Dual-targeted photodynamic synergistic ferroptosis diagnosis and treatment integrated probe for lung cancer and preparation method and application thereof

Abstract

The application discloses a lung cancer dual-targeting photodynamic synergistic ferroptosis diagnosis and treatment integrated probe and a preparation method and application thereof. The probe is dual-targeted on integrin receptors and high-order sulfonic acidization characteristics which are highly expressed in tumor cells, realizes efficient enrichment and long-term retention of the probe in a tumor site. Meanwhile, by integrating photodynamic therapy and ferroptosis therapy, the degree of lipid peroxidation of cells is promoted, the progress of tumors is effectively inhibited, and diagnosis and treatment integration of the tumors is realized.

Description

Technical Field

[0001] This invention belongs to the technical field of small molecule therapeutic probes for bioimaging and tumor treatment. Specifically, it relates to a photodynamic synergistic ferroptosis-based molecular probe for dual-targeting lung cancer, its preparation method, and its application in fluorescence imaging and tumor treatment. Background Technology

[0002] In recent years, photodynamic therapy (PDT) and ferroptosis have become research hotspots in the early diagnosis and treatment of non-small cell lung cancer (NSCLC) due to their high specificity and low side effects. PDT induces tumor cell death by generating reactive oxygen species (ROS) at specific wavelengths using photosensitizers, but its efficacy is limited by the hypoxic tumor microenvironment and compensatory activation of the antioxidant system. Ferroptosis, on the other hand, causes cell membrane damage through iron-dependent lipid peroxidation, but existing ferroptosis inducers suffer from poor targeting and metabolic instability. Furthermore, the tumor microenvironment exhibits highly expressed hyposulfonated proteins, and based on this, targeting and cross-linking hyposulfonated proteins provides a new direction for precision medicine. Summary of the Invention

[0003] This invention discloses a dual-target photodynamic therapy (PDT) synergistic ferroptosis (FFT) probe for lung cancer, its preparation method, and its application. By targeting integrin αvβ3 receptor and hyposulfonated protein, the probe enhances the enrichment and long retention of tumor-specific components, integrates PDT and ferroptosis mechanisms, and synergistically enhances the anti-tumor effect through ROS burst and lipid peroxidation. Simultaneously, near-infrared fluorescence imaging is used to monitor drug distribution and tumor treatment efficacy in real time, achieving integrated diagnostic and therapeutic functions.

[0004] The present invention adopts the following technical solution:

[0005] A dual-targeted photodynamic therapy-assisted ferroptosis diagnostic probe for lung cancer, the chemical structure of which is as follows:

[0006] .

[0007] This invention also provides a method for preparing the above-mentioned dual-targeted photodynamic therapy-assisted ferroptosis diagnostic and therapeutic probe for lung cancer, comprising the following steps:

[0008] I-CD was obtained by reacting dye IR780 with mercapto-β-cyclodextrin. I-CD was then reacted with RGD-azidocyclopeptide to obtain IR-CD. IR-CD was then reacted with azidocyclohexanedione in a CuSO4 / sodium ascorbate system to obtain dIR-CD. dIR-CD was then encapsulated with ferrocene under ultrasound to obtain dIR-CDF, a dual-targeted photodynamic synergistic ferroptosis diagnostic and therapeutic probe for lung cancer.

[0009] Furthermore, the azidocyclohexanedione is obtained by reacting 3,5-dioxanecarboxylic acid with 3-azidopropylamine.

[0010] Furthermore, the reaction of 3,5-dioxanecarboxylic acid with 3-azidopropylamine was carried out in the presence of EDC, N-hydroxysuccinimide, and N,N-diisopropylethylamine; the molar ratio of 3,5-dioxanecarboxylic acid, 3-azidopropylamine, 1-ethyl-3-(3-dimethylaminopropyl)carbonyldiimazole hydrochloride, N-hydroxysuccinimide, and N,N-diisopropylethylamine was 1:1:1 to 2:1 to 2:1; the reaction was carried out at room temperature.

[0011] Furthermore, the reaction of dye IR780 with mercapto-β-cyclodextrin was carried out in N,N-dimethylformamide, with the addition of triethylamine; the molar ratio of dye IR780, mercapto-β-cyclodextrin and triethylamine was 1:1~2:1~2, the reaction was carried out at room temperature, and the reaction time was 10~14 hours.

[0012] Furthermore, the reaction between I-CD and RGD-azidocyclic peptide was carried out in the presence of sodium ascorbate and copper sulfate; the molar ratio of I-CD, RGD-azidocyclic peptide, sodium ascorbate and copper sulfate was 1:1:1~2:1~2, the reaction was carried out at room temperature, and the reaction time was 1~3 hours.

[0013] Furthermore, the molar ratio of IR-CD, azidocyclohexanedione, sodium ascorbate, and copper sulfate is 1:1:1~2:1~2.

[0014] Furthermore, the molar ratio of dIR-CD to ferrocene is 1:1~4.

[0015] The present invention also provides a fluorescence imaging contrast agent, including the above-mentioned dual-targeting photodynamic synergistic ferroptosis diagnostic probe for lung cancer.

[0016] The present invention also provides the application of the above-mentioned dual-targeted photodynamic synergistic ferroptosis diagnostic and therapeutic probe for lung cancer in the preparation of reagents that improve the retention time of the probe in tumor tissue or inhibit tumors.

[0017] This invention discloses the application of the above-mentioned integrated probe for diagnosis and treatment of lung cancer with dual-target photodynamic therapy and ferroptosis in the preparation of fluorescent imaging agents; or the application of the above-mentioned integrated probe for diagnosis and treatment of lung cancer with dual-target photodynamic therapy and ferroptosis in improving the retention time of the probe in tumor tissue or inhibiting tumors; or the application of the above-mentioned integrated molecular probe for diagnosis and treatment of lung cancer with dual-target photodynamic therapy and ferroptosis in the cross-linking of protein sulfinic acid in tumor cells to prolong the fluorescence imaging of tumor cells; or the application of the above-mentioned integrated probe for diagnosis and treatment of lung cancer with dual-target photodynamic therapy and ferroptosis in the targeted killing of tumor cells; or the application of the integrated probe for diagnosis and treatment of lung cancer with dual-target photodynamic therapy and ferroptosis in the inhibition of tumor growth.

[0018] Beneficial effects

[0019] This invention designs and synthesizes a novel dual-targeting photodynamic therapy-assisted ferroptosis diagnostic probe for lung cancer, which can target non-small cell lung cancer with stronger specificity, thereby enabling fluorescence imaging and anti-tumor treatment.

[0020] In this invention, the target probe specifically reacts with sulfinic acid, a protein in the mitochondria of tumor cells, to form a probe-protein conjugate, which prolongs the probe retention time to up to 96 hours and increases the probe enrichment effect.

[0021] In this invention, the target probe is cross-linked in vivo, extending the fluorescence imaging window of the animal to 96 hours.

