CN103865069B - There is active target star polymer carrier of physiological environment response function and preparation method thereof - Google Patents

Abstract

The invention relates to an active targeting star-shaped polymer carrier with a physiological environment response function and a preparation method thereof. The carrier has a specific targeting function for tumor cells and can rapidly degrade in response to a reducing environment in the cell. The carrier material is an amphiphilic star-shaped polymer, and the hydrophilic segment material is polyethylene glycol with an active targeting ligand at the end; the hydrophobic segment is degradable and absorbable star-shaped polycaprolactone; hydrophilic and hydrophobic The segments are chemically connected by disulfide bonds with physiological environment response functions to form functionalized star-shaped block copolymers. The polymer of the present invention can be used as a drug carrier, self-assembled in water to form drug-loaded nanoparticles, and can be used for targeted therapy of tumors.

Description

Active targeting star polymer carrier with physiological environment response function and preparation method thereof

technical field

The invention relates to a preparation method of an active targeting star-shaped polymer carrier with a physiological environment response function, and belongs to the technical field of drug controlled release.

Background technique

The side effects of anticancer drugs seriously hinder their clinical application. In order to solve this problem, researchers currently use nanoparticles as drug carriers, such as nanomicelles to carry drugs. The main basis for this application is that polymer micelles carry anticancer drugs through endocytosis of cells. Entering cells, bypassing P-glycoprotein, avoiding the outflow of drugs through extracellular secretion, so that drugs can be absorbed in large quantities by tumor cells with multi-drug resistance, improving the therapeutic effect and greatly reducing toxic and side effects.

However, polymer micelles still face many challenges in the application of anticancer drugs. It is expected that the drug-loaded micelles will have good stability in the blood circulation before reaching tumor cells, and they must also be selectively enriched in tumor sites. , and the drug can be rapidly released inside the tumor cells. Controlled release of anticancer drugs through cell targeting and cytoplasm is the key to reducing the damage of tumor therapy to normal cells and overcoming the problem of drug resistance. Therefore, the preparation of polymer carriers with stable structure, targeting and rapid release of drugs in response to the environment of tumor cells is a hot spot in the research and technology development of tumor therapy.

In the structural design of block copolymers forming micelles, amphiphilic star polymers not only have the properties of general amphiphilic linear block copolymers, but also have multiple chain segments connected to a multifunctional core by covalent bonds. In addition, it also has its special properties. Star polymers are multi-arm branched polymers formed by at least three linear polymer chains connected to the same central core through chemical bonds. Compared with linear polymers, they have unique properties including: high molecular surface functionality, The end of the molecular chain has a large number of functional groups that can be further modified; the molecular chain is not easy to entangle, the viscosity of the solution and the bulk is significantly lower than that of the linear polymer of the same molecular weight, and the degree of crystallinity is lower; the molecule is more prone to microphase separation, etc. Based on these characteristics, compared with linear amphiphilic block copolymers, star copolymers are more likely to form micelles in water, and also have better stability.

Compared with normal human cells, the surface of most tumor cells exhibits high expression of some specific receptors, such as folate receptors and galactosyl receptors. Therefore, for the special receptors on the surface of tumor cells, some corresponding ligands are connected to the drug carrier material through chemical bonds. Through the specific binding of this receptor-ligand, the active targeting function of the drug carrier can be realized. In addition, inside the tumor cells, the content of glutathione is much higher than that in the blood outside the cells, and also higher than that in normal cells. According to the characteristics of this physiological environment, we can link disulfide bonds with physiological environment response functions between the hydrophilic and hydrophobic segments of the polymer, so that the carrier can be rapidly cleaved and broken under reducing conditions in tumor cells, releasing drugs to cause tumors Cells undergo rapid apoptosis.

