Macrophage drug delivery system for enhancing photodynamic therapy as well as preparation method and application of macrophage drug delivery system

Abstract

The invention relates to the technical field of nano medicines, in particular to a macrophage drug delivery system for enhancing photodynamic therapy as well as a preparation method and application of the macrophage drug delivery system. The macrophage drug delivery system provided by the invention has the advantages of macrophages, can penetrate through body barriers such as blood vessels and the like, reduces the immunogenicity of the nanoparticles, enhances the tumor targeting of the nanoparticles through the tumor tropism of the macrophages, and enhances the uptake of the nanoparticles by tumor cells through drug release mechanisms such as nanotubes, efflux and the like; secondly, endogenous hydrogen peroxide of macrophages can be catalyzed into oxygen by the BMB nanoparticles, so that an anoxic environment is improved, and possibility is provided for enhancing a photodynamic therapy; besides, by utilizing the innate immune effect and adaptive immune stimulation capability of macrophages, photodynamic killing of tumor cells can be assisted, and infiltration and maturation of dendritic cells and T cells can be recruited and stimulated under the action of antigens generated by tumor killing, so that the immune microenvironment in tumors is improved.

Description

Technical Field

[0001] The present invention relates to the field of nano-medicine technology, and particularly to a macrophage drug-loading system for enhancing photodynamic therapy, its preparation method and application. Background Art

[0002] Photodynamic therapy is a safe and effective method for ablating melanoma, and has unique advantages compared with other therapies such as chemotherapy and immunotherapy. First of all, photodynamic therapy is minimally invasive, and on the premise of effectively killing tumor cells, it causes less damage to normal tissues. In addition, photodynamic therapy has the characteristics of good tolerance and low toxicity, that is, repeated use will not aggravate the toxic side effects. Due to the excellent therapeutic properties of photodynamic therapy, it has become one of the important means for clinical treatment of melanoma.

[0003] Photodynamic therapy requires the participation of photosensitizers and oxygen. After being irradiated by light, the photosensitizer absorbs energy and transitions from the ground state to the triplet excited state, and the energy released by it converts oxygen into highly reactive reactive oxygen species. And the reactive oxygen species will directly kill tumor cells. However, most solid tumors have the characteristic of local hypoxia inside. The hypoxia of tumor tissues accelerates the expression of hypoxia-inducible factor, thereby promoting the expression of vascular endothelial growth factor by tumor cells and glycolytic metabolism. The expression of vascular endothelial growth factor, etc. will promote the invasion and metastasis of tumor cells, and the glycolytic metabolite lactic acid will lead to an acidic tumor microenvironment. If photodynamic therapy is carried out in a hypoxic tumor microenvironment, it may exacerbate hypoxia and even promote tumor development. Therefore, oxygen is very necessary for promoting photodynamic efficacy and photodynamic killing of cells.

[0004] There are mainly two ways to supply oxygen to the tumor site: directly supply oxygen to the tumor site and indirectly generate oxygen through catalytic reaction. MnO2 can be used as an oxygen supply material to decompose the abundant H2O2 in the tumor microenvironment into oxygen and perform magnetic resonance imaging of the tumor site. Therefore, manganese dioxide is a good oxygen supply material. As a solid metal oxide, manganese dioxide lacks biocompatibility when used alone. If it is prepared into nanoparticles with a size of 100-150 nm, it will not only improve its biocompatibility but also promote cell uptake. Bovine serum albumin (BSA), as a common raw material for nanoparticles, has good biocompatibility. BSA has metal binding sites and can bind to manganese ions to in-situ generate manganese dioxide under alkaline conditions. In addition, the tumor microenvironment is rich in glutathione, and glutathione will reduce manganese dioxide to manganese ions. Manganese ions are paramagnetic substances and can perform magnetic resonance imaging of the tumor site, thus realizing the integration of diagnosis and treatment.

[0005] Photosensitizers can be attached to albumin-manganese dioxide nanoparticles to form albumin-manganese dioxide-photosensitizer nanoparticles. Manganese dioxide provides sufficient oxygen for photodynamic therapy, which not only ensures the photodynamic efficacy but also avoids exacerbating the hypoxic tumor microenvironment.

[0006] However, the above-mentioned nanoparticles have defects during transportation, such as being degraded before reaching the target; nanoparticles with immunogenicity being cleared by the immune system; and the tumor targeting ability of the nanoparticles themselves being limited, etc. Summary of the Invention

[0007] To solve the above problems, the present invention provides a macrophage drug-loading system for enhancing photodynamic therapy, its preparation method and application. The macrophage drug-loading system provided by the present invention has the advantages of macrophages, that is, it can penetrate body barriers such as blood vessels, reduce the immunogenicity of nanoparticles, enhance the tumor targeting of nanoparticles through the tumor tropism of macrophages themselves, and enhance the uptake of tumor cells by nanoparticles through drug release mechanisms such as nanopores and efflux; secondly, the endogenous hydrogen peroxide of macrophages can be catalyzed by BMB nanoparticles into oxygen, thereby improving the hypoxic environment and providing the possibility for enhancing photodynamic therapy; in addition, by utilizing the innate immune effect of macrophages and the ability to stimulate adaptive immunity, it can assist in photodynamically killing tumor cells, and under the action of antigens generated by killing tumors, it can recruit and stimulate the infiltration and maturation of dendritic cells and T cells, thereby improving the immune microenvironment inside the tumor and achieving continuous killing of tumors.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention provides a macrophage carrying nanoparticles, including BMB nanoparticles and M1 macrophages; the BMB nanoparticles are located inside the M1 macrophages; the BMB nanoparticles include albumin, manganese dioxide and I-BDP photosensitizer; the albumin and manganese dioxide are connected through metal binding sites, and the I-BDP photosensitizer is attached to the manganese dioxide.

