A selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeting preparation and a preparation method thereof

Abstract

The application provides a selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeted preparation and a preparation method thereof. The preparation method synthesizes carbon dots from ethylenediamine and citric acid, improves catalytic activity through selenium doping, and obtains selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeted preparation Se-CDs@SS31 through SS31 to realize precise mitochondrial targeting. The Se-CDs@SS31 has the functions of efficient ROS removal, precise mitochondrial targeting, mitochondrial autophagy activation and immune regulation, provides a new artificial carbon dot-based nanoscale enzyme preparation for myocarditis treatment, and simultaneously expands the application scene of selenium-doped artificial carbon dots in the treatment of myocarditis-related diseases.

Description

Technical Field

[0001] This invention relates to a selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeted formulation and its preparation method. Background Technology

[0002] The application of nanozymes in the biotherapy of inflammatory diseases is becoming increasingly mature. Carbon dots (CDs), as zero-dimensional carbon nanomaterials, have become the core carrier for nanozyme research and development due to their small particle size, easy surface functionalization, and excellent biocompatibility. Ethylenediamine-citric acid-based artificial carbon dots are a classic synthetic system. Citric acid provides the carboxyl group as the carbon source, and ethylenediamine introduces the amino group as the nitrogen source. The synthesized carbon dots are rich in N and O dual heteroatoms on their surface, naturally possessing basic antioxidant and ROS scavenging capabilities. Moreover, the raw materials are readily available, the preparation process is controllable, and the structure is more uniform than that of natural carbon dots, making it an excellent matrix for constructing functionalized anti-inflammatory nanozymes.

[0003] Myocarditis, a common inflammatory disease of the myocardium, has a complex pathogenesis and lacks specific treatment. Its core pathological mechanism involves oxidative stress triggered by mitochondrial dysfunction in cardiomyocytes, leading to the massive release of inflammatory factors and inhibition of the mitochondrial autophagy pathway. Excessive reactive oxygen species (ROS) further damage the myocardial cell membrane and mitochondrial structure, exacerbating myocardial inflammation and necrosis, forming a vicious cycle of "oxidative stress-inflammatory damage-mitochondrial failure." Selenium (Se), an essential trace element for the human body, possesses excellent antioxidant and anti-inflammatory properties. Doping it into carbon dots can significantly enhance their catalytic scavenging ability against various types of ROS, such as ·OH, ·O2⁻, and H2O2, by regulating electron transfer efficiency. Simultaneously, selenium doping can synergistically strengthen the inhibitory effect of carbon dots on inflammatory factors. The mitochondrial-targeting peptide SS31, a classic mitochondrial-targeting molecule, can achieve precise delivery via the mitochondrial membrane potential gradient, solving the problem of insufficient targeting in nanomaterials. This allows functionalized carbon dots to specifically accumulate in damaged myocardial mitochondria, fundamentally intervening in the pathological process of myocarditis.

[0004] Currently, although ethylenediamine-citrate carbon dots have been applied in biosensing and basic antioxidant research, there are few studies on their selenium-doped modification and combined with SS31 mitochondrial-targeted modification for mediating mitophagy in the treatment of myocarditis. Existing technologies do not integrate a unified treatment model of "selenium-doped enhanced catalysis - SS31 precise targeting - mitophagy activation - immune regulation," thus failing to meet the treatment needs of the complex pathological state of myocarditis.

[0005] Existing research mainly focuses on the following aspects:

[0006] 1. Basic applications of ethylenediamine-citrate carbon dots: These carbon dots are mostly used in fluorescence sensing and general drug carriers. They rely solely on surface functional groups to achieve basic ROS removal. Without selenium doping modification, their catalytic activity is limited and they cannot efficiently remove the large amounts of various types of ROS generated in myocarditis. Furthermore, without targeted modification, they are difficult to accumulate in damaged mitochondria of the myocardium and cannot achieve specific treatment of myocarditis.

[0007] 2. Anti-inflammatory research on selenium-doped nanomaterials: Some studies have used selenium doping in inorganic nanoparticles and natural carbon dots for inflammation suppression, but there are problems such as complex selenium doping process, poor biocompatibility of carrier, lack of precise mitochondrial targeting design, and failure to design for the core mechanism of mitochondrial autophagy inhibition in myocarditis, which cannot improve myocardial mitochondrial dysfunction and has limited therapeutic effect.

[0008] 3. Mitochondrial-targeting peptide SS31 modified nanomaterials: Existing technologies modify SS31 into liposomes, polymer carriers, etc. to achieve mitochondrial targeting. However, such carriers have no enzyme-like catalytic activity and can only achieve drug delivery. They cannot simultaneously complete ROS clearance and mitochondrial autophagy activation. Furthermore, they do not combine the antioxidant advantages of selenium doping, resulting in a single treatment mechanism and difficulty in breaking the vicious cycle of myocarditis.

[0009] 4. Nanozyme-mediated treatment of myocardial inflammation: Current nanozyme treatments for myocardial inflammation mostly focus on single ROS clearance or anti-inflammation, without integrating core links such as mitochondrial targeting and mitochondrial autophagy activation. Furthermore, they do not use ethylenediamine-citrate carbon dots as a matrix, and cannot utilize the natural activity and easy modification characteristics of their N and O dual heteroatoms. It is difficult to achieve both targeting and therapeutic efficiency, and thus cannot realize multi-dimensional intervention for myocarditis.

[0010] In summary, current technologies have not yet developed an integrated myocarditis treatment agent that integrates "selenium doping-enhanced catalysis, SS31 mitochondrial precise targeting, mitochondrial autophagy activation, and immune regulation" using ethylenediamine-citric acid-based artificial carbon dots as the matrix.

