A macrophage drug delivery system loaded with nifurquinazone and application thereof

Abstract

The application discloses a macrophage drug delivery system loaded with nitrofurazone and application thereof, and belongs to the technical field of antitumor drugs. The drug delivery system is formed by co-incubation of nitrofurazone in macrophages. The application uses macrophages as natural drug delivery carriers to effectively load nitrofurazone and realize tumor-targeted delivery. In-vivo antitumor research shows that the macrophages carrying nitrofurazone can significantly inhibit tumor growth and reshape the tumor immune microenvironment; mechanism research reveals that nitrofurazone can significantly increase the M1 / M2 macrophage ratio to realize antitumor immune activation. The macrophage carrying strategy provides a new idea for the clinical conversion and application of nitrofurazone.

Description

Technical Field

[0001] This invention relates to the field of antitumor drug technology, specifically to a macrophage drug delivery system loaded with nifurazolidone and its application. Background Technology

[0002] With advancements in technology, the treatment of hepatocellular carcinoma (HCC) has made significant progress. Current clinical interventions include liver resection, liver transplantation, radiofrequency ablation (RFA), transarterial chemotherapy and embolization (TACE), radiotherapy, and comprehensive treatment. Surgery is the ideal treatment for HCC; however, many patients are diagnosed too late, missing the optimal surgical window. Therefore, drug-based TACE and targeted immunotherapy-based comprehensive treatment are crucial for HCC patients who have lost the opportunity for surgery. However, the prognosis for HCC remains less than ideal. For patients with advanced-stage disease, TACE and targeted drug therapy can extend median survival by approximately 6 to 16 months. Furthermore, HCC exhibits high heterogeneity and is prone to drug resistance, with some patients showing low responsiveness to existing drug therapies. Therefore, it is necessary to develop new, safe, and effective drug treatment regimens to complement existing HCC treatment strategies.

[0003] Drug repurposing is a strategy that utilizes drugs already proven effective in one area of ​​medicine for use in other areas. By applying established non-oncology drugs to cancer treatment, the significant time, human, and material costs associated with developing new drugs can be avoided. Furthermore, the well-known pharmacological properties and side effects allow these drugs to quickly enter clinical trials. In addition, these drugs often have complete industrial manufacturing systems, resulting in relatively lower prices, which aligns with the economic interests of cancer patients. Based on these advantages, drug repurposing has become a crucial method in the development of anti-tumor drugs.

[0004] In the field of liver cancer treatment, numerous studies have explored the anti-liver cancer activity of commonly used drugs. For example, metformin, used to treat type II diabetes, has been shown to inhibit liver cancer cell proliferation, induce apoptosis, and regulate the tumor immune microenvironment. Other drugs, such as the nonsteroidal anti-inflammatory drug aspirin, the lipid-lowering drug simvastatin, and the mTOR inhibitor rapamycin, have also shown certain anti-liver cancer activity. Therefore, repurposing existing drugs has great potential in liver cancer treatment, and further screening for potential anti-liver cancer drugs among commonly used medications is a key research focus.

[0005] Nifurazide is a widely used antibacterial drug that has shown good therapeutic effects in treating infectious diarrhea. Subsequent studies have shown that nifurazide possesses highly potent STAT3 pathway inhibitory activity and exerts antitumor effects. For example, Yang et al. reported that nifurazide significantly downregulated the phosphorylation level of STAT3 at tyrosine 705 (p-STAT3Tyr705), thereby inhibiting breast cancer cell activity (YANG F, et al., 2015). Similarly, nifurazide inhibits the growth of colon cancer, melanoma, and osteosarcoma (YE TH, et al., 2017; ZHU Y, et al., 2016; LUO Y, et al., 2019). Given the important role of abnormal STAT3 pathway activation in the development and progression of liver cancer, the application of nifurazide in liver cancer treatment is expected to further enrich the comprehensive treatment system for liver cancer, thus providing strategies and ideas for developing novel drug regimens.

[0006] However, nifurazolidone also has certain limitations, such as its low water solubility, which means it can only be taken orally, resulting in low bioavailability and occasional gastrointestinal side effects (BAILLY C, 2019). To improve the treatment efficacy for extraintestinal tumors, it is necessary to design more rational nifurazolidone administration strategies, which will help improve its antitumor efficacy and reduce the occurrence of toxic side effects. Summary of the Invention

[0007] The purpose of this invention is to provide a drug delivery system loaded with nifurazolidone for the treatment of extraintestinal tumors, so as to achieve targeted and efficient delivery of nifurazolidone, overcome the disadvantages of poor water solubility and low bioavailability of nifurazolidone itself, and improve its anti-tumor performance.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The present invention provides a macrophage drug delivery system loaded with nifurazolidone, wherein the drug delivery system is formed by co-incubating nifurazolidone within macrophages.

[0010] This invention utilizes macrophages as drug carriers for nifurazolidone. Studies have shown that macrophages have the ability to phagocytose and release drugs as well as target tumors, and can accumulate and release nifurazolidone in the tumor area, thus exerting a significant anti-tumor effect.

[0011] Furthermore, the drug delivery system contains 370–450 ng of nifurazolidone per 10,000 macrophages.

[0012] Furthermore, the preparation method of the drug delivery system includes: firstly, activating and culturing macrophages and then starving them in a serum-free culture medium for 1 hour; then removing the serum-free culture medium and adding a complete culture medium containing nifurazolidone for co-incubation for 2-4 hours to obtain the nifurazolidone-loaded macrophage drug delivery system.

[0013] Specifically, the culture medium is DMEM medium, and the complete culture medium is DMEM medium containing 10% fetal bovine serum.

[0014] This invention demonstrates that macrophages can effectively carry nifurazolidone, and co-incubation of the two for 2–4 hours has little effect on macrophage activity.

[0015] Furthermore, the macrophages are RAW264.7 cells or bone marrow-derived macrophages.

[0016] The present invention also provides the application of the aforementioned nifurazolidone-loaded macrophage drug delivery system in the preparation of antitumor drugs.

[0017] This invention utilizes macrophages loaded with nifurazolidone to form a drug delivery system, which not only achieves targeted drug delivery but also leverages nifurazolidone's polarization regulation of macrophages to activate anti-tumor immunity. Specifically, macrophages possess strong plasticity and can polarize into M1, M2, or M2-biased forms under different environmental stimuli, thus performing different functions. For example, during liver cancer formation, macrophages gradually become immunosuppressive tumor-associated macrophages (biased towards M2) in the tumor-specific microenvironment, participating in mediating angiogenesis, tumor metastasis, drug resistance formation, and malignant transformation of tumor stem cells. In this study, an in vitro macrophage polarization induction system was constructed using tumor supernatant, and nifurazolidone was used for intervention. The results showed that nifurazolidone can promote M1 macrophage polarization and significantly upregulate the M1 / M2 macrophage ratio. Therefore, the nifurazolidone-loaded macrophage drug delivery system provided by this invention can reshape the tumor immune microenvironment, activate anti-tumor immune responses, and significantly inhibit tumor growth.

