In-vivo editing and metabolism reprogramming nano drug-loaded material for targeting macrophages as well as preparation method and application of in-vivo editing and metabolism reprogramming nano drug-loaded material

Abstract

The invention relates to the technical field of biology, in particular to an in-vivo editing and metabolism reprogramming nano drug-loaded material for targeting macrophages as well as a preparation method and application of the in-vivo editing and metabolism reprogramming nano drug-loaded material. On the basis of a metal organic framework (MOF) material, CAR mRNA molecules targeting DLL3 are loaded in the MOF material, a macrophage-derived cell membrane is coated outside the MOF material, and the nano drug-loading material with the macrophage targeting capability is constructed. The material has the effects of specifically swallowing and killing tumor cells, improves the immunosuppression effect of a tumor microenvironment by utilizing the immune activation effects of succinic acid and manganese, and synergistically promotes the killing effect on small cell lung cancer. The macrophage in-vivo editing technology designed by the invention is a novel safe, efficient, simple and convenient tumor immunotherapy technology, overcomes the defect that CAR-T cannot enter the interior of a solid tumor, and solves the problem of tedious cell in-vitro amplification and gene editing procedures in the conventional CAR immunotherapy process; and a new strategy is provided for immunotherapy of small cell lung cancer with limited treatment means.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a nano-drug delivery material for in vivo editing and metabolic reprogramming targeting macrophages, and a preparation method and application thereof. Background Art

[0002] Lung cancer is the leading cause of cancer death globally, and small cell lung cancer (SCLC) accounts for approximately 15% of all lung cancer types. SCLC is an aggressive, high-grade neuroendocrine carcinoma with a very poor prognosis, a 5-year survival rate of less than 7%, and the characteristic of early metastasis and spread. Compared with non-small cell lung cancer, SCLC lacks effective targeted therapeutic drugs. DLL3 is highly selectively expressed on the surface of SCLC cells and rarely expressed in normal lung tissue, making DLL3 a potential specific target for treating small cell lung cancer.

[0003] Currently, chimeric antigen receptor-T cell (CAR-T) is the mainstream of tumor immunocyte therapy and has shown potential application value in clinical treatments such as hematological tumors. However, it has disadvantages such as weak ability to penetrate solid tissues and easy exhaustion, thus limiting the therapeutic effect in solid tumors. Macrophages account for an important proportion in the tumor microenvironment and have superior tissue penetration, chemotactic ability, and phagocytic and killing functions. In addition, macrophages have the functions of antigen presentation and activation of T cells, and can play an immune activation role by secreting cytokines and chemokines. Therefore, macrophages have become a new force in the treatment of solid tumors. By constructing chimeric antigen receptor-macrophage (CAR-M) to infiltrate into solid tumors, they can play a role in specifically targeting and killing antigen-specific solid tumors and regulating the immune microenvironment, and are expected to achieve the goal of inhibiting tumor development and removing tumors.

[0004] However, currently, cell therapies such as CAR-T have complex processes, requiring in vitro isolation and enrichment of immune cells and transfection of the CAR gene using viral or non-viral vectors, etc. They have high technical requirements, high costs, and there are pollution and safety hazards. In Vivo CAR Editing directly achieves gene editing of immune cells in patients through vector-targeted delivery. This treatment method provides a new way to directly endow immune effector cells with tumor-targeted recognition and killing, simplifies the manufacturing process, is expected to solve the problems of standardization and large-scale production of in vitro CAR-M cell manufacturing processes, and is expected to overcome the limitations of traditional CAR-T cell therapies.

[0005] Based on the above background, the present invention aims to provide a new technology for tumor immunotherapy that is safe, efficient, and simple, with the expectation of providing a new strategy for the immunotherapy of small cell lung cancer with limited treatment options. Summary of the Invention

[0006] The first technical problem to be solved by the present invention is to provide a nano-drug delivery material with macrophage targeting, the ability to reshape the tumor microenvironment, and the ability to synergistically enhance the tumor treatment effect, as well as its preparation method and application, aiming at the deficiencies of CAR technology in tumor immunotherapy.

