A kind of amphiphilic graft copolymer based on hyaluronic acid and its preparation method and application

Abstract

The present invention specifically relates to an amphiphilic graft copolymer based on hyaluronic acid, and a preparation method and application thereof. The copolymer in the present invention uses hyaluronic acid as the main skeleton, and methoxy polyethylene glycol, octadecanoic acid and 2,4-dinitrobenzene sulfonic acid are grafted thereon; the present invention also relates to a preparation method of the above copolymer, and a drug carrier and a pharmaceutical composition prepared by the copolymer. The polymer prepared by the present invention has good biocompatibility, active and passive dual-targeting capabilities, redox conversion and release of SO2, and can therefore be used to prepare a release carrier for anticancer drugs, achieve high drug uptake and rapid drug release in the body, and has broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of polymer materials technology, specifically relating to an amphiphilic graft copolymer based on hyaluronic acid, its preparation method, and its application. Background Technology

[0002] Cancer, as the leading cause of death worldwide, has always been a focus of research in treatment and diagnosis. Currently, the main treatments for cancer include surgery, radiotherapy, and chemotherapy, with chemotherapy being the most prevalent clinical treatment. However, chemotherapy has many drawbacks, such as poor water solubility, low biocompatibility, and significant toxic side effects. Therefore, developing a drug carrier that can overcome these shortcomings of chemotherapy drugs is essential.

[0003] Patent No. 201910046763.2, entitled "A Derivative of Hyaluronic Acid and Its Preparation Method and Application", discloses the use of hyaluronic acid as the parent skeleton and the modification of hyaluronic acid with fatty alcohol to obtain an amphiphilic hyaluronic acid derivative. On this basis, it is further modified with disulfide bonds and mPEG to give it the characteristics of tumor targeting, long circulation, and reduction-sensitive drug release. When used as a carrier for anti-tumor drugs, it can achieve targeted and precise release of drugs at the tumor site, achieving the effect of synergistic effect and reduced toxicity. This patent has a significant effect on improving the tumor inhibition rate and has high clinical application value.

[0004] The patent with patent number 202010280704.4 and invention title "An amphiphilic graft copolymer based on hyaluronic acid and its preparation method and application" includes: (1) hyaluronic acid as the parent skeleton; (2) deoxycholic acid group grafted onto the first primary alcohol group of hyaluronic acid; (3) methoxy polyethylene glycol unit grafted onto the carboxyl group of hyaluronic acid; and (4) N-acetylcysteine ​​group grafted onto the second primary alcohol group of hyaluronic acid. This copolymer has good biocompatibility, active and passive dual targeting and redox conversion, so it can be used as an anticancer drug release carrier to achieve prolonged blood circulation, high uptake and rapid drug release, effectively improve the drug concentration in tumor cells, and has broad application prospects.

[0005] However, the above research results cannot achieve the effect of assisting chemotherapy drugs in inhibiting tumor growth by releasing sulfur dioxide, which leads to the use of higher doses. In particular, the drug dose of the derivative prepared by the above patented technology in the experiment was 15mg / kg. As those skilled in the art know, the higher the dose, the greater the side effects, treatment costs, and drug resistance of the body. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention proposes an amphiphilic graft copolymer based on hyaluronic acid, its preparation method, and its applications.

[0007] Specifically, this is achieved through the following technical solutions:

[0008] The first object of the present invention is to provide: an amphiphilic graft copolymer based on hyaluronic acid, comprising:

[0009] (1) Hyaluronic acid as the parent skeleton;

[0010] (2) Methoxylated polyethylene glycol units grafted onto the carboxyl group of hyaluronic acid;

[0011] (3) A 2,4-dinitrobenzenesulfonic acid group grafted onto the first primary alcohol group of hyaluronic acid;

[0012] (4) An octadecanoic acid group grafted onto the second primary alcohol group of hyaluronic acid.

[0013] An amphiphilic graft copolymer based on hyaluronic acid has the following structural formula:

[0014]

[0015] Where m ranges from 7 to 323, and n ranges from 12 to 1241.

[0016] The average molecular weight of the methoxy polyethylene glycol unit is 370–20000 Da.

[0017] The average molecular weight of the hyaluronic acid is 5100 to 500000 Da.

[0018] A second objective of this invention is to provide a method for preparing the aforementioned amphiphilic graft copolymer based on hyaluronic acid, comprising the following steps:

[0019] (1) Dissolve hyaluronic acid in the first reaction solvent, add the first catalyst and mix evenly, stir at 0-60℃ for 0.5-24h, then add methoxy polyethylene glycol, stir at 0-60℃ for 2-96h, dialyze the product with distilled water and freeze dry to obtain the first reaction product methoxy polyethylene glycol-hyaluronic acid derivative, denoted as mPEG-HA;

[0020] (2) Dissolve 2,4-dinitrobenzenesulfonic acid in the second reaction solvent, add the second catalyst and mix evenly, stir at 20-100℃ for 3-9h, then add mPEG-HA dissolved in DMSO / pyridine mixed solution and continue stirring for 6-24h to obtain the second reaction product, freeze dry the second reaction product to obtain the freeze-dried product methoxy polyethylene glycol-hyaluronic acid-2,4-dinitrobenzenesulfonic acid derivative, denoted as mPEG-HA-DNs;

[0021] (3) Dissolve octadecanoic acid in the third reaction solvent, add the third catalyst, and catalyze the reaction at 0-60℃ for 0.5-24h. Then add mPEG-HA-DNs and continue the reaction for 2-48h to obtain the third reaction product. Freeze-dry the third reaction product to obtain the freeze-dried product methoxy polyethylene glycol-hyaluronic acid-2,4-dinitrobenzenesulfonic acid-octadecanoic acid derivative, which is designated as mPEG-HA(SA)-DNs, which is an amphiphilic graft copolymer based on hyaluronic acid.