[0022] In this invention, the target probe enters tumor cells and produces a photodynamic killing effect on the tumor cells under laser irradiation. Together with ferroptosis therapy, it causes mitochondrial damage and has a good ability to promote tumor cell apoptosis. The cell killing rate reaches 32.2%, realizing the integration of diagnosis and treatment. Attached Figure Description

[0023] Figure 1 A schematic diagram illustrating the preparation of a dual-targeted photodynamic therapy-assisted ferroptosis diagnostic and therapeutic probe for lung cancer;

[0024] Figure 2 For probe characterization; among which Figure 2 In the image, A represents the absorption and fluorescence spectra of the probe dIR-CDF in aqueous solution. Black represents the absorption spectrum, and red represents the fluorescence spectrum. Figure 2 In the image, B represents the UV-Vis absorption spectrum of a mixture of TMB solution, probe dIR-CDF, and hydrogen peroxide of different concentrations. Figure 2 C represents the ultraviolet absorption spectrum of DPBF solution after laser irradiation for different times with probe dIR-CDF added; Figure 2 D in the diagram represents the cross-linking of the probe dIR-CDF with hyposulfonated protein; Figure 2 E in the image represents the protein gel Coomassie brilliant blue staining and fluorescence imaging of the probe dIR-CDF (10 μM) and the protein conjugate of IR-CDF and BSA. Figure 2 In the middle F, the protein gel Coomassie brilliant blue staining image and fluorescence imaging image of the probe dIR-CDF (10 μM) with IR-CDF and BSA protein conjugate under different H2O2 conditions are shown.

[0025] Figure 3 To study the targeting and retention capabilities of the probe; Figure 3 Image A is a confocal image showing the uptake and retention of probes dIR-CDF and IR-CDF in NCI-H1975 cells; Figure 3In Figure B, the uptake of probe dIR-CDF in BEAS-2B cells is shown. Figure 3 In the middle C, the uptake of the experimental probe dIR-CDF and the control probe IR-CDF in NCI-H1975 cells is shown. Figure 3 In the middle D, the protein gel Coomassie brilliant blue staining image and fluorescence imaging image are shown after the probes IR-CDF and dIR-CDF were incubated with cells for 48 hours and then retained. Figure 3 E in the middle is a confocal image showing the retention of probes dIR-CDF and IR-CDF in NCI-H1975 cells; Figure 3 The F in the middle section represents a quantitative analysis of the retention of probes dIR-CDF and IR-CDF in NCI-H1975 cells;

[0026] Figure 4 For the study of the antitumor ability of the probe; Figure 4 In Figure A, the cytotoxicity assays of NCI-H1975 against different probes and treatment methods are shown. Figure 4 In Figure B, we present the cytotoxicity experiments of BEAS-2B using different probes and treatment methods. Figure 4 C represents the quantitative analysis of apoptosis in NCI-H1975 cells treated with different methods (10 μM); Figure 4 D represents the live-dead experiment of NCI-H1975 treated with different methods (10 μM). Figure 4 E represents the flow cytometry analysis of NCI-H1975 processed with different methods (10 μM); Figure 4 In the middle F, the subcellular distribution of probe dIR-CDF in NCI-H1975 cells is shown. Figure 4 In the middle G, NCI-H1975 cells were treated with different methods (10 μM) to perform mitochondrial membrane potential assays using the JC-1 probe. Figure 4 In the middle, H represents ROS fluorescence images of NCI-H1975 cells treated with different methods (10 μM);

[0027] Figure 5 To study the antitumor mechanism of the probe; Figure 5 In Figure A, the levels of GSH and MDA in NCI-H1975 cells were detected after treatment with different methods (10 μM); (Ⅰ: blank group, Ⅱ: dIR-CD+L, Ⅲ: IR-CDF, Ⅳ: dIR-CDF, Ⅴ: IR-CDF+L, Ⅵ: dIR-CDF+L); Figure 5 In Figure B, the quantitative analysis of GSH content in NCI-H1975 cells after treatment with different methods (10 μM) is presented. Figure 5In Figure 5, C represents the quantitative analysis of MDA content in NCI-H1975 cells after treatment with different methods (10 μM); in Figure 5, D represents the analysis of MDA content in NCI-H1975 cells after treatment with different methods (10 μM) using BODIPY. TM Confocal fluorescence microscopy detection of lipid peroxides in cells using the 581 / 591 C11 probe; Figure 5 E represents the different treatment methods (10 μM) used to treat NCI-H1975 cells before applying BODIPY. TM Quantitative statistical analysis of lipid peroxides in cells using the 581 / 591 C11 probe; Figure 5 In the middle F, NCI-H1975 cells were treated with different methods (10 μM) before being processed with BODIPY. TM Flow cytometry analysis of lipid peroxides in cells using the 581 / 591 C11 probe;

[0028] Figure 6 To study the targeted retention of probes at the living cell level; Figure 6 In Figure A, fluorescence imaging of mice at different time points was performed after tail vein injection. Figure 6 Image B shows a side view of fluorescence imaging of mice at different time points after tail vein injection. Figure 6 C represents the quantitative analysis of fluorescence intensity at different time points in mice after tail vein injection. Figure 6 The middle image (D) shows the fluorescence image of the distribution of dIR-CDF in mouse tumors and major organs 12 h after tail vein injection of the probe. Figure 6 The middle section (E) represents the quantitative statistical analysis of the distribution of dIR-CDF in mouse tumors and major organs 12 h after tail vein injection of the probe.

[0029] Figure 7 This demonstrates the antitumor effect of the probe at the in vivo level. Figure 7 A diagram in Figure A illustrates the drug administration regimens for treating NCI-H1975 tumors using different probes. Figure 7 Figure B shows the statistical analysis of tumor growth curves in mice from different groups. Figure 7 Image C shows isolated tumors from mice in different treatment groups; Figure 7 D represents the statistical analysis of the weight change curves of mice in different groups; Figure 7 Image E shows images of tumor tissue apoptosis (TUNEL), cell proliferation (Ki 67), and GPX 4 fluorescence staining in mice of each group after treatment.

[0030] Figure 8 Bioluminescence imaging of mice in different treatment groups during monitoring period; gray boxes indicate mouse deaths.

[0031] Figure 9Images of H&E staining of tumor tissues and major organs (heart, liver, spleen, lungs, and kidneys) in mice of each group after treatment; metastatic lesions in the liver are circled in red.

