A composition for treating bladder cancer, its preparation method and application

Abstract

The present invention relates to a composition for treating bladder cancer, which is prepared from raw materials in the following weight ratios: 40-100 parts of elemene, and 1 part of chlorin e6. By combining elemene and chlorin e6 in a specific ratio and using the thin film dispersion method to prepare liposomes, the present invention solves the problems of strong hydrophobicity, weak tumor targeting ability of the photosensitizer Ce6, and insufficient efficacy when used alone, providing an innovative solution for the application of photodynamic therapy in the treatment of non-muscle invasive bladder cancer, and having practical popularization and application value.

Description

Technical Field

[0001] The present invention relates to the field of chemical pharmacy, and more particularly to a composition for treating bladder cancer, a preparation method thereof and an application thereof. Background Art

[0002] Malignant tumors currently pose one of the greatest threats to human life and health, and cancer prevention and treatment have become a major issue that urgently needs to be addressed in the global medical field. Globally, bladder cancer ranks ninth among malignant tumors and sixth among men. In my country, bladder cancer is also one of the most common malignant tumors of the urinary system. Clinically, bladder cancer is primarily divided into non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC); NMIBC accounts for approximately 70% of all newly diagnosed bladder cancer cases. Surgery combined with chemotherapy is a common treatment option for NMIBC. However, while chemotherapy drugs kill tumor cells, they can also cause serious toxic side effects to normal tissues and organs, impacting patients' quality of life. Therefore, finding more effective methods to enhance the treatment efficacy of NMIBC, improve patients' quality of life, and prolong their survival is a daunting challenge we currently face.

[0003] Photodynamic therapy (PDT) is a new and highly effective method for treating tumors. Its principle is to utilize photosensitizers to receive light energy of a specific wavelength, which in turn triggers a series of reactions in the body to produce reactive oxygen species (ROS), ultimately inducing apoptosis in tumor cells. Compared to intravesical chemotherapy, PDT has the advantages of being minimally invasive, having fewer toxic side effects, being reusable, and not prone to developing drug resistance. Anatomically, the bladder is a hollow organ with a unique sac-like structure, and clinically, the commonly used excitation light wavelength for PDT is 630-650nm. At this wavelength, the laser can penetrate tissue to a depth of 5-10mm, theoretically completely killing tumor cells in the bladder mucosa or submucosa. These factors make PDT very suitable for the treatment of superficial bladder cancer.

[0004] Chlorin e6 (Ce6) is one of the most widely used photosensitizers in PDT. It is a chlorophyll degradation product made from natural chlorophyll and is a second-generation photosensitizer with high efficiency and low dark toxicity. Currently, there are still many problems in the treatment of Ce6: due to singlet oxygen ( 1 The half-life of O2 in the PDT process is short (<40ns). 1 O2 molecules can only exert their therapeutic effects within regions less than 20 nm in diameter, necessitating the precise delivery of Ce6 to tumor cells. However, free Ce6 has weak tumor-targeting capabilities, and its nonspecific activation in normal tissues can also cause potential cytotoxicity. Furthermore, Ce6 is highly hydrophobic, making its tumor-suppressing effect insufficient when used alone, all of which limit its clinical application. Therefore, effective strategies to enhance the therapeutic efficacy of Ce6 are urgently needed. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a composition for treating bladder cancer, which is prepared from raw materials in the following weight ratios:

[0006] Elemene 40 - 100 parts, Chlorin e6 1 part.

[0007] Further, it is prepared from raw materials in the following weight ratios:

[0008] Elemene 40 - 80 parts, Chlorin e6 1 part.

[0009] Further, it is a preparation prepared from elemene and Chlorin e6 as active ingredients plus a pharmaceutical carrier.

[0010] Further, the preparation is an injection; the injection is a solution, ointment, gel or liposome, preferably a liposome.

[0011] Even further, the liposome is prepared from the following raw materials:

[0012] Elemene 40 - 80 parts, Chlorin e6 1 part, soybean lecithin 150 - 200 parts, PC - 98T 150 - 200 parts, cholesterol 10 - 20 parts, DSPE - PEG 2000 10 - 30 parts, L - histidine 15 - 16 parts.

