PH-responsive ferrocene-modified oxidized dextran micelle and preparation method thereof

Abstract

The invention discloses a pH-responsive ferrocene-modified oxidized dextran micelle and a preparation method thereof, and relates to the technical field of biological materials and nano-medicines. The structural general formula of the pH-responsive ferrocene modified oxidized dextran micelle is as follows: Fc-ODex. The preparation method comprises the following steps: carrying out a Schiff base reaction on N-(4-aminobutyl) ferrocene formamide and oxidized dextran to obtain an Fc-ODex polymer, and then obtaining the pH-responsive ferrocene modified oxidized dextran micelle by using a dialysis method. The pH-responsive ferrocene-modified oxidized dextran micelle prepared by the method provided by the invention has stable chemical properties and good biocompatibility, and can be subjected to Fenton reaction with high active oxygen in tumor cells so as to cause ferroptosis to cooperate with chemotherapeutic drugs to treat tumors; the problems of insufficient targeting, complex preparation process and uncontrollable drug release in the prior art are effectively solved.

Description

Technical Field

[0001] The present invention relates to the technical fields of biomaterials and nano-medicine, and particularly relates to a pH-responsive ferrocene-modified oxidized dextran micelle and a preparation method thereof. Background Art

[0002] Cancer, also known as malignant tumor, is a disease of malignant cell hyperplasia caused by chemical substances, physical factors, viruses, genetics and other factors, ranking first or second in the total human mortality rate. There are various treatment methods for cancer. Among them, chemotherapy is the main method. Early chemotherapy drugs with specific cytotoxicity to cancer cells generally have disadvantages such as large toxic side effects, poor water solubility, and lack of specific recognition. Targeted drug delivery systems are advanced drug delivery systems that selectively deliver drugs to specific target sites such as diseased tissues and organs through carriers while keeping healthy tissues unaffected. This drug delivery system significantly improves the efficacy of drugs and reduces side effects.

[0003] In 2003, researchers discovered a new anti-cancer compound, erastin, which can selectively cause the death of tumor cells. And the mode of death is significantly different from apoptosis and autophagy. In 2012, this mode of death was named ferroptosis. Ferroptosis is an iron-dependent programmed cell death mode, and the pathways leading to it involve multiple complex biochemical processes, such as the accumulation of reactive oxygen species, lipid peroxidation, and iron metabolism pathways. In cancer treatment, ferroptosis has become a research hotspot that has attracted much attention. Some chemotherapy drugs and targeted drug delivery systems can kill tumor cells by inducing ferroptosis, especially for cancer cells that are resistant to traditional apoptotic pathways. In addition, ferroptosis can also act synergistically with traditional treatment methods such as chemotherapy, radiotherapy, and immunotherapy to improve the treatment effect.

[0004] Dextran widely exists in nature, in the mucus secreted by many microorganisms during growth, and also in the cell walls of plants (such as algae, seaweeds), fungi, and certain bacteria. It is a neutral polysaccharide composed of glucose monomers. In medicine, dextran is a natural hydrophilic polysaccharide with good biodegradability, biocompatibility, and multifunctionality that is easy to chemically modify. Due to the above characteristics, dextran is widely used in the field of drug targeted delivery. However, as a targeted drug delivery carrier, it has limitations in actual application, such as insufficient targeting, complex preparation processes, and uncontrollable drug release. Summary of the Invention

[0005] In view of the above deficiencies in the prior art, the present invention provides a pH-responsive ferrocene-modified oxidized dextran micelle and a preparation method thereof. The pH-responsive ferrocene-modified oxidized dextran micelle prepared by the method provided by the present invention has stable chemical properties and good biocompatibility, effectively solving the problems of insufficient targeting, complex preparation process and uncontrollable drug release existing in the prior art.

[0006] To achieve the above object, the technical solution adopted by the present invention to solve its technical problems is: to provide a pH-responsive ferrocene-modified oxidized dextran micelle, and its structural formula is:

[0007]

[0008] Among them, n is 37-123.

[0009] Further, the molecular weight of dextran is 6000-20000 Da.

[0010] Further, the general structural formula of the pH-responsive ferrocene-modified oxidized dextran micelle is: Fc-ODex, where ODex is oxidized dextran and Fc is ferrocene.

[0011] Further, the structural formula of the pH-responsive ferrocene-modified oxidized dextran micelle is shown as follows.

