Targeted antioxidant nanovesicle delivery system for treatment of drug-induced liver injury

Abstract

The invention discloses preparation and application of a hepatic targeting polymer nano-vesicle. The hepatic targeting polymer nano-vesicle comprises a water inner shell arginine nonapeptide, a hydrophobic membrane layer polylactic acid-glycolic acid copolymer, a hydrophilic shell polyethylene glycol and zwitterionic groups embedded in the hydrophilic shell, the polymer nano-vesicle has a vesicle structure, and by embedding zwitterionic groups in a hydrophilic shell, the nano-vesicle has an active liver targeting function; the polymer nano vesicles are loaded with natural enzymes, namely catalase and superoxide dismutase. The polymer nano-vesicles can effectively protect antioxidant enzymes, increase removal of ROS by the liver and improve acetaminophen-induced hepatotoxicity and liver injury.

Description

Technical Field

[0001] The present invention belongs to the technical field of polymer nano-drugs, and particularly relates to a liver-targeting polymer nanovesicle system, a preparation method thereof, and an application thereof. Background Art

[0002] Drug-induced liver injury (DILI) is a disease in which the liver is damaged by drugs (including pharmacological treatments, traditional drugs, herbal medicines, or dietary supplements), and is a liver injury associated with the ingestion of hepatotoxic drugs. Currently, it has been confirmed that more than 1,200 drugs can cause DILI. In addition, hepatotoxicity is also a common reason for restricting drug use and stopping clinical development programs. DILI not only occurs in some special treatment processes such as anti-tuberculosis or anti-cancer treatments, but also some commonly used drugs such as acetaminophen and amoxicillin can cause severe liver damage.

[0003] Oxidative stress plays a key role in all types of liver injury, and among them, reactive oxygen species (ROS) are crucial. Hepatocytes are rich in mitochondria. When hepatotoxicity occurs, the damaged mitochondria will produce excessive ROS. Although there is a powerful antioxidant enzyme system in the cells, it is not sufficient to cope with liver injury.

[0004] With the application of drug delivery systems in mRNA, small nucleic acids, proteins, and polypeptide drugs in recent years, drug delivery systems (DDS) have shown great potential and advantages. According to the different drugs carried by the nanocarriers, they can be applied in different fields, including the application of vaccines in tumors, the treatment of rare diseases, and the development of gene therapy. Therefore, the delivery of nanoparticles has great application prospects. Summary of the Invention

[0005] The present invention aims to provide a liver-targeting polymer nanovesicle delivery system, a preparation method thereof, and an application thereof, aiming to solve the problems of organ targeting, treatment of liver injury, and improvement of liver function in the prior art.

[0006] The targeting of nanovesicles is increased by modifying PEG-PLGA. The nanovesicles encapsulate the natural enzymes superoxide dismutase (SOD) and catalase (CAT) inside. The technical solution of the present invention is as follows:

[0007] The polymer in the present invention is composed of two parts, PEG-PLGA-PA9 and ZP3-PEG-PLGA. Among them, the positive charge of the guanidine group in PEG-PLGA-PA9 is used to load antioxidant enzymes, and the zwitterion at the head of ZP-PEG-PLGA regulates the surface potential of the nanocarrier, so that the nanocarrier has the function of actively targeting the liver. The preparation method of the liver-targeting polymer nanovesicles of the present invention can be as follows:

[0008] Catalase (CAT) and superoxide dismutase (SOD) were dissolved in ultrapure water to obtain an antioxidant enzyme mixture (mass ratio 1:4), which was mixed evenly with PBS buffer (pH 7.4, 10 mM). Then, a mixed solution of PEG-PLGA-PA9 and ZP3-PEG-PLGA polymers (DMSO, 20 mg / mL, mass ratio 50:50) was added dropwise thereto under stirring conditions. After stirring for 30 minutes, dialysis was carried out with PBS (pH 7.4, 10 mM) to obtain CS@PS.

[0009] Beneficial effects:

[0010] Preparation of a polymer nanovesicle with liver targeting and its application in drug-induced liver injury according to the present invention. The polymer nanovesicle includes: a hydrophilic inner shell of arginine nonapeptide, a hydrophobic membrane layer of poly(lactic-co-glycolic acid), a hydrophilic outer shell of polyethylene glycol, and an amphoteric ion group embedded in the hydrophilic outer shell; the polymer nanovesicle has a vesicle structure, and by embedding an amphoteric ion group in the hydrophilic outer shell, the nanovesicle has the function of actively targeting the liver; the hydrophilic outer shell of polyethylene glycol can effectively protect catalase and superoxide dismutase, and at the same time allow small molecule ROS to pass through, protecting the liver from ROS-mediated damage and reducing the inflammatory response.

