Preparation method and application of zinc-cerium double-doped resveratrol carbon dots
Zinc-cerium co-doped resveratrol carbon dots were prepared by hydrothermal method. By combining the synergistic effect of zinc and cerium ions, the limited efficacy of resveratrol carbon dots in tissue regeneration was solved, achieving high biocompatibility and antioxidant activity, and significantly promoting angiogenesis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-04
- Publication Date
- 2026-03-27
Smart Images
Figure CN121736742A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical nanomaterials and regenerative medicine, and in particular relates to a method for preparing zinc-cerium dual-doped resveratrol carbon dots and their application. Background Technology
[0002] Regenerative angiology is a core area of research in the treatment of ischemic heart disease, peripheral artery disease, and tissue engineering repair. Promoting "therapeutic angiogenesis" by introducing exogenous therapeutic molecules or cells has been a focus of research in recent years, such as pro-angiogenic factors, gene therapy, and cell therapy. However, the clinical translation of these strategies is severely limited by factors such as short half-life, low gene transfection efficiency, high cost, and immunogenicity.
[0003] Resveratrol, a natural polyphenol containing resorcinol-type phenolic hydroxyl groups, possesses a styrene conjugated skeleton that combines carbon source precursor function with antioxidant activity. Studies have shown that it can protect vascular endothelium by promoting nitric oxide production and promoting microvascular angiogenesis and remodeling. However, resveratrol has poor water solubility and stability, resulting in low bioavailability. In recent years, carbon quantum dots (CDs) have been widely used in the biomedical field due to their excellent biocompatibility and tunable optical properties. Resveratrol carbon dots exhibit good biocompatibility, fluorescence properties, and certain antioxidant properties. However, the effect of carbon dots prepared solely from resveratrol on tissue regeneration is limited. Summary of the Invention
[0004] To address the aforementioned technical problems, this invention provides a method for preparing zinc-cerium dual-doped resveratrol carbon dots and their application.
[0005] The technical solution adopted in this invention is:
[0006] A method for preparing zinc / cerium dual-doped resveratrol carbon dots, using resveratrol as the carbon source and zinc acetate and cerium nitrate as dopants, is used to synthesize zinc / cerium dual-doped carbon dots in one step by hydrothermal method.
[0007] Preferably, the molar ratio of resveratrol zinc acetate to cerium nitrate is 1:0.1-0.3:0.05-0.15.
[0008] Preferably, the concentration of resveratrol in the reaction system is 5-20 mM, the concentration of zinc acetate is 1-5 mM, and the concentration of cerium nitrate is 0.5-3 mM.
[0009] Preferably, resveratrol, zinc acetate, and cerium nitrate are dissolved in deionized water, sonicated until completely dissolved, and transferred to a reaction vessel; hydrothermal reaction is carried out at 160-200℃ for 8-16 hours; the reaction system is cooled to room temperature, centrifuged, and filtered through a membrane. The filtrate is purified by dialyzing and then freeze-dried to obtain zinc / cerium dual-doped resveratrol carbon dots.
[0010] Preferably, a gradient cooling procedure is adopted: first maintain at 120℃ for 1 hour, then maintain at 100℃ for 1 hour, and finally maintain at 50℃ for 1 hour, and then cool to room temperature.
[0011] A method for preparing zinc-cerium co-doped resveratrol carbon dots.
[0012] Preferably, it exhibits blue-green fluorescence under 365nm ultraviolet light irradiation.
[0013] Preferably, the particle size is 2-8 nm.
[0014] Application of zinc-cerium dual-doped resveratrol carbon dots in the preparation of angiogenesis-promoting drugs.
[0015] The advantages and positive effects of this invention are: it provides a zinc-cerium dual-doped resveratrol carbon dot, which has high biocompatibility, antioxidant activity and excellent angiogenesis-promoting properties, and can be used in angiogenesis-promoting drugs;
[0016] Zinc / cerium co-doped resveratrol carbon dots promote angiogenesis through a synergistic effect of zinc / cerium bimetallic action. Zinc ions activate the p38 MAPK, HIF-1α, and PI3K / VEGF signaling pathways, while cerium ions promote angiogenesis through... / Reversible scavenging of reactive oxygen species, the two work synergistically to significantly improve the angiogenesis efficiency of oxidatively damaged endothelium. Attached Figure Description
[0017] Figure 1 UV-Vis absorption spectra of Zn / Ce-RCDs;
[0018] Figure 2 Cytotoxicity results of Zn / Ce-RCDs;
[0019] Figure 3 Intracellular antioxidant activity assays for different carbon dots;
[0020] Figure 4 The effects of different carbon dots on endothelial cell angiogenesis activity. Detailed Implementation
[0021] The embodiments of the present invention will now be described with reference to the accompanying drawings.
