Polymer nanoparticle with GSH / ROS dual response as well as preparation method and application of polymer nanoparticle

Abstract

The invention relates to a polymer nanoparticle with GSH / ROS dual response as well as a preparation method and application of the polymer nanoparticle. The GSH / ROS double-response type polymer nanoparticle is used for a drug loading system, can have the characteristics of high drug loading capacity, high responsiveness, high tumor targeting, high tumor cell inhibition and stability without a stabilizer, can be rapidly disintegrated in an environment similar to a tumor microenvironment, rapidly releases an entrapped drug, and has a good application prospect. The toxicity of chemotherapeutic drugs to normal tissues is reduced, and the composition is green and safe. The invention provides a preparation method of polymer nanoparticles which are good in stability and have GSH / ROS double responses. In addition, the invention further provides a freeze-drying method of the drug loading system, and the stability of the preparation is improved. The technology solves the problems of complex synthesis and high cost of the existing nano-drug carrier, and has wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology. Specifically, it relates to a polymer nanoparticle with dual GSH / ROS response, its preparation method, and its applications. Background Technology

[0002] Cancer has become a major disease threatening people's lives and health. Although extensive treatment strategies have been adopted to improve treatment outcomes, the incidence of malignant tumors continues to rise year by year. Chemotherapy is an indispensable treatment method in the clinical treatment of cancer.

[0003] Paclitaxel, a highly effective, low-toxicity, and broad-spectrum natural anticancer drug, is widely used clinically for the treatment of breast cancer, ovarian cancer, and some head and neck cancers and lung cancers. As a diterpenoid alkaloid compound with anticancer activity, paclitaxel's novel and complex chemical structure, broad and significant biological activity, and unique mechanism of action have made it highly sought after by chemists, pharmacologists, and molecular biologists. It is one of the most commonly used chemotherapy drugs approved by regulatory agencies and is a broad-spectrum chemotherapy agent. However, its severe toxic side effects, including cumulative cardiotoxicity and bone marrow suppression, acute nausea and vomiting, gastrointestinal disturbances, and hair loss, not only limit its dosage but also significantly impact patients' quality of life and reduce patient compliance. Currently, four paclitaxel drugs (original drugs) have been approved in the United States, Japan, and China: paclitaxel injection, paclitaxel liposomes for injection, paclitaxel for injection (albumin-bound), and paclitaxel polymer micelles for injection. However, no formulation has yet demonstrated tumor microenvironment responsiveness, improving efficacy while reducing toxic side effects. Current research has yielded monofunctional nanomedicine formulations for drug delivery and tumor therapy. For example, CN110804178 B discloses a glutathione-responsive monofunctional polymer for drug delivery and tumor therapy, and CN110746598 B also discloses a bifunctional polymer similar to that of this invention for drug delivery. However, the relatively complex synthesis process and high cost limit the large-scale use of functional polymers and the widespread application of nanomedicine formulations. Therefore, developing low-carbon and economical multifunctional polymer synthesis strategies has significant scientific importance and application prospects. Summary of the Invention

[0004] This application introduces cysteine ​​into methionine and copolymerizes it to form a ROS and GSH dual-responsive polyester amide copolymer (poly(ester-amine), PEA). Using paclitaxel (PTX) as a model drug, a nanomedicine delivery system (PTX@PEANPs) is prepared via nanoprecipitation or thin-film hydration. This carrier exhibits good stability, high drug loading capacity, high tumor aggregation, and high penetration. It rapidly releases the drug in the presence of high concentrations of ROS at the tumor site and high concentrations of GSH within tumor cells, thereby exerting a therapeutic effect. Furthermore, the nanomedicine carrier of this application, while consuming GSH within tumor cells to inhibit chemotherapeutic drug resistance, induces an increase in intracellular ROS, thus promoting further degradation of the carrier. Therefore, to some extent, this indicates that the prepared carrier has a targeting effect, not only improving drug bioavailability but also potentially reducing the sensitization, skin toxicity, and insufficient drug release problems of excipients used in marketed liposomes, thereby achieving the goal of reducing side effects and even achieving superior efficacy.

[0005] To address one of the aforementioned technical problems in the prior art, this invention provides a method for preparing polymer nanoparticles with dual GSH / ROS responses and their applications.

[0006] The first objective of this invention is to provide a polymer nanoparticle with dual GSH / ROS response.

[0007] A second objective of this invention is to provide a method for preparing polymer nanoparticles with dual GSH / ROS responses.

[0008] A third objective of this invention is to provide applications of the aforementioned polymer nanoparticles with dual GSH / ROS responses.

[0009] The fourth objective of this invention is to provide a polymer nanoparticle drug delivery system with dual GSH / ROS responsiveness.

[0010] The fifth objective of this invention is to provide a method for preparing a polymer nanoparticle drug delivery system with dual GSH / ROS responsiveness.

[0011] The sixth objective of this invention is to provide a freeze-drying method for preparing polymer nanoparticle drug delivery systems.

[0012] The above-mentioned objectives of this invention are achieved through the following technical solutions.

[0013] In a first aspect, the present invention provides polymer nanoparticles with dual GSH / ROS responses, comprising the polymer shown in formula (I):

[0014]

[0015] Where x is an integer selected from 2 to 15, such as 2-12, 4-12, 2-6 or 6-15; y is an integer selected from 0 to 13, such as 0-12, 4-10, 0-6 or 6-13; m:n = (1:19) ~ (19:1);

[0016] R1 and R2 are each independently selected from H, substituted or unsubstituted C. 1-12 Alkyl groups (e.g., substituted or unsubstituted C4) 1-10 Alkyl or C 1-5 Alkyl), substituted or unsubstituted C 3-12 cycloalkyl, substituted or unsubstituted C 6-12 aryl, substituted or unsubstituted C 5-12 heteroaryl, or substituted or unsubstituted C 4-12 Heterocyclic group.

[0017] In some implementations, the C 1-12 Alkyl groups are optionally selected from halogens, OH, NH2, CN, and C. 1-12 Alkyl (preferably C) 1-5 Alkyl groups, preferably C 1-3 Alkyl), C 3-12 One or more substituents in a cycloalkyl group are substituted.

[0018] In some implementations, the C 3-12 The cycloalkyl group is optionally selected from halogens, OH, NH2, CN, C. 1-12 Alkyl (preferably C) 1-5 Alkyl groups, preferably C 1-3 Alkyl), C 3-12 One or more substituents in a cycloalkyl group are substituted.

[0019] In some implementations, the C 6-12 The aryl group is optionally selected from halogens, OH, NH2, CN, and C. 1-12 Alkyl (preferably C) 1-5 Alkyl groups, preferably C 1-3 Alkyl), C 3-12 One or more substituents in a cycloalkyl group are substituted.

[0020] In some implementations, the C 5-12 Heteroaryl groups are optionally selected from halogens, OH, NH2, CN, and C. 1-12 Alkyl (preferably C) 1-5 Alkyl groups, preferably C 1-3 Alkyl), C 3-12 One or more substituents in a cycloalkyl group are substituted.

[0021] In some implementations, the C 4-12The heterocyclic group is optionally selected from halogen, OH, NH2, CN, C. 1-12 Alkyl (preferably C) 1-5 Alkyl groups, preferably C 1-3 Alkyl), C 3-12 One or more substituents in a cycloalkyl group are substituted.

[0022] In some implementations, the C 1-12 Alkyl groups include straight-chain or branched C4 groups. 1-12 Alkyl group. In some embodiments, C 1-12 The alkyl group is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, etc. In some embodiments, the C 3-12 Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, the C... 6-12 Aryl groups include, but are not limited to, phenyl and naphthyl groups. In some embodiments, the C... 5-12 Heteroaryl groups include, but are not limited to, pyridyl, quinolinyl, purine, and adenine groups. In some embodiments, the C... 4-12 Heterocyclic groups include, but are not limited to, furanyl, thiophene, pyrrole, pyrazolyl, oxazolyl, and thiazolyl.

[0023] In some implementations, x = 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; preferably, x = 2, 4, 6, 8, 10 or 12.

[0024] In some implementations, y = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13; preferably, y = 0, 2, 4, 6, 8, 10 or 12.

[0025] In some implementations, m:n = 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or any value between them.

[0026] Preferably, m:n = (2-9):1; more preferably, m:n = (4-6):1.

[0027] In some embodiments, the polymer exhibits GSH / ROS dual responsiveness. In some embodiments, the positive and negative charges of the polymer are tunable. In some embodiments, the hydrophilicity / hydrophobicity of the polymer is tunable.

[0028] In some embodiments, the degree of polymerization of the polymer is 4 to 20. In some embodiments, the degree of polymerization of the polymer is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0029] The degree of polymerization is the sum of the number of repeating units m and n in the polymer obtained by the reaction.

[0030] In some embodiments, the weight-average molecular weight of the polymer ranges from 2700 to 100000 Da, for example, it can be 2700 Da, 3000 Da, 4000 Da, 5000 Da, 6000 Da, 7000 Da, 8000 Da, 9000 Da, 10000 Da, 15000 Da, 20000 Da, 25000 Da, 30000 Da, 35000 Da, 40000 Da, 45000 Da, 50000 Da, 55000 Da, 60000 Da, 65000 Da, 70000 Da, 75000 Da, 80000 Da, 85000 Da, 90000 Da, 95000 Da, 100000 Da or any value between them.

