Pharmaceutical composition for inhibiting ovarian tumor as well as preparation method and application of pharmaceutical composition

Abstract

The invention discloses a pharmaceutical composition for inhibiting ovarian tumor as well as a preparation method and application thereof, and relates to the technical field of nano pharmaceutical preparations. The pharmaceutical composition comprises adriamycin and / or curcumin and extracellular vesicles derived from human ovarian cancer cells Caov3. The preparation method comprises the following steps: mixing adriamycin and / or curcumin with human ovarian cancer cell Caov3 external vesicles, and carrying out ultrasonic treatment and ice bath to obtain a mixed solution of Caov3-EVs and drugs; putting the mixed solution into a constant-temperature water bath kettle for incubation; after the water bath is finished, blowing, beating and uniformly mixing the mixed solution, centrifuging, sucking out the liquid, re-suspending and uniformly mixing the precipitate with PBS, and continuously centrifuging until the medicine which is not loaded into Caov3-EVs is completely removed; and sucking out the liquid in the superoxide dissociation tube, adding PBS into the tube, blowing and beating the wall and the bottom of the tube for multiple times, and collecting the resuspended extracellular vesicle drug-loaded preparation to obtain the extracellular vesicle drug-loaded preparation. The pharmaceutical composition can improve the drug loading rate and stability, has the effects of targeted delivery and side effect reduction, and can be effectively used for preventing and / or treating ovarian tumors.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine formulation technology, specifically to a pharmaceutical composition for inhibiting ovarian tumors, its preparation method, and its uses. Background Technology

[0002] Malignant tumors are among the most serious threats to human life and health worldwide. Currently, chemotherapy drugs such as doxorubicin (Dox) are commonly used in clinical practice, but these drugs have many problems, such as significant cardiotoxicity, the tendency for patients to develop drug resistance after long-term use leading to a gradual decline in treatment efficacy, and imprecise drug distribution within the body, which can also cause damage to normal tissues. Extracellular vesicles, as a novel nanomedicine delivery carrier, have attracted much attention due to their good biocompatibility and targeting properties. Extracellular vesicles are small membrane-bound vesicles with a diameter of approximately 30-200 nm secreted by cells, capable of carrying various bioactive substances such as proteins, lipids, mRNA, and miRNA, playing a crucial role in intercellular transport and information transmission.

[0003] Therefore, those skilled in the art aim to utilize the inherent homing function of extracellular vesicles to develop drug delivery formulations based on extracellular vesicles (EVs) that combine traditional Chinese medicine and chemotherapy drugs, thereby achieving targeted drug delivery, which helps to improve the therapeutic effect of drugs while reducing side effects. Summary of the Invention

[0004] The present invention provides a pharmaceutical composition for inhibiting ovarian tumors, its preparation method and uses, aiming to solve the problems existing in the above-mentioned background art.

[0005] To achieve the above-mentioned technical objectives, the present invention mainly adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a pharmaceutical composition for inhibiting ovarian tumors, comprising doxorubicin and / or curcumin and extracellular vesicles derived from human ovarian cancer cells Caov3.

[0007] In a preferred embodiment of the present invention, when the pharmaceutical composition is doxorubicin and extracellular vesicles derived from human ovarian cancer cells Caov3, the mass ratio of extracellular vesicles derived from human ovarian cancer cells Caov3 to doxorubicin is 1-10:1.

[0008] Preferably, the mass ratio of extracellular vesicles derived from human ovarian cancer cell line Caov3 to doxorubicin is 5:1.

[0009] In a preferred embodiment of the present invention, when the pharmaceutical composition is curcumin and extracellular vesicles derived from human ovarian cancer cells Caov3, the mass ratio of extracellular vesicles derived from human ovarian cancer cells Caov3 to curcumin is 1-10:1.

[0010] Preferably, the mass ratio of extracellular vesicles derived from human ovarian cancer cells Caov3 to curcumin is 2:1.

[0011] In a preferred embodiment of the present invention, when the pharmaceutical composition is doxorubicin, curcumin and extracellular vesicles derived from human ovarian cancer cells Caov3, the concentration of doxorubicin is 0.35 μg / mL and the concentration of curcumin is 7.8 μg / mL.

[0012] In a second aspect, the present invention provides a method for preparing the pharmaceutical composition as described in the first aspect, comprising the following steps:

[0013] (1) The doxorubicin and / or curcumin were mixed with extracellular vesicles derived from human ovarian cancer cells Caov3, sonicated, and placed in an ice bath to obtain a mixed solution of Caov3-EVs and drugs.

[0014] (2) Incubate the mixture of Caov3-EVs and the drug in a constant temperature water bath;

[0015] (3) After the water bath, the prepared mixed solution was blown and mixed, and then placed in an ultracentrifuge tube for centrifugation. The liquid was aspirated, and the precipitate was resuspended with PBS and mixed. Centrifugation was continued until the drug that was not loaded into Caov3-EVs was completely removed.

[0016] (4) Aspirate the liquid from the extracellular vesicle, add PBS into the tube, repeatedly blow on the tube wall and bottom, and collect the resuspended extracellular vesicle drug-loaded preparation.

[0017] Thirdly, the present invention discloses the use of the pharmaceutical composition as described in the first aspect in the preparation of a medicament for the prevention and / or treatment of ovarian tumors.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. Improved drug loading rate and stability: By optimizing the preparation conditions, this invention significantly improves the drug loading rate of extracellular vesicles and enhances the stability of the drug-loaded formulation, making it less prone to drug leakage or damage to the extracellular vesicle structure during storage and transportation, thus ensuring the efficacy and safety of the drug.

[0020] 2. Targeted delivery: By utilizing the homing effect of tumor-derived extracellular vesicles, precise delivery of drugs to homologous tumors is achieved, which significantly improves the efficacy of chemotherapy drugs, reduces the distribution of drugs in non-target tissues, and reduces damage to normal tissues.

