CN106729745B - Preparation method and application of micelle capable of delivering nitric oxide based on hyaluronic acid modification - Google Patents

Preparation method and application of micelle capable of delivering nitric oxide based on hyaluronic acid modification Download PDF

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CN106729745B
CN106729745B CN201611237444.2A CN201611237444A CN106729745B CN 106729745 B CN106729745 B CN 106729745B CN 201611237444 A CN201611237444 A CN 201611237444A CN 106729745 B CN106729745 B CN 106729745B
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hyaluronic acid
nitric oxide
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CN106729745A (en
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侯琳
张振中
张银玲
郝丽莎
郑亚珍
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Zhengzhou University
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    • A61K33/00—Medicinal preparations containing inorganic active ingredients
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    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
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    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract

The invention relates to a preparation method and application of micelles capable of delivering nitric oxide based on hyaluronic acid modification, which can effectively solve the problems of poor targeting property and large toxic and side effects in vivo of anticancer drugs, and solves the technical scheme that two fluorine atoms on 1, 5-difluoro-2, 4-dinitrobenzene are respectively nucleophilic-substituted by hyaluronic acid and 2- (N, N-diethylamino) -diazene-2-oxygen sodium salt to form a polymer, and the polymer can spontaneously form micelles in an aqueous medium; the hyaluronic acid is low molecular weight hyaluronic acid with molecular weight equal to or lower than 100kDa and equal to or higher than 1000 Da; the invention has the advantages of high targeting property, enzyme sensitivity, good biocompatibility, low toxicity and the like, and is an innovation in a medicinal preparation for treating tumors.

Description

Preparation method and application of micelle capable of delivering nitric oxide based on hyaluronic acid modification
Technical Field
The invention relates to the field of medicines, in particular to a preparation method and application of a micelle which is modified by hyaluronic acid and can deliver nitric oxide.
Background
Cancer has become the leading cause of death in china. The related data show that only 2015 years are enough, 429.2 ten thousand new tumor cases and 281.4 ten thousand cancer death cases exist in China, the morbidity and the mortality are high, the health of human beings is seriously threatened, and therefore, the prevention, the diagnosis and the treatment of the cancer are not slow.
Nitric Oxide (NO) is a bioregulatory mediator in the organism, involved in the pathological and physiological processes of various diseases, and is closely associated with the development and progression of tumors. The existing data show that low concentration of NO can overcome multidrug resistance (MDR) of tumor cells by inhibiting the activity of P-glycoprotein (P-gP) and multidrug resistance related proteins (MRPs); when the concentration of NO is increased to the level of mu mol/L, the NO has the antitumor effect against the growth of tumor, and the antitumor mechanism comprises: (1) combine with superoxide anion in cells to generate nitrogen/oxygen free radicals, damage DNA, and thus cause cytotoxicity; (2) inducing apoptosis of tumor cells by activating expression of p53 and the like; (3) affecting the energy metabolism of cells, and leading tumor cells to die due to energy metabolism disorder; (4) inhibiting tumor metastasis by inhibiting platelet aggregation; (5) increasing the sensitivity of the tumor cells to the chemotherapeutic drugs. In addition, a large number of researches find that NO also participates in the chemotherapy and immunotherapy process of tumors widely, interacts with chemotherapeutic drugs, cytokines and the like, and influences the killing effect of the drugs on the tumors.
Hyaluronic Acid (HA) is an important component of an intercellular matrix of vertebrates, is a linear biological polysaccharide, and HAs the characteristics of good biocompatibility, biodegradability, no toxicity, no immunogenicity and the like. The CD44 receptor is a transmembrane glycoprotein, the most important HA receptor on the cell surface, and is overexpressed on the surface of most tumor cells. HA interacts with CD44 receptors on the surface of tumor cells, promoting the endocytosis of HA by the tumor cells.
GSTs mainly comprises α subtypes such as mu, pi, theta, etc., wherein the pi subtype (GST-pi) is most widely distributed and abundant, the physiological function of GST-pi is mainly to catalyze the combination of harmful polar compounds in organisms and glutathione, or to discharge various potential toxic chemical substances, carcinogens, lipophilic compounds, etc. from the organisms in a non-enzyme combination mode, thereby achieving the purposes of eliminating toxic substances, carcinogenic substances and protecting the stability of DNA genetic substances.
