Polypeptide self-assembly nano diagnosis and treatment preparation based on NIR-II image and preparation method of polypeptide self-assembly nano diagnosis and treatment preparation

Abstract

The invention discloses a polypeptide self-assembled nano diagnosis and treatment preparation based on an NIR-II image and a preparation method thereof, a brand-new aggregation-induced emission luminophor TPA-Se based on a selenodiazole structure is prepared, the TPA-Se is used as a fluorescent probe and is combined with a mitochondrial targeting prodrug LND-1-PEG-24, and the preparation method of the polypeptide self-assembled nano diagnosis and treatment preparation based on the NIR-II image is used for preparing a mitochondrial targeting drug. The invention relates to an image navigation-immunotherapy-tumor microenvironment remodeling synergistic nano system constructed by coating an immune cell membrane. According to the invention, not only are tumor site specific targeting enrichment and accurate biological imaging of the preparation realized, but also targeted release of chemotherapeutic drugs in a tumor area can be realized to accelerate pyroptosis of tumor cells, and a photo-thermal treatment physical killing effect and a chemical treatment biological killing effect are synchronously realized; and a new scheme is provided for realizing accurate imaging-guided obvious killing of tumor cells and promotion of pyroptosis of the tumor cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical engineering and nanomedicine, and particularly relates to a nano diagnosis and treatment system based on polypeptide self-assembly technology, which has NIR-II fluorescence imaging function and can realize pyroptosis treatment and immune activation effect in cooperation, and is suitable for precise diagnosis and efficient treatment of tumors. BACKGROUND

[0002] Cancer is a disease that seriously threatens human life and health, and has become the focus of global medical research in recent years. With the progress of science and technology, the treatment methods for cancer are increasingly diversified, and the survival period of cancer patients has been significantly prolonged. However, traditional cancer treatment methods such as surgery, chemotherapy and radiotherapy still face many insurmountable bottlenecks. Surgical treatment is often difficult to eradicate lesions that have occurred micro-metastasis, and its application is limited by the particularity of the location of the tumor; radiotherapy has problems such as dose-limiting toxicity and tumor radiation resistance, which affect the efficacy; traditional small molecule chemotherapy drugs such as doxorubicin and cisplatin usually lack targeting, and cannot effectively distinguish between cancer cells and normal cells, thereby causing serious damage to rapidly proliferating normal tissues.

[0003] Although these traditional methods can inhibit tumor growth to some extent, due to the heterogeneity of tumors, metastasis and the complexity of the tumor microenvironment, the effect of a single treatment mode varies greatly, and it is often difficult to achieve complete cure.

[0004] Unlike normal tissues, the tumor microenvironment (TME) in which most solid tumors are located has a series of unique characteristics. Scientific research has confirmed that the expression level of cathepsin B in tumor cells is significantly increased, and this protease plays a key role in various malignant processes such as tumor metastasis, angiogenesis and treatment resistance, and is a major factor leading to the failure of many therapies. Therefore, the unique high cathepsin B characteristic exhibited by tumor cells also makes it a highly potential molecular target, providing an important opportunity for the development of new targeted cancer treatment strategies.

[0005] To achieve precision medicine, cathepsin B-responsive prodrugs have been developed as a promising treatment strategy. Such prodrugs can remain stable and non-toxic in healthy tissues where cathepsin B levels are normal; however, in tumor areas where cathepsin B is overexpressed, they are specifically activated and converted into cytotoxic compounds, thereby achieving selective release and killing effect of drugs at tumor sites.

[0006] However, the clinical application of this strategy faces a major obstacle, which is that the distribution of cathepsin B in the tumor microenvironment is usually highly heterogeneous. This uneven distribution hinders the effective and rapid activation of prodrugs throughout the tumor, resulting in insufficient activated drug concentration. Therefore, single chemotherapy drugs that simply rely on cathepsin B activation mechanisms often fail to achieve the desired anti-tumor effect.

[0007] With the increasing demand for cancer treatment, optical therapy (including photothermal therapy and photodynamic therapy) as a treatment method with the advantages of non-invasiveness, high spatiotemporal accuracy, and low systemic toxicity, has shown great potential for clinical translation and has been widely used in the biomedical field. Among them, the combination of phototherapy and chemotherapy plays an increasingly important role in overcoming the limitations of single therapy and significantly improving anti-tumor effects. Phototherapy can effectively induce cancer cell apoptosis or necrosis through local irradiation of tumors with specific wavelength lasers; while chemotherapy drugs can enhance the overall treatment response and eliminate residual or metastatic lesions. This combined strategy not only improves the accuracy of treatment, but also helps to reverse or delay the occurrence of tumor drug resistance.

[0008] The combination of phototherapy and nanotechnology has also become a research hotspot. The development of nanotechnology provides new opportunities for cancer treatment, especially the construction of nanodiagnostic and therapeutic systems, which provides a new effective strategy for clinical treatment. By integrating different functions into nanoparticles, targeted diagnosis and treatment can be achieved, further improving the accuracy and effectiveness of treatment. Using nanocarriers, drugs can be precisely delivered to tumor sites, minimizing damage to normal tissues (High-Drug-Loading Mesoporous Silica Nanorods with Reduced Toxicity for Precise Cancer Therapy against Nasopharyngeal Carcinoma [J]. Advanced Functional Materials , 2017, 27: 1703313.;Photothermal Conversion and Transferin Photothermal Therapy: From Macroscale to Nanoscale [J]. Advances in Colloid and Interface Science . 2022, 308: 102753.;Dilute solution routes tovarious controllable morphologies of MCM-41 silica with a basic medium [J].Chemistry of materials , 2001, 13(2): 258-263.). The development of efficient and safe nanotherapeutic agents is of vital importance for improving the level of tumor diagnosis and treatment and promoting the development of cancer treatment.

[0009] Therefore, how to cleverly utilize the specific characteristic of high expression of cathepsin B in the tumor microenvironment and integrate the unique advantages of photothermal therapy to design and construct a novel multifunctional diagnostic and therapeutic nanoplatform is a key technical problem that urgently needs to be solved.

[0010] High-performance diagnostic and therapeutic systems are key to achieving precise imaging-guided cancer immunotherapy. Traditional organic photosensitive materials are constrained by rigid planar π-conjugated frameworks, and in practical applications, they are often limited by the aggregation-induced fluorescence quenching (ACQ) effect. They can exhibit bright fluorescence emission in dilute solutions (non-aggregated state), but when they are at high concentrations or in unfavorable solvent environments and aggregate, the fluorescence intensity decreases sharply or is even completely quenched (Cd45-Pet Is a Robust, Non-Invasive Tool for Imaging Inflammation [J]). Nature . 2025, 639 (8053): 214-224.), which severely limits further improvements in its imaging quality and treatment efficiency.

[0011] To overcome the limitations of the ACQ effect, the team led by Academician Benzhong Tang of the Hong Kong University of Science and Technology pioneered the report of aggregation-induced emission (AIE) in 2001, as evidenced by their review article published in Advanced Materials (Aggregation-Induced Emission: The Whole Is More Brilliant Than the Parts [J]). Advanced Materials . 2014, 26(31): 5429-5479.) This paper explores the latest developments in the AIE phenomenon and its applications in different fields.

[0012] The emergence of AIE materials offers a revolutionary solution to fundamentally overcome the challenges of traditional photosensitizer ACQ. Developing novel AIE probes (AIEgens) that combine excellent optical properties, high reactive oxygen species yield, and good biocompatibility is key to advancing multimodal imaging-guided synergistic therapy, especially photoimmunotherapy. AIEgens-based diagnostic and therapeutic systems hold the promise of simultaneously achieving high-resolution tumor imaging, precise photodynamic / photothermal therapy, and subsequently activating anti-tumor immune responses through inducing immunogenic cell death (ICD), ultimately resulting in highly efficient synergistic cancer immunotherapy.

[0013] In the construction process of AIEgens, nano-precipitation method is the mainstream technology at present, which encapsulates hydrophobic AIE molecules by amphiphilic polymers such as DSPE-PEG and Pluronic F-127 to form nanoparticles with core-shell structure (Blood Circulation Assessment by Steadily Fluorescent near-Infrared-IiAggregation-Induced Emission Nano Contrast Agents [J. ACS Nano . 2023, 17(19): 19265-19274.). However, the nanoparticles prepared by traditional nano-precipitation method have defects such as residual motion of encapsulated molecules, dynamic leakage of surfactants, and limited drug loading capacity, which restricts the further improvement of probe fluorescence brightness and phototherapy efficiency. SUMMARY

[0014] The purpose of the present application is to provide a polypeptide self-assembled nano-diagnosis and treatment integrated preparation based on NIR-II image guided pyroptosis-immune synergistic therapy by specifically combining AIEgens with selenadiazole structure and cathepsin B responsive drug-loaded polypeptide nanoparticles, and a preparation method of the preparation.

[0015] To achieve the above-mentioned purpose of the application, the present application first provides a new aggregation-induced emission luminophore (AIEgen), which has a structure as shown in structural formula (I) with selenadiazole as the core functional unit:

[0016] The AIEgen is referred to as TPA-Se, and its chemical name is 4-hexyloxy-N-4-hexyloxyphenyl-N-4-7-tributylstannyl-2,3-dihydrothiophene[3,4-b][1,4]dioxane-5-ylphenylphenylamine-4,8-dibromo[1,2,5]selenadiazolo[3,4-f]benzo[c][1,2,5]thiadiazole, which is a dark green solid.

[0017] The TPA-Se described in the application adopts triphenylamine as an electron donor (D) unit, selenadiazolobenzothiadiazole as an acceptor (A) unit, forms a D-A-D type architecture, and presents a highly twisted geometric structure. The three-dimensional topological configuration effectively inhibits intermolecular π-π stacking through a steric hindrance effect, while maintaining the structural integrity of the intramolecular conjugated network, thereby exhibiting typical aggregation-induced emission (AIE) characteristics; in addition, the electron band gap of TPA-Se is 1.15 eV, which can respond to long-wavelength near-infrared two-region (NIR-II) laser, and has excellent light-heat conversion performance under 808 nm laser irradiation, and is suitable for deep tissue imaging and treatment.

[0018] The TPA-Se described in the application can be prepared by a standard Stille reaction (Stille coupling reaction), which is a cross-coupling reaction of a tributyl tin-containing donor unit and a bromine-containing halogenated aromatic hydrocarbon acceptor unit under the action of a palladium catalyst, and belongs to a mature and reliable conventional synthesis method in organic synthesis.

[0019] To achieve the above-mentioned application purposes, the application further constructs a pyroptosis-immune synergistic treatment polypeptide self-assembled nano-diagnosis and treatment integrated preparation based on near-infrared two-region (NIR-II) image guidance and cathepsin B activation, with the above-mentioned aggregation-induced emission light emitter (AIEgen) as the core.

