Tumor targeting compound as well as preparation and application thereof

Abstract

The invention provides a tumor targeting compound. The compound comprises a compound shown in a formula I and a compound shown in a formula II: # imgabs 0 #, I # imgabs 1 # and II. According to the compound disclosed by the invention, a monomeric compound can be selectively transferred into tumor cells, so that the monomeric compound is accurately assembled into nanofibers in lysosome, and a strong anti-tumor immune response is excited. The programmable assembly strategy provides a new possibility for creating an intelligent nano assembly as a pyroptosis inducer for precise cancer treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a tumor-specific complex and its preparation and application. Background Art

[0002] Pyroptosis is a type of programmed inflammatory cell death mediated by gasdermin-like proteins. It initiates inflammatory responses by forming pores in the cell membrane and triggering the release of proinflammatory cytokines, providing a new perspective for cancer immunotherapy. 1-4 To date, a variety of pyroptosis inducers have been reported, such as ions, small molecule drugs, and nanomaterials, highlighting their ability to activate pyroptotic death of cancer cells. 5-9 However, these inducers usually lack selectivity for cancer cells and may induce pyroptosis in normal tissue cells, causing systemic toxic side effects. Recently, photodynamic therapy has shown the potential to overcome the side effects and drug resistance of traditional pyroptosis inducers by specifically inducing pyroptosis of cancer cells through the generation of cytotoxic reactive oxygen species controlled by light. 10-15 However, its effectiveness is limited by the demand for light and oxygen, especially in the treatment of deep tumors and their metastases, mainly due to the limited penetration depth of light in biological tissues and the hypoxic characteristics of the tumor microenvironment. 16-18 Therefore, there is an urgent need to develop a strategy for selectively activating tumor pyroptosis that is independent of light and oxygen and can induce irreversible cell death, so as to better apply it to tumor immunotherapy.

[0003] Supramolecular interactions are involved in many key biological processes and regulate cellular functions essential for life. 19,20 Direct synthesis of supramolecules within living cells has emerged as a versatile strategy to control cell function and manipulate cell fate. 21-39 Specifically, the controlled self-assembly of synthetic small molecules into supramolecular structures within cancer cell lysosomes has been shown to be an effective cancer therapy. By causing irreversible lysosomal membrane damage, the formation of supramolecular structures within lysosomes can induce severe lysosomal dysfunction and lysosome-dependent cell death, thereby effectively overcoming cross-resistance. 40-43 At present, a variety of small molecule compounds of amphiphilic peptides that can be assembled into supramolecular structures have been developed as LMP inducers, but most of them induce cancer cell death through apoptosis, which usually inhibits inflammatory response, resulting in a weakened immune response to "cold" tumors. 44-47 Therefore, the controlled synthesis of supramolecular structures with high stability and efficiency within lysosomes to induce selective pyroptosis of cancer cells and subsequently elicit potent antitumor immune responses remains a challenge. Summary of the invention

[0004] The purpose of the present invention is to provide a complex for tumor targeting, which can stably target tumor cells, selectively induce pyroptosis of tumor cells, and further induce a strong anti-tumor immune response.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a tumor targeting complex comprising a compound of formula I and a compound of formula II:

[0007]

[0008] Formula I

[0009]

[0010] Formula II.

[0011] Preferably, the molar ratio of the compound of formula I to the compound of formula II in the complex is 1:1 to 100:1.

[0012] More preferably, the molar ratio of the compound of formula I to the compound of formula II in the composite is 5:1.

[0013] In a second aspect, the present invention provides a method for preparing the complex as described above, the method comprising separately preparing the compound of formula I and the compound of formula II, and then mixing them according to a proportion.

[0014] Preferably, the preparation method of the compound of formula I comprises:

[0015] 3,5-bis(trifluoromethyl)benzamide is connected to the detection group with a first flexible chain, and then the terminal amino group is connected on the other side of the detection group through a second flexible chain to obtain a compound of formula I, wherein the first flexible chain is an alkyl chain and the second flexible chain is a hydrophilic PEG chain.

[0016] Also preferably, the preparation method of the compound of formula II comprises:

[0017] 3,5-bis(trifluoromethyl)benzamide is connected to the detection group via a third flexible chain, and then the GALGLP peptide chain is connected on the other side of the detection group via a fourth flexible chain to obtain a compound of formula II, wherein the third flexible chain is a flexible chain containing a disulfide bond, and the fourth flexible chain is a hydrophilic PEG chain.

[0018] In a third aspect, the present invention discloses use of the complex as described above for targeting tumors.

[0019] In a fourth aspect, the present invention provides a drug for treating tumors, comprising the complex as described above.

[0020] Preferably, the medicament further comprises an immunotherapeutic agent.

[0021] More preferably, the immunotherapeutic agent is anti-PD-L1.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention discloses a tumor-specific lysosome destroyer and pyroptosis inducer, NP-NH-D5. NP-NH-D5 can sequentially respond to extracellular MMP-2 and intracellular GILT enzymes and GSH and other reducing substances, and selectively transport F-C6-NH2 into tumor cells, thereby accurately assembling into nanofibers in lysosomes. Experimental results show that NP-NH-D5 can induce GSDMD-mediated pyroptosis in different tumor cells, and stimulate a strong anti-tumor immune response by causing lysosomal membrane permeability and lysosomal destruction. In addition, the combined use of NP-NH-D5 and anti-PD-L1 significantly improved the survival rate of mice in immunotherapy for the treatment of advanced 4T1 lung metastasis and in situ Pan02 pancreatic tumors, without any systemic side effects. This strategy demonstrates the feasibility of inducing tumor cell pyroptosis and promoting anti-tumor immunity by finely regulating the charge and morphology of nanoassemblies. This programmable assembly strategy provides new possibilities for creating "intelligent" nanoassemblies as pyroptosis inducers for precise cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The diagram shows the induction of cell pyroptosis and anti-tumor immune response by NP-NH-D5 and the design principle of the present invention;

[0025] Figure 2 and Figure 3 Demonstrate the characterization of the cascade response of NP-NH-D5 to MMP-2 / GILT and GSH;

[0026] Figure 4 demonstrated that NP-NH-D5 was selectively taken up by tumor cells;

[0027] Figure 5 Demonstrate the lysosomal disruptive effect and cytotoxicity of NP-NH-D5;

[0028] Figure 6 The fluorescence imaging of NP-NH-D5 / Cy5.5 and NP-ctrl-1 / Cy5.5 treated groups in the tumor area is shown;

[0029] Figure 7 Shows H&E staining, Ki-67 staining, and TUNEL staining of tumor tissue sections resected from orthotopic 4T1 tumor model mice after receiving the indicated treatments;

[0030] Figure 8 Bio-TEM images of tumor tissues in mice with orthotopic 4T1 tumor model injected via tail vein are shown. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] In the description of the present invention, it should be noted that the terms "upper", "lower", "inner", "outer", "front end", "rear end", "two ends", "one end", "the other end" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0033] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "provided with", "connected", etc. should be understood in a broad sense. For example, "connected" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0034] In this work, we developed a programmable nanoparticle NP-NH-D5 through a co-assembly strategy, aiming to construct functional nanostructures in situ within cancer cell lysosomes and activate inflammatory forms of cell death on demand to achieve efficient antitumor immunotherapy.

[0035] Figure 1 A schematic diagram showing NP-NH-D5-induced cell pyroptosis and anti-tumor immune response. Figure 1 a is the chemical structure of F-C6-NH2 and F-SS-NH-GALGLP-D5. Figure 1 b shows the process of preparing NP-NH-D5 by coassembling F-C6-NH2 and F-SS-NH-GALGLP-D5, and the schematic diagram of the transformation of spherical nanoparticles (NPs) into nanofibers (NFs) by the tandem response of MMP-2 and GSH to induce GSDMD-mediated cell pyroptosis. Figure 1 c Schematic diagram showing that NP-NH-D5 selectively delivers F-C6-NH2 to the tumor site for cancer immunotherapy. After systemic administration, negatively charged NP-NH-D5 enters the tumor tissue from the blood circulation. In tumor tissues where MMP-2 is overexpressed, NP-NH-D5 is first converted into positively charged NPs (NP-NH2) through MMP-2-mediated removal of the GLP-D5 sequence. These positively charged nanoparticles then efficiently enter tumor cells through enhanced endocytosis and accumulate in lysosomes. Once NP-NH2 enters the lysosome, reducing substances such as GILT enzymes and GSH reduce the disulfide bonds in NP-NH2, resulting in the release of small molecule products F-SH and NH-GAL from the cleavage. In this process, the released F-C6-NH2 is reassembled into nanofibers with high rigidity and rich amino groups in the lysosome, strongly inducing LMP, leading to severe lysosomal damage and promoting the release of mature cathepsin B (Cathepsin B, CatB) into the cytoplasm. Subsequently, mature CatB induces the assembly of NLRP3 inflammasome and the activation of Casp-1, thereby initiating pyroptosis of tumor cells through Casp-1-mediated cleavage of GSDMD-FL into GSDMD-N. Figure 1 d shows the chemical structures of the cleavage products F-SH, NH-GAL, and GLP-D5.

[0036] In the present invention, we synthesized two molecules, one of which is a small molecule non-peptide amphiphilic compound (F-C6-NH2), which can accumulate in lysosomes and self-assemble into nanofibers, thereby causing cell pyroptosis. Here, in order to improve the selectivity of F-C6-NH2 for tumor cells, we further synthesized F-SS-NH-GALGLP-D5 (1a) containing five negatively charged aspartic acid (D5). Unlike F-C6-NH2, F-SS-NH-GALGLP-D5 can be hydrolyzed by two biomarkers upregulated in tumor sites—extracellular matrix metalloproteinase-2 (MMP-2) and reduced by intracellular GILT enzymes and glutathione (GSH). F-C6-NH2 and F-SS-NH-GALGLP-D5 were co-assembled to form spherical nanoparticles (NPs, NP-NH-D5) with negative charges on the surface. The conversion of NPs to NFs was achieved through the tandem response to extracellular MMP-2 and intracellular GILT enzymes and GSH and other reducing substances ( Figure 1b). Due to the negative charge on the surface, NP-NH-D5 is difficult to enter normal tissue cells. However, in tumor tissues, through MMP-2-mediated hydrolysis, NP-NH-D5 is converted into NP-NH2, which is positively charged and easily absorbed by tumor cells, and then accumulates in lysosomes. Subsequently, the abundant endogenous GSH can continue to reduce the disulfide bonds in NP-NH2, releasing the cleavage small molecule products (F-SH and NH-GAL). In addition, the released F-C6-NH2 can reassemble into rigid and amino-rich nanofibers, causing lysosomal damage and GSDMD-mediated cell pyroptosis. This pyroptosis can induce the secretion of proinflammatory factors, stimulate a strong ICD effect, and reverse the immunosuppressive tumor microenvironment, which helps to shape immunogenic "hot" tumors for effective anti-tumor immunotherapy ( Figure 1 cd). We demonstrated that NP-NH-D5 could effectively eradicate orthotopic 4T1 breast tumors, inhibit tumor metastasis and recurrence, and prolong the survival of mice. In addition, NP-NH-D5 could synergize with anti-PD-L1 antibody (α-PD-L1) immunotherapy in advanced lung metastasis and orthotopic Pan02 pancreatic cancer models to stimulate a strong anti-tumor immune response.

