Mitochondrial nano-robot as well as preparation method and application thereof
Mitochondrial nanorobots that construct polymer layers on the mitochondria surface have achieved targeting and activity protection of mitochondria, solved the problem of targeting and activity loss in ROS environments in mitochondrial transplantation treatment, and restored the energy metabolism function of liver cells.
Patent Information
- Application Number
- CN202510728885.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-03
- Publication Date
- 2025-09-02
AI Technical Summary
When existing mitochondrial transplantation treats liver metabolic diseases, mitochondria lacks targeting ability and is difficult to resist the microenvironment of high ROS diseases, resulting in loss of activity.
Mitochondrial nanorobots were prepared, and polymer layers were constructed through the mitochondrial surface, and enzyme-substrate-specific affinity was used to achieve enzyme-substrate-specific affinity with the concentration gradient of CDO and AST enzymes, targeting damaged sites, and degrading responses in the ROS environment to protect mitochondrial activity.
Mitochondrial nanorobots can efficiently target the disease microenvironment, protect mitochondrial activity, restore the energy metabolism function of damaged liver cells, and have good biocompatibility and motility.
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Figure CN120570862A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to a novel mitochondrial biological nano preparation, and specifically relates to a mitochondrial nano robot and a preparation method and application thereof. Background Art
[0002] Mitochondria are the cell's energy factories, primarily responsible for producing ATP through oxidative phosphorylation to provide energy for cellular activity. In the liver, mitochondria are not only central to energy production but also participate in a variety of metabolic processes, including fatty acid and amino acid metabolism, gluconeogenesis, detoxification, and ROS regulation. When the liver is compromised by factors such as viruses, alcohol, drugs and toxins, cholestasis, and parasitic infections, hepatocyte mitochondrial function is significantly impaired, severely impacting the liver's metabolic and detoxification functions, leading to a range of liver metabolic diseases, such as chronic viral hepatitis, non-alcoholic fatty liver disease, drug-induced liver injury, and liver fibrosis. Factors such as oxidative stress and metabolic disorders associated with mitochondrial dysfunction contribute to the pathogenesis of these diseases. Therefore, restoring mitochondrial homeostasis has the potential to have a therapeutic effect on these diseases and their complications. Mitochondrial transplantation, which relies on exogenous supplementation of healthy mitochondria, is emerging as a promising treatment approach.
[0003] Current mitochondrial transplantation approaches for treating liver metabolic diseases primarily involve extracting fully functional mitochondria from normal cells through specialized methods. These mitochondria are then delivered intravenously to the damaged area, where they are taken up by damaged host cells and fuse with the damaged mitochondria, thereby repairing mitochondrial function and reestablishing host cell metabolism. However, free mitochondria lack the ability to target damaged areas and are unable to withstand the high ROS-producing microenvironment, which can lead to loss of activity. Therefore, designing an engineered mitochondrial approach that can effectively protect mitochondrial activity and efficiently target mitochondria to damaged areas has significant research value and application prospects. Summary of the Invention
[0004] Purpose of the invention: In response to the problems existing in the prior art, the present invention provides a mitochondrial nanorobot that not only protects mitochondrial activity and resists disease microenvironment, but can also effectively target the disease microenvironment, effectively solving the technical problems of unsatisfactory activity and targeting of mitochondrial therapy in the prior art.
[0005] The present invention also provides a preparation method and application of a mitochondrial nanorobot.
[0006] Technical solution: In order to achieve the above-mentioned purpose, the present invention provides a mitochondrial nanorobot, which includes mitochondria and a polymer layer wrapped on the surface of the mitochondria. The polymer layer is formed by a polymerization reaction of a monomer and a cross-linker initiated by an initiator. The monomer is a compound with a thiol functional group, and the cross-linker is a degradable cross-linker.
[0007] Wherein, the mitochondria are derived from any one of stem cells, hepatocytes, cardiomyocytes or skeletal muscle cells.
[0008] The monomer compound is a compound containing a double bond and a thiol functional group, and is any one of an L-cysteine derivative, a homocysteine derivative, a glutathione derivative or an N-acetylcysteine derivative; and the degradable cross-linking agent is a compound containing a diselenide bond, a disulfide bond, a thioether bond or a peroxide bond.
[0009] The initiator is any one or more of a persulfate-tetramethylethylenediamine initiation system, a hydrogen peroxide-ferrous salt Fenton reaction system, and an azobisisobutyronitrile-ascorbic acid initiation system.
