WO2021169744A1 - 一种荧光碳量子点及其制备方法和在制备抗肿瘤药物增敏剂中的用途 - Google Patents

一种荧光碳量子点及其制备方法和在制备抗肿瘤药物增敏剂中的用途 Download PDF

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WO2021169744A1
WO2021169744A1 PCT/CN2021/074781 CN2021074781W WO2021169744A1 WO 2021169744 A1 WO2021169744 A1 WO 2021169744A1 CN 2021074781 W CN2021074781 W CN 2021074781W WO 2021169744 A1 WO2021169744 A1 WO 2021169744A1
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oil
sugar
ecnds
preparation
carbon quantum
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French (fr)
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黄容琴
王�义
霍涛涛
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复旦大学
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    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/65—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing carbon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0065—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle
    • A61K49/0067—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle quantum dots, fluorescent nanocrystals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B32/00—Carbon; Compounds thereof
    • C01B32/15—Nano-sized carbon materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the invention belongs to the technical field of medicine, and relates to a biologically safe anti-tumor drug sensitizer, in particular to a fluorescent carbon quantum dot, a preparation method thereof, and use in the preparation of an anti-tumor drug sensitizer.
  • the fluorescent carbon quantum dots prepared by the invention have uniform size, stable and adjustable fluorescence emission, high water dispersibility, high safety and good biocompatibility, and can competitively inhibit the uptake of glucose by tumor cells and interfere with the growth of tumor cells Proliferation, increase the sensitivity of tumor cells to various anti-tumor agents, improve tumor treatment effects, and can be used for combined treatment of various tumors.
  • Adjuvant first appeared in traditional Chinese medicine. Adjuvants can enhance the therapeutic effects of the monarch and minister drugs and eliminate or slow down the toxicity and potency of the monarch and minister drugs. At the same time, they play an auxiliary role in the treatment of the monarch drugs.
  • the "adjuvant” concept has applications in both vaccines and chemotherapy. Studies have reported that under the combined application of vaccines and adjuvants, the body's immune response to antigens is enhanced, which improves the efficacy of vaccines, reduces the amount of antigens and the number of immunizations; anti-tumor drug sensitizers are an extension of adjuvants in tumor treatment .
  • An anti-tumor drug sensitizer refers to the use of a small dose of the substance in combination with an anti-tumor drug to increase the sensitivity of tumor cells to the drug and reduce the toxic and side effects of the drug through different coordination mechanisms.
  • most of the sensitizers currently used in tumor treatment are another type of preparations or traditional Chinese medicine extracts that have toxic side effects that are different from those of anti-tumor drugs, and other side effects may occur during use.
  • most of the current sensitizers play a synergistic effect on specific drugs themselves, and have little effect on the development of metabolic behavior of tumor cells, and their use has limitations, that is, only work on individual drugs or cancer patients, and individual differences are large. Therefore, the development of new anti-tumor drug sensitizers that can be widely used in tumor treatment with high safety and good biocompatibility is of vital importance to tumor treatment.
  • Sugar is composed of carbon, hydrogen and oxygen, which provides a carbon skeleton for the synthesis of proteins, nucleic acids and lipids and participates in cellular communication. It is the most important energy source for living organisms. For tumor cells, an adequate supply of sugar is the prerequisite for their rapid growth and proliferation and resistance to external stimuli. In recent years, the Warburg effect based on sugar and sugar metabolism has been widely concerned about the development of tumors and cancer treatment. That is, tumor cells mainly rely on glycolysis to produce energy when oxygen is sufficient. Most of the glucose absorbed by tumor cells undergoes glycolysis to produce lactic acid, and a large number of intermediate products produced during glycolysis provide a material basis for the synthesis of nucleotides, phospholipids and proteins required for the abnormal proliferation of tumor cells.
  • Tumor cells have far more sugar requirements than normal cells.
  • Tumor cells usually use glucose transporters (GLUT1, etc.) overexpressed on their surface to achieve their sugar uptake. Therefore, it is necessary to study how to prevent GLUT1 from mediating tumor sugar uptake to inhibit tumor cell growth or change the growth state of tumor cells to make tumors.
  • GLUT1 glucose transporters
  • the inventor of the present application intends to provide a biologically safe anti-tumor drug sensitizer and its preparation method and application.
  • the present invention uses biologically safe raw materials to develop a simple, green and fully biocompatible
  • the synthesis method prepares carbon nanodots, and further develops the biological effects of carbon dots to realize their wider biological applications.
  • the purpose of the present invention is to provide a biologically safe anti-tumor drug sensitizer based on the current state of the art, in particular to a fluorescent carbon quantum dot, a preparation method thereof, and use in the preparation of an anti-tumor drug sensitizer.
  • the present invention adopts biologically safe raw materials to develop a simple, green and completely biocompatible synthesis method to prepare carbon nanodots.
  • the prepared fluorescent carbon quantum dots have uniform size, stable and adjustable fluorescence emission, high water dispersibility, and safety High and good biocompatibility, it can competitively inhibit the uptake of glucose by tumor cells, interfere with the growth and proliferation of tumor cells, improve the sensitivity of tumor cells to various antitumor agents, and improve the effect of tumor treatment. It can be used for various types of tumors. Combination therapy.
  • the present invention uses green food processing technology, preferably a simple heating and stirring method, and uses sugar as a raw material and oil as a reaction solvent to prepare fluorescent carbon quantum dots (eCNDs).
  • eCNDs fluorescent carbon quantum dots
  • the fluorescent carbon quantum dot of the present invention has a particle size between 2 and 14 nm.
  • the fluorescent carbon quantum dot contains four elements of C, N, O, and H, and the atomic content of C, O, and N on the surface is respectively 33% ⁇ 70at%, 28% ⁇ 35at%, 1% ⁇ 5%at.
  • the mass ratio of C is 40% to 58%
  • the mass ratio of H is 5% to 8%
  • the mass ratio of O is 39% to 55%
  • the mass ratio of N is 2% to 5 %.
  • the fluorescent carbon quantum dots of the present invention have a GPC test molecular weight of 8,000 to 20,000.
  • the present invention provides a simple eCNDs synthesis method, which includes the steps:
  • step (2) After removing the unreacted sugar and oil solvent from the crude reaction product obtained in step (1), fluorescent carbon quantum dots (eCNDs) are obtained.
  • eCNDs fluorescent carbon quantum dots
  • the sugar in step (1) is edible sugar, and the oil is edible oil;
  • the edible sugar includes white granulated sugar, soft white sugar, red granulated sugar, polycrystalline rock sugar, monocrystalline rock sugar, cube sugar, and borneol sugar And brown sugar;
  • the edible oils include corn oil, rapeseed oil, peanut oil, hemp oil, corn oil, olive oil, camellia oil, palm oil, canola oil, sunflower oil, soybean oil, sesame oil, grape seed oil
  • the round-bottomed flask in step (1) is a type commonly used in laboratories.
  • the reaction temperature in step (1) is 150-199°C.
  • reaction time in step (1) is 3-9 minutes.
  • the mass ratio of the solvent to the reactant in step (1) is between 1:1 and 5:1.
  • the method for removing unreacted sugar and oil in step (2) is selected from any one or any combination of extraction, vacuum distillation, dialysis, or high-speed centrifugation.
  • the method for removing unreacted sugar and oil is extraction-assisted double-layer membrane dialysis combined with vacuum distillation, and the specific steps are as follows:
  • step (1) Wash the crude reaction product obtained in step (1) several times with a mixed solution of one or more reagents such as dichloromethane, n-hexane, butyl acetate and chloroform;
  • one or more reagents such as dichloromethane, n-hexane, butyl acetate and chloroform;
  • step a) the crude product obtained by washing is extracted with a mixed solution of dichloromethane, n-hexane, butyl acetate or chloroform and water to obtain an aqueous solution of eCNDs;
  • step c) Vacuum rotary evaporation of the eCNDs aqueous solution obtained in step b) to obtain a concentrated carbon quantum dot aqueous solution; preferably, the reaction conditions of step c) are 35-55°C water bath rotary evaporation, and the vacuum pressure is below -100KPa;
  • step c) Dialysis the product obtained in step c); the dialysis conditions are: 300K/18mm and 3500K/45mm dialysis bags, the dialysis time is 48-96h, preferably, the dialysis time is 72h, and the reaction temperature is room temperature;
  • the eCNDs provided by the present invention have a size distribution of 2-14 nm.
  • the core has obvious carbon lattice stripes, the size of the crystalline carbon core is 2-8nm, and the hydration diameter after adding surface groups is 5-14nm. The results are shown in Figures 1 and 2.
  • the zeta potential of the eCNDs provided by the present invention is -17-28mV, and the result is shown in FIG. 3.
  • the eCNDs provided by the present invention contain carbon, hydrogen, oxygen and nitrogen in their skeleton, and the mass ratio of C is 40% to 58%, the mass ratio of H is 5% to 8%, and the mass ratio of O is 39% to 39%.
  • the mass ratio of 55% to N is 2% to 5%.
  • the atomic content of carbon, oxygen, and nitrogen on the surface are 33 to 70 at%, 28 to 35 at%, and 1 to 5 at%, respectively.
  • the eCNDs provided by the present invention have a molecular weight of 8000 to 20000, and the eCNDs can be split into ion fragments with mass-to-charge ratios of 200-480, 750-920, and 1000-1600 through MALDI-TOF.
  • the eCNDs provided by the present invention are irradiated with a salt concentration (K+, Na+, etc.) of 0-5Mol/L, an aqueous solution with a pH of 4-9, or strong ultraviolet light (2.5W/cm 2 ) for 0-48h, There is no obvious change in the fluorescence emission from 375 to 580 nm, and it has good fluorescence stability.
