A supramolecular coordination complex with sonodynamic activity, and a preparation method and application thereof

Abstract

The application belongs to the technical field of biomedical materials, and particularly relates to a supramolecular coordination complex with sonodynamic activity and a preparation method and application thereof. The application proposes a new supramolecular coordination complex formed by self-assembly of a phthalocyanine compound and Ru-dbq, and finds that the supramolecular coordination complex has excellent ROS yield in response to US, and also has the characteristics of multifunctional integration such as CAT enzyme-like activity and antioxidant activity, which endows the material with great application potential in response to complex biological environments and excellent sonodynamic therapy prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a supramolecular coordination complex with acoustic dynamic activity, its preparation method, and its application. Background Technology

[0002] Sonodynamic therapy (SDT) is an emerging non-invasive treatment method following photodynamic therapy (PDT). Depending on the frequency used, ultrasound (US) can be precisely focused on the lesion site, thereby achieving targeted activation of sonosensitive agents. Therefore, it can selectively kill target cells without harming surrounding healthy organs, offering advantages such as deep tissue penetration, high precision, and fewer side effects, demonstrating unique potential in the fields of anti-infection and anti-tumor therapy. Sonosensitive agents, as the foundation of SDT, are responsible for converting O2 into reactive oxygen species (ROS) under US stimulation. Furthermore, the cavitation, mechanical, and thermal effects associated with US further enhance the killing effect of SDT. However, commonly used sonosensitive agents (such as protoporphyrin, dihydroporphyrin, curcumin, and methylene blue), mainly derived from traditional photosensitizers, still exhibit unsatisfactory ROS yields under US excitation, and the structure-activity relationship between the molecular structure of sonosensitive agents and ROS generation efficiency remains unclear. This severely restricts the improvement of SDT efficacy and its clinical translation. Therefore, designing highly efficient sonodynamic formulations through molecular engineering strategies has become an urgent research need.

[0003] In recent years, inorganic sonosensitive agents have seen significant development due to their excellent catalytic activity and stability. Materials such as metal oxides, metal carbides, metal hydroxides, and organometallic frameworks (MOFs) have been successively developed. They mainly generate reactive oxygen species (ROS) through ultrasonic cavitation effects or enhance therapeutic effects through other mechanisms (such as Fenton reactions and sonothermal effects). For example, TiO2 and ZnO can generate electron-hole pairs under ultrasound, which then react with O2 to produce ROS. Fe3O4 can generate ·OH through the Fenton reaction, enhancing the ROS-killing function of sonosensitive catalytic therapy (SDT). Au NPs can enhance sonothermal conversion and assist in enhancing SDT. However, some materials have poor biodegradability and may remain in the body for a long time, leading to long-term biosafety issues. At the same time, the relatively wide band gap of inorganic sonosensitive agents may also limit their potential clinical applications (e.g., the band gap of TiO2 is ~3.2 eV, and the band gap of ZIF-8 is 4.94 eV). Compared to inorganic sonosensitive agents, organic sonosensitive agents have greater potential for clinical translation due to their well-defined structures, controllable metabolism, and ease of detection. Several porphyrin-based drugs have been approved by the FDA for clinical research, such as hematoporphyrin monomethyl ether (HMME) and protoporphyrin (PpIX). However, most currently developed organic sonosensitive agents suffer from poor water solubility, insufficient sonodynamic efficiency, and difficulty in coping with the complex microenvironments of tumors or bacterial biofilms (such as hypoxia and antioxidant defense mechanisms). To overcome these limitations, researchers have used nanotechnology to incorporate organic sonosensitive agents into mesoporous materials, microbubbles, liposomes, artificial nanozymes, and other nanoformations, leveraging the unique advantages of nanoformations to overcome these shortcomings. Encapsulated nanoparticles can be used as energy converters to improve SDT efficiency. For example, gas-filled microbubbles (MBs) can transfer the sonoluminescence energy generated by gas rupture to the encapsulated sonosensitive agent, or provide more nucleation sites for the generation of cavitation bubbles by lowering the cavitation threshold, a technique widely used in microbubbles. Furthermore, catalase-mimicking nanomaterials (CAT) as a loading substrate can alleviate the problem of O2 deficiency in the tumor microenvironment. Although combining with nanotechnology can partially improve the shortcomings of organic sonosensitive agents, such strategies still face problems such as insufficient sonosensitive agent loading and imprecise regulation of release behavior. In addition, the preparation routes of related nanoplatforms are usually complex and technically challenging, which increases the difficulty of their clinical translation. Therefore, developing sonodynamic materials with high sonodynamic activity, multifunctional integrated properties, and simple preparation to address the challenges of complex disease microenvironments has become an urgent research need. Summary of the Invention

[0004] To address the aforementioned issues, this invention proposes a novel supramolecular coordination complex, such as ZnPC-Ru, which is self-assembled from metal phthalocyanine and aromatic hydrocarbon Ru. It has been found to exhibit excellent US-responsive ROS yield and also possesses multifunctional properties such as CAT-like enzyme activity and antioxidant activity, demonstrating promising prospects for sonodynamic therapy.

[0005] Specifically, in a first aspect, the present invention provides a supramolecular coordination complex with acoustic dynamic activity, the supramolecular coordination complex being formed by coordination self-assembly of a phthalocyanine compound and Ru-dbq;

[0006] The structure of the phthalocyanine compounds is shown in formula (I):

[0007] (I),

[0008] Where M is absent or selected from Zn, Al, Ga, Ti or Ru, preferably M is Zn;

[0009] The structure of Ru-dbq is shown in equation (II):

[0010] (II).

