A cell membrane-coated drug-loaded vesicle, preparation method and application thereof

Abstract

The present invention discloses a cell membrane-coated drug-loaded vesicle, a preparation method and an application, which mainly relate to the field of anti-tumor technology. It includes co-incubating a macrophage membrane solution with a TLR4 agonist RS09, and then extruding it back and forth 10 to 20 times using a 200nm polycarbonate membrane to obtain a cell membrane-coated nanovesicle loaded with a TLR4 agonist. The beneficial effect of the present invention is that it is used to target the delivery of TLR4 agonists to osteosarcoma tumor tissues, induce TAMs polarization, reshape the immunosuppressive TME, and efficiently kill osteosarcoma. This study achieved the dual purpose of regulating TME and killing tumors, providing a new strategy for the treatment of osteosarcoma.

Description

Technical Field

[0001] The present invention relates to the field of anti-tumor technology, and in particular to a cell membrane-coated drug-loaded vesicle, a preparation method and an application thereof. Background Art

[0002] Osteosarcoma is the most common primary malignant bone tumor. However, the tumor microenvironment (TME) of osteosarcoma is often in an immunosuppressive state, which leads to poor osteosarcoma progression and treatment effects. Currently, there is no anti-osteosarcoma treatment that can effectively regulate TME and kill tumors. Tumor associated macrophages (TAMs) are important participants in the tumor immunosuppressive microenvironment. TAMs can be divided into two subtypes: M1 TAMs mainly play an anti-tumor role, while M2 promotes tumor progression. Based on the functional characteristics of TAMs, inducing polarization from M2 to M1 is an important strategy to reshape the tumor immunosuppressive TME and treat tumors.

[0003] Toll-like receptor (TLR) is one of the most widely studied pattern recognition receptors in the innate immune system. TLR4 exists on the surface of many cells, such as macrophages, endothelial cells, epithelial cells, etc., and plays an important role in infiltrating immune cells or tumor cells in the TME. The intracellular domain of TLR4 is responsible for recruiting downstream adaptor proteins and participating in inflammatory responses. Studies have shown that TLR agonists can induce M1 polarization of TAMs and exert anti-tumor effects. Paclitaxel can activate the TLR4 signaling pathway to reprogram TAMs to M1 type and inhibit tumor growth. Therefore, the TLR4 signaling pathway is involved in regulating the M1 polarization of macrophages.

[0004] If tumor targeted therapy is developed based on the above theory, then how to effectively deliver effective drug ingredients to the target is a difficult problem to achieve targeted therapy. If the delivered TLR agonist has an "off-target" effect, it will cause greater systemic toxicity and cannot accurately target the local tumor tissue, and cannot fully exert its anti-tumor effect.

[0005] Tumor targeted therapy strategies rely on the construction of an efficient targeted drug delivery system. Existing traditional nanocarriers are mostly synthetic nanomaterials or exosomes, which have the following problems: (1) Synthetic nanomaterials are easily recognized and cleared by the immune system, have large toxic side effects, and have complex functional designs; the preparation method of exosomes is cumbersome, with low yield and high cost, which is not conducive to clinical promotion and application. (2) The drug loading method is usually physical adsorption or chemical modification. The physical adsorption method has the problem of low drug loading rate, and the chemical modification method has the problem of complex modification and difficult drug release. (3) The targeting effect is single, and can only target tumor cells, but cannot target other cells in the tumor microenvironment such as macrophages to play a dual regulatory role. Summary of the invention

[0006] The purpose of the present invention is to provide a cell membrane-coated drug-loaded vesicle, a preparation method and an application thereof, which is used to deliver TLR4 agonists to osteosarcoma tumor tissues in a targeted manner, induce TAMs polarization, reshape the immunosuppressive TME, and efficiently kill osteosarcoma. This study achieves the dual purpose of regulating TME and killing tumors, providing a new strategy for the treatment of osteosarcoma.

[0007] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0008] A method for preparing cell membrane-coated drug-loaded vesicles comprises the following steps: incubating a macrophage membrane solution with a TLR4 agonist RS09, and then extruding the solution back and forth 10 to 20 times using a 200 nm polycarbonate membrane to obtain cell membrane-coated nanovesicles loaded with the TLR4 agonist.

[0009] Preferably, the method for preparing the macrophage membrane fluid is: stimulate RAW264.7 macrophages to differentiate into M1 type by LPS, collect 1×10 8 The M1 macrophages were washed three times with PBS, resuspended with hypotonic cell lysis buffer at 4°C, and placed on ice for 15 minutes. The resuspended solution was disrupted with an ultrasonic disruptor for 10 minutes, and the obtained solution was purified by differential centrifugation to obtain a macrophage membrane solution.

