Selenium-based nanoparticle-modified macrophage, drug and application thereof

Abstract

The application discloses a selenium-based nanoparticle modified macrophage, a drug and application thereof, and relates to the technical field of biological medicines.The selenium-based nanoparticle modified macrophage provided by the application has good heart targeting and the effect of promoting the repair of myocardial ischemia-reperfusion injury, and provides a novel strategy for the precise intervention of MIRI.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a selenium-based nanoparticle-modified macrophage, a drug, and its application. Background Technology

[0002] Myocardial ischemia-reperfusion injury (MIRI) is a serious complication following myocardial infarction revascularization, characterized by refractory immune activation, oxidative damage, and abnormal energy metabolism, which can lead to infarct enlargement, malignant arrhythmias, and even sudden cardiac death. The immune response plays a crucial regulatory role in MIRI and mediates the exacerbation of other pathological processes. Although anti-inflammatory drugs, antioxidants, and antithrombotic therapies are used clinically, they suffer from poor targeting, narrow therapeutic windows, and systemic toxicity, and lack effective means for precise regulation of immune-inflammatory homeostasis. Therefore, there is an urgent need to develop novel targeted therapeutic strategies.

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

[0004] The present invention aims to provide selenium-based nanoparticle-modified macrophages, drugs, and their applications to address at least one of the following problems in the existing treatment of myocardial ischemia-reperfusion injury:

[0005] 1) Insufficient targeting: Systemic administration of traditional drugs (such as anti-inflammatory agents and antioxidants) results in low drug concentrations at the lesion site and is prone to systemic toxic side effects; 2) Imbalanced immune regulation: CAR cell therapy carries the risk of cytokine storms and graft-versus-host disease, and is difficult to precisely 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 therapy: Single mechanisms of action (such as only inhibiting inflammation or clearing ROS) cannot fully reverse the multi-pathway pathological damage of MIRI.

[0006] This invention is implemented as follows:

[0007] On one hand, the present invention provides a selenium-based nanoparticle-modified macrophage, comprising macrophages, wherein liposome particles are bound to the surface of the macrophages, and the liposome particles contain selenium-based nanoparticles.

[0008] Cell-based delivery platforms have become an important research direction in drug delivery due to their advantages such as low immunogenicity, long circulation period, natural homing ability, and receptor integration capability. T-cell and macrophage therapies based on chimeric antigen receptor (CAR) technology can achieve local immune regulation by specifically recognizing myocardial injury-related antigens. However, CAR cell therapy suffers from drawbacks such as complex manufacturing processes, cytokine release syndrome, and graft-versus-host disease, limiting its clinical translational potential. M2 macrophages, as key effector cells in the repair phase of MIRI, can be actively recruited to the injury site and improve prognosis through anti-inflammatory-repair mechanisms. Constructing a drug delivery platform using M2 macrophages can retain their natural targeting and anti-inflammatory capabilities while enhancing efficacy by loading exogenous drugs, providing a new approach for MIRI treatment.

[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, selenium has a narrow therapeutic window, and excessive absorption of selenium compounds in organic and inorganic forms can easily lead to 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 this invention have good cardiac targeting and can promote the repair of myocardial ischemia-reperfusion injury, providing a new strategy for 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, such as C-178, H-151, SN-011, Astin C, or GSK-626, etc., 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, which may be selected by those skilled in the art according to their therapeutic purpose.

[0015] Optionally, in some embodiments of the present invention, the selenium-based nanoparticles include selenium nanospheres and platinum nanoparticles bonded to the surface of the selenium nanospheres.

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

[0017] Optionally, 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. They can be phospholipids of natural origin, such as egg yolk lecithin (PC) and soybean lecithin (Soy PC), or synthetic and semi-synthetic lecithin, such as DSPE, DOPE, and hydrogenated lecithin.

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

[0020] It should be noted that PEG-modified lipids can be modified with various components as needed. These modifications can include polymer modifications such as polysorbate, polyethylene glycol-polylactic acid (PEG-PLA), polyvinylpyrrolidone (PVP), and polyhydroxyethyl acrylate (PHEA); polysaccharide modifications such as hyaluronic acid (HA), chitosan, and heparin; protein or peptide modifications such as transferrin, RGD peptides, and antibody fragments (e.g., Fab); lipid derivative modifications such as cholesterol-PEG, stearylamine, and DSPE (distearate phosphatidylethanolamine); small molecule ligand modifications such as folic acid, galactose, and mannose; and environmentally responsive modifications such as pH-sensitive lipids (e.g., DOPE) and temperature-sensitive polymers (e.g., poly-N-isopropylacrylamide).

