An anti-free radical polymer capable of alleviating ischemia-reperfusion injury of organs, and a preparation method and use thereof

Abstract

The application discloses an anti-free radical polymer capable of reducing organ ischemia-reperfusion injury, a preparation method and application thereof, and a structural formula is shown as follows: wherein x represents a proportion of a tetramethylpiperidine modification part in the polymer, x is 0.01-0.3; n represents a chain number of the polymer, n is 10-100, and R is selected from one of a methyl group, an ethyl group, a normal propyl group, an isopropyl group and a normal butyl group. The application can target mitochondria, has a function of combining free radicals, has an obvious effect of reducing liver ischemia-reperfusion injury, and has an effect of reducing cold ischemia injury of the liver when being added into an organ preservation solution.

Description

Technical Field

[0001] This invention relates to the field of organ transplantation technology, and in particular to an anti-free radical polymer that can alleviate organ ischemia-reperfusion injury, its preparation method, and its uses. Background Technology

[0002] Organ transplantation is hailed as a major life science advancement of the 20th century that has changed human lives, and it is a globally recognized effective method for treating various end-stage diseases. The fundamental purpose of organ preservation is to maintain the viability of organs after transplantation, minimize ischemic damage, make them more suitable for transport, and enable them to recover function more quickly after surgery. UW solution, invented by the University of Wisconsin in the United States, is recognized as the standard preservation solution for the liver, pancreas, and kidneys. Clinical studies have shown that it can preserve kidneys for 72 hours and livers for 20-24 hours. However, this preservation solution is high in potassium and low in sodium, which can easily cause damage to vascular endothelial cells. Currently, the vast majority of preservation solutions used in organ transplantation surgery are imported from abroad, leaving key technologies dependent on foreign sources and incurring high costs, thus increasing the burden on patients.

[0003] The ischemia / reperfusion process during organ transplantation generates a large number of reactive oxygen species, which aggravates oxidative damage to donor organs, especially marginal donor organs, thereby reducing the utilization rate of transplanted organs and exacerbating organ shortages. How to mitigate organ ischemia-reperfusion injury and thus improve the utilization rate of transplanted organs has become an urgent problem to be solved. Summary of the Invention

[0004] The purpose of this invention is to provide an anti-free radical polymer that can reduce organ ischemia-reperfusion injury, its preparation method and uses, which can target mitochondria, has the function of binding free radicals, has a significant effect on reducing liver ischemia-reperfusion injury, and can reduce liver cold ischemia injury when added to organ preservation fluid.

[0005] The technical solution adopted by this invention to solve its technical problem is:

[0006] An anti-free radical polymer capable of mitigating organ ischemia-reperfusion injury has the following structural formula:

[0007]

[0008] Where x represents the proportion of tetramethylpiperidine oxynitride modified in the polymer, x is 0.01-0.3; n represents the number of repeating units in the polymer, n is 10-100; and R is selected from methyl, ethyl, n-propyl, isopropyl, and n-butyl.

[0009] In the polymer of this invention, tetramethylpiperidine oxynitride (TEMPO) is mainly used to capture and scavenge oxygen free radicals. TEMPO is an orange-red sublimed crystal or liquid, readily soluble in water and organic solvents such as ethanol and benzene. It is toxic, corrosive, can be absorbed through the skin, and is highly irritating. Due to its strong toxicity, TEMPO is not conventionally used as a pharmaceutical raw material. Through long-term research, the inventors discovered that when TEMPO is randomly copolymerized with OPDEA, the hydrophilicity of OPDEA weakens its interaction with platelets, significantly reducing the cytotoxicity of TEMPO. Thus, this invention utilizes the oxygen free radical capture and scavenging properties of TEMPO while overcoming the toxicity issues associated with its application. To minimize the toxicity of the TEMPO composite, x is taken as 0.01-0.3, preferably 0.1; the polymer molecular weight is preferably 10,000, and n is preferably 50.

