A preparation method of a pH / anaerobic dual response type anti-tumor nano drug carrier

Abstract

A preparation method of a pH / hypoxia dual response type anti-tumor nano drug carrier, adopts sulfonamide zwitterionic copolymer modified COF nanoparticles, which is used as a nano drug carrier, and further loads doxorubicin to obtain a nano drug. The nano drug prepared by the method has the dual response type ability of acid and hypoxia in the tumor microenvironment; in the tumor acid environment, the surface charge of the nano carrier can be stimulated to change responsively, which can effectively improve the endocytosis effect of tumor cells, and then improve the enrichment and drug efficiency of the drug in the tumor site. At the same time, the drug is degraded in response to the hypoxic environment, so as to realize the controlled release of the drug in the tumor site.

Description

Technical Field

[0001] This invention belongs to the field of drug carrier technology, specifically a method for preparing a pH / hypoxia dual-responsive antitumor nanomedicine carrier. Background Technology

[0002] The unique advantages of nanocarriers in cancer therapy have attracted widespread attention. These advantages include loading insoluble drugs, reducing the dosage of small molecule drugs and thus improving biosafety, and nanocarriers are more likely to accumulate at tumor sites, leading to better therapeutic effects. However, traditional nanocarrier delivery principles are based on passive transport. Non-targeted drug delivery not only easily causes toxicity to normal tissues but is also easily cleared rapidly by the mononuclear phagocyte system through the liver and spleen. Designing nanocarriers with unique functions to enable targeted drug delivery to tumor sites and controlled release is a hot research topic.

[0003] Tumors exhibit different local tissue conditions compared to normal tissues due to their lesions. For example, the "Warburg effect" causes tumor cells to produce large amounts of lactic acid through glycolysis, resulting in a slightly acidic tumor environment (pH 6.5–6.8). Rapid tumor growth also creates a hypoxic environment, leading to the overexpression of azoreductases. Utilizing these characteristics of the tumor microenvironment to design stimulus-responsive nanomedicine carriers is an effective means to improve the efficacy of cancer treatment.

[0004] Currently, various carriers for in vivo drug delivery have been developed in existing technologies, such as liposomes, micelles, iron oxide (Fe3O4), covalent organic frameworks (COFs), and carbon nanomaterials (CNASs). Among them, COFs, as a novel type of ordered porous material formed by covalent bonds, have shown great potential for drug delivery due to their high chemical stability and tunable porosity. However, their stable chemical structure makes it difficult to release the loaded drug, and the low dispersibility and poor cell permeability of COFs limit their application in drug therapy. Therefore, surface modification of COFs to change their surface charge, particle size, and assembly structure to enable them as nanomedicine carriers for targeted antitumor delivery is of great significance for tumor treatment. Summary of the Invention

[0005] The technical objective of this invention is to provide a sulfonyl zwitterionic monomer and a sulfonamide zwitterionic copolymer, and to use the sulfonamide zwitterionic copolymer to modify the surface of a COF with a specific structure to prepare a pH / hypoxia dual-responsive antitumor nanomedicine carrier. This carrier can accelerate the uptake of antitumor nanomedicine loaded with DOX by tumor cells through surface charge flipping, azo bond breaking, etc., and promote the disintegration of the COF carrier to release the chemotherapeutic drug DOX, thereby achieving drug enrichment at the tumor site and improving the endocytosis effect and drug delivery efficiency of tumor cells.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by this invention is: a sulfonyl zwitterionic monomer with the following structural formula:

[0007] A method for preparing sulfonyl zwitterionic monomers includes the following steps: Step (1): Mix the sulfonamide compound with toluene at a ratio of (0.12-0.24) mol / L, heat at 100-120℃ to fully dissolve, and prepare a mixed solution A. Add bromoacetyl bromide dropwise to mixed solution A at a molar ratio of 3.5:1 to bromoacetyl bromide, and stir at 100-120℃ for 5-10 h. After cooling, place the resulting reaction product at -20-10℃ for 12-24 h, and then filter, wash and vacuum dry to obtain 2-bromo-NR-(sulfonyl)acetamide for later use. The sulfonamide compound is benzenesulfonamide or 4-hydroxybenzenesulfonamide, and R is benzene or 4-hydroxybenzene. Step (2): Take 2-bromo-NR-(sulfonyl)acetamide and allyl dimethylamino ester compounds 2-dimethylaminoethyl methacrylate or dimethylaminoethyl acrylate respectively according to a molar ratio of 1:(2-4). Then, dissolve the weighed 2-bromo-NR-(sulfonyl)acetamide in deionized water, and then add the weighed allyl dimethylamino ester compounds and polymerization inhibitors to it. Stir the reaction at 60-80℃ for 24-72h. After the reaction product is freeze-dried to remove water, it is purified by column chromatography and dried in sequence to obtain sulfonyl zwitterionic monomers.