Currently, Chinese patent applications 200910273157.0 and 201110004873.6 disclose preparation methods of copolymers containing disulfide bonds, but do not involve the targeting of the carrier, and the carrier polymer is not a star polymer. According to the previous analysis, these polymer materials cannot achieve the stability of the carrier in the blood circulation, nor can it achieve the targeted therapeutic properties for tumor treatment. However, in our system, according to the characteristics of the tumor's physiological environment, we designed a high-efficiency protein that can not only maintain stability in blood circulation, but also target tumor cells and rapidly lyse in response to intracellular reducing conditions. Molecular chains, star-shaped polymer carriers for rapid release of antitumor drugs. The carrier is also completely biodegradable in vivo.

Contents of the invention

The purpose of the present invention is to provide a preparation method of an active targeting star-shaped polymer carrier with physiological environment response function. The resulting star polymer ensures the stability of the carrier in the blood circulation.

A method for preparing an active targeting star-shaped polymer carrier with a physiological environment response function, comprising the following steps:

(1) Preparation of star-shaped polycaprolactone: take stannous chloride as a catalyst, add polyol and caprolactone, and after vacuuming for 3 hours, ring-opening polymerization at a temperature of 150 degrees, react for 6 hours to synthesize polycaprolactone with a theoretical molecular weight of 8000 Star-shaped polycaprolactone, the product is cooled, dissolved in dichloromethane, precipitated in a large amount of ice ethanol, filtered with suction, and the product is vacuum-dried to obtain the preparation of star-shaped polycaprolactone;

(2) Disulfide bonds are introduced at the end of star-shaped polycaprolactone: cyclic dithiodipropionic anhydride reacts with terminal hydroxyl groups of star-shaped polycaprolactone obtained in (1), using dimethylformamide DMF as solvent, adding The catalyzed reaction of 4-dimethylaminopyridine DMAP and triethylamine is carried out, and the reaction is stirred for 24 hours under the condition of a 35-degree water bath; after the reaction, the reaction solution is acidified with 0.1M dilute hydrochloric acid, and the reaction solution is poured into six times the volume of distilled water , extracted three times with double the volume of dichloromethane, collected the dichloromethane layer, added anhydrous sodium sulfate to remove water, and filtered the clarified filtrate by rotary evaporation, precipitation with ice ether, and suction filtration to obtain the molecular formula PER-(PCL- SS-COOH)4 is the amphiphilic block copolymer of the main body;

(3) Formation of amphiphilic star-shaped block copolymer: Weigh 1 equivalent of the hydrophobic segment product (PER-(PCL-SS-COOH)4) and dissolve it in a certain amount of DMF. The dropping funnel was slowly dropped into the DMF solution of 8-10 equivalents of aminated PEG. In the presence of catalysts NHS and DCC, amphiphilic block copolymers were formed through amide bonds. The reaction liquid was filtered, rotated and evaporated on ice. Ethanol precipitation to obtain amphiphilic star-shaped block copolymer;

(4) Preparation of active targeting star-shaped polymer carrier with physiological environment response function: the activated targeting material reacts with the amphiphilic block copolymer prepared in the previous step at an equivalent ratio of 4:1, and stirs at room temperature for 24 hours. The solvent is dimethyl sulfoxide DMSO; after the reaction, dialyze with a dialysis bag with a molecular weight cut-off of 1000 g/mole for 3 days, and after the dialysis, freeze-dry to obtain the final target product.

The raw materials of the method of the invention are cheap and easy to obtain, the method is mature and simple, and the application prospect is wide. All involved steps and methods can be realized by those skilled in the art.

The object of the present invention is also the object obtained by the above means.

An active targeting star polymer carrier with physiological environment response function, the carrier material is an amphiphilic star polymer with a four-arm or six-arm star structure, and the hydrophilic segment material is terminated with an active targeting The polyethylene glycol of the ligand; the hydrophobic segment is a degradable and absorbable star-shaped polycaprolactone; the hydrophilic and hydrophobic segments are connected by a disulfide bond with a physiological environment response function to form a functional star-shaped insert Segment copolymers, this polymer can be used as a drug carrier, and self-assemble in water to form drug-loaded nanoparticles for targeted therapy of tumors.

