阅读说明：本技术 一种基于双硫仑的两亲性嵌段共聚物前药及其制备方法和应用 (Disulfiram-based amphiphilic block copolymer prodrug and preparation method and application thereof ) 是由 王林格 方宇煌 于倩倩 徐蒙蒙 于 2021-07-09 设计创作，主要内容包括：本发明属于纳米医药及新材料领域,公开了一种双硫仑前药单体和基于该单体合成的两亲性嵌段共聚物前药及其制备方法和应用。该制备方法首先合成双硫仑前药单体,然后通过可逆加成断裂转移(RAFT)法将亲水性的聚乙二醇丙烯酸甲酯单体(PEGA)和疏水性的双硫仑前药单体(DTCM)共聚,得到所述两亲性嵌段共聚物前药(PPEGA-PDTCM),进而构建其自组装聚合物囊泡。本发明制备的聚合物囊泡可提高双硫仑的溶解性和稳定性,避免双硫仑在体内过早释放,以克服双硫仑临床治疗的局限性,并且这种聚合物囊泡能同时负载另一种药物,实现两种药物联合治疗。该聚合物囊泡具有较好的还原响应释药性,并具有抑制肿瘤细胞生长的活性。(The invention belongs to the field of nano medicines and new materials, and discloses a disulfiram prodrug monomer, an amphiphilic block copolymer prodrug synthesized based on the monomer, and a preparation method and application of the amphiphilic block copolymer prodrug. The preparation method comprises the steps of firstly synthesizing a disulfiram prodrug monomer, then copolymerizing a hydrophilic polyethylene glycol methyl acrylate monomer (PEGA) and a hydrophobic disulfiram prodrug monomer (DTCM) by a Reversible Addition Fragmentation Transfer (RAFT) method to obtain the amphiphilic block copolymer prodrug (PPEGA-PDTCM), and further constructing the self-assembly polymer vesicle of the amphiphilic block copolymer prodrug. The polymersome prepared by the invention can improve the solubility and stability of disulfiram, avoid the premature release of disulfiram in vivo, overcome the limitation of clinical treatment of disulfiram, and can simultaneously load another drug to realize the combined treatment of the two drugs. The polymersome has good reduction response drug release property and activity of inhibiting the growth of tumor cells.)

1. A disulfiram prodrug monomer characterized by having the structure of formula (I):

2. an amphiphilic block copolymer prodrug based on a disulfiram prodrug monomer of claim 1, characterized by having the structure of formula (II):

in the formula (II), x is 1-20; y is 5-20; and z is 8-17.

3. A process for preparing the disulfiram prodrug monomer of claim 1 comprising the specific steps of:

(a) in dichloromethane, mixing diethylamine, mercaptoethanol, carbon disulfide, triethylamine and carbon tetrabromide, and then reacting to obtain a product HDTC;

(b) mixing HDTC, methacryloyl chloride and triethylamine in dichloromethane, and then carrying out esterification reaction to obtain a disulfiram prodrug monomer DTCM with a structure shown in formula (I).

4. The method of claim 3, wherein:

in the step (a), the molar ratio of diethylamine to mercaptoethanol to carbon disulfide to triethylamine to carbon tetrabromide is 1:1:1 (1-2) to (1-2); the reaction temperature is 20-32 ℃; the reaction time is 1-4 h;

in the step (b), the molar ratio of HDTC, methacryloyl chloride and triethylamine is 1 (1-2) to 1-2; the reaction temperature is 20-32 ℃; the reaction time is 8-15 h.

5. A method for preparing the amphiphilic block copolymer prodrug as claimed in claim 2, which comprises the following specific steps:

(a) in N, N-dimethylformamide, 4-azobis (4-cyanovaleric acid) is used as an initiator, 4-cyano-4- [ (dodecyl sulfanyl thiocarbonyl) sulfanyl ] valeric acid is used as a transfer agent, and a disulfiram prodrug monomer DTCM with a structure shown in a formula (I) is subjected to polymerization reaction to obtain a polymer prodrug PDTCM;

(b) in N, N-dimethylformamide, polymer prodrug PDTCM is used as a macromolecular chain transfer agent, 4-azobis (4-cyanovaleric acid) is used as an initiator, PEGA is polymerized, and amphiphilic block copolymer prodrug PPEGA-PDTCM with a structure of a formula (II) is obtained.