[0007] The second technical problem to be solved by the present invention is to provide a preparation method for the above nano-drug delivery material.

[0008] The third technical problem to be solved by the present invention is to provide an in vivo editing and DLL3-targeting delivery system.

[0009] The fourth technical problem to be solved by the present invention is to provide the application of the above nano-drug delivery material or the nano-drug delivery material prepared by the above preparation method in the preparation of drugs targeting small cell lung cancer.

[0010] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0011] In the first aspect, the present invention provides a nano-drug delivery material for in vivo editing and metabolic reprogramming targeting macrophages, which comprises manganese succinate nanoparticles loaded with DLL3-mRNA and macrophage cell membranes coated on the surface of the manganese succinate nanoparticles.

[0012] Among them, the DLL3-mRNA is a chimeric antigen receptor-encoding mRNA targeting DLL3, and its sequence is as shown in SEQ ID NO.1.

[0013] Among them, the nano-drug delivery material has a mesoporous spherical structure, and it is measured that its particle size is 200-300 nm.

[0014] In the second aspect, the present invention provides a preparation method for the nano-drug delivery material, comprising the following steps:

[0015] (1) Adding sodium succinate crystals and manganese chloride tetrahydrate to an ethanol aqueous solution with a concentration of 5-40%, mixing evenly, standing, and centrifuging to obtain a white precipitate. After washing the white precipitate, freeze-drying is carried out to obtain manganese succinate nanoparticles SaMn;

[0016] (2) Transcribing the plasmid carrying DLL3 in vitro to obtain DLL3-mRNA, and infiltrating the DLL3-mRNA into the manganese succinate nanoparticles SaMn obtained in step (1) by a one-pot method to obtain manganese succinate nanoparticles loaded with DLL3-mRNA DLL3-mRNA@SaMn;

[0017] (3) Mix the manganese succinate nanoparticles DLL3-mRNA@SaMn loaded with DLL3-mRNA obtained in step (2) with macrophage membranes, and obtain a homogeneous solution through ultrasonic treatment and extrusion treatment. Then, centrifuge the homogeneous solution to remove the supernatant to obtain the coated manganese succinate nanoparticles DLL3-mRNA@SaMn@Mm loaded with DLL3-mRNA, which is the nano drug delivery material.

[0018] Among them, in step (1), the sodium succinate crystals are obtained by dissolving succinic acid and sodium hydroxide in deionized water, forming a supersaturated solution by evaporation (>80% moisture) at room temperature, and then precipitating.

[0019] Among them, in step (1), the mass ratio of the sodium succinate crystals to manganese chloride tetrahydrate is 0.5-1:1.

[0020] Among them, in step (1), the standing condition is: standing at room temperature for 20-40 min; the centrifugation condition is: centrifuging at 8000-12000 rpm for 5-15 min.

[0021] Among them, in step (2), the plasmid carrying DLL3 is transcribed in vitro to obtain DLL3-mRNA. The specific process is as follows: First, clone the DLL3 CAR gene from the pLVX-EGFP-F2A-DLL3 CAR plasmid by means of molecular cloning technology, transfer the gene into the GS-mK-100A plasmid by means of enzyme digestion and ligation, and identify the correctness of the DLL3 CAR gene by enzyme digestion and sequencing; then linearize the GS-mK-100A plasmid constructed with the DLL3-CAR molecule with BspQI enzyme, and synthesize DLL3-mRNA by in vitro transcription using an in vitro transcription kit.

[0022] Among them, in step (2), the mass ratio of the DLL3-mRNA to the manganese succinate nanoparticles SaMn is 1.2-1.5:100.

[0023] Among them, in step (3), the macrophage membranes are obtained by inducing monocytes derived from the human leukemia cell line with PMA; preferably, the macrophage membranes are derived from THP1 monocytes and induced into macrophages with PMA.

