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

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

A kind of quercetin-low molecular weight heparin-paclitaxel conjugate, preparation method and application

technical field

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

Background technique

The information disclosed in this Background section is only for enhancement of understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information forms the prior art 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 Taxus chinensis, is mainly used in clinical treatment of ovarian cancer, small cell lung cancer and breast cancer; for other types of cancer, Such as prostate cancer, nasopharyngeal cancer, etc. also have a certain effect. However, the solubility of PTX in water is less than 4 μg/mL, which greatly limits its application. At present, Taxol, a taxol injection widely used clinically, is prepared from polyoxyethylene castor oil/ethanol as a solubilizing carrier, but polyoxyethylene castor oil can cause severe allergic reactions, causing inconvenience to patients.

Quercetin (QT), a polyphenol flavonoid extracted from natural plants, has a wide range of pharmacological effects and can be used as an effective antioxidant for the treatment of diseases such as inflammation, tumors, and viral infections. Studies have shown that quercetin is a broad-spectrum anticancer drug, which has a good inhibitory effect on a variety of cancers such as liver cancer, lung cancer, breast cancer, and ovarian cancer. However, quercetin has extremely low water solubility (1 μg/mL solubility in water), is unstable in the gastrointestinal tract, and has low oral bioavailability, which greatly limits its clinical application.

Heparin is a natural, heterogeneous polysaccharide that has been used clinically for decades as an anticoagulant drug. However, the use of heparin often brings many adverse reactions, such as allergic reactions, hemorrhagic complications and thrombocytopenia. In order to avoid these problems, low molecular weight heparin (LWMH) is often used as a substitute for heparin. Low-molecular-weight heparin is a general term for a class of heparin with lower molecular weight depolymerized from unfractionated heparin, which has the advantages of high bioavailability and less toxic effects. In recent years, it has become a new trend to use heparin/low molecular weight heparin as carrier materials to construct various nanocarriers for tumor therapy. The reasons mainly depend on the following properties of heparin: (1) non-toxicity and good biocompatibility; (2) anti-tumor activity; (3) anti-tumor metastasis activity; (4) targeting.

SUMMARY OF THE INVENTION

According to the current research results in this field, the co-treatment of chemotherapeutic drugs with different mechanisms can effectively improve the tumor killing effect and reduce the probability of tumor drug resistance. The present invention is to provide a drug delivery system that simultaneously delivers paclitaxel and quercetin, improves the adverse biological properties of paclitaxel and quercetin, improves their solubility and bioavailability, and at the same time can play a "multi-pronged" anti-tumor effect Effect. Based on this technical purpose, the present invention designs a paclitaxel-low molecular weight heparin-quercetin conjugate, and connects two drugs to the low molecular weight heparin skeleton at the same time to obtain an amphiphilic conjugate, which can be self-assembled in water into a Nanoparticles with a core-shell structure, on the one hand, the invention improves the solubility of the two drugs, and on the other hand, the two drugs can exert a combined anti-tumor effect.

Based on the above object, the present invention provides the following technical solutions:

A first aspect of the present invention provides a combination of paclitaxel-low molecular weight heparin-quercetin, wherein the combination uses low molecular weight heparin as a skeleton, and the paclitaxel and quercetin are linked to the low molecular weight heparin through covalent bonds.

A second aspect of the present invention provides a method for preparing the taxol-low molecular weight heparin-quercetin conjugate described in the first aspect, the preparation method comprising the steps of: synthesizing adipic acid dihydrazide-modified low molecular weight heparin (LWMH- ADH), quercetin-NHS ester (QT-NHS) and carboxylated paclitaxel (PTX-COOH), adipic acid dihydrazide modified low molecular weight heparin and quercetin-NHS ester were used as raw materials to synthesize low molecular weight heparin-quercetin Cortexin (LWMH-QT); the paclitaxel-low molecular weight heparin-quercetin conjugate (PTX-LWMH-QT) was obtained by mixing low molecular weight heparin-quercetin with carboxylated paclitaxel.

The third aspect of the present invention provides the application of the taxol-low molecular weight heparin-quercetin conjugate described in the first aspect in the preparation of antitumor drugs.