[0010] Preferably, the albumin includes bovine serum albumin.

[0011] The present invention provides a preparation method of the macrophage according to the above technical solution, including the following steps:

[0012] Mix the albumin solution and the salt solution containing manganese ions, adjust the pH value of the mixture to 9-10, and stir until neutral to obtain a BSA-MnO2 nanoparticle solution;

[0013] Mix the BSA-MnO2 nanoparticle solution and the I-BDP photosensitizer to obtain BMB nanoparticles;

[0014] Mix the BMB nanoparticles and M1 macrophages, and incubate for more than 4 hours to obtain the macrophage carrying nanoparticles.

[0015] Preferably, the salt containing manganese ions includes manganese chloride.

[0016] Preferably, the mass ratio of the albumin to the manganese chloride is 10-1000:0.5-5.

[0017] Preferably, the mass ratio of the I-BDP photosensitizer to the albumin is 0.1-20:100.

[0018] Preferably, based on the mass of the I-BDP photosensitizer, in the mixture of the BMB nanoparticles and the M1 macrophages, the concentration of the BMB nanoparticles is 1-200 μg / mL.

[0019] Preferably, the incubation temperature is 37°C.

[0020] The present invention provides the use of the macrophages described in the above technical solution or the macrophages prepared by the preparation method described in the above technical solution in the preparation of a drug for photodynamic therapy.

[0021] Preferably, the diseases for photodynamic therapy include melanoma.

[0022] Beneficial effects:

[0023] The present invention provides a macrophage carrying nanoparticles, including BMB nanoparticles and M1 macrophages; the BMB nanoparticles are located inside the M1 macrophages; the BMB nanoparticles include albumin, manganese dioxide, and an I-BDP photosensitizer; the albumin and the manganese dioxide are connected through a metal binding site, and the I-BDP photosensitizer is attached to the manganese dioxide. The macrophage carrying nanoparticles provided by the present invention has the following advantages:

[0024] 1) The macrophage drug delivery system has many advantages such as good tumor targeting, multiple drug release mechanisms, innate immune effects, stimulation of adaptive immunity, and high phagocytosis efficiency of nanoparticles. Using it as a carrier can protect nanoparticles from immune system degradation and destruction, can cross blood vessels, the blood-brain barrier, and other body barriers to deliver nanoparticles to the tumor site, can reduce the free nanoparticles in the body, and reduce the toxic side effects of nanoparticles. The endogenous hydrogen peroxide in M1 macrophages can form an oxygen-rich "backpack" under the catalysis of BMB nanoparticles to provide exogenous oxygen supply for tumors.

[0025] 2) Macrophages, as nanoparticle carriers, have the natural ability to target tumor cells and deliver nanoparticles and oxygen into tumor cells in various ways such as efflux and nanopipelines. The targeting efficiency of tumors is higher than that of nanoparticles that only target tumors through the enhanced permeability and retention effect. M1 macrophages carrying oxygen enhance the innate immune effect and can effectively kill tumors before photodynamic therapy.

[0026] 3) Photodynamic therapy can cause tumor antigens and macrophages to synergistically further stimulate adaptive immunity and recruit T lymphocyte infiltration into tumors. The supplementation of oxygen can extend the lifespan of T cells in the tumor microenvironment, enhance the immune effect of photodynamic therapy sequence, and make up for the deficiencies of existing photodynamic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the embodiments.

[0028] Figure 1 Transmission electron microscope images of BSA-MnO2 and BMB nanoparticles in Example 1;

[0029] Figure 2 Images of M1 macrophages carrying BMB nanoparticles under bright field and fluorescence fields;

[0030] Figure 3 Fluorescence field images and drug loading analysis of M1 macrophages at different incubation times;

[0031] Figure 4 Drug loading analysis of M1 macrophages at different drug concentrations;

[0032] Figure 5 Cell viability graphs of M1 macrophages at different drug concentrations;

[0033] Figure 6 Drug leakage rate analysis of M1 macrophages carrying BMB nanoparticles within 24 hours;

[0034] Figure 7 Fluorescence field images and relative fluorescence intensity analysis of M1 macrophages carrying BMB nanoparticles in relieving hypoxia;

[0035] Figure 8 Experimental procedures and cell migration of the chemotaxis of M1 macrophages carrying BMB nanoparticles to tumor cells;

[0036] Figure 9 Changes in nitric oxide concentration produced by M1 macrophages before and after carrying BMB nanoparticles;

[0037] Figure 10 Fluorescence field images and analysis of the amount of drug transported by M1 macrophages carrying BMB nanoparticles to tumor cells;

[0038] Figure 11 Fluorescence field images and analysis of the production of reactive oxygen species under the action of M1 macrophages carrying BMB nanoparticles;

[0039] Figure 12Analysis of the contents of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin-12 (IL-12) produced by M1 macrophages carrying BMB nanoparticles before and after photodynamic therapy;

[0040] Figure 13 Analysis of the in vivo targeting of tumor sites by M1 macrophages carrying BMB nanoparticles;

[0041] Figure 14 Analysis of the in vivo inhibition of tumor growth by M1 macrophages carrying BMB nanoparticles;

[0042] Figure 15 Schematic diagrams of the preparation process and treatment process of macrophages carrying BMB nanoparticles;

[0043] Figure 16 Schematic diagrams of the binding of albumin-manganese dioxide-photosensitizer nanoparticles and drug release;

[0044] Among them, ns indicates no significant difference, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Detailed implementation manners

[0045] The present invention provides a macrophage carrying nanoparticles, including BMB nanoparticles and M1 macrophages; the BMB nanoparticles are located inside the M1 macrophages; the BMB nanoparticles include albumin, manganese dioxide, and I-BDP photosensitizer; the albumin and manganese dioxide are connected through a metal binding site, and the I-BDP photosensitizer is attached to the manganese dioxide.

