Macrophage-loaded nanoparticles and their preparation method and application

Abstract

A macrophage-loaded drug-loaded nanoparticle, its preparation method, and its application belong to the field of drug-targeted carrier technology. To address the problem that the therapeutic efficacy of drugs such as lenvatinib for HCC after minimally invasive ablation is limited by their nonspecific accumulation and distribution in the body, the LEN-loaded MΦs prepared by the present invention are constructed by engulfing Escherichia coli membrane-coated LEN-loaded nanoparticles by natural M1-type MΦs. The E. coli membrane camouflage enhances the phagocytic efficiency of MΦs, prevents the leakage of LEN within the MΦs, and maintains the M1 type of LEN@MΦs within the immunosuppressive tumor microenvironment. The natural inflammatory gradient induced by ablation is used to guide LEN-loaded macrophages to target tumors, increasing the in vivo delivery efficiency of LEN in postoperative HCC by 10-fold. This method significantly inhibits tumor cell proliferation and angiogenesis, and this enhanced LEN delivery stimulates systemic immune responses and induces long-lasting immune memory.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug targeting carriers, and in particular relates to a macrophage-loaded nano drug-loaded particle and a preparation method and application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) accounts for approximately 90% of all liver cancer cases and is the fourth most lethal cancer in clinical oncology. Multifocal HCC, in particular, is characterized by its aggressive nature and poor prognosis. Local therapies, including minimally invasive ablation, play a key role in treating 50%-60% of HCC cases (Han S, Bao X, Zou Y, et al. d-lactate modulates M2tumor-associated macrophages and remodels immunosuppressive tumor microenvironment for hepatocellular carcinoma. Science Advances 2023;9:eadg2697.). However, for patients with multifocal HCC who have more than three tumor nodules, minimally invasive ablation often carries the risk of inadequate ablation, leading to a 5-year recurrence rate of up to 52% (Zeng X, Liao G, Li S, et al. Eliminating METTL1-mediated accumulation of PMN-MDSCs prevents hepatocellular carcinoma recurrence after radiofrequency ablation. Hepatology 2023;77:1122-1138.). The persistence of residual tumor cells can induce immunosuppression, accelerate tumor progression, and affect patient prognosis (Huang Z, Guo Z, Ni J, et al. Four types of tumor progression after microwaveablation of single hepatocellular carcinoma of ≤5 cm: incidence, risk factors and clinical significance. International journal of Hyperthermia: The Official Journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group 2021;38:1164–1173.).In clinical practice, patients with advanced multifocal HCC can receive conventional systemic therapy after local treatment (Ogasawara S, Ooka Y, Koroki K, et al. Switching to systemic therapy after locoregional treatment failure: Definition and best timing. Clinical and Molecular Hepatology 2020;26:155–162.), such as lenvatinib, sorafenib, and regorafenib, especially lenvatinib (LEN). LEN is a multikinase inhibitor that primarily inhibits tumor proliferation and angiogenesis and has been used as a first-line treatment. Although LEN is effective in some patients with advanced multifocal HCC (~24.1%), its therapeutic effect on HCC after minimally invasive ablation is limited by its nonspecific accumulation and distribution in the body (Yang C, Zhang H, Zhang L, et al. Evolving therapeutic landscape of advanced hepatocellular carcinoma. Nature Reviews Gastroenterology & Hepatology 2023;20:203–222.). Therefore, more advanced therapeutic drug delivery strategies are urgently needed.

[0003] Macrophages (MΦs) have attracted attention in precision medicine for their ability to load drugs and target tumors and inflamed tissues. Numerous MΦ-based delivery systems have recently been developed based on the ability of monocytes to act as hitchhikers on MΦ particles (Hou J, Yang X, Li S, et al. Accessing neuroinflammation sites: Monocyte / neutrophil-mediated drug delivery for cerebral ischemia. Science Advances 2019;5:eaau8301.). These MΦ particles also exhibit chemotactic motility along gradients of inflammatory factors (Wang S, Li F, Ye T, et al. Macrophage-tumor chimeric exosomes accumulate in lymph nodes and tumors to activate the immune response and the tumor microenvironment. Science Translational Medicine 2021;13:eabb6981.). Although considerable progress has been made in the use of innate immune cells for biomedicine, there has been no report combining this MΦ hitchhiking strategy with minimally invasive ablation to target HCC (Targeting drugs to tumours using cellmembrane-coated nanoparticles. Nature Reviews Clinical Oncology 2023;20:33–48.). Summary of the Invention

[0004] In order to solve the problem that the therapeutic effect of lenvatinib and other systemic therapeutic drugs suitable for local treatment of patients with advanced multifocal HCC after minimally invasive ablation is limited by their nonspecific accumulation and distribution in the body, the present invention provides a nanoparticle loaded with drug by macrophages and a more efficient drug delivery strategy based on the nanoparticle for the prognosis of patients with advanced multifocal HCC after local treatment.

[0005] In order to solve the above technical problems, the present invention specifically provides the following technical solutions:

[0006] The first object of the present invention is to provide a macrophage-loaded nanoparticle, wherein the nanoparticle comprises Escherichia coli vesicles and a small molecule active ingredient of a drug encapsulated in the Escherichia coli vesicles. The nanoparticle is obtained by incubating M1 macrophages with Escherichia coli vesicles encapsulated with the small molecule active ingredient of the drug; the small molecule active ingredient of the drug is any one of lenvatinib, sorafenib, and regorafenib.

[0007] Lenvatinib, sorafenib, and regorafenib are all drugs that can be used for systemic treatment of patients with advanced multifocal HCC after local treatment. They are all hydrophobic drugs. Based on the properties of the drugs and PLGA, they can be prepared into macrophage-loaded nanoparticles using the preparation method of the nanoparticles described in the present invention, and used to treat hepatocellular carcinoma and improve the prognosis of hepatocellular carcinoma after ablation.

[0008] A second object of the present invention is to provide a method for preparing the above-mentioned drug-loaded nanoparticles, the method comprising the following steps:

[0009] S1. Preparation of nanoparticles containing small molecule active ingredients of drugs;

[0010] S2. Mix the E. coli vesicles with the nanoparticles obtained in S1, and extrude them through a polycarbonate membrane with a pore size of 200 nm for at least 21 times (the number of times should be an odd number), and obtain the E. coli vesicles encapsulating the active ingredient of the small molecule drug after washing;

[0011] S3 and M1 macrophages were incubated with E. coli vesicles encapsulated with small molecule active ingredients of drugs obtained from S2 at 37°C for 60 min, and the drug-loaded nanoparticles loaded by macrophages were obtained after washing.

[0012] In one embodiment of the present invention, the method for preparing the nanoparticles in S1 is to dissolve the small molecule active ingredient of the drug and PLGA in DMSO, add the surfactant TPGS after mixing, and mix to form an oil phase; the oil phase is added dropwise to deionized water, continuously stirred at room temperature, and purified and resuspended to obtain a solution containing nanoparticles.

[0013] In one embodiment of the present invention, the mass ratio of the small molecule active ingredient of the drug to PLGA is 1:4, the concentration of the small molecule active ingredient of the drug in the oil phase is 0.25 mg / mL, and the concentration of TPGS in the oil phase is 2 mg / mL.

[0014] In one embodiment of the present invention, the volume ratio of the oil phase to the water phase is 1:7.

[0015] In one embodiment of the present invention, the continuous stirring time is 0.5-1 h.

[0016] In one embodiment of the present invention, the content of the small molecule active ingredient of the drug in the Escherichia coli vesicles encapsulating the small molecule active ingredient of the drug obtained in S2 is 0.2~0.225 mg / mL.

[0017] In one embodiment of the present invention, the concentration of M1 macrophages in the incubation system of S3 is 2×10 6 cells / mL.

[0018] The third object of the present invention is to provide the use of the above-mentioned nano drug-loaded particles in the preparation of drugs for treating hepatocellular carcinoma.

[0019] The fourth object of the present invention is to provide the use of the nano drug-loaded particles in the preparation of drugs for improving the prognosis of hepatocellular carcinoma after ablation.

[0020] In one embodiment of the present invention, the drug has the effect of inhibiting tumor cell proliferation and angiogenesis, or stimulating systemic immune response and inducing long-lasting immune memory.

[0021] Beneficial effects of the present invention:

[0022] The present invention prepares a macrophage-loaded nanoparticle. The LEN-loaded MΦs (LEN@MΦs) are constructed by natural M1-type MΦs engulfing Escherichia coli membrane-coated LEN-loaded nanoparticles. The E. coli membrane camouflage enhances the phagocytic efficiency of MΦs, prevents the leakage of LEN inside MΦs, and ensures that LEN@MΦs can still maintain the macrophage M1 phenotype in the immunosuppressive tumor microenvironment.

[0023] Based on the chemotaxis of tumor-infiltrating macrophages, this study applied LEN@MΦs to treat multifocal HCC after minimally invasive ablation. This strategy, termed microinvasive ablation-navigated macrophage hitch (MAMH), provides a targeted therapy for HCC. This strategy leverages the natural inflammatory gradient induced by ablation to guide LEN-loaded macrophages to tumors, resulting in an approximately 10-fold increase in the delivery efficiency of LEN to post-operative HCC. MAMH demonstrated significant antitumor activity in multiple HCC models, including a hydrodynamic tail vein injection multifocal HCC mouse model and an orthotopic xenograft HCC rabbit model. It systematically inhibited the progression of residual tumors after ablation and prolonged the median survival of tumor-bearing mice. Furthermore, the study explored its potential antitumor mechanism using flow cytometry, ELISA, immunohistochemistry, and immunoblotting. The results revealed that this strategy significantly inhibited tumor cell proliferation and angiogenesis, and that this enhanced LEN delivery stimulated systemic immune responses and induced long-lasting immune memory.

