RGD-modified crocetin-paclitaxel co-drug-loaded compound liposome as well as preparation method and application thereof

Abstract

The invention discloses an RGD modified crocetin-paclitaxel co-drug-loaded compound liposome and a preparation method and application thereof, and belongs to the technical field of biological medicines.The RGD modified crocetin-paclitaxel co-drug-loaded compound liposome comprises the following components: an RGD modified liposome, paclitaxel and crocetin, wherein the RGD modified lipidosome is used as a drug carrier, paclitaxel and crocetin are delivered to prostate tumor cells in a targeted manner, and the RGD modified lipidosome is prepared through a film dispersion method or a reverse evaporation method. The prostate cancer targeted compound liposome prepared by the invention can increase the uptake of prostate cancer cells, improve the in-vitro cytotoxicity of the prostate cancer cells to the prostate cancer cell line RM-1, inhibit the growth of RM-1 ectopic solid tumors in mice, and do not cause obvious damage to other tissues.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to an RGD-modified crocetin-paclitaxel co-drug-loaded composite liposome for treating prostate cancer and a preparation method and application thereof. Background Art

[0002] Although some progress has been made in the treatment of prostate cancer, the vast majority of patients will eventually develop castration-resistant prostate cancer (CRPC). Castration-resistant prostate cancer is a very stubborn type of cancer that gradually develops resistance to traditional androgen deprivation therapy (ADT), and the therapeutic effect is significantly reduced. The occurrence and development of CRPC involves complex biological mechanisms, including mutation or amplification of androgen receptors, reprogramming of androgen synthesis pathways, and changes in the tumor microenvironment. Due to the diversity and complexity of these mechanisms, the current treatment of CRPC still faces many challenges, and there is a lack of treatment options that can effectively control the progression of the disease in the clinic.

[0003] Paclitaxel (PTX) is a natural organic compound extracted from the bark of the American yew (Taxus brevifolia), belonging to the taxane class of compounds. Paclitaxel is widely used in the treatment of various malignant tumors such as ovarian cancer, breast cancer, non-small cell lung cancer, esophageal cancer, prostate cancer, etc., and is one of the important anticancer drugs used clinically. In the treatment of prostate cancer, paclitaxel has shown good efficacy, especially in combating advanced and metastatic prostate cancer. However, adverse reactions are common during paclitaxel treatment, including leukopenia, neurotoxicity, and cardiovascular adverse reactions.

[0004] Crocetin (CRT) is an important active ingredient in saffron. It is an amphiphilic low molecular weight carotenoid compound that shows significant anti-tumor potential in animal models and cell culture systems. Its anti-tumor effect is mainly achieved through multiple pathways such as inhibiting tumor cell proliferation, inducing cell apoptosis, inhibiting tumor metastasis, and enhancing the effects of radiotherapy and chemotherapy (Koch, Wojciech, et al. "Exploring the therapeutic efficacy of crocetin in oncology: an evidence-based review." Naunyn-Schmiedeberg's Archives of Pharmacology, vol. 397, no. 3, 2023, pp. 1-12. DOI: 10.1007 / s00210-023-02714-z.). Other studies have shown that crocetin has the potential to be used in combination with other drugs, which can significantly enhance the efficacy. Because crocetin itself has good biocompatibility and low toxic side effects, it can be used in combination with traditional chemotherapy drugs or targeted drugs to synergistically enhance the anti-cancer effect. For example, when crocetin is used in combination with chemotherapy drugs such as cisplatin and paclitaxel, it can increase the sensitivity of tumor cells to chemotherapy drugs and improve the patient's clinical prognosis.

