Pharmaceutical composition and method for enhancing chemotherapy immunogenicity of ovarian cancer

Abstract

The invention discloses a pharmaceutical composition and a method for enhancing chemotherapy immunogenicity of ovarian cancer, and relates to the technical field of medicines, the pharmaceutical composition comprises human serum albumin, paclitaxel and a histone deacetylase inhibitor; according to the pharmaceutical composition and the method for enhancing the chemotherapy immunogenicity of the ovarian cancer, human serum albumin is adopted as a stabilizer and a carrier, and nanoparticles with a specific ternary mass ratio (human serum albumin to paclitaxel to a histone deacetylase inhibitor is (8-15): 1: (0.1-0.3)) are prepared by using a high-pressure homogenization method; therefore, a delivery system which is stable in physicochemical property and can carry paclitaxel and immunomodulatory small molecules together is constructed. The specific mass ratio is combined with the preparation process, so that the technical problem that two drugs with different physicochemical properties are stably, efficiently and jointly entrapped in the same nanoparticle is effectively solved, the synergistic effect basis of the drugs in the delivery process is ensured, and a material guarantee is provided for subsequent enhancement of chemotherapy immunogenicity.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a pharmaceutical composition and method for enhancing the immunogenicity of chemotherapy for ovarian cancer. Background Technology

[0002] Ovarian cancer, as one of the gynecological malignancies with a poor prognosis, has always had a focus of clinical research in optimizing its treatment strategies. Chemotherapy, as the core of comprehensive treatment for ovarian cancer, depends not only on the direct cytotoxic effects of the drugs but also on its ability to effectively induce immunogenic cell death in tumor cells. Intracellular cytotoxicity (ICD) activates the host's anti-tumor immune response by releasing damage-related molecular patterns such as calreticulin, ATP, and HMGB1, thereby transforming "cold tumors" into "hot tumors" and enhancing the long-term efficacy of chemotherapy. However, in current technologies, the combined use of classic chemotherapeutic drugs such as paclitaxel with immunomodulators (such as histone deacetylase inhibitors) often results in poor stability of the co-delivery system and asynchronous drug release due to significant differences in the physicochemical properties of the drugs, making it difficult to achieve synergistic induction of ICD in the tumor microenvironment.

[0003] However, current nanodelivery systems face challenges in co-loading hydrophobic chemotherapeutic drugs (such as paclitaxel) and small molecule immunomodulators. The challenge lies in achieving efficient encapsulation, stable release, and simultaneous enrichment of both drugs within the same nanoparticle through innovative carrier materials and optimized preparation processes. Traditional methods either suffer from low encapsulation rates due to insufficient interaction between the carrier and the drug, or exhibit uneven nanoparticle size and excessively high polydispersity index due to rough preparation processes. This leads to drug leakage, uncontrolled in vivo distribution, and ultimately weakens the co-delivery system's ability to induce ICD in ovarian cancer cells, directly limiting the clinical translational potential of chemotherapy-immunotherapy combination therapy. Therefore, improvements are needed. Summary of the Invention

[0004] The purpose of this invention is to provide a pharmaceutical composition and method for enhancing the immunogenicity of chemotherapy for ovarian cancer, in order to solve the problem in the prior art that the co-delivery system of paclitaxel and immunomodulators (such as histone deacetylase inhibitors) is unstable and the drug release is asynchronous due to differences in physicochemical properties, which makes it impossible to efficiently and synergistically induce immunogenic cell death (ICD) in the tumor microenvironment.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a pharmaceutical composition for enhancing the immunogenicity of ovarian cancer chemotherapy, comprising: human serum albumin, paclitaxel, and histone deacetylase inhibitor;

[0006] The mass ratio of the human serum albumin, paclitaxel, and histone deacetylase inhibitor is (8-15):1:(0.1-0.3), and the pharmaceutical composition is a nanoparticle suspension with an average particle size of 100-160 nm.

[0007] Furthermore, the histone deacetylase inhibitor is vorinostat or pabistat.

[0008] Furthermore, the polydispersity index of the nanoparticle suspension is less than 0.18, and the Zeta potential is -10mV to -25mV.

[0009] A method for using a pharmaceutical composition to enhance the immunogenicity of ovarian cancer chemotherapy includes the following steps:

[0010] S1. Paclitaxel and histone deacetylase inhibitor are dissolved together in an organic solvent that is immiscible with water to form an organic phase;

[0011] S2. Dissolve human serum albumin in a buffer solution with a pH of 6.8-7.4 to form an aqueous phase;

[0012] S3. Under high-speed shear conditions, the organic phase is injected into the aqueous phase and emulsified to obtain the primary emulsion;

[0013] S4. Immediately transfer the colostrum into a high-pressure homogenizer and cycle it 3-8 times at a pressure of 10,000-25,000 psi.

