Selenium-based targeted protein degradation nano material as well as preparation method and application thereof

Abstract

The invention belongs to the field of biomedical technology and nano material preparation, and discloses a selenium-based targeted protein degradation nano material as well as a preparation method and application thereof. An antibody of a target protein is connected to the surface of nano-selenium through dopamine hydrochloride, and the universal selenium-based targeted protein degradation nano-material is obtained. The preparation method is simple in step and easy to implement, and the development process of the traditional TPD technology is simplified. In addition, the selenium-based targeted protein degradation nano material can also degrade membrane protein and cytoplasm protein at the same time, and the limitation that the membrane protein or cytoplasm protein can only be selectively degraded in the existing TPD technology is overcome, so that the curative effect of immunotherapy is enhanced. In addition, under X rays, the selenium-based targeted protein degradation nano material has a radiation sensitization effect. Therefore, the selenium-based targeted protein degradation nano material can be used for preparing antitumor drugs or used as an X-ray sensitizer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine and nanomaterial preparation, and particularly relates to a selenium-based targeted protein degradation nanomaterial and a preparation method and application thereof. BACKGROUND

[0002] Targeted protein degradation (TPD) technologies, such as proteolysis-targeting chimeras (PROTACs) and molecular glues, have emerged as an effective approach to selectively eliminate disease-associated proteins. Compared with traditional small molecule inhibitors and antibodies, this technology mainly relies on the ubiquitin-proteasome system (UPS) to promote the degradation of intracellular proteins, thus opening up new avenues for various proteins that are difficult to administer. However, this technology can only degrade intracellular proteins containing cytoplasmic domains, and cannot be used for the degradation of non-cytoplasmic proteins (about 40% of human disease-associated proteins). In order to achieve the targeted degradation of these proteins, people have been committed to the development of lysosome-targeting chimeras (LYTACs), thus promoting the direct lysosomal transport of non-cytoplasmic proteins to achieve TPD. However, traditional small molecule-based or antibody / aptamer-based LYTACs have low delivery efficiency in tumor tissues, and there are significant off-target effects, and their therapeutic efficacy is still limited. In addition, significant differences exist in target proteins (POIs) and LTRs among different patients or even cells of the same patient. This intercellular difference forces LYTACs to be designed in a complex individualized manner for different disease types, and also seriously restricts their large-scale production and clinical translation.

[0003] In recent years, nanotechnology has provided innovative solutions for optimizing tissue penetration, tumor-targeted accumulation and biodistribution modulation of therapeutic drugs due to its unique physicochemical properties. More importantly, the inherent lysosomal self-targeting ability and highly programmable surface functionalization characteristics of nanomaterials provide a key technical basis for the development of LYTACs platform. Although researchers have made a series of important progress in the TPD research of Nano-LYTACs based on nanomaterials, its clinical research is still in the stage of empirical research, and there are still some key technical problems to be solved: (1) As the disease progresses, POIs are usually dynamically distributed and abnormally expressed in different locations of cells, for example, PD-L1 protein is not only expressed on the membrane of tumor cells, but also expressed in the cytoplasm, thereby mediating the occurrence of radiation resistance and immune tolerance, however, the current Nano-LYTACs based on nanomaterials are still mainly limited to the degradation of membrane-associated protein targets; (2) The current research lacks comprehensive evaluation of the synergistic treatment of Nano-LYTACs with clinical treatment, including radiotherapy and immunotherapy; (3) The encapsulation efficiency of Nano-LYTACs for target proteins is relatively low, and most of them are achieved through non-covalent interactions, which is easy to cause off-target effects; (4) The safety and clinical transformation potential of most reported Nano-LYTAC platforms need to be further considered. Therefore, there is an urgent need for the development of a transformed Nano-LYTAC platform that can covalently bind to a targeting ligand and then synergistically degrade membrane and cytoplasmic proteins.

