WO2021253470A1 - 一种碳纳米复合生物制剂及制备方法和应用 - Google Patents

一种碳纳米复合生物制剂及制备方法和应用 Download PDF

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WO2021253470A1
WO2021253470A1 PCT/CN2020/097723 CN2020097723W WO2021253470A1 WO 2021253470 A1 WO2021253470 A1 WO 2021253470A1 CN 2020097723 W CN2020097723 W CN 2020097723W WO 2021253470 A1 WO2021253470 A1 WO 2021253470A1
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carbon
biological
carbon nanoparticles
preparation
biological material
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王先玉
王晓筠
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广东量子墨滴生物科技有限公司
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    • 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
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/13—Tumour cells, irrespective of tissue of origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/38—Albumins
    • A61K38/385—Serum albumin
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/52—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an inorganic compound, e.g. an inorganic ion that is complexed with the active ingredient
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • 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
    • C—CHEMISTRY; METALLURGY
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    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
    • 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
    • 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

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  • the invention relates to the fields of nano materials and biomedicine, in particular to a carbon nano composite biological preparation and a preparation method and application.
  • Carbon Nano Particles are spherical-like, oblate cylindrical, or other irregularly shaped carbon-based nanoparticles with three dimensions all less than 100 nm in size.
  • CNPs have many characteristics such as good fluorescence characteristics, non-toxicity, good biocompatibility, and easy absorption and emission by organisms.
  • CNPs as nanomaterials have huge application prospects in the field of biomedicine.
  • Pure carbon nanoparticles are easy to aggregate in aqueous solvents, so there are potential risks when applied in vivo, which limits their applications in biomedicine fields such as drug carriers, imaging reagents, and tumor immune activation.
  • the purpose of the present invention is to overcome the technical problems of carbon nano-particles in the prior art that are easily agglomerated in aqueous solvents, unable to obtain stable carbon nanocomposite biomaterials, and limited application in the field of biomedicine, and provide a carbon nanocomposite Biological agents.
  • Another object of the present invention is to provide a method for preparing a carbon nano composite biological preparation.
  • Another object of the present invention is to provide an application of carbon nanocomposite biological preparation.
  • a carbon nano composite biological preparation comprising carbon nano particles.
  • the surface of the carbon nano particles contains multiple functional groups.
  • the functional groups are one or more of hydroxyl, carboxyl, sulfhydryl or amino;
  • the surface of the carbon nanoparticle is wrapped around the composite biological material, and the functional group on the surface of the carbon nanoparticle is the binding site of the carbon nanoparticle and the biological material;
  • the biological material includes biological macromolecules, cells or cell fragments, bacteria or bacterial fragments, and viruses Or one or more of virus fragments.
  • Biological materials mainly include biological macromolecules, cells or cell fragments, bacteria or bacterial fragments, viruses or viral fragments.
  • the biological macromolecules interact with the functional groups on the surface of the carbon nanoparticles, and the biological macromolecules are wound around the surface of the carbon nanoparticles under the thermal process.
  • the surfaces of cells, bacteria, and viruses are rich in proteins and other substances.
  • cells, bacteria, viruses and their fragments can also be compounded with carbon nanoparticles through biological macromolecules such as proteins on the surface.
  • the composite of biomaterials on the surface of carbon nano-particles helps to improve the dispersibility of carbon nano-particles in aqueous solution, prevents the agglomeration of carbon nano-particles in water, forms a stable carbon nano-composite biological material, and improves the bio-materials coated on the surface of carbon nano-particles.
  • the immunogenicity of carbon nanocomposite has better immune activation characteristics, and improves the effectiveness of the application of carbon nanocomposite biomaterials in tumor immune activation, drug carriers, and bioimaging agents.
  • the biological macromolecule includes one or more of protein, polypeptide, nucleic acid, polynucleotide, carbohydrate, dextran, polysaccharide or lipid.
  • the cells or cell fragments are cancer cells or cancer cell fragments.
