Selenium-based nanoparticle modified macrophage, medicine and application of selenium-based nanoparticle modified macrophage

Abstract

The invention discloses a selenium-based nanoparticle modified macrophage, a medicine and application of the selenium-based nanoparticle modified macrophage, and relates to the technical field of biological medicine. According to the selenium-based nanoparticle modified macrophage disclosed by the invention, the selenium-based nanoparticles are loaded by the liposome particles, so that the selenium-based nanoparticle modified macrophage has good heart targeting property and an effect of promoting myocardial ischemia reperfusion injury repair, and a brand new strategy is provided for precise intervention of MIRI.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a selenium-based nanoparticle-modified macrophage, a drug, and applications thereof. Background Art

[0002] Cardiac ischemia-reperfusion injury (MIRI) is a serious complication following myocardial infarction revascularization, characterized by persistent immune activation, oxidative damage, and abnormal energy metabolism, which can lead to an expansion of the infarct size, malignant arrhythmias, and even sudden cardiac death. The immune response plays an important regulatory role in MIRI and also mediates the exacerbation of other pathological processes. Although anti-inflammatory drugs, antioxidants, and antithrombotic agents have been used clinically, they suffer from poor targeting, a narrow therapeutic window, and systemic toxic side effects. In addition, there is a lack of effective means to precisely regulate immune-inflammatory homeostasis. Therefore, the development of new targeted therapeutic strategies is urgently needed.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] The present invention provides a selenium-based nanoparticle-modified macrophage, a drug, and its application to solve at least one of the following problems in the current treatment of myocardial ischemia-reperfusion injury:

[0005] 1) Insufficient targeting: Systemic administration of traditional drugs (such as anti-inflammatory agents and antioxidants) leads to low drug concentrations in the lesion site and is prone to systemic toxic side effects; 2) Imbalance in immune regulation: CAR cell therapy carries the risk of cytokine storm and graft-versus-host disease, making it difficult to accurately regulate immune-inflammatory homeostasis; 3) Uncontrollable drug metabolism: Selenium-based compounds have a narrow therapeutic window, are prone to toxicity due to cumulative doses, and lack a lesion-responsive release mechanism; 4) Lack of synergistic treatment: A single mechanism of action (such as only inhibiting inflammation or clearing ROS) cannot fully reverse the multi-pathway pathological damage of MIRI.

[0006] The present invention is achieved in that:

[0007] In one aspect, the present invention provides a macrophage modified with selenium-based nanoparticles, comprising a macrophage, wherein the surface of the macrophage is bound to liposome particles, and the liposome particles contain selenium-based nanoparticles.

[0008] Cell-based delivery platforms have become an important research direction in the field of drug delivery due to their advantages such as low immunogenicity, long circulation period, natural homing ability, and receptor integration capacity. T cell and macrophage therapies based on chimeric antigen receptor (CAR) technology can achieve local immune regulation by specifically recognizing myocardial injury-associated antigens. However, CAR cell therapy has drawbacks such as complex preparation process, cytokine release syndrome, and graft-versus-host disease, which limit its potential for clinical translation. M2 macrophages, as key effector cells in the repair phase of MIRI, can be actively recruited to the site of injury and improve prognosis through anti-inflammatory-repair mechanisms. Utilizing M2 macrophages to construct a drug delivery platform can not only retain their natural targeting and anti-inflammatory capabilities, but also enhance efficacy by loading exogenous drugs, providing a new approach for the treatment of MIRI.

[0009] Selenium (Se) has attracted much attention due to its unique antioxidant and immunomodulatory properties. As an essential trace element for the human body, selenium can scavenge reactive oxygen species (ROS) and inhibit cardiomyocyte apoptosis by activating the glutathione peroxidase (GPx) pathway. However, the therapeutic window of selenium is narrow, and excessive absorption of organic and inorganic selenium compounds can easily induce toxic reactions. In contrast, nanoscale selenium drugs have low toxicity and good biocompatibility. By combining selenium-based nanoparticles with cell carriers, their targeted delivery efficiency can be further improved and the risk of systemic exposure can be reduced.

[0010] The selenium-modified macrophages provided by the present invention have good cardiac targeting and promote the repair of myocardial ischemia-reperfusion injury, providing a new strategy for the precise intervention of MIRI.

[0011] Optionally, in some embodiments of the present invention, the liposome particles further contain a STING inhibitor.

[0012] Optionally, in some embodiments of the present invention, the STING inhibitor is c176.

[0013] It should be noted that, in some other embodiments, the STING inhibitor can be selected as needed, for example, it can be: C-178, H-151, SN-011, Astin C, or GSK-626, etc., it can be one of them or a combination of several of them.

[0014] It should be noted that, in some other embodiments, the STING inhibitor may also be other drugs, and those skilled in the art may select the drug according to the purpose of treatment.

