A near-infrared cyclometalated iridium (III) photosensitizer and a method for synthesizing the same

Abstract

This invention discloses a near-infrared cyclic metallic iridium (III) photosensitizer and its synthesis method. The BDP-Ir prepared by the method of this invention can be used at low doses (IC50). 50 =68nM) at low power (10mW·cm) ‑2 It exhibits high photosensitivity under 808nm excitation, successfully solving the problem of sacrificing both excitation wavelength and photosensitivity efficiency; at the same time, its synergistic biomimetic artificial nano-hybrid delivery system can effectively enhance the tumor targeting and immune escape ability of photosensitizers.

Description

Technical Field

[0001] This invention relates to a near-infrared cyclic iridium (III) photosensitizer, and also to a method for synthesizing the aforementioned cyclic iridium (III) photosensitizer. Background Technology

[0002] Photodynamic therapy (PDT) is a promising non-invasive cancer treatment method. The development of photosensitizers (PSs) has been a crucial factor in the advancement of PDT over the past few decades. Numerous reports document the widespread application of organic PSs in PDT. Porphyrins and their derivatives, the first PS approved by the U.S. Food and Drug Administration (FDA), have been shown to kill cancer cells and ablate tumors. However, the hydrophobicity of various porphyrin photosensitizers leads to insufficient tumor localization and aggregation, thus reducing the efficiency of PDT. Heptamethyl cyano dyes, due to their long-wavelength properties, are widely used as photosensitizers in fluorescence diagnostics and PDT. However, the electron-rich polyimide chains of cyano dyes are oxidized by ROS generated under light, leading to decomposition or photobleaching, thus limiting their clinical application. Fluoroboropyridine (BODIPY) dyes, renowned for their high fluorescence emission and photostability, are excellent fluorescent dyes with broad practical applications. The photophysical properties of BODIPY dyes, including redshifts in absorption and emission and excited-state characteristics, can be modulated through structural modification. However, they typically have weak spin-orbit coupling and low triplet excited-state efficiency, resulting in low reactive oxygen species (ROS) generation efficiency.

[0003] Cyclic iridium complexes significantly enhance spin-orbit coupling due to the heavy atom effect, thereby achieving efficient energy conversion of triplet excited states and promoting the generation of reactive oxygen species. However, their inherently short absorption wavelength greatly limits tissue penetration. Several studies have explored combining cyclic iridium with organic dyes to modulate the wavelength redshift, but their molecular design remains limited to the red light region. According to the Planck-Einstein relation, increasing wavelength is associated with decreasing energy. Therefore, near-infrared (NIR) (>700 nm) excited cyclic iridium photosensitizers have low photodynamic efficiency, limiting therapeutic effects. The trade-off between excitation wavelength and photosensitivity efficiency is a significant bottleneck in the development of cyclic iridium photosensitizers. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a near-infrared cyclic iridium (III) photosensitizer, which can effectively solve the problem of sacrificing excitation wavelength and photosensitivity efficiency, and exhibits superior phototoxicity under low-power 808nm laser irradiation; this invention also provides a method for synthesizing the above-mentioned cyclic iridium (III) photosensitizer.

[0005] Technical solution: The near-infrared ring metallic iridium (III) photosensitizer of the present invention has the following structural formula:

[0006] .

[0007] The synthesis method of the above-mentioned cyclic iridium(III) photosensitizer includes the following steps:

[0008] (1) Under an inert atmosphere, the bipyridine ligand modified with fluoroboron dipyrrole (BODIPY) and the cyclic metal iridium dimer were heated and refluxed in an organic solvent, and the solvent was removed by vacuum distillation.

[0009] (2) The crude product was obtained by substituted with NH4PF6, and the crude product was separated and purified by column chromatography to obtain a fluoroboron dipyrrole-modified cyclic metal iridium (III) photosensitizer;

[0010] The structural formula of the fluoroboron dipyrrole-modified bipyridine ligand is shown below:

[0011] ;

[0012] The structural formula of the cyclic metallic iridium dimer is shown below:

[0013] .

[0014] The fluoroboron dipyrrole-modified bipyridine ligand was prepared by the following method, with the specific steps as follows: A fluoroboron dipyrrole derivative (prepared based on the method reported in this literature, Org. Lett. 2023, 25, 1638-1642), 2,2'-bipyridine-5-carboxaldehyde, piperidine, and p-toluenesulfonic acid (catalyst) were dissolved in an organic solvent and reacted at reflux temperature; after the reaction was completed, the solvent was removed by vacuum distillation, and the crude product was purified by column chromatography to obtain the fluoroboron dipyrrole-modified bipyridine ligand; the molar ratio of the fluoroboron dipyrrole derivative to 2,2'-bipyridine-5-carboxaldehyde was 1:1~3.

[0015] In step (1), the molar ratio of the fluoroboron dipyrrole-modified bipyridine ligand to the cyclic metal iridium dimer is 2:1 to 1.2; the organic solvent is a mixture of dichloromethane and methanol; in the mixed solvent, the volume ratio of dichloromethane to methanol is 2:1 to 1.5; the reflux reaction time is 12 to 13 h, and the temperature is 45 to 48 °C.

[0016] In step (2), the crude product is obtained by substitution with NH4PF6. Specifically, a methanol solution saturated with NH4PF6 is added and stirred for 2-3 hours.

[0017] This invention relates to a cyclic iridium (III) photosensitizer for deep diagnosis and treatment of triple-negative breast cancer. The fluoroboron dipyrrole backbone, through the introduction of a bipyridine ligand to prolong conjugation, can redshift the spectrum to the near-infrared region. An 808nm laser can effectively enhance its tissue penetration and reduce photodamage to the skin. To further improve the tumor selectivity and in vivo circulation time of the photosensitizer, a biomimetic artificial nanohybrid delivery system is used to encapsulate it. Fluorescence / photoacoustic imaging demonstrates its precise positioning at the tumor site, effectively improving the photosensitizer's targeting to the tumor and thus enhancing its safety, while also significantly improving the therapeutic effect. Simultaneously, the delivery system can be activated by ultrasound to break down and release the drug. This dual-mode photodynamic chemotherapy therapy can effectively inhibit triple-negative breast cancer.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The iridium (III) ring photosensitizer of the present invention has good anti-tumor cell proliferation ability, and can efficiently kill cancer cells under low-power 808nm laser, and induce cell apoptosis and necrosis. (2) Its synergistic biomimetic artificial nano-hybrid delivery system can effectively improve the tumor targeting of the photosensitizer. (3) The present invention can characterize the in vivo tumor site using fluorescence / photoacoustic dual-modal imaging. Attached Figure Description

[0019] Figure 1 The diagram shows the theoretical calculation results for the coordination compound of Example 1;

[0020] Figure 2 This is a simulated absorption spectrum obtained from theoretical calculations of the coordination compound in Example 1;

[0021] Figure 3 The absorption and emission spectra of the complex in Example 1 are shown below.

