A lysosome-oriented nanofiber monomer and its preparation and application

Abstract

The present invention provides a lysosome oriented nanofiber monomer and its preparation and application, wherein the monomer has a structure of formula I: #imgabs0#; wherein A1=S, O or C, A2=C or N, B1=C or N, B2=S, O or C, X=0-6, Y=C or S, Z=C or O. The fiber monomer can be taken up by cells and enter lysosomes, and assembled in the lysosomes to form nanofibers with strong rigidity, thereby destroying the lysosomes and further causing cell pyroptosis.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a lysosome oriented nanofiber monomer and the preparation and application thereof. Background Art

[0002] In recent years, constructing supramolecular assemblies inside living cells has been shown to be a versatile strategy that is essential for precisely controlling cell fate. As a complex nonequilibrium system, cells rely on compartmentalization and energy-driven concentration gradients within them to maintain physiological functions. In such an environment, self-assembling monomers activated by chemical conversion must exhibit a sufficiently low critical aggregation concentration (usually at the micromolar level) to effectively self-assemble under physiological conditions. Intermolecular interactions, namely self-assembly tendency, are the key factor in determining the critical aggregation concentration reached by these active monomers. Moreover, in the intracellular environment, especially the macromolecular crowding phenomenon in organelles, such as soluble proteins, dense fiber structures, and phase separation, not only increase the diffusion barriers between molecules, but also further enhance the self-aggregation tendency of molecules. Therefore, the Organelle-localization Induced Supramolecular Self-assembly (OLISA) strategy induced by specific organelle localization has been proposed, that is, to promote the self-assembly process by increasing the local concentration of molecules in the target organelle without the need for additional external stimuli. Small molecules designed using this strategy can easily penetrate the cell membrane, precisely locate to specific organelles or subcellular structures, and achieve self-assembly by increasing the local concentration of molecules.

[0003] Nanofibers, as a key form of molecular self-assembly, especially the formation of nanofibers in specific organelles, have become a widely used strategy to selectively affect cellular functions. The formation of these nanofibers depends on the balanced assembly process between individual fiber-forming units and their intermolecular interactions. In the intracellular self-assembly process, commonly used assembly units include peptides, polyaromatic compounds, polymers, and metal nanoparticles. Among them, most molecules that can directly self-assemble on subcellular organelles are usually peptide-based amphiphilic compounds, and their assembly sequences are usually derived from the active part of the protein structure. For example, Lys-Leu-Val-Phe-Phe (KLVFF) is a β-folding peptide derived from β-amyloid peptide (Aβ), while amino acids such as Phenylalanine (Phe, F), Tryptophan (Trp, W) and Tyrosine (Tyr, Y) are also commonly used to construct fiber-forming units. These simple fiber-forming units can self-assemble into ordered structures, such as parallel or antiparallel β-folds, driven by non-covalent interactions such as hydrogen bonding, hydrophobic interactions, and π-π stacking. However, these sequences face challenges of proteolysis and metabolism when used in vivo, especially when constructing nanofiber assemblies in lysosomes rich in proteolytic enzymes, which limits their stability and application range. Therefore, although nanofibers show great potential in regulating cell functions, it is particularly important to solve the problem of their stability in complex biological environments in order to obtain wider applications in vivo. Summary of the invention

[0004] The purpose of the present invention is to provide a lysosome-oriented nanofiber monomer and the preparation and application of the monomer, thereby providing a new strategy for tumor treatment.

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

[0006] In a first aspect, the present invention provides a lysosomal oriented nanofiber monomer having a structure of Formula I:

[0007]

[0008] Formula I

[0009] Among them, A1=S, O or C, A2=C or N, B1=C or N, B2=S, O or C, X=0~6, Y=C or S, Z=C or O.

[0010] Preferably, the monomer is the following compound:

[0011] .

[0012] In a second aspect, the present invention provides a method for preparing a compound of formula I, comprising the following steps:

[0013] The compound of formula I is obtained by connecting 3,5-bis(trifluoromethyl)benzamide to the detection group via a first flexible chain, and then connecting the terminal amino group on the other side of the detection group via a second flexible chain.

[0014] Preferably, the first flexible chain is an alkyl chain, the second flexible chain is a hydrophilic PEG chain, and the detection group is fluorescein.

[0015] In a third aspect, the present invention provides use of a compound of formula I for destroying intracellular lysosomes.

[0016] Preferably, in said use, a cell is contacted with a compound of formula I, the compound of formula I enters and assembles in the cell lysosome, and destroys the lysosome.

[0017] In a fourth aspect, the present invention further provides a drug for treating tumors, wherein the drug comprises a compound of formula I.

[0018] Compared with the prior art, the beneficial effects of the present invention are: the fiber monomer can be taken up by cells into lysosomes, assembled in the lysosomes to form nanofibers with strong rigidity, destroying the lysosomes and further causing cell pyroptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The design principle of the lysosome-oriented nanofibers of the present invention is shown, wherein Figure 1 a shows the structure of compound 2-1, Figure 1 b shows a schematic diagram of compound 2-1 assembled into nanofibers, Figure 1 c is a schematic diagram showing the reaction caused by compound 2-1 after assembly in tumor lysosomes;

[0020] Figure 2 The chemical structures and cartoon diagrams of the control probes 2-2, 2-3 and 2-4;

[0021] Figure 3 Schematic diagram of the stacking mode study of compound 2-1;

[0022] Figure 4 The atomic force microscopy results of the dense structure of nanofibers of the present invention;

[0023] Figure 5 In vitro characterization of compounds 2-3 is shown;

[0024] Figure 6 Shows the co-localization study of compound 2-1 and lysosomal probe (red) in HeLa cells;

[0025] Figure 7 Showing the co-localization study of compound 2-1 in 4T1 cells;

[0026] Figure 8 Shows the results of studying lysosomal disruption in HeLa cells by AO staining;

[0027] Fig. 9 Shows Bio-TEM images of cells treated with compound 2-1;

[0028] Fig.10 The antitumor effects of the compounds were demonstrated against orthotopic 4T1 breast tumor model mice. DETAILED DESCRIPTION

[0029] 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.

