Sound-sensitive agent based on metal-pyridine coordination structure as well as preparation method and application of sound-sensitive agent

Abstract

The invention belongs to the field of biological catalytic materials, particularly relates to a sound-sensitive agent based on a metal-pyridine coordination structure as well as a preparation method and application of the sound-sensitive agent, and more particularly relates to a sound-sensitive agent doped with second metal and based on the metal-pyridine coordination structure. Compared with other most reported sound-sensitive agents based on micromolecules, the sound-sensitive agent based on the metal-pyridine coordination structure and the doped second metal and based on the metal-pyridine coordination structure, especially the latter, has higher ultrasonic mediated < 1 > O2 generation activity; in addition, the effects of relieving oxygen deficit, promoting polarization of macrophages to M1 and enhancing immunotherapy are achieved, and therefore the tumor microenvironment of immunosuppression is reversed. When the sonosensitizer provided by the invention is used in combination with a PD-L1 antibody, satisfactory sonodynamic triggered synchronous immune activation is realized, and original and distant metastatic tumors are eradicated impressively.

Description

Technical Field

[0001] The invention belongs to the field of biocatalytic materials, and in particular relates to a sonosensitizer based on a metal-pyridine coordination structure and a preparation method and application thereof. Background Art

[0002] Immune checkpoint blockers programmed cell death protein-1 (anti-PD-L1) and its ligands can restore the ability of tumor-infiltrating cytotoxic T lymphocytes (CTLs) to kill tumor cells by binding to the corresponding receptors on tumor cells, showing great potential in tumor treatment. However, for tumors with suppressed tumor microenvironment and insufficient anti-tumor immune activation, such as triple-negative breast cancer, one of the most deadly cancers in women worldwide, the immunotherapy effect of immune checkpoint blocker treatment is poor. The key to achieving good immunotherapy effects lies in improving the immunogenicity of the tumor microenvironment. Cancer cell apoptosis can induce immunogenic cell death, and the released tumor-associated antigens, damage-associated molecular patterns, and proinflammatory cytokines have been shown to play a role in triggering the tumor microenvironment from immunosuppression to immune activation. On the other hand, improving the hypoxia state of the tumor microenvironment can promote the polarization of macrophages from anti-inflammatory subtypes to proinflammatory subtypes, thereby promoting the production of cytotoxic T lymphocytes and promoting anti-tumor immunotherapy. Therefore, enhancing the immunogenicity in tumor cells and reversing the immunosuppression of the tumor microenvironment are the best strategies for tumor immunotherapy.

[0003] Reactive oxygen species (ROS) can kill tumor cells and promote immunogenic death of tumor cells. At the same time, the regulation of reactive oxygen species can simply and effectively regulate the tumor microenvironment. These two effects together enhance the effect of immunotherapy. Among them, the sonodynamic therapy (SDT) developed in recent years, which uses ultrasound (US) to activate sonosensitizers to produce cytotoxic reactive oxygen species, has become an emerging non-invasive and in situ activation method for inducing or amplifying immune responses during cancer treatment, showing great clinical application potential.

[0004] Sonosensitizers play an important role in ultrasound immunotherapy. Like photosensitizers used in photodynamic therapy, sonosensitizers stimulated by ultrasound irradiation will undergo redox reactions with oxygen-containing substrates (such as oxygen and other oxygen-containing biomolecules) to produce reactive oxygen species or directly transfer energy to oxygen to produce singlet oxygen; the reactive oxygen species formed easily react with the phospholipid bilayer, destroy the cell membrane, and ultimately lead to cell apoptosis and immunogenic death. More importantly, since the ultrasound-mediated reactive oxygen generation mode has strong controllability, reactive oxygen species can be generated locally by applying ultrasound irradiation, minimizing the impact on normal tissues. More importantly, designing sonosensitizers with oxygen generation capabilities can not only improve the efficiency of reactive oxygen generation by increasing oxygen substrates, but also help reprogram the immunosuppressive tumor microenvironment by alleviating hypoxia and inhibiting the polarization of macrophages to proinflammatory subtypes, thereby increasing the recruitment of regulatory T lymphocytes. From the perspective of structural design, the reactive oxygen and oxygen generation properties of ultrasound sensitizers can be effectively improved through engineering, and ultrasound immunotherapy has broad application prospects in anti-tumor immunotherapy.

[0005] Most of the sonosensitizers currently used are composed of small organic molecules, such as porphyrins, phthalocyanines and their derived metal complexes. However, there are two major challenges in using them for tumor ultrasound immunotherapy: a) their highly conjugated structure is prone to aggregation-induced quenching, resulting in insufficient ultrasound energy conversion efficiency; b) due to the hypoxia of the tumor microenvironment, the reactive oxygen species generation performance is poor, resulting in low efficiency of immunogenic cell death. Therefore, the development of sonosensitizers with high ultrasound-mediated reactive oxygen species production activity is the key to achieving effective sonosensitizers. Similar to porphyrin or phthalocyanine molecules, the pyridine group exhibits strong complexation with transition metal ions, and the formed coordination structure is a non-planar structure, which is not easy to form π-π stacking and theoretically has a strong energy transfer efficiency. Despite the great development potential, there are still few reports on pyridine metal complex-based sonosensitizers. Summary of the invention

[0006] In order to solve the above problems, the present invention has designed a metal-pyridine coordination structure-based sonosensitizer in an original way. The metal is Au, Ru, Pd, Os, Ir, Pt, etc.; the pyridine ligand is tris(pyridin-4-yl)amine, tris(4-(pyridin-4-yl)phenyl)amine, 2,4,6-tris-(4-pyridinyl)pyridine, 2,4,6-tris(2-pyridinyl)triazine, etc. Further, in order to improve the generation of ultrasound-mediated reactive oxygen species, the present invention has designed a M2@M1-pyridine coordination structure-based sonosensitizer in an original way, wherein the M1-pyridine coordination polymer is the above-mentioned metal-pyridine coordination structure-based sonosensitizer (for the convenience of description, the metal is referred to as the first metal and marked as M1 in the following text), and M2 is the second metal, and M2 can be Os, Ru, Ir, Pt, etc.

