Macrophage membrane liposome-packaged metformin hydrochloride drug delivery system, its preparation method and use in tumor treatment

Abstract

The present application relates to the technical field of medicine preparation, and discloses a macrophage membrane liposome packaged metformin drug delivery system, a preparation method thereof and application in tumor treatment, which comprises a ZIF-8 carrier carrying a medical active substance of metformin hydrochloride, wherein the metformin hydrochloride is encapsulated in ZIF-8 nanoparticles, and the outer layer is packaged with tumor-related macrophage membrane liposomes; in application examples, it is found through in-vitro experiments that the killing effect of the system on human osteosarcoma cell lines is strong, the system can more effectively kill tumor cells and induce immunogenic death; compared with free metformin hydrochloride monomer drugs, the aforementioned delivery system has a more obvious inhibitory effect on tumor cells and can more effectively induce immunogenic death.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug preparation, and in particular relates to a macrophage membrane liposome-packaged metformin hydrochloride drug delivery system, a preparation method thereof, and an application thereof in tumor treatment. Background Art

[0002] Osteosarcoma (OS) is one of the most common primary musculoskeletal malignancies, often seen in adolescents and characterized by high malignancy. Patients face repeated tumor invasion due to recurrence and lung metastasis. Osteosarcoma is highly drug-resistant and immunosuppressive, resulting in only a minority of patients responding to immunotherapy. Therefore, finding strategies to mitigate resistance to immunotherapy is urgently needed in clinical practice.

[0003] Metformin has the ability to inhibit tumor cells by generating ROS and inducing endoplasmic reticulum stress, but its intracellular accumulation capacity is low and its hydrophilicity is too high. Encapsulating metformin in a drug delivery system allows for precise delivery and controlled release of the active ingredient, promoting precise release within lesions and inducing immunogenic death via the type II pathway, thereby improving resistance to immunotherapy. The high specific surface area, rich pore structure, and pH responsiveness of the degradable polymer cage, ZIF-8, a zeolite imidazolate framework, enable controlled drug encapsulation and delivery. Furthermore, macrophage membrane liposomes are packaged on the surface of ZIF-8, giving it excellent tumor homing ability and reticuloendothelial system (RES) escape properties that enhance the drug's resistance to degradation. Summary of the Invention

[0004] The purpose of the present invention is to provide a macrophage membrane liposome-encapsulated metformin hydrochloride drug delivery system, a preparation method thereof, and use in tumor treatment. The nanoparticles prepared according to the preparation method of the present invention can prevent metformin hydrochloride from dissolving in water prematurely, increase its intracellular accumulation ability, enable it to better target tumor cells, promote endoplasmic reticulum stress-induced immunogenic death of osteosarcoma cells, and increase the efficacy of the drug.

[0005] To achieve the above-mentioned purpose, the macrophage membrane liposome-encapsulated metformin hydrochloride precise drug delivery system of the present invention is characterized in that the drug delivery system is composed of drug-loaded nanoparticles made of ZIF-8 loaded with metformin hydrochloride and biomimetic nanoparticles formed by macrophage membrane liposomes encapsulated on the outer surface of the drug-loaded nanoparticles.

[0006] The drug-loaded nanoparticles are also loaded with rhodamine 6G.

[0007] The preparation method of the macrophage membrane liposome-encapsulated metformin hydrochloride precise drug delivery system of the present invention comprises the following steps:

[0008] Step 1, preparation of ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles: adding metformin hydrochloride to a 2.5 mol / L methylimidazole solution to prepare a metformin hydrochloride-methylimidazole solution having a metformin hydrochloride concentration of 5-10 mg / mL, then mixing a 0.1-0.5 mol / L zinc nitrate solution and the metformin hydrochloride-methylimidazole solution at a volume ratio of 1-1.5:1, stirring at room temperature to allow for full reaction, collecting the product by centrifugation, rinsing with deionized water, and then drying under vacuum at room temperature to obtain ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles;

[0009] Step 2, preparation of macrophage ghost liposomes: Incubate the cells in a 37°C, 95% humidity, 5% CO2 incubator with a volume of 5 × 10 macrophages per well. 4 3×10 osteosarcoma cells in the lower chamber 5 The co-cultured macrophages were digested from the Transwell plate, frozen and revived, and then resuspended in 0.25× PBS buffer. After washing and purification, the macrophage ghost liposomes were collected by low-temperature centrifugation.

