Preparation method of docetaxel-crocetin-loaded pegylated liposome as well as product and application of docetaxel-crocetin-loaded pegylated liposome
By preparing pegylated liposomes, docetaxel and saffron acid were co-loaded, which solved the problems of poor solubility and drug resistance of docetaxel, and achieved sustained release effect with small particle size and high stability and coordinated anti-tumor activity.
Patent Information
- Application Number
- CN202510815291.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-18
- Publication Date
- 2025-08-15
AI Technical Summary
Docetaxel has poor solubility in aqueous solution, commonly used solubilizers cause adverse reactions, and drug resistance is prominent. How to co-encapsulate lipophilic docetaxel with saffron acid into liposomes to achieve synergistic anti-tumor effect.
Succinyl cholesterol is prepared by reaction of succinic anhydride, cholesterol and 4-dimethylaminopyridine, and then coupled with methoxy polyethylene glycol to prepare pegylated cholesterol. Finally, co-soluble with lecithin, docetaxel and saffronic acid and dispersed ultrasonically to prepare pegylated liposomes.
The prepared liposomes have small particle size and high stability, significant sustained release effects of docetaxel and saffronic acid, good biocompatibility and anti-tumor activity, and coordinated killing tumor cells by activating the apoptosis pathway.
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Figure CN120478282A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, and in particular to a preparation method of docetaxel-crocetin-loaded PEGylated liposomes and a product and application thereof. Background Art
[0002] Docetaxel is the most active cytotoxic drug for the treatment of cancer and is widely used clinically. Docetaxel has poor solubility in aqueous solutions, and Tween 80 and ethanol are commonly used clinically to solubilize docetaxel. These solubilizers can cause allergic reactions in most patients, including hypersensitivity reactions, fluid retention, and nausea, ultimately leading to serious adverse reactions and suboptimal therapeutic effects. With the advancement of clinical application, docetaxel resistance has gradually emerged, becoming a major factor restricting its application.
[0003] Liposomes are vesicles with a lipid bilayer and are a new dosage form for targeted drug delivery systems. As drug carriers, liposomes significantly enhance the therapeutic efficacy of drugs and reduce their toxicity by improving their targeting and bioavailability. A large number of researchers have found that extracting effective auxiliary drugs from natural plants can enhance the anti-tumor activity of cancer drugs. Crocetin is a compound extracted from gardenia yellow, which has multiple pharmacological activities, including anti-inflammatory, antioxidant, and anti-tumor. How to co-encapsulate lipophilic docetaxel and crocetin in liposomes to achieve co-delivery of docetaxel and crocetin and synergistically enhance the anti-tumor effect is a key technical issue that needs to be solved. Summary of the Invention
[0004] Against this background, the present invention proposes a method for preparing docetaxel-crocetin-loaded PEGylated liposomes, its products, and applications. The docetaxel-crocetin-loaded PEGylated liposomes prepared by this method have many characteristics, including small particle size, high liposome stability, and the ability to enhance the sustained-release effect of docetaxel and crocetin.
[0005] In order to overcome at least one of the above-mentioned shortcomings of the prior art, the first aspect of the present invention provides a method for preparing PEGylated liposomes loaded with docetaxel-crocetin, comprising: S1. Dissolving succinic anhydride, cholesterol, and 4-dimethylaminopyridine in a first non-polar solvent, heating for reaction, and performing post-treatment to obtain white solid succinylated cholesterol; S2. Dissolving the succinyl cholesterol, methoxypolyethylene glycol, 4-dimethylaminopyridine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride prepared in step S1 in a second non-polar solvent, heating and stirring to react, and performing post-treatment to obtain PEGylated cholesterol. S3: dissolving the PEGylated cholesterol, lecithin, docetaxel and crocetin prepared in step S2 in a third non-polar solvent. After removing the solvent, adding a mixed solution of lysine and sucrose, sonicating, and centrifuging to obtain a supernatant to obtain a drug-loaded liposome solution.
