TW201534321A - Carbon nanodots, method for synthesizing the same and use of producing pharmaceutical agents for treating liver tumor - Google Patents

Abstract

Carbon nanodots and method for synthesizing the same, wherein the method at least comprises the following steps: providing a ginger juice, heating the ginger juice with a predetermined temperature for a predetermined time period to make the ginger juice carbonized and transformed to carbon nanomaterials containing carbides; centrifuging the carbon nanomaterials containing the carbides to obtain a supernatant; and after filtrating the supernatant, dialyzing the supernatant to obtain a solution of carbon nanodots. In addition, each one of the carbon nanodots synthesized by the above method residues in some curcumin molecules, exhibiting a growth inhibition effect to liver cancer cell. Accordingly, use of producing pharmaceutical agents for treating liver tumor can also be derived from the same.

Description

Carbon nano point and its synthesis method and its use as a medicine for treating liver tumors

The invention relates to a carbon nano point and a synthetic method thereof, and a use as a medicine for manufacturing a liver tumor, in particular to a carbon nano point obtained by synthesizing ginger as a carbon source, and a synthetic method thereof The use of carbon nano-dots as a medicine for the treatment of liver tumors.

A carbon nano-dots are one type of quantum dots. In general, carbon nano-dots have photoluminescent properties that can be excited by light of different wavelengths to make the carbon nano-dots themselves different. Fluorescent color. Moreover, carbon nano-dots are also widely used in bio-imaging systems and their related industries, such as high water solubility, quantum yield and light stability. Compared to other highly toxic quantum dots, such as quantum dots derived from toxic heavy metals such as cadmium (Cd ²⁺ ) and lead (Pb ²⁺ ), carbon nanotubes have relatively low toxicity and high levels of biology. Compatibility, so it is more suitable for biological applications. However, the biological application of carbon nano-points is currently only used for development and detection purposes. In fact, the biological aspects of carbon nano-dots Application, yet to be further developed, as well as anti-cancer use, but currently anti-cancer is no more than the use of surgical resection, radiation or target drugs and other chemotherapy, each with its medical high risk, and during the course of treatment It will also bring many side effects to patients, increase the pain, and later advocate the use of natural drugs (or herbs) to fight cancer, but it has many problems and bottlenecks to overcome in the development of dosage forms and the extraction of active ingredients. The development of drugs based on natural medicines as an anti-cancer has been slow to enter the stage of maturity.

Moreover, conventional methods for fabricating carbon nanodots can be divided into two major types: chemical and physical methods. Chemical methods mainly include: microwave pyrolysis, combustion routes, and electrochemical oxidation. Physical methods include laser ablation, arc discharge, high-energy ion beam radiation, and plasma treatment. However, the steps of the above methods are more complicated, and the technical difficulty is high, and it is difficult to control the quality of the manufactured carbon nano-dots. In particular, when using the above methods, it takes a lot of money to purchase expensive instruments, or needs to be used. A large number of unfavorable environmental oxidative acids. Even in order to make the carbon nano-dots have photoluminescence characteristics, it takes a long time to wait in the manufacturing process, and sometimes it is necessary to add surface passivating agents or additionally modify the carbon nano-dots themselves. During this long waiting period, it still needs to consume a lot of energy continuously. Therefore, it can be understood that the conventional carbon nano-point has room for improvement in terms of process simplification, cost reduction or environmental protection.

In addition, natural plant components have many anti-cancer or tumor-inhibiting components, such as catechin or curcumin. For example, curcumin is known to induce apoptosis and promote liver cancer cells. Compounds that die, but their poor solubility in water also lead to difficulties in formulation development, and their active ingredients are more difficult to enter the lesion. In 2006, J. Cao et al., in Toxicological Sceince, published the results of inhibition of curcumin on HepG2 cell line (Human hepatocellular liver carcinoma cell line), and the results showed that the action time range was 24 hours, 48 hours and 72 hours. Under the condition of an hour, the minimum inhibitory concentration (50%, IC ₅₀ ) of curcumin on HepG2 needs to be as high as 31.29±0.90μg/ml, 22.36±1.45μg/ml and 16.43±1.81μg/ml, respectively. The concentration can be reached. Curcumin is mostly used in the preparation of silica gel column chromatography. This method is technically difficult and difficult to handle, and requires a large amount of organic solvent to be used for eluting and recrystallization of curcumin. Very inconsistent with cost and environmental benefits.

The reason is that the present inventors have felt that the above problems can be improved, and that the present invention has been deliberately studied and used in conjunction with the theory, and a present invention which is reasonable in design and effective in improving the above problems has been proposed.

The main object of the present invention is to provide a carbon nano point and a manufacturing method thereof, so as to improve the problems of high cost, complicated process and unfavorable environmental protection during the manufacturing process of the carbon nano point. Carbon nano-dots are derived from anti-cancer applications to provide another new technological solution for today's anti-cancer strategy.

To achieve the above object, the present invention provides a method for producing a carbon nano point, comprising at least the following steps: (A) providing a ginger juice, heating the ginger juice at a predetermined temperature for a predetermined time to carbonize the ginger juice, And forming a carbon nanomaterial containing carbide; (B) centrifuging the carbide-containing carbon nanomaterial to obtain a supernatant; and (C) filtering the supernatant, and then performing the supernatant Dialysis treatment to obtain a carbon nano point suspension solution.