[0031] The theoretical molecular weight of the polymer can be obtained by multiplying the molecular weight of the repeating unit by the number of repeating units or the molecular weight of the structural unit by the number of structural units.

[0032] In practice, the weight-average molecular weight of the prepared polymer can be measured using any conventional method in the art, such as light scattering, ultracentrifugation sedimentation rate, and gel permeation chromatography. In some embodiments, the following measurement method is used: the polymer is dissolved in tetrahydrofuran (THF) and dimethylformamide (DMF) to a concentration of 1 mg / mL, and then detected by gel permeation chromatography (GPC). The molecular weight is calculated using polyethylene oxide (PEO) as a standard curve.

[0033] When the degree of polymerization of the polymer is 4 to 20 and / or the weight-average molecular weight is in the range of 2700 to 100000 Da, all the properties of the polymer are suitable, including good GSH / ROS dual responsiveness, adjustable positive and negative charges, and adjustable hydrophilicity and hydrophobicity, etc., without affecting the polymer's response characteristics and encapsulation effect.

[0034] The polymer represented by formula (I) according to the first aspect of the present invention is prepared by the following steps:

[0035] S1. Methionine or its derivatives, or salts of said methionine or its derivatives, diols, and benzenesulfonic acid compounds are reacted in a solvent to obtain intermediate Met-x;

[0036] S2. Dissolve cystine or its derivatives or salts of said cystine or its derivatives in a solvent with the intermediate Met-x, and react with diacyl chloride in the presence of an acid-binding agent to obtain the polymer.

[0037] In some embodiments, in step S1, the methionine or its derivatives include, but are not limited to: methionine, S-adenosylmethionine, methionine methyl ester, methionine ethyl ester, methionine n-propyl ester, methionine isopropyl ester, or any combination thereof. The methionine contained in the above substances may be in the L-configuration, D-configuration, or a mixture thereof.

[0038] In some embodiments, the salt of methionine or its derivatives is selected from hydrochloride, sulfonate, 1,4-butanedisulfonate, or p-toluenesulfonate. For example, the salt of methionine or its derivatives may be methionine hydrochloride, methionine sulfonate, methionine methyl ester hydrochloride, methionine methyl ester sulfonate, S-adenosylmethionine 1,4-butanedisulfonate, S-adenosylmethionine p-toluenesulfonate, methionine ethyl ester hydrochloride, methionine ethyl ester sulfonate, methionine n-propyl ester hydrochloride, methionine n-propyl ester sulfonate, methionine isopropyl ester hydrochloride, methionine isopropyl ester sulfonate, or any combination thereof.

[0039] In some embodiments, the methionine or its derivatives, or salts of the methionine or its derivatives, are selected from L-methionine, D-methionine, L-methionine hydrochloride, D-methionine hydrochloride, L-methionine methyl ester hydrochloride, D-methionine methyl ester hydrochloride, S-adenosylmethionine, L-methionine ethyl ester hydrochloride, D-methionine ethyl ester hydrochloride, L-methionine n-propyl ester hydrochloride, D-methionine n-propyl ester hydrochloride, L-methionine isopropyl ester hydrochloride, D-methionine isopropyl ester hydrochloride, or any combination thereof. Compared to phenylalanine, L-methionine (methionine, Met) is a sulfur-containing amino acid closely related to the metabolism of various sulfur-containing compounds in organisms and can change from hydrophobic to hydrophilic under the action of a certain concentration of hydrogen peroxide (H2O2). Studies have shown that tumor cells can increase their uptake of L-methionine through a thiol / methyl-mediated process. Therefore, methionine-based carriers can deliver drugs to tumor cells to a certain extent, and release the drugs rapidly under the action of high concentrations of H2O2 in tumor tissue and tumor cells, thereby achieving the purpose of killing tumors. As a result, polymers based on methionine or its derivatives have a wider range of applications than polymers based on phenylalanine.

[0040] In some embodiments, in step S1, the diol comprises one or more of ethylene glycol, propylene glycol, butanediol, pentanediol, hexanediol, heptanediol, octanediol, nonanediol, decanediol, undecanediol, dodecanediol, tridedecanediol, tetradecanediol, and pentadecanediol. The hydroxyl group on the diol may be located at any substitution position of the alkyl group. Preferably, the diol comprises one or more of 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 1,7-heptanediol, 1,8-octanediol, 1,9-nonanediol, 1,10-decanediol, 1,11-undecanediol, and 1,12-dodecanediol.

[0041] In some embodiments, in step S1, the benzenesulfonic acid compound includes one or more of toluenesulfonic acid, aminobenzenesulfonic acid, fluorobenzenesulfonic acid, chlorobenzenesulfonic acid, bromobenzenesulfonic acid, and hydroxybenzenesulfonic acid. The substituents on the benzenesulfonic acid can be ortho, meta, or para positions relative to each other; for example, o-toluenesulfonic acid, m-toluenesulfonic acid, p-toluenesulfonic acid, and so on.

[0042] In some embodiments, in step S1, the molar ratio of the methionine or its derivatives, or the salts of the methionine or its derivatives, the diol, and the benzenesulfonic acid compound is (2-4):(1-1.2):(2.4-5); for example, the molar ratio can be (2.1-4):(1-1.2):(2.4-5), (2.1-2.5):(1-1.2):(2.4-5), (2-2.5):(1-1.5):(2.5-3.2), (2.1-2.5):(1-1.2):(2.4-2.5), (2.1-2.5):(1-1.1):(2.4-2.5), (2-4):1:(2.4-5), 2.1:1:2.4, or any value between them.

[0043] In some embodiments, in step S1, the solvent is an organic solvent, preferably one or more of toluene, ethylbenzene, xylene, and benzene.

[0044] In some embodiments, the reaction in step S1 is carried out under reflux conditions. The reflux temperature in step S1 is 85–145°C. In some embodiments, the reflux temperature in step S1 is 85°C, 90°C, 95°C, 100°C, 105°C, 110°C, 115°C, 118°C, 120°C, 125°C, 130°C, 135°C, 140°C, 145°C, or any value between these values ​​(the appropriate reflux temperature can be selected according to the solvent used). In some embodiments, the reflux time in step S1 is 6–12 hours. In some embodiments, the reflux time in step S1 is 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours, or any value between these values. During the preparation of intermediates, the reaction conditions (reflux temperature, reflux time, and system airtightness) must be strictly controlled according to the solvent used; otherwise, the yield and purity of the intermediates will be significantly affected. If the reflux temperature is too high or the time is too long, the intermediate groups will be oxidized, resulting in more byproducts; while if the reflux temperature is too low or the time is too short, the yield will be low and the purity will decrease, which will also affect the polymer properties.

[0045] In some embodiments, the reflux is carried out under stirring. In some embodiments, the reflux is carried out under mechanical or magnetic stirring.

[0046] In some embodiments, step S1 further includes purifying the obtained intermediate Met-x using a combination of cold and boiling water. The purpose of this purification is to remove unreacted starting materials and residual solvents. The purified product, after vacuum drying, yields a white or off-white powdered intermediate (Met-x).

[0047] In some implementations, in step S1, the structure of the intermediate (Met-x) is as shown in formula (II):

[0048]

[0049] Where x is defined in the same way as in equation (I);

[0050] R is selected from methyl, amino, hydroxyl, and halogen; among which halogens include fluorine, chlorine, and bromine, with chlorine being preferred.

[0051] In some implementations, x = 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or any value therein; preferably, x = 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0052] In this document, the intermediate is simply referred to as Met-x. Met-x represents the structure of the polymer unit of the intermediate, which consists of two methionine (Met) groups linked by x methylene (-CH2-) via ester bonds. For example, Met-6 represents the intermediate whose polymer unit consists of two methionine (Met) groups linked by six methylene (-CH2-) via ester bonds, Met-8 represents the intermediate whose polymer unit consists of two methionine (Met) groups linked by eight methylene (-CH2-) via ester bonds, Met-10 represents the intermediate whose polymer unit consists of two methionine (Met) groups linked by ten methylene (-CH2-) via ester bonds, and so on. In some embodiments, in step S2, the cystine or its derivative has the structure of formula (III):

[0053]

[0054] The definitions of R1 and R2 are the same as in equation (I).

[0055] In some implementations, R1 and R2 are each independently selected from H and C. 1-12 Alkyl group. In some embodiments, R1 and R2 are the same and selected from H or C. 1-5 Alkyl group. In some embodiments, R1 and R2 are different and independently selected from H and C. 1-12 alkyl.

[0056] In some embodiments, the cystine or its derivative is selected from cystine, dimethyl cystine, diethyl cystine, dipropyl cystine, dibutyl cystine, dipentyl cystine, N,N-diacetylcystine, or any combination thereof.

[0057] In some embodiments, the salt of the cystine or its derivative is selected from hydrochloride, sulfonate or sulfate.

[0058] In some embodiments, the salt of cystine or its derivative is selected from cystine hydrochloride, cystine dimethyl ester dihydrochloride, cystine dimethyl ester disulfate, N,N-diacetylcystine hydrochloride, or any combination thereof.

[0059] The cystine contained in the above substances can be L-configuration, D-configuration, or a mixture thereof. For example, L-cystine, D-cystine, L,D-cystine, N,N-diacetyl-L-cystine, and N,N-diacetyl-L-cystine hydrochloride.