[0011] The main shortcomings of existing research are as follows:

[0012] 1. The intrinsic catalytic activity of ethylenediamine-citrate carbon dots is limited: These carbon dots without selenium doping rely solely on surface N / O functional groups to achieve basic ROS removal. They cannot efficiently catalyze the decomposition of various types of ROS generated in large quantities during myocarditis, resulting in poor oxidative stress relief and difficulty in intervening in the core pathological process of myocarditis.

[0013] 2. Insufficient targeting and low enrichment efficiency at lesion sites: Existing artificial carbon dots and selenium-doped nanomaterials lack mitochondrial targeting modification. After administration, they are easily cleared by the reticuloendothelial system and are difficult to specifically enrich in the mitochondria of myocardial tissue damaged by myocarditis. The therapeutic dose is insufficient and may have potential effects on normal myocardial tissue.

[0014] 3. Unregulated core mitophagy pathway: Existing technologies only focus on ROS clearance or simple anti-inflammation, without addressing the core issue of suppressed mitophagy pathway in myocarditis. They cannot clear damaged mitochondria, cannot fundamentally improve myocardial mitochondrial dysfunction, and are unable to break the vicious cycle of "mitochondrial damage-oxidative stress-intensified inflammation".

[0015] 4. Single treatment mechanism without synergistic effect: Existing treatments mostly rely on a single treatment mechanism and do not integrate multiple pathological links such as ROS clearance, mitochondrial function repair, myocardial tissue protection, and immune regulation. They cannot achieve multi-dimensional intervention in the progression of myocarditis, which easily leads to recurrence of the disease and limited treatment effect.

[0016] 5. Some selenium-doped carriers have poor biocompatibility: Most existing selenium-doped anti-inflammatory nanomaterials use inorganic particles or unknown carbon sources as carriers, which pose risks of cytotoxicity and in vivo accumulation. The selenium-doped modification potential of ethylenediamine-citrate carbon dots has not been fully explored, and it is impossible to balance therapeutic efficacy and myocardial tissue safety. Summary of the Invention

[0017] The technical problem to be solved by this invention is to provide a method for preparing a selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeted formulation. This method uses ethylenediamine and citric acid as raw materials to synthesize carbon dots, enhances catalytic activity through selenium doping, and achieves precise mitochondrial targeting via SS31 to obtain the selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeted formulation Se-CDs@SS31. This Se-CDs@SS31 possesses efficient ROS scavenging, precise mitochondrial targeting, mitochondrial autophagy activation, and immunomodulatory functions, providing a novel artificial carbon dot-based nanozyme formulation for the treatment of myocarditis, while also expanding the application scenarios of selenium-doped artificial carbon dots in the treatment of myocardial inflammation-related diseases.

[0018] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0019] A method for preparing a selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeted formulation includes the following steps:

[0020] S1. Mix citric acid, ethylenediamine, and deionized water, and stir magnetically for 25-35 minutes until completely dissolved to obtain the precursor solution;

[0021] S2. Place the precursor liquid obtained in step S1 in a high-pressure reactor and hydrothermally react for 5-7 hours. After the reaction is completed, cool to room temperature to obtain a carbon dot mixture.

[0022] S3. Centrifuge the carbon dot mixture obtained in step S2, discard solid impurities and keep the supernatant. Put the supernatant into a dialysis bag and place the dialysis bag in ultrapure water for dialysis for 2-4 days. Change the water every 8 hours to obtain dialysis solution one. Freeze-dry dialysis solution one for 45-51 hours to obtain carbon dot CDs powder.

[0023] S4. Add the carbon dot CDs powder obtained in step S3 to deionized water, and ultrasonically disperse for 15-25 minutes to obtain a carbon dot suspension. Add sodium selenite to the carbon dot suspension, and magnetically stir for 50-70 minutes. Then add hydrazine hydrate solution dropwise and continue magnetically stirring for 21-27 hours to obtain reaction solution one.

[0024] S5. Place the reaction solution I obtained in step S4 into a dialysis bag, place the dialysis bag in ultrapure water for dialysis for 2-4 days, change the water every 8 hours to obtain dialysis solution II, freeze-dry dialysis solution II for 45-51 hours to obtain selenium-doped carbon dots Se-CDs powder.

[0025] S6. Add the selenium-doped carbon dot Se-CDs powder obtained in step S5 to PBS buffer, and sonicate for 15-25 minutes to obtain a Se-CDs solution. Add the thiol-polyethylene glycol-mitochondrial targeted antioxidant peptide to the Se-CDs solution and stir for 1-3 hours to obtain reaction solution two.

[0026] S7. Centrifuge the reaction solution obtained in step S6 to obtain precipitate one. Disperse precipitate one in PBS buffer and centrifuge again to obtain precipitate two. Disperse precipitate two in PBS buffer and centrifuge again to obtain precipitate three. Freeze-dry precipitate three for 21-27 hours to obtain the selenium-doped ethylenediamine-citric acid carbon dots-mitochondrial targeting formulation Se-CDs@SS31.

[0027] Furthermore, in step S1 of the present invention, the ratio of citric acid, ethylenediamine, and deionized water is 2g:1mL:(40-60)mL; the magnetic stirring speed is 500rpm.

[0028] Furthermore, in step S2 of this invention, the lining of the high-pressure reactor is made of polytetrafluoroethylene, and the hydrothermal reaction temperature is 190-210℃. The hydrothermal reaction parameters defined in step S2 can prepare carbon dots with a surface rich in N and O dual heteroatoms, exhibiting uniform structure and good dispersibility. This lays the foundation for selenium doping and SS31 modification, solving the problems of poor controllability of functional groups and inconsistent preparation processes in traditional artificial carbon dots.

