Ratio-type fluorescent carbon dots for tracing nicotinamide adenine dinucleotide in liver cancer glycolysis, preparation method, and application thereof

Abstract

The present invention discloses a ratiometric fluorescent carbon dot for tracing nicotinamide adenine dinucleotide in liver cancer glycolysis, and its preparation method and application, belonging to the field of pharmaceutical preparation technology. The ratiometric fluorescent carbon dot uses aniline derivatives and aromatic disulfide as carbon and nitrogen sources and is obtained by a solvent thermal method. The carbon dots prepared by the present invention have a small particle size and are evenly distributed, are basically non-cytotoxic, and are activated by NAD. + As the concentration increases, the carbon dots migrate to red fluorescence, and the ratio fluorescence intensity F 640 / F 520 With NAD + The linear relationship between the two groups was good. After the action of glycolysis inhibitor 2-DG, the uptake of carbon dots in HepG2 cells was significantly reduced, which will provide a scientific basis for the development of integrated diagnosis and treatment nanomedicines.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and more particularly to a ratiometric fluorescent carbon dot for tracing nicotinamide adenine dinucleotide in liver cancer glycolysis, and a preparation method and application thereof. Background Art

[0002] Liver cancer is an insidious disease with a high mortality rate. Therefore, it is urgent to develop specific early diagnostic agents for liver cancer. Compared with normal liver cells, liver cancer cells have a higher sensitivity to nicotinamide adenine dinucleotide (oxidized coenzyme I, NAD + ) is more dependent on NAD + As an initiator of glycolysis metabolism in cancer cells, it is mainly involved in regulating biological metabolic processes such as DNA damage repair, inflammation and epigenetic regulation. + Targeted identification of NAD is beneficial for tracing the aerobic glycolysis process in cancer cells and is used for early diagnosis of spontaneous liver cancer. + / NADH bioluminescent probe for analyzing NAD in cells or in vivo + / NADH content and level changes, the study of NAD + / NADH-related physiological and pathological processes are of great significance.

[0003] In recent years, new fluorescent carbon nanoparticles - carbon dots have attracted extensive attention from researchers due to their advantages such as easy functionalization, adjustable fluorescence emission and strong photostability, and have shown significant advantages in the fields of bioimaging, optical diagnosis and treatment, and drug delivery. O-phenylenediamine has a structure similar to phenazine. Studies have found that fluorescent carbon dots prepared with 2,3-diaminophenazine as raw material, NAD + (excitation / emission wavelength 423 / 476nm) and carbon dots (excitation / emission wavelength 495 / 530nm) undergo fluorescence resonance energy transfer. + In the presence of carbon dots, the green fluorescence intensity is enhanced, which is beneficial for identifying aerobic glycolysis in cells and can be used for early tumor diagnosis of spontaneous liver cancer. However, in bioimaging studies, most fluorescent diagnostic agents are randomly distributed in the cytoplasm and cell membranes, lacking specificity; and the blue fluorescence background of living tissues interferes with bioimaging resolution.

[0004] Therefore, how to provide a fluorescent diagnostic and therapeutic agent and its preparation method that can avoid interference from the living body's own tissues and target distribution in liver cancer cells to achieve early diagnosis of liver cancer is an urgent problem that technicians in this field need to solve. Summary of the Invention

[0005] In view of this, the present invention provides a ratiometric fluorescent carbon dot for tracing nicotinamide adenine dinucleotide in liver cancer glycolysis, as well as a preparation method and application thereof.

[0006] The present invention uses aniline derivatives and aromatic disulfides as carbon and nitrogen sources, adopts a solvent thermal method and separation and purification procedures to prepare ratiometric fluorescent carbon dots. The preparation method is simple, the obtained carbon dots have uniform particle size distribution, stable physical and chemical properties, basically no cytotoxicity, and good selectivity, and can be used for nicotinamide adenine dinucleotide NAD in the glycolysis metabolism of liver cancer. + Concentration detection can achieve the purpose of early diagnosis of liver cancer.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A ratiometric fluorescent carbon dot for tracing nicotinamide adenine dinucleotide in liver cancer glycolysis, the structure of which is shown in Formula 1:

[0009]

[0010] Furthermore, the carbon dots are obtained by a solvothermal method using aniline derivatives and aromatic disulfides as carbon and nitrogen sources.

[0011] Furthermore, the aniline derivative is o-phenylenediamine, m-phenylenediamine, p-phenylenediamine or 2,3-diaminophenazine.

[0012] Furthermore, the aromatic disulfide is 2,2'-dithiosalicylic acid, 2,2'-dithiobis(6-fluorobenzoic acid), 4,4'-dithiodibenzoic acid, 3-(2-pyridyldithio)propionic acid or 5,5'-dithiobis(2-nitrobenzoic acid).

[0013] Furthermore, the aniline derivative is o-phenylenediamine, and the aromatic disulfide is 2,2'-dithiosalicylic acid.

