CN108014346A - A kind of preparation method of methotrexate (MTX) prodrug and its double targeted nano-particles - Google Patents

Abstract

The invention discloses the preparation method of a kind of methotrexate (MTX) prodrug and its double targeted nano-particles, hyaluronic acid is dissolved in the first organic solvent, octadecylamine is dissolved in the second organic solvent, and hybrid reaction obtains hyaluronic acid acidoctadecylamine conjugate in the presence of having activator；Hyaluronic acid acidoctadecylamine conjugate is dissolved in the 3rd organic solvent, then the methotrexate (MTX) for the activation for being dissolved in the 3rd organic solvent is added thereto, and reaction obtains methotrexate (MTX) hyaluronic acid acidoctadecylamine conjugate, is methotrexate (MTX) prodrug.Then methotrexate (MTX) prodrug is added in deionized water, ultrasound is self-assembled into the double targeted nano-particles of methotrexate (MTX) prodrug in ice bath, for the spherical of 60~120nm of particle diameter, stability is good in water, methotrexate (MTX) had not only played therapeutic effect but also had played targeting, hyaluronic acid plays a part of targeting ligand so that the double targeted nano-particles of the methotrexate (MTX) prodrug have the effect of targeting and synergistic treatment, are with a wide range of applications.

Description

A kind of preparation method of methotrexate prodrug and its dual targeting nanoparticles

technical field

The invention belongs to the field of pharmaceutical anti-tumor and anti-rheumatic drug preparations, and in particular relates to a preparation method of methotrexate prodrug and its dual-targeting nanoparticles.

Background technique

Methotrexate (MTX) is a folic acid anti-metabolite agent that can bind to folic acid receptors on the surface of tumor cells and play a good targeting role. It is often used in the treatment of various malignant tumors and is also a commonly used antirheumatic drug . MTX also has many disadvantages in medicine, which can increase the toxicity of liver and kidney function, and because the mechanism of action of the drug is to act on the cytoplasm of tumor cells rather than the nucleus, it can block the DNA of tumor cells by inhibiting dihydrofolate reductase. The purpose of synthesis, so it also has a drug effect on normal biological cells, and the toxic and side effects are increased.

Hyaluronic acid (HA) is a natural biomacromolecule substance that widely exists in synovial fluid and interstitial cells. It has good biocompatibility, biodegradability, non-toxicity, non-immunogenicity, and chemical modification. Moreover, hyaluronic acid receptors are highly expressed in a variety of tumor cells, and using ligand-receptor interactions, hyaluronic acid can target the CD44 receptors overexpressed in tumor cells and improve the therapeutic effect of tumors. It is widely used in the delivery of anticancer drugs. Natural HA is easily degraded in the body, and its retention time in the body as a drug or biological material is relatively short, which hinders its development and application in new biological materials. The chemical modification of HA not only improves the ability of HA to resist degradation by hyaluronidase, but also endows HA with other special properties, which expands the application field of HA.

Nanoparticles enter the body as a drug carrier, which can effectively reduce the phagocytosis of human reticuloendothelial system (RES) macrophages. As nanoscale particles, they can pass through the intercellular space and pass through the smallest capillaries and blood-brain barrier (BBB) in the human body. , and absorbed by cells and tissues, nanoparticle drug carriers can control the release of drugs at targeted sites, reduce drug dosage, enhance drug efficacy and reduce drug toxicity. At the same time, the particle system can avoid the loss of drug activity, which is beneficial to the storage and transportation of drugs. The many advantages of nanoparticles make them a promising new drug dosage form.

The targeting of methotrexate and hyaluronic acid has a certain limit, and the combination of the two will inevitably increase the targeting of the carrier, but there is no combination of the two to prepare dual-targeting preparations or to prepare nanoparticle drug carriers. reports.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, and provide a method for preparing methotrexate prodrug and its dual-targeted nanoparticles.