[0024] Among them, in step (3), the mass ratio of the manganese succinate nanoparticles DLL3-mRNA@SaMn loaded with DLL3-mRNA to the macrophage membranes is 1:0.5-1.5; in some embodiments of the present invention, the mass ratio is 1:1.

[0025] Specifically, the DLL3-mRNA@SaMn@Mm nano-drug delivery material is taken up by macrophages through cell membrane homologous targeting and delivers DLL3-mRNA into macrophages. Macrophages in the tumor microenvironment are transformed into CAR-M cells by in vivo editing of DLL3-mRNA@SaMn@Mm, and perform phagocytosis and killing functions in a manner specific to targeting tumor cells, further regulating immune cells such as T and NK cells. Through the effective penetration of CAR-M and regulation of the tumor microenvironment, it promotes the transformation of the tumor immune microenvironment from an anti-inflammatory direction to a pro-inflammatory direction, thereby effectively inhibiting tumor growth.

[0026] Specifically, the DLL3-mRNA@SaMn@Mm nano-drug delivery material has pH responsiveness. Under acidic conditions (pH = 5.7), it has the fastest mRNA release behavior, with the drug release reaching 43% at 6 hours, 76% at 48 hours, and the final release amount being approximately 81% at 72 hours.

[0027] Specifically, macrophages have a high uptake efficiency for the DLL3-mRNA@SaMn@Mm nano-drug delivery material. When incubated for 8 hours, the DLL3-mRNA@SaMn@Mm nano-drug delivery material can be efficiently taken up by macrophages.

[0028] In a third aspect, the present invention provides an in vivo editing and DLL3-targeting delivery system.

[0029] Specifically, the in vivo editing and DLL3-targeting delivery system contains the above-mentioned nano-drug delivery material.

[0030] In a fourth aspect, the present invention provides the use of the above-mentioned nano-drug delivery material or the nano-drug delivery material prepared by the above-mentioned preparation method in the preparation of drugs targeting small cell lung cancer.

[0031] Specifically, the small cell lung cancer is small cell lung cancer with high expression of DLL3.

[0032] Specifically, the DLL3-mRNA@SaMn@Mm nano-drug delivery material can effectively improve the tumor microenvironment and treat small cell lung cancer in tumor-bearing mice with small cell lung cancer.

[0033] Beneficial effects:

[0034] (1) The present invention targets small cell lung cancer, takes DLL3 as a specific target, designs a macrophage-targeted mRNA delivery and in vivo editing strategy based on MOF nanomaterials, and endows monocytes / macrophages with specific phagocytosis and killing effects on tumor cells by using chimeric antigen receptors, solving the drawback that CAR-T cannot enter the interior of solid tumors, and at the same time solving the cumbersome in vitro cell expansion and gene editing procedures in the conventional CAR immunotherapy process; and through the synergistic delivery effect of succinic acid and manganese ions, activates the immune activation signal pathway, relieves the inhibitory effect of the immune microenvironment, so as to enhance the safety, effectiveness and specificity of CAR cell therapy, and brings a new potential treatment plan for small cell lung cancer patients with poor traditional treatment effects.

[0035] (2) The present invention combines the immune metabolic regulation advantage of manganese succinate nanomaterials with the tumor targeting ability endowed by DLL3-mRNA, and designs the following nano-drug-loading material for treating small cell lung cancer: First, transcribe DLL3-mRNA in vitro and load it into SaMn nanoparticles by a one-pot method, and after multiple ultrasonic extrusions with cell membranes, the coated DLL3-mRNA@SaMn@Mm nanoparticles are obtained. This nano-drug-loading material enters macrophages through homologous targeting and releases mRNA, realizing in vivo editing of macrophages, obtaining CAR-M cells with the activity of phagocytosing and killing tumor cells, and at the same time strengthening the anti-tumor activity of CAR-M cells by improving the tumor microenvironment, thereby realizing targeted treatment of small cell lung cancer.