[0046] The present invention uses a monoiodo-substituted BODIPYs (4,4-difluoro-4-bora-3a,4-diaza-s-indacene) photosensitizer, namely I-BDP, which can provide more singlet oxygen compared to other BODIPY photosensitizers. The preparation method of the I-BDP photosensitizer of the present invention can be referred to in the literature [Yu, Zhiliang et al. “Discovery of a Monoiodo Aza-BODIPY Near-Infrared Photosensitizer: in vitro and in vivo Evaluation for Photodynamic Therapy.” Journal of medicinal chemistry vol. 63, 17 (2020): 9950-9964.].

[0047] The nanoparticles of the present invention use albumin as a carrier. Manganese dioxide can be bound to the metal-binding sites of albumin to form albumin-manganese dioxide nanoparticles. The albumin-manganese dioxide nanoparticles adsorb photosensitizers through electrostatic adsorption. Finally, albumin-manganese dioxide-photosensitizer nanoparticles are formed (the binding schematic diagram is shown in Figure 16 ). The encapsulation efficiency of the photosensitizer in the nanoparticles is 86.6±2.73%, and its particle size is 112.83±3.89 nm. The albumin described in the present invention preferably includes bovine serum albumin.

[0048] The present invention utilizes the natural tumor tropism of macrophages to penetrate into the hypoxic microenvironment of tumors, and macrophages can release drugs into tumor cells through nanopipelines and efflux ([[]] Figure 16 ), thereby increasing the tumor accumulation degree of the nanoparticles; in addition, classically activated M1 macrophages are used, and the endogenous hydrogen peroxide of the nanoparticles and M1 macrophages is utilized to form oxygen-rich macrophages, and the oxygen present inside the macrophages avoids the polarization of macrophages in the tumor microenvironment. And the oxygen-rich macrophages can also provide exogenous oxygen supply to the tumor site to further improve the tumor hypoxic microenvironment. Since the oxygen content in the tumor microenvironment increases, the reactive oxygen species generated by subsequent photodynamic therapy will also increase accordingly, thereby exerting a stronger anti-tumor effect. Using macrophages as a drug carrier can also significantly reduce the immunogenicity of the nanoparticles, which is beneficial to reducing the toxic and side effects of the drug.

[0049] The present invention provides a preparation method of the macrophages described in the above technical solution, including the following steps:

[0050] Mix the albumin solution and the manganese ion-containing salt solution, adjust the pH value of the mixture to 9-10, and stir until neutral to obtain a BSA-MnO2 nanoparticle solution;

[0051] Mix the BSA-MnO2 nanoparticle solution and the I-BDP photosensitizer to obtain BMB nanoparticles;

[0052] Mix the BMB nanoparticles and M1 macrophages, and incubate for more than 4 h to obtain the macrophages carrying the nanoparticles.

[0053] In the present invention, an albumin solution and a salt solution containing manganese ions are mixed, and the pH value of the mixture is adjusted to 9-10 and stirred to neutrality to obtain a BSA-MnO2 nanoparticle solution. In the present invention, the salt containing manganese ions preferably includes manganese chloride; the mass ratio of the albumin to manganese chloride is preferably 10-1000:0.5-5, more preferably 50-100:3-5, and still more preferably 100:5; the reagent for adjusting the pH value of the mixture preferably includes sodium hydroxide solution; the concentration of the sodium hydroxide solution is preferably 0.1M. In the present invention, manganese ions bind to metal binding sites such as the sulfhydryl group of albumin and are in-situ mineralized to manganese dioxide under alkaline conditions. Manganese dioxide is uniformly dispersed in the solution under the action of albumin to form albumin-manganese dioxide nanoparticles.

[0054] After obtaining the BSA-MnO2 nanoparticle solution, the present invention mixes the BSA-MnO2 nanoparticle solution with an I-BDP photosensitizer to obtain BMB nanoparticles. In the present invention, the mass ratio of the I-BDP photosensitizer to albumin is preferably 0.1-20:100, more preferably 3-10:100, and still more preferably 4:100; the I-BDP photosensitizer is preferably dissolved in acetone and then mixed with the BSA-MnO2 nanoparticle solution. The photosensitizer solution dissolved in acetone forms albumin-manganese dioxide-photosensitizer nanoparticles by adhering to manganese dioxide in nanomorphology after the acetone has completely volatilized.

[0055] After obtaining the BMB nanoparticles, the present invention preferably mixes the obtained BMB nanoparticles with DMEM medium to obtain a DMEM-nanoparticle medium. In the present invention, based on the mass of the I-BDP photosensitizer, in the DMEM-nanoparticle medium, the concentration of BMB nanoparticles is preferably 1-200 μg / mL, more preferably 10-100 μg / mL, and still more preferably 20 μg / mL.

