WO2023274297A1 - 一种量子点增强型肿瘤灭活疫苗及制备方法和应用 - Google Patents

一种量子点增强型肿瘤灭活疫苗及制备方法和应用 Download PDF

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WO2023274297A1
WO2023274297A1 PCT/CN2022/102375 CN2022102375W WO2023274297A1 WO 2023274297 A1 WO2023274297 A1 WO 2023274297A1 CN 2022102375 W CN2022102375 W CN 2022102375W WO 2023274297 A1 WO2023274297 A1 WO 2023274297A1
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tumor
quantum dot
protein
enhanced
quantum dots
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French (fr)
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曲松楠
汤子康
梁桃
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澳门大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • 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
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen

Definitions

  • the disclosure relates to the fields of nanomaterials and biomedical technology, in particular, to a quantum dot-enhanced tumor inactivation vaccine, its preparation method and application.
  • Personalized cancer vaccines are currently one of the more popular cancer immunotherapies. It aims to modulate the innate and adaptive immune systems to broadly activate antitumor immunity with individual tumor-specific antigens (neoantigens).
  • neoantigens tumor-specific antigens
  • the process of how massively parallel sequencing and machine learning identify neoantigens is very expensive and time-consuming, and changes in neoantigens during this time will lead to ineffective anticancer responses.
  • Tumor cells are a natural source of a variety of tumor neoantigens, including those that have not yet been identified. However, whole-tumor cell vaccination always lacks strong immune stimulation, and the anti-tumor response is very limited. The main reason may be that most of the proteins contained in tumor cells are self-proteins, which themselves have low immunogenicity and express weak "eat me” signals to antigen-presenting cells such as macrophages and dendritic cells. Tumor cells derived from normal cell variants contain multiple immunosuppressive proteins that express "don't eat me” signals to prevent autophagy.
  • the purpose of the present disclosure is to provide a quantum dot-enhanced tumor inactivation vaccine and its preparation method and application in order to overcome the above-mentioned defects in the prior art.
  • the present disclosure provides a quantum dot-enhanced tumor inactivation vaccine, which includes tumor cells and quantum dots located in the protein space structure of tumor cells, and the quantum dots interact with protein molecules on tumor cells through biocompatible functional groups on their surfaces The groups on the chain are hydrogen bonded.
  • the present disclosure also provides a method for preparing the quantum dot-enhanced tumor inactivation vaccine described above, including: mixing quantum dots with tumor cells, and using heat to make the functional groups on the surface of the quantum dot particles and the protein molecular chains on the tumor cells groups interact to form hydrogen bonds.
  • the disclosure also provides an application of the above-mentioned quantum dot-enhanced tumor inactivation vaccine as personalized tumor immunotherapy and prevention.
  • the disclosure provides a quantum dot-enhanced tumor inactivation vaccine and its preparation method and application.
  • the quantum dot-enhanced tumor inactivation vaccine includes tumor cells and quantum dots located in the protein space structure of the tumor cells, and the quantum dots pass through
  • the biocompatible functional groups on the surface are hydrogen bonded to the groups on the protein molecular chains on the tumor cells. Due to its small surface size, quantum dots can enter the protein gap of tumor proteins, and the groups on the surface of quantum dots further interact with proteins on the surface of tumor cells to form hydrogen bonds.
  • the quantum dot particles only changed the conformation of the protein, but did not destroy its primary structure, so that the "don't eat me” signal expressed by it was reduced, and the "eat me” signal was increased.
  • the immunogenicity of the tumor protein is greatly improved, thereby exhibiting better immune activation properties, and can be applied to enhanced cellular vaccines.
  • FIG. 1 is a schematic diagram of the structure of the interaction between carbon quantum dot particles and tumor cells in an embodiment of the present disclosure (protein 1 "don't eat me” protein, protein 2 tumor-specific protein, protein 3 normal protein);
  • Fig. 2 is the zeta potential diagram of the carbon quantum dot particles and the bovine serum albumin inactivated vaccine of Experimental Example 1 of the present disclosure
  • Fig. 3 is the electrophoresis gel run diagram of the carbon quantum dot particles and the bovine serum albumin inactivated vaccine of Experimental Example 1 of the present disclosure
  • Fig. 4 is the infrared absorption spectrogram of the carbon quantum dot particles and the cell inactivated vaccine of Experimental Example 1 of the present disclosure
  • FIG. 6 is a confocal comparison diagram of macrophages in Experimental Example 3 of the present disclosure.
  • Fig. 7 is a comparison diagram of the mouse inguinal lymph of Experimental Example 4 of the present disclosure.