[0010] Preferably, the differential centrifugation purification comprises centrifuging at 700 g for 10 min to obtain the supernatant; and centrifuging at 15,000 g for 30 min to obtain the precipitate to purify the membrane.

[0011] Preferably, the macrophage RAW264.7 is cultured in a high-glucose DMEM medium containing 10% fetal bovine serum and in an environment of 37°C and 5% CO 2 Culture in an incubator.

[0012] Preferably, the TLR4 agonist is used in an amount of 0.05 mg and the macrophage membrane solution is used in an amount of 0.1 mg for co-incubation.

[0013] A cell membrane-coated drug-loaded vesicle is prepared by the above method.

[0014] A method for preparing cell membrane coated drug-loaded vesicles and application of the prepared cell membrane coated nano vesicles in drugs for treating osteosarcoma tumors.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] (1) Develop a bionic macrophage membrane-coated nanovesicle that has no systemic toxic side effects, and the preparation method (extruder extrusion) is simple, efficient, and has a large output.

[0017] (2) TLR4 agonists can be efficiently loaded into cell membrane vesicles by co-extrusion of drugs and cell membranes, and the drugs are immediately released when the vesicles are internalized by cells.

[0018] (3) The drug-loaded cell membrane-coated vesicles retain the cell membrane surface-related proteins and antigens. The vesicles in the present invention have the same antigenic properties as macrophages, indicating that they have the functions of homologous targeting of macrophages and targeting of tumor cells. The drug-loaded vesicles can target the delivery of TLR4 agonists to osteosarcoma tumor tissues, and simultaneously target tumor cells and macrophages in the tumor microenvironment.

[0019] (4) The bionic macrophage membrane-coated nano-drug-loaded vesicles can induce TAMs polarization, reshape the immunosuppressive TME, and efficiently kill osteosarcoma, achieving the dual purpose of regulating TME and killing tumors, providing a new strategy for the treatment of osteosarcoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the preparation and structure of the cell membrane-coated nanovesicles carrying TLR4 agonist of the present invention.

[0021] Figure 2 It is a TEM electron microscope image of the TLR4 agonist-loaded nanovesicle of the present invention (scale: 100 nm).

[0022] Figure 3 is the particle size of the nanovesicles of the present invention and the nanovesicles loaded with TLR4 agonist (ns: no statistical difference).

[0023] Figure 4 The present invention discloses a cell membrane-coated nanovesicle having a drug loading performance for TLR4 agonists.

[0024] Figure 5 This is the targeting effect of the TLR4 agonist-loaded nanovesicles of the present invention on macrophages (DiD marks the cell membrane, scale: 50 μm).

[0025] Figure 6After the untreated group, TLR4 agonist group and TLR4 agonist-loaded nanovesicle group were treated with the present invention, the fluorescence intensity of macrophage markers (iNOS marks M1 macrophages, CD163 marks M2 macrophages, scale: 50 μm) was detected.

[0026] Figure 7-Figure 8 The relative proportions of M1 and M2 macrophages were analyzed after treatment with the untreated group, TLR4 agonist group and TLR4 agonist-loaded nanovesicle group of the present invention (**P<0.01).

[0027] Fig. 9 This is a schematic diagram of the present invention constructing a cell co-culture system, wherein the untreated group, the TLR4 agonist group, and the TLR4 agonist-loaded nanovesicle group respectively treat the upper layer macrophages, and after 24 hours, the lower layer osteosarcoma cells are collected for subsequent experiments.

[0028] Fig.10 The present invention detected the viability of osteosarcoma cells through CCK-8 experiment (**P<0.01).

[0029] Fig.11 This is the experimental result of clone formation experiment.

[0030] Figure 12-13 The present invention detected the proliferation ability of osteosarcoma cells through clone formation experiment (**P<0.01).

[0031] Fig.14 The number, morphology and adhesion of osteosarcoma cells observed by light microscopy (scale: 100 μm)

[0032] Fig.15 The nanovesicles loaded with TLR4 agonist in the mice of the present invention are enriched in tumor tissues.

[0033] Figure 16-Figure 18 After treatment with the untreated group, TLR4 agonist group, and TLR4 agonist-loaded nanovesicle group of the present invention, the volume of tumor tissue was detected and calculated (**P<0.01). (D, E, F) The fluorescence intensity of macrophage markers in tumor tissue sections was detected, and the relative proportion of M1 and M2 macrophages was analyzed (iNOS marked M1 macrophages, CD163 marked M2 macrophages, scale: 50 μm, **P<0.01).