[0020] Specifically, the nano-diagnosis and treatment integrated preparation described in the application is an image navigation-immunotherapy-tumor microenvironment remodeling synergistic nanosystem constructed by coating an immune cell membrane after combining the aggregation-induced emission light emitter TPA-Se as a fluorescent probe with the mitochondria-targeting prodrug LND-1-PEG-24.

[0021] Therefore, the nano-diagnosis and treatment integrated preparation described in the application is composed of three components:

[0022] 1) The aggregation-induced emission light emitter TPA-Se is a NIR-II imaging guide and a photothermal therapeutic agent.

[0023] 2) The mitochondria-targeting prodrug LND-1-PEG-24 is an intelligent response unit, which is connected to a hydrophilic group PEG at the C-terminal of the polypeptide through an amidation reaction 24 , and a hydrophobic antitumor drug lonidamide at the N-terminal, and the drug-loaded polypeptide nanocarrier constructed therefrom has the following structural formula: LND-Phe-Phe-Arg-Phe-Lys-Gly-Phe-Leu-Gly-PEG 24 .

[0024] The more specific structural formula of the mitochondria-targeting prodrug LND-1-PEG-24 is as follows:

[0025] Due to the drug-loaded polypeptide nanocarrier is connected with the enzyme-responsive linker and polyethylene glycol chain which can be cut by cathepsin B on the antitumor drug lonidamide, it has cathepsin B responsiveness, and after entering the tumor microenvironment, it can be specifically cut by the high expression of cathepsin B in tumor cells into a hydrophobic fragment LND-Phe-Phe-Arg-Phe-Lys (LND-1) and a hydrophilic fragment Gly-Phe-Leu-Gly-PEG 24 Two parts, the LND-1 part can specifically target mitochondria through the mitochondria-targeting peptide sequence Phe-Arg-Phe-Lys connected on the antitumor drug lonidamide (LND), thereby accelerating the release of pyroptosis nanoinductor LND and realizing on-demand release and precise activation of the drug.

[0026] Among them, the antitumor drug lonidamide is named Lonidamine in English, the chemical name is 1-[(2,4-dichlorophenyl) methyl]-1H-indazole-3-carboxylic acid, the molecular formula is C 15 H 10 Cl2N2O2.

[0027] 3) The immune cell membrane is used as an immune targeting camouflage component to coat the above-mentioned TPA-Se and LND-1-PEG-24 core components, so as to endow the nanodiagnosis and treatment integrated preparation with long blood circulation time, good immune escape ability and active targeting property to tumor tissues.

[0028] Further, the immune cell membrane in the application is preferably M1 macrophage membrane.

[0029] In order to achieve the above-mentioned application purposes, the application further provides a preparation method of the polypeptide self-assembled nanodiagnosis and treatment preparation based on NIR-II image, which specifically comprises:

[0030] 1) The organic phase containing the mitochondria-targeting prodrug LND-1-PEG-24 and the aggregation-induced emissive luminophore TPA-Se is injected into the water phase to form a nanometer particle dispersion liquid by using the nanometer precipitation method, and after removing the organic solvent, the drug-loaded polypeptide nanoparticles ALNPs dispersion liquid is prepared;

[0031] 2) The drug-loaded polypeptide nanoparticles ALNPs dispersion liquid is mixed with the immune cell membrane, and the immune cell membrane is coated on the surface of the drug-loaded polypeptide nanoparticles ALNPs by ultrasonic co-cultivation to form the polypeptide self-assembled nanodiagnosis and treatment preparation M1-ALNPs.

[0032] Further, the preparation method can further include extruding and homogenizing the formed polypeptide self-assembled nanodiagnosis and treatment preparation.

[0033] More specifically, the obtained polypeptide self-assembled nanodiagnostic and nanotherapeutic preparation M1-ALNPs is subjected to multiple extrusion treatments through polycarbonate membranes with different pore sizes in sequence to obtain M1-ALNPs with uniform particle size and stable structure.

[0034] Further preferably, the organic solvent used by the present application to form the organic phase is tetrahydrofuran. After the organic phase is injected into the aqueous phase to form a nanoparticle dispersion, the present application removes the organic solvent in the nanoparticle dispersion by stirring overnight and concentrates the dispersion by centrifugal filtration.

[0035] Further, in the preparation method of the polypeptide self-assembled nanodiagnostic and nanotherapeutic preparation, the mass ratio of the mitochondrial-targeting prodrug LND-1-PEG-24 to the aggregation-induced emissive luminophore TPA-Se is preferably 4-6:1, and more preferably 5:1; the mass ratio of the drug-loaded polypeptide nanoparticle ALNPs dispersion to the immune cell membrane is preferably 1:1.5-2.5, and more preferably 1:2; and the volume ratio of the organic phase to the aqueous phase is preferably 1:8-12, and more preferably 1:10.

[0036] The polypeptide self-assembled nanodiagnostic and nanotherapeutic preparation based on NIR-II imaging utilizes the natural difference between tumor tissues and normal tissues, and proposes a triple combination treatment strategy of "NIR-II imaging + mitochondrial-targeting pyroptosis + immune activation", which has clear multimodal diagnosis and treatment integration characteristics and multiple technical effects.

[0037] Precise diagnosis: thanks to the camouflage function of the immune cell membrane and the response to cathepsin B, the preparation achieves tumor site-specific targeting enrichment and accurate bioimaging, and the NIR-II fluorescence of TPA-Se in the body can realize real-time imaging with high resolution and high signal-to-noise ratio, which helps to sensitively delineate the tumor area and provide accurate guidance for tumor treatment.

[0038] On-demand drug release: in the tumor area, cathepsin B specifically cleaves LND-1-PEG24, changes its structure, realizes targeted release of the chemotherapeutic drug, and reduces systemic toxicity; the specific activation of the prodrug accelerates the release of the pyroptosis inducer LND, improving the effect of anti-tumor treatment.

[0039] Synergistic treatment: under the external laser remote control, the efficient photothermal treatment physical killing effect of TPA-Se directly kills tumor cells, while the released LND molecules play a chemical treatment biological killing effect to effectively induce pyroptosis of tumor cells, and the two produce a strong synergistic anti-tumor effect, promoting tumor cell apoptosis and inhibiting tumor growth, realizing efficient and precise diagnosis and treatment integration of complex tumors.

[0040] Immune activation: pyroptosis belongs to a kind of inflammatory programmed cell death, which can release a large number of tumor associated antigens and inflammatory factors, significantly activate the anti-tumor immune response, and through the synergistic effect with the immune regulation function of the cell membrane, a strong immunotherapy effect can be achieved.

[0041] Therefore, the nano diagnosis and treatment preparation of the application proposes a new diagnosis and treatment strategy, which utilizes the cathepsin B characteristics of the tumor microenvironment and the advantages of photothermal therapy, so that the preparation can not only realize accurate biological imaging and real-time tumor diagnosis, but also can exert strong anti-tumor effect through photothermal and chemotherapy, and realizes the conversion of cathepsin B activated pyroptosis nano inducer, which provides a new scheme for realizing accurate imaging guided tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of the aggregation-induced emission luminophore TPA-Se.

[0043] Figure 2 is the nuclear magnetic resonance carbon spectrum of the aggregation-induced emission luminophore TPA-Se.

[0044] Figure 3 is the high-resolution mass spectrum of the aggregation-induced emission luminophore TPA-Se.

[0045] Figure 4 is the spectral property characterization comparison diagram of TPA-Se and ANPs.

[0046] Figure 5 is the photoluminescence property change trend of TPA-Se under different aggregation states.

[0047] Figure 6 is the photothermal response performance determination result of TPA-Se.

[0048] Figure 7 is the molecular optical mechanism diagram of TPA-Se.

[0049] Figure 8 is the synthesis flow chart of the drug-loaded polypeptide nanocarrier LND-1-PEG24.

[0050] Figure 9 is the CTSB enzymatic reaction result of LND-1-PEG-24.

[0051] Figure 10 is the mitochondrial targeting verification diagram of LND-1-PEG-24.

[0052] Figure 11 is the DLS and TEM diagram of ALNPs and M1-ALNPs.

[0053] Figure 12is the colloidal stability plot of M1-ALNPs.

[0054] Figure 13 is the in vitro hemolysis experiment result of M1-ALNPs composite nanoparticles.

[0055] Figure 14 is the cellular uptake and in vitro targeting experiment result of ALNPs and M1-ALNPs.

[0056] Figure 15 is the bio-TEM images of 4T1 cells treated with M1-ALNPs for different time periods.

[0057] Figure 16 is the heatmap of M1-ALNPs treated RAW264.7 cells for specific genes related to M1 / M2 phenotypes and KEGG enrichment analysis plot.

[0058] Figure 17 is the western blotting plot of 4T1 cells treated with different nanoparticles for specific proteins related to MAPK signaling pathway and the quantification plot of inflammatory cytokines.

[0059] Figure 18 is the in vitro cytotoxicity assay result of 4T1 tumor cells treated with different nanoparticles.

[0060] Figure 19 is the analysis plot of the results of plate colony formation experiment.

[0061] Figure 20 is the bright field view images of in vitro pyroptosis detection of 4T1 cells after different treatments.

[0062] Figure 21 is the morphological features of 4T1 cells treated with M1-ALNPs+NIR at different time points.

[0063] Figure 22 is the expression of pyroptosis related proteins (Caspase-3, Cleaved caspase-3, GSDME-FL and GSDME-N) in 4T1 cells after different treatments detected by western blotting.

[0064] Figure 23 is the lactate secretion detection result of 4T1 cells after different treatments.

[0065] Figure 24 is the LDH release and cell ATP level of 4T1 cells after different treatments.

[0066] Figure 25 is the CLSM images of mitochondrial membrane potential changes and quantification analysis of membrane potential changes of 4T1 cells after different treatments.

[0067] Figure 26are the results of the mitochondrial permeability transition pore assay of 4T1 cells after different treatments.

[0068] Figure 27 are the CLSM images and quantification of HMGB1 release of 4T1 cells after different treatments.

[0069] Figure 28 are the CLSM images and quantification of etco-CRT expression on the surface of 4T1 cells after different treatments.

[0070] Figure 29 are the results of flow cytometry analysis of BMDCs maturation in vitro.

[0071] Figure 30 are the images of NIR-II imaging ability of ALNPs and M1-ALNPs at the tumor site of mice.

[0072] Figure 31 are the images of photothermal effect of ALNPs and M1-ALNPs at the tumor site of mice in vivo.

[0073] Figure 32 are the images of fluorescence imaging and quantification of fluorescence intensity of the internal organs and tumors of mice ex vivo after injection of ALNPs and M1-ALNPs.

[0074] Figure 33 are the results of comparison of anti-tumor effects of M1-ALNPs and different treatments on 4T1 tumor-bearing mice.