[0010] Step 3, packaging drug-loaded nanoparticles in macrophage membrane liposomes: adding ZIF-8-loaded metformin hydrochloride-loaded nanoparticles to double-distilled water to form a 4 mg / ml nanoparticle dispersion, adding macrophage ghost liposomes to double-distilled water to form a 10-20 mg / ml macrophage ghost liposome suspension, mixing the nanoparticle dispersion and the macrophage ghost liposome suspension at a volume ratio of 1 to 1.5:1 at 4°C or in an ice-water mixing bath, and then ultrasonically treating the mixture. The mixture is extruded through a polycarbonate porous membrane using an Avanti mini extruder at 4°C or in an ice-water mixing bath to obtain nanoparticles packaged in macrophage ghost liposomes.

[0011] The methylimidazole solution in step 1 is further added with rhodamine 6G, and the concentration of rhodamine 6G is 5-10 mg / mL.

[0012] The centrifugation in step 1 is performed at a rotation speed of 8000-12000 rpm for 10-15 minutes.

[0013] The 0.25×PBS buffer in step 2 also contains PMSF with a concentration of 0.1-1 mmol / L.

[0014] The low-temperature centrifugation in step 2 is to collect macrophage ghost liposomes by centrifugation at 5000-8000 rpm at 4°C.

[0015] In step 3, the ultrasonic treatment is performed for 5 to 10 minutes.

[0016] The pore size of the polycarbonate porous membrane in step 3 is 200-300 nm.

[0017] The application of the macrophage membrane liposome-encapsulated metformin hydrochloride precision drug delivery system obtained by the above preparation method in the treatment of osteosarcoma cells.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention obtains ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles through the full reaction of a zinc nitrate solution and a methylimidazole solution containing metformin hydrochloride. The ZIF-8 has a high specific surface area of ​​a degradable polymer cage and a rich pore structure, and can adsorb metformin hydrochloride molecules in the pores on the surface to achieve a drug-loading function. The ZIF-8 nanoparticles also have a pH-responsive release capability, which compensates for the excessive water solubility of metformin hydrochloride and can prevent metformin hydrochloride from dissolving in water too early. The ZIF-8 nanoparticles can significantly increase their intracellular accumulation capability, can better target tumor cells, and reduce the side effects of metformin hydrochloride, reduce the drug load on the digestive tract, liver, kidneys, etc., and increase the drug efficacy. Then, macrophages are activated by co-culturing with a tumor to be treated to express immune markers, tumor-associated macrophages are obtained, and cell membranes of the macrophages are extracted to obtain cell membrane liposomes containing specific immune markers. The ZIF-8 nanoparticles are packaged with the macrophage membrane liposomes to enable the macrophages to have tumor targeting and the ability to avoid degradation by the reticuloendothelial system. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a cell viability curve of an osteosarcoma cell line treated with ZIF-8 nanoparticles of the present invention;

[0021] Figure 2 This is a comparative example of the ZIF-8 nanoparticles administration treatment of the present invention (free metformin treatment);

[0022] Figure 3 This is a graph showing protein expression changes in osteosarcoma HOS cell lines after treatment with ZIF-8 nanoparticles in the present invention;

[0023] Figure 4 This is a graph showing changes in ATP concentration inside and outside the osteosarcoma HOS cell line after treatment with ZIF-8 nanoparticles in the present invention;

[0024] Figure 5 This is a comparison of the changes in intracellular and extracellular ATP concentrations in the osteosarcoma HOS cell line after treatment with ZIF-8 nanoparticles in the present invention for 24 and 48 hours;

[0025] Figure 6This is a fluorescence staining comparison of protein expression changes in the osteosarcoma HOS cell line after treatment with ZIF-8 nanoparticles in the present invention for 24 and 48 hours;

[0026] Figure 7 This is an electron microscope image of the nanoparticles of the present invention;