One of the technical solutions adopted by the present invention to solve its technical problems is:

A preparation method of methotrexate prodrug, comprising:

1) Dissolve hyaluronic acid (HA) in the first organic solvent, heat at 20-40°C until the hyaluronic acid dissolves, then cool to room temperature, then add 1-(3-dimethylaminopropyl)-3-ethyl Carbodiimide (EDC) and N-hydroxysuccinimide (NHS), stirred for at least 1h; octadecylamine (OCA) was dissolved in the second organic solvent, and added to the above solution; the octadecylamine The molar ratio of amine to hyaluronic acid monomer is 5:1～1:1; the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to hyaluronic acid monomer , The molar ratio of N-hydroxysuccinimide to hyaluronic acid monomer is 8:1～1:1; then under the condition of cutting off oxygen, stir at 55～65℃ for at least 5h, and then stir at room temperature for 22～ 26h; the obtained mixed solution was dialyzed with water-ethanol mixed solution with a volume ratio of 1:3~1:1 for 45~50h, then dialyzed with water for 45~50h, filtered, and freeze-dried to obtain hyaluronic acid-octadecylamine conjugate ( HA-OCA);

2) Dissolve methotrexate (MTX) in a third organic solvent, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 4-dimethylaminopyridine (DMAP), stirred and reacted for at least 30min; the hyaluronic acid-octadecylamine conjugate obtained in step 1) was dissolved in the third organic solvent, and then added to the above solution; The molar ratio of the hyaluronic acid monomer in the octaamine conjugate is 5:1 to 1:1; the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and methotrexate The molar ratio of 4-dimethylaminopyridine and methotrexate is 4:1 to 1:1; then it is protected from light, reacted at room temperature for 45 to 50 hours, dialyzed with water for 45 to 50 hours, and freeze-dried to obtain methylamine The pterin prodrug is methotrexate-hyaluronic acid-octadecylamine conjugate (MTX-HA-OCA).

In one embodiment: the first organic solvent is at least one of anhydrous formamide and water.

In one embodiment: the second organic solvent is at least one of dimethylformamide and ethanol.

In one embodiment: the third organic solvent is at least one of tetrahydrofuran, dimethylsulfoxide, dimethylformamide, dimethylacetamide, anhydrous formamide, methanol, ethanol, acetonitrile, and acetone.

In one embodiment: the molecular weight cut-off of the dialysis bag used in the dialysis is 1000-10000.

Two of the technical solutions adopted by the present invention to solve the technical problems are:

A methotrexate prodrug prepared according to the above preparation method.

The third technical solution adopted by the present invention to solve the technical problems is:

A method for preparing dual-targeted nanoparticles of methotrexate prodrugs, comprising: preparing methotrexate-hyaluronic acid-octadecylamine conjugates according to the above-mentioned preparation method, dispersing them in water, and -5 Ultrasonic treatment at ~5°C, then stirring at room temperature for 22-26 hours, self-assembled to obtain methotrexate-hyaluronic acid-octadecanine dual-targeted nanoparticle dispersion, which is the dual-targeted methotrexate prodrug to nanoparticles.

In one embodiment: the power of the ultrasonic treatment is 100-300W, the duration is 3-6s, and the interval is 2-4s.

In one embodiment: the time of the ultrasonic treatment is 8-20 minutes.

Four of the technical solutions adopted by the present invention to solve the technical problems are:

A dual-targeted nanoparticle of methotrexate prodrug prepared according to the above preparation method, wherein the dual-targeted nanoparticle of methotrexate prodrug is spherical and has a particle diameter of 60-120 nm.

Compared with the background technology, this technical solution has the following advantages:

The present invention prepares methotrexate prodrug and its dual-target slow-release nanoparticles by chemically linking methotrexate, hyaluronic acid and octadecylamine, and octadecylamine forms the hydrophobic core of micelles during the self-assembly process, Methotrexate, a drug and targeting molecule, acts as the hydrophilic end of the MTX-HA-OCA molecule to form a hydrophilic shell of micelles, which makes the nanoparticles stable in water, with an average particle size of about 100nm, suitable for intravenous injection preparations , methotrexate has both therapeutic and targeting effects, hyaluronic acid acts as a targeting ligand, and has dual targeting, strong targeting, sustained release, long circulation, low toxicity and synergistic treatment to enhance The characteristics of curative effect have broad application prospects in anti-tumor, anti-rheumatism and other medical fields.