[0036] (3) The present invention provides a safe, efficient and simple new technology for tumor immunotherapy, providing a new strategy for the immunotherapy of small cell lung cancer with limited treatment means. The present invention helps to analyze and explore new targets and new mechanisms of small cell lung cancer, and provides new methods and new technologies for the development of biological agents for treating tumors and other related diseases. Brief Description of the Drawings

[0037] The following further specifically describes the present invention in conjunction with the drawings, and the above and / or other advantages of the present invention will become clearer.

[0038] Figure 1 Scanning electron microscope images, particle size and potential analysis diagrams of the constructed nano-drug-loading material. Among them, a is the scanning electron microscope image of SaMn@Mm; b is the dynamic light scattering particle size distribution diagram of SaMn, Mm, and SaMn@Mm; c is the potential analysis diagram of SaMn, Mm, and SaMn@Mm.

[0039] Figure 2 Responsive in vitro drug release diagram of mRNA in the DLL3-mRNA@SaMn@Mm nano-drug-loading material.

[0040] Figure 3 Evaluation of the uptake efficiency of DLL3-mRNA@SaMn@Mm nanomedicine by macrophages. Among them, a is the fluorescence microscopy images at different time points (2h, 4h, 8h, 12h); b is the distribution of intracellular fluorescence intensity at different time points; c is the proportion of positive cells at different time points.

[0041] Figure 4 Construction process of the tumor-bearing model of small cell lung cancer-bearing mice, and the tumor volume and body weight of small cell lung cancer-bearing mice after treatment with nanomedicine. Among them, a is the diagram of the tumor-bearing model construction process; b is the physical map of the tumor volume of different experimental groups; c is the line graph of the body weight change of mice in different experimental groups; d is the graph of the tumor volume change of different experimental groups.

[0042] Figure 5 HE staining results of tumors of small cell lung cancer-bearing mice after treatment with DLL3-mRNA@SaMn@Mm nanomedicine.

[0043] Figure 6 HE staining of organ sections of small cell lung cancer-bearing mice after treatment with DLL3-mRNA@SaMn@Mm nanomedicine.

[0044] Figure 7 Schematic diagram of the remodeling of the tumor immune microenvironment and targeted therapy of small cell lung cancer by in vivo editing and metabolic reprogramming nanomedicine of macrophages. Detailed implementation manners

[0045] The following further specific descriptions of the present invention are made in conjunction with the detailed implementation manners, and the above and / or other advantages of the present invention will become clearer.

[0046] In the following examples, the experimental methods are all conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0047] In the following examples, the GS-mK-100A plasmid was purchased from GenScript Biotech Corporation.

[0048] Example 1 Preparation of coated manganese succinate nanoparticles (abbreviation: SaMn@Mn)

[0049] 1. Preparation of sodium succinate crystals

[0050] In a 10 cm glass petri dish, 192 mg of succinic acid and 136 mg of sodium hydroxide were quickly dissolved in 16 mL of deionized water, and then a supersaturated solution was formed by evaporation (>80% water) at room temperature to promote the precipitation of sodium succinate crystals.

[0051] 2. Preparation of SaMn composite

[0052] Weigh 24.1 mg of the obtained succinate sodium crystals and add them to 1.0 mL of deionized water. Weigh 27.84 mg of manganese chloride tetrahydrate and add it to 1 mL of an aqueous ethanol solution with a concentration of 25%. Vigorously shake and mix well. After mixing these two mixed solutions, let them stand at room temperature for 30 minutes, and white precipitation can be observed. Collect the white precipitate, centrifuge at 10000 rpm for 10 minutes, and then wash it with a large amount of deionized water to remove excess impurities, obtaining the SaMn composite.

[0053] 3. Preparation of manganese succinate nanoparticles

[0054] Take the above-prepared SaMn composite, resuspend and dilute it with 1 mL of deionized water, and freeze-dry the solution in a freeze dryer for 1 day to obtain manganese succinate nanoparticles, abbreviated as SaMn.