  • Fig. 8 is the tumor growth curve of the mice treated with the injection of the tumor immune activator and the mice of the control group in Experimental Example 5 of the present disclosure
  • FIG. 9 is an electrophoresis run gel diagram of Comparative Example 1 of the present disclosure.
  • FIG. 10 is a fluorescence spectrum diagram of Comparative Example 2 of the present disclosure.
  • the purpose of the present disclosure is to overcome the technical problems in the prior art, such as difficult screening of neoantigens and poor immunogenicity of tumor antigens, and provide a quantum dot-enhanced tumor inactivation vaccine.
  • Another object of the present disclosure is to provide a method for preparing a quantum dot-enhanced inactivated tumor vaccine.
  • Another object of the present disclosure is to provide an application of a quantum dot-enhanced tumor inactivation vaccine.
  • An embodiment of the present disclosure provides a quantum dot-enhanced tumor inactivation vaccine, which includes tumor cells and quantum dots located in the protein space structure of tumor cells, and the quantum dots interact with tumor cells through biocompatible functional groups on their surfaces. Groups on protein molecular chains are hydrogen bonded.
  • the embodiment of the present disclosure provides a schematic diagram of the structure of a quantum dot-enhanced tumor inactivation vaccine.
  • all of the above three kinds of proteins contain quantum dot materials in their spatial structure, small-sized quantum dots enter the spatial conformation of the secondary structure of the protein, and through the above-mentioned functional groups on the surface of the quantum dots and the protein molecular chain The groups of the quantum dots form hydrogen bonds, and then form the inactivated cancer cell complex modified by quantum dots.
  • quantum dot materials can reduce the function of immunosuppressive proteins ("don't eat me” signal proteins) on the surface of cancer cells and enhance the immunogenicity of normal proteins ("eat me” signal proteins) , on the basis of maintaining the primary structure of cancer cell antigens, that is, the amino acid sequence, to improve the capture, presentation and induction of specific anti-tumor immune responses of cancer cell antigen proteins by the immune system.
  • immunosuppressive proteins Don't eat me” signal proteins
  • normal proteins eat me signal proteins
  • the quantum dots are selected from carbon quantum dots, and the particle size of the quantum dots is less than 15nm;
  • the biocompatible functional group is one or more of amino group, imine group, carbon-nitrogen double bond, pyridine nitrogen, pyrrole nitrogen, graphene nitrogen, carboxyl group, hydroxyl group and carbonyl group.
  • the proteins on tumor cells include one or more of normal proteins, immunosuppressive proteins, and tumor antigen proteins.
  • the tumor cells are derived from one or more of in vitro cultured or proliferated cancer cell lines, in vitro digested or proliferated tumor tissue cells.
  • the tumor cells or cell fragments are cancer cells or cancer cell fragments.
  • the embodiment of the present disclosure also provides a preparation method of the above-mentioned quantum dot-enhanced tumor inactivation vaccine, including: mixing quantum dots with tumor cells, and using heat to make the biocompatible functional groups on the surface of quantum dot particles and tumor cells Groups on protein molecular chains interact to form hydrogen bonds.
  • the quantum dot particles are mixed with the tumor cells of the biological material, and under the action of external energy, the biocompatible functional groups on the surface of the quantum dot particles interact with the groups on the protein molecular chains on the surface of the tumor cells through the action of heat to form hydrogen key.
  • Tumor cells mainly include one or more of in vitro cultured or proliferated cancer cell lines, in vitro digested or proliferated tumor tissue cells. Through the action of heat, the secondary structure of the antigen protein is opened, and the protein chain is loosened to form a gap, which is convenient for quantum dot particles to enter and form hydrogen bonds.
  • the spatial structure of the protein on the tumor cell is opened, and the quantum dots with biocompatible functional groups on the surface can enter the tumor cell without destroying the primary structure of the tumor protein.
  • the quantum dots with biocompatible functional groups on the surface can enter the tumor cell without destroying the primary structure of the tumor protein.
  • the secondary structure protein space structure In the secondary structure protein space structure.
  • the thermal action includes any one of thermal conduction, thermal radiation, photothermal, magnetic thermal or microwave heating;
  • the thermal action is to react at 50-90° C. for 1-30 minutes.
  • the mass ratio of quantum dots to tumor cells is 1:0.1-10000.
  • the reason why the mass ratio of quantum dots and tumor cells is relatively large here is that since most of the cells are water, by estimation, the embodiments of the present disclosure can realize the present disclosure within a relatively large mass ratio range.
