A pharmaceutical preparation for reversing cisplatin resistance of ovarian cancer and a preparation method thereof

Abstract

The application belongs to the technical field of medicines, and particularly relates to a medicine preparation for reversing cisplatin drug resistance of ovarian cancer and a preparation method thereof; wherein the medicine preparation for reversing cisplatin drug resistance of ovarian cancer comprises the following raw materials in parts by weight: 5-15 parts of cisplatin and 80-100 parts of a lipid film; the synergistic effect of upstream and downstream pathway blockage formed by the SPHK1 inhibitor and the S1PR1 antagonist is combined with cisplatin, which directly enhances the killing of the chemotherapy drug on the drug-resistant cells, reduces the drug resistance by improving the tumor immune microenvironment through reducing the immunosuppressive macrophages, and realizes more efficient reversal of cisplatin drug resistance. A double-targeted delivery system is constructed in the lipid film: folate can specifically recognize the folate receptor highly expressed by the ovarian cancer cells, and mannose targets the mannose receptor on the surface of the M2 type macrophages, so that the drug is double-enriched in the tumor cells and the tumor microenvironment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicines, and particularly relates to a medicine preparation for reversing cisplatin resistance of ovarian cancer and a preparation method thereof. BACKGROUND

[0002] Cisplatin, as a classic platinum-based chemotherapy drug, has become an important part of the chemotherapy regimen for ovarian cancer and has been widely used in clinical treatment due to its mechanism of destroying the DNA structure of cancer cells and inhibiting the proliferation of cancer cells. However, with the progress of treatment, a serious problem gradually emerges-cisplatin resistance. The mechanism of cisplatin resistance is extremely complex and involves multiple aspects. For example, cancer cells may enhance their DNA damage repair ability to quickly repair the DNA damage caused by cisplatin, thereby escaping the killing effect of the drug; abnormal activation of the sphingosine-1-phosphate (S1P) signaling pathway: high expression of sphingosine kinase 1 (SPHK1) in tumor cells catalyzes the generation of a large amount of S1P, which is secreted by exosomes into the tumor microenvironment, binds to S1P receptor 1 (S1PR1) on the surface of macrophages, induces the polarization of macrophages to the immunosuppressive M2 type, and forms a vicious cycle, further weakening the anti-tumor effect of cisplatin.

[0003] Existing targeted preparations mostly use single targeting modification, only targeting tumor cells, ignoring the regulation of immunosuppressive cells in the tumor microenvironment, resulting in insufficient enrichment efficiency and depth of drug action in the lesion site. At the same time, there are problems such as low drug encapsulation rate, poor preparation stability, short in vivo circulation time, etc., which limit the clinical application. Therefore, developing a safe and effective drug preparation that can specifically reverse cisplatin resistance of ovarian cancer has become a key problem to be solved in the current field of ovarian cancer treatment. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application provides a medicine preparation for reversing cisplatin resistance of ovarian cancer and a preparation method thereof; the present application uses cisplatin as the chemotherapy basis, combines SPHK1 inhibitors and S1PR1 antagonists, and uses a lipid film containing HSPC, cholesterol, DSPE-PEG2000 and folate and mannose modified PEG lipids to play a role. PF-543 inhibits SPHK1 to reduce S1P generation, FTY720 blocks S1P-mediated M2 polarization of macrophages, and the two drugs cooperate with cisplatin to form a triple action of “pathway inhibition-immune regulation-chemotherapy killing” to reverse drug resistance; the folate and mannose modification of the lipid film constructs double-targeted delivery, improves drug lesion enrichment and reduces the toxic side effects on normal tissues; solves the problems of poor targeting of traditional chemotherapy and difficulty in reversing drug resistance, and provides an innovative solution for the clinical treatment of drug-resistant ovarian cancer.

[0005] The application provides a drug preparation for reversing cisplatin drug resistance of ovarian cancer, and the drug preparation for reversing cisplatin drug resistance of ovarian cancer comprises the following raw materials in parts by weight: 5-15 parts of cisplatin and 80-100 parts of a lipid film.

[0006] The lipid film comprises the following raw materials in a mass ratio: SPHK1 inhibitor (PF-543):S1PR1 antagonist (FTY720):HSPC (oxidized soybean lecithin):DSPE-PEG2000 (distearoylphosphatidyl ethanolamine-polyethylene glycol 2000):DSPE-PEG2000-FA (folic acid-polyethylene glycol 2000-distearoylphosphatidyl ethanolamine):DSPE-PEG2000-Man (mannose-polyethylene glycol 2000-distearoylphosphatidyl ethanolamine)=1-4:1-4:20-30:2.5:2.5:2:2.