[0027] Figure 8 This is a UV absorption detection curve diagram of metformin and rhodamine 6G in the nanoparticles of the present invention after release in vitro solution. DETAILED DESCRIPTION

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0029] Example 1: Step 1, preparation of ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles: adding metformin hydrochloride to a 2.5 mol / L methylimidazole solution to prepare a metformin hydrochloride-methylimidazole solution having a metformin hydrochloride concentration of 5 mg / mL, then mixing 0.2 mol / L zinc nitrate solution and the metformin hydrochloride-methylimidazole solution in a volume ratio of 1:1, stirring at room temperature to allow for sufficient reaction, and then centrifuging at 8000 rpm for 15 minutes to collect the product, rinse with deionized water, and then vacuum dry at room temperature to obtain ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles;

[0030] Step 2, preparation of macrophage ghost liposomes: Incubate the cells in a 37°C, 95% humidity, 5% CO2 incubator with a volume of 5 × 10 macrophages per well. 4 3×10 osteosarcoma cells in the lower chamber 5 The co-cultured macrophages were digested from the Transwell plate, frozen and revived, and then resuspended in 0.25× PBS buffer. After washing and purification, the macrophage ghost liposomes were collected by centrifugation at 5000 rpm at 4°C.

[0031] Step 3, packaging drug-loaded nanoparticles in macrophage membrane liposomes: ZIF-8-loaded metformin hydrochloride-loaded nanoparticles were added to double-distilled water to form a 4 mg / ml nanoparticle dispersion, and macrophage ghost liposomes were added to double-distilled water to form a 15 mg / ml macrophage ghost liposome suspension. The nanoparticle dispersion and the macrophage ghost liposome suspension were mixed in a 1:1 volume ratio at 4°C or in an ice-water mixing bath, and then ultrasonically treated for 5 minutes. The mixture was extruded through a polycarbonate porous membrane with a pore size of 200 nm using an Avanti mini extruder at 4°C or in an ice-water mixing bath to obtain nanoparticles packaged in macrophage ghost liposomes.

[0032] Example 2: Step 1, preparation of ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles: metformin hydrochloride was added to a 2.5 mol / L methylimidazole solution to prepare a metformin hydrochloride-methylimidazole solution having a metformin hydrochloride concentration of 8 mg / mL, and then 0.1 mol / L zinc nitrate solution and the metformin hydrochloride-methylimidazole solution were mixed at a volume ratio of 1.4:1. After stirring at room temperature to allow for sufficient reaction, the product was collected by centrifugation at 10,000 rpm for 13 minutes, rinsed with deionized water, and then dried in vacuo at room temperature to obtain ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles;

[0033] Step 2, preparation of macrophage ghost liposomes: Incubate the cells in a 37°C, 95% humidity, 5% CO2 incubator with a volume of 5 × 10 macrophages per well. 4 3×10 osteosarcoma cells in the lower chamber 5 The co-cultured macrophages were digested from the Transwell plate, frozen and revived, and then resuspended in 0.25× PBS buffer. After washing and purification, the macrophage ghost liposomes were collected by centrifugation at 7000 rpm at 4°C.

[0034] Step 3, packaging drug-loaded nanoparticles in macrophage membrane liposomes: ZIF-8-loaded metformin hydrochloride-loaded nanoparticles were added to double-distilled water to form a 4 mg / ml nanoparticle dispersion, and macrophage ghost liposomes were added to double-distilled water to form a 12 mg / ml macrophage ghost liposome suspension. The nanoparticle dispersion and the macrophage ghost liposome suspension were mixed in a volume ratio of 1.4:1 at 4°C or in an ice-water mixing bath, and then ultrasonically treated for 8 minutes. The mixture was extruded through a polycarbonate porous membrane with a pore size of 230 nm using an Avanti mini extruder at 4°C or in an ice-water mixing bath to obtain nanoparticles packaged in macrophage ghost liposomes.