[0055] 4. Extraction of macrophage cell membrane (Mm)

[0056] Induce THP-1 cells with 100 ng / mL of PMA for 48 hours to differentiate them into macrophages, then centrifuge to collect 1×10 7 cells and suspend them in 1 mL of lysis buffer to obtain a cell suspension. Use an ultrasonic instrument to ultrasonically lyse the cell suspension at 600 W for 15 minutes. Centrifuge the ultrasonically treated cell suspension at 4 °C and 800 g for 10 min, discard the precipitate to remove nuclear components and unbroken cells. Then use an ultracentrifuge to centrifuge at 100,000 g for 60 min to obtain a macrophage cell membrane precipitate. Freeze-dry the macrophage cell membrane precipitate, use the BCA method to measure the protein concentration, and after quantifying the protein concentration, store it in a -80 °C refrigerator for later use.

[0057] Among them, the lysis buffer has the following components: 0.5% (w / v) BSA, 225 mM mannitol, 30 mM Tris-HCl, 0.5 mM EDTA, 75 mM sucrose, protease inhibitor (20 μL / 4 mL, purchased from Beyotime Biotechnology Co., Ltd.) and 1% (v / v) phosphatase inhibitor mixture (pH 7.4, purchased from Beyotime Biotechnology Co., Ltd.).

[0058] 5. Preparation of coated manganese succinate nanoparticles (abbreviated as SaMn@Mn)

[0059] Mix 1 mg of the manganese succinate nanoparticles (SaMn) obtained in Step 3 and 1 mg of the macrophage cell membrane derived from THP-1 cells obtained in Step 4, and ultrasonicate the mixture in a water bath at 26 °C for 3 min. After the ultrasonication, use an Avanti mini extruder to extrude the above mixture through a 200-nm polycarbonate porous membrane, and repeat the extrusion 40 times to obtain a homogeneous solution. Further centrifuge the homogeneous solution at 100,000 g for 60 min, and the obtained precipitate is the manganese succinate nanoparticles coated with a membrane (abbreviated as SaMn@Mm), and this nanoparticle can be used as a nano drug delivery material.

[0060] Example 2: Characterization of manganese succinate nanoparticles coated with a membrane (abbreviated as SaMn@Mn)

[0061] Resuspend SaMn@Mm in PBS buffer for observing the morphology and particle size of the nanoparticles. Use SaMn and Mn as controls.

[0062] 1. Observe the morphology of SaMn@Mm by scanning electron microscopy (SEM)

[0063] Place the powder prepared by freeze-drying SaMn@Mm on the conductive adhesive, and observe its morphology and particle size using a scanning electron microscope. The results are shown as a in Figure 1 It can be seen from the figure that the SaMn@Mn synthesized based on the method of Example 1 is spherical and has a uniform particle size.

[0064] 2. Measure the particle size distribution and Zeta potential using a Malvern nano particle size analyzer

[0065] Resuspend SaMn@Mm in PBS buffer (pH 7.4) to obtain a resuspended solution, and use SaMn and Mn as controls. Take 1 mL of the resuspended solution and add it to the sample cell of the Malvern nano particle size analyzer to measure its particle size distribution and Zeta potential.

[0066] Figure 1 b in is the dynamic light scattering particle size distribution diagram, and its particle size is 231.53 ± 46.3 nm. It can be seen from the figure that the particle size distributions of SaMn (blue curve) and Mm (red curve) are relatively wide, indicating that their particle size distributions are not uniform; the particle size distribution of SaMn@Mm (green curve) is relatively narrow, indicating that its particle size is more uniform. This shows that the coating process helps to control the size of the nanoparticles. Figure 1 c in shows the Zeta potential of the three samples (SaMn, Mm, SaMn@Mm). It can be seen that the Zeta potential value of SaMn@Mm is lower, indicating that the surface charge property of the nanoparticles has changed after coating.