[0056] After obtaining the DMEM-nanoparticle medium, the present invention preferably adds the DMEM-nanoparticle medium to a container containing M1 macrophages in the form of a medium change and incubates for 4 hours or more to obtain the macrophages carrying nanoparticles. In the present invention, the incubation temperature is preferably 37°C; after the incubation, it is preferably washed with PBS to obtain the macrophages carrying nanoparticles. M1 macrophages have a strong internalization effect on nanoparticles with a particle size of 100-150 nm and finally form macrophages carrying nanoparticles through the cell phagocytosis mechanism.

[0057] The present invention provides the use of the macrophages described in the above technical solution or the macrophages prepared by the preparation method described in the above technical solution in the preparation of a drug for photodynamic therapy. In the present invention, the diseases treated by photodynamic therapy include melanoma. The macrophages carrying nanoparticles of the present invention can be used to enhance the photodynamic efficacy and its sequential immune effect.

[0058] To further illustrate the present invention, a macrophage drug delivery system for enhancing photodynamic therapy, its preparation method and application provided by the present invention will be described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0059] Example 1

[0060] Preparation of macrophages carrying BMB nanoparticles (BMB@M1), the schematic diagram of the preparation process is as Figure 1 shown, and the specific method is as follows:

[0061] 1) Preparation of BSA-MnO2: At 25 °C, first add 100 mg of bovine serum albumin (BSA) to 10 mL of double-distilled water to prepare a 10 mg / mL solution, and stir for 5 min; while stirring, add 5 mL of a 1 mg / mL manganese chloride solution to the above BSA solution at a rate of 1 mL / min, and dropwise add 0.1 M sodium hydroxide at the same rate until the solution pH reaches 10; continue to stir for 3 h until the BSA-MnO2 nanoparticle solution is neutral, and an albumin-manganese dioxide nanoparticle solution is obtained.

[0062] 2) Dissolve 4 mg of photosensitizer (I-BDP) in 8 mL of acetone to prepare a 0.5 mg / mL solution, and slowly add it to the albumin-manganese dioxide nanoparticle solution obtained in step 1); stir overnight to obtain a clear albumin-manganese dioxide-photosensitizer nanoparticle (BMB nanoparticle) solution.

[0063] 3) Seed unpolarized RAW264.7 macrophages (1×10 5 cells / mL) into a culture dish and culture them in a cell culture incubator at 37 °C with 5% CO2 for 24 h. Add DMEM medium containing 2 ng / mL IFN-γ, 0.5 μg / mL LPS and 10% fetal bovine serum to the RAW264.7 cell culture dish in the form of medium replacement, and incubate for 24 h to obtain M1 macrophages.

[0064] 4) Prepare the DMEM-nanoparticle medium, which is composed of the albumin-manganese dioxide-photosensitizer nanoparticles obtained in step 2) and the DMEM medium; based on the content of the photosensitizer, the content of the albumin-manganese dioxide-photosensitizer nanoparticles in the DMEM-nanoparticle medium is 20 μg / mL; add the DMEM-nanoparticle medium to the culture dish containing the M1 macrophages obtained in step 3) in the form of medium replacement, place it in a 37 °C cell culture incubator containing 5% CO2 and incubate for 4 h, and wash twice with PBS to obtain BMB@M1.

[0065] Test Example 1

[0066] The transmission electron microscope images of the BSA-MnO2 and BMB nanoparticles prepared in Example 1 are as Figure 2 shown, and the scale bar is 100 nm. It can be seen from Figure 2 that both the BSA-MnO2 and BMB nanoparticles have uniform particle sizes, and the surface of the BMB nanoparticles is rougher than that of BSA-MnO2.

[0067] The DMEM complete medium containing 20 μg / mL BMB nanoparticles with photosensitizer was co-incubated with macrophages for 4 h. Since BMB is dark green, the cytoplasm color of the loaded macrophages can be seen to deepen in the bright field image ( Figure 3 , scale bar is 100 μm), which confirmed that BMB was successfully phagocytosed by M1 cells. The fluorescence field image shows that BMB appears red under the action of a 688 nm wavelength laser. The bright field and fluorescence field images indicate that the BMB nanoparticles were successfully internalized into the cytoplasm of macrophages ( Figure 3 ).

[0068] Comparative Example 1

[0069] A preparation method similar to that in Example 1, except that in step 4), the incubation times are 0 h (con), 1 h, 2 h, 4 h, and 5 h respectively. After different incubations, fluorescence field pictures of the drug-loaded cells were taken, and the drug-loaded macrophages were digested and resuspended in PBS solution, and flow cytometry was used to analyze the intracellular fluorescence intensity.

[0070] The results are as Figure 4 shown. As the incubation time prolongs, the intracellular fluorescence intensity increases accordingly, and the intracellular fluorescence intensity remains unchanged after 4 h. This indicates that the concentration of BMB nanoparticles inside M1 macrophages reaches saturation after 4 h.

[0071] Comparative Example 2

[0072] A preparation method similar to that in Example 1, except that the BMB nanoparticles prepared in Example 1 were added to the 6-well plate containing M1 macrophages (1×10 5(cells / well), and the final concentrations were 0, 10, 20, 30, and 40 μg / mL respectively. After incubation in a 37°C cell culture incubator containing 5% CO2 for 4 h, the drug-loaded macrophages were digested and resuspended in PBS solution, and flow cytometry was used to analyze the intracellular fluorescence intensity.