[0022] Figure 4 The fluorescence spectrum of reactive oxygen species generated by the complex in Example 1 under light irradiation in aqueous solution is shown.

[0023] Figure 5 The absorption spectrum of the complex in Example 1 generated singlet oxygen in aqueous solution under light irradiation;

[0024] Figure 6 The following are experimental diagrams of the complex in Example 1 loaded with a biomimetic artificial nano-hybrid delivery system: where a is a schematic diagram of the experiment; b is a protein electrophoresis diagram; cf is a TEM image of the nanoparticles and their particle size distribution; gh is the absorption spectrum of the nanoparticles and their changes over 7 days; i is the Zeta potential and average particle size of the nanoparticles; j is the change in average particle size over 7 days.

[0025] Figure 7 This is a confocal fluorescence imaging image of the uptake of nanoparticles in MDA-MB-231 and A549 cells in Example 5;

[0026] Figure 8 ICP-MS statistics, flow cytometry, and confocal fluorescence imaging of nanoparticle uptake in RAW264.7 cells in Example 5;

[0027] Figure 9 This is a photoacoustic imaging image of nanoparticles in aqueous solution in Example 5;

[0028] Figure 10 This is a fluorescence imaging image of the nanoparticles in Example 5 in MDA-MB-231 tumor-bearing mice;

[0029] Figure 11 This is a photoacoustic imaging image of the nanoparticles in Example 5 in MDA-MB-231 tumor-bearing mice;

[0030] Figure 12 This is a colocalization map of the nanoparticles in Example 5 with a commercial dye in MDA-MB-231 cells;

[0031] Figure 13 Example 5: ROS flow cytometry image of nanoparticles in MDA-MB-231 cells under light irradiation;

[0032] Figure 14 This is a flow cytometry image of light-induced mitochondrial membrane potential depolarization in MDA-MB-231 cells, based on nanoparticles from Example 5.

[0033] Figure 15 Flow cytometry and WB protein diagrams of light-induced apoptosis and necrosis in MDA-MB-231 cells, as shown in Example 5.

[0034] Figure 16 This is a schematic diagram of the surface film structure rupture of nanoparticles under ultrasonic treatment in Example 5.

[0035] Figure 17 This is a flow cytometry image of the nanoparticles in Example 5 induced by ultrasound in MDA-MB-231 cells;

[0036] Figure 18 This is a flow cytometry image of ultrasound-induced apoptosis in MDA-MB-231 cells using nanoparticles from Example 5.

[0037] Figure 19 Example 5: Cell viability and 3D cell spheroids of nanoparticles in MDA-MB-231 cells after being blocked by chicken breast of different thicknesses and subjected to light / ultrasound.

[0038] Figure 20The following are images from Example 5: a) a schematic diagram of the experiment; b) an image after tumor removal; c) a graph showing changes in tumor volume; and d) a graph showing tumor volume on day 16. Detailed Implementation

[0039] Example 1

[0040] The method for preparing the cyclic metallic iridium(III) complex (BDP-Ir) of the present invention includes the following steps:

[0041]

[0042] (1) Preparation of compound BDP-bpy: BDP-CH3 (196 mg, 0.5 mmol), 2,2'-bipyridine-5-carboxaldehyde (276 mg, 1.5 mmol), piperidine (1 mL) and p-toluenesulfonic acid (5 mg) were dissolved in toluene (20 mL), a water separator and a spherical condenser were connected, and the mixture was heated under reflux for 8 hours. After the reaction was completed, the solvent was removed by vacuum distillation, and the crude product was further purified by column chromatography to obtain a black solid (fluoroboron dipyrrole-modified bipyridine ligand BDP-bpy), with a yield of 53%. 1 H NMR (600 MHz, CDCl3) δ 8.81 (s, 1H), 8.70 (d, J = 4.3 Hz, 1H), 8.47 - 8.45 (m, 2H), 8.13 (d, J = 9.1 Hz, 2H), 8.11 - 8.09 (m, 1H), 7.88 - 7.82 (m, 2H), 7.40 (d, J =2.2 Hz, 1H), 7.35-7.29 (m, 2H), 7.21 (s, 1H), 7.00 (d, J = 4.5 Hz, 1H), 6.95(d, J = 4.8 Hz, 1H), 6.82 (d, J = 9.1 Hz, 2H), 3.15 (s, 6H). 13C NMR (151 MHz, CDCl3) δ 162.68, 155.74, 152.54, 149.34, 149.17, 137.16, 136.18, 134.25,132.67, 132.07, 131.73, 130.32, 126.40, 123.98, 123.60, 121.59, 121.45,121.17, 118.34, 116.96, 111.88, 40.18.

[0043] (2) Preparation of complex BDP-Ir: Under an argon protective atmosphere, the fluoroboron dipyrrole modified bipyridine ligand BDP-bpy (48 mg, 0.08 mmol) and the cyclic metal iridium dimer (50 mg, 0.04 mmol) were dissolved in a mixed solvent of anhydrous dichloromethane and anhydrous methanol (the volume ratio of anhydrous dichloromethane to anhydrous methanol in the mixed solvent was 2:1). The mixture was heated and stirred under reflux at 45 °C for 12 h. After the reaction was completed, the solvent was removed by vacuum distillation, and a methanol solution of saturated NH4PF6 was added. After stirring for 2 h, the solid was collected by centrifugation. The crude product was further purified by column chromatography to obtain black solid BDP-Ir with a yield of 20%. 1 H NMR (400 MHz, CD2Cl2) δ 8.49 - 8.43 (m, 2H), 8.35 (d, J = 8.4 Hz, 1H), 8.20(d, J = 9.1 Hz, 2H), 8.12 (t, J = 7.8 Hz, 1H), 8.03 – 7.94 (m, 4H), 7.82-7.67 (m,5H), 7.59 (d, J = 5.7 Hz, 1H), 7.52 (d, J = 5.6 Hz, 1H), 7.48-7.42 (m, 2H), 7.14-6.94 (m, 9H), 6.91 (d, J = 3.7 Hz, 1H), 6.89-6.83 (m, 2H), 6.33 (t, J = 6.7 Hz, 2H), 3.17 (s, 6H). 13C NMR (126 MHz, CD2Cl2) δ 168.18, 168.13, 164.61, 155.88,154.39, 153.50, 151.12, 150.25, 150.23, 150.20, 149.01, 148.94, 144.07,144.04, 139.77, 138.65, 138.61, 138.00, 135.14, 133.91, 133.87, 133.82,132.16, 132.03, 131.97, 131.51, 131.26, 131.12, 128.40, HR-MS (CH3OH): calcd for [BDP-Ir-PF6 - ] + m / z = 1060.2909, found m / z = 1060.2892.

[0044] Comparative Example 1 is a bipyridine-coordinated cyclic metallic iridium (Ir-0, prepared based on the method reported in the literature Chem. Eng. J. 2024, 497, 155022), whose structural formula is shown below:

[0045] .