[0010] Mitochondria are the main source of reactive oxygen species (ROS) in cells. Targeting mitochondria to scavenge ROS can minimize oxidative damage to transplanted organs. The inventors discovered that the anti-free radical polymer of this invention can rapidly enter cells, target mitochondria, effectively capture and scavenge ROS, and significantly reduce organ ischemia-reperfusion injury.

[0011] The method for preparing the anti-free radical polymer includes the following steps:

[0012] Step 1: The 2,2,6,6-tetramethyl-4-piperidinyl methacrylate monomer is polymerized using free radical polymerization, atom radical transfer polymerization, or reversible addition-fragmentation chain transfer polymerization.

[0013] Step 2: After the polymerization reaction is completed, the tertiary amine groups in the product are oxidized to obtain an anti-free radical polymer.

[0014] Preferably, the polymerization solvent is selected from at least one of methanol, isopropanol, toluene, N,N-dimethylformamide, and dioxane.

[0015] Preferably, the polymerization reaction is carried out in nitrogen atmosphere at a temperature of 25-80°C for 4-24 hours.

[0016] Preferably, the oxidation of the tertiary amine group in the product is specifically carried out by adding an oxidant to the product and stirring the reaction at room temperature for 4-12 hours; the oxidant is m-chloroperoxybenzoic acid or hydrogen peroxide.

[0017] Preferably, the amount of oxidant used is 1-5 times the molar amount of tertiary amine groups in the product.

[0018] The aforementioned free radical scavenging polymer is used as an additive in organ preservation solutions that can reduce organ ischemia-reperfusion injury during organ transplantation.

[0019] Preferably, the organ includes the liver.

[0020] An organ preservation solution capable of mitigating organ ischemia-reperfusion injury, the organ preservation solution comprising the aforementioned anti-free radical polymer. The amount of the anti-free radical polymer in the organ preservation solution is 2-3 mg / mL.

[0021] As one embodiment, the organ preservation solution of the present invention has the following composition:

[0022] NaCl 12-18 mmol / L, KCl 6-10 mmol / L, MnCl2 6-10 mmol / L, Histidine 160-200 mmol / L, Histidine-hydrochloric acid 12-18 mmol / L, Tryptophan 2-4 mmol / L, Mannitol 15-25 mmol / L, Anti-free radical polymer 2-3 mg / mL, Water balance.

[0023] Preferably, the organ preservation solution is stored at a temperature of 0-8℃.

[0024] The free radical scavenging polymer described in this invention is a nitrogen oxide tertiary amine polymer that can target mitochondria and has the function of binding free radicals. It significantly reduces liver ischemia-reperfusion injury and, when added to organ preservation fluid, alleviates cold ischemia injury to the liver. This free radical scavenging polymer shows promise as an additive for novel organ transplant perfusion fluids (organ preservation fluids), benefiting organ transplant patients and possessing excellent application prospects. Attached Figure Description

[0025] Figure 1 The 1H NMR spectrum of OP-10;

[0026] Figure 2 The 1H NMR spectrum of OP-20;

[0027] Figure 3 The results show the changes in serum ALT, AST, IL-6, and TNF-α after liver ischemia for 75 min and reperfusion for 60 min.

[0028] Figure 4 HE staining image of the liver after 75 min of ischemia and 60 min of reperfusion (200x);

[0029] Figure 5 TUNEL staining image (200x) of the liver after 75 min of ischemia and 60 min of reperfusion;

[0030] Figure 6Oil Red staining images (200x) of livers after preservation in organ preservation solutions at 4°C for 24 hours in each group;

[0031] Figure 7 Results of in vitro hemolysis experiment of OP10;

[0032] Figure 8 A confocal microscope image of OP10 entering the mitochondria;

[0033] Figure 9 This is a graph showing the ABTS method's effect on OP10's free radical scavenging properties.

[0034] Figure 10 These are the free radical scavenging rates of TM, OP, OP10, and OP20 at different time points;

[0035] Figure 11 These are fluorescence microscopy data showing that OP10 and OP20 reduced ROS in normal hepatocytes of AML12 mice.