[0022] The first reaction solvent, the second reaction solvent, and the third reaction solvent are independently selected from at least one of formamide, sulfonium chloride, N,N-dimethylformamide, N,N-dimethylacetamide, water, DMSO, and pyridine.

[0023] The first catalyst and the third catalyst are independently selected from at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), N-hydroxysuccinimide (NHS), 4-dimethylaminopyridine (DMAP), and dicyclohexylcarbodiimide (DCC).

[0024] The first catalyst is a composition of EDC·HCl and NHS; the molar ratio of EDC·HCl to NHS in the first catalyst is (1:10)-(10:1); the molar ratio of the first catalyst to hyaluronic acid is (1:100)-(100:1); the molar ratio of methoxy polyethylene glycol to hyaluronic acid is (1:10)-(100:1).

[0025] The second catalyst is N,N-dimethylformamide (DMF); the molar ratio of the second catalyst to 2,4-dinitrobenzenesulfonic acid is (1:20)-(20:1); the molar ratio of 2,4-dinitrobenzenesulfonic acid to mPEG-HA is (1:10)-(110:1).

[0026] The third catalyst is a composition of EDC·HCl and NHS; the molar ratio of EDC·HCl to NHS in the third catalyst is (1:10)-(10:1); the molar ratio of the third catalyst to octadecanoic acid is (1:5)-(5:1); and the molar ratio of octadecanoic acid to mPEG-HA-DNs is (1:10)-(200:1).

[0027] A third object of the present invention is to provide the use of the aforementioned hyaluronic acid-based amphiphilic graft copolymer or the hyaluronic acid-based amphiphilic graft copolymer prepared by the methods thereof in the preparation of anticancer drugs.

[0028] Furthermore, the aforementioned hyaluronic acid-based amphiphilic graft copolymers or hyaluronic acid-based amphiphilic graft copolymers prepared by the aforementioned methods are used as anticancer drug carriers.

[0029] Furthermore, the aforementioned amphiphilic graft copolymer based on hyaluronic acid, or the amphiphilic graft copolymer based on hyaluronic acid prepared by the aforementioned method, is used in the preparation of anticancer drugs. Specifically, the amphiphilic graft copolymer based on hyaluronic acid is used as a coating material to encapsulate the anticancer active ingredient, thereby forming an anticancer drug with tumor-targeted release.

[0030] The anticancer active ingredient is at least one of docetaxel, paclitaxel, epirubicin, and camptothecin.

[0031] Beneficial effects:

[0032] (1) The present invention is based on an amphiphilic graft copolymer of hyaluronic acid, which has the characteristics of high drug loading, high encapsulation rate, good biocompatibility, high safety and biodegradability in vivo.

[0033] (2) This invention utilizes an amphiphilic graft copolymer of hyaluronic acid as a drug carrier. This carrier has a small particle size, enabling passive targeting through the EPR effect. The modification with hyaluronic acid allows the copolymer to specifically bind to the CD44 receptor overexpressed on tumor cells, thereby achieving active targeting. Due to the modification with 2,4-dinitrobenzenesulfonic acid, this polymer can selectively release the loaded drug and SO2 at high glutathione concentrations, significantly increasing drug utilization and reducing drug toxicity and dosage. This copolymer carrier can rapidly undergo redox reactions within tumor cells, precisely releasing the drug and sulfur dioxide. Sulfur dioxide assists in chemotherapy, thereby achieving long-term circulation, high uptake, and rapid release of the drug in vivo. This maintains high therapeutic efficacy while reducing drug dosage, demonstrating significant application potential in controlled drug release.

[0034] (3) This invention is based on an amphiphilic graft copolymer of hyaluronic acid, using octadecanoic acid and 2,4-dinitrobenzenesulfonic acid groups as hydrophobic ends. It utilizes the characteristic of 2,4-dinitrobenzenesulfonic acid groups to open sulfonate bonds and release SO2 through redox mechanisms in the tumor tissue microenvironment. This enables rapid and targeted release of anticancer drugs in vivo, while increasing the ability of anticancer drugs to inhibit tumors. At the same time, the octadecanoic acid groups are used to increase the stability of micelles.

[0035] (4) In this invention, polyethylene glycol and hyaluronic acid are used as the main hydrophilic ends of the amphiphilic polymer carrier. Hyaluronic acid is the target, which binds to the CD44 receptor on the surface of tumor cells and enters the tumor cells through endocytosis, thus overcoming the problem of low cell uptake capacity of ordinary carrier micelles.