  • a method for preparing the carbon nano composite biological preparation includes the following steps:
  • step S2 Mix the carbon nano particles obtained in step S1 with the biological material in a mass ratio of 1: (0.1 to 10000), react at 25 to 100° C. for 2 to 30 minutes, and obtain after purification.
  • the preparation of carbon nanoparticles in step S1 specifically includes: dissolving citric acid and urea in dimethyl sulfoxide or dimethyl formamide, putting them in an autoclave, and heating at 110-220°C for reaction 2 ⁇ 10h, and then centrifuge and clean the obtained carbon nanoparticles with methanol or ethanol, and the centrifugal speed is preferably 6000-10000rpm.
  • the preparation of carbon nanoparticles is specifically: mixing citric acid and Disperse Blue-1 in deionized water or ultrapure water, putting them in an autoclave, heating and reacting at 110-220°C for 2-10 hours, and then The obtained carbon nano particles are centrifuged and cleaned with methanol or ethanol, and the centrifugal rotation speed is preferably 6000-10000 rpm.
  • nanoparticles in the present invention are not limited to the above-mentioned methods.
  • the heating method used in step S2 includes one of thermal conduction, thermal radiation, photothermal, magnetic heating, or microwave heating.
  • the present invention has the following technical effects:
  • the present invention provides a carbon nanoparticle biological composite preparation.
  • the biological material interacts with the active groups (one or more of hydroxyl, sulfhydryl, carboxyl, and amino) on the surface of the carbon nanoparticle to generate energy. Under the action of the heat, it is wound onto the surface of the carbon nano-particles to form a biological composite preparation of carbon nano-particles and biological materials.
  • the coating of biological materials on the surface of carbon nanoparticles will effectively improve the dispersibility of carbon nanoparticles in the aqueous solution and prevent the agglomeration of carbon nanoparticles in water.
  • Figure 1 Fluorescence spectra of carbon nanoparticles and their compound preparations with bovine serum albumin
  • Figure 5 Changes in the absorption spectrum of carbon nanoparticles and their compound preparations with bovine serum albumin after being stored at room temperature for 1 month;
  • Figure 6 Changes in fluorescence spectra of carbon nanoparticles and their compound preparations with bovine serum albumin after being stored at room temperature for 1 month;
  • Fig. 11 is a schematic diagram of the structure of the interaction between carbon nanoparticles of the present invention and biomaterials (a-hydroxyl, b-carboxyl, c-sulfhydryl, d-amino; 1-biomaterial, 2-carbon nanoparticle).
  • the equipment used in this embodiment is conventional experimental equipment, and the materials and reagents used are all commercially available unless otherwise specified, and the experimental methods without special instructions are also conventional experimental methods.
  • Dissolve the obtained carbon nanoparticles in deionized water configure a solution with a concentration of (0.1 ⁇ 1.5) mg/mL, mix the carbon nanoparticles and bovine serum albumin at a mass ratio of 1:10, use light heating, hot plate heating, and water bath (Oil bath) heating, oven heating or ultrasonic heating and other heating methods to raise the temperature to 25-100°C, react for 2-30min, and stir at 40-250rpm at the same time.
  • the bovine serum albumin (CQD-BSA) composite preparation of carbon nanoparticles is obtained.
  • Dissolve citric acid and Disperse Blue-1 in water or ultrapure water, PBS buffer, etc.
  • water or ultrapure water, PBS buffer, etc.
  • a large amount of ethanol was added to the reaction solution to obtain a black solid, which was washed with water, centrifuged (8000 rpm, 5 min), and dried to obtain a dark blue powder.
  • the carbon nanoparticles are dissolved in deionized water at a concentration of (0.1-1.2) mg/mL, and 1 ⁇ 10 4 to 1 ⁇ 10 10 4T1 breast cancer cells are added to each milliliter of the above solution.
  • the breast cancer cell (CQD-cell) composite preparation of carbon nanoparticles is obtained.
  • CQD-BSA bovine serum albumin
  • the carbon nanoparticle (CQD) and carbon nanoparticle bovine serum albumin (CQD-BSA) composite preparation obtained in Example 1 was put into a dialysis bag for dialysis, and the dialysis time was 2 days.