6. The method of claim 5, wherein:

in the step (a), the molar ratio of 4, 4-azobis (4-cyanovaleric acid), 4-cyano-4- [ (dodecylsulfanylthiocarbonyl) sulfanyl ] pentanoic acid and DTCM is (0.1-0.5) to 1 (10-100); the reaction temperature is 60-80 ℃; the reaction time is 12-36 h;

in the step (b), the molar ratio of the 4, 4-azobis (4-cyanopentanoic acid), the PDTCM and the PEGA is (0.1-0.5) to 1 (10-100); the reaction temperature is 60-80 ℃, and the reaction time is 12-36 h.

7. A method for preparing polymersomes based on the amphiphilic block copolymer prodrug of claim 2, characterized by comprising the following specific steps:

(a) the method comprises the following steps Dissolving the amphiphilic block copolymer prodrug PPEGA-PDTCM in an organic solvent, injecting water, and stirring the solution;

(b) the method comprises the following steps And dialyzing the obtained mixed solution, and then performing freeze drying to obtain the polymersome based on the amphiphilic block copolymer prodrug.

8. Use of polymersomes obtained according to claim 7 for the preparation of pharmaceutical carriers.

9. A method for preparing a drug carrier based on the polymersome of claim 7, which comprises the following steps:

when the hydrophilic drug is loaded, the method comprises the following steps:

(a) the method comprises the following steps Dissolving the amphiphilic block copolymer prodrug PPEGA-PDTCM in an organic solvent to obtain a polymer solution, dissolving a hydrophilic drug in water to obtain a drug solution, injecting the drug solution into the polymer solution, and stirring the solution;

(b) the method comprises the following steps Dialyzing the obtained mixed solution, and then carrying out freeze drying to obtain the polymersome-based hydrophilic drug-loaded drug carrier;

when the hydrophobic drug is loaded, the method comprises the following steps:

(a) the method comprises the following steps Dissolving the hydrophobic drug and the amphiphilic block copolymer prodrug PPEGA-PDTCM together in an organic solvent, injecting water, and stirring the solution;

(b) the method comprises the following steps Dialyzing the obtained mixed solution; and then freeze-drying to obtain the polymersome-based hydrophobic drug-loaded drug carrier.

10. Use of a polymersome-based pharmaceutical carrier prepared according to the method of claim 9 for the preparation of a medicament for the treatment of tumors.

Technical Field

The invention belongs to the field of nano medicines and new materials, and relates to a disulfiram prodrug monomer, an amphiphilic block copolymer prodrug synthesized based on the disulfiram prodrug monomer, a polymer vesicle prepared based on the amphiphilic block copolymer prodrug, and application of the polymer vesicle in the aspect of a drug carrier.

Background

Disulfiram (disulfiram) is a drug that has received FDA approval for the treatment of alcoholism. Since the last 70 s, a number of clinical studies have found that disulfiram exhibits good anti-tumor effects on a variety of cancers (Cvek B. drug discovery today,2012,17(9-10): 409-412). The antitumor effect of disulfiram is copper ion dependent. Disulfiram is metabolized in the body and converted into the disulfiram derivative Diethyldithiocarbamate (DTC), which chelates copper ions and forms Cu (DTC)₂Chelate (LIU P, et al British Journal of Cancer,2012,107(9): 1488-97). It is currently believed that Cu (DTC)₂Plays an important role in disulfiram-based tumor therapy. Recent studies have shown that Cu (DTC)₂Can target a p97-NPL4-UFDI signaling pathway (SKROTT Z, et al. Nature,2017,552(7684): 194-9). Cu (DTC)₂Binding to thiol sites in the NPL4 protein that bind zinc ions results in aggregation of NPL 4. Aggregation of NPL4 results in inactivation of p97 segregase, leading to accumulation of misfolded proteins in the endoplasmic reticulum, ultimately leading to apoptosis.

Although oral dosage forms of disulfiram have long been used in alcohol withdrawal, they do not work in cancer therapy because disulfiram is poorly stable in the gastric environment and readily degrades in vivo into low potency molecules. Therefore, the development of more effective drug delivery systems is urgently needed to meet the clinical application requirement of disulfiram as an anticancer drug. The disulfiram is loaded by the nano-drug carrier, so that the degradation of the disulfiram in-vivo circulation can be minimized, the circulation half-life period of the disulfiram can be obviously improved, the tumor can be passively targeted by utilizing the high-permeability long-retention effect (EPR effect) of a tumor part, the enrichment of the disulfiram in tumor tissues is promoted, the tumor inhibition effect is improved, and the toxic and side effects of the disulfiram on normal tissues are reduced.