[0008] In step (2), the polymerization inhibitor is any one of 4-methoxyphenol, p-tert-butylcatechol and hydroquinone, and the amount of polymerization inhibitor is 0.01-1% of the molar amount of double bonds in the reaction raw materials.

[0009] A sulfonamide zwitterionic copolymer, prepared by RAFT polymerization or free radical polymerization of sulfonyl zwitterionic monomers with 2-aminoethyl methacrylate hydrochloride, has the following structural formula: The above-mentioned sulfonamide zwitterionic copolymers were prepared by RAFT polymerization as follows: According to the molar ratio of (1-5):1, sulfonyl zwitterionic monomers and 2-aminoethyl methacrylate hydrochloride were dissolved in an organic solvent. Then, a chain transfer agent and an initiator were added to the reaction system. The amount of chain transfer agent added was 1 / 30 of the total molar amount of the reaction raw materials, and the molar ratio between the chain transfer agent and the initiator was (3-5):1. Then, nitrogen gas was introduced into the reaction system for 30 min. After that, the polymerization reaction was carried out at a temperature of 65-85℃ for 12-48 h. The resulting reaction product was first dialyzed with deionized water for 3-5 days, and then freeze-dried to obtain the sulfonamide zwitterionic copolymer. The above-mentioned sulfonamide zwitterionic copolymers were prepared by free radical polymerization as follows: According to a molar ratio of (1-5):1, sulfonyl zwitterionic monomers and 2-aminoethyl methacrylate hydrochloride were dissolved in an organic solvent. Then, 0.1% to 1% of the total molar amount of the reactants as an initiator was added to the reaction system. Nitrogen gas was then introduced into the reaction system for 30 minutes. After that, the polymerization reaction was carried out at a temperature of 65-85℃ for 12-48 hours. The resulting reaction product was first dialyzed with deionized water for 3-5 days, and then freeze-dried to obtain the sulfonamide zwitterionic copolymer.

[0010] Furthermore, the initiator is AIBN or ACVA, and the chain transfer agent is any one of 4-cyano-4-(thiobenzoylthio)valerate, 4-cyano-4-(thiobenzoyl)valerate, 2-(dodecylthiothiocarbonylthio)-2-methylpropionic acid, 4-cyano-4-[[(dodecylthio)thionylmethyl]thio]valerate, benzyl dithiobenzoate, and S-thiobenzoyl mercaptoacetic acid.

[0011] A method for preparing a pH / hypoxia dual-responsive antitumor nanomedicine carrier, characterized by comprising the following steps: According to the mass ratio of (2-4):1, sulfonamide zwitterionic copolymer and COF nanoparticles were weighed separately. Then, the weighed sulfonamide zwitterionic copolymer was dissolved in deionized water and ultrasonically dispersed for 2-10 min. Then, under continuous stirring, the weighed COF nanoparticles were added to the sulfonamide zwitterionic copolymer solution and stirred at 35-37℃ for 24-72 h. After centrifugation, washing and freeze-drying, the pH / hypoxia dual-responsive antitumor nanomedicine carrier was obtained. The COF nanoparticles are constructed from p-diaminoazobenzene as the backbone monomer and trialdehyde phloroglucinol, and their structural formula is as follows:

[0012] Furthermore, the concentration of the sulfonamide zwitterionic copolymer solution is 2-4 mg / mL, the number of centrifugation cycles is 3-5, and the washing solvent is deionized water.

[0013] An antitumor nanomedicine is prepared by encapsulating doxorubicin in a pH / hypoxia dual-responsive antitumor nanomedicine carrier.

[0014] Furthermore, according to the ratio of pH / hypoxia dual-responsive antitumor nanomedicine carrier to doxorubicin solution of 10-20 mg / mL, the pH / hypoxia dual-responsive antitumor nanomedicine carrier was added to the doxorubicin solution and stirred continuously for 2-12 h. Then, the obtained product was placed in deionized water for dialysis purification for 24-72 h, and after filtration and freeze-drying, the finished antitumor nanomedicine was obtained.

[0015] Furthermore, the concentration of the doxorubicin solution is 2–8 mg / mL.

[0016] The beneficial effects of this invention are as follows: 1. This invention provides a method for preparing a pH / hypoxia dual-responsive antitumor nanomedicine carrier. The method involves modifying COF nanoparticles with a sulfonamide zwitterionic copolymer to obtain a pH / hypoxia dual-responsive antitumor nanomedicine carrier. This carrier is further coated with DOX to obtain the antitumor nanomedicine. The antitumor nanomedicine prepared by this invention is neutral under physiological conditions and can achieve long-term circulation in vivo. In the tumor microenvironment, a charge reversal occurs on the particle surface, i.e., the COF surface-modified copolymer undergoes charge flipping, interacting electrostatically with tumor cells and accelerating the uptake of the antitumor nanomedicine by tumor cells. Furthermore, under the action of azo reductase, the azo bonds in the antitumor nanomedicine structure break, the COF carrier disintegrates, and the chemotherapeutic drug DOX is released, exhibiting pH / hypoxia dual responsiveness, achieving targeted and controlled drug delivery. Compared with single-responsive carriers, the pH / hypoxia dual-responsive antitumor nanomedicine carrier of this application can better control drug release and significantly improve therapeutic efficacy.