Description of drawings

The present invention will be further described below in conjunction with drawings and embodiments.

Figure 1 is a synthetic route map of MTX-HA-OCA dual-targeting nanoparticles.

Figure 2 is the Fourier transform infrared characterization spectrum of MTX-HA-OCA dual-targeting nanoparticles.

Figure 3 is a 400MHz H NMR spectrum characterization diagram of MTX-HA-OCA dual-targeted nanoparticles.

Fig. 4 is a particle size distribution diagram of MTX-HA-OCA dual targeting nanoparticles.

Fig. 5 is the Zeta potential distribution diagram of MTX-HA-OCA dual targeting nanoparticles.

Fig. 6 is a transmission electron microscope image of MTX-HA-OCA dual targeting nanoparticles.

Detailed ways

Below by embodiment the content of the present invention is specified:

Example 1

1) Weigh 100 mg of hyaluronic acid with a molecular weight of 8000 Da (the molecular weight of the hyaluronic acid monomer is 776.65), add 5 mL of anhydrous formamide, slowly heat to 20-40 ° C until the hyaluronic acid dissolves, cool to room temperature, and add 96mg EDC and 58mg NHS were stirred and activated for 2h; 75mg octadecylamine was dissolved in 7mL dimethylformamide, and slowly added to the above-mentioned activated hyaluronic acid solution, stirred at 60°C for 12h under the protection of argon, and then heated at room temperature Stir for 24 hours; the obtained mixed solution was sequentially dialyzed with deionized water-ethanol mixture with a volume ratio of 1:3, 1:2, and 1:1 for 48 hours, and the time for each gradient dialyzed was 6 hours, 18 hours, and 24 hours in sequence, and then used Dialyze with deionized water for 48 hours, and the molecular weight cut-off of the dialysis bag used in the dialysis is 3500; after the dialysis is completed, filter and vacuum freeze-dry to obtain the hyaluronic acid-octadecylamine conjugate (HA-OCA);

2) Dissolve 84 mg of methotrexate in 5 mL of dimethyl sulfoxide, add 84 mg of EDC and 84 mg of DMAP, and activate with gentle stirring for 30 minutes; dissolve 114 mg of HA-OCA in 10 mL of anhydrous formamide, and add to the activated formamide In the aminopterin solution, avoid light, stir and react at room temperature for 48 hours, then dialyze with deionized water for 48 hours, the molecular weight cut-off of the dialysis bag used in the dialysis is 3500, and vacuum freeze-dry to obtain the methotrexate prodrug, which is methotrexate Phosphine-hyaluronic acid-octadecylamine conjugate (MTX-HA-OCA);

3) Disperse the obtained methotrexate-hyaluronic acid-octadecylamine conjugate in deionized water, sonicate in an ice bath for 10min, the power of sonication is 200W, the duration is 5s, and the interval is 2s; Stir slowly at room temperature for 24 hours, self-assemble to obtain methotrexate-hyaluronic acid-octadecylamine dual-targeted nanoparticle dispersion.

Example 2

1) Weigh 100mg of hyaluronic acid with a molecular weight of 8000Da, add 5mL of anhydrous formamide, slowly heat to 20-40°C until the hyaluronic acid dissolves, cool to room temperature, add 96mg of EDC and 58mg of NHS and stir for 2h; Octadecylamine was dissolved in 7mL dimethylformamide, and slowly added to the above-mentioned activated hyaluronic acid solution, stirred at 60°C for 12h under the protection of argon, and then stirred at room temperature for 24h; Gradient dialysis of deionized water-ethanol mixture with a ratio of 1:3, 1:2, and 1:1 for 48 hours, each gradient dialysis time is 6 hours, 18 hours, and 24 hours in sequence, and then dialyzed with deionized water for 48 hours. The dialysis bag used for dialysis The molecular weight cut-off is 3500; After dialysis is completed, filter and vacuum freeze-dry to obtain hyaluronic acid-octadecylamine conjugate (HA-OCA);