A fourth aspect of the present invention provides an anti-tumor drug, comprising the paclitaxel-low-molecular-weight heparin-quercetin conjugate described in the first aspect and pharmaceutically necessary excipients.

The beneficial effects of the above one or more technical solutions are:

Quercetin and paclitaxel have poor water solubility, are unstable in the gastrointestinal tract, and have low oral bioavailability, which greatly limits their clinical applications. Therefore, the development of quercetin drug delivery systems with good solubility has attracted much attention. Heparin is highly water-soluble, and as a carrier, while improving the water-solubility of the drug, it can also prolong the residence time of the drug in the body, so as to achieve the targeted therapeutic effect of the preparation on tumors. The present invention combines quercetin, paclitaxel and heparin for the first time, which is a quercetin-heparin-paclitaxel conjugate preparation which can effectively improve the solubility of the drug and enhance its tumor-inhibiting effect.

In addition, the preparation method adopted in the present invention is simple and easy to implement, and the conditions are mild, which effectively avoids the destruction of the medicinal activities of quercetin and paclitaxel under severe reaction conditions. And the method has good versatility, and can be widely used in the preparation process of combining heparin with other medicines.

Description of drawings

The accompanying drawings forming a part of the present invention are used to provide further understanding of the present invention, and the exemplary embodiments of the present invention and their descriptions are used to explain the present invention, and do not constitute an improper limitation of the present invention.

1 is a schematic diagram of the synthesis method of PTX-LWMH-QT described in Example 1;

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

FIG. 2 is the ¹ H-NMR spectrum of PTX-LWMH-QT described in Example 2. FIG.

Figure 3 is the result of live and dead cell staining of MCF-7 cells treated with free PTX, QT or PTX-LWMH-QT in Example 3;

Among them, Figure 3(A) is the staining result of the free drug group; Figure 3(B) is the staining result of the PTX-LWMH-QT solution group.

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the invention. Unless otherwise defined, 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 should be noted that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural as well, furthermore, it is to be understood that when the terms "comprising" and/or "including" are used in this specification, it indicates that There are features, steps, operations, devices, components and/or combinations thereof.

Terminology Explanation:

EDCI: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, CAS number: 25952-53-8.

HOBt: 1-Hydroxybenzotriazole, CAS number: 2592-95-2.

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

NHS: N-hydroxysuccinimide, CAS number: 6066-82-6.

As described in the background art, in view of the problems of low solubility, low bioavailability and limited clinical application of quercetin and paclitaxel, the present invention proposes a combination of paclitaxel-low molecular weight heparin-quercetin, which is prepared by The method is simple, can well retain the medicinal activities of quercetin and paclitaxel, has good active targeting, and has good clinical therapeutic significance.

A first aspect of the present invention provides a combination of paclitaxel-low molecular weight heparin-quercetin, wherein the combination uses low molecular weight heparin as a skeleton, and the paclitaxel and quercetin are linked to the low molecular weight heparin through covalent bonds.

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

A second aspect of the present invention provides a method for preparing the taxol-low molecular weight heparin-quercetin conjugate described in the first aspect, the preparation method comprising the steps of: synthesizing adipic acid dihydrazide-modified low molecular weight heparin (LWMH- ADH), quercetin-NHS ester (QT-NHS) and carboxylated paclitaxel (PTX-COOH), adipic acid dihydrazide modified low molecular weight heparin and quercetin-NHS ester were used as raw materials to synthesize low molecular weight heparin-quercetin Cortexin (LWMH-QT); the paclitaxel-low molecular weight heparin-quercetin conjugate (PTX-LWMH-QT) was obtained by mixing low molecular weight heparin-quercetin with carboxylated paclitaxel.

Preferably, the synthesis method of the adipic acid dihydrazide modified low molecular weight heparin is as follows: adding adipic acid amide (ADH), EDCI, HOBt to the aqueous solution of low molecular weight heparin, and adding lye to adjust the pH value of the solvent system To 6-7, the crude product was obtained by stirring at room temperature, and the LWMH-ADH was obtained after dialysis.

