Ovarian cancer cell membrane modified co-loaded DOX / IND nanoparticles and preparation method thereof
By constructing liposomes co-loaded with DOX/IND using thin-film dispersion ultrasound and ammonium sulfate gradient methods, and then preparing ovarian cancer cell membranes using gradient centrifugation, the problems of residual immunogenicity, limited targeting efficiency, and high toxicity in ovarian cancer cell membrane modification in existing technologies have been solved, achieving highly efficient ovarian cancer treatment.
Patent Information
- Application Number
- CN202511592251.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-03
- Publication Date
- 2026-02-10
AI Technical Summary
Existing methods for modifying ovarian cancer cell membranes suffer from problems such as residual immunogenicity, limited targeting efficiency, and high toxicity, making them difficult to effectively treat ovarian cancer.
Liposomes co-loaded with DOX/IND were constructed using thin-film dispersion ultrasound and ammonium sulfate gradient methods. Ovarian cancer cell membranes were then prepared using gradient centrifugation. These membranes were then modified with DOX/IND nanoparticles to achieve simultaneous spatiotemporal release of both drugs, reducing the toxicity of DOX to normal tissues.
It improves drug encapsulation rate and targeting, enhances therapeutic effect, reduces toxicity to normal tissues, reverses immunosuppression in the tumor microenvironment, and provides a new approach for combination therapy of drug-resistant tumors such as ovarian cancer.
Smart Images
Figure CN121489906A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of ovarian cancer cell membrane modification, in particular to an ovarian cancer cell membrane modified co-loaded DOX / IND nanoparticle and a preparation method thereof. BACKGROUND
[0002] Ovarian cancer cell membrane refers to the biological structure composed of lipid bilayer, membrane protein and sugar complex on the surface of ovarian malignant tumor cells, and its functional abnormality is closely related to tumor progression and drug resistance. The specific definition includes the following key features: Lipid bilayer: rich in cholesterol and sphingomyelin, forming a dynamic flow barrier involved in signal transduction and drug resistance;Membrane protein: including transmembrane transporters (such as ABC / SLC family), receptors and adhesion molecules, regulating drug influx / efflux and intercellular communication.
[0003] Ovarian cancer cell membrane modification refers to extracting the natural membrane structure of ovarian cancer cells, wrapping it on the surface of synthetic nanoparticles (such as PLGA or mesoporous silica carriers), and forming a core-shell structure nanoparticle delivery system with biomimetic function.
[0004] The existing ovarian cancer cell membrane modification method has the following disadvantages: Immune residue: the extracted cell membrane may retain tumor-associated antigens, posing a potential risk of immune activation and possibly triggering non-specific inflammatory reactions; Limited targeting efficiency: dependent on the recognition of homologous tumor cell membrane proteins, the targeting efficiency may be insufficient for heterogeneous tumors or metastases; High toxicity: easily leading to the killing of normal cells in the heart and bone marrow. SUMMARY
[0005] (I) Technical problems solved In view of the shortcomings of the prior art, the present application provides an ovarian cancer cell membrane modified co-loaded DOX / IND nanoparticle and a preparation method thereof, which has the advantages of reversing tumor microenvironment immunosuppression, enhancing DOX efficacy, co-loaded nanoparticles achieving simultaneous release of double drugs, overcoming single drug resistance, and reducing the toxicity of DOX to normal tissues, solving the problems of immune residue, limited targeting efficiency and high toxicity of the existing ovarian cancer cell membrane modification method.
[0006] (II) Technical solutions In order to achieve the above-mentioned purposes of reversing tumor microenvironment immunosuppression, enhancing DOX efficacy, co-loaded nanoparticles achieving simultaneous release of double drugs, overcoming single drug resistance, and reducing the toxicity of DOX to normal tissues, the present application provides the following technical solutions: an ovarian cancer cell membrane modified co-loaded DOX / IND nanoparticle, comprising IND, DOX and ID8 cell membrane.
