Rgd-modified safflor yellow a-paclitaxel co-loaded liposome and preparation method and application thereof

Abstract

The application discloses RGD modified crocetin-paclitaxel co-loaded drug compound liposomes and a preparation method and application thereof, and belongs to the technical field of biological medicines. The RGD modified crocetin-paclitaxel co-loaded drug compound liposomes comprise RGD modified liposomes, paclitaxel and crocetin, wherein the RGD modified liposomes are used as a drug carrier to target and deliver the paclitaxel and the crocetin to prostate tumor cells, and the RGD modified liposomes are prepared by a film dispersion method or a reverse evaporation method. The prostate cancer targeted compound liposomes prepared by the application can increase the uptake of the prostate cancer cells, improve the in-vitro cytotoxicity of the prostate cancer cell line RM-1, and inhibit the growth of RM-1 ectopic solid tumors in mice, and does not cause obvious damage to other tissues.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an RGD-modified crocin-paclitaxel co-loaded compound liposome for the treatment of prostate cancer, its preparation method and application. Background Technology

[0002] While significant progress has been made in prostate cancer treatment, the vast majority of patients eventually develop castration-resistant prostate cancer (CRPC). CRPC is a highly intractable type of cancer, characterized by gradual resistance to traditional androgen deprivation therapy (ADT), resulting in significantly reduced treatment efficacy. The occurrence and development of CRPC involve complex biological mechanisms, including mutations or amplifications of androgen receptors, reprogramming of androgen synthesis pathways, and changes in the tumor microenvironment. Due to the diversity and complexity of these mechanisms, current treatment of CRPC still faces many challenges, and there is a lack of effective treatment options to control disease progression in clinical practice.

[0003] Paclitaxel (PTX) is a natural organic compound extracted from the bark of the American yew (Taxus brevifolia) and belongs to the taxane class of compounds. Paclitaxel is widely used in the treatment of various malignant tumors, including ovarian cancer, breast cancer, non-small cell lung cancer, esophageal cancer, and prostate cancer, 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, common adverse reactions during paclitaxel treatment include leukopenia, neurotoxicity, and cardiovascular adverse reactions.

[0004] Crocetin (CRT) is an important active ingredient in saffron, an amphiphilic low-molecular-weight carotenoid compound that has shown significant anti-tumor potential in animal models and cell culture systems. Its anti-tumor effects are achieved primarily through multiple pathways, including inhibiting tumor cell proliferation, inducing 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.). Studies have also shown that crocetin has the potential to be used in combination with other drugs to significantly enhance efficacy. Due to its good biocompatibility and low toxicity, crocetin can be used in combination with traditional chemotherapy drugs or targeted therapies to synergistically enhance anti-cancer effects. For example, when crocin 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 clinical prognosis of patients.

[0005] RGD peptide is a polypeptide containing the arginine-glycine-aspartic acid (Arg-GLy-ASP) sequence. It can recognize integrin α, which is highly expressed on the surface of tumor cells and tumor blood vessels. v The drug binds to the β3 receptor. Using RGD-modified liposomes as antitumor drug carriers can enhance their targeting of tumors, thereby improving the antitumor effect of the drug. This invention explores for the first time the synergistic inhibitory effect of CRT and PTX in combination on prostate cancer, and prepares an RGD-modified co-loaded liposome of CRT and PTX, evaluating its efficiency in anti-prostate tumors. Summary of the Invention

[0006] The purpose of this invention is to provide an RGD-modified crocin-paclitaxel co-loaded compound liposome, its preparation method, and its application. This liposome targets tumor cells and can treat prostate cancer, and therefore can be used to prepare pharmaceutical preparations for treating prostate cancer.

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

[0008] This invention provides an RGD-modified crocin-paclitaxel co-loaded compound liposome, comprising RGD-modified liposomes, paclitaxel (PTX), and crocin (CRT). The RGD-modified liposomes serve as drug carriers to target and deliver paclitaxel and crocin to prostate tumor cells. The RGD-modified liposomes are prepared by thin-film dispersion or reverse evaporation.

