Mitochondrial nano-enzyme compound and preparation method thereof
By preparing a complex of mitochondria and ceria nanoparticles, the toxic side effects caused by the use of high-dose drugs by conventional inflammatory treatment methods are solved, and faster and safer inflammatory treatment effects are achieved.
Patent Information
- Application Number
- CN202411929781.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-26
- Publication Date
- 2025-03-28
AI Technical Summary
Although conventional inflammatory treatment methods in the prior art can play an anti-inflammatory effect, large doses and long-term medications will cause toxicity to human organs and have greater side effects.
Mitochondrial nanoenzyme complex is used, which consists of mitochondria and ceria nanoparticles, and is formed by self-assembly through chemical reactions and has the ability to scavenge reactive oxygen species.
This complex can effectively remove reactive oxygen species, improve the therapeutic effect on inflammation, and reduce toxic side effects on human organs.
Smart Images
Figure CN119700817A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technology, and in particular to a mitochondrial nanoenzyme complex and a preparation method thereof. Background Art
[0002] With the emergence of nanozymes with enzyme-like activities, nanozymes with peroxidase activity, catalase activity and oxidase activity have been widely used in the treatment of various inflammatory diseases.
[0003] In the treatment of inflammation, conventional treatment methods such as large amounts of hormone therapy can also play an anti-inflammatory role, but large doses and long-term use of drugs will be toxic to human organs and have significant side effects. Therefore, it is necessary to provide a treatment method to replace conventional treatment methods in the prior art. Summary of the invention
[0004] The purpose of the present invention is to solve the technical problem that although conventional treatment methods in the prior art can have an anti-inflammatory effect, large doses and long-term use of the drugs will cause toxicity to human organs and have significant side effects.
[0005] In order to achieve the above object, the present invention adopts the following technical solutions:
[0006] A mitochondrial nanozyme complex, which is a complex of mitochondria and cerium dioxide, and the mitochondrial nanozyme complex is in the form of self-assembly through chemical reactions.
[0007] Preferably, the preparation method is as follows:
[0008] S1: preparation of cerium dioxide nanoparticles;
[0009] S2: preparing a functionalized cerium dioxide solution: dissolving the cerium dioxide nanoparticles, distearoylphosphatidylethanolamine-polyethylene glycol and dipalmitoylphosphatidylethanolamine-polyethylene glycol-maleimide in S1 in chloroform, performing ultrasonic treatment after mixing, and then removing chloroform from the sample, adding deionized water to the sample and performing ultrasonic treatment, and after the treatment, centrifuging and filtering to obtain a maleimide-functionalized cerium dioxide solution;
[0010] S3: Extract mitochondria from mouse heart tissue;
[0011] S4: Preparation of thiolated mitochondrial solution: Incubate the mitochondria obtained in S3 with Traut's reagent, stir at room temperature, and then wash the mitochondria three times with Tyrode's buffer to obtain thiolated mitochondria;
[0012] S5: Preparation of mitochondrial ceria nanozyme complex solution: The thiolated mitochondria of S4 were mixed with an excess of maleimide-functionalized ceria solution in Tyrode's buffer, gently stirred at room temperature for 3 h, and washed with PBS several times to obtain a mitochondrial ceria nanozyme complex solution.
[0013] Preferably, the preparation method in S1 is as follows: dissolving cerium nitrate hexahydrate and sodium citrate dihydrate in deionized water respectively, mixing the two solutions, then adding ammonia water and stirring for 24 hours, centrifuging after stirring to obtain the supernatant, dialyzing the supernatant, centrifuging again, and freeze-drying the supernatant to obtain cerium dioxide nanoparticles.
[0014] Preferably, the dialysis in S1 is performed using a 3 kDa dialysis bag for 24 hours, and after the dialysis is completed, the dialysis is centrifuged at 15000 g for 10 minutes, and the obtained cerium dioxide nanoparticles are stored at 4°C.
[0015] Preferably, the mass ratio of distearoylphosphatidylethanolamine-polyethylene glycol, dipalmitoylphosphatidylethanolamine-polyethylene glycol-maleimide and cerium dioxide nanoparticles in S2 is 2:1:2, and after the mixing in S2 is completed, ultrasonic treatment is performed for 5 minutes.
