CN112138001A

CN112138001A - Quercetin-low molecular weight heparin-paclitaxel conjugate, preparation method and application

Info

Publication number: CN112138001A
Application number: CN202011023817.2A
Authority: CN
Inventors: 翟光喜; 杨小叶; 程慧玲
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2020-09-25
Filing date: 2020-09-25
Publication date: 2020-12-29

Abstract

The invention particularly relates to a quercetin-low molecular weight heparin-paclitaxel conjugate, a preparation method and application. Quercetin and paclitaxel have good antitumor activity, but the water solubility of the two is poor, the bioavailability is low, and the clinical application condition is not ideal. The invention provides a paclitaxel-low molecular heparin-quercetin conjugate, which takes low molecular heparin sodium as a framework and connects paclitaxel and quercetin through covalent bonds. The conjugate as an anti-tumor active component effectively improves poor solubility of paclitaxel and quercetin. The conjugate obtained by the preparation method basically does not destroy the active structures of the paclitaxel and the quercetin, well retains the pharmacological activities of the paclitaxel and the quercetin, is applied to antitumor drugs, is expected to realize the treatment effect of 'double administration', and has important clinical treatment significance.

Description

Quercetin-low molecular weight heparin-paclitaxel conjugate, preparation method and application

Technical Field

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a quercetin-low molecular heparin-paclitaxel conjugate, and a preparation method of the conjugate records application in preparation of antitumor drugs.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Paclitaxel (PTX), a diterpene alkaloid compound isolated and purified from the bark of the gymnosperm yew, is used clinically mainly for the treatment of ovarian cancer, small cell lung cancer, breast cancer, and the like; it also has therapeutic effect on other types of cancers, such as prostate cancer, nasopharyngeal carcinoma, etc. However, the solubility of PTX in water is less than 4. mu.g/mL, which greatly limits its use. At present, Taxol (Taxol) which is a paclitaxel injection widely used clinically is prepared by taking polyoxyethylene castor oil/ethanol as a cosolvent carrier, but the polyoxyethylene castor oil can cause serious anaphylactic reaction, which causes inconvenience to patients.

Quercetin (QT), a polyphenol flavonoid extracted from natural plants, has a wide range of pharmacological actions, and can be used as an effective antioxidant for the treatment of inflammation, tumor, viral infection, etc. Research shows that quercetin is a broad-spectrum anti-cancer drug and has good inhibition effect on various cancers such as liver cancer, lung cancer, breast cancer, ovarian cancer and the like. However, quercetin has very low water solubility (solubility in water of 1 μ g/mL), is unstable in the gastrointestinal tract, has low oral bioavailability, and has a great limitation in clinical application.

Heparin is a natural, heterogeneous polysaccharide that has been used clinically as an anticoagulant for decades. However, heparin is often used to cause many adverse reactions such as allergic reactions, hemorrhagic complications, thrombocytopenia, etc., and low molecular weight heparin (LWMH) is often used clinically as an alternative to heparin in order to avoid these problems. The low molecular heparin is a general name of heparin which is depolymerized by common heparin and has lower molecular weight, and has the advantages of high bioavailability, low toxic effect and the like. In recent years, a new trend is to use heparin/low-molecular heparin as a carrier material to construct various nano-carriers for tumor treatment. The reasons for this depend mainly on the following properties of heparin: (1) no toxicity and better biocompatibility; (2) anti-tumor activity; (3) anti-tumor metastasis activity; (4) targeting property.

Disclosure of Invention

According to the current research results in the field, the chemotherapeutic drugs with different mechanisms are adopted for co-treatment, so that the tumor killing effect can be effectively improved, and the occurrence probability of tumor drug resistance is reduced. The invention aims to provide a drug delivery system for simultaneously delivering paclitaxel and quercetin, which improves the adverse biological characteristics of the paclitaxel and the quercetin, improves the solubility and the bioavailability of the quercetin, and can also play a role in multi-tube anti-tumor effect. Based on the technical purpose, the invention designs a paclitaxel-low molecular heparin-quercetin conjugate, two drugs are simultaneously connected to a low molecular heparin framework to obtain an amphiphilic conjugate, and the conjugate can be self-assembled into nanoparticles with a core-shell structure in water.