  • the eCNDs provided by the present invention can perform in vivo tumor imaging in all directions and multiple angles through the excitation of different fluorescence channels.
  • the fluorescence emission wavelength increases with the increase of the excitation light wavelength, and it has the properties of multicolor (blue, green, red) light emission.
  • the wavelength of maximum excitation light and emission light are 440nm and 599nm respectively.
  • the eCNDs when the aqueous solution concentration is less than 120 mg/mL, the eCNDs are dispersed in different media including water, phosphate buffer, cell culture fluid and fetal bovine serum. When the centrifugal speed is less than 16000 rpm and the centrifugation is within 10 minutes, There is no precipitation in the sample solution, which proves that the prepared eCNDs have good water solubility and dispersibility.
  • the eCNDs provided by the present invention have good biological safety, and when the concentration is less than 4 mg/mL, the co-incubation with cells for less than 6 hours does not affect the growth and proliferation of normal cells (such as HA 1800 and MCF 10). After feeding SD rats at a dose of less than 200 mg/kg, within 12 weeks of monitoring, no inflammation and lesions occurred in all major organs and tissues (heart, liver, spleen, lung, kidney, and brain).
  • Blood routine average red blood cell hemoglobin concentration (MCHC), hematocrit (HCT), red blood cell (RBC), average red blood cell volume (MCV), platelet (PLT), average red blood cell hemoglobin (MCH), white blood cell (WBC), hemoglobin ( HGB) and blood biochemical indicators: creatinine (CK), creatinine acid (Crea), urea (Urea), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), albumin (ALB), alanine aminotransferase (ALT) and total bilirubin (TBIL) are normal, consistent with the indicators of normal rats, indicating that the carbon quantum dots have high safety and biocompatibility.
  • the eCNDs provided by the present invention have abundant surface functional groups such as carboxyl groups, carbonyl groups, hydroxyl groups and amino groups on the surface.
  • the eCNDs provided by the present invention can be targeted and enriched at tumor sites for tumor imaging in vivo.
  • the eCNDs provided by the present invention due to the presence of sugar residues on the surface, have physical and chemical properties similar to glucose, and can react with Fehling's reagent to produce brick red precipitates.
  • the eCNDs provided by the present invention have sugar residues on the surface, but do not have the energy supply properties of glucose, and will not cause the increase of blood glucose concentration in the organism and the increase of animal weight.
  • the eCNDs provided by the present invention due to the presence of sugar residues on the surface, are similar in structure to glucose, and can competitively bind to cell surface receptors (GLUT1, GLUT2 or GLUT4, etc.), inhibiting the uptake of glucose by tumor cells and reducing intracellular
  • GLUT1, GLUT2 or GLUT4, etc. cell surface receptors
  • the production of ATP reduces the body's energy supply to tumor cells, disturbs the growth and proliferation of tumor cells, thereby increasing the sensitivity of tumor cells to drugs and improving the therapeutic effect.
  • the eCNDs provided by the present invention are based on the Warburg effect. By interfering with the sugar uptake of tumor cells, it affects the production of tumor cell ATP and reduces energy supply, thereby affecting the growth and proliferation of tumor cells, reducing the drug resistance of tumor cells, and improving tumors. Chemosensitivity of cells can improve the effect of chemotherapy, has broad-spectrum characteristics, can be used to prepare various anti-tumor drug sensitizers, is suitable for the sensitization of various cancer treatment methods, and improves the efficiency of tumor treatment.
  • the present invention provides the application of eCNDs in tumor imaging in vivo.
  • the present invention provides that eCNDs affect tumor cell proliferation, enhance the sensitivity of treatment, and the effect is more obvious when the tumor cells are starved (glucose deficiency).
  • the eCNDs of the present invention can be prepared as sensitizers for clinical anti-tumor drug treatment.
  • the eCNDs are used as sensitizers of anti-tumor drugs such as doxorubicin, sorafenib, temozolomide, etc., and can significantly improve the therapeutic effect of anti-tumor drugs on tumors.
  • anti-tumor drugs include chemotherapy drugs and/or immunotherapy drugs and other physical therapy drugs.
  • the present invention provides an anti-tumor drug composition, including an anti-tumor drug and eCNDs; the anti-tumor drug composition may also include a pharmaceutically acceptable carrier.
  • the tumors include glioma, liver cancer, breast cancer, lymphoma, ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testis At least one of cancer, gastric cancer or other solid tumors.
  • the outstanding advantages and characteristics of the present invention are that edible sugar is used as a raw material, and edible oil is used as a reaction solvent, and a chemotherapeutic sensitizer fluorescent carbon quantum dots (eCNDs) are prepared through green food processing technology (simple heating and stirring).
  • the preparation method of the present invention is simple, time-saving and easy to implement, low cost, can effectively avoid the addition of toxic raw materials and reagents, and has high safety.
  • the prepared eCNDs have the characteristics of small particle size, good dispersion, stable fluorescence, and high safety. , ECNDs can be enriched in tumor sites through the EPR effect.
  • chemotherapeutic sensitizer fluorescent carbon quantum dots are a sensitizer that broadly enhances the therapeutic effect of tumors, and can be widely applied to the combined treatment of various tumors.
  • the invention provides new research concepts and ideas for tumor treatment.
  • Figure 11 Dispersibility evaluation: Disperse eCNDs in different media, and observe the changes in dispersion before and after centrifugation at 16000 rpm/10 min.
  • Figure 12 A fluorescence stability evaluation: the influence of salt (Na/K) on the fluorescence stability of eCNDs;
  • Figure 15 Time-dependent cellular uptake of eCNDs.
  • Figure 19 The results of eCNDs interfering with cell proliferation.
  • Figure 20 The results of eCNDs inhibiting the glucose uptake of tumor cells.
  • PET/CT imaging research results of the effect of eCNDs concentration on glucose uptake in tumor tissues.
  • Figure 25 The results of the study on the effect of eCNDs on the body weight of SD rats.
  • Figure 28 The pharmacodynamic evaluation of eCNDs combined with temozolomide for the treatment of glioma (eCNDs intravenously and temozolomide orally) cell apoptosis experiment results.
  • Figure 29 The pharmacodynamic evaluation of eCNDs combined with temozolomide for the treatment of glioma (eCNDs intravenously and temozolomide orally) results, where A: body weight of tumor-bearing mice, B: survival curve.
  • Figure 32 The effect of eCNDs on the proliferation and growth of tumors and normal cells.
  • Figure 33 The effect of eCNDs on ATP production in tumor and normal cells.
  • Figure 35 The results of the pharmacodynamic evaluation of eCNDs combined with sorafenib for the treatment of liver cancer (eCNDs intravenously and sorafenib orally),
  • A tumor volume
  • B body weight of tumor-bearing mice.
  • Figure 37 The pharmacodynamic evaluation of eCNDs combined with sorafenib for the treatment of liver cancer (both eCNDs and sorafenib are administered orally),
  • Figure 38 The pharmacodynamic evaluation of eCNDs combined with sorafenib for the treatment of liver cancer (both eCNDs and sorafenib are administered orally) cell apoptosis experiment results.
  • Figure 39 Biological safety evaluation of eCNDs: main organ tissue sections and HE staining results.
  • oil corn oil, rapeseed oil, peanut oil, hemp oil, corn oil, olive oil, camellia oil, palm oil, canola oil, sunflower oil, soybean oil, sesame oil, grape seed oil, walnut oil, and peony seed
  • sugar one or more of white sugar, soft white sugar, red sugar, polycrystalline rock sugar, single crystal rock sugar, cube sugar, borneol sugar, yellow sugar, etc.
  • Oil corn oil, rapeseed oil, peanut oil, hemp oil, corn oil, olive oil, camellia oil, palm oil, canola oil, sunflower oil, soybean oil, sesame oil, grape seed oil, walnut oil, and peony seed
  • sugar white sugar, soft white sugar, red sugar, polycrystalline rock sugar, single crystal rock sugar, cube sugar, borneol sugar, yellow sugar, etc.
  • sugar white sugar, soft white sugar, red sugar, polycrystalline rock sugar, single crystal rock sugar, cube sugar, borneol sugar, yellow sugar, etc.
  • oil corn oil, rapeseed oil, peanut oil, hemp oil, corn oil, olive oil, camellia oil, palm oil, canola oil, sunflower oil, soybean oil, sesame oil, grape seed oil, walnut oil, and peony seed oil Etc.
  • the resulting product is extracted, rotary steamed and dialyzed to remove residual sugar and oil to obtain eCNDs.
  • sugar white sugar, soft white sugar, red sugar, polycrystalline rock sugar, single crystal rock sugar, cube sugar, borneol sugar, yellow sugar, etc.
  • sugar white sugar, soft white sugar, red sugar, polycrystalline rock sugar, single crystal rock sugar, cube sugar, borneol sugar, yellow sugar, etc.
  • oil corn oil, rapeseed oil, peanut oil, hemp oil, corn oil, olive oil, camellia oil, palm oil, canola oil, sunflower oil, soybean oil, sesame oil, grape seed oil, walnut oil, and peony seed oil Etc.
  • oil corn oil, rapeseed oil, peanut oil, hemp oil, corn oil, olive oil, camellia oil, palm oil, canola oil, sunflower oil, soybean oil, sesame oil, grape seed oil, walnut oil, and peony seed oil Etc.
  • Example 5 The eCNDs prepared in Example 5 were observed through JEM-2010 transmission electron microscope, and the results showed that the nanodots had a uniform size, as shown in FIG. 1.
