Mannose-modified organic metal framework-coated nano-selenium as well as preparation method and application of mannose-modified organic metal framework-coated nano-selenium

Abstract

The invention relates to the field of medical nano-materials, in particular to mannose-modified nano-selenium wrapped by an organic metal framework as well as a preparation method and application of the mannose-modified nano-selenium. Mixing the ultra-small nano-selenium solution, a 2-methylimidazole solution, polyvinylpyrrolidone and a zinc nitrate solution, and carrying out standing reaction to obtain a standing reactant; standing, centrifuging the reactant, washing the obtained solid, and mixing with water to obtain a Se-Z8 solution; the preparation method comprises the following steps: mixing PEG-COOH, water, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, then mixing with D-mannose, carrying out a stirring reaction to obtain a stirring reactant, and preparing the mannose-modified nano-selenium system Se (at) Z8 (at) Man wrapped by the organic metal framework. The mannose-modified nano-selenium coated with the organic metal framework provided by the invention can be used for effectively treating lupus nephritis.

Description

Technical Field

[0001] This invention relates to the field of medical nanomaterials, and in particular to a mannose-modified organometallic framework-encapsulated selenium nanoparticles, their preparation method, and their applications. Background Technology

[0002] Lupus nephritis (LN) is a serious complication of systemic lupus erythematosus (SLE), affecting approximately half of all SLE patients within 5 years of diagnosis, and has a poor prognosis. Macrophages are abundant in inflammatory lesions and participate in the progression of LN. Targeting macrophage-related functions may be a specific therapeutic approach for controlling LN. Macrophages are involved in multiple processes of LN initiation and inflammatory development, with different polarization types playing different functions. Inhibiting abnormal macrophage polarization has been shown to treat LN.

[0003] Mounting evidence suggests that M2b macrophages play a crucial role in the development and progression of inflammatory diseases, particularly autoimmune diseases. In patients with kidney disease (LN), the number of M2b macrophages infiltrating the kidneys is significantly increased, and this is associated with elevated proteinuria, severity of glomerulosclerosis, and impaired renal function. Within the renal microenvironment, M2b macrophages release various pro-inflammatory factors, promoting immune system activation and renal tissue damage. Therefore, inhibiting macrophage polarization towards the M2b phenotype while reducing their overall number is a feasible strategy for treating LN; however, safe and effective methods for targeting and inhibiting M2b macrophage polarization are currently lacking.

[0004] Selenium is an essential micronutrient for the health of humans, animals, and microorganisms. Physiologically active selenium is primarily bound to proteins in the forms of selenocysteine ​​and selenomethionine, forming various selenoproteins. Therefore, selenium plays a crucial role in many important enzymatic processes, participating in cellular redox reactions and regulating immune system function. Studies have shown that selenium can inhibit the activation and polarization of macrophages, thereby suppressing their immune activation capacity. However, traditional selenium preparations have limitations in clinical treatment due to the toxicity and instability of selenium. Therefore, finding a new, safe, and effective method to utilize the therapeutic effects of selenium is imperative.

[0005] In recent years, selenium nanoparticles (Se NPs) have attracted researchers' attention due to their high biocompatibility, bioavailability, and low toxicity. Compared with traditional selenium preparations, selenium nanoparticles exhibit higher bioactivity and better valence stability, thus Se NPs are widely used in various biomedical fields. While Se NPs have been reported to regulate macrophage polarization, their application in spinal cord injury (SLE) and lymphocyte neuropathy (LN) is lacking. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a mannose-modified organometallic framework-encapsulated selenium nanoparticles, their preparation method, and applications. The mannose-modified organometallic framework-encapsulated selenium nanoparticles provided by this invention can effectively treat lupus nephritis.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for preparing mannose-modified organometallic framework-encapsulated selenium nanoparticles, comprising the following steps:

[0009] 1) Mix ultra-small nano-selenium solution, 2-methylimidazole solution, polyvinylpyrrolidone and zinc nitrate solution, and let them stand to react, to obtain the reactants after standing.

[0010] 2) Centrifuge the static reactant obtained in step 1), wash the resulting solid and mix it with water to obtain a Se@Z8 solution;

[0011] 3) Mix PEG-COOH, water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, then mix with D-mannose and stir to obtain the stirred reaction product;

[0012] 4) Dialyze the stirred reaction mixture obtained in step 3) to obtain a PEG-mannose solution;

[0013] 5) The Se@Z8 solution obtained in step 2) is mixed with the PEG-mannose solution obtained in step 4) and reacted. The reactants are centrifuged and the precipitate obtained is mannose-modified organometallic framework-encapsulated selenium nanoparticles.

