Application of protein nano-selenium in preparation of medicine for preventing and treating acute monocyte leukemia

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of protein nano-selenium in preparation of a medicine for preventing and treating acute monocyte leukemia. The protein nano-selenium prepared by the invention has better stability and safety than inorganic nano-selenium prepared by a traditional method, has higher bioavailability than selenium yeast tablets and inorganic nano-selenium after oral administration, has stronger capability of promoting ferroptosis of leukemia cells, and has a better treatment effect on acute mononuclear leukemia.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to use of protein nano-selenium in preparing a drug for preventing and treating acute monocytic leukemia. Background Art

[0002] Compared to other acute myeloid leukemia (AML) subtypes, acute monocytic leukemia (AML-M5) is often associated with higher aggressiveness, a tendency for extramedullary infiltration, a higher relapse rate, and poor prognosis. Currently, the clinical treatment of AML-M5 relies primarily on chemotherapy and hematopoietic stem cell transplantation. Commonly used chemotherapy drugs include daunorubicin, cytarabine, and homoharringtonine. These drugs kill cancer cells by interfering with their DNA synthesis. However, chemotherapy drugs lack specificity and, while killing cancer cells, can also damage normal cells, leading to serious adverse reactions such as bone marrow suppression leading to leukopenia and thrombocytopenia, and gastrointestinal effects leading to nausea and vomiting. These reactions can cause significant distress to patients, and some patients may develop drug resistance, leading to chemotherapy failure. In recent years, new approaches such as targeted therapy and immunotherapy have emerged, but most remain in the clinical research stage and have yet to completely replace traditional treatments. In addition, the market for oral dosage forms that are easy to administer is growing slowly, with an estimated compound annual growth rate of only 7.60% from 2022 to 2029. This highlights the urgency of developing highly effective, low-toxic, and highly specific oral anti-AML-M5 drugs.

[0003] Selenium, as an essential trace element for the human body, plays a key role in physiological processes such as anti-oxidation and immune regulation. In recent years, a number of studies have shown that selenium deficiency is closely related to the occurrence and development of leukemia, but currently no effective selenium supplementation strategy has been established clinically to treat malignant tumors such as leukemia. The high heterogeneity of AML results in the difference in significance between different subtypes and selenium deficiency levels, and it is still unclear. At the same time, the commonly used selenium-containing supplements / drugs currently available on the market include inorganic selenium salts, organic selenium and nano-selenium (NSe), among which NSe has the best safety and activity. However, inorganic selenium salts, organic selenium and NSe still have limitations such as narrow therapeutic window or easy aggregation and easy oxidation. Therefore, based on the background that the inventors first discovered that the plasma and bone marrow supernatant selenium deficiency levels of AML-M5 patients were the most significant, the development of highly efficient and low-toxic selenium-containing drugs (especially oral convenient administration) provides a better choice for the personalized treatment of AML-M5 patients. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present invention is to provide a use of protein nano-selenium in the preparation of drugs for the prevention and treatment of acute monocytic leukemia; based on the fact that albumin can maintain good stability at a certain temperature and pH, and has the advantages of being non-toxic, low immunogenic and biodegradable, the present invention constructs orally administrable protein nano-selenium (also known as protein-bound selenium nano, protein-bound nano-elemental selenium, protein-bound nano-elemental selenium, ASe) through protein self-assembly technology; compared with NSe, ASe exhibits more excellent stability and safety; compared with selenium yeast tablets and NSe, oral ASe has higher bioavailability; in addition, ASe exerts its anti-AML-M5 effect through a unique mechanism of driving ferroptosis, and its therapeutic effect is significantly better than NSe.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] Disclosed is a use of protein nano-selenium in the preparation of a drug for preventing and treating acute monocytic leukemia. The preparation steps of the protein nano-selenium include: reducing albumin with a protein reducing agent at 37°C, then adding a sodium selenite solution or a sodium selenate solution containing chitosan or chitosan oligosaccharide, stirring and reacting at room temperature or 4°C for 1-12 hours, and dialyzing or centrifuging to remove unreacted substances to obtain protein nano-selenium particles.

[0007] The protein nano-selenium has the effect of driving the ferroptosis of acute monocytic leukemia cells.

[0008] The protein reducing agent is glutathione, and tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol, β-mercaptoethanol or cysteine ​​can also be used.

[0009] The albumin can be one or more of human serum albumin, recombinant human serum albumin, bovine serum albumin, ovalbumin, donkey serum albumin, transferrin or similar disulfide bond-rich proteins.

[0010] The protein nano-selenium is prepared specifically according to the following steps:

[0011] Human serum albumin is dissolved in a phosphate buffer solution containing a protein reducing agent at a pH of 5.0 to 9.0, wherein the final concentration of human serum albumin is 0.01 to 200 mg / mL and the final concentration of the protein reducing agent is 0.1 to 100 mM; the mixture is reacted at 37°C for 30 to 120 minutes with stirring to obtain a homogeneous protein solution with a spatially unfolded structure; then, sodium selenite or sodium selenate solution containing chitosan oligosaccharide or chitosan is added to the homogeneous protein solution with a spatially unfolded structure, and the chitosan oligosaccharide or chitosan solution is added to the homogeneous protein solution with a spatially unfolded structure. The final concentration of sugar is 0.05-50 mg / mL, and the final concentration of sodium selenite or sodium selenate is 0.1-30 mM. The reaction is stirred at room temperature or 4°C for 1-12 hours to obtain a crude protein nano-selenium solution; the crude protein nano-selenium solution is placed in a dialysis bag and dialyzed against a PBS solution at 0-20°C overnight, or the crude protein nano-selenium solution is centrifuged at 8000-30000 rpm for 10-60 minutes and repeatedly resuspended with deionized water and centrifuged several times to finally obtain protein nano-selenium particles.

