Application of DPPA encapsulated AMD3100 nanoparticles in immunotherapy of residual cancer after IRFA of liver cancer

Abstract

The application provides application of DPPA loaded AMD3100 nanoparticles in immunotherapy of residual cancer after liver cancer IRFA. The application finds that CXCR4 up-regulation in residual cancer after IRFA causes MDSC cell recruitment and further mediates insufficient T cell function, and DPPA loaded AMD3100 nanoparticles can effectively activate T cell anti-tumor immunity in residual cancer after IRFA, and the DPPA loaded AMD3100 nanoparticles have strong tumor targeting ability and good safety. Further, when the DPPA loaded AMD3100 nanoparticles are combined with a PD-1 antibody, the DPPA loaded AMD3100 nanoparticles and the PD-1 antibody can synergistically enhance the anti-tumor effect and inhibit residual cancer growth.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of DPPA-encapsulated AMD3100 nanoparticles in the immunotherapy of residual liver cancer after IRFA. Background Technology

[0002] Radiofrequency ablation (RFA) is currently the main method for local ablation treatment of early-stage liver cancer in clinical practice. However, in some patients, due to the large size of the lesion or its proximity to important tissues or blood vessels, incomplete radiofrequency ablation (IRFA) occurs in practice. Studies have shown that residual cancer after IRFA can progress rapidly, leading to poor prognosis for patients.

[0003] The instantaneous high temperature during radiofrequency ablation can generate a large number of tumor antigens, inducing the recruitment of immune cells and providing sufficient conditions for the establishment of innate immunity. Literature indicates that post-RFA procedures can cause... CD8+ T cell infiltration was observed, but IFNγ levels in the residual tumor microenvironment only increased in the first 3 days and decreased significantly within 3-8 days, suggesting that the T-cell immune response induced by antigen exposure after IRFA was suppressed. Currently, the mechanism of residual tumor immunosuppression after radiofrequency ablation of liver cancer is unclear, and effective treatment options are still lacking.

[0004] Dipalmitoyl phospatidic acid (DPPA) is a naturally occurring anionic phospholipid in human cells and a minor metabolite in the synthesis of glycerophospholipids. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide the application of DPPA-encapsulated AMD3100 nanoparticles in the immunotherapy of residual tumor after radiofrequency ablation of liver cancer. DPPA-encapsulated AMD3100 nanoparticles can effectively improve the immunosuppressive state of residual tumor after radiofrequency ablation of liver cancer and exert an anti-residual tumor effect.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] Application of dipalmitoyl phosphatidyl acid-encapsulated AMD3100 nanoparticles in the preparation of immunotherapy drugs for residual tumors after radiofrequency ablation of liver cancer.

[0008] In some embodiments, the dipalmitoyl phosphatidyl acid-encapsulated AMD3100 nanoparticles have a particle size of 30 nm to 450 nm and a potential of -8 mV to 5 mV.

[0009] In some embodiments, the DPPA-encapsulated AMD3100 nanoparticles are prepared by the following method: (1) dissolving DPPA in methanol to obtain a DPPA methanol solution; (2) mixing an aqueous solution of AMD3100 and a G0C14 N,N-dimethylformamide solution, then adding a DSPE-PEG3400 solution and the DPPA methanol solution, and mixing well; (3) slowly adding the mixed solution obtained in step (2) to ultrapure water under high-speed stirring to obtain a nanoparticle solution; and (4) centrifuging the nanoparticle solution through an ultrafiltration tube to obtain the DPPA-encapsulated AMD3100 nanoparticles.

[0010] In some embodiments, the mass ratio of AMD3100, G0C14, DSPE-PEG3400 and DPPA is (1-3):(10-20):(90-110):(70-90).

[0011] In some preferred embodiments, the mass ratio of AMD3100, G0C14, DSPE-PEG3400 and DPPA is 2:15:100:80.

[0012] In some embodiments, the rotation speed is 1000 rpm-1500 rpm.