[0014] Preferably, in step 1), the volume ratio of the ultra-small nano-selenium solution, the volume of the 2-methylimidazole solution, the mass of polyvinylpyrrolidone, and the volume of the zinc nitrate solution is 4 mL:25 mL:250 mg:25 mL.

[0015] The concentration of the nano-selenium solution is 18 nM;

[0016] The 2-methylimidazole solution is prepared by dissolving 26.175 mg of 2-methylimidazole in 25 mL of water.

[0017] The zinc nitrate solution is prepared by dissolving 93.6 mg of zinc nitrate hexahydrate in 25 mL of water.

[0018] Preferably, the preparation method of the ultra-small nano-selenium solution includes the following steps: mixing polyethylene glycol 400 and selenium powder, and maintaining the mixture at a temperature of 220°C for 1 hour to obtain a nano-selenium solution;

[0019] The volume ratio of polyethylene glycol 400 to selenium powder is 20 mL: 40 mg.

[0020] Preferably, the static reaction time in step 1) is 6 hours.

[0021] Preferably, the centrifugation conditions in step 2) include: a rotation speed of 12,000 rpm and a time of 10 min.

[0022] Preferably, in step 3), the mass ratio of PEG-COOH, water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 100 mg: 10 mL: 10 mg: 10 mg.

[0023] The molecular weight of the PEG-COOH is 5000 Da.

[0024] Preferably, in step 3), the mass ratio of PEG-COOH to D-mannose is 1:1; and the stirring reaction time is 12 hours.

[0025] Preferably, in step 5), the volume ratio of the Se@Z8 solution to the PEG-mannose solution is 10:1.

[0026] The concentration of the Se@Z8 solution is 3-5 mg / mL, and the concentration of the PEG-mannose solution is 0.3-0.5 mg / mL.

[0027] This invention provides a mannose-modified organometallic framework-encapsulated selenium nanoparticles prepared by the preparation method described above.

[0028] The present invention also provides the application of the mannose-modified organometallic framework-encapsulated nano-selenium described in the above-described technical solution in the preparation of drugs for treating lupus nephritis.

[0029] The beneficial effects of this invention are:

[0030] 1. This invention overcomes the shortcomings of traditional treatments that cannot specifically target M2b macrophages. Based on the high specific uptake of mannose by M2b macrophages, a mannose-targeting nanomedicine was designed to treat LN efficiently and effectively.

[0031] 2. Through relevant experiments, the mannose-modified organometallic framework-encapsulated selenium nanoparticles exhibit excellent stability and dispersibility, as well as good biocompatibility. Furthermore, these selenium nanoparticles can significantly inhibit M2b macrophage polarization and suppress the release of inflammatory factors generated by polarization.

[0032] 3. Animal experiments have demonstrated that this nano-selenium drug can effectively improve the condition of LN in mice and inhibit the deterioration of kidney function. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0034] Figure 1 Electron microscopy images of selenium nanoparticles Se@ZIF-8@Man and their components encapsulated in a mannose-modified organometallic framework, as well as Zeta and 2Θ potentials of the nanoparticles; where SeZ NPs represent Se@ZIF-8 NPs and SeZMNPs represent Se@ZIF8@Man NPs.

[0035] Figure 2 The diagram shows the biocompatibility and biosafety of the selenium nanoparticle system; A represents the effect of different concentrations of SeZM NPs on peripheral blood erythrocytes; B and C represent the effects of different concentrations of SeZM on macrophage cell viability and apoptosis, detected by CCK8 assay and apoptosis flow cytometry, respectively.

[0036] Figure 3 The image shows the targeting of the selenium nanoparticle system to macrophages; where A represents the specific phagocytosis of SeZNPs and SeZMNPs by M2b macrophages; B represents the specific phagocytosis of SeZNPs and SeZMNPs by monocytes and non-monocytes / macrophages in mouse peripheral blood; both types of selenium nanoparticles are labeled with PE.

[0037] Figure 4 The effects of the selenium nanoparticle system on the polarization of M2b macrophages are shown in Figure A. Figure A shows the effect of adding ZMNPs (representing ZIF8@Man) or SeZMNPs on the polarization direction of M2b macrophages; Figure B shows the effect of SeZMNPs on cytokine expression in M2b macrophages.