[0012] The dialysis molecular cut-off of the dialysis bag is not less than 1000.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0014] This invention utilizes protein nanoselenium (ASe), which is more stable and safer than NSe and has higher oral bioavailability than selenium yeast tablets and NSe. It has a superior effect on driving ferroptosis in AML-M5 cells compared to NSe and can be used as a drug treatment for acute monocytic leukemia. In addition to oral administration, the ASe prepared in this invention can also be used for intravenous injection and topical administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Figure 3 is an evaluation of trace element characteristics of bone marrow supernatants (BM) from HD (healthy volunteers) and patients with various AML subtypes; A is a schematic diagram of BM collection and trace element detection from HD and AML patients; BM represents the levels of various trace elements in BM supernatants from HD (n=37), AML-M1 patients (n=5), AML-M2 patients (n=15), AML-M3 patients (n=16), AML-M4 patients (n=15), AML-M5 patients (n=18), and patients with other AML subtypes (n=6); N is a heat map showing the fold difference in trace element levels between HD and various AML subtypes.

[0016] Figure 2Figure 5 is the trace element characteristics of PB in patients with different FAB subtypes of HD and AML; A is a schematic diagram of PB collection and trace element detection in HD and AML patients; BM is the levels of various trace elements in the serum of HD (n=37), AML-M1 patients (n=5), AML-M2 patients (n=15), AML-M3 patients (n=16), AML-M4 patients (n=15), AML-M5 patients (n=18), and patients with other AML subtypes (n=6); N is a heat map of the fold difference in trace element levels between HD and each AML subtype.

[0017] Figure 3 Figure 1 is the preparation and characterization diagram of ASe; A is the preparation process diagram of ASe; B is the stability diagram of NSe and ASe; C is the UV / visible absorption spectrum diagram of NSe and ASe; D is the Fourier transform infrared spectrum diagram of NSe and ASe; E is the particle size diagram of NSe and ASe; F is the Zeta potential diagram of NSe and ASe; G is the transmission electron microscopy (TEM) diagram of NSe and ASe and the energy dispersive X-ray spectroscopy (EDS) diagram of ASe, scale bar = 100 nm.

[0018] Figure 4 It is an evaluation of the cytotoxicity of ASe against normal cell lines and AML subtype cell lines.

[0019] Figure 5 Figure 3 is the targeting and anti-proliferative effect of ASe on AML-M5 cells; A is the cell viability of MNCs derived from PB and BM samples of HD and AML-M5 patients after treatment with different concentrations of NSe or ASe for 48 h (n=18); B is the cell viability of L02, 293T, MV-4-11, and U937 cells after treatment with different concentrations of NSe or ASe for 2 h or 4 h, respectively (n=5); C is the number of colony formation in MV-4-11 and U937 cell lines after treatment with NSe or ASe (n=3); D is a representative CLSM image of Calcein-AM / PI staining in MV-4-11 and U937 cells.

[0020] Figure 6 Figure 3 is a diagram of gastrointestinal retention and trans-intestinal epithelial transport of ASe after oral administration; A is a schematic diagram of fluorescence resonance energy transfer (FRET) of ASe in artificial gastric fluid (SGF) and artificial intestinal fluid (SIF); B is a diagram of the FRET ratio of ASe in SGF and SIF; C is a diagram of the distribution of ASe in the gastrointestinal tract of living mice after oral administration; D is a diagram of the distribution of ASe in the isolated gastrointestinal tract of mice after oral administration; E is a quantitative diagram of the distribution of ASe in the isolated gastrointestinal tract of mice after oral administration; F is a laser confocal image of the trans-intestinal epithelial transport of ileal segments at different time points after oral administration of ASe.

[0021] Figure 7Figure 1 is a pharmacokinetic and safety evaluation diagram of ASe; A is the plasma drug concentration-time curve and main pharmacokinetic parameter diagram after oral administration of selenium yeast, NSe and ASe (selenium equivalent is 1 mg / kg) to mice; B is a safety evaluation diagram of oral administration of NSe and ASe to mice every day for 14 consecutive days (selenium equivalent is 1 mg / kg and 4 mg / kg); C is a graph of mouse body weight; D is a graph of mouse tissue weight; E is a graph of mouse white blood cell (WBC) count; F is a graph of mouse hematocrit (HCT) count; G is a graph of mouse platelet (PLT) count; H is a graph of mouse liver tissue morphology; I is a graph of mouse alanine aminotransferase (ALT) level; J is a graph of mouse aspartate aminotransferase (AST) level; K is a graph of mouse creatinine (CREA) level; L is a graph of mouse urea (UREA) level; M is a graph of H&E staining of various mouse tissues.

[0022] Figure 8 Figure 1 is a diagram showing the effect of oral administration of ASe against AML-M5; A is a schematic diagram showing the treatment of AML-M5 xenograft model with oral administration of ASe; B is a diagram showing bioluminescence imaging of AML-M5 mice at different time points after oral administration of PBS, 0.5 mg / kg NSe (NSe-0.5), 1 mg / kg NSe (NSe-1), 0.5 mg / kg ASe (ASe-0.5), 1 mg / kg ASe (ASe-1) and 75 mg / kg Venetoclax (Ven-75); C is a diagram showing the fluorescence quantification of mouse bioluminescence imaging; D is a diagram showing the white blood cell count of mice; E is a curve showing the change in mouse body weight; F is a diagram showing the survival curve of mice; G is a diagram showing the spleen weight of mice; H is a diagram showing the Wright-Giemsa staining of mouse peripheral blood (PB) and bone marrow (BM); I is a diagram showing the proportion of AML-M5 cells in mouse PB, BM, liver, spleen and lung tissues.

[0023] Figure 9 Figure 1 is a diagram of ASe-induced ferroptosis in AML-M5 cells; A is a diagram of the death pathway of AML-M5 cells induced by ASe; B is a diagram of the MDA content in MV-4-11 and U937 cells after NSe or ASe treatment; C is a diagram of the Fe 2+ D is the content of MDA in peripheral blood mononuclear cells (PBMCs) or bone marrow mononuclear cells (BMMCs) of normal donors (HD) and AML-M5 patients after NSe or ASe treatment; E is the content of MDA and Fe in BMMCs after oral administration of NSe-1 or ASe-1 in mice 2+ F is a laser confocal image of lipid peroxides in AML-M5 cells after treatment with NSe or ASe; G is a quantitative image of lipid peroxides in AML-M5 cells after treatment with NSe or ASe. DETAILED DESCRIPTION

[0024] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0025] Example 1: Assessment of trace element characteristics of bone marrow (BM) in patients with various AML subtypes

[0026] 1. Experimental methods

[0027] (1) Place the bone marrow cell blood sample after standing in a centrifuge, set the centrifuge temperature to 4°C, the speed to 3000 rpm, and the centrifugation time to 10 min; after centrifugation, collect the supernatant in a 1.5 mL centrifuge tube and store at -80°C.

