Preparation and application of STING agonist bionic nano delivery system based on ferroptosis cell membrane

Abstract

The invention belongs to the field of medical biology, and relates to an STING agonist bionic nano delivery system and a preparation method and application thereof. The bionic nanoparticle containing the STING agonist comprises a shell, a drug carrier and an active component, the shell wraps the drug carrier, the drug carrier loads the active component, and the shell is a tumor cell membrane after ferroptosis induction; wherein the drug carrier is PLGA; wherein the active component is an STING agonist, and the STING agonist comprises a small molecule non-CDN (Content Delivery Network) type STING agonist; the bionic nano-particles are prepared by adopting a nano-precipitation method. The problem that an existing small molecule non-CDN STING agonist drug delivery system is poor in selectivity is solved.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and biology, and relates to a STING agonist bionic nano delivery system based on ferroptotic cell membranes, and a preparation method and application thereof. Background Art

[0002] Poly(lactic-co-glycolic acid) (PLGA) is a degradable functional polymer organic compound with good biocompatibility and non-toxicity, so it is widely used as a drug carrier. Ferroptosis is a new immunogenic cell death form that is different from apoptosis, necrosis and autophagy, and ferroptosis cells can activate the immune system in vivo and in vitro, causing a similar effect to vaccination. Therefore, the cell membrane after ferroptosis can be used to biomimetic modify PLGA nanoparticles.

[0003] Non-CDN STING agonist drugs have inherent disadvantages: (1) poor solubility, which makes administration difficult; (2) poor tumor targeting and toxicity, which greatly reduce their therapeutic efficacy.

[0004] Disadvantages of previous drug delivery systems: (1) low drug loading and encapsulation efficiency; (2) poor selectivity, which can easily lead to off-target effects of drugs, and systemic administration can easily cause systemic immune system toxicity; (3) the construction of bionic nano-drug delivery systems based on low-immunogenic tumor cell membranes still has problems such as insufficient tumor targeting. Summary of the invention

[0005] The purpose of the present invention is to provide a STING agonist biomimetic nano delivery system based on ferroptotic cell membranes, also known as STING agonist biomimetic nanoparticles based on ferroptotic cell membranes.

[0006] The present invention solves the problem of poor selectivity of existing non-CDN STING agonist drug delivery systems. This study uses tumor cell membranes that have been induced to undergo ferroptosis to coat drug-loaded nanoparticles, which allows the drug to have homologous targeting, improves the immunogenicity of immune "cold" tumor cells, achieves specific aggregation at the tumor site, improves the efficacy, and reduces systemic toxicity.

[0007] The bionic nanoparticles containing the STING agonist based on the ferroptotic cell membrane of the present invention include: a bionic shell, a drug carrier, and an active component, wherein the bionic shell wraps the drug carrier, and the drug carrier carries the active component.

[0008] The bionic shell is a tumor cell membrane after induced ferroptosis; specifically, it is a tumor cell membrane after induced ferroptosis by Erastin; wherein Erastin can be purchased on the market;

[0009] Among them, the drug carrier is PLGA, and the biomimetic nanoparticle core is prepared by nanoprecipitation method;

[0010] The active component is a STING agonist, including a non-CDN small molecule STING agonist. Preferably, the STING agonist is MSA-2; PLGA and MSA-2 can be purchased on the market.

[0011] Another object of the present invention is to provide a method for preparing STING agonist biomimetic nanoparticles based on ferroptotic cell membranes.

[0012] This method solves the problems that the existing drug delivery system has poor selectivity, which easily leads to off-target effects of drugs, and systemic administration is prone to systemic immune system toxicity; the low immunogenic tumor cell membrane is used to construct a bionic nano drug delivery system, but there is still insufficient tumor targeting. This is achieved by using the co-extrusion method to prepare bionic drug-loaded nanoparticles.