To achieve the above object, the present invention provides a carbon nano point comprising at least: a plurality of curcumin molecules bound to a surface of the carbon nano point.

In order to achieve the above object, the present invention also provides a use of a carbon nano point as a medicine for treating a liver tumor, wherein the carbon nano point comprises at least: a plurality of curcumin molecules bound to the carbon nano point, wherein the The carbon nano point is dissolved in an aqueous carrier.

In summary, the present invention can achieve the advantages of greatly reducing the cost required for manufacturing a carbon nano point, simplifying the process, and taking into consideration environmental protection advantages by the above-described manufacturing method, and by the above-mentioned manufacturing method, The carbon nano-dots produced/synthesized by the carbon source have been naturally combined with curcumin molecules on the surface at the time of manufacture, and are not lost during dialysis, and exhibit anti-cancer which inhibits tumor growth. The activity, apparently by the above-mentioned manufacturing method, the carbon nano-dots produced, with unpredictable anti-cancer activity, can be another important option in today's medical community to improve the high risk of currently known anti-cancer therapies, Shortcomings such as high side effects.

For a better understanding of the features and technical aspects of the present invention, reference should be made to the accompanying drawings.

1 is a flow chart showing the steps of a method for producing a carbon nano point of the present invention; FIG. 2A is a transmission electron microscope image of a carbon nano point of the present invention; and FIG. 2B is a carbon nano point of the present invention at an excitation wavelength of 325 nm to Fluorescence spectroscopy spectrum between 445 nm; FIG. 2C is a map of the results of using the Fourier transform infrared spectrometer to detect functional groups present on the carbon nano-dots of the present invention; FIG. 2D is an example using X-ray spectrometer Figure 3A is an X-ray diffraction pattern of a carbon nano-point of the present invention; Figure 3B is a UV-Vis absorption spectrum of a carbon nano-point of the present invention; Figure 4A The result of the X-ray photoelectron spectroscopy of the 1s orbital domain of the carbon of the carbon nano-point of the present invention; FIG. 4B is a result of the Raman spectrum of the carbon nano-dots of the present invention; FIG. 4C is the carbon nano-point of the present invention; Figure ^13A is a graph showing the results of fluorescence luminescence stability of the carbon nano-dots at pH values of 3, 4, 5, 6, 7, 8, 9, 10; Figure 5B The result of the fluorescence light emission stability of the carbon nano point of the present invention at a sodium chloride concentration of 0, 100, 200, 300, 400, 500 mM; FIG. 5C is a carbon nene of the present invention; Figure 5 is a graph showing the results of fluorescence luminescence after 0 to 360 minutes of ultraviolet light irradiation; Figure 6 is a carbon nano point (1.11 mg/mL) added to A549, HeLa, HepG2, MCF10A and MDA. The results of the bright field (field A) and the fluorescent image (field B) exhibited by the four cell lines of MB-231 at 4 hours; Fig. 7A shows the carbon nano spot (0-2.8 mg/) of the present invention. mL) results of cell viability measured after 24 hours of action of five cell lines added to A549, HeLa, HepG2, MCF10A and MDA-MB-231; Figure 7B shows the carbon nano-dots of the present invention (0-2.8) Mg/mL) results of the western blot method of p53 expression measured after 24 hours of action on five cell lines of A549, HeLa, HepG2, MCF10A and MDA-MB-231; FIG. 7C is the carbon of the present invention Nano-point (0-2.8mg/mL) was added to the cell cycle distribution of the five cell lines of A549, HeLa, HepG2, MCF10A and MDA-MB-231 after 24 hours; Figure 8 is at different concentrations The ginger cell juice was added to the cell viability of the five cell lines of A549, HeLa, HepG2, MCF10A and MDA-MB-231 for 24 hours; Figure 9 is compared with the carbon nanoparticle of the present invention. Tumor effect In addition, the carbon nano-dots made by EDTA, glycine and green tea were added to HepG2 cells, and the cell viability was measured after 24 hours. Figure 10 shows the analysis of A549, HeLa, HepG2, and MCF10A by image cytometry. And the cellulite effect (horizontal axis) and ROS production (vertical axis) of the five cell lines of MDA-MB-231 on the carbon nano-point material, wherein the left A column is the control group without the carbon nano-dots The B column on the right is the experimental group adding 1.11 mg/mL carbon nano-dots; Figure 11 is the SALDI mass spectrum measured at the concentration of 0.011 mg/mL of the carbon nano-dots of the present invention, wherein the smaller insertion map is prediction [curcumin + H] ⁺ isotope pattern; FIG. 12A is 10 ⁷ cells were seeded in HepG2 tumors in mice dorsal formed, respectively, and at 7 and 14 days, the carbon nano dot 220μg (440 μg in total) was injected into the tumor, the arrow pointed to the location of the injection; Figure 12B shows the diameter measurement of the tumor taken on the 16th day after inoculation of HepG2, and the left half shows the control of the uninjected carbon nanopore Group, the right half is the experimental group with the injected carbon nano-dots; and Figure 12C is the tumor taken on the 16th day after the inoculation of HepG2 Tumor weighing result of the histogram.