In the prior art, a chinese patent with publication number CN109700782A discloses a disulfiram nanoparticle, a chinese patent with publication number CN108513543A discloses a polymer nanoparticle encapsulating disulfiram, and a chinese patent with publication number CN105125495B discloses a nanoparticle carrying disulfiram with a polyester material, and the above techniques all adopt a delivery mode of physically encapsulating disulfiram with nanoparticles, which improves the solubility and stability of disulfiram, but physical encapsulation cannot avoid premature release of the drug. Premature release of the drug during the in vivo circulation process can result in reduced drug exposure at the tumor site, which can reduce the anti-tumor effect and increase the damage to healthy organs. Aiming at the problem of premature release of the drug, the drug molecules are combined with the carrier through covalent bonds, so that the premature release of the drug can be effectively inhibited. For example, the drug and the amphiphilic block copolymer are combined through covalent bonds to obtain the amphiphilic block copolymer prodrug, and the amphiphilic block copolymer prodrug is self-assembled through solution to form a nano-sized prodrug carrier (such as a micelle or a vesicle), so that the solubility and the stability of the drug are more effectively improved compared with the drug carrier for physically coating the drug, and the premature release of the drug in the blood circulation process is minimized. In addition, prodrug carriers can control the release behavior of drugs by the design of covalent bonds between the drug and the carrier. For example, the carrier and the drug are connected through a responsive covalent bond (such as a reduction responsive disulfide bond), and the characteristic that glutathione (a biological reducing agent) in tumor cells is higher than that in blood and normal cells is utilized, so that the drug release can be triggered in the tumor cells, and the stability of the drug can be maintained in the blood and normal cells, thereby effectively inhibiting the premature release of the drug and the damage to normal tissues.

Disclosure of Invention

Aiming at the problems of poor water solubility, easy degradation and premature release of disulfiram, the invention aims to provide a disulfiram prodrug monomer and an amphiphilic block copolymer prodrug synthesized based on the monomer, and the amphiphilic block copolymer prodrug is self-assembled to form polymer vesicles; the polymersome can improve the solubility and stability of disulfiram, avoid the premature release of disulfiram in human body, and can be used as a drug carrier to realize the co-delivery and combined anti-tumor of disulfiram and other drugs.

Another object of the present invention is to provide the above disulfiram prodrug monomer and amphiphilic block copolymer prodrug synthesized based on the monomer, and a method for synthesizing and preparing polymer vesicles based on the amphiphilic block copolymer prodrug.

The invention further aims to provide application of the polymersome in preparation of a drug carrier. .

The invention is realized by the following modes:

a disulfiram prodrug monomer having the structure of formula (I):

an amphiphilic block copolymer prodrug based on a disulfiram prodrug monomer having the structure of formula (II):

in the formula (I), x is 1-20; y is 5-20; and z is 8-17.

A synthetic method of a disulfiram prodrug monomer comprises the following specific steps:

(a) in dichloromethane, mixing diethylamine, mercaptoethanol, carbon disulfide, triethylamine and carbon tetrabromide, and then reacting to obtain a product HDTC;

(b) mixing HDTC, methacryloyl chloride and triethylamine in dichloromethane, and then carrying out esterification reaction to obtain a disulfiram prodrug monomer DTCM with a structure shown in formula (I).

In the step (a), the molar ratio of diethylamine to mercaptoethanol to carbon disulfide to triethylamine to carbon tetrabromide is 1:1:1 (1-2) to (1-2); the reaction temperature is 20-32 ℃; the reaction time is 1-4 h, preferably 2 h.

In the step (b), the molar ratio of HDTC, methacryloyl chloride and triethylamine is 1 (1-2) to 1-2; the reaction temperature is 20-32 ℃; the reaction time is 8-15 h, preferably 12 h.

A synthetic method of an amphiphilic block copolymer prodrug based on disulfiram prodrug monomer comprises the following specific steps:

(a) in N, N-dimethylformamide, 4-azobis (4-cyanovaleric acid) is used as an initiator, 4-cyano-4- [ (dodecyl sulfanyl thiocarbonyl) sulfanyl ] valeric acid is used as a transfer agent, and a disulfiram prodrug monomer DTCM with a structure shown in a formula (I) is subjected to polymerization reaction to obtain a polymer prodrug PDTCM;

(b) in N, N-dimethylformamide, polymer prodrug PDTCM is used as a macromolecular chain transfer agent, 4-azobis (4-cyanovaleric acid) is used as an initiator, PEGA is polymerized, and amphiphilic block copolymer prodrug PPEGA-PDTCM with a structure of a formula (II) is obtained.

