Use of an anti-free radical polymer to enhance the therapeutic effect of stem cells

Abstract

This invention discloses a novel use of an anti-free radical polymer for enhancing the efficacy of stem cell therapy. The anti-free radical polymer has the following structural formula: #imgabs0#, where x represents the proportion of tetramethylpiperidinium nitroxide-modified moieties in the polymer, and is between 0.01 and 0.3; n represents the number of polymer segments, and is between 10 and 100; and R is selected from methyl, ethyl, n-propyl, isopropyl, and n-butyl. This invention can target mitochondria and bind free radicals, significantly enhancing the efficacy of stem cell therapy. Stem cells pre-treated with the anti-free radical polymer exhibit more pronounced organ repair and regeneration.

Description

Technical Field

[0001] The present invention relates to the technical field of organ repair and regeneration, and in particular to use of an anti-free radical polymer for improving the therapeutic effect of stem cells. Background Art

[0002] Stem cells (SCs) are a type of cell with the ability to self-renew and multidirectionally differentiate. Under certain conditions, they can differentiate into a variety of functional cells. Based on their developmental stage, stem cells are categorized as embryonic stem cells (ES cells) and adult stem cells. Based on their developmental potential, they are divided into three types: totipotent stem cells (TSCs), pluripotent stem cells, and unipotent stem cells. Simply put, stem cells are primitive, unspecialized cells that are not fully differentiated and have the potential to regenerate various tissues and organs. Stem cells are present in all multicellular tissues and can divide into a variety of specialized cells through mitosis and differentiation. They can also self-renew to provide more stem cells. This characteristic of stem cells has given them immense significance in medicine.

[0003] Organs in the body are composed of cells that originate from stem cells and then differentiate (change) into different forms. Stem cells promote tissue development, renewal, and regeneration. In theory, stem cells or their derivatives can be used to restore any tissue in the body that has been lost or damaged due to disease or injury. The potential of human pluripotent stem cells in cell therapy and other applications is enormous. The most potent stem cells are embryonic stem cells, which are discovered in the early stages of pregnancy or can be created outside the body through in vitro fertilization. Adult stem cells, present throughout life, are less potent but still crucial for maintaining overall health. They were originally discovered in the bone marrow but are now thought to be present in nearly every organ in the body. These adult stem cells also include newborn stem cells found in the umbilical cord and placenta. Finally, induced pluripotent stem cells are adult cells that have been reprogrammed in the laboratory to regenerate into stem cells similar to embryonic stem cells. Stem cells have many medical uses. Stem cells in the bone marrow, called hematopoietic stem cells, are used in bone marrow transplants for patients with cancer or other blood disorders. Currently, my country relies overwhelmingly on imported drugs critical for improving the efficacy of stem cell transplants. This limited access to key technologies and the high cost of these drugs increases the burden on patients.

[0004] Research has found that ROS control plays a crucial role in cell differentiation in vivo. Inhibition of mitochondrial superoxide promotes the development and differentiation of induced pluripotent stem cells, and inhibiting mitochondrial dysfunction can delay the aging of mesenchymal stem cells. Improving the efficacy of stem cell therapy and, in turn, enhancing the ability to repair and regenerate organs has become an urgent issue. Summary of the Invention

[0005] The purpose of the present invention is to provide an anti-free radical polymer for improving the therapeutic effect of stem cells. The polymer can target mitochondria and has the function of binding free radicals, thereby improving the therapeutic effect of stem cells by pre-treating stem cells.

[0006] The technical solution adopted by the present invention to solve its technical problem is:

[0007] An anti-free radical polymer is used as a synergist to enhance the therapeutic effect of stem cells, wherein the anti-free radical polymer has the following structural formula:

[0008]

[0009] Wherein, x represents the proportion of tetramethylpiperidinium nitrogen oxide modified parts in the polymer, and x is 0.01-0.3; n represents the number of chain segments of the polymer, and n is 10-100, and R is selected from one of methyl, ethyl, n-propyl, isopropyl, and n-butyl.

