Redox dual-responsive docetaxel targeted prodrug nanoparticles and preparation method thereof

Abstract

The present invention provides a redox dual-responsive docetaxel targeted prodrug nanoparticle and a preparation method thereof. The redox dual-responsive docetaxel targeted prodrug nanoparticle is self-assembled in an aqueous solution by a polymer represented by a general structural formula (I).

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a redox dual-responsive docetaxel targeted prodrug nanoparticle and a preparation method thereof. Background Art

[0002] Docetaxel is a commonly used chemotherapeutic drug in clinical practice. It is mainly used to treat advanced breast cancer, ovarian cancer, and non-small cell lung cancer. Docetaxel also has certain efficacy on head and neck cancer, small cell lung cancer, gastric cancer, pancreatic cancer, etc. Due to its unique polyene anticancer mechanism and broad-spectrum and efficient anticancer activity, docetaxel has become the most popular anticancer drug after doxorubicin and cisplatin. The pharmacological activity is higher than that of paclitaxel. In in vitro anti-tumor effect studies, the tumor inhibition effect of docetaxel has been confirmed to be 1.3 to 12 times that of paclitaxel.

[0003] Although docetaxel is effective against a variety of cancers, it has also encountered various inevitable difficulties during its clinical use. First, docetaxel has extremely poor water solubility and is almost insoluble in water. Therefore, when used clinically, ethanol and Tween 80 are needed to increase its water solubility, and Tween 80 can cause severe hypersensitivity reactions in patients. Therefore, patients need to undergo desensitization when receiving such drug treatment, which will aggravate the patient's pain to a certain extent and increase the burden on medical staff. Secondly, due to the lack of targeting to tumor tissues, such drugs often have serious toxic side effects on normal tissues and organs of patients when administered at therapeutic doses, such as neutropenia, bone marrow suppression, cardiotoxicity, and severe neurotoxicity, which limits the dosage of the drug, which restricts the clinical use of the drug. In order to overcome the above defects, researchers have done a lot of work in the past few decades, such as developing new cosolvents or developing new drug delivery systems.

[0004] With the rapid development of nanomaterials, the construction of nano drug delivery systems has attracted widespread attention in the research of anticancer drugs. Researchers have developed a variety of docetaxel nano drug delivery systems to improve the water solubility and in vivo process of docetaxel, but these nano drug delivery systems still have limitations, such as low drug loading, uncontrollable drug release, and insufficient targeting. How to construct a docetaxel nano drug delivery system with selective killing effect on tumor cells still faces great technical challenges. Summary of the invention

[0005] One of the purposes of the present invention is to provide a redox dual-responsive docetaxel targeted prodrug nanoparticle and a preparation method thereof, wherein the redox dual-responsive docetaxel targeted prodrug nanoparticle has a selective killing effect on tumor cells with high expression of biotin receptors.

[0006] To achieve this purpose, the present invention adopts the following technical solutions:

[0007] A redox dual-responsive docetaxel targeted prodrug nanoparticle, which is self-assembled in an aqueous solution by a polymer having a general structural formula (I)

[0008]

[0009] In one embodiment, the compound of formula (I) self-assembles in aqueous solution to form nanoparticles with a hydrated diameter of 60 to 200 nm.

[0010] In one embodiment, the preparation reaction formula of the compound of formula (I) is as follows:

[0011]

[0012] In one embodiment, the preparation method of the compound of formula (I) comprises the following steps:

[0013] (1) Preparation of amino hydroxyethyl starch HES-NH2:

[0014] Hydroxyethyl starch and 3-bromopropylamine hydrobromic acid are used as raw materials, reacted in an aqueous solution containing sodium hydroxide for 24 hours, then added to methanol for precipitation, centrifuged and separated, washed with methanol for 3 times, dissolved in ultrapure water, adjusted the pH to 5-6 with dilute hydrochloric acid, dialyzed in ultrapure water for 3 days, and freeze-dried to obtain amino hydroxyethyl starch;

[0015] (2) Preparation of 3,3'-diselenedipropionic acid-docetaxel conjugate DTX-DSeDPA:

[0016] Docetaxel DTX and 3,3'-diselenide dipropionate DSeDPA were used as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDCI was used as a condensation agent, and the mixture was heated to 40°C in dichloromethane for reaction for 24 hours. The reaction solution was then spin-dried, dissolved in dimethyl sulfoxide, dialyzed in ultrapure water for 3 days, and freeze-dried to obtain a 3,3'-diselenide dipropionate-docetaxel conjugate.