[0018] Furthermore, the drug activates the anti-tumor immune response by upregulating the ratio of M1 / M2 macrophages, thereby achieving a tumor-killing effect.

[0019] The drug delivery system provided by this invention utilizes nifurazolidone to exert anti-tumor effects by inhibiting the STAT3 pathway; and by inducing M1 macrophage polarization in the tumor microenvironment, thereby activating the immune response and achieving tumor killing. This drug delivery system achieves a synergistic anti-tumor effect through the combined action of different pathways.

[0020] Furthermore, the tumor is an extraintestinal solid tumor, which may be, but is not limited to, hepatocellular carcinoma.

[0021] Furthermore, the active ingredient in anti-hepatocellular carcinoma drugs also includes oxaliplatin. This invention demonstrates that macrophage-loaded nifurazolidone significantly enhances the anti-hepatocellular carcinoma effect of oxaliplatin.

[0022] The present invention also provides a combination drug for treating hepatocellular carcinoma, comprising a first formulation formed by the macrophage drug delivery system loaded with nifurazolidone and a pharmaceutically acceptable carrier, and a second formulation formed by oxaliplatin and a pharmaceutically acceptable carrier.

[0023] The drug can be in the form of, but is not limited to, an injectable formulation; further, the first formulation is an intravenous injection formulation.

[0024] In this invention, the macrophage drug delivery system loaded with nifurazolidone, when used in combination with oxaliplatin, exhibits a synergistic anti-hepatocellular carcinoma effect. The first and second formulations can be administered at different times or simultaneously. The dosage and frequency of administration can be adjusted according to actual circumstances.

[0025] Furthermore, the mass ratio of nifurazolidone to oxaliplatin in the drug is 2:1. Specifically, the first formulation is administered at a dose of 5 mg / kg based on nifurazolidone, and the second formulation is administered at a dose of 2.5 mg / kg based on oxaliplatin.

[0026] The beneficial effects of this invention are as follows:

[0027] This invention provides a macrophage-based drug delivery system loaded with nifurazolidone. Macrophages, as natural drug delivery carriers, can effectively load nifurazolidone and achieve tumor-targeted delivery. In vivo anti-tumor studies have shown that macrophages loaded with nifurazolidone significantly inhibit tumor growth and remodel the tumor immune microenvironment, synergistically exerting anti-tumor effects. Mechanistic studies reveal that nifurazolidone can significantly upregulate the M1 / M2 macrophage ratio, thereby activating anti-tumor immunity. Animal experiments show that macrophages treated with nifurazolidone did not experience significant changes in body weight, indicating high biocompatibility. This invention provides a new approach for the clinical translation of nifurazolidone. Attached Figure Description

[0028] Figure 1 The UV-Vis spectrophotometer was used to detect that nifurazolidone reached its peak UV absorbance at a wavelength of 388 nm.

[0029] Figure 2 This is the absorbance / concentration standard curve for nifurazolidone.

[0030] Figure 3 The dosage of nifurazolidone for macrophages after different incubation times.

[0031] Figure 4Mass spectrometry detection of Cy5-labeled nifurazolidone.

[0032] Figure 5 Confocal image of macrophages loaded with Cy5-labeled nifurazolidone. Scale bar: 200 μm; Green fluorescence: CFSE-labeled macrophages; Red fluorescence: Cy5-labeled nifurazolidone; Blue fluorescence: DAPI-labeled cell nuclei.

[0033] Figure 6 The cell activity of macrophages treated with different incubation times at various time points after drug loading.

[0034] Figure 7 The in vitro drug release rate of nifurazolidone loaded onto macrophages.

[0035] Figure 8 Laser confocal microscopy image of drug delivery between macrophages and tumor cells. Scale bar: 200 μm; Green fluorescence: CFSE-labeled tumor cells; Red fluorescence: Cy5-labeled nifurazolidone; Blue fluorescence: DAPI-labeled cell nuclei.

[0036] Figure 9 This is a flow cytometry plot showing drug delivery between macrophages and tumor cells. In plot A, the flow cytometry gating circles sequentially indicate sampled cells, single cells, live cells, and CFSE-labeled tumor cells. Plot B shows the Cy5 fluorescence signal intensity in the two groups of tumor cells. Macrophages carrying Cy5-labeled nifurazolidone; Macrophages were used as a control group, with untreated macrophages co-incubated with CFSE-labeled tumor cells.

[0037] Figure 10 Effect of supernatant containing nifurazolidone on Huh7 cell viability; NC: normal culture medium; Macrophages carry conditional supernatant containing nifurazolidone.

[0038] Figure 11 The in vivo distribution of Cy5-labeled nifurazolidone loaded onto macrophages was shown, with A representing fluorescence signals in tumor regions detected by an in vivo imaging system; B representing fluorescence imaging of isolated organs and tumors; and C representing quantitative analysis of fluorescence signals in tumors and kidneys. n=3; Macrophages carrying Cy5-labeled nifurazolidone; Free-Cy5: Cy5 without any treatment; He: heart; Lu: lung; Li: liver; Sp: spleen; Ki: kidney; Tu: tumor.

[0039] Figure 12 The gating procedure for macrophage flow cytometry detection. The flow cytometry gating sequentially circles lymphocytes, single cells, live cells, and CD45. + Cells, F4 / 80+ Cells and CD11b + cell.

[0040] Figure 13 Nifurazolidone upregulated the polarization ratio of M1 / M2 macrophages. In this study, A represents the polarization of RAW264.7 cells after induction; B represents the polarization of bone marrow-derived macrophages after induction; BMDM: bone marrow-derived macrophages; TCM: tumor conditional supernatant; TCM-NFX: tumor conditional supernatant containing nifurazolidone.

[0041] Figure 14 To investigate the inhibition of H22 cell-derived subcutaneous tumor growth by nifurazolidone-induced macrophages, A shows tumor images for each group; B shows tumor weight for each group; and C shows tumor volume change curves for each group. n=5; Macrophages induced by tumor conditional supernatant; Macrophages induced by tumor conditional supernatant containing nifurazolidone.