[0013] Even further, the liposome is prepared from the following raw materials:

[0014] Elemene 50 parts, Chlorin e6 1 part, soybean lecithin 175 parts, PC - 98T 175 parts, cholesterol 15 parts, DSPE - PEG 2000 20 parts, L - histidine 15.52 parts.

[0015] The present invention also provides a method for preparing the aforementioned composition, which includes the following steps:

[0016] 1) Weigh the raw materials according to the ratio, take L - histidine, dissolve it in water, and then adjust the pH value to 6.5 to obtain an aqueous phase;

[0017] 2) Take soybean lecithin, PC - 98T, cholesterol, DSPE - PEG 2000 、elemene and Chlorin e6, dissolve them in ethanol, and dry to obtain a lipid film;

[0018] 3) Mix the lipid film obtained in step 2) and the aqueous phase obtained in step 1), stir, ultrasonicate, and filter to obtain the product.

[0019] Further, the mass-volume ratio of L-histidine to water in step 1) is 15-16 mg: 10 ml; the reagent for adjusting the pH value is hydrochloric acid.

[0020] Further, the addition amount of ethanol in step 2) is 1 / 5 of the amount of elemene used, ml / mg; the drying is to rotary evaporate under reduced pressure in a 55 °C water bath until a lipid film is formed; and / or, the temperature of stirring in step 3) is 55 °C, the time is 45 min; the ultrasonic treatment is 2 min; the filtration is through a 0.22 μm microporous membrane.

[0021] Finally, the present invention provides the use of the aforementioned composition in the preparation of a drug for treating bladder cancer; the bladder cancer includes non-muscle invasive bladder cancer.

[0022] A composition for treating non-muscle invasive bladder cancer according to the present invention, by combining elemene and chlorin e6 in a specific ratio and preparing liposomes by the thin film dispersion method, solves the problems that the photosensitizer Ce6 has strong hydrophobicity, weak tumor targeting ability, and insufficient single use efficacy, provides an innovative solution for the application of photodynamic therapy in the treatment of non-muscle invasive bladder cancer, and has practical popularization and application value.

[0023] Obviously, according to the above content of the present invention, according to the common general technical knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can also be made.

[0024] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Description of the Drawings

[0025] Figure 1 Effects of ELE, Ce6 and Ce6(0.3 w·cm -2 ) on the proliferation of T24 cells and the combination index (CI) of ELE / Ce6;

[0026] Figure 2 Optimization of the preparation process of Lipo-ELE / Ce6 by single factor experiments;

[0027] Figure 3 Transmission electron micrographs of Lipo-ELE / Ce6 before and after near-infrared light irradiation (A: before irradiation, B: after irradiation, scale bar: 200 nm);

[0028] Figure 4 Detection of the absorbance of different preparations of Ce6 by ultraviolet-visible spectrophotometer;

[0029] Figure 5 The ability of DPBF to detect the generation of singlet oxygen by Ce6 and Lipo-ELE / Ce6 at different concentrations;

[0030] Figure 6 Changes in tumor volume, tumor weight, and body weight of T24 subcutaneous tumor-bearing mice after treatment with different drugs (A: Representative images of tumors and changes in tumor volume after treatment with different drugs; B: Results of weighing tumor tissues; C: Body weight of mice after treatment);

[0031] Figure 7 Results of HE staining, TUNEL apoptosis, and immunohistochemistry (Cleaved Caspase-3 and Ki67) of tumor tissues (Scale bar: 50 μm);

[0032] Figure 8 Preliminary safety evaluation of T24 subcutaneous tumor-bearing mice after treatment with different drugs (Scale bar: 50 μm). Specific implementation mode

[0033] Example 1 Preparation of elemol / Ce6 liposomes of the present invention

[0034] Formula: 50 mg of elemol (ELE), 1 mg of chlorin e6 (Ce6), 175 mg of soybean lecithin, 175 mg of PC-98T, 15 mg of cholesterol, DSPE-PEG 2000 20 mg, 15.52 mg of L-histidine;

[0035] Preparation method

[0036] 1) Weigh the raw materials according to the ratio. Take L-histidine, add it to 10 mL of water, and then adjust the pH to 6.5 with hydrochloric acid to obtain the aqueous phase;

[0037] 2) Take soybean lecithin, PC-98T, cholesterol, DSPE-PEG 2000 , elemol, and chlorin e6, add them to 10 mL of absolute ethanol, place them in a 55 °C water bath and rotate under reduced pressure to evaporate to form a lipid film; add the L-histidine solution with a pH of 6.5 to the rotating flask, stir and hydrate at 55 °C for 45 min, sonicate for 2 min, and filter through a 0.22 μm microporous filter membrane to obtain the product.