[0017] (3) Preparation of Hydroxyethyl Starch-Docetaxel Conjugate HES-SeSe-DTX:

[0018] Using the amino hydroxyethyl starch prepared in step (1) and the 3,3'-diselenidedipropionic acid-docetaxel conjugate prepared in step (2) as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as a condensation agent, 4-dimethylaminopyridine as a catalyst, and dimethyl sulfoxide as a solvent, heating to 50° C. for reaction for 24 hours, then cooling to room temperature, dialyzing in ultrapure water for 3 days, and freeze-drying to obtain a hydroxyethyl starch-docetaxel conjugate;

[0019] (4) Preparation of the compound of formula (I) Biotin-HES-SeSe-DTX:

[0020] The hydroxyethyl starch-docetaxel conjugate prepared in step (3) and D-biotin are used as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is used as a condensation agent, 4-dimethylaminopyridine is used as a catalyst, and dimethyl sulfoxide is used as a solvent. The mixture is heated to 50° C. for reaction for 24 hours, then cooled to room temperature, dialyzed in ultrapure water for 3 days, and freeze-dried to obtain a biotin-modified hydroxyethyl starch-docetaxel conjugate, i.e., a compound of formula (I).

[0021] A method for preparing redox dual-responsive docetaxel targeted prodrug nanoparticles comprises the following steps:

[0022] (1) Using hydroxyethyl starch and 3-bromopropylamine hydrobromic acid as raw materials, a nucleophilic substitution reaction occurs under alkaline conditions to prepare amino hydroxyethyl starch;

[0023] (2) using docetaxel and 3,3'-diselenidepropionic acid as raw materials, preparing 3,3'-diselenidepropionic acid-docetaxel conjugate by condensation reaction;

[0024] (3) using amino hydroxyethyl starch and 3,3'-diselenidedipropionic acid-docetaxel conjugate as raw materials, preparing hydroxyethyl starch-docetaxel conjugate by condensation reaction;

[0025] (4) Using hydroxyethyl starch-docetaxel conjugate and D-biotin as raw materials, a biotin-modified hydroxyethyl starch-docetaxel conjugate is prepared by condensation reaction. The conjugate can self-assemble in an aqueous solution to form nanoparticles.

[0026] In one embodiment, the molecular weight of the hydroxyethyl starch described in step (1) is 20kDa-450kDa, the degree of hydroxyethyl substitution is 0.4-0.6, the molar ratio of anhydroglucose unit, 3-bromopropylamine hydrobromic acid and sodium hydroxide in the hydroxyethyl starch is 1:1-2:7-9, and the amount of methanol used is 10 times the volume of the reaction solution.

[0027] In one embodiment, the molar ratio of docetaxel, 3,3'-diselenodipropionic acid and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in step (2) is 1:1.2-1.5:10-20.

[0028] In one embodiment, the mass ratio of the amino-hydroxyethyl starch and 3,3'-diselenide dipropionic acid-docetaxel conjugate in step (3) is 1:0.1-0.3, and the molar ratio of 3,3'-diselenide dipropionic acid-docetaxel conjugate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:8-16:8-16.

[0029] In one embodiment, the mass ratio of the hydroxyethyl starch-docetaxel conjugate and D-biotin in step (4) is 1:0.1-0.2, and the molar ratio of D-biotin, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:8-16:8-16.

[0030] Application of the redox dual-responsive docetaxel targeted prodrug nanoparticles prepared by any of the methods described above or the redox dual-responsive docetaxel targeted prodrug nanoparticles described in any of the methods described above in the field of tumors.

[0031] The redox dual-responsive docetaxel targeted prodrug nanoparticles have redox dual-responsive drug release characteristics.

[0032] The redox dual-responsive docetaxel targeted prodrug nanoparticles are targeted to tumor cells with high expression of biotin receptors.

[0033] The redox dual-responsive docetaxel targeted prodrug nanoparticles have a selective killing effect on tumor cells with high expression of biotin receptors.

[0034] In one embodiment, the preparation method of the compound of formula (I) is carried out according to the following steps:

[0035] (1) Preparation of amino hydroxyethyl starch (HES-NH2)

[0036] Hydroxyethyl starch (HES) and 3-bromopropylamine hydrobromic acid are used as raw materials, reacted in an aqueous solution containing sodium hydroxide for 24 hours, then added to methanol for precipitation, centrifuged and separated, washed with methanol for 3 times, dissolved in ultrapure water, adjusted the pH to 5-6 with dilute hydrochloric acid, dialyzed in ultrapure water for 3 days, and freeze-dried to obtain amino hydroxyethyl starch.

