A magnetic nanoparticle targeting carrier for liver cancer and a method of using the same

Abstract

The present application relates to the technical field of targeting carrier, in particular to a kind of magnetic nanoparticle targeting carrier for liver cancer and its use method, comprising: the superparamagnetic magnetite magnetic inner core with, the inner core is surface modified, and modification group is carboxyl;Platelet membrane layer that is tightly wrapped the inner core, and the platelet membrane layer is derived from the platelet of healthy individual;Gal and anti-PD-L1 antibody modified on the surface of the platelet membrane layer;Chemotherapeutic drug is loaded in the inner core, and the platelet membrane layer is combined with inner core by ultrasound fusion;The magnetic nanoparticle targeting carrier of the present application has significant therapeutic effect in the treatment of liver cancer, and the superparamagnetic magnetite magnetic inner core in the technical scheme of the present application not only provides magnetism for carrier, so that it can be guided to move directionally under external magnetic field, and accurately gather in tumor site, improve the concentration of drug in tumor tissue.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of targeted carrier, in particular to a magnetic nanoparticle targeted carrier for liver cancer and a use method thereof. BACKGROUND

[0002] Liver cancer is a common and highly malignant tumor disease worldwide, which seriously threatens human health. Its onset is occult, and most patients are in the middle and advanced stages when diagnosed, with poor cure effect. Liver cancer cells have strong invasion and metastasis ability, easily invading surrounding tissues and distant organs, leading to rapid deterioration of the disease. Among the many causes of cancer-related deaths, liver cancer accounts for a high proportion, causing heavy burden to patient families and society.

[0003] Currently, chemotherapy is one of the common means of liver cancer treatment. Chemotherapeutic drugs exert their effects by inhibiting the division and growth of cancer cells, but in the treatment process, chemotherapeutic drugs lack specific recognition ability for cancer cells, and while attacking cancer cells, they also cause damage to normal cells. This causes patients to experience a series of serious side effects, such as nausea, vomiting, hair loss, bone marrow suppression, etc. Bone marrow suppression can lead to decreased immunity in patients, increasing the risk of infection; long-term chemotherapy can also cause toxic reactions to important organs such as heart, liver, and kidney, further damaging the physical health of patients, reducing the quality of life, and even affecting the smooth progress of subsequent treatment.

[0004] In order to reduce the side effects of traditional treatment methods such as chemotherapy on liver cancer patients and improve treatment effect, more safe and effective treatment plans are being explored. Against this background, the present application proposes a magnetic nanoparticle targeted carrier for liver cancer treatment and a use method thereof.

[0005] The technical solution aims to achieve precise delivery of chemotherapeutic drugs, allowing the drugs to specifically act on liver cancer cells and reduce damage to normal tissues, thereby improving treatment effect while reducing side effects. SUMMARY

[0006] In view of the problems in the prior art, the present application provides a magnetic nanoparticle targeted carrier for liver cancer and a use method thereof.

[0007] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0008] A magnetic nanoparticle targeted carrier for liver cancer treatment, comprising:

[0009] A ferroferric oxide magnetic inner core with superparamagnetic property, the inner core is surface-modified, and the modification group is carboxyl to enhance the binding ability of the inner core with other components;

[0010] a platelet membrane layer tightly wrapping the inner core, the platelet membrane layer being derived from platelets of a healthy individual and being purified multiple times to remove impurities during preparation;

[0011] galactose (Gal) and anti-PD-L1 antibodies modified on the surface of the platelet membrane layer;

[0012] chemotherapeutic drugs loaded in the inner core, the platelet membrane layer being combined with the inner core by ultrasonic fusion, and a surfactant being added during the fusion process to promote the fusion effect.

[0013] As a further technical solution, the chemotherapeutic drugs are selected from at least one of doxorubicin, cisplatin or paclitaxel, and the mass ratio of the chemotherapeutic drugs to ferroferric oxide is 1:6-10. When the chemotherapeutic drugs are doxorubicin, the dispersion state of doxorubicin in the carrier is uniform dispersion, and there is a weak interaction between doxorubicin and ferroferric oxide, such as hydrogen bond or van der Waals force.