Referring to FIG. 1 , the present invention provides a method for manufacturing a carbon nano point, comprising at least the following steps: Step S101: providing a ginger juice, heating the ginger juice at a predetermined temperature for a predetermined time to carbonize the ginger juice. And forming a carbon nanomaterial containing carbide, wherein preferably, the predetermined temperature may be 300 degrees Celsius, and the predetermined time may be 120 minutes. In this demonstration, a calciner is used to perform the above-mentioned Heating step.

Step S103: The carbide-containing carbon nanomaterial is centrifuged to obtain a supernatant, and the condition of centrifugation may be 12000 g for 10 minutes to remove large residue particles generated after calcination.

Step S105: The supernatant can be filtered by a filter membrane having a pore size of 0.1 micrometer (μm) to 0.22 micrometer, and then the filtered supernatant is dialyzed to prepare a carbon nano point suspension solution, wherein the dialysis conditions and details are as follows. The dialysis method is to collect the filtered supernatant into the dialysis tube (in order to increase the ratio of the carbon nano-dots contained in the supernatant after dialysis and the amount of curcumin it is combined, the dialysis used in the dialysis tube) Membrane, its preferred molecular weight cut-off (MWCO) can be 3.5-5kD, Float-A-Lyzer G2, Spectral Laboratory, Rancho Dominguez, California, USA), and let the whole The dialysis tube containing the ginger juice supernatant is placed in an ultrapure water of about 2 liters (Ultra-pure water definition: generally refers to the removal of the main impurities in the water by ion exchange resin, activated carbon, and membrane method, so that it is at 25 ° C The resistance value at a time of 18.2 million ohms ^* cm was carried out for two hours of dialysis (replacement of ultrapure water every half hour) to obtain the above-mentioned carbon nano point suspension solution. The carbon nano point suspension solution can then be freeze-dried for about 24 hours to obtain a carbon nano point of about 11.1 mg.

Please refer to the larger graph in Fig. 2A for the observation of the transmission electron microscope image of the carbon nano-dots. The particle size of the carbon nano-dots is determined by a transmission electron microscope (TEM, model: H7100, Hitachi High-Tech Co., Ltd., Tokyo, Japan) to carry out the measurement. Before the measurement, the manufactured carbon nano-dots (11.1 mg/ml) were diluted 10-fold, and the diluted carbon nano-dots were deposited on a copper mesh coated with 400-mesh carbon particles. On the top, the excess solvent is dried after the deposition is completed. For the observation of the particle size of the carbon nano point. As can be seen from the larger graph of FIG. 2A, the carbon nano point of the obtained carbon nano point suspension solution is spherical and uniformly dispersed in the solution, and the diameter (particle diameter) of the carbon nano point is about 3 From the rice to 8 nm, the result of the lattice structure (pitch d is 0.34 nm) observed by the high-resolution transmission electron microscope of the smaller image inserted in Fig. 2A can be further compared with Fig. 3A. As a result of the X-ray diffraction of the carbon nano-point, the peak near the diffraction angle of about 23.5 degrees shows that the carbon nano-point is the 002 plane (002 plane), which is consistent with the results of the past literature.

Please refer to FIG. 2B again, which is a fluorescence emission spectrum of a carbon nanometer at an excitation wavelength of 325 nm to 445 nm, which is a fluorescence spectrometer using Cary Eclipse (Varian, California, USA). ), the fluorescence intensity of the produced carbon nano-dots was measured. The horizontal axis of Fig. 2B is the wavelength of the excitation light given to the carbon nano-dots, referred to as the excitation wavelength, in nanometers, showing that the carbon nano-dots have a light-emitting property that can be excited by photons to emit fluorescence, at 325 nm. At the excitation wavelength range of 445 nm, the carbon nano-dots produce blue to green fluorescence, while at an excitation wavelength of 325 nm, the carbon nano-dots exhibit the strongest fluorescence intensity. And as the excitation wavelength increases, the wavelength of the fluorescent light emitted by the carbon nano-point will gradually drift to the long-wavelength band, and gradually reduce its fluorescence intensity. Therefore, the results of the above-mentioned electron microscope observation and the qualitative analysis of the fluorescence emission characteristics, the results of the particle size range and the fluorescence emission characteristics, prove that the method of the present invention can be used as a raw material for the production of carbon nanotubes. point. In addition, the quantum yield Q of the manufactured carbon nano-dots is calculated by the following formula: Q = Q _R (I / I _R ) (OD _R / OD) (n ² / n ² _R ), using Cary Eclipse The fluorescence spectrometer measures and calculates, firstly, quinine sulfate as a reference, wherein Q _R is the quantum yield reference value obtained by looking up the quinine sulphate: = 0.54 as a reference to the reference, and 0.54 into Q _R, Q _R = corresponds to the value 0.54, quinine sulfate absorbance (optical density, OD _R) light at a wavelength of 365 nm can be 0.1 or less [ At this time, quinine sulfate is dissolved in 0.1M sulfuric acid, and the n _R (refractive index) value of quinine sulfate is 1.33], and the above 0.54, 1.33, and 0.1 are respectively brought into Q _R , n _R , OD _R in the above formula. And I _R and I are respectively the integral area/integral intensity of the fluorescence of quinine sulfate and carbon nano-dots, which can be measured and utilized by Cary Eclipse fluorescence spectrometer under the excitation of 365 nm wavelength light. Origin 6.0 software performs integral calculations. In addition, the UV-Vis absorption spectrometer was used to measure the OD value of the carbon nano-dots at a wavelength of 365 nm. The n value can be found from the literature. Finally, the quantum yield Q of the carbon nano-point is about 0.134 ( 13.4%).

