Artificial mitochondrial nano motor and preparation method and application thereof

Abstract

The invention discloses an artificial mitochondrial nano motor and a preparation method and application thereof, the artificial mitochondrial nano motor comprises a monomer and a cross-linking agent and is formed by initiating polymerization reaction through an initiator, the monomer is a compound with high-density active guanidyl functional groups and high-energy phosphate bonds, and the cross-linking agent is a compound with high-density active guanidyl functional groups and high-energy phosphate bonds. The cross-linking agent is a compound containing a disulfide bond or a diselenide bond. The artificial mitochondrial nano motor not only can generate energy in cells, but also can chemotactically target to a disease microenvironment to achieve an ideal disease treatment effect, and has a wide application prospect in the field of biological medicines.

Description

Technical Field

[0001] This invention pertains to novel biomedical nanomaterials, specifically relating to an artificial mitochondrial nanomotor, its preparation method, and its applications. Background Technology

[0002] Mitochondria play a vital role in cells, particularly in providing ATP through oxidative phosphorylation and central carbon metabolism pathways. Disruptions in their function can severely impact cellular activity, ion homeostasis, and multiple processes related to cell signaling. Impaired ATP production directly affects cellular energy supply, hindering various life activities and triggering a range of diseases, including cardiovascular diseases, neurodegenerative diseases, and metabolic diseases. Therefore, developing a method to enhance mitochondrial ATP production is crucial for treating diseases characterized primarily by mitochondrial damage.

[0003] Existing systems for enhancing mitochondrial ATP production mainly fall into two categories: direct transplantation of viable mitochondria and the construction of artificial systems capable of generating ATP. The former involves extracting fully functional mitochondria from normal cells using specialized methods and transplanting them to the damaged area. The damaged cells then take up the mitochondria and fuse with them to repair their function. This method theoretically offers the advantage of completely replacing the structure and function of damaged mitochondria and is currently the most widely used direct mitochondrial energy repair system in vivo. However, due to the fragility of mitochondrial isolation, the complexity and difficulty of purification and preservation, immunocompatibility during transplantation, and potential ethical issues, its future clinical translation will face significant challenges. As for the latter, some researchers have attempted to construct artificial systems to generate ATP, partially mimicking mitochondrial function. This strategy is highly efficient in vitro; however, most of these artificial systems require additional energy input, such as visible light irradiation, to drive proton transmembrane transport. Their limited in vivo penetration depth restricts their efficiency in generating ATP in vivo. Therefore, despite the promising prospects of artificial ATP synthesis systems in the field of disease treatment, no artificial ATP synthesis system has yet been reported that is stable, applicable in vivo, does not rely on external energy sources, and can effectively target the disease microenvironment. Summary of the Invention

[0004] Purpose of the invention: To address the problems existing in the prior art, this invention provides an artificial mitochondrial nanomotor with stable performance that can effectively target the disease microenvironment in vivo without relying on an external energy source, effectively solving the problem that the currently artificially synthesized ATP system cannot be applied to the disease microenvironment in vivo.

[0005] The present invention also provides a method for preparing an artificial mitochondrial nanomotor and its application.

[0006] Technical Solution: To achieve the above objectives, this invention discloses an artificial mitochondrial nanomotor. The artificial mitochondrial nanomotor comprises a monomer and a cross-linking agent, formed by a polymerization reaction initiated by an initiator. The monomer is a compound with high-density active guanidine functional groups and high-energy phosphate bonds, and the cross-linking agent is a compound containing disulfide or diselenyl groups. This artificial mitochondrial nanomotor not only generates energy within cells but can also chemotactically target the disease microenvironment, achieving ideal therapeutic effects and possessing broad application prospects in the biomedical field.

[0007] The monomeric compound is one or more of L-arginine creatine phosphate derivatives, L-lysine creatine phosphate derivatives, and L-cysteine ​​creatine phosphate derivatives.