[0007] Preferably, the preparation method of the ovarian cancer cell membrane modified co-loaded DOX / IND nanoparticle comprises the following steps: Step one: preparation of DOX / IND@cmLPs, co-loaded DOX / IND liposomes are constructed by thin film dispersion ultrasonic method and ammonium sulfate gradient method; Step two: preparation of ovarian cancer cell membrane by gradient centrifugation method, the cell line used in this patent is mouse ovarian cancer ID8 cell line; Step three: preparation of ovarian cancer cell membrane fused co-loaded Indoximod and DOX liposomes (DOX / IND@cmLPs); Step four: characterization and detection verification of DOX / IND@cmLPs.
[0008] Preferably, the step one of preparing DOX / IND@cmLPs further comprises the following steps: Step 1: using a precision electronic balance, weigh 2.0 mg of IND, 4 mg of DSPE-PEG2000, 4 mg of cholesterol and 12 mg of HSPC in a clean glass bottle, add 15 mL of mixed solution of chloroform and methanol, and ultrasonically shake and dissolve in an ultrasonic water bath for half an hour; Step 2: after the solid is completely dissolved and the mixed liquid is uniformly dispersed, vacuum rotary evaporation is carried out at 40°C and 0.06kPa negative pressure for 15 minutes until the solvent is evaporated; Step 3: then, add 4 mL of 250 mM ammonium sulfate solution to hydrate the phospholipid membrane, ultrasonically shake for ten minutes in a water bath, and obtain a milky white suspension; Step 4: after the obtained suspension is ultrasonically shaken by an ultrasonic cell crusher at 20% power for 10 minutes (3 seconds on and 3 seconds off), it is filtered through 0.45 nm and 0.22 nm filter membranes respectively, and the liquid obtained after filtration is collected, which is the IND-loaded liposome (IND@LPs); Step 5: remove the unencapsulated ammonium sulfate in the liquid after dialysis for 3 hours using a dialysis bag with a molecular weight of 3.5KD; Step 6: under an oil bath at 55°C, add 1.25 mg / mL doxorubicin hydrochloride solution to the liquid obtained in the previous step, and stir on a magnetic stirrer under dark conditions for 15 minutes, then put the above mixture into a Merck ultrafiltration tube (with a molecular weight cutoff of 100K); Step 7: centrifuge at 7000 rpm for half an hour, add a certain amount of ultrapure water and continue to centrifuge at 7000 rpm for half an hour, to obtain co-loaded DOX / IND liposomes (DOX / IND@LPs), and collect the supernatant for subsequent determination of the encapsulation efficiency of IND and DOX.
[0009] Preferably, in step two, the ID8 cell line is one of 10 clonal lines established from late-passage C57BL / 6 mouse ovarian surface epithelial cells (MOSEC). Intraperitoneal injection of each of the 10 clonal lines into C57BL / 6 mice leads to the formation of peritoneal tumors and ascites. Among the 10 clonal lines, ID8 exhibits the highest tumor burden.
[0010] Preferably, the ID8 cell line is a highly published and well-characterized cell line that is commonly used as a syngeneic mouse model of ovarian cancer.
[0011] Preferably, in step two, the method for preparing the ovarian cancer cell membrane includes the following steps: Step (1): Mouse ovarian cancer cell line ID8 cells were cultured in 90% DMEM (1×, 4.5g / LD-Glucose) medium + 10% FBS + 1% (v / v) penicillin-streptomycin. All culture operations were performed in a clean bench and aseptic operation principles were followed. Step (2): After the cells have grown to the logarithmic growth phase, observe the cells under a microscope to ensure they are in good condition. Digest the cells with trypsin, wash the cells with 1×PBS (pH=7.4), and separate ID8 cells with EDTA (pH=7.4). Step (3): Then centrifuge all collected cells at 900 rpm for 5 minutes. Resuspend the precipitated cell particles and mix with 1 mL of lysis buffer (20 mmol / L Tris-HCl, 10 mmol / L KCl, 2 mmol / L MgCl2 and 1 tablet of EDTA-free protease inhibitor) for 2–3 minutes; Step (4) Then centrifuge the cells at 6000 rpm for 5 minutes to obtain the supernatant, and add another 250 μL of lysis buffer to resuspend the precipitate, and centrifuge the cells at 6000 rpm for 5 minutes to obtain the supernatant again; Step (5): Centrifuge the supernatant in an ultracentrifuge at 2.7×104 rpm for 20 minutes, and then collect the supernatant by centrifuging at 6.1×104 rpm for 10 minutes using the same apparatus, thus obtaining the ovarian cancer cell membrane.
