阅读说明：本技术 快速氧化/还原双重响应性含双硒键的嵌段共聚物的制备方法 (Preparation method of block copolymer containing double selenium bonds with rapid oxidation/reduction dual responsiveness ) 是由 倪沛红 孙月 何金林 张明祖 于 2017-11-01 设计创作，主要内容包括：本发明公开了一种快速氧化/还原双重响应性含双硒的嵌段共聚物制备方法及其应用。具体制备方法为首先分别制备二叠氮基封端的双硒小分子(N<Sub>3</Sub>-SeSe-N<Sub>3</Sub>)和二炔丙基封端的聚乙二醇(PA-PEG-PA)。利用它们结构中的叠氮基与炔基的反应,获得两端炔基封端的含双硒聚乙二醇交替共聚物(PA-PEG-<I>alt</I>-SeSe-PA)。然后,与叠氮基单封端的聚己内酯(PCL-N<Sub>3</Sub>)反应,获得具有快速氧化/还原双重响应性含双硒的嵌段共聚物(PCL-PEGSeSe-PCL)。利用这种两亲性共聚物在水溶液中自组装成纳米粒子,具有良好的生物相容和生物可降解性。包载疏水性抗癌药物,可以在肿瘤细胞环境中,聚合物纳米粒子被破坏,快速释放出包载的药物,因此,在癌症治疗方面具有潜在的应用前景。(The invention discloses a preparation method and application of a quick oxidation/reduction dual-responsiveness block copolymer containing diselenide. The specific preparation method comprises the steps of firstly respectively preparing the bis-selenium micromolecules (N) blocked by the diazide groups 3 ‑SeSe‑N 3 ) And dipropargyl-terminated polyethylene glycol (PA-PEG-PA). By stacks in their constructionThe reaction of the nitrogen group and the alkynyl group to obtain the diselenide-containing polyethylene glycol alternating copolymer (PA-PEG- alt -SeSe-PA). Then, polycaprolactone (PCL-N) mono-terminated with azido 3 ) And reacting to obtain the double-selenium-containing block copolymer (PCL-PEGSeSe-PCL) with quick oxidation/reduction dual responsiveness. The amphiphilic copolymer is self-assembled into nano particles in aqueous solution, and has good biocompatibility and biodegradability. The hydrophobic anticancer drug is entrapped, so that the polymer nanoparticles can be destroyed in the tumor cell environment to quickly release the entrapped drug, and the preparation method has potential application prospect in the aspect of cancer treatment.)

1. A preparation method of a block copolymer containing a double selenium bond with rapid oxidation/reduction dual responsiveness is characterized by comprising the following steps:

(1) reacting and preparing the azido-terminated diselenide micromolecule by taking selenocysteine hydrochloride, acyl chloride compound and azido compound as raw materials;

(2) polyethylene glycol, potassium hydride and a propine compound are used as raw materials to prepare polyethylene glycol with propargyl end capping at two ends through reaction;

(3) reacting the azido-terminated diselenide micromolecules prepared in the step (1) with the two-end propargyl-terminated polyethylene glycol prepared in the step (2) to prepare two-end propargyl-terminated diselenide bond-containing polyethylene glycol alternating copolymers;

(4) polymerizing e-caprolactone, and reacting with azide to prepare azide single-ended polycaprolactone;

(5) reacting the propargyl-terminated polyethylene glycol alternating copolymer containing the diselenide bond at two ends prepared in the step (3) with the azido-single-terminated polycaprolactone prepared in the step (4) to prepare the rapid oxidation/reduction dual-responsiveness block copolymer containing the diselenide bond;

the fast oxidation/reduction dual-responsive double-selenium bond-containing block copolymer is expressed by the following chemical structural formula:

wherein m is 5-15, n is 4-114, and x is 15-45.

2. The method for preparing a rapid oxidation/reduction dual-responsive diselenide bond-containing block copolymer according to claim 1, wherein in step (1), the reaction product of cysteamine seleno hydrochloride and an acid chloride compound is reacted with an azide compound to prepare an azide-terminated diselenide small molecule; in the step (2), reacting a reaction product of polyethylene glycol and potassium hydride with a propine compound to prepare polyethylene glycol with propargyl groups at two ends blocked; in the step (3), the reaction is carried out in the presence of a copper salt catalyst and a catalyst ligand; in the step (4), initiating the polymerization of e-caprolactone by using small molecular alcohol in the presence of an organic catalyst; in step (5), the reaction is carried out in the presence of a copper salt catalyst and a catalyst ligand.