[0008] The crosslinking agent is a disulfide crosslinking agent N,N'-bis(acryloyl)cystamine or a diselenyl ether compound crosslinking agent.

[0009] The initiator includes one or more of the following: ammonium persulfate-tetramethylethylenediamine initiating system, benzoin, benzoin ethyl ether and benzoin butyl ether, benzoin dimethyl ether, benzophenone, thioxanthone, camphor porone, or diimidazole.

[0010] The artificial mitochondrial nanomotor is prepared by the following steps:

[0011] (1) Dissolve creatine phosphate in a buffer solution, add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, then add L-arginine derivative or L-lysine derivative or L-cysteine ​​derivative, stir the reaction to obtain L-arginine creatine phosphate derivative, L-lysine creatine phosphate derivative or L-cysteine ​​creatine phosphate derivative;

[0012] (2) The L-arginine creatine phosphate derivative, L-lysine creatine phosphate derivative or L-cysteine ​​creatine phosphate derivative obtained in step (1) are dissolved in a buffer solution with a crosslinking agent, an initiator is added to carry out a polymerization reaction, and artificial mitochondrial nanomotors are obtained by centrifugation and washing.

[0013] In step (1), the molar ratio of creatine phosphate to N-hydroxysuccinimide is 2-50:1, the molar ratio of N-hydroxysuccinimide to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1-50:1, the molar ratio of creatine phosphate to L-arginine derivative, L-lysine derivative, or L-cysteine ​​derivative is 1-10:1, the stirring reaction temperature is 20-30℃, and the reaction time is 10-48h.

[0014] In step (2), the molar ratio of the L-arginine creatine phosphate derivative, L-lysine creatine phosphate derivative, or L-cysteine ​​creatine phosphate derivative to the crosslinking agent is 16:1-1:1, the molar ratio of the L-arginine creatine phosphate derivative, L-lysine creatine phosphate derivative, or L-cysteine ​​creatine phosphate derivative to the initiator is 1:1-50:1, the reaction temperature is 0-30℃, and the reaction time is 1-48h.

[0015] The centrifugation washing process involves collecting nanoparticles through ultracentrifugation, washing them, and freeze-drying them to obtain artificial mitochondrial nanomotors. The ultracentrifugation speed is 5000-12000 rpm, and the centrifugation time is 1-30 min.

[0016] The application of the artificial mitochondrial nanomotor described in this invention in the preparation of drugs, reagents, or health products for treating energy deficiency diseases.

[0017] The energy deficiency diseases mentioned above refer to diseases caused by impaired ATP production capacity affecting cellular energy supply, including cardiovascular diseases, neurodegenerative diseases, and metabolic diseases. This invention utilizes a covalent bond between arginine derivatives and creatine phosphate to prepare a novel monomer containing both arginine and creatine phosphate, which is then constructed into an artificial mitochondrial nanomotor via free radical polymerization. The mechanism of action is that the creatine phosphate portion of the artificial mitochondrial nanomotor structure provides sufficient high-energy phosphate bonds to damaged mitochondria, reacting with ADP in the cytoplasm under enzymatic catalysis to generate ATP. Simultaneously, the arginine portion of its structure can specifically recognize inducible nitric oxide synthase (iNOS), which is highly expressed in damaged mitochondria, thereby achieving chemotactic targeting of the damaged site. The artificial mitochondrial nanomotor reaching the damaged site can not only rapidly generate large amounts of ATP to reduce the burden on damaged mitochondria and restore cellular activity, but also react with reactive oxygen species in the damaged microenvironment to produce NO, effectively alleviating inflammation; the generated NO can also promote mitochondriogenesis, further repairing mitochondrial function.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following advantages.

