Preparation method and application of E5 polypeptide functionalized platinum nano-enzyme

Abstract

The invention discloses a preparation method and application of an E5 polypeptide functionalized platinum nano enzyme. A chemical precipitation method is adopted as a basis, sodium borohydride is dropwise added into a mixed reaction system of a platinum-containing precursor and E5 polypeptide (modified with cysteine) at a constant speed, reduction precipitation of platinum atoms is achieved, and therefore the E5 polypeptide functionalized platinum nano-enzyme assembly (Pt at E5) is synthesized in one step. The synthesis method has the advantages of low toxicity, simplicity and convenience in operation and the like, and effectively solves the problems of low coupling efficiency of platinum nanoparticles and polypeptide, complex material preparation process and the like in the prior art. The prepared Pt-coated E5 nano delivery system has excellent enzyme-like activity in vitro, shows good tumor targeting toxicity on the cellular level, and shows an excellent treatment effect in a B cell lymphoma animal model. The implementation of the invention provides a new thought and method for the application of the platinum-based nano-enzyme material in the field of lymphoma treatment.

Description

Technical Field

[0001] The present invention relates to a preparation method and application of an E5 polypeptide-functionalized platinum nanozyme, belonging to the technical field of nanomedicine. Background Art

[0002] Lymphoma, as a malignant tumor with a global incidence ranking among the top ten, has a treatment history dating back to the 1940s when nitrogen mustard was first used to treat the disease. In 1997, the US Food and Drug Administration (FDA) approved rituximab for the treatment of relapsed or refractory B-cell lymphoma, marking the beginning of the era of targeted therapy for lymphoma. In 2016, the FDA approved PD-1 inhibitors for the treatment of Hodgkin lymphoma (HL). In 2018, the FDA approved CAR-T cell therapy for the treatment of relapsed or refractory large B-cell lymphoma, opening a new era of immunotherapy and cell therapy for lymphoma. Currently, these existing treatment strategies have achieved certain efficacy to some extent, but there are still some patients who relapse after standard treatment regimens, leading to difficulties in further treatment and poor prognosis. As an emerging treatment modality, nanomedicine has proven to have significant advantages in improving drug bioavailability and reducing toxicity. The progress in the field of nanomedicine has provided new treatment approaches for the treatment of various major diseases, including lymphoma. In particular, the targeted application of nanotechnology in the treatment of blood cancers has become a promising treatment strategy for such malignancies.

[0003] Nanozymes exhibit significant application potential in the field of tumor treatment. They can bidirectionally regulate the level of reactive oxygen species (ROS) and play a key role in maintaining the redox balance of organisms. In a specific microenvironment, nanozymes can intervene and regulate the catalytic processes in organisms, providing new strategies for the diagnosis and treatment of various diseases, especially playing a crucial role in cancer treatment. In particular, platinum-based nanozymes have shown remarkable effects in chemodynamic therapy, photodynamic therapy, photothermal therapy, sonodynamic therapy, and radiotherapy sensitization of tumors. However, due to insufficient tumor specificity, their therapeutic effects in vivo are limited to a certain extent.

[0004] Chemokine receptor 4 (CXCR4), as a key member of the G protein-coupled receptor (GPCRs) family, is highly expressed in at least 23 different types of human cancers, covering malignancies such as breast cancer and leukemia. The binding of CXCR4 to its cognate ligand CXCL12 (also known as stromal cell-derived factor 1α, SDF-1α) plays a crucial role in regulating the directed migration, invasion, and metastasis of tumor cells. The CXCR4-CXCL12 biological axis is closely related to the initiation and development of tumors. Therefore, blocking this biological axis may provide new ideas for cancer immunotherapy. Existing studies have confirmed that CXCR4-CXCL12 is involved in the pathogenesis of diffuse large B-cell lymphoma (DLBCL). Thus, blocking the CXCR4-CXCL12 biological axis is considered a potential target for lymphoma treatment. Currently, only two CXCR4 antagonists, Plerixafor and Mocetinostat, have been approved for marketing in the global market. At the same time, more than 20 CXCR4 antagonists are in the research and development stage. These candidate drugs include small molecule compounds, polypeptides, and antibodies, aiming to treat various cancers. A research report pointed out that the E5 polypeptide can effectively block the CXCR4 / CXCL12 axis, enhance the efficacy of chemotherapy drugs in a mouse model of acute myeloid leukemia (AML), and improve the therapeutic effects of various chemotherapy drugs in a mouse model of breast cancer. These findings provide a scientific basis for the application of CXCR4 antagonists in cancer treatment and point the way for future drug development.

