WO2014206044A1 - Amphiphilic 4/6 miktoarm star-shaped ph-responsive copolymer and preparation method thereof - Google Patents
Amphiphilic 4/6 miktoarm star-shaped ph-responsive copolymer and preparation method thereof Download PDFInfo
- Publication number
- WO2014206044A1 WO2014206044A1 PCT/CN2013/090020 CN2013090020W WO2014206044A1 WO 2014206044 A1 WO2014206044 A1 WO 2014206044A1 CN 2013090020 W CN2013090020 W CN 2013090020W WO 2014206044 A1 WO2014206044 A1 WO 2014206044A1
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- WO
- WIPO (PCT)
- Prior art keywords
- amphiphilic
- responsive
- hetero
- star copolymer
- arm star
- Prior art date
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Classifications
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- C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
- C08G63/06—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from hydroxycarboxylic acids
- C08G63/08—Lactones or lactides
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/10—Esters
- C08F220/26—Esters containing oxygen in addition to the carboxy oxygen
- C08F220/28—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety
- C08F220/285—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety and containing a polyether chain in the alcohol moiety
- C08F220/286—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety and containing a polyether chain in the alcohol moiety and containing polyethylene oxide in the alcohol moiety, e.g. methoxy polyethylene glycol (meth)acrylate
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
- C08F293/005—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/10—Esters
- C08F220/34—Esters containing nitrogen, e.g. N,N-dimethylaminoethyl (meth)acrylate
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F2438/00—Living radical polymerisation
- C08F2438/01—Atom Transfer Radical Polymerization [ATRP] or reverse ATRP
Definitions
- the invention belongs to the technical field of high molecular polymer materials for biomedicine, and particularly relates to an amphiphilic pH response 4/6 heteroarm star copolymer and a preparation method thereof. Background technique
- Cancer is one of the three major diseases of the 21st century, which seriously jeopardizes human health.
- the World Health Organization's Global Cancer Report shows that it currently kills more than 7 million people worldwide each year.
- the number of cancer deaths worldwide is expected to rise rapidly, reaching more than 13.1 million by 2030.
- the treatment of cancer drugs still faces enormous challenges.
- chemotherapy plays a very important role in the treatment of cancer.
- the ideal chemotherapeutic drug should only act on cancer cells in the lesion, with little or no toxic side effects on normal cells.
- the biodegradable amphiphilic polymer forms a core/shell structure micelle by self-assembly in an aqueous solution, and its use as an anticancer drug carrier has received increasing attention from researchers all over the world. It has the following advantages: (1) The polymer has a large molecular weight, and when used as a carrier, the drug can stay in the lesion for a long time; the cell permeability of the tumor site is hyperthyroidized, and the time for the anticancer drug to exist in the blood circulation is more Long, the more chances of entering the tumor site; (2) the drug can achieve sustained release or controlled release through diffusion or degradation of the polymer itself in the polymer micelle; (3) can have some targeting effects Or the functional component controlling the release of the drug is bonded to the surface of the polymer particle by chemical bonding; (4) The biodegradable polymer material can prevent the carrier material from accumulating in the human organ tissue and causing toxic side effects after the drug is released.
- a star polymer refers to a polymer in which at least three polymer arms are drawn from one core. Generally divided into two categories: One is a star polymer (AB) with the same chemical structure for each arm, and each arm may be a homopolymer, a copolymer or a triblock. Copolymer; another type of heteroarm star polymer with at least two arms different chemical structures, commonly A 2 B 2 , A 3 B 3 , A 2 B, A 3 B, ABC, A 5 B, AB 2 C 2 , ABCD, etc.
- AB star polymer
- star polymer makes it have some special physicochemical properties, such as small hydrodynamic radius, low intrinsic viscosity, low crystallinity, easy formation of stable micelles, etc., which will be widely used in more fields. Applications. Because of its similar topology to polymer micelles, it improves the thermodynamic stability of micelles, and star polymers are well suited for controlled release of anticancer drugs.
- star polymers are synthesized primarily by core-first or arm-first methods.
- the first nuclear rear arm method requires the synthesis of a multifunctional initiator.
- the synthesized star polymer has the same arm length and the number of arms is determined, and the degree of polymerization can be precisely controlled.
- the hind-arm nucleus method requires the synthesis of monofunctional active linear macromolecules.
- the synthesized star-shaped polymer has the same arm length but the number of arms is difficult to determine.
- the aggregates formed by the self-assembly of the star block copolymers have been reported to have spherical, tubular, vesicular, worm-like and complex forms, thereby promoting the potential of the star block copolymer in many fields. Applications such as drug release, separation, optoelectronic materials and catalysis.
- Patent application WO2003078489-A1 discloses a process for preparing a hydrophilic diblock, triblock and star polymer which is transferred by at least one ethylene monomer, macromolecular degradable chain under inert gas protection The agent and the initiator are prepared by polymerization.
- Patent Application 200810107054.2 discloses a three-arm star copolymer tri-arm P (NIPAAm-co-DMAEMA) and its self-assembled micelles that utilize controlled external or in vivo environmental changes to achieve controlled release of the drug.
- Patent application 200910024899.X discloses a star-shaped block acid-sensitive nanomicelle, which is activated by ring-opening polymerization of D,L-lactide by cage-type octasil silsesquioxane, and then with hydrazine, hydrazine -Dimethylaminoethyl-methacrylate (DMAEMA) is synthesized by ATRP reaction, and the controlled release of the drug is achieved by sensitivity to temperature and pH. Summary of the invention
- the primary object of the present invention is to provide an amphipathic pH response. 4/6 heteroarm star copolymer.
- the star-shaped copolymer structure is: a pentaerythritol or dipentaerythritol as a core, followed by a hydrophobic group, a pH-responsive group and a hydrophilic group to form an amphiphilic pH-responsive 4/6 heteroarm star copolymer .
- Another object of the present invention is to provide a process for the preparation of the above star polymer.
- the method comprises the following steps: acylating two or three hydroxyl groups of pentaerythritol (or dipentaerythritol) with an acylating agent to obtain a heterofunctional initiator, then performing ROP polymerization of caprolactone, and sequentially performing a pH response by sequential polymerization.
- ARGET ATRP polymerization of bulk and hydrophilic macromonomers yielded an amphiphilic pH-responsive 4/6 heteroarm star polymer.
- Still another object of the present invention is to provide an application of the above star polymer in the preparation of a water-insoluble drug micelle system.
- the nano-layer polymer micelles in which the inner layer is a hydrophobic group, the intermediate layer is a pH-responsive group, and the outer shell is a hydrophilic group are prepared by dialysis method, and an anticancer drug is wrapped.
- the nano drug carrier enters the cell, it can utilize the acidic environment in the tumor cell to promote the protonation of the pH response layer in the middle, thereby realizing the rapid and controlled release of the drug in the tumor cell.
- the pH-responsive hybrid arm star copolymer structure can increase the pH response sensitivity of the micelle while maintaining high drug loading, more effectively control drug release, and improve drug treatment efficiency.
- Ri is:
- the amphiphilic pH-responsive 4/6 heteroarm star copolymer has a number average molecular weight of 30,000 to 54000 g/moL.
- the preparation method of the amphiphilic pH-responsive 4/6 heteroarm star copolymer includes the following specific methods. Steps:
- polycaprolactone macroinitiator mixing ⁇ -caprolactone, stannous octoate and small molecule initiator, reacting at 110 ⁇ 140 °C for 24 ⁇ 48 h, distillation under reduced pressure, precipitation, filtration, Drying to obtain a polycaprolactone macroinitiator;
- the weight fraction of the reactants in the step (1) is formulated as follows:
- the weight fraction of the reactants in the step (2) is formulated as follows:
- the amount of the toluene used is 2 to 4 mL of toluene per 1 g of the polycaprolactone macroinitiator.
- the precipitation in the step (1) means that the solution is subjected to spin evaporation, and 10 times by volume of a methanol solution having a volume fraction of 0 ° C of 50 ° is precipitated.
- Step (2) The removal of the catalyst means that the reaction product is dissolved in tetrahydrofuran, and the catalyst is removed by passing through a neutral alumina chromatography column and tetrahydrofuran.
- the post-treatment of the filtrate described in the step (2) means that the filtrate is subjected to rotary evaporation, precipitated by adding 10 volumes of n-hexane, filtered, and dried.
- the small molecule initiator refers to pentaerythritol 2-bromoisobutyrate.
- the diester is prepared by the following method: pentaerythritol, 2-bromoisobutyryl bromide and triethylamine in a molar ratio of 1:2:2 are dissolved in tetrahydrofuran (THF), and reacted in an ice water bath for 4-6 hours. Then, the reaction is carried out at room temperature for 16 to 30 h, and after rotary evaporation, precipitation, filtration, and drying, a pentaerythritol diester of 2-bromoisobutyric acid is obtained.
- THF tetrahydrofuran
- the tetrahydrofuran is used in an amount of 20 to 25 mL of THF per 1 g of pentaerythritol.
- the small molecule initiator refers to dipentaerythritol 2-bromoisobutyrate, which is prepared by the following method: a molar ratio of 1:3:3 Dipentaerythritol, 2-bromoisobutyryl bromide and triethylamine are dissolved in THF, reacted in an ice water bath for 4-6 h, then reacted at room temperature for 16-30 h, subjected to rotary evaporation, precipitation, filtration and drying to obtain Dipentaerythritol triester of 2-bromoisobutyrate.
- the amount of THF used is 10 to 15 mL of THF per 1 g of dipentaerythritol.
- the above amphiphilic pH-responsive 4/6 heteroarm star copolymer is used in the preparation of a water-insoluble drug micelle system.
- the water-insoluble drug micelle system is prepared by the following method: dissolving the amphiphilic pH-responsive 4/6 hybrid arm star copolymer and the poorly water-soluble drug in an organic solvent, and stirring at room temperature for 4-6 hours. The deionized water was dialyzed for 24 ⁇ 48 h, and lyophilized to obtain a water-insoluble drug micelle system.
- the poorly water-soluble drug refers to a drug having a solubility of less than or equal to 1 g in 1 L of water.
- the organic solvent refers to at least one of dimethylformamide and dimethyl sulfoxide.
- the water-insoluble drug micelle system can control the slow release of the loaded drug in normal tissues (pH 7.4) and the rapid and controlled release under the weak acidic conditions (pH 5-6) of the tumor cells.
- the mechanism of the invention is:
- the amphiphilic pH-responsive 4/6 heteroarm star copolymer disclosed in the present invention is a drug-loading material having excellent performance and pH sensitivity.
- Polycaprolactone (PCL) acts as a hydrophobic core for micelles to solubilize poorly soluble anticancer drugs.
- the polyethylaminoethyl methacrylate structure (PDEAEMA) obtained by polymerization is a pH-responsive intermediate layer, which is hydrophobic at physiological pH (7.4), and shrinkage on the surface of PCL as a core, which can effectively prevent drug burst phenomenon;
- PDEAEMA polyethylaminoethyl methacrylate structure
- PPEGMA is beneficial to increase the density of micelle shell, enhance the hydrophilicity of the micelle surface, anti-protein and platelet adsorption capacity, improve the stability of micelles, and prolong the cycle time of micelles in the body, improve Controlled release properties of micellar drug delivery systems.
- the release rate of the drug can be regulated by adjusting the content of each group in the polymer molecular material to meet the release requirements of different drugs.
- the preparation method of the present invention is simple in operation, mild in reaction conditions, and easy to adjust the amphiphilic pH response.
- the arm length of the 4/6 heteroarm star copolymer is adjustable in a wide range.
- amphiphilic pH-responsive 4/6 heteroarm star copolymer prepared by the invention is applied to prepare a water-insoluble drug micelle system, which can meet the release requirements of different drugs.
- Figure 1 is a GPC elution curve of (PCL) 2 (PDEAEMA-b-PPEGMA) 2 in Example 1.
- PCL 2 is an NMR spectrum of (PCL) 2 (PDEAEMA-b-PPEGMA) 2 in Example 1, and the solvent is d-CDC13.
- 3 is a GPC elution curve of (PCL) 3 (PDEAEMA-b-PPEGMA) 3 in Example 4.
- FIG. 4 is an NMR spectrum of (PCL) 3 (PDEAEMA-b-PPEGMA) 3 in Example 4, and the solvent is d-CDC13.
- Figure 5 is a graph showing the critical micelle concentration of CPCL) 3 CPDEAEMA-b-PPEGMA) 3 in Example 8.
- Figure 6 is a scanning electron microscope (SEM) image of (PCL) 3 (PDEAEMA-b-PPEGMA) 3 drug-loaded micelles of Example 9.
- Figure 7 is an in vitro release profile of doxorubicin micelles of (PCL) 2 (PDEAEMA-b-PPEGMA) 2 (product of Example 2) in Example 10.
- Figure 8 is an in vitro release profile of doxorubicin-loaded micelles of (PCL) 3 (PDEAEMA-b-PPEGMA) 3 (product of Example 4) in Example 10.
- Figure 9 is an in vitro cytotoxicity of CPCL) 3 CPDEAEMA-b-PPEGMA) 3 blank micelles in Example 11.
- Figure 10 is an in vitro cytotoxicity of (PCL) 2 (PDEAEMA-b-PPEGMA) 2 and (PCL) 3 - (PDEAEMA-b-PPEGMA) 3 doxorubicin micelles in Example 11. detailed description
- Example 1 Amphiphilic pH response Preparation of 4 heteroarm star copolymer
- step (2) prepared (PLC) 2 -Br 2 , bromination Copper (CuBr 2 , Shanghai Xinbao) In an eggplant-shaped bottle, sealed, vacuumed - argon gas 3 times, followed by 18 mL of toluene, monomeric diethylaminoethyl methacrylate (DEAEMA, TCI-EP), The ligand hexamethyltriethylenetetramine (HMTETA, Sigma-Aldrich) was injected into the reaction flask, and the catalyst complex Cu/HMTETA was formed by stirring for 10 min.
- HMTETA ligand hexamethyltriethylenetetramine
- step (2) prepared (PLC) 2 -Br 2 , CuBr 2 (Shanghai Xinbao) In the eggplant bottle, sealed, vacuumed - argon gas 3 times, sequentially inject 18 mL of anhydrous toluene, monomer DEAEMA (TCI-EP), ligand HMTETA (Sigma-Aldrich) into the reaction bottle
- the catalyst complex Cu/HMTETA was formed by stirring for 10 min.
- Example 8 Amphiphilic pH response 6 adjacent micelle concentration of the hybrid arm star copolymer
- the adjacent micelle concentration of the amphiphilic pH-responsive 6-arm star-type copolymer (PCL) 3 - (PDEAEMA-b-PPEGMA) 3 prepared in Example 4 was tested by a fluorescent probe method.
- the DOX drug-loaded micelle I was prepared by using the amphiphilic pH-responsive 4-arm star-type copolymer (PCL) 2 (PDEAEMA-b-PPEGMA) 2 prepared in Example 2 .
- Example 9 produced DOX drug-loaded micelles II.
- Blank micelle preparation Blank micelles of the copolymer of Example 4 were prepared in the same manner as in Example 9 without adding doxorubicin.
- Toxicity test A 96-well flat-bottomed tissue culture plate was taken, and 200 cells (DMEM) were added to the surrounding plates as a blank group. Inoculate HepG2 at a cell concentration of lxlO 4 cells/well (200 ⁇ ) per well in the middle 60 wells Cells (purchased in ATCC), column 2 as a control, 96-well plates were placed in a 37 ° C, saturated humidity, 5 % CO 2 incubator for 48 h.
