A carbon dot based on proto-catechuic aldehyde as a precursor and a preparation method and application thereof

Abstract

The present application relates to a kind of carbon dots based on protoanemonin as precursor and its preparation method and application, the carbon dots are protoanemonin after dehydration carbonization and form carbon dots.Preparation, protoanemonin and citric acid are mixed in ultrapure water, react at 200 DEG C for 24 hours, after reaction is complete, in natural cooling and reduces to room temperature, the reaction liquid is centrifuged, filtered and dialyzed, finally freeze-dried and protoanemonin carbon dots are obtained.Studies show that the carbon dots have good biocompatibility in cells, and have good performance in mitochondrial fluorescence imaging, and have certain antioxidant capacity.In addition, the preparation process of the present application is simple, low toxicity, low cost, and strong applicability.

Description

Technical Field

[0001] This invention relates to the field of medical carbon nanomaterials technology, specifically to a carbon dot based on protocatechuic aldehyde as a precursor, its preparation method, and its application. Background Technology

[0002] In recent years, with continuous research and exploration of traditional medicines, Chinese medicine has developed into a vast repository of medicinal materials. Many Chinese medicines possess unimaginable rich functions and remarkable efficacy, attracting increasing attention from scientists. Protocatechuic aldehyde (PCA) is the main product of the traditional Chinese medicine Danshen (Salvia miltiorrhiza), exhibiting good water solubility and reported to possess various effects such as anti-inflammatory and antibacterial properties. However, protocatechuic aldehyde is metabolized too quickly in vivo, making it difficult to fully exert its therapeutic effects, and its biocompatibility is generally poor, limiting its further clinical application. Therefore, there is a need in this field for a drug with good biocompatibility and the related efficacy of protocatechuic aldehyde. Summary of the Invention

[0003] The purpose of this invention is to overcome the defects of the prior art and provide a drug with good biocompatibility and protocatechuic aldehyde-related effects.

[0004] The purpose of this application is also to provide methods for preparing and applying the aforementioned substances.

[0005] To achieve the objectives of this invention, the following technical solutions are provided in this application.

[0006] In a first aspect, this application provides a carbon dot based on protocatechuic aldehyde as a precursor, wherein the carbon dot is formed by dehydration and carbonization of protocatechuic aldehyde.

[0007] In one embodiment of the first aspect, the diameter of the carbon dots is ≤10nm, and the diameter distribution coefficient of the carbon dots is [value missing].

[0008] In a second aspect, this application provides a method for preparing carbon dots as described above, the method comprising the following steps:

[0009] (1) Citric acid and protocatechuic aldehyde were dissolved in ultrapure water in sequence to obtain a reaction solution;

[0010] (2) Place the reaction solution in a closed reactor, heat and pressurize it to react, and then let it cool naturally to obtain crude product solution;

[0011] (3) The crude solution is centrifuged, filtered, dialyzed and freeze-dried in sequence to obtain the carbon dots.

[0012] In one embodiment of the second aspect, the mass ratio of citric acid, protocatechuic aldehyde, and ultrapure water is (1-2):1:(15-30).

[0013] In one embodiment of the second aspect, step (2) includes at least one of the following technical features:

[0014] 21) The reactor is made of polystyrene;

[0015] 22) The reaction temperature is 180–200℃;

[0016] 23) The reaction pressure is 1.0–2.0 MPa;

[0017] 24) The reaction time is 24 to 30 hours.

[0018] In one embodiment of the second aspect, step (3) includes at least one of the following technical features:

[0019] 31) The centrifugation speed is 5000-15000 rpm, and the centrifugation time is 10-15 min;

[0020] 32) The filtration is performed using a needle filter with a pore size of 0.22 μm;

[0021] 33) The dialysis bag used for the dialysis has a molecular weight cutoff of 300–1000 Da;

[0022] 34) The freeze-drying temperature is -20 to -10°C, and the freeze-drying time is 48 to 72 hours.