[0012] Step (6): Examine the above-mentioned ovarian cancer cell membrane under an optical microscope and store the sample at -80°C.
[0013] Preferably, the cells used in this patent are free of mycoplasma infection.
[0014] Preferably, the cell culture chamber conditions are: 95% air, 5% carbon dioxide, and a temperature of 37°C.
[0015] Preferably, the preparation method of the liposomes co-loaded with Indoximod and DOX (DOX / IND@cmLPs) of ovarian cancer cell membrane fusion further includes: extruding the DOX / IND@LPs obtained above with ID8 cell membrane at a ratio of phospholipid content to cell membrane protein content of 200:1 using an Avanti liposome cell extruder through a 200nm filter membrane 20 times to obtain DOX / IND@cmLPs.
[0016] (III) Beneficial Effects Compared with the prior art, the present invention provides a method for preparing ovarian cancer cell membrane-modified co-loaded DOX / IND nanoparticles, which has the following beneficial effects: 1. This invention relates to membrane-modified DOX / IND nanoparticles for ovarian cancer cells and their preparation method. Liposomes co-loaded with DOX / IND were constructed using a thin-film dispersion ultrasonic method and an ammonium sulfate gradient method. This strategy, through process optimization, solved the technical bottleneck of DOX / IND co-loading and combined high encapsulation efficiency, synergistic efficacy, and intelligent release characteristics, providing a new approach for the combined treatment of drug-resistant tumors such as ovarian cancer.
[0017] 2. The method for preparing ovarian cancer cell membrane modified with co-loaded DOX / IND nanoparticles: The ovarian cancer cell membrane is prepared by gradient centrifugation, which makes the cell membrane more intact, the separation purity higher and the compatibility wider. Attached Figure Description
[0018] Fig. 1 This is a schematic diagram of the preparation method of ovarian cancer cell membrane-modified co-loaded DOX / IND nanoparticles according to the present invention; Fig. 2 This is a schematic diagram of the ovarian cancer cell membrane modification and co-loading of DOX / IND nanoparticles according to the present invention. Detailed Implementation
[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.
[0020] Please see Figs. 1-2 An ovarian cancer cell membrane-modified co-loaded DOX / IND nanoparticle, comprising IND, DOX, and ID8 cell membrane.
[0021] Example 1 A method for preparing ovarian cancer cell membrane-modified co-loaded DOX / IND nanoparticles includes the following steps: Step 1: Preparation of DOX / IND@cmLPs: Liposomes co-loaded with DOX / IND were constructed using thin-film dispersion sonication and ammonium sulfate gradient methods. Step 2: Ovarian cancer cell membranes are prepared using gradient centrifugation. The cell line used in this patent is the mouse ovarian cancer ID8 cell line. Step 3: Prepare liposomes co-loaded with Indoximod and DOX for ovarian cancer cell membrane fusion (DOX / IND@cmLPs); Step 4: Detect and verify the characterization of DOX / IND@cmLPs.