[0036] (5) The amphiphilic graft copolymer based on hyaluronic acid disclosed in this invention is simple and convenient to prepare, with a wide range of raw material sources, easy structure modification, and low requirements for preparation conditions. Attached Figure Description

[0037] Figure 1 This is a synthetic route diagram of the methoxy polyethylene glycol-hyaluronic acid-2,4-dinitrobenzenesulfonic acid-octadecanoic acid derivative in Example 1;

[0038] Figure 2 The infrared spectrum of the third product in Example 1;

[0039] Figure 3 The NMR spectrum is that of the third product in Example 1. Detailed Implementation

[0040] The specific embodiments of the present invention will be described in further detail below, but the present invention is not limited to these embodiments. Any improvements or substitutions based on the basic spirit of these embodiments shall still fall within the scope of protection claimed by the claims of the present invention.

[0041] The first object of the present invention is to provide: an amphiphilic graft copolymer based on hyaluronic acid, comprising:

[0042] (1) Hyaluronic acid as the parent skeleton;

[0043] (2) Methoxylated polyethylene glycol units grafted onto the carboxyl group of hyaluronic acid;

[0044] (3) A 2,4-dinitrobenzenesulfonic acid group grafted onto the first primary alcohol group of hyaluronic acid;

[0045] (4) An octadecanoic acid group grafted onto the second primary alcohol group of hyaluronic acid.

[0046] In this invention, the positions of the first primary alcohol group grafted with 2,4-dinitrobenzenesulfonic acid group, the carboxyl group grafted with methoxy polyethylene glycol unit, and the second primary alcohol group grafted with octadecanoic acid group on the skeleton are not particularly limited. The first primary alcohol group and the second primary alcohol group are only used to distinguish each other, and there are no restrictions on their order of appearance or importance on the skeleton.

[0047] In some preferred embodiments, the hyaluronic acid-based amphiphilic graft copolymer has the following structural formula:

[0048]

[0049] Where m is 7 to 323 (e.g., 50, 65, 80) and n is 12 to 1241 (e.g., 100, 150, 200).

[0050] In some preferred embodiments, the value of m is such that the average molecular weight of the methoxy polyethylene glycol unit is 370 to 20000 Da; for example, 100, 1500, 3000, 4000, 15000 or 20000 Da.

[0051] In some preferred embodiments, the value of n is such that the average molecular weight of the hyaluronic acid is 5100 to 500000 Da; for example, 20000, 25000, 30000 or 35000 Da.

[0052] A second objective of this invention is to provide a method for preparing the aforementioned amphiphilic graft copolymer based on hyaluronic acid, comprising the following steps:

[0053] (1) Dissolve hyaluronic acid in the first reaction solvent, add the first catalyst and mix evenly, stir for 0.5-24h (e.g. 0.5, 1, 2, 5, 8, 10, 12, 15, 18, 21 or 24h) at 0-60℃ (e.g. 0, 10, 20, 30, 40, 50 or 60℃), then add methoxy polyethylene glycol, stir and react for 2-96h (e.g. 2, 3, 6, 10, 14, 18, 24, 36, 72, 84 or 96h) at 0-60℃ (e.g. 0, 10, 20, 30, 40, 50 or 60℃), dialyze the product with distilled water and freeze-dry to obtain the first reaction product methoxy polyethylene glycol-hyaluronic acid derivative, denoted as mPEG-HA;

[0054] (2) Dissolve 2,4-dinitrobenzenesulfonic acid in the second reaction solvent, add the second catalyst and mix evenly, stir for 3-9 h (e.g. 3, 4, 6, 7, 8 or 9 h) at 20-100℃ (e.g. 20, 30, 40, 60, 80, 90 or 100℃), then add mPEG-HA dissolved in DMSO / pyridine mixed solution and continue stirring for 6-24 h (e.g. 6, 7, 9, 12, 18, 21 or 24 h) to obtain the second reaction product, freeze-dry the second reaction product to obtain the freeze-dried product methoxy polyethylene glycol-hyaluronic acid-2,4-dinitrobenzenesulfonic acid derivative, denoted as mPEG-HA-DNs;

[0055] (3) Dissolve octadecanoic acid in the third reaction solvent, add the third catalyst, and catalyze the reaction for 0.5-24h (e.g., 0.5, 1, 2, 5, 8, 10, 12, 15, 18, 21 or 24h) at 0-60℃ (e.g., 0, 10, 20, 30, 40, 50 or 60℃). Then add mPEG-HA-DNs and continue the reaction for 2-48h (e.g., 2, 3, 6, 10, 14, 18, 24, 36, 45 or 48h) to obtain the third reaction product. Freeze-dry the third reaction product to obtain the freeze-dried product methoxy polyethylene glycol-hyaluronic acid-2,4-dinitrobenzenesulfonic acid-octadecanoic acid derivative, denoted as mPEG-HA(SA)-DNs, which is an amphiphilic graft copolymer based on hyaluronic acid.