3. The method of claim 1, wherein the fast oxidation/reduction dual-responsive block copolymer containing diselenide linkages comprises: in the step (1), the acyl chloride compound is chloracetyl chloride, and the azide compound is sodium azide; in the step (2), the propyne compound is 3-bromopropyne; in the step (3), the copper salt catalyst is selected from copper sulfate pentahydrate, cuprous chloride or cuprous bromide, and the catalyst ligand is selected from one of sodium ascorbate, bipyridyl, pentamethyldiethylenetriamine, tetramethylethylenediamine or hexamethyltriethylenetetramine; in the step (4), the catalyst is selected from stannous octoate or 1, 8-diazabicycloundecen-7-ene; in the step (5), the copper salt catalyst is selected from copper sulfate pentahydrate, cuprous chloride or cuprous bromide, and the catalyst ligand is selected from one of sodium ascorbate, bipyridyl, pentamethyldiethylenetriamine, tetramethylethylenediamine or hexamethyltriethylenetetramine.

4. The method of claim 1, wherein the fast oxidation/reduction dual-responsive block copolymer containing diselenide linkages comprises: in the step (1), the reaction temperature is 25-60 ℃, and the reaction time is 12-40 h; in the step (2), the reaction temperature is 0-50 ℃, and the reaction time is 1-40 h; in the step (3), the reaction temperature is 35-45 ℃, and the reaction time is 24-48 h; in the step (4), the reaction temperature is 60-90 ℃, and the reaction time is 4-24 h; in the step (5), the reaction temperature is 35-45 ℃, and the reaction time is 24-48 h.

5. A preparation method of a block copolymer nanoparticle containing a double selenium bond with rapid oxidation/reduction dual responsiveness is characterized by comprising the following steps:

(1) reacting and preparing the azido-terminated diselenide micromolecule by taking selenocysteine hydrochloride, acyl chloride compound and azido compound as raw materials;

(2) polyethylene glycol, potassium hydride and a propine compound are used as raw materials to prepare polyethylene glycol with propargyl end capping at two ends through reaction;

(3) reacting the azido-terminated diselenide micromolecules prepared in the step (1) with the two-end propargyl-terminated polyethylene glycol prepared in the step (2) to prepare two-end propargyl-terminated diselenide bond-containing polyethylene glycol alternating copolymers;

(4) polymerizing e-caprolactone, and reacting with azide to prepare azide single-ended polycaprolactone;

(5) reacting the propargyl-terminated polyethylene glycol alternating copolymer containing double selenium bonds at two ends prepared in the step (3) with the azido-singly-terminated polycaprolactone prepared in the step (4) to prepare a rapid oxidation/reduction dual-responsiveness block copolymer containing double selenium bonds;

(6) and (3) self-assembling and dialyzing the block copolymer containing the double selenium bonds with the fast oxidation/reduction dual responsiveness prepared in the step (5) to prepare the block copolymer nano particles containing the double selenium with the fast oxidation/reduction dual responsiveness.

6. A preparation method of a rapid oxidation/reduction dual-responsiveness anticancer nano-drug system is characterized by comprising the following steps:

(1) reacting and preparing the azido-terminated diselenide micromolecule by taking selenocysteine hydrochloride, acyl chloride compound and azido compound as raw materials;

(2) polyethylene glycol, potassium hydride and a propine compound are used as raw materials to prepare polyethylene glycol with propargyl end capping at two ends through reaction;

(3) reacting the azido-terminated diselenide micromolecules prepared in the step (1) with the two-end propargyl-terminated polyethylene glycol prepared in the step (2) to prepare two-end propargyl-terminated diselenide bond-containing polyethylene glycol alternating copolymers;

(4) polymerizing e-caprolactone, and reacting with azide to prepare azide single-ended polycaprolactone;

(5) reacting the propargyl-terminated polyethylene glycol alternating copolymer containing double selenium bonds at two ends prepared in the step (3) with the azido-singly-terminated polycaprolactone prepared in the step (4) to prepare a rapid oxidation/reduction dual-responsiveness block copolymer containing double selenium bonds;

(6) and (3) mixing the block copolymer with the rapid oxidation/reduction dual responsiveness and containing the double selenium bonds and the anticancer drug, and then carrying out self-assembly and dialysis to prepare the rapid oxidation/reduction dual responsiveness anticancer nano-drug system.

Technical Field

The invention belongs to the field of biomedical high polymer materials, and particularly relates to a preparation method and application of a rapid oxidation/reduction dual-responsiveness block copolymer containing a double selenium bond.

