A neutrophil-mimicking vesicle nanoplatform, its preparation method and application in the preparation of gastric cancer-targeted drugs

Abstract

The present invention provides a neutrophil biomimetic vesicle nanoformulation, a preparation method thereof, and an application in the preparation of gastric cancer targeted drugs, belonging to the field of biopharmaceutical technology. The present invention uses RGD cyclic peptide to modify neutrophil biomimetic vesicles. RGD cyclic peptide is a tumor-targeting peptide that can bind to αvβ3 receptors and is a highly efficient and selective inhibitor of αvβ3 integrin receptors. The neutrophil biomimetic vesicles modified with RGD cyclic peptide have good gastric cancer cell targeting. The present invention uses neutrophil biomimetic vesicles modified with RGD cyclic peptide to wrap drug-loaded photosensitive liposomes. The drug-loaded photosensitive liposomes contain photosensitizers. The photosensitizers have photothermal corresponding properties. Under near-infrared zone II laser irradiation, the photothermal effect of the photosensitizer causes the drug-loaded photosensitive liposomes to decompose, releasing the active ingredients PD-L1 inhibitor and doxorubicin, thereby inhibiting the proliferation of gastric cancer cells, delaying the occurrence and development of gastric cancer, and achieving the effect of targeted treatment of gastric cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to a neutrophil biomimetic vesicle nano preparation, a preparation method thereof, and application thereof in the preparation of gastric cancer targeted drugs. Background Art

[0002] The latest data from the National Cancer Center indicates that gastric cancer ranks third in both incidence and mortality in my country, and there remains a long way to go in the diagnosis and treatment of gastric cancer. In recent years, clinical translation, precision medicine, and immunotherapy have become hot topics in the field of gastric cancer. Currently, gastric cancer treatment primarily relies on surgery and chemoradiotherapy. Surgical resection of the primary tumor, supplemented with chemoradiotherapy, is highly effective for patients with early-stage cancer. However, 90% of cancer patients are diagnosed in the advanced or late stages of the disease with distant metastases. Traditional chemotherapy lacks targeted therapy, killing tumor cells while also causing severe toxic side effects on normal tissues, a common cause of chemotherapy failure in cancer patients.

[0003] Extracellular vesicles (EVs) hold great promise for targeted drug delivery in tumor therapy due to their inherent low immunogenicity, biocompatibility, and long-lived circulation. Immune cell-derived nanovesicles can specifically deliver proteins, nucleic acids, and lipids, achieving certain anti-tumor effects. However, nanoformulations based on neutrophil-inspired vesicles are relatively understudied in the oncology field. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a neutrophil biomimetic vesicle nanoformulation, a preparation method thereof, and an application thereof in the preparation of gastric cancer targeted drugs. The neutrophil biomimetic vesicle nanoformulation provided by the present invention has good efficacy in the targeted treatment of gastric cancer.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing a neutrophil biomimetic vesicle nanoformulation, comprising the following steps:

[0007] The peripheral blood neutrophils are subjected to a first physical extrusion and a first density gradient separation to obtain neutrophil biomimetic vesicles;

[0008] Mixing the neutrophil biomimetic vesicles with RGD cyclic peptide for a first co-incubation to obtain RGD-modified neutrophil biomimetic vesicles;

[0009] Phosphatidylcholine, phospholipid-polyethylene glycol-peptide, cholesterol, a photosensitizer, and an organic solvent are mixed to form a film, and the resulting film is mixed with a PD-L1 inhibitor and a doxorubicin hydrochloride solution, followed by extrusion to obtain drug-loaded photosensitive liposomes;

[0010] The RGD-modified neutrophil biomimetic vesicles and the drug-loaded photosensitive liposomes are subjected to a second co-incubation, a second physical extrusion, and a second density gradient separation to obtain a neutrophil biomimetic vesicle nanoformulation.

[0011] Preferably, the RGD cyclic peptide is DSPE-PEG2000-cRGD;

[0012] The mass ratio of the neutrophil biomimetic vesicles to the RGD cyclic peptide is 1×10 6 ~5×10 6 Pieces: 100μg.