[0056] In some preferred embodiments, the first reaction solvent, the second reaction solvent, and the third reaction solvent are independently selected from at least one of formamide, sulfonium chloride, N,N-dimethylformamide, N,N-dimethylacetamide, water, DMSO, and pyridine.

[0057] In some preferred embodiments, the first catalyst and the third catalyst are independently selected from at least one of EDC·HCl, NHS, DMAP, and DCC.

[0058] In some other preferred embodiments, the first catalyst is preferably a composition of EDC·HCl and NHS; the molar ratio of EDC·HCl to NHS in the first catalyst is (1:10)-(10:1), for example, 1:2, 1:5, 1:10, 8:1, 5:1, 2:1 or 1:1; the molar ratio of the first catalyst to hyaluronic acid is (1:100)-(100:1), for example, 1:2, 1:5, 5:1, 2:1 or 1:1; the molar ratio of methoxy polyethylene glycol to hyaluronic acid is (1:10)-(100:1), for example, 1:2, 1:5, 1:8, 1:1, 2:1, 5:1, 6:1 or 8:1.

[0059] In some preferred embodiments, the second catalyst is N,N-dimethylformamide (DMF); the molar ratio of the second catalyst to 2,4-dinitrobenzenesulfonic acid is (1:20)-(20:1), for example, 1:2, 1:5, 5:1, 2:1 or 1:1; the molar ratio of 2,4-dinitrobenzenesulfonic acid to mPEG-HA is (1:10)-(110:1), for example, 1:2, 1:5, 1:10, 1:1, 2:1, 5:1, 6:1 or 8:1.

[0060] In some other preferred embodiments, the third catalyst is preferably a composition of EDC·HCl and NHS; the molar ratio of EDC·HCl to NHS in the third catalyst is (1:10)-(10:1), for example, 1:3, 1:6, 1:1, 8:1, 5:1 or 2:1; the molar ratio of the third catalyst to octadecanoic acid is (1:5)-(5:1), for example, 1:2, 1:5, 5:1, 2:1 or 1:1; the molar ratio of octadecanoic acid to mPEG-HA-DNs is (1:10)-(200:1), for example, 1:2, 1:5, 1:8, 1:1, 2:1, 5:1, 6:1 or 8:1.

[0061] In a specific embodiment, the amounts of the first reaction solvent and the third reaction solvent are at least the minimum amount required to fully dissolve hyaluronic acid; the amount of the second reaction solvent is at least the minimum amount required to fully dissolve 2,4-dinitrobenzenesulfonic acid; the amount of the third reaction solvent is at least the minimum amount required to fully dissolve octadecanoic acid; and the amount of the DMSO / pyridine mixed solution is at least the minimum amount required to fully dissolve the first product mPEG-HA, wherein DMSO and pyridine are in any volume ratio.

[0062] A third object of the present invention is to provide the use of the aforementioned hyaluronic acid-based amphiphilic graft copolymer or the hyaluronic acid-based amphiphilic graft copolymer prepared by the methods thereof in the preparation of anticancer drugs.

[0063] Furthermore, an amphiphilic graft copolymer based on hyaluronic acid is used as a coating material to encapsulate anticancer active ingredients, thereby forming an anticancer drug with tumor-targeted release.

[0064] The anticancer active ingredient is at least one of docetaxel, paclitaxel, epirubicin, and camptothecin.

[0065] Furthermore, a method for preparing an anticancer drug formulation based on an amphiphilic graft copolymer of hyaluronic acid includes the following steps:

[0066] (1) Dissolve paclitaxel in acetone solution to obtain paclitaxel solution;

[0067] (2) Using an ultrasonic method, paclitaxel solution was slowly added dropwise to a hyaluronic acid-based amphiphilic graft copolymer. After removing unencapsulated paclitaxel using a dialysis bag, the mixture was lyophilized to obtain an anticancer drug formulation.

[0068] In some other preferred embodiments, in step (1), the concentration of paclitaxel in the paclitaxel solution is 0.01 mg / mL to 10 mg / mL (e.g., 0.1, 0.5, 1.0, 5.0 mg / mL).

[0069] In some other preferred embodiments, in step (2), the ultrasound time is 1-8 min (e.g., 1, 2, 4, 7 or 8 min).

[0070] In some other preferred embodiments, in step (2), the mass ratio of the hyaluronic acid-based amphiphilic graft copolymer to paclitaxel is 1:20-20:1, for example, 1:7, 1:5, 1:2, 1:1, 2:1, 5:1 or 7:1.

[0071] The aforementioned hyaluronic acid-based amphiphilic graft copolymers, or those prepared by the same method, exhibit excellent biocompatibility and can be used for drug encapsulation. In the delivery of anticancer drugs, this invention utilizes active CD44 receptor targeting, EPR-mediated passive receptor targeting, and SO2 release as adjuvant therapy to enhance drug uptake in tumor tissues and increase therapeutic efficacy. Therefore, the novel materials of this invention have broad application prospects.