[0007] The preparation method of the lipid film comprises the following steps:

[0008] A1, HSPC, cholesterol, DSPE-PEG2000, DSPE-PEG2000-FA, DSPE-PEG2000-Man, SPHK1 inhibitor (PF-543) and S1PR1 antagonist (FTY720) are weighed and dissolved in a mixed solvent of chloroform and methanol, the volume ratio of chloroform to methanol is 2:1, the volume ratio of the mixed solvent to the total solute is 5-25 mL:1 g, and vortex oscillation is performed to fully dissolve them;

[0009] A2, under the condition of a 50 ℃ water bath, an organic solvent is removed by using a rotary evaporator under reduced pressure, vacuum drying is performed to completely remove residual solvents, and the lipid film is obtained.

[0010] The application further provides a preparation method of the drug preparation for reversing cisplatin drug resistance of ovarian cancer, and the preparation method specifically comprises the following steps:

[0011] S1, 250 mM ammonium sulfate solution preheated at 50-65 ℃ is added to the lipid film, the pH is 4.0, the container is subjected to violent vortex oscillation in a 55 ℃ water bath for 1 h, and a multi-compartment liposome suspension is formed;

[0012] S2, the multi-compartment liposome suspension is subjected to polycarbonate membrane extrusion filtration by using a high-pressure extruder under the condition that the temperature is maintained at 50-65 ℃, and a single-compartment liposome suspension with uniform particle size distribution is obtained;

[0013] S3, the single-chambered liposome suspension is loaded into a dialysis bag with a molecular weight cut-off of 12-14 kDa, and dialysis is carried out in PBS (phosphate buffer) with a pH of 7.4 at 4 DEG C, and the dialysis solution is replaced every 2 h, and the dialysis is repeated multiple times, so that the outer water phase of the liposome is replaced by PBS, and a liposome suspension I with a neutral buffer as the outer water phase is obtained;

[0014] S4, cisplatin is added to the liposome suspension I, and incubation is carried out in a 55 DEG C water bath for 90 min in the dark, so that cisplatin is encapsulated in the inner water phase of the liposome by an active drug loading mechanism, and a liposome suspension II is obtained, and the liposome suspension II is loaded into a dialysis bag again, and dialysis is carried out in PBS with a pH of 7.4 at 2-8 DEG C overnight, so that free cisplatin that is not encapsulated is completely removed, and a purified liposome suspension is formed.

[0015] S5, a freeze-drying protective agent is added to the purified liposome suspension, and the mass percentage concentration is 3-7%, and after oscillation and mixing, the liposome suspension is divided into small portions, and pre-freezing is carried out at-80 DEG C for 4 h, and then the liposome suspension is transferred to a freeze-drying machine, and freeze-drying is carried out at-50 DEG C and a vacuum degree of less than 0.1 mbar for 24 h, and a drug preparation for reversing cisplatin resistance of ovarian cancer is obtained.

[0016] Further, the ratio of the use amount of the lipid film to the use amount of the ammonium sulfate solution is 1 g:5-15 mL.

[0017] Further, the average particle size of the single-chambered liposome suspension is between 80 nm and 120 nm.