[0060] In some embodiments, dimethyl cysteine ​​dihydrochloride is preferred. In some embodiments, L-cysteine ​​dimethyl ester dihydrochloride is more preferred, as it is widely available and inexpensive.

[0061] The cystine or its derivatives or salts can provide a stable source of disulfide bonds and can also be used to synthesize polymers with high yields and stable properties with diacyl chlorides.

[0062] In some implementations, in step S2, the solvent is an organic solvent, preferably one or more of chloroform and dichloromethane.

[0063] In some embodiments, in step S2, the acid-binding agent includes an organic base or an inorganic base.

[0064] In some embodiments, the organic base includes one or more of pyridine, piperidine, triethylamine, and N,N-dimethylformamide.

[0065] In some embodiments, the inorganic base includes one or more of sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate, calcium bicarbonate, and calcium carbonate.

[0066] In some embodiments, the acid-binding agent is triethylamine or sodium bicarbonate, which are inexpensive, readily available, and low in cost.

[0067] In some embodiments, in step S2, the diacyl chloride includes one or more of oxaloyl chloride, malonyl chloride, succinoyl chloride, glutaryl chloride, adipyl chloride, heptayl chloride, octanoyl chloride, azeloyl chloride, sebacyl chloride, undecanediol chloride, dodecanediol chloride, tridecanediol chloride, tetradecanediol chloride, and pentadecanediol chloride; preferably, the diacyl chloride includes one or more of succinoyl chloride, glutaryl chloride, adipyl chloride, heptayl chloride, octanoyl chloride, azeloyl chloride, and sebacyl chloride. The acyl chloride group on the diacyl chloride can be located at any position on the skeletal alkyl group. In some embodiments, the acyl chloride group on the diacyl chloride is located at both ends of the skeletal alkyl group. In some embodiments, the diacyl chloride includes one or more of 1,4-succinyl chloride, 1,5-glutaryl chloride, 1,6-adiacyl chloride, 1,7-heptyl chloride, 1,8-octyl chloride, 1,9-azelyl chloride, and 1,10-sepaidyl chloride.

[0068] In some embodiments, in step S2, the diacyl chloride is diluted with chloroform and / or dichloromethane, and the dilution amount is 5 to 15 times the volume of the diacyl chloride, preferably 8 to 12 times; for example, it can be 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times or any value between them.

[0069] Adding undiluted diacyl chloride directly can cause localized, violent reactions and produce numerous byproducts. Diluting with a volume greater than 5 times (critical value) can reduce byproducts, but excessive dilution (e.g., more than 15 times) may increase subsequent purification time and costs. Therefore, using diluted diacyl chloride avoids the formation of dark brown byproducts due to high concentrations, which reduces product yield and purity. It also reduces the amount of organic solvent needed, shortens reaction time, and lowers costs. In some embodiments, in step S2, the molar ratio of the intermediate Met-x to cystine or its derivative or salt is (1:19) to (19:1); for example, the molar ratio can be 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1 or any value between them.

[0070] The molar ratio of intermediate Met-x to cystine or its derivatives or salts significantly affects the degree of polymerization, yield, reaction time, and GSH / ROS response characteristics of the synthesized polymer. Experiments revealed that a molar ratio of intermediate Met-x to cystine or its derivatives or salts of 19:1 to 1:19, preferably (1–19):1, further preferably (2–9):1, and even more preferably (4–6):1, resulted in higher polymer yields, shorter reaction times, and enhanced GSH / ROS response characteristics, with a more pronounced hydrophilic-hydrophobic conversion.

[0071] In some embodiments, in step S2, the molar ratio of the total molar number of the intermediate Met-x and cystine or its derivative or salt to the diacyl chloride is (0.5–1.5):1, preferably (0.5–1.2):1, and more preferably (0.8–1.2):1; for example, the molar ratio can be 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, or any value between them. With the molar ratio within the above range, the potential value of the prepared polymer can be adjusted within a suitable molecular weight range.

[0072] In some embodiments, the reaction raw material cystine or its derivatives or salts can be commercially available or synthesized from cysteine ​​or its derivatives or salts.

[0073] In some embodiments, when the reaction raw material is cysteine ​​or its derivatives or salts, it can be dissolved in DMSO, then stirred in air to convert it into cysteine ​​or its derivatives or salts, and then crystallized and dried before use.

[0074] In some implementations, the reaction in step S2 needs to be carried out under anhydrous conditions, as the presence of water will react with the diacyl chloride, reducing the yield.

[0075] In some embodiments, in step S2, after adding the acid-binding agent, the mixture is stirred for 5 to 15 minutes to obtain a mixed solution; for example, the stirring time can be 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes or any value between them.

[0076] In some embodiments, in step S2, diluted diacyl chloride is added dropwise to the mixed solution to carry out the reaction.

[0077] In some implementations, the reaction temperature in step S2 is 0–25°C. Reactions below 0°C take too long, and the degree of polymerization does not change significantly with extended time, which is not conducive to energy saving and cost reduction. Reactions that are too short cannot guarantee a complete reaction, resulting in lower polymer purity and yield, and poorer polymer performance. When the reaction time is 5–210 min, preferably 1–3 h, and more preferably 1.5–2.5 h, at a temperature of 0–25°C, the reaction can proceed fully and rapidly.

[0078] In some implementations, step S2 is carried out at a temperature of 0 to 25°C. For example, the reaction temperature can be 0°C, 5°C, 10°C, 15°C, 20°C, 25°C or any value in between.

[0079] In some embodiments, in step S2, after adding diluted diacyl chloride, the reaction is carried out for 5 to 210 min; for example, the reaction time is 5 min, 30 min, 60 min, 75 min, 90 min, 105 min, 120 min, 135 min, 150 min, 165 min, 180 min, 195 min, 210 min or any value between these values.

[0080] In some embodiments, step S2 further includes purifying the reaction solution using boiling water or an organic solvent. The purpose of this purification is to remove the solvent, unreacted diacyl chloride, and intermediates. The purified product is then vacuum dried to obtain the polymer.

[0081] In some implementations, in step S2, the organic solvent used for purification includes one or more of isopropanol, n-propanol, methanol, ethanol, and ethyl acetate.

[0082] In some implementations, boiling water is used for purification in step S2.

[0083] In some implementations, the organic solvents used in steps S1 and S2 are recovered and reused by rotary evaporation or further distillation, with a recovery rate of 85%-97%. The recovery and reuse of organic solvents reduces production costs, while also reducing environmental pollution and wastewater treatment costs.

[0084] In some embodiments, when the degree of polymerization of the polymer prepared by the method is 4 to 20 and the molecular weight is in the range of 2700 to 100000, the polymer's various properties are suitable, including good GSH / ROS dual responsiveness, adjustable positive and negative charges, adjustable hydrophilicity and hydrophobicity, and moderate ease of synthesis.

[0085] In the above method, in step S1, after obtaining crude Met-x by rotary evaporation, unreacted starting materials and residual solvents are removed by a combination of cold and boiling water circulation. In step S2, after the reaction liquid is rotary evaporated to a certain extent, chloroform, unreacted diacyl chloride and intermediates are removed by hot water extraction or organic solvent method, preferably hot water extraction. Then, the polymer is obtained by vacuum drying. The use of water extraction in steps S1 and S2 can greatly reduce production costs and is low-carbon and environmentally friendly.

[0086] In some implementations, the polymer nanoparticles with GSH / ROS dual responsiveness also include stabilizers.

[0087] In some embodiments, the stabilizer is selected from polyvinyl alcohol (PVA), amphoteric surfactants, povidone, or DSPE-PEG, preferably DSPE-PEG. 2000 And / or the mass of the stabilizer is 0-50% of the polymer mass, preferably 0-30%, more preferably 0-10%.

[0088] Secondly, the present invention provides a method for preparing polymer nanoparticles with dual GSH / ROS responses, the method comprising the following steps:

[0089] Step 1): Provide a polymer having GSH / ROS dual responsiveness as shown in formula (I), wherein the polymer shown in formula (I) is as defined in the first aspect of the present invention;

[0090]

[0091] Step 2): The polymer with GSH / ROS dual responsiveness is prepared into polymer nanoparticles with GSH / ROS dual responsiveness by nanoparticle preparation technology, wherein the nanoparticle preparation technology is selected from: nanoprecipitation method, homogenization method, and microfluidic method.

[0092] In some implementations, the nanoparticle preparation technique is a nanoprecipitation method, and this method includes the following steps:

[0093] Step 2a): Dissolve the polymer with GSH / ROS dual responsiveness shown in formula (I) provided in step 1) in an organic solvent to form an oil phase, wherein the concentration of the polymer in the oil phase is less than 200 mg / mL, preferably 5 to 100 mg / mL, even more preferably 10 to 50 mg / mL, and more preferably 20 to 50 mg / mL;

[0094] Step 2b): Under stirring conditions (e.g., 100 rpm to 2000 rpm), the oil phase obtained in step 2a) is added dropwise to the aqueous phase to form a nanoparticle solution;

[0095] Step 2c): Remove the organic solvent from the nanoparticle solution obtained in step 2) to obtain polymer nanoparticles with GSH / ROS dual response.

