WO2024087273A1 - Polymer prodrug releasable in response to redox environment, and preparation and use thereof - Google Patents
Polymer prodrug releasable in response to redox environment, and preparation and use thereof Download PDFInfo
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- WO2024087273A1 WO2024087273A1 PCT/CN2022/133438 CN2022133438W WO2024087273A1 WO 2024087273 A1 WO2024087273 A1 WO 2024087273A1 CN 2022133438 W CN2022133438 W CN 2022133438W WO 2024087273 A1 WO2024087273 A1 WO 2024087273A1
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- polymer prodrug
- telmisartan
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- redox environment
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
- A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
Definitions
- the present invention relates to the field of pharmaceutics, and in particular to a polymer prodrug released in response to a redox environment and the preparation and application thereof.
- Cancer is one of the most serious diseases that threaten the quality of human life. More than 90% of cancer patients die from metastasis, mainly due to the inefficiency of conventional treatments such as surgery, radiotherapy and chemotherapy. Among them, the hypoxic microenvironment of tumor tissue is the key to tumor metastasis and low patient survival rate. 50% to 60% of solid tumors have hypoxic areas. Therefore, alleviating the tumor hypoxic microenvironment and inhibiting tumor metastasis have gradually become the focus of researchers. Due to the lack of in-depth research on the interactions and related mechanisms of multiple pathological barriers during tumor development, the clinical efficacy of tumor treatment is still unsatisfactory.
- telmisartan is an angiotensin receptor inhibitor, and related studies have reported that telmisartan has anti-tumor effects.
- thiol groups which are also prodrugs to a certain extent, which will affect the spatial binding of telmisartan and AT1R receptors, reducing or even losing the efficacy of the drug. Therefore, there is an urgent need for a polymer prodrug based on a drug delivery system that responds to release in a redox environment.
- the present invention provides a polymer prodrug that is released in response to a redox environment and the preparation and application thereof.
- the present invention adopts the following technical solutions:
- the present invention provides a redox-responsive release polymer prodrug, the structural formula of the polymer prodrug is as follows:
- n 16 to 52.
- the polymer prodrug is a polymer prodrug obtained by connecting heparin sodium to telmisartan through an esterification reaction of 2,2'-dithiodiethanol, and the polymer prodrug responds to release the telmisartan prototype drug under redox conditions.
- the present invention provides a method for preparing a polymer prodrug that is released in response to a redox environment, which specifically comprises the following steps:
- telmisartan Dissolving telmisartan in a mixed solution of dimethyl sulfoxide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine, stirring for reaction, adding 2,2'-dithiodiethanol, and stirring for 12 hours to obtain a coupling product of telmisartan and 2,2'-dithiodiethanol;
- heparin sodium Dissolving heparin sodium with a molecular weight of 8000-25000 in a mixed solution of dimethyl sulfoxide and water, then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine to the mixed solution, stirring and reacting to obtain activated heparin sodium;
- the coupling product of telmisartan and 2,2'-dithiodiethanol is mixed with activated sodium heparin for reaction for 24 hours to obtain a reaction solution, the reaction solution is dialyzed in pure water for 3 days, and the polymer prodrug is obtained after freeze-drying.
- step (1) the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and telmisartan is 5-8:0.5-1:1-2.
- step (1) the molar ratio of 2,2'-dithiodiethanol to telmisartan is 1:1.
- step (2) the volume ratio of dimethyl sulfoxide to water is 8-9:2-1.
- step (2) the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and heparin sodium is 5-8:0.5-1:1-2.
- steps (1) and (2) the stirring reaction is carried out under a nitrogen atmosphere at 60° C. for 1 hour.
- step (3) in the reaction between the coupled product of telmisartan and 2,2'-dithiodiethanol and activated heparin sodium, the molar ratio of telmisartan in the coupled product of telmisartan and 2,2'-dithiodiethanol to heparin sodium in the activated heparin sodium is 7.8:24.3.
- the present invention uses the redox-responsive release polymer prodrug prepared by the above method to prepare drugs for treating tumors.
- the polymer prodrug realizes redox-responsive release of original telmisartan in the tumor microenvironment to intervene in tumor-associated fibroblasts, destroy the tumor matrix barrier, and increase intratumor permeability.
- the present invention provides a polymer prodrug that intervenes in the tumor pathological barrier in response to the redox environment in the cytoplasm in the tumor microenvironment.
- the present invention concentrates the two functions of reversing the phenotype of tumor-associated fibroblasts and anticoagulation on the nanoparticles formed by the same polymer prodrug by chemical synthesis.
- the polymer prodrug is composed of three parts, the hydrophobic part is the pharmacological structure of telmisartan, the hydrophilic part is composed of the pharmacological structure of heparin sodium, and the hydrophilic part and the hydrophobic part are connected by a chemical bond that responds to the redox environment.
- the nanoparticles formed by the polymer prodrug have a stable structure, can self-aggregate to form micelles in aqueous media, have strong passive tumor targeting effects, and have high in vivo stability.
- the polymer prodrug releases the prototype telmisartan drug in response to the redox environment in the human body to enhance the permeation and retention effects, downregulate ⁇ -smooth muscle actin, reverse the phenotype of tumor-associated fibroblasts, inhibit the tumor matrix barrier, improve the drug distribution ability in the tumor, increase the drug concentration at the intracellular target, and improve the therapeutic efficacy of patients.
- FIG1 is a diagram showing the synthesis mechanism of polymer prodrugs
- FIG2 is a particle size, potential and scanning electron microscopy image of the polymer prodrug
- FIG3 shows the survival rate of activated mouse fibroblasts after being treated with different concentrations of telmisartan and polymer prodrugs for 48 hours;
- FIG4 shows the expression level of ⁇ -smooth muscle actin in activated mouse fibroblasts after treatment with different concentrations of telmisartan and polymer prodrugs
- FIG5 is a graph showing the response spectrum of telmisartan released from polymer prodrug to glutathione measured by HPLC.