In the step (a), the molar ratio of 4, 4-azobis (4-cyanovaleric acid), 4-cyano-4- [ (dodecylsulfanylthiocarbonyl) sulfanyl ] pentanoic acid and DTCM is (0.1-0.5) to 1 (10-100); the reaction temperature is 60-80 ℃; the reaction time is 12-36 h, preferably 24 h.

In the step (b), the molar ratio of the 4, 4-azobis (4-cyanopentanoic acid), the PDTCM and the PEGA is (0.1-0.5) to 1 (10-100); the reaction temperature is 60-80 ℃, the reaction time is 12-36 h, and preferably 24 h.

A method for preparing polymersomes based on amphiphilic block copolymer prodrugs comprises the following specific steps:

(a) the method comprises the following steps Dissolving the amphiphilic block copolymer prodrug PPEGA-PDTCM in an organic solvent, injecting water, and stirring the solution;

(b) the method comprises the following steps And dialyzing the obtained mixed solution, and then performing freeze drying to obtain the polymersome based on the amphiphilic block copolymer prodrug.

In the step (a), the organic solvent is at least one of tetrahydrofuran, dimethyl sulfoxide, N-dimethylformamide and 1, 4-dioxane; the volume ratio of the organic solvent to the water is 1 (2-10); the water injection rate is 0.5-5 mL/h; the stirring speed is 600-1000 rpm.

In the step (b), the cut-off molecular weight of the dialysis bag is 1000-3500 Da; the dialysis time is 12-48 h.

The application of the polymersome in preparing a drug carrier.

A preparation method of a drug carrier based on polymersome comprises the following specific steps:

when the hydrophilic drug is loaded, the method comprises the following steps:

(a) the method comprises the following steps Dissolving the amphiphilic block copolymer prodrug PPEGA-PDTCM in an organic solvent to obtain a polymer solution, dissolving a hydrophilic drug in water to obtain a drug solution, injecting the drug solution into the polymer solution, and stirring the solution;

(b) the method comprises the following steps Dialyzing the obtained mixed solution, and then carrying out freeze drying to obtain the polymersome-based hydrophilic drug-loaded drug carrier;

in the step (a), the organic solvent is at least one of tetrahydrofuran, dimethyl sulfoxide, N-dimethylformamide and 1, 4-dioxane; the hydrophilic medicine is at least one of doxorubicin hydrochloride, epirubicin hydrochloride and gemcitabine hydrochloride; the mass ratio of the hydrophilic drug to the amphiphilic block copolymer prodrug is 1: (1-20); the volume ratio of the organic solvent to the water is 1 (2-10); the injection rate of the medicine solution is 0.5-5 mL/h; the stirring speed is 600-1000 rpm.

In the step (b), the cut-off molecular weight of the dialysis bag is 1000-3500 Da; the dialysis time is 12-48 h.

When the hydrophobic drug is loaded, the method comprises the following steps:

(a) the method comprises the following steps Dissolving the hydrophobic drug and the amphiphilic block copolymer prodrug PPEGA-PDTCM together in an organic solvent, injecting water, and stirring the solution;

(b) the method comprises the following steps Dialyzing the obtained mixed solution; and then freeze-drying to obtain the polymersome-based hydrophobic drug-loaded drug carrier.

In the step (a), the hydrophobic drug is at least one of adriamycin, gemcitabine and paclitaxel; the mass ratio of the hydrophobic drug to the amphiphilic block copolymer prodrug is 1: (1-20); the organic solvent is at least one of tetrahydrofuran, dimethyl sulfoxide, N-dimethylformamide and 1, 4-dioxane; the volume ratio of the organic solvent to the water is 1 (2-10); the water injection rate is 0.5-5 mL/h; the stirring speed is 600-1000 rpm.

In the step (b), the cut-off molecular weight of the dialysis bag is 1000-3500 Da; the dialysis time is 12-48 h.

The application of the polymersome-based drug carrier in preparing a drug for treating tumor.

The invention has the beneficial effects that:

the invention provides a disulfiram prodrug monomer DTCM, which is copolymerized with another monomer PEGA to obtain an amphiphilic block copolymer prodrug PPEGA-PDTCM, and then the PPEGA-PDTCM-based polymer vesicle is prepared through solution self-assembly. The polymersome breaks through a delivery mode of physically encapsulating disulfiram by nanoparticles, effectively improves the solubility and stability of the disulfiram, can avoid premature release of the disulfiram in the in vivo circulation process, and can respond to and trigger the release of the disulfiram monomer in the reductive environment of a tumor part. In addition, the polymer vesicle can entrap other hydrophilic or hydrophobic drugs, so that co-delivery of disulfiram and other drugs is realized for combined antitumor, and the tumor treatment effect is further improved.