[0037] Experimental Section

[0038] 1. Reagents and Instruments

[0039] All materials were purchased from qualified reagent suppliers and were of analytical grade without further purification. Matrix metalloproteinase-2 (MMP-2) and SB-3CT were obtained from R&D Systems, USA. Cy5.5-NHS and Lyso-Tracker@RedDND-99 were obtained from Thermo Fisher Scientific Inc., USA. MTT kit, trypsin, DAPI and Annexin V-FITC / PI apoptosis staining kit were purchased from KeyGen Biotech. Co. Ltd., Nanjing, China. Anti-PD-L1 antibody was obtained from BioCell (BE0101, 832023M2). Anti-CD11c-FITC (C0114081320363, 35-0114-U100), anti-CD4-APC (C0041043019202, 20-0041-U025), and anti-CD8a-PE (C1886080519502, 50-1886-U025) antibodies were purchased from Tonbo, San Diego, USA. Anti-CD80-PE (B282893, 104707), anti-CD86-APC (B346111, 105011), Zombie (B384493, 77143), anti-CD3-FITC (B3 61053, 100204), anti-CD8a-PE-Cy7 (B295389, 100722), anti-CD8a-APC (B308247, 100711), anti-CD25-PB (B365395, Antibodies: anti-CD3 (100201, B292651, 1:1000) antibody was purchased from Thermo Fisher Scientific Inc. Anti-CD4 (SC-13573, #L0519, 1:1000) antibody was purchased from Santa Cruz Biotechnology, Texas, USA.Anti-Calprotectin (CRT) (ab92516, GR3287998-30, 1:1000), anti-CD8 (ab217344, GR3366917-14, 1:1000) and AlexaFluor-488 conjugated goat anti-rabbit (ab150077, GR3365766-1, 1:1000) antibodies were purchased from Abcam, UK. ELISA kits for detecting HMGB1, IL-18, IL-1β, IL6, IFN-γ and TNF-α were purchased from CUSABIO, China. ELISA kits for detecting ATP were purchased from Bio-Tech, China. Western blotting antibodies: GSDMD (ab219800, GR3424478-14, 1:1000), Caspase-1 (ab184787, GR3396760-9, 1:1000), β-actin (ab8226, GR3249122-19, 1:1000) were obtained from abcam, UK. Anti-mouse IgG HRP-labeled antibody (7076S, 1:1000) and anti-rabbit IgG HRP-labeled antibody (7074P2, 1:1000) were obtained from Cell Signaling Technology, USA.

[0040] Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry and liquid chromatography-mass spectrometry (LC-MS) were acquired on an ABSCIEX 4800 Plus MALDITOF / TOFTM mass spectrometer and an Agilent 6540 UHDQ-TOF LC / MS mass spectrometer, respectively. Analytical and semi-preparative HPLC were performed on a Thermo Scientific Dionex Ultimate 3000 instrument with a mixture of acetonitrile and water containing 1‰ trifluoroacetic acid as the mobile phase. UV-visible absorption spectra were measured using an OceanOptics Maya 2000Pro UV-Visible spectrometer. Fluorescence spectra were measured using a HORIBA JobinYvon Fluoromax-4 fluorescence spectrometer. Dynamic light scattering analysis and zeta potential analysis were performed on a 90Plus / BI-MAS particle size analyzer (Brookhaven, USA). Transmission electron microscopy images were acquired on a JEM-2800 transmission electron microscope. Circular dichroism spectra were measured by an Applied Photophysis Chirascan. Fluorescence imaging of cells and tissue sections was performed using an SP8 confocal laser scanning microscope or an Olympus IX73 fluorescence inverted microscope. The MTT corresponding absorption value was read using a microplate reader (Tcan) to complete the cytotoxicity experiment. Flow cytometry analysis was performed on a Coulter FC-500 flow cytometer or a NovoCyte Quanteon (Agilent Technologies, Inc., USA). In vivo fluorescence and bioluminescence (BLI) images were captured and stored using the IVIS Lumina XR III system. ROIs in the images were quantitatively analyzed using Living image software (PerkinElmer, USA).

[0041] 2. Synthesis Example

[0042] 1. Synthesis of Compound 2-1

[0043]

[0044] Synthesis route of compound 2-1. Reaction conditions: (a) (i) DMAP, 90-120℃, triphosgene, toluene, 4 h; (ii) dry DCM, overnight, 63%; (b) (i) Cysteine, DCM / MeOH (1 / 1), DIPEA, 3 h; (ii) MnO2, overnight, 83%; (c) (i) HBTU, Boc-NH-PEG4-CH2CH2NH2, 4 h; (ii) 30% TFA, DCM, rt, 1 h, 75%.

[0045] 1.1. Synthesis of compound 2-6:

[0046] Compound CBT-NH2 (220 mg, 1.25 mmol) and 4-dimethylaminopyridine (DMAP, 152 mg, 1.25 mmol) were dissolved in 45 mL of dry toluene, heated to 90-120 ° C under nitrogen protection, and stirred for 15 minutes. The reaction solution was then transferred to an ice-water bath and stirred at 0 ° C for 15 minutes. Then, triphosgene (326 mg, 1.10 mmol) dissolved in 10 mL of dry toluene was added dropwise to the above reaction solution under nitrogen protection. After the addition was completed, the reaction solution was moved to an oil bath and heated and stirred at 120 ° C for 4 hours. After the reaction was completed, the solvent was dried, and the residue was protected by nitrogen and then 20 mL of ultra-dry dichloromethane was added to dissolve it. Then, compound 2-5 (357 mg, 1.00 mmol) was dissolved in 10 mL of ultra-dry dichloromethane solution and slowly added to the reaction solution at 0 ° C. After the addition was completed, the reaction solution was protected by nitrogen and stirred at room temperature overnight. The next day, the solvent was dried by spin drying. The obtained product was eluted by silica gel column chromatography with a dichloromethane / methanol mixture at a slowly increasing gradient. After this purification treatment, a white solid compound 2-6 (350 mg, yield: 63%) was obtained. 1H NMR (400 MHz, DMSO-d6) δ 10.20 (s, 1H), 8.95 (t, J = 5.4Hz, 1H), 8.50 (d, J = 3.9 Hz, 3H), 8.30 (s, 1H), 8.15 (d, J = 9.0 Hz, 1H), 7.68 (dd, J = 9.1, 2.1 Hz, 1H), 4.15 (t, J = 6.6 Hz, 2H), 3.34 – 3.28 (m,2H), 1.72 – 1.63 (m, 2H), 1.63 –1.52 (m, 2H), 1.49 – 1.28 (m, 4H). 13 C NMR(101 MHz, DMSO-d6) δ 163.53, 154.04, 147.57, 140.57, 137.36, 137.16, 134.76,131.07, 130.74, 128.45, 128.41, 125.26, 122.25, 120.47, 114.07, 110.14,65.06, 29.24, 28.84, 26.59, 25.55. MS: calcd. for C 24 H 20 F6N4NaO3S+ [M + Na]+ :581.1053; HRMS found: m / z 581.1030.

[0047] 1.2. Synthesis of compound 2-7:

[0048] At room temperature, compound 2-6 (307 mg, 0.55 mmol), L-cystine (73 mg, 0.6 mmol) and N,N-diisopropylethylamine (DIPEA, 142 mg, 1.1 mmol) were added to a mixed solvent of dichloromethane / methanol (1 / 1, 10 mL) and stirred at a uniform speed. After waiting patiently for 3 hours, the reaction progress was monitored by thin layer chromatography. After the reaction was completed, manganese dioxide (MnO2, 860 mg, 10 mmol) was added and stirred overnight. Subsequently, the reaction mixture was filtered and dried. The residue was purified by silica gel column chromatography using a mixed solvent of dichloromethane / methanol to obtain a light yellow solid compound 1-3 (301 mg, yield: 83%). 1H NMR (400 MHz, DMSO-d6) δ 10.05 (s, 1H), 8.96 (d, J= 10.9 Hz, 1H), 8.69 (s, 1H), 8.50 (s, 2H), 8.38 (s, 1H), 8.30 (s,1H), 8.03 (d, J = 8.9 Hz, 1H), 7.58 (d, J = 6.8 Hz, 1H), 4.14 (t, J = 6.6 Hz, 2H), 3.33 (dd, J =12.8, 6.8 Hz, 2H), 1.75 – 1.63 (m, 2H), 1.62 – 1.53 (m,2H), 1.42 (m,4H). 13 C NMR (101 MHz, DMSO-d6) δ 163.53, 162.05, 161.57,158.72, 154.11, 148.88, 148.71, 138.81, 137.16, 136.54, 131.91, 131.07,130.73, 128.45, 128.41, 125.17, 124.96, 124.04, 122.25, 119.34, 110.59,64.90, 29.24, 28.88, 26.61, 25.57. MS: calcd. for C 27 H 22 F6N4NaO5S2+ [M + Na]+ :683.0828; HRMS found: m / z 683.0826.

[0049] 1.3. Synthesis of compound 2-1:

[0050] Compound 2-7 (165 mg, 0.25 mmol), 1,1-dimethylethyl 16-amino-5,8,11,14-tetraoxa-2-azahexadecanoate (Boc-NH-PEG4-CH2CH2NH2, 101 mg, 0.3 mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 142 mg, 0.38 mmol) and DIPEA (113 mg, 0.88 mmol) were stirred at room temperature for 4 hours in 10 mL of anhydrous tetrahydrofuran (THF). The progress of the reaction was monitored by thin layer chromatography. After the reaction was completed, the solvent was removed by spin drying. The obtained residue was purified by silica gel column chromatography with DCM / MeOH as the eluent. The purified solid was then dissolved in a dichloromethane solution (6 mL) containing 30% trifluoroacetic acid and stirred for 1 hour to remove the tert-Butyloxycarbonyl (Boc). After that, the liquid was dried by spin drying and further purified by semi-preparative HPLC. After treatment with liquid nitrogen and freeze drying, a light yellow solid compound 2-1 (165 mg, yield: 75%) was obtained. 1H NMR (400 MHz, DMSO-d6) δ10.06 (s, 1H), 8.97 (t, J =5.4 Hz, 1H), 8.51 (s, 1H), 8.50 (s, 2H), 8.45 (d, J = 5.8 Hz, 1H), 8.42 (d, J= 1.8 Hz, 1H), 8.30 (s, 1H), 8.04 (d, J = 8.9 Hz, 1H), 7.56 (dd, J = 9.0, 2.1Hz, 1H), 4.14 (t, J = 6.6 Hz, 2H), 3.66 – 3.43 (m, 18H), 3.33 (dd, J = 12.9,6.8 Hz, 2H), 3.04 – 2.87 (m, 2H), 1.67 (dq, J = 13.4, 6.7 Hz, 2H), 1.58 (dd,J = 13.7, 7.0 Hz, 2H), 1.48 – 1.35 (m, 4H). 13C NMR (126 MHz, DMSO-d6) δ163.50, 161.34, 160.43, 158.42, 154.11, 151.51, 148.65, 138.82, 137.11,136.57, 131.01, 130.75, 128.43, 127.56, 125.16, 124.67, 124.04, 122.50,119.37, 110.40, 70.23, 70.20, 70.09, 70.06, 70.02, 69.24, 67.13, 64.90,39.26, 39.11, 29.25, 28.88, 26.61, 25.58. MS: calcd. for C 37 H 45 F6N6O8S2+ [(M +H)] + : 879.2639; found ESI-MS: m / z 879.2622.

[0051] 2. Synthesis of Compound 3-1

[0052]

[0053] Synthesis of compound F-SS-NH-GALGLP-D5 (3-1). Reaction conditions: (a) SPPS procedure, rt, 36h, 74%; (b) HBTU, DIPEA, THF, rt, 4h, 73%; (c) TFA, TIPSH, DCM, rt, 3h, 82%; (d) i: 5%piperidine / DMF, rt, 10min; ii: HCl (1M), adjusted pH to 7.0, rt, 72%.