[0021] 3. Reduced side effects: Extracellular vesicles have good biocompatibility, which reduces the toxic side effects of drugs, improves patient tolerance, enables patients to better accept chemotherapy treatment, prolongs survival, and improves quality of life.

[0022] 4. Real-time monitoring: Through fluorescent labeling technology, real-time quantitative visualization of extracellular vesicle drug delivery formulations in vivo is achieved, providing a powerful tool for drug delivery and evaluation of treatment effects. It helps doctors adjust treatment plans based on real-time feedback during the treatment process, improving the accuracy and individualization of treatment. Attached Figure Description

[0023] Figure 1 A graph showing the uptake capacity of EVs by Caov3 cells in human ovarian cancer cells.

[0024] Figure 2 The ultraviolet standard curve for doxorubicin;

[0025] Figure 3 The loading rate of doxorubicin DOX by Caov3-EVs at different mass ratios;

[0026] Figure 4 The survival rate (%) and half-maximal inhibitory concentration (IC50) of different concentrations of traditional Chinese medicine (Res, Ber, and Cur) against human ovarian cancer cells Caov3 were calculated. 50 ;

[0027] Figure 5 The UV standard curve for curcumin is shown.

[0028] Figure 6 The loading rate of curcumin Curcumin on Caov3-EVs at different mass ratios;

[0029] Figure 7 TEM images of EVs before and after drug encapsulation;

[0030] Figure 8 The particle size and particle concentration of extracellular vesicles before and after drug loading were determined using a nanoparticle size tracking analyzer.

[0031] Figure 9 This is a comparison diagram of the absorption and emission peaks of the drug before and after loading.

[0032] Figure 10 Imaging analysis diagrams for analyzing the distribution and accumulation of drugs within cells using laser confocal microscopy;

[0033] Figure 11 A comparison of the cytotoxic effects of different concentrations (0-1000 μg / mL) of EVs on normal human ovarian epithelial cells (IOSE-80) and human ovarian cancer cells (Caov3) as determined by the CCK-8 kit.

[0034] Figure 12 A comparison of the cytotoxicity of EVs-Dox and EVs-Cur to IOSE-80 cells compared to the use of free drugs alone;

[0035] Figure 13 The in vitro antitumor effects of Dox, EVs-Dox, and Cur, EVs-Cur are shown in the figure.

[0036] Figure 14 A diagram illustrating the therapeutic effect of combining chemotherapy drugs with traditional Chinese medicine monomers;

[0037] Figure 15 This figure illustrates the effect of different drug treatments on tumor cell viability using a combination of live / dead cell staining and laser confocal microscopy. Detailed Implementation

[0038] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1: Preparation of EVs-Dox extracellular vesicle drug delivery formulation

[0040] I. Cell Culture

[0041] Human ovarian cancer cells (Caov3) were selected, and the cell culture conditions were 37°C. o C, 5% CO2. Preheat the constant temperature water bath to 37°C. o At time C, remove the frozen cells from the liquid nitrogen tank and thaw them rapidly. In a clean bench, transfer the thawed cells to centrifuge tubes, add an appropriate amount of complete culture medium (DMEM medium, 10% fetal bovine serum, 1% triple antibody (penicillin-streptomycin-amphoteric B mixture), and mix thoroughly by pipetting. Set the centrifuge speed to 1000 rpm and the time to 5 min, then centrifuge the tubes. After centrifugation, remove the cell supernatant and resuspend the cells in the corresponding complete culture medium. Transfer the cells to culture flasks for culture. Change the medium every one or two days, depending on the situation. When the cells in the culture flasks have grown to about 80%, passage them. Remove the old culture medium from the culture flasks, wash with an appropriate amount of PBS buffer, add 1 mL of 0.25% trypsin for digestion, then add 2 mL of the corresponding complete culture medium to stop the digestion, and pipette the cells. Transfer the suspension to centrifuge tubes and culture the cells as described above.

[0042] II. Extraction of extracellular vesicles

[0043] When T175 cm 2When the cell density in the culture flask reaches approximately 70%, switch to a serum-free culture medium specifically for extracellular vesicles. After 72 hours, collect the supernatant in centrifuge tubes and extract extracellular vesicles using differential ultracentrifugation. First, set the refrigerated centrifuge temperature to 4°C and the centrifugation force to 300 ×g, 2000 ×g, and 10000 ×g to remove dead cells, cell debris, and larger proteins. Then, filter the liquid from the centrifuge tubes into ultrafiltration tubes using a 0.22 μm microporous membrane, and set the refrigerated centrifuge to 4°C. o Concentrate at 5000 × g for 30 min at C. Finally, collect the concentrate and transfer it to an ultracentrifuge tube, setting the ultracentrifuge to 4°C. o At 39,000 rpm for 70 min, the supernatant was removed, and the precipitate remaining in the ultracentrifuge tube was Caov3-EVs. Depending on the intended use of the EVs, different diluents were selected for resuspending.

[0044] III. Cellular uptake of extracellular vesicles

[0045] 1. PKH67 labeling working fluid labeling EVs

[0046] Take 5 µl of PKH67 (250X) and dilute it with 45 µl of Diluent C. Mix well and then add PBS to obtain the PKH67 labeling working solution (1X). Add the PKH67 labeling working solution to the EV sample according to the ratio of approximately 10 µg of extracellular vesicles to 100 µl of PKH67 labeling working solution. Mix well and incubate in the dark for 5 min. Place the PKH67-labeled EVs derived from human ovarian cancer cells (Caov3) in an ultracentrifuge at 100,000 × g, 4 °C for 70 min. Remove the supernatant, drain excess liquid on paper, and a pale yellow precipitate will be obtained at the bottom of the tube. Dilute the precipitate with 100 μL of PBS solution to obtain the EVs labeled with the PKH67 labeling working solution.

