Self-assembly nano material for targeting liver and improving fatty liver as well as preparation method and application of self-assembly nano material

Abstract

The invention discloses a self-assembled nano material for targeting liver and improving fatty liver as well as a preparation method and application of the self-assembled nano material, and belongs to the technical field of preparation of novel nano materials. According to the invention, a self-assembled nano material is formed by MOF, thyroid hormone and an ASGR1 antibody. The MOF material has good preservation performance, can effectively remove active oxygen in cells and intercellular substances, and reduces damage of the active oxygen to liver cells. The thyroid hormone is gathered in the MOF, the thyroid hormone can be accurately released after the MOF is targeted to the liver, the effect of the thyroid hormone on relieving the fatty liver degree is efficiently achieved, and toxic and side effects of the thyroid hormone on other organs of the whole body are effectively avoided. The surface of MOF is modified with an ASGR1 antibody, the antibody can accurately target the self-assembled nano-material to the surface of a liver cell membrane, and the ASGR1 antibody can effectively inhibit ASGR1 on the surface of the liver cell membrane, so that the self-assembled nano-material can effectively promote discharge of cholesterol in cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of novel nanomaterial preparation, and specifically relates to a self-assembling nanomaterial that targets the liver and improves fatty liver, and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Nanomaterials, especially metal organic framework (MOF) materials, have received extensive attention in targeted drug delivery systems in recent years. The advantages of these materials include large specific surface area, adjustable pore structure and good biocompatibility, making them ideal drug carriers. The current research progress of MOF materials is mainly focused on targeting functionality, controlled release performance, imaging and treatment. By modifying the surface of MOF materials, specific targeting ligands (such as antibodies, peptide chains, etc.) can be introduced, so that these materials can recognize and bind to specific cells or tissues to achieve their targeting functionality. In addition, MOF materials can be designed to respond to specific physiological environments (such as pH, temperature, enzyme activity, etc.) to achieve controlled release of drugs, thereby improving therapeutic effects and reducing side effects, and exerting their controlled release properties. Some MOF materials also have the ability of fluorescence imaging, which can be used to monitor the distribution and release of drugs in the body in real time.

[0004] However, although MOF materials have shown certain potential in organ targeting, how to improve targeting efficiency and reduce the impact on healthy cells remains a research hotspot today, and a lot of research is still needed to overcome existing challenges and achieve clinical application.

[0005] Liver transplantation (LT) provides the only effective treatment option for patients with end-stage liver disease. The current shortage of liver donor sources has greatly limited the development of liver transplantation. Although living donor liver transplantation has expanded the liver source, cadaveric liver sources still account for the vast majority of liver donors. Due to the shortage of donors, the liver donation criteria have been expanded to include fatty liver and elderly liver. Some scholars have proposed the concept of marginal donors or extended criteria donors (ECD). Although the previous strict donor selection criteria can ensure the safety of recipients, they also exclude many livers that can actually be used. Expanding the use of ECD transplantation can increase donor supply and alleviate the demand gap. However, improper use of ECD livers may increase the risk of graft dysfunction, such as primary nonfunction (PNF) of the transplanted liver after surgery and early allograft dysfunctional (EAD) after liver transplantation. Therefore, the rational use of ECD livers is one of the current research focuses. The main risk factors for ECD liver donation include fatty liver, hepatitis B or C, elderly donors, unstable respiratory circulation of brain-dead donors, malignant tumor donors, and livers of donors with prolonged cold ischemia time. With the increasing incidence of fatty liver worldwide, the possibility of accepting fatty liver as a non-standard donor organ has also increased significantly. Fatty degeneration of donor liver (macrovesicular steatosis>30%) accounts for 30% and 20% of cadaveric liver and living donor liver, respectively.

