A biological catalyst based on palladium monatomic atom, a biological catalytic complex thereof, and a preparation method and application thereof

Abstract

The application belongs to the field of biological catalytic materials, and particularly relates to a biological catalyst based on a palladium single atom, a biological catalytic complex thereof, and a preparation method and application thereof. A coordination complex (Pd-Tpb) based on a palladium single atom is designed from scratch, and it is found for the first time that the coordination complex can be used as a biological catalyst with ROS generation activity and tumor killing performance with radioenhancement. On this basis, the Pd-Tpb is used to modify lactobacillus probiotics, and the obtained biological catalytic complex is used for biological catalysis and radiosensitization treatment, and it is found that the biological catalytic complex has a significantly improved radiosensitization effect and has the effect of eradicating primary and metastatic tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological catalytic materials, and particularly relates to a biological catalyst based on a palladium single atom, a biological catalytic complex thereof, and a preparation method and application thereof. BACKGROUND

[0002] Cancer is the leading cause of death worldwide, causing millions of deaths each year, and poses a major challenge to global public health. Radiotherapy (RT) as a widely used cancer treatment, produces reactive oxygen species (ROS) and induces double-strand DNA breaks, and shows significant efficacy in refractory, recurrent and metastatic tumors when combined with immune checkpoint inhibitors, marking a major breakthrough in cancer treatment. However, radiation resistance, tumor heterogeneity and tumor microenvironment (TME) and other factors still pose a serious challenge to the clinical effect of radiotherapy-based immune checkpoint inhibitor therapy in some solid tumors. Recent studies have shown that developing radiation-sensitive drugs or materials to simultaneously activate local and systemic immune responses can maximize the killing effect on primary, locally recurrent and metastatic tumors. However, the currently developed radiosensitizing materials (such as lanthanides, hafnium and gold) have insufficient tumor killing effect and poor biodegradability, which may lead to chronic toxicity, limiting their clinical translation potential.

[0003] There are two major challenges in designing a new generation of radio-activated materials to meet the clinical needs of tumor radiotherapy: 1) developing radiosensitizing materials with high biocompatibility and biodegradability to enhance the energy deposition and reactive oxygen species (ROS) generation of X-ray radiation; 2) alleviating radioresistance, reducing drug dosage and activating systemic anti-tumor response by enhancing ROS, thereby maximizing therapeutic effect and preventing metastasis and recurrence. In recent years, artificial peroxidases that can generate ROS in the TME rich in acid and hydrogen peroxide have become increasingly popular, such as metal oxides, metal hydroxides and metal-coordinated carbon nanomaterials. However, due to the complex multi-electron reactions involving hydrogen peroxide molecules and oxygen radicals, the complex formation of intermediate bonds, and the energy-intensive desorption of oxygen species, it is still challenging to develop high-performance ROS artificial peroxidases. In addition, most artificial peroxidases contain a large amount of inorganic or metal components, which raises concerns about their biological safety as a new generation of radiosensitizing materials.

[0004] However, the ability of these nanoparticles to target the tumor microenvironment is still limited by low blood perfusion and active penetration of vascular endothelial cells into tumors. There is an urgent need for a more effective and safer tumor treatment regimen that can effectively target tumor sites and rapidly remodel the tumor immune microenvironment. SUMMARY

[0005] To solve the above problems, the application designs a coordination complex based on a palladium single atom, which is found to be used as a biological catalyst and has a radio-enhanced ROS generation activity and tumor killing performance. In addition, the application also utilizes the characteristics of lactic acid bacteria that can target the hypoxic tumor microenvironment (TME) to reduce the pH value in the TME, and the mechanism that acidic conditions can enhance the ROS generation efficiency of the biological catalyst, and loads the coordination complex based on the palladium single atom on lactic acid bacteria, so as to realize excellent tumor targeting and treatment effect.

[0006] Specifically, in the first aspect, the application provides a biological catalyst based on a palladium single atom, which is a coordination complex formed by Pd salt and 1,3,5-tris(pyridin-4-yl)benzene through Pd-N coordination.

[0007] Further, the Pd salt is a Pd salt containing [PdCl4] 2- .

[0008] Further, the Pd salt containing [PdCl4] 2- is Na2PdCl4.

[0009] Further, Pd is distributed in the form of a single atom in the biological catalyst.

[0010] Further, the biological catalyst has a radio-activated ROS generation property.

[0011] In the second aspect, the application provides a preparation method of the biological catalyst as described herein, which comprises reacting a Pd salt and 1,3,5-tris(pyridin-4-yl)benzene in an acidic aqueous solution to obtain the biological catalyst.

[0012] Further, the molar ratio of the Pd salt and 1,3,5-tris(pyridin-4-yl)benzene is 1-3:1.

[0013] Further, the Pd salt is a Pd salt containing [PdCl4] 2- .

[0014] Further, the Pd salt containing [PdCl4] 2- is Na2PdCl4.

[0015] Further, the reaction conditions are stirring the reaction at room temperature for 10-60 min.

[0016] Further, the acidic aqueous solution comprises 0.01M-0.1M hydrochloric acid.

[0017] Further, the preparation method further comprises adding a high molecular surfactant in the reaction system.

[0018] Further, the high-molecular surfactant is polyvinylpyrrolidone.

[0019] Further, the preparation method comprises dissolving 1,3,5-tris(pyridin-4-yl)benzene and a high-molecular surfactant in an acid to form solution A; dissolving a Pd salt in water to obtain solution B; then mixing and reacting solution A and solution B under stirring, collecting the reaction product, and washing and drying to obtain the biological catalyst. Further, the stirring speed is 200-800 rpm. Further, the reaction condition is stirring at room temperature for 10-60 min.

