Nanometer medicine for treating alcoholic liver injury

Abstract

The invention belongs to the technical field of biological medicines, and relates to a nano-drug for treating alcoholic liver injury. According to the present invention, the nano-drug ferr-1 / lincRNA-p21 (at) NP is constructed according to the influence of lincRNA-p21 on two programmed cell death approaches such as autophagy and ferroptosis during the alcoholic liver injury, the alcoholic liver injury is treated, the dual effects of damaged mitochondria and lipid accumulation reducing and cell oxidation level reducing are provided, and the alcoholic liver injury can be significantly improved. In the treatment of chronic liver injury, the nano-drug can be administered once every three days, and is convenient to use.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a nanomedicine for the treatment of alcoholic liver injury. Background Technology

[0002] Alcoholic liver injury (ALI) is a liver disease caused by long-term heavy drinking, including alcoholic hepatitis and cirrhosis. Approximately 8-20% of alcoholics will develop alcoholic cirrhosis, and about 2% of these patients will progress to hepatocellular carcinoma. It is the most common chronic liver disease globally and one of the leading causes of death from liver disease worldwide. The latest Global Alcohol and Health Report shows that approximately 3 million people die from alcohol abuse globally each year, accounting for 5.3% of all deaths, with economic losses exceeding $200 billion. Acute-on-chronic liver failure (ACLF) manifests as acute liver dysfunction on the basis of chronic liver disease or cirrhosis, characterized by organ failure and high short-term mortality. More than 25% of hospitalized cirrhosis patients have ACLF. Currently, there is no effective treatment for ACLF. Treatment options include treatment of precipitating events, organ support, and liver transplantation. The short-term mortality rate within 90 days for ACLF patients remains approximately 50%, with a poor prognosis. Given the persistently high prevalence and the urgent need to improve clinical treatment methods, further elucidating the regulatory mechanisms of alcoholic hepatocellular injury and finding new, safe, and effective targeted drugs is of paramount importance. Summary of the Invention

[0003] This invention proposes a novel nanomedicine for the treatment of alcoholic liver injury, addressing the problems existing in traditional treatments.

[0004] To achieve the above objectives, the present invention is implemented using the following technical solution:

[0005] This invention experimentally demonstrates that lincRNA-p21 plays a dual role in alcoholic liver injury: it protects cells by upregulating autophagy, but it also exacerbates Erastin-induced ferroptosis in alcoholic liver injury, which can be reversed by ferroptosis inhibitors. This invention proposes the development of a nanomedicine, ferr-1 / lincRNA-p21@NP, that co-delivers the ferroptosis inhibitor ferrostatin-1 (ferr-1) and a plasmid encoding lincRNA-p21, for the treatment of alcoholic liver injury.

[0006] The ferrostatin-1 inhibitor was delivered, with CAS number 347174-05-4.

[0007] The chemical formula is C 15 H 22 N2O2, structural formula as follows:

[0008]

[0009] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0010] 1. This invention constructs a nanomedicine, ferr-1 / lincRNA-p21@NP, based on the influence of lincRNA-p21 on two programmed cell death pathways, autophagy and ferroptosis, in alcoholic liver injury. It has the dual effects of enhancing autophagy to reduce damaged mitochondria and lipid accumulation and reducing cellular oxidation levels to inhibit ferroptosis, and can significantly improve alcoholic liver injury.

[0011] 2. In the treatment of chronic liver injury, nanomedicines can be administered once every three days, making them convenient to use. Attached Figure Description

[0012] Figure 1 This describes the synthesis process of the cationic polymer PBAE.

[0013] Figure 2 The results are from the transmission electron microscopy characterization of nanoparticles.

[0014] Figure 3 The results of experiments demonstrating the protective effect of ferr-1 / lincRNA-p21@NP on hepatocytes.

[0015] Figure 4 The results show the protective effect of ferr-1 / lincRNA-p21@NP on mouse liver. Detailed Implementation

[0016] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described below with reference to specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0017] Numerous specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways than those described herein, and therefore the invention is not limited to the specific embodiments disclosed in the following specification.

[0018] Example 1

[0019] Unless otherwise specified, all procedures in this embodiment are standard practices used in biological, cell, or animal experiments. Unless otherwise specified, the content of each substance in this embodiment is a mass percentage.

[0020] 1. Cell Culture and Passaging

[0021] HepG2 cells (purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai)) were routinely cultured in DMEM high-glucose complete medium (GIBCO, USA) containing 15% serum. The culture environment was a 37°C incubator containing 5% CO2. Subsequent experiments were performed after cell attachment. Cells were passaged after trypsin digestion, depending on the degree of cell confluence (above 80%) and experimental requirements.