In this way, the introduction of targeting molecules makes the drug-loaded micelles selectively enrich around the tumor cells and enter into the tumor cells through receptor-mediated endocytosis; linking between the hydrophilic and hydrophobic segments of the polymer The disulfide bond with the function of responding to the physiological environment can make the carrier rapidly cleaved and broken under reducing conditions in the tumor cells, releasing the drug and causing the rapid apoptosis of the tumor cells. The main component of the hydrophobic segment is polycaprolactone, which can be completely biodegraded in vivo. Therefore, the carrier can improve the therapeutic effect of tumors and reduce the toxic and side effects of drugs.

Description of drawings

The synthetic route of Fig. 1 star polycaprolactone.

Fig. 2 Synthetic route of amphiphilic star block copolymer.

Figure 3 The synthetic route of targeting materials.

detailed description

(1) Preparation of star-shaped polycaprolactone (the synthetic route is shown in Figure 1): stannous chloride is used as the catalyst, and polyol is the core of the star-shaped structure. Usually, for 1 equivalent of pentaerythritol, put 70 equivalents of caprolactone and 1% of the total mass of stannous chloride to synthesize star-shaped polycaprolactone with a theoretical molecular weight of 8000. After evacuating for 3 hours, ring-opening polymerization was carried out at a temperature of 150 degrees, and the reaction was carried out for 6 hours. After the product is cooled, it is dissolved in dichloromethane, precipitated in a large amount of ice ethanol, filtered with suction, and the product is vacuum-dried to obtain four-arm polycaprolactone.

In the above preparation process, the molecule used as the core of the star structure can be a kind of polyhydric alcohol such as glycerol, pentaerythritol and dipentaerythritol, which respectively form three-arm, four-arm and six-arm star structure. The theoretical molecular weight of the designed star-shaped polymer can be 500 to 10,000.

(2) Preparation of dithiodipropionic acid cyclic anhydride: A certain amount of dithiodipropionic acid is refluxed in acetyl chloride at a temperature of 65 degrees to obtain the cyclic internal anhydride of dithiodipropionic acid.

(3) Disulfide bonds are introduced at the end of the star-shaped polycaprolactone: the cyclic dithiodipropionic anhydride synthesized above can react with the terminal hydroxyl group of the star-shaped polycaprolactone, and dimethylformamide (DMF) is used as the solvent. Add 4-dimethylaminopyridine (DMAP) and triethylamine to catalyze the reaction, and stir the reaction for 24 hours under the condition of 35 degree water bath. After the reaction, the reaction solution was acidified with 0.1M dilute hydrochloric acid. The reaction solution was poured into six volumes of distilled water, and extracted three times with two volumes of dichloromethane. The dichloromethane layer was collected, and anhydrous sodium sulfate was added to remove water. After filtration, the clarified filtrate was rotary evaporated, precipitated with glacial ether, and suction filtered to obtain the product. The molecular formula of the product is PER-(PCL-SS-COOH)4.

(4) Preparation of bis-aminated polyethylene glycol: Gabriel method was used to aminate polyethylene glycol. For the method of amination of both ends of PEG, other mature amination methods can also be used, for example, after PEG is used to synthesize sulfonylated PEG, it is reacted with ammonia water in a high temperature and autoclave.

(5) Formation of amphiphilic star-shaped block copolymer (as shown in the synthetic route diagram 2): according to the above scheme, weigh 1 equivalent of the hydrophobic segment product (PER-(PCL-SS-COOH)4) In a certain amount of DMF, the solution is slowly dropped into the DMF solution of 8-10 equivalents of aminated PEG with a constant pressure dropping funnel, and in the presence of catalysts NHS and DCC, the amphiphilic intercalation is formed by amide bonds. segment copolymers. The reaction solution was filtered, rotary evaporated, and precipitated with ice ethanol to obtain the product.

(6) Preparation of targeting materials: The source of targeting molecules can be one of folic acid and galactose molecules, and their receptors are overexpressed on the surface of human tumor cells.

