A calcium ion-doped carbon dot and self-triggered sustained-release system, its preparation method and application

Abstract

This invention relates to the field of nanomedicine technology, providing a calcium ion-doped carbon dot and a self-triggered sustained-release system, its preparation method, and applications. Specifically, it involves heating a mixture of aspirin, metformin, and an aqueous solution of calcium chloride in a vacuum environment to form carbon dots. Based on this, the positive charge of calcium ions in the carbon dots attracts and cross-links with the negative charge of sodium alginate carboxyl groups, achieving a self-triggered gelation process to form a carbon dot-based hydrogel microsphere sustained-release system. This method, by adjusting the raw material ratio, can obtain fluorescent carbon dots and their microsphere sustained-release system with high calcium ion doping content, and can be further applied in biomedical fields such as inflammation and bone defects, solving the problems of poor water solubility and rapid release of existing non-steroidal anti-inflammatory drugs, which cannot remain at inflammatory defect sites for extended periods.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, and in particular to a calcium ion-doped carbon dot and self-triggered sustained-release system, its preparation method, and its application. Background Technology

[0002] Bone reconstruction following bone defects caused by trauma, infection, tumors, and other diseases is one of the major challenges in clinical treatment. Bone regeneration is a slow and complex physiological process. Controlling infection, eliminating inflammation, reshaping the immune microenvironment, and achieving tissue regeneration and functional reconstruction are the ultimate goals of treating inflammatory bone defects. While traditional bone substitutes and biological growth factors can provide guiding scaffolds for bone tissue regeneration, they suffer from drawbacks such as high cost, complex preparation, and the lack of anti-inflammatory and osteoinductive properties, requiring further research and development of controlled and orderly drug release systems.

[0003] Currently, carbon dot nanomaterials possess advantages such as low preparation cost, low biotoxicity, and ease of functionalization, making them highly promising for applications in the biomedical field, particularly in bioimaging, biological function regulation, and drug delivery. Various drugs also provide abundant raw materials for the preparation of carbon dots, retaining not only the pharmacological activity of their precursors but also their unique characteristics. However, nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin suffer from poor water solubility and are difficult to dissolve, which remains one of the technical challenges that need to be overcome in the preparation of nanomedicines. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a calcium ion-doped carbon dot, which is prepared by the following method:

[0005] A mixed aqueous solution of metformin and calcium chloride was added to a container containing aspirin. After the aspirin dissolved, the product was reacted at 200-300℃ for 110-150 min under vacuum to obtain yellow, fluffy, porous carbon dot spheres. After purification, a carbon dot solution based on calcium ion doping was obtained.

[0006] The mass ratio of aspirin, metformin, and calcium chloride is 1:(0.01-20):(0.01-4).

[0007] As a preferred option, the mass ratio of aspirin, metformin, and calcium chloride is 1:2:4.

[0008] The synthesized calcium-doped carbon dots contain calcium atoms accounting for over 9% of the total atoms.

[0009] This invention further applies calcium ion-doped carbon dots to the preparation of a self-triggered sustained-release system, namely a hydrogel microsphere sustained-release system.

[0010] This invention provides a self-triggered sustained-release system based on calcium ion doping of carbon dots, which is prepared by the following method:

[0011] The purified solution of the calcium ion-doped carbon dots synthesized above was added to sodium alginate solution at a uniform rate and stirred continuously until it gelled. The solution was washed alternately with deionized water and ethanol, centrifuged, the supernatant was discarded and the precipitate was retained to obtain the carbon dot-based hydrogel microsphere sustained-release system, namely the calcium ion-doped carbon dot self-triggered sustained-release system.

[0012] The mass ratio of carbon dots to sodium alginate is 1:(1-100).

[0013] Preferably, the sodium alginate solution has a mass fraction of 1%.