[0023] In one embodiment of the second aspect, the dialysis uses ultrapure water, each dialysis session lasts 12 hours, and at least 3 dialysis sessions are performed.

[0024] In a third aspect, this application also provides an application of the carbon dots as described above, wherein the carbon dots are used to prepare imaging agents for cell mitochondrial imaging or antioxidant and anti-inflammatory drugs that reduce the level of reactive oxygen species in cells.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] 1. The method for preparing protocatechuic aldehyde carbon dots in this invention is simple and environmentally friendly, with readily available raw materials, high yield, and low cost. The carbon dot particle size is within 10 nm, which can achieve stable fluorescence emission through size confinement effect and facilitates entry into cells and organelles.

[0027] 2. The protocatechuic aldehyde carbon dots described in this invention possess excellent biocompatibility, superior mitochondrial imaging capabilities, and the ability to regulate oxidation and inflammation. They can serve as a valuable cell imaging tool, aiding in early disease diagnosis and visual tracking of subsequent treatments. Furthermore, as an antioxidant and anti-inflammatory drug, they enable integrated diagnosis and treatment. Attached Figure Description

[0028] Figure 1a This is a TEM image of the carbon dots prepared in Example 1.

[0029] Figure 1b This is a diameter distribution diagram of the carbon dots prepared in Example 1.

[0030] Figure 2 The image shows the XRD pattern of the carbon dots prepared in Example 1.

[0031] Figure 3a The image shows the ultraviolet absorption spectrum of the carbon dots prepared in Example 1.

[0032] Figure 3b The fluorescence spectrum of the carbon dots prepared in Example 1.

[0033] Figure 4 The Fourier transform infrared spectrum of the carbon dots prepared in Example 1.

[0034] Figure 5 The image shows the nuclear magnetic resonance spectrum of the carbon dots prepared in Example 1.

[0035] Figure 6 The image shows the experimental results of CCK-8 carbon dots prepared in Example 1.

[0036] Figure 7 This is a cell viability / death experiment diagram of the carbon dots prepared in Example 1.

[0037] Figure 8 This is a laser confocal image of the mitochondria of the carbon dots prepared in Example 1.

[0038] Figure 9 This is a quantitative analysis image showing the inhibitory effect of the carbon dots prepared in Example 1 on intracellular reactive oxygen species levels. Detailed Implementation

[0039] Unless otherwise defined, the technical or scientific terms used in this specification and claims shall have the ordinary meaning understood by one of ordinary skill in the art to which this invention pertains. All values ​​listed herein, ranging from the minimum to the maximum, refer to all values ​​obtained by incrementing the minimum and maximum values ​​by one unit when the difference between the minimum and maximum values ​​is more than two units.

[0040] The following describes specific embodiments of the present invention. It should be noted that, in order to provide a concise description, this specification cannot provide a detailed description of all features of the actual embodiments. Without departing from the spirit and scope of the present invention, those skilled in the art can modify and substitute the embodiments of the present invention, and the resulting embodiments are also within the protection scope of the present invention.

[0041] Carbon dots (CDs) are an emerging carbon-based zero-dimensional nanomaterial with excellent photoluminescence properties, modifiability, and biocompatibility. Furthermore, the preparation process of carbon dots is relatively simple, thus they have wide applications in the biomedical field. Carbon dot fluorescence imaging is a non-invasive, highly sensitive, and high-resolution bioimaging technique that can be used for cell imaging, playing a role in diagnosis and adjuvant therapy. In recent years, the ability of carbon dots to inherit the properties of drug precursors has been widely reported, with some even exhibiting better medical efficacy. Therefore, using small molecule drugs to synthesize carbon dots can allow the therapeutic effects of the drugs to complement the fluorescence properties of the carbon dots, potentially achieving integrated diagnosis and treatment. The purpose of this application is to provide a carbon dot based on protocatechuic aldehyde as a precursor, wherein the carbon dot is formed by the dehydration and carbonization of protocatechuic aldehyde.