[0038] Example 2 Preparation of elemol / Ce6 liposomes of the present invention

[0039] Formula: 40 mg of elemol, 1 mg of chlorin e6, 175 mg of soybean lecithin, 175 mg of PC-98T, 15 mg of cholesterol, DSPE-PEG 2000 20 mg, 15.52 mg of L-histidine;

[0040] Preparation method: The same as that in Example 1.

[0041] Example 3 Preparation of Elemene / Ce6 Liposomes of the Present Invention

[0042] Formulation: 80 mg of elemene, 1 mg of chlorin e6, 175 mg of soybean lecithin, 175 mg of PC-98T, 15 mg of cholesterol, DSPE-PEG 2000 20 mg, 15.52 mg of L-histidine;

[0043] Preparation method: The same as that in Example 1.

[0044] Example 4 Preparation of Elemene / Ce6 Liposomes of the Present Invention

[0045] Formulation: 100 mg of elemene, 1 mg of chlorin e6, 175 mg of soybean lecithin, 175 mg of PC-98T, 15 mg of cholesterol, DSPE-PEG 2000 20 mg, 15.52 mg of L-histidine;

[0046] Preparation method: The same as that in Example 1.

[0047] The beneficial effects of the present invention are illustrated by the following test examples:

[0048] Test Example 1 Study on Elemene / Ce6 Liposomes

[0049] (1) Preparation and Characterization of Elemene / Ce6 Liposomes (Lipo-ELE / Ce6)

[0050] 1) Study on the Synergistic Effect of ELE and Ce6

[0051] The cytotoxicity of ELE, Ce6 (without light irradiation), and Ce6 (light irradiation, 660 nm, 0.3 W·cm -2 ) on T24 cells was detected by the MTT method. Briefly, cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum in a 96-well plate (5000 cells / well) at 37°C. After 12 hours, the cells were treated with different drugs at different concentrations for 24 h, and the absorbance was measured at 490 nm using an enzyme-linked immunosorbent assay reader. Based on the IC -2 values of ELE and Ce6 (0.3 W·cm 50 ), the combination index CI of different ratios of ELE / Ce6 was calculated using CompuSyn software: 0.1 ≤ CI < 0.7, highly synergistic effect; 0.7 ≤ CI < 0.85, moderately synergistic effect; 0.85 ≤ CI < 1.1, additive effect; CI ≥ 1.1, antagonistic effect.

[0052] 2) Preparation of Lipo-ELE / Ce6

[0053] ① Preparation process of Lipo-ELE / Ce6

[0054] Prescription: 175 mg of soybean lecithin, 175 mg of PC-98T, 15 mg of cholesterol, 20 mg of DSPE-PEG 2000 50 mg of elemene, 1 mg of Ce6

[0055] Lipo-ELE / Ce6 was prepared by the thin-film dispersion method: Weigh 15.52 mg of L-histidine and add it to 10 mL of water. Adjust the pH to 6.5 with hydrochloric acid to obtain the aqueous phase. Weigh the samples according to the prescription, add them to absolute ethanol, place them in a 40 °C water bath and rotate under reduced pressure to evaporate to form a lipid film. Add the aqueous phase to the rotating flask, stir and hydrate at 55 °C for 45 min, sonicate, and filter through a microporous membrane to obtain Lipo-ELE / Ce6.

[0056] Lipo-ELE / Ce6 was prepared by the ethanol injection method: Weigh 15.52 mg of L-histidine and add it to 10 mL of water. Adjust the pH to 6.5 with hydrochloric acid to obtain the aqueous phase. Add 1 mL of ethanol according to the prescription, fully dissolve it and then slowly inject it into the aqueous phase. Stir continuously at 1000 rpm for 45 min, perform fine homogenization with a probe-type high-speed dispersion homogenizer, sonicate for 2 min, and filter through a microporous membrane to obtain Lipo-ELE / Ce6.