[0035] Example 3: Step 1, preparation of ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles: metformin hydrochloride and tracer rhodamine 6G were added to a 2.5 mol / L methylimidazole solution to prepare a metformin hydrochloride / rhodamine 6G methylimidazole solution with a metformin hydrochloride concentration of 10 mg / mL and a rhodamine 6G concentration of 10 mg / mL, and then 0.5 mol / L zinc nitrate solution and the metformin hydrochloride / rhodamine 6G methylimidazole solution were mixed at a volume ratio of 1.2:1, stirred at room temperature for sufficient reaction, and then centrifuged at 9000 rpm for 14 minutes to collect the product, rinsed with deionized water, and then dried in vacuo at room temperature to obtain ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles;

[0036] Step 2, preparation of macrophage ghost liposomes: Incubate the cells in a 37°C, 95% humidity, 5% CO2 incubator with a volume of 5 × 10 macrophages per well. 4 3×10 osteosarcoma cells in the lower chamber 5 The co-cultured macrophages were digested from the Transwell plate, frozen and revived, and then resuspended in 0.25× PBS buffer. After washing and purification, the macrophage ghost liposomes were collected by centrifugation at 8000 rpm at 4°C.

[0037] Step 3, packaging drug-loaded nanoparticles in macrophage membrane liposomes: ZIF-8-loaded metformin hydrochloride-loaded nanoparticles were added to double-distilled water to form a 4 mg / ml nanoparticle dispersion, and macrophage ghost liposomes were added to double-distilled water to form an 18 mg / ml macrophage ghost liposome suspension. The nanoparticle dispersion and the macrophage ghost liposome suspension were mixed at a volume ratio of 1.2:1 at 4°C or in an ice-water mixing bath, and then ultrasonically treated for 6 minutes. The mixture was extruded through a polycarbonate porous membrane with a pore size of 280 nm using an Avanti mini extruder at 4°C or in an ice-water mixing bath to obtain nanoparticles packaged in macrophage ghost liposomes.

[0038] Example 4: Step 1, preparation of ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles: metformin hydrochloride and tracer rhodamine 6G were added to a 2.5 mol / L methylimidazole solution to prepare a metformin hydrochloride / rhodamine 6G methylimidazole solution with a metformin hydrochloride concentration of 7 mg / mL and a rhodamine 6G concentration of 7 mg / mL, and then 0.3 mol / L zinc nitrate solution and the metformin hydrochloride / rhodamine 6G methylimidazole solution were mixed at a volume ratio of 1.5:1, stirred at room temperature for sufficient reaction, and the product was collected by centrifugation at a speed of 11,000 rpm for 12 minutes, rinsed with deionized water, and then dried in vacuo at room temperature to obtain ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles;

[0039] Step 2, preparation of macrophage ghost liposomes: Incubate the cells in a 37°C, 95% humidity, 5% CO2 incubator with a volume of 5 × 10 macrophages per well. 4 3×10 osteosarcoma cells in the lower chamber 5 The co-cultured macrophages were digested from the Transwell plate, frozen and revived, and then resuspended in 0.25× PBS buffer containing 0.1 mmol / L PMSF. After washing and purification, the macrophage ghost liposomes were collected by centrifugation at 6000 rpm at 4°C.

[0040] Step 3, packaging drug-loaded nanoparticles in macrophage membrane liposomes: ZIF-8-loaded metformin hydrochloride-loaded nanoparticles were added to double-distilled water to form a 4 mg / ml nanoparticle dispersion, and macrophage ghost liposomes were added to double-distilled water to form a 20 mg / ml macrophage ghost liposome suspension. The nanoparticle dispersion and the macrophage ghost liposome suspension were mixed at a volume ratio of 1.5:1 at 4°C or in an ice-water mixing bath, and then ultrasonically treated for 9 minutes. The mixture was extruded through a polycarbonate porous membrane with a pore size of 300 nm using an Avanti mini extruder at 4°C or in an ice-water mixing bath to obtain nanoparticles packaged in macrophage ghost liposomes.