[0037] The molecular weight of hydroxyethyl starch is 20kDa-450kDa, the degree of hydroxyethyl substitution is 0.4-0.6, the molar ratio of anhydroglucose unit, 3-bromopropylamine hydrobromic acid and sodium hydroxide in hydroxyethyl starch is 1:1-2:7-9, and the amount of methanol used is 10 times the volume of the reaction solution.

[0038] (2) Preparation of 3,3'-diselenodipropionic acid-docetaxel conjugate (DTX-DSeDPA)

[0039] Docetaxel (DTX) and 3,3'-diselenodipropionic acid (DSeDPA) were used as raw materials, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) was used as a condensation agent. The reaction mixture was heated to 40°C in dichloromethane for 24 hours, and then the reaction solution was spin-dried, dissolved in dimethyl sulfoxide, dialyzed in ultrapure water for 3 days, and freeze-dried to obtain 3,3'-diselenodipropionic acid-docetaxel conjugate.

[0040] The molar ratio of docetaxel, 3,3'-diselenodipropionic acid and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:1.2-1.5:10-20.

[0041] (3) Preparation of hydroxyethyl starch-docetaxel conjugate (HES-SeSe-DTX)

[0042] The amino hydroxyethyl starch prepared in step (1) and the 3,3'-diselenodipropionic acid-docetaxel conjugate prepared in step (2) are used as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is used as a condensation agent, 4-dimethylaminopyridine (DMAP) is used as a catalyst, and dimethyl sulfoxide is used as a solvent. The mixture is heated to 50° C. for reaction for 24 hours, then cooled to room temperature, dialyzed in ultrapure water for 3 days, and freeze-dried to obtain a hydroxyethyl starch-docetaxel conjugate.

[0043] The mass ratio of amino hydroxyethyl starch to 3,3'-diselenodipropionic acid-docetaxel conjugate is 1:0.1-0.3, and the molar ratio of 3,3'-diselenodipropionic acid-docetaxel conjugate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:8-16:8-16.

[0044] (4) Preparation of the compound of formula (I) (Biotin-HES-SeSe-DTX)

[0045] The hydroxyethyl starch-docetaxel conjugate prepared in step (3) and D-biotin are used as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is used as a condensation agent, 4-dimethylaminopyridine is used as a catalyst, and dimethyl sulfoxide is used as a solvent. The mixture is heated to 50° C. for reaction for 24 hours, then cooled to room temperature, dialyzed in ultrapure water for 3 days, and freeze-dried to obtain a biotin-modified hydroxyethyl starch-docetaxel conjugate, i.e., a compound of formula (I).

[0046] The mass ratio of hydroxyethyl starch-docetaxel conjugate to D-biotin is 1:0.1-0.2, and the molar ratio of D-biotin, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:8-16:8-16.

[0047] The advantages of the present invention are:

[0048] The present invention first performs amino functional modification on hydroxyethyl starch, and then sequentially condenses the modified amino group with the carboxyl group in 3,3'-diselenidedipropionic acid-docetaxel conjugate and the carboxyl group in biotin to form amides; the present invention adopts condensation agent EDCI and catalyst DMAP to carry out condensation reaction, which can be carried out in one pot without pre-activation; the compound of formula (I) prepared by the present invention has a specific recognition effect on tumor cells with overexpression of biotin receptors due to the modification of biotin, and can selectively kill tumor cells with overexpression of biotin receptors.

[0049] In the present invention, the polysaccharide is firstly modified with amino functionalization, and then the molecules with carboxyl groups are successively condensed with the modified amino groups to form amides. Since the reaction activity of the amino groups and carboxyl groups to form amides is relatively high, the 3,3'-diselenidedipropionic acid-docetaxel conjugate and biotin in the present invention can be efficiently coupled to the amino-functionalized hydroxyethyl starch. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the amino hydroxyethyl starch prepared in Example 1 of the present invention.

[0051] Figure 2 This is the nuclear magnetic resonance hydrogen spectrum of the hydroxyethyl starch-docetaxel conjugate prepared in Example 3 of the present invention.

[0052] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of the compound of formula (I) prepared in Example 4 of the present invention.

[0053] Figure 4 The infrared spectra are of the amino hydroxyethyl starch prepared in Example 1 of the present invention, the hydroxyethyl starch-docetaxel conjugate prepared in Example 3, and the compound of formula (I) prepared in Example 4. DETAILED DESCRIPTION

[0054] See also Figure 1-4 The method of the present invention will be further described below in conjunction with specific embodiments.

