A nano-coordination polymer for treating liver injury and a preparation method thereof

Abstract

The application discloses a preparation method of a nano-coordination polymer for treating sepsis-induced liver injury, and comprises the following steps: 1, glycyrrhetinic acid and metal ions form glycyrrhetinic acid / metal ion coordination nanoparticles through coordination; 2, under alkaline conditions, dopamine monomers are self-polymerized on the surface of the glycyrrhetinic acid / metal ion coordination nanoparticles to form core-shell structure nanoparticles with the glycyrrhetinic acid / metal ion coordination nanoparticles as the core and polydopamine as the shell; 3, a gene silencing tool designed for HMGB1 protein is connected to the surface of the core-shell structure nanoparticles to obtain the nano-coordination polymer for treating sepsis-induced liver injury. The application further discloses the nano-coordination polymer for treating sepsis-induced liver injury prepared by the preparation method. The application realizes the combined application of drug prevention and treatment and gene silencing treatment for treating sepsis-induced liver injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nano-coordination polymer for treating sepsis-induced liver injury and its preparation method. Background Technology

[0002] Currently, the main methods for treating sepsis-induced liver injury include:

[0003] 1. Antibiotic treatment is the basic method for treating sepsis. It can effectively control infection and reduce the number of pathogens. However, it may lead to antibiotic resistance, making the disease untreatable or even worsening liver damage.

[0004] 2. Supportive therapies, including intravenous infusion, hemodialysis, and mechanical ventilation, are used to maintain the patient's vital signs and organ functions. They can stabilize the patient's vital signs and support the function of various organ systems, but they cannot directly treat liver damage; they can only provide support and cannot solve the root problem.

[0005] 3. Anti-inflammatory treatment: Using anti-inflammatory drugs such as corticosteroids to reduce the inflammatory response can alleviate inflammation and protect liver cells. However, long-term use may cause side effects and may not necessarily significantly improve liver function.

[0006] 4. Liver protectants, such as heparin and N-acetylcysteine ​​(NAC), are used to protect hepatocytes and reduce oxidative stress. They can directly protect hepatocytes, but their therapeutic effect is limited and they need to be used in combination with other treatment methods.

[0007] Therefore, there is a need to design better therapeutic drugs and preparation methods for sepsis-induced liver injury. Summary of the Invention

[0008] The purpose of this invention is to provide a nano-coordination polymer for treating sepsis-induced liver injury, thereby addressing the various shortcomings of the main methods for treating sepsis-induced liver injury as presented in the background art.

[0009] To achieve the above objectives, the present invention provides a method for preparing a nano-coordination polymer for treating sepsis-induced liver injury, comprising the following steps:

[0010] Step 1: Glycyrrhetinic acid and metal ions coordinate to form glycyrrhetinic acid / metal ion coordination nanoparticles; the metal ions include Zn. 2+ Mg 2+ and Ce 2+ ;

[0011] Step 2: Under alkaline conditions, dopamine monomers are allowed to self-polymerize on the surface of the glycyrrhetinic acid / metal ion coordination nanoparticles to form core-shell structured nanoparticles with glycyrrhetinic acid / metal ion coordination nanoparticles as the core and polydopamine as the shell.

[0012] Step 3: Connect a gene silencing tool designed for the HMGB1 protein to the surface of the core-shell structured nanoparticles to obtain a nano-coordination polymer for treating sepsis-induced liver injury; the gene silencing tool includes DNAzyme, siRNA, and antisense oligonucleotides.

[0013] In one specific embodiment, in step 3, a gene silencing tool designed for the HMGB1 protein is attached to the surface of the core-shell nanoparticle by utilizing the Michael addition or Schiff base reaction between catechol or amino-terminated molecules on the surface of the polydopamine shell.

[0014] In one specific implementation, the gene silencing tool in step 3 is DNAzyme.

[0015] In one specific embodiment, the DNAzyme sequence is: CTCTTCCTTCTTTTTAAGCTGGCCGAGCCTTGCTCTTTTCAGCCT.

[0016] In one specific embodiment, the metal ion in step 1 is Zn. 2+ .

[0017] In one specific embodiment, step 1, the specific steps for forming glycyrrhetinic acid / metal ion coordination nanoparticles, are as follows:

[0018] First, add glycyrrhetinic acid ethanol solution to the container, then add Tris buffer and polyvinylpyrrolidone, and stir at room temperature to obtain a homogeneous mixed solution; then, add zinc acetate solution dropwise to the above mixed solution through a constant flow pump to generate glycyrrhetinic acid / metal ion coordination nanoparticles, namely glycyrrhetinic acid / zinc nanocoordination polymer (GA / Zn nanocoordination polymer).

[0019] In one specific embodiment, the Tris buffer is a Tris buffer with a pH of 8.4 to 8.6.

[0020] In one specific embodiment, in step 2, the dopamine monomer is added in the form of a dopamine hydrochloride solution.