Please refer to FIG. 2C again, which is a map using a Fourier transform infrared spectrometer to detect the functional groups present on the carbon nano-dots of the present invention, using an ultraviolet-visible spectrometer (Cintra 10e, Dandenong, Victoria). , Australia) to measure the absorption of carbon nano-dots. The horizontal axis of the graph indicates the wavenumber of the infrared light used, and the unit is cm ^-1 . The wave number of the infrared light can be regarded as the frequency corresponding to the infrared light; the vertical axis of the figure indicates For transmittance, the unit is %, in other words, when the transmittance decreases, the carbon nano-dots absorb the infrared light at the infrared wave number/frequency. It can be seen from the map that the surface of the carbon nano-dots of the present invention has: OH/NH (3400 cm ^-1 ), CH (3000 cm ^-1 ), C=O (1700 cm ^-1 ), CO (1100 cm ^-1 ), and CH ₂ (1400 cm). ^-1 ) and other groups. In addition, the measured zeta potential value (about -35 mV) can also confirm the hydrophilic groups such as the -OH group and the -COOH group which are indeed present on the surface of the carbon nano-point, and therefore, further, the carbon of the present invention The surface of the nano-dots may have such hydrophilic groups, which may contribute to maintaining good dispersibility of the carbon nano-dots of the present invention in a carrier of a general aqueous solution, and help to avoid aggregation between carbon nano-dots. Larger particles, so that they lose the characteristics of nanocrystallization, the characteristics of nanocrystallization also contribute to the biological application of carbon nano points.

Please continue to refer to Figure 2D, which is the result of using the X-ray spectrometer to measure the four binding energies of the carbon nanotubes. The Varian 640 FT-IR spectrometer (Varian, USA) was used to analyze the possible presence of carbon naphthalene. Functional group of rice dots. The horizontal axis in the figure indicates the binding energy when the carbon atoms are bonded to other atoms, and the unit is electron volt (eV); and the vertical axis indicates the intensity/counts of the X-ray photoelectrons used. The ratios of carbon (C), oxygen (O) and nitrogen (N) are 0.72, 0.17 and 0.11, respectively. In addition, please cooperate with the results of the 1s X-ray photoelectron spectrometer of carbon of Figure 4A, which shows that the carbon nano-dots of the present invention have four main binding energies: 284.7, 286.5, 287.3, and 288.5 eV, respectively. The bonded form of the corresponding carbon is as follows: C=C/CC, CO, C=O, and C-NH _x .

Please refer to FIG. 4B again, which is a result of the Raman spectrum of the carbon nano-point, and the horizontal axis of the figure represents the Raman shift, and the unit is the wave number (cm ^{- 1);} the vertical axis represents intensity (intensity), an arbitrary unit (arbitrary unit, au), at a position where the frequency of 1337cm ^-1 and 1562cm ^-1, respectively, on behalf of SP ³ hybrid (SP ³ -hybridized) of D band (band) representing SP ² hybrid (SP ² -hybridized) present in the G band, where D represents the strip crystal defect level carbon atoms; G band represents the degree of crystallization carbon atoms, From the ratio of the relatively strong cross between the D strip and the G strip (ID/IG = 1.20), it is known that the carbon nanopoint of the present invention has a structure similar to graphene.

Please refer to Figure 4C, which is a spectrum of ¹³ C NMR analysis of the carbon nano-dots using ¹³ C NMR spectroscopy (AVIII-500 MHz FT-NMR, Bruker, Reinstetten, Germany) To measure the form of carbon hybridization of carbon nano-dots. Carbon nano dot may be learned by NMR analysis after, in the range 90-180ppm of 8-80ppm represent aliphatic (Aliphatic) carbon atoms (SP ³ hybrid, SP ³ -hybridized) and unsaturated (SP ² hybrid , SP ² -hybridized). In addition, the signal in the interval of 170-185 ppm mostly represents the presence of a carboxyl group (-COOH) or an amine group.

Please refer to FIG. 5A, FIG. 5B and FIG. 5C, which are the results of the fluorescence intensity test of the carbon nano point at different pH values, sodium chloride concentration and illumination time, respectively, as shown in FIG. 5A. The rice dots were placed in 5 mM phosphate buffers with a pH of 3.0 to 11.0. It was found that the carbon nanotubes produced by the method of the present invention maintained their fluorescing between pH 3.0 and pH 10.0. Stability of light intensity. As shown in FIG. 5B, the carbon nano point is disposed in a phosphate buffer solution (phosphate concentration: 5 mM, pH 7.4) containing 0 to 500 mM sodium chloride, and it can be understood that the carbon nano point of the present invention is Stable fluorescence emission characteristics are maintained in low salt or high salt environments. As shown in Fig. 5C, the carbon nano-dots were placed in a 5 mM phosphate solution having a pH of 7.4, and the carbon nano-dots were continuously irradiated with ultraviolet light of 6 watts (6 W/cm ² ) per square centimeter. Up to 6 hours, and with the Cary Eclipse fluorescence spectrometer to determine the stability of the fluorescence intensity of the carbon nano-point, you can find the fluorescence intensity of the carbon nano-dots after six hours of ultraviolet light irradiation. Only a slight weakening (about 8% reduction) shows that the carbon nano-dots of the present invention can withstand different pH changes, adapt to high and low salt environments, and have high stability to withstand reticle.