[0057] ② Single-factor investigation of the prescription and preparation process of Lipo-ELE / Ce6

[0058] Investigate the effects of different preparation methods (ethanol injection method and thin-film dispersion method), different phospholipid amounts (100, 125, 150, 175, 200, 250 mg), different Ce6 amounts (0.5, 0.8, 1, 1.5, 2 mg), different hydration temperatures (40, 45, 50, 55, 60 °C), and different cholesterol amounts (5, 10, 15, 20, 25, 30 °C) on the encapsulation efficiency of elemene and Ce6 in Lipo-ELE / Ce6, and optimize the best preparation process of Lipo-ELE / Ce6.

[0059] 3) Characterization of Lipo-ELE / Ce6

[0060] ① Observation of liposome morphology

[0061] a. After the liposomes were negatively stained with 2% phosphotungstic acid, their morphology was observed by transmission electron microscopy; b. The liposomes were irradiated with near-infrared light at 660 nm (0.3 w·cm -2 , 3 min), and their morphological changes were observed by transmission electron microscopy.

[0062] ② Particle size determination

[0063] a. The particle size distribution of liposomes was determined by a Malvern Nano ZS laser particle size analyzer; b. The liposomes were irradiated with near-infrared light at 660 nm (0.3 w·cm -2 , 3 min), and their particle size and zeta potential were determined by a Malvern Nano ZS laser particle size analyzer.

[0064] ③ Zeta potential determination

[0065] The zeta potential of liposomes was determined by a Malvern Nano ZS zeta potential analyzer.

[0066] ④ Detection of the absorbance of Ce6 in liposomes

[0067] Ce6, Ce6 liposomes (Lipo-Ce6), elemenol liposomes (Lipo-ELE), and Lipo-ELE / Ce6 were taken respectively, and the absorbance of Ce6 was detected using a UV-visible spectrophotometer to prove that Ce6 was successfully encapsulated in liposomes. Among them, the preparation methods of Ce6 liposomes and elemenol liposomes were the same as those of Lipo-ELE / Ce6, except that ELE and Ce6 were missing respectively.

[0068] ⑤ Determination of the encapsulation efficiency of elemenol and Ce6

[0069] After the liposomes were microfiltered and centrifuged, the concentration of elemenol in the liposomes was determined by HPLC; the absorbance was measured using a UV-visible spectrophotometer and the concentration of Ce6 was calculated, and the encapsulation efficiency of the drug was calculated according to the formula "encapsulation efficiency = amount of drug encapsulated in liposomes / total amount of drug × 100%".

[0070] ⑥ Detection of the singlet oxygen generation ability

[0071] Since the 1,3-diphenylisobenzofuran (DPBF) probe is easily oxidized by ROS and its structure is changed, resulting in a gradual decrease in its absorbance at 415 nm, therefore, the amount of singlet oxygen generated can be evaluated by measuring its absorbance at 415 nm. Using DPBF as a probe, the singlet oxygen generation ability of Ce6 and different concentrations of Lipo-ELE / Ce6 was investigated. It was divided into 5 groups: a. PBS, b. Lipo-ELE / Ce6 (Ce6, 1 μg·mL -1 ), c. Ce6 (2 μg·mL -1 ), d. Lipo-ELE / Ce6 (Ce6, 2 μg·mL -1 ), e. Lipo-ELE / Ce6 (Ce6, 4 μg·mL -1 ), and 20 μg·mL was added to each group of solutions respectively -1DPBF, and for each group of solutions, after being irradiated with near-infrared light at a power of 0.015 w·cm -2 for different times (0, 2, 4, 6, 8, 10 min) at a wavelength of 660 nm, the absorbance and absorption curve of each group of solutions at 415 nm under different irradiation times were measured using a UV-visible spectrophotometer.

