A crocetin-polyethylene glycol monomethyl ether conjugate nanoparticle, a preparation method and application thereof

Abstract

The application provides a crocetin-polyethylene glycol monomethyl ether conjugate nanoparticle, a structure formula of the conjugate nanoparticle is as follows: the conjugate nanoparticle is self-assembled in an aqueous solution to form a three-block conjugate, the conjugate has an amphiphilic structure, and the conjugate nanoparticle is self-assembled in the aqueous solution to form a conjugate nanoparticle, so that the water solubility is significantly improved, and the application also provides a preparation method of the crocetin-polyethylene glycol monomethyl ether conjugate nanoparticle and application thereof.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to crocin-polyethylene glycol monomethyl ether conjugate nanoparticles, their preparation method, and applications. Background Technology

[0002] Crocin (TC) possesses various pharmacological effects, including antioxidant, anti-inflammatory, neuroprotective, cardioprotective, hepatoprotective, antidepressant, anti-angiogenic, burn treatment, lipid-lowering, and improvement of asthma, Alzheimer's disease, diabetes, colitis, retinal damage, and anti-tumor effects. Recent studies have demonstrated that crocin exhibits good antioxidant activity, achieving therapeutic effects in liver protection, kidney protection, and Alzheimer's disease prevention through its potent antioxidant activity. However, crocin is a polyene compound with conjugated double bonds and is almost insoluble in water; its poor water solubility is one of the main factors limiting its medicinal applications. Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of the above-mentioned water solubility by providing crocin-polyethylene glycol monomethyl ether coupling nanoparticles, their preparation method, and applications. The aim is to prepare crocin-polyethylene glycol monomethyl ether couplings through esterification, and the couplings self-assemble into nanoparticles in water, further improving the water solubility of crocin-polyethylene glycol monomethyl ether coupling nanoparticles and enhancing their antioxidant capacity and antitumor activity.

[0004] This invention provides, in one aspect, crocin-polyethylene glycol monomethyl ether coupling nanoparticles, wherein the structural formula of the coupling is:

[0005]

[0006] The coupling nanoparticles are formed by self-assembly in an aqueous solution.

[0007] The second aspect of this invention provides a method for preparing crocin-polyethylene glycol monomethyl ether coupling nanoparticles, the specific steps of which are as follows:

[0008] 1) Dissolve crocin in an organic solvent, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and triethylamine, stir and heat to react, and obtain reaction solution 1;

[0009] 2) Add polyethylene glycol monomethyl ether to the reaction solution 1 from the previous step, continue the reaction, and remove the organic solvent by rotary evaporation after the reaction is completed to obtain intermediate 1;

[0010] 3) Add intermediate 1 to distilled water, dissolve and disperse it using ultrasound, and then put it into a dialysis bag for dialysis. After dialysis, centrifuge the mixture in the dialysis bag to separate the precipitate and the supernatant. Freeze-dry the supernatant to obtain crocin-polyethylene glycol monomethyl ether conjugate nanoparticles.

[0011] Furthermore, the organic solvent in step 1) is any one or a combination of dichloromethane, dichloroethane, petroleum ether, and cyclohexane.

[0012] Furthermore, in step 1), the molar ratio of crocin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, and triethylamine is 1:2.5:2.5:2.5, the heating reaction temperature is 35-50°C, and the reaction time is 30-60 min.

[0013] Furthermore, the molecular weight of the polyethylene glycol monomethyl ether in step 2) is 550-2000.

[0014] Furthermore, in step 2), the mass ratio of polyethylene glycol monomethyl ether to crocin is 1:2.5, and the reaction time is 24-36 hours.

[0015] Furthermore, in step 3), the mass ratio of distilled water to crocin is 50-100:1.

[0016] Furthermore, the specific dialysis conditions in step 3) are: dialysis for 3-4 days, with the water changed 3-4 times a day.