Background

Traditional nano-drug carriers are often prepared from non-degradable polymers such as polyacrylics, polyethylenes, polystyrenes, and the like. These polymeric carriers are not easily degraded after entering the body, are not easily excreted by the kidney, and finally remain and accumulate in the body, which in the past causes many side effects for patients.

To date, polylactic acid (PLA), polyethylene glycol (PEG), Polycaprolactone (PCL), polylactic-co-glycolic acid (PLGA), etc. have been approved by the Food and Drug Administration (FDA) for use in medical materials. Many nano-drug carriers based on such polymers have been developed and used. The polymer material has good biocompatibility and biodegradability, and can be hydrolyzed or enzymolyzed under physiological conditions to form molecules which can be absorbed by organisms or excreted by kidneys. However, they are still insufficient as nano drug carriers, i.e. the polymer itself degrades slowly, and the hydrolysis or enzymolysis rate cannot meet the controllable release of the drug, which may result in insufficient release of the drug at the tumor focus site, and affect the therapeutic effect to some extent. Therefore, in recent years, the synthesis of various biodegradable 'intelligent' nano-drug carriers with good biocompatibility has been proposed.

Compared with the traditional nano-drug carrier, the intelligent nano-drug carrier can be dissociated under the stimulation of in-vivo microenvironment (pH value, redox, enzyme and the like) or external (temperature, light, ultrasonic and the like) conditions, so that the controlled release of the drug is realized. The redox responsiveness of the support is currently a more studied class. The stimulation mainly comes from the reducing or oxidizing environment in the tumor cells, so that chemical bonds with redox responsiveness are broken, the hydrophilic and hydrophobic properties of the polymer are further influenced, finally, the drug-loaded nanoparticles are dissociated, and the drug is rapidly released.

Disclosure of Invention

The invention aims to provide a block copolymer containing double selenium bonds with quick oxidation/reduction dual responsiveness based on polyethylene glycol and polycaprolactone and a preparation method thereof; in the copolymer, selenium is used as a basic trace element in a human body and has an indispensable effect on the health of the human body, the selenium can clear free radicals in the human body, effectively inhibit the generation of lipid peroxide, stimulate the immune response of the human body, improve the protective capability of the immune system and prevent diseases, and more importantly, the selenium is closely and indiscriminately connected with cancer prevention; selenium mainly exists in the form of selenase and selenoprotein in a human body, the invention creatively designs the block copolymer containing double selenium bonds, has the effect of antioxidation, can well protect cell membranes from being damaged by peroxide, prevents the accumulation of the peroxide in the body, reduces the damage of the peroxide to DNA, prevents mutation and achieves the effect of preventing canceration; further, selenium and an element in the same main group as chalcogen are distinguished, however, in that selenium has a larger atomic radius than sulfur and is less electronegative than sulfur, so that the bond energy of the diselenide bond (172 kJ/mol) is lower than the bond energy of the disulfide bond (240 kJ/mol). Thus, the diselenide bond can be oxidized in addition to being reduced, and even has a radiation response characteristic. Therefore, the chemical bond energy of selenium is smaller, the chemical bond is more sensitive, and the application of the double selenium bond in the field of stimulus-responsive drug carriers is greatly promoted.

The specific technical scheme of the invention is as follows: a fast oxidation/reduction dual-responsive double selenium bond-containing block copolymer expressed by the following chemical structural formula:

wherein m is 5-15, n is 4-114, and x is 15-45.

In the technical scheme, the rapid oxidation/reduction dual-responsiveness block copolymer containing the double selenium bonds has the double selenium bonds in the structure, the alternating copolymer of the double selenium component and PEG is a hydrophilic section, and PCL is a hydrophobic section; the formed nano particles have good stability under normal physiological conditions, and under the oxidation and reduction conditions, the double selenium bond is broken, so that the nano particles are destroyed, and the hydrophobic anticancer drugs gathered in the nano particles are rapidly released.

In a preferred embodiment, the number of the fast oxidation/reduction dual-responsive double-selenium bond-containing block copolymersThe average molecular weight is 7220-98250 g × mol^-1(ii) a Wherein the polyethylene glycol repeating chain segment is 15-45, and the polycaprolactone repeating chain segment is 5-15.

The invention adopts a structure containing diselenide and polyethylene glycol (PA-PAG) with the end blocked by propynylaltSeSe-PA) as basic raw material reacts with azido single-terminated polycaprolactone (PCL-N) under the catalysis of copper salt and ligand₃) And preparing the rapid oxidation/reduction dual-responsiveness block copolymer PCL-PEGSeSee-PCL containing the diselenide based on the polyethylene glycol and the polycaprolactone.