[0067] Example 3: Preparation of DLL3-mRNA@SaMn@Mm nano drug delivery material

[0068] 1. In vitro transcription synthesis of DLL3-mRNA

[0069] First, use molecular cloning techniques to clone the DLL3 CAR gene from the pLVX-EGFP-F2A-DLL3 CAR plasmid autonomously constructed in the early stage of the laboratory. Transfer the gene into the GS-mK-100A plasmid by enzymatic digestion and ligation, and identify the correctness of the DLL3 CAR gene by enzymatic digestion and sequencing. Then, linearize the GS-mK-100A plasmid containing the DLL3-CAR molecule with BspQI, observe the success of linearization by agarose gel electrophoresis, and after success, purify the linearized DNA using a DNA gel extraction kit. Use the purified DNA as a template and synthesize DLL3-mRNA by in vitro transcription using the T7 High Yield RNA Transcription Kit transcription kit. The DLL3-mRNA is the chimeric antigen receptor-encoding mRNA targeting DLL3, and its sequence is shown in SEQ ID NO.1.

[0070] 2. Preparation of DLL3-mRNA@SaMn@Mm

[0071] (1) Preparation of DLL3-mRNA@SaMn

[0072] Detect the concentration of DLL3-mRNA by Nanodrop 2000 and calculate the DLL3-mRNA synthesized by in vitro transcription. Stir and synthesize 12 - 15 μg of DLL3-mRNA with 1 mg of the manganese succinate nanoparticles SaMn prepared in Example 1 using the one-pot method, that is, infiltrate the in vitro transcribed DLL3-mRNA solution into the manganese succinate nanoparticles SaMn prepared in Example 1 to obtain the manganese succinate nanoparticles DLL3-mRNA@SaMn loaded with DLL3-mRNA.

[0073] (2) Preparation of DLL3-mRNA@SaMn@Mm

[0074] To modify the cell membrane of the DLL3-mRNA@SaMn nanoparticles, ultrasonically extrude the DLL3-mRNA@SaMn nanoparticles and macrophage cell membranes (Mm) to obtain the coated DLL3-mRNA@SaMn@Mm nanoparticles. The process of cell membrane modification is the same as the preparation process of the coated manganese succinate nanoparticles (abbreviated as SaMn@Mn) in Example 1.

[0075] Example 4: In vitro responsive drug release of the DLL3-mRNA@SaMn@Mm nano-drug delivery material

[0076] 100 μL of DLL3-mRNA@SaMn@Mm aqueous solution was diluted in PBS solution with pH=7.4 or pH=5.7, and the supernatant was taken at 3, 6, 12, 24, 48, and 72 hours, respectively. The mRNA concentration was detected using an ultra-micro nucleic acid detector, and the mRNA release efficiency of each group was calculated.

[0077] like Figure 2 The results showed that under neutral conditions (pH = 7.4), mRNA can be slowly released from DLL3-mRNA@SaMn@Mm, and the total release amount in 72 hours is only about 19%; under acidic conditions (pH = 5.7), it can be observed that DLL3-mRNA@SaMn@Mm has the fastest mRNA release behavior, and the drug release reaches 43% at 6 hours, 76% at 48 hours, and the final release amount in 72 hours is about 81%. This shows that DLL3-mRNA@SaMn@Mm is pH responsive.

[0078] Example 5: Evaluation of the uptake efficiency of DLL3-mRNA@SaMn@Mm nanomaterials by macrophages

[0079] 3×10 5 THP-1 cells induced by PMA were inoculated in confocal microscopy and incubated with DLL3-mRNA@SaMn@Mm for 2, 4, 8, and 12 hours, with SaMn@Mm as a control. After the incubation, the cells were fixed with 4% paraformaldehyde, and the difference in the cell uptake efficiency of DLL3-mRNA@SaMn@Mm nanoparticles was observed by confocal microscopy.