[0021] Optionally, 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] Optionally, in some embodiments of the present invention, the macrophages are covalently bound to the liposome particles;

[0024] Optionally, in some embodiments of the present invention, the covalent bonding is achieved through 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 bonding can also be achieved through one or more of the following reactions: azido-yne cycloaddition (SPAAC), reverse electron demand Diels-Alder reaction (iEDDA), oxime / Hydrazone ligation, Staudinger ligation, thiol-ene / yne click, hexavalent sulfonium-fluorine exchange (SuFEx), cyanobenzothiazole coupling (CBT-Cys Reaction), photoclick reactions, bioorthogonal uncaging, and enzyme-mediated bioorthogonal reactions.

[0027] Optionally, in some embodiments of the present invention, the click chemistry reaction is selected from any one of cycloaddition reactions, nucleophilic ring-opening reactions, non-alcoholic carbonyl chemistry, and carbon-carbon multi-bond addition reactions.

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

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

[0030] It should be noted that, in other embodiments, the click chemical reaction can also be based on the following reactions: azide and alkyne, azide and dibenzocyclooctylene (DBCO), tetrazine and transcyclooctene (TCO), tetrazine and cyclopropene, thiol and alkene, thiol and alkyne, tetrazolium and alkene, hydroxylamine and ketone / aldehyde (Ketone / Aldehyde), phosphine and azide, hydrazine and aldehyde / ketone (Aldehyde / Ketone), cyanobenzothiazole (CBT) and cysteine, sulfonyl fluoride... Fluoride with amines / hydroxyl groups (Amine / Hydroxyl), or norbornene with tetrazine.

[0031] Optionally, in some embodiments of the present invention, a Tetrazine group is modified onto DSPE-PEG5k, and a Cyclooctene group is modified onto macrophages, wherein the Cyclooctene group is modified onto DSPE-PEG5k inserted onto the macrophages.

[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 macrophage is an M2 type polarized macrophage.

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

[0035] On the other hand, the present invention provides a method for preparing the above-mentioned macrophages, which includes one of the following steps:

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

[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 to form 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 through a TZ-TCO click reaction (25℃, 30 min) to form an engineered macrophage system with surface-modified selenium-based nanomedicine (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 injury site contains a large amount of ROS, which triggers the rupture of liposome membranes and releases nanomedicines Pt / Se and c176, which alleviate 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 part can be prepared, loaded, modified and coupled using conventional techniques in the art; this is easily achieved by those skilled in the art.

[0041] On the other hand, the present invention provides the use of the macrophages described in any of the above claims in the preparation of drugs for treating myocardial ischemia-reperfusion injury.

[0042] On the other hand, the present invention provides a medicament for treating myocardial ischemia-reperfusion injury, comprising macrophages as described in any of the preceding claims and a pharmaceutically acceptable carrier. Attached Figure Description

[0043] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 This is a TEM image of the α-Se nanospheres in Example 1 of the present invention. As can be seen from the image, the α-Se nanospheres exhibit a uniform spherical structure with a uniform size of approximately 80-100 nm.

[0045] Figure 2The image shows a TEM image of the Pt / Se nanoheterosome in Example 1 of this invention. As can be seen from the image, the Pt / Se is a uniformly distributed core-shell sphere, consisting of a central spherical Se nanosphere and dense, tiny Pt nanostructures dispersed on the Se surface, with a diameter of approximately 100 nm.

[0046] Figure 3 The X-ray diffraction (XRD) pattern of the Pt / Se nanoheterogeneous structure in Example 1 of this invention shows that the spectral lines of Se exhibit 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 The image shows the X-ray photoelectron spectroscopy (XPS) spectrum of Pt / Se in Example 1 of this invention. The peak positions are shifted to higher energies, approximately 70.6 eV and 74.6 eV, confirming that Pt achieves efficient chemical assembly on the surface of Se nanospheres.

[0048] Figure 5 The image shows a TEM image of Lipo-PS-c in Example 2 of this invention. As can be seen from the image, there is a membrane-like structure around Pt / Se with a thickness of about 5.5 nm, indicating that we have successfully constructed drug-loaded liposomes.