  • the preparation method of quantum dots is as follows: citric acid and urea are mixed and dissolved in dimethyl sulfoxide, placed in a high-pressure reactor, and heated for 2-10 hours at 110-220°C , and then use methanol or ethanol to centrifuge the obtained carbon quantum dot particles, and the centrifugation speed can be selected as 6000-10000rpm.
  • the above method for preparing quantum dots is only a method for preparing carbon quantum dots provided by the inventors, and the preparation of quantum dot particles in the present disclosure is not limited to the above preparation methods.
  • An embodiment of the present disclosure provides an application of the above-mentioned quantum dot-enhanced inactivated tumor vaccine as a tumor immune drug.
  • the quantum dot-enhanced inactivated tumor vaccine is used as a tumor immune drug.
  • the equipment used in this example is conventional experimental equipment, the materials and reagents used are commercially available unless otherwise specified, and the experimental methods without special instructions are also conventional experimental methods.
  • Dissolve the obtained carbon quantum dot particles in deionized water configure a solution with a concentration of (0.1-1.5) mg/mL, mix the carbon quantum dot particles and bovine serum albumin at a mass ratio of 1:50, and heat with light or a hot plate , water bath (oil bath) heating, oven heating or ultrasonic heating and other heating methods to raise the temperature to 60°C, react for 10min, and stir at 100rpm at the same time. That is, the bovine serum albumin (CQD-BSA) inactivated vaccine of carbon quantum dot particles is obtained.
  • CQD-BSA bovine serum albumin
  • Example 1 Dissolve the carbon quantum dot particles of Example 1 in deionized water at a concentration of (0.1-1.2) mg/mL, and add 1 ⁇ 10 4 -1 ⁇ 10 10 4T1 breast cancer cells per mL of the above solution. The temperature was raised to 60° C. by means of oven heating, and reacted for 10 minutes while stirring at 100 rpm. The breast cancer cell (CQD-cell) inactivated vaccine of carbon quantum dot particles is obtained.
  • the preparation of the carbon quantum dot enhanced cell inactivated vaccine is the same as in Example 2.
  • the preparation of the carbon quantum dot enhanced cell inactivated vaccine is the same as in Example 2.
  • the tumors of the breast cancer cell transgenic mice were excised, digested, proliferated, and cancer cells were obtained.
  • CQD-BSA bovine serum albumin
  • the carbon quantum dot particle (CQD) obtained in the embodiment and the electrophoresis running gel of the bovine serum albumin (CQD-BSA) inactivated vaccine of the obtained carbon quantum dot particle were measured, and the results are shown in FIG. 3 .
  • the CQD-BSA obtained after compounding corresponds to an electrophoresis band on the protein, showing fluorescence, indicating the effective bonding between the carbon dot and the protein.
  • the infrared absorption spectrum of the carbon quantum dot particle (CQD) obtained in the embodiment and the bovine serum albumin (CQD-BSA) inactivated vaccine of the obtained carbon quantum dot particle is measured, and the results are shown in Figure 4.
  • the CQD-BSA obtained after compounding has an obvious peak at 3300cm -1 , which proves that there is a hydrogen bond between the carbon quantum dots and the protein on the tumor cells.
  • the fluorescence electron microscope of the cell (CQD-cell) inactivated vaccine obtained by measuring the carbon quantum dot particles obtained in the embodiment is shown in FIG. 5 .
  • the CQD-cell obtained after compounding exhibits strong fluorescence, indicating that carbon dots can compound proteins on cells indiscriminately.
  • the carbon quantum dot-enhanced cell inactivated vaccine obtained in 0.05mL (the concentration of carbon quantum dots is 200ppm, the number of 4T1 cells is 1x104-1x1010 ), pure carbon quantum dots (the concentration of carbon quantum dots is 200ppm), DiD staining
  • the inactivated 4T1 breast cancer cells (the number of 4T1 cells is 1 ⁇ 10 4 -1 ⁇ 10 10 , which is the same as that of the experimental group) and PBS were respectively added to the culture dish of 6 cm macrophage RAW264.7. After 24 hours, the morphological changes of the macrophages were observed with confocal.
  • the macrophages cultured with the inactivated vaccine of tumor cells (CQD-cell) with carbon quantum dot particles had a significant increase in volume and dendritic cells Differentiation, indicating that CQD-cells can promote the maturation of macrophages and the engulfment of antigens.
  • the carbon quantum dot enhanced cell inactivated vaccine (the carbon quantum dot concentration is 200ppm, 4T1 cell number is 1x104-1x1010 ) that 0.05mL obtains in embodiment 1 , pure carbon quantum dots (the carbon quantum dot concentration is 200ppm ), DiD-stained inactivated 4T1 breast cancer cells (the number of 4T1 cells is 1 ⁇ 10 4 -1 ⁇ 10 10 , which is the same as that of the experimental group), and PBS were subcutaneously injected into mice respectively.