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

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

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

[0012] Furthermore, the RGD-modified crocin-paclitaxel co-loaded compound liposome of the present invention is used as a pharmaceutical composition to prepare a clinically acceptable formulation with a pharmaceutically acceptable carrier, wherein the formulation is an injection, an oral formulation, or a topical formulation.

[0013] The RGD-modified crocin-paclitaxel co-loaded compound liposomes of the present invention are prepared by thin-film dispersion method.

[0014] Furthermore, the preparation method of the RGD-modified crocin-paclitaxel co-loaded drug compound liposome includes the following steps:

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

[0016] (2) Mix paclitaxel and crocin and add them to the RGD-modified liposome solution;

[0017] (3) The RGD-modified liposomes loaded with paclitaxel and crocin were purified by ultrafiltration or centrifugation to obtain the composition.

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

[0019] The application of the RGD-modified crocin-paclitaxel co-loaded compound liposomes described in this invention in the preparation of drugs for treating cancer metastasis.

[0020] Furthermore, the cancer in question is prostate cancer. The RGD-modified crocin-paclitaxel co-loaded liposome delivers paclitaxel and crocin to prostate tumor cells, synergistically exerting an anti-tumor effect. Specifically, the RGD-modified crocin-paclitaxel co-loaded liposome is injected into the patient to inhibit tumor growth and induce tumor cell apoptosis.

[0021] The beneficial effects of this invention are:

[0022] 1. The co-loaded drug compound liposomes prepared by this invention can exert the synergistic effect of two drugs to inhibit the growth of prostate cancer solid tumors.

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

[0024] 3. The method of the present invention significantly improves the antitumor effects of paclitaxel and crocin through the targeted delivery of RGD-modified liposomes. Attached Figure Description

[0025] Figure 1 This invention illustrates the inhibitory effect of CRT, PTX, and the combined use of PTX-CRT on RM-1 cell proliferation in embodiments of the present invention.

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

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

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

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

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

[0031] Figure 7 The uptake efficiency of different liposomes in HaCaT cells is shown in the embodiments of the present invention.

[0032] Figure 8 This invention demonstrates the in vivo antitumor effect of compound liposomes in a prostate tumor animal model.

[0033] Figure 9 This invention provides an analysis of the toxic side effects of compound liposomes on normal tissues in an embodiment of the invention. Detailed Implementation

[0034] The following description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

[0035] Example 1

[0036] Preparation of crocin liposomes: Liposomes were prepared using a thin-film hydration method. 15.0 mg of DPPC, 2.0 mg of cholesterol, and 0.8 mg of CRT were weighed and dissolved in anhydrous ethanol. The solution was placed in a round-bottom flask, and an appropriate amount of anhydrous ethanol was added. The mixture was sonicated for 30 min to ensure complete dissolution of DPPC, cholesterol, and crocin. The solvent was removed under reduced pressure using a rotary evaporator, allowing the solids to form a thin film on the flask wall. The solids were then dried under reduced pressure at room temperature for 12 h to remove residual solvent. An appropriate amount of PBS solution (pH = 7.4) was added to the completely dried flask, and the mixture was hydrated at 50°C for 2 h. After hydration, the mixture was sonicated in an ice bath for 10 min using a probe-type ultrasonic instrument with pulses of 10 s / 5 s. The liposomes were then filtered through 0.45 μm and 0.22 μm filters to obtain crocin liposomes CRT-LIP.

[0037] Example 2

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

[0039] Example 3

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

[0041] Example 4

[0042] Preparation of PEG-modified long-circulating complex liposomes: DPPC 45.0 mg, cholesterol 6.5 mg, CRT 0.8 mg, PTX 2.0 mg, and PEG were weighed. 2000 -DSPE 2.0mg, prepared in the same way as in Example 1, yielded PEG-modified long-circulating compound liposome PEG-CO-LIP.