[0011] Furthermore, the coordination self-assembly is achieved by the coordination of the pyridine nitrogen in the phthalocyanine compound with the metallic Ru in the Ru-dbq.

[0012] Furthermore, the molar ratio of the phthalocyanine compound to the Ru-dbq is 1-2:1-3, for example 1:2, 2:1, 1:1 or 1:3, preferably 1:2.

[0013] Furthermore, the supramolecular coordination complex possesses multiple functions, including acoustic-induced ROS production, CAT-like enzyme activity, antioxidant properties, and promotion of glutathione and reduced coenzyme I oxidation.

[0014] Furthermore, the multiple functions of the supramolecular coordination complex are pH-dependent.

[0015] Furthermore, the supramolecular coordination complex exhibits better acoustic-induced ROS activity under acidic conditions (e.g., pH ≤ 6.5).

[0016] Furthermore, the supramolecular coordination complex exhibits CAT-like enzyme activity, antioxidant activity, and glutathione and reduced coenzyme I oxidation activity under neutral conditions.

[0017] Furthermore, the optimal pH range for the CAT-like activity of the supramolecular coordination complex is 6.5-8.

[0018] In a second aspect, the present invention provides a method for preparing a sonodynamically active supramolecular coordination complex, comprising the following steps:

[0019] (1) Under the protection of an inert gas, 3,4-pyridinedicarboxynitrile undergoes a cyclotetramerization reaction in the presence or absence of a metal salt in an alkaline catalyst and a high-boiling solvent to obtain phthalocyanine compounds as shown in formula (I).

[0020] The metal salt is a salt of a metal selected from Zn, Al, Ga, Ti, or Ru;

[0021] (2) Under the protection of an inert gas, bis(4-methylisopropylphenyl)ruthenium(II) and 2,5-dihydroxy-1,4-benzimidone were reacted in a weakly coordinating solvent to obtain Ru-dbq as shown in formula (II);

[0022] (3) The phthalocyanine compound obtained in step (1) and Ru-dbq obtained in step (2) are self-assembled by coordination between the pyridine nitrogen in the phthalocyanine compound and the metal Ru in the Ru-dbq to obtain a supramolecular coordination complex with acoustic dynamic activity.

[0023] Furthermore, the alkaline catalyst is 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

[0024] Furthermore, the high-boiling-point solvent is n-pentanol, dimethylaminoethanol, or quinoline.

[0025] Furthermore, the metal salt is an acetate or chloride salt of a metal selected from Zn, Al, Ga, Ti, or Ru.

[0026] Furthermore, in the presence of a metal salt, the molar ratio of the 3,4-pyridinedicarboxynitrile to the metal salt is 3-4:1.

[0027] Furthermore, the conditions for the cyclotetramerization reaction are: stirring at 150-180°C for 6-24 hours.

[0028] Furthermore, step (1) also includes collecting the reaction product by precipitation and filtration, and performing post-treatments such as washing, purification and drying to obtain phthalocyanine compounds as shown in formula (I).

[0029] Furthermore, the molar ratio of dichlorobis(4-methylisopropylphenyl)ruthenium(II) to 2,5-dihydroxy-1,4-benzimidone is 1:0.8-1.2.

[0030] Furthermore, the weakly coordinating solvent is an alcohol solvent, acetonitrile, or acetone.

[0031] Furthermore, the reaction conditions in step (2) are 25-60°C with stirring for 2-4 hours.

[0032] Furthermore, step (2) also includes, after the reaction is completed, concentrating the reaction mixture under reduced pressure, filtering to collect the reaction product, and washing with ethanol, water, acetone and diethyl ether to obtain Ru-dbq as shown in formula (II).

[0033] Further, step (3) involves first removing chloride ions from Ru-dbq using Ag⁺, and then reacting the phthalocyanine compound dissolved in an organic solvent with stirring at 25-80°C for 12-36 h under an inert gas atmosphere.

[0034] Furthermore, the molar ratio of the phthalocyanine compound to the Ru-dbq is 1-2:1-3, for example 1:2, 2:1, 1:1 or 1:3, preferably 1:2.

[0035] Furthermore, the organic solvent is dimethylformamide.

[0036] Further, the removal of chloride ions from Ru-dbq using Ag⁺ involves reacting Ru-dbq and AgCF3SO3 in methanol at room temperature with stirring, filtering to remove AgCl, and obtaining a filtrate containing Ru-dbq with chloride ions removed.

[0037] Furthermore, step (3) also includes filtering the reaction mixture after the reaction is completed, then adding diethyl ether to the filtrate to precipitate the reaction product, collecting the reaction product and washing and drying it.

[0038] Furthermore, the prepared supramolecular coordination complex has multiple functions, including acoustic-induced ROS production, CAT-like enzyme activity, antioxidant properties, and promotion of glutathione and reduced coenzyme I oxidation.

[0039] Furthermore, the multiple functions of the supramolecular coordination complex are pH-dependent.

[0040] Furthermore, the supramolecular coordination complex exhibits better acoustic-induced ROS activity under acidic conditions (e.g., pH ≤ 6.5).