[0013] In some embodiments, the drug can inhibit the chemotaxis of MDSCs by residual tumor after radiofrequency ablation of liver cancer.

[0014] In some embodiments, the drug can enhance the function and / or proliferative capacity of CD8 + T cells.

[0015] In some embodiments, the drug further comprises a PD-1 antibody.

[0016] The present application also provides an immunotherapy drug for residual tumor after radiofrequency ablation of liver cancer, which comprises DPPA-encapsulated AMD3100 nanoparticles and a PD-1 antibody.

[0017] In some embodiments, the DPPA-encapsulated AMD3100 nanoparticles have a particle size of 30 nm-450 nm and an electric potential of -8 mV-5 mV.

[0018] This invention has revealed that upregulation of CXCR4 in residual hepatocellular carcinoma cells after IRFA (Intra-Invasive Therapy for Cancer) leads to the recruitment of MDSC cells, which in turn mediates T cell insufficiency. Dipalmitoyl phosphatidyl acid-encapsulated AMD3100 nanoparticles can effectively activate T cell anti-tumor immunity in residual hepatocellular carcinoma cells after IRFA, exhibiting strong tumor-targeting ability and good safety. Furthermore, when the dipalmitoyl phosphatidyl acid-encapsulated AMD3100 nanoparticles are used in combination with PD-1 antibodies, they can synergistically enhance the anti-tumor effect and inhibit residual cancer cell growth. Attached Figure Description

[0019] Figure 1 CXCR4 expression, MDSCs infiltration, and CD8 expression in residual cancer cells after IRFA surgery. + Results of T-cell infiltration detection.

[0020] Figure 2 The results show the expression of CXCR4, chemotaxis of MDSCs, and the proliferation, invasion, and migration abilities of hepatocellular carcinoma cells in sublethal thermally damaged hepatocellular carcinoma cells.

[0021] Figure 3 The results are for characterizing DPPA (AMD3100) NPs.

[0022] Figure 4 DPPNPs and DPPA(AMD3100)NPs can inhibit the proliferation, invasion, and migration of tumor cells after sublethal thermal injury.

[0023] Figure 5 AMD3100 and DPPA (AMD3100) NPs can reduce the chemotaxis of tumor cells to MDSCs after sublethal thermal injury and restore CD8. + T cell function and proliferation capacity.

[0024] Figure 6 The AMD3100, anti-PD-1 antibody, DPPNPs, DPPA(AMD3100)NPs, DPPNPs+anti-PD-1 antibody, and DPPA(AMD3100)NPs+anti-PD-1 antibody have no significant toxic side effects on the heart, liver, spleen, lungs, and kidneys.

[0025] Figure 7 In a mouse hepatocellular carcinoma orthotopic tumor IRFA model, the DPPA (AMD3100) NPs + anti-PD-1 antibody treatment group achieved the strongest inhibition of residual tumor growth.

[0026] Figure 8The DPPA (AMD3100) NPs + anti-PD-1 antibody treatment group in the mouse liver cancer orthotopic tumor IRFA model can significantly increase the TUNEL level in residual cancer, reduce the expression of Ki67, increase the CD8 + T cell infiltration, and reduce Treg cell infiltration.

[0027] Figure 9 The AMD3100, anti-PD-1 antibody, DPPA (AMD3100) NPs, DPPA NPs + anti-PD-1 antibody, DPPA (AMD3100) NPs + anti-PD-1 antibody treatment groups in the mouse liver cancer lung metastasis tumor model can inhibit the MDSC infiltration level, CD8 + T cell GranzymeB and IFNγ levels, CD8 + CD44 + The infiltration level of CD8

[0028] Figure 10 The DPPA (AMD3100) NPs + anti-PD-1 antibody treatment group in the mouse liver cancer lung metastasis tumor model can most effectively inhibit tumor growth. DETAILED DESCRIPTION

[0029] The experimental methods in the following examples of the present application not specifically indicated are generally carried out according to conventional conditions, or according to the conditions recommended by the manufacturers. The various common chemical reagents used in the examples are commercially available products.

