A nanoparticle preparation for protecting liver from ischemia-reperfusion injury and its application

Abstract

The present invention discloses a nanoformulation for protecting against liver ischemia-reperfusion injury and its application. The nanoformulation comprises glucose-6-phosphate disodium, an esterase-responsive charge-reversal polymer (ERP), a lipid, and DSPE-PEG-Gal. The esterase-responsive charge-reversal polymer (ERP) serves as a nanocarrier to encapsulate glucose-6-phosphate disodium to form ERP / G6P. The lipid encapsulates ERP / G6P to form a spherical core-shell structure of Lipids / ERP / G6P. DSPE-PEG-Gal assembles with Lipids / ERP / G6P to form a PEG-Lipids / ERP / G6P nanoformulation. The present invention utilizes nanoscale size effects and active targeting to enrich G6P in the liver, improving its stability and bioavailability. The polymer ERP used can be hydrolyzed by esterases, thus exhibiting good biosafety.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a nano preparation for protecting liver ischemia-reperfusion injury and an application thereof. Background Art

[0002] Liver transplantation is an effective surgical treatment for end-stage liver disease (such as cirrhosis and liver cancer). However, the operation is inevitably affected by ischemia-reperfusion injury of the liver. Ischemia-reperfusion injury refers to the damage that occurs when blood flow (reperfusion) is restored to an organ or tissue after a period of interruption of blood supply (ischemia). During the process of liver preservation and liver transplantation, the liver needs to temporarily cut off its blood supply during surgery and then restore blood flow. This process may cause cell damage and inflammatory response, thereby damaging the transplanted liver. It is an important cause of graft dysfunction and graft rejection after liver transplantation.

[0003] The pathogenesis of hepatic ischemia-reperfusion injury (IRI) involves a multi-layered cytotoxic cascade. The generation of highly reactive oxygen free radicals (ROS) and the leakage of damage-associated molecular patterns (DAMPs) play key roles in this process, triggering redox imbalance, activation of inflammatory responses, and hepatocyte damage and cell death. Hepatocytes are parenchymal cells in the liver that are susceptible to metabolic disturbances, triggering early necrosis during the ischemic phase. During the reperfusion phase, activated immune cells release inflammatory cytokines into the liver tissue, exacerbating cellular damage.

[0004] To mitigate the effects of ischemia-reperfusion injury on liver transplantation, researchers have been exploring different methods and strategies, which may include the use of antioxidants to reduce oxidative stress, the use of anti-inflammatory drugs to reduce inflammatory responses, and the optimization of preoperative preparation and surgical techniques to minimize ischemia time. However, there is still a lack of effective strategies to prevent or treat liver ischemia-reperfusion injury in clinical practice. Therefore, in-depth exploration and elucidation of the cellular signaling pathways and molecular events of liver ischemia-reperfusion injury will help to find targeted therapeutic strategies to reduce or prevent ischemia-reperfusion injury in situations such as liver transplantation.

[0005] The rapid development of nanotechnology has provided promising opportunities for achieving safe and effective pharmacological interventions for liver ischemia-reperfusion injury. The unique properties of nanoscale materials, including high surface-to-volume ratios, unique surface chemistry, exceptional optical properties, and excellent free radical scavenging capabilities, provide strong support for mitigating liver ischemia-reperfusion injury. The ease of modification offered by nanotechnology is also one of its advantages. In particular, nanoparticles can be designed to create hydrophilic, amphiphilic, or hydrophobic microenvironments, which is crucial for developing drug delivery systems and improving therapeutic efficacy.

[0006] Specifically, the surface charge of nanoparticles affects their collection in the body. Nanoparticles with negative surface charge are more easily absorbed by liver sinusoidal endothelial cells, while nanoparticles with positive surface charge may be more easily internalized by liver cells. In addition, specific surface ligands on nanoparticles can bind to receptors or targets on liver cells to achieve precise liver-targeted therapy. Nanomaterials can also show sensitivity to specific stimuli, such as pH, temperature, light, magnetic fields, shear strength, and electrolyte or glucose concentrations. These properties help improve the efficacy of therapeutic and diagnostic agents while reducing the occurrence of adverse side effects.

