Near-infrared fluorescent carbon dots for cardiac targeting and preparation method and application thereof

Abstract

The application provides near-infrared fluorescent carbon dots for heart targeting and a preparation method and application thereof. By a one-step hydrothermal method, methylene blue and prussian blue are mixed and dissolved, then zinc salt and nickel salt are added, and after mixing, hydrothermal reaction is carried out, and through separation and purification, dialysis and freeze-drying, near-infrared fluorescent carbon dots with excellent performance are prepared. Through optimization of reaction conditions and doping strategy, the near-infrared fluorescence intensity of the carbon dots is significantly improved, and through heart-targeting peptide modification, the carbon dots have excellent heart-targeting performance. The method not only has the advantages of simple operation, low cost and environmental friendliness, but also can effectively improve the application value of the carbon dots in biomedical imaging, optical sensing and precise treatment.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to a near-infrared fluorescent carbon dot for cardiac targeting, its preparation method, and its application. Background Technology

[0002] Carbon dots (CDs) are a novel class of fluorescent nanomaterials that have attracted widespread attention in biomedicine, sensing, and photocatalysis in recent years due to their unique optical properties, good biocompatibility, and stability. Compared with traditional fluorescent dyes and quantum dots, carbon dots have advantages such as low toxicity, good water solubility, high chemical stability, and easy functionalization, making them a promising candidate for applications in fluorescence imaging, disease diagnosis, and optical probes. Among their many optical properties, near-infrared (NIR) fluorescence is particularly important in bioimaging and therapy due to its strong tissue penetration, lower background fluorescence interference, and higher signal-to-noise ratio. However, most carbon dots currently emit fluorescence in the ultraviolet to visible light range, while far-red and near-infrared emitting carbon dots are relatively scarce. More importantly, existing near-infrared carbon dot materials generally lack effective cardiac targeting capabilities for the specific technical requirement of cardiac targeting, which severely restricts their development in core application scenarios such as precise diagnosis and visualization of cardiovascular diseases. Therefore, developing a simple, efficient preparation method that can simultaneously endow carbon dots with excellent near-infrared luminescence properties and cardiac targeting function is a key problem that urgently needs to be solved in this technical field. Summary of the Invention

[0003] To address the aforementioned issues, this application proposes a near-infrared fluorescent carbon dot for cardiac targeting, obtained through a hydrothermal reaction of methylene blue, Prussian blue, zinc salt, and nickel salt. This application significantly improves the near-infrared fluorescence intensity of the carbon dot by optimizing the reaction conditions and doping strategy.

[0004] Furthermore, the mass ratio of methylene blue, Prussian blue, zinc salt, and nickel salt is 1:1:(2~4):(2~4).

[0005] Furthermore, the zinc salt includes one or more of zinc acetate, zinc chloride, zinc nitrate, and zinc sulfate;

[0006] The nickel salt includes one or more of nickel chloride, nickel nitrate, nickel acetate, and nickel sulfate.

[0007] Preferably, the zinc salt is zinc acetate and the nickel salt is nickel chloride.

[0008] Furthermore, near-infrared fluorescent carbon dots are combined with cardiac-targeting peptides. The method for combining near-infrared fluorescent carbon dots with cardiac-targeting peptides is as follows: the cardiac-targeting peptides, EDC and NHS are fully dissolved in water, stirred and reacted for 20-40 minutes, near-infrared fluorescent carbon dots are added, and the reaction is continued for 4-8 hours, followed by dialysis for 10-14 hours.

[0009] Furthermore, the mass ratio of near-infrared fluorescent carbon dots to cardiac-targeting peptides is (4~6):1.

[0010] Furthermore, the mass ratio of the cardiac-targeting peptide, EDC, and NHS is 1:(1~2):(0.5~1.5).

[0011] Furthermore, cardiac-targeting peptides include one or more of CTP, atrial natriuretic peptide (ANP), myocardial peptide, CRPPR (Cys-Arg-Pro-Pro-Arg, which binds to cardiac vascular endothelial receptor CRIP-2), and SS-31 (HD-Arg-Dmt-Lys-Phe-NH2, which binds to mitochondrial endometrial cardiolipin).