[0029] Furthermore, in step S3 of this invention, the centrifugation speed is 12000 rpm, the centrifugation time is 15-25 minutes, the molecular weight cutoff of the dialysis bag is 3500 Da, and the freeze-drying temperature is -50°C. The centrifugation operation in step S3 can remove a small amount of insoluble matter.

[0030] Further, in step S4 of this invention, the ratio of carbon dot CDs powder, deionized water, sodium selenite, and hydrazine hydrate solution obtained in step S3 is 30 mg: (25-35) mL: 25 mg: 0.8 mL; the ultrasonic dispersion power is 200 W, and the frequency is 40 kHz; the magnetic stirring speed is 500 rpm; the mass concentration of the hydrazine hydrate solution is 85%, and the dropping rate of the hydrazine hydrate solution is 0.1 mL / min. The magnetic stirring in step S4 allows selenium ions to fully complex with the surface functional groups of CDs; step S4 uses sodium selenite as the selenium source and hydrazine hydrate as the reducing agent to achieve in-situ reduction doping of selenium on the surface of ethylenediamine-citric acid carbon dots, with the doping ratio controlled at 9.86 ± 0.12%, significantly enhancing the multi-type ROS catalytic scavenging ability and anti-myocardial inflammation activity of the carbon dots, solving the problem of limited intrinsic catalytic activity of artificial carbon dots.

[0031] Furthermore, in step S5 of the present invention, the molecular weight cutoff of the dialysis bag is 3500 Da, and the freeze-drying temperature is -50°C.

[0032] Further, in step S6 of this invention, the pH value of the PBS buffer is 7.4, the ratio of selenium-doped carbon dot Se-CDs powder obtained in step S5 to PBS buffer is 30 mg: (18-22) mL; the ultrasonic dispersion power is 200 W, and the frequency is 40 kHz; the mass ratio of thiol-polyethylene glycol-mitochondrial targeted antioxidant peptide to selenium-doped carbon dot Se-CDs powder obtained in step S5 is 1:3; and the stirring speed is 500 rpm. Step S6 uses thiol-polyethylene glycol-mitochondrial targeted antioxidant peptide HS-PEG-SS31 at a mass ratio of 3:1 to achieve targeted modification of selenium-doped carbon dots through covalent binding of thiol groups to the carbon dot surface. Through the specific binding of SS31 peptide to the inner mitochondrial membrane, the formulation is precisely delivered to the damaged mitochondria of myocardium in myocarditis, improving the enrichment efficiency of the lesion site, reducing the impact on normal myocardial tissue, and solving the defect of insufficient targeting of existing nanomaterials.

[0033] Furthermore, in step S7 of this invention, the centrifugation speed is 15000 rpm each time, and the centrifugation time is 25-35 minutes each time; the pH value of the PBS buffer is 7.4, and the mass ratio of precipitate one / precipitate two to PBS buffer is 1:3; the freeze-drying temperature is -50℃. The repeated centrifugation and dispersion operations in step S7 can remove unbound SS31 peptides.

[0034] Another technical problem to be solved by the present invention is to provide a selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeted formulation prepared by the above preparation method.

[0035] This invention utilizes ethylenediamine and citric acid as raw materials, optimizing the synthesis process to prepare artificial carbon dots with uniform structure and rich N / O functional groups on the surface. Selenium doping significantly enhances their ability to catalytically scavenge multiple types of ROS and their anti-myocardial inflammation activity. The selenium-doped carbon dots are covalently modified with the mitochondrial-targeting peptide SS31 to achieve precise delivery of the formulation to damaged mitochondria in myocardial tissue during myocarditis, improving enrichment efficiency at the lesion site and reducing the impact on normal myocardial tissue. An integrated system of "selenium-doped carbon dots-SS31 mitochondrial targeting" is constructed, specifically activating the PINK1 / Parkin pathway of mitochondrial autophagy, achieving synergistic effects of ROS scavenging, mitochondrial function repair, myocardial inflammation suppression, and immune regulation, significantly improving the therapeutic effect of myocarditis. Optimized preparation process parameters ensure that the Se-CDs@SS31 formulation has excellent biocompatibility, dispersion stability, and in vivo safety, meeting the clinical application requirements for myocardial inflammation diseases. The invention also provides a method for applying this formulation in the treatment of myocarditis, offering a novel, efficient, and highly targeted artificial carbon dot-based therapeutic solution for myocarditis and other myocardial inflammation-related diseases.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1) High catalytic activity and excellent ROS scavenging efficiency: Through the synergistic catalytic effect of selenium doping and the N / O functional groups on the surface of ethylenediamine-citric acid carbon dots, it achieves efficient scavenging of multiple types of ROS such as ·OH, ·O2⁻, and H2O2. In in vitro experiments, the ·OH scavenging rate reached 46.85±1.52%, and the ·O2⁻ scavenging rate reached 79.23±0.38%, which are significantly higher than those of pure ethylenediamine-citric acid carbon dots. It can effectively alleviate oxidative stress damage in myocarditis and protect the structural integrity of cardiomyocytes and mitochondria.

[0038] 2) Precise targeting and high enrichment efficiency at the lesion site: SS31 peptide modification endows the formulation with precise myocardial mitochondrial targeting ability. After tail vein injection, it can specifically enrich in the mitochondria of myocardial tissue damaged by myocarditis. The enrichment time in myocardial tissue is up to 12 hours, and there is almost no enrichment in normal organs such as liver, spleen, and kidney, which greatly increases the drug concentration at the lesion site and avoids non-specific damage to normal myocardial tissue.