[0020] In a third aspect, the present application provides a biological catalysis complex comprising a lactobacillus and a biological catalyst as described herein supported on the surface thereof.

[0021] Various methods for supporting the biological catalyst on the surface of the lactobacillus are well known to those skilled in the art, for example, the biological catalyst as described herein can be co-incubated with the lactobacillus, and the lactobacillus supporting the biological catalyst on the surface can be obtained.

[0022] Further, the biological catalysis complex has the ROS production property activated by radiation.

[0023] Further, the biological catalysis complex can activate the immune response and the immune memory effect.

[0024] In a fourth aspect, the present application provides use of the biological catalyst or the biological catalysis complex as described herein in the preparation of a material having ROS production activity.

[0025] Further, the ROS production activity is enhanced under radiation treatment.

[0026] In a fifth aspect, the present application provides use of the biological catalyst or the biological catalysis complex as described herein in the preparation of a medicament for primary and metastatic tumor treatment.

[0027] Further, the effect of the tumor treatment is enhanced under radiation treatment.

[0028] Further, the medicament can activate the immune response and the immune memory effect.

[0029] In a sixth aspect, the present application provides use of the biological catalysis complex as described herein in combination with an immune checkpoint inhibitor in the preparation of a medicament for tumor treatment.

[0030] Further, the effect of the tumor treatment is enhanced under radiation treatment.

[0031] Further, the immune checkpoint inhibitor is a PD-L1 antibody.

[0032] Advantages of the present application

[0033] The present application designs a coordination complex based on palladium monatomic atom (referred to as Pd-Tpb or Pd-Tpb biocatalyst in the present specification) from scratch and first discovers that it can be used as a biocatalyst with ROS generation activity and tumor killing performance with radioenhancement. On this basis, the present application uses Pd-Tpb to modify lactobacillus probiotics, and uses the obtained biocatalytic complex (referred to as LA@Pd-Tpb or LA@Pd-Tpb biocatalyst in the present specification) for biocatalysis and radiosensitization treatment to eradicate primary and metastatic tumors. The probiotics can promote the targeted positioning and retention of the biocompatible biocatalyst, thereby generating active oxygen activated by radioactivation. The increased active oxygen generated by the biocatalyst activated by radioactivation can intensify cell membrane and DNA damage, trigger apoptosis through oxidative damage, significantly improve radiosensitization effect, and activate CD8 + T cells. When used in combination with anti-PD-1 therapy, this synergy produces a strong systemic anti-tumor response in female mice, promotes distant effects and establishes durable tumor immune memory, thereby reducing the risk of recurrence and metastasis. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Structural characterization of Pd-Tpb is shown: (a) scanning electron microscope (SEM) image; (b) energy dispersive spectroscopy (EDS) spectrum; (c) atomic resolution high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) imaging; (d) X-ray diffraction (XRD) analysis; (e) Pd 3d XPS analysis; (f) N 1s XPS analysis; (g) Cl 2p XPS analysis; (h) X-ray absorption near-edge structure (XANES) spectroscopy analysis; (i) k2-weighted Fourier transform (FT) spectrum in R space; (j) k-space fitting curve; (k) R-space fitting curve; (l) Pd K-edge wavelet transform analysis of Pd metal, (m) PdO and (n) Pd-Tpb; (o) schematic diagram of the coordination structure of Pd-Tpb.

[0035] Figure 2 Biocatalytic performance of Pd-Tpb is shown: (a) radioenhanced ROS biocatalytic activity under TME conditions at pH = 6.5; (b) radioenhanced ROS biocatalytic activity under TME conditions at different pH; (c) electron paramagnetic resonance (EPR) spectrum; (d) scanning electron microscope image of LA@Pd-Tpb.

[0036] Figure 3In vitro biocatalysis and radiolytic activation anti-tumor effects of LA@Pd-Tpb biocatalyst are shown: (a) fluorescence images of reactive oxygen species generation level of CT26 cells after different treatments and (b) relative mean fluorescence intensity (n=3 independent replicates). Scale bar = 50 pm; (c) pH values of culture medium after 48 hours of each treatment group; (d) probiotic colony images of LA and LA@Pd-Tpb; (e) cell cycle proportion of CT26 cells after different treatments (n=3 independent replicates); (f) y-H2AX positive cells of CT26 cells after different treatments (n=3 independent replicates) and (g) corresponding fluorescence images; (h) apoptosis proportion of live and dead cells of CT26 of each treatment group (n=3 independent replicates); (i) apoptosis cell proportion analyzed by flow cytometry (n=3 independent replicates); (j) fluorescence images of live and dead cells of CT26 of each treatment group; (k) images of CT26 cells after Annexin V-FITC / PI staining of different treatment groups. Pd-Tpb concentration: 80 pg mL −1 Statistical significance was calculated by Student's t-test for comparisons between two groups, and one-way ANOVA for comparisons among multiple groups, followed by two-tailed Tukey's post-hoc test to confirm the results of multiple comparisons. ** indicates p<0.01, *** indicates p<0.001.