[0024] The prepared LEN@MΦ is used in the targeted treatment of multifocal HCC after minimally invasive ablation, showing excellent therapeutic efficacy and biocompatibility among different species, and has broad prospects in clinical translation for controlling residual tumor progression and improving prognosis after HCC ablation.

[0025] In addition, sorafenib and regorafenib, which are hydrophobic drugs like lenvatinib, can also be used for systemic treatment of patients with advanced multifocal HCC after local treatment. Based on the properties of the drugs and PLGA, they can be prepared into macrophage-loaded nanoparticles using the preparation method of the nanoparticles described in the present invention, and used to treat hepatocellular carcinoma and improve the prognosis of hepatocellular carcinoma after ablation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Figure 3 is the characterization of LEN-NPs and LEN-EM@NPs and their effects on cell carriers; (A) is the transmission electron microscopy image and size distribution diagram of LEN-NPs, with a scale bar of 200 nm; (B) is the transmission electron microscopy image of LEN-EM@NPs, in which blue is LEN-NPs and yellow is E. coli membrane, with a scale bar of 200 nm in the left image and 100 nm in the right image; (C) is the confocal laser scanning microscopy image of LEN-NPs and E. coli membrane, with a scale bar of 20 μm; (D) is the statistical result of the cumulative release of LEN from LEN-EM@NPs and LEN-NPs at pH 5.0 and 7.4; (E) is the co-culture of LEN-EM@NPs and LEN-NPs with MΦs for 24 h. (h) Flow cytometric analysis results of cell surface CD80 expression levels; (F) Wright-Giemsa staining results of MΦs and LEN@MΦs, scale bar is 10 μm;

[0027] Figure 2Figure 2 is the characterization result of LEN@MΦs; (A) is the transmission electron microscopy image of LEN@MΦs, the scale bar is 3 μm, (B) is the representative flow cytometry histogram of C6-LEN-NPs@MΦs and C6-LEN@MΦs, (C) is the flow cytometry histogram of RAW264.7 cells cultured in MΦs, LEN-NPs@MΦs and LEN@MΦs for 6 days. h. (D) is the effect of treating RAW264.7 cells with MΦs, LEN-NPs@MΦs and LEN@MΦs on the expression level of cell surface CD80. Red and blue represent the expression levels of CD80 before and after different treatments, respectively. (E) is the statistical result of the fold change of the expression level of CD80 on the cell surface injected into the tumor after different treatments relative to the expression level of CD80 on the cell surface before injection. (F) is the analysis result of the LEN release amount of LEN@MΦs in the three stages of circulation, chemotaxis and release. (G) is the cell viability detection result of Hepa1-6 cells after incubation with NPs, EM@NPs, LEN-EM@NPs, LEN@MΦs, PMA-MΦs and PMA-LEN@MΦs for 72 h. (H) is the effect of Bcl-2, Bax and Cleaved leukemia on Hepa1-6 cells after treatment with MΦs and LEN@MΦs, respectively. The results of the effect of caspase-3 expression are shown in Figure 1. (I) is the result of relative quantitative analysis of the apoptosis level of Hepa1-6 cells after treating Hepa1-6 cells with MΦs and LEN@MΦs respectively using Annexin V-FITC / PI staining. (J) is a schematic diagram of the chemotactic and permeable behavior of LEN@MΦs in the transwell experiment. (K) is the result of counting MΦs in the lower chamber after transwell experiments with MΦs and LEN@MΦs respectively. *** P <0.001, **** P <0.0001, ns indicates no statistical significance;

[0028] Figure 3 Figure 2 shows the effect of LEN@MΦs on tumor cell proliferation and apoptosis. (A) shows the Ki-67 immunofluorescence staining results of Hepa1-6 cells in the lower chamber after 72 h of co-culture under different conditions. The scale bar is 10 μm. (B) shows the statistical results of the relative expression of Bcl-2, BAX, and Cleaved Caspase-3 proteins in Hepa1-6 cells in each group. P <0.05,** P <0.01;

[0029] Figure 4Figure 2 is an analysis of the distribution of MAMH in vivo; (A) is a statistical graph showing the fold change of CCL-2, IL-1β, IL-6, and TNF-α in tumor tissues of hepa1-6 tumor-bearing mice compared with normal tissues within 14 days; (B) is a schematic diagram of the treatment plan of MAMH in a subcutaneous transplanted liver cancer model; (C) is an IVIS image showing the biodistribution of LEN, LEN-EM@NPs, LEN@MΦs, and MAMH in the tumor area under different treatment groups, with the tumor represented by a blue dotted circle; (D) is an IVIS image of important organs 6 h and 24 h after different treatments; (E) is a statistical graph showing the radiation efficiency of important organs 6 h and 24 h after different treatments; Group 1 is LEN treatment, Group 2 is LEN-EM@NPs treatment, Group 3 is LEN@MΦs treatment, and Group 4 is MAMH treatment; (F) is a statistical graph showing the LEN concentration in important organs and tumor tissues 6 h after different treatments; * P <0.05, **** P <0.0001;

[0030] Figure 5 The figures are statistical diagrams of the therapeutic effect of MAMH in vivo; among them, (A) is an IVIS image monitoring the progression of residual tumors after different treatments, (B) is the tumor growth curve and digital photos of mice treated with different methods, (C) is the statistical result of the relative levels of VEGFR2 and VEGFA mRNA in the tumor tissues of mice treated with different methods, (D) is the quantitative analysis result of the microvessel density in the tumor tissues of mice treated with different methods, (E) is the statistical result of the expression level of ki-67 in the tumor tissues of mice treated with different methods, and (F) is the statistical result of the expression level of CD11b in the tumor tissues of mice treated with different methods. + f / 4 / 80 + The statistical results of the M1 / M2 ratio of cells, (G) is the CD8 + The statistical results of the CTL ratio of cells, (H) is the CD45 + Statistical results of NK ratio of cells, (I) is the CD4 + (J) is the statistical result of the Treg ratio of cells, and the result of the effect of different treatments on the blood biochemical indicators of mice; * P <0.05,** P <0.01,*** P <0.001, **** P <0.0001, ns indicates no statistical significance;

[0031] Figure 6The figures show the therapeutic mechanism and biosafety of MAMH in a mouse subcutaneous liver cancer transplant model; (A) shows the relative expression levels of VEGFR2, VEGFA, and p-ERK proteins in tumor tissues of different groups; (B) shows representative immunohistochemistry and TUNEL staining images of different groups, with a scale bar of 100 μm; (C) shows the quantitative analysis of TUNEL in tumor tissues of different groups; and (D) shows the HE staining results of the main organs of mice after different treatments, with a scale bar of 100 μm. * P <0.05,** P <0.01,*** P <0.001, **** P <0.0001;

[0032] Figure 7 Schematic diagram of the establishment of the mouse challenge model and the results of the effect of MAMH on immune memory effect; (A) is a schematic diagram of the complete elimination of residual tumors in mice using MAMH and the IVIS image monitoring the growth status of tumors; (B) is a representative flow cytometry scatter plot of spleen lymphocytes;

[0033] Figure 8 Figure 2 is the evaluation result of the immune response induced by MAMH in vivo; (A) is a representative IVIS image of monitoring the tumor progression of mice after treatment with PBS (group 1), minimally invasive ablation (MA) (group 2), LEN (group 3), LEN+MA (group 4), LEN-EM@NPs+MA (group 5), MAMH (group 6) and 10xLEN (group 7); (B) is a digital photo of the liver after treatment with PBS (group 1), minimally invasive ablation (MA) (group 2), LEN (group 3), LEN+MA (group 4), LEN-EM@NPs+MA (group 5), MAMH (group 6) and 10xLEN (group 7); the scale bar is 2 cm, (C) is a representative flow cytometry scatter plot and frequency statistical results of CD3+CD8+IFNγ+CTL cells and CD3+CD4+CD25+FoxP3+Treg cells in tumor tissues after different treatments, (D) is a representative flow cytometry scatter plot and frequency statistical results of CD3+CD8+T cells in peripheral blood after different treatments, (E) is a representative flow cytometry scatter plot and frequency statistical results of CD3+CD8+CD44+CD62L-TEM cells in spleen after different treatments; * P <0.05,** P <0.01, ns indicates no statistical significance;

[0034] Figure 9The figures are the statistical results of the relative liver weight and body weight of mice after different treatments; (A) is the statistical results of the ratio of liver weight to body weight of mice bearing orthotopic tumors treated with PBS (group 1), minimally invasive ablation (MA) (group 2), LEN (group 3), LEN+MA (group 4), LEN-EM@NPs+MA (group 5), MAMH (group 6), and 10-fold LEN (group 7); (B) is the statistical results of the body weight of mice bearing orthotopic tumors treated with PBS (group 1), minimally invasive ablation (MA) (group 2), LEN (group 3), LEN+MA (group 4), LEN-EM@NPs+MA (group 5), MAMH (group 6), and 10-fold LEN (group 7); * P <0.05,** P <0.01, **** P <0.0001, ns indicates no statistical significance;