[0034] Fig.19 It is a schematic diagram of the mechanism by which the cell membrane-coated nanovesicles of the present invention regulate the polarization of tumor-associated macrophages, reshape the tumor immunosuppressive microenvironment, and kill osteosarcoma. DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the application equally.

[0036] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0037] The osteosarcoma cell line (MNNG / HOS) and macrophage cell line (RAW264.7) in this example were purchased from the Cell Bank of the Chinese Academy of Sciences. Osteosarcoma cells were cultured in α-MEM medium, and macrophages were cultured in high-glucose DMEM medium containing 10% fetal bovine serum. All cells were cultured at 37°C and 5% CO. 2 Culture in an incubator.

[0038] Example 1: Preparation method of cell membrane drug-loaded vesicles

[0039] RAW264.7 macrophages were differentiated into M1 type by LPS stimulation, and 1×10 8 M1 macrophages were washed 3 times with PBS, resuspended with hypotonic cell lysis buffer at 4°C, and placed on ice for 15 minutes. The resuspended solution was crushed with an ultrasonic crusher for 10 minutes to destroy the cell structure. The obtained solution was subjected to differential centrifugation (700g, 10min to take the supernatant; 15000g, 30min to take the precipitate) to purify the membrane and obtain a macrophage membrane solution. After the collected macrophage membrane solution was incubated with the TLR4 agonist RS09, it was extruded back and forth 10 to 20 times with a 200nm polycarbonate membrane in an Avanti extruder to obtain cell membrane-coated nanovesicles carrying the TLR4 agonist.

[0040] Based on the dosage of the macrophage membrane solution and the TLR4 agonist RS09 for co-incubation, the following dosage experiments were performed:

[0041] The morphology of the cell membrane-coated nanovesicles obtained based on the above method was detected.

[0042] 1) Transmission electron microscopy (TEM) was used to examine the morphology of cell membrane-coated nano-drug-loaded vesicles using a JEM-2000EXII TEM instrument (JEOL, Tokyo, Japan). Figure 2TEM electron microscopy showed that the TLR4 agonist-loaded nanovesicles were round in shape and had a particle size of about 200 nm.

[0043] 2) Dynamic light scattering (DLS) was used to detect the particle size of cell membrane-coated nano-drug-loaded vesicles using a Zetasizer Nano ZS instrument (Malvern Zetasizer, Malvern, UK). Figure 3 There was no significant difference in the particle size of nanovesicles before and after loading with TLR4 agonist.

[0044] 3) Detect the drug loading performance of the nano drug-loaded vesicles, and the calculation formula is as follows: Encapsulation efficiency (%) = W 1 / W 2 × 100%, where W 1 is the encapsulated drug mass (mg), W 2 is the total mass of the drug used (mg). Drug loading (mg / mg) = W 1 / W 3 , where W 1 is the encapsulated drug mass (mg), W 3 is the total mass of the entire drug delivery system (mg). Figure 4 The optimal conditions for preparing drug-loaded nanovesicles were the co-incubation of 0.05 mg TLR4 agonist and 0.1 mg nanovesicles, with an encapsulation rate of about 35% and a drug loading of about 0.11 mg / mg. The cell membrane-coated nanovesicles could efficiently load and deliver TLR4 agonists.

[0045] Example 2: Targeting effect test of the nanovesicles obtained in Example 1

[0046] The prepared TLR4 agonist-loaded nanovesicles were labeled with DiD red fluorescent dye, and then the labeled nanovesicles were co-incubated with RAW264.7 macrophages for 24 hours. Confocal fluorescence microscopy revealed that RAW264.7 macrophages had strong DiD fluorescence (e.g. Figure 5 As shown in FIG. 4 , the nanovesicles have good targeting to macrophages and can be used for targeted delivery of TLR4 agonists, thereby achieving targeted drug delivery.

[0047] Compared with the untreated group and the TLR4 agonist group, the fluorescence of the M1 macrophage marker (iNOS) in the TLR4 agonist-loaded nanovesicle group was significantly enhanced ( Figure 6 and 7 ), while the fluorescence of M2 macrophage marker (CD163) decreased significantly ( Figure 6 and 7). Therefore, based on the good macrophage targeting effect, the nanovesicles can target and deliver TLR4 agonists to macrophages, which is more significant than the macrophage regulation effect of TLR4 agonists alone.

[0048] Example 3: In vitro anti-osteosarcoma effect test of the nanovesicles obtained in Example 1

[0049] A cell co-culture system was constructed. The untreated group, TLR4 agonist group, and TLR4 agonist-loaded nanovesicle group were treated with macrophages in the upper layer, and osteosarcoma cells in the lower layer were collected after 24 hours for subsequent experiments ( Fig. 9 ).