[0022] 2. Alcoholic hepatocellular injury

[0023] Alcoholic hepatocellular injury was induced by sealing and continuous culture in 0.1M ethanol medium for 72 hours. 0.1M ethanol concentration is equivalent to the blood alcohol concentration in a person experiencing intoxication. Cells were cultured routinely or after transfection at 37°C under 5% CO2 conditions. Cells were cultured at a rate of 6 × 10⁶ cells / mL according to the required total cell count. 4 Inoculate the appropriate volume of cell suspension (cells / mL) into 3cm diameter culture dishes (or 96-well cell culture plates) and incubate until adherent. Add 0.1M ethanol medium to each well according to experimental requirements and incubate at 37°C. Change the medium daily for 72 hours.

[0024] 3. Assessment of cell survival, intracellular lipid oxidation, and reactive oxygen species (ROS) production.

[0025] In step 1, cells were obtained at a concentration of 6 × 10⁶. 4 Cells were seeded at a density of 100 cells / mL in 96-well plates. A blank control group was set up with culture medium only and no cells. After 12 hours, the cells adhered and the initial cell count was measured. The alcoholic liver injury assay was then performed using the treatment method from step 2, treating the cells with 0.1M alcohol culture medium for 3 days. CCK8 reagent (Elabscience, China) was diluted 1:10 in complete cell culture medium, and the medium was changed. The cells were then incubated for another 1.5 hours. The absorbance of each well was measured (450 nm) using a microplate reader. The survival rate of the normal control group was set as 100%.

[0026] Intracellular lipid oxidation and ROS production were assessed using the fluorescence intensity of C11-BODIPY 581 / 591 and 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), respectively. Cells were treated with alcoholic liver injury, then incubated with 50 μM C11-BODIPY 581 / 591 at 37°C for 1 h, or with 10 μM DCFH-DA for 30 min. The labeled cells were then extensively washed with PBS, digested with trypsin into single cells, and immediately analyzed by flow cytometry to assess cellular oxidation levels.

[0027] 4. Ferr-1 / lincRNA-p21@NP preparation

[0028] Autophagy and ferroptosis are two major programmed cell death pathways in alcoholic hepatocellular injury. Previous studies have found that lincRNA-p21 plays a dual role in alcoholic liver injury: it protects cells by upregulating autophagy but exacerbates ferroptosis. This invention develops a nanodrug, ferr-1 / lincRNA-p21@NP, that co-delivers the ferroptosis inhibitor ferrostatin-1 (ferr-1) and a plasmid encoding lincRNA-p21 for the treatment of alcoholic liver injury. The specific steps for preparing ferr-1 / lincRNA-p21@NP are as follows:

[0029] 4.1 Synthesis of cationic polymer PBAE:

[0030] 1.1 mol of 1,4-butanediol diacrylate (purchased from Bide Pharmaceuticals) and 1 mol of 4-amino-1-butanol (purchased from Bide Pharmaceuticals) were mixed and stirred at 90 °C for 24 hours to generate a polymer. The polymer was dissolved in 20 mL of tetrahydrofuran (THF, purchased from Energy Chemical). To form a piperazine-terminated polymer, 1-(3-aminopropyl)-4-methylpiperazine (purchased from Bide Pharmaceuticals) was dissolved at a concentration of 0.2 M in 5 mL of THF and then added to the polymer solution. The polymerization reaction was carried out by stirring at room temperature for 2 hours. After the reaction was completed, the terminal polymer was precipitated with 5 volumes of diethyl ether. The ether was then poured off, and the collected pure polymer was washed with 2 volumes of fresh diethyl ether. The pure polymer was dissolved in DMSO (purchased from Energy Chemical) to a concentration of 100 mg / mL and stored at -20 °C (the process for obtaining the polymer is as follows). Figure 1 A), 1 H-NMR spectroscopy confirmed the successful synthesis of PBAE. Figure 1 B).

[0031] 4.2 Preparation of Ferr-1 and lincRNA-p21 co-loaded nanoparticles (ferr-1 / lincRNA-p21@NP)

[0032] First, the plasmid overexpressing lincRNA-p21 was dissolved in 10 mL of citrate buffer (pH 3.0-3.5) at a concentration of 5 mg / mL. The PBAE synthesized in step 4.1 was added to 10 mL of citrate buffer (pH 3.0-3.5) at a concentration of 20 mg / mL and mixed thoroughly. Under gentle vortexing, the plasmid-citrate buffer solution overexpressing lincRNA-p21 was added dropwise to the solution, and then incubated at room temperature for 30 minutes to ensure sufficient electrostatic complexation. Ferr-1 (purchased from MCE) and PLGA (purchased from MCE) were dissolved in 20 mL of DMSO at concentrations of 16.67 mg / mL and 50 mg / mL, respectively. Under sonication, the DMSO mixture of ferr-1 and PLGA was added dropwise to the electrostatically complexed solution, and sonication was continued for 30 minutes to obtain ferr-1 / lincRNA-p21@NP. Ferr-1 / lincRNA-p21@NP was freeze-dried after adding 1% mannitol as a cryoprotectant. Finally, the ferr-1 / lincRNA-p21@NP was reconstituted in 20 ml of pH 7.4 PBS buffer (Merck, USA). Particle size and potential were measured using DLS (Malvern Instruments, UK), and the results are shown in Table 1 below. The morphology of the nanoparticles was characterized by transmission electron microscopy, and the results are as follows. Figure 2 ,from Figure 2 It can be seen that the nanoparticles have a relatively regular morphology and a uniform particle size distribution.