[0014] As a further technical solution, the galactose (Gal) and anti-PD-L1 antibodies are covalently linked to the surface of the platelet membrane layer by a cross-linking agent SMCC, and the molar ratio of galactose (Gal) to anti-PD-L1 antibodies is 3-5:1.

[0015] During the linking process, the reaction temperature is controlled at 25℃±2℃, and the reaction time is 4-5 hours to ensure the stability and effectiveness of the linking.

[0016] As a further technical solution, the platelet membrane layer is prepared by the following steps:

[0017] Whole blood is collected and platelet-rich plasma is separated by density gradient centrifugation, with a centrifugal speed of 1500-2000 rpm and a centrifugal time of 10-15 minutes.

[0018] The separated platelets are subjected to multiple freeze-thaw lysis, with each freeze-thaw condition being: freezing temperature -80℃, freezing time 2-3 hours, thawing temperature 37℃, thawing time 18-20 minutes, followed by ultrasonic treatment to obtain membrane vesicles, with an ultrasonic power of 200-300 watts and an ultrasonic time of 10-12 minutes.

[0019] The membrane vesicles and drug-loaded ferroferric oxide magnetic inner core are ultrasonically fused under an ultrasonic frequency of 40-50 kHz.

[0020] As a further technical solution, the particle size is 80-150 nm, which is 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%, to ensure its stability.

[0021] As a further technical solution, the preparation method of the magnetic nanoparticle targeting carrier comprises the following steps:

[0022] a. Synthesizing carboxylated Fe3O4 nanoparticles by a solvothermal method, the reaction temperature is 180-200 DEG C, the reaction time is 8-12 hours, and nitrogen is introduced during the reaction process;

[0023] b. Mixing and stirring the chemotherapeutic drug with Fe3O4, the stirring speed is 300-500 revolutions / minute, the stirring time is 2-3 hours, and a drug-loaded inner core is formed;

[0024] c. Ultrasonic fusion of platelet membrane layer vesicles and drug-loaded inner core, the ultrasonic power is 250-350 watts, and the ultrasonic time is 15-20 minutes, forming a membrane-coated structure;

[0025] d. Gal and anti-PD-L1 antibody are coupled to the membrane surface using SMCC crosslinking agent, the coupling reaction is carried out in the dark, and the pH value of the reaction system is controlled at 7.2-7.6.

[0026] As a further technical solution, the coupling reaction conditions in step d are: room temperature reaction in the dark for 2-3 hours, ultracentrifugation is used for centrifugal purification, the centrifugal speed is 10000-12000 revolutions / minute, the centrifugal time is 20-30 minutes, and after purification, it is stored in a phosphate buffer solution with pH 7.4, and the storage temperature is 4 DEG C.

[0027] The use method of the magnetic nanoparticle targeting carrier comprises the following steps:

[0028] The carrier is introduced into the liver cancer matrix by tail vein injection, and the injection speed is 0.1-0.16 milliliters / minute;

[0029] A 0.6-1.0T external magnetic field is applied at the tumor site, and the magnetic field is applied for 30-40 minutes, and the magnetic field is applied by using an electromagnet to generate a uniform magnetic field;

[0030] The drug is administered once every 3 days, and the dose is 0.2-1.0 mg siRNA / kg or 5-20 mg chemotherapeutic drug / kg.

[0031] As a further technical solution, the carrier releases the chemotherapeutic drug in the tumor microenvironment through pH sensitivity, 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 the tumor cell surface PD-L1 to activate T cell immune response, and the number of activated T cells increases by more than 50% within 72 hours after administration.