[0022] This invention relates to a method for preparing zinc / cerium co-doped resveratrol carbon dots and their applications. Using resveratrol as the carbon source and zinc acetate and cerium nitrate as dopants, zinc / cerium co-doped resveratrol carbon dots (Zn / Ce-RCDs) are synthesized in one step via a controlled hydrothermal method. The prepared zinc / cerium co-doped resveratrol carbon dots (Zn / Ce-RCDs) exhibit high biocompatibility, antioxidant activity, and excellent pro-angiogenic properties, and can be used as biomaterials for promoting angiogenesis.
[0023] The preparation process of zinc / cerium co-doped resveratrol carbon dots Zn / Ce-RCDs specifically includes the following steps:
[0024] Resveratrol, zinc acetate ( ) and cerium nitrate ( Zinc resveratrol was dissolved in deionized water at a molar ratio of 1:0.1-0.3:0.05-0.15. In the reaction system, the concentrations of resveratrol, zinc acetate, and cerium nitrate were 5-20 mM, 1-5 mM, and 0.5-3 mM, respectively. The mixture was sonicated until completely dissolved, and then transferred to a reaction vessel with a filling density of 60-80%. The mixture was then subjected to hydrothermal reaction at 160-200℃ for 8-16 h to obtain zinc / cerium dual-doped resveratrol carbon dots (Zn / Ce-RCDs). After cooling the reaction system to room temperature, the reaction product was centrifuged. The filtrate was filtered through a 0.22 μm microporous membrane to obtain an aqueous solution of zinc / cerium dual-doped carbon dots. Centrifugation removes large precipitates and severely agglomerated particles, while membrane filtration removes submicron-sized fine particles and agglomerates that centrifugation could not completely remove. The above aqueous solution was purified by dialysis to remove free metal ions, small molecule salts, and organic byproducts, and then freeze-dried to obtain zinc / cerium dual-doped resveratrol carbon dots. The particle size of the zinc / cerium dual-doped resveratrol carbon dots was 2-8 nm, and the zinc / cerium dual-doped resveratrol carbon dots exhibited blue-green fluorescence under 365 nm ultraviolet light irradiation.
[0025] In some embodiments of this invention, the hydrothermal reaction employs a gradient cooling procedure: first maintaining the temperature at 120°C for 1 hour, then at 100°C for 1 hour, and finally at 50°C for 1 hour, before cooling to room temperature. Gradient cooling enhances the crystallinity and structural order of the carbon dots, promotes stable modification of surface functional groups, and improves size uniformity and monodispersity. After cooling to room temperature, the reaction product is centrifuged at 12000 rpm / min and filtered through a 0.22 μm microporous membrane. This centrifugation-membrane filtration process is repeated two to three times. Dialysis purification is performed using a dialysis bag with a molecular weight of 500 Da, with a dialysis time of 24-48 hours.
[0026] Zinc (Zn) is an essential transition metal element for the human body, and its unique electronic configuration ( This gives it both structural stability and redox activity in biological systems. Studies have found that... It can activate signaling pathways such as p38 MAPK, HIF-1α, and PI3K, significantly promoting the secretion of vascular endothelial growth factor (VEGF), demonstrating unique advantages in the treatment of ischemic diseases. However, the therapeutic window of free zinc ions is narrow; concentrations of free zinc ions at the cellular level greater than 100 μM produce cytotoxicity. Doping zinc into carbon dots fundamentally alters its delivery and metabolic kinetics compared to zinc ions alone, enabling therapeutic effects to be achieved at lower concentrations, far below the toxicity threshold of free zinc ions. Furthermore, cerium (Ce), as a lanthanide rare earth element, possesses a unique 4f electron layer structure that... / Reversible valence state transition capability; The site scavenges superoxide radicals through a single-electron transfer mechanism, while It can directly decompose hydrogen peroxide ( This "self-circulating" antioxidant property has significant value in anti-inflammatory treatment.