Please refer to FIG. 6 , which is a result of the bright field (A) and the fluorescent image (B) of the carbon nano-points among the five cell lines, wherein the five cell lines used are: normal cells. (MCF-10A) and tumor cells (A594, MDA-MB-231, HeLa, and HepG2), mainly from the American Type Culture Collection (Manassas, Virginia, USA). A549, HeLa, HepG2 and MDA-MB-231 were cultured in DMEM containing 10% fetal bovine serum, 1% antibiotic-antimycotic, 2mM L-glutamine and 1% non-essential amino acids, and let the cells The air composition was cultured in an environment of 5% by volume of carbon dioxide and 37 degrees Celsius (cell culture incubator). MCF10A was cultured in α-MEM containing 10% fetal bovine serum and 1% antibiotic-antimycotic, and was also cultured in 5% CO ₂ and 37 ° C. Before cytotoxicity test, cells were The density was controlled at 400 cells/100 microliters/culture wellbore (cells/100 μL/well) and seeded in 96-well plates, respectively. After the cytotoxicity test, the washing was performed three times with a buffer solution of 1X PBS (phosphate buffer saline containing 0.027 M KCl, 0.14 M NaCl, 1.76 mM KH ₂ PO ₄ and 0.01 M Na ₂ HPO ₄ , and pH 7.4). The above five cells can then be reacted with 1.11 mg/ml of carbon nano-dots for 4 hours, and then observed and imaged. The cells are fixed with 4% paraformaldehyde before cell observation. After 15 minutes, it was washed with a 1x concentration of PBS buffer solution and observed by optical and fluorescence microscopy. The imaging system used was an Olympus BX61 (Tokyo, Japan) microscope with a DP71 digital camera to observe and record images of cell endocytosed carbon nano-dots. It can be found that, in addition to the bright field image, (a) ultraviolet light (wavelength of 360-380 nm), (b) blue light (wavelength of 460-480 nm), and (c) green light (wavelength of 510) -530 nm) When excited, the cell images of blue, green and red can be clearly seen separately. Moreover, as can be seen from Figure 6, most of the carbon nano-dots are located in the cytoplasmic region of the cell.

Please refer to FIG. 7A, which is a result of toxicity test of carbon nano dots to cells. The survival rate of the cells was mainly analyzed by using Alamar Blue [Alamar Blue reagent purchased from Biosource (Camarillo, Calif., USA)], and the above five cells were respectively cultured in a cell culture well, each The initial cell concentration in the well plate was about 4000 cells/100 μL (cells/100 μl), and cultured for 24 hours under 5% carbon dioxide and 37 degrees Celsius, followed by concentrations of 0, 0.18, 0.35, 0.7, 1.4. The carbon nano-dots of 2.8 mg/ml (mg/ml) were added to the culture wells of the above five kinds of cells, and after being cultured for another 24 hours, they were washed three times with the PBS solution of 1 time, and then The Alamar Blue reagent was diluted 10 times in the original concentration with the culture solution, and 100 μl of the culture solution containing Alamar Blue was separately added to the well plate for the cultured cells for 4 hours, and then the microplate type fluorescence spectrometer was used. Synergy, purchased from BioTek (Wenuschi, Vermont, USA), was used to detect cell viability (excitation wavelength 545 nm, fluorescence emission wavelength 590 nm). In general, the fluorescence intensity of Alamar Blue after reaction with cells is proportional to living cells and can therefore be used to count cell viability. As shown in Fig. 7A, the horizontal axis represents the concentration of the carbon nanotubes added; and the vertical axis represents the survival rate of each cell. After the above five kinds of cells were subjected to the above-mentioned different concentrations of carbon nano-dots, it showed that HepG2 cells showed a significantly decreasing survival rate with the increase of the carbon nano-dots concentration; and for other normal cells (MCF- 10A) and tumor cells (A549 and HeLa), the growth condition remained stable, indicating that the carbon nano-dots have less cytotoxicity to normal cells and tumor cells A549 and HeLa. When the concentration of carbon nano-dots increased to 1.4 mg / ml, it began to cause relatively bright tumor cells MDA-MB-231 Significant cytotoxicity. Therefore, as shown in FIG. 7A above, it can be inferred that the 50% inhibition concentration (IC50) of the carbon nanoparticle to HepG2 is 0.32 mg/ml to 0.38 mg/ml (the HepG2 can be inhibited by 50%). The concentration of cells survived, and at the carbon nano spot concentration of 1.4 mg/ml, cells other than HepG2 cells still had more than 80% cell viability, indicating that the carbon nano-dots of the present invention were in addition to HepG2. In addition to a more specific inhibitory effect, it also has lower cytotoxicity than other cells.