[0096] In some embodiments, the concentration of the polymer in the oil phase is 200 mg / mL, for example, 2.5 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 40 mg / mL, 50 mg / mL, 100 mg / mL, or 200 mg / mL.

[0097] In some embodiments, the nanoparticles have a particle size of less than 200 nm, preferably 40–120 nm, and more preferably 55–100 nm.

[0098] In some embodiments, the oil phase or aqueous phase further contains a stabilizer, wherein the mass of the stabilizer is 0 to 50% of the polymer mass, preferably 0 to 30%, more preferably 0 to 10%.

[0099] In some embodiments, in step 2a), the polymer and stabilizer shown in formula (I) are dissolved in an organic solvent to form an oil phase containing the stabilizer.

[0100] In some implementations, prior to step 2b), the stabilizer is dissolved in the aqueous phase to form an aqueous phase containing the stabilizer.

[0101] In some implementations, the aqueous phase is an aqueous solution, preferably water.

[0102] In some embodiments, the stabilizer is polyvinyl alcohol (PVA), amphoteric surfactants (such as carboxybetaine), povidone series, or DSPE-PEG, preferably DSPE-PEG. 2000 .

[0103] In some embodiments, the organic solvent used is one or more of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF) or tetrahydrofuran (THF); preferably dimethyl sulfoxide (DMSO).

[0104] In some embodiments, in step 2c), the organic solvent is removed by placing the nanoparticle solution obtained in step 2b) in an ultrafiltration tube with a molecular weight cutoff (MWCO = 100 kD) and ultrafiltration (e.g., 3000 rpm, twice, 15 min each time) to obtain polymer nanoparticles.

[0105] Thirdly, the present invention provides the use of polymer nanoparticles with GSH / ROS dual response as described in the first aspect of the present invention and polymer nanoparticles prepared by the method for preparing polymer nanoparticles with GSH / ROS dual response as described in the second aspect of the present invention, in the use of these nanoparticles as or in the preparation of drug nanodelivery carriers.

[0106] In some implementations, polymer nanoparticles serve as carriers for drug delivery.

[0107] In some implementation schemes, the delivered drug is an anti-tumor drug.

[0108] In some implementations, the drug is an antitumor drug. Specific examples of antitumor drugs are not particularly limited, including both hydrophilic and hydrophobic drugs. The hydrophilic drugs include, but are not limited to, doxorubicin hydrochloride, gemcitabine hydrochloride, irinotecan hydrochloride, fluorouracil, or lentinan. The hydrophobic drugs include, but are not limited to, paclitaxel (PTX), docetaxel, methotrexate, camptothecin, doxorubicin, and curcumin.

[0109] Fourthly, the present invention provides a polymer nanoparticle drug delivery system with dual GSH / ROS responsiveness, comprising polymer nanoparticles as described in the first aspect of the present invention or polymer nanoparticles prepared by the method for preparing polymer nanoparticles with dual GSH / ROS responsiveness as described in the second aspect of the present invention, and a drug, preferably an antitumor drug.

[0110] In some embodiments, the polymer nanoparticle drug carrier is in the form of an aqueous solution or a lyophilized powder.

[0111] In some embodiments, the polymer nanoparticle drug carrier is in the form of a lyophilized powder, and the lyophilized powder further includes 20-80 wt% of a lyophilization protectant; optionally, the lyophilization protectant is selected from one or more of sucrose, trehalose, lactose, glycine, mannitol, etc.

[0112] Fifthly, this invention provides a method for preparing a polymer nanoparticle drug delivery system with dual GSH / ROS response, comprising the following steps:

[0113] Step c1): Dissolve the drug and the polymer with the structural formula shown in formula (I) in an organic solvent to obtain an oil phase with a concentration of 5 to 60 mg / mL, wherein the polymer shown in formula (I) is as defined in the first aspect of the present invention;

[0114]

[0115] Step c2): Under stirring conditions, the oil phase obtained in step c1) is added dropwise to the aqueous phase to form a polymer nanoparticle drug delivery system.

[0116] In some embodiments, the particle size of the polymer nanoparticle drug delivery system is below 200 nm, preferably 40–120 nm, and more preferably 55–100 nm.

[0117] In some embodiments, the mass ratio of the polymer to the drug added in step c1) is 1:0.05 to 0.50, preferably 1:0.2 to 0.4, and more preferably 1:0.3. If the mass ratio of the polymer to the drug is too large, the drug loading will be low, resulting in fewer nanoparticles; if it is too small, the encapsulation efficiency will be low, leading to drug waste, and the resulting nanoparticles will be unstable and prone to precipitation.

[0118] In some implementations, the mass ratio of polymer to drug in the obtained polymer nanoparticle drug delivery system is the same as the mass ratio of polymer to drug added in step c1).

[0119] In some embodiments, the oil phase or aqueous phase further contains a stabilizer, wherein the mass of the stabilizer is 0 to 50% of the total mass of the polymer and the drug, preferably 0 to 30%, more preferably 0 to 10%.

[0120] In some embodiments, in step c1), the polymer, drug and stabilizer shown in formula (I) are dissolved in an organic solvent to form an oil phase containing the stabilizer.

[0121] In some implementations, prior to step c2), the stabilizer is dissolved in the aqueous phase to form an aqueous phase containing the stabilizer.

[0122] In some implementations, the aqueous phase is an aqueous solution, preferably water.

[0123] In some embodiments, the stabilizer is DSPE-PEG, preferably DSPE-PEG. 2000 .

[0124] In some embodiments, the organic solvent used is one or more of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF) or tetrahydrofuran (THF); preferably dimethyl sulfoxide (DMSO).

[0125] In some embodiments, the polymer nanoparticle drug-loaded system is placed in an ultrafiltration tube with a molecular weight cutoff (MWCO = 100kD), ultrafiltered at 3000-8000 rpm for 15-25 min, washed with ultrapure water and ultrafiltered again, and repeated 2-3 times to remove unencapsulated drugs and reduce the organic solvent content to less than one part per thousand, thereby obtaining purified drug-loaded polymer nanoparticles.

[0126] The raw materials required for the nano-drug delivery system prepared by the method of this invention are readily available, the preparation process is mature and easy to operate, and it does not require expensive instruments. Moreover, the prepared nanocomposite has a moderate size and good biocompatibility. It not only realizes the controllable loading of hydrophobic drugs and improves the solubility of drugs, but also greatly improves the availability of hydrophobic drugs. It can also greatly increase the circulation time of nanoparticles in the blood, thereby increasing the accumulation of drugs at the tumor site and improving the therapeutic effect.

[0127] Sixthly, a lyophilization method for preparing a polymer nanoparticle drug delivery system with dual GSH / ROS response is provided, comprising the following steps:

[0128] Step d1): The polymer nanoparticle drug delivery system of aspect 5 is concentrated by ultrafiltration to remove organic solvent; Step d2): The concentrated polymer nanoparticle drug delivery system is mixed with a 5-20% (w / v) lyophilization protectant solution, and the mixed solution is vacuum freeze-dried to obtain lyophilized powder.

[0129] In some embodiments, the freeze-drying protectant is selected from one or more of sucrose, trehalose, lactose, glycine, mannitol, etc.

[0130] In some implementation schemes, the polymer nanoparticle drug delivery system is mixed with the lyophilization protectant at a volume ratio of 4:1 to 1:4.

[0131] In some implementations, the polymer nanoparticle drug delivery system and the lyophilization protectant are preferably mixed in a 1:1 volume ratio.

[0132] In some implementations, the lyophilized powder can be reconstituted using a diluent (e.g., water, glucose injection) to prepare an injection.

[0133] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0134] The materials of this invention are derived from biocompatible aliphatic amino acids, ensuring safety. This invention rapidly and easily synthesizes a series of polymers with dual GSH / ROS responses and tunable positive and negative charges and hydrophilicity / hydrophobicity using a one-pot method. The preparation method is simple and effectively solves the problems of complex synthesis steps and high costs associated with polymer materials with special applications and good biocompatibility, despite the high demand. Simultaneously, it cleverly improves upon the limitations of single polyamino acids in terms of structure and function. Compared to phenylalanine, L-methionine (Met) is a sulfur-containing amino acid closely related to the metabolism of various sulfur-containing compounds in organisms and can change from hydrophobic to hydrophilic under the action of a certain concentration of hydrogen peroxide (H2O2). Studies have shown that tumor cells can increase their uptake of L-methionine through a thiol / methyl-mediated process. Therefore, methionine-based carriers can deliver drugs to tumor cells to a certain extent, and the drugs can be rapidly released under the action of high concentrations of H2O2 in tumor tissue and tumor cells, thereby achieving the purpose of killing tumors. Therefore, the synthesis of methionine-based polymers will have a wider range of applications than phenylalanine-based polymers. In addition, this invention can also obtain polymers with different structures and physicochemical properties (redox responsiveness, stability, hydrophobicity, particle size) by controlling the ratio of each monomer and the reaction time and temperature. This novel polymer synthesis strategy, which is simple to synthesize and has tunable redox responsiveness, can broaden the application range of functional polymers to a certain extent and provide new ideas for controlling the structure-activity relationship of polymers, thus expanding the application range of functional materials and their potential value in the therapeutic field.