[0010] In the polymer of this invention, tetramethylpiperidinium nitroxide (TEMPO) is primarily used to capture and scavenge oxygen free radicals. TEMPO is an orange-red sublimed crystal or liquid that is readily soluble in water and organic solvents such as ethanol and benzene. However, it is toxic, corrosive, absorbable through the skin, and highly irritating. Due to its high toxicity, TEMPO is not conventionally used as a pharmaceutical ingredient. After extensive research, the inventors discovered that random copolymerization of TEMPO with OPDEA weakens its interaction with platelets due to the hydrophilicity of OPDEA, significantly reducing the cytotoxicity of TEMPO. This method leverages TEMPO's ability to capture and scavenge oxygen free radicals while overcoming the toxicity associated with its use. To minimize the toxicity of the TEMPO compound, x in this invention is 0.01-0.3, preferably 0.1; the polymer molecular weight is preferably 10,000; and n is preferably 50.

[0011] Mitochondria are the primary source of reactive oxygen species (ROS) within cells. Targeting mitochondria for scavenging ROS can minimize oxidative damage. The inventors have discovered that the anti-radical polymers of the present invention can rapidly enter cells, target mitochondria, and effectively capture and scavenge ROS. Stem cells treated with these anti-radical polymers can significantly mitigate organ ischemia-reperfusion injury. The anti-radical polymers enter stem cell mitochondria, improving their state and reducing mitochondrial loss during stem cell migration in vivo, thereby enhancing stem cell efficacy.

[0012] The anti-free radical polymer treats stem cells, thereby enhancing the therapeutic efficacy of stem cells. Improving the therapeutic efficacy of stem cells refers to enhancing the effectiveness of stem cells in treating injuries. More preferably, the invention enhances the effectiveness of stem cells in treating organ transplant injuries, including but not limited to the liver, kidneys, lungs, and brain.

[0013] When the anti-free radical polymer is used to treat stem cells, the concentration of the anti-free radical polymer is controlled to be 4 mg / mL.

[0014] The anti-free radical polymer treatment of stem cells specifically comprises adding the anti-free radical polymer into the stem cells and co-culturing the stem cells, and incubating the cells at 37° C. for 24 hours.

[0015] The stem cells include bone marrow-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells.

[0016] A synergist for improving the therapeutic effect of stem cells, comprising a therapeutically effective dose of the anti-free radical polymer.

[0017] The method for preparing the anti-free radical polymer comprises the following steps:

[0018] Step 1: using a synthesis method of free radical polymerization, atom radical transfer polymerization, or reversible addition-fragmentation chain transfer polymerization to polymerize 2,2,6,6-tetramethyl-4-piperidinyl methacrylate and amino methacrylate monomers;

[0019] Step 2: After the polymerization reaction is completed, the tertiary amine groups in the product are oxidized to obtain an anti-free radical polymer.

[0020] The polymerization reaction solvent is selected from at least one of methanol, isopropanol, toluene, N,N-dimethylformamide, and dioxane. The polymerization reaction is carried out under nitrogen, at a reaction temperature of 25-80°C, and for a polymerization time of 4-24 hours. The tertiary amine groups in the product are oxidized by adding an oxidizing agent to the product and stirring the reaction at room temperature for 4-12 hours. The oxidizing agent is m-chloroperbenzoic acid or hydrogen peroxide. The amount of the oxidizing agent is 1-5 times the molar amount of the tertiary amine groups in the product.

[0021] A drug that can enhance the therapeutic effect of stem cells, with a concentration of 4 mg / mL used for pre-treatment of stem cells.

[0022] The anti-free radical polymer described in the present invention is a tertiary amine nitrogen oxide polymer that can target mitochondria and has the function of binding free radicals; stem cells treated with this anti-free radical polymer can effectively improve the therapeutic effect of stem cell therapy, and this free radical capture polymer is expected to become a new treatment drug for stem cell transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results of the in vitro hemolysis experiment of OP10;

[0024] Figure 2 Confocal microscopy images of mitochondria after OP10 was co-incubated with bone marrow mesenchymal stem cells (BMSCs) for 1 hour; statistical analysis of the coincidence between OP10 and mitochondria;

[0025] Figure 3 This is the anti-free radical effect diagram of OP10 tested by ABTS method;

[0026] Figure 4 The free radical scavenging rate results of TM, OP, OP10, and OP20 at different time points;

[0027] Figure 5 Fluorescence microscopy data showing that OP10 and OP20 reduce ROS in normal hepatocytes of AML12 mice;

[0028] Figure 6 The results of in vitro cytotoxicity experiments of OP10 are shown;

[0029] Figure 7 This is the result of in vitro cytotoxicity experiment of TM;