- a redox-responsive release polymer prodrug, as shown in FIG1 is prepared by the following method:
- telmisartan 80 mg of telmisartan, 148 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 8 mg of 4-dimethylaminopyridine were dissolved in 3 mL of dimethyl sulfoxide, and stirred at 60° C. for 1 hour under a nitrogen atmosphere, and then 57 mg of 2,2’-dithiodiethanol was added, and stirred for another 12 hours to obtain a coupling product of telmisartan and 2,2’-dithiodiethanol;
- heparin sodium 160 mg was dissolved in 10 mL of a mixed solution of dimethyl sulfoxide and water (volume ratio 8:2), and then 752 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 48 mg of 4-dimethylaminopyridine were added to the mixed solution, and the mixture was stirred at 60° C. for 1 hour under a nitrogen atmosphere to obtain activated heparin sodium;
- the coupling product of telmisartan and 2,2'-dithiodiethanol obtained in step (1) and the activated sodium heparin obtained in step (2) are mixed and reacted for 24 hours to obtain a reaction solution, the reaction solution is dialyzed in pure water for 3 days, and the polymer prodrug is obtained after freeze-drying.
- the particle size, potential and scanning electron microscope image of the obtained polymer prodrug are shown in FIG2 .
- the present invention evaluates the micellar cell survival rate of polymer prodrugs by the inhibition rate of tumor-associated fibroblasts.
- the test adopts tetrazolium salt colorimetry. Using mouse fibroblast NIH/3T3 cells activated by mouse breast cancer 4T1 cells as a model, 200 ⁇ L of culture solution containing 2 ⁇ 10 3 activated NIH/3T3 cells is added to each well of a 96-well cell culture plate, and the plate is placed in an incubator at 37°C and 5% CO 2 for 12 hours. After the cells are completely attached to the wall, different concentrations of telmisartan and polymer prodrug (experimental group) are added to the cell wells of the 96-well cell culture plate, and untreated blank cells are used as controls.
- Each well is set up with duplicate wells; after incubation for 48 hours, 20 ⁇ L of 5 mg/mL thiazolyl blue solution is added to each well, and the supernatant is discarded after continued incubation for 4 hours, 200 ⁇ L of dimethyl sulfoxide is added to each well, and the absorbance at 570 nm is measured by an enzyme-linked detector.
- the cell survival rate is calculated according to the following formula:
- telmisartan and polymer prodrug The toxicity of telmisartan and polymer prodrug to activated mouse fibroblasts is shown in FIG3 . It can be seen from the figure that the survival rate of fibroblasts treated with polymer prodrug is lower than that of fibroblasts treated with telmisartan, indicating that polymer prodrug has a stronger efficacy on mouse fibroblasts than telmisartan.
- a redox-responsive release polymer prodrug, as shown in FIG1 is prepared by the following method:
- telmisartan Dissolve 100 mg of telmisartan, 190 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 12 mg of 4-dimethylaminopyridine in 4 mL of dimethyl sulfoxide, stir for 1 hour at 60° C. under nitrogen atmosphere, add 67 mg of 2,2’-dithiodiethanol, and stir for another 12 hours to obtain a coupling product of telmisartan and 2,2’-dithiodiethanol;
- heparin sodium 200 mg was dissolved in 16 mL of a mixed solution of dimethyl sulfoxide and water (volume ratio 8:2), and then 826 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 60 mg of 4-dimethylaminopyridine were added to the mixed solution, and the mixture was stirred at 60° C. for 1 hour under a nitrogen atmosphere to obtain activated heparin sodium;
- the coupling product of telmisartan and 2,2'-dithiodiethanol obtained in step (1) and the activated sodium heparin obtained in step (2) are mixed and reacted for 24 hours to obtain a reaction solution, the reaction solution is dialyzed in pure water for 3 days, and the polymer prodrug is obtained after freeze-drying.
- the present invention evaluates the ability of polymer prodrug micelles to reverse the phenotypic ability of tumor fibroblasts by using the expression level of ⁇ -smooth muscle actin in tumor-associated fibroblasts.
- the experiment adopted the Western blotting method, and mouse fibroblast NIH/3T3 cells activated by mouse breast cancer 4T1 cells were used as the model.
- 2.5 mL of culture medium containing 2 ⁇ 10 5 activated NIH/3T3 cells was added to each well of a 6-well cell culture plate, and the plates were cultured in a 37°C, 5% CO 2 incubator for 12 hours. After the cells were completely attached to the wall, different concentrations of polymer prodrugs were added to the cell wells of the 6-well cell culture plate.
- a redox-responsive release polymer prodrug, as shown in FIG1 is prepared by the following method:
- telmisartan 160 mg of telmisartan, 296 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 16 mg of 4-dimethylaminopyridine were dissolved in 6 mL of dimethyl sulfoxide, and stirred at 60° C. for 1 hour under a nitrogen atmosphere, and then 114 mg of 2,2’-dithiodiethanol was added, and stirred for another 12 hours to obtain a coupling product of telmisartan and 2,2’-dithiodiethanol;
- heparin sodium 320 mg was dissolved in 20 mL of a mixed solution of dimethyl sulfoxide and water (volume ratio 8:2), and then 1.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 96 mg of 4-dimethylaminopyridine were added to the mixed solution, and the mixture was stirred at 60° C. for 1 hour under a nitrogen atmosphere to obtain activated heparin sodium;
- the coupling product of telmisartan and 2,2'-dithiodiethanol obtained in step (1) and the activated sodium heparin obtained in step (2) are mixed and reacted for 24 hours to obtain a reaction solution, the reaction solution is dialyzed in pure water for 3 days, and the polymer prodrug is obtained after freeze-drying.