[0039] 3) Significantly activates mitophagy, fundamentally improving myocardial mitochondrial function: The formulation does not require external stimulation such as near-infrared radiation. Through the intrinsic synergy of selenium doping and SS31 targeting, it constructs an integrated treatment mode of "ROS clearance - mitophagy activation - myocardial inflammation suppression - immune regulation". It can precisely target damaged myocardial mitochondria, activate the PINK1 / Parkin mitophagy pathway, significantly upregulate PINK1 and Parkin protein expression, downregulate P62 protein expression, and effectively clear damaged mitochondria. In vivo experiments have confirmed that it can restore LPS-induced myocardial mitochondrial membrane potential to more than 90% of the normal level and ATP production to more than 97%, fundamentally breaking the vicious cycle of "mitochondrial damage - oxidative stress - increased inflammation" and restoring the energy metabolism function of myocardial cells.

[0040] 4) Synergistic effect of multiple mechanisms to significantly improve the treatment effect of myocarditis: Integrating four core mechanisms of ROS clearance, mitochondrial function repair, myocardial inflammation suppression and immune regulation, it can significantly reduce the expression of pro-inflammatory factors such as TNF-α and IL-6 in myocardial tissue of myocarditis mice (the serum concentration of IL-6 drops to near normal level) and increase the expression of anti-inflammatory factor IL-10; at the same time, it induces macrophage M2 polarization (CD206 / CD86 ratio 2.01±0.23), upregulates the number of CD4⁺T cells, achieves synergistic effect of inflammation suppression and immune regulation, significantly improves the myocardial pathological state of myocarditis mice and reduces the degree of myocardial damage.

[0041] 5) Good biocompatibility and high in vivo safety: Artificial carbon dots are prepared using ethylenediamine and citric acid as raw materials. The raw materials are low in toxicity and the preparation process is mild. Selenium is an essential trace element for the human body and the doping ratio can be controlled. Combined with the biocompatible SS31 peptide modification, the formulation has no obvious cytotoxicity (≤200μg / mL cell survival rate >95%). In vivo experiments have confirmed that after tail vein injection, mice have normal weight gain, no abnormalities in routine blood and biochemical indicators (myocardial enzymes, liver and kidney function, etc.), no pathological damage to major organs such as heart, liver, spleen, and kidney, and hemolysis rate is less than 5%, which meets the safety requirements of biomedical materials for myocardial diseases.

[0042] 6) The preparation process is simple and easy to scale up: ethylenediamine, citric acid, and sodium selenite are all commonly used chemical raw materials, which are inexpensive and readily available; the hydrothermal synthesis, selenium in-situ doping, and SS31 targeted modification processes of this invention are simple, reproducible, and have mild reaction conditions, requiring no complex equipment, making them easy to scale up and possessing industrial transformation potential; the preparation parameters of the core formulation Se-CDs@SS31 defined in this invention can ensure that the formulation has good dispersion stability, biocompatibility (≤200μg / mL cell viability >95%), and myocardial mitochondrial targeted therapeutic effect.

[0043] 7) No external stimulation required, strong clinical applicability: The formulation does not require external stimulation equipment such as near-infrared irradiation and can be administered via tail vein injection, which is simple to operate and suitable for bedside clinical application; the dosage and dosing cycle can be adjusted according to the severity of the patient's condition, making it suitable for patients with different types and severities of myocarditis and highly applicable to clinical promotion.

[0044] 8) Broad application prospects: The "artificial carbon dot-selenium doping-SS31 mitochondrial targeting" proposed in this invention is not only applicable to myocarditis, but can also be extended to the treatment of other inflammatory diseases related to myocardial mitochondrial dysfunction, such as viral myocarditis, ischemic myocarditis, and cardiomyopathy complicated with myocarditis. At the same time, it expands new directions for the application of selenium-doped artificial carbon dots in the field of nanozyme therapy for myocardial diseases. Attached Figure Description

[0045] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, do not constitute an undue limitation of the invention. In the drawings:

[0046] Figure 1 This is a figure showing the cytotoxicity test results of the selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeting formulation Se-CDs@SS31 prepared in Example 1 of this invention;

[0047] Figure 2 This is a diagram showing the intracellular ROS scavenging effect of the selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeted formulation Se-CDs@SS31 prepared in Example 1 of this invention.

[0048] Figure 3 Mitochondrial targeting colocalization image of the selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeting formulation Se-CDs@SS31 prepared in Example 1 of this invention (scale bar = 25 μm).

[0049] Figure 4 Mitochondrial functional JC-1 fluorescence expression of the selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeting formulation Se-CDs@SS31 prepared in Example 1 of this invention (scale bar = 50 μm).

[0050] Figure 5 H&E myocardial tissue staining image (scale bar = 100 μm) showing the in vivo safety of the selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeted preparation Se-CDs@SS31 for myocarditis prepared in Example 1 of this invention.

[0051] Figure 6 This is an immunohistochemical staining image of mitochondrial autophagy-related proteins in myocardial tissue of the selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeting formulation Se-CDs@SS31 prepared in Example 1 of this invention.

[0052] Figure 7This is a statistical chart of the macrophage M2 polarization (CD206 / CD86) ratio of the selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeting formulation Se-CDs@SS31 prepared in Example 1 of this invention;

[0053] Figure 8 This is a statistical chart showing the CD4+ / CD8+ T cell ratio in myocardial tissue of the selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeting formulation Se-CDs@SS31 prepared in Example 1 of this invention. Detailed Implementation

[0054] The present invention will now be described in detail with reference to specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0055] Example 1

[0056] Selenium-doped ethylenediamine-citric acid carbon dots-mitochondrial targeted formulations were prepared according to the following steps:

[0057] S1. Mix citric acid, ethylenediamine, and deionized water in a ratio of 2g:1mL:50mL, and stir magnetically at 500rpm for 30 minutes until completely dissolved to obtain the precursor solution;

[0058] S2. The precursor liquid obtained in step S1 is placed in a high-pressure reactor lined with polytetrafluoroethylene and hydrothermally reacted at 200°C for 6 hours. After the reaction is completed, it is cooled to room temperature to obtain a carbon dot mixture.