- Figure 9 shows the toxicity results of blank micelles (product of Example 4).
- (PCL) 3 PDEAEMA-b-PPEGMA 3 is essentially non-toxic to HepG2 cells, at a high concentration of 400 mg/L. Survival rates are still as high as 90%.
- Figure 10 shows the cytotoxicity results of free DOX, DOX-loaded micelle I and DOX-loaded micelle II. It can be seen from the figure that after 48 h incubation, low concentration (0.1 mg/L) of drug-loaded micelles can kill cells and have obvious enhancement; at high concentration (20 mg/L), drug-loaded gel The effect of bunch and free DOX killing cells is similar, with more than 80% of cells killed.
- the DOX drug-loaded micelles prepared by the product of Example 4 have similar cytotoxicity to free doxorubicin, indicating that the adriamycin can effectively maintain its anticancer activity after being encapsulated.
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Abstract
The present invention belongs to the technical field of biomedical high molecular polymer materials and discloses an amphiphilic 4/6 miktoarm star-shaped pH-responsive copolymer with a number-average molecular weight of 30000-54000 g/mol. The present invention also discloses a method for preparing the copolymer: mixing ε-caprolactone, stannous octoate and a small molecule initiator, reacting at 110-140°C for 24-48 h, distilling under reduced pressure, precipitating, filtering and drying to obtain a polycaprolactone macroinitiator; dissolving the polycaprolactone macroinitiator, diethylaminoethyl methacrylate, hexamethyl triethylenetetramine and copper bromide in toluene, adding stannous octoate after stirring, and reacting at 60-90°C for 5-12 h; and adding methacrylic acid ester of mono-methoxylated polyethylene glycol and continuously polymerizing for 5-12 h, removing the catalyst, filtering and postreating the filtrate to obtain the amphiphilic 4/6 miktoarm star-shaped pH-responsive copolymer, which is used to prepare a micellar system for loading water-insoluble drugs.
Description
说 明 书 一种两亲性 pH响应 4/6杂臂星型共聚物及其制备方法 技术领域 Amphiphilic pH response 4/6 heteroarm star copolymer and preparation method thereof
本发明属于生物医药用高分子聚合物材料技术领域， 特别涉及一种两亲性 pH响应 4/6 杂臂星型共聚物及其制备方法。 背景技术 The invention belongs to the technical field of high molecular polymer materials for biomedicine, and particularly relates to an amphiphilic pH response 4/6 heteroarm star copolymer and a preparation method thereof. Background technique
癌症是 21世纪三大疾病之一， 严重危害着人类的健康。 世界卫生组织的全球癌症报告 显示： 目前每年在全球夺去 700多万人的生命， 随着世界人口日趋老龄化， 预计全世界癌症 死亡人数还将快速上升， 到 2030年可能将超过 1310万。 由于癌症细胞的异质性、 多药耐药 性等， 癌药的治疗仍面临着巨大的挑战。 作为抗击癌症最重要的工具之一， 化疗对于癌症的 治疗起着非常重要的作用。理想的化疗药物应该只对病变部位的癌细胞发挥作用， 而不会或 少对正常细胞产生毒副作用。 为了降低毒副作用， 提高药物的利用度， 减少药物的降解及损 失， 人们近年来大力开发各种药物载体体系。这些药物载体体系能改变药物进入人体的方式 和在体内的分布、控制药物的释放速度并将药物输送到靶向器官。为了寻找合适的药物载体， 人们对各种载体体系如聚合物胶束、 水凝胶、 囊泡、 微球、 脂质体、 微乳液等进行了大量的 研究。 其中， 聚合物胶束以其特有优异性质而成为目前研究的热点。 Cancer is one of the three major diseases of the 21st century, which seriously jeopardizes human health. The World Health Organization's Global Cancer Report shows that it currently kills more than 7 million people worldwide each year. As the world's population ages, the number of cancer deaths worldwide is expected to rise rapidly, reaching more than 13.1 million by 2030. Due to the heterogeneity of cancer cells, multidrug resistance, etc., the treatment of cancer drugs still faces enormous challenges. As one of the most important tools to fight cancer, chemotherapy plays a very important role in the treatment of cancer. The ideal chemotherapeutic drug should only act on cancer cells in the lesion, with little or no toxic side effects on normal cells. In order to reduce toxic and side effects, improve drug utilization, and reduce drug degradation and loss, various drug carrier systems have been vigorously developed in recent years. These drug carrier systems can alter the manner in which the drug enters the body and its distribution in the body, control the rate of drug release, and deliver the drug to the targeted organ. In order to find a suitable pharmaceutical carrier, a great deal of research has been conducted on various carrier systems such as polymer micelles, hydrogels, vesicles, microspheres, liposomes, microemulsions and the like. Among them, polymer micelles have become a hot spot of research due to their unique and excellent properties.
可生物降解两亲性聚合物在水溶液中通过自组装形成核 /壳结构的胶束， 其作为抗癌药 物载体越来越受到各国研究者的普遍关注。 它具有以下优点： （1 )聚合物分子量大， 作为载 体使用时能使药物在病灶部位停留较长时间； 肿瘤部位的细胞透过性处于亢进状态， 抗癌药 物在血液循环中存在的时间越长， 进入肿瘤部位的机会就越多； （2)药物在聚合物胶束内能 通过扩散或聚合物自身的降解达到缓释或可控释放的目的； （3 )可以把一些具有靶向作用或 控制药物释放的功能性组分通过化学键合的方式结合到聚合物粒子表面； （4)可生物降解的 聚合物材料， 能够避免药物释放后载体材料在人体器官组织内聚积和产生毒副作用。 The biodegradable amphiphilic polymer forms a core/shell structure micelle by self-assembly in an aqueous solution, and its use as an anticancer drug carrier has received increasing attention from researchers all over the world. It has the following advantages: (1) The polymer has a large molecular weight, and when used as a carrier, the drug can stay in the lesion for a long time; the cell permeability of the tumor site is hyperthyroidized, and the time for the anticancer drug to exist in the blood circulation is more Long, the more chances of entering the tumor site; (2) the drug can achieve sustained release or controlled release through diffusion or degradation of the polymer itself in the polymer micelle; (3) can have some targeting effects Or the functional component controlling the release of the drug is bonded to the surface of the polymer particle by chemical bonding; (4) The biodegradable polymer material can prevent the carrier material from accumulating in the human organ tissue and causing toxic side effects after the drug is released.
星型聚合物是指一个核中至少引出三个聚合物臂的聚合物。一般分为两大类： 一类为每 条臂均具有相同化学结构的星型聚合物（AB)， 每条臂上可能是均聚物、 共聚物或者三嵌段
共聚物； 另一类为至少两条臂化学结构不相同的杂臂星型聚合物， 常见的有 A2B2、 A3B3、 A2B、 A3B、 ABC、 A5B、 AB2C2、 ABCD等。 星形聚合物其独特的化学结构使其具有一些特 殊的物理化学性能， 如流体力学半径小、 特性粘度低、 结晶度低、 易于形成稳定的胶束等， 将在更多的领域里得到广泛的应用。 因为具有与聚合物胶束相似的拓扑结构， 可提高胶束的 热力学稳定性， 星形聚合物非常适用于抗癌药物的控制释放。 A star polymer refers to a polymer in which at least three polymer arms are drawn from one core. Generally divided into two categories: One is a star polymer (AB) with the same chemical structure for each arm, and each arm may be a homopolymer, a copolymer or a triblock. Copolymer; another type of heteroarm star polymer with at least two arms different chemical structures, commonly A 2 B 2 , A 3 B 3 , A 2 B, A 3 B, ABC, A 5 B, AB 2 C 2 , ABCD, etc. The unique chemical structure of star polymer makes it have some special physicochemical properties, such as small hydrodynamic radius, low intrinsic viscosity, low crystallinity, easy formation of stable micelles, etc., which will be widely used in more fields. Applications. Because of its similar topology to polymer micelles, it improves the thermodynamic stability of micelles, and star polymers are well suited for controlled release of anticancer drugs.
作为树枝状聚合物的典型代表， 星形聚合物主要通过先核后臂法 （core-first) 或先臂后 核法 （arm-first) 方式合成。 先核后臂法需要先合成多功能引发剂， 合成的星型聚合物的臂 长一致且臂数确定， 可以精确控制聚合度。 先臂后核法需要先合成单官能活性线性大分子， 合成的星型聚合物的臂长一致但臂数难以确定。 As a typical representative of dendrimers, star polymers are synthesized primarily by core-first or arm-first methods. The first nuclear rear arm method requires the synthesis of a multifunctional initiator. The synthesized star polymer has the same arm length and the number of arms is determined, and the degree of polymerization can be precisely controlled. The hind-arm nucleus method requires the synthesis of monofunctional active linear macromolecules. The synthesized star-shaped polymer has the same arm length but the number of arms is difficult to determine.
目前已报道的星形嵌段共聚物经自组装所形成的聚集体的形态有球状、 管状、 囊泡状、 蠕虫状和复合状等，从而促进了星形嵌段共聚物在诸多领域的潜在应用，如药物缓释、分离、 光电材料和催化等。在药物缓释方面， Lang等（Journal of Colloid and Interface Science, 2011, 364: 92.) 采用直链聚乙二醇 -b-聚己内酯 （PEG-b-PCL) 和六臂星形聚己内酯 -b-聚甲基丙烯 酸二乙氨基乙酯 （S(PCL-b-PDEAEMA)) 制备了一种混合胶束， 有效提高胶束的稳定性。将 其包载硝苯地平后， 利用 PDEAEMA亲疏水性随 pH值的变化来控制硝苯地平的释放速率。 Cao等 （Biomacromolecules, 2011, 12(7): 2697. ) 制备了一种以聚丙交酯 （PLLA) 为内核， 聚乙二醇 （PEG) 为亲水外壳， 表面偶联叶酸的星形单分子胶束用于阿霉素的靶向给药， 该 胶束在 pH 7.4 时释放速率很慢， 而在 pH 5.0 时释放速率明显加快。 专利申请 WO2003078489-A1公布了一种制备亲水性两嵌段、 三嵌段及星型聚合物的制备方法， 在惰 性气体保护下， 由至少一种乙烯单体、 大分子可降解性链转移剂及引发剂聚合制备而得。专 利申请 200810107054.2公布了一种三臂星型共聚物 tri-arm P(NIPAAm-co-DMAEMA)及其自 组装胶束，利用外部的或体内固有的环境变化实现对药物的控释。专利申请 200910024899.X 公布了一种星型嵌段酸敏性纳米胶束，它是由笼型八聚硅倍半氧烷引发 D,L-丙交酯活性开环 聚合， 再与 Ν,Ν-二甲氨基乙基-甲基丙烯酸酯（DMAEMA)进行 ATRP反应所合成的， 通过 对温度和 pH敏感实现对药物的控释。 发明内容 The aggregates formed by the self-assembly of the star block copolymers have been reported to have spherical, tubular, vesicular, worm-like and complex forms, thereby promoting the potential of the star block copolymer in many fields. Applications such as drug release, separation, optoelectronic materials and catalysis. In drug release, Lang et al (Journal of Colloid and Interface Science, 2011, 364: 92.) used linear polyethylene glycol-b-polycaprolactone (PEG-b-PCL) and six-arm star clustering Caprolactone-b-polyethylaminoethyl methacrylate (S(PCL-b-PDEAEMA)) A mixed micelle was prepared to effectively improve the stability of the micelle. After it was loaded with nifedipine, the release rate of nifedipine was controlled by the change of hydrophilicity of PDEAEMA with pH. Cao et al. (Biomacromolecules, 2011, 12(7): 2697.) prepared a star-shaped single molecule with polylactide (PLLA) as the core, polyethylene glycol (PEG) as the hydrophilic shell, and surface-coupled with folic acid. The micelles were used for the targeted administration of doxorubicin, which released slowly at pH 7.4, and the release rate was significantly accelerated at pH 5.0. Patent application WO2003078489-A1 discloses a process for preparing a hydrophilic diblock, triblock and star polymer which is transferred by at least one ethylene monomer, macromolecular degradable chain under inert gas protection The agent and the initiator are prepared by polymerization. Patent Application 200810107054.2 discloses a three-arm star copolymer tri-arm P (NIPAAm-co-DMAEMA) and its self-assembled micelles that utilize controlled external or in vivo environmental changes to achieve controlled release of the drug. Patent application 200910024899.X discloses a star-shaped block acid-sensitive nanomicelle, which is activated by ring-opening polymerization of D,L-lactide by cage-type octasil silsesquioxane, and then with hydrazine, hydrazine -Dimethylaminoethyl-methacrylate (DMAEMA) is synthesized by ATRP reaction, and the controlled release of the drug is achieved by sensitivity to temperature and pH. Summary of the invention
为了克服上述现有技术的缺点与不足， 本发明的首要目的在于提供一种两亲性 pH响应
4/6杂臂星型共聚物。 In order to overcome the above disadvantages and disadvantages of the prior art, the primary object of the present invention is to provide an amphipathic pH response. 4/6 heteroarm star copolymer.
该星型共聚物结构为： 以季戊四醇或二季戊四醇为核， 分别依次接上疏水性基团， pH 响应基团和亲水性基团形成两亲性 pH响应 4/6杂臂星形共聚物。 The star-shaped copolymer structure is: a pentaerythritol or dipentaerythritol as a core, followed by a hydrophobic group, a pH-responsive group and a hydrophilic group to form an amphiphilic pH-responsive 4/6 heteroarm star copolymer .
本发明另一目的在于提供一种上述星型聚合物的制备方法。 Another object of the present invention is to provide a process for the preparation of the above star polymer.
该方法为先用酰化剂将季戊四醇（或二季戊四醇）的两个或三个羟基进行酰化得到杂官 能团引发剂，然后进行己内酯的 ROP聚合，再采用连续聚合法依次进行 pH响应单体和亲水 大分子单体的 ARGET ATRP聚合制得两亲性 pH响应 4/6杂臂星形聚合物。 The method comprises the following steps: acylating two or three hydroxyl groups of pentaerythritol (or dipentaerythritol) with an acylating agent to obtain a heterofunctional initiator, then performing ROP polymerization of caprolactone, and sequentially performing a pH response by sequential polymerization. ARGET ATRP polymerization of bulk and hydrophilic macromonomers yielded an amphiphilic pH-responsive 4/6 heteroarm star polymer.
本发明再一目的在于提供上述星型聚合物在制备装载水难溶性药物胶束系统中的应用。 采用透析法制备内层为疏水基团， 中间层为 pH响应基团、 外壳为亲水基团的纳米级聚 合物胶束， 并包裹抗癌药物。 这种纳米药物载体进入细胞后， 能利用肿瘤细胞内的酸性环境 促使其中间 pH响应层发生质子化， 从而实现药物在肿瘤细胞内的快速可控释放。 这种 pH 响应杂臂星形共聚物结构可以在维持高载药量的前提下提高胶束的 pH响应灵敏度， 更有效 的控制药物释放， 提高药物的治疗效率。 Still another object of the present invention is to provide an application of the above star polymer in the preparation of a water-insoluble drug micelle system. The nano-layer polymer micelles in which the inner layer is a hydrophobic group, the intermediate layer is a pH-responsive group, and the outer shell is a hydrophilic group are prepared by dialysis method, and an anticancer drug is wrapped. After the nano drug carrier enters the cell, it can utilize the acidic environment in the tumor cell to promote the protonation of the pH response layer in the middle, thereby realizing the rapid and controlled release of the drug in the tumor cell. The pH-responsive hybrid arm star copolymer structure can increase the pH response sensitivity of the micelle while maintaining high drug loading, more effectively control drug release, and improve drug treatment efficiency.