[0005] RGD peptide is a synthetic peptide containing the sequence of arginine-glycine-aspartic acid (Arg-GLy-ASP), which can recognize integrin α highly expressed on the surface of tumor cells and tumor blood vessels. v β 3 The RGD-modified liposomes can bind to the receptors. The use of RGD-modified liposomes as anti-tumor drug carriers can enhance their targeting to tumors, thereby improving the anti-tumor effect of the drug. The present invention explores for the first time the synergistic inhibitory effect of CRT and PTX on prostate cancer, and prepares a RGD-modified CRT and PTX co-drug-loaded liposome to evaluate its efficiency in anti-prostate tumors. Summary of the invention

[0006] The purpose of the present invention is to provide an RGD-modified crocetin-paclitaxel co-drug-loaded composite liposome and its preparation method and application. The liposome targets tumor cells and can treat prostate cancer, so it can be used to prepare a pharmaceutical preparation for treating prostate cancer.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The invention provides an RGD-modified crocetin-paclitaxel co-drug-loaded composite liposome, which comprises RGD-modified liposome, paclitaxel (PTX) and crocetin (CRT), wherein the RGD-modified liposome is used as a drug carrier to deliver paclitaxel and crocetin to prostate tumor cells in a targeted manner, and the RGD-modified liposome is prepared by a thin film dispersion method or a reverse evaporation method.

[0009] Furthermore, the ratio of paclitaxel to crocetin is between 1:3 and 1:1.

[0010] Furthermore, the paclitaxel and crocetin are loaded into the RGD-modified liposomes by passive loading or active loading.

[0011] Furthermore, in the RGD-modified liposome, the liposome carrier comprises the following auxiliary materials in parts by weight: 20-200 parts of phospholipids, 3-15 parts of cholesterol, 1-5 parts of PEG-DSPE, and 1-5 parts of RGD-PEG-DSPE.

[0012] Furthermore, the RGD-modified crocetin-paclitaxel co-drug-loaded composite liposome described in the present invention is used as a pharmaceutical composition and prepared into a clinically acceptable preparation with a pharmaceutically acceptable carrier. The preparation is an injection, an oral preparation or an external preparation.

[0013] The RGD-modified crocetin-paclitaxel co-drug-loaded composite liposome of the present invention is prepared by a thin film dispersion method.

[0014] Furthermore, the preparation method of the RGD-modified crocetin-paclitaxel co-drug-loaded composite liposome comprises the following steps:

[0015] (1) Phospholipids, cholesterol, and PEG-DSPE were mixed in proportion, RGD peptide was added, and RGD-modified liposomes (RGD-PEG-DSPE) were prepared by a thin film dispersion method;

[0016] (2) mixing paclitaxel and crocetin, and adding the mixture to the RGD-modified liposome solution;

[0017] (3) Purify by ultrafiltration or centrifugation to obtain a composition of RGD-modified liposomes loaded with paclitaxel and crocetin.

[0018] In the step (2), paclitaxel and crocetin are mixed in a ratio of 1:3 to 1:1.

[0019] The invention discloses an application of the RGD-modified crocetin-paclitaxel co-drug-loaded composite liposome in the preparation of a drug for treating cancer metastasis.

[0020] Furthermore, the cancer is prostate cancer. The RGD-modified crocetin-paclitaxel co-drug-loaded composite liposomes deliver paclitaxel and crocetin to prostate tumor cells in a targeted manner, and synergistically exert anti-tumor effects. The specific method of use is to inject the RGD-modified crocetin-paclitaxel co-drug-loaded composite liposomes into a patient's body to inhibit tumor growth and induce tumor cell apoptosis.

[0021] Beneficial effects of the present invention:

[0022] 1. The co-drug-loaded composite liposome prepared by the present invention can exert the synergistic effect of the two drugs and inhibit the growth of prostate cancer solid tumors.

[0023] 2. The co-drug-loaded composite liposomes prepared by the present invention can enhance the uptake of the liposomes by prostate cancer cells and do not cause nonspecific damage to the body.

[0024] 3. The method of the present invention significantly improves the anti-tumor effect of paclitaxel and crocetin through the targeted delivery effect of RGD-modified liposomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figures show the inhibition of RM-1 cell proliferation by CRT, PTX, and the combination of PTX-CRT in the examples of the present invention.

[0026] Figure 2 This is an electron micrograph of the composite liposome prepared in Example 5 of the present invention.

[0027] Figure 3 It is the in vitro release curve of PTX in the embodiment of the present invention.

[0028] Figure 4 It is the in vitro release curve of CRT in the embodiment of the present invention.

[0029] Figure 5 The in vitro cytotoxicity of different liposomes in the examples of the present invention.

[0030] Figure 6 The uptake efficiency of different liposomes in RM-1 cells in the examples of the present invention is shown in FIG.

