Preparation method and application of near-infrared carbon dots based on Simiao kaempferi

Abstract

The present invention discloses a preparation method of near-infrared carbon dots based on Simiangsu kaempferi and its application in the field of biological imaging. The Simiangsu kaempferi near-infrared carbon dots are prepared by a one-step solvent thermal method using Simiangsu kaempferi branches and leaves as a carbon source. The near-infrared carbon dots prepared by the method of the present invention have no excitation dependence, a quantum efficiency of 27.22% under 409 nm excitation, and good fluorescence properties. The process of the present invention is simple and the cost is low. The prepared near-infrared carbon dots realize fast and real-time fluorescence imaging of cells and living animals, and have extremely low toxicity to cells and living animals. The carbon dots of the present invention have high stability, low toxic side effects, and good biocompatibility, and have broad application prospects in the fields of biological imaging and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent nanomaterials, and in particular relates to a method for preparing near-infrared carbon dots based on Simiao kaempferi and the application of the near-infrared carbon dots in the field of biological imaging. Background Art

[0002] Carbon dots (Cdots) are a new type of fluorescent carbon nanoparticles (less than 10 nanometers in size). Due to their unique properties, such as low toxicity, good biocompatibility, and high photostability, they have been widely used in various fields, including sensors, bioimaging, catalysis, light-emitting diodes (LEDs), and optoelectronic devices. Currently reported Cdot fluorescence is primarily blue-green. Near-infrared fluorescence (650 nm–1000 nm) has been highly sought after due to its advantages, such as minimal background interference, strong tissue penetration, and minimal damage to organisms. Currently, Cdots prepared from plant resources have attracted extensive attention due to their abundant, renewable, environmentally friendly, and simple preparation methods. Their synthesis does not require the addition of additional oxidants or passivating agents. In contrast to chemical substances, plant resources contain abundant carbohydrates, proteins, amino acids, and other biomolecules, which provide sufficient elements for the surface functionalization of Cdots. However, current research has focused solely on preparation methods and performance characterization. Therefore, developing a simple, efficient, and green Cdot synthesis route that can be used in practical applications would be of great value.

[0003] Bioimaging is an important means of further interpreting the life activities in organisms through imaging observation. The development of bioimaging technology requires not only enhancing the continuity and real-time performance of tissue imaging, but also improving the imaging effect in order to achieve the observation and recording of life activities. Fluorescence imaging has developed into a powerful means of clinical diagnosis due to its advantages such as convenience, low cost, high sensitivity, non-invasiveness, and long observation time. However, traditional fluorescent agents, such as quantum dots and organic dyes, often have problems such as high toxicity, poor biocompatibility, and poor fluorescence performance. Plant-derived carbon dots have advantages such as good biocompatibility, flexible design, multi-color emission, red light / near-infrared light emission, and two-photon / multi-photon fluorescence, making them suitable for use as a new generation of fluorescent probes for in vivo / in vitro imaging. Summary of the Invention

[0004] The present invention provides a method for preparing near-infrared carbon dots based on Simiana kirilowii and the application of the near-infrared carbon dots in the field of biological imaging. The method of the present invention is simple and easy to implement, and realizes the preparation of plant-derived near-infrared carbon dots and their imaging applications in cells and living animals.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] 1. Preparation of near-infrared carbon dots based on Simiao tung

[0007] (1) Grind the dried Simiao vine branches and leaves into powder, mix with an organic solvent, and sonicate for 10-30 minutes;

[0008] The mass volume ratio of the powder to the organic solvent is 1g:5-30mL; the organic solvent is acetone;

[0009] (2) Transfer the mixture from step (1) to a reactor, heat at 90-220°C for 3-7 hours, and then cool naturally to room temperature;

[0010] (3) The reaction product of step (2) was centrifuged, and the supernatant was collected and filtered with a 0.22 μm organic phase filter membrane. The filtrate was purified by passing through a silica gel column and eluted with a petroleum ether-ethyl acetate mixture as a washing liquid. The eluate was collected and vacuum dried to obtain Simitake tung oil near-infrared carbon dots;

[0011] In the silica gel column chromatography purification, the product was first eluted with a petroleum ether-ethyl acetate mixture in a volume ratio of 4:1, the eluate was collected, and then eluted with a petroleum ether-ethyl acetate mixture in a volume ratio of 1:3, the eluate was collected, and the eluates were combined; the centrifugation was carried out at 9000-10000 r / min for 10 minutes.