  • the eCNDs prepared in Example 5 were dispersed in an aqueous solution, and their particle size distribution was characterized by a dynamic light scattering method. The results showed that the prepared eCNDs had a particle size of 2-14 nm, please see FIG. 2.
  • the prepared eCNDs core has obvious carbon lattice stripes, the size of the crystalline carbon core is 2-8nm, and the hydration diameter after adding surface groups is 5-14nm, as shown in Figure 1.
  • Example 5 The eCNDs prepared in Example 5 were dispersed in an aqueous solution, and the zeta potential of the particle size Zeta potential meter was measured. The result showed that the zeta potential of the prepared eCNDs solution was -18 to -24 mV, as shown in FIG. 3.
  • the eCNDs prepared in Example 5 were characterized by 13 C nuclear magnetic resonance, 1 H nuclear magnetic resonance and infrared spectroscopy.
  • the 13 C NMR spectrum shows (as shown in Figure 4A) that the surface of the eCNDs prepared in Example 5 has sp 3 hybridized aliphatic carbon (CO, CC, and CH) signals at a chemical shift ⁇ of 0-60 ppm; eCNDs are 1
  • the 1 H-NMR signal of C-OH appears at a chemical shift ⁇ of 4 to 5 ppm (as shown in Figure 4B); infrared spectroscopy is used to characterize the eCNDs prepared in Example 5, and the result is shown in Figure 4C.
  • the surface of the eCNDs prepared in Example 5 may have surface functional groups such as carboxyl, carbonyl, hydroxyl, and amino groups.
  • the carbon, hydrogen, oxygen, and nitrogen elements in the eCNDs prepared in Example 5 were characterized by an element analyzer.
  • the results show that the framework of eCNDs provided by the present invention contains carbon, hydrogen, oxygen and nitrogen elements, and the mass ratio of C is 40.53% to 43.82%, the mass ratio of H is 5.73% to 7.94%, and the mass ratio of O is The mass ratio of 51.86% to 54.97% to N is 2.57% to 4.32%, please see attached figure 5.
  • the atomic content of carbon, oxygen, and nitrogen on the surface are respectively: 60 to 70 at%, 28 to 35 at%, and 1 to 5 at%, as shown in Figure 6.
  • the molecular weight of the eCNDs prepared in Example 5 was characterized by gel chromatography (GPC). As shown in Figure 7, the molecular weight of the eCNDs was 8,000 to 20,000.
  • the molecular composition of the eCNDs prepared in Example 5 was characterized by MALDI TOF, and the result is shown in FIG. 8.
  • the resulting spectrum of eCNDs there are molecular peaks in the m/z ranges of 200 to 480, 750 to 920, and 1000 to 1600.
  • the eCNDs prepared in Example 5 were dispersed in an aqueous solution and tested by a fluorescence spectrometer. The results showed that the eCNDs aqueous solution can emit fluorescence (475 nm to 599 nm) after ultraviolet excitation (375 nm to 440 nm), and the fluorescence emission wavelength increases as the excitation wavelength increases.
  • the wavelengths of the maximum excitation light and emission light are 440nm and 599nm, respectively, as shown in Figure 9.
  • the eCNDs prepared in Example 5 were diluted with water in a concentration gradient, and the linear relationship between the absorbance and the concentration was investigated by an ultraviolet spectrophotometer (270nm). The results showed that the eCNDs in the concentration range of 25 ⁇ 300 ⁇ g/mL and its corresponding absorbance had good linearity. , Can be used for quantitative analysis of related eCNDs, as shown in Figure 10.
  • the eCNDs prepared in Example 5 were dispersed in different media including water, phosphate buffer, cell culture medium and fetal calf serum. The dispersion of the carbon quantum dot solution before and after centrifugation was observed. No obvious precipitation was observed in the media, and the color of the solution did not change significantly, indicating that the prepared eCNDs have good dispersibility in different media, as shown in Figure 11.
  • the eCNDs prepared in Example 5 were respectively exposed to ultraviolet light environment (2.5W/cm 2 , irradiation 0 ⁇ 48h), dispersed in different concentrations of salt (K+, Na+, etc., 0 ⁇ 5Mol/L) aqueous solution and pH (4 ⁇ 4 ⁇ 9)
  • the fluorescence stability was investigated, and the results showed that the fluorescence characteristics of 375 ⁇ 580nm did not change significantly under the interference of ultraviolet light, NaCl and pH, and it had good fluorescence stability, as shown in Figure 12A. , B and C are shown.
  • the eCNDs prepared in Example 5 were added to the culture medium of tumor cells (U87 and HepG2) and normal cells (1800 and HL 7702) at different concentrations, and incubated with a cell incubator for 6 hours.
  • the cells of eCNDs were investigated by CCK-8 toxicity. The results show that eCNDs have low cytotoxicity and high safety, as shown in Figure 13.
  • the eCNDs prepared in Example 5 were added to the U87 cell culture medium at different concentrations.
  • the cell uptake was investigated after incubation for 1 h. The results showed that the cell uptake of eCND was concentration-dependent. The better cellular uptake is shown in Figure 14.
  • the eCNDs prepared in Example 5 were added to the U87 cell culture medium at 200 ⁇ g/mL, and their cell uptake was investigated at different time periods. The results showed that the cell uptake of eCND is time-dependent, and eCND has the best 2h Cell uptake, as shown in Figure 15.
  • eCNDs prepared in Example 5 were co-incubated with different inhibitors to study the transmembrane transport mechanism of chemotherapeutic sensitizers.
  • the results show that eCND is mainly transported across the cell membrane through the glucose receptor GLUT1 in the form of passive transport, as shown in Figure 16.
  • Example 5 After the eCNDs prepared in Example 5 were incubated with U 87 cells, the cells were labeled with GLUT-1 fluorescent probe (TRITC-labeled GLUT1 probe), and the fluorescence distribution of eCNDs and GLUT-1 was observed under a confocal microscope . The results showed that the fluorescence of eCNDs mostly overlapped with the fluorescence of GLUT-1, which proved the mechanism of eCNDs transmembrane transport through GLUT1, as shown in Figure 17.
  • GLUT-1 fluorescent probe TRITC-labeled GLUT1 probe
  • the eCNDs (200 ⁇ g/mL) prepared in Example 5 were added to the U87 cell and 1800 cell culture medium and incubated with the cells.
  • the quartz crystal microbalance (QCM-D) was used to determine the frequency ( ⁇ f) and dissipation value of the quartz crystal.
  • ( ⁇ D) is an evaluation index to study the interaction between eCNDs and cells. The results showed that compared with normal cells, eCNDs are more likely to be adsorbed on the surface of tumor cells with high GLUT-1 expression, as shown in Figure 18.
  • the eCNDs (200 ⁇ g/mL) prepared in Example 5 were added to the cell culture medium and incubated with the cells (U87 and 1800) for 12 hours, then the medium was changed to a sugar-free medium and incubated for another 12 hours, and then changed back to containing eCNDs (200 ⁇ g/mL) culture medium to continue to incubate the cells.
  • the cell viability real-time monitoring system the state of the cells is monitored. The results show that compared with normal cells, eCNDs can significantly inhibit the growth and proliferation of tumor cells, as shown in Figure 19.
  • the eCNDs prepared in Example 5 were added to the culture dishes of U87 cells at different concentrations (100, 200, and 400 ⁇ g/mL). After 30 minutes, glucose fluorescent probes were added to observe the glucose uptake of the cells. The results show that eCNDs can inhibit the uptake of glucose by cells, and the inhibition is proportional to its concentration, as shown in Figure 20.
  • the eCNDs prepared in Example 5 were intravenously injected into mice bearing glioma in situ. At different time periods, the fluorescence distribution in vivo was observed by a small animal in vivo imaging instrument, indicating that fluorescent carbon quantum dots can accumulate in the brain through the EPR effect Perform intravital imaging of the glioma site, as shown in Figure 21.
  • the eCNDs prepared in Example 5 were injected into mice with in situ brain glia at different concentrations via the tail vein, and the glucose content at the tumor site was monitored by PET/CT.
  • the results show that eCNDs can effectively inhibit the glucose uptake of tumor cells, and the effect is most obvious when the concentration of eCNDs is 2 mg and intravenous injection for 30 minutes, as shown in Figure 22.
  • the eCNDs and glucose prepared in Example 5 were characterized by 1 H nuclear magnetic resonance method and infrared spectroscopy, respectively, and the structural characteristics were analyzed.
  • the result is shown in Figure 23, which shows that the chemical shift ⁇ of eCNDs is 2.7 ⁇ 3.7ppm, 4.2 ⁇ 4.8 ppm, glucose has a similar C 2-6 -H and C 1 H-NMR signals of 1-6 -OH;
  • IR spectra show, eCNDs in 3500 ⁇ 3700cm -1, 2850 ⁇ 2960cm -1, 1710 ⁇ 1750cm - 1.
  • the analysis of the above experimental results can indicate that there may be functional groups similar to the glucose surface groups on the surface of eCNDs.
  • the eCNDs and glucose prepared in Example 5 were respectively orally administered to SD rats (both doses are 200 mg/kg), and the trend of changes in the blood glucose concentration of the rats was monitored.
  • the phenomenon of large fluctuations in a short period of time, eCNDs has no effect on the blood glucose concentration of rats, indicating that eCNDs may not have the characteristics of glucose supply.
  • the eCNDs prepared in Example 5 were orally administered to SD rats (200 mg/kg), and fasted rats were used as negative controls. Rats that had eaten normal food and edible sugar (200 mg/kg) were used as negative controls. Monitor the change trend of rat body weight to study the biological characteristics of eCNDs. The results showed that compared with the rats that were eating and sugar, the weight change trend of the rats that consumed eCNDs was consistent with that of fasted rats, indicating that eCNDs could not provide energy for the body, as shown in Figure 25.