[0035] Figure 10 Figure 1 is an evaluation result diagram of the immune response induced by various treatment methods detected by flow cytometry; (A) is a representative flow cytometry scatter plot of M1 and M2 MΦs in tumor tissue and a statistical result diagram of the ratio of M1 to M2 MΦs; (B) is a CD3 − Representative flow cytometry scatter plots of NK1.1 in peripheral blood and CD45 in peripheral blood + Statistical results of NK cell ratio, (C) is CD3 − Representative flow cytometry scatter plots of NK1.1 in spleen and CD45 in spleen + Statistical results of NK cell ratio, (D) is CD3 − Representative flow cytometry scatter plots of NK1.1 in tumor tissues and CD45 in tumor tissues + Statistical results of NK ratio of cells;* P <0.05,** P <0.01, ns indicates no statistical significance;

[0036] Figure 11 Figure 2 shows the statistical results of the systemic tumor inhibition effect of MAMH on multifocal liver cancer in mice; (A) includes a schematic diagram of multifocal liver cancer constructed by tail vein hydrodynamic injection (HDTVi), pictures of HDTVi HCC tumors (P53 knockout and MYC overexpression) (scale bar 0.5 cm), HE sections (scale bar 50 μm) and immunoblotting results; (B) is a picture of the liver after different treatments (scale bar 2 cm) and HE sections (scale bar 500 μm); (C) is a statistical result of the number of tumor nodules in mice after treatment with different methods; (D) is a survival curve of mice after treatment with different methods; (E) is the TEM in the CD8 +The proportion of cells and TEM in CD4 + The statistical results of the proportion of cells in the peripheral blood of mice treated with different methods are shown in Figure 5. (F) shows the CD8 + In CD3 + The proportion of NK cells in CD45 + The statistical results of the proportion of M1 and M2 macrophages in the tumor of mice treated with different methods, the content of NK in CD45 + The proportion of cells, CTL in CD8 + The proportion of Treg cells in CD4 + Statistical results of the cell ratio, AT is ablated tumor, NAT is non-ablated tumor; * P <0.05,** P <0.01,*** P <0.001, **** P <0.0001, ns indicates no statistical significance;

[0037] Figure 12 Figures 1 and 2 show the results of the biosafety evaluation of MAMH in a mouse model of multifocal liver cancer. (A) shows the statistical results of the body weight of mice with multifocal liver cancer after different treatments, and (B) shows the statistical results of the blood biochemical indicators of mice after different treatments.

[0038] Figure 13 Figure 2 shows the results of flow cytometry detection of the proportion of immune cells in peripheral blood and spleen tissue; (A) is a representative flow cytometric scatter plot of spleen lymphocytes in different groups, (B) is a representative flow cytometric scatter plot of CD3+CD8+T cells in peripheral blood, (C) is a representative flow cytometric scatter plot of CD3-NK1.1+NK cells in peripheral blood, and (D) is a representative flow cytometric scatter plot of CD3+CD8+T cells and CD3-NK1.1+NK cells in spleen tissue and the corresponding statistical results of the proportions; * P <0.05,** P <0.01;

[0039] Figure 14 Flow cytometry was used to detect the proportion of immune cells in the tumor microenvironment; (A) is a representative flow cytometric scatter plot of M1 and M2 MΦs in tumor tissues, (B) is a representative flow cytometric scatter plot of CD3+CD8+IFNγ+CTL cells in tumor tissues, (C) is a representative flow cytometric scatter plot of CD3+CD4+CD25+FoxP3+Treg cells in tumor tissues, and (D) is a representative flow cytometric scatter plot of CD3−NK1.1+NK in tumor tissues;

[0040] Figure 15 Figures show the results of the clinical translational evaluation of MAMH in a rabbit orthotopic liver cancer xenograft model; (A) shows the experimental setup for MAMH, the experimental operation diagram, and a magnified image showing real-time ultrasound imaging of the liver tumor tissue ablation process; (B) shows a photograph of the rabbit liver after different treatments, with a scale bar of 2 cm; (C) shows the statistical results of mouse tumor volume and weight after different treatments; (D) shows the statistical results of blood biochemical indicators after different treatments; and (E) shows the HE staining results of the main organs of rabbits after different treatments. P <0.05,** P <0.01,*** P <0.001, **** P <0.0001;

[0041] Figure 16 The following are representative gating strategy diagrams for various immune cell populations, taking tumor, spleen, and blood samples from orthotopic hepatocellular carcinoma model mice as examples; among them, (A) is a representative gating strategy diagram for various immune cell populations in tumors, (B) is a representative gating strategy diagram for various immune cell populations in spleens, and (C) is a representative gating strategy diagram for various immune cell populations in blood. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and the accompanying drawings. The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, drugs and instruments used are conventional materials, reagents, drugs and instruments in the art unless otherwise specified, and can be obtained commercially by those skilled in the art.

[0043] The materials, reagents, drugs and instruments involved in the present invention are as follows:

[0044] Antibodies: Cleaved caspase-3 (Asp175) antibody (Product No. 9661) and p53 (7F5) rabbit monoclonal antibody (Product No. 2527) were purchased from Cell Signaling Technology; anti-CD34 antibody (Product No. ab81289), anti-VEGFA antibody (Product No. ab214424), anti-Bcl-2 antibody (Product No. ab182858), anti-p-ERK antibody (Product No. ab201015), anti-c-Myc antibody (Product No. ab185656), and anti-Ki-67 antibody (Product No. ab16667) were purchased from Abcam; anti-CD45 FITC (Product No. 103108), anti-CD3ε PerCP / Cyanine5.5 (Product No. 100328), anti-CD8a APC (Product No. 100712), and anti-CD4 PE / Cyanine7 (Product No. 100422), Anti-F4 / 80 PE / Cyanine7 (Product No. 123114), Anti-CD11b PerCP / Cyanine5.5 (Product No. 101228), Anti-CD80 PE (Product No. 104708), Anti-CD86 PE (Product No. 105008), Anti-NK 1.1 PE (Product No. 108708), Anti-CD25 PE (Product No. 108708), Anti-FoxP3 PE (Product No. 126404), Anti-IFN-γ PE (Product No. 505808), Anti-CD4 Cyanine7 (Product No. 100411), Anti-CD44 PE / Cyanine7 (Product No. 103030), Anti-CD62L PE (Product No. 104408), and Anti-CD8a APC / Cyanine7 (Product No. 100714) was purchased from Biolegend; anti-β-tubulin antibody (Product No. abs830032) and anti-GAPDH antibody (Product No. abs830030) were purchased from Absin.

[0045] Reagents: Poly(lactic-co-glycolic acid) (PLGA, product number: P133297), D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS, product number: T110277), and coumarin 6 (C6, product number: C100929) were purchased from Aladdin Chemical Co., Ltd.; Lenvatinib (LEN, product number: S1164) and CFSE (product number: S8269) were purchased from Selleck; fetal bovine serum (product number: 10091148), RPMI 1640 (product number: 11875093), and DMEM high glucose medium (product number: 11995065) were purchased from Gibco Invitrogen Corp; collagenase IV (Product No.: C8160), hyaluronidase (Product No.: H8030), and deoxyribonuclease I (Product No.: D8072) were purchased from Solarbio; formic acid (Product No.: F0507), acetonitrile (Product No.: 100029), myristate mononitrate (PMA, Product No.: 524400), and N-formyl-methylthio-acetylamino-phenylalanine (fMLP, Product No.: F3506) were purchased from Sigma-Aldrich.

[0046] [2H5]-Lenvatinib (Product No. IR-18688) was purchased from IsoReag.

[0047] Plasmids pT3-EFIa-MYC-IRES-luciferase, CMV Sleeping Beauty 13, and px330-sig-p53 were disclosed in the following article: Liu Y, Sun L, Guo H, et al. Targeting SLP2-mediated lipid metabolism reprograming restricts proliferation and metastasis of hepatocellular carcinoma and promotes sensitivity to Lenvatinib. Oncogene. 2023;42(5):374-388. doi:10.1038 / s41388-022-02551-z.

[0048] Kits: PrimeScript™ RT reagent Kit with gDNA Eraser Kit (Product No.: RR047A) were purchased from Takara; Cell Counting Kit-8 (CCK-8, Product No.: 10K1018) was purchased from Apexbio; TNF-α (Product No.: PMTA00B) and IFN-γ (Product No.: MIF00) ELISA kits were purchased from R&D Systems®; IL-1β (Product No.: SEA563Mu), IL-6 (Product No.: SEA079Mu), and MCP1 (CCL-2, Product No.: MEA087Mu) ELISA kits were purchased from Cloud-Clone Corporation; and a one-step TUNEL apoptosis detection kit (Product No.: C1086) was purchased from Beyotime Biotechnology.

[0049] Avanti microextruder (Cat. No. 610000), filter holder (Cat. No. 610014), and PC membranes (Cat. Nos. 610005, 610006, 610007, 610009, 610010) were purchased from Avanti; 12 mm Transwell® 0.4 µm (Cat. No. 3401) and 12 mm Transwell® 3 µm (Cat. No. 3402) were purchased from Corning.