[0012] 2. Application of Simiao Teng Near-Infrared Carbon Dots in the Preparation of Bioimaging Reagents

[0013] The present invention tested the imaging effect of Simiao Teng near-infrared carbon dots in cells (normal cells, tumor cells), living animals and animal organs. The results showed that the near-infrared carbon dots of the present invention can be used for imaging in cells and living animals at extremely low concentrations, and have extremely low toxicity to cells and living animals.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention uses the Simiao vine plant as a carbon source, which has the advantages of abundant sources, green environmental protection and renewable resources. A one-step solvent thermal method is used to prepare near-infrared light-emitting carbon dots. The near-infrared carbon dots of the present invention have no excitation dependence, and the fluorescence emission wavelength basically does not change with the change of the excitation wavelength. The quantum efficiency is 27.22% under 409nm excitation, and has good fluorescence performance.

[0016] The near-infrared carbon dots prepared by the present invention can be imaged in cells and living animals, and can be metabolized in mice, with good biocompatibility. The carbon dots of the present invention have high stability, low toxic side effects and good biocompatibility, and have broad application prospects in fields such as biological imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1Figure 1 is a morphological characterization of the near-infrared carbon dots of Simiao kaempferi prepared in Example 1; Figure a is a high-resolution transmission electron microscopy image, and Figure b is a statistical distribution diagram of the particle size obtained by counting 200 carbon dots;

[0018] Figure 2 This is the infrared spectrum of the near-infrared carbon dots of Simiaoteng prepared in Example 1;

[0019] Figure 3 This is an X-ray powder diffraction (XRD) spectrum of the near-infrared carbon dots of Simiaoteng prepared in Example 1;

[0020] Figure 4 This is the X-ray photoelectron spectrum (XPS) of the near-infrared carbon dots of Simiao Teng prepared in Example 1, where the upper left is the survey spectrum and the upper right is the C1 s Spectrum, lower left picture O1 s Spectrum, the lower right picture is N1 s spectrum;

[0021] Figure 5 This is the ultraviolet absorption spectrum of the near-infrared carbon dots of Simiao kaempferi prepared in Example 1;

[0022] Figure 6 The fluorescence emission spectra of the near-infrared carbon dots of Simiaoteng prepared in Example 1 at different excitation wavelengths;

[0023] Figure 7 This is a graph showing the fluorescence quantum yield of the Simiaoteng near-infrared carbon dots prepared in Example 1 at an excitation wavelength of 409 nm;

[0024] Figure 8 This is a graph showing the toxicity test results of the Simiaoteng near-infrared carbon dots prepared in Example 1 on HaCat and A549 cells;

[0025] Figure 9 This is a laser confocal imaging image of HaCat and A549 cells incubated with the Simiao kaempferi near-infrared carbon dots prepared in Example 1 for 12 hours;

[0026] Figure 10 In vivo imaging of nude mice (top) and in vivo organ imaging results (bottom) after subcutaneous injection of the Simiyon kirilowii near-infrared carbon dots prepared in Example 1 into the back of nude mice;

[0027] Figure 11 HE staining results of organs 7 days after subcutaneous injection of the Simiaki kaempferi near-infrared carbon dots prepared in Example 1 into the back of nude mice. DETAILED DESCRIPTION

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and examples. However, the contents should not be construed as limiting the present invention. In the present examples, the methods are conventionally performed unless otherwise specified, and the reagents used are conventional reagents or reagents prepared according to conventional methods unless otherwise specified.

[0029] Example 1: Preparation method of near-infrared carbon dots based on Simiao kaempferi

[0030] 1. Select discarded dried Simao truncatum branches and leaves and crush them into powder, weigh 1g of powder and add 30mL of acetone, ultrasonicate for 30min, transfer the ultrasonic mixture to a reactor with a polytetrafluoroethylene liner, heat at a constant temperature of 120°C for 5h, cool naturally to room temperature, centrifuge at 10000r / min for 10min, remove insoluble matter, and filter the supernatant with a 0.22μm organic phase filter membrane. The filtrate is purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate as the chromatographic liquid, first eluting with a petroleum ether-ethyl acetate mixture with a volume ratio of 4:1, collecting the eluate, and then eluting with a petroleum ether-ethyl acetate mixture with a volume ratio of 1:3, collecting the eluate, combining the eluates, and drying in a vacuum drying oven at 60°C to obtain black solid Simao truncatum near-infrared carbon dots;