  • eCNDs prepared in Example 5 were added to U87 cell culture dishes at different concentrations and (or) 20 ⁇ g/mL temozolomide, and the cytotoxicity was evaluated by CCK-8. The results are shown in Figure 26, eCNDs significantly improved the cytotoxicity of temozolomide.
  • eCNDs prepared in Example 5 were added to U87 cell culture dishes at different concentrations and (or) 20 ⁇ g/mL temozolomide, and the cytotoxicity was evaluated by LIVEDEAD. The results are shown in Figure 27, eCNDs significantly increased the cytotoxicity of temozolomide.
  • the eCNDs prepared in Example 5 were administered intravenously to mice bearing glioma in situ, and temozolomide was administered orally. The vital signs and life cycle of the mice were tested. The results showed that the combined application of eCNDs and temozolomide killed Glioma cells (as shown in Figure 28) improve the quality of life of tumor-bearing mice (as shown in Figure 29A) and life cycle (as shown in Figure 29B).
  • the eCNDs prepared in Example 5 were added to Hep G2, MCF-7, A549, H1299, HCT 116, 4T1 and PANC-1 cell culture dishes at a concentration of 200 ⁇ g/mL and/or 20 ⁇ g/mL DOX, respectively.
  • CCK-8 was evaluated for cytotoxicity. As shown in Figure 30, eCNDs significantly improved the cytotoxicity of DOX.
  • the eCNDs prepared in Example 5 were added to the cell culture dishes of MCF-7 and MCF-10 at a concentration of 200 ⁇ g/mL and/or 20 ⁇ g/mL DOX, respectively, and the cytotoxicity was evaluated by CCK-8. The results are shown in Figure 31, eCNDs significantly increased the toxicity of DOX to tumor cells.
  • the eCNDs prepared in Example 5 were added to the cell culture dishes of Hep G2 and HL 7702 at different concentrations, and the cytotoxicity was evaluated by CCK-8. The results are shown in Figure 32. Compared with normal cells, eCNDs significantly inhibited the growth and proliferation of tumor cells under long-term intervention.
  • the eCNDs prepared in Example 5 were added to the cell culture dishes of Hep G2 and HL 7702 at different concentrations, respectively, and the ATP content in the cells was evaluated by the ATP fluorescent probe. The results are shown in Figure 33. Compared with normal cells, eCNDs significantly inhibited tumor cell ATP production.
  • ECNDs 5 prepared in Example sorafenib administered in a concentration of 400 ⁇ g / mL with various concentrations of Hep G2 cells to the petri dish, the value of IC 50 was calculated for 24 hours were evaluated. The results are shown in Figure 34, eCNDs significantly increased the cytotoxicity of sorafenib.
  • eCNDs prepared in Example 5 and sorafenib were used in combination with different treatment schemes in mice with liver cancer subcutaneous tumors, through changes in tumor volume, body weight, and cell apoptosis.
  • the pharmacodynamic evaluation was carried out in the death experiment. The results are shown in Figures 35A and B, 36, sorafenib combined with eCNDs treatment significantly inhibited tumor growth.
  • Example 5 After the eCNDs prepared in Example 5 were fed SD at a dose of less than 200 mg/kg for 12 weeks, their main organs were stained for biochemical tissue sections, as shown in Figure 39, their blood routine and blood biochemical indicators were tested to investigate their biological safety sex. The results are shown in Figure 40. There were no inflammatory lesions in all organs of SD rats that consumed eCNDs. The blood routine and blood biochemical indexes were normal, which were consistent with those of normal rats, indicating that the carbon quantum dots have high safety and biological characteristics. compatibility.

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Abstract

本发明属医学技术领域，涉及一种生物安全的抗肿瘤药物增敏剂及其制备方法和应用。本发明以糖为原料、以油为反应溶剂通过类似于传统烹饪的加热搅拌制备荧光碳量子点。本发明的制备方法较传统碳量子点制备方法简单、省时、易行，制备条件要求简单，成本低，可大规模制备。制得的该荧光碳量子点尺寸均一、荧光发射稳定且可调、水分散性高、安全性高且生物相容性好。同时，该碳量子点的化学结构特征与葡萄糖相似，可竞争性抑制肿瘤细胞对葡萄糖的摄取，干扰肿瘤细胞的生长增殖，提高肿瘤细胞对各种抗肿瘤剂的敏感性，改善肿瘤治疗效果，是一种广泛增强肿瘤治疗效果的增敏剂，可推广应用于各类肿瘤的联合治疗。

Description

一种荧光碳量子点及其制备方法和在制备抗肿瘤药物增敏剂中的用途 技术领域