[0006] The degree of steatosis needs to be considered when evaluating ECD fatty donor livers, which may affect transplantation outcomes. Traditionally, liver steatosis is graded according to the percentage of adipocyte infiltration, and can be divided into mild (macrovesicular steatosis <30%), moderate (macrovesicular steatosis 30%-60%), and severe (macrovesicular steatosis >60%). Steatosis increases ischemia-reperfusion injury (IRI) in donor livers, especially for donor livers with moderate to severe steatosis, which are very sensitive to the ischemia-reperfusion injury process, which greatly affects the prognosis of recipients. Mild steatosis donor livers are generally believed to have no obvious adverse effects on the prognosis after liver transplantation; moderate steatosis donor livers are associated with transplant survival and EAD; severe steatosis donor livers significantly increase the incidence of PNF. Most centers use mild steatosis donor livers and consider severe steatosis to be an absolute contraindication for use, while there is controversy over the optimal use of moderate steatosis donor livers. Reducing the degree of liver steatosis is beneficial to alleviate ischemia-reperfusion injury, thereby improving recipient prognosis. It is also crucial for expanding the donor pool, reducing the abandonment of suitable organs, and reducing waiting list mortality.

[0007] The mechanism of the onset and progression of non-alcoholic fatty liver disease (NAFLD) has not been fully elucidated. It has been confirmed that oxidative stress damage, cholesterol metabolism, insulin resistance, hypothyroidism, etc. are all related to the onset and progression of NAFLD. In addition, in 2022, internationally renowned scholars discovered a single-transmembrane protein on the surface of the liver cell membrane, ASGR1, which is only expressed on the surface of the liver cell membrane and has an inhibitory effect on downstream LXRα, which has been shown to have a clear effect of promoting the excretion of intracellular cholesterol. Inhibition of ASGR1 is beneficial to promote cholesterol excretion and reduce lipid levels, thereby improving fatty liver. Thyroid hormone plays an important role in the development of fatty liver, and its effects on fatty liver are complex and involve multiple metabolic pathways. Thyroid hormone can enhance the oxidation of fatty acids and reduce the accumulation of fat in the liver. It can also improve insulin sensitivity and reduce the excessive production of glucose in the liver. Thyroid hormone also has certain anti-inflammatory properties, which helps to reduce liver inflammation associated with fatty liver.

[0008] Although there are currently a variety of nanomaterials targeting the liver, such as patent CN114569743A which discloses a liver-specific gene-editing nanomedicine, there are few reports of nanomaterials targeting the liver using MOF as a carrier, and there are even fewer reports of nanomaterials that target the liver with MOF, adrenal hormones and ASGR1 combined to improve fatty liver. Summary of the invention

[0009] In order to address the deficiencies of the prior art, the purpose of the present invention is to provide a self-assembled nanomaterial that targets the liver and improves fatty liver, and a preparation method and application thereof. The self-assembled nanomaterial provided by the present invention can target the liver, scavenge reactive oxygen species, and reduce oxidative stress, thereby reducing the degree of fatty degeneration of the liver. It has the characteristics of strong targeting, clear structure, high specific surface area and porosity, and easy chemical functionalization.

[0010] In order to achieve the above object, the technical solution of the present invention is:

[0011] The first aspect of the present invention provides a self-assembled nanomaterial that targets the liver and improves fatty liver, comprising: a metal organic framework (MOF), thyroid hormone aggregated in the MOF, and an ASGR1 antibody modified on the surface of the MOF.

[0012] The present invention uses MOF as a carrier, and Anti-ASGR1 is modified on the surface of the nanocarrier, so that the self-assembled nanomaterial can be accurately targeted to the surface of the liver cell membrane, inhibiting ASGR1 to promote cholesterol excretion and reduce lipid levels. In addition, thyroid hormones are aggregated in the MOF material. After reaching the liver, the self-assembled nanomaterial slowly and accurately releases thyroid hormones, which effectively reduce fatty degeneration of the liver by regulating lipid metabolism, activating thyroid hormone receptor β, and promoting autophagy. It can efficiently play its role and avoid the side effects of excessive thyroid hormones. At the same time, MOF itself has the function of removing reactive oxygen and reducing liver cell damage. In summary, the self-assembled nanomaterial formed by MOF, thyroid hormone and ASGR1 antibody in the present invention has the advantages of precise targeting, sustained release, large specific surface area, clear structure, high porosity, easy chemical functionalization, and is easy to store.

[0013] The second aspect of the present invention provides a method for preparing the above-mentioned self-assembled nanomaterial that targets the liver and improves fatty liver, comprising:

[0014] dissolving the metal organic framework and thyroid hormone in water, stirring and reacting to obtain a precursor material;

[0015] The precursor material and ASGR1 antibody are dissolved in water, and reacted by stirring at 0-5° C. to obtain a self-assembled nanomaterial that targets the liver and improves fatty liver.