- telmisartan in polymer prodrug was studied by dialysis. Equal amounts of telmisartan and polymer prodrug were placed in a dialysis bag and then dialyzed with 25 mL of phosphate buffered saline or 25 mL of phosphate buffered saline and different concentrations of glutathione for 72 hours. The dialysate was extracted at a predetermined time and a new phosphate buffered saline was added. The reaction profile of telmisartan released from the polymer prodrug to glutathione was determined by HPLC, and the results are shown in Figure 5.
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Abstract
A polymer prodrug releasable in response to a redox environment. The polymer prodrug is a polymer prodrug obtained by linking heparin sodium to telmisartan by means of an esterification reaction of 2,2'-dithioethanol, said polymer prodrug releasing the telmisartan prototype drug in response to a redox condition. Nanoparticles formed by the polymer prodrug have a stable structure and can be self-aggregated in an aqueous medium so as to form micelles, so that the polymer prodrug has a strong passive tumor-targeting effect and high in-vivo stability. The polymer prodrug releases in response to a redox environment the telmisartan prototype drug in human bodies so as to enhance the permeability and retention effects, down-regulate α-smooth muscle actin, reverse phenotypes of tumor-associated fibroblasts, and inhibit tumor matrix barriers, thus improving the drug distribution capability in tumors and increasing the drug concentration of intracellular targets, thereby improving the therapeutic efficacy.
Description
本发明涉及药剂学领域，具体涉及一种氧化还原环境响应释放的聚合物前药及其制备与应用。The present invention relates to the field of pharmaceutics, and in particular to a polymer prodrug released in response to a redox environment and the preparation and application thereof.
肿瘤是威胁人类生活质量的最严重的疾病之一。90％以上的肿瘤患者死于转移，其主要原因是手术、放疗和化疗等常规治疗手段效率低下。其中，肿瘤组织缺氧微环境是导致肿瘤转移和患者低生存率的关键，实体肿瘤中50％～60％存在缺氧区。因此，缓解肿瘤缺氧微环境和抑制肿瘤转移逐渐成为研究者关注的焦点。由于研究人员在肿瘤发展过程中对多种病理屏障的相互作用及相关机制缺乏深入研究，肿瘤治疗的临床疗效仍不理想。Cancer is one of the most serious diseases that threaten the quality of human life. More than 90% of cancer patients die from metastasis, mainly due to the inefficiency of conventional treatments such as surgery, radiotherapy and chemotherapy. Among them, the hypoxic microenvironment of tumor tissue is the key to tumor metastasis and low patient survival rate. 50% to 60% of solid tumors have hypoxic areas. Therefore, alleviating the tumor hypoxic microenvironment and inhibiting tumor metastasis have gradually become the focus of researchers. Due to the lack of in-depth research on the interactions and related mechanisms of multiple pathological barriers during tumor development, the clinical efficacy of tumor treatment is still unsatisfactory.
肿瘤相关成纤维细胞是肿瘤微环境中胶原和其他基质分泌的主要来源。替米沙坦是一种血管紧张素受体抑制剂，相关研究报道了替米沙坦具有抗肿瘤的作用。然而受替米沙坦自身难溶性的制约，其单独治疗的疗效并不理想，且传统药物递释系统释放出来的替米沙坦含有巯基，巯基在一定程度上也是前药，这会影响替米沙坦和AT1R受体的空间结合，降低甚至丢失药物疗效。因此，急需一种基于药物传递系统的，在氧化还原环境中响应释放的聚合物前药。Tumor-associated fibroblasts are the main source of collagen and other matrix secretion in the tumor microenvironment. Telmisartan is an angiotensin receptor inhibitor, and related studies have reported that telmisartan has anti-tumor effects. However, due to the poor solubility of telmisartan itself, its efficacy as a single treatment is not ideal, and the telmisartan released by the traditional drug delivery system contains thiol groups, which are also prodrugs to a certain extent, which will affect the spatial binding of telmisartan and AT1R receptors, reducing or even losing the efficacy of the drug. Therefore, there is an urgent need for a polymer prodrug based on a drug delivery system that responds to release in a redox environment.
发明内容Summary of the invention
本发明针对现有技术中的不足，提供一种氧化还原环境响应释放的聚合物前药及其制备与应用。In view of the deficiencies in the prior art, the present invention provides a polymer prodrug that is released in response to a redox environment and the preparation and application thereof.
为实现上述目的，本发明采用以下技术方案：To achieve the above object, the present invention adopts the following technical solutions:
一方面，本发明提供一种氧化还原环境响应释放的聚合物前药，所述聚合物前药的结构式如下：In one aspect, the present invention provides a redox-responsive release polymer prodrug, the structural formula of the polymer prodrug is as follows:
其中，n的范围是16～52。Among them, the range of n is 16 to 52.
进一步地，所述聚合物前药是肝素钠通过2,2’-二硫二乙醇的酯化反应连接替米沙坦得到的聚合物前药，该聚合物前药在氧化还原条件下响应释放替米沙坦原型药物。Furthermore, the polymer prodrug is a polymer prodrug obtained by connecting heparin sodium to telmisartan through an esterification reaction of 2,2'-dithiodiethanol, and the polymer prodrug responds to release the telmisartan prototype drug under redox conditions.