[0030] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0031] The terms "comprising" and "having" and any variations thereof used in the present application are intended to cover not exclusively containing. For example, a process, method, device, product or apparatus comprising a series of steps is not limited to the listed steps, but optionally also includes steps not listed, or optionally also includes other steps inherent to these processes, methods, products or apparatuses.

[0032] The following specific examples are described below. In the following examples, DPPA (AMD3100) NPs represent AMD3100 nanoparticles encapsulated by dipalmitoyl phosphatidic acid, and DPPA NPs represent dipalmitoyl phosphatidic acid nanoparticles.

[0033] G0C14 is an amphiphilic cationic solution obtained by dissolving an amphiphilic cationic lipid in dimethylformamide, wherein the G0C14 amphiphilic cationic lipid is an amphiphilic cationic lipid synthesized from a dendrimers generation 0 (G0) and a 1,2 epoxytetradecane (C14).

[0034] Example 1

[0035] 1. Experimental methods

[0036] 1) Collect 5 pairs of preoperative and postoperative tumor tissue sections of primary hepatocellular carcinoma patients with IRFA occurrence, and detect the MDSC infiltration level using immunofluorescence technology.

[0037] 2) Construct a mouse liver cancer orthotopic tumor IRFA model: anesthetize, skin, disinfect and expose the liver of 3-5 week old, 15-18 g weight C57BL / 6 male mice, and then close the abdominal cavity after injecting 30 μl of Hepa1-6 cell suspension containing 50% matrix glue with an insulin needle. When the orthotopic tumor volume reaches 50 mm 3 , perform radiofrequency ablation treatment under ultrasound guidance. After inserting the radiofrequency needle into one side of the tumor, stop after 3W treatment for 30s. After hemostasis, close the abdominal cavity.

[0038] 3) Verify the immune indicators in the mouse subcutaneous tumor before and after IRFA: inject 100ul of Hepa1-6 cell suspension containing 50% matrix glue into the left and right sides of the back of 3-5 week old, 15-18 g weight C57BL / 6 male mice. When the tumor grows to 500 mm 3 , remove the left tumor and perform IRFA treatment on the right tumor (conditions same as 2)). Half of the removed left tumor is enzymatically digested into a single cell suspension, and flow cytometry is used to analyze the proportion of MDSC cells and the expression levels of CD8 + T cells Granzyme B and IFNγ in the tumor. The other half is fixed in a 4% paraformaldehyde solution, paraffin-embedded and sectioned. The right tumor is removed on the fourth day, the seventh day and the tenth day, half of which is enzymatically digested into a single cell suspension, and flow cytometry is used to analyze the proportion of (CD11b + Gr-1 + ) MDSC cells and the expression levels of CD8 + T cells Granzyme B and IFNγ in the tumor. The other half is fixed in a 4% paraformaldehyde solution, paraffin-embedded and sectioned.

[0039] 4) Construction of DPPA(AMD3100) NPs: DPPA (purchased from Xi'an Ruishi Biological Technology Co., Ltd., Catalog No. LP-R4-025) was dissolved in methanol (2 mg / ml) and dissolved in water at 70°C. DSPE-PEG MW:3400 (purchased from Xi'an Ruishi Biological Technology Co., Ltd., Catalog No. R-1028-3.4K) was dissolved in methanol (10 mg / ml), G0C14 was dissolved in DMF (5 mg / ml), and AMD3100 (purchased from Selleck Company, Catalog No. S3013, CAS: 155148-31-5) was dissolved in water (2 mg / ml). 10 ul of AMD3100 solution was mixed with 30 ul of G0C14, followed by the addition of 100 ul of DSPE-PEG 3400 solution and 400 ul of DPPA solution. After mixing, 5 ml of ultrapure water was slowly added at a constant speed (1000 rpm / min). After centrifugation through an ultrafiltration tube, the upper liquid was DPPA(AMD3100) NPs.

[0040] 5) Characterization and verification of DPPA(AMD3100) NPs:

[0041] ① The particle size, zeta potential and morphology of the nanodrug were detected using dynamic light scattering and 120 kV transmission electron microscopy.