[0013] The method for preparing bionic nanoparticles comprises the following steps:

[0014] (1) Induce ferroptosis of tumor cells at 0h, 2h, 4h, and 8h respectively;

[0015] (2) collecting cells and preparing tumor cell membrane biomimetic shells of each group using ultrasound or repeated freezing and thawing methods;

[0016] (3) Preparation of PLGA drug-loaded nanoparticles by nanoprecipitation method:

[0017] (4) Preparation of biomimetic drug-loaded nanoparticles by co-extrusion method.

[0018] Wherein, step (1) induces ferroptosis of tumor cells for 0h, 2h, 4h and 8h respectively.

[0019] Preferably, inducing ferroptosis of tumor cells comprises the following steps:

[0020] 1) Cultivating cells: Cultivate tumor cells in a suitable complete culture medium at 37°C and 5% CO2;

[0021] 2) Preparation of drug working solution: First, use DMSO to prepare 10 mM Erastin stock solution, and then use tumor cell complete culture medium to dilute the Erastin stock solution to 10 μM Erastin working solution for later use;

[0022] 3) Drug-induced ferroptosis: Tumor cells were incubated with the above-mentioned Erastin working solution for 0 h, 2 h, 4 h, and 8 h.

[0023] Wherein, in step (2), cells are collected and each group of tumor cell membrane bionic shells are prepared by ultrasound or repeated freezing and thawing. The present invention adopts ultrasound or repeated freezing and thawing method for preparation.

[0024] Preferably, the preparation of the tumor cell membrane shell comprises the following steps:

[0025] 1) Collecting cells: Use a rubber scraper or a pipette to gently scrape or blow to absorb the above-mentioned tumor cells after induced ferroptosis, wash them twice with 4°C cold PBS, and centrifuge at 4°C to obtain tumor cells;

[0026] 2) Ice bath: Add PMSF to membrane protein extraction reagent A, resuspend the tumor cells, and then cool the suspension in an ice bath;

[0027] 3) Ultrasonic disruption: Use ultrasonic probe in an ice bath, and centrifuge the resulting solution at 1000 g for 10 min at 4°C to remove unbroken cells or cell debris;

[0028] 4) Collection by centrifugation: Carefully remove the upper layer by aspiration and collect by centrifugation.

[0029] Wherein, step (3) preparing PLGA drug-loaded nanoparticles by nanoprecipitation method comprises the following steps:

[0030] 1) Weigh MSA-2, PLGA, and F-68 respectively;

[0031] 2) Take acetone to dissolve MSA-2 and PLGA, and ultrapure water to dissolve F-68;

[0032] 3) F-68 aqueous solution needs to be prepared under ultrasound;

[0033] 4) Add the acetone solutions of MSA-2 and PLGA dropwise to the F-68 aqueous solution while stirring;

[0034] 5) Rotary evaporation to remove acetone to obtain the product.

[0035] Preferably,

[0036] The molar concentration of MSA-2 mother solution in step 1) is 1 to 10 mM,

[0037] The concentration of PLGA mother solution is 1-100 mg / mL.

[0038] The concentration of F-68 is 1 to 10 mg / mL;

[0039] In step 2), the volume of acetone is 1 to 2 mL, and the volume of ultrapure water is 1 to 10 mL;

[0040] In step 3), the power of ultrasound is 10 to 100 W, and the ultrasound time is 1 to 20 min;

[0041] The stirring time in step 4) is 1 to 20 minutes;

[0042] The rotary evaporation conditions in step 5) are 10-50° C. and the rotary evaporation time is 10-60 min.

[0043] Among them, (4) preparing biomimetic drug-loaded nanoparticles by coextrusion;

[0044] 1) Mix the prepared tumor cell membrane biomimetic shell and PLGA drug-loaded nanoparticles and perform ultrasound treatment for 5 to 10 minutes;

[0045] 2) The mixture in the previous step is mechanically repeatedly extruded 10 to 20 times using polycarbonate membranes with pore sizes of 1000 and 400 nm, and then repeatedly extruded 10 to 20 times using a polycarbonate membrane with a pore size of 200 nm to obtain.