[0030] 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 indicating the orientation or positional relationship 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 operate in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0031] In the research work of the present invention, we designed and synthesized a small molecule non-peptide amphiphilic compound 2-1 based on the supramolecular self-assembly strategy induced by organelle localization. Figure 1As shown in a, the structure of 2-1 is mainly composed of four parts: 1) hydrophobic 3,5-bis(trifluoromethyl)benzamide; 2) C6 alkyl chain resistant to GSH; 3) fluorescence quenching oxidized firefly luciferin (AO-Luc); 4) hydrophilic PEG3 chain fragment with amino group at the end. Driven by a series of strong intermolecular interactions, such as hydrogen bonds, van der Waals forces and π-π stacking, 2-1 can self-assemble into highly rigid nanofibers. After being taken up by cells, the molecule can quickly accumulate in lysosomes and reach a critical aggregation concentration to form a nanofiber structure. Such nanofibers have high rigidity and abundant amino groups, which can effectively increase the permeability of lysosomal membranes, thereby causing significant damage to lysosomes and promoting the release of mature cathepsin B into the cytoplasm. The release of mature cathepsin B activates the assembly of NLRP3 inflammasome and the activation of cysteine ​​protease-1 (Caspase-1), which in turn triggers the pyroptosis of tumor cells through the cleavage of GSDMD-FL into GSDMD-N mediated by Caspase-1. Figure 1 bc). In addition, the release of proinflammatory factors during pyroptosis stimulates a strong immunogenic cell death effect, which helps to create immunogenic “hot” tumors and achieve effective anti-tumor immunotherapy.

[0032] Experimental Section

[0033] 1. Reagents and Instruments

[0034] All chemical reagents were purchased from qualified reagent suppliers (Shanghai Bailingwei Technology Co., Ltd., Shanghai TCI Chemical Industry Development Co., Ltd., and Sigma-Aldrich, Germany) and were of analytical grade without further purification. Lyso-Tracker@RedDND-99 was purchased from Thermo Fisher Scientific, USA. Anti-CD11c-FITC, anti-CD80-PE, and anti-CD86-APC were purchased from BioLegend, San Diego, USA. High-glucose medium (Dulbecoo's Modified Eagle Medium, DMEM), fetal bovine serum (FBS), trypsin (Try), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) cell proliferation detection kit, DAPI and AnnexinV-FITC / PI apoptosis staining kits were all purchased from KeyGen Biotech Co., Ltd., China. Calprotectin (CRT) and AlexaFluor-488 conjugated goat anti-rabbit antibodies were purchased from Abcam, UK. ELISA kits for HMGB1, IL-18, and IL-1β were purchased from Huamei Biotechnology, China. ELISA kits for ATP detection were purchased from Beyotime, China. GSDMD, GSDME, and Caspase-1 antibodies were purchased from Abcam, UK. GPX4 and MLKL antibodies were purchased from Huaan Biotechnology, China. Anti-mouse IgG HRP-linked antibodies and anti-rabbit IgG HRP-linked antibodies were obtained from Cell Signaling Technology, USA. 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 normal stomach 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). BALB / c female mice aged 5-6 weeks and 6-8 weeks were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

[0035] NMR spectrum( 1 HNMR and 13C NMR was measured by 400 MHz Bruker Avance III 400 spectrometer or 500 MHz Bruker Avance spectrometer. The solvent was DMSO-d6. The chemical shift (δ) was in ppm. Singlet, doublet, triplet, quartet, dd (doublet of doublets), multiplet and broad peak were represented by s, d, t, q, dd, m and br respectively. The coupling constant (J) was in Hz. The number of hydrogen was determined by the integral value of the spectrum and was marked as nH.Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry and liquid chromatography-mass spectrometry (LC-MS) were acquired on AB SCIEX 4800Plus MALDITOF / TOFTM mass spectrometer and Agilent 6540 UHD Q-TOF LC / MS mass spectrometer, respectively. Analytical and semi-preparative high-performance liquid chromatography (HPLC) analyses were performed on ThermoScientific Dionex Ultimate 3000 with a mixture of acetonitrile and water (containing 1‰ trifluoroacetic acid) as the mobile phase. UV-visible absorption spectra were measured using Ocean Optics Maya 2000 Pro UV-Visible spectrometer. Fluorescence spectra were measured using HORIBA Jobin Yvon Fluoromax-4 fluorescence spectrometer. Dynamic light scattering (DLS) analysis was performed on a 90Plus / BI-MAS particle size analyzer (Brookhaven Instrument, USA). Transmission electron microscopy (TEM) was used to analyze the sample. Microscope (TEM) images were obtained on a JEM-2800 transmission electron microscope (JEOL Ltd.); atomic force microscopy (AFM) images were collected by a BioScopeResolve atomic force microscope (Bruker Corporation, USA); circular dichroism (CD) spectroscopy was performed on an Applied Photophysis Chirascan; fluorescence imaging analysis of cells and tissue sections was performed using a Leica TCS SP8 confocal laser scanning microscope or an Olympus IX73 fluorescence inverted microscope; the corresponding absorption value of MTT was read by an enzyme-labeled instrument (Tcan) to complete the cytotoxicity experiment; flow cytometry analysis was performed on a Coulter FC-500 flow cytometer or a NovoCyte Quanteon (Agilent Technologies, USA); an IVISLumina XRIII system was used to capture and store in vivo fluorescence images; and Livingimage software (PerkinElmer) was used to quantitatively analyze the regions of interest (ROI) in the images.