[0012]

[0013] The preparation method of the above-mentioned pH-responsive ferrocene-modified oxidized dextran micelle includes the following steps:

[0014] S1. Dissolve dextran in deionized water, drop in sodium periodate, stir, dialyze the mixed solution, and freeze-dry to obtain oxidized dextran;

[0015] S2. Dissolve the oxidized dextran obtained in step S1 in an aqueous solution of hydroxylamine hydrochloride containing methyl orange, let it stand, titrate with sodium hydroxide solution, and perform blank correction;

[0016] S3. Add ferrocene formic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and triethylamine to dichloromethane, stir and activate, then add tert-butyl(4-aminobutyl)carbamate and 4-dimethylaminopyridine to react, add water to quench the reaction, extract, wash, dry and filter and spin-dry, and perform column separation to obtain N-(4-tert-butoxycarbonylamino)ferrocenecarboxamide;

[0017] S4. Dissolve the N-(4-tert-butoxycarbonylamino)ferrocenecarboxamide obtained in step S3 in dichloromethane, add trifluoroacetic acid, stir, add water to stop the reaction, adjust the pH value after extraction and then extract, dry and filter and spin-dry to obtain N-(4-aminobutyl)ferrocenecarboxamide;

[0018] S5. Dissolve the oxidized dextran obtained in step S1 and N-(4-aminobutyl)ferrocenecarboxamide obtained in step S4 in dimethyl sulfoxide. After the reaction, wash with ice-cold methyl tert-butyl ether and freeze-dry to obtain a pH-responsive ferrocene-modified oxidized dextran micelle polymer, and then obtain pH-responsive ferrocene-modified oxidized dextran micelles (Fc-ODex) by dialysis.

[0019] Furthermore, in step S1, the mass-volume ratio of dextran, sodium periodate and deionized water is 5 - 6 g: 0.9 - 0.99 g: 45 - 50 mL.

[0020] Furthermore, in step S1, the mass-volume ratio of dextran, sodium periodate and deionized water is 6 g: 0.99 g: 50 mL.

[0021] Furthermore, in step S1, stir and react overnight in the dark, then transfer the mixed solution to a dialysis bag with a MWCO of 3500 Da and dialyze for 5 - 6 days.

[0022] Furthermore, in step S1, stir and react overnight in the dark, then transfer the mixed solution to a dialysis bag with a MWCO of 3500 Da and dialyze for 5 days.

[0023] Furthermore, in step S1, freeze-dry at -60 °C for 3 days.

[0024] Furthermore, in step S1, the oxidized dextran is a white pine needle-like solid.

[0025] Furthermore, in step S2, dissolve 1.47 g of sodium hydroxide in 250 mL of pure water to prepare a sodium hydroxide solution. Dissolve 0.1 g of potassium hydrogen phthalate in 25 mL of pure water to prepare a potassium hydrogen phthalate solution and add two drops of phenolphthalein. Take a 10 mL burette and titrate the potassium hydrogen phthalate solution with the sodium hydroxide solution to measure the concentration of the sodium hydroxide solution as 0.0929 mol / L. Dissolve the oxidized dextran in a hydrochloric hydroxylamine aqueous solution containing methyl orange, let it stand and then titrate the red color to yellow with the sodium hydroxide solution and perform a blank correction.

[0026] Furthermore, in step S2, the mass-volume ratio of oxidized dextran to hydrochloric hydroxylamine aqueous solution is 4.13 - 402.5 mg: 25 - 30 mL.

[0027] Furthermore, in step S2, the concentration of methyl orange in the hydrochloric hydroxylamine aqueous solution is 0.02 - 0.04 mg / mL.

[0028] Furthermore, in step S2, the concentration of methyl orange in the hydrochloric hydroxylamine aqueous solution is 0.03 mg / mL.

[0029] Further, in step S2, leave it to stand for 2.5 - 3.5 h.

[0030] Further, in step S2, leave it to stand for 3 h.

[0031] Further, in step S3, the mass - volume ratio of ferrocene carboxylic acid, 1-(3 - dimethylaminopropyl)-3 - ethylcarbodiimide hydrochloride, triethylamine, tert - butyl(4 - aminobutyl)carbamate, 4 - dimethylaminopyridine and dichloromethane is 3 - 5 g:3 - 4 g:1 - 2 g:3 - 4 g:0.2 - 0.3 g:35 - 45 mL.

[0032] Further, in step S3, the mass - volume ratio of ferrocene carboxylic acid, 1-(3 - dimethylaminopropyl)-3 - ethylcarbodiimide hydrochloride, triethylamine, tert - butyl(4 - aminobutyl)carbamate, 4 - dimethylaminopyridine and dichloromethane is 4 g:3.67 g:1.94 g:3.6 g:0.23 g:40 mL.

[0033] Further, the dichloromethane is at 0 °C.

[0034] Further, in step S3, stir and activate for 25 - 35 min and then react for 24 - 25 h, add 35 - 45 mL of water to quench, extract with dichloromethane 3 - 4 times, wash the combined organic layer with saturated brine 2 - 3 times, and add anhydrous magnesium sulfate to dry overnight.