[0007] Specifically, in one aspect, the present invention provides a sonosensitizer based on a metal-pyridine coordination structure, wherein the sonosensitizer is a coordination polymer formed by metal and a pyridyl-containing ligand through metal-N coordination, wherein the pyridyl-containing ligand includes tris(pyridin-4-yl)amine, tris(4-(pyridin-4-yl)phenyl)amine, 2,4,6-tri-(4-pyridyl)pyridine, 2,4,6-tris(2-pyridyl)triazine and other pyridyl-containing ligands of similar structures.

[0008] Furthermore, the metal in the coordination polymer exists in the form of metal nanoparticles.

[0009] Furthermore, the sonosensitizer further comprises a second metal doped in the coordination polymer.

[0010] Furthermore, the second metal is introduced into the coordination polymer by doping (lattice doping / lattice substitution, etc.) on the metal nanoparticles.

[0011] Furthermore, the first metal includes but is not limited to at least one of Au, Ru, Pd, Os, Ir, and Pt.

[0012] Furthermore, the second metal includes but is not limited to at least one of Os, Ru, Ir, and Pt.

[0013] In another aspect, the present invention provides a method for preparing a sonosensitizer based on a metal-pyridine coordination structure as described herein, characterized in that it comprises mixing a pyridine-containing ligand with a first metal compound and optionally a second metal compound in water to obtain the sonosensitizer.

[0014] More specifically, the present invention provides a method for preparing a sonosensitizer based on a metal-pyridine coordination structure as described herein, which comprises mixing a pyridine-containing ligand with a first metal compound in water to obtain the sonosensitizer.

[0015] Furthermore, the present invention provides a method for preparing a sonosensitizer based on a metal-pyridine coordination structure as described herein, which comprises mixing a pyridine-containing ligand with a first metal compound and a second metal compound in water to obtain the sonosensitizer.

[0016] Furthermore, the pyridyl-containing ligand includes tris(pyridin-4-yl)amine, tris(4-(pyridin-4-yl)phenyl)amine, 2,4,6-tri-(4-pyridyl)pyridine, 2,4,6-tris(2-pyridyl)triazine and other pyridyl-containing ligands of similar structures.

[0017] Furthermore, the first metal compound includes but is not limited to at least one of the salts of Au, Ru, Pd, Os, Ir or Pt, such as chloroauric acid.

[0018] Furthermore, the second metal compound includes but is not limited to at least one of salts of Os, Ru, Ir or Pt, such as osmium chloride.

[0019] Further, in the presence of the second metal compound, the molecular ratio of the second metal compound to the first metal compound in the blended solution is 3:7.

[0020] Furthermore, the molar ratio of the pyridyl-containing ligand to the metal ion in the blend solution is 1:0.5-4, preferably 2:3.

[0021] Furthermore, the reaction is carried out at room temperature for 3-6 hours.

[0022] Furthermore, the method also includes adding a polymer surfactant into the blended solution.

[0023] Furthermore, the polymer surfactant is polyvinyl pyrrolidone.

[0024] In another aspect, the present invention provides use of the metal-pyridine coordination structure-based sonosensitizer as described herein or the metal-pyridine coordination structure-based sonosensitizer prepared by the preparation method described herein in preparing a material having ROS generating activity.

[0025] Furthermore, the ROS generating activity is enhanced under ultrasound.

[0026] Further, the material comprises a catalase-like, peroxidase-like or oxidase-like material.

[0027] In another aspect, the present invention provides use of the sonosensitizer based on the metal-pyridine coordination structure as described herein or the sonosensitizer based on the metal-pyridine coordination structure prepared by the preparation method described herein in the preparation of a medicament for treating tumors.

[0028] Furthermore, the effect of the tumor treatment is enhanced under ultrasound.

[0029] In another aspect, the present invention provides a use of a sonosensitizer based on a metal-pyridine coordination structure as described herein or a sonosensitizer based on a metal-pyridine coordination structure prepared by the preparation method described herein in combination with a PD-L1 antibody in the preparation of a medicament for treating tumors.

[0030] Furthermore, the effect of the tumor treatment is enhanced under ultrasound.

[0031] Beneficial Effects of the Invention

[0032] The present invention successfully constructs a metal-pyridine coordination structure using pyridine ligands such as tris(pyridin-4-yl)amine, tris(4-(pyridin-4-yl)phenyl)amine, 2,4,6-tri-(4-pyridinyl)pyridine, 2,4,6-tris(2-pyridinyl)triazine, etc., and proves that the pyridine metal complex constructed in this way can be used as a sonosensitizer with excellent ROS generation activity.