[0013] Preferably, the photosensitizer is FD-1080;

[0014] The mass ratio of the phosphatidylcholine, phospholipid-polyethylene glycol-polypeptide, cholesterol and photosensitizer is 1-2:1-2:1-2:1-2.

[0015] Preferably, the PD-L1 inhibitor is trifluoroacetate;

[0016] The volume ratio of the film-forming material, the PD-L1 inhibitor and doxorubicin hydrochloride is 0.5-1:0.5-1:0.5-1.

[0017] Preferably, the extrusion is extrusion using a liposome extruder.

[0018] Preferably, the mass ratio of the RGD-modified neutrophil biomimetic vesicles to the drug-loaded photosensitive liposomes is 1×10 6 Pieces: 100~200μg.

[0019] Preferably, the pressure of the first physical extrusion and the second physical extrusion are independently 500-1000 psi, the temperature is independently 50-60° C., and the time is independently 5-10 min.

[0020] Preferably, the temperature of the first co-incubation is 25-37°C and the time is 25-30 minutes;

[0021] The temperature of the second co-incubation is 25-37° C., and the time is 10-20 minutes.

[0022] The present invention provides a neutrophil biomimetic vesicle nanopreparation prepared by the above-mentioned preparation method, comprising RGD-modified neutrophil biomimetic vesicles and drug-loaded photosensitive liposomes encapsulated inside the RGD-modified neutrophil biomimetic vesicles, wherein the drug-loaded photosensitive liposomes comprise a liposome membrane layer loaded with a photosensitizer and an active ingredient encapsulated inside the membrane layer, wherein the active ingredient comprises a PD-L1 inhibitor and doxorubicin.

[0023] The present invention provides the use of the neutrophil biomimetic vesicle nano preparation in the preparation of gastric cancer targeted drugs.

[0024] The present invention provides a method for preparing a neutrophil biomimetic vesicle nanoformulation, comprising the following steps: performing a first physical extrusion and a first density gradient separation on peripheral blood neutrophils to obtain neutrophil biomimetic vesicles (abbreviated as NNV); mixing the neutrophil biomimetic vesicles with RGD cyclic peptides and performing a first co-incubation to obtain RGD-modified neutrophil biomimetic vesicles (abbreviated as RGD-NNV); combining phosphatidylcholine, phospholipid-polyethylene glycol-peptide, cholesterol, a photosensitizer and an organic molecule; and The organic solvent is mixed to form a film, and the obtained film-forming material is mixed with a PD-L1 inhibitor and a doxorubicin hydrochloride solution, and extruded to obtain drug-loaded photosensitive liposomes (abbreviated as FD1080&DOX / TFA); the RGD-modified neutrophil biomimetic vesicles and the drug-loaded photosensitive liposomes are subjected to a second co-incubation, a second physical extrusion and a second density gradient separation to obtain a neutrophil biomimetic vesicle nanoformulation (abbreviated as RGD-NNV@FD1080&DOX / TFA). The present invention uses RGD cyclic peptide to modify the neutrophil biomimetic vesicles. RGD cyclic peptide is a tumor-targeting peptide that can bind to αvβ3 receptors and is a highly efficient and selective inhibitor of αvβ3 integrin receptors. The neutrophil biomimetic vesicles modified with RGD cyclic peptide have good gastric cancer cell targeting. The present invention uses RGD cyclic peptide-modified neutrophil biomimetic vesicles to encapsulate drug-loaded photosensitive liposomes. The drug-loaded photosensitive liposomes contain a photosensitizer, which has photothermal response characteristics. Under near-infrared laser irradiation in the second zone, the photothermal effect of the photosensitizer causes the drug-loaded photosensitive liposomes to decompose, releasing the active ingredients PD-L1 inhibitor and doxorubicin, thereby inhibiting the proliferation of gastric cancer cells, delaying the occurrence and development of gastric cancer, and achieving the effect of targeted treatment of gastric cancer. The results of the examples show that the neutrophil biomimetic vesicle nanoformulation provided by the present invention acts on gastric cancer cells, can specifically target gastric cancer cells, induce gastric cancer cell apoptosis, inhibit gastric cancer metastasis, prolong the survival time of mice, and improve the tumor immune microenvironment.

