Preparation method and application of traditional chinese medicine derived carbon dots

Abstract

The application aims to provide a preparation method and application of traditional Chinese medicine derived carbon dots, and belongs to the technical field of nano biological medical materials.The traditional Chinese medicine derived carbon dots are prepared by using eucommia bark extract and glycine as precursors through one-step hydrothermal method.The carbon dots take eucommia bark, which has the effect of promoting bone formation, as a carbon source precursor, retain the biological activity of the original traditional Chinese medicine, and have the effects of promoting bone regeneration and fluorescence of the carbon dots.The preparation method is simple, the synthesis cost is low, the prepared traditional Chinese medicine derived carbon dots have good biocompatibility and low cytotoxicity, and have potential application prospect in the field of bone tissue engineering.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanobiomedical materials, and particularly relates to a preparation method of a traditional Chinese medicine derived carbon dot and application thereof. BACKGROUND

[0002] Bone defects caused by inflammation, trauma, tumors and other reasons are common clinical problems in the medical field, which seriously affect the physical and mental health and quality of life of patients. Traditional autologous bone transplantation is still the gold standard for repairing bone defects. However, autologous bone transplantation has the problems of material limitation and donor site trauma, and allogeneic bone transplantation may cause serious immune rejection. Other common bone repair techniques such as distraction osteogenesis are difficult to popularize due to limited indications, complex operation, long treatment period and high cost. For the above reasons, bone tissue engineering has emerged as the times require, providing a new treatment idea for repairing bone defects. Seed cells, scaffold materials and cytokines constitute the three major elements of bone tissue engineering, among which scaffold materials play a key role as a template for cell growth, proliferation, adhesion and differentiation in bone tissue engineering. At present, there have been a large number of basic researches on scaffold materials promoting bone regeneration, but the problems such as how cells distribute after transplantation, whether they can proliferate and differentiate, and the related mechanisms still need to be solved, which have become the main bottleneck restricting its development. Therefore, finding a safe and non-invasive tracing method that can effectively regulate osteogenic differentiation is the key to solving the above problems. Traditional scaffold materials such as metals, ceramics and polymers mostly do not have fluorescent properties, meaning that without other fluorescent dyes and nanoprobes, cells cannot be labeled at the same time, which is not conducive to real-time monitoring of the morphological changes of cells and tissues through imaging.

[0003] Carbon dots are spherical carbon nanoparticles with a diameter of less than 10 nm. Compared with other semiconductor quantum dots, carbon dots have unique optical properties, good biocompatibility, low cytotoxicity and easy surface functionalization. As a new zero-dimensional carbon-based nanomaterial, carbon dots play an important role in multiple fields of biomedicine such as biological imaging, biological sensing and drug carriers. In addition, carbon dots also have excellent biodegradability, mineralization induction ability and antibacterial performance. Considering these advantages, carbon dots may have broad application prospects in tracing and promoting bone regeneration.

[0004] The research results have confirmed the great potential of CDs as a dual-function tool for tracing and promoting bone regeneration. However, the raw materials for preparing CDs currently contain some chemical reagents, which limits the further development of CDs in the biomedical field. In recent years, due to the increasing emphasis on the concept of "green chemistry", the carbon source precursors for preparing carbon dots are mostly biomass materials derived from nature, such as orange juice, milk, pomelo peel, chitosan, egg, etc. However, there are few reports on the preparation of carbon dots using drugs as carbon sources. It is found that traditional Chinese medicine-derived carbon dots can improve some inherent properties of the original traditional Chinese medicine, such as solubility, or retain the material properties of the original traditional Chinese medicine, or increase the new pharmacological activity. At present, the application of traditional Chinese medicine-derived carbon dots is mainly concentrated in the fields of biological imaging, pH sensing, molecular detection, etc., and there are few studies on osteogenic differentiation. Traditional Chinese medicine Eucommia ulmoides is a unique plant in Eucommia ulmoides family in China. Modern pharmacological studies have confirmed that the effective chemical components of Eucommia ulmoides have the effects of promoting the proliferation of some mesenchymal stem cells, regulating bone metabolism, and promoting bone formation, etc. Clinically, Eucommia ulmoides is mainly used for preventing and treating osteoporosis, osteoarthritis, and bone fracture, etc. Therefore, synthesizing carbon dots from Eucommia ulmoides which has the effect of promoting bone formation can not only retain the biological activity of the carbon source itself, but also have the effects of promoting bone regeneration and fluorescence, and realize the dual effects of tracing and promoting bone regeneration. SUMMARY

