Preparation method and application of biotin-modified pH-sensitive amphiphilic polymer

Abstract

The present application relates to a preparation method and application of a biotin-modified pH-sensitive amphiphilic polymer, and provides a biotin-modified pH-sensitive amphiphilic polymer, a preparation method and application of the amphiphilic polymer. The preparation method of the amphiphilic polymer comprises the following steps: biotin and a first catalyst are dissolved in a solvent to react, then polyethylene glycol is added to synthesize biotin-modified polyethylene glycol; alpha-tocopherol succinate and a second catalyst are dissolved in a solvent to react, then ethylene glycol vinyl ether is added to synthesize ethylene glycol vinyl ether-modified alpha-tocopherol succinate; under the condition of a nitrogen atmosphere, the ethylene glycol vinyl ether-modified alpha-tocopherol succinate, a third catalyst and the biotin-modified polyethylene glycol are dissolved in a solvent to react, and then the amphiphilic polymer is prepared. The amphiphilic polymer can be used as a drug carrier to load a plurality of antitumor drugs, can produce a high-efficiency antitumor treatment effect, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of polymer materials and drug carrier technology, and in particular to a method for preparing biotin-modified pH-sensitive amphiphilic polymers and their applications. Background Technology

[0002] Cancer is a serious disease that threatens human health. Appropriate treatment methods need to be selected based on the patient's physical condition, tumor pathology type, and extent of invasion. Currently, the main treatments for cancer include surgery, chemotherapy, radiation therapy, immunotherapy, and traditional Chinese medicine. Among these, chemotherapy is an essential and convenient treatment method in clinical practice. Chemotherapy achieves its therapeutic goal by infusing the body with chemical drugs that inhibit the growth of cancer cells. However, most commonly used chemical drugs are lipid-soluble, which have disadvantages such as poor specificity, low biocompatibility, and significant toxic side effects. Researchers are using drug carriers for treatment, and the effective delivery and release of chemical drugs within these carriers is crucial. With the rising incidence of cancer worldwide, there is an urgent need for an effective anti-tumor drug carrier to address the enormous challenge that cancer poses to humanity.

[0003] Current cancer treatments also rely on drugs from various biological sources, but these drugs are easily degraded and become inactive after entering the body due to the influence of the internal environment, thus failing to achieve effective treatment. The occurrence, growth, and metastasis of tumors are closely related to the internal and external environment of tumor cells. The tumor microenvironment has very distinct physiological characteristics. The pH value of blood and normal tissues is approximately 7.4, while due to the high rate of glycolysis in tumor cells, the pH value in the tumor environment fluctuates between 6.0 and 7.2. The pH value in endosomes and lysosomes within tumor cells can reach 4.5-6.5. Therefore, designing anti-tumor drug carriers that can effectively and precisely release chemical drugs in response to changes in the tumor microenvironment is a key focus of current drug carrier research.

[0004] Antitumor drug carriers require good biocompatibility and precise tumor targeting. Research on antitumor drug carriers with excellent pH-sensitive drug release is of great significance. Biotin (water-soluble vitamin B7), also known as vitamin H, coenzyme R, etc., is crucial for cell growth and development and is called a cell growth promoter. It plays an important role in the metabolism of carbohydrates, amino acids, and fatty acids. Studies have shown that biotin receptors are overexpressed in various cancer cells, including breast cancer cells. Polyethylene glycol, as a polymer material with good water solubility and biocompatibility, is widely used in the surface modification of various nanocarriers. Combining biotin and polyethylene glycol and introducing it into antitumor drug carriers as a hydrophilic component is a good choice. On the other hand, α-tocopherol succinate, as a well-known vitamin E derivative, is an excellent solvent for many poorly soluble drugs due to its excellent lipophilicity. Therefore, combining α-tocopherol succinate with hydrophilic materials can yield amphiphilic polymers that can self-assemble into micelles, improving the solubility of hydrophobic antitumor drugs.

[0005] Currently, various drug delivery carriers or systems have been disclosed. For example, patent document CN106133024B provides a novel polymer-based co-water solubilizer for the delivery of hydrophobic drugs. It discloses a polymer conjugate and a therapeutic composition. The polymer conjugate can be used to prepare polymer micelles to deliver hydrophobic drugs. The therapeutic composition is used to treat cancer, infectious diseases, hypertension, angina pectoris, gynecological diseases, and hyperlipidemia.

[0006] Patent document CN107141464B discloses an amphiphilic polycarbonate-polyethylene glycol polymer loaded with a Schiff base, its preparation method, and its application. Specifically, this invention relates to the preparation of a biodegradable polymer carrier material loaded with a drug via a pH-sensitive bond. Specifically, a hydrophobic drug is linked to a biodegradable amphiphilic block polymer carrier material. This invention overcomes the disadvantages of poor absorption and bioavailability caused by poor hydrophilicity of drugs. In addition, it can also prepare a polymer micelle nano-drug delivery system in aqueous solution, which has a passive targeting effect, further improving the bioavailability of the drug and reducing the toxic side effects of the drug.

[0007] Existing technologies also provide hydrogel drug-loaded pH-sensitive modified dextran-based nanohydrogels, which, as carriers, effectively load drugs through electrostatic interactions and physical encapsulation. Their in vitro release behavior (cumulative release of 35.85% in a weakly acidic environment at pH 5.0) exhibits significant sensitivity to acidic environments; the stronger the acidity of the environment, the faster the drug is released. Therefore, they can be used as pH-sensitive carriers in the field of drug delivery.