[0025] At the same time, the preparation method provided by the present invention is simple to operate, low in cost, and suitable for industrial batch production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Flow chart of the preparation of RGD-NNV@FD1080&DOX / TFA;

[0027] Figure 2 Schematic diagram of the structure of RGD-NNV@FD1080&DOX / TFA;

[0028] Figure 3Western blot image of RGD-NNV@FD1080&DOX / TFA;

[0029] Figure 4 Transmission electron microscopy image of RGD-NNV@FD1080&DOX / TFA;

[0030] Figure 5 is the particle size distribution diagram of RGD-NNV@FD1080&DOX / TFA;

[0031] Figure 6 The near-infrared warming effect of RGD-NNV@FD1080&DOX / TFA;

[0032] Figure 7 This is the effect of RGD-NNV@FD1080&DOX / TFA on the proliferation ability of MGC-803 cells. DETAILED DESCRIPTION

[0033] The present invention provides a method for preparing a neutrophil biomimetic vesicle nanoformulation, comprising the following steps:

[0034] The peripheral blood neutrophils are subjected to a first physical extrusion and a first density gradient separation to obtain neutrophil biomimetic vesicles;

[0035] Mixing the neutrophil biomimetic vesicles with RGD cyclic peptide for a first co-incubation to obtain RGD-modified neutrophil biomimetic vesicles;

[0036] Phosphatidylcholine, phospholipid-polyethylene glycol-peptide, cholesterol, a photosensitizer, and an organic solvent are mixed to form a film, and the resulting film is mixed with a PD-L1 inhibitor and a doxorubicin hydrochloride solution, followed by extrusion to obtain drug-loaded photosensitive liposomes;

[0037] The RGD-modified neutrophil biomimetic vesicles and the drug-loaded photosensitive liposomes are subjected to a second co-incubation, a second physical extrusion, and a second density gradient separation to obtain a neutrophil biomimetic vesicle nanoformulation.

[0038] Unless otherwise specified, the raw materials used in the present invention are all commercially available.

[0039] The present invention performs a first physical extrusion and a first density gradient separation on peripheral blood neutrophils to obtain neutrophil biomimetic vesicles. In the present invention, the density of the peripheral blood neutrophils is preferably (4-5)×10 6 In the present invention, the peripheral blood neutrophils are preferably obtained by culturing in RPMI1640 culture medium containing 10% fetal bovine serum. The culture temperature is preferably 25-37°C, more preferably 28-32°C; and the culture time is preferably 6-12 hours, more preferably 8-10 hours.

[0040] In the present invention, the first physical extrusion is preferably performed using a mini-extrader extruder. In the present invention, the pressure of the first physical extrusion is preferably 500-1000 psi, more preferably 600-800 psi; the temperature is preferably 50-60°C, more preferably 55°C; and the duration is preferably 5-10 minutes, more preferably 6-8 minutes. In the present invention, the first physical extrusion is preferably repeated 7-11 times, more preferably 8-10 times.

[0041] In the present invention, the temperature of the first density gradient separation is preferably 50-60°C, more preferably 55°C. In the present invention, the first density gradient separation preferably includes centrifugation and filtration performed sequentially. In the present invention, the centrifugal force of the centrifugation is preferably 800-1000g, and the time is preferably 5-10 minutes; the filtration is preferably performed through 2μm, 800nm, and 200nm PC filter membranes in sequence.

[0042] After the first density gradient separation, the present invention preferably washes, filters and centrifuges the obtained neutrophil biomimetic vesicles, resuspends the bottom buffer pad, and obtains a neutrophil biomimetic vesicle suspension. In the present invention, the washing detergent is preferably phosphate-balanced physiological saline; the present invention has no special requirements for the filtration, and a filtration method familiar to those skilled in the art can be used. In the present invention, the centrifugation is preferably differential centrifugation, and the centrifugal force of the differential centrifugation is preferably 800-1000g, more preferably 900g; the time is preferably 5-10min, more preferably 6-8min. The present invention preferably uses PBS buffer solution to resuspend the bottom buffer pad.