[0032] Compared with the prior art, the beneficial effects of the present application are:

[0033] The magnetic nanoparticle targeting carrier of the present application has a remarkable therapeutic effect in the treatment of liver cancer. In the technical scheme of the present application, the superparamagnetic ferroferric oxide magnetic core not only provides the carrier with magnetism, enabling it to move directionally under the guidance of an external magnetic field and precisely gather at the tumor site, thereby increasing the concentration of the drug in the tumor tissue, but also provides a basis for loading the chemotherapeutic drug. The carboxyl groups modified on the surface of the core increase the hydrophilicity and chemical reactivity of the core, which is conducive to the subsequent connection and reaction with other components.

[0034] The platelet membrane layer is derived from the platelets of healthy individuals. The platelet membrane layer has good biocompatibility and immune evasion properties, which can reduce the probability of the carrier being recognized and removed 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 wrapping of the platelet membrane layer can protect the core and the chemotherapeutic drug, reducing their premature release and degradation during blood circulation.

[0035] The 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, enabling the carrier to actively target liver cancer cells and further increasing the enrichment of the drug in the tumor tissue. 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 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.

[0036] The specific mass ratio of the chemotherapeutic drug to ferroferric oxide ensures the effective loading amount of the drug, while avoiding the influence of excessive or insufficient drug on the treatment effect and safety. In the tumor microenvironment, the carrier releases the chemotherapeutic drug through a pH-sensitive mechanism. When the environmental pH value is lower than 6.5, the structure of the carrier changes, triggering the release of the drug, achieving precise release of the chemotherapeutic drug at the tumor site, improving the killing effect of the drug on cancer cells, and reducing the toxic side effects on normal tissues.

[0037] The carrier is introduced into the liver cancer base by tail vein injection, and an external magnetic field is applied at the tumor site. The characteristics of the magnetic core further enhance the enrichment effect of the carrier at the tumor site. Reasonable drug administration frequency and dosage can ensure that the drug maintains an effective therapeutic concentration in the body, while avoiding the toxic side effects caused by excessive drug. The technical scheme of the present application can achieve the synergistic effect of chemotherapy and immunotherapy, improve the therapeutic effect of liver cancer, and effectively reduce the side effects of traditional chemotherapy, thereby providing an innovative and efficient solution for the treatment of liver cancer. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is a statistical diagram of tumor inhibition rate of each group in the test. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0040] The present application provides a magnetic nanoparticle targeting carrier for liver cancer treatment and a preparation and use method thereof, specifically as follows:

[0041] The magnetic nanoparticle targeting carrier has a superparamagnetic ferroferric oxide magnetic core, the surface of the core is modified with carboxyl, a platelet membrane layer tightly wrapping the core is derived from the platelets of healthy individuals, galactose (Gal) and anti-PD-L1 antibody are modified on the surface of the platelet membrane layer, a chemotherapeutic drug is loaded in the core, and the platelet membrane layer is combined with the core by ultrasonic fusion.

[0042] The chemotherapeutic drug is at least one selected from adriamycin, cisplatin or paclitaxel, and the mass ratio of the chemotherapeutic drug to the ferroferric oxide is 1:6-10.

[0043] Galactose (Gal) and anti-PD-L1 antibody connection: 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℃±2℃, and the reaction time is 4-5 hours.

[0044] Preparation of platelet membrane layer: whole blood is collected and platelet-rich plasma is separated by density gradient centrifugation, the centrifugal speed is 1500-2000 rpm / min, and the centrifugal time is 10-15 minutes; the separated platelets are subjected to multiple freeze-thaw lysis, each freeze-thaw condition is: freezing temperature-80℃, freezing time 2-3 hours, thawing temperature 37℃, thawing time 18-20 minutes, then ultrasonic treatment is performed to obtain membrane vesicles, the ultrasonic power is 200-300 watts, and the ultrasonic time is 10-12 minutes; the membrane vesicles and the drug-loaded ferroferric oxide magnetic core are ultrasonically fused under the condition that the ultrasonic frequency is 40-50 kHz.

[0045] Carrier properties: the particle size is 80-150 nm, which is 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%.