[0021] The present invention also provides a nano-coordination polymer for treating sepsis-induced liver injury, which is prepared by the method described above for preparing a nano-coordination polymer for treating sepsis-induced liver injury.

[0022] The present invention also provides a medicament for treating sepsis-induced liver injury, wherein the medicament for treating sepsis-induced liver injury contains the nano-coordination polymer for treating sepsis-induced liver injury as described above.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The glycyrrhetinic acid / zinc core in the nano-coordination polymer of this invention for treating sepsis-induced liver injury can degrade into glycyrrhetinic acid and Zn in the in vivo environment of septic acidosis. 2+ Glycyrrhetinic acid is a natural inhibitor of HMGB1, which can be used to inhibit sepsis-induced hepatocyte pyroptosis and has anti-inflammatory and antioxidant effects; Zn 2+ It can activate DNAzyme, downregulate the expression of HMGB1 protein, and enhance the therapeutic effect of sepsis-induced liver injury.

[0025] The present invention provides a nano-coordination polymer for treating sepsis-induced liver injury, which not only improves the bioavailability of glycyrrhetinic acid in vivo, but also achieves co-delivery of glycyrrhetinic acid and zinc ions, exerting a synergistic therapeutic effect; furthermore, it enhances the gene silencing effect by achieving efficient delivery and in-situ activation of DNases. This invention not only achieves targeted targeting of the liver with nanomedicine, but also realizes the combined application of drug prevention and gene silencing therapy.

[0026] In addition to the objectives, features, and advantages described above, the present invention has other objectives, features, and advantages. The present invention will now be described in further detail. Attached Figure Description

[0027] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0028] Figure 1 This is a particle size distribution diagram of the nano-coordination polymer in this invention;

[0029] Figure 2 This is a potential diagram of the nano-coordination polymer in this invention;

[0030] Figure 3 This is a transmission electron microscope image of the nano-coordination polymer in this invention;

[0031] Figure 4 This is the elemental analysis diagram of the nano-coordination polymer in this invention;

[0032] Figure 5 This is the energy dispersive X-ray spectrum of the nano-coordination polymer in this invention;

[0033] Figure 6 The results of the stability study of the nano-coordination polymer in this invention are as follows: Particle size variation diagram of the nano-coordination polymer under different conditions;

[0034] Figure 7 The results of the stability study of the nano-coordination polymer in this invention are as follows: a graph showing the variation of the polydispersity index of the nano-coordination polymer under different conditions;

[0035] Figure 8 This is a graph showing the release rate of the nano-coordination polymer in this invention under different conditions;

[0036] Figure 9 This is a comparative diagram of the cell delivery effect of the nano-coordination polymer on DNase in this invention (scale bar = 50 μm);

[0037] Figure 10 This is a comparison chart of the uptake of nano-coordination polymers by cells at different time points in this invention.

[0038] Figure 11 The results of the in vitro sepsis efficacy evaluation of the nano-coordination polymer in this invention: live and dead cell staining results;

[0039] Figure 12 The results of the in vitro sepsis efficacy evaluation of the nano-coordination polymer in this invention are shown in the figure: protein expression results of sepsis model cells induced by multiple modes;

[0040] Figure 13 The results of the in vitro sepsis efficacy evaluation of the nano-coordination polymer in this invention: a comparison of serum IL-1α in each group;

[0041] Figure 14 The results of the in vitro sepsis efficacy evaluation of the nano-coordination polymer in this invention: a comparison of serum IL-1β in each group;

[0042] Figure 15 This is a diagram showing the results of the in vivo distribution study of the nano-coordination polymer in this invention;

[0043] Figure 16 The following is an image showing the in vivo efficacy evaluation results of the nano-coordination polymer for sepsis in this invention: LPS model mouse liver protein expression results;

[0044] Figure 17 The following is an image showing the in vivo efficacy evaluation results of the nano-coordination polymer for sepsis in this invention: liver protein expression results in CLP model mice;

[0045] Figure 18 The following are the in vivo efficacy evaluation results of the nano-coordination polymer in this invention for sepsis: serum ALT graphs for each group;

[0046] Figure 19The following are the in vivo efficacy evaluation results of the nano-coordination polymer in this invention for sepsis: serum AST maps of each group;

[0047] Figure 20 The following are the in vivo efficacy evaluation results of the nano-coordination polymer in this invention for sepsis: serum IL-1α graphs for each group;

[0048] Figure 21 The following are the in vivo efficacy evaluation results of the nano-coordination polymer in this invention for sepsis: serum IL-1β graphs for each group;

[0049] Figure 22 The following are the in vivo efficacy evaluation results of the nano-coordination polymer in this invention for sepsis: serum TAT graphs for each group;

[0050] Figure 23 The following are the in vivo efficacy evaluation results of the nano-coordination polymer in this invention for sepsis: serum PAI-1 levels in each group;

[0051] Figure 24 The following is an evaluation result of the in vivo efficacy of the nano-coordination polymer in this invention for sepsis: H&E staining image of liver tissue;

[0052] Figure 25 The following is an evaluation result of the in vivo efficacy of the nano-coordination polymer in this invention for sepsis: Immunohistochemical image of HMGB1 protein in liver tissue;

[0053] Figure 26 The following is an evaluation result of the in vivo efficacy of the nano-coordination polymer in this invention for sepsis: survival curve of mice in the LPS model group;

[0054] Figure 27 The following is an evaluation result of the in vivo efficacy of the nano-coordination polymer in this invention for sepsis: survival curve of mice in the CLP model group;

[0055] Figure 28 The safety evaluation results of the nano-coordination polymer in this invention are shown in the figure: hemolysis test results.