本发明属医药技术领域，涉及生物安全的抗肿瘤药物增敏剂，具体涉及一种荧光碳量子点及其制备方法和在制备抗肿瘤药物增敏剂中的用途。本发明制得荧光碳量子点，尺寸均一、荧光发射稳定且可调、水分散性高、安全性高且生物相容性好，能竞争性抑制肿瘤细胞对葡萄糖的摄取，干扰肿瘤细胞的生长增殖，提高肿瘤细胞对各种抗肿瘤剂的敏感性，改善肿瘤治疗效果，可用于各类肿瘤的联合治疗。
背景技术
现有技术公开了临床实践中若干抗肿瘤药物在肿瘤部位的正常药效不能完全发挥且对正常组织具有明显的副作用，因此，研究如何提高肿瘤细胞对抗肿瘤药物的敏感性，同时降低药物对正常组织的毒副作用，对于肿瘤治疗具有十分重要的临床实用价值。
“佐”最早出现在中药中，佐剂在加强君臣两类药物的治疗作用以及消除或减缓君臣两药的毒性和烈性的同时，在治疗中对君药起辅助作用。“佐剂”理念在疫苗和化疗中均有应用。有研究报道，在疫苗与佐剂的联合应用下，机体对抗原的免疫应答增强，提高了疫苗的功效，减少抗原用量和免疫次数；抗肿瘤药物增敏剂是佐剂在肿瘤治疗中的延伸。抗肿瘤药物增敏剂指采用微小剂量的该物质与抗肿瘤药物合用，通过不同的协作机制，增加肿瘤细胞对药物敏感性，减少药物的毒副作用。但目前在肿瘤治疗中应用的增敏剂大部分是具有有别于抗肿瘤药物毒副作用的另一类制剂或中药提取物，在使用期间会产生其他一些其他副作用。而且目前增敏剂大多针对特定药物本身发挥协同作用，对肿瘤细胞发展代谢行为影响较小，使用具有局限性，即仅对个别药物或者癌症病人起作用，个体差异性较大。因此，开发新的能够广泛应用于肿瘤治疗且安全性高，生物相容性好的抗肿瘤药物增敏剂对于肿瘤的治疗具有至关重要的作用。
糖由碳氢氧元素构成，为蛋白质、核酸和脂类的合成提供碳骨架构并参与细胞通信，是生物体最主要的能源。对肿瘤细胞来说，充足的糖源供应是其快速生长增殖，对抗外界刺激干扰的前提。近几年，以糖和糖代谢为基础的沃伯格效应对肿瘤发生发展和癌症治疗的影响被广泛关注，即在氧气充足的情况下肿瘤细胞也主要依赖糖酵解产生能量。肿瘤细胞吸收的葡萄糖大多数经过糖酵解生成乳酸，而糖酵解过程中产生的大量中间产物，为合成肿瘤细胞异常增殖所需的核苷酸、磷脂和蛋白质等提供物质基础，所以，基于沃伯格效应的糖代谢过程使得肿瘤细 胞对糖的需求远超正常细胞。通常肿瘤细胞通过其表面过度表达的葡萄糖转运体(GLUT1等)来实现其对糖的摄取，因此，研究如何阻止GLUT1介导肿瘤糖摄取来抑制肿瘤细胞生长或改变肿瘤细胞的生长状态，使得肿瘤细胞在恶性增殖、转移以及外对界刺激干扰的抵抗力减弱，对于癌症临床靶向治疗具有十分重要的意义。
纳米技术在生物医学领域的深入发展，其增强的EPR效应为肿瘤的诊疗开辟新的道路。具有良好生物相容性和安全性的多功能纳米材料在肿瘤治疗的应用中已经受到广泛的关注并展现出了良好的潜力。其中，碳量子点作为一种荧光碳纳米材料，具有尺寸大小可调、光稳定性好以及荧光寿命长、毒性低、生物相容性好和成本低等优点，越来越多被开发应用于肿瘤诊疗的研究。但目前研究的碳量子点在生物医学应用方面仍存在一些亟待解决的问题：(1)原料具有毒性或者生物安全性低，限制其生物体内应用；(2)合成方法复杂，反应条件较为严格，绿色且无生物毒性的方法较少，严重阻碍了其应用发展；(3)目前对碳量子点研究主要集中在生物成像和作为药物载体等方面，其进一步的生物学效应，特别是在肿瘤治疗中碳点-生物相互作用的机制不明，限制了碳量子点在癌症治疗上的广泛应用。
基于现有技术的现状，本申请的发明人拟提供一种生物安全的抗肿瘤药物增敏剂及其制备方法和应用，本发明采用生物安全的原料，发展简易、绿色且完全生物相容的合成方法制备碳纳米点，且对碳点生物效应进行更深层次的开发以实现其更广泛的生物学应用。
发明内容
本发明的目的是基于现有技术的现状，提供一种生物安全的抗肿瘤药物增敏剂具体涉及一种荧光碳量子点及其制备方法和在制备抗肿瘤药物增敏剂中的用途。
本发明采用生物安全的原料，发展简易、绿色且完全生物相容的合成方法制备碳纳米点，制得的荧光碳量子点，尺寸均一、荧光发射稳定且可调、水分散性高、安全性高且生物相容性好，能竞争性抑制肿瘤细胞对葡萄糖的摄取，干扰肿瘤细胞的生长增殖，提高肿瘤细胞对各种抗肿瘤剂的敏感性，改善肿瘤治疗效果，可用于各类肿瘤的联合治疗。
本发明通过绿色食品加工技术，优选简易加热搅拌的方法，以糖为原料，油为反应溶剂制得荧光碳量子点(eCNDs)。
本发明的荧光碳量子点，其粒径在2～14nm之间，所述的荧光碳量子点包含C、N、O、H四种元素，其表面的C、O、N的原子含量分别为33％～70at％，28％～35at％，1％～5％at。
本发明的荧光碳量子点，C的质量比为40％～58％，H的质量比为5％～8％,O的质量比为39％～55％和N的质量比为2％～5％。
本发明的荧光碳量子点，其GPC测试分子量为8000～20000。
本发明的荧光碳量子点，在3500～3700cm -1、2850～2960cm -1、1710～1750cm -1、1660～1700cm -1、1507cm -1、1250～1300cm -1、1100～1200cm -1处分别具有v(O-H)、v(C-H)、v(C＝O)、v(C-C)、v(C-H)、v(C-N)和v(C-O)特征红外吸收峰，且在化学位移δ为0～60ppm出现sp3杂化的 13C(C-O，C-C，C-N)NMR信号，以及在化学位移δ为4～5ppm出现C-OH的 1H-NMR信号。
本发明提供了一种简单的eCNDs合成方法，其包括步骤：
(1)将糖为原料、油为反应溶剂加入到圆底烧瓶中，在一定的温度下加热搅拌一定时间，得到反应粗产物；
(2)将步骤(1)得到的反应粗产物，去除未反应的糖和油溶剂后，得到荧光碳量子点(eCNDs)。
本发明中，步骤(1)中所述糖为可食用糖，油为可食用油；所述可食用糖包括白砂糖、绵白糖、赤砂糖、多晶体冰糖、单晶体冰糖、方糖、冰片糖以及黄砂糖等；所述可食用油包括粟米油、菜籽油、花生油、火麻油、玉米油、橄榄油、山茶油、棕榈油、芥花子油、葵花子油、大豆油、芝麻油、葡萄籽油、核桃油和牡丹籽油等本发明中，步骤(1)中所述圆底烧瓶为实验室常用类型。
本发明中，步骤(1)中所述反应温度为150～199℃。
本发明中，步骤(1)中所述反应时间为3～9min。
本发明中，步骤(1)中所述溶剂与反应物的质量比为1:1～5:1之间。
本发明中，步骤(2)中所述去除未反应的糖和油的方法选自萃取、减压蒸馏、透析或高速离心中的任一种或其任意组合。优选地，所述去除未反应的糖和油的方法为萃取辅助的双层膜透析联合减压蒸馏，具体步骤如下：
a)用二氯甲烷、正己烷、乙酸丁酯和三氯甲烷等一种或几种试剂的混合溶液洗涤步骤(1)中得到的反应粗产物若干次；
b)经步骤a)洗涤所得粗产物通过二氯甲烷、正己烷、乙酸丁酯或三氯甲烷与水的混合溶液进行萃取，得到eCNDs水溶液；
c)将步骤b)所得的eCNDs水溶液经真空旋蒸得到浓缩的碳量子点水溶液；优选地，步骤c)反应条件为35～55℃水浴旋蒸，真空压为-100KPa以下；
d)将步骤c)所得产物进行透析；透析条件为：300K/18mm和3500K/45mm规格透析袋，透析时长为48-96h，优选地，透析时长为72h，反应温度为室温；
e)得到所述的eCNDs，最终为纯水溶液体系，储存温度为常温。
本发明所提供的eCNDs，尺寸分布为2～14nm。其内核具有明显的碳晶格条纹，其结晶的碳核尺寸为2～8nm，附加表面基团后的水合直径为5～14nm，结果如图1和图2所示。
本发明所提供的eCNDs，Zeta电位为-17～28mV，结果如图3所示。
本发明所提供的eCNDs，其骨架含碳、氢、氧和氮元素，并且C的质量比为40％～58％，H的质量比为5％～8％,O的质量比为39％～55％和N的质量比为2％～5％。其表面的碳、氧、氮的原子含量分别为：33～70at％，28～35at％，1～5at％。
本发明所提供的eCNDs，其分子量为8000～20000，并且可通过MALDI-TOF将eCNDs裂解为质荷比为200～480、750～920以及1000～1600的离子碎片。
本发明所提供的eCNDs，在盐浓度(K+，Na+等)为0～5Mol/L水溶液、pH为4～9的水溶液或强紫外光(2.5W/cm 2)照射0～48h的条件下，其375～580nm的荧光发射无明显变化，具有较好的荧光稳定性。
本发明所提供的eCNDs，通过不同荧光通道的激发可以全方位多角度进行体内肿瘤成像，其荧光发射波长随着激发光波长的增加而增加，具有多色(蓝，绿，红)发光的性质，最大激发光和发射光的波长分别440nm和599nm。
本发明所提供的eCNDs，其水溶液浓度小于120mg/mL时，将eCNDs分散于不同介质包括水、磷酸盐缓冲液、细胞培养液和胎牛血清中，当离心速度小于16000rpm，离心10min以内时，样品溶液中均无沉淀，证明所制备的eCNDs具有良好的水溶性和分散性。
本发明所提供的eCNDs生物安全性好，在浓度小于4mg/mL时与细胞共孵育小于6h不影响正常细胞(如HA 1800和MCF 10)生长增殖。在小于200mg/kg剂量喂食SD大鼠后，大于12周的监测内，各主要脏器组织(心、肝、脾、肺、肾和脑)均无炎症以及病灶发生。血常规：平均红细胞血红蛋白浓度(MCHC)，红细胞比容(HCT)，红细胞(RBC),红细胞平均容量(MCV)，血小板(PLT)，平均红细胞血红蛋白量(MCH)，白细胞(WBC)，血红蛋白(HGB)和血液生化指标：肌酐(CK)，肌酐酸(Crea),尿素(Urea)，天冬氨酸转氨酶(AST)，乳酸脱氢酶(LDH)，白蛋白(ALB)，丙氨酸转氨酶(ALT)和总胆红素(TBIL)均正常，与正常大鼠指标一致，表明该碳量子点具有较高的安全性和生物相容性。
本发明所提供的eCNDs，其表面具有羧基、羰基、羟基和氨基等丰富的表面官能团。