  • the absorption spectra of carbon nano-particles before (CQD) and after (CQD dialys); and before (CAD-BSA) and after (CQD-BSA dialys) of the bovine serum albumin compound preparation of carbon nanoparticles As shown in Figure 2, the absorption of the pure carbon nanoparticle solution after dialysis is close to 0, and almost all of it leaks out.
  • the absorption spectrum of the composite preparation is basically unchanged, indicating that the carbon nanoparticles and bovine serum albumin are effectively combined.
  • the carbon nanoparticles and bovine serum albumin obtained in Example 1 were dissolved in deionized water at a mass ratio of 1:10, stirred and heated for 10 minutes, and the reaction temperature was set to 25°C, 40°C, 60°C, 80°C, and 90°C, respectively. Fluorescence test was performed on CQD-BSA obtained at different reaction temperatures. The results are shown in Figure 3. As the compounding temperature increases, the fluorescence intensity of the compound preparation increases significantly. The CQD-BSA obtained under different temperature conditions was dialyzed for 2 days, and the absorption spectra before and after the dialysis were measured. As shown in Figure 4, as the composite temperature increased, the carbon quantum particles that were dialyzed out gradually decreased.
  • the carbon nanoparticle (CQD) and carbon nanoparticle bovine serum albumin (CQD-BSA) composite preparation obtained in Example 1 was dispersed in an aqueous solution to prepare a 1 mg/mL solution. Store the solution at room temperature for 30 days.
  • the absorption spectra of CQD and CQD-BSA were measured before and after placement. The results are shown in Figure 5. After placement, the absorption peak near 600nm in the red band of carbon nanodots disappeared, leaving only the ultraviolet absorption peak near 350nm, while the absorption spectrum of the composite preparation The line remained basically unchanged, indicating that the structure of the pure carbon nanoparticles changed after 30 days of storage, while the composite formulation was slightly affected.
  • the fluorescence characteristics of the breast cancer (CQD-cell) composite preparation of carbon nanoparticles and the carbon nanoparticles obtained in Example 2 were observed by fluorescence spectroscopy, and the results are as shown in FIG. 7. After recombination, the dispersibility of carbon nanoparticles is increased, and the emission peak intensity is enhanced, accompanied by a blue shift of the emission peak position.
  • the absorption spectra of CQD and CQD-cell as shown in Figure 8, it can be found that the absorption spectra of carbon nanoparticles before and after recombination have not substantially changed.
  • the carbon nanoparticles obtained in Example 2 were combined with 4T1 breast cancer cells, and 1 mL of carbon nanoparticles with a mass concentration of 0.2 mg/mL and 4T1 cells with a quantity of 1 ⁇ 10 6 were stirred for 10 minutes, and the reaction temperature was set to 25 °C, 40°C, 70°C, 90°C.
  • the composite preparation proved the composite stability of carbon nanoparticles and 4T1 cells under the applied energy through the changes of the absorption spectra before and after dialysis.
  • the results are shown in FIG. 9. It is found from Fig. 9 that with the increase of the recombination temperature, the number of carbon nano-particles dialyzed out gradually decreases, the absorption intensity gradually increases, and the shape of the absorption spectrum curve basically remains unchanged.
  • the CQD-cell preparation obtained by compounding in a certain amount of experimental example 6 at 80°C was injected into mice with 4T1 breast cancer tumors.
  • the injection dose is 200ul
  • the injection method is intravenous injection, subcutaneous injection and ascites injection, preferably subcutaneous injection.
  • Figure 10 shows that the growth of the tumor in the experimental group a was significantly inhibited and then cured, while the tumor in the control group b gradually increased. It shows that the CQD-cell preparation effectively activates the tumor immune function of mice, inhibits and kills the tumors in the mice.