[0056] Figure 29 The safety evaluation results of the nano-coordination polymer in this invention are as follows: pathological sections of the heart, liver, spleen, lung, and kidney of mice in each group;

[0057] Figure 30 The safety evaluation results of the nano-coordination polymer in this invention are shown in the graph of weight changes in mice in each group.

[0058] Figure 31 The results of the safety evaluation of the nano-coordination polymer in this invention are shown in the graph of liver and kidney injury indicators in mice of each group.

[0059] Figure 32 This is a flowchart illustrating the preparation process of a nano-coordination polymer according to the present invention. Detailed Implementation

[0060] The embodiments of the present invention will be described in detail below. The specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0061] A method for preparing a nano-coordination polymer for treating sepsis-induced liver injury according to the present invention includes the following steps:

[0062] Step 1: Glycyrrhetinic acid and metal ions coordinate to form glycyrrhetinic acid / metal ion coordination nanoparticles; the metal ions include Zn. 2+ Mg 2+ and Ce 2+ .

[0063] Preferably, the metal ion in step 1 is Zn. 2+ .

[0064] In step 1, the specific steps for forming glycyrrhetinic acid / metal ion coordination nanoparticles are as follows:

[0065] First, add glycyrrhetinic acid ethanol solution to the container, then add Tris buffer solution with a pH of 8.4-8.6 and polyvinylpyrrolidone, and stir at room temperature to obtain a homogeneous mixed solution; then, add zinc acetate solution dropwise to the above mixed solution through a constant flow pump to generate glycyrrhetinic acid / metal ion coordination nanoparticles, namely glycyrrhetinic acid / zinc nanocoordination polymer (GA / Zn nanocoordination polymer).

[0066] Step 2: Under alkaline conditions, dopamine monomers are allowed to self-polymerize on the surface of the glycyrrhetinic acid / metal ion coordination nanoparticles to form core-shell structured nanoparticles with glycyrrhetinic acid / metal ion coordination nanoparticles as the core and polydopamine as the shell.

[0067] In step 2, the dopamine monomer is added in the form of dopamine hydrochloride solution.

[0068] Step 3: Using the Michael addition or Schiff base reaction between catechol or amino-amino-terminated molecules on the surface of the polydopamine shell, a gene silencing tool designed for the HMGB1 protein is attached to the surface of the core-shell nanoparticles to obtain a nano-coordination polymer for treating sepsis-induced liver injury; the gene silencing tool includes DNAzyme, siRNA, and antisense oligonucleotides.

[0069] Preferably, the gene silencing tool in step 3 is DNAzyme. More preferably, the sequence of the DNAzyme is: CTCTTCCTTCTTTTTAAGCTGGCCGAGCCTTGCTCTTTTCAGCCT.

[0070] The present invention also provides a nano-coordination polymer for treating sepsis-induced liver injury, which is prepared by the method described above for preparing a nano-coordination polymer for treating sepsis-induced liver injury.

[0071] The present invention also provides a medicament for treating sepsis-induced liver injury, wherein the medicament for treating sepsis-induced liver injury contains the nano-coordination polymer for treating sepsis-induced liver injury as described above.

[0072] Example 1

[0073] 1. Preparation of GA / Zn@PDA / DZ nano-coordination polymers:

[0074] First, 0.1 mL of a 25 mM glycyrrhetinic acid ethanol solution was added to a 5 mL flask, followed by 0.8 mL of a 200 mM Tris buffer solution (pH 8.5) and 0.1 mL of 20 wt% polyvinylpyrrolidone. The mixture was stirred at room temperature for 1 minute to obtain a homogeneous solution. Next, 0.5 mL of a 10 mM zinc acetate solution was drawn up using a 1 mL syringe and added dropwise to the above mixture at a rate of 40 μL / min using a constant flow pump to generate a glycyrrhetinic acid / zinc nanocoordination polymer (GA / Zn nanocoordination polymer).