[0016] The third aspect of the present invention provides a use of the self-assembled nanomaterial or the self-assembled nanomaterial prepared by the above preparation method in preparing a drug for treating fatty liver.

[0017] The beneficial effects of the present invention are:

[0018] The present invention forms a self-assembled nanomaterial by MOF, thyroid hormone and ASGR1 antibody, with MOF as a nanocarrier. MOF material has good preservation, and MOF material can effectively remove reactive oxygen species (ROS) in cells and interstitial cells, catalytically decompose them into water and oxygen, reduce the concentration of ROS, and reduce the damage of ROS to liver cells. Thyroid hormone is aggregated in MOF nanocarriers, and the self-assembled nanomaterials are accurately released after being targeted to the liver, and the thyroid hormone is effectively exerted to reduce the degree of fatty liver, and the toxic and side effects of thyroid hormone on other organs of the body are effectively avoided. The surface of MOF nanocarrier is modified with ASGR1 antibody, which can accurately target the self-assembled nanomaterial to the surface of liver cell membrane, and ASGR1 antibody can effectively inhibit ASGR1 on the surface of liver cell membrane. ASGR1 has been proven to inhibit multiple pathways such as downstream LXRα, and the latter pathway has the effect of promoting the excretion of cholesterol from cells. The self-assembled nanomaterial of the present invention can effectively promote the excretion of cholesterol in cells.

[0019] The synthesis process of the self-assembling nanomaterial provided by the present invention is simple, time-saving, and the raw materials are cheap and readily available, the cost is low, the material is universal, and the material is easy to produce on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0021] Figure 1 The diagram is a preparation method and functional mechanism diagram of MIL@T4 / ASGR1 in Example 1 of the present invention;

[0022] Figure 2 The XRD pattern of MIL@T4 / ASGR1 obtained in Example 1 of the present invention;

[0023] Figure 3 This is the XPS spectrum of MIL@T4 / ASGR1 obtained in Example 1 of the present invention;

[0024] Figure 4 The MIL@T4 / ASGR1 obtained in Example 1 of the present invention reduces the degree of fat change in cells to improve cell survival rate;

[0025] Figure 5 The targeting of MIL@T4 / ASGR1 to the liver in mice obtained in Example 1 of the present invention;

[0026] Figure 6 The MIL@T4 / ASGR1 obtained in Example 1 of the present invention reduces the degree of fatty degeneration in the liver of mice with fatty liver. DETAILED DESCRIPTION

[0027] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0028] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0029] The MOF described in the present invention, namely the metal organic framework compound, is a coordination polymer with a one-dimensional, two-dimensional or three-dimensional structure formed by metal ions or clusters and organic ligands.

[0030] The present invention provides a self-assembled nanomaterial MIL@T4 / ASGR1 that targets the liver and improves fatty liver, and a preparation method and application thereof, which are used to solve the problems of poor targeting performance of nanomaterials in the prior art and improve their accuracy.

[0031] The first typical embodiment of the present invention provides a self-assembled nanomaterial that targets the liver and improves fatty liver, including: a metal organic framework (MOF), thyroid hormone aggregated in the metal organic framework, and an ASGR1 antibody modified on the surface of the metal organic framework.

[0032] The present invention uses MOF as a carrier, and Anti-ASGR1 is modified on the surface of the nanocarrier, so that the self-assembled nanomaterial can be accurately targeted to the surface of the liver cell membrane, inhibiting ASGR1 to promote cholesterol excretion and reduce lipid levels. In addition, thyroid hormones are aggregated in the MOF material. After reaching the liver, the self-assembled nanomaterial slowly and accurately releases thyroid hormones, which effectively reduce fatty degeneration of the liver by regulating lipid metabolism, activating thyroid hormone receptor β, and promoting autophagy. It can efficiently play its role and avoid the side effects of excessive thyroid hormones. At the same time, MOF itself has the function of removing reactive oxygen and reducing liver cell damage. In summary, the self-assembled nanomaterial formed by MOF, thyroid hormone and ASGR1 antibody in the present invention has the advantages of precise targeting, sustained release, large specific surface area, clear structure, high porosity, easy chemical functionalization, and is easy to store.