[0006] Furthermore, the post-treatment in step S1 is specifically as follows: removing the solvent by rotary evaporation, recrystallizing with a mixed solution of ethanol and ethyl acetate, washing with dilute hydrochloric acid, and drying to obtain white solid succinyl cholesterol.
[0007] Furthermore, in step S1, the non-fluorine material matrix is selected from any one or more of polybenzimidazole, polyetheretherketone, and sulfonated polyetheretherketone.
[0008] Furthermore, the post-treatment in step S2 is specifically as follows: removing the solvent by rotary evaporation, transferring the obtained reaction system to ultrapure water for dialysis for 2 to 3 days, and then centrifuging the reaction system. The supernatant obtained by centrifugation is collected and freeze-dried to obtain a white solid, namely, PEGylated cholesterol.
[0009] Furthermore, the post-treatment in step S2 is specifically as follows: removing the solvent by rotary evaporation, transferring the obtained reaction system to ultrapure water for dialysis for 2 to 3 days, and then centrifuging the reaction system. The supernatant obtained by centrifugation is collected and freeze-dried to obtain a white solid, namely, PEGylated cholesterol.
[0010] Furthermore, in step S3, the method for removing the solvent is rotary evaporation, the ultrasonic equipment used is an ultrasonic crusher, the ultrasonic crusher has an operating power of 90W, and the time is 10 to 30 minutes; the centrifugal speed is 5500 to 6000 rpm, and the centrifugation time is 10 to 20 minutes.
[0011] Furthermore, in step S1, the molar ratio of succinic anhydride, cholesterol and 4-dimethylaminopyridine is 1:3:0.5, the heating and stirring reaction temperature is 38-41° C., the reaction time is 24-36 hours, and the first non-polar solvent is dichloromethane.
[0012] Furthermore, in step S2, the molar ratio of succinyl cholesterol, methoxypolyethylene glycol, EDCI and DMAP is 1:1:15:2, the heating and stirring reaction temperature is 38-41° C., the reaction time is 24-36 hours, and the second non-polar solvent is dichloromethane.
[0013] Furthermore, in step S3, the mass ratio of PEGylated cholesterol, lecithin, docetaxel and crocetin is 20:60:1:1; and the third non-polar solvent is dichloromethane.
[0014] The second aspect of the present invention provides a docetaxel-crocetin-loaded PEGylated liposome, wherein the docetaxel-crocetin-loaded PEGylated liposome is prepared by the above-mentioned preparation method.
[0015] The third aspect of the present invention provides an application of docetaxel-crocetin loaded PEGylated liposomes for use in anti-tumor drugs.
[0016] The beneficial effects of the present invention are: The docetaxel-crocetin-loaded PEGylated liposomes prepared by the present invention have a small particle size, high liposome stability, can enhance the sustained release of docetaxel and crocetin, have good biocompatibility, and have a higher killing effect on tumor cells. Experimental results show that co-loading crocetin and docetaxel into liposomes can significantly enhance the in vitro anti-tumor effect. This is because docetaxel activates the caspase-3 / 9-dependent mitochondrial apoptosis pathway, and crocetin upregulates the Bax / Bcl-2 ratio and inhibits NF-κB survival signaling. The two cross-amplify the apoptotic effect through the endogenous / exogenous apoptosis pathway, synergistically killing tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The present invention will be further described below with reference to the accompanying drawings and examples.
[0018] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of succinyl cholesterol prepared in Example 1 of the present invention.
[0019] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the PEGylated cholesterol prepared in Example 1 of the present invention.
[0020] Figure 3 This is the infrared spectrum of the PEGylated cholesterol prepared in Example 1 of the present invention.
[0021] Figure 4 The particle size distribution diagrams of docetaxel-crocetin-loaded PEGylated liposomes, crocetin-loaded PEGylated liposomes, and docetaxel PEGylated liposomes prepared in Example 1 of the present invention and Comparative Examples 1-2, respectively.
