Magnetic nano-particle targeting carrier for liver cancer and use method of magnetic nano-particle targeting carrier

Abstract

The invention relates to the technical field of targeting carriers, in particular to a magnetic nanoparticle targeting carrier for liver cancer and a use method thereof.The magnetic nanoparticle targeting carrier comprises a ferroferric oxide magnetic inner core with superparamagnetism, the inner core is subjected to surface modification, and a modification group is carboxyl; the platelet membrane layer tightly wraps the inner core, and the source of the platelet membrane layer is platelet of a healthy individual; the galactose (Gal) and the anti-PD-L1 antibody are modified on the surface of the platelet membrane layer; a chemotherapeutic drug is loaded in the inner core, and the platelet membrane layer is combined with the inner core through ultrasonic fusion; the magnetic nanoparticle targeting carrier has a remarkable treatment effect in the aspect of liver cancer treatment, and the ferroferric oxide magnetic core with superparamagnetism in the technical scheme provides magnetism for the carrier, so that the carrier can directionally move under the guidance of an external magnetic field and accurately gather at a tumor site, and the treatment effect is greatly improved. The concentration of the medicine in tumor tissues is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of targeted carriers, and specifically provides a magnetic nanoparticle targeted carrier for liver cancer and its usage method. Background Art

[0002] Liver cancer is a common and highly malignant tumor disease globally, seriously threatening human health. Its onset is latent, and most patients are diagnosed at the middle or advanced stage, with poor curative effects. Hepatocellular carcinoma cells have extremely strong invasion and metastasis abilities, easily invading surrounding tissues and distant organs, leading to rapid deterioration of the condition. Among the many causes of cancer-related deaths, liver cancer accounts for a relatively high proportion, bringing a heavy burden to the families of patients and society.

[0003] Currently, chemotherapy is one of the commonly used means for the treatment of liver cancer. Chemotherapeutic drugs act by inhibiting the division and growth of cancer cells. However, during the treatment process, chemotherapeutic drugs lack the ability to specifically recognize cancer cells and will also damage normal cells while attacking cancer cells. This causes a series of serious side effects in patients, such as nausea, vomiting, hair loss, bone marrow suppression, etc. Bone marrow suppression will lead to a decrease in the patient's immunity and an increased risk of infection; long-term chemotherapy may also cause toxic reactions in important organs such as the heart, liver, and kidneys, further damaging the patient's physical health, reducing the quality of life, and even affecting the smooth progress of subsequent treatment.

[0004] In order to reduce the side effects caused by traditional treatment methods such as chemotherapy to liver cancer patients and improve the treatment effect, safer and more effective treatment regimens have been continuously explored. Against this background, the present invention proposes a magnetic nanoparticle targeted carrier and usage method for the treatment of liver cancer.

[0005] This technical solution aims to achieve the precise delivery of chemotherapeutic drugs, enabling the drugs to specifically act on hepatocellular carcinoma cells and reducing damage to normal tissues, thereby improving the treatment effect while reducing side effects. Summary of the Invention

[0006] Aiming at the problems existing in the prior art, the present invention provides a magnetic nanoparticle targeted carrier for liver cancer and its usage method.

[0007] To achieve the above object, the present invention provides the following technical solution: A magnetic nanoparticle targeted carrier for the treatment of liver cancer, comprising: A superparamagnetic magnetite magnetic core, the core is surface-modified, and the modifying group is carboxyl to enhance the binding ability of the core to other components; A platelet membrane layer tightly wrapping the core, the platelet membrane layer is derived from platelets of healthy individuals and is purified multiple times during the preparation process to remove impurities; Galactose (Gal) and anti-PD-L1 antibody modified on the surface of the platelet membrane layer; The core is loaded with chemotherapeutic drugs. The platelet membrane layer and the core are combined by ultrasonic fusion, and a surfactant is added during the fusion process to promote the fusion effect. As a further technical solution, the chemotherapeutic drug is selected from at least one of doxorubicin, cisplatin or paclitaxel, and the mass ratio of the chemotherapeutic drug to iron oxide is 1:6 - 10. When the chemotherapeutic drug is doxorubicin, its dispersion state in the carrier is uniform dispersion, and there is a weak interaction between doxorubicin and iron oxide, such as hydrogen bond or van der Waals force. As a further technical solution, the galactose (Gal) and anti-PD-L1 antibody are covalently linked to the surface of the platelet membrane layer through a crosslinking agent SMCC, and the molar ratio of galactose (Gal) to anti-PD-L1 antibody is 3 - 5:1; During the linking process, the reaction temperature is controlled at 25°C ± 2°C, and the reaction time is 4 - 5 hours to ensure the stability and effectiveness of the linking.