The biggest treatment defect of the traditional anti-tumor medicine is that most of the medicine effects are non-selective, and the traditional anti-tumor medicine has stronger killing effect on normal tissues while killing cancer cells. Moreover, the existing drug delivery system has the defects of poor targeting property, short in-vivo circulation time, large toxic and side effects and the like, so that the drug delivery system which can actively target cancer cells, has long in-vivo circulation and small toxic and side effects is imperative to invent.
Disclosure of Invention
Aiming at the problems and overcoming the defects of the prior art, the invention provides a preparation method of HA modified micelle capable of delivering NO, the micelle is sensitive to GST-pi abundantly existing in tumor cells, and under the condition that GST-pi exists, the micelle can release a large amount of NO for killing the tumor cells. The system can realize long circulation in vivo, has tumor targeting property, and the released NO can generate an anti-tumor effect in cooperation with the anti-cancer drug loaded in the micelle.
The invention solves the technical scheme that a polymer (HA-DNB-DEA/NO) capable of delivering nitric oxide based on hyaluronic acid modification is formed by nucleophilic substitution of two fluorine atoms on 1, 5-difluoro-2, 4-dinitrobenzene (FFDNB) by HA and 2- (N, N-diethylamino) -diazene-2-oxygen sodium salt (DEA/NO), the polymer spontaneously forms micelles in water, and the molecular weight of HA is 1-100 kDa (namely 1000-100000 daltons), and the specific preparation method is as follows:
(1) preparation of HA-FDNB
Placing 10 mg-1 g of HA and 50 mg-1 g of FFDNB in a mortar, adding 10 mg-1 g of alkaline reagent, and grinding for 1-20 min; then transferring the mixture into a 50mL flask, heating and stirring the mixture in oil bath at 78 ℃ for 12 hours, washing away free FFDNB by using dichloromethane after the reaction is finished, centrifuging, and naturally drying precipitate to obtain an HA monofluoro substituted product (HA-FDNB);
the alkaline reagent is anhydrous sodium carbonate or sodium bicarbonate, or a mixture of other similar alkaline reagents and other similar alkaline reagents;
(2) preparation of HA-DNB-DEA/NO micelle
① placing 10 mg-1 g HA-FDNB into a three-neck flask, dissolving with 500 ul-10 mL reaction solvent, adding 500 ul-10 mL tertiary butanol, and cooling to 0 ℃ under the protection of nitrogen;
② dissolving 10-500 mg of 2- (N, N-diethylamino) -diazene-2-oxygen sodium salt hydrate (DEA/NO) in 500 ul-10 mL of methanol;
③ mixing the solutions ① and ②, stirring for 48h at 23-27 ℃, precipitating with 3-5 times of precooled acetone after the reaction is finished, centrifuging at 2000rpm, washing the precipitate twice with acetone, redissolving with water, dialyzing in a dialysis bag for 8h, and freeze-drying the dialysate to obtain HA-DNB-DEA/NO micelles for later use;
the reaction solvent is N, N-dimethylformamide, or dimethyl sulfoxide, or other similar solvents and mixtures of other similar solvents.
The anti-tumor drug composition is prepared by preparing micelles capable of delivering nitric oxide based on hyaluronic acid modification and anti-tumor drugs into drug-carrying nano-micelles, wherein the anti-tumor drugs are paclitaxel, adriamycin, vincristine and hydroxycamptothecin.
The method for preparing the drug-loaded nano-micelle based on the micelle modified by hyaluronic acid and capable of delivering nitric oxide and the anti-tumor drug comprises the following operation steps:
the preparation of the micelle loaded with the antitumor drug comprises the following steps: dissolving the freeze-dried HA-DNB-DEA/NO and water in the ratio of 1-20:1000 in water to form a micelle solution; dissolving the antitumor drug in a proper solvent, then dropwise adding the antitumor drug into the micelle solution, stirring for 30min at 23-27 ℃, performing ultrasonic treatment for 30min by using a probe under ice bath, dialyzing to remove free drug, centrifuging dialysate, filtering by using a 0.45-micrometer filter membrane, and freeze-drying to obtain the drug-loaded nano-micelle; the proper solvent is a pharmaceutically used solvent capable of dissolving the medicine.