Based on the above purpose, the invention provides the following technical scheme:

in a first aspect of the present invention, a paclitaxel-low molecular heparin-quercetin conjugate is provided, wherein the conjugate uses low molecular heparin as a skeleton, and paclitaxel and quercetin are linked to the low molecular heparin through covalent bonds.

In a second aspect of the present invention, there is provided a method for preparing the paclitaxel-low molecular heparin-quercetin conjugate according to the first aspect, the method comprising the steps of: respectively synthesizing adipic acid dihydrazide modified low molecular heparin (LWMH-ADH), quercetin-NHS ester (QT-NHS) and carboxylated paclitaxel (PTX-COOH), and synthesizing the low molecular heparin-quercetin (LWMH-QT) by using the adipic acid dihydrazide modified low molecular heparin and the quercetin-NHS ester as raw materials; mixing low molecular heparin-quercetin with carboxylated paclitaxel, and reacting to obtain paclitaxel-low molecular heparin-quercetin conjugate (PTX-LWMH-QT).

In a third aspect of the present invention, there is provided a use of the paclitaxel-low molecular heparin-quercetin conjugate of the first aspect in the preparation of an antitumor drug.

In a fourth aspect of the present invention, an antitumor agent is provided, wherein the antitumor agent comprises the paclitaxel-low molecular heparin-quercetin conjugate of the first aspect and pharmaceutically necessary excipients.

The beneficial effects of one or more technical schemes are as follows:

quercetin and paclitaxel have poor water solubility, are unstable in gastrointestinal tracts and have low oral bioavailability, and the clinical application of the quercetin and the paclitaxel is greatly limited, so that the development of a quercetin administration system with good dissolving performance is concerned. The heparin has strong water solubility, and can be used as a carrier, so that the water solubility of the drug is improved, the retention time of the drug in vivo can be prolonged, and the targeted therapy effect of the preparation on tumors is realized. The invention combines quercetin, paclitaxel and heparin for the first time, and is a quercetin-heparin-paclitaxel conjugate preparation which can effectively improve the solubility of the medicine and enhance the tumor inhibition effect of the medicine.

In addition, the preparation method adopted by the invention is simple and easy to implement, has mild conditions, and effectively avoids the damage of severe reaction conditions on the activity of the quercetin and paclitaxel medicaments. The method has good universality and can be widely used in the preparation process of combining heparin as a carrier with other medicines.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a schematic diagram of the synthesis of PTX-LWMH-QT described in example 1;

wherein, (1) is the synthesis of QT-NHS; (2) for the synthesis of PTX-Phe, (3) for the synthesis of PTX-LWMH-QT.

FIG. 2 is a diagram of PTX-LWMH-QT as described in example 2¹H-NMR spectrum.

FIG. 3 shows the staining results of the living and dead cells of MCF-7 cells treated with free PTX, QT or PTX-LWMH-QT in example 3;

wherein, FIG. 3(A) shows the staining results of the free drug group; FIG. 3(B) is a graph showing the results of staining in the PTX-LWMH-QT solution group.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

Interpretation of terms:

EDCI: 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride, CAS No.: 25952-53-8.

HOBt: 1-hydroxybenzotriazole, CAS No.: 2592-95-2.

DMAP 4-dimethylaminopyridine, CAS number: 1122-58-3.

NHS: n-hydroxysuccinimide, CAS No.: 6066-82-6.

As introduced in the background technology, aiming at the problems of low solubility, low bioavailability and limited clinical application of quercetin and paclitaxel, the invention provides a paclitaxel-low molecular heparin-quercetin conjugate which is simple and convenient in preparation method, can well retain the medicinal activity of quercetin and paclitaxel, has good active targeting property and has good clinical treatment significance.