[0028] (2) 100 μL of sample was dissolved in 3.9 μL HNO 3 , 3.9 μL Triton X-100, and 3892.2 μL ddH 2 O. Subsequently, the contents of aluminum (Al), titanium (Ti), vanadium (V), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), copper (Cu), zinc (Zn), gallium (Ga), arsenic (As), Se, rubidium (Rb), strontium (Sr), silver (Ag), cadmium (Cd), cesium (Cs), barium (Ba), lead (Pb), and uranium (U) in each sample were determined using ICP-MS (inductively coupled plasma mass spectrometry).

[0029] 2. Conclusion

[0030] The progression of AML is correlated with the homeostasis of trace elements, but the matching relationship between each subtype and biological trace elements is unclear, which seriously restricts the implementation of the trace element prevention and treatment strategy for AML. In order to further study the trace element characteristics in patients with various AML subtypes, this study conducted a trace element omics analysis on a cohort consisting of 37 HD (healthy volunteers) and 75 AML (acute myeloid leukemia) patients with different FAB subtypes. This analysis systematically evaluated the levels of 20 trace elements, including metal elements and non-metal elements, in individual BM supernatants ( Figure 1 The results showed that the Zn element in AML-M2 subtype was 0.77 times that of HD, while the Se element in AML-M5 patients was 0.43 times that of HD, and no difference was found in other elements ( Figure 1 Notably, Se was most significantly decreased in the BM of AML-M5 patients.

[0031] Example 2: Evaluation of trace element characteristics of peripheral blood (PB) plasma of patients with various AML subtypes

[0032] 1. Experimental methods

[0033] (1) Place the rested venous peripheral blood sample in a centrifuge, set the centrifuge temperature to 4°C, the speed to 3000 rpm, and the centrifugation time to 10 min; after centrifugation, collect the supernatant in a 1.5 mL centrifuge tube and store at -80°C.

[0034] (2) 100 μL of sample was dissolved in 3.9 μL HNO 3 , 3.9 μL Triton X-100, and 3892.2 μL ddH 2 O. Subsequently, the contents of aluminum (Al), titanium (Ti), vanadium (V), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), copper (Cu), zinc (Zn), gallium (Ga), arsenic (As), Se, rubidium (Rb), strontium (Sr), silver (Ag), cadmium (Cd), cesium (Cs), barium (Ba), lead (Pb), and uranium (U) in each sample were determined using ICP-MS (inductively coupled plasma mass spectrometry).

[0035] 2. Conclusion

[0036] The trace element characteristics of PB serum in HD and AML patients were evaluated ( Figure 2 The results showed that the Mn level in AML-M2, AML-M4 and other subtypes was 0.66, 0.74 and 0.37 times that of HD, respectively; the Cu level in AML-M1 and AML-M3 patients was 1.26 and 1.16 times that of HD, respectively; the Zn level in AML-M2 patients was 0.76 times that of HD; the Se level in AML-M4 and AML-M5 patients was 0.78 times and 0.39 times that of HD, respectively; the Sr level in AML-M5 patients increased by 1.25 times; the Pb level in AML-M2 patients increased by 1.52 times; no significant differences were observed in other trace elements such as Al, Cr, Fe, As, Rb and Ba ( Figure 2 Notably, the serum Se level decreased most significantly in patients with AML-M5.

[0037] Example 3: Preparation and Characterization of NSe and ASe

[0038] (1) Preparation of inorganic nano-selenium NSe

[0039] 46.05 mg of glutathione (GSH) and 30 mg of human serum albumin (HAS) were weighed and added to a beaker. Subsequently, 7.06 mL of ddH2O was slowly added to the beaker and stirred to fully dissolve the solute. Next, 0.32 mL of a 20 mg / mL Na2SeO3 solution was added dropwise with stirring, and 120 μL of a 1 M NaOH solution was slowly added dropwise. The mixture was transferred to an 8-14 kDa dialysis bag and dialyzed for 24 hours to prepare inorganic nanoselenium NSe.

[0040] (2) Preparation of protein nanoselenium ASe (the preparation process of ASe is shown in the figure Figure 3 A)

[0041] Human serum albumin (HSA) and the protein reducing agent glutathione (GSH) were dissolved in phosphate buffered saline (PBS) solution or deionized water ddH2O preheated to 37°C, with the final concentration of albumin being 60 mg / mL and the final concentration of the protein reducing agent being 80 mM. The HSA and GSH mixed solution was stirred by a magnetic stirrer for 80 min in a 37°C water bath to obtain a homogeneous protein solution with a spatially unfolded structure. Subsequently, a sodium selenite solution containing chitosan oligosaccharides was slowly added to the homogeneous protein solution with a final concentration of chitosan oligosaccharides of 5 mg / mL and a final concentration of sodium selenite of 10 mM. The mixture was stirred at 4°C for 12 h or at room temperature for 3 h to obtain a crude ASe solution. The crude ASe solution was placed in a dialysis bag and dialyzed against PBS or ddH2O solution at 0-20°C overnight to remove excess unreacted reagents, including oxidized GSH byproducts, to finally obtain a mother solution of protein nanoselenium particles (ASe).

[0042] (3) Characterization of NSe and ASe

[0043] In order to verify the stability of ASe, the NSe and ASe prepared above were dispersed in PBS and placed at room temperature for 28 days. The stability of ASe was better than that of NSe ( Figure 3 UV-visible spectroscopy and Fourier transform infrared spectroscopy confirmed that HSA and NSe were components of ASe ( Figure 3 The hydrated particle size and zeta potential of ASe were determined by dynamic light scattering (DLS). The size of ASe was about 40 nm, while that of NSe was about 120 nm ( Figure 3 The Zeta potential of ASe is -18mV and that of NSe is -8mV, both of which are negatively charged ( Figure 3 F); TEM results show that both ASe and NSe are quasi-spherical, and EDS images show that the elemental composition of ASe contains selenium ( Figure 3 G).