[0004] In the nano platform, selenium (Se) has a good ability to maintain human health as an essential trace element for the human body. Previous studies have found that selenium nanomaterials (SeNPs) can not only act as radiosensitizers for a variety of tumors, but also enhance immune cells, including NK cells, γδT cells and CIK cells, thereby inducing a strong anti-tumor immunity. In addition, more and more evidence shows that after selenium is internalized in cells, it inhibits the expression of disease-related proteins including cytoplasmic PD-L1 by promoting the synthesis of selenium proteins, thereby inhibiting the growth of cancer cells. These signs indicate that engineered SeNPs can solve the above challenges in terms of extracellular and intracellular POIs degradation. Therefore, further construction of a targeted protein degradation system based on nano-selenium is expected to solve the limitations of current TPD technology, expand the biological utilization range of TPD technology, and realize its clinical application in tumor treatment. SUMMARY

[0005] The present application aims to overcome the shortcomings and deficiencies of the prior art, and provides a preparation method of selenium-based targeted protein degradation nanomaterials.

[0006] Another object of the present application is to provide a selenium-based targeted protein degradation nanomaterial obtained by the above preparation method.

[0007] Still another object of the present application is to provide the application of the selenium-based targeted protein degradation nanomaterial.

[0008] The object of the present application is achieved by the following technical solutions:

[0009] A preparation method of a selenium-based targeted protein degradation nanomaterial comprises the following steps:

[0010] (1) Adjust the pH of a nanoseelenium aqueous solution to alkaline, then add a dopamine hydrochloride solution under stirring, and stir to react; after the reaction is completed, solid-liquid separation is performed, and the solid is taken out;

[0011] (2) Resuspend the solid obtained in step (1) with water to obtain a Se / PDA nanocarrier solution;

[0012] (3) Add an antibody solution targeting the target protein to the Se / PDA nanocarrier solution, stir to react, perform solid-liquid separation, resuspend the obtained solid with water, and obtain a selenium-based targeted protein degradation nanomaterial.

[0013] The nanoseelenium in step (1) is preferably prepared by the following steps: adding an aqueous solution of a reducing agent to an aqueous solution of a soluble inorganic selenium salt under stirring, stirring to react in an ice bath, and then dialyzing to obtain nanoseelenium.

[0014] The reducing agent is preferably ascorbic acid.

[0015] The soluble inorganic selenium salt is preferably sodium selenite.

[0016] The amount of the reducing agent is excessive relative to the soluble inorganic selenium salt; preferably, the molar ratio of the reducing agent to the soluble inorganic selenium salt is 4:1.

[0017] The stirring speed of the stirring reaction is preferably 200-400 rpm; more preferably, the stirring speed is 300 rpm.

[0018] The stirring time of the stirring reaction is preferably 8-16 h.

[0019] The dialysis time is preferably 24-72 h; more preferably, the dialysis time is 40-60 h.

[0020] The pH in step (1) is preferably 8.0-10.0; more preferably, the pH is 9.0.

[0021] The amount of dopamine hydrochloride in step (1) is preferably calculated as 3.5-4.5 mg of dopamine hydrochloride per 100 μmol of nanoseelenium; more preferably, the amount of dopamine hydrochloride is calculated as 4 mg of dopamine hydrochloride per 100 μmol of nanoseelenium.

[0022] The stirring speed in step (1) is preferably 200-400 rpm; more preferably 300 rpm.

[0023] The stirring reaction time in step (1) is preferably 8-16 h; more preferably 12 h.

[0024] The solid-liquid separation mode in step (1) is preferably centrifugation.

[0025] The centrifugation condition is preferably 10000-12000 rpm for 15-30 min.

[0026] The target protein in step (3) is preferably a surface receptor of tumor cells; more preferably PD-L1 or Her2.

[0027] The antibody in step (3) is a monoclonal antibody; the amount thereof is preferably 9-11 mg of antibody per 100 μmol of nano-selenium; more preferably 10 mg of antibody per 100 μmol of nano-selenium.