[0014] Furthermore, the molar ratio of o-phenylenediamine to 2,2'-dithiosalicylic acid is 1:1.

[0015] Furthermore, the particle size of the ratiometric fluorescent carbon dots is 8 to 10 nm.

[0016] Beneficial effects achieved: The present invention uses aniline derivatives and aromatic disulfides as carbon sources and nitrogen sources, and obtains them through a solvent thermal method. The selection of carbon sources and nitrogen sources is crucial to the preparation of multicolor fluorescent carbon dots. Among them, aniline derivatives are very easy to undergo polymerization reactions. Blue, green and orange-red fluorescent carbon dots can be prepared using o-phenylenediamine, m-phenylenediamine and p-phenylenediamine as carbon sources, respectively. Heteroatom doping or increasing the area of ​​conjugated π domains will be beneficial to the long-wavelength emission of carbon dots. Aromatic disulfides are beneficial to the red shift of the molecular ultraviolet absorption wavelength due to the conjugation effect of the aromatic ring. In addition, the polarity, pH value and solubility of the solvent will have a significant effect on the fluorescence emission behavior of the carbon dots. However, short-wavelength fluorescence such as blue and green cannot penetrate deeper into biological tissues, and the interference of autofluorescence cannot be avoided. The synthesis of long-wavelength emission, especially red fluorescent carbon dots, is still one of the difficult problems for scientific researchers. Therefore, there is an urgent need to analyze the solvatochromic effect of samples by studying solvents as reaction media or post-treatment methods, and to utilize quantum size effects, heteroatom doping, and surface functional groups (-OH, -COOH, -NH2) to jointly improve the fluorescence properties of carbon dots.

[0017] Nicotinamide adenine dinucleotide (oxidized coenzyme I, NAD + ) acts as an initiator of glycolysis metabolism in cancer cells and is mainly involved in regulating biological metabolic processes such as DNA damage repair, inflammation, and epigenetic regulation. + Targeted identification of NAD is beneficial for tracing aerobic glycolysis in cancer cells and is used for early diagnosis of spontaneous liver cancer. + The metabolic effect is affected by many factors, such as reaction solvent, NAD + Factors such as concentration, functional group structure of carbon and nitrogen sources, and the interaction between polar groups of solvent and solvent molecules will affect the surface emission state of carbon dots.

[0018] In order to solve the above problems, the present invention effectively slows down or prevents the occurrence of chemical reactions by adding reaction solvents, studies the regulation rules of carbon source, nitrogen source and solvation effect, and thus realizes the long wavelength carbon dots to NAD + high selectivity.

[0019] The above-mentioned method for preparing ratiometric fluorescent carbon dots comprises the following steps:

[0020] (1) dissolving an aniline derivative and an aromatic disulfide in a reaction solvent so that the concentration of each in the mixed solution is 10 mM (here, the aniline derivative and the aromatic disulfide are added to a sample bottle, the reaction solvent is added, and the mixture is placed in a reactor for reaction);

[0021] (2) The mixed solution is transferred to a polytetrafluoroethylene autoclave for solvothermal reaction, and the collected sample is centrifuged, dialyzed and freeze-dried to obtain a finished product.

[0022] Furthermore, the reaction solvent is water, ethanol, acetic acid, phosphoric acid or N,N-dimethylformamide.

[0023] Furthermore, the reaction solvent is phosphoric acid.

[0024] Furthermore, the reaction solvent is phosphoric acid with a volume fraction of 80%.

[0025] Furthermore, the reaction temperature of the solvent thermal reaction in step (2) is 180° C. and the reaction time is 12 h;

[0026] The molecular weight cut-off of the dialysis bag used for dialysis was 500 Da, and the dialysis time was 48 h.

[0027] Application of the ratiometric fluorescent carbon dots prepared by the above preparation method in the preparation of early diagnosis reagents for liver cancer.

[0028] In bioimaging research, most fluorescent diagnostic and therapeutic agents are randomly distributed in the cytoplasm and cell membrane, lacking specificity; and the blue fluorescence background of the living body's own tissues interferes with the resolution of bioimaging. How to avoid the interference of the living body's own tissues and make the fluorescent diagnostic and therapeutic agents targeted in liver cancer cells has become the key to liver cancer diagnosis. O-phenylenediamine has a structure similar to phenazine, and the phenazine structure can be used as the fluorescent luminescence center of carbon dots. In addition, aromatic disulfides themselves have photoinitiation ability, and can undergo a reversible reaction of "breaking and recovering" of sulfur-sulfur bonds under the long emission wavelength of the light source, and initiate monomer polymerization. The present invention uses o-phenylenediamine and 2,2'-dithiosalicylic acid, the reaction raw materials of 2,3-diaminophenazine, as carbon sources and nitrogen sources to prepare fluorescent carbon dots with long emission wavelengths; nicotinamide adenine dinucleotide (oxidized coenzyme I, NAD + ) affects the ability of the donor or acceptor to push and pull electrons, and the carbon dots migrate from green fluorescence to red fluorescence. The ratio fluorescence intensity F 640 / F 520 With NAD + The concentration showed a good linear relationship.