(a) Preparation of folic acid-targeting materials (as shown in the synthetic route diagram 3): folic acid was activated by equivalent amounts of NHS and DCC under ice-water bath conditions. The activated folic acid was reacted with the amphiphilic block copolymer prepared in the previous step at an equivalent ratio of 4:1, stirred at room temperature for 24 h, and the solvent was dimethyl sulfoxide (DMSO). After the reaction was finished, dialyze for 3 days with a dialysis bag with a molecular weight cut-off of 1000 g/mol. After dialysis, freeze-dry to obtain the final product, a star polymer with folic acid targeting groups and disulfide bonds.

(b) Preparation of galactose-targeting material: the preparation method is the same as above, replacing folic acid with lactobionic acid to obtain a star-shaped polymer with galactose-targeting groups and disulfide bonds.

Example 1

Take by weighing 0.17 gram of pentaerythritol, 10 gram of caprolactone and 0.1 gram of tin protochloride, place in a one-necked bottle, after vacuumizing for 3 hours, ring-opening polymerization under the condition of temperature 150 degrees, take out after 6 hours, treat that the product cools from melting to In solid state, add 5 mL of dichloromethane to dissolve, drop into 250 mL of ice ethanol for precipitation, filter with suction, and dry in vacuum to obtain four-arm polycaprolactone.

Weigh 2 grams of dithiodipropionic acid, add 20 grams of acetyl chloride, reflux reaction at a temperature of 65 degrees, stir magnetically for 4 hours, remove most of the organic solvent by rotary evaporation, precipitate with glacial ether, filter with suction, and dry in vacuum, namely have to.

Weigh 1 part of dithiodipropionic anhydride and 5.2 grams of the above-mentioned star-shaped polycaprolactone, add them to 70 mL of DMF, catalyst DMAP 0.3176 grams, triethylamine 0.4 mL, and stir for 24 hours under the condition of a water bath at a temperature of 35 degrees . After the reaction, adjust the pH value to 5 with the prepared 0.1M hydrochloric acid, then pour the reaction solution into six times the volume of distilled water, and extract with two times the volume of dichloromethane for a total of three extractions. The lower dichloromethane layer was collected, and anhydrous sodium sulfate was added to remove water. After the liquid layer was clarified, sodium sulfate was filtered off, the filtrate was rotary evaporated, a large amount of glacial ether was precipitated, and the product was obtained by suction filtration. The molecular formula of the product is PER-(PCL-SS-COOH)4.

Dissolve 3 grams of diaminated polyethylene glycol (PEG) in 50 mL of DMF and place it in a three-necked flask. In addition, weigh 1.2 grams of the hydrophobic segment product PER-(PCL-SS-COOH)4 and dissolve it in 30 mL of DMF. The solution was slowly dropped into the DMF solution of aminated polyethylene glycol, and a catalytic amount of NHS and DCC was added, and the reaction was carried out at room temperature for 24 hours. Filtration of the reaction solution, rotary evaporation, precipitation with a large amount of ice ethanol, and suction filtration were repeated three times, followed by vacuum drying to obtain a star-shaped amphiphilic block copolymer product.

Weigh 0.022 g of folic acid, 0.0103 g of DCC, and 0.0058 g of NHS, add them into 20 mL of dimethyl sulfoxide (DMSO) and stir magnetically for 12 h, and carry out the first 2 h in an ice-water bath. Weigh 3 grams of the star-shaped amphiphilic block copolymer product synthesized in the previous step, dissolve it in 50 mL of DMSO, and drop the activated folic acid solution into the polymer solution, and react for 24 hours. After the reaction, it was dialyzed for 3 days with a dialysis bag with a molecular weight cut-off of 1000 g/mole, and then freeze-dried to obtain the final product: an active targeting four-armed star-shaped polymer carrier with a physiological environment response function.

Example 2

In this example, the pentaerythritol in Example 1 was replaced with dipentaerythritol, and the ratio of dipentaerythritol to caprolactone was 1:105. Other implementation methods in Example 1 were used to obtain a six-armed star-shaped copolymer.