[0014] S5. The homogeneous emulsion obtained in step S4 is subjected to vacuum distillation to completely remove the organic solvent, resulting in a nanoparticle suspension.

[0015] Further, in step S1, the organic solvent is dichloromethane, chloroform, or ethyl acetate.

[0016] Furthermore, in step S3, the high-speed shearing speed is 15,000-25,000 rpm, and the emulsification time is 2-5 minutes.

[0017] Furthermore, in step S2, the buffer aqueous solution also contains poloxamer 188 at a concentration of 0.2%–1.0% of human serum albumin.

[0018] Furthermore, after step S5, the method further includes a step of filtering the nanoparticle suspension through a 0.22 μm filter membrane for sterilization.

[0019] Compared with existing technologies, this invention provides a pharmaceutical composition and method for enhancing the immunogenicity of ovarian cancer chemotherapy. By using human serum albumin as a stabilizer and carrier, and employing a high-pressure homogenization method to prepare nanoparticles with a specific ternary mass ratio (human serum albumin: paclitaxel: histone deacetylase inhibitor = 8-15:1:0.1-0.3), a physicochemically stable delivery system capable of co-loading paclitaxel and immunomodulatory small molecules is constructed. This specific mass ratio, combined with the preparation process, effectively solves the technical challenge of stably and efficiently co-encapsulating two drugs with different physicochemical properties within the same nanoparticle, ensuring the synergistic effect of the drugs during delivery and providing material support for subsequent enhancement of chemotherapy immunogenicity.

[0020] By co-encapsulating paclitaxel and histone deacetylase inhibitors (such as vorinostat) in albumin nanoparticles in a specific ratio, the co-delivery and synergistic effect of the two active ingredients at the tumor site was achieved. In vitro experiments showed that, compared with single-drug nanoparticles and physical mixtures of the two drugs, the co-loaded nanoparticles significantly enhanced the induction of immunogenic cell death in ovarian cancer cells, specifically manifested as more efficient exposure of calreticulin, release of ATP and HMGB1 danger signaling molecules.

[0021] By designing a specific preparation method that includes high-speed shear emulsification and high-pressure homogenization cycling, and optimizing process parameters (such as shear speed, homogenization pressure and number of cycles), the resulting nanoparticles were ensured to have a uniform particle size distribution (polydispersity index less than 0.18) and high encapsulation efficiency (encapsulation efficiency of paclitaxel and vorinostat both greater than 85%). Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0023] Figure 1 This is a flowchart of a method provided in an embodiment of the present invention. Detailed Implementation

[0024] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0025] As attached Figure 1 As shown:

[0026] Example 1:

[0027] This invention provides a pharmaceutical composition and method for enhancing the immunogenicity of chemotherapy for ovarian cancer. The raw materials and reagents include: paclitaxel (purity ≥99.0%), vorinostat (purity ≥98.5%), human serum albumin (for injection), poloxamer 188 (pharmaceutical excipient grade), sodium dihydrogen phosphate (analytical grade), disodium hydrogen phosphate (analytical grade) for preparing buffer solutions, dichloromethane (chromatographic grade), and water for injection (compliant with the Chinese Pharmacopoeia).

[0028] S1. Preparation of buffer solution: Weigh 2.96 g of sodium dihydrogen phosphate and 29.0 g of disodium hydrogen phosphate, place them in a 1000 mL volumetric flask, add approximately 900 mL of water for injection, stir until completely dissolved, and dilute to the mark with water for injection. Mix well to obtain a 0.2 M phosphate buffer stock solution with a pH of approximately 7.0. Take 50 mL of this stock solution, dilute to 1000 mL with water for injection, mix well, and obtain a 0.01 M phosphate buffer solution with a pH of 7.0 for later use.

[0029] S2. Preparation of the organic phase: Accurately weigh 20.0 mg of paclitaxel and 4.0 mg of vorinostat, and place them together in a clean 10 mL glass bottle. Add 2.0 mL of dichloromethane to the bottle, gently shake and let stand at room temperature until the drugs are completely dissolved, resulting in a clear organic phase solution.

