Nanometer preparation for inhibiting tumor metastasis, preparation method and application

Abstract

The invention belongs to the technical field of pharmaceutical preparations. The invention discloses a nano preparation for inhibiting tumor metastasis, a preparation method and application. The nano preparation for inhibiting tumor metastasis comprises liposome spheres and nano spheres. The liposome sphere provided by the invention comprises an inner-layer core structure and a shell wrapping the outer layer of the inner-layer core structure, wherein the inner-layer core structure comprises lecithin, dioleoyl phosphatidyl ethanolamine, cholesterol succinic acid monoester, cholesterol, imidazolyl-cholesterol and DSPE-PEG2000-CS; the core structure of the inner layer is loaded with aspirin and DNaseI; the housing includes platelets. The nano preparation for inhibiting tumor metastasis can dissolve NET, inhibit the platelet function and disintegrate tumor cell-platelet-NET aggregate, and the nanosphere for inhibiting the far-end metastasis of tumors can deeply permeate into the tumors to play a role in killing the tumors.

Description

Technical Field

[0001] The present application belongs to the technical field of pharmaceutical preparations, and in particular relates to a nano preparation for inhibiting tumor metastasis, a preparation method and an application thereof. Background Art

[0002] Doxorubicin (DOX), as a chemotherapeutic drug, has good anti-tumor effects because it can inhibit the synthesis of tumor cell DNA and RNA. However, free drugs are limited in clinical application due to their rapid clearance rate, poor targeting, and strong toxic side effects in the body. Recent studies have shown that lecithin and cholesterol-based nanoparticles have the advantages of good biocompatibility, non-toxicity, non-immunogenicity, and easy synthesis, and have been widely used in the delivery of anti-tumor drugs. Summary of the invention

[0003] Purpose of the invention: The present application provides a nanoformulation, preparation method and application for inhibiting tumor metastasis. The nanoformulation for inhibiting tumor metastasis of the present application includes liposome spheres, which can dissolve neutrophil extracellular traps (NETs), inhibit platelet function, disintegrate tumor cell-platelet-NET aggregates, and inhibit the distal metastasis of tumors.

[0004] Technical solution: The embodiment of the present application provides a nanoformulation for inhibiting tumor metastasis, wherein the nanoformulation for inhibiting tumor metastasis comprises a liposome sphere, wherein the liposome sphere comprises an inner core structure and a shell wrapped in the outer layer of the inner core structure, wherein the inner core structure comprises lecithin, dioleoylphosphatidylethanolamine, cholesterol succinate monoester, cholesterol, imidazole-cholesterol and DSPE-PEG 2000 -CS; the inner core structure is loaded with aspirin and DNase Ⅰ; the shell includes platelets; the lecithin, dioleoylphosphatidylethanolamine, cholesterol succinate, cholesterol, imidazole-cholesterol and DSPE-PEG 2000 -CS mass ratio is (35-45): (55-65): (10-20): (10-20): (5-15): (1-5); based on the total mass of the liposome sphere, the mass percentage of aspirin loaded in the liposome sphere is 1%-5%; based on the mass of the inner core structure per milligram, the amount of DNase I loaded in the inner core structure is 0.6U / mg-2U / mg; The amount of platelets meets the requirement: M1= (M2×M0) / 250, where M1 is the mass of platelets in mg, M2 is the mass of lecithin in mg, and M0 is the mass of platelets extracted from 1 mL of whole blood in mg.

[0005] In some embodiments, the phosphatidylcholine, dioleoylphosphatidylethanolamine, cholesterol succinate, cholesterol, imidazolyl-cholesterol and DSPE-PEG 2000 -The mass ratio of CS is 40:60:15:15:10:5.

[0006] In some embodiments, based on the total mass of the liposome sphere, the mass percentage of the aspirin loaded in the liposome sphere is any value of 1%, 2%, 3%, 4%, 5%, or a range consisting of any two values.

[0007] In some embodiments, the amount of DNase I loaded on the inner core structure per milligram of the mass of the inner core structure is any value of 0.6 U / mg, 1 U / mg, 1.5 U / mg, 2 U / mg, or a range consisting of any two values.

[0008] In some embodiments, the average particle size of the liposome spheres is 150 nm to 160 nm, such as the average particle size of the liposome spheres is any value of 150 nm, 151 nm, 152 nm, 153 nm, 154 nm, 155 nm, 156 nm, 157 nm, 158 nm, 159 nm, 160 nm, or a range consisting of any two values.

[0009] In some embodiments, the DSPE-PEG 2000 The structural formula of -CS is as follows: .

[0010] In some embodiments, the nanoformulation for inhibiting tumor metastasis further comprises nanospheres (D / GN), wherein the nanospheres comprise glycocholic acid, phosphatidylcholine, DSPE-PEG 2000 -GA and doxorubicin, the glycocholic acid, phosphatidylcholine and DSPE-PEG 2000 -GA mass ratio is (15-20): (20-25): 1; based on the total mass of the nanospheres, the mass percentage of the doxorubicin loaded on the nanospheres is 1%-5%.

[0011] In some embodiments, the mass ratio of glycocholic acid to phosphatidylcholine is 1:1.2.

[0012] In some embodiments, the average particle size of the nanospheres is in the range of 25 nm to 30 nm, such as the average particle size of the nanospheres is any value of 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, or a range consisting of any two values.

[0013] In some embodiments, glycocholic acid, phosphatidylcholine and DSPE-PEG 2000The mass ratio of -GA is 18:22:1. In some embodiments, based on the total mass of the nanospheres, the mass percentage of doxorubicin loaded on the nanospheres is any value of 1%, 2%, 3%, 4%, 5%, or a range consisting of any two values.

[0014] In some embodiments, the liposome spheres are prepared by the following method: (S11) DSPE-PEG 2000 Synthesis of -CS:DSPE-PEG 2000 -NH2 was activated by EDC and NHS, and then cross-linked with chondroitin sulfate under nitrogen to obtain DSPE-PEG 2000 -CS; (S12) Phosphatidylcholine, dioleoylphosphatidylethanolamine, cholesterol succinate, cholesterol, imidazole-cholesterol, DSPE-PEG 2000 -CS and aspirin were dissolved in ethanol, and the ethanol was removed using a rotary evaporator in a water bath at 50°C to 60°C and a rotation speed of 10 rpm to 30 rpm; (S13) adding DNase I to a PBS solution, dissolving it by ultrasonication, adding it to the system formed in step (S12), and hydrating it by rotary evaporation for 30 min to 60 min; (S14) The product obtained in step (S13) is ultrasonically crushed in an ice bath, platelets are added and mixed, and liposome spheres wrapped by platelet membrane are obtained through an extruder.

[0015] In some embodiments, the nanospheres are prepared by the following method: (S21) dissolving glycocholic acid in a PBS solution to obtain a first solution; (S22) Phosphatidylcholine, doxorubicin, DSPE-PEG 2000 -GA is dissolved in ethanol by ultrasonication to obtain a second solution; (S23) dripping the second solution into the first solution, and after the ethanol is completely evaporated, ultrasonically crushing the product in an ice bath, and sequentially passing through polyethersulfone membranes with pore sizes of 0.45 μm and 0.22 μm to obtain nanospheres.