[0012] This application provides a method for preparing near-infrared fluorescent carbon dots for cardiac targeting, comprising the following steps:

[0013] S1. Mix and dissolve methylene blue and Prussian blue, and stir to obtain mixed solution A;

[0014] S2. Mix mixed solution A with zinc salt and nickel salt and dissolve them completely to obtain mixed solution B;

[0015] S3. Perform a hydrothermal reaction on mixed solution B at 130-150℃ for 8-10 hours to obtain mixed solution C;

[0016] S4. The resulting mixed solution C was purified by separation, dialyzed, and freeze-dried to obtain near-infrared fluorescent carbon dots.

[0017] Preferably, the hydrothermal reaction heating temperature in S3 is 140℃, and the preferred reaction time is 9h.

[0018] Furthermore, in step S1, the solvent used to dissolve methylene blue and Prussian blue is water.

[0019] Furthermore, in step S1, the methylene blue needs to be fully dissolved under heating conditions.

[0020] Furthermore, the specific steps for separation and purification in S4 are as follows: the separation and purification methods are aqueous membrane filtration (0.22μm) and centrifugation (10000rpm, 10min).

[0021] Furthermore, the specific steps of dialysis in S4 are as follows: the obtained purified solution is placed in a dialysis bag and dialyzed for 24 hours, with the dialysis solution being replaced every 12 hours. The dialysis solution is deionized water, and the molecular weight cutoff of the dialysis bag is 1000 KD.

[0022] The near-infrared fluorescent carbon dots obtained in this application for cardiac targeting have applications in biomedical imaging, optical sensing, and the preparation of drugs for treating diseases.

[0023] Furthermore, the near-infrared fluorescent carbon dots obtained in this application for cardiac targeting can be used in in vitro and in vivo cardiac targeting imaging and in the preparation of drugs for treating cardiovascular diseases.

[0024] Furthermore, the cardiac-targeted application includes both cellular and in vivo levels.

[0025] This application can bring the following beneficial effects:

[0026] 1. This application significantly improves the near-infrared fluorescence intensity of carbon dots by optimizing reaction conditions and doping strategies, and exhibits excellent cardiac targeting performance through modification with cardiac-targeting peptides. This method not only has the advantages of simple operation, low cost, and environmental friendliness, but also effectively enhances the application value of carbon dots in biomedical imaging, optical sensing, and precision medicine.

[0027] 2. In this application, methylene blue is used as both a carbon and nitrogen source. Its conjugated aromatic structure can be carbonized under high-temperature hydrothermal conditions to form fluorescent carbon dots, while the benzothiazole structure contributes to near-infrared fluorescence emission. Prussian blue provides an iron / cyano framework, which decomposes at high temperatures to form iron oxide nanoparticles or iron-doped carbon dots. The cyano group promotes nitrogen doping during carbonization, modulating the energy level structure. The combination of methylene blue and Prussian blue in this application forms a precursor complex through π-π stacking or coordination, affecting the final carbon dot size and surface states.

[0028] 3. The introduction of zinc ions in this application promotes carbon dot nucleation, and the formation of zinc oxide at high temperatures can generate mesoporous structures, increasing the specific surface area. The introduction of nickel ion doping in this application can introduce defect states, broadening fluorescence emission into the near-infrared region, while simultaneously enhancing photostability. Zinc / nickel co-doping in this application can form a metal-nitrogen-carbon structure, improving electron transfer efficiency.

[0029] 4. Methylene blue and Prussian blue undergo dehydration and condensation at high temperatures to form carbon cores. Incompletely carbonized molecules adhere to the surface of the carbon cores, forming functional groups such as amino / carboxyl groups, which impart water solubility and fluorescence properties. Iron / zinc / nickel ions are embedded in the matrix to adjust the band gap and achieve near-infrared emission.