[0018] Further, the freeze-drying protective agent is sucrose.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] The application adopts the synergistic effect of SPHK1 inhibitor PF-543 and S1PR1 antagonist FTY720 in blocking the upstream and downstream pathways: PF-543 inhibits SPHK1 to catalyze the generation of S1P from the source, reduces the content of S1P in tumor cells and exosomes; FTY720 competitively binds to S1PR1 on the surface of macrophages, blocks the M2-type polarization signal mediated by S1P, and the combination of the two and cisplatin not only directly enhances the killing of chemotherapeutic drugs on drug-resistant cells, but also reduces the drug resistance by improving the immune microenvironment of tumor through reducing immunosuppressive macrophages, to achieve more efficient reversal of cisplatin resistance. The introduction of DSPE-PEG2000-FA and DSPE-PEG2000-Man in the lipid film constructs a double-targeted delivery system: folic acid can specifically recognize the high expression of folic acid receptor on ovarian cancer cells, and mannose can target and bind to the mannose receptor on the surface of M2-type macrophages, so that the drug is double-enriched in tumor cells and tumor microenvironment. This precise delivery not only improves the concentration of cisplatin and pathway inhibitors at the lesion site, but also reduces the exposure to normal tissues, reduces the nephrotoxicity and systemic side effects of cisplatin, and solves the problem of insufficient targeting of traditional chemotherapeutic drugs. The active drug loading mechanism of the ammonium sulfate gradient method can efficiently load cisplatin, and cisplatin forms a stable precipitate in the aqueous phase of the liposome, reducing leakage during storage. The addition of lyophilization protectants further improves the long-term stability of the preparation, and after reconstitution, the structure integrity and drug activity of the liposome can still be maintained. In the preparation process of the application, the dialysis step controls the replacement of the outer phase to establish a transmembrane gradient to drive the active loading of cisplatin, and removes free ammonium sulfate to ensure the physiological compatibility of the preparation. It not only maintains the fluidity of the lipid membrane to facilitate drug loading, but also avoids the damage to the activity of the drug caused by high temperature. In summary, the application realizes the efficient reversal of cisplatin resistance of ovarian cancer by multi-target drug combination, double-targeted delivery system and optimized preparation process, and provides an innovative solution for the treatment of clinically drug-resistant tumors. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Schematic diagram of mRNA transcription level in platinum-resistant ovarian cancer cells after applying the drug preparation prepared by the application to reverse cisplatin resistance of ovarian cancer;

[0022] Figure 2 Staining result map of plate colony formation experiment. DETAILED DESCRIPTION

[0023] In order to enable the person skilled in the art to better understand the technical solutions of the present application, and to make the above-mentioned features, objects and advantages of the present application more clear and easy to understand, the present application will be further described below in conjunction with examples. The examples are only used to illustrate the present application and not to limit the scope of the present application.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present application. The exemplary methods and materials described herein are illustrative only and not intended to be limiting.

[0025] In the following examples, unless otherwise stated, conventional methods were used; in the following examples, unless otherwise stated, the materials used were commercially available new materials.

[0026] Example 1: The present example provides a drug preparation for reversing cisplatin resistance of ovarian cancer, the drug preparation for reversing cisplatin resistance of ovarian cancer comprises the following raw materials by weight: 5 parts of cisplatin, 80 parts of lipid film;

[0027] The lipid film comprises the following raw materials in mass ratio: SPHK1 inhibitor:S1PR1 antagonist:HSPC:cholesterol:DSPE-PEG2000:DSPE-PEG2000-FA:DSPE-PEG2000-Man=1:1:20:2.5:2.5:2:2;

[0028] The preparation method of the lipid film comprises the following steps:

[0029] A1, HSPC, cholesterol, DSPE-PEG2000, DSPE-PEG2000-FA, DSPE-PEG2000-Man, and SPHK1 inhibitor and S1PR1 antagonist are weighed and dissolved in a mixed solvent of chloroform and methanol, the volume ratio of chloroform to methanol is 2:1, the amount ratio of mixed solvent to total solute is 5 mL:1 g, and vortex oscillation is used to make it fully dissolved;

[0030] A2, under the condition of 50℃ water bath, rotary evaporator is used to remove organic solvent under reduced pressure, vacuum drying is used to completely remove residual solvent, and the lipid film is obtained.

[0031] The present example also provides a preparation method of a drug preparation for reversing cisplatin resistance of ovarian cancer, which specifically comprises the following steps:

[0032] S1, 250 mM ammonium sulfate solution preheated at 50℃ is added to the lipid film, the pH is 4.0, the amount ratio of lipid film to ammonium sulfate solution is 1 g:5 mL, vortex oscillation is performed in a 55℃ water bath for 1 h, and a multi-compartment liposome suspension is formed;

[0033] S2, the multi-chambered liposome suspension is extruded and filtered using a high-pressure extruder, sequentially flowing through polycarbonate membranes with pore sizes of 400 nm, 200 nm, and 100 nm, with the temperature maintained at 50 ℃, and the extrusion frequency of each pore size membrane being 10 times, to obtain a single-chambered liposome suspension with an average particle size of 102.3 nm;