And (3) determining the content of the loaded antitumor drug in the micelle:
the drug loading of the micelle was calculated by the formula (1):
Figure BDA0001195617980000031
the HA-modified nano micelle capable of delivering NO loaded with the antitumor drug prepared by the invention HAs a strong killing effect on tumors, the targeted therapy on the tumors can be achieved through the antitumor drug loaded into the micelle core, and the drug delivery system HAs the advantages of high targeting property, good biocompatibility, enzyme sensitivity, low toxicity and the like, and is an innovation in a pharmaceutical preparation for tumor therapy.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
Example 1
In the specific implementation, the method can be realized by the following steps:
(1) preparation of HA-FDNB
Placing HA 100mg and FFDNB 150.27mg in a mortar, adding 104.1mg of anhydrous sodium carbonate, and grinding for 5 min; then transferring the mixture into a 50mL flask, heating and stirring the mixture in oil bath at 78 ℃ for 12 hours; after the reaction is finished, washing off free FFDNB by using 30mL of dichloromethane, centrifuging, and naturally drying precipitate to obtain HA-FDNB;
(2) preparation of HA-DNB-DEA/NO micelle
① placing HA-FDNB 17.6mg into 25mL three-neck flask, adding 700ul N, N-dimethylformamide for dissolution, adding 700ul tert-butanol, and cooling to 0 ℃ under nitrogen protection;
② DEA/NO 10mg is taken and dissolved in 500ul of methanol, and then added into ①;
③ is gradually increased to 23-27 ℃, stirred for 48h at 23-27 ℃, and after the reaction is finished, precooled acetone with 3 times is used for precipitation, the centrifugation is carried out at 2000rpm, the precipitation is washed twice by acetone, the water is redissolved and is placed in a dialysis bag for dialysis for 8h, and the dialysate is ultrapure water and is freeze-dried to obtain HA-DNB-DEA/NO micelle for standby;
(3) preparation of doxorubicin loaded in micelle
① weighing 12mg HA-DNB-DEA/NO micelle and dissolving in 3mL water to form micelle solution;
② weighing 6mg doxorubicin hydrochloride, desalting, dissolving in 700ul N, N-Dimethylformamide (DMF), and adding the doxorubicin solution dropwise into micelle;
③ 23-27 deg.C, stirring for 30min, performing ultrasonic treatment with probe in ice bath for 30min, dialyzing to remove free adriamycin, centrifuging at 3500rpm, filtering with 0.45 μm filter membrane, and lyophilizing to obtain adriamycin-loaded nano micelle.
(4) Determination of doxorubicin loading in micelles
The content of adriamycin is measured at 481nm by adopting an ultraviolet spectrophotometry, and the drug loading of the micelle is calculated according to the formula (1), wherein the drug loading reaches 28.4%.
Example 2
(1) Preparation of HA-FDNB
Placing HA 100mg and FFDNB 150.27mg in a mortar, adding 123.68mg of sodium bicarbonate, and grinding for 5 min; then transferring the mixture into a 50mL flask, heating and stirring the mixture in oil bath at 78 ℃ for 12 hours; after the reaction is finished, washing off free FFDNB by using 30mL of dichloromethane, centrifuging, and naturally drying precipitate to obtain HA-FDNB;
(2) preparation of HA-DNB-DEA/NO micelle
① placing HA-FDNB 35.2mg into 25mL three-neck flask, adding 1.4mL N, N-dimethylformamide to dissolve, adding 1.4mL tert-butanol, and cooling to 0 deg.C under nitrogen protection;
② DEA/NO 20mg is taken and dissolved in 1mL of methanol, and then is dripped into ①;
③ gradually increasing to 23-27 deg.C, stirring at 23-27 deg.C for 48h, precipitating with 4 times of precooled acetone after reaction, centrifuging at 2000rpm, washing the precipitate with acetone twice, dissolving with water, dialyzing in dialysis bag for 8h, and lyophilizing;
(3) preparation of loaded paclitaxel in micelle
① weighing 18mg of HA-DNB-DEA/NO and dissolving in 3mL of water to form a micelle solution;
② weighing 10mg of paclitaxel, and dissolving in 330ul ethanol to obtain paclitaxel ethanol solution;
③ mixing, stirring at 23-27 deg.C for 30min, performing ultrasonic treatment with ice bath probe for 30min, dialyzing to remove free paclitaxel, centrifuging at 3500rpm, filtering with 0.45 μm filter membrane, and lyophilizing to obtain paclitaxel-loaded nanometer micelle.