[0036] Figure 12 These are the results of the in vitro cytotoxicity assay for OP10;

[0037] Figure 13 These are the results of TM's in vitro cytotoxicity assay;

[0038] Figure 14 These are the results of the in vitro cytotoxicity experiment of OP20. Detailed Implementation

[0039] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0040] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. Unless otherwise specified, the methods in the following embodiments are conventional methods in the field.

[0041] Example 1 (OP10 synthesis):

[0042]

[0043] OP10 was synthesized by atomic radical random copolymerization of diethylaminoethyl methacrylate (DEA) and 2,2,6,6-tetramethyl-4-piperidinyl methacrylate (TEMP) in a molar ratio of 9:1. 2-((tert-Butoxycarbonyl)amino)ethyl 2-bromo-2-methylpropionate ethyl ester was used as the initiator, 2,2-bipyridine as the ligand, and cuprous bromide as the catalyst. 3.34 g of DEA and 0.45 g of TEMP were dissolved in 4 mL of methanol, and 53 mg of cuprous bromide and 116 mg of 2,2-bipyridine were added. The mixture was subjected to a triple freeze-thaw cycle under N2 gas. Finally, 115 mg of the initiator was added, and the reaction was carried out in an oil bath at 40 °C for 4 hours. The polymerization was terminated after the reaction was complete. Copper was removed by passing the product through a neutral alumina column, and the crude product was precipitated in n-hexane.

[0044] Take 1g of the above polymer, add 4mL of 30% hydrogen peroxide solution, and stir at room temperature for 4 hours. The product is a viscous solution. After the reaction, dialyze to remove the hydrogen peroxide. After lyophilization, OP-10 (0.92g, yield: 89%) is obtained.

[0045] Figure 1 NMR data analysis: 1 H NMR(400MHz, CDCl3)δ:ppm 5.10(s,-CH),4.12(s,-CH2),3.66(s,-CH2),2.72(s,-CH2),2.54(s,-CH3),1.75(s,-CH2),1.56(s,-CH3),1.38(s,-CH3),0.93(s,-CH3).

[0046] Example 2 (OP20 synthesis):

[0047]

[0048] OP20 was synthesized by atomic radical random copolymerization of DEA and TEMP at a molar ratio of 8:2. 2.96 g of DEA and 0.92 g of TEMP were dissolved in 4 mL of methanol, and 53 mg of cuprous bromide and 116 mg of 2,2-bipyridine were added. The mixture was subjected to a triple freeze-thaw cycle under N2 gas. Finally, 115 mg of the initiator ethyl 2-((tert-butyloxycarbonyl)amino)ethyl 2-bromo-2-methylpropionate was added, and the reaction was carried out in an oil bath at 40 °C for 4 hours. The polymerization was terminated after the reaction was completed. Copper was removed by passing the product through a neutral alumina column, and the crude product was precipitated in n-hexane.

[0049] Take 1g of the above polymer, add 4mL of 30% hydrogen peroxide solution, and stir at room temperature for 4 hours. The product is a viscous solution. After the reaction, dialyze to remove the hydrogen peroxide. After lyophilization, OP-20 (0.88g, yield: 85%) is obtained.

[0050] Figure 2 NMR data analysis: 1 H NMR(400MHz, CDCl3)δ:ppm 5.11(s,-CH),4.15(s,-CH2),3.56(s,-CH2),2.70(s,-CH2),2.51(s,-CH3),1.70(s,-CH2),1.56(s,-CH3),1.38(s,-CH3),0.93(s,-CH3).

[0051] Example 2 (Organ Preservation Solution):

[0052] The concentrations of each component in the organ preservation solution of this invention are shown in Table 1.