另一方面，本发明提供一种氧化还原环境响应释放的聚合物前药的制备方法，具体包括以下步骤：In another aspect, the present invention provides a method for preparing a polymer prodrug that is released in response to a redox environment, which specifically comprises the following steps:
(1)替米沙坦耦合2,2’-二硫二乙醇：(1) Telmisartan coupled with 2,2'-dithiodiethanol:
将替米沙坦溶解在二甲基亚砜、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐和4-二甲氨基吡啶的混合溶液中，搅拌反应后加入2,2’-二硫二乙醇，再搅拌12小时得到替米沙坦和2,2’-二硫二乙醇的耦合物；Dissolving telmisartan in a mixed solution of dimethyl sulfoxide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine, stirring for reaction, adding 2,2'-dithiodiethanol, and stirring for 12 hours to obtain a coupling product of telmisartan and 2,2'-dithiodiethanol;
(2)肝素钠的激活：(2) Activation of heparin sodium:
将分子量为8000-25000的肝素钠溶解于二甲基亚砜和水的混合溶液中，然后向混合溶液中加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐和4-二甲氨基吡啶，搅拌反应后得到活化的肝素钠；Dissolving heparin sodium with a molecular weight of 8000-25000 in a mixed solution of dimethyl sulfoxide and water, then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine to the mixed solution, stirring and reacting to obtain activated heparin sodium;
(3)肝素钠耦合替米沙坦聚合物前药的合成：(3) Synthesis of heparin sodium coupled telmisartan polymer prodrug:
将替米沙坦和2,2’-二硫二乙醇的耦合物与活化的肝素钠混合反应24h得到反应液，将反应液在纯水中透析3天，冷冻干燥后得到聚合物前药。The coupling product of telmisartan and 2,2'-dithiodiethanol is mixed with activated sodium heparin for reaction for 24 hours to obtain a reaction solution, the reaction solution is dialyzed in pure water for 3 days, and the polymer prodrug is obtained after freeze-drying.
进一步地，步骤(1)中，1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐、4-二甲氨基吡啶和替米沙坦的摩尔比为5～8:0.5～1:1～2。Furthermore, in step (1), the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and telmisartan is 5-8:0.5-1:1-2.
进一步地，步骤(1)中，2,2’-二硫二乙醇和替米沙坦的摩尔比为1:1。Furthermore, in step (1), the molar ratio of 2,2'-dithiodiethanol to telmisartan is 1:1.
进一步地，步骤(2)中，二甲基亚砜和水的体积比为8～9:2～1。Furthermore, in step (2), the volume ratio of dimethyl sulfoxide to water is 8-9:2-1.
进一步地，步骤(2)中，1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐、4-二甲氨基吡啶和肝素钠的摩尔比为5～8:0.5～1:1～2。Furthermore, in step (2), the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and heparin sodium is 5-8:0.5-1:1-2.
进一步地，步骤(1)和(2)中，所述搅拌反应的反应条件为在60℃的氮气气氛下搅拌1小时。Furthermore, in steps (1) and (2), the stirring reaction is carried out under a nitrogen atmosphere at 60° C. for 1 hour.
进一步地，步骤(3)中，替米沙坦和2,2’-二硫二乙醇的耦合物与活化的肝素钠的反应中，替米沙坦和2,2’-二硫二乙醇的耦合物中的替米沙坦与活化的肝素钠中的肝素钠的摩尔比为7.8∶24.3。Furthermore, in step (3), in the reaction between the coupled product of telmisartan and 2,2'-dithiodiethanol and activated heparin sodium, the molar ratio of telmisartan in the coupled product of telmisartan and 2,2'-dithiodiethanol to heparin sodium in the activated heparin sodium is 7.8:24.3.
又一方面，本发明将通过上述方法制备得到的氧化还原环境响应释放的聚合物前药用于制备治疗肿瘤的药物，该聚合物前药在肿瘤微环境中实现氧化还原响应性释放原形替米沙坦干预肿瘤相关成纤维细胞，破坏肿瘤基质屏障，增加瘤内通透性。On the other hand, the present invention uses the redox-responsive release polymer prodrug prepared by the above method to prepare drugs for treating tumors. The polymer prodrug realizes redox-responsive release of original telmisartan in the tumor microenvironment to intervene in tumor-associated fibroblasts, destroy the tumor matrix barrier, and increase intratumor permeability.
本发明的有益效果是：The beneficial effects of the present invention are:
本发明提供一种在肿瘤微环境中，响应胞浆中氧化还原环境干预肿瘤病理屏障的聚合物前药。本发明通过化学合成的方式，将具备逆转肿瘤相关成纤维细胞表型和抗凝两个功能集中于同一聚合物前药形成的纳米粒上。该聚合物前药由三部分组成，疏水部分为替米沙坦药理作用结构，亲水部分则由肝素钠药理作用结构组成，亲水部分和疏水部分通过响应氧化还原环境的化学键连接。The present invention provides a polymer prodrug that intervenes in the tumor pathological barrier in response to the redox environment in the cytoplasm in the tumor microenvironment. The present invention concentrates the two functions of reversing the phenotype of tumor-associated fibroblasts and anticoagulation on the nanoparticles formed by the same polymer prodrug by chemical synthesis. The polymer prodrug is composed of three parts, the hydrophobic part is the pharmacological structure of telmisartan, the hydrophilic part is composed of the pharmacological structure of heparin sodium, and the hydrophilic part and the hydrophobic part are connected by a chemical bond that responds to the redox environment.
