WO2021046966A1 - 含氟石墨烯量子点与制备及其作为光动力治疗光敏剂的应用 - Google Patents

含氟石墨烯量子点与制备及其作为光动力治疗光敏剂的应用 Download PDF

Info

Publication number
WO2021046966A1
WO2021046966A1 PCT/CN2019/111622 CN2019111622W WO2021046966A1 WO 2021046966 A1 WO2021046966 A1 WO 2021046966A1 CN 2019111622 W CN2019111622 W CN 2019111622W WO 2021046966 A1 WO2021046966 A1 WO 2021046966A1
Authority
WO
WIPO (PCT)
Prior art keywords
fluorine
containing graphene
graphene quantum
quantum dots
oxidized
Prior art date
Application number
PCT/CN2019/111622
Other languages
English (en)
French (fr)
Inventor
毕红
李真真
克劳迪奥 特德斯科安东尼奥
Original Assignee
安徽大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 安徽大学 filed Critical 安徽大学
Publication of WO2021046966A1 publication Critical patent/WO2021046966A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • 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
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • 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
    • C01B32/182—Graphene
    • C01B32/184—Preparation
    • C01B32/19—Preparation by exfoliation
    • 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
    • C01B32/182—Graphene
    • C01B32/198—Graphene oxide

Definitions

  • the invention relates to a fluorine-containing graphene quantum dot, its preparation and its application as a photodynamic therapy photosensitizer, and belongs to the technical field of nano-medical materials.
  • Graphene quantum dots represent a new type of carbon quantum dots with unique properties. They are zero-dimensional materials with characteristics derived from graphene and carbon dots. They can be regarded as small pieces of graphene with a size of less than 10 nanometers and will become traditional semiconductors. Quantum dots and organic dyes are potential substitutes in the field of life sciences. Graphene quantum dots have some superior properties, such as better biocompatibility, excellent photobleaching resistance, good fluorescence properties, and easy surface modification, making them useful in biomarking, bioimaging and photodynamic therapy, etc. The application of this aspect has attracted much attention.
  • Photodynamic therapy is a trigger strategy, mainly used in the treatment of superficial tumors.
  • photosensitizers play a key role in PDT.
  • the photosensitizer absorbs the photon energy of the appropriate wavelength and transfers to its excited state.
  • Active oxygen species such as singlet oxygen are generated by the energy transferred from the excited photosensitizer, and finally, the cancer cells are killed by the generated cytotoxic active oxygen.
  • PDT has been used to treat a variety of tumors, esophageal cancer, skin cancer, and early stage lung cancer. Compared with traditional tumor therapy, the advantage of PDT is that it can be treated accurately and effectively, and the side effects of this therapy are very small.
  • fluorinated graphene quantum dots (Gong, Pei-Wei, et al. "To lose is to gain: Effective synthesis of water-soluble graphene fluoroxide quantum dots by sacrificing certain fluorine atoms from exfoliated fluorinated graphene.” , 83, 152-161) uses fluorinated graphite as raw material and uses solvothermal to synthesize fluorine-containing graphene quantum dots with a size of 2.5 nanometers to 3.5 nanometers, but the fluorine content is as high as 17%-25%. In the synthesis process, the operation process is complicated and cumbersome, which is not conducive to practical applications. Unfortunately, the fluorinated graphene quantum dots reported above have not been used in the study of PDT photosensitizers.
  • the present invention provides a fluorine-containing graphene quantum dot, which has small size, good water solubility, high singlet oxygen generation efficiency, stable structure, and can be better used in PDT.
  • the fluorinated graphene quantum dots of the present invention are prepared by using fluorinated graphite as a raw material and using the Hummers method to obtain oxidized fluorinated graphene, and then chemically oxidized and cut to obtain an average thickness of 1.0-3.0 nanometers and a size of 2.0 -3.0 nanometers, fluorine content of 1%-2%, oxygen content of 20%-30%, carbon content of 60%-70%, and singlet oxygen yield of 0.4-0.5 fluorine-containing graphene quantum under visible light irradiation point.
  • the method for preparing fluorine-containing graphene quantum dots of the present invention includes the following steps:
  • the strong alkaline substance in step 1) can be sodium hydroxide, potassium hydroxide, etc., which can be prepared into an alkaline solution, which can be easily soaked in fluorine.
  • concentration of the alkali solution can generally be between 10% and 80%, and more preferably between 20% and 50%.
  • the high concentration soaking and peeling time is short, and the low concentration soaking and peeling The time is slightly longer.
  • Step 1) The fluorine content of the obtained oxidized fluorine-containing graphene is 6%-8%, the oxygen content is 6%-8%, and the carbon content is 80%-90%; this step can be used to immerse the fluorinated graphite in strong alkaline In solution to achieve peeling.
  • the oxidized fluorine-containing graphene prepared by the Hummers method can refer to the literature: ACS Nano, 2010, 4(8), 4806–4814.
  • the strong oxidant in step 2) can be a mixture of one or more of hydrogen peroxide, concentrated sulfuric acid, concentrated nitric acid, and potassium persulfate.