[0031] Figure 7 : is the uptake efficiency of different liposomes in HaCaT cells in the examples of the present invention.

[0032] Figure 8 The figure shows the in vivo antitumor effect of the composite liposome in the example of the present invention in a prostate tumor animal model.

[0033] Fig. 9 This is an analysis of the toxic and side effects of the compound liposomes on normal tissues in the embodiments of the present invention. DETAILED DESCRIPTION

[0034] What is described below is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

[0035] Example 1

[0036] Preparation of crocetin liposomes: The liposomes were prepared by the thin film hydration method. 15.0 mg DPPC, 2.0 mg cholesterol and 0.8 mg CRT were weighed and dissolved in anhydrous ethanol. After placing them in an eggplant-shaped bottle, an appropriate amount of anhydrous ethanol was added and ultrasonicated for 30 minutes to completely dissolve DPPC, cholesterol and crocetin. The solvent was removed under reduced pressure by a rotary evaporator to form a thin film on the wall of the eggplant-shaped bottle. The solid was then dried under reduced pressure in a vacuum drying oven at room temperature for 12 hours to remove the residual solvent. An appropriate amount of PBS solution (pH = 7.4) was added to the completely dried eggplant-shaped bottle and hydrated at 50°C for 2 hours. After hydration, the solution was ultrasonicated in an ice bath on a probe ultrasonicator for 10 minutes with a pulse of 10s / 5s. The crocetin liposomes CRT-LIP were obtained after passing through 0.45μm and 0.22μm filter membranes respectively.

[0037] Example 2

[0038] Preparation of paclitaxel liposomes: Liposomes were prepared by thin film hydration method, 30.0 mg of DPPC, 4.5 mg of cholesterol and 2.0 mg of PTX were weighed, and the preparation method was the same as that in Example 1 to obtain paclitaxel liposomes PTX-LIP.

[0039] Example 3

[0040] Preparation of composite liposomes: 30.0 mg DPPC, 4.5 mg cholesterol, 0.8 mg CRT and 2.0 mg PTX were weighed and prepared in the same manner as in Example 1 to obtain composite liposomes CO-LIP.

[0041] Example 4

[0042] Preparation of PEG-modified long-circulation compound liposomes: weigh 45.0 mg DPPC, 6.5 mg cholesterol, 0.8 mg CRT, 2.0 mg PTX, 2000 -DSPE 2.0 mg, prepared by the same method as in Example 1, to obtain PEG-modified long-circulation composite liposome PEG-CO-LIP.

[0043] Example 5

[0044] Preparation of RGD-modified crocetin-paclitaxel co-drug-loaded composite liposomes: 45.0 mg DPPC, 6.5 mg cholesterol, 0.8 mg CRT, 2.0 mg PTX, RGD-PEG 2000 -DSPE 2.0 mg, the preparation method is the same as Example 1, to obtain RGD modified long circulation targeted compound liposomes. The specific steps are as follows:

[0045] (1) Take an appropriate amount of DSPE-PEG 2000 -Mal was dissolved in a small amount of chloroform, and the organic solvent was removed under reduced pressure to form a thin film. It was hydrated with 0.01 mol / L phosphate buffer (PBS, pH 7.4), ultrasonicated in a water bath for 2 min, and 1.5 times the equivalent of cRGDyk-cys was added. The reaction was carried out at 4 ° C for 48 h. The product was placed in a dialysis bag and dialyzed with distilled water for 2 days to remove excess cRGDyk-cys. After the dialysis was completed, it was freeze-dried to obtain the product RGD-PEG 2000 -DSPE;

[0046] (2) DPPC 45.0 mg, cholesterol 6.5 mg, RGD-PEG 2000 -DSPE 2.0 mg, add anhydrous ethanol to dissolve, and ultrasonicate for 30 min to make DPPC, cholesterol and RGD-PEG 2000 -DSPE is completely dissolved to prepare RGD-modified liposome solution;

[0047] (3) Add 2.0 mg of PTX and 0.8 mg of CRT to the RGD-modified liposome solution of step (2) and stir evenly;

[0048] (4) The solvent was removed under reduced pressure using a rotary evaporator, so that the solid formed a thin film on the wall of the eggplant-shaped bottle; then the residual solvent was removed by drying under reduced pressure in a vacuum drying oven at room temperature for 12 h; an appropriate amount of PBS solution (pH = 7.4) was added to the completely dried eggplant-shaped bottle and hydrated at 50°C for 2 h. After hydration, the solution was sonicated in an ice bath on a probe ultrasonic instrument for 10 min with a pulse of 10s / 5s. After filtering through 0.45μm and 0.22μm filter membranes, RGD-CO-LIP loaded with paclitaxel and crocetin was obtained.