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Abstract

涉及纳米材料和生物医学领域，具体涉及一种碳纳米复合生物制剂及制备方法和应用，包括碳纳米粒子，所述碳纳米粒子表面包含有多个官能团，所述官能团为羟基、羧基、巯基或氨基中的一种或多种；通过热复合作用，所述碳纳米粒子表面缠绕复合生物材料，所述碳纳米粒子表面的官能团为所述碳纳米粒子与生物材料的结合位点；所述生物材料包括生物大分子、细胞或细胞碎片、细菌或细菌碎片、病毒或病毒碎片中的一种或多种。生物材料在碳纳米粒子表面的包覆，可稳定包覆在碳纳米粒子表面的生物材料。大幅度提高碳纳米粒子的发光性能，提高碳纳米粒子表面所包覆的生物材料的免疫源性，进而具有更佳的免疫激活特性，以及能够应用到癌症的诊断和治疗中。

Description

一种碳纳米复合生物制剂及制备方法和应用 技术领域
本发明涉及纳米材料和生物医学领域，具体涉及一种碳纳米复合生物制剂及制备方法和应用。
技术背景
碳纳米粒子(Carbon Nano Particles，以下简称CNPs)是三个维度的尺寸均小于100nm的类球状、扁柱状、或其它不规则形状的碳基纳米粒子。CNPs具有良好荧光特性，无毒性，生物相容性好，容易被生物体吸收和排放等诸多特点。CNPs作为纳米材料在生物医学领域有巨大的应用前景。
纯的碳纳米粒子在水性溶剂中容易聚集成团，因而在生物体内应用时存在潜在风险，限制了其作为药物载体、影像试剂、肿瘤免疫激活等生物医药领域中的应用。
发明内容
本发明的目的在于克服现有技术中的碳纳米粒子在水性溶剂中容易团聚，无法得到稳定的碳纳米复合生物材料以及在生物医药领域的应用受到限制等技术问题，提供了一种碳纳米复合生物制剂。
本发明的另一个目的在于提供一种碳纳米复合生物制剂的制备方法。
本发明的另一个目的在于提供一种碳纳米复合生物制剂的应用。
本发明的目的通过以下技术方案予以实现：
一种碳纳米复合生物制剂，包括碳纳米粒子，所述碳纳米粒子表面包含有多个官能团，所述官能团为羟基、羧基、巯基或氨基中的一种或多种；通过热复合作用，所述碳纳米粒子表面缠绕复合生物材料，所述碳纳米粒子表面的官能团为碳纳米粒子与生物材料的结合位点；所述生物材料包括生物大分子、细胞或细胞碎片、细菌或细菌碎片、病毒或病毒碎片中的一种或多种。
将碳纳米粒子与生物材料混合，在外加能量的作用下，通过热过程使碳纳米粒子表面的羟基、巯基、羧基和氨基的一种或多种基团与生物材料相互作用，使生物材料缠绕到碳纳米粒子表面。生物材料主要包括生物大分子、细胞或者细胞碎片、细菌或细菌碎片、病毒或病毒碎片。生物大分子与碳纳米粒子表面的官能团相互作用，在热过程下，使得生物大分子缠绕在碳纳米粒子的表面。细胞、细菌、病毒表面含有丰富的蛋白等物质，因此细胞、细菌、病毒及其碎片也可以通过表面的蛋白等生物大分子与碳纳米粒子复合。生物材料复合到碳纳 米表面有助于提高碳纳米粒子在水溶液中的分散性，阻止碳纳米粒子在水中的团聚，形成稳定的碳纳米复合生物材料，提高碳纳米粒子表面所包覆的生物材料的免疫源性，进而具有更佳的免疫激活特性，提高碳纳米复合生物材料在肿瘤免疫激活、药物载体以及生物影像剂等领域的应用的有效性。
优选地，所述生物大分子包括蛋白、多肽、核酸、多核苷酸、碳水化合物、葡聚糖、多糖或脂类中的一种或多种。
优选地，所述细胞或细胞碎片为癌细胞或癌细胞碎片。
一种所述碳纳米复合生物制剂的制备方法，包括以下步骤：
S1.制备碳纳米粒子；
S2.将步骤S1得到的碳纳米粒子和生物材料按照质量比为1:(0.1～10000)混合，在25～100℃条件下反应2～30min，纯化后即得。