[0022] Figure 5 Graph showing the particle size stability in ultrapure water at 25° C. of docetaxel-crocetin PEGylated liposomes, crocetin PEGylated liposomes, and docetaxel PEGylated liposomes prepared in Example 1 of the present invention and Comparative Examples 1-2, respectively.
[0023] Figure 6Graph showing the particle size stability of docetaxel-crocetin-loaded PEGylated liposomes, crocetin-loaded PEGylated liposomes, and docetaxel-loaded PEGylated liposomes prepared in Example 1 of the present invention and Comparative Examples 1-2, respectively, in ultrapure water at 37°C.
[0024] Figure 7 The graph shows the particle size stability of docetaxel-crocetin-loaded PEGylated liposomes, crocetin-loaded PEGylated liposomes, and docetaxel-loaded PEGylated liposomes prepared in Example 1 of the present invention and Comparative Examples 1-2, respectively, in PBS buffer at 25°C.
[0025] Figure 8 The graphs show the particle size stability of docetaxel-crocetin PEGylated liposomes, crocetin PEGylated liposomes, and docetaxel PEGylated liposomes prepared in Example 1 of the present invention and Comparative Examples 1-2, respectively, in PBS buffer at 37°C.
[0026] Figure 9 This is the drug combination index curve of crocetin and docetaxel. DETAILED DESCRIPTION
[0027] Before discussing the exemplary embodiments in more detail, it should be mentioned that some exemplary embodiments are described as processes or methods depicted as flow charts. Although the flow charts describe the various operations as sequential processes, many of the operations therein can be implemented in parallel, concurrently, or simultaneously. In addition, the order of the various operations can be rearranged. The process can be terminated when its operation is completed, but can also have additional steps not included in the accompanying drawings. The process can correspond to a method, function, procedure, subroutine, subprogram, etc.
[0028] It should be understood that although the terms "first," "second," and the like may be used herein to describe various elements, these elements should not be limited by these terms. These terms are used solely to distinguish one element from another. For example, a first element may be referred to as a second element, and similarly, a second element may be referred to as a first element, without departing from the scope of the exemplary embodiments. The term "and / or" as used herein includes any and all combinations of one or more of the listed associated items.
[0029] The present invention provides a method for preparing docetaxel-crocetin loaded PEGylated liposomes, comprising: S1. Dissolving succinic anhydride, cholesterol, and 4-dimethylaminopyridine in a first non-polar solvent, heating for reaction, and performing post-treatment to obtain white solid succinylated cholesterol; The preparation reaction formula of succinyl cholesterol is as follows:
[0030] The molar ratio of succinic anhydride, cholesterol and 4-dimethylaminopyridine is 1:3:0.5.
[0031] S2. Dissolving the succinyl cholesterol, methoxypolyethylene glycol, 4-dimethylaminopyridine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride prepared in step S1 in a second non-polar solvent, heating and stirring to react, and performing post-treatment to obtain PEGylated cholesterol. The reaction formula for preparing PEGylated cholesterol is as follows:
[0032] The molar ratio of succinyl cholesterol, methoxypolyethylene glycol, EDCI and DMAP is 1:1:15:2.
[0033] S3: dissolving the PEGylated cholesterol, lecithin, docetaxel and crocetin prepared in step S2 in a third non-polar solvent. After removing the solvent, adding a mixed solution of lysine and sucrose, sonicating, and centrifuging to obtain a supernatant to obtain a drug-loaded liposome solution.
[0034] The mass ratio of pegylated cholesterol, lecithin, docetaxel and crocetin is 20:60:1:1.
[0035] Example 1 Preparation of S1 succinylated cholesterol (CHOL-SAA) In a 50 ml round-bottom flask, add 10 ml of dichloromethane, 2.0 g of cholesterol, 1.56 g of succinic anhydride, and 317 mg of 4-dimethylaminopyridine, stir to dissolve, heat to 40°C and stir to react for 24 hours. After the reaction, remove the solvent by rotary evaporation, recrystallize with ethanol and ethyl acetate, wash twice with dilute hydrochloric acid, wash twice with ultrapure water, and dry to obtain a white solid (yield 77.5%).