[0046] Preparation method

[0047] The carboxylated Fe3O4 nanoparticles are synthesized by a solvothermal method, the reaction temperature is 180-200 DEG C, the reaction time is 8-12 hours, and nitrogen is introduced during the reaction.

[0048] The chemotherapeutic drug is mixed with Fe3O4 and stirred, the stirring speed is 300-500 rpm, and the stirring time is 2-3 hours, so as to form a drug-loaded core.

[0049] The platelet membrane layer vesicle is ultrasonically fused with the drug-loaded core, the ultrasonic power is 250-350 W, and the ultrasonic time is 15-20 minutes, so as to form a membrane-coated structure.

[0050] Gal and anti-PD-L1 antibody are coupled to the membrane surface by using SMCC crosslinking agent, the coupling reaction is carried out in the dark, the pH value of the reaction system is controlled at 7.2-7.6, the amount of SMCC crosslinking agent used is 1.5-2 micromoles per milligram of platelet membrane protein, the coupling reaction condition is: room temperature, dark reaction for 2-3 hours, the ultracentrifugation method is used in centrifugal purification, the centrifugal speed is 10,000-12,000 rpm, the centrifugal time is 20-30 minutes, and after purification, the product is stored in a phosphate buffer solution with pH 7.4, and the storage temperature is 4 DEG C.

[0051] Use method

[0052] The carrier is introduced into the liver cancer matrix by tail vein injection, and the injection speed is 0.1-0.16 ml / min.

[0053] An external magnetic field of 0.6-1.0 T is applied to the tumor site, and the magnetic field acts for 30-40 minutes, and the magnetic field is generated by an electromagnet.

[0054] The drug is administered once every 3 days, and the dose is 0.2-1.0 mg siRNA / kg or 5-20 mg chemotherapeutic drug / kg; the carrier releases the chemotherapeutic drug in the tumor microenvironment through pH sensitivity, 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 the PD-L1 on the surface of the 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 administration.

[0055] In order to further illustrate the present application, the following examples are used to illustrate the present application in detail. The reagents and materials used in the following examples of the present application are commercially available, unless otherwise specified.

[0056] Example 1: Preparation of magnetic nanoparticle targeted carrier

[0057] The carboxylated Fe3O4 nanoparticles are synthesized by a solvothermal method: sodium acetate trihydrate and sodium citrate are dissolved in ethylene glycol, and The carboxylated Fe3O4 nanoparticles were prepared by mixing the Fe3O4 nanoparticles and the ethylene glycol solution, stirring for 30 minutes, and then reacting at 180°C under nitrogen protection for 12 hours. After cooling to room temperature, the product was washed with ethanol and dried at 60°C to obtain the carboxylated Fe3O4 nanoparticles.

[0058] Loading of the chemotherapeutic drug: doxorubicin was selected and mixed with Fe3O4 at a mass ratio of 1:6. The mixture was stirred at a speed of 300 rpm for 3 hours to form a drug-loaded core.

[0059] Preparation of platelet membrane layer vesicles: whole blood was collected from healthy individuals, and platelet-rich plasma was separated by density gradient centrifugation at a speed of 1500 rpm for 15 minutes. The platelets were subjected to freeze-thaw lysis, frozen at -80°C for 2.5 hours, thawed at 37°C for 18 minutes, and repeated 3 times. The membrane vesicles were obtained by ultrasonic treatment at an ultrasonic power of 200 watts for 12 minutes.

[0060] Ultrasonic fusion: the membrane vesicles and the drug-loaded core were ultrasonically fused at an ultrasonic frequency of 40 kHz and an ultrasonic power of 250 watts for 15 minutes to form a membrane-coated structure.

[0061] Coupling modification: SMCC crosslinking agent was used to couple galactose (Gal) and anti-PD-L1 antibody to the membrane surface at a molar ratio of 3:1 at 25°C, in the dark, and at pH 7.2 for 4 hours. The amount of SMCC crosslinking agent used was 1.5 micromoles per milligram of platelet membrane protein. After the reaction, the product was purified by ultracentrifugation at a speed of 10000 rpm for 30 minutes, and then stored in a phosphate buffer at pH 7.4 at 4°C.