[0027] In-situ construction of zinc / cerium co-doped resveratrol carbon dots (Zn / Ce-RCDs) using a hydrothermal method was performed. The coordination effect of the resveratrol carbon framework stabilized the metal ions, thus preserving the... The pro-angiogenic activity of the carbon dots is enhanced by cerium doping, which solves the problems of poor controllability, limited biological function, and narrow therapeutic window associated with bimetallic doping. Through synergistic doping of zinc and cerium bimetallic ions, zinc ions promote the secretion of vascular endothelial growth factor, while cerium ions provide reversible redox activity.
[0028] Based on the high biocompatibility, antioxidant activity, and excellent angiogenesis-promoting properties of zinc / cerium co-doped resveratrol carbon dots, they can be used as bioactive nanomaterials or directly in nanomedicines required for angiogenesis medicine. For example, injecting a solution containing zinc / cerium co-doped resveratrol carbon dots directly into a specific location on a human or animal limb can promote angiogenesis at that location. Mouse validation experiments showed that injecting a zinc / cerium co-doped resveratrol carbon dot solution (concentration of 10-100 μg / mL) into the limbs of mice at a dose ranging from 2.5 to 5 μL / g (mouse body weight) can promote angiogenesis in the mouse limbs.
[0029] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.
[0030] Example 1: Preparation of Zinc / Cerium Co-doped Resveratrol Carbon Dots (Zn / Ce-RCDs)
[0031] 0.228 g resveratrol, 0.183 g zinc acetate, and 0.434 g cerium nitrate were dissolved in 20 mL of deionized water. The mixture was placed in an ultrasonic processor and ultrasonically treated at 300 W for 30 min until completely dissolved. The mixture was then transferred to a 50 mL polytetrafluoroethylene reactor and heated to 180 °C in a muffle furnace at a rate of 5 °C / min. After reacting for 12 h, a gradient cooling program was executed (120 °C for 1 h → 100 °C for 1 h → 50 °C for 1 h).
[0032] After centrifuging the reaction solution at 12000 rpm for 15 min, the filtrate was collected and filtered through a 0.22 μm filter membrane. The centrifugation-filtration step was repeated three times. The filtrate was then placed in a dialysis bag with a molecular weight of 500 Da and dialyzed with double-distilled water at pH 7.4 for 36 h, with the dialysate being replaced every 6 h. The dialyzed solution was freeze-dried at -80℃ for 24 h to obtain a solid powder, which is Zn / Ce-RCDs.
[0033] Example 2: Ultraviolet Spectral Evaluation of Zn / Ce-RCDs
[0034] The Zn / Ce-RCDs carbon dot solution prepared in Example 1 was diluted with ultrapure water to a concentration of 10 μg / mL, ensuring that the absorbance value was within the linear detection range of the instrument. The diluted carbon dot solution was injected into a quartz cuvette, and a baseline scan was performed using a UV-Vis spectrophotometer with ultrapure water as a reference within the same wavelength range. The spectral scan was initiated, and the absorption spectrum data were recorded. The results are as follows: Figure 1 As shown, a distinct characteristic absorption peak can be observed at around 265 nm, corresponding to the π-π* electronic transition of the aromatic carbon skeleton, confirming the successful synthesis of the carbon dot structure.
[0035] Example 3: Detection of the cytotoxicity of Zn / Ce-RCDs to HUVECs
[0036] The cytotoxicity of Zn / Ce-RCDs prepared in Example 1 was tested using HUVEC cells.
[0037] HUVEC cells were seeded at 8 × 10³ / well in 96-well plates and cultured overnight in complete medium. Different concentrations of Zn / Ce-RCDs were then added, with concentration gradients of 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL. Wells containing an equal volume of PBS served as a control group (assuming 100% cell viability). A zero-entry well was also included, containing only culture medium without cell seeding. After 24 h of further culture, CCK-8 reagent was added and incubated for 2 hours. The absorbance at 450 nm was measured using a microplate reader.