[0038] Figure 5 Figure 1 shows the effect of the selenium nanoparticle system SeZMNPs on the mitochondrial oxidative respiration function of M2b macrophages.

[0039] Figure 6 To verify the mechanism by which the selenium nanoparticle system SeZMNPs affects M2b macrophages using RNA-seq detection, it was found that the main mechanism by which it exerts its effects is by inhibiting JAK1 / JAK2-STAT1.

[0040] Figure 7 The distribution and metabolism of the selenium nanoparticle system SeZMNPs in mice with LN disease;

[0041] Figure 8 Figure showing the improvement in glomerular sclerosis and fibrosis levels in LN mice three weeks after tail vein injection of the selenium nanoparticle system SeZMNPs. Detailed Implementation

[0042] This invention provides a method for preparing mannose-modified organometallic framework-encapsulated selenium nanoparticles, comprising the following steps:

[0043] 1) Mix ultra-small nano-selenium solution, 2-methylimidazole solution, polyvinylpyrrolidone and zinc nitrate solution, and let them stand to react, to obtain the reactants after standing.

[0044] 2) Centrifuge the static reactant obtained in step 1), wash the resulting solid and mix it with water to obtain a Se@Z8 solution;

[0045] 3) Mix PEG-COOH, water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, then mix with D-mannose and stir to obtain the stirred reaction product;

[0046] 4) Dialyze the stirred reaction mixture obtained in step 3) to obtain a PEG-mannose solution;

[0047] 5) The Se@Z8 solution obtained in step 2) is mixed with the PEG-mannose solution obtained in step 4) and reacted. The reactants are centrifuged and the precipitate obtained is mannose-modified organometallic framework-encapsulated selenium nanoparticles.

[0048] This invention involves mixing an ultra-small nano-selenium solution, a 2-methylimidazole solution, a polyvinylpyrrolidone solution, and a zinc nitrate solution, and allowing them to stand for reaction to obtain a reactant.

[0049] In this invention, the volume ratio of the ultra-small nano-selenium solution, the volume of the 2-methylimidazole solution, the mass of polyvinylpyrrolidone, and the volume of the zinc nitrate solution is preferably 4 mL:25 mL:250 mg:25 mL. In this invention, the average molecular weight of the polyvinylpyrrolidone is preferably 58000 Da. In this invention, the concentration of the ultra-small nano-selenium solution is preferably 15-20 nM, more preferably 18 nM. In this invention, the preparation method of the 2-methylimidazole solution is preferably: dissolving 26.175 mg of 2-methylimidazole in 25 mL of water. In this invention, the preparation method of the zinc nitrate solution is preferably: dissolving 93.6 mg of zinc nitrate hexahydrate in 25 mL of water. In this invention, the preparation method of the ultra-small nano-selenium solution preferably includes the following steps: mixing polyethylene glycol 400 and selenium powder, and maintaining the mixture at 220°C for 1 hour to obtain a nano-selenium solution; the volume ratio of polyethylene glycol 400 to the mass ratio of selenium powder is preferably 20 mL:40 mg. In this invention, the settling time is preferably 6 hours.

[0050] In this invention, the obtained static reactants are centrifuged, the resulting solid is washed and mixed with water to obtain a Se@Z8 solution. In this invention, the centrifugation conditions preferably include: a rotation speed of 12000 rpm and a time of 10 min.

[0051] This invention involves mixing PEG-COOH, water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide, then mixing with D-mannose and stirring to obtain a stirred reaction product. In this invention, the preferred mass ratio of PEG-COOH, water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 100 mg:10 mL:10 mg:10 mg. The preferred molecular weight of PEG-COOH is 5000 Da. The preferred mass ratio of PEG-COOH to D-mannose is 1:1. The preferred stirring time is 12 hours.

[0052] The present invention dialyzes the obtained stirred reaction mixture to obtain a PEG-mannose solution. Preferably, the present invention uses a dialysis bag with a molecular weight cutoff of 10000 Da for dialysis for 24 hours to remove unreacted reagents.