[0042] In one specific embodiment, this application provides a carbon dot based on protocatechuic aldehyde as a precursor, wherein the carbon dot is formed after dehydration and carbonization of protocatechuic aldehyde.

[0043] Protocatechuic aldehyde has good water solubility, and the prepared carbon dots also have good water dispersibility, which facilitates further in vivo application. At the same time, as a drug, the active groups of protocatechuic aldehyde can be partially retained in the carbon dots after the reaction, so that the product inherits its antioxidant and anti-inflammatory effects.

[0044] In one specific embodiment, the diameter of the carbon dots is ≤10 nm. On the one hand, nanoparticles can obtain a certain fluorescence emission intensity due to the size effect; on the other hand, the small size facilitates the particles to pass through the cell membrane and enter organelles.

[0045] In one specific embodiment, a method for preparing carbon dots as described above includes the following steps:

[0046] (1) Citric acid and protocatechuic aldehyde were dissolved in ultrapure water in sequence to obtain a reaction solution;

[0047] (2) Place the reaction solution in a closed reactor, heat and pressurize it to react, and then let it cool naturally to obtain crude product solution;

[0048] (3) The crude solution is centrifuged, filtered, dialyzed and freeze-dried in sequence to obtain the carbon dots.

[0049] During the reaction, small molecules undergo dehydration, polymerization, and carbonization under high temperature and pressure conditions, eventually forming tiny spherical nanoparticles. Citric acid, as an auxiliary carbon source, not only helps in particle formation but also generates subfluorophores to provide some fluorescence emission.

[0050] In one specific embodiment, the mass ratio of citric acid, protocatechuic aldehyde, and ultrapure water is (1-2):1:(15-30). A certain amount of citric acid can serve as an auxiliary carbon source, ensuring a relatively regular spherical morphology of the particles and providing a certain fluorescence emission intensity. Water, as a dispersant, affects the water dispersibility of the product; when there are no safety concerns, an appropriate amount can be added after complete dissolution.

[0051] In one specific embodiment, step (2) includes at least one of the following technical features:

[0052] 21) The reactor is made of polystyrene;

[0053] 22) The reaction temperature is 180–200℃;

[0054] 23) The reaction pressure is 1.0–2.0 MPa;

[0055] 24) The reaction time is 24–30 hours. The temperature and time of the hydrothermal reaction affect particle size and nucleation, thereby affecting the ability to enter organelles and for imaging. In a preferred embodiment, the hydrothermal reaction temperature is 200°C and the reaction time is 24 hours.

[0056] In one specific embodiment, step (3) includes at least one of the following technical features:

[0057] 31) The centrifugation speed is 5000-15000 rpm, and the centrifugation time is 10-20 min;

[0058] 32) The filtration is performed using a needle filter with a pore size of 0.22 μm;

[0059] 33) The dialysis bag used for the dialysis has a molecular weight cutoff of 300–1000 Da;

[0060] 34) The freeze-drying temperature is -20 to -10°C, and the freeze-drying time is 48 to 72 hours. Centrifugation and filtration are used to remove larger particles from excessive reaction. Dialysis is used to remove unreacted reagents and other small byproducts; the dialysis bag size is limited to 300-500 Da to prevent carbon dots from leaking out. In some embodiments of the present invention, a brownish-yellow solution can be obtained by passing through a 0.22 μm filter membrane.

[0061] In one specific embodiment, the dialysis uses ultrapure water, each dialysis session lasts 12 hours, and at least 3 dialysis sessions are performed.

[0062] In one specific embodiment, an application of the carbon dots described above is used to prepare imaging agents for cell mitochondrial imaging or (antioxidant and anti-inflammatory drugs that reduce the level of reactive oxygen species in cells). The former utilizes the small size and photoluminescence properties of the carbon dots, enabling them to enter organelles and generate fluorescence under external excitation; the latter is that the carbon dots inherit the antioxidant active groups of the reaction precursor protocatechuic aldehyde, giving them antioxidant and anti-inflammatory effects as well.