[0062] Use of 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 0.6T was applied to the tumor site using an electromagnet for 30 minutes. The drug was administered every 3 days at a dose of 0.2 mg siRNA / kg and 5 mg chemotherapeutic drug / kg.

[0063] Example 2: Preparation of magnetic nanoparticle targeting carrier

[0064] Synthesis of carboxylated Fe3O4 nanoparticles: the reaction temperature was set to 190°C, the reaction time was 10 hours, and the other conditions were the same as in Example 1.

[0065] Loading of the chemotherapeutic drug: cisplatin was selected, and the mass ratio of cisplatin to Fe3O4 was 1:8. The stirring speed was 400 rpm, and the stirring time was 2.5 hours.

[0066] Preparation of platelet membrane vesicles: centrifugal speed 1800 rpm, centrifugal time 12 min; freezing time 2 h, thawing time 20 min; ultrasonic power 250 W, ultrasonic time 11 min.

[0067] Ultrasonic fusion: ultrasonic frequency 45 kHz, ultrasonic power 300 W, ultrasonic time 18 min.

[0068] Coupling modification: molar ratio of galactose (Gal) to anti-PD-L1 antibody 4:1, reaction temperature 25℃, reaction time 4.5 h, pH 7.4; ultracentrifugal speed 11000 rpm, centrifugal time 25 min; SMCC crosslinking agent was used in an amount of 1.8 micromole per milligram of platelet membrane protein.

[0069] Use of magnetic nanoparticle targeting carrier: another batch of mouse models with liver cancer was selected, and the tail vein injection speed was 0.13 ml / min; the additional magnetic field strength was 0.8 T, and the action time was 35 min; the drug was administered once every 3 days, and the dose was 0.6 mg siRNA / kg and 12 mg chemotherapeutic drug / kg.

[0070] Example 3: Preparation of magnetic nanoparticle targeting carrier

[0071] Synthesis of carboxylated Fe3O4 nanoparticles: reaction temperature 200℃, reaction time 8 h.

[0072] Loading of chemotherapeutic drugs: paclitaxel to Fe3O4 mass ratio 1:10, stirring speed 500 rpm, stirring time 2 h.

[0073] Preparation of platelet membrane vesicles: centrifugal speed 2000 rpm, centrifugal time 10 min; freezing time 3 h, thawing time 19 min; ultrasonic power 300 W, ultrasonic time 10 min.

[0074] Ultrasonic fusion: ultrasonic frequency 50 kHz, ultrasonic power 350 W, ultrasonic time 20 min.

[0075] Coupling modification: molar ratio of galactose (Gal) to anti-PD-L1 antibody 5:1, reaction temperature 23℃, reaction time 5 h, pH 7.6; ultracentrifugal speed 12000 rpm, centrifugal time 20 min; SMCC crosslinking agent was used in an amount of 1.6 micromole per milligram of platelet membrane protein.

[0076] Use of magnetic nanoparticle targeting carrier: a new mouse model of liver cancer was selected, and the tail vein injection speed was 0.16 ml / min; the additional magnetic field strength was 1.0 T, and the action time was 40 min; the drug was administered once every 3 days, and the dose was 1.0 mg siRNA / kg and 20 mg chemotherapeutic drug / kg.

[0077] Example 4: Preparation of magnetic nanoparticle targeting carrier

[0078] Synthesis of carboxylated Fe3O4 nanoparticles: reaction conditions were the same as in Example 2.

[0079] Loading of chemotherapeutic drugs: doxorubicin and cisplatin were used simultaneously, with a total mass ratio of 1:7, and a mass ratio of doxorubicin to cisplatin of 1:1, a stirring speed of 350 rpm, and a stirring time of 2.8 hours.

[0080] Preparation of platelet membrane layer vesicles: conditions were the same as in Example 3.

[0081] Ultrasonic fusion: conditions were the same as in Example 1.

