CN104555980B - A kind of carbon quantum dot based on aspirin and biologic applications thereof - Google Patents

Abstract

The application of aspirin-based carbon quantum dots and cell imaging and anti-inflammatory treatment belongs to the field of nano-medicine technology. It specifically relates to the preparation of carbon quantum dots with strong fluorescent properties by using microwave heating method under the condition that hydrazine hydrate solution promotes the dissolution of aspirin. The carbon quantum dots prepared by this method are very stable under high salt concentration and physiological pH conditions, maintain blue fluorescence, and can enter the nucleus to effectively trace the cells; meanwhile, the original anti-inflammatory effect of aspirin is retained In the acute inflammation model constructed with carrageenan, the production of inflammatory cells can be effectively inhibited; in addition, the prepared aspirin carbon quantum dots are less toxic, and the application in vivo will not affect liver function, kidney function and heart, liver, spleen, Kidney and other organs are affected, and the goal of nanomaterials with dual functions of diagnosis and treatment can be realized.

Description

A carbon quantum dot based on aspirin and its biological application

technical field

The invention belongs to the technical field of nanomedicine, and specifically relates to the preparation of an aspirin-based carbon quantum dot, which can be obtained by heating aspirin and hydrazine hydrate with microwaves, has strong fluorescent properties, and retains the drug activity of aspirin, so it can be Applied to cell imaging and anti-inflammatory therapy.

Background technique

In recent years, nanotechnology has been used in the transfer and treatment of genes/drugs, or as a diagnostic probe in medicine. The combination of nanotechnology and medicine is called nanomedicine. The core problem facing this field is how to design a multifunctional nanomaterial that can be used not only for disease diagnosis but also for disease treatment. As a new member of the family of carbon nanomaterials, carbon quantum dots with fluorescent properties have attracted widespread attention from scholars in the field of nanotechnology. These surface-passivated carbon quantum dots are usually less than 10nm in size and have been applied to biomarkers due to their stable fluorescence properties, wide excitation spectrum and tunable emission peak. Compared with traditional organic dyes and semiconductor quantum dots, carbon quantum dots have good biocompatibility and low toxicity, so they have good application prospects in nanomedicine.

As the main representative of non-steroidal anti-inflammatory drugs, aspirin (acetylsalicylic acid) has a history of hundreds of years. Although its effect has been recognized, there are still problems such as damage to the gastrointestinal tract and poor water solubility. Although the modification of aspirin has never stopped, there are very few that can be actually applied. Therefore, finding one that can improve the water solubility of aspirin and reduce its damage to the gastrointestinal tract is the key to solving this problem.

At present, carbon quantum dots are mainly used in biomedicine for bioimaging or drug/gene transfer, but the research on making drugs directly into carbon quantum dots has not been reported. Whether the biological activity of drugs is preserved after making carbon quantum dots No research has been seen. If aspirin can be prepared into carbon quantum dots, it can not only improve its water solubility, but also have application prospects in tracing, anti-inflammation, and prevention and treatment of cardiovascular and cerebrovascular diseases.

Contents of the invention

The object of the present invention is to provide a multifunctional carbon quantum dot based on aspirin and its biological application. By modifying the traditional drug aspirin, the water solubility is improved. The carbon quantum dots prepared by the present invention are very stable under high salt concentration and physiological pH conditions, maintain blue fluorescence, and can enter the nucleus to effectively trace the cells; meanwhile, they also retain the original anti-inflammatory effect of aspirin. In the acute inflammation model constructed with carrageenan, the production of inflammatory cells can be effectively inhibited; in addition, the prepared aspirin carbon quantum dots are less toxic, and the application in vivo will not affect liver function, kidney function and heart, liver, spleen, Kidneys and other organs can be affected, and the goal of nanomaterials with dual functions of diagnosis and treatment can be realized.

The invention utilizes a microwave heating method to dissolve aspirin in deionized water with the assistance of hydrazine hydrate in the early stage, and then obtain strong fluorescent and pharmaceutically active carbon quantum dots with a size distribution of 2 to 6 nm after microwave heating. After separation and purification, this carbon quantum dot can be used in cell imaging and anti-inflammatory treatment.

The raw materials used in the present invention are all commercially available substances, without further treatment, and can be directly mixed according to a certain ratio, so the experimental operation is simple, the risk is small, and it has good experimental repeatability, and can be produced in batches. At the same time, the obtained carbon quantum dots have high fluorescence quantum efficiency.

The preparation of the aspirin-based carbon quantum dots of the present invention comprises the following steps: (1) preparing the aspirin-based carbon quantum dots by a microwave method; (2) separating and purifying the prepared carbon quantum dots.