[0073] The results are as Figure 5 shown. As the concentration of incubated BMB increased, the intracellular fluorescence intensity increased accordingly, and the intracellular fluorescence intensity remained unchanged after 10 μg / mL. This indicates that when M1 macrophages are co-incubated with BMB nanoparticle solution at a concentration of 10 μg / mL or higher for 4 h, the intracellular saturation concentration can be reached.

[0074] Comparative Example 3

[0075] A preparation method similar to Example 1, except that the BSA-MnO2 solution, BMB nanoparticle solution, and I-BDP solution were respectively added to a 96-well plate containing M1 macrophages (5×10 3 (cells / well). The final concentrations of the three solutions in the 96-well plate were 0.2, 0.5, 1.0, 2.0, 10.0, 20.0, 30.0, 40.0, and 50.0 μg / mL respectively (where the concentration of BMB nanoparticles was calculated based on I-BDP). After 24 h, a DMEM medium containing 10% cytotoxicity test reagent (CCK8) was added in the form of medium replacement for toxicity testing. Cell viability = experimental group cell viability / blank group cell viability × 100%. The results are as Figure 6 shown.

[0076] The results show that BMB nanoparticles have no toxicity to macrophages, and when the concentration of the photosensitizer is 20 μg / mL, the cell viability reaches about 150%. However, under the treatment of the I-BDP solution at the same concentration, the viability of macrophages was significantly inhibited. This indicates that BMB nanoparticles counteract the toxicity of free photosensitizers to macrophages and have a promoting effect on macrophage viability.

[0077] Comparative Example 4

[0078] To detect the ability of the macrophage drug-loading system to retain BMB nanoparticles before reaching the target, the present invention detected the 24 h leakage rate of BMB@M1 as follows:

[0079] According to the method of Example 1, M1 cells were seeded at 2×10 per well 5Inoculate at a density of cells into 6-well plates and culture for 24 h. Add DMEM medium containing BMB nanoparticles (at a concentration of 20 μg / ml in terms of I-BDP) to the 6-well plates in the form of medium replacement, and culture for 4 hours to obtain BMB@M1. After washing twice with PBS, replace with fresh DMEM medium without BMB nanoparticles, and collect cell supernatants at different time points (i.e., 1, 2, 4, 6, 12, 24 h after medium replacement). Use a fully automatic microplate reader to detect the content of BMB in the supernatant, which is the leakage amount of BMB@M1.

[0080] The leakage curve of BMB@M0 was obtained using the same method; the preparation method of BMB@M0 was similar to that of Example 1, except that M1 cells were replaced with M0 macrophages. The leakage rate of BMB was determined as the percentage of the leakage amount compared to the amount of BMB loaded in BMB@M1 or BMB@M0. Leakage rate = A leakage amount / A drug loading amount × 100%.

[0081] The results are as Figure 7 shown. The leakage rates of both BMB@M1 and BMB@M0 are relatively low (less than 20%), and the leakage rate of BMB@M1 is even lower. This indicates that M1 as a carrier of BMB nanoparticles has good drug-carrying and retention ability, reducing the leakage of BMB nanoparticles before reaching the tumor site and reducing the burden of the drug in the body.

[0082] Comparative Example 5

[0083] To detect the ability of BMB@M1 to relieve the hypoxic environment, BMB@M1 and BMB@M0 were prepared using the method of Comparative Example 4 in the present invention, and untreated M1 cells and BMB@M0 were used as control groups. Next, use a hypoxic green fluorescent probe (purchased from Thermo Fisher Scientific, catalog number l14834) and Hoechst 33342 to stain the cells for 40 min, wash the cells 3 times with PBS to remove free probes, and place the 6-well plates in a cell culture incubator with an oxygen partial pressure of 5% for 4 h. Observe the cell staining conditions through a fluorescence microscope. The results are as Figure 8 shown.

[0084] As Figure 8 can be seen, untreated M1 macrophages without carrying BMB nanoparticles and M0 macrophages carrying BMB nanoparticles emit strong green fluorescence under hypoxic conditions, indicating that the cells are in a hypoxic environment. When M1 cells carry BMB nanoparticles, hydrogen peroxide in M1 macrophages is catalyzed by manganese dioxide in BMB nanoparticles to generate oxygen, which quenches the hypoxic probe, so the emitted green fluorescence is weak. This shows that BMB@M1 generates oxygen, which can improve the hypoxic environment, and the generated oxygen can also enhance the subsequent photodynamic therapy.

[0085] Comparative Example 6

[0086] To verify the ability of BMB@M1 to target tumor cells, the present invention uses a transwell experiment. A transwell device consisting of a 24-well plate and a polycarbonate membrane chamber with an 8.0 μm pore size is used. 1×10 5 B16F10 cells in the logarithmic growth phase are resuspended in 600 μL of DMEM medium. The cell suspension is transferred to a 24-well plate and incubated for 24 h. The negative control group only adds DMEM medium to the bottom well. After 24 h, 1×10 5 BMB@M1 are resuspended in 100 μL of DMEM medium and inoculated into the chamber, and blank macrophages are used as the blank control group. After incubating for another 24 h, the cells in the chamber are fixed and stained. The specific method is to add a methanol solution containing 0.1% crystal violet to the chamber and the 24-well plate for fixation and staining for 30 min, wash twice with PBS, and wipe off the non-migrated cells (i.e., the cells remaining inside the chamber) with a cotton swab. Due to crystal violet staining, the migrated cells appear purple under bright field, and at this time, the targeting of BMB@M1 can be observed through a fluorescence microscope.

