A controllable catalytic oncolytic complex that can activate tumor immunity

Abstract

This application relates to a controllable catalytic oncolytic complex that can activate tumor immunity, comprising a carrier and an exogenous activating small molecule linked to the carrier. The controllable catalytic oncolytic complex of this application not only significantly reduces the release of inflammatory factors from cells but also possesses natural targeting specificity to tumor cells, enabling specific recognition of the tumor cell membrane. Under exogenous stimulation, it can controllably and precisely perforate the tumor cell membrane, resulting in the massive release of intracellular inflammatory factors, thereby altering the tumor microenvironment and ultimately achieving highly effective immunotherapy.

Description

Technical Field

[0001] This application relates to a controllable catalytic oncolytic complex that can activate tumor immunity, belonging to the field of biotechnology. Background Technology

[0002] Cancer is a leading cause of death worldwide, and the vast majority of cancer patients undergo chemotherapy and radiotherapy. These treatments typically offer only partial efficacy and cause various serious side effects. In recent years, cancer immunotherapy has brought great hope to the fight against cancer. In clinical treatment strategies for tumors, immunotherapy has been widely used, either alone or in combination with other conventional treatments. Numerous clinical data show that the use of immunotherapies such as immune checkpoint inhibitors (ICBs) and CAR-T therapy can significantly reduce the risk of cancer recurrence and metastasis.

[0003] Exogenous activation of small molecules can also help people controllably conduct anti-tumor immune activities. PDT (Photodynamic Therapy) is a method that uses light energy to destroy abnormal and malignant lesions. It includes three main elements: photosensitizers, light of specific wavelengths, and oxygen molecules, with the photosensitizer being the most crucial. Photosensitizers absorb energy from specific wavelengths of light and transfer it to surrounding molecules, exerting cytotoxic effects by producing reactive oxygen species (ROS) and other toxic substances to induce tumor cell death. They can even expose or release danger-related molecular patterns through the PDT effect, thereby activating the body's anti-tumor immunity. SDT (Sonic Dynamic Therapy) uses ultrasound to activate acoustic sensitizers accumulated in tumors and convert them into an activated state. The activated sensitizer converts surrounding molecular oxygen into reactive oxygen species, primarily singlet oxygen (O2), through energy transfer. These reactive oxygen species, under the physical and mechanical action of ultrasound, synergistically kill tumor cells. After tumor cell death, the released tumor-associated antigens activate the immune system, enabling SDT to exert subsequent anti-tumor immune responses.

[0004] However, immunotherapy also presents many challenges. First, immunotherapy eliminates cancer cells by reactivating the body's own anti-tumor immune system, making effective activation of this immune system a significant challenge. One key technical issue is the rapid clearance of drugs by the body's immune response to externally delivered complexes, preventing the drugs from effectively activating tumor cells. Second, excessive immunization can lead to severe "immune storms," ​​causing serious damage and even death. Therefore, controlling the body's own anti-tumor immune system is another critical challenge. Thus, reducing the release of inflammatory factors from the carrier or complex itself and developing precise and controllable anti-tumor immune induction strategies are essential. Summary of the Invention

[0005] To address the aforementioned technical problems, this application provides a recombinant compound characterized by comprising a carrier and an exogenous activating small molecule linked to the carrier. In one embodiment, the carrier can target tumor cells or tissues; preferably, the carrier is an immune cell or a carrier with a core-shell structure. In a preferred embodiment, the immune cell is a phagocytic autologous or allogeneic natural cell, such as a monocyte, macrophage, or neutrophil, or the immune cell contains an engineered membrane, such as the cell membrane of an immune cell stimulated by a gene or drug, designed to contain relevant recognition proteins capable of interacting with cancer cells. In a particularly preferred embodiment, the immune cell is a RAW 264.7 macrophage. In one embodiment, the core-shell structured carrier comprises a core and a shell, the core being a drug-loaded carrier, the shell being a cell membrane covering the outer surface of the core, the cell membrane being a phagocytic autologous or allogeneic natural cell membrane, such as the cell membrane of a monocyte, macrophage, or neutrophil, or an engineered phagocytic cell membrane, the carrier being a nanocarrier, and / or the carrier being a carrier for the controlled release of the loaded drug. In one embodiment, the carrier is cryogenically frozen after being linked to the exogenous activating small molecule; optionally, it is then washed after cryogenic freezing. In one embodiment, the exogenous activating small molecule is a photosensitizer, a sonosensitizer, or a photothermal agent, etc. In one embodiment, the photosensitizer may be hematoporphyrin; hematoporphyrin derivatives such as hematoporphyrin monomethyl ether, 5-aminolevulinic acid; phthalocyanine photosensitizers such as glucose conjugated phthalocyanine, aluminum phthalocyanine; dihydroporphyrin derivatives such as pyrophyllite-a, erythropoietin-18 methyl ester, 2-(1-hexyloxyethyl)-2-devinylpyrophyllite, dihydroporphyrin e6 (Ce6), bacterial dihydroporphyrin; polycyclic quinone photosensitizers such as baicalin, hypericin; phenothiazine photosensitizers such as 3,7-bis(dimethylamino)phenothiazine-5-onium chloride; the sonosensitive agent may be hematoporphyrin, photoporphyrin II, hematoporphyrin monomethyl ether, 5-aminolevulinic acid, porphyrin derivatives, xanthracene compounds such as fluorescein rose red, phthalocyanine dye phthalimide zinc phthalocyanine, or [Ru(bpy)3] 2+ The photothermal agent can be a noble metal material such as gold, platinum, and palladium; or a transition metal material such as transition metal oxides, sulfides, carbides, nitrides, and dichlorides, including TiS2, FeS, and Cu. x S y Cu 2-x S, MoS2, Ag2S, WS2, FeSe2, Cu 2-x Se, TiO2, Ti8O 15 MoO x W x O yInorganic non-metallic photothermal agents include carbon-based materials, silicon-based materials, and black phosphorus, as well as some carbides, nitrides, and dichlorides. Organic small molecules include indocyanine green (ICG), indoles such as IR780, IR820, IR825, cypate, and porphyrins. Photothermal agents based on polymeric materials include artificially synthesized semiconductor polymer nanoparticles, such as polyaniline, polythiophene, polypyrrole, and polydopamine (PDA). In one embodiment, the carrier and the exogenous small molecule are linked via a click reaction. Preferably, the exogenous activated small molecule undergoes a click reaction with tetraacetyl-N-azidoacetylmannosamine (Ac4ManNAz) to connect to the carrier.

[0006] This application also provides a method for preparing the above-mentioned complex, the method comprising: (1) linking an exogenous activating small molecule to a carrier; (2) subjecting the prepared carrier-exogenous small molecule complex to cryogenic freezing; optionally, the complex is washed after cryogenic freezing. This application also provides the use of the drug in the preparation of a medicament for treating tumors. This application also provides the use of the drug alone or in combination with a PD1 antibody in the preparation of a medicament for treating tumors.

[0007] In one embodiment, washing is any method that elutes inflammatory factors from the cell membrane, such as centrifugation with a buffer solution (e.g., PBS). In another embodiment, the cryogenic freezing creates pores on the cell membrane surface, altering the cell membrane structure of the carrier (but not the basic or main structure of the cell membrane), allowing inflammatory factors to be removed from the cell while retaining a greater extent of surface antibodies to preserve their associated functions.

[0008] Advantages of this application: 1. Utilizing the natural targeting ability of autologous immune cells to tumor cells to achieve specific recognition of tumor cell membranes. 2. Using low-temperature rapid freezing technology to freeze-treat modified immune cells can maximize the preservation of membrane components and their functions, reduce immunogenicity, and enable rapid large-scale preparation. 3. Through the connection of exogenous activating small molecules to the immune cells, the tumor cell membrane can be precisely and controllably perforated under exogenous stimulation, resulting in the large-scale release of intracellular inflammatory factors, thereby altering the tumor microenvironment and achieving immunotherapy. 4. By connecting tetraacetyl-N-azidoacetylmannosamine (Ac4ManNAz), more precise targeting of tumor cells can be achieved. 5. When used in combination with immune checkpoint inhibition (ICB) therapy, it can effectively inhibit tumor recurrence and metastasis, expanding the applicability of immune checkpoint inhibition (ICB) therapy. Attached Figure Description

[0009] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application and to make other features, objectives and advantages of this application more apparent.

[0010] Figure 1 Confocal fluorescence images of Ac4ManNAz_Raw 264.7 and Ce6-Ac4ManNAz-LNT_Raw 264.7.

[0011] Figure 2 Flow cytometry assays of normal and liquid nitrogen-frozen modified RAW264.7 membrane surfaces for cell recognition and phagocytosis of antibodies by phagocytes.

[0012] Figure 3 Comparison of inflammatory factor release levels in normal and liquid nitrogen-frozen modified RAW264.7 cells.

[0013] Figure 4 Measurement of singlet oxygen production.

[0014] Figure 5 Ce6-Ac4ManNAz-LNT_Raw 264.7 In vitro confocal imaging of targeted cancer cells.

[0015] Figure 6 Detection of light-controlled cell death, cell viability (CCK-8), and cell membrane rupture death (LDH).

[0016] Figure 7 Confocal image of Ce6-Ac4ManNAz-LNT_Raw 264.7 causing cancer cell death.

[0017] Figure 8 Ce6-Ac4ManNAz-LNT_Raw 264.7 In vivo targeting capability assay.

[0018] Figure 9 In vivo antitumor potency assay of Ce6-Ac4ManNAz-LNT_Raw 264.7 alone and in combination with PD1 antibody.

[0019] Figure 10 Results of an immune memory relapse experiment using Ce6-Ac4ManNAz-LNT_Raw 264.7.

[0020] Figure 11 Results of in situ and tail vein immunophotoinfiltration experiments on Ce6-Ac4ManNAz-LNT_Raw 264.7.

[0021] Figure 12 The reaction process for preparing the acoustic sensitizer Ce6-Ac4ManNAz-LNT_Raw 264.7.

[0022] Figure 13 UV-Vis characterization of the acoustic sensor Ru(bpy)32+ -NHS.

[0023] Figure 14 Characterization of singlet oxygen generation in response to ultrasound in Cryo-MC@Ru(bpy)3.

[0024] Figure 15 Flow cytometry assay of antibodies on the membrane surfaces of MC@Ru(bpy)3 and Cryo-MC@Ru(bpy)3 for cell recognition and avoidance of phagocytosis by phagocytes.

[0025] Figure 16 Results of in vitro tumor cell-targeting confocal microscopy.

[0026] Figure 17 Comparison of the release levels of cytoinflammatory factors from MC@Ru(bpy)3 and Cryo-MC@Ru(bpy)3.

[0027] Figure 18 Cell viability (CCK-8), cell membrane rupture and death (LDH) assay and MDA assay.