[0050] Cell lines and animals: RAW 264.7 cells were purchased from Procell Life Science Co., Ltd. Mouse hepatocellular carcinoma cell lines (Hepa1-6 and Hepa1-6-luc) were generously provided by Professor Lianxin Liu of the Department of Hepatobiliary Surgery (The First Affiliated Hospital of University of Science and Technology of China) and cultured in Dulbecco's Modified Eagle's Medium (Gibco, MA, USA) supplemented with 10% fetal bovine serum (Gibco, MA, USA) and 1% penicillin-streptomycin solution (Beyotime, China) at 37°C in a 5% CO2 atmosphere. Rabbit tumor cells VX2 were purchased from Jennio Biotechnology Co., Ltd. in Guangzhou. 6- to 8-week-old C57BL / 6 mice and Japanese white rabbits weighing 2.5 to 3 kg were purchased from Changsheng Biotechnology Co., Ltd. Alveolar macrophages were prepared from Japanese white rabbits.

[0051] All animal experiments in the present invention were approved by the Experimental Animal Committee of Harbin Medical University (HMUIRB2023034) and the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (230620-01-GR) and complied with the Regulations on Laboratory Animal Management.

[0052] The experimental method involved in the present invention is as follows:

[0053] LEN@MΦs cell viability detection method:

[0054] Three groups (MΦs, LEN@MΦs, and LEN-NPs@MΦ) were seeded at a density of 5,000 cells per well in 96-well plates (independent experiments were repeated three times). Exposure times were set to 0.5, 1, 2, 4, and 6 hours. Subsequently, the cells were treated with serum-free medium containing 10% CCK-8 for 2 hours in the dark. The plates were then shaken and the absorbance at 450 nm was measured using a microplate reader. Relative cell viability was determined by calculating the optical density values obtained from a microplate reader. GraphPad Prism was used to calculate relative cell viability.

[0055] In vitro cytotoxicity assay:

[0056] The CCK-8 assay was used to quantitatively evaluate the in vitro cytotoxicity of different experimental groups. Hepa1-6 cells (1×10 4 Cells were seeded in 96-well plates (100 cells / well) and cultured for 24 hours. The experiment was divided into seven groups: control, empty-NPs, empty-EM@NPs, LEN-EM@NPs, LEN@MΦs, PMA-treated MΦs, and PMA-treated LEN@MΦs (100 nM, 4 hours). Each of the LEN@MΦs, PMA-treated MΦs, and PMA-treated LEN@MΦs was centrifuged at 2000 rpm for 5 minutes. The nanoparticles and supernatant were then incubated with Hepa1-6 cells for 72 hours. The cells were incubated in serum-free medium containing 10% CCK-8 for 2 hours in the dark. The plates were shaken, and the absorbance at 450 nm was measured using a microplate reader. Subsequent calculations were performed as described above.

[0057] In vitro tumor cell proliferation inhibition ability detection method:

[0058] Hepa1-6 cells were grown at 10 5 The cells were seeded at a density of 10 cells / mL in 12-well plates. After 24 h, PMA (as a control, 100 nM) was added to 0.4 μm Transwell plates. Then, 3×10 5 PMA-MΦs and PMA-LEN@MΦs were co-cultured in the upper Transwell chamber at 100 cells / mL. The Transwell plates were removed, and Hepa1-6 cells in the lower chamber were washed and fixed with 4% paraformaldehyde for 30 minutes. Ki-67 immunofluorescence staining was then performed according to the manufacturer's instructions. To evaluate the effect of LEN@MΦs on tumor cell apoptosis, Hepa1-6 cells were washed and collected after co-culture in different groups for immunoblotting and flow cytometry.

[0059] In vitro and in vivo tumor targeting ability assays:

[0060] The chemotaxis of LEN@MΦs was studied using an in vitro Transwell migration assay (3 μm pore size, Corning). 5 MΦs and LEN@MΦs (pretreated with 50 μg / mL EM@NPs for 1 hour) were co-cultured in the upper Transwell chamber at a concentration of 10 cells / mL. The lower chamber was filled with fresh DMEM, Hepa1-6 conditioned medium collected from DMEM cells after 48 hours of culture, or a mixture of Hepa1-6 conditioned medium and fMLP. After incubation at 37°C for 6 hours, cells migrating into the lower chamber of the Transwell were examined under a fluorescence microscope (BX53, Olympus Corporation, Tokyo, Japan).

[0061] In vivo biodistribution detection method of LEN@MΦs:

[0062] MΦs were incubated with Cy5.5-LEN-EM@NPs at 37°C for 1 h to obtain MΦs carrying Cy5.5-LEN-EM@NPs. Considering that the black hair of C57BL / 6 mice may affect the fluorescence signal, the tumor tissue area was shaved. When the tumor grew to about 200 mm 3 At 37 ℃, mice were intravenously injected with free Cy5.5-LEN, Cy5.5-LEN-EM@NPs, Cy5.5-LEN@MΦs, and MAMH (the dose of Cy5.5-LEN in all the above treatments was 1 mg kg −1 Mice were anesthetized 1, 6, and 24 hours after injection and observed using IVIS spectroscopy. For further in vitro evaluation, mice were sacrificed at 6 or 24 hours, and tumors or organs were harvested.

[0063] Determination of inflammatory cytokines:

[0064] Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of inflammatory cytokines IL-1β, IL-6, TNF-α, and CCL-2 in tumor tissues of tumor-bearing mice. Appropriate amounts of tissue were collected and washed with pre-chilled PBS (pH 7.0-7.2) to remove blood. The weighed tissue was then cut into small pieces and placed in a centrifuge tube containing ice-cold fresh lysis buffer (mass-to-volume ratio = 1:50; for example, 50 mg of tissue sample was placed in 1 mL of lysis buffer). The tissue suspension was processed using an ultrasonic cell disruptor until clear. The homogenate was centrifuged at 10,000 × g for 5 minutes, and the pellet was discarded. The supernatant was assayed according to the kit's protocol or stored at -20°C. Serum levels of TNF-α and IFN-γ were also measured to assess the antitumor effects of LEN@MΦs. Blood samples were collected from mice in different groups and allowed to clot naturally at room temperature. Serum was then centrifuged at 10,000 rpm for 20 minutes to obtain serum.

[0065] TQ-S method for determining the content of LEN in tissue samples:

[0066] Chromatographic separation and analysis were performed using a Xevo TQ-S system. The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile (B). Gradient elution was performed at a flow rate of 0.4 mL / min using the following gradient program: 5% B (0-0.5 min); 5%-95% B (0.5-4 min); and 5% B (4-6 min). The injection volume was 1 µL, and the injection temperature was 40°C. The mass spectrometer was operated in positive ion multiple reaction monitoring mode. The precursor and product ion targets for LEN and IS were m / z 427.1→370 and m / z 432.1→370, respectively. The cone and capillary voltages were set to 30 V and 2.0 kV, respectively. Other experimental conditions for mass spectrometry were as follows: desolvation temperature, 350 °C; desolvation gas flow rate, 650 L / h; cone gas flow rate, 150 l / h; nebulizer gas flow rate, 7.0 Bar; and collision gas flow rate, 0.25 mL / min.

[0067] C57BL / 6 mice were inoculated into the right axilla with Hepa1-6 cells (1 × 10 6 cells / 100 μL PBS). The tumor volume reached approximately 200 mm 3Treatment began at 4:00 p.m., and mice were randomly divided into groups: free LEN, LEN-EM@NPs, LEN@MΦs, and MAMH (the LEN dose was 1 mg / kg in all treatments). Six hours after treatment, mice were sacrificed, and liver, kidney, and tumor tissues were gently removed. Approximately 100 mg of each tissue was accurately weighed into a 2 mL grinding tube, to which 0.5 mL of acetonitrile was added. The samples were homogenized using an automated tissue homogenizer (70 Hz) for 2 minutes, followed by centrifugation at 13,000 rpm for 15 minutes at 4°C. Finally, 100 µL of the resulting supernatant was transferred to an autosampler vial for analysis.

[0068] Animal Treatment:

[0069] Mice bearing subcutaneous Hepa1-6-luc tumors were randomly divided into four groups (n=5 per group) after minimally invasive ablation (MA): PBS, LEN, LEN-EM@NPs, and MAMH. Mice received a single intravenous dose of LEN (1 mg / kg) every three days for a total of three injections. The ablation power and duration in this mouse model were controlled between 5 and 10 W and 1 to 1.5 minutes, respectively. Mice bearing orthotopic Hepa1-6-luc tumors were randomly divided into seven groups (n=5 per group): PBS, MA, LEN, MA+LEN, MA+LEN-EM@NPs, MAMH, and 10x LEN (10 mg / kg). Mice bearing multifocal HCC were randomly divided into four groups (n=5 per group): PBS, MA, LEN, and MAMH. To better simulate the actual application of potential clinical translation, orthotopic tumor-bearing rabbits were established and randomly divided into four groups (n=3 per group): PBS, MA, LEN+MA, and MAMH (LEN, 0.3 mg / kg). MA was performed under ultrasound guidance with a power of 30 W and a duration of 30–40 s.

[0070] Animal model construction:

[0071] Subcutaneous HCC Mouse Model:

[0072] C57BL / 6 mice were inoculated in the right axillary region with Hepa1-6 cancer cells (1 × 10 6 cells / 100 μL PBS). When the tumor volume reached approximately 200 mm 3 Treatment was started at 4 hr. Subsequently, mice were randomized into groups for further treatment.