[0008] As a further technical solution, the platelet membrane layer is prepared by the following steps: Collect whole blood and centrifuge to separate platelet-rich plasma by density gradient centrifugation, with a centrifugation speed of 1500 - 2000 revolutions per minute and a centrifugation time of 10 - 15 minutes; Perform multiple freeze-thaw lyses on the separated platelets. Each freeze-thaw condition is: freezing temperature -80°C, freezing time 2 - 3 hours, thawing temperature 37°C, thawing time 18 - 20 minutes, and then perform ultrasonic treatment to obtain membrane vesicles, with an ultrasonic power of 200 - 300 watts and an ultrasonic time of 10 - 12 minutes; Ultrasonically fuse the membrane vesicles with the drug-loaded iron oxide magnetic core under the condition of an ultrasonic frequency of 40 - 50 kHz. As a further technical solution, its particle size is 80 - 150 nm, measured by dynamic light scattering method, the surface Zeta potential is +20 to +35 mV, and after the carrier is placed in physiological saline for 72 hours, the particle size change rate is less than 10%, and the Zeta potential change rate is less than 5% to ensure its stability. As a further technical solution, the preparation method of the magnetic nanoparticle targeting carrier includes the following steps: a. Synthesize carboxylated Fe3O4 nanoparticles by solvothermal method, with a reaction temperature of 180 - 200°C, a reaction time of 8 - 12 hours, and nitrogen protection is introduced during the reaction process; b. Mix and stir the chemotherapeutic drug and Fe3O4 at a stirring speed of 300 - 500 revolutions per minute for 2 - 3 hours to form a drug-loaded core; c. Ultrasonically fuse the platelet membrane vesicles with the drug-loaded core, with the ultrasonic power being 250 - 350 watts and the ultrasonic time being 15 - 20 minutes to form a membrane-coated structure; d. Use the SMCC crosslinking agent to conjugate galactose (Gal) and anti-PD-L1 antibody to the membrane surface. The conjugation reaction is carried out under light avoidance conditions, and the pH value of the reaction system is controlled at 7.2 - 7.6. As a further technical solution, the conditions for the conjugation reaction in step d are: reacting at room temperature under light avoidance for 2 - 3 hours. When centrifugally purifying, the ultracentrifugation method is adopted, with the centrifugal speed being 10,000 - 12,000 revolutions per minute and the centrifugal time being 20 - 30 minutes. After purification, it is stored in a phosphate buffer solution with a pH of 7.4, and the storage temperature is 4°C. The usage method of the magnetic nanoparticle targeting carrier includes the following steps: Introduce the carrier into the liver cancer body through tail vein injection, with the injection speed being 0.1 - 0.16 ml / minute; Apply an external magnetic field of 0.6 - 1.0 T at the tumor site and continuously act for 30 - 40 minutes. The magnetic field application method is to generate a uniform magnetic field using an electromagnet; Administer the drug once every 3 days, with the dose being 0.2 - 1.0 mg siRNA / kg or 5 - 20 mg chemotherapy drug / kg.

[0009] As a further technical solution, the carrier releases chemotherapy drugs through pH-sensitive release in the tumor microenvironment. When the environmental pH value is lower than 6.5, the structure of the carrier changes, thereby triggering drug release. And the anti-PD-L1 antibody binds to PD-L1 on the surface of tumor cells to activate the T cell immune response, and the number of activated T cells increases by more than 50% within 72 hours after drug administration.

[0010] Compared with the prior art, the beneficial effects of the present invention are: The magnetic nanoparticle targeting carrier of the present invention has a significant therapeutic effect in the treatment of liver cancer. In the technical solution of the present invention, the superparamagnetic magnetite magnetic core not only provides magnetism for the carrier, enabling it to move directionally under the guidance of an external magnetic field and accurately aggregate at the tumor site, thereby increasing the drug concentration in the tumor tissue; at the same time, as the core structure of the carrier, it provides a basis for loading chemotherapy drugs. The carboxyl groups modified on its surface increase the hydrophilicity and chemical reactivity of the core, which is beneficial for subsequent connection and reaction with other components.

[0011] The platelet membrane layer is derived from the platelets of healthy individuals. The platelet membrane layer has good biocompatibility and immune evasion characteristics, which can reduce the probability of the carrier being recognized and cleared by the immune system, prolong the circulation time of the carrier in the body, and enable the drug to have more opportunities to reach the tumor site. In addition, the encapsulation of the platelet membrane layer can protect the core and chemotherapy drugs, reducing their premature release and degradation during blood circulation.