[0016] Preferably, in S2, a rotary evaporator is used to remove chloroform in the sample, and the sample is further dried in a vacuum oven at 60° C. for 3 h to ensure complete removal of chloroform.
[0017] Preferably, the mitochondria in S3 are extracted from mouse heart using a tissue mitochondrial isolation kit.
[0018] Preferably, the specific steps of S3 are as follows:
[0019] 1) Use fresh mouse heart tissue, usually no more than one hour after the animal was sacrificed and kept on ice;
[0020] 2) Weigh the cut heart tissue in a 1.5 mL centrifuge tube and wash once with PBS;
[0021] 3) Place the heart tissue in a centrifuge tube or culture dish on ice and cut the heart tissue into very small tissue fragments using scissors or a blade;
[0022] 4) Centrifuge to precipitate the tissue sample and discard the supernatant;
[0023] 5) Add pre-cooled trypsin digestion solution and place on ice;
[0024] 6) Centrifuge at 600 g for 10-20 seconds to pellet the tissue sample and discard the supernatant;
[0025] 7) Add mitochondrial isolation reagent and resuspend the tissue to wash away residual trypsin;
[0026] 8) Centrifuge at 600 g for 10-20 seconds to pellet the tissue sample and discard the supernatant;
[0027] 9) Add mitochondrial isolation reagent and homogenize on ice for 20-30 times;
[0028] 10) Centrifuge the homogenate at 1000g, 4°C for 5 minutes;
[0029] 11) Carefully transfer the supernatant to another centrifuge tube and centrifuge again;
[0030] 12) Carefully remove the supernatant; the precipitate is the isolated mitochondria.
[0031] In the present application, a mitochondrial nanozyme complex and a preparation method thereof are constructed, which connects two key components, cerium dioxide nanoparticles and mitochondria, so that the two can work alone or in conjunction with each other to produce a synergistic effect, providing a faster treatment for inflammatory diseases; specifically, the present application utilizes the ability of mitochondria and cerium dioxide to scavenge reactive oxygen species to prepare a simple mitochondrial nanozyme complex, which has a significant effect of scavenging reactive oxygen species, effectively improving the therapeutic effect on inflammation. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 is an electron microscope image of cerium dioxide nanoparticles in one embodiment of the present invention;
[0033] Figure 2 1 is a characterization diagram of ceria nanoparticles and mitochondrial ceria nanozyme complexes in one embodiment of the present invention, wherein A and B are particle size diagrams of the two, respectively, and C is a potential diagram of the two;
[0034] Figure 3 Schematic diagram demonstrating the enzyme activity of cerium dioxide nanoparticles in one embodiment of the present invention, including hydroxyl radical scavenging rate (A) and SOD inhibition rate (B).
[0035] Figure 4 Schematic diagram demonstrating the ROS-scavenging ability of ceria nanoparticles and mitochondrial ceria nanozyme complexes in one embodiment of the present invention. DETAILED DESCRIPTION
[0036] The present invention is further described in detail below in conjunction with specific embodiments.
[0037] A mitochondrial nanozyme complex, which is a complex of mitochondria and cerium dioxide, and the mitochondrial nanozyme complex is in the form of self-assembly through chemical reactions.
[0038] The preparation method of the mitochondrial nanozyme complex is as follows:
[0039] S1: Preparation of cerium dioxide nanoparticles:
[0040] Weigh 108.5 mg of cerium nitrate hexahydrate and 100 mg of sodium citrate dihydrate and dissolve them in 2 mL and 1 mL of deionized water, respectively, and then mix them (the solid particles need to be completely dissolved, and the purpose of adding sodium citrate dihydrate is to prevent the aggregation of cerium dioxide nanoparticles that have just precipitated, so as to ensure that the particle size of the prepared cerium dioxide nanoparticles is small enough and evenly distributed), then add 50 mL of 0.4 M ammonia water as a precipitant, stir for 24 hours (to slowly precipitate the cerium dioxide nanoparticles), then centrifuge to remove the aggregated particles, take the supernatant, dialyze with a 3 kDa dialysis bag for 24 hours, centrifuge at 15000 g for 10 minutes, take the supernatant and freeze-dry, finally obtaining cerium dioxide nanoparticles, which are stored at 4°C.