[0022] Example 2 Step one, the preparation of DOX / IND@cmLPs, also includes the following steps: Step 1: Using a precision electronic balance, weigh 2.0 mg IND, 4 mg DSPE-PEG2000, 4 mg cholesterol and 12 mg HSPC into a clean glass bottle, add 15 mL of a mixed solution of chloroform and methanol, and dissolve by ultrasonic vibration in an ultrasonic water bath for half an hour. Step 2: After the solid has completely dissolved and the mixed liquid has formed a uniformly dispersed solution, vacuum rotary evaporate at 40℃ and 0.06kPa negative pressure for 15 minutes until the solvent evaporates; Step 3: Subsequently, add 4 mL of 250 mM ammonium sulfate solution to hydrate the phospholipid membrane, sonicate in a water bath for ten minutes to obtain a milky white suspension; Step 4: The obtained suspension was ultrasonically disrupted at 20% power for 10 minutes (3 seconds on, 3 seconds off) using an ultrasonic cell disruptor. Then, it was filtered through 0.45nm and 0.22nm filter membranes, respectively. The filtered liquid was collected as IND-loaded liposomes (IND@LPs). Step 5: After dialysis for 3 hours using a dialysis bag with a molecular weight of 3.5KD, remove unencapsulated ammonium sulfate from the liquid; Step 6: In an oil bath at 55°C, add 1.25 mg / mL doxorubicin hydrochloride solution to the liquid obtained in the previous step, stir for 15 minutes on a magnetic stirrer under light-protected conditions, and then put the mixture into a Merck ultrafiltration tube (molecular weight cutoff of 100K). Step 7: Centrifuge at 7000 rpm for half an hour, add a certain amount of ultrapure water and continue centrifuging at 7000 rpm for half an hour to obtain liposomes co-loaded with DOX and IND (DOX / IND@LPs), and collect the supernatant for subsequent determination of IND and DOX loading rates.
[0023] In addition, in step two, the ID8 cell line is one of 10 clone lines established from late-passage C57BL / 6 mouse ovarian surface epithelial cells (MOSEC). Intraperitoneal injection of each of the 10 clone lines into C57BL / 6 mice resulted in peritoneal tumor and ascites formation. Among the 10 clone lines, ID8 showed the highest tumor burden.
[0024] It also includes the ID8 cell line, a highly published and well-characterized cell line commonly used as an syngeneic mouse model of ovarian cancer.
[0025] In this embodiment: 1. Advantages of the preparation process Thin-film dispersion ultrasound can form uniform small monolayer liposomes (30-100nm), improving drug encapsulation efficiency and enhancing tumor penetration.
[0026] The ammonium sulfate gradient method drives active drug loading of DOX through transmembrane pH / ion gradient, achieving an encapsulation efficiency of over 90%, which is significantly better than passive drug loading methods.
[0027] The combination of these two methods enables the simultaneous and efficient loading of DOX (weakly basic) and IND (weakly acidic) drugs, solving the technical challenge of traditional liposome co-loading of acidic and basic drugs.
[0028] 2. Synergistic effect of drugs DOX exerts its cytotoxic effect by inserting into DNA, while IND inhibits the COX-2 / PGE2 pathway and reverses the tumor immunosuppressive microenvironment. The two work synergistically to enhance the anti-tumor effect.
[0029] The sustained-release properties of liposomes can prolong the blood circulation time of DOX, reduce cardiotoxicity, and maintain the local anti-inflammatory concentration of IND.
[0030] 3. Innovative Design Dual-response release: The acidic internal aqueous phase (pH 4-5) formed by the ammonium sulfate gradient can trigger the dissolution and release of DOX, while IND is released through lipid bilayer diffusion, achieving spatiotemporal controlled release.
[0031] Targeted modification potential: The liposomes prepared by this method can be further coupled with targeting peptides (such as porogens) or tumor cell membranes to enhance active targeting.