[0043] Example 5

[0044] Preparation of RGD-modified crocin-paclitaxel co-loaded liposomes: DPPC 45.0 mg, cholesterol 6.5 mg, CRT 0.8 mg, PTX 2.0 mg, RGD-PEG... 2000 -DSPE 2.0mg, prepared using the same method as in Example 1, yielded RGD-modified long-circulating targeted compound liposomes. 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. The film was then hydrated with 0.01 mol / L phosphate buffer (PBS, pH 7.4), sonicated in a water bath for 2 min, and 1.5 equivalents of cRGDyk-cys were 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 dialysis, the product was lyophilized to obtain RGD-PEG. 2000 -DSPE;

[0046] (2) DPPC 45.0mg, cholesterol 6.5mg, RGD-PEG 2000 -DSPE 2.0mg, dissolved in anhydrous ethanol, and sonicated for 30 minutes to remove DPPC, cholesterol, and RGD-PEG. 2000 -DSPE was completely dissolved to prepare RGD-modified liposome solutions;

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

[0048] (4) The solvent was removed by rotary evaporator under reduced pressure, so that the solid formed a film on the wall of the eggplant-shaped flask; then the residual solvent was removed by vacuum drying at room temperature for 12 h in a vacuum drying oven; an appropriate amount of PBS solution (pH=7.4) was added to the completely dried eggplant-shaped flask and hydrated at 50℃ for 2 h. After hydration, the flask was ultrasonically treated in an ice bath for 10 min with pulses of 10 s / 5 s. After passing through 0.45 μm and 0.22 μm filter membranes, respectively, RGD-CO-LIP modified liposomes loaded with paclitaxel and crocin were obtained.

[0049] Example 6

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

[0051] The effects of different concentrations of PTX, CRT, and various combinations thereof on the cell viability of RM-1 cells were investigated using the CCK-8 assay. Figure 1As shown, crocin concentrations within the 0-10 μM range showed no significant cytotoxicity to RM-1 cells; therefore, further studies on CRT-LIP were not considered. Compared to the control group, paclitaxel concentrations greater than 10 nM exhibited significant cytotoxicity to RM-1 cells; the inhibitory effect on RM-1 cell viability reached its peak at a paclitaxel concentration of 100 nM. At a paclitaxel concentration of 10 nM, cell viability decreased in both the crocin and paclitaxel treatment groups compared to the paclitaxel-only group; when the crocin concentration was greater than 10 nM, the combined treatment group showed a significant difference compared to the paclitaxel-only group, and the cell viability of RM-1 cells decreased in a dose-dependent manner with increasing crocin dosage. This indicates that the combined treatment produced a better inhibitory effect on RM-1 prostate cancer cells compared to either crocin or paclitaxel alone.

[0052] As shown in Table 1, except for the crocin 1nM combined administration group, the IC50 values ​​of the other combined administration groups were... 50 The combined administration of saffron acid and paclitaxel was less than that of the paclitaxel monotherapy group, which also demonstrates that the combined administration produced a greater cytotoxic effect on RM-1 prostate cancer cells compared to treatment with crocin or paclitaxel alone.

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

[0054]

[0055] Example 7

[0056] Quality evaluation of compound liposomes:

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

[0058] After successfully preparing RGD-modified crocin-paclitaxel co-loaded compound liposomes (RGD-CO-LIP) according to the method described in Example 5, accurately measure 0.5 mL of RGD-CO-LIP suspension into a 5 mL volumetric flask, add 4.5 mL of methanol, and sonicate for 15 min to fully demulsify. The total drug content is recorded as m1. Then, measure 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 from the centrifuge tube and place it into a 5 mL volumetric flask, dilute to the mark with methanol, sonicate for 15 min to fully demulsify, and the free drug content is recorded as m2.