[0037] Figure 4(a) Schematic diagram of CT26 tumor model construction and intratumoral treatment schedule; (b) Near-infrared fluorescence imaging results of mice at different time points (4, 8, 12 and 24 hours) after administration; (c) Cy7 fluorescence intensity in different organs and tumor tissues of mice injected with Pd-Tpb and LA@Pd-Tpb; (d) Accumulation of LA@Pd-Tpb in different tissues 4 hours after intravenous injection; (e) Average body weight of each group (n=5 independent repeats); (f) Representative photos of tumors in each group on day 12 after treatment (n=5 biologically independent mice per group); (g) CT26 tumor growth curves of each group (n=5 independent repeats); (h) Average tumor weight of each group on day 12 after treatment (n=5 independent repeats); (i) Representative results of H&E staining of tumors after different treatments, H&E experiments were repeated three times independently; (j) TUNEL immunofluorescence images of tumors after different treatments and their corresponding semi-quantitative analysis results (m), immunofluorescence experiments were repeated three times independently, scale bar = 50 pm; (k) Ki-67 immunofluorescence images of tumors after different treatments and their corresponding semi-quantitative analysis results (n), immunofluorescence experiments were repeated three times independently, scale bar = 50 pm shown in the figure; (l) CD31 immunofluorescence staining images of tumors after different treatments and their corresponding semi-quantitative analysis results (o), immunofluorescence experiments were repeated three times independently, scale bar = 50 pm shown in the figure. The results are expressed as mean ± standard deviation. Statistical significance was compared between groups by one-way ANOVA, followed by two-tailed Tukey's post-hoc test for multiple comparisons. **: p<0.01, ***: p<0.001.

[0038] Figure 5 (a) Flow cytometry sorting detection of live / dead intratumoral lymphocytes (n=3 independent repeats); (b) Flow cytometry sorting detection of CD4 + and CD8 + intratumoral lymphocytes (n=3 independent repeats); (c) Flow cytometry sorting detection of CD206 + and CD86 + intratumoral lymphocytes (n=3 independent repeats); (d-f) Quantitative analysis results after different treatments (n=3 independent repeats); (g) IL-6, IFN-β, IFN-γ and TNF-β levels in serum after treatment of CT26 cancer cells (n=3 independent repeats). Data are expressed as mean ± standard deviation, and statistical significance was calculated using Student's t-test. *: p<0.05, **: p<0.01; ***: p<0.001.

[0039] Figure 6The synergistic effect of biocatalysis and radio-activated LA@Pd-Tpb combined with immune checkpoint blockade therapy against tumor metastasis is shown: (a) Experimental flow chart showing the establishment of CT26 tumor model and the treatment scheme after intratumoral administration; (b) Representative photos of primary tumors and distant metastases of each group on day 10 after treatment (5 biologically independent mice per group); (c) Primary tumor growth curve of each group of CT26 tumor-bearing mice (5 biologically independent mice per group); (d) Average weight of primary tumors of each treatment group on day 10 after treatment (5 biologically independent mice per group); (e) Distant metastasis growth curve of each group of CT26 tumor-bearing mice (5 biologically independent mice per group); (f) Average weight of distant metastases of each treatment group on day 10 after treatment (5 biologically independent mice per group); (g) TUNEL immunofluorescence images and (h) corresponding semi-quantitative analysis (3 independent repeated experiments), scale bar = 50 pm; (i) Ki-67 immunofluorescence images and (j) corresponding semi-quantitative analysis (3 independent repeated experiments), scale bar = 50 pm; (k) CD31 immunofluorescence images and (l) corresponding semi-quantitative analysis (3 independent repeated experiments), scale bar = 50 pm; (m) Gamma-H2AX fluorescence images and (n) corresponding semi-quantitative analysis (3 independent repeated experiments), scale bar = 50 pm. Fluorescence image data are from representative results of three independent repeated experiments. Experimental data are expressed as mean ± standard deviation. Statistical significance analysis uses Student's t-test for comparison between two groups, and one-way analysis of variance (ANOVA) for comparison among multiple groups, supplemented by two-tailed Tukey post-hoc test for multiple comparisons. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

[0040] Figure 7 Whole-body immune activation analysis after LA@Pd-Tpb + X-ray combined with immune checkpoint inhibitor therapy is shown: (a) CD4 + and CD8 + tumor interstitial lymphocyte flow cytometry (n = 3 independent repeats); (b) CD69 + tumor interstitial lymphocyte flow cytometry (n = 3 independent repeats); (c) CD206 + and CD86 + tumor interstitial lymphocyte flow cytometry (n = 3 independent repeats); (d) CD8 + PD-1 + tumor interstitial lymphocyte detection (n = 3 independent repeats); (e-h) Corresponding semi-quantitative analysis (n = 3 independent repeats); (i-l) Serum IL-6, IFN-β, IFN-γ and TNF-β levels detected after CT26 cancer cell treatment (n = 3 independent repeats); (m) CD4 + and CD8 +Representative multicolor immunofluorescence images of lymphocytes, which are representative results of two independent repeat experiments; (n) Representative multicolor immunofluorescence images of F4 / 80, CD163 + and CD80 + Representative multicolor immunofluorescence images of lymphocytes, which are representative results of two independent repeat experiments. Data are presented as mean ± s.d. Statistical significance was determined by Student's t-test for two-group comparisons, and one-way ANOVA followed by two-tailed Tukey's post-hoc test for multiple comparisons. *: p < 0.05, **: p < 0.01. DETAILED DESCRIPTION

[0041] The present application is further described in conjunction with the following specific examples, which in no way should be interpreted as being limiting. The reagents, methods, and apparatuses employed in the present application are routine in the art unless specifically indicated otherwise.

[0042] The materials and experimental methods employed in the examples and test examples are described below.

[0043] Ethanol and 2,2,6,6-tetramethylpiperidine (TEMP) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China); palladium(II) chloride sodium salt (Na2PdCl4) was purchased from Energy Chemical (Shanghai, China); 1,3,5-tris(pyridin-4-yl)benzene (Tpb) was purchased from Yunnan Pharmaceutical Group (Jilin, China); anti-mouse PD-1 antibody was purchased from Biocan Biotech (Beijing, China); all other chemicals were used directly without purification. Deionized water (conductivity 18.2 MΩ·cm) used in the experiments was prepared by a Milli-Q Academic system (Millipore, Billerica, MA, USA).