  • the mass ratio of the oxidized fluorine-containing graphene and the strong oxidizing agent in the step 2) may be (1-3): (1000-3000), preferably (1 -3): 1000; the mass ratio of the oxidized fluorine-containing graphene to ultrapure water can be (1-3): (1000-3000), preferably 1: (1000-3000).
  • the ultrasonic power in the step 2) can be 500-800 watts;
  • the strong oxidant can be hydrogen peroxide, concentrated sulfuric acid, and concentrated nitric acid.
  • the lye can be potassium hydroxide solution, sodium hydroxide solution, ammonia water, etc., which is mainly to promote the oxidative cutting reaction, has a catalytic effect, and its concentration can generally be 0.5-2 moles per liter .
  • the purification in step 3) can be suction filtration, chromatography, dialysis, filtration, extraction, distillation and fractionation, etc.; the drying can be vacuum Drying, freeze drying, high temperature drying, etc.
  • the fluorine-containing graphene quantum dots of the present invention can be used as a photosensitizer for photodynamic therapy.
  • the surface of the fluorine-containing graphene quantum dots is rich in hydroxyl and carboxyl groups, and the interior is mainly composed of sp 2 hybridized carbon atoms. It has good water solubility (see Figure 1 and Figure 3); it has good properties in the ultraviolet-visible light region. Absorption (see Figure 2) and non-excitation-dependent luminescence properties; and under visible light irradiation, the singlet oxygen yield can reach 0.4-0.5.
  • the fluorine-containing graphene quantum dots of the present invention have stable structure, high singlet oxygen generation efficiency, low cytotoxicity, good water solubility and excellent biocompatibility. They are ideal photosensitizers for PDT and can Better application in PDT. Generally, graphene quantum dots have poor cytotoxicity and biocompatibility and are difficult to use as PDT photosensitizers.
  • the fluorine-containing graphene quantum dots prepared by the present invention are biomedical nanomaterials with hydrophilic groups such as hydroxyl, carboxyl, and amino groups on the surface.
  • the material is smaller in size, uniform in morphology, stable optical performance, and easy Surface modification, and can be well dispersed in water, phosphate buffer solution, biological culture medium and other aqueous systems.
  • the fluorine-containing graphene quantum dots prepared by the present invention have good absorption in the ultraviolet-visible light region.
  • singlet oxygen can be generated (see Figure 5), and The singlet oxygen production rate is as high as 0.4-0.5.
  • the singlet oxygen production rate of Rose Bengal (RB) is 0.75.
  • the singlet oxygen yield reaches 0.4-0.5. Based on this, the fluorine-containing graphene quantum dots of the present invention can effectively kill under visible light irradiation. Tumor cells can be used as photosensitizers for photodynamic therapy (see Figure 7).
  • fluorine-containing graphene quantum dots prepared by the present invention are used in photodynamic therapy, they can also be used for biological imaging, and the operation process is simple, which is beneficial to practical applications.
  • Figure 1 is a transmission electron microscope image of the fluorine-containing graphene quantum dots of the present invention
  • Fig. 2 is an ultraviolet-visible absorption spectrum and fluorescence emission spectrum diagram of the fluorine-containing graphene quantum dots in ultrapure water (excitation wavelength is 320 nanometers).
  • Fig. 3 is an X-ray photoelectron spectrogram of the fluorine-containing graphene quantum dots of the present invention.
  • Fig. 4 is a scanning electron microscope picture of the oxidized fluorine-containing graphene of the present invention.
  • Figure 5 shows the electron paramagnetic resonance of the fluorine-containing graphene quantum dots of the present invention with and without LED laser irradiation, respectively, of the 2,2,6,6-tetramethylpiperidine (TEMP) trapping agent Spectrometer (EPR) spectrum.
  • TEMP 2,2,6,6-tetramethylpiperidine
  • Figure 6 shows the results of testing the cytotoxicity of the fluorine-containing graphene quantum dots of the present invention with esophageal cancer cells for 12 hours after co-cultivation with MTT method;
  • Fig. 7 is the cytotoxicity test result of the MTT method after 12 hours of co-cultivation of the fluorine-containing graphene quantum dots of the present invention with esophageal cancer cells and then light for 12 minutes.
  • a method for preparing fluorine-containing graphene quantum dots that can be used for photodynamic therapy specifically includes the following steps:
  • the fluorinated graphite treated in step (1) is prepared by Hummers method to obtain oxidized fluorine-containing graphene; the fluorine content is 6%-8%, the oxygen content is 6%-8%, and the carbon content is 80%-90 %, it can be seen that it is a two-dimensional nano film (see Figure 4);
  • the above solution is purified by vacuum filtration with an organic microporous filter membrane with a pore size of 0.22 microns to remove bulk impurities, and then dried to obtain fluorine-containing graphene quantum dots with a smaller size (The lateral size is about 2.0-3.0 nanometers), uniform morphology, non-excited light dependent luminescence and other characteristics;