[0036] The chemical structure of the target compound prepared in Example 1 was confirmed by H NMR. Figure 1 As shown, the H NMR spectrum showed characteristic peaks of methylene hydrogen in succinic anhydride at chemical shifts of 2.61 and 2.67 ppm, and a characteristic peak of cholesterol at 4.65 ppm, indicating that succinic anhydride and cholesterol were successfully coupled.
[0037] Preparation of S2 PEGylated cholesterol (mPEG-CHOL) In a 50 ml round-bottom flask, add 10 ml of dichloromethane, 909 mg of succinyl cholesterol, 4 g of methoxypolyethylene glycol (molecular weight 2000), 488 mg of 4-dimethylaminopyridine, and 5.75 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, stir to dissolve, heat to 40°C and stir to react for 24 hours. After the reaction, remove the solvent by rotary evaporation, transfer the resulting reaction system into ultrapure water and dialyze for 3 days, then centrifuge the reaction system, collect the supernatant obtained by centrifugation, and freeze-dry to obtain a white solid, namely PEGylated cholesterol (yield 83.5%).
[0038] like Figure 2 As shown in the figure, the H NMR spectrum of PEGylated cholesterol showed characteristic peaks of methylene hydrogen in succinic anhydride at chemical shifts of 2.60 and 2.65 ppm, characteristic peaks of polyethylene glycol at 3.51-3.78 ppm, and characteristic peaks of cholesterol at 4.62 ppm, indicating that PEGylated cholesterol was successfully coupled. Figure 3 As shown, the infrared spectrum of PEGylated cholesterol copolymer is at 1730 cm -1 A strong peak appeared at the ester bond, which was the characteristic absorption peak of C=O in the ester bond, while mPEG itself did not have this peak, which proved that the coupling of monocholesterol succinate with the terminal hydroxyl group of mPEG was successful.
[0039] S3 Preparation of docetaxel-crocetin PEGylated liposomes (DTX-CRO-Lip) To a 50-ml round-bottom flask, add 30 ml of dichloromethane, 1 g of PEGylated cholesterol, 3 g of lecithin, and 50 mg each of docetaxel and crocetin, stirring to dissolve. The solvent was removed by rotary evaporation, and a film was formed. 2.5 g of sucrose and 0.05 g of lysine were dissolved in 25 ml of ultrapure water and added in batches to the spin-dried round-bottom flask. Ultrasonic dispersion was performed for 15 minutes (90 W), followed by sonication in a sonicator for 15 minutes. The supernatant was then centrifuged at 6000 rpm, and the docetaxel encapsulation efficiency was determined to be 81.44% by HPLC, and 92.09% by microplate reader.
[0040] The encapsulation efficiency calculation formula is: Encapsulation efficiency =
[0041] Comparative Example 1 Preparation of Crocetin PEGylated Liposomes (CRO-Lip) To a 50-ml round-bottom flask, add 30 ml of dichloromethane, 1 g of PEGylated cholesterol, 3 g of lecithin, and 50 mg of crocetin, stirring to dissolve. The solvent was removed by rotary evaporation, and a film was formed. 2.5 g of sucrose and 0.05 g of lysine were dissolved in 25 ml of ultrapure water and added in batches to the spin-dried round-bottom flask. Ultrasonic dispersion was performed for 15 minutes (90 W), followed by sonication in a sonicator for 15 minutes. The supernatant was centrifuged at 6000 rpm, and the encapsulation efficiency of crocetin was measured by a microplate reader, indicating a 92.95% encapsulation efficiency.