[0049] Example 6

[0050] Effects of crocetin, paclitaxel alone and their combination on prostate cancer cells RM-1:

[0051] The CCK-8 method was used to investigate the effects of different concentrations of PTX, CRT, and their combination on the cell survival rate of RM-1 cells. Figure 1As shown in the figure, crocetin concentrations in the range of 0-10 μM had no obvious cytotoxicity to RM-1 cells, so subsequent studies on CRT-LIP were not considered. Compared with the control group, paclitaxel produced obvious cytotoxicity to RM-1 cells when the concentration was greater than 10 nM; when the concentration of paclitaxel reached 100 nM, the inhibitory effect on the cell activity of RM-1 cells reached the highest. When the concentration of paclitaxel was 10 nM, the cell viability of the crocetin combined with paclitaxel treatment group decreased compared with the paclitaxel alone treatment group; when the concentration of crocetin was greater than 10 nM, the combined administration group showed significant differences compared with the paclitaxel alone treatment group, and with the increase of the dose of crocetin, the cell activity of RM-1 cells showed a dose-dependent decrease. This shows that combined administration has a better inhibitory effect on prostate cancer cells RM-1 than crocetin or paclitaxel alone.

[0052] As shown in Table 1, except for the 1 nM crocetin combined administration group, the IC 50 Both were smaller than that of the paclitaxel alone group, which also proved that the combined administration produced a greater cytotoxic effect on prostate cancer cell RM-1 compared with crocetin or paclitaxel alone.

[0053] Table 1 Effects of CRT, 10nM PTX and different concentrations of CRT+10nM on prostate cancer cells RM-1

[0054]

[0055] Example 7

[0056] Quality evaluation of compound liposomes:

[0057] 1. Determination of encapsulation efficiency and drug loading:

[0058] After the RGD-modified crocetin-paclitaxel co-loaded composite liposome (RGD-CO-LIP) was successfully prepared according to the method described in Example 5, 0.5 mL of RGD-CO-LIP suspension was accurately measured in a 5 mL volumetric flask, 4.5 mL of methanol was added, and the emulsion was fully broken by ultrasonication for 15 min. The total drug content was measured and recorded as m 1 , then take 0.5 mL of RGD-CO-LIP suspension into a 1.5 mL ultrafiltration centrifuge tube, centrifuge at 6000 rpm for 10 min, take the lower layer of liquid in the centrifuge tube and place it in a 5 mL volumetric flask, dilute to the mark with methanol, and perform ultrasonic demulsification for 15 min. The free drug content was measured and recorded as m 2 .

[0059] Encapsulation efficiency = (m 1 -m 2 ) / m 1

[0060] Drug loading = (amount of encapsulated drug / total amount of encapsulated drug and encapsulating excipients) × 100%

[0061] Table 2 Measured values ​​of encapsulation efficiency and drug loading

[0062]

[0063] 2. Determination of particle size and potential

[0064] The particle size (effective hydrodynamic diameter) of micelles was determined by photon correlation spectroscopy using a laser particle size analyzer (Nano-zs zen 3700, Malvern, UK). The experimental method is as follows: 1 mL of RGD-CO-LIP prepared in Example 5 was taken, shaken thoroughly, and then filtered through a 0.22 μm filter membrane and measured in a constant temperature cell at a temperature of 25°C and a scattering angle of 173° (backscattering, NIBS default value). The effective hydrodynamic diameter value and Zeta potential value were calculated using the software provided by the manufacturer.