[0041] S2: Preparation of functionalized cerium dioxide solution:
[0042] Distearoylphosphatidylethanolamine-polyethylene glycol and dipalmitoylphosphatidylethanolamine-polyethylene glycol-maleimide were dissolved in chloroform and then mixed with the cerium dioxide nanoparticles prepared in S1 in chloroform, wherein the mass ratio of distearoylphosphatidylethanolamine-polyethylene glycol, dipalmitoylphosphatidylethanolamine-polyethylene glycol-maleimide and cerium dioxide nanoparticles was 2:1:2.
[0043] After the mixing was completed, the samples were ultrasonically treated for 5 minutes, and the chloroform in the samples was removed using a rotary evaporator. The samples were further dried in a vacuum oven at 60°C for 3 h to ensure the complete removal of chloroform.
[0044] Next, deionized water was added to the sample and ultrasonicated to obtain a transparent colloidal suspension. The obtained cerium dioxide sample was centrifuged at 5000 g to remove excess PEG and purified using a 0.22 μm filter to finally obtain a maleimide-functionalized cerium dioxide solution.
[0045] S3: Extraction of mitochondria from mouse heart tissue:
[0046] The mitochondria were extracted from the heart of BALB / C mice using a tissue mitochondrial isolation kit; the specific steps are as follows:
[0047] (1) Use fresh mouse heart tissue, usually no more than one hour after the animal was sacrificed, and keep it on ice.
[0048] (2) Weigh the excised heart tissue in a 1.5 mL centrifuge tube and wash once with PBS.
[0049] (3) Place the heart tissue in a centrifuge tube or culture dish on ice, cut the heart tissue into very small tissue fragments with scissors or a blade, add 10 times the volume of pre-cooled PBS, and place on ice for 3 minutes.
[0050] Note: If the weight of the tissue is 80 mg, it can be roughly assumed that the volume of the tissue is close to 80 μl, and 800 μl of PBS should be added.
[0051] (4) Centrifuge at 600 g for 10-20 seconds to pellet the tissue sample and discard the supernatant.
[0052] (5) Add 8 times the volume of pre-cooled trypsin digestion solution and place on ice for 20 minutes.
[0053] Note: If the tissue weight is 80 mg, then add about 640 μL of the corresponding trypsin digestion solution.
[0054] (6) Centrifuge at 600 g for 10-20 seconds to pellet the tissue sample and discard the supernatant.
[0055] (7) Add 2 times the volume of the corresponding mitochondrial isolation reagent and resuspend the tissue to wash away residual trypsin.
[0056] Note: If the tissue weight is 80 mg, add about 160 μL of mitochondrial isolation reagent at this time.
[0057] (8) Centrifuge at 600 g for 10-20 seconds to pellet the tissue sample and discard the supernatant.
[0058] (9) Add 8 times the volume of pre-cooled mitochondrial isolation reagent and homogenize on an ice bath for 20-30 times.
[0059] Note: If the weight of the tissue is 80 mg, it can be roughly assumed that the volume of the tissue is close to 80 μL, and 640 μL of mitochondrial isolation reagent should be added.
[0060] (10) Centrifuge the homogenate at 1000 g, 4°C for 5 min.
[0061] (11) Carefully transfer the supernatant to another centrifuge tube and centrifuge at 3500g, 4°C for 10 min.
[0062] (12) Carefully remove the supernatant. The precipitate is the isolated mitochondria.
[0063] S4: Preparation of thiolated mitochondrial solution:
[0064] The mitochondrial pellet prepared in S3 was resuspended in PBS at pH 8 and incubated with Traut's reagent according to the manufacturer's protocol. After gentle agitation at room temperature for 30 min, the mitochondria were washed three times with Tyrode's buffer to obtain thiolated mitochondria.