[0041] Furthermore, the supramolecular coordination complex exhibits CAT-like enzyme activity, antioxidant activity, and glutathione and reduced coenzyme I oxidation activity under neutral conditions.

[0042] Furthermore, the optimal pH range for the CAT-like activity of the supramolecular coordination complex is 6.5-8.

[0043] As used in this article, inert gases are gases that are chemically very inert and do not readily react with other elements or compounds, including nitrogen and argon.

[0044] In a third aspect, the present invention provides the use of supramolecular coordination complexes as described herein or supramolecular coordination complexes prepared by the preparation methods described herein in the preparation of sound-sensitive agents.

[0045] Furthermore, the sonosensitive agent can be used in sonodynamic therapy, for example, for anti-infection and anti-tumor purposes.

[0046] Beneficial effects of the present invention

[0047] This invention proposes a novel supramolecular coordination complex formed by the coordination self-assembly of a metal phthalocyanine and an aromatic hydrocarbon Ru. For example, the supramolecular coordination complex formed by the coordination self-assembly of zinc phthalocyanine and aromatic hydrocarbon Ru, as described in the examples, is referred to herein as ZnPC-Ru. As demonstrated in Example 3 of this specification, both ZnPC-Ru and PC-Ru (PC-Ru is a supramolecular coordination complex prepared from a metal-free phthalocyanine) exhibit acoustic stimulation-induced ROS production activity and H2O2 scavenging activity, demonstrating that the metal Zn at the phthalocyanine center has a relatively small impact on the activity of the material. This implies that the metal ion at the phthalocyanine center can be expanded.

[0048] This invention further demonstrates that, under US stimulation, ZnPC-Ru produces more ROS than commonly used commercial sonostimulants (hematoporphyrin, curcumin). ZnPC-Ru synergistically amplifies the therapeutic effect of sound through a triple regulatory mechanism: (1) ZnPC coordinates with aromatic Ru to exhibit an enhanced conjugated system, narrowing the band gap and increasing ROS yield; (2) It exhibits CAT enzyme-mimicking activity, catalyzing the conversion of H2O2 to O2 to enhance ROS production, which can address diseases with hypoxic microenvironments; (3) It protects the generated ROS by consuming the oxidation of GSH and NADH to disrupt redox homeostasis. In addition, ZnPC-Ru also has ROS scavenging activity under neutral conditions, which is crucial for the anti-inflammatory and repair-promoting period in the later stages of disease treatment, revealing the great application potential of this material in dealing with complex biological environments. Therefore, this invention proposes a novel supramolecular coordination complex with excellent US-responsive ROS yield and multifunctional integration, providing a framework for the development of novel sonodynamic materials. Attached Figure Description

[0049] Figure 1 The diagram shows the synthesis of ZnPC, Ru-dbq, and ZnPC-Ru.

[0050] Figure 2 The EIS-MS plot of ZnPC-Ru is shown, with the inset being [ZnPC-Ru-3OTf]. - ]5+ [ZnPC-Ru-4OTf] - ] 4+ Comparison of experimental values ​​and simulated isotope distributions.

[0051] Figure 3 The Fourier transform infrared spectra of ZnPC, Ru-dbq and ZnPC-Ru are shown; the Raman spectra of ZnPC, Ru-dbq and ZnPC-Ru are shown; and the UV-vis spectra of ZnPC, Ru-dbq and ZnPC-Ru are shown.

[0052] Figure 4 The figures show (a) the ROS content and (b) catalase activity of materials with different raw material ratios as determined by UV-Vis spectroscopy based on TMB color development, and (c) the ROS content and (d) catalase activity of PC, ZnPC, PC-Ru, and ZnPC-Ru as determined by time.

[0053] Figure 5 The results show (a) determination of reactive oxygen species (ROS) content (mean ± SD, n=3) in the material using UV-Vis spectroscopy based on TMB colorimetry; (b) the effect of pH on ROS generation performance; (c) free radical quenching experiment; and (d) DPA test. 1 O2 generation capacity; (e) O2⁻ generation capacity by HE test; (f) O2⁻ and (g) 1 EPR spectrum of O2; in-situ infrared spectrum (h) and contour line color-filled map (i) of the reaction of ZnPC-Ru with hydrogen peroxide.

[0054] Figure 6 The following data are presented: (a) the catalase-like activities of Ru-dbq, ZnPC, and ZnPC-Ru over time; (b) the real-time kinetics of catalytic decomposition of H2O2 to produce oxygen; (c) the effect of pH on the catalase-like activities of ZnPC-Ru; (d) the scavenging rate of DPPH free radicals by Ru-dbq, ZnPC, and ZnPC-Ru (15 min); and (e) the oxidation performance of GSH to GSSH and (f) NADH at different ZnPC-Ru concentrations.