[0046] According to one embodiment, the preparation method of the present invention comprises the following steps:

[0047] (1) Induce ferroptosis of 9464D tumor cells at 0h, 2h, 4h, and 8h respectively;

[0048] 1) Cultivating cells: The tumor cells were cultured in DMEM complete medium (DMEM medium, 10% FBS, 1% penicillin-streptomycin mixture (100×)) at 37° C. and 5% CO 2 ;

[0049] 2) Preparation of drug working solution: First, use DMSO to prepare 10 mM Erastin stock solution, then use DMEM complete medium to dilute the Erastin stock solution to 10 μM Erastin working solution for later use;

[0050] 3) Drug-induced ferroptosis: Tumor cells were incubated with the above-mentioned Erastin working solution for 0 h, 2 h, 4 h, and 8 h.

[0051] (2) collecting cells and preparing tumor cell membrane biomimetic shells of each group using ultrasound or repeated freezing and thawing methods;

[0052] 1) Collecting cells: Use a rubber scraper or a pipette to gently scrape or blow to absorb the above-mentioned tumor cells after ferroptosis induction, wash twice with 4°C cold PBS, and centrifuge at 4°C and 1500g to obtain tumor cells;

[0053] 2) Ice bath: Add PMSF to membrane protein extraction reagent A (purchased from Bio-Tech Biotech Co., Ltd.), resuspend the tumor cells, and then cool the suspension in an ice bath;

[0054] 3) Ultrasonic disruption: ultrasonically apply the probe in an ice bath at a power of 60-100 W for 2-5 minutes, and centrifuge the resulting solution at 1000 g for 10 minutes at 4°C;

[0055] 4) Centrifugal collection: Carefully aspirate the liquid to remove the upper layer, and centrifuge at 4℃ for 30 minutes to obtain the product.

[0056] (3) Preparation of PLGA drug-loaded nanoparticles by nanoprecipitation method:

[0057] 1) Prepare acetone solution of MSA-2 and PLGA for later use;

[0058] 2) preparing an aqueous solution of F-68, dissolving it with ultrasound at a power of 10 to 100 W and an ultrasound time of 1 to 20 min;

[0059] 3) Add the acetone solution of MSA-2 and PLGA dropwise to the F-68 aqueous solution while stirring for 1 to 20 minutes;

[0060] 4) At 10-50°C, rotary evaporate for 10-60 min to remove acetone.

[0061] (4) Preparation of biomimetic drug-loaded nanoparticles by co-extrusion method.

[0062] 1) Mix the prepared tumor cell membrane biomimetic shell and PLGA drug-loaded nanoparticles and perform ultrasound treatment for 5 to 10 minutes;

[0063] 2) The mixture in the previous step is mechanically repeatedly extruded 10 to 20 times using polycarbonate membranes with pore sizes of 1000 and 400 nm, and then repeatedly extruded 10 to 20 times using a polycarbonate membrane with a pore size of 200 nm to obtain.

[0064] Another object of the present invention is to provide pharmaceutical applications of STING agonist biomimetic nanoparticles based on ferroptotic cell membranes.

[0065] STING agonist biomimetic nanoparticles are used in tumor treatment, and at the same time they solve the problems of poor solubility of existing drugs, insufficient tumor targeting, and difficulty in clinical transformation. By inducing tumor cell membrane coating after ferroptosis, the immunogenicity of immune "cold" tumor cells is increased, and specific aggregation at the tumor site is achieved. This improves efficacy while reducing systemic toxic reactions, making clinical transformation easier.

[0066] Application of the STING agonist biomimetic nanoparticles based on ferroptotic cell membranes of the present invention in treating tumors.