[0042] Figure 15 To investigate the inhibition of Hepa1-6 cell-derived subcutaneous tumor growth by nifurazolidone-induced macrophages, A shows tumor images for each group; B shows tumor weight for each group; and C shows tumor volume change curves for each group. n=5; Macrophages induced by tumor conditional supernatant; Macrophages induced by tumor conditional supernatant containing nifurazolidone.

[0043] Figure 16 For the flow cytometry detection of tumor-infiltrating T cells, the gating process sequentially circles lymphocytes, single cells, live cells, and CD45. + Cells, CD3 + Cells and CD8 + cell.

[0044] Figure 17 To promote the upregulation of intratumoral IFN-γ in macrophages induced by nifurazolidone + CD8 + The proportion of T cell infiltration, where A represents IFN-γ in subcutaneous tumors derived from H22 cells. + CD8 + T cell percentage; B represents IFN-γ in subcutaneous tumors derived from Hepa1-6 cells. + CD8 + T cell ratio. Macrophages induced by tumor conditional supernatant; Macrophages induced by tumor conditional supernatant containing nifurazolidone.

[0045] Figure 18Macrophages were loaded with nifurazolidone to inhibit the growth of H22 cell-derived subcutaneous tumors. Figure A shows tumor images for each group; Figure B shows tumor volume change curves for each group; and Figure C shows tumor weight for each group. n=5; NC: saline control; NFX: nifurazolidone. Untreated macrophages; Macrophages carry nifurazolidone.

[0046] Figure 19 To inhibit the growth of Hepa1-6 cell-derived subcutaneous tumors, macrophages were loaded with nifurazolidone. Figure A shows tumor images for each group; Figure B shows tumor volume change curves for each group; and Figure C shows tumor weight for each group. n=5; NC: saline control; NFX: nifurazolidone. Untreated macrophages; Macrophages carry nifurazolidone.

[0047] Figure 20 The graph shows the change in body weight of mice after drug administration. NC: saline control; NFX: nifurazolidone; Untreated macrophages; Macrophages carry nifurazolidone.

[0048] Figure 21 To inhibit the growth of H22 cell-derived hepatocellular carcinoma in situ by loading macrophages with nifurazolidone combined with oxaliplatin, the following data are presented: A: images of hepatocellular carcinoma in situ in each group; B: tumor volume in each group; C: tumor weight in each group. n=6; NC: saline control; OXA: oxaliplatin. Macrophages carrying nifurazolidone; Macrophages were loaded with nifurazolidone in combination with oxaliplatin. Detailed Implementation

[0049] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0051] Pharmaceuticals: Nifurazolidone (HY-B1436) and oxaliplatin (HY-17371) were purchased from MCE.

[0052] Experimental cell lines: Human hepatocellular carcinoma cell line Huh7, mouse hepatocellular carcinoma cell line H22, and mouse macrophage cell line RAW264.7 were all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Mouse hepatocellular carcinoma cells Hepa1-6 were purchased from the American Type Culture Collection (ATCC) cell bank in the United States.

[0053] Laboratory animals: C57BL / 6 mice (5 weeks old, male) and BALB / C mice (5 weeks old, male) were purchased from Hangzhou Ziyuan Biotechnology Co., Ltd. All animals were housed in an SPF-grade environment in the animal laboratory of the First Affiliated Hospital of Zhejiang University. All animal experiments were conducted in accordance with relevant ethical guidelines and approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine.

[0054] The following examples involve common experimental methods:

[0055] 1. RAW264.7 cell culture and passage

[0056] (1) RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum.

[0057] (2) Observe whether the cell morphology is round, and start passage when the cell growth density reaches about 70%.

[0058] (3) Remove the original culture medium, slowly add 2 mL of PBS to the culture dish, and gently shake the culture dish to ensure that the PBS is in full contact with the cells.

[0059] (4) Remove the PBS, gently add 3 mL of complete culture medium to the culture dish, blow off the cells (if the cells are difficult to blow off, they may have been polarized), and collect them into a 15 mL centrifuge tube.

[0060] (5) Pre-cool the centrifuge to 4°C and centrifuge the cells at 1000 rpm for 5 minutes.

[0061] (6) Discard the supernatant, resuspend the cells in 3 mL of complete culture medium, and then inoculate 1 mL of the cell suspension into a new culture dish.

[0062] (7) Add the required complete culture medium, gently blow and shake to mix, and then transfer to an incubator (37°C, 5% CO2) for incubation.

[0063] 2. H22 cell culture and passage

[0064] (1) H22 cells were cultured as suspension cells in 1640 medium containing 10% fetal bovine serum.

[0065] (2) After seeding the cells in a culture dish, closely observe their morphology and growth density. When the growth density reaches 40%-50%, passage them.

[0066] (3) Take the culture medium containing cells into a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes.

[0067] (4) After discarding the supernatant, resuspend the cells in 4-5 mL of complete culture medium, and take 1 mL of the cell suspension and seed it in a new culture dish.

[0068] (5) Add the required complete culture medium, gently blow and shake to mix, and then transfer to an incubator (37°C, 5% CO2) for incubation.

[0069] 3. Determination of nifurazolidone UV absorbance / concentration standard curve

[0070] (1) Nifurazide was dissolved in dimethyl sulfoxide to prepare a 10 mg / mL nifurazide stock solution.

[0071] (2) Dilute the mother liquor with dimethyl sulfoxide to prepare gradient concentrations of nifurazide (1.25 μg / mL, 5 μg / mL, 10 μg / mL, 12.5 μg / mL).

[0072] (3) The absorbance of nifurazolidone at a wavelength of 388 nm at each concentration was measured using an ultraviolet spectrophotometer.

[0073] (4) Plot a concentration / absorbance standard curve based on the concentration and absorbance.

[0074] As measured by a UV spectrophotometer, nifurazolidone exhibits an absorption peak at a wavelength of 388 nm. An absorbance / concentration standard curve was calculated based on this absorption peak, as follows: Figure 1 and Figure 2 As shown.

[0075] 4. Extraction and culture of bone marrow-derived macrophages

[0076] (1) Prepare scissors, tweezers, 1mL syringe, sterile gauze, 40μm cell filter, culture dish, red blood cell lysis buffer, complete culture medium, centrifuge tubes, and 75% alcohol and sterilize them in a laminar flow hood.

[0077] (2) After euthanizing the mice, soak them in alcohol for 5 minutes.

[0078] (3) Transfer the mouse to sterile gauze in the laminar flow hood, carefully remove the mouse's hind limbs with scissors and tweezers, and remove the muscles to obtain the bone.