[0015] Alternatively, in some embodiments of the present invention, the selenium-based nanoparticles include selenium nanospheres and platinum nanoparticles bound to the surfaces of the selenium nanospheres.

[0016] Alternatively, in some embodiments of the present invention, the liposome particles include phospholipids, cholesterol or its derivatives, and PEG-modified lipids.

[0017] Alternatively, in some embodiments of the present invention, the phospholipid is selected from DPPC;

[0018] It should be noted that, in some other embodiments, the phospholipids can be selected as needed and can be natural phospholipids, such as egg yolk phosphatidylcholine (PC), soybean phosphatidylcholine (Soy PC), etc., or synthetic and semi-synthetic lecithins, such as DSPE, DOPE, and hydrogenated lecithin.

[0019] Alternatively, in some embodiments of the present invention, the PEG-modified lipid is selected from DSPE-PEG.

[0020] It should be noted that the PEG-modified lipids can be modified with modified components as needed, for example, polymer modifications such as polysorbate, polyethylene glycol-polylactic acid (PEG-PLA), polyvinyl pyrrolidone (PVP), polyhydroxyethyl acrylate (PHEA), etc.; polysaccharide modifications such as hyaluronic acid (HA), chitosan, heparin, etc.; protein or polypeptide modifications such as transferrin, RGD peptide, antibody fragments (such as Fab); lipid derivative modifications: such as cholesterol-PEG, stearylamine, DSPE (distearoylphosphatidylethanolamine); small molecule ligand modifications: such as folic acid, galactose, mannose; environmental responsive modifications: such as pH-sensitive lipids (such as DOPE), temperature-sensitive polymers (such as poly (N-isopropylacrylamide), etc.

[0021] Alternatively, in some embodiments of the present invention, the phospholipid is DPPC and the PEG-modified lipid is DSPE-PEG5k.

[0022] It should be noted that, in other embodiments, the molecular weight of PEG in DSPE-PEG can be selected as needed, for example, it can be 0.5k, 1k, 1.5k, 2k, 2.5k, 3k, 3.5k, 4k, 4.5k, 5k, 5.5k, 6k, 6.5k, 7k, 7.5k, 8k, 8.5k, 9k, 9.5k, or 10k, etc.

[0023] Alternatively, in some embodiments of the present invention, the macrophages are covalently bound to the liposome particles;

[0024] Alternatively, in some embodiments of the present invention, the covalent binding is achieved by a bioorthogonal coupling reaction;

[0025] Optionally, in some embodiments of the present invention, the bioorthogonal coupling reaction is a click chemistry reaction.

[0026] It should be noted that, in other embodiments, covalent binding can also be achieved by one or more of the following reactions: azide-alkyne cycloaddition (SPAAC), inverse electron demand Diels-Alder reaction (iEDDA), oxime / hydrazone bond formation (Oxime / Hydrazone Ligation), Staudinger ligation (Staudinger Ligation), thiol-ene / yne click (Thiol-ene / yne), hexavalent sulfur fluoride exchange (SuFEx), cyanobenzothiazole coupling (CBT-Cys Reaction), phototriggered reaction (Photoclick Reactions), bioorthogonal uncaging (Bioorthogonal Uncaging), enzyme-mediated bioorthogonal reaction.

[0027] Alternatively, in some embodiments of the present invention, the click chemistry reaction is selected from any one of a cycloaddition reaction, a nucleophilic ring-opening reaction, a non-aldol carbonyl chemistry, and a carbon-carbon multiple bond addition reaction.

[0028] Alternatively, in some embodiments of the present invention, the click chemistry reaction is achieved based on a Tetrazine (TZ)-Trans-Cyclooctene (TCO) specific cycloaddition reaction.

[0029] Through the Tetrazine (TZ)-Trans-Cyclooctene (TCO) bioorthogonal reaction, the loaded selenium-based nanoparticles and STING inhibitor liposomes are efficiently anchored on the cell surface, ensuring the controlled release of the drug at the lesion site.

[0030] It should be noted that, in other embodiments, the click chemistry reaction can also be achieved based on the following reactions: azide and alkyne, azide and dibenzocyclooctyne (DBCO), tetrazine and trans-cyclooctene (TCO), tetrazine and cyclopropene, thiol and alkene, thiol and alkyne, tetrazole and alkene, hydroxylamine and ketone / aldehyde (Ketone / Aldehyde), phosphine and azide, hydrazide and aldehyde / ketone (Aldehyde / Ketone), cyanobenzothiazole (CBT) and cysteine, sulfonyl fluoride (Sulfonyl fluoride) and hydroxylamine. Fluoride) and amine / hydroxyl (Amine / Hydroxyl), or norbornene (Norbornene) and tetrazine (Tetrazine).

[0031] Alternatively, in some embodiments of the present invention, the Tetrazine group is modified on DSPE-PEG5k, the Cyclooctene gene is modified on the macrophage, and the Cyclooctene gene is modified on the DSPE-PEG5k inserted on the macrophage.