Preferably, the molecular weight of the low molecular heparin is 4000-4500 Da; more preferably, the molecular weight of the low molecular heparin is 4350 Da.

Preferably, the synthesis method of the adipic acid dihydrazide modified low-molecular heparin is as follows: adding adipic acid Amide (ADH), EDCI and HOBt into a low molecular heparin aqueous solution, adding alkali liquor to adjust the pH value of the solvent system to 6-7, stirring at room temperature to obtain a crude product, and dialyzing to obtain the LWMH-ADH.

Further preferably, the mass ratio of the low molecular heparin to the ADH is 1: 2 to 16.

Further preferably, the mass ratio of the low molecular heparin to the EDCI is 1: 1-4.

Further preferably, the mass ratio of the low molecular heparin to the HOBt is 1: 1-4.

Further preferably, the reaction time is 12-48 h under stirring at room temperature.

Further preferably, the dialysis is water dialysis, the dialysis time is 2.5-3 days, and the water is changed every 6 hours.

Preferably, the synthesis method of the quercetin-NHS ester comprises the following steps: adding a catalyst into a tetrahydrofuran solution of quercetin and succinic anhydride, reacting in an inert gas atmosphere to obtain a quercetin hemisuccinic acid intermediate, dissolving the intermediate in acetonitrile, adding N-hydroxysuccinimide diphenyl phosphate (SDPP) and the catalyst, and stirring in the inert gas atmosphere to react to obtain the succinic anhydride modified quercetin, namely a QT-NHS ester crude product.

Further preferably, the molar ratio of the quercetin to the succinic anhydride is 1: 1-5.

Further preferably, the dosage of the quercetin and the tetrahydrofuran is 1-5 mg/mL.

More preferably, the molar ratio of the quercetin to the catalyst is 1: 1-20.

Further preferably, the catalyst is selected from any one or a mixture of pyridine, triethylamine and carbodiimide hydrochloride.

Preferably, the synthesis method further comprises a step of purifying the intermediate, wherein the purification operations comprise removing the solvent, alkali washing, ethyl acetate extraction, saturated salt water washing and adding anhydrous Na₂SO₄Drying, filtering and evaporating the solvent.

Further preferably, the molar ratio of the intermediate to the SDPP is 1: 1-5.

Further preferably, the dosage of the intermediate and acetonitrile is 1-5 mg/L.

More preferably, the molar ratio of the intermediate to the catalyst is 1: 1-10.

Preferably, the method for synthesizing the low molecular heparin-quercetin (LWMH-QT) comprises the following steps: adding QT-NHS ester into phosphate buffer solution of LWMH-ADH, reacting for a period of time under inert gas atmosphere at room temperature, and dialyzing the reaction solution to obtain the low molecular heparin-quercetin.

Further preferably, the mass ratio of QT-NHS to LWMH-ADH is 1: 3 to 20.

Further preferably, the reaction time is 24-48 h.

Further preferably, after the reaction is finished, the DMSO is dialyzed for 2 days, the solution is changed every 6h, and then the water is dialyzed for 3 days, and the water is changed every 6 h.

Preferably, the hydroxylated paclitaxel is synthesized as follows: dissolving paclitaxel, succinic anhydride and DMAP into an organic solvent together, reacting under the condition of inert gas atmosphere and room temperature, removing the organic solvent after the reaction is finished to obtain the product, and washing the product to obtain PTX-COOH.

Further preferably, the mass ratio of the paclitaxel to the succinic anhydride is 1-2: 1.

further preferably, the mass ratio of the paclitaxel to the DMAP is 20-50: 1.

further preferably, the reaction time is 12-48 h.

Preferably, the paclitaxel-low molecular heparin-quercetin conjugate (PTX-LWMH-QT) is synthesized as follows: adding PTX-COOH, EDCI and NHS into a formamide solution of LWMH-QT, and stirring at room temperature for reaction to obtain the PTX-LWMH-QT.