[0031] 2. The high-resolution transmission electron microscopy characterization results of the near-infrared carbon dots of Simiao kaempferi obtained in the above embodiment are shown in FIG. Figure 1 From the figure, it can be seen that the near-infrared carbon dots prepared by using Simao vine are spherical, with small particle size, and no obvious lattice fringes can be observed, indicating an amorphous structure; the particle size distribution is 0.2~3nm, showing a narrow and symmetrical distribution. The average particle size is about 1.34±0.29nm after statistical calculation;

[0032] Near infrared absorption spectrum of Simiaoteng carbon dots Figure 2 , it can be seen from the figure that 3423cm -1 The absorption peak at 2916 cm corresponds to the stretching vibration of -OH and -NH, and the stretching vibration of CH methyl leads to the absorption peak at 2916 cm -1 and 2847cm -1 There are two peaks at 1725cm -1 The peak at 1495 cm is attributed to the stretching vibration of O=C. -1 、1380-1450cm -1 The absorption peaks at 1195cm correspond to the stretching vibration of C=C and CN, respectively. -1 and 975cm -1 The absorption peaks at correspond to the stretching vibrations of COC and NH. The above results indicate that the prepared near-infrared carbon dots are rich in groups such as carbonyl, amide, methyl, hydroxyl and ether bonds.

[0033] Figure 3This is the X-ray powder diffraction spectrum of Simao Teng near-infrared carbon dots. The near-infrared carbon dots have a broad X-ray diffraction peak between 10 and 32 degrees, which is attributed to highly disordered carbon atoms. The results show that a disordered carbon structure is formed inside the prepared near-infrared carbon dots, and there is a large amount of amorphous carbon. Figure 4 This is the X-ray photoelectron spectrum of near-infrared carbon dots. The near-infrared carbon dots are mainly composed of three elements: C (81.14%, atomic percentage), N (1.03%, atomic percentage), and O (17.83%, atomic percentage); the high-resolution C1s spectrum analyzes the existence of five types of carbon: CC / C=C, CN, CO, C=N, and -COOH; the high-resolution N1s spectrum shows three different types of nitrogen: pyridinic N, amino N, and pyrrolic N; the high-resolution O1s spectrum shows two types of oxygen-containing chemical bonds, C=O and CO; this is consistent with the infrared spectrum and XRD results.

[0034] Figure 5 This is the ultraviolet-visible absorption spectrum of the near-infrared carbon dots of Simiao teng, which has a very wide light absorption range, from the ultraviolet region to the near-infrared region. Figure 6 This is the fluorescence emission spectrum of the Simiaoteng near-infrared carbon dots at different excitation wavelengths. Its fluorescence emission peak basically does not change with the change of the excitation light wavelength, that is, the fluorescence excited by light with a wavelength of 380-640nm is between 650-800nm, belonging to the near-infrared region, and the optimal emission peak is located at around 671nm and 717nm. Figure 7 When the excitation wavelength is 409nm, the fluorescence emission intensity of the Simiaoteng near-infrared carbon dots is the highest, and the measured quantum yield is about 27.22%, which has good fluorescence properties and application potential.

[0035] Example 2: Application of Simiao Teng near-infrared carbon dots in cell imaging experiments

[0036] 1. Preparation of experimental materials

[0037] Solution preparation: The Simiao Teng near-infrared carbon dots were prepared into a 1 mg / mL stock solution with cell-grade DMSO under sterile conditions, sealed and stored in a -20°C refrigerator in the dark for later use. In the experiment, the solution was diluted into sample solutions of different concentrations according to the final concentration required after drug addition.

[0038] Human immortalized epidermal cells Hacat and human non-small cell lung cancer cells A549 were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences. Both cell lines were cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin and streptomycin in a 37°C, 5% CO2 incubator until ready for use.