[0082] Coupling modification: the molar ratio of galactose (Gal) to anti-PD-L1 antibody was 3.5:1, the reaction temperature was 24°C, the reaction time was 4.2 hours, the pH was 7.3, the ultracentrifugation speed was 10500 rpm, the centrifugation time was 28 minutes, and the amount of SMCC crosslinking agent used was 2 micromoles per milligram of platelet membrane protein.

[0083] Use of the magnetic nanoparticle targeting carrier: a new mouse model of liver cancer was selected, and the tail vein injection speed was 0.12 ml / min; the external magnetic field strength was 0.7 T, and the action time was 32 minutes; the drug was administered once every 3 days, with a dose of 0.4 mg siRNA / kg and 8 mg chemotherapeutic drug / kg.

[0084] Comparative Example 1

[0085] Preparation of the comparative carrier

[0086] The preparation process was the same as in Example 1, but no chemotherapeutic drugs were loaded, and only a carrier containing an Fe3O4 core, a platelet membrane layer, and surface modification with galactose (Gal) and anti-PD-L1 antibody was prepared.

[0087] Use of the comparative carrier: the same type of mouse model of liver cancer as in Example 1 was selected, and the tail vein injection speed, external magnetic field conditions, and drug administration frequency were the same as in Example 1, but no chemotherapeutic drug dose was given, and only 0.2 mg siRNA / kg was given.

[0088] Comparative Example 2

[0089] Preparation of the comparative carrier

[0090] The preparation process was the same as in Example 1, but no anti-PD-L1 antibody was modified, and only galactose was modified.

[0091] Use of the comparative carrier: a mouse model of liver cancer was selected, and the tail vein injection speed, external magnetic field conditions, and drug administration frequency and dose were the same as in Example 1.

[0092] Preparation before the test

[0093] I. Experimental animals

[0094] Selection of experimental animals: 6-8 week old, 20-25 g C57BL / 6 female mice were selected.

[0095] Raising environment: The mice were raised in a SPF level animal room with a temperature of 22±2℃, a relative humidity of 50±5%, 12 hours of light / 12 hours of darkness, free access to food and water, and adaptive feeding for 1 week before the experiment.

[0096] Mouse screening criteria

[0097] The body weight was within the standard body weight ±10%.

[0098] The appearance was healthy, without obvious skin damage, hair loss, abnormal breathing, etc.

[0099] The behavior was normal, without abnormal performance such as slow movement, huddling, and convulsions.

[0100] After pathogen detection, it was confirmed that there was no specific pathogen infection.

[0101] II. Liver cancer tumor modeling method

[0102] Cell strain: Hepa1-6 liver cancer cell strain was selected.

[0103] Cell culture: Hepa1-6 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and placed in a 37℃, incubator for culture, with subculture every 2-3 days, and logarithmic growth phase cells were used for modeling.

[0104] Modeling method

[0105] Logarithmic growth phase Hepa1-6 cells were washed with PBS for 2 times, and the cell concentration was adjusted to cells / mL.

[0106] After the mice were anesthetized with 1% sodium pentobarbital (50 mg / kg) intraperitoneally, they were fixed on the operating table, and the skin was routinely disinfected.

[0107] A 0.5 cm long incision was made about 0.5 cm below the right axillary line of the mouse, and the subcutaneous tissue and muscle were bluntly separated to expose the liver.

[0108] 100 μL of cell suspension (containing Hepa1-6 cells) was slowly injected into the left lobe of the liver with a 1 mL sterile syringe, and the injection depth was about 3 mm.

[0109] After the injection was completed, the liver was reset, and the muscle and skin were sutured layer by layer.

[0110] Postoperative mice were placed in an incubator until they woke up, and were routinely fed, with their mental state, diet, and activity observed daily.

[0111] Modeling success criteria: 7-10 days after inoculation, a lump can be felt in the upper right abdomen of the mouse by palpation; 14 days after inoculation, an ultrasound imaging system is used for detection, and it is confirmed that there is a space-occupying lesion in the liver, with a tumor volume of about 50-100 mm³, which is considered to be a successful modeling.