[0063] Example

[0064] The embodiments of the present invention will be described in detail below. These embodiments are implemented based on the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the scope of protection of the present invention is not limited to the following embodiments.

[0065] Example 1

[0066] (1) Mixing raw materials: Mix 0.4g citric acid with 4g ultrapure water and sonicate to dissolve completely. Then mix the resulting citric acid solution with 0.2g protocatechuic aldehyde and sonicate to dissolve completely.

[0067] (2) Hydrothermal reaction: The clarified precursor solution was transferred to a reaction vessel made of polyphenylene (PPL) material, and the mixture was sealed and heated to 200°C for 24 hours under high temperature and high pressure.

[0068] (3) Post-processing: The obtained reaction solution was purified by centrifugation, filtration, dialysis and other measures, and then freeze-dried to obtain solid powder, which was stored in a refrigerator at 4°C. The speed of the high-speed centrifuge was 10,000 rpm, the pore size of the needle filter was 0.22 μm, and dialysis was performed using a 500 Da dialysis bag and ultrapure water for 48 hours, with the water changed every 12 hours.

[0069] Example 2

[0070] (1) Mixing raw materials: Mix 0.28g of citric acid with 4g of ultrapure water and dissolve it completely by sonication. Then mix the resulting citric acid solution with 0.2g of protocatechuic aldehyde and dissolve it completely by sonication.

[0071] (2) Hydrothermal reaction: The clarified precursor solution was transferred to a polyphenylene (PPL) reactor, sealed and heated to 180°C, and then reacted under high temperature and high pressure for 30 hours.

[0072] (3) Post-processing: The obtained reaction solution was purified by centrifugation, filtration, dialysis and other measures, and then freeze-dried to obtain solid powder, which was stored in a refrigerator at 4°C. The speed of the high-speed centrifuge was 5000 rpm, the pore size of the needle filter was 0.22 μm, and dialysis was performed using a 300 Da dialysis bag and ultrapure water for 48 hours, with the water changed every 12 hours.

[0073] Example 3

[0074] (1) Mixing raw materials: Mix 0.2g citric acid with 6g ultrapure water and sonicate to dissolve completely. Then mix the resulting citric acid solution with 0.2g protocatechuic aldehyde and sonicate to dissolve completely.

[0075] (2) Hydrothermal reaction: The clarified precursor solution was transferred to a reaction vessel made of polyphenylene (PPL) material, and the mixture was sealed and heated to 200°C for 24 hours under high temperature and high pressure.

[0076] (3) Post-processing: The obtained reaction solution was purified by centrifugation, filtration, dialysis and other measures, and then freeze-dried to obtain solid powder, which was stored in a refrigerator at 4°C. The speed of the high-speed centrifuge was 15000 rpm, the pore size of the needle filter was 0.22 μm, and dialysis was performed using a 1000 Da dialysis bag and ultrapure water for 48 hours, with the water changed every 12 hours.

[0077] Test Implementation Examples

[0078] I. Basic Performance Testing

[0079] The carbon dot powder prepared in Example 1 was subjected to transmission electron microscopy (TEM) scanning, and the results are as follows: Figure 1a , Figure 1b As shown. From Figure 1a , Figure 1b As we can see, the carbon dot powder has a small size and good dispersibility, with an average diameter of 3.5 nm.

[0080] The carbon dot powder prepared in Example 1 was subjected to X-ray diffraction testing, and the results are as follows: Figure 2 As shown, we can see that the particles have a broad peak at 20-30°, which is characteristic of amorphous carbon, indicating that the carbon dots were successfully synthesized.

[0081] The carbon dot powder prepared in Example 1 was tested for its ultraviolet light absorption and fluorescence properties, and the results are as follows: Figure 3a , Figure 3b As shown, we can see that its maximum excitation wavelength is 390 nm, its maximum emission wavelength is 460 nm, and it exhibits yellow-green fluorescence.

[0082] The carbon dot powder prepared in Example 1 was tested for its infrared spectrum. The Fourier transform infrared spectrum is shown below. Figure 4 As shown, this indicates that it has abundant functional groups.

