CN113548656B - Carbon dots with anticancer bioactivity and preparation method thereof - Google Patents

Abstract

The invention discloses a carbon dot and a preparation method and application thereof. The carbon dots are prepared according to a method comprising the following steps: preparing carbon dots by adopting a pulse electrolysis method or a laser irradiation method; the carbon dots have a particle size of less than 1000nm; the number of carbon atoms in the composition is more than 50%. The carbon dots have the effect of inducing apoptosis of tumor cells. Has no toxic and side effect on normal human peripheral lymphocytes. In vivo and in vitro researches prove that the material can obviously inhibit the growth of tumor cells and induce cancer cell death; in a nude mouse transplanted tumor model, the growth of the tumor is obviously inhibited, and important organs of the mouse are not obviously influenced.

Description

Carbon dots with anticancer bioactivity and preparation method thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to an anticancer active carbon dot, and a preparation method and application thereof.

Background

The diagnosis and treatment of cancer has been a major research area in all countries for many years, and although there is a constant progress and breakthrough in both anti-cancer drugs and methods, cancer is still one of the major diseases threatening human life and health. At present, the treatment means of cancer mainly comprises surgical excision, radiotherapy, chemotherapy, immunotherapy and the like, and the expected effect cannot be achieved. Surgical resection not only causes a variable degree of damage to the body, but also often does not provide a condition for surgical treatment for patients already in a state of cancer cell spread. Radiotherapy, chemotherapy, immunotherapy and the like have limited curative effects and generally have toxic and side effects in the treatment process. Therefore, in the face of increasing incidence of cancer, development of anticancer drugs with low or no toxic side effects is required.

Research literature reports that carbon dots smaller than a certain size can reach cell membranes and cytoplasm, carbon dots with different particle sizes can reach intracellular positions, and different positions of cells can be marked by carbon dots with different particle sizes. The experimental results of the research after the fusion imaging of the carbon dots and the cancer cells show that: the carbon spots were able to successfully enter the cells after incubation with the cells. After the surface of the carbon dots is specially modified, the carbon dots can be better combined with cancer cells. Current research is limited to using these properties of carbon dots to label and image cancer cells. Due to the general action characteristics of carbon spot cancer cell organs and sites, it will also have an impact on the physiological processes of the cells. However, no studies on the influence of carbon points on the biological activity of cancer cells have been reported.

Disclosure of Invention

It is an object of the present invention to provide a carbon dot having physiological activity against cancer.

The carbon dots are prepared by preparing graphite into the carbon dots through laser irradiation or an electrolytic method.

The laser irradiation method can be pulse laser irradiation; the electrolysis process may in particular be a pulse electrolysis process.

The graphite can be natural graphite or artificial graphite, and the graphite can be in a shape of a sheet, a block or a powder without limitation.

The grain diameter of the carbon dots prepared by the invention is less than 1000 nanometers, and the carbon dots are mainly grains.

The carbon dots prepared by the method have the components with carbon atom percentage of more than 50 percent. The carbon dots can contain water, other elements or functional groups (such as oxygen-containing functional groups-OH, C-O-C, etc.) inside and on the surface.

According to one embodiment of the invention, the carbon colloid solution is prepared by the following method, which comprises the following steps:

and preparing the graphite structure carbon into an electrode, immersing the electrode into water or aqueous solution containing electrolyte, adopting 20KHz-40KHz pulse current or electrochemical cyclic voltammetry scanning with average voltage of 1-10V and average current of 1-30A, and stripping carbon points from the defects of the graphite structure raw material electrode material by utilizing ionic hydrogen ions, hydroxyl ions or other ions with positive electricity or negative electricity in distilled water or drinking water to obtain the carbon point sol.

According to one embodiment of the invention, the carbon colloid solution is prepared by the following method, which comprises the following steps:

laser is used for irradiating graphite powder suspension of organic solvents such as water or ethanol and the like, the graphite powder is used as a laser irradiation target material, the laser irradiation is carried out at the interface between the target material and the solution in the solvent, and pulsed laser is used for ablating the target material to prepare the carbon dot sol.

The concentration of the graphite powder in the graphite powder suspension is 10-1000mg/l.

The laser wavelength is 532nm,10ns laser; the irradiation time is 0.5-10 hours, and continuous irradiation can also be carried out. The irradiation time is adjusted according to the amount of graphite irradiated. If the amount of irradiation is large, the irradiation time can be appropriately prolonged.

It is still another object of the present invention to provide the carbon dots and the application of the carbon dot colloidal solution.