[0038] Cell viability = (Experimental group absorbance - Zeroing well absorbance) / (Control group absorbance - Zeroing well absorbance) × 100%
[0039] Organize and calculate the results, as follows: Figure 2 As shown, even at an addition level of 400 μg / mL, the cell viability of HUVECs remained above 88%, indicating that the zinc / cerium dual-doped resveratrol carbon dots Zn / Ce-RCDs have excellent cell compatibility.
[0040] Comparative Example 1: Preparation of Resveratrol Carbon Dots (RCDs)
[0041] 0.228 g of resveratrol was dissolved in 20 mL of deionized water. The mixture was placed in an ultrasonic processor and ultrasonically treated at 300 W for 30 min until completely dissolved. The mixture was transferred to a 50 mL polytetrafluoroethylene (PTFE) reactor and heated to 180 °C in a muffle furnace at a rate of 5 °C / min. After reacting for 12 h, a gradient cooling program was executed (120 °C for 1 h → 100 °C for 1 h → 50 °C for 1 h). The reaction solution was centrifuged at 12000 rpm for 15 min and filtered through a 0.22 μm filter membrane. This step (centrifugation and filtration) was repeated three times. The filtrate was then placed in a dialysis bag with a molecular weight of 500 Da and dialyzed with double-distilled water at pH 7.4 for 36 h, with the dialysate replaced every 6 h. The dialyzed solution was freeze-dried at -80 °C for 24 h to obtain a solid powder, which is RCDs.
[0042] Comparative Example 2: Preparation of Zinc-Doped Resveratrol Carbon Dots (Zn-RCDs)
[0043] 0.228 g of resveratrol and 0.183 g of zinc acetate were dissolved in 20 mL of deionized water. The mixture was placed in an ultrasonic processor and ultrasonically treated at 300 W for 30 min until completely dissolved. The mixture was transferred to a 50 mL polytetrafluoroethylene reactor and heated to 180 °C in a muffle furnace at a rate of 5 °C / min. After reacting for 12 h, a gradient cooling program was executed (120 °C for 1 h → 100 °C for 1 h → 50 °C for 1 h). The reaction solution was centrifuged at 12000 rpm for 15 min and filtered through a 0.22 μm filter membrane. This step (centrifugation and filtration) was repeated three times. The filtrate was then placed in a dialysis bag with a molecular weight of 500 Da and dialyzed with double-distilled water at pH 7.4 for 36 h, with the dialysate replaced every 6 h. The dialyzed solution was freeze-dried at -80 °C for 24 h to obtain a solid powder, which is Zn-RCDs.
[0044] Comparative Example 3: Preparation of Cerium-Doped Resveratrol Carbon Dots (Ce-RCDs)
[0045] 0.228 g of resveratrol and 0.434 g of zinc cerium nitrate were dissolved in 20 mL of deionized water. The mixture was placed in an ultrasonic processor and ultrasonically treated at 300 W for 30 min until completely dissolved. The mixture was transferred to a 50 mL polytetrafluoroethylene reactor and heated to 180 °C in a muffle furnace at a rate of 5 °C / min. After reacting for 12 h, a gradient cooling program was executed (120 °C for 1 h → 100 °C for 1 h → 50 °C for 1 h). The reaction solution was centrifuged at 12000 rpm for 15 min and filtered through a 0.22 μm filter membrane. This step (centrifugation and filtration) was repeated three times. The filtrate was then placed in a dialysis bag with a molecular weight of 500 Da and dialyzed with double-distilled water at pH 7.4 for 36 h, with the dialysate replaced every 6 h. The dialyzed solution was freeze-dried at -80 °C for 24 h to obtain a solid powder, which is Ce-RCDs.
[0046] Example 4: ROS scavenging capability test of Zn / Ce-RCDs
[0047] The differences in ROS scavenging ability between Zn / Ce-RCDs prepared in Example 1, RCDs prepared in Comparative Examples 1-3, Zn-RCDs and Ce-RCDs were compared.
[0048] HUVEC cells were seeded in 24-well plates (2 × 10⁻⁶ cells per well). 4 After overnight culture in an incubator, CoCl2 (1200 μM) was added to simulate hypoxia. After 12 hours, 20 μg / mL RCDs, Zn-RCDs, Ce-RCDs, or Zn / Ce-RCDs were administered, with wells containing an equal volume of PBS serving as a control. After another 12 hours of culture, DMEM containing DCFH-DA dye was added and incubated for 20 minutes. The intensity of green fluorescence under 488 nm excitation light was observed using a fluorescence microscope to reflect intracellular reactive oxygen species levels.