[0055] Figure 7 The diagrams show (a) Tauc plots of ZnPC and ZnPC-Ru; (b) Mott-Schottky curves of ZnPC and (c) ZnPC-Ru at different AC frequencies; (d) schematic diagrams of the band structures of ZnPC and ZnPC-Ru; and (e) schematic diagrams of the potential mechanism for ROS generation in ZnPC-Ru under US excitation. Detailed Implementation

[0056] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0057] Example 1: Synthesis of supramolecular coordination complexes

[0058] In this embodiment, a supramolecular coordination complex, referred to as ZnPC-Ru, was prepared by coordination self-assembly of a Zn-coordinated phthalocyanine compound (ZnPC) and Ru-dbq. Figure 1 As shown, ZnPC-Ru was synthesized in three steps. First, using 3,4-pyridinedicarboxynitrile and zinc acetate as starting materials, tetra-(4-pyridyl)phthalocyanine zinc was synthesized in one step using DBU as a base catalysis. Ru-dbq was synthesized by replacing the -Cl in dichlorobis(4-methylisopropylphenyl)ruthenium(II) with 2,5-dihydroxy-1,4-benzylquinone. Subsequently, ZnPC and Ru-dbq coordinated and self-assembled at a molar ratio of 1:2 to form a supramolecular coordination complex ZnPC-Ru 1:2. Simultaneously, by changing the feed ratio of ZnPC and Ru-dbq, ZnPC-Ru 2:1 (ZnPC:Ru-dbq=2:1), ZnPC-Ru 1:1 (ZnPC:Ru-dbq=1:1), and ZnPC-Ru 1:3 (ZnPC:Ru-dbq=1:3) were synthesized in the same manner. Metal-free phthalocyanine (abbreviated as PC) was also synthesized and synthesized with Ru-dbq at a molar ratio of 1:2 to form PC-Ru. This was used to test its ultrasonic response ROS generation activity and hydrogen peroxide decomposition performance under neutral conditions.

[0059] Specifically, the synthesis of supramolecular coordination complex materials is described below.

[0060] ZnPC Synthesis: A 100 mL double-necked flask was connected to a reflux condenser. 8 mL of n-pentanol, Zn(OAc)₂·2H₂O (26.1 mg, 0.21 mmol), and 3,4-pyridinedicarboxynitrile (100 mg, 0.775 mmol) were added. The mixture was stirred at 100 °C under a N₂ atmosphere for 10 min. DBU (0.12 mL, 1.55 mmol) was added dropwise, and the mixture was stirred at 160 °C for 18 h. After the reaction was complete, the mixture was cooled to room temperature, and 20 mL of cyclohexane was added and stirred at room temperature for 2 h. The mixture was filtered and washed with 20 mL of H₂O and 20 mL of n-hexane to obtain a dark solid. Further purification was performed by dissolving the solid in methanol (10 mL) and stirring at 40 °C for 1 h. H₂O was then added dropwise at room temperature to precipitate the product. The precipitate was filtered, washed with a small amount of cold methanol, and the solid was dried overnight in a hollow chamber to obtain a dark blue solid. The synthesis method of PC is similar to the above scheme, but without the addition of Zn(OAc)2·2H2O.

[0061] Synthesis of Ru-dbq: Dichlorobis(4-methylisopropylphenyl)ruthenium(II) (100 mg, 0.163 mmol) and 2,5-dihydroxy-1,4-benzylquinone (22.9 mg, 0.163 mmol) were dissolved in anhydrous methanol and stirred at room temperature for 3 h under a nitrogen atmosphere. After the reaction was completed, the reaction mixture was concentrated under reduced pressure, filtered, and washed with ethanol, water, acetone and diethyl ether to obtain the product Ru-dbq as a brown solid.

[0062] ZnPC-Ru:Ru-dbq (100 mg, 0.12 mmol) and AgCF3SO3 (62 mg, 0.24 mmol) were dissolved in 25 mL of methanol and stirred at room temperature for 3 h. AgCl was removed by filtration, and the filtrate was obtained. ZnPC (34.8 mg, 0.06 mmol) was dissolved in 12 mL of dimethylformamide (DMF), and the above filtrate was added. The mixture was reacted at 60 °C for 24 h under nitrogen atmosphere. The mixture was filtered, and excess diethyl ether was added to the filtrate to precipitate a blue-black solid. The solid was then filtered and dried under vacuum to obtain the product. The synthesis scheme of PC-Ru is similar to the above scheme, except that ZnPC is replaced with PC. In the synthesis of ZnPC-Ru 2:1, ZnPC-Ru 1:1, and ZnPC-Ru 1:3, the molar ratio of ZnPC:Ru-dbq was changed to 2:1, 1:1, and 1:3, respectively.

[0063] Example 2: Characterization and structural testing of supramolecular coordination complexes

[0064] 1. Testing instruments and equipment

[0065] Electrospray ionization mass spectrometry (ESI-MS) was performed on a triple quadrupole mass spectrometer (Thermo Finnigan TSQ Quantum Ultra, USA) with an ESI source of 1 pg reserpine >500:1 (calculated by peak-to-peak ratio, m / z 609 >195).

[0066] Fourier transform infrared spectroscopy (FTIR) was performed using a Fourier transform infrared spectrometer (Nicolet iS50, ThermoFisher Scientific Inc., USA).

[0067] Raman spectroscopy was measured using a Raman spectrometer (Horiba XploRA PLUS, Japan) at an excitation wavelength of 633 nm.

[0068] The ultraviolet spectrum (UV-vis) was measured using a UV spectrophotometer (Shimadzu UV-1750, Japan) (5×10⁻⁶). -6M).

[0069] The Mott-Schottky curves were measured using an electrochemical workstation (Gamry reference 600 workstations, USA).

[0070] In-situ Fourier transform infrared spectroscopy was performed on an infrared spectrometer (Thermo Scientific, iS50 FTIR) equipped with an in-situ reaction cell (Shanghai Yuanfang Technology Co., Ltd., SPECEL-III).