[0028] The stirring speed in step (3) is preferably 200-400 rpm; more preferably 300 rpm.

[0029] The stirring reaction time in step (3) is preferably 8-16 h; more preferably 12 h.

[0030] The solid-liquid separation mode in step (3) is preferably centrifugation.

[0031] The centrifugation condition is preferably 10000-12000 rpm for 15-30 min.

[0032] The water used in the present application is preferably ultrapure water.

[0033] A selenium-based targeted protein degradation nanomaterial is obtained by the above preparation method: the average particle size thereof is about 150 nm.

[0034] The above selenium-based targeted protein degradation nanomaterial can simultaneously degrade multiple target proteins in the cytoplasm and cell membrane in various diseases, thereby inducing a strong radioimmunotherapy effect. At the same time, the selenium-based targeted protein degradation nanomaterial enhances the absorption of X-rays, generates abundant ROS, and induces a large amount of DNA damage, thereby playing a radiosensitization role.

[0035] Therefore, the above selenium-based targeted protein degradation nanomaterial is used in the preparation of an antitumor drug.

[0036] The tumor is preferably a digestive system cancer or a reproductive system cancer.

[0037] The digestive system cancer is preferably esophageal cancer or intestinal cancer.

[0038] The intestinal cancer is preferably colon cancer.

[0039] The reproductive system cancer is preferably cervical cancer; more preferably, cervical squamous cell carcinoma.

[0040] The selenium-based targeted protein degradation nanomaterial is applied as an X-ray radiotherapy sensitizer.

[0041] The present application has the following advantages and effects relative to the prior art:

[0042] 1. The selenium-based targeted protein degradation nanomaterial provided by the present application can introduce various target ligand antibodies at any time to achieve targeted degradation of target proteins of various diseases, and has universality. Meanwhile, the synthesis process is simple and fast, and large-scale production can be realized, which makes the selenium-based targeted protein degradation nanomaterial have a potential clinical transformation application prospect.

[0043] 2. The selenium-based targeted protein degradation nanomaterial provided by the present application improves the tumor penetration and the ability to target tumor tissues of the existing TPD technology.

[0044] 3. The selenium-based targeted protein degradation nanomaterial provided by the present application can realize simultaneous degradation of membrane proteins and cytoplasmic proteins, overcoming the limitation that the existing TPD technology can only selectively degrade membrane proteins or cytoplasmic proteins.

[0045] 4. With the photoelectric effect and Compton scattering characteristics of selenium, the selenium-based targeted protein degradation nanomaterial provided by the present application can generate abundant ROS, induce a large amount of DNA damage, and thus play a radiation sensitization role.

[0046] 5. The selenium-based targeted protein degradation nanomaterial provided by the present application synergizes with other clinical treatment methods (radiotherapy, immunotherapy), improves the efficacy of radioimmunotherapy, realizes effective inhibition of tumor malignant progression, and provides a reference basis for the efficacy and mechanism of the existing TPD technology combined with other therapies. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 A transmission electron microscope image of the selenium-based targeted protein degradation nanomaterial prepared by the present application.

[0048] Figure 2 A detection result graph of the degradation ability of the selenium-based targeted protein degradation nanomaterial prepared by the present application on different tumor cell membrane proteins.

[0049] Figure 3 A detection result graph of the degradation ability of the selenium-based targeted protein degradation nanomaterial prepared by the present application on tumor cytoplasmic proteins.

[0050] Figure 4The image shows the detection results of the radiosensitizing ability of the selenium-based targeted protein degradation nanomaterials prepared in this invention.

[0051] Figure 5 The figure shows the detection results of the in vivo PD-L1 inhibition ability of the selenium-based target protein degradation nanomaterials prepared in this invention.