[0135] Compared with existing marketed products, the drug-loaded nanoparticles of this invention have a particle size of less than 200 nm (preferably 100 nm), a high specific surface area, high drug loading capacity, and degradation products that are non-toxic amino acids, making them an excellent drug delivery system that can enhance the efficacy of drugs. In addition, the high GSH / ROS ratio in the tumor microenvironment compared to normal tissue can promote the release of drugs by the drug carrier in the tumor microenvironment (tissue targeting). For unreleased drug carriers, the EPR effect can be used to enhance the targeting of drugs to the tumor site. Attached Figure Description

[0136] Figure 1 The present invention illustrates the polymer monomer and polymer synthesis reaction equations and processes: where (A) represents the synthesis of the monomer; and (B) represents the synthesis of the polymer.

[0137] Figure 2 .1H NMR spectrum analysis of the polymers prepared in Examples 1 and 2: (A) is the 1H NMR spectrum analysis of the polymer prepared in Example 1; (B) is the 1H NMR spectrum analysis of the polymer prepared in Example 2.

[0138] Figure 3Glass transition temperatures of the polymers prepared in Examples 1 and 2: (A) is the glass transition temperature of the polymer prepared in Example 1; (B) is the glass transition temperature of the polymer prepared in Example 2.

[0139] Figure 4 The redox potential changes of the polymer prepared in Example 3 were determined using cyclic voltammetry.

[0140] Figure 5 .Graph showing the changes in hydrophilicity and hydrophobicity of polymers under different redox conditions.

[0141] Figure 6 Particle size data of polymer nanoparticles in Example 7.

[0142] Figure 7 The release curves of Example 10 under the action of GSH(A) and ROS(B) were determined by liquid chromatography.

[0143] Figure 8 The lyophilization of drug-loaded nanoparticles in 50ml vials (top row from left to right corresponds to lyophilized product numbers 1, 2, 3, 4, 5; bottom row from left to right corresponds to lyophilized product numbers 6, 7, 8, 9, 10).

[0144] Figure 9 The hemolysis experiment was used to verify the blood compatibility of the lyophilized powder after reconstitution and dilution into drug-loaded nanoparticles of different concentrations. Detailed Implementation

[0145] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0146] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0147] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0148] definition

[0149] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0150] Unless the context clearly indicates otherwise, all references to quantity not explicitly stated in this text include their plural forms.

[0151] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0152] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0153] Example

[0154] Test method:

[0155] The tests in the embodiments of the present invention are performed using the methods described below.

[0156] 1. Nuclear Magnetic Resonance (NMR): The chemical structures of the monomers and polymers synthesized in this invention were characterized by 1H NMR spectroscopy, using deuterated dimethyl sulfoxide (DMSO-d6) containing tetramethylsilane (TMS) as the solvent. The measurement results were analyzed using MestReNova software. Furthermore, FT-IR spectroscopy was used in the range of 400-4000 cm⁻¹. -1 The monomers and polymers were scanned and analyzed within the wavelength range.

[0157] 2. Determination of glass transition temperature of polymer: Characterization was performed by differential scanning calorimetry (DSC). The glass transition temperature (Tg) of the polymer was measured by DSC under the conditions of a temperature range of -20.0℃ to 100.0℃, a heating rate of 10.0℃ / min, and a nitrogen flow rate of 20.0mL / min.

[0158] 3. Redox Response of the Polymer: The redox potential of the polymer was tested using an electrochemical workstation. Specifically, the redox potential (OPR) of the polymer synthesized in Example 1 was determined by cyclic voltammetry. The polymer was dissolved in dichloromethane, coated onto one end of an electrode, and immersed in an electrolyte solution. The electrochemical workstation then scanned the current, with the potential range designed to allow alternating reduction and oxidation reactions at the electrode, and the current-potential curves were recorded. The workstation electrode consisted of three parts: an Ag / AgCl reference electrode, a Pt wire as an auxiliary electrode, and a glassy carbon electrode as the working electrode.

[0159] 4. Hydrophilicity / Hypersensitivity and GSH / ROS Responsiveness of the Polymer: Nile red was used to determine the hydrophilicity / hydropersensitivity and GSH / ROS responsiveness of the polymer. Nile red is a lipophilic oxazine fluorescent dye that is photosensitive. The hydrophilicity / hydropersensitivity of the polymer was determined by the difference in color in water after the polymer was combined with Nile red. The redder the color, the stronger the hydrophobicity. The synthesized polymer was dissolved in dimethyl sulfoxide, dimethylformamide, or tetrahydrofuran to prepare a concentration of approximately 20 mg / ml, and then mixed with Nile red (1%). Nanoparticle solutions were then prepared using a nanoprecipitation method. The hydrophilicity / hydropersensitivity of the polymer was then indirectly characterized by photographs.

[0160] 5. Nanoparticle size determination: A certain amount of polymer nanoparticles were taken and dissolved in dimethyl sulfoxide, dimethylformamide, or tetrahydrofuran to prepare a concentration of about 20 mg / mL. Then, the solution was slowly added dropwise to an aqueous solution to obtain a final sample concentration of about 1.0 mg / mL. The particle size of the solution was then determined by dynamic light scattering diffraction. The sample was measured three times and the average value was taken.

[0161] 6. Hemolysis Experiment of Polymer Nanoparticles: The hemolysis experiment was used to verify the blood compatibility of drug-loaded nanoparticles diluted to different concentrations after reconstitution of the lyophilized powder. Preparation of 2% Red Blood Cell Suspension: Blood from healthy rabbits was placed in an Erlenmeyer flask containing glass beads and shaken for 10 minutes, or stirred with a glass rod to remove fibrinogen and obtain defibrinated blood. Approximately 10 times the volume of 0.9% sodium chloride solution was added, and the mixture was shaken well. The mixture was centrifuged at 1000–1500 rpm for 15 minutes, and the supernatant was removed. The precipitated red blood cells were washed 2–3 times with 0.9% sodium chloride solution as described above until the supernatant no longer appeared red. The resulting red blood cells were then used to prepare a 2% suspension with 0.9% sodium chloride solution for experimental use.

[0162] Preparation of test solution: Unless otherwise specified, prepare the test solution according to the concentration specified under the variety description. Test method: Take five clean glass test tubes, number them, and add 2% red blood cell suspension to tubes 1-4 sequentially. Then, add the test solution of different concentrations (0.45-2 mg / mL) to tubes 1 and 2 respectively; tube 3 is the negative control; tube 4 is the positive control; and tube 5 is the test control. Add the corresponding sample, mix well, and immediately incubate at 37℃±0.5℃. Observe the hemolysis and agglutination reactions after 3 hours.

[0163] 7. Liquid Chromatography: In this application, liquid chromatography is performed under the following conditions:

[0164] Mobile phase: acetonitrile:water (62:38), flow rate: 1 mL / min; chromatographic column: Nano chrom chromcore 120C 18 4.6 × 150 mm, 5 μm; column temperature: 30℃; detection wavelength: 227 nm; injection volume: 10 μL; run time: 8 min. The PTX standard curve was plotted after measuring PTX at different concentration gradients.

[0165] In this embodiment, the polymer monomers and the general synthesis schemes of the polymer are as follows: Figure 1 As shown in (A) and (B).

[0166] Example 1: Synthesis of a polymer with GSH / ROS dual responsiveness

[0167] (1) Weigh 94.03g of methionine, 35.45g of 1,6-hexanediol and 136.99g of p-toluenesulfonic acid and mix them in a 1000mL flask containing toluene. Stir and reflux at 115℃ for 10h. The molar ratio of methionine to 1,6-hexanediol and p-toluenesulfonic acid is 2.1:1:2.4.

[0168] (2) Most of the toluene in the reactants was recovered by rotary evaporation. Then, boiling water was added to the reaction flask to dissolve it and remove the residual toluene in the reactants. The product was then placed in an ice-water bath to precipitate the product. The product was filtered at room temperature to remove unreacted methionine monomer and p-toluenesulfonic acid. This process was repeated twice. The product was then vacuum dried for 1 day to obtain a white powder intermediate (Met-6).

[0169] (3) After dissolving the obtained intermediate (Met-6) and cystine methyl ester dihydrochloride (Cys) in chloroform, a certain amount of triethylamine was added, and the mixture was stirred rapidly for 10 min to make it fully mixed. The insoluble matter was removed by filtration. Adipic acid chloride diluted with chloroform (1.05 times the total molar amount of the two intermediates (this is recorded as the theoretical amount)) was slowly added dropwise to the above solution and the reaction was continued for 60 min to obtain a polymer solution.

[0170] (4) The crude polymer product obtained from the reaction is rotary evaporated to recover the solvent chloroform. Then the reactants are slowly added dropwise to boiling water to remove chloroform and unreacted adipic acid chloride. This process is repeated 3 times and the product is dried under vacuum for 24 hours to obtain the polymer.

[0171] Example 2: Synthesis of polymers with GSH / ROS dual responsiveness

[0172] (1) Weigh 94.02g of methionine, 44.75g of 1,8-octanediol and 136.99g of p-toluenesulfonic acid and mix them in a 1000mL flask containing toluene. Stir and reflux at 118℃ for 11h. The molar ratio of methionine to 1,8-octanediol and p-toluenesulfonic acid is 2.1:1.02:2.4.