[0030] Figure 8 This is the result of in vitro cytotoxicity experiment of OP20;

[0031] Figure 9 The results of serum ALT and AST changes after 75 minutes of partial liver ischemia and 24 hours of reperfusion;

[0032] Figure 10 This is the HE staining result after partial liver ischemia for 75 minutes and reperfusion for 24 hours;

[0033] Figure 11 This is the TUNEL staining result after 75 min of partial liver ischemia and 24 h of reperfusion;

[0034] Figure 12 This is the Ly6G staining result of a portion of the liver after 75 minutes of ischemia and 24 hours of reperfusion;

[0035] Figure 13 This is the result of F4 / 80 staining after 75 minutes of partial liver ischemia and 24 hours of reperfusion; DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described in detail below through specific embodiments.

[0037] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following examples, unless otherwise specified, are all conventional methods in the art.

[0038] Example 1 (OP10 synthesis):

[0039] OP10

[0040] OP10 was synthesized by atom-free radical random copolymerization of diethylaminoethyl methacrylate (DEA) and 2,2,6,6-tetramethyl-4-piperidinyl methacrylate (TEMP) in a 9:1 molar ratio. 2-((tert-Butoxycarbonyl)amino)ethyl 2-bromo-2-methylpropanoate served as the initiator, 2,2-bipyridine served as the ligand, and cuprous bromide served 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 reaction was performed under N2 atmosphere with three freeze-and-pump steps. Finally, 115 mg of initiator was added, and the reaction was continued in an oil bath at 40°C for 4 hours. The polymerization was terminated after the reaction. Copper was removed by passing through a neutral alumina column, and the crude product was precipitated in n-hexane.

[0041] To 1 g of the polymer, 4 mL of 30% hydrogen peroxide solution was added and stirred at room temperature for 4 hours. The product became a viscous solution. The solution was then dialyzed to remove the hydrogen peroxide. After freeze-drying, OP-10 (0.92 g, 89% yield) was obtained.

[0042] OP-10 NMR data: 1 H NMR (400 MHz, CDCl3) δ: ppm 5.10 (s, -CH ), 4.12 (s, - CH 2 ), 3.66 (s, -CH 2 ), 2.72 (s, -CH 2 ), 2.54 (s, -CH 3 ), 1.75 (s, -CH 2), 1.56 (s, - CH 3 ), 1.38 (s, -CH 3 ), 0.93 (s, -CH 3 ).

[0043] Example 2 (OP20 synthesis):

[0044] OP20

[0045] OP20 was synthesized by atom-free radical random copolymerization of DEA and TEMP in 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 then cooled and cooled under N2 atmosphere, with three freeze-and-drain steps. Finally, 115 mg of the initiator, 2-((tert-butoxycarbonyl)amino)ethyl 2-bromo-2-methylpropionate, was added, and the mixture was reacted in an oil bath at 40°C for 4 hours. After completion of the reaction, the polymerization was terminated. Copper was removed by passing the mixture through a neutral alumina column, and the crude product was precipitated in n-hexane.

[0046] To 1 g of the polymer, add 4 mL of 30% hydrogen peroxide solution and stir at room temperature for 4 hours. The product becomes a viscous solution. After dialysis, the hydrogen peroxide is removed. After freeze-drying, OP-20 (0.88 g, 85% yield) is obtained.

[0047] OP-20 NMR data: 1 H NMR (400 MHz, CDCl3) δ: ppm 5.11 (s, -CH ), 4.15 (s, - CH 2 ), 3.56 (s, -CH 2 ), 2.70 (s, -CH 2 ), 2.51 (s, -CH 3 ), 1.70 (s, -CH 2 ), 1.56 (s, - CH 3 ), 1.38 (s, -CH 3 ), 0.93 (s, -CH 3 ).

[0048] Experimental part:

[0049] Test Example 1: Safety Effect Test of Anti-Free Radical Polymer Materials:

[0050] 1. Security testing methods

[0051] Hemolysis test

[0052] Fresh blood was collected from the mouse orbital cavity into sodium heparin-pretreated tubes. Red blood cells were washed three times with wash buffer (containing 150 mM NaCl and 6 mM glucose) to separate erythrocytes from serum components. Erythrocytes were incubated with varying concentrations of OP10 in PBS (10 mM, pH 7.4). The mixture was incubated at 37°C for 1 hour. After incubation, the erythrocyte suspension was centrifuged at 300 g for 5 minutes. As a positive control, erythrocytes were lysed with 0.01% (w / v) Triton X-100. The absorbance of the supernatant was measured at 541 nm using a microplate spectrophotometer.