[0079] (4) Soak the bone in alcohol for 1 minute, then transfer it to a new petri dish and wash it twice with PBS.

[0080] (5) Use another clean pair of scissors to cut open both ends of the bone, then use a 1mL syringe to draw up PBS and insert the needle into the bone marrow to flush it.

[0081] (6) Collect the flushed bone marrow into a new culture dish and gently blow it to disperse it into individual cells.

[0082] (7) Place a 40μm cell filter on a 50mL centrifuge tube and aspirate bone marrow cells to pass through the filter into the centrifuge tube to remove bone fragments or fascia and other impurities.

[0083] (8) Centrifuge the cells at 400g for 5 minutes and discard the supernatant.

[0084] (9) Resuspend the cells in 5 mL of red blood cell lysis buffer and let stand at room temperature for 5 minutes.

[0085] (10) Add 15 mL of complete culture medium to stop red cracking, centrifuge at 400 g for 5 minutes, and discard the supernatant.

[0086] (11) Add 20 mL of complete culture medium to resuspend the cells, and repeat the centrifugation operation to remove residual red blood cell lysate.

[0087] (12) Add an appropriate amount of complete culture medium to resuspend the cells, and add mouse recombinant M-CSF protein to make the final concentration 20 ng / mL. Seed the cells in a culture dish or six-well plate and then incubate them in an incubator.

[0088] (13) Change the medium on the third day after extraction. Mature macrophages can generally be formed one week after M-CSF stimulation for subsequent experiments.

[0089] 5. Statistical Analysis

[0090] Statistical analysis was performed using GraphPad Prism 8.0 software. The Shapiro-Wilk test was used to assess whether the data conformed to a normal distribution. Differences between two normally distributed groups were compared using the Student's t-test; comparisons among multiple groups were performed using a one-way ANOVA test with Tukey's HSD correction. For non-normally distributed data, the Mann-Whitney U test was used. Data from at least three independent experiments were presented as mean ± standard deviation. A p-value < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, #p > 0.05).

[0091] Example 1

[0092] This embodiment investigates the characterization of nifurazolidone loading onto macrophages. RAW264.7 is a widely used macrophage line, and this study used RAW264.7 cells for nifurazolidone loading.

[0093] 1. Experiment on macrophages loaded with nifuroslid in specific quantities

[0094] (1) RAW264.7 cells were blown off with DMEM containing 10% fetal bovine serum and collected into 15 mL centrifuge tubes for cell counting.

[0095] (2) Take 1.0 × 10⁻⁶ cells based on cell density. 7 Each cell was seeded into a new culture dish and cultured overnight.

[0096] (3) Remove the original culture medium of RAW264.7 cells and add serum-free culture medium for starvation treatment for 1 hour.

[0097] (4) Prepare a nifurate solution with a concentration of 50 μg / mL using DMEM containing 10% fetal bovine serum.

[0098] (5) Remove the serum-free culture medium and replace it with 10 mL of the above nifurazolidone solution for incubation.

[0099] (6) After the drug incubation reaches the specified time (2 hours, 4 hours and 6 hours), remove the drug solution, add 2 mL of PBS to the wall, and gently shake the culture dish to rinse the cells.

[0100] (7) After rinsing once, blow off the cells and collect them in a 15mL centrifuge tube. Centrifuge at 1000rpm for 5 minutes at 4℃ and discard the supernatant.

[0101] (8) Resuspend the cells in 1 mL of dimethyl sulfoxide solution and vortex for 1 minute to break the cell membrane and release nifurazolidone.

[0102] (9) The absorbance at a wavelength of 388 nm was measured using an ultraviolet spectrophotometer, and the concentration of nifurazolidone was calculated based on the ultraviolet absorbance / concentration standard curve.

[0103] (10) Formula for calculating drug loading capacity on macrophages:

[0104] Cellular drug loading = Nifurazolidone concentration (ng / mL) × 1 mL / number of macrophages.

[0105] The results are as follows Figure 3 As shown, RAW264.7 cells were starved for 1 hour and then co-incubated with nifurazide for 2 hours, 4 hours and 6 hours to enable them to engulf nifurazide and achieve drug loading. Subsequently, based on the UV absorbance peak and standard curve, the nifurazide dosage carried by macrophages was calculated to be 370±5ng, 422±3ng and 451±12ng per 10,000 cells, respectively.

[0106] To more intuitively reflect the ability of macrophages to carry nifurazolidone, we used the near-infrared fluorescent dye Cy5 to label nifurazolidone.

[0107] 2. Experiment with Cy5-labeled nifurazide on macrophages

[0108] 2.1 Cy5 fluorescent dye labeling of nifurazolidone

[0109] (1) Add equimolar amounts of nifurazolidone, Cy5, and catalysts DISC and DMAP to the reaction vessel, dissolve them in anhydrous DCM, and then place them at 45°C in the dark and stir overnight.

[0110] (2) Use thin-layer chromatography to track the degree of reaction.

[0111] (3) After the reaction is complete, the reaction system is washed with citric acid solution (5%), saturated sodium bicarbonate solution and saturated saline solution in sequence, and the reactant DCM solution is obtained by extraction.

[0112] (4) Use anhydrous sodium sulfate to completely remove the moisture from the DCM.

[0113] (5) After filtering the product, perform vacuum distillation to remove the solvent.

[0114] (6) The product was separated and purified by column chromatography, and the solvent was removed by repeated vacuum distillation.

[0115] (7) Take a portion of the product for mass spectrometry detection, and store the remaining portion in a -80℃ freezer for subsequent experiments.

[0116] The molecular equation for the reaction of near-infrared fluorescent dye Cy5 labeled with nifurazide is as follows:

[0117]

[0118] Wherein, NFX: Nifurazide; Cy5-NFX: Cy5-labeled nifurazide.

[0119] The mass spectrometry results of the obtained product are as follows: Figure 4 As shown.

[0120] 2.2 Macrophages carrying Cy5-labeled nifurazolidone

[0121] (1) RAW264.7 cells were inoculated at 5.0 × 10⁻⁶ cells per cell line. 4 Seeds were placed at a density of 100 individuals per dish in confocal dishes and cultured for 12 hours.

[0122] (2) Remove the original cell culture medium and replace it with serum-free culture medium for starvation treatment for 1 hour.

[0123] (3) Dissolve Cy5-labeled nifurazide in dimethyl sulfoxide to prepare a stock solution containing 10 mg / mL of nifurazide.

[0124] (4) Dilute the above stock solution with complete culture medium to prepare a solution containing 50 μg / mL of nifurazolidone.