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

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

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

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

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

Preferably, the method for synthesizing the quercetin-NHS ester is as follows: adding a catalyst to a tetrahydrofuran solution of quercetin and succinic anhydride, reacting under an inert gas atmosphere to obtain a quercetin hemisuccinic acid intermediate, and adding the intermediate The body is dissolved in acetonitrile, N-hydroxysuccinimide diphenyl phosphate (SDPP) and a catalyst are added, and the reaction is stirred under an inert gas atmosphere to obtain the succinic anhydride-modified quercetin, that is, the crude product of QT-NHS ester.

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

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

Further 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.

Further preferably, the synthetic method also includes the step of purifying the intermediate, and the purification operations are sequentially followed by removing the solvent, washing with alkali, extracting with ethyl acetate, washing with saturated brine, adding anhydrous Na ₂ SO ₄ to dry, filtering, The step of evaporating the solvent.

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

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

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

Preferably, the low-molecular-weight heparin-quercetin (LWMH-QT) synthesis method is as follows: adding QT-NHS ester to the phosphate buffer of LWMH-ADH, reacting the reaction solution for a period of time at room temperature in an inert gas atmosphere The low molecular weight heparin-quercetin was obtained by dialysis.

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

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

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

Preferably, the synthesis method of the hydroxylated paclitaxel is as follows: co-dissolving paclitaxel, succinic anhydride and DMAP in an organic solvent, reacting in an inert gas atmosphere at room temperature, removing the organic solvent after the reaction to obtain the product, washing the The product is PTX-COOH.

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

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

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

Preferably, the synthetic method of the paclitaxel-low molecular weight heparin-quercetin conjugate (PTX-LWMH-QT) is as follows: adding PTX-COOH, EDCI and NHS to the formamide solution of LWMH-QT, stirring and reacting at room temperature to obtain The PTX-LWMH-QT.

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

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

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

Further preferably, after the synthesis reaction is completed, it also includes the step of using DMF/H ₂ O mixed solvent to dialyze the product against water.

In some embodiments with better effects of the present invention, the specific steps of the preparation method are as follows:

(1) Synthesis of adipic acid dihydrazide modified low molecular weight heparin (LWMH-ADH):

Weigh a certain amount of low molecular weight heparin and dissolve it in distilled water, stir to fully dissolve it, and then add ADH, EDCI, and HOBt. The pH of the system was adjusted to 6.8 with 0.1M NaOH solution, and the crude product was obtained after stirring and reacting at room temperature for a period of time, which was fully dialyzed against water, and the dialysate was filtered and freeze-dried to obtain LWMH-ADH.

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

Quercetin and succinic anhydride are dissolved in tetrahydrofuran, a catalyst is added, and the reaction is stirred at room temperature for 1 to 3 days under the protection of N ₂ , and the reaction intermediate is obtained by removing impurities and purifying to obtain the intermediate quercetin hemisuccinic acid. The obtained intermediate is dissolved in acetonitrile , adding N-hydroxysuccinimide diphenyl phosphate (SDPP) and catalyst, stirring at room temperature for 2-10 h under the protection of N ₂ , and post-processing the reaction solution to obtain quercetin modified with succinic anhydride, namely QT-NHS Crude ester.

(3) Synthesis of low molecular weight heparin-quercetin (LWMH-QT):

A certain amount of LWMH-ADH was dissolved in phosphate buffer and QT-NHS ester was added. After a period of time after stirring the reaction at room temperature under the protection of _N2 , the reaction solution was dialyzed against DMSO for 2-3 days, fully dialyzed against pure water, and the dialyzed solution was filtered and freeze-dried to obtain LWMH-QT.

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

Paclitaxel, succinic anhydride and DMAP were co-dissolved in DCM and reacted at room temperature under nitrogen protection. After the reaction, DCM was removed by rotary evaporation, the product was dissolved in ethyl acetate, the product was washed with dilute hydrochloric acid and water respectively, anhydrous sodium sulfate was added to the organic phase, filtered, the ethyl acetate was removed by rotary evaporation, and the product was dried in vacuo, That is, PTX-COOH is obtained.