本发明的目的通过下述方案实现： The object of the invention is achieved by the following scheme:
一种两亲性 pH响应 4/6杂臂星型共聚物， 具有公式 （1 ) 或公式 （2) 所示结构： An amphiphilic pH-responsive 4/6 heteroarm star copolymer having the structure shown in formula (1) or formula (2):
Ri为: Ri is:
R2为:
x=17~53, y=10~41 , z=12~29。 R 2 is: x=17~53, y=10~41, z=12~29.
所述两亲性 pH响应 4/6杂臂星型共聚物的数均分子量为 30000~54000 g/moL 所述两亲性 pH响应 4/6杂臂星型共聚物的制备方法， 包括以下具体步骤： The amphiphilic pH-responsive 4/6 heteroarm star copolymer has a number average molecular weight of 30,000 to 54000 g/moL. The preparation method of the amphiphilic pH-responsive 4/6 heteroarm star copolymer includes the following specific methods. Steps:
( 1 )制备聚己内酯大分子引发剂：将 ε-己内酯、辛酸亚锡及小分子引发剂混合， 110〜140 °C下反应 24~48 h， 减压蒸馏、 沉淀、 过滤、 干燥， 得到聚己内酯大分子引发剂； (1) Preparation of polycaprolactone macroinitiator: mixing ε-caprolactone, stannous octoate and small molecule initiator, reacting at 110~140 °C for 24~48 h, distillation under reduced pressure, precipitation, filtration, Drying to obtain a polycaprolactone macroinitiator;
(2)制备两亲性 pH响应 4/6臂杂臂星型共聚物： 将步骤 （1 ) 制备的聚己内酯大分子引 发剂、 甲基丙烯酸二乙氨基乙酯、 六甲基三亚乙基四胺、 溴化铜溶于甲苯中， 搅拌后加入 辛酸亚锡， 60~90 °C下反应 5~12 h; 再加入甲基丙烯酸单甲氧基聚乙二醇酯连续聚合 5~12 h， 去除催化剂， 过滤， 滤液后处理， 得到两亲性 pH响应 4/6杂臂星型共聚物。 (2) Preparation of amphiphilic pH-responsive 4/6-arm heteroarm star copolymer: Polycaprolactone macroinitiator prepared in step (1), diethylaminoethyl methacrylate, hexamethyltriphenyl The tetraamine and copper bromide are dissolved in toluene, stirred and then added with stannous octoate, reacted at 60~90 °C for 5~12 h ; then added with monomethoxypolyethylene glycol methacrylate for continuous polymerization 5~12 h, removing the catalyst, filtering, and post-treatment of the filtrate to obtain an amphiphilic pH-responsive 4/6 heteroarm star copolymer.
所述步骤 （1 ) 中反应物的重量份数配方如下： The weight fraction of the reactants in the step (1) is formulated as follows:
小分子引发剂 2.35-13.43份 Small molecule initiator 2.35-13.43 parts
ε-己内酯 86.51-97.52 Ε-caprolactone 86.51-97.52
辛酸亚锡 0.06-0.13份； Stannous octoate 0.06-0.13 parts;
所述步骤 （2) 中反应物的重量份数配方如下： The weight fraction of the reactants in the step (2) is formulated as follows:
聚己内酯大分子引发剂 12.41-26.75份 Polycaprolactone macroinitiator 12.41-26.75
溴化铜 0.04-0.08份 Copper bromide 0.04-0.08 parts
六甲基三亚乙基四胺 0.24-0.83份 Hexamethyltriethylenetetramine 0.24-0.83 parts
辛酸亚锡 0.42~1.44份 Stannous octoate 0.42~1.44 parts
甲基丙烯酸二乙氨基乙酯 17.72-31.03份 Diethylaminoethyl methacrylate 17.72-31.03 parts
甲基丙烯酸单甲氧基聚乙二醇酯 49.09-58.97份 Monomethoxy polyethylene glycol methacrylate 49.09-58.97 parts
所述甲苯的用量为每 1 g聚己内酯大分子引发剂使用 2〜4 mL甲苯。 The amount of the toluene used is 2 to 4 mL of toluene per 1 g of the polycaprolactone macroinitiator.
步骤 （1 ) 所述的沉淀均指往旋转蒸发后溶液加入 10倍体积的 0 °C体积分数为 50 %的 甲醇水溶液进行沉淀。 The precipitation in the step (1) means that the solution is subjected to spin evaporation, and 10 times by volume of a methanol solution having a volume fraction of 0 ° C of 50 ° is precipitated.
步骤 （2) 所述的去除催化剂指将反应产物溶解于四氢呋喃后， 过中性氧化铝层析柱， 四氢呋喃洗脱， 除去催化剂。 Step (2) The removal of the catalyst means that the reaction product is dissolved in tetrahydrofuran, and the catalyst is removed by passing through a neutral alumina chromatography column and tetrahydrofuran.
步骤（2)所述的滤液后处理指将滤液旋蒸蒸发、加入 10倍体积的正己烷中沉淀、过滤、 干燥。 The post-treatment of the filtrate described in the step (2) means that the filtrate is subjected to rotary evaporation, precipitated by adding 10 volumes of n-hexane, filtered, and dried.
当制备两亲性 pH响应 4杂臂星型共聚物时， 所述小分子引发剂指 2-溴异丁酸季戊四醇
二酯， 由以下方法制备得到： 将摩尔比为 1:2:2的季戊四醇、 2-溴异丁酰溴、 三乙胺溶解于 四氢呋喃 （THF) 中， 在冰水浴中反应 4~6 h， 然后在室温下反应 16~30 h， 经旋转蒸发、沉 淀、 过滤、 干燥， 得到 2-溴异丁酸季戊四醇二酯。 When preparing an amphiphilic pH-responsive 4-arm star-type copolymer, the small molecule initiator refers to pentaerythritol 2-bromoisobutyrate. The diester is prepared by the following method: pentaerythritol, 2-bromoisobutyryl bromide and triethylamine in a molar ratio of 1:2:2 are dissolved in tetrahydrofuran (THF), and reacted in an ice water bath for 4-6 hours. Then, the reaction is carried out at room temperature for 16 to 30 h, and after rotary evaporation, precipitation, filtration, and drying, a pentaerythritol diester of 2-bromoisobutyric acid is obtained.
所述四氢呋喃的用量为每 1 g季戊四醇使用 20~25 mL THF。 The tetrahydrofuran is used in an amount of 20 to 25 mL of THF per 1 g of pentaerythritol.
当制备两亲性 pH响应 6杂臂星型共聚物时， 所述小分子引发剂指 2-溴异丁酸二季戊四 醇三酯， 由以下方法制备得到： 将摩尔比为 1:3:3的二季戊四醇、 2-溴异丁酰溴、 三乙胺溶 解于 THF中， 在冰水浴中反应 4~6 h， 然后在室温下反应 16~30 h， 经旋转蒸发、 沉淀、 过 滤、 干燥， 得到 2-溴异丁酸二季戊四醇三酯。 When preparing an amphiphilic pH-responsive 6-arm star-type copolymer, the small molecule initiator refers to dipentaerythritol 2-bromoisobutyrate, which is prepared by the following method: a molar ratio of 1:3:3 Dipentaerythritol, 2-bromoisobutyryl bromide and triethylamine are dissolved in THF, reacted in an ice water bath for 4-6 h, then reacted at room temperature for 16-30 h, subjected to rotary evaporation, precipitation, filtration and drying to obtain Dipentaerythritol triester of 2-bromoisobutyrate.
所述 THF的用量为每 1 g二季戊四醇使用 10~15 mL THF。 The amount of THF used is 10 to 15 mL of THF per 1 g of dipentaerythritol.
上述两亲性 pH响应 4/6杂臂星型共聚物在制备装载水难溶性药物胶束系统中的应用。 所述装载水难溶性药物胶束系统由以下方法制备得到： 将两亲性 pH响应 4/6杂臂星型 共聚物和水难溶性药物溶于有机溶剂中， 室温下搅拌 4~6 h， 去离子水透析 24~48 h， 冷冻干 燥， 得到装载水难溶性药物胶束系统。 The above amphiphilic pH-responsive 4/6 heteroarm star copolymer is used in the preparation of a water-insoluble drug micelle system. The water-insoluble drug micelle system is prepared by the following method: dissolving the amphiphilic pH-responsive 4/6 hybrid arm star copolymer and the poorly water-soluble drug in an organic solvent, and stirring at room temperature for 4-6 hours. The deionized water was dialyzed for 24~48 h, and lyophilized to obtain a water-insoluble drug micelle system.
所述的水难溶性药物指在 1 L水中溶解度小于或等于 1 g的药物。 The poorly water-soluble drug refers to a drug having a solubility of less than or equal to 1 g in 1 L of water.
所述的有机溶剂指二甲基甲酰胺和二甲基亚砜中的至少一种。 The organic solvent refers to at least one of dimethylformamide and dimethyl sulfoxide.
所述的装载水难溶性药物胶束系统可控制装载的药物在正常组织（pH 7.4)时缓慢释放， 在肿瘤细胞弱酸性条件 （pH 5~6) 下快速可控释放。 The water-insoluble drug micelle system can control the slow release of the loaded drug in normal tissues (pH 7.4) and the rapid and controlled release under the weak acidic conditions (pH 5-6) of the tumor cells.
本发明的机理为： The mechanism of the invention is:
本发明公开的两亲性 pH响应 4/6杂臂星型共聚物是性能优异、 具有 pH敏感性的载药 材料。 聚己内酯 （PCL) 作为胶束的疏水内核， 用于增溶难溶性抗癌药物。 聚合得到的聚甲 基丙烯酸二乙氨基乙酯结构 （PDEAEMA) 为 pH响应中间层， 在生理 pH ( 7.4) 下是疏水 状态， 收缩在 PCL表面共同作为内核， 可以有效防止药物突释现象； 在肿瘤组织弱酸性条 件下侧链的氨基发生质子化作用带正电， 易与带负电的细胞膜发生吸附作用， 进而被胞吞到 细胞内； 进入溶酶体的强酸性环境后， 进一步质子化， 胶束发生溶胀， 将药物释放到细胞质 和细胞核中。 PPEGMA作为亲水外层， 有利于提高胶束壳层密度， 增强胶束表面的亲水性、 抗蛋白和血小板吸附能力， 提高胶束的稳定性， 从而延长胶束在体内的循环时间， 改善胶束 载药体系的控释性能。 可通过调节聚合物分子材料中各基团的含量来调控药物的释放速率， 满足不同药物的释放要求。
本发明相对于现有技术具有如下的优点及有益效果： The amphiphilic pH-responsive 4/6 heteroarm star copolymer disclosed in the present invention is a drug-loading material having excellent performance and pH sensitivity. Polycaprolactone (PCL) acts as a hydrophobic core for micelles to solubilize poorly soluble anticancer drugs. The polyethylaminoethyl methacrylate structure (PDEAEMA) obtained by polymerization is a pH-responsive intermediate layer, which is hydrophobic at physiological pH (7.4), and shrinkage on the surface of PCL as a core, which can effectively prevent drug burst phenomenon; Under the weak acid condition of tumor tissue, the protonation of the amino group of the side chain is positively charged, and it is easy to adsorb with the negatively charged cell membrane, and then is swallowed into the cell; after entering the strong acidic environment of lysosome, further protonation, The micelles swell and release the drug into the cytoplasm and nucleus. As a hydrophilic outer layer, PPEGMA is beneficial to increase the density of micelle shell, enhance the hydrophilicity of the micelle surface, anti-protein and platelet adsorption capacity, improve the stability of micelles, and prolong the cycle time of micelles in the body, improve Controlled release properties of micellar drug delivery systems. The release rate of the drug can be regulated by adjusting the content of each group in the polymer molecular material to meet the release requirements of different drugs. The present invention has the following advantages and advantageous effects over the prior art:
( 1 )本发明的制备方法操作简单， 反应条件温和， 易于调控两亲性 pH响应 4/6杂臂星 型共聚物的臂长， 分子量在较宽的范围内可调。 (1) The preparation method of the present invention is simple in operation, mild in reaction conditions, and easy to adjust the amphiphilic pH response. The arm length of the 4/6 heteroarm star copolymer is adjustable in a wide range.
(2)本发明制备得到的两亲性 pH响应 4/6杂臂星型共聚物应用于制备装载水难溶性药 物胶束系统， 可满足不同药物的释放要求。 附图说明 (2) The amphiphilic pH-responsive 4/6 heteroarm star copolymer prepared by the invention is applied to prepare a water-insoluble drug micelle system, which can meet the release requirements of different drugs. DRAWINGS
图 1为实施例 1中 (PCL)2(PDEAEMA-b-PPEGMA)2的 GPC洗脱曲线。 Figure 1 is a GPC elution curve of (PCL) 2 (PDEAEMA-b-PPEGMA) 2 in Example 1.
图 2为实施例 1中 (PCL)2(PDEAEMA-b-PPEGMA)2的 NMR谱， 溶剂为 d-CDC13。 图 3为实施例 4中 (PCL)3(PDEAEMA-b-PPEGMA)3的 GPC洗脱曲线。 2 is an NMR spectrum of (PCL) 2 (PDEAEMA-b-PPEGMA) 2 in Example 1, and the solvent is d-CDC13. 3 is a GPC elution curve of (PCL) 3 (PDEAEMA-b-PPEGMA) 3 in Example 4.
图 4为实施例 4中 (PCL)3(PDEAEMA-b-PPEGMA)3的 NMR谱， 溶剂为 d-CDC13。 图 5为实施例 8中 CPCL)3CPDEAEMA-b-PPEGMA)3的临界胶束浓度测试曲线。 4 is an NMR spectrum of (PCL) 3 (PDEAEMA-b-PPEGMA) 3 in Example 4, and the solvent is d-CDC13. Figure 5 is a graph showing the critical micelle concentration of CPCL) 3 CPDEAEMA-b-PPEGMA) 3 in Example 8.
图 6为实施例 9中 (PCL)3(PDEAEMA-b-PPEGMA)3载药胶束的扫描电镜 （SEM) 图。 图 7为实施例 10中 (PCL)2(PDEAEMA-b-PPEGMA)2 (实施例 2产物） 载阿霉素胶束的 体外释放曲线。 Figure 6 is a scanning electron microscope (SEM) image of (PCL) 3 (PDEAEMA-b-PPEGMA) 3 drug-loaded micelles of Example 9. Figure 7 is an in vitro release profile of doxorubicin micelles of (PCL) 2 (PDEAEMA-b-PPEGMA) 2 (product of Example 2) in Example 10.
图 8为实施 10中 (PCL)3(PDEAEMA-b-PPEGMA)3 (实施例 4产物） 载阿霉素胶束的体 外释放曲线。 Figure 8 is an in vitro release profile of doxorubicin-loaded micelles of (PCL) 3 (PDEAEMA-b-PPEGMA) 3 (product of Example 4) in Example 10.