(4) Determination of loaded paclitaxel content in micelle
Determining the content of paclitaxel by High Performance Liquid Chromatography (HPLC), calculating the drug loading of micelle according to the formula (1) to reach 31.4%,
example 3
(1) Preparation of HA-FDNB
Placing HA 60mg and FFDNB 90.16mg in a mortar, adding anhydrous sodium carbonate 62.46mg, and grinding for 5 min; then transferring the mixture into a 50mL flask, heating and stirring the mixture in oil bath at 78 ℃ for 12 hours; after the reaction is finished, washing off free FFDNB by using 30mL of dichloromethane, centrifuging, and naturally drying precipitate to obtain HA-FDNB;
(2) preparation of HA-DNB-DEA/NO micelle
① placing HA-FDNB 44mg into 25mL three-neck flask, adding 1.5mL N, N-dimethylformamide to dissolve, adding 1.5mL tert-butanol, and cooling to 0 deg.C under nitrogen protection;
② DEA/NO 25mg is taken and dissolved in 1mL of methanol, and then is dripped into ①;
③ gradually increasing to 23-27 deg.C, stirring at 23-27 deg.C for 48h, precipitating with 5 times of precooled acetone after reaction, centrifuging at 2000rpm, washing the precipitate with acetone twice, dissolving with water, dialyzing in dialysis bag for 8h, and lyophilizing;
(3) preparation of vincristine loaded in micelle
① weighing 10mg of HA-DNB-DEA/NO and dissolving in 2mL of water to form a micelle solution;
② dissolving vincristine 5mg in 200ul ethanol, and mixing with micelle solution;
③ 23-27 deg.C, stirring for 30min, performing ultrasonic treatment with probe in ice bath for 30min, dialyzing to remove free vincristine, centrifuging at 3500rpm, filtering with 0.45 μm filter membrane, and lyophilizing to obtain vincristine-loaded nano micelle.
(4) Determination of vincristine loading in micelles
Measuring vincristine content by HPLC, calculating drug loading of micelle according to the formula (1), wherein the drug loading is 26.7%
Example 4
(1) Preparation of HA-FDNB
Placing HA 50mg and FFDNB 75.29mg in a mortar, adding 61.84mg of sodium bicarbonate, and grinding for 5 min; then transferring the mixture into a 50mL flask, heating and stirring the mixture in oil bath at 78 ℃ for 12 hours; after the reaction is finished, washing off free FFDNB by using 30mL of dichloromethane, centrifuging, and naturally drying precipitate to obtain HA-FDNB;
(2) preparation of HA-DNB-DEA/NO micelle
① placing HA-FDNB 52.8mg into 25mL three-neck flask, adding 2.1mL N, N-dimethylformamide to dissolve, adding 2.1mL tert-butanol, and cooling to 0 deg.C under nitrogen protection;
② DEA/NO 30mg is taken and dissolved in 1.5mL of methanol, and then is dripped into ①;
③ gradually increasing to 23-27 deg.C, stirring at 23-27 deg.C for 48h, precipitating with 3 times of precooled acetone after reaction, centrifuging at 2000rpm, washing the precipitate with acetone twice, dissolving with water, dialyzing in dialysis bag for 8h, and lyophilizing;
(3) preparation of loaded hydroxycamptothecin in micelle
① weighing 12mg of HA-DNB-DEA/NO and dissolving in 3mL of water to form a micelle solution;
② weighing 6mg of hydroxycamptothecin, dissolving in 50ul of dimethyl sulfoxide, and mixing with micelle solution;
③ 23-27 deg.C, stirring for 30min, performing ultrasonic treatment with probe under ice bath for 30min, dialyzing overnight, centrifuging at 3500rpm, filtering with 0.45 μm filter membrane, and lyophilizing to obtain the final product.