[0046] The complex BDP-Ir prepared in Example 1, Comparative Example 1 Ir-0, and raw materials BDP-CH3 and BDP-bpy were subjected to the following experiments: cytotoxicity against human triple-negative breast cancer cells MDA-MB-231 and normal human breast epithelial cells MCF-10A:

[0047] The MTT assay was used to analyze the antiproliferative effects of fluoroboron dipyrrole-modified cyclic iridium (III) complexes BDP-Ir, Ir-0, BDP-CH3, and BDP-bpy, the commercial photosensitizer Ce6, and cisplatin CDDP. MTT (thiazolyl blue) is a tetrazolium salt that can be reduced by succinate dehydrogenase in the mitochondria of living cells to a blue-violet product, formazan (the product is soluble in DMSO), which has an absorption peak at 490 nm. Therefore, it can be used as an indicator. 490 nm To analyze cell proliferation.

[0048] The specific experimental steps are as follows:

[0049] (1) First, revive a tube of tumor cells, culture them in fresh culture medium (DMEM medium + 10% fetal bovine serum + 1% penicillin and streptomycin), and use them after passage 3 times;

[0050] (2) When the cells reach the logarithmic growth phase, seed them into 96-well plates at a density of 5000 cells / well (100 μL of culture medium per well) and incubate them in a constant oxygen (21% O2) incubator (37℃, 5% CO2);

[0051] (3) After the cells adhered, 100 μL of fresh culture medium containing different concentration gradients of compounds BDP-Ir, Ir-0, BDP-CH3, BDP-bpy, Ce6 and CDDP was added to each well, and the cells were then placed in an incubator for further incubation. The light-illuminated group was incubated for 6 hours and then treated with an 808 nm laser (10 mW / cm²). 2 Irradiate for 1 hour and continue incubation;

[0052] (4) After incubation for 48 hours, add 20 μL MTT (5 mg / mL) to each well and continue incubation at 37°C for 4 hours. After removing the supernatant, add 150 μL dimethyl sulfoxide (DMSO) to each well and use an enzyme-linked immunosorbent assay (ELISA) reader to detect A. 490nm Calculate the cell proliferation inhibition rate and determine the IC50. 50 Value (drug concentration corresponding to an inhibition rate of 50%). The MTT test results of compounds BDP-Ir, Ir-0, BDP-CH3, BDP-bpy, Ce6 and CDDP are shown in Table 1.

[0053] Table 1 shows the IC50 values ​​of compounds BDP-Ir, Ir-0, BDP-CH3, BDP-bpy, Ce6, and CDDP. 50 Value (μM)

[0054]

[0055] The results show that at low power 808 nm (10 mW·cm⁻¹), -2 Under laser irradiation, BDP-CH3 exhibits almost no phototoxicity, while BDP-bpy shows some degree of phototoxicity (IC50). 50 = 2.8 µM), while BDP-Ir is compatible with the IC of MDA-MB-231. 50 The concentration is as low as 68 nM, far lower than a series of reported near-infrared ringed iridium photosensitizers. These results demonstrate that the present invention successfully constructs a low-power, low-dose, and highly phototoxic photosensitizer.

[0056] Example 2

[0057] Applications of theoretical calculations to the cyclic metallic iridium(III) complexes BDP-Ir, Ir-0, BDP-CH3, and BDP-bpy prepared in Example 1:

[0058] Methods: The ground-state geometry of the compounds was optimized using time-dependent density functional theory (TDDFT) and B3LYP functions, with the LANL2DZ basis set used for Ir and the 6-31G(d,p) basis set used for the remaining atoms. The ground-state energy was calculated based on the geometry optimization. All calculations were performed using Gaussian 09.

[0059] The theoretical calculation results for the cyclic metallic iridium(III) complexes BDP-Ir, Ir-0, BDP-CH3, and BDP-bpy prepared in Example 1 are as follows: Figure 1 and 2 As shown, the results indicate that the electron cloud of the BDP-Ir complex is mainly distributed on the BODIPY backbone. The energy difference (ΔE) between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) decreases with the elongation of the conjugated chain. As a control, the ΔE of Ir-0 coordinated with bipyridine is the largest, at 2.28 eV, corresponding to its shortest absorption, while the ΔE of BDP-Ir (1.88 eV) is much smaller than that of Ir-0. Simultaneously, the simulated UV absorption spectrum shows that the absorption is effectively redshifted, which is attributed to the contribution of the conjugated BODIPY backbone.

[0060] Example 3

[0061] Applications of the spectroscopic analysis of the cyclic iridium(III) complexes BDP-Ir, Ir-0, BDP-CH3 and BDP-bpy prepared in Example 1:

[0062] Methods: Concentrated stockpiles (10 mM) of Ir-0, BDP-CH3, BDP-bpy, and BDP-Ir were prepared in DMSO, diluted to 10 µM with ultrapure water, and transferred to 1 cm path length quartz cuvettes (3 mL). UV spectra (wavelength range 200–1000 nm) were detected at 25°C using a Lambda 365 UV-Vis spectrophotometer. Fluorescence emission spectra (wavelength range 500–1100 nm) were detected using an FS5 fluorescence spectrometer.

[0063] The spectroscopic results of the cyclic iridium(III) complexes BDP-Ir, Ir-0, BDP-CH3 and BDP-bpy prepared in Example 1 are as follows: Figure 3As shown, the results indicate that BDP-Ir exhibits a broad absorption band in aqueous solution, covering the wavelength range of 600–1000 nm, with strong light absorption at 808 nm. Simultaneously, the emission peak of BDP-Ir in water is located at 870 nm, with most of its emission spectrum in the near-infrared region. In addition to near-infrared absorption and emission, BDP-Ir also possesses a Stokes shift as high as 160 nm, a property that helps reduce interference from biological autofluorescence background, providing a basis for in vivo NIR fluorescence imaging.

[0064] Example 4

[0065] Application of reactive oxygen species generation in solutions of the cyclic iridium(III) complexes BDP-Ir, Ir-0, BDP-CH3, and BDP-bpy prepared in Example 1:

[0066] Method: Dilute 16 µL of DCFH-DA in 144 µL of ethanol solution, and add 640 µL of sodium hydroxide aqueous solution (10 -2 Stirring at room temperature in the dark for 30 min, then diluting and neutralizing with PBS to obtain DCFH stock solution for later use in the dark. Prepare mixed aqueous solutions containing BDP-Ir, Ir-0, BDP-CH3, and BDP-bpy (10 µM) and DCFH (10 µM), respectively, and measure the emission spectra (wavelength range 495-625 nm, λ) in the dark. ex = 488 nm), using an 808 nm laser (10 mW·cm). -2 The fluorescence intensity was measured every 30 seconds, and the change at 527 nm was recorded. A sample solution was prepared by mixing 1,3-diphenylisobenzofuran (DPBF) with the sample (10 μM) in ultrapure water. After measuring the UV-Vis spectrum in the dark, the sample was exposed to an 808 nm light source (10 mW·cm⁻¹). -2 After 5 seconds of irradiation, record the change in absorbance at 414 nm.