[0049] The results are as follows Figure 3 As shown, the intracellular fluorescence of cells in the untreated group (Unactivated) was very weak, while the green fluorescence intensity increased significantly after CoCl2 treatment (PBS group). In cells treated with different carbon dots, varying degrees of fluorescence intensity reduction were observed. The fluorescence intensity of carbon dots doped with cerium alone (Ce-RCD) and those doped with both cerium and zinc (Zn / Ce-RCDs) was the lowest, approaching that of the untreated group. These results strongly suggest that zinc / cerium co-doped resveratrol carbon dots (Zn / Ce-RCDs) possess outstanding antioxidant properties and can significantly reduce reactive oxygen species levels in hypoxia-induced endothelial cells.
[0050] Example 5: Study on the angiogenesis activity of Zn / Ce-RCDs in endothelial cells
[0051] HUVEC cells were seeded in 6-well plates (2.5 × 10⁻⁶). 5 After overnight culture in an incubator, CoCl2 (1200 μM) was added to simulate hypoxia. After 12 hours, cells were treated with 20 μg / mL RCDs, Zn-RCDs, Ce-RCDs, or Zn / Ce-RCDs, with wells containing an equal volume of PBS serving as a control. After 24 hours of culture, the pretreated HUVEC cells were digested with trypsin and then subjected to 3.5 × 10⁻⁶ ppm of PBS. 4 The wells were seeded at a density of / wells onto Matrigel (pre-laid and fixed on a 96-well plate), and observed after incubation in an incubator for 6 hours.
[0052] The results are as follows Figure 4 As shown, the number of tubular structures in the Zn / Ce-RCDs group was significantly increased, which was 3 times that of the control group (PBS group). Compared with the different carbon dots prepared in comparative examples 1-3, the zinc / cerium dual-doped resveratrol carbon dots Zn / Ce-RCDs could significantly promote the angiogenesis activity of endothelial cells.
[0053] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.
Claims
1. A method for preparing zinc-cerium dual-doped resveratrol carbon dots, characterized in that: Zinc / cerium dual-doped carbon dots were synthesized in one step using a hydrothermal method with resveratrol as the carbon source and zinc acetate and cerium nitrate as dopants.
2. The method for preparing zinc-cerium dual-doped resveratrol carbon dots according to claim 1, characterized in that: The molar ratio of resveratrol zinc acetate to cerium nitrate is 1:0.1-0.3:0.05-0.
15.
3. The method for preparing zinc-cerium dual-doped resveratrol carbon dots according to claim 2, characterized in that: The concentrations of resveratrol, zinc acetate, and cerium nitrate in the reaction system are 5-20 mM, 1-5 mM, and 0.5-3 mM, respectively.
4. The method for preparing zinc-cerium dual-doped resveratrol carbon dots according to any one of claims 1-3, characterized in that: Resveratrol, zinc acetate, and cerium nitrate were dissolved in deionized water, sonicated until completely dissolved, and transferred to a reaction vessel. The mixture was then subjected to hydrothermal reaction at 160-200℃ for 8-16 hours. The reaction system was cooled to room temperature, centrifuged, and filtered through a membrane. The filtrate was purified by dialyzing and then freeze-dried to obtain zinc / cerium dual-doped resveratrol carbon dots.
5. The method for preparing zinc-cerium dual-doped resveratrol carbon dots according to claim 4, characterized in that: A gradient cooling program was used: first, maintain the temperature at 120℃ for 1 hour, then at 100℃ for 1 hour, and finally at 50℃ for 1 hour, before cooling to room temperature.
6. Zinc-cerium dual-doped resveratrol carbon dots prepared by the method described in any one of claims 1-5.
7. The zinc-cerium dual-doped resveratrol carbon dots according to claim 6, characterized in that: It exhibits blue-green fluorescence under 365nm ultraviolet light.
8. The zinc-cerium dual-doped resveratrol carbon dots according to claim 6, characterized in that: The particle size is 2-8 nm.
9. The use of zinc-cerium dual-doped resveratrol carbon dots as described in any one of claims 6-8 in the preparation of angiogenic drugs.