[0005] Immunogenic Cell Death (ICD) is a cutting-edge anti-tumor treatment strategy that specifically targets secondary tumors that are insensitive to traditional treatments such as chemotherapy and radiotherapy. ICD inducers can induce immunogenic death of cancer cells, thereby triggering a systemic immune response. Currently, a series of organic compounds, including doxorubicin, cyclophosphamide, bleomycin, digoxin, saharitine, and mitoxantrone, have been identified as potential ICD inducers. In particular, the ICD induction potential of doxorubicin and cyclophosphamide, as conventional chemotherapy drugs for diffuse large B-cell lymphoma (DLBCL), has received extensive attention. In addition, the research on metal-based compounds as new therapeutic agents is also increasing, covering metal complexes such as platinum (Pt), gold (Au), copper (Cu), ruthenium (Ru), and iridium (Ir). Among them, platinum complexes are used in more than 50% of global chemotherapy. Although these metal complexes have shown good ICD induction effects in in vitro cancer cell experiments and mouse models, they still have deficiencies in pharmacological properties, especially in terms of tumor tissue targeting and structure-activity relationships. To address these issues, conjugating metal nanozymes capable of inducing ICD to active tumor-targeting moieties (such as antibodies, polypeptides) is considered promising to improve the therapeutic effect.

[0006] Although nanozymes have shown preliminary potential in the field of cancer treatment, the effectiveness and feasibility of their clinical applications have not been fully evaluated. Although the research literature on nanoparticles for cancer treatment is abundant, the types of nanoparticles approved by the FDA are limited, which has led to a serious disconnect between clinical applications and academic outputs in the field of nanomedicine, which is partly attributed to the complexity of the nanozyme material design and preparation process. Therefore, to promote the application of nanozymes in lymphoma treatment, the synthesis process of nanozymes must be optimized to achieve the efficient synthesis of nanoparticles and smooth clinical translation. Summary of the Invention

[0007] Object of the Invention: The object of the present invention is to provide a preparation method of an E5 polypeptide-functionalized platinum nanozyme. The platinum nanozyme is prepared by a one-step synthesis method and can achieve precise targeted treatment of diseases related to the CXCR4-CXCL12 biological axis.

[0008] Technical solution: The present invention provides a preparation method of an E5 polypeptide-functionalized platinum nanozyme, and the method includes the following steps: mixing a platinum precursor solution and a polypeptide E5 solution modified with cysteine, and then dropwise adding a sodium borohydride solution while mechanically stirring under ultrasonic conditions. After the reaction is completed, ultrafiltration is carried out and washed with pure water to obtain the E5 polypeptide-functionalized platinum nanozyme.

[0009] Further, the platinum precursor solution is a chloroplatinic acid solution.

[0010] Further, the molar concentration of the chloroplatinic acid solution is 18 - 20 mM.

[0011] Further, the molar concentration of the polypeptide E5 solution is 0.03 - 0.05 mM.

[0012] Further, the molar concentration of the sodium borohydride solution is 263 - 265 mM.

[0013] Further, the H 2 PtCl 6 ·6H 2 O solution, polypeptide E5 solution and sodium borohydride solution have a volume ratio of 1:8:1.

[0014] Further, the speed of the mechanical stirring is 900 - 1100 rpm, the reaction temperature is 20 - 30 °C, and the ultrasonic power is 90 - 110 W.

[0015] Further, the molecular weight of the ultrafiltration centrifuge tube is 30 KDa, the centrifugation speed is 1000 - 3000 rpm, the centrifugation time is 5 - 15 min, and the number of centrifugation times is 3 - 5 times.

[0016] The present invention also provides a platinum nanozyme prepared by the above preparation method of the E5 polypeptide-functionalized platinum nanozyme.

[0017] The present invention also provides the application of the above platinum nanozyme in the preparation of a drug for treating diseases related to the CXCR4-CXCL12 biological axis.

[0018] Further, the diseases related to the CXCR4-CXCL12 biological axis include acute myeloid leukemia, breast cancer or lymphoma.

[0019] Advantages: Compared with the prior art, the present invention has the following outstanding and remarkable advantages: The preparation process of the present invention is simple and easy to purify, and has good development prospects. The nanozyme prepared by the present invention has good stability, water solubility and biocompatibility, can be used for targeted therapy in the tumor microenvironment, effectively reduces the toxic and side effects on normal cells, and provides a new idea for constructing safe, intelligent and efficient nanozyme drugs. The nanozyme of the present invention can achieve the "Fenton reaction" through platinum nanozyme, disrupt the redox balance in cells and further cause immunogenic cell death. After the nanozyme of the present invention is injected into B-cell lymphoma mice through the tail vein, it has a significant anti-tumor effect, can achieve chemodynamic / immunogenic cell death, and the E5 polypeptide can also block the CXCR4-CXCL12 biological axis and prevent the infiltration of tumor cells into the bone marrow, having potential clinical application value. Description of the Drawings

[0020] Figure 1 This is a physical photograph of Pt@E5 prepared by the present invention.