The preparation method of the rapid oxidation/reduction dual-responsiveness block copolymer containing diselenide comprises the following steps:

(1) reacting and preparing the azido-terminated diselenide micromolecule by taking selenocysteine hydrochloride, acyl chloride compound and azido compound as raw materials;

(2) polyethylene glycol, potassium hydride and a propine compound are used as raw materials to prepare polyethylene glycol with propargyl end capping at two ends through reaction;

(3) reacting the azido-terminated diselenide micromolecules prepared in the step (1) with the two-end propargyl-terminated polyethylene glycol prepared in the step (2) to prepare two-end propargyl-terminated diselenide bond-containing polyethylene glycol alternating copolymers;

(4) polymerizing e-caprolactone, and reacting with azide to prepare azide single-ended polycaprolactone;

(5) and (3) reacting the propargyl-terminated polyethylene glycol alternating copolymer containing the diselenide bond at two ends prepared in the step (3) with the azido-single-terminated polycaprolactone prepared in the step (4) to prepare the rapid oxidation/reduction dual-responsiveness block copolymer containing the diselenide bond.

In the technical scheme, in the step (1), a reaction product of selenocysteine hydrochloride and an acyl chloride compound is reacted with an azide compound to prepare an azide-terminated diselenide micromolecule; in the step (2), reacting a reaction product of polyethylene glycol and potassium hydride with a propine compound to prepare polyethylene glycol with propargyl groups at two ends blocked; in the step (3), the reaction is carried out in the presence of a copper salt catalyst and a catalyst ligand; in the step (4), initiating the polymerization of e-caprolactone by using small molecular alcohol in the presence of an organic catalyst; in step (5), the reaction is carried out in the presence of a copper salt catalyst and a catalyst ligand. In the step (1), the acyl chloride compound is chloracetyl chloride, and the azide compound is sodium azide; in the step (2), the propyne compound is 3-bromopropyne; in the step (3), the copper salt catalyst is selected from copper sulfate pentahydrate, cuprous chloride or cuprous bromide, and the catalyst ligand is selected from one of sodium ascorbate, bipyridyl, pentamethyldiethylenetriamine, tetramethylethylenediamine or hexamethyltriethylenetetramine; in the step (4), the catalyst is selected from stannous octoate or 1, 8-diazabicycloundecen-7-ene; in the step (5), the copper salt catalyst is selected from copper sulfate pentahydrate, cuprous chloride or cuprous bromide, and the catalyst ligand is selected from one of sodium ascorbate, bipyridyl, pentamethyldiethylenetriamine, tetramethylethylenediamine or hexamethyltriethylenetetramine.

In the technical scheme, in the step (1), the reaction temperature is 25-60 ℃, and the reaction time is 12-40 h; in the step (2), the reaction temperature is 0-50 ℃, and the reaction time is 1-40 h; in the step (3), the reaction temperature is 35-45 ℃, and the reaction time is 24-48 h; in the step (4), the reaction temperature is 60-90 ℃, and the reaction time is 4-24 h; in the step (5), the reaction temperature is 35-45 ℃, and the reaction time is 24-48 h.

