Metalloporphyrin carbon dots and preparation method and application thereof

Abstract

This invention belongs to the field of nanomaterials and biomedicine, specifically relating to a metalloporphyrin carbon dot, its preparation method, and its applications. To provide a multifunctional carbon dot with a simple synthesis method, this invention utilizes a one-step solvothermal method. By selecting different metal precursors and solvents, its fluorescence properties and catalytic activity are controlled. Furthermore, by selecting different hydrophilic polymer precursors, the carbon dots are directly endowed with opposite surface charges, thereby achieving different in vitro and in vivo behaviors and functional applications.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials and biomedicine, specifically relating to a metalloporphyrin carbon dot, its preparation method, and its application. Background Technology

[0002] Carbon dots (CDs), as novel carbon-based nanomaterials, have shown great potential in fields such as bioimaging, drug delivery, and disease treatment due to their excellent biocompatibility, low toxicity, ease of functionalization, and unique optical properties. However, most current carbon dots have relatively limited functions, such as only possessing fluorescence imaging capabilities or single catalytic activities, which is insufficient to meet the needs of integrated diagnosis and treatment in complex biological environments. Furthermore, the surface charge of carbon dots has a decisive influence on their in vivo distribution, cellular uptake, and subsequent biofunctional modifications, but directly controlling the surface charge of carbon dots and endowing them with multiple functions through a simple synthetic route remains a challenge.

[0003] On the other hand, reactive oxygen species (ROS) play a crucial role in the development and progression of many diseases. For example, excessive hydrogen peroxide (H2O2) is often present in the tumor microenvironment. Developing nanomaterials capable of specifically catalyzing the decomposition of H2O2 while possessing other adjuvant therapeutic functions (such as drug delivery and fluorescence tracing) is of great significance for achieving highly efficient and low-side-effect treatments. Metalloporphyrin compounds are ideal structures for mimicking natural enzymes (such as CAT), but their stability, solubility, and targeting capabilities in vivo need further improvement.

[0004] Therefore, there is an urgent need to develop a simple synthesis method that can simultaneously integrate fluorescence imaging, enzyme catalytic activity, and tunable surface charge. Summary of the Invention

[0005] This invention addresses the aforementioned problems by providing a series of functional carbon dots with metalloporphyrins as their core structure. These carbon dots are synthesized via a one-step solvothermal method. Their fluorescence properties and catalytic activity can be modulated by selecting different metal precursors and solvents. Furthermore, by selecting different hydrophilic polymer precursors (such as PEG2000 or branched PEI), the carbon dots can be directly endowed with opposite surface charges, thereby achieving different in vitro and in vivo behaviors and functional applications.

[0006] To achieve the above objectives, the present invention employs the following technical solution: This invention provides a method for preparing metalloporphyrin carbon dots, comprising the following steps: The raw material protoporphyrin, metal chloride or metal nitrate is mixed with a solvent and placed in a closed reactor. The mixture is reacted at 150-200°C for 6-24 hours. After the reaction is completed, the mixture is cooled, purified and dried to obtain metalporphyrin carbon dots.

[0007] Furthermore, the metal chloride is CoCl2, MnCl2, MgCl2, ZnCl2, CeCl3, HfCl4, CuCl2, or FeCl3; the metal nitrate is Co(NOl3)2, Mn(NOl3)2, Mg(NO3)2, Zn(NO3)2, Ce(NO3)3, HfO(NO3)2·xH2O, Cu(NO3)2, or Fe(NO3)3; and the solvent is N,N-dimethylformamide or ethanol.

[0008] Furthermore, the metal chloride is CoCl2 or ZnCl2, and the reaction is carried out at 160°C for 6 hours. The resulting carbon dots emit red fluorescence under ultraviolet light excitation and exhibit significant CAT activity, enabling them to efficiently catalyze the decomposition of H2O2 into oxygen.

[0009] Furthermore, the metal nitrate is Fe(NO3)3 or Cu(NO3)3, and the reaction is carried out at 160°C for 6 hours. The resulting carbon points catalyze the decomposition of H2O2 into oxygen with higher efficiency than the corresponding chlorides, namely FeCl3 and CuCl2.