[0059] Encapsulation efficiency = (m1-m2) / m1

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

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

[0062]

[0063] 2. Determination of particle size and potential

[0064] The particle size (effective hydrodynamic diameter) of the micelles was determined using a laser particle size analyzer (Nano-ZS Zen 3700 Malvern, UK) via photon correlation spectroscopy. The experimental method was as follows: 1 mL of the RGD-CO-LIP prepared in Example 5 was taken, thoroughly mixed, filtered through a 0.22 μm filter membrane, and measured in a constant-temperature cell at 25°C and a scattering angle of 173° (backscattering, NIBS default value). The effective hydrodynamic diameter and Zeta potential 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 morphological characterization of liposomes

[0068] The morphology of the RGD-CO-LIP prepared in Example 5 was observed using a transmission electron microscope (Hitachi HT7800, Japan). The experimental method was as follows: 1 mL of the RGD-CO-LIP prepared in Example 5 was taken, thoroughly mixed, filtered through a 0.22 μm filter membrane, and 10 μL was applied to a copper grid. When the droplet on the grid was almost dry, a drop of 3% phosphotungstic acid solution was added for negative staining. After a period of time, the copper grid was lifted with tweezers, and excess staining solution was absorbed with filter paper. The copper grid was allowed to dry naturally at room temperature, and then observed under a transmission electron microscope. The accelerating voltage for the experiment was 80 kV. Figure 2 As shown, the prepared RGD-CO-LIP has a double-layered 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 assay of RGD-CO-LIP employed dynamic dialysis. PBS (pH 7.4) containing 0.5% Tween 80 was selected as the release medium. One end of a pretreated dialysis bag (molecular weight 4000) was tightly sealed, and 2 mL of liposome suspension was precisely pipetted into the bag. The bag opening was then tied tightly with a string, and the dialysis bag was placed in a beaker containing 20 mL of the release medium. After being protected from light, the beaker was placed at 37°C and 500 rpm. -1 In a transdermal diffusion apparatus, 0.5 mL of dialysate was periodically aspirated, and an equal volume of fresh, constant-temperature release medium was added promptly to maintain a constant release volume. Three parallel samples were prepared for each group. The collected dialysate was diluted with an equal volume of methanol and the content was determined by HPLC. Results are as follows: Figure 3-4 As shown, within 24 hours, the cumulative release rate of PTX and CRT in different liposome formulations was 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] Using mouse prostate cancer cells RM-1 as an in vitro model, different groups of liposomes were used, with paclitaxel injection (PTX Injection) as a positive control. After 24 hours of treatment, the inhibitory effects of different formulations on the RM-1 cell line were investigated using the CCK-8 assay kit.

[0076] Using mouse prostate cancer cells RM-1 and human immortalized epidermal cells HaCaT as in vitro models, we treated patients with drug-free blank liposomes CO@LIP and RGD-CO@LIP containing the fluorescent ingredient coumarin-6 in complete culture medium for 1 h, 2 h and 4 h, respectively. We then used laser confocal microscopy to observe and examine the uptake of liposomes by different cells.

[0077] 1.2 Animals:

[0078] Male SD rats were used as a pharmacokinetic model. PTX injection, PTX-LIP, CO-LIP, PEG-modified long-circulating CO-LIP, and RGD-modified crocin-paclitaxel co-loaded CO-LIP were administered intravenously at doses of 10 mg / kg PTX and 4 mg / kg CRT. Blood samples of 0.5 mL were collected from the orbital venous plexus at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h post-administration. Blood samples were collected in 1.5 mL EP tubes pre-soaked in heparin sodium. The collected blood samples were centrifuged at 4000 rpm for 10 min, and plasma was separated. The contents of PTX and CRT were determined using liquid chromatography (AgiLent 1290infinity-AB Sciex API). The DAS2.0 pharmacokinetic software was used to process the blood drug concentration data of rats injected with different liposomes at various time points, and the pharmacokinetic parameters were calculated using a non-compartmental model.