[0008] Existing technologies provide drug carriers or systems that can reduce drug toxicity and enhance targeting effects. However, existing technologies using drug carriers to deliver therapeutic drugs cannot simultaneously solve problems such as low drug loading, low release, and low tumor inhibition rate. Therefore, this invention provides a method for preparing a biotin-modified pH-sensitive amphiphilic polymer and its application to overcome the above problems.

[0009] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0010] To address the shortcomings of existing technologies, this invention provides a biotin-modified pH-sensitive amphiphilic polymer, the structural formula of which is as follows:

[0011]

[0012] Where n is 5-200.

[0013] Preferably, n is 40-170.

[0014] More preferably, the amphiphilic polymer is prepared from biotin, polyethylene glycol, α-tocopherol succinate, and ethylene glycol ethylene ether.

[0015] This invention also provides a method for preparing a biotin-modified pH-sensitive amphiphilic polymer, comprising the following steps:

[0016] Biotin and the first catalyst were dissolved in a solvent and reacted, followed by the addition of polyethylene glycol to synthesize biotin-modified polyethylene glycol.

[0017] After reacting α-tocopherol succinate and a second catalyst in a solvent, ethylene glycol ethylene ether is added to synthesize ethylene glycol ethylene ether-modified α-tocopherol succinate.

[0018] The amphiphilic polymer is prepared by reacting the ethylene glycol ethylene ether-modified α-tocopherol succinate, the third catalyst, and the biotin-modified polyethylene glycol in a solvent under a nitrogen atmosphere.

[0019] Preferably, the method for synthesizing the biotin-modified polyethylene glycol is as follows: biotin and a first catalyst are dissolved in a solvent and activated at 15-50°C for 1-24 hours, then polyethylene glycol is added and the reaction is continued at the same temperature for 12-48 hours. The reaction product is obtained by cold ether precipitation, filtration and washing, and vacuum drying.

[0020] Preferably, the synthesis method of the ethylene glycol ethylene ether modified α-tocopherol succinate is as follows: α-tocopherol succinate and a second catalyst are dissolved in a solvent and activated at 15-50°C for 1-24 h, then ethylene glycol ethylene ether is added, and the reaction is continued at 20-50°C for 12-48 h. The reaction product is purified by dialysis and freeze-dried to obtain the product.

[0021] Preferably, the synthesis method of the biotin-modified pH-sensitive amphiphilic polymer is as follows: under a nitrogen atmosphere, ethylene glycol ethylene ether-modified α-tocopherol succinate and a third catalyst are dissolved in a solvent, and then a biotin-modified polyethylene glycol solution is slowly added dropwise to the solvent. The reaction solution is stirred at 15-50°C for 6-72 hours. The reaction product is obtained by cold ether precipitation, filtration and washing, dialysis purification, and freeze drying.

[0022] Preferably, the first catalyst, the second catalyst, and the third catalyst are independently selected from any one or more combinations of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), N-hydroxysuccinimide (NHS), 4-dimethylaminopyridine (DMAP), dicyclohexylcarbodiimide (DCC), p-toluenesulfonic acid (p-TSA), and pyridine p-toluenesulfonate (PPTS).

[0023] Preferably, the first catalyst is a composition of EDC·HCl and NHS in a molar ratio of (1:5) to (5:1). Particularly preferably, the first catalyst is a composition of EDC·HCl and NHS in a molar ratio of (1:2) to (2:1).

[0024] Preferably, the second catalyst is a composition of EDC·HCl and NHS in a molar ratio of (1:5) to (5:1). Particularly preferably, the second catalyst is a composition of EDC·HCl and NHS in a molar ratio of (1:2) to (2:1).

[0025] Preferably, the third catalyst is p-TSA.

[0026] Preferably, the polyethylene glycol used in the preparation method has a molecular weight of 238-8818 Da.

[0027] Preferably, the solvent used in the preparation method is selected from one or more of water, formamide, dimethyl sulfoxide, N,N-dimethylformamide, tetrahydrofuran, and N,N-dimethylacetamide.

[0028] Preferably, the molar ratio of the carboxyl group in biotin to the hydroxyl group in polyethylene glycol used in the preparation method is (1:3) to (3:1). Preferably, the molar ratio of the carboxyl group in biotin to the total amount of EDC·HCl and NHS is (1:4) to (4:1). Preferably, the molar ratio of EDC·HCl and NHS is (1:5) to (5:1).

[0029] Preferably, the molar ratio of the carboxyl group in α-tocopherol succinate to the hydroxyl group in ethylene glycol ether used in the preparation method is (1:3) to (3:1). Preferably, the molar ratio of the carboxyl group in α-tocopherol succinate to the total amount of EDC·HCl and NHS is (1:4) to (4:1). Preferably, the molar ratio of EDC·HCl and NHS is (1:5) to (5:1).

[0030] Preferably, the molar ratio of the hydroxyl groups in the biotin-modified polyethylene glycol to the vinyl groups in the α-tocopherol succinate modified with ethylene glycol ether in the preparation method is (1:5) to (5:1). Preferably, the molar ratio of the biotin-modified polyethylene glycol to p-TSA is (1:20) to (20:1).