[0030] S3. Preparation of the aqueous phase: Accurately weigh 260.0 mg of human serum albumin and 2.6 mg of poloxamer 188, and place them in a 50 mL glass beaker. Measure 20 mL of the 0.01 M, pH 7.0 phosphate buffer prepared in step S1 and add it to the beaker. Stir the mixture on a magnetic stirrer at 500 rpm at room temperature for 30 minutes until the albumin and poloxamer 188 are completely dissolved, yielding a clear aqueous solution.

[0031] S4. Formation of the colostrum: Transfer the aqueous solution prepared in step S3 to the sample container of a high-speed shear disperser. While stirring, slowly and uniformly inject the organic phase solution prepared in step S2 into the aqueous solution using a disposable syringe. After all the organic phase has been added, immediately turn on the high-speed shear disperser and continuously shear at 20,000 rpm for 3 minutes. The entire process is carried out in an ice-water bath, yielding a milky white colostrum.

[0032] S5. High-Pressure Homogenization: Immediately transfer the colostrum obtained in step S4 to the feed tank of a high-pressure homogenizer. Set the homogenizer pressure to 18000 psi and perform circulating homogenization at the set pressure, repeating 5 times. The process is also carried out under ice-water bath cooling.

[0033] S6. Removal of Organic Solvents and Post-treatment: Transfer the emulsion obtained after homogenization in step S5 to a round-bottom flask and connect it to a rotary evaporator. Perform vacuum distillation at a water bath temperature of 35°C and a rotation speed of 60 rpm until no dichloromethane droplets flow in the flask and the volume no longer decreases, which takes about 20 minutes, to obtain a crude suspension of nanoparticles with obvious opalescence. Add the crude suspension to the initial aqueous phase volume (20 mL) with 0.01 M, pH 7.0 phosphate buffer and gently mix.

[0034] S7. Filtration and sterilization: The nanoparticle suspension obtained in step S6 is filtered using a 0.22μm polyethersulfone sterile filter membrane. The filtrate is collected in a sterile container to obtain the drug composition for enhancing the immunogenicity of ovarian cancer chemotherapy, denoted as nanoparticle suspension NP-PT / V.

[0035] In the nanoparticle suspension NP-PT / V prepared in this embodiment, the mass ratio of human serum albumin, paclitaxel and vorinostat is 13:1:0.2.

[0036] Example 2:

[0037] This embodiment is basically the same as the previous embodiment, except that the raw materials and reagents are as follows: the nanoparticle suspension NP-PT / V prepared in Example 1, sodium dihydrogen phosphate (analytical grade), disodium hydrogen phosphate (analytical grade) are used to prepare the buffer solution, water for injection conforms to the Chinese Pharmacopoeia standard, and ultrafiltration centrifuge tubes have a molecular weight cutoff of 10 kDa.

[0038] S1. Determination of Particle Size, Polydispersity Index, and Zeta Potential: An appropriate amount of the NP-PT / V nanoparticle suspension prepared in Example 1 was diluted 50-fold with 0.01M, pH 7.0 phosphate buffer to make the sample concentration suitable for instrument detection. The diluted sample was transferred to a dedicated sample cell and placed in a dynamic light scattering particle size analyzer. After equilibration at 25°C for 2 minutes, measurements were taken. Instrument parameters were set as follows: detection angle 173°, wavelength 633nm. Three consecutive measurements were performed, each lasting at least 60 seconds, and the average value was taken as the result. The measurements showed that the average particle size of the NP-PT / V nanoparticle suspension was 128nm, and the polydispersity index was 0.11. The same sample was transferred to the dedicated folded capillary electrophoresis cell of the Zeta potential analyzer and measured using phase analysis light scattering at the same temperature. Five consecutive measurements were performed, and the average value was taken as the result. The measurements showed that the Zeta potential of the NP-PT / V nanoparticle suspension was -18.2mV.

[0039] S2. Morphological observation by transmission electron microscopy: Take 20 μL of the NP-PT / V nanoparticle suspension and dilute it 10-fold with 0.01M, pH 7.0 phosphate buffer. Add 10 μL of the diluted sample to a special copper mesh (coated with a carbon support film) and let it stand for 1 minute to absorb the adsorption. Gently blot away excess liquid from the edge of the copper mesh with filter paper. Then, add 10 μL of phosphotungstic acid negative staining solution to the copper mesh, let it stand for 30 seconds to stain, and blot away excess staining solution again with filter paper. Place the treated copper mesh in a 50℃ oven to dry for 5 minutes. Install the dried copper mesh into the sample holder of a transmission electron microscope and observe it under an accelerating voltage of 80 kV. The electron microscopy images show that the nanoparticles are round or nearly round, uniformly distributed, without significant adhesion, and the particle size distribution is consistent with the dynamic light scattering results.