[0029] It can be seen from the above technical solutions that, compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The present invention uses o-phenylenediamine as the carbon source and nitrogen source to carry out the solvent thermal reaction, and the carbon dots react with NAD + Fluorescence resonance energy transfer occurs, enhancing the red / green fluorescence ratio of carbon dots, which can be used for early tumor diagnosis of spontaneous liver cancer;

[0031] (2) The present invention uses 2,2'-dithiosalicylic acid with photoinitiation ability to initiate monomer polymerization, generating aromatic thiol radicals through the reversible reaction of sulfur-sulfur bond "breakage-recovery", and attacking the monomer to generate primary carbon radicals, ultimately initiating polymerization;

[0032] (3) The present invention adopts a solvent thermal method to prepare carbon dots. The reaction solvent effectively slows down or prevents the occurrence of chemical reactions, thereby obtaining carbon dots with uniform particle size distribution and good dispersion. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0034] Figure 1 Figure 1 shows the effects of solvents on the fluorescence imaging of carbon dots in Example 1 of the present invention, where A shows the effects of different solvents on the UV-visible absorption spectra of carbon dots; B shows the effects of different volume fractions of H3PO4 on the UV-visible absorption spectra of carbon dots; C shows digital photographs of carbon dots prepared with different solvents under 365 nm UV light; and D shows digital photographs of carbon dots prepared with different volume fractions of H3PO4 under 365 nm UV light.

[0035] Figure 2 3D fluorescence excitation-emission wavelength analysis of carbon dots prepared with 80% H3PO4 by volume in Example 1 of the present invention;

[0036] Figure 3 This is an infrared spectrum of carbon dots prepared with 80% H3PO4 by volume in Example 1 of the present invention;

[0037] Figure 4 This is an X-ray photoelectron spectroscopy (XPS) analysis of carbon dots prepared with 80% H₃PO₄ by volume in Example 1 of the present invention, wherein A is the full-scan XPS spectrum of the carbon dots; B is the high-resolution XPS spectrum of C₁s; C is the high-resolution XPS spectrum of N₁s; and D is the high-resolution XPS spectrum of S₂p.

[0038] Figure 5 This is a graph showing the particle size of carbon dots prepared with 80% H3PO4 by volume in Example 1 of the present invention;

[0039] Figure 6 This is a transmission electron micrograph of carbon dots prepared with 80% H3PO4 by volume in Example 1 of the present invention;

[0040] Figure 7NAD in Example 2 of the present invention + Effect of concentration on the fluorescence excitation-emission wavelength of carbon dots, where A is the emission wavelength at an excitation wavelength of 390 nm; B is the emission wavelength at an excitation wavelength of 566 nm; C is the emission wavelength at an excitation wavelength of 615 nm; D is the ratiometric fluorescence intensity fitting curve of carbon dots; E is a digital photograph of carbon dots under 365 nm ultraviolet light irradiation;

[0041] Figure 8 This is a cytotoxicity analysis diagram of carbon dots in Application Example 1 of the present invention;

[0042] Figure 9 Figure 2 shows the hemolytic analysis of carbon dots in Application Example 2 of the present invention, where A is the hemolytic analysis of carbon dots; B is the hemolytic rate analysis;

[0043] Figure 10 This is the uptake diagram of carbon dots in HepG2 cells in Application Example 3 of the present invention;

[0044] Figure 11 The carbon dots and NAD in Application Example 4 of the present invention + Uptake diagram in HepG2 cells after co-culture;

[0045] Figure 12 NAD in Application Example 4 of the present invention + Effect of concentration on intracellular uptake of carbon dots;

[0046] Figure 13 The fluorescence intensity of carbon dots in application examples 3 and 4 of the present invention and the time relationship analysis diagram, wherein A is the addition of NAD + Red fluorescence intensity of carbon dots before and after addition; B is the red ... + The red / green fluorescence intensity ratio of the front and rear carbon dots;

[0047] Figure 14 NAD in Application Example 4 of the present invention + Analysis of the effect of concentration on the red fluorescence intensity and red / green fluorescence intensity ratio of carbon dots.

[0048] Figure 15 Figure 5 shows the effect of the glycolysis inhibitor 2-deoxy-d-glucose (2-DG) on the migration ability of HepG2 cells in Application Example 5 of the present invention. Figure A shows the cell migration of HepG2 cells co-cultured with carbon dots at 0 and 7 hours without the addition of 2-DG; Figure B shows the cell migration of HepG2 cells co-cultured with carbon dots at 0 and 7 hours after pretreatment with 2-DG for 5 hours.