[0035] Further, in step S3, stir and activate for 30 min and then react for 24 h, add 40 mL of water to quench, extract with dichloromethane 3 times, wash the combined organic layer with saturated brine 2 times, and add anhydrous magnesium sulfate to dry overnight.

[0036] Further, in step S4, the mass - volume ratio of N-(4 - tert - butoxycarbonylamino)ferrocenecarboxamide, dichloromethane and trifluoroacetic acid is 2 - 3 g:25 - 30 mL:5 - 10 mL.

[0037] Further, in step S4, the mass - volume ratio of N-(4 - tert - butoxycarbonylamino)ferrocenecarboxamide, dichloromethane and trifluoroacetic acid is 2 g:25 mL:5 mL.

[0038] Further, in step S4, adjust the pH to 9.

[0039] Further, in step S4, stir in an ice - water bath, gradually warm up to room temperature and react for 12 - 13 h, add water to stop the reaction, extract with saturated brine 2 - 3 times, adjust the pH value with sodium hydroxide, and then extract with dichloromethane 3 - 4 times, and add anhydrous magnesium sulfate to dry.

[0040] Further, in step S4, stir under an ice-water bath, gradually raise the temperature to room temperature and react for 12 h, add water to stop the reaction, extract twice with saturated brine, adjust the pH value with sodium hydroxide, and then extract three times with dichloromethane, and add anhydrous magnesium sulfate for drying.

[0041] Further, in step S5, the mass-to-substance-to-volume ratio of oxidized dextran, N-(4-aminobutyl)ferrocenecarboxamide, and dimethyl sulfoxide is 0.8 - 0.9 g: 0.2 - 0.3 g: 25 - 30 mL.

[0042] Further, in step S5, the mass-to-substance-to-volume ratio of oxidized dextran, N-(4-aminobutyl)ferrocenecarboxamide, and dimethyl sulfoxide is 0.8 g: 0.29 g: 25 mL.

[0043] Further, in step S5, react at 49 - 51 °C for 48 - 50 h and then wash 3 - 4 times with ice-cold methyl tert-butyl ether.

[0044] Further, in step S5, react at 50 °C for 48 h and then wash 3 times with ice-cold methyl tert-butyl ether.

[0045] Further, in step S5, when the pH-responsive ferrocene-modified oxidized dextran micelle is a blank micelle, the dialysis method includes the following steps: Dissolve 30 - 32 mg of the pH-responsive ferrocene-modified oxidized dextran micelle polymer in 2 - 3 mL of dimethyl sulfoxide and stir overnight, add 8 - 9 mL of water, then transfer to a dialysis bag with a molecular weight cut-off of 3500 Da, dialyze for 24 - 25 h, and freeze-dry.

[0046] Further, in step S5, when the pH-responsive ferrocene-modified oxidized dextran micelle is a blank micelle, the dialysis method includes the following steps: Dissolve 30 mg of the pH-responsive ferrocene-modified oxidized dextran micelle polymer in 2 mL of dimethyl sulfoxide and stir overnight, add 8 mL of water, then transfer to a dialysis bag with a molecular weight cut-off of 3500 Da, dialyze for 24 h, and freeze-dry.

[0047] Further, in step S5, when the pH-responsive ferrocene-modified oxidized dextran micelle polymer is a drug-loaded micelle, the dialysis method includes the following steps: Dissolve 30 - 32 mg of the pH-responsive ferrocene-modified oxidized dextran micelle polymer in 2 - 3 mL of DMSO, add 3 - 4 mg of doxorubicin hydrochloride, stir overnight, add 8 - 9 mL of water, then transfer to a dialysis bag with a molecular weight cut-off of 3500 Da, dialyze for 24 - 25 h, and freeze-dry.

[0048] Further, in step S5, when the pH-responsive ferrocene-modified oxidized dextran micelle polymer is a drug-loaded micelle, the dialysis method includes the following steps: Dissolve 30 mg of the pH-responsive ferrocene-modified oxidized dextran micelle polymer in 2 mL of DMSO, add 3 mg of doxorubicin hydrochloride, stir overnight, add 8 mL of water, then transfer it to a dialysis bag with a molecular weight cut-off of 3500 Da, dialyze for 24 h, and freeze-dry.

[0049] Furthermore, the synthesis route of a preparation method of a pH-responsive ferrocene-modified oxidized dextran micelle provided by the present invention is as follows.

[0050]

[0051] The present invention has the following beneficial effects:

[0052] 1. For the pH-responsive ferrocene-modified oxidized dextran micelle prepared by the present invention, dextran, which is common in nature and has good biocompatibility, is selected as the hydrophilic phase, and N-(4-aminobutyl) ferrocenecarboxamide is used as the hydrophobic phase, and they are connected together through a Schiff base reaction. Among them, the Schiff base structure in the micelle breaks the bond in the acidic environment inside and outside the tumor cells, so as to achieve targeted controlled release of the encapsulated drug.