[0021] Figure 2 This is a schematic diagram of the synthesis and application of Pt@E5 prepared by the present invention.

[0022] Figure 3 This is a transmission electron microscope image of Pt@E5 prepared in the present invention.

[0023] Figure 4 This is a hydrodynamic particle size distribution diagram of Pt@E5 prepared by the present invention.

[0024] Figure 5 This is a graph showing the change of hydrodynamic size of Pt@E5 prepared by the present invention over time in water, PBS and RPMI-1640 medium.

[0025] Figure 6 This is a potential distribution diagram of Pt@E5 prepared by the present invention.

[0026] Figure 7 This is an X-ray photoelectron spectroscopy diagram of Pt@E5 prepared by the present invention.

[0027] Figure 8 This is a graph of the analysis of the peroxidase-like activity of Pt@E5 prepared by the present invention.

[0028] Figure 9 This is a graph of the analysis of the catalase-like activity of Pt@E5 prepared by the present invention.

[0029] Figure 10 This is an electron paramagnetic resonance spectroscopy analysis diagram of Pt@E5 prepared by the present invention.

[0030] Figure 11Cell viability graph after co-incubating Pt@E5 prepared according to the present invention with A20 cells for 24 h.

[0031] Figure 12 Flow cytometry analysis graphs of material uptake by cells after co-incubating Pt@E5 of the present invention with A20 cells for 6 h and 12 h.

[0032] Figure 13 Analysis graph of blocking the migration of Pt@E5 of the present invention to MS-5 cells after co-incubating with A20 cells.

[0033] Figure 14 Analysis graph of intracellular ROS level after co-incubating Pt@E5 of the present invention with A20 cells for 24 h.

[0034] Figure 15 Analysis graph of intracellular GSH level after co-incubating Pt@E5 of the present invention with A20 cells for 24 h.

[0035] Figure 16 Flow cytometry analysis graph of cell apoptosis after co-incubating Pt@E5 of the present invention with A20 cells for 24 h.

[0036] Figure 17 Analysis graph of intracellular ATP level after co-incubating Pt@E5 of the present invention with A20 cells for 24 h.

[0037] Figure 18 Analysis graph of intracellular HMGB-1 level after co-incubating Pt@E5 of the present invention with A20 cells for 24 h.

[0038] Figure 19 Flow cytometry analysis graph of the maturation level of DC cells induced in bone marrow after co-incubating Pt@E5 of the present invention with A20 cells for 24 h.

[0039] Figure 20 Graph of the body weight change of mice with B-cell lymphoma treated by tail vein injection of PBS and different doses of Pt@E5, recorded within 15 days.

[0040] Figure 21 Graph of the treatment effect of mice on the 15th day recorded by tail vein injection of PBS and different doses of Pt@E5 to treat mice with B-cell lymphoma. Detailed implementation manners

[0041] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0042] Unless otherwise specified, all chemical reagents are commercially available and can be used directly without further purification.

[0043] H 2 PtCl 6 ·6H2 O and 3,3',5,5'-tetramethylbenzidine (TMB) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. E5 polypeptide was purchased from Anhui Guoping Pharmaceutical Co., Ltd. Sodium borohydride and hydrogen peroxide were purchased from Sinopharm Chemical Reagent Co., Ltd. A20 cells (murine B cell lymphoma cell line) were purchased from Guangzhou Saiku Biotechnology Co., Ltd. RPMI-1640 medium was purchased from Jiangsu KeyGen Biotech Co., Ltd. Fetal bovine serum was purchased from Thermo Fisher Scientific. Cell Counting Kit-8 (CCK8) was purchased from Jiangsu KeyGen Biotech Co., Ltd. GSH detection kit was purchased from Yacoin Biotechnology Co., Ltd. ROS detection kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Shanghai, China). Cell apoptosis detection kit was purchased from Jiangsu KeyGen Biotech Co., Ltd. ATP kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Shanghai, China). HMGB1 kit was purchased from Nanjing Cyberscience Biotechnology Co., Ltd. (Nanjing, China). Anti-CD80-PE and Anti-CD86-APC antibodies were purchased from Shanghai Univ-Bio Biotechnology Co., Ltd. BALB / c mice were purchased from Jiangsu Qinglongshan Experimental Animal Center. Water with a resistivity higher than 18.2 MΩ·cm used in all experiments was purified through a laboratory water purification system (AW-2002-H, Aikepu Co., Ltd.).