[0019] The artificial mitochondrial nanomotor of this invention is primarily composed of polymeric segments rich in active guanidine groups and high-energy phosphate bonds. This covalent bonding ensures a sufficiently high density of active groups, enabling it to sensitively respond to the high iNOS concentration gradient in the disease microenvironment and achieve chemotactic targeting of damaged tissues. This overcomes the limitation of currently reported artificial ATP-generating systems being difficult to apply in vivo, and also provides a solution to the problem of artificial mitochondria being unable to target damaged tissues in vivo. Attached Figure Description

[0020] Figure 1 The structural formula of the monomer containing arginine and phosphocreatine in Example 1;

[0021] Figure 2 The monomer containing arginine and phosphocreatine in Example 1 1 HNMR spectrum;

[0022] Figure 3 Transmission electron microscopy of the artificial mitochondrial nanomotor in Example 1 (scale bar: 500 nm);

[0023] Figure 4 The particle size distribution of the artificial mitochondrial nanomotor in Example 2;

[0024] Figure 5 The trajectory of the artificial mitochondrial nanomotor in Example 3 under a damaged and stimulated cellular environment;

[0025] Figure 6 The velocity distribution of the artificial mitochondrial nanomotor in Example 3 under a damaged and stimulated cellular environment;

[0026] Figure 7 This is the trajectory of the artificial mitochondrial nanomotor in a normal cellular environment in Example 4;

[0027] Figure 8 The velocity distribution of the artificial mitochondrial nanomotor in a normal cellular environment in Example 4;

[0028] Figure 9 These are representative images of the chemotactic behavior of the artificial mitochondrial nanomotors in Example 5 towards the damaged cellular environment;

[0029] Figure 10 The fluorescence quantitative normalization results are representative images of the chemotactic behavior of the artificial mitochondrial nanomotors towards the damaged cellular environment in Example 5.

[0030] Figure 11 To evaluate the biocompatibility of artificial mitochondrial nanomotors for the treatment of ischemic diseases, the cell viability of different concentrations of artificial mitochondrial nanomotors in Example 6 after incubation with normal cells for 24 hours was assessed.

[0031] Figure 12 This refers to the situation in Example 7 where the artificial mitochondrial nanomotor generates ATP under cellular stress caused by damage. Detailed Implementation

[0032] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0033] The structure of the L-arginine derivative N-methacryloylarginine is as follows:

[0034]

[0035] For details of the synthesis method, please refer to the literature: A nitric-oxide driven chemotactic nanomotor for enhanced immunotherapy of glioblastoma. Nature Communications, 2023, 14, 941.

[0036] Example 1

[0037] Preparation of artificial mitochondrial nanomotors:

[0038] (1) Creatine phosphate (1.0 g, 4.0 mmol) was dissolved in 20 mL of PBS solution (pH = 7.4). N-hydroxysuccinimide (0.8 g, 4.0 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (0.5 g, 4.0 mmol) were added, followed by N-methacryloylarginine (0.9 g, 4.0 mmol). The mixture was stirred and reacted at 25 °C for 24 h to obtain a monomer containing arginine and phosphocreatine. Figure 1 );like Figure 2 As shown, 1 HNMR spectra showed that monomers containing arginine and phosphocreatine were successfully prepared.

[0039] (2) The monomer containing arginine and phosphocreatine obtained in step (1), namely L-arginine creatine phosphate derivative (140 mg, 0.3 mmol), and the GSH-responsive disulfide compound BAC (N,N'-bis(acryloyl)cysteine, 10 mg, 0.04 mmol) were dissolved in 10 mL of PBS (pH = 7.4). An ammonium persulfate-tetramethylethylenediamine initiator system (molar ratio of L-arginine creatine phosphate derivative to ammonium persulfate was 50:1, and molar ratio of ammonium persulfate to tetramethylethylenediamine was 2:1) was added for polymerization (25 °C, 6 h). After centrifugation and washing at 10,000 rpm for 10 min, the supernatant was discarded to obtain the artificial mitochondrial nanomotor (lower precipitate). Figure 3 As shown, the synthesized artificial mitochondrial nanomotors have a particle size of approximately 110 nm and exhibit uniformly dispersed and irregularly shaped spherical nanoparticles.