[0075] Figure 34 are the tumor images, H&E, TUNEL and Cleaved-caspase-3 staining results of tumor sections of 4T1 tumor-bearing mice treated with M1-ALNPs and different treatments.

[0076] Figure 35 are the results of anti-metastasis experiments of M1-ALNPs in situ mouse models.

[0077] Figure 36 are the results of in vivo biological safety experiments of M1-ALNPs. Embodiments

[0078] The embodiments of the present application will be described in detail below with reference to the accompanying drawings and specific examples. It should be noted that the examples are for illustrative purposes only, and are intended to provide a thorough understanding of the technical solutions of the present application and to guide those skilled in the art to implement and apply the present application. It should be understood that these descriptions do not constitute any limitation on the scope of protection of the present application.

[0079] Unless otherwise expressly stated, in embodiments of the present application, the production processes, experiments, detection or analysis methods involved are considered to be conventional methods known to those skilled in the art, which only need to be carried out according to conventional conditions or relevant product instructions, and the steps and names involved are also generally clear and unambiguous in the art.

[0080] The instruments, equipment, raw materials, reagents or samples used in the examples do not have special restrictions on the source, and are conventional products that can be purchased through normal commercial channels or prepared by known methods, and the source does not substantially affect the implementation results of the present application.

[0081] The scientific and technical terms used in the present application, unless otherwise expressly defined, have the meanings generally understood by those skilled in the art of the technology described in the present application. If there is a conflict, the definition in the specification shall prevail.

[0082] The terms "include", "contain", "have" and the like used in the present application should be understood as open, i.e. "including but not limited to". The term "and / or" includes any and all combinations of one or more associated listed items. The quantity terms such as "one", "a" and the like do not exclude the case of multiple; "multiple" or "plurality" means greater than or equal to two in number.

[0083] The terms "preferably", "more preferably", "exemplary" and the like used in the present application are only used to describe a particular solution or effect, and are not necessary limitations on the solution or limitations on the protection scope.

[0084] The present application relates to the description of numerical parameters (such as quantity, concentration, temperature, time, etc.), which should be understood to have a reasonable deviation caused by measuring instruments, operation errors, statistical fluctuations, etc. The range of this deviation should be within the limits acceptable by those skilled in the art according to common sense.

[0085] Term explanation:

[0086] Near-infrared two-region fluorescence imaging (NIR-II FI): a fluorescence imaging technology based on the near-infrared light region, with a wavelength range of about 1000-1700 nm. In this wavelength range, the light scattering and absorption of biological tissues are low, and the tissue depth penetration is good, which is suitable for in vivo imaging. NIR-II FI usually uses fluorescent probes or fluorescent markers to specifically bind to tumor tissues or to label specific cells or molecules. When these fluorescent probes or markers are excited, they will emit NIR-II fluorescence signals, which are captured and recorded by imaging equipment. Near-infrared two-region fluorescence imaging has a wide range of applications in the biomedical field, including tumor diagnosis, tumor microenvironment research, tumor treatment monitoring, etc.

[0087] Tumor microenvironment (TME): The surrounding microenvironment in which tumor cells exist, including surrounding blood vessels, immune cells, fibroblasts, myeloid-derived inflammatory cells, various signal molecules and extracellular matrix. In solid tumors, due to the rapid growth of tumor tissue, and the high swelling and incomplete vascular system inside the tumor tissue, it will cause insufficient oxygen supply in the tumor tissue, and the tumor microenvironment presents the characteristics of overall hypoxia. Due to insufficient oxygen supply, tumor cells can only undergo anaerobic glycolysis for energy metabolism, which will cause lactic acid accumulation; at the same time, the ion exchange protein on the tumor cell membrane also continuously transports H + + ions inside the cell to the outside of the cell to avoid causing its own acidosis. These cellular responses also cause the pH of the tumor microenvironment to decrease to varying degrees, and the overall environment presents an acidic environment. Under the hypoxic and acidic microenvironment of tumor occurrence and development, a large number of apoptosis of tumor tissue and peripheral tissue cells will occur, releasing cell fragments and chemotactic factors, leading to inflammatory cell infiltration and inflammatory factor secretion. At the same time, the development of tumors itself will also trigger an immune response of the immune system, causing inflammatory cells to accumulate in the area, triggering a severe inflammatory response.

[0088] Stille reaction: also known as Stille coupling reaction, Stille coupling reaction, is the cross-coupling reaction of organotin compounds with halogenated hydrocarbons without β-hydrogen under palladium catalysis. The reaction is generally carried out in a solvent free of water and oxygen and an inert environment. The addition of equimolar Cu(I) or Mn(II) salt can improve the specificity and reaction rate of the reaction. However, oxygen will cause the oxidation of the palladium catalyst and the self-coupling of the organotin compound. Tetra(triphenylphosphine) palladium is the most commonly used palladium catalyst, and other catalysts include PdCl2(PPh3)2, PdCl2(MeCN)2, etc. The halogenated hydrocarbon used is generally vinyl or aryl trifluoromethanesulfonate or chloro, bromo, iodo hydrocarbon. Examples

[0089] Example 1

[0090]

[0091] Weigh 4-iodophenol (5 g, 22.73 mmol), 1-bromohexane (4.83 g, 25 mmol) and potassium carbonate (2.07 g, 15 mmol) into a 250 mL two-necked round-bottom flask, add 50 mL DMF, vacuum pump to dry the air in the flask, then fill with argon to protect, repeat this operation three times, then heat the mixture to 65°C and reflux for 24 h.

[0092] After the reaction liquid is cooled to room temperature, quench the reaction with water, extract with dichloromethane for 3 times, combine the organic phase, and dehydrate with anhydrous magnesium sulfate, then concentrate by rotary evaporation to obtain the crude product.

[0093] The crude product was purified by silica gel column chromatography with dichloromethane as the eluent to prepare 1-hexyloxy-4-iodobenzene colorless solid with a yield of 85%.

[0094]

[0095] A 250 mL two-necked round-bottom flask was charged with bromoaniline (3 g, 17.44 mmol), 1-hexyloxy-4-iodobenzene (14.49 g, 43.6 mmol), palladium acetate catalyst (39.15 mg, 0.1744 mmol), and 150 mL of toluene, and ultrasonication was performed to fully dissolve the contents. The air in the flask was pumped out and replaced with argon to protect the contents. The contents were heated to reflux and continuously stirred for 24 h to prepare the crude product.

[0096] The crude product was purified by silica gel column chromatography with dichloromethane as the eluent to prepare 4-bromo-N,N-bis(4-hexyloxyphenyl)aniline colorless solid with a yield of 65%.

[0097]

[0098] A 100 mL two-necked round-bottom flask was charged with 4-bromo-N,N-bis(4-hexyloxyphenyl)aniline (3.23 g, 4.08 mmol), tributyl(2,3-dihydrothiopheno[3,4-b][1,4]dioxan-5-yl)stannane (500 mg, 2.04 mmol), and 40 mL of anhydrous THF, and tetrakis(triphenylphosphine)palladium catalyst (92.44 mg, 0.08 mmol) was quickly added. The contents were heated to reflux at 65 °C in an inert anhydrous and oxygen-free environment to perform the Stille reaction, and the crude product was prepared.

[0099] The crude product was purified by silica gel column chromatography with dichloromethane as the eluent to prepare dihydrothiopheno[3,4-b][1,4]dioxan-5-yl-N,N-bis(4-hexyloxyphenyl)aniline light yellow solid with a yield of 50%.

[0100]

[0101] Into a 100 mL two-necked round bottom flask, 4-hexyloxy-N-4-hexyloxyphenyl-N-4-7-tributylstannyl-2,3-dihydrothiopheno[3,4-b][1,4]dioxin-5-ylphenylamine (3.33 g, 5 mmol) was added, 25 mL THF was added, the air in the flask was pumped out by vacuum, then argon was filled to protect, the operation was repeated three times, then the mixture was cooled to -78 °C for 30 min, n-butyllithium (2.0 mL, 5 mmol) was slowly added dropwise, the reaction was stirred at -78 °C for 3 h, then tributyltin chloride (1.36 mL, 5 mmol) was continuously added, and the reaction was carried out at room temperature to obtain compound 4-hexyloxy-N-4-hexyloxyphenyl-N-4-7-tributylstannyl-2,3-dihydrothiopheno[3,4-b][1,4]dioxin-5-ylphenylamine in the form of yellow oil, the yield was 100%.

[0102] The product prepared above does not need to be purified and can be directly used in the following reaction.

[0103]

[0104] Into a 100 mL two-necked round bottom flask, 4-hexyloxy-N-4-hexyloxyphenyl-N-4-7-tributylstannyl-2,3-dihydrothiopheno[3,4-b][1,4]dioxin-5-ylphenylamine (3.33 g, 5 mmol) was added, 25 mL THF was added, the air in the flask was pumped out by vacuum, then argon was filled to protect, the operation was repeated three times, then the mixture was cooled to -78 °C for 30 min, n-butyllithium (2.0 mL, 5 mmol) was slowly added dropwise, the reaction was stirred at -78 °C for 3 h, then tributyltin chloride (1.36 mL, 5 mmol) was continuously added, and the reaction was carried out at room temperature to obtain compound 4-hexyloxy-N-4-hexyloxyphenyl-N-4-7-tributylstannyl-2,3-dihydrothiopheno[3,4-b][1,4]dioxin-5-ylphenylamine in the form of yellow oil, the yield was 100%.

[0105] The air in the flask was pumped out by vacuum, then argon was filled to protect, the operation was repeated three times, then the mixture was heated to 110 °C and refluxed, and the reaction was continuously stirred for 24 h in the dark.

[0106] The reaction product was cooled to room temperature, water was added, extracted with dichloromethane for 3 times, the organic phases were combined, dried with anhydrous magnesium sulfate, and then concentrated under reduced pressure to remove the solvent to obtain a crude product.

[0107] The crude product was purified by silica gel column chromatography with dichloromethane as the eluent to obtain dark green 4-hexyloxy-N-4-hexyloxyphenyl-N-4-7-tributylstannyl-2,3-dihydrothiopheno[3,4-b][1,4]dioxin-5-ylphenylamine-4,8-dibromo[1,2,5]selenadiazolo[3,4-f]benzo[c][1,2,5]thiadiazole solid compound, the yield was 40%.

[0108] The final prepared dark green solid compound is as an aggregation-induced emission luminogen (AIEgen), which is abbreviated as TPA-Se.