[0044] Example 1 Preparation of Pt@E5 nanozyme

[0045] Method 1: Weigh 10 mg of hexahydrate chloroplatinic acid (H 2 PtCl 6 ·6H 2 O) and dissolve it in 1 mL of pure water. Weigh 1 mg of E5 polypeptide (Gly-Gly-Arg-Ser-Phe-Phe-Leu-Leu-Arg-Arg-Ile-Gln-Gly-Cys-Arg-Phe-Arg-Asn-Thr-Val-Asp-Asp) and dissolve it in 8 mL of pure water, then mix the two evenly and pour them into a reactor. Weigh 10 mg of sodium borohydride and dissolve it in 1 mL of pure water, then add it dropwise to the above reactor. During the dropping process, the mechanical stirring speed is 1000 rpm, the reaction temperature is 25 °C, the ultrasonic power is 100 W, and the time for the liquid-phase chemical precipitation reaction is 30 min. Finally, centrifuge with an ultrafiltration tube with a molecular weight of 30 KDa at 2000 rpm for 10 min and repeat 3 times to obtain an aqueous solution of Pt@E5 nanozyme assembly. As shown in Figure 1 a, it can be seen that the prepared Pt@E5 nanozyme assembly is unstable, stratified after standing, and extremely easy to precipitate.

[0046] Method 2: A cysteine is additionally modified on the left side of the E5 polypeptide (Cys-Gly-Gly-Arg-Ser-Phe-Phe-Leu-Leu-Arg-Arg-Ile-Gln-Gly-Cys-Arg-Phe-Arg-Asn-Thr-Val-Asp-Asp), and other steps are the same as those in Method 1. The results are as Figure 1 shown in b. The E5 polypeptide with added cysteine can be directly modified on the surface of platinum nanoparticles, and the material has strong stability. The principle is to use the thiol group (-SH) on cysteine in the E5 polypeptide to bind to platinum atoms to form a Pt-S bond ( Figure 1 c).

[0047] In the following examples, unless otherwise specified, the experimental materials used are all aqueous solutions of Pt@E5 nanozyme assemblies prepared by Method 2, and the concentration is adjusted with pure water according to the usage requirements.

[0048] Example 2 Morphology and particle size of Pt@E5 nanozyme assemblies

[0049] Drop the aqueous solution of Pt@E5 nanozyme assemblies synthesized in Example 1 (50 μg / mL) onto the surface of a copper grid with a carbon film, and dry it at room temperature to obtain a sample. The TEM image of Pt@E5 is as Figure 3 shown. Pt@E5 is in the form of an assembly, and the size of a single Pt nanoparticle in the nanozyme assembly is 2.54 nm. Then, another 1 mL of the aqueous solution of Pt@E5 nanozyme assemblies (100 μg / mL) is taken for measuring the surface potential and hydrodynamic size. The results are as Figure 4 shown. The hydrodynamic size of the Pt@E5 nanozyme assembly is 107.57 ± 4.09 nm. Figure 6 It shows that the potential of the Pt@E5 nanozyme assembly is -38.4 ± 0.56 mV. The hydrodynamic size of the Pt@E5 nanozyme assembly in various solutions (water, PBS, RPMI-1640 medium) hardly changes within a week (as Figure 5 shown), which proves that the Pt@E5 nanozyme assembly has good colloidal stability.

[0050] Example 3 X-ray photoelectron spectroscopy

[0051] Take the aqueous solution of Pt@E5 nanozyme assemblies synthesized in Example 1 and dry it into a powder for X-ray photoelectron spectroscopy testing. The results are as Figure 7 shown. The formation of chemical bonds between Pt elements and S and N elements on the E5 polypeptide proves that the E5 polypeptide is successfully modified on the surface of the Pt nanozyme assembly.

[0052] Example 4 Determination of peroxidase-like activity

[0053] The aqueous solution of the Pt@E5 nanozyme assembly synthesized in Example 1 was subjected to peroxidase-like activity testing. Using 3,3',5,5'-tetramethylbenzidine (TMB) as the chromogenic substrate, the peroxidase-like activity of the Pt@E5 nanozyme assembly was measured under different pH conditions. The test sample solution contained 160 μL of buffer solutions with different pH values [pH 3.92 (acetic acid / sodium acetate buffer), 5.03 (acetic acid / sodium acetate buffer), 5.60 (acetic acid / sodium acetate buffer), 6.54 (citric acid / sodium citrate buffer), 7.40 (PBS phosphate buffer), 8.00 (MES buffer)], 20 μL of the aqueous solution of the Pt@E5 nanozyme assembly (80 μg / mL), and 20 μL of the TMB (0.04 M) solution. After reacting in a well plate for 2 min, the absorbance value at 650 nm was measured. The results are shown in Figure 8. The absorbance gradually decreased with the increase of pH, indicating that the Pt@E5 nanozyme assembly had good peroxidase-like activity under acidic conditions.