[0071] 2. Test Results

[0072] The materials synthesized in Example 1 were characterized using Fourier transform infrared spectroscopy (FTIR), electrospray mass spectrometry (ESI-MS), ultraviolet-vis spectroscopy (UV-vis), and Raman spectroscopy. It is worth noting that, due to the very low solubility of zinc tetra-(4-pyridyl)phthalocyanine in common solvents (no NMR spectra have been reported in the literature), although the formation of the supramolecular coordination complex reduces the π-π stacking of phthalocyanine molecules, it is still difficult to obtain high-quality NMR data. The EIS-MS spectrum of ZnPC-Ru shows three distinct peaks at m / z = 649.4354, 808.8440, and 1048.8774, corresponding to [ZnPC-Ru-2OTf], respectively. - ] 6+ [ZnPC-Ru-3OTf] - ] 5+ [ZnPC-Ru-4OTf] - ] 4+ The isotope analysis distribution is consistent with the theoretical value. Figure 2 ).

[0073] Fourier transform infrared spectroscopy (FTIR) Figure 3 a) This indicates that the ZnPC-Ru spectrum contains characteristic peaks of two precursors, namely the peak at 1518 cm⁻¹ in Ru-dbq. -1 (ν C=O ), 1253 cm -1 (ν C-F ), ZnPC 2842-2955 cm -1 (ν C-H ), ~1624 cm -1 (ν C=N In the study, a significant blue shift and broadening of the pyridine C=N stretching vibration peak in ZnPC-Ru was observed, which is likely due to the coordination of pyridine nitrogen with the metal. Raman spectroscopy (Raman, Figure 3b) ZnPC and ZnPC-Ru exhibit phthalocyanine characteristic vibrational modes (A 1g B 1g This demonstrates the successful synthesis of the phthalocyanine structure, and simultaneously, the 385 cm⁻¹ in Ru-dbq... -1 The Ru-O peak shifts slightly to 379 cm⁻¹ after complex formation. -1 This is likely due to the formation of Ru-N coordination bonds. Ultraviolet spectroscopy further characterized the structural features ( Figure 3 c) After ZnPC coordinates with Ru-dbq, a new strong absorption peak appears at 621 nm in ZnPC-Ru. This new absorption peak is generated by the MLCT of Ru with phthalocyanine, confirming the coordination of Ru-dbq with the pyridine nitrogen of phthalocyanine. Furthermore, a significant red shift (from 667 nm to 677 nm) is observed in the Q band of phthalocyanine. This is likely due to the participation of both the π electrons of the aromatic hydrocarbon in Ru-dbq and the d electrons of the transition metal Ru in the conjugation of the system, forming a more effective conjugated framework, leading to the significant red shift of the Q band. Simultaneously, a slight blue shift of the B band and a significant increase in absorption intensity are observed, demonstrating enhanced π→π* electronic transitions. These results indicate that the formation of the ZnPC-Ru complex significantly enhances the π-π conjugated system of the phthalocyanine molecule.

[0074] Example 3: Performance testing of supramolecular coordination complexes

[0075] 1. Performance Testing Methods

[0076] TMB-based ROS generation assay: TMB was used as the substrate for ROS detection. Ru-dbq, ZnPC, and ZnPC-Ru were prepared into 8 mM, 4 mM, and 2 mM sample stock solutions for subsequent testing. 5 μL of each solution was added to 1.94 mL of acetate-sodium acetate buffer (0.1 M, pH 4.5), followed by 25 μL of H2O2 (final concentration 1.25 mM) and 25 μL of TMB (DMF, final concentration 0.5 mM). After reacting for 10 min, the color change of TMB was detected at 652 nm. The US group followed the same conditions, with sonication at 1.5 W / cm². -2 1 MHz, 30% duty cycle. The pH dependence of ROS generation of ZnPC-Ru under US excitation was tested by replacing the acetate-sodium acetate buffer solution at pH 4.5 with buffer solutions at pH 4.5–8.

[0077] HE probe detection of superoxide anion: HE is a specific probe that reacts with ·O₂⁻ to produce fluorescent ethidium. First, 15 μL of sample stock solution was added to 1.485 mL of pH 4.5 acetate-sodium acetate buffer (ZnPC 0.04 mM, ZnPC-Ru 0.02 mM), followed by 1.5 μL of 0.1 M H₂O₂. The mixture was stirred at 37 ℃ for 40 min, and then sonicated for 10 min (1.5 W cm⁻¹). -2 1 MHz, 30% duty cycle), then 1.5 mL of HE-ethanol solution (1 mg / mL) was added to the system. The solution was then vortexed and kept undisturbed for 40 minutes before fluorescence measurement (Ex: 470 nm, Em: 610 nm).

[0078] DPA detection of singlet oxygen: Prepare a 1 mL reaction system by mixing 5 μL of sample stock solution (containing ZnPC: 4 mM or ZnPC-Ru: 2 mM) with 995 μL of acetate-sodium acetate buffer (pH 4.5) and 50 μL of DPA solution (1 mg / mL). Then, in a 1.5 W cm⁻¹ atmosphere... -2 The samples were treated for different durations under ultrasonic conditions of 1 MHz and 30% duty cycle. Immediately after the reaction was complete, the absorbance change at 378 nm was measured using a UV-Vis spectrophotometer.