[0010] The method for preparing the mitochondrial nanorobot of the present invention comprises the following steps:
[0011] (1) Dispersing mitochondria in a buffer solution, adding N-acryloyloxysuccinimide for reaction, and centrifuging and washing to obtain mitochondria with modified carbon-carbon double bonds;
[0012] (2) dispersing the mitochondria modified with carbon-carbon double bonds obtained in step (1) into a buffer solution containing a monomer and a cross-linking agent, adding an initiator to carry out a polymerization reaction, and centrifuging and washing to obtain mitochondrial nanorobots.
[0013] Wherein, the concentration of mitochondria in step (1) is 5×10 7 -5×10 9 / mL, the concentration of N-acryloyloxysuccinimide is 0.1-2 mg / mL, the reaction temperature is 0-10°C, and the reaction time is 1-8 h.
[0014] Preferably, the monomer in step (2) has the following structural formula:
[0015]
[0016] Preferably, the cross-linking agent in step (2) has the following structural formula:
[0017]
[0018] The mitochondrial concentration of the modified carbon-carbon double bond in step (2) is 5×10 7 -5×109 The reaction temperature is 0-10°C, the reaction time is 1-24h, the centrifugal speed is 9000-12000g, the centrifugal time is 1-30min, and the temperature is 0-10°C.
[0019] The invention relates to the use of the mitochondrial nanorobot in the preparation of medicines for treating mitochondrial dysfunction diseases.
[0020] The present invention utilizes monomers and degradable cross-linking agents to construct a polymer layer on the surface of mitochondria through free radical polymerization to form mitochondrial nanorobots. The working principle is that the L-cysteine portion in the polymer layer of the mitochondrial nanorobot can generate enzyme-substrate specific affinity with the concentration gradient of CDO and AST enzymes released from damaged liver parts in the body, thereby achieving the purpose of chemotaxis targeting damaged parts. When the mitochondrial nanorobot reaches the damaged part, the polymer layer can be degraded in response to ROS in the damaged microenvironment, and the L-cysteine structure produces SO2 under the catalytic action of the enzyme, which plays a role in vasodilation and collagen remodeling, and the mitochondria play a role in rebuilding the energy metabolism of the host cells. The mitochondrial nanorobot of the present invention can not only protect the activity of mitochondria and resist the harsh disease microenvironment, but also can chemotaxis target the disease microenvironment, and achieve the ideal disease treatment effect through mitochondrial transplantation. As a mitochondrial biological preparation, it has broad application prospects in the field of biomedicine.
[0021] Beneficial effects: Compared with the prior art, the present invention has the following advantages:
[0022] This invention proposes specific mitochondrial nanorobots for the first time. The introduction of nanorobot technology can not only protect mitochondrial activity from the influence of oxidative stress in the disease microenvironment, but also enable mitochondria to efficiently chemotaxis target the damaged liver under the action of the CDO and AST enzyme concentration gradient field, further alleviating the pathological characteristics of the disease microenvironment, which is impossible for free mitochondria to do.
[0023] The preparation method of the present invention is simple and efficient, the synthesis conditions are mild, the material dispersion performance is good, the synthesized mitochondrial nanorobots have excellent biocompatibility and motility, can restore the ability of damaged liver cells to produce ATP, and have broad application prospects in the field of biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 is the structural formula of the L-cysteine derivative monomer in Example 1;
[0025] Figure 2 is the L-cysteine derivative monomer in Example 1 1 HNMR spectrum;
[0026] Figure 3 is the structural formula of the diselenide crosslinking agent in Example 1;
[0027] Figure 4 is the diselenide crosslinking agent in Example 1 1 HNMR spectrum;
[0028] Figure 5 This is a transmission electron microscope image of the mitochondrial nanorobot in Example 1;
[0029] Figure 6 The particle size distribution of the mitochondrial nanorobot in Example 2;
[0030] Figure 7 This is the motion trajectory of the mitochondrial nanorobot in the liver cell environment in Example 3;
[0031] Figure 8 The velocity distribution of the mitochondrial nanorobot in the liver cell environment in Example 3;
[0032] Figure 9 The motion trajectory of the mitochondrial nanorobot in the endothelial cell environment in Example 4;
[0033] Figure 10 is the velocity distribution of the mitochondrial nanorobot in the endothelial cell environment in Example 4;
[0034] Figure 11 This is a representative picture of the chemotactic behavior of the mitochondrial nanorobot toward the liver cell environment in Example 5;
[0035] Figure 12 The normalized fluorescence quantitative results of the representative images of the chemotactic behavior of the mitochondrial nanorobot toward the hepatocyte environment in Example 5;
[0036] Figure 13 The cell activity after incubation of normal liver cells with different concentrations of mitochondrial nanorobots for 24 hours in Example 6;
[0037] Figure 14 The mitochondrial nanorobot of Example 7 restores the ability of damaged liver cells to generate ATP;
[0038] Figure 15 This is an evaluation of the anti-fibrotic efficacy of the mitochondrial nanorobot of Example 8 in a mouse model of liver fibrosis. DETAILED DESCRIPTION
[0039] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.