[0041] Example 5: Step 1, preparation of ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles: metformin hydrochloride and tracer rhodamine 6G were added to a 2.5 mol / L methylimidazole solution to prepare a metformin hydrochloride / rhodamine 6G methylimidazole solution with a metformin hydrochloride concentration of 9 mg / mL and a rhodamine 6G concentration of 9 mg / mL, and then 0.4 mol / L zinc nitrate solution and the metformin hydrochloride / rhodamine 6G methylimidazole solution were mixed at a volume ratio of 1.3:1, stirred at room temperature for sufficient reaction, and then centrifuged at 12,000 rpm for 10 minutes to collect the product, rinsed with deionized water, and then dried in vacuo at room temperature to obtain ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles;

[0042] Step 2, preparation of macrophage ghost liposomes: Incubate the cells in a 37°C, 95% humidity, 5% CO2 incubator with a volume of 5 × 10 macrophages per well. 4 3×10 osteosarcoma cells in the lower chamber 5 The co-cultured macrophages were digested from the Transwell plate, frozen and revived, and then resuspended in 0.25× PBS buffer containing 0.3 mmol / L PMSF. After washing and purification, the macrophage ghost liposomes were collected by centrifugation at 7000 rpm at 4°C.

[0043] Step 3, packaging drug-loaded nanoparticles in macrophage membrane liposomes: ZIF-8-loaded metformin hydrochloride-loaded nanoparticles were added to double-distilled water to form a 4 mg / ml nanoparticle dispersion, and macrophage ghost liposomes were added to double-distilled water to form a 13 mg / ml macrophage ghost liposome suspension. The nanoparticle dispersion and the macrophage ghost liposome suspension were mixed at a volume ratio of 1.3:1 at 4°C or in an ice-water mixing bath, and then ultrasonically treated for 7 minutes. The mixture was extruded through a polycarbonate porous membrane with a pore size of 250 nm using an Avanti mini extruder at 4°C or in an ice-water mixing bath to obtain nanoparticles packaged in macrophage ghost liposomes.

[0044] Example 6: Step 1, preparation of ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles: metformin hydrochloride was added to a 2.5 mol / L methylimidazole solution to prepare a metformin hydrochloride-methylimidazole solution with a metformin hydrochloride concentration of 6 mg / mL, and then 0.3 mol / L zinc nitrate solution and the metformin hydrochloride-methylimidazole solution were mixed at a volume ratio of 1.1:1. After stirring at room temperature to allow for sufficient reaction, the product was collected by centrifugation at 8000 rpm for 15 minutes, and rinsed with deionized water and then dried in vacuo at room temperature to obtain ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles;

[0045] Step 2, preparation of macrophage ghost liposomes: Incubate the cells in a 37°C, 95% humidity, 5% CO2 incubator with a volume of 5 × 10 macrophages per well. 4 3×10 osteosarcoma cells in the lower chamber 5 The co-cultured macrophages were digested from the Transwell plate, frozen and revived, and then resuspended in 0.25× PBS buffer containing 0.8 mmol / L PMSF. After washing and purification, the macrophage ghost liposomes were collected by centrifugation at 8000 rpm at 4°C.

[0046] Step 3, packaging drug-loaded nanoparticles in macrophage membrane liposomes: ZIF-8-loaded metformin hydrochloride-loaded nanoparticles were added to double-distilled water to form a 4 mg / ml nanoparticle dispersion, and macrophage ghost liposomes were added to double-distilled water to form a 17 mg / ml macrophage ghost liposome suspension. The nanoparticle dispersion and the macrophage ghost liposome suspension were mixed in a volume ratio of 1.1:1 at 4°C or in an ice-water mixing bath, and then ultrasonically treated for 10 minutes. The mixture was extruded through a polycarbonate porous membrane with a pore size of 220 nm using an Avanti mini extruder at 4°C or in an ice-water mixing bath to obtain nanoparticles packaged in macrophage ghost liposomes.