2) Dissolve 84 mg of methotrexate in 5 mL of dimethyl sulfoxide, add 84 mg of EDC and 84 mg of DMAP, and activate with gentle stirring for 30 minutes; dissolve 114 mg of HA-OCA in 10 mL of anhydrous formamide, and add to the activated formamide In the aminopterin solution, avoid light, stir and react at room temperature for 48 hours, then dialyze with deionized water for 48 hours, the molecular weight cut-off of the dialysis bag used in the dialysis is 3500, and vacuum freeze-dry to obtain the methotrexate prodrug, which is methotrexate Phosphine-hyaluronic acid-octadecylamine conjugate (MTX-HA-OCA);

3) Disperse the obtained methotrexate-hyaluronic acid-octadecylamine conjugate in deionized water, sonicate in an ice bath for 10min, the power of sonication is 200W, the duration is 5s, and the interval is 2s; Stir slowly at room temperature for 24 hours, self-assemble to obtain methotrexate-hyaluronic acid-octadecylamine dual-targeted nanoparticle dispersion.

Example 3

1) Weigh 100mg of hyaluronic acid with a molecular weight of 8000Da, add 5mL of anhydrous formamide, slowly heat to 20-40°C until the hyaluronic acid dissolves, then cool to room temperature, add 192mg of EDC and 116mg of NHS and stir for activation for 1 hour; Octadecylamine was dissolved in 7mL dimethylformamide, and slowly added to the above-mentioned activated hyaluronic acid solution, stirred at 60°C for 12h under the protection of argon, and then stirred at room temperature for 24h; Gradient dialysis of deionized water-ethanol mixture with a ratio of 1:3, 1:2, and 1:1 for 48 hours, each gradient dialysis time is 6 hours, 18 hours, and 24 hours in sequence, and then dialyzed with deionized water for 48 hours. The dialysis bag used for dialysis The molecular weight cut-off is 3500; After dialysis is completed, filter and vacuum freeze-dry to obtain hyaluronic acid-octadecylamine conjugate (HA-OCA);

2) Dissolve 84 mg of methotrexate in 5 mL of dimethyl sulfoxide, add 84 mg of EDC and 84 mg of DMAP, and activate with gentle stirring for 30 minutes; dissolve 114 mg of HA-OCA in 10 mL of anhydrous formamide, and add to the activated formamide In the aminopterin solution, avoid light, stir and react at room temperature for 48 hours, then dialyze with deionized water for 48 hours, the molecular weight cut-off of the dialysis bag used in the dialysis is 3500, and vacuum freeze-dry to obtain the methotrexate prodrug, which is methotrexate Phosphine-hyaluronic acid-octadecylamine conjugate (MTX-HA-OCA);

3) Disperse the obtained methotrexate-hyaluronic acid-octadecylamine conjugate in deionized water, sonicate in an ice bath for 10min, the power of sonication is 200W, the duration is 5s, and the interval is 2s; Stir slowly at room temperature for 24 hours, self-assemble to obtain methotrexate-hyaluronic acid-octadecylamine dual-targeted nanoparticle dispersion.

Example 4

1) Weigh 100 mg of hyaluronic acid with a molecular weight of 30,000 Da, add 5 mL of anhydrous formamide, slowly heat to 20-40 ° C until the hyaluronic acid dissolves, cool to room temperature, add 96 mg of EDC and 58 mg of NHS and stir for 2 hours; Octadecylamine was dissolved in 7mL dimethylformamide, and slowly added to the above-mentioned activated hyaluronic acid solution, stirred at 60°C for 12h under the protection of argon, and then stirred at room temperature for 24h; Gradient dialysis of deionized water-ethanol mixture with a ratio of 1:3, 1:2, and 1:1 for 48 hours, each gradient dialysis time is 6 hours, 18 hours, and 24 hours in sequence, and then dialyzed with deionized water for 48 hours. The dialysis bag used for dialysis The molecular weight cut-off is 10000; filter after the dialysis is completed, and freeze-dry in vacuum to obtain hyaluronic acid-octadecylamine conjugate (HA-OCA);