本发明所提供的eCNDs，可以靶向富集于在肿瘤部位，进行体内肿瘤成像。
本发明所提供的eCNDs，由于其表面存在糖残基，具有与葡萄糖类似的理化性质，可与斐 林试剂发生反应产生砖红色沉淀。
本发明所提供的eCNDs，其表面存在糖残基，但不具有葡萄糖供能特性，不会引起生物体内血糖浓度的升高和动物体重增加。
本发明所提供的eCNDs，由于其表面存在糖残基，与葡萄糖结构类似，可竞争性地与细胞表面受体(GLUT1、GLUT2或GLUT4等)结合，抑制肿瘤细胞对葡萄糖的摄取，降低细胞内ATP的生成，减少机体对肿瘤细胞的能量供给，扰肿瘤细胞的生长增殖，进而提高肿瘤细胞对药物的敏感性，改善治疗效果。
本发明所提供的eCNDs是基于沃伯格效应，通过干扰肿瘤细胞的糖摄取，影响肿瘤细胞ATP的生成，降低能量供给，进而影响肿瘤细胞的生长增殖，降低肿瘤细胞的耐药性，提高肿瘤细胞的化疗敏感性，改善化疗效果，具有广谱特性，能够用于制备各种抗肿瘤药物的增敏剂，适用于各种癌症治疗方式的增敏，提高肿瘤治疗的效率。本发明提供eCNDs在肿瘤体内成像中的应用。
本发明提供eCNDs影响肿瘤细胞增殖，增强治疗的敏感性，在肿瘤细胞处于饥饿(葡萄糖缺乏)的情况下效果更加明显。
进一步，本发明的eCNDs可制备用作临床抗肿瘤药物治疗的增敏剂。所述eCNDs作为抗肿瘤药物如多柔比星、索拉菲尼和替莫唑胺等的增敏剂，可显著提高抗肿瘤药物对肿瘤的治疗效果。所述抗肿瘤药物包括化疗药物和/或免疫治疗药物以及其它物理治疗药物。
本发明提供了一种抗肿瘤药物组合物，包括抗肿瘤药物和eCNDs；所述抗肿瘤药物组合物还可包含药学上可接受的载体。
本发明中，所述肿瘤包括神经胶质瘤、肝癌、乳腺癌、淋巴瘤、卵巢癌、软组织肉瘤、成骨肉瘤、横纹肌肉瘤、膀胱癌、甲状腺癌、前列腺癌、头颈部鳞癌、睾丸癌、胃癌或者其他实体瘤中的至少一种。
本发明的突出优点及特征在于，以食用糖作为原料，食用油为反应溶剂，通过绿色食品加工技术(简易加热搅拌)，制备化疗增敏剂荧光碳量子点(eCNDs)。本发明的制备方法简单、省时易行、成本低，能有效避免有毒原料和试剂的加入，安全性高，制备的eCNDs具有粒径小、分散性好、荧光稳定以及安全性高等特点，而且，eCNDs能够通过EPR效应富集于肿瘤部位，同时，由于eCNDs表面糖残基的存在，可以竞争性抑制肿瘤细胞对葡萄糖的摄取，干扰肿瘤细胞的生长增殖，提高肿瘤细胞对各种抗肿瘤剂的敏感性，改善肿瘤治疗效果，所述的化疗增敏剂荧光碳量子点(eCNDs)为一种广泛增强肿瘤治疗效果的增敏剂，可推广应用于各 类肿瘤的联合治疗。本发明为肿瘤治疗提供了新的研究理念和思路。
附图说明
图1，eCNDs透射电子显微镜图。
图2，eCNDs粒径分布图。
图3，eCNDs Zeta电位图。
图4，eCNDsA： 13C核磁共振图谱，B： 1H核磁共振图谱，C：红外光谱分析图谱。
图5，eCNDs表面元素分析图谱。
图6，eCNDs元素质量比分析图谱。
图7，eCNDs GPC图谱。
图8，eCNDs MODILTOF分析图谱。
图9，eCNDs激发光(Ex)和发射光(Em)。
图10，eCNDs浓度与其紫外吸光度(A)线性结果。
图11分散性评价：将eCNDs分散于不同介质中，16000rpm/10min离心后观察其前后分散性变化。
图12，A荧光稳定性评价：盐(Na/K)对eCNDs荧光稳定性的影响；图12B荧光稳定性评价：溶液pH对eCNDs荧光稳定性的影响，图12C荧光稳定性评价：紫外光(2.5W/cm 2)对eCNDs荧光稳定性的影响。
图13，eCNDs的细胞毒性(6h)结果。
图14，eCNDs浓度依赖性细胞摄取结果。
图15 eCNDs时间依赖性细胞摄取结果。
图16 eCNDs跨膜转运机制研究结果。
图17 eCNDs-GLUT-1跨膜转运机制研究结果。
图18 QCM-D法考察eCNDs与不同细胞膜表面作用力差异性。
图19 eCNDs干预细胞增殖的研究结果。
图20 eCNDs抑制肿瘤细胞葡萄糖摄取的研究结果。
图21 eCNDs体内分布研究结果。
图22 PET/CT成像：eCNDs浓度对肿瘤组织葡萄糖摄取影响的研究结果。
图23 eCNDs与葡萄糖结构对比研究结果。
图24 eCNDs对SD大鼠血糖浓度影响研究结果。
图25 eCNDs对SD大鼠体重影响研究结果。
图26 eCNDs联合化疗药替莫唑胺的胶质瘤细胞毒性评价(CCK-8)。
图27 eCNDs联合化疗药替莫唑胺的胶质瘤细胞毒性评价(LIVEDEAD)。
图28 eCNDs联合替莫唑胺用于脑胶质瘤治疗的药效学评价(eCNDs静脉给药和替莫唑胺口服给药)细胞凋亡实验结果。
图29 eCNDs联合替莫唑胺用于脑胶质瘤治疗的药效学评价(eCNDs静脉给药和替莫唑胺口服给药)结果，其中，A：荷瘤小鼠体重，B：生存曲线。
图30 eCNDs提高DOX化疗效果的普适性细胞毒性评价(CCK-8)。
图31 eCNDs联合DOX不同给药方案的细胞毒性评价(CCK-8)。
图32 eCNDs对肿瘤和正常细胞增殖生长干预的影响结果。
图33 eCNDs对肿瘤和正常细胞内ATP生成的影响结果。
图34 eCNDs联合索拉菲尼的IC 50评价结果。
图35 eCNDs联合索拉菲尼用于肝癌治疗的药效学评价(eCNDs静脉给药和索拉菲尼口服给药)结果，
其中，A：肿瘤体积，B：荷瘤小鼠体重。
图36 eCNDs联合索拉菲尼用于肝癌治疗的药效学评价(eCNDs静脉给药和索拉菲尼口服给药)细胞凋亡研究结果。
图37 eCNDs联合索拉菲尼用于肝癌治疗的药效学评价(eCNDs和索拉菲尼均口服给药)结果，
其中，A：给药方案B：肿瘤体积变化C：荷瘤小鼠体重。
图38 eCNDs联合索拉菲尼用于肝癌治疗的药效学评价(eCNDs和索拉菲尼均口服给药)细胞凋亡实验结果。
图39 eCNDs生物安全性评价：主要脏器组织切片并进行HE染色结果。
图40 eCNDs生物安全性评价：血常规与血液生化指标。
具体实施方式
实施例1.
将6g的油(粟米油、菜籽油、花生油、火麻油、玉米油、橄榄油、山茶油、棕榈油、芥 花子油、葵花子油、大豆油、芝麻油、葡萄籽油、核桃油和牡丹籽油等中的一种或者几种)和3g的糖(白砂糖、绵白糖、赤砂糖、多晶体冰糖、单晶体冰糖、方糖、冰片糖以及黄砂糖等中的一种或者几种)置入25mL的圆底烧瓶内，160℃油浴锅中反应6min，所得产物经萃取、旋蒸和透析去除残余的糖和油后得到eCNDs。
实施例2.
将6g的油(粟米油、菜籽油、花生油、火麻油、玉米油、橄榄油、山茶油、棕榈油、芥花子油、葵花子油、大豆油、芝麻油、葡萄籽油、核桃油和牡丹籽油等)和3g的糖(白砂糖、绵白糖、赤砂糖、多晶体冰糖、单晶体冰糖、方糖、冰片糖以及黄砂糖等)置入25mL的圆底烧瓶内，199℃油浴锅中反应6min，所得产物经萃取、旋蒸和透析去除残余的糖和油后得到eCNDs。
实施例3.
将8g的油(粟米油、菜籽油、花生油、火麻油、玉米油、橄榄油、山茶油、棕榈油、芥花子油、葵花子油、大豆油、芝麻油、葡萄籽油、核桃油和牡丹籽油等)置入25mL的圆底烧瓶内在油浴锅中加热至160℃，然后加入3g的糖(白砂糖、绵白糖、赤砂糖、多晶体冰糖、单晶体冰糖、方糖、冰片糖以及黄砂糖等)反应6min，所得产物经萃取、旋蒸和透析去除残余的糖和油后得到eCNDs。
实施例4.
将8g的油(粟米油、菜籽油、花生油、火麻油、玉米油、橄榄油、山茶油、棕榈油、芥花子油、葵花子油、大豆油、芝麻油、葡萄籽油、核桃油和牡丹籽油等)置入25mL的圆底烧瓶内在油浴锅中加热至199℃，然后加入3g的糖(白砂糖、绵白糖、赤砂糖、多晶体冰糖、单晶体冰糖、方糖、冰片糖以及黄砂糖等)反应6min，所得产物经萃取、旋蒸和透析去除残余的糖和油后得到eCNDs。
实施例5.
将10g的油(粟米油、菜籽油、花生油、火麻油、玉米油、橄榄油、山茶油、棕榈油、芥花子油、葵花子油、大豆油、芝麻油、葡萄籽油、核桃油和牡丹籽油等)置入25mL的圆底烧瓶内在油浴锅中加热至160℃，同时，将3g的糖(白砂糖、绵白糖、赤砂糖、多晶体冰糖、单晶体冰糖、方糖、冰片糖以及黄砂糖等)置入另一个25mL的圆底烧瓶内在油浴条件下加热至160℃。然后，将油加入到含有糖的反应瓶中反应6min，所得产物经萃取、旋蒸和透析去除残余的糖和油后得到eCNDs。
实施例6.
将10g的油(粟米油、菜籽油、花生油、火麻油、玉米油、橄榄油、山茶油、棕榈油、芥花子油、葵花子油、大豆油、芝麻油、葡萄籽油、核桃油和牡丹籽油等)置入25mL的圆底烧瓶内在油浴锅中加热至199℃，同时，将3g的糖(白砂糖、绵白糖、赤砂糖、多晶体冰糖、单晶体冰糖、方糖、冰片糖以及黄砂糖等)置入另一个25mL的圆底烧瓶内在油浴条件下加热至199℃。然后，将油加入到含有糖的反应瓶中反应6min，所得产物经萃取、旋蒸和透析去除残余的糖和油后得到eCNDs。
实施例7.
将6g的糖(白砂糖、绵白糖、赤砂糖、多晶体冰糖、单晶体冰糖、方糖、冰片糖以及黄砂糖等)置入25mL的圆底烧瓶内在油浴锅中加热至160℃，然后加入12g的油(粟米油、菜籽油、花生油、火麻油、玉米油、橄榄油、山茶油、棕榈油、芥花子油、葵花子油、大豆油、芝麻油、葡萄籽油、核桃油和牡丹籽油等)反应6min，所得产物经萃取、旋蒸和透析去除残余的糖和油后得到eCNDs。
实施例8.
将6g的糖(白砂糖、绵白糖、赤砂糖、多晶体冰糖、单晶体冰糖、方糖、冰片糖以及黄砂糖等)置入25mL的圆底烧瓶内在油浴锅中加热至199℃，然后加入12g的油(粟米油、菜籽油、花生油、火麻油、玉米油、橄榄油、山茶油、棕榈油、芥花子油、葵花子油、大豆油、芝麻油、葡萄籽油、核桃油和牡丹籽油等)反应6min，所得产物经萃取、旋蒸和透析去除残余的糖和油后得到eCNDs。
实施例9.