[0033] More specifically, the present invention has demonstrated that the Os@Au-TPA and Au-TPA prepared by the present invention exhibit higher singlet oxygen production ( 1 O2) activity, especially the ultrasound-mediated production of singlet oxygen ( 1 Os@Au-TPA has an activity of about 41.6 times that of hexacarboxytriphenyl porphyrin (ce6), 19.5 times that of protoporphyrin (PpIX), 12.0 times that of indocyanine green (ICG), and about 11.1 times that of iron phthalocyanine (Pc(Fe)). Os@Au-TPA can not only produce abundant reactive oxygen species (ROS) for sonodynamic therapy (SDT) under ultrasound (US) irradiation, stimulate cell apoptosis and further immunogenic cell death, but also produce oxygen (O2) to relieve hypoxia, promote the polarization of M2 to M1 macrophages, and enhance the immunogenicity of tumors. Therefore, when used in combination with PD-L1 antibody, it reshapes the immunosuppressive tumor microenvironment and simultaneously achieves sonodynamically triggered immune activation, effectively eradicating primary and distant metastatic tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the synthesized Os@Au-TPA coordination structure is shown.

[0035] Figure 2 Structural characterization of Au-TPA and Os@Au-TPA is shown: a) SEM image of Au TPA and b) corresponding size distribution; c) SEM image of Os@Au-TPA and d) corresponding size distribution; e) FTIR spectrum; f) high-resolution XPS spectrum of N1s; g) the amounts of Au and Os calculated based on ICP-OES measurement; h) high-resolution XPS spectra of Au and i) Os; j) PXRD results.

[0036] Figure 3Shown are a) high-resolution transmission electron microscopy image of Au-TPA; b) micromorphology of Au nanoparticles; c) size statistics of Au nanoparticles; d) selected area electron diffraction pattern of Au-TPA; e) HR-STEM image of Os@Au-TPA; f) micromorphology of Os@Au nanoparticles; g) size statistics of Os@Au nanoparticles; h) selected area electron diffraction pattern of Os@Au-TPA; i) elemental distribution maps of Au-TPA and j) Os@Au-TPA.

[0037] Figure 4 Figure 2 shows the analysis of US-mediated ROS generation activity: a) Schematic diagram of the US-mediated ROS generation mechanism of Os@Au-TPA; b) Evaluation of the ROS generation activity of Os@Au-TPA by SOSG probe under US irradiation. 1 Generation of O2; c) Absorbance intensity around 526 nm; d) Generation of O2 by CAT-simulated activity; e) Band gap measured by solid UV-visible spectroscopy; f) Energy band diagram calculated based on band gap and XPS results; g) Comparison of Os@AuTPA with other commonly used ultrasound sensitizers by SOSG probe 1 h) UV-visible absorption spectrum and absorbance at 652 nm (pH 4.5) measured by TMB method; i) EPR spectrum showing the generation of ·OH using DMPO as a spin trapping agent. (n=3 independent replicates).

[0038] Figure 5 Shown is a UV-visible absorption spectrum showing OXD activity measured by the TMB method.

[0039] Figure 6 Shown are a) fluorescence images of [Ru(dpp)3]Cl2 expression after immunofluorescence staining (scale bar = 20 μm); c) MFI analysis and quantification. b) Fluorescence images of HIF-α expression after immunofluorescence staining (scale bar = 20 μm); d) MFI analysis and quantification. The results are expressed as mean ± SD, and the p values ​​were analyzed by one-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001.

[0040] Figure 7 Shown are a) Schematic diagram of the mechanism of producing ICD effect and activating DCs; b) Live / dead staining after different treatments; c) Representative flow cytometry images of 4T1 cell apoptosis rate after different treatments by Annexin V-FITC / PI staining; d) ROS detected by FCM; e) Fluorescence image of CRT expression after immunofluorescence staining (scale bar = 20 μm); f) Fluorescence image of HMGB1 expression after immunofluorescence staining (scale bar = 20 μm); g) Flow cytometry results of dendritic cell maturation.

[0041] Figure 8 Shown are a) representative flow cytometry plots of in vitro M2 macrophages (CD206+ in F4 / 80+) and c) corresponding quantitative data. b) in vitro M1 macrophages (F4 / 80 + CD86 + ) and d) corresponding quantitative data. In the above experiments, n = 3 for each group. The results are expressed as mean ± SD, and the p values ​​were evaluated by one-way analysis of variance (ANVA), *p < 0.05, **p < 0.01, **p < 0.001, ****p < 0.001, Os@Au-TPA (60 μg mL-1), US irradiation (1.0 MHz, 1.0 W cm -2 ,1min,30% duty cycle).

[0042] Fig. 9 Shown are a) schematic diagram of anti-tumor treatment of primary tumors; b) weight of primary tumors in each group; c, g) images of HIF-α immunostaining of in situ tumors and their quantitative analysis; d, h) images of TUNEL immunostaining of in situ tumors and their quantitative analysis; e, i) images of CRT immunostaining of in situ tumors and their quantitative analysis; f, j) images of HMGB1 immunostaining of in situ tumors and their quantitative analysis.

[0043] Fig.10 a) FCM detection of mature DCs (CD80 + CD86 + CD11c + ) and b) corresponding quantitative data; c) M1 macrophages (CD11b + F4 / 80 + CD86 + ) and M2 macrophages (CD11b + F4 / 80 + CD206 + ) Representative flow cytometry images and d) corresponding quantitative data; e) Treg T cells (CD45 + CD4 + ) Representative flow cytometry images and f) corresponding quantitative data; g) CD8 + T cells (CD45 + CD3 + ) Representative flow cytometry images and h) corresponding quantitative data; i) CD206 + and j) CD8 + Representative images of primary tumors after immunofluorescence staining (scale bar = 50 μm).