[0055] Example 1: Preparation of amino-hydroxyethyl starch

[0056] Weigh 4.0g of hydroxyethyl starch 130 / 0.4 in a 100mL round-bottom flask, add 20mL of ultrapure water to dissolve, stir in an ice-water bath, add 32mL of NaOH aqueous solution (6.0mol / L), continue stirring in an ice-water bath for 30 minutes, then add 6.0g of 3-bromopropylamine hydrobromide, stir at room temperature for 24 hours. After the reaction is completed, add the reaction solution to 520mL of methanol, precipitate, centrifuge and separate the precipitate, wash it 3 times with methanol, then dissolve it with about 20mL of distilled water, adjust the pH to 5-6 with hydrochloric acid solution, dialyze in ultrapure water for 3 days, and freeze-dry to obtain a white solid with a yield of 72%.

[0057] The degree of substitution of the amino groups in the synthesized amino hydroxyethyl starch was characterized by hydrogen nuclear magnetic resonance spectroscopy (solvent: deuterated water). Figure 1 As shown, characteristic peaks of methylene in propylamine appeared in the H NMR spectrum of amino hydroxyethyl starch (a and b), indicating that the propylamine group was successfully coupled to the hydroxyethyl starch. By comparing with the integral of acetal hydrogen (c) in hydroxyethyl starch, the degree of substitution of the propylamine group was calculated to be 15%.

[0058] Example 2: Preparation of 3,3'-diselenidedipropionic acid-docetaxel conjugate

[0059] 103.7 mg of docetaxel was weighed into a 100 mL round-bottom flask, 10 mL of dichloromethane was added to dissolve it, and then 53.3 mg of diselenide dipropionic acid and 336.6 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added, and the mixture was heated to 40°C and refluxed with stirring for 24 hours. After the reaction was completed, the reaction solution was spin-dried, 5 mL of dimethyl sulfoxide was added to dissolve it, and it was dialyzed in ultrapure water for 3 days. After freeze-drying, 3,3'-diselenide dipropionic acid-docetaxel conjugate was obtained with a yield of 92%.

[0060] The synthesized 3,3'-diselenodipropionic acid-docetaxel conjugate was characterized by hydrogen nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. 1H NMR (400MHz, DMSO-D6): δ7.98(t,J=6.8Hz,2H),7.71(t,J=7.4Hz,1H),7.64 (dt,J=15.0,7.5Hz,2H),7.41(ddd,J=20.3,13.9,8.4Hz,3H),7.30(d,J=7.4 Hz,1H),7.19(t,J=7.1Hz,1H),6.20(m,1H),5.93–5.75(m,1H),5.41(d,J=7 .2Hz,1H),5.10(s,2H),4.91(d,J=9.2Hz,2H),4.52(s,1H),4.33(d,J=6.9Hz ,1H),4.04(s,1H),4.02(d,J=4.8Hz,2H),3.66(d,J=7.2Hz,1H),3.59–2.74 (m,8H),2.66(ddd,J=19.4,10.9,4.2Hz,1H),2.28(s,1H),2.23(d,J=14.5Hz ,3H),1.87(dd,J=14.7,6.2Hz,1H),1.74(s,3H),1.52(s,3H),1.38(s,3H), 1.35(s,3H),1.23(s,3H),1.01(s,3H),0.98(s,3H).HRMS(ESI):m / zof[M+H] + calculated for C 50 H 63 NO 17 Se2 1110.2499, found 1110.2520.

[0061] Example 3: Preparation of Hydroxyethyl Starch-Docetaxel Conjugate

[0062] 582 mg of amino hydroxyethyl starch was placed in a 100 mL round-bottom flask, 15 mL of dimethyl sulfoxide was added to dissolve, and then 64.2 mg of 3,3'-diselenidedipropionic acid-docetaxel coupling, 111.1 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 7.6 mg of 4-dimethylaminopyridine were added, and the mixture was heated to 50°C and stirred for 24 hours. After the reaction, the reaction solution was cooled to room temperature, dialyzed in ultrapure water for 3 days, and freeze-dried to obtain a dark yellow solid with a yield of 89%.