[0067] Compared with the prior art, the present invention has the following beneficial effects:

[0068] (1) Strong tumor targeting: The surface of tumor cell membranes expresses surface adhesion molecules with homologous adhesion domains. Tumor cell membrane coatings can enhance the homologous targeting ability of nanoparticles. By inducing ferroptosis-induced tumor cell membrane coatings, the immunogenicity of immune “cold” tumor cells is increased, specific aggregation at the tumor site is achieved, systemic toxicity and side effects are reduced, and the therapeutic effect is enhanced.

[0069] (2) It can be injected intravenously, providing more possibilities for clinical transformation: Biofilms have good biocompatibility, which can protect drugs from degradation and improve drug stability. Based on this, this study has achieved a breakthrough in improving the solubility of non-CDN STING agonists by coating the tumor cell membrane on the surface of drug-loaded nanoparticles, and added intravenous injection as a drug delivery method, providing more possibilities for clinical transformation.

[0070] Further explanation and description of some technical terms or abbreviations appearing in the article:

[0071] CDN: Cyclic dinucleotides;

[0072] PBS: Phosphate buffered saline;

[0073] FBS: Fetal bovine serum;

[0074] PLGA: Poly(lactide-co-glycolide);

[0075] F-68: Pluronic 188, Pluronic 188;

[0076] PMSF: Phenyl methane sulfonyl fluoride;

[0077] DMEM medium: Dulbecco's modified eagle medium;

[0078] DMSO: Dimethyl sulfoxide;

[0079] Erastin: Erastin, Ferroptosis activator;

[0080] MSA-2: an oral non-nucleotide STING agonist. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 .MSA-2 liquid phase test results

[0082] Figure 2 .MSA-2 liquid phase standard curve

[0083] Table 1. Drug loading and encapsulation efficiency of STING agonist biomimetic nanoparticles based on ferroptotic cell membrane

[0084] Figure 3 .STING agonist biomimetic nanoparticles based on ferroptotic cell membranes

[0085] Figure 4 .Particle size and Zeta potential detection diagram of STING agonist biomimetic nanoparticles based on ferroptotic cell membrane Figure 5 .BMDC maturation and cytokine secretion detected by Elisa

[0086] Figure 6 . Changes in mouse weight and tumor size

[0087] Figure 7 .HE staining results of mouse organs and tissues DETAILED DESCRIPTION

[0088] The product and preparation method of the present invention are further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The methods are conventional methods unless otherwise specified. The raw materials can be obtained from public commercial channels unless otherwise specified.

[0089] The advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0090] Example 1 Preparation of STING agonist biomimetic nanoparticles based on ferroptotic cell membranes and determination of drug loading and encapsulation efficiency

[0091] 1. Induce ferroptosis of tumor cells at 0h, 2h, 4h, and 8h respectively:

[0092] 1) Cell culture: Tumor cells were cultured in DMEM complete medium (DMEM medium, 10% FBS, 1% penicillin-streptomycin mixture (100×)) at 37°C and 5% CO 2 environment;

[0093] 2) Preparation of drug working solution: First, use DMSO to prepare 10 mM Erastin stock solution, then use DMEM complete medium to dilute the Erastin stock solution to 10 μM Erastin working solution for later use;

[0094] 3) Drug-induced ferroptosis: Tumor cells were incubated with the above-mentioned Erastin working solution for 0 h, 2 h, 4 h, and 8 h.

[0095] 2. Collect cells and prepare biomimetic shells of tumor cell membranes in each group using ultrasound or repeated freezing and thawing methods;

[0096] 1) Collecting cells: Use a rubber scraper or a pipette to gently scrape or blow to absorb the above-mentioned tumor cells after induced ferroptosis, wash them twice with 4°C cold PBS, and centrifuge at 4°C and 1500g to obtain tumor cells;

[0097] 2) Ice bath: Add PMSF to membrane protein extraction reagent A (purchased from Bio-Tech Biotech Co., Ltd.), resuspend the tumor cells, and then cool the suspension in an ice bath;

[0098] 3) Ultrasonic disruption: ultrasonically apply the probe in an ice bath at a power of 60-100 W for 2-5 minutes, and centrifuge the resulting solution at 1000 g for 10 minutes at 4°C;

[0099] 4) Centrifugal collection: Carefully aspirate the liquid to remove the upper layer, and centrifuge at 4℃ for 30 minutes to obtain the product.