In the step (1), selenocysteine hydrochloride is used as a raw material to react with chloroacetyl chloride with high activity, and dichloro-terminated diselenide micromolecules (Cl-SeSe-Cl) are prepared in the presence of a catalyst; further reacting with sodium azide to obtain double selenium micromolecules blocked by azide groups; in the step (2), the KH with high activity is reacted with polyethylene glycol to prepare an oxyanion initiator; further carrying out nucleophilic reaction with 3-bromopropyne to prepare polyethylene glycol (PA-PEG-PA) with propargyl end caps at two ends; in the step (3), in the presence of a copper salt catalyst and a ligand, the bis-selenium micromolecule (N) blocked by azide group₃-SeSe-N₃) Reacting with polyethylene glycol (PA-PEG-PA) with propargyl end caps at two ends to obtain the polyethylene glycol alternating copolymer (PA-PAG) containing diselenide with alkynyl end caps at two endsalt-SeSe-PA); in the step (4), 2-bromoethanol is used as an initiator, and Sn (Oct)₂Initiating the polymerization of e-caprolactone (e-CL) by using ring-opening polymerization as a catalyst to prepare the mono-bromo-terminated polycaprolactone (PCL-Br); further modifying the end group of PCL-Br by sodium azide to prepare azido single-ended polycaprolactone (PCL-N)₃) (ii) a In the step (5), under the existence of a copper salt catalyst and a ligand, the double-selenium-bond-containing polyethylene glycol alternating copolymer with two end alkynyl end caps reacts with the azido single-end-capped polycaprolactone to obtain the quick oxidation/reduction double-responsiveness double-selenium-bond-containing block copolymer. In the step (1), the molar ratio of the selenocysteine hydrochloride to the chloracetyl chloride is 1: (2-10); the molar ratio of dichloro-terminated diselenide small molecules (Cl-SeSe-Cl) to sodium azide is 1: (2-10); in the step (2), the molar ratio of the polyethylene glycol to the potassium hydride to the 3-bromopropyne is 1: (2-6): (2-10); in the step (3), the double selenium micromolecules (N) blocked by the azide group₃-SeSe-N₃) The molar ratio of the catalyst to polyethylene glycol and copper salt catalyst with propargyl end capping at two ends is 1 to (1.05-1.3) to (0.5-1.5); the molar ratio of the copper salt catalyst to the ligand is 1: 1-2; in the step (4), the molar ratio of the 2-bromoethanol to the e-caprolactone is 1: (15-45); in the step (5), the molar ratio of the diselenide-containing polyethylene glycol alternating copolymer with two end alkynyl end caps to the azido single end capped polycaprolactone is 1: (2-4).

The invention also discloses the application of the rapid oxidation/reduction dual-responsiveness block copolymer containing the double selenium bond in the preparation of nano-drugs; or the block copolymer with quick oxidation/reduction dual responsiveness and containing the double selenium bonds is used as a nano-drug carrier.

The invention also discloses a fast oxidation/reduction dual-responsiveness block copolymer nano particle containing a double selenium bond and a preparation method thereof, and the fast oxidation/reduction dual-responsiveness block copolymer nano particle comprises the following steps:

(1) reacting and preparing the azido-terminated diselenide micromolecule by taking selenocysteine hydrochloride, acyl chloride compound and azido compound as raw materials;

(2) polyethylene glycol, potassium hydride and a propine compound are used as raw materials to prepare polyethylene glycol with propargyl end capping at two ends through reaction;

(3) reacting the azido-terminated diselenide micromolecules prepared in the step (1) with the two-end propargyl-terminated polyethylene glycol prepared in the step (2) to prepare two-end propargyl-terminated diselenide bond-containing polyethylene glycol alternating copolymers;

(4) polymerizing e-caprolactone, and reacting with azide to prepare azide single-ended polycaprolactone;

(5) reacting the propargyl-terminated polyethylene glycol alternating copolymer containing double selenium bonds at two ends prepared in the step (3) with the azido-singly-terminated polycaprolactone prepared in the step (4) to prepare a rapid oxidation/reduction dual-responsiveness block copolymer containing double selenium bonds;

(6) and (3) self-assembling and dialyzing the block copolymer containing the double selenium bonds with the fast oxidation/reduction dual responsiveness prepared in the step (5) to prepare the block copolymer nano particles containing the double selenium with the fast oxidation/reduction dual responsiveness. For example, the rapid oxidation/reduction dual-responsive diselenide-containing block copolymer is self-assembled in an aqueous solution to form nanoparticles; the fast oxidation/reduction dual-responsiveness double-selenium-containing segmented copolymer nanoparticles are prepared by a good solvent dialysis method.

The invention also discloses the application of the block copolymer nano particle containing double selenium bonds with quick oxidation/reduction dual responsiveness in preparing nano medicaments; or as a nano-drug carrier.

The invention also discloses a rapid oxidation/reduction dual-responsiveness anticancer nano-drug system and a preparation method thereof, and the preparation method comprises the following steps:

(1) reacting and preparing the azido-terminated diselenide micromolecule by taking selenocysteine hydrochloride, acyl chloride compound and azido compound as raw materials;

(2) polyethylene glycol, potassium hydride and a propine compound are used as raw materials to prepare polyethylene glycol with propargyl end capping at two ends through reaction;

(3) reacting the azido-terminated diselenide micromolecules prepared in the step (1) with the two-end propargyl-terminated polyethylene glycol prepared in the step (2) to prepare two-end propargyl-terminated diselenide bond-containing polyethylene glycol alternating copolymers;

(4) polymerizing e-caprolactone, and reacting with azide to prepare azide single-ended polycaprolactone;

