Nano preparation for inhibiting tumor metastasis, preparation method and application

Abstract

This application belongs to the technical field of pharmaceutical preparations. This application discloses a nano - preparation for inhibiting tumor metastasis, a preparation method and an application. The nano - preparation for inhibiting tumor metastasis of this application includes a liposome sphere and a nano - sphere. The liposome sphere of this application includes an inner core structure and a shell wrapped around the outer layer of the inner core structure. The inner core structure includes lecithin, dioleoyl phosphatidylethanolamine, cholesterol succinate monoester, cholesterol, imidazolyl - cholesterol and DSPE - PEG 2000 -CS; the inner core structure is loaded with aspirin and DNase Ⅰ; the shell includes a platelet membrane. The nano - preparation for inhibiting tumor metastasis of this application can dissolve NET, inhibit platelet function, and disintegrate tumor cell - platelet - NET aggregates. The nano - sphere for inhibiting distal metastasis of tumors of this application can deeply penetrate into the tumor interior and play a role in killing tumors.

Description

Technical Field

[0001] This application belongs to the technical field of pharmaceutical preparations, and particularly 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 fast clearance rate, poor targeting and strong toxic and side effects in vivo. Recent studies have shown that nanoparticles based on lecithin and cholesterol have advantages such as good biocompatibility, non - toxicity, non - immunogenicity and easy synthesis, and have been widely used for the delivery of anti - tumor drugs. Summary of the Invention

[0003] Object of the Invention: This application provides a nano - preparation for inhibiting tumor metastasis, a preparation method and an application thereof. The nano - preparation for inhibiting tumor metastasis of this application includes liposome spheres, and the liposome spheres of this application can dissolve neutrophil extracellular traps (NETs), inhibit platelet function, disintegrate tumor cell - platelet - NET aggregates, and inhibit distant metastasis of tumors.

[0004] Technical Solution: The embodiments of this application provide a nano - preparation for inhibiting tumor metastasis. The nano - preparation for inhibiting tumor metastasis includes liposome spheres. The liposome spheres include an inner core structure and a shell wrapped around the outer layer of the inner core structure. The inner core structure includes lecithin, dioleoyl phosphatidylethanolamine, cholesteryl succinate, cholesterol, imidazolyl - cholesterol and DSPE - PEG 2000 -CS; the inner core structure is loaded with aspirin and DNase Ⅰ; the shell includes a platelet membrane; the mass ratio of lecithin, dioleoyl phosphatidylethanolamine, cholesteryl succinate, cholesterol, imidazolyl - cholesterol and DSPE - PEG 2000 -CS is (35 - 45):(55 - 65):(10 - 20):(10 - 20):(5 - 15):(1 - 5); based on the total mass of the liposome spheres, the mass percentage of the aspirin loaded by the liposome spheres is 1% - 5%; based on the mass of each milligram of the inner core structure, the amount of DNase Ⅰ loaded by the inner core structure is 0.6 U / mg - 2 U / mg;

[0005] The dosage of the platelet membrane satisfies: M 1 =(M 2 ×M 0 ) / 250, where M 1 is the mass of the platelet membrane, in mg, and M 2 is the mass of lecithin, in mg, M0 The mass of platelet membranes extracted from 1 mL of whole blood, with the unit of mg.

[0006] In some embodiments, the mass ratio of the lecithin, dioleoyl phosphatidylethanolamine, cholesteryl succinate, cholesterol, imidazolyl-cholesterol, and DSPE-PEG 2000 -CS is 40:60:15:15:10:5.

[0007] In some embodiments, based on the total mass of the liposome spheres, the mass percentage of the aspirin loaded in the liposome spheres is any value among 1%, 2%, 3%, 4%, 5% or a range composed of any two values.

[0008] In some embodiments, based on the mass of each milligram of the inner nuclear structure, the amount of DNase I loaded in the inner nuclear structure is any value among 0.6 U / mg, 1 U / mg, 1.5 U / mg, 2 U / mg or a range composed of any two values.

[0009] 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 among 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 composed of any two values.

[0010] In some embodiments, the DSPE-PEG 2000 -CS has the following structural formula:

[0011] .

[0012] In some embodiments, the nano-formulation for inhibiting tumor metastasis further includes nano-spheres (D / GN), and the nano-spheres include glycocholic acid, lecithin, DSPE-PEG 2000 -GA, and doxorubicin. The mass ratio of the glycocholic acid, lecithin, and DSPE-PEG 2000 -GA is (15 - 20):(20 - 25):1; based on the total mass of the nano-spheres, the mass percentage of the doxorubicin loaded in the nano-spheres is 1% - 5%.

[0013] In some embodiments, the mass ratio of the glycocholic acid and lecithin is 1:1.2.

[0014] In some embodiments, the average particle size range of the nano-spheres is 25 nm to 30 nm, such as the average particle size of the nano-spheres is any value among 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm or a range composed of any two values.

[0015] In some embodiments, the mass ratio of glycocholic acid, lecithin, and DSPE-PEG 2000 -GA is 18:22:1. In some embodiments, based on the total mass of the nanospheres, the mass percentage of doxorubicin loaded in the nanospheres is any value or a range composed of any two values among 1%, 2%, 3%, 4%, and 5%.

[0016] In some embodiments, the liposome spheres are prepared by the following method:

[0017] (S11) Synthesis of DSPE-PEG 2000 -CS: After DSPE-PEG 2000 -NH 2 is activated by EDC and NHS, it is cross-linked with chondroitin sulfate in a nitrogen environment to obtain DSPE-PEG 2000 -CS;

[0018] (S12) Dissolve lecithin, dioleoyl phosphatidylethanolamine, cholesteryl succinate, cholesterol, imidazolyl-cholesterol, DSPE-PEG 2000 -CS, and aspirin in ethanol, and remove ethanol using a rotary evaporator in a water bath at 50°C to 60°C and a rotation speed of 10 rpm to 30 rpm;

[0019] (S13) Add DNase I to the PBS solution, dissolve it by ultrasonic treatment, add it to the system formed in step (S12), and perform rotary evaporation and hydration for 30 min to 60 min;

[0020] (S14) Ultrasonically disrupt the product obtained in step (S13) in an ice bath, mix it with platelet membranes, and obtain liposome spheres wrapped with platelet membranes through an extruder.

[0021] In some embodiments, the nanospheres are prepared by the following method:

[0022] (S21) Dissolve glycocholic acid in the PBS solution to obtain a first solution;

[0023] (S22) Ultrasonically dissolve lecithin, doxorubicin, DSPE-PEG 2000 -GA in ethanol to obtain a second solution;

[0024] (S23) Drop the second solution into the first solution. After the ethanol is completely evaporated, ultrasonically disrupt the product in an ice bath, and sequentially pass it through polyethersulfone membranes with pore sizes of 0.45 μm and 0.22 μm to obtain nanospheres.