[0054] 2.1. Synthesis of compound 3-4:

[0055] This peptide was synthesized by standard solid phase peptide synthesis (SPPS) technology, using 2-chlorotrityl chloride resin and the corresponding 9-fluorenylmethyloxycarbonyl (9-Fluorenylmethyloxycarbonyl, Fmoc) protecting amino acids. The specific steps are as follows: First, the starting amino acid Fmoc-Gly was loaded onto the resin at a ratio of approximately 1.2 equivalents. After loading, it was treated with a capping reagent (DCM: MeOH: DIPEA = 17: 2: 1) to ensure that all active sites on the resin participated in the reaction. Next, a 20% piperidine / DMF solution was used to remove the Fmoc protecting group, and then HBTU was used as a coupling reagent to achieve the next step of connecting the Fmoc protected amino acid to the exposed free amino group. Fmoc-GALGLPD(OtBu)D(OtBu)D(OtBu)D(OtBu)D(OtBu)-OH was synthesized by sequential coupling of Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Pro-OH and Fmoc-Asp(OtBu)-OH (5 equivalents each) by SPPS strategy. Afterwards, the polypeptide chain was released from the resin using a 1% TFA / DCM mixed solvent, and the reaction product was collected and spin-dried. Finally, cold ether (Et2O, 20 mL) was added to the dry residue, and the white solid product 3-4 (601 mg, yield: 74%) was obtained by centrifugation. MS: calcd. For C 79 H 117 N 11 NaO 24 + [(M+Na) + ]:1626.8165; found ESI-MS: m / z 1626.8104.

[0056] 2.2. Synthesis of compound 3-5:

[0057] Compound 2-3 (synthesized according to the previously reported method (Research2020, 4087069), 30 mg, 0.033 mmol), compound 3-4 (79.4 mg, 0.05 mmol), HBTU (19 mg, 0.05 mmol) and DIPEA (15 µL, 0.066 mmol) were mixed and added to 3 mL of anhydrous THF, and then stirred at room temperature for 4 hours. After the reaction was completed, the solvent was removed by rotary evaporation. The residue was then purified by semi-preparative HPLC, and finally freeze-dried and vacuum-dried to obtain a light yellow solid 3-5 (60 mg, yield: 73%). MS: calcd. For C114 H 157 F6N 17 O 31 S 42 +[(M+2H) 2+ ]: 1251.5021; found ESI-MS: m / z 1251.5088.

[0058] 2.3. Synthesis of Compound 3-6:

[0059] Compound 3-5 (50 mg, 0.02 mmol) was dissolved in a mixed solution of DCM, TFA and TIPSH (volume ratio = 2.5 / 90 / 2.5, 10 mL) and stirred at room temperature for 3 hours to remove the tert-butoxy group. The subsequent operation was the same as before, that is, after the reaction was complete, the TFA was removed as much as possible by rotating the evaporator and vacuuming the oil pump for several hours. After that, the residue was dissolved in a small amount of N,N-dimethylformamide (DMF) and a trace amount of deionized water was added as appropriate. Finally, the mixed solution was purified by semi-preparative HPLC using a suitable elution method. After freeze-drying, a light yellow solid 3-6 (36 mg, yield: 82%) was obtained. MS calcd.For C 94 H 113 F6N 17 O 31 S4 2- [(M-2H) 2- ]: 1109.3310; found ESI-MS: m / z 1109.3335.

[0060] 2.4. Synthesis of compound 3-1:

[0061] To remove the Fmoc protecting group in compound 3-6, the compound (30 mg, 0.014 mmol) was first dissolved in 5 mL of 5% piperidine / DMF solution and stirred at room temperature for 10 minutes. Then, the pH of the reaction solution was adjusted to 7.00 using 1 M hydrochloric acid under ice-water bath conditions. Next, the resulting mixed solution was purified by semi-preparative HPLC. After freeze-drying, a light yellow solid F-SS-NH-GALGLP-D5 (19 mg, yield: 72%) was obtained. MS: calcd. For C 79 H 107 F6N 17 NaO 29 S4 3+ [(M+2H+Na) 3+ ]:674.2030; found ESI-MS: m / z 674.1913.

[0062] 3. Synthesis of Compound 3-2

[0063]

[0064] Synthesis of compound F-SS-NH-galglp-D5. Reaction conditions: (a) SPPS procedure, rt, 36h, 75%; (b) HBTU, DIPEA, THF, rt, 4h, 69%; (c) TFA, TIPSH, DCM, rt, 3h, 84%; (d) i: 5%piperidine / DMF, rt, 10min; ii: HCl (1M), adjusted pH to 7.0, rt, 76%.

[0065] 3.1. Synthesis of Compound 3-7:

[0066] Through SPPS technology, using 2-chlorotrityl chloride resin and the corresponding Fmoc protected amino acid, a peptide molecule containing a D-amino acid sequence was successfully synthesized: Fmoc-galglp-D(OtBu)D(OtBu)D(OtBu)D(OtBu)D(OtBu)-OH (galglp represents a peptide chain containing a D-amino acid). First, Fmoc-Gly was used as the starting amino acid and loaded onto the resin at about 1.2 equivalents. After loading, it was treated with a capping reagent (DCM: MeOH: DIPEA = 17: 2: 1) to ensure that all active sites on the resin were fully reacted. Subsequently, a 20% piperidine / DMF solution was used to remove the 9-fluorenylmethoxycarbonyl (Fmoc), and then HBTU was used as a coupling reagent to achieve the connection between the next Fmoc protected amino acid and the exposed free amino group. According to the SPPS strategy, (D)-Fmoc-Ala-OH, (D)-Fmoc-Leu-OH, Fmoc-Gly-OH, (D)-Fmoc-Leu-OH, (D)-Fmoc-Pro-OH and Fmoc-Asp(OtBu)-OH (5 equivalents each) were gradually added to 2-chlorotrityl chloride (1 equivalent) to construct the target peptide chain. Next, the peptide chain was released from the resin using a 1% TFA / DCM mixed solvent, and the reaction solution was collected and the solvent was removed by rotary evaporation under vacuum. Finally, Et2O (20 mL) was added to the dried residue, and the white solid product 3-7 (611 mg, yield: 75%) was obtained by centrifugation. MS: calcd. For C 79 H 117 N 11 NaO 24 + [(M+Na) +]:1626.8165; found ESI-MS: m / z 1626.8077.

[0067] 3.2. Synthesis of Compound 3-8:

[0068] 2-3 (30 mg, 0.033 mmol), compound 3-7 (79.4 mg, 0.05 mmol), HBTU (19 mg, 0.05 mmol) and DIPEA (15 µL, 0.066 mmol) were added to 3 mL of anhydrous THF and stirred at room temperature for 4 hours. After the reaction was completed, the solvent was removed by rotary evaporation. Finally, semi-preparative HPLC was used for purification to obtain a light yellow solid 3-8 (57 mg, yield 69%). MS: calcd. For C 114 H 157 F6N 17 O 31 S4 2+ [(M+2H) 2+ ]: 1251.5021; found ESI-MS: m / z1251.5077.

[0069] 3.3. Synthesis of Compound 3-9:

[0070] To remove the tert-butyloxy protecting group, compound 3-8 (55 mg, 0.022 mmol) was dissolved in 10 mL of a mixed solvent consisting of dichloromethane, trifluoroacetic acid and TIPSH in a volume ratio of 2.5 / 90 / 2.5. Subsequently, the mixed solution was stirred at room temperature for 3 hours to ensure that the reaction was complete. After the reaction was completed, the solvent was removed by spin drying. The residue was purified by semi-preparative HPLC to obtain a light yellow solid 3-9 (41 mg, yield: 84%). MS: calcd. for:C 94 H 113 F6N 17 O 31 S4 2- [(M-2H) 2- ]: 1109.3310; found ESI-MS: m / z 1109.3336.

[0071] 3.4. Synthesis of compound 3-2:

[0072] The Fmoc protecting group in compound 3-9 (40 mg, 0.019 mmol) was removed using 5 mL of 5% piperidine / DMF solution (volume ratio). After stirring at room temperature for 10 minutes, the pH of the reaction solution was adjusted to 7.00 using 1 M aqueous hydrochloric acid. Subsequently, the residue was purified by semi-preparative HPLC and lyophilized to obtain a light yellow solid F-SS-NH-galglp-D5 (28 mg, yield: 76%). MS: calcd. for: C 79 H 107 F6N 17 NaO 29 S4 3+ [(M+2H+Na) 3+ ]: 674.2030; found ESI-MS: m / z674.1910.

[0073] 4. Synthesis of Compound 3-3

[0074]

[0075] Synthesis of compound F-C6-NH-GALGLP-D5. Reaction conditions: (a) HBTU, DIPEA, THF, rt, 4h, 75%; (b) TFA, TIPSH, DCM, rt, 3h, 79%; (c) i: 5%piperidine / DMF, rt, 10min; ii: HCl (1M), adjusted pH to 7.0, rt, 79%.

[0076] 4.1. Synthesis of Compound 3-10:

[0077] Compound 2-1 (40 mg, 0.046 mmol), compound 3-4 (110 mg, 0.069 mmol), HBTU (26 mg, 0.69 mmol) and DIPEA (21 µL, 0.092 mmol) were mixed in 3 mL of anhydrous THF and stirred at room temperature for 4 hours. After the reaction was complete, the solvent was dried and the residue was purified by semi-preparative HPLC to obtain a light yellow solid 3-10 (57 mg, yield: 69%). MS: calcd. for C 114 H 157 F6N 17 O 31 S4 2+ [(M+2H) 2+ ]: 1251.5021; found ESI-MS: m / z 1251.5077.

[0078] 4.2. Synthesis of Compound 3-11:

[0079] Compound 3-10 (40 mg, 0.016 mmol) was dissolved in 10 mL of a solvent prepared by DCM, TFA, and TIPS in a volume ratio of 2.5 / 90 / 2.5 and stirred at room temperature for 3 hours to remove the tert-butyl ester (OtBu) group. After the reaction was completed, the solvent was removed under vacuum and the residue was purified by semi-preparative HPLC to obtain the desired product as a light yellow solid 3-11 (28 mg, yield: 79%). MS: calcd. for: C 96 H 121 F6N 17 O 31 S2 2+ [(M+2H) 2+ ]: 1093.3892; found ESI-MS: m / z1093.3936.

[0080] 4.3. Synthesis of compound 3-3:

[0081] Compound 3-11 (25 mg, 0.011 mmol) was treated with 3 mL of DMF solution containing 5% piperidine by volume to remove the Fmoc protecting group. After stirring at room temperature for 10 minutes, the pH of the reaction solution was adjusted to 7.0 by adding 1 M hydrochloric acid. Next, the mixture was purified by semi-preparative HPLC and lyophilized to obtain a light yellow solid F-C6-NH-GALGLP-D5 (17 mg, yield: 79%). MS calcd. for: C 81 H 112 F6N 17 O 29 S2 2+ [(M+2H) 2+ ]:982.2573; found ESI-MS: m / z 981.8559.

[0082] 5. Synthesis of Compound 3-12

[0083]

[0084] Synthesis of compound F-C6-NH-Cy5.5. Reaction conditions: (a) DIPEA, DMF, rt, 1 h, 69%;

[0085] Synthesis of compound F-C6-NH-Cy5.5:

[0086] Compound 2-1 (0.36 mg, 0.41 μmol), Cy5.5-NHS (0.51 mg, 0.45 μmol) and DIPEA (0.5 μL, 0.002 mmol) were added to 0.5 mL of anhydrous DMF and stirred at room temperature for 1 hour. The reaction progress was monitored in real time by HPLC. After the reaction was completed, the residue was purified by semi-preparative HPLC. Finally, after freeze-drying, a blue solid F-C6-NH-Cy5.5 (0.5 mg, yield: 69%) was obtained. MS: calcd. ForC 78 H 87 F6N8O 21 S6 + [M + ]: 1777.4209; found MALDI-MS: m / z 1777.1877.

[0087] 3. Experimental Design

[0088] 1. Confirmation of the co-assembly ratio of nanoparticles NP-NH-D5

[0089] Compounds F-C6-NH2 and F-SS-NH-GALGLP-D5 were dissolved in DMSO to prepare stock solutions. To determine their specific concentrations, the molecular UV absorption was measured based on the amino-oxidized firefly luciferin group in each molecular structure, and the calculation was unified by the Lambert-Beer law. Subsequently, compounds F-C6-NH2 and F-SS-NH-GALGLP-D5 with different molar ratios were mixed in different molar ratios (0 / 1, 1 / 1, 2 / 1, 5 / 1, 10 / 1, 20 / 1, 100 / 1, 1 / 0), and the concentration of each mixture was adjusted to 10 mM. Then, the above DMSO solution was slowly added to PBS buffer with the assistance of 40 kHz ultrasound. After 10 minutes of ultrasonic treatment, assembled nanoparticles were obtained at a concentration of 100 μM.