[0040] The mitochondrial isolation kit was purchased from Thermo Fisher Scientific (cat. 89874).
[0041] Human umbilical cord mesenchymal stem cells (huMSCs) are provided by Nanjing Taisheng Biotechnology Co., Ltd. The cells in the present invention are all commercially available. The human umbilical cord mesenchymal stem cells of the present invention can also replace other mesenchymal stem cells such as bone marrow mesenchymal stem cells.
[0042] Among them, the structure of L-cysteine derivative N-methacryloyl-L-cysteine is:
[0043]
[0044] Among them, the crosslinking agent diselenide crosslinking agent structure is:
[0045]
[0046] Example 1
[0047] Preparation of mitochondrial nanorobots:
[0048] (1) 5.0 g of L-cysteine hydrochloride and 0.2 g of NaNO2 were dissolved in 30 mL of K2CO3 (mass fraction 5%) solution. 4.0 mL of methacryloyl chloride was added in an N2 atmosphere and an ice-water bath, and the mixture was stirred magnetically at room temperature for 2 h. After the reaction, the pH value of the solution was adjusted to 7.0 with K2CO3 solution, and the unreacted methacryloyl chloride was extracted with ethyl acetate. The aqueous phase was dried using a rotary evaporator. The residue was recrystallized with a mixed solvent of ethyl acetate / ethanol (v / v = 1 / 50) and freeze-dried to obtain an L-cysteine derivative monomer ( Figure 1 ), 1 HNMR spectrum proves the successful preparation of L-cysteine derivative monomer ( Figure 2 ).
[0049] (2) 1.0 g of 2,2'-diselenane diethylbis(1-ethylamine) dihydrochloride was dissolved in 60 mL of anhydrous dichloromethane. 3.48 mL of triethylamine was added and the mixture was reacted in an ice-water bath under N2 atmosphere for 30 min. 1.2 mL of methacryloyl chloride was added and the mixture was reacted at room temperature for 24 h. The product was purified by silica gel column (ethyl acetate and petroleum ether, v / v = 1:2-2:1) and finally dried by rotary evaporation to obtain the diselenide crosslinker ( Figure 3 ), 1 The HNMR spectrum shows that the diselenide crosslinker was successfully prepared ( Figure 4 ).
[0050] (3) According to the mitochondrial isolation kit, 2×10 7One unit of umbilical cord mesenchymal stem cells is extracted. Add 1 ml of mitochondrial isolation reagent supplemented with 1% PMSF, gently resuspend the cells, and place on ice for 15 minutes. Homogenize using a glass homogenizer for approximately 30 strokes. Centrifuge the homogenate at 600g for 10 minutes at 4°C. Remove the supernatant and centrifuge it again at 11,000g for 10 minutes at 4°C to obtain mitochondria.
[0051] (4) Mitochondrial suspension (5×10 9 / mL, 100μL) was mixed with N-acryloyloxysuccinimide (1mg / mL, 100μL) and reacted at 4°C for 6h. After the reaction was completed, centrifuged at 11000g for 10min to obtain carbon-carbon double bond modified mitochondria. Then 500μL PBS (pH7.4) was added, in which 1.5mg L-cysteine derivative monomer and 0.4mg diselenide crosslinker were dissolved. Tetramethylethylenediamine (1mg / mL, 50μL) and ammonium persulfate (1mg / mL, 25μL) were added at 4°C and N2 atmosphere, and the reaction was carried out for 12h. After the reaction was completed, centrifuged at 11000g for 10min to obtain mitochondrial nanorobots ( Figure 5 ).
[0052] Example 2
[0053] DLS size detection of mitochondrial nanorobots:
[0054] The mitochondrial nanorobots prepared in Example 1 were dispersed in PBS (pH 7.4) solution to a concentration of 5×10 8 / mL, take 1mL of suspension and add it to the cuvette, then put it into the laser particle size analyzer for testing. Figure 6 As shown, the DLS particle size of the mitochondrial nanorobot was approximately 700 nm.