  • Figure 1 is a transmission electron microscope image of the fluorine-containing graphene quantum dots. It can be seen that the fluorine-containing graphene quantum dots have good monodispersity and are flat round particles with a size of about 2 nanometers.
  • Figure 2 shows the ultraviolet-visible absorption spectrum and fluorescence emission spectrum of the fluorine-containing graphene quantum dots in ultrapure water prepared by the present invention (excitation wavelength is 320 nanometers). The analysis shows that it shows bright green fluorescence under a 365-nanometer ultraviolet lamp. Under the excitation of 320nm light, the aqueous solution of fluorine-containing graphene quantum dots has a fluorescence emission peak at 510nm.
  • Figure 6 shows the cytotoxicity of the prepared fluorinated graphene quantum dots under dark conditions using the MTT method. After co-cultivating esophageal cancer cells (provided by Anhui Medical University) with fluorinated graphene quantum dots for 12 hours, the cell survival rate remained above 90%, indicating that the prepared fluorinated graphene quantum dots have very low toxicity .
  • EPR electron paramagnetic resonance spectrometer
  • ⁇ sam ⁇ RB ⁇ (K sam ⁇ A RB )/(K RB ⁇ A sam )
  • K sam and K RB are the ABDA decomposition rate constants of the test sample and Rose Bengal (RB), respectively.
  • a sam and A RB represent the light absorbed by the sample and Rose Bengal (RB), respectively, and are determined by integrating the optical absorption band in the wavelength range of 400 to 700 nm.
  • ⁇ RB is the 1 O 2 quantum yield of Rose Red (RB)
  • ⁇ RB 0.75 in water.
  • the fluorine-containing graphene quantum dots prepared by the present invention are used as photosensitizers in photodynamic therapy, which can produce a large amount of singlet oxygen under light, and the yield is 0.4-0.5, thereby improving the effect of photodynamic therapy. It is used in the treatment of various tumors, esophageal cancer, skin cancer and early lung cancer.
  • Esophageal cancer cell culture conditions esophageal cancer cells were placed in 1640 medium containing 8% fetal bovine serum, and then placed in an incubator at 37 degrees Celsius and 5% CO 2 for culture. When the cell culture proliferates to approximately fill the bottom of the culture flask, remove the old medium, add 2 ml of phosphate buffer solution that has been preheated to 37 degrees Celsius to wash the cells, and then add 1 ml of 0.25% trypsin solution to infiltrate the cells , followeded by centrifugation to remove pancreatin, digestion at 37 degrees Celsius for about 4 minutes, and place it under an optical microscope to observe the morphological changes of cells.
  • Detecting the cytotoxicity of the prepared fluorinated graphene quantum dots by the tetramethylazolium salt (MTT) method it is a common method for detecting cell survival and growth.
  • the detection principle is: the succinate dehydrogenase in the mitochondria of living cells can reduce the exogenous MTT into water-insoluble blue-purple crystal formazan and deposit it in the cells. Dead cells do not have this ability. Then use dimethyl sulfoxide. To dissolve the crystalline formazan in the cells, use 630 nm as the reference wavelength and 563 nm as the test wavelength, and test the absorbance with a microplate reader. The cell viability was standardized as the cell viability cultivated in the culture medium.
  • a method for preparing fluorine-containing graphene quantum dots specifically includes the following steps:
  • the fluorinated graphite treated in step (1) is prepared by Hummers method to obtain oxidized fluorine-containing graphene; the fluorine content is 6%-8%, the oxygen content is 6%-8%, and the carbon content is 80%-90 %, it can be seen that it is a two-dimensional nano film;
  • the above solution is subjected to extraction and purification treatment, and then freeze-drying treatment to obtain fluorine-containing graphite with an average thickness of 1.0-3.0 nanometers, a size of 2.0 nanometers-3.0 nanometers, and a fluorine content of 1%-2% Ene quantum dots.
  • a method for preparing fluorine-containing graphene quantum dots specifically includes the following steps:
  • the fluorinated graphite treated in step (1) is prepared by Hummers method to obtain oxidized fluorine-containing graphene; the fluorine content is 6%-8%, the oxygen content is 6%-8%, and the carbon content is 80%-90 %, it can be seen that it is a two-dimensional nano film;
  • the fluorine-containing graphene quantum dots prepared by the invention have the advantages of simple reaction steps, low cost, environmental protection, etc., are easily dispersed in aqueous systems such as water, phosphate buffer solution, biological culture medium, etc.; have good biocompatibility and low cost. toxicity.
  • the fluorine-containing graphene quantum dots prepared by the invention can generate singlet oxygen under the light irradiation of visible light, can be used as photosensitizers in photodynamic therapy, and are suitable for the treatment process of esophageal cancer, skin cancer and early lung cancer. Wide application prospects.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Nanotechnology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Materials Engineering (AREA)
  • Inorganic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Optics & Photonics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Composite Materials (AREA)
  • Condensed Matter Physics & Semiconductors (AREA)
  • General Physics & Mathematics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)