[0044] Fig.11The figure shows a) schematic diagram of in vivo anti-tumor treatment of distant tumors; b) heat map of distant tumor volume in each group; c) distant tumor weight at the end of treatment; d) mechanism of distant tumor immune activation; e) CD8 + T cells and CD4 + T cells (CD45 + CD3 + ) Representative flow cytometry images, f) CD8 + Corresponding quantitative data of T cells, h) CD4 + Corresponding quantitative data of T cells; g) Representative flow cytometry plots showing CD8 + T cells (CD45 + CD3 + ) and j) corresponding quantitative data; i) CD8 + GZM + Representative flow cytometry images of T cell activation and k) corresponding quantitative data. l) IFN-γ content in serum isolated from mice, n=4. m) TNF-α content in serum isolated from mice, n=4. DETAILED DESCRIPTION

[0045] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0046] Example: Synthesis of Au-TPA and Os@Au-TPA coordination structures

[0047] Taking advantage of the strong coordination tendency between metal ions and pyridine groups, a facile and green one-pot synthetic method was adopted to synthesize Au-TPA coordination structures in aqueous solution at room temperature, such as Figure 1 shown.

[0048] Preparation of Au-TPA: Pour 13.3 mg of chloroauric acid (HAuCl4) into an aqueous solution of 5.44 mg of tri(pyridin-4-yl)amine (TPA) and stir for about 30 minutes to allow the metal ions to completely coordinate with the TPA molecules; then add 10 mg of polyvinylpyrrolidone (PVP) and react for 4 hours to make the coordination structure morphology and size uniform; after purification and drying, yellow powder Au-TPA is obtained.

[0049] Preparation of Os@Au-TPA: Pour osmium chloride (OsCl3) and chloroauric acid (HAuCl4) in a molecular ratio of 3:7 into an aqueous solution of tri(pyridin-4-yl)amine (TPA) and stir for about 30 minutes to allow the metal ions to completely coordinate with the TPA molecules; then add polyvinylpyrrolidone (PVP) and react for 4 hours to make the coordination structure morphology and size uniform; after purification and drying, olive green powder Os@Au-TPA is obtained.

[0050] Experimental Example 1: Characterization of the coordination structures of Au-TPA and Os@Au-TPA

[0051] The Au-TPA and Os@Au-TPA prepared in Example 1 were both spherical, with sizes of 149.8 nm and 125.0 nm, respectively. Figure 2 ad). In a phosphate buffered saline solution simulating a biological environment, dynamic light scattering was performed to evaluate the particle size, which showed that the particle sizes of Au-TPA and Os@Au-TPA were 107.7 nm and 98.8 nm, respectively.

[0052] To determine the coordination structure, we performed FTIR measurements ( Figure 2 e). Obviously, the pyridine ring is at 1600-1400cm -1 The spectrum of the stretching vibrations associated with the pyridine ring is in the range of 995–833 cm -1 The plane bending peaks in the range show the most significant changes in the FTIR spectrum. TPA at 1578 cm -1 The peaks at 1500 and 1420 cm-1 belong to the C=N stretching vibration of the pyridine ring, while the peaks at 1500 and 1420 cm-1 belong to the C=N stretching vibration of the pyridine ring. -1 The peak at 3038 cm belongs to the C=C stretching vibration of the pyridine ring. -1 The peaks at 1349, 1295 and 1217 cm are also related to the CN vibration of the pyridine ring. -1 The three bands are due to the stretching of aromatic CN. - After the reaction, the energy bands associated with the pyridine ring decreased significantly and shifted to higher directions, to 3100 and 1600 cm -1 ; 1440, 1638 and 1075cm -1 A new band appeared at 1638 and 3441 cm -1 The broad line at is the characteristic peak of hydrogen bond water shearing and OH stretching vibration caused by the increased hydrophilicity of Au-TPA compared with pure TPA monomer. Os-TPA shows a similar but weaker spectrum to Au-TPA. However, it is difficult for Os ions to coordinate with TPA, and few products are generated under the same synthetic route. Therefore, Os@Au-TPA and Au-TPA show similar spectra ( Figure 2 e). The coordination structure was studied by XPS. From the N1s spectrum, it can be seen that the characteristic of pyridine N changes from 398.2eV of pure TPA monomer to 400.3eV of Au-TPA and Os@Au-TPA ( Figure 2 f), clearly indicating the formation of Au / Os-pyridinic N coordination structure. The high-resolution spectrum of gold can be divided into two peaks, the Au 4f / 2 region at 84.0, the yellow marker at 87.7 eV, and the blue marker at 87.7 eV, which are attributed to Au(0) and Au(I), respectively ( Figure 2 h), indicating that Au metal / Au ions form a coordination structure with TPA. On the contrary, doped Os does not have an obvious zero valence. 3+ and Os 4+ ( Figure 2 i) As mentioned above, it is difficult for Os ions to coordinate directly with TPA. 3+ AuCl4 is positively charged - The interaction between the negative valence and the high doping content detected by ICP-OES ( Figure 2 g), it can be considered that the doped Os is attached to the Au nanoparticles (Os@Au). In addition, the doping of Os has a slight effect on the Au-TPA coordination structure, and the Au(0) ratio increases from 44.2 to 66.6% ( Figure 2 h). The results show that the PXRD spectrum of Os@Au-TPA has peaks at 38.2 and 44.5, which belong to the (111) and (222) lattices of Au metal; while Au-TPA has no obvious metal peaks ( Figure 2 j). These results indicate that Au-TPA and Os@Au-TPA coordination structures were successfully synthesized.