[0073] Orthotopic HCC Mouse Model:

[0074] After the mice were anesthetized, the abdominal area was cleaned alternately with 70% alcohol irrigation and povidone-iodine scrubbing. Subsequently, an incision was made in the midline of the abdomen, and the falciform ligament and hepatogastric ligament were separated to expose the liver. Then, 25 μL of matrix was mixed with 1×10 6The Hepa1-6-luc cell mixture was injected into the subhepatic peritoneum, taking care to prevent damage to other organs. The abdominal wall and skin incisions were closed using Vicryl 5 / 0 sutures, respectively. Approximately 5 days later, mice were randomly assigned to different treatment groups based on bioluminescence values. Tumor volume was assessed weekly using a bioluminescent IVIS imaging system, and mice were euthanized after completion of dosing.

[0075] Multifocal HCC Mouse Model:

[0076] A multifocal HCC model was established using the HDTVi mouse HCC model. C57BL / 6 mice were administered an intravenous (iv) injection of a plasmid mixture diluted in saline at a total injection volume equivalent to 10% of their body weight over 6-8 seconds via the tail vein. The plasmid mixture consisted of 30 µg of px330-sg-p53 (sgP53), 10 µg of pt3-efia-myc-ires-luciferase (MYC-luc), and 2 µg of CMV Sleeping Beauty 13 (SB13).

[0077] Orthotopic HCC Rabbit Model:

[0078] Rabbits were injected intramuscularly with thiazide hydrochloride (0.2 ml kg -1 body weight) anesthesia, and then 3% sodium pentobarbital (0.7 ml kg) was injected intravenously through the ear vein after an interval of 5 minutes. -1 At the onset of anesthesia, the area below the xiphoid process was shaved and disinfected. After the anesthesia was fully effective, the area below the xiphoid process was disinfected with a scraper, and a minimally invasive laparotomy was performed under aseptic conditions. VX2 rabbit tumor tissue without central necrosis was dissected into small fragments (1 or 2 mm 3 ), implanted in the liver lobe, and a single tumor was established. The abdominal wall was then sutured. To prevent infection, 200,000 units of penicillin were injected intramuscularly daily for three consecutive days.

[0079] Application of minimally invasive ablation

[0080] After anesthetizing Hepa1-6-luc tumor-bearing mice, the cold-tip MWA needle tip was percutaneously placed in the middle of the tumor's long axis. Ablation parameters, including power and duration, were maintained at 5-10 W and 0.5-1 min, respectively. Subsequently, residual tumor burden was monitored by photography and bioluminescence signals. For the subcutaneous HCC model, precise measurements were performed using a digital caliper, and the formula (long diameter × short diameter) was used. 2 ) / 2Calculate volume (mm 3 For the HDTVi mouse HCC model, the above power and time settings were used, and the tumor with the largest diameter in the left lobe of the liver was selected for ablation.

[0081] Immunoblotting

[0082] Cells and tumor tissues after various treatments were harvested, and total protein was extracted from the cultured cells using ice-cold RIPA lysis buffer. Total protein concentration was quantified using a BCA assay kit. Protein lysates were then separated by 7.5%-15% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The transferred membranes were blocked with 5% BSA for 90 minutes. Appropriate membranes were incubated with antibodies specific for Bcl-2, Bax, Cleaved caspase 3, VEGFR2, VEGFA, p-ERK, C-myc, p53, β-tubulin, and β-GAPDH overnight at 4°C. The following day, after washing with TBST, the membranes were incubated with the corresponding secondary antibodies for 1 hour at 25°C. Finally, visualization and quantification were performed using ImageJ software.

[0083] Histological examination:

[0084] At the designated time points, mice in each treatment group were euthanized, and major organs (heart, liver, spleen, lung, and kidney) and tumors were dissected and fixed overnight in 4% paraformaldehyde. Fixed tissues were then embedded and sectioned. Some sections were stained with H&E to assess tissue damage, while others were analyzed by TUNEL and IHC. For IHC, slides were deparaffinized with xylene, rehydrated with graded alcohols, and subjected to antigen retrieval and blocking with citric acid. Sections were then incubated with different primary antibodies (Ki-67 and CD34) overnight at 4°C. Slides were then incubated with corresponding secondary antibodies at 25°C, washed with PBS, and developed with diaminobenzidine. Finally, sections were counterstained with hematoxylin, visualized, and collected using a Motic EasyScan scanning system. Apoptotic cells were identified using a one-step TUNEL apoptosis assay kit according to the manufacturer's instructions. Cell nuclei were stained with DAPI, and fluorescence microscopy was performed.

[0085] Three independent pathologists, unaware of experimental data or group assignment, reviewed all cases. Ki-67 staining was assessed based on intensity and extent. Staining intensity was categorized as negative, weak, moderate, or strong (0, 1, 2, 3). The extent of staining was scored based on the percentage of positive cells: none, <25%, 25–50%, 50%–75%, and >75% (0, 1, 2, 3, 4). The final IHC staining score was calculated as the product of the intensity and extent scores. After CD34 staining, sections were fully examined at low magnification to identify areas of high vascular density, and the number of microvessels was counted at high magnification for scoring. The number of microvessels within three fields of view was recorded; each individual endothelial cell or group of endothelial cells that appeared brown was considered a single vessel; vessels with a luminal diameter exceeding eight red blood cells were excluded from the count.

[0086] RT-PCR:

[0087] Total RNA from tumor tissue was extracted using Trizol reagent. Reverse transcription was performed using the PrimeScript™ RTreagent Kit with gDNA Eraser. Real-time PCR was performed using the SYBR Green PCR kit. Target gene expression was determined using the comparative cycle threshold (Ct) method, with β-actin expression as a reference. The primers used are listed in Table 1.

[0088] Table 1 Primer information

[0089]

[0090] Flow cytometry:

[0091] Tumors extracted from mice were sliced into small fragments. Tumor sections were homogenized in a staining buffer containing digestive enzymes (collagenase IV, hyaluronidase, and deoxyribonuclease I) to generate a single-cell suspension. Cells were then stained with fluorescently labeled antibodies according to the manufacturer's instructions. Cells were treated with True-Nuclear™ Transcription Factor Buffer and then stained for intracellular factors. Spleens were aseptically harvested using sterile surgical instruments. The spleen mixture was filtered through a filter into a 50 mL conical tube and centrifuged at 500 × g for 5 minutes. After washing, the cell pellet was resuspended in red blood cell lysis buffer for 5 minutes. Cells were then stained with fluorescently labeled antibodies according to the manufacturer's protocol. Stained cells were analyzed by flow cytometry and evaluated using FlowJo software. Blood was collected from mice for intravascular T and NK cell analysis, and red blood cells were lysed. Cells were then stained with fluorescently labeled antibodies according to the manufacturer's protocol. Stained cells were monitored by flow cytometry and analyzed using FlowJo software. Flow cytometry gating strategies for different immune cell subsets in different samples (including blood, spleen, and tumor tissue) Figure 16 shown.

[0092] All statistical analyses in this paper were performed using the GraphPad Prism software package (Prism 9.1.0; GraphPad Software). The results of this study are presented as mean ± SEM and are derived from at least three independent experiments. Sample sizes were not predetermined using statistical methods but are comparable to those reported in previous publications. For scenarios involving multiple comparisons, one-way or two-way analysis of variance and Tukey's multiple comparison test were used. Student's t-test was used for single comparisons. P Values less than or equal to 0.05 were considered statistically significant. Statistical significance is indicated as follows:* P <0.05,** P <0.01,*** P <0.001, **** P<0.0001, ns indicates no statistical significance.

[0093] Example 1: Preparation of drug-loaded nanoparticles loaded by macrophages

[0094] Preparation of drug-loaded nanoparticles loaded by macrophages:

[0095] (1) Preparation of LEN-NPs: LEN and PLGA were dissolved in DMSO at a mass ratio of 1:4, and the surfactant TPGS was added after mixing to form an oil phase. The concentration of LEN in the oil phase was 0.25 mg / mL, the concentration of PLGA was 1 mg / mL, and the concentration of TPGS was 2 mg / mL. The oil phase was added dropwise to a certain amount of deionized water and stirred continuously at 25°C for 30 min to promote the self-assembly of the nuclei. During the entire experiment, the volume ratio of the oil phase to the water phase was kept constant at 1:7. During purification, the LEN-NPs were collected by centrifugation at 25°C and 12,000 rpm for 30 min. Subsequently, the LEN-NPs were resuspended in sterile PBS for subsequent experiments.

[0096] (2) Extraction of Escherichia coli membrane vesicles: Escherichia coli was cultured in nutrient broth at 37°C for 12 h, and then the Escherichia coli culture was centrifuged at 5000 rpm for 10 min to remove Escherichia coli, and the resulting supernatant containing bacterial outer membrane vesicles was collected; the supernatant was further filtered through a 0.45 μm membrane and concentrated using a 100 kDa ultrafiltration centrifuge tube; the concentrated suspension containing bacterial outer membrane vesicles was centrifuged at 4°C and 150 g for 2 h to precipitate the bacterial outer membrane vesicles, and the resulting bacterial outer membrane vesicle precipitate was dispersed in PBS and stored at -80°C; the resulting bacterial membrane vesicles were extruded 11 times through a polycarbonate membrane with a pore size of 200 nm using a micro extruder to obtain Escherichia coli membrane vesicles (EM vesicles).

[0097] (3) Preparation of LEN-EM@NPs: The obtained EM vesicles were mixed with LEN-NPs and extruded 21 times through a polycarbonate membrane with a pore size of 200 nm using a microextruder to obtain LEN-EM@NPs. Subsequently, the LEN-EM@NPs were centrifuged at 10,000 rpm, washed with PBS three times, and then stored at 4°C. The LEN content in the prepared LEN-EM@NPs was 0.2 mg / mL.