[0043] After obtaining the neutrophil biomimetic vesicles, the present invention mixes the neutrophil biomimetic vesicles with RGD cyclic peptide and performs a first co-incubation to obtain RGD-modified neutrophil biomimetic vesicles, which are recorded as RGD-NNV. In the present invention, the RGD cyclic peptide is preferably DSPE-PEG2000-cRGD, purchased from Xi'an Ruixi Biological Co., Ltd.

[0044] In the present invention, the mass ratio of the number of neutrophil biomimetic vesicles to the mass ratio of the RGD cyclic peptide is preferably 1×10 6 ~5×10 6 In the present invention, the temperature of the first co-incubation is preferably 25-37° C., more preferably 28-35° C.; the time is preferably 25-30 min, more preferably 26-28 min.

[0045] After the first co-incubation, the present invention preferably washes, filters and centrifuges the obtained first co-incubation liquid, resuspends the bottom buffer pad, and obtains an RGD-modified neutrophil biomimetic vesicle suspension. In the present invention, the washing detergent is preferably phosphate-balanced physiological saline; the present invention has no special requirements for the filtration, and a filtration method familiar to those skilled in the art can be used. In the present invention, the centrifugation is preferably ultracentrifugation, and the centrifugal force of the ultracentrifugation is preferably 10,000 to 12,000 g, more preferably 11,000 g; the time is preferably 60 to 80 min, more preferably 70 min. The present invention preferably uses PBS buffer solution to resuspend the bottom buffer pad.

[0046] The present invention mixes phosphatidylcholine (SPC), phospholipid-polyethylene glycol-polypeptide, cholesterol, a photosensitizer, and an organic solvent to form a film. The resulting film is mixed with a PD-L1 inhibitor and a doxorubicin hydrochloride solution and extruded to obtain drug-loaded photosensitive liposomes, designated as FD1080 & DOX / TFA. In the present invention, the phospholipid-polyethylene glycol-polypeptide model is preferably DSPE-PEG-2K, which is preferably purchased from Xi'an Ruixi Biological Co., Ltd. In the present invention, the organic solvent is preferably chloroform.

[0047] In the present invention, the photosensitizer is preferably FD-1080; the mass ratio of the phosphatidylcholine, phospholipid-polyethylene glycol-polypeptide, cholesterol and photosensitizer is preferably 1-2:1-2:1-2:1-2, more preferably 1:1:1:1.

[0048] In the present invention, the film forming method is preferably rotary evaporation. In the present invention, the rotation temperature is preferably 55 to 65° C., and the rotation speed is preferably 120 to 200 rpm.

[0049] In the present invention, the PD-L1 inhibitor is preferably trifluoroacetate TFA; the volume ratio of the film-forming substance, the PD-L1 inhibitor and doxorubicin hydrochloride is preferably 0.5-1 mL:0.5-1 mL:0.5-1 mL, more preferably 1:1:1.

[0050] In the present invention, the film-forming material is preferably mixed with the PD-L1 inhibitor and the doxorubicin hydrochloride solution by ultrasonic mixing, the power of the ultrasonic mixing is preferably 50 to 100 W, and the time is preferably 1 to 5 minutes. In the present invention, the extrusion is preferably extrusion by a liposome extruder.

[0051] The present invention performs a second co-incubation, a second physical extrusion, and a second density gradient separation on the RGD-modified neutrophil biomimetic vesicles and the drug-loaded photosensitive liposomes to obtain a neutrophil biomimetic vesicle nanoformulation. In the present invention, the mass ratio of the RGD-modified neutrophil biomimetic vesicles to the drug-loaded photosensitive liposomes is 1×106 Pieces: 100~200μg.

[0052] In the present invention, the temperature of the second co-incubation is preferably 25-37°C, more preferably 28-35°C; the time is preferably 10-20 minutes, more preferably 15 minutes. In the present invention, the pressure of the second physical extrusion is preferably 500-1000 psi, more preferably 600-800 psi; the temperature is preferably 50-60°C, more preferably 55°C; the time is preferably 5-10 minutes, more preferably 6-8 minutes. In the present invention, the number of repetitions of the second physical extrusion is preferably 7-11 times, more preferably 8-10 times.