图 9为实施 11中 CPCL)3CPDEAEMA-b-PPEGMA)3空白胶束的体外细胞毒性。 Figure 9 is an in vitro cytotoxicity of CPCL) 3 CPDEAEMA-b-PPEGMA) 3 blank micelles in Example 11.
图 10为实施 11中（PCL)2(PDEAEMA-b-PPEGMA)2和 (PCL)3- (PDEAEMA-b-PPEGMA)3 载阿霉素胶束的体外细胞毒性。 具体实施方式 Figure 10 is an in vitro cytotoxicity of (PCL) 2 (PDEAEMA-b-PPEGMA) 2 and (PCL) 3 - (PDEAEMA-b-PPEGMA) 3 doxorubicin micelles in Example 11. detailed description
下面结合实施例和附图对本发明作进一步详细的描述， 但本发明的实施方式不限于此。 实施例 1 : 两亲性 pH响应 4杂臂星型共聚物的制备 The present invention will be further described in detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto. Example 1 : Amphiphilic pH response Preparation of 4 heteroarm star copolymer
( 1 )小分子引发剂 2-溴异丁酸季戊四醇二酯的制备： 于 250 mL三口烧瓶中称取 6.08 g ( 0.05 mol) 季戊四醇 （Alfa-Aesar)， 加入 150 mL无水 THF， 通氩气 lO min除氧。 密封烧 瓶后， 注入 10.12 g ( 0.1 mol) 三乙胺 （TEA， AR， 江苏永华）。 0 °C冰水浴中， 逐滴注入 22.98 g ( 0.1 mol) 2-溴异丁酰溴 （ Alfa-Aesar)， 搅拌反应 4 h， 再在室温 25 °C下搅拌反应
30 ho 将反应液转入分液漏斗中， 加入 300 mL乙醚， 并依次用 200 mL水、 200 mL0.5 M的 碳酸氢钠溶液和 200 mL水萃取， 有机相用无水硫酸镁干燥后过滤， 溶液旋蒸得到白色固体 (产率为 56 %)， 乙醚重结晶两次后在 40 °C、 35 mb真空干燥 24 h，合成反应式见公式（3 )， 得到 2-溴异丁酸季戊四醇二酯。 (1) Preparation of small molecule initiator pentaerythritol diester of 2-bromoisobutyrate: 6.08 g (0.05 mol) of pentaerythritol (Alfa-Aesar) was weighed into a 250 mL three-necked flask, and 150 mL of anhydrous THF was added thereto, and argon gas was passed. lO min to remove oxygen. After the flask was sealed, 10.12 g (0.1 mol) of triethylamine (TEA, AR, Jiangsu Yonghua) was injected. In a 0 °C ice water bath, 22.98 g (0.1 mol) of 2-bromoisobutyryl bromide (Alfa-Aesar) was injected dropwise, and the reaction was stirred for 4 h, and then stirred at room temperature at 25 ° C. 30 ho The reaction solution was transferred to a separatory funnel, 300 mL of diethyl ether was added, and the mixture was extracted with 200 mL of water, 200 mL of 0.5 M sodium hydrogencarbonate solution and 200 mL of water. The organic phase was dried over anhydrous magnesium sulfate and filtered. The solution was obtained by rotary evaporation to give a white solid (yield: 56%). Recrystallized from diethyl ether twice and dried at 40 ° C, 35 mb under vacuum for 24 h. The reaction was carried out by the formula (3) to obtain 2-bromoisobutyric acid pentaerythritol. Diester.
(2) 聚己内酯大分子引发剂 ((PLC)2-Br2) 的制备： 将 0.43 g步骤 ( 1 ) 制备得到的 2- 溴异丁酸季戊四醇二酯加入烧瓶中， 橡皮塞密封， 抽真空-通氩气 3次， 氩气保护下加入 6 g ε-己内酯 （ε-CL, Sigma-Aldrich) 和 0.01 g辛酸亚锡 （Sn(Oct)2， 国药集团）， 用液氮进行三 次冷冻-抽气-升温循环后， 在氩气保护下置于 130 °C油浴中反应 24 h后用液氮终止反应。减 压蒸馏后， 用 50 mL THF溶解， 加入 500 mL体积分数为 50 %的冷甲醇水溶液， 沉淀后过 滤， 产物在 40 °C、 35 mb真空干燥 24 h， 得到白色粉末， 合成反应式见公式 （4)。 产率为 83 %， „=5007, PDI=1.49。 (2) Preparation of polycaprolactone macroinitiator ((PLC) 2 -Br 2 ): 0.43 g of pentaerythritol 2-bromoisobutyrate prepared in step (1) was added to the flask, and the rubber stopper was sealed. Vacuuming - argon gas 3 times, adding 6 g ε-caprolactone (ε-CL, Sigma-Aldrich) and 0.01 g stannous octoate (Sn(Oct) 2 , Sinopharm Group) under argon protection, using liquid nitrogen After three freeze-pump-heat-up cycles were carried out, the reaction was carried out in an oil bath at 130 ° C for 24 h under argon atmosphere and then quenched with liquid nitrogen. After distillation under reduced pressure, it was dissolved in 50 mL of THF, and 500 mL of a 50% cold methanol aqueous solution was added. The precipitate was precipitated and filtered. The product was dried under vacuum at 40 ° C and 35 mb for 24 h to obtain a white powder. (4). The yield was 83%, „=5007, PDI=1.49.
(3 )两亲性 pH响应 4杂臂星型共聚物（CPCL)2CPDEAEMA-b-PPEGMA)2) 的制备： 取 4.8 g步骤 （2) 制备得到的 (PLC)2-Br2、 溴化铜 （CuBr2， 上海新宝） 于茄形瓶中， 密封， 抽真空-通氩气 3次， 依次将 18 mL甲苯、 单体甲基丙烯酸二乙氨基乙酯 （DEAEMA, TCI-EP)、配体六甲基三亚乙基四胺（HMTETA, Sigma-Aldrich)注入反应瓶中，搅拌 lO min 使催化剂配合物 Cu/HMTETA形成。 将 Sn(Oct)2 (0.26 g) 溶于 2 mL甲苯中， 注入反应瓶。 搅拌 5 min后转入 70 °C油浴中反应 7 h; 再注入甲基丙烯酸单甲氧基聚乙二醇酯（PEGMA， „=475, Sigma-Aldrich)进行连续聚合， 反应 7 h后， 冷却至室温， 加入 50 mL THF并搅拌 溶解， 过中性氧化铝柱子， 去除催化剂， 得到反应液浓缩至约 50 mL后加入到 500 mL正己 烷中沉淀， 过滤。 产物在 40 °C、 35 mb真空干燥 24 h， 得到白色粉末， 合成反应式见公式(3) Preparation of amphiphilic pH response 4 heteroarm star copolymer (CPCL) 2 CPDEAEMA-b-PPEGMA) 2 ): Take 4.8 g of step (2) prepared (PLC) 2 -Br 2 , bromination Copper (CuBr 2 , Shanghai Xinbao) In an eggplant-shaped bottle, sealed, vacuumed - argon gas 3 times, followed by 18 mL of toluene, monomeric diethylaminoethyl methacrylate (DEAEMA, TCI-EP), The ligand hexamethyltriethylenetetramine (HMTETA, Sigma-Aldrich) was injected into the reaction flask, and the catalyst complex Cu/HMTETA was formed by stirring for 10 min. Dissolve Sn(Oct) 2 (0.26 g) in 2 mL of toluene and inject into the reaction flask. After stirring for 5 min, it was transferred to a 70 °C oil bath for 7 h . Then, methacrylic acid monomethoxypolyethylene glycol ester (PEGMA, „=475, Sigma-Aldrich) was injected to carry out continuous polymerization. After 7 h, After cooling to room temperature, add 50 mL of THF and stir to dissolve, pass through a neutral alumina column, remove the catalyst, and concentrate the reaction solution to about 50 mL, then add to 500 mL of n-hexane to precipitate and filter. The product is at 40 ° C, 35 mb. Dry under vacuum for 24 h to obtain a white powder.
(5 )， 利用 GPC测定其分子量， 并进行核磁分析， 见图 1和图 2。产率为 93 %， „=28200, PDI=1.28。 (5), the molecular weight was measured by GPC, and nuclear magnetic analysis was performed, as shown in Fig. 1 and Fig. 2. The yield was 93%, „=28200, PDI=1.28.
其中， 各反应物的重量份数配方如下： Wherein, the weight fraction of each reactant is formulated as follows:
(PCL)2-Br2 19.65份 (PCL) 2 -Br 2 19.65 parts
CuBr2 0.06份 CuBr 2 0.06 parts
HMTETA 0.61份 HMTETA 0.61 servings
Sn(Oct)2 1.06份 Sn(Oct) 2 1.06 parts
DEAEMA 19.65份
PEGMA 58.96份 DEAEMA 19.65 PEGMA 58.96 parts
公式 (3 ) Formula (3)
公式 （4) Formula (4)
公式 (5 ) 实施例 2: 两亲性 pH响应 4杂臂星型共聚物的制备 Formula (5) Example 2: Amphiphilic pH response Preparation of 4 heteroarm star copolymer
( 1 )小分子引发剂 2-溴异丁酸季戊四醇二酯的制备： 于 250 mL三口烧瓶中称取 6.08 g ( 0.05 mol) 季戊四醇 （Alfa-Aesar)， 加入 150 mL无水 THF， 通氩气 lO min除氧。 密封烧 瓶后， 注入 10.12 g (0.1 mol) TEA (AR， 江苏永华）。 0 °C冰水浴中， 逐滴注入 22.98 g (0.1 mol) 2-溴异丁酰溴 （Alfa-Aesar)， 搅拌反应 6 h， 再在室温 25 °C下搅拌反应 16 h。 将反应 液转入分液漏斗中， 加入 300 mL乙醚， 并依次用 200 mL水、 200 mL0.5 M的碳酸氢钠溶液 和 200 mL水萃取，有机相用无水硫酸镁干燥后过滤，溶液旋蒸得到白色固体（产率为 56 % )， 乙醚重结晶两次后在 40 °C、 35 mb真空干燥 24 h， 得到 2-溴异丁酸季戊四醇二酯。 (1) Preparation of small molecule initiator pentaerythritol diester of 2-bromoisobutyrate: 6.08 g (0.05 mol) of pentaerythritol (Alfa-Aesar) was weighed into a 250 mL three-necked flask, and 150 mL of anhydrous THF was added thereto, and argon gas was passed. lO min to remove oxygen. After sealing the flask, 10.12 g (0.1 mol) TEA (AR, Jiangsu Yonghua) was injected. In a 0 °C ice water bath, 22.98 g (0.1 mol) of 2-bromoisobutyryl bromide (Alfa-Aesar) was added dropwise, the reaction was stirred for 6 h, and the reaction was stirred at room temperature 25 ° C for 16 h. The reaction solution was transferred to a separatory funnel, and 300 mL of diethyl ether was added thereto, and extracted with 200 mL of water, 200 mL of 0.5 M sodium hydrogencarbonate solution and 200 mL of water, and the organic phase was dried over anhydrous magnesium sulfate and filtered. The mixture was evaporated to give a white solid (yield: 56%). The crystals were recrystallized twice and then dried at 40 ° C, 35 mb under vacuum for 24 h to give the 2-bromoisobutyric acid pentaerythritol diester.
(2) (PCL)2-Br2的制备： 将 0.6 g步骤 （1 ) 制备得到的 2-溴异丁酸季戊四醇二酯加入 烧瓶中，橡皮塞密封，抽真空-通氩气 3次，氩气保护下加入 9 g ε-CL ( Sigma- Aldrich)和 0.01 g Sn(Oct)2 (国药集团）， 用液氮进行三次冷冻-抽气-升温循环后， 在氩气保护下置于 140 °C 油浴中反应 24 h后用液氮终止反应。减压蒸馏后， 用 50 mL THF溶解， 加入 500 mL体积分
数为 50 %的冷甲醇水溶液， 沉淀后过滤， 产物在 40 °C、 35 mb真空干燥 24 h， 得到白色粉 末。 产率为 96 %， „=8633, PDI=1.53。 (2) Preparation of (PCL) 2 -Br 2 : 0.6 g of the pentaerythritol dibromide 2-bromoisobutyrate prepared in the step (1) was added to the flask, the rubber stopper was sealed, and vacuum was applied - argon gas 3 times, argon Under the protection of gas, 9 g ε-CL (Sigma-Aldrich) and 0.01 g Sn(Oct) 2 (Zhongguo Group) were added, and after three times of freezing-exhausting-heating cycle with liquid nitrogen, it was placed under 140 ° under argon protection. After reacting for 24 hours in the C oil bath, the reaction was terminated with liquid nitrogen. After distillation under reduced pressure, dissolve with 50 mL of THF, add 500 mL of volume A 50% aqueous solution of cold methanol was added, and the mixture was filtered, and the product was dried under vacuum at 40 ° C for 24 h to give a white powder. The yield was 96%, „=8633, PDI=1.53.
(3 )两亲性 pH响应 4杂臂星型共聚物（CPCL)2CPDEAEMA-b-PPEGMA)2) 的制备： 取 7.2 g步骤 （2) 制备得到的 (PLC)2-Br2、 CuBr2 (上海新宝） 于茄形瓶中， 密封， 抽真空-通 氩气 3次， 依次将 18 mL甲苯、单体 DEAEMA (TCI-EP)、配体 HMTETA ( Sigma- Aldrich) 注入反应瓶中， 搅拌 lO min使催化剂配合物 Cu/HMTETA形成。 将 Sn(Oct)2 (0.32 g) 溶于 2 mL甲苯中，注入反应瓶。搅拌 5 min后转入 60 °C油浴中反应 5 h;再注入 PEGMA(Mn=475， Sigma- Aldrich) 进行连续聚合， 反应 12 h后， 冷却至室温， 加入 50 mL THF并搅拌溶解， 过中性氧化铝柱子， 去除催化剂， 得到反应液浓缩至约 50 mL后加入到 500 mL正己烷中沉 淀， 过滤。 产物在 40 °C、 35 mb真空干燥 24 h， 得到白色粉末。 产率为 98 %， „=28524, PDI=1.43。 (3) Preparation of amphiphilic pH response 4 heteroarm star copolymer (CPCL) 2 CPDEAEMA-b-PPEGMA) 2 ): Take 7.2 g of (PLC) 2 -Br 2 , CuBr 2 prepared in step (2) (Shanghai Xinbao) In the eggplant bottle, sealed, vacuumed - argon gas 3 times, in turn 18 mL of toluene, monomer DEAEMA (TCI-EP), ligand HMTETA (Sigma-Aldrich) into the reaction bottle, The catalyst complex Cu/HMTETA was formed by stirring for 10 min. Sn(Oct) 2 (0.32 g) was dissolved in 2 mL of toluene and poured into a reaction flask. After stirring for 5 min, transfer to a 60 °C oil bath for 5 h; then inject PEGMA (M n = 475, Sigma-Aldrich) for continuous polymerization. After 12 h of reaction, cool to room temperature, add 50 mL of THF and stir to dissolve. After passing through a neutral alumina column, the catalyst was removed, and the reaction liquid was concentrated to about 50 mL, and then added to 500 mL of n-hexane to precipitate, and filtered. The product was dried under vacuum at 40 ° C for 24 h at 40 ° C to give a white powder. The yield was 98%, „=28524, PDI=1.43.