[0053] 2. The ferrocene structure contained in the pH-responsive ferrocene-modified oxidized dextran micelle prepared by the present invention is an iron-containing organic compound with stable chemical properties and good biocompatibility. It can undergo a Fenton reaction with the highly reactive oxygen in tumor cells, resulting in ferroptosis and synergistically treating tumors with chemotherapeutic drugs. Description of the Drawings

[0054] Figure 1 is the nuclear magnetic resonance spectrum of oxidized dextran;

[0055] Figure 2 is the nuclear magnetic resonance spectrum of N-(4-tert-butoxycarbonylamino) ferrocenecarboxamide;

[0056] Figure 3 is the nuclear magnetic resonance spectrum of the pH-responsive ferrocene-modified oxidized dextran micelle polymer;

[0057] Figure 4 is the nuclear magnetic resonance comparison spectrum of oxidized dextran, Fc-ODex and N-(4-aminobutyl) ferrocenecarboxamide;

[0058] Figure 5 is the infrared comparison spectrum of Fc-ODex, oxidized dextran and dextran;

[0059] Figure 6 is the Tyndall effect diagram of the pH-responsive ferrocene-modified oxidized dextran micelle;

[0060] Figure 7 HPLC comparative spectra of Fc-ODex, A-Fc-ODex, and N-(4-tert-butoxycarbonylamino) ferrocenecarboxamide hydrochloride (A-Fc-NH3);

[0061] Figure 8 Particle size comparison diagrams of Fc-ODex at different pH values. Specific implementation manners

[0062] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. For those not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0063] Example 1

[0064] A pH-responsive ferrocene-modified oxidized dextran micelle (Fc-ODex), and the molecular weight of the above dextran is 20000 Da.

[0065] The preparation method of the above pH-responsive ferrocene-modified oxidized dextran micelle includes the following steps:

[0066] S1. Dissolve 6 g of dextran with a molecular weight of 20000 Da in 50 mL of deionized water, slowly add 0.99 g of sodium periodate dropwise, stir and react overnight in the dark, then transfer the mixed solution into a dialysis bag with a MWCO of 3500 Da, dialyze for 5 - 6 days, and freeze-dry at -60 °C for 3 days to obtain oxidized dextran. The 1H NMR spectrum of the oxidized dextran is as Figure 1 shown;

[0067] S2. Dissolve 1.47 g of sodium hydroxide in 250 mL of pure water to prepare a sodium hydroxide solution. Dissolve 0.1 g of potassium hydrogen phthalate in 25 mL of pure water to prepare a potassium hydrogen phthalate solution and add two drops of phenolphthalein. Take a 10 mL burette for alkali, titrate the potassium hydrogen phthalate solution with the sodium hydroxide solution, and measure the concentration of the sodium hydroxide solution to be 0.0929 mol / L. Take 402.5 mg of oxidized dextran with a molecular weight of 20000 Da in 25 mL of 0.25 mol / L hydroxylamine hydrochloride aqueous solution containing 3 drops of 0.05% methyl orange, and let it stand for 3 h; titrate the red color to yellow with the sodium hydroxide solution and make a blank correction. The aldehyde group content C (aldehyde group content of oxidized dextran) of the oxidized dextran with a molecular weight of 20000 Da is measured to be 0.8027 mmol / g;

[0068] S3. Add 4 g of ferrocene carboxylic acid, 3.67 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 1.94 g of triethylamine to 40 mL of dichloromethane at 0 °C. Stir and activate for 30 min, then add 3.6 g of tert-butyl (4-aminobutyl) carbamate and 0.23 g of 4-dimethylaminopyridine, and react at room temperature for 24 h. Add 40 mL of water to quench the reaction, extract with dichloromethane 3 times, wash the combined organic layer with saturated brine 2 times, dry over anhydrous magnesium sulfate overnight, filter and rotary evaporate to dryness, and perform column chromatography separation to obtain N-(4-tert-butoxycarbonylaminomethyl)ferrocene carboxamide;

[0069] S4. Dissolve 2 g of the N-(4-tert-butoxycarbonylaminomethyl)ferrocene carboxamide obtained in step S3 in 25 mL of dichloromethane, add 5 mL of trifluoroacetic acid, stir in an ice-water bath, gradually warm up to room temperature and react for 12 h, add water to stop the reaction, extract with saturated brine 2 times, adjust the pH value to 9 with sodium hydroxide, then extract with dichloromethane 3 times, add anhydrous magnesium sulfate to dry and filter and rotary evaporate to dryness to obtain N-(4-aminobutyl)ferrocene carboxamide. The nuclear magnetic resonance hydrogen spectrum of N-(4-aminobutyl)ferrocene carboxamide is as follows Figure 2 shown;

[0070] S5. Dissolve 0.8 g of the oxidized dextran obtained in step S1 and 0.29 g of the N-(4-aminobutyl)ferrocene carboxamide obtained in step S4 in 25 mL of dimethyl sulfoxide, react at 50 °C for 48 h, then wash with ice-cold methyl tert-butyl ether 3 times, and freeze-dry to obtain a pH-responsive ferrocene-modified oxidized dextran micelle polymer (Fc-ODex). The nuclear magnetic resonance spectrum of the above pH-responsive ferrocene-modified oxidized dextran micelle polymer is as follows Figure 3 shown.