[0044] The generation of O2was evaluated using 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMP). 1 The mixture was prepared by adding catalyst (10 μL, 10 mg mL −1 ) and hydrogen peroxide (10 μL, 10 M) into NaOAc / HOAc buffer (500 μL, 100 mM, pH 4.5), followed by the addition of 20 μL of TEMP.

[0045] Cell lines: Mouse colorectal cancer cell line CT26 and mouse head and neck squamous cell carcinoma cell line MOC2 were kindly provided by Professor Lei Dai of the State Key Laboratory of Biotherapy, West China Hospital, Sichuan University. CT26 cells were cultured in RPMI-1640 medium (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% certified fetal bovine serum (FBS, Shanghai Zelbio Technology Co., Ltd., Cat. No. Z7185FBS-500). MOC2 cell line was also maintained under the same conditions. In addition, human keratinocyte cell line HaCaT was also provided by Professor Lei Dai and was cultured in DMEM medium (Gibco) with similar ingredients.

[0046] Animal model: Female BALB / c mice (6-8 weeks old) were purchased from Jinpukai Co., Ltd. (Nanjing, China). All animal experimental procedures were in accordance with the Regulations on the Administration of Experimental Animals. The animals were fully fed under specific pathogen-free (SPF) conditions, with an environmental temperature controlled at about 22 °C, humidity of 50%, and a 12-hour light / dark cycle. After measuring the tumor size, the volume was calculated using the formula Tumor volume = length x width2x 0.5. Mice with primary tumor volumes reaching 2000 mm3or tumor burden affecting animal health were euthanized under carbon dioxide anesthesia. No tumor size exceeded the maximum value in this study. The animal experiments were approved by the Institutional Animal Care and Use Committee of West China Hospital, Sichuan University (Approval No. 20231012013).

[0047] Antitumor effect. CT26 tumor models were established by subcutaneously injecting 1 x 106 CT26 cancer cells into the right flank of each mouse. Experimental animals were randomly divided into six groups: control group, palladium-triphenyltin (Pd-Tpb) group, LA@Pd-Tpb group, X-ray irradiation group (6 Gy), Pd-Tpb + X-ray irradiation group (6 Gy), and LA@Pd-Tpb + X-ray irradiation group (6 Gy). Each mouse received a dose of 100 μL (100 μg mL−1) of Pd-Tpb or LA@Pd-Tpb. Tumor volume was monitored every two days, and euthanasia was performed when the ethical endpoint (tumor volume < 2000 cubic millimeters) was reached. After euthanasia, tumor photographs were taken and tumor weight was measured.

[0048] Abscopal effect evaluation. To study the abscopal effect of LA@Pd-Tpb, the present invention established a bilateral CT26 tumor model as primary tumor by injecting 5 x 105CT26 cells into the right flank of each mouse. At the distal site, tumor inoculation was performed by injecting 5 x 104CT26 cells into the left flank of the mice. The aPD-1 inhibitor was used as an immune checkpoint inhibitor in the experiment. The groups were set as follows: control group, LA@Pd-Tpb + aPD-1 group, X-ray (6 Gy) group, aPD-1 + X-ray (6 Gy) group, LA@Pd-Tpb + X-ray (6 Gy) group, and LA@Pd-Tpb + X-ray + aPD-1 (6 Gy) group. Seven days after inoculation, when the primary tumor volume reached about 100 cubic millimeters, the mice received a single intratumoral injection of 100 μL PBS, 100 μL LA@Pd-Tpb (100 μg / mL), and 6 Gy X-ray. At the same time, the aPD-1 inhibitor was injected intraperitoneally three times in the left lower abdomen after irradiation: 10 mg / kg on day 0, 5 mg / kg on day 1, and another 5 mg / kg on day 2. The tumor volume was measured every two days, and euthanasia was performed when the ethical criteria were met. After euthanasia, the tumor images were taken and weighed.

[0049] Software. Data were recorded using Microsoft Office 2019. Statistical analysis was performed using GraphPad Prism software (version 8.0). Flow cytometry data were analyzed using Flow Jo software (version 10.8.1). Quantitative analysis of immunofluorescence and immunological data was performed using Image J software (version 1.52).

[0050] Statistical analysis method. Quantitative and semi-quantitative data were collected at least three times, and the results were expressed as mean ± standard deviation (SD). Student's t-test was used for comparison between two groups, and one-way analysis of variance (ANOVA) with two-tailed Tukey post-hoc test was used for multiple comparisons between multiple groups. Log-rank test was used for comparison of survival curves. The sample size (n), probability (P) value, data standardization process, and specific statistical methods are described in the figure legend. Statistical significance was set at p < 0.05, and NS indicates no significant difference. The significance levels are marked as: *p < 0.05, **p < 0.01, ***p < 0.001.

[0051] Example 1: Preparation of Pd-Tpb

[0052] Tpb (30.9 mg, 0.1 mmol) and polyvinylpyrrolidone (PVP) (25 mg, molecular weight: 58,000) were dissolved in hydrochloric acid (0.1 M, 25 mL) to form solution A. Na2PdCl4(44.13 mg, 0.15 mmol) was dissolved in 25 mL deionized water to form solution B. Solution A was rapidly added to solution B under vigorous stirring (500 rpm). The mixture was stirred at 500 rpm for 30 min at room temperature, then centrifuged at 11,000 rpm, washed with deionized water for three times, and dried under vacuum at 60 °C for 24 h to obtain the single-atom-based palladium biocatalyst Pd-Tpb.