[0072] (2) Study on the in vivo antitumor effect of Lipo-ELE / Ce6

[0073] ① Animal grouping and drug administration

[0074] T24 cells were inoculated into the right axilla of female BALB / c nude mice at 5×10 7 cells / mL / rat to establish a subcutaneous bladder cancer model in BALB / c nude mice, and the effects of Lipo-ELE / Ce6 on the proliferation and apoptosis of bladder cancer cells were investigated. A total of 5 groups were divided, with 5 mice in each group: a. PBS group, b. ELE / Ce6 group (the drug in this group is a mixture of ELE and Ce6 with a mass ratio of 50:1), c. Lipo-ELE group (the preparation method of the drug in this group is the same as that of Lipo-ELE / Ce6, except that Ce6 is missing), d. Lipo-Ce6 group (the preparation method of the drug in this group is the same as that of Lipo-ELE / Ce6, except that ELE is missing), e. Lipo-ELE / Ce6 group. According to the grouping, intratumoral administration was performed on days 0, 3, 6, 9, and 12 (the administration dose of Ce6 was 2 mg / kg, and the administration dose of ELE was 100 mg / kg). After administration, groups b, d, and e were irradiated with near-infrared light (660 nm, 0.3 w·cm -2 ) and irradiated immediately for 10 min after administration. The long diameter and short diameter of the tumor were measured every 2 days, the change in tumor volume of the mice was monitored, and the body weight of the mice was recorded. After the treatment ended on day 14, tumor tissues and main organs (heart, liver, spleen, lung, kidney) were collected.

[0075] ② Pathological examination of tumor tissues and main organs

[0076] Tumor tissues and main organs (heart, liver, spleen, lung, kidney) were collected, fixed with 4% paraformaldehyde, dehydrated with ethanol, embedded in paraffin, sectioned, stained with HE, and the pathological examination of tumor tissues and main organs was performed.

[0077] ③ Apoptosis of tumor tissues

[0078] Tumor tissues were collected, fixed with 4% paraformaldehyde, dehydrated with ethanol, embedded in paraffin, sectioned, and after processing the sections according to the TUNEL cell apoptosis detection kit, the apoptosis of tumor tissue cells was observed under a fluorescence microscope.

[0079] ④ Immunohistochemical determination of the expression of Ki-67 and Cleaved Caspase-3 in tumor tissues

[0080] Collect tumor tissues, fix them with 4% paraformaldehyde, dehydrate them with ethanol, embed them in paraffin. After sectioning, perform antigen repair → block endogenous peroxidase → serum blocking → primary antibody incubation → secondary antibody incubation → DAB staining → counterstain the cell nuclei → dehydrate, clear, and mount the slides, and observe the expression of Ki-67 and Cleaved Caspase-3 in tumor tissues under a microscope.

[0081] ◆ Experimental results

[0082] (1) Study on the synergistic effect of ELE and Ce6

[0083] It can be seen from Figure 1 that the IC -2 values of ELE, Ce6, and Ce6 (0.3 w·cm 50 ) are 109.04 μg·mL -1 , 19.78 μg·mL -1 , and 2.175 μg·mL -1 respectively; based on the IC -2 values of ELE and Ce6 (0.3 w·cm 50 ), use CompuSyn software to screen whether the combination of ELE and Ce6 (10:1 - 150:1) has a synergistic effect; the results show that the combination index (CI) values of ELE and Ce6 at 10:1, 20:1, 40:1, 60:1, 80:1, 100:1, 120:1, and 150:1 are 1.20, 0.94, 0.76, 0.70, 0.76, 0.82, 0.89, and 0.90 respectively; the CI values of ELE and Ce6 at 40:1, 60:1, 80:1, and 100:1 are all less than 0.85, indicating that ELE / Ce6 has a synergistic effect in the range of 40:1 - 100:1.