[0031] This invention provides an antitumor pharmaceutical composition comprising the biotin-modified pH-sensitive amphiphilic polymer and an antitumor drug. Preferably, the antitumor drug is selected from any one or more of docetaxel, paclitaxel, doxorubicin, and camptothecin.

[0032] The present invention relates to the application of the biotin-modified pH-sensitive amphiphilic polymer and the preparation method of the amphiphilic polymer in the preparation of drug carriers.

[0033] The biotin-modified pH-sensitive amphiphilic polymer and the preparation method of the amphiphilic polymer provided by this invention are used in antitumor drug release carriers.

[0034] The present invention relates to the application of the biotin-modified pH-sensitive amphiphilic polymer and the preparation method of the amphiphilic polymer in the preparation of drugs for treating cancer.

[0035] The present invention relates to the application of the biotin-modified pH-sensitive amphiphilic polymer and the preparation method of the amphiphilic polymer in the preparation of polymer micelle formulations for antitumor drugs.

[0036] This invention provides a polymeric micelle formulation of an antitumor drug, the preparation method of which is as follows:

[0037] Antitumor drugs are dissolved in organic solvents to prepare antitumor drug solutions. Then, during the ultrasonic dispersion of amphiphilic polymers, the antitumor drug solutions are added dropwise to the amphiphilic polymer solutions. After the addition is complete, the mixture is ultrasonically dispersed for another 5 minutes. After dialysis, centrifugation and freeze-drying, an antitumor drug polymer micelle formulation is prepared.

[0038] Preferably, the antitumor drug is selected from any one or more of docetaxel, paclitaxel, doxorubicin, and camptothecin.

[0039] The present invention relates to the application of the biotin-modified pH-sensitive amphiphilic polymer and the preparation method of the amphiphilic polymer in drug encapsulation.

[0040] Technical principle of the invention:

[0041] Acetal bonds are a common type of acid-sensitive chemical bond, characterized by stability under normal physiological conditions but cleavage under the acidic conditions of tumor tissue. Introducing acetal bonds into amphiphilic polymers can achieve precise release of antitumor drugs. This invention links hydrophilic biotin-modified polyethylene glycol with modified hydrophobic α-tocopherol succinate via pH-sensitive acetal bonds, creating a novel biotin-modified pH-sensitive amphiphilic polymer. When formulated into drug-loaded polymer micelles with antitumor drugs, this polymer prolongs circulation time in the blood, actively targets the surface of tumor cells, and cleaves under the low pH conditions of tumor tissue, thereby releasing the encapsulated antitumor drugs.

[0042] The beneficial effects of this invention are:

[0043] The biotin-modified pH-sensitive amphiphilic polymer disclosed in this invention has good water solubility; as a drug carrier, it can encapsulate a variety of antitumor drugs and has excellent biocompatibility; after being formulated into antitumor drug preparations, it has good stability and high drug loading capacity, and the preparations have long blood circulation time, strong active targeting ability and pH-sensitive drug release characteristics, which can produce highly effective antitumor therapeutic effects and have broad application prospects.

[0044] (1) This invention uses biotin, polyethylene glycol, and α-tocopherol succinate as the main components. The raw materials are widely available, and the preparation process is simple, convenient, safe, and non-toxic. Biotin, as a water-soluble vitamin, is an essential micronutrient for cell growth and is overexpressed in many cancer cells. α-Tocopherol succinate has good lipophilicity and biodegradability. Polyethylene glycol has excellent biocompatibility and does not accumulate in vivo. Therefore, these three are widely used in the medical field. The biotin-modified pH-sensitive amphiphilic polymer prepared by reacting biotin, polyethylene glycol, and α-tocopherol succinate can serve as an in vivo delivery carrier. The amphiphilic polymer interacts with the therapeutic drug to form stable polymer micelles. These polymer micelles can be used to deliver the therapeutic drug to the tumor site in vivo. Under the microenvironmental conditions of the tumor cells, the polymer micelles cleave and release the encapsulated therapeutic drug. Preferably, the therapeutic drug is an antitumor drug.

[0045] (2) Based on the difference in pH between tumor tissue and normal physiological environment, this invention uses acid-sensitive acetal bonds to connect the hydrophilic and hydrophobic parts to obtain an amphiphilic polymer. When the anti-tumor drug is made into a drug formulation, the anti-tumor drug can be rapidly released when it reaches the acidic environment of the tumor tissue. This can achieve precise release of the drug at the tumor site, reduce the toxic side effects of the drug formulation at non-target sites, and improve the efficacy.

[0046] (3) The antitumor drug formulation prepared by the present invention using amphiphilic polymers and therapeutic drugs has strong stability. Its appearance does not change significantly after being stored at room temperature for many days. Moreover, the stability indicators fluctuate little as the storage time increases, which is beneficial to the storage and clinical application of the formulation. At the same time, the formulation has a high drug loading capacity, which can efficiently load antitumor drugs and improve the therapeutic efficiency of tumor chemotherapy. In addition, due to the high drug loading capacity of the formulation, the volume of administration can be reduced and it is suitable for large-dose administration.