[0049] Figure 16 This is the analysis of the migration ability of HepG2 cells by the glycolysis inhibitor 2-DG in Application Example 5 of the present invention;

[0050] Figure 17 Figure 6 shows the uptake of carbon dots in HepG2 cells under the action of 2-deoxy-d-glucose (2-DG), a glycolysis inhibitor, in Application Example 6 of the present invention. Figure A shows the cell uptake of carbon dots after 7 hours of co-culture with HepG2 cells without the addition of 2-DG; Figure B shows the cell uptake of carbon dots after 7 hours of co-culture with HepG2 cells after 5 hours of 2-DG pretreatment.

[0051] Figure 18 This is the uptake analysis of carbon dots in HepG2 cells under the action of glycolysis inhibitor 2-deoxy-d-glucose (2-DG) in Application Example 6 of the present invention. DETAILED DESCRIPTION

[0052] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0053] The reagents required for the present invention are conventional experimental reagents, purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0054] Example 1

[0055] A method for preparing ratiometric fluorescent carbon dots for tracing nicotinamide adenine dinucleotide in liver cancer glycolysis, the main steps comprising:

[0056] Carbon dots were prepared using a solvothermal method. 3 mg of o-phenylenediamine (OPD) and 9 mg of 2,2'-dithiosalicylic acid (DTSA) were dissolved in 3 mL of a reaction solvent (water, ethanol, acetic acid, 5% by volume phosphoric acid, 20% by volume phosphoric acid, 50% by volume phosphoric acid, 80% by volume phosphoric acid, 100% by volume phosphoric acid, and N,N-dimethylformamide). The solution was then transferred to a polytetrafluoroethylene autoclave and reacted at 180°C for 12 hours. After cooling, the sample was collected. The powder was centrifuged at 10,000 rpm and dialyzed through a 500 Da molecular weight cutoff dialysis bag for 48 hours. Finally, it was freeze-dried in a vacuum oven to obtain the carbon dots.

[0057] Depend on Figure 1 The effects of solvent and H3PO4 concentration on the UV absorption of carbon dots are as follows: Figure 1As shown in Figure A, the solvent has a greater effect on the ultraviolet absorption wavelength of carbon dots. The carbon dots prepared with H3PO4 as solvent tend to be more excited / emitted at long wavelengths, with strong ultraviolet absorption peaks at 390 and 615 nm. At the same time, there is a shoulder peak at 566 nm at 615 nm. Similarly, under a 365 nm ultraviolet lamp, the carbon dots prepared with H3PO4 as solvent emit fluorescence at long wavelengths, and the fluorescence color is yellow-green ( Figure 1 C). Figure 1 As shown in Figure B, with the increase of H3PO4 volume fraction, the absorption intensity of carbon dots at 390 and 615 nm gradually increases. Among them, the carbon dots prepared with 80% H3PO4 volume fraction have the highest ultraviolet absorption intensity at 615 nm, while the absorption intensity at 390 nm is relatively weak, which can be used as a basis for selecting long-wavelength ratio fluorescent carbon dots. Figure 1 As shown in D, under a 365nm ultraviolet lamp, the carbon dots prepared with o-phenylenediamine (OPD) and 2,2'-dithiosalicylic acid (DTSA) as carbon sources and H3PO4 as solvent were blue-green in color, among which the fluorescence intensity of the carbon dots prepared with o-phenylenediamine as the carbon source was significantly enhanced; when OPD and DTSA were used as carbon sources, the fluorescence of the carbon dots gradually shifted to yellow / red with the increase of the volume fraction of H3PO4, among which the red / green fluorescence ratio of the carbon dots prepared with a volume fraction of 80% H3PO4 was more significant, so the subsequent carbon dots used 80% H3PO4 as the solvent.

[0058] Figure 2 3D fluorescence excitation-emission wavelength analysis of carbon dots prepared with 80% H3PO4 by volume. As shown in the figure, the carbon dots prepared with 80% H3PO4 by volume as solvent have excitation wavelengths of 566 and 615 nm, and emission wavelengths of 640 and 680 nm. Their fluorescence intensity is significantly higher than that after excitation at 390 nm.

[0059] Figure 3 The infrared spectrum analysis of carbon dots prepared with 80% H3PO4 volume fraction is shown in the figure. -1 There is a broad peak at 2973 cm, indicating the presence of OH and NH bonds on the surface of carbon dots. -1 The characteristic peaks at 1682 and 1261 cm are attributed to the stretching vibration peaks of CH. -1 The stretching vibration peaks at 685 and 491 cm are C=O and CN respectively. -1 The 3 and 4 represent the stretching vibration peaks of CS and SS, respectively. This shows that the carbon dots were successfully synthesized.