[0028] Figure 19 Fluorescence microscopy was used to detect cell apoptosis.

[0029] Figure 20 In vivo targeting ability assay of Cryo-MC@Ru(bpy)3.

[0030] Figure 21 In vivo antitumor efficacy assay of Cryo-MC@Ru(bpy)3.

[0031] Figure 22 Immune cell infiltration in tumors after Cryo-MC@Ru(bpy)3+ ultrasound treatment. Detailed Implementation

[0032] Embodiments of this application will now be described with reference to the accompanying drawings. Elements and features described in one drawing or embodiment of this application may be combined with elements and features shown in one or more other drawings or embodiments. It should be noted that, for clarity, representations and descriptions unrelated to this application and known to those skilled in the art have been omitted from the drawings and description. The application will now be further described with reference to the accompanying drawings.

[0033] This application provides a complex comprising a carrier and an exogenous activating small molecule on the carrier, characterized in that the carrier has cell or tissue targeting properties. In one embodiment, the carrier is an immune cell or a carrier with a core-shell structure. In one embodiment, the immune cell is a phagocytic autologous or allogeneic natural cell, such as a monocyte, macrophage, or neutrophil, or the immune cell comprises an engineered membrane, such as the cell membrane of an immune cell stimulated by a gene or drug, which is designed to contain relevant recognition proteins capable of interacting with cancer cells. In one embodiment, the core-shell structured carrier comprises a core and a shell, the core being loaded with a drug, the shell being a cell membrane covering the outer surface of the core, the cell membrane being a phagocytic autologous or allogeneic natural cell membrane, such as the cell membrane of a monocyte, macrophage, or neutrophil, or an engineered phagocytic cell membrane, the carrier being a nanocarrier, and / or the carrier being a carrier for the controlled release of the loaded drug. In one embodiment, the cell membrane has a cell recognition factor or contains at least one heterologous extracellular active protein. In one embodiment, the complex or carrier is cryogenically frozen before use; optionally, the complex or carrier is washed after cryogenic freezing. In one embodiment, the exogenous activating molecule is directly or indirectly linked to the interior or exterior of the carrier. In one embodiment, the exogenous activating molecule is a photosensitizer, a sonosensitizer, or a photothermal agent, etc.

[0034] This application also provides a method for preparing a complex, characterized by: (1) linking an exogenous activating small molecule with a carrier having cell or tissue targeting properties to form a complex; (2) preferably, freezing the carrier or the complex at low temperature before use; (3) optionally, washing the complex or carrier after freezing at low temperature. This application also provides the use of the complex alone or in combination with a PD1 antibody in the preparation of a drug for treating tumors. The complex in this application can be referred to by suitable names such as drug delivery system, drug, controlled catalytic oncolytic drug, drug complex, etc.

[0035] definition

[0036] The term "oncolysis" refers to the ability of a controlled-catalytic oncolysis complex to specifically cause the rupture of the cell membrane of identified cancer cells under exogenous stimulation, thereby leading to cancer cell death. The term "catalysis" refers to the fact that, due to the excellent cell membrane targeting ability of the controlled-catalytic oncolysis complex, it can maintain its integrity after being ruptured by exogenous stimulation and can be reused multiple times.

[0037] The term "cryogenic freezing," also known as rapid cryogenic freezing, includes, but is not limited to, liquid nitrogen freezing, dry ice freezing, or other rapid freezing methods that instantly lower the temperature to below -80°C. Cryogenic freezing alters the cell membrane structure of the carrier without damaging the recognition factors on the cell surface. Therefore, inflammatory factors can be gently removed from the cells by washing, while drugs with larger volumes or surface areas remain in the cells. The inventors hypothesize that after rapid cryogenic freezing, ice crystals form in the carrier cell membrane, creating extremely tiny pores, allowing inflammatory factors to be naturally removed during subsequent routine washing, effectively preventing inflammatory responses in the body.

[0038] The term "washing" refers to any method that washes inflammatory factors out of the cell membrane. It is a routine step in the drug delivery process, such as using, but not limited to, PBS, to repeatedly centrifuge and resuspend materials to remove corresponding components such as inflammatory factors.

[0039] The term "encapsulation" refers to any method of covering the outer surface of a carrier with a cell membrane, such as by using phagocytosis by phagocytes to allow the carrier to enter the cell and be encapsulated by the cell membrane.

[0040] The term "click reaction" refers to the generation of heteroatom-containing compounds through a series of reliable, efficient, selective, and modular chemical reactions, thereby achieving the linkage of carbon heteroatoms (CXC).

[0041] The term "carrier" refers to a compound, composition, substance, or structure that, when combined with a compound or composition, contributes to or facilitates the preparation, storage, administration, delivery, effectiveness, selectivity, or any other characteristic of that compound or composition, according to its intended use or purpose. For example, a carrier may be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects on the subject. In this application, the carrier may be a cell or a carrier having a core-shell structure.

[0042] The term "nanocarrier" refers to a device with a diameter less than about 5000 nm, such as less than about 4000 nm, less than about 3000 nm, less than about 2000 nm, from about 10 nm to about 2000 nm, from about 20 nm to about 2000 nm, from about 50 nm to about 2000 nm, from about 100 nm to about 2000 nm, from about 200 nm to about 2000 nm, from about 250 nm to about 2000 nm, and from about 300 nm. From approximately 350nm to approximately 2000nm, from approximately 400nm to approximately 2000nm, from approximately 10nm to approximately 1000nm, from approximately 20nm to approximately 1000nm, from approximately 50nm to approximately 1000nm, from approximately 100nm to approximately 1000nm, from approximately 200nm to approximately 1000nm, from approximately 250nm to approximately 1000nm, from approximately 300nm to approximately 1000nm, from approximately 350nm to approximately 2000nm, from approximately 300nm to approximately 1000nm, from approximately 350nm to approximately 2000nm, from approximately 300nm to approximately 2 ... m to approximately 1000 nm, from approximately 400 nm to approximately 1000 nm, less than 5000 nm, less than 4000 nm, less than 3000 nm, less than 2000 nm, from 10 nm to 2000 nm, from 20 nm to 2000 nm, from 50 nm to 2000 nm, from 100 nm to 2000 nm, from 200 nm to 2000 nm, from 250 nm to 2000 nm, from 300 nm to 2000 nm Particles ranging from 350nm to 2000nm, from 400nm to 2000nm, from 10nm to 1000nm, from 20nm to 1000nm, from 50nm to 1000nm, from 100nm to 1000nm, from 200nm to 1000nm, from 250nm to 1000nm, from 300nm to 1000nm, from 350nm to 1000nm, and from 400nm to 1000nm, etc. The shape of the particles is not particularly important: spherical particles are typical. When using non-spherical nanoparticles, "diameter" refers to the diameter of an imaginary sphere with the same volume as the non-spherical nanoparticle. For the purposes of this application, a “majority” of a nanoparticle is considered to have a specific diameter or a specific diameter range when more than 50% (e.g., more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, etc.) has a specific diameter or a diameter within a specific diameter range. For example, the nanoparticle diameter range is in the range of 1 to 1000 nm, or has a size in the range of 2 to 200 nm, preferably in the range of 2 to 150 nm, or even more preferably in the range of 2 to 100 nm.In addition, the nanocarriers can have an average zeta potential (surface charge) ranging from -1 mV to -40 mV, including approximately -1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, -18, -19, -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, -31, -32, -33, -34, -35, -36, -37, -38, -39, or -40 mV.

[0043] The term "drug" refers to a compound, mixture, or composition that can be used to achieve physiological changes in a subject, including but not limited to small molecules, peptides, and nucleic acids. Examples include antitumor drugs such as paclitaxel, camptothecin, docetaxel, doxorubicin, cisplatin, and 5-fluorouracil.

[0044] In some embodiments, the compound may be formulated as the sole active pharmaceutical ingredient in the composition, or it may be combined with other active ingredients. For example, the compound may be formulated or combined with known NSAIDs, anti-inflammatory compounds, steroids, and / or antibiotics.