[0047] (4) The amphiphilic polymer provided by the present invention can encapsulate a variety of antitumor drugs. Its good biocompatibility, biodegradability, safety and non-toxicity enable the amphiphilic polymer to be widely used in the medical field. Furthermore, the preparation method of the biotin-modified pH-sensitive amphiphilic polymer provided by the present invention is simple and easy to operate and promote. In addition, the biotin-modified pH-sensitive amphiphilic polymer can break down under low pH conditions in tumor tissue, thereby releasing the encapsulated antitumor drugs and achieving the purpose of precise and targeted release.

[0048] (5) The amphiphilic polymer provided by the present invention has high safety and good anti-tumor effect. Attached Figure Description

[0049] Figure 1 This is the infrared spectrum of the biotin-modified pH-sensitive amphiphilic polymer provided by the present invention;

[0050] Figure 2 This is a schematic diagram of the molecular structure of the biotin-modified pH-sensitive amphiphilic polymer provided by the present invention. Detailed Implementation

[0051] The following is in conjunction with the appendix Figures 1-2 The present invention will be described in detail below. Figure 1 This is the infrared spectrum of a biotin-modified pH-sensitive amphiphilic polymer. Figure 2 This is a schematic diagram of the molecular structure of a biotin-modified pH-sensitive amphiphilic polymer.

[0052] This invention connects hydrophilic biotin-modified polyethylene glycol with modified hydrophobic α-tocopherol succinate via pH-sensitive acetal bonds to create a novel biotin-modified pH-sensitive amphiphilic polymer. When formulated into drug-loaded polymer micelles with an antitumor drug, this polymer prolongs circulation time in the bloodstream, actively targets the surface of tumor cells, and cleaves under low pH conditions within tumor tissue, releasing the encapsulated antitumor drug for highly effective treatment. The biotin-modified pH-sensitive amphiphilic polymer provided in this application can be used in novel drug delivery systems for tumor treatment. Preferably, the antitumor drug formulation prepared from the biotin-modified pH-sensitive amphiphilic polymer and the antitumor drug can be used for cancer treatment. Preferably, the drug formulation prepared from the amphiphilic polymer and the therapeutic drug can be administered via enteral or parenteral routes, such as intravenous injection. In this invention, the drug carrier refers to a carrier that binds to the active pharmaceutical ingredient through physical encapsulation, chemical bonding, and electrostatic interactions, facilitating drug entry into the body and exerting its effect through different administration methods; it does not contain the active pharmaceutical ingredient. Drug carriers include polymeric micelles, liposomes, and nanoparticles. Polymeric micelles are a type of drug carrier. They are thermodynamically stable colloidal solutions formed by the self-assembly of synthesized amphiphilic block copolymers in solution. Polymer micelle formulations refer to monolithic drug formulations formed by encapsulating drugs in amphiphilic polymers through physical embedding, chemical bonding, and electrostatic interactions; that is, they include both the amphiphilic polymer and the active pharmaceutical ingredient encapsulated in the amphiphilic polymer.

[0053] Example 1

[0054] This embodiment provides a method for synthesizing a biotin-modified pH-sensitive amphiphilic polymer, wherein the structural formula of the amphiphilic polymer is:

[0055]

[0056] Where n is 80-100.

[0057] (1) Synthesis of biotin-modified polyethylene glycol

[0058] 20 mg of biotin and a certain amount of catalyst (composed of 24 mg of EDC·HCl and 14 mg of NHS) were weighed and placed in a round-bottom flask. A certain volume of solvent was added, and the reaction solution was stirred at 25 °C for 4 h. Then 820 mg of polyethylene glycol was added, and the reaction was stirred at 25 °C for another 24 h. The reaction product was obtained by cold ether precipitation, filtration and washing, and vacuum drying.

[0059] In the synthesis of biotin-modified polyethylene glycol, the average molecular weight of the polyethylene glycol is 4000 Da, and the solvent is N,N-dimethylformamide.

[0060] (2) Synthesis of α-tocopherol succinate modified with ethylene glycol ethylene ether

[0061] 100 mg of α-tocopherol succinate and a certain amount of catalyst (composed of 54 mg of EDC·HCl and 33 mg of NHS) were weighed and placed in a round-bottom flask. A certain volume of solvent was added, and the reaction solution was stirred at 25 °C for 4 h. Then 20 μL of ethylene glycol ether was added, and the reaction was stirred at 35 °C for 24 h. The reaction product was purified by dialysis and freeze-dried to obtain the product.

[0062] In the synthesis of ethylene glycol ethylene ether modified α-tocopherol succinate, the solvent is N,N-dimethylformamide.

[0063] (3) Synthesis of biotin-modified pH-sensitive amphiphilic polymers

[0064] Under a nitrogen atmosphere, 396 mg of biotin-modified polyethylene glycol was dissolved in a solvent and then added dropwise to a solution containing 180 mg of ethylene glycol ether-modified α-tocopherol succinate and 2 mg of p-TSA. The reaction was continued at 25 °C for 48 h. The reaction product was then precipitated with cold ether, filtered, washed, purified by dialysis, and freeze-dried to obtain the amphiphilic polymer.

[0065] In the synthesis of biotin-modified pH-sensitive amphiphilic polymers, the solvent is N,N-dimethylformamide.

[0066] Example 2

[0067] This embodiment provides another method for synthesizing a biotin-modified pH-sensitive amphiphilic polymer, the structural formula of which is:

[0068]

[0069] Where n is 40-50.