(1) Preparation of aspirin-based carbon quantum dots: Add 1.0 to 2.5 g of aspirin to an aqueous solution containing 0.5 to 2 mL of hydrazine hydrate with a mass concentration of 98%, and the total volume of the entire system is controlled at 10 to 20 mL; then the solution is Put it into a microwave oven and heat it at 80-100W for 6-10 minutes to obtain carbon quantum dots with blue fluorescence;

(2) Separation and purification of carbon quantum dots: add 50 to 100 mL of deionized water to the carbon quantum dots prepared in step (1), ultrasonically disperse the product, and then separate and purify the prepared carbon with a molecular weight of 1800 to 10,000. Quantum dots, after 3-7 days of dialysis, filter the product in the dialysis bag with a water-based filter head with a pore size of 0.22-0.45 μm, and concentrate the product to 10-20 mL after rotary evaporation, and finally freeze-dry the concentrate to obtain a brown-yellow Carbon quantum dot powder for aspirin.

Description of drawings

Figure 1: Transmission electron micrograph (a), particle size distribution (b), high-resolution transmission electron micrograph (c), and powder diffraction spectrum (d) of aspirin-based carbon quantum dots prepared by microwave method, illustrating The prepared carbon quantum dots have narrow size distribution, good monodispersity, and obvious crystal lattice;

Figure 2: The ultraviolet absorption spectrum (a) of aspirin-based carbon quantum dots prepared by microwave method, the fluorescence excitation spectrum (solid line in b) and emission spectrum (dotted line in b), and the carbon quantum dots under room light ( Photos of left inset in b) and photos under ultraviolet light (right inset in b), indicating that the prepared carbon quantum dots have strong blue fluorescence;

Figure 3: The infrared spectrum of aspirin-based carbon quantum dots prepared by the microwave method, indicating that the prepared carbon quantum dots retain the acetyl group in aspirin;

Figure 4: Stability curves (a) of aspirin-based carbon quantum dots in different concentrations of KCl salt solutions prepared by microwave method, and stability curves (b) in different pH solutions, indicating that carbon quantum dots have good Stability, can be applied to biological systems;

Figure 5: Confocal microscope images of the aspirin-based carbon quantum dots co-cultured with cervical cancer cells. The dark field picture (a), the bright field picture (b) and the superimposed picture (c) of the bright field and dark field under the excitation of light with a wavelength of 400nm indicate that carbon quantum dots can be used for cell imaging;

Figure 6: Histological section results of the anti-inflammatory effect of the prepared carbon quantum dots in rats. The blank control group (a, b, c) had a thicker inflammatory cell layer with a large number of inflammatory cells, while the aspirin group (d, e, f) and aspirin-based carbon quantum dots group (g, h, i) had significantly reduced inflammation, The relative reduction of inflammatory cells;

Figure 7: Effects of aspirin and aspirin-based carbon quantum dots on serum biochemical indicators. Aspirin-based carbon quantum dots had no significant effect on liver function indexes alanine aminotransferase (a) and aspartate aminotransferase (b), and had no significant effect on kidney function indexes urea nitrogen (c) and creatinine (d);

Figure 8: Effects of aspirin and aspirin-based carbon quantum dots on organs. Heart (i), liver (j), spleen (k), kidney (l) of rats in intraperitoneal injection of carbon quantum dots group and blank control group (a, b, c, d) and aspirin group (e, f, g , h) There were no obvious histological differences in the corresponding organs.

detailed description

The present invention will be further described below in conjunction with the examples, rather than limiting the present invention.

Example 1

(1) Preparation of aspirin-based carbon quantum dots: 2 g of acetylsalicylic acid was added to 1.08 mL of an aqueous solution of hydrazine hydrate with a mass concentration of 98%, and the total volume of the entire system was 10 mL. The microwave power is 100W, and the time is 8 minutes. Aspirin-based carbon quantum dots with blue fluorescence were obtained.

(2) Separation and purification of carbon quantum dots: add 80 mL of deionized water to the carbon quantum dots prepared in step (1), ultrasonically disperse the product, separate and purify the prepared carbon quantum dots with a molecular weight of 3500, and dialysis for 7 Days later, the obtained product was filtered with a water-based filter head with a particle size of 0.22 μm, concentrated to 10 mL after rotary evaporation, and then freeze-dried to obtain aspirin-based carbon quantum dot powder (brown yellow), with a yield of about 50 mg.

Observation under a transmission electron microscope shows that the obtained aspirin-based carbon quantum dots have a size distribution of 2-6 nm, as shown in FIG. 1 .