[0033] Table 1. Particle size and potential of ferr-1 / lincRNA-p21@NP

[0034]

[0035] 5. In vitro assays to verify the hepatocyte protective ability of ferr-1 / lincRNA-p21@NP

[0036] This embodiment of the in vitro experiment set up five experimental groups: Group 1: PBS group; Group 2: Nanoparticle (NP) group alone (synthesized in step 4.1); Group 3: ferr-1@NP (ferr-1: 330 μg / mL) group (synthesized in step 4.2); Group 4: lincRNA-p21@NP (lincRNA-p21: 100 μg / mL) group (synthesized in step 4.2); Group 5: ferr-1 / lincRNA-p21@NP (containing ferr-1: 330 μg / mL and lincRNA-p21: 100 μg / mL) group (synthesized in step 4.2). HepG2 cells were used as the experimental subjects, and 96-well plates were used for culture. The treatment method was as follows: 200 μL of cell culture medium containing 100 mM ethanol and 5 μM esterin was added, and 0.5 μL of the corresponding nanoparticle drug was added to each well according to the group. After 48 hours, cell viability was measured using the CCK8 assay. The results are as follows: Figure 3 As shown in Figure A, it can be seen that in the case of alcoholic hepatocyte injury combined with Erastin-induced ferroptosis, the cell survival rate of lincRNA-p21@NP was reduced compared with the control group (control@NP). Compared with the first group (PBS), both the third group (ferr-1@NP) and the fifth group (ferr-1 / lincRNA-p21@NP) nanomedicines could protect hepatocytes. Compared with the second group (NP alone), only the fifth group (ferr-1 / lincRNA-p21@NP) nanomedicine could significantly protect cells.

[0037] HepG2 cells were cultured in 6-well plates, and 2 mL of cell culture medium containing 100 mM ethanol and 5 μM methanotin was added, followed by 5 μL of the various nanomedicines synthesized in step 4. Cellular ROS levels and cellular lipid oxidation levels were detected by flow cytometry using DCFH-DA and C11-BODIPY 581 / 591 probes. Results are as follows: Figure 3 As shown in Figure B, the figure reveals that lincRNA-p21@NP alone, in the presence of the ferroptosis inducer Erastin, increased cellular ROS and lipid oxidation levels, exacerbating cellular oxidative damage. Ferr-1@NP alone reduced cellular ROS levels, but its effect on lipid oxidation was not significant. In contrast, the ferr-1 / lincRNA-p21@NP combination significantly reduced both cellular ROS and lipid oxidation levels, effectively protecting cells.

[0038] 6. Hepatoprotective effect of Ferr-1 / lincRNA-p21@NP on chronic alcoholic liver injury in mice

[0039] Following the guidelines of the National Institutes of Health (NIH), the experimental animals were kept in an environment of 21±2°C, 50±10% relative humidity, and 12h light / 12h dark alternation. C57BL / 6cnc mice were randomly assigned to five experimental groups (n=6-7). Each group received a different treatment regimen, including PBS, nanoparticles (NP) alone, ferr-1@NP (ferr-1: 5 mg / kg), lincRNA-p21@NP (lincRNA-p21: 1.5 mg / kg), and ferr-1 / lincRNA-p21@NP (co-delivery of 5 mg / kg ferr-1 and 1.5 mg / kg lincRNA-p21). Mice in each group received the corresponding formulation intraperitoneally every three days for four consecutive weeks. Chronic alcoholic liver injury was induced in mice using an ALD mouse model (National Institute on Alcohol Abuse and Alcoholism, NIAAA model) induced by feeding mice with a liquid diet containing 5% (v / v) alcohol followed by acute alcohol gavage for the last three days. Mice were fed a liquid diet (Liberty-Decalley diet) for 4 weeks, with alcohol administered via gavage for the last 3 days. Twelve hours after the last gavage, blood was collected from the fundus venous plexus under anesthesia, and the mice were sacrificed. Liver tissue was collected and stored at -80°C, and serum was separated from the blood and stored at -80°C.