[0090] Dynamic light scattering analysis and Zeta potential analysis of each solution were performed using a 90Plus / BI-MAS analyzer produced by Brookhaven, USA. For NP-NH-D5 nanoparticles with a molar ratio of 5 / 1 (F-C6-NH2 / F-SS-NH-GALGLP-D5), the solution was dropped onto a carbon-coated copper grid and immediately freeze-dried for transmission electron microscopy analysis. Subsequently, the samples were observed on a JEM-1011 transmission electron microscope.

[0091] 2. Stability characterization of nanoparticles

[0092] To evaluate the stability of NP-NH-D5 under physiological conditions, 1 mL of NP-NH-D5 solution (100 μM) was prepared in PBS buffer and DMEM medium containing 10% FBS, respectively, and stored at room temperature for 1 to 7 days. The hydrodynamic size and fluorescence properties of NP-NH-D5 were monitored daily by DLS and fluorescence spectrometer, respectively.

[0093] 3. Study on the cascade response of nanoparticles to MMP-2, GILT enzyme and GSH

[0094] 2 mL of NP-NH-D5 solution (100 μM) was incubated with MMP-2 (8 nM) in enzyme reaction buffer (TNCB buffer, 150 mM NaCl, 10 mM CaCl2, 100 mM Tris·HCl, 0.05% Brij35, pH = 7.4) at 37 °C for 8 h. Subsequently, GSH (10 mM) was added and incubated at 37 °C for 0.5, 2, and 6 h. Appropriate amounts of solution were taken out at different time points for corresponding DLS, HPLC, CD, FL, Zeta potential, and TEM analysis.

[0095] Similarly, NP-NH-D5 (100 μM) was incubated with MMP-2 (8 nM) at 37 °C and then incubated with GSH (1.5 mM) or GILT enzyme (2.5 μg + 20 μM cysteine) for 2 h. Appropriate amount of solution was taken out for HPLC analysis.

[0096] The relevant characterizations of the other three control probes were similar to those of NP-NH-D5.

[0097] 4. Cell culture

[0098] Mouse breast cancer cells (4T1 and 4T1-Luc cells), mouse colon cancer CT-26 cells, mouse pancreatic cancer cells (Pan02 and Pan02-Luc), human breast cancer MCF-7 cells, human gastric epithelial cells GES-1 cells, human cervical cancer HeLa cells, human brain glioma U87MG cells, human hepatocellular carcinoma HepG2 cells, human gastric cancer MKN45 cells, and human lung cancer A549 cells were purchased from the Stem Cell Bank of the Chinese Academy of Sciences (Shanghai). 4T1, 4T1-Luc, HeLa, HepG2, U87MG, Pan02, Pan02-Luc, and A549 cells were cultured in DMEM supplemented with 10% FBS and appropriate amounts of penicillin and streptomycin. MKN45, GES-1, and CT-26 cells were cultured in RPMI-1640 supplemented with 10% FBS and appropriate amounts of penicillin and streptomycin. The cells were cultured at 37°C in a humidified atmosphere containing 5% CO2 and 95% air.

[0099] 5. Cell fluorescence imaging and cell co-localization experiments

[0100] 5×10 4 4T1 cells were seeded in four-well glass-bottomed confocal dishes and cultured overnight before use. To evaluate the uptake of NP-NH-D5 by 4T1 cells over time, 4T1 cells were incubated with NP-NH-D5 (30 μM, 0.5 mL) at 37°C for 0.5, 1, 2, and 4 hours. The old medium was aspirated and the cells were rinsed with PBS. In the subsequent operation, fresh medium was added and fluorescence images were taken using a Leica TCS SP8 confocal laser scanning microscope. Fluorescence images in the F-C6-NH2 channel were acquired with an excitation light of 350 ± 22.5 nm and an emission wavelength of 420-460 nm. Fluorescence images in the AO-Luc channel were acquired with an excitation light of 350 ± 22.5 nm and an emission wavelength of 510-550 nm.

[0101] To evaluate the uptake of NP-NH-D5 by 4T1 cells, 4T1 cells were incubated with different concentrations of NP-NH-D5 (5, 10, 20, 30, and 50 μM) for 4 hours. After the incubation, the medium was removed and the cells were washed three times with PBS. Finally, fresh medium was added and fluorescence images were taken using an inverted microscope. Fluorescence images in the F-C6-NH2 channel were acquired with an excitation wavelength of 350 ± 22.5 nm and an emission wavelength of 420-460 nm.

[0102] To examine the intracellular localization, the original culture medium was first removed, and then 4T1 cells were incubated with NP-NH-D5 (30μM) for 30 minutes. After the incubation was completed, the cells were washed three times with PBS. A dilution of Lyso-TrakerRedDND-99 (1μM) was prepared and incubated with the cells for 15 minutes. After the incubation was completed, the culture medium was carefully removed. Similarly, the cells were washed several times with PBS to ensure that the residual dye in the culture medium was removed. After adding fresh culture medium to the dish, the fluorescence image was immediately collected using an inverted microscope. Among them, the excitation wavelength of the fluorescence channel corresponding to NP-NH-D5 is 350±22.5nm, and the emission wavelength range is 420-460nm. 350±22.5nm, the emission wavelength range is 420-460nm. The excitation wavelength of the corresponding fluorescence channel of LysoTrackerRed is 570nm excitation, and the emission wavelength range is 600-650nm.

[0103] 6. Specific imaging experiment of probe on tumor cells

[0104] 4T1, A549, HeLa and GES-1 cells (density 5×10 4Cells / well) were seeded in glass-bottomed culture dishes and cultured overnight at 37°C until the cells attached to the wall. The next day, after removing the culture medium, fresh culture medium containing NP-NH-D5 (20μM, 0.5mL) was added to the cells and incubated at 37°C for 4 hours. In order to study the effect of MMP-2 enzyme activity on NP-NH-D5 uptake, 4T1 cells were pretreated with SB-3CT (20µM) 1 hour before incubation with NP-NH-D5 to inhibit the activity of MMP-2. After the incubation, the culture medium was removed. Similarly, the cells were washed with PBS several times to ensure that the residues in the culture medium were removed. After replacing the fresh culture medium, the fluorescence images were captured using an inverted microscope. The fluorescence images of the F-C6-NH2 channel were acquired with an excitation wavelength of 350±22.5nm and an emission wavelength range of 420-460nm.

[0105] 7. HPLC study of the cascade response of intracellular NP-NH-D5

[0106] First, 1×10 6 4T1 cells were inoculated in a 6 cm cell culture dish and incubated overnight at 37 °C in an incubator. The next day, the original culture medium was replaced with 2 mL of culture medium containing NP-NH-D5 (30 μM) and incubated for different times (0.5, 1, 2, and 4 hours). After the incubation, the extracellular medium was collected first. At the same time, the cells were digested with trypsin, and the cell pellet was obtained after counting and centrifugation. Subsequent operations should be carefully performed according to the following steps. That is, the cells were first lysed with 250 μL of DMSO, and then 250 μL of cold methanol and 500 μL of deionized water were added to the lysate. Then, the mixture was centrifuged at 14,000 rpm for 10 minutes at 4 °C and set aside. Finally, 250 μL of the supernatant of the cell lysate or 500 μL of the culture medium were injected into HPLC for analysis.

[0107] 8. Assessment of lysosomal rupture

[0108] To assess lysosomal disruption, 4T1 cells were stained with acridine orange (AO). First, 4T1 cells were seeded in a 4-well confocal dish and incubated overnight at 37.0°C. Then, 4T1 cells were incubated with NP-NH-D5 or other control nanoparticles (30 μM) for 4 hours. The cells were washed three times with PBS and 5.0 μM AO was added, and then incubated in an incubator for another 15 minutes. Subsequent operations were as described above, that is, the cells were washed with PBS for multiple rounds to ensure that the residual dye in the culture medium was removed. After replacing fresh culture medium in the dish, fluorescence images were immediately collected. The excitation wavelength of the green channel was 488 nm and the emission wavelength was 515-545 nm; the excitation wavelength of the red channel was 550 nm and the emission wavelength was 610-640 nm.

[0109] 9. Cytotoxicity assay

[0110] 4T1, CT-26, Pan02, MCF-7, GES-1, HeLa, U87MG, HepG2, MKN45, A549, and GES-1 cells were seeded in 96-well plates (1 × 10 4 cells) to ensure that they grow in the incubator for 24 hours. Subsequently, they are incubated with a concentration gradient of NP-NH-D5 for 24 hours. Next, 50 μL of PBS buffer containing MTT is added to each well, and the culture plate is returned to the incubator. After 4 hours, the well plate is slightly moved sideways, the culture medium is carefully removed, and DMSO (150 μL / well) is added. Before testing on the machine, the well plate is carefully shaken several times to ensure complete dissolution. The absorbance (OD) at 490 nm is recorded using a microplate reader.

[0111] Untreated cells were used as the control group (ODcontrol), and the cell viability in each well was calculated as OD / ODcontrol × 100%. Each experiment was repeated three times to ensure the reliability of the results.

[0112] 10. Biological transmission electron microscopy experiment of cells

[0113] 4T1 cells were seeded in 6-well plates at a density of 5 × 10 cells per well. 5cells and cultured at 37°C for 24 hours. Then, the cells were incubated with blank DMEM medium or DMEM medium containing NP-NH-D5 (30μM) for 4 hours. After incubation, the cells were washed three times with cold PBS (1×, pH7.4), and the cells were scraped off with a cell scraper and collected by centrifugation. Subsequently, the cell pellets were dehydrated by acetone solutions with a concentration gradient (50%, 70%, 80%, 96% for 10 minutes each, 100% three times), and then treated with a mixed solution containing embedding agent and acetone (volume ratio = 1 / 1) for 1 hour. After that, the mixed solution containing a higher proportion of embedding agent (volume ratio = 3 / 1) was treated for 3 hours. Finally, the pure embedding agent was treated overnight. After completing the above steps, to ensure the complete solidification of the embedding agent, the sample was heated at 70°C overnight. Finally, a completely solidified and stable sample was obtained.

[0114] Next, the cell sections were cut into approximately 70 nm. After being placed on a copper grid, they were stained with a lead citrate solution and an ethanol saturated solution of uranyl acetate for 5-10 minutes. Finally, they were rinsed with deionized water and dried before being observed using an HT7700 transmission electron microscope.

[0115] 11. Flow cytometry analysis of cell life and death

[0116] In a 6-well plate, approximately 2 × 10 5 4T1 cells were cultured with 2 mL of DMEM medium, and the plates were then incubated at 37°C overnight to promote cell adhesion and growth. Subsequently, the cells were incubated with NP-NH-D5 or its control probe (at a concentration of 30 μM) for 6 hours. After incubation, the medium was removed and washed three times with PBS. After that, trypsin (500 μL per well) was used to gently detach the cells from the surface of the culture plate, and then centrifuged at 4°C and 161 × g for 4 minutes to collect the cell pellet. After washing the pellet with PBS once, it was resuspended in 500 μL PBS and double-stained with Annexin V-FITC and PI. All flow cytometry analyses were performed in strict accordance with the manufacturer's instructions, and the cell viability was analyzed using Annexin V-FITC and PI channels. The sample count for flow cytometry was approximately 10,000 cells, and the data was processed using NovoCyte Quanteon software.