[0053] In this invention, the obtained Se@Z8 solution is mixed with the obtained PEG-mannose solution and reacted. The resulting reactants are centrifuged, and the precipitate is mannose-modified organometallic framework-encapsulated selenium nanoparticles. In this invention, the preferred volume ratio of the Se@Z8 solution to the PEG-mannose solution is 10:1. The preferred concentration of the Se@Z8 solution is 3–5 mg / mL, and the preferred concentration of the PEG-mannose solution is 0.3–0.5 mg / mL.

[0054] This invention provides a mannose-modified organometallic framework-encapsulated selenium nanoparticles prepared by the preparation method described above.

[0055] The present invention also provides the application of mannose-modified organometallic framework-encapsulated nano-selenium as described in the above-described technical solution in the preparation of drugs for treating lupus nephritis.

[0056] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0057] Example 1

[0058] Preparation of Se@ZIF-8@Man (SeZMNPs) nanosystem

[0059] 1. Synthesis of ultra-small selenium nanoparticles: A high-temperature heating plate was set to 220℃ and preheated for half an hour. Then, 20 mL of polyethylene glycol 400 and 40 mg of selenium powder were added to a 50 mL beaker. A magnetic stirrer was added, and the mixture was stirred at 500 rpm for 10 minutes. After ultrasonic dispersion for 5 minutes using an ultrasonic cleaner, the beaker was transferred to the high-temperature heating plate and heated at 220℃ for 1 hour, while simultaneously stirring with a glass rod to ensure uniform heating. The reaction temperature was monitored and maintained at 220℃. As the reaction proceeded, the color gradually deepened. After the reaction was complete, the product was cooled to room temperature and stored at room temperature to obtain a selenium nanoparticle solution (concentration 5 mg / mL).

[0060] 2. Synthesis of Se@Z8 (SeZNPs): 26.175 mg of 2-methylimidazole was dissolved in 25 mL of water, and 93.9 mg of zinc nitrate hexahydrate was dissolved in 25 mL of water, with sonication as the dissolution aid. Then, 250 mg of polyvinylpyrrolidone (average molecular weight 58000, K29-32) was weighed into a 200 mL beaker, and 25 mL of the dissolved 2-methylimidazole solution was added to the beaker. The mixture was stirred and sonicated until the polyvinylpyrrolidone was completely dissolved. Then, 4 mL of the nano-selenium solution prepared in reaction 1 was taken using a pipette and stirred until homogeneous. Finally, 25 mL of the prepared zinc nitrate hexahydrate solution was added, and the mixture was stirred with a glass rod until homogeneous. The reaction was then carried out in a quiet, vibration-free environment for 6 hours. After the reaction was complete, the precipitate was centrifuged at 12000 rpm for 10 min. The precipitate was washed three times with methanol and twice with ultrapure water, and finally stored in 10 mL of aqueous solution to obtain the Se@Z8 solution (concentration 5 mg / mL).

[0061] 3. Preparation of PEG-mannose: Weigh 100 mg of PEG-COOH (molecular weight: 5000 Da) and dissolve it in 10 mL of water. Then add 10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 10 mg of N-hydroxysuccinimide, stir and dissolve on a magnetic stirrer, and react for 30 min. Then add 100 mg of D-mannose and stir and react at room temperature for 12 h. Finally, dialyze the reaction product for 24 h using a dialysis bag with a molecular weight of 10000 Da to remove unreacted D-mannose, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide. Finally, obtain 10 mL of PEG-mannose solution (10 mg / mL) and store at room temperature.

[0062] 4. Preparation of Se@Z8@mannose (SeZMNPs): 1 mL of the PEG-mannose solution from reaction 3 was added to 10 mL of the Se@Z8 solution obtained in reaction 2. The mixture was stirred at 300 rpm for 24 h at room temperature to target and modify the surface of Se@Z8. After the reaction was complete, the mixture was centrifuged at 12000 rpm for 10 min, washed three times with water, and finally stored in 10 mL of water to obtain SeZMNPs.

[0063] Zeta potential detection

[0064] 1. Sample Preparation: Weigh equal masses of ultra-small nano-selenium, ZIF-8, SeZNPs, and SeZMNPs samples and place them in an equal volume of physiological saline. Use an ultrasonic processor to sonicate each sample for 10 minutes to ensure thorough particle dispersion. Simultaneously, perform preliminary analysis of the dispersion using a laser particle size analyzer to ensure that the particle size distribution is within the instrument's detectable range and that the dispersion is uniform.