[0060] Figure 4 The X-ray photoelectron spectroscopy XPS analysis of carbon dots prepared with a volume fraction of 80% H3PO4 is shown in Figure 2. Figure 4As shown in A, carbon dots are mainly composed of five elements: C, N, O, S, and P, with contents of 60.07%, 27.57%, 3.25%, 4.57%, and 4.54%, respectively. From the high-resolution spectrum of C1s ( Figure 4 B) shows four distinct peaks at 284.4 eV (CC, C=C), 286.2 eV (CN, CS), 287.9 ​​eV (C=O), and 288.9 eV (-COO-). High-resolution spectrum of N 1s ( Figure 4 C) shows two different peaks at 399.4 and 400.9 eV, corresponding to pyrrolic N and pyridinic N, respectively, indicating that the carbon dots have a phenazine-like structure. The high-resolution spectrum of S2p ( Figure 4 D) shows three different peaks at 163.7eV (SC), 164.1eV (SH) and 164.9eV (SS). The above results indicate the successful synthesis of carbon dots.

[0061] Figure 5 and Figure 6 The particle size distribution and transmission electron microscopy images of carbon dots prepared with a volume fraction of 80% H3PO4 show that the particle size of the carbon dots is small, averaging 8 to 10 nm, and has good dispersion.

[0062] Example 2

[0063] Fluorescent carbon dots and nicotinamide adenine dinucleotide NAD + The linear relationship:

[0064] 0.02 g of carbon dots prepared with 80% H3PO4 by volume was weighed and dissolved in PBS to prepare a 2 mg / mL carbon dot solution. 0.2 mL of the 2 mg / mL carbon dot solution was then added to 2 mL of NAD solutions with concentrations of 0.01, 0.1, 1, 10, 20, and 40 mM / L, respectively. + Fluorescence spectrometer was used to measure the effect of adding different concentrations of NAD + After that, the excitation-emission wavelength and fluorescence intensity of carbon dots at 390, 566 and 615 nm changed.

[0065] Figure 7 NAD + The concentration affects the fluorescence excitation-emission wavelength of carbon dots, as shown by Figure 7 A~ Figure 7 D shows that no NAD was added + When the carbon dots are excited at 390, 566 and 615 nm, they emit fluorescence at 450, 520, 640 and 680 nm respectively. The fluorescence emission intensities at 520 and 640 nm are relatively strong, so the relative fluorescence intensity (F) of the carbon dots at 520 and 640 nm is used to calculate the fluorescence intensity of the carbon dots. 640 / F 520) as the basis for detection. + Concentration ([NAD + ]) In the range of 0~40mM / L, the fluorescence intensity ratio F 640 / F 520 With NAD + The concentration has a good linear relationship, and the linear equation is F 640 / F 520 =4.9834+0.1931×[NAD + ](R 2 =0.987). Calculated based on detection limit = 3δ / slope, NAD + The detection limit (LOD) of the ratiometric fluorescence system was as low as 67 μM. In addition, under 365 nm UV light ( Figure 7 E), the red fluorescence of the carbon dots gradually increases. This indicates that the ratiometric carbon dots constructed have good selectivity and can be used for NAD in the aerobic glycolysis metabolic pathway of tumors. + Concentration detection.

[0066] Based on the above characterization results, it can be seen that the present invention successfully prepared NAD + The fluorescent carbon dots have a uniform particle size distribution and good dispersion, and are sensitive to NAD. + The selection ability is good, and it is expected to provide a feasible solution for the construction of carbon dots and their imaging in liver cancer cells and tissues.

[0067] Application Example 1

[0068] Cytotoxicity experimental verification of carbon dots

[0069] The cytotoxicity of ratiometric fluorescent carbon dots was investigated by MTT assay. HepG2 cells in logarithmic growth phase were collected, digested and counted. 4 Cells were seeded into 96-well plates at 100 μg / well and incubated at 37°C for 24 hours to allow attachment. The old medium was removed, and the wells were gently washed twice with 100 μL of 37°C preheated PBS buffer to remove dead cells. Carbon dots prepared with 80% H₃PO₄ were dispersed in fresh DMEM complete medium to give final concentrations of 1, 10, 100, 250, and 500 μg / mL. These were then added to the 96-well plates with five replicates for each concentration. After further incubation at 37°C in the dark for 24 and 48 hours, the old medium was removed, and the plates were washed twice with preheated PBS to remove any residual carbon dots. Subsequently, 100 μL of 20% MTT was added to the 96-well plates. After incubation at 37°C for 4 hours, the MTT was aspirated, and 100 μL of DMSO was added to each well. After shaking for 10 minutes, the absorbance of each well was measured at 490 nm and compared to that of the blank control.

[0070] The cell survival rate was calculated according to formula (1):

[0071] Cell survival rate (%) = (Abs sample -Abs control ) / (Abs cell -Abs control )×100%(1)

[0072] Abs sample —Absorbance at 490 nm after carbon dots were incubated with cells, Abs control —Absorbance of culture medium at 490 nm, Abs cell —Absorbance of cells in the untreated group at 490 nm.