[0053] Table 1. Components of organ preservation solutions containing anti-free radical polymers

[0054] Element concentration NaCl 15mmol / l KCl 8mmol / l <![CDATA[MnCl2]]> 8mmol / l Histidine 180mmol / l Histidine hydrochloride (Histidine-HCl) 15mmol / l Tryptophan 2mmol / l Mannitol 20mmol / l OP10 3mg / ml water margin

[0055] The following specific experimental examples demonstrate the beneficial effects of the present invention:

[0056] Experimental Example 1: Test on the effect of anti-free radical polymer materials in reducing ischemia-reperfusion injury:

[0057] 1. Methods to reduce liver ischemia-reperfusion injury

[0058] An OP10 PBS solution at a concentration of 6 mg / ml was used to test the reduction of liver ischemia-reperfusion injury. A 0.9% NaCl aqueous solution (CTRL group) served as a control group. The specific methods for treating liver ischemia-reperfusion injury are as follows:

[0059] A 70% hepatic ischemia-reperfusion model was established in C57BL / 6 male mice. The portal vein was clamped for 75 min and reperfused for 6 h. The treatment group was injected intraperitoneally with the material solution 5 h before modeling and the material solution was injected on the liver surface at the upper edge of the liver before reperfusion. The control group was replaced with 0.9% NaCl aqueous solution.

[0060] 2. Reduces liver ischemia-reperfusion injury

[0061] like Figure 3 In the CTRL group and the OP10 group, the portal vein was clamped for 75 minutes, and after reperfusion for 6 hours, blood was collected from the retro-orbital venous plexus of C57BL / 6. Serum was obtained by separation at 3000 rpm, and changes in ALT, AST, IL-6, and TNF-α were measured. Under the same portal vein clamping time, the serum ALT, AST, IL-6, and TNF-α levels in the OP10 group were significantly lower than those in the CTRL group.

[0062] like Figure 4Hematologic and epithelial (H&E) staining was performed on liver tissue in the CTRL and OP10 groups after clamping for 75 min and reperfusion for 6 h. In the CTRL group, hepatocytes showed significant nuclear pyknosis and fragmentation; hepatocyte edema with loose, pale cytoplasm; vacuolar degeneration of hepatocytes with small, round vacuoles visible in the cytoplasm; and dilation and compression of the sinusoids. Compared to the CTRL group (with the same ischemia and reperfusion time), the OP10 group showed significantly reduced hepatocyte nuclear pyknosis and fragmentation; clear liver lobule structure; neatly arranged hepatic cords; and no significant dilation or compression of the sinusoids, nor obvious inflammation.

[0063] like Figure 5 TUNEL staining was performed on the CTRL and OP10 groups after clamping for 75 min and reperfusion for 6 h. With the same ischemia and reperfusion time, the number of apoptotic hepatocytes in the OP10 group was significantly lower than that in the CTRL group.

[0064] Experimental Example 2: Test on the effect of the present invention in reducing cold ischemia injury:

[0065] 1. Organ preservation methods

[0066] The organ preservation solution prepared in Example 2 of this invention was used to test the reduction of liver cold ischemia injury, while the HTK solution alone (CTRL group) was used as a control group. The specific method for resisting liver cold ischemia injury is as follows:

[0067] A cold ischemia 24h model was constructed in the fatty liver of Bama pigs. The livers were cut into small pieces and randomly divided into control and experimental groups. The livers were obtained by portal vein perfusion with 4L of heparinized saline and then placed in HTK solution and a novel organ preservation solution for 24h. The area of ​​red lipid droplets in the oil red stained sections of the livers was then observed.

[0068] 2. Organ preservation effect

[0069] like Figure 6 The organ preservation solution prepared in Example 2, used as the experimental group, showed a significantly smaller red lipid droplet area in its oil red sections compared to the control group consisting solely of HTK solution. These results indicate that the anti-free radical polymer material prepared in this invention has a good mitigating effect on liver ischemia-reperfusion injury. Furthermore, the HTK solution containing the anti-free radical polymer material OP10, when stored at 0–4°C for 24 hours, showed significantly better results than the standard HTK solution.