[0042] Comparative Example 2 Preparation of docetaxel PEGylated liposomes (DTX-CRO-Lip) To a 50-ml round-bottom flask, add 30 ml of dichloromethane, 1 g of PEGylated cholesterol, 3 g of lecithin, and 50 mg of docetaxel, stirring to dissolve. The solvent was removed by rotary evaporation and film formation was completed. 2.5 g of sucrose and 0.05 g of lysine were dissolved in 25 ml of ultrapure water and added in batches to the spin-dried round-bottom flask. Ultrasonic dispersion was performed at 90 W for 10 minutes, followed by sonication in a sonicator for 15 minutes. The supernatant was centrifuged at 6000 rpm and the docetaxel encapsulation efficiency was 82.54% as determined by HPLC.
[0043] The docetaxel-crocetin PEGylated liposomes, crocetin PEGylated liposomes, and docetaxel PEGylated liposomes prepared in the above examples and comparative examples 1-2, respectively, were tested.
[0044] Test Example 1 Determination of particle size and potential Preparation of test solution: The liposomes prepared in Example 1 and Comparative Examples 1-2 were diluted 5 times with ultrapure water to obtain test solution.
[0045] Particle size and potential measurement: 1 ml of each test solution prepared above was taken and the particle size and potential were measured using a Malvern particle size analyzer.
[0046] Figure 4 Figure 2 shows the particle size distribution of docetaxel-PEGylated liposomes, crocetin-PEGylated liposomes, and docetaxel-crocetin-PEGylated liposomes prepared in Example 1 and Comparative Examples 1 and 2. The experimental results show that the particle size of the liposomes prepared in Example 1, Comparative Examples 1, and 2 is comparable, all less than 100 nm. The polydispersity index of all three liposomes is approximately 0.2, indicating a narrow particle size distribution. The zeta potential of the prepared liposomes is negative, which promotes long-term blood circulation.
[0047] Table 1. Particle size and potential of liposomes prepared in Example 1 and Comparative Examples 1-2
[0048] Test Example 2 Stability test of PEGylated liposomes The stability of docetaxel-loaded liposomes, crocetin liposomes, and docetaxel-crocetin liposomes in PBS buffer and deionized water at different temperatures was determined by dynamic light scattering, and a coordinate diagram was drawn ( Figures 5 to 8 ).Depend on Figure 5 and Figure 6 As shown, after 7 days of incubation, the particle size of the liposomes prepared in Example 1, Comparative Example 1 and Comparative Example 2 in deionized water at different temperatures did not change significantly; Figure 7 and Figure 8 As shown, after 7 days of incubation, the particle sizes of the liposomes prepared in Example 1, Comparative Example 1 and Comparative Example 2 did not change significantly in PBS buffer at different temperatures, indicating that they had good stability in deionized water and PBS buffer. Test Example 3 In vitro simulated drug release evaluation Based on the encapsulation efficiency and particle size test results, the liposomes prepared in Example 1 and Comparative Examples 1-2 were selected for in vitro simulated drug release evaluation.
[0049] Preparation of pH 5.0 release solution: Weigh 0.24 g of potassium dihydrogen phosphate and 3.58 g of disodium hydrogen phosphate dodecahydrate, add 500 ml of ultrapure water and 5 ml of Tween 80 to dissolve, and then adjust the pH to 5.0.
[0050] Preparation of pH 7.4 release solution: Weigh 0.24 g potassium dihydrogen phosphate and 3.58 g disodium hydrogen phosphate dodecahydrate, add 500 ml ultrapure water and 5 ml Tween 80 to dissolve, and then adjust the pH to 7.4.
[0051] In vitro simulated drug release evaluation: 1 mL of liposomes prepared in Example 1 and Comparative Examples 1-2 was placed in a dialysis bag (molecular weight cutoff: 3500), sealed, and then immersed in 30 mL of the aforementioned release solution. The bag was shaken at 37°C and 180 rpm. 1 mL of the release solution was removed at time points of 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, and 96 h, and replaced with 1 mL of the corresponding blank release solution to maintain the same volume. Three sets of release experiments were performed in parallel for each release solution. Release experiments were conducted in the dark. The absorbance of the removed release solution was measured to calculate the cumulative release of docetaxel and crocetin. The cumulative release results for the liposome solutions prepared in Example 1 and Comparative Examples 1-2 are shown in Tables 2-3.