[0032] Optionally, in some embodiments of the present invention, the macrophages are M1 or M2 polarized macrophages.

[0033] Optionally, in some embodiments of the present invention, the macrophages are M2 polarized macrophages.

[0034] By utilizing the natural targeting and immune escape capabilities of M2 macrophages, precise targeting of damaged areas can be achieved.

[0035] In another aspect, the present invention provides a method for preparing the above-mentioned macrophages, comprising one of the following steps:

[0036] Step a, liposome particle preparation: liposomes loaded with Pt / Se nanoparticles (100 mg / L) and the STING inhibitor c176 (drug loading 5-10 wt%) were synthesized using a thin film hydration method and surface modified with DSPE-PEG5k-TCO;

[0037] Step b, M2 macrophage induction: Monocytes were extracted from mouse bone marrow and polarized with IL-4 / IL-13 (20 ng / mL) for 48 hours to obtain M2 phenotype macrophages; DSPE-PEG5k-TZ (50 μg / mL) was inserted into the M2 macrophage membrane and anchored to the phospholipid bilayer through hydrophobic interactions, forming engineered cells with exposed TZ groups;

[0038] Step c, bioorthogonal coupling: TZ-modified M2 macrophages were mixed with TCO-drug-loaded liposomes at a particle ratio of 1:50 and covalently linked via a TZ-TCO click reaction (25°C, 30 min) to form an engineered macrophage system with surface-modified selenium-based nanomedicines (PS-c@M).

[0039] Step d, targeted delivery and responsive release: M2 macrophages actively migrate to the ischemia-reperfusion injury area through their own chemotaxis. The damaged area contains a large amount of ROS, which triggers the rupture of the liposome membrane and releases nanodrugs Pt / Se and c176, alleviating MIRI through multiple mechanisms.

[0040] It should be noted that, based on the selenium-modified macrophage structure provided in the embodiments of the present invention, each component and assembly can be prepared, loaded, modified and coupled using conventional techniques in the art; this is easily achievable for those skilled in the art.

[0041] In another aspect, the present invention provides use of any of the above-mentioned macrophages in the preparation of a medicament for treating cardiac ischemia-reperfusion injury.

[0042] In another aspect, the present invention provides a drug for treating cardiac ischemia-reperfusion injury, comprising the macrophages as described in any one of the above items and a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0044] Figure 1 This is a TEM image of the α-Se nanospheres in Example 1 of the present invention. From the image, it can be seen that the α-Se nanospheres have a uniform spherical structure and a uniform size of about 80-100 nm.

[0045] Figure 2This is a TEM image of the Pt / Se nanoheterogeneous structure in Example 1 of the present invention. From the image, it can be seen that Pt / Se is a uniformly distributed core-shell sphere, consisting of a central spherical Se nanosphere and dense and tiny Pt nanostructures dispersed on the Se surface, with a diameter of about 100 nm.

[0046] Figure 3 Figure 3 is the X-ray diffraction (XRD) pattern of the Pt / Se nanoheterogeneous structure in Example 1 of the present invention. The results in the figure show that the spectrum of Se is characterized by an amorphous structure. The XRD characteristic peaks of the Pt / Se nanospheres are located at approximately 39.8°, 46.2°, 67.5°, 81.3° and 85.7°, respectively, corresponding to the (111), (200), (220), (311) and (222) crystal planes of the Pt fluorite phase (JCPDS No. 04-0802).

[0047] Figure 4 This is the X-ray photoelectron spectroscopy (XPS) diagram of Pt / Se in Example 1 of the present invention. The binding energy peaks shift toward the high energy direction to about 70.6 eV and 74.6 eV, confirming that Pt achieves efficient chemical assembly on the surface of Se nanospheres.

[0048] Figure 5 This is the TEM image of Lipo-PS-c in Example 2 of the present invention. From the figure, we can see that there is a membrane structure around Pt / Se with a thickness of about 5.5 nm, indicating that we have successfully constructed drug-loaded liposomes.

[0049] Figure 6 Dynamic light scattering (DLS) graphs of Se, Pt / Se, and Lipo-PS-c in Examples 1 and 2 of the present invention show that the average particle sizes of Se, Pt / Se, and Lipo-PS-c are 96.33, 108.6, and 127.3 nm, respectively, and the polymer dispersibility index (PDI) is 0.023, 0.092, and 0.112, respectively.

[0050] Figure 7 The Zeta potential diagrams of Se, Pt / Se and Lipo-PS-c in Examples 1 and 2 of the present invention show that the Zeta potentials of Se, Pt / Se and Lipo-PS-c are -32.76, -33.83 and -27.27 mV, respectively.

[0051] Figure 8 Se, Pt / Se and Lipo-PS-c obtained in Examples 1 and 2 removed O 2- The ESR graphs of ·OH and Pt / Se and Lipo-PS-c showed that the ·O 2- and ·OH, and is superior to Se nanospheres.