[0016] The present application further provides a method for preparing a nanoformulation for inhibiting tumor metastasis, comprising preparing liposome spheres; the liposome spheres are prepared by the following method: (1) Phosphatidylcholine, dioleoylphosphatidylethanolamine, cholesterol succinate, cholesterol, imidazole-cholesterol, DSPE-PEG 2000 -CS and aspirin were dissolved in ethanol, and the ethanol was removed using a rotary evaporator in a water bath at 50°C to 60°C and a rotation speed of 10 rpm to 30 rpm; (2) Add DNase I to PBS solution, dissolve by ultrasonication, add to the system formed in step (1), and hydrate by rotary evaporation for 30 min to 60 min; (3) The product obtained in step (2) is ultrasonically crushed in an ice bath, platelets are added and mixed, and liposome spheres wrapped by platelet membrane are obtained through an extruder.

[0017] In some embodiments, the method for preparing the nanoformulation for inhibiting tumor metastasis further comprises preparing nanospheres, wherein the nanospheres are prepared by the following method: Dissolving glycocholic acid in PBS solution to obtain a first solution; Phosphatidylcholine, doxorubicin, DSPE-PEG 2000 -GA is dissolved in ethanol by ultrasonication to obtain a second solution; The second solution was dripped into the first solution. After the ethanol was completely evaporated, the product was ultrasonically crushed in an ice bath and passed through polyethersulfone membranes with pore sizes of 0.45 μm and 0.22 μm in sequence to obtain nanospheres.

[0018] The embodiments of the present application further provide the use of the above-mentioned nanoformulation for inhibiting tumor metastasis or the nanoformulation prepared by the above-mentioned method for preparing the nanoformulation for inhibiting tumor metastasis in the preparation of drugs for treating tumors and drugs for inhibiting tumor metastasis.

[0019] In some embodiments, the tumor described herein is liver cancer.

[0020] Beneficial effects: The present application discloses a nanoformulation, preparation method and application for inhibiting tumor metastasis. The nanoformulation for inhibiting tumor metastasis of the present application comprises liposome spheres and nanospheres. The liposome spheres of the present application comprise an inner core structure and a shell wrapped in the outer layer of the inner core structure, wherein the inner core structure comprises lecithin, dioleoylphosphatidylethanolamine, cholesterol succinate monoester, cholesterol, imidazole-cholesterol and DSPE-PEG 2000 -CS; the inner core structure is loaded with aspirin and DNase I; the shell includes platelets. The nanoformulation for inhibiting tumor metastasis of the present application can dissolve NET, inhibit platelet function, disintegrate tumor cell-platelet-NET aggregates, and inhibit the distal metastasis of the tumor. The nanospheres in the nanoformulation for inhibiting tumor metastasis of the present application can deeply penetrate into the tumor and play a role in killing the tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1The electron micrographs of liposomes wrapped by platelet membranes at three different feed ratios in the examples of the present application, wherein Figures A, B, and C are platelet membrane-wrapped lipid spheres formed after extrusion of liposomes prepared by extruding platelets extracted from 1 mL of whole blood and 100 mg, 250 mg, and 350 mg of lecithin, respectively; Figure 2 Schematic diagram of the preparation of lipid spheres (A&E / CPL) and nanospheres (D / GN) in the examples of this application; Figure 3 The physicochemical properties of A&E / CPL and D / GN prepared in the examples of the present application are characterized, wherein Figure A is a particle size distribution diagram of A&E / CPL; Figure B is a particle size distribution diagram of D / GN; Figure 4 TEM images of A&E / CPL prepared in the examples of the present application, wherein Figure A, Figure B, and Figure C are TEM images of A&E / CPL under the conditions of pH=7.4, pH=6.0, and pH=5.5, respectively; Figure 5 This is a TEM image of D / GN prepared in an example of the present application at pH = 7.4; Figure 6 The 7-day stability of A&E / CPL prepared in the examples of the present application in PBS solution and DMEM medium, wherein the left ordinate is the particle size measured by dynamic light scattering (DLS), and the right ordinate is the dispersion index (PDI); Figure 7 The 7-day stability of D / GN prepared in the examples of the present application in PBS solution and DMEM medium, wherein the left ordinate is the particle size measured by dynamic light scattering (DLS), and the right ordinate is the dispersion index (PDI); Figure 8 Coomassie Brilliant Blue image of A&E / CPL prepared for the examples of this application; Fig. 9 The in vitro drug release diagram of A&E / CPL prepared in the examples of this application under different pH conditions; Fig.10 This is the in vitro drug release diagram of D / GN prepared in the examples of this application; Fig.11 Horizontal gel electrophoresis image of A&E / CPL prepared for the examples of the present application; Fig.12 The in vitro cellular uptake results of D / GN prepared in the examples of the present application, wherein Figure A is a laser confocal image of D / GN uptake in vitro; Figures B and C are the quantitative results of D / GN uptake in vitro by flow cytometry; Fig.13The cytotoxicity evaluation model of D / GN combined with A&E / CPL prepared in the examples of the present application, wherein Figure A is a HuH-7 model alone, and Figure B is a schematic diagram of a HuH-7+PLT+NET co-culture model; Fig.14 The cytotoxicity evaluation results of D / GN combined with A&E / CPL prepared in the examples of the present application, wherein Figure A is the MTT test result of the HuH-7 model alone; Figure B is the MTT test result of the HuH-7+PLT+NET co-culture model; Fig.15 The results of the investigation of the cell migration mechanism by D / GN combined with A&E / CPL prepared in the examples of the present application, wherein Figure A is the Transwell cell invasion result of the HuH-7+PLT+NET co-culture model; Figure B is the Transwell invasion quantitative result of the HuH-7+PLT+NET co-culture model; Figure C is the Western blot analysis result of E-Cadherin and Vimentin; Figure D is the quantitative analysis result of E-Cadherin and Vimentin; Fig.16 The biodistribution test results of A&E / CPL and D / GN prepared in the examples of this application in tumor-bearing mice. Figure A is the NIR images of A&E / CPL and D / GN at different time points; Figure B is the fluorescence image of each group of organs and tumors at 48 hours; Figures C and D are the fluorescence intensity analysis of each group of organs and tumors at DID and DIR wavelengths; Fig.17 The results of the effect of A&E / CPL prepared in the examples of the present application on the tumor penetration of D / GN, wherein Figure A is a frozen section image of the tumor of each treatment group at different depths; Figure B is a quantitative image of the DID fluorescence intensity in each group of tumors at different depths; Fig.18 This application uses D / GN combined with A&E / CPL to evaluate the anti-tumor effect of H22+PLT+NET subcutaneous transplanted tumors, wherein Figure A is a schematic diagram of H22+PLT+NET subcutaneous transplanted tumors; Figure B is a photo of tumors in different treatment groups; Figure C is the change in tumor volume in different treatment groups; Figure D is the change in mouse weight in different treatment groups; Figure E is the inhibition rate of tumor growth in different treatment groups; Fig.19The evaluation results of the therapeutic effect of D / GN combined with A&E / CPL on H22+PLT+NET subcutaneous transplanted tumors in this application, wherein Figure A is the H&E image of tumors in different treatment groups; Figure B is the CD31 immunohistochemistry image of tumors in different treatment groups; Figure C is the α-SMA immunohistochemistry image of tumors in different treatment groups; Figure D is the Masson image of tumors in different treatment groups; Figure E is the Western blot analysis result of E-Cadherin and Vimentin in tissues; Figure F is the quantitative analysis result of E-Cadherin and Vimentin; Fig. 20 H&E images of major organs including heart, liver, spleen, lung, and kidney in different treatment groups for this application; Fig.21 The hemolysis test results of A&E / CPL and D / GN prepared in the examples of this application; Fig. 22 The evaluation results of the treatment effect of lung metastasis by D / GN combined with A&E / CPL in this application, wherein Figure A is a schematic diagram of the establishment and treatment of lung metastasis; Figure B is the weight change of mice in each treatment group; Figure C is the number of metastatic lung nodules in each treatment group; Figure D is a photo of the lungs of each treatment group; Figure E is an H&E image of the lungs of each treatment group; Fig.23 For this application, the effects of D / GN combined with A&E / CPL on lung neutrophil and NET colonization were investigated. DETAILED DESCRIPTION

[0022] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used in the present application have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0023] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present application. In addition, in the description of the present application, the term "including" means "including but not limited to". The terms first, second, third, etc. are used only as labels, and no numerical requirements or order are imposed. Various embodiments of the present application may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and simplicity, and should not be understood as a hard limit to the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single values ​​within the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the numbered range, such as 1, 2, 3, 4, 5 and 6, which are applicable regardless of the range. Additionally, whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in the field described in the present application. Although the present application only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present application. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. When conflicting with any incorporated documents, the content of this specification shall prevail. Unless otherwise stated, "%" is a percentage based on mass, and the solvent ethanol used refers to an ethanol solution of mass percentage content ≥95%.