[0083] The carbon dot powder prepared in Example 1 was tested, and its NMR spectrum was obtained as follows. Figure 5 As shown, the phenolic hydroxyl group, an anti-inflammatory and antioxidant functional group of the protocatechuic aldehyde precursor, is successfully retained in the carbon dot.

[0084] II. Detection of cell viability by carbon dots

[0085] The carbon dot powder obtained in Example 1 was used to prepare culture media with different concentrations of protocatechuic aldehyde carbon dots, namely 0 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL.

[0086] P3 chondrocytes were harvested and inoculated into 96-well plates, approximately 5 × 10⁶ cells per well. 3 Cells were incubated at 37°C and 5% ELISA for 24 hours. The culture medium was then aspirated, and 200 μL of PBS was added to each well to wash the cells. The PBS solution was discarded, and 100 μL of culture medium containing different concentrations of protocatechuic aldehyde carbon dots was added. Cells were incubated for 24, 48, and 96 hours, respectively. After incubation, the culture medium was aspirated under dark conditions, and 100 μL of fresh culture medium and 10 μL of CCK-8 solution were added to each well. The cells were then incubated at 37°C and 5% ELISA for another 2 hours. The absorbance was then measured at 450 nm using a microplate reader.

[0087] Experimental results are as follows Figure 6 As shown, this indicates that cells can grow normally at concentrations below 0.2 mg / mL of protocatechuic aldehyde carbon dots.

[0088] III. Cell viability detection and co-culture with protocatechuic aldehyde carbon dots

[0089] P3 chondrocytes were harvested and inoculated into 6-well plates, approximately 1 × 10⁶ cells per well. 5 Cells were cultured at 37°C and 5% HCl for 24 hours. The culture medium was then aspirated, and 1 mL of PBS was added to each well to wash the cells. The PBS solution was discarded, and 2 mL of culture medium containing 0.1 mg / mL protocatechuic aldehyde and protocatechuic aldehyde carbon dots were added to each well. Growth medium was used as a blank control. Cells were cultured for 24 hours. The culture medium was discarded under dark conditions, and the cells were washed three times with PBS. Calcein-AM / PI was added, and the cells were co-cultured with the precipitate at 37°C and 5% HCl for 30 minutes. Cell viability was then observed using a laser confocal microscope.

[0090] Experimental results are as follows Figure 7 As shown, protocatechuic aldehyde carbon dots have better biocompatibility than protocatechuic aldehyde and can significantly promote cell proliferation.

[0091] IV. Imaging test of protocatechuic aldehyde carbon dots on mitochondria

[0092] P3 chondrocytes were harvested, and 200,000 cells were inoculated into a confocal microplate and grown at 37°C for 24 hours. The culture medium was discarded, and the cells were fixed with paraformaldehyde for 20 min, followed by washing twice with PBS. Under light-protected conditions, 200 μL of the prepared Hoechst dye was added, and the cells were stained at 37°C for 30 min, followed by washing twice with PBS. Then, 200 μL of the prepared mitochondrial dye was added, and the cells were stained at 37°C for 20 min, followed by washing twice with HBSS. Finally, 200 μL of protocatechuic aldehyde carbon dots were added, and the cells were incubated at 37°C for 15 min. The targeting of the particles to the cells was observed under a laser confocal microscope.

[0093] The results of the above experiment are as follows Figure 8 As shown, from Figure 8 As we can see, carbon dots can enter cells and have good co-localization with mitochondria.

[0094] V. Test on the Regulation of Reactive Oxygen Species in Cells by Protocatechuic Aldehyde Carbon Dots

[0095] P3 chondrocytes were harvested, and 10,000 chondrocytes were inoculated into each well of a 96-well plate. After 24 hours of growth in a 37°C, 5% ELISA incubator, the culture medium was discarded. Hydrogen peroxide was added to induce the generation of reactive oxygen species (ROS), and then the medium was replaced with PCA and PCA-CDs solution for co-culturing. The group without the addition of particles and hydrogen peroxide served as a blank control group. After another 24 hours, the working solution of the DCFH-DA ROS detection kit was added and co-cultured for 30 minutes. The cells were washed twice with HBSS solution. Finally, fluorescence intensity was detected by excitation at 490 nm and emission at 530 nm using a microplate reader. The differences in fluorescence intensity were statistically analyzed, with * indicating p < 0.05 and ** indicating p < 0.01.