[0007] Given these advantages, nanomaterials are widely considered a promising option for the effective and safe treatment of liver ischemia-reperfusion injury. Using nanotechnology, we can design precise drug delivery systems to precisely direct drugs to the liver areas in need of treatment, thereby minimizing the adverse effects of ischemia-reperfusion injury. Research in this area offers new hope for patients undergoing liver transplantation, potentially significantly improving postoperative recovery and transplant survival. Summary of the Invention

[0008] The purpose of the present invention is to provide a lipid esterase-responsive charge reversal polymer PEG-Lipids / ERP / G6P nanoformulation with liver targeting, which utilizes active targeting to enrich glucose-6-phosphate in the liver, that is, it can be selectively enriched in the liver. This property can ensure that the therapeutic substance acts where it is needed, reducing distribution in other organs or tissues, thereby reducing unnecessary side effects, and achieving slow and long-lasting release to protect the liver from ischemia-reperfusion injury.

[0009] This treatment is expected to effectively protect damaged liver tissue after organ transplantation, alleviate or prevent problems caused by ischemia-reperfusion injury, help improve the success rate of organ transplant surgery, and reduce patient discomfort and complications.

[0010] The present invention also provides a method for preparing the nanoformulation, which can directly prepare a high-concentration polymer nanoformulation and can achieve the preparation of a high-concentration formulation using simple steps. This can reduce production costs, simplify production technology, and has good feasibility.

[0011] A nanoformulation for protecting liver ischemia-reperfusion injury comprises disodium glucose-6-phosphate, an esterase-responsive charge reversal polymer ERP, lipids and DSPE-PEG-Gal. The esterase-responsive charge reversal polymer ERP is used as a nanocarrier to encapsulate disodium glucose-6-phosphate to form ERP / G6P. The lipids encapsulate ERP / G6P to form Lipids / ERP / G6P having a spherical core-shell structure. DSPE-PEG-Gal and Lipids / ERP / G6P are assembled to form a PEG-Lipids / ERP / G6P nanoformulation, which is the nanoformulation for protecting liver ischemia-reperfusion injury.

[0012] Preferably, the esterase-responsive charge-reversal polymer (ERP) is prepared by reacting a polymer containing a primary or secondary amino group with a carboxylic ester, followed by quaternization. The preparation of the esterase-responsive charge-reversal polymer (ERP) of the present invention is described in the patent application number CN106146834B, entitled "An Esterase-Responsive Cationic Polymer."

[0013] Preferably, the mass ratio of the esterase-responsive charge reversal polymer ERP to glucose-6-phosphate disodium is 0.1 to 5:1;

[0014] The mass ratio of lipid to ERP / G6P was 0.1 to 5:1;

[0015] The molar ratio of DSPE-PEG-Gal to the lipid is 1:9-19.

[0016] Preferably, the lipid is a mixture of a neutral auxiliary lipid DOPC and a cationic lipid DC-Chol, and the molar ratio of the neutral auxiliary lipid DOPC to the cationic lipid DC-Chol is 3:1.

[0017] The present invention further provides a method for preparing the nanoformulation, comprising the following steps:

[0018] S1, esterase-responsive charge reversal polymer ERP was used as a nanocarrier to encapsulate disodium glucose-6-phosphate to form ERP / G6P;

[0019] S2, encapsulating the ERP / G6P obtained in step S1 with lipids to form Lipids / ERP / G6P with a spherical core-shell structure;

[0020] S3 adds DSPE-PEG-Gal as a target head to the product obtained in step S2 to assemble and form a PEG-Lipids / ERP / G6P nanoformulation.

[0021] Preferably, in step S1, the method for preparing the esterase-responsive charge reversal polymer ERP comprises the following steps:

[0022] S1.1, dissolving polyethyleneimine and 2,6-di-tert-butyl-p-cresol in N,N-dimethylformamide, and then adding 4-acetoxybenzyl acrylate to react;

[0023] S1.2, adding methyl iodide to the reaction product obtained in step S1.1, and performing a quaternization reaction in the dark;

[0024] S1.3, precipitating the reaction product obtained in step S1.2 with diethyl ether, and separating and purifying the precipitate to obtain the esterase-responsive charge reversal polymer ERP.

[0025] The present invention also provides application of the nano preparation in preparing medicine for protecting liver ischemia-reperfusion injury.