[0075] The GA / Zn nanocoordination polymer prepared by the above method is further modified through the following steps to prepare a core-shell structured nanocoordination polymer containing a polydopamine coating layer and a DNase: 2 mL of GA / Zn nanocoordination polymer (GA / Zn NPs) is added to a 5 mL flask. Under stirring, 40 μL of a 10 mg / mL dopamine hydrochloride solution and 30 μL of a 100 μM DNase solution (DNAzyme solution) are added. The DNAzyme sequence is CTCTTCCTTCTTTTTAAGCTGGCCGAGCCTTGCTCTTTTCAGCCT. After the addition is complete, the mixture is stirred at room temperature for 12 hours to obtain the core-shell structured nanocoordination polymer, namely GA / Zn@PDA / DZ nanocoordination polymer (GA / Zn@PDA / DZ NPs), which is also the nanocoordination polymer used to treat sepsis-induced liver injury.

[0076] 2. Measurement of particle size and potential of nano-coordination polymers:

[0077] The particle size of GA / Zn NPs and GA / Zn@PDA / DZ NPs was determined using dynamic light scattering. Samples were placed in a Marlven Nano ZS instrument, with the measurement cell temperature set to 25℃. Each sample was measured three times. The experimental results are as follows: Figure 1-2As shown, the particle size of the GA / Zn nano-coordination polymer is 220 nm and the potential is -19 mV, while the particle size of the GA / Zn@PDA / DZ nano-coordination polymer coated with polydopamine increases to 280 nm and the potential decreases to -22 mV.

[0078] 3. Observation of the morphology of nano-coordination polymers:

[0079] The GA / Zn@PDA / DZ nano-coordination polymer was dropped onto a 400-mesh copper grid covered with a carbon film. After natural drying, its morphology was observed using a transmission electron microscope (Titan G2-F20). The results are as follows: Figure 3 As shown, the GA / Zn@PDA / DZ nano-coordination polymer exhibits an amorphous polymer morphology. Figure 3 Figure A in the image was observed at a scale bar of 500 nm. Figure 3 Image B in the image was observed at a scale bar of 200 nm.

[0080] 4. X-ray photoelectron spectroscopy analysis of nano-coordination polymers:

[0081] XPS scans were performed on the GA / Zn@PDA / DZ nanocoordination polymers, and the results are as follows: Figure 4-5 As shown.

[0082] 5. Stability testing of nano-coordination polymers:

[0083] GA / Zn@PDA / DZ NPs were placed in pure water (Water), physiological saline (Saline), fetal bovine serum (FBS), and DMEM complete medium containing 10% FBS at 37℃, and their particle size changes were measured at different time points. Results are as follows: Figure 6-7 As shown, the particle size of the GA / Zn@PDA / DZ nano-coordination polymer did not change significantly in various media, demonstrating good stability.

[0084] 6. Detection of release rate of nano-coordination polymer:

[0085] GA / Zn@PDA / DZ nano-coordination polymers were diluted 2-fold and placed in dialysis bags with a molecular weight cutoff of 3500 (21 samples of each type). The dialysis bags were then placed in 50 mL centrifuge tubes, and release media at pH 7.4, pH 7.0, and pH 6.5 were added, respectively. Samples were taken at 1, 2, 4, 8, 12, 24, and 48 hours, and the glycyrrhetinic acid content in the dialysate was determined by HPLC to confirm its release behavior. The results are as follows: Figure 8 As shown, the GA / Zn@PDA nano-coordination polymers all exhibit certain acid sensitivity under acidic conditions.

[0086] 7. Preparation of GA / Zn@PDA / DZ-FAM nano-coordination polymers:

[0087] In this embodiment, a nano-coordination polymer (GA / Zn@PDA / DZ-FAM NPs) loaded with FAM-modified DNase was also prepared to investigate its targeting effect on AML12 cells. The specific steps are as follows:

[0088] Add 0.1 mL of 25 mM glycyrrhetinic acid ethanol solution to a 5 mL flask, then add 0.8 mL of 200 mM Tris buffer (pH 8.5) and 0.1 mL of 20 wt% polyvinylpyrrolidone. Stir at room temperature for 1 minute to obtain a homogeneous mixture. Using a 1 mL syringe, add 0.5 mL of 10 mM zinc acetate solution dropwise to the mixture at a constant flow rate of 40 μL / min to generate glycyrrhetinic acid / zinc nanocoordination polymers (GA / Zn NPs). Add 2 mL of GA / Zn NPs to a 5 mL flask, and while stirring, add 40 μL of 10 mg / mL dopamine hydrochloride solution and 30 μL of 1 mM FAM-modified DNase solution. After the addition is complete, stir at room temperature for 12 hours to obtain the core-shell structured nanocoordination polymer GA / Zn@PDA / DZ-FAM NPs.