[0012] Galactose (Gal) modified on the surface of the platelet membrane layer and the galactose in the anti-PD-L1 antibody can specifically recognize the asialoglycoprotein receptor highly expressed on the surface of liver cancer cells, achieving the active targeting of the carrier to liver cancer cells and further improving the enrichment degree of the drug in tumor tissues. The anti-PD-L1 antibody can block the binding of PD-L1 on the surface of tumor cells to PD-1 on the surface of T cells, activate the T cell immune response, enhance the body's own anti-tumor immune ability, and enable the immune system to better recognize and attack cancer cells.

[0013] The specific mass ratio of the chemotherapy drug to iron oxide ensures the effective payload of the drug, while avoiding the influence of too much or too little drug on the treatment effect and safety. In the tumor microenvironment, the carrier releases the chemotherapy drug through a pH-sensitive mechanism. When the environmental pH value is lower than 6.5, the structure of the carrier changes to trigger drug release, realizing the precise release of the chemotherapy drug at the tumor site, improving the killing effect of the drug on cancer cells, and reducing the toxic and side effects on normal tissues at the same time.

[0014] The carrier is introduced into the liver cancer body through tail vein injection, and an external magnetic field is applied at the tumor site. Utilizing the characteristics of the magnetic core, the enrichment effect of the carrier at the tumor site is further enhanced. A reasonable dosing frequency and dose can not only ensure that the drug maintains an effective therapeutic concentration in the body, but also avoid the toxic and side effects caused by excessive drug. The technical solution of the present invention can achieve the synergistic effect of chemotherapy and immunotherapy, while improving the treatment effect of liver cancer, effectively reducing the side effects of traditional chemotherapy, and providing an innovative and efficient solution for the treatment of liver cancer. Description of the Drawings

[0015] Figure 1 It is a statistical chart of the tumor inhibition rate of each experimental group. Detailed Embodiments

[0016] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0017] The present invention provides a magnetic nanoparticle targeting carrier for the treatment of liver cancer, and its preparation method and usage method, which are as follows: Magnetic nanoparticle targeting carrier: It has a superparamagnetic Fe₃O₄ magnetic core, and the surface of the core is modified with carboxyl groups; a platelet membrane layer tightly wrapping the core, which is derived from platelets of healthy individuals; galactose (Gal) and anti-PD-L1 antibody modified on the surface of the platelet membrane layer; chemotherapy drugs are loaded in the core, and the platelet membrane layer and the core are combined by ultrasonic fusion.

[0018] Chemotherapy drugs: The chemotherapy drugs are selected from at least one of doxorubicin, cisplatin or paclitaxel, and the mass ratio of the chemotherapy drug to Fe₃O₄ is 1:6 - 10.

[0019] Connection of galactose (Gal) and anti-PD-L1 antibody: Galactose (Gal) and anti-PD-L1 antibody are covalently connected to the surface of the platelet membrane layer through a cross-linking agent SMCC, and the molar ratio of galactose (Gal) to anti-PD-L1 antibody is 3 - 5:1; during the connection process, the reaction temperature is controlled at 25°C ± 2°C, and the reaction time is 4 - 5 hours.

[0020] Preparation of platelet membrane layer: Whole blood is collected and platelet-rich plasma is centrifugally separated by density gradient centrifugation, with a centrifugal speed of 1500 - 2000 revolutions per minute and a centrifugation time of 10 - 15 minutes; the separated platelets are subjected to multiple freeze-thaw lysis, and each freeze-thaw condition is: freezing temperature -80°C, freezing time 2 - 3 hours, thawing temperature 37°C, thawing time 18 - 20 minutes, and then ultrasonic treatment is carried out to obtain membrane vesicles, with an ultrasonic power of 200 - 300 watts and an ultrasonic time of 10 - 12 minutes; the membrane vesicles and the drug-loaded Fe₃O₄ magnetic core are ultrasonically fused under the condition of an ultrasonic frequency of 40 - 50 kHz.

[0021] Carrier characteristics: The particle size is 80 - 150 nm, determined by dynamic light scattering method, the surface Zeta potential is +20 to +35 mV, and after the carrier is placed in physiological saline for 72 hours, the particle size change rate is less than 10%, and the Zeta potential change rate is less than 5%.

[0022] Preparation method Carboxylated Fe₃O₄ nanoparticles are synthesized by a solvothermal method, with a reaction temperature of 180 - 200°C and a reaction time of 8 - 12 hours, and nitrogen protection is introduced during the reaction process.