Drawings

FIG. 1 is a diagram of the disulfiram prodrug monomer of the present invention¹H NMR spectrum.

FIG. 2 shows the preparation of the amphiphilic block copolymer prodrug of the present invention¹H NMR spectrum.

Fig. 3 is a schematic view of the polymersome of the present invention.

FIG. 4 is a graph of the morphology (TEM) and particle size Distribution (DLS) of the polymersome of the present invention.

Fig. 5 shows the morphology (TEM) of the doxorubicin-loaded polymersome (left) and the doxorubicin hydrochloride-loaded polymersome (right) according to the present invention.

FIG. 6 shows that the amphiphilic block copolymer prodrug releases disulfiram monomer under the condition of 10mM glutathione¹H NMR spectrum.

Fig. 7 is a graph showing the reduction-triggered doxorubicin hydrochloride cumulative release curves of the doxorubicin hydrochloride-loaded polymersome of the present invention under different reduction conditions.

FIG. 8 shows the inhibitory effect of the doxorubicin hydrochloride-loaded polymersome of the present invention on tumor cell growth (upper: no copper chloride was added; lower: 0.25. mu.g/mL copper chloride was added).

Detailed Description

For the clear understanding of the technical solutions of the present invention, the following detailed descriptions of the technical solutions of the present invention are provided with reference to the accompanying drawings and the embodiments, but the technical solutions of the present invention are not to be construed as being limited thereto. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially. The room temperature of the invention is 20-32 ℃.

EXAMPLE 1 Synthesis of DTCM a Dithioren prodrug monomer

2.19g of diethylamine (30.0mmol) and 2.34g of mercaptoethanol (30.0mmol) were weighed into a single-neck flask, 30.0mL of anhydrous dichloromethane were added, and ice-bath was performed. 2.28g of carbon disulfide (30.0mmol), 3.03g of triethylamine (30.0mmol) and 9.94g of carbon tetrabromide (30.0mmol) were successively added thereto, and the reaction was stirred at room temperature for 2 hours. After the reaction was completed, the reaction mixture was washed 3 times with water and dried over anhydrous sodium sulfate overnight. Concentration by rotary evaporation, followed by purification by column chromatography using n-hexane/ethyl acetate as eluent gave the product as a yellow oil, which was designated HDTC.

1.0g of HDTC (4.4mmol) and 0.44g of triethylamine (4.4mmol) were weighed into a single-neck flask, and 20.0mL of anhydrous dichloromethane was added in an ice bath. 0.46g of methacryloyl chloride (4.4mmol) was added and the reaction was stirred at room temperature for 12 h. After the reaction was completed, the reaction mixture was washed 3 times with a saturated sodium chloride solution and deionized water, and dried overnight with anhydrous sodium sulfate. Concentration by rotary evaporation and purification by column chromatography using n-hexane/ethyl acetate as eluent gave the product as a pale yellow oil (DTCM).

EXAMPLE 2 Synthesis of DTCM a Dithioren prodrug monomer

2.19g of diethylamine (30.0mmol) and 2.34g of mercaptoethanol (30.0mmol) were weighed into a single-neck flask, 30.0mL of anhydrous dichloromethane were added, and ice-bath was performed. 2.28g of carbon disulfide (30.0mmol), 3.03g of triethylamine (30.0mmol) and 14.92g of carbon tetrabromide (45.0mmol) were successively added thereto, and the reaction was stirred at room temperature for 2 hours. After the reaction was completed, the reaction mixture was washed 3 times with water and dried over anhydrous sodium sulfate overnight. Concentration by rotary evaporation, followed by purification by column chromatography using n-hexane/ethyl acetate as eluent gave the product as a yellow oil, which was designated HDTC.

1.0g of HDTC (4.4mmol) and 0.67g of triethylamine (6.6mmol) were weighed into a single-neck flask, and 20.0mL of anhydrous dichloromethane was added in an ice bath. 0.69g of methacryloyl chloride (6.6mmol) was added and the reaction was stirred at room temperature for 12 h. After the reaction was completed, the reaction mixture was washed 3 times with a saturated sodium chloride solution and deionized water, and dried overnight with anhydrous sodium sulfate. Concentrating by rotary evaporation, and purifying by chromatography with n-hexane/ethyl acetate as eluent to obtain yellowish oily product (DTCM),¹the H NMR spectrum is shown in FIG. 1.