[0089] 8. Experiment on the cell delivery of DNase by nano-coordination polymers:

[0090] After digesting and counting AML12 cells (mouse hepatocytes, purchased from the Cell Bank of the Chinese Academy of Sciences) in logarithmic growth phase, they were diluted to 2×10⁻⁶ with an appropriate amount of DMEM / F12 complete medium. 5 Cells were seeded at a concentration of 2 mL / mL in confocal culture dishes, with a total of 4 wells. After 24 hours of adherent culture, the culture medium was aspirated and the cells were washed three times with PBS. The following solutions were added to each well: 2 mL of 10 μM free DZ-FAM solution; 2 mL of 100 mg / mL GA / Zn@PDA nanopolymer solution; and 2 mL of 100 mg / mL GA / Zn@PDA / DZ-FAM nanopolymer solution. The cells were incubated at 37°C for 6 hours. After incubation, the culture medium was aspirated and the cells were washed three times with PBS. 1 mL of paraformaldehyde was added to each well for fixation in the dark for 20 minutes. The supernatant was aspirated and the cells were washed three times with PBS. 0.5 mL of 1 μg / mL phalloidin solution was added to each well for staining the cytoskeleton in the dark for 30 minutes. The supernatant was aspirated and the cells were washed three times with PBS. Add 0.5 mL of 1 μg / mL DAPI solution to each well, stain cell nuclei in the dark for 15 minutes, discard the supernatant, and wash three times with PBS. Finally, observe the fluorescence signal of each well under a laser confocal microscope. The results are as follows: Figure 9As shown, compared with the blank group, the free DNAzyme group, and the GA / Zn@PDA group, the GA / Zn@PDA / Dz NPs treatment group showed a strong green fluorescent signal, located around the cell nucleus, which preliminarily confirms that GA / Zn@PDA / Dz NPs can deliver nucleic acids into cells.

[0091] 9. Experiment on cell uptake of nano-coordination polymers at different time points:

[0092] After digesting and counting AML12 cells (mouse hepatocytes, purchased from the Cell Bank of the Chinese Academy of Sciences) in logarithmic growth phase, they were diluted to 2×10⁻⁶ with an appropriate amount of DMEM / F12 complete medium. 5 Cell suspension at a concentration of [number] cells / mL was seeded at 2 mL per well in a confocal culture dish, for a total of 4 wells. After 24 hours of adherent culture, the culture medium was aspirated, and the cells were washed three times with PBS. 2 mL of 100 mg / mL GA / Zn@PDA / DZ-FAM nanopolymer solution was added to each well, and the cells were incubated at 37°C for 0, 1, 3, and 6 hours. After incubation, the culture medium was aspirated, and the cells were washed three times with PBS. 1 mL of paraformaldehyde was added to each well for fixation in the dark for 20 minutes. The supernatant was aspirated, and the cells were washed three times with PBS. 0.5 mL of 1 μg / mL phalloidin solution was added to each well, and the cytoskeleton was stained in the dark for 30 minutes. The supernatant was aspirated, and the cells were washed three times with PBS. 0.5 mL of 1 μg / mL DAPI solution was added to each well, and the cell nuclei were stained in the dark for 15 minutes. The supernatant was aspirated, and the cells were washed three times with PBS. Finally, the fluorescence signal of each well was observed under a laser confocal microscope. The results are as follows: Figure 10 As shown, the amount of DNase delivered by GA / Zn@PDA NPs increases with increasing treatment time, exhibiting a significant concentration dependence.

[0093] 10. Evaluation results of in vitro sepsis treatment efficacy of nano-coordination polymers:

[0094] After digestion and counting of primary mouse peritoneal macrophages, they were diluted to 2 × 10⁻⁶ with an appropriate amount of 1640 complete culture medium. 5Cell suspension at 1000 cells / mL was seeded at 2 mL per well in a confocal culture dish, for a total of 5 wells. Except for the control group, all cells were induced for 4 hours with 1 μg / mL LPS + 400 ng / mL HMGB1 + 10 μg / mL OMV, followed by treatment with 0, 50, 100, and 200 mg / mL nano-coordination polymers, respectively, for another 4 hours. 1 mL of Calcein AM / PI detection working solution was added to each well, and the cells were incubated at 37°C in the dark for 30 min. After washing three times with PBS, the staining effect was observed under a fluorescence microscope (Calcein AM Ex / Em = 494 / 517 nm; PI Ex / Em = 535 / 617 nm). Results are as follows: Figure 11 As shown, the nano-coordination polymer can block LPS, HMGB1 and OMV-induced cell death in a dose-dependent manner.

[0095] Cells were induced using the same stimulation method. After cell drug administration according to the experimental group design, the culture plates were observed under a microscope. Based on the number of viable cells in each well, the required volume of lysis buffer was estimated. The required volume of lysis buffer was prepared according to the ratio of enhanced RIPA lysis buffer to protease inhibitor PMSF = 100:1. The old culture medium was discarded, and the cells were washed twice with PBS. After removing the PBS, an appropriate amount of lysis buffer containing PMSF was added to each well according to the cell density. Cells were scraped from each well using a cell scraper and transferred to centrifuge tubes, labeled, and lysed on ice for 30 min. After complete lysis, the total protein extract from each tube was centrifuged, and the protein was analyzed by Western blotting. The results are as follows: Figure 12 As shown, the nano-coordination polymer can reduce the cleavage of GSDMD-NT by decreasing the expression of HMGB1.