[0072] Example 1

[0073] A method for preparing an amphiphilic graft copolymer based on hyaluronic acid includes the following steps:

[0074] (1) Synthesis of the first product mPEG-HA

[0075] 88.3 mg EDC·HCl, 53.0 mg NHS, and 183 mg hyaluronic acid were weighed separately and placed in a round-bottom flask. The hyaluronic acid was dissolved in 50 mL of the first reaction solvent, and then EDC·HCl and NHS were added and mixed thoroughly. The mixture was stirred and activated at room temperature for 5 h. After the reaction was complete, 550 mg of methoxy polyethylene glycol was added, and the mixture was stirred at room temperature for another 14 h. After the reaction was completed, the mixture was dialyzed with distilled water and then freeze-dried to obtain the first product.

[0076] In the synthesis of the first product, the hyaluronic acid used had a molecular weight of 20,000, and the first reaction solvent was formamide; the methoxy polyethylene glycol had a molecular weight of 1,200.

[0077] (2) Synthesis of the second product mPEG-HA-DNs

[0078] Measure 240 mg of 2,4-dinitrobenzenesulfonic acid and 180 mg of the first product. In a round-bottom flask, dissolve 2,4-dinitrobenzenesulfonic acid in 9 mL of the second reaction solvent, add 1 mL of the second catalyst DMF, and stir at 60 °C for 5 h. Then, dissolve 180 mg of the first product in 61.5 mL of a DMSO / pyridine (60:1.5, v / v) mixed solution and slowly add it dropwise to the reaction solution under ice bath conditions. Stir at room temperature for 24 h, dialyze with distilled water for 48 h, and freeze dry to obtain the second product.

[0079] In the synthesis of the second product, the second reaction solvent is thionyl chloride, which, along with the second catalyst DMF, is used to activate the sulfonic acid group;

[0080] (3) Synthesis of the third product mPEG-HA(SA)-DNs

[0081] Weigh out 192 mg EDC·HCl, 115 mg NHS, and 190 mg octadecanoic acid, respectively. In a round-bottom flask, dissolve octadecanoic acid in 50 mL of the third reaction solvent, then add EDC·HCl and NHS and mix well. Stir and activate at room temperature for 10 h. After the reaction is complete, add 150 mg of the second product and continue stirring at room temperature for 14 h. After the reaction is complete, dialyze the mixture with distilled water and freeze-dry to obtain the third product.

[0082] In the synthesis of the third product, the solvent for the third reaction is dimethyl sulfoxide;

[0083] Finally, the inventors further performed infrared and nuclear magnetic resonance (NMR) analyses on the final product mPEG-HA(DNs)-SA, and the results are shown in [reference needed]. Figure 2 and 3 .

[0084] exist Figure 2 In the infrared spectrum shown, located at 1616 cm⁻¹ -1 The absorption peak at 1738 cm⁻¹ is attributed to the C=O (carboxyl group) absorption peak on hyaluronic acid (HA). -1 The absorption peak belongs to the C=O (ester carbonyl group) of the second product, while at 934 cm⁻¹... -1 The absorption peak at 2985 cm⁻¹ is the absorption peak of SO on DNs. -1 The absorption peak of the -CH2 stretching vibration is a characteristic absorption peak of SA, which proves that mPEG-HA(SA)-DNs have been successfully synthesized.

[0085] Example 2

[0086] A method for preparing an amphiphilic graft copolymer based on hyaluronic acid includes the following steps:

[0087] (1) Synthesis of the first product mPEG-HA

[0088] 94 mg of DCC and 183 mg of hyaluronic acid were weighed separately and placed in a round-bottom flask. The hyaluronic acid was dissolved in 50 mL of the first reaction solvent, and then DCC was added and mixed thoroughly. The mixture was stirred and activated at room temperature for 5 h. After the reaction was completed, 550 mg of methoxy polyethylene glycol was added and the mixture was stirred at room temperature for another 14 h. After the reaction was completed, the mixture was dialyzed with distilled water and then freeze-dried to obtain the first product.

[0089] In the synthesis of the first product, the hyaluronic acid used had a molecular weight of 20,000, and the first reaction solvent was formamide; the methoxy polyethylene glycol had a molecular weight of 1,200.

[0090] (2) Synthesis of the second product mPEG-HA-DNs

[0091] Measure 240 mg of 2,4-dinitrobenzenesulfonic acid and 180 mg of the first product. In a round-bottom flask, dissolve 2,4-dinitrobenzenesulfonic acid in 9 mL of the second reaction solvent, add 1 mL of the second catalyst DMF, and stir at 60 °C for 5 h. Dissolve 180 mg of the first product in 61.5 mL of a DMSO / pyridine (60:1.5, v / v) mixed solution and slowly add it dropwise to the reaction solution under ice bath conditions. Stir at room temperature for 24 h, dialyze with distilled water for 48 h, and freeze dry to obtain the second product.

[0092] In the synthesis of the second product, the second reaction solvent is thionyl chloride, which, along with the second catalyst DMF, is used to activate the sulfonic acid group;

[0093] (3) Synthesis of the third product mPEG-HA(SA)-DNs

[0094] Weigh out 207 mg DCC and 190 mg octadecanoic acid respectively; in a round-bottom flask, dissolve octadecanoic acid in 50 mL of the third reaction solvent, then add DCC and mix well, and stir to activate at room temperature for 10 h; after the reaction is complete, add 150 mg of the second product, and continue stirring at room temperature for 14 h; after the reaction is complete, dialyze the mixture with distilled water and freeze-dry to obtain the third product;

[0095] In the synthesis of the third product, the solvent for the third reaction is dimethyl sulfoxide.