[0052] Table 2. Cumulative release of crocetin from PEGylated liposome solutions prepared in Example 1 and Comparative Example 1
[0053] Table 3. Cumulative release of docetaxel from the PEGylated liposome solutions prepared in Example 1 and Comparative Example 2
[0054] As shown in Tables 2-3, Example 1 and Comparative Examples 1-2 have similar drug release behaviors in release media at pH 7.4 and pH 5.0. Example 1 and Comparative Example 1 both released only about 13% of crocetin within 24 h, and Example 1 and Comparative Example 2 both released only about 14% of docetaxel within 24 h. Subsequently, crocetin and docetaxel were released slowly. The slower release rates of crocetin and docetaxel were beneficial to the stability of the two drugs. There was no significant change in the release behaviors of crocetin and docetaxel under different pH conditions, indicating that Example 1 and Comparative Examples 1-2 both had good sustained-release effects under different pH conditions.
[0055] Test Example 4 In vitro antitumor test 4T1 cells were cultured at 5 × 10 3 The cells were seeded at a density of 10 cells per well in a 96-well plate, and four parallel experiments were performed for each concentration. Free docetaxel, free crocetin, and the liposomes prepared in Example 1 and Comparative Examples 1-2 were diluted with complete culture medium to a series of concentrations (the docetaxel concentrations were 3.125, 6.25, 12.5, 25, 50, and 100 μg / mL, and the saffron concentrations were 6.875, 13.75, 27.5, 55, 110, and 220 μg / mL, respectively) and added to a 96-well plate. 4T1 cells were co-incubated for 48 h, and then 20 μL of MTT was further added to each well. The cells were incubated at 37° C. for 4 hours, and the absorbance at 490 nm was measured in a multi-function microplate reader.
[0056] In all cell lines, the tested drugs showed dose- and time-dependent cytotoxicity. The maximum half-inhibitory concentrations (IC50) of free docetaxel, crocetin, Example 1, and Comparative Examples 1-2 on 4T1 cells were 50 ) The data are shown in Table 4. The anti-tumor activity of Example 1 is stronger than that of Comparative Example 1 and Comparative Example 2. The average IC values of free docetaxel, crocetin, Example 1 and Comparative Example 1 after incubation with 4T1 for 48 hours are 50The values were 43.94, 83.69, 1.527, 63.66, and 32.46 μg / mL, respectively. The results showed that compared with Comparative Examples 1 and 2, the co-loading of crocetin and docetaxel into liposomes in Example 1 significantly enhanced the in vitro anti-tumor effect of Example 1. Docetaxel activates the caspase-3 / 9-dependent mitochondrial apoptosis pathway. Crocetin upregulates the Bax / Bcl-2 ratio and inhibits NF-κB survival signaling. The two cross-amplify the apoptotic effect through the intrinsic / extrinsic apoptosis pathway and synergistically kill tumor cells.
[0057] Table 4. In vitro antitumor activity of liposomes prepared in Example 1 and Comparative Examples 1-2
[0058] According to the cell viability results of Example 1 and Comparative Examples 1 and 2, the cell viability data of crocetin in Comparative Example 1 and docetaxel in Comparative Example 2 at different concentrations were input into GraphPad 9.0 software to calculate the IC values of single administration and combined administration. 50 The combination index (CI) of the drugs was further calculated using CompuSyn 1.0 software. The experimenter drew a curve showing the relationship between the CI value and the effect (fa) of crocetin and docetaxel at the combined ratio ( Figure 9 When the CI value is greater than 1, it indicates that the two drugs have an antagonistic effect; when the CI value is less than 1, it indicates that the two drugs have a synergistic effect, and the smaller the CI value, the stronger the synergistic effect. Figure 9 As shown, the CI values are all less than 1, indicating that the crocetin-docetaxel-loaded PEGylated liposomes prepared in Example 1 of the present invention have a strong synergistic anti-tumor effect.