[0025] The embodiments of the present application further provide a method for preparing a nano - preparation for inhibiting tumor metastasis, including preparing liposome spheres; the liposome spheres are prepared by the following method:

[0026] (1) Dissolve lecithin, dioleoyl phosphatidylethanolamine, cholesterol succinate monoester, cholesterol, imidazolyl - cholesterol, DSPE - PEG 2000 - CS and aspirin in ethanol, and remove ethanol using a rotary evaporator in a water bath at 50 °C to 60 °C and a rotation speed of 10 rpm to 30 rpm;

[0027] (2) Add DNase Ⅰ to the PBS solution, dissolve it by ultrasound, add it to the system formed in step (1), and perform rotary evaporation and hydration for 30 min to 60 min;

[0028] (3) Ultrasonically disrupt the product obtained in step (2) in an ice bath, mix it with platelet membranes, and obtain liposome spheres wrapped with platelet membranes through an extruder.

[0029] In some embodiments, the method for preparing the nano - preparation for inhibiting tumor metastasis further includes preparing nano - spheres, and the nano - spheres are prepared by the following method:

[0030] Dissolve glycocholic acid in the PBS solution to obtain a first solution;

[0031] Dissolve lecithin, doxorubicin, DSPE - PEG 2000 - GA in ethanol by ultrasound to obtain a second solution;

[0032] Drop the second solution into the first solution. After the ethanol has completely evaporated, ultrasonically disrupt the product in an ice bath, and sequentially pass it through polyethersulfone membranes with pore sizes of 0.45 μm and 0.22 μm to obtain nano - spheres.

[0033] The embodiments of the present application further provide the application of the above - mentioned nano - preparation for inhibiting tumor metastasis or the nano - preparation prepared by the above - mentioned method for preparing the nano - preparation for inhibiting tumor metastasis in the preparation of drugs for treating tumors and drugs for inhibiting tumor metastasis.

[0034] In some embodiments, the tumor in the present application is liver cancer.

[0035] Beneficial effects: The present application discloses a nano - preparation for inhibiting tumor metastasis, a preparation method, and an application. The nano - preparation for inhibiting tumor metastasis in the present application includes liposome spheres and nano - spheres. The liposome spheres in the present application include an inner core structure and a shell wrapped around the outer layer of the inner core structure. The inner core structure includes lecithin, dioleoyl phosphatidylethanolamine, cholesterol succinate monoester, cholesterol, imidazolyl - cholesterol, and DSPE - PEG 2000-CS; the inner core structure is loaded with aspirin and DNase I; the shell includes a platelet membrane. The nano - preparation for inhibiting tumor metastasis of the present application can dissolve NET, inhibit platelet function, disintegrate tumor cell - platelet - NET aggregates, and inhibit distant metastasis of tumors. The nano - spheres in the nano - preparation for inhibiting tumor metastasis of the present application can deeply penetrate into tumors and play a role in killing tumors. Description of the Drawings

[0036] Figure 1 It is the electron micrograph of liposomes wrapped with platelet membranes under three different feeding ratios in the examples of the present application. Among them, Figure A, Figure B, and Figure C are liposome - like spheres wrapped with platelet membranes formed after extrusion of liposomes prepared from platelet membranes extracted from 1 mL of whole blood and 100 mg, 250 mg, and 350 mg of lecithin respectively;

[0037] Figure 2 It is the schematic diagram for the preparation of liposome - like spheres (A&E / CPL) and nano - spheres (D / GN) in the examples of the present application;

[0038] Figure 3 It is the physicochemical property characterization of A&E / CPL and D / GN prepared in the examples of the present application. Among them, Figure A is the particle size distribution diagram of A&E / CPL; Figure B is the particle size distribution diagram of D / GN;

[0039] Figure 4 It is the TEM image of A&E / CPL prepared in the examples of the present application. Among them, Figure A, Figure B, and Figure C are the TEM images of A&E / CPL under the conditions of pH = 7.4, pH = 6.0, and pH = 5.5 respectively;

[0040] Figure 5 It is the TEM image of D / GN prepared in the examples of the present application under the condition of pH = 7.4;

[0041] Figure 6 It is the 7 - day stability of A&E / CPL prepared in the examples of the present application in PBS solution and DMEM medium. Among them, the left vertical coordinate is the particle size measured by dynamic light scattering (DLS), and the right vertical coordinate is the polydispersity index (PDI);

[0042] Figure 7 It is the 7 - day stability of D / GN prepared in the examples of the present application in PBS solution and DMEM medium. Among them, the left vertical coordinate is the particle size measured by dynamic light scattering (DLS), and the right vertical coordinate is the polydispersity index (PDI);

[0043] Figure 8 It is the Coomassie Brilliant Blue image of A&E / CPL prepared in the examples of the present application;

[0044] Figure 9In vitro drug release profiles of A&E / CPL prepared in the embodiments of the present application under different pH conditions;

[0045] Figure 10 In vitro drug release profile of D / GN prepared in the embodiments of the present application;

[0046] Figure 11 Horizontal gel electrophoresis image of A&E / CPL prepared in the embodiments of the present application;

[0047] Figure 12 In vitro cellular uptake results of D / GN prepared in the embodiments of the present application. Among them, Figure A is the in vitro uptake laser confocal image of D / GN; Figures B and C are the quantitative results of flow cytometry for the in vitro cellular uptake of D / GN;

[0048] Figure 13 Cytotoxicity evaluation model of D / GN combined with A&E / CPL prepared in the embodiments of the present application. Among them, Figure A is the single HuH-7 model, and Figure B is the schematic diagram of the HuH-7+PLT+NET co-culture model;

[0049] Figure 14 Cytotoxicity evaluation results of D / GN combined with A&E / CPL prepared in the embodiments of the present application. Among them, Figure A is the MTT detection result of the single HuH-7 model; Figure B is the MTT detection result of the HuH-7+PLT+NET co-culture model;

[0050] Figure 15 Results of exploring the cell migration mechanism of D / GN combined with A&E / CPL prepared in the embodiments of the present application. Among them, Figure A is the Transwell cell invasion result of the HuH-7+PLT+NET co-culture model; Figure B is the quantitative result of Transwell invasion 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;

[0051] Figure 16 Results of biodistribution tests of A&E / CPL and D / GN in tumor-bearing mice prepared in the embodiments of the present application. Among them, Figure A is the NIR image 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 h; Figures C and D are the fluorescence intensity analysis of each group of organs and tumors at DID and DIR wavelengths;

[0052] Figure 17Results of the effect of A&E / CPL prepared in the embodiments of the present application on the tumor penetration of D / GN. Among them, Figure A is the tumor frozen section images of each treatment group at different depths; Figure B is the quantitative graph of DID fluorescence intensity in tumors of each group at different depths.