[0042] ② DPPA(Cy5) NPs and an equal amount of Cy5 solution were placed in dialysis tubes (100 kDa) and soaked in PBS at room temperature and 37°C, respectively. Samples were taken at multiple time points and the Cy5 fluorescence intensity was measured to determine the drug release level of the nanoliposomes. The particle size of the nanodrug at different time points was also detected to determine its stability.

[0043] ③ DPPA(Cy5) NPs and an equal amount of Cy5 solution were added to a six-well plate seeded with Hepa1-6 cells. Cells were collected at different time points and the level of Cy5 inside the cells was detected.

[0044] ④ Six 3-5 week old male C57BL / 6 mice weighing 15-18 g were prepared for skin disinfection, and 1 million Hepa1-6 cells were injected into the skin with a 1 ml syringe. When the tumor volume reached 200 mm 3 The mice were randomly divided into two groups (n=3) and injected with Cy5 and DPPA(Cy5) NPs via the tail vein, respectively. The heart, liver, spleen, lung, kidney, tumor and muscle tissues of the mice were analyzed for tumor enrichment of the nanomaterials.

[0045] ⑤Twenty-one 6-8-week-old male C57BL / 6 mice weighing 19-22 g were randomly divided into seven groups (n = 3): PBS, AMD3100, anti-PD-1 antibody, DPPANPs, DPPA(AMD3100)NPs, DPPANPs + anti-PD-1 antibody, and DPPA(AMD3100)NPs + anti-PD-1 (In Vivo Mab anti-mouse PD-1, manufacturer: BioXcell, Cat# BE0146). After continuous injection of drugs through the tail vein for 3 days, serum and heart, liver, spleen, lung, and kidney were collected to analyze the toxicity of nanomedicines in vivo.

[0046] 6) Sublethal heat-injured tumor cell construction: Hepa1-6 cells were cultured in a 10 cm dish, and when the cell confluence reached about 70-80%, they were heated in a water bath at 45°C for 10 min to obtain sublethal heat-injured tumor cells.

[0047] 7) Mouse primary MDSCs extraction: The hind limbs of 6-8-week-old male C57BL / 6 mice were taken, and after the skin and muscle tissue were stripped, the leg bones were exposed. The bone marrow was flushed with DMEM high-glucose medium and filtered through a 70-μm filter. Mouse bone marrow primary cells were obtained by centrifugation. Mouse primary MDSCs were obtained by flow sorting and cultured in RMI1640 medium containing GM-CSF (20 ng / ml) and IL-4 (20 ng / ml).

[0048] 8) Mouse primary CD8 + T cell extraction and activation: The spleen of the mouse in 7) was taken, ground, and passed through a 40-μm filter. After centrifugation at 400 g / min for 5 min, red blood cells were removed using red blood cell lysis solution, washed once with PBS, and centrifuged again at 400 g / min for 5 min. CD8 + T cells were sorted by magnetic beads and cultured in 1640 medium containing CD3 and CD28 antibodies for 24 h.

[0049] 9) CXCR4 level and chemotactic ability of sublethal heat-injured tumor cells to MDSCs:

[0050] ① The control group and Hepa1-6 cells at different time points after sublethal heat injury were collected, and their CXCR4 expression levels were detected by PCR, WB, and immunofluorescence.

[0051] ②Sublethal heat-injured Hepa1-6 cells were seeded in the lower chamber of Transwell. After the cells were completely adhered, complete medium, AMD3100, DPPANPs, DPPA(AMD3100)NPs were added respectively. After the same amount of MDSCs were added in the upper chamber, the mixture was cultured in the incubator for 8 hours. After fixation and staining with 4% paraformaldehyde, the number of MDSCs crossing the transwell was observed.

[0052] 10) Verify the proliferation, invasion and migration ability of tumor cells after sublethal heat injury by CCK8 test, plate cloning, invasion and migration test after treatment with complete medium, AMD3100, DPPANPs, DPPA(AMD3100)NPs.