[0032] Figure 10 High-resolution mass spectrometry characterization of compound dIR-CDF. Detailed Implementation

[0033] The steps of synthesizing the dIR-CDF probe for dual-targeting photodynamic therapy and synergistic ferroptosis diagnosis and treatment of lung cancer in this invention are as follows:

[0034] 3,5-Dioxocyclohexanecarboxylic acid (compound 1) reacts with 3-azidopropylamine to yield azidocyclohexanedione (compound 2); dye IR780 (compound 3) reacts with mercapto-β-cyclodextrin cyclic peptide to yield I-CD (compound 4); N3-RGD reacts with I-CD (compound 4) to yield IR-CD (compound 5); IR-CD (compound 5) then undergoes a click chemistry reaction with azidocyclohexanedione (compound 2) to yield dIR-CD (compound 6); dIR-CD (compound 6) is encapsulated with ferrocene to obtain the final probe, namely the lung cancer dual-targeting photodynamic synergistic ferroptosis diagnostic and therapeutic probe dIR-CDF. IR-CDF and dIR-CD serve as control probes.

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are only used to explain and illustrate the technical solutions in the present invention, and are not intended to limit the scope of the present invention. In addition, unless otherwise stated, the materials, reagents, instruments, etc. used in the following embodiments can be obtained by commercial means, and the specific preparation operations and testing methods of the present invention are conventional techniques.

[0036] Example 1

[0037] The method for preparing the lung cancer dual-targeting photodynamic therapy-assisted ferroptosis diagnostic and therapeutic integrated probe of the present invention includes the following specific synthesis steps:

[0038] Compound 2 (azidocyclohexanedione): Weigh 30 mg (0.192 mmol) of 3,5-dioxocyclohexanecarboxylic acid (compound 1), 44.2 mg (0.384 mmol) of N-hydroxysuccinimide (NHS), and 73.5 mg (0.384 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbonyldiimidazolium hydrochloride (EDC). Dissolve the above raw materials in 3 mL of N,N-dimethylformamide (DMF), mix and add to a reaction flask and stir at room temperature for 2 h. Monitor the reaction progress by HPLC. After the reaction is complete, add 19.3 mg (0.192 mmol) of 3-azidopropylamine and 24.8 mg (0.192 mmol) of N,N-diisopropylethylamine, and continue stirring. Monitor by HPLC. The reaction is basically complete after stirring for 2 h. The reaction solvent DMF was removed using a rotary evaporator, and compound 2 (azidocyclohexanedione, 26.50 mg, yield 58.1%) was purified by silica gel column chromatography. Its molecular formula is C2. 10 H 14 The molecular ion peak of N4O3 was measured to be 237.1 by ESI-MS. 1 H NMR (600 MHz, DMSO) δ 8.01 (s, 1H), 3.23 – 3.00 (m, 4H), 2.44 – 2.18 (m, 4H), 1.33 – 1.23 (m, 5H).

[0039] Compound 4 (I-CD): 32 mg (0.054 mmol) of compound 3 (IR780, purchased from Aladdin) and 62.7 mg (0.054 mmol) of mercapto-β-cyclodextrin were weighed and dissolved in 2 ml of DMF. 5.5 mg (0.054 mmol) of triethylamine was added dropwise, and the mixture was stirred at room temperature for 12 h. The reaction progress was monitored by HPLC. The solvent DMF was removed by rotary evaporation, and the mixture was purified by silica gel column chromatography to obtain compound 4 (I-CD, 55 mg, yield 118.64%). MS (MALDI-TOF) Calcd for: C 82 H 13 N2O 34 S + ([M+H)) + ): 1701.69, found: 1701.72).

[0040] Compound 5 (IR-CD): Compound 4 (I-CD, 39.1 mg, 0.023 mmol) and RGD-azidocyclic peptide (14.86 mg, 0.023 mmol) (purchased from Gir Chemical) were weighed and dissolved in 2 mL of dimethyl sulfoxide (DMSO). Copper sulfate pentahydrate (4.55 mg, 0.023 mmol) and sodium ascorbate (5.76 mg, 0.023 mmol) were weighed and dissolved separately in 100 μL of ultrapure water. The two solutions were mixed and repeatedly blown and stirred until the solution turned into an orange suspension. This mixture was then added dropwise to the reaction solution and stirred at room temperature for 3 h. The reaction was monitored by HPLC. After the reaction was completed, the compound was separated and purified using preparative high performance liquid chromatography to obtain compound 5, namely probe IR-CD (23 mg, yield 59.4%; MS (MALDI-TOF) Calcd for: C109H152N13O42S+([M+H]+):2346.99, found: 2346.99).

[0041] Compound 6 (dIR-CD): Compound 5 (IR-CD, 23 mg, 0.0098 mmol) and Compound 2 (azidocyclohexanedione, 2.33 mg, 0.0098 mmol) were weighed and dissolved in 1 mL DMSO. Copper sulfate pentahydrate (2.45 mg, 0.0098 mmol) and sodium ascorbate (1.94 mg, 0.0098 mmol) were weighed and dissolved separately in 50 μL ultrapure water. The two mixtures were combined and repeatedly blown until the solution changed from black to orange suspension. This mixture was then added dropwise to the reaction solution and stirred at room temperature for 3 h. The reaction was monitored by HPLC. After the chemical reaction was completed, the compound was purified using preparative high-performance liquid chromatography to obtain compound 6, namely probe dIR-CD (15.68 mg, yield 68.26%). MS (MALDI-TOF) Calcd for: C 119 H 166 N 17 O 45 S + ([M+H)) + ):2585.09, found: 2585.62).

[0042] Preparation of probe dIR-CDF: Compound 6 dIR-CD (12.15 mg, 0.0047 mmol) was weighed and dissolved in 5 mL of ultrapure water. Ferrocene (Fc) 1.75 mg was weighed and dissolved in 50 μL of DMSO. The filtrate was slowly added dropwise to the aqueous solution of dIR-CD under ultrasonic conditions, followed by stirring at room temperature for 24 h. After inclusion, the mixture was filtered, and the filtrate was lyophilized for 48 h to obtain the product dIR-CDF.

[0043] Preparation of probe IR-CDF: Compound 5 IR-CD (11.03 mg, 0.0047 mmol) was weighed and dissolved in 5 mL of ultrapure water. Ferrocene (Fc) 1.75 mg was weighed and dissolved in 1 mL of 50 μL DMSO. The IR-CD was slowly added dropwise to the aqueous solution of IR-CD under ultrasonic conditions, followed by stirring at room temperature for 24 h. After inclusion, the mixture was filtered, and the filtrate was lyophilized for 48 h to obtain the product IR-CDF.

[0044] Example 2

[0045] Physicochemical properties of the probe:

[0046] UV absorption and fluorescence spectroscopy determination: Prepare 1 mL of probe dIR-CDF (10 μM) aqueous solution, add 400 μL to a micro-quartz cuvette, measure the absorption spectrum of the probe using a UV-Vis spectrophotometer, measure the fluorescence spectrum of the probe using a fluorescence spectrophotometer, and perform statistical analysis of the spectral data using statistical software (Graphpad Prism 8).