[0109] The chemical structure of compound TPA-Se was confirmed by the nuclear magnetic resonance hydrogen spectrum, carbon spectrum and mass spectrum data shown by Figure 1 、 Figure 2 and Figure 3 respectively. The specific nuclear magnetic spectrum data are as follows:

[0110] 1 H NMR (500 MHz, CDCl3) δ 7.06 (d, J = 7.0 Hz, 4H), 7.02 (d, J = 8.0Hz, 12H), 6.87 (d, J = 8.0 Hz, 8H), 4.34 – 4.32 (m, 8H), 3.99 (t, J = 6.5 Hz,8H), 1.80 – 1.74 (m, 8H), 1.48 – 1.43 (m, 8H), 1.34 – 1.27 (m, 16H), 0.89 (t, J = 6.5 Hz, 12H)。

[0111] 13 C NMR (125 MHz, CDCl3) δ 160.83, 153.19, 150.72, 150.17, 148.67,144.61, 142.96, 130.32, 128.50, 127.45, 127.27, 124.93, 122.51, 116.19,109.34, 68.06, 65.51, 31.46, 29.18, 25.75, 22.56, 14.09。

[0112] HRMS (ESI) m / z: calcd for C 78 H 84 N6O8S3Se [M+H] + 1408.7158, found1408.7188。

[0113] Example 2

[0114] The above prepared TPA-Se (1 mg) was mixed with the conventional carrier material DSPE-PEG 2000(4mg) were dissolved in 1mL THF, and then added dropwise into 9mL ultrapure water, and left to stand at room temperature. After the THF was removed by volatilization, the concentrated TPA-Se-loaded nanoparticles suspension was prepared by nanoseeding method at 5000rpm / min, and was recorded as ANPs.

[0115] Figure 4 The visible-near infrared absorption spectra and fluorescence emission spectra of TPA-Se and ANPs in THF solution are shown in the figures. In the figure a, the maximum absorption wavelength of TPA-Se in THF solution is 850nm, while the maximum absorption peak of ANPs is red-shifted to 920nm. Similarly, in the figure b, the maximum fluorescence emission wavelength of TPA-Se is 1100nm, and the maximum emission wavelength of ANPs is also red-shifted to 1170nm.

[0116] The above spectral results show that the absorption and emission spectra of TPA-Se are basically stable after nanocrystallization to form ANPs, and there is no quenching or significant broadening, only the expected red-shift effect, which shows that TPA-Se can still maintain its good optical properties in nanoparticles, and the nanocrystallization process has no adverse effect on the core optical properties of TPA-Se, which lays a foundation for its subsequent biological applications.

[0117] THF as a solvent can provide a good solubility environment for TPA-Se, while water is a poor solvent for TPA-Se. This change in solvent environment can promote the behavior of TPA-Se in different aggregation states to change. Therefore, the present application dissolves TPA-Se in THF / water mixed solutions with different volume ratios, and measures TPA-Se in different mixed solutions by fluorescence spectrophotometer to observe the change of photoluminescence intensity of TPA-Se in different solvent systems.

[0118] Figure 5 In the figures, a is the fluorescence emission spectrum of TPA-Se in THF / water mixed solutions with different water mass fractions, and b is the curve of fluorescence emission peak intensity versus water mass fraction in THF / water mixed solutions, wherein I0 and I represent the fluorescence emission intensity in pure THF and THF / water mixed solutions with different water mass fractions, respectively. It can be seen that the fluorescence emission intensity of TPA-Se increases with the increase of water mass fraction in the mixed solution, which proves that the TPA-Se prepared in the present application has the property of aggregation-induced emission (AIE).

[0119] The change of temperature can be used to evaluate the thermal effect and photothermal conversion efficiency of TPA-Se under light. Based on the strong absorption characteristics of TPA-Se in the near-infrared region, the present application further investigates the stability of the photothermal response of TPA-Se under different light conditions.

[0120] The experiment used a TPA-Se solution with a concentration of 100 μmol, irradiated under an 808 nm laser (1 W / cm 2 ) for 5 min, stopped for 5 min, for a total of six cycles, and the temperature change was recorded every 15 s during the entire 60 min process, with an indocyanine green (ICG) solution of the same concentration as a control. The results are shown in Fig. a, which shows that TPA-Se exhibits better stability of photothermal response than ICG in six consecutive laser cycles. Figure 6

[0121] Figure 6 Fig. b is a temperature response curve of TPA-Se solutions with different concentrations after irradiation under an 808 nm laser (1 W / cm²) for 5 min. When the concentration is 100 μg / mL, the solution temperature rises to 79°C and remains stable. According to this, the photothermal conversion efficiency of TPA-Se at this concentration is calculated to be 53.93%, showing good potential for photothermal therapy. In addition, Figure 6 Fig. c is a temperature response curve under different laser powers, which shows that the heating effect of TPA-Se increases with the increase of solution concentration and laser power.

[0122] Figure 6 Fig. d shows the energy level structure of TPA-Se and the intersystem crossing (ISC) process, with a minimum energy gap (Δ E ST ) of 0.102 eV between the singlet and triplet states, which theoretically supports the efficient and stable photothermal performance of TPA-Se, consistent with the experimental results, thereby laying the foundation for achieving optimal photothermal therapy (PTT).

[0123] To further analyze the molecular structure and electronic properties of TPA-Se, density functional theory (DFT) was used for systematic calculations, and the specific results are shown in Fig. Figure 7 , which comprehensively shows the molecular geometry, frontier orbital distribution, and corresponding optical mechanism of TPA-Se.

[0124] Theoretical calculations show that TPA-Se molecules exhibit a highly twisted three-dimensional helical topological configuration. This structure effectively suppresses intermolecular π-π stacking through steric hindrance effects, while maintaining the conjugation channel of the intramolecular donor-acceptor (D-A) unit, which helps to stabilize and regulate its optical properties.

[0125] Frontier orbital analysis shows that the electron cloud distribution of the highest occupied molecular orbital (HOMO) is present throughout the molecular skeleton, exhibiting delocalization characteristics; while the lowest unoccupied molecular orbital (LUMO) is mainly concentrated in the electron acceptor unit, indicating an effective charge separation trend within the molecule.

[0126] ​Furthermore, TPA-Se has a calculated electronic bandgap of 1.15 eV. This narrow bandgap characteristic enables it to achieve a highly efficient near-infrared II (NIR-II) optical response, which theoretically explains the source of its strong near-infrared absorption and excellent photothermal performance.

[0127] The aforementioned DFT calculations revealed the structure-activity relationship of TPA-Se at the atomic and electronic levels, providing an important theoretical basis for its application in photothermal therapy (PTT) and other fields.

[0128] Example 3

[0129] according to Figure 8 The synthetic route shown employs a typical standard solid-phase peptide synthesis (SPPS) method to prepare the self-assembling deformable peptide nanocarrier LND-1-PEG-24. The peptide nanocarrier was synthesized by Nanjing Jietai Biotechnology Co., Ltd.

[0130] Weigh 2g of CTC resin (0.2mmol / g) into the reactor, add 10mL of anhydrous dichloromethane, stir at room temperature to allow the resin to fully swell, and then remove the solvent.

[0131] Weigh out Fmoc-PEG 24 -COOH (0.4 mmol) was dissolved in 10 mL of anhydrous N,N-dimethylformamide (DMF), and then N,N-diisopropylethylamine (DIEA, 0.6 mmol) was added to dissolve the mixture to obtain a mixed solution.

[0132] The mixed solution was transferred to a reactor containing swollen resin and reacted at room temperature for 2 hours. After washing with DMF, Fmoc-PEG was prepared. 24 -CTC resin.

[0133] Fmoc-PEG treated with 20% piperidine solution 24 -CTC resin, by removing the Fmoc protecting group at the end of PEG, exposes free amino groups (-NH2), which can be used as the starting material for subsequent SPPS.

[0134] Weigh out Fmoc-Gly-OH (0.6 mmol) and HOBt (0.6 mmol) and dissolve them in 10 mL of anhydrous DMF. After activation with DIEA (0.6 mmol), the mixture is transferred to a reactor and reacted at room temperature for 1 h. The product is then deprotected from Fmoc using 20% ​​piperidine solution.

[0135] According to the same method, amino acids Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Arg(Mtr)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH were sequentially coupled and deprotected to obtain the target polypeptide H-Phe-Phe-Arg(Mtr)-Phe-Lys(Boc)-Gly-Phe-Leu-Gly-PEG 24 -Resin.

[0136] Finally, Lonidamine (LND, 0.6 mmol) and HOBt (0.6 mmol) were weighed and dissolved in 10 mL of anhydrous DMF, and then N,N'-diisopropylcarbodiimide (DIC, 0.6 mmol) was added dropwise to activate it. After that, it was transferred to the reactor and reacted with the target polypeptide at room temperature for 1 h. The reaction solution was dried by suction, washed with anhydrous DMF 6 times, 10 mL each time, and then washed with anhydrous methanol 5 times, 10 mL each time. The resin was dried by suction.

[0137] The resin was loaded into a 50 mL centrifuge tube, and a pre-cooled cutting solution (TFA: water: benzyl thioether: ethanedithiol = 92.5: 2.5: 2.5: 2.5, v / v) was added. The polypeptide was cut from the resin and all the protecting groups were removed simultaneously by shaking the reaction for 3 h. The polypeptide was filtered into a new 50 mL centrifuge tube and precipitated with pre-cooled ether. The crude polypeptide was separated by low-speed centrifugation and washed with pre-cooled ether to obtain the polypeptide nanocarrier LND-1-PEG-24 white solid.

[0138] The structure of the polypeptide nanocarrier is LND-Phe-Phe-Arg-Phe-Lys-Gly-Phe-Leu-Gly-PEG 24 , which is composed of a hydrophobic fragment LND-Phe-Phe-Arg-Phe-Lys (LND-1) and a hydrophilic fragment Gly-Phe-Leu-Gly-PEG 24 , wherein the hydrophobic fragment part is connected to the mitochondria-targeting peptide sequence Phe-Arg-Phe-Lys on LND, and the hydrophilic fragment is connected to the cathepsin B (CTSB) cleavable peptide sequence Gly-Phe-Leu-Gly on PEG 24 , so that the LND-1-PEG-24 nanocarrier can be disassembled to release LND-1 after exposure to intracellular CTSB, guiding LND to specifically target mitochondria.

[0139] To evaluate the enzyme-responsive property of LND-1-PEG-24 under the action of Cathepsin B (CTSB), 1 pmol / mL of CTSB enzyme was added to the reaction buffer containing 0.5 mg / mL LND-1-PEG-24, and the reaction buffer was composed of 10 mM phosphate buffer (PBS) and 1 mM EDTA, and the pH value was adjusted to 5.5 to simulate the acidic microenvironment of lysosomes and maintain the optimal activity of CTSB enzyme. Through this system, quantitative analysis by high performance liquid chromatography can accurately monitor the hydrolysis products of LND-1-PEG-24 and the kinetic behavior of enzyme reaction.