[0005] In view of the above problems, the purpose of the present application is to provide a preparation method of traditional Chinese medicine-derived carbon dots and application thereof.

[0006] The present application adopts the following technical solutions:

[0007] A preparation method of traditional Chinese medicine-derived carbon dots, comprising the following steps:

[0008] In the first step, Eucommia ulmoides extract and glycine are dissolved in deionized water respectively, and ultrasonic oscillation is performed at room temperature to obtain uniformly dispersed Eucommia ulmoides extract solution and glycine solution;

[0009] In the second step, the Eucommia ulmoides extract solution and the glycine solution are mixed in proportion and placed in a reaction kettle for hydrothermal reaction.

[0010] In the third step, after the reaction is completed, carbon dot powder is obtained through centrifugation, filtration, dialysis, and freeze-drying.

[0011] Further, the ultrasonic oscillation time in the first step is 10-30 min.

[0012] Further, the concentrations of the Eucommia ulmoides extract solution and the glycine solution in the first step are 50 mg / mL and 10 mg / mL respectively.

[0013] Further, the volume ratio of the Eucommia ulmoides extract solution to the glycine solution in the second step is 3:1.

[0014] Further, the temperature of the hydrothermal reaction in the second step is 180-220 DEG C, and the time of the hydrothermal reaction is 8-12 hours.

[0015] Further, the centrifugal speed in the third step is 8000-11000 rpm, the centrifugal time is 15-30 minutes, the supernatant collected after centrifugation is filtered through a 0.22 mu m filter membrane, and the filtrate after filtration is dialyzed through a 500-1000 Da dialysis bag for 48-72 hours, and the product after dialysis is vacuum freeze-dried at -40 DEG C to obtain carbon dot powder.

[0016] A traditional Chinese medicine derived carbon dot in osteogenesis.

[0017] The application successfully prepares a traditional Chinese medicine derived carbon dot, which has good nano size structure and excellent fluorescence properties, is rich in hydrophilic groups such as hydroxyl, amino and carboxyl groups, has good biological safety, has the effects of tracing and promoting osteogenic differentiation on cells, and has important research significance as a new type of bifunctional nanomaterial in the field of bone tissue engineering.

[0018] The application uses eucommia bark extract and glycine as precursors to prepare a traditional Chinese medicine derived carbon dot through one-step hydrothermal method, and the carbon dot has potential application prospect in the field of bone tissue engineering.

[0019] The effect of eucommia bark extract: eucommia bark is a special eucommia ulmoides oliver plant in China, has the effects of strengthening bones and muscles and tonifying liver and kidney, and is one of the commonly used traditional Chinese medicinal materials in orthopedics. Modern pharmacological research results show that active compounds in eucommia ulmoides oliver can promote osteoblast proliferation, regulate bone metabolism and promote bone formation, and are mainly used in the prevention and treatment of osteoporosis, osteoarthritis and bone fracture and other diseases.

[0020] The effect of adding glycine: the two ends of a glycine molecule are amino and carboxyl groups, the amino group can be connected with the carboxyl group and the carbonyl group, and the carboxyl group can be connected with the hydroxyl group and the amino group, so that more active sites are provided for the prepared carbon dot, and the yield of the carbon dot is significantly improved. In addition, the introduction of the amino group also causes the emission peak of the carbon dot to be red-shifted, and the carbon dot exhibits better optical performance.