Example 2

(1) Preparation of aspirin-based carbon quantum dots: 1 g of acetylsalicylic acid was added to an aqueous solution containing 1.08 mL of 98% hydrazine hydrate, and the total volume of the entire system was 10 mL. The microwave power is 100W, and the time is 7 minutes. Aspirin-based carbon quantum dots with blue fluorescence were obtained.

(2) Separation and purification of carbon quantum dots: add 50 mL of deionized water to the carbon quantum dots prepared in step (1), ultrasonically disperse the product, separate and purify the prepared carbon quantum dots with a molecular weight of 3500, and dialysis for 7 Days later, the obtained product was filtered with a water-based filter head with a particle size of 0.22 μm, concentrated to 5 mL after rotary evaporation, and freeze-dried to obtain aspirin-based carbon quantum dot powder (brown yellow), with a yield of about 30 mg. The resulting carbon quantum dots have strong fluorescent properties, as shown in Figure 2b.

Example 3

(1) Preparation of aspirin-based carbon quantum dots: 2 g of acetylsalicylic acid was added to an aqueous solution containing 0.54 mL of 98% hydrazine hydrate, and the total volume of the entire system was 10 mL. The microwave power is 100W, and the time is 8 minutes. Aspirin-based carbon quantum dots with blue fluorescence were obtained.

(2) Separation and purification of carbon quantum dots: add 60 mL of deionized water to the carbon quantum dots prepared in step (1), ultrasonically disperse the product, separate and purify the prepared carbon quantum dots with a molecular weight of 3500, and dialysis for 7 Days later, the obtained product was filtered with a water-based filter head with a particle size of 0.45 μm, concentrated to 5 mL after rotary evaporation, and then freeze-dried to obtain aspirin-based carbon quantum dot powder (brown yellow), with a yield of about 40 mg. After infrared testing, it can be seen that carbon quantum dots retain the acetyl group in aspirin, as shown in Figure 3

Example 4

(1) Preparation of aspirin-based carbon quantum dots: 2 g of acetylsalicylic acid was added to an aqueous solution containing 2.16 mL of 98% hydrazine hydrate, and the total volume of the entire system was 10 mL. The microwave power is 100W, and the time is 8 minutes. Aspirin-based carbon quantum dots with blue fluorescence were obtained.

(2) Separation and purification of carbon quantum dots: add 80 mL of deionized water to the carbon quantum dots prepared in step (1), ultrasonically disperse the product, separate and purify the prepared carbon quantum dots with a molecular weight of 3500, and dialysis for 7 Days later, the obtained product was filtered with a water-based filter head with a particle size of 0.22 μm, concentrated to 10 mL after rotary evaporation, and then freeze-dried to obtain aspirin-based carbon quantum dot powder (brown yellow), with a yield of about 50 mg. Then preparation concentration is the KCl salt solution of 0, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75 and 2.0mol/L, and utilizes hydrochloric acid and sodium hydroxide to adjust the pH obtained by deionized water to be 1.8, 2.3, 3.2, 4.2, 4.9, 6.1, 7.2, 8.1, 8.9, 9.8, 10.8 and 12.5 aqueous solutions, then add 0.1g of carbon quantum dots to each of the above solutions (3mL), and finally detect the fluorescence intensity of the solution, as shown in Figure 4.

Example 5

(1) The preparation and purification of aspirin-based carbon quantum dots are as described in Example 1.

(2) Since the prepared aspirin-based carbon quantum dots have fluorescent properties, it is considered that they can be used for cell labeling. Human cervical cancer cells were inoculated at 200,000/well in a confocal microscope special plate, and cultured in a 37°C cell culture incubator containing 5% carbon dioxide. After overnight cell attachment, the medium was replaced and carbon quantum dots were added to a final concentration of 20 μg/mL. After co-cultivation for 6 hours, the medium was discarded, washed twice with phosphate buffered saline, fixed with 4% paraformaldehyde for 10 minutes, and washed twice with phosphate buffered saline. When the excitation wavelength is 400nm, it can be seen under the laser confocal microscope that the whole cell is blue (Figure 5), indicating that the carbon quantum dots enter the cytoplasm, and can pass through the nuclear membrane and enter the nucleus to effectively label the cells.

Example 6

(1) The preparation and purification of aspirin-based carbon quantum dots are as described in Example 1.

(2) The mechanism of aspirin's anti-inflammatory effect is that its ester bond undergoes an acetylation reaction with the serine residue of cyclooxygenase, thereby inhibiting the formation of prostaglandin E2 and exerting an anti-inflammatory effect, but we did not destroy it in the reaction Aspirin's ester bond, so theoretically its biological effects should be preserved. In order to investigate the biological activity of the prepared aspirin-based carbon quantum dots, we selected male Wistar rats (160-180g) and injected equal volumes of phosphate-buffered saline, aspirin and aspirin-based carbon quantum dots (20mg/ kg) solution. After half an hour, a polyester fiber sponge with a size of 2.0×1.0×0.5 cm was soaked in a carrageenan solution with a mass concentration of 1%, taken out, implanted subcutaneously in the back of the rat, and finally closed with a 3/0 surgical suture suture. Five hours after the operation, the heart was perfused with 4% paraformaldehyde and sacrificed, and the subcutaneous tissue was taken, fixed, and dehydrated step by step for histological staining.