[0071] Example 2

[0072] A pH-responsive ferrocene-modified oxidized dextran micelle (Fc-ODex), wherein the molecular weight of the above dextran is 6000 Da.

[0073] The preparation method of the above pH-responsive ferrocene-modified oxidized dextran micelle comprises the following steps:

[0074] S1. Dissolve 5 g of dextran with a molecular weight of 6000 Da in 45 mL of deionized water, slowly add 0.9 g of sodium periodate dropwise, stir and react overnight in the dark, then transfer the mixed solution to a dialysis bag with a MWCO of 3500 Da, dialyze for 5 days, and freeze-dry at -60 °C for 3 days to obtain oxidized dextran;

[0075] S2. Dissolve 1.47 g of sodium hydroxide in 250 mL of pure water to prepare a sodium hydroxide solution. Dissolve 0.1 g of potassium hydrogen phthalate in 25 mL of pure water to prepare a potassium hydrogen phthalate solution and add two drops of phenolphthalein. Take a 10 mL burette for alkali, and titrate the potassium hydrogen phthalate solution with the sodium hydroxide solution. The concentration of the sodium hydroxide solution is measured to be 0.0929 mol / L. Take 212.3 mg of oxidized dextran with a molecular weight of 6000 Da and add it to 25 mL of 0.25 mol / L hydroxylamine hydrochloride aqueous solution containing 3 drops of 0.05% methyl orange. Let it stand for 3 h, and titrate the red color to yellow with the sodium hydroxide solution and perform a blank correction. The aldehyde group content C (aldehyde group content of oxidized dextran) of oxidized dextran with a molecular weight of 6000 Da is measured to be 0.6183 mmol / g;

[0076] S3. Add 3 g of ferroceneformic acid, 3 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 1 g of triethylamine to 35 mL of dichloromethane at 0 °C. After stirring and activating for 25 min, add 3 g of tert-butyl (4-aminobutyl) carbamate and 0.2 g of 4-dimethylaminopyridine and react at room temperature for 24 h. Add 35 mL of water to quench the reaction, extract with dichloromethane 3 times, wash the combined organic layer with saturated brine 2 times, add anhydrous magnesium sulfate to dry overnight and filter and concentrate by rotary evaporation, and perform column separation to obtain N-(4-tert-butoxycarbonylamino)ferrocenecarboxamide;

[0077] S4. Dissolve 2 g of N-(4-tert-butoxycarbonylamino)ferrocenecarboxamide obtained in step S3 in 25 mL of dichloromethane, add 5 mL of trifluoroacetic acid, stir in an ice-water bath, and gradually warm up to room temperature and react for 12 h. Add water to stop the reaction, extract with saturated brine 2 times, adjust the pH value to 9 with sodium hydroxide, and then extract with dichloromethane 3 times. Add anhydrous magnesium sulfate to dry and filter and concentrate by rotary evaporation to obtain N-(4-aminobutyl)ferrocenecarboxamide;

[0078] S5. Dissolve 0.8 g of oxidized dextran obtained in step S1 and 0.24 g of N-(4-aminobutyl)ferrocenecarboxamide obtained in step S4 in 25 mL of dimethyl sulfoxide, react at 49 °C for 49 h, then wash with ice-cold methyl tert-butyl ether 3 times, and freeze-dry to obtain a pH-responsive ferrocene-modified oxidized dextran micelle polymer. Then take 30 mg of the pH-responsive ferrocene-modified oxidized dextran micelle polymer, dissolve it in 2 mL of dimethyl sulfoxide and stir overnight, add 8 mL of water, then transfer it to a dialysis bag with a molecular weight cut-off of 3500 Da, dialyze for 24 h, and freeze-dry to obtain a pH-responsive ferrocene-modified oxidized dextran micelle.

[0079] Example 3

[0080] A pH-responsive ferrocene-modified oxidized dextran micelle (Fc-ODex), and the molecular weight of the above dextran is 10000 Da.