[0045] In some implementations, the drug is a hydrophilic or hydrophobic antitumor agent. For example, the drug may be selected from abiraterone acetate, abitrexate, abraxane (albumin-stabilized paclitaxel nanoparticles), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris, ADE, Ado-Trastuzumab Emtansine, Adriamycin (doxorubicin hydrochloride), Adrucil (fluorouracil), afatinib maleate, everolimus (afinil), Akynzeo (netoripantan and palonosetron hydrochloride), idarubicin (imvamod), interleukin, alemtuzumab, pemetrexed disodium, and Alox. i (palonosetron hydrochloride), Ambochlorin (chlorambucil), Amboclorin (chlorambucil), aminoacetylpropionic acid, anastrozole, aprepitant, acetamiprid (disodium benzoate), rennet (anastrozole), Aromasin (exemestane), Arranon (neurabin), arsenic trioxide, Arzerra (Ofatumumab), Erwinia chrysanthemi asparaginase, Avastin (bevacizumab), axitinib, azacitrap, BEACOPP, Becenum (carmustine), Beleodaq (belistat), belistat Bendamustine hydrochloride, BEP, bevacizumab, bexarotine, cyclophosphamide (tosimomab and iodine-131 tosimomab), bicalutamide, BiCNU (carmustine), bleomycin, bonatumab, Blincyto (bonatumab), bortezomib, bosulif (bosutinib), bosutinib, Brentuximab Vedotin, busulfan, busulfan, cabazitaxel, cabozantinib S-malate, CAF, Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, carboplatin, carboplatin-paclitaxel, carfilzomib, Carmubris ( Carmustine, carmustine implant film, Cascade (bicalutamide), CeeNU (lomustine), ceritinib, daunorubicin (daunorubicin hydrochloride), Cervarix (recombinant bivalent HPV vaccine), cetuximab, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, clafen (cyclophosphamide), clorapine, Clofarex (clorapine), Clolar (clorapine), CMF, Cometriq (cazozantinib S-malate), COPP, COPP-ABV, Cosmegen (actinomycin D), cristatinib, CVP, cyclophosphamide, Cyfos (ificosylphosphamide).Cyramza (ramucirumab), Cytarabine, Cytarabine liposome, Cytosar-U (cytarabine), Cyclophosphamide, Darafenib, Dacarbazol, Decitabine, Actinomycin D, Dasatinib, Daunoromycin Hydrochloride, Decitabine, Degarelix, Denisulin-2, Dinosema, DepoCyt (cytarabine liposome), DepoFoam (cytarabine liposome), Dexamethasone Hydrochloride, Dinutuximab, Docetaxel, Doxil (doxorubicin hydrochloride liposome), Doxorubicin Hydrochloride, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazol), Efudex (fluorouracil), Elitek (raburicase), Ellence (epimacin hydrochloride), Oxaliplatin (oxaliplatin) Eltrombopag ethanolamine, Emend (Aprepitant), Enzalutamide, Epirubicin Hydrochloride, EPOCH, Elbidos (Cetuximab), Eribulin Mesylate, Vismodegib, Erlotinib Hydrochloride, Erwinaze (Erwinia chrysanthum asparaginase), Vanbex (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposome), Everolimus, Iveret (Raloxifene Hydrochloride), Exemestane, Fareston (Toremifene), Farydak (Papirostat), Faslodex (Flushexant), FEC, Letromethorphan, Figex, Fludarabine (Fluorabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluoroplex), Fluoroplex, Foles (Methotrexate), Foles PFS (Methotrexate), FOLFIRI, FOLFIRI-Bevacizumab, FOLFIRI-Cetuximab, FOLFIRINOX, Oxaliplatin, Folotyn (Platroxadu), FU-LV, Fulvestrant, Gardex (Recombinant HPV Quadrivalent Vaccine), Gardex 9 (Recombinant HPV No. 9 Vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, Gemcitabine-Cisplatin, Gemcitabine-Oxaliplatin, Gemtuximab, Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Maleate), Gleevec Imatinib Sulfate, Gliadel (Carmustine Implantable Film).Gliadelwafer (carmustine implant film), fucoidase, goserelin acetate, Halaven (eribulin mesylate), Herceptin (trastuzumab), recombinant bivalent HPV vaccine, recombinant nine-valent HPV vaccine, recombinant quadrivalent HPV vaccine, Hycamtin (topotecan hydrochloride), Hyper-CVAD, Ibrance (bocinib), tivamoxicillin, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Idamycin (idarubicin hydrochloride), edalaris, ifosfamidum (ifosfamidum), imatinib mesylate, Imbruvica (ibrutinib), mivamoxicillin, Inlyta (axitinib), recombinant interferon Alfa-2b, Intron A (recombinant interferon Alfa-2b), iodine-131 tosimomumab and tosimomumab, Iprimumab, Iressa (gefitinib), irinotecan hydrochloride, Istodax (romidesin), ixaprilone, Ixempra (ixaprilone), Jakafi (ruxolitinib phosphate), Jevtana (cabazitaxel), Kadcyla (Ado-Trastuzumab) Emtansine, Keoxifene (raloxifene hydrochloride), Kepivance, Pembrolizumab, Kyprolis (carfilzomib), Lanrefu acetate, Lapatinib besylate, Lenalidomide, Lenvatinib mesylate, Lenvima (lenvatinib mesylate), Letrozole, Calcium leucovorin, Leukeran (chlorambucil), Leuprolide acetate, Levulan (aminoacetylpropionic acid), Linfolizin (chlorambucil), LipoDox (doxacin hydrochloride liposome), Cytarabine liposome, Lomustine, Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot-Ped (leuprolide acetate). Depot-3Month (Leuprolide Acetate), LupronDepot-4Month (Leuprolide Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane (Methylparaben Hydrochloride), Nitrogen Mustard Hydrochloride, Megace (Megestrol Acetate), Mekinist (Trametinib), Lupron, Mesna, Mesnex, Methazolastone (Temolimid), Methotrexine, Methotrexine LPF (Methotrexine), Mexate (Methotrexine), Mexate-AQ (Methotrexine), Mitomycin C, Mitozytrex Hydrochloride, Mitozytrex (Mitomycin C), MOPP,Mozobil (Plesafox), Mustargen (Nitrogen Mustard Hydrochloride), Mustamycin (Mitomycin C), Mylosar (Azacitrap), Mylotarg (Gilotumab), Paclitaxel Nanoparticles (Albumin-Stabilized Paclitaxel Nanoparticles), Navelbine (Vinorelbine Tartrate), Nerapine, Neosar (Cyclophosphamide), Netopitam and Palonosetron Hydrochloride, Neupogen (Figrex), Nexavar (Sorafenib Tosylate), Nilotinib, Nivolumab, Tamoxifen Citrate, Nplate (Romilastine). Obinutuzumab, Odomzo (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Homoharringtonine, Oncaspar (pegaspargase), Ondansetron Hydrochloride, Ontak (interleukin-2), Opdivo (Nivolumab), OPPA, Oxaliplatin, Paclitaxel, Albumin-stabilized Paclitaxel Nanoparticles, PAD, Palbociclib, Palivmin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netopitam, Disodium Pamirnate, Panitumumab, Pabistat, Paraplatin (Carboplatin), Paraplatin (Carboplatin), Peripanib Hydrochloride Pembrolizumab, pemetrexed disodium, Perjeta, pertuzumab, cisplatin, cisplatin-AQ, Plerixafor, Pomalidomide, Pomalyst, ponatinib hydrochloride, Pralatrexate, prednisone, methylparaben hydrochloride, Proleukin, Prolia, Promacta, eltrombopag ethanolamine Provenge (Sipuleucel-T), Purinethol, Purixan, Radium-223 chloride, Raloxifene Hydrochloride, Ramucirumab, Raburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonvalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, R-EPOCH, Revlimid, Rheumatrex, Rituximab, Rituximab, RomidepsinRomiplostim, ruxolitinib hydrochloride, ruxolitinib phosphate, Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, Somatuline Depot (Lanrefu acetate), Sonidegib, Sorafenib tosylate, Sprycel (dasatinib), STANFORD V, sterile Talc powder (Talc), Steritalc (Talc), Stivarga (regorafenib), sunitinib malate, Sutent (sunitinib malate), Sylatron (pegylated interferon Alfa-2b), Sylvant (Siltuximab), Synovir (thalidomide), Synribo (homoharringtonine), TAC, Tafinlar (dabrafenib), Talc, tamoxifen citrate, Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotin), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Temodar (Temolid), Temolid, Sirolimus Lipids, Thalidomide, Thalomid, Toposar (Etoposide), Topotecan Hydrochloride, Toremifene, Torisel (Sirolimus Lipids), Tosimomab and Iodine-131 Tosimomab, Toted (Dexazosone Hydrochloride), TPF Trametinib, Trastuzumab, Treanda (bendamustine hydrochloride), Trisenox (arsenic trioxide), Tykerb, Unituxin (Dinutuximab), Vandetanib, VAMP, Vectibix (panitumumab), VeIP, Velban (vincrine sulfate), Velcade (bortezomib), Velsar (vincrine sulfate), Vemurafenib, VePesid (etoporoplastin), Viadur (leuprolide acetate), Vidaza (azacytoplastin), Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposomes, Vinorelbine Tartrate, VIP, Vismodegib, Voraxaze, Vorinostat, Votrient (Paripanil Hydrochloride), Wellcovorin (Calcium Leucovorin), Xalkori (Critinib), Xeloda (Capecitabine), XELIRI, XELOX, Xgeva (Denosema), Xofigo (Radium-223 Chloride), Xtandi (Enzalutamide), Yervoy (Iprimumab), Zaltrap (Ziv-Aflibercept), Zelboraf (Vemurafenib).Zevalin (Ibritumomab Tiuxetan), Zinecard (dexrazoxan hydrochloride), Ziv-abflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), levofloxacin, Zolinza (vorinostat), Zometa (levofloxacin), Zydelig (ederaris), Zykadia (ceritinib), and Zytiga (abiraterone acetate).

[0046] The term "small molecule" refers to molecules with a molecular weight of less than 2,000 Daltons, more preferably less than 1,500 Daltons, and most preferably less than 1,000 Daltons, such as organic or organometallic compounds. Small molecules can be hydrophilic, hydrophobic, or amphiphilic compounds.

[0047] The term "treatment" refers to the medical management of a patient with the intent to cure, improve, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, which is treatment specifically aimed at improving a disease, pathological condition, or disorder, and also includes etiological treatment, which is treatment aimed at eliminating the cause of the related disease, pathological condition, or disorder. Furthermore, the term includes palliative treatment, which is treatment designed to alleviate symptoms rather than cure a disease, pathological condition, or disorder; preventative treatment, which is treatment aimed at minimizing or partially or completely suppressing the development of a related disease, pathological condition, or disorder; and supportive treatment, which is treatment used to complement another specific therapy aimed at improving a related disease, pathological condition, or disorder.

[0048] "Inflammatory factors" (or inflammatory cytokines) are various cytokines that participate in the inflammatory response, such as IL-6, IL-8, IL-10, IL-12, IL-1β, TNFα, etc.

[0049] The term "cell recognition factor" refers to factors that can recognize target cells, such as self-recognition immune regulatory proteins like CD47, CD24, SIRPα / SHPS1, CD55, or CD5.

[0050] The term "heterogeneous extracellular active protein" can refer to any natural or synthetic protein that will have a therapeutic effect when in contact with target cells. For example, the extracellular active protein could be a tumor necrosis factor (TNF)-associated apoptosis-inducing ligand (TRAIL), which binds to DR4 and DR5 death receptors on tumor cells and induces apoptosis. Alternatively, the extracellular active protein could be a therapeutic antibody, such as cetuximab, trastuzumab, bevacizumab, panitumumab, ipilimumab, rituximab, alemtuzumab, offatumumab, gemtuzumab ozogamicin, brentuximab vedotin, pembrolizumab (Keytruda), nivolumab (Opdivo), or combinations thereof.

[0051] "Photosensitizers" utilize their special chemical structure to convert light of a specific wavelength into chemical energy to generate ROS and complete photodynamic therapy (SDT). Photosensitizers can be hematoporphyrin; hematoporphyrin derivatives such as hematoporphyrin monomethyl ether, 5-aminolevulinic acid; phthalocyanine photosensitizers such as glucose conjugated phthalocyanine, aluminum phthalocyanine; dihydroporphyrin derivatives such as pyrophyllite-a, erythropoietin-18 methyl ester, 2-(1-hexyloxyethyl)-2-devinylpyrophyllite, dihydroporphyrin e6 (Ce6), bacterial dihydroporphyrin; polycyclic quinone photosensitizers such as baicalin, hypericin; and phenothiazine photosensitizers such as 3,7-bis(dimethylamino)phenothiazine-5-onium chloride.

[0052] "Sound sensitizers" are substances that convert sound energy into chemical energy through a special chemical structure to generate ROS and complete sonodynamic therapy (SDT). Sound sensitizers can be hematoporphyrin, photoporphyrin II, hematoporphyrin monomethyl ether, 5-aminolevulinic acid, porphyrin derivatives, xanthracene compounds such as fluorescein rose red, phthalocyanine dye phthalimide zinc phthalocyanine, or [Ru(bpy)3]2+.

[0053] “ABDA” stands for 9,10-anthratridimyl-bis(methylene)dicarboxylic acid, a probe for detecting singlet oxygen production, where reactive oxygen species cause a decrease in the corresponding absorption peak.