[0079] EPR Detection: The generation of ·O₂⁻ was assessed by EPR spectroscopy using DMPO spin-capture adduct in DMSO solvent. 10 μL of material stock solution and 10 μL of H₂O₂ (10 M) were added to 0.5 mL of DMSO, followed by the addition of 10 μL of LDMPO.

[0080] The TEMP spin-trapping adduct in NaOAc-HOAc buffer (100 mM, pH 4.5) was evaluated by EPR spectroscopy. 1 O2 generation. Add 10 μL of material stock solution and 10 μL of H2O2 (10 M) to 0.5 mL of buffer solution, and then add 10 μL of TEMP.

[0081] CAT-like enzyme activity assay: Prepare PBS buffer (pH 7.4) containing 10 mM H2O2 and different catalysts (Ru-dbq: 0.04 mM, ZnPC: 0.02 mM, ZnPC-Ru: 0.01 mM), with a total reaction volume of 2 mL. Mix 100 μL of the above reaction solution with 100 μL of Ti(SO4)2 solution (13.9 mM) and start timing immediately. Measure the absorbance at 405 nm every 5 minutes for 30 minutes. To investigate the effect of pH on catalase-like activity, repeat the above measurement process using different buffer systems in the pH range of 4.5-8.0. Prepare a mixture of 200 mM H2O2 and catalysts with concentrations of 2 μM ZnPC-Ru, 4 μM ZnPC, and 8 μM Ru-dbq in 20 mL PBS (pH = 7.4). O2 concentration was monitored using a dissolved oxygen meter (INESA, JPSJ-605F), with measurements recorded every 5 seconds until 600 seconds.

[0082] DPPH radical scavenging assay: Prepare a 50 μg / mL DPPH (2,2-diphenyl-1-trinitrophenylhydrazine) ethanol solution, mix an appropriate amount with 20 μL of stock solution, and adjust the final reaction volume to 2 mL. Incubate the mixture in the dark for 30 minutes, and then measure the absorbance at 517 nm using a microplate reader. The DPPH radical scavenging rate is calculated using the following formula: Scavenging rate (%) = [(A0 - A1) / A0] × 100%, where A0 is the absorbance of the blank control and A1 is the absorbance of the sample.

[0083] GSH consumption assay: Prepare 3 mg / mL DTNB (PBS) and 10 mM GSH solution. Mix 240 μL of DTNB solution and 30 μL of GSH solution, add ZnPC-Ru samples of different concentration gradients, and add PBS to bring the final volume to 1 mL. Stir magnetically at 25℃ for 60 minutes. After centrifugation, transfer 200 μL of the supernatant to each well in a 96-well plate. Use a microplate reader to scan the entire wavelength range of 250-500 nm and record the changes in the characteristic absorption peak at 412 nm.

[0084] NADH oxidation experiment: Prepare a 10 mM N-hydroxyethylpiperazine-2-ethanesulfonic acid (HEPES) buffer solution with pH=7.4. Weigh an appropriate amount of NADH and dissolve it in the HEPES buffer to obtain a 0.4 mM NADH solution. Add ZnPC-Ru of different concentrations to 2 mL of NADH solution (0.4 mM), allow the reaction to stand for 30 min, and measure the absorption peak (wavelength range 250-500 nm) using a UV spectrophotometer. Record the change in the characteristic absorption peak of NADH at 334 nm.

[0085] Mott-Schottky assay: An electrochemical workstation was used to measure the Mott-Schottky curve of the material, using a 0.5 M Na₂SO₄ aqueous solution (pH = 6.8) as the electrolyte. An ITO glass plate coated with the material served as the working electrode, platinum foil as the counter electrode, and saturated Ag / AgCl as the reference electrode. Mott-Schottky curves were measured at AC frequencies of 2000 Hz, 2500 Hz, and 3000 Hz. Working electrode preparation: 10 mg of photocatalyst, 1 mL of ethanol, and 10 μL of Nafion were mixed and sonicated for 20 min. 50 μL of the material solution was uniformly deposited onto an ITO glass plate (1 × 1 cm⁻¹). 2 Dry it under xenon lamp irradiation.

[0086] In-situ infrared spectroscopy: A catalyst / Nafion solution with a concentration of 10 mg / mL was prepared. The Nafion solution consisted of 210 μL isopropanol, 750 μL deionized water, and 40 μL Nafion. During the experiment, 40 μL of the catalyst solution was deposited onto the surface of ZnSe crystals, dried, and then the crystals were mounted in an in-situ cell. Subsequently, 5 mL of 0.5 M H2O2 solution was added to the in-situ cell, and in-situ FTIR spectra were collected at specific intervals, maintaining a reaction time of 10 minutes.

[0087] 2. Performance Test Results

[0088] After verifying the chemical structure of ZnPC-Ru, we then examined the material's ROS production activity under US stimulation. Figure 4 This paper demonstrates the detection of total free radical generation (ROS) in materials under acidic conditions with different feed ratios using 3,3',5,5'-tetramethylbenzidine (TMB) oxidation colorimetric assay. The material with a ZnPC-Ru feed ratio of 1:2 (unless otherwise specified, ZnPC-Ru in the accompanying figures refers to ZnPC-Ru 1:2 (ZnPC:Ru-dbq=2:1)) exhibited the best ultrasonic response to ROS generation, along with excellent CAT-like enzyme activity. Figure 4b). Furthermore, PC-Ru and ZnPC-Ru exhibit similar activities, demonstrating that the metal Zn at the phthalocyanine center has little impact on the material's activity, implying that the metal ions at the phthalocyanine center can be expanded.