  • the lymphatic conditions were observed at 4h and 24h, and the results are shown in Figure 7. It shows that the inactivated vaccine of tumor cells (CQD-cell) with carbon quantum dot particles can increase the lymphatic presentation of antigens.
  • the tumors of the breast cancer cell transgenic mice were excised, digested, proliferated, and cancer cells were obtained.
  • a certain amount of carbon quantum dot particle-enhanced tumor cell vaccine was injected back into the transgenic mouse.
  • the injection volume is 200ul
  • the injection method is one of intravenous injection, subcutaneous injection and ascites injection, and optionally, subcutaneous injection.
  • the tumor growth curve of the mice was measured, and the results are shown in FIG. 8 .
  • 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 carbon quantum dot particle-enhanced tumor cell vaccine preparation effectively activates the tumor immune function of the mice, and inhibits and kills the tumors in the mice.
  • the thermal recombination temperature is the key to the recombination of quantum dots and tumor cells.
  • the temperature of thermal recombination should not be too low. If the temperature is too low, the protein structure cannot be opened, and the two cannot be recombined. .
  • the temperature of thermal compounding should not be too high. If the temperature is higher than 100° C. and heated for a long time, the protein will be completely denatured, and the fluorescence will not be enhanced, so the vaccine cannot be obtained. That is, the quantum dot-enhanced inactivated cancer cell vaccine provided by the embodiments of the present disclosure can be successfully prepared only when the quantum dots are combined with the protein without denaturing the protein.
  • the embodiments of the present disclosure provide a quantum dot-enhanced inactivated cancer cell vaccine and its preparation method and application.
  • the functional groups are one or more of amino group, imine group, carbon-nitrogen double bond, pyridine nitrogen, pyrrole nitrogen, graphene nitrogen, carboxyl group, hydroxyl group, carbonyl group;