(5) Synthesis of paclitaxel-low molecular weight heparin-quercetin conjugate (PTX-LWMH-QT)

Weigh a certain amount of LWMH-QT, add formamide, dissolve under magnetic stirring, add PTX-COOH, EDCI, NHS, and react at room temperature for 1-2 days under magnetic stirring to obtain the crude product of PTX-LWMH-QT. The crude product was first dialyzed against DMF/H ₂ O mixed solvent, and then fully dialyzed against distilled water. The liquid obtained after dialysis was filtered and lyophilized to obtain PTX-LWMH-QT.

The third aspect of the present invention provides the application of the taxol-low molecular weight heparin-quercetin conjugate described in the first aspect in the preparation of antitumor drugs.

Preferably, the antitumor drugs include but are not limited to oral dosage forms, parenteral dosage forms, topical dosage forms and rectal dosage forms.

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

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

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

Further preferably, the preparation method of the nanoparticle preparation is as follows: the PTX-LWMH-QT is dispersed in a buffered saline solution, and the nanoparticle preparation is obtained by ultrasonic filtration for a period of time.

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

Further, the ultrasound adopts probe ultrasound, the probe ultrasound power is 30-40W, the pulse is 2s on/4s off, and the ultrasound time is 2-30min.

Further, the sonicated sample was filtered through a 0.45 or 0.8 μm microporous membrane to obtain a PTX-LWMH-QT self-assembled nanoparticle solution.

A fourth aspect of the present invention provides an anti-tumor drug, comprising the paclitaxel-low-molecular-weight heparin-quercetin conjugate described in the first aspect and pharmaceutically necessary excipients.

Preferably, the antitumor drug is composed of the paclitaxel-low molecular weight heparin-quercetin conjugate described in the first aspect and pharmaceutically necessary excipients.

Further preferably, the paclitaxel-low molecular weight heparin-quercetin conjugate accounts for 1-99% of the total amount of anti-tumor drugs, and the anti-tumor drugs are in a unit dosage form suitable for single administration of precise doses.

In some embodiments of the above preferred technical solutions, the amount of the antitumor drug is in the range of about 0.001 mg/kg body weight/day to about 1000 mg/kg body weight/day.

In other embodiments, the amount of the antineoplastic drug ranges from about 0.5 mg/kg body weight/day to about 50 mg/kg body weight/day.

In some embodiments, the amount of the antineoplastic drug is from about 0.001 g/day to about 7 g/day.

In other embodiments, the amount of the antineoplastic drug is from about 0.002 g/day to about 6 g/day.

In other embodiments, the amount of the antineoplastic drug is from about 0.005 g/day to about 5 g/day.

In other embodiments, the amount of the antineoplastic drug is from about 0.01 g/day to about 5 g/day.

In other embodiments, the amount of the antineoplastic drug is from about 0.02 g/day to about 5 g/day.

In other embodiments, the amount of the antineoplastic drug is from about 0.05 g/day to about 2.5 g/day.

In other embodiments, the amount of the antineoplastic drug is from about 0.1 g/day to about 1 g/day.

In other embodiments, dosage levels below the lower limit of the above ranges may be sufficient.

In other embodiments, dosage levels above the upper limit of the above ranges may be required.

Preferably, the antitumor drug includes the paclitaxel-low molecular weight heparin-quercetin conjugate described in the first aspect and pharmaceutically necessary excipients, and further includes other components with antitumor activity or auxiliary antitumor activity.

Further preferably, the other components with antitumor activity include quercetin and paclitaxel chemical entities with different therapeutic mechanisms.

Further preferably, the components with auxiliary anti-tumor activity include but are not limited to tumor auxiliary drugs, immunomodulatory drugs, anti-inflammatory drugs, radiosensitizing drugs, antiemetic drugs and the like specified in the National Health Insurance Catalogue.

In order to enable those skilled in the art to understand the technical solutions of the present invention more clearly, 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

1. Synthesis of LWMH-QT

(1) Weigh 0.25 g of LWMH and dissolve it in 50 mL of distilled water, stir to fully dissolve it, and add 4 g of ADH, 0.48 g of EDCI, and 0.48 g of HOBt. The pH of the system was adjusted to 6.8 with 0.1M NaOH solution, and the crude product was obtained after stirring and reacting at room temperature for 24 hours. The crude product was dialyzed against water for 3 days. The water was changed every 6 hours.