[0040] S3. Determination of encapsulation efficiency and drug loading:

[0041] Determination of total drug content: Accurately measure 0.5 mL of the NP-PT / V nanoparticle suspension and place it in a 10 mL volumetric flask. Add 8 mL of methanol and vortex for 5 minutes to completely destroy the nanoparticle structure and dissolve the drug. Dilute to the mark with methanol and mix well. Take an appropriate amount of this solution and filter it through a 0.22 μm organic phase filter membrane. Discard the initial filtrate and collect the subsequent filtrate as test solution A. Determine the concentrations of paclitaxel and vorinostat separately using high performance liquid chromatography (HPLC). Chromatographic conditions: C18 column, mobile phase of acetonitrile and 0.1% phosphoric acid aqueous solution, gradient elution, flow rate 1.0 mL / min, column temperature 30℃, detection wavelength of paclitaxel 227 nm and vorinostat 241 nm. Calculate the concentrations of paclitaxel and vorinostat in test solution A according to the standard curve, and convert them to the total content of the two drugs in 0.5 mL of suspension, denoted as Wtotal (PTX) and Wtotal (VOR), respectively.

[0042] Determination of free drug content: Accurately measure 1.0 mL of the NP-PT / V nanoparticle suspension and add it to a pre-treated ultrafiltration centrifuge tube (molecular weight cutoff 10 kDa). Centrifuge at 4℃ and 14000×g for 30 minutes. Collect the ultrafiltrate at the bottom of the tube and accurately measure its volume. Take an appropriate amount of the ultrafiltrate, directly or appropriately dilute it, and determine the concentrations of paclitaxel and vorinostat under the same high-performance liquid chromatography conditions as for (total drug content determination). Based on the standard curve and the volume of ultrafiltrate, calculate the content of free (i.e., unencapsulated) paclitaxel and vorinostat in 1.0 mL of suspension, denoted as W_free (PTX) and W_free (VOR), respectively.

[0043] Calculation: The encapsulation efficiency and drug loading are calculated using the following formulas.

[0044] Paclitaxel encapsulation efficiency = [(total (PTX) × 2 - free (PTX)) / (total (PTX) × 2)] × 100%;

[0045] Vorinostat encapsulation rate = [(total W(VOR) × 2 - free W(VOR)) / (total W(VOR) × 2)] × 100%;

[0046] Paclitaxel loading = (total (PTX) × 2 - free (PTX)) / dry weight of nanoparticles × 100%;

[0047] Vorinostat loading = (total VOR × 2 - free VOR) / dry weight of nanoparticles × 100%.

[0048] The dry weight of the nanoparticles was obtained by precisely measuring a certain volume of suspension, freeze-drying it, and then weighing it. Calculations showed that the encapsulation efficiency of paclitaxel in the NP-PT / V nanoparticle suspension was 92.5%, with a drug loading of 7.8%; the encapsulation efficiency of vorinostat was 88.7%, with a drug loading of 1.6%.

[0049] Example 3:

[0050] This embodiment is basically the same as the previous embodiment, except that the raw materials and reagents are as follows: Paclitaxel: purity ≥99.0%, Vorinostat: purity ≥98.5%, human serum albumin: for injection, Poloxamer 188: pharmaceutical excipient grade, Sodium dihydrogen phosphate, analytical grade, Disodium hydrogen phosphate, analytical grade, used to prepare buffer solution, Dichloromethane: chromatographic grade, Water for injection: conforms to the standards of the Chinese Pharmacopoeia, RPMI-1640 medium, fetal bovine serum, penicillin-streptomycin double antibody solution, trypsin-EDTA digestion solution, SKOV-3 human ovarian cancer cell line, calreticulin primary antibody, FITC-labeled secondary antibody, ATP detection kit, HMGB1 ELISA detection kit, 96-well cell culture plate, 6-well cell culture plate, flow cytometry tubes.

[0051] S1. Preparation of experimental sample solutions:

[0052] Preparation and dilution of single paclitaxel albumin nanoparticles (NP-PTX): Following the method and formulation of Example 1, NP-PTX suspension was prepared using only paclitaxel (20.0 mg) and human serum albumin (260.0 mg), without the addition of vorinostat. The suspension was then diluted with complete culture medium to a working solution with a paclitaxel concentration of 10 μM.