Further preferably, the mass ratio of PTX-COOH to LWMH-QT is 1: 3 to 20.

Further preferably, the molar ratio of PTX-COOH to EDCI and NHS is 1: 1-5: 1 to 5.

Further preferably, the reaction time is 12-48 h.

Further preferably, the synthesis reaction also comprises the step of using DMF/H after the synthesis reaction is finished₂And (4) carrying out dialysis on the product by using the mixed solvent.

In some embodiments with better effects, the preparation method comprises the following specific steps:

(1) synthesis of adipic acid dihydrazide-modified Low molecular heparin (LWMH-ADH):

weighing a certain amount of low molecular weight heparin, dissolving the low molecular weight heparin in distilled water, stirring to fully dissolve the low molecular weight heparin, and adding ADH, EDCI and HOBt. And (3) adjusting the pH value of the system to 6.8 by using 0.1M NaOH solution, stirring and reacting for a period of time at room temperature to obtain a crude product, fully dialyzing water, filtering the dialyzate, and freeze-drying to obtain the LWMH-ADH.

(2) Synthesis of Quercetin-NHS ester (QT-NHS):

dissolving quercetin and succinic anhydride in tetrahydrofuran, adding catalyst, N₂Stirring and reacting at room temperature for 1-3 days under protection, removing impurities, purifying to obtain a reaction intermediate quercetin hemisuccinic acid, dissolving the obtained intermediate in acetonitrile, adding N-hydroxysuccinimide diphenyl phosphate (SDPP) and a catalyst, and reacting N₂Stirring and reacting for 2-10 h at room temperature under protection, and carrying out aftertreatment on the reaction liquid to obtain succinic anhydride modified quercetin, namely a QT-NHS ester crude product.

(3) Synthesis of Low molecular heparin-Quercetin (LWMH-QT):

a certain amount of LWMH-ADH was dissolved in phosphate buffer and QT-NHS ester was added. N is a radical of₂Stirring at room temperature under protection, reacting for a while, dialyzing the reaction solution against DMSO for 2-3 days, dialyzing with pure water, filtering the dialyzed solution, and lyophilizing to obtain LWMH-QT.

(4) Synthesis of carboxylated paclitaxel (PTX-COOH):

dissolving paclitaxel, succinic anhydride and DMAP together in DCM, and reacting at room temperature under the protection of nitrogen. And after the reaction is finished, removing DCM by rotary evaporation, dissolving the product in ethyl acetate, washing the product with dilute hydrochloric acid and water respectively, adding anhydrous sodium sulfate into an organic phase, filtering, removing ethyl acetate by rotary evaporation, and drying the product in vacuum to obtain PTX-COOH.

(5) Synthesis of paclitaxel-Low molecular heparin-Quercetin conjugate (PTX-LWMH-QT)

Weighing a certain amount of LWMH-QT, adding formamide, dissolving under magnetic stirring, adding PTX-COOH, EDCI and NHS, and reacting at room temperature for 1-2 days under magnetic stirring to obtain a crude product of the PTX-LWMH-QT. The crude product is initially para to DMF/H₂Dialyzing with mixed solvent O, and dialyzing with distilled water. Filtering the dialyzed liquid, and freeze-drying to obtain PTX-LWMH-QT.

Preferably, the anti-tumor drug is in a dosage form including, but not limited to, oral dosage form, parenteral dosage form, external dosage form and rectal dosage form.

Further preferably, the anti-tumor drug is an oral tablet, capsule, pill, powder, sustained release preparation, solution or suspension.

Further preferably, the anti-tumor drug is a sterile solution, suspension or emulsion for parenteral injection.

Preferably, in the antitumor drug, the paclitaxel-low molecular heparin-quercetin conjugate is a nanoparticle preparation.