[0033] In some examples of this embodiment, the metal organic framework material is self-assembled from 1,4-terephthalic acid and iron ions and copper ions through coordination bonds.

[0034] In some examples of this embodiment, the thyroid hormone is thyroid hormone T4.

[0035] In some examples of this embodiment, in the self-assembled nanomaterial, the loading amount of thyroid hormone is 13-17% of the mass of the metal organic framework, and the loading amount of ASGR1 antibody is 0.1-0.2% of the mass of the metal organic framework.

[0036] In some examples of this embodiment, in the self-assembled nanomaterial, the loading amount of thyroid hormone is 14.6% of the mass of the metal organic framework, and the loading amount of ASGR1 antibody is 0.17% of the mass of the metal organic framework.

[0037] A second typical embodiment of the present invention provides a method for preparing the above-mentioned self-assembled nanomaterial that targets the liver and improves fatty liver, comprising:

[0038] dissolving the metal organic framework and thyroid hormone in water, stirring and reacting to obtain a precursor material;

[0039] The precursor material and ASGR1 antibody are dissolved in water, and reacted by stirring at 0-5° C. to obtain a self-assembled nanomaterial that targets the liver and improves fatty liver.

[0040] The present invention uses a metal organic framework to load thyroid hormone, which enables the self-assembled nanomaterial to slowly and accurately release the thyroid hormone after reaching the liver, effectively exerting its effect and avoiding the side effects of excessive thyroid hormone.

[0041] ASGR1 is only expressed on the surface of liver cell membranes. The surface of the self-assembled nanomaterial of the present invention is modified with ASGR1 antibodies, which can be precisely targeted to the surface of liver cell membranes, promote the excretion of intracellular cholesterol, reduce lipid levels, and thus improve fatty liver.

[0042] In some examples of this embodiment, the mass ratio of the metal organic framework to the thyroid hormone is 4.5-5.5:1, preferably 5:1.

[0043] In some examples of this embodiment, the mass ratio of the precursor material to the ASGR1 antibody is 4.5-5.5:0.01, preferably 5:0.01.

[0044] In some examples of this embodiment, the preparation method further comprises the following steps:

[0045] 1,4-Terephthalic acid and iron and copper ions self-assemble to form a metal-organic framework through coordination bonds.

[0046] In some examples of this embodiment, 1,4-terephthalic acid, iron salt and copper salt are dissolved in an organic solvent, stirred and subjected to a solvothermal reaction to obtain a metal organic framework.

[0047] In some examples of this embodiment, the molar ratio of 1,4-terephthalic acid, iron salt and copper salt is (2.8-3):1:1.

[0048] In some examples of this embodiment, the iron salt includes, but is not limited to, one of ferric chloride, ferric nitrate, ferric sulfate, and ferric acetate.

[0049] In some examples of this embodiment, the copper salt includes, but is not limited to, one of copper chloride, copper nitrate, copper sulfate, and copper acetate.

[0050] In some examples of this embodiment, the organic solvent includes but is not limited to N,N-dimethylformamide (DMF).

[0051] In some examples of this embodiment, the solvothermal reaction is carried out at a temperature of 90-110° C. and for a time of 10-15 h.

[0052] In some examples of this embodiment, after the solvothermal reaction is completed, the product is separated and washed and dried to obtain a metal organic framework.

[0053] In some examples of this embodiment, the drying is vacuum drying, preferably vacuum drying at 70-90° C. for 10-15 hours.

[0054] A third typical embodiment of the present invention provides a use of the self-assembled nanomaterial or the self-assembled nanomaterial prepared by the above preparation method in the preparation of a drug for treating fatty liver.

[0055] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0056] Example 1

[0057] A method for preparing a self-assembled nanomaterial MIL@T4 / ASGR1 that targets the liver and improves fatty liver, the steps are as follows:

[0058] (1) FeCl3·6H2O (2.1 mmol), CuCl2·6H2O (2.1 mmol) and 1,4-terephthalic acid (6 mmol) were dissolved in 60 mL DMF and stirred for 30 min. Then, the mixture was transferred to a 100 mL polytetrafluoroethylene-lined autoclave and reacted at 100 °C for 12 h. The obtained sample was separated by centrifugation. It was repeatedly rinsed with DMF, ethanol and deionized water and dried in vacuum at 80 °C for 12 h to obtain MIL·MOF.