[0080] like Figure 3 As shown in a, the FITC fluorescence intensity in the cells gradually increased with time, and reached the highest fluorescence intensity at 8 hours. Figure 3 As shown in b, as time goes by, the distribution of intracellular fluorescence intensity moves to the right. At 8 and 12 hours, the fluorescence intensity distribution is obviously to the right. Figure 3 As shown in c, the proportion of positive cells gradually increased with the increase of time, and at 8 and 12 hours, the proportion of positive cells increased significantly. The above results show that the uptake of DLL3-mRNA@SaMn@Mm increased, indicating that THP-1 cells have a high uptake efficiency of DLL3-mRNA@SaMn@Mm, and can be efficiently taken up by macrophages when incubated for 8 hours. Example 6: Evaluation of the therapeutic effect of DLL3-mRNA@SaMn@Mm nano-drug carrier material in a small cell lung cancer tumor-bearing mouse model

[0081] 1. Construction and treatment of subcutaneous tumor model

[0082] Nude mice aged 6 - 8 weeks and weighing 18 - 22 g (purchased from Hangsi Biotechnology Co., Ltd.) were randomly divided into 5 groups, with 6 mice in each group, namely: (1) PBS group; (2) αPD-1 group; (3) SaMn@Mm group; (4) DLL3-mRNA@SaMn@Mm group; (5) DLL3-mRNA@SaMn@Mm + αPD-1 group. According to Figure 4 , each mouse was injected subcutaneously with 5×10 6 H146 cells to establish a tumor-bearing model according to the procedure of a in 3 . When the tumor volume increased to 50 mm 3 , unified drug administration was carried out once every three days for a total of four injections. The drug administration method for all groups was intravenous injection through the tail vein. Starting from the first drug administration, the tumor volume of the mice was measured every two days, and the body weight of the mice was measured every three days. The mice were sacrificed after 30 days.

[0083] The tumor tissues of mice in different groups were collected, fixed with 4% paraformaldehyde (Beyotime, P0099), embedded in paraffin and sectioned after 24 h, and stained with H&E to observe the pathological conditions of the tumor sections in each group. As shown in b in Figure 4 , in the single-drug treatment groups (αPD-1, SaMn@Mm, DLL3-mRNA@SaMn@Mm) and the combination drug treatment group (DLL3-mRNA@SaMn@Mm + αPD-1), the tumor volume decreased significantly, while the body weight of the tumor-bearing mice did not change significantly ( Figure 4 c in Figure 4 and d in 4), indicating that with combination treatment, the therapeutic effect was further enhanced and the tumor volume decreased significantly. Figure 5 The HE staining results showed that in the mice treated with the αPD-1 group, SaMn@Mm group, DLL3-mRNA@SaMn@Mm group and DLL3-mRNA@SaMn@Mm + αPD-1 group, compared with the Ctrl group (i.e., the PBS group), the degree of tumor cell apoptosis was significantly enhanced, indicating that the therapeutic effect of the drug on the tumor gradually increased. In addition, the DLL3-mRNA@SaMn@Mm combined with αPD-1 group also showed the best anti-tumor effect.

[0084] 2. HE staining of each organ after treatment

[0085] The liver, spleen, lung and kidney organs of the tumor-bearing mice collected were fixed with 4% paraformaldehyde (Beyotime, P0099), embedded in paraffin and sectioned after 24 h, and stained with H&E. The results are as shown in Figure 6As shown, compared with the Ctrl group (i.e., the PBS group), the alveolar structure of other groups was intact, without inflammatory cell infiltration; there was no inflammatory cell infiltration in the liver; there was no vacuolar degeneration in the renal tubules, the white pulp of the spleen was intact, the splenic corpuscles had clear structures, and there was no obvious biological toxicity. Therefore, the above results indicate that SaMn@Mm, DLL3-mRNA@SaMn@Mm, and DLL3-mRNA@SaMn@Mm+αPD-1 developed in the present invention all have good biosafety.