[0100] 3. Preparation of PLGA drug-loaded nanoparticles by nanoprecipitation method:

[0101] 1) Prepare acetone solution of MSA-2 and PLGA for later use;

[0102] 2) preparing an aqueous solution of F-68, dissolving it with ultrasound at a power of 10 to 100 W and an ultrasound time of 1 to 20 min;

[0103] 3) Add the acetone solution of MSA-2 and PLGA dropwise to the F-68 aqueous solution while stirring for 1 to 20 minutes;

[0104] 4) At 10-50°C, rotary evaporate for 10-60 min to remove acetone.

[0105] 4. Preparation of bionic drug-loaded nanoparticles by co-extrusion method.

[0106] 1) Mix the prepared tumor cell membrane bionic shell and PLGA drug-loaded nanoparticles and perform ultrasound treatment for 5 to 10 minutes;

[0107] 2) The mixture in the previous step is mechanically repeatedly extruded 10 to 20 times using polycarbonate membranes with pore sizes of 1000 and 400 nm, and then repeatedly extruded 10 to 20 times using a polycarbonate membrane with a pore size of 200 nm to obtain.

[0108] 5. Add appropriate amount of acetonitrile to dissolve the prepared biomimetic nanoparticles containing STING agonist to release them, centrifuge to remove impurities, and use high performance liquid chromatography to detect the MSA-2 concentration after passing through a 0.22 μm filter membrane, and use the following formula to calculate the drug loading DL and encapsulation efficiency EE:

[0109] DL% = (actual drug mass loaded into nanoparticles / total mass of nanoparticles) × 100%;

[0110] EE% = (actual drug mass measured / total drug mass added during preparation) × 100%

[0111] Results Figure 1 , Figure 2 And Table 1. As shown in the figure, the drug loading of the STING agonist biomimetic nanoparticles prepared by the present invention is 7.8%±0.1, and the encapsulation efficiency reaches 86.3%±0.5.

[0112] Table 1

[0113]

[0114] Example 2. Characterization of the physicochemical properties of STING agonist biomimetic nanoparticles based on ferroptotic cell membranes 1. Test materials: STING agonist biomimetic nanoparticles and PLGA drug-loaded nanoparticles prepared in Example 1; 2. Test methods and results

[0115] (1) Morphological characterization: Use a transmission electron microscope (TEM) to observe the morphology. First, use tweezers to clamp the edge of the TEM-specific copper mesh, and gently clamp it to prevent the copper mesh from being broken; then take 10μL of M@P liquid and drop it on the copper mesh covered with a support film, dry it naturally, and carefully absorb the excess liquid with the tip of filter paper. Then drop 10μL of tungsten phosphate negative staining solution, dry it naturally, and absorb the excess staining liquid with filter paper. Use TEM to observe its morphology, size, and dispersion. Transmission electron microscopy results are shown in Figure 3 .

[0116] TEM images showed that the STING agonist biomimetic nanoparticles had a uniform particle size distribution without aggregation, and there was an obvious membrane structure on the surface, demonstrating a typical core-shell structure, proving the successful coating of the cell membrane; the membrane-coated PLGA nanoparticles showed a typical spherical shape with a smooth surface.

[0117] (2) Particle size and Zeta potential detection: The particle size and Zeta potential of STING agonist biomimetic nanoparticles were detected. The test results are shown in Figure 4 .