The application provided by the invention is the application of the carbon dot and/or the carbon dot colloidal solution in the preparation of medicines for preventing and/or treating cancers or the application in the preparation of medicines for inhibiting the proliferation of cancer cells.

Such cancers include various cancers (solid or non-solid) known in the art, including but not limited to: liver cancer, osteosarcoma, lymph cancer, myeloma, cervical cancer, colon cancer, non-small cell lung cancer, breast cancer, esophageal cancer, and leukemia.

The cancer cells comprise liver cancer cells, osteosarcoma cells, lymphoma cells, myeloma cells, cervical cancer cells, colon cancer cells, non-small cell lung cancer cells, breast cancer cells, esophageal cancer cells and leukemia cells.

The liver cancer cell can be a human liver cancer HepG2 cell; the osteosarcoma cell can be osteosarcoma 143B cell.

The medicament for preventing and/or treating cancer, which is prepared by taking the carbon dots and/or the carbon dot colloidal solution as the active ingredient, also belongs to the protection scope of the invention.

The agent for preventing and/or treating cancer can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spray, nasal drop, eye drop, penetration, absorption, physical or chemical mediated method; or mixed or coated with other materials and introduced into body.

If necessary, one or more pharmaceutically acceptable carriers can be added into the medicine. The carrier includes diluent, excipient, filler, binder, wetting agent, disintegrating agent, absorption enhancer, surfactant, adsorption carrier, lubricant, etc. which are conventional in the pharmaceutical field.

The above medicine can be made into various forms such as injection, tablet, powder, granule, capsule, oral liquid, paste, cream, etc. The medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field.

The invention has the advantages that: the carbon dots are prepared from simple materials, are low in manufacturing cost and fast in synthesis, have the advantages of good water solubility, low biotoxicity, good biocompatibility, environmental friendliness and the like, are fast in migration in cells due to small particle size, can be discharged out of the body through kidneys, and do not show toxic reaction in the whole experimental process. The invention has a plurality of advantages, thus having wide application prospect in the field of anticancer.

In vivo and in vitro experimental studies prove that the carbon dots can obviously inhibit the growth of tumor cells and induce cancer cell death, and obviously inhibit the growth of tumors in a nude mouse transplanted tumor model, but have no obvious influence on important organs of mice, and can provide important protection on the contrary. Has no toxic and side effect on normal human peripheral lymphocytes; the medicament is taken by a large number of patients with various terminal tumors in a voluntary form, and a good clinical effect is achieved.

The research shows that the inhibition mechanism of the compound on cancer cells is as follows: in vitro cell experiments can show that the carbon dots show time-dose dependent cytotoxicity to tumor cells with different degrees of malignancy, which indicates that the carbon dots can cause the death of the tumor cells through a certain way, and the carbon dots can induce the apoptosis of the tumor cells through the classical mitochondrial apoptosis pathway by combining the existing research on the carbon dots (figure 4).

Drawings

FIG. 1 is a transmission electron microscope photograph of carbon dots prepared by graphite pulse electrolysis

FIG. 2 is a graph of carbon point infrared absorption spectrum (Fourier infrared spectrum) prepared by graphite pulse electrolysis.

FIG. 3 is the Emission spectrum of carbon dot Emission wavelet prepared by pulse electrolysis.

FIG. 4 is a carbon point Excitation wavelet Excitation spectrum prepared by a pulse electrolysis method.

FIG. 5 is a transmission electron micrograph of a carbon dot prepared by a graphite pulse laser irradiation method

FIG. 6 is a carbon dot infrared absorption spectrum (Fourier infrared spectrum) prepared by graphite pulse laser irradiation.

FIG. 7 shows an Emission spectrum of carbon dots Emission wavelet prepared by pulsed laser irradiation.

FIG. 8 is a carbon spot Excitation wavelet Excitation spectrum prepared by a pulsed laser irradiation method.

FIG. 9 influence of concentration and treatment time of carbon quantum dot sol on in vitro cytotoxicity of human hepatoma cells (HepG 2).

FIG. 10 shows a carbon-dotted mitochondrial apoptotic pathway prepared by pulse electrolysis.

FIG. 11 is a CCK-8 method for determining cytotoxicity of carbon dots prepared by a pulse electrolysis method to HepG2 cells.

FIG. 12 shows the CCK-8 method for determining cytotoxicity of 143B cells with respect to carbon dots prepared by the pulse electrolysis method.

FIG. 13 is a photograph of pathological changes (HE staining) of each mouse tissue after gastric perfusion of carbon spots prepared by pulse electrolysis.