[0053] In order to clarify the catalytic center, the fine morphology of Au-TPA and Os@Au-TPA was further studied by high-resolution scanning transmission electron microscopy (HR-STEM). The average particle sizes of Au-TPA and Os@Au-TPA were approximately 150.0nm and 132.0nm, respectively, which is consistent with the SEM results ( Figure 3 a, e). Due to the synergistic effect of TPA, e.g. Figure 3 As shown in b and f, Au or Os@Au particles are nanosized. Interestingly, the size of Au nanoparticles (Au NPs) in Os@Au-TPA (4.4 nm) is slightly larger than that in Au-TPA (3.2 nm, Figure 3 c, g). These indicate that the Os element has been successfully added, and the PXRD spectrum shows the characteristic peaks of Au, which explains the formation of crystals. Selected area electron diffraction (SAED) shows obvious bright spots. After calculation, we confirmed that they are Au NPs ( Figure 3d, h). Figure 3 Elemental mapping of Au (purple), N (blue), Cl (green), C (red), and Os (yellow), as shown in i, j, shows that the atoms under TPA confinement are uniformly distributed throughout the sphere.

[0054] Experimental Example 2: Ultrasound-mediated ROS generation performance of Au-TPA and Os@Au-TPA

[0055] In order to confirm whether Au-TPA and Os@Au-TPA can effectively enhance SDT ( Figure 4 a), we first studied the ROS generation activity under US irradiation. The singlet oxygen green fluorescent probe (SOSG) was first used to evaluate 1 O2 production. Os@Au-TPA produced under US irradiation 1 O2 is higher than Au-TPA( Figure 4 b); It is worth noting that Os@Au-TPA produced in the presence of hydrogen peroxide (H2O2) 1 O2 increased dramatically from 12,874 to 61,545, an increase of about 4.8 times, while Au-TPA showed no significant change ( Figure 4 b,c) To explain the obvious enhancement of Os@Au-TPA production by H2O2 1 To investigate the mechanism of O2 removal, we evaluated its catalase (CAT)-like activity, because CAT catalyzes the conversion of H2O2 to O2, providing the most important substrate for SDT. The CAT-like activity was evaluated by monitoring the decomposition of H2O2 and the generation of O2. In a time-dependent H2O2 scavenging experiment, Os@Au-TPA showed significantly improved H2O2 scavenging efficiency and O2 generation efficiency compared with Au-TPA ( Figure 4 d). Besides, it is well known that band gap plays an important role in SDT-induced ROS generation, and narrow band gap is preferred. To explain the mechanism of enhanced ROS generation by Os@Au-TPA, we performed solid UV-vis measurements on Os@Au-TPA and Au-TPA. The band gap of Au-TPA is about 4.7 eV, while that of Os@AuTPA is about 3.2 eV ( Figure 4 e, f), are much smaller, so Os@Au-TPA has a higher 1 O2 generation activity. However, the large increase in ROS generation in the presence of H2O2 illustrates the importance of the O2 substrate. In summary, we attribute the significant improvement in SDT performance to the reduction in the band gap and the abundant supply of oxygen. We further compared the US-mediated generation of Os@Au-TPA in the presence of H2O2. 1The activity of O2 is comparable to that of the most reported traditional sonosensitizers such as ce6, PpIX, ICG, and Pc(Fe) under the same conditions. Impressively, both Au-TPA and Os@Au-TPA showed higher 1 O2 generation performance; in particular, as measured by SOSG, Os@Au-TPA has a catalytic activity of about 41.6 times for ce6, about 19.5 times for PpIX, about 12.0 times for ICG, and about 11.1 times for Pc(Fe) ( Figure 4 g), indicating that the well-designed Os@Au-ATP-based sonosensitizer possesses efficient catalytic activity.

[0056] We further investigated the US-enhanced ROS generation activity of Os@Au-TPA through enzyme mimetic activity. Figure 4 h showed peroxidase (POD)-like activity. In the presence of H2O2, the catalytic efficiency of Os@Au-TPA was slightly higher than that of Au-TPA, as evidenced by the characteristic peak of blue oxidized-3,3',5,5'-tetramethylbenzidine (ox-TMB) at 652 nm. In order to determine the free radical signals generated during the catalytic process, electron paramagnetic resonance (EPR) measurements were performed using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a capture agent. Both Au-TPA and Os@Au-TPA showed typical hydroxyl radical (·OH) signals with an intensity of 1:2:2:1, but the enzyme-mimicking performance of Os@Au-TPA was significantly enhanced under US irradiation ( Figure 4 i). In addition, both Au-TPA and Os@Au-TPA exhibited excellent OXD-like oxidase (OXD) performance, which can catalyze the oxidation of oxygen into ROS, as indicated by the strong peak of ox-TEM at 652 nm ( Figure 5 ). In conclusion, our results validated our concept that Os@AU-TPA-based sonosensitizers exhibited efficient ROS generation performance under US irradiation.

[0057] Experimental Example 3: In vitro hypoxia alleviation, anti-tumor and immune activation effects of Au-TPA and Os@Au-TPA

[0058] We first investigated in vitro whether Au-TPA and Os@Au-TPA could alleviate hypoxia at the cellular level. We explored the production of O2 at the cellular level in vitro by cell-permeable [Ru(dpp)3]Cl2. Under normoxic conditions, the red fluorescence of [Ru(dpp)3]Cl2 was completely quenched ( Figure 6a, d). Under hypoxic conditions, the red fluorescence of [Ru(dpp)3]Cl2 was restored in 4T1 cells treated with PBS and Au-TPA, but it was still completely quenched in 4T1 cells treated with Os@Au-TPA. We further evaluated whether Os@Au-TPA could reverse hypoxia in 4T1 cells by in vitro HIF-1α fluorescence staining. Figure 6 As shown in b and c, no HIF-1α fluorescence signal was observed in 4T1 cells in the PBS group under normoxic conditions, and the HIF-1α fluorescence signal of 4T1 cells treated with PBS and Au-TPA under hypoxic conditions was high, while no HIF-1α fluorescence signal was observed in the Os@Au-TPA group. These results indicate that Os@Au-TPA treatment can alleviate the hypoxic state of 4T1 cells in vitro under hypoxic conditions and produce oxygen through CAT mimicking activity.