  • the spatial structure of the protein molecular chain on the cancer cell is opened, the protein molecular chain is loose to form gaps, and the small-sized quantum dots enter the spatial conformation of the protein, and form hydrogen bonds with the groups on the protein molecular chain through the above-mentioned functional groups on the surface of the quantum dots , and then form the inactivated cancer cell complex modified by quantum dots.
  • quantum dot materials can reduce the function of immunosuppressive proteins ("don't eat me” signal proteins) on the surface of cancer cells and enhance the immunogenicity of normal proteins ("eat me” signal proteins) , on the basis of maintaining the primary structure of cancer cell antigens, that is, the amino acid sequence, to improve the capture, presentation and induction of specific anti-tumor immune responses of cancer cell antigen proteins by the immune system.
  • the proposed quantum dot-enhanced inactivated cancer cell vaccine can be applied in personalized cancer immunotherapy.

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Abstract

提供了一种量子点增强型肿瘤灭活疫苗及制备方法和应用，在热作用下，肿瘤细胞上蛋白分子链的空间结构打开，蛋白分子链松散形成空隙，小尺寸量子点进入蛋白的空间构象中，并通过量子点表面的上述官能团与蛋白分子链上的基团形成氢键，进而形成量子点修饰后的灭活癌细胞复合物。提供的灭活疫苗在保持癌细胞抗原一级结构，即氨基酸序列不被破坏的基础上，提高癌细胞抗原蛋白的被免疫系统捕获、递呈及诱导特异性抗肿瘤免疫反应。所提出的量子点增强型癌细胞灭活疫苗可以应用到个性化的癌症免疫治疗中。

Description

一种量子点增强型肿瘤灭活疫苗及制备方法和应用
相关申请的交叉引用
本公开要求于2021年06月30日提交中国专利局的申请号为CN202110731659.4、名称为“一种量子点增强型肿瘤灭活疫苗及制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及纳米材料和生物医学技术领域，具体而言，涉及一种量子点增强型肿瘤灭活疫苗及制备方法和应用。
背景技术
个性化癌症疫苗是目前比较受欢迎的癌症免疫疗法之一。其旨在调节先天性和适应性免疫系统，以广泛激活具有个别肿瘤特异性抗原(新抗原)的抗肿瘤免疫力。然而，大规模并行测序和机器学习何如鉴定新抗原的过程是非常昂贵且耗时的，而且在此期间新抗原的变化将导致无效的抗癌反应。
肿瘤细胞是多种肿瘤新抗原的天然来源，包括尚未鉴定的那些。但是，全肿瘤细胞疫苗接种始终缺乏强有力的免疫刺激，抗肿瘤反应非常有限。主要原因可能是肿瘤细胞中包含的大多数蛋白质是自身蛋白质，其自身的免疫原性很低，并且向巨噬细胞和树突状细胞等抗原呈递细胞表达微弱的“吃我”信号。源自正常细胞变异的肿瘤细胞包含多种免疫抑制蛋白，它们表达“不吃我”信号以防止自身吞噬作用。所有这些导致抗原呈递细胞对新抗原的细胞吸收效率低，并且它们在淋巴结中的低效率积累导致无效的抗肿瘤T细胞活化。在最近的进展中，提出了许多通过与佐剂偶联或阻断肿瘤细胞上免疫抑制蛋白的方法来增强肿瘤细胞的免疫原性的策略。然而，这些方法不能在很大程度上改变或增强肿瘤细胞的免疫原性。
发明内容
本公开的目的是为了克服上述现有技术存在的缺陷而提供一种量子点增强型肿瘤灭活疫苗及制备方法和应用。
本公开提供一种量子点增强型肿瘤灭活疫苗，包括肿瘤细胞和位于肿瘤细胞的蛋白 空间结构中的量子点，且该量子点通过其表面的生物相容性官能团与肿瘤细胞上的蛋白分子链上的基团以氢键键合。
本公开还提供一种根据上述的量子点增强型肿瘤灭活疫苗的制备方法，包括：将量子点与肿瘤细胞混合，通过热作用，使量子点粒子表面的官能团与肿瘤细胞上的蛋白分子链上的基团相互作用，形成氢键。