[0054] Photothermal therapy primarily utilizes photothermal agents (PTA) to convert light energy into heat energy, killing tumor cells through localized heating and the triggering of biochemical processes. Photothermal agents can be noble metal materials such as gold, platinum, and palladium; or transition metal materials such as transition metal oxides, sulfides, carbides, nitrides, and dichlorides, including TiS2, FeS, and Cu. x S y Cu 2-x S, MoS2, Ag2S, WS2, FeSe2, Cu 2-x Se, TiO2, Ti8O 15 MoO x W x O y Inorganic non-metallic photothermal agents include carbon-based materials, silicon-based materials, and black phosphorus, as well as some carbides, nitrides, and dichlorides. Organic small molecules include indocyanine green (ICG), indoles such as IR780, IR820, IR825, cypate, and porphyrins. Photothermal agents based on polymer materials include artificially synthesized semiconductor polymer nanoparticles such as polyaniline, polythiophene, polypyrrole, and polydopamine (PDA).

[0055] The complex of this application can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intrathoracically, intratracheally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intracysticly, mucosally, intraperitoneally, intraumbilically, intraocularly, intrathecally, locally, by injection, by infusion, by continuous infusion, by direct local perfusion of target cells, via catheter, or by irrigation. For example, the complex can be administered by injection or infusion.

[0056] In some embodiments, the complex is administered externally by injecting it near the tumor site. As used herein, "near the tumor site" means locally targeting and delivering the complex to the tumor site, and is intended to include direct injection into the tumor as well as injection into a region approximately 1 cm from the tumor (e.g., within 1 cm, within approximately 5 mm, within 5 mm, within approximately 2 mm, within 2 mm, etc.). The complex can be administered, for example, via a single injection or via multiple injections, such as by injecting the complex into the tumor and around the tumor margin. In some embodiments, such as in the case of intravenous administration, the complex is administered systemically to the subject, for example, by injecting the complex into the subject's circulatory system. In some embodiments, the complex is administered intravenously, for example, to infuse the tumor in the gastrointestinal tract. For better therapeutic benefit, the complex of this application can be administered in combination with at least one additional agent selected from radiotherapy agents, hormone therapy agents, immunotherapy agents, chemotherapy agents, cryotherapy agents, and gene therapy agents.

[0057] This application discloses a macrophage-photosensitizer complex and a macrophage-soundsensitizer complex, which have cell membrane targeting capabilities to specifically recognize tumor cells and catalyze tumor dissolution through exogenous stimulation. By modifying immune cells, exogenous stimulation such as lasers, radiation, and ultrasound can be used to controllably destroy cancer cells by disrupting their membranes. Cold-frozen immune cells retain their membrane structure and associated functional proteins to the greatest extent possible. Utilizing the high affinity between immune cells and cancer cells, this complex can effectively aggregate on the surface of cancer cells, enabling interaction. Through drug recognition of the cancer cell membrane and control via exogenous stimulation, catalytic tumor dissolution can be achieved, significantly improving treatment efficacy and reducing drug dosage. Macrophage-photosensitizer complexes and macrophage-soundsensitizer complexes, through their specific recognition of tumor cells and exogenous stimulation, can precisely induce membrane-rupture-type death of cancer cells, leading to the massive release of intracellular inflammatory factors and activating the body's own anti-tumor immunity. Furthermore, macrophage-photosensitizer complexes and macrophage-soundsensitizer complexes can effectively eliminate primary tumor cells and significantly inhibit tumor metastasis and recurrence.

[0058] The advantages of the macrophage-photosensitizer complex and the macrophage-soundsensitizer complex in the embodiments of this application are as follows: 1. Utilizing the natural targeting ability of autologous immune cells to tumor cells, specific recognition of tumor cell membranes is achieved. 2. Using low-temperature rapid freezing technology to freeze-treat modified immune cells can maximize the preservation of membrane components and their functions, reduce immunogenicity, and allow for rapid large-scale preparation. 3. Through exogenous activating small molecules linked to immune cells, precise membrane perforation of tumor cells can be controlled under exogenous stimulation, resulting in the large-scale release of intracellular inflammatory factors, thereby altering the tumor microenvironment to achieve immunotherapy. 4. The macrophage-photosensitizer complex, by linking tetraacetyl-N-azidoacetylmannosamine (Ac4ManNAz), achieves more precise targeting of tumor cells. 5. It can be used alone or in combination with immune checkpoint inhibition (ICB) therapy, effectively inhibiting tumor recurrence and metastasis, and expanding the applicability of immune checkpoint inhibition (ICB) therapy.

[0059] The following examples specifically describe a macrophage-photosensitizer complex and a macrophage-soundsensitizer complex, wherein the macrophage membrane can be a natural or engineered membrane containing macrophages capable of interacting with cancer cells. In some cases, the macrophages are generated through bone marrow isolation, such as from autologous bone marrow from a subject to be treated. In other cases, the cell membrane is an engineered membrane, such as the cell membrane of macrophages stimulated by genes or drugs, which is designed to contain relevant recognition proteins capable of interacting with cancer cells. Whether natural or synthetic, the outer shell macrophage membrane can be designed to contain at least one heterologous extracellular active protein. This extracellular active protein can be any natural or synthetic protein that will have a therapeutic effect upon contact with cancer cells. For example, the extracellular active protein could be a tumor necrosis factor (TNF)-associated apoptosis-inducing ligand (TRAIL), which binds to DR4 and DR5 death receptors on tumor cells and induces apoptosis. Alternatively, the extracellular active protein could be a therapeutic antibody, such as cetuximab, trastuzumab, bevacizumab, panitumumab, ipilimumab, rituximab, alemtuzumab, offatumumab, gemtuzumabozogamicin, brentuximab vedotin, pembrolizumab (Keytruda), nivolumab (Opdivo), or combinations thereof.

[0060] This application also specifically discloses a method for treating cancer in a subject, the method involving administering to the subject the macrophage-sound-sensitizer complex or macrophage-photosensitizer complex disclosed herein. In some cases, the macrophage membrane is autologous, i.e., produced from bone marrow obtained from the subject. In some cases, the cancer is a primary cancer cell. In some cases, the cancer is a metastatic cancer cell. This application also specifically discloses a method for treating cancer in a subject, the method involving administering to the subject simultaneously the macrophage-sound-sensitizer complex or macrophage-photosensitizer complex disclosed herein, and an immune checkpoint inhibitor, in some cases, the immune checkpoint inhibitor being a PD1 antibody. This application more specifically provides a method for preparing macrophage-sound-sensitizer complex and macrophage-photosensitizer complex by cryogenic freezing, the method comprising: (a) conjugating a photosensitizer and / or a sound-sensitizer to macrophages; (2) cryogenically freezing the prepared macrophage-exogenous small molecule complex. The methods for linking photosensitizers and / or sonosensitizers to macrophages can be varied. In one specific embodiment, macrophage-soundsensitizer complexes and macrophage-photosensitizer complexes are prepared via click reactions. Preferably, the photosensitizer is linked to macrophages via a click reaction with the azide group of Ac4ManNAz. In some embodiments, the cryogenic freezing method includes rapid freezing techniques such as liquid nitrogen freezing and dry ice freezing, which instantly lower the temperature to below -80°C. This application also specifically provides the use of macrophage-photosensitizer complexes or macrophage-soundsensitizer complexes in the preparation of medicaments for treating cancer in a subject. This application also discloses a method for treating cancer in a subject, the method involving administering the macrophage-photosensitizer complex or macrophage-soundsensitizer complex disclosed herein to the subject. In some cases, the macrophage membrane is autologous, i.e., generated from macrophages obtained from the subject. The cancer is either in situ cancer cells or metastatic cancer cells.

[0061] "Cancer" can be a solid tumor, metastatic cancer, or non-metastatic cancer. In some implementations, cancer can originate in the bladder, blood, bones, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, rectum, anus, teeth, head, kidneys, liver, lungs, nasopharynx, neck, ovaries, prostate, skin, stomach, testicles, tongue, or uterus. In some implementations, the cancer is ovarian cancer. In certain aspects, the cancer may be chemotherapy-resistant cancer. In some cases, the cancer is an in situ cancer cell.

[0062] "Cancer" may specifically refer to, but is not limited to, the following histological types: growths, malignant; malignant epithelial cell tumors; malignant epithelial cell tumors, undifferentiated; giant cell and spindle cell carcinomas; small cell carcinomas; papillary carcinomas; squamous cell carcinomas; lymphoepithelial carcinomas; basal cell carcinomas; pilomatal carcinomas; transitional cell carcinomas; papillary transitional cell carcinomas; adenocarcinomas; gastrinomas, malignant; cholangiocarcinomas; hepatocellular carcinomas; mixed hepatocellular carcinomas and cholangiocarcinomas; trabecular adenocarcinomas; adenoid cystic carcinomas; adenocarcinomas in adenomatous polyps; adenocarcinomas, familial adenomatous polyposis; solid tumors; carcinoid tumors, malignant; bronchioloalveolar adenocarcinomas; papillary adenocarcinomas; chromophobe carcinomas; eosinophilic cell carcinomas; eosinophilic adenocarcinomas; basophilic cell carcinomas; clear cell adenocarcinomas; granular cell carcinomas; follicular adenocarcinomas. Papillary and follicular adenocarcinoma; non-surrounded sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous gland carcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; theca cell tumor, malignant; granulosa cell tumor, malignant; male cell tumor, malignant; Sertoli cell carcinoma; stromal cell tumor, malignant; lipocytoma, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; Glomerular sarcoma; Malignant melanoma; Amelanoma; Superficial diffuse melanoma; Giant pigmented nevus malignant melanoma; Epithelioid cell melanoma; Blue nevus, malignant; Sarcoma; Fibrosarcoma; Fibrohistocytoma, malignant; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Mixed tumor, malignant; Uterine mesodermal mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Mesenchymal tumor, malignant; Brunnelli's tumor, malignant; Phyllodes tumor, malignant; Synovial sarcoma; Mesothelioma, malignant; Dysgerminoma; Embryonic carcinoma; Teratoma, malignant; Ovarian thyroid tumor, malignant; Choriocarcinoma; Mesonephric tumor, malignant; Vascular Sarcoma; Hemangioendothelioma, malignant; Kaposi's sarcoma; Hemangiopericytoma, malignant; Lymphangiosarcoma; Osteosarcoma; Cortical osteosarcoma; Chondrosarcoma; Chondroblastoma, malignant; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Odontogenic tumor, malignant; Ameloblastic osteosarcoma; Ameloblastoma, malignant; Ameloblastic fibrosarcoma; Pineal tumor, malignant; Chordoma; Glioma, malignant; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibroblastic astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal tumor; Cerebellar sarcoma; Ganglioblastoma; Neuroblastoma; Retinoblastoma; Oligosacral neurogenic tumor;Meningioma, malignant; neurofibrosarcoma; schwannoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; grass granuloma; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0063] Example

[0064] Example 1: Preparation and in vitro / in vivo activity verification of photosensitizer Ce6-Ac4ManNAz-LNT_Raw 264.7

[0065] 1.1 Preparation of Ce6-Ac4ManNAz-LNT_Raw 264.7

[0066] First, well-grown RAW 264.7 cells were added to DMEM complete medium containing 125 μM Ac4ManNAz and cultured for 72 h. Ac4ManNAz can serve as a metabolic glycogen source and participate in the synthesis of cell membrane surface glycoproteins, resulting in a high concentration of Ac4ManNAz on the cell membrane surface. After the Ac4ManNAz-prepared RAW 264.7 cells were cultured, they were aspirated from the bottom of the dish and resuspended in PBS to achieve a cell concentration of 5 x 10⁻⁶ cells / mL. 6 / ml.