[0017] 2. The pH / hypoxia dual-responsive antitumor nanomedicine carrier prepared by this invention can achieve long-term circulation in vivo; in the tumor microenvironment, the particle surface undergoes charge transformation, which can accelerate the uptake of drugs by tumor cells; and under the action of azo reductase, the COF carrier disintegrates, releasing the chemotherapy drug DOX, achieving the purpose of targeted drug delivery and controlled release, and improving the therapeutic effect of the drug. Attached Figure Description

[0018] Figure 1 This is the 1H NMR spectrum of the sulfonamide zwitterionic monomer prepared in Example 1 of this invention; Figure 2This is the carbon NMR spectrum of the sulfonamide zwitterionic monomer prepared in Example 1 of this invention; Figure 3 This is a schematic diagram illustrating the working principle of Embodiment 1 of the present invention; Figure 4 This is a comparison of the surface potential changes of the pH / hypoxia dual-responsive antitumor nanomedicine carrier prepared in Example 1 of this invention and COF-PEG under different pH conditions; Figure 5 This is a graph showing the particle size change of the pH / hypoxia dual-responsive antitumor nanomedicine carrier prepared in Example 1 of the present invention under hypoxic conditions. Figure 6 This is a structural diagram showing the stability test of the pH / hypoxia dual-responsive antitumor nanomedicine carrier prepared in Example 1 of the present invention in different solution environments. Figure 7 This is a biocompatibility test diagram of the pH / hypoxia dual-responsive antitumor nanomedicine carrier prepared in Example 1 of the present invention; Figure 8 A comparative graph showing the test results of the anti-tumor nanomedicine prepared in Example 1 of this invention regarding the change in body weight of mice after 14 days of treatment; Figure 9 Comparison of test results of tumor volume changes after 14 days of treatment with the anti-tumor nanomedicine prepared in Example 1 of the present invention; Figure 10 Comparison of test results on the changes in tumor quality after 14 days of treatment with the anti-tumor nanomedicine prepared in Example 1 of the present invention; Figure 11 The particle size distribution and TEM image of the antitumor nanomedicine prepared in Example 1 of this invention. Detailed Implementation

[0019] The present invention will be further explained and described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are based on the technical solutions of the present invention and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0020] This invention provides a sulfonyl zwitterionic monomer, which can be used to prepare sulfonamide zwitterionic copolymers. The sulfonamide zwitterionic copolymers can be used to prepare pH / hypoxia dual-responsive antitumor nanomedicine carriers. At the same time, antitumor nanomedicines can be prepared using the pH / hypoxia dual-responsive antitumor nanomedicine carriers.

[0021] Sulfonyl zwitterionic monomers, with the following structural formula:

[0022] The preparation method of the above-mentioned sulfonyl zwitterionic monomer includes the following steps: Step (1) Take sulfonamide compound benzenesulfonamide or 4-hydroxybenzenesulfonamide and toluene at a ratio of (0.12-0.24) mol / L, heat at 100-120℃ to fully dissolve it, and prepare mixed solution A. Take bromoacetyl bromide and sulfonamide compound at a molar ratio of 3.5:1, add bromoacetyl bromide dropwise to mixed solution A, stir and react at 100-120℃ for 5-10h, cool, and place the obtained reaction product at -20-10℃ for 12-24h. After filtration, washing and vacuum drying, 2-bromo-NR-(sulfonyl)acetamide is obtained, wherein R is benzene or 4-hydroxybenzene, for later use. Step (2): Take 2-bromo-NR-(sulfonyl)acetamide and allyl dimethylamino ester compounds methacrylate-2-dimethylaminoethyl ester or dimethylaminoethyl acrylate respectively according to a molar ratio of 1:(2-4). Then, dissolve the weighed 2-bromo-NR-(sulfonyl)acetamide in deionized water, and then add the weighed allyl dimethylamino ester compounds and polymerization inhibitors. The polymerization inhibitors are any one of 4-methoxyphenol, p-tert-butylcatechol and hydroquinone, and the amount of polymerization inhibitor is 0.01-1% of the molar amount of double bonds in the reaction raw materials. Stir the reaction at 60-80℃ for 24-72h. After the obtained reaction product is freeze-dried to remove water, it is purified by column chromatography and dried in sequence to obtain sulfonyl zwitterionic monomers.