公开还提供一种根据上述的量子点增强型肿瘤灭活疫苗作为个性化肿瘤免疫治疗及预防的应用。
本公开具有以下有益效果：
本公开提供一种量子点增强型肿瘤灭活疫苗及制备方法和应用，该量子点增强型肿瘤灭活疫苗包括肿瘤细胞和位于肿瘤细胞的蛋白空间结构中的量子点，且该量子点通过其表面的生物相容性官能团与肿瘤细胞上的蛋白分子链上的基团以氢键键合。量子点由于其表面尺寸小，能够进入肿瘤蛋白的蛋白空隙内，量子点表面的基团进一步和肿瘤细胞表面的蛋白发生作用产生氢键。量子点颗粒只是改变了蛋白的构象，并未破坏其一级结构，使其表达的“不吃我”信号降低，“吃我”信号增加。大幅度提高肿瘤蛋白的免疫原性，进而表现出更佳的免疫激活特性，以及能够应用到增强型的细胞疫苗中。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本公开实施例的碳量子点粒子与肿瘤细胞相互作用的结构示意图(蛋白①“不吃我”蛋白，蛋白②肿瘤特异性蛋白，蛋白③正常蛋白)；
图2为本公开实验例1的碳量子点粒子及其与牛血清蛋白灭活疫苗的zeta电位图；
图3为本公开实验例1的碳量子点粒子及其与牛血清蛋白灭活疫苗的电泳跑胶图；
图4为本公开实验例1的碳量子点粒子及其与细胞灭活疫苗的红外吸收光谱图；
图5为本公开实验例2的碳量子点粒子及其与细胞灭活疫苗的荧光电镜图；
图6为本公开实验例3的巨噬细胞的confocal对比图；
图7为本公开实验例4的小鼠腹股沟淋巴的对比图；
图8为本公开实验例5的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9为本公开对比例1的电泳跑胶图；
图10为本公开对比例2的荧光光谱图。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本公开的目的在于克服现有技术中的新抗原难以筛选、肿瘤抗原免疫原性差等技术问题，提供了一种量子点增强型肿瘤灭活疫苗。
本公开的另一个目的在于提供一种量子点增强型肿瘤灭活疫苗的制备方法。
本公开的另一个目的在于提供一种量子点增强型肿瘤灭活疫苗的应用。
本公开的目的通过以下技术方案予以实现：
本公开实施例提供一种量子点增强型肿瘤灭活疫苗，包括肿瘤细胞和位于肿瘤细胞的蛋白空间结构中的量子点，且该量子点通过其表面的生物相容性官能团与肿瘤细胞上的蛋白分子链上的基团以氢键键合。
本公开实施例提供一种量子点增强型肿瘤灭活疫苗的结构示意图参见图1，可以看出，在生物材料的肿瘤细胞上包含有3种蛋白:①“不吃我”蛋白，②肿瘤特异性蛋白，③正常蛋白，在上述3种蛋白的空间结构中均含有量子点材料，小尺寸量子点进入蛋白的二级结构的空间构象中，并通过量子点表面的上述官能团与蛋白分子链上的基团形成氢键，进而形成量子点修饰后的灭活癌细胞复合物。量子点材料通过改变癌细胞上蛋白分子的构象，可使癌细胞表面免疫抑制蛋白(“不吃我”信号蛋白)功能的降低，并增强正常蛋白的免疫原性(“吃我”信号蛋白)，在保持癌细胞抗原一级结构，即氨基酸序列不被破坏的基础上，提高癌细胞抗原蛋白的被免疫系统捕获、递呈及诱导特异性抗肿瘤免疫反应。
在可选的实施方式中，量子点选自碳量子点，且量子点的粒径小于15nm；
可选地，生物相容性官能团为氨基、亚胺基、碳氮双键、吡啶氮、吡咯氮，石墨烯氮、羧基、羟基及羰基中的一种或多种。
在可选的实施方式中，肿瘤细胞上蛋白包括正常蛋白、免疫抑制蛋白，肿瘤抗原蛋 白中的一种或多种。
在可选的实施方式中，肿瘤细胞来源于体外培养或者增殖的癌细胞系、体外消化或者增殖的肿瘤组织细胞中的一种或多种。
可选地，肿瘤细胞或细胞碎片为癌细胞或癌细胞碎片。
本公开实施例还供一种上述量子点增强型肿瘤灭活疫苗的制备方法，包括：将量子点与肿瘤细胞混合，通过热作用，使量子点粒子表面的生物相容性官能团与肿瘤细胞上的蛋白分子链上的基团相互作用形成氢键。
将量子点粒子与生物材料的肿瘤细胞混合，在外加能量的作用下，通过热作用使量子点粒子表面的生物相容性官能团与肿瘤细胞表面的蛋白分子链上的基团相互作用，形成氢键。肿瘤细胞主要包括外培养或者增殖的癌细胞系、体外消化或者增殖的肿瘤组织细胞中的一种或多种。通过热作用，抗原蛋白的二级结构打开，蛋白质链松散形成空隙，方便量子点粒子进入形成氢键。
在可选的实施方式中，在热作用下，肿瘤细胞上蛋白空间结构打开，在保持肿瘤蛋白一级结构不破坏的基础上，使表面包含有生物相容性官能团的量子点进入肿瘤细胞上的二级结构蛋白空间结构中。
在可选的实施方式中，热作用包括热传导、热辐射、光热、磁热或微波加热中的任意一种；
可选地，热作用是在50-90℃条件下反应1-30min。
在可选的实施方式中，量子点和肿瘤细胞的质量比为1:0.1-10000。此处量子点和肿瘤细胞的质量比的范围较大的原因在于：由于细胞的中大部分是水，通过估算，本公开实施例中的可以在较大的质量比的范围内，实现本公开实施例提出的方案。
在可选的实施方式中，量子点的制备方法如下：将柠檬酸和尿素混合溶于二甲基亚砜中，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量子点粒子进行离心清洗，离心转速可选为6000-10000rpm。