Abstract

一种含氟石墨烯量子点与制备方法及其作为光动力治疗光敏剂的应用，属于生物医用材料领域。制得的含氟石墨烯量子点平均厚度为1.0-3.0纳米，尺寸为2.0-3.0纳米，氟含量为1％-2％，氧含量为20％-30％，碳含量为60％-70％。所述含氟石墨烯量子点尺寸更小，形貌均一，结构稳定，在可见光照射下，单线态氧产率高，而且具有极低细胞毒性、良好的水溶性和更加优异的生物相容性，是一种用于光动力治疗的理想光敏剂，适用于食道癌、皮肤癌和肺癌等的早期治疗过程，具有广泛的应用前景。

Description

含氟石墨烯量子点与制备及其作为光动力治疗光敏剂的应用 技术领域
本发明涉及一种含氟石墨烯量子点与制备及其作为光动力治疗光敏剂的应用，属于纳米医用材料技术领域。
背景技术
石墨烯量子点代表了一类具有独特性质的新型碳量子点，具有源自石墨烯和碳点的特征的零维材料，其可被视为尺寸小于10纳米的小片石墨烯，将成为传统半导体量子点和有机染料在生命科学领域中潜在的替代物。石墨烯量子点具有一些优越的性能，例如较好的生物相容性，优异的抗光漂白能力，良好的荧光性质，易于被表面修饰等优点，使其在生物标记、生物成像和光动力治疗等方面的应用倍受关注。
光动力疗法(PDT)是一种触发策略，主要是应用在浅表性肿瘤的治疗中。在光动力治疗中光敏剂在PDT中起着关键作用。在PDT的过程中，光敏剂吸收适当波长的光子能量并转移到其激发态。通过从激发的光敏剂转移的能量产生诸如单线态氧的活性氧物质，最后，癌细胞被产生的细胞毒性活性氧杀死。PDT已被用于治疗多种肿瘤、食道癌、皮肤癌和早期肺癌等疾病。与传统肿瘤疗法相比，PDT的优势在于能够进行精确有效地治疗，且这种疗法的副作用很小。
现有报道的石墨烯量子点做PDT光敏剂的报道(Ge,Jie-Chao,et al."A graphene quantum dot photodynamic therapy agent with high singlet oxygen generation."Nat.Commun.2014,5,4596-4603.)其荧光发射波峰位置为700纳米，单线态氧( 1O 2)产率为1.3(相对产率)，但合成的方法复杂，而且是通过改变噻吩单体末端结构而制备出的石墨烯量子点，亲水性略差，细胞毒性也较大。
现有报道的氟化石墨烯量子点(Gong,Pei-Wei,et al."To lose is to gain:Effective synthesis of water-soluble graphene fluoroxide quantum dots by sacrificing certain fluorine atoms from exfoliated fluorinated graphene."Carbon 2015,83,152-161)是采用氟化石墨为原料，利用溶剂热合成尺寸为2.5纳米-3.5纳米的含氟石墨烯量子点，但其含氟量高达17％-25％。在合成的过程中，操作过程复杂繁琐，不利于实际应用。遗憾的是，上述报道的氟化石墨烯量子点并没有用于PDT光敏剂的研究。
另有报道的氟化石墨烯量子点(Sun,Han-Jun,et al."Synthesis of Fluorinated and Nonfluorinated Graphene Quantum Dots through a New Top-Down Strategy for Long-Time Cellular Imaging."Chem.Eur.J.2015,21,3791-3797)也是采用氟化石墨为原料，通过微波辅助水热法合成的，其含氟量虽然低至1.2％，但尺寸稍大(3.0纳米-10.0纳米)，且该合成过程比较复杂。此外，上述报道的氟化石墨烯量子点也没有用于PDT光敏剂的研究。
另外，现有中国发明专利(CN201610935474.4)报道的氟化石墨烯量子点，由于原料、合成的方法不同，所合成的氟化石墨烯量子点尺寸不是很均匀，含氟量不明确，而且也没有用于PDT光敏剂的研究。
发明内容
针对上述现有技术存在的问题，本发明提供一种含氟石墨烯量子点，尺寸小、水溶性好、产生单线态氧效率高，结构稳定，可以更好地应用在PDT中。
本发明的含氟石墨烯量子点，其是通过采用氟化石墨为原料，使用Hummers法制备得到氧化含氟石墨烯，再经化学氧化切割而得到的平均厚度为1.0-3.0纳米，尺寸为2.0-3.0纳米，氟含量为1％-2％，氧含量为20％-30％，碳含量为60％-70％，在可见光照射下单线态氧产率达到0.4-0.5的含氟石墨烯量子点。
本发明所述的含氟石墨烯量子点的制备方法，包括以下步骤：
1)使用强碱性物质对氟化石墨进行超声剥离处理，再使用Hummers法制备得到氧化含氟石墨烯。
2)称取氧化含氟石墨烯，溶解在超纯水中，超声处理使分散充分；向上述溶液中加入强氧化剂，然后再加入碱液，在60-100摄氏度下回流5-9小时，让化学氧化切割反应充分进行；
3)待回流结束后，过滤、提纯、干燥，即得；
上述所述的含氟石墨烯量子点的制备方法中，所述步骤1)中强碱性物质可采用氢氧化钠、氢氧化钾等，可将其制备成碱溶液，则可方便的浸泡氟化石墨，所述碱溶液浓度不做特别要求，质量浓度一般可在10％-80％之间，更佳的可在20％-50％之间，高浓度浸泡剥离时间短，低浓度浸泡剥离则时间稍长。
步骤1)所得氧化含氟石墨烯的氟含量为6％-8％，氧含量为6％-8％，碳含量为80％-90％；该步骤可采用将氟化石墨浸泡于强碱性溶液中以实现剥离。所 述使用Hummers法制备得到氧化含氟石墨烯可参照文献：ACS Nano,2010,4(8),4806–4814。
上述所述的含氟石墨烯量子点的制备方法中，所述步骤2)中的强氧化剂可采用过氧化氢、浓硫酸、浓硝酸、过硫酸钾中的一种或多种的混合。