[0079] A mouse model of ectopic tumor carrying RM-1 tumors was established using male C57BL / 6 mice (2-3 weeks old, 18-20g). Different formulations of liposome preparations were injected into the tail vein (once every 3 days). The experiment was terminated after 9 days of administration. The mice were euthanized and the tumors and major organs were collected for weighing.

[0080] 1.3 Histopathological examination:

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

[0082] 2. Experimental Results:

[0083] 2.1 Pharmacokinetic assessment results:

[0084] As shown in Table 4, the pharmacokinetics of PTX in rats were significantly altered in the four liposome groups compared to the PTX injection group. The area under the curve (AUC) during the drug-time reaction was also significantly different. 0-∞ It can be seen that all four liposome groups showed significant improvements compared to the PTX injection group, with the PEG-modified compound liposome group and the RGD-modified compound liposome group showing the most significant improvements, increasing by 3.84 and 3.87 times, respectively. The drug half-life T... 1 / 2 It can also be seen that, compared with the PTX injection group, the four liposome groups significantly increased the circulating half-life of paclitaxel in vivo. Similarly, the PEG-modified and RGD-modified compound liposome groups showed the most significant increases. Furthermore, in terms of clearance (CL), the four liposome groups showed a significant decrease compared with the injection group, which greatly contributes to improving the antitumor effect of paclitaxel in vivo.

[0085] As shown in Table 5, for CRT, the area under the curve (AUC) of the PEG-modified compound liposome group compared to the RGD-modified compound liposome group compared to the compound liposome group is as follows: 0-∞ The levels were increased by 1.38 and 1.33 times, respectively, and the circulation time of crocin in vivo was also significantly increased. In terms of clearance (CL), the PEG-modified compound liposome group and the RGD-modified compound liposome group also showed significant reductions compared to the compound liposome group, which greatly contributes to the anti-tumor effect of crocin in vivo.

[0086] Table 4. Pharmacokinetic parameters of PTX in each treatment group (n=6)

[0087]

[0088] Table 5. Pharmacokinetic parameters of CRT in each treatment 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, RGD-CO-LIP groups) showed significant inhibitory effects on RM-1 cells (P<0.0001, P<0.01), demonstrating that the synergistic use of the two drugs has a better inhibitory effect on tumor cells. Figure 5 Compared with other groups, the RGD-CO-LIP group showed the strongest inhibitory effect on RM-1 cells, which was statistically significant, meaning that RGD-CO-LIP has the strongest ability to inhibit tumor cell growth.

[0092] 2.3 Results of in vitro uptake:

[0093] Using mouse prostate cancer cells RM-1 and human immortalized epidermal cells HaCaT as in vitro models, we treated patients with drug-free blank liposomes CO@LIP and RGD-CO@LIP containing the fluorescent ingredient coumarin-6 in complete culture medium for 1 h, 2 h, and 4 h, respectively. Laser confocal microscopy was used to observe the uptake of liposomes by different cell types. 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, compared with the CO-LIP group, the uptake capacity of RM-1 cells by the RGD-CO-LIP group increased by 1.62 times, which was statistically significant (P<0.05), indicating that RM-1 cells have a stronger uptake capacity for RGD-CO-LIP. With the increase of incubation time between liposomes and cells, the fluorescence intensity inside RM-1 cells gradually increased, indicating that the uptake of liposomes by RM-1 cells is time-dependent. Figure 7 As shown, in normal human epidermal cells (HaCaT cells), although the fluorescence intensity increased with the incubation time of liposomes and cells, there was no statistically significant difference in 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 liposomes by normal cells between the two groups.