2) Dissolve 84 mg of methotrexate in 5 mL of dimethyl sulfoxide, add 84 mg of EDC and 84 mg of DMAP, and activate with gentle stirring for 30 minutes; dissolve 114 mg of HA-OCA in 10 mL of anhydrous formamide, and add to the activated formamide In the aminopterin solution, avoid light, stir and react at room temperature for 48 hours, then dialyze with deionized water for 48 hours, the molecular weight cut-off of the dialysis bag used in the dialysis is 10000, vacuum freeze-dry to obtain the methotrexate prodrug, which is methotrexate Phosphine-hyaluronic acid-octadecylamine conjugate (MTX-HA-OCA);

3) Disperse the obtained methotrexate-hyaluronic acid-octadecylamine conjugate in deionized water, sonicate in an ice bath for 10min, the power of sonication is 200W, the duration is 5s, and the interval is 2s; Stir slowly at room temperature for 24 hours, self-assemble to obtain methotrexate-hyaluronic acid-octadecylamine dual-targeted nanoparticle dispersion.

Example 5

1) Weigh 100mg of hyaluronic acid with a molecular weight of 8000Da, add 5mL of anhydrous formamide, slowly heat to 20-40°C until the hyaluronic acid dissolves, cool to room temperature, add 96mg of EDC and 58mg of NHS and stir for 2h; Octadecylamine was dissolved in 7mL dimethylformamide, and slowly added to the above-mentioned activated hyaluronic acid solution, stirred at 60°C for 12h under the protection of argon, and then stirred at room temperature for 24h; Gradient dialysis of deionized water-ethanol mixture with a ratio of 1:3, 1:2, and 1:1 for 48 hours, each gradient dialysis time is 6 hours, 18 hours, and 24 hours in sequence, and then dialyzed with deionized water for 48 hours. The dialysis bag used for dialysis The molecular weight cut-off is 3500; After dialysis is completed, filter and vacuum freeze-dry to obtain hyaluronic acid-octadecylamine conjugate (HA-OCA);

2) Dissolve 168 mg of methotrexate in 5 mL of dimethyl sulfoxide, add 84 mg of EDC and 84 mg of DMAP, and activate with gentle stirring for 30 minutes; dissolve 114 mg of HA-OCA in 10 mL of dimethyl sulfoxide, add to the activated In the methotrexate solution, avoid light, stir and react at room temperature for 48h, then dialyze with deionized water for 48h, the molecular weight cut-off of the dialysis bag used in the dialysis is 3500, and vacuum freeze-dry to obtain the methotrexate prodrug, which is methotrexate Pterin-hyaluronic acid-octadecylamine conjugate (MTX-HA-OCA);

3) Disperse the obtained methotrexate-hyaluronic acid-octadecylamine conjugate in deionized water, sonicate in an ice bath for 10min, the power of sonication is 200W, the duration is 5s, and the interval is 2s; Stir slowly at room temperature for 24 hours, self-assemble to obtain methotrexate-hyaluronic acid-octadecylamine dual-targeted nanoparticle dispersion.

Example 6

1) Weigh 100mg of hyaluronic acid with a molecular weight of 8000Da, add 5mL of deionized water, slowly heat to 20-40°C until the hyaluronic acid dissolves, cool to room temperature, add 192mg of EDC and 116mg of NHS and stir for 2h; Octamine was dissolved in 5 mL of absolute ethanol, and slowly added to the above-mentioned activated hyaluronic acid solution, stirred at 60°C for 12 hours under the protection of argon, and then stirred at room temperature for 24 hours; 3. Gradient dialysis of 1:2, 1:1 deionized water-ethanol mixed solution for 48 hours, each gradient dialysis time is 6 hours, 18 hours, 24 hours in turn, and then dialysis with deionized water for 48 hours, the molecular weight cut-off of the dialysis bag used for dialysis It was 3500; after the dialysis was completed, it was filtered and vacuum freeze-dried to obtain hyaluronic acid-octadecylamine conjugate (HA-OCA);