[0065] Table 3 Measured values ​​of particle size and potential

[0066]

[0067] 3. Transmission electron microscopy morphology characterization of liposomes

[0068] A transmission electron microscope (Hitachi HT7800, Japan) was used to observe the morphology of the RGD-CO-LIP prepared in Example 5. The experimental method is as follows: take 1 mL of the RGD-CO-LIP prepared in Example 5, shake it thoroughly, filter it through a 0.22 μm filter membrane, take 10 μL and apply it to a copper mesh. When the liquid droplets on the mesh are about to dry but not dry, add a drop of 3% phosphotungstic acid solution for negative staining. After a period of time, pick up the copper mesh with tweezers, absorb the excess dye with filter paper, let the copper mesh dry naturally at room temperature, and then observe it on a transmission electron microscope. The accelerating voltage of the experiment is 80 kV. Figure 2 As shown, the prepared RGD-CO-LIP has a double-layer spherical vesicle structure with uniform size, which is basically consistent with the data measured by the particle size analyzer.

[0069] 4. Investigation of in vitro release behavior

[0070] The in vitro release experiment of RGD-CO-LIP adopted the dynamic dialysis method. PBS with pH = 7.4 was selected as the release medium (containing 0.5% Tween 80), one end of the pretreated dialysis bag (molecular weight 4000) was tied tightly, 2mL of liposome suspension was accurately drawn, the bag mouth was tied tightly with a string, and then the dialysis bag was placed in a beaker containing 20mL of release medium, and the beaker was placed at 37°C and 500r / min in a dark place. -1In the transdermal diffusion instrument, 0.5 mL of dialysate was drawn regularly, and an equal amount of constant temperature fresh release medium was added in time to maintain a constant release volume. Three parallel samples were made for each group. The dialysate was diluted with an equal amount of methanol and the content was determined by HPLC. The results are shown in Figure 3-4 As shown, within 24 h, the cumulative release rates of PTX and CRT in different liposome preparations were less than 75%.

[0071] Example 8

[0072] Application of compound liposomes in the treatment of prostate cancer:

[0073] 1. Experimental methods:

[0074] 1.1 Cells:

[0075] Mouse prostate cancer cell RM-1 was used as an in vitro model, different groups of liposomes were used, and paclitaxel injection (PTX Injection) was used as a positive drug control group. After 24 hours of treatment, the inhibitory effects of different preparations on the RM-1 cell line were investigated using the CCK-8 kit.

[0076] Mouse prostate cancer cells RM-1 and human immortalized epidermal cells HaCaT were used as in vitro models. The drug-free blank liposomes CO@LIP and RGD-CO@LIP encapsulating the fluorescent raw material coumarin-6 were treated with complete culture medium for 1h, 2h and 4h, respectively. Laser confocal microscopy was used to observe and photograph, and the uptake of liposomes by different cells was investigated.

[0077] 1.2 Animals:

[0078] Male SD rats were used as pharmacokinetic models, and PTX injection (PTXInjection), PTX liposomes (PTX-LIP), compound liposomes (CO-LIP), PEG-modified long-circulation compound liposomes (PEG-CO-LIP), and RGD-modified crocetin-paclitaxel co-loaded compound liposomes (RGD-CO-LIP) were intravenously injected, respectively, with a dose of PTX 10 mg / kg and CRT 4 mg / kg. 0.5 mL of blood was collected from the orbital venous plexus at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h after administration, and the blood samples were collected in 1.5 mL EP tubes soaked in sodium heparin in advance. The collected blood samples were centrifuged at 4000 rpm for 10 min to separate plasma. Liquid chromatography (AgiLent 1290infinity-AB Sciex API) was used to detect the content of PTX and CRT. DAS2.0 pharmacokinetic software was used to process the blood drug concentration data at each time point after injection of different liposomes in rats, and the pharmacokinetic parameters were calculated using the non-compartmental model.

[0079] Male C57BL / 6 mice (2-3 weeks, 18-20g) were used to establish an in vivo ectopic tumor model in mice bearing RM-1 tumors. Liposome preparations of different prescriptions were injected through the tail vein (once every 3 days). The experiment was terminated 9 days after administration, and the mice were euthanized, and the tumors and major organs were collected and weighed.

[0080] 1.3 Histopathological examination:

[0081] After treatment, organ tissues of mice were collected for histopathological examination, including H&E staining and immunohistochemical staining.