[0036] 2. Synthesis Example

[0037] 1. Synthesis of Compound 2-1

[0038]

[0039] 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%.

[0040] 1.1. Synthesis of compound 2-6:

[0041] 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.

[0042] 1.2. Synthesis of compound 2-7:

[0043] 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 F6N4NaO5 S2+ [M + Na]+ :683.0828; HRMS found: m / z 683.0826.

[0044] 1.3. Synthesis of compound 2-1:

[0045] 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.

[0046] 2. Synthesis of Compound 2-2

[0047]

[0048] Synthesis route of compound 2-2. Reaction conditions: (a) Aceticchloride, DIPEA, THF, rt, 2h, 85%.

[0049] Synthesis of compound 2-2:

[0050] Compound 2-1 (30 mg, 0.034 mmol), acetyl chloride (5 mg, 0.068 mmol) and DIPEA (16 µL, 0.068 mmol) were mixed in 1 mL of anhydrous THF and stirred at room temperature for 2 hours. The reaction progress was monitored by HPLC. After the reaction was completed, the liquid was dried by spin drying. Then, it was purified by semi-preparative HPLC. After liquid nitrogen treatment and freeze drying, a light yellow solid compound 2-2 (26.7 mg, yield: 85%) was obtained. 1H NMR (500 MHz, DMSO-d6) δ10.05 (s, 1H), 8.96 (t, J = 5.4Hz, 1H), 8.51 (s, 1H), 8.49 (d, J = 5.5 Hz, 2H), 8.43 (t, J = 5.8 Hz, 1H), 8.40 (s, 1H), 8.30 (s, 1H), 8.02 (dd, J = 8.9, 4.8 Hz, 1H), 7.87 (s, 1H), 7.56 (d, J = 8.9 Hz, 1H), 4.13 (t, J = 6.6 Hz, 2H), 3.53 (t, J= 7.9 Hz, 18H),3.36 (t, J = 5.9 Hz, 2H), 3.15 (q, J = 5.8 Hz, 2H), 1.78 (s, 3H), 1.71 – 1.62(m, 2H), 1.58 (dd, J = 13.8, 6.9 Hz, 2H), 1.48 – 1.31 (m, 4H). 13 C NMR (101MHz, DMSO-d6) δ 169.76, 163.87, 163.58, 161.32, 160.44, 158.60, 158.49,154.14, 153.75, 151.52, 148.79, 148.70, 138.79, 137.15, 136.79, 136.58,131.41, 131.08, 130.75, 130.46, 128.43, 127.53, 125.17, 124.95, 124.04,122.23, 119.41, 110.51, 70.27, 70.25, 70.21, 70.16, 70.04, 69.98, 69.57,69.23, 64.92, 39.00, 29.23, 28.87, 26.59, 25.56, 22.96. MS: calcd. forC 39 H 46 F6N6NaO9 S2+ [(M+Na)+]: 943.2564; found ESI-MS: m / z 943.2540.

[0051] 3. Synthesis of Compound 2-4

[0052]

[0053] Synthesis route of compound 2-4. Reaction conditions: (a) TCEP, NaHCO3, PBS, 37℃, 2h, 79%.

[0054] Synthesis of compound 2-4:

[0055] Compound 2-3 was synthesized according to a previously reported method (Research 2020, 4087069). Compound 2-3 (20 mg, 0.022 mmol), TCEP (6 mg, 0.024 mmol) and NaHCO3 (18 mg, 0.22 mmol) were dissolved in 2 mL PBS and stirred at 37 °C for 2 hours. The reaction was monitored by HPLC. After completion, the residue was purified by semi-preparative HPLC. A dark yellow oil, compound 2-4 (8.5 mg, yield: 79%), was obtained by lyophilization. 1 H NMR (500 MHz, DMSO-d6) δ8.42 (s, 1H), 8.39 (t, J = 5.8 Hz, 1H), 7.79 (d, J = 8.8 Hz, 2H), 7.16 (s,1H), 6.89 (dd, J = 8.8, 1.4 Hz, 1H), 3.59 – 3.45 (m, 18 H), 3.01 – 2.93 (m, 3H), 1.28 (s, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 161.90, 160.51, 158.37,153.62, 151.20, 144.98, 137.79, 126.54, 124.44, 116.64, 104.20, 70.24, 70.20,70.19, 70.11, 70.06, 70.01, 69.24, 67.12, 39.06, 28.90.MS: calcd. forC 21 H 30 N5O5S2+ [(M+H)+]: 496.1683; found ESI-MS: m / z 496.1658.

[0056] 3. Experimental Design

[0057] 1. Characterization of self-assembly performance

[0058] Compounds (2-1, 2-2, 2-3, 2-4) were dissolved in DMSO to obtain stock solutions. To determine their specific concentrations, the molecular UV absorption was measured. At the same time, the Lambert-Beer law was used to calculate the amino-oxidized firefly luciferin group in each molecular structure. Then, 2-1, 2-2, 2-3, and 2-4 were added to 1 mL of PBS buffer under ultrasonic treatment at a concentration of 100 μM. Subsequent characterization included: DLS analysis of each solution at different time points. For TEM analysis, the solution was gently dropped on a copper grid (carbon support film coated), immediately frozen in liquid nitrogen and vacuum dried overnight, and then the image was collected on the machine. Circular dichroism spectroscopy was collected using an Applied Photophysis Chirascan spectrometer, using a 1 mm quartz cuvette to measure samples with a concentration of 100 μM. For AFM analysis, 100 μM, 50 μL of 2-1 was dropped on a mica surface (TedPella, USA). After 5 minutes, the mica surface was gently washed twice with deionized water. Subsequently, 2 mL of deionized water was added to the detection chamber and imaging was performed using an atomic force microscope.