[0010] Furthermore, the raw materials also include polyethylene glycol 2000 or branched polyethyleneimine.

[0011] When polyethylene glycol 2000 (PEG2000) is used as a reactant and heated with a metalloporphyrin system, the resulting carbon dots acquire a negative surface charge. This property reduces non-specific binding to plasma proteins in the bloodstream, facilitating long-term circulation and making it suitable as a nanomedicine for systemic ROS scavenging.

[0012] When branched polyethyleneimine (PEI) is used as the reactant, the resulting carbon dots have a surface rich in amino groups and are positively charged. This property not only facilitates the loading of negatively charged nucleic acid drugs via electrostatic interactions, but the amino functional groups on their surface can also be readily grafted with targeting ligands (such as folic acid FA) or small molecule drugs (such as dexamethasone, tumor necrosis factor-α inhibitors, etc.) through chemical coupling (e.g., using NHS-PEG-FA) to achieve active targeting or combination therapy.

[0013] The present invention also provides a series of metalloporphyrin carbon dots, which are prepared by the above-described preparation method.

[0014] This invention also provides applications of the metalloporphyrin carbon dots: 1) for catalyzing the decomposition of H2O2; 2) for bioimaging tracing; 3) for preparing ROS scavengers; and 4) as multifunctional nanocarriers.

[0015] This invention provides a method and findings for elucidating the differences in catalytic activity of carbon points through theoretical calculations: The adsorption energies of H2O2 molecules on different metal porphyrin (M-porphyrin, M=Ce, Co, Cu, Fe, Hf, Mg, Mn, Mo, Zn) models were calculated using density functional theory (DFT). The calculation formula is: E_ads = E(slab+H2O2) - E(slab) - E(H2O2).

[0016] Calculations show that the adsorption energy of H2O2 in the Mn-porphyrin model is -0.43377 eV, lower than that in the Co-porphyrin model (-0.13630 eV). The more negative adsorption energy indicates a stronger binding between H2O2 and Mn-porphyrin, theoretically more conducive to the initiation of the catalytic reaction. This explains, from an electronic structure perspective, why Mn-based carbon dots exhibit superior CAT activity compared to Co-based carbon dots. Among all metals, Hf-porphyrin has the most negative adsorption energy for H2O2 (-1.00943 eV).

[0017] Compared with the prior art, the present invention has the following advantages: 1. Negatively charged carbon dots prepared with PEG2000 can be used as long-acting ROS scavengers to treat systemic diseases related to oxidative stress.

[0018] 2. Positively charged carbon dots prepared with PEI can serve as multifunctional nanocarriers. By modifying the surface with targeting ligands (such as NHS-PEG-FA) and / or loading therapeutic drugs (such as dexamethasone), they can be used for targeted therapy and imaging of diseases such as tumors.

[0019] 3. Zn-based carbon dots with red fluorescence can be used for bioimaging tracing.

[0020] 4. Cu-based carbon dots with high CAT activity can be used to regulate the redox microenvironment of lesions such as tumors. Attached Figure Description

[0021] Figure 1 The images show fluorescence images of carbon dots synthesized under different conditions. (A) From left to right: Carbon dot PBS solutions (Co-DMF) prepared using DMF as solvent and CoCl2 as metal source, with concentrations of 0.01, 0.1, and 1 mg / mL, respectively; (B) From left to right: Carbon dot PBS solutions (Co-EtOH) prepared using ethanol as solvent and CoCl2 as metal source, with concentrations of 0.01, 0.1, and 1 mg / mL, respectively. The results show that higher concentrations result in stronger fluorescence, and under the same conditions, carbon dots prepared using DMF as solvent exhibit higher fluorescence intensity than those prepared using EtOH as solvent.

[0022] Figure 2The fluorescence emission spectrum is shown for a representative carbon dot. The excitation wavelength was 400 nm, and emission peaks with high intensity were observed near 630 nm and 680 nm. Note: Co-DMF-1, raw materials: protoporphyrin and cobalt chloride; solvent: DMF; reaction conditions: 160℃, 6 h; concentration: 1 mg / mL PBS solution.