[0044] Example 4: Evaluation of the cytotoxicity of ASe prepared in Example 3 on normal and AML cell lines and its targeting and anti-proliferative effects on AML-M5 cells

[0045] 1. Experimental methods

[0046] (1) Cell culture and recovery: 5 mL of culture medium was pre-added to a 15 mL centrifuge tube. Subsequently, the cell cryovial was quickly removed from liquid nitrogen and immediately thawed at 37°C. After thawing, the cells were transferred to a pre-prepared centrifuge tube, mixed evenly, and centrifuged at 1000 rpm for 5 min. After centrifugation, the supernatant was discarded, 1 mL of fresh culture medium was added to the precipitate to resuspend the cells and transfer them to a culture flask. All cell lines were cultured at 37°C in a constant temperature incubator containing 5% CO2. KG-1α, Kasumi-1, HL-60, NB4, HEL, MV-4-11, and U937 cells were cultured in RPMI 1640 medium. OCI-AML2 cells were cultured in α-MEM medium. 293T and L02 cells were cultured in DMEM medium. All culture media were supplemented with 10% fetal bovine serum and 1% P / S.

[0047] (2) Isolation and extraction of PBMCs (peripheral blood mononuclear cells) and BMMNCs (bone marrow mononuclear cells): The collected PB (peripheral blood) or BM (bone marrow) extract was centrifuged at 400×g for 10 min at 4°C. After centrifugation, the upper serum layer was collected and stored at -80°C. An equal volume of 0.9% NaCl was used to dilute the remaining blood and inverted at least 20 times to mix it thoroughly. First, Ficoll separation solution was carefully added to the bottom of a 50 mL centrifuge tube to minimize adhesion to the tube wall. 8 mL of Ficoll separation solution was added to every 10 mL of blood sample. Then, the diluted blood sample was carefully added slowly to the Ficoll layer (tilted at 45°C), taking care to avoid mixing the blood sample into the Ficoll. Centrifuge at 400×g for 20 min at 4°C, following a 5-up and 0-down procedure. Use a new 50 mL centrifuge tube to collect the separated mononuclear cell (MNC) layer. Subsequently, add 0.9% NaCl to the tube to make up to 40 mL and centrifuge at 450 × g for 10 minutes at 4°C. After removing the supernatant, add 2 mL of red blood cell lysis buffer to the MNC tube, mix thoroughly by pipetting, and lyse at 4°C for 5 minutes. Add 0.9% NaCl to the MNC tube to make up to 20 mL and centrifuge at 450 × g for 10 minutes at 4°C. After removing the supernatant, add 0.9% NaCl to the MNC tube to make up to 10 mL and count the cells. Centrifuge again at 450 × g for 10 minutes at 4°C. After removing the supernatant, add pre-prepared freezing solution and freeze at -80°C. After overnight, transfer to liquid nitrogen for long-term storage.

[0048] (3) Cell viability assay: AML cell lines were assayed at 1×10 4 293T and L02 cells were seeded at a density of 1×10 3The cells were seeded at a density of 100 μM / well in a 96-well plate. Subsequently, NSe or ASe at final concentrations of 0, 0.25, 5, 10, 20, 40, and 80 μM were added thereto and incubated in a cell culture incubator for 24 or 48 h. Afterwards, 10 μL of CCK-8 solution was added to the 96-well plate and incubated at 37°C for 2 h. The absorbance (OD) value of each group was measured at 450 nm using a microplate reader. The half-maximal inhibitory concentration (IC50) of each cell to the drug was calculated using GraphPad Prism 8 software. 50 )value.

[0049] MNCs were measured at 1×10 4 Cells were seeded at a density of 1 μM per well in a 96-well plate. NSe or ASe were then added to final concentrations of 10 and 20 μM, respectively, and incubated in a cell culture incubator for 48 hours. Afterwards, 10 μL of CCK-8 solution was added to the 96-well plate and incubated at 37°C for 2 hours. The OD value of each group was measured at 450 nm using a microplate reader.

[0050] Cell viability (%) = (A1 / A0) x 100%, where A1 refers to the OD value in the presence of drugs, and A0 refers to the OD value in the absence of drugs.

[0051] (4) Methylcellulose-based colony formation: MV-4-11 and U937 cells were seeded into 12-well plates at a density of 1000 cells per well. Drugs were added directly to the methylcellulose-containing medium and the cells were cultured. The leukemia cells were then incubated in a 37°C, 5% CO2 incubator for 14 days. The number of colonies was counted under a microscope.

[0052] (5) Calcein-AM / PI staining: MV-4-11 and U937 cells were plated at 5×10 5 Cells were seeded at a density of 100 cells / mL in 12-well plates and incubated with NSe or ASe for 48 hours. Subsequently, 2 μL of Calcein-AM was added per 1 mL of cell suspension and incubated at 37°C in the dark for 25 minutes. Furthermore, 5 μL of PI stock solution was added to the cell suspension and incubated at room temperature in the dark for 5 minutes. After washing three times with PBS, the cells were examined by CLSM.

[0053] (6) Statistical analysis: GraphPad Prism 8.0.1 software was used. Data are presented as mean ± standard deviation. For significance tests between two groups of data, the t-test was used; for data from more than two groups, one-way analysis of variance (ANOVA) was used. Differences were considered significant when the P value was less than 0.05.

[0054] 2. Conclusion

[0055] (1) To evaluate the toxicity of ASe on normal cell lines and AML subtype cell lines, the IC values ​​of normal human hepatocytes L02, normal human renal epithelial cell line 293T, and eight different AML subtype cell lines, including KG-1α, Kasumi-1, HL-60, NB4, OCI-AML2, MV-4-11, U937, and HEL, were determined after 24 h and 48 h of ASe treatment. 50 It is noteworthy that AML-M5 cell lines (MV-4-11 and U937) showed the highest sensitivity when incubated with ASe for 48 h, with IC 50 The values ​​were 9.14 μM and 11.32 μM, respectively, which were significantly lower than 35.08 μM and 51.94 μM of NSe. Importantly, even at a concentration of up to 80 μM, ASe showed no toxicity to normal cell lines (L02 and 293T), indicating that ASe has excellent anti-AML-M5 activity and safety in vitro ( Figure 4 shown).