[0021] The application has the following beneficial effects:

[0022] 1. The traditional Chinese medicine eucommia ulmoides oliver is used as a carbon source precursor, which meets the concept of "green chemistry", the preparation method is simple, the synthesis cost is low, the prepared carbon dot has regular shape, uniform size, low cytotoxicity, excellent optical performance and good biological safety.

[0023] 2. The traditional Chinese medicine derived carbon dot provided by the application retains the biological activity of the traditional Chinese medicine eucommia ulmoides oliver itself, has the optical properties of carbon dots and the effect of promoting bone regeneration, has good biocompatibility, has wide application prospect in the field of bone tissue engineering, and can be used as a new type of bone formation nanomaterial in the field of bone tissue engineering. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Transmission electron microscopy image and size distribution histogram of the traditional Chinese medicine derived carbon dots of the present application.

[0025] Figure 2 Ultraviolet absorption spectrum of the traditional Chinese medicine derived carbon dots of the present application.

[0026] Figure 3 Photoluminescence spectrum of the traditional Chinese medicine derived carbon dots of the present application.

[0027] Figure 4 Fourier transform infrared spectrum of the traditional Chinese medicine derived carbon dots of the present application.

[0028] Figure 5 X-ray diffraction pattern of the traditional Chinese medicine derived carbon dots of the present application.

[0029] Figure 6 X-ray photoelectron spectroscopy of the traditional Chinese medicine derived carbon dots of the present application.

[0030] Figure 7 Cytotoxicity experiment results of the traditional Chinese medicine derived carbon dots of the present application.

[0031] Figure 8 Cell uptake images of the traditional Chinese medicine derived carbon dots of the present application.

[0032] Figure 9 Cell fluorescence imaging results of the traditional Chinese medicine derived carbon dots of the present application.

[0033] Figure 10 Alkaline phosphatase activity detection results of the traditional Chinese medicine derived carbon dots of the present application. DETAILED DESCRIPTION

[0034] In order to make the objectives, technical solutions and advantages of the present application more apparent, more comprehensible, the specific embodiments of the present application are further described in detail below in combination with the drawings and examples. The examples are intended to explain the present application, and are not a limitation of the present application. The experimental methods are conventional methods if not specifically stated, and the reagents and materials are ordinary commercially available products if not specifically stated.

[0035] Example 1: Preparation of traditional Chinese medicine derived carbon dots

[0036] A traditional Chinese medicine (TCM) derived carbon dot was prepared by a one-step hydrothermal method using Eucommia ulmoides and glycine as raw materials. Specifically, a 50 mg / mL solution of Eucommia ulmoides extract and a 10 mg / mL solution of glycine were prepared, mixed in a volume ratio of 3:1, and then ultrasonically shaken for 30 minutes. The mixture was then transferred to a high-pressure reaction kettle and reacted at 200°C for 10 hours. After natural cooling to room temperature, the mixture was transferred to a high-speed centrifuge and centrifuged at 10,000 rpm for 15 minutes. The supernatant was collected and filtered through a 0.22 µm filter membrane. The filtrate was dialyzed in a 1000 Da dialysis bag for 48 hours, with the dialysis solution being replaced every 6 hours. The dialyzed product was freeze-dried to obtain a carbon dot powder.

[0037] Example 2: Characterization of the TCM derived carbon dot

[0038] The morphology of the carbon dot was observed by transmission electron microscopy. The carbon dot powder was dissolved in deionized water and ultrasonically treated for 5 minutes before being slowly dropped onto an ultrathin carbon film. The morphology of the carbon dot was observed at an acceleration voltage of 200 kV. As shown in Figure 1 , the carbon dot was a regular spherical particle with uniform size and good dispersibility, with an average particle size of 3.83 nm. The carbon dot had clear lattice fringes, with a lattice spacing of 0.21 nm, which was attributed to the (100) crystal plane of graphite.