该聚合物前药形成的纳米粒结构稳定，在水性介质中可以自聚集形成胶束，被动靶向肿瘤作用强，体内稳定性高。该聚合物前药在人体内通过响应氧化还原环境释放替米沙坦原型药物以增强渗透与滞留效应，下调α-平滑肌肌动蛋白，逆转肿瘤相关成纤维细胞表型，抑制肿瘤基质屏障，提高肿瘤内药物分布能力，增加了细胞内靶点的药物浓度，提高患者的治疗药效。The nanoparticles formed by the polymer prodrug have a stable structure, can self-aggregate to form micelles in aqueous media, have strong passive tumor targeting effects, and have high in vivo stability. The polymer prodrug releases the prototype telmisartan drug in response to the redox environment in the human body to enhance the permeation and retention effects, downregulate α-smooth muscle actin, reverse the phenotype of tumor-associated fibroblasts, inhibit the tumor matrix barrier, improve the drug distribution ability in the tumor, increase the drug concentration at the intracellular target, and improve the therapeutic efficacy of patients.
图1为聚合物前药的合成机理图；FIG1 is a diagram showing the synthesis mechanism of polymer prodrugs;
图2为聚合物前药的粒径、电位及扫描电镜图；FIG2 is a particle size, potential and scanning electron microscopy image of the polymer prodrug;
图3为激活的小鼠成纤维细胞在不同浓度的替米沙坦和聚合物前药处理48小时后的存活率；FIG3 shows the survival rate of activated mouse fibroblasts after being treated with different concentrations of telmisartan and polymer prodrugs for 48 hours;
图4为激活的小鼠成纤维细胞在不同浓度的替米沙坦和聚合物前药处理后α-平滑肌肌动蛋白的表达水平；FIG4 shows the expression level of α-smooth muscle actin in activated mouse fibroblasts after treatment with different concentrations of telmisartan and polymer prodrugs;
图5为高效液相色谱测定的聚合物前药释放的替米沙坦对谷胱甘肽的反应谱。FIG5 is a graph showing the response spectrum of telmisartan released from polymer prodrug to glutathione measured by HPLC.
实施例1Example 1
一种氧化还原环境响应释放的聚合物前药，如图1所示，通过以下方法制备：A redox-responsive release polymer prodrug, as shown in FIG1 , is prepared by the following method:
(1)替米沙坦耦合2,2’-二硫二乙醇(即2，2’-二硫代乙醇)：(1) Telmisartan coupled with 2,2'-dithiodiethanol (i.e. 2,2'-dithioethanol):
将80mg替米沙坦、148mg的1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐和8mg的4-二甲氨基吡啶溶解在3mL二甲基亚砜中，60℃氮气气氛下搅拌1小时后，加入57mg的2,2’-二硫二乙醇，再搅拌12小时得到替米沙坦和2,2’-二硫二乙醇的耦合物；80 mg of telmisartan, 148 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 8 mg of 4-dimethylaminopyridine were dissolved in 3 mL of dimethyl sulfoxide, and stirred at 60° C. for 1 hour under a nitrogen atmosphere, and then 57 mg of 2,2’-dithiodiethanol was added, and stirred for another 12 hours to obtain a coupling product of telmisartan and 2,2’-dithiodiethanol;
(2)肝素钠的激活：(2) Activation of heparin sodium:
将160mg的肝素钠溶解于10mL的二甲基亚砜和水(体积比8:2)的混合溶液中，然后向混合溶液中加入752mg的1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐和48mg的4-二甲氨基吡啶，60℃氮气气氛下搅拌1小时后，得到活化的肝素钠；160 mg of heparin sodium was dissolved in 10 mL of a mixed solution of dimethyl sulfoxide and water (volume ratio 8:2), and then 752 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 48 mg of 4-dimethylaminopyridine were added to the mixed solution, and the mixture was stirred at 60° C. for 1 hour under a nitrogen atmosphere to obtain activated heparin sodium;
(3)肝素钠耦合替米沙坦聚合物前药的合成：(3) Synthesis of heparin sodium coupled telmisartan polymer prodrug:
将步骤(1)得到的替米沙坦和2,2’-二硫二乙醇的耦合物与步骤(2)得到的活化的肝素钠混合反应24h得到反应液，将反应液在纯水中透析3天，冷冻干燥后得到聚合物前药。The coupling product of telmisartan and 2,2'-dithiodiethanol obtained in step (1) and the activated sodium heparin obtained in step (2) are mixed and reacted for 24 hours to obtain a reaction solution, the reaction solution is dialyzed in pure water for 3 days, and the polymer prodrug is obtained after freeze-drying.
得到的聚合物前药的粒径、电位及扫描电镜图如图2所示。The particle size, potential and scanning electron microscope image of the obtained polymer prodrug are shown in FIG2 .