[0125] (5) After the starvation treatment, remove the serum-free culture medium and add 1 mL of PBS to the wall for rinsing.

[0126] (6) Add the above-mentioned nifurazolidone solution containing Cy5 labeling to the wall and incubate in an incubator for 2 hours.

[0127] (7) Remove the drug solution, add 1 mL of PBS to the wall for rinsing, and repeat the rinsing twice.

[0128] (8) Add 500 μL of 4% paraformaldehyde to fix the cells for 15 minutes to adhere to the wall.

[0129] (9) Remove the paraformaldehyde, add 1 mL of PBS to the wall for rinsing, and repeat the rinsing twice.

[0130] (10) Add DAPI working solution and incubate for 10 minutes to perform nuclear labeling.

[0131] (11) After the nuclear staining is completed, the sample is rinsed with PBS and observed using a confocal microscope.

[0132] The results are as follows Figure 5 As shown, after starved macrophages were co-incubated with Cy5-labeled nifurazolidone for 2 hours, confocal microscopy revealed a large amount of Cy5 fluorescence signal in the cells, confirming that macrophages could effectively carry nifurazolidone.

[0133] To determine whether nifurazolidone loading has toxic side effects on macrophages themselves, we tested the activity of macrophages after drug loading.

[0134] 3. Macrophage activity detection

[0135] (1) After incubating macrophages with 50 μg / mL nifurazide solution for 2 hours, 4 hours and 6 hours respectively, the solution was removed.

[0136] (2) Add complete culture medium and continue culturing. Collect cells from each group into 15mL centrifuge tubes at 12 hours, 24 hours, 36 hours and 48 hours respectively.

[0137] (3) The cell activity detection function of the Beckman Coulter cell counter was used to determine the activity ratio of each group of cells at each time point.

[0138] The results are as follows Figure 6As shown, cell viability tends to decrease over time. Co-incubation for 2 hours and 4 hours has little effect on macrophage activity, with activity still reaching over 85% after 48 hours of drug loading. However, co-incubation for 6 hours has a significant impact on cell viability, with activity decreasing to around 75% after 48 hours of drug loading. Based on this, this study adopted a 2-hour co-incubation method to reduce the influence of the drug itself on macrophage activity.

[0139] Furthermore, we investigated whether macrophages could successfully release nifurazolidone, and also used absorbance values ​​to detect drug concentrations in cell supernatants at different time points.

[0140] 4. Macrophage drug release experiment

[0141] (1) Obtain macrophages loaded with nifurazolidone according to steps 1-6 in Experiment 1, resuspend them in 5 mL of complete culture medium and seed them in a new culture dish.

[0142] (2) Take 1 mL of culture medium at the specified time (6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours) and replenish 1 mL of culture medium at the same time.

[0143] (3) The absorbance of the extracted nifurazide solution at a wavelength of 388 nm was detected by ultraviolet spectrophotometer, and the concentration of nifurazide was calculated according to the ultraviolet absorbance / concentration standard curve.

[0144] (4) Formula for calculating drug release rate:

[0145] Drug release rate = concentration of nifurazolidone released (ng / mL) × 1mL / total amount of nifurazolidone.

[0146] The results are as follows Figure 7 As shown, the in vitro drug release rate of macrophages carrying nifurazolidone is a relatively slow and gradual process.

[0147] Example 2

[0148] This embodiment investigates the drug delivery between macrophages and tumor cells, using the following method:

[0149] 1. Intercellular drug delivery experiments

[0150] (1) Huh7 cells were inoculated at 5.0 × 10⁻⁶ cells per cell line. 4 Seeds were placed at a density of 100 individuals per dish in confocal dishes and cultured overnight.

[0151] (2) Dilute CFSE (1000×) with PBS at a ratio of 1:1000 to prepare working solution.

[0152] (3) Remove the original culture medium of Huh7 cells, and add 1 mL of PBS to rinse the adherent cells.

[0153] (4) After rinsing once, add CFSE working solution to the wall and incubate in an incubator for 7 minutes.

[0154] (5) Remove the CFSE working solution, add 1 mL of PBS to the wall for rinsing, and repeat the rinsing twice.

[0155] (6) Macrophages loaded with Cy5-labeled nifurazolidone were obtained according to the experimental method in 2.2 of Example 1. After cell counting, the cells were counted at 5.0 × 10⁻⁶ cells per cell. 4 The cells were added to Huh7 cells at a density of 1 cell per dish.

[0156] (7) After co-incubating the cells for 6 hours, remove the culture medium, add 1 mL of PBS to the adherent cells for rinsing, and repeat the rinsing twice.

[0157] (8) Cell fixation and nuclear staining were performed according to the experimental method in 2.2 of Example 1, and then observed using confocal microscopy, or the following operations were performed for flow cytometry detection.

[0158] (9) Add 500 μL of pancreatic enzyme and digest at 37°C for 2-3 minutes.

[0159] (10) Add 3 mL of PBS, blow the cells off and collect them in a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 5 minutes and discard the supernatant.

[0160] (11) Add 3 mL of PBS to resuspend the cells and repeat the centrifugation process to remove residual trypsin.

[0161] (12) Add 100 μL PBS to resuspend the cells and transfer the cells to a flow cytometer.

[0162] (13) Add 1 μL of live / dead dye (APC-Cy7 channel) to the cells, gently mix by pipetting, and incubate at room temperature in the dark for 15 minutes.

[0163] (14) Add 1 mL of PBS to the cells and centrifuge at 1000 rpm for 5 minutes, then discard the supernatant.

[0164] (15) Add 200 μL PBS to resuspend the cells, and analyze them using a flow cytometer. The data obtained are analyzed using Flowjo software.

[0165] Confocal detection results as follows Figure 8 As shown, a large amount of Cy5 fluorescence signal appeared in Huh7 cells, indicating that the drug can enter Huh7 cells from macrophages.

[0166] The above cells were analyzed by flow cytometry, and single cells, live cells, and Huh7 cells expressing CFSE were sequentially delineated using gating. Results are as follows: Figure 9As shown in Figures A and B, Huh7 cells highly express Cy5 fluorescence signals, a result consistent with that obtained under a confocal microscope.

[0167] 2. Tumor cell viability detection

[0168] (1) Macrophages loaded with Cy5-labeled nifurazide were obtained according to the experimental method in 2.2 of Example 1.

[0169] (2) Add 5 mL of complete culture medium and continue culturing for 6 hours until the macrophages release nifurazolidone into the culture medium.

[0170] (3) Collect the culture medium for later use.