[0047] Example 7: Step 1, preparation of ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles: metformin hydrochloride and tracer rhodamine 6G were added to a 2.5 mol / L methylimidazole solution to prepare a metformin hydrochloride / rhodamine 6G methylimidazole solution with a metformin hydrochloride concentration of 8 mg / mL and a rhodamine 6G concentration of 5 mg / mL, and then 0.4 mol / L zinc nitrate solution and the metformin hydrochloride / rhodamine 6G methylimidazole solution were mixed at a volume ratio of 1.3:1. After stirring at room temperature to allow for sufficient reaction, the product was collected by centrifugation at 10,000 rpm for 12 minutes, rinsed with deionized water, and then dried in vacuo at room temperature to obtain ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles;

[0048] Step 2, preparation of macrophage ghost liposomes: Incubate the cells in a 37°C, 95% humidity, 5% CO2 incubator with a volume of 5 × 10 macrophages per well. 4 3×10 osteosarcoma cells in the lower chamber 5 The co-cultured macrophages were digested from the Transwell plate, frozen and revived, and then resuspended in 0.25× PBS buffer containing 0.5 mmol / L PMSF. After washing and purification, the macrophage ghost liposomes were collected by centrifugation at 6000 rpm at 4°C.

[0049] Step 3, packaging drug-loaded nanoparticles in macrophage membrane liposomes: ZIF-8-loaded metformin hydrochloride-loaded nanoparticles were added to double-distilled water to form a 4 mg / ml nanoparticle dispersion, and macrophage ghost liposomes were added to double-distilled water to form a 10 mg / ml macrophage ghost liposome suspension. The nanoparticle dispersion and the macrophage ghost liposome suspension were mixed in a volume ratio of 1.3:1 at 4°C or in an ice-water mixing bath, and then ultrasonically treated for 5 minutes. The mixture was extruded through a polycarbonate porous membrane with a pore size of 260 nm using an Avanti mini extruder at 4°C or in an ice-water mixing bath to obtain nanoparticles packaged in macrophage ghost liposomes.

[0050] Example 8: Step 1, preparation of ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles: metformin hydrochloride and tracer rhodamine 6G were added to a 2.5 mol / L methylimidazole solution to prepare a metformin hydrochloride / rhodamine 6G methylimidazole solution with a metformin hydrochloride concentration of 6 mg / mL and a rhodamine 6G concentration of 8 mg / mL, and then 0.2 mol / L zinc nitrate solution and the metformin hydrochloride / rhodamine 6G methylimidazole solution were mixed at a volume ratio of 1.2:1, stirred at room temperature for sufficient reaction, and the product was collected by centrifugation at 12,000 rpm for 10 minutes, rinsed with deionized water, and then dried in vacuo at room temperature to obtain ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles;

[0051] Step 2, preparation of macrophage ghost liposomes: Incubate the cells in a 37°C, 95% humidity, 5% CO2 incubator with a volume of 5 × 10 macrophages per well. 4 3×10 osteosarcoma cells in the lower chamber 5The co-cultured macrophages were digested from the Transwell plate, frozen and revived, and then resuspended in 0.25× PBS buffer containing 1 mmol / L PMSF. After washing and purification, the macrophage ghost liposomes were collected by centrifugation at 5000 rpm at 4°C.

[0052] Step 3, packaging drug-loaded nanoparticles in macrophage membrane liposomes: ZIF-8-loaded metformin hydrochloride-loaded nanoparticles were added to double-distilled water to form a 4 mg / ml nanoparticle dispersion, and macrophage ghost liposomes were added to double-distilled water to form a 16 mg / ml macrophage ghost liposome suspension. The nanoparticle dispersion and the macrophage ghost liposome suspension were mixed in a volume ratio of 1.2:1 at 4°C or in an ice-water mixing bath, and then ultrasonically treated for 10 minutes. The mixture was extruded through a polycarbonate porous membrane with a pore size of 270 nm using an Avanti mini extruder at 4°C or in an ice-water mixing bath to obtain nanoparticles packaged in macrophage ghost liposomes.

[0053] The macrophage membrane liposome-encapsulated metformin hydrochloride precise drug delivery system prepared according to the above embodiment comprises drug-loaded nanoparticles made of ZIF-8 loaded with metformin hydrochloride and biomimetic nanoparticles formed by macrophage membrane liposomes encapsulated on the outer surface of the drug-loaded nanoparticles.