[0053] Figure 18 Antitumor evaluation results of the present application using D / GN combined with A&E / CPL for H22+PLT+NET subcutaneous transplanted tumors. Among them, Figure A is the schematic diagram of H22+PLT+NET subcutaneous transplanted tumors; Figure B is the tumor photos of different treatment groups; Figure C is the change in tumor volume of different treatment groups; Figure D is the change in body weight of mice in different treatment groups; Figure E is the inhibition rate of tumor growth in different treatment groups.

[0054] Figure 19 Evaluation results of the treatment effect of the present application using D / GN combined with A&E / CPL on H22+PLT+NET subcutaneous transplanted tumors. Among them, Figure A is the H&E images of tumors in different treatment groups; Figure B is the immunohistochemical images of CD31 in tumors of different treatment groups; Figure C is the immunohistochemical images of α-SMA in tumors of different treatment groups; Figure D is the Masson images of tumors in different treatment groups; Figure E is the Western blot analysis results of E-Cadherin and Vimentin in tissues; Figure F is the quantitative analysis results of E-Cadherin and Vimentin.

[0055] Figure 20 H&E images of the main organs including the heart, liver, spleen, lungs, and kidneys of different treatment groups in the present application.

[0056] Figure 21 Hemolysis experiment results of A&E / CPL and D / GN prepared in the embodiments of the present application.

[0057] Figure 22 Evaluation results of the treatment effect of the present application using D / GN combined with A&E / CPL on lung metastasis. Among them, Figure A is the schematic diagram of the establishment and treatment of lung metastasis; Figure B is the change in body weight of mice in each treatment group; Figure C is the number of metastatic lung nodules in each treatment group; Figure D is the lung photos of each treatment group; Figure E is the H&E images of the lungs in each treatment group.

[0058] Figure 23 Effect of D / GN combined with A&E / CPL on the colonization of neutrophils and NETs in the lungs in the present application. Detailed implementation manners

[0059] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present application. Unless otherwise specified, all technical and scientific terms used in the present application have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0060] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part rather than all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative efforts shall fall within the protection scope of the present application. Additionally, in the description of the present application, the term "including" means "including but not limited to". The use of terms such as first, second, and third is merely for labeling purposes and does not impose numerical requirements or establish an order. The various embodiments of the present application may exist in a range format; it should be understood that the description in a range format is only for convenience and brevity and should not be construed as a rigid limitation on the scope of the present application; therefore, it should be considered that the described range description has specifically disclosed all possible sub-ranges and individual values within that 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., and individual numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any cited number (fraction or integer) within the indicated range.

[0061] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present application pertains. 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. In case of conflict with any incorporated document, the content of this specification shall prevail. Unless otherwise specified, "%" is a mass-based percentage, and the solvent ethanol used refers to an ethanol solution with a mass percentage content of ≥95%.

[0062] I. Materials and Methods

[0063] 1. Materials

[0064] Lecithin (PC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol were all purchased from Shanghai A.V.T. Pharmaceutical Technology Co., Ltd. Aspirin (ASP), deoxyribonuclease I (DNase Ⅰ), and doxorubicin (DOX) were all purchased from Dalian Meilun Biotechnology Co., Ltd. Glycocholic acid (GAH) was purchased from MedChemExpress (USA). Antibodies such as E-cadherin, vimentin, and platelet-endothelial cell adhesion molecule (CD31) were all purchased from Abcam (Massachusetts, USA). Other chemical reagents were all of analytical grade or chromatographic grade.

[0065] 2. Cells and Animals

[0066] The human hepatocellular carcinoma cell line (HuH-7) and murine hepatocarcinoma cells (H22) were both purchased from the Beijing Institute of Life Sciences. BALB / c mice (female, 5 weeks old, 18 g - 22 g) were purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. All animal experiments were approved by the Animal Experiment Ethics Committee of Shandong First Medical University (SDSMU, 2019 - 045), and the care and use of experimental animals strictly complied with the "Animal Management Regulations" of the Ministry of Health of the People's Republic of China (No. 55, 001).

[0067] II. Test Methods

[0068] 1. Physicochemical Property Characterization of A&E / CPL and D / GN

[0069] The particle size distribution and zeta potential of A&E / CPL and D / GN were measured using a Malvern particle size and zeta potential analyzer. A&E / CPL and D / GN were diluted 10-fold 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-fold with PBS and negatively stained with uranyl acetate. After the samples were dried, the morphology of A&E / CPL and D / GN was observed using a transmission electron microscope (TEM). The content of DOX in D / GN was measured using a UV spectrophotometer at 480 nm, and the content of ASP in A&E / CPL was measured using a high-performance liquid chromatograph (HPLC) at 276 nm. The calculation formulas for encapsulation efficiency (EE) and drug loading (LE) are as follows:

[0070] 。

[0071] 2. In Vitro Drug Release of A&E / CPL and D / GN

[0072] In PBS buffer solutions with different pH values (pH = 7.4, 6.5 or 5.5), the in vitro drug release of ASP in A&E / CPL was evaluated. 1 mL of A&E / CPL was added into a dialysis bag (MWCO = 3500), and after sealing, it was placed in a 50 mL centrifuge tube containing 40 mL of PBS buffer solution with different pH values (containing 0.5% Tween-80) as the release medium. The release system was placed in a constant temperature shaking incubator, and the culture conditions were 37 °C and 100 rpm. At the established time points (0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h), 2 mL of the release medium was taken out, and at the same time, 2 mL of fresh release medium was supplemented. Then, the amount of released ASP was determined by high performance liquid chromatography at 276 nm respectively.

[0073] 3. Hemolysis evaluation

[0074] 1 mL of blood from healthy mice was collected by enucleation and placed in a centrifuge tube. After centrifugation at 3500 rpm for 10 min, the supernatant was removed, and physiological 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 physiological saline to obtain a 2% red blood cell suspension for standby. The nanometer preparation was mixed with the red blood cell suspension to obtain samples with different concentrations (0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, 5 mg / mL), and Triton X-100 and physiological saline were used as positive and negative controls respectively. All samples were placed in a constant temperature shaking incubator and incubated for 2 h, and then the samples were centrifuged (3500 rpm, 10 min). Observe and take pictures, and take the supernatant and put it in a 96-well plate, and measure its absorbance value (OD value) at 545 nm with an enzyme-linked immunosorbent assay instrument. The hemolysis rate (%) was calculated as follows:

[0075] Among them, A, A 1 and A 2 represent the absorbance values of the positive control group, the experimental group and the negative control group respectively.

[0076] 4. Construction of an in vitro co-culture research model for simulating the microenvironment

[0077] In the tumor microenvironment, there are not only cancer cells, but also platelets (PLT) and neutrophil extracellular traps (NET), and the latter two can promote the proliferation and metastasis of tumors. In order to simulate the real tumor microenvironment, we established a co-culture model composed of HuH-7 cells, PLT and NET. HuH-7 cells, PLT and induced NET were added to the culture plate at a quantity ratio of 2:20:1 and cultured for 16 h to establish a co-culture cell model.