[0173] (2) Most of the toluene in the reactants was recovered by rotary evaporation. Then, boiling water was added to the reaction flask to dissolve it and remove the residual toluene in the reactants. The product was then placed in an ice-water bath to precipitate the product. The product was filtered at room temperature to remove unreacted methionine monomer and p-toluenesulfonic acid. This process was repeated twice. The product was then vacuum dried for 1 day to obtain a white powder intermediate (Met-8).

[0174] (3) After dissolving the obtained intermediate (Met-8) and cystine methyl ester dihydrochloride (Cys) in chloroform, a certain amount of triethylamine was added, and the mixture was stirred rapidly for 10 min to make it fully mixed. The insoluble matter was removed by filtration, and adipic acid chloride diluted with chloroform (1.05 times the total molar amount of the two intermediates) was slowly added dropwise to the above solution and reacted for 90 min to obtain a polymer solution.

[0175] (4) The crude polymer product obtained from the reaction is rotary evaporated to recover the solvent chloroform. Then the reactants are slowly added dropwise to boiling water to remove chloroform and unreacted adipic acid chloride. This process is repeated 3 times and the product is dried under vacuum for 24 hours to obtain the polymer.

[0176] Example 3: Synthesis of polymers with GSH / ROS dual responsiveness

[0177] (1) Weigh 94.05g of methionine, 35.45g of 1,6-hexanediol and 136.99g of p-toluenesulfonic acid and mix them in a flask containing 1000mL of toluene. Stir and reflux at 115℃ for 10.5h. The molar ratio of methionine to 1,6-hexanediol and p-toluenesulfonic acid is 2.1:1:2.4.

[0178] (2) Most of the toluene in the reactants was recovered by rotary evaporation. Then, boiling water was added to the reaction flask to dissolve the reactants and remove the residual toluene in the reactants. The product was then placed in an ice-water bath for precipitation. The unreacted methionine monomer and p-toluenesulfonic acid were removed by filtration at room temperature. This process was repeated twice. The product was then vacuum dried for 1 day to obtain a white powder intermediate (Met-6).

[0179] (3) Dissolve the obtained intermediate (Met-6) and cystine dimethyl ester dihydrochloride in dichloromethane, add a certain amount of triethylamine, stir rapidly for 12 min to mix thoroughly, filter to remove insoluble matter, and slowly add sebacate chloride (theoretically the amount of which is 1.05 times the total molar amount of intermediate (Met-6) and cystine dimethyl ester dihydrochloride) diluted 5 times with dichloromethane to the above solution at 5°C, and react for 75 min to obtain a polymer solution;

[0180] (4) The crude polymer obtained from the reaction was rotary evaporated to recover the solvent dichloromethane. Then the reactants were slowly added dropwise to boiling water to remove dichloromethane and unreacted sebacate chloride. This process was repeated 3 times and the product was dried under vacuum for 24 hours to obtain the polymer.

[0181] Example 4: Polymer Performance Characterization

[0182] The polymers prepared in Examples 1-3 were characterized using the methods described in the test methods of the examples.

[0183] The present invention uses a 400M superconducting nuclear magnetic resonance spectrometer (Ascend™ 400) and differential scanning calorimetry (DSC) to characterize the structure and glass transition temperature of the polymers prepared in Example 1 and Example 2, respectively.

[0184] Figure 2 Figures A and B show the 1H NMR spectra of the polymers prepared in Examples 1 and 2, respectively. The peaks around 1.5 and 2.1 ppm correspond to the hydrogen atoms on the methylene group of adipic acid chloride, the peaks around 3.0 and 4.5 ppm correspond to the hydrogen atoms on the methylene and methylene groups of dimethyl cysteine, respectively, and the peaks from right to left between 8.0 and 8.5 ppm correspond to the NH peaks on the bis(L-methionine) alkylene diester (monomer) and dimethyl cysteine. The positions of other polymer peaks are not significantly different from those of the polymers described above. These results indicate that the synthesized polymer consists of three compounds: bis(L-methionine) alkylene diester (monomer), diacyl chloride, and dimethyl cysteine. Furthermore, screening purification solvents revealed that water is superior to ethyl acetate as a purification solvent due to its environmental and economical nature, as the latter leaves some residue.

[0185] Figure 3 Example 1, characterized by differential scanning calorimetry (DSC), is shown. Figure 3 Example A) and Example 2 ( Figure 3 The glass transition temperature of the polymer prepared in step B). Figure 3 As shown, with the increase of the number of methylene groups used, the glass transition temperature increases significantly, and the polymer changes from a viscous gel state to a solid state, which is beneficial to increasing the storage temperature of the polymer and provides a reference for the synthesis of polymers with controllable glass transition temperature.

[0186] The present invention also used an electrochemical workstation to characterize the redox responsiveness of the polymer prepared in Example 3. For example... Figure 4 As shown, the voltammetric curve contains both oxidation and reduction peaks, which indicates that the synthesized polymer does indeed have redox potential.

[0187] Furthermore, this invention also uses Nile Red to determine the hydrophilicity / hydrophobicity and redox responsiveness of the polymers prepared in Examples 1-3. This is because Nile Red is a lipophilic oxazine fluorescent dye that is photosensitive. The difference in color in water after the polymer binds to Nile Red is used to determine the hydrophilicity / hydrophobicity of the polymer. The redder the color, the stronger the hydrophobicity.

[0188] Figure 5 The hydrophilicity / hydrophobicity and GSH of the polymers prepared in Examples 1-3 are... Figure 5 (A) / ROS( Figure 5 (B) Responsive images (three bottles per group, and from left to right, the colors exhibited by the polymers corresponding to Examples 3, 2 and 1 after interaction with Nile Red under different GSH (20 μM and 10 mM) and ROS (20 nM and 100 μM) conditions).

[0189] (1) Judging from the color, when the number of methylene groups in the polymer structure increases, the hydrophobicity will also be enhanced to a certain extent. For example, the polymer aqueous solution synthesized with sebacate chloride as the starting material is the reddest (Example 3), and the hydrophobicity is stronger.

[0190] (2) GSH responsiveness: The chain polymer synthesized from aliphatic amino acids in this invention becomes turbid after about 15 hours under the action of 10 mM GSH, indicating that the polymer has redox responsiveness and is degraded, causing the solution to become turbid (Example 1). However, the clarity of the solution does not change much under the action of 20 μM GSH, indicating that the reducing agent that destroys the polymer structure needs to reach a certain concentration.

[0191] (3) ROS responsiveness: After 17 hours of exposure to 100 μM hydrogen peroxide, the hydrophilicity of the polymer was significantly enhanced. The polymer synthesized in Example 1 showed the most significant effect, while the polymer synthesized in Example 3 did not show obvious oxidation responsiveness. This may be because Example 3 is highly hydrophobic and the thiomethyl group is encapsulated inside and is not easily oxidized. After 17 hours of exposure to 20 nM hydrogen peroxide, the color change of the polymer was not obvious. This also indicates that the reducing power of the polymer is related to the polymer structure. A certain concentration of oxidant is required for complete oxidation.

[0192] Example 5: Effect of starting materials on polymer intermediate yield

[0193] Using a method similar to that in Example 1, 1,4-butanediol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol, and 1,12-dodecanediol were used as starting materials for the intermediate product. The proportions of each starting material were adjusted, and the effects on the yield of the intermediate product were observed. The results are shown in Table 1. Table 1 shows that when the molar ratio of methionine, the diol, and p-toluenesulfonic acid is between (2–4):(1–1.2):(2.4–5), the yield of the intermediate (Met-x) is relatively high.

[0194] Table 1. Effect of starting material ratio on intermediate product yield

[0195]

[0196]

[0197] Example 6: Effect of starting materials on polymer yield and potential

[0198] Using a method similar to that in Example 1, the proportions of each material were adjusted, and the effects of the starting material on the polymer yield and potential were observed. The results are shown in Table 2.

[0199] Table 2 Effect of monomer ratio on polymer yield (Synthesis method in Example 1)

[0200]

[0201] #Note: The diacyl chloride is taken from one or more of succinyl chloride, adipyl chloride, octyl chloride and sebacate. The theoretical amount of diacyl chloride is 1.05 times the sum of the molar amounts of Met and Cys. The amount of diacyl chloride used in this invention is 0.5 to 1.20 times the theoretical amount.

[0202] Example 7: Preparation and characterization of a polymer nanoparticle with GSH / ROS dual responsiveness (i.e., a GSH / ROS dual responsive drug delivery carrier).

[0203] 1. Preparation of GSH / ROS dual-responsive polymer nanoparticles:

[0204] (1) The polymer prepared in Example 1 was dissolved in DMSO to obtain an oil phase, wherein oil phases with polymer concentrations of 2.5 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 40 mg / mL, 50 mg / mL, 100 mg / mL and 200 mg / mL were prepared for later use;

[0205] (2) The above oil phase was slowly added dropwise to the aqueous solution at a speed of 1500 rpm. The polymer self-assembled into a nano system in the aqueous solution by nanoprecipitation.

[0206] (3) The obtained nanoparticle solution was placed in an ultrafiltration tube with a molecular weight cutoff (MWCO = 100kD) and ultrafiltered twice at 3000rpm for 15min each time, so that the DMSO content was less than one part per thousand, and finally polymer nanoparticles were obtained.