(4) Determination of content of hydroxycamptothecin loaded in micelle
The content of hydroxycamptothecin is measured at 369nm by ultraviolet spectrophotometry, and the drug loading of the micelle is calculated according to the formula (1) and reaches 27.9 percent.
According to the invention, one fluorine atom on FFDNB is nucleophilic-substituted by HA, and the other fluorine atom is nucleophilic-substituted by DEA/NO to form HA-DNB-DEA/NO polymer, and the polymer spontaneously forms micelle in water, wherein the particle size is 100-200 nm. The HA is used as a hydrophilic end of the micelle, so that the micelle can realize long circulation in vivo, and can be combined with a CD44 receptor highly expressed on the surface of a tumor cell to realize active targeting of the tumor; DNB-DEA/NO is used as the hydrophobic end of the micelle, DEA/NO is wrapped in the hydrophobic core of the micelle, and the stability is enhanced. Under the action of highly expressed GST-pi in tumor cells, a large amount of NO can be released, and the NO and the anticancer drug loaded in the micelle cooperate to generate an antitumor effect. The invention is a novel drug system which has the functions of active targeting of tumor and the fixed-point release of NO by tumor cells, and the NO and the drug generate the synergistic anti-tumor effect. The test results show that the method has very satisfactory beneficial technical effects, and the relevant test data are as follows:
1. the particle size and potential of the HA-DNB-DEA/NO micelle are characterized as follows:
an appropriate amount of HA-DNB-DEA/NO polymer is diluted by water to an appropriate concentration, and the particle size and the potential of the polymer are respectively 157.3nm and-39.5 +/-0.30 mv measured by a Nano-ZS90 type laser Nano particle size analyzer.
2. Proliferation inhibition assay of doxorubicin-loaded micelles (DOX/HA-DNB-DEA/NO) on MCF-7 cells:
MCF-7 cells were processed into single cell suspensions according to the cell passaging procedure, and after counting, 5X 10 cells were seeded per well3The cells were cultured in a 96-well plate in an incubator (37 ℃ C., 5% CO)2)24h, after the cells are completely attached to the wall, removing the original culture medium and adding the medicine, wherein the experiment is divided into a DOX/HA-DNB-DEA/NO group, an HA-DNB-DEA/NO group and a DOX group; the concentration of the added medicine is in a series of gradients according to the concentration of DOX, the medicine is prepared by a culture medium without serum, the concentration gradient of DOX is 0,0.039,0.078,0.156,0.313,0.625,1.25,2.5,5 and 10 mu g/ml in sequence, the culture plate is taken out after continuous culture for 48 hours, 50 mu l of pre-cooled 50% trichloroacetic acid is added into each hole, the final concentration is 10%, and the culture plate is kept still for 5 min; moving to a refrigerator at 4 ℃ for standing for 1h, taking out, washing with ultrapure water for 5 times, drying in air at 23-27 ℃ completely, adding 50 mu l of SRB prepared by 1% acetic acid into each hole, dyeing at 23-27 ℃ for 20min, pouring off dye liquor, washing with 1% acetic acid for 5 times, removing unbound dye in each hole, drying in air at 23-27 ℃, dissolving with 150 mu l of 10mmol/l Tris alkali liquor with pH of 10.5, oscillating in an air heating shaking table for 10min, and measuring the light absorbance value of each hole at 515nm on an enzyme linked immunosorbent assay detector. The calculated inhibition ratio (%) × 100% (1-experimental group a/control group a) gave half inhibition concentrations (IC50) of the above samples in the order of: DOX/HA-DNB-DEA/NO groupThe HA-DNB-DEA/NO group and the DOX group were 0.5376, 2.3428 and 1.538. mu.g/ml, respectively.