[0047] Singlet oxygen determination using a probe: Weigh an appropriate amount of DPBF and dissolve it in DMSO to prepare a 40 μM DPBF working solution. Add the probe dIR-CDF and mix to achieve a concentration of 10 μM. Add 2 ml of the solution to a four-way cuvette. Adjust the 808 nm laser parameters to (0.5 W cm⁻¹). -2 After irradiating the sample, samples were taken every ten seconds, mixed thoroughly, and their ultraviolet spectra were measured using a UV-Vis spectrophotometer in the wavelength range of 300-500 nm. Statistical analysis of the spectral data was then performed using statistical software (Graphpad Prism 8).

[0048] The ability of the probe to generate ·OH under different H2O2 concentrations was demonstrated by preparing PBS solutions with H2O2 concentrations of 0 mM, 1 mM, 5 mM, 10 mM, 25 mM, and 50 mM, respectively. The probe dIR-CDF (10 μM) was added, and the reaction was carried out at room temperature for 30 min. Then, the same volume of TMB chromogenic solution was added, and the OD value at 370 nm was measured using a microplate reader after 2 h. Statistical analysis of the OD values ​​was performed using Graphpad Prism 8 software.

[0049] In vitro cross-linking reaction of the probe: To investigate whether the probe dIR-CDF can effectively cross-link with hyposulfonated proteins, this study first verified this using in vitro protein cross-linking. Tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) was weighed and prepared into a 50 mM DMSO solution. 16 mg of fetal bovine serum albumin (BSA) was dissolved in 1 mL of PBS, and 40 μL was added to the BSA solution. The mixture was shaken at room temperature for 30 min. After 30 min, TCEP was removed using a 30 KD ultrafiltration tube. Before each centrifugation, double the volume of PBS solution was added, and the mixture was centrifuged at 4000 rpm for 10 min. This washing process was repeated three times, and the volume was adjusted to 2 mL for later use. PBS buffers for dIR-CDF and IR-CDF (40 μM) were prepared separately. The probe was added to the prepared BSA solution, and H2O2 (5 μM 1 mM) was added. Based on the type of probe added, three groups were designated: control group, IR-CDF group, and dIR-CDF group. The mixed reaction solution was placed in a shaker and reacted at 37°C for 1 h. After shaking, the mixture was dialyzed through a 3000 MW dialysis membrane for 24 h. 40 μM of the sample from the dialysis bag was mixed with 10 μL of loading buffer and loaded onto the gel for electrophoresis. After gel electrophoresis, the gel was imaged using an upconversion luminescence imaging system, followed by Coomassie brilliant blue staining for 4 h. After 4 h, the gel was destained using a destaining agent, and after 12 h of destaining, the gel was photographed against a white background. NCI-H1975 cells were seeded into 6-well plates, shaken well, and cultured in a cell culture incubator. When the cell confluence reached 70%, the old culture medium was discarded, and 1 mL of 10 μM probe dIR-CD or 10 μM IR-CDF in 1640 medium solution was added. After culturing for 12 h, the probe was discarded, and the cells were washed three times with PBS. Fresh culture medium was added, and the old culture medium was discarded and fresh culture medium was added at 0 h, 6 h, 12 h, and 24 h. At 48 h, the old culture medium was washed away, and 1 mL of lysis buffer was mixed with 10 μM of protease inhibitor. 300 μL of the prepared lysis buffer was added to each well for lysis for 15 min under ice bath conditions. After lysis, cells were scraped off using a cell scraper and transferred to 1.5 mL centrifuge tubes. The tubes were centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was collected after centrifugation and stored temporarily at -20°C or long-term at -80°C if not used immediately. The extracted proteins were used for gel electrophoresis. After electrophoresis, the gel was fluorescently imaged, followed by Coomassie Brilliant Blue staining and destaining.

[0050] Experimental Results: The experimental probe dIR-CDF and the control probe IR-CDF were successfully prepared and characterized by high-resolution mass spectrometry. Figure 10 As shown. Figure 2 As shown in Figure A, its UV-Vis absorption and fluorescence emission spectra peak at 789 nm and 816 nm, respectively, which gives the probe deep tissue penetration and low autofluorescence background. Figure 2 As shown in Figure B, the efficiency of the probe in catalyzing the generation of ·OH in the Fenton reaction is H2O2 concentration-dependent, demonstrating that it can accumulate more ·OH in the weakly acidic tumor microenvironment, thereby enhancing tumor-killing ability. Figure 2 As shown in Figure C, the efficiency of the probe in generating ¹O₂ is positively correlated with the laser irradiation time, providing experimental basis for setting subsequent laser irradiation parameters. Figure 2 As shown in E and F in Figure 2, in vitro BSA model experiments revealed that, in the presence of H2O2, the probe dIR-CDF can effectively undergo covalent cross-linking with hyposulfonate groups, and the fluorescence signal intensity after cross-linking is significantly higher than that of the control group.

[0051] Conclusion: This embodiment successfully prepared the experimental probe dIR-CDF and the control probe IR-CDF. The probe dIR-CDF has good optical properties, stability and excellent reactive oxygen species generation ability. The probe dIR-CDF has good targeting labeling efficiency with hyposulfonated protein, providing a molecular biological basis for the cross-linking strategy.

[0052] Example 3

[0053] Cell-killing effect of probes

[0054] Cytotoxicity assay: NCI-H1975 cells in logarithmic growth phase were collected, resuspended in culture medium, and then 100 μL (approximately 1000-3000 cells) was added to each well of a 96-well plate using a pipette. The 96-well plates were then incubated for 24 hours. After the cells had fully adhered and reached a suitable growth density, the old culture medium was removed, and culture medium containing different concentrations (0, 1, 5, 10, 20, 40, 80 μM) of probe was added according to the groups, with 5 replicates for each concentration. The 96-well plates were returned to the cell culture incubator, and after 12 hours of incubation, the culture medium was changed, and the 808 nm laser parameters were adjusted to 0.5 W cm⁻¹. -2 Cells were irradiated in 96-well plates for 3 min, and then incubated for 48 h. The prepared CCK-8 solution was added to each well, and the cells were returned to the incubator for further incubation. The 96-well plates were then removed to observe the degree of color change. The OD value of each well at 450 nm was detected using a multi-functional microplate reader, and the data were exported. Statistical analysis of the OD values ​​was performed using statistical software (Graphpad Prism 8).

[0055] Cell uptake experiment: Healthy NCI-H1975 cells were taken up at approximately 5 × 10⁻⁶ cells per cell. 4Cells were seeded at a density of [number] cells / well in confocal microscopy dishes with a glass bottom. After mixing, the dishes were placed in a cell culture incubator. When the cells reached approximately 80% confluence, the original culture medium was discarded, and 1 mL of 10 μM dIR-CDF and IR-CDF 1640 medium solution was added. Cells were cultured for 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h, respectively. After incubation at the set time points, the old culture medium was discarded, and the cell nuclei were stained and located using a nucleoside staining reagent (Hoechst 33342). After staining for 20 min, the staining agent was washed off with PBS, a small amount of PBS was added, and the uptake of the probe by the cells was observed using a confocal fluorescence microscope. The uptake of the dIR-CDF probe by BEAS-2B cells was observed and statistically analyzed using the same method.