[0040] Example 2

[0041] DLS particle size determination of artificial mitochondrial nanomotor solution:

[0042] Weigh 4 mg of the artificial mitochondrial nanomotor solution prepared in Example 1 and disperse it in 4 mL of PBS solution to obtain a 1 mg / mL solution. Take 1 mL of this solution and add it to a cuvette, then place it in a laser particle size analyzer for testing. Figure 4 As shown, the DLS particle size of the artificial mitochondrial nanomotor is approximately 105 nm.

[0043] Example 3

[0044] Study on the motility of artificial mitochondrial nanomotors in a damaged cellular environment:

[0045] (1) Weigh 4 mg of the artificial mitochondrial nanomotor prepared in Example 1, add 4 mL of deionized water, and sonicate for 30 min to disperse it fully. Then add 4 mg of N-hydroxysuccinimide and 8 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide. After reacting at room temperature for 3 h, add 20 μL of 1 mg / mL Cy5-NH2 DMSO dye solution and react in the dark for 12 h to obtain Cy5-labeled artificial mitochondrial nanomotor.

[0046] (2) Cardiac cells (H9c2) were prepared at a concentration of 5*10 5 Cells were seeded at a density of 1 / mL in 14mm cell culture dishes with 1mL of complete culture medium and placed in a 37℃ incubator overnight to allow the cells to adhere.

[0047] (3) Take 1 μL of 1 mg / mL lipopolysaccharide DMSO solution, add it to the above culture dish, and place it in a constant temperature incubator at 37℃ for 24 h to stimulate inflammation;

[0048] (3) Take 10 μL of artificial mitochondrial nanomotors loaded with Cy5-NH2 dye at 200 μg / mL and add them to the above adherent cell culture dish. Immediately use a fluorescence microscope to observe and record the movement of the nanomotors.

[0049] (4) Figure 5 The trajectory of the artificial mitochondrial nanomotor in damaged cells demonstrates its ability to move in the damaged cell environment, and its velocity distribution is calculated based on the trajectory. Figure 6 As shown, this demonstrates that the artificial mitochondrial nanomotor exhibits significant motility under damaged conditions. The artificial mitochondrial nanomotor prepared in this invention gains motility under iNOS catalysis in damaged cell environments with high iNOS levels, exhibiting stronger cell permeability and thus enhancing its therapeutic effect.

[0050] Example 4

[0051] Study on the motility of artificial mitochondrial nanomotors in a normal cellular environment:

[0052] (1) Weigh 4 mg of the artificial mitochondrial nanomotor prepared in Example 1, add 4 mL of deionized water, and sonicate for 30 min to disperse it fully. Then add 4 mg of N-hydroxysuccinimide and 8 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide. After reacting at room temperature for 3 h, add 20 μL of 1 mg / mL Cy5-NH2 DMSO dye solution and react in the dark for 12 h to obtain Cy5-labeled artificial mitochondrial nanomotor.

[0053] (2) H9c2 cells were fed at a concentration of 5*10 5 Cells were seeded at a density of 1 / mL in 14mm cell culture dishes with 1mL of complete culture medium and placed in a 37℃ incubator overnight to allow the cells to adhere.

[0054] (3) Take 10 μL of artificial mitochondrial nanomotor solution loaded with Cy5-NH2 dye and add it to the above adherent cell culture dish. Immediately use a fluorescence microscope to observe and record the movement of the nanomotor.

[0055] (4) Figure 7 The trajectory of the artificial mitochondrial nanomotor in a normal cell demonstrates that this artificial mitochondrial nanomotor lacks autonomous movement capability in a normal cellular environment. Its velocity distribution was calculated based on the trajectory. Figure 8 As shown, this demonstrates that the artificial mitochondrial nanomotor moves in a random Brownian motion within a normal cellular environment. The nanorobots prepared in this invention do not exhibit autonomous movement in normal cells with normal iNOS levels, but possess autonomous movement capabilities in damaged cells.