(5) reacting the propargyl-terminated polyethylene glycol alternating copolymer containing double selenium bonds at two ends prepared in the step (3) with the azido-singly-terminated polycaprolactone prepared in the step (4) to prepare a rapid oxidation/reduction dual-responsiveness block copolymer containing double selenium bonds;

(6) and (3) mixing the block copolymer with the rapid oxidation/reduction dual responsiveness and containing the double selenium bonds and the anticancer drug, and then carrying out self-assembly and dialysis to prepare the rapid oxidation/reduction dual responsiveness anticancer nano-drug system. For example, the anti-cancer nano drug-carrying system with the rapid oxidation/reduction dual-responsiveness and containing the double selenium bonds is prepared by mixing the block copolymer with the rapid oxidation/reduction dual-responsiveness and the anti-cancer drug, dissolving the block copolymer with the rapid oxidation/reduction dual-responsiveness and containing the double selenium bonds in a good solvent and dialyzing the mixture in a water phase.

The invention also discloses application of the rapid oxidation/reduction dual-responsiveness anticancer nano drug system in preparation of anticancer drugs, in particular application in preparation of stimulus-responsiveness anticancer nano drug-loaded systems.

In the invention, the anti-cancer drug is selected from one of adriamycin, paclitaxel, camptothecin and curcumin.

Specifically, the following scheme can be adopted by the invention as an example:

(1) synthesizing a dichloro-terminated diselenide micromolecule by taking selenocysteine hydrochloride and chloroacetyl chloride as raw materials, taking dichloromethane as a solvent and triethylamine, pyridine or ethylenediamine as an acid-binding agent through an amide-type reaction; further reacts with sodium azide to prepare the bis-selenium micromolecule (N) blocked by diazide₃-SeSe-N₃）；

Wherein the molar ratio of the selenocysteine hydrochloride to the chloroacetyl chloride is 1: (2-10);

the chemical structural formula of the dichloro-terminated diselenide micromolecule is as follows:

the chemical structural formula of the diazido-terminated diselenide micromolecule is as follows:

(2) polyethylene glycol is used as a raw material, tetrahydrofuran is used as a solvent, and under the anhydrous and oxygen-free conditions, terminal hydroxyl reacts with potassium hydride (KH) to form oxyanions; further reacting with 3-bromopropyne to prepare dialkynyl terminated polyethylene glycol (PA-PEG-PA);

wherein the molar ratio of the reaction of the polyethylene glycol, the potassium hydride and the 3-bromopropyne is 1: (2-6): (2-10);

the chemical structural formula of the oxyanion is as follows:

the chemical structural formula of the dipropargyl-terminated polyethylene glycol is as follows:

n is 4 to 114;

under the condition of inert gas atmosphere, under the existence of copper salt catalyst and ligand, propargyl-terminated polyethylene glycol and diazido-terminated diselenide micromolecules are used as raw materialsAs a ligand, toN,N,Dimethyl formamide as solvent, and preparing polyethylene glycol alternating copolymer (PA-PAG) with two end alkynyl end caps through reactionalt-SeSe-PA)；

Wherein the molar ratio of the propargyl terminated polyethylene glycol to the diazido terminated diselenide micromolecule to the copper salt catalyst is 1: (1.05-1.3): (0.5 to 1.5); the mole ratio of copper salt catalyst and ligand is 1: (1-2);

the chemical structural formula of the diselenide-containing polyethylene glycol alternating copolymer with two end alkynyl end closures is as follows:

m is 5 to 15, n is 4 to 114;

(4) 2-bromoethanol is used as an initiator to initiateRing-opening polymerization is carried out, and modification is carried out to obtain azido single-end-capped polycaprolactone (PCL-N)₃)；

Wherein the content of the first and second substances,and sodium azide at a 1: (15-45): (2-4);

the chemical structural formula of the azido single-terminated polycaprolactone is as follows:

x is 15-45;

(5) under the condition of inert gas atmosphere, in the presence of a copper salt catalyst and a ligand, taking a polyethylene glycol alternating copolymer with two end alkynyl end caps and azido group single end capped polycaprolactone as raw materials

As a ligand, toN,N,Dimethyl formamide is used as a solvent, and a rapid oxidation/reduction dual-responsiveness block copolymer (PCL-PEGSeSE-PCL) containing a dual selenium bond is prepared through reaction;

wherein, the mol ratio of the selenium-containing polyethylene glycol alternating copolymer with two end alkynyl end-capped ends, the azido single end-capped polycaprolactone and the copper salt is 1: (2-4): (0.5 to 1.5); the mole ratio of copper salt catalyst and ligand is 1: (1-2);

the structural formula of the rapid oxidation/reduction dual-responsiveness double-selenium-containing block copolymer is as follows:

m is 5 to 15, n is 4 to 114, and x is 15 to 45.