需要说明的是，上述量子点的制备方法仅是发明人提供的一种碳量子点的制备方法，本公开中的量子点粒子的制备不限于上述制备方法。
本公开实施例提供一种上述量子点增强型灭活肿瘤疫苗作为肿瘤免疫药物的应用。
在可选的实施方式中，量子点增强型灭活肿瘤疫苗作为肿瘤免疫药物。
实施例
以下结合实施例对本公开的特征和性能作进一步的详细描述。
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1:4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。测定获得的碳量子点粒子的直径为3-5nm。碳量子点粒子的C、N、O、S元素质量比分别为59.6％，16.2％，23.0％和1.2％。
将得到的碳量子点粒子溶于去离子水中，配置浓度为(0.1-1.5)mg/mL溶液，将碳量子点粒子和牛血清蛋白的质量比以1:50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60℃，反应10min，并同时以100rpm转速搅拌。即得到碳量子点粒子的牛血清蛋白(CQD-BSA)灭活疫苗。
实施例2
将实施例1的碳量子点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。采用烘箱加热方式升温至60℃，反应10min，并同时以100rpm转速搅拌。即得碳量子点粒子的乳腺癌细胞(CQD-cell)灭活疫苗。
实施例3
碳量子点增强型细胞灭活疫苗的制备同实施例2。
将0.05mL得到的碳量子点增强型细胞灭活疫苗(碳量子点浓度为200ppm，4T1细胞数为1x10 4-1x10 10个)、纯的碳量子点(碳量子点浓度为200ppm)、DiD染色的灭活的4T1乳腺癌细胞(4T1细胞数为1x10 4-1x10 10个，具体和实验组一样)以及PBS分别加入到6cm巨噬细胞RAW264.7的培养皿中，24h后用confocal观察巨噬细胞的形态变化。
实施例4
碳量子点增强型细胞灭活疫苗的制备同实施例2。
将0.1mL得到的碳量子点增强型细胞灭活疫苗(碳量子点浓度为200ppm，4T1细 胞数为1x10 4-1x10 10个)、纯的碳量子点(碳量子点浓度为200ppm)、DiD染色的灭活的4T1乳腺癌细胞(4T1细胞数为1x10 4-1x10 10，具体和实验组一样)以及PBS分别皮下注射到balb/c小鼠中，4h和24h分别取出其第四对乳腺，进行成像，观察淋巴的荧光情况。
实施例5
碳量子点粒子的制备同实施例1。
将乳腺癌细胞转基因小鼠的肿瘤摘除，进行消化，增殖，得到癌细胞。
将得到的碳量子点粒子溶于去离子水中，配置浓度为(0.1-1.5)mg/mL溶液，将碳量子点粒子和消化的癌细胞的质量比以1.0:10混合，采用热板加热升温至60℃，反应10min，并同时以100rpm转速搅拌。即得到碳量子点粒子增强型肿瘤细胞疫苗。
实验例1
测定实施例获得的碳量子点粒子(CQD)以及得到的碳量子点粒子的牛血清蛋白(CQD-BSA)灭活疫苗的zeta电位，结果如图2所示。经过复合后得到的CQD-BSA，表现为zeta电位的增强。
测定实施例获得的碳量子点粒子(CQD)以及得到的碳量子点粒子的牛血清蛋白(CQD-BSA)灭活疫苗的电泳跑胶，结果如图3所示。经过复合后得到的CQD-BSA，在蛋白质对应一条电泳带，显示出荧光，表明碳点和蛋白的有效键合。
测定实施例获得的碳量子点粒子(CQD)以及得到的碳量子点粒子的牛血清蛋白(CQD-BSA)灭活疫苗的红外吸收光谱，结果如图4所示。经过复合后得到的CQD-BSA，在3300cm -1有明显的尖峰，证明碳量子点和肿瘤细胞上的蛋白之间是氢键键合。
实验例2
测定实施例获得的碳量子点粒子的细胞(CQD-cell)灭活疫苗的荧光电镜，结果如图5所示。经过复合后得到的CQD-cell，表现为强的荧光，说明碳点可以无差别复合细胞上的蛋白。
实验例3
将0.05mL得到的碳量子点增强型细胞灭活疫苗(碳量子点浓度为200ppm，4T1 细胞数为1x10 4-1x10 10个)、纯的碳量子点(碳量子点浓度为200ppm)、DiD染色的灭活的4T1乳腺癌细胞(4T1细胞数为1x10 4-1x10 10个，具体和实验组一样)以及PBS分别加入到6cm巨噬细胞RAW264.7的培养皿中。24h后用confocal观察巨噬细胞的形态变化，如图6所示，经过碳量子点粒子的肿瘤细胞(CQD-cell)灭活疫苗培养的巨噬细胞有着明显的体积增大以及树突状细胞分化的情况，表明CQD-cell可以促进巨噬细胞的成熟以及对抗原的吞食。
实验例4
将实施例1中0.05mL得到的碳量子点增强型细胞灭活疫苗(碳量子点浓度为200ppm，4T1细胞数为1x10 4-1x10 10个)、纯的碳量子点(碳量子点浓度为200ppm)、DiD染色的灭活的4T1乳腺癌细胞(4T1细胞数为1x10 4-1x10 10个，具体和实验组一样)以及PBS，分别皮下注射进小鼠。4h和24h观察其淋巴情况，结果如图7所示。表明经过碳量子点粒子的肿瘤细胞(CQD-cell)灭活疫苗可以增加抗原的淋巴呈递。