[0055] Example 3
[0056] Study on the Motility of Mitochondrial Nanorobots in Liver Cell Environment:
[0057] (1) Take 100 μL of the artificial mitochondrial nanomotor (5×10 8 / mL), and then added 100 μL MitoTracker Deep Red probe (10 μM) and reacted at 4°C in the dark for 30 min to obtain MitoTracker Deep Red labeled mitochondrial nanorobots;
[0058] (2) Take 10 μL of MitoTracker Deep Red labeled mitochondrial nanorobots (5×10 8cells / mL) were added into 200 μL L-02 cell lysate (1×10 6 The movement of the nanorobots was immediately observed and recorded using a fluorescence microscope.
[0059] (3) Figure 7 This is the movement trajectory of the mitochondrial nanorobot in the L-02 cell environment, showing obvious displacement and different movement directions. Figure 8 The velocity distribution is calculated based on the motion trajectory, and the motion speed is about 3.4 μm s -1 The mitochondrial nanorobots prepared by the present invention acquire movement ability by enzyme catalysis in a cellular environment with high concentrations of CDO and AST, which is conducive to the uptake of mitochondrial nanorobots by cells and can better exert therapeutic effects.
[0060] Example 4
[0061] Study on the motility performance of mitochondrial nanorobots in endothelial cell environment:
[0062] (1) Take 100 μL of the artificial mitochondrial nanomotor (5×10 8 / mL), and then added 100 μL MitoTracker Deep Red probe (10 μM) and reacted at 4°C in the dark for 30 min to obtain MitoTracker Deep Red labeled mitochondrial nanorobots;
[0063] (2) Use cell lysate to obtain HUVECs cell lysate (1×10 6 / mL), take 200 μL HUVECs cell lysate and add it to the confocal dish;
[0064] (3) Take 10 μL of MitoTracker Deep Red labeled mitochondrial nanorobots (5×10 8 / mL) were added into the above-mentioned confocal dish, and the movement of the nanorobots was immediately observed and recorded using a fluorescence microscope.
[0065] (4) Figure 9 This is the motion trajectory of the mitochondrial nanorobot in the HUVECs cell environment, showing an obvious Brownian motion trajectory. Figure 10 The velocity distribution is calculated based on the motion trajectory, and the motion speed is about 1.5 μm s -1 The mitochondrial nanorobots prepared by the present invention have no autonomous movement ability in a cell environment without high concentrations of CDO and AST.
[0066] Example 5
[0067] Evaluation of the chemotactic behavior of mitochondrial nanorobots toward the hepatocyte environment:
[0068] (1) Take 100 μL of the artificial mitochondrial nanomotor (5×10 8 / mL), and then added 100 μL MitoTracker Deep Red probe (10 μM) and reacted at 4°C in the dark for 30 min to obtain MitoTracker Deep Red labeled mitochondrial nanorobots;
[0069] (2) Cell lysate was used to obtain L-02 cell lysate and HUVECs cell lysate (1×10 6 / mL);
[0070] (3) The chemotactic behavior of mitochondrial nanorobots toward the hepatocyte environment was observed using a Y-shaped glass substrate microchannel. The main channel was 1 cm long and 0.4 cm wide, and the branch channel was 0.7 cm long and 0.3 cm wide. 25 μL agarose gel (1%, w / v) and 25 μL L-02 or HUVECs cell lysate were added to chambers II and III of the branch channel, respectively, to allow the chemoattractant to diffuse in the channel and form a concentration gradient. The entire channel was filled with 400 μL PBS. Then, 10 μL MitoTracker Deep Red probe-labeled mitochondrial nanorobots (2×10 9 mL -1 ) were gently dripped into chamber I. Fluorescence images of chambers II and III were taken at specific time points using a 10× inverted fluorescence microscope, and the fluorescence intensity was analyzed using Image J. Figure 11 and Figure 12 It can be seen that the fluorescence intensity in the chamber containing L-02 cell lysate is significantly higher than that containing HUVECs cell lysate, indicating that the mitochondrial nanorobots exhibit chemotactic behavior towards the CDO / AST concentration gradient, proving that mitochondrial nanorobots can chemotactically attract environments containing CDO / AST concentration gradients and can target damaged areas for precise treatment.
[0071] Example 6
[0072] Biocompatibility assessment of mitochondrial nanorobots:
[0073] L-02 cells were cultured at 5*10 4 cell / mL were inoculated into a 96-well plate. After the cells adhered to the wall, the mitochondrial nanorobots prepared in Example 1 were dispersed in the culture medium. Mitochondrial nanorobots of different concentrations were prepared and incubated with L-02 cells at 37°C for 24 h. The cell activity was detected using MTT reagent. Figure 13It can be seen that with the increase of material concentration, the activity of L-02 cells is almost unaffected, which proves that the mitochondrial nanorobot has good biocompatibility.
