding 5mL of deionized water into the mixed solution which is stirred at a high speed for self-assembly for 12h, dialyzing with the deionized water for 24 h to obtain a polymer mixed solution, and freeze-drying to obtain the PLLA-g-Dex polymer self-assembly drug-loaded micelle.

Example 3:

1. l-lactide (32.5.0mmol) and purified toluene (13mL) were added separately to a round-bottomed flask, the mixture was stirred well to dissolve L-lactide sufficiently, and a chain transfer agent (BSTSE, 0.60mmol) and Sn (Oct) were added to the solution₂(200. mu.L) and a rotor, performing three freeze-thaw cycles under nitrogen atmosphere to remove oxygen in the reaction tube, performing oil bath reaction at 110 ℃ for 12h, finally terminating the reaction by liquid nitrogen quenching, precipitating the reacted solution with glacial methanol three times, drying in vacuum at 40 ℃ overnight to obtain a white powdery product, namely PLLA-CTA, sealing and storing at 4 ℃ for the next reaction.

2. Dextran with a molecular weight of 2000 (Dex, 0.5mmol) was dissolved in DMSO (250mL) in a round-bottomed flask, and a mixed solution of DMSO/DMF (7mL/3mL) and 1.5mL triethylamine were added thereto. The round bottom flask was then placed in an ice-water bath, followed by 750 μ L of acryloyl chloride dissolved in 2mL of DMF in an isopiestic dropping funnel and added dropwise to the round bottom flask at a rate of one drop per three seconds with stirring until the addition was complete. After a subsequent reaction time of 12h at room temperature. The solution was removed and dialyzed against dialysis bag (Mw ═ 1,000Da) for 72h, and lyophilized to obtain a white powder sample.

3. PLLA-CTA (500mg) and AC-Dex (1.0g) were added to the reaction tube, and finally AIBN (3.1mg) was added thereto under N₂After three freeze-thaw cycles under ambient conditions, the reaction was stopped by exposure to air at 70 ℃ for 12 hours, followed by removal. Dialyzing with dialysis bag (Mw ═ 5,000Da) for 72h, taking out the dialyzate, lyophilizing to obtain white powder sample, sealing, and storing at 4 deg.C.

4. Dissolving 20mg of PLLA-g-Dex and 5mg of gallic acid in 2mL of DMSO, then dropwise adding 5mL of deionized water into the mixed solution stirred at a high speed for self-assembly for 12h, then dialyzing with the deionized water for 24 h to obtain a polymer mixed solution, and freeze-drying to obtain the PLLA-g-Dex polymer self-assembly drug-loaded micelle.

Example 4:

1. l-lactide (35.0mmol) and purified toluene (15mL) were added to a round-bottomed flask, and stirred sufficiently to dissolve L-lactide sufficiently, and a chain transfer agent (BSTSE, 0.63mmol) and Sn (Oct) were added to the solution₂(200. mu.L) and a rotor, performing three freeze-thaw cycles under nitrogen atmosphere to remove oxygen in the reaction tube, performing oil bath reaction at 110 ℃ for 12h, finally terminating the reaction by liquid nitrogen quenching, precipitating the reacted solution with glacial methanol three times, drying in vacuum at 40 ℃ overnight to obtain a white powdery product, namely PLLA-CTA, sealing and storing at 4 ℃ for the next reaction.

2. Dextran with a molecular weight of 2000 (Dex, 0.15mmol) was dissolved in DMSO (250mL) in a round-bottom flask, and a mixed solution of DMSO/DMF (5mL/2mL) and 310. mu.L triethylamine were added thereto. The round bottom flask was then placed in an ice-water bath, and 155. mu.L of acryloyl chloride was then dissolved in 2mL of DMF in an isopiestic dropping funnel and added dropwise to the round bottom flask at a rate of one drop per three seconds with stirring until the addition was complete. After a subsequent reaction time of 12h at room temperature. The solution was removed and dialyzed against dialysis bag (Mw ═ 1,000Da) for 72h, and lyophilized to obtain a white powder sample.

3. PLLA-CTA (250mg) and AC-Dex (900mg) were added to the reaction tube, and finally AIBN (2.5mg) was added thereto under N₂Under the atmospheric conditionAfter three cycles of freeze-thaw, the reaction was stopped by exposure to air at 70 ℃ for 12 hours, and then removed. Dialyzing with dialysis bag (Mw ═ 5,000Da) for 72h, taking out the dialyzate, lyophilizing to obtain white powder sample, sealing, and storing at 4 deg.C.

4. Dissolving 25mg of PLLA-g-Dex and 5mg of citric acid in 2mL of DMSO, dropwise adding 5mL of deionized water into the mixed solution stirred at a high speed for self-assembly for 12h, dialyzing with the deionized water for 24 h to obtain a polymer mixed solution, and freeze-drying to obtain the PLLA-g-Dex polymer self-assembly drug-loaded micelle.