[0059] 2. Molecular simulation experiments

[0060] For each molecule, the most favorable crystal packing was predicted using the Polymorph module running on Materials Studio. The crystal structure prediction protocol consists of three parts: packing, geometry optimization, and clustering. The main part of the process is the packing phase, during which the space of possible crystal structures is searched for low-energy candidate structures. In addition, the space groups of interest must be provided (usually a small subset is required, rather than all 230 space groups). In this study, the 10 most common space groups (P21 / c, P-1, P212121, C2 / c, P21, Pbca, Pna21, Cc, Pbcn, and C2) were provided in the packing phase, and Monte Carlo simulated annealing was used as the search algorithm. Intelligent algorithms were used in the geometry optimization phase. Ewald summation and atom-based summation methods were used for charge and van der Waals interactions, respectively. All energy calculations were performed using the Universal force field.

[0061] 3. Cell culture

[0062] 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 mucosal cells GES-1 cells, human cervical cancer HeLa cells, human glioma U87MG cells, human hepatocellular carcinoma HepG2 cells, human gastric cancer MKN45 cells and human lung cancer A549 cells were purchased from the cell bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. 4T1, 4T1-Luc, HeLa, HepG2, U87MG, Pan02, Pan02-Luc and A549 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 units / ml penicillin and 100 units / ml streptomycin). MKN45, GES-1, and CT-26 cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI-1640) supplemented with 10% FBS, 100 units / ml penicillin, and 100 units / ml streptomycin. All cells were cultured at 37°C in a humidified atmosphere containing 5% carbon dioxide and 95% air.

[0063] 4. Cell co-localization experiment

[0064] HeLa cells were plated at 5 × 10 4 The cells were plated in four wells of a glass-bottomed culture dish and cultured overnight. To examine the intracellular localization, the original culture medium was removed, and 2-1 (10 μM) was then dissolved in DMEM medium without fetal bovine serum and incubated with HeLa cells at 37 degrees for 0.5, 1, 2, and 4 hours. After incubation, multiple rounds of washing were performed with PBS. Then, Lyso-Traker Red DND-99 (1 μM) was treated for 15 minutes. Fresh culture medium was replaced before shooting. Finally, images were taken using a fluorescent inverted microscope. Each experiment was repeated three times. Among them, the excitation wavelength of the fluorescence channel corresponding to 2-1 was 350 ± 22.5 nm, and the emission wavelength range was 420-460 nm. The excitation wavelength of the fluorescence channel corresponding to LysoTracker Red was 570 nm, and the emission wavelength range was 600-650 nm. After shooting, the cell images were analyzed using ImageJ software.

[0065] 5. Cytotoxicity assay

[0066] 4T1, CT-26, Pan02, MCF-7, GES-1, HeLa, U87MG, HepG2, MKN45, and A549 cells were seeded in 96-well plates (density of 1 × 10 4 / well), and cultured in a constant temperature in an incubator for 24 hours. Subsequently, compound 2-1 with a concentration gradient was incubated with the cells for 24 hours. Then, MTT PBS buffer solution (50μL, 1mg / mL) was added to each well and continued to be placed in the incubator for 4 hours. In subsequent operations, the well plate needs to be slightly shifted sideways, the culture medium needs to be carefully removed, and then DMSO (150μL / well) needs to be added. Before testing on the machine, carefully shake the well plate several times to ensure complete dissolution. Finally, use an enzyme reader to read the absorbance (OD) at 490nm.

[0067] The cell viability can be determined by the formula OD / ODcontrol×100%. The cells incubated with compounds 2-2, 2-3, and 2-4 were used to evaluate toxicity in the same manner as described above.

[0068] 6. Assessment of lysosomal rupture

[0069] To assess lysosomal disruption, HeLa and 4T1 cells were stained with AO. HeLa and 4T1 cells were seeded in glass-bottomed confocal culture dishes and cultured overnight. Subsequently, HeLa cells were incubated with 10 μM of 2-1, 2-2, 2-3, or 2-4 for 2 hours. After washing three times with PBS, 5.0 μM of AO dye dilution was added and incubated for 15 minutes. The culture medium was then removed and gently washed several times with PBS. In addition, the sterile culture medium was replaced before shooting, and the imaging of specific cells was captured using an inverted microscope. Specifically, its green channel: excitation wavelength is 488nm, emission wavelength is 515-545nm. Red channel: excitation wavelength is 550nm, emission wavelength is 610-640nm. Its detection was completed by a TRITC filter set equipped with a 590nm dichroic mirror and a 590nm long-pass emission filter.

[0070] 7. Biological transmission electron microscopy experiments on cells

[0071] To verify that it can be assembled into nanofibers in cells, HeLa cells were seeded in a 6-well culture dish at a density of 5×10 5cells / well, overnight incubation for 24 hours. Then, the cells were incubated with blank DMEM medium or DMEM medium containing compound 2-1 (10 μM) at 37°C for 1 hour and 4 hours, respectively. After washing each group of cells three times with cold PBS (pH 7.4), the cells were scraped with a cell scraper and collected by centrifugation. Subsequently, the cell pellet was dehydrated by using a concentration gradient acetone solution (50%, 70%, 80%, 96% for 10 minutes each, 100% three times). Subsequently, the sample was first treated with a mixed solution containing embedding agent and acetone (volume ratio = 1 / 1) for 1 hour. Then it was treated with a mixed solution of embedding agent and acetone with a volume ratio of 3:1 for 3 hours, and finally treated with pure embedding agent 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.

[0072] Next, the cells were sliced ​​to obtain slices about 70 nm thick. Before photographing, the slices were placed on a copper grid and stained with a saturated solution of lead citrate and uranyl acetate in ethanol for 5-10 minutes. After being rinsed with deionized water, the copper grid was dried. Finally, TEM analysis was performed using an HT7700 transmission electron microscope.