[0014] Working principle of the invention:

[0015] The calcium-doped carbon dots provided by this invention retain their pharmacological activity and exert anti-inflammatory effects while also promoting bone tissue regeneration by incorporating calcium doping. They can exert anti-inflammatory and bone-promoting biological effects in the microenvironment of inflammatory bone defects.

[0016] A self-triggered carbon dot-based hydrogel microsphere sustained-release system is constructed by mixing calcium ion-doped carbon dot solutions with sodium alginate solutions. This is achieved through the attraction and cross-linking between the positive charge of calcium ions and the negative charge of sodium alginate carboxyl groups, utilizing the chelating effect of the doped metal ions. This prolongs drug retention time, making it more suitable for application in the slow and complex physiological process of bone regeneration. It can be applied to the microenvironment of inflammatory bone defects to exert anti-inflammatory and osteopromoting biological effects.

[0017] Based on the above principles, this invention provides an application of calcium ion-doped carbon dots in the preparation of anti-inflammatory drugs.

[0018] Furthermore, this invention provides the application of calcium ion-doped carbon dots in the preparation of drugs for treating inflammatory bone defects.

[0019] The present invention also provides the application of a calcium ion-doped carbon dot self-triggered sustained-release system in the preparation of drugs for treating inflammatory bone defects.

[0020] Furthermore, this invention provides the application of a calcium ion-doped carbon dot self-triggered sustained-release system in the preparation of drugs that promote bone regeneration.

[0021] The beneficial effects of this invention are:

[0022] The synthesis process of this invention is simple and low-cost, with a high calcium doping ratio, which is beneficial to promoting bone regeneration. On the one hand, carbon dots are used to exert anti-inflammatory and osteogenic biological effects in the microenvironment of inflammatory bone defects. On the other hand, the constructed hydrogel microsphere sustained-release system is used to prolong the drug action time. The two work together to regulate bone tissue regeneration. Attached Figure Description

[0023] Figure 1 The images show the following: a photograph of a carbon dot sphere under sunlight (a), a photograph under UV light (b), and the photoluminescence spectrum of the carbon dot sphere (c) of Embodiment 1 of the present invention.

[0024] Figure 2 The following are the UV-Vis absorption spectrum (black line) and photoluminescence spectrum (red line) of the carbon dot solution in Example 2 of the present invention. The illustrations are: the upper left is a photograph of the carbon dot solution under sunlight, the upper right is a photograph of the carbon dot solution under UV light (a) and the EDS energy spectrum of the carbon dots (b).

[0025] Figure 3 The following are examples of the expression of inflammatory factors at the gene and protein levels in RAW264.7 cells after treatment with carbon dot solution in Example 3 of this invention: mRNA level of iNOS (a1), mRNA level of IL-1β (a2), mRNA level of TNF-α (a3), and Western blot detection of the expression of iNOS, IL-1β, and TNF-α (b) and quantitative analysis (c).

[0026] Figure 4 Example 4 of the present invention: ALP staining of rBMSC cells under normal conditions after carbon dot treatment for 7 days;

[0027] Figure 5 These are microscopic photographs of the carbon dot-based hydrogel microsphere sustained-release system and Micro-CT three-dimensional reconstruction images of a rat periodontitis animal model in Example 5 of the present invention: a1 is a 600μm microscopic photograph, a2 is a 150μm microscopic photograph; b1 is a normal rat periodontitis animal model, b2 is a rat periodontitis animal model, b3 is a rat periodontitis animal model treated with carbon dot solution alone, and b4 is a rat periodontitis animal model treated with the carbon dot hydrogel microsphere sustained-release system.

[0028] Figure 6 The carbon dot photoluminescence spectrum of Example 6 of the present invention;

[0029] Figure 7 This is the photoluminescence spectrum of carbon dots in Example 7 of the present invention. Detailed Implementation

[0030] Example 1

[0031] Weigh 1g of metformin and 2g of calcium chloride and dissolve them in 3mL of deionized water to obtain a clear solution; add the solution to a spherical flask containing 0.5g of aspirin, heat at 120℃ for 110min to dissolve, and react the product at 250℃ under vacuum for 120min to obtain yellow, fluffy, porous carbon spheres. Figure 1 a) The sphere emits green fluorescence when excited at 405 nm. Figure 1b) The emission peak is located at 524 nm. Figure 1 c).