According to the invention, the selenium-containing polyethylene glycol alternating copolymer raw material with two end alkynyl end closures and the quick oxidation/reduction dual-responsiveness double-selenium-containing block copolymer are prepared by limiting the raw material and parameters in the presence of a copper salt catalyst and a ligand for the first time, so that the problem that the existing reaction system cannot prepare the copolymer with the main chain containing the double-selenium alternating is solved, and the method is a novel, efficient and quick synthesis method.

According to a further technical scheme, after the steps (1) to (5) are finished, products are respectively purified, and the purification process comprises the following steps:

(i) purifying the bisazido-terminated diselenide micromolecules: after the reaction was completed, the crude product was filtered to remove unreacted sodium azide. The DMF solvent was removed under reduced pressure using an oil pump. Adding CH₂Cl₂The concentrated product was dissolved sufficiently and extracted. Collecting organic phase, drying with anhydrous sodium sulfate, filtering, and rotary evaporating to remove CH₂Cl₂A solvent. Putting the obtained product into a vacuum drying oven to be dried to constant weight to obtain a dark yellow solid product;

(ii) purification of propargyl terminated polyethylene glycol: after the reaction was complete, the crude product was filtered and the THF solvent was removed by rotary evaporation. By CH₂Cl₂Extracting with solvent, collecting organic phase, drying with anhydrous sodium sulfate, filtering, and rotary evaporating to remove most CH₂Cl₂A solvent. Precipitating in n-hexane for three times, and drying the product in a vacuum drying oven to constant weight to obtain a light yellow viscous liquid product;

(iii) purifying the diselenide-containing polyethylene glycol alternating copolymer with two end alkynyl end closures: after the reaction is finished, the crude product is passed through neutral Al₂O₃Short chromatographic column of (4). Transferring the solution into dialysis bag (MWCO 7000 Da), dialyzing in secondary water for 48-72 h, periodically changing water, and freeze drying to obtain light yellow solid product;

(iv) purification of azido single-terminated polycaprolactone: after the reaction was completed, the crude product was filtered to remove unreacted sodium azide. Concentrating the reaction solution under reduced pressure with an oil pump, and using CH₂Cl₂And (4) solvent extraction. The organic layer was collected, and dried by adding anhydrous sodium sulfate. Then filtered and rotary evaporated to remove CH₂Cl₂A solvent. Putting the obtained product into a vacuum drying oven to be dried to constant weight to obtain a white solid product;

(v) purification of fast oxidation/reduction dual-responsive diselenide-containing block copolymers: after the reaction is finished, the crude product is passed through neutral Al₂O₃Short chromatographic column of (4). Transferring the solution into dialysis bag (MWCO 7000 Da), dialyzing in secondary water for 48-72 h, periodically changing water, and freeze drying to obtain light yellow solid product.

The invention discloses a rapid oxidation/reduction dual-responsiveness block copolymer PCL-PEGSeSee-PCL containing diselenide, which can be self-assembled in an aqueous solution to form nanoparticles. The hydrophobic polycaprolactone block forms the core of the nanoparticle; the hydrophilic polyethylene glycol chain segment forms the shell of the nano particle, and plays a role in stabilizing the nano particle. The double selenium bond is easy to break under the condition of rapid oxidation/reduction, and the nano particles are destroyed, thereby rapidly releasing the encapsulated hydrophobic anticancer drug. Therefore, the invention requests to protect the application of the rapid oxidation/reduction dual-responsive diselenide-containing block copolymer in the preparation of a stimulus-responsive anticancer nano-drug system.

Due to the implementation of the scheme, compared with the prior art, the invention has the following advantages:

1. according to the invention, hydrophilic polyethylene glycol and hydrophobic polycaprolactone with good biocompatibility are used as raw materials, a double selenium bond is introduced into a main chain of a block copolymer for the first time through a chemical reaction, and the fast response and the dissociation degree of a copolymer carrier in vivo are very excellent; in aqueous solution, the block copolymer can self-assemble to form nano particles and is used as a carrier of a hydrophobic anticancer drug;