[0207] 2. Results

[0208] The effect of polymer concentration on particle size is shown in Table 3. As the initial polymer concentration increases, the particle size gradually increases. After being placed in a sealed container at room temperature for 90 days, the particle size of the polymer nanoparticles all showed a certain increase. When the concentration increases to a certain level, such as 200 mg / mL, its stability gradually decreases, and flocculent precipitation occurs later. At low initial concentrations, such as below 10 mg / mL, although the particle size is relatively good, there is more organic solvent, which increases the cost of subsequent organic solvent removal and also increases the equipment maintenance cost. Figure 6 This shows the particle size data of polymer nanoparticles determined using DLS. For example... Figure 6 As shown, the polymer nanoparticles (polymer concentration of 20 mg / mL) prepared in Example 7 have a particle size of approximately 89 nm.

[0209] Table 3. Effect of polymer concentration (Example 1) on nanoparticle size.

[0210]

[0211] Example 8: Preparation of a polymer nanoparticle with GSH / ROS dual responsiveness (i.e., a GSH / ROS dual responsive drug delivery carrier). Except that DMSO was replaced with N,N-dimethylformamide (DMF), polymer nanoparticles were prepared under the same conditions as in Example 7, and the polymer concentration was controlled at 10 mg / mL, 20 mg / mL, and 50 mg / mL, respectively, finally obtaining polymer nanoparticles with a diameter below 200 nm and good stability.

[0212] Example 9: Preparation and characterization of a polymer nanoparticle drug delivery system with GSH / ROS dual responsiveness

[0213] 1. Preparation of polymer nanoparticle drug delivery systems:

[0214] (1) PTX and the polymer prepared in Example 1 were dissolved in DMSO to prepare solutions of the same concentration (20 mg / mL); DSPE-PEG 2000 As a surface stabilizer, DSPE-PEG 2000 Dissolve in water to prepare a solution with a concentration of 0.5 mg / mL for later use;

[0215] (2) Two solutions of equal concentration were mixed in different proportions (polymer to antitumor drug mass ratios of 1:0.05, 1:0.10, 1:0.20, 1:0.30, and 1:0.50), and then slowly added dropwise at 1000 rpm until the solution containing DSPE-PEG was obtained. 2000 In aqueous solution, the drug-nanoparticle complex self-assembles into a polymer nanoparticle drug-carrying system via nanoprecipitation, wherein DSPE-PEG... 2000 The mass is 10% of the total mass of the polymers and antitumor drugs used.

[0216] In addition to dissolving DSPE-PEG in water 2000 Except for controlling the mass to be 30%, 50%, and 0% of the total mass of polymer and drug, respectively, polymer nanoparticle drug delivery systems with GSH / ROS dual responsiveness were prepared using the same method as in Example 9, and were respectively used as Examples 10, 11, and 12.

[0217] The drug-loaded nanoparticles were examined using the methods described in the examples.

[0218] result

[0219] Examples 9-12 all yielded drug-loaded nanoparticles with good particle size (as shown in Table 4). However, with the increase of stabilizer, the polydispersity index of the drug-loaded nanoparticles increased, and their particle size also increased accordingly. In addition, if the mass ratio of polymer to antitumor drug is too high, the drug loading will be low, resulting in drug waste, and the obtained nanoparticles will be unstable and prone to precipitation (as shown in Table 5).

[0220] Table 4. Effect of stabilizer dosage on product particle size

[0221] Polymers: Drugs Stabilizer percentage (%) Particle size (nm) PDI 10:3 10％ 70.71 0.161 10:3 30％ 79.26 0.119 10:3 50％ 93.44 0.21 10:3 0％ 73.86 0.227

[0222] Table 5. Effect of polymer to drug ratio on product particle size and encapsulation efficiency.

[0223] Polymers: Drugs Stabilizer percentage (%) Particle size (nm) PDI Encapsulation efficiency (%) 20:1 10％ 109.35 0.079 99.6 10:1 10％ 96.51 0.113 99.4 10:2 10％ 81.6 0.2185 98.7 10:3 10％ 73.17 0.2025 98.4 10:4 10％ 91.82 0.351 91.3 10:5 10％ 163.56 0.699 80.6

[0224] Example 13: Preparation and characterization of a polymer nanoparticle drug delivery system with GSH / ROS dual responsiveness

[0225] 1. Preparation of polymer nanoparticle drug delivery systems:

[0226] (1) PTX, the polymer prepared in Example 1 and the surface stabilizer DSPE-PEG were added. 2000 Dissolve each separately in DMSO to prepare solutions of the same concentration (20 mg / mL) for later use;

[0227] (2) Three solutions of equal concentration were prepared according to the following mass ratios of polymer to antitumor drug: 1:0.05, 1:0.10, 1:0.20, 1:0.30, and 1:0.50, with DSPE-PEG... 2000 After mixing the polymer and the antitumor drug at a ratio of 10% of the total mass, the mixture was slowly added dropwise to water at a speed of 1000 rpm. The drug-nanoparticle complex self-assembled in the aqueous solution through a nanoprecipitation method to form a polymer-loaded drug nanosystem with a diameter of less than 200 nm and good stability.

[0228] result

[0229] As shown in Table 6, when the polymer:drug ratio is 10:3, the nanoparticles obtained by dissolving the stabilizer in DMSO and adding it dropwise have a larger particle size than those obtained by dissolving the stabilizer in water in Example 9. This may be because the amount of DMSO in the system increases, and the stabilizing effect of the stabilizer decreases, resulting in a larger final particle size.

[0230] Table 6. Effect of stabilizer solubility in different solvents on particle size.

[0231] Polymers: Drugs Stabilizer percentage (%) Particle size (nm) PDI 10:3 10% (water) 70.71 0.161 10:3 20% (water) 72.89 0.125 10:3 30% (water) 77.89 0.2 10:3 10% (DMSO) 83.6 0.169 10:3 20% (DMSO) 91.6 0.251 10:3 30% (DMSO) 95.3 0.239

[0232] Example 14: Effect of dilution solvent on lyophilized powder

[0233] 1. Preparation of lyophilized powder samples of polymer nanoparticle drug delivery system:

[0234] (1) Dissolve the polymer and PTX obtained in Example 1 in DMSO respectively to prepare 20 mg / mL solutions (oil phase) for later use;

[0235] (2) The above oil phases are mixed in proportion and then slowly added to an aqueous solution under mechanical stirring at 400 rpm. The polymer self-assembles into a nano system in the aqueous solution by nanoprecipitation.

[0236] (3) The obtained nanoparticle solution was placed in an ultrafiltration membrane with a molecular weight cutoff (MWCO = 100kD) and concentrated by ultrafiltration at 90 rpm five times to make the DMSO content less than one part per thousand, and finally the polymer nanoparticle concentrate was obtained.

[0237] (4) Mix the drug-loaded nanoparticle concentrate with 5-30% (w / v) sucrose or trehalose aqueous solution at a volume ratio of 1:1, fill the mixed solutions into 50mL vials, half-stop, freeze dry in a vacuum freeze dryer, fill with nitrogen, stopper, and cap.

[0238] The lyophilized powder was obtained using the method described above, and then diluted with diluent at different ratios before being tested. Table 7 lists the composition of the test samples and their processing conditions.

[0239] Table 7

[0240]

[0241] 2. Results

[0242] The dilution factor has little effect on the particle size of the finished product (Table 8), and the product is relatively stable. In addition, the effects of different manufacturers' glucose dilution on the potential are shown in Table 9: (1) The absolute value of the potential of the dilute solution is slightly larger than that of the concentrated solution, and the stability is higher (when the particle concentration is high, the interaction between particles increases, leading to an increase in particle aggregation and sedimentation); (2) The overall potential is relatively stable. Therefore, this product can be diluted with 5% glucose from different manufacturers (Sichuan Kelun Pharmaceutical Co., Ltd., Shijiazhuang No. 4 Pharmaceutical Co., Ltd. and Guangxi Yuyuan Pharmaceutical Co., Ltd.) and the dilution does not affect its stability.

[0243] Table 8. Investigation of the effect of dilution factor on particle size.

[0244]

[0245] Table 9. Effects of different manufacturers' compatible solvents (glucose) and dilution factors on zeta potential determination.

[0246]

[0247]

[0248] Example 15: Effect of lyophilization protectant on lyophilized powder and stability test of lyophilized powder

[0249] 1. Preparation of lyophilized powder samples of polymer nanoparticle drug delivery system:

[0250] (1) Dissolve the polymer and PTX obtained in Example 1 in DMSO to prepare a 20 mg / mL solution (oil phase) for later use;

[0251] (2) The above oil phase was slowly added dropwise to the aqueous solution under mechanical stirring at 400 rpm. The polymer self-assembled into a nano system in the aqueous solution by nanoprecipitation.

[0252] (3) The obtained nanoparticle solution was placed in an ultrafiltration membrane with a molecular weight cutoff (MWCO = 100kD) and concentrated by ultrafiltration at 90 rpm five times to make the DMSO content less than one part per thousand, and finally the polymer nanoparticle concentrate was obtained.

[0253] (4) Mix the drug-loaded nanoparticle concentrate with 5-20% (w / v) trehalose solution at a volume ratio of 1:1, then fill the mixed solutions into 50mL vials, half-stop, freeze dry in a vacuum freeze dryer, fill with nitrogen, stopper, and cap.