[0025] 1. Materials and Methods 1. Materials Phosphatidylcholine (PC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol were purchased from Shanghai AVT Pharmaceutical Technology Co., Ltd. Aspirin (ASP), deoxyribonuclease I (DNase Ⅰ), and doxorubicin (DOX) were purchased from Dalian Meilun Biotechnology Co., Ltd. Glycosaminoglycan (GAH) was purchased from MedChemExpress (USA). Antibodies such as E-cadherin, vimentin, and platelet-endothelial cell adhesion molecule (CD31) were purchased from Abcam (Massachusetts, USA). Other chemical reagents were of analytical or chromatographic grade.

[0026] 2. Cells and Animals Human hepatoma cell line (HuH-7) and mouse hepatoma cell line (H22) were purchased from Beijing Institute of Life Sciences. BALB / c mice (female, 5 weeks old, 18 g-22 g) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. All animal experiments were approved by the Animal Experiment Ethics Committee of Shandong Second Medical University (SDSMU, 2019-045), and the care and use of experimental animals strictly complied with the Animal Management Regulations (No. 55, 001).

[0027] 2. Test Method 1. Characterization of physical and chemical properties of A&E / CPL and D / GN The particle size distribution and zeta potential of A&E / CPL and D / GN were measured by Malvern particle size potential analyzer. A&E / CPL and D / GN were diluted 10 times with PBS or DMEM medium and stored at 4°C for 7 days, and the particle size changes of A&E / CPL and D / GN in PBS or DMEM medium were measured respectively. A&E / CPL and D / GN were diluted 30 times with PBS and negatively stained with uranyl acetate. After the samples were dried, the morphology of A&E / CPL and D / GN was observed by transmission electron microscopy (TEM). The content of DOX in D / GN was determined by ultraviolet spectrophotometer at 480nm, and the content of ASP in A&E / CPL was determined by high performance liquid chromatography (HPLC) at 276nm. The encapsulation efficiency (EE) and drug loading (LE) were calculated as follows: .

[0028] 2. In vitro drug release of A&E / CPL and D / GN The in vitro drug release of ASP from A&E / CPL was evaluated in PBS buffer with different pH values ​​(pH=7.4, 6.5 or 5.5). 1 mL of A&E / CPL was added to a dialysis bag (MWCO=3500), which was sealed and placed in a 50 mL centrifuge tube containing 40 mL of PBS buffer with different pH values ​​(containing 0.5% Tween-80) as the release medium. The release system was placed in a constant temperature shaking incubator at 37°C and 100 rpm. At the specified time points (0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 36h, 48h), 2 mL of the release medium was taken out and 2 mL of fresh release medium was added at the same time. The amount of released ASP was then measured by HPLC at 276 nm.

[0029] 3. Hemolytic evaluation 1 mL of blood from healthy mice was collected by eye removal and placed in a centrifuge tube. After centrifugation at 3500 rpm for 10 min, the supernatant was removed and saline was added to wash the red blood cells. After the cells were resuspended, the above centrifugation steps were repeated until the supernatant was colorless. The red blood cells were diluted with saline to obtain a 2% red blood cell suspension for use. The nanoparticle preparation was mixed with the red blood cell suspension to obtain samples of different concentrations (0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, 5 mg / mL), and Triton X-100 (Triton X-100) and saline were used as positive and negative controls, respectively. All samples were placed in a constant temperature shaking incubator for incubation for 2 h, and then the samples were centrifuged (3500 rpm, 10 min). Observe and take pictures, and take the supernatant in a 96-well plate. The absorbance value (OD value) was measured at 545 nm on an ELISA reader. The hemolysis rate (Hemolysis, %) was calculated as follows: Among them, A, A1 and A2 represent the absorbance values ​​of the positive control group, experimental group and negative control group, respectively.

[0030] 4. Construction of in vitro co-culture research model simulating microenvironment In the tumor microenvironment, there are not only cancer cells, but also platelets (PLT) and neutrophil extracellular traps (NET), which can promote tumor proliferation and metastasis. In order to simulate the real tumor microenvironment, we established a co-culture model consisting of HuH-7 cells, PLT and NET. HuH-7 cells, PLT and induced NET were added to the culture plate in a ratio of 2:20:1 and cultured for 16 hours to establish a co-culture cell model.

[0031] 5. Cellular uptake assay To investigate the uptake of nanoparticles by tumor cells, HuH-7 cells were inoculated in a cell culture dish (5 × 10 4 Cells / culture dish), after the cells adhered and grew, free DOX, DOX-loaded nanospheres (D / N) and GA-modified nanospheres (D / GN) (DOX concentration was 10 μg / mL) were added and treated with drugs for 1 hour. After washing three times with PBS, the tissue cells were fixed with fixative for 10 minutes and stained with DAPI (concentration of 1 μg / mL) nuclear dye for 10 minutes. After washing three times with PBS, the cells were photographed with a laser confocal microscope and quantitatively detected by AccuriC6 Plus flow cytometer.

[0032] 6. In vitro cytotoxicity assay The MTT method was used to detect the toxicity of different preparations to tumor cells. In order to better evaluate the effect of drugs in the tumor microenvironment, we established two cell models: (1) HuH-7 cells; (2) "HuH-7+PLT+NET" co-culture cell model. First, these two cell models were inoculated in 96-well plates, and 6×10 3 cells and cultured them in a 37°C, 5% CO2 incubator for 16 h. Add DOX, DOX+ASP+DNase Ⅰ, D / GN, A&E / CPL, D / GN+A&E / CPL and other drugs, the DOX concentration was set at 0.01μg / mL~10μg / mL, and the drugs were treated for 48 h. Add MTT solution (concentration of 5 mg / mL) to each well and incubate for 4 h. Remove the culture medium and add 150μL dimethyl sulfoxide (DMSO) to each well. Use an ELIX800, BioTek, USA to detect the OD value of each well at 490nm, and calculate the cell survival rate of each group.

[0033] 7. Cell migration and invasion analysis The in vitro anti-migration and anti-invasion abilities of DOX, ASP+DNase Ⅰ, DOX+ASP+DNase Ⅰ, D / GN, A&E / CPL, and D / GN+A&E / CPL were evaluated by scratch healing assay and Transwell cell invasion assay.