[0034] S3, the single-chambered liposome suspension is placed in a dialysis bag with a molecular weight cut-off of 12 kDa, and dialyzed in PBS with a pH of 7.4 at 2 ℃, with the dialysis external solution being replaced every 2 h, for a total of 3 times, to replace the inner water phase of the liposome with PBS, to obtain a liposome suspension I with a neutral buffer as the outer water phase;

[0035] S4, cisplatin is added to the liposome suspension I, and incubated in a 55 ℃ water bath for 90 min in the dark, to allow cisplatin to be encapsulated in the inner water phase of the liposome by an active drug loading mechanism, to obtain a liposome suspension II, which is again placed in a dialysis bag and dialyzed in PBS with a pH of 7.4 at 2 ℃ overnight, to completely remove unencapsulated free cisplatin, to form a purified liposome suspension;

[0036] S5, sucrose is added to the purified liposome suspension to a mass percentage concentration of 3%, and after being mixed uniformly by oscillation, it is first pre-frozen at -80 ℃ for 4 h, and then transferred to a freeze-drying machine, and freeze-dried at -50 ℃ under a vacuum degree of less than 0.1 mbar for 24 h, to obtain a drug preparation for reversing cisplatin resistance of ovarian cancer.

[0037] Example 2: The present embodiment provides a drug preparation for reversing cisplatin resistance of ovarian cancer, which comprises the following raw materials by weight: 10 parts of cisplatin, and 90 parts of a lipid film;

[0038] The lipid film comprises the following raw materials in a mass ratio: SPHK1 inhibitor:S1PR1 antagonist:HSPC:cholesterol:DSPE-PEG2000:DSPE-PEG2000-FA:DSPE-PEG2000-Man=2:2.5:25:2.5:2.5:2:2;

[0039] The preparation method of the lipid film comprises the following steps:

[0040] A1, HSPC, cholesterol, DSPE-PEG2000, DSPE-PEG2000-FA, DSPE-PEG2000-Man, SPHK1 inhibitor, and S1PR1 antagonist are weighed, and dissolved in a mixed solvent of chloroform and methanol, with the volume ratio of chloroform to methanol being 2:1, and the use amount ratio of the mixed solvent to the total solute being 20 mL:1 g, and vortex oscillation is performed to fully dissolve them;

[0041] A2, under the condition of 50℃ water bath, the organic solvent was removed by rotary evaporator under reduced pressure, and the residue was dried under vacuum to completely remove the residual solvent to obtain a lipid film.

[0042] The embodiment also provides a preparation method of the drug preparation for reversing cisplatin resistance of ovarian cancer, and specifically includes the following steps:

[0043] S1, adding a 250 mM ammonium sulfate solution preheated at 55℃ to the lipid film, the pH is 4.0, the dosage ratio of the lipid film to the ammonium sulfate solution is 1 g:10 mL, and the multi-compartment liposome suspension is formed by vigorously vortexing in a 55℃ water bath for 1 h;

[0044] S2, the multi-compartment liposome suspension is filtered by a high-pressure extruder under the condition that the temperature is maintained at 50-65℃, and sequentially flows through polycarbonate membranes with pore sizes of 400 nm, 200 nm and 100 nm, the extrusion times of each pore size membrane are 10 to 20 times, and a single-compartment liposome suspension with an average particle size of 105.2 nm is obtained;

[0045] S3, the single-compartment liposome suspension is loaded into a dialysis bag with a molecular weight cut-off of 14 kDa, and is dialyzed in PBS with a pH of 7.4 at 4℃, and the dialysis external solution is replaced every 2 h, and the dialysis is performed for a total of 4 times, so that the outer water phase of the liposome is replaced by PBS, and a liposome suspension I with a neutral buffer as the outer water phase is obtained;

[0046] S4, adding cisplatin to the liposome suspension I, and incubating in a 55℃ water bath in the dark for 90 min, so that the cisplatin is encapsulated in the inner water phase of the liposome by an active drug loading mechanism, to obtain a liposome suspension II, and the liposome suspension II is loaded into a dialysis bag again, and is dialyzed in PBS with a pH of 7.4 at 4℃ overnight, so as to completely remove the unencapsulated free cisplatin, and form a purified liposome suspension;

[0047] S5, adding sucrose to the purified liposome suspension to make the mass percentage concentration of sucrose be 5%, and then the mixture is uniformly oscillated, pre-frozen for 4 h at-80℃, transferred to a freeze-drying machine, and freeze-dried at-50℃ under a vacuum degree lower than 0.1 mbar for 24 h, to obtain the drug preparation for reversing cisplatin resistance of ovarian cancer.