[0056] Probe subcellular organelle distribution assay: Well-grown NCI-H1975 cells were introduced at approximately 5 × 10⁻⁶ cells per cell line. 4 Cells were seeded at a density of 1 cell / well in confocal microscopy dishes with a glass bottom. After mixing, the dishes were placed in a cell culture incubator. When the cells reached approximately 80% confluence, the original culture medium was aspirated, and 1 mL of 1640 medium solution containing 10 μM probe dIR-CDF was added. The cells were then incubated for 12 h. The original culture medium was aspirated, and the cells were washed three times with PBS buffer. Mitotracker-Green mitochondrial staining agent or Lysotracker-Green working solution prepared with 1640 medium was added, and the cells were returned to the incubator for 30 min. After 30 min, the original culture medium was aspirated, and the cells were washed with PBS buffer. An appropriate amount of PBS buffer was added, and the distribution of the probe in the organelles was observed under a confocal fluorescence microscope.

[0057] Intracellular retention assay: NCI-H1975 cells were seeded into confocal microscopy dishes, agitated, and cultured in a cell culture incubator. When cell confluence reached 70%, the old culture medium was discarded, and 1 mL of either dIR-CDF (10 μM) or IR-CDF (10 μM) in 1640 medium solution was added. After 12 h of incubation, the probe was discarded, and the cells were washed three times with PBS. Fresh culture medium was added, and the cell nuclei were stained (Hoechst 33342) at 0 h, 6 h, 12 h, 24 h, 48 h, and 96 h, and the fluorescence distribution was observed under a confocal fluorescence microscope. Quantitative analysis and statistical analysis were performed using ImageJ software.

[0058] Live-dead cell assay: Well-grown NCI-H1975 cells were subjected to a 5 × 10⁻⁶ ppm incubation. 4Seeds were placed in confocal dishes with glass bottoms at a density of approximately 80%. After mixing, the dishes were placed in a cell culture incubator. When the cells reached approximately 80% confluence, the original culture medium was discarded, and 1 mL of a prepared 1640 medium solution containing dIR-CDF (10 μM), dIR-CD (10 μM), IR-CDF (10 μM), or IR-CD (10 μM) was added to each dish. The dishes were then returned to the cell culture incubator for 24 hours. The 808 nm laser parameters were then adjusted to 0.5 W / cm². -2 Following the above group settings, each dish of cells was irradiated for 3 minutes. The cells were then returned to the cell culture incubator for 24 hours. After 24 hours, the cells were stained using a cell viability (Calcein / PI) and cytotoxicity assay kit. Cell viability was observed using a confocal fluorescence microscope after staining. Live cells stained with Calein showed green fluorescence, while dead cells stained with PI showed red fluorescence.

[0059] Apoptosis assay: Healthy NCI-H1975 cells were evenly seeded into 6-well plates, shaken well, and then placed in a cell culture incubator. When the cell confluence reached 80%, the original culture medium was discarded, and 1 mL of a prepared 1640 medium solution containing dIR-CDF (10 μM), dIR-CD (10 μM), IR-CDF (10 μM), or IR-CD (10 μM) was added to each well. After incubation for 24 h, the parameters of the 808 nm laser were adjusted to 0.5 W / cm². -2 Following the above group settings, cells were irradiated for 3 min per dish. Cells were then returned to the cell culture incubator for 24 h. After 24 h, cells were digested with trypsin and collected. Cells were stained using an Annexin V-FITC apoptosis kit, resuspended in 1 mL PBS, and transferred to EP tubes. The treated cells were placed on ice and analyzed by flow cytometry within 2 h.

[0060] Cellular uptake experiments verified that the probe dIR-CDF exhibits long retention characteristics and tumor targeting in tumor cells. Figure 3 As shown in Figures A, B, and C, the fluorescence signal of the probe in NCI-H1975 cells peaks at 12 h. Using long-stay cell assays, such as... Figure 3 D, Figure 3As shown in Figures E and F, the cross-linking efficiency of probe dIR-CDF with mitochondria at the cellular level is demonstrated. This indicates that the specific cross-linking of probe dIR-CDF with hyposulfonated proteins in tumor cells can significantly promote the enrichment of probe dIR-CDF in tumor cells and prolong its retention time to up to 96 hours. This overcomes the shortcomings of traditional lung cancer diagnostic and therapeutic drugs, such as poor targeting of tumor cells and short enrichment retention time, and lays a good foundation for subsequent imaging and treatment experiments in animal tumor models.

[0061] Cytotoxicity assays such as Figure 4 Figures A and B demonstrate that under 808 nm laser irradiation, the dIR-CDF group exhibited superior killing effect on NCI-H1975 cells compared to other treatment groups, while maintaining a low level of toxicity to normal cells and achieving a survival rate greater than 90%, highlighting the selectivity and targeting of the probe for tumor cell therapy. Live / dead cell assays and apoptosis assays revealed the synergistic therapeutic mechanism of the dIR-CDF probe, as shown in the experimental results. Figure 4 As shown in C, D, and E, the dIR-CDF+808 nm group exhibited significant red fluorescence and apoptosis rate of 32.2%, indicating that the synergistic effect of photodynamic therapy and ferroptosis promoted the killing effect of the probe on tumor cells. Organelle localization experiments were performed as follows... Figure 4 As shown in Figure F, the probe dIR-CDF mainly targets and accumulates in mitochondria, and generates reactive oxygen species locally that directly damage mitochondria, leading to cell dysfunction and apoptosis.

[0062] Conclusion: The probe dIR-CDF exhibits selectivity for NCI-H1975 cells. Due to the high degree of protein hyposulfonation in mitochondria, the probe accumulates primarily in the mitochondria after entering the cells, with the optimal uptake time being 12 h. The probe overcomes the efflux defects of small molecule drugs and amplifies the cell-damaging effect by covalently cross-linking tumor hyposulfonated proteins with cyclohexanedione groups through a photodynamic synergistic ferroptosis mechanism.