[0171] (4) After counting the Huh7 cells, use 3.0 × 10⁻⁶ cells. 3 Seeds were planted at a density of 100 cells / well in 96-well plates and cultured for 12 hours.

[0172] (5) Remove the original culture medium from the cells, add the culture medium collected above, and use the normal complete culture medium as a control group, and place it in an incubator to continue culturing.

[0173] (6) Set time points (6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours) to detect CCK8 cell activity.

[0174] The results are as follows Figure 10 As shown, when the culture supernatant of macrophages loaded with nifurazolidone was used to treat Huh7 cells, cell activity was significantly inhibited, suggesting that macrophages can exert anti-tumor effects by releasing nifurazolidone.

[0175] The above results indicate that the drug can successfully enter tumor cells from macrophages, thereby exerting an anti-tumor effect.

[0176] Example 3

[0177] To investigate whether macrophages loaded with nifurazolidone can effectively enter tumors, this embodiment examined their in vivo distribution in a subcutaneous tumor model derived from H22 cells. Cy5, as a near-infrared fluorescent dye, can be characterized for in vivo distribution using an in vivo imaging system. Macrophages loaded with Cy5-labeled nifurazolidone were infused in vivo, with an equal dose of Free-Cy5 as a control. In vivo imaging was performed 24 hours after injection to detect the fluorescence signal intensity in the tumor region.

[0178] 1. In vivo distribution of macrophages carrying Cy5-labeled nifurazolidone

[0179] (1) Investigate the in vivo distribution of macrophages carrying Cy5-labeled nifurazolidone in a subcutaneous tumor model derived from H22 hepatocellular carcinoma cells.

[0180] (2) Collect H22 cells for counting, and prepare 1.0×10⁻⁶ cells with PBS. 7 Cell suspension with a density of cells / mL.

[0181] (3) 200 μL of cell suspension was injected subcutaneously into the lateral ventral wall of each mouse to form a small mound.

[0182] (4) Closely observe the growth of the tumor and conduct a study on the distribution of the drug in vivo after the tumor diameter reaches 1 cm.

[0183] (5) Macrophages loaded with Cy5-labeled nifurazide were obtained by referring to the experimental method in 2.2 of Example 1.

[0184] (6) After resuspending the cells in PBS, a portion of the cells were used for Cy5 quantification.

[0185] (7) Prepare an equal dose of Cy5 with PBS for the control experiment.

[0186] (8) Prepared macrophages loaded with Cy5-labeled nifurazolidone or control group Cy5 were injected intravenously into mice, 200 μL per mouse.

[0187] (9) After 24 hours, the intensity of fluorescence signal in the tumor area was detected using a mouse in vivo imaging system.

[0188] (10) Tumor tissue and major organs (heart, liver, spleen, lung and kidney) were obtained from euthanized mice for in vitro organ imaging.

[0189] like Figure 11 As shown in Figure A, macrophages loaded with Cy5-labeled nifuran showed stronger fluorescence signals in the subcutaneous tumor area compared to the Free-Cy5 group, suggesting greater drug accumulation.

[0190] like Figure 11 As shown in BC, in vitro organ imaging of tumor tissue and major organs revealed that the intratumoral fluorescence signal in the macrophage-loaded group was significantly stronger than that in the Free-Cy5 group. Furthermore, the fluorescence signal in the kidneys of the macrophage-loaded group was significantly lower than that of the Free-Cy5 group, suggesting that it can slow down the renal metabolism rate of the drug, thus improving the drug's circulation time and pharmacokinetics.

[0191] Example 4

[0192] This embodiment utilizes tumor cell supernatant to construct an in vitro macrophage polarization induction system to simulate the formation process of tumor-associated macrophages, and then uses nifurazolidone for intervention. The method is as follows:

[0193] 1. In vitro macrophage polarization induction experiment

[0194] (1) Macrophages were charged at 5.0 × 10⁻⁶ 5 Seeds were planted in six-well plates at a density of 100 cells / well and cultured overnight.

[0195] (2) Collect the supernatant of Hepa1-6 liver cancer cells, centrifuge at 3000 rpm for 10 minutes, and discard the precipitate (dead cells or cell debris).

[0196] (3) Prepare 5 μM nifurazolidone using the supernatant of liver cancer cells.

[0197] (4) Remove the original culture medium of macrophages, add liver cancer cell supernatant with or without nifurazolidone, and continue to culture in an incubator for 48 hours to induce polarization.

[0198] 2. Macrophage phenotype detection

[0199] (1) Collect macrophages in flow cytometry tubes, add 1 μL LCD16 / 32 antibody to each tube, mix well by pipetting, and incubate at 4°C in the dark for 15 minutes to block macrophages.

[0200] (2) After sealing, add 1 μL of dead or live dye to each tube, mix well by blowing and incubate at room temperature in the dark for 15 minutes.

[0201] (3) Prepare PBS containing 10% fetal bovine serum.

[0202] (4) Add 1 mL of the above PBS to each tube, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant.

[0203] (5) Resuspend the cells in 100 μL PBS, add 1 μL of CD45 antibody, F4 / 80 antibody and CD11b antibody to each tube to label the macrophage surface, mix well by pipetting and incubate at 4°C in the dark for 30 minutes.

[0204] (6) Repeat step 4.

[0205] (7) Add 250 μL of membrane breaking reagent A to each tube, mix well by pipetting, and incubate in a refrigerator at 4°C in the dark for 20 minutes.

[0206] (8) Dilute 10× membrane rupture reagent B with ultrapure water at a ratio of 9:1 to the working concentration. Add 1 mL of membrane rupture reagent B to each tube and centrifuge at 2000 rpm for 5 minutes. Discard the supernatant.

[0207] (9) Resuspend the cells in each tube with 100 μL of membrane rupture reagent B, and then add 1 μL of iNOS and CD206 antibody to label M1 / M2 macrophages. Mix well by pipetting and incubate at 4°C in the dark for 30 minutes.

[0208] (10) Add 1 mL of membrane-breaking reagent B to each tube, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant.

[0209] (11) Add 200 μL of cell permeabilization reagent B to each tube to resuspend the cells, and then perform flow cytometry analysis. The specific gating steps for macrophage flow cytometry analysis are as follows: Figure 12 As shown.

[0210] (12) The obtained data were analyzed using Flowjo software.

[0211] M1 and M2 macrophages were classified based on the markers iNOS and CD206, respectively. Results are as follows: Figure 13 As shown, nifurazolidone significantly upregulated the M1 / M2 macrophage ratio in both RAW264.7 cells and bone marrow-derived macrophages (BMDM). Further observation revealed that nifurazolidone primarily promotes the upregulation of M1 / M2 macrophages. + Macrophage polarization is dominant. Therefore, nifurazolidone may awaken a macrophage-mediated anti-tumor immune response.