[0032] Summarize This strategy overcomes the technical bottleneck of DOX / IND co-loading through process optimization, combining high encapsulation efficiency, synergistic efficacy, and intelligent release characteristics, providing a new approach for the combination therapy of drug-resistant tumors such as ovarian cancer. Example 3 Step two, the method for preparing ovarian cancer cell membranes includes the following steps: Step (1): Mouse ovarian cancer cell line ID8 cells were cultured in 90% DMEM (1×, 4.5g / LD-Glucose) medium + 10% FBS + 1% (v / v) penicillin-streptomycin. All culture operations were performed in a clean bench and aseptic operation principles were followed. Step (2): After the cells have grown to the logarithmic growth phase, observe the cells under a microscope to ensure they are in good condition. Digest the cells with trypsin, wash the cells with 1×PBS (pH=7.4), and separate ID8 cells with EDTA (pH=7.4). Step (3): Then centrifuge all collected cells at 900 rpm for 5 minutes. Resuspend the precipitated cell particles and mix with 1 mL of lysis buffer (20 mmol / L Tris-HCl, 10 mmol / L KCl, 2 mmol / L MgCl2 and 1 tablet of EDTA-free protease inhibitor) for 2–3 minutes; Step (4) Then centrifuge the cells at 6000 rpm for 5 minutes to obtain the supernatant, and add another 250 μL of lysis buffer to resuspend the precipitate, and centrifuge the cells at 6000 rpm for 5 minutes to obtain the supernatant again; Step (5): Centrifuge the supernatant in an ultracentrifuge at 2.7×104 rpm for 20 minutes, and then collect the supernatant by centrifuging at 6.1×104 rpm for 10 minutes using the same apparatus, thus obtaining the ovarian cancer cell membrane.
[0033] Step (6): Examine the above-mentioned ovarian cancer cell membrane under an optical microscope and store the sample at -80°C.
[0034] Specifically, the cells used in this patent are all free of mycoplasma infection.
[0035] In this embodiment, the cell culture incubator conditions are: 95% air, 5% carbon dioxide, and a temperature of 37°C.
[0036] In this embodiment: Technological advantages High-purity separation: Gradient centrifugation can effectively enrich cell membrane components, increasing HCG binding rate by 1750 times, which is significantly better than simple homogenization.
[0037] Structural integrity protection: The buffering effect of the gradient medium can avoid mechanical damage to membrane proteins caused by high-speed centrifugation and maintain the native conformation of membrane receptors.
[0038] Wide compatibility: Applicable to different samples (primary cells, cell lines), and can simultaneously isolate membrane-derived structures such as exosomes.
[0039] Example 4 The preparation method of liposomes co-loaded with Indoximod and DOX for ovarian cancer cell membrane fusion (DOX / IND@cmLPs) further includes: mixing the DOX / IND@LPs prepared above with ID8 cell membrane at a ratio of phospholipid content to cell membrane protein content of 200:1, and extruding them back and forth 20 times using an Avanti liposome cell extruder through a 200nm filter membrane to obtain DOX / IND@cmLPs.
[0040] Example 5 DOX / IND@cmLPs characterization The particle size of DOX / IND@cmLPs was measured to be 111.7±2.7 nm and the zeta potential was -22.4±4.00 mV using a particle size potentiometer.
[0041] The encapsulation efficiencies of DOX and IND in DOX / IND@cmLPs were determined by UPLC and UV spectrophotometry, respectively, and were 85.1%±3.4% and 23.9%±1.3%.
[0042] At pH 7.4 and 5.5, the DOX in DOX / IND@cmLPs was released at a relatively fast rate in the first 24 hours, followed by a slow and steady release.
[0043] Protein Coomassie Brilliant Blue staining and Western Blot experiments were performed on DOX / IND@cmLPs, which confirmed the successful cell membrane fusion modification.
[0044] CellTiter-Glo assays confirmed the good in vitro tumor-killing activity of DOX / IND@cmLPs. Flow cytometry, confocal microscopy, and small animal imaging experiments demonstrated that liposomes modified with ovarian cancer cell membrane fusion technology could enhance their targeting.
[0045] The expression of intracellular CRT, HMGB1, and ATP was significantly increased after treatment with DOX / IND@cmLPs, as detected by ELISA, demonstrating the ability of DOX / IND@cmLPs to induce ICD.