步骤S1中碳纳米粒子的制备具体为：将柠檬酸和尿素混合溶于二甲基亚砜或二甲基甲酰胺中，放入高压反应釜中，在110～220℃条件下，加热反应2～10h，然后用甲醇或者乙醇对得到的碳纳米粒子进行离心清洗，离心转速优选为6000～10000rpm。
或者碳纳米粒子的制备具体为：将柠檬酸和分散蓝-1混合在去离子水或者超纯水中，放入高压反应釜中，在110～220℃条件下，加热反应2～10h，然后用甲醇或者乙醇对得到的碳纳米粒子进行离心清洗，离心转速优选为6000～10000rpm。
当然本发明中的纳米粒子不限于上述方法制备。
步骤S2中采用的加热方法包括热传导、热辐射，光热，磁热或微波加热中的一种。
所述碳纳米复合生物制剂作为生物影像剂的应用。
所述碳纳米复合生物制剂作为药物载体的应用。
所述碳纳米复合生物制剂作为肿瘤免疫药物的应用。
与现有技术相比，本发明具有以下技术效果：
本发明提供的一种碳纳米粒子生物复合制剂，所述的生物材料与碳纳米粒子表面的活性基团(羟基、巯基、羧基、氨基中的一种或多种)发生作用，在外加能量产生的热的作用下缠绕到碳纳米粒子表面，形成碳纳米粒子与生物材料的生物复合制剂。生物材料在碳纳米粒子表面的包覆，将有效地提高碳纳米粒子在水溶液里面的分散性，防止碳纳米粒子在水中的团聚。其效果表现为大幅度提高碳纳米粒子的发光强度及发光稳定性，提高碳纳米粒子表面所包覆的生物材料的免疫源性，进而具有更佳的免疫激活特性，提高碳纳米复合生物材料在肿瘤免疫激活、药物载体以及生物影像剂等领域的应用。
附图说明
图1碳纳米粒子及其与牛血清蛋白复合制剂的荧光光谱；
图2碳纳米粒子及其与生物复合制剂透析前后的吸收光谱；
图3不同反应温度下碳纳米粒子及其与牛血清蛋白复合制剂的荧光光谱；
图4不同反应温度下碳纳米粒子及其与牛血清蛋白复合制剂透析后的吸收光谱；
图5碳纳米粒子及其与牛血清蛋白复合制剂常温存放1个月后，吸收光谱的变化；
图6碳纳米粒子及其与牛血清蛋白复合制剂常温存放1个月后，荧光光谱的变化；
图7碳纳米粒子及其与细胞复合制剂的荧光光谱；
图8碳纳米粒子及其与细胞复合制剂的吸收光谱；
图9不同反应温度下，碳纳米粒子及其与细胞复合制剂透析前后的吸收光谱；
图10注射肿瘤免疫激活剂治疗的小鼠以及对照组小鼠的肿瘤生长曲线；
图11本发明碳纳米粒子与生物材料相互作用的结构示意图(a-羟基、b-羧基、c-巯基、d-氨基；1-生物材料，2-碳纳米粒子)。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面结合具体实施例和对比例将对本发明的技术方案进行详细的描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式，都属于本发明所保护的范围。
除特殊说明，本实施例中所用的设备均为常规实验设备，所用的材料、试剂若无特殊说明均为市售得到，无特殊说明的实验方法也为常规实验方法。
实施例1
将柠檬酸和尿素按质量比1:3溶解于DMSO溶剂中，得到透明溶液置于50ml聚四氟乙烯高压反应釜中，160℃下反应4h，反应后的溶液加入大量乙醇洗涤，得到黑色固体，将固体水洗，离心(8000rpm，5min)，干燥后得到深蓝色粉末。测定获得的碳纳米粒子的直径为3～10nm。碳纳米粒子的C、N、O、S元素质量比分别为50.1％，29.3％，19.1％和1.5％。
将得到的碳纳米粒子溶于去离子水中，配置浓度为(0.1～1.5)mg/mL溶液，将碳纳米粒子和牛血清蛋白的质量比是1:10混合，采用光照加热、热板加热、水浴(油浴)加热、烘箱加热或超声加热等加热方式升温至25～100℃，反应2～30min，并同时以40～250rpm转速搅拌。即得到碳纳米粒子的牛血清蛋白(CQD-BSA)复合制剂。