[0073] 8. Flow cytometry analysis of cell life and death

[0074] Approximately 2 × 10 5 4T1 cells were seeded in 6-well plates containing 2 mL of DMEM medium and cultured overnight. The original medium was then replaced with 1 mL of fresh medium containing different concentrations of compound 2-1 (10, 20, and 40 μM, respectively). After 2 hours, the medium was removed and the cells were washed three times with PBS. After gently washing with PBS (pH 7.4), the cells were detached from the plate with trypsin (500 μL per well) and the cell pellets were collected after centrifugation at 4 °C and 161 × g for 4 minutes. The washed pellets were resuspended in 500 μL of PBS buffer and stained with Annexin V-FITC and PI. All flow cytometry experiments were performed according to the manufacturer's instructions, and the cell population was analyzed by Annexin V-FITC and PI channels. The counts for each flow cytometric analysis were approximately 10,000, and the data were processed using NovoCyte Quanteon.

[0075] 9. Western Blot Analysis

[0076] The expression levels of MLKL, GPX4, GSDME, GSDMD, NLRP3, CTSB, ACS, and Caspase-1 were detected by Western Blot (WB) analysis. 4T1 cells were incubated with different concentrations of compound 2-1 (10, 20, and 40 μM) for 2 h. To induce pyroptosis, 4T1 cells were first treated with lipopolysaccharide (LPS, 5 μg / mL, 4 h), followed by the addition of Nigericin (15 μM, 15 min). Then, the cells were washed three times with PBS (pH 7.4), harvested using a cell scraper, and the cell pellets were collected by centrifugation. After lysing on ice, the cells were allowed to stand, vortexed once every 15 min, and vortexed four times before centrifugation at 13,000 × g for 15 min at 4 °C to obtain the lysate. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membranes (PVDF). The membrane was blocked for 1 hour in Tris Buffered Saline with Tween®20 (TBST) with 5% skim milk powder or bovine serum albumin. The membrane was then sealed with a plastic bag and incubated at room temperature for 30 minutes and then 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 horseradish peroxidase (HRP)-labeled secondary antibody for 1 hour. Finally, the membrane was washed four times with TBST and the signal was detected in a Tanon-4600 developer using an enhanced chemiluminescence (ECL) developer. After the target band was exposed, the exposed membrane was placed in an excess of TBST solution and shaken vigorously on a shaker overnight. On the second day, the membrane was blocked for a 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 slowly on a shaker. After washing four times with TBST, the membrane was incubated with the HRP-labeled secondary antibody buffer for 1 hour. After washing four times, the band signal was displayed using a Tanon-4600 imager and ECL developer. Finally, the band intensity was quantitatively analyzed using ImageJ software.

[0077] 10. Immunofluorescence staining of cells

[0078] 4T1 cells were seeded in four-well glass bottom dishes at a rate of approximately 5 × 10 cells per well. 4Cells. Next, the cells were incubated with compound 2-1 (at concentrations of 10, 20, and 40 μM, respectively) for 2 hours. Subsequently, the compound-containing medium was removed and the cells were washed three times with cold PBS. Next, the cells were fixed with 4% formaldehyde for 10 minutes. After fixation, they were washed again three times with cold PBS and incubated with the primary antibody dilution for CRT overnight. After washing, they were incubated with AlexaFluor-488-conjugated monoclonal secondary antibodies for 1 hour. After complete washing, immunofluorescence imaging was performed using an inverted microscope.

[0079] For the immunofluorescence staining experiment of the expression level of intracellular NLRP3, CTSB, and ASC, all steps were consistent with the previous description before fixation with 4% paraformaldehyde. After fixation, the cells were treated with 0.1% TritonX-100 for 20 minutes to enhance cell permeability, followed by overnight incubation with the corresponding primary antibody, washing, and incubation with AlexaFluor488-labeled secondary antibody for 1 hour. Finally, propidium iodide was used for nuclear staining, and the incubation was ensured for 15 minutes. Subsequent operations were as before. After light washing with PBS and replacing fresh culture medium, cell immunofluorescence images were collected using an inverted microscope.

[0080] 11. Cytokine detection experiment

[0081] The secretion of IL-18 and IL-1β was detected by enzyme-linked immunosorbent assay (ELISA). 4T1 cells were incubated with different concentrations of compound 2-1 (10, 20, and 40 μM) for 2 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 medium was detected using ELISA kits according to the instructions provided by the manufacturer.

[0082] 12 Evaluation of the immunogenic cell death effect

[0083] The release of HMGB1 and ATP was detected by ELISA kit. 4T1 cells were plated in 6-well plates at 5 × 10 5 The cells were seeded at a density of 10 μM, 20 μM, and 40 μM at 37 °C overnight. Subsequently, the cells were treated with compound 2-1 (at concentrations of 10 μM, 20 μM, and 40 μM) for 2 hours. After the incubation, the cell culture medium was collected and the release of HMGB1 and ATP in the culture medium was measured using ELISA kits according to the instructions provided by the manufacturer.

[0084] 13. In vitro assessment of dendritic cell maturity

[0085] To study the maturation of dendritic cells in vitro, bone marrow-derived dendritic cells (BMDCs) were collected and cultured from the bone marrow of 5- to 6-week-old BALB / c mice. Subsequently, 4T1 cells were incubated with different concentrations of compound 2-1 (10, 20, and 40 μM) for 2 hours, and untreated cells were set as a negative control group. After treatment, the cells were cultured overnight in fresh culture medium. Subsequently, 1×10 6 Immature DCs were co-cultured with 4T1 cells pretreated overnight with 2-1. The maturation of DCs was examined by flow cytometry after staining with anti-CD11c-FITC, anti-CD80-PE, and anti-CD86-APC antibodies.