Example 5:

1. l-lactide (40.0mmol) and purified toluene (15mL) were added to a round-bottomed flask, and stirred sufficiently to dissolve L-lactide sufficiently, and a chain transfer agent (BSTSE, 0.65mmol) and Sn (Oct) were added to the solution₂(200. mu.L) and a rotor, performing three freeze-thaw cycles under nitrogen atmosphere to remove oxygen in the reaction tube, performing oil bath reaction at 110 ℃ for 12h, finally terminating the reaction by liquid nitrogen quenching, precipitating the reacted solution with glacial methanol three times, drying in vacuum at 40 ℃ overnight to obtain a white powdery product, namely PLLA-CTA, sealing and storing at 4 ℃ for the next reaction.

2. Dextran with a molecular weight of 2000 (Dex, 0.2mmol) was dissolved in DMSO (250mL) in a round-bottomed flask, and a mixed solution of DMSO/DMF (5mL/2mL) and 400. mu.L of triethylamine were added thereto. The round bottom flask was then placed in an ice-water bath, and 200 μ L of acryloyl chloride was then dissolved in 2mL of DMF in an isopiestic dropping funnel and added dropwise to the round bottom flask at a rate of one drop per three seconds with stirring until the addition was complete. After a subsequent reaction time of 12h at room temperature. The solution was removed and dialyzed against dialysis bag (Mw ═ 1,000Da) for 72h, and lyophilized to obtain a white powder sample.

3. PLLA-CTA (350mg) and AC-Dex (1g) were added to the reaction tube, and finally AIBN (2.5mg) in N₂After three freeze-thaw cycles under ambient conditions, the reaction was stopped by exposure to air at 70 ℃ for 12 hours, followed by removal. Dialyzing with dialysis bag (Mw ═ 5,000Da) for 72h, and collecting dialysateAnd (4) freeze-drying to obtain a white powder sample, and sealing and storing the white powder sample at 4 ℃ for later use.

4. Dissolving 25mg of PLLA-g-Dex and 5mg of vitamin c in 2mL of DMSO, dropwise adding 5mL of deionized water into the mixed solution which is stirred at a high speed for self-assembly for 12h, dialyzing with the deionized water for 24 h to obtain a polymer mixed solution, and freeze-drying to obtain the PLLA-g-Dex polymer self-assembly drug-loaded micelle.

Example 6:

1. l-lactide (50.0mmol) and purified toluene (15mL) were added to a round-bottomed flask, and stirred sufficiently to dissolve L-lactide sufficiently, and a chain transfer agent (BSTSE, 0.70mmol) and Sn (Oct) were added to the solution₂(250 mu L) and a rotor, performing three times of freeze-thaw cycles under nitrogen atmosphere to remove oxygen in the reaction tube, then performing oil bath reaction at 110 ℃ for 12 hours, finally terminating the reaction by means of liquid nitrogen quenching, precipitating the reacted solution by using glacial methanol for three times, drying in vacuum at 40 ℃ overnight to obtain a white powdery product, namely PLLA-CTA, sealing and storing at 4 ℃ for waiting for the next reaction.

2. Dextran with a molecular weight of 2000 (Dex, 0.5mmol) was dissolved in DMSO (250mL) in a round-bottomed flask, and a mixed solution of DMSO/DMF (7.5mL/2.5mL) and 600. mu.L of triethylamine were added thereto. The round bottom flask was then placed in an ice-water bath, and then 300 μ L of acryloyl chloride was dissolved in 2mL of DMF in an isopiestic dropping funnel and added dropwise to the round bottom flask at a rate of one drop per three seconds with stirring until the addition was complete. After a subsequent reaction time of 12h at room temperature. The solution was removed and dialyzed against dialysis bag (Mw ═ 1,000Da) for 72h, and lyophilized to obtain a white powder sample.

3. PLLA-CTA (350mg) and AC-Dex (900mg) were added to the reaction tube, and finally AIBN (2.5mg) was added thereto under N₂After three freeze-thaw cycles under ambient conditions, the reaction was stopped by exposure to air at 70 ℃ for 12 hours, followed by removal. Dialyzing with dialysis bag (Mw ═ 5,000Da) for 72h, taking out the dialyzate, lyophilizing to obtain white powder sample, sealing, and storing at 4 deg.C.

4. Dissolving 25mg PLLA-g-Dex and 5mg gallic acid in 2mL DMSO, then dropwise adding 5mL deionized water into the mixed solution stirred at high speed for self-assembly for 12h, then dialyzing with deionized water for 24 h to obtain a polymer mixed solution, and freeze-drying to obtain the PLLA-g-Dex polymer self-assembly drug-loaded micelle.