[0039] The ultraviolet-visible absorption spectrum of the carbon dot was detected by an ultraviolet-visible spectrophotometer. The carbon dot powder was dissolved in deionized water and diluted to an effective measurable concentration before being placed in a cuvette for detection in the ultraviolet-visible spectrophotometer. Deionized water was used as a blank control, with a scanning speed of 1 nm / step and a range of 200-800 nm. As shown in the inset of Figure 2 , the carbon dot aqueous solution appeared light brown under sunlight and bright yellow fluorescence under 365 nm ultraviolet light, indicating that the prepared carbon dot had good fluorescence properties. The ultraviolet-visible absorption spectrum results showed that the carbon dot had two absorption peaks at 218 nm and 261 nm, corresponding to the π-π* transition of C=C bond and the n-π* transition of C=O bond, respectively.

[0040] The photoluminescence spectrum of the carbon dot was detected by a fluorescence spectrophotometer. The carbon dot powder was dissolved in deionized water and diluted to an effective measurable concentration before being placed in a cuvette for detection in the fluorescence spectrophotometer. The excitation and emission slit width was 5 nm, the scanning speed was 240 nm / min, and the emission wavelength measurement range was 200-800 nm. As shown in Figure 3 (a), the optimal excitation wavelength of the carbon dot was 508 nm, and the corresponding optimal emission wavelength was 576 nm. The photoluminescence spectrum was scanned every 20 nm within a range of 430 nm-570 nm, and the results are shown in Figure 3(b) shows that the carbon dots have obvious excitation dependence, and the size of the excitation wavelength has a direct impact on the position and intensity of the emission wavelength. With the excitation wavelength gradually increasing from 430 nm to 570 nm, the intensity of the emission peak first shows an upward trend, and then gradually decreases.

[0041] The chemical bonds and functional groups of the carbon dots were analyzed by Fourier transform infrared spectroscopy. The carbon dot powder and potassium bromide were mixed in a ratio of 1:100, uniformly ground, formed in a tablet press, and detected in a Fourier transform infrared spectroscopy analyzer with a detection wavelength of 400-4000 cm -1 . The results are shown in Figure 4 Figure 2 (b), where 3422 cm -1 is the O-H / N-H stretching vibration; 2932 cm -1 is the CH2stretching vibration; 1625 cm -1 is the C=C / C=O stretching vibration, and the conjugation effect may cause the double bond order to decrease, resulting in red shift; 1392 cm -1 is the C-O-H in-plane bending vibration / C-N stretching vibration, and the CH2may form a hyperconjugation effect with the conjugated electrons, causing the C-N bond order to increase, resulting in blue shift; 1036 cm -1 is the C-OH stretching vibration; 601 cm -1 may be the H2O rocking vibration. It can be seen that the carbon dots contain a large number of hydroxyl, amino, carboxyl and other groups on the surface, which endow them with good water solubility and dispersibility, as well as the possibility of further modification.

[0042] The crystal structure of the carbon dots was characterized by X-ray diffractometer, and the scanning speed was set to 4° / min and the scanning range was 10-80°. The results are shown in Figure 5 Figure 3 (b), where there is a clear diffraction peak at 19.54°, corresponding to the (002) crystal plane of graphite carbon, proving that the center of the carbon dots has a highly carbonized graphite structure.