Detailed Description

The present invention is described below by way of examples, but the present invention is not limited thereto, and any modifications, equivalents, and improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

Example 1 preparation of carbon dots by graphite pulse Electrolysis and solution containing carbon dots of Panax ginseng

Preparing an electrode by graphite, immersing the electrode in water, and peeling off carbon points from the defects of the graphite structure raw material target by using the vibration action of water ions by using 20KHz pulse current with the average voltage of 5V and the current variation range of 3-10A to prepare carbon point sol with the particle size of less than 1000nm;

FIG. 1 is a transmission electron micrograph of carbon dots prepared by graphite pulse electrolysis, wherein the carbon dots are spherical, have a diameter of less than 10nm, and are mostly monodisperse. Most carbon dots have diameters ranging from 5 to 10nm, as measured. FIG. 2 is a Fourier transform infrared spectrum of carbon dots prepared electrochemically. The atoms constituting the chemical bond or the functional group are in a constantly vibrating state, and the vibration frequency thereof is equivalent to that of infrared light. Therefore, when the organic molecules are irradiated by infrared light, the chemical bonds or functional groups in the molecules can absorb vibration, different chemical bonds or functional groups have different absorption frequencies and are located at different positions on the infrared spectrum, and therefore information about which chemical bonds or functional groups are contained in the molecules can be obtained. 3436cm as shown in FIG. 2 ^-1 The peak of the stretching vibration at the hydroxyl (-OH) position is 2956cm ^-1 And 1453cm ^-1 Is the vibration peak of C-H, 1629cm ^-1 The absorption peak at (A) indicates the presence of amide groups 1045cm ^-1 The absorption signal at (A) indicates the presence of a C-O-C functional group, 538cm ^-1 And (b) represents a bending vibration of C-C = O. C = O telescopic vibration at 1714cm-1Peak 1253cm ^-1 The absorption peak at (A) indicates the presence of a C-O functional group, 866cm ^-1 And (b) indicates the presence of C-C stretching vibration. Therefore, the surface of the carbon dot has a plurality of oxygen-containing functional groups (such as-OH, C-O-C and the like), and the oxygen-containing functional groups enable the carbon dot to have good hydrophilicity and stability in water.

Fig. 3 is an Emission spectrum of Emission Wavelength of a carbon dot under the condition that the Wavelength of the excitation light is not changed, which is also called a fluorescence spectrum. The fluorescence emitted by the sample is measured at different wavelengths, the wavelength being plotted on the abscissa and the intensity F on the ordinate. Fluorescence spectrum of carbon dots at an excitation wavelength of 325 nm. It can be seen that the maximum emission wavelength of fluorescence at this excitation wavelength is about 460nm.

FIG. 4 is a carbon site Excitation wavelet Excitation spectrum with a fluorescence emission Wavelength kept constant. The fluorescence intensity F obtained under excitation by excitation light of different wavelengths was measured. Then, the excitation light wavelength is plotted on the abscissa and the fluorescence intensity F is plotted on the ordinate, and the excitation spectrum of the fluorescent substance is obtained. The wavelength corresponding to the maximum value of the fluorescence intensity in the excitation spectrum is the maximum excitation wavelength, and is the most sensitive wavelength for exciting fluorescence. FIG. 3 is an excitation spectrum of nanoparticles at an emission wavelength of 400nm, and it can be seen that the maximum excitation wavelength is about 390nm. It can be seen that the nanoparticles have photoluminescence.

Substances that are generally fluorescent contain a structural system of conjugated double bonds (pi-bonds) in their molecules, since conjugated systems contain delocalized pi-electrons that are easily excited. The larger the conjugated system is, the larger the delocalization of the electron is, the easier it is to excite, and the stronger the fluorescence intensity is. As can be seen from fig. 3 and 4, the particles have photoluminescence, and in conjunction with the conclusion of fig. 2, the molecules of the fluorescent substance are in an excited state and are extremely unstable after absorbing the energy of the exciting light under specific conditions, and when they rapidly return to the ground state, all the light energy is released as electromagnetic radiation. Fluorescent substances contain a strongly absorbing group such as a conjugated double bond in the molecule, and the larger the conjugated system is, the stronger the delocalization of pi electrons is, and the more easily it is excited to generate fluorescence. The substituent of the fluorescent substance has a large influence on the fluorescence intensity. Electron donating substituents (such as-OH) increase the conjugation system, resulting in increased fluorescence. Whereas electron-withdrawing substituents such as (-COOH) attenuate fluorescence. As can be seen from FIG. 1, the particle surface contains-OH and-COOH, and these groups interact with each other to possibly make the fluorescence intensity of the particles not high.