[0117] 12. Protein immunoblotting analysis

[0118] The expression levels of pyroptosis-related indicators: Caspase-1, GSDMD, NLRP3 and ASC were analyzed by WB. To evaluate the pyroptosis effect of NP-NH-D5 and its control nanoparticles on 4T1 cells, cells were incubated with NP-NH-D5 or the corresponding control probe (30 μM) for 6 hours. Among them, the cells in the positive control group were treated with LPS (5 μg / mL) for 4 hours and then briefly treated with Nigericin (15 μM) for 15 minutes. After treatment in each group, the cells were washed three times with PBS. Subsequently, the cells were collected in lysis buffer, incubated on ice for 30 minutes, and the lysates were collected by centrifugation at 13,000 × g and 4 ° C for 15 minutes. The obtained protein samples were transferred to PVDF membranes by electrophoretic transfer technology after SDS-PAGE electrophoresis. The membrane was blocked with 5% skim milk powder or 5% bovine serum albumin in TBST buffer containing 0.1% Tween 20 for 1 hour at room temperature, and then the primary antibody buffer and membrane were sealed in a plastic bag, incubated at room temperature for 30 minutes and placed at 4°C overnight. The next day, the membrane was gently shaken at room temperature for 30 minutes. After washing four times with TBST, the membrane was incubated with HRP-labeled secondary antibody buffer for 1 hour. Similarly, after washing four times with TBST, the secondary antibody signal was displayed using the Tanon-4600 imaging system and ECL developer. After the target band was exposed, the exposed membrane was placed in excess TBST solution and shaken vigorously on a shaker overnight. On the second day, the membrane was blocked for the second time using TBST solution containing 5% skim milk. One hour later, the membrane was placed in TBST solution containing the primary antibody of the internal reference protein and shaken slowly on a shaker. After washing four times with TBST, the membrane was incubated with the buffer of the horseradish HRP-labeled secondary antibody for 1 hour. After washing four times, the target bands were displayed using the Tanon-4600 imaging system and ECL developer. Finally, the band intensity was quantitatively analyzed using ImageJ software.

[0119] 13. Cytokine detection experiment

[0120] The secretion of IL-18 and IL-1β was measured by ELISA kits. 4T1 cells were incubated with NP-NH-D5 or its control nanoparticles (30 μM) for 6 h. After incubation, the cell culture medium was collected. Untreated cells served as negative controls. The secretion of proinflammatory cytokines, including IL-1β and IL-18, in the culture medium was detected using ELISA kits according to the manufacturer's instructions.

[0121] 14. Evaluation of cell immunogenic death effects

[0122] The exposure level of calreticulin (CRT) on the cell surface was detected by immunofluorescence staining and flow cytometry. The specific operation is as follows: In the immunofluorescence staining experiment, 4T1 cells were plated at 5×104 The cells were seeded at a density of 10 μM in a four-well glass-bottomed culture dish. Then, the cells were incubated with NP-NH-D5 (30 μM) for 6 hours. After incubation, the cells were washed three times with pre-cooled PBS and fixed with 4% paraformaldehyde for 10 minutes. After washing with cold PBS, they were incubated overnight with a dilution containing the CRT primary antibody. The next day, the cells were gently washed to remove unbound primary antibodies. The cells were then placed in a dilution solution containing AlexaFluor-488-labeled monoclonal secondary antibodies and incubated for another hour. Similarly, after washing three times, immunofluorescence images of the cells were taken using an inverted microscope.

[0123] For flow cytometry experiments, approximately 2 × 10 5 4T1 cells were seeded at a cell density of 100 μM and then incubated at 37°C overnight to promote cell adhesion and growth. Then, the cells were incubated with NP-NH-D5 or its control probe (30 μM) for 6 hours. After incubation, the cells were washed three times with PBS and detached from the culture plate surface by trypsin. Next, the cell pellets were collected by centrifugation at 4°C and 161×g for 4 minutes. Subsequently, the cells were fixed with 4% formaldehyde for 10 minutes and washed again with cold PBS three times. The cells were then incubated with primary antibodies against CRT overnight. After primary antibody treatment, the cells were washed three times and continued to be incubated with AlexaFluor-488-labeled monoclonal secondary antibodies for 1 hour. After incubation, the cell pellets were washed again with PBS three times and resuspended in 500 μL PBS for flow cytometric analysis. Flow cytometric analysis was performed strictly according to the manufacturer's operating instructions, the sample count was approximately in the range of 10,000 cells, and the data processing was completed using NovoCyteQuanteon software.

[0124] The secretion of high mobility group box B1 (HMGB1) and ATP was tested by ELISA kits. For NP-NH-D5, 4T1 cells were incubated with NP-NH-D5 or its control probe (30 μM) for 6 h. The cell peripheral culture medium was collected and the secretion of HMGB1 and ATP in the culture medium was detected using ELISA kits according to the manufacturer's instructions.

[0125] To evaluate the maturation of dendritic cells (DCs) in vitro, BMDCs were generated and collected from the bone marrow of 5- to 6-week-old BALB / c mice. 4T1 cells were first incubated with NP-NH-D5 or its control probe (30 μM) for 6 h. Untreated cells served as negative controls. Subsequently, cells were treated with fresh medium overnight. 1×10 6Immature DCs were co-cultured with 4T1 cells pretreated with NP-NH-D5 overnight. The cells were gently blown and stained with anti-CD11c-FITC, anti-CD80-PE and anti-CD86-APC antibodies, followed by multiple rounds of centrifugation and washing. The maturation of DCs in each group was detected by flow cytometry.

[0126] 15. Animal experiments and establishment of various tumor models

[0127] Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing University and were performed in accordance with the guidelines of the IACUC of Nanjing University. BALB / c female mice aged 5–6 weeks and 6–8 weeks were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. All mice were housed under a 12-h light-dark cycle, 50–70% humidity, and an ambient temperature of 18–22°C with free access to food and water.

[0128] To establish an orthotopic 4T1 breast tumor model, 5×10 5 4T1 cells were resuspended in 50 μL of a 50% volume ratio of Matrigel and DMEM mixture and injected subcutaneously into the right side of the mammary gland of 5-6 week-old mice. The length (L) and width (W) of the tumor were measured with a caliper and the formula V = (L × W 2 ) / 2 to calculate the tumor volume. When the tumor grows to about 90mm 3 Mice were subjected to fluorescence imaging and treatment at the same time.

[0129] To establish the late-stage 4T1 lung metastasis model, 4T1-Luc cells (100 µL, approximately 2 × 10 5 ) was injected into 5-6 week old BALB / c female mice via the tail vein. About 7 days later, the mice successfully established a lung metastasis model by bioluminescence imaging and monitored by intraperitoneal injection of D-luciferin (150 mg / kg).

[0130] To establish an orthotopic Pan02-Luc pancreatic tumor model, Pan02-Luc cell suspension (5 × 10 5 Cells, 50% Matrigel, 30 µL) were injected into the pancreatic tail of C57BL / 6 mice (5-6 weeks old). Five days later, D-luciferin (150 mg / kg) was injected intraperitoneally, and BLI images (IVISSpectrum, PerkinElmer, USA) were acquired to verify the successful establishment of the orthotopic pancreatic tumor model in mice.

[0131] 16. In vivo fluorescence imaging

[0132] BALB / c mice with established 4T1 orthotopic tumor model or advanced 4T1-Luc lung metastasis model, and C57BL / 6 mice with orthotopic Pan02-Luc pancreatic tumors were injected intravenously (iv) with NP-NH-D5 / Cy5.5 or NP-ctrl-1 / Cy5.5 (200μM, 200μL, containing 8μM F-C6-NH-Cy5.5). Then, at different time points, mice were lightly anesthetized and fluorescence images were acquired using the IVIS Lumina XRIII imaging system (excitation wavelength: 660nm; emission wavelength: 710nm emission filter set). ROIs were quantified by using LivingImage software to determine the fluorescence intensity of the tumor. Each experiment was performed in three mice.

[0133] 17. Fluorescence imaging of ex vivo tumors and major organs

[0134] NP-NH-D5 / Cy5.5 or NP-ctrl-1 / Cy5.5 (200 μM, 200 μL) was injected intravenously (iv) in mice with established orthotopic 4T1 tumor models or advanced 4T1 lung metastasis models. Eight hours later, the mice were euthanized. Tumors and major organs, including liver, kidneys, heart, lungs, and spleen, were subsequently removed. Fluorescence imaging of these organs and tumors was performed using an IVIS Lumina XRIII imaging system equipped with a 660 nm excitation filter and a 710 nm emission filter. ROIs were quantified by using LivingImage software to determine the fluorescence intensity of tumors and organs. Each experiment was performed on three mice.

[0135] 18. Tumor treatment plan

[0136] For mice bearing orthotopic 4T1 tumors, when the tumor volume reached about 90 mm³, each mouse was randomly divided into 9 groups and received intravenous injections of I: PBS; II: NP-NH-D5 (200 μM, 200 μL); III: NP-NH-D5 (400 μM, 200 μL); IV: NP-ctrl-1 (400 μM, 200 μL); V: NP-ctrl-2 (400 μM, 200 μL); VI: NP-ctrl-3 (400 μM, 200 μL); VII: NP-ctrl-4 (400 μM, 200 μL); VIII: anti-PD-L1 (100 μg); IX: NP-NH-D5 (400 μM, 200 μL) + anti-PD-L1 (100 μg). NP-NH-D5 or various control nanoparticles were injected intravenously on days 0, 3, and 6. For combination therapy, anti-PD-L1 antibody (100 μg per mouse) was injected intraperitoneally on days 1, 4, and 7. Tumor volume was calculated, and the relative tumor volume of each mouse was calculated as V / V0, where V0 was the tumor volume at the start of treatment and V was the volume during treatment. Relative body weight was normalized to the body weight at the initial treatment. On day 21, all mice were euthanized. Tumor tissues were resected, weighed, and imaged. Lung metastases in treated mice were further analyzed by taking photographs of dissected lungs and counting the number of lung metastases. Then, lung tissues were fixed, sectioned, and examined by H&E staining. Five mice were set up for each experiment.

[0137] For the advanced 4T1-Luc lung metastasis model mice, each mouse was randomly divided into 2 groups and received intravenous injection of I: PBS; II: NP-NH-D5 (400μM, 200μL) + anti-PD-L1 (100μg). Mice in group II received intravenous injection of NP-NH-D5 (400μM, 200μL) on days 0, 3, and 6, respectively, followed by intraperitoneal injection of 100μg anti-PD-L1 on days 1, 4, and 7. To monitor the therapeutic effect, bioluminescent images of mice were acquired using the bioluminescence imaging mode of the IVISLumina XRIII system within 10 minutes after intraperitoneal injection of D-luciferin (150mg / kg). The average intensity of the lungs was quantified using ROI. For ex vivo imaging of the lungs, mice treated with PBS were euthanized on day 6, while mice treated with NP-NH-D5 + anti-PD-L1 were euthanized on day 36. The excised lungs were fixed with 4% formaldehyde and photographed. Then, lung tissues were cut into 10 μm thickness and subjected to H&E staining analysis.

[0138] For mice with orthotopic Pan02 pancreatic cancer, each mouse was randomly divided into 2 groups and received intravenous injection of I: PBS; II: NP-NH-D5 (400μM, 200μL) + anti-PD-L1 (100μg). The first group of mice received five administrations of PBS buffer (200μL) by intravenous injection on days 0, 3, 6, 9, and 12. The second group of mice received five treatments of NP-NH-D5 (400μM, 200μL) by intravenous injection on days 0, 3, 6, 9, and 12, followed by five treatments of anti-PD-L1 treatment (100μg per mouse) by intraperitoneal injection on days 1, 4, 7, 10, and 13. During the treatment period, both groups of mice were injected with D-luciferin (150mg / kg) by intraperitoneal injection. After 10 minutes, BLI images of mice (front-up and side-on) were collected using the BLI mode (open mode) of the IVIS Lumina XRIII system. The BLI intensity of the pancreatic region of each mouse was quantified by ROI. The body weight of mice in each group was measured every three days.