其中， 各反应物的重量份数配方如下： Wherein, the weight fraction of each reactant is formulated as follows:
(PCL)2-Br2 23.05份 (PCL) 2 -Br 2 23.05 servings
CuBr2 0.05份 0.05 parts of CuBr 2
HMTETA 0.29份 HMTETA 0.29 parts
Sn(Oct)2 1.04份 Sn(Oct) 2 1.04 parts
DEAEMA 19.21份 DEAEMA 19.21
PEGMA 56.36份。 实施例 3: 两亲性 pH响应 4杂臂星型共聚物的制备 56.36 parts of PEGMA. Example 3: Amphiphilic pH response Preparation of 4 heteroarm star copolymer
( 1 )小分子引发剂 2-溴异丁酸季戊四醇二酯的制备： 于 250 mL三口烧瓶中称取 6.08 g ( 0.05 mol) 季戊四醇 （Alfa-Aesar)， 加入 150 mL无水 THF， 通氩气 10 min除氧。 密封烧 瓶后， 注入 10.12 g (0.1 mol) TEA (AR， 江苏永华）。 0 °C冰水浴中， 逐滴注入 22.98 g (0.1 mol) 2-溴异丁酰溴 （Alfa-Aesar)， 搅拌反应 5 h， 再在室温 25 °C下搅拌反应 24 h。 将反应 液转入分液漏斗中， 加入 300 mL乙醚， 并依次用 200 mL水、 200 mL0.5 M的碳酸氢钠溶液 和 200 mL水萃取，有机相用无水硫酸镁干燥后过滤，溶液旋蒸得到白色固体（产率为 56 %)， 乙醚重结晶两次后在 40 °C、 35 mb真空干燥 24 h， 得到 2-溴异丁酸季戊四醇二酯。 (1) Preparation of small molecule initiator pentaerythritol diester of 2-bromoisobutyrate: 6.08 g (0.05 mol) of pentaerythritol (Alfa-Aesar) was weighed into a 250 mL three-necked flask, and 150 mL of anhydrous THF was added thereto, and argon gas was passed. Oxygen removal for 10 min. After sealing the flask, 10.12 g (0.1 mol) TEA (AR, Jiangsu Yonghua) was injected. In a 0 °C ice water bath, 22.98 g (0.1 mol) of 2-bromoisobutyryl bromide (Alfa-Aesar) was added dropwise, the reaction was stirred for 5 h, and the reaction was stirred at room temperature 25 ° C for 24 h. The reaction solution was transferred to a separatory funnel, and 300 mL of diethyl ether was added thereto, and extracted with 200 mL of water, 200 mL of 0.5 M sodium hydrogencarbonate solution and 200 mL of water, and the organic phase was dried over anhydrous magnesium sulfate and filtered. The mixture was evaporated to give a white solid (yield: 56%). The crystals were recrystallized twice and then dried at 40 ° C, 35 mb vacuum for 24 h to give the 2-bromoisobutyric acid pentaerythritol diester.
(2) (PCL)2-Br2的制备： 将 0.29 g步骤 （1 ) 制备得到的 2-溴异丁酸季戊四醇二酯加入
烧瓶中，橡皮塞密封，抽真空-通氩气 3次，氩气保护下加入 6 g ε-CL ( Sigma- Aldrich)和 0.01 g Sn(Oct)2 (国药集团）， 用液氮进行三次冷冻-抽气-升温循环后， 在氩气保护下置于 120 °C 油浴中反应 48 h后用液氮终止反应。减压蒸馏后， 用 50 mL THF溶解， 加入 500 mL体积分 数为 50 %的冷甲醇水溶液， 沉淀后过滤， 产物在 40 °C、 35 mb真空干燥 24 h， 得到白色粉 末。 产率为 96 %， „=5741 , PDI= 1.38 ο (2) Preparation of (PCL) 2 -Br 2 : 0.29 g of pentaerythritol diester of 2-bromoisobutyric acid prepared in the step (1) In the flask, the rubber stopper was sealed, vacuumed - argon gas was applied 3 times, and 6 g ε-CL (Sigma-Aldrich) and 0.01 g Sn(Oct) 2 (Nippon Pharmaceutical Group) were added under argon protection, and the mixture was frozen three times with liquid nitrogen. - After the pumping-heating cycle, the reaction was carried out in an oil bath at 120 °C for 48 h under argon atmosphere and then quenched with liquid nitrogen. After distillation under reduced pressure, it was dissolved in 50 mL of THF, and 500 mL of a 50% aqueous solution of cold methanol was added thereto, and the mixture was precipitated and filtered. The product was dried under vacuum at 40 ° C for 35 h to give a white powder. The yield is 96%, „=5741, PDI= 1.38 ο
(3 )两亲性 ρΗ响应 4杂臂星型共聚物（CPCL)2CPDEAEMA-b-PPEGMA)2) 的制备： 取 4.8 g步骤 （2) 制备得到的 (PLC)2-Br2、 CuBr2 (上海新宝） 于茄形瓶中， 密封， 抽真空-通 氩气 3次，依次将 18 mL无水甲苯、单体 DEAEMA (TCI-EP)、配体 HMTETA( Sigma- Aldrich) 注入反应瓶中， 搅拌 lO min使催化剂配合物 Cu/HMTETA形成。 将 Sn( ct)2 (0.16 g) 溶于 2 mL甲苯中，注入反应瓶。搅拌 5 min后转入 90 °C油浴中反应 10 h;再注入 PEGMA(Mn=475， Sigma- Aldrich) 进行连续聚合， 反应 8 h后， 冷却至室温， 加入 50 mL THF并搅拌溶解，过 中性氧化铝柱子，去除催化剂，得到反应液浓缩至约 50 mL后加入到 500 mL 正己烷中沉淀， 过滤。产物在 40 °C、35 mb真空干燥 24 h，得到白色粉末。产率为 76 %, „=40470, PDI=1.37。 (3) Preparation of amphiphilic ρΗ response 4 heteroarm star copolymer (CPCL) 2 CPDEAEMA-b-PPEGMA) 2 ): Take 4.8 g of step (2) prepared (PLC) 2 -Br 2 , CuBr 2 (Shanghai Xinbao) In the eggplant bottle, sealed, vacuumed - argon gas 3 times, sequentially inject 18 mL of anhydrous toluene, monomer DEAEMA (TCI-EP), ligand HMTETA (Sigma-Aldrich) into the reaction bottle The catalyst complex Cu/HMTETA was formed by stirring for 10 min. Dissolve Sn( ct) 2 (0.16 g) in 2 mL of toluene and inject into the reaction flask. After stirring for 5 min, transfer to a 90 °C oil bath for 10 h; then inject PEGMA (M n = 475, Sigma-Aldrich) for continuous polymerization. After 8 h of reaction, cool to room temperature, add 50 mL of THF and stir to dissolve. After passing through a neutral alumina column, the catalyst was removed, and the reaction liquid was concentrated to about 50 mL, and then added to 500 mL of n-hexane to precipitate, and filtered. The product was dried under vacuum at 40 ° C for 24 h at 40 ° C to give a white powder. The yield was 76%, „=40470, PDI=1.37.
其中， 各反应物的重量份数配方如下： Wherein, the weight fraction of each reactant is formulated as follows:
(PCL)2-Br2 12.41份 (PCL) 2 -Br 2 12.41
CuBr2 0.04份 CuBr 2 0.04 parts
HMTETA 0.24份 HMTETA 0.24 parts
Sn(Oct)2 0.42份 Sn(Oct) 2 0.42 parts
DEAEMA 31.03份 DEAEMA 31.03
PEGMA 55.86份。 实施例 4: 两亲性 pH响应 6杂臂星型共聚物的制备 55.86 parts of PEGMA. Example 4: Amphiphilic pH response Preparation of 6 heteroarm star copolymer
( 1 )小分子引发剂 2-溴异丁酸二季戊四醇三酯的制备：于 250 mL三口烧瓶中称取 12.72 g (0.05 mol)二季戊四醇 （Alfa-Aesar)， 加入 150 mL无水 THF， 通氩气 10 min除氧。 密封 烧瓶后， 注入 15.17 g (0.15 mol) TEA (AR， 江苏永华）。 0 °C冰水浴中， 逐滴注入 34.48 g (1) Preparation of small molecule initiator 2-bromoisobutyric acid dipentaerythritol triester: 12.72 g (0.05 mol) of dipentaerythritol (Alfa-Aesar) was weighed into a 250 mL three-necked flask, and 150 mL of anhydrous THF was added. Argon was deaerated for 10 min. After sealing the flask, 15.17 g (0.15 mol) TEA (AR, Jiangsu Yonghua) was injected. In a 0 °C ice water bath, drop in. 34.48 g
( 0.15 mol) 2-溴异丁酰溴 （Alfa-Aesar)， 搅拌反应 4 h， 再在室温 25 °C下搅拌反应 30 h。 将反应液转入分液漏斗中， 加入 300 mL乙醚， 并依次用 200 mL水、 200 mL0.5 M的碳酸氢 钠溶液和 200 mL水萃取， 有机相用无水硫酸镁干燥后过滤， 溶液旋蒸得到白色固体 （产率
为 43 %)， 乙醚重结晶两次后在 40 °C、 35 mb真空干燥 24 h， 得到 2-溴异丁酸二季戊四醇 三酯。 (0.15 mol) 2-Bromoisobutyryl bromide (Alfa-Aesar), stirred for 4 h, and stirred at room temperature for 25 h at 25 °C. The reaction solution was transferred to a separatory funnel, and 300 mL of diethyl ether was added thereto, and successively extracted with 200 mL of water, 200 mL of 0.5 M sodium hydrogencarbonate solution and 200 mL of water, and the organic phase was dried over anhydrous magnesium sulfate and filtered. Rotary distillation to give a white solid (yield 43%), diethyl ether was recrystallized twice and dried under vacuum at 40 ° C, 35 mb for 24 h to obtain dipentaerythritol triester of 2-bromoisobutyric acid.
(2) (PCL)3-Br3的制备： 将 0.93 g步骤 （1 ) 制备得到的 2-溴异丁酸二季戊四醇三酯加 入烧瓶中， 橡皮塞密封， 抽真空-通氩气 3次， 氩气保护下加入 6 g ε-CL (Sigma-Aldrich)和 0.01 g Sn(OCt)2 (国药集团）， 用液氮进行三次冷冻-抽气-升温循环后， 在氩气保护下置于 130 °C油浴中反应 24 h后用液氮终止反应。减压蒸馏后， 用 50 mL THF溶解， 加入 500 mL体积 分数为 50 %的冷甲醇水溶液， 沉淀后过滤， 产物在 40 °C、 35 mb真空干燥 24 h， 得到白色 粉末。 产率为 100 %， „=6090, PDI= 1.28ο (2) Preparation of (PCL) 3 -Br 3 : 0.93 g of the dipentaerythritol 2-bromoisobutyrate prepared in the step (1) was added to the flask, the rubber stopper was sealed, and the vacuum was applied - argon gas 3 times. Under the protection of argon, 6 g ε-CL (Sigma-Aldrich) and 0.01 g Sn(O C t) 2 (Zhongguo Group) were added, and after three times of freezing-exhausting-heating cycle with liquid nitrogen, under argon protection After reacting for 24 h in a 130 ° C oil bath, the reaction was quenched with liquid nitrogen. After distillation under reduced pressure, it was dissolved in 50 mL of THF, and 500 mL of a 50% aqueous solution of cold methanol was added thereto, and the mixture was precipitated and filtered. The product was dried under vacuum at 40 ° C for 35 h to give a white powder. The yield is 100%, „=6090, PDI= 1.28ο
(3 )两亲性 ρΗ响应 6杂臂星型共聚物（(PCL)3(PDEAEMA-b-PPEGMA)3) 的制备： 取 4.8 g步骤 （2) 制备得到的 (PLC)3-Br3、 CuBr2 (上海新宝） 于茄形瓶中， 密封， 抽真空-通 氩气 3 次， 依次将 18 mL无水甲苯、 单体 DEAEMA ( TCI-EP )、 配体 HMTETA(3) Preparation of amphiphilic ρΗ response 6-arm star copolymer ((PCEA) 3 (PDEAEMA-b-PPEGMA) 3 ): Take 4.8 g of step (2) prepared (PLC) 3 -Br 3 , CuBr 2 (Shanghai Xinbao) in an eggplant-shaped bottle, sealed, vacuumed - argon gas 3 times, followed by 18 mL of anhydrous toluene, monomer DEAEMA (TCI-EP), ligand HMTETA
( Sigma-Aldrich)注入反应瓶中，搅拌 10 min使催化剂配合物 Cu/HMTETA形成。将 Sn(Oct)2 ( 0.26 g) 溶于 2 mL甲苯中， 注入反应瓶。 搅拌 5 min后转入 80 °C油浴中反应 8 h; 再注 入 PEGMA (Mn=475， Sigma-Aldrich)进行连续聚合， 反应 6 h后， 冷却至室温， 加入 50 mL THF并搅拌溶解， 过中性氧化铝柱子， 去除催化剂， 得到反应液浓缩至约 50 mL后加入到 500 mL 正己烷中沉淀， 过滤。 产物在 40 °C、 35 mb真空干燥 24 h， 得到白色粉末， 利用 GPC测定其分子量， 并进行核磁分析， 见图 3和图 4。 产率为 67 %， „=23100, PDI=1.31。 (Sigma-Aldrich) was injected into the reaction flask and stirred for 10 min to form a catalyst complex Cu/HMTETA. Sn(Oct) 2 (0.26 g) was dissolved in 2 mL of toluene and poured into a reaction flask. After stirring for 5 min, transfer to an 80 °C oil bath for 8 h; then inject PEGMA (M n = 475, Sigma-Aldrich) for continuous polymerization. After 6 h, cool to room temperature, add 50 mL of THF and stir to dissolve. After passing through a neutral alumina column, the catalyst was removed, and the reaction solution was concentrated to about 50 mL, and then added to 500 mL of n-hexane to precipitate and filtered. The product was dried under vacuum at 40 ° C for 24 h at 40 ° C to give a white powder. The molecular weight was determined by GPC and subjected to nuclear magnetic analysis, see Fig. 3 and Fig. 4. The yield was 67%, „=23100, PDI=1.31.