[0059] Encouraged by the excellent ROS generation performance of Os@Au-TPA under US induction, we then evaluated its in vitro antitumor therapeutic performance (the mechanism of which is Figure 7 a). The cytotoxicity of Os@Au-TPA biocatalyst to human umbilical vein endothelial cells (HUVECs) was first determined. During anti-tumor treatment, Os@Au-TPA showed good biocompatibility to normal tissue cells when the concentration was below 200ug / mL. In addition, the hemolysis rate of Os@Au-TPA was less than 5% even at a concentration of 200μg / mL. The above results indicate that Os@Au-TPA has ideal in vitro biocompatibility and biosafety.

[0060] Subsequently, we used the 4T1 cell model to conduct anti-tumor experiments on Os@Au-TPA to verify its biocatalytic killing ability under hypoxic conditions. First, calcein-AM and PI were used to co-stain the treated 4T1 cells. Neither the control group nor the US group showed obvious cell damage, while Au-TPA, Os@Au-TPA, Au-TPA+US, and Os@Au-TPA+US groups all showed varying degrees of cell damage. Compared with the other groups, the Os@Au-TPA+US group showed the most dead cells ( Figure 7 b). Flow cytometry (FCM) results also demonstrated that Os@Au-TPA combined with US irradiation can effectively kill 4T1 cells in vitro ( Figure 7 c). Subsequently, the intracellular ROS level was measured by 2,7-dichlorofluorescein diacetate (DCFH-DA) flow cytometry; compared with the Au-TPA+US group and other groups, the ROS intensity in the Os@Au-TPA+US group was higher ( Figure 7 d). Therefore, Os@Au-TPA can not only effectively alleviate the hypoxic state under US irradiation, but also increase the intracellular ROS level, leading to tumor cell apoptosis.

[0061] Considering its excellent tumor killing performance, we conducted a comprehensive evaluation of the immunohistochemical performance of Os@Au-TPA+US. First, the expression of calreticulin (CRT), high mobility group box 1 (HMGB1), and ATP markers were detected in 4T1 cells to verify the ICD activation pathway. Immunofluorescence was used to detect the exposure of CRT and HMGB1, and enzyme-linked immunosorbent assay (ELISA) was used to detect ATP. Figure 7 As shown in e, the CRT exposure in the Os@Au-TPA+US treatment group was much higher than that in the other groups. At the same time, due to the release from the cell nucleus into the culture medium, the HMGB1 exposure in the Os@Au-TPA+US treatment group was the lowest compared with the other groups ( Figure 7 f). In addition, the Os@Au-TPA+US group showed the highest extracellular ATP release compared with the other groups. Due to the important role of dendritic cells in innate and adaptive immunity, it should be activated and verified in vitro. We examined the activation of bone marrow-derived DCs after different treatments in vitro. The maturation of DCs was confirmed by flow cytometry (FCM) staining (CD86+CD80+), which are typical markers on the surface of mature DCs. As expected, the Os@Au-TPA+US group had the highest proportion of DC maturation ( Figure 7 g). The above results indicate that Os@Au-TPA-enhanced SDT effectively promoted the ICD effect, thereby accelerating the activation of DCs.

[0062] As is well known, the hyperpolarization of M2 macrophages is one of the main causes of immunosuppressive TME, so repolarizing the M2 subtype to the immunostimulatory M1 subtype has become a promising technique to reverse immunosuppression in cancer immunotherapy, and O2 provides a feasible way to favorably regulate the immunosuppressive hypoxic TME. In addition, it has been demonstrated that overexpressed ROS-induced oxidative stress also favors this conversion. Therefore, we used Os@Au-TPA+US to improve the polarization of macrophages from M2 to M1 phenotype due to its excellent O2 and ROS generation activities. RAW264.7 macrophages were treated with interleukin 4 (IL-4) to establish M2 macrophages, which were co-cultured with 4T1 cells treated differently in a transwell insert system under hypoxic conditions; the ratio of M2 and M1 macrophages was then analyzed by flow cytometry (FCM). The proportion of macrophages in the M2 group decreased significantly, while the proportion of macrophages in the M1 group increased significantly. When the Os@Au-TPA+US group was compared with the other groups ( Figure 8 ). These results suggest that Os@Au-TPA+US treatment can effectively stimulate M2 to M1 subtypes, thereby reversing the immunosuppressive TME.

[0063] Experimental Example 4: In vivo hypoxia relief and anti-tumor effects of Au-TPA and Os@Au-TPA

[0064] We evaluated the efficacy of Os@Au-TPA+US and aPD-L1 combined therapy in vivo for primary tumors in Balb / c female mice with 4T1 orthotopic tumors; the treatment route was as follows Fig. 9 Compared with other groups, the tumor weight of Os@Au-TPA+US+aPD-L1 group was the lowest ( Fig. 9 b), with the lowest volume (results not shown), indicating that the growth of primary tumors in this group was effectively inhibited. Afterwards, considering its efficiency in alleviating hypoxia in vitro, we evaluated the performance of Os@Au-TPA in improving tumor hypoxia. Fig. 9 As shown in c, the HIF-1α level in the Os@Au-TPA group was significantly reduced, the immunofluorescence of tumor sections was reduced, and the MFI quantitative analysis was as shown in Fig. 9 As shown in g, Os@Au-TPA significantly improved the tumor hypoxic microenvironment.