[0118] The average particle size of STING agonist biomimetic nanoparticles measured by dynamic light scattering (DLS) was about 120 nm, and the PDI was 0.068, indicating that the STING agonist biomimetic nanoparticles had a good particle size distribution; the Zeta potential of the STING agonist biomimetic nanoparticles was about -10 mV.

[0119] Example 3: Experiment on in vitro induction of dendritic cell maturation by STING agonist biomimetic nanoparticles based on ferroptotic cell membranes

[0120] Experimental steps:

[0121] 1. Extract mouse BMDC cells and induce them with GM-CSF and IL-4. Incubate at 37°C and 5% CO 2 Incubator culture;

[0122] 2. On the 2nd, 4th and 6th days, change the medium by half, and incubate the cells on the 6th day;

[0123] 3. After 24 hours of drug incubation, cells were extracted and CD11c, CD80, and CD86 were analyzed by FCM to determine the percentage of mature BMDCs; the supernatant was temporarily stored at -80°C and the IL-6 and IFN-β contents were analyzed by ELISA kit. Figure 5 .

[0124] The maturation of dendritic cells is a key driving force for T cell activation. High expression of stimulatory factors such as CD80 and CD86 is a sign of dendritic cell maturation. The present invention detected the in vitro immunostimulatory activity of dendritic cells after treatment with each preparation, and found that the percentage of CD80-positive and CD86-positive dendritic cells after treatment with STING agonist biomimetic nanoparticles was significantly higher than that of other groups, indicating that STING agonist biomimetic nanoparticles based on ferroptotic cell membranes have a stronger ability to induce maturation of dendritic cells.

[0125] Example 4: Pharmacodynamic evaluation test of STING agonist biomimetic nanoparticles based on ferroptotic cell membranes Experimental steps:

[0126] 1. Establishment of tumor-bearing mouse model: Mouse high-risk neuroblastoma 9464D cell line was cultured in DMEM (high glucose) culture medium containing 10% fetal bovine serum and 1% double antibody. After adherence, cells in the logarithmic growth phase were taken, washed with PBS, and then digested with trypsin containing 0.25% EDTA. After terminating digestion, centrifuged at 1000 rpm for 5 minutes, and resuspended in basal culture medium to adjust the cell concentration to 1.0×10^6×50μL -1 / mouse, inoculated into the right hind hip of C57BL / 6N female mice (6 weeks old).

[0127] 2. Dosage regimen: When the tumor volume reaches ~50mm 3 At the same time, the mice were randomly divided into groups and 100 μL of each group of preparations were injected into the tail vein of the mice, wherein the STING agonist biomimetic nanoparticles based on ferroptotic cell membrane were prepared according to the method of Example 1. The tumor volume was monitored and the survival status of the mice was observed; the body weight was measured at the same time, and the curve of body weight change over time was drawn. The changes of mouse body weight and tumor over time are shown in Figure 6 .

[0128] According to the experimental results, by coating the tumor cell membrane after ferroptosis on the surface of PLGA drug-loaded nanoparticles, the targeting of the STING agonist to the tumor site is increased, the effect of tumor treatment is significantly improved, and the weight of mice is not affected.

[0129] Example 5: In vivo safety test of STING agonist biomimetic nanoparticles based on ferroptotic cell membranes Experimental steps:

[0130] 1. Establishment of tumor-bearing mouse model: Mouse high-risk neuroblastoma 9464D cell line was cultured in DMEM (high glucose) culture medium containing 10% fetal bovine serum and 1% double antibody. After adherence, cells in the logarithmic growth phase were taken, washed with PBS, and then digested with trypsin containing 0.25% EDTA. After terminating digestion, centrifuged at 1000 rpm for 5 minutes, and resuspended in basal culture medium to adjust the cell concentration to 1.0×10^6×50μL -1 / mouse, inoculated into the right hind hip of C57BL / 6N female mice (6 weeks old).