[0065] S5: Preparation of mitochondrial ceria nanozyme complex solution:
[0066] The thiolated mitochondrial solution was mixed with an excess of maleimide-functionalized ceria solution in Tyrode's buffer, gently stirred for 3 h at room temperature, and washed several times with PBS to obtain a mitochondrial ceria nanozyme complex solution.
[0067] The above contents are described below in conjunction with specific embodiments:
[0068] Example 1: Preparation of mitochondrial cerium oxide nanocomposite:
[0069] (1) Preparation of 3-5 nm cerium oxide nanoparticles
[0070] 108.5 mg of cerium nitrate hexahydrate and 100 mg of sodium citrate dihydrate were weighed and dissolved in 2 mL and 1 mL of deionized water, respectively, and then mixed.
[0071] Subsequently, 50 mL of 0.4 M ammonia water was added as a precipitant and stirred for 24 h to allow the cerium dioxide nanoparticles to slowly precipitate, and then the aggregated particles were removed by centrifugation.
[0072] The supernatant was taken and dialyzed with a 3 kDa dialysis bag for 24 h, then centrifuged at 15000 g for 10 min, and the supernatant was freeze-dried to finally obtain cerium dioxide nanoparticles. The scanning electron microscopy image of cerium dioxide nanoparticles is shown in FIG. Figure 1 As shown, stored at 4°C, the particle size is Figure 2 A, its potential is Figure 2 As shown in C.
[0073] (2) Preparation of maleimide functionalized cerium dioxide solution
[0074] Weigh 20 mg of the cerium dioxide nanoparticles prepared in step (1) and dissolve it in 2 mL of chloroform. Dissolve 20 mg of distearoylphosphatidylethanolamine-polyethylene glycol and 10 mg of dipalmitoylphosphatidylethanolamine-polyethylene glycol-maleimide in 10 mL of chloroform. Mix the two and treat with ultrasound for 5 minutes.
[0075] The chloroform in the sample was removed using a rotary evaporator, and the sample was further dried in a vacuum oven at 60 °C for 3 h to ensure complete removal of the chloroform.
[0076] Next, 4 mL of deionized water was added to the sample and ultrasonicated to obtain a transparent colloidal suspension. The obtained cerium dioxide sample was centrifuged at 5000 g to remove excess PEG and purified using a 0.22 μm filter to finally obtain a maleimide-functionalized cerium dioxide solution.
[0077] (3) Extraction of mitochondria from mouse heart tissue
[0078] Use a tissue mitochondrial isolation kit to extract heart tissue from BALB / C mice, place the tissue in a centrifuge tube or culture dish on ice, cut the tissue into very small tissue fragments with scissors or a blade, add 10 times the volume of pre-cooled PBS, and bathe on ice for 3 minutes.
[0079] Centrifuge at 600g for 10-20s to pellet the tissue sample and discard the supernatant.
[0080] Add 8 times the volume of pre-cooled trypsin digestion solution and place on ice for 20 minutes. Centrifuge at 600g for 10-20 seconds to precipitate the tissue sample and discard the supernatant.
[0081] Add 2 times the volume of the corresponding mitochondrial isolation reagent, resuspend the tissue to wash away the residual trypsin. Centrifuge at 600g for 10-20s to precipitate the tissue sample and discard the supernatant. Add 8 times the volume of pre-cooled corresponding mitochondrial isolation reagent and homogenize on an ice bath for 20-30 times.
[0082] Centrifuge the homogenate at 1000g, 4°C for 5 minutes. Carefully transfer the supernatant to another centrifuge tube and centrifuge at 3500g, 4°C for 10 minutes. Carefully remove the supernatant and the precipitate is the isolated mitochondria.
[0083] (4) Preparation of thiolated mitochondrial solution
[0084] Mitochondria resuspended in PBS at pH 8 were incubated with Traut's reagent according to the manufacturer's protocol. After gentle agitation at room temperature for 30 minutes, the mitochondria were washed three times with Tyrode's buffer to obtain a thiolated mitochondrial solution.