(2) Weigh 302 mg of QT and 200 mg of succinic anhydride, dissolve them together in 10 mL of tetrahydrofuran, add 805 μL of pyridine, and react under nitrogen protection for 3 days. The solvent in the reaction solution was removed by rotary evaporation, washed with alkali, washed with saturated brine, dried, and passed through a silica gel column to obtain the intermediate quercetin hemisuccinic acid.

(3) 1.15 g of N-hydroxysuccinimide was dissolved in 6 mL of dichloromethane, 1.4 mL of triethylamine was added thereto under an ice bath, and 2.7 mg of dichlorophosphoric acid dissolved in dichloromethane was added dropwise Phenyl ester, after reacting under ice bath for 2h, the product was dissolved in ethyl acetate, washed with water, dried, filtered to obtain diphenyl N-hydroxysuccinimide phosphate (SDPP)

(4) Dissolve 230 mg of quercetin hemi-succinic acid obtained in (2) in 15 mL of acetonitrile, add 260 mg of SDPP and 280 μL of triethylamine, react under nitrogen protection for 8 h in the dark, dissolve the product in ethyl acetate, wash with water, and dry , filtered, and the organic solvent was evaporated to obtain QT-NHS ester.

(5) Weigh 50 mg of LWMH-ADH and dissolve it in 50 mL of phosphate buffer (pH 6.5); dissolve 6 mg of QT-NHS in 100 mL of DMSO. The DMSO solution of QT-NHS was added to the LWMH-ADH solution, reacted under nitrogen protection for 2 days, first dialyzed DMSO for 2 days, and then dialyzed against distilled water for 3 days. QT solid.

2. Synthesis of PTX-LWMH-QT

(1) 100 mg of paclitaxel, 65 mg of succinic anhydride and 3 mg of DMAP were co-dissolved in 5 mL of DCM, and reacted at room temperature for one day under nitrogen protection. After the reaction, DCM was removed by rotary evaporation, the product was dissolved in ethyl acetate, the product was washed with dilute hydrochloric acid and water (3 times respectively), anhydrous sodium sulfate was added to the organic phase, filtered, and the ethyl acetate was removed by rotary evaporation The product was then placed in a vacuum drying oven overnight to obtain PTX-COOH.

(2) Weigh 100 mg of LWMH-QT, add 40 mL of formamide to dissolve. Weigh 20 mg of PTX-COOH, add 40 mL of DMF to dissolve, add EDCI and NHS with 3 times the molar equivalent of PTX-COOH, stir for 30 min, add the above LWMH-QT formamide solution, react at room temperature for 2 days, the reaction solution is first dialyzed against DMSO 2 days, and then dialyzed against distilled water for 3 days, the dialyzed liquid was filtered and then freeze-dried to obtain PTX-LWMH-QT solid.

Example 2 Preparation of PTX-LWMH-QT Self-Assembled Nanoparticles

Weigh 5 mg of PTX-LWMH-QT, disperse it in 3 mL of PBS (pH=7.4, 10 mM), stir evenly, and perform probe sonication on the sample (40 W, 2s on/4s off, 4min in total). The PTX-LWMH-QT self-assembled nanoparticle solution was obtained by filtering the sonicated sample through a 0.8 μm microporous membrane.

Weigh 10 mg of PTX-LWMH-QT, dissolve it in a mixed solvent of d ₆ -DMSO/D ₂ O, and perform ¹ H-NMR analysis to obtain the spectrum shown in Figure 2. The characteristic absorption peaks of PTX and QT appear, which proves the polymerization was successfully synthesized.

Example 3 Investigation of the cytotoxicity of PTX-LWMH-QT

The PTX-LWMH-QT solution was prepared as described in Example 2, and the obtained solution was diluted to a PTX concentration of 5 μg/mL; the same concentration of free PTX, QT solution was prepared separately. After incubating MCF-7 cells with PTX-LWMH-QT solution or free PTX, QT solution for 12 h, the cells were stained with Calcein-AM/PI live/dead cell double staining kit, observed under an inverted fluorescence microscope, and photographed. (See Figure 3 for the results). The results showed that PTX-LWMH-QT could enhance the cytotoxicity of the drug.