本发明以肿瘤相关成纤维细胞抑制率评价聚合物前药的胶束细胞存活率。试验采用四唑盐比色法测定。以小鼠乳腺癌4T1细胞激活的小鼠成纤维NIH/3T3细胞为模型，在96孔细胞培养板每孔中加入200μL含2×10
3个激活的NIH/3T3细胞的培养液，置37℃、5％CO
2孵箱培养12小时，待细胞完全贴壁后，在96孔细胞培养板的细胞孔中分别加入不同浓度的替米沙坦和聚合物前药(实验组)，以未经处理的空白细胞为对照，每孔设复孔；孵育48小时后，每孔加入20μL浓度为5mg/mL的噻唑兰溶液，继续孵育4h后弃去上清液，每孔加入二甲基亚砜200μL，用酶联检测仪测定570nm处吸光度，按下式计算细胞存活率：
The present invention evaluates the micellar cell survival rate of polymer prodrugs by the inhibition rate of tumor-associated fibroblasts. The test adopts tetrazolium salt colorimetry. Using mouse fibroblast NIH/3T3 cells activated by mouse breast cancer 4T1 cells as a model, 200 μL of culture solution containing 2×10 3 activated NIH/3T3 cells is added to each well of a 96-well cell culture plate, and the plate is placed in an incubator at 37°C and 5% CO 2 for 12 hours. After the cells are completely attached to the wall, different concentrations of telmisartan and polymer prodrug (experimental group) are added to the cell wells of the 96-well cell culture plate, and untreated blank cells are used as controls. Each well is set up with duplicate wells; after incubation for 48 hours, 20 μL of 5 mg/mL thiazolyl blue solution is added to each well, and the supernatant is discarded after continued incubation for 4 hours, 200 μL of dimethyl sulfoxide is added to each well, and the absorbance at 570 nm is measured by an enzyme-linked detector. The cell survival rate is calculated according to the following formula:
细胞存活率(％)＝实验组吸光度/对照组吸光度×100％Cell survival rate (%) = absorbance of experimental group/absorbance of control group × 100%
替米沙坦及聚合物前药对激活的小鼠成纤维细胞的毒性如图3所示，从图中可以看出聚合物前药处理的成纤维细胞的生存率低于替米沙坦处理的成纤维细胞，表明聚合物前药相比替米沙坦对小鼠成纤维细胞有更强的药效。The toxicity of telmisartan and polymer prodrug to activated mouse fibroblasts is shown in FIG3 . It can be seen from the figure that the survival rate of fibroblasts treated with polymer prodrug is lower than that of fibroblasts treated with telmisartan, indicating that polymer prodrug has a stronger efficacy on mouse fibroblasts than telmisartan.
实施例2Example 2
一种氧化还原环境响应释放的聚合物前药，如图1所示，通过以下方法制备：A redox-responsive release polymer prodrug, as shown in FIG1 , is prepared by the following method:
(1)替米沙坦耦合2,2’-二硫二乙醇：(1) Telmisartan coupled with 2,2'-dithiodiethanol:
将100mg替米沙坦溶、190mg的1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐和12mg的4-二甲氨基吡啶溶解在4mL二甲基亚砜中，60℃氮气气氛下搅拌1小时后，加入67mg的2,2’-二硫二乙醇，再搅拌12小时得到替米沙坦和2,2’-二硫二乙醇的耦合物；Dissolve 100 mg of telmisartan, 190 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 12 mg of 4-dimethylaminopyridine in 4 mL of dimethyl sulfoxide, stir for 1 hour at 60° C. under nitrogen atmosphere, add 67 mg of 2,2’-dithiodiethanol, and stir for another 12 hours to obtain a coupling product of telmisartan and 2,2’-dithiodiethanol;
(2)肝素钠的激活：(2) Activation of heparin sodium:
将200mg的肝素钠溶解于16mL的二甲基亚砜和水(体积比8:2)的混合溶液中，然后向混合溶液中加入826mg的1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐和60mg的4-二甲氨基吡啶，60℃氮气气氛下搅拌1小时后，得到活化的肝素钠；200 mg of heparin sodium was dissolved in 16 mL of a mixed solution of dimethyl sulfoxide and water (volume ratio 8:2), and then 826 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 60 mg of 4-dimethylaminopyridine were added to the mixed solution, and the mixture was stirred at 60° C. for 1 hour under a nitrogen atmosphere to obtain activated heparin sodium;
(3)肝素钠耦合替米沙坦聚合物前药的合成：(3) Synthesis of heparin sodium coupled telmisartan polymer prodrug:
将步骤(1)得到的替米沙坦和2,2’-二硫二乙醇的耦合物与步骤(2)得到的活化的肝素钠混合反应24h得到反应液，将反应液在纯水中透析3天，冷冻干燥后得到聚合物前药。The coupling product of telmisartan and 2,2'-dithiodiethanol obtained in step (1) and the activated sodium heparin obtained in step (2) are mixed and reacted for 24 hours to obtain a reaction solution, the reaction solution is dialyzed in pure water for 3 days, and the polymer prodrug is obtained after freeze-drying.
本发明以肿瘤相关成纤维细胞中α-平滑肌肌动蛋白的表达水平评价聚合物前药胶束逆转肿瘤成纤维细胞的表型能力。试验采用蛋白质印迹法，以小鼠乳腺癌4T1细胞激活的小鼠成 纤维NIH/3T3细胞为模型，在6孔细胞培养板的每孔中加入2.5mL含2×10
5个激活的NIH/3T3细胞的培养液，置37℃、5％CO
2孵箱培养12小时，待细胞完全贴壁后，在6孔细胞培养板的细胞孔中分别加入不同浓度的聚合物前药，孵育48小时后，弃去上清液，用磷酸缓冲盐溶液洗2次，将6孔细胞培养板置于冰上，每孔加入100μL细胞完全裂解液充分裂解细胞后，4℃，12000rpm，20分钟离心弃沉淀，测定蛋白浓度并定量计算加样量，对蛋白质进行高温水浴10分钟(100℃)变性，进行电泳后转膜封闭孵育一抗二抗后曝光，结果如图4所示。图4结果表明聚合物前药影响肿瘤相关成纤维细胞中α-平滑肌肌动蛋白的表达水平，且表达水平与药物浓度相关。
The present invention evaluates the ability of polymer prodrug micelles to reverse the phenotypic ability of tumor fibroblasts by using the expression level of α-smooth muscle actin in tumor-associated fibroblasts. The experiment adopted the Western blotting method, and mouse fibroblast NIH/3T3 cells activated by mouse breast cancer 4T1 cells were used as the model. 2.5 mL of culture medium containing 2×10 5 activated NIH/3T3 cells was added to each well of a 6-well cell culture plate, and the plates were cultured in a 37°C, 5% CO 2 incubator for 12 hours. After the cells were completely attached to the wall, different concentrations of polymer prodrugs were added to the cell wells of the 6-well cell culture plate. After incubation for 48 hours, the supernatant was discarded, and the plates were washed twice with phosphate buffered saline. The 6-well cell culture plates were placed on ice, and 100 μL of complete cell lysis solution was added to each well to fully lyse the cells. The plates were centrifuged at 4°C, 12000 rpm, and the precipitate was discarded. The protein concentration was determined and the sample addition amount was quantitatively calculated. The protein was denatured in a high temperature water bath for 10 minutes (100°C), and after electrophoresis, the plates were transferred to the membrane for blocking and incubation with primary and secondary antibodies, and then exposed. The results are shown in Figure 4. The results in Figure 4 show that the polymer prodrug affects the expression level of α-smooth muscle actin in tumor-associated fibroblasts, and the expression level is related to the drug concentration.