In addition, please refer to FIG. 7B, which is the result of the performance of the above five cell lines on the addition of p53 protein at a concentration of 2.8 mg/ml for 24 hours, which is represented by Western blot. As a result, the western ink dot is executed by separately breaking the above-mentioned cells, and electrophoresis is carried out by 10% polypropylene guanamine colloid electrophoresis (SDS/PAGE), followed by dissociation of the resolved protein in the electric field, and colloid Transfer to a polyvinylidene difluoride (PVDF) membrane and react for 1 hour in a blocking buffer containing 1x Tris-buffered saline (TBS), 0.1% Tween 20, 5% w/v skim milk powder. The membrane was then immersed in a 4 ° C environment with primary antibody: anti-p53 [from Santa Cruz Biotechnology (Santa Cruz, CA)] and anti-α-tubulin antibody [from Oncogene Science (Cambridge, MA)] Reaction overnight. All primary antibodies were diluted in 1X Tris-buffered saline (TBS; 25 mM Tris, 150 mM NaCl, 2 mM KCl, pH 7.4) containing 0.1% Tween 20 with 5% w/v BSA. After the PVDF membrane was sufficiently washed, it was placed in a horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G antibody (1:2000 dilution, Rockland Immunochemicals, Gilbertsville, PA), and reacted at room temperature for 1 hour. From the data of the western ink spots, it can be found that in the case where the carbon nano-dots are added, the expression level of the p53 protein of HepG2 does have a relatively significant increase relative to the case where the carbon nano-dots are not added.

Please continue to refer to Figure 7C, which shows the cells of HepG2 cells after the addition of a carbon nanoparticle concentration of 1.11 mg/ml and after 24 hours of action. The periodic distribution, in the left panel of Fig. 7C, is the HepG2 cells (control group) to which no carbon nanotubes are added; the right panel of Fig. 7C is the experimental group of HepG2 cells with carbon nanotubes added, and the data shows that no carbon is added. At the nano-point HepG2 cells, the SubG1 fraction was only 7.4%, and when the carbon nano-dots were added, as shown in the experimental group on the right in Fig. 7C, the proportion of the SubG1 phase in the cell cycle in HepG2 cells was increased to 43.1%. In addition, it can be understood from the data in Fig. 7C that in the presence of carbon nano-dots, the ratio of HepG2 cells in the cell cycle G0/G1 and G2/M is significantly decreased, but the proportion of SubG1 is increased, meaning Carbon nanospots can induce HepG2 pathways toward apoptosis.

In addition, please refer to FIG. 8 , which is the survival rate of the above five cell strains after adding fresh ginger juice of 0, 1.6, 3.1, 6.3, 12.5 and 25% by weight respectively, after 24 hours, wherein The fresh ginger juice used is obtained by some pretreatment. The pretreatment method is as follows. Fresh ginger juice is provided in a volume of about 20 ml. The ginger juice can be peeled and sliced from ginger which is generally purchased in the market. After grinding, fresh ginger juice was centrifuged at 12,000 g for 10 minutes to obtain a supernatant of the fresh ginger juice. Adjusting the pH value (pH) of the supernatant of the fresh ginger juice to be weakly alkaline. Preferably, the pH of the fresh ginger juice is adjusted to 7.1 to 7.6 by NaOH. In the present demonstration, the supernatant of fresh ginger juice at pH 7.4 was selected for subsequent experiments. The supernatant of the fresh ginger juice is then filtered again to remove excess solid residue. Preferably, the filter membrane having a pore size of 0.1 μm to 0.22 μm can also be used to filter the supernatant of the fresh ginger juice, which can be removed by the above method. In addition to the excess solid residue in the first ginger juice supernatant, microorganisms such as harmful parasites or bacteria can be effectively removed, so that it can be used for the experiment as a carbon nanopore relative to the present invention. Control group. It can be seen from Fig. 8 that HepG2 also has a significantly lower survival rate as the concentration of fresh ginger juice increases. However, it can be found that normal cell MCF-10A also has a significantly lower level with the increase of fresh ginger juice concentration. Survival rate, and under the same fresh ginger juice concentration, normal cell MCF-10A has a lower survival rate than HepG2 tumor cells, showing fresh ginger Juice is more likely to bring toxicity to normal cells, but the carbon nano-dots of the present invention do not have this disadvantage. Obviously, the carbon nano-dots of the present invention are more specific to HepG2 cells than ordinary fresh ginger juice. The selective inhibition effect is less harmful to other normal cells.

In addition, the carbon nano-dots of the present invention are manufactured by ginger, in other words, the present invention is a carbon nano-powder using natural ginger as a carbon source, and has a better selectivity for hepatoma cells of HepG2. The inhibitory effect, as shown in Fig. 9, some carbon nano-dots not using ginger as a carbon source, such as carbon nano-dots made of green tea, glycine and EDTA, have a poor inhibitory effect on HepG2, for example, At the same concentration of 2.8 mg / ml, the above-mentioned carbon nano-dots with non-ginger as a carbon source have the best inhibitory effect on HepG2, which can only achieve about 40% inhibition of cell viability (green tea), while others For example, glycine and EDTA have a survival rate of 80% and 60%, respectively, at the same carbon nano point concentration. However, in the case of the carbon nano point of the present invention using ginger as a carbon source, the survival rate of HepG2 cells can be about 5%-10% at a concentration of 2.8 mg/ml (Fig. 7A), showing the present invention. The carbon nano-points using ginger as a carbon source do have an unexpected growth-inhibiting effect on HepG2 cells compared to other carbon nano-points that do not use ginger as a carbon source.

