Multi-organ regulation and control combined nano preparation and application thereof

Abstract

The invention discloses a multi-organ regulation and control combined nano preparation and application thereof, and belongs to the technical field of medicines. The combined nano preparation consists of a nano preparation A and a nano preparation B, the nano preparation A is a parenchymal hepatic cell regulation and control nano delivery system and is prepared from phospholipid, cholesterol, PEGylated phospholipid, DSPE-PEG-Glactase and a THR-beta agonist; and the nano preparation B is an adipose cell regulation and control nano delivery system and is prepared from phospholipid, cholesterol, PEGylated phospholipid, DSPE-PEG-CKGGRAKDC and a PPAR gamma agonist. According to the invention, malignant crosstalk formed by liver-adipose tissue axes is utilized for the first time, and a strategy for treating fatty hepatitis and hepatic fibrosis related to metabolic dysfunction by cooperatively regulating lipid metabolism balance between two organs of liver and fat by using two types of liposomes is innovatively provided; the invention provides a new strategy and way for treating metabolic liver diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a multi-organ regulatory combination nano-formulation and its application. Background Technology

[0002] Liver fibrosis is an inevitable stage in the progression of various chronic liver diseases to irreversible and fatal conditions, posing a serious threat to human health. With changing lifestyles leading to the widespread obesity epidemic, metabolic-associated steatohepatitis (MASH) has become the most prevalent chronic liver disease. As a vital organ for lipid metabolism, the liver is often accompanied by lipid metabolism disorders in fibrotic pathology. Damage to hepatocytes from metabolically imbalanced lipids is a key event in initiating and exacerbating liver fibrosis. Therefore, effectively regulating lipid metabolism is crucial for alleviating liver fibrosis.

[0003] Studies have shown that white adipose tissue (WAT) and the liver form a lipid metabolism unit, namely the hepatic-adipose axis, used to store and distribute energy to maintain metabolic homeostasis. However, under the pathological conditions of MASH and liver fibrosis, lipid imbalance occurs in the hepatic-adipose axis: WAT dysfunction releases inflammatory factors and excessive free lipids, increasing the lipid burden on the liver and exacerbating liver fibrosis; liver inflammation intensifies, releasing inflammatory factors and promoting WAT inflammation and insulin resistance. This malignant crosstalk between fat and the liver further promotes the progression of MASH and liver fibrosis. Therefore, coordinating lipid metabolism in the hepatic-adipose axis is a feasible strategy for treating MASH and liver fibrosis. Summary of the Invention

[0004] One objective of this invention is to provide a combined nano-formulation, consisting of nano-formulation A and nano-formulation B; The nano-formulation A is a hepatocyte-regulated nanodelivery system, which is made of phospholipids, cholesterol, PEGylated phospholipids, DSPE-PEG-Galactose, and THR-β agonists; The nanoformulation B is an adipocyte-regulated nanodelivery system, composed of phospholipids, cholesterol, PEGylated phospholipids, DSPE-PEG-CKGGRAKDC, and PPARγ agonists.

[0005] Furthermore, the THR-β agonist is remetiro, and the PPARγ agonist is rosiglitazone.

[0006] Furthermore, the phospholipid is soybean phospholipid or lecithin.

[0007] Furthermore, the PEGylated phospholipid is DSPE-PEG, preferably DSPE-mPEG.2000 .

[0008] In one specific embodiment of the present invention, in nanoformulation A, the mass ratio of phospholipids, cholesterol, PEGylated phospholipids, DSPE-PEG-Galactose, and THR-β agonist is 12:1.2:1:1:0.4; in nanoformulation B, the mass ratio of phospholipids, cholesterol, PEGylated phospholipids, DSPE-PEG-CKGGRAKDC, and PPARγ agonist is 12:1.2:2:2:0.4. The mass ratio of nanoformulation A to nanoformulation B is 1:1.

[0009] In this invention, both nano-formulation A and nano-formulation B can be prepared using conventional methods of the prior art, such as solvent injection, thin film dispersion, direct titration, or solvent exchange.

[0010] In a specific embodiment of the present invention, the preparation process of nano-formulation A is as follows: soybean phospholipids, cholesterol, and DSPE-PEG are taken. 2000 DSPE-PEG 2000 Gal and Remetidine RMT were dissolved in a benign organic solvent to obtain an organic phase; under 40°C oil bath conditions, the organic phase was added dropwise to a continuously stirred physiological saline solution, stirred, sonicated, and centrifuged to obtain the hepatocyte-regulated nanodelivery system (G-Lip / RMT); the preparation process of nano-formulation B was as follows: soybean lecithin, cholesterol, DSPE-PEG were taken. 2000 DSPE-PEG 2000 -CKGGRAKDC and rosiglitazone RGL were dissolved in a benign organic solvent, incubated, and then rotary evaporated. Physiological saline solution was added for hydration, the suspension was collected by pipetting, sonicated, and centrifuged to obtain the supernatant, yielding the adipocyte-regulated nanodelivery system (P-Lip / RGL). The benign organic solvent is one or more of dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, methanol, ethanol, acetonitrile, and acetone.