[0086] 14. Animal experiments and establishment and treatment of orthotopic 4T1 breast tumor model

[0087] BALB / c female mice aged 5–6 weeks and 6–8 weeks were purchased from Beijing Weitonglihua Experimental 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.

[0088] To establish an orthotopic 4T1 tumor model, 5×10 5 4T1 cells were resuspended in 50 μL of a 50% volume ratio of matrix gel and DMEM mixture and then injected orthotopically into the right side of the mammary gland of 5-6 week-old mice. The length (L) and width (W) of each 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 90 mm 3 When the orthotopic 4T1 tumor volume was about 90 mm³, the mice were divided into two groups: one group received direct intratumoral injection of PBS (1×, 50 μL), and the other group received direct intratumoral injection of compound 2-1 (200 μM, 50 μL), once every 3 days for a total of five times. The tumor volume and body weight of each mouse were measured every three days for 21 days. Each experiment was performed in five mice.

[0089] 15. Data Processing

[0090] 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.

[0091] 4. Results and Discussion

[0092] 1. Design of compound 2-1 and its control compound

[0093] Compound 2-1 was synthesized according to the above-mentioned synthetic route. In order to verify the role of the terminal amino group and 3,5-bis(trifluoromethyl)benzamide in controlling the formation of nanofibers (NFs), we designed two control compounds: one is 2-2 in which the amino group is blocked by acetyl (Ac), and the other is 2-4 lacking the 3,5-bis(trifluoromethyl)benzamide part. In addition, we also designed 2-3, which introduced a disulfide bond that can be cleaved by GSH at the C6 chain of 2-2 (the specific structure is shown in Figure 2 The chemical structures of all intermediates and probes have been 1 HNMR, 13 The samples were characterized by CNMR, MALDI-TOFMS or ESI-MS mass spectrometry.

[0094] 2. Characterization of self-assembly performance

[0095] We studied the process of 2-1 self-assembly into nanofibers in aqueous solution. Through simulations using the polymorphic prediction module, we found that 2-1 molecules are stacked in an ordered β-sheet structure ( Figure 3 In addition, the positive peak at 195 nm and the negative peak at 204 nm in the circular dichroism (CD) analysis further confirmed the formation of the β-sheet structure ( Figure 3 b).

[0096] Figure 3 The stacking mode study of compound 2-1 is shown. Figure 3 a shows a simulation of the stacking pattern of compound 2-1 assembled into nanofibers. The upper figure (top view) shows the shape of NFs, and the lower figure (side view) shows the ordered β-fold structure. Figure 3 b shows the circular dichroism spectrum of compound 2-1 (100 μM) after dispersion in deionized water for 4 h.

[0097] Subsequently, we conducted an in-depth analysis of the molecular interactions in the simulated stacking mode and found that there are multiple strong interactions between the tightly packed molecules, including hydrogen bonds, van der Waals forces, and π-π stacking. These interactions work together to promote the effective self-assembly of the molecules, thus forming structurally stable nanofibers.

[0098] The results of DLS and TEM analysis further showed that compound 2-1 could quickly self-assemble into spherical nanoparticles (NPs) and short nanofibers (NFs). Over time, it gradually developed into longer nanofibers with an average diameter of about 12 nm within 4 hours. This process clearly demonstrated the dynamic changes of 2-1's self-assembly in aqueous solution and its ability to form a stable structure.

[0099] In addition, the results of atomic force microscopy (AFM) clearly show the dense structure of the nanofibers, with an average height of about 3nm. High-resolution AFM images further show that these nanofibers can be tightly entangled with each other to form a structure similar to horsehair. At the same time, the average Young's modulus was found to be 464±75MPa, showing high mechanical strength ( Figure 4 ).

[0100] Figure 4 a shows a schematic diagram of atomic force microscopy measurement. Figure 4 b shows the AFM image of compound 2-1 (100 μM) dispersed in deionized water for 4 h (left image, scale bar: 400 nm. Right image is a high-resolution AFM image, scale bar: 200 nm). Figure 4 c is shown in Figure 4 Height (top) and Young's modulus (bottom) maps of the cross sections marked in b. White dashed lines indicate the cross sections marked in the surface topography, labeled with numbers 1–7. Figure 4 c shows the Gaussian distribution statistics of Young’s modulus measured by AFM.

[0101] Compared with the free state of 2-1 molecules in methanol, it self-assembles in PBS buffer solution, and the corresponding UV absorption peak becomes broad. Although the fluorescence intensity at 450nm decreases, the fibrous structure can still be clearly observed under confocal fluorescence microscopy. In addition, its CAC is 9.3µM measured and calculated by fluorescence method.

[0102] In addition, we investigated the self-assembly behavior of 2-2, 2-3, and 2-4 to evaluate the effect of the structural features of 2-1 on its self-assembly ability. DLS and TEM analysis showed that 2-2, which was protected by acetyl groups, could only self-assemble into spherical nanoparticles of approximately 120 nm with a CAC concentration of 4.6 µM, which emphasizes the key role of free amino groups in the formation of nanofibers. In contrast, since 2-3 has a similar structure to 2-1, it can also self-assemble into a similar nanofiber structure ( Figure 5). In addition, the responsiveness of 2-3 to GSH was observed by HPLC and fluorescence spectroscopy. However, compound 2-4 lacked the hydrophobic 3,5-bis(trifluoromethyl)benzamide and therefore failed to form any nanoparticles or nanofibers.