[0030] 5. Large particles or unreacted precursors are removed through separation and purification steps, while small fluorescent carbon dots are retained; free metal ions and small molecule impurities are removed through dialysis, improving biocompatibility.

[0031] 6. The freeze-drying step can preserve the activity of surface functional groups.

[0032] 7. This application enables the binding of cardiac-targeting peptides to fluorescent carbon dots via carboxyl-amino coupling or electrostatic adsorption. Under the action of EDC, the carboxyl groups on the carbon dot surface generate unstable O-acylisourea intermediates. NHS reacts with these intermediates to form more stable NHS esters, significantly improving the efficiency of subsequent reactions with amino groups. The free amino group of the cardiac-targeting peptide nucleophilically attacks the activated NHS ester, forming stable amide bonds, thus achieving covalent linkage between the peptide and carbon dots. In this process, EDC / NHS selectively activates the carboxyl groups and reacts with the amino groups, avoiding random cross-linking and preserving the targeting properties of the peptide and the fluorescence properties of the carbon dots.

[0033] 8. The reaction can be completed in aqueous phase with stirring at room temperature, avoiding damage to the structure of peptides or carbon dots by high temperature or organic solvents; by adjusting the EDC / NHS ratio or the peptide / carbon dot feed ratio, the modification density of peptides on the carbon dot surface can be controlled, balancing targeting and fluorescence efficiency; the active sequence of the cardiac targeting peptide retains its conformation after coupling, ensuring targeting, and small molecule byproducts are removed through purification steps. Attached Figure Description

[0034] Figure 1 This is a TEM image of the carbon dots prepared in Example 5 of the present invention;

[0035] Figure 2 The image shown is the AFM pattern of the carbon dots prepared in Example 5 of this invention.

[0036] Figure 3 The fluorescence emission spectrum of the carbon dots prepared in Example 5 of this invention;

[0037] Figure 4 The fluorescence stability diagram of the carbon dots prepared in Example 5 of this invention;

[0038] Figure 5 This is a targeted imaging diagram of cardiomyocytes using carbon dots from the present invention;

[0039] Figure 6 This is an in vivo cardiac targeted imaging image of the carbon dots of the present invention. Detailed Implementation

[0040] Unless otherwise specified, the reagents and instruments used in the following examples are all commercially available products.

[0041] A specific embodiment provides a method for preparing near-infrared fluorescent carbon dots for cardiac targeting, as follows:

[0042] S1. Weigh out methylene blue and Prussian blue;

[0043] S2. Using deionized water as a solvent, mix and fully dissolve methylene blue and Prussian blue to obtain mixed solution A;

[0044] S3. Mix the mixed solution A obtained in S2 with nickel salt and zinc salt and dissolve them completely to obtain mixed solution B;

[0045] S4. Transfer the mixed solution B obtained in S3 to a polytetrafluoroethylene hydrothermal reactor for hydrothermal reaction to obtain mixed solution C;

[0046] S5. Filter the mixed solution C obtained in S4 through a 0.22 μm aqueous filter membrane and centrifuge (10000 rpm, 10 min) to obtain solution D;

[0047] S6. Place the solution D obtained in S5 into a dialysis bag with a molecular weight cutoff of 1000KD and dialyze for 24 hours, changing the dialysate every 12 hours. After the process is completed, freeze-dry to obtain near-infrared fluorescent carbon dots.

[0048] Specific implementation examples and comparisons are shown in Table 1 below:

[0049] Table 1. Specific parameters of the detailed embodiments and comparative examples.

[0050]

[0051] The above embodiments and comparative examples were characterized, and the characterization results are shown in Table 2:

[0052] 1. Carbon dot morphology and particle size detection: Tested using transmission electron microscopy;

[0053] 2. Carbon dot dispersion: Tested using atomic force microscopy;

[0054] 3. Fluorescence emission spectrum: Take an appropriate amount of carbon dot powder, dissolve it fully in PBS solution, and use a fluorescence spectrophotometer to detect the fluorescence emission properties of near-infrared fluorescent carbon dots (CDs) at different excitation wavelengths (550 nm-600 nm).