[0078] 5. Cell uptake assay

[0079] To investigate the uptake of tumor cells by the nanoplatforms, HuH-7 cells were seeded in cell culture dishes (5×10 4 cells / 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 the cells were treated with the drugs for 1 h. After washing three times with PBS, the cells were fixed with tissue cell fixative for 10 min and stained with DAPI (concentration was 1 μg / mL) for 10 min to stain cell nuclei. After washing three times with PBS, the cells were photographed with a laser confocal microscope and quantitatively detected by an Accuri C6 Plus flow cytometer.

[0080] 6. In vitro cytotoxicity assay

[0081] The MTT method was used to detect the toxicity of different formulations to tumor cells. To better evaluate the effect of the drugs in the tumor microenvironment, two cell models were established: (1) HuH-7 cells; (2) "HuH-7 + PLT + NET" co-culture cell model. First, these two cell models were seeded in 96-well plates, with 6×10 3 cells added to each well, and the cells were cultured in an incubator at 37 °C and 5% CO 2 for 16 h. Drugs such as DOX, DOX + ASP + DNase I, D / GN, A&E / CPL, and D / GN + A&E / CPL were added, and the DOX concentration was set to 0.01 μg / mL - 10 μg / mL. The cells were treated with the drugs for 48 h. MTT solution (concentration was 5 mg / mL) was added to each well and incubated for 4 h. The culture medium was removed, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. The OD values of each well at 490 nm were measured with an enzyme-linked immunosorbent assay (ELISA) reader (ELX800, BioTek, USA), and the cell survival rate of each group was calculated.

[0082] 7. Cell migration and invasion analysis

[0083] 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 wound healing assay and Transwell cell invasion assay.

[0084] (1)Scratch assay. HuH-7 cells were incubated in six-well plates. When the cell confluence reached 80%, the cell layer was scratched with the tip of a 200-µL sterile pipette, 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. After washing three times with PBS, the wound width at 24 h was observed again, and the migration rate was quantified using ImageJ software. The calculation is as shown in the following formula:

[0085] where d 0h is the initial wound width in mm, and d 24h is the wound width at 24 h in mm.

[0086] (2)Transwell cell invasion assay. First, Matrigel matrix glue was diluted with serum-free medium (1:5) on ice. The prepared matrix glue was added to the Transwell chamber and incubated at 37 °C for 1 h for later use. Cells were pretreated with different preparation groups. After 24 h, the cells were collected and resuspended with serum-free medium (cell density, 1×10 5 cells / mL). 200 µL of the cell suspension was added to the chamber, and a medium containing 10% FBS (volume: 600 µL) was added to the 24-well plate under the chamber as an inducer. After incubating at 37 °C for 24 h, the medium in the upper chamber was discarded, and the upper-layer matrix glue of the chamber was gently wiped off with a sterile cotton swab. After washing the cells three times with PBS, the cells migrated to the lower layer of the chamber were fixed with tissue cell fixative for 20 min, then rinsed three times with PBS, stained with crystal violet for 20 min, and observed under a microscope after washing three times with PBS. Finally, the cells migrated to the lower surface of the chamber were quantitatively analyzed using ImageJ software.

[0087] 8. Western blot analysis of the anti-hepatocellular carcinoma mechanism

[0088] The expression levels of proteins such as E-cadherin and Vimentin in hepatocellular carcinoma cells were detected by Western blot. Tumor cells and hepatocellular carcinoma tissues were lysed to extract proteins, which were loaded and electrophoresed on a 12% SDS-PAGE gel and transferred to a PVDF membrane. Then, the PVDF membrane was blocked with 5% milk in TBST at room temperature for 2 h, and then incubated with the primary antibody overnight at 4 °C. Subsequently, the secondary antibody was added and incubated at room temperature for 1 h. The protein expression was detected using a hypersensitive ECL chemiluminescence solution.

[0089] 9. In vivo imaging and tissue penetration analysis

[0090] Two fluorescent substances, DID and DIR, were selected to replace the 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 an H22+PLT+NET tumor-bearing model. When the tumor volume was approximately 180 mm 3 ³, the mice were divided into groups of DID, DIR, DID-GN, DIR-CPL, and DID-GN+DIR-CPL. A real-time fluorescence imaging system (IVIS) was used to monitor the biodistribution of DID and DIR in the mice at different time points. After 48 h, the organs and tumors were taken for ex vivo fluorescence intensity analysis.

[0091] To evaluate the permeability of D / GN, an H22+PLT+NET tumor-bearing mouse model was established. When the tumor volume was approximately 180 mm 3 ³, the mice were divided into 5 groups: DID, DID-D / GN, DID-D / GN (pretreatment with DNase Ⅰ), DID-D / GN (pretreatment with ASP+DNase Ⅰ), and DID-D / GN (pretreatment with A&E / CPL). All pretreatment groups were continuously treated via the tail vein for 3 days. The mice were sacrificed 12 h after injecting DID, and the tumors were dissected. Then the tumor tissues were cut into 10-μm-thick sections. The tumor sections were stained with DAPI and then photographed using a laser confocal microscope (TCSSP8, Leica).

[0092] 10. Evaluation of tumor suppression of subcutaneous transplanted tumors

[0093] To evaluate the therapeutic effects of A&E / CPL and D / GN on liver cancer, an H22+PLT+NET subcutaneous tumor-bearing mouse model was established with a tumor volume of approximately 150 mm 3 ³, and the mice were divided into 8 groups (n = 5): (1) control group; (2) normal 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 drug doses injected via the tail vein were: DOX (3 mg / kg), ASP (3 mg / kg), and DNase Ⅰ (200 U / mouse), and a total of 7 administrations were given. The tumor size and body weight of the mice were monitored every other day after drug treatment. After two weeks, the mice were sacrificed, and the heart, liver, spleen, lung, kidney, and tumor were removed and weighed. The tumors and important organs were fixed with 4% paraformaldehyde. Then, the tumor sections were subjected to H&E staining, immunohistochemical analysis, Masson's trichrome staining, and Western blot analysis.

[0094] 11. Lung metastasis experiment

[0095] Intravenous injection of 2×10 6H22 cells were used to establish a liver cancer lung metastasis model. Then, the mice were randomly divided into 7 groups (n = 3): (1) normal saline; (2) DOX; (3) DOX + ASP + DNase Ⅰ; (4) D / GN; (5) A&E / CPL; (6) D / GN + A&E / CPL. Treatment was given once every 2 days for a total of 7 times. The mice were sacrificed 14 days later. The lungs were dissected and fixed with 4% paraformaldehyde. Then, the number of metastatic nodules was measured, and the lung tissue sections were stained with H&E and immunofluorescently stained with His H3 and Ly6G.