[0063] Example 4

[0064] Mechanism of probe-induced tumor cell killing effect

[0065] Intracellular ROS measurement: Well-grown NCI-H1975 cells were subjected to a 5 × 10⁻⁶ ppm incubator. 4Cells were seeded at a density of 1 cell / well in confocal microspheres with a glass bottom, well mixed, and then placed in a cell culture incubator. When the cells reached approximately 80% confluence, the original culture medium was discarded, and 1 mL of a prepared 1640 medium solution containing 10 μM dIR-CDF, 10 μM dIR-CD, 10 μM IR-CDF, or 10 μM IR-CD was added to each well. The cells were then returned to the cell culture incubator and incubated for 12 h. Afterward, the DCFH-DA probe was added and the cells were incubated in the dark for 30 min. The probe was then washed off, and the cells were washed three times with PBS. Subsequently, the 808 nm laser parameters were adjusted to 0.5 W / cm². -2 Following the above group settings, cells in each dish were irradiated for 3 min. Subsequently, images were taken using a confocal laser microscope. For flow cytometry, well-grown NCI-H1975 cells were seeded into 6-well plates, shaken well, and placed in a cell culture incubator. When the cells reached approximately 80% confluence, the original culture medium was discarded, and 1 mL of a prepared 10 μM dIR-CDF, 10 μM dIR-CD, or 10 μM IR-CDF 1640 medium solution was added to each dish. After incubation for 12 h, the DCFH-DA probe was added for incubation in the dark for 30 min. The probe was then washed off, and the cells were washed three times with PBS. The 808 nm laser parameters were then adjusted to 0.5 W / cm². -2 Following the above group settings, cells in each dish were irradiated for 3 minutes. Then, the cells were digested with trypsin and collected into centrifuge tubes. After centrifugation and discarding the supernatant, the cells were resuspended in 1 mL of PBS and placed in an ice bath for flow cytometry analysis within two hours.

[0066] Mitochondrial membrane potential assay: Well-grown NCI-H1975 cells were subjected to a mitochondrial membrane potential assay at approximately 5 × 10⁻⁶ m³ / h. 4 Seeds were placed in confocal microspheres at a density of cells / well, mixed well, and then placed in a cell culture incubator. When the cells reached approximately 80% confluence, the original culture medium was discarded, and 1 mL of a prepared 10 μM dIR-CDF, 10 μM dIR-CD, 10 μM IR-CDF, or 10 μM IR-CD medium solution was added to each well. The cells were then returned to the cell culture incubator and incubated for 12 h. The 808 nm laser parameters were then adjusted to 0.5 W / cm². -2 Cells were irradiated for 3 minutes per dish according to the above group settings. After irradiation, the cells were returned to the incubator and cultured for another 24 hours. After 24 hours, the probes were washed and stained using a mitochondrial membrane potential detection kit. After staining for 25 minutes, the JC-1 working solution was washed off, and the cells were observed and photographed using a confocal fluorescence microscope to assess the damage to the mitochondria.

[0067] GSH assay: NCI-H1975 cells were seeded into 6-well plates, shaken well, and incubated in a cell culture incubator. When the cell confluence reached 80%, the old culture medium was discarded, and 1 mL of 10 μM probe IR-CD, dIR-CD (10 μM), or IR-CDF (10 μM) in 1640 medium solution was added. After culturing in the incubator for 12 h, the 808 nm laser parameters were adjusted to 0.5 W cm⁻¹. -2 Cells were irradiated for 3 minutes per dish according to the above group settings. After irradiation, the cells were returned to the incubator and cultured for another 24 hours. After 24 hours, the cells were disrupted using an ultrasonic homogenizer, and a reduced glutathione assay kit was used for colorimetric detection. After colorimetric detection, the absorbance at 412 nm was measured using a multi-mode microplate reader. Finally, statistical analysis of the OD values ​​was performed using statistical software (GraphpadPrism 8).

[0068] MDA assay: After cell lysis, reagents were added according to the order specified in the malondialdehyde (MDA) assay kit. The samples were then placed flat in a shaker and shaken at 100°C for 90 min. After cooling in an ice bath, the samples were centrifuged at 10,000 rpm for 10 min using a refrigerated centrifuge. The supernatant was transferred to a 96-well plate, and the absorbance at 532 nm and 600 nm was measured using a multi-mode microplate reader. Finally, the differences in OD values ​​were statistically analyzed using Graphpad Prism 8 software.

[0069] Measurement of lipid peroxides at the cellular level: Confocal fluorescence microscopy imaging: Well-grown NCI-H1975 cells were imaged at approximately 5 × 10⁻⁶ cells per cell. 4 Seeds were placed in confocal microspheres at a density of cells / well, mixed well, and then placed in a cell culture incubator. When the cells reached approximately 80% confluence, the original culture medium was discarded, and 1 mL of a prepared 10 μM dIR-CDF, 10 μM dIR-CD, 10 μM IR-CDF, or 10 μM IR-CD medium solution was added to each well. The cells were then returned to the cell culture incubator and incubated for 12 h. The 808 nm laser parameters were then adjusted to 0.5 W / cm². -2Cells were irradiated for 3 min per dish according to the above group settings. After irradiation, the cells were returned to the incubator and cultured for another 24 h. A 7.5 μM working solution (BODIPY™ 581 / 591 C11) was prepared using culture medium and co-incubated with NHC-H1975 cells for 35 min. The cells were then washed three times with HBSS buffer, and fluorescence was captured at 488 nm and 585 nm using a confocal fluorescence microscope. For flow cytometry experiments, cells were treated differently according to the group settings, labeled with the working solution (BODIPY™ 581 / 591 C11), digested with trypsin, collected, and placed on ice for flow cytometry analysis. Cell detection was performed using the FITC channel.

[0070] Western blotting experiment: (1) Cell collection: NCI-H1975 cells were seeded into 6-well plates, shaken well, and then placed in a cell culture incubator. When the cell confluence reached 80%, the old culture medium was discarded, and 1 mL of 1640 culture medium solution containing probes IR-CD (10 μM), dIR-CD (10 μM), or IR-CDF (10 μM) was added respectively. After culturing in the incubator for 12 h, the parameters of the 808 nm laser were adjusted to 0.5 W cm⁻¹. -2(1) Irradiate cells for 3 min per dish according to the above group settings. After irradiation, put the cells back into the incubator and continue culturing for 48 h. (2) Extract total protein: After 48 h, wash away the old culture medium in the 6-well plate. Add lysis buffer containing 1% PMSF to each well of the 6-well plate and lyse for 15 min under ice bath conditions. Shake during lysis. After lysis, scrape the cells off with a cell scraper and aspirate them into a 1.5 mL centrifuge tube. Centrifuge for 10 min at 4°C using a refrigerated centrifuge at a speed of 12000 rpm. After centrifugation, aspirate the supernatant. If not used immediately, store temporarily in a -20°C freezer or for long-term storage in a -80°C freezer. (3) Protein quantification: Quantify the protein extracted in the previous step using the BCA protein quantification kit and calculate the loading volume. (4) Protein sample preparation: Add 5× loading buffer to the protein sample to dilute it to 1× loading buffer. Mix well and then boil the sample at 95°C for 10 min in a shaker to denature it. (5) SDS-PAGE electrophoresis: After preparing a 10% PAGE gel, samples were loaded for electrophoresis. After electrophoresis, the membrane was transferred using rapid transfer buffer at a constant current of 400 mA for 25 min. After transfer, the PVDF membrane was blocked with rapid blocking buffer for 10 min. After blocking, the membrane was washed three times with TBST solution for 10 min each time. Then, the membrane was cut according to the protein molecular weight standard into bands of GPX 4 (19 KD), xCT (55 KD), Bcl-2 (26 KD), and GAPDH (36 KD). The corresponding bands were incubated with the primary antibody of the corresponding protein and placed on a shaker. o Incubate overnight under C conditions. After the primary antibody incubation is complete, the primary antibody is recovered and the PVDF membrane is washed three times with TBST solution for 10 min each time. Then add the corresponding secondary antibody and incubate on a shaker at room temperature for 1 h. After the incubation is complete, wash the PVDF membrane three times with TBST for 10 min each time. (6) Development: Prepare the developing working solution by mixing chemiluminescent developing solution A and solution B in a 1:1 ratio, and drop the developing solution onto the washed PVDF membrane for development using a chemiluminescent developing instrument.