[0096] Example 3

[0097] A method for preparing an amphiphilic graft copolymer based on hyaluronic acid includes the following steps:

[0098] (1) Synthesis of the first product mPEG-HA

[0099] Weigh out 56 mg DMAP and 183 mg hyaluronic acid respectively; in a round-bottom flask, dissolve hyaluronic acid in 50 mL of the first reaction solvent, then add DMAP and mix well, and stir to activate at room temperature for 5 h; after the reaction is complete, add 550 mg of methoxy polyethylene glycol, and continue stirring at room temperature for 14 h; after the reaction is complete, dialyze the mixture with distilled water and freeze-dry to obtain the first product;

[0100] In the synthesis of the first product, the hyaluronic acid used had a molecular weight of 20,000, and the first reaction solvent was formamide; the methoxy polyethylene glycol had a molecular weight of 1,200.

[0101] (2) Synthesis of the second product mPEG-HA-DNs

[0102] Measure 240 mg of 2,4-dinitrobenzenesulfonic acid and 180 mg of the first product; in a round-bottom flask, dissolve 2,4-dinitrobenzenesulfonic acid in 9 mL of the second reaction solvent, add 1 mL of the second catalyst, and stir at 60 °C for 5 h; dissolve 180 mg of the first product in 61.5 mL of a DMSO / pyridine (60:1.5, v / v) mixed solution, and slowly add it dropwise to the reaction solution under ice bath conditions; stir at room temperature for 24 h, dialyze with distilled water for 48 h, and freeze dry to obtain the second product;

[0103] In the synthesis of the second product, the second reaction solvent is thionyl chloride, which, along with the second catalyst DMF, is used to activate the sulfonic acid group;

[0104] (3) Synthesis of the third product mPEG-HA(SA)-DNs

[0105] Weigh out 122 mg DMAP and 190 mg octadecanoic acid respectively; in a round-bottom flask, dissolve octadecanoic acid in 50 mL of the third reaction solvent, then add DMAP and mix well, and stir to activate at room temperature for 10 h; after the reaction is complete, add 150 mg of the second product, and continue stirring at room temperature for 14 h; after the reaction is complete, dialyze the mixture with distilled water and freeze-dry to obtain the third product;

[0106] In the synthesis of the third product, the solvent for the third reaction is dimethyl sulfoxide.

[0107] Comparative Example 1

[0108] The same method as in Example 1 was used to proceed to step (1) to obtain mPEG-HA.

[0109] Comparative Example 2

[0110] Steps (1) and (3) were performed using the same method as in Example 1 to obtain mPEG-HA-SA.

[0111] Comparative Example 3

[0112] The same method as in Example 1 was used to proceed to step (2) to obtain mPEG-HA-DNs.

[0113] Comparative Example 4

[0114] Step (2) was performed using the same method as in Example 1, except that the first product was replaced with hyaluronic acid to obtain HA-DNs.

[0115] Application Examples 1-9

[0116] These application examples relate to the use of the hyaluronic acid-based amphiphilic graft copolymer of the present invention as an anticancer drug release carrier, wherein an anticancer drug release composition comprising the amphiphilic graft copolymer and an anticancer drug encapsulated by the amphiphilic graft copolymer is prepared. The anticancer drug release composition is nanoparticles containing mPEG-HA(SA)-DNs encapsulating paclitaxel (PTX). First, different masses of PTX are dissolved in 0.5 mL of acetone to prepare a PTX solution; separately, 10 mg of each of the mPEG-HA(SA)-DNs prepared in Examples 1 to 3 are dissolved in 10 mL of distilled water. Under ultrasonic conditions, the PTX solution is added dropwise, and ultrasonication is continued for 5 min to ensure that PTX fully enters the mPEG-HA(SA)-DNs. Then, the suspension is dialyzed using a dialysis bag (molecular weight cutoff: 3500 Da) to remove excess PTX. Finally, the dialysis product is lyophilized to obtain the anticancer drug release composition. Then, the particle size, encapsulation efficiency, and drug loading of the drug-loaded nanoparticles in the anticancer drug release composition, as well as the weight percentage of the anticancer drug in the anticancer drug release composition, were determined, and the results are shown in Table 1.

[0117] Comparative Application Example 1

[0118] The anticancer drug release composition was prepared using a method essentially the same as that used in Application Example 1, except that in Comparative Application Example 1, the mPEG-HA prepared in Comparative Example 1 was used instead of mPEG-HA(SA)-DNs.

[0119] Comparative Application Example 2

[0120] The anticancer drug release composition was prepared using a method that was essentially the same as that used in Application Example 1. The difference was that in Comparative Application Example 2, the mPEG-HA-SA prepared in Comparative Example 2 was used instead of mPEG-HA(SA)-DNs.

[0121] Comparative Application Example 3

[0122] The anticancer drug release composition was prepared using a method essentially the same as that used in Application Example 1, except that in Comparative Application Example 3, the mPEG-HA-DNs prepared in Comparative Example 3 were used instead of mPEG-HA(SA)-DNs.

