A drug targeting delivery system targeting liver and a drug for treating and / or adjuvant treating metabolic-related fatty liver disease

Abstract

The application discloses a drug targeting delivery system targeting liver and a drug for treating and / or assisting in treating metabolic associated fatty liver disease, and achieves the targeting of estradiol to the liver, enhances the treatment effect of estradiol on metabolic associated fatty liver disease, and avoids the high concentration distribution of estradiol in non-liver tissues to reduce side effects by modifying a drug delivery system with a polypeptide specifically targeting different liver cells. The nano drug prepared by the application can effectively treat MASLD, the treatment effect is remarkable, and there is no side effect, so that a new direction and means for treating MASLD can be provided.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nanomedicine, in particular to a drug targeting delivery system targeting liver and a drug for treating and / or adjuvant treating metabolic dysfunction-associated steatotic liver disease. BACKGROUND

[0002] Metabolic dysfunction-associated steatotic liver disease (MASLD) as a metabolic disease closely related to obesity has a sharp increase in the global prevalence. MASLD is a disease spectrum, covering simple steatosis and metabolic dysfunction-associated steatohepatitis (MASH). Simple steatosis is characterized by massive deposition of triglycerides in hepatocytes; MASH is accompanied by increased liver inflammation, hepatocyte damage and apoptosis on this basis. The pathogenesis of MASLD is multifactorial, and it is currently generally believed that environmental factors (lifestyle, dietary choices, etc.) will lead to obesity, insulin resistance, thereby causing increased de novo lipogenesis and fatty acid uptake in hepatocytes, decreased fatty acid oxidation and very low density lipoprotein output, causing liver lipid accumulation; lipid toxicity leads to mitochondrial dysfunction in hepatocytes, producing reactive oxygen species and endoplasmic reticulum stress, thereby causing hepatocyte damage and even apoptosis; hepatocyte damage and apoptosis in turn trigger Kupffer cell activation, immune cell infiltration, and hepatic stellate cell activation and fibrosis. At the same time, insulin resistance will also lead to adipose tissue dysfunction and exacerbate systemic inflammation by increasing pro-inflammatory cytokines, adipokines, etc. Due to the complex pathogenesis of MASLD and its easy progression to cirrhosis and liver cancer, effective intervention measures are urgently needed. However, so far the treatment measures for MASLD are very limited.

[0003] In recent years, the number of nanomedicine-based drugs has increased significantly. Their advantages include targeted delivery to improve efficacy, reduce toxicity, making them ideal candidates for the treatment of complex diseases. Lipid-polymer nanoparticles (LNP) have excellent serum stability due to strong hydrophobic interaction between polymers and lipids; by adding a targeting peptide / small molecule to the end of the lipid coating on the core polymer surface, cell-specific targeted delivery can be achieved. Estradiol (E2) is the main type of estrogen, mainly secreted by the ovary, and its main physiological functions are to promote the development of female reproductive organs and the development of secondary sexual characteristics, and to promote endometrial hyperplasia. Clinical studies have reported that the prevalence of MASLD in men of reproductive age is significantly higher than that in women; in women, the prevalence of MASLD after menopause has steadily increased. And high levels of estrogen have serious side effects in both women and men, such as breast development, sexual dysfunction and reproductive function decline in men, and increased risk of breast and ovarian cancer, endometrial lesions and mood changes in women. However, there is no research on using nanocarriers to load estrogen for the treatment of MASLD in current research. SUMMARY

[0004] Therefore, based on the above background, the present application provides a drug targeting delivery system targeting the liver and its drug for treating and / or adjuvant treating metabolic-related fatty liver disease, which achieves the targeting of estradiol to the liver by modifying the drug delivery system with polypeptides that specifically target different liver cells, enhances the therapeutic effect of estradiol on metabolic-related fatty liver disease, and avoids high concentration distribution of estradiol in non-liver tissues to reduce side effects.

[0005] One of the technical solutions of the present application is:

[0006] A drug targeting delivery system targeting the liver, comprising a drug delivery carrier and a targeting peptide, the targeting peptide comprising a targeting peptide GalNAc that specifically targets hepatocytes and Kupffer cells, and a targeting peptide AEAA that specifically targets hepatic stellate cells;

[0007] The drug delivery carrier comprises a polymer and a lipid layer encapsulating the polymer.

[0008] The polypeptide AEAA and the polypeptide GalNAc are modified on the lipid layer.

[0009] Further, the polymer is PLGA polylactic acid-glycolic acid copolymer (50:50, MW 38000-54000).

[0010] Further, the liposome is formed using DSP-PEG(2000)-NH2 as raw material.

[0011] Further, the molecular formula of the targeting peptide GalNAc is C 56 H 85 N 15 O 31 The molecular structure is shown in the following formula (1):

[0012]

[0013] The molecular formula of the targeting peptide AEAA is C 21 H 29 N5O 10， The molecular structure is shown in the following formula (2):

[0014]

[0015] Based on the same inventive concept, the second technical solution of the present application is:

[0016] The application of the above-mentioned liver-targeted drug targeted delivery system in the preparation of a drug for treating and / or adjuvant treating metabolic-related fatty liver disease, wherein the drug is an injection.

[0017] Based on the same inventive concept, the third technical solution of the present application is:

[0018] A nano-drug for treating and / or adjuvant treating metabolic-related fatty liver disease, comprising the liver-targeted drug targeted delivery system and estradiol, wherein the estradiol is grafted on the polymer.

[0019] Further, the drug loading rate of the estradiol is 55%-80%.

[0020] Further, it comprises the following steps:

[0021] S1: preparing a lipid film modified by the targeting peptide AEAA and the targeting peptide GalNAc;

[0022] S2: adding the lipid film of step S1 into a solvent to prepare a lipid dispersion;

[0023] S3: modifying estradiol on the polymer;

[0024] S4: adding the lipid dispersion of step S2 into the polymer solution to encapsulate the polymer particles with a lipid layer to obtain a nano-drug dispersion.

[0025] Further, step S1 specifically comprises the following steps:

[0026] S1.1: adding the targeting peptide AEAA, the targeting peptide GalNAc and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide into a solvent for dissolution;

[0027] S1.2 Take DSPE-PEG(2000)-NH2, pyridine into the solvent to dissolve;

[0028] S1.3 Mix the solutions of step S1.1 and step S1.2, stir, freeze-dry to obtain a solid sample;

[0029] S1.4 The solid sample of step S1.3 is dissolved in a solvent and evaporated to obtain a lipid film.

[0030] Further, the targeting peptide AEAA, the targeting peptide GalNAc and

[0031] The molar ratio of DSPE-PEG(2000)-NH2 is 1:1:2.

[0032] The effective effects achieved by adopting the present application are:

[0033] The present application realizes the targeting of estradiol to the liver by modifying the drug delivery system with polypeptides specifically targeting different liver cells, enhances the treatment effect of estradiol on metabolic-related fatty liver disease, and avoids the high concentration distribution of estradiol in non-liver tissues to reduce side effects.

[0034] The present application utilizes the strong hydrophobic interaction between polymers (poly(lactic-co-glycolic acid)) and lipids to make the nanomedicine have excellent serum stability, which can avoid the burst release of the pharmaceutical ingredient estradiol of the polymer nanoparticle. And the drug loading rate of the pharmaceutical ingredient estradiol in the nanomedicine of the present application is high, which can realize long-term sustained release and effective pharmaceutical ingredients to improve the treatment effect.

[0035] And the present application is verified by mouse experiments, the nanomedicine prepared by the present application can effectively treat MASLD, the treatment effect is remarkable, and there is no side effect, so as to provide a new direction and means for the treatment of MASLD.

[0036] Drawings of the specification

[0037] Appendix Figure 1 Preparation and characterization of liver-targeting nanomedicine carrier:

[0038] Figure 1 a is a schematic diagram of the synthesis of hepatocyte, Kupffer cell and hepatic stellate cell targeting peptide;

[0039] Figure 1 b is a schematic diagram of the combination of hepatocyte, Kupffer cell and stellate cell targeting peptide with liposome;

[0040] Figure 1c is a schematic diagram of the E2@LNP-AEAA / GalNAc system synthesized from liposomes and drug-loaded nanoparticles.

[0041] Appendix Figure 2 Particle size and drug loading of targeted nanocarriers for drug delivery:

[0042] Figure 2 a represents the particle size and potential of E2@LNP and E2@LNP-AEAA / GalNAc;

[0043] Figure 2 b represents the drug loading of E2@LNP-AEAA / GalNAc.

[0044] Appendix Figure 3 The nanocarrier achieves specific targeting of various liver cells in vitro and in vivo through its targeting peptides:

[0045] Figure 3 a represents the in vitro uptake of E2@LNP and E2@LNP-AEAA / GalNAc by HepG2 hepatocytes, THP1 macrophages, and LX2 stellate cells;

[0046] Figure 3 b represents the in vivo uptake of E2@LNP and E2@LNP-AEAA / GalNAc by different tissues in male and female mice.

[0047] Appendix Figure 4 The therapeutic effect of estradiol-loaded nanocarriers in male mice with metabolic-associated fatty liver disease:

[0048] Figure 4 a represents the effect of estradiol-loaded nanocarriers on serum ALT in male mice;

[0049] Figure 4 b represents the effect of estradiol loading on serum AST in male mice;

[0050] Figure 4 c represents the effect of estradiol loading on liver pathology in male mice.

[0051] Appendix Figure 5 The therapeutic effect of estradiol-loaded nanocarriers on female metabolic-associated fatty liver disease in mice:

[0052] Figure 5 a represents the effect of estradiol loading on serum ALT in female mice;

[0053] Figure 5 b represents the effect of estradiol loading on serum AST in female mice; Figure 5 c represents the effect of estradiol loading on liver pathology in female mice.

[0054] AppendixFigure 6 Toxicity of liposome-nanoparticles in vivo and in vitro:

[0055] Figure 6 a is the toxicity of E2@LNP-AEAA / GalNAc to hepatocytes HepG2, macrophages THP1 and stellate cells LX2

[0056]

[0057] Figure 6 b is the in vivo toxicity of E2@LNP-AEAA / GalNAc.

[0058] Figure 1 shows the protection of free estradiol on metabolic-related fatty liver disease: Figure 7

[0059] Figure 7 a is the effect of free estradiol on serum ALT of male mice;

[0060] Figure 7 b is the effect of free estradiol on serum AST of female mice;

[0061] Figure 7 c is the effect of free estradiol on liver pathology of male mice. DETAILED DESCRIPTION

[0062] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below in combination with its embodiments; it should be understood that the specific embodiments described herein are only used to explain the present application, and are not used to limit the present application.

[0063] Example 1: Preparation of a nanomedicine for treating and / or assisting in treating metabolic-related fatty liver disease includes the following steps (as shown in Figure 1 Figure 1):

[0064] S1: Preparation of targeting peptide AEAA and targeting peptide GalNAc

[0065] Targeting peptide GalNAc:

[0066] 1) 0.2 mmol (153 mg) of resin (Fmoc-amino acid-Wang resin, purchased from Gilbio, item number 48101) was swelled with 4 mL of anhydrous dichloromethane (DCM) (CAS: 27639), and activated for 10 min before being pressed dry;

[0067] 2) Reaction of resin (Fmoc-amino acid-Wang resin, purchased from Gilbio, item number 48101) and amino acid:

[0068] ​​0.3 mmol of glycine (G) (89.78 mg) (CAS 29022-11-5): in a glass bottle, add solvent DCM 4 mL and 104 uL of N,N-diisopropylethylamine (DIPEA) (CAS: 7087-68-5) (2 times the amount of amino acid, 0.6 mmol), nitrogen bubbling, mix well, react for 60 min.

[0069] 3) Close the resin that is not completely reacted:

[0070] 2 mL of methanol + 2 mL of DCM (1:1) + 200 uL of DIPEA, react for 15 min, then rinse with dimethylformamide (DMF) (CAS: 25174) 5 times, nitrogen bubbling, mix well for 1 min each time.

[0071] 4) Fmoc removal:

[0072] Configure 20% (v / v 4 mL DMF + 1 mL piperidine) of piperidine (CAS: 110-89-4), add to the reactor, nitrogen bubbling, mix well, treat for 20 min. Rinse with DMF 5 times, nitrogen bubbling, mix well for 1 min each time.

[0073] 5) Two amino acid reactions:

[0074] Take 0.3 mmol of serine (S) (CAS: 71989-33-8), 0.3 mmol of HBTU (CAS: 94790-37-1), 0.3 mmol of HOBT (CAS: 2592-95-2) and 0.3 mmol of DIPEA in a glass bottle, dissolve with 4 mL of DMF, nitrogen bubbling, mix well, react for 2 h.

[0075] 6) Repeat steps 4-5, connect different amino acids (GSGS) in sequence according to the sequence.

[0076] 7) Succinic anhydride (SA) (CAS: 108-30-5) connection: after Fmoc removal, rinse with DMF 5 times, add 0.6 mmol of SA, dissolve in 4 mL of DMF, nitrogen bubbling, mix well, react for 4 h, then rinse with DMF and DCM 4 times respectively.

[0077] 8) N,N'-Diisopropylcarbodiimide (DIC) (CAS: 693-13-0), N-Hydroxysuccinimide (NHS) (CAS: 6066-82-6), DIPEA, DMF as catalyst to add -COOH: 0.4 mmol DIC and 0.4 mmol NHS were bubbled in 3 mL DMF for 1 h, then 0.4 mmol Tris was dissolved in 1 mL DMF, and then 0.4 mmol DIPEA was added. After 4 h of reaction, the resin was washed with DMF for 5 times, each time for 1 min.

[0078] 9) 4-Dimethylaminopyridine (DMAP) (CAS: 1122-58-3), DIC, DIPEA, DMF as catalyst to introduce 5-hexynoic acid (HA) (CAS: 53293-00-8): 1.2 mmol DIC, 1.2 mmol HA and 1.2 mmol DMAP were dissolved in DMF and added to the vial with stirring, then 1.2 mmol DIPEA was added. After 24 h of reaction under magnetic stirring (rotation speed < 500 rpm), the reaction mixture was transferred to the synthesis column and washed with DMF and DCM for 4 times, respectively.

[0079] 10) 0.6 mmol N3-galactose (CAS: 869186-83-4), 0.6 mmol copper(I) iodide (CAS: 7681-65-4) were dissolved in 100 uL DMF, 18 mmol DIPEA was added, and the reaction was carried out under stirring at room temperature for 16 h. The resin was washed with saturated aqueous sodium ascorbate, water, methanol, DMF, and DCM for 3 times.

[0080] 11) 95% Trifluoroacetic acid (TFA) (CAS: 27881) (2.5% water and 2.5% triisopropylsilane) was prepared, 1 mL of the mixture was taken in the reactor, and nitrogen was bubbled for 1 min to mix. The cut amino acid was collected by pressing dry. Then 1 mL of the mixture was taken in the reactor, and nitrogen was bubbled for 3 min to mix. The cut amino acid was collected by pressing dry. Finally, the remaining mixture was taken in the reactor, and nitrogen was bubbled for 3 min to mix. The cut amino acid was collected by pressing dry. The reaction was carried out on a magnetic stirrer for 2 h.

[0081] 12) The collected polypeptide was rotary evaporated to near dry state. 10-20 mL of ice ethyl ether was added, and white precipitate appeared. Centrifugation was carried out at 5000 rpm for 5 min, and a small amount of the precipitate was dissolved in methanol for MS. The synthesized polypeptide was a targeting peptide targeting hepatocellular and Kupffer cells in the liver, named GalNAc, and its molecular structure is shown in the following formula (1).

[0082]

[0083] Targeting peptide GalNAc:

[0084] 1) 0.2 mmoL resin (Fmoc-amino acid-Wang resin, purchased from Gilbio, item number 48101) was swelled with 4 mL of anhydrous DCM, activated for 10 min and then drained.

[0085] 2) Reaction of the resin with the amino acid:

[0086] 0.3 mmoL of glycine in a glass bottle, 4 mL of solvent DCM and 104 uL of DIPEA (2 times the amount of amino acid, 0.6 mmoL) were added, mixed with nitrogen bubbling and left to react for 60 min.

[0087] 3) Blocking of the resin not completely reacted:

[0088] 2 mL of methanol + 2 mL of DCM (1 : 1) + 200 uL of DIPEA were added for 15 min, then washed 5 times with DMF, mixed with nitrogen bubbling for 1 min each time.

[0089] 4) Fmoc removal: 20% piperidine was prepared and added to the reactor, mixed with nitrogen bubbling and left to act for 20 min. Washed 5 times with DMF, mixed with nitrogen bubbling for 1 min each time.

[0090] 5) Two amino acid reactions:

[0091] 0.3 mmoL of serine (Serine, S), 0.3 mmoL of HBTU, 0.3 mmoL of HOBT and 0.3 mmoL of DIPEA were taken in a glass bottle, dissolved in 4 mL of DMF, mixed with nitrogen bubbling and left to react for 2 h.

[0092] 6) Steps 4-5 were repeated, connecting the different amino acids in sequence according to the sequence (GSGS-beta alanine).

[0093] 7) When connecting to the last amino acid, methoxybenzoic acid (Methoxybenzoic acid, MA) (CAS 100-09-4), the following coupling agents were used: 0.6 mmoL of MA, 0.6 mmoL of DIC, 0.6 mmoL of DIPEA and 0.06 mmoL of DMAP were dissolved separately in DMF and added to the reactor, left to react for 4 h and washed 4 times with DMF and DCM, respectively.

[0094] 8) 50 mL of 1% TFA + 2.5% TPS were prepared in DCM, 4 mL of the mixture were added at a time, mixed with nitrogen bubbling for 1 min, the liquid was drained into a rotary evaporator flask and the operation was repeated 10 times.

[0095] 9) The collected polypeptide was rotary evaporated to near dry state. 10-20 mL of ice-ethanol was added, and a white precipitate appeared. Centrifugation at 5000 rpm for 5 min, and a small amount of the precipitate was dissolved in methanol for MS. The synthesized polypeptide, i.e., the targeted peptide hepatic stellate cell, was named AEAA, and its molecular structure is shown in the following formula (2):

[0096]

[0097] S2: Synthesis of nanoparticles

[0098] 1) P1, P2, NHS (N-hydroxysuccinimide), 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC) were taken in a 1.5 mL sample bottle according to the corresponding proportions shown in Table 1 (the proportions are shown in the following figure), 200 uL of DMF was added for dissolution, a magnetic stirring rotor was added to the sample bottle, and it was placed on a magnetic stirrer for stirring for 1 h.

[0099] Table 1: Material taking amount ratio

[0100]

[0101] 2) DSPE-PEG (2000)-NH2 was taken in a sample bottle, 82.5 uL of pyridine and 100 uL of DMF were added for dissolution, and magnetic stirring was performed for 12 h.

[0102] 3) One end of the dialysis bag was clamped with a clamp, and the sample in step 2) was moved into the dialysis bag with a yellow gun head, the reactants in the sample bottle were added to the dialysis bag, 10 times the volume of distilled water was added, and the other end of the dialysis bag was clamped.

[0103] 4) The dialysis bag was placed in a beaker filled with pure water, and magnetic stirring was performed for 3 days. The freeze-dried sample obtained DSP-PEG (2000)-(GalNAc / AEAA).

[0104] 5) The lipid component DSP-PEG (2000)-(GalNAc / AEAA) 15 mg was taken in a 5 mL tomato-shaped bottle, 200 uL of methanol was added for dissolution, and rotary evaporation was performed to obtain a lipid film.

[0105] 6) 2 mL of water and Tris-HCl (20 mM, pH 7.4) were added, and ultrasonic hydration was performed for 10 min to obtain a lipid dispersion.

[0106] 7) The liposome dispersion solution described above was placed in a 5 mL glass bottle, 3 mg of PLGA (50:50, MW 38000-54000) and 0.06 mg of estradiol (E2) (CAS No: 50-28-2, molecular formula: C 18 H 24 O2, molecular weight: 272.4 g / mol) was dissolved in 1 mL of acetonitrile (DID dye can be added to 2% of the mass of PLGA for subsequent imaging observation), and the PLGA acetonitrile solution was quickly injected into the bottom of the centrifuge tube with a pipette.

[0107] 8) Continue vortexing for 30 s to obtain the targeted liposome-nanoparticle-estradiol dispersion E2@LNP-AEAA / GalNAc.

[0108] Comparative Example: The same as Example 1 above, omit the synthesis steps of polypeptide P1 and polypeptide P2 in step S1, and the step of dissolving the polypeptide in step S2, and the same as above, to prepare a liposome-nanoparticle-estradiol dispersion E2@LNP without modified polypeptide.

[0109] Next, the hydrated particle size and zeta potential of the nanoparticles E2@LNP-AEAA / GalNAc and E2@LNP were detected by Malvern nanoparticle size analyzer, and the results are shown in Figure 1 a. As can be seen from the figure, the targeting peptide does not affect the particle size and potential of the nanoparticles, and the size of the nanoparticles prepared is between 40-50 nm.

[0110] The fenestrations of liver sinusoidal endothelial cells limit the accessibility of functional cells in the liver, such as hepatocytes and hepatic stellate cells, so it is essential to control the size of LNP below 100 nm for targeting these cells, and the particle size of the nanoparticle drug prepared in the above example is 40-50 nm, which fully meets the requirements.

[0111] Next, the drug loading capacity of E2@LNP-AEAA / GalNAc was explored, and the results are shown in Figure 2 b.

[0112] The specific operation is as follows:

[0113] 1) The liposome dispersion solution was placed in a 5 mL glass bottle, 3 mg of PLGA and different amounts of estradiol (0, 0.03, 0.06, 0.15, 0.3, 0.6, 1.2 mg) were dissolved in 1 mL of acetonitrile, and the PLGA acetonitrile solution was quickly injected into the bottom of the centrifuge tube with a pipette.

[0114] 2) Continue vortexing for 30 s to obtain the liposome-polymer nanoparticle-estradiol dispersion (E2@LNP-AEAA / GalNAc).

[0115] 3) Ultrafiltration membrane filtration, HPLC detection of estradiol in the filtrate.

[0116] 4) Drug loading efficiency (DLE) and encapsulation efficiency (EE) of liposome-nanoparticles were calculated according to the following formula respectively

[0117]

[0118] From Figure 2 It can be seen from b that the estradiol drug loading rate EE of the nanoparticles can reach 70%-80%, and the drug loading rate is high, which is conducive to long-term drug release.

[0119] Next, the targeting effect of estradiol-loaded liposome-nanocarriers in vivo and in vitro was detected (results are shown in Figure 3 )

[0120] (1) In vitro uptake of liposome-nanoparticles by hepatocytes HepG2, macrophages THP1 and stellate cells LX2

[0121] 1) In vitro targeted uptake of hepatocytes HepG2

[0122] ① 12-well plates were pre-placed with cell slides.

[0123] ② 2.0 x 10^5 hepatocytes were plated in 12-well plates and incubated at 37°C in a 5% CO2 incubator for 12 hours.

[0124] ③ Equal volume of normal saline and liposome-nanoparticles were added, respectively, and the groups were divided into normal group (NC), non-targeting peptide liposome-nanoparticle group (E2@LNP) and targeting peptide liposome-nanoparticle group (E2@LNP-AEAA / GalNAc) (3 wells were repeated for each condition), and incubated at 37°C in a 5% CO2 incubator for 12 hours.

[0125] ④ Each well was carefully washed with PBS for 6 times, fixed with 4% paraformaldehyde for 5 minutes, then carefully washed with PBS for 2 times, stained with DAPI for 5 minutes, and then mounted, and laser confocal imaging was performed.

[0126] 2) In vitro targeted uptake of macrophages THP1

[0127] ① 12-well plates were pre-placed with cell slides.

[0128] ② 4.0 x 10^5 hepatocytes were plated in 12-well plates and incubated at 37°C in a 5% CO2 incubator for 12 hours.

[0129] ③ 12 hours later, 100 ng / mL PMA was added to each well to polarize THP1 cells, and the cells were incubated in a 37 °C 5% CO2 incubator for 24 hours.

[0130] ④ Equal volumes of normal saline and liposome-nanoparticles were added, respectively, and the groups were divided into normal group (NC), non-targeting peptide liposome-nanoparticle group (E2@LNP), and targeting peptide liposome-nanoparticle group (E2@LNP-AEAA / GalNAc) (3 wells were repeated for each condition), and the cells were incubated in a 37 °C 5% CO2 incubator for 4 hours.

[0131] ⑤ Each well was carefully washed 6 times with PBS, fixed with 4% paraformaldehyde for 5 minutes, carefully washed 2 times with PBS, stained with DAPI for 5 minutes, and then mounted for laser confocal imaging.

[0132] 3) In vitro targeted uptake of hepatic stellate cells LX2 (from the Chinese Academy of Sciences Cell Bank)

[0133] ① Cell slides were pre-placed in 12-well plates.

[0134] ② 2.0 x 10^5 hepatocytes were plated in 12-well plates and incubated in a 37 °C 5% CO2 incubator for 12 hours.

[0135] ③ 12 hours later, 10 ng / mL TGF-β was added to each well to activate LX2 cells (blank control group without TGF-β), and the cells were incubated in a 37 °C 5% CO2 incubator for 24 hours.

[0136] ④ Equal volumes of normal saline and liposome-nanoparticles were added, respectively, and the groups were divided into normal group (NC), non-targeting peptide liposome-nanoparticle group (E2@LNP), and targeting peptide liposome-nanoparticle group (E2@LNP-AEAA / GalNAc) (3 wells were repeated for each condition), and the cells were incubated in a 37 °C 5% CO2 incubator for 12 hours.

[0137] ⑤ Each well was carefully washed 6 times with PBS, fixed with 4% paraformaldehyde for 5 minutes, carefully washed 2 times with PBS, stained with DAPI for 5 minutes, and then mounted for laser confocal imaging.

[0138] 4) In vivo uptake of liposome-nanoparticles in different tissues of male and female mice

[0139] Injection dose of nanoparticles: 25 mg / kg (based on the mass of PLGA)

[0140] 8-week-old male and female mice, 8 in each, were depilated on the abdomen, and injected with targeted peptide liposome-nanoparticles or the same volume of normal saline through the tail vein. The groups were as follows: male group 1 (STC, 2 mice), male group 2 (CDAHF60+E2@LNP, 3 mice), male group 3 (CDAHF60+E2@LNP-AEAA / GalNAc, 3 mice); female group 1 (STC, 2 mice), female group 2 (CDAHF60+E2@LNP, 3 mice), female group 3 (M-CDAHF60+E2@LNP-AEAA / GalNAc, 3 mice). After 24 hours, the male mice were taken out of the heart, liver, spleen, lung, kidney and testis, and the female mice were taken out of the heart, liver, spleen, lung, kidney, subcutaneous fat and ovary, and fluorescence imaging was performed.

[0141] By Figure 3 a The uptake rate of the E2@LNP-AEAA / GalNAc group loaded with targeted peptide nanoparticles was significantly higher than that of the E2@LNP group without targeted peptide in HepG2, macrophage THP1 and stellate cell LX2;

[0142] By Figure 3 b It can be seen that the E2@LNP-AEAA / GalNAc group loaded with targeted peptide nanoparticles was mainly taken up in the liver, and the uptake rate was significantly higher than that of the E2@LNP group without targeted peptide.

[0143] Next, the prepared targeted peptide liposome-nanomedicine E2@LNP-AEAA / GalNAc was used for animal experiments on metabolic-related fatty liver disease.

[0144] The specific operation is as follows:

[0145] (1) A total of 15 6-week-old C57BL / 6J male mice were divided into 3 groups, namely normal diet (Stand chow, STC) 5 mice, L-amino acid-deficient high-fat diet (Choline-deficient and low methionine high-fat, CDAHFD) model group 5 mice and drug administration group E2@LNP-AEAA / GalNAc 5 mice, and the total diet lasted for 12 weeks. When the model group and the drug administration group were given 4-week CDAHF60 diet, the drug administration group started to inject nanomedicine (0.25 mg / kg) through the tail vein, 2 times a week, for 8 weeks, and the model group was injected with normal saline (saline) through the tail vein, and each group was tested respectively.

[0146] (2) 15 C57BL / 6J female mice aged 6 weeks were divided into 3 groups, 5 in each group, namely normal diet (Stand chow, STC) group, L-amino acid deficient high-fat diet (Choline-deficient and low methionine high-fat, CDAHFD) model group and drug administration group E2@LNP-AEAA / GalNAc, and the total diet duration was 12 weeks. When the model group and the drug administration group were given 4 weeks of CDAHFD60 diet, the drug administration group started to inject nanomedicines (0.25 mg / kg) via tail vein injection twice a week for 8 weeks, and the model group was injected with saline via tail vein injection, and each group was subjected to experiments respectively.

[0147] At 12 weeks, the blood, heart, liver, spleen, lung and kidney samples of male and female mice were collected respectively. The liver injury related indicators ALT and AST in mouse serum were detected, and the degree of liver fat accumulation, inflammation and fibrosis was judged by histopathological H&E, F4 / 80 and Sirius red staining, and the results are shown in Figures 4 to 6 .

[0148] ①Detection of ALT and AST in mouse serum

[0149] Take out the prepared mouse serum, and fresh hemolysis-free blood samples (venous blood) are needed. Take 2 uL of serum in a 96-well plate, add 100 uL of reaction reagent (configured according to the instructions of the kit), and immediately read under the enzyme label meter at wavelength 340 nm and temperature 37℃ for 8 min, once a minute. Calculate the absorbance change rate of each minute △A sample / min, OD = AVE (absorbance change rate of each minute △A sample / min).

[0150] ②Staining of histopathological sections

[0151] H&E staining

[0152] 1) Reagent preparation: prepare 3 250 mL glass bottles with xylene, labeled xylene 1, 2, 3; two 250 mL bottles of 100% ethanol, labeled 100% ethanol 1, 2; 90% ethanol, 80% ethanol, 70% ethanol each.

[0153] 2) Place the liver sections in the slide rack, and place the slide rack in the xylene No. 1 bottle for 15 min, and then place it in the xylene No. 2 and No. 3 bottles in turn, so that the paraffin is completely soaked and dissolved.

[0154] 3) Place the slide rack in the No. 1 bottle containing 100% ethanol, and after 5 min, place the slide rack in the No. 2 bottle containing 100% ethanol, and take out the slide rack.

[0155] 4) Put the slide rack into 90%, 80% and 70% ethanol for 2 min.

[0156] 5) Rinse with ddH2O for 3 times, 2 min each time.

[0157] 6) Gently drain the water drops on the paper towel, put the slide rack into the staining jar with hematoxylin and stain for 15 min, then gently rinse the slide with running water for 15 min.

[0158] 7) After the running water rinse is completed, put the slide rack into ddH2O and rinse for 2 min each time, 3 times.

[0159] 8) Put the slide rack into the staining jar with eosin and stain for 1 min.

[0160] 9) Put the slide rack into 100%-1 ethanol, 100%-2 ethanol for 5 min, then into xylene-1 for 30 min, xylene-2 for 1 h, mount, and dry overnight.

[0161] ③ Immunohistochemical staining

[0162] 1) Rehydrate the tissue section, and refer to the steps 1)-5) of H&E staining in 3.23.2 Staining of histopathological section.

[0163] 2) Antigen repair: Put the slide rack into a high-temperature-resistant plastic cup, pour the antigen repair solution (TE) into the plastic cup, which should cover the section, put it into the microwave oven and heat to boiling, then continue to heat for 10 min at medium heat.

[0164] 3) After natural cooling to room temperature, wash with PBS for 2 times, 5 min each time.

[0165] 4) Circle the tissue with the groupware, and add 3% H2O2 and incubate for 30 min.

[0166] 5) Wash with PBS for 2 times, 5 min each time, and block with 10% BSA for 1 h.

[0167] 6) Wash with PBST for 2 times, 5 min each time, add 2% BSA containing the primary antibody, and incubate overnight at 4°C.

[0168] 7) Wash with PBST for 3 times, 5 min each time, add 2% BSA containing the secondary antibody, and incubate at room temperature for 1 h.

[0169] 8) Wash with PBST for 3 times, 5 min each time, and develop with DBA for 2 min.

[0170] 9) Wash with PBST for 3 times, 5 min each time, stain with hematoxylin for 2 min, and rinse with running water for 10 min.

[0171] 10) Wash three times with ddH2O for 2 minutes each time, then immerse in 70%, 80%, 90%, and 100% ethanol for 30 minutes each time, followed by immersion in xylene for 1 hour. After sealing, blow dry.

[0172] ④Sirius Red staining

[0173] 1) Rehydrate the tissue sections, referring to steps 1)-5) of H&E staining in 3.23.2 Histopathological section staining.

[0174] 2) Stain with Sirius red staining solution for 30 minutes.

[0175] 3) Place the slide in 70%, 80%, 90%, and 100% ethanol for 30 minutes in sequence, then place it in xylene for 1 hour, seal it, and blow it dry.

[0176] 5) Detection of estradiol toxicity in liposome-nanocarriers

[0177] ① CCK8 assay for the toxicity of liposome-nanocarrier estradiol to HepG2 hepatocytes, THP1 macrophages, and LX2 stellate cells.

[0178] 1) 5.0 x 10^3 hepatocytes (HepG2), 8.0 x 10^3 macrophages (THP1), and 5.0 x 10^3 stellate cells (LX2) respectively

[0179] The plates were laid in 96-well plates and incubated in a 37°C, 5% CO2 incubator for 12 hours.

[0180] 2) On the second day, three types of cells were treated with different concentrations of P1+P2-PLGA@E2, with nanoparticle concentrations of 0, 50, 100, 200, 400, and 1000 μg / mL, respectively.

[0181] 3) The cytotoxicity of liposome-nanoparticles was detected by the CCK8 kit after 24 hours.

[0182] ② H&E staining was used to detect the toxicity of E2@LNP-AEAA / GalNAc to the heart, spleen, lungs and kidneys of mice.

[0183] The heart, spleen, lungs, and kidneys of mice were stained with H&E. The staining steps are the same as those for H&E staining in the staining of histopathological sections above.

[0184] For male mice, by Figure 4 As can be seen, the E2@LNP-AEAA / GalNAc group significantly reduced serum ALT compared to the disease model CDAHF60 group; Figure 4 b. As can be seen, the E2@LNP-AEAA / GalNAc group significantly reduced serum AST compared to the CDAHF60 group; Figure 4c. The anatomical pathology showed that the E2@LNP-AEAA / GalNAc group could significantly reduce liver fat accumulation, liver inflammation and fibrosis compared with the CDAHF60 group.

[0185] For female mice, by Figure 5 a. It can be seen that the E2@LNP-AEAA / GalNAc group can significantly reduce serum ALT compared with the disease model CDAHF60 group, by Figure 5 b. It can be seen that the E2@LNP-AEAA / GalNAc group can significantly reduce serum AST compared with the CDAHF60 group, by Figure 5 c. The anatomical pathology showed that the E2@LNP-AEAA / GalNAc group could significantly reduce liver fat accumulation, liver inflammation and fibrosis compared with the CDAHF60 group.

[0186] by Figure 6 a. It can be seen that different concentrations of E2@LNP-AEAA / GalNAc have no significant effect on the viability of HepG2, macrophages THP1 and stellate cells LX2, indicating that E2@LNP-AEAA / GalNAc has no in vitro toxic side effects;

[0187] by Figure 6 b. It can be seen that the pathological changes of heart, spleen, lung and kidney of the healthy control group STC, the disease model group CDAHF60 and the treatment group E2@LNP-AEAA / GalNAc have no significant difference, indicating that E2@LNP-AEAA / GalNAc has no toxic side effects on mice.

[0188] 6) The protective effect of free estradiol on the liver, the results are shown in Figure 7 .

[0189] 6-week-old C57BL / 6J male mice, a total of 10, were divided into 2 groups, 5 in the L-amino acid deficient high-fat diet (Choline-deficient and low methionine high-fat, CDAHFD) model group and 5 in the drug administration group E2. The total diet duration is 12 weeks. When the model group and the drug administration group are given 4 weeks of CDAHF60 diet, the drug administration group starts to inject estradiol drug (0.4 mg / kg) every 2 days for 8 weeks, and the model group injects saline, and each group is tested respectively.

[0190] At 12 weeks, the blood and liver samples of the mice were collected. The liver damage related indicators ALT and AST in the serum of the mice were detected, and the liver fat accumulation, inflammation and fibrosis degree were judged by histopathology H&E, F4 / 80 and Sirius red staining, and the results are shown in Figure 7 .

[0191] byFigure 7 and Figure 4 Comparing with the free estradiol, the ALT level, AST level and other indicators of liver disease of the modified estradiol-loaded nanoparticles with the targeting peptide are all lower, which indicates that the nanoparticles of the present application have better improvement effect on liver injury.

[0192] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. A nanomedicine for the treatment and / or adjunctive treatment of metabolic-related fatty liver disease, characterized in that, It includes a liver-targeted drug delivery system and estradiol grafted onto a polymer; The targeted drug delivery system for the liver includes a drug delivery carrier and a targeting peptide; The targeting peptides include GalNAc, a targeting peptide that specifically targets hepatocytes and Kupffer cells, and AEAA, a targeting peptide that specifically targets hepatic stellate cells. The drug delivery carrier comprises a polymer and a lipid layer encapsulating the polymer. The targeting peptides GalNAc and AEAA are modified on the lipid layer; The polymer is a polylactic acid-glycolic acid copolymer; The lipid layer was formed using DSP-PEG(2000)-NH2 as a raw material; The molecular formula of the targeting peptide GalNAc is C2 56 H 85 N 15 O 31， Its molecular structure is shown in equation (1): ； The molecular formula of the targeting peptide AEAA is C 21 H 29 N5O 10 Its molecular structure is shown in equation (2): ； Its preparation includes the following steps: S1: Preparation of lipid films modified with targeting peptides GalNAc and AEAA; S2: Add the lipid film from step S1 to the solvent to prepare a lipid dispersion; S3: Estradiol is modified onto the polymer; S4: Add the lipid dispersion from step S2 to the polymer solution to encapsulate the polymer particles with a lipid layer, thereby obtaining a nano-drug dispersion. Step S1 specifically includes the following steps: S1.1 Dissolve the target peptide GalNAc, the target peptide AEAA, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in a solvent; S1.2 Dissolve DSPE-PEG(2000)-NH2 and pyridine in a solvent; S1.3 Mix the solutions from steps S1.1 and S1.2, stir, and freeze-dry to obtain a solid sample; S1.4 The solid sample from step S1.3 is dissolved in a solvent and then evaporated to dryness to prepare a lipid film. In steps S1.1 and S1.2, the molar ratio of the targeting peptide GalNAc, the targeting peptide AEAA, and DSPE-PEG(2000)-NH2 is 1:1:

2.

2. The nanomedicine for treating and / or adjuvant treatment of metabolic-related fatty liver disease according to claim 1, characterized in that, The loading rate of estradiol is 55%-80%.

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