[0070] (1) Synthesis of biotin-modified polyethylene glycol

[0071] 20 mg of biotin and a certain amount of catalyst (composed of 24 mg of EDC·HCl and 14 mg of NHS) were weighed and placed in a round-bottom flask. A certain volume of solvent was added, and the reaction solution was stirred at 25 °C for 4 h. Then 410 mg of polyethylene glycol was added, and the reaction was stirred at 25 °C for another 24 h. The reaction product was obtained by cold ether precipitation, filtration and washing, and vacuum drying.

[0072] In the synthesis of biotin-modified polyethylene glycol, the average molecular weight of the polyethylene glycol is 2000 Da; the solvent is N,N-dimethylformamide.

[0073] (2) Synthesis of α-tocopherol succinate modified with ethylene glycol ethylene ether

[0074] 100 mg of α-tocopherol succinate and a certain amount of catalyst (composed of 54 mg of EDC·HCl and 33 mg of NHS) were weighed and placed in a round-bottom flask. A certain volume of solvent was added, and the reaction solution was stirred at 25 °C for 4 h. Then 20 μL of ethylene glycol ether was added, and the reaction was stirred at 35 °C for 24 h. The reaction product was purified by dialysis and freeze-dried to obtain the product.

[0075] In the synthesis of ethylene glycol ethylene ether modified α-tocopherol succinate, the solvent is N,N-dimethylformamide.

[0076] (3) Synthesis of biotin-modified pH-sensitive amphiphilic polymers

[0077] Under a nitrogen atmosphere, 209 mg of biotin-modified polyethylene glycol was dissolved in a solvent and then added dropwise to a solution containing 180 mg of ethylene glycol ether-modified α-tocopherol succinate and 2 mg of p-TSA. The reaction was continued at 25 °C for 48 h. The reaction product was then precipitated with cold ether, filtered, washed, purified by dialysis, and freeze-dried to obtain the amphiphilic polymer.

[0078] In the synthesis of biotin-modified pH-sensitive amphiphilic polymers, the solvent is N,N-dimethylformamide.

[0079] Example 3

[0080] This embodiment provides another method for synthesizing a biotin-modified pH-sensitive amphiphilic polymer, the structural formula of which is:

[0081]

[0082] Where n is 125-170.

[0083] (1) Synthesis of biotin-modified polyethylene glycol

[0084] 20 mg of biotin and a certain amount of catalyst (composed of 24 mg of EDC·HCl and 14 mg of NHS) were weighed and placed in a round-bottom flask. A certain volume of solvent was added, and the reaction solution was stirred at 25 °C for 4 h. Then 1230 mg of polyethylene glycol was added, and the reaction was stirred at 25 °C for another 24 h. The reaction product was obtained by cold ether precipitation, filtration and washing, and vacuum drying.

[0085] In the synthesis of biotin-modified polyethylene glycol, the average molecular weight of the polyethylene glycol is 6000 Da; the solvent is N,N-dimethylformamide.

[0086] (2) Synthesis of α-tocopherol succinate modified with ethylene glycol ethylene ether

[0087] 100 mg of α-tocopherol succinate and a certain amount of catalyst (composed of 54 mg of EDC·HCl and 33 mg of NHS) were weighed and placed in a round-bottom flask. A certain volume of solvent was added, and the reaction solution was stirred at 25 °C for 4 h. Then 20 μL of ethylene glycol ether was added, and the reaction was stirred at 35 °C for 24 h. The reaction product was purified by dialysis and freeze-dried to obtain the product.

[0088] In the synthesis of ethylene glycol ethylene ether modified α-tocopherol succinate, the solvent is N,N-dimethylformamide.

[0089] (3) Synthesis of biotin-modified pH-sensitive amphiphilic polymers

[0090] Under a nitrogen atmosphere, 584 mg of biotin-modified polyethylene glycol was dissolved in a solvent and then added dropwise to a solution containing 180 mg of ethylene glycol ether-modified α-tocopherol succinate and 2 mg of p-TSA. The reaction was continued at 25 °C for 48 h. The reaction product was then precipitated with cold ether, filtered, washed, purified by dialysis, and freeze-dried to obtain the amphiphilic polymer.

[0091] In the synthesis of biotin-modified pH-sensitive amphiphilic polymers, the solvent is N,N-dimethylformamide.

[0092] Comparative Example 1

[0093] Synthesis of pH-sensitive amphiphilic polymer 2, wherein the structural formula of the amphiphilic polymer is:

[0094]

[0095] Where n is 80-100.

[0096] (1) Synthesis of α-tocopherol succinate modified with ethylene glycol ethylene ether

[0097] 100 mg of α-tocopherol succinate and a certain amount of catalyst (composed of 54 mg of EDC·HCl and 33 mg of NHS) were weighed and placed in a round-bottom flask. A certain volume of solvent was added, and the reaction solution was stirred at 25 °C for 4 h. Then 20 μL of ethylene glycol ether was added, and the reaction was stirred at 35 °C for 24 h. The reaction product was purified by dialysis and freeze-dried to obtain the product.