Observation under a microscope showed that the blank control group (injected with phosphate buffered saline) had a thicker inflammatory cell layer, with a large number of inflammatory cells, such as neutrophils and lymphocytes infiltrating (Figure 6); while the injection of aspirin and aspirin-based carbon quantum dots group (Figure 6), the inflammation was significantly reduced, and the inflammatory cells were relatively reduced. It shows that the aspirin-based carbon quantum dots still retain the original anti-inflammatory activity, and the effect is better than that of aspirin alone. Analysis of the reasons, on the one hand, due to the relatively large specific surface area of nanomaterials, because there are relatively more surface binding sites per unit volume; on the other hand, carbon quantum dots have improved water solubility compared with the simple drug aspirin, and are slightly positively charged. Can promote its circulation in the body and into cells.

Example 7

(1) The preparation and purification of aspirin-based carbon quantum dots are as described in Example 1.

(2) To investigate the biological toxicity of the prepared aspirin-based carbon quantum dots. Through animal experiments in vivo, we have observed its effects on serum biochemical indicators such as liver function and kidney function. Male Wistar rats (160-180 g) were intraperitoneally injected with equal volumes of phosphate buffered saline, aspirin and aspirin-based carbon quantum dots (20 mg/kg) solution. Eye blood was collected on days 1, 3 and 7, collected in heparin-free blood collection tubes, left to stand for 1 to 2 hours, centrifuged at 3000 rpm for 5 minutes, and the supernatant was used for detection of biochemical indicators.

Alanine aminotransferase and aspartate aminotransferase are important indicators of liver function. Compared with the blank control group (injection of phosphate-buffered saline), the aspirin group and the aspirin-based carbon quantum dot group were slightly different (Figure 7), but the difference There was no statistical significance, and the indicators in each group were within the normal range, indicating that there was no significant effect on liver function. Blood urea nitrogen and creatinine are important evaluation indicators of renal function. Compared with the blank control group, the aspirin group and the aspirin-based carbon quantum dots group were both within the normal range (Figure 7), with no significant difference. The above serum biochemical tests preliminarily indicate that the prepared aspirin-based carbon quantum dots have good biological safety and have no significant effect on liver and kidney functions.

Example 8

(1) The preparation and purification of aspirin-based carbon quantum dots are as described in Example 1.

(2) Regarding the biological toxicity investigation of aspirin-based carbon quantum dots, in addition to taking eyeball blood for serum biochemical analysis, at the same time, 4% paraformaldehyde was perfused and fixed to rats (in Example 7), and heart, liver, and spleen were taken. Fixation, step-by-step dehydration, and histological staining of kidneys and other organs to clarify whether aspirin-based carbon quantum dots can cause organ damage, inflammation, or other lesions.

Observation under a microscope shows that compared with the blank control group, the aspirin group and the aspirin-based carbon quantum dots group have no obvious histological changes (Figure 8), indicating that the biological safety is better and will not cause organ damage, inflammation or other damage. lesion.

Claims (2)

1. A carbon quantum dot based on aspirin, characterized in that: it is prepared by the following steps, (1) Preparation of aspirin-based carbon quantum dots: Add 1.0 to 2.5 g of aspirin to an aqueous solution containing 0.5 to 2 mL of hydrazine hydrate with a mass concentration of 98%, and the total volume of the entire system is controlled at 10 to 20 mL; then the solution is Put it into a microwave oven and heat it at 80-100W for 6-10 minutes to obtain carbon quantum dots with blue fluorescence; (2) Separation and purification of carbon quantum dots: add 50 to 100 mL of deionized water to the carbon quantum dots prepared in step (1), disperse the product ultrasonically, and then use a dialysis bag with a molecular weight of 1800 to 10000 to separate and purify the prepared carbon quantum dots After dialysis for 3-7 days, filter the obtained product with a water-based filter head with a pore size of 0.22-0.45 μm, concentrate it to 10-20 mL after rotary evaporation, and finally freeze-dry the concentrated solution to obtain brown-yellow aspirin-based carbon quantum dots powder. 2. the application of a kind of aspirin-based carbon quantum dots according to claim 1 in the preparation of cell imaging or anti-inflammatory drugs.