[0043] The elemental composition and chemical bonds of the carbon dots were characterized by X-ray photoelectron spectroscopy, and the excitation source was a monochromatic X-ray source Al Kα with a maximum power of 300 W. The detection results are shown in Figure 6 (a). The XPS spectrum of the carbon dots has three obvious binding energy peaks at 285.0 eV, 400.0 eV and 532.0 eV, corresponding to C, N and O elements, respectively. The high-resolution spectrum is shown in Figure 6(b)-(d) shown. The high-resolution C1s spectrum can be fitted into three peaks, 284.8 eV corresponding to C-C, accounting for 40.9%; 286.14 eV corresponding to C-O / C-N, accounting for 51.4%; 287.96 eV corresponding to C=O, accounting for 7.7%. The high-resolution N1s spectrum can be fitted into two peaks, 399.6 eV corresponding to C-N, accounting for 53.0%; 400.9 eV corresponding to N-H, accounting for 47%. The high-resolution O1s spectrum can be fitted into two peaks, 531.1 eV corresponding to C=O, accounting for 15.2%; 532.5 eV corresponding to C-O, accounting for 84.8%. The XPS spectrum analysis results are basically consistent with the FTIR analysis results, further proving that the prepared traditional Chinese medicine derived carbon dots contain functional groups such as amino, hydroxyl and carboxyl groups, and can be endowed with good water solubility and stability without further modification.

[0044] Example 3: Biological safety of traditional Chinese medicine derived carbon dots

[0045] Take the MC3T3-E1 cells in the logarithmic growth phase and inoculate them in a 96-well plate, with the number of cells per well being 5×10 3 The carbon dot group, the eucommia ulmoides group, the blank group and the zero setting group are set. The carbon dot group is set to have a concentration gradient of 25, 50, 100, 200 and 400 µg / mL of carbon dots, the eucommia ulmoides group is set to have a concentration gradient of 25, 50, 100, 200 and 400 µg / mL of eucommia ulmoides extract, the blank group is set to have only culture medium, and the zero setting group is set to have no cells in the culture medium. After the cells adhere, 100 µL is added to each well. On the 1st, 3rd and 7th day after co-culture, 100 µL of culture medium containing 10% CCK-8 is added to each well, the absorbance value (OD) at 450 nm is measured on an enzyme marker, and the relative survival rate of the cells is calculated. The results are shown in Figure 7 Fig. 2, which shows the relative survival rate of the cells on the 1st (a), 3rd (b) and 7th (c) day at different concentrations of carbon dots and eucommia ulmoides extract. Even when the concentration of carbon dots is 400 µg / mL, the survival rate of the cells is still more than 80%, and when the concentration of carbon dots is 200 µg / mL, the survival rate of the cells is more than 85%. Compared with the eucommia ulmoides extract, the carbon dots at different concentrations show better biological safety on the 1st, 3rd and 7th day, which is suitable for long-term growth of the cells. Therefore, 200 µg / mL is selected as the appropriate concentration for the subsequent experiments.

[0046] In order to further observe the cell morphology of MC3T3-E1 after co-culture with carbon dots, rhodamine-labeled phalloidin and DAPI are used to stain the actin cytoskeleton and nucleus of MC3T3-E1. Take the MC3T3-E1 cells in the logarithmic growth phase and inoculate them in a laser confocal special dish, with the number of cells being 5×10 4The number of cells was 5 x 105, and the cells were cultured for 24 h; after the cells adhered, the medium was replaced with a medium containing carbon dots at a concentration of 200 pg / mL, and the cells were incubated for 24 h. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and then treated for 10 min, followed by treatment with 1% BSA for 20 min at room temperature. Rhodamine-labeled phalloidin solution was added in the dark for 30 min, and then DAPI was added for 10 min. The cell morphology was observed by laser confocal microscopy. Figure 8 For carbon dots entering MC3T3-E1, the fluorescence images labeled with DAPI (a), rhodamine-labeled phalloidin (b), and carbon dots (c) were observed to have good cell morphology, and the cell outline was clearly visible, showing an irregular spindle shape, indicating that the carbon dots did not cause adverse reactions such as deformation and inactivation after entering the cells, further proving that they have good biological safety.