[0131] 2. Dosage regimen: When the tumor volume reaches ~50mm 3 At the same time, the mice were randomly divided into groups, and 100 μL of each preparation was injected into the tail vein of the mice, wherein the STING agonist biomimetic nanoparticles based on ferroptotic cell membrane were prepared according to the method of Example 1.

[0132] 3. After the last dosing cycle, the mice were killed and the mouse tissues were taken out for HE staining to study the in vivo safety of STING agonist biomimetic nanoparticles based on ferroptotic cell membranes. The results of tissue HE staining are shown in Figure 7 .

[0133] According to the experimental results, the tumor cell membrane after ferroptosis is coated on the surface of PLGA drug-loaded nanoparticles, which has good biosafety.

[0134] The above description is only a preferred embodiment of the present invention. It should be noted that the embodiments of the present invention are not limited to the embodiments. Without departing from the principle of the present invention, several changes, modifications, substitutions, combinations, and simplifications can be made, all of which should be equivalent replacement methods, and these should also be regarded as the scope of protection of the present invention.

Claims

1. A biomimetic nanoparticle containing a STING agonist based on ferroptotic cell membrane, characterized in that: The bionic nanoparticles include a bionic shell, a drug carrier, and an active component, wherein the bionic shell wraps the drug carrier, and the drug carrier carries the active component, wherein the bionic shell is a tumor cell membrane after induced ferroptosis; wherein the drug carrier is PLGA, and the bionic nanoparticle core is prepared by a nanoprecipitation method; wherein the active component is a STING agonist, including a small molecule STING agonist.

2. The biomimetic nanoparticle according to claim 1, characterized in that: The STING agonist is MSA-2.

3. The biomimetic nanoparticle according to claim 1, characterized in that: The following steps are involved: (1) Induce ferroptosis of tumor cells at 0h, 2h, 4h, and 8h respectively; (2) collecting cells and preparing tumor cell membrane biomimetic shells of each group using ultrasound or repeated freezing and thawing methods; (3) Preparation of PLGA drug-loaded nanoparticles by nanoprecipitation method; (4) Preparation of biomimetic drug-loaded nanoparticles by co-extrusion method.

4. The biomimetic nanoparticle according to claim 3, characterized in that: (1) Inducing ferroptosis of tumor cells for 0 h, 2 h, 4 h, and 8 h, respectively, comprising the following steps: 1) Cultivating cells: Cultivate tumor cells in a suitable complete culture medium at 37°C and 5% CO2; 2) Preparation of drug working solution: First, use DMSO to prepare 10 mM Erastin stock solution, and then use tumor cell complete culture medium to dilute the Erastin stock solution to 10 μM Erastin working solution for later use; 3) Drug-induced ferroptosis: Tumor cells were incubated with the above-mentioned Erastin working solution for 0 h, 2 h, 4 h, and 8 h.

5. The biomimetic nanoparticle according to claim 3, characterized in that: (2) collecting cells and preparing the tumor cell membrane biomimetic shell of each group by ultrasonic method or repeated freezing and thawing method, including the following steps: 1) Collecting cells: Use a rubber scraper or a pipette to gently scrape or blow to absorb the above-mentioned tumor cells after induced ferroptosis, wash them twice with 4°C cold PBS, and centrifuge at 4°C to obtain tumor cells; 2) Ice bath: Add PMSF to membrane protein extraction reagent A, resuspend the tumor cells, and then cool the suspension in an ice bath; 3) Ultrasonic disruption: Use ultrasonic probe in an ice bath, and centrifuge the resulting solution at 1000 g for 10 min at 4°C to remove unbroken cells or cell debris; 4) Collection by centrifugation: Carefully remove the upper layer by aspiration and collect by centrifugation.