[0053] Example 2: Preparation of LA@Pd-Tpb

[0054] The Pd-Tpb prepared in Example 1 was co-incubated with Lactobacillus (CGMCC No. 1.1878) to obtain Lactobacillus loaded with Pd-Tpb on the surface, referred to as LA@Pd-Tpb. Briefly, the prepared Pd-Tpb was dispersed in PBS buffer (concentration of 1 mg / mL). Then, 0.25 mL of Pd-Tpb solution was added to the PBS solution (0.75 mL, colony concentration of 10 8 CFU / mL) of Lactobacillus under magnetic stirring at room temperature. After incubation at 37 °C for 4 h, the obtained LA@Pd-Tpb complex was collected by centrifugation at 6000 rpm for 5 min and stored in PBS buffer at 4 °C.

[0055] Test Example 1: Structural characterization of Pd-Tpb

[0056] The catalytic site of Pd-Tpb prepared in Example 1 of the present application is formed by coordination of palladium single atoms with Tpb ligands. It can be inferred that this developed biocatalytic Pd-Tpb complex may have the characteristics of Pd-Tpb with spatial organic ligands, and can efficiently realize the generation of radioactively activatable reactive oxygen species (ROS). In order to characterize its structure, the present application carried out the following tests to analyze its structural characteristics.

[0057] Scanning electron microscope (SEM) images show that Pd-Tpb exhibits nanostructure characteristics (Fig. 1a). Figure 1 a). Energy dispersive spectroscopy (EDS) spectrum confirms the presence of palladium, carbon, nitrogen and chlorine elements in the material (Fig. 1b). Figure 1 a). Energy dispersive spectroscopy (EDS) spectrum confirms the presence of palladium, carbon, nitrogen and chlorine elements in the material (Fig. 1b). Figure 1c). X-ray diffraction (XRD) analysis shows that no diffraction peaks related to crystalline palladium are detected in Pd-Tpb, which can be attributed to the dispersion of palladium in the form of single atoms in the material Figure 1 d).

[0058] To explore the valence state and electronic configuration of the biocatalytic Pd-Tpb complex, the present application adopts X-ray photoelectron spectroscopy (XPS) technology. The Pd 3d spectrum can be divided into two regions of 3d 3 / 2 and 3d 5 / 2 Two main peaks at 342.1 eV and 336.8 eV correspond to Pd 2+ species Figure 1 e). Through N 1s and Cl 2p XPS analysis, it is found that there is a Pd 2+ valence state in the Pd-Tpb complex, in which the PdN2Cl2 center is coordinated with the chlorine and nitrogen atoms of the Tpb ligand to form a +2 oxidation state, and no Pd metal state is detected Figure 1 f-g), which will be conducive to the exertion of its enzyme activity.

[0059] Subsequently, X-ray absorption near-edge structure (XANES) spectroscopy and extended X-ray absorption fine structure (EXAFS) spectroscopy are used to elucidate the atomic coordination environment of the Ru center in the biocatalytic Pd-Tpb complex. Analysis of the Pd K-edge XANES spectrum shows that the pre-edge peak of Pd-Tpb is between Pd foil and PdO Figure 1 h). The Fourier transform (FT) EXAFS curve of Pd-Tpb at the Pd K-edge presents two peaks at 1.50 Å and 1.87 Å, which is in good agreement with the Cl2−Pd−N2 configuration Figure 1 i). These spectra are also highly consistent with the fitting spectrum of the Pd-Tpb structure, providing strong verification for the accurate structure model and facilitating further research on catalytic reactions Figure 1 j,k). In addition, the wavelet transform (WT) and Fourier transform of the k2-weighted EXAFS spectrum prove the existence of Pd-N and Pd-Cl bonds in Pd-Tpb, indicating that the Pd single atom is successfully coordinated with the Tpb ligand Figure 1 l-n). The coordination structure of Pd-Tpb is shown in the schematic diagram Figure 1 o).

[0060] Test Example 2: Radioactivation of the active oxygen biocatalytic activity of Pd-Tpb

[0061] After the morphology and electronic structure of Pd-Tpb were characterized, the present application verified the radio-enhanced biological catalytic performance thereof. Research shows that the composite has a dual catalytic function: it can not only generate active oxygen, but also generate a large amount of active oxygen under X-ray irradiation, thereby enhancing the radio-sensitization effect, reducing radiation resistance, and significantly improving the overall anti-tumor efficacy.

[0062] Firstly, the radio-enhanced ROS biological catalytic activity of the biological catalyst was evaluated under the condition of different pH values of TME using 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay, and the results show that Pd-Tpb has the ability to generate active ROS under radiation (Fig. 2a). Figure 2 a) and has a certain pH dependence (b). Figure 2 b). The type of ROS generated by Pd-Tpb in the presence of hydrogen peroxide was determined by electron paramagnetic resonance (EPR) spectrum, which confirmed that O2and 1 O2are the main ROS products (c), which reveals the enhanced ROS generation ability of Pd-Tpb in the presence of X-ray radiation. Figure 2

[0063] Test Example 3: Structure characterization of LA@Pd-Tpb

[0064] The scanning electron microscope image (d) of LA@Pd-Tpb shows that Pd-Tpb can be effectively loaded on the surface of LA. Figure 2

[0065] Test Example 4: In vitro biological catalysis and radio-activated tumor killing ability of Pd-Tpb and LA@Pd-Tpb