[0103] Figure 5 In vitro characterization of compounds 2-3 is shown. Figure 5 a Schematic diagram showing the molecular self-assembly of compound 2-3 into nanofibers through GSH reduction to small molecule products F-SH and AO-Luc-NH2. Figure 5 b shows the DLS analysis of compounds 2-3 (100 μM) in PBS buffer (pH 7.4), Figure 5 c is TEM analysis, Figure 5 d is CD analysis. Figure 5 eg shows HPLC analysis (e), DLS analysis (f) and fluorescence spectroscopy analysis (g) of compound 2-3 before and after incubation with GSH (10 mM) for 2 hours.

[0104] 3. Lysosomal destruction and cytotoxicity studies

[0105] After confirming that the probe can self-assemble into nanofibers, we further explored its activity at the cellular level. Fluorescence imaging results showed that compound 2-1 accumulated mainly in lysosomes after being co-incubated with HeLa or 4T1 tumor cells for 0.5 hours. When the incubation time reached 2 hours, although the green fluorescence emitted by compound 2-1 in the cells was brighter, the red fluorescence of the lysosomes labeled by the lysosomal probe (Lyso-trackerred) disappeared. This change clearly indicated the rupture of the lysosomes ( Figure 6 and Figure 7 ). In addition, with the increase of incubation time, the morphology of the cells changed significantly, especially after 4 hours, obvious vacuoles were generated around the cells, further confirming the damage to the cell structure after lysosomal rupture. In contrast, after incubation of HeLa cells with compounds 2-2 and 2-3 for 2 hours, the lysosomal probe was still able to label the intracellular lysosomes, indicating that no lysosomal damage was caused. In addition, the results of acridine orange (AO) staining also showed that in HeLa cells treated with compound 2-1 (10 μM) for 2 hours, the red fluorescence inside was weakened compared with the control group, while the green fluorescence intensity increased, which further confirmed that compound 2-1 can cause increased lysosomal membrane permeability, ultimately leading to lysosomal damage ( Figure 8 ).

[0106] Figure 6 Co-localization study of compound 2-1 and lysosomal probe (red) in HeLa cells, with incubation time of 0.5, 1, 2, and 4 hours. The red arrow indicates the pyroptotic body. Scale bar: 20µm.

[0107] Figure 7 Colocalization study of compound 2-1 in 4T1 cells is shown. 4T1 cells were incubated with compound 2-1 (10 μM, green) for 0.5 h and then co-stained with a lysosomal probe (red) for 15 min. Scale bar: 20 µm.

[0108] Figure 8 Shown is the study of lysosomal disruption in HeLa cells by AO staining. Figure 8 a shows fluorescence imaging of HeLa cells after the indicated treatments. HeLa cells were incubated with I: blank medium, II: 2-1 (10 µM), III: 2-2 (10 µM), or IV: 2-3 (10 µM) for 2 h, washed three times with PBS, and then co-stained with acridine orange (5 µM) for 15 min. Green channel: λ em =515-545nm; red channel λ em =610-640nm. Scale bar: 20μm. Figure 8 b shows flow cytometry analysis after AO staining. Red channel λ em =610-640nm (top) and green channel λ em =515-545nm (below). Figure 8 cDisplay Figure 8 Quantification analysis of the mean fluorescence intensity of the red and green channels in b. Data are expressed as mean ± SD (n = 3).

[0109] In addition, when tumor cells were incubated with preformed nanofibers, the green fluorescence from the nanofibers was mainly distributed on the cell membrane. Lysosomal probe staining and AO staining results showed that there was no significant difference between the imaging of cells treated with preformed nanofibers and those in the blank control group, indicating that the lysosomes were not significantly damaged. These results indicate that, unlike free compound 2-1, preformed nanofibers mainly adhere to the outside of the cell membrane and are difficult to enter the cell to cause lysosomal damage.

[0110] To investigate whether lysosomal damage is caused by self-assembled nanofibers inside lysosomes, we conducted experiments using biological transmission electron microscopy (Bio-TEM) ( Fig. 9). In the untreated blank control group, no nanofiber structure was found and the lysosomal membrane of the cells was intact. However, after 1 hour of treatment with compound 2-1, dense nanofibers appeared in the lysosomes of HeLa cells, and the lysosomal membrane became loose. After 4 hours, the cytoplasm was filled with long nanofibers, a large number of damaged lysosomes appeared, the cell structure was destroyed, and obvious signs of lysosome-dependent cell death were shown. This result fully demonstrates the ability of compound 2-1 to form nanofibers through self-assembly in lysosomes, thereby causing lysosomal damage and leading to cell death.

[0111] Fig. 9 Bio-TEM images show blank HeLa cells and HeLa cells incubated with compound 2-1 (10 μM) for 1 hour or 4 hours. The red box marks the magnified area. The red arrow indicates the loose lysosomal membrane with dense nanofibers inside. The green arrow shows the swollen cell membrane. The blue arrow points to the nanofibers.

[0112] Next, we further evaluated the specific effect of 2-1 on cell activity through MTT assay. Compound 2-1 showed obvious concentration-dependent cytotoxicity to HeLa and 4T1 cells. The half inhibitory concentration (IC 50 ) were 2.40±0.14μM and 4.05±0.12μM, respectively, showing significant anticancer activity. In contrast, compounds 2-2, 2-3, 2-4 and preformed nanofibers showed almost no cytotoxicity to HeLa and 4T1 cells at a concentration of 50μM.

[0113] It is worth mentioning that compound 2-1 also showed strong cytotoxicity to other tumor cells, showing its potential to fight against multiple types of cancer. This result not only verifies the effectiveness of compound 2-1 as a new anticancer agent, but also demonstrates its potential as a broad-spectrum anti-tumor drug.