[0048] Embodiment 3: The embodiment provides a drug preparation for reversing cisplatin resistance of ovarian cancer, and the drug preparation for reversing cisplatin resistance of ovarian cancer includes the following raw materials by weight: 15 parts of cisplatin and 100 parts of a lipid film.

[0049] The lipid film comprises raw materials in the following mass ratio: SPHK1 inhibitor:S1PR1 antagonist:HSPC:cholesterol:DSPE-PEG2000:DSPE-PEG2000-FA:DSPE-PEG2000-Man = 4:4:30:2.5:2.5:2:2;

[0050] The preparation method of the lipid film comprises the following steps:

[0051] A1, HSPC, cholesterol, DSPE-PEG2000, DSPE-PEG2000-FA, DSPE-PEG2000-Man, and SPHK1 inhibitor and S1PR1 antagonist are weighed and dissolved in a mixed solvent of chloroform and methanol, the volume ratio of chloroform to methanol is 2:1, the amount ratio of the mixed solvent to the total solute is 25 mL:1 g, and vortex oscillation is used to fully dissolve them;

[0052] A2, under the condition of a 50 ℃ water bath, a rotary evaporator is used to evaporate and remove the organic solvent under reduced pressure, vacuum drying is performed to completely remove the residual solvent, and a lipid film is obtained.

[0053] The embodiment also provides a preparation method of a drug preparation for reversing cisplatin resistance of ovarian cancer, and specifically comprises the following steps:

[0054] S1, 250 mM ammonium sulfate solution preheated at 65 ℃ is added to the lipid film, the pH is 4.0, the amount ratio of the lipid film to the ammonium sulfate solution is 1 g:15 mL, vortex oscillation is performed at 55 ℃ in a water bath for 1 h, and a multi-compartment liposome suspension is formed;

[0055] S2, the multi-compartment liposome suspension is subjected to extrusion filtration through polycarbonate membranes with pore sizes of 400 nm, 200 nm and 100 nm in sequence using a high-pressure extruder under the condition that the temperature is maintained at 65 ℃, the extrusion times of each pore size membrane are 10 to 20 times, and a single-compartment liposome suspension with an average particle size of 112.5 nm is obtained;

[0056] S3, the single-compartment liposome suspension is loaded into a dialysis bag with a molecular weight cut-off of 14 kDa, dialysis is performed in PBS with a pH of 7.4 at 8 ℃, the dialysis external solution is replaced every 2 h, the dialysis is performed for 5 times, the outer water phase of the liposome is replaced with PBS, and a liposome suspension I with a neutral buffer as the outer water phase is obtained;

[0057] S4, cisplatin was added to the liposome suspension I, and the mixture was incubated in a 55 ℃ water bath for 90 min in the dark to allow cisplatin to be encapsulated in the inner water phase of the liposome by active drug loading mechanism, to obtain liposome suspension II, and the liposome suspension II was again loaded into a dialysis bag and dialyzed against PBS at pH 7.4 at 8 ℃ overnight to completely remove unencapsulated free cisplatin, to form a purified liposome suspension;

[0058] S5, sucrose was added to the purified liposome suspension to a mass percentage concentration of 7%, and the mixture was mixed and then pre-frozen at -80 ℃ for 4 h, transferred to a freeze-drying machine, and freeze-dried at -50 ℃ under a vacuum of less than 0.1 mbar for 24 h to obtain a drug preparation for reversing cisplatin resistance in ovarian cancer.

[0059] Comparative Example 1 differs from Example 2 in that no lipid film is added, and the rest is exactly the same as Example 2.

[0060] Comparative Example 2 differs from Example 2 in that no SPHK1 inhibitor and S1PR1 antagonist are added, and the rest is exactly the same as Example 2.

[0061] Comparative Example 3 differs from Example 2 in that a common cisplatin injection is used.