[0059] S3. Centrifuge the carbon dot mixture obtained in step S2 at 12000 rpm for 20 minutes, discard solid impurities and keep the supernatant. Put the supernatant into a dialysis bag with a molecular weight cutoff of 3500 Da, place the dialysis bag in ultrapure water for dialysis for 3 days, change the water every 8 hours to obtain dialysis solution one, freeze-dry dialysis solution one at -50℃ for 48 hours to obtain carbon dot CDs powder;

[0060] S4. Add the carbon dot CDs powder obtained in step S3 to deionized water and ultrasonically disperse it for 20 minutes at 200W power and 40kHz frequency to obtain a carbon dot suspension. Add sodium selenite to the carbon dot suspension and magnetically stir at 500rpm for 60 minutes. Then, add 85% hydrazine hydrate solution dropwise at a rate of 0.1mL / min and continue magnetically stirring at 500rpm for 24 hours to obtain reaction solution one. The ratio of carbon dot CDs powder, deionized water, sodium selenite, and hydrazine hydrate solution obtained in step S3 is 30mg:30mL:25mg:0.8mL.

[0061] S5. Place the reaction solution I obtained in step S4 into a dialysis bag with a molecular weight cutoff of 3500 Da, place the dialysis bag in ultrapure water for dialysis for 3 days, change the water every 8 hours to obtain dialysis solution II, freeze-dry dialysis solution II at -50℃ for 48 hours to obtain selenium-doped carbon dots Se-CDs powder.

[0062] S6. The selenium-doped carbon dot Se-CDs powder obtained in step S5 was added to PBS buffer with a pH of 7.4 at a ratio of 30 mg: 20 mL. The mixture was ultrasonically dispersed at 200 W power and 40 kHz frequency for 20 minutes to obtain a Se-CDs solution. The thiol-polyethylene glycol-mitochondrial targeted antioxidant peptide (HS-PEG-SS31, commercially available product) was added to the Se-CDs solution. The mass ratio of the thiol-polyethylene glycol-mitochondrial targeted antioxidant peptide to the selenium-doped carbon dot Se-CDs powder obtained in step S5 was 1:3. The mixture was stirred at 500 rpm for 2 hours to obtain reaction solution two.

[0063] S7. Centrifuge the reaction solution 2 obtained in step S6 to obtain precipitate 1. Disperse precipitate 1 in PBS buffer and centrifuge again to obtain precipitate 2. Disperse precipitate 2 in PBS buffer and centrifuge again to obtain precipitate 3. Freeze-dry precipitate 3 at -50℃ for 24 hours to obtain the selenium-doped ethylenediamine-citric acid carbon dots-mitochondrial targeting preparation Se-CDs@SS31. The centrifugation speed is 15000 rpm and the centrifugation time is 30 minutes each time. The pH value of PBS buffer is 7.4, and the mass ratio of precipitate 1 / precipitate 2 to PBS buffer is 1:3.

[0064] Example 2

[0065] Selenium-doped ethylenediamine-citric acid carbon dots-mitochondrial targeted formulations were prepared according to the following steps:

[0066] S1. Mix citric acid, ethylenediamine, and deionized water in a ratio of 2g:1mL:40mL, and stir magnetically at 500rpm for 35 minutes until completely dissolved to obtain the precursor solution;

[0067] S2. The precursor liquid obtained in step S1 is placed in a high-pressure reactor lined with polytetrafluoroethylene and hydrothermally reacted at 190°C for 7 hours. After the reaction is completed, it is cooled to room temperature to obtain a carbon dot mixture.

[0068] S3. Centrifuge the carbon dot mixture obtained in step S2 at 12000 rpm for 25 minutes, discard solid impurities and keep the supernatant. Put the supernatant into a dialysis bag with a molecular weight cutoff of 3500 Da, place the dialysis bag in ultrapure water for dialysis for 4 days, change the water every 8 hours to obtain dialysis solution one, freeze dry dialysis solution one at -50℃ for 51 hours to obtain carbon dot CDs powder;

[0069] S4. Add the carbon dot CDs powder obtained in step S3 to deionized water and ultrasonically disperse it for 25 minutes at 200W power and 40kHz frequency to obtain a carbon dot suspension. Add sodium selenite to the carbon dot suspension and magnetically stir at 500rpm for 70 minutes. Then, add 85% hydrazine hydrate solution dropwise at a rate of 0.1mL / min and continue magnetically stirring at 500rpm for 27 hours to obtain reaction solution one. The ratio of carbon dot CDs powder, deionized water, sodium selenite, and hydrazine hydrate solution obtained in step S3 is 30mg:25mL:25mg:0.8mL.

[0070] S5. Place the reaction solution I obtained in step S4 into a dialysis bag with a molecular weight cutoff of 3500 Da, place the dialysis bag in ultrapure water for dialysis for 4 days, change the water every 8 hours to obtain dialysis solution II, freeze-dry dialysis solution II at -50℃ for 51 hours to obtain selenium-doped carbon dots Se-CDs powder.

[0071] S6. The selenium-doped carbon dot Se-CDs powder obtained in step S5 was added to PBS buffer with a pH of 7.4 at a ratio of 30 mg: 18 mL. The mixture was ultrasonically dispersed at 200 W power and 40 kHz frequency for 25 minutes to obtain a Se-CDs solution. The thiol-polyethylene glycol-mitochondrial targeted antioxidant peptide was added to the Se-CDs solution. The mass ratio of the thiol-polyethylene glycol-mitochondrial targeted antioxidant peptide to the selenium-doped carbon dot Se-CDs powder obtained in step S5 was 1:3. The mixture was stirred at 500 rpm for 3 hours to obtain reaction solution two.