2) Dissolve 168 mg of methotrexate in 5 mL of dimethyl sulfoxide, add 84 mg of EDC and 84 mg of DMAP, and activate with gentle stirring for 30 minutes; dissolve 114 mg of HA-OCA in 10 mL of dimethyl sulfoxide, add to the activated In the methotrexate solution, avoid light, stir and react at room temperature for 48h, then dialyze with deionized water for 48h, the molecular weight cut-off of the dialysis bag used in the dialysis is 3500, and vacuum freeze-dry to obtain the methotrexate prodrug, which is methotrexate Pterin-hyaluronic acid-octadecylamine conjugate (MTX-HA-OCA);

3) Disperse the obtained methotrexate-hyaluronic acid-octadecylamine conjugate in deionized water, and perform ultrasonic treatment in an ice bath for 10 minutes, the power of the ultrasonic treatment is 300W, the duration is 5s, and the interval is 2s; Stir slowly at room temperature for 24 hours, self-assemble to obtain methotrexate-hyaluronic acid-octadecylamine dual-targeted nanoparticle dispersion.

Example 7

1) Weigh 100mg of hyaluronic acid with a molecular weight of 8000Da, add 5mL of anhydrous formamide, slowly heat to 20-40°C until the hyaluronic acid dissolves, cool to room temperature, add 96mg of EDC and 58mg of NHS and stir for 2h; Octadecylamine was dissolved in 7mL dimethylformamide, and slowly added to the above-mentioned activated hyaluronic acid solution, stirred at 60°C for 12h under the protection of argon, and then stirred at room temperature for 24h; Gradient dialysis of deionized water-ethanol mixture with a ratio of 1:3, 1:2, and 1:1 for 48 hours, each gradient dialysis time is 6 hours, 18 hours, and 24 hours in sequence, and then dialyzed with deionized water for 48 hours. The dialysis bag used for dialysis The molecular weight cut-off is 3500; After dialysis is completed, filter and vacuum freeze-dry to obtain hyaluronic acid-octadecylamine conjugate (HA-OCA);

2) Dissolve 84mg of methotrexate in 5mL of DMSO, add 84mg of EDC and 84mg of DMAP, and activate with gentle stirring for 30min; dissolve 114mg of HA-OCA in 10mL of DMSO, add to the activated In the methotrexate solution, avoid light, stir and react at room temperature for 48h, then dialyze with deionized water for 48h, the molecular weight cut-off of the dialysis bag used in the dialysis is 3500, and vacuum freeze-dry to obtain the methotrexate prodrug, which is methotrexate Pterin-hyaluronic acid-octadecylamine conjugate (MTX-HA-OCA);

3) Disperse the obtained methotrexate-hyaluronic acid-octadecylamine conjugate in deionized water, and perform ultrasonic treatment in an ice bath for 15 minutes, the power of the ultrasonic treatment is 300W, the duration is 5s, and the interval is 2s; Stir slowly at room temperature for 24 hours, self-assemble to obtain methotrexate-hyaluronic acid-octadecylamine dual-targeted nanoparticle dispersion.

Please refer to Figure 1, Figure 2, and Figure 3. Figure 1 is the synthesis route of MTX-HA-OCA dual-targeted nanoparticles, and Figures 2 and 3 are the Fourier transforms of MTX-HA-OCA dual-targeted nanoparticles. From the infrared characterization spectrum and 400MHz hydrogen NMR spectrum characterization graph, it can be seen that MTX-HA-OCA dual-targeted nanoparticles were successfully synthesized according to the synthesis method shown in Figure 1.

Please refer to Figure 4, Figure 5 and Figure 6. Figure 4 and Figure 5 are the particle size distribution and Zeta potential distribution of MTX-HA-OCA dual-targeting nanoparticles respectively, and Figure 6 is the MTX-HA-OCA dual-targeting From the transmission electron microscope images of the nanoparticles, it can be seen that the MTX-HA-OCA dual-targeting nanoparticles of the present invention are spherical, the particle diameter is distributed between 60-120nm, the average particle diameter is about 100nm, and the Zeta potential is about -17mV.

In summary, the present invention provides a method for preparing dual-targeted nanoparticles by chemically linking methotrexate, hyaluronic acid and octadecylamine. The particle size of the dual-targeted nanoparticles is between 60 and 120 nm. Distribution, good stability, non-toxic, good targeting and sustained release characteristics, and good therapeutic effect.