[0053] Preparation of Vorinostat single solution (VOR-Sol): Accurately weigh 0.355 mg of vorinostat, dissolve it in 10 μL of dimethyl sulfoxide, vortex to completely dissolve, add 990 μL of complete culture medium to prepare a 200 μM stock solution, and then dilute it with complete culture medium to a 2 μM vorinostat concentration for use as a working solution.

[0054] Preparation of the physical mixture (NP-PTX+VOR-Sol) working solution: Take an equal volume of the NP-PTX standby working solution prepared in the single step of preparing and diluting paclitaxel albumin nanoparticles (NP-PTX) and the VOR-Sol standby working solution prepared in the single step of preparing vorinostat solution (VOR-Sol), so that the final concentration of paclitaxel in the mixture is 10 μM and the final concentration of vorinostat is 2 μM. Prepare fresh each time you use it.

[0055] Preparation of working solution for co-loaded nanoparticles (NP-PT / V): Take the nanoparticle suspension NP-PT / V prepared in Example 1 and dilute it with complete culture medium to make the concentration of paclitaxel 10 μM and vorinostat 2 μM, as the backup working solution.

[0056] Blank control: using complete culture medium.

[0057] S2. Cell Culture and Grouping: SKOV-3 cells were cultured routinely in an incubator at 37°C and 5% CO2 using RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics. Cells in the logarithmic growth phase were digested, centrifuged, resuspended, counted, and then seeded at an appropriate density in culture plates.

[0058] Calcium reticulin exposure assay grouping: Cells were seeded in 6-well plates at a density of 3 × 10⁶ cells per well, 2 mL per well. 5 Cells were cultured for 24 hours to allow cell adhesion. Five groups were set up: a blank control group (2 mL of complete culture medium), an NP-PTX group (2 mL of NP-PTX working solution), a VOR-Sol group (2 mL of VOR-Sol working solution), a physical mixture group (2 mL of NP-PTX + VOR-Sol working solution), and a group containing the co-loaded nanoparticles of this invention (2 mL of NP-PT / V working solution). Each group had three replicates. The culture plates were returned to the incubator and cultured for another 24 hours.

[0059] ATP release assay grouping: Cells were seeded in 96-well white opaque culture plates, 100 μL per well, at a density of 1 × 10⁶ cells / well. 4 Cells were cultured for 24 hours. Grouping was performed using the same steps as for calreticulin exposure detection, with 6 replicates per group. Each well was replaced with 100 μL of the corresponding drug-containing working solution or complete culture medium, and cultured for another 24 hours.

[0060] HMGB1 release assay group: Cells were seeded in 6-well plates, and the same procedure as the calreticulin exposure assay group was followed. After culturing for 24 hours, the medium was replaced with the corresponding drug-containing working solution or complete medium, and cultured for another 48 hours.

[0061] S3. Flow cytometry detection of calreticulin exposure: After 24 hours of treatment, collect the cell supernatant from each well of the calreticulin exposure detection group into centrifuge tubes for later use. Gently wash the cells in the wells twice with pre-chilled PBS, add EDTA-free trypsin digestion solution to digest the cells, stop digestion with the corresponding supernatant collected in the previous step, and pipette to form a single-cell suspension. Transfer to flow cytometry tubes, centrifuge at 300×g for 5 minutes at 4°C, and discard the supernatant. Wash the cell pellet once with pre-chilled PBS, centrifuge, and discard the supernatant. Add 100 μL of PBS containing 1% bovine serum albumin to each tube to resuspend the cells, add calreticulin primary antibody, and incubate at 4°C in the dark for 30 minutes. Wash twice with PBS, centrifuge, and discard the supernatant. Add 100 μL of PBS containing 1% bovine serum albumin to each tube to resuspend the cells, add FITC-labeled secondary antibody, and incubate at 4°C in the dark for 30 minutes. Wash twice with PBS, centrifuge, and discard the supernatant. Finally, resuspend the cells in 300 μL of PBS and immediately perform flow cytometry detection. The percentage of calreticulin-positive cells in each group was recorded. The results showed that the percentage of calreticulin-positive cells in the co-loaded nanoparticle group was significantly higher than that in the NP-PTX group, VOR-Sol group, and physical mixture group.