As can be seen from Figure 3, after the free drug was incubated for a period of time, the number of viable cells was significantly more than that in the PTX-LWMH-QT conferring group, proving that PTX-LWMH-QT had stronger tumor cytotoxicity than the free drug.

Example 4

Weigh 15 mg of PTX-LWMH-QT, disperse it in 3 mL of PBS (pH=7.4, 10 mM), stir evenly, and perform probe sonication on the sample (40 W, 2s on/4s off, 10min in total). The PTX-LWMH-QT self-assembled nanoparticle solution was obtained by filtering the sonicated sample through a 0.8 μm microporous membrane. The PTX concentration was determined to be 275 μg/mL by HPLC, and the QT concentration was determined to be 262 μg/mL by UV spectrophotometry.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. A taxol-low-molecular-weight heparin-quercetin conjugate, characterized in that, the conjugate uses low-molecular-weight heparin as a skeleton, and the taxol and quercetin are connected to low-molecular-weight 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; further preferably, the molecular weight of the low molecular weight heparin is 4350 Da. 3 . 3. the preparation method of the taxol-low molecular weight heparin-quercetin conjugate described in claim 1 or 2, is characterized in that, described preparation method comprises the following steps: respectively synthesizing adipic acid dihydrazide modified low molecular weight heparin, quercetin Cortex-NHS ester and carboxylated paclitaxel, using adipic acid dihydrazide modified low molecular weight heparin and quercetin-NHS ester as raw materials to synthesize low molecular weight heparin-quercetin; low molecular weight heparin-quercetin and carboxylated The paclitaxel-low molecular weight heparin-quercetin conjugate is obtained by the mixed reaction of paclitaxel. 4. the preparation method of the described taxol-low molecular weight heparin-quercetin conjugate of claim 3, it is characterised in that the adipic acid dihydrazide modified low molecular weight heparin synthesis method is as follows: add to the aqueous solution of low molecular weight heparin Adipic acid and amide, EDCI, HOBt, adding alkaline solution to adjust the pH value of the solvent system to 6-7, stirring at room temperature to obtain a crude product, and obtaining the LWMH-ADH after dialysis; Preferably, the mass ratio of the low molecular weight heparin to ADH is 1:2 to 16; Preferably, the mass ratio of the low molecular weight heparin to EDCI is 1:1-4; Preferably, the mass ratio of the low molecular weight heparin to HOBt is 1:1-4; Preferably, the stirring reaction time at room temperature is 12-48h; Preferably, the dialysis is dialysis against water, the time is 2.5 to 3 days, and the water is changed every 6 hours. 5. the preparation method of the described taxol-low molecular weight heparin-quercetin conjugate of claim 3, is characterized in that, the synthetic method of described quercetin-NHS ester is as follows: to the tetrahydrofuran solution of quercetin and succinic anhydride A catalyst is added to the medium, and the quercetin hemi-succinic acid intermediate is obtained by the reaction in an inert gas atmosphere. The reaction obtains the quercetin modified by the succinic anhydride, i.e. the QT-NHS ester crude product; Preferably, the molar ratio of the quercetin to succinic anhydride is 1:1 to 5; Preferably, the dosages of the quercetin and tetrahydrofuran are 1-5 mg/mL; Preferably, the molar ratio of the quercetin to the catalyst is 1:1 to 20; Preferably, the catalyst is selected from any one or a mixture of pyridine, triethylamine and carbodiimide hydrochloride; Preferably, the synthesis method further includes the step of purifying the intermediate, and the purification operations are sequentially followed by removing the solvent, washing with alkali, extracting with ethyl acetate, washing with saturated brine, adding anhydrous Na ₂ SO ₄ to dry, filtering, and evaporating. the step of removing solvent; Preferably, the molar ratio of the intermediate to SDPP is 1:1 to 5; Preferably, the amount 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 preparation method of the described taxol-low molecular weight heparin-quercetin conjugate of claim 3, is characterized in that, described low molecular weight heparin-quercetin synthesis method is as follows: in the phosphate buffer solution of LWMH-ADH, add QT-NHS ester, the reaction solution