[0085] (5) Preparation of mitochondrial ceria nanozyme complex solution
[0086] The thiolated mitochondria were mixed with an excess of maleimide-functionalized cerium oxide in Tyrode's buffer, gently stirred for 3 h at room temperature, and washed with PBS several times to obtain a mitochondrial ceria nanozyme complex solution.
[0087] like Figure 2 B and Figure 2 As shown in C, the particle size of the prepared mitochondrial cerium oxide nanozyme complex is about 1600 nm and the potential is about -11 mV.
[0088] Example 2: Verification of enzyme activity of cerium dioxide nanoparticles
[0089] (1) Prepare the equipment
[0090] Any specification of microplate reader (450±10nm wavelength), ordinary disposable 96-well microplate, micropipette (single-channel pipette, multi-channel pipette), 37℃ constant temperature incubator (or water bath), ordinary low-speed centrifuge.
[0091] (2) Sample addition test
[0092] According to the Nanjing Jiancheng test kit operation table, add different concentrations of the sample to be tested (cerium dioxide solution), distilled water, enzyme working solution, enzyme diluent, and substrate application solution, gently shake the well plate to mix, incubate at 37°C for 20 minutes, take out the microplate reader reading at 450nm, and calculate according to the formula described in the manual.
[0093] (3) Experimental results
[0094] The results are as follows Figure 3 As shown, the cerium dioxide nanoparticle enzyme sample prepared in the present application has enzyme activity, including hydroxyl radical scavenging rate, SOD inhibition rate, and has anti-inflammatory properties.
[0095] Example 3: In vitro ROS elimination ability of mitochondrial ceria
[0096] (1) Raw 264.7 cells were evenly plated in a six-well plate, and a positive control group, a blank control group, and an experimental group were set up until the cell density reached 60%-70%.
[0097] (2) 10 mL of basal culture medium and 2.83 μL of 3% hydrogen peroxide were mixed to prepare solution A, and a ceria solution and a mitochondrial ceria nanozyme complex solution were prepared.
[0098] (3) The old culture medium was discarded and corresponding treatments were performed respectively, i.e., 2 mL of basal culture medium was added to each well of the blank control group, and 2 mL of LA solution was added to each well of the positive control group and the experimental group, respectively, to construct an inflammatory cell model. Then, ceria solution and mitochondrial ceria nanozyme complex solution were added to the wells of different experimental groups at the same time and incubated with Raw264.7 cells for 4 h.
[0099] (4) Wash with PBS and collect the incubated cells in each group in the six-well plate, transfer them to 1.5 mL EP tubes, and mark them. Then, add the diluted probe DCFH-DA to the EP tubes of different groups according to the instructions. The dilution ratio is 1:3000. Be careful to avoid light during operation.
[0100] (5) After incubation at 37°C in the dark for 20 minutes, the cells were washed twice with PBS, resuspended, and the intracellular ROS levels of each group were immediately detected by flow cytometry. The experimental results are shown in the following table and Figure 4 shown.
[0101] The groups and results are shown in the following table:
[0102] Group Name Mean:FITC-A <![CDATA[H2O2+CeO2-Mito]]> 585 <![CDATA[H2O2+CeO2]]> 1119 <![CDATA[H2O2]]> 1219 Blank control 22.3
[0103] The flow cytometry results showed that both ceria and mitochondrial ceria nanozyme complexes could eliminate ROS in Raw264.7 cells, and compared with ceria, mitochondrial ceria nanozyme complexes had a stronger ability to eliminate ROS.
[0104] The experimental materials, animals and their sources used in the above examples of this application are:
[0105]
[0106] In summary, the present application provides a method for preparing a mitochondrial nanozyme complex. It utilizes the ability of mitochondria and ceria to remove reactive oxygen species, and prepares a mitochondrial nanozyme complex, so that the two can work alone and synergistically to produce a synergistic effect, and the complex has a significant effect of removing reactive oxygen species, which can effectively improve the therapeutic effect on inflammation.
Claims
1. A mitochondrial nanozyme complex, characterized in that: The mitochondrial nanozyme complex is a complex of mitochondria and cerium dioxide, and the mitochondrial nanozyme complex is in the form of self-assembly through chemical reactions.