[0065] 2. Zeta potential detection: The instrument should be warmed up for 30 minutes to reach a stable operating state, and initialization operations such as optical path calibration and background noise detection should be performed. Each sample dispersion should be slowly and carefully injected into the sample cell, ensuring no air bubbles are generated. Measurements should be taken three times at 25°C, with a 2-minute interval between each measurement, and the results recorded.

[0066] X-ray diffraction (XRD) detection

[0067] 1. Turn on the XRD diffractometer and allow it to warm up for 60 minutes.

[0068] 2. Centrifuge the dispersed SeNPs, ZIF-8, and SeZ NPs sample solutions at 7000 rpm for 10 min, and discard the supernatant. Then, dry the samples in a 60℃ oven for 3 h to obtain dried powder samples. Weigh appropriate amounts of the dried samples, spread them evenly on the sample stage, and gently press them down with tweezers.

[0069] 3. Set the scanning range to 5-80°, the scanning speed to 0.03° / s, the tube voltage to 40kV, and the tube current to 30mA, and perform XRD detection.

[0070] Example 2:

[0071] Safety Study of SeZMNPs

[0072] (1) Biocompatibility detection of SeZMNPs

[0073] Hemolysis was detected using an assay: Morphologically analyzed red blood cells were centrifuged at 1500g for 10 min at room temperature, and the supernatant and leukocyte membranes were removed. The red blood cell pellet was washed three times with PBS, and then diluted to 10% with PBS for further observation. Red blood cells were diluted with distilled water until hemolysis occurred, serving as a hemolysis control. Different concentrations of SeZMNPs (0, 10, 20, 40, 80 μg / mL) were added to 1 mL of 10% red blood cell solution, gently mixed, and incubated at 37℃ for 24 h. After incubation, the diluted 10% red blood cells were centrifuged at 1500g for 10 min, and the supernatant was transferred to a 96-well plate. The absorbance was measured at 540 nm. The formula for calculating the hemolysis rate using absorbance is as follows:

[0074] Experimental group - negative control

[0075] Hemolysis rate = Positive control - Negative control * 100%

[0076] The results are as follows Figure 2 As shown in Figure A, after treatment with various concentrations of SeZM, the hemolysis rate of erythrocytes remained below 0.05%, with almost no hemolysis, indicating that the nano-selenium system SeZM has good biocompatibility.

[0077] (2) Detection of cytotoxicity of SeZMNPs

[0078] In this embodiment, the cytotoxicity of SeZM was detected by CCK-8 staining and flow cytometry. For the CCK-8 assay, mouse bone marrow primary macrophages (BMDMs) were cultured at 2.5 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 cells / well in 96-well plates and incubated overnight in DMEM medium. The next day, the medium was removed, and medium containing different concentrations of SeZMNPs (0, 5, 10, 15, 20, 30 μg / ml) was added and incubated for 12 or 24 hours. After incubation, the medium was removed, and the cells were washed three times with PBS. 63.75 μl of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 1 hour. The absorbance at 450 nm was measured using a microplate reader, and cell viability was assessed by observing changes in absorbance.

[0079] For flow cytometry analysis, BMDMs were prepared at 8 x 10⁸ cells per well. 5Cells were seeded at a density of [number] cells / well in six-well plates and incubated overnight. The next day, the culture medium was removed, and SeZMNP-containing medium was added to the six-well plates using the same SeZMNP concentration gradient as in the CCK-8 experiment. The plates were incubated for 12 hours. After incubation, the culture medium was removed, and the cells were washed three times with PBS. 1 ml of trypsin was added to each well, and the plates were incubated at 37°C for 4 minutes to digest the cells. Twice the volume of culture medium was then added to stop digestion. The cells were then aspirated and centrifuged at 1000 rpm for 5 minutes to obtain a cell pellet. The cells were resuspended in 100 μl of pre-chilled PBS and centrifuged at 1000 rpm for 5 minutes to obtain another cell pellet. The cells were resuspended again in 100 μl of pre-chilled PBS, and 2 μl of Annexin V and 7AAD were added. After mixing, the cells were incubated at room temperature in the dark for 20 minutes, centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, and the cells were resuspended in PBS. This centrifugation and resuspension process was repeated twice. Finally, the cells were analyzed using flow cytometry.

[0080] The results are as follows Figure 2 As shown in Figures B and C, at concentrations up to 30 μg / ml, SeZMNPs do not induce significant apoptosis and exhibit very low cytotoxicity. Compared to traditional lineage drugs, SeZMNPs have a good safety profile.