Further preferably, the preparation method of the nanoparticle preparation is as follows: and (3) dispersing PTX-LWMH-QT in a buffer salt solution, and filtering for a period of time by ultrasonic treatment to obtain the nanoparticle preparation.

Further, the PTX-LWMH-QT is dissolved in PBS (pH 7.4,10mM) at a concentration of 0.1-10 mg/mL.

Furthermore, the ultrasonic is probe ultrasonic, the ultrasonic power of the probe is 30-40W, the pulse is 2s on/4 s off, and the ultrasonic time is 2-30 min.

Furthermore, the sample after ultrasonic treatment is filtered by a 0.45 or 0.8 μm microporous membrane to obtain PTX-LWMH-QT self-assembled nanoparticle solution.

Preferably, the anti-tumor drug comprises the paclitaxel-low molecular weight heparin-quercetin conjugate of the first aspect and pharmaceutically necessary excipients.

Further preferably, the paclitaxel-low molecular heparin-quercetin conjugate accounts for 1-99% of the total amount of the antitumor drug, and the antitumor drug is in a unit dosage form suitable for single administration of precise dosage.

In some embodiments of the above preferred embodiments, the amount of said anti-neoplastic agent ranges from about 0.001mg/kg body weight/day to about 1000mg/kg body weight/day.

In other embodiments, the amount of the anti-neoplastic agent ranges from about 0.5mg/kg body weight/day to about 50mg/kg body weight/day.

In some embodiments, the amount of the anti-neoplastic agent is from about 0.001 g/day to about 7 g/day.

In other embodiments, the amount of the anti-neoplastic agent is from about 0.002 g/day to about 6 g/day.

In other embodiments, the amount of the anti-neoplastic agent is from about 0.005 g/day to about 5 g/day.

In other embodiments, the amount of the anti-neoplastic agent is from about 0.01 g/day to about 5 g/day.

In other embodiments, the amount of the anti-neoplastic agent is from about 0.02 g/day to about 5 g/day.

In other embodiments, the amount of the anti-neoplastic agent is from about 0.05 g/day to about 2.5 g/day.

In other embodiments, the amount of the anti-neoplastic agent is from about 0.1 g/day to about 1 g/day.

In other embodiments, dosage levels below the lower limit of the aforesaid range may be more than adequate.

In other embodiments, dosage levels above the upper limit of the range recited above may be desired.

Preferably, the anti-tumor drug comprises the paclitaxel-low molecular weight heparin-quercetin conjugate in the first aspect, pharmaceutically necessary auxiliary materials, and other components with anti-tumor activity or auxiliary anti-tumor activity.

Further preferably, the other ingredients with anti-tumor activity include chemical entities with different treatment mechanisms of quercetin and paclitaxel.

Further preferably, the component having auxiliary anti-tumor activity includes, but is not limited to, tumor auxiliary drugs, immunomodulatory drugs, anti-inflammatory drugs, radiosensitizing drugs, antiemetic drugs, etc. specified in the national health insurance catalogue.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

EXAMPLE 1 preparation of PTX-LWMH-QT

Synthesis of LWMH-QT

(1) 0.25g of LWMH was weighed out and dissolved in 50mL of distilled water, stirred to dissolve it sufficiently, and then 4g of ADH, 0.48g of EDCI, and 0.48g of HOBt were added. Adjusting the pH value of the system to 6.8 by using 0.1M NaOH solution, stirring and reacting for 24h at room temperature to obtain a crude product, dialyzing for 3 days with water, changing water every 6h, filtering the dialyzate, and freeze-drying to obtain LWMH-ADH.

(2) QT 302mg and succinic anhydride 200mg were weighed, dissolved together in 10mL tetrahydrofuran, and 805. mu.L pyridine was added and reacted under nitrogen for 3 days. And (3) removing the solvent in the reaction solution by rotary evaporation, washing with alkali, washing with saturated salt water, drying, and passing through a silica gel column to obtain an intermediate quercetin hemisuccinic acid.