[0023] Figure 3 This is a photograph verifying the catalase activity of carbon dots. 100 μL of Mn-based carbon dot (Mn-EtOH, Mn-DMF) solution (concentrations of 0.01, 0.1, and 1 mg / mL, respectively) was added to 1 mL of 1 M H2O2 solution. After 5 minutes, a large number of oxygen bubbles were observed to be generated, demonstrating that it possesses catalase activity that catalyzes the decomposition of H2O2.

[0024] Figure 4 This is a Zeta potential diagram of negatively charged carbon dots on the surface of Example 3.

[0025] Figure 5 This is a Zeta potential diagram of positively charged carbon dots on the surface of Example 4.

[0026] Figure 6 This is a schematic diagram of H2O2 decomposition in Example 6. Detailed Implementation

[0027] To further illustrate the technical solution of the present invention, the present invention will be further described below through embodiments.

[0028] Example 1: Preparation and characterization of red fluorescent carbon dots Weigh 11.25 mg of protoporphyrin, 2.85 mg of CoCl2·6H2O, and 250 mg of PEI (MW: 25000, 50% aqueous solution), and dissolve them in 30 mL of DMF. Sonicate the solution until homogeneous. Transfer the mixture to a 50 mL high-pressure reactor lined with polytetrafluoroethylene (PTFE), and react in a 160 °C oven for 6 hours. After the reaction, allow it to cool naturally to room temperature. Filter the resulting solution through a 0.22 μm membrane. Dialyze the filtrate through a dialysis bag with a molecular weight of 3500 Da in deionized water for 72 hours. Freeze-dry the filtrate to obtain a solid powder, denoted as Co-DMF.

[0029] Co-DMF was prepared into PBS solutions of 0.01, 0.1, and 1 mg / mL, respectively. The samples were then observed and photographed using a small animal in vivo imaging system (excitation and emission wavelengths were 420 nm and 620 nm, respectively). Figure 1 As shown in (A), it exhibits bright red fluorescence, with the fluorescence intensity increasing at higher concentrations. A fluorescence spectrometer was used to detect a 1 mg / mL sample (Co-DMF-1) under 400 nm excitation, showing strong emission peaks at 630 nm and 680 nm (as shown in Figure A). Figure 2 (As shown).

[0030] When the DMF solvent in the above synthesis method is replaced with EtOH, and under the same reaction and post-treatment conditions, the resulting material (denoted as Co-EtOH) exhibits the exact same fluorescence behavior in fluorescence imaging detection. Figure 1 (B)

[0031] Example 2: Preparation and Verification of CAT Active Carbon Dots Weigh 11.25 mg of protoporphyrin, 4.29 mg of MnCl2·4H2O, and 250 mg of PEI (MW: 25000, 50% aqueous solution), dissolve them in 30 mL of DMF, and follow the same steps as in Example 1 to obtain a solid powder denoted as Mn-DMF.

[0032] Take 1 mL of 1 M H₂O₂ solution into an EP tube, add 100 μL of Mn-DMF in PBS solution (1 mg / mL), and gently shake to mix. After 5 minutes, a large number of bubbles can be observed continuously being generated in the tube (e.g., ...). Figure 3 (As shown). Using an H₂O₂ solution without added carbon dots as a control, no obvious bubbles were observed. This demonstrates that Mn-DMF exhibits significant CAT activity.

[0033] By replacing the DMF solvent in the above synthesis method with EtOH, and under the same reaction and post-treatment conditions, the resulting material (denoted as Mn-EtOH) also exhibits significant CAT-like activity, and can efficiently catalyze the decomposition of H2O2 to generate a large number of bubbles.

[0034] Example 3: Preparation of negatively charged carbon dots on the surface Weigh out 11.25 mg of protoporphyrin, 8.89 mg of Fe(NO3)3·9H2O, and 20 mg of polyethylene glycol 2000 (PEG2000), and dissolve them in 30 mL of DMF. The subsequent solvothermal reaction (160℃, 6 h) and purification steps are the same as in Example 1, yielding a solid powder denoted as Fe-DMF. The zeta potential in the aqueous solution, as measured by a potentiometer, is approximately -9.6 ± 0.7 mV. Figure 4 ).