[0017] A third aspect of the present invention also provides an application of crocin-polyethylene glycol monomethyl ether coupling nanoparticles, wherein the coupling nanoparticles are used to inhibit the proliferation and oxidation of liver cancer cells.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention utilizes the carboxyl groups at both ends of hydrophobic crocin to couple with the hydrophilic polymer polyethylene glycol monomethyl ether via esterification to form a triblock conjugate. This conjugate has an amphiphilic structure, and then self-assembles into conjugate nanoparticles in aqueous solution, significantly improving the water solubility of the active groups in crocin. Due to the increased water solubility, it is easier to capture reactive oxygen free radicals in the solution, thus improving the antioxidant capacity of the crocin-polyethylene glycol monomethyl ether conjugate nanoparticles. At the same time, the crocin-polyethylene glycol monomethyl ether conjugate nanoparticles can be taken up by tumor cells through endocytosis, which is beneficial to tumor cell uptake, thus improving the antitumor activity of the crocin-polyethylene glycol monomethyl ether conjugate nanoparticles. Attached Figure Description

[0020] Figure 1 The NMR spectrum of TC-mPEG550 prepared in Example 1 of this invention;

[0021] Figure 2 The NMR spectrum of TC-mPEG1000 prepared in Example 2 of this invention;

[0022] Figure 3 The NMR spectrum of TC-mPEG2000 prepared in Example 3 of this invention;

[0023] Figure 4 Infrared images of crocin (TC), TC-mPEG550, TC-mPEG1000, and TC-mPEG2000 used in this invention;

[0024] Figure 5 This is a standard curve of crocin (TC) in this invention;

[0025] Figure 6 This is a graph showing the DPPH free radical scavenging rates of crocin (TC), TC-mPEG550, TC-mPEG1000, and TC-mPEG2000 in this invention. Detailed Implementation

[0026] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification. Unless otherwise specified, the methods described are conventional methods, and the raw materials described are available from publicly available commercial sources unless otherwise specified.

[0027] The term "an embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the invention. The phrase "in one embodiment" appearing in different places throughout this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0028] The sources of the pharmaceuticals and reagents described in the examples are as follows:

[0029] Crocin (TC): TCI, >99.0%

[0030] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: Anage Chemicals, >98.0%

[0031] 4-Dimethylaminopyridine: Anage Chemicals, >98.0%

[0032] Dichloromethane: Shanghai Lingfeng Chemical Reagent Co., Ltd., ≥99.9%

[0033] Dichloroethane: Shanghai Lingfeng Chemical Reagent Co., Ltd., ≥99.9%

[0034] n-Hexane: Shanghai Lingfeng Chemical Reagent Co., Ltd., ≥99.9%

[0035] Triethylamine: Sinopharm Chemical Reagent Co., Ltd., ≥99.5%

[0036] Polyethylene glycol monomethyl ether (mPEG550): TCI, >99.0%

[0037] Polyethylene glycol monomethyl ether (mPEG1000): TCI, >99.0%

[0038] Polyethylene glycol monomethyl ether (mPEG2000): TCI, >99.0%

[0039] 1,1-Diphenyl-2-trinitrophenylhydrazine: TCI, >99.0%

[0040] Ethanol: Sinopharm Chemical Reagent Co., Ltd., ≥99.9%

[0041] The specific preparation method is as follows:

[0042] 1) Dissolve crocin in an organic solvent, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and triethylamine, stir and heat to react, and obtain reaction solution 1;

[0043] 2) Add polyethylene glycol monomethyl ether to the reaction solution 1 from the previous step, continue the reaction, and remove the organic solvent by rotary evaporation after the reaction is completed to obtain intermediate 1;

[0044] 3) Add intermediate 1 to distilled water, dissolve and disperse using ultrasound, then place it in a dialysis bag for dialysis. After dialysis, centrifuge the mixture in the dialysis bag to separate the precipitate and supernatant. Freeze-dry the supernatant to obtain crocin-polyethylene glycol monomethyl ether coupling nanoparticles. For this step, different dialysis bag models are selected based on the molecular weight of the polyethylene glycol monomethyl ether used. When using mPEG550 and mPEG1000, a dialysis bag with a molecular weight cutoff of 1000 is selected; when using mPEG2000, a dialysis bag with a molecular weight cutoff of 3500 is selected.