[0063] The synthesized hydroxyethyl starch-docetaxel conjugate was characterized by hydrogen nuclear magnetic resonance spectroscopy (solvent: deuterated dimethyl sulfoxide) and infrared spectroscopy. Figure 2As shown in FIG. 1 , in addition to the characteristic signal of hydroxyethyl starch, the H NMR spectrum of the hydroxyethyl starch-docetaxel conjugate also showed the signals of aromatic hydrogen (a) and saturated fatty hydrogen (b) in docetaxel, indicating that docetaxel was successfully coupled to hydroxyethyl starch. Figure 4 As shown in the figure, compared with amino hydroxyethyl starch, the infrared spectrum of hydroxyethyl starch-docetaxel conjugate is 1645 cm -1 and 1246cm -1 The peaks at 3,3'-dithiodipropionic acid-docetaxel were enhanced, which were attributed to the C=O stretching vibration and CN stretching vibration in the amide bond, indicating that 3,3'-diselenide dipropionic acid-docetaxel was successfully coupled with amino hydroxyethyl starch through the amide bond. The diselenide bond in the hydroxyethyl starch-docetaxel conjugate was cut by dithiothreitol, and the docetaxel loading was determined to be 9.3% by high performance liquid chromatography.

[0064] Example 4: Preparation of the compound of formula (I)

[0065] 104.9 mg of hydroxyethyl starch-docetaxel conjugate was weighed into a 50 mL round-bottom flask, 5 mL of dimethyl sulfoxide was added to dissolve, 79.8 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 5.0 mg of 4-dimethylaminopyridine and 10.4 mg of D-biotin were added, and the mixture was heated to 50° C. and stirred for 24 hours. After the reaction, the reaction solution was cooled to room temperature, dialyzed in ultrapure water for 3 days, and freeze-dried to obtain a dark yellow solid with a yield of 91%.

[0066] The synthesized compound of formula (I) was characterized by H NMR (solvent: deuterated dimethyl sulfoxide) and infrared spectroscopy, and the substitution degree of biotin was calculated by H NMR. Figure 3 As shown, in addition to the characteristic signals (a and b) of docetaxel, the characteristic signal (c) of biotin active hydrogen appeared in the H NMR spectrum of the compound of formula (I), indicating that the compound of formula (I) was successfully prepared. Figure 4 As shown, compared with the hydroxyethyl starch-docetaxel conjugate, the infrared spectrum of the compound of formula (I) at 1645 cm -1 and 1246cm -1 The peak at further enhanced, indicating that the hydroxyethyl starch-docetaxel conjugate was successfully coupled to biotin through an amide bond. By comparing the integral of the active hydrogen of hydroxyethyl starch (4.25-6.00 ppm) and the integral of the active hydrogen of biotin (6.25-6.50 ppm) in the H NMR spectrum, the substitution degree of biotin in the compound of formula (I) was calculated to be 10.9%. The diselenide bond was cut by dithiothreitol, and the docetaxel loading was determined to be 8.5% by high performance liquid chromatography.

[0067] Example 5: Particle size and potential measurement

[0068] Preparation of test solution: The hydroxyethyl starch-docetaxel conjugate prepared in Example 3 and the compound of formula (I) prepared in Example 4 were added into ultrapure water and dispersed by ultrasound for 5 minutes to obtain 10 mg / ml nanoparticle dispersions.

[0069] Particle size and potential measurement: Take 1 ml of each of the above-prepared nanoparticle dispersions and measure the particle size and potential using a laser particle size analyzer and a zeta potential analyzer.

[0070] As shown in Table 1, the hydration diameters of hydroxyethyl starch-docetaxel conjugate nanoparticles and nanoparticles of the compound of formula (I) are 124.4±4.6 nm and 135.2±5.4 nm, respectively, indicating that after modification with biotin, the particle size of the nanoparticles increases slightly. The polydispersity index is lower than 0.3, indicating that both nanoparticles have a narrow particle size distribution. The potential of the hydroxyethyl starch-docetaxel conjugate nanoparticles is positive, which is due to the modified amino group in the hydroxyethyl starch. After further modification of biotin on the amino group, the potential of the nanoparticles of the compound of formula (I) is nearly neutral.

[0071] Table 1. Particle size and potential of hydroxyethyl starch-docetaxel conjugate and compound of formula (I)

[0072] sample Particle size (nm) Polydispersity index Zeta potential (mV) Hydroxyethyl starch-docetaxel conjugate nanoparticles 124.4±4.6 0.213±0.023 5.4±0.2 Nanoparticles of the compound of formula (I) 135.2±5.4 0.198±0.036 0.4±0.2

[0073] Example 6: In vitro drug release evaluation

[0074] Preparation of pH 7.4 release solution: weigh 0.2 g potassium dihydrogen phosphate, 2.16 g disodium hydrogen phosphate dodecahydrate, and 5 g Tween-80, add ultrapure water to dissolve, and make up to 1000 ml, then adjust the pH to 7.4 with dilute hydrochloric acid solution.