[0098] (4) Preparation of LEN@MΦs: LEN@MΦs were constructed by incubating M1-type MΦs with LEN-EM@NPs. The specific construction method was to culture RAW 264.7 cells at 10 5The cells were inoculated at a density of 10 cells / mL in cell culture medium. After 24 hours, the culture medium was removed and replaced with M1 medium (DMEM medium containing 10 ng / mL LPS + 10 ng / mL IFN-γ). The cells were cultured for another 24 hours to obtain M1-type MΦs. The M1-type MΦs were incubated with LEN-EM@NPs in a sterile tube at 37°C for 60 min. The concentration of MΦs in the incubation system was 2×10 6 cells / mL, the content of LEN in LEN-EM@NPs was 0.2 mg / mL, and LEN@MΦs were obtained after washing twice with PBS.

[0099] Characterization of drug-loaded nanoparticles loaded by macrophages:

[0100] Transmission electron microscopy was used to evaluate the morphology of LEN@MΦs, and flow cytometry was used to investigate the cellular uptake of C6-LEN-NPs and C6-LEN-EM@NPs by MΦs. After incubation, excess C6-LEN-NPs or C6-LEN-EM@NPs were removed, and the C6-LEN-NPs- or C6-LEN-EM@NP-loaded MΦs were collected and analyzed for fluorescence. The resulting fluorescence signal was compared with that of pristine MΦs as a control group to determine the relative fluorescence intensity of MΦs under different incubation conditions.

[0101] Transmission electron microscopy revealed that LEN-NPs were nanoparticles with an average size of 93.5 ± 4.0 nm ( Figure 1 (A) in the figure), E. coli membrane vesicles were wrapped on LEN-NPs by co-extrusion to form nanoparticles (LEN-EM@NPs) with a diameter of 200 nm. Transmission electron microscopy images showed that the prepared LEN-EM@NPs had a spherical geometry with an average diameter of 102.9±5.0 nm and a core-shell structure ( Figure 1 Confocal laser scanning microscopy images showed that LEN-NPs and the E. coli outer membrane were successfully colocalized ( Figure 1 HPLC showed that at pH 7.4 and 5.0, the release of LEN from LEN-NPs within 48 h was 28.16% and 45.45%, respectively. In contrast, the release of LEN from LEN-EM@NPs decreased significantly, reaching 9.45% and 16.61% at pH 7.4 and pH 5.0, respectively. Figure 1 These data indicate that complete coverage of the E. coli membrane prevents leakage of LEN, which is critical for macrophage hitchhiking induced by minimally invasive ablation.

[0102] LEN-EM@NPs were incubated with M1-type MΦs to construct LEN@MΦs. Ultrastructural analysis showed that there were approximately 18 aggregates with an average size of 500 nm in the cytoplasm of LEN@MΦs. Considering the changes in the concentration of LEN-EM@NPs before and after phagocytosis, the average number of LEN-EM@NPs loaded in each MΦ was approximately 91 ( Figure 2 (A) in the figure). The high loading of LEN is due to the coating of E. coli membrane on LEN-EM@NPs, which disguises LEN-NPs as natural E. coli, thereby increasing the phagocytic ability of MΦs. Next, we quantified the phagocytic efficiency of MΦs. By flow cytometry, we evaluated the loading rate of MΦs after 1 h of pre-incubation of LEN-NPs and LEN-EM@NPs. The results showed that 98.36% of MΦs engulfed LEN-EM@NPs, which was significantly higher than the percentage of MΦs that engulfed LEN-NPs (41.31%) ( Figure 2 (B)). The cell viability of RAW264.7 cells after culturing for 6 h in the systems containing M1-type MΦs, LEN-NPs@MΦs (MΦs that engulfed LEN-NPs), and LEN@MΦs were 85.3%, 51.0%, and 77.7%, respectively ( Figure 2 In addition, flow cytometry and Wright-Giemsa staining showed that LEN@MΦs had good cell polarity and shape after 6 hours of in vitro culture ( Figure 1 (E) and (F) in the figure). Subsequently, in vivo cell polarity was evaluated by injecting LEN@MΦs into mouse tumors. After inoculation with MΦs and LEN-NPs@MΦs, the expression of CD80 (an M1 marker) was significantly reduced (85.16%~13.72% for MΦs and 80.10%~11.50% for LEN-NPs@MΦs) ( Figure 2 In contrast, the expression of CD80 in tumors before and after LEN@MΦs inoculation changed less, from 82.33% to 77.36%, which is equivalent to 94% of LEN@MΦs maintaining the tumor M1 type ( Figure 2 This phenomenon demonstrates the efficient interaction of E. coli membrane integration in directing cell polarization, thereby maintaining an anti-tumor phenotype in the immunosuppressive tumor microenvironment.

[0103] We further studied the release kinetics of LEN in LEN@MΦs at different stages, including the blood circulation stage (circulation stage), the chemotaxis stage along the inflammatory factor gradient induced by ablation to the tumor tissue (chemotaxis stage), and the release of LEN in a local hyperinflammatory environment (release stage), using N-formyl-methylthio-acetylamino-phenylalanine (fMLP) and myristate (PMA) as model inflammatory factors. LEN from LEN@MΦs released less than 1.1% within 6 h in the untreated group (circulation stage), while in the presence of 10 nM fMLP (chemotaxis stage), the release was slightly higher at 14.3% within 6 h. In the presence of 100 nM PMA (release stage), LEN from LEN@MΦs was low (1.0%) within 2 h, increased significantly to 58.2% within 4 h, and finally reached 63.0% within 6 h ( Figure 2 These data indicate that E. coli membrane coverage improves the biocompatibility of LEN-NPs, prevents intracellular leakage of LEN before use, and maintains M1-type polarity, providing sufficient biological functionality for MAMH in vivo.

[0104] Example 2: Application of drug-loaded nanoparticles loaded by macrophages in the treatment of HCC after minimally invasive ablation

[0105] (1) In vitro anti-tumor and chemotactic ability detection of LEN@MΦs

[0106] First, the in vitro therapeutic effect of LEN@MΦs was evaluated by measuring the viability of Hepa1-6 cells under different treatments. Compared with LEN-EM@NPs treatment, which directly induces cancer cell apoptosis (27.3% cell viability), LEN@MΦs treatment had lower toxicity to Hepa1-6 cells (74.1% cell viability), while the cell viability decreased to 39.5% after PMA induction ( Figure 2 (G) in the figure) indicates that LEN@MΦs has controllable release ability. Correspondingly, the expression of Ki-67 decreased after co-culture, indicating that LEN@MΦs has a significant inhibitory effect on the proliferation of Hepa1-6 cells ( Figure 3 (A) in the figure).

[0107] The study of the apoptosis mechanism of Hepa1-6 cells found that LEN@MΦs treatment could increase the expression of pro-apoptotic BAX and Cleaved caspase-3 in Hepa1-6 cells and inhibit the expression of anti-apoptotic Bcl-2 ( Figure 2 (H) and Figure 3In contrast, MΦ treatment had a smaller effect on inducing apoptosis in Hepa1-6 cells. Further quantitative flow cytometry analysis revealed that the apoptosis rate of Hepa1-6 cells in the LEN@MΦs group was 59.9%, while that in the MΦs group was 20.4% ( Figure 2 (I) in ), indicating that the therapeutic effect of LEN@MΦs is mainly due to the release of LEN rather than MΦs.

[0108] The chemotactic permeation behavior of LEN@MΦs was studied using a transwell assay. The lower chamber was filled with DMEM medium, hepa1-6 conditioned medium, and a mixture of conditioned medium and fMLP, respectively, and the number of cells that chemotactically permeated from the upper chamber to the lower chamber was observed. Figure 2 (J)). Similar to native MΦs, LEN@MΦs permeated along the fMLP gradient and reached the Hepa1-6 cell sheet within 6 hours. Quantitative analysis showed that the number of LEN@MΦs in the lower chamber increased approximately 2-fold under the influence of fMLP compared to conditioned medium without fMLP, indicating that LEN@MΦs have inflammatory chemotactic motility ( Figure 2 (K) in the ).

[0109] (2) Application of LEN@MΦs in the treatment of HCC after minimally invasive ablation (minimally invasive ablation-induced macrophage hitchhiking, MAMH)

[0110] A subcutaneous xenograft HCC model was established in C57BL / 6 mice using luciferase-transfected Hepa1-6 cells (Hepa1-6 Luc) to investigate the effects of MAMH on HCC in vivo. Enzyme-linked immunosorbent assay was first used to measure primary inflammatory cytokines in tumors associated with MAMH infiltration, such as TNF-α, IL-1β, IL-6, and CCL-2 (MCP-1). These inflammatory factors are rapidly produced after ablation, forming a significant inflammatory gradient with surrounding healthy tissues and exceeding normal levels for more than 12 days ( Figure 4 Importantly, CCL-2, primarily produced by tumor cells and tumor-associated stromal cells, showed varying degrees of decrease in the following days and gradually increased on day 5 after ablation, likely due to insufficient ablation leading to the persistence and growth of residual tumor cells. Therefore, a MAMH treatment regimen for HCC in vivo was designed, with LEN@MΦs administered intravenously once every 3 days after ablation for a total of 3 doses ( Figure 4(B)). It is worth noting that minimally invasive ablation has a shorter operation time (0.5-1.0 min, compared to 3-5 min in tumor-bearing mice in the same ablation study), which is designed to trigger an inflammatory response while having a smaller direct killing effect on HCC. Parallel comparisons were conducted and residual tumors after local treatment were simulated in the clinical setting. As an example of minimally invasive ablation therapy, the role of MAMH in HCC treatment was studied in vivo. Given that the LEN content in LEN-EM@NPs is 0.1 pg and each MΦ contains approximately 91 LEN-EM@NPs, the intravascular administration dose of LEN@MΦs was set to 10 according to previous reports. 8 Subsequently, after ablation surgery, all HCC-bearing mice were divided into LEN group, LEN-nps group, LEN-EM@NPs group, and MAMH group.