[0067] Secondly, alkynyl-Ce6 was added to the cell suspension to a concentration of 20 μg / m, along with ascorbic acid and CuSO4 in a 1:1 ratio to a concentration of 0.2 μM. The mixture was then incubated at room temperature for 1 hour in the dark. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) + Catalyzed by [catalyst], the alkynyl-Ce6 group undergoes a click reaction with the azide group of Ac4ManNAz, ultimately resulting in Ce6 conjugation on the RAW 264.7 cell membrane. The Ce6-conjugated cells were centrifuged, the supernatant was removed, and the cells were resuspended and washed twice with PBS. The final cell pellet was resuspended in serum-free DMEM medium at a cell density of 1 x 10⁻⁶ cells / mL. 7 / ml, loaded into cryovials, and rapidly frozen in liquid nitrogen for more than 12 hours to obtain Ce6-Ac4ManNAz-LNT_Raw 264.7 (i.e. LCS).

[0068] The results showed that Ac4ManNAz_Raw 264.7 and Ce6-Ac4ManNAz-LNT_Raw264.7 were successfully prepared. After flash freezing in liquid nitrogen, the cell membranes remained intact, and significant Ce6 fluorescence was observed on the cell membrane surface. Figure 1 ).

[0069] 1.2 Flow cytometry detection

[0070] Flow cytometry analysis was performed using conventional methods in the art. Flow cytometry tests on the surface antibodies for cell recognition and phagocytosis prevention on the cell surface of normal and liquid nitrogen-frozen modified RAW264.7 cells with the same cell number revealed that liquid nitrogen freezing caused less damage to cell surface antibodies, indicating that liquid nitrogen freezing can preserve surface antibodies to a greater extent, thereby retaining their related functions. Figure 2 ).

[0071] 1.3 Release of cytoinflammatory factors

[0072] The release of inflammatory factors from modified RAW264.7 cells (normal and liquid nitrogen frozen) was compared. Supernatant was collected from each group of cells after 24 hours, and IL-1β, IL-6, IL-10, IL-12, TNF-α, TGF-β, and HMGB1 were detected using a double-antibody sandwich ELISA method (n=3). The results show that modified live cells released a large number of inflammatory factors under laser stimulation. Direct use of these cells in vivo would cause a systemic inflammatory response, negatively impacting health. However, the rapid freezing method effectively removes these inflammatory factors during the washing process, thus preventing a systemic inflammatory response. Figure 3 ).

[0073] 1.4 Photo-controlled generation of reactive oxygen species

[0074] 4T1 cells were seeded in confocal microplates (1×10⁻⁶). 6 In a dish, incubate at 37°C for 6 hours until the sample adheres to the wall, then add 1 ml of Ce6-Ac4ManNAz-LNT_Raw 264.7 (5×10⁻⁶) solution. 6 The medium ( / dish) was incubated for 6 hours in an incubator, and the sample was washed three times with medium to remove free material. Then, SOSG probe (2 μl, 5 mM) was added and incubated for 5 min. Ce6 was excited with a 660 nm laser at 60x objective to observe FITC (525 nm) green fluorescence and bright-field images. Finally, confocal microscopy (Olympus FV3000) was used for imaging. 1 ml of Ce6-Ac4ManNAz-LNT_Raw 264.7 was added to a confocal culture dish (1×10⁻⁶). 6After culturing the cells in a 3-hour incubator until sedimentation, SOSG probe (2 μl, 5 mM) was added and incubated for 5 minutes. Ce6 cells were excited with a 660 nm laser at 60 x objective to observe FITC (525 nm) green fluorescence and bright-field images. Finally, confocal microscopy (Olympus FV3000) was used for imaging.

[0075] The results showed that Ce6-Ac4ManNAz-LNT_Raw 264.7 generated a large amount of singlet oxygen after being irradiated by a 660nm laser. ABDA captured the singlet oxygen, causing its characteristic absorption peak to decrease, and this decrease continued with increasing illumination time. The singlet oxygen green probe exhibited green fluorescence after oxidation (see...). Figure 4 ; where 4a indicates that the characteristic absorption peak of ABDA continuously decreases with increasing illumination time; 4b indicates that the singlet oxygen green probe exhibits green fluorescence after oxidation).

[0076] 1.5 In vitro tumor cell targeting

[0077] 1 ml of culture medium containing tumor cells (EMT6, 4T1, and CT26) was added to a confocal culture dish (1×10⁻⁶). 6 After culturing in a medium containing Calcein-AM (10 μL, 1 mM) for 6 h until cells adhered, the cells were washed twice with PBS. Then, 500 μL of medium containing Calcein-AM (10 μL, 1 mM) was added to stain the cells for 30 min, followed by washing three times with PBS. After staining, 1 ml of Dil-labeled Ce6-Ac4ManNAz-LNT_Raw 264.7 (5 × 10⁻⁶ mM) was added. 6 The culture medium ( / dish) was incubated in an incubator for 6 hours and washed three times with the culture medium to remove unidentified materials. Finally, the images were imaged using a confocal microscope (Olympus FV3000).

[0078] The results showed that Ce6-Ac4ManNAz-LNT_Raw 264.7 targeted cancer cells in vitro using confocal microscopy. After co-incubating the Dil-labeled Ce6-Ac4ManNAz-LNT_Raw 264.7 with cancer cells, it clearly adhered to the surface of the cancer cells, and multiple Ce6-Ac4ManNAz-LNT_Raw264.7 molecules could be identified and adhered to the surface of a single cancer cell. Figure 5 ).

[0079] 1.6 Light-controlled cell death

[0080] 1.6.1CCK8 LDH

[0081] To assess the efficacy of Ce6-Ac4ManNAz-LNT_Raw 264.7 in vitro, 4T1 cells were seeded in 48-well plates (2 × 10⁻⁶ cells per well). 4 After the cells adhered to the wells, Ce6-Ac4ManNAz-LNT_Raw264.7 with different effector-to-target ratios (Cell index, Ce6-Ac4ManNAz-LNT_Raw 264.7: 4T1 = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) were added and incubated at 37°C for 6 hours. Unbound Ce6-Ac4ManNAz-LNT_Raw 264.7 was then washed away with PBS, followed by irradiation with a 660nm (1W, 5 min) laser and incubation for 6 hours. A commercially available CCK8 reagent was prepared to contain 10% CCK8 (v / v) in culture medium. The 48-well plate was removed, the culture medium was collected, and the plates were washed three times with PBS. The CCK8 reagent was added to each well, and the plates were incubated for another 2 hours. OD450 was measured using a microplate reader. Simultaneously, the collected culture medium was used for LDH release assay. 100 μL of each medium was added to a new 96-well plate, along with 100 μL of LDH Working Solution. After incubation at room temperature in the dark for 30 minutes, 50 μL of Stop Solution was added, and OD490 was immediately measured using a microplate reader. Data are expressed as mean ± SD (n = 3 independent experiments). To investigate the effects of laser irradiation and Ce6-Ac4ManNAz-LNT_Raw 264.7, and their combined use, on tumor cells, the same method was used to test the efficacy of Ce6-Ac4ManNAz-LNT_Raw 264.7 (Cell index, Ce6-Ac4ManNAz-LNT_Raw 264.7:tumor cells = 5) on 4T1, CT26, and EMT6 cells under different conditions.

[0082] The results were measured using cell viability (CCK-8) and cell membrane rupture death (LDH). Untreated 4T1, CT26, and EMT6 cells and cancer cells + Ce6-Ac4ManNAz-LNT_Raw 264.7 served as controls (n=3). The figures show that Ce6-Ac4ManNAz-LNT_Raw 264.7 itself is not cytotoxic; only under 660nm red light stimulation can it induce large-scale cell death, and it can cause cancer cells to undergo membrane rupture death. This indicates that Ce6-Ac4ManNAz-LNT_Raw 264.7 has a good and controllable cancer cell killing effect. Figure 6 ).

[0083] 1.6.2 Confocal Microscopy Detection

[0084] To observe cell morphology under different treatments, tumor cells 4T1, EMT6, and CT26 were seeded in confocal microplates (1×10⁻⁶ cells / mL). 6 In a 37°C container, incubate for 6 hours until the sample adheres to the vessel wall. Then add 1 ml of a solution containing Ce6-Ac4ManNAz-LNT_Raw264.7 (5×10⁻⁶ ppm). 6 The culture medium ( / dish) was incubated for 6 hours and washed three times with the culture medium to remove free material. Ce6 was then excited with a 660-channel laser using a confocal microscope (Olympus FV3000), and bright-field and fluorescence images were captured in real-time on a 60x objective lens. Images were processed using Olympus FV31S software. Confocal imaging of Ce6-Ac4ManNAz-LNT_Raw 264.7 targeting cancer cells in vitro was performed. After co-incubating Ce6-Ac4ManNAz-LNT_Raw 264.7 labeled with Dil with cancer cells, the cells clearly adhered to the surface of the cancer cells, and multiple Ce6-Ac4ManNAz-LNT_Raw 264.7 cells could be identified and adhered to the surface of a single cancer cell. Figure 7 ).

[0085] 1.7 In vivo targeting capability determination

[0086] 1.7.1 Preparation of Ce6-Ac4ManNAz-LNT_Raw 264.7@DiI. Ce6-Ac4ManNAz-_Raw 264.7 was first co-incubated with the membrane dye DiI (1 mM) at 37 °C for 30 min. The prepared (Ce6-Ac4ManNAz-_Raw264.7@DiI) was then rapidly frozen in liquid nitrogen.

[0087] 1.7.2. Establishing a tumor model. 1×10⁻⁶ tumor cells were subcutaneously injected onto the right back of 7-week-old female BALB / c mice. 6 4T1 cells.