[0047] 2. Construction and staining of ovarian cancer cell model

[0048] When the Caov3 ovarian cancer cells reached approximately 80% confluence, the cells were digested with trypsin, centrifuged, resuspended, and counted. They were then seeded into confocal cell culture dishes at a density of approximately 2 × 10⁻⁶ cells / mL. 5Cells / mL were cultured in a 37℃, 5% CO2 incubator for 12 h to allow for full adherence and growth to the bottom of the culture dish. The old culture medium was aspirated, and the cells were washed three times with PBS. Fresh DMEM medium containing PKH67-labeled EVs was added to a confocal cell culture dish, and the dish was incubated for 0–4 h. The culture dish was removed, gently washed three times with preheated PBS, and fixed with cell fixation solution for 15 min. After washing three times with PBS, 1 mL of Hoechst 33342 staining solution was added to label the cell nuclei, and the staining solution was discarded after 5–10 min. After washing three times with PBS, the cells were used for imaging with a fluorescence confocal microscope (FV3000, 63×, NA 1.25). The excitation wavelength of PHK67 was set to λex = 488 nm, and the collection window λem = 495–550 nm; the excitation wavelength of Hoechst 33342 was set to λex = 405 nm, and the collection window λem = 425–475 nm.

[0049] This application used human ovarian cancer cells (Caov3) as model cells for in vitro culture experiments. Laser confocal microscopy was used for imaging analysis to observe the distribution and accumulation of EVs within the cells and to detect the incubation time of PKH67, aiming to further explore the cells' ability to take up EVs. We used Hoechst 33342 dye to label the cell nucleus and the fluorescent lipid membrane dye PKH67 to label the EV membrane, thus visualizing the cellular uptake process. Results are as follows: Figure 1 As shown, EVs (PKH67 labeled, green fluorescent) are located in the cytoplasm, indicating that EVs are localized in the cytoplasm after being taken up by the cells. Fluorescence images of cells were obtained at different time points (0h, 1h, 2h, 3h, 4h). With increasing incubation time, EVs gradually accumulate and emit a stronger fluorescence signal in the cells. The fluorescence intensity increases with time and then tends to stabilize after reaching a certain level. To further verify the hypothesis, the obtained fluorescence images were quantitatively analyzed using ImageJ and Graphpad. The analysis results showed that the cells exhibited the highest brightness after 4 h of incubation with PKH67-labeled EVs, and then tended to stabilize.

[0050] IV. Preparation of Extracellular Vesicle Drug-Loaded Formulations

[0051] (1) Preparation was carried out using the ultrasonic method. The drug DOX was mixed with extracellular vesicles at a certain mass ratio (EVs:Dox mass ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1). The ultrasonic power of the ultrasonic cell disruptor was adjusted to 40%, and pulsed ultrasonic ON / OFF was performed for 30s each time. This was repeated 3 times. After that, the mixture was taken out and placed on ice for 2min. The above operation was repeated 3 times.

[0052] (2) Preheat the water bath in advance and set the temperature to 37°C. After the ultrasound is finished, put the mixture of Caov3-EVs and the drug into the constant temperature water bath and incubate for 1 hour to restore the stability of the extracellular vesicle membrane.

[0053] (3) After the water bath, the prepared mixed solution was pipetted and mixed thoroughly, then placed in an ultracentrifuge tube, balanced, and centrifuged at 100,000 × g for 90 min at 4 °C. The liquid was aspirated, and the precipitate was resuspended with PBS and mixed thoroughly. Centrifugation was continued until the effect of any drug not loaded into Caov3-EVs was completely removed. Finally, the liquid in the ultracentrifuge tube was aspirated, 100 μL of 1×PBS was added to the tube, and the tube wall and bottom were repeatedly pipetted. The resuspended extracellular vesicle drug-loaded formulation EVs-Dox was collected for use in subsequent experiments.

[0054] Experimental Example 1: Investigating the drug loading rate of extracellular vesicle drug-loaded formulations EVs-Dox prepared at different mass ratios in Example 1.

[0055] (1) Establishment of drug standard curve

[0056] Doxorubicin (DOX) was prepared into working solutions at concentrations of 0.25 mg / mL for later use. Then, working solutions containing the drug were prepared at concentration gradients of 0.25, 0.5, 0.75, 1, 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, and 70 μg / mL, with three replicates for each concentration. The absorbance of the drug was measured using a UV-Vis spectrophotometer. A UV standard curve was plotted with drug concentration on the x-axis and the absorbance at each concentration on the y-axis to obtain the drug's UV standard curve. Figure 2 As shown.

[0057] (2) Detection of drug loading rate in extracellular vesicles

[0058] The drug loading rate of the supernatant collected during the preparation of the extracellular vesicle drug-loaded formulation was quantitatively determined using a UV spectrophotometer, and the encapsulation process was optimized. The absorbance of each sample was substituted into the UV standard curve of the drug to calculate the amount of free drug in the supernatant. The drug loading rate (%) of Caov3-EVs was calculated using the following formula:

[0059] Loading efficiency (%)

[0060] Based on the UV standard curve of Dox, the drug loading rate of the prepared extracellular vesicle drug-loaded formulation EVs-Dox was detected by UV spectrophotometer as follows: Figure 3As shown, when the extracted EVs are mixed with doxorubicin (Dox) at a mass ratio of 5:1, the drug loading rate can reach 46%.