[0023] A sulfonamide zwitterionic copolymer, prepared by RAFT polymerization or free radical polymerization of sulfonyl zwitterionic monomers with 2-aminoethyl methacrylate hydrochloride, has the following structural formula: The above-mentioned sulfonamide zwitterionic copolymers were prepared by RAFT polymerization as follows: According to a molar ratio of (1-5):1, sulfonyl zwitterionic monomers and 2-aminoethyl methacrylate hydrochloride were dissolved in an organic solvent. Then, a chain transfer agent and an initiator were added to the reaction system. The initiator was AIBN or ACVA, and the chain transfer agent was 4-cyano-4-(thiobenzoylthio)valerate, 4-cyano-4-(thiobenzoyl)valerate, 2-(dodecylthio-thiocarbonylthio)-2-methylpropionic acid, or 4-cyano-4-[[(dodecylthio)thione] The reaction mixture contains any one of [methyl]thio[pentanoic acid], benzyl dithiobenzoate, and S-thiobenzoyl mercaptoacetic acid, with the amount of chain transfer agent added being 1 / 30 of the total molar amount of the reaction raw materials. The molar ratio between the chain transfer agent and the initiator is (3-5):1. Then, nitrogen gas is introduced into the reaction system for 30 min. After that, the polymerization reaction is carried out at a temperature of 65-85℃ for 12-48 h. The resulting reaction product is first dialyzed with deionized water for 3-5 days, and then freeze-dried to obtain a sulfonamide zwitterionic copolymer. The above-mentioned sulfonamide zwitterionic copolymers were prepared by free radical polymerization as follows: According to a molar ratio of (1-5):1, sulfonyl zwitterionic monomers and 2-aminoethyl methacrylate hydrochloride were dissolved in an organic solvent. Then, 0.1% to 1% of the total molar amount of the reaction raw materials as initiator AIBN or ACVA was added to the reaction system. Nitrogen gas was then introduced into the reaction system for 30 minutes. After that, the polymerization reaction was carried out at a temperature of 65-85℃ for 12-48 hours. The resulting reaction product was first dialyzed with deionized water for 3-5 days, and then freeze-dried to obtain the sulfonamide zwitterionic copolymer.

[0024] A method for preparing a pH / hypoxia dual-responsive antitumor nanomedicine carrier, characterized by comprising the following steps: According to a mass ratio of (2-4):1, sulfonamide zwitterionic copolymer and COF nanoparticles were weighed separately. Then, the weighed sulfonamide zwitterionic copolymer was dissolved in deionized water and ultrasonically dispersed for 2-10 min to obtain a sulfonamide zwitterionic copolymer solution with a concentration of 2-4 mg / mL. Then, under continuous stirring, the weighed COF nanoparticles were added to the sulfonamide zwitterionic copolymer solution and stirred at 35-37℃ for 24-72 h. After centrifugation, washing and freeze-drying, a pH / hypoxia dual-responsive antitumor nanomedicine carrier was obtained. The COF nanoparticles are constructed from p-diaminoazobenzene as the backbone monomer and trialdehyde phloroglucinol, and their structural formula is as follows:

[0025] An antitumor nanomedicine is prepared by encapsulating doxorubicin with a pH / hypoxia dual-responsive antitumor nanomedicine carrier. Specifically, the pH / hypoxia dual-responsive antitumor nanomedicine carrier is added to a doxorubicin solution with a concentration of 2-8 mg / mL at a ratio of 10-20 mg / mL, and the mixture is stirred continuously for 2-12 h. After that, the product is placed in deionized water for dialysis purification for 24-72 h. After filtration and freeze-drying, the finished antitumor nanomedicine is obtained.

[0026] Example 1: This embodiment provides a method for preparing anti-tumor nanomedicines, specifically including the following steps: I. Preparation of Sulfonamide Zwitterions First, benzenesulfonamide (1.1 g, 7 mmol) and 50 mL of toluene were placed in a 250 mL three-necked flask and heated at 110 °C for 10 min to promote the dissolution of benzenesulfonamide in toluene. Then, 2.2 mL of bromoacetyl bromide was slowly added dropwise to the mixture, and the reaction was carried out at 110 °C for 5 h. After cooling, the reaction mixture was placed at -20 °C overnight, filtered, washed, and dried under vacuum to obtain the product 2-bromo-N-(benzenesulfonyl)acetamide. Then, 2-bromo-N-(benzenesulfonyl)acetamide (400 mg, 1.44 mmol) was dissolved in 3 mL of deionized water. Add 2-dimethylaminoethyl methacrylate (800 mg, 5.09 mmol) and polymerization inhibitor 4-methoxyphenol (6 mg, 0.048 mmol), stir at 70 °C for 48 h, and purify by column chromatography to obtain the pure product, a sulfonamide zwitterionic monomer—(2-((2-(methacryloyloxy)ethyl)dimethylamino)acetyl)(benzenesulfonyl)amide (BSPAD).

[0027] The reaction principle of the sulfonamide zwitterionic monomers prepared in this embodiment is as follows:

[0028] The sulfonamide zwitterionic monomers prepared in this embodiment were analyzed by 1H NMR and 1C NMR, and the results are attached. Figure 1 and attached Figure 2 As shown.