[0056] (2) To verify the targeting and anti-proliferative ability of ASe on AML-M5 cells, ASe intervention was performed on MNCs from 18 HD and 18 AML-M5 patients. The results showed that ASe showed no obvious toxicity to MNCs of normal subjects, but showed a strong anti-leukemia effect on MNCs of AML-M5 patients ( Figure 5 A). In addition, ASe was co-incubated with MV-4-11 or U937 cells for 2 hours or 4 hours, respectively. Subsequently, the medium containing ASe was removed and replaced with fresh medium without drug, and cultured for another 48 hours. The results showed that ASe had no significant effect on the survival rate of normal cells and AML-M5 cells when co-incubated with cells for 2 hours. However, when the co-incubation time was extended to 4 hours, ASe significantly inhibited the viability of MV-4-11 and U937 cells, indicating that ASe has excellent targeting ability for AML-M5 cells and is non-toxic to normal cells ( Figure 5 Furthermore, the inhibitory effect of ASe on the proliferation of MV-4-11 and U937 was verified by cell colony formation assay and Calcein-AM / PI staining ( Figure 5 C and D).

[0057] Example 5: Gastrointestinal retention and trans-intestinal epithelial transport of the ASe prepared in Example 3 after oral administration

[0058] 1. Experimental methods

[0059] (1) Artificial gastric juice (SGF) according to the pharmacopoeia standard: Take 16.4 mL of dilute hydrochloric acid, add 800 mL of ddH2O and 10 g of pepsin, mix thoroughly, adjust the solution pH to 1.2 with HCl, and dilute to 1000 mL with ddH2O.

[0060] (2) Pharmacopoeia standard artificial intestinal fluid (SIF): Dissolve 6.8 g of KH2PO4 in 500 mL of ddH2O and adjust the pH to 6.8 with 0.1 M NaOH solution. Dissolve 10 g of trypsin in ddH2O, mix the two solutions, and dilute to 1000 mL with ddH2O.

[0061] (3) Integrity evaluation of ASe in SGF (artificial gastric fluid of pharmacopoeia standard) and SIF (artificial intestinal fluid of pharmacopoeia standard): DiO (donor) and DiI (acceptor) were used as FRET pairs for the experiment. The physical mixture of DiO+DiI (2μM) and DiO / DiI@ASe (2μM) were incubated with SGF at 37°C for 0, 0.5, 1, 2 and 6 h, respectively. Similarly, they were incubated with SIF at 37°C for 0, 0.5, 1, 2, 6, 12 and 24 h, respectively. At each time point, 200uL of the mixed solution was placed in a quartz cell and detected using a fluorescence spectrometer. The experimental setting was an excitation wavelength of 475nm and an emission wavelength scanning range of 495 to 650nm to capture the changes in the FRET signal. At the same time, the excitation and emission slit widths were adjusted to 10nm. The FRET ratio was calculated as I FRET / (I FRET +I DiO ), where I FRET represents the intensity of the FRET signal, I DiO Indicates the intensity of DiO signal.

[0062] (4) Distribution of ASe in the gastrointestinal tract of mice and evaluation of trans-intestinal epithelial transport: Male Balb / C mice (5-7 weeks old) were randomly divided into 6 groups (n=3) and fasted for 12 hours before the experiment with free access to water. The abdominal hair of the mice was removed using a depilatory cream to minimize hair-derived autofluorescence. Subsequently, free Cy5 and Cy5@ASe were orally administered at a dose equivalent to 1.5 mg / kg Cy5. At predetermined time points (0, 0.5, 2, 6, 12, and 24 hours), the mice were anesthetized with 2% isoflurane and imaged using the IVIS Spectrum Imaging System. At the same time, the mice were euthanized after imaging at each time point, and their stomachs and intestines were dissected and imaged ex vivo using IVIS. At the same time, the fluorescence intensity of the stomach, duodenum, jejunum, ileum, cecum / colon / rectum was quantified using Image J. At the same time, ileum samples were obtained at 0, 0.5, 2, 6, 12, and 24 h after administration, and quickly frozen in liquid nitrogen. The intestinal sections were then frozen and sectioned, and the cell nuclei were marked with DAPI staining. After sealing, the sections were photographed using CLSM.

[0063] 2. Conclusion

[0064] (1) During the 6-h exposure of ASe to SGF and SIF, the ratio of the FRET fluorescence pairs remained basically stable, proving that the ASe structure remained intact within 6 h in SGF and SIF. However, a significant decrease in the FRET ratio of DiO / DiI@ASe was observed after 6 h of exposure to SIF, indicating that the integrity of the ASe structure was destroyed to some extent after 6 h. Figure 6 AB).

[0065] (2) In vivo and in vitro imaging showed that after oral administration of free Cy5, the fluorescence in the mid-abdomen area of ​​the mouse was the strongest at 0.5 h, and the fluorescence disappeared after 6 h. However, after oral administration of Cy5@ASe, the fluorescence in the mid-abdomen area of ​​the mouse was the strongest at 2 h, and the drug retention time was as long as 12 h ( Figure 6 C). In vitro fluorescence imaging and quantitative images of the mouse gastrointestinal tract showed that free Cy5 was only distributed in the cecum / colon / rectum at 6 hours after oral administration, and the fluorescence disappeared in all intestinal segments at 12 hours. After oral administration of Cy5@ASe, the fluorescence intensity reached the highest in the ileum at 6 hours, and was mostly distributed in the cecum / colon / rectum at 12 hours. Figure 6 DE). The ileum segments were frozen and sectioned to verify the absorption and uptake capacity of the intestinal epithelial cells for the drug. Laser confocal imaging showed that 0.5 hours after oral administration, free Cy5 quickly reached the free side of the intestine, and by 6 hours, the free Cy5 in the ileum segments was almost completely cleared. Cy5@ASe was taken up by intestinal epithelial cells 2 hours after oral administration and distributed simultaneously on the free side and basolateral side. By 6 hours, the fluorescence intensity on the basolateral side reached its peak and could be retained for up to 12 hours ( Figure 6 F).

[0066] Example 6: Pharmacokinetics and safety evaluation of ASe prepared in Example 3

[0067] 1. Experimental methods

[0068] (1) Pharmacokinetics of ASe: Nine SD rats (5-7 weeks old) were fasted for 12 hours with free access to water. Subsequently, they were randomly divided into three groups: selenium yeast group, NSe group and ASe group. Each group was gavaged with 1 mg / kg of selenium equivalent. 0.3 ml of blood samples were collected from the orbital venous plexus of the rats at time points of 0, 0.25, 0.5, 1, 2, 4, 8, 12, 24 and 48 hours, and centrifuged at 3500 rpm for 10 minutes at 4°C to separate the plasma. Subsequently, the plasma was digested using a microwave digester, and the selenium concentration in the plasma was determined by ICP-MS. The peak time (T max ), peak concentration (C max ), half-life (T 1 / 2 ), the area under the curve from 0 to t (AUC 0-t ) and pharmacokinetic parameters such as relative bioavailability.