[0062] S4. Detection of extracellular ATP release: After 24 hours of treatment, the 96-well plate from the ATP release detection group was removed from the incubator and allowed to equilibrate at room temperature for 10 minutes. Following the ATP detection kit instructions, the detection substrate and buffer were mixed in the correct proportions to prepare the working solution. 100 μL of the ATP detection working solution was added to each well of the culture plate, and the plate was incubated at room temperature in the dark for 5 minutes. The luminescence value of each well was measured using a chemiluminescence detector. The absolute concentration of ATP in the culture medium of each well was calculated based on the standard curve. The results showed that the ATP concentration in the culture medium of the co-loaded nanoparticle group of this invention was significantly higher than that of the NP-PTX group, the VOR-Sol group, and the physical mixture group.

[0063] S5. Detection of extracellular HMGB1 release: After 48 hours of treatment, the cell supernatant from each well in the HMGB1 release detection group was collected and centrifuged at 1000×g for 10 minutes at 4°C to remove cell debris. The supernatant was carefully aspirated as the test sample. The procedure was performed according to the HMGB1 ELISA kit instructions: Standards and test samples were added sequentially to the antibody-coated wells of the ELISA plate, and incubated at room temperature for 2 hours; after washing, biotinylated detection antibody was added, and incubated at room temperature for 1 hour; after washing again, horseradish peroxidase-labeled streptavidin was added, and incubated at room temperature for 30 minutes; after washing, substrate chromogenic solution was added, and incubated at room temperature in the dark for 15 minutes; stop solution was added, and the absorbance of each well was immediately measured at 450 nm using an ELISA reader. The concentration of HMGB1 in each sample was calculated based on the standard curve. The results showed that the HMGB1 concentration in the supernatant of the co-loaded nanoparticle group was significantly higher than that of the NP-PTX group, VOR-Sol group, and physical mixture group.

[0064] Comparative Example 1:

[0065] Raw materials and reagents: Paclitaxel: purity ≥99.0%, Vorinostat: purity ≥98.5%, Human serum albumin: for injection, Poloxamer 188: pharmaceutical excipient grade, Sodium dihydrogen phosphate, analytical grade, Disodium hydrogen phosphate, analytical grade, used for preparing buffer solution, Dichloromethane: chromatographic grade, Water for injection: conforms to the standards of the Chinese Pharmacopoeia.

[0066] S1. Preparation of buffer solution: Weigh 2.96 g of sodium dihydrogen phosphate and 29.0 g of disodium hydrogen phosphate, place them in a 1000 mL volumetric flask, add approximately 900 mL of water for injection, stir until completely dissolved, and dilute to the mark with water for injection. Mix well to obtain a 0.2 M phosphate buffer stock solution with a pH of approximately 7.0. Take 50 mL of this stock solution, dilute to 1000 mL with water for injection, mix well, and obtain a 0.01 M phosphate buffer solution with a pH of 7.0 for later use.

[0067] S2. Preparation of the organic phase: Accurately weigh 20.0 mg of paclitaxel and 8.0 mg of vorinostat, and place them together in a clean 10 mL glass bottle. Add 2.0 mL of dichloromethane to the bottle, gently shake and let stand at room temperature until the drugs are completely dissolved, resulting in a clear organic phase solution.

[0068] S3. Preparation of the aqueous phase: Accurately weigh 100.0 mg of human serum albumin and 1.0 mg of poloxamer 188 and place them in a 50 mL glass beaker. Measure 20 mL of the 0.01 M, pH 7.0 phosphate buffer prepared in step S1 and add it to the beaker. Stir the mixture on a magnetic stirrer at 500 rpm at room temperature for 30 minutes until the albumin and poloxamer 188 are completely dissolved, yielding a clear aqueous solution.

[0069] S4. Formation of the colostrum: Transfer the aqueous solution prepared in step S3 to the sample container of a high-speed shear disperser. While stirring, slowly and uniformly inject the organic phase solution prepared in step S2 into the aqueous solution using a disposable syringe. After all the organic phase has been added, immediately turn on the high-speed shear disperser and continuously shear at 20,000 rpm for 3 minutes. The entire process is carried out in an ice-water bath, yielding a milky white colostrum.

[0070] S5. High-Pressure Homogenization: Immediately transfer the colostrum obtained in step S4 to the feed tank of a high-pressure homogenizer. Set the homogenizer pressure to 18000 psi and perform circulating homogenization at the set pressure, repeating 5 times. The process is also carried out under ice-water bath cooling.

[0071] S6. Removal of Organic Solvents and Post-treatment: Transfer the emulsion obtained after homogenization in step S5 to a round-bottom flask and connect it to a rotary evaporator. Perform vacuum distillation at a water bath temperature of 35°C and a rotation speed of 60 rpm until no dichloromethane droplets flow in the flask and the volume no longer decreases, which takes about 20 minutes, to obtain a crude suspension of nanoparticles with obvious opalescence. Add the crude suspension to the initial aqueous phase volume (20 mL) with 0.01 M, pH 7.0 phosphate buffer and gently shake to obtain the control sample suspension, designated as control sample DC-1.