[0254] The lyophilized powder was obtained according to the above method, and after being reconstituted with a solution, the sample was examined. The sample formulation and the examination results are shown in Table 10.

[0255] Table 10. Effects of different types and dosages of freeze-drying protectants on the reconstitution effect of freeze-dried products.

[0256]

[0257]

[0258] 2. Results

[0259] As shown in Table 10 and Figure 8 As shown, both trehalose and sucrose can yield good freeze-dried products. As shown in Tables 11-12, after being stored at 25℃ for 20 days, the drug content remained essentially unchanged, and although the nanoparticle encapsulation rate decreased slightly, the overall stability was good. Furthermore, Figure 9 Hemolysis experiments showed that the polymer nanoparticle drug delivery system has good blood compatibility at different concentrations and can be used for intravenous injection.

[0260] Table 11 shows the effect of storage time on content at 25℃.

[0261]

[0262] Table 12 shows the effect of storage time on content at 25℃.

[0263]

[0264] Note: In Tables 11 and 12, 0d represents the sample on day 0; 25℃-10d represents the sample placed at 25℃ for 10 days; and 25℃-20d represents the sample placed at 25℃ for 20 days.

[0265] The above specific embodiments of the present invention are preferred embodiments described to facilitate understanding of the present invention. However, the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any technical modifications made according to the technical solution of the present invention, any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, and additions of auxiliary components, and specific methods thereof, are all considered improvements to the present invention.

Claims

1. A polymer nanoparticle having GSH / ROS dual responsiveness, comprising a polymer having GSH / ROS dual responsiveness as shown in formula (I), and optionally a stabilizer; in, x is an integer selected from 2 to 15, such as 2-12, 4-12, 2-6 or 6-15; y is an integer selected from 0 to 13, such as 0-12, 4-10, 0-6 or 6-13; m:n = (1:19) ~ (19:1); R1 and R2 are each independently selected from H, substituted or unsubstituted C. 1-12 Alkyl groups (e.g., substituted or unsubstituted C4) 1-10 Alkyl or C 1-5 Alkyl), substituted or unsubstituted C 3-12 cycloalkyl, substituted or unsubstituted C 6-12 aryl, substituted or unsubstituted C 5-12 heteroaryl, or substituted or unsubstituted C 4-12 Heterocyclic groups; Preferably, the degree of polymerization of the polymer is 4 to 20; Preferably, the weight-average molecular weight of the polymer is in the range of 2700 to 100000 Da.

2. The polymer nanoparticles with GSH / ROS dual responsiveness according to claim 1, wherein, The stabilizer is selected from polyvinyl alcohol (PVA), amphoteric surfactants, povidone, or DSPE-PEG, preferably DSPE-PEG. 2000 The stabilizer is 0-50% of the polymer mass, preferably 0-30%, more preferably 0-10%.

3. The polymer nanoparticles with GSH / ROS dual responsiveness according to claim 1, wherein, The polymer nanoparticles have a particle size of less than 200 nm, preferably 40–120 nm, and more preferably 55–100 nm.

4. A method for preparing polymer nanoparticles with dual GSH / ROS response, the method comprising: Step 1): Provide a polymer with GSH / ROS dual responsiveness as shown in formula (I), Where x is an integer selected from 2 to 15, such as 2-12, 4-12, 2-6 or 6-15; y is an integer selected from 0 to 13, such as 0-12, 4-10, 0-6 or 6-13; m:n = (1:19) ~ (19:1); R1 and R2 are each independently selected from H, substituted or unsubstituted C. 1-12 Alkyl groups (e.g., substituted or unsubstituted C4) 1-10 Alkyl or C 1-5 Alkyl), substituted or unsubstituted C 3-12 cycloalkyl, substituted or unsubstituted C 6-12 aryl, substituted or unsubstituted C 5-12 heteroaryl, or substituted or unsubstituted C 4-12 Heterocyclic groups; Preferably, the degree of polymerization of the polymer is 4 to 20; Preferably, the polymer is a polymer with a weight-average molecular weight range of 2700–100000 Da; and Step 2) The polymer with GSH / ROS dual responsiveness is prepared into polymer nanoparticles with GSH / ROS dual responsiveness by nanoparticle preparation technology, wherein the nanoparticle preparation technology is selected from: nanoprecipitation method, homogenization method, and microfluidic method.

5. The method according to claim 4, wherein the nanoparticle preparation technology is a nanoprecipitation method, and step 2) includes the following steps: Step 2a): Dissolve the polymer with GSH / ROS dual responsiveness shown in formula (I) provided in step 1) in an organic solvent to form an oil phase, wherein the concentration of the polymer in the oil phase is less than 200 mg / mL, preferably 5 to 100 mg / mL, even more preferably 10 to 50 mg / mL, and more preferably 20 to 50 mg / mL; Step 2b): Under stirring conditions (e.g., 100 rpm to 2000 rpm), the oil phase obtained in step 2a) is added dropwise to the aqueous phase to form a nanoparticle solution; Step 2c): Remove the organic solvent from the nanoparticle solution obtained in step 2b) to obtain polymer nanoparticles with GSH / ROS dual response.

6. The method according to claim 5, wherein in step 2c), the organic solvent is removed by placing the nanoparticle solution obtained in step 2b) in an ultrafiltration tube with a molecular weight cutoff (MWCO = 100 kD) and ultrafiltration (e.g., 3000 rpm, twice, 15 min each time) to obtain polymer nanoparticles.

7. The method according to claim 5, wherein the organic solvent used in step 1) is one or more of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF) or tetrahydrofuran (THF); preferably dimethyl sulfoxide (DMSO).

8. The method according to claim 5, wherein the oil phase or aqueous phase further comprises a stabilizer, wherein the stabilizer is polyvinyl alcohol (PVA), a zwitterionic surfactant, povidone, or DSPE-PEG, preferably DSPE-PEG. 2000 The stabilizer is 0-50% of the polymer mass, preferably 0-30%, more preferably 0-10%.

9. The method according to claim 5, wherein the aqueous phase in step 2b) is an aqueous solution, preferably water.

10. The use of a polymer nanoparticle according to any one of claims 1-3 or a polymer nanoparticle prepared by the method according to any one of claims 4-8, for use as a drug delivery carrier or for preparing a drug delivery carrier.

11. A polymer nanoparticle drug delivery system with GSH / ROS dual responsiveness, comprising polymer nanoparticles according to any one of claims 1-3 or polymer nanoparticles prepared by any one of claims 4-9 and a drug, preferably the drug being an antitumor drug.

12. The polymer nanoparticle drug delivery system according to claim 11, wherein the polymer nanoparticle drug delivery system is in the form of an aqueous solution or a lyophilized powder.

13. The polymer nanoparticle drug delivery system according to claim 12, wherein the polymer nanoparticle drug delivery system is in the form of a lyophilized powder, and the lyophilized powder further comprises 20-80 wt% of a lyophilization protectant; optionally, the lyophilization protectant is selected from one or more of sucrose, trehalose, lactose, glycine, mannitol, etc.

14. A method for preparing a polymer nanoparticle drug delivery system with GSH / ROS dual responsiveness, comprising the following steps: Step c1): Dissolve the drug and the polymer in an organic solvent, wherein the total concentration of the polymer and the drug in the oil phase is 5-60 mg / mL; Where x is an integer selected from 2 to 15, such as 2-12, 4-12, 2-6 or 6-15; y is an integer selected from 0 to 13, such as 0-12, 4-10, 0-6 or 6-13; m:n = (1:19) ~ (19:1); R1 and R2 are each independently selected from H, substituted or unsubstituted C. 1-12 Alkyl groups (e.g., substituted or unsubstituted C4) 1-10 Alkyl or C 1-5 Alkyl), substituted or unsubstituted C 3-12 cycloalkyl, substituted or unsubstituted C 6-12 aryl, substituted or unsubstituted C 5-12 heteroaryl, or substituted or unsubstituted C 4-12 Heterocyclic groups; Step c2): Under stirring conditions, the oil phase obtained in step c1) is added dropwise to the aqueous phase to form a polymer nanoparticle drug delivery system.

15. The method for preparing the polymer nanoparticle drug delivery system according to claim 14, wherein the mass ratio of the polymer to the drug is 1:0.05 to 0.50, preferably 1:0.2 to 0.4, and more preferably 1:0.

3.

16. A lyophilization method for preparing a polymer nanoparticle drug delivery system with dual GSH / ROS response, comprising the following steps: Step d1): The polymer nano-drug delivery system obtained by the preparation method of the polymer nano-drug delivery system according to claim 14 or 15 is concentrated by ultrafiltration to remove organic solvents; Step d2): The concentrated polymer nanoparticle drug delivery system is mixed with a 5-20% (w / v) lyophilization protectant solution, and the mixed solution is subjected to vacuum freeze-drying to obtain lyophilized powder.

17. The freeze-drying method according to claim 16, wherein the freeze-drying protectant is selected from one or more of sucrose, trehalose, lactose, glycine, mannitol, etc.; and / or The polymer nanoparticle drug delivery system is mixed with the lyophilization protectant solution at a volume ratio of 4:1 to 1:4 (preferably 1:1).

18. The freeze-drying method according to claim 16 or 17, wherein the freeze-dried powder is reconstituted with a diluent to prepare an injection, preferably, the diluent is water or glucose injection.

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