[0045] The following examples 1-3 elaborate on the amount of raw materials fed and the specific reaction conditions. The beneficial effects of the present invention are verified through experiments in Examples 1-5, including water solubility evaluation, particle size potential measurement, crocin standard curve measurement, DPPH free radical scavenging ability, and tumor cell inhibitory activity.

[0046] Example 1

[0047] 1) Dissolve 100 mg of crocin (TC) in 100 mL of dichloromethane. Add 10 times the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 2.5 times the amount of 4-dimethylaminopyridine, and 2.5 times the amount of triethylamine in molar proportion based on crocin (TC). Stir and heat to about 35°C for 30 min to obtain reaction solution 1.

[0048] 2) Add 2.5 times the molar amount of polyethylene glycol monomethyl ether (TC) to the reaction solution 1 from the previous step, maintain the temperature at around 35°C, and continue the reaction for 24 hours. After the reaction is complete, remove dichloromethane under reduced pressure using a rotary evaporator to obtain intermediate 1.

[0049] 3) Add intermediate 1 to 5 ml of distilled water, dissolve and disperse it by ultrasound, and then put it into a dialysis bag with a molecular weight cutoff of 1000 for dialysis. Dialyze for 3 days, changing the water three times a day. After dialysis, centrifuge the mixture in the dialysis bag to separate the precipitate and the supernatant. Freeze-dry the supernatant to obtain crocin-polyethylene glycol monomethyl ether conjugate nanoparticles TC-mPEG550.

[0050] like Figure 1 The NMR spectrum shows signals at 1-4 ppm, which are the signals from the four methyl groups on the crocin conjugated polyene chain and the methyl group on polyethylene glycol monomethyl ether; at 2.5 ppm is the signal from dimethyl sulfoxide; at 3.3 ppm is the signal from water; at 3.6 ppm is the methylene signal from polyethylene glycol monomethyl ether; and at 6-7 ppm is the signal from the crocin conjugated polyene chain. Figure 4 The absorption peak at approximately 1700 cm⁻¹ in the infrared spectrum of the TC-mPEG550 curve is a characteristic peak of ester bonds, which proves that crocin and polyethylene glycol monomethyl ether (mPEG550) were successfully coupled.

[0051] Example 2

[0052] 1) Dissolve 100 mg of crocin (TC) in 100 mL of dichloroethane. Add 10 times the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 2.5 times the amount of 4-dimethylaminopyridine, and 2.5 times the amount of triethylamine in molar proportion based on crocin (TC). Stir and heat to about 40°C for 30 min to obtain reaction solution 1.

[0053] 2) Add 2.5 times the molar amount of polyethylene glycol monomethyl ether (TC) to the reaction solution 1 from the previous step, maintain the temperature at around 35°C, and continue the reaction for 36 hours. After the reaction is complete, remove dichloroethane under reduced pressure using a rotary evaporator to obtain intermediate 1.

[0054] 3) Add intermediate 1 to 8 ml of distilled water, dissolve and disperse it by ultrasound, and then put it into a dialysis bag with a molecular weight cutoff of 1000 for dialysis. Dialyze for 3 days, changing the water three times a day. After dialysis, centrifuge the mixture in the dialysis bag to separate the precipitate and the supernatant. Freeze-dry the supernatant to obtain crocin-polyethylene glycol monomethyl ether conjugate nanoparticles TC-mPEG1000.

[0055] like Figure 2 The signals at 1-4 ppm represent the four methyl groups on the crocin conjugated polyene chain and the methyl group on polyethylene glycol monomethyl ether; 2.5 ppm represents the signal of dimethyl sulfoxide; 3.3 ppm represents the signal of water; 3.6 ppm represents the methylene signal of polyethylene glycol monomethyl ether; and 6-7 ppm represents the signal of the crocin conjugated polyene chain. Figure 4 The infrared image of TC-mPEG1000 shows a depth of approximately 1700 cm⁻¹. -1 The absorption peak is a characteristic peak of ester bond, which proves that crocin and polyethylene glycol monomethyl ether (mPEG1000) were successfully coupled.