[0098] In the synthesis of ethylene glycol vinyl ether modified α-tocopherol succinate, the solvent is N,N-dimethylformamide;

[0099] (2) Synthesis of pH-sensitive amphiphilic polymer 2

[0100] Under a nitrogen atmosphere, 375 mg of polyethylene glycol was dissolved in a solvent and then added dropwise to a solution containing 180 mg of α-tocopherol succinate modified with ethylene glycol ether and 2 mg of p-TSA. The reaction was continued at 25 °C for 48 h. The reaction product was then precipitated with cold ether, filtered, washed, purified by dialysis, and freeze-dried to obtain amphiphilic polymer 2.

[0101] In the synthesis of pH-sensitive amphiphilic polymer 2, the average molecular weight of the polyethylene glycol is 4000 Da; the solvent is N,N-dimethylformamide.

[0102] Comparative Example 2

[0103] Synthesis of biotin-modified amphiphilic polymer 3, wherein the structural formula of the amphiphilic polymer is:

[0104]

[0105] For example, n is 80-100.

[0106] (1) Synthesis of biotin-modified polyethylene glycol

[0107] 20 mg of biotin and a certain amount of catalyst (composed of 24 mg of EDC·HCl and 14 mg of NHS) were weighed and placed in a round-bottom flask. A certain volume of solvent was added, and the reaction solution was stirred at 25 °C for 4 h. Then 820 mg of polyethylene glycol was added, and the reaction was stirred at 25 °C for another 24 h. The reaction product was obtained by cold ether precipitation, filtration and washing, and vacuum drying.

[0108] In the synthesis of biotin-modified polyethylene glycol, the average molecular weight of the polyethylene glycol is 4000 Da; the solvent is N,N-dimethylformamide.

[0109] (2) Synthesis of biotin-modified amphiphilic polymer 3

[0110] 30 mg of α-tocopherol succinate and a certain amount of catalyst (composed of 16 mg of EDC·HCl and 10 mg of NHS) were weighed and placed in a round-bottom flask. A certain volume of solvent was added, and the reaction solution was stirred at 25 °C for 4 h. Then, 200 mg of biotin-modified polyethylene glycol was added, and the reaction was stirred at 25 °C for another 36 h. The reaction product was purified by dialysis and freeze-dried to obtain amphiphilic polymer 3.

[0111] In the synthesis of biotin-modified amphiphilic polymer 3, the solvent is N,N-dimethylformamide.

[0112] Experimental Example 1: Formulation Stability Study and Drug Loading Determination

[0113] To evaluate the stability of the drug-loaded polymer micelles, amphiphilic polymers prepared by any of the methods in Examples 1-3 and Comparative Examples 1-2 were mixed with paclitaxel to form polymer micelle solutions. The solutions were placed in vials and stored at room temperature (25°C) for one month. Samples were taken on days 1, 15, and 30 to observe the appearance of the samples and to determine their particle size and drug loading.

[0114] Table 1. Appearance, particle size, and drug loading of different formulations

[0115] Example 1 1 No significant changes 110.0±0.6 16.2 Example 1 15 No significant changes 115.8±1.1 14.9 Example 1 30 No significant changes 120.3±1.8 13.4 Example 2 1 No significant changes 120.2±1.1 17.6 Example 2 15 No significant changes 124.4±0.9 16.7 Example 2 30 No significant changes 127.5±1.6 15.5 Example 3 1 No significant changes 101.3±1.0 15.0 Example 3 15 No significant changes 105.5±1.3 13.7 Example 3 30 No significant changes 111.5±1.5 12.6 Comparative Example 1 1 No significant changes 108.2±0.9 17.0 Comparative Example 1 15 No significant changes 113.1±1.5 16.5 Comparative Example 1 30 No significant changes 119.8±1.8 15.2 Comparative Example 2 1 No significant changes 111.9±0.7 16.1 Comparative Example 2 15 No significant changes 115.6±1.0 15.0 Comparative Example 2 30 No significant changes 120.2±1.4 13.4

[0116] The results showed that the amphiphilic polymers prepared in Examples 1-3 and Comparative Examples 1-2, when mixed with paclitaxel to form polymer micelle solutions, did not show significant changes in appearance after being stored at room temperature for 30 days. Furthermore, the stability indicators did not fluctuate much over time, indicating that the formulations have strong stability. In addition, after 30 days of storage, the minimum drug loading of the formulations could still reach more than 12%, indicating that the formulations have a high drug loading capacity.

[0117] Experimental Example 2: pH Sensitivity Test

[0118] To determine the pH-sensitive properties of the pH-sensitive amphiphilic polymer solution, amphiphilic polymers prepared by any of the methods in Examples 1-3 and Comparative Examples 1-2 were selected for experiments, as follows: 2 mg of the amphiphilic polymer was added to a certain volume of PBS 5.0 (or PBS 7.4) to prepare an amphiphilic polymer solution; then, the amphiphilic polymer solution was shaken at 37°C for 12 h in a water bath constant temperature shaker; the change in polymer solution particle size was measured by dynamic light scattering (DLS).

[0119] The results showed that the amphiphilic polymers prepared in Examples 1-3 and Comparative Example 1 all exhibited the following pattern: after shaking in PBS 7.4 for 12 hours, no significant change in particle size was observed in the amphiphilic polymer solution. However, when the pH decreased to 5.0, the particle size of the amphiphilic polymer solution increased significantly and the size distribution became uneven. These experimental results indicate that the amphiphilic polymers containing acetal bonds prepared in Examples 1-3 and Comparative Example 1 possess pH-sensitive properties, while the amphiphilic polymer without acetal bonds prepared in Comparative Example 2 does not exhibit pH sensitivity.