EXAMPLE 3 Synthesis of Dithioren prodrug monomer DTCM

2.19g of diethylamine (30.0mmol) and 2.34g of mercaptoethanol (30.0mmol) were weighed into a single-neck flask, 30.0mL of anhydrous dichloromethane were added, and ice-bath was performed. 2.28g of carbon disulfide (30.0mmol), 3.03g of triethylamine (30.0mmol) and 19.89g of carbon tetrabromide (60.0mmol) were successively added thereto, and the reaction was stirred at room temperature for 2 hours. After the reaction was completed, the reaction mixture was washed 3 times with water and dried over anhydrous sodium sulfate overnight. Concentration by rotary evaporation, followed by purification by column chromatography using n-hexane/ethyl acetate as eluent gave the product as a yellow oil, which was designated HDTC.

1.0g of HDTC (4.4mmol) and 0.89g of triethylamine (8.8mmol) were weighed into a single-neck flask, and 20.0mL of anhydrous dichloromethane was added and ice-cooled. 0.92g of methacryloyl chloride (8.8mmol) was added and the reaction was stirred at room temperature for 12 h. After the reaction was completed, the reaction mixture was washed 3 times with a saturated sodium chloride solution and deionized water, and dried overnight with anhydrous sodium sulfate. Concentration by rotary evaporation and purification by column chromatography using n-hexane/ethyl acetate as eluent gave the product as a pale yellow oil (DTCM).

Example 4 Synthesis of amphiphilic Block copolymer prodrugs

0.29g of the DTCM synthesized in example 2 (1.0mmol), 4.0mg of 4, 4-azobis (4-cyanovaleric acid) (0.01mmol) and 40.3mg of 4-cyano-4- [ (dodecylsulfanylthiocarbonyl) sulfanyl ] pentanoic acid (0.1mmol) were weighed out in a Schlenk tube, and 4.0mL of N, N-dimethylformamide was added. The reaction system is subjected to deoxidization procedures of liquid nitrogen freezing, vacuumizing and unfreezing cycle for 3 times, and finally vacuum sealing is carried out. The reaction was carried out in an oil bath at 65 ℃ for 24 h. After the reaction the product was precipitated in glacial ethyl ether (dissolve-precipitate, repeat 3 times) and dried under vacuum overnight to give the product as a yellow solid, named PDTCM.

0.2g of PDTCM (0.1mmol), 4.0mg of 4, 4-azobis (4-cyanovaleric acid) (0.01mmol) and 0.48g of PEGA (1.0mmol) were weighed into a Schlenk tube, and 4.0mL of N, N-dimethylformamide was added. The reaction system is subjected to deoxidization procedures of liquid nitrogen freezing, vacuumizing and unfreezing cycle for 3 times, and finally vacuum sealing is carried out. The reaction was carried out in an oil bath at 65 ℃ for 24 h. After the reaction, the product was precipitated in glacial ethyl ether (dissolve-precipitate, repeat 3 times) and dried under vacuum overnight to give the product as a yellow solid (PPEGA-PDTCM).

Example 5 Synthesis of amphiphilic Block copolymer prodrugs

0.73g of the DTCM synthesized in example 2 (2.5mmol), 10.0mg of 4, 4-azobis (4-cyanovaleric acid) (0.025mmol) and 40.3mg of 4-cyano-4- [ (dodecylsulfanylthiocarbonyl) sulfanyl ] pentanoic acid (0.1mmol) were weighed out in a Schlenk tube, and 4.0mL of N, N-dimethylformamide was added. The reaction system is subjected to deoxidization procedures of liquid nitrogen freezing, vacuumizing and unfreezing cycle for 3 times, and finally vacuum sealing is carried out. The reaction was carried out in an oil bath at 65 ℃ for 24 h. After the reaction the product was precipitated in glacial ethyl ether (dissolve-precipitate, repeat 3 times) and dried under vacuum overnight to give the product as a yellow solid, named PDTCM.

0.3g of PDTCM (0.1mmol) and 4.0mg of 4, 4-azobis (azobis) were weighed out(4-Cyanovaleric acid) (0.01mmol) and 1.2g of PEGA (2.5mmol) were placed in a Schlenk tube, and 4.0mL of N, N-dimethylformamide was added. The reaction system is subjected to deoxidization procedures of liquid nitrogen freezing, vacuumizing and unfreezing cycle for 3 times, and finally vacuum sealing is carried out. The reaction was carried out in an oil bath at 65 ℃ for 24 h. After the reaction, the product was precipitated in glacial ethyl ether (dissolution-precipitation, repeated 3 times), dried overnight in vacuo to give a yellow solid product (PPEGA-PDTCM),¹the H NMR spectrum is shown in FIG. 2.