[0039] 2. Human epidermal keratinocytes (HaCat) and human non-small cell lung cancer cells (A549) in exponential growth phase were cultured in complete culture medium at a concentration of 1×10 4The cell suspension was seeded in 96-well plates, with 100 μL per well. The experiment was divided into drug group and negative control group, with 6 replicates per group (or each drug concentration). After 24 h of incubation in a cell culture incubator at 37°C, 5% CO2, and saturated humidity, the original culture medium was replaced with 100 μL of culture medium containing different concentrations of near-infrared carbon dots (final concentrations of 0, 0.05, 0.1, 0.2, 0.3, and 0.4 mg / mL) in the drug group, and the original culture medium was replaced with 100 μL of fresh complete culture medium in the negative control group. After 24 h of incubation, 10 μL of CCK-8 reagent was added to each well, and the plates were returned to the incubator for another 4 h. The absorbance of each well was then measured at a wavelength of 450 nm using a fluorescence microplate reader (OD value). The effect of different drug concentrations on cell survival was calculated according to the formula: survival rate = (OD value of drug group / OD value of negative control group) × 100%.

[0040] See the results Figure 8 As can be seen from the figure: when the concentration of Simiyon kaempferi near-infrared carbon dots is 0.4μg / mL, the cell survival rate is still over 95%, and there is no significant difference among the concentration groups, indicating that Simiyon kaempferi near-infrared carbon dots have no obvious toxicity and are cell compatible with HaCat and A549 cells;

[0041] 3. Human epidermal keratinocytes (HaCat) and human non-small cell lung cancer cells (A549) in exponential growth phase were cultured in complete culture medium at a concentration of 1×10 5 The cell suspension was seeded into confocal microplates (2 mL per dish) and cultured in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for 12 h. The original culture medium was then replaced with 2 mL of complete culture medium containing 0.4 mg / mL near-infrared carbon dots. After 12 h of culture, the culture was terminated, the original culture medium was removed, and the cells were washed twice with 1 mL of pre-cooled 1× PBS buffer (pH 7.4) and discarded. 1 mL of 4% paraformaldehyde fixative was added to each dish and fixed at room temperature for 10 min. The cells were then washed twice with 1 mL of pre-cooled 1× PBS buffer (pH 7.4) and discarded. The cells were then stained with 1 mL of 0.1 μg / mL DAPI solution at room temperature in the dark for 20 min. The cells were then washed twice with 1 mL of pre-cooled 1× PBS buffer (pH 7.4) and discarded. 0.5 mL of cold 1× PBS buffer (pH 7.4) was added to moisten the cells. Images were taken using a laser confocal fluorescence microscope at an excitation wavelength of 402 nm.

[0042] The results of laser confocal imaging are shown in Figure 9, the excitation wavelength is 402nm, and bright red light (CDs) and blue light (DAPI) are obtained respectively. It can be seen from the figure that near-infrared carbon dots can enter cells and mark living cells. The Ex: 402nm picture shows that the bright red fluorescence is mainly concentrated in the cytoplasm area, and the Merge picture shows that the CDs fluorescence imaging and the DAPI fluorescence imaging overlap slightly, indicating that a small part of the near-infrared carbon dots are concentrated in the cell nucleus; these preliminary results show that near-infrared carbon dots are candidates for living cell imaging.

[0043] Example 3: Simiao Teng near-infrared carbon dots used in in vivo mouse imaging experiments

[0044] 1. Preparation of experimental materials

[0045] The Simiao Teng near-infrared carbon dots were prepared into a 1 mg / mL stock solution with cell-grade DMSO under sterile conditions, sealed and stored in a -20°C refrigerator in the dark for later use. The 1 mg / mL stock solution was then diluted to 0.1 mg / mL with normal saline.

[0046] Ten 6-week-old Balb / c male nude mice weighing 18-22 g were purchased from Kunming Shuangxin Biotechnology Co., Ltd.

[0047] 2. Seven 6-week-old Balb / c male nude mice weighing 18-22g were divided into two groups: a treatment group of 6 mice and a control group of 1 mouse. They were housed in an SPF-grade animal breeding room at 23±2℃, 50-60% humidity, and a 12-h light and dark cycle per day. They were given free access to water and food. After one week of adaptation, 100μL of a diluted 0.1mg / mL Simitake vine near-infrared carbon dot solution was injected subcutaneously into the back of the nude mice. The control group was injected with the same volume of solvent (a mixture of cell-grade DMSO and saline). The experimental nude mice were anesthetized with isoflurane gas at 0.5h, 1h, 2h, 4h, 8h, and 24h, respectively. The mice were then photographed alive using a small animal in vivo imaging device. The in vivo imaging effect was observed at an excitation wavelength of 630nm and an emission wavelength of 670nm. The results are shown in Figure 2. Figure 10 Above: 2 hours after injection, a clear and strong fluorescence signal was detected throughout the mouse's body. Over time, the signal gradually weakened, reaching its weakest point at 24 hours, with bright fluorescence clearly visible throughout the mouse's body. After 24 hours, the fluorescence signal became very weak, indicating that the synthesized carbon dots were rapidly excreted from the mouse's body. In vivo imaging experiments demonstrate that the Simao Teng near-infrared carbon dots possess good biocompatibility, strong tissue penetration, and excellent imaging performance.