[0069] (2) Safety evaluation

[0070] Twenty-five Balb / C mice (4-6 weeks old, half male and half female) were randomly divided into five groups: PBS group, 1 mg / kg NSe group (NSe-1), 4 mg / kg NSe group (NSe-4), 1 mg / kg ASe group (ASe-1) and 4 mg / kg ASe group (ASe-4), and were gavage-administered daily for 14 days. The body weight of the mice was recorded every two days. After 2 weeks, blood samples were collected from the mouse eyeballs, and the WBC, HCT and PLT levels were measured using a hematology analyzer. ALT, AST, CREA and UREA levels were detected using a biochemical analyzer. Subsequently, the liver was photographed and weighed. In addition, the main tissues were weighed and histologically observed using H&E staining

[0071] 2. Conclusion

[0072] (1) The plasma selenium concentration in the ASe group increased over time within the first 2 h and then decreased. In contrast, the plasma concentrations of oral administration of selenium yeast or NSe were much lower at all time points ( Figure 7 A). The selenium yeast group showed a C of about 0.73 μg / mL. max , and was rapidly eliminated. In contrast, ASe showed the highest C max Value and the longest T 1 / 2. Through calculation, the bioavailability of ASe is as high as 232.83%, far exceeding the 117.37% of NSe, which proves that ASe is significantly better than selenium yeast tablets and NSe in promoting selenium absorption.

[0073] (2) Evaluate the safety of oral administration of different doses of ASe or NSe ( Figure 7 The results of body weight showed that high dose (4 mg / kg) of NSe caused the mice to lose weight, while the same dose of ASe had no significant effect on the mice's body weight ( Figure 7 In addition, high-dose NSe also increased liver weight, suggesting that NSe may have a hepatotoxic risk at higher doses ( Figure 7 Hematological analysis further showed that ASe had no significant effect on hematological parameters, while high-dose NSe reduced the number of HCT and PLT in mice, indicating that NSe may have an adverse effect on the hematopoietic system ( Figure 7 In liver function tests, oral administration of NSe at a dose of 4 mg / kg resulted in significant changes in liver morphology in mice, with ALT and AST values ​​significantly higher than those in the normal group, further confirming the hepatotoxicity of NSe. In contrast, ASe did not significantly affect liver function even at high doses ( Figure 7 The results of renal function tests showed that ASe did not cause nephrotoxicity even at high doses, whereas NSe at the same dose had significant nephrotoxicity, manifested as abnormal renal function indicators ( Figure 7 The results of H&E staining showed that after oral administration of NSe-4 (4 mg / kg), the volume of hepatocytes in the mouse liver increased, and round vacuoles of varying sizes and with clear boundaries appeared in the cytoplasm. The normal structure of the hepatic lobules was destroyed, and the hepatic cords were arranged irregularly. In addition, the renal tubular epithelial cells also showed an increase in volume and vacuolar degeneration, further confirming the toxic effects of NSe on the liver and kidneys ( Figure 7 Therefore, compared with NSe, ASe showed good safety and had no significant effect on mouse body weight, hematological parameters, liver function and kidney function.

[0074] Example 7: Anti-AML-M5 effect of oral administration of ASe prepared in Example 3

[0075] 1. Experimental methods

[0076] (1) Establishment of AML-M5 xenograft mouse model: Female NOD / SCID mice (5-6 weeks old) were fed adaptively for one week. Mice were intraperitoneally injected with cyclophosphamide (100 mg / kg). 24 hours later, 8×10 6Each MV-4-11-EGFP / Luc cell was suspended in 200 μL of PBS and injected into the mouse via the tail vein. Two weeks after transplantation, 20 μL of peripheral blood was collected via tail tip bleeding. 2 mL of red blood cell lysis buffer was added and lysed on ice for 5 minutes. The cells were resuspended in PBS and the proportion of EGFP-positive cells was determined by flow cytometry. The AML-M5 mouse xenograft model was successfully established when the proportion of EGFP-positive cells exceeded 1%.

[0077] (2) Grouping and dosing of AML-M5 mice: AML-M5 mice were randomly divided into 6 groups, with 9 mice in each group, and received the following treatments by oral gavage every day for 3 consecutive weeks: PBS, 0.5 mg / kg NSe (NSe-0.5), 1 mg / kg NSe (NSe-1), 0.5 mg / kg ASe (ASe-0.5), 1 mg / kg ASe (ASe-1), and 75 mg / kg Venetoclax (Ven-75).

[0078] (3) Monitoring indicators: The weight of mice was recorded every other day. In addition, mice were intraperitoneally injected with 150 mg / kg D-luciferin potassium solution every week. Immediately after the injection, the mice were placed in the induction box of the respiratory anesthesia machine and anesthetized by inhalation of 2.5% isoflurane gas. The anesthetized mice were placed in a fluorescent imaging device, and their body positions were adjusted to capture the fluorescent signal by the in vivo imaging device to detect the spread of mouse leukemia cells and quantify the bioluminescence imaging (BLI) value. PB was collected by tail tip blood sampling every other week, and the number of white blood cells was detected by a blood cell counter. The survival status of mice was recorded during the entire treatment process to draw a survival curve. At the end of the treatment, the PB and BM of the mice were collected and the inhibition of leukemia cells by each group of drugs was detected by Wright-Giemsa staining. At the end of the treatment, the PB, BM, liver, spleen and lung tissues of the mice were collected, and the proportion of leukemia cells was detected by flow cytometry after lysis of red blood cells.