[0034] (1) Scratch test. HuH-7 cells were incubated in a six-well plate. When the cell confluence rate reached 80%, the cell layer was scratched with a 200 µL sterile pipette tip, and three straight lines were drawn in each well. After washing with PBS, the wound width at 0 h was observed under an inverted microscope. Then, the cells were treated with different preparations and cultured in an incubator for 24 h. The cells were washed with PBS three times, and the wound width at 24 h was observed again. The migration rate was quantified using ImageJ software. The calculation is shown in the following formula:

[0035] Among them, d 0h Initial wound width, in mm, d 24h It is the 24h wound width in mm.

[0036] (2) Transwell cell invasion assay. First, dilute Matrigel with serum-free medium (1:5) on ice. Add the prepared Matrigel into the Transwell chamber and incubate at 37°C for 1 h. Pre-treat cells with different preparations, collect cells 24 h later, and resuspend in serum-free medium (cell density, 1×10 5 / mL), take 200µL of cell suspension and add it to the chamber, add medium containing 10% FBS (volume: 600µL) as an inducer to the 24-well plate under the chamber, incubate at 37℃ for 24h, discard the upper chamber medium, gently wipe off the upper layer of matrix gel in the chamber with a sterile cotton swab, wash the cells with PBS three times, fix the cells that migrated to the lower layer of the chamber with tissue cell fixative for 20min, then rinse with PBS three times, stain with crystal violet for 20min, and observe under a microscope after rinsing with PBS three times. Finally, use ImageJ software to quantitatively analyze the cells that migrated to the lower surface of the chamber.

[0037] 8. Western blot analysis of anti-liver cancer mechanism Western blot was used to detect the expression of E-cadherin, Vimentin and other proteins in liver cancer cells. Tumor cells and liver cancer tissues were lysed to extract proteins, spotted on 12% SDS-PAGE gel electrophoresis, and transferred to PVDF membrane. Then, the PVDF membrane was sealed with 5% emulsion TBST at room temperature for 2 hours, and then incubated with primary antibodies at 4°C overnight. Subsequently, secondary antibodies were added and incubated at room temperature for 1 hour. Protein expression was detected using ultrasensitive ECL chemiluminescent solution.

[0038] 9. In vivo imaging and tissue penetration analysis Two fluorescent substances, DID and DIR, were selected to replace drugs to detect the biodistribution of D / GN and A&E / CPL in H22+PLT+NET tumor-bearing mice. The mixed cell suspension was subcutaneously injected into BALB / c mice to establish the H22+PLT+NET tumor-bearing model. When the tumor volume was about 180mm 3 At the same time, the mice were divided into DID, DIR, DID-GN, DIR-CPL and DID-GN+DIR-CPL. The biodistribution of DID and DIR in mice was monitored at different time points using a real-time fluorescence imaging system (IVIS). Organs and tumors were harvested 48 hours later for in vitro fluorescence intensity analysis.

[0039] In order to evaluate the permeability of D / GN, an H22+PLT+NET tumor-bearing mouse model was established. 3 At the same time, the mice were divided into 5 groups: DID, DID-D / GN, DID-D / GN (DNase Ⅰ pretreatment), DID-D / GN (ASP+DNase Ⅰ pretreatment), DID-D / GN (A&E / CPL pretreatment). All pretreatment groups were treated continuously for 3 days via tail vein. Mice were killed 12 h after DID injection, and the tumors were dissected. The tumor tissues were then cut into 10 μm thick sections. Tumor sections were stained with DAPI and then photographed using a laser confocal microscope (TCSSP8, Leica).

[0040] 10. Evaluation of tumor inhibition in subcutaneous transplanted tumors To evaluate the therapeutic effect of A&E / CPL and D / GN liver cancer, a H22+PLT+NET subcutaneous tumor-bearing mouse model was established with a tumor volume of approximately 150 mm 3 , divided into 8 groups (n=5): (1) control group; (2) saline; (3) DOX; (4) ASP+DNase Ⅰ; (5) DOX+ASP+DNase Ⅰ; (6) D / GN; (7) A&E / CPL; (8) D / GN+A&E / CPL. The following drug doses were injected into the tail vein: DOX (3 mg / kg), ASP (3 mg / kg), and DNase Ⅰ (200 U / mouse), for a total of 7 doses. The tumor size and body weight of the mice were monitored every other day after drug treatment. Two weeks later, the mice were killed, and the heart, liver, spleen, lung, kidney and tumor were removed and weighed. The tumor and important organs were fixed with 4% paraformaldehyde. The tumor sections were then subjected to H&E staining, immunohistochemical analysis, Masson trichrome staining, and Western blot analysis.

[0041] 11. Lung metastasis experiment Tail vein injection 2×10 6 H22 cells were used to construct a liver cancer lung metastasis model. Then, the mice were randomly divided into 7 groups (n=3): (1) saline; (2) DOX; (3) DOX+ASP+DNase I; (4) D / GN; (5) A&E / CPL; (6) D / GN+A&E / CPL. The treatment was once every 2 days for a total of 7 times. The mice were killed after 14 days. The lungs were dissected and fixed with 4% paraformaldehyde. The number of metastatic nodules was then measured, and the lung tissue sections were subjected to H&E staining and His H3 and Ly6G immunofluorescence staining.

[0042] 12. Detection of NET formation by neutrophils in the lungs To evaluate the effects of A&E / CPL and D / GN on neutrophils and neutrophil extracellular trap (NET) formation in the lungs, lung immunofluorescence experiments were performed. 6H22 cells were injected into the tail vein of mice, and then the mice were randomly divided into 7 groups (n=3): (1) normal saline; (2) DOX; (3) ASP+DNase Ⅰ; (4) DOX+ASP+DNase Ⅰ; (5) A&E / CPL; (6) D / GN; (7) A&E / CPL+D / GN. The tail vein was administered every other day from the second day of tumor bearing, and the treatment was performed 7 times in total. After the treatment, the mice were killed, and the lungs were dissected and cryoembedded in OCT cryoembedding medium (optimal cutting temperature compound). The tissues were cryosectioned with a Leica cryostat at a thickness of 10μm. Subsequently, His H3 and Ly6G protein immunofluorescence staining was performed, and the tissues were observed and photographed using a laser confocal microscope.

[0043] 13. Statistical analysis Data are presented as mean ± standard deviation. Prism 8.0 (GraphPad) was used for data plotting and statistical analysis. The differences between the two groups were analyzed by student's t test or analysis of variance, and p < 0.05 was considered statistically significant.

[0044] 3. Sample preparation and experimental results 1. Sample preparation Example 1: Preparation of A&E / CPL liposome spheres 1. Preparation of platelet membrane 1 mL of whole blood from BALB / c mice was collected by eyeball blood sampling and placed in a centrifuge tube containing 50 μL of sodium heparin anticoagulant. The blood was centrifuged at 300×g for 10 min at 4°C, and the supernatant and the white cell layer at the liquid boundary were collected. The supernatant was centrifuged at 2000×g for 10 min at 4°C, and the white platelet precipitate was collected and washed twice with PBS containing anticoagulant. After repeated freezing and thawing, platelet membranes were obtained.