Please continue to refer to FIG. 10, which is a result of analyzing the effect of the above-mentioned five cell lines on the endocytosis effect (horizontal axis) and ROS (vertical axis) of the carbon nano-points by using an image cytometer, wherein the left half ( A) The field is the control group, and the carbon nano-point is not added; the right half (B) field is the experimental group, and the carbon nano-dots are added. Use DHE (dihydroethidine) dye to detect superoxide in cells. Once reacted with ROS in cells, it will generate 2-hydroxyethidium which can be inserted into DNA and produce red fluorescence to know the cell endocytosis. The main experimental method for ROS produced after the nano-point is that the above five cell lines are cultured in a cell culture medium at an initial cell concentration of 2 × 10 ⁵ cells/cells/well, and placed. The carbon dioxide containing 5% by volume was cultured in a cell incubator at 37 ° C for 24 hours. Thereafter, a 1.11 mg/ml carbon nanopoint material was added to each of the culture solutions used to react with the plated cells for 4 hours, and then washed three times with a 1-fold concentration of PBS solution. Then, freshly prepared 100 μl of DHE and 5 μM of DHE were added to the culture wells of the cells for 0.5 hour, then washed three times with 1 time of PBS solution, and then the cells were stopped by 1 time of trypsin. At the bottom of the culture well plate, it is sent to the image cytometry system for analysis ((NucleoCounter NC-3000, Chemometec, Aller) d, Denmark). The carbon nano-dots and DHE were excited at 365 and 475 nm, respectively. The emission of 475 and 530 nm was used to know the cell endocytosis of the material and the production of ROS. Therefore, it can be seen from the right half of the field in Figure 10. After the addition of a carbon nanoparticle concentration of 1.11 mg/ml, the proportion of endocytic behavior in HepG2 cells was increased from 15.72% of the carbon nano-dots. 83.79% of the carbon nano-dots were added, and the proportion of cells producing ROS was also increased from 0.86% of the carbon-free point to 15.66% of the added carbon nano-dots, compared to HepG2 without the carbon-doped point. After the addition of the carbon nano-dots, about 18.2 times of ROS was induced in HepG2 cells, whereas for other cells, there was no significant increase in ROS. In addition, ROS may be one of the pathways and indicators for inducing programmed cell death, causing cell DNA and cell damage to cause apoptosis, and further, from the results of the Western blot method shown in FIG. 7B, The p53 of HepG2 can be expressed by the ginger-derived carbon nanopoint of the present invention.

Therefore, it is clear that HepG2 can achieve cell-derived ROS to induce HepG2 cells to enter apoptosis after adding carbon nano-dots, and when the ROS of the cells rise, it shows that the cells are experiencing pressure, thus triggering HepG2 cells. The performance of p53, however, other carbon nano-dots made from EDTA, glycine and green tea could not achieve significant value-added inhibition of HepG2 cells. It is apparent that the carbon nano-dots of the present invention using ginger as a carbon source should be different from other carbon nano-dots not using ginger as a carbon source. In order to further understand whether the surface of the carbon nano-point has a special component that can exert a value-adding inhibitory effect on HepG2 cells, please refer to FIG. 11 , which is a SALDI (surface- at a concentration of 0.011 mg/mL of carbon nano point material. Assisted laser description/ionization time-of-flight mass spectrometry) is a spectrometer that operates in a reflector positive ion mode with pulsed laser light (Nd:YAG, 355 nm wavelength, 100 Hz, pulse width 6 ns). Analysis of carbon naphthalene using 13 nm gold nanoparticles as matrix and surface assisted laser desorption free time-of-flight mass spectrometer (SALDI-TOF MS; Brooke Dalton, Bremen, Germany) Rice spots may have biologically active ingredients. The instrument is calibrated using gold clusters ([Au _x ] ⁺ ; x = 1-5) and the laser energy is approximately 102.5 W cm ^-2 . The above-mentioned gold nanoparticles having a concentration of 7.5 nM were used as a substrate, and reacted in an ammonium citrate buffer of 0.5 mM and pH 7.0. Among them, the three groups of values of mass-to-charge ratio (m/z): 369.888, 370.900 and 371.889 indicate [curcumin + H] ⁺ isotope; insertion map: predicted [curcumin + H] ⁺ isotope, for utilization Measured by SALDI-TOF MS. In the case of mass-to-charge ratios of 369.888, 370.900 and 371.889, the relative peaks of the isotope [curcumin + H] ⁺ can be corresponding, indicating that the carbon nano-dots can still be combined after the dialysis (or With / attached) curcumin molecule. In other words, the present invention successfully uses ginger as a carbon source to manufacture/synthesize carbon nano-dots, and after it is made, curcumin can naturally coexist with carbon nano-dots and does not disappear due to dialysis. It solves the problem that curcumin has poor water solubility and is difficult to be prepared into a suitable water-soluble preparation, and is difficult to be fed into cells such as liver cancer cells and liver tumor cells.