[0089] Figure 5 This study demonstrates the preliminary detection of total free radical generation (ROS) in the material under acidic conditions using 3,3',5,5'-tetramethylbenzidine (TMB) oxidation colorimetric assay. Results show that, compared to Ru-dbq and ZnPC without sonication, ZnPC-Ru exhibits enhanced ROS production activity. With sonication, ZnPC-Ru shows the greatest improvement, increasing by 4.6 times. Furthermore, compared to commercial sonosensitive agents hematoporphyrin and curcumin, its sonodynamic activity is increased by 23 times. By detecting ROS generated by the material under US stimulation at different pH conditions, it was found that the ROS generation efficiency in an acidic environment (pH ≤ 6.5) is significantly higher than that under neutral and alkaline conditions. Figure 5 b) indicates that the ROS generation performance of this material is pH-dependent, and superior under acidic conditions. We then further tested the types of free radicals generated by the material, using sodium azide (NaN3), p-benzoquinone (BQ), and tert-butanol (TBA) to capture them through free radical quenching experiments. 1 O2, ·O2⁻, ·OH( Figure 5 c) The results show that under US conditions, the material mainly produces 1 O2 and ·O2⁻. Subsequently, we systematically detected reactive oxygen species generated under US irradiation using specific probes: singlet oxygen was detected using the 9,10-diphenylanthracene (DPA) probe. 1 O2)( Figure 5 d) Detection of superoxide anions (·O₂⁻) using a dihydroethidium (HE) probe. Figure 5 e). Furthermore, using electron paramagnetic resonance (EPR) technology with DMPO and TEMP as specific trapping agents, the effects of ultrasound stimulation were further confirmed. 1 Significant generation of O2 and ·O2⁻ ( Figure 5 f and 5g). Subsequently, the intermediates formed during the reaction were detected by in-situ Fourier transform infrared spectroscopy (f and 5g). Figure 5 h), proving that the intermediate ·OOH (1266 cm) -1 ), O2⁻ (1172 cm) -1 O2 (839 cm) -1 ) and adsorbed H2O2 (1385 cm) -1 The generation of intermediates is further visualized through contour line color-filled maps. Figure 5i). These results indicate that the supramolecular coordination complex, under ultrasonic irradiation, simultaneously undergoes both type I (electron transfer generating ·O₂⁻ radicals) and type II (energy transfer generating ·O₂⁻ radicals). 1 Reactive oxygen species (ROS) are generated via the O2 pathway. Of particular note is that, compared to free ZnPC, the formation of supramolecular structures significantly enhances sonodynamic activity, likely due to: 1) supramolecular assembly promoting intermolecular energy / electron transfer; and 2) the cage-like structure enhancing the energy capture efficiency of ultrasonic cavitation. This dual ROS generation mechanism is similar to the type I / II pathway observed in photodynamic therapy, but is achieved through ultrasound, a completely physical stimulation method, offering new possibilities for the treatment of deep tumors.

[0090] In the hypoxic environment of tumors and bacterial biofilms, the lack of oxygen often leads to unsatisfactory effects of sound-sensing agents. Therefore, considering the oxidoreductase activity exhibited by Ru-based compounds, we tested the CAT-like enzyme activity of the material. Hydrogen peroxide content was determined by Ti(SO4)2 colorimetry. Compared with Ru-dbq, ZnPC-Ru showed a significant improvement in hydrogen peroxide decomposition performance. Figure 6 a) Simultaneously, dissolved oxygen meter detection confirmed the generation of O2, with ZnPC-Ru exhibiting the highest O2 production ( Figure 6 b). As the pH of the buffer solution decreases, CAT performance declines, indicating that the optimal pH range for ZnPC-Ru CAT enzyme activity is 6.5-8. Figure 6 c). Simultaneously, we tested the material's antioxidant capacity under neutral conditions and found that it can efficiently scavenge DPPH free radicals (c). Figure 6 d).

[0091] Furthermore, considering the rich redox properties of Ru(II) ions, we tested whether ZnPC-Ru could promote the oxidation of glutathione (GSH) and reduced coenzyme I (NADH), thereby disrupting the redox balance in the microenvironment. We used 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a GSH indicator to confirm the ability of ZnPC-Ru to consume GSH. The results showed that ZnPC-Ru induced GSH consumption in a concentration-dependent manner. Figure 6 e) At a low concentration of 25 ug / mL, 62.6% of GSH was consumed, demonstrating that ZnPC-Ru has excellent GSH-consuming ability. The catalytic oxidation ability of ZnPC-Ru for NADH was studied by detecting the characteristic absorption peak (336 nm). Similarly, the characteristic absorption peak of NADH (336 nm) decreased with increasing material concentration. Figure 6 f) demonstrates that ZnPC-Ru induces NADH oxidation in a concentration-dependent manner.