[0065] Hematoxylin and eosin (H&E) results showed that Os@Au-TPA+US and Os@Au-TPA+US+aPD-L1 treatments destroyed most tumor cells. In addition, we stained tumor blood vessels with CD31 antibodies to verify the effects of different treatments on tumor angiogenesis. The Os@AuTPA+US+aPD-L1 group had the least brown areas in the tumor sections, indicating that its activity in inhibiting vascular proliferation was prominent. We then performed terminal deoxynucleotidyl transferase dUTP nickel-end labeling (TUNEL) staining to explore the level of cell apoptosis. Obviously, the Os@Au-TPA+US+aPD-L1 treatment group had the strongest green fluorescence signal and the strongest fluorescence quantitative intensity ( Fig. 9 d,h), indicating a higher rate of tumor apoptosis.

[0066] Inspired by the high ICD activation performance in vitro, we also evaluated the expression of CRT exposure and HMGB1 release in vivo. The Os@Au-TPA+US+aPD-L1 group had the highest signal ( Fig. 9 e), CRT quantitative MFI as Fig. 9 i. At the same time, the HMGB1 signal of this group is also the strongest among all groups ( Fig. 9 f, j). During the treatment period, the body weight of mice did not change significantly. The potential toxicity evaluation of the main organs of mice revealed its in vivo biosafety; H&E staining showed no obvious tissue damage; in addition, serum biochemical results showed no significant damage to kidney and liver function (data not shown). In summary, we expect Os@Au-TPA+US+aPD-L1 to be effective and safe in anti-tumor.

[0067] Experimental Example 5: SDT using Os@Au-TPA and in vivo immune activation of aPD-L1

[0068] Given its significant hypoxia-relieving and anti-tumor effects, we studied the corresponding in situ tumor immune activation in vivo. After various treatments, the primary tumor lymph node draining lymph was collected to obtain single cell suspensions, and flow cytometry (FCM) was used to detect DC (CD80 + CD86 + ) The proportion of mature DCs in the Os@AuTPA+US+aPD-L1 group was the highest among all groups ( Fig.10 a, b), indicating that the combination of SDT and aPD-L1 can effectively activate the anti-tumor immune response in vivo. In addition, Os@Au-TPA+US treatment can effectively improve the M1 / M2 ratio of the TME that reverses immunosuppression, thereby amplifying the reversal effect of the Os@Au-TPA+US+aPD-L1 group in vivo ( Fig.10 c, d). It has been demonstrated that innate immune suppression in the TME overexpresses Tregs (CD4 + Foxp3 + ), thereby hindering the immune response. Impressively, the CD4 + Treg T cells decreased the most in all groups ( Fig.10 e,f), indicating successful TME reprogramming. We then evaluated the T cell population in tumor tissues and further assessed immune activation by flow cytometry. After Os@Au-TPA+US treatment, CD8+ T cells in tumor tissues increased significantly, which was further promoted after combination with aPD-L1 ( Fig.10 g, h). Immunofluorescence staining also confirmed this activation. Fig.10 As shown in i, the CD206 immunofluorescence signals in the Os@Au-TPA+US and Os@Au-TPA+US+aPD-L1 groups were significantly less than those in the other groups, especially in the Os@Au-TPA+US+aPD-L1 group. + T cells were the most abundant, which was consistent with the results of flow cytometry ( Fig.10 j). In summary, Os@Au-TPA+US+aPD-L1 treatment can significantly reverse the immunosuppressive TEM and induce the infiltration of CTLs within tumors, thereby promoting immunotherapy.

[0069] Experimental Example 6: In vivo immune activation of distant tumors using SDT of Os@Au-TPA and aPD-L1

[0070] Ideal anti-tumor therapy requires not only effective elimination of the primary tumor, but also successful colonization of distant tumors, and immunotherapy is expected to be one of the most effective methods to inhibit tumor metastasis. Therefore, we explored the therapeutic effect of our therapeutic strategy on tumor metastasis and elucidated the mechanism behind it. A bilateral orthotopic tumor model was established, in which 4T1 cells were subcutaneously inoculated on both sides of the chest pad of Balb / c mice every 5 days to observe its anti-distant tumor performance. Os@Au-TPA was then injected into the primary tumor, followed by US irradiation and intraperitoneal injection of aPD-L1( Fig.11 a). The results showed that the body weight of mice did not change significantly during the treatment period. The distal tumors of each group were removed and collected on day 14. As expected, the Os@Au-TPA+US+aPD-L1 group showed the smallest distal tumors, while the group receiving aPD-L1 alone showed distal tumors that were almost the same as those of the control group ( Fig.11 b, c). These results suggest that aPD-L1 inhibition alone is poor, whereas Os@Au-TPA combined with US can effectively inhibit the growth of distant tumors.

[0071] Subsequently, we investigated whether Os@Au-TPA+US+aPD-L1 treatment could effectively activate systemic immune responses ( Fig.11 d). The spleen is an important peripheral immune organ and plays a vital role in systemic immunity. Therefore, the CTL cells in the spleen are worth evaluating. Fig.11 As shown in e, f, and h, CD8 + T cells accounted for the highest proportion; at the same time, CD4 + The proportion of T cell infiltration was significantly reduced, indicating that the spleen immune response was further activated. These results show that the treatment method of enhancing SDT combined with aPD-L1 can activate the immune response through CTL expansion and proliferation of peripheral immune organs.