[0139] 19. Fluorescence imaging of tumor tissue sections

[0140] For fluorescence imaging analysis of tumor tissue sections, mice bearing orthotopic 4T1 tumors were injected with NP-NH-D5 / Cy5.5 or NP-ctrl-1 / Cy5.5 (200 μM, 200 μL) via the tail vein. Eight hours after injection, the mice were sacrificed and the tumor tissues were removed. The obtained tumor tissues were processed by a vibrating knife microtome to prepare tissue sections with a thickness of 10 μm. Then, these tissue sections were imaged by fluorescence inverted microscope. The blue channel fluorescence images of F-C6-NH2 were acquired under 350±22.5 nm excitation, and the emission wavelength range was set to 420-460 nm. For AO-Luc, the green channel fluorescence images were acquired under 350±22.5 nm excitation, and the emission wavelength range was 510-550 nm. The red channel fluorescence images of Cy5.5 were acquired by 650±22.5 nm excitation, and the emission wavelength range was 690-750 nm.

[0141] 20. H&E, TUNEL and Ki-67 staining analysis of tumor tissue sections

[0142] One day after treatment, mice bearing 4T1 and Pan02 tumor models were euthanized and tumor tissues were removed. Tumors were fixed with 4% formaldehyde solution, embedded in paraffin and cut into 10 μm thick sections. H&E, TUNEL and Ki-67 staining analysis were performed using standardized staining protocols, and color images were captured using an Olympus vs200 scanner.

[0143] In addition, to evaluate potential side effects, healthy BALB / c mice received a series of intravenous injections of the agents on days 0, 3, and 6. 21 days after the first injection, the mice were euthanized and major organs including the liver, spleen, kidney, heart, and lungs were removed. The organs were fixed with 4% formaldehyde and embedded in paraffin, and the sections were sliced ​​at a thickness of 10 μm. Tissue samples were stained with H&E staining, and images were acquired using an Olympus vs200 slide scanner. The reagents used in the experiment include: I: PBS; II: NP-NH-D5 (200μM, 200μL); III: NP-NH-D5 (400μM, 200μL); IV: NP-ctrl-1 (400μM, 200μL); V: NP-ctrl-2 (400μM, 200μL); VI: NP-ctrl-3 (400μM, 200μL); VII: NP-ctrl-4 (400μM, 200μL); VIII: anti-PD-L1 antibody (100μg); IX: NP-NH-D5 (400μM, 200μL) + anti-PD-L1 antibody (100μg).

[0144] 21. Western blotting of tumor tissue

[0145] After the indicated treatment, 4T1 and Pan02 tumor tissues were resected from mice. Tumor tissues were homogenized by grinding in lysis buffer. After incubation on ice for 30 min, the homogenate was centrifuged at 13,000 × g for 15 min at 4°C to obtain the supernatant. The relative protein concentration in the supernatant was then determined. The membrane was blocked in 5% bovine serum albumin (dispersed in TBST buffer containing 0.1% Tween 20) for 2 h at room temperature. The membrane was then sealed with the primary antibody buffer of GSDMD and shaken on a shaker for 30 min at room temperature and then stored at 4°C overnight. The next day, the membrane was gently shaken at room temperature for 30 min. After washing four times with TBST, the membrane was incubated with HRP-labeled secondary antibody for 1 h and washed again four times. Finally, the secondary antibody signal was visualized using a Tanon-4600 imager and ECL developer. After the target band was exposed, the exposed membrane was placed in excess TBST solution and shaken vigorously on a shaker overnight. On the second day, the membrane was blocked for the second time using a TBST solution containing 5% skim milk. After 1 hour, the membrane was placed in a TBST solution containing the primary antibody of the internal reference protein and shaken on a shaker. After washing four times with TBST, the membrane was incubated with the HRP-labeled secondary antibody for 1 hour and washed again four times. The band signal was displayed and recorded using a Tanon-4600 imager and ECL developer. Finally, the band intensity was quantitatively analyzed using ImageJ software.

[0146] 22. Evaluation of the anti-tumor immune memory effect of NP-NH-D5+anti-PD-L1

[0147] To investigate the antitumor immune memory effect, mice bearing orthotopic 4T1 tumors were treated with NP-NH-D5 and anti-PD-L1 when the tumor volume reached approximately 90 mm³. Specifically, NP-NH-D5 (400 μM, 200 μL) was administered intravenously on days 0, 3, and 6, and anti-PD-L1 antibody (100 μg per mouse) was administered intraperitoneally on days 1, 4, and 7. On day 29 after treatment, 5×10 5 Treated mice were re-challenged with 5 × 10 4T1-Luc cells (suspended in 50 μL of a 50 v / v% mixture of Matrigel and DMEM). 5 The growth of tumors in the two groups of mice was monitored by bioluminescence imaging.

[0148] 23. Evaluation of the anti-tumor immune effect of NP-NH-D5+anti-PD-L1

[0149] To evaluate immune responses in orthotopic 4T1 tumors, mice were euthanized after receiving the indicated treatments to extract tumors and lymph nodes (LNs), and blood was collected from mice. To evaluate the adaptive antitumor immunity of NP-NH-D5 in orthotopic 4T1 tumor-bearing mice, spleens were dissected from mice on day 29 after the indicated treatments. In addition, C57BL / 6 mice bearing orthotopic pancreatic tumors were euthanized after receiving the indicated treatments to extract tumors and lymph nodes, and blood was collected.

[0150] For flow cytometric analysis of dendritic cell maturation, the excised lymph nodes were ground. After filtering through a 70-μm cell strainer, the cells were washed twice with PBS to obtain a DC cell suspension. Subsequently, DC cells from the lymph nodes were stained with fluorescent antibodies specific for mature DC (anti-mouse CD11c, CD80, and CD86).

[0151] For flow cytometric analysis of tumor-infiltrating T lymphocytes, excised tumors were ground and resuspended in culture medium. After filtration and washing twice with PBS, T cell suspensions were obtained and subsequently stained with fluorescent antibodies specific for infiltrating T lymphocytes (anti-mouse CD3, CD4, and CD8). For flow cytometric analysis of regulatory T cells (Tregs), T cell suspensions from tumors were stained with fluorescent antibodies specific for Tregs (anti-mouse CD3, CD4, Foxp3, CD25). For intracellular staining, cells were permeabilized with fixation / permeabilization working solution before incubation with relevant antibodies.

[0152] For flow cytometric analysis of memory T cells, spleens were mechanically ground and resuspended in red blood cell (RBC) lysis buffer to remove erythrocytes. After filtration and addition of culture medium, the suspension was centrifuged at 500 g for 5 min at 4 °C to obtain splenocytes. Finally, splenocytes were stained with fluorescent antibodies for memory T cells (anti-mouse CD3, CD8, CD62L, CD44). Details of the antibodies used are provided in Supplementary Materials and Methods.

[0153] To analyze inflammatory cytokines in serum, including IL-6, TNF-α, and IFN-γ, the collected blood was placed in a serum separator tube (SST) at 4°C overnight. Subsequently, the blood was centrifuged at 1000×g for 15 minutes to obtain serum. Inflammatory cytokines in serum were measured using ELISA kits for mouse IL-6, TNF-α, and IFN-γ.

[0154] 24. Biosafety Analysis

[0155] Healthy BALB / c mice were intravenously injected with NP-NH-D5 (400 μM, 200 μL). On days 0, 1, 7, and 28 after injection, approximately 1.0 mL of blood was collected from the venous sinus and stored in 1.5-mL ETA-coated centrifuge tubes placed on ice. The collected blood samples were divided into two parts. The first part was used for blood count analysis. The second part was centrifuged at 2,400 × g for 20 minutes. Biochemical related biomarkers including alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptidase (GGT), total bilirubin (TBil), albumin (Alb), total protein (TP), blood urea nitrogen (BUN), and creatinine (CRE) were determined using ELISA kits according to the manufacturer's protocol.

[0156] 25. Data Processing

[0157] Data are expressed as mean ± standard deviation. Statistical comparisons between the two groups were evaluated by Student's t-test. All results were analyzed using Prism9 (GraphPad Software, Inc, CA, USA). P < 0.05 indicated a statistically significant difference.

[0158] 4. Results and Discussion

[0159] 1. Design of compound F-SS-NH-GALGLP-D5 and its control compound

[0160] The compound F-SS-NH-GALGLP-D5 consists of four key parts: hydrophobic 3,5-bis(trifluoromethyl)benzamide, AO-Luc fluorophore, GSH-reducible disulfide bond, and a peptide sequence that can respond to MMP-2, with five aspartic acid units (GALGLP-D5) connected to the end of the sequence. The detailed synthesis steps of this compound are shown in the above synthesis examples. In addition, two control molecules were designed and synthesized: one is F-SS-NH-galglp-D5 that does not respond to MMP-2 (compound 3-2); the other is F-C6-NH-GALGLP-D5 that does not respond to GSH (compound 3-3). In order to further carry out near-infrared fluorescence imaging studies in vivo, F-C6-NH-Cy5.5 was also synthesized. The chemical structures of all intermediates and compounds have been confirmed and characterized by MALDI-TOFMS or ESI-MS mass spectrometry.

[0161] 2. Study on the co-assembly ratio of NP-NH-D5

[0162] In order to optimize the co-assembly ratio of F-C6-NH2 and F-SS-NH-GALGLP-D5, we co-assembled F-C6-NH2 and F-SS-NH-GALGLP-D5 into different nanoparticles at different molar ratios, and conducted DLS and Zeta potential studies. The results showed that with the increase of the molar ratio between F-C6-NH2 and F-SS-NH-GALGLP-D5, the average particle size and Zeta potential of the nanoparticles showed an increasing trend. To ensure that the nanoparticles exhibit excellent in vivo performance after systemic injection, three key factors are mainly considered: the nanoparticle size needs to be less than 100nm for biodistribution, the Zeta potential should be negative to reduce the absorption of normal tissue cells, and at the same time, the content of F-C6-NH2 in the nanoparticles is maximized to enhance the killing effect on tumor cells. Based on the above considerations, the optimal ratio (molar ratio) of F-C6-NH2 and F-SS-NH-GALGLP-D5 co-assembly was determined to be 5:1 to prepare the nanoparticles NP-NH-D5 with the best performance. It exists as spherical nanoparticles with an average hydrodynamic size of approximately 60 nm and a zeta potential of -4.39 ± 1.52 mV.

[0163] In addition, stability studies showed that no significant changes were observed in the particle size or fluorescence intensity of the co-assembled nanoparticles in PBS and cell culture medium containing 10% FBS. This result indicates that the co-assembly can maintain its stability under physiological conditions.

[0164] 3. Characterization of the cascade response of NP-NH-D5 to MMP-2 / GSH

[0165] Next, we continued to investigate the specific response performance of NP-NH-D5 to MMP-2 enzyme, GILT enzyme and GSH. Figure 2 As shown in the figure, after incubation with MMP-2, NP-NH-D5 was converted to NP-NH2 due to the removal of the terminal negatively charged aspartic acid sequence, which resulted in a slight decrease in its particle size to about 55 nm, while the Zeta potential increased significantly to +25.58±1.40 mV, reflecting the change in the surface charge properties of the nanoparticles. It is worth noting that no significant increase in the Zeta potential was observed after incubation of NP-NH-D5 with other proteases such as γ-glutamyltransferase (GGT), ALP, and cathepsin B (CatB), which emphasized the high selectivity of NP-NH-D5 for MMP-2 enzyme. After HPLC and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-MS) analysis, we further confirmed that F-SS-NH-GALGLP-D5 (tR=17.6min) in NP-NH-D5 could be effectively recognized and cleaved by MMP-2 enzyme to form F-SS-NH-GAL (tR=18.2min). In the subsequent incubation with GILT enzyme or GSH, F-SS-NH-GAL can be effectively reduced to NH-GAL. In this process, the fluorescence of the probe at 515nm is significantly enhanced due to the departure of the quenching group of amino-oxidized firefly luciferin, showing GSH responsiveness. At the same time, the results of DLS and TEM analysis also showed that the spherical nanoparticles can rapidly grow into granular nanofiber structures after 30 minutes, and further form long nanofibers after 6 hours. In addition, circular dichroism spectroscopy analysis also further confirmed the gradual formation of β-folded structure. These research results fully demonstrate the sensitivity and specific response of NP-NH-D5 to the dual stimulation of MMP-2 and GSH, as well as its morphological transformation ability under biological stimulation.