[0081] Example 3

[0082] Targeting studies of SeZMNPs

[0083] To better demonstrate the targeting effect of selenium nanoparticles on macrophages, Rhodamine B was used to label SeZNPs and SeZMNPs, causing them to show a bright red color under the PE channel of a fluorescence microscope.

[0084] (1) Detection of the targeting ability of the nano-selenium system to M2b cells

[0085] BMDMs at 8*10 per orifice 5 Cells were seeded at a density of [number] cells / well in six-well plates and incubated overnight. To construct M2b macrophages, 2 ng / ml LPS and 2% serum from female lupus model mice (MRL / lpr) were added to the culture medium, and the cells were incubated for 12 h. After incubation, the culture medium was removed, and medium containing 10 μg / ml SeZMNPs was added, and the cells were incubated at 37°C for 3 h. Subsequently, the culture medium was removed, and 5 μg / ml Hoechst 33342 PBS was added, and the cells were incubated at 37°C for 30 min to label the cell nuclei. Finally, the culture medium was removed, and the cells were washed three times with PBS before being observed under a fluorescence microscope.

[0086] The results are as follows Figure 3As shown in Figure A, compared to SeZM NPs, M2b macrophages significantly engulf more SeZM NPs containing mannose.

[0087] (2) Detection of the targeting ability of the nano-selenium system in peripheral blood

[0088] Peripheral blood was collected from 6-8 week old female C57BL / 6 mice. After centrifugation at 1500g for 10 min, the supernatant was removed. 10 ml of erythrocyte lysis buffer was added to each milliliter of cell pellet, gently mixed, and incubated at 4°C for 10 min. Finally, the pellet was centrifuged at 1500 rpm for 10 min. Alternatively, peripheral blood leukocytes (PBMCs) were collected. The PBMCs were then resuspended in culture medium and incubated with rhodamine b-labeled SeZ NPs and SeZM NPs (10 μg / ml) for 3 h. After incubation, the culture medium was removed, and immune cells were labeled with CD45 and monocytes / macrophages with CD11b. The cells were incubated at room temperature in the dark for 20 min, and washed three times with PBS. The samples were then analyzed by flow cytometry.

[0089] Example 4

[0090] Effects of SeZM NPs on M2b macrophage polarization and inflammatory cytokine expression

[0091] BMDMs at 8*10 per orifice 5 Cells were seeded at a density of [number] cells per well in a six-well plate and incubated overnight in an incubator to allow for proper adhesion. The experiment consisted of four groups: control group M0 (normal macrophages); M2b cells (cells supplemented with 2 ng / ml LPS and 2% serum from female MRL / lpr diseased mice, incubated for 12 h); M2b+ZM (M2b macrophages supplemented with 10 μg / ml ZMNPs, incubated for 12 h; ZMNPs are selenium-free empty-loaded nanomedicines); and M2b+SeZM (M2b macrophages supplemented with 10 μg / ml SeZMNPs, incubated for 12 h).

[0092] (1) Effects of the nano-selenium system on the polarization of M2b macrophages

[0093] After cell incubation in each group, the supernatant was removed, and the cells were gently washed twice with PBS. 500 μL of trypsin was added to each well, and the cells were incubated at 37°C for 3 min to digest the cells. After adding an equal volume of culture medium to terminate digestion, the cells were centrifuged at 1500 rpm for 5 min to obtain the cell pellet. The cells were resuspended in 100 μL of PBS, and 5 μL each of anti-CD11b, anti-CD86, and anti-CD206 flow cytometry antibodies were added in the dark. After mixing, the cells were incubated at room temperature in the dark for 20 min. Subsequently, the cell suspension was centrifuged at 1500 rpm for 5 min, the supernatant was removed, and the cells were resuspended in PBS and centrifuged twice to wash away unbound antibodies. Finally, the cells were resuspended in 200 μL of PBS for flow cytometry analysis. The proportion of cells expressing CD86 and CD206 in each group was observed among CD11b-positive cells.

[0094] The results are as follows Figure 4 As shown in Figure A, M2b macrophages highly express CD86 and CD206, but their expression is significantly inhibited after the addition of SeZMNPs, while ZMNPs do not have this effect. This indicates that SeZMNPs can inhibit M2b macrophage polarization, and their function is mainly achieved through the selenium encapsulation within them.