[0070] 3. Experimental Example 3: Safety Effect Test of Anti-Free Radical Polymer Materials:

[0071] 1. Security Testing Methods

[0072] hemolysis test

[0073] Fresh blood was collected from the mouse orbital cavity into heparin sodium pretreated tubes. Red blood cells were separated from serum components by washing three times with washing buffer (containing 150 mM NaCl and 6 mM glucose). Red blood cells were incubated with different concentrations of OP10 in PBS (10 mM, pH 7.4). The mixture was incubated at 37°C for 1 hour. After incubation, the red blood cell suspension was centrifuged at 300 g for 5 minutes. As a positive control, red blood cells were lysed with 0.01% (w / v) Triton X-100. The absorbance of the supernatant was measured at 541 nm using a microplate spectrophotometer.

[0074] 2. Security test results

[0075] like Figure 7 Red blood cells co-incubated with OP10 at concentrations of 1 mg / ml, 5 mg / ml, and 13.5 mg / ml did not show significant hemolysis, while red blood cells co-incubated with 0.1% Triton (as a positive control) did show hemolysis. The hemolysis rate of red blood cells co-incubated with OP10 at concentrations of 1 mg / ml, 5 mg / ml, and 13.5 mg / ml was less than 5%.

[0076] Experimental Example 4: OP10 Targeting Mitochondria Assay

[0077] 1. OP10 co-localization method with mitochondria

[0078] AML12 cells in 1 mL of culture medium were divided into 1 × 10⁻⁶ cells per culture dish. 5 Cells were placed at a density of [number] cells in glass-bottomed culture dishes and incubated for 24 h. The culture medium was then replaced with fresh medium containing 1 mL of OP10-Cy5 (Cy5 dose: 0.5 μg / mL). After timed incubation, the cells were further incubated with Mito-Tracker Green (Invitrogen, 200 nM) for 0.5 h. The nuclei were stained with Hoechst 33342 (Invitrogen, 2 drops) for 15 min. The culture medium was then removed, and the cells were washed three times with cold PBS for confocal imaging (FV3000 system; Olympus, Japan). The excitation and emission wavelengths for each dye were set according to the manufacturer's instructions. Hoechst 33342 was blue, Mito-Tracker Green was green, and OP10-Cy5 was red.

[0079] 2. Results of OP10 co-localization with mitochondria

[0080] like Figure 8 The red OP10-Cy5 region overlaps with the green mitochondrial region, appearing yellow, indicating that OP10 has entered the mitochondria.

[0081] Experimental Example 5: In vitro determination of total antioxidant capacity of anti-free radical polymers

[0082] 1. In vitro test method for determining the total antioxidant capacity of free radical scavenging polymers

[0083] ABTS (2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) was incubated overnight with 2.45 mM potassium persulfate. The solution then turned deep blue, yielding ABTS radical cations (ABTS+). The solution was then diluted with 200 μL of the diluted solution and mixed with materials of different concentrations. The radical scavenging efficiency was observed to vary with absorbance at 734 nm.

[0084] 2. Results of in vitro determination of total antioxidant capacity of anti-free radical polymers

[0085] like Figure 9 Compared to the CONTROL group, the ABTS free radical solution with added OP10 showed a lighter blue color, indicating that OP10 has free radical scavenging activity and is an antioxidant.

[0086] like Figure 10 The free radical scavenging efficiency of TM, OP10, and OP20 was concentration-dependent, while OP did not show a significant free radical scavenging effect.

[0087] Experimental Example 6: Experiment on the Reduction of Intracellular ROS by Anti-Free Radical Polymers

[0088] 1. Assay method for reducing intracellular ROS using anti-free radical polymers

[0089] DCFH-DA freely passes through the cell membrane and is hydrolyzed by lipases inside the cell. DCF is impermeable to the cell. DCF is oxidized by ROS and emits green fluorescence, which can be detected by the FITC channel. AML12 cells were pretreated with the specified drug for 1 h, followed by incubation with hydrogen peroxide (300 μM) for 1 h to induce oxidative stress. The cells were then washed with PBS and stained with DCFH-DA according to the manufacturer's instructions. Finally, after washing away the free dye, the cells were analyzed using a confocal microscope.