[0053] In the present invention, the second density gradient separation preferably comprises centrifugation and filtration performed sequentially. In the present invention, the centrifugal force of the centrifugation is preferably 800-1000 g, and the time is preferably 5-10 min; and the filtration is preferably performed sequentially through 2 μm, 800 nm, and 200 nm PC filter membranes.

[0054] After the second gradient density separation, the present invention preferably washes, filters, and centrifuges the obtained neutrophil biomimetic vesicle nanoformulation, resuspends the bottom buffer pad, and obtains a neutrophil biomimetic vesicle suspension. In the present invention, the washing detergent is preferably phosphate-balanced physiological saline; the present invention has no special requirements for the filtration, and a filtration method familiar to those skilled in the art can be used. In the present invention, the centrifugation is preferably ultracentrifugation, and the centrifugal force of the ultracentrifugation is preferably 10,000 to 12,000 g, more preferably 11,000 g, and the centrifugation time is preferably 60 to 80 min, more preferably 70 min.

[0055] The present invention provides a neutrophil biomimetic vesicle nanopreparation prepared by the above-mentioned preparation method, comprising RGD-modified neutrophil biomimetic vesicles and drug-loaded photosensitive liposomes encapsulated inside the RGD-modified neutrophil biomimetic vesicles, wherein the drug-loaded photosensitive liposomes comprise a liposome membrane layer loaded with a photosensitizer and an active ingredient encapsulated inside the membrane layer, wherein the active ingredient comprises a PD-L1 inhibitor and doxorubicin.

[0056] In the present invention, the loading amount of the PD-L1 inhibitor in the neutrophil biomimetic vesicle nanoformulation is preferably 5-10 wt%, more preferably 6-8 wt%; the loading amount of doxorubicin is preferably 5-10 wt%, more preferably 6-8 wt%.

[0057] In the present invention, the particle size of the neutrophil biomimetic vesicle nanoformulation is preferably 50 to 60 nm, more preferably 55 nm.

[0058] The present invention provides the use of the neutrophil biomimetic vesicle nano preparation in the preparation of gastric cancer targeted drugs.

[0059] The neutrophil biomimetic vesicle nanoformulation provided by the present invention, its preparation method and its application in the preparation of gastric cancer targeted drugs are described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0060] Example 1

[0061] The raw materials and instruments used in Example 1 are as follows:

[0062] Human peripheral blood neutrophil culture reagents: polymorphonuclear leukocyte separation medium (PolymorphPrep separation medium, Norway), RPMI1640 (Bioind, USA), fetal bovine serum (Gibco, USA), trypsin (Sigma, USA), carbon dioxide incubator (Forma), serum-free culture medium (Excell, China);

[0063] Clean bench, desktop centrifuge (Eppendorf, Germany), ultracentrifuge (Beckman, USA).

[0064] Extraction reagents for RGD-NNV@FD1080&DOX / TFA biological preparations: RGD material, nanocomplex encapsulating doxorubicin, doxorubicin, PDL1 inhibitor and near-infrared second-region fluorescent dye (Xi'an Ruixi Biological Technology, China), Mini-extrader extruder (Avanti Polar Lipids, USA), PC filter membranes with different pore sizes of 2μm, 800nm, and 200nm (Xi'an Ruixi Biological Technology, China), transmission electron microscope (FEI Tecnai 12, Philips), ultracentrifuge (Beckman, USA), and nanoparticle tracking analysis (nanosight tracking analysis, UK).

[0065] according to Figure 1 The process shown below is used to prepare neutrophil biomimetic vesicle nanoformulations:

[0066] 1. Human peripheral blood neutrophils (5×10 6 / mL) suspension was subjected to the first high-temperature physical extrusion and then passed through PC filter membranes with different pore sizes of 2μm, 800nm, and 200nm to prepare neutrophil biomimetic vesicles;

[0067] 2. Centrifuge at 1000 g for 10 min (Eppendorf Centrifuge 5804 / 5804 centrifuge), collect the buffer at the bottom, and resuspend in PBS to prepare a neutrophil biomimetic vesicle suspension;

[0068] 3. The neutrophil biomimetic vesicle suspension was incubated with RGD cyclic peptide at 37°C for 30 minutes to obtain RGD-modified neutrophil biomimetic vesicles (RGD-NNV);

[0069] 4. Wash, filter and centrifuge the RGD-modified neutrophil biomimetic vesicle suspension, and resuspend the bottom buffer to obtain the RGD-modified neutrophil biomimetic vesicle suspension;

[0070] 5. SPC, DSPE-PEG-2K, cholesterol, and FD-1080 were dissolved in 4 mL of chloroform and rotary evaporated in a flask to form a film at the bottom of the flask. 0.5 mL of PD-L1 inhibitor and 0.5 mL of DOX·HCl solution were added. The liposomes were treated by sonication and a liposome extruder to prepare the dual-drug loaded photosensitive material FD1080 & DOX / TFA liposomes.