[0059] The above is only an embodiment of the present invention. Common knowledge such as the known specific structures and characteristics in the scheme is not described in detail here. Ordinary technicians in the field are aware of all common technical knowledge in the technical field of the invention before the application date or priority date, can obtain all existing technologies in the field, and have the ability to apply conventional experimental means before that date. Ordinary technicians in the field can improve and implement this scheme in combination with their own abilities under the inspiration given by this application. Some typical known structures or known methods should not become obstacles for ordinary technicians in the field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.
Claims
1. A method for preparing PEGylated liposomes loaded with docetaxel-crocetin, characterized in that: The method comprises the following steps S1. Dissolving succinic anhydride, cholesterol, and 4-dimethylaminopyridine in a first non-polar solvent, heating for reaction, and performing post-treatment to obtain white solid succinylated cholesterol; S2. Dissolving the succinyl cholesterol, methoxypolyethylene glycol, 4-dimethylaminopyridine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride prepared in step S1 in a second non-polar solvent, heating and stirring to react, and performing post-treatment to obtain PEGylated cholesterol. S3: dissolving the PEGylated cholesterol, lecithin, docetaxel and crocetin prepared in step S2 in a third non-polar solvent. After removing the solvent, adding a mixed solution of lysine and sucrose, sonicating, and centrifuging to obtain a supernatant to obtain a drug-loaded liposome solution.
2. The method for preparing docetaxel-crocetin loaded PEGylated liposomes according to claim 1, characterized in that: The post-treatment in step S1 specifically includes removing the solvent by rotary evaporation, recrystallizing with a mixed solution of ethanol and ethyl acetate, washing with dilute hydrochloric acid, and drying to obtain white solid succinylated cholesterol.
3. The preparation method of the PEGylated liposome loaded with docetaxel-crocetin according to claim 1 The method is characterized by: The post-treatment in step S2 is specifically to remove the solvent by rotary evaporation, transfer the resulting reaction system into ultrapure water for dialysis for 2 to 3 days, and then centrifuge the reaction system. The supernatant obtained by centrifugation is collected and freeze-dried to obtain a white solid, namely, PEGylated cholesterol.
4. The preparation method of the PEGylated liposome loaded with docetaxel-crocetin according to claim 1 The method is characterized by: In step S3, the method for removing the solvent is rotary evaporation, and the equipment used for ultrasound is an ultrasonic crusher with an operating power of 90W and a time of 10 to 30 minutes; the centrifugal speed is 5500 to 6000 rpm and the centrifugal time is 10 to 20 minutes.
5. The preparation method of the PEGylated liposome loaded with docetaxel-crocetin according to claim 1 The method is characterized by: In step S1, the molar ratio of succinic anhydride, cholesterol and 4-dimethylaminopyridine is 1:3:0.5, the heating and stirring reaction temperature is 38-41° C., the reaction time is 24-36 hours, and the first non-polar solvent is dichloromethane.
6. The preparation method of the PEGylated liposome loaded with docetaxel-crocetin according to claim 1 The method is characterized by: In step S2, the molar ratio of succinyl cholesterol, methoxypolyethylene glycol, EDCI and DMAP is 1:1:15:2, the heating and stirring reaction temperature is 38-41° C., the reaction time is 24-36 hours, and the second non-polar solvent is dichloromethane.
7. The preparation method of the PEGylated liposome loaded with docetaxel-crocetin according to claim 1 The method is characterized by: In step S3, the mass ratio of PEGylated cholesterol, lecithin, docetaxel, and crocetin is 20:60:1:1; and the third non-polar solvent is dichloromethane.
8. A PEGylated liposome loaded with docetaxel-crocetin prepared by the method for preparing PEGylated liposome loaded with docetaxel-crocetin according to any one of claims 1 to 7.
9. Use of the docetaxel-crocetin loaded PEGylated liposomes according to claim 8, characterized in that: The PEGylated liposome loaded with docetaxel-crocetin is used in anti-tumor drugs.