[0045] 2. Preparation of platelet membrane-encapsulated liposomes co-loaded with ASP and DNase I (A&E / CPL) (1) Screening of the feed ratio of platelet membrane and liposome: fix the content of platelet membrane, change the content of liposome, and examine the effect of different feed ratios on the platelet membrane encapsulation efficiency. First, determine the ratio of platelet membrane and liposome. Mix the platelets taken from 1 mL of whole blood with 100 mg, 250 mg, and 350 mg of liposomes prepared with lecithin, respectively. Then extrude them through an extruder to prepare platelet membrane-encapsulated liposomes. Observe the morphology of the three liposomes and the platelet membrane encapsulation under an electron microscope to determine the best feed ratio and obtain the highest encapsulation efficiency. The results are as follows: Figure 1 As shown, Figure 1Figures A, B, and C are electron micrographs of platelet membrane-wrapped lipid spheres formed after platelets extracted from 1 mL of whole blood were extruded into liposomes prepared with 100 mg, 250 mg, and 350 mg of lecithin, respectively. Figure 1 The results show that under the condition of constant platelet membrane content, adding too much or too little lecithin will result in poor encapsulation efficiency. At the feed ratio of platelets taken from 1 mL of whole blood: 250 mg of lecithin, the platelet membrane encapsulation efficiency is the highest. This feed ratio was finally selected to prepare platelet membrane-encapsulated lipid spheres.

[0046] (2) Preparation of A&E / CPL liposomes Preparation of imidazole-cholesterol (AIM-Chol): Dissolve cholesteryl chloroformate (0.45 g, 1 mmol) in dichloromethane (10 mL). Add N-(3-aminopropyl)imidazole (0.6 mL, 4 mmol) dropwise, then add 200 μL triethylamine, stir in an ice bath for 10 h, and remove the solvent by vacuum distillation after the reaction. Purify the product by column chromatography. Under the catalytic action of triethylamine, cholesteryl chloroformate and N-(3-aminopropyl)imidazole generate AIM-Chol.

[0047] DSPE-PEG 2000 Preparation of -CS:DSPE-PEG 2000 -NH2 was activated by EDC and NHS, and then cross-linked with chondroitin sulfate (CS) under nitrogen to obtain DSPE-PEG 2000 -CS.

[0048] Phosphatidylcholine (PC) 40 mg, dioleoylphosphatidylethanolamine (DOPE) 60 mg, cholesterol succinate monoester (CHEMS) 15 mg, cholesterol 15 mg, imidazolyl-cholesterol (AIM-Chol) 10 mg, DSPE-PEG 2000 -CS 5mg, aspirin (ASP) 5mg were dissolved in 5mL ethanol. Ethanol was removed by rotary evaporator at 55°C and 18rpm in a water bath. 200U DNase I was added to 5mL PBS and dissolved by ultrasonic. When a uniform film was formed on the bottom of the round-bottom flask, the above PBS solution was added and hydrated by rotary evaporation for 1h. After that, the product was ultrasonically broken at a power of 120W for 10min in an ice bath. Finally, the platelets were mixed with the above product in the ratio of platelets taken from 1mL blood: liposomes prepared from 250mg lecithin, and platelet membrane-wrapped liposome spheres (A&E / CPL) were obtained by extruder with a pore size of 200nm.

[0049] Example 2: Preparation of DOX-loaded liver-targeted nanospheres (D / GN) DSPE-PEG 2000 - Preparation of GA: synthesized by the method of Example 1 of Chinese patent CN2018113191397.

[0050] (1) Optimization of the preparation process of nanospheres D / GN DOX-loaded nanospheres were prepared by ethanol injection method ( Figure 2 ). First, GAH was dissolved in PBS and the pH was adjusted to 5.5-6.0 with 1 mol / L NaOH solution. Secondly, DOX, PC and DSPE-PEG were added according to different mass ratios of GAH and lecithin (1:1.2, 1:1.4, 1:1.7, 1:2.2 and 1:3.2). 2000 -GA ethanol solution (DSPE-PEG 2000 -GA / PC ratio of 1:20) was dropped into the GAH solution. After ethanol was evaporated, it was sonicated in an ice bath and passed through 0.45μm and 0.22μm polyethersulfone membranes in sequence to obtain D / GN.

[0051] Table 1 Effects of different ratios of lecithin and glycocholic acid on particle size and PDI of D / GN

[0052] (2) Preparation of nanospheres D / GN 90 mg of glycocholic acid was added to 5 mL of PBS solution, and the pH of the solution was adjusted to 5.5-6.0 with 1 mol / L NaOH to obtain the first solution. 110 mg of lecithin, 5 mg of doxorubicin (DOX), and 5 mg of DSPE-PEG 2000 -GA was dissolved in 2 mL of ethanol and sonicated to obtain a second solution, which was slowly dripped into the first solution. After the ethanol was completely evaporated, the product was sonicated in an ice bath at a power of 120 W for 10 min and passed through 0.45 μm and 0.22 μm polyethersulfone membranes in sequence to obtain nanospheres (D / GN).

[0053] Comparative Example: Preparation of DOX-loaded nanospheres (D / N): The preparation method is the same as that of nanospheres (D / GN), except that DSPE-PEG is not added. 2000 -GA.

[0054] (II) Experimental results 1. Characterization and analysis of the physical and chemical properties of A&E / CPL and D / GN The physical and chemical properties of A&E / CPL and D / GN are shown in Table 2 and Figure 3 As shown. The average particle size of A&E / CPL is about 155.6nm ( Figure 3 As shown in Figure A), it has negative potential characteristics. Figure 3 As shown in Figure B, the average particle size of D / GN is 27.5nm, which not only retains the function of nano-particles to penetrate deep into the tumor, but also avoids high renal metabolism caused by too small particle size, improves its bioavailability, and has a negative zeta potential, which prolongs the drug circulation time in the body. The drug loading and encapsulation efficiency of ASP and DOX are 2.52%, 2.20% and 82.51%, 90.20%, respectively, with high drug loading and encapsulation efficiency.

[0055] Table 2 Physical and chemical properties of A&E / CPL and D / GN

[0056] The physicochemical properties of A&E / CPL prepared in this application are as follows Figure 4 shown. Figure 4 TEM images of A&E / CPL prepared in the examples of the present application, wherein Figure A, Figure B, and Figure C are TEM images of A&E / CPL under the conditions of pH=7.4, pH=6.0, and pH=5.5, respectively; Figure 4 The results show that the A&E / CPL prepared in the present application is in a regular spherical shape at pH=7.4, while the particle size of A&E / CPL gradually increases at pH=6.0 and pH=5.5, and the shape becomes irregular and disintegrates, indicating that it is pH sensitive. Figure 5 As shown, the TEM image of D / GN prepared in the example of the present application under the condition of pH=7.4, the D / GN prepared in the present application is a regular sphere at pH=7.4.

[0057] Figure 6 The 7-day stability of A&E / CPL prepared in the examples of this application in PBS solution and DMEM medium, wherein the left ordinate is the particle size measured by dynamic light scattering (DLS), and the right ordinate is the dispersion index (PDI). Figure 6 The results show that the particle size of A&E / CPL in PBS and DMEM culture medium is stable and uniform within 7 days, indicating that it has good stability.

[0058] Figure 7 The 7-day stability of D / GN prepared in the examples of the present application in PBS solution and DMEM medium, wherein the left ordinate is the particle size measured by dynamic light scattering (DLS), and the right ordinate is the dispersion index (PDI); Figure 7 The results show that the particle size of D / GN prepared in the present application is stable and uniform in PBS and DMEM culture media within 7 days, indicating that it has good stability.

[0059] Figure 8This is the Coomassie Brilliant Blue image of A&E / CPL prepared in the embodiment of the present application. The Coomassie Brilliant Blue result shows that A&E / CPL carries most of the proteins on the platelet membrane, including key functional proteins such as CD11b and CD47.

[0060] Fig. 9 The in vitro drug release diagram of A&E / CPL prepared in the examples of this application under different pH conditions. This application once again verifies its sensitivity to pH by measuring the release rate of ASP in A&E / CPL under different pH conditions. Fig. 9 As shown in the figure, under the condition of pH = 5.5, the cumulative release rate of ASP within 48 h was 75.9%, which was significantly higher than pH = 6.0 (48.8%) and pH = 7.4 (35.6%), indicating that A&E / CPL is pH sensitive and is more likely to release drugs under acidic conditions.