[0023] The chemotherapy drug and Fe₃O₄ are mixed and stirred at a stirring speed of 300 - 500 revolutions per minute for 2 - 3 hours to form a drug-loaded core.

[0024] The platelet membrane vesicles and the drug-loaded core are ultrasonically fused with an ultrasonic power of 250 - 350 watts and an ultrasonic time of 15 - 20 minutes to form a membrane-coated structure.

[0025] Galactose (Gal) and anti-PD-L1 antibody are conjugated to the membrane surface using an SMCC crosslinker. The conjugation reaction is carried out in the dark, and the pH value of the reaction system is controlled at 7.2 - 7.6; the usage amount of the SMCC crosslinker is 1.5 - 2 micromoles per milligram of platelet membrane protein; the conjugation reaction conditions are: reacting in the dark at room temperature for 2 - 3 hours. Ultracentrifugation is used for centrifugal purification, with a centrifugal speed of 10,000 - 12,000 revolutions per minute and a centrifugal time of 20 - 30 minutes. After purification, it is stored in a phosphate buffer solution with a pH of 7.4 at a storage temperature of 4°C.

[0026] Usage The carrier is introduced into the liver cancer body by tail vein injection at an injection speed of 0.1 - 0.16 ml / minute.

[0027] An external magnetic field of 0.6 - 1.0 T is applied at the tumor site for 30 - 40 minutes continuously. The magnetic field application method is to generate a uniform magnetic field using an electromagnet.

[0028] The drug is administered once every 3 days, with a dose of 0.2 - 1.0 mg siRNA / kg or 5 - 20 mg chemotherapeutic drug / kg; the carrier releases chemotherapeutic drugs through pH-sensitive release in the tumor microenvironment. When the environmental pH value is lower than 6.5, the carrier structure changes to trigger drug release, and the anti-PD-L1 antibody binds to PD-L1 on the surface of tumor cells to activate the T cell immune response, and the number of activated T cells increases by more than 50% within 72 hours after drug administration.

[0029] To further illustrate the present invention, the following detailed description is given through the following examples. The reagents and materials used in the following examples of the present invention are commercially available unless otherwise specified.

[0030] Example 1: Preparation of magnetic nanoparticle targeting carrier Carboxylated Fe3O4 nanoparticles are synthesized by the solvothermal method: Sodium acetate trihydrate and sodium citrate are dissolved in ethylene glycol and mixed with an ethylene glycol solution, stirred for 30 minutes, and then reacted at 180°C under nitrogen protection for 12 hours. After cooling to room temperature, it is washed with ethanol and dried at 60°C to obtain carboxylated Fe3O4 nanoparticles.

[0031] Loading chemotherapeutic drug: Doxorubicin is selected and mixed according to the mass ratio of doxorubicin to Fe3O4 of 1:6, and stirred at a stirring speed of 300 revolutions per minute for 3 hours to form a drug-loaded core.

[0032] Preparation of platelet membrane vesicles: Whole blood from healthy individuals was collected and subjected to density gradient centrifugation at a speed of 1500 revolutions per minute for 15 minutes to separate platelet-rich plasma. The platelets were subjected to freeze-thaw lysis at a freezing temperature of -80 °C for 2.5 hours and a thawing temperature of 37 °C for 18 minutes, repeated 3 times, and then sonicated for 12 minutes under the condition of a sonication power of 200 watts to obtain membrane vesicles.

[0033] Sonication fusion: The membrane vesicles and the drug-loaded core were sonicated and fused for 15 minutes under the conditions of a sonication frequency of 40 kHz and a sonication power of 250 watts to form a membrane-coated structure.

[0034] Coupling modification: Using SMCC crosslinker, according to the molar ratio of galactose (Gal) to anti-PD-L1 antibody of 3:1, reacting for 4 hours at 25 °C, in the dark, and at pH 7.2 to couple galactose (Gal) and anti-PD-L1 antibody to the membrane surface; the usage amount of SMCC crosslinker was 1.5 micromoles per milligram of platelet membrane protein; after the reaction, ultracentrifugation was used to centrifuge at a speed of 10,000 revolutions per minute for 30 minutes for purification, and after purification, it was stored in phosphate buffer at pH 7.4 and stored at 4 °C.

[0035] Using magnetic nanoparticle targeting carrier: A mouse model with liver cancer was selected and the prepared magnetic nanoparticle targeting carrier was injected through the tail vein at a speed of 0.1 ml / min; an external magnetic field of 0.6 T was applied at the tumor site using an electromagnet for 30 minutes continuously; the drug was administered once every 3 days, and the dosage was 0.2 mg siRNA / kg and 5 mg chemotherapeutic drug / kg.