[0112] III. Experimental grouping

[0113] The successfully modeled mice were randomly divided into 6 groups, with 10 mice in each group:

[0114] Example 1 group: received magnetic nanoparticle targeted carrier prepared in Example 1 for treatment.

[0115] Example 2 group: received magnetic nanoparticle targeted carrier prepared in Example 2 for treatment.

[0116] Example 3 group: received magnetic nanoparticle targeted carrier prepared in Example 3 for treatment.

[0117] Example 4 group: received magnetic nanoparticle targeted carrier prepared in Example 4 for treatment.

[0118] Comparative Example 1 group: received comparative carrier prepared in Comparative Example 1 for treatment.

[0119] Comparative Example 2 group: received comparative carrier prepared in Comparative Example 2 for treatment.

[0120] IV. Preparation before administration

[0121] Carrier solution preparation: the prepared magnetic nanoparticle targeted carrier or comparative carrier was diluted with sterile PBS to the required concentration, filtered through a 0.22 μm filter to remove bacteria, and stored for use.

[0122] Preparation of administration equipment: a 1 mL sterile syringe and a 26G sterile needle were used, and each administration was subjected to high-pressure sterilization treatment before administration.

[0123] Magnetic field application equipment: a custom-made electromagnet device was used to generate a uniform magnetic field of 0.6-1.0 T, and the magnetic field strength and action time could be accurately controlled.

[0124] V. Administration scheme

[0125] Administration route: tail vein injection.

[0126] Injection speed: injection was performed at the speed set in each example and comparative example (0.1-0.16 mL / min).

[0127] Frequency of administration: once every 3 days, for a total of 5 administrations.

[0128] Dose of administration: administration was performed according to the doses set in the examples and comparative examples (0.2-1.0 mg siRNA / kg or 5-20 mg chemotherapeutic drug / kg).

[0129] Test 1: Tumor inhibition effect test

[0130] Test method: At different time periods (7th day, 14th day, 21st day) after the treatment of the examples and comparative examples, the growth of tumors in mice was observed using a live imaging system, and the change in tumor volume was measured; the mice were dissected, and the tumor weight was measured, and the tumor inhibition rate was calculated, tumor inhibition rate = (tumor weight of the control group - tumor weight of the experimental group) / tumor weight of the control group x 100%; test results:

[0131] Table 1

[0132] Tumor volume (mm3) on day 7 Tumor volume (mm3) on day 14 Tumor volume (mm3) on day 21 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

[0133] Tumor inhibition 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 examples 1 and 2. Comparative example 1 had the worst tumor inhibition effect because it was not loaded with chemotherapeutic drugs; although comparative example 2 was loaded with chemotherapeutic drugs, it lacked the modification of anti-PD-L1 antibodies, and its tumor inhibition effect was not as good as that of the example groups. This shows that the magnetic nanoparticle targeting carrier of the present application can more effectively inhibit tumor growth when loaded with chemotherapeutic drugs and modified with anti-PD-L1 antibodies at the same time. The tumor inhibition effect of example 3 was the best, which may be due to the fact that the preparation process parameters are more conducive to the loading and release of drugs, and the function of the targeting molecules.

[0134] Test 2: Immune activation effect test

[0135] Test method: At 72 hours after administration, the tumor tissue and peripheral blood of the mice were collected, and the number of activated T cells in the tumor tissue and peripheral blood was detected by flow cytometry; the expression level of related immune factors (such as IFN-γ, TNF-α, etc.) in the tumor tissue was detected by ELISA method; test results:

[0136] Table 2

[0137]

[0138] Immune activation effect: As can be seen from Table 2, the number of activated T cells and the expression level of immune factors in the groups of Examples 1-4 are significantly higher than those in the groups of Comparative Examples 1 and 2. Comparative Example 1 is not loaded with a chemotherapeutic drug and cannot effectively activate the immune response; Comparative Example 2 lacks anti-PD-L1 antibody modification and has weak T cell activation and immune factor secretion capacity. The immune activation effect of Example 3 is 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, enabling it to more effectively block the PD-L1 / PD-1 signaling pathway and activate T cell immune response.