4. Uptake experiment of MCF-7 cells on DOX/HA-DNB-DEA/NO, HA-DNB-DEA/NO nano-micelle
The experiment was divided into DOX/HA-DNB-DEA/NO group, DOX group, and blank cell group. Processing MCF-7 cells in logarithmic growth phase into single cell suspension according to the cell passage step, and spreading a six-hole plate after counting, wherein the cell number of each hole is 3 multiplied by 105Cell/well, incubator (37 ℃, 5% CO)2) Culturing for 24h, removing culture medium after adherence is complete, adding medicine prepared from serum-free culture medium, performing post-treatment in time periods of 0.5h, 1h, 2h, 4h and 6h respectively, sucking medicinal liquid into corresponding EP tube, washing with PBS for 2 times, then digesting with 0.5ml of pancreatin without EDTA for 1min, adding 1ml of culture medium, sucking into corresponding EP tube, centrifuging at 1000rpm/10min, discarding supernatant, adding 2ml PBS into the precipitate, blowing and beating uniformly, centrifuging at 1000rpm/10min, discarding the supernatant, adding 0.5ml PBS into the precipitate, blowing uniformly, transferring to 1.5ml EP tube, placing in ice box, measuring with flow cytometer, measuring time point 6h, the DOX uptake of MCF-7 cell is 90.2%, the uptake of DOX/HA-DNB-DEA/NO was 79.6%, the amount of HA-DNB-DEA/NO taken up was 82.7%.
5. Pharmacodynamic test of DOX/HA-DNB-DEA/NO nano-micelle:
35 nude mice (female, 3-4 weeks old) were purchased and inoculated with MCF-7 cells in logarithmic growth phase, when the tumor volume reached 100mm3In the above, the tumor model was successfully inoculated, and 24 tumor-bearing nude mice with similar tumor volume and body weight were randomly divided into 4 groups of 8 mice each, and the grouping was as follows: blank group, DOX group, HA-DNB-DEA/NO group, and DOX/HA-DNB-DEA/NO group. Then, the administration is carried out every other day, the administration dose is 4mg/kg according to the conversion of the human mouse dose, and the administration is carried out by tail vein injection. Mice were observed daily for survival and weighed (by tumor volume (V) ═ a × B)2/2), then by relative tumor volume R ═ V/V0Change (V) of0The size of tumor volume for the day before administration) was evaluated. The data recorded indicate the end of the administrationCompared with the prior art, the tumor volume of the nude mice in the blank group is increased by 70 percent, the tumor volume of the nude mice in the DOX group is increased by 11 percent, the tumor volume of the nude mice in the DOX/HA-DNB-DEA/NO group is reduced by 78 percent, the tumor volume of the nude mice in the HA-DNB-DEA/NO group is reduced by 54 percent, and the composition HAs the effect of obviously inhibiting the tumor volume and can be used for preparing anti-tumor drugs.
Experiments show that compared with the prior art, the invention has the following outstanding beneficial effects:
(1) based on the over-expressed GST-pi in the tumor cells, under the action of the enzyme, DEA/NO in the micelle quickly releases a large amount of NO, and the triggering mechanism can realize the fixed-point and quick release of NO in the tumor cells and generate a synergistic effect with anticancer drugs; in addition, HA is a hydrophilic substance, can prolong the circulation time in the micelle, and HAs the function of actively targeting tumor cells by recognizing CD44 receptors on the surface of the tumor so as to reduce toxic and side effects in normal tissues.
(2) The nano micelle formed by the invention has the advantages of good biocompatibility, high tumor targeting property, long circulation, small toxic and side effects, various drug-loading forms and the like, has strong killing effect on tumors, is a great innovation on tumor treatment drug preparations, and has great economic and social benefits.