[0046] In vivo studies showed that DOX / IND@cmLPs significantly inhibited the growth of subcutaneous tumors in mice without significant toxicity.
[0047] Flow cytometry and immunofluorescence staining both indicated an increased ratio of CD8+ T cells and Treg cells in the tumor tissue, suggesting that DOX / IND@cmLPs may have reversed the local immunosuppression to some extent. Lower Ki67 expression and higher TUNEL staining positivity rates in tumor sections suggest that DOX / IND@cmLPs treatment can inhibit tumor proliferation and promote apoptosis.
[0048] Immunohistochemical detection of mTOR protein changes in tumors suggests that DOX / IND@cmLPs may exert an immunosuppressive effect by downregulating the mTOR pathway.
[0049] In summary, this method for preparing DOX / IND nanoparticles modified with ovarian cancer cell membranes utilizes a thin-film dispersion ultrasonic method and an ammonium sulfate gradient method to construct DOX / IND-coated liposomes. This strategy overcomes the technical bottleneck of DOX / IND co-loading through process optimization, achieving high encapsulation efficiency, synergistic efficacy, and intelligent release characteristics. It provides a new approach for the combined treatment of drug-resistant tumors such as ovarian cancer. Furthermore, the use of gradient centrifugation to prepare ovarian cancer cell membranes results in more intact cell membranes, higher separation purity, and better compatibility.
[0050] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes the element.
[0051] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. An ovarian cancer cell membrane-modified co-loaded DOX / IND nanoparticle, comprising IND, DOX, and ID8 cell membrane.
2. A method for preparing ovarian cancer cell membrane-modified co-loaded DOX / IND nanoparticles, comprising the ovarian cancer cell membrane-modified co-loaded DOX / IND nanoparticles of claim 1 and the method for preparing the same, characterized in that: Includes the following steps: Step 1: Preparation of DOX / IND@cmLPs: Liposomes co-loaded with DOX / IND were constructed using thin-film dispersion sonication and ammonium sulfate gradient methods. Step 2: Ovarian cancer cell membranes are prepared using gradient centrifugation. The cell line used in this patent is the mouse ovarian cancer ID8 cell line. Step 3: Prepare liposomes co-loaded with Indoximod and DOX for ovarian cancer cell membrane fusion (DOX / IND@cmLPs); Step 4: Detect and verify the characterization of DOX / IND@cmLPs.
3. The method for preparing ovarian cancer cell membrane-modified co-loaded DOX / IND nanoparticles according to claim 2, characterized in that: Step one, the preparation of DOX / IND@cmLPs, also includes the following steps: Step 1: Using a precision electronic balance, weigh 2.0 mg IND, 4 mg DSPE-PEG2000, 4 mg cholesterol and 12 mg HSPC into a clean glass bottle, add 15 mL of a mixed solution of chloroform and methanol, and dissolve by ultrasonic vibration in an ultrasonic water bath for half an hour. Step 2: After the solid has completely dissolved and the mixed liquid has formed a uniformly dispersed solution, vacuum rotary evaporate at 40℃ and 0.06kPa negative pressure for 15 minutes until the solvent evaporates; Step 3: Subsequently, add 4 mL of 250 mM ammonium sulfate solution to hydrate the phospholipid membrane, sonicate in a water bath for ten minutes to obtain a milky white suspension; Step 4: The obtained suspension was ultrasonically disrupted at 20% power for 10 minutes (3 seconds on, 3 seconds off) using an ultrasonic cell disruptor. Then, it was filtered through 0.45nm and 0.22nm filter membranes, respectively. The filtered liquid was collected as IND-loaded liposomes (IND@LPs). Step 5: After dialysis for 3 hours using a dialysis bag with a molecular weight of 3.5KD, remove unencapsulated ammonium sulfate from the liquid; Step 6: In an oil bath at 55°C, add 1.25 mg / mL doxorubicin hydrochloride solution to the liquid obtained in the previous step, stir for 15 minutes on a magnetic stirrer under light-protected conditions, and then put the mixture into a Merck ultrafiltration tube (molecular weight cutoff of 100K). Step 7: Centrifuge at 7000 rpm for half an hour, add a certain amount of ultrapure water and continue centrifuging at 7000 rpm for half an hour to obtain liposomes co-loaded with DOX and IND (DOX / IND@LPs), and collect the supernatant for subsequent determination of IND and DOX loading rates.