上述所述的含氟石墨烯量子点的制备方法中，所述步骤2)中的氧化含氟石墨烯和强氧化剂的质量比可为(1-3)：(1000-3000)，优选(1-3)：1000；所述氧化含氟石墨烯与超纯水的质量比可为(1-3)：(1000-3000)，优选1：(1000-3000)。
作为改进，对于上述所述含氟石墨烯量子点的制备方法，所述步骤2)中的超声功率可以为500-800瓦；所述强氧化剂可采用过氧化氢、浓硫酸、浓硝酸中的一种或多种的混合；所述碱液可以为氢氧化钾溶液、氢氧化钠溶液、氨水等，其主要是促进氧化切割反应，具催化作用，其浓度一般可在0.5-2摩尔每升。
作为改进，对于上述所述含氟石墨烯量子点的制备方法，所述步骤3)中的提纯可采用抽滤、层析、透析、过滤、萃取、蒸馏分馏等；所述的干燥可采用真空干燥、冷冻干燥、高温干燥等。
本发明所述的含氟石墨烯量子点可作为光动力治疗光敏剂应用。
所述的含氟石墨烯量子点表面富含羟基、羧基，内部主要由sp 2杂化的碳原子组成，其具有良好的水溶性(参见图1和图3)；在紫外-可见光区域具有良好吸收(参见图2)和非激发光依赖性发光性质；而且在可见光照射下，单线态氧产率可达到0.4-0.5。本发明所述含氟石墨烯量子点，结构稳定，产生单线态氧效率高，具有低的细胞毒性、良好的水溶性和优异的生物相容性，是用于PDT的理想光敏剂，并可更好地应用在PDT中。而一般石墨烯量子点细胞毒性、生物相容性都较差，难以用作PDT光敏剂。
与现有技术相比，本发明的有益效果是：
1)本发明制备的含氟石墨烯量子点为生物医用纳米材料，表面带有羟基、羧基、氨基等亲水基团，该材料尺寸更小，形貌均一，具有稳定的光学性能，且易于进行表面修饰，而且能较好地分散于水、磷酸盐缓冲溶液、生物培养基等水溶液体系中。
2)通过MTT法(四甲基偶氮唑盐)测试本发明制得含氟石墨烯量子点的 细胞毒性，测试结果显示采用食道癌细胞与不同浓度的含氟石墨烯量子点和1640培养基溶液共培养12小时后，细胞的存活率仍维持在90％以上，表明本发明制得的含氟石墨烯量子点具有非常低的毒性(参见图6)。
3)本发明制得含氟石墨烯量子点在紫外-可见光区域具有良好吸收，采用可见光的光照射本发明制备的含氟石墨烯量子点时，可产生单线态氧(参见图5)，且单线态氧产率高达0.4-0.5，与目前用于光动力治疗的理想光敏剂玫瑰红(RB)相比，玫瑰红(RB)的单线态氧产率为0.75。此外，与已有的石墨烯量子点做PDT光敏剂的报道相比，单线态氧产率达到0.4-0.5，基于此，本发明的含氟石墨烯量子点，在可见光照射下能有效杀死肿瘤细胞，可用作光动力治疗的光敏剂(参见图7)。
4)本发明制得的含氟石墨烯量子点用于光动力治疗时，而且也可以进行生物成像，操作过程简单，有利于实际应用。
附图说明
图1为本发明的含氟石墨烯量子点的透射电子显微镜图像；
图2为本发明的含氟石墨烯量子点在超纯水中的紫外-可见吸收光谱和荧光发射光谱图(激发波长为320纳米)。
图3为本发明的含氟石墨烯量子点的X-射线光电子能谱谱图。
图4为本发明的氧化含氟石墨烯的扫描电子显微镜图片。
图5为本发明的含氟石墨烯量子点分别在有LED激光照射和无LED激光照射的情况下，2,2,6,6-四甲基哌啶(TEMP)捕获剂的电子顺磁共振波谱仪(EPR)光谱。
图6为本发明的不同浓度含氟石墨烯量子点与食道癌细胞共培养12小时后，采用MTT法测试细胞毒性的结果；
图7为本发明的不同浓度含氟石墨烯量子点与食道癌细胞共培养12小时后，再经光照12分钟的MTT法细胞毒性测试结果。
具体实施方式
下述实施例是对于本发明内容的进一步说明以作为对本发明技术内容的阐释，但本发明的实质内容并不仅限于下述实施例所述，本领域的普通技术人员可以且应当知晓任何基于本发明实质精神的简单变化或替换均应属于本发明所要 求的保护范围。
实施例1
一种可用于光动力治疗的含氟石墨烯量子点的制备方法，具体包括以下步骤：
1)将氟化石墨浸泡于30％的氢氧化钠溶液中，超声搅拌分散12小时使充分剥离，再经过滤、干燥处理；
2)将步骤(1)处理后的氟化石墨，采用Hummers法制备得到氧化含氟石墨烯；氟含量为6％-8％，氧含量为6％-8％，碳含量为80％-90％，可见其为二维纳米薄膜(参见图4)；
3)称取60毫克上述氧化含氟石墨烯，溶解在60毫升超纯水中，超声功率为500瓦处理，再加入60毫升过氧化氢充分溶解；
4)向上述溶液中加入400微升浓度为1摩尔每升的氢氧化钾溶液，然后将上述溶液转移到圆底烧瓶中，在60摄氏度的油浴锅中回流6小时；
5)回流结束后，将上述溶液用孔径为0.22微米的有机微孔滤膜真空抽滤进行提纯处理，除去大块的杂质，再进行干燥处理，得到含氟石墨烯量子点，其尺寸更小(横向尺寸约2.0-3.0纳米)、形貌均一、非激发光依赖性发光等特点；
6)将上述反应后的溶液装入截留分子量为500-1000的透析袋透析，旋蒸、冻干处理，制得平均厚度为1.0-3.0纳米、尺寸为2.0纳米-3.0纳米，氟含量为1％-2％的含氟石墨烯量子点。
图1为该含氟石墨烯量子点的透射电子显微镜图像。可以看到含氟石墨烯量子点具有良好的单分散性，为尺寸约2纳米的扁平状圆形颗粒。
图2为本发明制得含氟石墨烯量子点在超纯水中的紫外可见吸收光谱和荧光发射光谱图(激发波长为320纳米)，分析可知在365纳米的紫外灯下呈明亮的绿色荧光，在320纳米光激发下，含氟石墨烯量子点水溶液在510纳米有一个荧光发射峰。
图6为采用MTT法检测制备的含氟石墨烯量子点在黑暗条件下的细胞毒性。将食道癌细胞(由安徽医科大学提供)与含氟石墨烯量子点共培养12小时后，细胞的存活率仍维持在90％以上，表明制得的含氟石墨烯量子点具有很低的毒性。
最后，检测所制备的含氟石墨烯量子点在水溶液中产生的单线态氧，步骤如下：
(a)称取合成好的含氟石墨烯量子点，用超纯水配成浓度为0.2毫克每毫升的含氟石墨烯量子点溶液；
(b)向2毫升离心管A中加入上述所制取的0.2毫克每毫升的含氟石墨烯量子点溶液1毫升，再向离心管A中加入2,2,6,6-四甲基哌啶(TEMP)10微升，离心管A用LED灯(激发波长400-700纳米)光照12分钟；