[0026] The present invention also provides a medicine for protecting liver ischemia-reperfusion injury, the active ingredient of which is the nano preparation.

[0027] Preferably, the drug is administered by intravenous injection, and the drug is administered twice within 24 hours before ischemia-reperfusion.

[0028] The beneficial effects of the present invention are: the present invention utilizes nano-size effect and active targeting to enrich G6P in the liver, thereby improving its stability and bioavailability; the polymer ERP used can be hydrolyzed by esterase and has good biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Particle size and potential of ERP / G-6P complex at different mass ratios.

[0030] Figure 2 The particle size and potential of ERP / G-6P complex (mass ratio = 0.1) coated with liposomes and DSPC-PEG-Gal at different concentrations.

[0031] Figure 3 The chemical structure and synthesis process of G6P nanoparticles (A) and the in vitro bioimaging fluorescence intensity of various organs (B).

[0032] Figure 4 This is a graph showing the test results of serum liver function levels.

[0033] Figure 5 This is the result of detecting the expression level of inflammatory cell chemokines in liver tissue.

[0034] Figure 6 This is the result of ELISA detection of the secretion level of inflammatory factors in serum.

[0035] Figure 7 Figure 2 shows the detection results of apoptotic proteins and anti-apoptotic proteins in liver tissue.

[0036] Figure 8 The results of histological HE staining (A) and immunohistochemistry / immunofluorescence staining (B) are shown. DETAILED DESCRIPTION

[0037] Example 1

[0038] Synthesis of esterase-responsive charge-reversal polymer ERP:

[0039] Weigh 0.1g of 10KDa polyethyleneimine and 0.01g of 2,6-di-tert-butyl-p-cresol, add 1mL of dried, dehydrated N,N-dimethylformamide (DMF), and stir to dissolve. Add 0.7g of 4-acetoxybenzyl acrylate dropwise to the reaction system. After reacting at room temperature for 48 hours, transfer to a 45°C oil bath and heat for 24 hours. Add 1.0g of iodomethane dropwise to the reaction system and allow to react overnight in the dark. Precipitate the reaction system with 20 volumes of diethyl ether, dissolve it in chloroform, and remove any remaining diethyl ether by rotary evaporation. Place it in a vacuum oven to remove any remaining organic solvents. Purify the mixture by dialyzing it against a 0.9% NaCl solution and then pure water. Freeze-dry the mixture to obtain the final ERP product as a milky white powder. Seal it and store it in a desiccator.

[0040] Example 2

[0041] Preparation of lipid esterase-responsive charge-reversal polymer PEG-Lipids / ERP / G6P nanoformulations:

[0042] (1) G6P disodium salt (glucose-6-phosphate disodium) was dissolved at a concentration of 5 mg / mL in a 10 mM HEPES buffer solution with a pH of 7.4 to obtain a G6P solution.

[0043] (2) ERP was prepared into a 50 mg / mL solution with DMSO and used as the stock solution.

[0044] (3) HEPES buffer (10 mM) with a pH of 7.4 was diluted with the above ERP stock solution to prepare ERP solutions of different concentrations according to the mass ratio.

[0045] (4) At room temperature, equal volumes of ERP solution and G6P solution were mixed and allowed to stand for 30 min, vortexed for 10 s. The mass ratio of ERP to G6P was 0.1 to 5:1 ( Figure 1 ) to prepare ERP / G6P composite mixed solution. The optimal mass ratio of ERP to G6P was 0.1:1, which was used in subsequent experiments.

[0046] (5) The neutral helper lipid DOPC and the cationic lipid DC-Chol (molar ratio 1:1) were dissolved in chloroform, and the chloroform was evaporated to obtain a thin lipid film. HEPES buffer (10 mM) at pH 7.4 was added in a volume corresponding to the volume required for a neutral helper lipid concentration of 1 mg / mL. The mixture was stirred at room temperature for 8 h and then sonicated for 10 min to obtain a lipid solution.

[0047] (6) The ERP / G6P mixed solution was added dropwise to the lipid solution, and different dosage forms were prepared according to a DOPC / G6P (μM / μg) ratio of 0.05 to 0.2:1. The mixture was further stirred at room temperature for 2 h to obtain a Lipids / ERP / G6P solution. Finally, DSPE-PEG-Galactose was added to the Lipids / ERP / G6P solution at a molar mass of 5% or 10% of the total lipid mass and stirred at 50°C for 15 min to obtain a PEG-Lipids / ERP / G6P nanoformulation.