[0096] Cells were induced using the same stimulation method. After cell drug administration according to the experimental group design, the concentrations of IL-1α and IL-1β in the cell supernatant were detected using an enzyme-linked immunosorbent assay (ELISA) kit. 10× Coating Buffer was diluted to 1× with ddH2O. 250× Capture antibody for IL-1β was prepared with 1× Coating Buffer to a final concentration of 1×, and 50 μL / well was added to a 96-well ELISA plate. The plate was incubated overnight at 4°C. After washing three times with 0.005% PBST, 200 μL of 1× Assay Buffer was added to each well for blocking at room temperature for 1 h. 50 μL of the cell supernatant to be tested was added to each well. Standards were started at 1000 pg / mL and serially diluted with 1× Assay Buffer to a final concentration of 62.5 pg / mL, with a minimum concentration of 0 pg / mL. Each well was incubated for 2 h at room temperature. After washing three times with PBST, Detection antibody was diluted to 1× with 1× Assay Buffer. Each well was incubated for 1 h at room temperature. Wash three times with PBST, then dilute 250×HRP to 1× with 1×AssayBuffer and incubate at room temperature for 30 min. After washing five times with PBST, add 50 μL of TMB for color development, stop the color development with 2M H2SO4, and measure the OD value at 450 nm using a microplate reader. Calculate the final concentration of the sample according to the formula derived from the standard curve. Results are as follows. Figure 13-14 As shown, the nano-coordination polymer can inhibit the release of IL-1α and IL-1β by multimodal induced pyroptosis in a dose-dependent manner.

[0097] 11. Results of in vivo distribution study of nano-coordination polymers:

[0098] GA / Zn@PDA / DZ-Cy5.5 nano-coordination polymer was injected into mice via tail vein. The dosage of GA / Zn@PDA / DZ-Cy5.5 nano-coordination polymer was 20 mg / kg. A control group (Ctrl) was also included, receiving an equivalent dose of GA / Zn@PDA / DZ-Cy5.5 nano-coordination polymer via tail vein injection. Mice were sacrificed at 0, 1, 2, 6, 12, and 24 hours post-injection, and the hearts, livers, spleens, lungs, and kidneys were removed for imaging.

[0099] The results are as follows Figure 15 As shown, after tail vein injection of GA / Zn@PDA / DZ-Cy5.5 nano-coordination polymer, the fluorescence intensity of the mouse liver injected with nanoparticles was significantly stronger than that injected with free fluorescein in the isolated organ, indicating that the nano-coordination polymer of the present invention can accumulate in the liver and has targeting properties to the liver.

[0100] 12. Evaluation results of the efficacy of nano-coordination polymers in treating sepsis in vivo:

[0101] Mice were randomly divided into 10 groups (n=8). All groups were injected with GA / Zn@PDA / DZ NPs (10mg / kg) on ​​the first day. Groups 2-5 were injected with LPS (10mg / kg) on ​​the first day. Groups 7-10 underwent cecal ligation and puncture (CLP) on the first day. On the third day, the mice were anesthetized, the eyeballs were enucleated and blood was collected. The blood was centrifuged and the serum was stored at 4°C. After the mice were sacrificed, the livers were removed, washed with physiological saline, dried with filter paper, and fixed with 4% paraformaldehyde for 24 hours.

[0102] After rinsing and draining the dissected liver in pre-chilled PBS, it was transferred to 2 mL centrifuge tubes and placed in a pre-chilled homogenizer on ice. An appropriate amount of lysis buffer was prepared according to a ratio of enhanced RIPA lysis buffer to protease inhibitor PMSF of 100:1. Based on the tissue volume, an appropriate amount of lysis buffer (200 μL / 50 mg) was added, ensuring complete coverage of the tissue. The tissue was homogenized on ice using a homogenizer to ensure thorough tissue disruption for efficient protein extraction. The homogenized tissue sample was transferred to a pre-chilled centrifuge tube and incubated on ice for 30 min for lysis, gently shaking the tube for a few seconds every 10 min to promote lysis. After lysis, the sample was centrifuged at 4°C and 13000 rpm for 15 min, and proteins were analyzed by Western blotting. The results are as follows: Figure 16-17 As shown, in both the LPS and CLP models, the nano-coordination polymers can reduce the cleavage of GSDMD-NT by decreasing the expression of HMGB1.

[0103] Fresh serum collected from mice was used to measure ALT and AST levels using an automated liver function analyzer. Results are as follows: Figure 18-19 As shown, the serum ALT and AST levels in both the LPS and CLP modeling groups increased significantly, indicating successful modeling. In contrast, the serum ALT and AST levels in the nano-coordination polymer treatment group were significantly lower than those in the injury group, indicating that the nano-coordination polymer can reduce the level of liver enzymes in the serum of septic mice and play a certain role in liver protection.