[0029] II. Preparation of COF nanoparticles 9.55 mg of p-diaminoazobenzene was dispersed in 100 mL of anhydrous dichloromethane (DCM), and 20 μL of trifluoroacetic acid was added as a catalyst. Then, 6.3 mg of trialdehyde phloroglucinol was dispersed in 100 mL of anhydrous DCM and slowly added to the mixture. The reaction was carried out at 35 °C for 6 h. After the reaction was complete, n-hexane was added, the product was allowed to precipitate, the precipitate was collected, centrifuged at 10000 rpm for 10 min, washed three times successively with dimethylacetamide (DMAC) and acetone, and dried overnight under vacuum at 80 °C to obtain the COF nanoparticles, the structural formula of which is:

[0030] III. Preparation of Sulfonamide Zwitterionic Copolymers 284.71 mg of [2-((2-(methacryloyloxy)ethyl)dimethylamino)acetyl](benzenesulfonyl)amide (BSPAD) and 66.52 mg of 2-aminoethyl methacrylate hydrochloride (AEHM) were dissolved in 2 mL of methanol. Then, 11.17 mg of chain transfer agent 4-cyano-4-(thiobenzoyl)valerate and 2.8 mg of polymerization initiator ACVA were added. The mixture was purged with nitrogen for 30 min and polymerized at 70 °C for 24 h. The above system was dialyzed in deionized water for 3 days and then lyophilized to obtain the sulfonamide zwitterionic copolymer P(BSPAD-co-AEHM) (PBA).

[0031] IV. pH / Hypoxia Dual-Response Antitumor Nanomedicine Carrier: 20 mg of sulfonamide zwitterionic copolymer PBA and 10 mg of COF particles were added to 10 mL of deionized water and stirred at room temperature for 24 h. Then, the mixture was dialyzed in deionized water to remove unreacted copolymers. After freeze-drying, modified COF-PBA was obtained, which is the pH / hypoxia dual-response antitumor nanomedicine carrier.

[0032] V. Anti-tumor nanomedicines 4 mg of COF-PBA was placed in 0.4 mL of a 2 mg / mL doxorubicin (DOX) solution and stirred continuously for 4 h. After dialysis for 72 h to remove free DOX, COF-PBA@DOX was obtained by freeze drying, which is the anti-tumor nanomedicine. The reaction principle of the sulfonamide zwitterionic copolymer PBA-modified COF and DOX encapsulation prepared in this embodiment is attached. Figure 3 As shown.

[0033] Example 2: This embodiment provides a method for preparing anti-tumor nanomedicines, specifically including the following steps: I. Preparation of Sulfonamide Zwitterions First, 12 mmol of 4-hydroxybenzenesulfonamide and 50 mL of toluene were placed in a 250 mL three-necked flask and heated at 120 °C for 8 min to promote the dissolution of 4-hydroxybenzenesulfonamide in toluene. Then, 3.8 mL of bromoacetyl bromide was slowly added dropwise to the mixture, and the reaction was carried out at 120 °C for 8 h. After cooling, the reaction mixture was allowed to stand at -10 °C for 24 h, filtered, washed, and dried under vacuum to obtain the product 2-bromo-N-(4-hydroxybenzenesulfonyl)acetamide. Then, 1.5 mmol of 2-bromo-N-(4-hydroxybenzenesulfonyl)acetamide was dissolved in 3 mL of deionized water, and 3 mmol of 2-dimethylaminoethyl methacrylate and 0.03 mmol of hydroquinone as a polymerization inhibitor were added. The mixture was stirred at 80 °C for 24 h, and purified by column chromatography to obtain the pure product, a sulfonamide zwitterionic monomer—2-((4-hydroxyphenyl)sulfonamide)-N-(2-(methacryloyloxy)ethyl)-N,N-dimethyl-2-oxy-1-amino (HSMED).

[0034] II. Preparation of COF nanoparticles The preparation of COF nanoparticles in this embodiment is the same as in Example 1.

[0035] III. Preparation of Sulfonamide Zwitterionic Copolymers 297 mg HSMED and 66.52 mg 2-aminoethyl methacrylate hydrochloride (AEHM) (molar ratio 2:1) were dissolved in 2 mL of methanol. Then, 11.17 mg of chain transfer agent 4-cyano-4-(thiobenzoyl)valerate and 2.8 mg of polymerization initiator ACVA were added. The mixture was purged with nitrogen for 30 min and polymerized at 70 °C for 24 h. The above system was dialyzed in deionized water for 3 days and then lyophilized to obtain the sulfonamide zwitterionic copolymer P(HSMED-co-AEHM)(PHA).

[0036] IV. pH / Hypoxia Dual-Response Antitumor Nanomedicine Carrier: 40 mg of sulfonamide zwitterionic copolymer PHA and 10 mg of COF particles were added to 15 mL of deionized water, ultrasonically dispersed for 10 min, stirred at room temperature for 24 h, and then dialyzed in deionized water to remove unreacted copolymer. After freeze-drying, modified COF-PHA was obtained, which is the pH / hypoxia dual-response antitumor nanomedicine carrier.