(c)用直径0.55毫米的毛细管抽取30-50微升待测样品，将毛细管放入测试管底部，将样品调节到测试管中央再放入电子顺磁共振波谱仪(EPR)中，EPR管放置在谐振腔中心位置；EPR测定参数如下：中心磁场3430.00高斯，扫场宽度60.00高斯，扫描次数10，时间常数10.49毫秒；
(d)另外，向2毫升离心管B中加入上述所制取的0.2毫克每毫升的含氟石墨烯量子点溶液1毫升，再向离心管B中加入2,2,6,6-四甲基哌啶(TEMP)10微升，离心管B置于黑暗条件下，重复操作步骤(c)，所得出的结果如图3所示。分析图3可知，含氟石墨烯量子点在LED(激发波长400-700纳米)照射下有单线态氧产生的信号，而在黑暗条件只有少量甚至没有单线态氧的产生，其单线态氧产率为0.4-0.5。
单线态氧产率计算方法如下：
使用化学方法进行单线态氧( 1O 2)量子产率评估。水溶性9,10-蒽二基-双(亚甲基)二酮酸(ABDA)用作 1O 2捕获剂，玫瑰红(RB)用作光敏剂标准。简而言之，将120微升ABDA溶液(2.5毫克每毫升)加入到400微升含氟石墨烯量子点溶液中，并使用设定为40毫瓦每平方厘米的LED灯(400～700纳米)作为光源。为了消除内部滤波效应，将玫瑰红(RB)和测试样品的吸收最大值调整为小于0.2。在各种照射时间记录ABDA的吸收，以获得光敏化过程的衰减速率。通过下式计算水中样品的 1O 2量子产率：
Ф sam＝Ф RB×(K sam×A RB)/(K RB×A sam)
其中K sam和K RB分别是测试样品和玫瑰红(RB)的ABDA分解速率常数。A sam和A RB分别代表样品和玫瑰红(RB)吸收的光，通过积分400～700纳米波长范围内的光学吸收带来确定。Ф RB是玫瑰红(RB)的 1O 2量子产率，在水中Ф RB＝0.75。
将本发明制得的含氟石墨烯量子点是作为光敏剂应用在光动力治疗中，在光 照下可以产生大量的单线态氧，其产率在0.4-0.5，从而提高光动力治疗效果，可以应用在多种肿瘤、食道癌、皮肤癌和早期肺癌等疾病的治疗。
食道癌细胞培养条件：将食道癌细胞放置于含有8％的胎牛血清的1640培养基中，然后再置于37摄氏度，5％CO 2的培养箱中培养。当细胞培养增殖至大约铺满培养瓶底部时，去除老旧的培养基，加入2毫升已经预热至37摄氏度的磷酸盐缓冲溶液对细胞进行清洗，然后加入1毫升0.25％胰蛋白酶溶液浸润细胞，随后离心去除胰酶，置于37摄氏度下消化大约4分钟，放置于光学显微镜下观察细胞的形态变化。最后，加入2毫升1640培养液，轻轻吹打细胞使得其脱壁，形成单细胞悬液，并加入1640培养基继续培养细胞。按照所需浓度将细胞转移到96孔板中继续培养。
采用四甲基偶氮唑盐(MTT)法检测制得的含氟石墨烯量子点的细胞毒性：是一种常见的检测细胞存活和生长的方法。检测原理是：活细胞线粒体中的琥珀酸脱氢酶能使外源性MTT还原成不溶于水的蓝紫色结晶甲瓒并沉积在细胞中，死细胞没有此能力，然后用二甲基亚砜溶解细胞中的结晶甲瓒，使用630纳米作为参比波长，同时使用563纳米为测试波长，用酶标仪测试吸光度。将细胞活力标准化为在培养基中培养的细胞活力。
MTT细胞毒性测试结果显示，食道癌细胞与不同浓度的含氟石墨烯量子点共培养12小时后，再光照12分钟，由图6和图7可知，不同浓度的含氟石墨烯量子点经LED灯光照后，其细胞毒性明显大于在黑暗中的细胞毒性，说明含氟石墨烯量子可有效并安全的应用于光动力治疗中。
实施例2
一种含氟石墨烯量子点的制备方法，具体包括以下步骤：
1)将氟化石墨浸泡于40％氢氧化钠溶液中，超声搅拌分散12小时使充分剥离，再经过滤、干燥处理；
2)将步骤(1)处理后的氟化石墨，采用Hummers法制备得到氧化含氟石墨烯；氟含量为6％-8％，氧含量为6％-8％，碳含量为80％-90％，可见其为二维纳米薄膜；
3)称取60毫克上述氧化石墨烯，溶解在60毫升超纯水中，超声功率为800瓦处理，再加入20克浓硫酸和浓硝酸的混合液充分溶解；
4)向上述溶液中加入600微升浓度为1摩尔每升的氢氧化钾溶液，然后将上述溶液转移到圆底烧瓶中，在70摄氏度的油浴锅中回流6小时；
5)回流反应结束后，将上述溶液进行萃取提纯处理，再进行冷冻干燥处理，得到平均厚度为1.0-3.0纳米、尺寸为2.0纳米-3.0纳米，氟含量为1％-2％的含氟石墨烯量子点。
实施例3
一种含氟石墨烯量子点的制备方法，具体包括以下步骤：
1)将氟化石墨浸泡于30％氢氧化钠溶液中，超声搅拌分散12小时使充分剥离，再经过滤、干燥处理；
2)将步骤(1)处理后的氟化石墨，采用Hummers法制备得到氧化含氟石墨烯；氟含量为6％-8％，氧含量为6％-8％，碳含量为80％-90％，可见其为二维纳米薄膜；
3)称取60毫克上述氧化含氟石墨烯，溶解在60毫升超纯水中，超声功率为600瓦处理，再加入60毫升过氧化氢充分溶解；
4)向上述溶液中加入400微升、1摩尔每升的氢氧化钠溶液，然后将上述溶液转移到圆底烧瓶中，在80摄氏度的油浴锅中回流5小时；
5)回流反应结束后，将上述溶液进行抽滤、提纯处理，除去大块的杂质，再进行真空干燥处理，得到平均厚度为1.0-3.0纳米、尺寸为2.0纳米-3.0纳米，氟含量为1％-2％的含氟石墨烯量子点含氟石墨烯量子点；
本发明制备的含氟石墨烯量子点具有反应步骤简单、成本低、绿色环保等优点，易分散于水、磷酸盐缓冲溶液、生物培养基等水溶液体系中；具有良好的生物相容性和低毒性。
本发明制得含氟石墨烯量子点在可见光的光照射下，可产生单线态氧，可作为光敏剂应用在光动力治疗中，适用于食道癌、皮肤癌和早期肺癌等的治疗过程，有着广泛的应用前景。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换或改进等，均应包含在本发明的保护范围之内。