[0096] 12. Detection experiment of neutrophil NET formation in the lungs

[0097] To evaluate the effects of A&E / CPL and D / GN on neutrophil and neutrophil extracellular trap NET formation in the lungs, a lung immunofluorescence experiment was conducted. 1×10 6 H22 cells were injected into the mice via the tail vein. 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. Tail vein administration was given every other day starting from the day after tumor inoculation for a total of 7 times. After the treatment, the mice were sacrificed, the lungs were dissected and cryo-embedded with OCT compound (optimal cutting temperature compound). The tissue was cryo-sectioned using a Leica cryostat at a thickness of 10 μm. Subsequently, immunofluorescent staining of His H3 and Ly6G proteins was performed, and observation and photography were carried out using a laser confocal microscope.

[0098] 13. Statistical analysis

[0099] Data are expressed as mean ± standard deviation. Prism 8.0 (GraphPad) was used for data plotting and statistical analysis. The differences between two groups were analyzed by student't test or analysis of variance, and p < 0.05 was considered statistically significant.

[0100] III. Sample preparation and experimental results

[0101] (I) Sample preparation

[0102] Example 1: Preparation of A&E / CPL liposome spheres

[0103] 1. Preparation of platelet membranes

[0104] Collect 1 mL of whole blood from BALB / c mice by eyeball blood collection method into a centrifuge tube containing 50 μL of heparin sodium anticoagulant, centrifuge at 300×g centrifugal force for 10 min at 4°C, and take the supernatant and the white cell layer at the liquid level boundary. Centrifuge the supernatant at 2000×g centrifugal force for 10 min at 4°C, take the white platelet precipitate, and wash it twice with PBS containing anticoagulant. After repeated freezing and thawing, platelet membranes are obtained.

[0105] 2. Preparation of liposome spheres (A&E / CPL) co-loaded with ASP and DNase Ⅰ wrapped by platelet membranes

[0106] (1)Screening of the feeding ratio of platelet membranes to liposomes: Fix the content of platelet membranes, change the content of liposomes, and investigate the effect of different feeding ratios on the encapsulation efficiency of platelet membranes. First, determine the ratio of platelet membranes to liposomes. After mixing the platelets taken from 1 mL of whole blood with the liposomes prepared from 100 mg, 250 mg, and 350 mg of lecithin respectively according to the ratio, extrude them through an extruder to prepare liposomes wrapped by platelet membranes. Observe the morphology of the three liposomes and the encapsulation situation of platelet membranes by electron microscopy to determine the optimal feeding ratio and obtain the highest encapsulation efficiency. The results are as Figure 1 shown, Figure 1 Figures A, B, and C in it are the electron micrographs of liposome spheres wrapped by platelet membranes formed after extrusion of liposomes prepared from platelet membranes extracted from 1 mL of whole blood and 100 mg, 250 mg, and 350 mg of lecithin respectively. From Figure 1 the results, it can be seen that under the condition of fixed platelet membrane content, too much or too little addition of lecithin will result in poor encapsulation efficiency. At the feeding ratio of platelet membranes taken from 1 mL of whole blood: 250 mg of lecithin, the encapsulation efficiency of platelet membranes is the highest. Finally, this feeding ratio is selected to prepare large liposomes wrapped by platelet membranes.

[0107] (2)Preparation of A&E / CPL liposome spheres

[0108] Preparation of imidazolyl-cholesterol (AIM-Chol): Dissolve cholesterol chloroformate (0.45 g, 1 mmol) in dichloromethane (10 mL). Dropwise add N-(3-aminopropyl)imidazole (0.6 mL, 4 mmol), then add 200 μL of 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, cholesterol chloroformate and N-(3-aminopropyl)imidazole generate AIM-Chol.

[0109] DSPE-PEG 2000 -CS preparation: DSPE-PEG 2000 -NH 2After activation with EDC and NHS, it was cross-linked with chondroitin sulfate (CS) under a nitrogen atmosphere to obtain DSPE-PEG 2000 -CS.

[0110] Dissolve 40 mg of lecithin (PC), 60 mg of dioleoyl phosphatidylethanolamine (DOPE), 15 mg of cholesterol succinate monoester (CHEMS), 15 mg of cholesterol, 10 mg of imidazolyl-cholesterol (AIM-Chol), 5 mg of DSPE-PEG 2000 -CS and 5 mg of aspirin (ASP) in 5 mL of ethanol. Remove the ethanol using a rotary evaporator at a water bath temperature of 55 °C and a rotation speed of 18 rpm. Add 200 U of DNase Ⅰ to 5 mL of PBS and dissolve it by sonication. When a uniform thin film forms at the bottom of the round-bottom flask, add the above PBS solution and hydrate it by rotary evaporation for 1 h. Then, ultrasonically disrupt the product in an ice bath at a power of 120 W for 10 min. Finally, mix the platelet membrane with the above product in a ratio of platelet membrane taken from 1 mL of blood:liposomes prepared from 250 mg of lecithin, and obtain liposome spheres (A&E / CPL) encapsulated with platelet membranes through an extruder with a pore size of 200 nm.

[0111] Example 2: Preparation of doxorubicin-loaded liver-targeted nanospheres (D / GN)

[0112] Preparation of DSPE-PEG 2000 -GA: Synthesized by the method of Example 1 of Chinese Patent CN2018113191397.

[0113] (1) Optimization of the preparation process of nanospheres D / GN

[0114] Prepare doxorubicin-loaded nanospheres ( Figure 2 ) by the ethanol injection method. First, dissolve GAH in PBS and adjust the pH to 5.5 - 6.0 with 1 mol / L NaOH solution. Secondly, according to different mass ratios of glycocholic acid (GAH) to lecithin of 1:1.2, 1:1.4, 1:1.7, 1:2.2, 1:3.2, add the ethanol solutions of DOX, PC and DSPE-PEG 2000 -GA (the ratio of DSPE-PEG 2000 -GA / PC is 1:20) dropwise into the GAH solution. After the ethanol evaporates, sonicate it in an ice bath and sequentially pass through 0.45 μm and 0.22 μm polyethersulfone membranes to obtain D / GN.

[0115] Table 1 Effects of different ratios of lecithin to glycocholic acid on the particle size and PDI of D / GN

[0116]

[0117] (2)Preparation of Nanospheres D / GN

[0118] Dissolve 90 mg of glycocholic acid in 5 mL of PBS solution, and adjust the pH of the solution to 5.5 - 6.0 with 1 mol / L NaOH to obtain the first solution. Dissolve 110 mg of lecithin, 5 mg of doxorubicin (DOX), and 5 mg of DSPE-PEG 2000 -GA in 2 mL of ethanol, and dissolve it by ultrasonic treatment to obtain the second solution. Slowly drip the second solution into the first solution. After the ethanol has completely evaporated, ultrasonically disrupt the product in an ice bath at a power of 120 W for 10 min, and sequentially pass it through 0.45 μm and 0.22 μm polyethersulfone membranes to obtain nanospheres (D / GN).