[0072] S7. Centrifuge the reaction solution 2 obtained in step S6 to obtain precipitate 1. Disperse precipitate 1 in PBS buffer and centrifuge again to obtain precipitate 2. Disperse precipitate 2 in PBS buffer and centrifuge again to obtain precipitate 3. Freeze-dry precipitate 3 at -50℃ for 27 hours to obtain the selenium-doped ethylenediamine-citric acid carbon dots-mitochondrial targeting preparation Se-CDs@SS31. The centrifugation speed is 15000 rpm and the centrifugation time is 35 minutes each time. The pH value of PBS buffer is 7.4, and the mass ratio of precipitate 1 / precipitate 2 to PBS buffer is 1:3.

[0073] Example 3

[0074] Selenium-doped ethylenediamine-citric acid carbon dots-mitochondrial targeted formulations were prepared according to the following steps:

[0075] S1. Mix citric acid, ethylenediamine, and deionized water in a ratio of 2g:1mL:60mL, and stir magnetically at 500rpm for 25 minutes until completely dissolved to obtain the precursor solution;

[0076] S2. The precursor liquid obtained in step S1 is placed in a high-pressure reactor lined with polytetrafluoroethylene and hydrothermally reacted at 210°C for 5 hours. After the reaction is completed, it is cooled to room temperature to obtain a carbon dot mixture.

[0077] S3. Centrifuge the carbon dot mixture obtained in step S2 at 12000 rpm for 15 minutes, discard solid impurities and keep the supernatant. Put the supernatant into a dialysis bag with a molecular weight cutoff of 3500 Da, place the dialysis bag in ultrapure water for dialysis for 2 days, change the water every 8 hours to obtain dialysis solution one, freeze dry dialysis solution one at -50℃ for 45 hours to obtain carbon dot CDs powder;

[0078] S4. Add the carbon dot CDs powder obtained in step S3 to deionized water and ultrasonically disperse it for 15 minutes at 200W power and 40kHz frequency to obtain a carbon dot suspension. Add sodium selenite to the carbon dot suspension and magnetically stir at 500rpm for 50 minutes. Then, add 85% hydrazine hydrate solution dropwise at a rate of 0.1mL / min and continue magnetically stirring at 500rpm for 21 hours to obtain reaction solution one. The ratio of carbon dot CDs powder, deionized water, sodium selenite, and hydrazine hydrate solution obtained in step S3 is 30mg:35mL:25mg:0.8mL.

[0079] S5. Place the reaction solution I obtained in step S4 into a dialysis bag with a molecular weight cutoff of 3500 Da, place the dialysis bag in ultrapure water for dialysis for 2 days, change the water every 8 hours to obtain dialysis solution II, freeze-dry dialysis solution II at -50℃ for 45 hours to obtain selenium-doped carbon dots Se-CDs powder.

[0080] S6. The selenium-doped carbon dot Se-CDs powder obtained in step S5 was added to PBS buffer with a pH of 7.4 at a ratio of 30 mg: 22 mL. The mixture was ultrasonically dispersed at 200 W power and 40 kHz frequency for 15 minutes to obtain a Se-CDs solution. The thiol-polyethylene glycol-mitochondrial targeted antioxidant peptide was added to the Se-CDs solution. The mass ratio of the thiol-polyethylene glycol-mitochondrial targeted antioxidant peptide to the selenium-doped carbon dot Se-CDs powder obtained in step S5 was 1:3. The mixture was stirred at 500 rpm for 1 hour to obtain reaction solution two.

[0081] S7. Centrifuge the reaction solution 2 obtained in step S6 to obtain precipitate 1. Disperse precipitate 1 in PBS buffer and centrifuge again to obtain precipitate 2. Disperse precipitate 2 in PBS buffer and centrifuge again to obtain precipitate 3. Freeze-dry precipitate 3 at -50℃ for 21 hours to obtain the selenium-doped ethylenediamine-citric acid carbon dots-mitochondrial targeting preparation Se-CDs@SS31. The centrifugation speed is 15000 rpm and the centrifugation time is 25 minutes each time. The pH value of PBS buffer is 7.4, and the mass ratio of precipitate 1 / precipitate 2 to PBS buffer is 1:3.

[0082] Experimental Example 1: Cytotoxicity Experiment

[0083] RAW264.7 macrophages were used at a rate of 1×10⁻⁶. 4 Cells were seeded at a density of [number] cells / mL in 96-well plates and cultured at 37°C with 5% CO2 for 24 hours. The original culture medium was discarded, and fresh culture medium containing different concentrations (0 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 500 μg / mL) of CDs, Se-CDs, and Se-CDs@SS31 was added, and the cells were cultured for another 24 hours. Cell viability was assessed using the CCK-8 assay. The results showed that at concentrations ≤200 μg / mL, the cell viability in the Se-CDs@SS31 treatment group was higher than 95%, indicating that the Se-CDs@SS31 prepared in this invention has good biocompatibility, no significant cytotoxicity, and is suitable for in vivo application in myocardial tissue.

[0084] Experiment Example 2: Intracellular ROS Scavenging Experiment

[0085] The specific steps to verify the multi-type ROS removal capability of Se-CDs@SS31 are as follows:

[0086] RAW264.7 cells were seeded in confocal culture dishes and stimulated with 1 μg / mL LPS for 30 minutes to construct a cellular oxidative stress model. Cells were divided into a control group, a CDs group, a Se-CDs group, and a Se-CDs@SS31 group, with each carbon dot formulation at a concentration of 200 μg / mL. After 24 hours of culture, the cells were stained with the DCFH-DA probe, and the intracellular ·OH and ·O2⁻ levels were detected by fluorescence microscopy and flow cytometry. The results showed that the ROS scavenging rate in the Se-CDs@SS31 group was significantly higher than that in the other groups, with ·OH scavenging reaching 46.85±1.52% and ·O2⁻ scavenging reaching 79.23±0.38%. This indicates that the synergistic effect of selenium doping and SS31 modification can significantly improve the scavenging efficiency of various ROS types and effectively alleviate oxidative stress damage in cardiomyocytes.