[0061] Fig.10 The in vitro drug release diagram of D / GN prepared in the examples of this application. From the release curve of D / GN, it can be seen that compared with free DOX, the in vitro cumulative release rate of D / GN within 6 hours is lower, indicating that it has long in vitro circulation. At 48 hours, its in vitro cumulative release rate is as high as 73.3%, which is close to 78.9% of free DOX, indicating that it has good in vitro drug release characteristics.

[0062] Fig.11 This is a horizontal gel electrophoresis image of A&E / CPL prepared in the examples of the present application. The SDS-PAGE gel electrophoresis results show that A&E / CPL can successfully encapsulate DNase I and highly retain the characteristics of solubilizing plasmids.

[0063] 2. In vitro cellular uptake Fig.12 The in vitro cellular uptake results of D / GN prepared in the examples of this application. Fig.12 Figure A in the middle is a confocal laser image of D / GN uptake in vitro; Fig.12 Figures B to C in the middle are the quantitative results of D / GN in vitro cell uptake by flow cytometry. The preparation method of D / N nanospheres is similar to that of D / GN nanospheres, except that there is no DSPE-PEG in D / N nanospheres. 2000 -GA. from Fig.12 As can be seen from Figure A, the D / GN group prepared in the present application showed a stronger red fluorescence signal than the free DOX group, indicating that compared with the free drug, nanospheres with nanoscale particle size can be more easily taken up by cells, thereby achieving a stronger anti-cancer effect. Secondly, compared with the D / N group, the D / GN group still showed a stronger red fluorescence signal, suggesting that D / GN may increase the uptake of nanomicelles by liver cancer cells through endocytosis mediated by GA-GA receptors on the surface of liver cancer cell membranes. Fig.12The flow cytometry quantitative experiments in Figures B and C also verified that D / GN increased the DOX content in cells, which was consistent with the qualitative results of confocal laser scanning microscopy (CLSM).

[0064] 3. In vitro cytotoxicity Fig.13 The cytotoxicity evaluation model of D / GN combined with A&E / CPL prepared in the examples of this application, Fig.14 These are the cytotoxicity evaluation results of the D / GN combined with A&E / CPL prepared in the examples of this application. Fig.13 Figure A in the middle is a separate HuH-7 model. Fig.13 Figure B is a schematic diagram of the HuH-7+PLT+NET co-culture model. Fig.14 Figure A in the middle shows the MTT test results of the HuH-7 model alone; Fig.14 Figure B shows the MTT test results of the HuH-7+PLT+NET co-culture model. The tumor microenvironment (TME) plays a vital role in the occurrence and development of tumors. Tumor-associated platelets (PLT) and neutrophil extracellular traps (NET) in the tumor microenvironment can promote tumor cell immune escape and help tumors metastasize to distant sites. Therefore, the traditional in vitro research model, that is, simple tumor cell culture, can no longer simulate the real and complex TME. Therefore, in this application, based on simple liver cancer cells (HuH-7), we established a HuH-7+PLT+NET co-culture cell model to simulate the real tumor microenvironment, so as to detect the in vitro cytotoxic effects of different drug preparations ( Fig.13 As shown in Figures A and B in the figure. Fig.14 As shown in Figures A and B, both cell models showed significant concentration dependence on various drug preparations. Compared with simple HuH-7 cells, the IC50 of the free DOX group in the co-culture system increased significantly, which was due to the protective effect of PLT and NET on tumor cells, leading to tumor resistance. In the co-culture system, the A&E / CPL+D / GN group had a stronger cytotoxic effect than the DOX+ASP+DNase Ⅰ group. This may be due to the mediation of nanospheres with nanoparticle size, namely GA-GA receptors, which made anticancer drugs easier to be taken up by cells, and CS targeted tumor-associated platelets, ASP inhibited its function, and the degradation of DNase Ⅰ caused tumor cells to remove their protective shells and fully expose them to the environment, increasing drug uptake, which was also confirmed in cell uptake detection.

[0065] 4. Cell migration and invasion assay Fig.15 The results of studying the cell migration mechanism by combining D / GN and A&E / CPL prepared for this application. Fig.15Figure A shows the Transwell cell invasion results of the HuH-7+PLT+NET co-culture model; Fig.15 Figure B shows the results of the Transwell invasion quantitative test of the HuH-7+PLT+NET co-culture model; Fig.15 Figure C in the middle shows the Western blot analysis results of E-Cadherin and Vimentin; Fig.15 Figure D in the middle shows the quantitative analysis results of E-Cadherin and Vimentin. The Transwell cell invasion experiment used the HuH-7 single cell model as the control group to explore the anti-invasion ability of different drug preparations on the HuH-7+PLT+NET co-cultured cell line. Fig.15 A picture and Fig.15 As shown in Figure B, compared with HuH-7 cells alone, the number of cell invasions in the co-cultured cell line increased significantly, indicating that PLT and NET promoted tumor cell invasion and that the in vitro model was successfully established. The number of cell invasions in the A&E / CPL+D / GN group was the least, and tumor cell invasion was effectively inhibited. This may be because the combination of A&E / CPL and D / GN can effectively inhibit the tumor cell-platelet-neutrophil extracellular trap transfer pathway. This application detected the expression of EMT-related proteins, E-Cadherin and Vimentin through Western blot experiments. Fig.15 As shown in Figures C and D, compared with the HuH-7 single cell model, the expression of E-Cadherin in the HuH-7+PLT+NET co-cultured cell line was significantly downregulated, and the expression of Vimentin was significantly upregulated, indicating that PLT and NET promoted the epithelial-mesenchymal transformation of tumor cells to M type. Compared with the control group, the expression of E-Cadherin in the A&E / CPL+D / GN group was significantly upregulated, and the expression of Vimentin was significantly downregulated, indicating that A&E / CPL combined with D / GN can effectively inhibit the epithelial-mesenchymal transformation (EMT) of tumor cells induced by PLT and NET, and inhibit tumor cell migration.

[0066] 5. Biodistribution in the body Fig.16 Biodistribution test results of A&E / CPL and D / GN prepared for this application in tumor-bearing mice. Fig.16 Figure A in the middle shows the NIR images of A&E / CPL and D / GN at different time points; Fig.16 Figure B in the middle shows the fluorescence images of organs and tumors in each group at 48 h; Fig.16Figures C to D in the middle are the fluorescence intensity analysis results of each group of organs and tumors at DID and DIR wavelengths. In order to study the in vivo targeting of liver cancer by nanospheres and liposomes, the cell membrane fluorescent probes DID and DIR were encapsulated in nanospheres and liposomes, respectively, and the biological distribution of DID-GN and DIR-CPL in vivo was tracked and observed by real-time fluorescence imaging system (IVIS). Fig.16 As shown in Figure A, under the DID excitation wavelength (544nm), DID-GN was observed to be concentrated in the tumor site. This is because the modification of GA improves the liver cancer targeting ability of the nanomicelles. Under the DIR excitation wavelength (754nm), DIR-CPL was also observed to be concentrated in the tumor site. This may be because the liposome spheres also have a nanoscale size, which can exert their passive effects of permeability and retention (EPR) and promote their accumulation in the tumor site. After 48 hours, the mice were killed, and the main organs (heart, liver, spleen, lung, kidney) and tumors were dissected and analyzed by in vitro fluorescence imaging. Fig.16 Figure C and Fig.16 Figure D in the middle is the fluorescence quantitative analysis at DID and DIR wavelengths. Fig.16 In Figure B, compared with the free DID and free DIR groups, the DID-GN group and the DIR-CPL group showed stronger fluorescence signals in the tumor tissue, and the fluorescence intensity of the DID-GN + DIR-CPL dual-drug combination group was the highest. This is due to the passive effect of EPR and the active targeting effect of GA modification, which enhances the drug's targeting to liver cancer in vivo.