[0075] Preparation of a pH 7.4 releasing solution containing 10 mM reduced glutathione: Take the pH 7.4 releasing solution prepared above, add reduced glutathione, and then adjust the pH to 7.4 with a sodium hydroxide solution to prepare a pH 7.4 releasing solution containing 10 mM reduced glutathione.

[0076] Preparation of a pH 7.4 releasing solution containing 0.1% hydrogen peroxide: Take the pH 7.4 releasing solution prepared above, add hydrogen peroxide, and prepare a pH 7.4 releasing solution containing 0.1% hydrogen peroxide.

[0077] In vitro drug release evaluation: 1 ml of 10 mg / ml of each nanoparticle dispersion prepared in Example 5 of the present invention was placed in a dialysis bag (molecular weight cutoff: 3500Da), then immersed in 30 ml of each release solution configured above, placed in a shaker for shaking at a temperature of 37°C and a speed of 150 rpm. 2 ml of release solution was taken out at 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 ​​hours, and 96 hours, and then supplemented with 2 ml of the corresponding blank release solution. The release experiment in each release solution was performed in parallel in three groups. The released solution taken out was tested and the cumulative release amount of docetaxel was calculated.

[0078] As shown in Table 2 and Table 3, the hydroxyethyl starch-docetaxel conjugate nanoparticles and the nanoparticles of the compound of formula (I) release the drug slowly in the pH 7.4 release solution, and the cumulative release amount in 96 hours is less than 11%, while the release rate of both is significantly improved in the pH 7.4 release solution containing 10mM reduced glutathione and the pH 7.4 release solution containing 0.1% hydrogen peroxide, and the cumulative release amount in 96 hours is higher than 80% and 50%, respectively, indicating that the hydroxyethyl starch-docetaxel conjugate nanoparticles and the nanoparticles of the compound of formula (I) have redox dual responsive drug release characteristics. Due to the high concentration of reduced glutathione and hydrogen peroxide in tumor cells, this release characteristic is conducive to the selective release of drugs by the above nanoparticles in tumor cells, improving the killing effect on tumor cells and reducing toxic side effects.

[0079] Table 2. Drug release from HES-docetaxel conjugate nanoparticles

[0080]

[0081] Table 3. Drug release of nanoparticles of compound of formula (I)

[0082]

[0083]

[0084] Example 7: Evaluation of cellular uptake

[0085] Preparation of test solution: The hydroxyethyl starch-docetaxel conjugate prepared in Example 3 and the compound of formula (I) prepared in Example 4 were respectively used to prepare dimethyl sulfoxide solutions with a docetaxel concentration of 2 mM, and then 10 mL of each solution was added with 2 mg of fluorescent probe Nile Red, stirred at room temperature overnight, dialyzed in ultrapure water for three days, centrifuged at 8000 rpm for 10 minutes, and the supernatant was taken and used as the mother solution. RPMI 1640 complete medium (containing 10% fetal bovine serum and 1% penicillin & streptomycin) was used as the diluent to prepare each nanoparticle test solution containing 1 μg / mL Nile Red.

[0086] Evaluation of cell uptake: Mouse breast cancer cells 4T1 and mouse embryonic fibroblasts NIH3T3 were seeded in 12-well plates at a density of 100,000 cells per well, cultured overnight to adhere to the wall, and then the culture medium was replaced with the above-prepared nanoparticle test solution (n=3), and cultured in a cell culture incubator at 37°C for 6 hours, then the culture medium was aspirated, the cells were washed three times with phosphate buffer (pH7.4, 6.7mM), trypsin was added to digest the cells, and then the cells were separated by centrifugation, and the cell uptake amount was measured by flow cytometry.

[0087] As shown in Tables 4 and 5, the uptake of nanoparticles of the compound of formula (I) by 4T1 cells (high expression of biotin receptor) was significantly higher than that of hydroxyethyl starch-docetaxel conjugate nanoparticles, while the uptake of nanoparticles of the compound of formula (I) by NIH3T3 cells (negative expression of biotin receptor) was comparable to that of hydroxyethyl starch-docetaxel conjugate nanoparticles, indicating that nanoparticles of the compound of formula (I) can be selectively taken up by tumor cells with high expression of biotin receptor, which is beneficial for selective killing of tumor cells with high expression of biotin receptor.