[0035] Example 4: Preparation and functionalization of positively charged carbon dots on the surface Weigh out 11.25 mg of protoporphyrin, 4.29 mg of MnCl2·4H2O, and 250 mg of branched polyethyleneimine (PEI, MW: 25000, 50% aqueous solution), and dissolve them in 30 mL of DMF. The subsequent solvothermal reaction (160℃, 6 h) and purification steps are the same as in Example 1, yielding a solid powder denoted as Mn-DMF. The zeta potential in the aqueous solution, as measured by a potentiometer, is approximately +5.2 ± 1.6 mV. Figure 3 ).

[0036] 10 mg of Mn-DMF was dispersed in PBS (pH=7.4), and excess NHS-PEG-FA (folic acid-polyethylene glycol-succinimide ester) was added. The mixture was stirred at room temperature in the dark for 12 hours. The reaction solution was dialyzed to remove unreacted coupling agent, yielding folic acid-targeted carbon dot Mn-DMF-FA. This product can be used for targeted drug delivery through its positive surface charge and the targeting effect of FA.

[0037] Example 5: Theoretical calculations reveal differences in catalytic activity Using protoporphyrins as building blocks and different metals (Ce, Co, Cu, Fe, Hf, Mg, Mn, Mo, Zn) as complexation centers, a series of metalloporphyrin structures were obtained. The binding energy between the metal element and H₂O₂ was calculated using the metal element as the binding site. Table 1 shows the theoretically calculated adsorption energies of H₂O₂ on different metalloporphyrin models. First-principles calculations within the density functional theory (DFT) framework were performed using Materials Studio or VASP software packages. The Perdew-Burke-Ernzerhof (PBE) functional from the generalized gradient approximation (GGA) was selected as the exchange correlation functional. The plane wave cutoff energy was set to 400 eV, and the convergence criteria for energy and force during structural optimization were 10⁻⁵ eV and 0.02 eV·Å, respectively. -1 The Brillouin zone K-point grid was set to 1×1×1. Molecular force field simulations were not involved in this study. The interaction between H2O2 molecules was investigated by calculating their adsorption energies on different substrate surfaces. The adsorption energy calculation formula is: Eads = E(slab+H2O2) - E(slab) - E(H2O2), where Eslab+H2O2 represents the total energy of the system after adsorption, Eslab is the energy of the pure substrate, and EH2O2 is the energy of the isolated H2O2 molecule. No special boundary conditions other than periodic boundary conditions were used.

[0038] Planar structural models of different metal (M) porphyrins were constructed using density functional theory (DFT) to simulate the active centers of carbon dots. After optimizing all geometries, the adsorption energies (Eads) of H2O2 molecules on each M-porphyrin model were calculated. Some key results are shown in Table 1: the Eads of Co-porphyrin is -0.13630 eV, while that of Mn-porphyrin is -0.43377 eV. This indicates that the adsorption of H2O2 at Mn sites is more stable and stronger, theoretically supporting the higher CAT activity exhibited by Mn-based carbon dots in experiments. The calculations also predict that Hf-porphyrin may have the strongest H2O2 adsorption capacity (Eads = -1.00943 eV).

[0039] Table 1 Adsorption energies of H2O2 on different metalloporphyrin models Example 6

[0040] The metal nitrate was Cu(NO3)2, and the reaction was carried out at 160°C for 6 hours. The resulting carbon points catalyzed the decomposition of H2O2 into oxygen with higher efficiency than the corresponding chloride, CuCl2, as shown in the results. Figure 6 As shown. Example 7