Claims (8)

1. A micelle capable of delivering nitric oxide based on hyaluronic acid modification is characterized in that a hyaluronic acid-modified polymer capable of delivering nitric oxide is formed through nucleophilic substitution of two fluorine atoms on 1, 5-difluoro-2, 4-dinitrobenzene by hyaluronic acid and 2- (N, N-diethylamino) -diazene-2-oxygen sodium salt, wherein the polymer spontaneously forms micelles in water, and the molecular weight of the hyaluronic acid is 1-100 kDa.
2. The method for preparing micelles capable of delivering nitric oxide based on hyaluronic acid modification according to claim 1, comprising the following steps: 1) putting 10 mg-1 g of hyaluronic acid and 50 mg-1 g of 1, 5-difluoro-2, 4-dinitrobenzene into a mortar, adding 10 mg-1 g of alkaline reagent, grinding for 1-20 min, transferring into a 50mL flask, heating and stirring in an oil bath at 78 ℃ for 12h, washing free 1, 5-difluoro-2, 4-dinitrobenzene with dichloromethane after the reaction is finished, centrifuging, precipitating and naturally drying to obtain a monofluoro substituted product of hyaluronic acid; the alkaline reagent is anhydrous sodium carbonate or sodium bicarbonate;
2) ① g of a hyaluronic acid monofluoro substitution product is taken and placed in a three-neck flask, 500 ul-10 mL of reaction solvent is used for dissolving, 500 ul-10 mL of tert-butyl alcohol is added, the mixture is cooled to 0 ℃ under the protection of nitrogen, ② g of 2- (N, N-diethylamino) -diazene-2-oxygen sodium salt 10-500 mg is dissolved in 500 ul-10 mL of methanol, ③ the solutions obtained in the step ① and ② are mixed, the mixture is stirred for 48 hours at the temperature of 23-27 ℃, precooled acetone is used for precipitation and extraction after the reaction is finished, the precipitation is centrifuged at 2000rpm and washed twice with acetone, the mixture is re-dissolved with water and placed in a dialysis bag for dialysis for 8 hours, and the dialysate is ultrapure water and is freeze-dried to obtain the micelle capable of delivering the nitric oxide based on the modification of the hyaluronic acid, wherein the reaction solvent is N, N-dimethylformamide or dimethyl sulfoxide.
3. The method for preparing micelles capable of delivering nitric oxide based on hyaluronic acid modification according to claim 2, comprising the following steps: 1) placing 100mg of hyaluronic acid and 150.27mg of 1, 5-difluoro-2, 4-dinitrobenzene in a mortar, adding 104.1mg of anhydrous sodium carbonate, grinding for 5min, transferring into a 50mL flask, and heating and stirring in an oil bath at 78 ℃ for 12 h; after the reaction is finished, washing off free 1, 5-difluoro-2, 4-dinitrobenzene by using 30mL of dichloromethane, centrifuging, precipitating and naturally drying to obtain a monofluoro substituted product of hyaluronic acid;
2) ① placing 17.6mg of hyaluronic acid monofluoro substitution product into a 25mL three-neck flask, adding 700ul of N, N-dimethylformamide for dissolving, adding 700ul of tert-butyl alcohol, cooling to 0 ℃ under the protection of nitrogen, ② taking 10mg of 2- (N, N-diethylamino) -diazene-2-oxygen sodium salt for dissolving into 500ul of methanol, then dropwise adding into ①, gradually heating ③ to 23-27 ℃, stirring for 48h at 23-27 ℃, after the reaction is finished, using 3 times of precooled acetone for precipitation, centrifuging at 2000rpm, washing the precipitate twice with acetone, redissolving with water, placing into a dialysis bag for dialysis for 8h, using ultrapure water as dialysate, and freeze-drying to obtain the hyaluronic acid modification based micelle capable of delivering nitric oxide.