[0095] (2) Effects of the nano-selenium system on the expression of inflammatory factors in M2b macrophages

[0096] After incubation of each cell group, the cells were gently washed twice with PBS, then RIPAlysis buffer containing protease inhibitors was added. The mixture was incubated on ice and lysed for 30 min, followed by centrifugation at 12000 rpm for 30 min to remove the precipitate. Protein quantification was performed using the BCA method, followed by SDS protein denaturation. Finally, samples of equal protein concentration and quantity were added to SDS-PAGE gels for electrophoresis and Western blotting, and exposed to ultrasensitive chromogenic reagent.

[0097] The results are as follows Figure 4 As shown in Figure B, M2b macrophages highly express TNF-α, IL-6, and CIL-1β, but their expression is significantly inhibited after the addition of SeZMNPs, while ZMNPs do not have this significant inhibitory effect. This indicates that SeZMNPs can inhibit M2b macrophage polarization, and their function is mainly achieved through the selenium encapsulation within them.

[0098] Example 5

[0099] Effects of selenium nanoparticle system on mitochondrial stress in M2b macrophages

[0100] This experiment used Seahorse technology to detect cellular oxygen consumption rate (OCR). 5 x 10⁵ cells / well were added to each well of a SeahorseXF 96-well cell culture microplate. 4 Raw264.7 cells were fully settled and adhered to the culture medium. 50 ng / ml LPS and 2% LN mouse serum were added, and the cells were incubated for 12 h to polarize them into M2b macrophages. Subsequently, 10 μg / ml SeZMNPs were added to the culture medium, gently mixed, and incubated for another 12 h. Simultaneously, the day before the experiment, a sensor probe plate was hydrated overnight with SeahorseXF calibration solution in a CO2-free incubator at 37°C. Detection solutions were prepared by adding 1 mmol / L sodium pyruvate, 2 mmol / L glutamine, and 10 mmol / L glucose to SeahorseXFDMEM or RPMI medium. The samples were then placed in the Seahorse detection system for analysis.

[0101] Experimental results are as follows Figure 5 As shown, M2b macrophages exhibit significant mitochondrial stress and reduced mitochondrial oxidative metabolism, while SeZM NPs effectively alleviate this stress state and protect mitochondria.

[0102] Example 6

[0103] SeZM NPs inhibit M2b macrophage polarization by suppressing the JAK1 / JAK2-STAT1 pathway.

[0104] The experimental grouping and drug treatment of cells were the same as in Experiment 3. The methods for collecting, denaturing, and analyzing cell proteins by Western blot were the same as in Experiment 3(2).

[0105] Experimental results are as follows Figure 6 As shown, the expression ratios of p-JAK1 / JAK1, p-JAK2 / JAK2, and p-STAT1 / STAT1 in M2b macrophages were significantly reduced after the addition of SeZMNPs, indicating that the activation of JAK1, JAK2, and STAT1 was significantly inhibited. ZM NPs, however, did not significantly inhibit the activation of these proteins. The JAK-STAT pathway is an important pathway dependent on macrophage activation and inflammatory cytokine secretion, and these experimental results can be mutually explained with previous cell characterization findings.

[0106] Experimental Example 7

[0107] Distribution of the nano-selenium system in lupus mice

[0108] The method for manufacturing ICG-labeled SeZM NPs differs slightly from the method for synthesizing SeZM NPs. During the synthesis of SeZM NPs, 0.5 mg / mL ICG was added to a 2-Mi solution to encapsulate the ICG during the metal-organic framework synthesis. After centrifugation at 12000 rpm for 10 min and washing with methanol, ICG-labeled SeZM NPs and SeZNPs were finally obtained. (The last sentence appears to be incomplete and possibly refers to a different process.)

[0109] Rats were intravenously injected with 0.4 mg / kg of ICG-labeled SeZMNPs. Fluorescence was monitored using an imaging system (NightOWL II LB 983) at 0 h, 0.25 h, 1 h, 8 h, and 12 h.

[0110] Experimental results are as follows Figure 7 As shown, both SeZM NPs and SeZ NPs are effectively distributed in vivo. However, compared to SeZ NPs, SeZM NPs accumulate more in the skin and kidneys, especially in the kidneys (white circle area at 12h). SeZM NPs have a longer residence time and are deposited in greater quantities. These sites are the main sites of damage in the mouse model of SLE and are also the two most common target organs of SLE in humans.