[0056] Example 3

[0057] 1) Dissolve 100 mg of crocin (TC) in 100 mL of n-hexane. Add 10 times the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 2.5 times the amount of 4-dimethylaminopyridine, and 2.5 times the amount of triethylamine in molar proportion based on crocin (TC). Stir and heat to about 40°C for 30 min to obtain reaction solution 1.

[0058] 2) Add 2.5 times the molar amount of polyethylene glycol monomethyl ether (TC) to the reaction solution 1 from the previous step, maintain the temperature at around 45°C, and continue the reaction for 28 hours. After the reaction is complete, remove n-hexane under reduced pressure using a rotary evaporator to obtain intermediate 1.

[0059] 3) Add intermediate 1 to 8 ml of distilled water, dissolve and disperse it by ultrasound, and then put it into a dialysis bag with a molecular weight cutoff of 1000 for dialysis. Dialyze for 3 days, changing the water three times a day. After dialysis, centrifuge the mixture in the dialysis bag to separate the precipitate and the supernatant. Freeze-dry the supernatant to obtain crocin-polyethylene glycol monomethyl ether conjugate nanoparticles TC-mPEG2000.

[0060] like Figure 3 The signals at 1-4 ppm represent the four methyl groups on the crocin conjugated polyene chain and the methyl group on polyethylene glycol monomethyl ether; 2.5 ppm represents the signal of dimethyl sulfoxide; 3.3 ppm represents the signal of water; 3.6 ppm represents the methylene signal of polyethylene glycol monomethyl ether; and 6-7 ppm represents the signal of the crocin conjugated polyene chain. Figure 4The infrared image of TC-mPEG2000 shows a depth of approximately 1700 cm⁻¹. -1 The absorption peak is a characteristic peak of ester bond, which proves that crocin and polyethylene glycol monomethyl ether (mPEG2000) were successfully coupled.

[0061] Experimental Example 1

[0062] Water solubility evaluation

[0063] Excess crocin and crocin-polyethylene glycol monomethyl ether coupling nanoparticles (M1) were placed in volumetric flasks, and a certain amount of distilled water was added to obtain supersaturated solutions. The solutions were then placed in a water bath constant temperature shaker (37°C, 120 rpm) for 24 hours and filtered through a 0.2 μm microporous membrane. The mass of undissolved sample (M0) was calculated, and its water solubility was calculated according to the following formula.

[0064] C = (M1 - M0) / V

[0065] Three parallel samples were prepared for each sample's saturated solution, and the average value was calculated. The saturated water solubility results for each sample are shown in Table 1. The water solubility results show that the crocin-polyethylene glycol monomethyl ether coupling nanoparticles have significantly improved water solubility compared to crocin. The order of water solubility is TC-mPEG2000, TC-mPEG1000, and TC-mPEG550.

[0066] Table 1. Saturated water solubility of each sample

[0067]

[0068] Experiment Example 2

[0069] Particle size potential measurement

[0070] The particle size, polydispersity index, and Zeta potential of TC-mPEG550, TC-mPEG1000, and TC-mPEG2000 were determined using a Zetasizer Nano ZS90 laser particle size analyzer. The results are shown in Table 2. Table 2 shows that all crocin-polyethylene glycol monomethyl ether coupling nanoparticles self-assembled in aqueous solution to form nanoparticles with sizes ranging from 400 to 500 nm. The particle size of TC-mPEG550 was 485 nm, TC-mPEG1000 was 429 nm, and TC-mPEG2000 was 462 nm. TC-mPEG550 had the best polydispersity index (PDI) at 0.4, followed by TC-mPEG2000 at 0.595, while TC-mPEG1000 had the worst PDI at 0.985. All the self-assembled nanoparticles of each conjugate are positively charged, with TC-mPEG550 having the lowest charge and TC-mPEG2000 having the highest charge.

[0071] Table 2. Particle size, potential, and PDI data of crocin-polyethylene glycol monomethyl ether couplings.

[0072]

[0073] Experimental Example 3

[0074] Determination of crocin standard curve

[0075] Dissolve a certain amount of crocin completely in anhydrous ethanol, and dilute it to 10ug / ml, 20ug / ml, and 30ug / ml. Measure its OD value using a microplate reader and plot its standard curve. Figure 5 As shown.