[0084] (2) Preparation and characterization of Lipo-ELE / Ce6

[0085] 1) Preparation of Lipo-ELE / Ce6

[0086] First, the effects of the ethanol injection method and the thin-film dispersion method on the drug encapsulation efficiency were investigated. The encapsulation efficiencies of elemene and Ce6 by the ethanol injection method were 76.12% and 80.6% respectively, while those by the thin-film dispersion method were 90.5% and 88.17% respectively. The results showed that the thin-film dispersion method had a higher drug encapsulation efficiency. Then, the effects of different amounts of phospholipids on the drug encapsulation efficiency were investigated through single-factor experiments. The results showed that the highest encapsulation efficiencies of elemene and Ce6 were achieved when 175 mg of soybean lecithin and PC-98T were added respectively, reaching 96.09% and 94.97% respectively. The effects of different amounts of cholesterol on the drug encapsulation efficiency were investigated. The results showed that the highest encapsulation efficiencies of elemene and Ce6 were achieved when 15 mg of cholesterol was added, reaching 93.06% and 89.25% respectively. The effects of different amounts of Ce6 on the drug encapsulation efficiency were investigated. The results showed that the highest encapsulation efficiencies of elemene and Ce6 were achieved when 1 mg of Ce6 was added, reaching 94.12% and 90.17% respectively. The effects of different hydration temperatures on the drug encapsulation efficiency were investigated. The results showed that the highest encapsulation efficiencies of elemene and Ce6 were achieved at a hydration temperature of 55 °C, reaching 93.18% and 92.25% respectively( Figure 2 ).

[0087] Determine the preparation process of Lipo-ELE / Ce6:

[0088] 1) Weigh 15.52 mg of L-histidine and add it to 10 mL of water. Adjust the pH to 6.5 with hydrochloric acid to obtain an L-histidine solution;

[0089] 2) Take 175 mg of soybean lecithin, 175 mg of egg yolk lecithin (PC-98T), 15 mg of cholesterol, 20 mg of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000 ), 50 mg of elemene, and 1 mg of Ce6. Add them to 10 mL of absolute ethanol, place them in a 55 °C water bath and rotate under reduced pressure to evaporate to form a lipid film. Add the L-histidine solution with a pH of 6.5 to the rotating flask, stir and hydrate at 55 °C for 45 min, sonicate, and filter through a 0.22 μm microporous membrane to obtain the product.

[0090] 2) Characterization of Lipo-ELE / Ce6

[0091] ① Liposome morphology, particle size, and zeta potential

[0092] Observed by transmission electron microscopy, Lipo-ELE / Ce6 was circular( Figure 3(A), the average particle size was 158.53 ± 1.33 nm, the polydispersity index (PDI) was 0.226 ± 0.0025, the zeta potential was -17.13 ± 1.08 mV, and the encapsulation efficiencies of elemicin and Ce6 were 94.12 ± 1.21% and 91.66 ± 2.20% respectively; after Lipo-ELE / Ce6 was irradiated with near-infrared light at 660 nm (0.3 w·cm -2 , 3 min), it ruptured ( Figure 3 (B), the particle size became 244.03 ± 0.623 nm, the PDI became 0.412 ± 0.062, and the zeta potential became -5.44 ± 0.443.

[0093] ② Detection of the absorbance of Ce6 in liposomes

[0094] Through the ultraviolet absorption spectra of different samples ( Figure 4 ), it was found that the Ce6 solution, Lipo-Ce6 solution and Lipo-ELE / Ce6 solution all had absorption peaks at wavelengths of 403 nm and 665 nm, while the Lipo-ELE solution did not show absorption peaks at the above two wavelengths, indicating that Ce6 was successfully encapsulated in the liposomes.

[0095] ③ Detection of the ability to generate singlet oxygen

[0096] As Figure 5 shown, the yield of singlet oxygen of Lipo-ELE / Ce6 was concentration-dependent; and the singlet yield of Lip-ELE / Ce6 at the same concentration was significantly higher than that of free Ce6 at the same concentration.

[0097] (3) Study on the in vivo antitumor effect of Lipo-ELE / Ce6

[0098] 1) In vivo pharmacodynamic evaluation

[0099] The representative images of tumors and the changes in tumor volume after treatment with different drugs were as Figure 6 shown in A. The results showed that compared with other groups, the tumor size in the Lipo-ELE / Ce6 group was the smallest; the results of weighing the tumor tissue are shown in Figure 6 B. Compared with other groups, the tumor mass in the Lipo-ELE / Ce6 group was also the lightest. After the treatment ended, the tumor inhibition rates of the ELE / Ce6, Lipo-ELE, Lipo-Ce6 and Lipo-ELE / Ce6 groups were 23.2%, 15.9%, 36.2% and 59.7% respectively, indicating that Lipo-ELE / Ce6 could more significantly inhibit tumor growth; in addition, the body weights of the mice in all groups did not decrease significantly after treatment ( Figure 6 C).