[0079] 2. Conclusion

[0080] (1) 14 days after the injection of MV-4-11-EGFP / Luc cells via the tail vein, the positive ratio of AML-M5 cells in PB was >1%, and the orthotopic xenograft model was successfully established. Subsequently, different therapeutic drugs were orally administered ( Figure 8A). In vivo imaging results showed that the leukemia cells of mice treated with PBS spread rapidly and widely throughout the body, resulting in only one mouse surviving on day 35. In contrast, the disease progressed just as rapidly in the NSe-0.5 treatment group, with only one mouse surviving on day 35. However, when the NSe dose was increased to 1 mg / kg, it showed a similar effect to the low-dose ASe treatment group in alleviating disease progression, with four mice surviving at the end of treatment. The ASe-1 treatment group and the positive drug control group, Ven-75, significantly slowed the spread of leukemia cells in the body. All mice in the Ven-75 group survived at the end of the experiment, and only one mouse died in the ASe-1 group ( Figure 8 of BC).

[0081] (2) WBC counts showed that ASe could significantly reduce the number of WBCs, and this reduction was dose-dependent. Specifically, the number of WBCs in the PBS control group was 14.6×10 9 / L, while the WBC count of mice treated with ASe-1 decreased to 9.4×10 9 / L, compared with the Ven-75 treatment group (9.3×10 9 / L) similar( Figure 8 D).

[0082] (3) In terms of body weight changes, ASe treatment effectively alleviated the weight loss of mice, and its effect was better than that of NSe ( Figure 8 E).

[0083] (4) Survival curve analysis confirmed that the survival rate of mice treated with ASe-1 was as high as 83.3% compared with the PBS control group, while the survival rates of the NSe treatment group and the ASe low-dose treatment group at the same dose were both 66.7% ( Figure 8 F).

[0084] (5) ASe also significantly inhibited splenomegaly in AML, with an effect comparable to that of Ven-75 ( Figure 8 G).

[0085] (6) PB, BM, liver, spleen and lung tissues were collected for leukemia cell detection. The results of Wright-Giemsa staining showed that except for the NSe-0.5 treatment group, the number of leukemia cells in the other treatment groups decreased ( Figure 8H). The proportion of AML-M5 cells in the tissues was quantified by flow cytometry. In PB, the proportion of AML-M5 cells in the PBS group was as high as 23.4%. After 21 days of low-dose and high-dose NSe treatment, the proportion of AML-M5 cells decreased to 23.9% and 15.8%, respectively. Low-dose and high-dose ASe treatment significantly reduced the proportion of AML-M5 cells to 16.0% and 5.6%, respectively. This suggests that ASe has a more significant effect in reducing the proportion of AML-M5 cells in PB. In BM, the reduction in the proportion of AML-M5 cells is particularly prominent. Low-dose and high-dose NSe reduced the proportion of AML-M5 cells by 1.0 and 1.2 times, respectively, while low-dose and high-dose ASe achieved a reduction of 1.3 and 3.1 times, respectively. The proportion of leukemia cells in the liver, spleen and lungs was also significantly reduced after ASe treatment ( Figure 8 I).

[0086] Example 8: ASe prepared in Example 3 induces ferroptosis in AML-M5 cells

[0087] 1. Experimental methods

[0088] (1) CCK-8 detection of cell death: AML-M5 cell line was detected by 1×10 4 Cells were seeded at a density of 100 μg / well in a 96-well plate. Subsequently, ASe and 10 μM Ferrostatin-1, 50 μM Necrostatin-1, or 10 μM Z-VAD-FMK were added to the plates at a final concentration of 20 μM. The cells were incubated in a cell culture incubator for 48 hours. Afterwards, 10 μL of CCK-8 solution was added to the 96-well plate and incubated at 37°C for 1-4 hours. The OD value of each group was measured at 450 nm using a microplate reader.

[0089] (2) Malondialdehyde (MDA) content detection: Cells were seeded into 6 cm culture dishes and incubated with different concentrations of NSe or ASe for 48 h. After the cells were collected, they were washed twice with PBS and then homogenized and lysed using Western and IP cell lysis buffer. The cell lysate was centrifuged at 12,000 × g for 10 min, and the supernatant was taken for use. For protein quantification, 2 μL was taken from each group. At the same time, MDA detection working solution was prepared. 18.5 mg of thiobarbituric acid (TBA) was dissolved in 5 mL of TBA preparation solution and heated in a 70 °C water bath until completely dissolved to form a 0.37% TBA solution. In addition, 150 μL of antioxidant, 7.5 mL of TBA dilution solution and 2.5 mL of TBA stock solution were mixed and dissolved in a 70 °C water bath to prepare MDA working solution. To prepare the standard curve, appropriate amounts of standards were diluted to concentrations of 1, 2, 5, 10, 20, and 50 μM using ddH2O. To each centrifuge tube, add 0.1 mL of lysate as a blank control, standards of varying concentrations to create a standard curve, and 0.1 mL of sample for testing. Next, add 0.2 mL of MDA working solution to each tube, mix thoroughly, and heat at 100°C for 15 minutes. After cooling to room temperature, centrifuge at 1000 × g for 10 minutes. Transfer 200 μL of the supernatant to a 96-well plate and measure absorbance at 532 nm using a microplate reader.

[0090] (3) Ferrous ions (Fe 2+ ) Content detection: Take 10 mg of FeSO4·7H2O, add 900 μL ddH2O and 20 μL concentrated sulfuric acid, mix thoroughly and prepare a 40 mM standard solution. Next, use ddH2O to dilute the 40 mM standard to obtain a series of standard concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0.78125 μM. Subsequently, the cells were ultrasonically disrupted in an ice bath and centrifuged at 10,000 × g for 10 minutes at 4°C. The supernatant was placed on ice for testing. During the measurement process, 0.2 mL ddH2O was added to the centrifuge tube as a blank control, 0.2 mL of standard at each concentration gradient, and 0.2 mL of the sample to be tested. Then, 0.1 mL of reagent 1 was added to each tube, mixed thoroughly, and allowed to stand at 37°C for 10 minutes. Next, add 0.1 mL of chloroform to the sample tube, vortex thoroughly for 5 minutes, centrifuge at 12,000 × g for 10 minutes, draw 0.2 mL of the upper layer into a 96-well plate, and measure the absorbance value A at 593 nm using a microplate reader. At the same time, measure the absorbance value A of the standard tube and blank tube. 标准 and A 空白, used to draw the standard curve. Ferrous ion content (μmoL / mg protein) = 0.001x ÷ Cpr. Where x is the sample concentration (μM) and Cpr is the protein concentration (mg / mL).