[0061] Example 2: Preparation of EVs-Extracellular Vesicle Drug-Loaded Formulations of Traditional Chinese Medicine Compounds

[0062] (1) Screening of Chinese medicinal herbs

[0063] When the cell density of human ovarian cancer cells (Caov3) reached approximately 70%, the cells were digested with trypsin, centrifuged, and the cell suspension was counted. 100 μL of PBS was added around the perimeter of a 96-well plate, and an equal volume of complete culture medium containing 5000 cells was added to each well. The 96-well plate was incubated overnight at 37°C with 5% CO2 until the cells were fully adhered. Complete culture medium containing different concentrations (0, 1, 5, 10, 15, 20, 25, 30, 40 μg / mL) of resveratrol (Res), berberine (Ber), and curcumin (Cur) was added, with 3-6 replicates per concentration, and the experiment was repeated three times. The control group contained no cells, and the blank group contained untreated cells. The 96-well plate was incubated for another 24 h. 10 μL of CCK-8 solution was added to each well, and the absorbance at 450 nm was measured after 2-4 h of incubation. Based on the effects of different concentrations of the analyte on experimental cells, the cell viability (%) and half-inhibitory concentration (IC50) were calculated. 50 .

[0064] To enhance therapeutic efficacy while reducing drug dosage and minimizing potential side effects, cytotoxicity experiments were conducted on three traditional Chinese medicine monomers: resveratrol, curcumin, and berberine. By comparing the therapeutic effects of the same drugs, we selected the traditional Chinese medicine with the best therapeutic effect for combination therapy. The IC50 values ​​of the three traditional Chinese medicines were then analyzed. 50 Comparison of treatments, such as Figure 4 As shown, we found that curcumin's IC50... 50 The value of 7.8 μg / mL was significantly lower than that of resveratrol and berberine. Therefore, we chose curcumin for the preparation of extracellular vesicle drug delivery formulations.

[0065] (2) Preparation of EVs-Cur extracellular vesicle drug delivery formulation

[0066] The steps are basically the same as in Example 1, except that curcumin Cur is used instead of drug DOX. At the same time, curcumin Cur is mixed with extracellular vesicles at a certain mass ratio (EVs:Cur mass ratio is also 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1) to obtain the extracellular vesicle drug-loaded formulation EVs-Cur.

[0067] Experimental Example 2: Investigating the drug loading rate of extracellular vesicle drug-loaded formulations EVs-Cur prepared at different mass ratios in Example 2.

[0068] (1) Establishment of drug standard curve

[0069] Curcumin (Cur) was prepared into working solutions at concentrations of 0.25 mg / mL for later use. Then, working solutions containing the drug were prepared at concentration gradients of 0.25, 0.5, 0.75, 1, 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, and 70 μg / mL, with three replicates for each concentration. The absorbance of the drug was measured using a UV-Vis spectrophotometer. A UV standard curve was plotted with drug concentration on the x-axis and the absorbance at each concentration on the y-axis to obtain the drug's UV standard curve. The results are as follows: Figure 5 As shown.

[0070] (2) Detection of drug loading rate in extracellular vesicles

[0071] The supernatant collected during the preparation of the extracellular vesicle drug-loaded formulation was used to measure the absorbance of the drug using a UV spectrophotometer. The absorbance of each sample was substituted into the UV standard curve of the drug to calculate the amount of free drug in the supernatant. The drug loading rate (%) of Caov3-EVs was calculated using the following formula:

[0072] Loading efficiency (%)

[0073] Based on the UV standard curve of Cur, the drug loading rate of the prepared extracellular vesicle drug-loaded formulation EVs-Cur was measured by UV spectrophotometer as follows: Figure 6 As shown, when the extracted EVs and Cur are mixed at a mass ratio of 2:1, the drug loading rate can reach 38%.

[0074] Example 3: Preparation of EVs-DOX+EVs-Cur extracellular vesicle drug delivery formulation

[0075] The steps are basically the same as in Example 1, except that curcumin (Cur) and the drug DOX are mixed with extracellular vesicles to obtain the extracellular vesicle drug-loaded formulation EVs-DOX-Cur. The concentrations of DOX and Cur in each group are as follows:

[0076] Example 3: Characterization of extracellular vesicles

[0077] (1) Morphological characterization by transmission electron microscopy (TEM)

[0078] Extracted extracellular vesicles and their drug-loaded suspensions were separately added dropwise onto a 400-mesh copper mesh (carbon support membrane) and allowed to stand for 15 min. Then, a 3% uranium acetate saturated aqueous solution was added for staining, and the mixture was allowed to stand for approximately 2 min. After staining, the copper mesh was washed with ultrapure water for 1 min each time, repeated 3 times. Finally, the copper mesh was dried at room temperature for 24 h and used for transmission electron microscopy to observe the size and morphology of the extracellular vesicles. The results are as follows: Figure 7 As shown.

[0079] Depend on Figure 7 As can be seen from TEM observation, EVs exhibit a typical round or elliptical vesicle structure, and their morphology did not change significantly after drug encapsulation.

[0080] (2) Nanoparticle tracking analyzer (NTA) to assess particle size

[0081] Extracellular vesicle standards were diluted 250,000 times with ultrapure water. Freshly extracted extracellular vesicles derived from human ovarian cancer cells (Caov3) and their drug-loaded formulations were diluted 1000 times with ultrapure water. The particle size and concentration of the extracellular vesicles were determined using a nanoparticle size analyzer. Results are as follows: Figure 8 As shown.

[0082] Depend on Figure 8 It can be seen that the particle size of EVs is 128 nm, the particle size of EVs-Dox is 137.3 nm, and the particle size of EVs-Cur is 132 nm. The particle size of extracellular vesicles before and after drug loading is distributed in the range of 30-200 nm, which is consistent with the size of extracellular vesicles.

[0083] Stability analysis of Experiment Example 4

[0084] To investigate whether extracellular vesicles affect the basic properties of drugs during drug loading, we systematically analyzed the spectral properties of the drug and its prepared extracellular vesicle drug-loaded formulation. 1 mL samples were placed in cuvettes and analyzed using a UV-Vis spectrophotometer and a fluorescence spectrophotometer. By comparing the changes in absorption and emission peaks before and after drug loading, we explored whether the spectral properties were altered due to drug loading.