[0055] The fluorescence intensity of all examples and comparative examples was measured at an excitation wavelength of 600 nm. The carbon dots of Example 5 were set as the base fluorescence intensity (normalized intensity was 1). The normalized fluorescence intensity was obtained by dividing the fluorescence intensity of all carbon dots by the base fluorescence intensity.

[0056] 4. Fluorescence stability: The fluorescence stability of the material was reflected by the effects of pH, ionic strength, irradiation time, and storage time on the fluorescence intensity of CDs. (1) Effect of pH on the fluorescence intensity of CDs: BR buffer solutions of varying concentrations were prepared, and the CDs prepared in Example 5 were thoroughly mixed with buffer solutions of different pH values. The fluorescence intensity of different samples was then measured using a fluorescence spectrophotometer. (2) Effect of ionic strength on the fluorescence intensity of CDs: NaCl solutions of varying concentrations were prepared and mixed thoroughly with the CDs prepared in Example 5. The fluorescence intensity of each sample was measured using a fluorescence spectrophotometer to observe the effect of ionic strength on its fluorescence. (3) Effect of irradiation time on the fluorescence intensity of carbon dots: The CDs prepared in Example 5 were mixed thoroughly with deionized water and continuously irradiated under a xenon lamp. The fluorescence intensity was measured at different irradiation time points to observe the effect of irradiation time on the fluorescence of the synthesized CDs. (4) Effect of storage time on the fluorescence intensity of carbon dots: The CDs prepared in Example 5 were mixed thoroughly with deionized water and stored for different times to measure their fluorescence intensity. The effect of storage time on the fluorescence intensity of CDs was observed.

[0057] Table 2 Characterization results of the examples and comparative examples

[0058]

[0059] This application utilizes a synergistic effect of methylene blue, Prussian blue, zinc salt, and nickel salt to create the most efficient and stable near-infrared luminescent centers. Simultaneously, the passivation effect of zinc salt reduces defects, resulting in the highest fluorescence intensity. Comparative Example 1 lacks Prussian blue, resulting in NIR luminescent centers constructed from iron and cyanide ions. The carbon dot emission wavelength is short, hindering biological applications, and the nickel / zinc combination primarily acts as a quencher, leading to extremely weak fluorescence. Comparative Example 2 lacks nickel, resulting in poor efficiency or stability in luminescent center generation and decreased intensity. Comparative Example 3 lacks zinc, leading to more surface defects, increased non-radiative transitions, and decreased intensity. Comparative Example 4 has a short carbon dot emission wavelength, hindering biological applications.

[0060] from Figure 1 TEM results show that the carbon dot material prepared in this application is quasi-spherical with an average particle size of less than 10.00 nm.

[0061] from Figure 2 The AFM results show that the carbon dots in this application have good dispersion and no obvious aggregation, with their thickness mainly concentrated at about 2.00 nm.

[0062] like Figure 1 , Figure 2 As shown, the carbon dot material obtained in this application is uniform in size, spherical, has good crystallinity and dispersibility, and does not aggregate over a long period of time. The carbon dots obtained in Comparative Examples 1-4 cannot achieve the technical effects of the embodiments in terms of morphology and dispersibility.

[0063] from Figure 3 Fluorescence spectroscopy analysis showed that the carbon dots prepared in this application have a fluorescence emission performance of 640 nm, belonging to near-infrared fluorescent carbon dots.

[0064] like Figure 4 As shown, the carbon dot materials of this application exhibit excellent stability in fluorescence and photothermal properties under different ionic strengths and pH environments. Furthermore, prolonged light exposure and storage have no significant impact on their performance, indicating that CDs possess excellent stability. The reaction system of this application maintains stable fluorescence intensity over a wide pH range (4-10). Comparative Examples 1 and 4 lack a robust framework and are more sensitive to pH; at acidic pH, fluorescence may be quenched due to protonation; at alkaline pH, it may be altered or quenched due to deprotonation or structural changes.