[0059] (2) 5 mg of MIL·MOF and 1 mg of thyroid hormone T4 were added to 5 mL of water and stirred for 5 h. After washing and centrifugation, the mixture was dispersed in 5 mL of water to obtain MIL@T4.

[0060] (3) The product obtained in step (2) (5 mg) and 10 μL Anti-ASGR1 (~10 μg) were stirred in 4°C water for 5 h, washed and centrifuged to obtain MIL@T4 / ASGR1.

[0061] In the obtained self-assembled nanomaterial MIL@T4 / ASGR1, 5 mg of MIL·MOF was used, and finally 0.73 mg of thyroid hormone was retained, and 8.5 μg of ASGR1 antibody (i.e., Anti-ASGR1) was preserved.

[0062] MIL@T4 / ASGR1 was administered to mice via tail vein injection. The material flowed through the mouse liver through the blood flow, and the surface-modified Anti-ASGR1 specifically bound to the ASGR1 receptor on the surface of the liver cell membrane, thereby precisely targeting the liver cells. On the one hand, Anti-ASGR1 inhibits ASGR1 after binding to ASGR1, thereby promoting cholesterol excretion and reducing lipid levels to improve fatty liver. On the other hand, thyroid hormones are accumulated in the material, and are slowly and accurately released after reaching the liver. By regulating lipid metabolism, activating thyroid hormone receptor β, and promoting autophagy, it effectively reduces liver fatty degeneration, efficiently exerts its effects, and avoids the side effects of excessive thyroid hormones. At the same time, MOFs themselves can remove reactive oxygen species and convert them into O2 and H2O, thereby reducing liver cell damage.

[0063] Example 2

[0064] A method for preparing a self-assembled nanomaterial MIL@T4 / ASGR1 that targets the liver and improves fatty liver is different from Example 1 in that FeCl36H2O (2 mmol), CuCl26H2O 2 mmol) and 1,4-terephthalic acid (5.6 mmol) are dissolved in 60 mL DMF, and the remaining steps are consistent with those of Example 1.

[0065] Example 3

[0066] A method for preparing a self-assembled nanomaterial MIL@T4 / ASGR1 that targets the liver and improves fatty liver is different from that in Example 1 in that FeCl36H2O (2 mmol), CuCl2 6H2O 2 mmol) and 1,4-terephthalic acid (6 mmol) are dissolved in 60 mL DMF, and the remaining steps are consistent with those in Example 1.

[0067] Performance Verification

[0068] Experimental Example 1

[0069] The diffraction data of MIL@T4 / ASGR1 obtained in Example 1 in the range of 5-35° were collected by X-ray diffraction (XRD) with a step frequency of 0.01°. The results show that ( Figure 2 ), MIL@T4 / ASGR1 showed diffraction crystal planes of

[002] and

[101] , which proved that the structure of MIL·MOF was stable and not destroyed during the synthesis process, and MIL@T4 / ASGR1 was successfully prepared.

[0070] Experimental Example 2

[0071] The element spectrum of MIL@T4 / ASGR1 obtained in Example 1 was collected by X-ray photoelectron spectroscopy (XPS). The results show that ( Figure 3 ), MIL@T4 / ASGR1 contains not only C, O, Fe and Cu elements of MIL·MOF, but also I and N elements of T4. In addition, the S element in the energy spectrum comes from -SH in ASGR1. Therefore, T4 and ASGR1 are successfully loaded in MIL·MOF.

[0072] Experimental Example 3

[0073] Study on in vitro cell experiments of MIL@T4 / ASGR1. The MIL@T4 / ASGR1 used in this experiment is the MIL@T4 / ASGR1 prepared in Example 1.