EXAMPLE 6 Synthesis of amphiphilic Block copolymer prodrugs

2.92g of the DTCM synthesized in example 2 (10.0mmol), 10.0mg of 4, 4-azobis (4-cyanovaleric acid) (0.025mmol) and 40.3mg of 4-cyano-4- [ (dodecylsulfanylthiocarbonyl) sulfanyl ] pentanoic acid (0.1mmol) were weighed into a Schlenk tube, and 4.0mL of N, N-dimethylformamide was added. The reaction system is subjected to deoxidization procedures of liquid nitrogen freezing, vacuumizing and unfreezing cycle for 3 times, and finally vacuum sealing is carried out. The reaction was carried out in an oil bath at 65 ℃ for 24 h. After the reaction the product was precipitated in glacial ethyl ether (dissolve-precipitate, repeat 3 times) and dried under vacuum overnight to give the product as a yellow solid, named PDTCM.

0.5g of PDTCM (0.1mmol), 4.0mg of 4, 4-azobis (4-cyanovaleric acid) (0.01mmol) and 2.4g of PEGA (5.0mmol) were weighed into a Schlenk tube, and 4.0mL of N, N-dimethylformamide was added. The reaction system is subjected to deoxidization procedures of liquid nitrogen freezing, vacuumizing and unfreezing cycle for 3 times, and finally vacuum sealing is carried out. The reaction was carried out in an oil bath at 65 ℃ for 24 h. After the reaction, the product was precipitated in glacial ethyl ether (dissolve-precipitate, repeat 3 times) and dried under vacuum overnight to give the product as a yellow solid (PPEGA-PDTCM).

Example 7 preparation of polymersomes

5.0mg of the amphiphilic block copolymer prodrug prepared in example 5 was weighed out finely and dissolved in 200. mu.L of tetrahydrofuran. 800. mu.L of ultrapure water was injected into the above solution at a rate of 1mL/h while vigorously stirring the solution at a stirring rate set at 800 rpm. After the injection of ultrapure water is finished, transferring the solution into a dialysis bag with the molecular weight cutoff of 1000Da, and dialyzing the ultrapure water for 24h to obtain the polymer vesicle, wherein the structure of the polymer vesicle is shown in figure 3, PPEGA forms a hydrophilic outer layer and an inner layer of a membrane, PDTCM forms a hydrophobic intermediate layer of the membrane, and the disulfiram monomer is connected with a polymer chain through a disulfide bond. The hollow spherical shape is observed by a Transmission Electron Microscope (TEM), and the average particle size of the hollow spherical shape is about 180nm measured by Dynamic Light Scattering (DLS).

The experimental result is shown in fig. 4, the particle size and the distribution of the polymersome measured by DLS are consistent with the TEM result, and most of the polymersome is distributed at about 180nm, which is in accordance with the characteristic that the nano-drug carrier has the characteristics required by the passive targeting of the tumor part, i.e. the polymersome with the particle size range of 5-500 nm can be gathered in the tumor tissue through the passive targeting.

Example 8 preparation of polymersomes

5.0mg of the amphiphilic block copolymer prodrug prepared in example 5 was weighed out finely and dissolved in 200. mu.L of tetrahydrofuran. 800. mu.L of ultrapure water was injected into the above solution at a rate of 2mL/h while vigorously stirring the solution at a stirring rate set at 800 rpm. And after the ultrapure water is injected, transferring the solution into a dialysis bag with the molecular weight cutoff of 1000Da, and dialyzing the ultrapure water for 24 hours to obtain the polymer vesicle.

Example 9 preparation of polymersomes

10.0mg of the amphiphilic block copolymer prodrug prepared in example 5 was weighed out accurately and dissolved in 200. mu.LN, N-dimethylformamide. 1800. mu.L of ultrapure water was injected into the above solution at a rate of 2mL/h while vigorously stirring the solution at a stirring rate set at 800 rpm. And after the ultrapure water is injected, transferring the solution into a dialysis bag with the molecular weight cutoff of 1000Da, and dialyzing the ultrapure water for 24 hours to obtain the polymer vesicle.

Example 10 polymersome Loading of hydrophobic Adriamycin

2.0mg of doxorubicin and 10.0mg of the amphiphilic block copolymer prodrug prepared in example 5 were weighed out closely and dissolved together in 500. mu.L of tetrahydrofuran. 1.5mL of ultrapure water was injected into the above solution at a rate of 2mL/h while vigorously stirring the solution at a stirring rate set at 800 rpm. After the injection of ultrapure water is finished, the solution is transferred into a dialysis bag with the molecular weight cutoff of 3500Da, and the ultrapure water is dialyzed for 24h to obtain the adriamycin-loaded polymersome, wherein a TEM photograph is shown in FIG. 5.