[0096] The above experimental results are as follows Figure 9 As shown, from Figure 9 It can be seen that both protocatechuic aldehyde and protocatechuic aldehyde carbon dots reduce the level of reactive oxygen species in cells. Among them, the antioxidant effect of protocatechuic aldehyde carbon dots is more significant, indicating that protocatechuic aldehyde carbon dots inherit the effects of the precursor and have the potential to become an antioxidant and anti-inflammatory drug.

[0097] In summary, the protocatechuic aldehyde carbon dots synthesized using protocatechuic aldehyde as a precursor exhibit uniform ultra-small size, good biocompatibility, and photoluminescence properties. They also inherit the antioxidant function of the precursor with even better results, not only capable of imaging mitochondria but also inhibiting intracellular reactive oxygen species levels, thus achieving anti-inflammatory and antioxidant effects. Furthermore, the preparation method is simple and low-cost.

[0098] The above description of the embodiments is intended to enable those skilled in the art to understand and apply this application. It will be apparent to those skilled in the art that various modifications can be easily made to these embodiments, and the general principles described herein can be applied to other embodiments without creative effort. Therefore, this application is not limited to the embodiments described herein, and any improvements and modifications made by those skilled in the art based on the disclosure of this application without departing from the scope and spirit of this application are within the scope of this application.

Claims

1. A method for preparing carbon dots based on protocatechuic aldehyde as a precursor, characterized in that, The preparation method includes the following steps: (1) Citric acid and protocatechuic aldehyde are dissolved in ultrapure water in sequence to obtain a reaction solution, and the mass ratio of citric acid, protocatechuic aldehyde and ultrapure water is (1~2):1:(15~30). (2) Place the reaction solution in a closed reactor, heat and pressurize it to react, and then let it cool naturally to obtain the crude product solution; (3) The crude solution is centrifuged, filtered, dialyzed and freeze-dried in sequence to obtain the carbon dots.

2. The method for preparing carbon dots as described in claim 1, characterized in that, Step (2) includes at least one of the following technical features: 21) The reactor is made of polystyrene; 22) The reaction temperature is 180~200℃; 23) The pressure of the reaction is 1~2 MPa; 24) The reaction time is 24~30h.

3. The method for preparing carbon dots as described in claim 1, characterized in that, Step (3) includes at least one of the following technical features: 31) The centrifugation speed is 5000~15000 rpm, and the centrifugation time is 10~20 min; 32) The filtration is performed using a needle filter with a pore size of 0.22 μm; 33) The dialysis bag used for the dialysis has a molecular weight cutoff of 300~1000 Da; 34) The freeze-drying temperature is -20 to -10 ℃, and the freeze-drying time is 48 to 72 h.

4. The method for preparing carbon dots as described in claim 3, characterized in that, The dialysis uses ultrapure water, each dialysis session lasts 12 hours, and at least 3 dialysis sessions are required.

5. A carbon dot based on protocatechuic aldehyde as a precursor, characterized in that, The carbon dots are prepared by any one of the preparation methods described in claims 1 to 4.

6. The carbon dots based on protocatechuic aldehyde as a precursor as described in claim 5, characterized in that, The diameter of the carbon dots is ≤10nm.

7. An application of carbon dots based on protocatechuic aldehyde as a precursor, characterized in that, The carbon dots are prepared by any one of the preparation methods described in claims 1 to 4, and the carbon dots are used to prepare imaging agents for cell mitochondrial imaging or antioxidant and anti-inflammatory drugs that reduce the level of reactive oxygen species in cells.

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