[0048] Example 3

[0049] Measuring the Characterization of PEG-Lipids / ERP / G6P Nanoformulations:

[0050] The particle size, PDI average dispersion coefficient, and Zeta potential of the PEG-Lipids / ERP / G6P prepared in Example 2 were measured using a Zetasizer Nano-ZS particle size analyzer. 40 μL of the nanoformulation was dripped into the sample detection cell to measure the particle size and average dispersion coefficient; 40 μL of the nanoformulation was diluted with 600 μL of HEPES solution and dripped into the sample detection cell to measure the potential. The results are shown in Figure 2. Figure 2 As shown, by testing the lipid solution and DSPE-PEG-Galactose concentration ratio, the nanoformulation under the conditions of DOPC / G6P (μM / μg) = 0.05 and PEG 5% had a uniform particle size of 167.7±1.57nm and a positive potential of 17.2±0.46mV, all within the biosafety range. Nanoformulations under other conditions were not considered optimal dosage forms due to excessively large particle size (over 200nm) or excessively high potential (over 20mV), as well as the principle of minimal material addition.

[0051] Example 4

[0052] Analysis of the distribution of PEG-Lipids / ERP / G6P nanoformulation in various organs of mice:

[0053] Fluorescent dye DiD was added to the PEG-Lipids / ERP / G6P nanoformulation at a molar ratio of 1:100 and stirred at 37°C for 30 minutes. The in vivo distribution of DiD-loaded PEG-Lipids / ERP / G6P nanoformulations was investigated by tracking mice at different times after tail vein injection (5 μg G6P / mouse in a volume of 200 μL / mouse).

[0054] The main organs (heart, liver, spleen, lung, and kidney) of mice were dissected at 2, 6, 12, 24, 48, and 72 hours after injection, and DiD fluorescence imaging was performed. The fluorescence intensity of different organs was quantitatively analyzed ( Figure 3 The results showed that the DiD-loaded PEG-Lipids / ERP / G6P nanoformulation effectively accumulated in the liver, with fluorescence intensity significantly stronger than in other organs, and maintained its strongest signal at 6 and 12 hours after injection. Due to the relatively short half-life of these nanoparticles, we chose to administer the drug via two intravenous injections over two days to maintain effective drug concentrations in the liver.

[0055] Example 5

[0056] Treatment of liver ischemia-reperfusion injury in mice:

[0057] The mice were divided into groups and administrated on average. They were divided into G6P nanoformulation group, G6P free drug group and normal saline group, with 6 mice in each group. The administration was the same as in Example 4, and the administration method was intravenous injection of the drug twice within 2 days before surgery, 200 μl each time. After the operation, the mice were anesthetized by intraperitoneal injection of 50 mg / kg of sodium pentobarbital, and then laparotomy was performed. The portal vein, hepatic artery and bile duct above the bifurcation point of the right lateral lobe of the liver were clamped with microvascular clamps to block the blood vessels of the left lateral lobe / median lobe of the liver. After 90 minutes of ischemia and 6 hours of reperfusion, the mice were anesthetized with ether, the eyeballs were removed and the blood collected was placed in a sterile centrifuge tube, and finally the mice were killed by cervical dislocation. After the death of the mice, the mice were immediately dissected and liver tissue was obtained. After the whole blood samples were placed on ice for 1-2 hours, they were centrifuged at 4000g for 10 minutes at 4°C, serum samples were taken, and the serum biochemical indicators AST, ALT and inflammatory factor levels were measured. The liver was washed twice in PBS solution, placed on filter paper, and the water was removed. The liver was fixed with paraformaldehyde, dehydrated, and embedded in paraffin. HE, TUNEL, immunohistochemistry, and fluorescent staining were then performed.