[0053] 2. Safety test results

[0054] like Figure 1 No significant hemolysis was observed in erythrocytes incubated with 1 mg / ml, 5 mg / ml, and 13.5 mg / ml OP10. However, hemolysis was observed in erythrocytes incubated with 0.1% Triton as a positive control. The hemolysis rate of erythrocytes incubated with 1 mg / ml, 5 mg / ml, and 13.5 mg / ml OP10 was less than 5%.

[0055] Experimental Example 2: OP10 Targeted Mitochondria Experiment

[0056] 1. OP10 co-localization with mitochondria

[0057] AML12 cells were plated at 1 × 10 cells per dish in 1 mL of culture medium. 5 Cells were plated at a density of 100 μg / mL in glass-bottom culture dishes and incubated for 24 hours. The culture medium was replaced with 1 mL of fresh culture medium containing OP10-Cy5 at a dose of 0.5 μg / mL. After the timed incubation, cells were further incubated with Mito-Tracker Green (Invitrogen, 200 nM) for 0.5 hours. Cell nuclei were stained with Hoechst 33342 (Invitrogen, 2 drops) for 15 minutes. 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 is blue, Mito-Tracker Green is green, and OP10-Cy5 is red.

[0058] 2. OP10 colocalization with mitochondria

[0059] like Figure 2 The red OP10-Cy5 region overlaps with the green mitochondrial region and appears yellow, indicating that OP10 enters the mitochondria. Line drawing statistical analysis shows that the overlapping lines of the red OP10-Cy5 region and the green mitochondrial region are highly overlapped, indicating that OP10 targets the mitochondria.

[0060] Test Example 3: In vitro determination of total antioxidant capacity of anti-free radical polymers

[0061] 1. In vitro total antioxidant capacity test method for anti-free radical polymers

[0062] ABTS (2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) was treated with 2.45 mM potassium persulfate overnight. The solution then turned dark blue, yielding the ABTS radical cation (ABTS+). 200 µL of this diluted solution was then diluted and mixed with various concentrations of materials. Radical scavenging efficiency was measured by measuring the absorbance at 734 nm.

[0063] 2. Results of in vitro total antioxidant capacity test of anti-free radical polymers

[0064] like Figure 3 The blue color of the ABTS free radical solution with OP10 added became lighter than that of the CONTROL group, indicating that OP10 has free radical scavenging activity and is an antioxidant.

[0065] like Figure 4 The free radical scavenging efficiency of TM, OP10, and OP20 was concentration-dependent, and OP had no obvious free radical scavenging effect.

[0066] Test Example 4: Anti-free radical polymer reduces intracellular ROS test

[0067] 1. Experimental method for reducing intracellular ROS by anti-radical polymers

[0068] DCFH-DA freely crosses the cell membrane and is hydrolyzed by lipases within the cell, leaving DCF impermeable to the cell. DCF is oxidized by ROS, emitting green fluorescence that can be detected using the FITC channel. AML12 cells were pretreated with the indicated drugs for 1 hour and then incubated with hydrogen peroxide (300 μM) for 1 hour to induce oxidative stress. The cells were then rinsed with PBS (10 mM, pH 7.4) and stained with DCFH-DA according to the manufacturer's instructions. Finally, after washing away the free dye, the cells were analyzed using confocal microscopy.

[0069] 2. Results of the Anti-Free Radical Polymer Experiment on Reducing Intracellular ROS

[0070] like Figure 5 The green fluorescence of OP10 and OP20 groups decreased significantly, which significantly reduced the level of intracellular free radicals. Glutathione (GSH), TM, and OP groups did not significantly reduce ROS.

[0071] Test Example 5: In vitro cytotoxicity test of anti-free radical polymers

[0072] In vitro cytotoxicity test method for anti-free radical polymers

[0073] Mouse hepatocyte AML12 cell line or mouse bone marrow-derived mesenchymal stem cells (BMSC) were seeded into 96-well plates (1×10 4 Cells were plated in 4% paraformaldehyde (400 cells / well) and cultured overnight. Subsequently, the cells were incubated with various concentrations of TEMPO, OP10, or OP20 for 24 hours. Cell viability was assessed using the CCK-8 assay.