[0119] 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 2000 -GA is not added.

[0120] (II)Experimental Results

[0121] 1. Physicochemical Property Characterization and Analysis of A&E / CPL and D / GN

[0122] The physicochemical properties of A&E / CPL and D / GN are shown in Table 2 and Figure 3 as follows. The average particle size of A&E / CPL is about 155.6 nm ( Figure 3 as shown in Figure A in Figure 3 ), and it has a negative potential property. As shown in Figure B in Figure 3 , the average particle size of D / GN is 27.5 nm, which not only retains the function of deep penetration of nanoscale particles into the tumor, but also avoids high renal metabolism caused by too small particle size, improves its bioavailability, and has a negative zeta potential, prolonging the in vivo drug circulation time. The drug loading and encapsulation efficiency of ASP and DOX are 2.52%, 2.20% and 82.51%, 90.20% respectively, with relatively high drug loading and encapsulation efficiency.

[0123] Table 2 Physicochemical Property Characterization of A&E / CPL and D / GN

[0124]

[0125] The physicochemical property characterization of A&E / CPL prepared in this application is as shown in Figure 4 . Figure 4 is the TEM image of A&E / CPL prepared in the example of this application. Among them, Figure A, Figure B, and Figure C are the TEM images of A&E / CPL under the conditions of pH = 7.4, pH = 6.0, and pH = 5.5 respectively; from Figure 4As can be seen from the results, the A&E / CPL prepared in this application is in the form of regular spheres at pH = 7.4, while the particle size of A&E / CPL gradually increases at pH = 6.0 and pH = 5.5, and its shape becomes irregular and disintegrates, indicating its pH sensitivity. As Figure 5 shown, the TEM image of D / GN prepared in the example of this application under the condition of pH = 7.4. The D / GN prepared in this application is in the form of regular spheres at pH = 7.4.

[0126] Figure 6 This is the 7-day stability of A&E / CPL prepared in the example of this application in PBS solution and DMEM medium. Among them, the left vertical coordinate is the particle size measured by dynamic light scattering (DLS), and the right vertical coordinate is the polydispersity index (PDI). From Figure 6 the results, it can be seen that the particle size of A&E / CPL is stable and uniform within 7 days in PBS and DMEM medium, indicating its good stability.

[0127] Figure 7 This is the 7-day stability of D / GN prepared in the example of this application in PBS solution and DMEM medium. Among them, the left vertical coordinate is the particle size measured by dynamic light scattering (DLS), and the right vertical coordinate is the polydispersity index (PDI); from Figure 7 the results, it can be seen that the particle size of D / GN prepared in this application is stable and uniform within 7 days in PBS and DMEM medium, indicating its good stability.

[0128] Figure 8 This is the Coomassie brilliant blue image of A&E / CPL prepared in the example of this application. The Coomassie brilliant blue results show that A&E / CPL carries the vast majority of proteins on the platelet membrane, including key functional proteins such as CD11b and CD47.

[0129] Figure 9 This is the in vitro drug release graph of A&E / CPL prepared in the example of this application under different pH conditions. In this application, by measuring the release rate of ASP in A&E / CPL under different pH conditions, its sensitivity to pH is verified again. As Figure 9 shown, under the condition of pH = 5.5, the cumulative release rate of ASP within 48 h is 75.9%, which is significantly higher than that at pH = 6.0 (48.8%) and pH = 7.4 (35.6%), indicating that A&E / CPL has pH sensitivity and is more likely to release drugs under acidic conditions.

[0130] Figure 10In vitro drug release profile of D / GN prepared in the embodiments of the present application. It can be seen from the release curve of D / GN that, compared with free DOX, the in vitro cumulative release rate of D / GN within 6 h is lower, indicating its long circulation property in vitro. At 48 h, its in vitro cumulative release rate is as high as 73.3%, which is relatively close to 78.9% of free DOX, indicating its good in vitro drug release characteristics.

[0131] Figure 11 Horizontal gel electrophoresis image of A&E / CPL prepared in the embodiments of the present application. The SDS-PAGE gel electrophoresis results show that A&E / CPL can successfully encapsulate DNase Ⅰ and highly retain the property of dissolving plasmids.

[0132] 2. In vitro cell uptake

[0133] Figure 12 In vitro cell uptake results of D / GN prepared in the embodiments of the present application. Figure 12 Figure A in it is the in vitro intake laser confocal image of D / GN; Figure 12 Figures B - C in it are the quantitative results of the in vitro cell intake of D / GN by flow cytometry. The preparation method of D / N nanospheres is similar to that of D / GN nanospheres, with the difference that there is no DSPE-PEG 2000 -GA in D / N nanospheres. It can be seen from Figure 12 Figure A that the D / GN group prepared in the present application shows stronger red fluorescence signals than the free DOX group, indicating that compared with the free drug, the nanospheres with nanoscale particle size can be more easily taken up by cells, thus achieving a stronger anti-cancer effect. Secondly, compared with the D / N group, the D / GN group still shows stronger red fluorescence signals, suggesting that D / GN may increase the uptake of nanomicelles by liver cancer cells through the endocytosis mediated by the GA-GA receptor on the surface of the liver cancer cell membrane. Figure 12 The flow cytometry quantitative experiments in Figures B and C of also verified that D / GN increased the content of DOX in cells, which was consistent with the qualitative results of the confocal laser scanning microscope (CLSM).

[0134] 3. In vitro cytotoxicity

[0135] Figure 13 Cytotoxicity evaluation model of D / GN combined with A&E / CPL prepared in the embodiments of the present application, Figure 14 Cytotoxicity evaluation results of D / GN combined with A&E / CPL prepared in the embodiments of the present application. Figure 13 Figure A in it is the diagram of the single HuH-7 model, Figure 13 Figure B in it is the schematic diagram of the HuH-7 + PLT + NET co-culture model. Figure 14 Figure A in it is the MTT detection result of the single HuH-7 model; Figure 14Figure B in this shows the MTT test results of the HuH-7+PLT+NET co-culture model. The tumor microenvironment (TME) plays a crucial 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 with distant tumor metastasis. Therefore, traditional in vitro research models, namely simple tumor cell culture, can no longer simulate the real and complex TME. Therefore, in this application, on the basis of 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 ( Figure 13 as shown in Figures A and B in this). As Figure 14 shown in Figures A and B in this, both cell models showed obvious concentration dependence on various drug preparations. Compared with the 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, resulting in tumor drug resistance. In the co-culture system, the A&E / CPL+D / GN group had stronger cytotoxic effects than the DOX+ASP+DNase Ⅰ group. This may be because the nanospheres with nano-sized particles, that is, mediated by the GA-GA receptor, made the anti-cancer drugs more easily taken up by cells. And CS targeted tumor-associated platelets, ASP inhibited their functions, plus the degradation effect of DNase Ⅰ, which made the tumor cells shed their protective shells and were fully exposed to the environment, increasing drug uptake, which was also confirmed in the cell uptake detection.