[0104] Serum IL-1α, IL-1β, PAI-1, and TAT concentrations were detected using an enzyme-linked immunosorbent assay (ELISA) kit. 10× Coating Buffer was diluted to 1× with ddH2O, and 250× Capture antibody prepared with 1× Coating Buffer was added to 1×. 50 μL / well of each plate was added to a 96-well ELISA plate and incubated overnight at 4°C. After washing three times with 0.005% PBST, 200 μL of 1× Assay Buffer was added to each well for blocking at room temperature for 1 h. 50 μL of the target animal serum was added to each well. Standards were started at 1000 pg / mL and serially diluted with 1× Assay Buffer to 62.5 pg / mL, with a minimum concentration of 0 pg / mL. 50 μL of each standard was added to each well and incubated at room temperature for 2 h. After washing three times with PBST, Detection antibody was diluted to 1× with 1× Assay Buffer. 50 μL of each antibody was added to each well and incubated at room temperature for 1 h. Wash three times with PBST, then dilute 250×HRP to 1× with 1×AssayBuffer and incubate at room temperature for 30 min. After washing five times with PBST, add 50 μL of TMB for color development, stop the color development with 2M H2SO4, and measure the OD value at 450 nm using a microplate reader. Calculate the final concentration of the sample according to the formula derived from the standard curve. Results are as follows. Figure 20-23 As shown, the serum levels of IL-1α, IL-1β, PAI-1, and TAT in both the LPS and CLP modeling groups were significantly increased, indicating successful modeling. In contrast, the serum levels of IL-1α, IL-1β, PAI-1, and TAT in the nano-coordination polymer treatment group were significantly lower than those in the injury group, indicating that the nano-coordination polymer can reduce the levels of inflammatory factors and coagulation molecules in the serum of septic mice.

[0105] Liver tissue was paraffin-embedded, sectioned, and stained with hematoxylin and eosin (HE). Pathological changes were observed using an optical microscope. Results are as follows: Figure 24As shown, H&E staining revealed that the liver tissue in the blank group had a normal structure, with hepatocytes arranged neatly and nuclei clearly visible, showing no obvious signs of inflammatory cell infiltration or necrosis. In the LPS and CLP treatment groups, however, red-stained fibrin deposits and erythrocyte extravasation were observed in the sinusoids or small blood vessels, representing microthrombus formation. Because microthrombi induced by DIC obstruct normal blood flow, hepatocytes may undergo ischemic degeneration, manifested as hepatocyte swelling, increased eosinophilic cytoplasm, or nuclear pyknosis (deeper nuclear staining). These changes were particularly pronounced in the sepsis model group, especially in tissues treated with CLP, where the lobular structure was disordered, and hepatocytes around the central lobular veins showed degeneration or necrosis. The liver tissue in the GA treatment group showed relatively fewer microthrombi, reduced ischemic damage to hepatocytes, and a more intact structure. In the GA / ZN@PD / DZ NPs treatment group, the hepatocytes were arranged more regularly, exhibiting a more complete lobular structure, with relatively less inflammatory cell infiltration and tissue necrosis, and almost no thrombus formation was observed. This suggests that nanoparticles may have a protective effect against liver damage caused by sepsis.

[0106] In addition, such as Figure 25 As shown, treatment with GA / ZN@PD / DZ NPs can significantly reduce the expression of HMGB1 in the liver, indicating that HMGB1 DNAase can play a role in reducing HMGB1 expression in vivo.

[0107] 13. The effect of nano-coordination polymers on prolonging the survival of septic mice:

[0108] Mice were randomly divided into 10 groups (n=10). Groups 2-5 were injected with LPS (10 mg / kg) on ​​day 1, and groups 7-10 underwent cecal ligation and puncture (CLP) on day 1. All groups were injected with 10 mg / kg GA / Zn@PDA / DZ nanocoordination polymer at the same time each day. The number of mice dying each day was recorded at the same time point for 7 days after modeling, and survival curves were plotted. Results are as follows... Figure 26-27 As shown, all LPS-induced septic mice died within 3 days, and all CLP-induced septic mice died within 2 days, while the survival rate of the group treated with the nano-coordination polymer was above 50% at 7 days after modeling. This demonstrates that the GA / Zn@PDA / DZ nanocomposite significantly improved the survival rate of septic model mice, indicating that the GA / Zn@PDA / DZ nanocomposite has a good life-protective effect on LPS / CLP-induced severe septicemia in mice.

[0109] 14. Safety evaluation results of nano-coordination polymers:

[0110] Hemolysis was observed and the hemolysis rate was calculated by incubating different concentrations of GA / Zn@PDA / DZ nanocomposite with 2% erythrocyte suspension at 37°C for 4 hours. 0.5 mL of mouse blood was placed in a 1.5 mL EP tube coated with sodium heparin, shaken well, and allowed to stand at room temperature for 5 minutes. Then, it was centrifuged at 800 rpm for 5 minutes, and the supernatant serum was discarded. 1 mL of physiological saline was then added, mixed well, and centrifuged again at 800 rpm for 15 minutes, discarding the supernatant serum. This process was repeated 2-3 times until the supernatant was no longer red. The erythrocytes obtained at the bottom of the EP tube were then diluted to a 2% erythrocyte suspension. Different concentrations of GA / Zn@PDA / DZ nanocomposite were added to this suspension, with physiological saline as a negative control and pure water as a positive control. After gentle mixing, the suspension was incubated at 37°C for 3 hours, and the hemolytic reaction was observed. After observation, the sample was centrifuged at 800 rpm for 15 minutes, the supernatant was collected, and the absorbance at 576 nm was measured using a microplate reader. The results are as follows: Figure 28 As shown, compared with the pure water group, physiological saline and nanocomposite materials of different concentrations did not cause significant hemolysis.