[0050] The CCK-8 experiment showed that the viability of osteosarcoma cells in the TLR4 agonist-loaded nanovesicle group was significantly reduced compared with the untreated group and the TLR4 agonist group ( Fig.10 ). The clone formation experiment found that compared with the untreated group and the TLR4 agonist group, the number of osteosarcoma cell clones in the TLR4 agonist nanovesicle group was significantly reduced, indicating that the cell proliferation ability was weakened ( Fig.11 and 13 ). Under the light microscope, it was found that the number of osteosarcoma cells in the TLR4 agonist nanovesicle group was significantly reduced, and a large number of cells shrank and became round, and floated due to poor adhesion. The number of cells in the untreated group and TLR4 agonist group was large, and the morphology was basically normal ( Fig.12 ).

[0051] Therefore, nanovesicles loaded with TLR4 agonist can significantly inhibit the viability and proliferation of osteosarcoma cells by inducing macrophage polarization, and have a strong in vitro anti-osteosarcoma effect.

[0052] Example 4: In vivo anti-osteosarcoma effect test of the nanovesicles obtained in Example 1

[0053] The in vivo anti-osteosarcoma effect and mechanism of cell membrane-coated nanovesicles were investigated by using a subcutaneous transplant tumor model. After successfully constructing a mouse osteosarcoma subcutaneous transplant tumor model, nanovesicles loaded with TLR4 agonists were injected through the tail vein. The distribution of DiD-labeled nanovesicles in mice was detected by small animal in vivo imaging experiments. The results showed that nanovesicles loaded with TLR4 agonists were locally enriched in tumor tissues, but not distributed in other organs throughout the body ( Fig.14 ). Therefore, the cell membrane-coated nanovesicles have a good tumor targeting effect in vivo, avoiding the systemic organ toxicity caused by the "off-target effect".

[0054] After tail vein injection of the untreated group, TLR4 agonist group, and TLR4 agonist-loaded nanovesicle group, the volume of tumor tissue was continuously detected and calculated. The results showed that the growth of tumor tissue in the TLR4 agonist-loaded nanovesicle group was significantly inhibited, and the tumor volume was the smallest compared with the untreated group and TLR4 agonist group, and was most significant on the 21st day ( Fig.15 ).

[0055] Subcutaneous transplanted tumors were sectioned and stained. Compared with the untreated group and TLR4 agonist group, the fluorescence of M1 macrophage marker (iNOS) in the TLR4 agonist-loaded nanovesicle group was significantly enhanced ( Fig.16 and 17 ), while the fluorescence of M2 macrophage marker (CD163) decreased significantly ( Fig.16 and 18 ). Therefore, based on the good in vivo tumor targeting effect, the nanovesicles can target the delivery of TLR4 agonists to tumor tissues in vivo, regulate the polarization of tumor-associated macrophages, and reshape the tumor immunosuppressive microenvironment, thereby achieving the purpose of significantly killing osteosarcoma ( Fig.19 ).

Claims

1. A method for preparing cell membrane-coated drug-loaded vesicles, characterized in that: After the M1 macrophage membrane solution was co-incubated with the TLR4 agonist RS09, it was extruded back and forth 10 to 20 times using a 200 nm polycarbonate membrane to obtain cell membrane-coated nanovesicles loaded with the TLR4 agonist. The co-incubation was performed with 0.05 mg of the TLR4 agonist and 0.1 mg of the macrophage membrane solution.

2. The method for preparing a cell membrane-coated drug-loaded vesicle according to claim 1, characterized in that: The method for preparing the macrophage membrane solution is as follows: LPS is used to stimulate RAW264.7 macrophages to differentiate into M1 type, and 1×10 8 The M1 macrophages were washed three times with PBS, resuspended with hypotonic cell lysis buffer at 4°C, and placed on ice for 15 minutes. The resuspended solution was disrupted with an ultrasonic disruptor for 10 minutes, and the obtained solution was purified by differential centrifugation to obtain a macrophage membrane solution.

3. The method for preparing a cell membrane-coated drug-loaded vesicle according to claim 2, characterized in that: The differential centrifugation purification includes centrifuging at 700 g for 10 min to obtain a supernatant; and centrifuging at 15,000 g for 30 min to obtain a precipitate to purify the membrane.

4. The method for preparing a cell membrane-coated drug-loaded vesicle according to claim 2, characterized in that: The macrophage RAW264.7 cells were cultured in a high-glucose DMEM medium containing 10% fetal bovine serum in a 37° C., 5% CO 2 incubator.

5. A cell membrane drug-loaded vesicle prepared according to the preparation method according to any one of claims 1 to 4.

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