[0076] Drug loading

[0077] An appropriate amount of crocin-polyethylene glycol monomethyl ether coupling nanoparticles (M1) were dissolved in anhydrous ethanol, and their OD values ​​were measured. If the OD values ​​were within the range of the standard curve, the content of crocin in the crocin-polyethylene glycol monomethyl ether coupling nanoparticles (M2) was obtained.

[0078] Drug loading = M2 / M1

[0079] The drug loadings are shown in Table 3. The results in Table 3 show that the drug loading of TC-mPEG550 is 12.5%, that of TC-mPEG is 6.2%, and that of TC-mPEG2000 is 1.2%. As the molecular weight of the coupled mPEG increases, its drug loading gradually decreases. It is speculated that this is because the increased molecular weight of mPEG makes coupling with crocin more difficult.

[0080] Table 3 Drug loading of each sample

[0081]

[0082] Experiment Example 4

[0083] DPPH free radical scavenging ability

[0084] Weigh 6 mg of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) and prepare a 100 mL solution with anhydrous ethanol. Sonicate for 5 minutes and store in the dark. Dilute crocin (TC), TC-mPEG550, TC-mPEG1000, and TC-mPEG2000 to prepare sample solutions of 1.06 μg / mL, 3.12 μg / mL, 6.25 μg / mL, 12.5 μg / mL, and 25 μg / mL, respectively. Take 2 mL of each solution and place it in a 5 mL test tube. Accurately add 2 mL of DPPH solution to each tube, mix thoroughly, and store at 25°C in the dark for 40 min. Measure the absorbance (Ai) at 517 nm using a microplate reader. Simultaneously, measure the absorbance (Aj) of 2 mL of sample solution + 2 mL of anhydrous ethanol and the absorbance (A0) of 2 mL of anhydrous ethanol + 2 mL of DPPH solution. Calculate the free radical scavenging rate using the following formula.

[0085] Clearance rate (%) = [1 - (Ai - Aj) / A0] × 100%

[0086] get Figure 6 DPPH free radical scavenging rate. From Figure 6 As can be seen, the DPPH radical scavenging rates of TC-mPEG550 and TC-mPEG1000 are higher than those of TC at different concentrations, while the DPPH radical scavenging rate of TC-mPEG2000 is slightly higher than that of TC. The DPPH radical scavenging rates, from highest to lowest, are TC-mPEG550, TC-mPEG1000, TC-mPEG2000, and TC.

[0087] Experimental Example 5

[0088] Tumor cell inhibitory activity

[0089] 1. Experimental reagents

[0090] Crocin (TC), TC-mPEG550, TC-mPEG1000, TC-mPEG2000.

[0091] 2. Cell lines

[0092] Human hepatocellular carcinoma cells (HepG2) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. The medium was changed after 48 hours. When cells reached saturation, they were passaged using 0.25% trypsin. Passages were performed every 2-3 days. Cells in the logarithmic growth phase were used in the experiment.

[0093] 3. Preparation of DMEM culture medium

[0094] Add 5 mL of fetal bovine serum, 0.5 mL of penicillin solution (200,000 U / mL) and streptomycin solution (200,000 U / mL), and finally adjust the volume to 50 mL using DMEM medium.

[0095] 4. Cell viability detection

[0096] The effects of crocin and crocin-polyethylene glycol monomethyl ether conjugate on the proliferation activity of human hepatocellular carcinoma cells HePG2 were determined using the MTT assay [3-(4,5)-bismethyl-2-thiazole-(2,5)-phenyltetrazozolium bromide blue].

[0097] Drug addition: The test drug solutions were added to each well according to the final concentration gradient, with four parallel wells for each concentration. The experiment was divided into a drug test group (different concentrations of test drug were added), a control group (only culture medium and cells were added, without test drug), and a blank group (only culture medium was added, without cells and test drug). The 96-well plates with drug addition were incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0098] Assay for viable cells: After 24 hours of culture, 20 μL of MTT (prepared to 5 mg / mL with PBS buffer) was added to each well of a 96-well plate. After incubation at 37°C for 4 hours, the supernatant was removed. 200 μL of DMSO was added to each well, and the plate was shaken for 30 minutes. Finally, the optical density (OD value) of each well was measured at 490 nm using an automated microplate reader.