[0087] The results are as Figure 9 shown. When B16F10 is not inoculated at the bottom of the 24-well plate, only a few M1 cells migrate, indicating that the migration ability of M1 cells is weak without any stimulation. When M1 macrophages carry BMB nanoparticles, the migration of M1 cells increases under the stimulation of the drug. When B16F10 cells are inoculated at the bottom of the 24-well plate, it can be clearly observed that the migration number of M1 macrophages increases, indicating the natural targeting of M1 cells to tumor cells, and the migration ability of M1 cells carrying the drug is even stronger than that of non-drug-loaded M1 cells. This shows that BMB nanoparticles can promote the migration of M1 cells, so BMB@M1 is superior to M1 in terms of tumor tropism.

[0088] Comparative Example 7

[0089] To detect the change in the level of nitric oxide produced by macrophages before and after carrying nanoparticles, the present invention cultured BMB@M1 cells, M1 cells, and M0 cells. These cells were seeded in 96-well plates at a density of 5000 cells per well and incubated for 24 h. Finally, there were 3 groups of cells in the culture plate, and the levels of NO in BMB@M1, M1, and M0 in the culture plate were detected using a nitric oxide detection kit. The specific operation method is to take 50 μL of the cell supernatant medium, add it to the detection plate, add 50 μL of reagent 1 to each well in sequence, and then add 50 μL of reagent 2, and measure the absorbance at 540 nm.

[0090] As a pro-inflammatory macrophage, M1 cells produce a large amount of NO by expressing iNOS. High-concentration (1 μM) NO induces apoptosis of tumor cells through nitrosation of mitochondria and DNA. This natural tumor-killing ability of M1 cells is also the mechanism by which BMB@M1 drug-loaded cells kill tumors.

[0091] The results are as Figure 10 shown. The concentration of NO produced by M0 cells is the lowest, while the concentrations of NO produced by M1 cells and BMB@M1 are higher, and BMB@M1 is higher than M1 cells. This not only shows the ability of M1 cells to kill tumor cells, but also indicates that after carrying nanoparticles, the toxicity to tumors still exists and is even stronger.

[0092] Comparative Example 8

[0093] To verify the amount of drug transported by BMB@M1 to tumor cells and the transport mode, the present invention adopted methods such as flow cytometry, transwell experiment, and fluorescence microscopy imaging of the nanoparticle transport process between cells. Macrophages were inoculated at a concentration of 100,000 cells per well in a 6-well plate containing 2 mL of medium and incubated for 24 h. Then, M0 macrophages were induced into M1 macrophages as needed. Meanwhile, 100,000 B16F10-GFP cells were inoculated into a 6-well plate containing 2 mL of medium. After 24 h, BMB (final concentration of 20 μg / ml in terms of I-BDP) and I-BDP (final concentration of 20 μg / ml) were added to the macrophages in the 6-well plate and incubated for 4 h. Finally, the macrophages were divided into four groups: BMB@M1, BMB@M0, I-BDP@M1, and I-BDP@M0. The four groups of macrophages were digested and resuspended in 2 mL of DMEM medium, and then added to the 6-well plate for culturing B16F10-GFP in the form of medium replacement. After co-incubation for 24 h, the cells were washed and resuspended in a flow cytometry sample tube, and the average fluorescence intensity of BMB contained in B16F10-GFP cells was detected by flow cytometry.

[0094] To verify that BMB@M1 transports drugs through an efflux mechanism, the present invention used a transwell device composed of a 24-well plate and a 0.4-μm pore size polycarbonate membrane chamber for detection. 600 μL of medium containing 1×10 5 B16F10 cells was added to a 24-well plate and incubated for 24 h. 100 μL of DMEM medium containing 1×10 5 BMB@M1 was inoculated into the chamber, and unloaded M1 macrophages were used as the control group. After incubation for 24 h, the cells in the 24-well plate were washed twice, and the tumor cells were collected for flow cytometry detection.

[0095] To verify the phenomenon that BMB@M1 delivers drugs to tumor cells through nanochannels, in this invention, BMB@M1 was co-incubated with B16F10-GFP for 24 h. After removing the culture medium and washing twice with PBS and fixing, it was incubated with DAPI for 30 min and then placed under a laser confocal microscope to observe the formation of nanochannels.

[0096] The flow cytometry results are as Figure 11 shown. The presence of drugs was detected in the tumor cells of all groups. Therefore, macrophages can deliver drugs. By comparing the I-BDP@M1 group and the BMB@M1 group, it can be concluded that macrophages carrying BMB nanoparticles deliver more drugs to tumor cells, indicating that the albumin structure improves the biocompatibility of I-BDP. The same result can also be obtained by comparing the BMB@M0 group and the I-BDP@M0 group, indicating that the biocompatibility of BMB nanoparticles is independent of the type of carrier cells. By comparing the BMB@M1 and BMB@M0 groups, it can be seen that compared with M0 cells, M1 cells have the superiority in transporting drugs to tumor cells because M1 cells can transport drugs through the nano-tunnels that attack tumors.