[0088] Table 4.4T1 cell uptake

[0089] sample Average fluorescence intensity of cells (au) Hydroxyethyl starch-docetaxel conjugate nanoparticles 134±12 Nanoparticles of compound of formula (I) 198±26

[0090] Table 5. NIH3T3 cell uptake

[0091] sample Average fluorescence intensity of cells (au) Hydroxyethyl starch-docetaxel conjugate nanoparticles 121±14 Nanoparticles of compound of formula (I) 115±13

[0092] Example 8: Evaluation of cell killing effect

[0093] Preparation of test solution: 2 mM free docetaxel dimethyl sulfoxide solution was prepared, and the 2 mM free docetaxel dimethyl sulfoxide solution and the hydroxyethyl starch-docetaxel conjugate with a docetaxel concentration of 2 mM prepared in Example 6 and the dimethyl sulfoxide solution of the compound of formula (I) were used as mother solutions, and RPMI 1640 complete medium (containing 10% fetal bovine serum and 1% penicillin & streptomycin) was used as diluent to prepare test solutions of each compound with docetaxel concentrations of 1 μM, 10 μM, 50 μM and 100 μM, respectively.

[0094] Prepare 5 mg / mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in phosphate buffer (pH 7.4, 6.7 mM) and filter through a 0.22 μm filter to obtain an MTT solution.

[0095] Evaluation of cell killing effect: 4T1 cells and NIH3T3 cells were seeded in 96-well plates at a density of 5000 cells per well, cultured overnight to adhere to the wall, and then the culture medium was replaced with the above-prepared test solution of each compound at different concentrations (n=5), cultured in a cell culture incubator at 37°C for 24 hours, and then 20 μL of MTT solution was added, and cultured in a cell culture incubator at 37°C for 4 hours, the culture medium was aspirated, 150 μL of dimethyl sulfoxide was added, and then the absorbance of each well at 490 was measured by a microplate reader, and the survival rate of the cells incubated with blank culture medium was calculated as 100%.

[0096] As shown in Tables 6 and 7, on 4T1 cells, the killing effect of hydroxyethyl starch-docetaxel conjugate was weaker than that of free docetaxel, and the killing effect of the compound of formula (I) was stronger than that of free docetaxel. On NIH3T3 cells, the killing effects of hydroxyethyl starch-docetaxel conjugate and the compound of formula (I) were both weaker than that of free docetaxel, and the killing effect of the compound of formula (I) on 4T1 cells was significantly higher than that on NIH3T3 cells, indicating that the compound of formula (I) had a selective killing effect on 4T1 cells with high expression of biotin receptors, which was mainly attributed to the redox dual-responsive drug release characteristics of the compound of formula (I) and its targeting effect on cells with high expression of biotin receptors.

[0097] Table 6.4T1 cell survival rate

[0098]

[0099] Table 7. NIH3T3 cell survival rate

[0100]

[0101] In the present invention, the polysaccharide is firstly modified with amino functionalization, and then the molecules with carboxyl groups are successively condensed with the modified amino groups to form amides. Since the reaction activity of the amino groups and carboxyl groups to form amides is relatively high, the 3,3'-diselenidedipropionic acid-docetaxel conjugate and biotin in the present invention can be efficiently coupled to the amino-functionalized hydroxyethyl starch.

[0102] The redox-responsive docetaxel targeted prodrug nanoparticles provided by the present invention have redox-responsive drug release characteristics, and have a targeting effect and a selective killing effect on tumor cells with high expression of biotin receptors. Specifically, the coupling of 3,3'-diselenodipropionic acid and docetaxel can make the conjugate have a redox-responsive drug release characteristic, remain relatively stable under normal physiological conditions, and selectively release drugs under the oxidation of high levels of hydrogen peroxide or the reduction of high levels of reduced glutathione in tumor cells.

[0103] The above-described embodiments are merely illustrative, and all equivalent changes and modifications made within the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims

1. A redox dual-responsive docetaxel targeted prodrug nanoparticle, characterized in that: The polymer with the general structural formula (I) is self-assembled in aqueous solution to form 2. The redox dual-responsive docetaxel targeted prodrug nanoparticles according to claim 1, characterized in that: The compound of formula (I) self-assembles in aqueous solution to form nanoparticles with a hydrated diameter of 60 to 200 nm.