[0111] To visualize the biodistribution of MAMH in vivo, LEN in all groups was labeled with a Cy5.5 fluorescent probe. Fluorescence images obtained using an in vivo imaging system (IVIS) showed that the fluorescence intensity of the tumor area in the MAMH group was higher than that in the LEN, LEN-NPs, and LEN-EM@NPs groups within 24 hours ( Figure 4 (C)). Analysis of normalized fluorescence intensity of important organs showed that compared with the LEN group, the fluorescence intensity of tumor tissue in the MAMH group increased by 11.5 times after 6 hours and by 8.5 times after 24 hours ( Figure 4 (D) and (E) in the figure). Subsequently, the concentrations of LEN in liver, kidney and tumor tissues were quantitatively determined using a triple quadruple mass spectrometer. It was found that the concentration of LEN in tumor tissue of the MAMH group was 392.4 ng / g, while the concentration of LEN in tumor tissue of the LEN group was 39.9 ng / g ( Figure 4 (F)). The LEN concentration increased by about 10 times, indicating that MAMH can effectively improve the delivery efficiency to tumors.

[0112] After characterizing the delivery efficiency of MAMH to HCC, an HCC model was established using hepa1-6 Luc in C57BL / 6 mice to evaluate the efficacy of MAMH in inhibiting tumor progression. Mice carrying Hepa1-6 Luc after ablation surgery served as the control group. The tumor fluorescence signal increased from day 8 (ablation surgery) to day 21 ( Figure 5 (A)). Similar to the control group, the fluorescence signals of the LEN and LEN-EM@NPs groups were enhanced. In contrast, the fluorescence signal change in the MAMH group was negligible. In addition, the corresponding resected tumors in the MAMH group were smaller than those in the other groups ( Figure 5 In particular, it was found that the tumors of mice in the MAMH group disappeared. The tumor volume of the control group increased to 1459 mm on day 21. 3In contrast, the tumor volume of the MAMH group remained at 265 mm 3 , indicating that the tumor was substantially inhibited after MAMH treatment ( Figure 5 (B) in the figure).

[0113] LEN treatment exerts its anti-tumor effect mainly by inhibiting angiogenesis and tumor cell proliferation and regulating immune response. The mRNA and protein expression of angiogenesis-related factors such as vascular endothelial growth factor receptor 2 (VEGFR2) and vascular endothelial growth factor A (VEGFA) protein expression were detected to reveal the therapeutic mechanism of MAMH. Compared with the LEN and LEN-EM@NP groups, the relative mRNA levels and protein expression of VEGFR2 and VEGFA in the MAMH group were reduced ( Figure 5 (C) and Figure 6 This reduction is consistent with the decrease in microvessel density marked by CD34, effectively reflecting the anti-angiogenic ability of MAMH ( Figure 5 (D) and Figure 6 (B)). p-ERK protein expression level was decreased, Ki-67 + The brown staining area decreased and the TUNEL green staining area increased, which together demonstrated the effectiveness of MAMH in inhibiting the proliferation of residual tumor cells ( Figure 5 (E) and Figure 6 (A), (B), (C)). Histological analysis of major organs (such as heart, lung, spleen, liver, and kidney) 10 and 20 days after MAMH revealed negligible histopathological abnormalities. This indicates no significant toxic effects ( Figure 6 (D) in the figure).

[0114] (3) Emergency systemic immune response and immune memory of MAMH

[0115] To further investigate the effect of MAMH on immune function in vivo, we evaluated the infiltrating immune cells in tumor tissue. In addition to the control group, LEN group, LEN-EM@NPs group, and MAMH group, an M1-type MΦs group was used as an MΦs group for comparison. Considering that the delivery efficiency of MAMH is approximately 10 times, a 10-fold dose of LEN was used as a 10-fold LEN group for comparison. + f / 4 / 80 + Significant responses were observed in the M1 / M2 ratio of cells and the frequencies of tumor-infiltrating cytotoxic T lymphocytes (CTL), natural killer cells (NK), and immunosuppressive regulatory T cells (Treg). + f / 4 / 80 +The M1 / M2 ratios of the cells were 1.2, 1.6, 1.9, and 1.32 ( Figure 5 In contrast, CD11b in the MAMH group + f / 4 / 80 + The M1 / M2 ratio of the cells was 3.3, which was comparable to that of the 10-fold LEN group (3.1). The difference in the M1 / M2 ratio indicates that the use of MAMH to deliver LEN causes the MΦs in the tumor microenvironment to shift from the M2 type to the M1 type. + CTL and CD45 + Similar to the 10-fold LEN group (9.9% of CTL and 0.8% of NK), the frequencies of CTL (10.8%) and NK (0.8%) in the MAMH group were higher than those in the other groups ( Figure 5 (G) and (H)). Compared with the control group, CD4 Treg cells with immune response suppression + In the LEN group and LEN-EM@NPs group, the decrease was 22% and 46%, respectively. In contrast, the MAMH group and 10-fold LEN group decreased by 67% and 66%, respectively, compared with the control group ( Figure 5 Notably, the MΦs in these projects were similar to those in the control group, suggesting that MΦs in MAMH play a minor role in tumor management.

[0116] The above data indicate that MAMH induces a significant immune response against tumors that is comparable to that of 10-fold LEN. However, 10-fold LEN causes significant hepatotoxicity and nephrotoxicity, especially increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities ( Figure 5 (J) in Figure 1). In contrast, despite achieving substantial therapeutic benefits, the toxicity of LEN delivered using MAMH was similar to that of normal doses of LEN and was negligible. In summary, the LEN@MΦs prepared in Example 1 for minimally invasive ablation of post-HCC HCC not only demonstrated a 10-fold higher administration efficiency and therapeutic efficacy than free LEN, but also exhibited minimal side effects in the human body.

[0117] MAMH has excellent antitumor efficacy in a subcutaneous xenograft liver cancer model, which is further demonstrated by the presence of cured HCC mice after minimally invasive ablation of HCC by LEN@MΦs (see Figure 5 To explore the potential stimulation of immune memory, a rechallenge model was established ( Figure 7 The results showed that mice treated with MAMH did not develop new tumor growth. The spleens of mice were collected and the CD8 + and CD4 +The percentage of effective memory T cells (Tem) in the T cell population was significantly increased. This observation suggests that the MAMH treatment strategy establishes a strong long-term immune memory effect ( Figure 7 (B) in the figure).

[0118] To further evaluate the immune response induced by MAMH in vivo, we established an orthotopic xenograft HCC model. Notably, MAMH effectively inhibited the growth of residual tumors, with an effect comparable to that of LEN at a 10-fold higher concentration ( Figure 8 (A) and (B) in Figure 9 Mice in the 10-fold LEN group began to lose weight 4 days after treatment, indicating potential toxic side effects ( Figure 9 Subsequent evaluation of the immune responses elicited by various treatments revealed that in the MAMH group, CD11b + f / 4 / 80 + The M1 / M2 ratio in the cells was significantly increased, indicating that this strategy can promote the transition of MΦs M2 to M1 phenotype in the tumor microenvironment ( Figure 10 At the same time, CTL infiltration in tumor tissues increased significantly, while Treg cells decreased significantly, which is consistent with the strong anti-tumor effect observed in the MAMH group ( Figure 8 In addition, CD8 + T cells increased slightly, while CD8 T cells increased significantly after MAMH treatment ( Figure 8 In addition, the administration of MAMH significantly increased Tem, indicating that it can reduce the recurrence of tumor tissue ( Figure 8 The proportion of NK cells in peripheral blood, spleen, and tumor tissue was always the highest in the MAMH group ( Figure 10 In summary, MAMH significantly improved the suppressive tumor microenvironment in an orthotopic xenograft HCC model, hindered the progression of residual tumors, induced systemic and long-term antitumor immune memory, and effectively prevented tumor recurrence.