[0054] Determination of peroxidase-like activity in Example 5

[0055] The aqueous solution of the Pt@E5 nanozyme assembly synthesized in Example 1 was subjected to peroxidase-like activity testing. Using 3,3',5,5'-tetramethylbenzidine (TMB) as the chromogenic substrate, the peroxidase-like activity of the Pt@E5 nanozyme assembly was measured under different pH conditions. The test sample solution contained 180 μL of buffer solutions with different pH values [pH 3.92 (acetic acid / sodium acetate buffer), 5.03 (acetic acid / sodium acetate buffer), 5.60 (acetic acid / sodium acetate buffer), 6.54 (citric acid / sodium citrate buffer), 7.40 (PBS phosphate buffer), 8.00 (MES buffer)], 5 μL of the aqueous solution of the Pt@E5 nanozyme assembly (80 μg / mL), 5 μL of H 2 O 2 solution (3.75%), and 20 μL of the TMB (0.04 M) solution. After reacting in a well plate for 2 min, the absorbance value at 650 nm was measured. The results are as Figure 9 shown. The absorbance gradually decreased with the increase of pH, indicating that the Pt@E5 nanozyme assembly had good peroxidase-like activity under acidic conditions.

[0056] Example 6 Detection of ·OH and 2 O 2 produced by the nanoparticle catalysis of H 1 O 2

[0057] The ESR spectrometer of Bruker Company, Germany, model A300-10 / 12 was used to detect the catalysis of H by the Pt@E5 nanozyme assembly2 O 2 The formed ·OH and 1 O 2 . Under the condition of H 2 O 2 , 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was used as a scavenger to capture ·OH to form the DMPO / ·OH adduct. 2,2,6,6-Tetramethylpiperidine (TEMP) was used as a scavenger to capture 1 O 2 to form TEMP / 1 O 2 . When testing ·OH, the sample solution contained 1.25 M H 2 O 2 , 100 mM DMPO, and 200 μg / mL of the Pt@E5 nanozyme assembly were placed in a glass capillary and sealed. After 1 min, the spectral characteristic lines were recorded. When testing 1 O 2 , the sample solution contained 1.25 M H 2 O 2 , 100 mM TEMP, and 200 μg / mL of the Pt@E5 nanozyme assembly were placed in a glass capillary and sealed. After 1 min, the spectral characteristic lines were recorded, and H 2 O 2 and the Pt@E5 nanozyme assembly were separately reacted with the scavengers TEMP and DMPO as control groups. The results are as Figure 10 shown. When the Pt@E5 nanozyme assembly alone or H 2 O 2 alone existed, there were no ESR characteristic signals of the DMPO / ·OH adduct (characteristic peak signals of 1:2:2:1) and the TEMP / 1 O 2 adduct (characteristic peak signals of 1:1:1). In contrast, after adding the Pt@E5 nanozyme assembly and H 2 O 2 simultaneously, the ESR characteristic signals of the DMPO / ·OH adduct and the TEMP / 1 O 2 adduct were significantly enhanced. Thus, it can be seen that the Pt@E5 nanozyme assembly can catalyze H 2 O 2 to produce ·OH and 1 O 2 .

[0058] Example 7 Determination of relative cell viability by CCK8 method

[0059] Using A20 cells as a model to detect the cytotoxicity of the Pt@E5 nanozyme assembly prepared in Example 1. A20 cells in the logarithmic growth phase were collected and seeded in 96-well plates at a density of 1×10 4 cells per well, and then different concentrations of the Pt@E5 nanozyme assembly (final Pt concentrations were 0, 2.5, 5, 10, 20, 40, 60 μg / mL) were added and incubated at 5% CO 2 , 37 °C for 24 h. The medium used was RPMI-1640 complete medium supplemented with 100 U / mL streptomycin and 10% FBS. Then 10% (V / V) CCK-8 was added and incubation continued for 2 - 4 h. Finally, the absorbance of each well was measured at a wavelength of 450 nm using a multifunctional microplate reader. A20 cells treated with PBS were used as the control group, and the cell viability was recorded as 100%. The medium treated with PBS was used as blank control 1, and the medium treated with Pt@E5 was used as blank control 2. The results are as Figure 11 shown. Within the experimental concentration range, the cytotoxicity gradually increased with the increase of Pt concentration. This demonstrated that the Pt@E5 nanozyme assembly had good anti-tumor effects. Cell survival percentage (%) =

(absorbance of the experimental group - blank control 2) / (absorbance of the control group - absorbance of blank control 1)

[0060] Example 8 Uptake of Pt@E5 nanoparticles by A20 cells

[0061] A20 cells were seeded into 6-well plates at a certain density (5 * 10 6 cells / well), and then the NHS-FAM modified Pt@E5 nanozyme assembly was added and incubated for 6 h and 12 h. Then the cells were collected and washed, and green fluorescence detection was performed using flow cytometry. To compare the uptake effects of platinum nanoparticles with different surface modifications by A20 cells, platinum nanoparticles modified with SH-PEG-NH 2 (Pt@PEG-NH 2 ) were used as the control. The surface of Pt@PEG-NH 2 nanoparticles was also modified with NHS-FAM. The results are as Figure 12 shown. Compared with Pt@PEG-NH 2 nanoparticles, the E5-functionalized platinum nanozyme assembly (Pt@E5) was taken up more by A20 cells at 12 h of incubation, and the difference was significant, which was beneficial for the subsequent therapeutic effects of the nanoparticles to play.