The above is only a preferred embodiment of the present invention, so the scope of the present invention cannot be limited accordingly, that is, the equivalent changes and modifications made according to the patent scope of the present invention and the content of the specification should still be covered by the present invention within range.

Claims (10)

1. A preparation method of methotrexate prodrug, characterized in that: comprising: 1) Dissolve hyaluronic acid in the first organic solvent, heat at 20-40°C until the hyaluronic acid dissolves, then cool to room temperature, then add 1-(3-dimethylaminopropyl)-3-ethylcarbadiene imine and N-hydroxysuccinimide, stirring and reacting for at least 1 h; dissolving octadecylamine in a second organic solvent and adding it to the above solution; the molar ratio of octadecylamine to hyaluronic acid monomer is 5 :1～1:1; the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to hyaluronic acid monomer, N-hydroxysuccinimide to hyaluronic acid The molar ratio of the monomers is 8:1～1:1; then under the condition of cutting off oxygen, stir at 55～65°C for at least 5h, and then stir at room temperature for 22～26h; the volume ratio of the obtained mixed solution is 1:3 ~1:1 water-ethanol mixed solution dialyzed for 45~50h, then water dialyzed for 45~50h, filtered, freeze-dried to obtain hyaluronic acid-octadecylamine conjugate; 2) Dissolving methotrexate in a third organic solvent, then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 4-dimethylaminopyridine, and stirring for at least 30 minutes; After dissolving the hyaluronic acid-octadecylamine conjugate obtained in step 1) in the third organic solvent, add it to the above solution; the hyaluronic acid in the methotrexate and hyaluronic acid-octadecylamine conjugate The molar ratio of monomers is 5:1～1:1; the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to methotrexate, 4-dimethylamino The molar ratio of pyridine to methotrexate is 4:1 to 1:1; then avoid light, react at room temperature for 45 to 50 hours, dialyze with water for 45 to 50 hours, and freeze-dry to obtain the prodrug of methotrexate, which is methotrexate Glycerin-hyaluronic acid-stearylamine conjugate. 2. The preparation method of methotrexate prodrug according to claim 1, characterized in that: the first organic solvent is at least one of anhydrous formamide and water. 3. The preparation method of methotrexate prodrug according to claim 1, characterized in that: the second organic solvent is at least one of dimethylformamide and ethanol. 4. the preparation method of methotrexate prodrug according to claim 1, is characterized in that: described the 3rd organic solvent is tetrahydrofuran (THF), dimethylsulfoxide, dimethylformamide, dimethylacetamide, At least one of anhydrous formamide, methanol, ethanol, acetonitrile, and acetone. 5. The method for preparing methotrexate prodrug according to claim 1, characterized in that: the dialysis bag used in the dialysis has a molecular weight cut-off of 1000-10000. 6. A methotrexate prodrug prepared by the preparation method according to any one of claims 1 to 5. 7. A method for preparing dual-targeted nanoparticles of methotrexate prodrug, characterized in that: comprising: preparing methotrexate-hyaluronic acid according to the preparation method described in any one of claims 1 to 5 Acid-octadecylamine conjugates, dispersed in water, ultrasonically treated at -5-5°C, then stirred at room temperature for 22-26 hours, self-assembled to obtain methotrexate-hyaluronic acid-octadecylamine dual-targeted nanoparticle dispersion. 8. The method for preparing double-targeted nanoparticles of methotrexate prodrug according to claim 7, characterized in that: the power of the ultrasonic treatment is 100-300W, the duration is 3-6s, and the interval is 2～4s. 9 . The method for preparing dual-targeted nanoparticles of methotrexate prodrug according to claim 7 , characterized in that: the ultrasonic treatment time is 8-20 minutes. 10. A dual-targeted nanoparticle of the methotrexate prodrug prepared by the preparation method according to any one of claims 7 to 9, characterized in that: the dual-targeted nanoparticle of the methotrexate prodrug The nanoparticles are spherical, with a particle size of 60-120nm.