[0071] 6. The RGD-modified neutrophil biomimetic vesicle suspension was co-incubated with the FD1080 & DOX / TFA liposome suspension at 37°C for 20 minutes. The mixed suspension was subjected to a second high-temperature physical extrusion, passing through PC filter membranes with different pore sizes of 2 μm, 800 nm, and 200 nm at 60°C in sequence to obtain the RGD-modified neutrophil biomimetic vesicle-loaded FD1080 & DOX / TFA nanoformulation.

[0072] 7. Ultracentrifuge at 12000g for 30 min in a Beckman ultracentrifuge, collect the bottom buffer pad by centrifugation, and resuspend in PBS to obtain RGD-modified neutrophil biomimetic vesicles loaded with FD1080&DOX / TFA nanoformulation suspension, recorded as RGD-NNV@FD1080&DOX / TFA.

[0073] The structural diagram of the obtained RGD-NNV@FD1080&DOX / TFA is shown in the figure. Figure 2 As shown. Figure 2 It can be seen that it has a photosensitizer structure that is encapsulated by RGD-modified neutrophil nanovesicles and loaded with dual drugs.

[0074] Agarose gel electrophoresis was used to detect the protein spectrum of RGD-NNV@FD1080&DOX / TFA. The Western blot image of RGD-NNV@FD1080&DOX / TFA was shown in the figure below. Figure 3 As shown. Figure 3It can be seen that RGD-NNV@FD1080&DOX / TFA has similar biological properties to RGD-NNV, and the protein profile expression is consistent.

[0075] Transmission electron microscopy and nanoparticle tracking analysis were used to observe the morphological characteristics of RGD-NNV@FD1080&DOX / TFA. The methods are as follows:

[0076] 20 μL of RGD-NNV@FD1080&DOX / TFA solution was mixed and then dropped onto a 2 mm diameter sample-loaded copper grid. After standing for 5 minutes, the residual liquid was removed with filter paper. The copper grid was then inverted on a drop of 30 g / L phosphotungstic acid (pH 6.8). Negative staining was performed at 25°C for 5 minutes, and the cells were dried under an incandescent lamp. The resulting transmission electron microscopy image of RGD-NNV@FD1080&DOX / TFA is shown below. Figure 4 As shown. Figure 4 It can be seen that RGD-NNV@FD1080&DOX / TFA presents a bilayer lipid membrane structure.

[0077] The particle size distribution of RGD-NNV@FD1080&DOX / TFA is shown in the figure below. Figure 5 As shown. Figure 5 It can be seen that RGD-NNV@FD1080&DOX / TFA is a vesicle-like structure with a particle size of about 180 nm.

[0078] The RGD-NNV@FD1080&DOX / TFA (1.5W / cm2) in the EP tube was irradiated by near-infrared laser. 2 , 3min) for infrared heating, the near infrared heating effect of RGD-NNV@FD1080&DOX / TFA is as follows Figure 6 As shown, it can be seen that the infrared heating effect of RGD-NNV@FD1080&DOX / TFA gradually increases with the extension of irradiation time, indicating that it has good photoresponse characteristics.

[0079] Example 2 In vitro antitumor effect of RGD-NNV@FD1080&DOX / TFA on tumor cells

[0080] The raw materials and instruments used in Example 2 are as follows:

[0081] 96-well cell culture plates (JET Biofil), RPMI1640 (Bioind, USA), trypsin (Sigma, USA), CCK8 detection kit (Vazyme, China), and microplate reader (FLX800, United States).