[0036] Example 2: Preparation of magnetic nanoparticle targeting carrier Synthesis of carboxylated Fe3O4 nanoparticles: The reaction temperature was set at 190 °C and the reaction time was 10 hours, and other conditions were the same as in Example 1.

[0037] Loading chemotherapeutic drug: Cisplatin was selected, and the mass ratio of cisplatin to Fe3O4 was 1:8, with a stirring speed of 400 revolutions per minute and a stirring time of 2.5 hours.

[0038] Preparation of platelet membrane vesicles: The centrifugation speed was 1800 revolutions per minute and the centrifugation time was 12 minutes; during freeze-thaw lysis, it was frozen for 2 hours and thawed for 20 minutes; the sonication power was 250 watts and the sonication time was 11 minutes.

[0039] Sonication fusion: The sonication frequency was 45 kHz, the sonication power was 300 watts, and the sonication time was 18 minutes.

[0040] Coupling modification: The molar ratio of galactose (Gal) to anti-PD-L1 antibody is 4:1, the reaction temperature is 25 °C, the reaction time is 4.5 hours, and the pH is 7.4; the ultracentrifugation speed is 11,000 revolutions per minute, and the centrifugation time is 25 minutes; the amount of SMCC cross-linking agent used is 1.8 micromoles per milligram of platelet membrane protein.

[0041] Using magnetic nanoparticle targeting carriers: Select another batch of mouse models with liver cancer, and the tail vein injection speed is 0.13 ml / min; the external magnetic field strength is 0.8 T, and the action time is 35 minutes; administer the drug once every 3 days, and the dose is 0.6 mg siRNA / kg and 12 mg chemotherapeutic drug / kg.

[0042] Example 3: Preparation of magnetic nanoparticle targeting carriers Synthesis of carboxylated Fe3O4 nanoparticles: The reaction temperature is 200 °C, and the reaction time is 8 hours.

[0043] Loading chemotherapeutic drugs: The mass ratio of paclitaxel to Fe3O4 is 1:10, the stirring speed is 500 revolutions per minute, and the stirring time is 2 hours.

[0044] Preparation of platelet membrane vesicles: The centrifugation speed is 2000 revolutions per minute, and the centrifugation time is 10 minutes; freeze-thaw lysis, freeze for 3 hours, and thaw for 19 minutes; the ultrasonic power is 300 watts, and the ultrasonic time is 10 minutes.

[0045] Ultrasonic fusion: The ultrasonic frequency is 50 kHz, the ultrasonic power is 350 watts, and the ultrasonic time is 20 minutes.

[0046] Coupling modification: The molar ratio of galactose (Gal) to anti-PD-L1 antibody is 5:1, the reaction temperature is 23 °C, the reaction time is 5 hours, and the pH is 7.6; the ultracentrifugation speed is 12,000 revolutions per minute, and the centrifugation time is 20 minutes; the amount of SMCC cross-linking agent used is 1.6 micromoles per milligram of platelet membrane protein.

[0047] Using magnetic nanoparticle targeting carriers: Select a new mouse model with liver cancer, and the tail vein injection speed is 0.16 ml / min; the external magnetic field strength is 1.0 T, and the action time is 40 minutes; administer the drug once every 3 days, and the dose is 1.0 mg siRNA / kg and 20 mg chemotherapeutic drug / kg.

[0048] Example 4: Preparation of magnetic nanoparticle targeting carriers Synthesis of carboxylated Fe3O4 nanoparticles: The reaction conditions are the same as those in Example 2.

[0049] Loading chemotherapeutic drugs: Simultaneously use doxorubicin and cisplatin, and the total mass ratio of the two to Fe3O4 is 1:7, the mass ratio of doxorubicin to cisplatin is 1:1, the stirring speed is 350 revolutions per minute, and the stirring time is 2.8 hours.

[0050] Preparation of platelet membrane vesicles: The conditions are the same as those in Example 3.

[0051] Ultrasonic fusion: The conditions are the same as those in Example 1.

[0052] Conjugation modification: The molar ratio of galactose (Gal) to anti-PD-L1 antibody is 3.5:1, the reaction temperature is 24 °C, the reaction time is 4.2 hours, and the pH is 7.3; the ultracentrifugation speed is 10,500 revolutions per minute, and the centrifugation time is 28 minutes; the usage amount of SMCC cross-linker is 2 micromoles per milligram of platelet membrane protein.