[0090] 2. Results of assays showing that anti-free radical polymers reduce intracellular ROS.

[0091] like Figure 11 The green fluorescence in the OP10 and OP20 groups was significantly reduced, indicating a significant decrease in intracellular free radical levels. Glutathione (GSH), TM, and OP groups did not show a significant reduction in ROS.

[0092] Experimental Example 7: In vitro cytotoxicity test of anti-free radical polymers

[0093] In vitro cytotoxicity assays for anti-free radical polymers

[0094] Mouse hepatocyte AML12 cell line was seeded into 96-well plates (1×10⁶ cells / well). 4 Cells were cultured overnight in wells (10 cells / well). Subsequently, the cells were incubated for 24 hours with different concentrations of TEMPO, OP10, or OP20. Cell viability was assessed using the CCK-8 assay.

[0095] Results of in vitro cytotoxicity assays of anti-free radical polymers

[0096] like Figure 12 13, 14, TEMPO IC50 is approximately 4.5 μM. OP10 and OP20 showed no significant decrease in cell viability at the corresponding TEMPO equivalent, and their IC50 increased significantly, indicating that the toxicity of the novel anti-free radical polymer is much lower than that of TEMPO.

[0097] In summary, this invention provides an anti-free radical polymer that significantly reduces liver ischemia-reperfusion injury and, when added to organ preservation solutions, alleviates cold ischemia-related liver damage. This free radical scavenging polymer shows promise as an additive for novel organ transplant perfusion solutions (organ preservation solutions), benefiting organ transplant patients and possessing excellent application prospects.

[0098] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A free radical scavenging polymer capable of mitigating organ ischemia-reperfusion injury, characterized in that, Its structural formula is shown below: Where x represents the proportion of tetramethylpiperidine oxynitride modified in the polymer, x is 0.01-0.3; n represents the number of repeating units in the polymer, n is 10-100; and R is selected from methyl, ethyl, n-propyl, isopropyl, and n-butyl.

2. The method for preparing the anti-free radical polymer according to claim 1, characterized in that, Includes the following steps: Step 1: The 2,2,6,6-tetramethyl-4-piperidinyl methacrylate monomer is polymerized using free radical polymerization, atom radical transfer polymerization, or reversible addition-fragmentation chain transfer polymerization. Step 2: After the polymerization reaction is completed, the tertiary amine groups in the product are oxidized to obtain an anti-free radical polymer.

3. The preparation method according to claim 2, characterized in that, The solvent used in the polymerization reaction is selected from at least one of methanol, isopropanol, toluene, N,N-dimethylformamide, and dioxane.

4. The preparation method according to claim 2, characterized in that, The polymerization reaction is carried out in nitrogen atmosphere at a temperature of 25-80℃ for 4-24 hours.

5. The preparation method according to claim 2, characterized in that, The oxidation of the tertiary amine group in the product is specifically carried out by adding an oxidant to the product and stirring the reaction at room temperature for 4-12 hours; the oxidant is m-chloroperoxybenzoic acid or hydrogen peroxide.

6. The preparation method according to claim 5, characterized in that, The amount of oxidant used is 1-5 times the molar amount of tertiary amine groups in the product.

7. Use of the anti-free radical polymer as described in claim 1 as an additive in organ preservation solutions that can reduce organ ischemia-reperfusion injury during organ transplantation.

8. The use according to claim 7, characterized in that, The organs mentioned include the liver.

9. An organ preservation solution capable of mitigating organ ischemia-reperfusion injury, characterized in that, The organ preservation solution comprises the anti-free radical polymer as described in claim 1.

10. The organ preservation solution according to claim 9, characterized in that, The preservation temperature of organ preservation solution is 0-8℃.

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