[0037] V. Anti-tumor nanomedicines 5 mg of COF-PHA was placed in 0.25 mL of a 6 mg / mL doxorubicin (DOX) solution and stirred continuously for 10 h. After dialysis for 72 h to remove free DOX, COF-PHA@DOX was obtained by freeze drying, which is the anti-tumor nanomedicine.

[0038] Example 3: The preparation method of the antitumor nanomedicine in this embodiment is the same as that in Example 1, except that the preparation method of the sulfonamide zwitterionic copolymer in Example 1 is changed from RAFT polymerization to free radical polymerization.

[0039] The preparation method of the sulfonamide zwitterionic copolymer is as follows: 284.71 mg BSPAD and 66.52 mg AEHM were dissolved in 2 mL of methanol, and then 2.8 mg of polymerization initiator ACVA was added. Nitrogen gas was purged for 30 min, and polymerization was carried out at 70 °C for 24 h. The above system was dialyzed in deionized water for 3 days, and after lyophilization, the sulfonamide zwitterionic copolymer P(BSPAD-AEHM)-2 (PBA-2) was obtained.

[0040] Example 4: The preparation method of the antitumor nanomedicine in this embodiment is the same as that in Example 2, except that the preparation method of the sulfonamide zwitterionic copolymer in Example 2 is changed from RAFT polymerization to free radical polymerization.

[0041] The preparation method of the sulfonamide zwitterionic copolymer is as follows: 297 mg HSMED and 66.52 mg AEHM (molar ratio 2:1) were dissolved in 2 mL of methanol, and then 2.8 mg of polymerization initiator ACVA was added. Nitrogen gas was purged for 30 min, and polymerization was carried out at 70 °C for 24 h. The above system was dialyzed in deionized water for 3 days, and after lyophilization, a sulfonamide zwitterionic copolymer P(HSMED-AEHM)-2 (PHA-2) was obtained.

[0042] Example 5: The preparation method of the antitumor nanomedicine in this embodiment is the same as that in Example 1, except that the raw material 2-dimethylaminoethyl methacrylate in the preparation method of sulfonamide zwitterionic monomer in Example 1 is replaced with dimethylaminoethyl acrylate.

[0043] This embodiment provides a method for preparing anti-tumor nanomedicines, specifically including the following steps: I. Preparation of Sulfonamide Zwitterions First, benzenesulfonamide (1.1 g, 7 mmol) and 50 mL of toluene were placed in a 250 mL three-necked flask and heated at 110 °C for 10 min to promote the dissolution of benzenesulfonamide in toluene. Then, 2.2 mL of bromoacetyl bromide was slowly added dropwise to the mixture, and the reaction was carried out at 110 °C for 5 h. After cooling, the reaction mixture was placed at -20 °C overnight, filtered, washed, and dried under vacuum to obtain the product 2-bromo-N-(benzenesulfonyl)acetamide. Then, 2-bromo-N-(benzenesulfonyl)acetamide (400 mg, 1.44 mmol) was dissolved in 3 mL of deionized water. Add 2-dimethylaminoethyl acrylate (720 mg, 5.04 mmol) and polymerization inhibitor 4-methoxyphenol (6 mg, 0.048 mmol), stir at 70 °C for 48 h, and purify by column chromatography to obtain the pure product, a sulfonamide zwitterionic monomer—N-(2-(propyleneoxy)ethyl)-N,N-dimethyl-2-oxo-2-(benzenesulfonyl)ethylamide (EDPEA).

[0044] II. Preparation of COF nanoparticles The preparation of COF nanoparticles in this embodiment is the same as in Example 1.

[0045] III. Preparation of Sulfonamide Zwitterionic Copolymers 279.62 mg EDPEA and 66.52 mg AEHM (molar ratio 2:1) were dissolved in 2 mL of methanol, then 11.17 mg of chain transfer agent 4-cyano-4-(thiobenzoyl)valerate and 2.8 mg of polymerization initiator ACVA were added. The mixture was purged with nitrogen for 30 min and polymerized at 70 °C for 24 h. The resulting system was dialyzed in deionized water for 3 days and then lyophilized to obtain the sulfonamide zwitterionic copolymer P(EDPEA-co-AEHM)(PEA).

[0046] IV. pH / Hypoxia Dual-Response Antitumor Nanomedicine Carrier: This step is the same as in Example 1, and the modified COF-PEA is finally obtained, which is the pH / hypoxia dual-response antitumor nanomedicine carrier.

[0047] V. Anti-tumor nanomedicines This step is the same as in Example 1, and the final product is COF-PEA@DOX, which is an anti-tumor nanomedicine.