[0067] The reactive oxygen species generation in the solutions of the cyclic iridium(III) complexes BDP-Ir, Ir-0, BDP-CH3, and BDP-bpy prepared in Example 1 is as follows: Figure 4 and 5As shown, the results indicate that the fluorescence intensity of the BDP-Ir group increased sixfold at 527 nm within 150 seconds, and the BDP-bpy group also showed some fluorescence enhancement, while BDP-CH3 showed almost no significant change. In the control group, without the presence of the photosensitizer, the fluorescence intensity of DCFH remained almost unchanged after 150 seconds of illumination. These results all demonstrate that the photosensitizer BDP-Ir has excellent near-infrared photosensitivity. Subsequently, the fluorescence intensity of BDP-Ir was measured using the singlet oxygen probe DPBF. 1 The results showed that after 30 seconds of laser irradiation, the absorption of DPBF at 414 nm in BDP-Ir almost completely disappeared, while the changes in the control compounds BDP-CH3 and BDP-bpy were very small. This indicates that BDP-Ir can generate a large amount of singlet oxygen under near-infrared light excitation and has excellent photosensitivity.

[0068] Example 5

[0069] Application of the cyclic iridium(III) complex BDP-Ir prepared in Example 1 with a biomimetic artificial nanohybrid delivery system:

[0070] Method 1: Fresh rabbit blood was collected into centrifuge tubes and centrifuged at 3000 r for 5 min to remove plasma. The blood was then washed with physiological saline and centrifuged three times. The blood was then allowed to stand in deionized water at 4°C for 1 hour to lyse the blood. The blood was then centrifuged at 12000 r for 10 min at 4°C, and the supernatant was discarded. The precipitate was washed with DD water until it was colorless. The precipitate was redispersed in water to obtain the red blood cell membrane (Rm). The protein concentration was detected using a BCA kit and stored at -80°C for later use. MDA-MB-231 cells were cultured in T75 culture flasks. After the cells reached confluence, the culture medium was removed, and the cells were washed with PBS, digested with T+E, and collected by centrifugation. Membrane protein extraction reagent was added, and the cells were homogenized on ice. The supernatant was collected by centrifugation (4°C, 700 g, 10 min). Further centrifugation was performed at 4°C, 14000 g for 30 min. The supernatant was discarded, and the precipitate was washed with DD water and resuspended in DD water to obtain the MDA-MB-231 cell membrane (Mm). The protein concentration was detected and stored at -80°C for later use. Equal amounts of protein from erythrocyte membranes and MDA-MB-231 cell membranes were sonicated for 30 min to obtain a mixed membrane (Hm). 1 mg of BDP-Ir was dissolved in 0.5 mL of dichloromethane, and 10 mg of DSPE-PEG was added. 3400 - Dissolve Biotin (purchased externally) in 10 mL of ultrapure water, then add the above-mentioned dichloromethane (containing BDP-Ir) to the above-mentioned ultrapure water (containing DSPE-PEG). 3400In a liposome extract, BDP-Ir-NP was sonicated (360 W, 20 min) to obtain a clear, transparent brown solution. BDP-Ir-NP and cell membrane were mixed at a mass:protein ratio of 1:1 and extruded sequentially through 800 nm, 400 nm, and 200 nm polycarbonate membranes 15 times using a liposome extruder to obtain Mm-NP, Rm-NP, and Hm-NP.

[0071] Method 2: The average particle size and zeta potential of the nanoparticles were measured using a Malvern Zetasizer Nano ZS90 laser particle size analyzer. A small amount of sample was dropped onto a copper grid, and the solvent was allowed to evaporate at room temperature while maintaining a dry environment. The morphology of the prepared sample was observed using transmission electron microscopy (TEM). The surface membrane proteins were characterized using protein gel electrophoresis.

[0072] The characterization of the cyclic iridium(III) complex BDP-Ir prepared in Example 1, which is carried by a biomimetic artificial nanohybrid delivery system, is as follows: Figure 6 As shown, BDP-Ir is encapsulated in DSPE-PEG via ultrasound. 3400 In the biotin, core nanoparticles BDP-Ir-NP are formed. TEM characterization of BDP-Ir-NP reveals uniform spherical nanoparticles. The particle size distribution shows that the average particle size of BDP-Ir-NP in aqueous solution is approximately 106 nm, indicating its ability to form uniformly sized nanoparticles in water. After coating the surface of the core nanoparticles with a membrane coating, Mm-NP, Rm-NP, and Hm-NP were obtained. These nanoparticles have similar morphologies, all being spherical structures with average particle sizes of 101.5 nm, 115.2 nm, and 121.7 nm, respectively. Nanoparticles of this size can be taken up by cells. TEM images show that the nanoparticle surface has a uniform core-shell structure, consistent with the reported thickness of the phospholipid bilayer (5-10 nm). Further protein gel electrophoresis analysis of Mm, Rm, Hm, and Mm-NP, Rm-NP, and Hm-NP confirmed the presence of membrane proteins in the nanoparticles with protein luminescence in the corresponding lanes. In summary, a cell membrane-coated nanodelivery system has been successfully constructed. After encapsulation with PEG and Hm, an absorption peak at 770 nm was detected, a redshift of approximately 60 nm compared to unencapsulated BDP-Ir. These NPs exhibited significant stability in an aqueous environment for up to seven days, ensuring leak-free drug delivery in vivo. Dynamic light scattering (DLS) analysis revealed that the Hm-coated nanoparticles had a larger particle size compared to uncoated BDP-Ir-NPs. The Zeta potential increased from -6.29 mV to -14.77 mV. This increase suggests that Hm may have reduced particle size growth by enhancing interparticle repulsion and preventing deposition.

[0073] Example 6

[0074] Applications of the isomorphic targeting capabilities of the nanoparticles Mm-NP, Rm-NP, and Hm-NP prepared in Example 5:

[0075] Methods: MDA-MB-231 and A549 cells were cultured separately in confocal dishes (50,000 cells / mL). After 24 hours, medium containing BDP-Ir-NP, Mm-NP, Rm-NP, and Hm-NP (2 µg / mL) was added and incubated for 6 hours. The drug-containing medium was aspirated, washed twice with PBS, and then HBSS was added. Imaging was immediately performed using a confocal microscope (excitation wavelength λ). ex = 641nm).