[0136] 4. Cell migration and invasion experiments

[0137] Figure 15 This shows the results of exploring the cell migration mechanism of the D / GN combined with A&E / CPL prepared in this application. Figure 15 Figure A in this is the Transwell cell invasion result of the HuH-7+PLT+NET co-culture model; Figure 15 Figure B in this is the Transwell invasion quantitative test result of the HuH-7+PLT+NET co-culture model; Figure 15 Figure C in this is the Western blot analysis result of E-Cadherin and Vimentin; Figure 15 Figure D in this is the quantitative analysis result 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-culture cell line. As Figure 15 shown in Figure A in this and Figure 15As shown in Figure B, compared with single HuH-7 cells, the number of invasive cells in the co-culture cell line increased significantly, indicating that PLT and NET promoted tumor cell invasion and suggesting that the in vitro model was successfully established. The number of invasive cells in the A&E / CPL+D / GN group was the least, effectively inhibiting tumor cell invasion, which may be because the combination of A&E / CPL and D / GN can effectively inhibit the tumor cell-platelet-neutrophil extracellular trap transfer pathway. In this application, Western blot experiments were used to detect the expression of EMT-related proteins, E-Cadherin and Vimentin. As Figure 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-culture cell line was significantly down-regulated, and the expression of Vimentin was significantly up-regulated, indicating that PLT and NET promoted the epithelial-mesenchymal transformation of tumor cells into the M type. Compared with the control group, the expression of E-Cadherin in the A&E / CPL+D / GN group was significantly up-regulated, and the expression of Vimentin was significantly down-regulated, indicating that the combination of A&E / CPL and D / GN can effectively inhibit the transformation of epithelial cells to mesenchymal cells (EMT) of tumor cells induced by PLT and NET, and inhibit tumor cell migration.

[0138] 5. In vivo biodistribution

[0139] Figure 16 This is the test result of the in vivo biodistribution of A&E / CPL and D / GN prepared in this application in tumor-bearing mice. Figure 16 Figure A shows the NIR images of A&E / CPL and D / GN at different time points; Figure 16 Figure B shows the fluorescence images of each group of organs and tumors at 48 h; Figure 16 Figures C to D show the fluorescence intensity analysis results of each group of organs and tumors at DID and DIR wavelengths. To study the in vivo liver cancer targeting of nanospheres and liposome spheres alone, the cell membrane fluorescent probes DID and DIR were respectively encapsulated in nanospheres and liposome spheres, and the in vivo biodistribution of DID-GN and DIR-CPL was tracked and observed through a real-time fluorescence imaging system (IVIS). As Figure 16 As shown in Figure A, at the DID excitation wavelength (544 nm), it was observed that DID-GN concentrated in the tumor site, which was because the modification of GA improved the liver cancer targeting ability of the nanomicelles. At the DIR excitation wavelength (754 nm), it was observed that DIR-CPL also concentrated in the tumor site, which may be because the liposome spheres also have a nanoscale size, which can exert its passive effect of enhanced permeability and retention (EPR) and promote its accumulation in the tumor site. After 48 h, the mice were sacrificed, the main organs (heart, liver, spleen, lung, kidney) and tumors were dissected, and ex vivo fluorescence imaging analysis was performed on them, Figure 16 Figure C andFigure 16 Figure D shows the fluorescence quantitative analysis at DID and DIR wavelengths. In Figure 16 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 tumor tissues. 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 enhanced the in vivo liver cancer targeting of the drugs.

[0140] 6. Drug penetration in vivo

[0141] Figure 17 This is the result of the influence of A&E / CPL prepared in the examples of this application on the tumor penetration effect of D / GN. Figure 17 Figure A shows the tumor cryosection images of each treatment group at different depths; Figure 17 Figure B shows the quantitative graph of the DID fluorescence intensity in tumors of each group at different depths. To evaluate the penetration ability of different drug formulations in tumor tissues, DID, DID-D / GN, DID-D / GN (pretreatment with DNase Ⅰ), DID-D / GN (pretreatment with ASP + DNase Ⅰ), and DID-D / GN (pretreatment with A&E / CPL) were respectively injected into the tail veins of H22+PLT+NET tumor-bearing mouse models, and the fluorescence images of the tumor tissue cryosections of different formulation groups were observed by CLSM and photographed. As Figure 17 shown in Figure A, compared with DID-D / GN, the fluorescence signal of the free drug DID group was mainly distributed at the edge of the tumor tissue, indicating that the free drug could not reach the core part of the tumor, resulting in a weaker anti-tumor effect. In addition, after the combination of DID-D / GN and A&E / CPL, obvious red fluorescence signals were shown from the tumor edge to the core, indicating that it could effectively penetrate the internal area of the tumor tissue, which was beneficial to the deep penetration of the drug in the tumor.

[0142] 7. Anti-tumor effect in vivo

[0143] Figure 18 This is the anti-tumor evaluation result of D / GN combined with A&E / CPL prepared in the examples of this application on the H22+PLT+NET subcutaneous transplanted tumor. Figure 18 Figure A shows the schematic diagram of the H22+PLT+NET subcutaneous transplanted tumor; Figure 18 Figure B shows the tumor photos; Figure 18 Figure C shows the change in tumor volume of different treatment groups; Figure 18 Figure D shows the change in body weight of different treatment groups; Figure 18 Figure E shows the tumor inhibition rate of different treatment groups. To evaluate the in vivo anti-tumor effect and mechanism study of A&E / CPL and D / GN, an H22+PLT+NET tumor-bearing mouse model was established ( Figure 18as shown in Figure A). As Figure 18 As shown in Figure D, compared with the saline group, the body weights of the mice in the A&E / CPL and D / GN groups did not significantly decrease, indicating that the carrier has good biosafety. While the body weight of the free DOX group decreased slightly, indicating that the free anticancer drug has high toxicity and side effects. The mice were sacrificed after 7 times of tail vein injection, and the tumor tissues were observed and photographed. As Figure 18 As shown in Figure B, the drug treatment groups showed varying degrees of tumor suppression effects. The tumor volume of the combination treatment group (DOX + ASP + DNase Ⅰ) was smaller than that of the single drug groups (DOX or ASP + DNase Ⅰ), indicating that combination therapy improved the anti-HCC treatment effect. Among them, the tumor volume of the A&E / CPL + D / GN combination 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 antitumor effect.