实施例3Example 3
一种氧化还原环境响应释放的聚合物前药，如图1所示，通过以下方法制备：A redox-responsive release polymer prodrug, as shown in FIG1 , is prepared by the following method:
(1)替米沙坦耦合2,2’-二硫二乙醇：(1) Telmisartan coupled with 2,2'-dithiodiethanol:
将160mg替米沙坦、296mg的1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐和16mg的4-二甲氨基吡啶溶解在6mL二甲基亚砜中，60℃氮气气氛下搅拌1小时后，加入114mg的2,2’-二硫二乙醇，再搅拌12小时得到替米沙坦和2,2’-二硫二乙醇的耦合物；160 mg of telmisartan, 296 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 16 mg of 4-dimethylaminopyridine were dissolved in 6 mL of dimethyl sulfoxide, and stirred at 60° C. for 1 hour under a nitrogen atmosphere, and then 114 mg of 2,2’-dithiodiethanol was added, and stirred for another 12 hours to obtain a coupling product of telmisartan and 2,2’-dithiodiethanol;
(2)肝素钠的激活：(2) Activation of heparin sodium:
将320mg的肝素钠溶解于20mL的二甲基亚砜和水(体积比8:2)的混合溶液中，然后向混合溶液中加入1.5g的1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐和96mg的4-二甲氨基吡啶，60℃氮气气氛下搅拌1小时后，得到活化的肝素钠；320 mg of heparin sodium was dissolved in 20 mL of a mixed solution of dimethyl sulfoxide and water (volume ratio 8:2), and then 1.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 96 mg of 4-dimethylaminopyridine were added to the mixed solution, and the mixture was stirred at 60° C. for 1 hour under a nitrogen atmosphere to obtain activated heparin sodium;
(3)肝素钠耦合替米沙坦聚合物前药的合成：(3) Synthesis of heparin sodium coupled telmisartan polymer prodrug:
将步骤(1)得到的替米沙坦和2,2’-二硫二乙醇的耦合物与步骤(2)得到的活化的肝素钠混合反应24h得到反应液，将反应液在纯水中透析3天，冷冻干燥后得到聚合物前药。The coupling product of telmisartan and 2,2'-dithiodiethanol obtained in step (1) and the activated sodium heparin obtained in step (2) are mixed and reacted for 24 hours to obtain a reaction solution, the reaction solution is dialyzed in pure water for 3 days, and the polymer prodrug is obtained after freeze-drying.
采用透析法研究聚合物前药中替米沙坦的释放谱。将等量的替米沙坦和聚合物前药装入透析袋，然后与25mL磷酸缓冲盐溶液或25mL磷酸缓冲盐溶液与不同浓度谷胱甘肽透析72小时，在预定的时间提取透析液并加入新的磷酸缓冲盐溶液，用高效液相色谱测定聚合物前药释放的替米沙坦对谷胱甘肽的反应谱，结果如图5所示。图5结果表明聚合物前药在谷胱甘肽环境与替米沙坦在28-30有同样的峰值，证明聚合物前药在谷胱甘肽环境下可释放出替米沙坦原 形药物。The release profile of telmisartan in polymer prodrug was studied by dialysis. Equal amounts of telmisartan and polymer prodrug were placed in a dialysis bag and then dialyzed with 25 mL of phosphate buffered saline or 25 mL of phosphate buffered saline and different concentrations of glutathione for 72 hours. The dialysate was extracted at a predetermined time and a new phosphate buffered saline was added. The reaction profile of telmisartan released from the polymer prodrug to glutathione was determined by HPLC, and the results are shown in Figure 5. The results in Figure 5 show that the polymer prodrug has the same peak value as telmisartan at 28-30 in the glutathione environment, proving that the polymer prodrug can release the prototype telmisartan drug in the glutathione environment.
以上仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，应视为本发明的保护范围。The above are only preferred embodiments of the present invention. The protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should be regarded as the protection scope of the present invention.
Claims (9)
- 一种氧化还原环境响应释放的聚合物前药，其特征在于，所述聚合物前药的结构式如下：A polymer prodrug that is released in response to a redox environment, characterized in that the structural formula of the polymer prodrug is as follows:
其中，n的范围是16～52。Among them, the range of n is 16 to 52. - 根据权利要求1所述的一种氧化还原环境响应释放的聚合物前药，其特征在于，所述聚合物前药是肝素钠通过2,2’-二硫二乙醇的酯化反应连接替米沙坦得到的聚合物前药，该聚合物前药在氧化还原条件下响应释放替米沙坦原型药物。A polymer prodrug that is released in response to a redox environment according to claim 1, characterized in that the polymer prodrug is a polymer prodrug obtained by connecting heparin sodium to telmisartan through an esterification reaction of 2,2'-dithiodiethanol, and the polymer prodrug releases the telmisartan prototype drug in response to redox conditions.