[0166] Figure 2 (ae) TEM images (a), DLS analysis (b), HPLC analysis (c), fluorescence spectrum (d), and circular dichroism spectrum (e) of NP-NH-D5 (100 μM) before and after incubation with MMP-2 (8 nM, 12 h), and after complete incubation with MMP-2 and then incubated with GSH (10 mM) for 0.5, 2, and 6 h. Figure 3 HPLC analysis of NP-NH-D5 (100 μM) before and after incubation with MMP-2 (8 nM, 12 h), and after complete incubation with MMP-2 and incubation with GSH (1.5 mM) or GILT enzyme (2.5 μg + 20 μM cysteine) for 2 h is shown.

[0167] 4. Design and characterization of NP-NH-D5 control probe

[0168] In order to better verify the feasibility of NP-NH-D5 design, we designed four other control probes. 1) NP-ctrl-1: Co-assembled by F-C6-NH2 and F-SS-NH-galglp-D5 at a molar ratio of 5:1. The MMP-2 recognition sequence in this design uses a D-type peptide, so it cannot be recognized and cleaved by the MMP-2 enzyme. The relevant results confirmed that after incubation with the MMP-2 enzyme overnight, the hydrated particle size and Zeta potential did not change significantly. 2) NP-ctrl-2: Co-assembled by F-C6-NH2 and F-C6-NH-GALGLP-D5 at a molar ratio of 5:1. Due to the lack of disulfide bond structure, the nanoparticle cannot react with GSH. After treatment with MMP-2, we observed that its hydrated particle size was slightly reduced, while the Zeta potential was significantly increased, indicating that NP-ctrl-2 can be effectively recognized and cleaved by MMP-2. After further incubation with GSH, no reduction products were found by HPLC analysis, and the hydrated particle size of the nanoparticles did not change much, further confirming the resistance of NP-ctrl-2 to GSH. 3) NP-ctrl-3: Co-assembled by F-SS-NH2 and F-C6-NH-GALGLP-D5 at a molar ratio of 5:1. DLS results confirmed that the structure could be responded to by MMP-2 and GSH in sequence, but could not be reassembled into nanofibers. 4) NP-ctrl-4: Co-assembled by F-C6-NHAc and F-C6-NH-GALGLP-D5 at a molar ratio of 5:1. DLS results confirmed that the structure could be responded to by MMP-2 and GSH in sequence, but the F-C6-NHAc released after the response could only be reassembled into nanoparticles.

[0169] 5. Study on the selective uptake of NP-NH-D5 by tumor cells

[0170] Before imaging various cell types, we first co-incubated NP-NH-D5 with 4T1 cells to optimize imaging conditions. Figure 4As shown in the figure, with the increase of probe concentration and incubation time, the green fluorescence intensity of the intracellular Oxy-Luciferin fluorophore also increased. In particular, when the probe concentration was 20μM and the incubation time was 4 hours, the fluorescence intensity reached saturation. Then, NP-NH-D5 (20μM) was co-incubated with different cells for 4 hours. The results showed that obvious F-C6-NH2 fluorescence appeared in MMP-2-positive tumor cells such as 4T1, HeLa and A549, and this fluorescence could be significantly inhibited by the MMP-2 inhibitor SB-3CT. In contrast, almost no obvious fluorescence was observed in human gastric epithelial cells GES-1 lacking MMP-2. In addition, in 4T1 tumor cells, the fluorescence intensity of the dissociated AO-Luc fluorophore (510-550nm) also gradually increased with the extension of incubation time. These experimental results indicate that NP-NH-D5 can be efficiently taken up by MMP-2-positive tumor cells compared with MMP-2-deficient GES-1 cells, thus demonstrating its selectivity for tumor cells.

[0171] Figure 4 Demonstrated optimization of NP-NH-D5 incubation time and concentration in 4T1 cells. Figure 4 a shows fluorescence imaging after incubation of 4T1 cells with 10 μM NP-NH-D5 for 0.5, 1, 2, and 4 h. Figure 4 b shows fluorescence imaging of 4T1 cells after incubation with 5, 10, 20, 30, and 50 μM NP-NH-D5 for 4 h. Scale bar: 25 μm.

[0172] On this basis, we performed HPLC analysis on the cell lysate and culture medium of 4T1 cells after incubation with NP-NH-D5, and found that the lysate not only contained F-C6-NH2, but also the cleavage product NH-GAL. Only F-C6-NH2 and F-SS-NH-GALGLP-D5 were detected in the culture medium. This result proves that NP-NH-D5 can selectively enter MMP-2-positive tumor cells through specific protease hydrolysis mediated by extracellular MMP-2. Subsequently, the intramolecular disulfide bond is broken after intracellular GSH reduction, which further promotes the release of the active ingredient F-C6-NH2 in NP-NH-D5, demonstrating its selectivity for MMP-2-positive tumor cells.

[0173] 6. Study on lysosomal destruction and cytotoxicity of NP-NH-D5

[0174] After confirming that NP-NH-D5 can specifically respond to extracellular MMP-2 and intracellular GILT enzymes and GSH and be selectively taken up by tumor cells, we further explored the feasibility of this strategy to form nanofibers in situ within lysosomes and then trigger lysosomal destruction. Preliminary experimental results showed that when 4T1 cells were incubated with NP-NH-D5 for 0.5 hours, the fluorescence of intracellular F-C6-NH2 and Lyso-tracker had a good co-localization effect ( Figure 5 ). As the incubation time was extended to 4 hours, the fluorescence signal of Lyso-tracker was significantly weakened, reflecting the rupture of cell lysosomes. The results of AO staining experiments showed that 4T1 cells treated with NP-NH-D5 showed weak fluorescence in the red channel and strong fluorescence in the green channel, further verifying the damage of lysosomes. Bio-TEM analysis further confirmed that a large number of nanofibers appeared in 4T1 cells after 4 hours of incubation with NP-NH-D5, accompanied by significant lysosomal rupture. These results confirm that NP-NH-D5 can achieve the conversion of nanoparticles into nanofibers in the lysosomes of tumor cells, thereby causing severe lysosomal damage in cells. In contrast, since NP-ctrl-1 cannot respond to extracellular MMP-2, it cannot enter 4T1 cells and therefore cannot cause obvious lysosomal damage. In addition, although NP-ctrl-2, NP-ctrl-3, and NP-ctrl-4 can be taken up by 4T1 cells, they cannot reorganize into nanofibers in cells and therefore cannot cause lysosomal damage.

[0175] Figure 5 The lysosomal disruption in 4T1 cells after treatment with NP-NH-D5 and other control probes is shown. 4T1 cells were treated with the probes (30µM, 4h) and then incubated with Lyso-Tracker (1µM, 15min). Scale bar: 20μm.

[0176] Based on the above experimental results, we used the MTT method to determine the toxicity of NP-NH-D5 on various cells. The results showed that NP-NH-D5 showed a significant killing effect on MMP-2 positive tumor cells, and its half inhibitory concentration (IC 50 ) ranged from 8.29 ± 1.83 to 26.89 ± 1.62 μM. Compared with F-C6-NH2, NP-NH-D5 showed significantly lower cytotoxicity on GES-1 cells, indicating that it has lower toxicity to normal cells.

[0177] 7. Study on the anti-tumor mechanism of NP-NH-D5

[0178] After confirming that NP-NH-D5 can selectively kill tumor cells, we verified its anti-tumor mechanism. Compared with other control groups, cells treated with NP-NH-D5 showed cell membrane rupture and nuclear damage. The results of immunofluorescence staining and protein immunoblotting experiments showed that NP-NH-D5 could significantly upregulate the expression levels of key proteins of the intracellular inflammatory pathway, such as cathepsin B (CTSB), ASC and NLRP3, reflecting the effective activation of the inflammatory pathway inside the cell. Subsequently, we further confirmed by protein immunoblotting analysis that NP-NH-D5 could induce the activation and cleavage of the key proteins of pyroptosis, Caspase-1 and GSDMD, and in particular, the activated fragment of GSDMD-N was observed in the NP-NH-D5-treated group. These findings indicate that NP-NH-D5 can effectively trigger the death of tumor cells by inducing the activation of the inflammatory pathway inside the cell and the cleavage of the key proteins of pyroptosis, which is also strongly supported by the results of flow cytometry experiments.

[0179] At the same time, the exocytic levels of pyroptosis-related cytokines (IL-18 and IL-1β) and lactate dehydrogenase in the NP-NH-D5-treated group were significantly increased, further confirming its effectiveness in selectively inducing tumor cell pyroptosis and stimulating inflammatory response.

[0180] In addition, NP-NH-D5 can not only directly kill tumor cells, but also promote the release of inflammatory factors and LDH, causing the immune system to respond, thereby enhancing the overall immune surveillance and clearance of tumors. To this end, we monitored the exposure level of CRT, the expression levels of HMGB1 and ATP, and found that the levels of the three indicators in 4T1 cells treated with NP-NH-D5 were significantly increased, indicating that NP-NH-D5 can effectively stimulate immunogenic cell death effects. Next, the maturity of DCs was evaluated by flow cytometry experiments. We found that 4T1 cells treated with NP-NH-D5 can significantly promote the expression of costimulatory molecules CD80 and CD86 on the surface of DCs. These upregulated molecules are key indicators of DCs maturation, which means that they are ready to activate T cells and trigger a stronger immune response.

[0181] 8. In vivo fluorescence imaging and immunotherapy study of orthotopic 4T1 breast tumor model mice

[0182] First, in order to visualize NP-NH-D5 in mice, we prepared Cy5.5-labeled NP-NH-D5 (NP-NH-D5 / Cy5.5) and MMP-2-unresponsive control nanoparticles (NP-ctrl-1 / Cy5.5) for in vivo fluorescence imaging. The fluorescence imaging results showed that 8 hours after tail vein injection, the fluorescence intensity of NP-NH-D5 / Cy5.5 in the tumor area was significantly higher than that of the NP-ctrl-1 / Cy5.5 treatment group, about 4 times higher ( Figure 6 ). Fluorescence imaging of the main ex vivo organs and tumors of mice showed that compared with the control group, the fluorescence intensity of NP-NH-D5 / Cy5.5 at the tumor location was much higher than that of other organs, while the NP-ctrl-1 / Cy5.5 group mainly showed accumulation in the liver. In addition, fluorescence imaging of tumor tissue sections further confirmed the uniform distribution of F-C6-NH2, Cy5.5, and uncaged AO-Luc in tumor tissue. These results prove that NP-NH-D5 can achieve effective accumulation and long-term retention in tumor tissue.

[0183] Figure 6 Fluorescence imaging of orthotopic 4T1 tumor model mice is shown. Tumor-bearing mice were injected with NP-NH-D5 / Cy5.5(I) or NP-ctrl-1 / Cy5.5(II) via tail vein at a dose of (200μM, 200μL). Figure 6 a shows the near-infrared fluorescence imaging before injection and at different times (2h, 4h, 6h, 8h, 12h, 24h and 48h) after injection. Figure 6 b shows the mean fluorescence intensity value of the tumor site.

[0184] After confirming that NP-NH-D5 can effectively accumulate in the tumor site, we evaluated its therapeutic effect on mice with orthotopic 4T1 breast tumor model. Treatment was performed every three days, and treatment was stopped after three injections until day 21, during which the tumor size and mouse body weight were recorded. The relevant experimental results showed that on the 21st day of treatment, the average tumor weight of the NP-NH-D5 (200μM) group and the NP-NH-D5 (400μM) group was significantly lower than that of the PBS control group, which was reduced by 19.3 times and 62.0 times, respectively, showing a concentration dependence of the therapeutic effect. In contrast, the tumor growth rate of 4T1 tumor mice treated with NP-ctrl-1, NP-ctrl-2, NP-ctrl-3 or NP-ctrl-4 was similar to that of the PBS group, and the tumor size increased by about 20 times after 21 days.