[0073] Figure 8 The cytotoxicity of carbon dots on HepG2 cells is shown in the figure. After 24 hours of incubation, carbon dots exhibited low cytotoxicity at concentrations of 1, 10, 100, 250, and 500 μg / mL. Extending the incubation time of carbon dots with HepG2 cells to 48 hours slightly decreased cell viability, but the survival rate of HepG2 cells remained above 80%, demonstrating that carbon dots are essentially non-toxic.

[0074] Application Example 2

[0075] Biocompatibility verification experiment of carbon dots

[0076] 5 mL of mouse blood was collected and immediately added with EDTA-2Na to prevent coagulation. The blood was stirred to remove fibrin and then centrifuged at 1000 rpm to precipitate the blood. A 2% cell suspension was prepared with physiological saline.

[0077] 0.5 mL of normal saline (negative control, 0% hemolysis), 1% Triton X-100 (positive control, 100% hemolysis), and different concentrations (1, 10, 100, 250, and 500 μg / mL) of carbon dots (prepared with 80% H₃PO₄) were added to 0.5 mL of the cell suspension and incubated in a 37°C incubator for 2 h. The cells were then centrifuged at 3000 rpm for 5 min, and 100 μL of the supernatant was transferred to a 96-well plate. The absorbance was measured at 540 nm, and the hemolysis rate was calculated according to formula (2).

[0078] Hemolytic Rate (%) = (A sample -A negative control ) / (A positive control -A negative

[0079] control)×100%(2)

[0080] Among them, A sample is the absorbance of the experimental group, A negative is the absorbance of the negative control group, A positive is the absorbance of the positive control group, with the carbon dot concentration as the horizontal axis and the hemolysis rate as the vertical axis, see Figure 9 .

[0081] Figure 9 This is a graph analyzing the hemolysis of carbon dots. A hemolysis rate below 5% indicates good biocompatibility. The experimental data show that within the experimental concentration range of carbon dots, the hemolysis rate remains below 5%, demonstrating the good biocompatibility of ratiometric fluorescent carbon dots used to track nicotinamide adenine dinucleotide in liver cancer glycolysis.

[0082] Application Example 3

[0083] Experimental verification of the distribution of carbon dots in cells

[0084] HepG2 cells were cultured at 10 5 Cells were seeded at a density of 100 μg / well in a 6-well plate, and 1 mL of culture medium was added to each well. After the cells were completely attached, fresh culture medium was replaced, and then fluorescent carbon dots prepared with a volume fraction of 80% H3PO4 prepared in Example 1 were added to a concentration of 75 μg / mL. After culturing for 1, 3, 7, 12, 24, and 48 h, the cells were stained with the nuclear dye Hoechst 33342 staining solution for 15 min. The culture medium was then discarded, and the cells were washed twice with PBS. The stained cells were placed under a fluorescence microscope to observe cell morphology, fluorescence distribution of the carbon dots in the cells, and changes in fluorescence intensity.

[0085] Figure 10 Figure 1 shows the uptake of carbon dots after incubation with HepG2 cells for different times. As shown, the red emission of the carbon dots is primarily located in and around the HepG2 cell nucleus, indicating that the carbon dots can penetrate the cell membrane and nuclear membrane and enter the cell interior. The incubation time comparison shows that the carbon dots rapidly aggregate within 1 to 3 hours, with a high fluorescence intensity at 3 hours. The fluorescence intensity gradually decreases from 7 to 48 hours, indicating that the carbon dots are gradually expelled from the cell after 3 hours.

[0086] Application Example 4

[0087] Nicotinamide adenine dinucleotide NAD + Effects of concentration and reaction time on the intracellular uptake of carbon dots

[0088] Investigating NAD + The concentration of NAD +The cells were incubated with culture medium containing carbon dots (fluorescent carbon dots prepared with 80% H3PO4 by volume in Example 1, 75 μg / mL) at different concentrations (0.01, 0.1, 1, 10, 20, 40 mM / L) and co-cultured with HepG2 cells for 3 h. The cells were then stained with Hoechst 33342, and the fluorescence distribution and fluorescence intensity changes of the carbon dots in the cells were observed using a fluorescence microscope.

[0089] Visiting NAD + When the reaction time affects the intracellular uptake of carbon dots in the presence of + The cells were co-cultured with HepG2 cells for 1, 3, 7, 12, 24 and 48 h, and then stained with Hoechst33342. The fluorescence distribution and fluorescence intensity changes of the carbon dots in the cells were observed by fluorescence microscopy.

[0090] Figure 11 Carbon dots (75 μg / mL) and NAD + (20mM / mL) after co-culture for 1 to 48h, the uptake diagram in HepG2 cells is shown in the figure. + After treatment, the red fluorescence in HepG2 cells was enhanced and the green fluorescence was slightly reduced, which was basically consistent with the trend of fluorescence intensity in vitro.