[0212] Example 5

[0213] To clarify whether the upregulation of the M1 / M2 macrophage ratio induced by nifurazolidone can activate an anti-tumor immune response, this embodiment mixed the induced macrophages with H22 mouse liver cancer cells or Hepa1-6 mouse liver cancer cells at a 1:1 ratio to construct H22 cell-derived subcutaneous tumor models and Hepa1-6 cell-derived subcutaneous tumor models, respectively.

[0214] 1. Constructing a subcutaneous liver cancer model by mixing macrophages and tumor cells.

[0215] (1) Macrophages after induced polarization were obtained according to the experimental method in Example 4.

[0216] (2) Count the macrophages and prepare a solution of 3.0 × 10⁶ cells using PBS. 7 Density of cells / mL.

[0217] (3) Collect tumor cells (H22 cells or Hepa1-6 cells) for counting, and prepare a solution of 1.0 × 10⁻⁶ cells with PBS. 7 Density of cells / mL.

[0218] (4) Mix macrophage suspension and tumor cell suspension at a volume ratio of 1:1, gently blow and mix well, and then seed into the subcutaneous tissue of the lateral abdominal wall of mice (200 μL / mouse) to form a small mound.

[0219] (5) Closely observe the condition of the mice and the growth of the tumor. Wait until the tumor volume reaches 2000 mm. 3The experiment was terminated after the mice were euthanized to obtain tumor tissue. The tissue was weighed, photographed, and then stored in culture medium and placed on ice for subsequent extraction of immune cells from the tumor.

[0220] like Figure 14 and Figure 15 As shown, compared with macrophages induced by tumor supernatant alone, macrophages treated with nifurazolidone significantly inhibited tumor growth in both liver cancer models, resulting in reduced tumor volume and weight.

[0221] Since macrophages primarily intervene in tumor immunity by regulating T cell function, we further examined intratumoral IFN-γ. + T cell infiltration status.

[0222] 2. Tumor Immune Cell Extraction Experiment

[0223] (1) Tumor tissue was obtained after euthanizing tumor-bearing mice.

[0224] (2) Preserve the tumor tissue in a culture medium and place it on ice.

[0225] (3) Cut the tissue into small pieces of 0.10cm×0.1cm with scissors.

[0226] (4) Tissue digestion solution was prepared using type IV collagenase (2 mg / mL), deoxyribonuclease I and KREBS-RINGER buffer.

[0227] (5) Transfer the tumor fragments to 2mL cryovials, add 2mL of tissue digestion solution to each tube, and place them on a shaker at 37°C for 30 minutes to digest.

[0228] (6) Place a 40μm cell filter on a 50mL centrifuge tube and transfer the digested tumor tissue into the filter.

[0229] (7) Gently squeeze the tissue fragments with the bottom of the syringe to allow the cells to pass through the filter and enter the centrifuge tube, rinsing with PBS during the process.

[0230] (8) Centrifuge the washed-off cells at 400g for 5 minutes, discard the supernatant, resuspend the cells in 2.5mL PBS and transfer them to a 15mL centrifuge tube.

[0231] (9) Prepare a working solution for separating tumor immune cells using tumor separation solution, 10×PBS and ultrapure water.

[0232] (10) Add 2.5 mL of separation solution to each tube and gently blow to mix the separation solution with PBS.

[0233] (11) Tilt the centrifuge tube and slowly inject 2 mL of PBS along the tube wall.

[0234] (12) After centrifuging at 1000g for 10 minutes, carefully remove the centrifuge tube (layering can be observed).

[0235] (13) Take the intermediate layer cells into a flow cytometer, add an equal volume of PBS, and gently pipette to mix.

[0236] (14) Centrifuge at 2000 rpm for 5 minutes, discard the supernatant, and the resulting precipitate is the tumor immune cells, which can be further labeled and stained with antibodies.

[0237] 3. T cell phenotype detection

[0238] (1) Obtain tumor T cells using the above-mentioned method for extracting tumor-intratumor immune cells, and then store the cells at a concentration of 1.0 × 10⁻⁶. 6 Seeds were planted at a density of 100 cells / well in 24-well plates, with 1 mL of culture medium per well.

[0239] (2) Add 2 μL of T cell activation reagent to each well and incubate in an incubator for 6 hours.

[0240] (3) Collect the cells into a flow cytometer, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant.

[0241] (4) Resuspend the cells in 100 μL PBS, add 1 μL of live and dead dye to each tube, mix well by pipetting, and incubate at room temperature in the dark for 15 minutes.

[0242] (5) Prepare PBS containing 10% fetal bovine serum.

[0243] (6) Add 1 mL of the above PBS to each tube, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant.

[0244] (7) Resuspend the cells in 100 μL PBS, add 1 μL of CD45 antibody, CD3 antibody and CD8 antibody to each tube to label the surface of T cells, mix well by pipetting and incubate in a refrigerator at 4°C in the dark for 30 minutes.

[0245] (8) Repeat step 6.

[0246] (9) Add 250 μL of membrane breaking reagent A to each tube, mix well by pipetting, and incubate in a refrigerator at 4°C in the dark for 20 minutes.

[0247] (11) Dilute 10× membrane breaking reagent B with ultrapure water at a ratio of 9:1 to the working concentration. Add 1 mL of membrane breaking reagent B to each tube, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant.

[0248] (12) Add 100 μL of cell permeabilizing reagent B to each tube to resuspend the cells, then add 1 μL of IFN-γ antibody, mix well by pipetting, and incubate at 4°C in the dark for 30 minutes.

[0249] (13) Add 1 mL of membrane-breaking reagent B to each tube, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant.

[0250] (14) Add 200 μL of cell permeabilization reagent B to each tube to resuspend the cells, and then perform flow cytometry analysis. The specific gating steps for T cell flow cytometry analysis are as follows: Figure 16 As shown.

[0251] (15) The obtained data were analyzed using Flowjo software.

[0252] like Figure 17 As shown, nifurazolidone-induced macrophages can significantly upregulate intratumoral IFN-γ. + CD8 + The proportion of T cells indicates that anti-tumor immunity has been activated.

[0253] Based on the above results, nifurazolidone can activate anti-tumor immunity and achieve liver cancer killing by upregulating the ratio of M1 / M2 macrophages.