[0074] The experimental process is as follows:

[0075] L-02 cell recovery: Ensure that the working environment is sterile and prepare equipment such as a biosafety cabinet, centrifuge, and microscope. Prepare appropriate culture medium (DMEM high-glucose culture medium) and 20% fetal bovine serum (FBS). Take out the cryovial containing L-02 cells from the -80℃ freezer and immediately place it in a 37℃ water bath and shake gently to accelerate thawing. Thaw the cells completely within 30s (avoid prolonged thawing) and then quickly transfer them to a sterile culture bottle. Add pre-warmed culture medium to the cell suspension, usually 5-10 times the volume, to help dilute DMSO. Place the cell suspension in a centrifuge tube and centrifuge at 800rpm for 5min to remove DMSO. Carefully remove the supernatant and resuspend the cells with appropriate culture medium. Transfer the resuspended cells to a 10cm culture dish and place it in an incubator, setting the appropriate temperature and CO2 concentration (usually 37℃, 5% CO2). After 24h, observe the cell morphology and adhesion to ensure successful recovery.

[0076] L-02 cell passaging: Make sure all materials and equipment are sterilized, including culture medium, pipette tips, etc. Make sure that the cells grow well and reach 70%-80% confluence. Rinse the cells with PSB buffer and remove the old culture medium. Add an appropriate amount of trypsin (containing EDTA) and place in the incubator for 3 minutes to allow the cells to be fully digested. Add culture medium containing FBS to terminate the digestion reaction, and then gently blow to suspend the cells. Transfer the cell suspension to a centrifuge tube and centrifuge at 800rpm for 5 minutes. Remove the supernatant and resuspend the cells with fresh culture medium. Depending on the cell density, distribute the cells to 2-3 10cm culture dishes to maintain an appropriate cell density. Place in the incubator and continue culturing, and change the culture medium regularly. Conduct subsequent experiments after the cells have been passaged 3-5 times.

[0077] Preparation of fatty acid culture medium: Dissolve 256.5 mg palmitic acid (PA) in 10 mL anhydrous sterile PBS, heat in a 75°C water bath for 2 hours until it becomes a colorless transparent liquid, obtain a mother solution with a concentration of 100 mM, and store it in a -20°C freezer; Dissolve 100 mg fatty acid-free bovine serum albumin (BSA) in 1 mL PBS, centrifuge at 3000 rpm / min for 10 min in a high-speed centrifuge to prepare a 10% clear and transparent light yellow solution; Mix the PA solution and BSA solution prepared above in a ratio of 3:1, then heat in a 55°C water bath for 30 min to become a light yellow transparent liquid PA-BSA mixed solution, filter with a 0.22 μm filter in a clean bench, and store in a -20°C freezer. Mix an appropriate amount of PA-BSA mixed solution and DMEM high glucose culture medium in a ratio of 1:10 to obtain fatty acid cell culture medium, which is stored in a 4°C refrigerator.

[0078] Induction and experiment of fatty liver cells: Ensure that the cells grow well and reach 70%-80% confluence. Rinse the cells with PSB buffer and remove the old culture medium. Add an appropriate amount of trypsin (containing EDTA) and place in an incubator for 3 minutes to allow the cells to be fully digested. Add culture medium containing FBS to terminate the digestion reaction, and then gently blow to suspend the cells. Transfer the cell suspension to a centrifuge tube and centrifuge at 800rpm for 5 minutes. Discard the supernatant, add 10mL DEME high-glucose culture medium, and gently resuspend the cells to ensure that the cells are evenly dispersed. Pipette 100μL of the cell suspension and evenly distribute it to 30 wells in a 96-well plate. Place the inoculated 96-well plate in an incubator at 37℃ and 5% CO2 for 6 hours to allow the cells to adhere to the wall. The 30 wells were evenly divided into 5 groups, namely negative control group, positive control group, MOF group, Anti-ASGR1 group and MIL@T4 / ASGR1 group, with 6 replicates in each group. After 6 hours, carefully aspirate the culture medium in the culture dish, taking care to avoid inhalation or damage to the cells. The negative control group added 100 μL DMEM high-glucose culture medium, and the other four groups added 100 μL fatty acid culture medium, all placed in an incubator at 37°C and 5% CO2, and continued to culture for 24 hours. After 24 hours, carefully aspirate the culture medium in the culture dish, taking care to avoid inhalation or damage to the cells. All duplicate wells were added with 100 μL DMEM high-glucose culture medium, and in addition, an appropriate amount of MOF material was added to each of the 6 duplicate wells in the MOF group, and an appropriate amount of Anti-ASGR1 was added to each of the 6 duplicate wells in the Anti-ASGR1 group (the MOF concentration and Anti-ASGR1 concentration were the same as the MIL concentration and Anti-ASGR1 concentration in MIL@T4 / ASGR1, respectively), and 1 μL MIL@T4 / ASGR1 was added to the 6 duplicate wells in the MIL@T4 / ASGR1 group (the concentration of MIL@T4 / ASGR1 in the cell culture medium was 10 μg / mL). Continue to culture it in an incubator at 37°C and 5% CO2 for 24 hours. After 24 hours, take out the 96-well plate and perform the Oil Red O staining experiment in the clean bench. The Oil Red O staining uses the Biotech Modified Oil Red O Staining Kit. Slowly aspirate the cell culture medium and wash once with PBS. Add 4% paraformaldehyde fixative to fix for 10 minutes and rinse twice with PBS. Aspirate the staining wash solution, add an appropriate amount of modified Oil Red O staining solution, and stain for 40 minutes. Remove the modified Oil Red O staining solution, add an appropriate amount of staining wash solution, let it stand for 30 seconds, then remove the staining wash solution, and wash with PBS for 20 seconds. Add an appropriate amount of PBS, evenly cover the cells, and observe and take pictures under a microscope. After Oil Red O staining, clear and distinct staining of lipid droplets can be seen in the cells, such as Figure 4 shown.