[0011] A second objective of this invention is to provide the application of the above-mentioned combined nano-formulations in the preparation of therapeutic drugs for metabolic dysfunction-related steatohepatitis and / or liver fibrosis.

[0012] This invention provides a combination formulation for combating MASH and liver fibrosis, comprising a first therapeutic agent and a second therapeutic agent; the first therapeutic agent is a hepatocyte-regulated nanodelivery system that targets hepatocytes and contains a thyroxine receptor β (THR-β) agonist; the second therapeutic agent is an adipocyte-regulated nanodelivery system that targets adipocytes and contains a peroxisome proliferator-activated receptor γ (PPARγ) agonist; the hepatocyte-regulated nanodelivery system and the adipocyte-regulated nanodelivery system are liposomes with independent structural forms.

[0013] The THR-β agonist includes retimerrol; the PPARγ agonist includes rosiglitazone. The hepatocyte-mediated nanodelivery system and the adipocyte-mediated nanodelivery system synergistically regulate the malignant crosstalk formed between the liver and adipose tissue, achieving the treatment of MASH and liver fibrosis.

[0014] The preferred hepatocyte-modifying nanodelivery system is a galactose (G)-modified resmetirom (RMT) liposome, hereinafter referred to as G-Lip / RMT; the G-Lip / RMT achieves targeting and regulation of hepatocytes by utilizing the function of galactose.

[0015] The adipocyte-regulated nanodelivery system is rosiglitazone (RGL) modified with an adipose tissue-targeting peptide (CKGGRAKDC peptide, P); the P-Lip / RGL achieves targeting and regulation of adipocytes by utilizing the function of the adipose tissue-targeting peptide. Beneficial effects

[0016] Given the limited efficacy of current clinical drug treatments for liver fibrosis, and the limitations of previous treatments that only addressed the symptoms of liver fibrosis and the effectiveness of single-target therapies, this invention proposes for the first time a strategy to treat MASH and liver fibrosis by using a combination formulation, including a hepatocyte-regulated nanodelivery system and an adipocyte-regulated nanodelivery system, to synergistically regulate the lipid metabolism balance between the liver and adipose tissue. Furthermore, this combination formulation has been found to have superior anti-MASH and anti-liver fibrosis effects, with no significant difference in treatment efficacy compared to the normal group, thus achieving a cure.

[0017] Furthermore, this invention includes a first therapeutic agent and a second therapeutic agent. The first therapeutic agent is a hepatocyte-regulating nanodelivery system, G-Lip / RMT, which targets hepatocytes and contains a THR-β agonist. It can target and act on hepatocytes with excessive lipid accumulation, promoting mitochondrial development and β-oxidation, reducing lipotoxicity to improve lipid metabolism and alleviate hepatocyte damage. The second therapeutic agent is an adipocyte-regulating nanodelivery system, P-Lip / RGL, which targets adipocytes and contains a PPARγ agonist. It can target and act on adipocytes with abnormal lipid metabolism, promoting the browning of white adipose tissue into brown adipose tissue, promoting energy consumption, and reducing the content of free fatty acids from adipose tissue, thereby alleviating the pressure on lipid metabolism in hepatocytes. The two agents have a synergistic effect, synergistically regulating lipid metabolism homeostasis in the liver and adipose tissue, and inhibiting malignant crosstalk between the liver and adipose tissue during MASH and liver fibrosis.

[0018] The anti-liver fibrosis combination formulation provided by this invention, through the synergistic regulatory effect of two nanodelivery systems on the liver and adipose tissue, two major organs in the disease process, during MASH and liver fibrosis, improves lipid metabolism of hepatocytes and promotes energy consumption of adipocytes. It has the advantages of good targeting, few toxic side effects, and better efficacy; it inhibits malignant crosstalk between organs in MASH and liver fibrosis and reverses the pathological microenvironment of liver fibrosis, and has broad application value, providing an innovative drug treatment strategy for the clinical treatment of liver fibrosis. Attached Figure Description

[0019] Figure 1 Particle size distribution and transmission electron microscope image of the hepatocyte-regulated nanodelivery system G-Lip / RMT prepared in Example 1.

[0020] Figure 2 The particle size distribution and transmission electron microscope image of the adipocyte-regulated nanodelivery system P-Lip / RGL prepared in Example 1 are shown.