[0111] Experimental Example 8

[0112] The therapeutic effect of selenium nanoparticle system SeZM NPs on LN mice

[0113] The experiment consisted of three groups: a control group (13-week-old female MRL / MPJ non-pathogenic control mice); an LN group (13-week-old female MRL / lpr pathogenic mice); and an LN+SeZM group (13-week-old female MRL / lpr pathogenic mice, receiving daily tail vein injections of 1 mg / kg SeZMNPs for 21 consecutive days). As controls, mice in the control and LN groups received daily tail vein injections of 1 mg / kg physiological saline during the experiment. At the end of the experimental period, the mice were euthanized, and the kidneys were removed and fixed with 4% paraformaldehyde at 4°C for 24 hours. Subsequently, the kidneys were paraffin-embedded and sectioned, and stained with HE, PAS, Masson's stain, and Sirus red stain to observe the pathological changes.

[0114] Experimental results are as follows Figure 8 As shown, LN mice exhibited significant glomerular sclerosis and fibrosis, with marked destruction of glomerular structure. SeZMNPs treatment significantly improved glomerular damage and mitigated the deterioration of renal function. These results indicate that SeZMNPs can significantly alleviate the progression of LN and reduce kidney damage.

[0115] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing selenium nanoparticles encapsulated in a mannose-modified organometallic framework, characterized in that, Includes the following steps: 1) Mix ultra-small nano-selenium solution, 2-methylimidazole solution, polyvinylpyrrolidone and zinc nitrate solution, and let them stand to react, to obtain the reactants after standing. 2) Centrifuge the static reactant obtained in step 1), wash the resulting solid and mix it with water to obtain a Se@Z8 solution; 3) Mix PEG-COOH, water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, then mix with D-mannose and stir to obtain the stirred reaction product; 4) Dialyze the stirred reaction mixture obtained in step 3) to obtain a PEG-mannose solution; 5) The Se@Z8 solution obtained in step 2) is mixed with the PEG-mannose solution obtained in step 4) and reacted. The reactants are centrifuged and the precipitate obtained is mannose-modified organometallic framework-encapsulated selenium nanoparticles.

2. The preparation method according to claim 1, characterized in that, In step 1), the volume ratio of the ultra-small nano-selenium solution, the volume of the 2-methylimidazolium solution, the mass of polyvinylpyrrolidone, and the volume of the zinc nitrate solution is 4 mL:25 mL:250 mg:25 mL. The concentration of the nano-selenium solution is 18 nM; The 2-methylimidazole solution is prepared by dissolving 26.175 mg of 2-methylimidazole in 25 mL of water; The zinc nitrate solution is prepared by dissolving 93.6 mg of zinc nitrate hexahydrate in 25 mL of water.

3. The preparation method according to claim 1 or 2, characterized in that, The preparation method of the ultra-small nano selenium solution includes the following steps: after mixing polyethylene glycol 400 and selenium powder, the mixture is kept at a temperature of 220°C for 1 hour to obtain the ultra-small nano selenium solution. The volume ratio of polyethylene glycol 400 to selenium powder is 20 mL: 40 mg.

4. The preparation method according to claim 1, characterized in that, The static reaction time in step 1) is 6 hours.

5. The preparation method according to claim 1, characterized in that, The centrifugation conditions in step 2) include: a rotation speed of 12,000 rpm and a time of 10 min.

6. The preparation method according to claim 1, characterized in that, In step 3), the mass ratio of PEG-COOH, water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 100 mg: 10 mL: 10 mg: 10 mg. The molecular weight of the PEG-COOH is 5000 Da.

7. The preparation method according to claim 1, characterized in that, In step 3), the mass ratio of PEG-COOH to D-mannose is 1:1; the stirring reaction time is 12 hours.

8. The preparation method according to claim 1, characterized in that, In step 5), the volume ratio of Se@Z8 solution to PEG-mannose solution is 10:

1. The concentration of the Se@Z8 solution is 3-5 mg / mL, and the concentration of the PEG-mannose solution is 0.3-0.5 mg / mL.

9. A mannose-modified organometallic framework-encapsulated selenium nanoparticles prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the mannose-modified organometallic framework-encapsulated selenium nanoparticles as described in claim 9 in the preparation of a drug for treating lupus nephritis.

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