[0079] Fatty liver cells were induced in the same way, and Calcein / PI cell activity was detected using the Calcein / PI cell activity and cytotoxicity detection kit from Biotech. According to the system of 100 μL Calcein AM / PI detection working solution per well of a 96-well plate, an appropriate amount of Calcein AM / PI detection working solution was prepared and mixed thoroughly. The culture medium was removed and the cells were washed once with PBS. 100 μL of Calcein AM / PI detection working solution was added and incubated at 37°C in the dark for 30 minutes. After incubation, the staining effect was observed under a fluorescence microscope. Calcein AM stains living cells with green fluorescence, while propidium iodide (PI) stains dead cells with red fluorescence. Figure 4 shown.

[0080] Induce fatty liver cells in the same induction method and perform Hoechst staining using Solebo's cell apoptosis-Hoechst staining kit. Aspirate the culture medium, add 100μL of Hoechst fixative and fix for 10 minutes, remove the fixative, wash twice with PBS, 3 minutes each time, aspirate the liquid, add 100μL of Hoechst staining solution, and incubate for 5 minutes. Discard the staining solution, wash twice with PBS, 3 minutes each time, aspirate the liquid. Add an appropriate amount of PBS and observe under a fluorescence microscope. The nuclei of apoptotic cells are densely stained, such as Figure 4 shown.

[0081] Compared with the positive control group, the MOF group and Anti-ASGR1 group had fewer lipid droplets and better cell survival rates, while the MIL@T4 / ASGR1 group showed better effects.

[0082] Experimental Example 4

[0083] Study on the targeting of MIL@T4 / ASGR1 to the liver in mice. The MIL@T4 / ASGR1 used in this experiment is the MIL@T4 / ASGR1 prepared in Example 1.

[0084] The experimental process is as follows:

[0085] DiR dyes are widely used in in vivo imaging or tracing because the infrared light they emit can efficiently pass through cells and tissues. After the DiR dyes were fused with MIL@T4 / ASGR1, they were injected into C57 wild-type mice through the tail vein for in vivo 3D imaging of small animals.

[0086] After 6 hours, MIL@T4 / ASGR1 was observed to be enriched in the liver, and the enrichment reached a peak after 1 day. After 7 days, the fluorescence basically disappeared, showing excellent liver targeting. Figure 5 shown.

[0087] Experimental Example 5

[0088] Study on MIL@T4 / ASGR1 to reduce the degree of fatty liver in mice. The MIL@T4 / ASGR1 used in this experimental example is the MIL@T4 / ASGR1 prepared in Example 1.