[0082] 1. MGC-803 cells were digested with trypsin and precipitated by centrifugation. MGC-803 cells were seeded into 96-well cell culture plates. After the cells attached, different substances (RGD-NNV (40 μg / mL), RGD-NNV@FD1080+NIR (40 μg / mL), RGD-NNV@FD1080&DOX+NIR (40 μg / mL), RGD-NNV@FD1080&TFA+NIR (40 μg / mL), RGD-NNV@FD1080&DOX / TFA (40 μg / mL), RGD-NNV@FD1080&DOX / TFA+NIR (40 μg / mL)) were added to treat MGC-803 cells.

[0083] 2. After 24 hours of treatment as above, add CCK8 detection reagent to each 96-well cell culture plate and place the culture plate in a CO2 incubator for further culture;

[0084] 3. Use an enzyme-labeled instrument to detect the absorbance of MGC-803 cells at 450 nm.

[0085] The CCK8 experiment was used to observe the effect of RGD-NNV@FD1080&DOX / TFA on the proliferation ability of MGC-803 cells. Figure 7 As shown. Figure 7 It can be seen that the dual targeting effect of RGD and NNV promotes RGD-NNV@FD1080&DOX / TFA to enter tumor cells, and the active ingredients such as NNV, DOX, and TFA are controllably released under the triggering of near-infrared zone II laser, exerting multiple anti-tumor effects and significantly inhibiting the proliferation of MGC-803 cells.

[0086] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a neutrophil biomimetic vesicle nanoformulation, comprising the following steps: Performing a first physical extrusion and a first density gradient separation on peripheral blood neutrophils to obtain neutrophil biomimetic vesicles; Mixing the neutrophil biomimetic vesicles with RGD cyclic peptide for a first co-incubation to obtain RGD-modified neutrophil biomimetic vesicles; Phosphatidylcholine, DSPE-PEG-2K, cholesterol, a photosensitizer, and an organic solvent are mixed to form a film, and the resulting film is mixed with a PD-L1 inhibitor and a doxorubicin hydrochloride solution, followed by extrusion to obtain drug-loaded photosensitive liposomes; The RGD-modified neutrophil biomimetic vesicles and the drug-loaded photosensitive liposomes are subjected to a second co-incubation, a second physical extrusion, and a second density gradient separation to obtain a neutrophil biomimetic vesicle nanoformulation; The RGD cyclic peptide is DSPE-PEG2000-cRGD; The photosensitizer is FD-1080.

2. The preparation method according to claim 1, characterized in that The mass ratio of the neutrophil biomimetic vesicles to the RGD cyclic peptide is 1×10 6 ~5×10 6 Pieces: 100μg.

3. The preparation method according to claim 1, characterized in that The mass ratio of the phosphatidylcholine, DSPE-PEG-2K, cholesterol and photosensitizer is 1-2:1-2:1-2:1-2.

4. The preparation method according to claim 1, characterized in that The extrusion is performed using a liposome extruder.

5. The preparation method according to claim 1, characterized in that The mass ratio of the RGD-modified neutrophil biomimetic vesicles to the drug-loaded photosensitive liposomes is 1×10 6 Pieces: 100~200μg.

6. The preparation method according to claim 1, characterized in that The pressure of the first physical extrusion and the second physical extrusion are independently 500-1000 psi, the temperature is independently 50-60° C., and the time is independently 5-10 min.

7. The preparation method according to claim 1, characterized in that The first co-incubation temperature is 25-37°C and the time is 25-30 minutes; The temperature of the second co-incubation is 25-37° C., and the time is 10-20 minutes.

8. The neutrophil biomimetic vesicle nanoformulation prepared by the preparation method according to any one of claims 1 to 7, comprising RGD-modified neutrophil biomimetic vesicles and drug-loaded photosensitive liposomes encapsulated within the RGD-modified neutrophil biomimetic vesicles, wherein the drug-loaded photosensitive liposomes comprise a liposome membrane layer loaded with a photosensitizer and an active ingredient encapsulated within the membrane layer, wherein the active ingredient comprises a PD-L1 inhibitor and doxorubicin.

9. Use of the neutrophil biomimetic vesicle nanoformulation according to claim 8 in the preparation of gastric cancer targeted drugs.

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