[0053] Using magnetic nanoparticle targeting carriers: Select a new mouse model of liver cancer, and the tail vein injection speed is 0.12 mL / min; the external magnetic field strength is 0.7 T, and the action time is 32 minutes; administer the drug once every 3 days, and the dose is 0.4 mg siRNA / kg and 8 mg chemotherapy drug / kg.

[0054] Comparative Example 1 Preparation of a comparative carrier The preparation process is the same as that in Example 1, but without loading chemotherapy drugs, only preparing a carrier containing an Fe3O4 core, a platelet membrane layer, and surface-modified galactose (Gal) and anti-PD-L1 antibody.

[0055] Using the comparative carrier: Select a mouse model of liver cancer of the same type as that in Example 1, and the tail vein injection speed, external magnetic field conditions, and drug administration frequency are the same as those in Example 1, but without giving the chemotherapy drug dose, only giving 0.2 mg siRNA / kg.

[0056] Comparative Example 2 Preparation of a comparative carrier The preparation process is the same as that in Example 1, but without modifying the anti-PD-L1 antibody, only modifying galactose.

[0057] Using the comparative carrier: Select a mouse model of liver cancer, and the tail vein injection speed, external magnetic field conditions, drug administration frequency, and dose are the same as those in Example 1.

[0058] Preparation before the experiment I. Experimental animals Selection of experimental animals: Female C57BL / 6 mice aged 6 - 8 weeks and weighing 20 - 25 g are selected.

[0059] Feeding environment: The mice are housed in an SPF-class animal room with a temperature of 22 ± 2 °C, a relative humidity of 50 ± 5%, and a 12-hour light / 12-hour dark cycle. They are allowed to eat and drink freely and are acclimated for 1 week before the experiment.

[0060] Mouse screening criteria The body weight is within the range of the standard weight ± 10%.

[0061] Appear healthy, without obvious skin damage, hair loss, abnormal breathing, etc.

[0062] Behavior and activities are normal, without abnormal manifestations such as sluggish movement, huddling, convulsions, etc.

[0063] After pathogen detection, it is confirmed that there is no specific pathogen infection.

[0064] II. Hepatocellular carcinoma tumor modeling method Cell line: The Hepa1-6 hepatocellular carcinoma cell line was selected.

[0065] Cell culture: Hepa1-6 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and placed in an incubator at 37°C. Cultured, passaged every 2 - 3 days, and cells in the logarithmic growth phase were used for modeling.

[0066] Modeling method Take Hepa1-6 cells in the logarithmic growth phase, wash them 2 times with PBS, and then adjust the cell concentration to cells / mL.

[0067] After anesthetizing the mice by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), fix them on the operating table and routinely disinfect the skin.

[0068] Make an incision about 0.5 cm long at about 0.5 cm below the costal arch in the right midclavicular line of the mice, bluntly separate the subcutaneous tissue and muscle to expose the liver.

[0069] Slowly inject 100 μL of cell suspension (containing Hepa1-6 cells) into the left lobe parenchyma of the liver with a 1 mL sterile syringe, and the injection depth is about 3 mm.

[0070] After the injection, compress the bleeding site with a sterile cotton ball for 2 minutes, replace the liver, and suture the muscle and skin layer by layer.

[0071] After the operation, place the mice in an incubator until they wake up, and raise them routinely. Observe the mental state, diet, and activities of the mice every day.

[0072] Judgment criteria for successful modeling: 7 - 10 days after inoculation, a mass can be felt in the upper right abdomen of the mice by palpation; 14 days after inoculation, detected by an ultrasonic imaging system, it is confirmed that there is a space-occupying lesion in the liver, and the tumor volume is about 50 - 100 mm³, then it can be considered that the modeling is successful.

[0073] III. Experimental grouping Randomly divide the mice with successful modeling into 6 groups, with 10 mice in each group: Example 1 group: Receive the treatment with the magnetic nanoparticle targeting carrier prepared in Example 1.

[0074] Example 2 group: Treated with the magnetic nanoparticle targeting carrier prepared in Example 2.

[0075] Example 3 group: Treated with the magnetic nanoparticle targeting carrier prepared in Example 3.

[0076] Example 4 group: Treated with the magnetic nanoparticle targeting carrier prepared in Example 4.

[0077] Control group 1: Treated with the control carrier prepared in Control Example 1.

[0078] Control group 2: Treated with the control carrier prepared in Control Example 2.