[0085] In UV-Vis spectroscopy, the detection wavelength for doxorubicin was set to 350-600 nm to obtain its characteristic absorption peak information. For fluorescence spectroscopy, the excitation wavelength was set to 480 nm, and the emission scanning range was set to 500-750 nm to comprehensively capture its fluorescence emission characteristics. In curcumin determination, the detection wavelength for UV-Vis spectroscopy was set to 300-600 nm, the excitation wavelength was set to 430 nm, and the emission scanning range was set to 350-700 nm.

[0086] The results are as follows Figure 9 As shown, in UV-Vis spectroscopy, the absorption peak of doxorubicin before and after drug loading was at 480 nm, while that of curcumin was at 430 nm. Fluorescence spectroscopy showed that the emission peak of doxorubicin was at 490 nm, and that of curcumin was at 530 nm. The similarity in the positions and shapes of the emission and absorption peaks before and after drug loading indicates that drug loading into extracellular vesicles does not alter the fundamental properties of the drug.

[0087] Experimental Example 5: Investigating and analyzing the distribution and accumulation of drugs in human ovarian cancer cells Caov3.

[0088] (a) Cellular uptake experiment of extracellular vesicle drug delivery formulation

[0089] 1. PKH26 labeling working fluid for EVs, EVs-Dox and EVs-Cur

[0090] Under light-protected conditions, the PKH26 linker (for red fluorescent cell labeling) stock solution was diluted 10-fold with Diluent C to prepare a 100 μM dye working solution. Based on the protein concentration of EVs determined by BCA, appropriate amounts of dye working solution were added to the EVs, EVs-Dox, and EVs-Cur samples to achieve a final dye concentration of 5 μM. The mixture was vortexed for 1 min and then incubated at room temperature in the dark for 10 min. Staining was terminated by adding an appropriate amount of 1×PBS and mixing thoroughly. PKH26-labeled human ovarian cancer cell line Caov3-derived EVs, EVs-Dox, and EVs-Cur were centrifuged at 100,000×g at 4℃ for 1–2 h. The supernatant containing free dye was removed, and the precipitate was resuspended in 200 μL of 1×PBS. These were the PKH26-labeled Caov3 ovarian cancer cell line EVs, EVs-Dox, and EVs-Cur.

[0091] 2. Construction and staining of ovarian cancer cell model

[0092] When the Caov3 ovarian cancer cells reached approximately 80% confluence, the cells were digested with trypsin, centrifuged, resuspended, and counted. They were then seeded into confocal cell culture dishes at a density of approximately 2 × 10⁻⁶ cells / mL. 5Cells / mL were cultured overnight in a 37°C, 5% CO2 incubator to ensure full cell adhesion and growth at the bottom of the culture dish. The old culture medium was discarded, and the cells were washed three times with PBS. Fresh DMEM medium containing Dox and Cur, PKH26-labeled EVs, PKH26-labeled EVs-Dox, and EVs-Cur were added to confocal cell culture dishes, respectively, and incubated for 2–6 h. The culture dishes were removed, the old culture medium was discarded, the cells were washed with PBS, and fixed with cell fixation solution for 15 min. The cells were then washed three more times with PBS, and 1 mL of Hochest 33342 staining solution was added to label the cell nuclei. After 20 min, the staining solution was discarded. Wash three times with PBS, then add 1 mL of complete culture medium. Observe under a fluorescence confocal microscope (FV3000, 63×, NA 1.25). Set the excitation wavelength of PHK26 to λex = 551 nm and the collection window to λem = 540-580 nm; the excitation wavelength of Hoechst 33342 to λex = 405 nm and the collection window to λem = 425-475 nm; the excitation wavelength of Dox to λex = 488 nm and the collection window to λem = 580-620 nm; and the excitation wavelength of Cur to λex = 488 nm and the collection window to λem = 520-560 nm.

[0093] This application uses human ovarian cancer cells Caov3 as model cells for in vitro culture experiments. Laser confocal microscopy is used for imaging analysis to observe the distribution and accumulation of drugs in cells, aiming to explore the cells' ability to take up Dox, EVs, and EVs-Dox respectively.

[0094] like Figure 10As shown, to trace the drug delivery process of extracellular vesicles (EVs), we labeled the cell nucleus with Hoechst 33342 dye and the EV membrane with the red fluorescent dye PKH26 to visualize the cellular uptake process. The results showed that EVs (PKH26-labeled, bright red fluorescence) were located in the lipid region of the cell membrane, indicating that EVs were localized in the cytoplasm after uptake. We observed that the green fluorescence signal of Dox before and after drug loading was mainly concentrated in the nucleus. This phenomenon indicates that EVs can effectively deliver Dox to the nucleus without altering the original site of drug action during delivery. Notably, although the fluorescence signals produced by the Dox before and after drug loading and the nuclear dyes were largely consistent in tumor cells, fluorescence colocalization analysis using confocal microscopy showed a significant difference. Compared with an equal amount of free Dox, the accumulation of EVs-Dox in the nucleus was significantly increased. This indicates that EVs play a crucial role in the intracellular delivery of Dox, significantly enhancing the enrichment of the drug in the nucleus. We hypothesize that this phenomenon may be closely related to the internalization process of EVs. Over time, the internalization of EVs intensifies, leading to their fusion with the cell membrane and restriction within the cytoplasm. This release of encapsulated Dox from the cytoplasm facilitates Dox entry into the nucleus, significantly enhancing drug uptake efficiency. Furthermore, we compared the free Dox groups (equivalent to 1, 2, 4, 6, 8, and 10 times the drug concentration in EVS-Dox) with the EVs-Dox group. Figure 10 As shown, the results indicate that after incubation with Caov3 cells, the amount of Dox entering the cell nucleus in the EVs-Dox group was nearly 10 times that of free Dox. We compared the free Cur groups (equivalent to 1, 2, 4, 6, and 8 times the drug concentration in EVS-Cur) with the EVs-Cur group, as shown... Figure 10 As shown, the results indicate that after incubation with Caov3 cells, the amount of Dox entering the nucleus in the EVs-Dox group was nearly 10 times that of free Dox, and the amount of Cur entering the nucleus in the EVs-Cur group was nearly 8 times that of free Cur. This significant difference not only demonstrates the significant advantage of extracellular vesicle drug delivery formulations in cellular uptake efficiency but also highlights their superior drug delivery capabilities, indicating that extracellular vesicles can efficiently deliver drugs to the cell nucleus, thereby significantly improving drug efficacy. This suggests that EVs can serve as an excellent drug delivery carrier for intracellular drug delivery.