[0100] 2) Study on the mechanism of in vivo antitumor effect

[0101] The results of HE staining, TUNEL apoptosis, and immunohistochemistry (Cleaved Caspase-3 and Ki67) of tumor tissues are shown in Figure 7 . The results of TUNEL staining showed that Lipo-ELE / Ce6 could significantly increase the apoptosis rate of tumor cells; the results of HE staining showed that the degree of tumor damage in the Lipo-ELE / Ce6 group was the greatest; the immunohistochemical results indicated that compared with other groups, the Lipo-ELE / Ce6 group could further reduce the expression of Ki-67 and increase the expression of Cleaved Caspase-3, thereby more effectively inhibiting the proliferation of tumor cells and promoting the apoptosis of tumor cells.

[0102] 3) Preliminary evaluation of in vivo safety

[0103] After different drug treatments of T24 tumor-bearing mice, there were no obvious pathological changes in the heart, liver, spleen, lungs, and kidneys, indicating that the preparation had no obvious toxicity to the main organs at this dosage, and the preliminary safety was good ( Figure 8 ).

[0104] In summary, by combining elemene and chlorin e6 in a specific ratio and preparing liposomes by the thin-film dispersion method, the present invention solves the problems of strong hydrophobicity, weak tumor targeting ability, and insufficient efficacy of the photosensitizer Ce6 alone, providing an innovative solution for the application of photodynamic therapy in the treatment of non-muscle-invasive bladder cancer.

Claims

1. A composition for treating bladder cancer, characterized in that, It is prepared from raw materials with the following weight ratios: Elemene 40 - 100 parts, Chlorin e6 1 part.

2. The composition according to claim 1, characterized in that, It is prepared from raw materials with the following weight ratios: Elemene 40 - 80 parts, Chlorin e6 1 part.

3. The composition according to claim 1 or 2, characterized in that, It is a preparation prepared with elemene and Chlorin e6 as active ingredients and a pharmaceutical carrier.

4. The composition according to claim 3, wherein The preparation is an injection; the injection is a solution, a gel or a liposome.

5. The composition according to claim 4, characterized in that, The liposome is prepared from the following raw materials: Elemene 40 - 80 parts, Chlorin e6 1 part, Soybean Lecithin 150 - 200 parts, Egg Yolk Lecithin PC - 98T 150 - 200 parts, Cholesterol 10 - 20 parts, DSPE - PEG 2000 10 - 30 parts, L - Histidine 15 - 16 parts.

6. The composition according to claim 4, wherein The liposome is prepared from the following raw materials: Elemene 50 parts, Chlorin e6 1 part, Soybean Lecithin 175 parts, Egg Yolk Lecithin PC-98T 175 parts, Cholesterol 15 parts, DSPE-PEG 2000 20 parts, L-Histidine 15.52 parts.

7. A method for preparing the composition according to claim 5 or 6, characterized in that, It includes the following steps: 1) Weigh the raw materials according to the ratio, take L - histidine, dissolve it in water, and then adjust the pH value to 6.5 to obtain the aqueous phase; 2) Take soybean lecithin, egg yolk lecithin PC-98T, cholesterol, DSPE-PEG 2000 , elemene and chlorin e6, dissolve them in ethanol, dry to obtain a lipid film; 3) Mix the lipid film obtained in step 2) and the aqueous phase obtained in step 1), stir, ultrasonicate, and filter to obtain the product.

8. The method according to claim 7, wherein: In step 1), the mass - to - volume ratio of the L - histidine to water is 15 - 16 mg:10 ml; the reagent for adjusting the pH value is hydrochloric acid.

9. The method according to claim 7, wherein: In step 2), the addition amount of ethanol is 1 / 5 of the dosage of elemene, ml / mg; the drying is to rotate and evaporate under reduced pressure in a 55 °C water bath until a lipid film is formed; in step 3), the stirring temperature is 55 °C, the time is 45 min; the ultrasonication time is 2 min; the filtration is through a 0.22 μm microporous membrane.

10. Use of the composition according to any one of claims 1 - 6 in the preparation of a medicament for treating bladder cancer; the bladder cancer includes non - muscle - invasive bladder cancer.

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