实验例5
碳量子点粒子的制备同实施例1。
将乳腺癌细胞转基因小鼠的肿瘤摘除，进行消化，增殖，得到癌细胞。
将得到的碳量子点粒子溶于去离子水中，配置浓度为(0.1-1.5)mg/mL溶液，将碳量子点粒子和消化的癌细胞的质量比是1.0:10混合，采用热板加热方式升温至60℃，反应10min，并同时以100rpm转速搅拌。即得到碳量子点粒子增强型肿瘤细胞疫苗。
将一定计量的碳量子点粒子增强型肿瘤细胞疫苗注射回该转基因小鼠体内。可选地，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，可选地，皮下注入。
测量小鼠的肿瘤生长曲线，结果如图8所示。图8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。说明碳量子点粒子增强型肿瘤细胞疫苗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。
对比例1
与实施例2的步骤相似，不同之处仅在于：热作用的温度为25℃，发现荧光未能增强，在蛋白质对应一条电泳带未显示出荧光，结果如图9所示。
对比例2
与实施例1的步骤相似，不同之处仅在于：热作用的温度为120℃，发现荧光大幅度减弱，结果如图10所示。
从以上的对比例1-2的实验结果可以看出，热复合温度是量子点和肿瘤细胞能否复合的关键，热复合的温度不能过低，温度太低蛋白结构不能打开，两者无法复合。热复合的温度也不能过高，大于100℃且长时间加热，蛋白完全变性，荧光也不会增强，得不到所述的疫苗。即在不让蛋白变性的情况下，让量子点和蛋白结合，才可以成功制取本公开实施例所提供的量子点增强型癌细胞灭活疫苗。
综上，本公开实施例提供了一种量子点增强型癌细胞灭活疫苗及制备方法和应用，该量子点增强型癌细胞灭活疫苗具有以下的特征：量子点粒子粒径小于15nm，表面包含有多个生物相容性的官能团，官能团为氨基、亚胺基、碳氮双键、吡啶氮、吡咯氮，石墨烯氮，羧基，羟基，羰基中的一种或多种；在热作用下，癌细胞上蛋白分子链的空间结构打开，蛋白分子链松散形成空隙，小尺寸量子点进入蛋白的空间构象中，并通过量子点表面的上述官能团与蛋白分子链上的基团形成氢键，进而形成量子点修饰后的灭活癌细胞复合物。量子点材料通过改变癌细胞上蛋白分子的构象，可使癌细胞表面免疫抑制蛋白(“不吃我”信号蛋白)功能的降低，并增强正常蛋白的免疫原性(“吃我”信号蛋白)，在保持癌细胞抗原一级结构，即氨基酸序列不被破坏的基础上，提高癌细胞抗原蛋白的被免疫系统捕获、递呈及诱导特异性抗肿瘤免疫反应。所提出的量子点增强型癌细胞灭活疫苗可以应用到个性化的癌症免疫治疗中。
以上仅为本公开的可选的实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。

Claims (10)

  1. 一种量子点增强型肿瘤灭活疫苗，其特征在于，包括肿瘤细胞和位于肿瘤细胞的蛋白空间结构中的量子点，且所述量子点通过其表面的生物相容性官能团与肿瘤细胞上的蛋白分子链上的基团以氢键键合。
  2. 根据权利要求1所述的量子点增强型肿瘤灭活疫苗，其特征在于，所述量子点选自碳量子点，且所述碳量子点的粒径小于15nm；
    优选地，所述量子点表面的生物相容性官能团为氨基、亚胺基、碳氮双键、吡啶氮、吡咯氮、石墨烯氮、羧基、羟基及羰基中的一种或多种。
  3. 根据权利要求1所述的量子点增强型肿瘤灭活疫苗，其特征在于，所述肿瘤细胞上的蛋白包括正常蛋白、免疫抑制蛋白，肿瘤抗原蛋白中的一种或多种。
  4. 根据权利要求1所述的量子点增强型肿瘤灭活疫苗，其特征在于，所述肿瘤细胞来源于体外培养或者增殖的癌细胞系、体外消化或者增殖的肿瘤组织细胞中的一种或多种；
    优选地，所述肿瘤细胞或肿瘤细胞碎片为癌细胞或癌细胞碎片。
  5. 一种根据权利要求1-4中任一项所述的量子点增强型肿瘤灭活疫苗的制备方法，其特征在于，包括：将量子点与肿瘤细胞混合，通过热作用，使量子点表面的生物相容性官能团与肿瘤细胞上的蛋白分子链上的基团相互作用，形成氢键。
  6. 根据权利要5所述的制备方法，其特征在于，在热作用下，所述肿瘤细胞上蛋白空间结构打开，在保持肿瘤蛋白一级结构不破坏的基础上，使表面包含有生物相容性官能团的量子点进入肿瘤细胞上的二级结构蛋白空间结构中。
  7. 根据权利要求5所述的制备方法，其特征在于，热作用包括热传导、热辐射、光热、磁热或微波加热中的任意一种；
    优选地，热作用是在50-90℃条件下反应1-30min。
  8. 根据权利要求5所述的制备方法，其特征在于，所述量子点和所述肿瘤细胞的质量比为1:0.1-10000。
  9. 根据权利要求1-4任一项所述量子点增强型灭活癌细胞疫苗或根据权利要求5-8中任一项所述量子点增强型灭活癌细胞疫苗作为个性化肿瘤免疫治疗及预防的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述量子点增强型灭活肿瘤疫苗作为肿瘤免疫药物。
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