[0067] 6. In vivo tumor drug penetration Fig.17 The results of the effect of A&E / CPL prepared in the examples of the present application on the tumor penetration of D / GN. Fig.17 Figure A in the middle shows frozen section images of tumors in each treatment group at different depths; Fig.17 Figure B is a quantitative graph of DID fluorescence intensity in each group of tumors at different depths. In order to evaluate the penetration ability of different drug preparations in tumor tissue, the H22+PLT+NET tumor-bearing mouse model was injected with DID, DID-D / GN, DID-D / GN (DNase Ⅰ pretreatment), DID-D / GN (ASP+DNase Ⅰ pretreatment), and DID-D / GN (A&E / CPL pretreatment) through the tail vein, and the fluorescence images of frozen sections of tumor tissues in different preparation groups were observed by CLSM and photographed. Fig.17As shown in Figure A, compared with DID-D / GN, the fluorescence signal of the free drug DID group is mainly distributed at the edge of the tumor tissue, indicating that the free drug cannot reach the core of the tumor, resulting in a weak anti-tumor effect. In addition, after DID-D / GN was combined with A&E / CPL, obvious red fluorescence signals were shown from the edge to the core of the tumor, indicating that it can effectively penetrate the internal area of ​​the tumor tissue, which is conducive to the deep penetration of the drug in the tumor.

[0068] 7. Anti-tumor effect in vivo Fig.18 These are the anti-tumor evaluation results of D / GN combined with A&E / CPL prepared in the examples of this application on H22+PLT+NET subcutaneous transplanted tumors. Fig.18 Figure A in the middle is a schematic diagram of H22+PLT+NET subcutaneous transplanted tumor; Fig.18 Figure B in the middle is a photo of the tumor; Fig.18 Figure C in the middle shows the changes in tumor volume in different treatment groups; Fig.18 Figure D in the middle shows the changes in body weight in different treatment groups; Fig.18 Figure E in the middle shows the tumor inhibition rate of different treatment groups. To evaluate the in vivo anti-tumor effect and mechanism of A&E / CPL and D / GN, an H22+PLT+NET tumor-bearing mouse model was established ( Fig.18 As shown in Figure A). Fig.18 As shown in Figure D, compared with the saline group, the weight of mice in the A&E / CPL and D / GN groups did not decrease significantly, indicating that the vector has good biosafety. The weight of the free DOX group decreased slightly, indicating that the free anticancer drug has high toxicity and side effects. After 7 doses of tail vein administration, the mice were killed, and the tumor tissues were observed and photographed. Fig.18 As shown in Figure B, the drug treatment groups showed different degrees of tumor inhibition. The tumor volume of the combined drug group (DOX+ASP+DNase Ⅰ) was smaller than that of the single drug group (DOX or ASP+DNase Ⅰ), indicating that the combined treatment improved the anti-HCC treatment effect. Among them, the tumor volume of the A&E / CPL+D / GN combined preparation group was significantly smaller than that of the A&E / CPL group and the D / GN group, and the tumor inhibition rate was the highest, showing the strongest in vivo anti-tumor effect.

[0069] Fig.19 Evaluation of the therapeutic effect of D / GN combined with A&E / CPL prepared in the examples of this application on H22+PLT+NET subcutaneous transplanted tumors. Fig.19 Figure A in the middle shows H&E images of tumors in different treatment groups; Fig.19 Middle panel B shows CD31 immunohistochemistry images of tumors in different treatment groups; Fig.19 Figure C in the middle shows the α-SMA immunohistochemistry images of tumors in different treatment groups; Fig.19Figure D in the middle shows the Masson images of tumors in different treatment groups; Fig.19 Figure E in the middle is the Western blot analysis of E-Cadherin and Vimentin in tissues; Fig.19 Figure F in the middle shows the quantitative analysis results of E-Cadherin and Vimentin. Fig.19 The H&E results in Figure A showed that the A&E / CPL+D / GN group showed obvious nuclear dissolution and shrinkage and cytoplasmic vacuolation, indicating a stronger effect of promoting tumor apoptosis. Fig.19 The results of Figure B in the middle show that CD31 immunohistochemistry experiments showed that the control group showed obvious tumor microvascular formation, indicating that the model was successfully established. Compared with the free drug group, the tumor microvascular formation in the A&E / CPL+D / GN group was significantly reduced, indicating that the combined treatment can inhibit the formation of new blood vessels in tumor foci. Fig.19 α-SMA immunofluorescence staining experiment in Figure C and Fig.19 The results of Masson trichrome staining in Figure D show that compared with other drug preparation groups, A&E / CPL+D / GN has fewer α-SMA positive areas and less collagen deposition, indicating that the combined use of drug preparations can reduce the generation of collagen fibrils. To further study the anti-liver cancer mechanism of the combined nanoformulation, Western blot experiments were performed to detect the expression of E-cadherin and Vimentin proteins in tumor tissues. The results are shown in Figure 2. Fig.19 As shown in Figure E, the expression levels of E-cadherin and Vimentin in the tumor tissues of "H22+PLT+NET" tumor-bearing mice were downregulated and upregulated, respectively, indicating that the addition of PLT and NET can promote EMT of liver cancer cells. Fig.19 As shown in Figure F, the ratio of E-cadherin to GAPDH in the tumor tissue of the A&E / CPL+D / GN group was 87.12%, which was significantly higher than that of the A&E / CPL group (67.39%) or the D / GN group (73.3%). The ratio of Vimentin to GAPDH in the tumor tissue of the A&E / CPL+D / GN group was 26.44%, which was significantly lower than that of the A&E / CPL group (41.03%) or the D / GN group (34.74%), indicating that the combined use of the two nanoformulations in this application can more effectively inhibit the EMT process of liver cancer.

[0070] Fig. 20 H&E images of the main organs (heart, liver, spleen, lung, kidney) of different treatment groups prepared in the examples of this application. This application evaluates the in vivo biosafety of the nanoformulation by H&E staining and hemolysis experiments. The H&E staining of the main organs (heart, liver, spleen, lung, kidney) of mice treated with each drug formulation is shown in Figure 2. Fig. 20As shown in the figure, compared with the saline group, the A&E / CPL+D / GN group had no obvious pathological damage to the organs of the mice, indicating that the combined nanoformulation has good biosafety. Fig.21 As shown, there was no obvious hemolysis in the A&E / CPL group or the D / GN group at each concentration, indicating that the combined nanoformulation can be used as a delivery vehicle for anti-liver cancer drugs.

