阅读说明：本技术 一种量子点增强型肿瘤灭活疫苗及制备方法和应用 (Quantum dot enhanced tumor inactivated vaccine and preparation method and application thereof ) 是由 曲松楠 汤子康 梁桃 于 2021-06-30 设计创作，主要内容包括：本发明公开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。本发明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。(Under the action of heat, the space structure of a protein molecular chain on a tumor cell is opened, the protein molecular chain is loosened to form a gap, small-size quantum dots enter a space conformation of protein, and hydrogen bonds are formed between the functional groups on the surfaces of the quantum dots and groups on the protein molecular chain, so that a quantum dot modified inactivated cancer cell compound is formed. The inactivated vaccine provided by the invention improves the capturing and presenting of cancer cell antigen protein by an immune system and induces specific anti-tumor immune response on the basis of keeping the primary structure of the cancer cell antigen, namely, the amino acid sequence is not damaged. The quantum dot enhanced cancer cell inactivated vaccine can be applied to personalized cancer immunotherapy.)

1. A quantum dot enhanced tumor inactivated vaccine is characterized by comprising tumor cells and quantum dots positioned in a protein space structure of the tumor cells, wherein the quantum dots are bonded with groups on a protein molecular chain on the tumor cells through biocompatible functional groups on the surfaces of the quantum dots by hydrogen bonds.

2. The quantum dot enhanced tumor inactivated vaccine as claimed in claim 1, wherein the quantum dots are selected from carbon quantum dots, and the particle size of the carbon quantum dots is less than 15 nm;

preferably, the biocompatible functional group on the surface of the quantum dot is one or more of amino, imino, carbon-nitrogen double bond, pyridine nitrogen, pyrrole nitrogen, graphene nitrogen, carboxyl, hydroxyl and carbonyl.

3. The quantum dot enhanced inactivated tumor vaccine of claim 1, wherein the proteins on the tumor cells comprise one or more of normal proteins, immunosuppressive proteins and tumor antigen proteins.

4. The quantum dot enhanced inactivated tumor vaccine as claimed in claim 1, wherein the tumor cells are derived from one or more of cancer cell lines cultured or proliferated in vitro, tumor tissue cells digested or proliferated in vitro;

preferably, the tumor cell or tumor cell fragment is a cancer cell or cancer cell fragment.

5. A method for preparing the quantum dot enhanced inactivated tumor vaccine according to any one of claims 1 to 4, comprising the following steps: the quantum dots and the tumor cells are mixed, and the biocompatible functional groups on the surfaces of the quantum dots interact with groups on protein molecular chains on the tumor cells through the thermal action to form hydrogen bonds.

6. The method according to claim 5, wherein the space structure of the protein on the tumor cell is opened under the action of heat, and the quantum dot having the biocompatible functional group on the surface is allowed to enter the space structure of the secondary structure protein on the tumor cell while the primary structure of the tumor protein is maintained.

7. The production method according to claim 5, wherein the thermal action includes any one of thermal conduction, thermal radiation, photothermal, magnetocaloric, or microwave heating;

preferably, the heat is applied at 50-90 deg.C for 1-30 min.

8. The method according to claim 5, wherein the mass ratio of the quantum dot to the tumor cell is 1: 0.1-10000.

9. Use of the quantum dot enhanced fire-fighting cancer cell vaccine according to any one of claims 1-4 or the quantum dot enhanced fire-fighting cancer cell vaccine according to any one of claims 5-8 as personalized tumor immunotherapy and prevention.

10. The use according to claim 9, wherein the quantum dot enhanced fire-fighting tumor vaccine is used as a tumor immunity drug.

Technical Field

The invention relates to the technical field of nano materials and biomedicine, in particular to a quantum dot enhanced tumor inactivated vaccine and a preparation method and application thereof.

Background

Personalized cancer vaccines are one of the more popular cancer immunotherapies today. It aims to regulate the innate and adaptive immune system to widely activate anti-tumor immunity with individual tumor-specific antigens (neoantigens). However, massively parallel sequencing and machine learning the process of how to identify neoantigens is very expensive and time consuming, and during this time the changes in neoantigens will lead to ineffective anti-cancer 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 is always lacking in powerful immune stimulation and has very limited anti-tumor response. The main reason may be that most of the proteins contained in tumor cells are self-proteins, which are themselves poorly immunogenic, and express weak "eat me" signals to antigen presenting cells such as macrophages and dendritic cells. Tumor cells derived from normal cellular variation contain a variety of immunosuppressive proteins that express a "no-eat-me" signal to prevent autophagy. All of these result in inefficient cellular uptake of neoantigens by antigen presenting cells, and their inefficient accumulation in lymph nodes results in ineffective anti-tumor T cell activation. In recent developments, a number of strategies have been proposed to enhance the immunogenicity of tumor cells by coupling to adjuvants or blocking immunosuppressive proteins on the tumor cells. However, these methods do not alter or enhance the immunogenicity of tumor cells to a large extent.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a quantum dot enhanced tumor inactivated vaccine, a preparation method and application thereof.