(3) Dissolving 1.15g N-hydroxysuccinimide in 6mL of dichloromethane, adding 1.4mL of triethylamine under ice bath, adding 2.7mg of diphenyl chlorophosphate dissolved in dichloromethane dropwise, reacting for 2h under ice bath, dissolving the product with ethyl acetate, washing with water, drying, and filtering to obtain N-hydroxysuccinimide diphenyl phosphate (SDPP)

(4) And (3) dissolving 230mg of quercetin hemisuccinic acid obtained in the step (2) in 15mL of acetonitrile, adding 260mg of SDPP and 280 mu L of triethylamine, reacting for 8 hours in a dark place under the protection of nitrogen, dissolving the product with ethyl acetate, washing with water, drying, filtering, and evaporating the organic solvent to obtain QT-NHS ester.

(5) 50mg of LWMH-ADH was weighed out and dissolved in 50mL of phosphate buffer (pH 6.5); 6mg QT-NHS was dissolved in 100mL DMSO. Adding a DMSO solution of QT-NHS into an LWMH-ADH solution, reacting for 2 days under the protection of nitrogen, dialyzing DMSO for 2 days, dialyzing distilled water for 3 days, filtering the dialyzed liquid, and freeze-drying to obtain LWMH-QT solid.

Synthesis of PTX-LWMH-QT

(1) 100mg of paclitaxel, 65mg of succinic anhydride and 3mg of DMAP were dissolved together in 5mL of DCM and reacted at room temperature for one day under nitrogen. And after the reaction is finished, removing DCM by rotary evaporation, dissolving the product in ethyl acetate, washing the product with dilute hydrochloric acid and water respectively (washing for 3 times respectively), adding anhydrous sodium sulfate into an organic phase, filtering, removing ethyl acetate by rotary evaporation, and placing the product in a vacuum drying oven overnight to obtain PTX-COOH.

(2) LWMH-QT (100 mg) was weighed and dissolved in 40mL of formamide. Weighing 20mg of PTX-COOH, adding 40mL of DMF for dissolving, adding EDCI and NHS with 3 times of molar equivalent of PTX-COOH, stirring for 30min, adding the LWMH-QT formamide solution, reacting for 2 days at room temperature, dialyzing the DMSO for 2 days, dialyzing distilled water for 3 days, filtering the dialyzed liquid, and freeze-drying to obtain the PTX-LWMH-QT solid.

Example 2 preparation of PTX-LWMH-QT self-assembled nanoparticles

5mg of PTX-LWMH-QT was weighed, dispersed in 3mL of PBS (pH 7.4,10mM), and after stirring well, the sample was sonicated (40W, 2s on/4 s off for 4 min). And filtering the sample subjected to ultrasonic treatment by a 0.8-micron microporous filter membrane to obtain the PTX-LWMH-QT self-assembled nanoparticle solution.

Weighing 10mg of PTX-LWMH-QT, using d₆-DMSO/D₂Dissolving it in O mixed solvent, and¹H-NMR analysis gave the spectrum shown in FIG. 2, with the appearance of characteristic absorption peaks for PTX and QT, confirming the successful synthesis of the polymer.

Example 3 cytotoxicity examination of PTX-LWMH-QT

A PTX-LWMH-QT solution was prepared as described under example 2, and the solution obtained was diluted to a PTX concentration of 5. mu.g/mL; separately prepared is free PTX, QT solution with the same concentration. After MCF-7 cells are respectively incubated with PTX-LWMH-QT solution or free PTX and QT solution for 12h, cells are stained by using a Calcein-AM/PI living cell/dead cell double staining kit, observed under an inverted fluorescence microscope and photographed (the result is shown in figure 3). The results show that PTX-LWMH-QT can enhance the cytotoxicity of the drug.

As can be seen from FIG. 3, the number of viable cells after a period of free drug incubation was significantly greater than that of the PTX-LWMH-QT-conferred group, demonstrating that PTX-LWMH-QT had greater tumor cytotoxicity than free drug.