[0076] The isomorphic targeting capabilities of the nanoparticles Mm-NP, Rm-NP, and Hm-NP prepared in Example 5 are as follows: Figure 7 As shown, the results indicate that laser confocal imaging revealed that in MDA-MB-231 cells, the fluorescence intensity of Mm-NP and Hm-NP after 6 hours of incubation was significantly higher than that of BDP-Ir-NP, while the fluorescence intensity of the Rm-NP group showed little increase, indicating that uptake increases with the increase of the Mm proportion. However, no significant differences were observed among the four cell groups in A549 cells. These results suggest that Mm-NP and Hm-NP, which are membrane-coated with homologous cancer cells, can selectively target MDA-MB-231 cells.

[0077] Example 7

[0078] Applications of the immune evasion capabilities of the nanoparticles Mm-NP, Rm-NP, and Hm-NP prepared in Example 5:

[0079] Method 1: RAW264.7 cells were cultured in 10 cm culture dishes and incubated overnight at 37°C. Then, culture medium containing BDP-Ir-NP, Mm-NP, Rm-NP, and Hm-NP (2 µg / mL) was added. After 6 h and 24 h of incubation, the culture medium was removed, and the cells were digested and collected, then washed three times with PBS. Protein lysis buffer was added, and protein concentration was determined using a BCA assay kit. Subsequently, the cells were digested sequentially with HNO3 (100 μL) for 1 h, 30% H2O2 (50 μL) for 1 h, and HCl (100 μL) at 95°C for 0.5 h. The remaining solution was diluted to 2 mL with double-distilled water, and the Ir content was determined by inductively coupled plasma mass spectrometry (ICP-MS).

[0080] Method 2: RAW264.7 cells were seeded in 6-well plates and cultured for 24 hours, then co-cultured for 6 hours with BDP-Ir-NP, Mm-NP, Rm-NP, and Hm-NP (2 µg / mL), respectively. Cells were collected and washed with PBS. Flow cytometry was performed using a BD C6 instrument, and data were analyzed using FlowJo 7.6.1 software. 10,000 cells were collected from each sample.

[0081] Method 3: RAW264.7 cells were seeded in confocal culture dishes and cultured overnight at 37°C. The cells were then treated with BDP-Ir-NP, Mm-NP, Rm-NP, and Hm-NP (2 µg / mL) for 6 hours, respectively. After washing twice with PBS, the cells were observed using a confocal microscope (excitation wavelength λ). ex = 641 nm).

[0082] The immune evasion abilities of the nanoparticles Mm-NP, Rm-NP, and Hm-NP prepared in Example 5 are as follows: Figure 8 As shown, the results indicated that ICP-MS and flow cytometry analyses revealed a significant reduction in Rm-NP uptake by macrophages compared to BDP-Ir-NP, suggesting that incorporation into the erythrocyte membrane can evade immune recognition and reduce macrophage phagocytosis. Furthermore, the MDA-MB-231 cell membrane also exhibited a certain immune evasion effect, which was further confirmed by confocal imaging. The results of the cell internalization studies demonstrate that the introduction of a biomimetic artificial nanohybrid delivery system can effectively promote precise tumor targeting of NPs, enhance immune evasion, and thus improve therapeutic efficacy.

[0083] Example 8

[0084] Application of Hm-NP nanoparticles prepared in Example 5 for photoacoustic imaging:

[0085] Methods: Prepare aqueous solutions of Hm-NP at different concentrations and measure the photoacoustic signal at 808 nm using the Vevo LAZR photoacoustic imaging system.

[0086] The photoacoustic imaging capability of the Hm-NP nanoparticles prepared in Example 5 in aqueous solution is as follows: Figure 9 As shown, the results indicate that Hm-NP exhibits a strong photoacoustic signal at a wavelength of 808 nm, and the PA intensity is linearly related to the concentration, suggesting that Hm-NP has the potential for photoacoustic imaging in biological tissues.

[0087] Example 9

[0088] Application of Hm-NP nanoparticles prepared in Example 5 for in vivo multidimensional imaging (fluorescence / photoacoustic):

[0089] Method 1: MDA-MB-231 cells were injected into the right axilla of nude mice to establish a tumor model. When the tumor reached approximately 100-200 cm... 3 Mice were randomly divided into 3 groups (n=3 per group): a: intravenous injection of BDP-Ir (5 mg·kg⁻¹); b: intravenous injection of BDP-Ir-NP (5 mg·kg⁻¹). -1 c: Intravenous injection of Hm-NP (5 mg·kg⁻¹). Mice were then placed in a fluorescence imaging system for imaging at different time points (0, 1, 4, and 10 h) (λ). ex / λ em = 745 / 820 nm). After 10 hours, major organs (heart, liver, kidney, spleen, and lungs) and tumors were collected, washed with physiological saline, and then subjected to in vitro imaging. The data were analyzed using LivingImage 4.5 software, and ROIs of equal area were obtained in the region of interest for further analysis.

[0090] Method 2: MDA-MB-231 cells were injected into the right axilla of nude mice to establish a tumor model. When the tumor reached approximately 100-200 cm... 3 Mice were randomly divided into 3 groups (n=3 per group): a: Intravenous injection of BDP-Ir (5 mg / kg) -1 b: Intravenous injection of BDP-Ir-NP (5 mg / kg) -1 c: Intravenous injection of Hm-NP (5 mg·kg) -1 The mice were then placed in the Vevo LAZR in vivo photoacoustic imaging system for imaging at specified time intervals (0, 1, 4, 6, and 10 h) (λ). ex = 808 nm).

[0091] Applications of Hm-NP nanoparticles prepared in Example 5 for in vivo fluorescence imaging, such as... Figure 10 As shown, the results indicated that the tumor sites in the control group exhibited only weak fluorescence signals that quickly disappeared. In contrast, the Hm-NP group showed significant fluorescence accumulation at the tumor sites 1 hour after injection, reaching a peak at 4 hours and persisting for 10 hours. Organ isolation analysis confirmed that the fluorescence of both BDP-Ir and BDP-Ir-NP accumulated in the mouse liver, while Hm-NP was abundant in the tumor tissue. This enhanced tumor targeting is attributed to the successful construction of the biomimetic artificial nanohybrid delivery system.

[0092] Applications of Hm-NP nanoparticles prepared in Example 5 for in vivo photoacoustic imaging, such as Figure 11As shown, the results indicated that within 1 hour after tail vein injection, almost no photoacoustic signal was observed inside the tumor. The photoacoustic intensity of Hm-NP gradually increased over time, increasing 30-fold after 4 hours, exhibiting a similar trend to the fluorescence results, indicating that Hm-NP was enriched at the tumor site. In contrast, the photoacoustic signal inside the tumors of mice in the BDP-Ir and BDP-Ir-NP groups showed no significant change, indicating no targeting ability. Both fluorescence and photoacoustic in vivo imaging demonstrate the excellent in vivo tumor targeting ability and multidimensional imaging capability of Hm-NP.