[0056] Example 5

[0057] Assessment of the chemotactic behavior of artificial mitochondrial nanomotors toward the damaged cellular environment:

[0058] (1) Weigh 4 mg of the artificial mitochondrial nanomotor prepared in Example 1, add 4 mL of deionized water, and sonicate for 30 min to disperse it fully. Then add 4 mg of N-hydroxysuccinimide and 8 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide. After reacting at room temperature for 3 h, add 20 μL of 1 mg / mL Cy5-NH2 DMSO dye solution and react in the dark for 12 h to obtain Cy5-labeled artificial mitochondrial nanomotor.

[0059] (2) H9c2 cells were fed at a concentration of 5*10 5 Cells were seeded at a density of 1 / mL in 14mm cell culture dishes with 1mL of complete culture medium and placed in a 37℃ incubator overnight to allow the cells to adhere.

[0060] (3) Take 1 μL of 1 mg / mL lipopolysaccharide DMSO solution, add it to the above culture dish, and place it in a constant temperature incubator at 37℃ for 24 h to stimulate inflammatory damage. Then, use 200 μL of lysis buffer to lyse the cells to obtain lipopolysaccharide pre-stimulated H9c2 cell lysate.

[0061] (3) To observe the collective chemotactic behavior of artificial mitochondrial nanomotors for target cell identification, Y-shaped glass substrate microchannels were used. The main channel was 1 cm long and 0.4 cm wide, and the branch channels were 0.7 cm long and 0.3 cm wide. The chemotactic concentration gradient was generated by different types of cell lysates placed in reservoirs (ii) or (iii) within the branch channels. Briefly, 5 mg of agarose was completely dissolved in 500 μL of PBS at 90 °C. When the melted agarose cooled to room temperature but did not solidify, 50 μL of lipopolysaccharide-prestimulated H9c2 cell lysate was added, and the mixture was then transferred to reservoir (ii) at 4 °C for gelation, or 50 μL of positive PBS was added, and the mixture was then transferred to reservoir (iii) at 4 °C for gelation. Before evaluating the chemotactic movement of the artificial mitochondrial nanomotors, the Y-shaped channels were pre-filled with PBS, and 50 μL of Cy5-labeled artificial mitochondrial nanomotor solution was gently dropped into the reservoir (i). At specific times, fluorescence images of reservoirs (ii) and (iii) were captured using an inverted fluorescence microscope equipped with a 10× objective lens, such as Figure 9 .like Figure 10 As shown, ImageJ was used to quantify the corresponding fluorescence intensity. It was observed that the fluorescence intensity of the reservoir containing pre-stimulated H9C2 cell lysate was significantly higher than that containing PBS, indicating that the artificial mitochondrial nanomotors exhibit chemotactic behavior towards the iNOS concentration gradient. This demonstrates that the artificial mitochondrial nanomotors can chemotact with environments containing iNOS concentration gradients, enabling them to target damaged sites for precise treatment.

[0062] Example 6

[0063] Biocompatibility assessment of artificial mitochondrial nanomotors:

[0064] H9c2 cells were used at a rate of 5*10 4 Cells were seeded at 100 μg / mL in 96-well plates. After cell adhesion, the artificial mitochondrial nanomotors prepared in Example 1 were dispersed in the culture medium. Different concentrations of artificial mitochondrial nanomotors (20, 50, 100, 200, 400 μg / mL) were prepared and incubated with cardiomyocytes at 37°C for 24 h. Cell viability was then detected using MTT assay. Figure 11 It can be seen that the increase in material concentration has almost no effect on the activity of cardiomyocytes, thus proving that the artificial mitochondrial nanomotor has good biocompatibility.