[0119] (4) Systemic immune response and immune memory of MAMH treatment suppress multifocal HCC

[0120] This study investigated the potential of using MAMH to deliver LEN to promote immune activation against multifocal HCC, which is characterized by complex intratumoral heterogeneity and clonal evolution. The primary HCC model established by tail vein injection (HDTVi) not only morphologically resembles the multifocal features of most clinical HCCs, but also closely resembles the histological, transcriptomic, and genetic alterations found in similar human HCCs. Among them, the HCC model induced by the combination of TP53 and MYC is an immunologically "cold" tumor, which remains a therapeutic challenge ( Figure 11 (A) in the figure). In this study, four groups of multifocal HCC-bearing mice were intravenously injected with equal volumes of normal saline (control group), normal saline combined with minimally invasive ablation (MA group), LEN (LEN group), and LEN@MΦs combined with ablation (MAMH group). Tumor photos in the control group showed multifocal HCC with separated nodules. The multifocal HCC nodules in the MA group were larger and more numerous, confirming that inflammation caused by incomplete radiofrequency ablation accelerated tumor progression. Compared with the control group, the multifocal HCC in the LEN group showed less difference, reflecting the limited therapeutic effect of LEN on multifocal HCC. Compared with the other groups, the tumor nodules in the MAMH group were the smallest and fewer ( Figure 11 (B) Hematoxylin and eosin (H&E) staining to evaluate the efficacy of combined treatment showed that the number of tumor nodules in the liver tissue of mice in the MAMH group was the lowest compared with the control group. In contrast, the ablation group and the LEN group showed a large number of tumor nodules, some of which were fused, consistent with the visual photographs. The number of tumor nodules in the control group, MA group, LEN group, and MAMH group were 12.0, 19.0, 10.1, and 3.6, respectively ( Figure 11 (C)). The therapeutic effect of MAMH on multifocal HCC was evaluated by recording the survival time of multifocal HCC-bearing mice. The median survival time of the control group, MA group, LEN group, and MAMH group was 44, 30, 48, and 73 days, respectively. Figure 11 Compared with the limited therapeutic effect of free LEN, the survival time under MAMH treatment increased by 65.1%, which is consistent with the systemic immunity and immune memory of the MAMH strategy. In addition, there were no significant changes in the body weight or liver and kidney function of mice after MAMH treatment, indicating its good safety ( Figure 12 ).

[0121] Flow cytometry studies showed that the MAMH group induced long-term immune memory, spleen CD4 + and CD8 + Increased frequency of TEM in T cells ( Figure 11 (E) and Figure 13 In addition, compared with the other groups, the MAMH group had a higher number of CD8 +The percentages of T cells and NK cells were higher in the MAMH group, and the levels of TNF-α and IFN-γ, key cytokines for T cells and NK cells to exert anti-tumor effects, were significantly increased in the serum of MAMH-treated mice ( Figure 11 (F) and Figure 13 In addition, compared with the control group, the CTL and NK cells in the tumor tissues of mice in the MAMH group were significantly increased, and the Treg cells were significantly decreased. Notably, the proportion of immune cells infiltrating the tumor in the non-ablated liver lobe was almost the same as that in the ablated liver lobe tumor, indicating that MAMH systematically reshapes the tumor immune microenvironment ( Figure 11 (G) and Figure 14 These findings are consistent with the macroscopic antitumor results and together indicate that MAMH treatment induces effective systemic and long-lasting antitumor immune memory against multifocal HCC.

[0122] (5) Clinical translation evaluation of MAMH in an orthotopic xenograft HCC rabbit model

[0123] The clinical MAMH system was implemented in an orthotopic rabbit HCC model to evaluate its potential for clinical application. Figure 15 As shown in (A). Tumor tissue degeneration was observed in real-time ultrasound imaging during the ablation process. Similar to the therapeutic effect in mice, the tumors in rabbits treated with MAMH were smaller than those in other groups, indicating that MAMH has a better inhibitory effect on residual tumors than free LEN ( Figure 15 Quantification of the harvested tumors showed that the MAMH group had the largest volume and weight (9.5 cm 3 and 10.3 g, respectively, which were higher than those in the control group (49.1 cm 3 and 49.2 g), MA group (80.0 cm 3 and 78.5 g) and LEN group (40.5 cm 3 and 38.8 g) ( Figure 15 (C) in the figure).

[0124] Tissues and blood were collected 14 days after treatment, and serum and major organ biosafety of MAMH were used to evaluate the tumor biosafety. In the MAMH treatment group, all serum biochemical substances (AST, ALT, creatinine, urea) levels remained at normal levels compared with the untreated control group ( Figure 15 No abnormal or inflammatory cells were observed in any of the important organs of each group ( Figure 15 (E) These results demonstrate the effectiveness of MAMH in suppressing residual tumors in a rabbit orthotopic HCC model with minimal toxic side effects.

[0125] To address the dire clinical prognosis of HCC, we introduced a novel, localized, minimally invasive ablation-induced MΦ hitchhiking system, in which minimally invasive ablation of HCC guides the chemoattractive delivery of drug-loaded MΦs. This strategy involves the formation of LEN@MΦs, where LEN-carrying nanoparticles bind to Escherichia coli membranes and are engulfed by MΦs. Furthermore, the E. coli membrane coating enhances the drug-loading efficiency of MΦs while maintaining their M1 phenotype and improves their ability to modify the tumor immune microenvironment.

[0126] Given that ablation directly destroys tumor tissue, generating an inflammatory gradient centered at the ablation site, native MΦs are capable of chemotaxis along this inflammatory gradient. To this end, a MAMH strategy was designed, leveraging the chemotaxis of LEN@MΦs. Tumor tissue was ablated under ultrasound imaging guidance during surgery, followed by intravenous injection of LEN@MΦs. Because LEN@MΦs share the chemotaxis of native MΦs, they confer chemotaxis along the inflammatory gradient generated by the ablation procedure. The chemotaxis of LEN@MΦs and the subsequent release of LEN enhanced the efficacy of LEN. Overall apoptosis rates indicate that LEN delivery is primarily responsible for the therapeutic effect. In addition to its role in inhibiting angiogenesis, LEN has also been implicated in immunomodulation. However, we observed that LEN administration alone was insufficient to activate a robust antitumor immune response and suppress residual tumors in resected HCC, likely due to its limited accumulation at the tumor site. In contrast, we found that the enhanced drug delivery efficiency in MAMHs resulted in a qualitative shift in LEN's antitumor effects, stimulating systemic immune responses and inducing long-lasting immune memory. LEN showed significant therapeutic effects on the progression of multifocal HCC in both a mouse model and an orthotopic rabbit model using the MΦ hitchhiking strategy.

[0127] This study proposes a universal treatment approach for various HCC types, leveraging ablation-induced natural inflammatory gradients to guide MΦ hitchhiking drug delivery. Its superior targeted delivery and antitumor efficacy were validated in multiple HCC models in mice and rabbits. Current treatments, such as intravenous injection, deliver therapeutic drugs via diffusion throughout the circulatory system, resulting in inefficient tumor delivery. In contrast, the MAMH approach leverages the natural chemotaxis of macrophages to tumors, addressing current limitations in therapeutic delivery. The use of LEN is an example of a clinically effective therapeutic agent for the treatment of HCC. A wide range of first-line drugs, such as sorafenib and reflenib, can also be targeted with the MAMH strategy. Notably, localized minimally invasive ablation, while the preferred approach for early-stage HCC patients, still carries a high recurrence rate. Therefore, our system offers a promising approach in real-world clinical practice, capable of preventing progression and recurrence after ablation. In patients with HCC whose tumors are large, irregular in shape, or near high-risk sites, minimally invasive ablation is often not the preferred treatment due to the risk of incomplete ablation, limiting its widespread application in clinical practice. Our strategic innovations in ablation approaches may contribute to the effective treatment of early-stage HCC, initially considered "unablative," and in some cases, may expand the criteria for ablation beyond early-stage HCC, enabling more patients to be treated with curative approaches. Furthermore, the therapeutic efficacy of the MΦ hitchhiking platform remains poised to be significantly improved through the adoption of more advanced systems, such as cryoablation and radiotherapy.

[0128] Although the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Use of drug-loaded nanoparticles loaded by macrophages in the preparation of a drug for improving the prognosis of hepatocellular carcinoma after ablation, characterized in that: The nano drug-loaded particles include E. coli vesicles and a small molecule active ingredient of a drug encapsulated in the E. coli vesicles. The nano drug-loaded particles are obtained by incubating M1 macrophages with the E. coli vesicles encapsulating the small molecule active ingredient of the drug; the small molecule active ingredient of the drug is any one of lenvatinib, sorafenib, and regorafenib; The preparation method of the drug-loaded nanoparticles comprises the following steps: S1. Preparation of nanoparticles containing small molecule active ingredients of drugs; S2. Mix the E. coli vesicles with the nanoparticles obtained in S1, and extrude them through a polycarbonate membrane with a pore size of 200 nm for at least 21 times (the number of times should be an odd number), and obtain the E. coli vesicles encapsulating the active ingredient of the small molecule drug after washing; S3 and M1 macrophages were incubated with E. coli vesicles encapsulated with small molecule active ingredients obtained from S2 at 37°C for 60 min, and the drug-loaded nanoparticles loaded by macrophages were obtained after washing. The preparation method of the nanoparticles in S1 is to dissolve the small molecule active ingredient of the drug and PLGA in DMSO, add the surfactant TPGS after mixing, and mix to form an oil phase; the oil phase is added dropwise to deionized water, continuously stirred at room temperature, and purified and resuspended to obtain a solution containing nanoparticles.

2. The use according to claim 1, characterized in that The mass ratio of the small molecule active ingredient of the drug to PLGA in S1 is 1:4, the concentration of the small molecule active ingredient of the drug in the oil phase is 0.25 mg / mL, and the concentration of TPGS in the oil phase is 2 mg / mL.

3. The use according to claim 1, characterized in that The volume ratio of the oil phase to the water phase in S1 was 1:

7.

4. The use according to claim 1, characterized in that The content of the drug small molecule active ingredient in the Escherichia coli vesicles encapsulated with the drug small molecule active ingredient obtained by S2 is 0.2~0.225 mg / mL.

5. The use according to claim 1, characterized in that The concentration of M1 macrophages in the S3 incubation system was 2×10 6 cells / mL.

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