[0088] 1.7.3. In vivo targeted therapy. When the tumor volume reaches 150mm... 3 Subsequently, inject 100 μL of 2×10 [drugs] into the tail vein. 6Mice were injected with Ce6-Ac4ManNAz-LNT_Raw 264.7@DiI. At 12h, 24h, 48h, 72h, and 96h, mice were dissected, and the heart, liver, spleen, lungs, kidneys, and tumor tissues were removed. The fluorescence intensity of DiI was detected using a small animal imaging system. Following tail vein injection of Ce6-Ac4ManNAz-LNT_Raw264.7, the fluorescence intensity of DiI in the heart, liver, spleen, lungs, kidneys, and tumors was detected at 12h, 24h, 48h, 72h, 96h, and 120h (n=3). The colors are: red (tumor), green (liver), orange (lung), blue (kidney), purple (heart), and black (spleen). It can be seen that Ce6-Ac4ManNAz-LNT_Raw264.7 still has a large amount of residue at the tumor site after 72 hours, and its deposition in other organs is very small, indicating that it has good in vivo circulation and tumor-specific targeting. Figure 8 (8a shows the fluorescence distribution of the drug in mouse tissues at different times; 8b shows the statistical graph of the average fluorescence intensity of the drug in mouse tissues at different times).

[0089] 1.8 In vivo antitumor efficacy assay

[0090] 1.8.1 Normal Treatment

[0091] Establish a tumor model. 1×10⁻⁶ mc²⁻¹ was subcutaneously injected into the right dorsal side of 7-week-old female BALB / c mice. 6 4T1 cells.

[0092] Groups: ①PBS group (n=8), ②PBS + 660nm light irradiation group (n=8), ③Ce6-Ac4ManNAz-LNT_Raw264.7 in situ injection group (n=8), ④Ce6-Ac4ManNAz-LNT_Raw 264.7 in situ injection + 660nm light irradiation group (n=8).

[0093] As mentioned above, when the tumor volume reaches 100mm 3 Then, the treatment experiment began: A. For groups ③ and ④, 25 μL of Ce6-Ac4ManNAz-LNT_Raw 264.7 PBS solution was injected into the tumor, with a cell count of 1.0 × 10⁻⁶ cells. 6 For groups ① and ②, 25 μL of PBS solution was injected intratumorally. B. 24 hours later, for groups ③ and ④, a laser was used ×10 6(Beijing Honglan Optoelectronics) The tumor site was irradiated with 660nm red light (1.0W) for 5 minutes. Tumor growth information and weight changes in the small animals were collected starting on the second day after treatment. The tumor size was measured using calipers, and the tumor volume V was calculated using the following formula: V = 0.5 × a × b × b (a is length, b is width). When the tumor volume reached 1500mm²... 3 Mice were sacrificed. E. Growth curves were plotted based on the tumor growth data. Tumor volume was measured every two days after treatment of mice in different groups. The graph shows that intratumoral injection of Ce6-Ac4ManNAz-LNT_Raw 264.7 into mice inoculated with 4T1, followed by treatment with 660° red light, effectively inhibited tumor growth. Figure 9 a).

[0094] 1.8.2. PD-1 combination therapy

[0095] A tumor model was established. 1×10⁻⁶ tumor cells were subcutaneously injected onto the right back of 7-week-old female BALB / c mice. 6 4T1 cells.

[0096] Groups: ①PBS group (n=8), ②intraperitoneal injection of anti-PD1 group, ③Ce6-Ac4ManNAz-LNT_Raw 264.7 tail vein injection + 660nm light irradiation group (n=8), ④Ce6-Ac4ManNAz-LNT_Raw 264.7 tail vein injection + intraperitoneal injection of anti-PD1 + 660nm light irradiation group (n=8).

[0097] As mentioned above, when the tumor volume reaches 100mm 3 Then, the treatment experiment began: A. For groups ③ and ④, 200 μL of Ce6-Ac4ManNAz-LNT_Raw 264.7 PBS solution was injected via the tail vein, with a cell mass of 1.5 × 10⁻⁶ cells. 7 For groups ① and ②, 200 μL of PBS solution was injected via the tail vein. B. 24 hours later, for groups ③ and ④, the tumor site was irradiated with 660 nm red light (1.0 W) for 5 minutes using a laser (Beijing Honglan Optoelectronics). C. 24 hours later, steps B and D were repeated. For groups ② and ④, anti-PD1 antibody (5 mg / kg) was injected intraperitoneally on the second day after treatment began, and the treatment was repeated 5 times (once every 2 days). E. Tumor growth information and changes in animal weight were collected starting on the second day after treatment. The tumor size was measured using calipers, and the tumor volume V was calculated using the following formula: V = 0.5 × a × b × b (a is length, b is width). When the tumor volume reached 1500 mm²... 3Mice were sacrificed. E. Growth curves were plotted based on the tumor growth data. Tumor volume was measured every two days after treatment of mice in different groups. It can be seen that intratumoral injection of Ce6-Ac4ManNAz-LNT_Raw 264.7 into mice inoculated with 4T1, followed by treatment with 660° red light, effectively inhibited tumor growth. Figure 9 b).

[0098] 1.9 Immune memory relapse

[0099] A tumor model was established. 1×10⁻⁶ tumor cells were subcutaneously injected onto the right back of 7-week-old female BALB / c mice. 6 4T1 cells.

[0100] Groups: ①PBS group (n=7), ②Ce6-Ac4ManNAz-LNT_Raw 264.7 orthotopic injection group (n=8)

[0101] As mentioned above, when the tumor volume reaches 100mm 3 Then, the treatment experiment began: A. In group ②, 25 μL of Ce6-Ac4ManNAz-LNT_Raw 264.7 PBS solution was injected into the tumor, with a cell count of 1.0 × 10⁻⁶ cells. 6 For group ①, 25 μL of PBS solution was injected intratumorally. B. 24 hours later, for group ②, the tumor site was irradiated with 660 nm red light (1.0 W) using a laser (Beijing Honglan Optoelectronics). C. After treatment, 25 μL of PBS solution containing 4T1 (5 × 10⁻⁶) was injected intratumorally. 5 A tumor recurrence model was established in mice in groups ① and ② by subcutaneous injection of PBS solution containing cancer cells into the left side of the tumor. When the tumor volume on the right side reached 1500 mm², a tumor recurrence model was established. 3 Mice were euthanized. E. The tumor-bearing mice were photographed, and the tumors on both sides of groups ① and ② were photographed. Data statistics: All results presented are mean ± standard deviation. Student's t-test was used for statistical analysis. A p-value < 0.05 was considered statistically significant between the experimental and control groups.

[0102] The results showed that mice treated with in situ injection had a significantly stronger ability to prevent tumor recurrence than untreated mice. Figure 10 ).

[0103] 1.10 Flow cytometry immunoassay

[0104] To analyze immune cells in tumors after Ce6-Ac4ManNAz-LNT_Raw 264.7 treatment, tumors were excised from 4T1 tumor-bearing mice on days 3–5. Tumors were weighed and homogenized using a Gentle MACSDissociator, followed by removal of cell clumps through a 100 μm cell filter to obtain a single-cell suspension. The resulting single-cell suspension was blocked with 100 μL (0.25 μg) of anti-CD16 / CD32 (BioLegend) at 4°C for 10 min, then resuspended in 100 μL of cell staining buffer containing 0.25 μg each of anti-CD45-FITC, anti-CD3-APC / Cy7, anti-CD8-PE / Cy7, and anti-CD4-APC, and incubated at 4°C for 20 min. Finally, the cells were permeabilized using the FOXP3 fixation / permeabilization kit (00-5521-00, Invitrogen), then stained with anti-FOXP3-PE (1 μg / test) at 4°C for 30 minutes, and analyzed by flow cytometry (Agilent Novocyte).

[0105] Tumor-bearing mice were injected orally or via the tail vein with Ce6-Ac4ManNAz-LNT_Raw 264.7 and then irradiated. Tumor single-cell suspensions were measured 3-8 days after treatment.

[0106] The results showed that analysis of T cell infiltration in tumors revealed that tail vein injection of LCS followed by 660nm red light irradiation significantly enhanced T cell infiltration into solid tumors and effectively increased CD4 count. + T,CD8 + The method effectively reduces the number of T cells and immunosuppressive Treg cells, strongly suggesting that it can effectively enhance the infiltration and killing of solid tumors by autoimmune cells, thus achieving immunotherapy for tumors. Specifically, FOXP3 represents Treg cells, which are immunosuppressive T cells; this reduction indicates an increase in the killing activity of T cells (see...). Figure 11 ).

[0107] Example 2: Preparation of the acoustic sensitizer Cryo-MC@Ru(bpy)3 and its in vitro and in vivo activity verification

[0108] 2.1 Preparation of sound-sensitive agent

[0109] To prepare a sonicator system with ultrasonic control properties, according to (Zhou et al. Anelectrochemiluminescence amplification strategy: a synergistic effect of electronspun Ru(bpy)3) 2+ A sonicating agent was prepared using the method described in / CNT / ionic liquid composite nanofibers.J.Mater.Chem.C,2014,2,9949–9956. Commercially available Ru bipyridine complexes 1 (201.9 mg, 0.35 mmol, 1 eq) and 2 (152.2 mg, 0.62 mmol, 1.8 eq), along with NaHCO3 (203.4 mg, 2.4 mmol, 7.0 eq), were added to a flask. A mixed solvent of methanol and water (V(MeOH) / V(H2O) = 4 / 1, 40 mL) was added, and the mixture was refluxed for 12 h. The reaction solution was carefully acidified with concentrated sulfuric acid in an ice-water bath to achieve a pH range of 4–5. The mixture was stirred for 2 h in the dark and in an ice-water bath. After filtering to remove the solid, the resulting brown solution of compound 3 was added to an aqueous solution of NaPF6 (2.5 g, 12.5 mL DI H2O), and the mixture was stirred for another 2 h in the dark and in an ice-water bath. The mixture was then centrifuged to obtain solid product 4, which was then lyophilized. Take the lyophilized product 4 (195.5 mg, 0.2 mmol, 1 eq), the dehydrating agent DCC (dicyclohexylcarbodiimide, 232.3 mg, 1.1 mmol, 5.8 eq), and NHS (N-hydroxysuccinimide, 107.4 mg, 1.0 mmol, 4.8 eq), dissolve them in anhydrous DMF (2 mL) in a flask, protect from light, and under nitrogen protection. React for 30 min in an ice-water bath, then react at room temperature for 5 h. Centrifuge, and the supernatant is the desired sound-sensitizing agent 5 solution (see reaction procedure below). Figure 12 Finally, the concentration of the sound-sensitizing agent was determined using the UV-Vis method. Figure 13 ).