[0079] IV. Preparation before administration Preparation of carrier solution: Dilute the prepared magnetic nanoparticle targeting carrier or control carrier with sterile PBS to the required concentration, filter and sterilize it through a 0.22 μm filter membrane, and reserve it for use.

[0080] Preparation of administration equipment: Use a 1 mL sterile syringe and a 26G sterile needle, and perform autoclaving before each administration.

[0081] Magnetic field application equipment: Adopt a customized electromagnet device, which can generate a uniform magnetic field of 0.6 - 1.0 T, and the magnetic field intensity and action time can be precisely controlled.

[0082] V. Administration protocol Route of administration: Intravenous injection via the tail vein.

[0083] Injection rate: Inject at the rate set in each example and control example (0.1 - 0.16 mL / min).

[0084] Administration frequency: Administer once every 3 days for a total of 5 times.

[0085] Administration dose: Administer according to the dose set in each example and control example (0.2 - 1.0 mg siRNA / kg or 5 - 20 mg chemotherapeutic drug / kg).

[0086] Experiment 1: Tumor inhibition effect experiment Experimental method: At different time periods (the 7th day, the 14th day, the 21st day) after the treatment in the examples and control examples, use an in vivo imaging system to observe the growth of tumors in mice, measure the change in tumor volume; dissect the mice, weigh the tumors, calculate the tumor inhibition rate, and the tumor inhibition rate = (tumor weight of the control group - tumor weight of the experimental group) / tumor weight of the control group × 100%; Experimental results: Table 1 Tumor volume on the 7th day (mm³) Tumor volume on the 14th day (mm³) Tumor volume on the 21st day (mm³) Tumor inhibition rate (%) Example 1 150±20 280±40 450±65 65.8 Example 2 130±15 220±30 350±50 73.5 Example 3 120±18 200±35 320±45 76.5 Example 4 140±22 250±38 400±60 69.4 Comparative Example 1 280±35 520±60 850±100 35.2 Comparative Example 2 250±30 480±55 780±90 42.1 Tumor suppression effect: As can be seen from the data in Table 1, the tumor volumes of the groups of Examples 1-4 at different time points were significantly smaller than those of the groups of Comparative Example 1 and Comparative Example 2. Since the chemotherapy drug was not loaded in Comparative Example 1, its tumor suppression effect was the worst; although the chemotherapy drug was loaded in Comparative Example 2, but lacked the modification of anti-PD-L1 antibody, its tumor suppression effect was also inferior to that of the Example groups. This indicates that the magnetic nanoparticle targeting carrier of the present invention can more effectively inhibit tumor growth when simultaneously loaded with a chemotherapy drug and modified with an anti-PD-L1 antibody. The tumor suppression effect of Example 3 was the best, probably because its preparation process parameters were more conducive to the loading and release of the drug, as well as the function of the targeting molecule.

[0087] Experiment 2: Immune activation effect experiment Experimental method: 72 hours after administration, the tumor tissues and peripheral blood of the mice were collected, and the number of activated T cells in the tumor tissues and peripheral blood was detected by flow cytometry; the expression levels of related immune factors (such as IFN-γ, TNF-α, etc.) in the tumor tissues were detected by ELISA method; Experimental results: Table 2

[0088] Immune activation effect: As can be seen from Table 2, the number of activated T cells and the expression levels of immune factors in the groups of Examples 1-4 were significantly higher than those of the groups of Comparative Example 1 and Comparative Example 2. Since the chemotherapy drug was not loaded in Comparative Example 1, it could not effectively activate the immune response; Comparative Example 2 lacked the modification of anti-PD-L1 antibody, and its ability to activate T cells and secrete immune factors was weak. The immune activation effect of Example 3 was the most prominent, which may be related to the modification ratio and activity of the anti-PD-L1 antibody on the surface of the carrier in this group, enabling it to more effectively block the PD-L1 / PD-1 signaling pathway and activate the T cell immune response.

[0089] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor limit the invention to the specific embodiments described. Obviously, many modifications and changes can be made according to the content of this specification.

Claims

1. A magnetic nanoparticle targeting carrier for liver cancer treatment, characterized in that, Comprising: A superparamagnetic magnetite magnetic core, the core being surface-modified with a carboxyl group; A platelet membrane layer tightly wrapping the core, the platelet membrane layer being derived from platelets of a healthy individual; Galactose (Gal) and anti-PD-L1 antibody modified on the surface of the platelet membrane layer; The core is loaded with a chemotherapeutic drug, and the platelet membrane layer and the core are combined by ultrasonic fusion.