Example 3

In this example, the folic acid molecule in Example 1 is replaced with lactobionic acid. Other methods are the same as in Example 1, and a star-shaped four-armed polymer with galactose targeting groups and disulfide bonds can be synthesized.

Example 4

In this example, the folic acid molecule in Example 1 is replaced by lactobionic acid, and the pentaerythritol is replaced by dipentaerythritol. The other methods are the same as in Example 1, and a star-shaped six-armed polymer with a galactose targeting group and a disulfide bond can be synthesized. things.

Claims (5)

1. A method for preparing an active targeting star-shaped polymer carrier with a physiological environment response function, comprising the steps of: (1) Preparation of star-shaped polycaprolactone: take stannous chloride as a catalyst, add polyol and caprolactone, and after evacuating for 3 hours, perform ring-opening polymerization at a temperature of 150°C, and react for 6 hours to synthesize polycaprolactone with a theoretical molecular weight of 8000. Star-shaped polycaprolactone, the product is cooled, dissolved in dichloromethane, precipitated in a large amount of ice ethanol, filtered with suction, and the product is vacuum-dried to obtain the preparation of star-shaped polycaprolactone; (2), introduction of disulfide bonds at the end of star-shaped polycaprolactone: cyclic dithiodipropionic anhydride reacts with terminal hydroxyl groups of star-shaped polycaprolactone obtained in (1), using dimethylformamide DMF as solvent, adding The catalyzed reaction of 4-dimethylaminopyridine DMAP and triethylamine was carried out, and the reaction was stirred for 24 hours under the condition of 35°C water bath; after the reaction, the reaction solution was acidified with 0.1M dilute hydrochloric acid, and the reaction solution was poured into six times the volume of distilled water , extracted three times with double the volume of dichloromethane, collected the dichloromethane layer, added anhydrous sodium sulfate to remove water, and filtered the clarified filtrate by rotary evaporation, precipitation with ice ether, and suction filtration to obtain the molecular formula PER-(PCL- SS-COOH)4 is the amphiphilic block copolymer of the main body; (3), the formation of amphiphilic star-shaped block copolymers: Weigh 1 equivalent of the hydrophobic segment product (PER-(PCL-SS-COOH)4) and dissolve it in a certain amount of DMF. The dropping funnel was slowly dropped into the DMF solution of 8-10 equivalents of aminated PEG. In the presence of catalysts NHS and DCC, amphiphilic block copolymers were formed through amide bonds. The reaction liquid was filtered, rotated and evaporated on ice. Ethanol precipitation to obtain amphiphilic star-shaped block copolymer; (4) Preparation of active targeting star-shaped polymer carrier with physiological environment response function: the activated targeting material reacts with the amphiphilic block copolymer prepared in the previous step at an equivalent ratio of 4:1, and stirs at room temperature for 24 hours. The solvent is dimethyl sulfoxide DMSO; after the reaction, dialyze with a dialysis bag with a molecular weight cut-off of 1000 g/mole for 3 days, and after the dialysis, freeze-dry to obtain the final target product. 2. The method according to claim 1, wherein the polyol is pentaerythritol, and the weight ratio of pentaerythritol to caprolactone in step (1) is 1.7:100. 3. The method according to claim 1, wherein the polyhydric alcohol is dipentaerythritol, and the weight ratio of dipentaerythritol to caprolactone in step (1) is 1:105. 4. The method according to claim 1, wherein the targeting material is one of folic acid and galactose. 5. An active targeting star-shaped polymer carrier with a physiological environment response function, the carrier material is an amphiphilic star-shaped polymer with a four-arm or six-arm star structure, and the hydrophilic segment material is terminated with active Polyethylene glycol targeting the ligand; the hydrophobic segment is a degradable and absorbable star-shaped polycaprolactone; the hydrophilic and hydrophobic segments are connected by a disulfide bond with a physiological environment response function to form a functional star Shaped block copolymer, this polymer can be used as a drug carrier, and self-assembles in water to form drug-loaded nanoparticles for targeted therapy of tumors, characterized in that, the method of claim 1 or 2 or 3 or 4 is adopted method made.