[0144] Figure 19 Evaluation of the therapeutic effect of D / GN combined with A&E / CPL prepared in the examples of this application on subcutaneous transplanted tumors of H22 + PLT + NET. Figure 19 Figure A in shows the H&E images of tumors in different treatment groups; Figure 19 Figure B in shows the CD31 immunohistochemical images of tumors in different treatment groups; Figure 19 Figure C in shows the α-SMA immunohistochemical images of tumors in different treatment groups; Figure 19 Figure D in shows the Masson images of tumors in different treatment groups; Figure 19 Figure E in shows the Western blot analysis of E-Cadherin and Vimentin in tissues; Figure 19 Figure F in shows the quantitative analysis results of E-Cadherin and Vimentin. Figure 19 The H&E results in Figure A show that the A&E / CPL + D / GN group showed obvious karyolysis and cytoplasmic vacuolization, indicating a stronger pro-apoptotic effect on tumors. Figure 19 The results in Figure B show that the CD31 immunohistochemical experiment showed that obvious tumor microvessel formation was shown in the control group, indicating the successful establishment of the model. Compared with the free drug group, the tumor microvessel formation in the A&E / CPL + D / GN group was significantly reduced, indicating that combination therapy can inhibit the formation of new blood vessels in tumor foci. Figure 19 The α-SMA immunofluorescence staining experiment in Figure C and Figure 19The Masson trichrome staining results of Figure D in the Chinese text showed that compared with other drug formulation groups, A&E / CPL+D / GN had fewer α-SMA positive areas and less collagen deposition, indicating that the combined use of drug formulations could reduce the production of collagen fibrin. To further investigate the anti-hepatocellular carcinoma mechanism of the combined nanoformulation, the expressions of E-cadherin and Vimentin proteins in tumor tissues were detected by Western blot assay, and the results were as Figure 19 shown in Figure E in the Chinese text. The expressions of E-cadherin and Vimentin in the tumor tissues of "H22+PLT+NET" tumor-bearing mice were down-regulated and up-regulated respectively, indicating that the addition of PLT and NET could promote the EMT of hepatocellular carcinoma cells. The relative expression levels of the two proteins were as Figure 19 shown in Figure F in the Chinese text. The ratio of E-cadherin to GAPDH in the tumor tissues 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 tissues 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 could more effectively inhibit the EMT process of hepatocellular carcinoma.

[0145] Figure 20 The H&E images of the main organs (heart, liver, spleen, lung, kidney) of different treatment groups prepared in the examples of this application. In this application, the in vivo biosafety of the nanoformulation was evaluated by H&E staining and hemolysis experiment. The H&E staining of the main organs (heart, liver, spleen, lung, kidney) of mice after treatment with each drug formulation was as Figure 20 shown. Compared with the normal saline group, there were no obvious pathological damages in the organs of mice in the A&E / CPL+D / GN group, indicating that the combined nanoformulation had good biosafety. The hemolysis experiment was as Figure 21 shown. There was no obvious hemolysis phenomenon in the A&E / CPL group or the D / GN group at each concentration, indicating that the combined nanoformulation could be used as a delivery carrier for anti-hepatocellular carcinoma drugs.

[0146] 8. Analysis of lung metastasis of hepatocellular carcinoma

[0147] Figure 22 This was the evaluation of the therapeutic effect of D / GN combined with A&E / CPL on lung metastasis prepared in the examples of this application. Figure 22 Figure A in it was the schematic diagram of the establishment and treatment of lung metastasis; Figure 22 Figure B in it was the body weight change of mice in each treatment group; Figure 22 Figure C in it was the number of lung nodules in each treatment group; Figure 22 Figure D in it was the lung photos of each treatment group; Figure 22Figure E in it is the lung H&E image of each treatment group. Distant metastasis is one of the main reasons for the high recurrence rate of HCC treatment. To evaluate the inhibitory effect of A&E / CPL combined with D / GN on lung metastasis of liver cancer in vivo, a lung metastasis model of liver cancer was established by injecting H22 cells through the tail vein ( Figure 22 as shown in Figure A in Figure 22 ). As shown in Figure B in Figure 22 , 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 nanoplatform is a safe carrier for anti-cancer drug delivery. As shown in Figure C in Figure 22 and Figure D in

[0148] , compared with the healthy group of mice, a large number of metastatic lung nodules were observed in the lung tissue of the saline group of mice, indicating that the lung metastasis model of liver cancer 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 nanoplatform can effectively inhibit the distant metastasis of liver cancer cells. 9. Pulmonary neutrophil NET formation experiment

[0149] Figure 23 This is the result of the effect of D / GN combined with A&E / CPL prepared in this application on pulmonary neutrophils and NET formation. To evaluate the effects of A&E / CPL and D / GN on pulmonary neutrophils and neutrophil extracellular trap NET colonization, a lung tissue immunofluorescence experiment was conducted. 1×10 6 H22 cells were injected into the mice through the tail vein, and different drug formulations were used for tail vein treatment 7 times. After the treatment, the mice were sacrificed, and the lung tissue was immunofluorescently stained with His H3 and Ly6G proteins. The results are as shown in Figure 23 . Compared with the A&E / CPL group and the D / GN group, the A&E / CPL+D / GN group showed less green fluorescence of His H3 and red fluorescence of Ly6G, indicating that the combined nanoplatform reduced the content of neutrophils in the lung tissue, inhibited the NET level, and showed a good effect of inhibiting pulmonary neutrophil colonization.

[0150] In the above embodiments, the descriptions of each embodiment have their own emphases. For the parts not detailed in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0151] The above has introduced in detail a nano - preparation for inhibiting tumor metastasis, its preparation method and application. Specific examples are used in this article to elaborate on the principle and implementation manner of this application. The description of the above embodiments is only used to help understand the method and its core idea of this application; at the same time, for those skilled in the art, according to the idea of this application, there will be changes in the specific implementation manner and application scope. In summary, the content of this specification should not be construed as a limitation to this 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 a platelet membrane; 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 platelet membrane meets the following requirement: M1=(M2×M0) / 250, where M1 is the mass of platelet membrane in mg, M2 is the mass of lecithin in mg, and M0 is the mass of platelet membrane extracted from 1 mL of whole blood in mg; The DSPE-PEG 2000 The structural formula of -CS is as follows: 。 2. 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%.

3. 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, platelet membrane is added and mixed, and platelet membrane-wrapped liposome spheres are obtained through an extruder.

4. The nanoformulation for inhibiting tumor metastasis according to claim 2, 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.

5. A method for preparing a nano preparation for inhibiting tumor metastasis as claimed in claim 1, 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, platelet membrane is added and mixed, and platelet membrane-wrapped liposome spheres are obtained through an extruder.

6. The method for preparing the nanoformulation for inhibiting tumor metastasis according to claim 5, 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.

7. The method for preparing the nano preparation for inhibiting tumor metastasis according to claim 6, characterized in that: The glycocholic acid, lecithin 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%.

8. Use of the nanoformulation for inhibiting tumor metastasis according to any one of claims 1 to 4 or the nanoformulation prepared by the method for preparing the nanoformulation for inhibiting tumor metastasis according to any one of claims 5 to 7 in preparing drugs for treating tumors and drugs for inhibiting tumor metastasis.

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