[0021] Figure 3 The images show stained tissue sections used for in vivo acute toxicity verification of the hepatocyte-regulated nanodelivery system and the adipocyte-regulated nanodelivery system in Example 2.

[0022] Figure 4 The ALT (A) and AST (B) levels of Xu Qing were used to verify the acute in vivo toxicity of the hepatocyte-regulated nanodelivery system and the adipocyte-regulated nanodelivery system of Example 2.

[0023] Figure 5The flow cytometry graph shows the cell uptake of the liver parenchyma cell-regulated nanodelivery system containing fluorescein isothiocyanate prepared in Example 3 after co-incubation with L02 for 2 hours.

[0024] Figure 6 The flow cytometry graph shows the uptake of the adipocyte-regulated nanodelivery system containing fluorescein isothiocyanate prepared in Example 3 after co-incubation with 3T3-L1 for 2 hours.

[0025] Figure 7 The image shows an inverted fluorescence microscope staining image of the DCFH-DA probe used in Example 4 to regulate ROS clearance in hepatocytes by the hepatocyte-mediated nanodelivery system.

[0026] Figure 8 This is an inverted fluorescence microscope image stained with Oil Red O to show the lipid accumulation in hepatocytes by the hepatocyte-regulated nanodelivery system of Example 4.

[0027] Figure 9 The image shows a FAOBlue laser confocal microscopy staining image of fatty acid β-oxidation in mitochondria of hepatocytes as described by the hepatocyte-mediated nanodelivery system of Example 4.

[0028] Figure 10 This is a laser confocal microscopy staining image showing the effect of the adipocyte-regulated nanodelivery system of Example 5 on UCP1 expression in adipocytes.

[0029] Figure 11 The image shows the in vivo immunofluorescence staining results of the liver distribution of the hepatocyte-regulated nanodelivery system encapsulated with DiI prepared in Example 6.

[0030] Figure 12 The image shows the in vivo immunofluorescence staining results of the adipocyte-regulated nanodelivery system encapsulated with DiI prepared in Example 6, distributed in adipose tissue.

[0031] Figure 13 The in vivo pharmacodynamic characterization of Example 7 shows the changes in body weight of mice in each experimental group.

[0032] Figure 14 For the in vivo pharmacodynamic characterization of Example 7, liver tissue sections from each experimental group were stained with hematoxylin and eosin, Sirius red, Masson's stain, α-SMA stain, and immunohistochemically analyzed for type I collagen.

[0033] Figure 15 Oil Red O stained sections of liver tissue from each experimental group were used for in vivo pharmacodynamic characterization in Example 7.

[0034] Figure 16For the in vivo pharmacodynamic characterization of Example 7, the adipose tissue sections of each experimental group were stained with hematoxylin and eosin.

[0035] Figure 17 For the in vivo pharmacodynamic characterization of Example 7, UCP1 immunofluorescence staining sections of adipose tissue sections from each experimental group.

[0036] Figure 18 The in vivo pharmacodynamic characterization of Example 7 shows the serum triglyceride levels in mice of each experimental group.

[0037] Figure 19 The in vivo pharmacodynamic characterization of Example 7 shows the serum ALT levels in mice of each experimental group.

[0038] Figure 20 The in vivo pharmacodynamic characterization of Example 7 shows the serum AST levels of mice in each experimental group.

[0039] Figure 21 The in vivo pharmacodynamic characterization of Example 7 is shown in the quantitative graph of hydroxyproline in the livers of mice in each experimental group. Detailed Implementation

[0040] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0043] It should be noted that the reagents involved in the following implementation are: DSPE-PEG 2000 -Gal and DSPE-PEG 2000 -CKGGRAKDC was purchased from Chongqing Yusi Pharmaceutical Technology Co., Ltd. Example 1

[0044] Preparation of hepatocyte-regulated nanodelivery system and adipocyte-regulated nanodelivery system 1. Preparation of a hepatocyte-regulated nanodelivery system Accurately weigh 12 mg of soybean lecithin, 1.2 mg of cholesterol, and 1 mg of DSPE-PEG using an analytical balance. 2000 1 mg DSPE-PEG 2000-Gal, 0.4 mg remetitrol RMT, were dissolved in 300 μL of anhydrous ethanol to obtain the organic phase. The organic phase was added dropwise to 2 mL of continuously stirred physiological saline solution under 40°C oil bath conditions, and stirred for 1 h. Subsequently, the mixture was sonicated at 200 W for 3 min; centrifuged at 3500 rpm for 15 min, and the supernatant was collected to obtain the hepatocyte-regulated nanodelivery system G-Lip / RMT.

[0045] Particle size distribution and transmission electron microscopy images of the hepatocyte-regulated nanodelivery system are shown below. Figure 1 As shown, its particle size is 118.78±1.12 nm, its potential is -2.54±0.47 mV, and it is electrically neutral. Its morphology is a uniformly distributed spherical shape.

[0046] Using an analytical balance, 12 mg of soybean phospholipids, 1.2 mg of cholesterol, and 2 mg of DSPE-PEG were precisely measured. 2000 0.4 mg of remetidine RMT was dissolved in 300 μL of anhydrous ethanol to obtain the organic phase. The organic phase was added dropwise to 2 mL of continuously stirred physiological saline solution at 40 °C in an oil bath for 1 h. The mixture was then sonicated at 200 W for 3 min; centrifuged at 3500 rpm for 15 min, and the supernatant was collected to obtain the control hepatocyte-regulated nanodelivery system G-Lip.

[0047] 2. Preparation of an adipocyte-regulated nanodelivery system Accurately weigh 12 mg of soybean lecithin, 1.2 mg of cholesterol, and 2 mg of DSPE-PEG using an analytical balance. 2000 2 mg DSPE-PEG 2000 -CKGGRAKDC and 0.4 mg rosiglitazone RGL were dissolved in 12 mL of methanol and incubated in a round-bottom flask for 5 min. The mixture was then rotary evaporated at 40 °C and 80 rpm for 20 min. 2 mL of physiological saline solution was added, and the mixture was hydrated at 20 rpm for 10 min. The suspension was then collected by pipetting and sonication at 150 W for 5 min. The mixture was then centrifuged at 3500 rpm for 15 min, and the supernatant was collected to obtain the adipocyte-regulated nanodelivery system P-Lip / RGL.

[0048] Particle size distribution and transmission electron microscopy images of the adipocyte-regulated nanodelivery system are shown below. Figure 2 As shown, its particle size is 109.42±1.39 mV, its potential is -0.24±0.64 mV, and it is electrically neutral. Its morphology is a uniformly distributed spherical shape.

[0049] Accurately weigh 12 mg of soybean lecithin, 1.2 mg of cholesterol, and 4 mg of DSPE-PEG using an analytical balance.2000 0.4 mg of rosiglitazone (RGL) was dissolved in 12 mL of methanol and incubated in a round-bottom flask for 5 min. The solution was then rotary evaporated at 40 °C and 80 rpm for 20 min. 2 mL of physiological saline solution was added, and the solution was hydrated at 20 rpm for 10 min. The suspension was then collected by pipetting. Subsequently, the suspension was sonicated at 150 W for 5 min; centrifuged at 3500 rpm for 15 min, and the supernatant was collected to obtain the control adipocyte-regulated nanodelivery system P-Lip. Example 2 In vivo acute toxicity validation of hepatocyte-regulated nanodelivery system and adipocyte-regulated nanodelivery system

[0050] Healthy, well-balanced male C57BL / 6J mice aged 6-8 weeks were randomly divided into three groups of five each. PBS, blank vector G-Lip or P-Lip (200 μL, with the vector concentration at 30 mg / kg), were injected via tail vein once daily for three consecutive days. Twenty-four hours after the last injection, the mice were enucleated to collect blood, then euthanized by cervical dislocation. The mice were dissected, and major organs were collected, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Blood samples and sections were used for subsequent analysis.

[0051] like Figure 3 As shown, the major organ structures of mice treated with blank vectors G-Lip and P-Lip were consistent with those of normal mice, with no obvious inflammatory infiltration or pathological changes.

[0052] like Figure 4 As shown, there were no significant differences in serum ALT and AST levels between mice treated with the blank vector G-Lip and P-Lip and mice treated with PBS. This demonstrates that both vectors have good biocompatibility and can be further used in animal efficacy experiments. Example 3

[0053] Targeting validation of hepatocyte-regulated nanodelivery system and adipocyte-regulated nanodelivery system

[0054] Two nanodelivery systems were prepared according to Example 1. FITC was used instead of RMT or RGL, and DSPE-PEG-Gal or DSPE-PEG-CKGGRAKDC were added to prepare different nanodelivery systems G-Lip / FITC and P-Lip / FITC, respectively. The FITC content in the nanodelivery systems was quantified using an enzyme-linked immunosorbent assay (ELISA) reader. After dilution with culture medium, the FITC content in the different groups of nanodelivery systems was made to 5 μg / mL. G-Lip / FITC encapsulated with FITC was added to L02 cells and incubated for 2 h. P-Lip / FITC encapsulated with FITC was added to 3T3-L1 cells and incubated for 2 h. The uptake of the nanodelivery systems by the cells was detected by flow cytometry.

[0055] Meanwhile, blank nanoformulations Lip(G) / FITC and Lip(P) / FITC were prepared using FITC as controls.

[0056] like Figure 5-6 As shown, after co-incubating the nano-formulation with cells for 2 h, the uptake efficiency of the control nano-formulation Lip(G) / FITC on L02 cells was significantly higher than that of the free FITC group. Furthermore, the fluorescence intensity of the G-Lip / FITC group was significantly increased compared to Lip(G) / FITC. This is mainly because the desialyl glycoprotein receptor is highly expressed on the surface of L02 cells, enabling efficient binding to galactose on the surface of G-Lip / FITC. Therefore, grafting galactose onto the surface of the nano-formulation can improve the drug delivery efficiency to hepatocytes. Similarly, the uptake efficiency of the control nano-formulation Lip(P) / FITC on 3T3-L1 cells was significantly higher than that of the free FITC group. Furthermore, the fluorescence intensity of the P-Lip / FITC group was significantly increased compared to Lip(P) / FITC. This is mainly because the adipose tissue-targeting peptide modified on the surface of P-Lip / FITC can bind to the inhibin receptor on the surface of 3T3-L1 cells. Therefore, grafting adipose tissue-targeting peptides onto the surface of nanoparticles can improve the drug's targeting efficiency to adipocytes. Example 4

[0057] 1. Experiment on the clearance of intracellular ROS in hepatocytes using a hepatocyte-regulated nanodelivery system

[0058] 200,000 L02 cells in logarithmic growth phase were seeded at 200,000 cells / well in 6-well plates and cultured overnight at 37 ℃ in a 5% CO2 cell culture incubator. The cells were randomly divided into three groups: (1) blank control group (Control group): cells were not stimulated with sodium palmitate or treated with the drug; (2) sodium palmitate stimulation model group (PA group): cells were stimulated with 0.25 mM sodium palmitate for 18 h; (3) treatment experimental group (PA+G-Lip / RMT group): cells were stimulated with 0.25 mM sodium palmitate for 18 h and then incubated with 10 μM G-Lip / RMT for 24 h. Next, intracellular ROS staining was performed. The specific staining method is as follows: after removing the old culture medium, wash three times with PBS for 5 min each time; add 1 mL of DCFH-DA probe diluted with 10 mM serum-free culture medium to each well and incubate at 37℃ in a 5% CO2 cell culture incubator for 20 min; after removing the old culture medium, wash three times with PBS for 5 min each time; add 1 mL of PBS and take pictures with an inverted fluorescence microscope.

[0059] like Figure 7 The results showed that hepatocytes stimulated with sodium palmitate exhibited a significant increase in ROS accumulation, and administration of G-Lip / RMT significantly reduced intracellular ROS levels. Therefore, the hepatocyte-regulated nanodelivery system possesses excellent ROS scavenging capabilities.

[0060] 2. Experiment on the use of a hepatocyte-regulated nanodelivery system to alleviate lipid accumulation in hepatocytes

[0061] 80,000 L02 cells in logarithmic growth phase were seeded per well in 24-well plates and cultured overnight at 37 ℃ in a 5% CO2 cell culture incubator. The cells were randomly divided into three groups: (1) Control group: cells were not stimulated with sodium palmitate and sodium oleate or treated with the drug; (2) Sodium palmitate + sodium oleate stimulation model group (PA + OA group): cells were stimulated with 0.25 mM sodium palmitate + 0.25 mM sodium oleate for 24 h; (3) Treatment experimental group (PA + OA + G-Lip / RMT group): cells were stimulated with 0.25 mM sodium palmitate + 0.25 mM sodium oleate for 24 h and then incubated with 10 μM G-Lip / RMT for 36 h. Next, intracellular lipid staining was performed. The specific staining method was as follows: After aspirating the old culture medium, the cells were washed three times with PBS for 5 min each time; 500 μL of 4% paraformaldehyde was added to each well and fixed at room temperature for 10 min; after aspirating the fixative, the cells were washed three times with PBS for 5 min each time; 500 μL of staining wash buffer was added to each well to cover the cells for 20 s, the wash buffer was aspirated, and then 500 μL of Oil Red O staining solution was added to stain for 20 min; after aspirating the staining solution, 500 μL of staining wash buffer was added to each well to cover the cells for 30 s, the wash buffer was aspirated, and then PBS was added to wash for 20 s; 500 μL of PBS was added to each well, and the cells were photographed using an inverted fluorescence microscope.

[0062] like Figure 8 The results showed that hepatocytes stimulated with sodium palmitate and sodium oleate exhibited significant lipid accumulation, which was significantly alleviated by G-Lip / RMT administration. Therefore, the hepatocyte-regulated nanodelivery system has a good effect on alleviating lipid accumulation.

[0063] 3. Experiment on restoring mitochondrial β-oxidation in hepatocytes using a nanodelivery system regulated by hepatocytes

[0064] 80,000 L02 cells in logarithmic growth phase were seeded per well in laser confocal dishes and cultured overnight at 37 ℃ in a 5% CO2 cell culture incubator. The cells were randomly divided into three groups: (1) Control group: cells were not stimulated with sodium palmitate and sodium oleate or treated with the drug; (2) Sodium palmitate + sodium oleate stimulation model group (PA + OA group): cells were stimulated with 0.25 mM sodium palmitate + 0.25 mM sodium oleate for 24 h; (3) Treatment experimental group (PA + OA + G-Lip / RMT group): cells were stimulated with 0.25 mM sodium palmitate + 0.25 mM sodium oleate for 24 h and then incubated with 10 μM G-Lip / RMT for 36 h. Next, the mitochondrial β-oxidation activity of cells was investigated. The specific method was as follows: after removing the old culture medium, the cells were washed three times with PBS for 5 min each time; 500 μL of fatty acid oxidation detection working solution FAOBlue diluted with 10 μM serum-free culture medium was added to each dish, and the cells were incubated at 37 ℃ in a 5% CO2 cell culture incubator for 30 min; the cells were washed three times with PBS for 5 min each time; 500 μL of PBS was added to each dish, and the cells were photographed using a laser confocal microscope.

[0065] like Figure 9 The results showed that hepatocytes stimulated with palmitic acid and sodium oleate exhibited decreased mitochondrial β-oxidation function, which was restored after administration of G-Lip / RMT. Therefore, the hepatocyte-regulated nanodelivery system has a good effect on restoring mitochondrial β-oxidation in hepatocytes and alleviating lipid burden in hepatocytes. Example 5

[0066] Experiments on the promotion of adipocyte browning by adipocyte-regulated nanodelivery systems

[0067] 80,000 3T3-L1 cells in logarithmic growth phase were seeded per well in 24-well plates pre-placed with cell spreaders and cultured overnight at 37 ℃ in a 5% CO2 cell culture incubator. The cells were randomly divided into three groups: (1) Control group: cells were not stimulated with sodium palmitate and sodium oleate or treated with the drug; (2) Sodium palmitate + sodium oleate stimulation model group (PA + OA group): cells were stimulated with 0.25 mM sodium palmitate + 0.25 mM sodium oleate for 24 h; (3) Treatment experimental group (PA + OA + P-Lip / RGL group): cells were stimulated with 0.25 mM sodium palmitate + 0.25 mM sodium oleate for 24 h and then incubated with 5 μM P-Lip / RGL for 48 h. Next, the expression of UCP1 in adipocytes was investigated using the following method: After removing the old culture medium, the cells were washed three times with PBS for 5 min each time; 500 μL of 4% paraformaldehyde was added to each well and fixed at room temperature for 20 min; after removing the fixative, the cells were washed three times with PBS for 5 min each time; 1 mL of 0.2% Triton X-100 was added to each well for permeabilization for 2-5 min; the cells were washed three times with PBS for 5 min each time; 400-500 μL of 5% donkey serum was added to each well and blocked at room temperature for 30 min; the blocking solution was removed, and 300 μL of primary antibody (Rabbit polyclonal to UCP1) diluted with 5% donkey serum was added to each well and incubated overnight at 4 ℃; the primary antibody was recovered the next day, and the cells were washed three times with PBS for 5 min each time; 200-300 μL of secondary antibody (Alexa Fluor-647-labeled goat anti-rabbit mouse IgG) diluted with 5% donkey serum was added to each well and incubated at 4 ℃ in the dark for 30 min; the cells were washed three times with PBS for 5 min each time. Add 300 μL of DAPI to each well and stain at room temperature for 15 min; remove the staining solution, wash three times with PBS for 5 min each time; remove the cell slides, mount them on a glass slide with mounting solution containing anti-fluorescence quencher, and fix the edges with clear nail polish; take pictures with a laser confocal microscope.

[0068] like Figure 10 The results showed that adipocytes stimulated with sodium palmitate and sodium oleate exhibited excessive hypertrophy, and the uncoupling protein UCP1 was inhibited. However, administration of P-Lip / RGL significantly increased UCP1 expression in adipocytes. This demonstrates that the adipocyte-regulated nanodelivery system promotes adipocyte browning and heat production, effectively alleviating metabolic disorders in adipocytes. Example 6

[0069] Immunofluorescence staining experiments of mouse liver and adipose tissue

[0070] Two nanodelivery systems prepared according to Example 1 were prepared by replacing RMT or RGL with DiI and adding DSPE-PEG-Gal or DSPE-PEG-CKGGRAKDC to prepare different nanodelivery systems G-Lip / DiI and P-Lip / DiI, respectively. The DiI content in the nanodelivery systems was quantified using an enzyme-linked immunosorbent assay (ELISA) reader.

[0071] Meanwhile, blank nanoformulations Lip(G) / DiI and Lip(P) / DiI were prepared using DiI as controls.

[0072] Immunofluorescence staining procedure for liver tissue: Healthy, well-balanced male C57BL / 6J mice aged 6-8 weeks were randomly divided into three groups. Free DiI, Lip(G) / DiI, and G-Lip / DiI were injected into the mice via the tail vein (Dil dose: 1 mg / kg). Four hours later, the mice were euthanized by cervical dislocation, and liver tissue was harvested. The tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Cell nuclei were stained with DAPI, and hepatocytes were labeled with Albumin for immunofluorescence staining. Full scans of the fluorescent sections were performed using a digital slide scanner, and the images were analyzed using image processing software.

[0073] Immunofluorescence staining procedure for adipose tissue: Healthy, well-balanced male C57BL / 6J mice aged 6-8 weeks were randomly divided into three groups. Free DiI, Lip(P) / DiI, and P-Lip / DiI were injected into the mice via the tail vein (DiI dose was 1 mg / kg). Four hours later, the mice were euthanized by cervical dislocation, and the epididymal white adipose tissue was collected. The tissue was digested with 0.1% collagenase for 30 min, filtered through a 100 μm cell filter to obtain the cell filtrate, and centrifuged at 1000-1500 rpm for 10 min. The top floating layer containing mature adipose tissue was collected. Lipid droplets in the adipocytes were stained with Bodipyr for live cell staining. The cells were then seeded on polylysine-coated confocal dishes and fixed with 4% paraformaldehyde. Images were taken using a laser confocal microscope.

[0074] Hepatocytes were labeled with Albumin, and the fluorescence colocalization of the nanoparticle formulation with hepatocytes was investigated using full-scan fluorescence imaging of tissue sections. The results are as follows: Figure 11 As shown, the control nanoformulation Lip(G) / Dil encapsulated with Dil exhibited higher hepatic accumulation compared to free Dil; and G-Lip / Dil showed more red fluorescence in hepatocytes compared to Lip(G) / Dil, demonstrating that galactose-modified nanoformulations can significantly improve the targeted accumulation of drugs in hepatocytes. Similarly, lipid droplets in adipocytes were labeled with Bodipy to examine the uptake efficiency of nanoformulations by adipocytes; as... Figure 12 As shown, the nanoformulation P-Lip / Dil modified with adipose tissue targeting peptides exhibits higher fluorescence intensity compared to the control nanoformulations Lip(P) / Dil and free Dil; demonstrating that nanoformulations modified with adipose tissue targeting peptides can significantly improve the targeted accumulation of drugs in adipocytes. Example 7

[0075] AMLN-induced MASH and treatment groups: histopathological analysis and serological tests of mouse liver and adipose tissue.

[0076] Male C57BL / 6J mice aged 6-8 weeks were used to establish MASH and liver fibrosis models. Mice were randomly divided into four groups: a normal control group (fed standard maintenance diet), a MASH group (fed AMLN), a free dual-drug group (AMLN fed diet + tail vein injection of RMT + RGL), a first-drug treatment group (AMLN fed diet + tail vein injection of G-Lip / RMT), a second-drug treatment group (AMLN fed diet + tail vein injection of P-Lip / RGL), and a combined treatment group (AMLN fed diet + tail vein injection of G-Lip / RMT + P-Lip / RGL). Mice were fed AMLN-rich diet at a dose of 4-6 g per day for 8 weeks, for a total of 24 weeks, to establish the MASH model. The therapeutic agents contained 5 mg / kg body weight of RMT and 3 mg / kg body weight of RGL. Injections were administered twice weekly for 6 weeks, with mouse weight recorded at fixed times each week. Liver tissue, adipose tissue, and blood were collected after 6 weeks for analysis.

[0077] like Figure 13 The figure shows the changes in body weight of mice in each experimental group. Compared with the free dual-drug group and the first treatment group, mice in the second treatment group and the combined treatment group all showed relatively significant weight loss; among them, the combined treatment group showed a stronger weight loss effect. At the treatment endpoint of week 6, the body weight decreased by 11.6% compared with the pre-treatment level, demonstrating that the combined treatment group had a good effect on improving obesity.

[0078] like Figure 14The images show stained sections of liver tissue from mice in each experimental group. Hematoxylin and eosin staining of liver sections from each experimental group revealed that, compared to the normal group, the MASH group showed infiltration of non-parenchymal cells, numerous fat vacuoles, and a coexistence of macrovesicular and microvesicular steatosis. In contrast, treatment with the free drug or nano-formulation improved the degree of liver steatosis; the combined treatment group showed a significant reduction in liver fat vacuoles, demonstrating that the combined treatment significantly improved liver steatosis. Masson staining, Sirius red staining, and type I collagen staining of liver sections from each experimental group showed that the MASH group mice had excessively deposited pathological collagen, forming bridging fibrosis; the first treatment group showed a significant reduction in liver collagen deposition, while the combined treatment group further reduced the level of liver collagen deposition. Immunohistochemical sections of the liver α-SMA showed that the MASH group mice had a large number of activated hepatic stellate cells, and both the first and second treatment groups showed a decrease in the degree of hepatic stellate cell activation; the combined treatment group effectively reversed the quiescent state of hepatic stellate cells. The combined treatment group demonstrated a significant therapeutic effect on liver fibrosis.

[0079] like Figure 15 The images show Oil Red O stained sections of mouse liver tissue from each experimental group. The results indicate that the livers of mice in the MASH and free dual-drug groups were extensively stained with Oil Red O, indicating severe lipid burden. In contrast, the combined treatment group significantly alleviated both macrovesicular and microvesicular steatosis in the liver. This demonstrates that the combined treatment group has a significant therapeutic effect on hepatic steatosis associated with MASH.

[0080] like Figure 16 The images show hematoxylin and eosin stained sections of adipose tissue from mice in each experimental group. The results show that in the MASH group, the adipocytes in the adipose tissue of mice were generally hypertrophic, indicating impaired lipid metabolism. In contrast, the adipose tissue of mice in the dual-drug combined treatment group showed smaller adipocyte units and a more rosy appearance, indicating that the blood vessels in the adipose tissue were more densely formed and that the number of beige adipocytes increased.

[0081] like Figure 17 The images shown are UCP1-stained sections of adipose tissue from mice in each experimental group. Adipose tissue, as an endocrine organ, plays an important role in systemic metabolic function. Thermogenic adipocytes that highly express UCP1 can improve systemic metabolic dysfunction by increasing energy expenditure and metabolism. Figure 17 The results showed that adipocytes in MASH mice were mainly characterized by hypertrophy and metabolic dysfunction, with low UCP1 expression. The second treatment group showed that P-Lip / RGL could effectively promote browning of adipose tissue. The combined treatment group further improved the thermogenesis function of adipose tissue. This demonstrates that the combined treatment group can significantly alleviate adipocyte hypertrophy and metabolize excess lipids through thermogenesis, showing good therapeutic potential for maintaining systemic metabolism.

[0082] like Figure 18 The results show the serum triglyceride levels. There was no significant difference in serum triglyceride levels between the combined treatment group and the normal group, demonstrating that the combined treatment can effectively alleviate the body's lipid metabolism burden.

[0083] like Figure 19 and 20 Serum ALT and AST levels were measured, and there was no significant difference between the combined treatment group and the normal group, demonstrating that the combined treatment has a significant therapeutic effect on liver fibrosis.

[0084] like Figure 21 The study measured the hydroxyproline content in the liver. Hydroxyproline, an important component of the extracellular matrix, is a biomarker for the severity of liver fibrosis. Results showed that the combined treatment group exhibited significantly reduced extracellular matrix deposition and a marked decrease in the degree of liver fibrosis. The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A combination nanoformulation, characterized in that, consisting of nano-preparation A and nano-preparation B; the nano-preparation A is made of phospholipid, cholesterol, PEGylated phospholipid, DSPE-PEG-Galactose and THR-β agonist; the nano-preparation B is made of phospholipid, cholesterol, PEGylated phospholipid, DSPE-PEG-CKGGRAKDC and PPARγ agonist.

2. The combination nanoformulation of claim 1, wherein, the THR-β agonist is resmetirom, and the PPARγ agonist is rosiglitazone.

3. The combination nanoformulation of claim 1, wherein, the phospholipid is soybean phospholipid or lecithin.

4. The combination nanoformulation of claim 1, wherein, the PEGylated phospholipid is DSPE-PEG.

5. The combination nanoformulation of claim 1, wherein, In the nano-preparation A, the mass ratio of phospholipid, cholesterol, PEGylated phospholipid, DSPE-PEG-Galactose and THR-β agonist is 12:1.2:1:1:0.4; in the nano-preparation B, the mass ratio of phospholipid, cholesterol, PEGylated phospholipid, DSPE-PEG-CKGGRAKDC and PPARγ agonist is 12:1.2:2:2:0.

4.

6. The combination nanoformulation of claim 5, wherein, The mass ratio of the nano-preparation A and the nano-preparation B is 1:

1.

7. Use of the combined nano-preparation according to any one of claims 1-6 in the preparation of a medicament for the treatment of metabolic dysfunction-associated steatohepatitis and / or liver fibrosis.

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