[0082] 2. Experimental results:

[0083] 2.1 Pharmacokinetic evaluation results:

[0084] As shown in Table 4, the pharmacokinetics of PTX in rats changed significantly in the four liposome groups compared with the PTX injection group. 0-∞ It can be seen that the four liposome groups and the PTX injection group have significantly improved, among which the PEG-modified compound liposome group and the RGD-modified compound liposome group have the most significant increase, increasing by 3.84 and 3.87 times respectively. 1 / 2 It can also be seen that compared with the PTX injection group, the four liposome groups also significantly increased the half-life of paclitaxel in the body. Similarly, the PEG-modified compound liposome group and the RGD-modified compound liposome group increased the most significantly. In addition, in terms of clearance rate (CL), the four liposome groups were significantly reduced compared with the injection group, which is very helpful for improving the anti-tumor effect of paclitaxel in the body.

[0085] As shown in Table 5, for CRT, the area under the drug-time curve (AUC) of the PEG-modified compound liposome group and the RGD-modified compound liposome group was significantly higher than that of the compound liposome group. 0-∞ The results showed that the PEG-modified liposome group and the RGD-modified liposome group had significantly lower clearance rate (CL) than the PEG-modified liposome group, which was also helpful for the anti-tumor effect of crocetin in vivo.

[0086] Table 4 PTX pharmacokinetic parameters in each administration group (n = 6)

[0087]

[0088] Table 5 Pharmacokinetic parameters of CRT in each drug administration group (n=6)

[0089]

[0090] 2.2 In vitro cytotoxicity:

[0091] Compared with the CRT-LIP group and the PTX-LIP group, the compound liposome groups (CO-LIP, PEG-CO-LIP, and RGD-CO-LIP groups) had a significant inhibitory effect on RM-1 cells (P<0.0001, P<0.01), proving that the synergistic use of the two drugs has a better effect in inhibiting tumor cells ( Figure 5 ). Compared with other groups, the RGD-CO-LIP group had the strongest inhibitory effect on RM-1 cells, with significant differences, which means that RGD-CO-LIP has the strongest ability to inhibit tumor cell growth.

[0092] 2.3 In vitro uptake results:

[0093] Mouse prostate cancer cells RM-1 and human immortalized epidermal cells HaCaT were used as in vitro models, and drug-free blank liposomes CO@LIP and RGD-CO@LIP encapsulating the fluorescent raw material coumarin-6 were treated with complete culture medium for 1h, 2h, and 4h, respectively. Laser confocal microscopy was used to observe and take pictures to investigate the uptake of liposomes by different cells. Figure 6 As shown, at each time point, the fluorescence of the RGD-CO-LIP group was stronger than that of the CO-LIP group. After 4 hours of co-incubation, the uptake capacity of RM-1 cells for the RGD-CO-LIP group increased by 1.62 times compared with the CO-LIP group, which was significantly different (P<0.05), indicating that RM-1 cells have a stronger uptake capacity for RGD-CO-LIP. As the incubation time of liposomes and cells increased, the fluorescence intensity in RM-1 cells gradually increased, indicating that the uptake of liposomes by RM-1 cells was time-dependent. Figure 7 As shown, in normal human epidermal cells (HaCaT cells), although the fluorescence intensity increased with the increase of the incubation time of liposomes and cells, there was no statistical difference in the fluorescence intensity between the RGD-CO-LIP group and the CO-LIP group at each time point (P>0.05), indicating that there was no significant difference in the uptake of the two groups of liposomes by normal cells.

[0094] 2.4 In vivo anti-tumor results:

[0095] like Figure 8As shown in the figure, compared with the blank group, the single liposome groups all had certain anti-tumor effects, among which the CRT-LIP group had a poor tumor treatment effect, with no significant difference in tumor volume and mass changes (P>0.05), and a tumor inhibition rate of 15.3%. The PTX-LIP group showed a stronger anti-tumor effect (P<0.05), with a tumor inhibition rate of 30.9%; compared with the single liposome groups (CRT-LIP group, PTX-LIP group), the tumor volume and mass of the CO-LIP group were significantly reduced (P<0.001, P<0.01), and the tumor inhibition rate was 54.6%, confirming that the compound liposome has a better effect on inhibiting tumor growth than the two single liposome groups, which is consistent with the cell experiment results mentioned above; compared with the CO-LIP group, the PEG-CO-LIP group had a significant tumor inhibition effect (P<0.05) due to its long circulation effect in mice, and the tumor inhibition rate was 79.0%. Compared with the PEG-CO-LIP group, the RGD-CO-LIP group had a better anti-tumor effect (P<0.01), with a tumor inhibition rate of 89.7%. This is because RGD-CO-LIP not only has a long circulation effect, but also has an active targeting function, which can deliver more crocetin and paclitaxel to the tumor site, achieving a better anti-tumor effect. From the results of the tumor inhibition rate, the tumor inhibition rates of the CO-LIP group were 3.57 times and 1.77 times that of the CRT-LIP group and the PTX-LIP group, respectively, further proving that the combination of CRT and PTX can achieve a synergistic anti-tumor effect. The tumor inhibition rates of the RGD-CO-LIP group were 5.86, 2.92, 1.64, and 1.14 times those of the other four drug-administered groups, respectively, further proving that the RGD-CO-LIP group had the best anti-tumor effect, and the results were consistent with the results of the cell experiment.

[0096] 2.5 In vivo safety assessment:

[0097] like Fig. 9 As shown in the figure, compared with the Saline group, no obvious abnormalities and organ damage were found in each drug-treated group; in the tumor sections, the nuclei of the tumor tissue in the Saline group were full and evenly distributed. In the tumor sections of each drug-treated group, the necrotic area increased, the nuclei were obviously wrinkled, and the distribution was uneven and irregular. Among them, the tumor cell necrosis in the RGD-CO-LIP group was the most significant, which further demonstrated that the RGD-CO-LIP group had good safety and the best anti-tumor therapeutic effect.

Claims

1. An RGD-modified crocetin-paclitaxel co-drug-loaded composite liposome, characterized in that: The components include RGD-modified liposomes, paclitaxel and crocetin, wherein the RGD-modified liposomes are used as drug carriers to deliver paclitaxel and crocetin to prostate tumor cells in a targeted manner, and the RGD-modified liposomes are prepared by a thin film dispersion method or a reverse evaporation method.

2. The RGD-modified crocetin-paclitaxel co-drug-loaded composite liposome according to claim 1, characterized in that: The ratio of paclitaxel to crocetin is between 1:3 and 1:

1.

3. The RGD-modified crocetin-paclitaxel co-drug-loaded composite liposome according to claim 1, characterized in that: In the RGD-modified liposome, the liposome carrier comprises the following auxiliary materials in parts by weight: 20-200 parts of phospholipids, 3-15 parts of cholesterol, 1-5 parts of PEG-DSPE, and 1-5 parts of RGD-PEG-DSPE.

4. A pharmaceutical composition, characterized in that The RGD-modified crocetin-paclitaxel co-drug-loaded composite liposomes according to any one of claims 1 to 3 are prepared into a clinically acceptable preparation with a pharmaceutically acceptable carrier, and the preparation is an injection, an oral preparation or an external preparation.

5. A method for preparing the RGD-modified crocetin-paclitaxel co-drug-loaded composite liposome according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) phospholipids, cholesterol, and PEG-DSPE were mixed in proportion, RGD peptide was added, and RGD-modified liposomes were prepared by a thin film dispersion method; (2) mixing paclitaxel and crocetin, and adding the mixture to the RGD-modified liposome solution; (3) Purify by ultrafiltration or centrifugation to obtain a composition of RGD-modified liposomes loaded with paclitaxel and crocetin.

6. The method for preparing a RGD-modified crocetin-paclitaxel co-drug-loaded composite liposome according to claim 5, characterized in that: In the step (2), paclitaxel and crocetin are mixed in a ratio of 1:3 to 1:

1.

7. Use of the RGD-modified crocetin-paclitaxel co-loaded composite liposome according to any one of claims 1 to 3 in the preparation of drugs for treating cancer metastasis.

8. Use of the pharmaceutical composition according to claim 4 in preparing a drug for treating cancer metastasis.

9. The use according to claim 7 or 8, characterized in that: The cancer is prostate cancer.

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