其中， 各反应物的重量份数配方如下： Wherein, the weight fraction of each reactant is formulated as follows:
(PCL)3-Br3 26.75份 (PCL) 3 -Br 3 26.75 parts
CuBr2 0.08份 CuBr 2 0.08 parts
HMTETA 0.82份 HMTETA 0.82 servings
Sn(Oct)2 1.44份 Sn(Oct) 2 1.44 parts
DEAEMA 17.72份 DEAEMA 17.72
PEGMA 53.17份。 实施例 5: 两亲性 pH响应 6杂臂星型共聚物的制备 PEGMA 53.17 parts. Example 5: Amphiphilic pH response Preparation of 6 heteroarm star copolymer
( 1 )小分子引发剂 2-溴异丁酸二季戊四醇三酯的制备：于 250 mL三口烧瓶中称取 12.72 g (0.05 mol)二季戊四醇 （Alfa-Aesar)， 加入 150 mL无水 THF， 通氩气 10 min除氧。 密封
烧瓶后， 注入 15.17 g (0.15 mol) TEA (AR， 江苏永华）。 0 °C冰水浴中， 逐滴注入 34.48 g (0.15 mol) 2-溴异丁酰溴 （Alfa-Aesar)， 搅拌反应 6 h， 再在室温 25°C下搅拌反应 16 h。将 反应液转入分液漏斗中， 加入 300 mL乙醚， 并依次用 200 mL水、 200 mL0.5 M的碳酸氢钠 溶液和 200 mL水萃取， 有机相用无水硫酸镁干燥后过滤， 溶液旋蒸得到白色固体 （产率为 43 %), 乙醚重结晶两次后在 40 °C、 35 mb真空干燥 24 h， 得到 2-溴异丁酸二季戊四醇三 酯。 (1) Preparation of small molecule initiator 2-bromoisobutyric acid dipentaerythritol triester: 12.72 g (0.05 mol) of dipentaerythritol (Alfa-Aesar) was weighed into a 250 mL three-necked flask, and 150 mL of anhydrous THF was added. Argon was deaerated for 10 min. Seal After the flask, 15.17 g (0.15 mol) TEA (AR, Jiangsu Yonghua) was injected. In a 0 °C ice water bath, 34.48 g (0.15 mol) of 2-bromoisobutyryl bromide (Alfa-Aesar) was added dropwise, the reaction was stirred for 6 h, and the reaction was stirred at room temperature 25 ° C for 16 h. The reaction solution was transferred to a separatory funnel, and 300 mL of diethyl ether was added thereto, and successively extracted with 200 mL of water, 200 mL of 0.5 M sodium hydrogencarbonate solution and 200 mL of water, and the organic phase was dried over anhydrous magnesium sulfate and filtered. The residue was evaporated to give a white solid (yield: 43%).
(2) (PCL)3-Br3的制备： 将 0.93 g步骤 （1 ) 制备得到的 2-溴异丁酸二季戊四醇三酯加 入烧瓶中， 橡皮塞密封， 抽真空-通氩气 3次， 氩气保护下加入 12 g s-CL ( Sigma-Aldrich) 和 0.01 g Sn(OCt)2 (国药集团）， 用液氮进行三次冷冻-抽气-升温循环后， 在氩气保护下置于 110 °C油浴中反应 48 h后用液氮终止反应。 减压蒸馏后， 用 50 mL THF溶解， 加入 500 mL 体积分数为 50 %的冷甲醇水溶液， 沉淀后过滤， 产物在 40 °C、 35 mb真空干燥 24 h， 得到 白色粉末。 产率为 92 %， „=11031 , PDI= 1.36 ο (2) Preparation of (PCL) 3 -Br 3 : 0.93 g of the dipentaerythritol 2-bromoisobutyrate prepared in the step (1) was added to the flask, the rubber stopper was sealed, and the vacuum was applied - argon gas 3 times. Add 12 g s-CL (Sigma-Aldrich) and 0.01 g Sn(O C t) 2 (Zhongguo Group) under argon protection, and perform three freeze-pump-heating cycles with liquid nitrogen, and then place under argon protection. After reacting for 48 h in an oil bath at 110 ° C, the reaction was terminated with liquid nitrogen. After distillation under reduced pressure, it was dissolved in 50 mL of THF, and 500 mL of a 50% aqueous solution of cold methanol was added thereto, and the mixture was precipitated and filtered. The product was dried at 40 ° C, 35 mb under vacuum for 24 h to give a white powder. The yield is 92%, „=11031, PDI= 1.36 ο
(3 )两亲性 ρΗ响应 6杂臂星型共聚物（(PCL)3(PDEAEMA-b-PPEGMA)3) 的制备： 取 6.36 g步骤 （2) 制备得到的 (PLC)3-Br3、 CuBr2 (上海新宝） 于茄形瓶中， 密封， 抽真空- 通氩气 3次， 依次将 18 mL无水甲苯、 单体 DEAEMA ( TCI-EP )、 配体 HMTETA(3) Preparation of amphiphilic ρΗ response 6-arm star-type copolymer ((PCL) 3 (PDEAEMA-b-PPEGMA) 3 ): Take 6.36 g of step (2) prepared (PLC) 3 -Br 3 , CuBr 2 (Shanghai Xinbao) in an eggplant-shaped bottle, sealed, vacuumed - argon gas 3 times, followed by 18 mL of anhydrous toluene, monomer DEAEMA (TCI-EP), ligand HMTETA
( Sigma-Aldrich)注入反应瓶中，搅拌 10 min使催化剂配合物 Cu/HMTETA形成。将 Sn(Oct)2 (0.16 g) 溶于 2 mL甲苯中， 注入反应瓶。 搅拌 5 min后转入 60 °C油浴中反应 12 h; 再注 入 PEGMA (Mn=475， Sigma-Aldrich)进行连续聚合， 反应 5 h后， 冷却至室温， 加入 50 mL THF并搅拌溶解， 过中性氧化铝柱子， 去除催化剂， 得到反应液浓缩至约 50 mL后加入到 500 mL 正己烷中沉淀， 过滤。 产物在 40 °C、 35 mb真空干燥 24 h， 得到白色粉末。 产率为 81 %， „=39270, PDI=1.43。 (Sigma-Aldrich) was injected into the reaction flask and stirred for 10 min to form a catalyst complex Cu/HMTETA. Dissolve Sn(Oct) 2 (0.16 g) in 2 mL of toluene and inject into the reaction flask. After stirring for 5 min, transfer to a 60 °C oil bath for 12 h ; then inject PEGMA (M n = 475, Sigma-Aldrich) for continuous polymerization. After 5 h of reaction, cool to room temperature, add 50 mL of THF and stir to dissolve. After passing through a neutral alumina column, the catalyst was removed, and the reaction solution was concentrated to about 50 mL, and then added to 500 mL of n-hexane to precipitate and filtered. The product was dried under vacuum at 40 ° C for 24 h at 40 ° C to give a white powder. The yield was 81%, „=39270, PDI=1.43.
其中， 各反应物的重量份数配方如下： Wherein, the weight fraction of each reactant is formulated as follows:
(PCL)3-Br3 24.72份 (PCL) 3 -Br 3 24.72
CuBr2 0.06份 CuBr 2 0.06 parts
HMTETA 0.36份 HMTETA 0.36 parts
Sn(Oct)2 0.63份 Sn(Oct) 2 0.63 parts
DEAEMA 18.66份 DEAEMA 18.66
PEGMA 55.58份。
实施例 6: 两亲性 pH响应 6杂臂星型共聚物的制备 55.58 parts of PEGMA. Example 6: Preparation of Amphiphilic pH Response 6 Heteroarm Star Copolymer
( 1 )小分子引发剂 2-溴异丁酸二季戊四醇三酯的制备：于 250 mL三口烧瓶中称取 12.72 g (0.05 mol)二季戊四醇 （Alfa-Aesar)， 加入 150 mL无水 THF， 通氩气 lOmin除氧。 密封 烧瓶后， 注入 15.17 g (0.15 mol) TEA (AR， 江苏永华）。 0 °C冰水浴中， 逐滴注入 34.48 g (1) Preparation of small molecule initiator 2-bromoisobutyric acid dipentaerythritol triester: 12.72 g (0.05 mol) of dipentaerythritol (Alfa-Aesar) was weighed into a 250 mL three-necked flask, and 150 mL of anhydrous THF was added. Argon gas was deoxidized at 10 min. After sealing the flask, 15.17 g (0.15 mol) TEA (AR, Jiangsu Yonghua) was injected. In a 0 °C ice water bath, drop in. 34.48 g
(0.15 mol) 2-溴异丁酰溴 （Alfa-Aesar)， 搅拌反应 5 h， 再在室温 25 °C下搅拌反应 24h。 将反应液转入分液漏斗中， 加入 300 mL乙醚， 并依次用 200 mL水、 200 mL0.5 M的碳酸氢 钠溶液和 200 mL水萃取， 有机相用无水硫酸镁干燥后过滤， 溶液旋蒸得到白色固体 （产率 为 43%)， 乙醚重结晶两次后在 40 °C、 35mb真空干燥 24h， 得到 2-溴异丁酸二季戊四醇 三酯。 (0.15 mol) 2-Bromoisobutyryl bromide (Alfa-Aesar), stirred for 5 h, and stirred at room temperature for 25 h at 25 °C. The reaction solution was transferred to a separatory funnel, and 300 mL of diethyl ether was added thereto, and successively extracted with 200 mL of water, 200 mL of 0.5 M sodium hydrogencarbonate solution and 200 mL of water, and the organic phase was dried over anhydrous magnesium sulfate and filtered. The residue was evaporated to give a white solid (yield: 43%).
(2) (PCL)3-Br3的制备： 将 0.07 g步骤 （1) 制备得到的 2-溴异丁酸二季戊四醇三酯加 入烧瓶中， 橡皮塞密封， 抽真空-通氩气 3次， 氩气保护下加入 12gs-CL (Sigma-Aldrich) 和 0.01gSn(OCt)2 (国药集团）， 用液氮进行三次冷冻-抽气-升温循环后， 在氩气保护下置于 130 °C油浴中反应 36 h后用液氮终止反应。 减压蒸馏后， 用 50mLTHF溶解， 加入 500 mL 体积分数为 50%的冷甲醇水溶液， 沉淀后过滤， 产物在 40 °C、 35mb真空干燥 24h， 得到 白色粉末。 产率为 88%， „=10500, PDI= 1.45ο (2) Preparation of (PCL) 3 -Br 3 : 0.07 g of the dipentaerythritol 2-bromoisobutyrate prepared in the step (1) was added to the flask, the rubber stopper was sealed, and the vacuum was passed through the argon gas 3 times. Under the protection of argon, 12gs-CL (Sigma-Aldrich) and 0.01gSn(O C t) 2 (Zhongguo Group) were added, and after three times of freezing-exhausting-heating cycle with liquid nitrogen, it was placed under 130 ° under argon protection. After reacting for 36 h in a C oil bath, the reaction was terminated with liquid nitrogen. After distillation under reduced pressure, it was dissolved in 50 mL of THF, and 500 mL of a 50% aqueous solution of cold methanol was added, and the mixture was precipitated and filtered, and the product was dried at 40 ° C, 35 mb under vacuum for 24 hours to give a white powder. The yield is 88%, „=10500, PDI= 1.45ο
(3)两亲性 pH响应 6杂臂星型共聚物（(PCL)3(PDEAEMA-b-PPEGMA)3) 的制备： 取 6.36 g步骤 （2) 制备得到的 (PLC)3-Br3、 CuBr2 (上海新宝） 于茄形瓶中， 密封， 抽真空- 通氩气 3次， 依次将 18 mL无水甲苯、 单体 DEAEMA ( TCI-EP )、 配体 HMTETA(3) Preparation of amphiphilic pH-responsive 6-arm star-type copolymer ((PCL) 3 (PDEAEMA-b-PPEGMA) 3 ): Take 6.36 g of the (PLC) 3 -Br 3 prepared in step (2), CuBr 2 (Shanghai Xinbao) in an eggplant-shaped bottle, sealed, vacuumed - argon gas 3 times, followed by 18 mL of anhydrous toluene, monomer DEAEMA (TCI-EP), ligand HMTETA
(Sigma-Aldrich)注入反应瓶中，搅拌 10 min使催化剂配合物 Cu/HMTETA形成。将 Sn(Oct)2 (0.32 g) 溶于 2 mL甲苯中， 注入反应瓶。 搅拌 5 min后转入 90 °C油浴中反应 5h; 再注 入 PEGMA ( „=475, Sigma-Aldrich)进行连续聚合， 反应 12 h后，冷却至室温，加入 50 mL THF并搅拌溶解， 过中性氧化铝柱子， 去除催化剂， 得到反应液浓缩至约 50 mL后加入到 500 mL 正己烷中沉淀， 过滤。 产物在 40 °C、 35 mb真空干燥 24 h， 得到白色粉末。 产率为 45%， „=33410, PDI=1.45。 (Sigma-Aldrich) was injected into the reaction flask and stirred for 10 min to form a catalyst complex Cu/HMTETA. Sn(Oct) 2 (0.32 g) was dissolved in 2 mL of toluene and injected into the reaction flask. After stirring for 5 min, transfer to a 90 °C oil bath for 5 h ; then inject PEGMA ( „=475, Sigma-Aldrich) for continuous polymerization. After 12 h, cool to room temperature, add 50 mL of THF and stir to dissolve. The alumina column was removed, and the catalyst was removed. The reaction solution was concentrated to about 50 mL, then added to 500 mL of n-hexane to precipitate and filtered. The product was dried at 40 ° C, 35 mb under vacuum for 24 h to give a white powder. , „=33410, PDI=1.45.
其中， 各反应物的重量份数配方如下： Wherein, the weight fraction of each reactant is formulated as follows:
(PCL)3-Br3 23.15份 (PCL) 3 -Br 3 23.15 parts
CuBr2 0.05份
HMTETA 0.42份 0.05 parts of CuBr 2 HMTETA 0.42 servings
Sn(Oct)2 1.18份 Sn(Oct) 2 1.18 parts
DEAEMA 23.15份 DEAEMA 23.15 copies
PEGMA 52.05份。 实施例 7: 两亲性 pH响应 6杂臂星型共聚物的制备 52.05 parts of PEGMA. Example 7: Amphiphilic pH response Preparation of 6 heteroarm star copolymer
( 1 )小分子引发剂 2-溴异丁酸二季戊四醇三酯的制备：于 250 mL三口烧瓶中称取 12.72 g (0.05 mol)二季戊四醇 （Alfa-Aesar)， 加入 150 mL无水 THF， 通氩气 lO min除氧。 密封 烧瓶后， 注入 15.17 g (0.15 mol) TEA (AR， 江苏永华）。 0 °C冰水浴中， 逐滴注入 34.48 g (1) Preparation of small molecule initiator 2-bromoisobutyric acid dipentaerythritol triester: 12.72 g (0.05 mol) of dipentaerythritol (Alfa-Aesar) was weighed into a 250 mL three-necked flask, and 150 mL of anhydrous THF was added. Argon gas was removed by oxygen for 10 min. After sealing the flask, 15.17 g (0.15 mol) TEA (AR, Jiangsu Yonghua) was injected. In a 0 °C ice water bath, drop in. 34.48 g
( 0.15 mol) 2-溴异丁酰溴 （Alfa-Aesar)， 搅拌反应 5 h， 再在室温 25 °C下搅拌反应 24 h。 将反应液转入分液漏斗中， 加入 300 mL乙醚， 并依次用 200 mL水、 200 mL0.5 M的碳酸氢 钠溶液和 200 mL水萃取， 有机相用无水硫酸镁干燥后过滤， 溶液旋蒸得到白色固体 （产率 为 43 %)， 乙醚重结晶两次后在 40 V、 35 mb真空干燥 24 h， 得到 2-溴异丁酸二季戊四醇 三酯。 (0.15 mol) 2-Bromoisobutyryl bromide (Alfa-Aesar), stirred for 5 h, and stirred at room temperature for 25 h at 25 °C. The reaction solution was transferred to a separatory funnel, and 300 mL of diethyl ether was added thereto, and successively extracted with 200 mL of water, 200 mL of 0.5 M sodium hydrogencarbonate solution and 200 mL of water, and the organic phase was dried over anhydrous magnesium sulfate and filtered. The mixture was evaporated to give a white solid (yield: 43%), which was twice recrystallized from diethyl ether and dried in vacuo at 40 V, 35 mb for 24 h to give di-pentaerythritol triester of 2-bromoisobutyric acid.