实施例2
将柠檬酸和分散蓝-1按质量比1:3溶解于水(或超纯水、PBS缓冲液等)中，得到透明溶液置于50ml聚四氟乙烯高压反应釜中，200℃下反应3h。反应后的溶液加入大量乙醇，得 到黑色固体，固体水洗，离心(8000rpm，5min)，干燥后得到深蓝色粉末。
将上述碳纳米粒子溶于去离子水中，配置浓度为(0.1～1.2)mg/mL，在上述溶液中每毫升中加入1x10 4～1x10 10个4T1乳腺癌细胞。采用光照加热、热板加热、水浴(油浴)加热、烘箱加热或超声加热等加热方式升温至25～100℃，反应2～30min，并同时以40～500rpm转速搅拌。即得碳纳米粒子的乳腺癌细胞(CQD-cell)复合制剂。
实施例3
碳纳米粒子的制备同实施例1。
将得到的碳纳米粒子溶于去离子水中，配置浓度为(0.1～1.5)mg/mL溶液，将碳纳米粒子和乳杆菌的质量比是1.0:10混合，采用光照加热、热板加热、水浴(油浴)加热、烘箱加热或超声加热等加热方式升温至25～100℃，反应2～30min，并同时以40～250rpm转速搅拌。即得到碳纳米粒子的乳酸杆菌复合制剂。
实施例4
碳纳米粒子的制备同实施例1。
将得到的碳纳米粒子溶于去离子水中，配置浓度为(0.1～1.5)mg/mL溶液，将碳纳米粒子和非洲猪瘟病毒(ASFV)的质量比是1.0:10混合，采用光照加热、热板加热、水浴(油浴)加热、烘箱加热或超声加热等加热方式升温至25～100℃，反应2～30min，并同时以40～250rpm转速搅拌。即得到碳纳米粒子的非洲猪瘟病毒(CQD-ASFV)复合制剂。
实验例1
测定实施例获得的碳纳米粒子(CQD)以及得到的碳纳米粒子的牛血清蛋白(CQD-BSA)复合制剂的荧光，结果如图1所示。经过复合后得到的CQD-BSA，增加了碳纳米粒子的分散性，具体表现为PL发射强度显著增强。
实验例2
将实施例1得到的碳纳米粒子(CQD)和碳纳米粒子的牛血清蛋白(CQD-BSA)复合制剂，分别放入透析袋中进行透析，透析时间为2天。通过测定碳纳米透析之前(CQD)和透析之后(CQD dialys)；以及碳纳米粒子的牛血清蛋白复合制剂透析前(CAD-BSA)和之后(CQD-BSA dialys)的吸收光谱。如图2所示，透析后的纯碳纳米粒子溶液的吸收接近0，几乎全部透出。而复合制剂的吸收谱线基本不变，说明碳纳米粒子和牛血清蛋白发生了有效复合。
实验例3
将实施例1获得的碳纳米粒子和牛血清蛋白按照质量比为1:10溶解在去离子水中，搅拌加热10min，反应温度分别设为25℃、40℃、60℃、80℃、90℃。对不同反应温度获得的 CQD-BSA进行荧光测试，结果如图3所示，随着复合温度的增加复合制剂的荧光强度显著增强。再对不同温度条件下获得的CQD-BSA透析2天，再测定透析前后的吸收光谱，结果如图4所示，随着复合温度的增加，透析出去的碳量子粒子逐渐减少。
实验例4
将实施例1获得碳纳米粒子(CQD)和碳纳米粒子的牛血清蛋白(CQD-BSA)复合制剂分散在水溶液中，配置成1mg/mL的溶液。将溶液在常温下保存，放置30天。测定放置前后CQD和CQD-BSA的吸收光谱，结果如图5所示，经过放置，碳纳米点的红色波段600nm附近的吸收峰消失，只保留350nm附近的紫外吸收峰，而复合制剂的吸收谱线基本保持不变，说明纯碳纳米粒子放置30天后结构发生了变化，而复合制剂受影响甚微。测定放置前后CQD和CQD-BSA的荧光光谱，结果如图6所示，经过长期放置，纯碳纳米点发生聚集，PL的峰值降低。而复合制剂非常稳定，光谱基本不变。