[0066] Tumor drug resistance and insufficient targeting are the main challenges faced by clinical treatment, which often leads to tumor recurrence and metastasis. In this context, the synthesis of LA@Pd-Tpb with unique structural advantages has attracted the attention of the present application to its potential as a targeted biological catalysis and radio-activated tumor treatment nanodrug. First, the in vitro cytotoxicity of Pd-Tpb and LA@Pd-Tpb was evaluated. The CCK-8 detection shows that even at a concentration as high as 100 μg mL −1 ​​In the case of Pd-Tpb and LA@Pd-Tpb, the cytotoxicity of epidermal cells was extremely low after 48 hours of co-culture, highlighting the excellent biological safety. Subsequently, to verify the synergistic effect of Pd-Tpb and LA@Pd-Tpb combined with X-rays on the destruction of tumor cells, the CT26 cells were incubated in different concentrations of Pd-Tpb and LA@Pd-Tpb for 4 hours, and then received a 6 Gy dose of X-ray irradiation, and then continued to be cultured for 48 hours. The results showed that the relative cell survival rates of the Pd-Tpb+X-rays group and the LA@Pd-Tpb+X-rays group (CT26 cell concentration was 80 μg / mL -1 ) were both reduced, and the reduction of the LA@Pd-Tpb+X-rays group was more significant, thereby confirming the enhanced anti-tumor effect of LA@Pd-Tpb.

[0067] Next, the influence of LA@Pd-Tpb on the generation of reactive oxygen species (ROS) was observed by fluorescence staining. Using the DCFH-DA probe, the amount of ROS generated in the CT26 cells was quantified. The oxidized DCFH in the X-ray treatment group and the control group showed weak green fluorescence, while the LA@Pd-Tpb+X-rays group showed strong bright green fluorescence, indicating that the level of ROS in the CT26 cells was increased (Fig. Figure 3 a, b). Therefore, the present application believes that the reason for the generation of a large amount of ROS by LA@Pd-Tpb is that the generation of lactic acid by LA reduces the concentration of phosphate (Ph) and provides H + In addition, since previous studies have shown that LA metabolism produces lactic acid, thereby reducing the environmental pH value, the present application also monitored the change of the environmental pH value with time during the in vitro culture of probiotics. As shown in Fig. Figure 3 c, after 48 hours of incubation, the pH values of the LA and LA@Pd-Tpb culture media were both significantly reduced. In addition, by coating LA and LA@Pd-Tpb on LB agar plates, the influence of Pd-Tpb modification on the activity of probiotics was further tested. The colony forming units (CFU) of pure LA and LA@Pd-Tpb were almost equal, indicating that Pd-Tpb modification would not affect the activity of LA (Fig. Figure 3 d).

[0068] The radiosensitivity of tumor cells is closely related to their proliferation cycle: the G1 phase cells have the lowest sensitivity to radiation, the M phase cells are the most sensitive, the G2 phase cells gradually enter the M phase, and the S phase cells show a defect in radiosensitivity. The present application used a cell cycle detection kit to analyze the cycle and detect the apoptosis of CT26 cells. The results showed that the proportion of cells in the G2 / M phase in the LA@Pd-Tpb+X-rays treated tumor cells was 64.09%, which was significantly higher than that in other groups, further confirming the potential of LA@Pd-Tpb as a biocatalytic radioactivation biocatalyst in anticancer therapy (Fig. Figure 3e) To verify the ability of LA@Pd-Tpb+X-ray to induce DNA damage, the present application conducted DNA damage experiments. After incubating tumor cells with different treatment groups for 1 hour, DNA damage markers were labeled with γ-H2AX staining, and cells were observed by inverted fluorescence microscopy to evaluate the damage effect. Notably, the DNA damage level of the LA@Pd-Tpb+X-ray group was the most significant, surpassing the effects of LA@Pd-Tpb, X-ray, or Pd-Tpb+X-ray treatment alone (Fig. 2e). Figure 3 f, g). Integrating the above results shows that LA@Pd-Tpb can be used as a radioactivated biocatalyst to produce a large amount of reactive oxygen species (ROS). It is confirmed by Live / Dead staining and apoptosis detection that the increase in the level of reactive oxygen species (ROS) is positively correlated with the increase in the mortality rate of tumor cells. Among them, the LA@Pd-Tpb+X-ray group always exhibits the strongest anti-tumor activity, characterized by the highest apoptosis rate and strong fluorescence signal (Fig. 2f, g). Figure 3 h-k). The comprehensive results show that this unique LA@Pd-Tpb biocatalyst improves radiosensitivity by changing the tumor microenvironment (TME) and enhancing ROS generation, significantly enhancing the synergistic effect of tumor killing. This leads to increased DNA damage and disruption of mitochondrial membrane potential, ultimately leading to greater apoptosis in tumor cells.

[0069] Test Example 5: In vivo biocatalysis and radioactivated anti-tumor effect of Pd-Tpb and LA@Pd-Tpb