[0092] To further investigate the mechanism by which the acoustically excited ROS performance of the ZnPC-Ru complex is significantly enhanced, we compared the band gaps of ZnPC-Ru and ZnPC. First, we performed UV spectroscopy measurements and converted the absorption spectral data based on the Kubelka-Munk equation to plot Tauc curves, as shown below. Figure 7 As shown in Figure a, the optical band gaps of ZnPC and ZnPC-Ru were determined to be 1.726 eV and 1.806 eV, respectively, by extrapolation. The band gap characteristics of the materials are crucial for the generation of reactive oxygen species (ROS) under ultrasonic (US) stimulation. Experimental results show that a narrower band gap is more conducive to US-induced ROS generation. Subsequently, Mott-Schottky tests were performed at frequencies of 2000, 2500, and 3000 Hz. Figure 7 b) and 7c) determine the flat band position (VFB) of the material, where the calculated VBF values ​​of ZnPC and ZnPC-Ru are -1.45 V and -0.59 V (vs Ag+ / Ag), respectively, based on the band structure E CB =V FB +E BG The conduction band positions E of both CB These can be calculated as -1.252 V and -0.392 V. The band structure diagram of ZnPC-Ru is shown below. Figure 7 As shown in Figure d, the conduction band positions of all materials are negative to the standard redox potential of the H₂O₂ / ·O₂⁻ pair (-0.33V vs NHE), which thermodynamically confirms the feasibility of ·O₂⁻ formation. It is noteworthy that only the valence band position of ZnPC-Ru simultaneously satisfies the ·O₂⁻ / ·O₂⁻ pair. 1 The oxidation potential requirement of the O2 pair (0.68 V vs NHE), this unique band structure feature explains its significantly enhanced singlet oxygen ( 1 O2) yield. We hypothesize that there may be O2⁻-oriented [O2⁻] in ZnPC-Ru. 1 The cascade reaction pathway of O2 conversion: First, US stimulation generates photogenerated electrons that reduce O2 to ·O2⁻; subsequently, ·O2⁻ is further converted into O2⁻ through an energy transfer process. 1 O2. Figure 7 e illustrates a possible reaction mechanism.

[0093] It should be noted that while the preferred embodiments of the present invention are given in the specification and accompanying drawings, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention specification. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A supramolecular coordination complex with acoustic dynamic activity, characterized in that, The supramolecular coordination complex is formed by coordination self-assembly of phthalocyanine compounds and Ru-dbq; The structure of the phthalocyanine compounds is shown in formula (I): (I)， Where M either does not exist or is Zn; The structure of Ru-dbq is shown in equation (II): (II); The coordination self-assembly is achieved by the coordination of the pyridine nitrogen in the phthalocyanine compound with the metallic Ru in the Ru-dbq; The molar ratio of the phthalocyanine compound to Ru-dbq is 1-2:1-3.

2. The supramolecular coordination complex according to claim 1, characterized in that, The supramolecular coordination complex has multiple functions, including acoustic-induced ROS production, CAT-like enzyme activity, antioxidant properties, and promotion of glutathione and reduced coenzyme I oxidation.

3. A method for preparing a sonodynamically active supramolecular coordination complex, characterized in that, Includes the following steps: (1) Under the protection of an inert gas, 3,4-pyridinedicarboxynitrile undergoes a cyclotetramerization reaction in the presence or absence of a metal salt in an alkaline catalyst and a high-boiling solvent to obtain a phthalocyanine compound as shown in formula (I); wherein the metal salt is a Zn salt. (2) Under the protection of an inert gas, bis(4-methylisopropylphenyl)ruthenium(II) and 2,5-dihydroxy-1,4-benzimidone were reacted in a weakly coordinating solvent to obtain Ru-dbq as shown in formula (II); (3) The phthalocyanine compound obtained in step (1) and Ru-dbq obtained in step (2) are self-assembled by coordination between the pyridine nitrogen in the phthalocyanine compound and the metal Ru in the Ru-dbq to obtain a supramolecular coordination complex with acoustic dynamic activity; The molar ratio of the phthalocyanine compound to Ru-dbq is 1-2:1-3.

4. The preparation method according to claim 3, characterized in that, The alkaline catalyst is 1,8-diazabicyclo[5.4.0]undec-7-ene; the high-boiling solvent is n-pentanol, dimethylaminoethanol, or quinoline; the metal salt is a Zn acetate or chloride salt; In the presence of a metal salt, the molar ratio of the 3,4-pyridinedicarboxynitrile to the metal salt is 3-4:1; The conditions for the cyclotetramerization reaction are: stirring at 150-180°C for 6-24 hours.

5. The preparation method according to claim 3, characterized in that, The molar ratio of dichlorobis(4-methylisopropylphenyl)ruthenium(II) to 2,5-dihydroxy-1,4-benzimidone is 1:0.8-1.2; the weakly coordinating solvent is an alcohol solvent, acetonitrile, or acetone. The reaction conditions in step (2) are: stirring at 25-60°C for 2-4 hours.

6. The preparation method according to claim 3, characterized in that, Step (3) includes: firstly removing chloride ions from Ru-dbq using Ag⁺, and then reacting the phthalocyanine compound dissolved in an organic solvent with stirring at 25-80°C for 12-36 h under an inert gas atmosphere.

7. The preparation method according to claim 6, characterized in that, The organic solvent is dimethylformamide.

8. The preparation method according to any one of claims 3-7, characterized in that, The prepared supramolecular coordination complex has multiple functions, including acoustic-induced ROS production, CAT-like enzyme activity, antioxidant properties, and promotion of glutathione and reduced coenzyme I oxidation.

9. The use of the supramolecular coordination complex as described in claim 1 or 2, or the supramolecular coordination complex prepared by the preparation method according to any one of claims 3-8, in the preparation of a somatosensitizer for sonodynamic therapy.

Images