[0065] Example 7

[0066] The production of ATP in the cellular environment stimulated by damage to artificial mitochondrial nanomotors:

[0067] (1) H9c2 cells were fed at a concentration of 5*10 5 Cells were seeded at a concentration of 1 / mL in 14 mm cell culture dishes with 1 mL of complete culture medium and incubated overnight at 37°C. The culture medium was then replaced with sugar-free DMEM and the dishes were incubated in a hypoxic incubator for 24 h. Artificial mitochondrial nanomotors prepared in Example 1 were added to the sugar-free DMEM at a concentration of 200 μg / mL.

[0068] (2) Following the instructions of the ATP assay kit (Beyotime, Cat No. S0026), the ATP production of H9c2 cells was detected. Figure 12 The results showed that the ability of cells to generate ATP after injury was significantly reduced, while the addition of artificial mitochondrial nanomotors enhanced the ability of cells to generate ATP after injury, restoring or even exceeding normal levels, indicating that artificial mitochondrial nanomotors have a good ability to generate ATP.

[0069] Example 8

[0070] The artificial mitochondrial nanomotor was prepared using the method described in Example 1, with the following differences:

[0071] In step (1), creatine (1.0 g, 7.6 mmol) was dissolved in 30 mL of PBS solution, and N-hydroxysuccinimide (1.6 g, 7.6 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (1.0 g, 7.6 mmol) were added. Then, L-arginine derivative N-methacryloylarginine (1.7 g, 7.6 mmol) was added, and the mixture was stirred. The reaction system was 25 °C and the reaction time was 24 h to obtain L-arginine creatine derivative. Subsequently, the reaction was carried out according to step (2) of Example 1 to obtain artificial mitochondrial nanomotor control sample 1, so as to study the difference between artificial mitochondrial nanomotor loaded with creatine phosphate as substrate and loaded with creatine as substrate.

[0072] Example 9

[0073] The artificial mitochondrial nanomotor was prepared using the method described in Example 1, with the following differences:

[0074] In step (1), creatine phosphate (10 g, 40 mmol) was dissolved in 30 mL of PBS solution, and N-hydroxysuccinimide (8.0 g, 40 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (5.0 g, 40 mmol) were added. Then, the L-arginine derivative N-methacryloylarginine (0.9 g, 4.0 mmol) was added, and the mixture was stirred. The reaction system was 25 °C and the reaction time was 24 h to obtain the L-arginine creatine derivative. Subsequently, the reaction was carried out according to step (2) of Example 1 to obtain artificial mitochondrial nanomotor control sample 2, so as to study the effect of the amount of creatine phosphate loaded on the artificial mitochondrial nanomotor on its performance.

[0075] Example 10

[0076] The artificial mitochondrial nanomotor was prepared using the method described in Example 1, with the following differences:

[0077] In step (2), the L-arginine creatine derivative and the GSH-responsive disulfide compound BAC were dissolved in 10 mL of PBS solvent at a molar ratio of 1. An initiator of ammonium persulfate-tetramethylethylenediamine (ammonium persulfate-tetramethylethylenediamine initiation system with a molar ratio of 50 for L-arginine creatine derivative and 2 for ammonium persulfate and tetramethylethylenediamine) was added to carry out the polymerization reaction. After centrifugation and washing at 10,000 rpm for 10 min, artificial mitochondrial nanomotor control sample 3 was obtained to study the effect of monomer dosage on artificial mitochondrial nanomotor generated by free radical polymerization.

[0078] Example 11

[0079] The artificial mitochondrial nanomotor was prepared using the method described in Example 1, with the following differences:

[0080] In step (2), the L-arginine creatine derivative and the GSH-responsive disulfide compound BAC were dissolved in 10 mL of PBS solvent at a molar ratio of 8. An initiator system of ammonium persulfate-tetramethylethylenediamine (the molar ratio of L-arginine creatine derivative to ammonium persulfate was 10, and the molar ratio of ammonium persulfate to tetramethylethylenediamine was 2) was added to carry out the polymerization reaction. The mixture was centrifuged and washed at 10,000 rpm for 10 min to obtain control sample 4 of the artificial mitochondrial nanomotor, so as to study the effect of the amount of initiator on the artificial mitochondrial nanomotor generated by free radical polymerization.