[0074] Example 7
[0075] Mitochondrial nanorobots restore the ability of damaged liver cells to produce ATP:
[0076] (1) L-02 cells were cultured at a rate of 5*10 5 Cells were seeded into 6-well plates with 2 mL of complete culture medium and placed in a 37°C incubator to allow the cells to adhere overnight. The culture medium was replaced with a culture medium containing 1.5 mM H2O2 and the cells were stimulated for 1 hour. 6 mL -1 The mitochondrial nanorobots prepared in Example 1 were incubated for 12 hours.
[0077] (2) According to the requirements of the ATP detection kit (Biyuntian, Cat No. S0026), the ATP production of L-02 cells was detected, such as Figure 14 Compared with normal cells, the ability of cells to generate ATP after damage stimulation decreased significantly, while the ability of cells to generate ATP returned to normal levels after the addition of mitochondrial nanorobots, indicating that mitochondrial nanorobots have a good ability to restore the ATP production of damaged host cells.
[0078] Example 8
[0079] Evaluation of the anti-fibrotic efficacy of mitochondrial nanorobots in a mouse model of liver fibrosis:
[0080] C57BL / 6 mice (male, 6-8 weeks old) were intraperitoneally injected with 1 mL kg -1 CCl4 (CCl4: olive oil = 1:3 v / v) was administered twice a week for six weeks to establish a CCl4-induced liver fibrosis mouse model. Mice were intraperitoneally injected with an equal volume of olive oil as a control group. In the fourth week of modeling, mice were injected with mitochondrial nanorobots (1×10 7 / mouse) or PBS, three times a week for two weeks. Masson staining results showed that compared with the normal group, the PBS group had more blue collagen fibers, and the area of collagen fibers was significantly reduced after mitochondrial nanorobot treatment ( Figure 15 ).
Claims
1. A mitochondrial nanorobot, characterized in that: The mitochondrial nanorobot includes mitochondria and a polymer layer wrapped on the surface of the mitochondria. The polymer layer is formed by a polymerization reaction between a monomer and a crosslinker initiated by an initiator. The monomer is a compound with a thiol functional group, and the crosslinker is a degradable crosslinker.
2. The mitochondrial nanorobot according to claim 1, characterized in that The mitochondria are preferably derived from any one of stem cells, hepatocytes, cardiomyocytes or skeletal muscle cells.
3. The mitochondrial nanorobot according to claim 1, characterized in that The monomer compound is a compound containing a double bond and a sulfhydryl functional group, and is any one of an L-cysteine derivative, a homocysteine derivative, a glutathione derivative or an N-acetylcysteine derivative. The degradable cross-linking agent is a compound containing a diselenide bond, a disulfide bond, a thioether bond or a peroxide bond.
4. The mitochondrial nanorobot according to claim 1, characterized in that The initiator is any one or more of a persulfate-tetramethylethylenediamine initiation system, a hydrogen peroxide-ferrous salt Fenton reaction system, and an azobisisobutyronitrile-ascorbic acid initiation system.
5. A method for preparing the mitochondrial nanorobot according to claim 1, characterized in that: The steps include: (1) Dispersing mitochondria in a buffer solution, adding N-acryloyloxysuccinimide for reaction, and centrifuging and washing to obtain mitochondria with modified carbon-carbon double bonds; (2) dispersing the mitochondria modified with carbon-carbon double bonds obtained in step (1) into a buffer solution containing a monomer and a cross-linking agent, adding an initiator to carry out a polymerization reaction, and centrifuging and washing to obtain mitochondrial nanorobots.
6. The preparation method according to claim 5, characterized in that The concentration of mitochondria in step (1) is 5×10 7 -5×10 9 / mL, the concentration of N-acryloyloxysuccinimide is 0.1-2 mg / mL, the reaction temperature is 0-10°C, and the reaction time is 1-8 h.
7. The preparation method according to claim 5, characterized in that The monomer in step (2) has the following structural formula:
8. The preparation method according to claim 5, characterized in that The cross-linking agent in step (2) has the following structural formula:
9. The preparation method according to claim 5, characterized in that The mitochondrial concentration of the modified carbon-carbon double bond in step (2) is 5×10 7 -5×10 9 The molar ratio of monomer to degradable cross-linker is 2-16:1, the reaction temperature is 0-10°C, and the reaction time is 1-24h.
10. Use of the mitochondrial nanorobot according to claim 1 in the preparation of a drug for treating mitochondrial dysfunction diseases.