[0062] Example 9 Testing of tumor cell migration ability

[0063] MS-5 cells (5 × 10 5(cells / well) were inoculated into the lower chamber of a 24-well plate and incubated overnight. A20 cells (2×10 4 (cells / well)) pretreated with different nanoparticles (final concentration of Pt at 5 μg / mL) for 12 h were inoculated into the upper chamber of Transwell-24. After incubating with MS-5 cells in the lower chamber for 24 h, the number of A20 cells migrated to the lower chamber was counted using a cell counter. And the relative migration rates of A20 cells in different treatment groups (Pt@E5, Pt@PEG-NH 2 ) were calculated. And treatment with PBS was used as the blank control group. The results are as Figure 13 shown. The migration degrees of A20 cells in different treatment groups were different. Among them, the migration degree of A20 cells to MS-5 cells was significantly inhibited in the presence of the Pt@E5 nanozyme assembly, which was significantly less than that of the nanoparticles without E5 modification (Pt@PEG-NH 2 ), indicating that the Pt@E5 nanozyme assembly can effectively block the CXCR4-CXCL12 biological axis, which is beneficial for the subsequent therapeutic effect of the nanoparticles.

[0064] Migration rate of A20 cells =

(C - A) / (B - A)

[0065] A = the number of cells that leaked naturally without any treatment of A20 cells when there were no MS-5 cells in the lower chamber;

[0066] B = the number of cells that migrated naturally downward without any treatment of A20 cells when there were MS-5 cells in the lower chamber;

[0067] C = the number of cells that migrated downward after treatment of A20 cells with PBS or platinum nanoparticles when there were MS-5 cells in the lower chamber.

[0068] Example 10 Reactive oxygen species level in cells

[0069] Using A20 cells as a cell model to evaluate the effect of the material on the intracellular ROS level. Cells were inoculated into a 6-well plate at a density of 2×10 5 cells per well, and then Pt@E5 nanozyme assembly or Pt@PEG-NH 2 (final concentration of Pt at 30 μg / mL) was added to each well and incubated in 5% CO 2, Incubate for 6 h at 37°C. The medium used was RPMI-1640 complete medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS. After incubation, the cells in all wells were collected, centrifuged to discard the original medium, washed 3 times with PBS, and then co-incubated with DCFH-DA probe diluted with serum-free medium for 30 min. Discard the medium, wash 3 times with PBS, and then detect the results by fluorescence microscopy. The DCFH-DA probe can be oxidized by ROS in cells to DCF showing green fluorescence. And treat with PBS as a blank control group. The results are as Figure 14 shown. Compared with the Pt@PEG-NH 2 group, stronger green fluorescence was shown in the cells of the Pt@E5 nanozyme assembly group, indicating that more Pt@E5 nanozyme assemblies were taken up by A20 cells, which would induce more ROS production in the cells.

[0070] Glutathione level in cells of Example 11

[0071] Using A20 cells as a cell model to evaluate the effect of the material on the intracellular glutathione (GSH) level. Seed the cells at a density of 2×10 5 cells per well into a 6-well plate, and then add Pt@E5 nanozyme assembly or Pt@PEG-NH 2 (final Pt concentration is 5 μg / mL) and incubate at 5% CO 2 2, 37°C for 24 h. The medium used was RPMI-1640 complete medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS. Then, collect the cells in the well plate, centrifuge, freeze-thaw and break them repeatedly under liquid nitrogen and 37°C conditions, and use a GSH detection kit (Yakeyin Biotechnology Co., Ltd., China) to detect the intracellular GSH content. And treat with PBS as a blank control group. The results are as Figure 15 shown. The Pt@E5 nanozyme assembly significantly reduced the intracellular GSH level, because more E5 polypeptide-functionalized platinum nanozyme assemblies were taken up by the cells, which could produce reactive oxygen species (ROS) through the "Fenton reaction" in the cells, thereby consuming GSH and further affecting the intracellular redox state.