[0054] like Figure 1 and Figure 2 The osteosarcoma cell lines HOS, MG63, and K7M2 were treated with the nanoparticles and free metformin for 24, 48, and 72 hours, respectively. The viability of the osteosarcoma cells was detected by CCK8 assay. The viability of the tumor cells in the nanoparticle and free metformin treatment groups was significantly reduced compared to the control group. 50 After concentration curve fitting, the IC of the nanoparticle group was measured. 50 (about 40-80ug / ml) and free metformin IC 50 (about 30-90 mg / ml) with a difference of about 1000 times, indicating that the nanoparticles have good intracellular accumulation ability.

[0055] like Figure 3As shown, the osteosarcoma HOS cell line was treated with nanoparticles at concentrations of 0, 40, and 80 μg / ml for 24 hours, and the osteosarcoma HOS cell line was treated with nanoparticles at a concentration of 40 μg / ml for 0, 2, 4, 6, 8, and 12 hours, respectively, and the total cell protein was extracted and detected by protein electrophoresis. It can be detected that the immunogenic death marker calreticulin CRT increased significantly after 8 hours of treatment, while the expression of endoplasmic reticulum stress markers BIP and PERK decreased, and the expression of endoplasmic reticulum-related cell death marker CASPASE12 increased significantly, indicating that the nanoparticles can induce endoplasmic reticulum stress-related death and promote immunogenic death after treatment of the osteosarcoma HOS line.

[0056] like Figure 4 and Figure 5 As shown, the osteosarcoma HOS cell line was treated with the nanoparticles at a concentration of 0, 40, and 80 ug / ml for 24 and 48 hours, respectively. The osteosarcoma HOS cell line was treated with the nanoparticles at a concentration of 40 ug / ml for 0, 2, 4, 6, 8, and 12 hours, respectively. The intracellular and extracellular ATP concentrations were detected, and the results showed that the intracellular ATP concentration decreased and the extracellular ATP concentration, an immunogenic death marker, increased, indicating that the nanoparticle treatment of the osteosarcoma HOS cell line promoted the occurrence of immunogenic death.

[0057] like Figure 6 As shown, the osteosarcoma HOS and MG63 cell lines were treated with the nanoparticles at concentrations of 0, 40, and 80 μg / ml for 24 and 48 hours, respectively, and then immunofluorescence staining of calreticulin CRT and PERK protein was performed. In both cell lines, it was observed that with the increase in the nanoparticle administration concentration and treatment time, the fluorescence intensity of calreticulin CRT increased, while the fluorescence intensity of PERK protein decreased, indicating that the nanoparticles can induce endoplasmic reticulum stress-related death and promote immunogenic death in the osteosarcoma HOS and MG63 lines after treatment.

[0058] like Figure 7 The morphology of the nanoparticles shown under an electron microscope is approximately 200 nm in size, which is a size with a high utilization rate in the body.

[0059] like Figure 8 The nanoparticles shown have obvious metformin hydrochloride and rhodamine 6G release peaks in simulated body fluid solution, indicating that the nanoparticles have the designed release capacity.

[0060] The present invention uses ZIF-8 to carry metformin hydrochloride. The presence of ZIF-8 can prevent metformin hydrochloride from dissolving in water too early, significantly increase its intracellular accumulation ability, and make metformin induce osteosarcoma cell death and even immunogenic death. 50The concentration was reduced by about 1000 times; and the method of packaging ZIF-8 nanoparticles using tumor-associated macrophage membrane liposomes was used. By co-culturing with the tumor to be treated, macrophages were activated to express immune markers, and tumor-associated macrophages were obtained. By extracting their cell membranes, cell membrane liposomes containing specific immune markers were obtained. After the drug-loaded nanoparticles were extruded and packaged, they had the ability to target tumors and avoid degradation by the reticuloendothelial system.

Claims

1. A macrophage vesicular liposome-encapsulated metformin hydrochloride drug delivery system, characterized in that: The drug delivery system comprises drug-loaded nanoparticles made of ZIF-8 loaded with metformin hydrochloride and biomimetic nanoparticles formed by macrophage vesicle liposomes packaged on the outer surface of the drug-loaded nanoparticles; Preparation of macrophage ghost liposomes: Incubate in a 37°C, 95% humidity, 5% CO2 incubator according to the number of macrophages in the chamber: 5×10 4 3×10 osteosarcoma cells in the lower chamber 5 The co-cultured macrophages were digested from the Transwell plate, frozen and revived, and then resuspended in 0.25× PBS buffer. After washing and purification, the macrophage ghost liposomes were collected by low-temperature centrifugation.