[0081] The preparation method of the above pH-responsive ferrocene-modified oxidized dextran micelles comprises the following steps:

[0082] S1. Dissolve 6 g of dextran with a molecular weight of 10,000 Da in 50 mL of deionized water, slowly dropwise add 0.99 g of sodium periodate, stir and react overnight in the dark, then transfer the mixed solution into a dialysis bag with a MWCO of 3500 Da, dialyze for 6 days, and freeze-dry at -60 °C for 3 days to obtain oxidized dextran;

[0083] S2. Dissolve 1.47 g of sodium hydroxide in 250 mL of pure water to prepare a sodium hydroxide solution, dissolve 0.1 g of potassium hydrogen phthalate in 25 mL of pure water to prepare a potassium hydrogen phthalate solution and add two drops of phenolphthalein. Take a 10 mL burette, titrate the potassium hydrogen phthalate solution with the sodium hydroxide solution, and measure the concentration of the sodium hydroxide solution to be 0.0929 mol / L. Take 305.9 mg of oxidized dextran with a molecular weight of 10,000 Da in 25 mL of 0.25 mol / L hydroxylamine hydrochloride aqueous solution containing 3 drops of 0.05% methyl orange, let it stand for 3 h, titrate the red color to yellow with the sodium hydroxide solution, and perform a blank correction. The aldehyde group content C (aldehyde group content of oxidized dextran) of oxidized dextran with a molecular weight of 10,000 Da is measured to be 0.4918 mmol / g;

[0084] S3. Add 5 g of ferrocene formic acid, 4 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 2 g of triethylamine to 45 mL of dichloromethane at 0 °C, stir and activate for 35 min, then add 4 g of tert-butyl(4-aminobutyl)carbamate and 0.3 g of 4-dimethylaminopyridine and react at room temperature for 25 h. Add 45 mL of water to quench the reaction, extract with dichloromethane 4 times, wash the combined organic layer with saturated brine 3 times, add anhydrous magnesium sulfate to dry overnight and filter and rotary evaporate, and perform column separation to obtain N-(4-tert-butoxycarbonylamino)ferrocenecarboxamide;

[0085] S4. Dissolve 3 g of N-(4-tert-butoxycarbonylamino)ferrocenecarboxamide obtained in step S3 in 30 mL of dichloromethane, add 10 mL of trifluoroacetic acid, stir in an ice-water bath, gradually warm up to room temperature and react for 13 h, add water to stop the reaction, extract with saturated brine 3 times, adjust the pH value to 9 with sodium hydroxide, then extract with dichloromethane 4 times, add anhydrous magnesium sulfate to dry and filter and rotary evaporate to obtain N-(4-aminobutyl)ferrocenecarboxamide;

[0086] S5. Dissolve 0.9 g of the oxidized dextran obtained in step S1 and 0.3 g of N-(4-aminobutyl)ferrocenecarboxamide obtained in step S4 in 30 mL of dimethyl sulfoxide, react at 51 °C for 50 h, then wash 4 times with ice-cold methyl tert-butyl ether, and freeze-dry to obtain a pH-responsive ferrocene-modified oxidized dextran micelle polymer. Then, take 30 mg of the pH-responsive ferrocene-modified oxidized dextran micelle polymer and dissolve it in 2 mL of DMSO, add 3 mg of doxorubicin hydrochloride, stir overnight, add 8 mL of water, then transfer it to a dialysis bag with a molecular weight cut-off of 3500 Da, dialyze for 24 h, and freeze-dry to obtain pH-responsive ferrocene-modified oxidized dextran micelles.

[0087] Example 4

[0088] The difference between Example 4 and Example 1 is that in Example 4, the molar ratio of sodium periodate to glucose units in dextran is 1:10, and the molecular weight of dextran is 20000 Da.

[0089] Test Example 1

[0090] Compare the nuclear magnetic resonance spectra of the oxidized dextran, pH-responsive ferrocene-modified oxidized dextran micelles, and N-(4-aminobutyl)ferrocenecarboxamide in Example 1, and the results are as Figure 4 shown.

[0091] It can be seen from Figure 4 that after the oxidized dextran is grafted with (4-aminobutyl)ferrocenecarboxamide, the aldehyde group peak of the oxidized dextran at 9.67 ppm disappears, and a ferrocene pentacyclic signal peak at 4.19 ppm appears.

[0092] Compare the infrared spectra of the pH-responsive ferrocene-modified oxidized dextran micelles, oxidized dextran, and dextran in Example 1, and the results are as Figure 5 shown.

[0093] It can be seen from Figure 5 that after grafting, a stretching vibration peak of C=O on the acyl group at 1545.88 cm -1 and a out-of-plane bending vibration peak of C-H on the pentacyclic ring of ferrocene at 949.66 cm -1 appear, indicating that the pH-responsive ferrocene-modified oxidized dextran micelles are successfully synthesized.