Claims (6)

  1. 含氟石墨烯量子点，其是通过采用氟化石墨为原料，使用Hummers法制备得到氧化含氟石墨烯，再经化学氧化切割而得到的平均厚度为1.0-3.0纳米，尺寸为2.0-3.0纳米，氟含量为1％-2％，氧含量为20％-30％，碳含量为60％-70％，在可见光照射下单线态氧产率达到0.4-0.5的含氟石墨烯量子点。
  2. 权利要求1所述的含氟石墨烯量子点的制备方法，其特征在于，包括以下步骤：
    1)使用强碱性物质对氟化石墨进行超声剥离处理，再使用Hummers法制备得到氧化含氟石墨烯。
    2)称取氧化含氟石墨烯，溶解在超纯水中，超声处理；向上述溶液中加入强氧化剂，然后再加入碱液，在60-100摄氏度下回流5-9小时，让化学氧化切割反应充分进行；
    3)待回流结束后，过滤、提纯、干燥，即得；
  3. 如权利要求2所述的含氟石墨烯量子点的制备方法，其特征在于，所述步骤1)，所述强碱性物质采用氢氧化钠、氢氧化钾，所述氧化含氟石墨烯的氟含量为6％-8％，氧含量为6％-8％，碳含量为80％-90％。
  4. 如权利要求2所述的含氟石墨烯量子点的制备方法，其特征在于，所述步骤2)中的强氧化剂采用过氧化氢、浓硫酸、浓硝酸、过硫酸钾中的一种或多种的混合。
  5. 如权利要求2所述的含氟石墨烯量子点的制备方法，其特征在于，氧化含氟石墨烯和强氧化剂的质量比为(1-3)：(1000-3000)；所述氧化含氟石墨烯与超纯水的质量比为(1-3)：(1000-3000)。
  6. 权利要求1所述的或者由权利要求1-5任一项所述制备方法得到的含氟石墨烯量子点，作为光动力治疗光敏剂的应用。
PCT/CN2019/111622 2019-09-10 2019-10-17 含氟石墨烯量子点与制备及其作为光动力治疗光敏剂的应用 WO2021046966A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910854472.6A CN111518552B (zh) 2019-09-10 2019-09-10 含氟石墨烯量子点与制备及其作为光动力治疗光敏剂的应用
CN201910854472.6 2019-09-10

Publications (1)

Publication Number Publication Date
WO2021046966A1 true WO2021046966A1 (zh) 2021-03-18

Family

ID=71900521

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/111622 WO2021046966A1 (zh) 2019-09-10 2019-10-17 含氟石墨烯量子点与制备及其作为光动力治疗光敏剂的应用

Country Status (3)

Country Link
CN (1) CN111518552B (zh)
NL (1) NL2026426B1 (zh)
WO (1) WO2021046966A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114479843B (zh) * 2021-12-20 2023-06-30 华南农业大学 一种具有光动力治疗和杀菌功能的新型荧光纳米材料的制备方法和应用
CN115403831B (zh) * 2022-09-13 2023-10-24 中国科学院深圳先进技术研究院 一种改性氟化石墨烯量子点、光敏复合材料及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012083534A1 (zh) * 2010-12-22 2012-06-28 海洋王照明科技股份有限公司 氟化氧化石墨烯及其制备方法
CN105460920A (zh) * 2014-09-05 2016-04-06 中国科学院兰州化学物理研究所 水溶性的氟含量可控的氟化石墨烯量子点的制备方法
CN108423655A (zh) * 2018-03-29 2018-08-21 中国科学院福建物质结构研究所 一种氟氧化石墨烯的制备方法
CN108545729A (zh) * 2018-04-24 2018-09-18 常州烯思新材料科技有限公司 高效石墨烯量子点的制备方法