[0052] Figure 9 This is a western blot image of M2 macrophage markers of PS-c@M in Example 3 of the present invention. The results show that the expression levels of M2 marker proteins such as MerTK, TGF-β, Arg1, and CD163 in PS-c@M and M2 macrophages are comparable, indicating that the modification of Lipo-PS-c does not affect the polarization state and function of M2 macrophages.

[0053] Figure 10 This is the TEM image of PS-c@M in Example 3 of the present invention, confirming that Lipo-PS-c was successfully connected to the surface of M2 macrophages.

[0054] Figure 11 This is a scanning electron microscope (SEM) image of PS-c@M in Example 3 of the present invention, which proves that M2 macrophages were successfully induced, showing a long spindle shape and loaded with Lipo-PS-c on the surface.

[0055] Figure 12 The drug release of Lipo-PS-c coupled to the PS-c@M in Example 3 of the present invention on the surface of M2 macrophages in a simulated infarcted myocardial microenvironment (ROS-rich acidic environment, pH = 6.8) was shown. The flow cytometry results showed that the drug release gradually increased with time and was completely released in about 8 hours.

[0056] Figure 13(a) shows the fluorescence staining results of Calcein AM / PI in cardiomyocytes, indicating that Lipo-PS-c has the strongest anti-cardiomyocyte apoptosis ability, which is significantly better than Pt / Se and Se.

[0057] Figure 13(b) shows the results of Annexin V-FITC / PI flow cytometry analysis, which shows that Lipo-PS-c has the strongest anti-cardiomyocyte apoptosis ability, which is significantly better than Pt / Se and Se.

[0058] Figure 14 In vivo fluorescence imaging of the hearts of mice in different groups showed that PS-c@M could be actively recruited to the damaged heart and persist for at least 96 hours. In addition, the fluorescence signal of FITC-Pt / Se decayed earlier than that of DID-M2 macrophages, indicating that nano-Pt / Se was continuously released from the surface of PS-c@M residing in the damaged area.

[0059] Figure 15 The results show that PS-c@M can significantly inhibit STING and its downstream related signaling pathways, resist cell death and regulate immune inflammatory responses, and its overall effect is better than Lipo-PS-c.

[0060] Figure 16The 28-day ultrasound data follow-up showed that Lipo-PS-c and PS-c@M significantly improved the cardiac function of mice, with a significant increase in left ventricular ejection fraction (EF) and left ventricular fractional shortening (FS). Furthermore, as time went on, the recovery effect of PS-c@M became increasingly better, and was overall superior to Lipo-PS-c.

[0061] Figure 17 Masson and HE staining were performed after 28 days of follow-up. The results showed that PS-c@M significantly alleviated the adverse ventricular remodeling caused by MIRI, reduced the area of ​​myocardial infarction, and the effect was better than Lipo-PS-c. DETAILED DESCRIPTION

[0062] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0063] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0064] Example 1

[0065] This example synthesizes Pt / Se nanoparticles, and the specific synthesis method is as follows:

[0066] 1) Weigh 100 mg of polyvinylpyrrolidone (PVP) and 387 mg of selenious acid (H2SeO3) and dissolve them in 100 mL of deionized water. Stir magnetically at 25°C for 10 min to form a homogeneous mixed solution.

[0067] 2) Add 1.16 mL of hydrazine hydrate (N2H4·H2O) to the solution obtained in step (1) at a uniform rate, maintain magnetic stirring conditions, and continue the reaction at 25°C for 3 h;

[0068] 3) After the reaction is completed, the mixture is placed in a centrifuge and centrifuged at 12000 rpm for 20 min to separate the solid phase product;

[0069] 4) washing the solid phase product three times with deionized water to remove unreacted impurities;

[0070] 5) The purified product was dispersed in 50 mL of deionized water to prepare an α-Se nanosphere suspension, which was stored at 4°C for later use.

[0071] 6) Add 5 mg of PVP and 4 mL of the prepared α-Se nanospheres to 4 mL of deionized water, stir at 25°C for 10 min, and uniformly add sodium hydroxide (NaOH) solution to adjust the pH to 7.0 to form a homogeneous mixed solution.

[0072] 7) To the mixed solution in step (6), 0.1 mL of potassium hexachloroplatinate (K2PtCl6) aqueous solution (100 mM) was added at a rate of 0.02 mL / min, and magnetic stirring was carried out at 25°C for 12 h to obtain Pt / Se nanoheteromorphs.

[0073] The Pt / Se nanoheterogeneous bodies (selenium-based nanoparticles) obtained in this example were tested by inductively coupled plasma optical emission spectrometer (ICP-OES), which showed that the mass ratio of Pt to Se elements in the Pt / Se nanocomposite was 1:48.277.