[0072] Detection of tumor cell apoptosis in Example 12

[0073] Using A20 cells as a cell model to evaluate the killing effect of the material prepared in Example 1 on cancer cells. Collect A20 cells in the logarithmic growth phase and seed them at a density of 2×10 5 cells per well into a 6-well plate, and then add Pt@PEG-NH 2 or Pt@E5 nanozyme assembly (final Pt concentration is 2 μg / mL) at 5% CO2 , Incubate for 24 h at 37 °C. The medium used was RPMI-1640 complete medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS. After incubation, the cells in all wells were collected, centrifuged, washed 3 times with PBS, and then co-incubated with AnnexinV-FITC and PI, a cell apoptosis detection reagent diluted 100-fold with PBS, for 10 min. The apoptosis and necrosis of the cells were detected by flow cytometry. Treatment with PBS was used as a blank control group. The results are as Figure 16 shown. There was no obvious cell apoptosis in the PBS group. Compared with the Pt@PEG-NH 2 group, the Pt@E5 nanozyme assembly group had a higher proportion of late apoptotic cells, indicating that more Pt@E5 nanozyme assemblies were taken up by A20 cells, which would cause the cells to produce more ROS and then induce apoptosis. This is due to the cell damage effect caused by chemodynamic therapy.

[0074] Example 13 Detection of immunogenic cell death of tumor cells

[0075] To verify the ICD effect caused by the nanomaterials prepared in Example 1 after treating tumors, A20 cells were used as a cell model to evaluate the expression levels of ICD markers high-mobility group protein (HMGB-1) and adenosine triphosphate (ATP) after treatment with Pt@E5 nanozyme assemblies or Pt@PEG-NH 2 , and the maturation level of dendritic cells in the bone marrow induced by ICD was evaluated by Transwell assay.

[0076] To evaluate the effect of the materials prepared in Example 1 on the release of HMGB-1 and ATP by cells, cancer cells in the logarithmic growth phase were collected and seeded into 6-well plates at a density of 2×10 5 cells. Then, Pt@PEG-NH 2 or Pt@E5 nanozyme assembly (final Pt concentration of 30 μg / mL) was added to each well and incubated at 5% CO 2 2, 37 °C for 24 h. The medium used was RPMI-1640 complete medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS. After incubation, the culture medium was collected, and the relative contents of HMGB-1 and ATP in the cell culture medium were calculated using an ELISA kit for HMGB-1 and an ATP detection kit, respectively. Treatment with PBS was used as a blank control group. The results are as Figure 17 and Figure 18 shown. The release amounts of HMGB-1 and ATP in the Pt@E5 nanozyme assembly group increased significantly. Pt@PEG-NH 2Compared with the PBS group, the release amounts of HMGB-1 and ATP in the nanomaterial group only increased to a certain extent but were much lower than those in the Pt@E5 nanozyme assembly group. This result indicates that the chemodynamic therapy induced by the drug Pt@E5 nanozyme assembly used in the present invention can promote the release of HMGB-1 and ATP to varying degrees, induce immunogenic cell death, and activate dendritic cells to achieve the activation of anti-tumor immune responses.

[0077] To evaluate the maturation level of dendritic cells induced by cancer cells treated with the materials prepared in Example 1, A20 cells and dendritic cells extracted from bone marrow were used as cell models, and the maturation level of dendritic cells was verified by Transwell experiments. Logarithmically growing A20 cells were inoculated into the upper chamber of a 12-well Transwell plate at a density of 1×10 5 cells, and then Pt@PEG-NH 2 or Pt@E5 nanozyme assembly (final Pt concentration of 30 μg / mL) was added to each well and incubated at 5% CO 2 2, 37 °C for 24 h. The medium used was RPMI-1640 complete medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS. Meanwhile, logarithmically growing dendritic cells were inoculated into the lower chamber of a 12-well Transwell plate at a density of 1×10 5 cells. The medium used was RPMI-1640 complete medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS, and it was cultured at 5% CO 2 2, 37 °C. After co-incubating and culturing A20 cells with the materials for 24 h, the original medium was collected. Subsequently, the medium in dendritic cells was replaced, and the original medium of A20 cells treated with different materials was transferred to the upper chamber of dendritic cells and co-cultured for 24 h. Then, the lower-layer dendritic cells were collected and co-incubated with dendritic cell maturation marker proteins CD11c-FITC, CD80-PE, and CD86-APC antibodies in the dark at room temperature for 30 min. Subsequently, they were washed 3 times with PBS and resuspended with PBS, and the maturation of dendritic cells was detected by flow cytometry. The results are as Figure 19 shown. The expression levels of CD80 and CD86 in the Pt@E5 nanozyme assembly-treated group increased significantly, indicating that the Pt@E5 nanozyme assembly can cause strong immunogenic cell death and further induce dendritic cell maturation. And the expression level of Pt@PEG-NH 2 was slightly lower than that of the Pt@E5 nanozyme assembly, indicating that cells showed stronger recognition and phagocytosis of the Pt@E5 nanozyme assembly modified with E5 polypeptide, which can enhance the effect of immunogenic cell death induced by nanodrugs, thus making the Pt@E5 nanozyme assembly group have a stronger dendritic cell maturation effect.