[0072] S7. Observation of Sample Stability: 2 mL of the nanoparticle suspension NP-PT / V prepared in Example 1 and the control sample DC-1 were placed in 5 mL transparent glass sample bottles and allowed to stand upright at room temperature. The initial state and the changes in sample appearance after 1 hour, 3 hours, and 6 hours of standing were observed and recorded. The nanoparticle suspension NP-PT / V maintained a uniform opalescence within 6 hours, with no obvious precipitation or stratification. After 1 hour of standing, a small amount of white flocculent matter was visible at the bottom of the bottle for the control sample DC-1; after 3 hours of standing, the flocculent precipitate increased, and the clarity of the upper liquid increased; after 6 hours of standing, obvious stratification occurred, with a clear upper layer and a dense precipitate at the bottom, which was difficult to disperse after gentle shaking.

[0073] S8. Particle size and polydispersity index determination: Take an appropriate amount of the control sample DC-1, which was shaken well before standing, and dilute it 50 times with 0.01M, pH 7.0 phosphate buffer. Place it in a dynamic light scattering particle size analyzer and measure it at 25°C using the same parameters and methods as in step S1 of Example 2. The measurement showed that the average particle size of the control sample DC-1 was 324 nm and the polydispersity index was 0.38.

[0074] Comparative Example 2:

[0075] Raw materials and reagents: Paclitaxel: purity ≥99.0%, Vorinostat: purity ≥98.5%, Human serum albumin: for injection, Poloxamer 188: pharmaceutical excipient grade, Sodium dihydrogen phosphate, analytical grade, Disodium hydrogen phosphate, analytical grade, used for preparing buffer solution, Dichloromethane: chromatographic grade, Water for injection: conforms to the Chinese Pharmacopoeia standard, Ultrafiltration centrifuge tubes: molecular weight cutoff 10 kDa.

[0076] S1. Preparation of buffer solution: Weigh 2.96 g of sodium dihydrogen phosphate and 29.0 g of disodium hydrogen phosphate, place them in a 1000 mL volumetric flask, add approximately 900 mL of water for injection, stir until completely dissolved, and dilute to the mark with water for injection. Mix well to obtain a 0.2 M phosphate buffer stock solution with a pH of approximately 7.0. Take 50 mL of this stock solution, dilute to 1000 mL with water for injection, mix well, and obtain a 0.01 M phosphate buffer solution with a pH of 7.0 for later use.

[0077] S2. Preparation of the organic phase: Accurately weigh 20.0 mg of paclitaxel and 4.0 mg of vorinostat, and place them together in a clean 10 mL glass bottle. Add 2.0 mL of dichloromethane to the bottle, gently shake and let stand at room temperature until the drugs are completely dissolved, resulting in a clear organic phase solution.

[0078] S3. Preparation of the aqueous phase: Accurately weigh 260.0 mg of human serum albumin and 2.6 mg of poloxamer 188 and place them in a 50 mL glass beaker. Measure 20 mL of the 0.01 M, pH 7.0 phosphate buffer prepared in step S1 and add it to the beaker. Stir the mixture on a magnetic stirrer at 500 rpm at room temperature for 30 minutes until the albumin and poloxamer 188 are completely dissolved, yielding a clear aqueous solution.

[0079] S4. High-speed shear emulsification to form a crude emulsion: Transfer the aqueous solution prepared in step S3 to the sample container of a high-speed shear disperser. While stirring, slowly and uniformly inject the organic phase solution prepared in step S2 into the aqueous solution using a disposable syringe. After all the organic phase has been added, immediately turn on the high-speed shear disperser and continuously shear at 25,000 rpm for 5 minutes. The entire process is carried out in an ice-water bath, yielding a milky white crude emulsion. This crude emulsion is directly labeled as control sample DC-2 and no further high-pressure homogenization is performed.

[0080] S5. Observation of Sample Appearance and Physical Stability: 2 mL of the nanoparticle suspension NP-PT / V prepared in Example 1 and the control sample DC-2 were placed in 5 mL transparent glass sample bottles and allowed to stand upright at room temperature. The initial state and changes in sample appearance after 30 minutes of standing were observed and recorded. The nanoparticle suspension NP-PT / V remained uniformly opalescent without change. The control sample DC-2 showed obvious opalescence initially, but was slightly turbid; after 30 minutes of standing, visible oil droplets appeared in the bottle, and a small amount of flocculent aggregates began to appear at the bottom, indicating that the system was unstable.