[0065] The reaction system in this application maintains stable fluorescence intensity in high-concentration salt solutions, indicating good surface inertness and resistance to aggregation. Comparative Examples 1 and 4, under high ionic strength, experience double-layer compression, leading to carbon dot aggregation and quenching, resulting in a sharp decrease in fluorescence intensity. Comparative Example 3, lacking zinc salt, exhibits a certain degree of concentration-dependent quenching.

[0066] In the reaction system of this application, the fluorescence intensity of the carbon dot material remains stable even after long-term irradiation. Comparative Examples 1 and 4 show poor stability, with fluorescence intensity decaying rapidly over time and weak resistance to photobleaching. Comparative Example 3 lacks the passivation effect of zinc salt, resulting in an increase in defect states and non-radiative transitions, making it more susceptible to damage under light irradiation.

[0067] The reaction system of this application showed no significant change in fluorescence intensity after storage for weeks or even months. Comparative Example 1 showed more significant attenuation, while Comparative Example 4 had a relatively unstable structure and may have undergone slow aggregation or chemical degradation.

[0068] The example provides another type of near-infrared fluorescent carbon dot. The near-infrared fluorescent carbon dots (CDs) prepared above are further combined with cardiac targeting peptides to prepare cardiac targeting near-infrared fluorescent carbon dots (targeting peptide-CDs). The specific implementation method is as follows:

[0069] The cardiac-targeting peptide, EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), and NHS (N-hydroxysuccinimide) were fully dissolved in water. After stirring for 20-40 min, infrared fluorescent carbon dots were added, and the reaction was continued for 4-8 h. After the reaction was completed, the mixture was dialyzed for 10-14 h (molecular weight cutoff of 1000D) to obtain cardiac-targeting near-infrared fluorescent carbon dots.

[0070] Cardiac-targeting peptides include CTP, atrial natriuretic peptide (ANP), myocardial peptide, CRPPR (Cys-Arg-Pro-Pro-Arg, which binds to cardiac vascular endothelial receptor CRIP-2), SS-31 (HD-Arg-Dmt-Lys-Phe-NH2, which binds to mitochondrial endometrial cardiolipin), etc., but are not limited to the above-mentioned cardiac-targeting peptides.

[0071] To further demonstrate the experimental results, cardiac-targeting near-infrared fluorescent carbon dots (CTP-CDs) were prepared. The specific preparation method is as follows:

[0072] 2 mg each of cardiac-targeting peptide CTP, EDC and NHS were fully dissolved in water and stirred for 30 min. Then, 10 mg of infrared fluorescent carbon dots (CDs) prepared in Example 5 were added and the reaction was continued for 6 h. After dialysis for 12 h (with a molecular weight cutoff of 1000D), cardiac-targeting near-infrared fluorescent carbon dots CTP-CDs were obtained.

[0073] The following tests were performed on cardiac-targeted near-infrared fluorescent carbon dots:

[0074] 1. Targeted imaging of cardiomyocytes using carbon dots: Cardiomyocytes were imaged at 1.00 × 10⁻⁶ pixels. 5 Cardiac cardiomyocytes were seeded at a density of 1 cell / well in confocal culture dishes. After 24 h, 1.00 mL of CDs and cardiac-targeted near-infrared fluorescent carbon dots CTP-CDs solution (concentration of 50 μg / mL) were added to each culture dish, and the cells were incubated in a cell culture incubator for another 24 h. After incubation, the cells were washed with PBS, and then fluorescence imaging was performed using a laser confocal microscope to observe the uptake of CDs and cardiac-targeted near-infrared fluorescent carbon dots by cardiomyocytes.

[0075] 2. In vivo cardiac-targeting imaging of carbon dots: Mice were injected via tail vein with 1.0 mg / mL of CDs and a cardiac-targeting near-infrared fluorescent carbon dot CTP-CDs solution. Drug circulation in mice was then monitored using a multimodal Xtreme imaging system, and fluorescence imaging was performed at different time points (0, 0.5, 3, and 5 h). Heart tissue was collected 5 h later and subjected to fluorescence imaging at the same excitation wavelength.