[0094] Test Example 2

[0095] Simultaneously irradiate the pH-responsive ferrocene-modified oxidized dextran micelles prepared in Example 1 and pure water with a laser pointer, and the results are as Figure 6 shown.

[0096] It can be seen from Figure 6It can be seen that there is no obvious change in pure water, while a bright path can be observed in the micelles, namely the Tyndall effect, which proves the successful preparation of pH-responsive ferrocene-modified oxidized dextran micelles.

[0097] Test Example 3

[0098] The pH-responsive ferrocene-modified oxidized dextran micelles prepared in Example 1 were used for pH sensitivity testing.

[0099] 1. Whether the imine bond is broken after Fc-ODex is acidified, where A-Fc-ODex refers to the Fc-ODex solution acidified with hydrochloric acid, and A-Fc-NH3 refers to N-(4-tert-butoxycarbonylamino)ferrocenecarboxamide hydrochloride. The result analysis is as follows:

[0100] (1) N-(4-tert-butoxycarbonylamino)ferrocenecarboxamide hydrochloride was obtained by reacting N-(4-tert-butoxycarbonylamino)ferrocenecarboxamide synthesized in Example 1 with hydrochloric acid dioxane: Weigh 0.2 g (0.5 mmol) of N-(4-tert-butoxycarbonylamino)ferrocenecarboxamide and react it in 5.8 mL of hydrochloric acid dioxane for 24 h, then rotary evaporate to obtain a black mucus, N-(4-tert-butoxycarbonylamino)ferrocenecarboxamide hydrochloride (A-Fc-NH3).

[0101] (2) Prepare Fc-ODex, A-Fc-ODex, and A-Fc-NH3 solutions: Take a little Fc-ODex and dissolve it in 1.5 mL of water and 1.5 mL of methanol. Take 1.5 mL of this solution and add a drop of hydrochloric acid to another reagent bottle to obtain the A-Fc-ODex solution, and let it stand for three days.

[0102] (3) Determine the pH sensitivity of Fc-ODex by HPLC: Set the column temperature of the high-performance liquid phase to 30 °C, and the mobile phase is an aqueous solution containing 0.5% acetic acid (A) and methanol (B); the gradient elution program is 90% A and 10% B for 1 min, gradually changing to 10% A and 90% B for 30 min; the detection wavelength is 321 nm; the injection volume is 20 μL.

[0103] The HPLC comparative spectra of A-Fc-ODex, A-Fc-NH3, and Fc-ODex are as Figure 7 shown.

[0104] From Figure 7 it can be seen that after the Fc-ODex polymer is acidified with hydrochloric acid, the signal peak of the original polymer at 14.5 min disappears, and at the same time, the signal peak of N-(4-tert-butoxycarbonylamino)ferrocenecarboxamide hydrochloride at 25.5 min is obtained. It is proved that after the Fc-ODex polymer is acidified, the Schiff base breaks, and the original N-(4-tert-butoxycarbonylamino)ferrocenecarboxamide is obtained.

[0105] 2. Particle size changes of Fc-ODex at different pH values. Fc-ODex was co-cultured for 36 h in buffer solutions with pH values of 7.4, 6.5, and 5.0 respectively. The particle size changes of Fc-ODex at different culture times and pH values are as Figure 8 shown.

[0106] It can be Figure 8 seen that in the buffer solution with pH 7.4, the particle size of Fc-ODex did not change significantly and remained between 300 - 400 nm, indicating that the nanoparticles tend to be stable in the normal physiological environment; when in the buffer solutions with pH 6.5 and 5.0, the particle size of Fc-ODex showed multimodality with the extension of time, and the particle distribution gradually became wider. This is due to the cleavage of imine bonds under acidic conditions, resulting in the dissociation of micelles, proving that Fc-ODex has pH-responsive properties.

[0107] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A pH-responsive ferrocene-modified oxidized dextran micelle, characterized in that: Its structural formula is: Wherein n is 37-123, and the molecular weight of the dextran is 6000-20000 Da.