[0011] Figure 1 Dynamic light scattering particle size diagram, Zeta potential diagram, and transmission electron microscope diagram of the polymer nanovesicle prepared according to the present invention and the nanovesicle loaded with natural enzymes (CS@PS);

[0012] Figure 2 SOD and CAT activities of CS@PS, superoxide dismutase (SOD), and catalase (CAT);

[0013] Figure 3 Cytotoxicity of the polymer and results of red blood cell hemolysis experiment;

[0014] Figure 4 Fluorescence images and quantitative analysis diagrams of FL83B cells untreated, treated with SOD and CAT, and treated with CS@PS under LPS stimulation;

[0015] Figure 5 Cell inflammatory factor expression diagrams of RAW264.7 cells untreated, treated with SOD and CAT, and treated with CS@PS under LPS stimulation;

[0016] Figure 6 Schematic diagram of the body weight change curve of mice with drug-induced liver injury caused by acetaminophen receiving various treatments and aspartate aminotransferase (AST), alanine aminotransferase (ALT)

[0017] Figure 7 It is a graph of cellular inflammatory factors for mice with drug-induced liver injury after treatment. Detailed implementation manners

[0018] The polymer nanovesicles in the present invention are composed of a hydrophilic inner shell amino acid nonapeptide, a hydrophobic membrane layer poly(lactic-co-glycolic acid) copolymer, a hydrophilic outer shell polyethylene glycol, a charge group embedded in the hydrophilic outer shell, and a natural enzyme; the polymer nanovesicles have a vesicle structure, and by embedding a charge group in the hydrophilic outer shell, the nanovesicles have the function of actively targeting the liver; positive charges are loaded with antioxidant enzymes, and the hydrophilic outer shell polyethylene glycol can effectively protect the antioxidant enzymes, and at the same time can allow small molecule ROS to pass through, protecting the liver from ROS-mediated damage and reducing the inflammatory response.

[0019] The following are example embodiments to illustrate the present invention in detail. The following examples are only used to further illustrate the present invention and cannot be construed as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention all fall within the protection scope of the present invention.

[0020] Example 1 Preparation of liver-targeted polymer nanovesicles

[0021] The liver-targeted polymer nanovesicles were prepared by the solution self-assembly method and loaded by means of the electrostatic interaction between the macromolecular drug and the amino acid nonapeptide. mPEG-PLGA-PA9 and ZP3-PEG-PLGA were used to load superoxide dismutase (SOD) and catalase (CAT). Specifically, 50 μL of DMSO solutions of mPEG-PLGA-PA9 and ZP3-PEG-PLGA polymers (total polymer concentration was 20 mg / mL) were taken and mixed evenly, and while stirring (500 rpm), it was dropped into 900 μL of PBS (10 mM, pH 7.4) buffer solution containing catalase (CAT) and superoxide dismutase (SOD), and continued to stir at room temperature for 30 minutes, and dialyzed with PBS (pH 7.4, 10 mM) for 8 hours to obtain the liver-targeted polymer nanovesicles CS@PS.

[0022] Dynamic light scattering (DLS) analysis showed that the average size of CS@PS was about 150 nm, and the polydispersity index (PDI = 1.2) was narrow. Using a single component PEG-PLGA-PA9 to load superoxide dismutase (SOD) and peroxidase (CAT), CS@PA9 was obtained as a control. The particle size and surface Zeta potential of CS@PA9 were similar to those of CS@PS. Transmission electron microscopy photographs showed that the particle size of CS@PS was uniform and the morphology was spherical.

[0023] Example 2 Enzymatic Activity Assay of Polymeric Nanovesicles

[0024] The catalase and superoxide dismutase activities of polymeric nanovesicles were determined using a detection kit. A 100 μmol / L hydrogen peroxide solution was added to a 96-well plate, and then different concentrations of CS@PS were added. After incubation at 37 °C for different times, 100 μl of hydrogen peroxide detection reagent was added to each well. Mix gently by shaking or tapping, leave at room temperature for 30 minutes, and then immediately measure the detection value at a wavelength of 560 nm. The concentration of hydrogen peroxide in the sample was calculated according to the standard curve. Different concentrations of the sample, SOD detection buffer, and WST-8 / enzyme working solution were added successively to a 96-well plate, and then the reaction starting working solution was added and mixed thoroughly. After incubation at 37 °C for 30 minutes, the results on the plate were immediately measured at a wavelength of 450 nm. Using free catalase and superoxide dismutase as controls, the CAT and SOD enzyme activities of CS@PS were calculated.