[0074] The structure of the obtained Pt / Se nanocomposite was identified, and the results are as follows:

[0075] Figure 1 This is the TEM image of α-Se nanospheres. From the image, we can see that the α-Se nanospheres have a uniform spherical structure and a uniform size of about 80-100nm.

[0076] Figure 2 This is the TEM image of the Pt / Se nanoheterogeneous structure. From the figure, we can see that Pt / Se is a uniformly distributed core-shell sphere, consisting of a central spherical Se nanosphere and dense and tiny Pt nanostructures dispersed on the Se surface, with a diameter of about 100 nm.

[0077] Figure 3 This is the X-ray diffraction (XRD) pattern of the Pt / Se nanoheterogeneous structure. The results in the figure show that the spectrum of Se is characterized by an amorphous structure. The XRD characteristic peaks of the Pt / Se nanospheres are located at approximately 39.8°, 46.2°, 67.5°, 81.3° and 85.7°, corresponding to the (111), (200), (220), (311) and (222) crystal planes of the Pt fluorite phase (JCPDS No. 04-0802).

[0078] Figure 4 The X-ray photoelectron spectroscopy (XPS) diagram of Pt / Se shows that the binding energy peaks shift toward the high energy direction to about 70.6 eV and 74.6 eV, confirming that Pt has achieved efficient chemical assembly on the surface of Se nanospheres.

[0079] Example 2

[0080] Based on the Pt / Se nanoheterosomes, this example further synthesized drug-loaded liposomes, i.e., liposomes loaded with small molecule drug c176 and nanodrug Pt / Se (Lipo-PS-c). The specific synthesis method is as follows:

[0081] 1) Liposome Dissolution and Film Formation: Weigh 20 mg of dipalmitoylphosphatidylcholine (DPPC), 5 mg of cholesterol (Chol), 2 mg of DSPE-PEG5k-TZ, and 5 mg of c176 (purchased from MedChemExpress) into a round-bottom flask. Add 5 mL of chloroform and stir magnetically at 25°C for 10 min to obtain a transparent, homogeneous mixed solution. Connect the flask to a rotary evaporator with a water bath temperature of 40°C, a vacuum of 200 mbar, and a rotation speed of 100 rpm for 30 min to ensure that the solvent is evaporated to dryness, no residual chloroform odor remains, and a uniform lipid film forms on the flask wall.

[0082] 2) Hydration and liposome formation: Before hydration, the Pt / Se solution was sonicated (30 s, 40 kHz) to eliminate agglomerates. Subsequently, Pt / Se (100 mg / L) was dispersed in preheated PBS buffer (60°C, above the lipid phase transition temperature to ensure sufficient hydration). 10 mL of preheated PBS buffer was added to the flask, and the mixture was placed in a constant temperature magnetic stirrer (60°C, 500 rpm) for 1 h to completely detach the film to form a mixed suspension. The suspension was then vortexed intermittently (10 s once, 30 s apart, repeated 5 times) to promote liposome homogenization.

[0083] 3) Particle Homogenization: Transfer the suspension to an ice bath and sonicate with a probe at 50W in pulse mode for 2 seconds, 3 seconds, and 5 minutes, until the suspension becomes translucent. Then, use a liposome extruder to pass the suspension through 0.45μm and 0.22μm polycarbonate membranes 10 times each to obtain uniformly loaded monolayer liposomes (particle size approximately 100-200nm).

[0084] 4) Purification and Storage: Ultracentrifuge (100,000 g, 4°C, 1 h) to remove unencapsulated material. Pass the product through a 0.22 μm sterile filter membrane. Store sterile in the dark and away from light. Store at 4°C for short-term storage (<7 days) or lyophilize (prefreeze at -80°C for 24 h and then freeze-dry) for long-term storage.

[0085] The results of high performance liquid chromatography (HPLC) analysis of the drug-loaded liposomes obtained in this example showed that the mass ratio of c176 in Lipo-PS-c was 5.962%.

[0086] Figure 5 This is the TEM image of Lipo-PS-c in this example. From the image, we can see that there is a membrane structure around Pt / Se with a thickness of about 5.5 nm, indicating that we have successfully constructed drug-loaded liposomes.

[0087] Figure 6 Dynamic light scattering (DLS) graphs of Se, Pt / Se, and Lipo-PS-c in Examples 1 and 2 of the present invention show that the average particle sizes of Se, Pt / Se, and Lipo-PS-c are 96.33, 108.6, and 127.3 nm, respectively, and the polymer dispersibility index (PDI) is 0.023, 0.092, and 0.112, respectively.

[0088] Figure 7 The Zeta potential diagrams of Se, Pt / Se and Lipo-PS-c in Examples 1 and 2 of the present invention show that the Zeta potentials of Se, Pt / Se and Lipo-PS-c are -32.76, -33.83 and -27.27 mV, respectively.