[0081] S6. Particle size and polydispersity index determination: Take an appropriate amount of the control sample DC-2, which was shaken well before standing, and dilute it 50 times with 0.01M, pH 7.0 phosphate buffer. Place it in a dynamic light scattering particle size analyzer and measure it at 25°C using the same parameters and methods as in step S1 of Example 2. The measurement showed that the average particle size of the control sample DC-2 was 265 nm, and the polydispersity index was 0.29.

[0082] S7. Encapsulation efficiency determination: The encapsulation efficiency of paclitaxel and vorinostat in the comparative sample DC-2 was determined according to the method in step S3 of Example 2.

[0083] Determination of total drug content: Accurately measure 0.5 mL of the crude emulsion of the control sample DC-2 and place it in a 10 mL volumetric flask. Add 8 mL of methanol, vortex for 5 minutes, and dilute to the mark with methanol. Take an appropriate amount of this solution, filter it through a 0.22 μm organic phase filter membrane, discard the initial filtrate, and collect the subsequent filtrate as the test solution. Determine the total content of paclitaxel and vorinostat using the same high-performance liquid chromatography method as in Example 2.

[0084] Determination of free drug content: Accurately measure 1.0 mL of the crude emulsion of the control sample DC-2 and add it to a pre-treated ultrafiltration centrifuge tube. Centrifuge at 4℃ and 14000×g for 30 minutes. Collect the ultrafiltrate and determine the concentration of free paclitaxel and vorinostat using high performance liquid chromatography.

[0085] Calculation: The encapsulation efficiency was calculated according to the formula in step S3 of Example 2. The calculated encapsulation efficiency of paclitaxel in the control sample DC-2 was 71.3%, and that of vorinostat was 65.8%.

[0086] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A pharmaceutical composition for enhancing immunogenicity of chemotherapy of ovarian cancer, characterized by, The pharmaceutical composition comprises: human serum albumin, paclitaxel and histone deacetylase inhibitor; the mass ratio of the three is (8-15):1:(0.1-0.3), and the pharmaceutical composition is a nanoparticle suspension with an average particle size of 100-160 nm.

2. The pharmaceutical composition for enhancing immunogenicity of chemotherapy for ovarian cancer according to claim 1, wherein The histone deacetylase inhibitor is vorinostat or pracinostat.

3. The pharmaceutical composition for enhancing immunogenicity of chemotherapy for ovarian cancer according to claim 1, wherein The nanoparticle suspension has a polydispersity index less than 0.18 and a Zeta potential of -10 mV to -25 mV.

4. A method for enhancing the immunogenicity of chemotherapy for ovarian cancer, suitable for a pharmaceutical composition for enhancing the immunogenicity of chemotherapy for ovarian cancer according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: S1. Dissolving paclitaxel and histone deacetylase inhibitor in a water-immiscible organic solvent to form an organic phase; S2. Dissolving human serum albumin in a buffer aqueous solution with a pH value of 6.8-7.4 to form an aqueous phase; S3. Under high-speed shearing conditions, injecting the organic phase into the aqueous phase to emulsify, obtaining a primary emulsion; S4. Immediately transferring the primary emulsion into a high-pressure homogenizer and processing it under a pressure of 10,000-25,000 psi for 3-8 cycles; S5. Distilling the homogeneous emulsion obtained in step S4 under reduced pressure to completely remove the organic solvent, obtaining a nanoparticle suspension.

5. The method of claim 4, wherein the pharmaceutical composition is administered in combination with a chemotherapeutic agent. In step S1, the organic solvent is dichloromethane, chloroform or ethyl acetate.

6. The method of claim 4, wherein the pharmaceutical composition is administered in combination with a chemotherapeutic agent. In step S3, the rotation speed of high-speed shearing is 15,000-25,000 rpm, and the emulsification time is 2-5 minutes.

7. The method for enhancing immunogenicity of chemotherapy for ovarian cancer according to claim 4, wherein, In step S2, the buffer aqueous solution further comprises 0.2%-1.0% poloxamer 188 by mass of human serum albumin.

8. The method for enhancing immunogenicity of chemotherapy for ovarian cancer according to claim 4, wherein, After step S5, the method further comprises a step of filtering the nanoparticle suspension through a 0.22 μm filter membrane to sterilize.

Images