[0091] Figure 12 NAD + The concentration of NAD increases the intracellular uptake of carbon dots. + With the increase of concentration, that is, from 0 to 20 mM / L, the red fluorescence intensity gradually becomes stronger, and the difference between the red fluorescence intensities at 20 and 40 mM / L is not much, indicating that 20 mM / L is the concentration of NAD + A more ideal concentration.

[0092] Figure 13 The relationship between the uptake fluorescence intensity of carbon dots and time is analyzed. The fluorescence intensity of carbon dots is quantitatively analyzed by ImageJ software. The red fluorescence intensity and the ratio of red and green fluorescence intensity are plotted over time. It can be observed that the curve first rises and then falls. Therefore, it is more intuitive to express that the red fluorescence intensity of carbon dots increases with time and then decreases. + The fluorescence intensity of carbon dots increased significantly, and then decreased with time ( Figure 13 A). In addition, the red / green fluorescence intensity ratio of carbon dots also showed a trend of first increasing and then decreasing, which was the same as the trend of the red fluorescence intensity curve, indicating that the red fluorescence of carbon dots gradually increased, while the green fluorescence intensity was relatively weakened; and the addition of NAD + After addition of NAD, the fluorescence intensity ratio of carbon dots was higher than that without NAD +The fluorescence intensity ratio of the carbon dots indicates that NAD + The red fluorescence of carbon dots is enhanced, while the green fluorescence is relatively weakened ( Figure 13 B).

[0093] Figure 14 NAD + The red fluorescence intensity analysis chart of carbon dots and concentration shows that the red fluorescence intensity curve of carbon dots increases sharply and then flattens, indicating that with the increase of NAD + As the concentration increases, the red fluorescence intensity of carbon dots increases. When the concentration is between 0 and 20 mM / L, the fluorescence intensity increases sharply, while when the concentration is between 20 and 40 mM / L, the fluorescence intensity does not change much. Therefore, 20 mM / L is also a relatively ideal experimental concentration. From the blue curve in the figure, that is, the red / green fluorescence intensity ratio curve, it can be observed that the curve also shows a trend of first increasing sharply, then flattening, and then slightly decreasing. As NAD + As the concentration increases, the red fluorescence intensity of the carbon dots increases, while the green fluorescence intensity gradually decreases. Red / F Green =-0.434×e [NAD+] / 1.111 +0.991,R 2 =0.991. The above experimental results show that carbon dots can quickly detect intracellular NAD + The detection limit LOD was 69 μM.

[0094] Application Example 5

[0095] Effect of glycolysis inhibition on cell migration in co-culture of carbon dots and HepG2 cells

[0096] The cell migration assay is a method to detect the growth and movement of adherent cells by statistically analyzing the migration distance. 5 The cells were seeded at a density of 1000 cells / well in a 6-well plate. When the cell confluence rate reached 80%, the cells were scratched with a 200 μL pipette tip, the old culture medium was removed, and the cells were washed with PBS. Then, the cells were added with different pH, GSH and NAD + (pH7.4, pH7.4+10mM GSH, pH7.4+20mM NAD + , pH 6.5, pH6.5+10mM GSH, pH6.5+10mM GSH+20mM NAD + ) carbon dots (fluorescent carbon dots prepared with 80% H3PO4 by volume in Example 1, 75 μg / mL) culture medium were added, and photos were taken using a fluorescence microscope after 0 h and 7 h to observe the scratch healing results.

[0097] 2-deoxy-d-glucose (2-DG) is a glucose antagonist that can competitively inhibit the phosphorylation of glucose transporters and hexokinase with glucose, effectively blocking the glycolysis metabolic pathway. Therefore, the cell migration ability after glycolysis inhibition was analyzed by adding 2-DG. HepG2 cells were cultured at 10 5 The cells were seeded at a density of 1000 cells / well in a 6-well plate. When the cell confluence rate reached 80%, 5 mM 2-DG-containing culture medium was added to the experimental group for pretreatment for 5 h. The culture medium was scratched with a 200 μL pipette tip, and then the culture medium containing different pH, GSH, and NAD was added. + (pH7.4, pH7.4+10mM GSH, pH7.4+20mM NAD + , pH6.5, pH6.5+10mM GSH, pH6.5+10mM GSH+20mMNAD + ) and carbon dots (fluorescent carbon dots prepared with 80% H3PO4 by volume in Example 1, 75 μg / mL) were cultured in the culture medium. After culturing for 0 h and 7 h, the scratches were photographed and recorded. The migration distance was statistically analyzed using Image J software to calculate the migration rate.