[0110] 2.2 Preparation of bone marrow-derived macrophages

[0111] Eight-week-old Balb / c mice were euthanized by cervical dislocation. The bone marrow was removed in a laminar flow hood after sterilization with 75% alcohol. The bone marrow was flushed out with PBS, centrifuged at 300°C for 5 min, and the supernatant was discarded. The cells were resuspended in 5 ml of culture medium (10% FBS, 1% antibiotic-containing DMEM high-glucose medium), and M-CSF (50 ng / ml) was added. The cells were then plated. Mature primary macrophages were obtained after 7 days of incubation.

[0112] 2.3 Preparation of macrophages modified with sonosensitive agent Cryo-MC@Ru(bpy)3

[0113] First, the culture medium was aspirated from the well-grown macrophages, and residual culture medium was washed away with PBS. Then, trypsin was added for 1 min, followed by neutralization with complete culture medium. The adherent cells were then pipetted off, centrifuged at 1000 rpm for 3 min, and washed. The cell pellet was resuspended in PBS to achieve a cell concentration of 5 × 10⁶ cells / mL. 6 / ml. Add Ru(bpy)3 to the cell suspension. 2+ -NHS was added to a concentration of 100 μM and incubated at room temperature for 12 h in the dark. After the reaction was complete, the cells were centrifuged to precipitate, and the cell pellet was resuspended in DMEM high-glucose medium and then rapidly frozen in liquid nitrogen for 18 h to obtain Cryo-MC@Ru(bpy)3.

[0114] 2.4 Characterization of singlet oxygen generation in response to ultrasound by Cryo-MC@Ru(bpy)3.

[0115] ABDA absorbs singlet oxygen. Add 1 x 10⁻⁶ ppm to each quartz cuvette. 7 Cryo-MC@Ru(bpy)3 cells and ABDA (20 μl, 5 mM), total volume 3 ml. Absorbance in the 300-450 nm range was measured using a UV spectrophotometer, and the cells were subjected to ultrasound therapy (EMS Physio) for 5 min (3 w / cm²). 2 Then, the absorbance curve of ABDA was measured again.

[0116] Singlet oxygen fluorescent probe. Add 1x10 to a 48-well plate. 5 / Kong Cryo-MC@Ru(bpy)3, used an ultrasound therapy device (EMS Physio) for 5 minutes (3w / cm) 2 Different ultrasonic intensities were set, and the generation of singlet oxygen was detected using a multifunctional microplate reader. The maximum excitation / emission wavelength was approximately 504 / 525 nm. Additionally, 1 x 10⁻⁶ μL of [unspecified substance] was added to each 48-well plate. 5 / Kong Cryo-MC@Ru(bpy)3, using an ultrasound therapy device (EMS Physio) for ultrasound (3w / cm) 2 The time gradients were set to 5, 10, 20, 30, 60, 120, 240, 360, 480, 600, 900, 1200, and 1800 s. Singlet oxygen generation was detected using a multi-functional microplate reader, with the maximum excitation / emission wavelengths approximately 504 / 525 nm.

[0117] Results: Cryo-MC@Ru(bpy)3 can rapidly generate singlet oxygen under ultrasonic stimulation, indicating that the prepared material has excellent ultrasonic control properties, which can further improve its therapeutic specificity. Figure 14 ).

[0118] 2.5 Flow cytometry test

[0119] Flow cytometry analysis of the surface antibodies for cell recognition and phagocytosis prevention on the membranes of normal and liquid nitrogen-frozen RAW264.7 (MC@Ru(bpy)3 and Cryo-MC@Ru(bpy)3) cells with the same cell number revealed that liquid nitrogen freezing caused less damage to cell surface antibodies, indicating that liquid nitrogen freezing can preserve surface antibodies to a greater extent, thereby retaining their related functions. Figure 15 ).

[0120] 2.6 Cell Culture

[0121] 4T1 tumor cells were obtained from the stem cell bank of the Chinese Academy of Sciences and cultured in Dulbecco's modified 1640 medium containing 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin.

[0122] 2.7 In vitro tumor cell targeting

[0123] Calcein-AM (Sigma, 50 μg / ml) was incubated with 4T1 tumor cells in a Thermo Scientific cell culture incubator at 37°C and 5% CO2 for 30 min, followed by gentle rinsing five times with 2 ml PBS. Cryo-MC@Ru(bpy)3 and 4T1@calcein-AM were incubated with 4T1@calcein-AM in a Thermo Scientific cell culture incubator at 37°C and 5% CO2 for 6 h, followed by gentle rinsing five times with 2 ml PBS. The binding of Cryo-MC@Ru(bpy)3 and 4T1@calcein-AM was observed under a confocal microscope (Olympus).

[0124] It can be seen that the treated material adheres extensively to the target cells, indicating that the low-temperature rapid freezing method can effectively preserve the original cells' ability to recognize cancer cells. Figure 16 ).

[0125] 2.8 In vitro release of inflammatory factors

[0126] The release of inflammatory factors from normal RAW264.7 (MC) cells and modified cells frozen in liquid nitrogen was compared. Supernatants from each group were collected at 6, 12, 24, and 48 hours, and IL-1β, IL-6, IL-10, IL-12, and TNF-α were detected using a double-antibody sandwich ELISA method (n=3). The results show that the modified live cells released a large number of inflammatory factors upon stimulation by the modifying molecules. Direct use of these cells in vivo would cause a systemic inflammatory response, negatively impacting health. However, the rapid freezing method effectively removes these inflammatory factors during the washing process, thus preventing a systemic inflammatory response. Figure 17 ).

[0127] 2.9 In vitro cytotoxicity

[0128] 100 μl of 4T1 cell suspension (cell count 5000) was seeded into 96-well plates, and the plates were pre-cultured at 37°C and 5% CO2 for 6 h. After the 4T1 cells adhered, 10 times the cell count of Cryo-MC@Ru(bpy)3 was added, and the plates were incubated at 37°C and 5% CO2 for 6 h. The cells were then gently washed five times with 2 ml of PBS. Ultrasound was then performed for 5 min (3 w / cm) using an EMS Physio ultrasound therapy device. 2 The plate was incubated at 37°C and 5% CO2 for 3 hours. Afterwards, the supernatant in the original 96-well plate was aspirated, and 100 μl of CCK-8 detection working solution (Cell Counting Kit-8, Dojin, Japan) was added to each well. The plate was then incubated at 37°C and 5% CO2 for 3 hours. The absorbance was measured at 450 nm using a microplate reader. Figure 18 a).

[0129] 2.10 Apoptosis assay

[0130] 2 ml of 4T1 cell suspension (cell count 1 × 10⁻⁶) was seeded into 6-well plates. 6 The culture plates were placed in an incubator and pre-cultured at 37°C and 5% CO2 for 6 hours. After 4T1 cells adhered, 10 times the cell count of Cryo-MC@Ru(bpy)3 was added, and the cells were incubated at 37°C and 5% CO2 for 6 hours. The cells were then gently washed five times with 2 ml of PBS. Ultrasound was then performed for 5 minutes (3 w / cm) using an EMS Physio ultrasound therapy device. 2 ), and incubated at 37℃ and 5% CO2 for 3 hours.

[0131] Flow cytometry detection: (1) Cell collection. Gently blow cells down with 2 ml of 1640 medium, centrifuge at 300 g, 4°C for 5 min to collect cells. (2) Wash cells twice with pre-cooled PBS, centrifuge at 300 g, 4°C for 5 min, 5 × 10⁻⁶ cells / mL. 5(3) Add 10 μL of FITC-Annexin V and 5 μL of PI working solution to each tube. (4) Incubate at room temperature in the dark for 15 min. (5) Add 400 μL of PBS to each tube and detect cell apoptosis as soon as possible using flow cytometry.

[0132] Fluorescence microscopy detection: (1) Gently wash cells with 2 ml of PBS. (2) Add 10 μL of FITC-Annexin V and 5 μL of PI to every 100 μL of Annexin V binding buffer. (3) Add 1 mL of 1× binding buffer to the cells to cover all cells, and incubate at room temperature in the dark for 30 min. (4) Wash cells with 2 ml of 1× binding buffer. (5) Observe cells under a fluorescence microscope using a suitable filter. Use a filter suitable for FITC for FITC-Annexin V, and use Cy3 or Texas channel detection for PI. Figure 19 ).

[0133] 2.11 Cell membrane rupture assay

[0134] LDH release. 100 μl of 4T1 cell suspension (cell count 5000) was seeded into 96-well plates, and the plates were pre-cultured at 37°C and 5% CO2 for 6 h. After 4T1 cells adhered, 10 times the cell count of Cryo-MC@Ru(bpy)3 was added, and the plates were incubated at 37°C and 5% CO2 for 6 h. The cells were then gently washed five times with 2 ml of PBS. Ultrasound was then performed for 5 min (3 w / cm) using an EMSPhysio ultrasound therapy device. 2 The culture was carried out at 37℃ and 5% CO2 for 3 hours. 100 μl of the supernatant from each well was transferred to a new 96-well plate, and 100 μl of Working Solution (Cytotoxicity LDH Assay Kit-WST, Dojin Chemicals, Japan) was added to each well. The plate was protected from light by wrapping it in aluminum foil and reacted at room temperature for 30 minutes. After adding 50 μl of Stop Solution (Cytotoxicity LDH Assay Kit-WST, Dojin Chemicals, Japan) to each well, the absorbance at 490 nm was immediately measured using a microplate reader. Figure 18 b).

[0135] 2.12 MDA Determination

[0136] 2 ml of 4T1 cell suspension (cell count 1 × 10⁻⁶) was seeded into 6-well plates. 6The culture plates were placed in an incubator and pre-cultured at 37℃ and 5% CO2 for 6 hours. After 4T1 cells adhered, 10 times the cell number of Cryo-MC@Ru(bpy)3 was added, and the cells were incubated at 37℃ and 5% CO2 for 6 hours. The cells were then gently washed 5 times with 2 ml of PBS. Ultrasound was performed for 5 minutes (3 w / cm2) using an EMS Physio sonicator, followed by 3 hours of culture at 37℃ and 5% CO2. Lysis was performed using PBS or Beyotime's Western lysis buffer and IP cell lysis buffer (P0013), using 0.1 ml of lysis buffer per million cells. After lysis, the cells were centrifuged at 10,000g-12,000g for 10 minutes, and the supernatant was collected to determine the protein concentration (Quick Start Bradford protein assay, Bio-Rad) for subsequent calculation of MDA content per unit weight of tissue or cell. An appropriate amount of TBA was weighed and prepared into a 0.37% TBA stock solution using TBA preparation solution. Prepare an appropriate amount of fresh MDA detection working solution immediately before testing. The solution includes TBA diluent, TBA stock solution, and an antioxidant. Add 0.1 ml of sample to a 1.5 ml EP tube for measurement, followed by 0.2 ml of MDA detection working solution. Mix well and heat at 100°C or in a boiling water bath for 15 minutes. Cool to room temperature in the water bath and centrifuge at 1000g for 10 minutes at room temperature. Add 200 μL of supernatant to a 96-well plate and measure the absorbance at 532 nm using a microplate reader. After calculating the MDA content in the sample solution, the initial MDA content (μmol / mg protein) can be expressed as the protein content per unit weight. Figure 18 c).