3. The redox dual-responsive docetaxel targeted prodrug nanoparticles according to claim 1, characterized in that: The preparation reaction formula of the compound of formula (I) is as follows:

4. The method for preparing redox dual-responsive docetaxel targeted prodrug nanoparticles according to claim 3, characterized in that: The preparation method of the compound of formula (I) comprises the following steps: (1) Preparation of amino hydroxyethyl starch HES-NH2: Hydroxyethyl starch and 3-bromopropylamine hydrobromic acid are used as raw materials, reacted in an aqueous solution containing sodium hydroxide for 24 hours, then added to methanol for precipitation, centrifuged and separated, washed with methanol for 3 times, dissolved in ultrapure water, adjusted the pH to 5-6 with dilute hydrochloric acid, dialyzed in ultrapure water for 3 days, and freeze-dried to obtain amino hydroxyethyl starch; (2) Preparation of 3,3'-diselenedipropionic acid-docetaxel conjugate DTX-DSeDPA: Docetaxel DTX and 3,3'-diselenide dipropionate DSeDPA were used as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDCI was used as a condensation agent, and the mixture was heated to 40°C in dichloromethane for reaction for 24 hours. The reaction solution was then spin-dried, dissolved in dimethyl sulfoxide, dialyzed in ultrapure water for 3 days, and freeze-dried to obtain a 3,3'-diselenide dipropionate-docetaxel conjugate. (3) Preparation of Hydroxyethyl Starch-Docetaxel Conjugate HES-SeSe-DTX: Using the amino hydroxyethyl starch prepared in step (1) and the 3,3'-diselenidedipropionic acid-docetaxel conjugate prepared in step (2) as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as a condensation agent, 4-dimethylaminopyridine as a catalyst, and dimethyl sulfoxide as a solvent, heating to 50° C. for reaction for 24 hours, then cooling to room temperature, dialyzing in ultrapure water for 3 days, and freeze-drying to obtain a hydroxyethyl starch-docetaxel conjugate; (4) Preparation of the compound of formula (I) Biotin-HES-SeSe-DTX: The hydroxyethyl starch-docetaxel conjugate prepared in step (3) and D-biotin are used as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is used as a condensation agent, 4-dimethylaminopyridine is used as a catalyst, and dimethyl sulfoxide is used as a solvent. The mixture is heated to 50° C. for reaction for 24 hours, then cooled to room temperature, dialyzed in ultrapure water for 3 days, and freeze-dried to obtain a biotin-modified hydroxyethyl starch-docetaxel conjugate, i.e., a compound of formula (I).

5. A method for preparing redox dual-responsive docetaxel targeted prodrug nanoparticles, comprising the following steps: (1) Using hydroxyethyl starch and 3-bromopropylamine hydrobromic acid as raw materials, a nucleophilic substitution reaction occurs under alkaline conditions to prepare amino hydroxyethyl starch; (2) using docetaxel and 3,3'-diselenidepropionic acid as raw materials, preparing 3,3'-diselenidepropionic acid-docetaxel conjugate by condensation reaction; (3) using amino hydroxyethyl starch and 3,3'-diselenidedipropionic acid-docetaxel conjugate as raw materials, preparing hydroxyethyl starch-docetaxel conjugate by condensation reaction; (4) Using hydroxyethyl starch-docetaxel conjugate and D-biotin as raw materials, a biotin-modified hydroxyethyl starch-docetaxel conjugate is prepared by condensation reaction. The conjugate can self-assemble in an aqueous solution to form nanoparticles.

6. The method for preparing redox dual-responsive docetaxel targeted prodrug nanoparticles according to claim 5, characterized in that: The molecular weight of the hydroxyethyl starch described in step (1) is 20kDa-450kDa, the degree of hydroxyethyl substitution is 0.4-0.6, the molar ratio of anhydroglucose unit, 3-bromopropylamine hydrobromic acid and sodium hydroxide in the hydroxyethyl starch is 1:1-2:7-9, and the amount of methanol used is 10 times the volume of the reaction solution.

7. The method for preparing redox dual-responsive docetaxel targeted prodrug nanoparticles according to claim 5, characterized in that: The molar ratio of docetaxel, 3,3'-diselenodipropionic acid and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in step (2) is 1:1.2-1.5:10-20.

8. The method for preparing redox dual-responsive docetaxel targeted prodrug nanoparticles according to claim 5, characterized in that: The mass ratio of the amino hydroxyethyl starch and 3,3'-diselenodipropionic acid-docetaxel conjugate described in step (3) is 1:0.1-0.3, and the molar ratio of the 3,3'-diselenodipropionic acid-docetaxel conjugate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:8-16:8-16.

9. The method for preparing redox dual-responsive docetaxel targeted prodrug nanoparticles according to claim 5, characterized in that: The mass ratio of the hydroxyethyl starch-docetaxel conjugate to D-biotin in step (4) is 1:0.1-0.2, and the molar ratio of D-biotin, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:8-16:8-16.

10. Use of the redox dual-responsive docetaxel targeted prodrug nanoparticles prepared according to the method according to any one of claims 5 to 8 or the redox dual-responsive docetaxel targeted prodrug nanoparticles according to any one of claims 1 to 4 in the preparation of anti-tumor drugs.

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