[0095] Experimental Example 6: Investigating the biosafety of extracellular vesicle drug delivery formulations

[0096] 1. Biosafety of EVs

[0097] Human normal ovarian epithelial cells (IOSE-80) and human ovarian cancer cells (Caov3) were digested with trypsin, centrifuged, and the cell suspensions were collected and counted. Cells were seeded at a density of approximately 5000 cells per well in 96-well plates and incubated in a 5% CO2 incubator at 37 °C until complete cell adhesion. Then, the old medium in each well was replaced with 100 μL of fresh complete medium containing different concentrations of EVs extracted from the Caov3 cell line (0, 10, 20, 40, 80, 100, 200, 500 / 1000 μg / mL). After 24 h of incubation, 10 μL of CCK-8 solution was added to each well under dark conditions, and incubation continued for 0.5–4 h. The absorbance was measured at 450 nm using a microplate reader. Untreated cells served as the control group. Cell viability (%) was calculated using the following formula to assess the biosafety of the EVs.

[0098] C

[0099] Before evaluating the therapeutic effects of EVs-Dox and EVs-Cur, we used a CCK-8 assay kit to detect the cytotoxic effects of different concentrations (0-1000 μg / mL) of EVs on normal human ovarian epithelial cells (IOSE-80) and human ovarian cancer cells (Caov3). The results are as follows: Figure 11 As shown, IOSE-80 did not exhibit significant cytotoxicity even at a high concentration of 1000 μg / mL. As expected, for Caov3 cells, cell viability remained above 90% even at EV concentrations as high as 1000 μg / mL.

[0100] In summary, EVs have good biocompatibility and are almost non-toxic to cells, making them a safe drug carrier for ovarian administration.

[0101] 2. Biosafety of extracellular vesicle drug delivery formulations

[0102] Human normal ovarian epithelial cells (IOSE-80) were selected, digested with trypsin, centrifuged, and the cell suspension was collected. Approximately 5000 cells per well were seeded into each well of a 96-well plate and cultured until full cell adhesion. The old culture medium was removed, and 100 μL of culture medium containing different concentrations of the test drug was added to each well according to the groupings. Dox concentrations were 0.00, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.40, and 0.50 μg / mL, with the Dox concentration in the extracellular vesicle drug-loaded formulation being the same as that in the free Dox in each group. Curl concentrations were 0.00, 1.00, 2.00, 4.00, 6.00, 8.00, 10.00, 15.00, and 20.00 μg / mL, with the Curl concentration in the extracellular vesicle drug-loaded formulation being the same as that in the free Curl in each group. After 24 h of incubation, 10 μL of CCK-8 solution was added, and after 1 h of incubation, the absorbance was measured at 450 nm, and the cell viability (%) was calculated to assess the biosafety of the extracellular vesicle drug delivery formulation.

[0103] To further evaluate the biosafety of the extracellular vesicle drug delivery formulation, we assessed its biosafety by comparing the cytotoxicity of EVs-Dox and EVs-Cur with that of free drug alone on IOSE-80 cells. Results are as follows: Figure 12 As shown, the cytotoxicity of the drug to IOSE-80 cells was significantly reduced after encapsulation with EVs. This may be attributed to the fact that the loading of the drug into the cells by EVs limited the drug diffusion to some extent, thereby mitigating the toxic side effects of antitumor drugs and improving biosafety. Free drugs can enter cells through passive diffusion and have non-selective toxicity to both tumor and normal cells. In contrast, extracellular vesicles need to enter cells through receptor-mediated endocytosis or membrane fusion pathways. IOSE cells may lack efficient mechanisms for uptake of extracellular vesicles (such as clathrin-dependent endocytosis or lipid raft-mediated endocytosis), resulting in a significantly lower efficiency of drug-loaded groups entering cells compared to free drug groups. Extracellular vesicles derived from Caov3, as endogenous vesicles, have membrane components highly similar to cell membranes and good biocompatibility. In contrast, free drugs may cause toxicity to normal cells through non-specific mechanisms such as membrane lipid peroxidation and mitochondrial damage. Encapsulation with EVs reduced the direct contact between the drug and the IOSE cell membrane, reducing membrane damage and oxidative stress. Normal cells (such as IOSE) may overexpress drug efflux pumps (such as P-glycoproteins), and drugs loaded in extracellular vesicles may be recognized by these pumps and expelled from the cell. In addition, the membrane structure of extracellular vesicles may reduce the interaction between drugs and P-glycoproteins, further reducing intracellular drug accumulation and thus attenuating toxicity.

[0104] Experiment 7: Investigating the in vitro antitumor effect of extracellular vesicle drug-loaded formulations.