[0139] The preferred embodiments of the application disclosed above are only used to help explain the application. The preferred embodiments do not describe all the details and limit the application to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of the specification.

Claims

1. A magnetic nanoparticle targeting carrier for liver cancer treatment, characterized in that, Comprise: A ferroferric oxide magnetic core with superparamagnetism, which is surface-modified with a carboxyl group; A platelet membrane layer tightly wrapped around the core, which is derived from the platelets of healthy individuals; Galactose (Gal) and anti-PD-L1 antibodies modified on the surface of the platelet membrane layer; A chemotherapeutic drug loaded in the core, and the platelet membrane layer is combined with the core by ultrasonic fusion; The platelet membrane layer is prepared by the following steps: Collect whole blood and centrifuge the platelet-rich plasma by density gradient centrifugation, with a centrifugal speed of 1500-2000 rpm and a centrifugal time of 10-15 minutes; Freeze-thaw lysis of the isolated platelets multiple times, with each freeze-thaw condition being: freezing temperature -80℃, freezing time 2-3 hours, thawing temperature 37℃, thawing time 18-20 minutes, followed by ultrasonic treatment to obtain membrane vesicles, with an ultrasonic power of 200-300 watts and an ultrasonic time of 10-12 minutes; Ultrasonic fusion of the membrane vesicles and the ferroferric oxide magnetic core loaded with a chemotherapeutic drug under an ultrasonic frequency of 40-50 kHz; The preparation method of the magnetic nanoparticle targeting carrier comprises the following steps: a. Synthesis of carboxylated Fe3O4 nanoparticles by solvothermal method, with a reaction temperature of 180-200℃ and a reaction time of 8-12 hours, and nitrogen protection during the reaction; b. Mix and stir the chemotherapeutic drug with Fe3O4, with a stirring speed of 300-500 rpm and a stirring time of 2-3 hours to form a drug-loaded core; c. Ultrasonic fusion of the platelet membrane layer vesicles and the drug-loaded core, with an ultrasonic power of 250-350 watts and an ultrasonic time of 15-20 minutes to form a membrane-coated structure; d. Coupling of galactose (Gal) and anti-PD-L1 antibodies to the surface of the platelet membrane layer using SMCC crosslinker, with the coupling reaction being carried out in the dark, and the pH value of the reaction system being controlled at 7.2-7.

6. 2.The magnetic nanoparticle targeting carrier for treating liver cancer according to claim 1, characterized in that, The chemotherapeutic drug is selected from at least one of doxorubicin, cisplatin or paclitaxel, and the mass ratio of the chemotherapeutic drug to ferroferric oxide is 1:6-10. 3.The magnetic nanoparticle targeting carrier for treating liver cancer according to claim 1, characterized in that, The galactose (Gal) and anti-PD-L1 antibodies are covalently linked to the surface of the platelet membrane layer through the crosslinking agent SMCC, and the molar ratio of galactose (Gal) to anti-PD-L1 antibodies is 3-5:1; During the linking process, the reaction temperature is controlled at 25℃±2℃, and the reaction time is 4-5 hours.

4. The 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, which is determined by dynamic light scattering method, a surface Zeta potential of +20 to +35 mV, and a particle size change rate of less than 10% and a Zeta potential change rate of less than 5% after being placed in physiological saline for 72 hours.

5. The magnetic nanoparticle targeting carrier for liver cancer treatment according to claim 1, wherein, The coupling reaction conditions in step d are: room temperature reaction in the dark for 2-3 hours, ultracentrifugation is used for centrifugal purification, with a centrifugal speed of 10000-12000 rpm and a centrifugal time of 20-30 minutes, and the purified product is stored in a phosphate buffer solution at pH 7.4, with a storage temperature of 4℃.

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