[0053] 11) Analysis of the proliferation level of CD8 + T cells and the expression level of Granzyme B and IFNγ after co-culture of MDSC cells with activated CD8 + T cells in different proportions.

[0054] 12) Evaluation of the therapeutic effect of DPPA(AMD3100)NPs on the progression of residual cancer after IRFA:

[0055] ①Mice with liver cancer IRFA model were randomly divided into seven groups: PBS, AMD3100, anti-PD-1 antibody, DPPANPs, DPPA(AMD3100)NPs, DPPANPs+anti-PD-1 antibody, DPPA(AMD3100)NPs+anti-PD-1. From the second day after ablation, drugs were administered via the tail vein once a day for a total of three times, and anti-PD-1 antibody was administered via intraperitoneal injection every three days. At the same time, the volume of residual cancer after liver cancer ablation was observed every three days by small animal ultrasonic three-dimensional scanning, and the body weight of the mice was recorded.

[0056] ②Verify the immune activation in the residual cancer after treatment: the mice were sacrificed and the residual cancer was removed. Half of the residual cancer was enzymatically digested into a single cell suspension, and the MDSC cell infiltration and the expression of CD8 + T cell granzyme B and IFNγ were observed by flow cytometry. The other half of the residual cancer was treated with 4% paraformaldehyde and paraffin-embedded and sectioned, and the infiltration of CD8 + T cells and Treg cells was observed by immunohistochemistry.

[0057] ③Verify the changes in proliferation and apoptosis of residual cancer: observe the proliferation (Ki67) and apoptosis (TUNEL) of residual cancer by immunohistochemical technique.

[0058] 13) DPPA(AMD3100)NPs inhibits lung metastasis of liver cancer:

[0059] ① Constructing a mouse model of liver cancer lung metastasis: 3-5 week old male C57BL / 6 mice weighing 15-18g were injected with 5 million luc-Hepa1-6 cells via the tail vein, and the luminescence of the lungs was observed by in vivo imaging one week later.

[0060] ② After observing lung luminescence, mice were randomly divided into seven groups: PBS, AMD3100, anti-PD-1 antibody, DPPNPs, DPPA(AMD3100)NPs, DPPNPs + anti-PD-1 antibody, and DPPA(AMD3100)NPs + anti-PD-1. Administered via tail vein for three consecutive days, and anti-PD-1 antibody was administered intraperitoneally every three days. Lung luminescence was detected by in vivo imaging every three days, and mouse weight and survival time in each group were recorded.

[0061] 2. Experimental Results

[0062] Experimental results are as follows Figures 1-10 As shown:

[0063] CXCR4 was upregulated and MDSCs infiltration was increased in residual cancer cells after IRFA; CD8 was also found in residual cancer cells after IRFA. + T cell infiltration increases, but their functional indicators decline over time, suggesting that their function is suppressed. Figure 1 ). Figure 1 In the middle, (A) the level of MDSC cell infiltration in recurrent tumors of liver cancer patients before and after ablation; (B) the level of MDSCs and CD8 in tumors of subcutaneous liver cancer in mice before and after incomplete radiofrequency ablation. + Quantitative statistical analysis of T cell infiltration levels at different time points; (C) Granzyme B before and after incomplete radiofrequency ablation of subcutaneous hepatocellular carcinoma in mice. + CD8 + T cells, IFN-γ + CD8 + (D) Quantitative statistics of T cells at different time points; (E) Protein expression level of CXCR4 in the tumor at different time points before and after incomplete radiofrequency ablation of subcutaneous hepatocellular carcinoma in mice; (D) Expression level of CXCR4 mRNA in the tumor at different time points before and after incomplete radiofrequency ablation of subcutaneous hepatocellular carcinoma in mice.