[0032] Example 2

[0033] After preparing carbon dot spheres according to the steps in Example 1, the carbon dot spheres were dissolved in 100 mL of deionized water. The dissolution was accelerated by sonication, followed by centrifugation at 10000 rpm at room temperature for 10 min to remove large particulate impurities. After rotary evaporation, the solution was transferred to a 100-500 Da dialysis bag and dialyzed for 12 h. The purified solution was a pale yellow carbon dot solution that emitted blue fluorescence under UV excitation, exhibiting a single emission peak at 475 nm. The UV-Vis absorption spectrum of this carbon dot solution showed absorption peaks at 270 nm and 300 nm. Figure 2 a). Characterization by EDS spectroscopy revealed that the elemental composition of the synthesized carbon dots included C, N, O, Cl, and Ca, with Ca accounting for 9.1% of the atoms. Figure 2 b) This confirms that the carbon dots are doped with calcium, and the resulting carbon dots are based on calcium ion doping.

[0034] Example 3

[0035] A calcium-doped carbon dot solution was prepared according to the steps in Examples 1 and 2. Cytotoxicity was first assessed, showing that carbon dots at concentrations below 300 μg / mL had no significant effect on the survival rate of RAW264.7 cells. RAW264.7 cells were induced with 100 ng / mL lipopolysaccharide (LPS) to establish an in vitro inflammatory cell model. Simultaneously, cells were treated with carbon dot solutions at concentrations of 100, 200, and 300 μg / mL. After 24 hours, the expression of pro-inflammatory cytokines at the mRNA level was detected by qPCR. The results showed that the mRNA expression levels of iNOS, IL-1β, and TNF-α significantly increased after LPS stimulation, indicating the successful establishment of the inflammatory cell model. The levels of related inflammatory cytokine mRNA after carbon dot addition were compared with those in the inflammatory model group using a relative quantification method. The results showed that the mRNA expression levels of these pro-inflammatory cytokines were significantly downregulated after carbon dot addition, exhibiting a clear concentration-dependent effect. Figure 3 The carbon dots (a1, a2, a3) showed a stronger anti-inflammatory effect compared to the group that simply added 300 μg / mL aspirin and metformin raw materials. Total cellular protein was collected 24 hours after treatment with the carbon dot solution, and the expression levels of iNOS, IL-1β, and TNF-α at the protein level were detected by Western blot (e.g., a1, a2, a3). Figure 3 (b) The results showed that the levels of iNOS, IL-1β, and TNF-α in the inflammation model group were significantly higher than those in the control group, indicating the successful establishment of the inflammation model. The expression levels of inflammatory factors decreased significantly after the addition of this carbon dot.

[0036] Example 4

[0037] A calcium-doped carbon dot solution was prepared according to the steps in Examples 1 and 2. The effect of carbon dot solution treatment on rat bone marrow mesenchymal cells (rBMSCs) under normal conditions was determined by alkaline phosphatase (ALP) staining. After 7 and 14 days of cell treatment, compared with the blank control group, the number of ALP-positive cells in the carbon dot group was significantly increased, the staining was darker, and the effect showed a concentration-dependent relationship. Figure 4 a).

[0038] Example 5

[0039] A calcium-doped carbon dot solution was prepared according to the steps of Examples 1 and 2. 1 mL of the 3 mg / mL calcium-doped carbon dot solution was added dropwise to 10 mL of a 1% sodium alginate solution at a rate of 0.1 mL / min using a syringe pump. The solution was stirred continuously at 500 rpm for 30 min until gelation occurred. The solution was then washed alternately with deionized water and ethanol 3-5 times. After centrifugation at 10000 rpm for 10 min, the supernatant was discarded, and the precipitate was retained to obtain the carbon dot hydrogel microsphere sustained-release system. Figure 5 a). Furthermore, it was applied to a rat model of periodontitis. Compared with the group treated with carbon dot solution alone, the hydrogel microsphere group could prolong the carbon dot residence time, effectively control periodontal inflammation, and promote tissue repair and regeneration. Figure 5 b).