[0072] In addition, whether Os@Au-TPA-mediated sonodynamic immunotherapy can induce the activation and proliferation of CTL cells in distant metastatic tumors, and the mechanism of combined killing of CTL cells with aPD-L1 were also explored. Flow cytometry was used to observe the proportion of CTL cells in distant tumors. The proportion of CD8+T cells in the Os@Au-TPA+US group increased significantly, indicating that Os@Au-TPA-mediated sonodynamic immunotherapy can promote the proliferation of CTL cells in distant tumors ( Fig.11 g, j). When combined with aPD-L1, CD8 +T cells further increased; Granzyme B (GZMB) secreted by cTL cells has an important killing effect on tumor / target cells, so we used flow cytometry (FCM) to evaluate it. Obviously, CD8 + GZM + The proportion of T cells was the highest, indicating that it has a higher efficiency in reducing TEM immunosuppression and enhancing CTL immune killing ( Fig.11 i, k). At the same time, the secretion levels of tumor necrosis factor-α and IFN-γ were detected by enzyme-linked immunosorbent assay to evaluate the tumor cooperative immune response. Fig.11 As shown in l and m, after Os@Au-TPA+US treatment, both cytokines were upregulated, and the immune response was further enhanced after combination with aPD-L1. The above results indicate that Os@AuTPA+US+aPD-L1 treatment can activate the immune response of distal tumors, promote the proliferation of CTLs, and alleviate the immunosuppression of distal tumors.

[0073] In summary, we synthesized Au-TPA and Os@Au-TPA, sonosensitizers based on metal-pyridine coordination structures, for the first time and explored their applications in anti-tumor sonoimmunotherapy. Au-TPA and Os@Au-TPA, especially Os@Au-TPA, are simple metal-pyridine coordination structures but are highly efficient sonosensitizers that show effective US-mediated ROS generation to fully realize SDT. In particular, its generation 1 O2 has significantly higher performance than conventional sonosensitizers, such as ~41.6-fold generation for ce6, ~19.5-fold generation for PpIX, ~12.0-fold generation for ICG, and ~11.1-fold generation for Pc(Fe). Moreover, it can generate O2 from H2O2, which not only provides sufficient substrate for SDT but also relieves hypoxia and promotes polarization of M2 to M1 macrophages. Impressively, the combination of US-enhanced ROS and tumor microenvironment reprogramming by Os@Au-TPA can ensure satisfactory eradication of primary tumors and distant metastases when synergistically acting with aPD-L1.

[0074] It should be noted that the preferred embodiments of the present invention are given in the specification of the present invention, but the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not intended to be additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the present invention specification; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims

1. A sonosensitizer based on a metal-pyridine coordination structure, characterized in that: The sonosensitizer is a coordination polymer formed by metal and pyridine-containing ligands through metal-N coordination. The pyridyl-containing ligand includes tris(pyridin-4-yl)amine, tris(4-(pyridin-4-yl)phenyl)amine, 2,4,6-tris-(4-pyridinyl)pyridine or 2,4,6-tris(2-pyridinyl)triazine.

2. The sonosensitizer according to claim 1, characterized in that: The metal in the coordination polymer exists in the form of metal nanoparticles.

3. The sonosensitizer according to claim 1 or 2, characterized in that: The sonosensitizer further comprises a second metal doped in the coordination polymer.

4. The sonosensitizer according to claim 3, characterized in that: The second metal is introduced into the coordination polymer by doping onto the metal nanoparticles.

5. The sonosensitizer according to any one of claims 1 to 4, characterized in that The metal includes at least one of Au, Ru, Pd, Os, Ir, and Pt; Furthermore, the second metal includes at least one of Os, Ru, Ir, and Pt.

6. A method for preparing a sonosensitizer based on a metal-pyridine coordination structure according to any one of claims 1 to 5, characterized in that: The method comprises mixing a pyridyl-containing ligand with a first metal compound and optionally a second metal compound in water to react and prepare the sonosensitizer.

7. The preparation method according to claim 6, characterized in that: In the presence of the second metal compound, the molecular ratio of the second metal compound to the first metal compound in the blended solution is 3:7; Further, the molar ratio of the pyridyl-containing ligand to the metal ion in the blend solution is 1:0.5-4, preferably 2:3; Further, the reaction conditions are to react at room temperature for 3-6 hours; Furthermore, the preparation method further comprises adding a polymer surfactant into the blend solution; Furthermore, the polymer surfactant is polyvinyl pyrrolidone.

8. Use of the metal-pyridine coordination structure-based sonosensitizer according to any one of claims 1 to 5 or the metal-pyridine coordination structure-based sonosensitizer prepared by the preparation method according to any one of claims 6 to 7 in the preparation of a material having ROS generating activity; Further, the ROS generating activity is enhanced under ultrasound; Further, the material includes a catalase-like, peroxidase-like or oxidase-like material.

9. Use of the sonosensitizer based on a metal-pyridine coordination structure according to any one of claims 1 to 5 or the sonosensitizer based on a metal-pyridine coordination structure prepared by the preparation method according to any one of claims 6 to 7 in the preparation of a medicament for treating tumors; Furthermore, the effect of the tumor treatment is enhanced under ultrasound.

10. Use of the sonosensitizer based on a metal-pyridine coordination structure according to any one of claims 1 to 5 or the sonosensitizer based on a metal-pyridine coordination structure prepared by the preparation method according to any one of claims 6 to 7 in combination with PD-L1 antibody in the preparation of a medicament for treating tumors; Furthermore, the effect of the tumor treatment is enhanced under ultrasound.

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