[0064] In sublethal thermally injured tumor cells, CXCR4 expression gradually increased over time; the chemotactic ability of tumor cells to MDSCs was enhanced after sublethal thermal injury; and the proliferation, invasion, and migration abilities of tumor cells were enhanced after sublethal thermal injury. Figure 2). Among them, (A) the change of CXCR4 at mRNA level at different time points after sublethal heat injury of Hepa1-6 cells; (B) the change of CXCR4 at different time points after sublethal heat injury of Hepa1-6 cells analyzed by WB experiment; (C) the change of CXCR4 at different time points after sublethal heat injury of Hepa1-6 cells analyzed by immunofluorescence experiment; (D) the schematic diagram of chemotaxis experiment of MDSCs to Hepa1-6 cells after sublethal heat injury; (E) the chemotaxis of MDSCs to Hepa1-6 cells after sublethal heat injury; (F) the quantitative statistical diagram of (E); (G) the proliferation of Hepa1-6 cells after sublethal heat injury; (H) the migration and invasion ability analysis of Hepa1-6 cells after sublethal heat injury; (I) the quantitative statistical diagram of (H).

[0065] As shown in Figure 3 , the AMD3100-loaded nanoliposomes were successfully constructed based on dipalmitoyl phosphatidic acid (DPPA), with particle size of 30 nm-450 nm, electric potential of-8 mV-5 mV, and drug stability and 48 h release were proved. Among them, (A) the mode diagram of DPPA(AMD3100)NPs; (B) the electron microscope diagram of DPPA(AMD3100)NPs; (C) the particle size diagram of DPPA(AMD3100)NPs; (D) the electric potential diagram of DPPA(AMD3100)NPs; (E) the uptake of Cy5 and DPPA(Cy5)NPs in Hepa1-6 cells at different time points; (F) the drug release of DPPA(Cy5)NPs at room temperature and 37℃ environment; (G) the stability of DPPA(AMD3100)NPs in room temperature PBS, 37℃ PBS and 37℃ complete medium.

[0066] As shown in Figure 4 , DPPANPs and DPPA(AMD3100)NPs can inhibit the proliferation, invasion and migration ability of tumor cells after sublethal heat injury. Among them, (A) plate colony experiment of Hepa1-6 cells after sublethal heat injury treated with complete medium, AMD3100, DPPANPs and DPPA(AMD3100)NPs; (B) CCK8 experiment of Hepa1-6 cells after sublethal heat injury treated with complete medium, AMD3100, DPPANPs and DPPA(AMD3100)NPs; (C) migration and invasion experiment of Hepa1-6 cells after sublethal heat injury treated with complete medium, AMD3100, DPPANPs and DPPA(AMD3100)NPs; (D) the quantitative statistical diagram of (C).

[0067] As shown in Figure 5As shown, AMD3100 and DPPA (AMD3100) NPs can reduce the chemotaxis of tumor cells to MDSCs after sublethal thermal injury, and the reduction of MDSCs can effectively restore CD8. + The function and proliferative capacity of T cells. Among them, (A) the chemotaxis of Hepa1-6 cells to MDSCs after treatment with complete culture medium, AMD3100, DPPNPs, and DPPA (AMD3100) NPs following sublethal heat injury; (B) a schematic diagram of (A); (C) a quantitative statistical graph of (A); (D) activated CD8+. + (E) shows the proliferation level of T cells co-cultured with different proportions of MDSCs; (D) is a quantitative statistical graph of (D); (F) shows the activated CD8+. + Expression of Granzyme B and IFNγ in T cells co-cultured with different proportions of MDSCs; (G) is a quantitative statistical graph of (F).