Claims

1. An artificial mitochondrial nanomotor, characterized in that, The artificial mitochondrial nanomotor comprises a monomer and a crosslinking agent, which are formed by a polymerization reaction initiated by an initiator. The monomer is a compound with high-density active guanidine functional groups and high-energy phosphate bonds, and the crosslinking agent is a compound containing disulfide bonds or diselenide bonds.

2. The artificial mitochondrial nanomotor according to claim 1, characterized in that, The monomeric compound is one or more of L-arginine creatine phosphate derivatives, L-lysine creatine phosphate derivatives, and L-cysteine ​​creatine phosphate derivatives.

3. The artificial mitochondrial nanomotor according to claim 1, characterized in that, The crosslinking agent is preferably a disulfide crosslinking agent N,N'-bis(acryloyl)cystamine or a diselenyl ether compound crosslinking agent.

4. The artificial mitochondrial nanomotor according to claim 1, characterized in that, The initiator includes one or more of the following: ammonium persulfate-tetramethylethylenediamine initiating system, benzoin, benzoin ethyl ether and benzoin butyl ether, benzoin dimethyl ether, benzophenone, thioxanthone, camphor porone, or diimidazole.

5. A method for preparing the artificial mitochondrial nanomotor according to claim 1, characterized in that, Includes the following steps: (1) Dissolve creatine phosphate in a buffer solution, add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, then add L-arginine derivative or L-lysine derivative or L-cysteine ​​derivative, stir the reaction to obtain L-arginine creatine phosphate derivative, L-lysine creatine phosphate derivative or L-cysteine ​​creatine phosphate derivative; (2) The L-arginine creatine phosphate derivative, L-lysine creatine phosphate derivative or L-cysteine ​​creatine phosphate derivative obtained in step (1) are dissolved in a buffer solution with a crosslinking agent, an initiator is added to carry out a polymerization reaction, and artificial mitochondrial nanomotors are obtained by centrifugation and washing.

6. The preparation method according to claim 5, characterized in that, In step (1), the molar ratio of creatine phosphate to N-hydroxysuccinimide is 2-50:1, the molar ratio of N-hydroxysuccinimide to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1-50:1, the molar ratio of creatine phosphate to L-arginine derivative, L-lysine derivative, or L-cysteine ​​derivative is 1-10:1, the stirring reaction temperature is 20-30℃, and the reaction time is 10-48h.

7. The preparation method according to claim 5, characterized in that, In step (2), the molar ratio of the L-arginine creatine phosphate derivative, L-lysine creatine phosphate derivative, or L-cysteine ​​creatine phosphate derivative to the crosslinking agent is 16:1:-1:1, the molar ratio of the L-arginine creatine phosphate derivative, L-lysine creatine phosphate derivative, or L-cysteine ​​creatine phosphate derivative to the initiator is 1:1-50:1, the reaction temperature is 0-30℃, and the reaction time is 1-48h.

8. The preparation method according to claim 5, characterized in that, The centrifugation washing in step (2) involves collecting nanoparticles through ultracentrifugation, washing them, and freeze-drying them to obtain artificial mitochondrial nanomotors. The ultracentrifugation speed is 5000-12000 rpm, and the centrifugation time is 1-30 min.

9. The use of the artificial mitochondrial nanomotor of claim 1 in the preparation of drugs, reagents or health products for treating energy deficiency diseases.

10. The application according to claim 9, characterized in that, The energy deficiency diseases mentioned above are diseases caused by damage to ATP production capacity that affects the energy supply of cells, including cardiovascular diseases, neurodegenerative diseases, and metabolic diseases.

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