2. The macrophage ghost liposome-packaged metformin hydrochloride drug delivery system according to claim 1, characterized in that: The drug-loaded nanoparticles are also loaded with rhodamine 6G.

3. A method for preparing the macrophage ghost liposome-encapsulated metformin hydrochloride drug delivery system according to claim 1, characterized in that: Step 1, preparation of ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles: adding metformin hydrochloride to a 2.5 mol / L methylimidazole solution to prepare a metformin hydrochloride-methylimidazole solution having a metformin hydrochloride concentration of 5-10 mg / mL, then mixing a 0.1-0.5 mol / L zinc nitrate solution and the metformin hydrochloride-methylimidazole solution at a volume ratio of 1-1.5:1, stirring at room temperature to allow for full reaction, collecting the product by centrifugation, rinsing with deionized water, and then drying under vacuum at room temperature to obtain ZIF-8-loaded metformin hydrochloride drug-loaded nanoparticles; Step 2, preparation of macrophage ghost liposomes: Incubate the cells in a 37°C, 95% humidity, 5% CO2 incubator with a volume of 5 × 10 macrophages per chamber. 4 3×10 osteosarcoma cells in the lower chamber 5 The co-cultured macrophages were digested from the Transwell plate, frozen and revived, and then resuspended in 0.25× PBS buffer. After washing and purification, the macrophage ghost liposomes were collected by low-temperature centrifugation. Step 3, packaging drug-loaded nanoparticles in macrophage vesicular liposomes: adding ZIF-8-loaded metformin hydrochloride-loaded nanoparticles to double-distilled water to form a 4 mg / ml nanoparticle dispersion, adding macrophage vesicular liposomes to double-distilled water to form a 10-20 mg / ml macrophage vesicular liposome suspension, mixing the nanoparticle dispersion and the macrophage vesicular liposome suspension at a volume ratio of 1 to 1.5:1 at 4°C or in an ice-water mixing bath, and then ultrasonically treating the mixture. The mixture is extruded through a polycarbonate porous membrane using an Avanti mini extruder at 4°C or in an ice-water mixing bath to obtain nanoparticles packaged in macrophage vesicular liposomes.

4. The method for preparing the macrophage ghost liposome-encapsulated metformin hydrochloride drug delivery system according to claim 3, wherein: The methylimidazole solution in step 1 is further added with rhodamine 6G, and the concentration of rhodamine 6G is 5-10 mg / mL.

5. The method for preparing the macrophage ghost liposome-encapsulated metformin hydrochloride drug delivery system according to claim 3, wherein: The centrifugation in step 1 is performed at a rotation speed of 8000-12000 rpm for 10-15 minutes.

6. The method for preparing the macrophage ghost liposome-encapsulated metformin hydrochloride drug delivery system according to claim 3, wherein: The 0.25×PBS buffer in step 2 also contains PMSF with a concentration of 0.1-1 mmol / L.

7. The method for preparing the macrophage ghost liposome-encapsulated metformin hydrochloride drug delivery system according to claim 3, wherein: The low-temperature centrifugation in step 2 is to collect macrophage ghost liposomes by centrifugation at 5000-8000 rpm at 4°C.

8. The method for preparing the macrophage ghost liposome-encapsulated metformin hydrochloride drug delivery system according to claim 3, wherein: In step 3, the ultrasonic treatment is performed for 5 to 10 minutes.

9. The method for preparing the macrophage ghost liposome-encapsulated metformin hydrochloride drug delivery system according to claim 3, wherein: The pore size of the polycarbonate porous membrane in step 3 is 200-300 nm.

10. Use of a precise drug delivery system of metformin hydrochloride packaged in macrophage ghost liposomes obtained by the preparation method according to any one of claims 3 to 9 in the preparation of a drug for treating osteosarcoma cells.

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