[0068] like Figure 6 As shown, DPPA(Cy5)NPs exhibited significantly higher enrichment capacity in tumor tissue than Cy5. Furthermore, AMD3100, anti-PD-1 antibody, DPPNPs, DPPA(AMD3100)NPs, DPPNPs + anti-PD-1 antibody, and DPPA(AMD3100)NPs + anti-PD-1 antibody showed no significant toxic side effects on the heart, liver, spleen, lungs, or kidneys. Among them, (A) the distribution of Cy5 and DPPA(Cy5)NPs in major organs and tumors in a mouse subcutaneous hepatocellular carcinoma model; (B) is a quantitative statistical graph of (A); (C) H&E staining of major organs in mice after treatment with PBS, AMD3100, anti-PD-1 antibody, DPPNPs, DPPA(AMD3100)NPs, DPPNPs + anti-PD-1 antibody, and DPPA(AMD3100)NPs + anti-PD-1; (D) serum ALT, AST, BUN, and Crea levels in mice after treatment with PBS, AMD3100, anti-PD-1 antibody, DPPNPs, DPPA(AMD3100)NPs, DPPNPs + anti-PD-1 antibody, and DPPA(AMD3100)NPs + anti-PD-1.

[0069] like Figure 7As shown, in the mouse hepatocellular carcinoma orthotopic tumor IRFA model, the DPPA(AMD3100)NPs + anti-PD-1 antibody treatment group achieved the strongest inhibition of residual tumor growth. Among them, (A) is a schematic diagram of the mouse hepatocellular carcinoma orthotopic tumor IRFA model treatment pattern; (B) ultrasound images of mice at different time points after treatment with PBS, AMD3100, anti-PD-1 antibody, DPPNPs, DPPA(AMD3100)NPs, DPPNPs + anti-PD-1 antibody, and DPPA(AMD3100)NPs + anti-PD-1; (C) changes in tumor volume in different groups of mice after treatment with PBS, AMD3100, anti-PD-1 antibody, DPPNPs, DPPA(AMD3100)NPs, DPPNPs + anti-PD-1 antibody, and DPPA(AMD3100)NPs + anti-PD-1; and (D) is a summary statistical chart of (C).

[0070] like Figure 8 As shown, in a mouse hepatocellular carcinoma orthotopic tumor IRFA model, the DPPA (AMD3100) NPs + anti-PD-1 antibody treatment group significantly increased TUNEL levels in residual tumor cells, decreased Ki67 expression, and increased CD8+ expression. + T cell infiltration and reduced Treg cell infiltration. Among them, (A) mouse hepatocellular carcinoma orthotopic tumor IRFA model after treatment with PBS, AMD3100, anti-PD-1 antibody, DPPA NPs, DPPA(AMD3100) NPs, DPPNPs + anti-PD-1 antibody, and DPPA(AMD3100) NPs + anti-PD-1, H&E staining and TUNEL, Ki67, CD8a and FoxP3 immunohistochemical staining images; (B) is the quantitative statistical graph of (A).

[0071] like Figure 9 As shown, in a mouse hepatocellular carcinoma orthotopic tumor (IRFA) model, the treatment groups of AMD3100, anti-PD-1 antibody, DPPA(AMD3100)NPs, DPPAMPs + anti-PD-1 antibody, and DPPA(AMD3100)NPs + anti-PD-1 antibody all reduced MDSC infiltration. The DPPA(AMD3100)NPs + anti-PD-1 antibody treatment group maximally activated CD8+ T cell Granzyme B and IFNγ levels and increased CD8+ T cell activity. + CD44 + T cell infiltration levels. Among them, (A) mouse hepatocellular carcinoma orthotopic tumor IRFA model after treatment with PBS, AMD3100, anti-PD-1 antibody, DPPNPs, DPPA(AMD3100)NPs, DPPNPs + anti-PD-1 antibody, and DPPA(AMD3100)NPs + anti-PD-1, showed CD11b+Gr1+ cells and CD8+ cells. +Granzyme B + T cells, CD8 + IFNγ + T cells, CD8 + CD44 + T cell levels; (B) is a quantitative statistical chart of (A).