(2) (PCL)3-Br3的制备： 将 0.29 g步骤 （1 ) 制备得到的 2-溴异丁酸二季戊四醇三酯加 入烧瓶中， 橡皮塞密封， 抽真空-通氩气 3次， 氩气保护下加入 12 g s-CL ( Sigma-Aldrich) 和 0.02 g Sn(OCt)2 (国药集团）， 用液氮进行三次冷冻-抽气-升温循环后， 在氩气保护下置于 120 °C油浴中反应 48 h后用液氮终止反应。 减压蒸馏后， 用 50 mL THF溶解， 加入 500 mL 体积分数为 50 %的冷甲醇水溶液， 沉淀后过滤， 产物在 40 °C、 35 mb真空干燥 24 h， 得到 白色粉末。 产率为 86 %， „=10376, PDI=1.59。 (2) Preparation of (PCL) 3 -Br 3 : 0.29 g of dipentaerythritol 2-bromoisobutyrate prepared in the step (1) was added to the flask, the rubber stopper was sealed, and vacuum-passing was performed for 3 times. Under the protection of argon, 12 g s-CL (Sigma-Aldrich) and 0.02 g Sn(O C t) 2 (Zhongguo Group) were added, and after three times of freezing-exhausting-heating cycle with liquid nitrogen, under argon protection After reacting for 48 h in an oil bath at 120 ° C, the reaction was terminated with liquid nitrogen. After distillation under reduced pressure, it was dissolved in 50 mL of THF, and 500 mL of a 50% aqueous solution of cold methanol was added thereto, and the mixture was precipitated and filtered. The product was dried at 40 ° C, 35 mb under vacuum for 24 h to give a white powder. The yield was 86%, „=10376, PDI=1.59.
(3 )两亲性 pH响应 6杂臂星型共聚物（(PCL)3(PDEAEMA-b-PPEGMA)3 ) 的制备： 取 6.36 g步骤 （2) 制备得到的 (PLC)3-Br3、 CuBr2 (上海新宝） 于茄形瓶中， 密封， 抽真空- 通氩气 3次， 依次将 18 mL无水甲苯、 单体 DEAEMA ( TCI-EP )、 配体 HMTETA(3) Preparation of amphiphilic pH-responsive 6-arm star-type copolymer ((PCL) 3 (PDEAEMA-b-PPEGMA) 3): Take 6.36 g of (PLC) 3 -Br 3 prepared in step (2), CuBr 2 (Shanghai Xinbao) in an eggplant-shaped bottle, sealed, vacuumed - argon gas 3 times, followed by 18 mL of anhydrous toluene, monomer DEAEMA (TCI-EP), ligand HMTETA
( Sigma-Aldrich)注入反应瓶中，搅拌 10 min使催化剂配合物 Cu/HMTETA形成。将 Sn(Oct)2 ( 0.32 g) 溶于 2 mL甲苯中， 注入反应瓶。 搅拌 5 min后转入 80 °C油浴中反应 7 h; 再注 入 PEGMA (Mn=475， Sigma-Aldrich))进行连续聚合， 反应 12 h后， 冷却至室温， 加入 50 mL THF并搅拌溶解， 过中性氧化铝柱子， 去除催化剂， 得到反应液浓缩至约 50 mL后加入 到 500 mL 正己烷中沉淀， 过滤。 产物在 40 °C、 35 mb真空干燥 24 h， 得到白色粉末。 产
率为 55 %， „=37295, PDI=1.59。 (Sigma-Aldrich) was injected into the reaction flask and stirred for 10 min to form a catalyst complex Cu/HMTETA. Sn(Oct) 2 (0.32 g) was dissolved in 2 mL of toluene and poured into a reaction flask. After stirring for 5 min, transfer to an 80 °C oil bath for 7 h; then inject PEGMA (M n = 475, Sigma-Aldrich)) for continuous polymerization. After 12 h of reaction, cool to room temperature, add 50 mL of THF and stir to dissolve. After passing through a neutral alumina column, the catalyst was removed, and the reaction solution was concentrated to about 50 mL, and then added to 500 mL of n-hexane to precipitate, and filtered. The product was dried under vacuum at 40 ° C for 24 h at 40 ° C to give a white powder. Production The rate is 55 %, „=37295, PDI=1.59.
其中， 各反应物的重量份数配方如下: Wherein, the weight fraction of each reactant is formulated as follows:
(PCL)3-Br3 21.83份 (PCL) 3 -Br 3 21.83
CuBr2 0.05份 0.05 parts of CuBr 2
HMTETA 0.63份 HMTETA 0.63 servings
DEAEMA 27.29份 DEAEMA 27.29 copies
PEGMA 49.09份。 实施例 8: 两亲性 pH响应 6杂臂星型共聚物的临近胶束浓度 49.09 parts of PEGMA. Example 8: Amphiphilic pH response 6 adjacent micelle concentration of the hybrid arm star copolymer
利用荧光探针法测试实施例 4制备得到的两亲性 pH响应 6杂臂星型共聚物 (PCL)3-(PDEAEMA-b-PPEGMA)3的临近胶束浓度。 The adjacent micelle concentration of the amphiphilic pH-responsive 6-arm star-type copolymer (PCL) 3 - (PDEAEMA-b-PPEGMA) 3 prepared in Example 4 was tested by a fluorescent probe method.
( 1 )芘溶液的配置：用丙酮溶解芘（Sigma-Aldrich)，配置浓度为 12 X 10- 5 M的芘溶液。(1) Configuration of hydrazine solution: Hydrazine (Sigma-Aldrich) was dissolved in acetone, and a hydrazine solution having a concentration of 12 X 10- 5 M was disposed.
(2) 样品溶液的配置： 称取 10 mg (PCL)3(PDEAEMA-b-PPEGMA)3溶于 5 mL丙酮中， 逐滴加入到 100 mL去离子水中，挥发丙酮后得到 0.1 mg/mL的溶液，稀释成一系列浓度（浓 度范围为 0.0001-0.1 mg/mL )0 取 20支 10 mL容量瓶， 分别加入 0.1 mL步骤（1 )配置的芘 溶液， 然后分别加上上述不同浓度的共聚物溶液定容， 摇匀， 得到样品溶液。 样品溶液中芘 的浓度为 12 X 10- 7 M o (2) Configuration of sample solution: Weigh 10 mg (PCL) 3 (PDEAEMA-b-PPEGMA) 3 and dissolve it in 5 mL of acetone, add it dropwise to 100 mL of deionized water, and evaporate acetone to obtain 0.1 mg/mL. The solution is diluted to a series of concentrations (concentration range 0.0001-0.1 mg/mL). 0 Take 20 10 mL volumetric flasks, add 0.1 mL of the hydrazine solution in step (1), and then add the above different concentrations of copolymer solution. Make up to volume, shake well, and get the sample solution. The concentration of cerium in the sample solution is 12 X 10- 7 M o
(3 ) 荧光光谱测试： 以 373 nm作为发射波长， 测试样品溶液在 300~350 nm的激发光 谱， 取波长为 339 nm和 336 nm的强度比值 （/339//336) 对浓度对数 logC作图， 见图 5， 曲 线转折点即为临界胶束浓度值。 测得实施例 4制备得到的 (PCL)3— (PDEAEMA-b-PPEGMA)3 的临近胶束浓度为 3.4 mg/L。 实施例 9: 两亲性 pH响应 6杂臂星型共聚物载药胶束的制备 (3) Fluorescence spectroscopy test: With 373 nm as the emission wavelength, test the excitation spectrum of the sample solution at 300-350 nm, take the intensity ratio of 339 nm and 336 nm (/ 339 // 336 ) to the logarithm of logC Figure, see Figure 5, the curve turning point is the critical micelle concentration value. The approximate micelle concentration of (PCL) 3 - (PDEAEMA-b-PPEGMA) 3 prepared in Example 4 was determined to be 3.4 mg/L. Example 9: Preparation of Amphiphilic pH Response 6 Heteroarm Star Copolymer Drug-loaded Micelles
采用透析法制备载药胶束： 称取 20 mg阿霉素 （DOX， 北京华奉联博） 溶于 20 mL二 甲基甲酰胺 （DMF) 中， 加入 2倍摩尔量的 TEA 20 μί， 搅拌 12 h。 称取 40 mg实施例 4制 备得到的两亲性 pH响应 6杂臂星型共聚物 （PCL)3(PDEAEMA-b-PPEGMA)3溶于 20 mL二 甲基亚砜 （DMSO) 中， 将两种溶液混合， 搅拌 4 h， 去离子水透析 24 h， 前 12 h每 4 h换
一次水， 随后 6 h换一次水。 经 0.45 m过滤头过滤后冻干， 即得到 D0X载药胶束粉末。 采 用 SEM观察其形貌为球形， 粒径范围为 100~180 nm， 见图 6。 实施例 10: 载药胶束的体外释放 Preparation of drug-loaded micelles by dialysis: Weigh 20 mg of doxorubicin (DOX, Beijing Huafeng Libo) dissolved in 20 mL of dimethylformamide (DMF), add 2 times molar amount of TEA 20 μί, stir 12 h. Weigh 40 mg of the amphiphilic pH-responsive 6-arm star copolymer (PCL) 3 (PDEAEMA-b-PPEGMA) 3 prepared in Example 4 and dissolve it in 20 mL of dimethyl sulfoxide (DMSO). Mixing solutions, stirring for 4 h, deionized water for 24 h, for the first 12 h every 4 h Once water, change the water 6 hours later. After filtering through a 0.45 m filter head and lyophilizing, D0X drug-loaded micelle powder was obtained. The morphology was spherical by SEM, and the particle size ranged from 100 to 180 nm, as shown in Figure 6. Example 10: In vitro release of drug-loaded micelles
按照实施例 9制备方法， 利用实施例 2制备得到的两亲性 pH响应 4杂臂星型共聚物 (PCL)2(PDEAEMA-b-PPEGMA)2制备得到 DOX载药胶束 I。 实施例 9制备得到 DOX载药 胶束 II。 According to the preparation method of Example 9, the DOX drug-loaded micelle I was prepared by using the amphiphilic pH-responsive 4-arm star-type copolymer (PCL) 2 (PDEAEMA-b-PPEGMA) 2 prepared in Example 2 . Example 9 produced DOX drug-loaded micelles II.
体外释放试验： 分别称取 5 mg DOX载药胶束 I和 DOX载药胶束 II分散于 5 mL缓冲 液中 （醋酸盐缓冲液和磷酸盐缓冲液）， 缓冲液 pH值分别为 5.0、 6.5和 7.4。 将上述分散于 置于透析袋中， 转入 40 mL相同 pH值的缓冲液中， 置于药物溶出仪， 在 37 °C， llO rpm转 速下进行体外释放。 定时取样 3 mL进行紫外分析， 并同时补加 3 mL新鲜缓冲液。 用紫外 分光光度法测定不同时间释芳液中 DOX浓度， 绘制体外释放曲线， 见图 7和图 8。 In vitro release test: 5 mg of DOX-loaded micelle I and DOX-loaded micelle II were separately dispersed in 5 mL of buffer (acetate buffer and phosphate buffer), and the pH of the buffer was 5.0. 6.5 and 7.4. The above dispersion was placed in a dialysis bag, transferred to 40 mL of the same pH buffer, placed in a drug dissolution apparatus, and released in vitro at 37 ° C, llO rpm. Sample 3 mL for UV analysis and add 3 mL of fresh buffer at the same time. The concentration of DOX in the arsenic solution at different times was determined by ultraviolet spectrophotometry, and the in vitro release profile was plotted, as shown in Fig. 7 and Fig. 8.
由图 7可见， 在正常组织的 pH 7.4环境下， DOX的释放速率非常慢， 24 h的累积释药 量只有 22 %左右， 96 h的累积释药量为 35 %。 当 pH降低至 6.5 (即肿瘤组织附近条件）时， DOX的释放速率加快， 24 h以及 96 h的累积释放量与 pH 7.4相比分别增加了 5 %和 10 %左 右。 而在肿瘤细胞内涵体的 pH 5.0环境下， DOX的释放速率明显加快， 24 h累积释放量增 力口至 IJ 50 %， 48 h达至 lj 62 %， 96 h释放了 91 %。 It can be seen from Fig. 7 that in the normal tissue pH 7.4 environment, the release rate of DOX is very slow, the cumulative release rate at 24 h is only about 22%, and the cumulative release at 96 h is 35 %. When the pH was lowered to 6.5 (ie, conditions near the tumor tissue), the release rate of DOX increased, and the cumulative release at 24 h and 96 h increased by 5 % and 10 %, respectively, compared with pH 7.4. In the pH 5.0 environment of tumor cell endosomes, the release rate of DOX was significantly accelerated. The cumulative release of 24 h increased to IJ 50%, reached 48% at 48 h, and 91% at 96 h.
同理， 由图 8可见， 在正常组织的 pH 7.4环境下， DOX的释放速率非常慢， 24 h的累 积释药量只有 25 %左右， 96 h的累积释药量为 38 %。 当 pH降低至 6.5 (即肿瘤组织附近条 件） 时， DOX的释放速率加快， 24 h以及 96 h的累积释放量与 pH 7.4相比都增加了 10 % 左右。 而在肿瘤细胞内涵体的 pH 5.0环境下， DOX的释放速率明显加快， 24 h累积释放量 增加到 65 %， 48 h达到 80 %， 96 h释放了 96 %。 实施例 11 : 两亲性 pH响应 6杂臂星型共聚物胶束的细胞毒性测试 Similarly, as can be seen from Fig. 8, in the normal tissue pH 7.4 environment, the release rate of DOX is very slow, the cumulative release rate at 24 h is only about 25 %, and the cumulative release at 96 h is 38%. When the pH was lowered to 6.5 (ie, near the tumor tissue), the release rate of DOX increased, and the cumulative release at 24 h and 96 h increased by about 10% compared to pH 7.4. In the pH 5.0 environment of tumor cell endosomes, the release rate of DOX was significantly accelerated. The cumulative release in 24 h increased to 65 %, reached 80 % in 48 h, and 96 % in 96 h. Example 11: Amphiphilic pH response 6 cytotoxicity test of heteroarm star copolymer micelles
( 1 ) 空白胶束制备： 按照实施例 9的方法， 不添加阿霉素， 制备得到实施例 4共聚物 的空白胶束。 (1) Blank micelle preparation: Blank micelles of the copolymer of Example 4 were prepared in the same manner as in Example 9 without adding doxorubicin.
(2) 毒性测试： 取 96孔平底组织培养板， 将四周孔板中分别加入 200 细胞培养基 (DMEM)作为空白组。中间 60个孔中每孔以 lxlO4细胞 /孔（200 μί)的细胞浓度接种 HepG2
细胞 （购买于 ATCC)， 其中第 2列作为对照， 将 96孔板放置到 37 °C， 饱和湿度， 5 %C02 培养箱中培养 48 h。 (2) Toxicity test: A 96-well flat-bottomed tissue culture plate was taken, and 200 cells (DMEM) were added to the surrounding plates as a blank group. Inoculate HepG2 at a cell concentration of lxlO 4 cells/well (200 μί) per well in the middle 60 wells Cells (purchased in ATCC), column 2 as a control, 96-well plates were placed in a 37 ° C, saturated humidity, 5 % CO 2 incubator for 48 h.