[0076] Depend on Figure 5 Cell imaging showed that after modification with cardiac-targeting peptides, the uptake of carbon dots by cardiomyocytes increased significantly, indicating its potential in cardiac-targeting imaging.

[0077] Figure 6 It is evident that the near-infrared fluorescent carbon dots of this application exhibit excellent cardiac targeting performance, laying the foundation for their application in cardiac targeted imaging.

[0078] The various embodiments in this specification are described in a progressive manner. Similar or identical parts between embodiments can be referred to interchangeably. Each embodiment focuses on describing the differences from other embodiments. In particular, the system embodiments are basically similar to the method embodiments, so the description is relatively simple; relevant parts can be referred to the descriptions in the method embodiments.

[0079] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A near-infrared fluorescent carbon dot for cardiac targeting, characterized in that, Near-infrared fluorescent carbon dots were obtained by hydrothermal reaction of methylene blue, Prussian blue, zinc salt, and nickel salt at 130–150 °C. The mass ratio of methylene blue, Prussian blue, zinc salt, and nickel salt is 1:1:(2~4):(2~4).

2. The near-infrared fluorescent carbon dot for cardiac targeting according to claim 1, characterized in that: The zinc salt includes one or more of zinc acetate, zinc chloride, zinc nitrate, and zinc sulfate; The nickel salt includes one or more of nickel chloride, nickel nitrate, nickel acetate, and nickel sulfate.

3. The near-infrared fluorescent carbon dot for cardiac targeting according to claim 1, characterized in that: The near-infrared fluorescent carbon dots are then combined with the cardiac-targeting peptide. The method for combining the near-infrared fluorescent carbon dots with the cardiac-targeting peptide is as follows: the cardiac-targeting peptide, EDC and NHS are fully dissolved in water, the mixture is stirred and reacted for 20-40 minutes, the near-infrared fluorescent carbon dots are added, the reaction is continued for 4-8 hours, and then dialyzed for 10-14 hours.

4. The near-infrared fluorescent carbon dot for cardiac targeting according to claim 3, characterized in that: The mass ratio of the near-infrared fluorescent carbon dots to the cardiac-targeting peptide is (4~6):

1.

5. A near-infrared fluorescent carbon dot for cardiac targeting according to claim 3, characterized in that: The mass ratio of the cardiac-targeting peptide, EDC, and NHS is 1:(1~2):(0.5~1.5).

6. The near-infrared fluorescent carbon dot for cardiac targeting according to claim 3, characterized in that: The cardiac-targeting peptides include one or more of CTP, atrial natriuretic peptide (ANP), cardiac peptide, CRPPR, and SS-31.

7. A method for preparing near-infrared fluorescent carbon dots for cardiac targeting, characterized in that, Includes the following steps: S1. Mix and dissolve methylene blue and Prussian blue, and stir to obtain mixed solution A; S2. Mix mixed solution A with zinc salt and nickel salt and dissolve them completely to obtain mixed solution B; S3. Perform a hydrothermal reaction on mixed solution B at 130-150℃ for 8-10 hours to obtain mixed solution C; S4. The resulting mixed solution C was purified by separation, dialyzed, and freeze-dried to obtain near-infrared fluorescent carbon dots; The mass ratio of methylene blue, Prussian blue, zinc salt, and nickel salt is 1:1:(2~4):(2~4).

8. The near-infrared fluorescent carbon dots for cardiac targeting as described in any one of claims 1-6, or the near-infrared fluorescent carbon dots for cardiac targeting prepared by the preparation method according to claim 7, are used in biomedical imaging, optical sensing, and the preparation of drugs for treating diseases.

9. The application according to claim 8, characterized in that: Applications of the near-infrared fluorescent carbon dots in in vivo and in vitro cardiac targeted imaging and in the preparation of drugs for treating cardiovascular diseases.

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