[0072] As Figure 10 shown, in the mouse liver cancer lung metastasis model, the DPPA (AMD3100) NP + anti-PD-1 antibody treatment group can inhibit tumor growth to the greatest extent. Among them, (A) is a mouse liver cancer lung metastasis model treatment mode chart; (B) is a body weight change chart of mice treated with PBS, AMD3100, anti-PD-1 antibody, DPPA NPs, DPPA (AMD3100) NPs, DPPA NPs + anti-PD-1 antibody, DPPA (AMD3100) NPs + anti-PD-1; (C) is a survival analysis chart of mice treated with PBS, AMD3100, anti-PD-1 antibody, DPPA NPs, DPPA (AMD3100) NPs, DPPA NPs + anti-PD-1 antibody, DPPA (AMD3100) NPs + anti-PD-1; (D) is a live imaging luminescence chart of mice treated with PBS, AMD3100, anti-PD-1 antibody, DPPA NPs, DPPA (AMD3100) NPs, DPPA NPs + anti-PD-1 antibody, DPPA (AMD3100) NPs + anti-PD-1 at different time points.

[0073] In summary, the DPPA encapsulated AMD3100 nanoparticle can effectively activate the T cell anti-tumor immunity in the residual cancer of the liver cancer after IRFA, and has strong tumor targeting ability and good safety. Moreover, when the DPPA encapsulated AMD3100 nanoparticle is combined with the PD-1 antibody, it can synergistically enhance the anti-tumor effect and inhibit the growth of residual cancer.

[0074] Each technical feature of the above-described embodiments can be combined arbitrarily. In order to make the description concise, each technical feature of the above-described embodiments is not described in all possible combinations, but it should be considered that any combination of these technical features is within the scope of the present disclosure, as long as the combination does not contradict.

[0075] The above-described embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it should not be understood as limiting the scope of the present patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the protection scope of the present patent should be subject to the appended claims.

Claims

1. Use of dipalmitoyl phosphatidic acid-encapsulated AMD3100 nanoparticles in the preparation of an immunotherapy drug for residual tumor after radiofrequency ablation of liver cancer.

2. Use according to claim 1, wherein The dipalmitoyl phosphatidic acid-encapsulated AMD3100 nanoparticles have a particle size of 30 nm to 450 nm and a potential of -8 mV to 5 mV.

3. The use according to claim 1, wherein The dipalmitoyl phosphatidic acid-encapsulated AMD3100 nanoparticles are prepared by the following method: (1) dissolving dipalmitoyl phosphatidic acid in methanol to obtain a dipalmitoyl phosphatidic acid methanol solution; (2) mixing an AMD3100 aqueous solution and a G0C14 N,N-dimethylformamide solution, then adding a DSPE-PEG3400 solution and the dipalmitoyl phosphatidic acid methanol solution, and mixing uniformly; (3) slowly and uniformly adding the mixed solution obtained in step (2) to high-speed stirring ultrapure water to obtain a nano solution; and (4) centrifuging the nano solution through an ultrafiltration tube to obtain the dipalmitoyl phosphatidic acid-encapsulated AMD3100 nanoparticles.

4. The use according to claim 3, wherein the compound is ###0002### The mass ratio of the AMD3100, G0C14, DSPE-PEG3400, and dipalmitoyl phosphatidic acid is (1-3):(10-20):(90-110):(70-90).

5. The use according to claim 4, wherein the compound is ###0002### The mass ratio of the AMD3100, G0C14, DSPE-PEG3400, and dipalmitoyl phosphatidic acid is 2:15:100:

80.

6. The use according to claim 3, wherein the compound is ###0002### In (3), the rotation speed of the high-speed stirring is 1000 rpm to 1500 rpm.

7. The use according to claim 1, wherein The drug can inhibit the chemotaxis of residual tumor after radiofrequency ablation of liver cancer to MDSCs; and / or, The medicaments can enhance the function and / or proliferative capacity of CD8 + T cells.

8. Use according to any one of claims 1 to 6, wherein The drug further comprises a PD-1 antibody.

9. An immunotherapeutic drug for residual tumor after radiofrequency ablation of liver cancer, characterized by, The immunotherapy drug comprises dipalmitoyl phosphatidic acid-encapsulated AMD3100 nanoparticles and a PD-1 antibody, wherein the dipalmitoyl phosphatidic acid-encapsulated AMD3100 nanoparticles have a particle size of 30 nm to 450 nm and a potential of -8 mV to 5 mV.

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