[0094] 2.4 In vivo anti-tumor results:

[0095] like Figure 8As shown, compared with the blank group, all single-component liposome groups had certain anti-tumor effects. Among them, 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 strong anti-tumor effect (P<0.05) with a tumor inhibition rate of 30.9%. Compared with the single-component liposome groups (CRT-LIP group and PTX-LIP group), the CO-LIP group showed a significant reduction in tumor volume and mass (P<0.001, P<0.01) and a tumor inhibition rate of 54.6%, confirming that the compound liposomes had a better inhibitory effect on tumor growth than the two single-component 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 due to its long circulation effect in mice (P<0.05) and a tumor inhibition rate of 79.0%. Compared with the PEG-CO-LIP group, the RGD-CO-LIP group showed better anti-tumor efficacy (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 an active targeting function, which can deliver more crocin and paclitaxel to the tumor site, achieving a better anti-tumor effect. In terms of tumor inhibition rate, the CO-LIP group's inhibition rate was 3.57 times that of the CRT-LIP group and 1.77 times that of the PTX-LIP group, further demonstrating that the combination of CRT and PTX can achieve a synergistic anti-tumor effect. The RGD-CO-LIP group's tumor inhibition rate was 5.86, 2.92, 1.64, and 1.14 times that of the other four treatment groups, further demonstrating that the RGD-CO-LIP group had the best anti-tumor effect, consistent with the results of cell experiments.

[0096] 2.5 In vivo safety assessment:

[0097] like Figure 9 As shown, compared with the Saline group, no significant abnormalities or organ damage were observed in any of the treatment groups. In tumor sections, the tumor cell nuclei in the Saline group were full and evenly distributed. In contrast, the necrotic areas increased and the cell nuclei were significantly shrunken and unevenly and irregularly distributed in the tumor sections of all treatment groups. The RGD-CO-LIP group showed the most significant tumor cell necrosis, further demonstrating that the RGD-CO-LIP group has good safety and the best anti-tumor therapeutic effect.

Claims

1. An RGD-modified crocin-paclitaxel co-loaded compound liposome, characterized in that, Its components include RGD-modified liposomes, paclitaxel, and crocin. The RGD-modified liposomes serve as drug carriers to target and deliver paclitaxel and crocin to prostate tumor cells. The RGD-modified liposomes are prepared by thin-film dispersion or reverse evaporation. The ratio of paclitaxel to crocin is between 1:3 and 1:

1.

2. The RGD-modified crocin-paclitaxel co-loaded compound liposome according to claim 1, characterized in that, The RGD-modified liposomes contain the following excipients in parts by weight: 20-200 parts phospholipids, 3-15 parts cholesterol, 1-5 parts PEG-DSPE, and 1-5 parts RGD-PEG-DSPE.

3. A pharmaceutical composition, characterized in that, The formulation comprises RGD-modified crocin-paclitaxel co-loaded liposomes as described in any one of claims 1-2, and is prepared into a clinically acceptable formulation with a pharmaceutically acceptable carrier, wherein the formulation is an injectable preparation.

4. A method for preparing RGD-modified crocin-paclitaxel co-loaded compound liposomes according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Take DSPE-PEG 2000 -Mal was dissolved in chloroform, and the organic solvent was removed under reduced pressure to form a thin film. The film was then hydrated with 0.01 mol / L phosphate buffer (pH 7.4), sonicated in a water bath for 2 min, and 1.5 equivalents of cRGDyk-cys were 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 dialysis, the product was lyophilized to obtain RGD-PEG. 2000 -DSPE; (2) Phospholipids, cholesterol, and RGD-PEG 2000 -DSPE was mixed and dissolved in anhydrous ethanol to prepare RGD-modified liposome solutions; (3) Paclitaxel and crocin were mixed and added to the RGD-modified liposome solution, and RGD-modified liposomes were prepared by thin film dispersion method; (4) The RGD-modified liposomes loaded with paclitaxel and crocin were purified by ultrafiltration or centrifugation to obtain the composition.

5. The use of the RGD-modified crocin-paclitaxel co-loaded compound liposome according to any one of claims 1-2 in the preparation of a drug for treating prostate cancer.

6. Use of the pharmaceutical composition of claim 3 in the preparation of a medicament for treating prostate cancer.

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