[0254] Example 6

[0255] To investigate whether macrophage-loaded nifurazolidone can be used in the treatment of liver cancer, this embodiment explores the antitumor efficacy of the drug delivery system in H22 cell subcutaneous tumor models and Hepa1-6 cell subcutaneous tumor models.

[0256] 1. Construction and processing of subcutaneous tumorigenesis model of liver cancer

[0257] (1) Collect liver cancer cells (H22 cells or Hepa1-6 cells) for counting, and prepare cell suspension with PBS.

[0258] (2) 200 μL of cell suspension was injected subcutaneously into the lateral ventral wall of each mouse to form a small mound.

[0259] (3) Mice were randomly divided into four groups: nifurazide treatment group, macrophage treatment group, macrophage-loaded nifurazide treatment group, and saline control group.

[0260] (4) Closely monitor tumor growth until the tumor volume reaches 100 mm. 3 Following this, drug administration began. Nifurazide was administered orally at a dose of 5 mg / kg, and macrophages carrying nifurazide were also intravenously infused at the same dose of 5 mg / kg (approximately 3.0 × 10⁻⁶). 6 (Number of cells), the number of individual macrophages reinfused was the same as the number of macrophages loaded with nifurazolidone. Administration lasted for one week, during which the body weight and tumor length and short axis of each group of mice were recorded.

[0261] (5) When the tumor volume reaches 2000 mm 3The experiment was terminated after the mice were euthanized to obtain tumor tissue. The tissue was weighed, photographed, and then stored in culture medium and placed on ice for subsequent extraction of immune cells from the tumor.

[0262] like Figure 18 and Figure 19 As shown, macrophages loaded with nifurazolidone significantly inhibited tumor growth in both liver cancer models. However, the oral nifurazolidone group did not show significant antitumor activity, which may be related to the low dosage and poor bioavailability of nifurazolidone. Furthermore, macrophage reinfusion alone also had no significant antitumor effect.

[0263] like Figure 20 As shown, no significant changes in mouse body weight were observed after macrophages were treated with nifurazolidone, indicating that this treatment method is relatively safe.

[0264] Example 7

[0265] This embodiment explores the combined anti-hepatocellular carcinoma treatment using macrophages loaded with nifurazolidone and oxaliplatin.

[0266] 1. Construction and treatment of H22 cell-derived BALB / c mouse liver carcinoma model in situ

[0267] (1) The H22 cell-derived BALB / C mouse liver in situ carcinoma model was constructed using the classic method. The tumor cell suspension was mixed with matrix gel at a volume ratio of 1:1, part of the peritoneum was opened, and the mixture was injected into the left lobe of the liver to construct the in situ tumor model.

[0268] (2) Three days after tumor implantation, the mice were randomly divided into an oxaliplatin treatment group, a macrophage-carried nifurazide treatment group, an oxaliplatin combined with macrophage-carried nifurazide treatment group, and a saline group, and the drugs were administered.

[0269] (3) Oxaliplatin was administered intraperitoneally at a dose of 2.5 mg / kg every two days; macrophages carrying nifurazide were infused intravenously at a dose of 5 mg / kg and administered daily for one week.

[0270] (4) After the experimental period reached three weeks, the mice were euthanized, tumor tissue was obtained, weighed and photographed and then preserved in formalin.

[0271] like Figure 21 As shown, macrophage-carrying nifurazolidone combined with oxaliplatin significantly inhibited carcinoma in situ growth compared to treatment alone.

[0272] Therefore, it is evident that using macrophages to carry nifurazolidone to optimize drug delivery can not only activate anti-tumor immune responses but also enhance the anti-hepatocellular carcinoma effects of clinical anti-tumor drugs, thus showing great potential in the treatment of hepatocellular carcinoma.

[0273] In summary, given the poor water solubility and low bioavailability of nifurazolidone, this study characterized the loading of nifurazolidone onto macrophages. Through in vitro experiments, we clarified the loading amount of nifurazolidone in macrophages and its intercellular transfer. Furthermore, we used near-infrared fluorescent dye labeling and in vivo imaging to characterize the in vivo distribution of nifurazolidone loaded onto macrophages, revealing its stronger tumor-region accumulation capacity. To investigate the effect of nifurazolidone on macrophage polarization, we constructed an in vitro macrophage polarization induction system using tumor supernatant and then intervened with nifurazolidone. The results showed that nifurazolidone significantly upregulated the M1 / M2 macrophage ratio, thus promoting macrophage-mediated anti-tumor immunity. We also mixed the induced macrophages with tumor cells to construct a subcutaneous hepatocellular carcinoma model to investigate the effect of these macrophages on tumor growth. The results showed that macrophages induced by nifurazolidone had a significant inhibitory effect on tumor growth. To clarify whether this inhibitory effect is related to anti-tumor immune activation, we investigated the infiltration of T cells within the tumor and found that IFN-γ + CD8 + The proportion of T cell infiltration increased significantly. Therefore, macrophage-loaded nifurazolidone not only enables drug delivery but also utilizes nifurazolidone's polarization regulation of macrophages to achieve anti-tumor immune activation. The macrophage drug delivery protocol used in this study is expected to provide some ideas and strategies for improving nifurazolidone pharmacokinetics and reshaping the tumor immune microenvironment.

Claims

1. The application of a macrophage drug delivery system loaded with nifurazolidone in the preparation of drugs for treating hepatocellular carcinoma, characterized in that, The drug delivery system is formed by co-incubating nifurazide within macrophages, wherein the macrophages are RAW264.7 cells or bone marrow-derived macrophages; nifurazide promotes the polarization of M1 macrophages, and the drug achieves its tumor-killing effect by upregulating the M1 / M2 macrophage ratio and activating the anti-tumor immune response.

2. The application as described in claim 1, characterized in that, The drug delivery system delivers 370-450 ng of nifurazolidone per 10,000 macrophages.

3. The application as described in claim 1, characterized in that, The preparation method of the drug delivery system includes: firstly, activating and culturing macrophages and then starving them in a serum-free culture medium for 1 h; then removing the serum-free culture medium and adding a complete culture medium containing nifurazolidone for co-incubation for 2-4 h to obtain the nifurazolidone-loaded macrophage drug delivery system.

4. The application as described in any one of claims 1-3, characterized in that, The active ingredient in the anti-hepatocellular carcinoma drug also includes oxaliplatin.

5. The application as described in claim 4, characterized in that, The drug comprises a first formulation formed from the macrophage delivery system loaded with nifurazolidone and a pharmaceutically acceptable carrier, and a second formulation formed from oxaliplatin and a pharmaceutically acceptable carrier.

6. The application as described in claim 5, characterized in that, The first formulation is in the form of an injectable formulation.

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