[0097] To verify the efflux effect of BMB@M1 by detecting the drug content in tumor cells in the transwell device by flow cytometry, the results are as Figure 11 shown. The presence of drugs in the tumor indicates that BMB@M1 can deliver drugs to tumor cells through the efflux mechanism.

[0098] To confirm that M1 macrophages transport drugs into tumor cells through nano-tunnels, the morphology of nano-tunnels was observed under a confocal microscope after co-incubating BMB@M1 with B16F10-GFP. The results are as Figure 11 shown. In the bright-field images, it can be seen that macrophages carrying drugs are green and tumor cells are colorless in the images co-incubated for 1 h. After co-incubating for 24 h, interlaced nano-tunnels can be clearly observed between cells, and at this time the green color of macrophages fades, indicating the successful release of drugs. In the dark-field images, green fluorescence can be observed in tumor cells. Cells that do not express green fluorescent protein are M1 cells, and the red fluorescence represents the emission light after excitation by BMB nanoparticles. The nano-tunnels are indicated by the white arrows, and M1 cells are transporting drugs into tumor cells through the nano-tunnels.

[0099] Comparative Example 9

[0100] To detect whether the efficacy of photodynamic therapy is enhanced, ROS detection was carried out in this invention. BMB@M1 and I-BDP@M1 (prepared by the same method as Comparative Example 8) were co-incubated with B16F10-GFP for 24 h. Fresh DMEM medium containing ROS probe was added in the form of medium change and incubation was continued for 30 min. The cells were washed and collected, and irradiated with 54 J / cm 2The cell suspension was irradiated with a light dose of 100 μg / cm2, and the fluorescence intensity of ROS in B16F10-GFP was detected by flow cytometry. In the same way, BMB@M1 was co-incubated with B16F10-GFP and a ROS probe was added. After incubation for 30 min, the unbound probe was washed away and irradiated. After irradiation, the cells were immediately fixed and then stained with DAPI to observe the intracellular ROS under a confocal microscope.

[0101] pass Figure 12 It can be seen that red ROS fluorescence exists in BMB@M1 and B16F10-GFP. In order to quantify ROS in tumor cells, the present invention detected the difference in intracellular ROS levels after co-incubation of B16F10-GFP with different groups (BMB@M1, I-BDP@M1, BMB@M0) by flow cytometry. Figure 12 As shown in the figure, the amount of ROS in the BMB@M1 group is higher than that in the I-BDP@M1 group. This is because the good biocompatibility of BMB increases the photosensitizer dose stored in tumor cells, and because M1 cells carry MnO2 to form an oxygen backpack, which can exogenously provide raw materials for generating ROS. The ROS generated in the BMB@M1 group is higher than that in the BMB@M0 group. This is because M1 cells have multiple drug transport mechanisms compared to M0 cells, and can transport drugs into tumor cells more efficiently.

[0102] Comparative Example 10

[0103] In order to verify the sequential immune effect of photodynamic therapy, the present invention detects cytokines such as IFN-γ and IL-12 by an enzyme-linked immunosorbent assay (ELISA) kit. The ELISA test is divided into 8 groups, as follows:

[0104] (Ⅰ) M1 cell group: RAW264.7 cells were seeded in 6-well plates (1×10 5 After culturing for 24 h, the cells were polarized to M1 according to the method in Example 1, and the cell supernatant was collected after culturing for 24 h and detected using the corresponding ELISA kit;

[0105] (Ⅱ) M1 cell cultured near-infrared irradiation group: Similar to the M1 cell group, the difference is that the M1 cells were cultured for 24 hours and then irradiated with 54 J / cm 2 The cells were irradiated with near-infrared light at a dose of 1.50 nm, and the cell supernatant was collected 12 h after irradiation and detected using the corresponding ELISA kit;

[0106] (III) M1 and B16F10 incubation group: Similar to the M1 cell group, the difference is that after obtaining M1 cells, B16F10 cells were inoculated in 6-well plates, with 2×10 cells per well. 5 Cells were co-cultured for 24 h, and the cell supernatant was collected and tested using the corresponding ELISA kit;

[0107] (IV) Co-incubation of M1 and B16F10 with near-infrared irradiation group; similar to the group of M1 and B16F10 co-incubation, the difference is that after co-culture for 24 h, near-infrared light irradiation is carried out with a light dose of 54 J / cm 2 , and the cell supernatant is collected 12 h after irradiation, and detected using the corresponding ELISA kit;

[0108] (V) BMB@M1 group: similar to the M1 cell group, the difference is that M1 is replaced by BMB@M1 prepared according to the method of Example 1;

[0109] (VI) Incubation group of BMB@M1 and B16F10: similar to the incubation group of M1 and B16F10, the difference is that M1 is replaced by BMB@M1 prepared according to the method of Example 1;

[0110] (VII) Co-incubation of BMB@M1 and B16F10 with near-infrared irradiation group: similar to the co-incubation of M1 and B16F10 with near-infrared irradiation group, the difference is that M1 is replaced by BMB@M1 prepared according to the method of Example 1;

[0111] (VIII) Near-infrared irradiation group of BMB@M1: similar to the near-infrared irradiation group after M1 cell culture, the difference is that M1 is replaced by BMB@M1 prepared according to the method of Example 1.