Example 11 Polymer vesicles Loading hydrophilic Adriamycin hydrochloride

2.0mg of doxorubicin hydrochloride and 10.0mg of the amphiphilic block copolymer prodrug prepared in example 5 were weighed out closely and dissolved together in 500. mu.L of tetrahydrofuran. 1.5mL of ultrapure water was injected into the above solution at a rate of 2mL/h while vigorously stirring the solution at a stirring rate set at 800 rpm. After the injection of the ultrapure water is finished, transferring the solution into a dialysis bag with the molecular weight cutoff of 3500Da, and dialyzing the ultrapure water for 24h to obtain the polymer vesicle loaded with the doxorubicin hydrochloride, wherein a TEM photograph is shown in FIG. 5.

Example 12 reduction-responsive drug Release behavior of drug-loaded polymersomes

By using¹H NMR researches the drug release performance of the disulfiram monomer of the polymersome in the reducing glutathione solution. The polymersome prepared in example 7 was dissolved in a solution containing 10mM glutathione, and then passed through¹H NMR detects the change of the chemical structure of the amphiphilic polymer prodrug.

The experimental result is shown in fig. 6, after the amphiphilic polymer prodrug reacts with glutathione, the signal peaks of methyl and methylene belonging to the disulfiram monomer disappear, and the amphiphilic polymer prodrug is proved to be capable of releasing the disulfiram monomer in a reducing environment.

The fluorescent spectrometry is utilized to determine the drug release performance of the doxorubicin hydrochloride in the phosphate buffer solution of the polymer vesicle loaded with the doxorubicin hydrochloride under the conditions of different glutathione concentrations and pH 7.4. The drug-loaded polymer vesicles prepared in example 11 were taken and placed in phosphate buffers with glutathione concentrations of 0, 2 and 10mM and pH 7.4 for dialysis for 48 hours (MWCO 1000), the doxorubicin hydrochloride content of each group after dialysis was measured using a fluorescence spectrophotometer, and the drug release amount of each group was calculated by comparison with the measured doxorubicin hydrochloride standard curve.

The experimental result is shown in fig. 7, the drug release amount of the drug-loaded polymer micelle and the concentration of glutathione present a certain dependency relationship, and when the concentration of glutathione is 2mM, the cumulative drug release amount of doxorubicin hydrochloride within 24h is about 40%; with the increase of the concentration of the glutathione to 10mM, the cumulative drug release amount of the doxorubicin hydrochloride in 24h reaches 80 percent; in the absence of glutathione, the cumulative release of doxorubicin hydrochloride over 48 hours was only about 20%. This experiment again demonstrates the reduction-sensitive nature of the disulfide bonds in such polymersomes. Considering that the glutathione concentration in tumor cells is increased compared with that in normal cells, the polymersome can realize the drug delivery targeting the tumor cells.

Example 13 Effect of doxorubicin hydrochloride-loaded polymersome on the inhibition of tumor cell growth

Mouse breast cancer cell 4T1 cell at 1X 10⁴The density of cells/well was seeded in 96-well culture plates. After overnight culture, the culture medium was replaced with a new one, and then Polymersome (PS) prepared in example 7, doxorubicin hydrochloride-loaded polymersome (PS-DOX) prepared in example 11, doxorubicin hydrochloride (DOX) and Disulfiram (DSF) were administered at different concentrations, corresponding to a disulfiram concentration of 0-12. mu.g/mL, a doxorubicin hydrochloride concentration of 0-1.6. mu.g/mL and a copper chloride concentration of 0.25. mu.g/mL. After continuing culturing for 24h, adding a CCK-8 reagent, incubating for 1h, detecting the absorbance value of each hole by using a microplate reader, setting the detection wavelength to be 450nm, and calculating the cell survival rate according to the following formula:

cell survival rate (%) < 100%

As shown in FIG. 8, PS-DOX showed some cytotoxicity without the addition of cupric chloride. After copper chloride is added, the cytotoxicity of PS-DOX is obviously improved, and the tumor inhibition effect of the PS-DOX is better than that of free small molecular drugs. Meanwhile, the cytotoxicity of PS-DOX is obviously higher than that of PS, which shows that disulfiram and doxorubicin hydrochloride have good combined anti-tumor effect. The experimental result shows that the drug-loaded polymer vesicle system designed by the invention can realize reduction response drug release and has the activity of inhibiting the growth of tumor cells.