[0120] Test Example 3: Drug Release Test

[0121] To evaluate the pH-sensitive drug release characteristics of polymer micelles containing antitumor drugs, amphiphilic polymers prepared by any of the methods in Examples 1-3 and Comparative Examples 1-2 were used to formulate drug products containing the antitumor drug paclitaxel. The specific steps were as follows: 5 mL of polymer micelles loaded with paclitaxel were transferred to a dialysis bag (MWCO 8000 Da) and then immersed in 150 mL of PBS 5.0 (or PBS 7.4) containing 0.5% (w / v) Tween 80. The release media under different pH conditions were maintained at 37°C and stirred at 100 rpm. At predetermined times, 5 mL of the dissolution medium containing paclitaxel was aspirated and immediately replenished with blank PBS solution to maintain a consistent total volume. The samples were analyzed by high-performance liquid chromatography (HPLC) to calculate the amount of paclitaxel released.

[0122] Table 2. Drug release rates of different samples at different pH values

[0123] Example 1 30.7 88.1 Example 2 29.9 88.4 Example 3 30.5 88.0 Comparative Example 1 30.3 79.8 Comparative Example 2 29.8 38.9

[0124] The results showed that the amphiphilic polymers prepared in Examples 1-3 and Comparative Example 1 all exhibited the following characteristics: Under PBS 7.4 conditions, the polymer micelles encapsulating paclitaxel maintained structural stability, with paclitaxel release remaining around 30% after 48 hours. However, under PBS 5.0 conditions, due to the breakage of acetal bonds, the polymer micelle structure became unstable, and paclitaxel release reached over 80%. In Comparative Example 2, due to the absence of acid-sensitive bonds, paclitaxel release was only 38.9% under PBS 5.0 conditions.

[0125] Experimental Example 4: Hemolysis Test

[0126] To assess the safety of intravenous injection of the amphiphilic polymer, an in vitro hemolysis test was performed. Rabbit blood was collected in heparinized centrifuge tubes and centrifuged at 3000 rpm for 5 min. The supernatant was discarded, and the lower layer of red blood cells was washed repeatedly with physiological saline until the supernatant was colorless. Finally, the red blood cells were collected and diluted with 0.9% NaCl solution to a concentration of 2% for later use. The amphiphilic polymers prepared in Examples 1-3 and Comparative Examples 1-2 were weighed and diluted with physiological saline to prepare a series of solutions with different concentrations. 2.5 mL of each solution was placed in a centrifuge tube, and 2.5 mL of red blood cell suspension was added and mixed well. The final solution concentration range was 10-200 μg / mL. All samples were incubated at 37°C for 2 h, centrifuged at 3000 rpm for 10 min, and the supernatant was collected. The absorbance was measured at 540 nm using a UV spectrophotometer. Physiological saline and distilled water were separately prepared and subjected to the same procedure as negative and positive controls, respectively, to determine the hemolysis rate. Hemolysis rate = (sample absorbance - negative control absorbance) / (positive control absorbance - negative control absorbance) × 100%.

[0127] Table 3. Hemolysis rate of erythrocytes in different samples at a final concentration of 200 μg / mL

[0128] Example 1 4.6 Example 2 4.7 Example 3 4.8 Comparative Example 1 4.9 Comparative Example 2 4.5

[0129] The results showed that the amphiphilic polymers prepared in Examples 1-3 and Comparative Example 1 had negligible hemolytic activity in the concentration range of 10-200 μg / mL, and the hemolysis rate did not exceed 5%, indicating that the amphiphilic polymers have excellent hemolytic safety.

[0130] Experimental Example 5: Cytotoxicity Test

[0131] The cytotoxicity of the amphiphilic polymer to MCF-7 cells was evaluated using the in vitro MTT assay. MCF-7 cells were cultured at 1.0 × 10⁶ cells per well. 4Cells were seeded at a concentration of [missing information] onto 96-well plates and cultured for one day. Then, the original culture medium was replaced with 200 μL of DMEM medium containing different concentrations of the amphiphilic polymers prepared in Examples 1-3 and Comparative Examples 1-2. After culturing for another 24 hours, the culture medium was removed, and 20 μL of MTT solution (5 mg / mL) was added, followed by 4 hours of culture. Finally, the culture medium was removed, and 200 μL of DMSO was added to dissolve the formazan crystals. The absorbance at 490 nm was measured using a microplate reader. Cell viability was calculated using the following formula:

[0132] Cell viability (%) = Absorbance of cells exposed to the sample / Absorbance of untreated cells × 100%.

[0133] Table 4. Cell viability of MCF-7 cells at different final concentrations of 50 μg / mL

[0134] Example 1 100.3 Example 2 99.9 Example 3 100.1 Comparative Example 1 99.6 Comparative Example 2 99.8

[0135] The results showed that the amphiphilic polymers prepared by any of the methods in Examples 1-3 and Comparative Examples 1-2 had negligible toxicity to MCF-7 cells at concentrations of 0.5-50 μg / mL, indicating that the amphiphilic polymers prepared by any of the methods in Examples 1-3 and Comparative Examples 1-2 all had good cell safety.