[0078] In summary, the nanomaterials prepared in Example 1 can effectively induce immunogenic death of cancer cells, promote the secretion of HMGB-1 and ATP by cells, and induce the maturation of dendritic cells. At the same time, the modification of E5 polypeptide enhances the recognition and phagocytosis of nanomaterials by A20 cells, further enhancing the level of immunogenic death induced by nanodrugs and activating the anti-tumor immune response.

[0079] Therapeutic effect on mice with Example 14 B-cell lymphoma.

[0080] All animal experiments were carried out strictly in accordance with the standards of the Animal Protection Association. Female BALB / c white mice aged 6-7 weeks used in the experiments were purchased from Jiangsu Qinglongshan Experimental Animal Center. A B-cell lymphoma model was constructed in mice by injecting 1×10 8 A20-Luc cells into the tail vein of mice. After waiting for one week, the mice were intraperitoneally injected with potassium D-luciferin (150 mg / kg), and then the disease status of the mice was detected by small animal in vivo imaging technology. If bioluminescence signals could be detected, the modeling was successful. The successfully modeled mice were randomly divided into 4 groups (n = 5 mice in each group). The specific grouping was as follows: control group (PBS, 200 μL); low-dose Pt group, medium-dose Pt group, high-dose Pt group (Pt doses were 1, 3, and 5 mg / kg respectively, 200 μL), and the injection method was tail vein injection. The day when the treatment started was recorded as day 1, and the treatment was carried out once every 3 days for a total of 5 times, and the body weight of the mice was recorded every 2 days. The results were as Figure 20 shown. The body weights of mice in each group of Pt@E5 nanozyme assemblies with different doses had a similar and stable change trend, indicating that the prepared Pt@E5 nanozyme assemblies had good biocompatibility in vivo and no obvious toxicity. The disease status of the mice during the treatment was as Figure 21 shown. Compared with the PBS group, each treatment group of Pt@E5 nanozyme assemblies with different doses showed different degrees of inhibitory effects. Compared with the PBS group and the low-dose and medium-dose Pt groups, the disease status of the mice treated with high-dose Pt was effectively alleviated ( Figure 21 b), indicating that the Pt@E5 nanozyme assemblies can effectively relieve tumors in vivo and improve the treatment effect.

Claims

1. A method for preparing a platinum nanozyme functionalized with an E5 polypeptide, characterized in that: The method comprises the following steps: mixing a platinum precursor solution and a cysteine-modified polypeptide E5 solution, then dripping a sodium borohydride solution under ultrasonic conditions while mechanically stirring, and after the reaction is completed, ultrafiltration and pure water washing are performed to obtain the E5 polypeptide functionalized platinum nanozyme.

2. The method for preparing the E5 polypeptide functionalized platinum nanozyme according to claim 1, characterized in that: The platinum precursor solution is a chloroplatinic acid solution.

3. The method for preparing the E5 polypeptide functionalized platinum nanozyme according to claim 1, characterized in that: The molar concentration of the polypeptide E5 solution is 0.03-0.05 mM.

4. The method for preparing the E5 polypeptide functionalized platinum nanozyme according to claim 1, characterized in that: The molar concentration of the sodium borohydride solution is 263-265 mM.

5. The method for preparing the E5 polypeptide functionalized platinum nanozyme according to claim 1, characterized in that: The volume ratio of the H2PtCl6·6H2O solution, the polypeptide E5 solution and the sodium borohydride solution is 1:8:

1.

6. The method for preparing the E5 polypeptide functionalized platinum nanozyme according to claim 1, characterized in that: The speed of the mechanical stirring is 900-1100 rpm, the reaction temperature is 20-30° C., and the ultrasonic power is 90-110W.

7. The method for preparing the E5 polypeptide functionalized platinum nanozyme according to claim 1, characterized in that: The molecular weight of the ultrafiltration centrifuge tube is 30KDa, the centrifugal speed is 1000-3000rpm, the centrifugal time is 5-15min, and the number of centrifugation is 3-5 times.

8. The platinum nanozyme prepared according to the method for preparing the E5 polypeptide functionalized platinum nanozyme according to any one of claims 1 to 7.

9. Use of the platinum nanozyme according to claim 8 in the preparation of a drug for treating diseases related to the CXCR4-CXCL12 biological axis.

10. The use according to claim 9, characterized in that: The diseases associated with the CXCR4-CXCL12 biological axis include acute myeloid leukemia, breast cancer or lymphoma.

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