[0071] 8. Analysis of lung metastasis of liver cancer Fig. 22 Evaluation of the therapeutic effect of D / GN combined with A&E / CPL on lung metastasis prepared in the examples of this application. Fig. 22 Figure A is a schematic diagram of lung metastasis establishment and treatment; Fig. 22 Figure B shows the weight changes of mice in each treatment group; Fig. 22 Figure C in the figure shows the number of lung nodules in each treatment group; Fig. 22 Panel D in the figure shows the lung photos of each treatment group; Fig. 22 Figure E in the figure is the H&E image of the lungs in each treatment group. Distant metastasis is one of the main reasons for the high recurrence rate of HCC in treatment. To evaluate the inhibitory effect of A&E / CPL combined with D / GN on liver cancer lung metastasis in vivo, a liver cancer lung metastasis model was established by tail vein injection of H22 cells ( Fig. 22 As shown in Figure A). Fig. 22 As shown in Figure B, during the treatment period, there was no significant change in body weight in the A&E / CPL+D / GN group compared with the control group, indicating that the combined nanoformulation is a safe carrier for anticancer drug delivery. Fig. 22 Figure C and Fig. 22 As shown in Figure D, a large number of metastatic lung nodules were observed in the lung tissues of mice in the saline group compared with those in the healthy group, indicating that the liver cancer lung metastasis model was successfully established. Compared with the A&E / CPL group and the D / GN group, the A&E / CPL+D / GN group showed fewer metastatic lung nodules, indicating that the combined nanoformulation can effectively inhibit the distal metastasis of liver cancer cells.

[0072] 9. Experiment on NET formation of neutrophils in lungs Fig.23 Results of the effects of D / GN combined with A&E / CPL prepared for this application on neutrophils and NET formation in the lungs. In order to evaluate the effects of A&E / CPL and D / GN on neutrophil and neutrophil extracellular trap NET colonization in the lungs, lung tissue immunofluorescence experiments were performed. 1×10 6 H22 cells were injected into the tail vein of mice, and different drug preparations were used for tail vein treatment 7 times. After the treatment, the mice were killed, and the lung tissues were immunofluorescently stained for His H3 and Ly6G proteins. Fig.23As shown, compared with the A&E / CPL group and the D / GN group, the A&E / CPL+D / GN group showed less His H3 green fluorescence and Ly6G red fluorescence, indicating that the combined nanoformulation reduced the content of neutrophils in lung tissue, inhibited the NET level, and showed a good effect in inhibiting neutrophil colonization in the lung.

[0073] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0074] The above is a detailed introduction to a nanoformulation, preparation method and application for inhibiting tumor metastasis provided in the examples of the present application. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea. At the same time, for technicians in this field, according to the ideas of the present application, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. A nanoparticle preparation for inhibiting tumor metastasis, characterized in that: The nanoformulation for inhibiting tumor metastasis comprises a liposome sphere, wherein the liposome sphere comprises an inner core structure and a shell wrapped in the outer layer of the inner core structure, wherein the inner core structure comprises lecithin, dioleoylphosphatidylethanolamine, cholesterol succinate monoester, cholesterol, imidazole-cholesterol and DSPE-PEG 2000 -CS; the inner core structure is loaded with aspirin and DNase Ⅰ; the shell includes platelets; the lecithin, dioleoylphosphatidylethanolamine, cholesterol succinate, cholesterol, imidazole-cholesterol and DSPE-PEG 2000 -CS mass ratio is (35-45): (55-65): (10-20): (10-20): (5-15): (1-5); based on the total mass of the liposome sphere, the mass percentage of aspirin loaded in the liposome sphere is 1%-5%; based on the mass of the inner core structure per milligram, the amount of DNase I loaded in the inner core structure is 0.6U / mg-2U / mg; The amount of platelets meets the requirement: M1= (M2×M0) / 250, where M1 is the mass of platelets in mg, M2 is the mass of lecithin in mg, and M0 is the mass of platelets extracted from 1 mL of whole blood in mg.

2. The nanoformulation for inhibiting tumor metastasis according to claim 1, characterized in that: The DSPE-PEG 2000 The structural formula of -CS is as follows: .

3. The nanoformulation for inhibiting tumor metastasis according to claim 1, characterized in that: The nanoformulation for inhibiting tumor metastasis also includes nanospheres, which include glycocholic acid, lecithin, DSPE-PEG 2000 -GA and doxorubicin, the glycocholic acid, phosphatidylcholine and DSPE-PEG 2000 -GA mass ratio is (15-20): (20-25): 1; based on the total mass of the nanospheres, the mass percentage of the doxorubicin loaded on the nanospheres is 1%-5%.

4. The nanoformulation for inhibiting tumor metastasis according to claim 1, characterized in that: The liposome spheres are prepared by the following method: (S11) DSPE-PEG 2000 Synthesis of -CS:DSPE-PEG 2000 -NH2 was activated by EDC and NHS, and then cross-linked with chondroitin sulfate under nitrogen to obtain DSPE-PEG 2000 -CS; (S12) Phosphatidylcholine, dioleoylphosphatidylethanolamine, cholesterol succinate, cholesterol, imidazole-cholesterol, DSPE-PEG 2000 -CS and aspirin were dissolved in ethanol, and the ethanol was removed using a rotary evaporator in a water bath at 50°C to 60°C and a rotation speed of 10rpm to 30rpm; (S13) adding DNase I to a PBS solution, dissolving it by ultrasonication, adding it to the system formed in step (S12), and hydrating it by rotary evaporation for 30 min to 60 min; (S14) The product obtained in step (S13) is ultrasonically crushed in an ice bath, platelets are added and mixed, and liposome spheres wrapped by platelet membrane are obtained through an extruder.

5. The nanoformulation for inhibiting tumor metastasis according to claim 3, characterized in that: The nanospheres are prepared by the following method: (S21) dissolving glycocholic acid in a PBS solution to obtain a first solution; (S22) Phosphatidylcholine, doxorubicin, DSPE-PEG 2000 -GA is dissolved in ethanol by ultrasonication to obtain a second solution; (S23) dripping the second solution into the first solution, and after the ethanol is completely evaporated, ultrasonically crushing the product in an ice bath, and sequentially passing through filter membranes with pore sizes of 0.45 μm and 0.22 μm to obtain nanospheres.

6. A method for preparing a nano preparation for inhibiting tumor metastasis as claimed in claim 3, characterized in that: The method comprises preparing liposome spheres; the liposome spheres are prepared by the following method: (1) Phosphatidylcholine, dioleoylphosphatidylethanolamine, cholesterol succinate, cholesterol, imidazole-cholesterol, DSPE-PEG 2000 -CS and aspirin were dissolved in ethanol, and the ethanol was removed using a rotary evaporator in a water bath at 50°C to 60°C and a rotation speed of 10rpm to 30rpm; (2) Add DNase I to PBS solution, dissolve by ultrasonication, add to the system formed in step (1), and hydrate by rotary evaporation for 30 min to 60 min; (3) The product obtained in step (2) is ultrasonically crushed in an ice bath, platelets are added and mixed, and liposome spheres wrapped by platelet membrane are obtained through an extruder.

7. The method for preparing the nano preparation for inhibiting tumor metastasis according to claim 6, characterized in that: Also included is the preparation of nanospheres, wherein the nanospheres are prepared by the following method: Dissolving glycocholic acid in PBS solution to obtain a first solution; Phosphatidylcholine, doxorubicin, DSPE-PEG 2000 -GA is dissolved in ethanol by ultrasonication to obtain a second solution; The second solution was slowly dripped into the first solution. After the ethanol was completely evaporated, the product was ultrasonically crushed in an ice bath and passed through filter membranes with pore sizes of 0.45 μm and 0.22 μm in sequence to obtain nanospheres.

8. Use of the nanoformulation for inhibiting tumor metastasis according to any one of claims 1 to 5 or the nanoformulation prepared by the method for preparing the nanoformulation for inhibiting tumor metastasis according to any one of claims 6 to 7 in preparing drugs for treating tumors and drugs for inhibiting tumor metastasis.

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