[0140] The reaction system was incubated at 37°C, and the sample was taken at different time points, and the content of the hydrolysis product of LND-1-PEG-24 was quantitatively detected by HPLC. The results are shown in Figure 9 As shown in

[0141] To verify the mitochondrial targeting ability of LND-1-PEG-24, the environmentally sensitive dye NBD was conjugated to the self-assembled peptide molecule to construct the NBD-labeled LND-1-PEG-24 probe (referred to as LND-1(NBD)-PEG-24) with strong green fluorescence, and cell imaging experiments were carried out using it.

[0142] LND-1(NBD)-PEG-24 solution was added to the cell culture medium to treat 4T1 cells for 6 h to ensure that the molecules were fully combined with the cell membrane and internalized, and the cells were washed with PBS buffer to remove free peptide molecules that were not combined or not entered the cells. At the same time, the mitochondria-specific fluorescent probe Mito-tracker Deep Red was used for dynamic tracking of live cell mitochondria, and with its membrane potential-dependent targeting property, high signal-to-noise ratio red fluorescent labeling was achieved.

[0143] As shown in Figure 10 After 6 h of treatment, the green fluorescent signal of LND-1(NBD)-PEG-24 was observed to gradually dissociate from the nanocarrier and be transported into the mitochondria in a filamentous structure, indicating that the peptide molecule was mainly located in the mitochondria. Figure 10The co-localization analysis of middle b shows that the signal of LND-1(NBD)-PEG-24 is highly overlapped with that of the mitochondrial probe Mito-tracker, further confirming the enrichment of LND-1-PEG-24 in mitochondria, suggesting that it achieves directional migration to mitochondria through a specific targeting sequence.

[0144] Example 4

[0145] RAW264.7 cells were cultured in medium containing LPS (100 ng / mL) and IFN-γ (50 ng / mL) for 24 h to stimulate the induction of RAW264.7 cells to polarize into M1 macrophages. After removing the supernatant by gradient centrifugation, a high-activity macrophage precipitate was obtained. The cell precipitate was resuspended in a cell freezing preservation solution containing 10% dimethyl sulfoxide and quickly frozen in liquid nitrogen for storage.

[0146] In use, a 37°C water bath is used for quick recovery, and low-temperature differential centrifugation is used to further purify the cell suspension to remove the supernatant to obtain extracted M1 macrophage membranes.

[0147] Example 5

[0148] 0.5 mg of LND-1-PEG-24 was weighed into a centrifuge tube, 1 mL of THF was added for dissolution, 1 mL of PBS buffer (50 mM, pH 7.4) was added and mixed uniformly, and then treated with an ultrasonic cell homogenizer (set power to 80%) for 10 min. After overnight standing in the dark, it was ensured that the solution was fully reacted to form stable LNPs.

[0149] 2 mg of TPA-Se and 10 mg of LND-1-PEG-24 were weighed and dissolved in 1 mL of THF to form a drug solution. The formed drug solution was gradually introduced into 10 mL of deionized water under rapid stirring to form a water / oil system. The system was stirred at room temperature in a fume hood overnight to remove THF from the solution. After further centrifugation of the solution using a centrifugal filter to remove most of the water, more stable ALNPs were obtained.

[0150] Freshly prepared ALNPs (2 mL, 1 mg / mL) were mixed with 4 mg of M1 macrophage membranes obtained in Example 4, and treated with ultrasonic waves at 100 W and 50 Hz for 10 min in an ice bath to promote the complete fragmentation of the cell membranes and self-assembly of the cell membranes on the surface of the ALNPs to form composite nanoparticles M1-ALNPs with M1 macrophage membrane biomimetic functions and targeting properties.

[0151] To further improve the monodispersity and stability of the particles, the above mixed system was subjected to multiple extrusion treatments through polycarbonate membranes with different pore sizes, and M1-ALNPs with uniform particle size and stable structure were obtained, which were stored in PBS buffer at 4°C.

[0152] The morphology of ALNPs and M1-ALNPs was observed by cryogenic transmission electron microscopy (TEM), and the average hydrodynamic diameter of ALNPs and M1-ALNPs was measured by dynamic light scattering (DLS) technology. The specific results are shown in FIG. 1. Figure 11 As shown in FIG. 1, both ALNPs (a) and M1-ALNPs (b) showed uniform spherical structure, and the average size was about 100 nm or so.

[0153] To evaluate the stability of M1-ALNPs in the actual application environment, the colloidal stability thereof was studied, as shown in FIG. 2. Figure 12 As shown in FIG. 2, the M1-ALNPs were incubated in PBS medium and cell culture medium containing 10% FBS (simulating blood environment) for 5 days, and the results showed that the hydrodynamic size remained stable throughout the process and did not increase significantly, proving that the M1 cell membrane coating effectively endowed the nanoparticles with excellent long-term stability and anti-aggregation ability. This excellent stability is an important prerequisite for the successful delivery of nanoparticles to the tumor site and the function of active targeting.

[0154] Example 6

[0155] In vitro hemolysis experiment is one of the important methods for evaluating the blood compatibility of nanoparticles, which can understand the influence of nanoparticles on red blood cells in blood and its potential biological safety.

[0156] Fresh blood was obtained from 6-week-old BALB / c mice, and the red blood cell suspension was obtained by centrifugation at 2000 rpm for 10 min.

[0157] 300 μL of M1-ALNPs composite nanoparticle solution of different concentrations was added to 1 mL of red blood cell suspension, and after mixing well, it was incubated at room temperature for 4 h. The red blood cell suspension treated with aqueous solution was used as the positive control group, and the red blood cell suspension treated with PBS buffer was used as the negative control group. The red blood cells and supernatant were separated by centrifugation at 15000 rpm for 10 min, and the absorbance of the supernatant was measured at 541 nm wavelength.

[0158] From the in vitro hemolysis analysis results of Figure 13 It can be seen that the red blood cells treated with M1-ALNPs in the concentration range of 0-100 μg / mL showed relatively low hemolysis rate, and M1-ALNPs showed good biological safety.

[0159] Example 7

[0160] To evaluate whether the encapsulation of Ml -type macrophage membrane enhances the uptake of complex nanoparticles by tumor cells, an in vitro model of 4T1 breast cancer cell line (malignant phenotype) was constructed, and laser confocal imaging special culture system was used to realize the synchronous three-dimensional culture of cells. After incubation at 37°C and 5% CO2 for 12 h, ALNPs and Ml -ALNPs were added to the cell culture medium for treatment, and the fluorescence images inside and outside the cells were obtained at 2 h, 4 h and 6 h.

[0161] The treated cells were digested, centrifuged and separated, and the fluorescence intensity of the cells was determined by flow cytometry. By recording the mean fluorescence intensity (MFI) of the cells, the uptake of complex nanoparticles and the response of cells to different complex nanoparticles under different conditions were accurately evaluated.

[0162] At the same time, an in vitro model of MCF-10a normal breast epithelial cells (benign control) was constructed, and Ml -ALNPs were added for treatment for comparison.

[0163] The experimental results are shown in Figure 14 , where a and b are representative confocal laser scanning images of 4T1 cells incubated with ALNPs and Ml -ALNPs for different periods of time; c is a representative confocal laser scanning image of MCF-10a cells incubated with Ml -ALNPs for different periods of time; d is a flow cytometry analysis of 4T1 cells treated with ALNPs and Ml -ALNPs; and e is the fluorescence intensity quantitative data in different cells.

[0164] As can be seen from a and b, Ml -ALNPs are significantly internalized by cancer cells at 6 h compared with ALNPs lacking Ml -type macrophage membrane modification, and the flow analysis results in d also show that the cell uptake of Ml -ALNPs treated for 6 h is nearly 3 times larger than that of ALNPs, which proves that Ml membrane modified ALNPs increase their internalization in tumor cells.

[0165] In addition, normal mouse breast epithelial cells (MCF-10a) treated with Ml -ALNPs significantly exhibit lower fluorescence signals (c and e), further verifying that Ml -ALNPs preferentially target cancer cells without affecting healthy epithelial cells.

[0166] Furthermore, the morphological changes of 4T1 cells after treatment with Ml -ALNPs were observed by transmission electron microscopy, and the morphological changes of cells treated with Ml -ALNPs were studied by in vitro targeting experiments.

[0167] The 4T1 cells were treated with M1-ALNPs for 24 h, the cell samples were fixed, and after stepwise dehydration, embedded by epoxy resin (Epon-812) gradient penetration, sectioned, double-stained with uranyl acetate and lead citrate, and then imaged by transmission electron microscopy (TEM) at high resolution for ultrastructure. All cells were observed at low magnification, and different areas were selected for high magnification image collection to observe the morphological changes of LND-1-PEG-24 in cells and its interaction with cell structures.

[0168] As shown in FIG. 8A, in untreated cells, most mitochondria showed normal morphological features with typical outer membranes and crista spaces (indicated by white arrows); while after treatment with M1-ALNPs and 808 nm laser irradiation (1 W / cm2, 2 min) for 4 h, dense nanofibers were found at the edges of mitochondria (indicated by red arrows), and partial mitochondrial damage was observed, including outer membrane and crista destruction, mitochondrial swelling, and abnormal vacuoles (indicated by yellow arrows); after 6 h of treatment, severe mitochondrial damage was observed, with severe destruction of the outer membrane and crista, and almost all mitochondria were swollen and produced abnormal vacuoles. 2 Figure 15

[0169] These nanofibers can be attributed to the transformation of M1-ALNPs nanoparticles to target organelles (i.e., mitochondria), indicating that the catalytic activity of CTSB is essential for the formation of nanofibers around mitochondria after M1-ALNPs treatment. These results also demonstrate that the aggregation of self-assembled peptide nanofibers around mitochondria disrupts the morphology of mitochondria.

[0170] Example 8

[0171] To reveal the mechanism of M1 macrophage membrane polarization, this experiment resorts to identify differential gene expression occurring in this process. RAW264.7 was co-incubated with M1-ALNPs, and a Control group was set up at the same time, and the cells were collected for transcriptome analysis.

[0172] A total of 15484 genes were identified in both groups, and the examination of genes related to M1 / M2 signals showed that the M1 phenotype was significantly enriched after M1-ALNPs treatment, and the M2 phenotype was lost, confirming the M1 polarization mediated by M1-ALNPs.

[0173] To elucidate the main pathways involved in this process, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed on the differentially expressed genes. The most significant enrichment involved genes in the mitogen-activated protein kinase (MAPK) and Toll-like receptor (TLR) signaling pathways. Figure 16 ​​Fig. 6 shows the heat map of specific genes related to Ml / M2 phenotypes extracted from RNA-seq data of Control and Ml-ALNPs treated RAW264.7, b is KEGG enrichment analysis of pathways involved in Ml-ALNPs induced macrophage polarization. Analysis shows that only TLR-2 is significantly up-regulated after Ml-ALNPs treatment, while the expression of other TLRs (TLR-4, TLR-7, TLR8 and TLR-9) is almost not up-regulated.