[0111] Mice were randomly divided into two groups (n=10). Each group was injected with saline and 10 mg / kg GA / Zn@PDA / DZ nano-coordination polymer for 14 consecutive days. Mouse weight was recorded daily, and a weight change curve was plotted. On day 14, mice from all four groups were sacrificed, and the heart, liver, spleen, lung, and kidney were removed. The tissues were washed with saline, blotted dry with filter paper, and fixed in 4% paraformaldehyde for 24 hours. The tissues were then embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and pathological changes were observed using an optical microscope. Results are as follows: Figures 29-30 As shown, compared with the saline group, the nano-coordination polymer group showed no significant pathological changes in any organ, and there was no significant difference in the weight change of mice. This indicates that the nano-coordination polymer has good in vivo safety.

[0112] Fresh serum collected from mice was used to measure ALT, AST, BUN, and CRE levels using an automated liver function analyzer. Results are as follows: Figure 31 As shown, no significant changes were observed in organ damage markers in the nano-coordination polymer group, indicating that the nano-coordination polymer has good in vivo safety.

[0113] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions and substitutions can be made without departing from the inventive concept, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a nano-coordination polymer for treating sepsis-induced liver injury, characterized in that, Includes the following steps: Step 1: Glycyrrhetinic acid and metal ions coordinate to form glycyrrhetinic acid / metal ion coordination nanoparticles; the metal ions include Zn. 2+ Mg 2+ and Ce 2+ ; Step 2: Under alkaline conditions, dopamine monomers are allowed to self-polymerize on the surface of the glycyrrhetinic acid / metal ion coordination nanoparticles to form core-shell structured nanoparticles with glycyrrhetinic acid / metal ion coordination nanoparticles as the core and polydopamine as the shell. Step 3: Connect a gene silencing tool designed for the HMGB1 protein to the surface of the core-shell structured nanoparticles to obtain a nano-coordination polymer for treating sepsis-induced liver injury; the gene silencing tool includes DNAzyme, siRNA, and antisense oligonucleotides.

2. The method for preparing the nano-coordination polymer for treating sepsis-induced liver injury according to claim 1, characterized in that, In step 3, a gene silencing tool designed for the HMGB1 protein is attached to the surface of the core-shell nanoparticle by utilizing the Michael addition or Schiff base reaction between catechol or amino-amino-terminated molecules on the surface of the polydopamine shell.

3. The method for preparing the nano-coordination polymer for treating sepsis-induced liver injury according to claim 1, characterized in that, The gene silencing tool in step 3 is DNAzyme.

4. The method for preparing the nano-coordination polymer for treating sepsis-induced liver injury according to claim 3, characterized in that, The DNAzyme sequence is: CTCTTCCTTCTTTTTAAGCTGGCCGAGCCTTGCTCTTTTCAGCCT.

5. The method for preparing the nano-coordination polymer for treating sepsis-induced liver injury according to claim 1, characterized in that, The metal ion in step 1 is Zn. 2+ .

6. The method for preparing the nano-coordination polymer for treating sepsis-induced liver injury according to claim 5, characterized in that, In step 1, the specific steps for forming glycyrrhetinic acid / metal ion coordination nanoparticles are as follows: First, add glycyrrhetinic acid ethanol solution to the container, then add Tris buffer and polyvinylpyrrolidone, and stir at room temperature to obtain a homogeneous mixed solution; then, add zinc acetate solution dropwise to the above mixed solution through a constant flow pump to generate glycyrrhetinic acid / metal ion coordination nanoparticles, namely glycyrrhetinic acid / zinc nanocoordination polymer (GA / Zn nanocoordination polymer).

7. The method for preparing the nano-coordination polymer for treating sepsis-induced liver injury according to claim 6, characterized in that, The Tris buffer solution is a Tris buffer solution with a pH value of 8.4 to 8.

6.

8. The method for preparing the nano-coordination polymer for treating sepsis-induced liver injury according to claim 1, characterized in that, In step 2, the dopamine monomer is added in the form of dopamine hydrochloride solution.

9. A nano-coordination polymer for treating sepsis-induced liver injury, characterized in that, The nano-coordination polymer for treating sepsis-induced liver injury was prepared using the preparation method described in any one of claims 1 to 8.

10. A drug for treating sepsis-induced liver injury, characterized in that, The medicament for treating sepsis-induced liver injury contains the nanocoordination polymer for treating sepsis-induced liver injury as described in claim 9.

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