Example 4

15mg of PTX-LWMH-QT was weighed out, dispersed in 3mL of PBS (pH 7.4,10mM), and after stirring well, the samples were sonicated (40W, 2s on/4 s off for 10 min). And filtering the sample subjected to ultrasonic treatment by a 0.8-micron microporous filter membrane to obtain the PTX-LWMH-QT self-assembled nanoparticle solution. The concentration of PTX was 275. mu.g/mL by HPLC and the concentration of QT was 262. mu.g/mL by UV spectrophotometry.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The conjugate of paclitaxel-low molecular heparin-quercetin is characterized in that the conjugate takes the low molecular heparin as a skeleton, and the paclitaxel and the quercetin are connected with the low molecular heparin through covalent bonds.

2. The paclitaxel-low molecular weight heparin-quercetin conjugate according to claim 1, wherein the molecular weight is 4000-4500 Da; more preferably, the molecular weight of the low molecular heparin is 4350 Da.

3. The method of preparing paclitaxel-low molecular weight heparin-quercetin conjugate according to claim 1 or 2, wherein the preparation method comprises the steps of: respectively synthesizing adipic acid dihydrazide modified low-molecular heparin, quercetin-NHS ester and carboxylated paclitaxel, and synthesizing the low-molecular heparin-quercetin by using the adipic acid dihydrazide modified low-molecular heparin and the quercetin-NHS ester as raw materials; mixing low molecular heparin-quercetin with carboxylated paclitaxel, and reacting to obtain paclitaxel-low molecular heparin-quercetin conjugate.

4. The method of claim 3, wherein the adipic acid dihydrazide-modified low molecular weight heparin is synthesized by the following steps: adding adipic acid amide, EDCI and HOBt into a low molecular heparin aqueous solution, adding alkali liquor to adjust the pH value of the solvent system to 6-7, stirring at room temperature to obtain a crude product, and dialyzing to obtain the LWMH-ADH;

preferably, the mass ratio of the low molecular heparin to the ADH is 1: 2-16;

preferably, the mass ratio of the low molecular heparin to the EDCI is 1: 1-4;

preferably, the mass ratio of the low molecular heparin to the HOBt is 1: 1-4;

preferably, the stirring reaction time at room temperature is 12-48 h;

preferably, the dialysis is water dialysis, the time is 2.5-3 days, and the water is changed every 6 hours.

5. The method of claim 3, wherein the quercetin-NHS ester is synthesized by the following steps: adding a catalyst into a tetrahydrofuran solution of quercetin and succinic anhydride, reacting under an inert gas atmosphere to obtain a quercetin hemisuccinic acid intermediate, dissolving the intermediate in acetonitrile, adding N-hydroxysuccinimide diphenyl phosphate and the catalyst, and stirring under the inert gas atmosphere to react to obtain the succinic anhydride modified quercetin, namely a QT-NHS ester crude product;

preferably, the molar ratio of the quercetin to the succinic anhydride is 1: 1-5;

preferably, the dosage of the quercetin and the tetrahydrofuran is 1-5 mg/mL;

preferably, the molar ratio of the quercetin to the catalyst is 1: 1-20;

preferably, the catalyst is selected from any one or a mixture of pyridine, triethylamine and carbodiimide hydrochloride;

preferably, the synthesis method further comprises the step of purifying the intermediate, wherein the purification operations comprise solvent removal, alkali washing, ethyl acetate extraction, saturated salt water washing and anhydrous Na addition in sequence₂SO₄Drying, filtering and evaporating the solvent;

preferably, the molar ratio of the intermediate to the SDPP is 1: 1-5;

preferably, the dosage of the intermediate and acetonitrile is 1-5 mg/L;

preferably, the molar ratio of the intermediate to the catalyst is 1: 1-10.