[0047] Example 4: In vitro fluorescence imaging of traditional Chinese medicine-derived carbon dots

[0048] MC3T3-E1 cells in the logarithmic growth phase were inoculated in laser confocal special dishes, and the number of cells inoculated was 5 x 105. 4 The cells were cultured for 24 h; after the cells adhered, the medium was replaced with a medium containing carbon dots at a concentration of 200 pg / mL, and the cells were incubated for 24 h. The cells were fixed with 4% paraformaldehyde for 10 min; the fixed solution was discarded, and the cells were washed with PBS for 2-3 times. The fluorescence imaging of the cells was observed by laser confocal microscopy at different excitation wavelengths. The results, as shown in Figure 9 The carbon dots showed different colors of fluorescence at different excitation wavelengths, and the fluorescence intensity was relatively stable. The results showed that the prepared traditional Chinese medicine-derived carbon dots had obvious excitation dependence in cells and could achieve fluorescence labeling.

[0049] Example 5: In vitro promotion of osteogenic differentiation by traditional Chinese medicine-derived carbon dots

[0050] MC3T3-E1 cells in the logarithmic growth phase were inoculated in 6-well plates, and the number of cells per well was 5 x 105. 4 After the cells were cultured for 24 h, the medium was replaced with osteogenic induction medium (containing sodium beta-glycerophosphate, ascorbic acid, and dexamethasone), the experimental group was replaced with osteogenic induction medium containing carbon dots at a concentration of 200 pg / mL, and the control group was replaced with osteogenic induction medium containing eucommia extract at a concentration of 200 pg / mL, and the cells were further cultured. The medium was replaced every 3 days. The activity of alkaline phosphatase (ALP) was measured according to the operation steps in the kit instructions on the 3rd, 7th, and 14th days after osteogenic induction. The results, as shown in Figure 10As shown, at 3, 7, 14 days after osteogenic induction, the ALP activity of the eucommia extract and carbon dot groups was significantly increased compared with the blank group, and the ALP activity of the carbon dot group was significantly higher than that of the eucommia extract group. The results show that the prepared carbon dots can significantly enhance the ALP activity in MC3T3-E1, and the effect is better than that of eucommia itself, proving that the prepared traditional Chinese medicine derived carbon dots can promote cell osteogenic differentiation in vitro.

Claims

1. A method for preparing carbon dots derived from traditional Chinese medicine, characterized in that: Includes the following steps: The first step involves dissolving Eucommia ulmoides extract and glycine in deionized water, and then ultrasonically vibrating them at room temperature to obtain a uniformly dispersed Eucommia ulmoides extract solution and glycine solution; the concentrations of the Eucommia ulmoides extract solution and the glycine solution are 50 mg / mL and 10 mg / mL, respectively. The second step involves mixing the Eucommia ulmoides extract solution and glycine solution in a specific ratio and placing them in a reaction vessel for a hydrothermal reaction; the volume ratio of the Eucommia ulmoides extract solution to the glycine solution is 3:

1. The third step involves centrifuging, filtering, dialysis, and freeze-drying to obtain carbon dot powder.

2. The method for preparing carbon dots derived from traditional Chinese medicine according to claim 1, characterized in that: The ultrasonic oscillation time mentioned in the first step is 10-30 minutes.

3. The method for preparing carbon dots derived from traditional Chinese medicine according to claim 1, characterized in that: The hydrothermal reaction in the second step is carried out at a temperature of 180-220℃ for 8-12 hours.

4. The method for preparing carbon dots derived from traditional Chinese medicine according to claim 1, characterized in that: The centrifugation speed in the third step is 8000-11000 rpm, and the centrifugation time is 15-30 minutes. The supernatant collected after centrifugation is filtered through a 0.22µm filter membrane. The filtered filtrate is dialyzed through a 500-1000Da dialysis bag for 48-72 hours. The dialysis product is then freeze-dried under vacuum at -40℃ to obtain carbon dot powder.

5. The application of a traditional Chinese medicine-derived carbon dot prepared by the preparation method according to any one of claims 1-4 in osteogenic processes.

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