[0052] Figure 6 The graph shows the in vivo immune activation ability of the selenium-based targeted protein degradation nanomaterials prepared in this invention. Detailed Implementation

[0053] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0054] Example 1: Preparation of selenium-based targeted protein degradation nanomaterials (SeTAC)

[0055] First, 0.07045 g of ascorbic acid and 0.01729 g of sodium selenite were weighed and dissolved in 10 mL of ultrapure water to prepare a 40 mM ascorbic acid solution and a 10 mM sodium selenite stock solution, which were stored at 4 °C for later use. Then, ascorbic acid was slowly added to the sodium selenite solution, and the mixture was stirred overnight in an ice bath at 300 rpm. After two days of dialyzing with ultrapure water, a nano-selenium (SeNPs) solution was obtained. Next, the pH of the above nano-selenium solution was adjusted to 9 with NaOH solution, and 1 mL of a 4 mg / mL dopamine hydrochloride solution was gradually added, while stirring at 300 rpm for 12 h. After the reaction was completed, the mixture was centrifuged at 12000 rpm for 20 minutes, and the supernatant was discarded. The resulting precipitate was resuspended in 5 mL of ultrapure water to obtain a Se / PDA nanocarrier solution, which forms the model chassis of a selenium-based targeted protein degradation nanoplatform. Finally, 1 mL of PD-L1 monoclonal antibody solution with a concentration of 10 mg / mL was added dropwise to the Se / PDA nanocarrier solution, and the mixture was stirred at 300 rpm for 12 h. After centrifugation, the solution was resuspended in ultrapure water to obtain a selenium concentration of 70 mg / L, which was named SeTAC.

[0056] Example 2 Characterization of selenium-based targeted protein degradation nanomaterials (SeTAC)

[0057] The transmission electron microscope image of the selenium-based targeted protein degradation nanomaterial prepared in Example 1 is shown below. Figure 1 ,Depend on Figure 1 It can be seen that the prepared selenium-based targeted protein degradation nanomaterials have uniform particle size and good dispersion, with a particle size of about 150 nm; moreover, dopamine is uniformly coated on the surface of the selenium nanoparticles.

[0058] Example 3: Degradation effect of selenium-based targeted protein degradation nanomaterials (SeTAC) on membrane proteins.

[0059] CT26 cells, KYSE-150 cells and Siha cells (purchased from American Type Culture Collection, ATCC) were incubated in 6-well plates at 1 x 10 5 cells per well and provided with 2 mL of RPMI-1640 medium containing 10% FBS. After 24 hours of culture, the medium was discarded. The group of each well added with 2 mL of RPMI-1640 medium was the control group (Control); the group of each well added with 2 mL of RPMI-1640 medium containing 0.007 mg / mL Anti-PD-L1 was the Anti-PD-L1 group; the group of each well added with 2 mL of RPMI-1640 medium containing 2 mg / L Se / PDA was the Se / PDA group; the group of each well added with 2 mL of RPMI-1640 medium containing 2 mg / L Se TAC was the Se TAC group. The X-Ray group was the control group which was irradiated with 4 Gy dose of rays after 4 h of incubation with fresh RPMI-1640 medium; the Se / PDA+X-Ray group, the Anti-PD-L1+X-Ray group and the Se TAC+X-Ray group were the Se / PDA group, the Anti-PD-L1 group and the Se TAC group which were irradiated with 4 Gy dose of rays after 4 h of incubation with drugs, respectively. Three parallels were set for each experimental group. After 24 h of incubation, the cells were collected and washed with PBS for three times. Finally, the washed cells were stained with PD-L1 flow cytometry antibody (BV510) and detected by flow cytometry. The results are shown in A-C of FIG. 6. Figure 2 In the CT26, KYSE150 and Siha cell models, compared with the Se / PDA group and the Anti-PD-L1 group, the Se TAC nanoparticles can cause a significant decrease in the expression of membrane protein PD-L1, regardless of whether there is X-ray irradiation, which reflects that the Se TAC can effectively hijack the membrane PD-L1 protein and guide it to lysosomal degradation through the nanoparticle-mediated endocytosis pathway. In contrast, neither the Se / PDA group nor the Anti-PD-L1 group can induce the degradation of PD-L1 in CT26 cells. This phenomenon may be due to the lack of specific POI binding ligands and functional lysosome targeting components, thereby preventing lysosome pathway-mediated targeted POI degradation.