[0093] Example 10

[0094] Intracellular localization application of Hm-NP nanoparticles prepared in Example 5 after co-incubation with commercial probes:

[0095] Methods: MDA-MB-231 cells were cultured overnight in confocal dishes, then cultured for another 24 h in medium containing Hm-NP (1 µg / mL). Cells were washed with PBS, and then stained with nuclear / endoplasmic reticulum / mitochondrial / lysosomal probes for 30 min each. After removing the staining solution, the cells were washed with PBS and then photographed using a confocal laser microscope. (Nuclear λ) ex = 405 nm, λ em = 488nm; Endoplasmic reticulum λ ex = 488 nm, λ em = 500-540 nm; mitochondrial λ ex = 488 nm, λ em = 500-540 nm; lysosome λ ex = 561 nm, λ em = 590 nm; Hm-NP λ ex = 641 nm, λ em = 750-1000 nm). Colocation coefficient analysis was performed using ImageJ software.

[0096] In Example 5, the intracellular localization of the Hm-NP nanoparticles prepared and co-incubated with a commercial probe was as follows: Figure 12 As shown, the results indicate that Hm-NP is mainly distributed in lysosomes after being taken up by MDA-MB-231 cells, with a colocalization coefficient of 0.88, indicating that Hm-NP has excellent ability to target lysosomes.

[0097] Example 11

[0098] Application of Hm-NP obtained in Example 5 to generate reactive oxygen species in cells upon photoexcitation:

[0099] Methods: ROS in tumor cells were detected by flow cytometry. MDA-MB-231 cells were seeded in 6-well plates and incubated in an incubator, then co-cultured with BDP-Ir-NP, Mm-NP, Rm-NP, and Hm-NP (3 µg / mL) for 6 hours. Cells were cultured at 808 nm and 10 mW·cm⁻¹. -2 Cells were collected 1 h after irradiation with a light source and washed with PBS. Subsequently, the ROS probe DCFH-DA (10 µM) was added to the cell samples and incubated for 30 min. After washing with PBS, cells were analyzed using a BD C6 flow cytometer with excitation wavelength of 488 nm and emission wavelength of 530 ± 30 nm. Data were analyzed and quantified using FlowJo 7.6.1 software. 10,000 cells were collected from each sample.

[0100] The result of Hm-NP generating reactive oxygen species under light is as follows: Figure 13 As shown in the figure. The results indicate that the green fluorescence of the 808 nm light-treated group was stronger, while the fluorescence of the BDP-Ir-NP group was relatively weaker, which may be due to lower cellular uptake. There were no significant changes in the dark group and the DCFH-DA group.

[0101] Example 11

[0102] Application of Hm-NP obtained in Example 5 in inducing changes in intracellular mitochondrial membrane potential:

[0103] Methods: Changes in mitochondrial membrane potential in tumor cells were detected by flow cytometry. MDA-MB-231 cells were cultured at a concentration of 2 × 10⁻⁶ cells / cells. 5 Cells were seeded at a density of 100 cells / well in 6-well plates and cultured overnight at 37°C. Subsequently, the cells were treated with BDP-Ir-NP, Mm-NP, Rm-NP, and Hm-NP (2 µg / mL) for 6 hours. The cells were then irradiated with an 808 nm laser (10 mW·cm⁻¹). -2 After an additional 17 hours of incubation, cells were collected, washed twice with PBS, and then resuspended in 500 μL of 1×JC-1 working solution at 37°C for 20 min. They were then washed twice with PBS and resuspended in 1× binding buffer. Samples were analyzed using flow cytometry, and data were analyzed using FlowJo 10 software. The detection fluorescence channel was λ. ex = 488 nm, λ em = 530±30 nm; λ ex = 488nm, λ em = 590±30nm.

[0104] The results of Hm-NP on induced changes in intracellular mitochondrial membrane potential are as follows: Figure 14As shown in the figure. The results indicate that, compared with the control group, the red fluorescence in cells was weakened and the green fluorescence was significantly enhanced after Hm-NP treatment, indicating that the compound Hm-NP effectively induced a decrease in mitochondrial membrane potential under light irradiation.

[0105] Example 12

[0106] Application of Hm-NP prepared in Example 5 to induce apoptosis and necrosis under light irradiation:

[0107] Method 1: Flow cytometry was used to detect tumor cell apoptosis and necrosis. MDA-MB-231 cells were seeded in 6-well plates and cultured overnight at 37°C. They were then treated with BDP-Ir-NP, Mm-NP, Rm-NP, and Hm-NP (1 µg / mL) for 6 hours, respectively. Finally, they were irradiated with an 808 nm laser (10 mW·cm⁻¹). -2 After another 17 hours of incubation, cells were collected, washed twice with PBS, resuspended in binding buffer, stained with Annexin V / PI, and analyzed by flow cytometry. Data were analyzed using FlowJo 10 software. (λ) ex = 488 / 561 nm)

[0108] Method 2: Western blotting (WB) was used to detect changes in the content of apoptotic and necrotic proteins. MDA-MB-231 cells were seeded in 100 mm culture dishes and incubated overnight. They were then treated with Hm-NP (1, 2 µg / mL) for 24 hours. The light-treated group received an 808 nm light source (10 mW·cm⁻¹) 6 hours after drug administration. -2 Cells were irradiated for 1 h and then cultured for another 17 h. Cells were collected and lysed on ice for 30 min using RIPA lysis buffer containing 100 μg / mL benzoylmethanesulfonyl fluoride (PMSF). The supernatant was obtained by centrifugation at 13,400 rpm for 20 min at 4 °C, and protein concentration was determined according to the BCA protein quantification kit. SDS-PAGE sample loading buffer was then added, and the sample was heated at 95 °C for 10 min and stored at -80 °C. Western blotting analysis was performed using 30 µg of protein. After electrophoresis, the protein was transferred to a PVDF membrane (200 mA, 1 h) and blocked for 2 h with PBST buffer containing 5% skim milk. The membrane was then incubated overnight at 4 °C with a specific primary antibody, followed by incubation at room temperature with an HRP-labeled secondary antibody for 1 h. After washing with PBST, the cells were developed using Tanon High-signal ECL Western Blotting substrate and visualized using a Tanon 5200 Multi.

[0109] Experimental results regarding the effects of Hm-NP on inducing apoptosis and necrosis under light irradiation are as follows: Figure 15 As shown in the results, no significant apoptotic signals were observed in either the control or dark groups under 808 nm light irradiation, with late apoptosis rates below 5%. In contrast, the apoptosis rate of MDA-MB-231 cells treated with Hm-NP increased from 4.1% to 40.4%, and the necrosis rate increased from 4.7% to 21.3%. Western blot analysis also showed that Hm-NP light treatment downregulated the anti-apoptotic protein Bcl-2 (B-lymphoma-2 protein) while upregulating the pro-apoptotic protein Bax (BCL-2-associated X protein). During necrosis, RIP1 activation triggered phosphorylation of RIP3 (receptor-interacting protein 3) and MLKL (mixed series kinase domain-like protein), manifested as upregulation of RIP3 and p-MLKL. All these results indicate that ROS generated by the photosensitizer Hm-NP induces cellular oxidative stress, leading to apoptosis and necrosis.