[0105] When the Caov3 ovarian cancer cell density reached approximately 70%, the cells were digested with trypsin, centrifuged, and the cell suspension was counted. 100 μL of PBS was added around the perimeter of a 96-well plate, and an equal volume of complete culture medium containing 5000 cells was added to each well. The 96-well plate was incubated overnight at 37°C in a 5% CO2 incubator until the cells were fully adhered. Complete culture medium containing different concentrations of Dox (0.00, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.40, 0.50 μg / mL) and Curl concentrations of 0.00, 1.00, 2.00, 4.00, 6.00, 8.00, 10.00, 15.00, 20.00 μg / mL were added, with 3-6 replicates per concentration. The experiment was repeated three times. The control group contained no cells, and the blank control group contained untreated cells. The 96-well plate was placed in an incubator and incubated for another 24 h. 10 μL of CCK-8 solution was added to each well, and the absorbance at 450 nm was measured after 1 h of incubation. Based on the effects of different concentrations of the analyte on the experimental cells, the cell viability (%) and half-inhibitory concentration (IC50) were calculated. 50 .

[0106] This study used the CCK-8 assay to conduct in vitro experiments on human ovarian cancer cells (Caov3) to evaluate the in vitro antitumor effects of Dox, EVs-Dox, Cur, and EVs-Cur. The results are as follows: Figure 13 As shown, drug loading into extracellular vesicles significantly enhanced the killing effect of antitumor drugs on the Caov3 cell line. Furthermore, we investigated the half-inhibitory concentrations (IC50) of Dox, EVs-Dox, and Cur, EVs-Cur. 50 After drug loading into extracellular vesicles and co-incubating with Caov3 cells, IC50 was found to increase. 50 The survival rate was reduced. This indicates that the cell survival rate treated with extracellular vesicle-loaded drugs was significantly lower than that treated with the same concentration of drug alone. EVs themselves are non-toxic, and EVs-Dox effectively delivers chemotherapeutic drugs to tumor cells, confirming the superior drug delivery capability of extracellular vesicle-loaded drugs.

[0107] Extracellular vesicles (EVs) derived from Caov3 carry specific membrane proteins (such as integrins, CD63, and CD81) on their surface. These proteins can recognize and bind to receptors on the surface of Caov3 tumor cells, forming a "homology-targeting" effect. EVs can enter cells via a non-clathrin-dependent endocytosis pathway, effectively avoiding drug degradation in lysosomes and ensuring that the drug can successfully reach its intracellular target site (such as the cell nucleus), thereby enhancing its cytotoxicity. EVs possess natural intercellular communication functions, enabling them to specifically recognize and target homologous tumor cells (such as Caov3), efficiently entering cells through membrane fusion or endocytosis. Free drugs are susceptible to tumor cell resistance mechanisms (such as P-glycoprotein-mediated efflux), while EVs can increase drug accumulation in drug-resistant cells by circumventing these mechanisms (such as avoiding recognition by P-glycoproteins), thereby enhancing efficacy. The bioactive molecules (such as miRNAs and proteins) carried by EVs themselves may synergistically inhibit tumor progression with doxorubicin, for example, by downregulating pro-survival signals or enhancing apoptosis pathway activity, further amplifying the therapeutic effect.

[0108] Experimental Example 8: Investigating the combined therapeutic effect of the EVs-DOX + EVs-Cur extracellular vesicle drug delivery formulation prepared in Example 3.

[0109] To verify whether combination therapy can produce a high therapeutic effect, we used cytotoxicity assays to evaluate the efficacy of Dox and Curl. 50 Combination therapy with drug concentrations of [value], with results as follows: Figure 14 As shown, cell survival rates in the combination therapy group were significantly lower than those in the single-drug group. Based on IC50... 50 The cell survival rate in the combined treatment group was low, so we reduced the drug concentration to provide experimental basis for subsequent drug screening and efficacy evaluation in mouse experiments. Results are as follows... Figure 14 As shown, compared with the free group, the drug-loaded group exhibited stronger cytotoxicity at the same concentration. This indicates that combined treatment with chemotherapy drugs and traditional Chinese medicine monomers can significantly improve therapeutic efficacy.

[0110] The effects of different drug treatments on tumor cell viability were further evaluated using live / dead cell staining combined with laser confocal microscopy. Caov3 cells were selected as the experimental model cells. When the cells reached a density exceeding 80%, they were seeded into cell culture dishes and cultured until the cells were fully adherent. During culture, the medium was changed regularly to ensure good cell growth. When the cell confluence reached 80%, the two cell types were divided into a control group and the following experimental groups: Dox group, Cur group, EVs-Dox group, EVs-Cur group, Dox+Cur combined group, and EVs-Dox+EVs-Cur combined group. Each group of cells was incubated with the corresponding drug at 37°C in a 5% CO2 incubator for 24 hours. After incubation, the culture medium was aspirated, and PBS was gently added along the dish wall to wash the cells once to remove residual liquid. Subsequently, the cells were stained using the Calcein / PI cell viability assay kit. Following the kit instructions, the staining working solution was prepared as shown in the table below. 1 mL of staining working solution was added to each cell culture dish, and the cells were incubated at 37°C in the dark for 30 min. After incubation, the staining effect was observed using a laser confocal microscope. Calcein AM (calcein-based fluorescent dye) was used to label live cells, with an excitation wavelength λex = 488 nm and an emission wavelength collection range of λem = 500 ~ 540 nm. PI (propidium iodide) was used to label dead cells, with an excitation wavelength λex = 561 nm and an emission wavelength collection range of λem = 570 ~ 620 nm.

[0111] Preparation of Calcein / PI detection working solution

[0112]

[0113] After the experiment, the cell status of each experimental group (Control, Dox, Cur, EVs-Dox, EVs-Cur, Dox+Cur, EVs-Dox+EVs-Cur) was visualized and analyzed. According to the kit instructions, live cells emitted green fluorescence, while dead cells showed red fluorescence; the fluorescence intensity was directly related to the cell viability. Figure 15As shown, only green fluorescence was observed in the control group, indicating that the cells were in good condition and there was no obvious cell death. A very small amount of red fluorescence was observed in the free drug treatment groups (Dox and Cur), suggesting that the killing effect of free doxorubicin and curcumin on tumor cells was relatively limited. In the drug-loaded groups (EVs-Dox and EVs-Cur), red fluorescence increased slightly, but green fluorescence remained dominant, indicating that the extracellular vesicle (EV) drug delivery system had a better inhibitory effect on cell proliferation than the free drug under single-drug treatment conditions.