[0087] Experiment Example 3: Mitochondrial Targeting Experiment

[0088] The specific steps for verifying the mitochondrial targeting ability of Se-CDs@SS31 are as follows:

[0089] Red fluorescent (Cy5) labeled Se-CDs@SS31 were co-incubated with RAW264.7 cells for 6 hours, with an unmodified Se-CDs group serving as a control. Mitochondria were stained with mito-tracker green and cell nuclei were stained with DAPI blue fluorescence. Fluorescence distribution was observed using laser confocal microscopy, and Pearson correlation coefficients were calculated. The results showed that the red fluorescence of Cy5 and the green fluorescence of mitochondria in the Se-CDs@SS31 group had a very high overlap, and the Pearson correlation coefficient was significantly higher than that in the unmodified Se-CDs group. This indicates that SS31 peptide modification endows the formulation with good mitochondrial targeting, enabling precise delivery to cardiomyocyte mitochondria.

[0090] Experimental Example 4: Mitochondrial Functional Fluorescence Expression

[0091] 1. Cell seeding and grouping: RAW264.7 macrophages were seeded at a rate of 1×10⁻⁶ cells / cells. 4 Cells were seeded at a density per well in confocal culture dishes and cultured for 24 hours to allow them to adhere. The experiment included a normal group, a model group, a CDs group, a Se-CDs group, and a Se-CDs@SS31 group. Except for the normal group, all other groups were stimulated with 1 μg / mL LPS for 12 hours to establish an acute myocarditis cell model, and the corresponding formulation was added to a final concentration of 200 μg / mL according to the group.

[0092] 2. After incubation with JC-1 staining solution, discard the culture medium and gently wash the cells twice with preheated PBS. Add 1 mL of JC-1 staining working solution to each dish, gently shake to mix, and incubate in a 37°C, 5% CO2 cell culture incubator in the dark for 20 minutes.

[0093] 3. Washing to remove free dye: After incubation, discard the JC-1 staining solution and gently wash the cells twice with preheated PBS buffer to fully remove unbound free dye.

[0094] 4. Fluorescence microscopy: Maintain dark conditions and use a confocal microscope or laser confocal microscope for observation and imaging.

[0095] Red fluorescence: JC-1 polymerized state, indicating normal mitochondrial membrane potential;

[0096] Green fluorescence: JC-1 monomer, representing a decrease in mitochondrial membrane potential.

[0097] The results showed that the normal group cells exhibited strong red fluorescence and weak green fluorescence; the model group showed significantly enhanced green fluorescence and weakened red fluorescence, indicating a significant decrease in mitochondrial membrane potential. The Se-CDs@SS31 group showed significantly enhanced red fluorescence and weakened green fluorescence, demonstrating that the Se-CDs@SS31 of this invention can effectively protect mitochondrial membrane potential and improve mitochondrial function.

[0098] Experimental Example 5: In vivo safety of myocarditis - H&E myocardial tissue staining images

[0099] 1. Experimental animals and grouping: SPF grade C57BL / 6 mice aged 6-8 weeks were randomly divided into normal group, CDs group, Se-CDs group, and Se-CDs@SS31 group, with n≥6 in each group.

[0100] 2. After the drug administration and treatment model was established, each group was administered the corresponding preparation via tail vein injection at a concentration of 200 μg / mL, with a dosage volume of 100 μL per animal, once daily for 3 consecutive days. The normal group and the model group were given an equal volume of physiological saline.

[0101] 3. Take samples, wrap them in wax, and send them to section. Use staining agents according to the H&E staining procedure.

[0102] Experiment 6: Verification of the therapeutic effect of myocarditis in vivo

[0103] The specific steps for verifying the therapeutic effect of Se-CDs@SS31 on a mouse model of myocarditis are as follows:

[0104] 1. Establishment of myocarditis model: 6-8 week old C57BL / 6 mice (18-22g) were anesthetized by intraperitoneal injection of sodium pentobarbital, and then injected intraperitoneally with 10mg / kg lipopolysaccharide (LPS) to establish an acute myocarditis mouse model. The criteria for successful model establishment were obvious inflammatory cell infiltration and myocardial fiber edema and necrosis in myocardial tissue.

[0105] 2. Grouping and Treatment: The model mice were randomly divided into 5 groups (n≥3), and an additional normal sham-operated group (Sham group) was set up: Sham group (normal mice, injected with physiological saline via tail vein), myocarditis group (model mice, injected with physiological saline via tail vein), CDs group (model mice, injected with 200μg / mL CDs via tail vein, 0.4mL / mouse), Se-CDs group (model mice, injected with 200μg / mL Se-CDs via tail vein, 0.4mL / mouse), and Se-CDs@SS31 group (model mice, injected with 200μg / mL Se-CDs@SS31 via tail vein, 0.4mL / mouse). The mice were administered the drugs for 3 consecutive days.

[0106] 3. Detection indicators: Mice were sacrificed after treatment and myocardial tissue samples were collected. Immunohistochemical staining showed that the expression of PINK1 and Parkin proteins in the Se-CDs@SS31 group was significantly upregulated and the expression of Lc3b was increased, indicating that the preparation can effectively activate the PINK1 / Parkin pathway of myocardial cell mitochondria and clear damaged mitochondria.