[0110] Example 13

[0111] Application of Hm-NP prepared in Example 5 under ultrasonic treatment:

[0112] Method 1: TEM detection of ultrasonic release. Hm-NP was dispersed in an aqueous solution and subjected to ultrasound (0.3 W·cm⁻¹). -2 After 10 minutes, the sample was prepared on a copper mesh and TEM images were taken.

[0113] Method 2: MTT assay for sonication release. MDA-MB-231 cells at an appropriate density (5000 cells per well) were pre-cultured in 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator. Culture medium containing different concentrations of the compound was then added. After 6 hours of incubation, the cells were exposed to 808 nm light (10 mW·cm⁻¹). -2 After incubation for another 41 hours, add 20 μL of MTT solution (5 mg / mL) to each well and incubate for 4 hours. After removing the culture medium, add 150 µL of dimethyl sulfoxide (DMSO) to each well. Measure the absorbance at 490 nm using a microplate reader (LabServ K3). Repeat each well three times to obtain an average value. The cited IC50 values ​​are... 50 The value is the mean ± standard deviation.

[0114] The release of Hm-NP prepared in Example 5 under ultrasonic treatment is as follows: Figure 16As shown in Table 2, the morphological changes of Hm-NP before and after ultrasound were observed using TEM. The results showed that the surface membrane structure of Hm-NP disappeared after ultrasound. Simultaneously, the toxicity of Hm-NP + ultrasound to MDA-MB-231 was detected using the MTT assay. The results are shown in Table 2: the toxicity of Hm-NP after ultrasound in both extracellular and intracellular environments was close to that of unencapsulated BDP-Ir-NP, indicating that ultrasound promoted drug release.

[0115] Table 2 IC50 values ​​of BDP-Ir-NP and Hm-NP nanoparticles 50 Value (μg / mL)

[0116]

[0117] In Table 2, Hm-NP 1 This refers to no processing; Hm-NP 2 This refers to drug administration following extracellular ultrasound; Hm-NP 3 This refers to intracellular ultrasound after drug administration; (ultrasound conditions: 0.3 W / cm²) 2 , 10 min).

[0118] Example 14

[0119] Application of Hm-NP ultrasound-induced intracellular oxidative stress and apoptosis obtained in Example 5:

[0120] Method 1: MDA-MB-231 cells were seeded in 6-well plates and cultured overnight at 37°C, followed by treatment with BDP-Ir-NP and Hm-NP (1 / 5 / 10 / 20 / 30 µg / mL) for 6 h, respectively. The sonication group was subjected to sonication for 10 min (0.3 W·cm⁻¹). -2 The control group received no treatment. Cells were cultured for another 18 h, then collected and washed twice with PBS. DCFH-DA was added and incubated at 37°C for 30 min. After washing, cells were analyzed by flow cytometry, and data were analyzed using FlowJo 10 software. (λ) ex = 488 nm)

[0121] Method 2: MDA-MB-231 cells were seeded in 6-well plates and cultured overnight at 37°C, followed by treatment with BDP-Ir-NP and Hm-NP (15 / 30 µg / mL) for 6 hours, respectively. The sonication group was subjected to sonication for 10 min (0.3 W·cm⁻¹). -2 After another 17 hours of incubation, cells were collected, washed twice with PBS, resuspended in binding buffer, stained with Annexin V / PI, and analyzed by flow cytometry. Data were analyzed using FlowJo 10 software. (λ) ex = 488 / 561 nm)

[0122] The results of intracellular oxidative stress induced by the ultrasonic drug release of Hm-NP prepared in Example 5 are as follows: Figure 17 As shown: After Hm-NP enters the cell, it releases BDP-Ir-NP upon sonication, inducing intracellular oxidative stress and significantly enhancing green fluorescence, consistent with the trend in the BDP-Ir-NP group, while the fluorescence change in the non-sonication group was not obvious. Cell apoptosis results are shown below. Figure 18 As shown, the apoptosis rate of the Hm-NP plus ultrasound group reached 53.1%, which was much higher than that of the non-ultrasound group (10.6%) and close to that of the group (46.5%). This indicates that Hm-NP effectively induced apoptosis after entering the cells and being acted upon by ultrasound.

[0123] Example 15

[0124] Application of Hm-NP simulated deep therapy obtained in Example 5:

[0125] Method 1: MTT assay for cell viability. MDA-MB-231 cells were cultured in 96-well plates and incubated overnight at 37°C. Hm-NP (2 / 60 µg / mL) was then added and incubated for 6 h. Chicken breast pieces of 5, 10, 15, and 20 mm diameter were cut and placed on the culture plates. The light-treated group was irradiated with 808 nm light (10 mW·cm⁻¹). -2 ) 1 hour, the ultrasound group used a handheld ultrasound device for 10 minutes (0.3 W·cm) -2 After incubation for 41 hours, add 20 μL of MTT solution (5 mg / mL) to each well and incubate for 4 hours. After removing the culture medium, add 150 μL of dimethyl sulfoxide (DMSO) to each well. Measure the absorbance at 490 nm using a LabServ K3 microplate reader. Calculate the viability, repeating the process three times per well to obtain the average value.

[0126] Method 2: 3D cell spheroid detection. 2000 MDA-MB-231 cells (100 μL) were added to each well of a 96-well plate. After 3 days, cell spheroids with a diameter of approximately 500 μm were formed. Culture medium containing Hm-NP (5 / 60 µg / mL) was added to each well. After 6 hours, chicken breast was used to simulate human muscle tissue, and irradiation (808 nm, 10 mW·cm⁻¹) was performed under different thicknesses of chicken breast covering. -2 (1h) or ultrasound (0.3 W·cm) -2 The cells were treated with a 10-minute incubation period and incubated for 17 hours, followed by photographing using a phase-contrast microscope. Photographs were then taken every 24 hours thereafter until the cell spheres broke apart.

[0127] The effect of Hm-NP simulated deep treatment obtained in Example 5 is as follows: Figure 19As shown, cell survival rate increased with increasing chicken breast thickness. When the chicken breast thickness was 20 mm, the light-treated group showed almost no killing effect, while the survival rate of the ultrasound-treated group dropped to 75%, indicating that ultrasound had a better penetration effect than light. In the 3D cell spheroid experiment, after five days of Hm-NP+ light treatment, the cell spheroids covered by 20 mm of chicken breast remained intact and were similar in size to the untreated group. In contrast, except for the control group, all cell spheroids in the Hm-NP+ ultrasound-treated group were broken on the fifth day, proving that Hm-NP+ ultrasound still had a significant killing effect under 20 mm chicken breast coverage.