[0123] Comparative Application Example 4

[0124] The anticancer drug release composition was prepared using a method essentially the same as that used in Application Example 1, except that in Comparative Application Example 4, the HA-DNs prepared in Comparative Example 4 were used instead of mPEG-HA(SA)-DNs.

[0125] Safety test

[0126] To evaluate the safety of the prepared anticancer drug-releasing composition for intravenous administration, an in vitro hemolysis test was conducted. Blood from the marginal ear vein of New Zealand white rabbits was collected, fibrinogen was removed, and the mixture was washed with physiological saline, centrifuged, and prepared into a 2% (v / v) suspension. 2.5 mL of the erythrocyte suspension and 2.5 mL of the anticancer drug-releasing composition (1 mg / mL) were mixed thoroughly. The mixture was incubated at 37°C for 4 hours, centrifuged at 3000 r / min for 10 min, and the supernatant was collected. The absorbance of the sample was measured at 540 nm. Physiological saline and distilled water were used in the same manner as negative and positive controls, respectively, and the hemolysis percentage was calculated. Hemolysis percentage = (sample absorbance - negative control absorbance) / (positive control absorbance - negative control absorbance) × 100%. The results are shown in Table 1 below.

[0127] Table 1. Anticancer drug encapsulation effect and safety of the prepared anticancer drug release composition

[0128]

[0129] The results showed that all formulations in Application Examples 1 to 9 had high encapsulation efficiency and drug loading, and the particle size was less than 200 nm. As the initial drug loading increased, the encapsulation efficiency decreased, while the particle size increased.

[0130] In addition, when the concentration of paclitaxel is in the range of 0-200 μg / mL, the hemolytic activity of the anticancer drug release composition is almost negligible, and its hemolytic percentage does not exceed 3.5%, indicating that the formulation has good hemolytic safety.

[0131] Antitumor efficacy trial:

[0132] A cervical cancer U14 tumor-bearing mouse model was established. The tumor-bearing mice were randomly divided into four groups of five mice each. Each group was labeled and numbered, and drug administration began. The treatment group received physiological saline, the anticancer drug-releasing composition (dose 10 mg / kg) prepared in Example 1, and the control groups in Examples 1 and 2. This day was designated as day 1 of drug administration. The formulation was administered via tail vein injection every three days for four consecutive cycles. After four drug administration cycles, the animals were sacrificed, the tumor masses were removed, weighed, and the tumor inhibition rate was calculated. Tumor inhibition rate = (average tumor weight in the saline group - average tumor weight in the treatment group) / average tumor weight in the saline group × 100%. The results showed that the tumor inhibition rate of the anticancer drug-releasing composition was 82.60%, thus demonstrating a significant inhibitory effect on cervical cancer tumor growth.

[0133] In addition, the inventors used H22 hepatocellular carcinoma mice in their experiments. First, the mice were randomly divided into 5 groups of 5 mice each, and each group was labeled and numbered. Then, drug administration began, with the control group receiving physiological saline, the treatment group receiving the anticancer drug-releasing composition (dose 10 mg / kg) prepared in Example 1 and Comparative Examples 1-4, respectively. This day was designated as Day 1 of drug administration. The drug was administered via tail vein injection every 3 days for a total of 4 cycles. After 4 drug administration cycles, the animals were sacrificed, the tumor was removed, weighed, and the tumor inhibition rate was calculated. The results showed that the average tumor inhibition rate of the anticancer drug-releasing composition prepared in Example 1 was 75.10%, thus demonstrating a significant inhibitory effect on hepatocellular carcinoma tumor growth.

[0134] In addition, Lewis lung cancer-bearing mice were used in the experiment. The mice were randomly divided into 5 groups of 5 mice each, and each group of animals was labeled and numbered. At the time of administration, the mice were given physiological saline, the anticancer drug-releasing composition prepared in Example 1, and the control group (Examples 1-4) (dose 10 mg / kg) (treatment group), and this day was designated as day 1 of administration. The drug was administered via tail vein injection every 3 days for a total of 4 cycles. After 4 administration cycles, the animals were sacrificed, the tumor was removed, weighed, and the tumor inhibition rate was calculated. The results showed that the average tumor inhibition rate of the anticancer drug-releasing composition prepared in Example 1 was 77.20%, thus demonstrating a significant inhibitory effect on lung cancer tumor growth.

[0135] Table 2. Tumor inhibition rate of anticancer drug release compositions

[0136]

[0137] As can be seen from the data results shown in Table 2 above, the anticancer drug release composition prepared in this invention has a significant inhibitory effect on U14 mouse tumor cells (tumor inhibition rate of 82.60%), and also has a significant inhibitory effect on other cancer cells such as liver cancer and lung cancer, with tumor inhibition rates of 75.10% and 77.20%, respectively.

[0138] Furthermore, this invention effectively reduces the dosage of anticancer drugs, which may be due to the adjuvant therapeutic effect and precise release of released sulfur dioxide. Therefore, while ensuring the tumor inhibition rate, the dosage of drugs is significantly reduced, greatly reducing drug side effects.