2. The magnetic nanoparticle targeting carrier for liver cancer treatment according to claim 1, wherein The chemotherapeutic drug is selected from at least one of doxorubicin, cisplatin or paclitaxel, and the mass ratio of the chemotherapeutic drug to magnetite is 1:6 - 10.

3. A magnetic nanoparticle targeting vector for liver cancer treatment according to claim 1, wherein The galactose (Gal) and anti-PD-L1 antibody are covalently linked to the surface of the platelet membrane layer through a cross-linking agent SMCC, and the molar ratio of galactose (Gal) to anti-PD-L1 antibody is 3 - 5:1; During the linking process, the reaction temperature is controlled at 25°C ± 2°C, and the reaction time is 4 - 5 hours.

4. A magnetic nanoparticle targeting carrier for liver cancer treatment according to claim 1, characterized in that, The platelet membrane layer is prepared by the following steps: Collect whole blood and centrifuge to separate platelet-rich plasma by density gradient centrifugation at a centrifugal speed of 1500 - 2000 revolutions per minute for 10 - 15 minutes; Perform multiple freeze-thaw lyses on the separated platelets. Each freeze-thaw condition is: freezing temperature -80°C, freezing time 2 - 3 hours, thawing temperature 37°C, thawing time 18 - 20 minutes, and then perform ultrasonic treatment to obtain membrane vesicles with an ultrasonic power of 200 - 300 watts and an ultrasonic time of 10 - 12 minutes; Ultrasonically fuse the membrane vesicles with the magnetite magnetic core loaded with the chemotherapeutic drug under the condition of an ultrasonic frequency of 40 - 50 kHz.

5. A magnetic nanoparticle targeting carrier for liver cancer treatment according to claim 1, characterized in that, The magnetic nanoparticle targeting carrier for liver cancer treatment has a particle size of 80 - 150 nm, determined by dynamic light scattering method, a surface Zeta potential of +20 to +35 mV, and after being placed in physiological saline for 72 hours, the particle size change rate is less than 10%, and the Zeta potential change rate is less than 5%.

6. The magnetic nanoparticle targeting carrier for liver cancer treatment according to claim 1, wherein The preparation method of the magnetic nanoparticle targeting carrier includes the following steps: a. Synthesize carboxylated Fe3O4 nanoparticles by solvothermal method at a reaction temperature of 180 - 200°C for 8 - 12 hours, and introduce nitrogen protection during the reaction; b. Mix and stir the chemotherapeutic drug with Fe3O4 at a stirring speed of 300 - 500 revolutions per minute for 2 - 3 hours to form a drug-loaded core; c. Ultrasonically fuse the platelet membrane layer vesicles with the drug-loaded core at an ultrasonic power of 250 - 350 watts and an ultrasonic time of 15 - 20 minutes to form a membrane-coated structure; d. Use the SMCC cross-linking agent to conjugate galactose (Gal) and anti-PD-L1 antibody to the surface of the platelet membrane layer. The conjugation reaction is carried out in the dark, and the pH value of the reaction system is controlled at 7.2 - 7.

6.

7. A magnetic nanoparticle targeting vector for liver cancer treatment according to claim 6, wherein, The conjugation reaction conditions in step d are: react in the dark at room temperature for 2 - 3 hours. When centrifuging and purifying, use ultracentrifugation at a centrifugal speed of 10000 - 12000 revolutions per minute for 20 - 30 minutes, and store after purification in a phosphate buffer solution with a pH of 7.4 at a storage temperature of 4°C.

8. A method for using a magnetic nanoparticle targeting carrier for liver cancer, applied to a magnetic nanoparticle targeting carrier for liver cancer as described in any one of claims 1-7, characterized in that, Including the following steps: The magnetic nanoparticle targeting carrier for liver cancer was introduced into the liver cancer matrix by tail vein injection at a rate of 0.1 - 0.16 ml / min; An external magnetic field of 0.6 - 1.0 T was applied at the tumor site for 30 - 40 minutes continuously. The magnetic field application method was to generate a uniform magnetic field using an electromagnet; The drug was administered once every 3 days at a dose of 0.2 - 1.0 mg siRNA / kg or 5 - 20 mg chemotherapeutic drug / kg.

9. The method for using a magnetic nanoparticle targeting carrier for liver cancer according to claim 8, wherein, The carrier releases chemotherapeutic drugs through pH-sensitive release in the tumor microenvironment. When the environmental pH value is lower than 6.5, the structure of the carrier changes to trigger drug release, and the anti-PD-L1 antibody binds to PD-L1 on the surface of tumor cells to activate the T cell immune response. The number of activated T cells increases by more than 50% within 72 hours after drug administration.

Images