[0062] Experimental Example:

[0063] 1. Human ovarian cancer SKOV3 and A2780 cell lines were used as adherent-dependent growth characteristics, and all cell lines were cultured in RPMI1640 complete medium containing 10% FBS and 1% penicillin-streptomycin. The culture conditions were set to a 37℃ constant temperature environment and a 5% CO2 concentration humidified incubator, and the A2780 and SKOV3 cells were induced to construct the resistant strains ADDP (A2780 / DDP) and SDDP (SKOV3 / DDP) by using the cisplatin (Cisplatin, DDP) concentration gradient increasing method. The cell density was adjusted to 5×10 3Cells were seeded per well in 96-well plates and cultured at 37°C with 5% CO2 for 24 h. The drug formulations for reversing cisplatin resistance in ovarian cancer prepared in Examples 1-3 and Comparative Examples 1-3 of this invention were divided into groups: Example 1-3 and Comparative Examples 1-3 groups, and a control group (medium containing 0.1% DMSO). Each group had three replicates, and the cells were cultured for 48 h. Cell-level response was then tested. Fresh medium containing 10% CCK-8 reagent was prepared beforehand, the old medium was discarded, and 10 μl of CCK-8 working solution was added to each well. After incubation for 2 h, the OD value at 450 nm was measured, and the cell proliferation inhibition rate was calculated. The dose-response curve was fitted using GraphPad Prism, and the half-maximal inhibitory concentration (IC50), i.e., the drug concentration that causes 50% cell death, was calculated. The results are recorded in Table 1.

[0064] Apoptosis rate detection: Cells treated as described above were stained with Annexin V-FITC / PI and the apoptosis rate was detected by flow cytometry. The induction effect of the preparation on apoptosis of drug-resistant cells was analyzed. The apoptosis rate results are recorded in Table 1.

[0065] Table 1: Cellular Level Response Test Table

[0066]

[0067] Table 1 shows that the IC50 of ADDP / SDDP cells in Examples 1-3 was much lower than that in Comparative Examples 1-3, and the apoptosis rate in Example 2 reached 60.6%. This indicates that the lipid film prepared in this invention can significantly enhance the killing effect of cisplatin on drug-resistant cells, reduce IC50, and promote apoptosis. Compared with ordinary cisplatin, the drug preparation prepared in this invention for reversing cisplatin resistance in ovarian cancer can significantly enhance the effect on drug-resistant cells.

[0068] 2. Take SDDP cells in the logarithmic growth phase, digest them with trypsin, and adjust the density to 1×10⁻⁶. 7 Cells / mL, 0.2 mL of cell suspension was subcutaneously injected into the right back of each nude mouse, until the tumor volume reached 100 mm². 3 The experiment was conducted using the drug formulations for reversing cisplatin resistance in ovarian cancer prepared in Examples 1-3 and Comparative Examples 1-3 of this invention. Model mice were randomly divided into Example 1-3 groups, Comparative Example 1-3 groups, and a control group (saline group). The drugs were administered continuously for 3 weeks to conduct in vivo tumor suppression experiments. The long diameter (a) and short diameter (b) of the tumor were measured with calipers every 3 days, and the tumor volume (V=ab) was calculated. 2 / 2), at the end of the experiment, the mice were sacrificed, the tumor weight was weighed, and the tumor inhibition rate (%) was calculated as (average tumor weight of control group - average tumor weight of experimental group) / average tumor weight of control group × 100%. The calculation results are recorded in Table 2.

[0069] Table 2: In vivo tumor suppression experiment data

[0070]

[0071] The results of Table 2 show that the tumor volume and tumor weight of Examples 1-3 are significantly lower than those of the control group and Comparative Examples 1-3, and the tumor inhibition rate is 69.6%-74.4%, indicating that the drug preparation prepared by the application for reversing cisplatin resistance of ovarian cancer can effectively inhibit tumor growth, and the synergistic effect of the pathway inhibitor and the liposome can further enhance the tumor inhibition effect.

[0072] Figure 1 The relative expression level of SPHK1 gene mRNA of Examples 1-3 is shown, which is significantly lower than that of the control group, indicating that the drug preparation prepared by the application for reversing cisplatin resistance of ovarian cancer can effectively down-regulate the expression of SPHK1 gene and cut off the key signal source of drug resistance; Figure 2 The colony formation of platinum-resistant ovarian cancer cells in Example 2 is significantly reduced compared with the control group, and the colony formation ability is highly related to the proliferation activity of cancer cells, indicating that the drug preparation prepared by Example 2 of the application for reversing cisplatin resistance of ovarian cancer further inhibits cancer cell proliferation on the basis of down-regulating SPHK1 gene expression and blocking the drug resistance pathway, so that the proliferation ability of cancer cells is weakened.