[0089] Figure 8 Se, Pt / Se and Lipo-PS-c obtained in Examples 1 and 2 removed O 2- The ESR graphs of ·OH and Pt / Se and Lipo-PS-c showed that the ·O 2- and ·OH characteristic peak intensities, and is superior to that of Se nanospheres;

[0090] Example 3

[0091] Based on the Lipo-PS-c drug-loaded liposomes, this example further synthesized an engineered macrophage system (PS-c@M) with surface-modified selenium-based nanomedicines. The specific synthesis steps are as follows:

[0092] The first step is to extract mouse bone marrow macrophages (BMDM) and induce M2 macrophages:

[0093] 1) Mouse euthanasia and bone isolation: C57BL / 6 male mice were euthanized and their entire bodies disinfected by soaking in alcohol. The skin of the hind limbs was cut open, and the femur and tibia were separated. Muscle and connective tissue were removed, and the intact bones were removed and soaked in pre-chilled 75% alcohol for 2 minutes. The bones were then rinsed three times with PBS.

[0094] 2) Bone marrow cell collection: Cut both ends of the bone and use a syringe (25G needle) to draw up pre-chilled PBS. Repeatedly flush the bone marrow cavity until the fluid becomes turbid. Pass the bone marrow suspension through a 70 μm cell sieve and collect it in a 50 mL centrifuge tube.

[0095] 3) Red blood cell lysis: Add 5 mL of red blood cell lysis buffer and incubate at room temperature for 5 min. Then add 10 mL of PBS to terminate the reaction. Centrifuge at 300 g for 5 min, discard the supernatant, and resuspend the cells in BMDM culture medium (DMEM / F12 + 10% FBS + 1% Pen / Strep + 20 ng / mL M-CSF).

[0096] 4) BMDM differentiation culture: Cell suspension was plated in a 10 cm culture dish and cultured in a 37°C, 5% CO2 cell culture incubator. On the third day, the medium was changed halfway, and on the sixth day, the medium was changed completely. Cell morphology and adherence were observed. On the seventh day, cells were harvested using a cell scraper, centrifuged (300 g, 5 min), and resuspended for later use.

[0097] 5) Induction of M2 macrophages: The cell suspension from (4) was inoculated into a 6-well plate, and 20 ng / mL IL-4 and 20 ng / mL IL-13 were added to the BMDM culture medium (cell density 1×10 6 cells / mL, 2 mL / well) and cultured for 48 h to induce BMDM polarization toward M2 macrophages.

[0098] The second step is the experimental procedure of click chemistry coupling between DSPE-PEG5k-TZ modified M2 macrophages and DSPE-PEG5k-TCO-Lipo-PS-c drug-loaded liposomes, based on the Tetrazine (TZ)-Trans-Cyclooctene (TCO) specific cycloaddition reaction:

[0099] 1) Cell pretreatment: M2 macrophages were seeded into 6-well plates and cultured in a cell culture incubator to 80% confluence;

[0100] 2) Preparation of TZ insertion solution: Dissolve DSPE-PEG5K-TZ in serum-free culture medium to a final concentration of 50 μg / mL (optimization range: 20-100 μg / mL);

[0101] 3) Incubation: Discard the original culture medium and add 2 mL of DSPE-PEG5K-TZ insertion solution. Incubate at 37°C in the dark for 2 h. Then wash three times with pre-cooled PBS (containing 1% BSA) to remove uninserted DSPE-PEG5K-TZ.

[0102] 4) Fluorescence labeling: FITC (10 μM) was incubated with drug-loaded liposomes at 25°C in the dark for 15 min, and DID (5 μM, a red fluorescent membrane dye) was incubated with M2 macrophages in the dark for 30 min. This step was used for subsequent drug release detection.

[0103] 5) Click reaction coupling: TZ-modified M2 macrophages were mixed with TCO-liposomes at a cell:liposome ratio of 1:50 in PBS and incubated in the dark at room temperature with shaking for 30 min to complete TZ-TCO covalent coupling;

[0104] 6) Washing and purification: Centrifuge at 300g for 5 minutes to remove unbound liposomes and resuspend in complete culture medium for later use.

[0105] Figure 9 This is a western blot image of M2 macrophage markers in PS-c@M in this example. The results show that the expression levels of M2 marker proteins such as MerTK, TGF-β, Arg1, and CD163 in PS-c@M and M2 macrophages are comparable, indicating that Lipo-PS-c modification does not affect the polarization state and function of M2 macrophages.

[0106] Figure 10 This is the TEM image of PS-c@M in this example, which confirms that Lipo-PS-c is successfully connected to the surface of M2 macrophages.

[0107] Figure 11 This is a scanning electron microscope (SEM) image of PS-c@M in this example, which proves that M2 macrophages were successfully induced, showing a long spindle shape and loaded with Lipo-PS-c on the surface.

[0108] Figure 12 Figure 3 shows the drug release of Lipo-PS-c coupled to the surface of M2 macrophages in the PS-c@M in this example in a simulated infarcted myocardial microenvironment (ROS-rich acidic environment, pH = 6.8). Flow cytometry results showed that drug release gradually increased with time and was completely released in about 8 hours.