2. A mitochondrial nanozyme complex according to claim 1, characterized in that: The preparation method is as follows: S1: preparation of cerium dioxide nanoparticles; S2: preparing a functionalized cerium dioxide solution: dissolving the cerium dioxide nanoparticles, distearoylphosphatidylethanolamine-polyethylene glycol and dipalmitoylphosphatidylethanolamine-polyethylene glycol-maleimide in S1 in chloroform, performing ultrasonic treatment after mixing, and then removing chloroform from the sample, adding deionized water to the sample and performing ultrasonic treatment, and after the treatment, centrifuging and filtering to obtain a maleimide-functionalized cerium dioxide solution; S3: Extract mitochondria from mouse heart tissue; S4: Preparation of thiolated mitochondrial solution: Incubate the mitochondria obtained in S3 with Traut's reagent, stir at room temperature, and then wash the mitochondria three times with Tyrode's buffer to obtain thiolated mitochondria; S5: Preparation of mitochondrial ceria nanozyme complex solution: The thiolated mitochondria of S4 were mixed with an excess of maleimide-functionalized ceria solution in Tyrode's buffer, gently stirred at room temperature for 3 h, and washed with PBS several times to obtain a mitochondrial ceria nanozyme complex solution.
3. A mitochondrial nanozyme complex according to claim 2, characterized in that: The preparation method in S1 is as follows: dissolving cerium nitrate hexahydrate and sodium citrate dihydrate in deionized water respectively, then mixing the two solutions, adding ammonia water and stirring for 24 hours, centrifuging after stirring to obtain the supernatant, dialyzing the supernatant, centrifuging again, and freeze-drying the supernatant to obtain cerium dioxide nanoparticles.
4. A mitochondrial nanozyme complex according to claim 3, characterized in that: In the S1, the dialysis was performed using a 3 kDa dialysis bag for 24 hours, and after the dialysis was completed, the dialysis was centrifuged at 15000 g for 10 minutes, and the obtained cerium dioxide nanoparticles were stored at 4°C.
5. A mitochondrial nanozyme complex according to claim 4, characterized in that: The mass ratio of distearoylphosphatidylethanolamine-polyethylene glycol, dipalmitoylphosphatidylethanolamine-polyethylene glycol-maleimide and cerium dioxide nanoparticles in S2 is 2:1:
2. After the mixing is completed, S2 is ultrasonically treated for 5 minutes.
6. The mitochondrial nanozyme complex according to claim 5, characterized in that: In S2, a rotary evaporator is used to remove chloroform from the sample, and the sample is further dried in a vacuum oven at 60° C. for 3 h to ensure complete removal of chloroform.
7. A mitochondrial nanozyme complex according to claim 6, characterized in that: The S3 mitochondria were extracted from mouse heart using a tissue mitochondria isolation kit.
8. The mitochondrial nanozyme complex according to claim 7, characterized in that: The specific steps of S3 are as follows: 1) Use fresh mouse heart tissue, usually no more than one hour after the animal was sacrificed and kept on ice; 2) Weigh the cut heart tissue in a 1.5 mL centrifuge tube and wash once with PBS; 3) Place the heart tissue in a centrifuge tube or culture dish on ice and cut the heart tissue into very small tissue fragments using scissors or a blade; 4) Centrifuge to precipitate the tissue sample and discard the supernatant; 5) Add pre-cooled trypsin digestion solution and place on ice; 6) Centrifuge at 600 g for 10-20 seconds to pellet the tissue sample and discard the supernatant; 7) Add mitochondrial isolation reagent and resuspend the tissue to wash away residual trypsin; 8) Centrifuge at 600 g for 10-20 seconds to pellet the tissue sample and discard the supernatant; 9) Add mitochondrial isolation reagent and homogenize on ice for 20-30 times; 10) Centrifuge the homogenate at 1000g, 4°C for 5 minutes; 11) Carefully transfer the supernatant to another centrifuge tube and centrifuge again; 12) Carefully remove the supernatant; the precipitate is the isolated mitochondria.