实验例5
通过荧光光谱观察碳纳米粒子和实施例2得到的碳纳米粒子的乳腺癌(CQD-cell)复合制剂的荧光特性，结果如图7所述。经过复合后，增加了碳纳米粒子的分散性，表现为发射峰强度增强，伴随发射峰位置蓝移。通过CQD和CQD-cell的吸收光谱，如图8所示，可以发现经过复合前后碳纳米粒子的吸收光谱没有发生实质性的变化。
实验例6
将实施例2获得的碳纳米粒子和4T1乳腺癌细胞复合，将1mL质量浓度为0.2mg/mL的碳纳米粒子和数量为1×10 6的4T1细胞，搅拌10min，反应温度分别设置为，25℃，40℃，70℃，90℃。复合制剂通过透析前后的吸收光谱的变化证明碳纳米粒子和4T1细胞在外加能量下的复合稳定性，结果如图9所示。由图9发现，随着复合温度的增加，透析出去的碳纳米粒子逐渐减少，吸收强度逐渐增强，吸收光谱曲线形状基本保持不变。
实验例7
将一定计量实验例6经80℃复合获得的CQD-cell制剂注射到带4T1乳腺癌肿瘤的小鼠体内。优选注射计量为200ul，注入方法为静脉注入、皮下注入和腹水注入的一种，优选皮下注入。
测量小鼠的肿瘤生长曲线，结果如图10所示。图10发现，实验组a肿瘤生长显著受到抑制进而治愈，而对照组b肿瘤逐渐增大。说明CQD-cell制剂有效地激活了小鼠的肿瘤免疫功能，抑制并灭杀了小鼠体内的肿瘤。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案，而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细地说明，本领域的普通技术人员应当理解， 可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。

Claims (8)

  1. 一种碳纳米复合生物制剂，其特征在于，包括碳纳米粒子，所述碳纳米粒子表面包含有多个官能团，所述官能团为羟基、羧基、巯基或氨基中的一种或多种；通过热复合作用，所述碳纳米粒子表面缠绕复合生物材料，所述碳纳米粒子表面的官能团为碳纳米粒子与生物材料的结合位点；所述生物材料包括生物大分子、细胞或细胞碎片、细菌或细菌碎片、病毒或病毒碎片中的一种或多种。
  2. 根据权利要求1所述碳纳米复合生物制剂，其特征在于，所述生物大分子包括蛋白、多肽、核酸、多核苷酸、碳水化合物、葡聚糖、多糖或脂类中的一种或多种。
  3. 根据权利要求1所述碳纳米复合生物制剂，其特征在于，所述细胞或细胞碎片包括癌细胞或癌细胞碎片。
  4. 一种权利要求1至3任一项所述碳纳米复合生物制剂的制备方法，其特征在于，包括以下步骤：
    S1.制备碳纳米粒子；
    S2.将步骤S1得到的碳纳米粒子和生物材料按照质量比为1:(0.10～10000)混合，在25～100℃条件下反应2～30min，纯化后即得。
  5. 根据权利要求4所述碳纳米复合生物制剂的制备方法，其特征在于，所述步骤S1中碳纳米粒子的制备，具体包括：将柠檬酸和尿素或者分散蓝-1，按照质量比为1：(1～6)混合溶于溶剂中，在110～220℃溶剂热反应条件下，反应2～10h。
  6. 权利要求1至3任一项所述碳纳米复合生物制剂作为生物影像剂的应用。
  7. 权利要求1至3任一项所述碳纳米复合生物制剂作为药物载体的应用。
  8. 权利要求1至3任一项所述碳纳米复合生物制剂作为肿瘤免疫激活剂的应用。
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