[0060] To further verify the role of the lysosome pathway in PD-L1 degradation, CT26 cells were incubated in 6-well plates at 1 x 10 6Cells were incubated in 10 cm dishes and provided with 10 mL of RPMI-1640 medium containing 10% FBS. After cell adhesion, 100 μL of 10 μM bafimycin A1 (Baf) was added for pretreatment for 2 h, followed by aspiration and washing three times with PBS. Baf is an effective pharmacological inhibitor of vacuolar H+-ATPase (V-ATPase), which can inhibit the acidification of organelles such as lysosomes, thereby leading to dysregulation of lysosomal degradation and inhibition of autolysosomes. Next, 10 mL of RPMI-1640 culture solution containing 5 mg / L SeTAC was added to obtain the SeTAC group; a control group was also set up using 10 mL of RPMI-1640 medium. The control group and the SeTAC group were incubated for 4 h and then irradiated with 4 Gy of X-rays to obtain the X-Ray group and the SeTAC+X-Ray group. After 24 h of drug incubation, CT26 cell proteins from different treatment groups were extracted with RIPA lysis buffer, and the protein samples were quantified using the BCA method. Next, the proteins were separated by 10% SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane. After blocking with 5% skim milk, the membrane was incubated with an antibody against the target protein, and the expression level of PD-L1 was then observed using a contrast agent and imaging system. The experiment was repeated three times. Figure 2 As shown in Figure D, Baf significantly reduced the PD-L1 degradation induced by the Se TAC group, confirming that Se TAC promotes PD-L1 proteolysis via the autophagy-lysosomal pathway. To apply Se TAC to a wider range of clinically relevant disease targets, the degradation of various oncoproteins was achieved by modularly replacing the POI-specific targeting ligand. 1 mL of a 10 mg / mL Her2 monoclonal antibody solution was added dropwise to 5 mL of a 60 mg / L Se / PDA nanocarrier solution, and stirred at 300 rpm for 12 h. After centrifugation and resuspending in ultrapure water, a second selenium-based protein-degrading nanomaterial was obtained, named Se / PDA-anti-Her2. CT26 cells were cultured at 1 × 10⁻⁶ cells per well. 6The cells were incubated in a 10 cm dish and provided with 10 mL of RPMI-1640 medium containing 10% FBS. After the cells adhered, the medium was discarded, and 10 mL of RPMI-1640 medium containing 5 mg / mL Se / PDA-anti-Her2 was added to obtain the Se / PDA-anti-Her2 group; at the same time, a control group was set up using only 10 mL of RPMI-1640 medium for culture. The control group and the Se / PDA-anti-Her2 group were incubated for 4 h and then given a 4 Gy dose of radiation to obtain the X-Ray group and the Se / PDA-anti-Her2+X-Ray group. After 24 h of drug incubation, the membrane proteins of the CT26 cells in the different treatment groups were extracted using a membrane protein extraction kit, and the sample proteins were quantified by the BCA method. Next, the proteins were separated by 10% SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane. After blocking with 5% skim milk, the membrane was incubated with the antibody of the target protein, and then the expression level of PD-L1 on the membrane was observed by developing agent and imaging system. The results are shown in Figure 2 As shown in E of FIG. 10, the intervention of Se / PDA-anti-Her2 effectively inhibited the expression level of HER2 on the membrane of tumor cells, demonstrating the versatility and membrane protein degradation ability of selenium-based targeted protein degradation nanomaterials.