2. The method for preparing the pH-responsive ferrocene-modified oxidized dextran micelles according to claim 1, characterized in that: The following steps are involved: S1. Dissolve dextran in deionized water, add sodium periodate dropwise, stir, dialyze the mixed solution, and freeze-dry to obtain oxidized dextran; S2, dissolving the oxidized dextran obtained in step S1 in an aqueous solution of hydroxylamine hydrochloride containing methyl orange, allowing to stand, titrating with a sodium hydroxide solution, and performing blank correction; S3, adding ferrocenecarboxylic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and triethylamine to dichloromethane, stirring and activating, adding tert-butyl (4-aminobutyl) carbamate and 4-dimethylaminopyridine to react, adding water to quench the reaction, extracting, washing, drying, filtering and spin-drying, and separating on a column to obtain N-(4-tert-butyloxycarbonylamino)ferrocenecarboxamide; S4, dissolving the N-(4-tert-butyloxycarbonylamino)ferrocenecarboxamide obtained in step S3 in dichloromethane, adding trifluoroacetic acid, stirring, adding water to stop the reaction, adjusting the pH value after extraction, re-extracting, drying, filtering and drying by suction, and obtaining N-(4-aminobutyl)ferrocenecarboxamide; S5. Dissolve the oxidized dextran obtained in step S1 and the N-(4-aminobutyl) ferrocenecarboxamide obtained in step S4 in dimethyl sulfoxide, wash with glacial methyl tert-butyl ether after the reaction, and freeze-dry to obtain pH-responsive ferrocene-modified oxidized dextran micelle polymer, and then use dialysis to obtain pH-responsive ferrocene-modified oxidized dextran micelles.

3. The method for preparing pH-responsive ferrocene-modified oxidized dextran micelles according to claim 2, characterized in that: In step S1, the mass volume ratio of dextran, sodium periodate and deionized water is 5-6 g:0.9-0.99 g:45-50 mL, and the reaction is stirred overnight in the dark, and then the mixed solution is transferred to a dialysis bag with MWCO 3500Da and dialyzed for 5-6 days.

4. The method for preparing pH-responsive ferrocene-modified oxidized dextran micelles according to claim 2, characterized in that: In step S2, the mass volume ratio of the oxidized dextran to the hydroxylamine hydrochloride aqueous solution is 4.13-402.5 mg:25-30 mL, and the concentration of the methyl orange in the hydroxylamine hydrochloride aqueous solution is 0.02-0.04 mg / mL.

5. The method for preparing pH-responsive ferrocene-modified oxidized dextran micelles according to claim 2, characterized in that: In step S3, the mass volume ratio of the ferrocenecarboxylic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, triethylamine, tert-butyl (4-aminobutyl) carbamate, 4-dimethylaminopyridine and dichloromethane is 3-5g:3-4g:1-2g:3-4g:0.2-0.3g:35-45mL.

6. The method for preparing pH-responsive ferrocene-modified oxidized dextran micelles according to claim 2, characterized in that: In step S3, after stirring and activating for 25-35 minutes, the mixture was reacted at room temperature for 24-25 hours, 35-45 mL of water was added to quench the reaction, and the mixture was extracted with dichloromethane for 3-4 times. The combined organic layer was washed with saturated brine for 2-3 times, and dried over anhydrous magnesium sulfate overnight.

7. The method for preparing pH-responsive ferrocene-modified oxidized dextran micelles according to claim 2, characterized in that: In step S4, the mass volume ratio of the N-(4-tert-butyloxycarbonylamino)ferrocenecarboxamide, dichloromethane and trifluoroacetic acid is 2-3 g:25-30 mL:5-10 mL.

8. The method for preparing pH-responsive ferrocene-modified oxidized dextran micelles according to claim 2, characterized in that: In step S4, the mixture is stirred in an ice-water bath, gradually heated to room temperature and reacted for 12-13 hours, water is added to stop the reaction, extracted with saturated brine 2-3 times, the pH value is adjusted with sodium hydroxide, extracted with dichloromethane 3-4 times, and dried with anhydrous magnesium sulfate.

9. The method for preparing pH-responsive ferrocene-modified oxidized dextran micelles according to claim 2, characterized in that: In step S5, the mass molar volume ratio of the oxidized dextran, N-(4-aminobutyl)ferrocenecarboxamide and dimethyl sulfoxide is 0.8-0.9 g:0.2-0.3 g:25-30 mL, and the mixture is reacted at 49-51° C. for 48-50 h and then washed 3-4 times with glacial methyl tert-butyl ether.

10. The method for preparing pH-responsive ferrocene-modified oxidized dextran micelles according to claim 2, characterized in that: In step S5, when the pH-responsive ferrocene-modified oxidized dextran micelles are blank micelles, the dialysis method comprises the following steps: 30-32 mg of pH-responsive ferrocene-modified oxidized dextran micelle polymer is dissolved in 2-3 mL of dimethyl sulfoxide and stirred overnight, 8-9 mL of water is added, and then transferred to a 3500 Da dialysis bag, dialyzed for 24-25 h, and freeze-dried; When the pH-responsive ferrocene-modified oxidized dextran micelle polymer is a drug-loaded micelle, the dialysis method includes the following steps: 30-32 mg of the pH-responsive ferrocene-modified oxidized dextran micelle polymer is dissolved in 2-3 mL of DMSO, 3-4 mg of doxorubicin hydrochloride is added, stirred overnight, 8-9 mL of water is added, and then transferred to a 3500Da dialysis bag, dialyzed for 24-25 hours, and freeze-dried.

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