4. The method for preparing micelles capable of delivering nitric oxide based on hyaluronic acid modification according to claim 2, comprising the following steps: 1) placing hyaluronic acid 100mg and 1, 5-difluoro-2, 4-dinitrobenzene 150.27mg in a mortar, adding sodium bicarbonate 123.68mg, and grinding for 5 min; then transferring the mixture into a 50mL flask, heating and stirring the mixture in oil bath at 78 ℃ for 12 hours; after the reaction is finished, washing off free 1, 5-difluoro-2, 4-dinitrobenzene by using 30mL of dichloromethane, centrifuging, precipitating and naturally drying to obtain a monofluoro substituted product of hyaluronic acid;
2) ① putting 35.2mg of a monofluoro hyaluronic acid substitution product into a 25mL three-neck flask, adding 1.4mL of N, N-dimethylformamide for dissolving, adding 1.4mL of tert-butyl alcohol, cooling to 0 ℃ under the protection of nitrogen, ② taking 20mg of 2- (N, N-diethylamino) -diazene-2-oxygen sodium salt for dissolving in 1mL of methanol, then dropwise adding into ①, gradually increasing ③ to 23-27 ℃, stirring for 48 hours at 23-27 ℃, after the reaction is finished, using 4 times of precooled acetone for precipitation, centrifuging at 2000rpm, washing the precipitate twice with acetone, redissolving with water, putting into a dialysis bag for dialysis for 8 hours, using ultrapure water as a dialysate, and freeze-drying to obtain the hyaluronic acid modification based micelle capable of delivering nitric oxide.
5. The method for preparing micelles capable of delivering nitric oxide based on hyaluronic acid modification according to claim 2, comprising the following steps: 1) placing hyaluronic acid 60mg and 1, 5-difluoro-2, 4-dinitrobenzene 90.16mg in a mortar, adding anhydrous sodium carbonate 62.46mg, and grinding for 5 min; then transferring the mixture into a 50mL flask, heating and stirring the mixture in oil bath at 78 ℃ for 12 hours; after the reaction is finished, washing off free FFDNB by using 30mL of dichloromethane, centrifuging, and naturally drying precipitate to obtain a hyaluronic acid monofluoro substitution product;
2) ① placing 44mg of hyaluronic acid monofluoro substitution product in a 25mL three-neck flask, adding 1.5mL of N, N-dimethylformamide for dissolving, adding 1.5mL of tert-butyl alcohol, cooling to 0 ℃ under the protection of nitrogen, ② dissolving 25mg of 2- (N, N-diethylamino) -diazene-2-oxygen sodium salt in 1mL of methanol, then dropwise adding the methanol into ①, gradually increasing ③ to 23-27 ℃, stirring for 48h at 23-27 ℃, after the reaction is finished, using 5 times of precooled acetone for precipitation, centrifuging at 2000rpm, washing the precipitate twice with acetone, redissolving with water, placing the mixture in a dialysis bag for dialysis for 8h, using ultrapure water as dialysate, and freeze-drying to obtain the hyaluronic acid modification based micelle capable of delivering nitric oxide.
6. The pharmaceutical composition containing the micelle capable of delivering nitric oxide modified by hyaluronic acid as claimed in claim 1, which is characterized in that the micelle capable of delivering nitric oxide modified by hyaluronic acid and an anti-tumor drug are prepared into drug-loaded nano-micelles, wherein the anti-tumor drug is paclitaxel, adriamycin, vincristine or hydroxycamptothecin; specifically, the freeze-dried micelle which is modified based on hyaluronic acid and can deliver nitric oxide and water are dissolved in water according to the ratio of 1-20:1000 to form a micelle solution; dissolving the antitumor drug in a proper solvent, then dropwise adding the antitumor drug into the micelle solution, stirring for 30min at 23-27 ℃, performing ultrasonic treatment for 30min by using a probe under ice bath, dialyzing to remove free drug, centrifuging dialysate, filtering by using a 0.45-micrometer filter membrane, and freeze-drying to obtain the drug-loaded nano-micelle; the proper solvent is a pharmaceutically used solvent capable of dissolving the medicine.
7. Use of micelles capable of delivering nitric oxide based on hyaluronic acid modifications as described in claim 1 for the preparation of antitumor pharmaceutical compositions.
8. Use of the hyaluronic acid-modified nitric oxide-based micelles capable of delivering nitric oxide prepared by the method for preparing hyaluronic acid-modified nitric oxide-based micelles of any one of claims 2 to 5 for preparing an antitumor pharmaceutical composition.
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