[0091] (4) Lipid peroxidation assay: Cells were seeded in 12-well plates and treated with different concentrations of NSe or ASe for 2 days. The cells were washed twice with PBS. The cells were collected and treated with C11-BODIPY at a final concentration of 3 μM. 581 / 591 The cells were incubated at 37°C for 30 min. After washing twice with PBS, the cells were stained with Hoechst dye for 10 min and observed using confocal laser scanning microscope.

[0092] (5) Isolation and extraction of PBMCs and BMMCs: The collected PB or BM extract was centrifuged at 400×g for 10 min at 4°C. After centrifugation, the upper serum was collected and stored at -80°C. An equal volume of physiological saline was used to dilute the remaining blood and inverted at least 20 times to mix it thoroughly. First, carefully add the Ficoll separation solution to the bottom of the 50mL centrifuge tube to minimize adhesion to the tube wall. Add 8mL of Ficoll separation solution for every 10mL of blood sample. Then, carefully add the diluted blood sample slowly to the Ficoll layer (tilted at 45 degrees), taking care to avoid mixing the blood sample into the Ficoll. Centrifuge at 400×g for 20 min at 4°C, following a 5-up and 0-down procedure. Use a new 50mL centrifuge tube to collect the separated mononuclear cell layer. Subsequently, physiological saline was added to the tube to 40mL and centrifuged at 450×g for 10 min at 4°C. After removing the supernatant, 2mL of red blood cell lysis solution was added to the MNCs tube and mixed thoroughly, and lysed at 4°C for 5min. Add physiological saline to the MNC tube to make up to 20 mL and centrifuge at 450 × g for 10 min at 4°C. After removing the supernatant, add physiological saline to the MNC tube to make up to 10 mL and count, then centrifuge at 450 × g for 10 min at 4°C. After removing the supernatant, add pre-prepared freezing solution and freeze at -80°C. After overnight, transfer to liquid nitrogen for long-term storage.

[0093] 2. Conclusion

[0094] (1) AML-M5 cells were treated with ferroptosis inhibitors (ferrostatin-1), necrosis inhibitors (necrostatin-1), and apoptosis inhibitors (Z-VAD-FMK). Under ASe treatment, only ferrostatin-1 could significantly inhibit cell death, while necrostatin-1 and Z-VAD-FMK had no significant effect on cell death ( Figure 9 A).

[0095] (2) In the study of the effect of ASe on cell mechanisms, it was found that the MDA content of MV-4-11 and U937 cells showed a dose-dependent accumulation after ASe treatment ( Figure 9 Specifically, the MDA levels of these two cells after high-dose ASe treatment reached 3.5 times and 2.7 times that of the untreated group, respectively, indicating that ASe treatment significantly enhanced the level of lipid peroxidation in the cells. When these cells were co-treated with Ferrostatin-1, the MDA levels were almost reduced to normal levels. In addition, after ASe was co-incubated with cells, the levels of Ferrostatin-1 were observed to be significantly increased. 2+ Gradually accumulate in leukemia cells. However, when treated with Ferrostatin-1, Fe 2+ This further supports that Ferrostatin-1 reduces Fe by inhibiting the ferroptosis process. 2 + The accumulation of Figure 9 ASe induced ferroptosis. Similar results were also observed in PBMCs and BMMCs extracted from AML-M5 patients and BMMCs from mice ( Figure 9 Then, C11-BODIPY 581 / 591 Fluorescence images showed that the green fluorescence of cells treated with NSe was weak. When treated with ASe at different doses, the green fluorescence gradually increased. When the ferroptosis inhibitor Ferrostatin-1 was added at the same time as ASe treatment, the green fluorescence almost disappeared, indicating that Ferrostatin-1 effectively inhibited the lipid peroxidation process induced by ASe. Figure 9 This further confirmed that ASe drives ferroptosis in AML-M5 cells, a process that can be effectively inhibited by Ferrostatin-1.

[0096] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A use of protein nano-selenium in the preparation of a drug for preventing and treating acute monocytic leukemia, characterized in that: The preparation steps of the protein nano-selenium include: reducing albumin with a protein reducing agent at 37°C, then adding a sodium selenite solution or a sodium selenate solution containing chitosan oligosaccharide or chitosan, stirring and reacting at room temperature or 4°C for 1-12 hours, and dialyzing or centrifuging to remove unreacted substances to obtain protein nano-selenium particles.

2. The use of a protein nano-selenium according to claim 1 in preparing a drug for preventing and treating acute monocytic leukemia, characterized in that: The protein nano-selenium has the effect of driving the ferroptosis of acute monocytic leukemia cells.

3. The use of a protein nano-selenium according to claim 1 in preparing a drug for preventing and treating acute monocytic leukemia, characterized in that: The protein reducing agent is glutathione.

4. The use of a protein nano-selenium according to claim 1 in preparing a drug for preventing and treating acute monocytic leukemia, characterized in that: The protein nano-selenium is prepared specifically according to the following steps: Human serum albumin is dissolved in a phosphate buffer solution containing a protein reducing agent at a pH of 5.0 to 9.0, wherein the final concentration of human serum albumin is 0.01 to 200 mg / mL and the final concentration of the protein reducing agent is 0.1 to 100 mM; the mixture is reacted at 37°C for 30 to 120 minutes with stirring to obtain a homogeneous protein solution with a spatially unfolded structure; then, sodium selenite or sodium selenate solution containing chitosan oligosaccharide or chitosan is added to the homogeneous protein solution with a spatially unfolded structure, and the chitosan oligosaccharide or chitosan solution is added to the homogeneous protein solution with a spatially unfolded structure. The final concentration of sugar is 0.05-50 mg / mL, and the final concentration of sodium selenite or sodium selenate is 0.1-30 mM. The reaction is stirred at room temperature or 4°C for 1-12 hours to obtain a crude protein nano-selenium solution; the crude protein nano-selenium solution is placed in a dialysis bag and dialyzed against a PBS solution at 0-20°C overnight, or the crude protein nano-selenium solution is centrifuged at 8000-30000 rpm for 10-60 minutes and repeatedly resuspended with deionized water and centrifuged several times to finally obtain protein nano-selenium particles.

5. The use according to claim 4, characterized in that: The dialysis molecular cut-off of the dialysis bag is not less than 1000.

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