[0048] 3. The experimental nude mice subjected to in vivo imaging at different time points (0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h) were killed by cervical dislocation. The organs (brain, heart, lung, liver, spleen, kidney, testicle, and bladder) of each experimental nude mouse were collected and placed on a black background cardboard. The mice were then imaged in vivo using a small animal in vivo imaging device. The in vivo imaging effects were observed at an excitation wavelength of 630 nm and an emission wavelength of 670 nm.

[0049] See the results Figure 10 The figure below shows fluorescence imaging of various organs (brain, heart, lung, liver, spleen, kidney, testis, and bladder) at different time points. Following subcutaneous injection into mice, near-infrared carbon dots from Simitake vine were primarily distributed in the brain, liver, kidney, testis, and bladder, with negligible concentrations in the heart and spleen. The strong light signal gradually weakened with increasing circulation time, suggesting that they are primarily metabolized in the liver and excreted through the kidneys. The results also suggest that Simitake vine near-infrared carbon dots can penetrate the blood-brain barrier, providing guidance for further research into their potential use in the diagnosis of brain diseases.

[0050] 4. Three 6-week-old Balb / c male nude mice weighing 18-22 g were divided into two groups: a treatment group of two mice and a blank group of one mouse. They were housed in an SPF-grade animal breeding room at 23±2°C, 50-60% humidity, and a 12-h light-dark cycle. They were given free access to water and food. After one week of adaptation, 100 μL of a diluted 0.1 mg / mL Simiao Teng near-infrared carbon dot solution was injected subcutaneously into the back of the nude mice. The blank group was injected with the same volume of solvent (a mixture of cell-grade DMSO and saline). After administration, the mice were sacrificed by cervical dislocation 7 days after the injection, and their organs (brain, heart, liver, spleen, lung, kidney, testis, and bladder) were dissected and fixed in 4% paraformaldehyde fixative. Tissue sections were prepared by routine paraffin embedding and stained with hematoxylin-eosin (H&E). All sections were observed under a microscope for histopathological analysis.

[0051] See the results Figure 11 Compared with the blank group (CON-7d), the Simiogensis cathayensis near-infrared carbon dots group (CDs-7d) did not show inflammation or necrotic cell aggregation, which indicates that Simiogensis cathayensis near-infrared carbon dots will not cause damage to the brain, heart, liver, spleen, lung, kidney, testicle and bladder of nude mice.

Claims

1. Application of near-infrared carbon dots based on Simiao kauri in the preparation of biological imaging reagents, characterized by: The dried Simi kaempferia truncatula branches and leaves were crushed into powder and mixed with an organic solvent. After ultrasonication for 10-30 minutes, the ultrasonic product was transferred to a reactor, heated at 90-220°C for 3-7 hours, and then naturally cooled to room temperature. The mixture was centrifuged, and the supernatant was collected and filtered through a 0.22 μm organic phase filter membrane. The filtrate was purified by silica gel column and eluted with a petroleum ether-ethyl acetate mixture. The eluate was collected and vacuum dried to obtain Simi kaempferia truncatula near-infrared carbon dots. The organic solvent is acetone; in the silica gel column chromatography purification, the mixture is first eluted with a petroleum ether-ethyl acetate mixture in a volume ratio of 4:1, the eluate is collected, and then eluted with a petroleum ether-ethyl acetate mixture in a volume ratio of 1:3, the eluate is collected, and the eluates are combined.

2. The use according to claim 1, characterized in that: The centrifugation was carried out at 9000-10000 r / min for 10 min.

3. The use according to claim 1, characterized in that: Biological imaging is cell imaging.

4. The use according to claim 1, characterized in that: Bioimaging is the imaging of living organisms.

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