6. The method of claim 3, wherein the paclitaxel-low molecular heparin-quercetin conjugate is synthesized by the following steps: adding QT-NHS ester into phosphate buffer solution of LWMH-ADH, reacting for a period of time at room temperature in an inert gas atmosphere, and dialyzing the reaction solution to obtain the low molecular heparin-quercetin;

further preferably, the mass ratio of QT-NHS to LWMH-ADH is 1: 3-20;

further preferably, the reaction time is 24-48 h;

7. The method of claim 3, wherein the hydroxylated paclitaxel is synthesized by: dissolving paclitaxel, succinic anhydride and DMAP into an organic solvent together, reacting under the condition of inert gas atmosphere and room temperature, removing the organic solvent after the reaction is finished to obtain a product, and washing the product to obtain PTX-COOH;

further preferably, the mass ratio of the paclitaxel to the succinic anhydride is 1-2: 1;

further preferably, the mass ratio of the paclitaxel to the DMAP is 20-50: 1;

further preferably, the reaction time is 12-48 h.

8. The method of claim 3, wherein the paclitaxel-low molecular heparin-quercetin conjugate is synthesized by the following steps: adding PTX-COOH, EDCI and NHS into a formamide solution of LWMH-QT, and stirring at room temperature for reaction to obtain the PTX-LWMH-QT;

preferably, the mass ratio of PTX-COOH to LWMH-QT is 1: 3-20;

preferably, the molar ratio of PTX-COOH to EDCI and NHS is 1: 1-5: 1-5;

preferably, the reaction time is 12-48 h;

preferably, the method also comprises the step of adopting DMF/H after the synthesis reaction is finished₂And (4) carrying out dialysis on the product by using the mixed solvent.

9. Use of the paclitaxel-low molecular heparin-quercetin conjugate according to claim 1 or 2 for preparing an antitumor drug;

preferably, the anti-tumor drug is in a dosage form including but not limited to oral dosage forms, parenteral dosage forms, external dosage forms and rectal dosage forms;

further preferably, the anti-tumor drug is an oral tablet, capsule, pill, powder, sustained-release preparation, solution or suspension;

further preferably, the anti-tumor drug is a sterile solution, suspension or emulsion for parenteral injection;

or, preferably, in the antitumor drug, the paclitaxel-low molecular heparin-quercetin conjugate is a nanoparticle preparation;

further preferably, the preparation method of the nanoparticle preparation is as follows: dispersing PTX-LWMH-QT in a buffer salt solution, and filtering for a period of time by ultrasonic treatment to obtain the nanoparticle preparation;

furthermore, the PTX-LWMH-QT is dissolved in PBS, and the concentration is 0.1-10 mg/mL;

furthermore, the ultrasonic is probe ultrasonic, the ultrasonic power of the probe is 30-40W, the pulse is 2s on/4 s off, and the ultrasonic time is 2-30 min;

10. An antitumor agent comprising the paclitaxel-low molecular heparin-quercetin conjugate according to claim 1 or 2 and a pharmaceutically necessary excipient;

preferably, the anti-tumor drug consists of the paclitaxel-low molecular weight heparin-quercetin conjugate in the first aspect and pharmaceutically necessary auxiliary materials;

further preferably, the paclitaxel-low molecular heparin-quercetin conjugate accounts for 1-99% of the total amount of the antitumor drug, and the antitumor drug is in a unit dosage form suitable for single administration of precise dosage;

or, preferably, the anti-tumor drug comprises the paclitaxel-low molecular weight heparin-quercetin conjugate according to claim 1 or 2, pharmaceutically necessary auxiliary materials, and other components with anti-tumor activity or auxiliary anti-tumor activity;

further preferably, the other ingredients with anti-tumor activity include chemical entities with different treatment mechanisms of quercetin and paclitaxel;

further preferably, the component with auxiliary anti-tumor activity includes, but is not limited to, tumor auxiliary drugs, immunomodulatory drugs, anti-inflammatory drugs, radiosensitizing drugs, and antiemetic drugs specified in the national health insurance catalogue.