[0137] Results: Untreated 4T1 cells and 4T1+Cryo-MC@Ru(bpy)3 served as controls (n=3). The figures show that Cryo-MC@Ru(bpy)3 itself has little cytotoxicity; only ultrasound stimulation induces significant cell death, and it leads to lipid peroxidation-induced membrane rupture and cell death in 4T1 cells. This indicates that Cryo-MC@Ru(bpy)3 has a good and controllable cancer cell killing effect. Figure 18 ).

[0138] 2.13 In vivo targeting capability.

[0139] Cryo-MC@Ru(bpy)3@DiI was prepared. Macrophages were first incubated with the membrane dye DiI (1 mM) at 37°C and 5% CO2 for 90 min, followed by gentle washing five times with 2 ml PBS. The prepared Ru(bpy)3 was then added to mature primary macrophages and co-cultured for 12 h to prepare macrophage membrane-coated Ru(bpy)3 (CryoMC@Ru(bpy)3@DiI).

[0140] A tumor model was established. 1×10⁻⁶ tumor cells were subcutaneously injected into the right shoulder of 7-week-old female BALB / c mice. 6 4T1 cells.

[0141] In vivo targeted therapy. Waiting until the tumor reaches a size of 150mm. 3 Subsequently, 100 μl of 2×10⁻⁶ solution was injected via the tail vein. 6 Cryo-MC@Ru(bpy)3@DiI was used to extract heart, liver, spleen, lung, kidney, and tumor tissue from mice after 6h, 12h, 24h, 48h, 72h, and 96h. The fluorescence intensity of DiI was detected using a small animal imaging system.

[0142] Results: Following tail vein injection of Cryo-MC@Ru(bpy)3@DiI, the fluorescence intensity of DiI was measured in the heart, liver, spleen, lung, kidney, and tumor of mice at 6, 12, 24, 48, 72, 96, and 120 hours (n=3). The colors are: red (tumor), green (liver), orange (lung), blue (kidney), purple (heart), and black (spleen). The graph shows that Cryo-MC@Ru(bpy)3 remained abundantly in the tumor site after 96 hours, with very little deposition in other organs, indicating good in vivo circulation and tumor-specific targeting. Figure 20 ).

[0143] 2.14 In vivo antitumor efficacy determination.

[0144] A tumor model was established. 1×10⁻⁶ tumor cells were subcutaneously injected into the right shoulder of 7-week-old female BALB / c mice. 6 4T1 cells.

[0145] Groups: ① Cryo-MC@Ru(bpy)3 + ultrasound therapy group (n=6), ② Cryo-MC@Ru(bpy)3 group (n=6), ③ PBS group (n=6)

[0146] Treatment. As mentioned above, when the tumor volume reaches 50mm... 3 Then, the treatment experiment began: A. For groups ① and ②, 100 μL of Cryo-MC@Ru(bpy)3 in PBS solution was injected via the tail vein, with a cell mass of 1 × 10⁻⁶ cells.7 For group ③, 100 μL of PBS solution was injected via the tail vein. B. 24 hours later, group ① underwent ultrasound therapy (EMS Physio) for 15 minutes (1.5 w / cm). 2 C. After another 24 hours, repeat the procedure in B. After another 24 hours, begin the second treatment cycle. D. On the third day after the start of treatment, collect tumor growth information and changes in the animal's weight. Measure the tumor size using calipers and calculate the tumor volume V using the following formula: V = 0.5 × a × b × b (a is the length, b is the width). When the tumor volume reaches 1000 mm²... 3 At that time, the mice were euthanized. E. Based on the tumor growth data above, a growth curve was plotted.

[0147] Statistics: All results are presented as mean ± standard deviation. Student's t-test was used for statistical analysis. A p-value < 0.05 was considered statistically significant between the experimental and control groups.

[0148] Results: After treatment of mice in different groups, tumor volume and body weight were measured every 2 days (n=6). The figures show that injecting Cryo-MC@Ru(bpy)3 into mice inoculated with 4T1 followed by ultrasound treatment effectively inhibited tumor growth, and monitoring of mouse body weight indicated that it caused no significant harm to the mice. Figure 21 ).

[0149] 2.15 Flow cytometry immunoassay

[0150] To analyze the immune cell infiltration in tumors after Cryo-MC@Ru(bpy)3+ ultrasound therapy, tumors were excised from 4T1 tumor-bearing mice on days 11-16. Tumors were weighed and ground using a GentleMACS Dissociator, followed by removal of cell clumps through a 100 μm cell filter to obtain a single-cell suspension. The obtained single-cell suspension was blocked with 100 μL (0.25 μg) of anti-CD16 / CD32 (BioLegend) at 4°C for 10 minutes, then resuspended in 100 μL of cell staining buffer containing 0.25 μg each of anti-CD45-FITC, anti-CD3-APC / Cy7, anti-CD8-PE / Cy7, and anti-CD4-APC, and incubated at 4°C for 20 minutes. Finally, the cells were permeabilized using the FOXP3 fixation / permeabilization kit (00-5521-00, Invitrogen), then stained with anti-FOXP3–PE (1 μg / test) at 4°C for 30 minutes, and analyzed by flow cytometry (Agilent Novocyte).

[0151] Results: After treatment of mice in different groups, tumor tissue was collected on day 11, prepared into single-cell suspensions, and subjected to flow cytometry analysis of its immune cells. The results showed that in the ultrasound-mediated sonosensitive agent-induced membrane rupture group, CD3+ was significantly higher in the tumor tissue. + T cells were significantly increased. Specifically, compared to the PBS group and the non-ultrasound-mediated sonositizer group, the ultrasound-mediated sonositizer group showed a significant increase in helper CD4+ cells in tumor tissue. + T cells, and cytotoxic CD8 + The number of T cells increased significantly. Simultaneously, analysis showed a significant decrease in the proportion of regulatory T cells (Tregs) in the tumor tissue of the ultrasound-mediated sonosensitive agent group. These results indicate that lipid peroxidation and cell membrane rupture caused by ROS generated by the ultrasound-controlled sonosensitive agent induce a strong immune response. Figure 22 ).

Claims

1. A complex or drug delivery system, characterized in that, The complex or drug delivery system is Cryo-MC@Ru(bpy)3, and the preparation method is as follows: well-grown macrophages are sucked to remove culture medium, residual culture medium is washed away by adding PBS, trypsin is added to digest for 1 min, complete culture medium is added to neutralize the trypsin, adherent cells are blown down by a gun, centrifugal washing is carried out at 1000 rpm for 3 min, and the cell precipitate is resuspended in PBS to make the cell concentration 5×10 6 / ml; Ru(bpy)3 2+ -NHS is added to the cell suspension to make the concentration 100 μM, and incubation is carried out at room temperature under dark conditions for 12 h; after the reaction is completed, the cells are centrifuged and precipitated, the cell precipitate is resuspended in DMEM high-sugar culture medium, and is rapidly frozen in liquid nitrogen for 18 h, so that Cryo-MC@Ru(bpy)3 is obtained.

2. A complex or drug delivery system, characterized in that, The complex or drug delivery system is LCS, and a preparation method thereof is as follows: first, well-grown RAW 264.7 cells are added into 125 μM Ac4ManNAz DMEM complete culture medium, and cultured for 72 h; after the Ac4ManNAz_Raw 264.7 culture is completed, the cells are blown from the bottom of a dish, and finally resuspended with PBS, so that the cell concentration is 5x10 6 / ml; second, alkynyl-Ce6 is added into the cell suspension, so that the concentration is 20 μg / m, and 1:1 ascorbic acid and CuSO4 are added, so that the concentration is 0.2 μM, and incubated for 1 h at room temperature in dark; under the catalysis of copper ions, click reaction occurs between alkynyl-Ce6 and the azido group of Ac4ManNAz, so that the RAW 264.7 cell membrane surface is finally provided with Ce6; the cells provided with Ce6 are centrifuged, the supernatant is removed, and then washed twice with PBS; the finally obtained cell precipitate is resuspended with serum-free DMEM culture medium, the cell density is 1x10 7 / ml, and then loaded into a cryopreservation tube, and rapidly frozen in liquid nitrogen for 12 h or more, and thus LCS is obtained.

3. Use of the complex or drug delivery system of claim 1 or 2, alone or in combination with a PD1 antibody, for the manufacture of a medicament for the treatment of breast cancer.

4. A method of preparing a composite or drug delivery system, characterized by, The complex or drug delivery system is Cryo-MC@Ru(bpy)3, and the preparation method is as follows: well-grown macrophages are sucked to remove culture medium, residual culture medium is washed away by adding PBS, trypsin is added to digest for 1 min, complete culture medium is added to neutralize the trypsin, adherent cells are blown down by a gun, centrifugal washing is carried out at 1000 rpm for 3 min, and the cell precipitate is resuspended in PBS to make the cell concentration 5×10 6 / ml; Ru(bpy)3 2+ -NHS is added to the cell suspension to make the concentration 100 μM, and incubation is carried out at room temperature under dark conditions for 12 h; after the reaction is completed, the cells are centrifuged and precipitated, the cell precipitate is resuspended in DMEM high-sugar culture medium, and is rapidly frozen in liquid nitrogen for 18 h, so that Cryo-MC@Ru(bpy)3 is obtained.

5. A method of preparing a composite or drug delivery system, characterized by, The complex or drug delivery system is LCS, and a preparation method thereof is as follows: first, well-grown RAW 264.7 cells are added into 125 μM Ac4ManNAz DMEM complete culture medium, and cultured for 72 h; after the Ac4ManNAz_Raw 264.7 culture is completed, the cells are blown from the bottom of a dish, and finally resuspended with PBS, so that the cell concentration is 5x10 6 / ml; second, alkynyl-Ce6 is added into the cell suspension, so that the concentration is 20 μg / m, and 1:1 ascorbic acid and CuSO4 are added, so that the concentration is 0.2 μM, and incubated for 1 h at room temperature in dark; under the catalysis of copper ions, click reaction occurs between alkynyl-Ce6 and the azido group of Ac4ManNAz, so that the RAW 264.7 cell membrane surface is finally provided with Ce6; the cells provided with Ce6 are centrifuged, the supernatant is removed, and then washed twice with PBS; the finally obtained cell precipitate is resuspended with serum-free DMEM culture medium, the cell density is 1x10 7 / ml, and then loaded into a cryopreservation tube, and rapidly frozen in liquid nitrogen for 12 h or more, and thus LCS is obtained.

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