[0109] Example 4

[0110] To verify the ability of Se, Pt / Se, and Lipo-PS-c in Examples 2 and 3 of the present invention to inhibit cardiomyocyte apoptosis, this example used Calcein AM / PI staining and Annexin V-FITC / PI flow cytometry to test cell survival in an oxygen-glucose deprivation / reperfusion (OGD / R) model. The specific experimental methods are as follows:

[0111] 1) Cardiomyocytes were seeded in a six-well plate and the cell density was observed to be 80-90%. Se, Pt / Se, and Lipo-PS-c (100 μg / mL) were added and co-cultured with the cardiomyocytes for 8 hours.

[0112] 2) The cells were placed in ischemia buffer (118 mmol / L NaCl, 24 mmol / L NaHCO3, 1 mmol / L NaH2PO4, 2.5 mmol / L CaCl2-2H2O, 1.2 mmol / L MgCl2, 20 mmol / L sodium lactate, 16 mmol / L KCl, and 10 mmol / L 2-deoxyglucose, pH adjusted to 6.2) and cultured in a 0.5% hypoxic incubator for 2 h. The ischemia buffer was then replaced with culture medium for reoxygenation for 3 h to induce an OGD / R model to simulate in vitro ischemia-reperfusion injury. The control group received no treatment.

[0113] 3) According to the reagent instructions, cells were fluorescently stained with Calcein-AM / PI and the proportion of apoptotic cells was analyzed by flow cytometry using Annexin V-FITC / PI.

[0114] Figure 13(a) shows the fluorescence staining results of Calcein AM / PI in cardiomyocytes, and Figure 13(b) shows the flow cytometry analysis results of Annexin V-FITC / PI. Both indicate that Lipo-PS-c has the strongest anti-cardiomyocyte apoptosis ability, which is significantly better than Pt / Se and Se.

[0115] Example 5

[0116] This example tests the PS-c@M obtained in Example 3 of the present invention to have good cardiac targeting and promote the repair of myocardial ischemia-reperfusion injury. The specific testing method is as follows:

[0117] 1) 8-week-old C57 / BL male mice were used to establish the MIRI model. The specific method was as follows: preoperatively, the mice underwent chest hair removal and were anesthetized with isoflurane inhalation. The left thoracotomy was then performed to expose the heart. The left anterior descending coronary artery was ligated through the left atrium with a 6-0 polypropylene suture, forming a sliding knot. The heart was returned to the chest cavity using a ribbed forceps. After 45 minutes, the mouse was reperfused, the sliding knot was released, and the mouse was allowed to recover. This established the myocardial ischemia-reperfusion injury model.

[0118] 2) To study the targeted distribution of PS-c@M in mice, mice were injected with DID-labeled M2 macrophages and FITC / DID-labeled PS-c@M via tail vein on the second day after surgery, with the injection dose of 3×10 6 cells / 200 μL. Experimental groups were as follows: control mice + M2 macrophages, control mice + PS-c@M, MIRI mice + M2 macrophages, and MIRI mice + PS-c@M. Mice were sacrificed at 24, 48, and 96 h, and hearts were harvested. The targeting ability of PS-c@M was assessed using in vivo fluorescence.

[0119] 3) Western blot analysis of STING and downstream related inflammatory signaling pathways was performed as follows: control group, MIRI group, MIRI+Lipo-PS-c group, and MIRI+PS-c@M group. 2 h after reperfusion, mice were given PS-c@M (3×10 6 cells / 200μL) or Lipo-PS-c (100ug / mL), and heart tissues were harvested 3 days later for WB analysis to detect changes in related signaling pathway proteins.

[0120] 4) The animal experiments for evaluating the therapeutic effect were divided into the following groups: control group, MIRI group, MIRI+Lipo-PS-c group, and MIRI+PS-c@M group. 2 h after reperfusion, mice were given PS-c@M (3×10 6 The mice were treated with 100 μg / mL of Lipo-PS-c (100 μg / mL) or 200 μg / mL of Lipo-PS-c. Echocardiography was performed before surgery and on days 3, 7, 14, and 28 after surgery. Masson's and HE staining were performed on day 28 to evaluate the cardiac repair.

[0121] Figure 14 In vivo fluorescence imaging of the hearts of mice from different groups in this example shows that PS-c@M can be actively recruited to the damaged heart and persist for at least 96 hours. In addition, the fluorescence signal of FITC-Pt / Se decays earlier than that of DID-M2 macrophages, indicating that nano-Pt / Se is continuously released from the surface of PS-c@M residing in the damaged area.

[0122] Figure 15 The results show that PS-c@M can significantly inhibit STING and its downstream related signaling pathways, play a role in resisting cell death and regulating immune inflammatory response, and its overall effect is better than Lipo-PS-c.