[0058] Compared with the control saline group and the G6P free drug group, the serum liver function level of mice in the G6P nanoformulation group was significantly decreased after ischemia injury ( Figure 4 ), and the expression levels of inflammatory cell chemokines IL6, IL1, TNF, CCL2 and CXCL10 in liver tissue were significantly decreased ( Figure 5), and the secretion levels of inflammatory factors IL6, IL1 and TNF in serum were significantly reduced by ELISA ( Figure 6 In liver tissue, the apoptotic protein Bax showed a down-regulation trend, while the anti-apoptotic protein Bcl2 showed an up-regulation trend ( Figure 7 The results of histological HE staining showed that the area of ​​liver necrosis in the nanoparticle group was significantly reduced ( Figure 8 TUNEL and c-caspase3 immunohistochemical staining of liver tissue showed that the apoptosis level of the nanoformulation group was significantly reduced, and immunofluorescence staining of MPO and F4 / 80 showed that the infiltration of immune cells was significantly reduced ( Figure 8 ). In summary, nanoformulation treatment induced a significant reduction in hepatocyte apoptosis, necrosis, and inflammatory cell recruitment.

Claims

1. A nanoparticle preparation for protecting liver from ischemia-reperfusion injury, characterized in that: The invention comprises disodium glucose-6-phosphate, an esterase-responsive charge reversal polymer ERP, lipids and DSPE-PEG-Galactose. The esterase-responsive charge reversal polymer ERP is used as a nanocarrier to encapsulate disodium glucose-6-phosphate to form ERP / G6P. The lipids encapsulate ERP / G6P to form Lipids / ERP / G6P with a spherical core-shell structure. The DSPE-PEG-Galactose and Lipids / ERP / G6P are assembled to form a PEG-Lipids / ERP / G6P nanoformulation, which is the nanoformulation for protecting liver ischemia-reperfusion injury. The preparation method of esterase-responsive charge reversal polymer (ERP) comprises the following steps: S1.1, dissolving polyethyleneimine and 2,6-di-tert-butyl-p-cresol in N,N-dimethylformamide, and then adding 4-acetoxybenzyl acrylate to react; S1.2, adding methyl iodide to the reaction product obtained in step S1.1, and performing a quaternization reaction in the dark; S1.3, precipitating the reaction product obtained in step S1.2 with diethyl ether, and separating and purifying the precipitate to obtain the esterase-responsive charge reversal polymer ERP.

2. The nanoparticle preparation for protecting liver from ischemia-reperfusion injury according to claim 1, characterized in that: The mass ratio of esterase-responsive charge reversal polymer ERP to glucose-6-phosphate disodium is 0.1-5:1; The mass ratio of lipid to ERP / G6P was 0.1–5:1; The molar ratio of DSPE-PEG-Galactose to the lipid is 1:9-19.

3. The nanoparticle preparation for protecting liver from ischemia-reperfusion injury according to claim 1, characterized in that: The lipid is a mixture of a neutral auxiliary lipid DOPC and a cationic lipid DC-Chol, and the molar ratio of the neutral auxiliary lipid DOPC to the cationic lipid DC-Chol is 3:

1.

4. The method for preparing the nanoformulation according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1, esterase-responsive charge reversal polymer ERP was used as a nanocarrier to encapsulate disodium glucose-6-phosphate to form ERP / G6P; S2, encapsulating the ERP / G6P obtained in step S1 with lipids to form Lipids / ERP / G6P with a spherical core-shell structure; S3 adds DSPE-PEG-Galactose as a target head to the product obtained in step S2 to assemble into PEG-Lipids / ERP / G6P nanoformulation, In step S1, the preparation method of the esterase-responsive charge reversal polymer ERP comprises the following steps: S1.1, dissolving polyethyleneimine and 2,6-di-tert-butyl-p-cresol in N,N-dimethylformamide, and then adding 4-acetoxybenzyl acrylate to react; S1.2, adding methyl iodide to the reaction product obtained in step S1.1, and performing a quaternization reaction in the dark; S1.3, precipitating the reaction product obtained in step S1.2 with diethyl ether, and separating and purifying the precipitate to obtain the esterase-responsive charge reversal polymer ERP.

5. Use of the nanoformulation according to any one of claims 1 to 3 in the preparation of a medicament for protecting liver ischemia-reperfusion injury.

6. A drug for protecting liver from ischemia-reperfusion injury, characterized in that: The active ingredient is the nanoformulation according to any one of claims 1 to 3.

7. The drug for protecting liver from ischemia-reperfusion injury according to claim 6, characterized in that: The drug was administered by intravenous injection twice within 24 hours before ischemia-reperfusion.

Images