[0099] Inhibition rate calculation: The cell growth inhibition rate is calculated according to the following formula:

[0100] Growth inhibition rate = (1 - survival rate) × 100% = [1 - (OD experiment - OD blank) / (OD control - OD blank)] × 100% (OD experiment represents the average optical density of the test drug group, OD control represents the average optical density of the control group, and OD blank represents the average optical density of the control group).

[0101] The inhibitory activity of crocin-PEG monomethyl ether conjugates on the proliferation of HepG2 cells is shown in Table 4. The results in Table 4 show that the crocin-PEG monomethyl ether conjugates have a strong inhibitory effect on the proliferation of HepG2 cells, and this effect is concentration-dependent. The IC50 values ​​of TC-mPEG550 and TC-mPEG1000 are 18.51 ug / mL and 19.28 ug / mL, respectively. The inhibitory effect of TC-mPEG2000 is weaker than that of TC-mPEG550 and TC-mPEG1000, and no IC50 value was found. However, the inhibitory effects of all conjugates are greater than those of crocin.

[0102] Table 4. Inhibition rate of tumor cells at different concentrations of each sample.

[0103]

[0104]

[0105] The various embodiments and experimental examples of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and technical principles of the described embodiments, and these modifications and variations should also be considered as protection of the present invention.

Claims

1. A crocin-polyethylene glycol monomethyl ether coupling nanoparticle, characterized in that, The structural formula of the coupling nanoparticles is: The coupling nanoparticles self-assemble in an aqueous solution. The molecular weight of polyethylene glycol monomethyl ether is 550-1000.

2. A method for preparing crocin-polyethylene glycol monomethyl ether coupling nanoparticles as described in claim 1, characterized in that, The specific steps are as follows: 1) Dissolve crocin in an organic solvent, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and triethylamine, stir, heat to 35-50°C, and react for 30-60 min to obtain reaction solution 1; 2) Add polyethylene glycol monomethyl ether to the reaction solution 1 from the previous step, and continue the reaction for 24-36 hours. After the reaction is completed, remove the organic solvent by rotary evaporation to obtain intermediate 1. 3) Add intermediate 1 to distilled water, dissolve and disperse it using ultrasound, and then put it into a dialysis bag for dialysis. After dialysis, centrifuge the mixture in the dialysis bag to separate the precipitate and the supernatant. Freeze-dry the supernatant to obtain crocin-polyethylene glycol monomethyl ether conjugate nanoparticles.

3. The method for preparing crocin-polyethylene glycol monomethyl ether coupling nanoparticles as described in claim 2, characterized in that, The organic solvent in step 1) is any one or a combination of dichloromethane, dichloroethane, petroleum ether, and cyclohexane.

4. The method for preparing crocin-polyethylene glycol monomethyl ether coupling nanoparticles as described in claim 2, characterized in that, The molar ratio of crocin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and triethylamine in step 1) is 1:2.5:2.5:2.

5.

5. The method for preparing crocin-polyethylene glycol monomethyl ether coupling nanoparticles as described in claim 2, characterized in that, In step 2), the mass ratio of polyethylene glycol monomethyl ether to crocin is 1:2.

5.

6. The method for preparing crocin-polyethylene glycol monomethyl ether coupling nanoparticles as described in claim 2, characterized in that, The mass ratio of distilled water to crocin in step 3) is 50-100:

1.

7. The method for preparing crocin-polyethylene glycol monomethyl ether coupling nanoparticles as described in claim 2, characterized in that, The specific dialysis conditions in step 3) are: dialysis for 3-4 days, with the water changed 3-4 times a day.

8. The application of the crocin-polyethylene glycol monomethyl ether coupling nanoparticles as described in any one of claims 1-7, characterized in that, The conjugate nanoparticles are used to prepare drugs that inhibit the proliferation of liver cancer cells.

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