将12g的油(粟米油、菜籽油、花生油、火麻油、玉米油、橄榄油、山茶油、棕榈油、芥花子油、葵花子油、大豆油、芝麻油、葡萄籽油、核桃油和牡丹籽油等)和6g的糖(白砂糖、绵白糖、赤砂糖、多晶体冰糖、单晶体冰糖、方糖、冰片糖以及黄砂糖等)置入25mL的圆底烧瓶内，超声破碎5min后，在油浴锅中加热至160℃条件下反应6min，所得产物经萃取、旋蒸和透析去除残余的糖和油后得到eCNDs。
实施例10.
将12g的油(粟米油、菜籽油、花生油、火麻油、玉米油、橄榄油、山茶油、棕榈油、芥花子油、葵花子油、大豆油、芝麻油、葡萄籽油、核桃油和牡丹籽油等)和6g的糖(白砂糖、绵白糖、赤砂糖、多晶体冰糖、单晶体冰糖、方糖、冰片糖以及黄砂糖等)置入25mL的 圆底烧瓶内，超声破碎5min后，在油浴锅中加热至199℃条件下反应6min，所得产物经萃取、旋蒸和透析去除残余的糖和油后得到eCNDs。
实施例11.
通过JEM-2010透射电镜观察实施例5所制备的eCNDs，结果显示该纳米点具有均一的尺寸，如图1所示。
实施例12.
实施例5所制备的eCNDs分散于水溶液中，通过动态光散射法表征其粒径分布情况，结果显示所制备的eCNDs粒径在2～14nm，请见附图2。所制备的eCNDs内核具有明显的碳晶格条纹，其结晶的碳核尺寸为2～8nm，附加表面基团后的水合直径为5～14nm，如图1所示。
实施例13.
实施例5所制备的的eCNDs分散于水溶液中，粒度Zeta电位测定仪测定其zeta电位，结果显示所制备的eCNDs溶液Zeta电位为-18～-24mV，如图3所示。
实施例14.
通过 13C核磁共振法、 1H核磁共振法及红外光谱法表征实施例5所制备的eCNDs。 13C核磁共振谱显示(如图4A所示)，实施例5所制备的eCNDs表面在化学位移δ为0～60ppm出现sp 3杂化的脂肪碳(C-O、C-C和C-H)信号；eCNDs的 1H核磁共振谱中在化学位移δ为4～5ppm出现C-OH的 1H-NMR信号(如图4B所示)；红外光谱法表征实施例5所制备的eCNDs，结果如图4C所示，eCNDs在3500～3700cm -1、2850～2960cm -1、1710～1750cm -1、1660～1700cm -1、1507cm -1、1250～1300cm -1、1100～1200cm -1处分别具有v(O-H)、v(C-H)、v(C＝O)、v(C-C)、v(C-H)、v(C-N)和v(C-O)特征吸收峰。通过以上3个图谱对比分析可得实施例5所制备的eCNDs表面可能具有羧基、羰基、羟基和氨基等表面官能团。
实施例15.
通过元素分析仪对实施例5所制备的eCNDs中的碳、氢、氧和氮元素进行表征。结果显示，本发明所提供的eCNDs，其骨架含碳、氢、氧和氮元素，并且C的质量比为40.53％～43.82％，H的质量比为5.73％～7.94％,O的质量比为51.86％～54.97％和N的质量比为2.57％～4.32％，请见附图5。其表面的碳，氧，氮的原子含量分别为：60～70at％，28～35at％，1～5at％，如图6所示。
实施例16.
通过凝胶色谱法(GPC)对实施例5所制备的eCNDs的分子量进行表征，结果如图7所示，eCNDs的分子量为8000～20000。
实施例17.
通过MALDI TOF对实施例5所制备的eCNDs的分子组成进行表征，结果如图8所示。在eCNDs的结果图谱中，在m/z为200～480、750～920以及1000～1600的范围内均存在分子峰。
实施例18.
实施例5制备的eCNDs分散于水溶液中，通过荧光光谱仪测试，结果显示eCNDs水溶液经紫外激发(375nm～440nm)能够发射荧光(475nm～599nm)，其荧光发射波长随着激发波长的增加而增加，最大激发光和发射光的波长分别440nm和599nm，如图9所示。
实施例19.
将实施例5制备的eCNDs等浓度梯度用水稀释，通过紫外分光光度仪(270nm)考察其吸光度与浓度之间的线性关系，结果显示eCNDs在25～300μg/mL的浓度范围与其对应的吸光度线性良好，可用于相关eCNDs的定量分析，如图10所示。
实施例20.
实施例5所制备的eCNDs分散于不同介质包括水、磷酸盐缓冲液、细胞培养液和胎牛血清中，观察碳量子点溶液离心前后的分散性，结果显示，16000rpm离心10min后，在各种介质中均未观察到明显的沉淀，且溶液颜色未发生明显变化，说明所制备的eCNDs在不同介质中分散性良好，如图11所示。
实施例21.
实施例5所制备的eCNDs分别暴露于紫外光环境(2.5W/cm 2，照射0～48h)，分散于不同浓度的盐(K+，Na+等，0～5Mol/L)水溶液和pH(4～9)的磷酸盐缓冲液中，考察其荧光稳定性，结果显示，其375～580nm的荧光特性在紫外光、NaCl以及pH的干扰下无明显变化，具有较好的荧光稳定性，如图12A、B和C所示。
实施例22.
实施例5所制备的eCNDs以不同浓度加入到肿瘤细胞(U87和HepG2)和正常细胞的(1800和HL 7702)的培养基中，与细胞培养箱共孵育6h之后通过CCK-8考察eCNDs的细胞毒性。结果显示，eCNDs细胞毒性低，安全性高，如图13所示。
实施例23.
实施例5所制备的eCNDs以不同浓度加入到U87细胞培养基中，孵育1h后对其细胞摄取情况进行考察，结果显示，eCND的细胞摄取存在浓度依赖性，200μg/mL以上的eCND在细胞具有较优的细胞摄取，如图14所示。
实施例24.
实施例5所制备的eCNDs以200μg/mL加入到U87细胞培养基中，在不同时间段对其细胞摄取情况进行考察，结果显示，eCND的细胞摄取存在时间依赖性，eCND在2h具有最优的细胞摄取，如图15所示。
实施例25.
实施例5所制备的eCNDs通过与不同抑制剂进行细胞共孵育，研究化疗增敏剂的跨膜转运机制。结果显示，eCND主要以被动转运的形式通过葡萄糖受体GLUT1进行细胞跨膜转运，如图16所示。
实施例26.
实施例5所制备的eCNDs与U 87细胞共孵育后，通过GLUT-1荧光探针(TRITC-labeled GLUT1探针)对细胞进行标记，在共聚焦显微镜下观察eCNDs与GLUT-1荧光的分布情况。结果显示，eCNDs的荧光与GLUT-1荧光大部分重合，证明eCNDs通过GLUT1进行细胞跨膜转运的机制，如图17所示。
实施例27.
实施例5所制备的eCNDs(200μg/mL)分别加入到U87细胞和1800细胞培养液中与细胞共孵育，通过石英晶体微天平(QCM-D)，以石英晶体频率(Δf)和耗散值(ΔD)为评价指标，对eCNDs和细胞之间的作用进行研究。结果显示，也正常细胞相比，eCNDs更容易吸附于GLUT-1高表达的肿瘤细胞表面，如图18所示。
实施例28.
实施例5所制备的eCNDs(200μg/mL)分别加入到细胞培养液中与细胞(U87和1800)共孵育12h后，将培养基换成无糖培养基继续孵育12h,之后再换回含eCNDs(200μg/mL)的培液继续孵育细胞。通过细胞活力实时监测系统，对细胞的状态进行监测。结果显示，与正常细胞相比，eCNDs能够明显抑制肿瘤细胞的生长和增殖，如图19所示。
实施例29.
实施例5所制备的eCNDs以不同浓度(100、200和400μg/mL)加入到U87细胞的培养皿中，30min后，加入葡萄糖荧光探针，观察细胞的葡萄糖摄取情况。结果显示，eCNDs可 以抑制细胞对葡萄糖的摄取，且抑制情况与其浓度成正比，如图20所示。
实施例30.
实施例5所制备的eCNDs静脉注射于荷原位脑胶质瘤小鼠体内，在不同时间段，通过小动物活体成像仪观察体内荧光分布情况，说明荧光碳量子点可以通过EPR效应蓄积于脑胶质瘤部位，进行活体成像，如图21所示。
实施例31.
实施例5所制备的eCNDs以不同浓度经尾静脉注射到荷原位脑胶质小鼠体内，通过PET/CT对于肿瘤部位的葡萄糖含量进行监测。结果显示，eCNDs能够有效抑制肿细胞的葡萄糖摄取，在eCNDs浓度为2mg，静脉注射30min时的效果最为明显，如图22所示。
实施例32.
通过 1H核磁共振法及红外光谱法分别表征实施例5所制备的eCNDs和葡萄糖，分析其结构的特点，结果如图23所示，显示eCNDs在化学位移δ为2.7～3.7ppm、4.2～4.8ppm处具有与葡萄糖类似的C 2-6-H和C 1-6-OH的 1H-NMR信号；红外光谱图显示，eCNDs在3500～3700cm -1、2850～2960cm -1、1710～1750cm -1、1250～1300cm -1、1100～1200cm -1处均有与葡萄糖重叠的红外特征吸收峰。对以上实验结果分析可以说明eCNDs表面可能存在与葡萄糖表面基团类似的官能基团。
实施例33.
将实施例5所制备的eCNDs和葡萄糖分别经口给SD大鼠(剂量均为200mg/kg)，监测大鼠血糖浓度的变化趋势，结果如图24所示，相比较于葡萄糖引起大鼠血糖短时间内大幅度的波动现象，eCNDs对大鼠的血糖浓度没有影响，表明eCNDs可能不具有葡萄糖供能的特性。
实施例34.