[0040] Liver function indicators such as ALT, AST, ALP, LDH, and GLDH in mouse serum were measured using a Roche cobas 8000Access analyzer. The liver function results of mice treated with nanomedicine are shown in Table 2. Table 2 shows that the serum ALT and AST levels in mice treated with ferr-1 / lincRNA-p21@NP were significantly lower than those in the PBS group. Furthermore, the serum ALT and GLDH levels in the ferr-1 / lincRNA-p21@NP group were significantly lower than those in the ferr-1@NP group, and the GLDH level was significantly lower in the lincRNA-p21@NP group. Figure 4 A). Hematoxylin-eosin (H&E) staining and Oil Red O staining results of liver tissue showed ( Figure 4 B) The degree of hepatocellular disorder and fatty liver were significantly improved in the ferr-1 / lincRNA-p21@NP group. Therefore, ferr-1 / lincRNA-p21@NP has the most significant liver-protective effect.

[0041] Table 2. Liver function in mice with alcoholic liver injury treated with the nanomedicine fer-1 / lincRNA-p21@NP

[0042]

[0043] 7. LincRNA-p21 small interference and overexpression mice

[0044] To further verify that overexpression of lincRNA-p21 alone does not provide hepatoprotection in alcoholic liver injury, this step involved transferring the short interfering RNA (siRNA) or overexpression plasmid corresponding to the lincRNA-p21 gene into the pAV-U6-GFP vector of adeno-associated virus (AAV) serotype 8, generating siRNA-lincRNA-p21-AAV8-U6-GFP (siRNA-lincRNA-p21) and over-lincRNA-p21-AAV8-U6-GFP (over-lincRNA-p21). Control AAV was prepared using the inserted nonsense sequence pAV-U6-GFP and an overexpression empty control vector. The relevant AAV virus was produced, packaged, and purified by Vigene Biosciences, USA. Six-week-old male C57BL / 6cnc mice were injected intravenously with AAV virus containing a lincRNA-p21 small interfering (siRNA-lincRNA-p21) plasmid, a lincRNA-p21 overexpression (over-lincRNA-p21) plasmid, or a corresponding control plasmid. The injection volume was 0.1 mL per mouse (AAV viral load: 5E11 v.g). Following NIH guidelines, the experimental environment for the animals was: 21±2℃, 50±10% relative humidity, 12h light / 12h dark alternation. Mice were routinely fed for 4 weeks until viral gene expression was achieved. Chronic alcoholic liver injury was induced using the NIAAA model, i.e., mice were acclimatized to a liquid diet (Liberty-Decalley diet containing 5% (v / v) alcohol) starting at 10 weeks, followed by an alcoholic diet for 4 weeks, and then administered alcohol via gavage for the last 3 days of the experiment. Twelve hours after the last gavage, blood was collected from the fundus venous plexus under anesthesia, and the mice were sacrificed. Liver tissue was collected and stored at -80℃. Liver function tests in mice revealed that overexpression of lincRNA-p21 exacerbated liver damage in mice with chronic alcoholic liver injury.

[0045] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments for application in other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. Use of co-delivery of ferrostatin-1 and lincRNA-p21 in the preparation of a drug for treating alcoholic liver injury, wherein the structure of ferrostatin-1 is as follows: The lincRNA-p21 sequence is shown in the sequence listing as SEQ ID No.

1.

2. Use of co-loaded nanoparticle ferr-1 / lincRNA-p21@NP of ferrostatin-1 and lincRNA-p21 in the preparation of a drug for treating alcoholic liver injury.

3. Use according to claim 2, characterized in that, The preparation steps of the co-loaded nanoparticle ferr-1 / lincRNA-p21@NP are as follows: dissolving ferrostatin-1 and PLGA in DMSO to obtain solution A; mixing cationic polymer PBAE in a citrate buffer solution to obtain solution B; dissolving a plasmid overexpressing lincRNA-p21 in a citrate buffer solution, and adding the solution B dropwise under mild vortex, then incubating at room temperature for 30 minutes to obtain solution C; under ultrasonic, solution A is added dropwise to solution C, and ultrasonic is continued to obtain ferr-1 / lincRNA-p21@NP.

4. Use according to claim 3, characterized in that, The preparation method of the cationic polymer PBAE is as follows: mixing 1,4-butanediol diacrylate and 4-amino-1-butanol at a molar ratio of 1.1:1, stirring at 90°C for 24 hours to generate a polymer solution P; dissolving the polymer in tetrahydrofuran; dissolving 1-(3-aminopropyl)-4-methylpiperazine in tetrahydrofuran at a concentration of 0.2M, and then adding to the polymer solution P, stirring at room temperature for 2 hours for polymerization reaction, and adding ether to precipitate the capped polymer after the reaction is completed; pouring out the ether, and then adding fresh ether to wash the precipitated polymer to obtain pure cationic polymer PBAE.

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