6. The biomimetic nanoparticle according to claim 3, characterized in that: (3) Preparation of PLGA drug-loaded nanoparticles by nanoprecipitation method: The molar concentration of MSA-2 mother solution in step 1) is 1 to 10 mM, The concentration of PLGA mother solution is 1-100 mg / mL. The concentration of F-68 is 1 to 10 mg / mL; In steps 2) and 4), the volumes of acetone and ultrapure water are 1-2 mL, 1-2 mL, and 1-10 mL, respectively; In step 3), the power of ultrasound is 10 to 100 W, and the ultrasound time is 1 to 20 min; The stirring time in step 4) is 1 to 20 minutes; The rotary evaporation conditions in step 5) are 10-50° C. and the rotary evaporation time is 10-60 min.

7. The biomimetic nanoparticle according to claim 1, characterized in that: (4) Preparation of biomimetic drug-loaded nanoparticles by co-extrusion; 1) Mix the prepared tumor cell membrane biomimetic shell and PLGA drug-loaded nanoparticles and perform ultrasound treatment for 5 to 10 minutes; 2) The mixture in the previous step is mechanically repeatedly extruded 10 to 20 times using polycarbonate membranes with pore sizes of 1000 and 400 nm, and then repeatedly extruded 10 to 20 times using a polycarbonate membrane with a pore size of 200 nm to obtain.

8. The biomimetic nanoparticle according to claim 1, characterized in that: The following steps are involved: (1) Induce ferroptosis of 9464D tumor cells at 0h, 2h, 4h, and 8h respectively; 1) Cultivate cells: Cultivate tumor cells in DMEM complete medium (DMEM medium, The cells were cultured in a 10% FBS, 1% penicillin-streptomycin mixture (100×) at 37°C and 5% CO2. 2) Preparation of drug working solution: First, use DMSO to prepare 10 mM Erastin stock solution, then use DMEM complete medium to dilute the Erastin stock solution to 10 μM Erastin working solution for later use; 3) Drug-induced ferroptosis: Tumor cells were incubated with the above-mentioned Erastin working solution for 0 h, 2 h, 4 h, and 8 h. (2) collecting cells and preparing tumor cell membrane biomimetic shells of each group using ultrasound or repeated freezing and thawing methods; 1) Collecting cells: Use a rubber scraper or a pipette to gently scrape or blow to absorb the above-mentioned tumor cells after induced ferroptosis, wash them twice with 4°C cold PBS, and centrifuge at 4°C and 1500g to obtain tumor cells; 2) Ice bath: Add PMSF to membrane protein extraction reagent A (purchased from Bio-Tech Biotech Co., Ltd.), resuspend the tumor cells, and then cool the suspension in an ice bath; 3) Ultrasonic disruption: ultrasonically perform ultrasonication in an ice bath at a power of 60 to 100 W for 2 to 5 minutes, and centrifuge the resulting solution at 1000 g for 10 minutes at 4°C; 4) Centrifugal collection: Carefully remove the upper layer by aspiration and centrifuge at 4°C for 30 minutes to obtain the liquid; (3) Preparation of PLGA drug-loaded nanoparticles by nanoprecipitation method: 1) Prepare acetone solution of MSA-2 and PLGA for later use; 2) preparing an aqueous solution of F-68, dissolving it with ultrasound at a power of 10 to 100 W and an ultrasound time of 1 to 20 min; 3) Add the acetone solution of MSA-2 and PLGA dropwise to the F-68 aqueous solution while stirring for 1 to 20 minutes; 4) 10-50°C, rotary evaporation for 10-60 min to remove acetone to obtain the product; (4) Preparation of biomimetic drug-loaded nanoparticles by co-extrusion; 1) Mix the prepared tumor cell membrane biomimetic shell and PLGA drug-loaded nanoparticles and perform ultrasound treatment for 5 to 10 minutes; 2) The mixture in the previous step is mechanically repeatedly extruded 10 to 20 times using polycarbonate membranes with pore sizes of 1000 and 400 nm, and then repeatedly extruded 10 to 20 times using a polycarbonate membrane with a pore size of 200 nm to obtain.

9. Use of the bionic nanoparticles according to claim 1 in drugs for treating tumors.

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