[0061] wherein the protein expression fold is calculated as follows: the gray value of Her2 / the gray value of β-actin is calculated for each group, and then each group is divided by the gray value of Her2 of the control group based on the calculated gray value of Her2 of the control group.

[0062] In addition, this selenium-based protein degradation nanoparticle with flexibility and versatility can also be suitable for a series of clinical treatments including tumor diseases.

[0063] Example 4 Degradation effect of cytoplasmic protein by selenium-based targeted protein degradation nanomaterial (Se TAC)

[0064] One million CT26 intestinal cancer cells were transplanted into a 10 cm dish and incubated overnight, and then different treatment groups of drugs were added, following the operation of Example 3, to obtain the control group, the Se TAC group, the X-Ray group, and the Se TAC combined with X-Ray group. The concentration of Se TAC solution was 5 mg / L, and the drug solvent was RPMI-1640 medium, with three repeated experiments, and one experiment counted as one parallel. After 12 h and 24 h of drug incubation, the cytoplasmic proteins of the CT26 cells in the different treatment groups were extracted using a cytoplasmic extraction kit, and the sample proteins were quantified by the BCA method. Next, the proteins were separated by 10% SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane. After blocking with 5% skim milk, the membrane was incubated with the antibody of the target protein, and then the expression level of cytoplasmic PD-L1 protein was observed by developing agent and imaging system. The results are shown inFigure 3 As shown, after 24 hours of X-ray irradiation with Se TAC, the expression of PD-L1 in the cytoplasm of CT26 cells was significantly reduced, reflecting the ability of Se TAC to degrade cytoplasmic proteins.

[0065] Example 5: Radiosensitizing effect of selenium-based targeted protein degradation nanomaterials (SeTAC)

[0066] Cytotoxicity assay of Se TAC: CT26 colon cancer cells were transplanted into 96-well plates at a density of 2000 cells / well. After 24 hours, different concentrations of SeNPs solution, Se / PDA solution, and Se TAC solution (0.3, 0.6, 1.3, 2.5, and 5 mg / L, in RPMI-1640 medium) were added to each well at a concentration of 100 μL, with three parallel wells. After 48 hours, 15 μL of 5 mg / mL MTT solution was added and incubated for 3 hours. The medium was then removed and 150 μL of DMSO solution was added. The absorbance at 490 nm was measured.

[0067] Cytotoxicity assay of Se TAC combined with X-ray: CT26 colon cancer cells were transplanted into 96-well plates at a rate of 2000 cells / well. After 24 hours, different concentrations of SeNPs solution, Se / PDA solution, and Se TAC solution (0.3, 0.6, 1.3, 2.5, and 5 mg / L, in RPMI-1640 medium) were added to each well at 100 μL. After incubation for 4 hours, the plates were irradiated with different doses of X-rays (2 Gy, 1 min and 4 Gy, 2 min) in three parallel wells. After 48 hours, 15 μL of 5 mg / mL MTT solution was added and incubated for 3 hours. Then, the medium was removed and 150 μL of DMSO solution was added, and the absorbance at 490 nm was measured.