将实施例5所制备的eCNDs经口给SD大鼠(200mg/kg)，同时以禁食的大鼠为阴性对照，正常进食和进食食用糖(200mg/kg)的大鼠为阴性对照，通过监测大鼠体重的的变化趋势研究eCNDs的生物学特性。结果显示，相比较于正行进食和糖的大鼠，食用eCNDs的大鼠体重变化趋势与禁食大鼠一致，表明eCNDs不能为机体提供能量，如图25所示。
实施例35.
实施例5所制备的eCNDs以不同浓度和(或)20μg/mL替莫唑胺加入到U87细胞培养皿中，通过CCK-8进行细胞毒性评价。结果如图26所示，eCNDs明显提高了替莫唑胺的细胞毒性。
实施例36.
实施例5所制备的eCNDs以不同浓度和(或)20μg/mL替莫唑胺加入到U87细胞培养皿中，通过LIVEDEAD进行细胞毒性评价。结果如图27所示，eCNDs明显提高了替莫唑胺的细胞毒性。
实施例37.
实施例5所制备的eCNDs静脉给药于荷原位脑胶质瘤小鼠体内，替莫唑胺经口服给药，对小鼠的生命体征以及生存周期进行检测，结果显示eCNDs和替莫唑胺的联合应用杀死脑胶质瘤细胞(如图28所示)、改善荷瘤小鼠的生存质量(如图29A所示)和生存周期(如图29B所示)。
实施例38.
实施例5所制备的eCNDs以200μg/mL的浓度和(或)20μg/mL的DOX分别加入到Hep G2，MCF-7，A549，H1299，HCT 116，4T1和PANC-1细胞培养皿中，通过CCK-8进行细胞毒性评价，结果如图30所示，eCNDs明显提高了DOX的细胞毒性。
实施例39.
实施例5所制备的eCNDs以200μg/mL的浓度和(或)20μg/mL的DOX分别加入到MCF-7和MCF-10的细胞培养皿中，通过CCK-8进行细胞毒性评价。结果如图31所示，eCNDs明显提高了DOX对肿瘤细胞的毒性。
实施例40.
实施例5所制备的eCNDs以不同浓度分别加入到Hep G2和HL 7702的细胞培养皿中，通过CCK-8进行细胞毒性评价。结果如图32所示，相比较于正常细胞来说，在长时间的干预下，eCNDs明显抑制肿瘤细胞的生长增殖。
实施例41.
实施例5所制备的eCNDs以不同浓度分别加入到Hep G2和HL 7702的细胞培养皿中，通过ATP荧光探针对细胞内的ATP含量进行评价。结果如图33所示，相比较于正常细胞来说，eCNDs明显抑制肿瘤细胞ATP的生成。
实施例42.
实施例5所制备的eCNDs以400μg/mL的浓度与不同浓度的索拉非尼联合给药于Hep G2细胞培养皿中,计算24小时的IC 50值进行评价。结果如图34所示，eCNDs明显提高了索拉非尼的细胞毒性。
实施例43.
实施例5所制备的eCNDs与索拉非尼(eCNDs静脉给药和索拉菲尼口服给药)以不同治疗方案联合用于肝癌皮下瘤小鼠体内，通过瘤体积变化、体重变化以及细胞凋亡实验进行药效学评价。结果如图35A和B，36所示，索拉非尼联合eCNDs的治疗明显抑制肿瘤的生长。
实施例44.
实施例5所制备的eCNDs与索拉非尼(eCNDs和索拉菲尼均口服给药)以不同治疗方案联合用于肝癌皮下瘤小鼠体内，通过瘤体积变化、体重变化以及细胞凋亡实验进行药效学评价进行药效学评价。结果如图37和38所示，索拉非尼联合eCNDs的治疗明显抑制肿瘤的生长。
实施例45.
实施例5所制备的eCNDs以小于200mg/kg剂量喂食SD 12周后，对其主要器官进行生化组织切片染色，如图39所示，对其血常规和血液生化指标进行检测，考察其生物安全性。结果如图40所示，食用eCNDs的SD大鼠各脏器均无炎症病灶发生，血常规和血液生化指标正常，与正常大鼠指标一致，表明该碳量子点具有较高的安全性和生物相容性。

Claims (24)

  1. 一种荧光碳量子点，其特征在于，其粒径在2～14nm之间，所述的荧光碳量子点包含C、N、O、H四种元素，其表面的C、O、N的原子含量分别为33％～70at％，28％～35at％，1％～5％at。
  2. 根据权利要求1所述的荧光碳量子点，其特征在于，C的质量比为40％～58％，H的质量比为5％～8％,O的质量比为39％～55％和N的质量比为2％～5％。
  3. 根据权利要求1所述的荧光碳量子点，其特征在于，所述荧光碳量子点的GPC测试分子量为8000～20000。
  4. 根据权利要求1所述的荧光碳量子点，其特征在于，所述荧光碳量子点在3500～3700cm -1、2850～2960cm -1、1710～1750cm -1、1660～1700cm -1、1507cm -1、1250～1300cm -1、1100～1200cm -1处分别具有v(O-H)、v(C-H)、v(C＝O)、v(C-C)、v(C-H)、v(C-N)和v(C-O)特征红外吸收峰，且在化学位移δ为0～60ppm出现sp3杂化的 13C(C-O，C-C，C-N)NMR信号，以及在化学位移δ为4～5ppm出现C-OH的 1H-NMR信号。
  5. 根据权利要求1-4所述的荧光碳量子点的制备方法，其特征在于，其包括步骤，
    (1)以糖为原料、油为反应溶剂，通过加热搅拌得到粗产物；(2)去除粗产物中的糖和油。
  6. 根据权利要求5所述的制备方法，其特征在于，所述步骤(1)中油与糖的质量比为1:1～5:1。
  7. 根据权利要求5所述的制备方法，其特征在于，所述步骤(1)中糖为可食用糖，油为可食用油。
  8. 根据权利要求7所述的制备方法，其特征在于，所述可食用糖选自白砂糖、绵白糖、赤砂糖、多晶体冰糖、单晶体冰糖、方糖、冰片糖以及黄砂糖中的一种或者几种。
  9. 根据权利要求7所述的制备方法，其特征在于，所述可食用油选自粟米油、菜籽油、花生油、火麻油、玉米油、橄榄油、山茶油、棕榈油、芥花子油、葵花子油、大豆油、芝麻油、葡萄籽油、核桃油和牡丹籽油中的一种或者几种。
  10. 根据权利要求5所述的荧光碳量子点的一种制备方法，其特征在于，所述步骤(1)中的反应温度为150～199℃，反应时间为3～9min。
  11. 根据权利要求5所述的制备方法，其特征在于，所述步骤(2)中的去除粗产物中的糖和油的方法选自萃取、减压蒸馏、透析或高速离心中的任一种或其任意组合。
  12. 根据权利要求5或11所述的制备方法，其特征在于，所述步骤(2)中的去除粗产物中的 糖和油的方法为萃取辅助的双层膜透析联合减压蒸馏。
  13. 根据权利要求12所述的制备方法，其特征在于，所述萃取辅助的双层膜透析联合减压蒸馏的步骤如下：a)洗涤步骤(1)中得到的反应粗产物若干次；b)将步骤a)洗涤所得粗产物进行萃取，得到荧光碳量子点水溶液；c)将步骤b)所得的荧光碳量子点水溶液经真空旋蒸得到浓缩的荧光碳量子点水溶液；d)将步骤c)所得产物进行透析；e)得到所述的荧光碳量子点。
  14. 根据权利要求13所述的制备方法，其特征在于，所述步骤a)中的洗涤剂为为二氯甲烷、正己烷、乙酸丁酯和三氯甲烷中的一种或几种试剂的混合。
  15. 根据权利要求13所述的制备方法，其特征在于，所述步骤b)中的萃取剂为二氯甲烷、正己烷、乙酸丁酯和三氯甲烷与水的混合溶液。
  16. 根据权利要求13所述的制备方法，其特征在于，所述步骤c)中的真空旋蒸为35～55℃水浴旋蒸，真空压为-100KPa以下。
  17. 根据权利要求13所述的制备方法，其特征在于，所述步骤d)中的透析时长为48-96h。
  18. 权利要求1-4所述的荧光碳量子点在制备肿瘤体内成像制剂中的应用。
  19. 权利要求1-4所述的荧光碳量子点在制备抗肿瘤药物的增敏剂中的用途。
  20. 根据权利要求19所述的用途，其特征在于，所述抗肿瘤药物包括化疗药物和/或免疫治疗药物及其它物理治疗药物。
  21. 根据权利要求20所述的用途，其特征在于，所述化疗药物选自多柔比星、索拉菲尼或替莫唑胺。
  22. 根据权利要求19-21所述的用途，其特征在于，所述肿瘤包括神经胶质瘤、肝癌、乳腺癌、淋巴瘤、卵巢癌、软组织肉瘤、成骨肉瘤、横纹肌肉瘤、膀胱癌、甲状腺癌、前列腺癌、头颈部鳞癌、睾丸癌或胃癌。
  23. 一种抗肿瘤药物组合物，其特征在于，包括权利要求1～4所述的荧光碳量子点和抗肿瘤药物。
  24. 根据权利要求23所述的抗肿瘤药物组合物，其特征在于，所述抗肿瘤药物组合物还包含药学上可接受的载体。
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CN116103041A (zh) * 2023-01-19 2023-05-12 河南大学 一种以药物功能保留策略合成的碳点及其制备方法和应用
CN116200192A (zh) * 2023-02-21 2023-06-02 广东工业大学 一种同位素标记的碳量子点及其制备方法

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