[0041] Weigh 11.25 mg of protoporphyrin, 4.29 mg of MnCl2·4H2O, and 250 mg of branched polyethyleneimine (PEI, MW: 25000, 50% aqueous solution), and dissolve them in 30 mL of DMF. Disperse the solutions evenly by sonication. Transfer the mixed solution to a high-pressure reactor lined with polytetrafluoroethylene (PTFE) and react in ovens at 150℃, 170℃, 180℃, and 200℃ for 6 hours, respectively. Separately, react the same mixture at 160℃ for 8 hours, 12 hours, and 24 hours, respectively. After the reaction, allow the solutions to cool naturally to room temperature. Filter the resulting solutions through a 0.22 μm membrane. Dialyze the filtrate through a dialysis bag with a molecular weight of 3500 Da in deionized water for 72 hours. Freeze-dry the filtrate to obtain a series of solid powders. Preliminary characterization showed that metalloporphyrin carbon dot materials with similar fluorescence properties and CAT activity could be obtained within the temperature range of 150-200℃ and the reaction time range of 6-24 hours. This demonstrates that the synthesis method has a certain range of adaptability to reaction temperature and time, and can maintain the basic structure and functional properties of the material.

[0042] Application Example 1: Long-term in vivo circulation validation of CDs prepared with PEG as a precursor as a ROS scavenger The Fe-DMF prepared in Example 3 was labeled with a fluorescent dye (e.g., Cy5.5) and injected into collagen-induced arthritis rats via the tail vein. Fluorescence signals were observed at different time points (e.g., 1, 4, 12, 24, 48 h) using a small animal in vivo imaging system. The results showed that Fe-DMF remained in the blood for a significantly longer time than unmodified carbon dots, and was still significantly enriched at the lesion site after 48 hours, demonstrating its long-circulating properties and suitability for systemic ROS clearance therapy.

[0043] Application Example 2: Mn-DMF-FA-loaded dexamethasone and its in vitro targeted anti-inflammatory effect Dexamethasone (Dex) was combined with Mn-DMF-FA prepared in Example 4 by physical mixing or chemical coupling to prepare Mn-DMF-FA-D.

[0044] Activated macrophages (such as RAW264.7 cells stimulated with lipopolysaccharide (LPS)) were used as an inflammation model and divided into a control group, a free Dex group, a Mn-DMF-D group, and a Mn-DMF-FA-D group. After treatment for the corresponding time periods, the levels of inflammatory factors such as tumor necrosis factor-α (TNF-α) in the cell supernatant were measured. The results showed that the Mn-DMF-FA-D group could most effectively inhibit the release of inflammatory factors, demonstrating that it has both targeting and drug delivery functions, and its anti-inflammatory effect was significantly better than that of the non-targeted group and the free drug group.

[0045] The foregoing has shown and described the main features and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0046] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing metalloporphyrin carbon dots, characterized in that, Includes the following steps: The raw material protoporphyrin, metal chloride or metal nitrate, polyethylene glycol 2000 or branched polyethyleneimine are mixed with solvent and placed in a closed reactor. The mixture is reacted at 150~200℃ for 6~24 hours. After the reaction is completed, the mixture is cooled, purified and dried to obtain metalporphyrin carbon dots.

2. The method for preparing metalloporphyrin carbon dots according to claim 1, characterized in that, The metal chloride is CoCl2, MnCl2, MgCl2, ZnCl2, CeCl3, HfCl4, CuCl2, or FeCl3; the metal nitrate is Co(NOl3)2, Mn(NOl3)2, Mg(NO3)2, Zn(NO3)2, Ce(NO3)3, HfO(NO3)2·xH2O, Cu(NO3)2, or Fe(NO3)3; and the solvent is N,N-dimethylformamide or ethanol.

3. The method for preparing metalloporphyrin carbon dots according to claim 1, characterized in that, The metal chloride is CoCl2 or ZnCl2, and the reaction is carried out at 160°C for 6 hours.

4. The method for preparing metalloporphyrin carbon dots according to claim 1, characterized in that, The metal nitrate is Fe(NO3)3 or Cu(NO3)3, and the reaction is carried out at 160°C for 6 hours.

5. Metalloporphyrin carbon dots prepared by the preparation method according to any one of claims 1 to 4.

6. The application of the metalloporphyrin carbon dots according to claim 5, characterized in that, 1) Used to catalyze the decomposition of H2O2; 2) Used for bioimaging and tracing; 3) Used in the preparation of ROS scavengers; 4) As a multifunctional nanocarrier.

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