[0098] Figure 15 and Figure 16 The effect of glycolysis inhibitor 2-deoxy-d-glucose (2-DG) on the migration ability of HepG2 cells. Figure 15 As shown in A, when 2-DG is not added, weak acid, reducing (GSH) and glycolysis metabolism initiator nicotinamide adenine dinucleotide (NAD + ) and other tumor microenvironments can promote the migration of HepG2 cells to the scratch area, and the migration rate ( Figure 16 ) increased from 3.82% (pH 7.4) to 11.96% (pH 6.5 + 10 mM GSH + 20 mM NAD + ), where NAD + The migration promoting effect of GSH> pH; after adding 2-DG ( Figure 15 B) The distance of cell migration toward the scratch area in each experimental group was reduced to varying degrees, especially in the condition of pH 6.5+10mM GSH+20mM NAD + Under these conditions, the migration rate ( Figure 16 ) decreased from 11.96% to 8.89%. The results showed that weak acid, reducing agent GSH and glycolysis metabolism initiator NAD + Tumor microenvironments such as TNF-α and TNF-α can significantly promote cell growth, while the addition of 2-DG can destroy glycolysis metabolism and inhibit cell migration ability.

[0099] Application Example 6

[0100] Verification of glycolysis inhibition on carbon dots uptake in HepG2 cells

[0101] The cells scratched 7 hours after the scratch in Application Example 5 were observed using a fluorescence microscope to observe the intracellular fluorescence distribution and fluorescence intensity changes of the carbon dots in the scratched area.

[0102] Figure 17 and Figure 18 This is the uptake analysis of carbon dots in HepG2 cells under the action of glycolysis inhibitor 2-deoxy-d-glucose (2-DG). Figure 17 As shown in A, when 2-DG is not added, weak acid, reducing (GSH) and glycolysis metabolism initiator nicotinamide adenine dinucleotide (NAD + ) and other tumor microenvironments can promote the uptake of carbon dots in HepG2 cells, and the ratio of red fluorescence to green fluorescence in cells is F Red / F Green Increased from 0.67 (pH 7.4) to 0.99 (pH 6.5 + 10 mM GSH + 20 mM NAD + , Figure 18 ), where NAD + The migration promoting effect of GSH> pH; after adding 2-DG ( Figure 17 B) The red fluorescence intensity in cells of each experimental group decreased to varying degrees, especially in the pH 6.5+10mM GSH+20mMNAD group. + Under these conditions, F Red / F Green From 0.99 to 0.61 ( Figure 18 ). The results showed that weak acid, reducing agent GSH and glycolysis metabolism initiator NAD + The addition of 2-DG can significantly promote the uptake of carbon dots into cells, and the addition of 2-DG can destroy glycolysis metabolism and reduce NAD + With the increase of the carbon content, the green fluorescence intensity of carbon dots increased slightly, but the red fluorescence intensity decreased significantly.

[0103] In summary, the present invention has constructed a ratiometric fluorescent carbon dot for tracing nicotinamide adenine dinucleotide in liver cancer glycolysis metabolism. O-phenylenediamine and 2,2-dithiosalicylic acid are used as carbon and nitrogen sources, and H3PO4 with a volume fraction of 80% is used as the reaction solvent. The carbon dots are prepared by a solvothermal method. The preparation method is simple, and the obtained ratiometric fluorescent carbon dots have uniform particle size distribution, stable physical and chemical properties, and are basically non-cytotoxic. NAD + The dependent carbon dots can produce "green-red" ratio fluorescence in HepG2 cells, which can quickly detect intracellular NAD +Fluorescence intensity changes at concentrations between 0 and 20 mM / L. Addition of the glycolysis inhibitor 2-DG significantly reduced cell migration and the red fluorescence intensity of the carbon dots. Therefore, the ratiometric fluorescent carbon dots prepared by the solvothermal method using o-phenylenediamine, 2,2-dithiosalicylic acid, and H₃PO₄ are an excellent and highly effective tool for the early diagnosis and treatment of liver cancer.

[0104] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0105] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a ratiometric fluorescent carbon dot for tracing nicotinamide adenine dinucleotide in liver cancer glycolysis in an early diagnosis reagent for liver cancer, characterized in that: The carbon dots are obtained by a solvothermal method using o-phenylenediamine and 2,2'-dithiosalicylic acid as carbon and nitrogen sources; The molar ratio of o-phenylenediamine to 2,2'-dithiosalicylic acid is 1:1; The reaction solvent of the solvothermal method is H3PO4 with a volume fraction of 80%.

2. The use according to claim 1, characterized in that The method for preparing the carbon dots comprises the following steps: (1) Dissolve o-phenylenediamine and 2,2'-dithiosalicylic acid in 80% H3PO4 to a concentration of 10 mM each in the mixture; (2) The mixed solution is transferred to a polytetrafluoroethylene autoclave for solvothermal reaction, and the collected sample is centrifuged, dialyzed and freeze-dried to obtain a finished product.

3. The use according to claim 2, characterized in that In the step (2), the reaction temperature of the solvent thermal reaction is 180°C and the reaction time is 12 h; The molecular weight cut-off of the dialysis bag used for dialysis was 500 Da, and the dialysis time was 48 h.

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