[0123] Figure 16 The 28-day ultrasound data follow-up of this example showed that Lipo-PS-c and PS-c@M significantly improved the cardiac function of mice, with a significant increase in left ventricular ejection fraction (EF) and left ventricular fractional shortening (FS). Furthermore, the recovery effect of PS-c@M became increasingly better over time, and was overall superior to Lipo-PS-c.

[0124] Figure 17 Masson and HE staining were performed after 28 days of follow-up in this example. The results showed that PS-c@M significantly alleviated the adverse ventricular remodeling caused by MIRI and reduced the area of ​​myocardial infarction, and the effect was better than that of Lipo-PS-c.

[0125] In summary, the engineered macrophage system of surface-modified selenium-based nanomedicines provided in the embodiment of the present invention connects the drug-loaded liposomes loaded with small molecule drugs c176 and nanomedicine Pt / Se to the surface of homologous M2 macrophages through bioorthogonal coupling (such as TZ-TCO click reaction). Through experiments, it was found that the PS-c@M provided in the embodiment of the present invention retains the characteristics of M2 macrophages, has high biosafety, can actively target myocardial ischemic areas and responsively release surface drugs. PS-c@M transplantation successfully inhibited STING and its downstream related signaling pathways, reshaped the complex immune inflammatory microenvironment, and reduced myocardial cell apoptosis. It can be seen that PS-c@M therapy can effectively promote the recovery of cardiac function and alleviate the adverse consequences of ventricular remodeling.

[0126] The innovative features of the engineered macrophage system of surface-modified selenium-based nanomedicine provided in the embodiments of the present invention are as follows:

[0127] 1. Functionalized M2 macrophage carrier: Utilizes the natural targeting and immune escape capabilities of M2 macrophages to achieve precise targeting of damaged areas;

[0128] 2. Click chemistry-mediated nanodrug conjugation: Through the Tetrazine (TZ)-Trans-Cyclooctene (TCO) bioorthogonal reaction, liposomes loaded with Pt / Se nanodrugs and the STING inhibitor c176 are efficiently anchored to the cell surface, ensuring controlled drug release at the lesion site.

[0129] 3. Multi-mechanism synergistic treatment: Pt / Se nanomedicines scavenge ROS and reduce oxidative damage; and cooperate with the STING inhibitor c176 to block excessive activation of the cGAS-STING pathway and inhibit related adverse immunopathological reactions; M2 macrophages themselves secrete anti-inflammatory factors to synergistically reshape the immune microenvironment.

[0130] This invention overcomes the shortcomings of existing therapies such as insufficient targeting, high immunotoxicity and uncontrollable pharmacokinetic properties, and provides a new strategy for precise intervention of MIRI.

[0131] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A macrophage modified with selenium-based nanoparticles, characterized in that: The invention comprises macrophages, the surface of the macrophages is combined with liposome particles, and the liposome particles contain selenium-based nanoparticles.

2. The macrophage according to claim 1, wherein The liposome particles also contain a STING inhibitor.

3. The macrophage according to claim 2, characterized in that The STING inhibitor is selected from c176, C-178, H-151, SN-011, Astin C, GSK-626, or a combination thereof.

4. The macrophage according to claim 1, wherein The selenium-based nanoparticles include selenium nanospheres and platinum nanoparticles combined on the surfaces of the selenium nanospheres.

5. The macrophage according to any one of claims 1 to 4, characterized in that The liposome particles include phospholipids, cholesterol or its derivatives, and PEG-modified lipids.

6. The macrophage according to claim 5, characterized in that The phospholipid is selected from DPPC; Optionally, the PEG-modified lipid is selected from DSPE-PEG; Optionally, the phospholipid is DPPC and the PEG-modified lipid is DSPE-PEG5k.

7. The macrophage according to claim 6, characterized in that The macrophages are covalently bound to the liposome particles; Optionally, the covalent binding is achieved by a bioorthogonal coupling reaction; Optionally, the bioorthogonal coupling reaction is a click chemistry reaction; Optionally, the click chemistry reaction is selected from any one of a cycloaddition reaction, a nucleophilic ring-opening reaction, a non-aldol carbonyl chemistry, and a carbon-carbon multiple bond addition reaction; Optionally, the click chemistry reaction is achieved based on a Tetrazine (TZ)-Trans-Cyclooctene (TCO) specific cycloaddition reaction; Optionally, the Tetrazine group is modified on DSPE-PEG5k, the Cyclooctene gene is modified on the macrophage, and the Cyclooctene gene is modified on the DSPE-PEG5k inserted on the macrophage.

8. The macrophage according to any one of claims 1 to 4, characterized in that The macrophages are M1 or M2 polarized macrophages.

9. Use of the macrophage according to any one of claims 1 to 8 in the preparation of a medicament for treating cardiac ischemia-reperfusion injury.

10. A drug for treating cardiac ischemia-reperfusion injury, characterized in that: The invention comprises the macrophage according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

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