[0040] Example 6

[0041] Weigh 1g of metformin and 1g of calcium chloride and dissolve them in 3mL of deionized water to obtain a clear solution; add the solution to a flask containing 0.5g of aspirin, heat at 120℃ for 110min to dissolve, and react the product at 250℃ under vacuum for 120min to obtain yellow, fluffy carbon dots that are not spherical and exhibit a single emission peak at 548nm under 405nm excitation. Figure 6 This confirms that changing the amount of calcium chloride used as a raw material can still synthesize fluorescent carbon dots.

[0042] Example 7

[0043] Weigh 0.5g metformin and 2g calcium chloride and dissolve them in 3mL of deionized water to obtain a clear solution; add the solution to a flask containing 0.5g aspirin, heat at 120℃ for 110min to dissolve, and react the product at 250℃ under vacuum for 120min to obtain yellow, fluffy carbon spheres, which exhibit a single emission peak under 405nm excitation, with the peak position at 520nm. Figure 7 It was confirmed that changing the amount of metformin as a raw material still allows for the synthesis of fluorescent carbon dots.

Claims

1. A type of carbon dot based on calcium ion doping, characterized in that: It is prepared by the following method: A mixed aqueous solution of metformin and calcium chloride was added to a container containing aspirin. After the aspirin dissolved, the product was reacted under vacuum at 200-300℃ for 110-150 min to obtain yellow, fluffy carbon spheres. After purification, a carbon dot solution based on calcium ion doping was obtained; The mass ratio of aspirin, metformin, and calcium chloride is 1:0.01-20:0.01-4.

2. The calcium ion-doped carbon dot according to claim 1, characterized in that: The mass ratio of aspirin, metformin, and calcium chloride is 1:2:

4.

3. The calcium ion-doped carbon dot according to claim 1, characterized in that: The synthesized calcium-doped carbon dots contain more than 9% calcium atoms.

4. A calcium ion-doped carbon dot according to any one of claims 1-3, characterized in that: It is used to prepare self-triggered sustained-release systems, namely hydrogel microsphere sustained-release systems.

5. An application of calcium ion-doped carbon dots according to any one of claims 1-3, characterized in that: It is used in the preparation of anti-inflammatory drugs.

6. An application of calcium ion-doped carbon dots according to any one of claims 1-3, characterized in that: Used to prepare drugs for treating inflammatory bone defects.

7. The application according to claim 5, characterized in that: Used to prepare drugs for treating inflammatory bone defects.

8. A self-triggered sustained-release system based on calcium ion-doped carbon dots, characterized in that: Prepared by the following method: A solution of purified carbon dots based on calcium ion doping as described in any one of claims 1-3 is added at a uniform rate to a sodium alginate solution and stirred continuously until it gels. The solution is then washed alternately with deionized water and ethanol, centrifuged, and the supernatant is discarded to obtain a carbon dot-based hydrogel microsphere sustained-release system, namely a self-triggered sustained-release system based on calcium ion doping carbon dots. The mass ratio of carbon dots to sodium alginate is 1:1-100.

9. The self-triggered sustained-release system based on calcium ion doping of carbon dots according to claim 8, characterized in that: In the preparation method, the mass fraction of sodium alginate solution is 1%.

10. The application of a calcium ion-doped carbon dot self-triggered sustained-release system according to claim 8 or 9, characterized in that: It is used in the preparation of drugs for treating inflammatory bone defects.

11. The application of a calcium ion-doped carbon dot self-triggered sustained-release system according to claim 8 or 9, characterized in that: It is used in the preparation of drugs that promote bone regeneration.

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