[0139] It should be noted that the above embodiments and test examples are only for further elaboration and understanding of the technical solutions of the present invention, and should not be construed as further limiting the technical solutions of the present invention. Inventions that do not highlight substantive features or make significant progress by those skilled in the art still fall within the protection scope of the present invention.

Claims

1. An amphiphilic graft copolymer based on hyaluronic acid, characterized in that: It includes: (1) Hyaluronic acid as the matrix; (2) methoxy polyethylene glycol units grafted onto the carboxyl groups of hyaluronic acid; (3) a 2,4-dinitrobenzenesulfonic acid group grafted onto the first primary alcohol group of hyaluronic acid; (4) an octadecanoic acid group grafted onto the second primary alcohol group of hyaluronic acid; The structural formula of the amphiphilic graft copolymer based on hyaluronic acid is as follows: ； Among them, m is 7~323, n is 12~1241; The average molecular weight of the methoxy polyethylene glycol unit is 370-20000 Da; The average molecular weight of the hyaluronic acid is 5100-500000 Da.

2. The method for preparing an amphiphilic graft copolymer based on hyaluronic acid according to claim 1, characterized in that: The steps include: (1) Dissolving hyaluronic acid in a first reaction solvent, adding a first catalyst and mixing evenly, stirring at 0-60° C. for 0.5-24 h, then adding methoxy polyethylene glycol, stirring at 0-60° C. for 2-96 h, dialyzing the product with distilled water and freeze-drying it to obtain a first reaction product, methoxy polyethylene glycol-hyaluronic acid derivative, which is referred to as mPEG-HA; (2) dissolving 2,4-dinitrobenzenesulfonic acid in a second reaction solvent, adding a second catalyst and mixing evenly, stirring at 20-100° C. for 3-9 hours, then adding mPEG-HA dissolved in a DMSO / pyridine mixed solution and continuing to stir and react for 6-24 hours to obtain a second reaction product, freeze-drying the second reaction product to obtain a freeze-dried product of methoxy polyethylene glycol-hyaluronic acid-2,4-dinitrobenzenesulfonic acid derivative, which is counted as mPEG-HA-DNs; (3) dissolving octadecanoic acid in a third reaction solvent, adding a third catalyst, and catalyzing the reaction at 0-60° C. for 0.5-24 h, then adding mPEG-HA-DNs, and continuing the reaction for 2-48 h to obtain a third reaction product, and freeze-drying the third reaction product to obtain a freeze-dried product of methoxy polyethylene glycol-hyaluronic acid-2,4-dinitrobenzenesulfonic acid-octadecanoic acid derivative, calculated as mPEG-HA(SA)-DNs, which is an amphiphilic graft copolymer based on hyaluronic acid.

3. The method for preparing an amphiphilic graft copolymer based on hyaluronic acid according to claim 2, characterized in that: The first reaction solvent, the second reaction solvent, and the third reaction solvent are independently selected from at least one of formamide, thionyl chloride, N,N-dimethylformamide, N,N-dimethylacetamide, water, DMSO, and pyridine; The first catalyst and the third catalyst are independently selected from at least one of EDC·HCl, NHS, DMAP, and DCC; The second catalyst is DMF.

4. The method for preparing an amphiphilic graft copolymer based on hyaluronic acid according to claim 3, characterized in that: The first catalyst is a composition of EDC·HCl and NHS; the molar ratio of EDC·HCl to NHS in the first catalyst is (1:10)-(10:1); the third catalyst is a composition of EDC·HCl and NHS; the molar ratio of EDC·HCl to NHS in the third catalyst is (1:10)-(10:1).

5. The method for preparing an amphiphilic graft copolymer based on hyaluronic acid according to claim 3, characterized in that: The molar ratio of the first catalyst to hyaluronic acid is (1:100)-(100:1); the molar ratio of the methoxy polyethylene glycol to hyaluronic acid is (1:10)-(100:1); the molar ratio of the second catalyst to 2,4-dinitrobenzenesulfonic acid is (1:20)-(20:1); the molar ratio of 2,4-dinitrobenzenesulfonic acid to mPEG-HA is (1:10)-(110:1); the molar ratio of the third catalyst to octadecanoic acid is (1:5)-(5:1); and the molar ratio of octadecanoic acid to mPEG-HA-DNs is (1:10)-(200:1).

6. Use of the amphiphilic graft copolymer based on hyaluronic acid as claimed in claim 1 or the amphiphilic graft copolymer based on hyaluronic acid obtained by the method according to any one of claims 2 to 5 in the preparation of anticancer drugs.

7. Use of the amphiphilic graft copolymer based on hyaluronic acid as claimed in claim 6 in the preparation of anticancer drugs, characterized in that: The anticancer active ingredients are encapsulated using amphiphilic graft copolymers based on hyaluronic acid as coatings to form anticancer drugs with tumor-targeted release.

8. The use of the amphiphilic graft copolymer based on hyaluronic acid as claimed in claim 7 in the preparation of anticancer drugs, characterized in that: The anticancer active ingredient is at least one of docetaxel, paclitaxel, epirubicin and camptothecin.

Images