[0025] Appendix Figure 2 This is the enzymatic activity of CS@PS. Over time, the concentration of hydrogen peroxide gradually decreased, and the addition of CS@PS accelerated the degradation of hydrogen peroxide. Similar results were also found in the SOD enzyme activity detection.

[0026] Example 3 Cytotoxicity and Hemolysis Experiments of Polymeric Nanovesicles

[0027] The cytotoxicity of polymeric nanovesicles was determined by the CCK8 method. First, FL83B cells were seeded in a 96-well plate (5×10 3 cells / well) and incubated in an incubator for 24 hours. Then, 10 μL of CS@PS solution (diluted with complete medium, and the final concentrations of nanovesicles in the wells were 12.5, 25, 50, 100 μg / mL respectively) was added to each well, and 10 μL of PBS was added to the control group. After incubation for 24 hours or 48 hours, 10 μL of CCK8 was added to each well and incubated in the incubator for another 2 hours. Finally, the absorbance at a wavelength of 450 nm was measured by an enzyme-linked immunosorbent assay reader. The test results showed that the cell survival rate was calculated as follows: (absorbance value of the experimental group - absorbance of the blank) / (absorbance value of the control group - absorbance of the blank) × 100%, and the experiment was carried out in three replicates in parallel. In the red blood cell hemolysis test, CS@PS in PBS (concentrations of 1, 2, 4 mg / mL) was co-cultured with 2% RBC at 37 °C for 1 hour. Deionized water and PBS were used as positive and negative controls for hemolysis respectively. The degree of RBC lysis was determined by measuring the amount of released hemoglobin (525 - 625 nm) by spectrophotometry.

[0028] Cytotoxicity and Hemolysis Experiments( Figure 3) It shows that CS@PS has no toxicity to cells even at a high concentration of 100 μg / ml, and has extremely little hemolytic effect on red blood cells even at a concentration as high as 2 mg / ml.

[0029] Example 4 Reactive Oxygen Species Scavenging of Polymer Nanovesicles

[0030] The in vitro reactive oxygen species (ROS) scavenging of polymer nanovesicles was detected using the 2,7-dichlorofluorescein diacetate (DCFH-DA) probe. After culturing FL83B cells in a 12-well plate (1×10 5 / well) for 24 hours, the cells were stimulated with LPS (500 ng / mL) for 3 hours, and then treated with CS@PS and free CAT / SOD for 12 hours respectively. DCFH-DA was added to each well and incubated at 37 °C for 30 minutes. The ROS levels of each group were observed by fluorescence microscopy.

[0031] The intracellular reactive oxygen species in the CS@PS group were almost the same as those in the normal cell group, while the intracellular reactive oxygen species in the free CAT / SOD group were similar to those in the hydrogen peroxide group, demonstrating the reactive oxygen species radical scavenging ability of CS@PS.

[0032] Example 5 Anti-Inflammatory Experiment of Polymer Nanovesicles

[0033] The anti-inflammatory ability of polymer nanovesicles was determined by detecting cell inflammatory factors using ELISA. First, RAW264.7 cells were seeded in a 6-well plate and cultured in an incubator at 37 °C for 24 hours. Then, 200 μl of CS@PS solution was added to each well, and 200 μl of CAT / SOD solution was added to each well in the control group. After continuing to incubate in the incubator for 6 hours, the cells were stimulated with LPS (100 ng / ml) for 24 hours, and then the cell supernatant was collected.

[0034] For each well on the ELISA plate, 300 μl of washing solution was added and allowed to stand for 30 seconds, then the washing solution was discarded and the microplate was patted dry on absorbent paper. 100 μl of cell supernatant and 50 μl of detection antibody working solution were added to each well and incubated at room temperature for 2 hours. The liquid in the well plate was discarded, and the wells were washed 6 times. 100 μl of streptavidin working solution was added and incubated at room temperature for 45 minutes. The liquid in the well plate was discarded, and the wells were washed 6 times. 100 μl of chromogenic substrate was added to each well, protected from light, and incubated at room temperature for 30 minutes. 100 μl of stop solution was added to each well. Finally, the absorbance at wavelengths of 450 nm and 570 nm was detected by an ELISA reader. The concentration was calculated according to the standard curve, and each experiment was performed in three parallel replicates for each group.