[0048] Example 6: The preparation method of the antitumor nanomedicine in this embodiment is the same as that in Example 5, except that the preparation method of the sulfonamide zwitterionic copolymer in Example 5 is changed from RAFT polymerization to free radical polymerization.

[0049] The preparation method of the sulfonamide zwitterionic copolymer is as follows: 279.62 mg EDPEA and 133.04 mg AEHM (molar ratio 1:1) were dissolved in 2 mL of methanol, and then 3.2 mg of polymerization initiator ACVA was added. Nitrogen gas was purged for 30 min, and polymerization was carried out at 70 °C for 24 h. The above system was dialyzed in deionized water for 3 days, and after lyophilization, the sulfonamide zwitterionic copolymer P(EDPEA-AEHM)-2 (PEA-2) was obtained.

[0050] Experimental measurement 1. pH responsiveness determination of pH / hypoxia dual-responsive antitumor nanocarriers: The COF-PBA prepared in Example 1 was dispersed in PBS buffer solutions with different pH values ​​(pH 6.0-8.0), with COF-PEG as a control group. The surface potential changes of these nanocarriers under different pH conditions were measured using a laser particle size analyzer. The results are attached. Figure 4 As shown, when the pH changes from 8.0 to 5.4, the surface potential of COF-PBA increases from -15.45mV to 12.24mV. This is because at pH 6.5, the protonation of the imine groups in PBA makes the surface potential of the carrier positive. In contrast, the zeta potential of the COF-PEG group does not change significantly.

[0051] 2. Determination of the hypoxia responsiveness of the pH / hypoxia dual-responsive antitumor nanomedicine carrier: The particle size change of COF-PBA prepared in Example 1 under hypoxia environment was studied, and the results are shown in the appendix. Figure 5 As shown: After mixing with 2M Na2S2O4 solution for 6 hours, the DLS measurement results of COF-PBA@DOX showed that the particle size of COF-PBA@DOX decreased significantly, and the distribution changed from an initial single peak to a three-peak distribution. This is because the azo bonds in the COF skeleton broke, causing the morphology of COF-PBA@DOX to change from a uniformly sized sphere to irregular fragments.

[0052] 3. Stability determination of pH / hypoxia dual-responsive antitumor nanomedicine carrier: The COF-PBA prepared in Example 1 was placed in water, physiological saline, and DMEM solution, respectively. Its size change was characterized by DLS to determine its structural stability. The test results are attached. Figure 6 As shown, after 7 days of cultivation in different media, the particle size of COF-PBA did not change significantly, indicating that it has excellent stability.

[0053] 4. The biocompatibility of the pH / hypoxia dual-responsive antitumor nanomedicine carrier was determined using the MTT assay. The cytotoxicity of COF-PBA prepared in Example 1 against the L02 cell line was investigated to determine its biocompatibility. The test results are attached. Figure 7As shown, even at a concentration of 200 μg / mL, the cell viability after co-culturing with COF-PBA was above 90% under both pH 7.4 and pH 6.5 conditions, indicating that COF-PBA has good biosafety.

[0054] 5. Determination of the antitumor effect of the antitumor nanomedicine: The therapeutic effect of the antitumor nanomedicine COF-PBA@DOX prepared in Example 1 was evaluated through in vivo experiments in mice. PBS was used as a blank control, and COF-PEG@DOX was used as a non-responsive control group. The results are shown in the appendix. Figure 8 Appendix Figure 9 and attached Figure 10 As shown, the body weight of mice in the PBS group, COF-PEG@DOX group and COF-PBA@DOX group did not fluctuate significantly. Only the free DOX group experienced a decrease in body weight due to the systemic toxicity of its chemotherapy. This indicates that COF-PBA@DOX nanomedicine has excellent biosafety in vivo and can effectively reduce the toxicity of free drugs.

[0055] like Figure 9 As shown, the tumors in the PBS group grew rapidly, reaching a volume of 700 mm² after 14 days. 3 Around 100 mmHg, while the tumors in the free DOX group grew more slowly, reaching a volume of 300 mmHg after 14 days. 3 This is because the chemotherapy drug DOX has a certain killing effect on tumors. The COF-PEG@DOX group, due to the enrichment of nanoparticles, was able to inhibit tumor growth to a certain extent, ultimately reducing the tumor volume to 180 mm². 3 However, due to its relatively short in vivo circulation time and low degree of tumor cell internalization, the treatment failed to produce a significant anti-tumor effect. In contrast, COF-PBA@DOX treatment in mice showed a significant inhibitory effect on tumor growth, significantly better than the COF-PEG@DOX group. COF-PBA@DOX completely inhibited tumor growth during the 14-day treatment period. Furthermore, analysis of tumor weight collected from dissections of each group after 14 days also verified the significant anti-tumor effect of COF-PBA@DOX.

[0056] The particle size and morphology of COF-PBA@DOX prepared in Example 1 were tested by DLS and TEM, and the results are as follows. Figure 11 As shown, the hydrodynamic diameter of COF-PBA@DOX (108.0±2.4nm) is a uniformly shaped sphere (inset).