- 一种氧化还原环境响应释放的聚合物前药的制备方法，其特征在于，包括以下步骤：A method for preparing a polymer prodrug that is released in response to a redox environment, characterized in that it comprises the following steps:(1)替米沙坦耦合2,2’-二硫二乙醇：将替米沙坦溶解在二甲基亚砜、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐和4-二甲氨基吡啶的混合溶液中，搅拌反应后加入2,2’-二硫二乙醇，再搅拌12小时得到替米沙坦和2,2’-二硫二乙醇的耦合物；(1) Coupling of telmisartan with 2,2'-dithiodiethanol: dissolving telmisartan in a mixed solution of dimethyl sulfoxide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine, stirring for reaction, adding 2,2'-dithiodiethanol, and stirring for 12 hours to obtain a coupling product of telmisartan and 2,2'-dithiodiethanol;(2)肝素钠的激活：将肝素钠溶解于二甲基亚砜和水的混合溶液中，然后向混合溶液中加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐和4-二甲氨基吡啶，搅拌反应后得到活化的肝素钠；(2) Activation of sodium heparin: Sodium heparin is dissolved in a mixed solution of dimethyl sulfoxide and water, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine are added to the mixed solution, and the mixture is stirred for reaction to obtain activated sodium heparin;(3)肝素钠耦合替米沙坦聚合物前药的合成：将替米沙坦和2,2’-二硫二乙醇的耦合物与活化的肝素钠混合反应24h得到反应液，将反应液在纯水中透析3天，冷冻干燥后得到聚合物前药。(3) Synthesis of heparin sodium coupled telmisartan polymer prodrug: The coupled product of telmisartan and 2,2'-dithiodiethanol was mixed with activated heparin sodium for 24 hours to obtain a reaction solution, which was dialyzed in pure water for 3 days and freeze-dried to obtain a polymer prodrug.
- 根据权利要求3所述的一种氧化还原环境响应释放的聚合物前药的制备方法，其特征 在于，The method for preparing a polymer prodrug that is released in response to a redox environment according to claim 3, characterized in that:步骤(1)中，1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐、4-二甲氨基吡啶和替米沙坦的摩尔比为5～8:0.5～1:1～2。In step (1), the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and telmisartan is 5-8:0.5-1:1-2.
- 根据权利要求3所述的一种氧化还原环境响应释放的聚合物前药的制备方法，其特征在于，The method for preparing a polymer prodrug that is released in response to a redox environment according to claim 3, characterized in that:步骤(1)中，2,2’-二硫二乙醇和替米沙坦的摩尔比为1:1。In step (1), the molar ratio of 2,2'-dithiodiethanol to telmisartan is 1:1.
- 根据权利要求3所述的一种氧化还原环境响应释放的聚合物前药的制备方法，其特征在于，The method for preparing a polymer prodrug that is released in response to a redox environment according to claim 3, characterized in that:步骤(2)中，二甲基亚砜和水的体积比为8～9:2～1。In step (2), the volume ratio of dimethyl sulfoxide to water is 8-9:2-1.
- 根据权利要求3所述的一种氧化还原环境响应释放的聚合物前药的制备方法，其特征在于，The method for preparing a polymer prodrug that is released in response to a redox environment according to claim 3, characterized in that:步骤(2)中，1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐、4-二甲氨基吡啶和肝素钠的摩尔比为5～8:0.5～1:1～2。In step (2), the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and heparin sodium is 5-8:0.5-1:1-2.
- 根据权利要求3所述的一种氧化还原环境响应释放的聚合物前药的制备方法，其特征在于，The method for preparing a polymer prodrug that is released in response to a redox environment according to claim 3, characterized in that:步骤(1)和(2)中，所述搅拌反应的反应条件为在60℃的氮气气氛下搅拌1小时。In steps (1) and (2), the stirring reaction is carried out under a nitrogen atmosphere at 60° C. for 1 hour.
- 如权利要求1～2任一项所述的氧化还原环境响应释放的聚合物前药在制备治疗肿瘤药物中的应用。Use of the redox environment-responsive released polymer prodrug as claimed in any one of claims 1 to 2 in the preparation of a drug for treating tumors.
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| CN106729749A (en) * | 2017-01-24 | 2017-05-31 | 四川大学 | It is a kind of to realize transmission system that gene and medicine are carried altogether and preparation method and application |
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| US20180250237A1 (en) * | 2017-03-01 | 2018-09-06 | National Tsing Hua University | Methods of preparing stimuli-responsive multifunctional nanoparticles |
| US20190091147A1 (en) * | 2016-12-26 | 2019-03-28 | Jiangnan University | Preparation Method for Charge Reversaland Reversibly Crosslinked Redox-Sensitive Nanomicelles |
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| US20190091147A1 (en) * | 2016-12-26 | 2019-03-28 | Jiangnan University | Preparation Method for Charge Reversaland Reversibly Crosslinked Redox-Sensitive Nanomicelles |
| CN106729749A (en) * | 2017-01-24 | 2017-05-31 | 四川大学 | It is a kind of to realize transmission system that gene and medicine are carried altogether and preparation method and application |
| US20180250237A1 (en) * | 2017-03-01 | 2018-09-06 | National Tsing Hua University | Methods of preparing stimuli-responsive multifunctional nanoparticles |
| US20190338051A1 (en) * | 2017-07-27 | 2019-11-07 | Dalian Minzu University | Preparation method for and use of redox-responsive chitosan-liposome |
| CN108003354A (en) * | 2017-12-28 | 2018-05-08 | 浙江大学 | A kind of polymer and its preparation and application for responding intracellular acidic and redox environment |
| CN112999359A (en) * | 2021-03-03 | 2021-06-22 | 中国药科大学 | Tumor-targeted redox-response prodrug nano preparation and preparation method and application thereof |
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