4. The method for preparing ovarian cancer cell membrane-modified co-loaded DOX / IND nanoparticles according to claim 2, characterized in that: In step two, the ID8 cell line is one of ten clone lines established from late-passage C57BL / 6 mouse ovarian surface epithelial cells (MOSEC). Intraperitoneal injection of each of the ten clone lines into C57BL / 6 mice leads to the formation of peritoneal tumors and ascites. Among the ten clone lines, ID8 exhibits the highest tumor burden.
5. The method for preparing ovarian cancer cell membrane-modified co-loaded DOX / IND nanoparticles according to claim 2, characterized in that: The ID8 cell line is a highly published and well-characterized cell line that is commonly used as an syngeneic mouse model of ovarian cancer.
6. The method for preparing ovarian cancer cell membrane-modified co-loaded DOX / IND nanoparticles according to claim 2, characterized in that: In step two, the method for preparing the ovarian cancer cell membrane includes the following steps: Step (1): Mouse ovarian cancer cell line ID8 cells were cultured in 90% DMEM (1×, 4.5g / LD-Glucose) medium + 10% FBS + 1% (v / v) penicillin-streptomycin. All culture operations were performed in a clean bench and aseptic operation principles were followed. Step (2): After the cells have grown to the logarithmic growth phase, observe the cells under a microscope to ensure they are in good condition. Digest the cells with trypsin, wash the cells with 1×PBS (pH=7.4), and separate ID8 cells with EDTA (pH=7.4). Step (3): Then centrifuge all collected cells at 900 rpm for 5 minutes. Resuspend the precipitated cell particles and mix with 1 mL of lysis buffer (20 mmol / L Tris-HCl, 10 mmol / L KCl, 2 mmol / L MgCl2 and 1 tablet of EDTA-free protease inhibitor) for 2–3 minutes; Step (4) Then centrifuge the cells at 6000 rpm for 5 minutes to obtain the supernatant, and add another 250 μL of lysis buffer to resuspend the precipitate, and centrifuge the cells at 6000 rpm for 5 minutes to obtain the supernatant again; Step (5): Centrifuge the supernatant in an ultracentrifuge at 2.7×104 rpm for 20 minutes, and then collect the supernatant by centrifuging at 6.1×104 rpm for 10 minutes using the same apparatus, thus obtaining the ovarian cancer cell membrane. Step (6): Examine the above-mentioned ovarian cancer cell membrane under an optical microscope and store the sample at -80°C.
7. The method for preparing ovarian cancer cell membrane-modified co-loaded DOX / IND nanoparticles according to claim 1, characterized in that: The cells used in this patent are all free of mycoplasma infection.
8. The method for preparing ovarian cancer cell membrane-modified co-loaded DOX / IND nanoparticles according to claim 2, characterized in that: The cell culture chamber conditions are: 95% air, 5% carbon dioxide, and a temperature of 37°C.
9. The method for preparing ovarian cancer cell membrane-modified co-loaded DOX / IND nanoparticles according to claim 2, characterized in that: The preparation method of the liposomes co-loaded with Indoximod and DOX (DOX / IND@cmLPs) for ovarian cancer cell membrane fusion further includes: extruding the DOX / IND@LPs obtained above with ID8 cell membrane at a ratio of phospholipid content to cell membrane protein content of 200:1 using an Avanti liposome cell extruder through a 200nm filter membrane 20 times to prepare DOX / IND@cmLPs.