Please continue to refer to FIG. 12A, FIG. 12B and FIG. 12C, which are the results of the test of the tumorigenicity test of the carbon nano-point in mice, and the mouse used is from the National Experimental Animal Center (Taipei). , Taiwan) purchased six-week-old BALB/c nude mice (BALB/cAnN. Cg-Foxn1nu/C-rlNarl ) and were housed in a specific pathogen-free animal room. Animal experiments in mice were obtained under license from the Animal Care and Use Committee (license number IACUC 2012-037). All procedures were performed under anesthesia with sodium pentobarbital to reduce pain in the mice. First, as shown by the arrow in the upper half of Fig. 7A, HepG2 cells of several 1 × 10 ⁷ cells were inoculated into the lateral back of female BALB/c mice to induce malignant liver tumor production. On the 7th day after the inoculation, the tumor volume will reach about 5 cubic centimeters, and then the drug injection, in which the drug is injected with PBS as the control group (-); with 220 micrograms (μg) of carbon nano point As the experimental group (+), the mice were injected into the tumor cells of the mice for testing, and the injection of the above drugs was further added on the 14th day. The test results are shown in Fig. 12B, and the results presented after the tumors were removed (on the 16th day after inoculation) were left control group; the right side was the experimental group. As shown in Fig. 12C, the quantified result of the excised tumor weighing (n = 3); ^*** indicates p < 0.001. After 14 days of injection and a total of 440 mg of carbon nano-dots, a significant tumor regression was observed, and the carbon nano-dots did achieve significant tumor growth inhibition in vivo, without injection of carbon nano-dots. The tumor has grown to about 104 mg, and the volume has continued to increase, whereas tumors with carbon-injected spots have only reached about 3.7 mg. Obviously, the carbon nano-dots of the present invention have high permeability and retention effect (EPR effect), and the carbon nano-dots can be retained for a long time and act on mice as long as the carbon nano-dots are injected twice during the 14-day experiment. The tumor. The inhibition effect of the tumorigenicity achieved can be quantified to a high tumorigenic effect of >95% and P<0.001. Therefore, it has been confirmed from the above results that the carbon nano point of the present invention using ginger as a carbon source has good anticancer activity, so that the use of natural ginger to produce a carbon nano point can be used as an effective anticancer drug. The invention has great potential for the treatment of liver cancer, and therefore the present invention also provides a use of the carbon nano point as a medicine for treating liver cancer diseases/suppressing growth of liver tumors, wherein the carbon nano point of the present invention includes at least several a curcumin molecule bound to the surface of the carbon nano-dots, and the carbon nano-dots are also fused to an aqueous carrier, the aqueous carrier being a pharmaceutically acceptable carrier, in particular, the aqueous carrier may be an organism An acceptable PBS buffer solution, deionized water or physiological saline solution, etc., but not limited thereto, preferably, the carbon nano point of the present invention can also be made into an injection type so as to be directly permeable. In a way, the tumor directly produces an inhibitory effect on proliferation.

In summary, the carbon nano-dots of the present invention can be produced from ginger, and can exhibit significant and highly selective HepG2 cell proliferation inhibitory effects in both in vitro and in vivo experiments, and the proliferation inhibition effect is increased by ROS. And caused by the expression of p53 protein, resulting in the cell cycle of HepG2, showing that it can develop into liver cancer / The potential of therapeutic drugs for liver tumors, as well as the results of cell experiments in vitro, also indicate that the carbon nanopoints of the present invention have relatively low cytotoxicity against other normal cells, especially after about one hour of injection into mice, ie The urine of the mouse can be excreted from the body, indicating that the carbon nano-dots of the present invention have excellent biosafety and biocompatibility. However, the above is only a preferred embodiment of the present invention, and all changes and modifications made to the scope of the present invention should be within the scope of the present invention.

Claims (10)

A method for synthesizing a carbon nano point, comprising at least the following steps: (A) providing a ginger juice, heating the ginger juice at a predetermined temperature for a predetermined time to carbonize the ginger juice, and forming a carbonaceous carbonaceous material (B) centrifuging the carbide-containing carbon nanomaterial to obtain a supernatant; and (C) filtering the supernatant, and dialysis the supernatant to obtain a carbon nano Point the suspension solution. The method for synthesizing a carbon nano-dots according to claim 1, wherein in the step (C), a filter having a pore diameter of 0.1 μm to 0.22 μm is used to filter the supernatant. The method for synthesizing a carbon nano-point according to claim 1, wherein the predetermined temperature is 280 to 320 degrees Celsius; and the predetermined time is 100 minutes to 140 minutes. The method for synthesizing a carbon nano-dot as described in claim 1, wherein in the step (C), the supernatant is subjected to dialysis treatment using a dialysis membrane having a molecular weight limit of 3.5 kD to 5 kD. A carbon nano-point comprising at least: a plurality of curcumin molecules bound to the carbon nano-dots. The carbon nano-point according to claim 5, wherein the carbon nano-point has a particle diameter of from 3 nm or more to 8 nm or less. The carbon nano-dots as claimed in claim 5, wherein the carbon nano-dots are dissolved in an aqueous carrier. The invention relates to a carbon nano point as a medicine for manufacturing a liver tumor, wherein the carbon nano point comprises at least: a plurality of curcumin molecules bound to the carbon nano point, wherein the carbon nano point is dissolved in an aqueous carrier . The use of the carbon nano point as the medicine for treating a liver tumor as described in claim 8, wherein the carbon nano point has a particle diameter of from 3 nm or more to 8 nm or less. The use of a carbon nano-dots as claimed in claim 8 or 9 for the manufacture of a medicament for treating liver tumors, wherein the aqueous carrier is PBS buffer solution, deionized water or physiological saline.