[0057] It should be noted that although the present invention has been described through the above embodiments, the present invention may have many other embodiments. Without departing from the spirit and scope of the present invention, those skilled in the art can obviously make various corresponding changes and modifications to the present invention, but all such changes and modifications should fall within the scope of protection of the appended claims and their equivalents.

Claims

1. A method for preparing a pH / hypoxia dual-responsive antitumor nanomedicine carrier, characterized in that, Includes the following steps: According to the mass ratio of (2-4):1, sulfonamide zwitterionic copolymer and COF nanoparticles were weighed separately. Then, the weighed sulfonamide zwitterionic copolymer was dissolved in deionized water and ultrasonically dispersed for 2-10 min. Then, under continuous stirring, the weighed COF nanoparticles were added to the sulfonamide zwitterionic copolymer solution and stirred at 35-37℃ for 24-72 h. After centrifugation, washing and freeze-drying, the pH / hypoxia dual-responsive antitumor nanomedicine carrier was obtained. The structural formula of the sulfonamide zwitterionic copolymer is: The COF nanoparticles are constructed from p-diaminoazobenzene as the backbone monomer and trialdehyde phloroglucinol, and their structural formula is as follows: 。 2. The method for preparing a pH / hypoxia dual-responsive antitumor nanomedicine carrier according to claim 1, characterized in that: The concentration of the sulfonamide zwitterionic copolymer solution is 2-4 mg / mL, the number of centrifugation cycles is 3-5, and the washing solvent is deionized water.

3. The method for preparing a pH / hypoxia dual-responsive antitumor nanomedicine carrier according to claim 1 or 2, characterized in that: The sulfonamide zwitterionic copolymer is prepared by RAFT polymerization or free radical polymerization of sulfonyl zwitterionic monomers with 2-aminoethyl methacrylate hydrochloride. The above-mentioned sulfonamide zwitterionic copolymers were prepared by RAFT polymerization as follows: According to the molar ratio of (1-5):1, sulfonyl zwitterionic monomers and 2-aminoethyl methacrylate hydrochloride were dissolved in an organic solvent. Then, a chain transfer agent and an initiator were added to the reaction system. The amount of chain transfer agent added was 1 / 30 of the total molar amount of the reaction raw materials, and the molar ratio between the chain transfer agent and the initiator was (3-5):

1. Then, nitrogen gas was introduced into the reaction system for 30 min. After that, the polymerization reaction was carried out at a temperature of 65-85℃ for 12-48 h. The resulting reaction product was first dialyzed with deionized water for 3-5 days, and then freeze-dried to obtain the sulfonamide zwitterionic copolymer. The above-mentioned sulfonamide zwitterionic copolymers were prepared by free radical polymerization as follows: According to a molar ratio of (1-5):1, sulfonyl zwitterionic monomers and 2-aminoethyl methacrylate hydrochloride were dissolved in an organic solvent. Then, 0.1% to 1% of the total molar amount of the reaction raw materials as an initiator were added to the reaction system. Nitrogen gas was then introduced into the reaction system for 30 minutes. After that, the polymerization reaction was carried out at a temperature of 65-85℃ for 12-48 hours. The resulting reaction product was first dialyzed with deionized water for 3-5 days, and then freeze-dried to obtain the sulfonamide zwitterionic copolymer.

4. The method for preparing a pH / hypoxia dual-responsive antitumor nanomedicine carrier according to claim 3, characterized in that: The initiator is AIBN or ACVA, and the chain transfer agent is any one of 4-cyano-4-(thiobenzoylthio)valerate, 4-cyano-4-(thiobenzoyl)valerate, 2-(dodecylthiothiocarbonylthio)-2-methylpropionic acid, 4-cyano-4-[[(dodecylthio)thiononemethyl]thio]valerate, benzyl dithiobenzoate, and S-thiobenzoyl mercaptoacetic acid.

5. The method for preparing a pH / hypoxia dual-responsive antitumor nanomedicine carrier according to claim 3, characterized in that: The structural formula of the sulfonyl zwitterionic monomer is: , or 。 6. An antitumor nanomedicine, characterized in that: The doxorubicin is prepared by the preparation method according to any one of claims 1-5, which encapsulates the pH / hypoxia dual-responsive antitumor nanomedicine carrier.

7. The method for preparing an antitumor nanomedicine according to claim 6, characterized in that: The pH / hypoxia dual-responsive antitumor nanomedicine carrier was added to the doxorubicin solution at a ratio of 10-20 mg / mL. The mixture was stirred continuously for 2-12 h. The resulting product was then purified by dialysis in deionized water for 24-72 h. After filtration and freeze-drying, the finished antitumor nanomedicine was obtained.

8. The method for preparing an antitumor nanomedicine according to claim 7, characterized in that: The concentration of the doxorubicin solution is 2–8 mg / mL.

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