[0071] Experimental Results: Mitochondria, as a core target of oxidative damage, play a crucial role in synergistic therapy due to their functional state. Mitochondrial membrane potential experiments showed that the dIR-CDF+808 nm group exhibited the most significant disruption in mitochondrial membrane potential. Figure 4 The presence of G in mitochondria is closely related to its specific accumulation within the mitochondria. Damage to the mitochondrial membrane potential not only inhibits ATP synthesis but also activates the caspase cascade, driving apoptosis. Simultaneously, lipid peroxidation of the cell membrane may exacerbate ROS generation; this targeted damage strategy can significantly enhance killing efficiency. Figure 4 As shown in Figure H, the probe dIR-CDF significantly enhanced intracellular ROS levels under irradiation with an 808 nm laser. Both fluorescence intensity and flow cytometry results showed that the ROS production level of the dIR-CDF+808 nm group was higher than that of other treatment groups.

[0072] like Figures 3-6 , Figures 3-7 As shown, Western blot and immunofluorescence assays revealed a significant downregulation of GPX4 and xCT protein expression in the dIR-CDF+808 nm group. Figure 5 In both A and B in group 5, Bcl-2 levels decreased synchronously. Figure 5 (A and 5C). The imbalance of the cellular redox system was validated using the BODIPY™ 581 / 591 C11 lipid peroxidation sensor. The dIR-CDF+808 nm group showed significantly enhanced green fluorescence intensity, as shown in the results. Figure 5 As shown in Figure D, flow cytometry quantification revealed that 95.3% of cells were undergoing lipid peroxidation, indicating widespread damage to the cell membrane system. Figure 5 Middle E and 5 Middle F.

[0073] Conclusions: 1. Photodynamic therapy (PDT) and ferroptosis synergistically enhance tumor cell killing. Exogenous ROS are generated by 808 nm laser excitation of the dIR-CDF probe, while Fc catalyzes the Fenton reaction in the tumor microenvironment to generate endogenous ·OH. This dual ROS accumulation significantly promotes cellular lipid peroxidation. 2. The synergistic effect of PDT and ferroptosis leads to a significant downregulation of key ferroptosis proteins, weakening cellular antioxidant defense. Mitochondrial membrane potential collapse and loss of Bcl-2's anti-apoptotic function drive the synergistic process of apoptosis and ferroptosis.

[0074] Example 5

[0075] Tumor fluorescence imaging and tumor inhibition effect of the probe

[0076] In vivo tumor long-window imaging: When the tumor volume of mouse NCI-H1975 reaches 80 mm 3 Imaging experiments were then performed, and probes dIR-CDF and IR-CDF were prepared using PBS solution at a concentration of 2.5 μg g. -1 The probe was injected into the tail vein of mice at the prescribed concentration. Fluorescence imaging of the mice was performed using a small animal IVIS in vivo imaging system at different time points before and after probe injection (pre, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h), and the data were processed using the in vivo imaging system software.

[0077] Tumor suppression experiment: When the tumor volume of mouse NCI-H1975 reached 80 mm... 3 Imaging experiments were then performed. Tumor-bearing mice were randomly divided into 6 groups (n=5): blank group, dIR-CD+L group, IR-CDF group, IR-CDF+L group, dIR-CDF group, and dIR-CDF+L group (n = 5). After gas anesthesia, the mice were administered 2.5 μg g of the drug according to their respective groups. -1 Mice were administered probes PBS, IR-CDF, and dIR-CDF via tail vein injection at the prescribed concentrations. Twenty-four hours after probe injection, an 808 nm laser with a power of 0.5 W / cm² was used. -2 The tumor sites in mice were irradiated for 3 minutes. Tumor volume and mouse weight were then measured and recorded over a 21-day period. Mice were sacrificed on day 21, and tumor tissue was removed and fixed in paraformaldehyde fixative. One mouse from each group was selected for dissection, and major organs (heart, liver, spleen, lungs, and kidneys) were removed and fixed in paraformaldehyde fixative. Tumor volume was later calculated using formulas, and tumor growth curves and weight change curves were plotted based on changes in tumor volume and weight. The animal experiments complied with the animal experiment regulations of Soochow University.

[0078] HE staining of ex vivo tumors, TUNNEL fluorescent labeling, Ki 67 immunofluorescence, GPX 4 immunofluorescence:

[0079] After treatment, tumor tissue was harvested from one mouse in each group, fixed in paraformaldehyde fixative, then embedded in paraffin, sectioned, and stained with hematoxylin and eosin. TUNEL, GPX 4, and Ki 67 immunofluorescence sections were also prepared. HE staining was observed and photographed using a fluorescence microscope, while TUNEL, GPX 4, and Ki 67 immunofluorescence sections were observed using a laser confocal fluorescence microscope.

[0080] Experimental Results: This embodiment further investigates the imaging and therapeutic effects of the dIR-CDF probe in vivo. In vivo fluorescence imaging results are as follows: Figure 6 Figures A, B, and C show that the fluorescence signal at the tumor site in the dIR-CDF group peaked 12 hours after injection and persisted until 96 hours, significantly better than the control group IR-CDF (signal disappeared after 48 hours). Simultaneously, the biodistribution experiment results are as follows... Figure 6 As shown in Figure E, the tumor fluorescence signal in the dIR-CDF group was significantly enhanced, and the metabolic rate of the kidney and liver was accelerated, demonstrating that the spatiotemporal specific distribution mediated by hyposulfonation reduced the non-specific accumulation of the probe in normal tissues, thereby reducing off-target effects.