Example 2 preparation of carbon dots by pulsed laser irradiation and solution containing carbon dots

The carbon dots prepared by the pulse laser irradiation method are similar to the carbon dots prepared by the pulse electrolysis method.

The preparation method comprises the following steps: irradiating 30L of graphite powder ethanol suspension (the concentration is 500 mg/L) by using laser, using the graphite powder as a laser irradiation target material, irradiating the laser at the interface between the target material and the solution in a solvent, and melting the target material by using pulse laser to prepare the carbon dot sol.

The laser wavelength is 532nm,10ns laser; the irradiation time was 1 hour.

FIG. 5 is a transmission electron micrograph of carbon dots prepared by pulsed laser irradiation, wherein the carbon dots are spherical, have a diameter within 10nm, and are mostly monodisperse. Most carbon dots have diameters ranging from 5 to 10nm, as measured. FIG. 6 is a Fourier infrared spectrum of carbon dots prepared by a pulsed laser irradiation method. 3439cm as shown in FIG. 6 ^-1 The peak of the stretching vibration at the hydroxyl (-OH) position is 2923cm ^-1 、1443cm ^-1 、860cm ^-1 And 625cm ^-1 Is the C-H vibration peak at 1629cm ^-1 The absorption peak at (a) indicates the presence of an amide group. 1725cm ^-1 Expansion vibration peak at C = O, 1235cm ^-1 The absorption peak at (a) indicates the presence of a C-O functional group. The surface of the obtained carbon dot has a plurality of oxygen-containing functional groups (such as-OH, C = O and the like), and the oxygen-containing functional groups enable the carbon dot to have good hydrophilicity and stability in water.

Fig. 7 is an Emission spectrum of Emission Wavelength, also called fluorescence spectrum, of a carbon dot with a constant excitation light Wavelength. The fluorescence emitted by the sample is measured at different wavelengths, the wavelength being plotted on the abscissa and the intensity F on the ordinate. Fluorescence spectrum of carbon spot at excitation wavelength of 325 nm. It can be seen that the maximum emission wavelength of fluorescence at this excitation wavelength is about 440nm.

FIG. 8 shows an Excitation spectrum of carbon site Excitation Wavelength with the fluorescence emission Wavelength kept constant. Excitation spectra of nanoparticles at 400nm emission wavelength, it can be seen that the maximum excitation wavelength is about 470nm. It can be seen that the nanoparticles have photoluminescence. As can be seen from fig. 7 and 8, the particles have photoluminescence, and in conjunction with the conclusion of fig. 6, the particle surface contains-OH and-COOH.

Example 3 in vitro efficacy test of carbon dots prepared by pulsed Electrolysis (example 1)

The carbon toxicity of human hepatoma cells (HepG 2) was evaluated by Cell Counting Kit-8 (CCK-8) and flow cytometer CCK-8 as shown in FIG. 9. After 72h of treatment, the C2 and C3 groups exhibited cytotoxicity higher than the other groups, while the low concentration group (C1 group) exhibited insignificant toxicity over the test period. The results show that the carbon dot sol has obvious toxicity to tumor cells under certain conditions (C2 and C3 treatment for 72 h). The effect of sol concentration on cytotoxicity was further verified by optical microscope observation (fig. 9 (b) - (d)). Figure 9 (a) effect of carbon dot sol concentration (C1 =69 μ gmL-1, C2=138 μ gmL-1, C3=276 μ gmL-1) and treatment time (24, 48, 72 h) on in vitro cytotoxicity of human hepatoma cells (HepG 2). Error bars indicate standard deviation (n = 3). * P <0.05, compared to control. Representative optical micrographs of HepG2 after incubation with carbon spot sols of varying concentrations for 72 hours. (b) a control group, (C) C2 and (d) C3. The data show that HepG2 cell activity is inhibited and that sol concentration and time can have an effect on this inhibition, validating the in vitro potency of the carbon dots prepared by the pulse electrolysis method (example 1).

Example 4 in vivo efficacy verification of carbon dots prepared by pulse electrolysis (example 1)

Preparing 143B human osteosarcoma cells into 1 × 10 cells ⁷ Sucking 0.2mL of cell suspension by using a 1mL syringe, injecting the cell suspension into the back subcutaneous side of the sterilized nude mouse, ensuring that the needle has penetrated the whole cortex of the nude mouse and does not enter the muscular layer and the abdominal cavity, and slightly clamping the cell suspension for 1min by using sterile forceps after the cell suspension is injected into the nude mouse, so as to prevent the cell suspension from leaking. Each nude mouse is injected with subcutaneous single injection at the dorsal side of the right hind limb until tumors at the injection site of the nude mouse are obviously formed (100 mm) ³ )。

Randomly dividing 30 BALB/c tumor-bearing nude mice into 2 groups, each group comprises 15 mice, and the experimental group comprises high-concentration 276 microgram/milliliter carbon point (CNC); control group was 0.9% physiological saline. The medicine is infused into the stomach for 0.5ml in each group, and the medicine is infused into the stomach for 30 days continuously 1 time every day according to the above groups, and the growth condition of the tumor is observed regularly.