[0136] Experimental Example 6: In vivo antitumor experiment

[0137] To conduct in vivo antitumor experiments, pathogen-free female Balb / c mice were selected, and 4 T1 cells were inoculated into the right axilla of each mouse to establish a mouse tumor model. When the tumor volume reached 100 mm², the tumor was investigated. 3 Mice were randomly divided into five groups of six each, and injected with saline, paclitaxel, paclitaxel-loaded polymeric micelles, doxorubicin-loaded polymeric drug micelles, and camptothecin-loaded polymeric drug micelles, respectively. Day 1 was defined as the day the mice received their first dose. Each sample was injected via the tail vein on days 1, 4, 7, and 10. After 12 days of treatment, on day 13, the animals were sacrificed, the tumors were removed, photographed, and weighed. Tumor inhibition rate (TIR%) = (tumor weight in saline group - tumor weight in treatment group) / tumor weight in saline group × 100%.

[0138] Table 5. Tumor inhibition rate in mice after injection of different formulations

[0139]

[0140]

[0141] As shown in the table, the saline group had no anti-tumor effect, and the tumor inhibition rate was 0%. The commercially available paclitaxel formulation showed some anti-tumor effect, but its tumor inhibition rate was only 62.4%. In contrast, Examples 1-3, loaded with paclitaxel, achieved higher tumor growth inhibition rates, while the comparative examples 1-2, loaded with paclitaxel, showed a significant decrease in tumor growth inhibition rates. This indicates that the lack of active targeting capability and pH-sensitive bonds significantly affect the anti-tumor effect. Furthermore, the examples and comparative examples loaded with doxorubicin and camptothecin showed similar trends to those loaded with paclitaxel. These experimental results suggest that biotin-modified pH-sensitive amphiphilic polymers hold promise as a novel drug delivery system for tumor treatment.

[0142] It should be noted that the specific embodiments described above are exemplary. Those skilled in the art can devise various solutions inspired by the disclosure of this invention, and these solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and do not constitute a limitation on the claims. The scope of protection of this invention is defined by the claims and their equivalents. This specification contains multiple inventive concepts; terms such as "preferredly," "according to a preferred embodiment," or "optionally" indicate that the corresponding paragraph discloses an independent concept. The applicant reserves the right to file divisional applications based on each inventive concept. Throughout the text, features introduced by "preferredly" are merely optional and should not be construed as mandatory. Therefore, the applicant reserves the right to abandon or delete relevant preferred features at any time.

Claims

1. A biotin-modified pH-sensitive amphiphilic polymer, characterized in that, The structural formula of the amphiphilic polymer is as follows: Where n is 5-200.

2. The method for preparing the biotin-modified pH-sensitive amphiphilic polymer according to claim 1, characterized in that, Includes the following steps: Biotin and the first catalyst were dissolved in a solvent and activated at 15-50°C for 1-24 h. Then, polyethylene glycol was added, and the reaction was continued at this temperature for 12-48 h. The reaction product was precipitated with cold diethyl ether, filtered, washed, and vacuum dried to obtain biotin-modified polyethylene glycol. α-Tocopherol succinate and a second catalyst were dissolved in a solvent and activated at 15-50°C for 1-24 h. Then, ethylene glycol ether was added, and the reaction was continued at 20-50°C for 12-48 h. The reaction product was purified by dialysis and freeze-dried to obtain ethylene glycol ether-modified α-tocopherol succinate. Under a nitrogen atmosphere, the ethylene glycol ethylene ether-modified α-tocopherol succinate and the third catalyst were dissolved in a solvent, and then the biotin-modified polyethylene glycol was added dropwise to the solution. The reaction solution was stirred at 15-50°C for 6-72 h. The reaction product was then precipitated with cold ether, filtered, washed, purified by dialysis, and freeze-dried to obtain the amphiphilic polymer.

3. The preparation method according to claim 2, characterized in that, The first catalyst, the second catalyst, and the third catalyst are selected from any one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, 4-dimethylaminopyridine, dicyclohexylcarbodiimide, p-toluenesulfonic acid, and pyridine p-toluenesulfonate.

4. The preparation method according to claim 2 or 3, characterized in that, The solvent used in the preparation method is selected from any one or more of dimethyl sulfoxide, N,N-dimethylformamide, tetrahydrofuran, and N,N-dimethylacetamide.

5. The use of the amphiphilic polymer as described in claim 1, or the amphiphilic polymer prepared by the preparation method according to any one of claims 2 to 4, as a drug carrier.

6. The use of the amphiphilic polymer as described in claim 1, or the amphiphilic polymer prepared by the preparation method according to any one of claims 2 to 4, in the encapsulation of antitumor drugs.

7. The application according to claim 6, characterized in that, The antitumor drug is selected from any one or more of docetaxel, paclitaxel, doxorubicin, and camptothecin.

8. The use of the amphiphilic polymer as described in claim 1, or the amphiphilic polymer prepared by the preparation method according to any one of claims 2 to 4, in the preparation of polymeric micelle formulations for antitumor drugs.

9. The application according to claim 8, characterized in that, The antitumor drug is selected from any one or more of docetaxel, paclitaxel, doxorubicin, and camptothecin.

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