[0081] In in vivo tumor treatment experiments, the photodynamic therapy combined with ferroptosis was verified to synergistically enhance tumor-killing effects. Treatment methods include... Figure 7 In Chinese A, the treatment results are as follows Figure 7 Middle B, 7 Middle C and Figure 8 As shown, the tumor volume inhibition rate in the dIR-CDF+808 nm group was significantly higher than that in the single treatment group. Figure 7 As shown in Figure E, HE-stained sections of tumors in the dIR-CDF+808 nm group exhibited extensive necrotic cavities, while TUNEL staining revealed a significantly increased apoptosis rate. Downregulation of Ki67 and GPX4 expression revealed the molecular mechanism of synergistic therapy from two dimensions: proliferation inhibition and disruption of the antioxidant defense system. The biosafety assessment of dIR-CDF provides crucial evidence for preclinical application research, such as... Figure 7 As shown in D. The mouse's body weight fluctuated stably, and there was no pathological damage to the major organs, as shown in Figure D. Figure 9 This indicates that the systemic toxicity of the probe is controllable.

[0082] Conclusions: 1. The probe dIR-CDF covalently cross-links with characteristic hyposulfonated proteins in tumors, significantly prolonging the probe's residence time at the tumor site and achieving high-specificity, long-term in vivo fluorescence imaging. 2. 808 nm laser excitation of the probe dIR-CDF generates exogenous ROS, which, in conjunction with Fc catalysis, generates endogenous ·OH. By downregulating key proteins in the ferroptosis pathway and increasing lipid peroxide accumulation, it significantly inhibits tumor growth and metastasis, verifying the effectiveness of the photodynamic-assisted ferroptosis strategy. 3. The probe dIR-CDF is rapidly metabolized in normal tissues of mice without causing abnormal weight fluctuations or organ damage, demonstrating its good biosafety and laying the foundation for clinical translation.

[0083] The probe of this invention is an integrated probe for the diagnosis and treatment of lung cancer through dual-target photodynamic therapy and ferroptosis. The structure and molecular weight of the probe dIR-CDF were characterized; its absorption and emission wavelengths were measured to be 789 nm and 816 nm, respectively. Under irradiation with an 808 nm laser, the probe generates a large amount of singlet oxygen (¹O2), resulting in a decrease in the absorption peak of 1,3-diphenylisobenzofuran (DPBF). Under the catalysis of hydrogen peroxide (H2O2) and an acidic environment, hydroxyl radicals (·OH) are released, which manifest as a gradual increase in the absorption peak of 3,3',5,5'-tetramethylbenzidine (TMB). The probe dIR-CDF showed a targeted aggregation in NCI-H1975 cells that was 9 times higher than that in normal cells, with a retention time of up to 96 h. It also exhibited significant cytotoxicity to NCI-H1975 cells, with a cell apoptosis rate of 32%, which was 8.3 times higher than that of the control group. The cytotoxic effect of the probe on NCI-H1975 cells stemmed from the accumulation of ROS and enhanced lipid peroxidation. In the synergistic treatment group, the proportion of cells undergoing lipid peroxidation reached 95.3%, with downregulation of proteins such as glutathione peroxidase 4 (GPX 4) and cysteine / glutamate reverse transporter (xCT). In in vitro protein cross-linking experiments, hyposulfonated proteins cross-linked with the probe exhibited strong fluorescence signals, validating the effectiveness of the probe dIR-CDF in cross-linking and labeling intracellular and extracellular hyposulfonated proteins. The probe dIR-CDF accumulated in tumor tissue, with a 5-fold increase in fluorescence imaging signal compared to the control group and a retention time extended to 96 hours. The cyclohexanedione group significantly enhanced the cross-linking efficiency of the probe on hyposulfonated proteins. In in vivo tumor inhibition experiments, the tumor inhibition rate reached 81%, indicating that the probe dIR-CDF effectively inhibited tumor growth while achieving dynamic fluorescence imaging of tumors.

Claims

1. A photodynamic synergistic ferroptosis diagnostic and therapeutic probe for lung cancer with dual targeting, characterized in that, The chemical structural formula of the probe is as follows: 。 2. The preparation method of the integrated probe for dual-targeted photodynamic therapy and ferroptosis diagnosis and treatment of lung cancer as described in claim 1, characterized in that, Includes the following steps: I-CD was obtained by reacting dye IR780 with mercapto-β-cyclodextrin. I-CD was then reacted with RGD-azidocyclopeptide to obtain IR-CD. IR-CD was then reacted with azidocyclohexanedione in a copper sulfate / sodium ascorbate system to obtain dIR-CD. dIR-CD was then encapsulated with ferrocene under ultrasound to obtain dIR-CDF, a dual-targeted photodynamic synergistic ferroptosis diagnostic and therapeutic probe for lung cancer.

3. The preparation method of the integrated probe for dual-targeted photodynamic therapy and ferroptosis diagnosis and treatment of lung cancer according to claim 2, characterized in that, The azidocyclohexanedione is obtained by reacting 3,5-dioxanecarboxylic acid with 3-azidopropylamine.

4. The preparation method of the integrated probe for dual-targeted photodynamic therapy and ferroptosis diagnosis and treatment of lung cancer according to claim 3, characterized in that, The reaction of 3,5-dioxanecarboxylic acid with 3-azidopropylamine was carried out in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbonyldiimidazolium hydrochloride, N-hydroxysuccinimide, and N,N-diisopropylethylamine in a molar ratio of 1~2∶1~2∶2~3∶2~3∶1~2; the reaction was carried out at room temperature.

5. The preparation method of the integrated probe for dual-targeted photodynamic therapy and ferroptosis diagnosis and treatment of lung cancer according to claim 2, characterized in that, The reaction of dye IR780 with mercapto-β-cyclodextrin was carried out in N,N-dimethylformamide with the addition of triethylamine. The molar ratio of dye IR780, mercapto-β-cyclodextrin and triethylamine was 1:1~2:1~2. The reaction was carried out at room temperature for 10~14 hours.

6. The preparation method of the integrated probe for dual-targeted photodynamic therapy and ferroptosis diagnosis and treatment of lung cancer according to claim 2, characterized in that, The reaction between I-CD and RGD-azidocyclic peptide was carried out in the presence of sodium ascorbate and copper sulfate; the molar ratio of I-CD, RGD-azidocyclic peptide, sodium ascorbate and copper sulfate was 1:1:1~2:1~2, the reaction was carried out at room temperature, and the reaction time was 1~3 hours.

7. The preparation method of the integrated probe for dual-targeted photodynamic therapy and ferroptosis diagnosis and treatment of lung cancer according to claim 2, characterized in that, The molar ratio of IR-CD, azidocyclohexanedione, sodium ascorbate, and copper sulfate is 1:1:1~2:1~2.

8. The preparation method of the integrated probe for dual-targeted photodynamic therapy and ferroptosis diagnosis and treatment of lung cancer according to claim 2, characterized in that, The molar ratio of dIR-CD to ferrocene is 1:1~4.

9. A fluorescent imaging contrast agent, characterized in that, This includes the photodynamic synergistic ferroptosis diagnostic and therapeutic probe for dual-targeted lung cancer as described in claim 1.

10. The application of the photodynamic synergistic ferroptosis diagnostic and therapeutic probe for dual-targeting lung cancer as described in claim 1 in the preparation of reagents for inhibiting tumors.

Images