2. in the polymer disclosed by the invention, the double selenium bond can be broken under the action of a reducing agent and even under the action of an oxidizing agent, so that the drug-loaded nano particle is endowed with a rapid oxidation/reduction double stimulation response behavior; under normal physiological conditions, the drug-loaded nanoparticles can exist stably and in an oxidizing environment (such as H)₂O₂) Or under reducing conditions (such as glutathione), the double selenium bond can be rapidly broken, and the polymer is dissociated, so that the polymer nanoparticles are rapidly destroyed, and the anticancer drug is rapidly released; in addition, selenium is used as an anticancer element, can be synergistically acted with the medicament, and can well play a role in preventing cancers, so that the medicament-carrying nano particles have potential application value in the aspect of cancer treatment;

3. the quick oxidation/reduction dual-responsiveness block copolymer containing diselenide provided by the invention has a definite structure and mild synthesis conditions, and has the following remarkable characteristics: (1) the raw materials and reagents are easy to obtain; (2) the reaction condition is simple and mild; (3) the yield is high; (4) the product is simple and convenient to separate; (5) the product has good stability and convenient purification.

Drawings

FIG. 1 shows an example of a bisazido-terminated diselenide small molecule (N)₃-SeSe-N₃) And nuclear magnetic resonance hydrogen spectra of the intermediate: (A) NH (NH)₂-SeSe-NH₂；(B) Cl-SeSe-Cl；(C) N₃-SeSe-N₃(ii) a The solvent is deuterated dimethyl sulfoxide;

FIG. 2 shows the bisazido-terminated diselenide small molecule (N) in example one₃-SeSe-N₃) And the infrared spectrogram of the intermediate: (A) NH (NH)₂-SeSe-NH₂; (B) Cl-SeSe-Cl; (C) N₃-SeSe-N₃；

FIG. 3 is the NMR spectrum of diynyl terminated polyethylene glycol (PA-PEG-PA) in example II with deuterated dimethyl sulfoxide as the solvent; (A) HO-PEG-OH; (B) PA-PEG-PA;

FIG. 4 shows two alkynyl-terminated diselenide-containing polyethylene glycol alternating copolymers (PA-PEG-alt-se-PA) nuclear magnetic resonance hydrogen spectrum;

FIG. 5 is a NMR chart of the azido single-capped polycaprolactone and the intermediate of example IV, with deuterated chloroform as the solvent; (A) PCL-Br; (B) PCL-N₃；

FIG. 6 is a gel permeation chromatography outflow curve of the azido single-capped polycaprolactone and intermediates of example IV, (A) PCL-Br; (B) PCL-N₃；

FIG. 7 is the IR spectrum of the azido single-terminated polycaprolactone and the intermediate of example IV, (A) PCL-Br; (B) PCL-N₃；

FIG. 8 is the NMR spectrum of the fast oxidation/reduction dual-responsive diselenide-containing block copolymer (PCL-PEGSeSE-PCL) synthesized in example five, with deuterated dimethyl sulfoxide as the solvent;

FIG. 9 is a gel permeation chromatography elution profile of the azido single-capped polycaprolactone, dialkynyl-capped polyethylene glycol, alkynyl-capped alternating copolymers of diselenide-containing polyethylene glycol, and diselenide-containing triblock copolymers of example V, (A) PA-PEG-PA; (B) PCL-N₃；(C) PA-PEG-alt-SeSe-PA ；(D) PCL-PEGSeSe-PCL；

FIG. 10 is the particle size distribution curve and nanoparticles formed by the fast oxidation/reduction dual-responsive diselenide-containing block copolymer (PCL-PEGSeSE-PCL) in the sixth embodiment in the aqueous solution;

FIG. 11 is a graph showing the particle size change of drug-loaded nanoparticles formed by the fast oxidation/reduction dual-responsive diselenide-containing block copolymer and doxorubicin under different conditions in example seven;

FIG. 12 is an in vitro drug release profile of the drug-loaded nanoparticles of example eight at different Glutathione (GSH) concentrations;

FIG. 13 shows the cell survival rates of the L929 cell and the HeLa cell in the ninth embodiment after culturing with different concentrations of diselenide polymer nanoparticles and drug-loaded nanoparticle solution thereof for 72h, respectively;

FIG. 14 shows the results of endocytosis observed using the living cell workstation in example ten: (A) fluorography of Doxorubicin (DOX) -entrapped nanoparticles with (B) free DOX into HeLa cells (DOX concentration of 1)) (ii) a The fluorescence signals from left to right are blue fluorescence of cell nucleus stained by blue dye Hoechst 33342, red fluorescence spontaneously generated by adriamycin and overlapping fluorescence of the two fluorescence signals respectively;

FIG. 15 is a flow cytometric curve of the loaded nanoparticles and free DOX of example ten.

Detailed Description

The invention is further described below with reference to examples and figures: