Drug targeting delivery system for targeting liver and drug thereof for treating and / or assisting in treating metabolism-related fatty liver diseases

Abstract

The invention discloses a liver-targeting drug targeting delivery system and a drug for treating and / or assisting in treating metabolism-related fatty liver diseases. Estradiol is targeted to the liver through the drug delivery system modified with polypeptides specifically targeting different liver cells; the treatment effect of estradiol on metabolism-related fatty liver diseases is enhanced, and high-concentration distribution of estradiol in non-liver tissues is avoided, so that side effects are reduced. Mouse experiments prove that the prepared nano-drug can be used for effectively treating MASLD, the treatment effect is remarkable, and no side effect exists, so that a new direction and means can be provided for treatment of MASLD.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-drug, and particularly relates to a drug targeting delivery system targeting the liver and a drug for treating and / or assisting in the treatment of metabolic associated fatty liver disease. Background Art

[0002] Metabolic dysfuction-associated steatotic liver disease (MASLD), as a metabolic disease closely related to obesity, has seen a sharp increase in prevalence globally. MASLD is a disease spectrum that encompasses simple steatosis and metabolic dysfunction-associated steatohepatitis (MASH). Simple steatosis is manifested as a large amount of triglyceride deposition in hepatocytes; MASH is accompanied by increased liver inflammation, hepatocyte injury and apoptosis on this basis. The pathogenesis of MASLD is multi-factorial. It is generally believed that environmental factors (lifestyle, dietary choices, etc.) can lead to obesity and insulin resistance, resulting in increased de novo lipid synthesis and fatty acid uptake in hepatocytes, reduced fatty acid oxidation and very low density lipoprotein output, causing liver lipid accumulation; lipotoxicity leads to mitochondrial dysfunction in hepatocytes, generating reactive oxygen species and endoplasmic reticulum stress, resulting in hepatocyte injury and even apoptosis; hepatocyte injury and apoptosis then trigger Kupffer cell activation, immune cell infiltration, as well as hepatic stellate cell activation and fibrosis. At the same time, insulin resistance also leads to adipose tissue dysfunction and exacerbates systemic inflammation by increasing pro-inflammatory cytokines, adipokines, etc. Due to the complex pathogenesis of MASLD and its tendency to progress to cirrhosis and liver cancer, effective intervention measures are urgently needed. However, to date, the treatment methods for MASLD are very limited.

[0003] In recent years, the number of formulations based on nanomedicine has increased significantly. Their advantages include targeted delivery to improve efficacy and reduced toxicity, making them ideal candidates for treating complex diseases. Lipid-polymer nanoparticles (LNPs) have excellent serum stability due to the strong hydrophobic interaction between the polymer and lipid; cell-specific targeted delivery can be achieved by adding targeting peptides / small molecules to the lipid ends coated on the surface of the core polymer. Estradiol (E2) is the main type of estrogen, mainly secreted by the ovaries, and its main physiological functions are to promote the development of female reproductive organs and 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 steadily increases after menopause. And excessive estrogen levels have serious side effects on both women and men. For example, in men, it can cause breast development, sexual dysfunction, and reduced reproductive function, while in women, it can increase the risk of breast cancer and ovarian cancer, cause endometrial lesions and mood changes, etc. However, there is no research on using nanocarriers to load estrogen for the treatment of MASLD in current studies. Summary of the Invention

[0004] Therefore, based on the above background, the present invention provides a drug targeted delivery system targeting the liver and a drug for treating and / or assisting in the treatment of metabolic associated fatty liver disease, which realizes the targeting of estradiol to the liver through a drug delivery system modified with polypeptides that specifically target different hepatocytes, enhances the therapeutic 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.

[0005] One of the technical solutions of the present invention:

[0006] A drug targeted delivery system targeting the liver, comprising a drug delivery carrier and a targeting peptide, wherein the targeting peptide comprises 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 poly(lactic-co-glycolic acid) copolymer (50:50, MW 38000 - 54000).

[0010] Further, the liposome is formed from DSP-PEG(2000)-NH2.

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

[0012]

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

[0014]

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

[0016] Use of the above-mentioned drug targeting delivery system targeting the liver in the preparation of drugs for the treatment and / or adjuvant treatment of metabolic associated fatty liver disease, and the drug is an injection.

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

[0018] A nano-drug for the treatment and / or adjuvant treatment of metabolic associated fatty liver disease, which comprises the above-mentioned drug targeting delivery system targeting the liver and estradiol, and the estradiol is grafted onto a polymer.

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

[0020] Further, it comprises the following steps:

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

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

[0023] S3: Modify estradiol onto a polymer;

[0024] S4: Add the lipid dispersion of step S2 into a polymer solution, and 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 Take the targeting peptide AEAA, the targeting peptide GalNAc, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and add them into a solvent for dissolution;

[0027] S1.2 Dissolve DSPE-PEG(2000)-NH2 and pyridine in a solvent.

[0028] S1.3 Mix the solutions from steps S1.1 and S1.2, stir, and then freeze-dry to obtain a solid sample.

[0029] S1.4 Dissolve the solid sample from step S1.3 in a solvent and then evaporate to dryness to prepare a lipid film.

[0030] Furthermore, in steps S1.1 and S1.2, the molar ratio of the targeting peptide AEAA, the targeting peptide GalNAc, and

[0031] DSPE-PEG(2000)-NH2 is 1:1:2.

[0032] The effective effects achieved by the present invention are as follows:

[0033] The present invention realizes the targeting of estradiol to the liver through a drug delivery system modified with polypeptides specifically targeting different hepatocytes, enhances the therapeutic effect of estradiol on metabolic-associated steatohepatitis, and avoids the high-concentration distribution of estradiol in non-liver tissues to reduce side effects.

[0034] The present invention utilizes the strong hydrophobic interaction between a polymer (polylactic-co-glycolic acid) and a lipid to endow the nano-drug with excellent serum stability, avoiding the burst release of the medicinal ingredient estradiol in the polymer nanoparticles. Moreover, the nano-drug of the present invention has a high drug loading rate of the medicinal ingredient estradiol, enabling long-term sustained release and its effective drug components to improve the therapeutic effect.

[0035] And through mouse experiments, it is verified that the nano-drug prepared by the present invention can effectively treat MASLD, with a significant therapeutic effect and no side effects, thus providing a new direction and means for the treatment of MASLD.

[0036] Description of the Drawings

[0037] Appendix Figure 1 For the preparation and characterization of a liver-targeted nano-drug carrier:

[0038] Figure 1 a is a schematic diagram of the synthesis of targeting peptides for hepatocytes, Kupffer cells, and hepatic stellate cells;

[0039] Figure 1 b is a schematic diagram of the binding of targeting peptides for hepatocytes, Kupffer cells, and stellate cells to liposomes;

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

[0041] Appendix Figure 2 For the particle size and drug loading of the targeted nanocarrier loaded with drugs:

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

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

[0044] Appendix Figure 3 For the results of the specific targeting of each cell in the liver by the nanocarrier through its targeting peptide in vitro and in vivo:

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

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

[0047] Appendix Figure 4 For the therapeutic effect of the nanocarrier loaded with estradiol in male mice with metabolic associated fatty liver disease:

[0048] Figure 4 a shows the effect of the nanocarrier loaded with estradiol on serum ALT in male mice;

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

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

[0051] Appendix Figure 5 For the therapeutic effect of the nanocarrier loaded with estradiol in female mice with metabolic associated fatty liver disease:

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

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

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

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

[0056] ;

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

[0058] Appendix Figure 7 Protective effect of free estradiol on metabolic associated fatty liver disease:

[0059] Figure 7 a shows the effect of free diol on serum ALT in male mice;

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

[0061] Figure 7 c shows the effect of free estradiol on liver pathology in male mice. Detailed implementation mode

[0062] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with its embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0063] Example 1: The preparation of a nano-drug for treating and / or assisting in the treatment of metabolic associated fatty liver disease includes the following steps (as Figure 1 shown):

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

[0065] Targeting peptide GalNAc:

[0066] 1) Soak 0.2 mmol (153 mg) of resin (Fmoc-amino acid - Wang resin, purchased from Gil Biotech, product number is 48101) in 4 mL of anhydrous dichloromethane (DCM) (CAS: 27639) for swelling, and press dry after activation for 10 min;

[0067] 2) Reaction of resin (Fmoc-amino acid - Wang resin, purchased from Gil Biotech, product number is 48101) and amino acid:

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

[0069] 3) Block the resin that has not reacted completely:

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

[0071] 4) Remove Fmoc:

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

[0073] 5) React two amino acids:

[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, mix well by bubbling with nitrogen, and react for 2 h.

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

[0076] 7) Connect succinic anhydride (SA) (CAS: 108-30-5): After removing Fmoc, rinse 5 times with DMF, add 0.6 mmol of SA, dissolve with 4 mL of DMF, mix well by bubbling with nitrogen, after reacting for 4 h, wash 4 times with DMF and DCM respectively.

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

[0078] 9) 4-Dimethylaminopyridine (DMAP) (CAS: 1122-58-3), DIC, DIPEA, and DMF were used as catalysts to introduce 5-hexynoic acid (HA) (CAS: 53293-00-8): 1.2 mmol of DIC, 1.2 mmol of HA, and 1.2 mmol of DMAP were dissolved in DMF and added to a vial and stirred. Then, 1.2 mmol of DIPEA was added, and it was magnetically stirred and reacted for 24 h (rotation speed < 500 rpm). Then, it was transferred back into the synthesis column and washed 4 times with DMF and DCM respectively.

[0079] 10) 0.6 mmol of N3-galactose (CAS: 869186-83-4) and 0.6 mmol of copper(I) iodide (CAS: 7681-65-4) were dissolved in 100 μL of DMF, 18 mmol of DIPEA was added, and it was sealed and stirred at room temperature for 16 h. The resin was washed with saturated sodium ascorbate aqueous solution, water, methanol, and DMF, and washed three times with DCM.

[0080] 11) 95% Trifluoroacetic acid (TFA) (CAS: 27881) (2.5% water and 2.5% triisopropylsilane) was prepared. 1 mL of the mixed solution was taken in a reactor, bubbled with nitrogen and mixed evenly for 1 min, the cut amino acids were pressed dry and collected. Then, 1 mL of the mixed solution was taken in the reactor again, bubbled with nitrogen and mixed evenly for 3 min, the cut amino acids were pressed dry and collected. Finally, the remaining mixed solution was taken in the reactor, bubbled with nitrogen and mixed evenly for 3 min, pressed dry, and the cut amino acids were collected and reacted on a magnetic stirrer for 2 h.

[0081] 12) The collected polypeptide was rotary evaporated to a nearly dry suspension state. 10 - 20 mL of ice-cold diethyl ether was added, and a white precipitate appeared. It was centrifuged at 5000 rpm for 5 minutes. A small amount of the precipitate was dissolved in methanol for MS. The synthesized polypeptide is a targeting peptide targeting Kupffer cells and hepatocytes in the liver, named GalNAc, and its molecular structural formula is shown in the following formula (1).

[0082]

[0083] Targeting peptide GalNAc:

[0084] 1) Swell 0.2 mmol of resin (Fmoc - amino acid - Wang resin, purchased from Gil Biotech, catalog number 48101) with 4 mL of anhydrous DCM, activate for 10 min and then press dry.

[0085] 2) Reaction of resin and amino acid:

[0086] Place 0.3 mmol of glycine in a glass bottle, add 4 mL of solvent DCM and 104 μL of DIPEA (2 times the amount of amino acid, 0.6 mmol), mix well by bubbling with nitrogen, and react for 60 min.

[0087] 3) Block the resin with unreacted residues:

[0088] React with 2 mL of methanol + 2 mL of DCM (1:1) + 200 μL of DIPEA for 15 min, then rinse with DMF 5 times, mix well by bubbling with nitrogen for 1 minute each time.

[0089] 4) Deprotect Fmoc: Prepare 20% piperidine, add it to the reactor, mix well by bubbling with nitrogen, and treat for 20 min. Rinse with DMF 5 times and mix well by bubbling with nitrogen for 1 minute each time.

[0090] 5) Reaction of two amino acids:

[0091] Take 0.3 mmol of serine (Serine, S), 0.3 mmol of HBTU, 0.3 mmol of HOBT, and 0.3 mmol of DIPEA in a glass bottle, dissolve with 4 mL of DMF, mix well by bubbling with nitrogen, and react for 2 h.

[0092] 6) Repeat steps 4 - 5 to sequentially link different amino acids (GSGS - β - alanine) according to the sequence.

[0093] 7) When linking to the last amino acid, methoxybenzoic acid (Methoxybenzoic acid, MA) (CAS 100 - 09 - 4), use the following coupling agent connection method: Dissolve 0.6 mmol of MA, 0.6 mmol of DIC, 0.6 mmol of DIPEA, and 0.06 mmol of DMAP separately with DMF, add them to the reactor, react for 4 h, and wash with DMF and DCM 4 times each.

[0094] 8) Prepare 50 mL of 1% TFA + 2.5% TPS with DCM as the solvent, add 4 mL of the mixed solution at a time, mix well by bubbling with nitrogen for 1 min, and press the liquid into a rotary evaporation flask, repeat 10 times.

[0095] 9) Rotate evaporate the collected polypeptide until it is nearly dry. Add 10 - 20 mL of ice-cold diethyl ether, and a white precipitate will appear. Centrifuge at 5000 rpm for 5 minutes, take a small amount of the precipitate and dissolve it in methanol for MS. The synthesized polypeptide, which is the targeting peptide for hepatic stellate cells, is named AEAA, and its molecular structural formula is shown in the following formula (2):

[0096]

[0097] S2: Synthesis of nanoparticles

[0098] 1) Take P1, P2, NHS (N-hydroxysuccinimide), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) according to the corresponding ratios shown in Table 1 (the ratios are shown in the following figure) in a 1.5 mL sample vial, add 200 μL of DMF to dissolve, add a magnetic stirring rotor to the sample vial, and stir on a magnetic stirrer for 1 h.

[0099] Table 1: Material dosage ratio

[0100]

[0101] 2) Take DSPE-PEG(2000)-NH2 in a sample vial, add 82.5 μL of pyridine and 100 μL of DMF to dissolve, and stir magnetically for 12 h.

[0102] 3) Clamp one end of the dialysis bag, and transfer the sample in step 2) into the dialysis bag using a yellow pipette tip. Add the reactants in the sample vial into the dialysis bag, add 10 times the volume of distilled water, and clamp the other end of the dialysis bag.

[0103] 4) Place the dialysis bag in a beaker filled with pure water and stir magnetically for 3 days. Lyophilize the sample to obtain DSP-PEG(2000)-(GalNAc / AEAA).

[0104] 5) Take 15 mg of the lipid component DSP-PEG(2000)-(GalNAc / AEAA) in a 5 mL eggplant-shaped flask, add 200 μL of methanol to dissolve, and evaporate to dryness on a rotary evaporator to obtain a lipid film.

[0105] 6) Then add 2 mL of water and Tris-HCl (20 mM, pH 7.4), and hydrate by ultrasound for 10 min to obtain a lipid dispersion.

[0106] 7) Place the above liposome dispersion in a 5 mL glass bottle. Dissolve 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) in 1 mL of acetonitrile (2% of the mass of PLGA of DID dye can be added for subsequent imaging observation), and quickly inject the PLGA acetonitrile solution to the bottom of the centrifuge tube with a pipette.

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

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

[0109] Next, detect the hydrated particle size and zeta potential of nanoparticles E2@LNP - AEAA / GalNAc and E2@LNP by a Malvern nanoparticle sizer. 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 prepared nanoparticles is between 40 - 50 nm.

[0110] The fenestrae of liver sinusoidal endothelial cells limit the accessibility of functional cells in the liver (such as hepatocytes and hepatic stellate cells). Therefore, controlling the LNP size below 100 nm is crucial for targeting these cells. The particle size of the nano - drug prepared in the above example is 40 - 50 nm, fully meeting the requirements.

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

[0112] The specific operation is as follows:

[0113] 1) Place the liposome dispersion in a 5 mL glass bottle. Dissolve 3 mg of PLGA and different masses of estradiol (0, 0.03, 0.06, 0.15, 0.3, 0.6, 1.2 mg) in 1 mL of acetonitrile, and quickly inject the PLGA acetonitrile solution to the bottom of the centrifuge tube with a pipette.

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

[0115] 3) Ultrafiltration membrane filtration, and estradiol in the filtrate was detected by HPLC.

[0116] 4) Calculate the drug loading efficiency (DLE) and encapsulation efficiency (EE) of liposome-nanoparticles according to the following formulas respectively

[0117]

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

[0119] Next, the in vitro and in vivo targeting effects of the estradiol-loaded liposome-nanocarrier were detected (the 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 targeting uptake of hepatocytes HepG2

[0122] ①Cell slides were pre-placed in 12-well plates.

[0123] ②2.0x 10^5 hepatocytes were plated in 12-well plates and cultured adherently in a 37°C 5% CO2 incubator for 12 hours.

[0124] ③Equal 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) (each condition was repeated in 3 wells), and cultured adherently in a 37°C 5% CO2 incubator for 12 hours.

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

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

[0127] ①Cell slides were pre-placed in 12-well plates.

[0128] ②4.0x 10^5 hepatocytes were plated in 12-well plates and cultured in a 37°C 5% CO2 incubator for 12 hours.

[0129] ③ After 12 hours, add 100 ng / mL of PMA to polarize THP1 cells in each well and culture them in a 37°C, 5% CO2 incubator for 24 hours.

[0130] ④ Add equal volumes of normal saline and liposome-nanoparticles respectively, and divide them into normal group (NC), liposome-nanoparticle group without targeting peptide (E2@LNP), and liposome-nanoparticle group with targeting peptide (E2@LNP-AEAA / GalNAc) (repeat 3 wells for each condition), and culture them adherently in a 37°C, 5% CO2 incubator for 4 hours.

[0131] ⑤ First, carefully wash each well 6 times with PBS, fix it with 4% paraformaldehyde for 5 minutes, then carefully wash it 2 times with PBS again, stain it with DAPI for 5 minutes, mount the slides, and take pictures with a laser confocal microscope.

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

[0133] ① Pre-place cell culture slides in a 12-well plate.

[0134] ② Seed 2.0x10^5 liver cells in a 12-well plate and culture them in a 37°C, 5% CO2 incubator for 12 hours.

[0135] ③ After 12 hours, add 10 ng / mL of TGF-β to activate LX2 cells in each well (do not add TGF-β to the blank control group), and culture them in a 37°C, 5% CO2 incubator for 24 hours.

[0136] ④ Add equal volumes of normal saline and liposome-nanoparticles respectively, and divide them into normal group (NC), liposome-nanoparticle group without targeting peptide (E2@LNP), and liposome-nanoparticle group with targeting peptide (E2@LNP-AEAA / GalNAc) (repeat 3 wells for each condition), and culture them adherently in a 37°C, 5% CO2 incubator for 12 hours.

[0137] ⑤ First, carefully wash each well 6 times with PBS, fix it with 4% paraformaldehyde for 5 minutes, then carefully wash it 2 times with PBS again, stain it with DAPI for 5 minutes, mount the slides, and take pictures with a laser confocal microscope.

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

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

[0140] Eight male and eight female mice at 8 weeks of age had their abdomens depilated and were injected with targeted peptide liposomes-nanoparticles or an equal volume of saline via the tail vein. The groups were: 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 hearts, livers, spleens, lungs, kidneys and testes were removed from male mice, and the hearts, livers, spleens, lungs, kidneys, subcutaneous fat and ovaries were removed from female mice for fluorescence imaging.

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

[0142] As Figure 3 b shows that the E2@LNP-AEAA / GalNAc group loaded with nanoparticles with targeted peptides was mainly taken up in the liver, and at the same time, the uptake rate was significantly higher than that of the E2@LNP group without targeted peptides.

[0143] Next, the prepared targeted peptide liposome-nanodrug E2@LNP-AEAA / GalNAc was used to conduct animal experiments on metabolic associated fatty liver disease.

[0144] The specific operation is as follows:

[0145] (1) A total of 15 six-week-old C57BL / 6J male mice were divided into 3 groups, namely 5 mice on a normal diet (Stand chow, STC), 5 mice in a choline-deficient and low methionine high-fat (CDAHFD) model group, and 5 mice in the administration group E2@LNP-AEAA / GalNAc. The total duration of the diet was 12 weeks. When the model group and the administration group were given the CDAHF60 diet for 4 weeks, the administration group started to inject the nanodrug (0.25 mg / kg) via the tail vein twice a week for 8 weeks, while the model group was injected with saline. Each group was experimented separately.

[0146] (2) A total of 15 six-week-old female C57BL / 6J mice were divided into 3 groups, namely 5 mice on a normal diet (Stand chow, STC), 5 mice in a choline-deficient and low methionine high-fat diet (CDAHFD) model group, and 5 mice in the administration group E2@LNP-AEAA / GalNAc. The total duration of the diet was 12 weeks. When the model group and the administration group were given the CDAHF60 diet for 4 weeks, the administration group started tail vein injection of the nanodrug (0.25 mg / kg) twice a week for 8 weeks, while the model group was injected with saline via the tail vein. Each group was subjected to the experiment separately.

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

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

[0149] Take out the pre-prepared mouse serum, which requires fresh non-hemolyzed blood samples (venous blood). Take 2 μL of serum into a 96-well plate, add 100 μL of the reaction reagent (prepared according to the instructions of the kit), and immediately read continuously for 8 minutes under an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 340 nm and a temperature of 37 °C, reading once per minute. Calculate the change rate of absorbance per minute ΔA sample / min, OD = AVE (change rate of absorbance per minute ΔA sample / min).

[0150] ② Staining of histopathological sections

[0151] H&E staining

[0152] 1) Reagent preparation: Prepare 3 covered 250 mL glass bottles filled with xylene, labeled as Xylene 1, 2, 3; two 250 mL bottles of 100% ethanol, labeled as 100% Ethanol 1, 2; one bottle each of 90% ethanol, 80% ethanol, and 70% ethanol.

[0153] 2) Place the liver sections in a section rack, place the section rack in the No. 1 bottle of xylene and let it stand for 15 minutes, and then place it in the No. 2 and No. 3 bottles of xylene in turn to allow the paraffin to be soaked and dissolved as much as possible.

[0154] 3) Place the section rack in the No. 1 bottle of 100% ethanol and let it stand. After 5 minutes, place the section rack in the No. 2 bottle of 100% ethanol and let it stand, and then take out the section rack.

[0155] 4) Place the section rack into 90%, 80% and 70% ethanol for 2 minutes successively.

[0156] 5) Rinse with ddH2O three times, 2 minutes each time.

[0157] 6) Gently drain the water droplets on the paper towel. After the section rack is placed in the hematoxylin staining jar for 15 minutes of staining, gently rinse the sections with running water for 15 minutes.

[0158] 7) After the rinsing with running water is completed, place the section rack into ddH2O for rinsing, 2 minutes each time, for 3 times.

[0159] 8) Place the section rack into the eosin staining jar for 1 minute of staining.

[0160] 9) Place it into 100%-1 ethanol and 100%-2 ethanol successively for 5 minutes, then into xylene-1 for 30 minutes and xylene-2 for 1 hour, cover the slides, and air dry overnight.

[0161] ③ Immunohistochemical staining

[0162] 1) Rehydrate the tissue sections. For rehydration, refer to steps 1)-5) of the H&E staining in 3.23.2 Staining of tissue pathological sections.

[0163] 2) Antigen retrieval: Place the section rack into a heat-resistant plastic cup, pour the antigen retrieval solution (TE) into the plastic cup. The antigen retrieval solution should cover the sections. Place it in the microwave oven and heat to boiling at high power, then switch to medium power and continue heating for 10 minutes.

[0164] 3) After cooling to room temperature naturally, wash with PBS twice, 5 minutes each time.

[0165] 4) Circle the tissue with a histochemical pen and add 3% H2O2 for incubation for 30 minutes.

[0166] 5) Wash with PBS twice, 5 minutes each time, and block with 10% BSA for 1 hour.

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

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

[0169] 8) Wash with PBST three times, 5 minutes each time, and develop color with DBA for 2 minutes.

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

[0171] 10) Wash with ddH2O three times, 2 minutes each time, and sequentially place in 70%, 80%, 90%, and 100% ethanol for 30 minutes, then place in xylene for 1 hour. After sealing the slides, air dry.

[0172] ④ Sirius red staining

[0173] 1) Rehydrate the tissue sections. For rehydration, refer to steps 1)-5) of the H&E staining in the staining of tissue pathological sections in 3.23.2.

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

[0175] 3) Sequentially place in 70%, 80%, 90%, and 100% ethanol for 30 minutes, then place in xylene for 1 hour. After sealing the slides, air dry.

[0176] 5) Detection of the toxicity of liposome-nanocarrier estradiol

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

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

[0179] separately in 96-well plates and incubate them in a 37°C, 5% CO2 incubator for 12 hours to allow cell attachment.

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

[0181] 3) After 24 hours, detect the cytotoxicity of the liposome-nanoparticles using a CCK8 kit.

[0182] ② Detect the toxicity of E2@LNP-AEAA / GalNAc to the heart, spleen, lung, and kidney of mice by H&E staining.

[0183] Perform H&E staining on the heart, spleen, lung, and kidney samples taken from mice. The staining steps are the same as those of the H&E staining in the staining of tissue pathological sections mentioned above.

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

[0185] For female mice, Figure 5 as shown in a, compared with the disease model CDAHF60 group, the E2@LNP-AEAA / GalNAc group could significantly reduce serum ALT. Figure 5 as shown in b, compared with the CDAHF60 group, the E2@LNP-AEAA / GalNAc group could significantly reduce serum AST. Figure 5 Anatomical pathology of c showed that compared with the CDAHF60 group, the E2@LNP-AEAA / GalNAc group could significantly reduce liver fat accumulation, liver inflammation and fibrosis.

[0186] As Figure 6 shown in a, different concentrations of E2@LNP-AEAA / GalNAc had no significant effect on the viability of HepG2, macrophages THP1 and stellate cells LX2, indicating that E2@LNP-AEAA / GalNAc had no in vitro toxic and side effects.

[0187] As Figure 6 shown in b, there were no significant differences in the pathology of the heart, spleen, lung and kidney between the healthy control group STC, the disease model group CDAHF60 and the treatment group E2@LNP-AEAA / GalNAc, indicating that E2@LNP-AEAA / GalNAc had no toxic and side effects on mice.

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

[0189] A total of 10 six-week-old C57BL / 6J male mice were divided into 2 groups, namely, a model group of 5 mice on a choline-deficient and low-methionine high-fat diet (CDAHFD) and a dosing group of 5 mice given E2. The total duration of the diet was 12 weeks. When the model group and the dosing group were given a CDAHF60 diet for 4 weeks, the dosing group began to inject estradiol (0.4 mg / kg) every 2 days for 8 weeks, while the model group was injected with saline. Each group was subjected to the experiment separately.

[0190] At 12 weeks, blood and liver samples of the mice were collected respectively. Liver injury-related indicators ALT and AST in the mouse serum were detected, and at the same time, histopathological H&E, F4 / 80 and Sirius red staining were used to judge the degree of liver fat accumulation, inflammation and fibrosis. The results are shown in Figure 7 .

[0191] AsFigure 7 and Figure 4 Comparing with it, it can be seen that although free estradiol can improve the levels of liver disease indicators such as ALT and AST, the ALT level, AST level, etc. of the estradiol-loaded nanoparticles modified with the targeting peptide of the present invention are all lower. This shows that the nanoparticles of the present invention have a better improvement effect on liver injury.

[0192] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A drug targeting delivery system targeting the liver, characterized in that, It includes a drug delivery carrier and a targeting peptide; The targeting peptide includes the targeting peptide GalNAc that specifically targets hepatocytes and Kupffer cells and the targeting peptide AEAA that specifically targets hepatic stellate cells; The drug delivery carrier includes a polymer and a lipid layer outside the encapsulated polymer; The polypeptide GalNAc and the polypeptide AEAA are modified on the lipid layer.

2. The drug targeting delivery system targeting the liver according to claim 1, wherein The polymer is a poly(lactic-co-glycolic acid) copolymer.

3. The drug targeting delivery system targeting the liver according to claim 1, characterized in that, The liposome is formed from DSP-PEG(2000)-NH2.

4. A drug targeting delivery system targeting the liver according to claim 1, characterized in that, The molecular general formula of the targeting peptide GalNAc is C 56 H 85 N 15 O 31， Its molecular structure is shown in the following formula (1): The molecular general formula of the targeting peptide AEAA is C 21 H 29 N5O 10 , and its molecular structure is shown in the following formula (2):

5. Use of a drug targeting delivery system targeting the liver according to claim 1 in the preparation of a drug for treating and / or assisting in the treatment of metabolic associated fatty liver disease, characterized in that, The drug is an injection.

6. A nano-drug for treating and / or adjuvant treating metabolic associated fatty liver disease, characterized in that, It includes the drug targeting delivery system for targeting the liver described in any one of claims 1 to 4 and estradiol, and the estradiol is grafted onto the polymer.

7. A nano-drug for treating and / or adjuvant treating metabolic associated fatty liver disease according to claim 6, characterized in that, The drug loading rate of the estradiol is 55%-80%.

8. A method for preparing a nanomedicine for treating and / or adjuvant treating metabolic associated fatty liver disease according to claim 6 or 7, characterized in that, It includes the following steps: S1: Prepare a lipid film modified with the targeting peptide GalNAc and the targeting peptide AEAA; S2: Add the lipid film in step S1 to a solvent to prepare a lipid dispersion; S3: Modify estradiol on the polymer; S4: Add the lipid dispersion in step S2 to the polymer solution, and encapsulate the polymer particles with the lipid layer to obtain a nano-drug dispersion.

9. The preparation method of a nano-drug for treating and / or assisting in treating metabolic associated fatty liver disease according to claim 8, characterized in that Step S1 specifically includes the following steps: S1.1 Take the targeting peptide GalNAc, the targeting peptide AEAA, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and add them to a solvent for dissolution; S1.2 Take DSPE-PEG(2000)-NH2 and pyridine and add them to a solvent for dissolution; S1.3 Mix the solutions in steps S1.1 and S1.2, stir, and freeze-dry to obtain a solid sample; S1.4 Add the solid sample in step S1.3 to a solvent for dissolution and then evaporate to dryness to prepare a lipid film.

10. The preparation method of a nano-drug for treating and / or assisting in treating metabolic associated fatty liver disease according to claim 9, characterized in that In steps S1.1 and S1.2, the molar amount ratio of the targeting peptide GalNAc, the targeting peptide AEAA, and DSPE-PEG(2000)-NH2 is 1:1:2.

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