[0070] Inspired by the significant anti-tumor activity exhibited by Pd-Tpb and LA@Pd-Tpb in vitro, the present application further evaluated their efficiency in inhibiting primary tumor progression using a BALB / c mouse colon cancer model (carrying CT26 colon cancer cells). Figure 4 a demonstrates the specific implementation process of the mouse model construction and treatment strategy. Through the live near-infrared fluorescence imaging technology, the present application observed the fluorescence aggregation in the tumor site after intravenous injection of Cy7-labeled Pd-Tpb and LA@Pd-Tpb (200 μL / each, equivalent to 1.35 x 10^8 CFU per mouse) and Pd-Tpb (200 μL / each, equivalent to 0.2 mg per mouse). Figure 4 b shows that the Cy7 fluorescence intensity of Pd-Tpb and LA@Pd-Tpb injection both peaked after 4 hours and gradually weakened. Consistent with expectations, the Cy7 fluorescence intensity in the tumor tissue of LA@Pd-Tpb injection was significantly higher than that in other organs, while no such targeting effect was observed in Pd-Tpb-injected mice (Fig. 3b). Figure 4c). These results indicate that the presence of LA effectively enhances the aggregation and retention capacity of LA@Pd-Tpb at tumor sites. To further analyze the colonization of LA@Pd-Tpb in tumor tissues, major organs, and tumors in mice after injection, the samples were ground, diluted, and plated on LB agar plates. Figure 4 The results showed that a large number of probiotic colonies appeared in the tumor area 4 hours after injection, while LA was not detected in the major organs, which fully demonstrates the excellent tumor targeting and colonization performance of LA@Pd-Tpb.

[0071] BALB / c female mice with subcutaneously implanted CT26 tumors of approximately 100 cubic millimeters were randomly divided into six treatment groups: control group, Pd-Tpb group, LA@Pd-Tpb group, X-ray group, Pd-Tpb+X-ray group, and LA@Pd-Tpb+X-ray group. On day 0, mice in each group received an equal dose (10 mg / kg) of Pd-Tpb or LA@Pd-Tpb intratumorally, followed by continuous low-dose X-ray irradiation 4 hours later. Twelve days after treatment, all mice were sacrificed, and the tumors were removed by weighing. The body weight of mice in each group remained stable throughout the treatment period. Figure 4 e). Although both RT + Pd-Tpb and RT alone can delay tumor growth, the combination therapy of RT + LA@Pd-Tpb has the most significant inhibitory effect on tumor growth. Figure 4 Histopathological evaluation confirmed that RT+ LA@Pd-Tpb treatment had a potent anti-tumor effect. Hematoxylin-eosin (H&E) staining results showed that the degree of tumor tissue necrosis in the RT+ LA@Pd-Tpb group was higher than that in other treatment groups. Figure 4 i). To further investigate the radiodynamic enhancement effect activated by LA@Pd-Tpb under irradiation, this invention employs TdT-mediated dUTP nick-end labeling (TUNEL), Ki-67, and CD31 immunofluorescence staining. Notably, LA@Pd-Tpb + X-ray treatment significantly enhanced apoptosis activity, with a significantly increased TUNEL positivity rate (i). Figure 4 j and m). Meanwhile, the Ki-67 and CD31 immunofluorescence positivity rates were significantly reduced in the LA@Pd-Tpb+X-ray group (j and m). Figure 5 (k, l, n, and o), which confirms that the drug has a significant in vivo inhibitory effect on tumor cell proliferation. Regarding the biocompatibility assessment of LA@Pd-Tpb, researchers removed major organs (heart, liver, spleen, lung, and kidney) from experimental mice for hematoxylin-eosin (H&E) staining. The results showed that no significant histopathological damage or inflammation was observed after LA@Pd-Tpb injection, highlighting its good biocompatibility.

[0072] Test Example 6: LA@Pd-Tpb induces immune response and memory effect

[0073] RNA transcriptome analysis of the various experimental groups in the in vivo experiment of Test Example 5 revealed that LA@Pd-Tpb irradiated with low-dose X-rays exhibited significant immunomodulatory effects in the tumor microenvironment. To verify these immunological changes, immunoflow cytometry was used to analyze samples from the four experimental groups. Notably, CD45 in the LA@Pd-Tpb + X-ray group was significantly higher than that in the LA@Pd-Tpb + X-ray group. + T cells and CD8 + The number of T cell subsets increased significantly. Figure 5 a, b, d, e). Furthermore, the proportion of M1-polarized macrophages in this group was significantly higher than in other treatment groups ( Figure 5 c, f). These findings indicate that under LA@Pd-Tpb+X-ray therapy, the tumor microenvironment shifts towards a pro-inflammatory phenotype, thereby enhancing the anti-tumor effect. Compared with the control group, the serum levels of pro-inflammatory cytokines such as IL-6, IFN-β, and IFN-γ were significantly increased in the LA@Pd-Tpb+X-ray group. Figure 6 g). This elevated level indicates that the immune response is activated, which helps promote the death of immunogenic cells within the tumor.

[0074] Test Case 7: Synergistic Effect of LA@Pd-Tpb Combined with Immune Checkpoint Blockade Therapy in Combating Tumor Metastasis

[0075] Immune checkpoint blockade therapy has been established as a highly effective strategy in oncology, but its efficacy against most cancers remains unsatisfactory. Novel nanomedicines or biomaterials that can synergize with immune checkpoint blockade therapy by enhancing T-cell toxicity and cytokine release hold significant potential for advancing precision cancer treatment. Against this backdrop, based on the aforementioned findings regarding the potent immunostimulatory effects of LA@Pd-Tpb, this invention further explores its ability to induce distant metastases and inhibit distant tumor growth when used in combination with immune checkpoint blockade therapy. In the experiment, after establishing a primary tumor (5 × 10^5 cells / 100µL), a secondary CT26 tumor (5 × 10^4 cells / 100µL) was transplanted contralaterally to simulate distant metastases. Subsequently, LA@Pd-Tpb was administered intravenously, followed by continuous low-dose X-ray irradiation (6 Gy) of the primary tumor. αPD-1 was injected intraperitoneally on days 0, 1, and 2 after the initial irradiation. Figure 6 a). The results showed that LA@Pd-Tpb+X-ray combined with αPD-1 not only maximally killed the primary tumor after irradiation, but also effectively inhibited the growth of distant tumors (a). Figure 6 (b, c, and e). Compared with other groups, the LA@Pd-Tpb+X-ray+αPD-1 group had the lowest tumor weight ( Figure 6 d, f).