In vivo animal experiments by gavage of carbon spots in normal mice showed: carbon dots lead to tumor apoptosis via the mitochondrial apoptotic pathway (as shown in figure 10). The carbon dots have no obvious influence on the weight, blood routine, biochemical indexes and important organ pathology of the mice (figure 13); a significant reduction in tumor volume was seen by gastric perfusion of the carbon spots in osteosarcoma nude mice (see Table 1). We speculate that carbon-point antibodies not only have direct antitumor properties, but also may be antitumor through immune regulation.

TABLE 1 influence of gastric perfusion carbon point on tumor volume change in nude mice

(. Compared with the control group P < 0.05)

And selecting a human liver cancer HepG2 cell line with lower malignancy degree to verify the action of the carbon point on osteosarcoma cells. The CCK-8 method was chosen to determine the cytotoxicity of carbon points towards HepG2 cells. It can be seen from fig. 11 that, as the concentration of carbon dots increases and the time elapses, the carbon dots at three concentrations all show significant proliferation inhibition to 143B cells at 72 hours, and the high concentration effect is the best. The carbon point toxicity to tumor cells was seen to be time-dose dependent.

And selecting an osteosarcoma 143B cell line with higher malignancy degree to verify the effect of the carbon point on osteosarcoma cells. The CCK-8 method was chosen to determine the cytotoxicity of carbon spots on 143B cells. As can be seen from fig. 12, the high-concentration carbon spot showed significant proliferation inhibition of 143B cells at 48h and 72h as the carbon spot concentration increased and the time passed. The toxicity of the carbon dots to tumor cells was also seen to be time-dose dependent.

Claims (2)

1. The application of the carbon dots in the preparation of medicaments for preventing and/or treating cancers;

the cancer is liver cancer or osteosarcoma;

the preparation method of the carbon dots comprises the following steps: preparing graphite into carbon dots by adopting a laser irradiation method or a pulse electrolysis method;

the specific method for preparing the carbon dots by adopting the pulse electrolysis method comprises the following steps:

immersing graphite electrode into water or aqueous solution containing electrolyte, adopting 10k Hz-60k Hz pulse current or electrochemical cyclic voltammetry scan with average voltage of 1-50V and average current of 1-30A, and stripping carbon points from the defects of the graphite structure raw material electrode material by utilizing ions in water to prepare carbon point sol;

the specific method for preparing the carbon dots by adopting the laser irradiation method comprises the following steps:

irradiating graphite powder suspension of water or an organic solvent by using laser, taking the graphite powder as a target material, irradiating the interface between the target material and the solution in the solvent by using the laser, and ablating the target material by using pulse laser to prepare carbon dot sol;

the laser wavelength of the pulse laser is 532nm and 10 ns; the irradiation time is 1-10 hours;

the grain diameter of the carbon dots is less than 1000 nanometers, and the carbon dots are mainly particles of carbon elements.

2. The application of the carbon dots in the preparation of the drugs for inhibiting the proliferation of cancer cells;

the cancer cell is a liver cancer cell or an osteosarcoma cell;

the preparation method of the carbon dots comprises the following steps: preparing graphite into carbon dots by adopting a laser irradiation method or a pulse electrolysis method;

the specific method for preparing the carbon dots by adopting the pulse electrolysis method comprises the following steps:

immersing graphite electrode into water or aqueous solution containing electrolyte, adopting 10k Hz-60k Hz pulse current or electrochemical cyclic voltammetry scan with average voltage of 1-50V and average current of 1-30A, and stripping carbon points from the defects of the graphite structure raw material electrode material by utilizing ions in water to prepare carbon point sol;

the specific method for preparing the carbon dots by adopting the laser irradiation method comprises the following steps:

irradiating graphite powder suspension of water or an organic solvent by using laser, taking the graphite powder as a target material, irradiating the interface between the target material and the solution in the solvent by using the laser, and melting the target material by using pulse laser to prepare carbon dot sol;

the laser wavelength of the pulse laser is 532nm and 10 ns; the irradiation time is 1-10 hours;

the grain diameter of the carbon dots is less than 1000 nanometers, and the carbon dots are mainly particles of carbon elements.

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