[0185] It is worth noting that although NP-NH-D5 (400μM) can effectively inhibit the growth of 4T1 tumors, after three doses of treatment, tumor growth did not remain suppressed and there was a slow recovery trend. Based on this, we introduced anti-PD-L1 for combined treatment. The treatment results showed that the combination of NP-NH-D5 (400μM) and anti-PD-L1 significantly improved the therapeutic effect of tumors, and three of the five tumors were completely eliminated. In contrast, the tumors of mice treated with anti-PD-L1 alone grew about 16.4 times on the 21st day after treatment. This result shows that NP-NH-D5 can enhance the immunotherapy efficacy of anti-PD-L1 on 4T1 tumors.

[0186] The results of hematoxylin and eosin staining (H&E staining) of tumor tissue sections, Ki-67 antibody (immunohistochemistry) (FLEX Monoclonal Mouse Anti-Human Ki-67 Antigen, Ki-67) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TdT-mediated dUTP nick-end Labeling, TUNEL) also strongly supported the above conclusions. Figure 7 As shown in the figure, compared with other treatment groups, large-area cell death can be observed in the tumor tissues of mice treated with NP-NH-D5, indicating that NP-NH-D5 can effectively inhibit tumor growth. In addition, given that orthotopic 4T1 tumors are very prone to metastasis in the late stage, we collected the lungs of mice in each experimental group at the end of treatment (day 21) to evaluate the anti-metastatic effect. Through white light observation of the lungs and corresponding lung section analysis, it was found that obvious metastatic tumor nodules appeared inside and outside the lungs of mice in PBS or other control groups, while there was no obvious sign of metastasis in the lungs of mice treated with NP-NH-D5, indicating that NP-NH-D5 has significant anti-tumor metastasis potential.

[0187] Subsequently, biological transmission electron microscopy was used to further study the changes in the tumor tissue. The tumor tissue of mice injected with PBS through the tail vein showed a compact structure, and the lysosomal structure was basically intact. In contrast, the tumor tissue treated with NP-NH-D5 (400μM) was loose inside, with a large number of long nanofibers entangled with each other. There were fine fibrous substances inside the lysosomes, most of which were damaged ( Figure 8 The above results indicate that after being taken up by tumors, NP-NH-D5 can be reassembled into nanofibers in tumor tissues and destroy lysosomes, effectively inhibiting 4T1 tumor growth and preventing lung metastasis.

[0188] exist Figure 8Figure 2 shows the Bio-TEM images of tumor tissues removed 24 hours after tail vein injection of NP-NH-D5 (400 μM, 200 μL) in an orthotopic 4T1 tumor model mouse. The red box indicates the magnified area. The red arrows point to the ruptured lysosomes in the tumor tissue. The blue arrows indicate the nanofibers in the cytoplasm.

[0189] 9. In vivo fluorescence imaging and immunotherapy study of late-stage 4T1-luc lung metastasis model mice

[0190] We tried to apply NP-NH-D5 to the treatment of advanced tumors of deep-seated 4T1 lung metastases. Similarly, we first injected Cy5.5-labeled NP-NH-D5 (NP-NH-D5 / Cy5.5) into healthy mice or tumor-bearing mice to determine whether it could reach the lung tumors. The results showed that 8 hours after intravenous injection of NP-NH-D5 / Cy5.5, the near-infrared fluorescence intensity of the lungs of tumor-bearing mice reached a peak, which was nearly 3.2 times higher than that of the lungs of healthy mice. Among them, the most noteworthy is that the near-infrared fluorescence of the ex vivo lung tissue can achieve good overlap with the bioluminescent signal of the lung metastasis, indicating that NP-NH-D5 / Cy5.5 is mainly accumulated in the metastatic foci of the lungs.

[0191] Tumor-bearing mice were injected with PBS or NP-NH-D5+anti-PD-L1 for treatment studies, and bioluminescence imaging was used to monitor the treatment effect. The imaging results showed that the bioluminescence intensity of the lungs of mice treated with PBS increased rapidly. At the same time, all mice died within 12 days due to the aggressive growth of 4T1-Luc tumors. In contrast, mice treated with NP-NH-D5+anti-PD-L1 showed significant attenuation and regression of lung tumor metastasis, and their lung bioluminescence intensity decreased by 31.9 times by day 36. In addition, white light photos of the lungs of each experimental group and H&E staining results of lung tissue further confirmed the effectiveness of NP-NH-D5+anti-PD-L1 in treating lung metastatic nodules. By monitoring the weight of each mouse during treatment, it was found that the mice treated with NP-NH-D5+anti-PD-L1 did not lose significant weight, and three of the five mice survived for more than 90 days. These results strongly indicate that the NP-NH-D5+anti-PD-L1 treatment strategy not only successfully cleared 4T1 lung metastatic nodules, but also significantly prolonged the survival of mice, demonstrating its excellent therapeutic effect on advanced lung metastatic tumors.

[0192] 10. In vivo fluorescence imaging and immunotherapy study of mice with orthotopic Pan02-Luc pancreatic cancer

[0193] Next, we applied NP-NH-D5 to the treatment of an orthotopic pancreatic cancer model, a highly invasive immune-cold tumor. Similar to the above two models, we first injected NP-NH-D5 / Cy5.5 into healthy mice or tumor-bearing mice for near-infrared fluorescence imaging to determine whether it could reach the tumor area. Eight hours after intravenous injection of NP-NH-D5 / Cy5.5, the fluorescence intensity of the tumor site of the tumor-bearing mouse gradually became stronger than that of the liver area, and its luminescent area and the bioluminescent area of ​​the lesion area could overlap well, indicating that the assembly can achieve accumulation and retention in pancreatic tumors.

[0194] Subsequently, we divided mice with the orthotopic Pan02-Luc pancreatic cancer model into two groups for treatment studies with PBS or NP-NH-D5+anti-PD-L1. The treatment results showed that the tumor bioluminescence intensity of the five mice treated with PBS increased sharply after treatment. After 12 days, significant bioluminescence enhancement was observed in the lungs, livers, and abdomens of multiple mice, reflecting the rapid growth of the tumor and the occurrence of distal metastasis. Within 22 days, five mice died one after another. In contrast, the tumor bioluminescence intensity and body weight of tumor-bearing mice treated with NP-NH-D5+anti-PD-L1 remained almost unchanged during the 60-day treatment process, indicating that the combined treatment strategy effectively inhibited the growth of orthotopic Pan02-Luc tumors. More importantly, the survival time of mice in this treatment group was significantly prolonged, with a median survival of nearly 82 days, which was much higher than that of the PBS treatment group. The H&E, Ki-67 and TUNEL-stained tumor tissue sections further verified that NP-NH-D5+anti-PD-L1 treatment could significantly promote the death of Pan02-Luc tumor cells. These results showed that the NP-NH-D5+anti-PD-L1 combined treatment strategy was effective in delaying the growth of orthotopic Pan02-Luc pancreatic tumors, preventing distant metastasis and prolonging the survival of mice.

[0195] Subsequently, we further verified the occurrence of cell pyroptosis in this process. Through protein immunoblotting analysis, we observed that the activation fragment of the key protein of pyroptosis, GSDMD-N, could be clearly detected in the tumor samples of the NP-NH-D5+anti-PD-L1 combined treatment group. Combined with the results that the levels of pyroptosis-related cytokines IL-18 and IL-1β were significantly upregulated in mouse serum. We thus confirmed that NP-NH-D5+anti-PD-L1 can induce obvious GSDMD-mediated pyroptosis in pancreatic tumors.

[0196] In addition, we evaluated the immunomodulatory effect of NP-NH-D5+anti-PD-L1 combined treatment. After NP-NH-D5+anti-PD-L1 combined treatment, the levels of immune-related cytokines TNF-α, IL-6, and IFN-γ in the serum were significantly upregulated. The maturity of DCs in the mouse lymph nodes was also improved. At the same time, the number of CD4+ and CD8+ T cells in the tumor microenvironment increased significantly, while the proportion of regulatory T cells (Treg) decreased significantly. NP-NH-D5+anti-PD-L1 treatment can also effectively promote the transformation of TAMs from the tumor-promoting M2 type to the anti-tumor M1 type, thereby significantly increasing the ratio of M1 to M2, far exceeding the PBS-treated group.

[0197] In summary, these results indicate that NP-NH-D5 is a specific inducer of tumor cell pyroptosis that not only effectively activates antitumor immune responses but also enhances the efficacy of anti-PD-L1 therapy against malignant 4T1 breast and Pan02 pancreatic tumors.

[0198] 11. Biosafety Research

[0199] To evaluate the biosafety of NP-NH-D5, we injected it into healthy mice and performed histological examinations of major organs. The results showed that no pathological abnormalities or other adverse changes were observed in the tissues. In addition, through routine blood tests and blood biochemical analysis, we did not find any abnormalities in blood or liver / kidney function, proving that NP-NH-D5 has good biocompatibility in mice. These results support the potential of NP-NH-D5 as a safe and effective anti-tumor therapeutic probe.

[0200] V. Conclusion

[0201] In the present study, we developed a tumor-specific lysosomal disruptor and pyroptosis inducer, NP-NH-D5. NP-NH-D5 can sequentially respond to extracellular MMP-2 and intracellular GILT enzymes and GSH to selectively transport F-C6-NH2 into tumor cells, thereby precisely assembling into nanofibers in lysosomes. The experimental results showed that NP-NH-D5 can induce GSDMD-mediated pyroptosis in different tumor cells and stimulate a strong anti-tumor immune response by causing lysosomal membrane permeability and lysosomal destruction. In addition, the combined use of NP-NH-D5 and anti-PD-L1 significantly improved the survival rate of mice in immunotherapy for the treatment of advanced 4T1 lung metastasis and orthotopic Pan02 pancreatic tumors without any systemic side effects. This strategy demonstrates the feasibility of inducing tumor cell pyroptosis and promoting anti-tumor immunity by finely regulating the charge and morphology of nanoassemblies. This programmable assembly strategy provides new possibilities for creating "intelligent" nanoassemblies as pyroptosis inducers for precise cancer treatment.

[0202] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

[0203] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified and replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A tumor targeting complex, characterized in that: The complex comprises a compound of formula I and a compound of formula II: Formula I Formula II.

2. The tumor targeting complex according to claim 1, characterized in that The molar ratio of the compound of formula I to the compound of formula II in the complex is 1:1 to 100:

1.

3. The tumor targeting complex according to claim 2, characterized in that The molar ratio of the compound of formula I to the compound of formula II in the complex is 5:

1.

4. The method for preparing the composite according to any one of claims 1 to 3, characterized in that: The method comprises preparing the compound of formula I and the compound of formula II separately and then mixing them according to a certain proportion.

5. The preparation method according to claim 4, characterized in that: The preparation method of the compound of formula I comprises: 3,5-bis(trifluoromethyl)benzamide is connected to the detection group with a first flexible chain, and then the terminal amino group is connected on the other side of the detection group through a second flexible chain to obtain a compound of formula I, wherein the first flexible chain is an alkyl chain and the second flexible chain is a hydrophilic PEG chain.

6. The preparation method according to claim 4, characterized in that: The preparation method of the compound of formula II comprises: 3,5-bis(trifluoromethyl)benzamide is connected to the detection group via a third flexible chain, and then the GALGLP peptide chain is connected on the other side of the detection group via a fourth flexible chain to obtain a compound of formula II, wherein the third flexible chain is a flexible chain containing a disulfide bond, and the fourth flexible chain is a hydrophilic PEG chain.

7. Use of the complex according to any one of claims 1 to 3 or the complex prepared by the method according to any one of claims 4 to 6 for targeting tumors.

8. A drug for treating tumors, characterized in that: The method comprises the compound according to any one of claims 1 to 3 or the compound prepared by the method according to any one of claims 4 to 6.

9. The drug according to claim 8, characterized in that Immunotherapeutic agents are also included.

10. The drug according to claim 9, characterized in that The immunotherapeutic agent is anti-PD-L1.

Images