[0174] The role of TLR-2 / MAPK signaling in this process was further tested, Figure 17 Fig. 7a is a specific protein immunoblotting related to MAPK signaling pathway after different groups of 4T1 cells were treated, which shows that TLR-2 plays an important role in initiating CSMl mediated macrophage polarization; Fig. 7b quantitatively measures the content of TNF-a, IL-12p40, IL-10 and TGF-bI by ELISA to study the effect of Ml polarization on inflammatory immune response, mainly including cytokine release. Compared with control macrophages, CSMl treated macrophages showed significantly increased levels of TNF-a and IL-12p40, and inhibited the secretion of IL-10 and TGF-bI.

[0175] Example 9

[0176] 4T1 tumor cells were incubated in different concentrations of various nanoparticle solutions to verify the tumor killing ability of different nanoparticles at the cellular level through in vitro cytotoxicity tests.

[0177] The nanoparticles used include LNPs, Ml-ALNPs, and laser irradiation treated ANPs+NIR, ALNPs+NIR and Ml-ALNPs+NIR, and a PBS control group, a total of 6 experimental groups for related in vitro cell experiments.

[0178] To further explore the effect of irradiation on cell viability, after the treatment was completed, the cells of the experimental groups that needed to be irradiated were exposed to 808 nm wavelength laser with an intensity of 10 mW / cm 2 for 5 min.

[0179] The relative viability of cells was tested by standard CCK-8 method, which can be converted to water-soluble yellow formazan salt by the action of intracellular dehydrogenase, and the absorbance is related to cell metabolic activity. Figure 18The results showed that, without light exposure, LNPs alone exhibited minimal cytotoxicity, indicating that the therapeutic effect of LNPs was not ideal. However, after the introduction of TPA-Se, the cytotoxicity of ALNPs+NIR exceeded that of LNPs and M1-ALNPs, and this enhanced tumor-killing efficacy was attributed to the photothermal therapy of TPA-Se. Importantly, M1-ALNPs, which are characterized by targeting the cell membrane, showed the highest tumor cell inhibition efficacy under 808nm laser irradiation.

[0180] To more intuitively demonstrate the cytotoxic effects of different nanoparticles on 4T1 tumor cells, a precise identification of live / dead cells was achieved using the Calcein-AM / PI dual-fluorescence labeling method. Spatiotemporal resolution imaging was completed by utilizing the difference in dye membrane permeability (Calcein-AM targets intracellular esterases in live cells to generate green fluorescence, while PI specifically labels damaged cell nuclei to release red signals). The fluorescence intensity after cell staining was observed using a fluorescence microscope to show the ratio of live to dead cells in different experimental groups.

[0181] Specific results are as follows Figure 18 As shown in Figure b, 4T1 cells treated with ANPs and light showed moderate cell death; increased cell death was observed in the ALNPs and light-treated group; and in the M1-ALNPs+NIR group, almost all 4T1 cells showed signs of cell death. Figure 18 Image J cell counting in c reached an astonishing 83%, consistent with previous CCK-8 experimental results.

[0182] The plate colony formation assay further verified that M1-ALNPs+NIR can effectively inhibit tumor cell activity and reduce cell proliferation through PTT and pyroptosis. Figure 19 As shown, the experimental results indicate that M1-ALNPs+NIR exhibits better anti-proliferative activity, while LNPs show a relatively weak inhibitory effect on cell proliferation.

[0183] Example 10

[0184] Pyroptosis is a form of programmed necrosis distinct from classical apoptosis. It relies on caspase cleavage of Gasdermin family proteins, such as GSDME, to generate an N-terminal active domain, mediating the formation of nanopores in the cell membrane and promoting the release of pro-inflammatory factors such as HMGB1 and IL-1β. This signaling cascade provides a novel molecular basis for tumor immunotherapy. During pyroptosis, cells swell due to water influx, eventually leading to membrane rupture and cell lysis. Simultaneously, this mechanism can activate specific immune signaling pathways, promoting the maturation and secretion of precursor cytokines such as IL-1β and IL-18, triggering a strong inflammatory response, recruiting immune cell aggregation, and thereby enhancing anti-tumor immunity.

[0185] Studies have shown that LND can promote pyroptosis of tumor cells by up-regulating the expression of GSDME, a key execution protein of pyroptosis; on the other hand, photothermal therapy (PTT) can activate caspase-3 expression, and then cut GSDME to generate GSDME-N active fragments, induce cell membrane perforation and trigger pyroptosis.

[0186] 4T1 tumor cells were incubated with LNPs, ANPs+NIR, ALNPs+NIR, M1-ALNPs and M1-ALNPs+NIR at a concentration of 80 μg / mL, respectively, to observe the morphology of 4T1 cells after treatment in each experimental group and analyze the in vitro pyroptosis of 4T1 cells.

[0187] The cells in each experimental group were collected for lysis and protein extraction, and then subjected to SDS-PAGE gel electrophoresis separation and Western blot detection on PVDF membrane. After transfer, the PVDF membrane was blocked with blocking solution for 1 h, and according to the experimental target, anti-GSDME (related to cell pyroptosis), anti-cleaved caspase-3 and anti-β-actin were used for primary antibody incubation to allow specific binding of target proteins to corresponding antibodies. After washing to remove non-specific binding, high-sensitivity development of target proteins was performed using ECL chemiluminescence detection system, and quantitative analysis of dynamic differences in protein expression in 4T1 cells in each experimental group was performed on a standardized Western blot technology platform to explore whether LND affects cell pyroptosis by regulating the expression of GSDME and caspase-3.

[0188] Figure 20 Representative bright field images of in vitro pyroptosis detection of 4T1 cells after different treatments in each experimental group are shown, in which the M1-ALNPs+NIR group of cells showed obvious pyroptosis characteristics, and the white arrows indicate cell swelling and bubble-like protrusions of the plasma membrane. In contrast, only a few pyroptotic cells were observed in the LNPs group, while more pyroptotic cells showing cell shrinkage and pyroptotic body formation were observed in the ANPs+NIR group, indicating that single LND or PTT treatment is not sufficient to effectively induce cell pyroptosis.

[0189] Furthermore, Figure 21 By observing 4T1 cells treated with M1-ALNPs+NIR at different time points, it was found that 4h after light exposure, most of the cells underwent pyroptosis, and 8h later the cells tended to rupture, indicating that LND-1-PEG-24 has a synergistic effect with ANPs in promoting pyroptosis.

[0190] Figure 22Western blot results showed that GSDME-N fragment expression was significantly increased in the M1-ALNPs + NIR group, indicating that pyroptosis levels were enhanced. Compared with the LNP alone group, the GSDME-N content in this group was significantly increased, highlighting the key role of AIEgen-based PTT in amplifying LND-1-PEG-24-induced pyroptosis. This enhanced effect may be related to the upregulation of cleaved caspase-3 expression on PTT, which in turn promotes the cleavage of GSDME into the GSDME-N domain. The above results again verified that the LND-1-PEG-24-mediated upregulation of GSDME combined with the PTT-promoted cleavage of GSDME enabled M1-ALNPs to induce a strong pyroptosis reaction in tumor cells under light conditions.

[0191] Example 11

[0192] As a glycolysis inhibitor, LND can block lactate efflux by inhibiting monocarboxylate transporter 4 (MCT4), which is believed to interfere with the polarization of tumor-associated macrophages (TAMs) to M2 type. Based on this, this study aims to explore whether M1-ALNPs can synergistically enhance M1 polarization induction when used in combination with LND, thereby jointly reshaping the immunosuppressive phenotype of TAMs.

[0193] Ensure that the 4T1 tumor cells are fully attached to the culture flask and are in the growth state. After 24h of adherent growth, use different nanoparticle solutions for light treatment or no light treatment. After the treatment is completed, collect the cell culture medium for lactate concentration determination.

[0194] Figure 23 In the meantime, the combination of M1-ALNPs + NIR resulted in the lowest lactate efflux into the extracellular region, which further prevented the M2 polarization of TAMs, verifying the synergistic effect of M1-ALNPs on the regulation of the immune environment of TAMs.

[0195] Lactate dehydrogenase (LDH) is a key enzyme in the glycolytic pathway, responsible for catalyzing the reversible conversion between pyruvate and lactate, maintaining the metabolic homeostasis of cells. When the structural integrity of the cell membrane is damaged, LDH will leak from the intracellular to the extracellular matrix, and therefore its release level is often used as an important indicator to evaluate cell damage and toxicity.

[0196] In the process of cell pyroptosis, a process of programmed necrosis, members of the Gasdermin protein family are activated to form pores on the cell membrane, causing abnormal increases in membrane permeability, thereby specifically promoting the release of LDH to the extracellular, which can be a typical biochemical marker of pyroptosis.

[0197] While ATP detection at the same time, the metabolic activity and energy status of cells can be reflected, to prove the decline of cell viability synchronously.

[0198] Figure 24 The LDH release and ATP levels of 4T1 cells after different experimental groups treatment were given. As a key indicator of the leakage of cell contents during pyroptosis, the LDH release content was the highest in the M1-ALNPs+NIR group, about 3 times and 1.8 times higher than that of PBS and ANPs groups, respectively. Similarly, the M1-ALNPs+NIR group showed a significant decrease in intracellular ATP content, proving the most effective ATP release in all treatment groups.

[0199] The decrease of mitochondrial membrane potential is a key event in the early stage of apoptosis and pyroptosis, and is also a marker of mitochondrial pathway activation. The mitochondrial membrane potential detection technology based on JC-1 fluorescent probe can realize the dynamic real-time quantitative monitoring of mitochondrial membrane potential through the change of J-aggregate and monomer fluorescence intensity ratio.

[0200] JC-1 reagent was added to 4T1 cells in different experimental groups for 6h, so that JC-1 dye could fully enter the cells and bind to mitochondria. PBS was used to wash away the unbound dye to ensure the accuracy of the staining results. The probe aggregates to form polymers (J-aggregates) in the mitochondria when the membrane potential is normal, emitting orange fluorescence; when the membrane potential decreases, it exists in the form of monomer, emitting green fluorescence. The change of red and green fluorescence was observed by confocal microscope, and the red / green fluorescence intensity ratio was calculated to objectively evaluate the membrane potential level.

[0201] As shown in Figure 25 , the mitochondria of cells in the PBS control group emitted strong orange fluorescence and weak green fluorescence, with a high red / green ratio. After ALNPs+NIR or M1-ALNPs+NIR treatment, the expression of JC-1 monomer increased, the expression of JC-1 aggregate decreased, the orange fluorescence of cell mitochondria significantly weakened, and the green fluorescence significantly enhanced, and the MFI ratio of monomer / polymer Figure 25 b) also increased in these groups, confirming that M1-ALNPs+NIR treatment can effectively induce mitochondrial membrane potential collapse, indicating that it successfully triggered mitochondrial dysfunction in cells, providing power for the subsequent occurrence of cell pyroptosis.