[0112] IL-12 is a cytokine for macrophages to attack tumors, and IFN-γ is a cytokine for macrophages to enhance the immune effect of Th1 cells. Therefore, in the present invention, IL-12 and IFN-γ ELISA kits are used to verify the sequential immune effect of photodynamic therapy. The results are as Figure 13 shown. The production of immune factors of BMB@M1 and M1 is similar and both are higher than that of M0 cells. Under the condition of not carrying a photosensitizer, the laser itself has no obvious effect on macrophages and B16F10 cells. When macrophages carry drugs, the inflammatory reaction caused by the photodynamic effect is manifested as an increase in the concentrations of IL-12 and IFN-γ. The increase in the concentration of immune factors confirms the sequential immune effect of photodynamic therapy.

[0113] Comparative Example 11

[0114] The present invention uses melanoma-bearing mice to verify the ability of BMB@M1 to target tumors in vivo. In order to establish a melanoma-bearing mouse model, the present invention uses 5×10 5100 μL of PBS was added to dissolve 5.0×10⁶ B16F10 cells, and the mixture was subcutaneously injected into the right side of the back of female C57BL / 6 mice. To detect the accumulation of BMB@M1 in tumors, a small portion of the photosensitizer in the present invention was replaced with DiR dye (BMB / DiR), that is, during the preparation process according to Example 1, the photosensitizer was replaced with a mixture of the photosensitizer and DiR dye (the mass ratio of the photosensitizer to DiR was 1:1). When the tumor mass reached a diameter of 1.0 cm, BMB / DiR@M1 was intravenously injected, and the distribution of BMB@M1 in mice was observed. The IVIS Spectrum imaging system was used to perform in vivo fluorescence imaging of the mice at multiple time points, and the mice were sacrificed 24 h after administration of BMB / DiR@M1. The tumors and major organs were collected and imaged on the IVIS Spectrum imaging system.

[0115] The results were as Figure 14 shown. Within 15 hours after intravenous injection of BMB / DiR@M1, the DiR fluorescence in melanoma gradually increased, indicating that BMB / DiR@M1 accumulated in the tumor site in a time-dependent manner. However, the DiR fluorescence intensity in the tumors of BMB / DiR mice was relatively low, indicating that the tumor targeting ability of M1 macrophages exceeded that of BMB / DiR nanoparticles. Twenty-four hours after injection of macrophages, ex vivo images of the heart, liver, spleen, lung, kidney, and tumor confirmed the tumor targeting ability of BMB / DiR@M1.

[0116] Each mouse was injected with 5.0×10⁶ 6 cells every 3 days for a total of 3 times. Twenty-four hours after each administration, light irradiation at 54 J / cm² 2 (λ = 660 nm) was given. During the injection process, the body weight and tumor volume of the mice were recorded every 2 - 3 days, and the tumor volume formula was calculated as: V = width 2 × length × π / 6. To verify the inhibitory effect of BMB@M1 on tumors, the present invention established a melanoma-bearing mouse model according to the above method. When the tumor size of the tumor-bearing mice reached 100 mm³ 3 , the mice were respectively injected with BMB@M1, BSA-MnO₂@M1, I-BDP@M1, M1 macrophages, or PBS. The present invention recorded the tumor volume every two days during the 10-day treatment cycle.

[0117] The results were shown in Figure 15 . The tumor growth rate in the PBS group was faster than that in other groups. The tumors of mice injected with M1 or I-BDP@M1 were smaller than those of mice injected with PBS, confirming the innate immune effect of macrophages. The anti-tumor effects of BSA-MnO₂@M1 and BMB@M1 were superior to those of I-BDP@M1, which was related to improving the viability of M1 cells and alleviating hypoxia in the TME.

[0118] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all of them. People can also obtain other embodiments based on these embodiments without creative work, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A macrophage carrying nanoparticles, characterized in that, It includes BMB nanoparticles and M1 macrophages; the BMB nanoparticles are located inside the M1 macrophages; the BMB nanoparticles include albumin, manganese dioxide, and I-BDP photosensitizer; the albumin and manganese dioxide are connected through metal binding sites, and the I-BDP photosensitizer is attached to the manganese dioxide.

2. The macrophage according to claim 1, wherein The albumin includes bovine serum albumin.

3. The method for preparing the macrophage according to claim 1 or 2, characterized in that, It includes the following steps: Mix the albumin solution and the salt solution containing manganese ions, adjust the pH value of the mixture to 9-10, and stir until neutral to obtain the BSA-MnO2 nanoparticle solution; Mix the BSA-MnO2 nanoparticle solution and the I-BDP photosensitizer to obtain the BMB nanoparticles; Mix the BMB nanoparticles and M1 macrophages, and incubate for more than 4 h to obtain the macrophages carrying the nanoparticles.

4. The preparation method according to claim 3, characterized in that, The salt containing manganese ions includes manganese chloride.

5. The preparation method according to claim 4, characterized in that, The mass ratio of the albumin to the manganese chloride is 10-1000:0.5-5.

6. The preparation method according to claim 3, characterized in that, The mass ratio of the I-BDP photosensitizer to the albumin is 0.1-20:

100.

7. The preparation method according to claim 3, characterized in that, Based on the mass of the I-BDP photosensitizer, in the mixture of the BMB nanoparticles and M1 macrophages, the concentration of the BMB nanoparticles is 1-200 μg / mL.

8. The preparation method according to claim 3, characterized in that, The temperature of the incubation is 37 °C.

9. Use of the macrophages according to claim 1 or 2 or the macrophages prepared by the preparation method according to any one of claims 3-8 in the preparation of a drug for photodynamic therapy.

10. The application according to claim 9, characterized in that, The diseases for photodynamic therapy include melanoma.

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