[0073] In summary, through the synergistic effect of the lipid film and the inhibitor, the application achieves strong in vivo tumor inhibition, and the absence of key components or the use of ordinary dosage forms greatly reduces the tumor inhibition effect, highlighting the innovation and effectiveness of the preparation; in terms of raw materials, cisplatin is used as the basis for chemotherapy, and SPHK1 inhibitors and S1PR1 antagonists are used, and the film containing HSPC, cholesterol and various targeted PEG modified lipids is used to form a multi-target synergistic and double-target delivery system. In the preparation process, steps such as film formation, hydration, extrusion, dialysis, drug loading and freeze-drying are used to ensure uniform particle size, high encapsulation efficiency and good stability. Through precise matching of raw materials and optimization of preparation process, the application realizes the synergistic effect of "pathway blocking-immune regulation-targeted chemotherapy", breaks through the limitations of traditional drug resistance intervention, provides an innovative and efficient solution for cisplatin-resistant ovarian cancer treatment, and has clinical transformation potential and application value.

[0074] The above describes the application and its embodiments, which are not limiting, and the embodiments shown in the drawings are only one of the embodiments of the application, and the actual application is not limited thereto. In summary, if a person skilled in the art is inspired by it, without departing from the spirit of the application, similar ways and embodiments can be designed without creative design, which should belong to the protection scope of the application.

Claims

1. A pharmaceutical preparation for reversing cisplatin resistance in ovarian cancer, characterized by, The raw materials include cisplatin 5-15 parts by weight and a lipid film 80-100 parts by weight; The lipid film comprises raw materials in a mass ratio of SPHK1 inhibitor:S1PR1 antagonist:HSPC:cholesterol:DSPE-PEG2000:DSPE-PEG2000-FA:DSPE-PEG2000-Man = 1-4:1-4:20-30:2.5:2.5:2:2; The preparation method of the lipid film, The method comprises the following steps: A1, HSPC, cholesterol, DSPE-PEG2000, DSPE-PEG2000-FA, DSPE-PEG2000-Man, and SPHK1 inhibitor and S1PR1 antagonist are weighed and dissolved in a mixed solvent of chloroform and methanol, and vortexed to obtain a mixed solution; A2, under water bath condition, the mixed solution is evaporated under reduced pressure to form a lipid film; The SPHK1 inhibitor is PF-543, and the S1PR1 antagonist is FTY720.

2. The pharmaceutical preparation according to claim 1, wherein the pharmaceutical preparation is characterized by comprising the following components: (a) a platinum compound; (b) a polyamine compound; (c) a reducing agent; and (d) a carrier. In step A1, the volume ratio of chloroform to methanol is 2:1, and the amount ratio of the mixed solvent to total solute is 5-25 mL:1 g.

3. A method for preparing a pharmaceutical preparation for reversing cisplatin resistance in ovarian cancer according to any one of claims 1 to 2, characterized in that, The method specifically comprises the following steps: S1, ammonium sulfate solution is added to the lipid film, and vortexed to form a multi-compartment liposome suspension; S2, the multi-compartment liposome suspension is extruded and filtered to obtain a single-compartment liposome suspension; S3, the single-compartment liposome suspension is dialyzed to obtain a dialyzed lipid suspension I; S4, cisplatin is added to the lipid suspension I, and incubated in the dark to obtain a lipid suspension II, which is dialyzed again overnight to form a purified liposome suspension; S5, a freeze-drying protective agent is added to the purified liposome suspension, which is mixed uniformly, then divided and packaged, and vacuum freeze-dried to obtain a drug preparation for reversing cisplatin resistance of ovarian cancer.

4. The method of claim 3, wherein the preparation of the pharmaceutical preparation for reversing cisplatin resistance of ovarian cancer is characterized by, In step S1, the amount ratio of the lipid film to the ammonium sulfate solution is 1 g:5-15 mL.

5. The method of claim 3, wherein the preparation of the pharmaceutical formulation for reversing cisplatin resistance in ovarian cancer is characterized by, In step S3, the dialysis process is as follows: the single-compartment liposome suspension is loaded into a dialysis bag with a molecular weight cut-off of 12-14 kDa, and dialyzed in PBS with a pH of 7.4 at 2-8 ℃, and the dialysis external solution is replaced during the dialysis.

6. The method of claim 3, wherein the preparation of the pharmaceutical formulation for reversing cisplatin resistance in ovarian cancer is characterized by, In step S5, the freeze-drying protective agent is sucrose.

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