[0086] Therefore, based on the metal-organic framework material SaMn loaded with DLL3-mRNA and macrophage-derived macrophage membranes, through means such as film coating and bioconjugation, a multifunctional targeted nanodelivery system with the functions of regulating the tumor immune microenvironment, in vivo editing of macrophages, and targeted delivery of DLL3-mRNA was prepared. The nanomaterial constructed in the present invention can target macrophages after tail vein injection, kill tumor cells by phagocytosing DLL3-mRNA, polarize M2 cells in the tumor microenvironment into M1-like cells, effectively improve the tumor microenvironment, and treat small cell lung cancer. Figure 7 Schematic diagram of the in vivo editing and metabolic reprogramming of macrophages by the nano-drug delivery material to achieve the remodeling of the tumor immune microenvironment and targeted treatment of small cell lung cancer.

[0087] In summary, the metal-organic framework material SaMn MOF and the macrophage-derived macrophage membrane nano-targeted delivery system constructed in the present invention can effectively enhance the in vivo delivery of DLL3-mRNA and in vivo macrophage editing, and have good therapeutic effects on small cell lung cancer.

[0088] The present invention provides an idea and method for an in vivo editing and metabolic reprogramming nano-drug delivery material targeting macrophages, its preparation method and application. There are many specific methods and ways to implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be realized by existing technologies.

Claims

1. A nano drug-carrying material for in vivo editing and metabolic reprogramming targeting macrophages, characterized in that: The nano drug-carrying material comprises manganese succinate nanoparticles loaded with DLL3-mRNA and macrophage membranes coated on the surface of the manganese succinate nanoparticles.

2. The nano drug-carrying material according to claim 1, characterized in that: The DLL3-mRNA is an mRNA encoding a chimeric antigen receptor targeting DLL3, and its sequence is shown in SEQ ID NO.

1.

3. The nano drug-carrying material according to claim 1, characterized in that: The nano drug-carrying material is a mesoporous spherical structure with a particle size of 200-300nm.

4. The method for preparing the nano drug-carrying material according to claim 1, characterized in that: The steps include: (1) adding sodium succinate crystals and manganese chloride tetrahydrate to an ethanol aqueous solution with a concentration of 5 to 40%, mixing, standing, and centrifuging to obtain a white precipitate, washing the white precipitate, and freeze-drying to obtain manganese succinate nanoparticles SaMn; (2) in vitro transcription of the plasmid carrying DLL3 to obtain DLL3-mRNA, and infiltration of the DLL3-mRNA into the manganese succinate nanoparticles SaMn obtained in step (1) using a one-pot method to obtain manganese succinate nanoparticles DLL3-mRNA@SaMn loaded with DLL3-mRNA; (3) The manganese succinate nanoparticles DLL3-mRNA@SaMn loaded with DLL3-mRNA obtained in step (2) are mixed with macrophage membranes, and a homogenous solution is obtained by ultrasonic and extrusion treatment. The homogenous solution is then centrifuged to remove the supernatant to obtain the coated manganese succinate nanoparticles DLL3-mRNA@SaMn@Mm loaded with DLL3-mRNA, i.e., the nano drug delivery material.

5. The preparation method according to claim 3, characterized in that: In step (1), the mass ratio of the sodium succinate crystals to manganese chloride tetrahydrate is 0.5 to 1:

1.

6. The preparation method according to claim 3, characterized in that: In step (2), the mass ratio of the DLL3-mRNA to the manganese succinate nanoparticles SaMn is 1.2-1.5:

100.

7. The preparation method according to claim 3, characterized in that: In step (3), the mass ratio of the manganese succinate nanoparticles DLL3-mRNA@SaMn loaded with DLL3-mRNA to the macrophage membrane is 1:0.5-1.5; the macrophage membrane is obtained by PMA-induced treatment of monocytes derived from a human leukemia cell line.

8. An in vivo editing and targeting DLL3 delivery system, characterized in that Contains the nano drug-carrying material according to any one of claims 1 to 3.

9. Use of the nano drug-carrying material according to any one of claims 1 to 3 or the nano drug-carrying material prepared by the preparation method according to any one of claims 4 to 7 in the preparation of drugs targeting small cell lung cancer.

10. The use according to claim 9, characterized in that: The small cell lung cancer is a small cell lung cancer with high expression of DLL3.

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