[0049] Figure 6 The images show the dynamic light scattering (DLS) patterns of Se, Pt / Se, and Lipo-PS-c in Examples 1 and 2 of this invention. The results 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 for Se, Pt / Se, and Lipo-PS-c in Examples 1 and 2 of this invention are shown. The results 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 •O was removed from Se, Pt / Se and Lipo-PS-c obtained in Examples 1 and 2. 2− The ESR plots of Pt / Se and Lipo-PS-c significantly reduced the effect of •OH. 2− The characteristic peak intensity of •OH is higher than that of Se nanospheres.

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

[0053] Figure 10 The image shown is a TEM image of PS-c@M in Example 3 of this invention, which confirms that Lipo-PS-c was successfully attached to the surface of M2 macrophages.

[0054] Figure 11 The image shown is a scanning electron microscope (SEM) image of PS-c@M in Example 3 of this invention, which proves that M2 macrophages were successfully induced, exhibiting an elongated spindle shape and being loaded with Lipo-PS-c on their surface.

[0055] Figure 12 To illustrate the drug release of PS-c@M coupled to Lipo-PS-c on the surface of M2 macrophages in a simulated infarcted myocardial microenvironment (acidic environment rich in ROS, pH=6.8) in Example 3 of this invention, flow cytometry results showed that drug release gradually increased over time, and was completely released at approximately 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-apoptotic ability of cardiomyocytes, which is significantly better than Pt / Se and Se.

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

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

[0059] Figure 15 The study investigated changes in protein levels of STING and downstream inflammatory signaling pathways in the hearts of mice treated for 3 days. The results showed that PS-c@M significantly inhibited STING and its downstream signaling pathways, exerting a role in resisting cell death and regulating immune inflammatory responses, with an overall effect superior to Lipo-PS-c.

[0060] Figure 16 The ultrasound data follow-up lasted for 28 days. The data showed that Lipo-PS-c and PS-c@M significantly improved cardiac function in 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 was better over time and was superior to that of Lipo-PS-c overall.

[0061] Figure 17 The results of 28-day follow-up with masson and HE staining showed that PS-c@M significantly reduced adverse ventricular remodeling caused by MIRI, reduced the area of ​​myocardial infarction, and was superior to Lipo-PS-c. Detailed Implementation

[0062] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

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

[0064] Example 1

[0065] This embodiment synthesizes Pt / Se nanoheterogeneous structures, and the specific synthesis method is as follows:

[0066] 1) Weigh 100 mg of polyvinylpyrrolidone (PVP) and 387 mg of selenite (H2SeO3) and dissolve them in 100 mL of deionized water. Stir magnetically for 10 min at 25 °C 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, and continue the reaction at 25°C for 3 h;

[0068] 3) After the reaction is complete, place the mixture in a centrifuge and centrifuge at 12,000 rpm for 20 minutes to separate the solid product;

[0069] 4) The solid product was washed three times with deionized water to remove unreacted impurities;

[0070] 5) Disperse the purified product in 50 mL of deionized water to prepare an α-Se nanosphere suspension, and store it at 4 °C for later use.

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

[0072] 7) Add 0.1 mL of potassium hexachloroplatinate (K2PtCl6) aqueous solution (100 mM) to the mixed solution in step (6) at a rate of 0.02 mL / min, and stir magnetically for 12 h at 25 °C to obtain Pt / Se nanoheterogeneous material.

[0073] The Pt / Se nanoheteromorphs (selenium-based nanoparticles) obtained in this embodiment were tested by inductively coupled plasma optical emission spectrometry (ICP-OES), which showed that the mass ratio of Pt to Se 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 The image shows a TEM image of α-Se nanospheres. As can be seen from the image, the α-Se nanospheres exhibit a uniform spherical structure with a size of approximately 80-100 nm.

[0076] Figure 2 The image shows a TEM image of a Pt / Se nanoheterosome. The Pt / Se nanoheterosome is a uniformly distributed core-shell sphere, consisting of a central spherical Se nanosphere and dense, tiny Pt nanostructures dispersed on the Se surface, with a diameter of approximately 100 nm.

[0077] Figure 3 The X-ray diffraction (XRD) pattern of the Pt / Se nanoheterogeneous structure is shown in the figure. The results show that the spectral lines of Se have an amorphous structure. The characteristic XRD 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).

[0078] Figure 4 The X-ray photoelectron spectroscopy (XPS) spectra of Pt / Se show that the energy peaks are shifted to higher energies, approximately 70.6 eV and 74.6 eV, confirming that Pt achieves efficient chemical assembly on the surface of Se nanospheres.