[0202] The opening of mitochondrial permeability transition pore (MPTP) is a key link leading to mitochondrial membrane potential collapse, cytochrome C release and ultimately triggering cell death. To determine whether cell pyroptosis is indeed initiated by MPT, mitochondrial permeability transition pore detection kit (K239-100, Biovision) was used for mitochondrial permeability transition pore detection.

[0203] Calcein-AM is a cell membrane permeable fluorescent dye that can enter cells and be hydrolyzed into Calcein inside the cells to produce green fluorescent signals. Dilute the Calcein-AM solution to 500 nM, take 50 μL and incubate with 4T1 cells in different experimental groups for 20 min, add 50 μL of CoCl2 solution with a concentration of 80 mM after staining, continue to incubate for 10 min, use a fluorescence reader to read the fluorescence signal at Ex490 / Em515 nm wavelength, and the strength of the fluorescence signal directly reflects the integrity of the mitochondrial membrane and the opening degree of MPTP.

[0204] Figure 26 The test results of M1-ALNPs+NIR show that the sustained opening of MPTP is triggered, which causes the leakage of mitochondrial content and the loss of inner membrane integrity, resulting in the sustained dissipation of mitochondrial membrane potential, which explains the reason for the collapse of mitochondrial membrane potential from the mechanism, and strongly proves that the pyroptosis process is closely related to the mitochondrial pathway.

[0205] The key feature of immunogenic cell death (ICD) is that the dead cells release or expose a series of "danger signals" (DAMPs), including high mobility group protein B1 (HMGB1) and ecto-calcium reticulum (ecto-CRT), thereby recruiting and activating immune cells. These DAMPs help antigen-presenting cells (APCs) phagocytose tumor cells and promote tumor-specific immune responses. The release of HMGB1 and the plasma membrane exposure of CRT are two classic gold standard biomarkers of ICD. By detecting the release of HMGB1 and ecto-CRT, it can be proved that the cell death induced by M1-ALNPs+NIR is not ordinary necrosis, but immunogenic cell death with immune activation.

[0206] Immunofluorescence staining method was used to label HMGB1 (usually located in the nucleus) and calreticulin (CRT) (usually located in the endoplasmic reticulum) with specific antibodies, and their changes in localization in cells were observed by confocal microscopy.

[0207] After incubation of 4T1 cells in different experimental groups for 6h, the irradiation group received 808nm wavelength laser irradiation with an intensity of 10mW / cm 2 for 2min, and continued to incubate with anti-HMGB1 antibody or anti-CRT antibody at 4°C overnight to specifically recognize and bind HMGB1 or CRT proteins expressed in cells or on the cell surface. After incubation, the cells were washed, anti-rabbit IgGH&L secondary antibody was added, and incubated at room temperature for 30 min. After DAPI staining, confocal laser scanning microscopy was used to observe the localization of intracellular HMGB1 and CRT and the position of the nucleus, and to explore the potential of M1-ALNPs to enhance anti-tumor immune response by activating the cell pyroptosis pathway.

[0208] Figure 27 In particular, HMGBl was only detected in the nucleus in the PBS control group, but after M1-ALNPs+NIR treatment, HIMGB1 was translocated to the cytoplasm and further released to the extracellular space, indicating the obvious release of HMGB1.

[0209] Figure 28 In particular, CRT signals were also located inside the cells in the PBS control group, while after M1-ALNPs+NIR treatment, CRT was exposed on the surface of cancer cells in large quantities, demonstrating that M1-ALNPs+NIR treatment can significantly increase the expression of etco-CRT on cancer cells.

[0210] Figure 27 and 28 Further quantitative analysis of b in the above-mentioned experiments showed that the amount of HMGB1 released and the amount of CRT exposed in the M1-ALNPs+NIR group were 6.1 times and 6.5 times that of the PBS group, respectively.

[0211] The above experimental results conclusively demonstrate that pyroptosis induced by M1-ALNPs+NIR is a typical immunogenic cell death, meaning that the killed tumor cells can actively send out "danger signals" to alert and activate the immune system in the body, so as to produce a long-term anti-tumor immune memory effect and inhibit tumor recurrence and metastasis.

[0212] Therefore, the above experiments collectively elucidate a core mechanism: M1-ALNPs+NIR induces mitochondrial MPTP opening leading to mitochondrial dysfunction, which in turn triggers cell pyroptosis, and the pyroptosis process releases HMGB1 and exposes CRT to activate the anti-tumor immune response, which provides a solid theoretical basis for M1-ALNPs+NIR as an efficient immunochemo-photothermal synergistic therapy.

[0213] Bone marrow-derived dendritic cells (BMDCs) were isolated from the bone marrow of 6-week-old BABL / c mice to study their maturation process under different conditions.

[0214] After centrifugation of mouse bone marrow with serum-free medium, bone marrow mononuclear cells were collected and extracted, and cultured in a medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF, 20 ng / mL) and interleukin-4 (IL-4, 10 ng / mL) for 4 days to promote dendritic cell differentiation. The differentiated immature dendritic cells (BMDCs) were also divided into 6 experimental groups, and were co-incubated with 4T1 breast cancer cells. After 24 h of incubation, flow cytometry was used to analyze the expression of dendritic cell surface markers CD11c, CD80 and CD86, and the effects of different treatments on the maturation process of BMDCs were evaluated by measuring the changes in these surface markers.

[0215] After that, 4T1 cancer cells were inoculated in the upper chamber of the transwell and exposed to different treatments, and BMDCs were co-cultured in the lower chamber of the transwell, and the maturation of BMDCs in each group was detected by flow cytometry.

[0216] As shown in Figure 29 compared with tumor cells treated with LNPs alone, tumor cells pretreated with M1-ALNPs+NIR showed significantly increased DC maturation, and the mature DCs (CD11c + CD80 + CD86 + ) were 4.3, 3.4, 2.8, 1.8 and 1.5 times more than the PBS, LNP, M1-ALNPs, ANPs+NIR and ALNPs+NIR groups, respectively. These results confirmed that pyroptosis effectively induced by M1-ALNPs+NIR can significantly promote the maturation of DCs.

[0217] The above Examples 7-11 carried out in vitro cell studies on the polypeptide self-assembled nanodiagnosis and nanotherapy preparation M1-ALNPs designed and prepared according to the present application, and verified that it can reprogram the immunosuppressive tumor microenvironment, induce persistent polarization of tumor-associated macrophages into immunostimulatory M1 phenotype through TLR-2 / MAPK signal transduction, and promote the targeted delivery of M1-ALNPs to tumor cells. Further, M1-ALNPs trigger tumor cell-specific pyroptosis through the caspase-3 / GSDME pathway, resulting in the release of damage-associated molecular patterns. Therefore, the combination of M1-ALNPs synergistically enhances immune infiltration and tumor cell immunogenicity within the TME, effectively reverses tumor immunosuppression, achieves effective anti-tumor effect, and induces long-term immune memory against tumor metastasis and recurrence.

[0218] Example 12

[0219] The incubated 1×10 6A 4T1 mouse breast cancer cell was suspended in 100 μL of pre-cooled PBS solution and injected into the right fourth inguinal mammary fat pad of 6-week-old female BALB / c mice to construct orthotopic tumor-bearing BALB / c mice. Seven days after injection, the tumor volume was measured to have grown to about 100 mm 3 , confirming that the 4T1 mouse breast cancer model was successfully constructed.

[0220] After the model was successfully constructed, ALNPs and M1-ALNPs were injected into the tail vein of mice, respectively, and fluorescence imaging was performed using a NIR-II in vivo imaging system to explore the dynamic distribution of ALNPs and M1-ALNPs in vivo and their imaging ability in tumor-bearing mice.

[0221] Figure 30 Fig. 8a shows representative NIR-II fluorescence images of 4T1 tumor-bearing mice after tail vein injection of ALNPs and M1-ALNPs at different times, and Fig. 8b shows the corresponding NIR-II fluorescence intensity of the tumor. It can be seen that the fluorescence intensity of the M1-ALNPs group reached a peak at about 24 h after injection, indicating that this is the best time for tumor imaging and treatment.

[0222] Notably, the fluorescence signal of the M1-ALNPs-treated mice was about 2.1 times that of the ALNPs group, indicating that M1 macrophage membrane coating promoted the increase in tumor accumulation. In addition, even 48 h after administration, strong NIR fluorescence was still observed at the tumor site, demonstrating that M1-ALNPs have effective long-term in vivo imaging capability due to the high sensitivity of fluorescence imaging.

[0223] Next, the in vivo photothermal effect of 4T1 tumor-bearing mice injected with ALNPs and M1-ALNPs and irradiated with an 808 nm laser (1 W / cm 2 ) for 24 h at the tumor site of the mice was studied. When the 808 nm laser was irradiated, a thermal infrared camera was used to perform photothermal imaging of the tumor area in the mice to monitor the temperature change at the tumor site due to the photothermal effect in real time.

[0224] Figure 31 Fig. 9a shows the temperature change of the tumor site of the mice under laser irradiation, and Fig. 9b shows the corresponding temperature change curve of the tumor site. Real-time thermal imaging proved that the temperature change in the mice injected with M1-ALNPs increased over time, and the local temperature rise of M1-ALNPs was more significant compared to the slight temperature rise of ALNPs, eventually reaching about 54°C, confirming the excellent in vivo photothermal properties of M1-ALNPs.

[0225] Finally, at 24 h after injection, the internal organs (heart, liver, spleen, lung, kidney) and tumor tissue of the mice were sampled for ex vivo fluorescence imaging and quantitative analysis.

[0226] Figure 32 Fig. 8a is representative ex vivo fluorescence imaging of major organs and tumors isolated from mice 24 h after intravenous injection of ALNPs and M1-ALNPs, and Fig. 8b is the corresponding fluorescence intensity of the isolated major organs and tumors. It is worth noting that compared with ALNPs-treated mice, bright NIR-II fluorescence signals were observed in the tumor region of M1-ALNPs-treated mice, even exceeding the signal in the liver.

[0227] There is sufficient evidence that the liver will usually exhibit high nanoparticle signals after systemic nanoparticle administration due to the isolation of the reticuloendothelial system to nanoparticles. The enhanced signal of M1-ALNPs observed in tumor tissues in the present application can be attributed to its effective accumulation in tumors and excellent NIR-II signals at the tumor site compared with the liver.

[0228] Example 13

[0229] The tumor-bearing mice constructed in Example 12 were randomly divided into PBS, LNPs, M1-ALNPs, ANPs+NIR, ALNPs+NIR and M1-ALNPs+NIR groups, a total of 6 experimental groups, 5 mice in each group, and the mice in each group received different treatments to evaluate the in vivo anti-tumor effect of M1-ALNPs.