[0068] The results are as follows Figure 4 As shown in Figure A, after treatment with Se TAC solution alone, the cell viability of CT26 cells gradually decreased with increasing concentration, reaching 50% at 1.1 mg / L. Furthermore, from... Figure 4 In Figure B (where a higher half-inhibitory concentration (WIC) value corresponds to a bluer color, and a lower WIC value to a more orange color), the results show that after exposure to 2 Gy and 4 Gy radiation doses, the WIC of Se TACs on CT26 cells (the drug concentration at which tumor cell survival is 50%) decreased from 1.1 mg / L to 0.5 mg / L and 0.2 mg / L, respectively. Meanwhile, the WICs of SeNPs and Se / PDA on CT26 cells at 2 Gy and 4 Gy radiation were 2.9 mg / L, 2.1 mg / L and 2.0 mg / L, 3.2 mg / L, respectively. Furthermore, the addition of anti-PD-L1 did not have significant toxic side effects on cells at 2 Gy and 4 Gy radiation (see Figure B).Figure 4 D) in FIG. 6. This result shows that Se TACs have the most significant inhibitory effect on CT26 intestinal cancer cell proliferation compared with SeNPs, Se / PDA and anti-PD-L1, and the addition of Se TACs can significantly enhance X-Ray-induced cytotoxicity after combined X-Ray treatment. The synergistic effect between Se TACs and X-rays was analyzed by reference isobologram. If the data points are below the straight line, it indicates that Se TACs have X-ray synergistic sensitization effect. If the data points are above the straight line, it means that Se TACs and X-rays are in an antagonistic relationship. If the data points are on the straight line, it is additive effect. The results are shown in FIG. 6. Figure 4 As shown in C in FIG. 6, Se TACs have superior radiosensitization effect.

[0069] Example 6 In vivo PD-L1 inhibition ability of selenium-based targeted protein degradation nanomaterials (Se TACs)

[0070] BALB / c female mice (4-6 weeks old) with an average weight of 20 g each were purchased from Guangdong Yaoke Biotechnology Co., Ltd. To establish a mouse subcutaneous tumor model, CT26 cells (6x10 5 were injected into the right lower limb subcutaneous site of female BALB / c mice (18-20 g). After 14 days of tumor formation in mice, the mice were randomly divided into 5 groups (8 mice in each group): (1) the control group was injected with normal saline, with a dose of 100 μL per mouse; (2) the X-Ray group, except that X-ray irradiation was performed once every 7 days, other operations were the same as the control group; (3) the Anti-PD-L1+X-Ray group; (4) the Se TAC+X-Ray group; (5) the Se / PDA combined with anti-PD-L1+X-Ray group. The method of tail vein injection was used to inject anti-PD-L1 solution, Se TAC solution and anti-PD-L1 combined with Se / PDA solution once every 2 days. The dosing amount of Se TAC and Se / PDA solution was 1 mg / kg (concentration was 200 mg / L), and the dosing amount of anti-PD-L1 was 0.686 mg / mL (anti-PD-L1 loading amount was 0.686 mg / mL in every 200 mg / L of Se TAC), and the dosing volume was 100 μL per mouse. X-ray irradiation was performed once every 7 days, and all groups were given normal drinking water and diet. After 21 days of administration, the mice were euthanized, and the tumor tissues of the mice were collected.

[0071] A portion of the tumor tissue was used for flow cytometry analysis of the expression of PD-L1 in tumor cell suspension, a portion was used for Western Blot to detect the expression of PD-L1 in tumor suspension in different treatment groups, and a portion was used for immunohistochemical analysis of PD-L1. The specific steps are as follows: (1) After fixation of the tumor tissue, paraffin-embedded sections were cut; (2) The sections were dewaxed with xylene and hydrated with ethanol of different grades; (3) Boil in citrate buffer or EDTA buffer, place the sections in it and keep it boiling for 10-15 minutes, then cool it to room temperature and wash it with PBS for 3 times; (4) Incubate with 3% H2O2 at room temperature for 10 minutes, wash with PBS, and add 5% BSA or normal serum (homologous to the second antibody) dropwise, and seal at room temperature for 30 minutes; (5) Add diluted first antibody dropwise, cover the tissue, and incubate at 4°C overnight or at 37°C for 1-2 hours, then wash with PBS for 3 times; (6) Add HRP / AP labeled second antibody dropwise, incubate at room temperature for 30-60 minutes, then wash with PBS for 3 times; (7) Add freshly prepared DAB solution dropwise, control the color development time under a microscope, and terminate with distilled water; (8) Mount the sections and examine them under a microscope. A portion was used for flow cytometry analysis of the expression of PD-L1 in tumor cell suspension. The flow cytometry results show (see Fig. A in Figure 5 , Se TACs combined with X-ray can reduce the expression of PD-L1 in tumor tissue, and the percentage of PD-L1 in tumor tissue decreases from 96.3% (control group) to 85.26%. Compared with the anti-PD-L1+X-Ray treatment group (93.1%) and the Se / PDA combined anti-PD-L1+X-Ray treatment group (89.5%), the inhibition effect of Se TACs combined with X-ray on PD-L1 is more obvious. The Western Blot results (see Fig. B in Figure 5 ) further show that Se TACs combined with X-ray can effectively reduce the PD-L1 level of tumor tissue. The quantitative immunohistochemical analysis as shown in Fig. C in Figure 5 shows that compared with the anti-PD-L1+X-Ray treatment group and the Se / PDA combined anti-PD-L1+X-Ray treatment group, Se TACs combined with X-ray have the strongest inhibitory effect on the expression of PD-L1 in tumor tissue. These results also mean that compared with the clinical radioimmunotherapy (anti-PD-L1+X-Ray treatment group), Se TACs exhibit unique protein degradation ability.