[0079] Example 2

[0080] Based on the Pt / Se nanoheterosome, this embodiment further synthesizes drug-loaded liposomes, namely, liposomes loaded with the small molecule drug c176 and the nanomedicine 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), place them in a round-bottom flask, add 5 mL of chloroform, and stir magnetically for 10 min at 25 °C to obtain a clear and homogeneous mixed solution. Connect the flask to a rotary evaporator, set the water bath temperature to 40 °C, the vacuum degree to 200 mbar, and the rotation speed to 100 rpm for 30 min, ensuring that the solvent is evaporated to dryness, there is no residual chloroform odor, 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 seconds, 40 kHz) to eliminate aggregation. Then, 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 placed in a thermostatic magnetic stirrer (60 °C, 500 rpm) for hydration for 1 h until the film was completely detached and a mixed suspension was formed. Then, the mixture was intermittently shaken with a vortex mixer (10 s once, 30 s interval, repeated 5 times) to promote liposome homogenization.

[0083] 3) Particle size homogenization: The suspension was transferred to an ice bath, and the ultrasonic probe was set to 50W power, pulse mode, sonicated for 2 seconds, paused for 3 seconds, and processed for 5 minutes until the suspension became translucent. Then, using a liposome extruder, the suspension was passed through 0.45μm and 0.22μm polycarbonate membranes 10 times each to obtain a monolayer of uniformly drug-loaded liposomes (particle size approximately 100-200nm).

[0084] 4) Purification and preservation: remove unencapsulated material by ultracentrifugation (100,000g, 4℃, 1h), filter through a 0.22μm sterile filter membrane, store aseptically protected from light, store at 4℃ for short-term storage (<7d) or freeze-dry (pre-freeze at -80℃ for 24h and then freeze-dry) for long-term storage.

[0085] The drug-loaded liposomes obtained in this embodiment were analyzed by high performance liquid chromatography (HPLC), which showed that the mass ratio of c176 in Lipo-PS-c was 5.962%.

[0086] Figure 5 The image shows a TEM image of Lipo-PS-c in this embodiment. As can be seen from the image, there is a membrane-like structure around Pt / Se with a thickness of about 5.5 nm, indicating that we have successfully constructed drug-loaded liposomes.

[0087] Figure 6 The images show the dynamic light scattering (DLS) patterns of Se, Pt / Se, and Lipo-PS-c in Examples 1 and 2 of this invention. The results 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 for Se, Pt / Se, and Lipo-PS-c in Examples 1 and 2 of this invention are shown. The results 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 •O was removed from Se, Pt / Se and Lipo-PS-c obtained in Examples 1 and 2. 2− The ESR plots of Pt / Se and Lipo-PS-c significantly reduced the effect of •O. 2− The characteristic peak intensity of •OH is higher than that of Se nanospheres;

[0090] Example 3

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

[0092] First, mouse bone marrow macrophages (BMDM) were extracted and M2 macrophages were induced:

[0093] 1) Euthanasia and bone separation of mice: C57BL / 6 male mice were euthanized, the mice were disinfected by soaking in alcohol, the skin of the hind limbs was cut open, the femur and tibia were separated, the muscles and connective tissues were removed, the intact bones were taken out and soaked in pre-cooled 75% alcohol for 2 minutes, and rinsed 3 times with PBS.

[0094] 2) Bone marrow cell collection: Cut off both ends of the bone marrow, draw pre-cooled PBS with a syringe (25G needle), rinse the bone marrow cavity repeatedly until the liquid becomes turbid, pass the bone marrow suspension through a 70μm cell sieve, and collect it into a 50mL centrifuge tube.

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

[0096] 4) BMDM differentiation culture: The cell suspension was seeded into a 10 cm culture dish and cultured in a 37℃, 5% CO2 cell culture incubator. Half of the medium was changed on the third day and the whole medium was changed on the sixth day. Cell morphology and whether the cells adhered to the wall were observed. On the seventh day, the cells were collected by cell scraper, centrifuged (300g, 5min), and resuspended for later use.

[0097] 5) M2 macrophage induction: The cell suspension from (4) above was seeded into 6-well plates, and 20 ng / mL IL-4 and 20 ng / mL IL-13 (cell density 1×10⁻⁶) were added to BMDM medium. 6 (cells / mL, 2mL / well), cultured for 48h to induce BMDM to polarize into M2 macrophages.

[0098] The second step involves click chemical coupling of DSPE-PEG5k-TZ modified M2 macrophages with 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 until 80% confluence.