[0089] Wild-type C57 mice were selected and fed with a compound high-fat diet from 4 weeks on. The model was successfully established after 14 weeks of continuous feeding. At the same time, mice from the same batch were selected and fed with ordinary diet. Finally, 24 fatty liver mice with uniform body weight and 6 ordinary mice were selected for subsequent experiments. The 6 ordinary mice were used as the negative control group, and the 24 fatty liver mice were randomly divided into 4 groups, namely the positive control group, MOF group, Anti-ASGR1 group and MIL@T4 / ASGR1 group. The MOF group, Anti-ASGR1 group and MIL@T4 / ASGR1 group were injected with 75μL MOF, 75μL Anti-ASGR1 and 75μL MIL@T4 / ASGR1 respectively by tail vein injection (the concentration of MOF and Anti-ASGR1 were the same as the concentration of MIL and Anti-ASGR1 in MIL@T4 / ASGR1, respectively). All mice were fed for another week and then killed. Serum was collected for testing of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG) and total cholesterol (T-CHO). Liver was collected for HE and Oil Red O staining.

[0090] Compared with the positive control group, the MOF group and the Anti-ASGR1 group showed decreased levels of alanine aminotransferase, aspartate aminotransferase, triglyceride and total cholesterol, and fewer fat vacuoles were seen in the HE-stained sections, and fewer lipid droplets were seen in the Oil Red O-stained sections. The MIL@T4 / ASGR1 group showed better effects than the MOF group and the Anti-ASGR1 group. Figure 6 shown.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A self-assembled nanomaterial that targets the liver and improves fatty liver, characterized in that: include: Metal-organic framework, thyroid hormone aggregated within the metal-organic framework, and ASGR1 antibody modified on the surface of the metal-organic framework.

2. The self-assembled nanomaterial according to claim 1, characterized in that: The metal organic framework material is self-assembled by 1,4-terephthalic acid, iron ions and copper ions through coordination bonds.

3. The self-assembled nanomaterial according to claim 1, characterized in that: The thyroid hormone is thyroid hormone T4; Preferably, in the self-assembled nanomaterial, the loading amount of thyroid hormone is 13-17% of the mass of the metal organic framework, and the loading amount of ASGR1 antibody is 0.1-0.2% of the mass of the metal organic framework; Further preferably, in the self-assembled nanomaterial, the loading amount of thyroid hormone is 14.6% of the mass of the metal organic framework, and the loading amount of ASGR1 antibody is 0.17% of the mass of the metal organic framework.

4. A method for preparing the self-assembled nanomaterial targeting the liver and improving fatty liver according to claim 1, characterized in that: include: dissolving the metal organic framework and thyroid hormone in water, stirring and reacting to obtain a precursor material; The precursor material and ASGR1 antibody are dissolved in water, and reacted by stirring at 0-5° C. to obtain a self-assembled nanomaterial that targets the liver and improves fatty liver.

5. The preparation method according to claim 4, characterized in that: The mass ratio of the metal organic framework to the thyroid hormone is 4.5-5.5:1, preferably 5:

1.

6. The preparation method according to claim 4, characterized in that: The mass ratio of the precursor material to the ASGR1 antibody is 4.5-5.5:0.01, preferably 5:0.

01.

7. The preparation method according to claim 4, characterized in that: The preparation method further comprises the following steps: 1,4-Terephthalic acid and iron and copper ions self-assemble to form a metal-organic framework through coordination bonds.

8. The preparation method according to claim 7, characterized in that: 1,4-terephthalic acid, iron salt and copper salt are dissolved in an organic solvent, stirred, and subjected to a solvothermal reaction to obtain a metal organic framework; Preferably, the molar ratio of 1,4-terephthalic acid, iron salt and copper salt is (2.8-3):1:1; Preferably, the iron salt includes one of ferric chloride, ferric nitrate, ferric sulfate and ferric acetate; Preferably, the copper salt includes one of copper chloride, copper nitrate, copper sulfate and copper acetate; Preferably, the organic solvent comprises N,N-dimethylformamide; The temperature of the solvent thermal reaction is 90-110° C. and the time is 10-15 hours.

9. The preparation method according to claim 8, characterized in that: After the solvothermal reaction is completed, the product is separated and washed and dried to obtain a metal organic framework; Preferably, the drying is vacuum drying.

10. Use of the self-assembled nanomaterial according to any one of claims 1 to 3 or the self-assembled nanomaterial prepared by the preparation method according to any one of claims 4 to 9 in preparing a drug for treating fatty liver.

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