[0076] Furthermore, apoptotic activity observed in TUNEL-positive regions indicated that the apoptosis level in the LA@Pd-Tpb+X-ray+αPD-1 group was significantly higher than that in other groups. Figure 6 g, h). Meanwhile, the expression levels of the proliferation marker Ki-67 and the angiogenesis marker CD31 were significantly reduced in both the primary tumor and distant metastases in the LA@Pd-Tpb+X-ray+αPD-1 group, consistent with observations at the primary treatment site. Figure 6 Notably, the expression level of the DNA damage marker γ-H2AX in distant metastases was also significantly elevated, highly similar to that of the primary tumor. These data strongly support the significant effect of enhanced synergistic immunotherapy in inhibiting distant tumor growth during the treatment of the primary tumor. Figure 7 m, n).

[0077] In addition to monitoring tumor growth patterns, this invention also analyzed the immune cell population within distant tumors seven days after treatment. In the LA@Pd-Tpb+X-ray+αPD-1 group, compared to other groups, CD8+ cells in distant tumors were significantly higher. + The proportion of T cells increased significantly ( Figure 7 a, e). Meanwhile, CD8, a key marker of systemic immune activation... + The CD69+ T cell population was also significantly increased in this treatment group. Figure 7 (b, f) These activated T cells are believed to be involved in inducing apoptosis in cancer cells. Notably, the proportion of pro-inflammatory M1 polarized macrophages was higher in the LA@Pd-Tpb+X-ray+αPD-1 group than in other groups. Figure 7 c, g), indicating that macrophage polarization is shifting towards a more anti-tumor phenotype.

[0078] In contrast, PD-1 in distant tumors was higher than in the control group. + CD8 + The proportion of T cells decreased significantly ( Figure 7 d, h). PD-1 + CD8 + T cells are considered a predictor of poor prognosis in cancer patients receiving immunotherapy, and their high expression levels are associated with CD8+. + Accelerated T cell depletion was associated with the LA@Pd-Tpb+X-ray+αPD-1 group, which showed significantly higher serum levels of pro-inflammatory cytokines (including IL-6, IFN-β, and IFN-γ) compared to the control group. Figure 7i-l). This elevation indicates that the immune response is activated, which helps to promote immunogenic cell death in the tumor. The results of multiplex immunohistochemistry (mIHC) analysis are consistent with this, showing that the proportion of CD8 + T cells in the tumor microenvironment of the LA@Pd-Tpb+X-ray+αPD-1 group of CT26 mice is higher than that of other groups Figure 7 m) In addition, the proportion of CD86+macrophages in the LA@Pd-Tpb+X-ray+αPD-1 group is higher ​ n) In summary, these data support the conclusion that biocatalytic and radioactivated LA@Pd-Tpb can induce a strong immune stimulation effect, thereby significantly enhancing the immunotherapy effect and further inhibiting the growth of distant tumors.

[0079] It should be noted that the preferred embodiments of the present application are given in the specification and drawings of the present application, but the present application can be implemented in many different forms, and is not limited to the embodiments described in the specification. These embodiments are not additional limitations on the content of the present application, and the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive. Furthermore, each of the above technical features continues to be combined, forming various embodiments not listed above, which are considered to be within the scope of the present application. Furthermore, for those skilled in the art, the above description can be improved or modified, and all such improvements and modifications shall fall within the scope of the appended claims of the present application.

Claims

1. A biocatalyst based on a palladium single atom, characterized in that, The biocatalyst is a coordination complex formed by Pd salt and 1,3,5-tris(pyridin-4-yl)benzene through Pd-N coordination, wherein the Pd salt is [PdCl4]. 2- Pd salt.

2. The biocatalyst according to claim 1, characterized in that, Pd is distributed in the biocatalyst in the form of single atoms.

3. A method for preparing a biocatalyst according to claim 1 or 2, characterized in that, The biocatalyst is prepared by reacting Pd salt and 1,3,5-tris(pyridin-4-yl)benzene in an acidic aqueous solution.

4. The preparation method according to claim 3, characterized in that, The molar ratio of Pd salt to 1,3,5-tris(pyridin-4-yl)benzene is 1~3:1; The reaction conditions are: stirring at room temperature for 10-60 minutes.

5. The preparation method according to claim 3, characterized in that, The acidic aqueous solution includes 0.01M-0.1M hydrochloric acid; The preparation method also includes adding a polymeric surfactant to the reaction system.

6. The preparation method according to claim 5, characterized in that, The polymeric surfactant is polyvinylpyrrolidone; The preparation method includes dissolving 1,3,5-tris(pyridin-4-yl)benzene and a polymeric surfactant in an acid to form solution A; dissolving a Pd salt in water to obtain solution B; then mixing solution A and solution B under stirring and reacting, collecting the reaction product, and obtaining the biocatalyst after washing and drying.

7. A biocatalytic complex, characterized in that, The biocatalytic complex comprises lactobacillus and a biocatalyst according to claim 1 or 2 supported on its surface.

8. Use of the biocatalyst according to claim 1 or 2 or the biocatalytic complex according to claim 7 in the preparation of materials with ROS generation activity.

9. Use of the biocatalyst according to claim 1 or 2 or the biocatalytic complex according to claim 7 in the preparation of a medicament for tumor treatment.

10. Use of the biocatalytic complex according to claim 7 in combination with an immune checkpoint inhibitor in the preparation of a pharmaceutical agent for the treatment of primary and metastatic tumors.

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