[0072] Example 7 In vivo immune activation ability of selenium-based targeted protein degradation nanomaterial (Se TAC)

[0073] The tumor tissues of mice were collected, and flow cytometry was used to detect the expression of immune cells including CD8 + T cells, CD4 + T cells in tumors in different treatment groups. As shown in Fig. A in Figure 6As shown, Se TACs combined with X-ray can effectively enhance T cell infiltration and trigger anti-tumor immune response.

[0074] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.

Claims

1. A method for preparing a selenium-based targeted protein degradation nanomaterial, the method comprising: The preparation method of the selenium-based targeted protein degradation nanomaterial according to any one of claims 1-6. ​ 2. The preparation method of the selenium-based targeted protein degradation nanomaterial according to claim 1, wherein the nanoseelenium in step (1) is prepared by adding an aqueous solution of a reducing agent into an aqueous solution of a soluble inorganic selenium salt under stirring, and then stirring in an ice bath and dialyzing to obtain the nanoseelenium.

3. The preparation method of the selenium-based targeted protein degradation nanomaterial according to claim 2, wherein the reducing agent is ascorbic acid, and the soluble inorganic selenium salt is sodium selenite.

4. The preparation method of the selenium-based targeted protein degradation nanomaterial according to claim 1, wherein the pH in step (1) is 8.0-10.0, the amount of dopamine hydrochloride in step (1) is 3.5-4.5 mg per 100 μmol of nanoseelenium, the target protein in step (3) is a ligand or a receptor of a tumor cell, and the antibody in step (3) is a monoclonal antibody.

5. The preparation method of the selenium-based targeted protein degradation nanomaterial according to claim 4, wherein the pH in step (1) is 9.0, the amount of dopamine hydrochloride in step (1) is 4 mg per 100 μmol of nanoseelenium, the target protein in step (3) is PD-L1 or Her2, and the amount of the antibody in step (3) is 10 mg per 100 μmol of nanoseelenium.

6. The preparation method of the selenium-based targeted protein degradation nanomaterial according to claim 1, wherein the stirring speed in steps (1) and (3) is 200-400 rpm, the stirring time in steps (1) and (3) is 8-16 h, and the solid-liquid separation mode in steps (1) and (3) is centrifugation.

7. The selenium-based targeted protein degradation nanomaterial obtained by the preparation method according to any one of claims 1-6.

8. The selenium-based targeted protein degradation nanomaterial according to claim 7 for use in the preparation of an anti-tumor drug.

9. The use according to claim 8, wherein the tumor is a cancer of the digestive system or a cancer of the reproductive system.

10. The selenium-based targeted protein degradation nanomaterial according to claim 7 for use as an X-ray radiotherapy sensitizer. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 7. A selenium-based targeted protein degradation nanomaterial, characterized by: ​ ​ ​ ​ ​

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