[0230] In terms of administration, all experimental groups received 200 μL of the corresponding solution with a concentration of 500 μg / mL; for the experimental groups that needed phototherapy, the tumor area was irradiated with light within 24 h after injection treatment, the light irradiation used 808 nm laser with a power of 1 W / cm 2 , and the irradiation time was 2 min.

[0231] The entire experimental period was 14 days, and the intravenous administration of different formulations and phototherapy treatment were repeated every 3 days, a total of 3 treatments. During the experiment, the body weight, tumor volume and survival rate of the mice were monitored regularly. On the 14th day, all mice were euthanized, tumor tissue sections were collected for H&E staining to observe the histological structure and pathological changes of the tumor; at the same time, TUNEL immunofluorescence staining and Cleaved-caspase-3 immunohistochemical staining were performed on the tumor tissue.

[0232] Figure 33 The tumor growth curve of Fig. 8a shows that the tumors in the PBS group exhibited rapid growth during the entire treatment period; the tumor volume in the LNPs group exhibited mild inhibition, indicating that LNPs alone produced a certain but unsatisfactory tumor inhibition effect. However, it is exciting that M1-ALNPs+NIR treatment significantly reduced tumor growth, and one-fifth of the mice exhibited complete regression of cancer.

[0233] Figure 33 Among the statistical results of b, c and d, the body weight of mice in each experimental group did not change significantly during the experimental period, but the tumor volume and weight changed significantly. On the 14th day, the average tumor weight of the M1-ALNPs+NIR group was about 18.8, 17.3, 12.8, 12.5 and 3.2 times smaller than that of the PBS, LNPs, M1-ALNPs, ANPs+NIR and ALNPs+NIR groups, respectively.

[0234] The above results show that LND-1-PEG-24 can target tumor cell mitochondria and exacerbate mitochondrial membrane permeability, triggering extensive pyroptosis, while LND-1-PEG-24 can reduce the expression of heat shock proteins during low-temperature photothermal therapy, enhancing the sensitivity of cells to heat, thereby promoting the PTT effect of TPA-Se molecules in tumors. Therefore, the photothermal therapy of TPA-Se combined with the pyroptosis induced by LND-1-PEG-24 synergistically enhances the anti-tumor effect, inhibits tumor cell proliferation, and accelerates tumor cell death.

[0235] After the treatment, the tumors of the mice in the six experimental groups were dissected and photographed, as shown in FIG. 6. Figure 34 As shown in a, the best treatment effect was also observed in the M1-ALNPs+NIR group.

[0236] Subsequently Figure 34 Further histopathological analysis of tumor tissues was performed in b. H&E staining showed that the PBS group had the highest cell density, while tumors from different anti-tumor drug treatments showed moderate cell density, and notably, M1-ALNPs+NIR-treated tumors showed the lowest cell density; TUNEL was used to detect the degree of tumor tissue necrosis, and also showed that the M1-ALNPs+NIR group had the highest green fluorescence of positive TUNEL staining, indicating that compared with other groups, M1-ALNPs+NIR caused the most significant degree of apoptosis and necrosis of tumor cells; Cleaved-caspase-3 results also showed that compared with other groups, M1-ALNPs+NIR successfully induced apoptosis of tumor cells. These results are consistent with the previously observed superior tumor inhibition effect of M1-ALNPs+NIR treatment.

[0237] Example 14

[0238] Further, the efficacy of M1-ALNPs in inhibiting breast cancer lung metastasis was studied through a metastasis model by further simulating the hematogenous metastasis of tumors.

[0239] The residual tumor tissues of tumor-bearing mice in each experimental group after different treatments were excised to simulate clinical surgical resection, and then 100 μL of 5×10 6The 4T1 mouse breast cancer cells were injected into the tail vein of the mice to mimic the process of tumor invasion and to establish a metastasis model of tumor cells entering the lung through the blood. Two weeks after injection, the lung tissues collected from the PBS group showed considerable metastatic tumors, even with some tissue necrosis, confirming the successful establishment of the lung metastasis model.

[0240] The lung metastasis model mice were also randomly divided into 6 experimental groups to receive different treatments to evaluate the in vivo anti-lung metastasis effect of M1-ALNPs. All mice were sacrificed on the 30th day of treatment, and lung tissues were collected for Bouin’s solution staining and H&E staining to observe the tumor metastasis in the lung tissues and evaluate the effect of different treatment regimens in preventing tumor metastasis.

[0241] Figure 35 Representative images of lung tissues and H&E staining of different experimental groups are shown, showing metastatic tumors in the lungs of tumor-bearing mice at the end of treatment (circled). The M1-ALNPs+NIR group showed the least lung metastatic tumors, while the other groups showed varying degrees of metastasis.

[0242] The PBS group had the most severe metastasis, followed by the LNP group. The remaining three groups showed slight improvement, but the H&E staining results still showed significant lung damage. Surprisingly, no significant lung metastasis was observed in the M1-ALNPs+NIR group, demonstrating that M1-ALNPs+NIR treatment had a significant inhibitory effect on tumor metastasis. The best performance of M1-ALNPs+NIR treatment can be attributed to the complete activation of anti-tumor immunity and the induction of long-lasting immune memory.

[0243] Example 15

[0244] Healthy mice were chosen as a model to evaluate the potential effects of M1-ALNPs on the physiological health status and organ function of mice in vivo. The experiment was divided into an experimental group and a control group. Mice in the experimental group received 200 μL of M1-ALNPs injection through intravenous injection at days 0, 3, and 6, while the control group received the same dose of PBS solution treatment.

[0245] The mice were euthanized after collecting blood samples on day 10, and the main organs (heart, liver, spleen, lung, and kidney) were collected for H&E staining for pathological analysis.

[0246] Routine blood tests were performed on whole blood extracted from mice after the end of the administration period, and all parameters were within the corresponding normal range. No significant differences were observed in serum biochemical analysis between the PBS and M1-ALNPs groups, indicating the absence of acute liver or kidney damage.

[0247] By Figure 36Microscopic image observation of the morphology and pathological changes of the tissues showed that the M1-ALNPs treatment group was similar to the PBS group, and no obvious histological abnormalities and tissue damage occurred.

[0248] The polypeptide self-assembled nanodiagnosis and nanotherapy preparation M1-ALNPs prepared by the polypeptide according to the application in the above embodiments 12-15 is applied to a mouse breast cancer model, and the application of M1-ALNPs in vivo is explored. After M1-ALNPs are intravenously injected into the breast cancer tumor-bearing mice, M1-ALNPs exhibit very good NIR-II fluorescence and photoacoustic imaging effects, which can provide comprehensive diagnostic information for subsequent photodynamic therapy; due to the enhanced phototherapy effect, M1-ALNPs can effectively inhibit tumor growth and cause a strong tumor-specific T cell immune response; on this basis, the TME is also successfully remodeled, TAM is converted from the M2 phenotype to the M1 phenotype, and the immune resistance is reduced. Based on the strong immunogenic cell death effect, M1-ALNPs can not only significantly inhibit breast cancer, but also produce a whole-cell therapeutic cancer vaccine to protect mice from tumor rechallenge, and provide a new idea for developing an efficient and multifunctional photodynamic therapy scheme.

[0249] The above embodiments of the application do not describe all the details, and the application is not limited to the above described embodiments. Various changes, modifications, replacements and variations of the embodiments made by those skilled in the art without departing from the principles and purposes of the application shall be included in the protection scope of the application.

Claims

1. An aggregation-induced emission luminescent material based on a selenodiazole structure, named TPA-Se, has the structure shown in formula (I): ， Chemical name: 4-hexyloxy-N-4-hexyloxyphenyl-N-4-7-tributyltin-2,3-dihydrothiophene[3,4-b][1,4]dioxane-5-ylphenylaniline-4,8-dibromo[1,2,5]selenodiazolo[3,4-f]benzo[c][1,2,5]thiadiazole.

2. A peptide self-assembled nanotherapeutic agent based on NIR-II imaging is an image navigation-immunotherapy-tumor microenvironment remodeling synergistic nanosystem constructed by using the aggregation-induced emission luminescent agent TPA-Se as described in claim 1 as a fluorescent probe, combining it with the mitochondrial targeted prodrug LND-1-PEG-24, and then coating it with immune cell membranes. in, The mitochondrial-targeting prodrug LND-1-PEG-24 is produced by linking a hydrophilic PEG group to the C-terminus of a peptide via an amidation reaction. 24 A drug-loaded peptide nanocarrier constructed by linking the N-terminus of the hydrophobic antitumor drug chlordamine has the following structural formula: LND-Phe-Phe-Arg-Phe-Lys-Gly-Phe-Leu-Gly-PEG 24 .

3. The polypeptide self-assembled nanotherapeutic agent according to claim 2, characterized in that... The immune cell membrane is the M1 macrophage membrane.

4. A method for preparing a peptide self-assembled nanotherapeutic agent based on NIR-II imaging, comprising: 1) Using the nanoprecipitation method, an organic phase containing the mitochondrial-targeting prodrug LND-1-PEG-24 and the aggregation-induced emission luminescent agent TPA-Se was injected into an aqueous phase to form a nanoparticle dispersion. After removing the organic solvent, a drug-loaded peptide nanoparticle ALNPs dispersion was prepared. 2) The drug-loaded polypeptide nanoparticles ALNPs dispersion is mixed with an immune cell membrane, and the immune cell membrane is coated onto the surface of the drug-loaded polypeptide nanoparticles ALNPs by ultrasonic co-extraction to form a polypeptide self-assembled nanotherapeutic preparation M1-ALNPs.

5. The method for preparing the polypeptide self-assembled nanotherapeutic agent according to claim 4, characterized in that: It also includes extrusion homogenization of the formed peptide self-assembled nanotherapeutic formulations.

6. The method for preparing the polypeptide self-assembled nanotherapeutic agent according to claim 4, characterized in that: The organic solvent is tetrahydrofuran.

7. The method for preparing the polypeptide self-assembled nanotherapeutic agent according to claim 4, characterized in that: Organic solvents in the nanoparticle dispersion were removed by stirring overnight, and the dispersion was then concentrated by centrifugation and filtration.

8. The method for preparing the polypeptide self-assembled nanotherapeutic agent according to claim 4, characterized in that: The mass ratio of the mitochondrial-targeting prodrug LND-1-PEG-24 to the aggregation-induced emission luminescent agent TPA-Se is 4–6:

1.

9. The method for preparing the polypeptide self-assembled nanotherapeutic agent according to claim 4, characterized in that: The mass ratio of drug-loaded polypeptide nanoparticles (ALNPs) dispersion to immune cell membranes was 1:1.5–2.

5.

10. The method for preparing the polypeptide self-assembled nanotherapeutic agent according to claim 4, characterized in that: The volume ratio of the organic phase to the aqueous phase is 1:8 to 12.

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