[0074] In vitro cytotoxicity test results of anti-free radical polymers

[0075] like Figure 6-8 , TEMPO IC50 is about 4.5 mM, OP10, OP20 have no obvious decrease in cell viability at the corresponding TEMPO equivalents, and IC50 is significantly increased, indicating that the toxicity of the new anti-free radical polymer is much less than TEMPO.

[0076] Test Example 6: Anti-free radical polymer material to enhance the effect of stem cell therapy:

[0077] The condition treated with stem cells is liver ischemia-reperfusion injury.

[0078] 1. Method

[0079] DMEM-F12 cell growth medium: DMEM / F12 (1:1) medium (Biosharp) was supplemented with 10% fetal bovine serum (FBS) by volume, and then 1% double-antibody storage solution (penicillin-streptomycin solution (100X), Biotime, China) was added by volume to achieve final concentrations of penicillin and streptomycin of 100 U / mL and 100 U / mL, respectively. The cells were stored in a refrigerator at 4°C.

[0080] Murine bone marrow mesenchymal stem cells (BMSCs, C57BL / 6 mouse BMSCs) were added to DMEM-F12 cell growth medium, and OP10 was added to a final concentration of 4 mg / mL. The cells were incubated at 37°C for 24 hours, then washed three times with PBS to remove OP10, and the stem cells were collected by trypsin digestion. The cells were then washed three times with PBS to remove the trypsin, and the BMSCs were collected for later use. A 0.9% NaCl aqueous solution (CTRL group) was used as a control group. The specific method is as follows:

[0081] A 70% hepatic ischemia-reperfusion model was established in 8-week-old C57BL / 6 male mice. The portal vein was clamped for 75 minutes and reperfused for 24 hours. The treatment group was injected with 1 million BMSCs or OP10-treated BMSCs into the tail vein immediately after the modeling surgery, while the control group was injected with 0.9% NaCl aqueous solution instead.

[0082] 2. Results

[0083] like Figure 9 The portal vein was clamped for 75 minutes in the CTRL group, the BMSC group, and the OP10-treated BMSC group. After 24 hours of reperfusion, blood was collected from the retro-orbital venous plexus of C57BL / 6 mice and separated at 3000 rpm to obtain serum. The changes in ALT and AST were measured. Figure 10-13 The portal vein was clamped for 75 minutes in the CTRL group, the BMSC group, and the OP10-treated BMSC group. After 24 hours of reperfusion, the damaged liver lobe was sectioned and stained to observe the area of ​​apoptosis and the fluorescence intensity of immune cells. Under the same portal vein clamping time, the serum ALT and AST levels in the OP10-treated BMSC group were significantly lower than those in the BMSC group. HE staining showed smaller areas of cell damage, TUNEL staining showed fewer areas of apoptosis, Ly6G staining showed fewer neutrophils, and F4 / 80 staining showed fewer macrophages. This indicates that OP10-treated stem cells are more effective in alleviating liver ischemia-reperfusion injury than stem cells alone.

[0084] The embodiment described above is only a preferred solution of the present invention and does not limit the present invention in any form. Other variations and modifications are possible without exceeding the technical solution described in the claims.

Claims

1. An anti-free radical polymer is used as a synergist for improving the therapeutic effect of stem cells, characterized in that: The structural formula of the anti-free radical polymer is shown below: Wherein, x represents the proportion of tetramethylpiperidinium nitroxide modified parts in the polymer, and x is 0.01-0.3; n represents the number of chain segments of the polymer, and n is 10-100; R is selected from one of methyl, ethyl, n-propyl, isopropyl, and n-butyl; After the anti-free radical polymer is treated with stem cells, the effect of stem cells in treating liver ischemia-reperfusion injury is enhanced; The stem cells are bone marrow-derived mesenchymal stem cells or umbilical cord-derived mesenchymal stem cells.

2. The use according to claim 1, characterized in that When the anti-free radical polymer is used to treat stem cells, the concentration of the anti-free radical polymer is controlled to be 4 mg / mL.

3. The use according to claim 1, characterized in that The anti-free radical polymer treatment of stem cells specifically comprises adding the anti-free radical polymer into the stem cells and co-culturing the stem cells, and incubating the cells at 37° C. for 24 hours.

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