随后将游离阿霉素、空白胶束、 DOX载药胶束粉末 I和 DOX载药胶束粉末 II用 DMEM 稀释成不同的聚合物浓度 （空白胶束 l~400 mg/L) 或药物浓度 （游离阿霉素或载药胶束， 0.1-20 mg/L)。 在移走 96孔板中从第 2列到第 11列所有孔中的细胞培养介质后， 在第 2列 中加入新鲜的培养介质， 作为对照。 从第 3列到第 10列， 向所有的孔中分别加入 200 的 样品溶液， 每个浓度的样品加入到 6个孔中进行重复。 Subsequently, the free doxorubicin, blank micelle, DOX drug-loaded micelle powder I and DOX drug-loaded micelle powder II were diluted with DMEM to different polymer concentrations (blank micelles l~400 mg/L) or drug concentration ( Free doxorubicin or drug-loaded micelles, 0.1-20 mg/L). After removing the cell culture medium from all the wells of column 2 to column 11 in the 96-well plate, fresh culture medium was added in the second column as a control. From column 3 to column 10, 200 sample solutions were added to each well, and each concentration of sample was added to 6 wells for repetition.
在经过 48 h的培养后， 吸走所有含有细胞的孔中的上清液， 加入 200 μL的 PBS润洗细 胞， 然后吸走 PBS。从第 2列到第 11列， 分别向每个孔中加入 20 μL的 MTT溶液和 180 μL 的培养介质， 然后将 96孔板放置于培养箱中培养 4 h。 随后吸走未还原的 MTT溶液和培养 介质。 每个孔用 200 的 PBS洗一遍， 并吸走 PBS。 向每个孔中加入 200 的 DMSO溶 解 ΜΤΤ结晶。 整个 96孔板放在 37 °C摇床中振荡 10 min， 然后利用酶标仪测定 490 nm处 每个孔的吸光度， 进而计算细胞存活率。 After 48 h of incubation, the supernatant in all wells containing cells was aspirated, 200 μL of PBS was added to wash the cells, and then PBS was aspirated. From column 2 to column 11, 20 μL of MTT solution and 180 μL of culture medium were added to each well, and then the 96-well plate was placed in an incubator for 4 h. The unreduced MTT solution and culture medium are then aspirated. Each well was washed once with 200 PBS and the PBS was aspirated. 200 DMSO was dissolved in each well to dissolve the ruthenium crystals. The entire 96-well plate was shaken for 10 min in a 37 °C shaker, and then the absorbance of each well at 490 nm was measured by a microplate reader to calculate the cell survival rate.
图 9 是空 白 胶束 （ 实施例 4 产物 ） 的毒性结果 ， 从 图 中可知 ， (PCL)3(PDEAEMA-b-PPEGMA)3对 HepG2细胞基本无毒， 在 400 mg/L的高浓度下细胞存活 率仍然高达 90 %。 Figure 9 shows the toxicity results of blank micelles (product of Example 4). As can be seen from the figure, (PCL) 3 (PDEAEMA-b-PPEGMA) 3 is essentially non-toxic to HepG2 cells, at a high concentration of 400 mg/L. Survival rates are still as high as 90%.
图 10是游离 DOX、 DOX载药胶束 I和 DOX载药胶束 II的细胞毒性结果。从图中可知， 经过 48 h培养后， 低浓度 （0.1 mg/L) 的载药胶束就可以杀死细胞， 有明显的增强作用；在 高浓度 （20 mg/L) 时， 载药胶束和游离 DOX杀死细胞的效果是相似的， 有超过 80 %的细 胞被杀死。 实施例 4产物制备的 DOX载药胶束 I与游离阿霉素有相近的细胞毒性， 说明阿 霉素经包载后可以有效的保持其抗癌活性。 上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制， 其他的任何未背离本发明的精神实质与原理下所作的改变、 修饰、 替代、 组合、 简化， 均应 为等效的置换方式， 都包含在本发明的保护范围之内。
Figure 10 shows the cytotoxicity results of free DOX, DOX-loaded micelle I and DOX-loaded micelle II. It can be seen from the figure that after 48 h incubation, low concentration (0.1 mg/L) of drug-loaded micelles can kill cells and have obvious enhancement; at high concentration (20 mg/L), drug-loaded gel The effect of bunch and free DOX killing cells is similar, with more than 80% of cells killed. The DOX drug-loaded micelles prepared by the product of Example 4 have similar cytotoxicity to free doxorubicin, indicating that the adriamycin can effectively maintain its anticancer activity after being encapsulated. The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and modifications may be made without departing from the spirit and scope of the invention. Simplifications, which are equivalent replacement means, are included in the scope of the present invention.
Claims
1、 一种两亲性 pH响应 4/6杂臂星型共聚物， 其特征在于具有公式 （1 ) 或公式 （2) 所示结构： 1. An amphiphilic pH-responsive 4/6 hetero-arm star copolymer, characterized by having a structure shown in formula (1) or formula (2):
2、 根据权利要求 1所述的两亲性 pH响应 4/6杂臂星型共聚物， 其特征在于： 所述两 亲性 pH响应 4/6杂臂星型共聚物的数均分子量为 30000~54000 g/moL 2. The amphiphilic pH-responsive 4/6 hetero-arm star copolymer according to claim 1, characterized in that: the number average molecular weight of the amphiphilic pH-responsive 4/6 hetero-arm star copolymer is 30,000 ~54000 g/moL
3、 一种根据权利要求 1或 2所述两亲性 pH响应 4/6杂臂星型共聚物的制备方法， 其 特征在于包括以下具体步骤： 3. A method for preparing the amphiphilic pH-responsive 4/6 hetero-arm star copolymer according to claim 1 or 2, which is characterized by including the following specific steps:
( 1 )制备聚己内酯大分子引发剂：将 ε-己内酯、辛酸亚锡及小分子引发剂混合， 110-140 °C下反应 24~48 h， 减压蒸馏、 沉淀、 过滤、 干燥， 得到聚己内酯大分子引发剂； (1) Preparation of polycaprolactone macromolecular initiator: Mix ε-caprolactone, stannous octoate and small molecule initiator, react at 110-140°C for 24-48 hours, distill under reduced pressure, precipitate, filter, Dry to obtain polycaprolactone macroinitiator;
(2) 制备两亲性 pH响应 4/6臂杂臂星型共聚物： 将步骤（1 )制备的聚己内酯大分子引 发剂、 甲基丙烯酸二乙氨基乙酯、 六甲基三亚乙基四胺、 溴化铜溶于甲苯中， 搅拌后加入 辛酸亚锡， 60-90 °C下反应 5~12 h;再加入甲基丙烯酸单甲氧基聚乙二醇酯连续聚合 5~12
h， 去除催化剂， 过滤， 滤液后处理， 得到两亲性 pH响应 4/6杂臂星型共聚物。 (2) Preparation of amphiphilic pH-responsive 4/6-arm hetero-arm star copolymer: Combine the polycaprolactone macroinitiator prepared in step (1), diethylaminoethyl methacrylate, and hexamethyltriethylene Dissolve tetramine and copper bromide in toluene, add stannous octoate after stirring, react at 60-90°C for 5 to 12 hours ; then add monomethoxypolyethylene glycol methacrylate for continuous polymerization for 5 to 12 hours h, remove the catalyst, filter, and post-process the filtrate to obtain an amphiphilic pH-responsive 4/6 hetero-arm star copolymer.
4、 根据权利要求 3所述的两亲性 pH响应 4/6杂臂星型共聚物的制备方法， 其特征在 小分子引发剂 2.35-13.43份 4. The preparation method of the amphiphilic pH-responsive 4/6 hetero-arm star copolymer according to claim 3, which is characterized by 2.35-13.43 parts of small molecule initiator
ε-己内酯 86.51-97.52 ε-Caprolactone 86.51-97.52
辛酸亚锡 0.06-0.13份； Stannous octoate 0.06-0.13 parts;
步骤 （2) 中反应物的重量份数配方如下: The formula of the weight parts of the reactants in step (2) is as follows:
聚己内酯大分子引发剂 12.41-26.75份 Polycaprolactone macroinitiator 12.41-26.75 parts
溴化铜 0.04-0.08份 Copper bromide 0.04-0.08 parts
六甲基三亚乙基四胺 0.24-0.83份 Hexamethyltriethylenetetramine 0.24-0.83 parts
辛酸亚锡 0.42~1.44份 Stannous octoate 0.42~1.44 parts
甲基丙烯酸二乙氨基乙酯 17.72-31.03份 Diethylaminoethyl methacrylate 17.72-31.03 parts
甲基丙烯酸单甲氧基聚乙二醇酯 49.09-58.97份 Monomethoxy polyethylene glycol methacrylate 49.09-58.97 parts
所述甲苯的用量为每 1 g聚己内酯大分子引发剂使用 2〜4 mL甲苯。 The amount of toluene used is 2 to 4 mL of toluene per 1 g of polycaprolactone macroinitiator.
5、 根据权利要求 3所述的两亲性 pH响应 4/6杂臂星型共聚物的制备方法， 其特征在 于： 步骤（1 )所述的沉淀均指往旋转蒸发后溶液加入 10倍体积的 0 °C体积分数为 50 %的 甲醇水溶液进行沉淀； 步骤（2)所述的去除催化剂指将反应产物溶解于四氢呋喃后， 过中 性氧化铝层析柱， 四氢呋喃洗脱， 除去催化剂； 步骤（2)所述的滤液后处理指将滤液旋蒸 蒸发、 加入 10倍体积的正己烷中沉淀、 过滤、 干燥。 5. The preparation method of amphiphilic pH-responsive 4/6 hetero-arm star copolymer according to claim 3, characterized in that: the precipitation described in step (1) refers to adding 10 times the volume to the solution after rotary evaporation Precipitate with a methanol aqueous solution with a volume fraction of 50% at 0°C; The removal of the catalyst described in step (2) refers to dissolving the reaction product in tetrahydrofuran, passing it through a neutral alumina chromatography column, eluting with tetrahydrofuran, and removing the catalyst; Steps (2) The post-treatment of the filtrate refers to evaporating the filtrate by rotary evaporation, adding 10 times the volume of n-hexane to precipitate, filtering, and drying.
6、 根据权利要求 3所述的两亲性 pH响应 4/6杂臂星型共聚物的制备方法， 其特征在 于： 当制备两亲性 pH响应 4杂臂星型共聚物时， 所述小分子引发剂指 2-溴异丁酸季戊四 醇二酯， 由以下方法制备得到： 将摩尔比为 1 :2:2的季戊四醇、 2-溴异丁酰溴、 三乙胺溶解 于四氢呋喃中， 在冰水浴中反应 4~6 h， 然后在室温下反应 16~30 h， 经旋转蒸发、 沉淀、 过滤、 干燥， 得到 2-溴异丁酸季戊四醇二酯； 6. The preparation method of the amphiphilic pH-responsive 4/6 hetero-arm star copolymer according to claim 3, characterized in that: when preparing the amphiphilic pH-responsive 4 hetero-arm star copolymer, the small The molecular initiator refers to 2-bromoisobutyric acid pentaerythritol diester, which is prepared by the following method: Dissolve pentaerythritol, 2-bromoisobutyryl bromide, and triethylamine in a molar ratio of 1:2:2 in tetrahydrofuran, and place on ice. React in a water bath for 4 to 6 hours, and then react at room temperature for 16 to 30 hours. After rotary evaporation, precipitation, filtration, and drying, 2-bromoisobutyric acid pentaerythritol diester is obtained;
当制备两亲性 pH响应 6杂臂星型共聚物时， 所述小分子引发剂指 2-溴异丁酸二季戊 四醇三酯， 由以下方法制备得到： 将摩尔比为 1 :3:3的二季戊四醇、 2-溴异丁酰溴、 三乙胺 溶解于四氢呋喃中， 在冰水浴中反应 4~6 h， 然后在室温下反应 16~30 h， 经旋转蒸发、 沉 淀、 过滤、 干燥， 得到 2-溴异丁酸二季戊四醇三酯。
When preparing an amphiphilic pH-responsive 6-arm star copolymer, the small molecule initiator refers to 2-bromoisobutyric acid dipentaerythritol triester, which is prepared by the following method: using a molar ratio of 1:3:3 Dipentaerythritol, 2-bromoisobutyryl bromide, and triethylamine were dissolved in tetrahydrofuran, reacted in an ice-water bath for 4 to 6 hours, and then reacted at room temperature for 16 to 30 hours, followed by rotary evaporation, precipitation, filtration, and drying to obtain 2-Bromoisobutyric acid dipentaerythritol triester.
7、 根据权利要求 6所述的两亲性 pH响应 4/6杂臂星型共聚物的制备方法， 其特征在 于： 当制备两亲性 pH响应 4杂臂星型共聚物时， 所述四氢呋喃的用量为每 l g季戊四醇使 用 20~25 mL 四氢呋喃； 当制备两亲性 pH响应 6杂臂星型共聚物时，所述四氢呋喃的用量 为每 1 g二季戊四醇使用 10~15 mL四氢呋喃。 7. The preparation method of the amphiphilic pH-responsive 4/6 hetero-arm star copolymer according to claim 6, characterized in that: when preparing the amphiphilic pH-responsive 4 hetero-arm star copolymer, the tetrahydrofuran The dosage of tetrahydrofuran is 20~25 mL of tetrahydrofuran per lg of pentaerythritol; when preparing an amphiphilic pH-responsive 6-arm star copolymer, the dosage of tetrahydrofuran is 10~15 mL of tetrahydrofuran for every 1 g of dipentaerythritol.
8、 根据权利要求 1或 2所述的两亲性 pH响应 4/6杂臂星型共聚物在制备装载水难溶 性药物胶束系统中的应用。 8. Application of the amphiphilic pH-responsive 4/6 hetero-arm star copolymer according to claim 1 or 2 in the preparation of a micelle system loaded with poorly water-soluble drugs.
9、 根据权利要求 8所述的两亲性 pH响应 4/6杂臂星型共聚物在制备装载水难溶性药 物胶束系统中的应用， 其特征在于所述装载水难溶性药物胶束系统由以下方法制备得到： 将两亲性 pH响应 4/6杂臂星型共聚物和水难溶性药物溶于有机溶剂中，室温下搅拌 4~6 h， 去离子水透析 24〜48 h， 冷冻干燥， 得到装载水难溶性药物胶束系统。 9. Application of the amphiphilic pH-responsive 4/6 hetero-arm star copolymer according to claim 8 in the preparation of a micelle system loaded with poorly water-soluble drugs, characterized in that the micelle system loaded with poorly water-soluble drugs It is prepared by the following method: Dissolve the amphiphilic pH-responsive 4/6 hetero-arm star copolymer and the poorly water-soluble drug in an organic solvent, stir at room temperature for 4 to 6 hours, dialyze with deionized water for 24 to 48 hours, and freeze. After drying, a micelle system loaded with poorly water-soluble drugs is obtained.
10、根据权利要求 9所述的两亲性 pH响应 4/6杂臂星型共聚物在制备装载水难溶性药 物胶束系统中的应用，其特征在于： 所述的水难溶性药物指在 1 L水中溶解度小于或等于 1 g的药物； 所述的有机溶剂指二甲基甲酰胺和二甲基亚砜中的至少一种。
10. Application of the amphiphilic pH-responsive 4/6 hetero-arm star copolymer according to claim 9 in the preparation of a micelle system loaded with poorly water-soluble drugs, characterized in that: the poorly water-soluble drug refers to Drugs with a solubility less than or equal to 1 g in 1 L of water; the organic solvent refers to at least one of dimethylformamide and dimethyl sulfoxide.
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