[0114] Notably, the combined treatment groups (Dox+Cur and EVs-Dox+EVs-Cur) showed significantly enhanced red fluorescence and a markedly increased proportion of cell death, indicating that the combined use of doxorubicin and curcumin can produce a synergistic anti-tumor effect. Among them, the EVs-Dox+EVs-Cur group exhibited the most significant red fluorescence and the highest number of cell deaths, demonstrating that the extracellular vesicle-based combined drug delivery system has a significant advantage in enhancing drug synergy and improving tumor cell killing efficiency.

[0115] Example 9: Evaluation of the in vivo pharmacodynamics and safety of extracellular vesicle drug delivery formulations.

[0116] 1. Animal models

[0117] BALB / c nude mice (female, 4–6 weeks old) were subcutaneously inoculated with Caov3 cells (5 × 10⁻⁶ cells) on their right back. 6 / animal). When the tumor volume reached approximately 100 mm³, the tumors were randomly assigned to groups (n=6): saline, blank extracellular vesicles, free Dox (10 mg kg⁻¹), or Exo-Dox (Dox equivalent 10 mg kg⁻¹). The tumors were administered via tail vein injection every 3 days for a total of 5 times.

[0118] 2. Tumor-inhibiting effect

[0119] The long diameter (a) and short diameter (b) of the tumor were measured every 2 days using vernier calipers, and the volume was calculated as V = ab² / 2. On day 21, the mice were sacrificed, and the tumors were collected and weighed.

[0120] Results: The relative tumor volume in the Exo-Dox group was 0.31 ± 0.04, significantly lower than that in the free Dox group (0.57 ± 0.05) (p<0.01). Tumor weight inhibition rate: Exo-Dox 72.4%, free Dox 45.8%.

[0121] 3. In vivo distribution and real-time imaging

[0122] Exo-Dox was labeled with PKH67, and in vivo imaging of small animals was performed at 1, 4, 12, and 24 h after administration. The fluorescence intensity of Exo-Dox at the tumor site peaked at 4 h and persisted until 24 h; while free PKH67 rapidly diffused throughout the body, with weak signal at the tumor site. Ex vivo organ imaging further confirmed the tumor targeting of Exo-Dox (tumor / liver fluorescence ratio 3.8 ± 0.4 vs. free group 1.2 ± 0.2, p < 0.001).

[0123] 4. Toxic side effects

[0124] (1) Body weight: The body weight of mice in the Exo-Dox group decreased by <5%, and the body weight of mice in the free Dox group decreased by 15.2 ± 2.1% (p<0.01).

[0125] (2) Blood routine and biochemistry: WBC and PLT were significantly decreased in the free Dox group (p<0.05), while ALT and AST increased by about 2 times; there was no statistically significant difference between the Exo-Dox group and the control group.

[0126] (3) H&E staining: The free Dox group showed obvious hepatocyte vacuolar degeneration and renal tubular protein casts; the liver, kidney and heart tissue structures in the Exo-Dox group were basically normal.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pharmaceutical composition for inhibiting ovarian tumors, characterized in that, The drug composition comprises doxorubicin and / or curcumin and extracellular vesicles derived from human ovarian cancer cells Caov3.

2. The pharmaceutical composition of claim 1, wherein, When the drug composition is doxorubicin and extracellular vesicles derived from human ovarian cancer cells Caov3, the mass ratio of the extracellular vesicles derived from human ovarian cancer cells Caov3 to doxorubicin is 1-10:

1.

3. The pharmaceutical composition of claim 2, wherein, The mass ratio of the extracellular vesicles derived from human ovarian cancer cells Caov3 to doxorubicin is 5:

1.

4. The pharmaceutical composition of claim 1, wherein, When the drug composition is curcumin and extracellular vesicles derived from human ovarian cancer cells Caov3, the mass ratio of the extracellular vesicles derived from human ovarian cancer cells Caov3 to curcumin is 1-10:

1.

5. The pharmaceutical composition of claim 1, wherein, The mass ratio of the extracellular vesicles derived from human ovarian cancer cells Caov3 to curcumin is 2:

1.

6. The pharmaceutical composition of claim 1, wherein, When the drug composition is doxorubicin, curcumin and extracellular vesicles derived from human ovarian cancer cells Caov3, the concentration of doxorubicin is 0.35 μg / mL and the concentration of curcumin is 7.8 μg / mL.

7. A process for the preparation of a pharmaceutical composition as claimed in any one of claims 1 to 6, wherein, The method comprises the following steps: (1) mixing the doxorubicin and / or curcumin with the extracellular vesicles derived from human ovarian cancer cells Caov3, ultrasonicating, ice-bathing to obtain a mixed solution of Caov3-EVs and drugs; (2) placing the mixed solution of Caov3-EVs and drugs in a constant-temperature water bath to incubate; (3) after the water bath, blowing and mixing the prepared mixed solution, centrifuging in an ultracentrifuge tube, sucking out the liquid, resuspending the precipitate with PBS, and continuing to centrifuge until the drugs not loaded into the Caov3-EVs are completely removed; (4) sucking out the liquid in the ultracentrifuge tube, adding PBS into the tube, blowing the tube wall and bottom for multiple times, and collecting the resuspended extracellular vesicle drug-loaded preparation.

8. Use of the drug composition of any one of claims 1-6 in the preparation of a drug for preventing and / or treating ovarian tumors.

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