[0035] The results showed ( Figure 5 ) that the addition of CS@PS significantly reduced the inflammatory cytokines and was close to the blank group, while the inflammatory cytokines in the control LPS group and the group treated with free CAT / SOD were significantly higher, demonstrating that CS@PS has excellent intracellular anti-inflammatory ability.

[0036] Example 6 Therapeutic Effect of Polymer Nanovesicles on Mice with Drug-Induced Liver Injury

[0037] To study the therapeutic effect of polymer nanovesicles on mice with drug-induced liver injury, a mouse model of drug-induced liver injury caused by acetaminophen (APAP) was established. All animal experiments and operations were approved by the Experimental Animal Center and the Animal Ethics Committee of Peking University Shenzhen Hospital. The mouse model was established using C57BL / 6 strain mice (6 - 8 weeks old, female). A drug-induced liver injury model was established by a single intraperitoneal injection of a large dose of acetaminophen (APAP) (350 mg / kg) to mice. Six hours later, the first drug intervention was carried out using CS@PS via the tail vein, and drug interventions were carried out at 24 hours and 48 hours later. The dose of superoxide dismutase was 175 μg / mouse, and the dose of catalase was 25 μg / mouse. The untreated group was injected with the same volume of normal saline.

[0038] The schematic diagram of the body weight change curve showed that the body weight of the mice in the acetaminophen group continued to decline, indicating that the drug-induced liver injury model was successfully established. The free CAT / SOD group and the CS@PS group did not prevent the body weight loss of mice within 24 hours, indicating that the acute hepatotoxicity of acetaminophen was more severe within 24 hours. In the next 48 hours, the body weight of the mice in the CS@PS group increased, indicating that the treatment with CS@PS effectively alleviated drug-induced liver injury. Serum aminotransferase levels, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT), were used as indicators of the occurrence of drug-induced liver injury. The levels of ALT and AST in the mice treated with acetaminophen (APAP) showed abnormal increases 30 h after intraperitoneal injection of APAP, verifying the successful establishment of the mouse model of drug-induced liver injury. After treatment with CS@PS, the levels of alanine aminotransferase and aspartate aminotransferase in the mice decreased to healthy levels, indicating that CS@PS had a good therapeutic effect on drug-induced liver injury, while the effect of free CAT / SOD was not as good as that of CS@PS.

[0039] Example 7 Polymer Nanovesicles Alleviated Inflammatory Injury in Mice with Drug-Induced Liver Injury A mouse model of drug-induced liver injury was established by a single intraperitoneal injection of a large dose of acetaminophen (APAP) (350 mg / kg) to mice. Three hours later, drug intervention was carried out using CS@PS via the tail vein. The dose of superoxide dismutase was 175 μg / mouse, and the dose of catalase was 25 μg / mouse. The untreated group was injected with the same volume of normal saline. Serum was taken to detect the levels of inflammatory cytokines TNF-α and IL-6 in the mice.

[0040] The results showed that the levels of TNF-α and IL-6 in the APAP group were significantly increased. Compared with the APAP group, the levels of TNF-α and IL-6 in the CS@PS treatment group were significantly decreased. It indicated that CS@PS reduced the expression levels of TNF-α and IL-6 and alleviated the inflammatory damage in mice.

Claims

1. A liver-targeted polymer nanovesicle, characterized in that: The nanovesicle comprises a hydrophilic inner shell arginine nonapeptide, a hydrophobic membrane layer polylactic acid-glycolic acid copolymer, a hydrophilic outer shell of polyethylene glycol and a zwitterionic group embedded in the hydrophilic outer shell.

2. The nanovesicle according to claim 1, characterized in that The following steps are involved: The amphiphilic polymer nanovesicles were prepared by a solution-based self-assembly method.

3. The nanovesicle according to claim 1 or 2, characterized in that: The drugs loaded into the nanovesicles are catalase and superoxide dismutase.

4. An application of a liver-targeted polymer nanovesicle carrier system, characterized in that: The nanovesicles are used in the treatment of liver damage.

5. The use according to claim 4, characterized in that: The liver injury is drug-induced liver injury caused by acetaminophen.

6. The application according to claim 5, characterized in that Nanovesicles effectively protect antioxidant enzymes, increase the liver's clearance of ROS, and improve liver damage.

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