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102660270A (zh) * 2012-05-03 2012-09-12 吉林大学 溶剂热法制备荧光石墨烯量子点的方法
CN104944403B (zh) * 2014-03-24 2017-04-26 中国科学院兰州化学物理研究所 一种水溶性双色氟化石墨烯量子点的制备方法
CN105271200B (zh) * 2015-11-06 2017-11-28 昆明物理研究所 氟掺杂石墨烯量子点及其制备方法
CN105567229B (zh) * 2016-01-29 2018-01-30 天津大学 氟掺杂荧光碳量子点制备的方法
CN105565310A (zh) * 2016-03-02 2016-05-11 桂林理工大学 一种具有优异光学性能的氟掺杂石墨烯量子点的制备方法
CN106477565B (zh) * 2016-10-24 2019-05-14 国家纳米科学中心 一种氟化石墨烯量子点、及其制备方法和用途
CN109486483B (zh) * 2017-09-11 2021-11-23 天津大学 氟氮双元素掺杂荧光碳量子点及其制备方法
CN108706579B (zh) * 2018-07-23 2020-11-10 广西师范大学 一种制备氟掺杂石墨烯量子点的方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012083534A1 (zh) * 2010-12-22 2012-06-28 海洋王照明科技股份有限公司 氟化氧化石墨烯及其制备方法
CN105460920A (zh) * 2014-09-05 2016-04-06 中国科学院兰州化学物理研究所 水溶性的氟含量可控的氟化石墨烯量子点的制备方法
CN108423655A (zh) * 2018-03-29 2018-08-21 中国科学院福建物质结构研究所 一种氟氧化石墨烯的制备方法
CN108545729A (zh) * 2018-04-24 2018-09-18 常州烯思新材料科技有限公司 高效石墨烯量子点的制备方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GONG PEIWEI; YANG ZHIGANG; HONG WEI; WANG ZHAOFENG; HOU KAIMING; WANG JINQING; YANG SHENGRONG: "To lose is to gain: Effective synthesis of water-soluble graphene fluoroxide quantum dots by sacrificing certain fluorine atoms from exfoliated fluorinated graphene", CARBON, ELSEVIER OXFORD, GB, vol. 83, 24 November 2014 (2014-11-24), GB, pages 152 - 161, XP029115883, ISSN: 0008-6223, DOI: 10.1016/j.carbon.2014.11.027 *
LI, XINCONG, ＭIAOＬEＩＹＩＮＧ: "Advances in the Research of Graphene Quantum Dots in Biomedica Science", MATERIALS REVIEW, vol. 32, 31 May 2018 (2018-05-31), pages 176 - 182, XP055790182 *

Also Published As

Publication number Publication date
CN111518552B (zh) 2021-08-10
CN111518552A (zh) 2020-08-11
NL2026426B1 (en) 2021-09-23
NL2026426A (en) 2021-05-12

Similar Documents

Publication Publication Date Title
Wu et al. Polymerization-enhanced photosensitization
Zhao et al. Preparation of N-doped yellow carbon dots and N, P co-doped red carbon dots for bioimaging and photodynamic therapy of tumors
Pan et al. Near-infrared emissive carbon dots for two-photon fluorescence bioimaging
CN108276996B (zh) 基于氮硫共掺杂石墨烯量子点材料、其制备方法及其应用
CN110339357B (zh) 铜离子掺杂碳点、制备及其作为光动力治疗光敏剂的应用
CN109321240B (zh) 一种橙色荧光碳点及其制备方法
WO2021046966A1 (zh) 含氟石墨烯量子点与制备及其作为光动力治疗光敏剂的应用
CN106629660A (zh) 一种n,p共掺杂碳量子点的制备方法及其产品、应用
Jiang et al. Fabrication of multifunctional fluorescent organic nanoparticles with AIE feature through photo-initiated RAFT polymerization
WO2022095131A1 (zh) 一种碳纳米粒子的制备方法及应用
CN114349756B (zh) 一种aie有机小分子及其制备方法和应用
Wan et al. Regulation of multi-color fluorescence of carbonized polymer dots by multiple contributions of effective conjugate size, surface state, and molecular fluorescence
CN113648414B (zh) 一种金属离子配位的碳点/二氧化钛异质结及其制备方法和应用
Kou et al. Self-assembled photosensitive carbon nanocrystals with broad-spectrum antibacterial bioactivity
Hu et al. Carbon-based dot nanoclusters with enhanced roles of defect states in the fluorescence and singlet oxygen generation
CN108421040B (zh) 兼具双光子成像和光动力疗效的共轭高分子的纳米光敏材料及制备与应用
CN113304280A (zh) 一种稀土上转换复合纳米材料用于肿瘤治疗
Li et al. NIR-II responsive PEGylated MoO 2 nanocrystals with LSPR for efficient photothermal and photodynamic performance enhancement
CN114681611B (zh) 一种聚3-噻吩乙酸修饰pcn-224复合材料及其制备方法和应用
CN111518274A (zh) 共轭聚合物量子点及其制备方法和应用
CN113999675B (zh) 一种细胞衍生荧光碳纳米片及其制备方法和应用
CN114209832B (zh) 一种光治疗试剂及其制备方法和应用
CN115353881B (zh) 一种荧光探针及其制备方法与应用
CN114933904A (zh) 一种用于光学诊疗的超薄壳层手性硒化镉/硫化镉材料及其制备方法与应用
CN114958363B (zh) 靶向高尔基体的红光发射荧光碳点及其制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19945183

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19945183

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 27/09/2022)

122 Ep: pct application non-entry in european phase

Ref document number: 19945183

Country of ref document: EP

Kind code of ref document: A1