is dialyzed to obtain the low molecular weight heparin-quercetin after reacting for a period of time in an inert gas atmosphere at room temperature; Further preferably, the mass ratio of QT-NHS to LWMH-ADH is 1:3-20; Further preferably, the reaction time is 24～48h; Further preferably, after the reaction is completed, DMSO is dialyzed for 2 days, and the medium is changed every 6h, and then the water is dialyzed for 3 days, and the water is changed every 6h. 7. the preparation method of the described paclitaxel-low molecular weight heparin-quercetin conjugate of claim 3, is characterized in that, the synthetic mode of described hydroxylated paclitaxel is as follows: paclitaxel, succinic anhydride and DMAP are jointly dissolved in organic solvent , react in an inert gas atmosphere and at room temperature, remove the organic solvent after the reaction to obtain the product, and wash the product to obtain PTX-COOH; Further preferably, the mass ratio of the paclitaxel to succinic anhydride is 1-2:1; Further preferably, the mass ratio of the paclitaxel to DMAP is 20-50:1; Further preferably, the reaction time is 12-48h. 8. the preparation method of the described taxol-low molecular weight heparin-quercetin conjugate of claim 3, is characterized in that, the described taxol-low molecular weight heparin-quercetin conjugate synthetic method is as follows: the formamide of LWMH-QT PTX-COOH, EDCI and NHS are added to the solution, and the reaction is stirred at room temperature to obtain the PTX-LWMH-QT; Preferably, the mass ratio of the PTX-COOH to LWMH-QT is 1:3-20; Preferably, the molar ratio of the PTX-COOH to EDCI and NHS is 1:1-5:1-5; Preferably, the reaction time is 12～48h; Preferably, after the synthesis reaction is completed, the step of performing dialysis against water on the product using a DMF/H ₂ O mixed solvent is also included. 9. the application of the taxol-low molecular weight heparin-quercetin conjugate described in claim 1 or 2 in the preparation of antitumor drugs; Preferably, the antitumor drug includes but is not limited to oral dosage forms, parenteral dosage forms, external dosage forms and rectal dosage forms; Further preferably, the antitumor drug is an oral tablet, capsule, pill, powder, sustained-release preparation, solution or suspension; Further preferably, the antitumor drug is a sterile solution, suspension or emulsion for parenteral injection; Or, preferably, in the antitumor drug, the paclitaxel-low molecular weight 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 buffered saline solution, and ultrasonically filtering for a period of time to obtain the nanoparticle preparation; Further, the PTX-LWMH-QT is dissolved in PBS at a concentration of 0.1-10 mg/mL; Further, the ultrasound adopts probe ultrasound, the probe ultrasound power is 30-40W, the pulse is 2s on/4s off, and the ultrasound time is 2-30min; Further, the sonicated sample was filtered through a 0.45 or 0.8 μm microporous membrane to obtain a PTX-LWMH-QT self-assembled nanoparticle solution. 10. An anti-tumor drug, characterized in that, the anti-tumor drug comprises the taxol-low molecular weight heparin-quercetin conjugate of claim 1 or 2 and pharmaceutically necessary excipients; Preferably, the antitumor drug is composed of the paclitaxel-low molecular weight heparin-quercetin conjugate described in the first aspect and pharmaceutically necessary excipients; Further preferably, the paclitaxel-low molecular weight heparin-quercetin conjugate accounts for 1-99% of the total amount of anti-tumor drugs, and the anti-tumor drugs are in a unit dosage form suitable for single administration of precise doses; Or, preferably, the anti-tumor drug includes the taxol-low molecular weight heparin-quercetin conjugate described in claim 1 or 2 and pharmaceutically necessary excipients, and also includes other anti-tumor activity or auxiliary anti-tumor activity. the ingredients; Further preferably, the other components with anti-tumor activity include chemical entities with different therapeutic mechanisms of quercetin and paclitaxel; Further preferably, the ingredients with auxiliary anti-tumor activity include but are not limited to tumor auxiliary drugs, immunomodulatory drugs, anti-inflammatory drugs, radiosensitizing drugs, and antiemetic drugs specified in the National Health Insurance Directory.

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