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

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

[0102] 4) Fluorescent labeling: FITC (10 μM) was incubated with drug-loaded liposomes at 25°C in the dark for 15 min, and DID (5 µM, 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 and TCO-liposomes were mixed in PBS at a cell:liposome ratio of 1:50 (particle ratio), and incubated at room temperature in the dark with shaking for 30 min to complete TZ-TCO covalent coupling.

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

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

[0106] Figure 10 The TEM image of PS-c@M in this embodiment confirms that Lipo-PS-c was successfully attached to the surface of M2 macrophages.

[0107] Figure 11 The image shown is a scanning electron microscope (SEM) image of PS-c@M in this embodiment, which proves that M2 macrophages were successfully induced, exhibiting an elongated spindle shape and being loaded with Lipo-PS-c on their surface.

[0108] Figure 12 To illustrate the drug release of PS-c@M coupled to Lipo-PS-c on the surface of M2 macrophages in a simulated infarcted myocardial microenvironment (acidic environment rich in ROS, pH=6.8) in this embodiment, flow cytometry results showed that drug release gradually increased over time, and was completely released at approximately 8 hours.

[0109] Example 4

[0110] To verify the anti-apoptotic ability of Se, Pt / Se, and Lipo-PS-c in Examples 2 and 3 of this invention, this example uses 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) Cardiac cells were seeded in six-well plates, 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 cardiac cells for 8 hours.

[0112] 2) 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. Then, the cells were reoxygenated for 3 h with culture medium instead of ischemia buffer to induce an OGD / R model to simulate in vitro ischemia-reperfusion injury. The control group was not treated.

[0113] 3) Following the reagent instructions, cells were stained with Calcein-AM / PI for fluorescence staining, and Annexin V-FITC / PI was used for flow cytometry staining to analyze the proportion of apoptotic cells.

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

[0115] Example 5

[0116] This embodiment demonstrates that the PS-c@M obtained in Example 3 of the present invention has good cardiac targeting properties and can promote the repair of myocardial ischemia-reperfusion injury. The specific test method is as follows:

[0117] 1) Eight-week-old male C57 / BL mice were selected for the experiment to construct the MIRI mouse model. The specific method is as follows: Before the operation, the mice were shaved on the chest, anesthetized with isoflurane, and then the left side of the chest was opened to expose the heart. The left anterior descending coronary artery was ligated from the left atrium with 6-0 polypropylene sutures to form a slip knot. The heart was returned to the thoracic cavity using a suture clamp. After 45 minutes, the mice were reperfused to loosen the slip knot and waited for the mice to recover. The mouse model of myocardial ischemia-reperfusion injury was completed.

[0118] 2) To investigate the targeted distribution of PS-c@M in mice, mice were injected via tail vein on the second day after surgery with DID-labeled M2 macrophages and FITC / DID-labeled PS-c@M at a dose of 3 × 10⁻⁶. 6 Cells / 200μL, 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 collected. The targeting ability of PS-c@M was assessed using in vivo fluorescence.

[0119] 3) Western blot grouping of STING and downstream related inflammatory signaling pathways was as follows: control group, MIRI group, MIRI+Lipo-PS-c group and MIRI+PS-c@M group. Two hours after reperfusion, mice were given PS-c@M (3×10⁻⁶). 6 After treatment with cells / 200μL) or Lipo-PS-c (100ug / mL), heart tissue was taken 3 days later for wb assay to detect changes in related signaling pathway proteins;

[0120] 4) The animal experimental groups for evaluating the therapeutic effect were as follows: control group, MIRI group, MIRI+Lipo-PS-c group, and MIRI+PS-c@M group. Two hours after reperfusion, mice were administered PS-c@M (3×10⁻⁶). 6 Mice were treated with either Lipo-PS-c (cells / 200μL) or Lipo-PS-c (100ug / mL) before surgery, and echocardiographic follow-up was performed at 3, 7, 14 and 28 days after surgery. Masson and HE staining were performed at 28 days to assess the repair of cardiac damage in mice.

[0121] Figure 14 This is an in vivo fluorescence imaging of the hearts of mice in different groups in this embodiment. The results show that PS-c@M can actively recruit to the damaged heart and remain there for at least 96 hours. In addition, the fluorescence signal of FITC-Pt / Se decays before that of DID-M2 macrophages, which indicates that the surface of PS-c@M residing in the damaged area continuously releases nano-Pt / Se.