[0107] Experiment Example 7: Verification of Immunomodulatory Effects

[0108] The immunomodulatory capacity of Se-CDs@SS31 was verified through the following steps:

[0109] 1. Macrophage polarization assay: RAW264.7 cells were stimulated with 1 μg / mL LPS for 30 minutes, and then 200 μg / mL Se-CDs@SS31 was added. After 24 hours of culture, the expression of M1 marker CD86 and M2 marker CD206 was detected by flow cytometry. The results showed that the CD206 / CD86 ratio in the Se-CDs@SS31 group was significantly higher than that in other groups, indicating that Se-CDs@SS31 can effectively induce macrophage polarization towards the anti-inflammatory M2 type and reduce myocardial inflammation.

[0110] 2. T-cell immune balance detection: Myocardial tissue from mice in each group of Experiment 5 was collected, and the number of CD4⁺ and CD8⁺ T cells was detected by flow cytometry. The results showed that the number of CD4⁺ T cells in the myocardial tissue of the Se-CDs@SS31 group was significantly increased, and the CD4⁺ / CD8⁺ ratio was also increased, indicating that Se-CDs@SS31 can effectively regulate the T-cell immune balance of myocardial tissue and enhance the body's own anti-myocardial inflammation ability.

[0111] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing a selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeted formulation, characterized in that: Includes the following steps: S1. Mix citric acid, ethylenediamine, and deionized water, and stir magnetically for 25-35 minutes until completely dissolved to obtain the precursor solution; S2. Place the precursor liquid obtained in step S1 in a high-pressure reactor and hydrothermally react for 5-7 hours. After the reaction is completed, cool to room temperature to obtain a carbon dot mixture. S3. Centrifuge the carbon dot mixture obtained in step S2, discard solid impurities and keep the supernatant. Put the supernatant into a dialysis bag and place the dialysis bag in ultrapure water for dialysis for 2-4 days. Change the water every 8 hours to obtain dialysis solution one. Freeze-dry dialysis solution one for 45-51 hours to obtain carbon dot CDs powder. S4. Add the carbon dot CDs powder obtained in step S3 to deionized water, and ultrasonically disperse for 15-25 minutes to obtain a carbon dot suspension. Add sodium selenite to the carbon dot suspension, and magnetically stir for 50-70 minutes. Then add hydrazine hydrate solution dropwise and continue magnetically stirring for 21-27 hours to obtain reaction solution one. S5. Place the reaction solution I obtained in step S4 into a dialysis bag, place the dialysis bag in ultrapure water for dialysis for 2-4 days, change the water every 8 hours to obtain dialysis solution II, freeze-dry dialysis solution II for 45-51 hours to obtain selenium-doped carbon dots Se-CDs powder. S6. Add the selenium-doped carbon dot Se-CDs powder obtained in step S5 to PBS buffer, and sonicate for 15-25 minutes to obtain a Se-CDs solution. Add the thiol-polyethylene glycol-mitochondrial targeted antioxidant peptide to the Se-CDs solution and stir for 1-3 hours to obtain reaction solution two. S7. Centrifuge the reaction solution obtained in step S6 to obtain precipitate one. Disperse precipitate one in PBS buffer and centrifuge again to obtain precipitate two. Disperse precipitate two in PBS buffer and centrifuge again to obtain precipitate three. Freeze-dry precipitate three for 21-27 hours to obtain the selenium-doped ethylenediamine-citric acid carbon dots-mitochondrial targeting formulation Se-CDs@SS31.

2. The method for preparing a selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeted formulation according to claim 1, characterized in that: In step S1, the ratio of citric acid, ethylenediamine, and deionized water is 2g:1mL:(40-60)mL; the magnetic stirring speed is 500rpm.

3. The method for preparing a selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeted formulation according to claim 1, characterized in that: In step S2, the lining of the high-pressure reactor is polytetrafluoroethylene, and the hydrothermal reaction temperature is 190-210℃.

4. The preparation method of a selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeted formulation according to claim 1, characterized in that: In step S3, the centrifugation speed is 12000 rpm, the centrifugation time is 15-25 minutes, the molecular weight cutoff of the dialysis bag is 3500 Da, and the freeze-drying temperature is -50℃.

5. The preparation method of a selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeted formulation according to claim 1, characterized in that: In step S4, the ratio of carbon dot CDs powder, deionized water, sodium selenite, and hydrazine hydrate solution obtained in step S3 is 30mg:(25-35)mL:25mg:0.8mL; the ultrasonic dispersion power is 200W and the frequency is 40kHz; the magnetic stirring speed is 500rpm; the mass concentration of hydrazine hydrate solution is 85%, and the dropping rate of hydrazine hydrate solution is 0.1mL / min.

6. The method for preparing a selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeted formulation according to claim 1, characterized in that: In step S5, the molecular weight cutoff of the dialysis bag is 3500 Da, and the freeze-drying temperature is -50°C.

7. The preparation method of a selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeted formulation according to claim 1, characterized in that: In step S6, the pH value of the PBS buffer is 7.4, the ratio of selenium-doped carbon dot Se-CDs powder obtained in step S5 to PBS buffer is 30 mg: (18-22) mL; the ultrasonic dispersion power is 200 W and the frequency is 40 kHz; the mass ratio of thiol-polyethylene glycol-mitochondrial targeted antioxidant peptide to selenium-doped carbon dot Se-CDs powder obtained in step S5 is 1:3; and the stirring reaction speed is 500 rpm.

8. The method for preparing a selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeted formulation according to claim 1, characterized in that: In step S7, the centrifugation speed is 15000 rpm and the centrifugation time is 25-35 minutes each time; the pH value of the PBS buffer is 7.4, and the mass ratio of precipitate one / precipitate two to PBS buffer is 1:3; the freeze-drying temperature is -50℃.

9. The selenium-doped ethylenediamine-citric acid carbon dot-mitochondrial targeted formulation prepared by the preparation method according to claims 1 to 8.

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