[0114] 4. Study on the anti-tumor mechanism of compound 2-1 and evaluation of its immunogenic cell death effect

[0115] In view of the above results, we further studied the mechanism of action of compound 2-1 in inducing cell death and triggering immunogenic cell death. 4T1 cells treated with compound 2-1 showed typical characteristics of pyroptosis, such as cell swelling and huge bubbles formed around the cell membrane. Immunofluorescence staining and western blotting experiments showed that cathepsin B was activated and diffused into the cytoplasm in 4T1 cells treated with compound 2-1. In addition, the relevant immunofluorescence staining results showed the assembly and activation of NLRP3 inflammasomes, that is, large areas of spotted fluorescence appeared inside the cells, indicating the activation of intracellular inflammatory pathways. Western blotting analysis results further confirmed that compound 2-1 can effectively promote the cleavage of Caspase-1 precursor, while cells treated with compounds 2-2 or 2-3 did not show similar changes. In addition, the expression levels of mixed lineage kinase domain-like protein (MLKL, a marker of necroptosis) and glutathione peroxidase 4 (GPX4, a marker of ferroptosis) did not change significantly, thus excluding the possibility of necroptosis and ferroptosis. The results of flow cytometry experiments also further verified that after 2 hours of treatment with compound 2-1, more than 70% of 4T1 cells were in a state of pyroptosis or late apoptosis (Annexin V + / PI + ), while almost no necrotic cells were observed (PI - / AnnexinV + ), thus demonstrating the ability of compound 2-1 in effectively inducing cell pyroptosis.

[0116] In addition, by western blot analysis of GSDMD and GSDME, the key executors of pyroptosis, we found that compound 2-1 could induce the cleavage of full-length GSDMD (GSDMD-FL) into GSDMD-N active fragments in a concentration-dependent manner, while having little effect on the cleavage of GSDME-FL. This result emphasizes that compound 2-1 induces cell death through the GSDMD-mediated pyroptosis pathway. Further studies showed that compound 2-1 could significantly promote the secretion of proinflammatory cytokines such as IL-1β and IL-18 by 4T1 cells. As the concentration of compound 2-1 increased, the release of lactate dehydrogenase (LDH) also increased, indicating that severe cell pyroptosis occurred in the 4T1 cells treated with it.

[0117] Subsequently, we used immunofluorescence staining to observe the expression of calreticulin (CRT) on the cell surface, and analyzed the release of high mobility group protein 1 (HMGB1) and ATP, and found that with the increase in the concentration of compound 2-1, the levels of these three indicators increased significantly. This result shows that compound 2-1 can strongly induce immunogenic cell death (ICD) effect. At the same time, this effect can effectively promote the maturation of DCs and stimulate a strong anti-tumor immune response.

[0118] 5. Study on the anti-tumor application of compound 2-1 at the in vivo level

[0119] After confirming that compound 2-1- can induce pyroptosis and stimulate immune response in tumor cells, we further conducted tumor treatment studies on mice with orthotopic 4T1 breast tumor models. In the experiment, PBS (50μL) and compound 2-1 (200μM, 50μL) were injected into the tumors of tumor-bearing mice by intratumoral injection to evaluate the anti-tumor effect of compound 2-1 ( Fig.10 a). During the 21-day observation period, the tumor volume and body weight of the mice were measured every three days. Compared with the control group (Group I) treated with PBS alone, the mice treated with Compound 2-1 (Group II) showed that their tumor growth was significantly inhibited within 21 days ( Fig.10 In addition, there was no significant change in the body weight of all treated mice during the observation period ( Fig.10 b). This result indicates that compound 2-1 exhibits lower biological toxicity in this treatment regimen.

[0120] Fig.10 The antitumor effect of compound 2-1 on orthotopic 4T1 breast tumor model mice was demonstrated. Fig.10 a Schematic diagram showing the treatment scheme of compound 2-1 in treating orthotopic 4T1 tumors in mice. Fig.10 b and c show the changes in relative body weight (m / m0) (b) and tumor volume (V / V0) (c) of mice after treatment with compound 2-1 or PBS (n=5). Fig.10 d shows representative photos of mice after indicated treatments on days 0, 3, 6, 9, 12, 15, 18, and 21. Red circles indicate tumor sites in mice. Mice were injected (it) with PBS (50 μL) or 2-1 (200 μM, 50 μL) on days 0, 3, 6, 9, and 12 and monitored for 21 days.

[0121] V. Conclusion

[0122] This study successfully synthesized a small molecule non-peptide amphiphilic compound 2-1, demonstrating its unique mechanism of self-assembly into nanofibers and inducing cell pyroptosis in living cell lysosomes, highlighting its great potential in anti-tumor therapy. By designing three control compounds, this study briefly explored the relationship between the self-assembly behavior of compound 2-1 and its molecular structure, laying the foundation for the design and structural optimization of new molecules in the future. It is worth noting that compound 2-1 can quickly aggregate in lysosomes after cell uptake, causing an increase in lysosomal membrane permeability, thereby triggering a series of intracellular reactions, including the release of mature cathepsin B, activation of NLRP3 inflammasomes, and cleavage of Caspase-1 precursors, ultimately leading to tumor cell pyroptosis through Caspase-1-mediated GSDMD-FL cleavage. Moreover, the release of pro-inflammatory factors during pyroptosis significantly promoted immunogenic cell death, providing a new strategy for constructing an immunogenic "hot" tumor environment and advancing anti-tumor immunotherapy. Future research will focus on improving its tumor-killing specificity, for example, by coupling targeting groups, in order to further improve the therapeutic efficiency and safety, and provide a more precise and effective solution for anti-tumor therapy. Organelle-specific supramolecular systems provide new opportunities for treatment and in-depth study of cell functions.

[0123] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

[0124] 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. Use of the following compound for the manufacture of a drug for destroying intracellular lysosomes, characterized in that: The compound has the following structure:

2. The use according to claim 1, characterized in that The cells are contacted with the compound, which enters and assembles in the lysosomes of the cells, disrupting the lysosomes.

3. Use of the compound according to claim 1 for the manufacture of a medicament for treating tumors.

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