[0122] Figure 15 This embodiment shows the changes in protein levels of STING and downstream related inflammatory signaling pathways in the hearts of mice treated for 3 days. 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 the overall effect is better than Lipo-PS-c.

[0123] Figure 16 The ultrasound data follow-up for 28 days in this embodiment showed that Lipo-PS-c and PS-c@M significantly improved cardiac function in 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 was better over time and was superior to that of Lipo-PS-c overall.

[0124] Figure 17 The results of the 28-day follow-up of this embodiment, including masson staining and HE staining, showed that PS-c@M significantly reduced adverse ventricular remodeling caused by MIRI, reduced the area of ​​myocardial infarction, and had a better effect than Lipo-PS-c.

[0125] In summary, the engineered macrophage system with surface-modified selenium-based nanomedicines provided in this invention connects drug-loaded liposomes containing the small molecule drug c176 and the nanomedicine Pt / Se to the surface of homologous M2 macrophages via bioorthogonal coupling (such as TZ-TCO click reaction). Experiments showed that the PS-c@M provided in this invention retains the characteristics of M2 macrophages, exhibits high biocompatibility, and can actively target ischemic areas of the myocardium and responsively release the surface drug. Successful PS-c@M transplantation inhibited STING and its downstream signaling pathways, remodeled the complex immune inflammatory microenvironment, and reduced cardiomyocyte apoptosis. It can be seen that PS-c@M therapy can effectively promote cardiac function recovery and mitigate the adverse consequences of ventricular remodeling.

[0126] The engineered macrophage system of surface-modified selenium-based nanomedicines provided in this invention is innovative in the following ways:

[0127] 1. Functionalized M2 macrophage vector: Utilizing the natural targeting and immune escape capabilities of M2 macrophages to achieve precise targeting of the damaged area;

[0128] 2. Click-chemical-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 the controlled release of drugs at the lesion site.

[0129] 3. Multi-mechanism synergistic therapy: Pt / Se nanomedicines remove ROS and reduce oxidative damage; and in conjunction with the STING inhibitor c176, they block the overactivation of the cGAS-STING pathway and inhibit related adverse immunopathological reactions; M2 macrophages secrete anti-inflammatory factors themselves, synergistically reshaping the immune microenvironment.

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

[0131] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A selenium-based nanoparticle-modified macrophage, characterized in that, It comprises macrophages, the surface of which is bound with liposome particles, the liposome particles contain selenium-based nanoparticles and STING inhibitors inside; the macrophages are M2 type polarized macrophages; the selenium-based nanoparticles are composed of selenium nanospheres and platinum nanoparticles bound on the surface of the selenium nanospheres, and the STING inhibitors are c176.

2. The selenium nanoparticle-modified macrophage of claim 1, wherein, The liposome particles comprise phospholipids, cholesterol and PEG-modified lipids.

3. The selenium nanoparticle-modified macrophage of claim 2, wherein, The phospholipids are DPPC, and the PEG-modified lipids are DSPE-PEG5k.

4. The selenium nanoparticle-modified macrophage of claim 3, wherein, The macrophages are covalently bound with the liposome particles.

5. The selenium nanoparticle-modified macrophage of claim 4, wherein, The covalent binding is achieved by a bio-orthogonal coupling reaction.

6. The selenium nanoparticle-modified macrophage of claim 5, wherein, The bio-orthogonal coupling reaction is a click chemistry reaction.

7. The selenium nanoparticle-modified macrophage of claim 6, wherein, The click chemistry reaction is selected from any one of a cycloaddition reaction, a nucleophilic ring-opening reaction, a non-alcohol aldehyde carbonyl chemistry and a carbon-carbon multiple bond addition reaction.

8. The selenium nanoparticle-modified macrophage of claim 7, wherein, The click chemistry reaction is achieved based on a Tetrazine (TZ)-Trans-Cyclooctene (TCO) specific cycloaddition reaction.

9. The selenium nanoparticle-modified macrophage of claim 8, wherein, A Tetrazine group is modified on DSPE-PEG5k, and a Cyclooctene group is modified on the macrophages, and the Cyclooctene group is modified on DSPE-PEG5k inserted on the macrophages.

10. Use of the selenium-based nanoparticle-modified macrophages of any one of claims 1-9 in the preparation of a drug for treating cardiac ischemia-reperfusion injury.

11. A medicament for treating cardiac ischemia-reperfusion injury, characterized by, It contains the selenium-based nanoparticle-modified macrophages of any one of claims 1-9 and a pharmaceutically acceptable carrier.

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