[0128] Example 16

[0129] Application of Hm-NP obtained in Example 5 in in vivo deep treatment of triple-negative breast cancer:

[0130] Methods: In vivo antitumor immunotherapy was conducted using the MDA-MB-231 cancer mouse model. Female nude mice (4-5 weeks old, 18-20 g) were selected. Cultured human breast cancer cells (MDA-MB-231) were collected and the cell concentration was adjusted to 1 × 10⁻⁶. 8 A tumor model was established by subcutaneous inoculation of the left forelimb axilla of Balb / c nude mice with 0.1 mL of cell culture and 0.1 mL of matrix gel per mouse. When the tumor grew to 100-200 mm... 3 Mice were randomly divided into nine groups (n=5 per group): 1) PBS; 2) PBS + Irradiation; 3) Hm-NP; 4) Hm-NP + Irradiation; 5) Hm-NP + 1cm chicken breast + Irradiation; 6) Hm-NP + 1cm chicken breast + Irradiation + Ultrasound; 7) Hm-NP + 2cm chicken breast + Irradiation; 8) PBS + 2cm chicken breast + Ultrasound; 9) Hm-NP + 2cm chicken breast + Ultrasound. The medication was administered via tail vein injection (0.75 mg / kg). -1 Ultrasound (0.5 W / cm) was used 4 hours later. 2 (5 minutes) or light therapy (808 nm, 0.5 W / cm²) 2 (5 minutes). Tumor volume is measured every two days. The tumor volume is calculated as follows: Tumor volume (V) = a × b 2 / 2, where a is the principal axis of the tumor and b is the lateral axis. Two weeks after drug administration, nude mice were sacrificed, and tumors and organs such as the heart, liver, lungs, spleen, and kidneys were collected for subsequent experiments.

[0131] Experimental results are as follows Figure 20 As shown in the figure. The results indicated that tumors in groups 1-3, 7, and 8 all exhibited exponential growth. In contrast, tumor growth in group 4 was significantly inhibited after Hm-NP treatment and subsequent light exposure; compared to the untreated Hm-NP group, the tumor volume decreased from 1318 mm². 3 Reduced to 264 mm 3 The tumor inhibition rate reached 79%, indicating that the near-infrared photosensitizer Hm-NP has excellent in vivo photodynamic therapy effects. In group 5, the addition of 1 cm of chicken breast reduced the photodynamic effect of Hm-NP, with the tumor inhibition rate decreasing to 41%. In group 7, the addition of 2 cm of chicken breast essentially eliminated the photodynamic therapy effect, indicating that the penetration depth of the 808 nm laser decreased with increasing chicken breast thickness, and a 2 cm thickness completely blocked the influence of the light source. In group 6, after the addition of ultrasound, compared with group 5, the tumor volume decreased from 790 mm². 3 Reduced to 305 mm 3 Even with a 2 cm chicken breast obstruction, the tumor inhibition rate of the Hm-NP + ultrasound treatment group 9 still reached 33%. All of the above results indicate that Hm-NP can realize deep controllable ultrasound-activated chemophotodynamic therapy, which has the potential to treat deep tumors in vivo.

[0132] The BDP-Ir prepared by the method of this invention can be produced at low doses (IC50). 50 = 68 nM) at low power (10 mW·cm) -2 The 808nm excitation exhibits high photosensitivity, successfully resolving the trade-off between excitation wavelength and photosensitivity efficiency. Furthermore, to enhance tumor targeting and immune evasion capabilities, an artificial nanohybrid delivery system was constructed, loading a photosensitizer to obtain Hm-NP. This system accurately delivers the photosensitizer to the tumor site, bypassing in vivo immune clearance, and allows for lesion observation using near-infrared fluorescence imaging, significantly improving biosafety. Hm-NP exerts photodynamic effects in superficial tissues, generating a ROS storm that kills a large number of cancer cells. In deeper tissues beyond the light source's penetration limits, it can rupture under ultrasound to release its active ingredients. For tumors at different depths, a combined photodynamic / chemotherapy approach can effectively inhibit the proliferation of triple-negative breast cancer.

Claims

1. A near-infrared cyclic metallic iridium(III) photosensitizer, characterized in that, Its structural formula is: 。 2. The method for synthesizing the cyclic iridium(III) photosensitizer according to claim 1, characterized in that, Includes the following steps: (1) Under an inert atmosphere, the fluoroboron dipyrrole-modified bipyridine ligand and the cyclic metal iridium dimer were heated and refluxed in an organic solvent, and the solvent was removed by vacuum distillation; (2) The crude product was obtained by substituted with NH4PF6, and the crude product was separated and purified by column chromatography to obtain the cyclic metal iridium (III) photosensitizer; The structural formula of the fluoroboron dipyrrole-modified bipyridine ligand is shown below: ； The structural formula of the cyclic metallic iridium dimer is shown below: 。 3. The method for synthesizing the cyclic metallic iridium(III) photosensitizer according to claim 2, characterized in that: In step (1), the fluoroboron dipyrrole-modified bipyridine ligand is prepared by the following method, the specific steps of which are as follows: fluoroboron dipyrrole derivative, 2,2'-bipyridine-5-carboxaldehyde, piperidine and catalyst are dissolved in an organic solvent and reacted at reflux temperature; after the reaction is completed, the solvent is removed by vacuum distillation, and the crude product is purified by column chromatography to obtain the fluoroboron dipyrrole-modified bipyridine ligand.

4. The method for synthesizing the cyclic metallic iridium(III) photosensitizer according to claim 3, characterized in that: The molar ratio of the fluoroboron dipyrrole derivative and 2,2'-bipyridine-5-carboxaldehyde is 1:1~3.

5. The method for synthesizing the cyclic metallic iridium(III) photosensitizer according to claim 3, characterized in that: The catalyst is p-toluenesulfonic acid; the amount of catalyst added is 2.5-3% of the mass of the fluoroboron dipyrrole derivative.

6. The method for synthesizing the cyclic metallic iridium(III) photosensitizer according to claim 2, characterized in that: In step (1), the reaction molar ratio of the fluoroboron dipyrrole-modified bipyridine ligand to the cyclic metal iridium dimer is 2:1~1.

2.

7. The method for synthesizing the cyclic metallic iridium(III) photosensitizer according to claim 2, characterized in that: In step (1), the organic solvent is a mixture of dichloromethane and methanol.

8. The method for synthesizing the cyclic metallic iridium(III) photosensitizer according to claim 7, characterized in that: In the mixed solvent, the volume ratio of dichloromethane to methanol is 2:1 to 1.

5.

9. The method for synthesizing the cyclic metallic iridium(III) photosensitizer according to claim 2, characterized in that: In step (1), the reflux reaction takes 12-13 hours and the temperature is 45-48°C.

10. The method for synthesizing the cyclic metallic iridium(III) photosensitizer according to claim 2, characterized in that: In step (2), the crude product is obtained by substitution with NH4PF6. Specifically, a methanol solution saturated with NH4PF6 is added and stirred for 2-3 hours.

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