The invention is realized by the following steps:

the invention provides a quantum dot enhanced tumor inactivated vaccine, which comprises tumor cells and quantum dots positioned in a protein space structure of the tumor cells, wherein the quantum dots are bonded with groups on a protein molecular chain on the tumor cells through biocompatible functional groups on the surfaces of the quantum dots by hydrogen bonds.

The invention also provides a preparation method of the quantum dot enhanced tumor inactivated vaccine, which comprises the following steps: the quantum dots and the tumor cells are mixed, and functional groups on the surfaces of the quantum dots interact with groups on protein molecular chains on the tumor cells through the thermal action to form hydrogen bonds.

The invention also provides application of the quantum dot enhanced tumor inactivated vaccine as personalized tumor immunotherapy and prevention.

The invention has the following beneficial effects:

the invention provides a quantum dot enhanced tumor inactivated vaccine, a preparation method and an application thereof. The quantum dots can enter protein gaps of tumor proteins due to small surface sizes of the quantum dots, and radicals on the surfaces of the quantum dots further react with the proteins on the surfaces of tumor cells to generate hydrogen bonds. The quantum dot particles only change the conformation of the protein and do not destroy the primary structure of the protein, so that the expressed 'eat-free' signal is reduced and the 'eat-free' signal is increased. Greatly improves the immunogenicity of the tumor protein, further shows better immune activation characteristics, and can be applied to enhanced cell vaccines.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a schematic diagram of the interaction structure of carbon quantum dot particles and tumor cells in an embodiment of the present invention (proteins: "do not eat me" protein, proteins: "tumor specific protein", proteins: "normal protein");

FIG. 2 is a zeta potential diagram of the carbon quantum dot particles and the inactivated vaccine of bovine serum albumin in Experimental example 1;

FIG. 3 is an electrophoresis gel-casting diagram of the carbon quantum dot particle and the inactivated vaccine of bovine serum albumin in Experimental example 1;

FIG. 4 is a chart of an infrared absorption spectrum of carbon quantum dot particles and inactivated vaccines of cells thereof according to Experimental example 1;

FIG. 5 is a fluorescence electron microscope image of the carbon quantum dot particle and the inactivated vaccine cell according to Experimental example 2;

FIG. 6 is a comparative graph showing confocal of macrophages in Experimental example 3 of the present invention;

FIG. 7 is a graph showing a comparison of inguinal lymph of a mouse in Experimental example 4 of the present invention;

FIG. 8 is a graph showing tumor growth curves of mice treated with the tumor immunoactivator injection and control mice according to Experimental example 5 of the present invention;

FIG. 9 is a graph of running gel for electrophoresis of comparative example 1 of the present invention;

FIG. 10 is a fluorescence spectrum of comparative example 2 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The invention aims to overcome the technical problems of difficult screening of new antigens, poor immunogenicity of tumor antigens and the like in the prior art, and provides a quantum dot enhanced tumor inactivated vaccine.

The invention also aims to provide a preparation method of the quantum dot enhanced tumor inactivated vaccine.

The invention also aims to provide application of the quantum dot enhanced tumor inactivated vaccine.

The purpose of the invention is realized by the following technical scheme:

in a first aspect, an embodiment of the present invention provides a quantum dot enhanced inactivated tumor vaccine, including a tumor cell and a quantum dot located in a protein space structure of the tumor cell, where the quantum dot is hydrogen bonded to a group on a protein molecular chain on the tumor cell through a biocompatible functional group on a surface of the quantum dot.

The embodiment of the invention provides a structural schematic diagram of a quantum dot enhanced tumor inactivated vaccine, and referring to fig. 1, it can be seen that tumor cells of a biological material comprise 3 proteins, namely a 'no-eat-me' protein, a tumor specific protein and a normal protein, wherein the 3 proteins all contain quantum dot materials in spatial structures, small-size quantum dots enter spatial conformations of secondary structures of the proteins, and hydrogen bonds are formed between functional groups on the surfaces of the quantum dots and groups on protein molecular chains, so that an inactivated cancer cell compound modified by the quantum dots is formed. The quantum dot material can reduce the function of the cancer cell surface immunosuppressive protein (the ' don't eat me ' signal protein) by changing the conformation of the protein molecules on the cancer cells, enhance the immunogenicity of the normal protein (the ' eat me ' signal protein), and improve the capture and presentation of the cancer cell antigen protein by an immune system and induce specific anti-tumor immune response on the basis of keeping the primary structure of the cancer cell antigen, namely, an amino acid sequence is not damaged.

In an alternative embodiment, the quantum dots are selected from carbon quantum dots, and the particle size of the quantum dots is less than 15 nm;

preferably, the biocompatible functional group is one or more of amino, imino, carbon-nitrogen double bond, pyridine nitrogen, pyrrole nitrogen, graphene nitrogen, carboxyl, hydroxyl and carbonyl.

In alternative embodiments, the protein on the tumor cell comprises one or more of a normal protein, an immunosuppressive protein, a tumor antigen protein.

In alternative embodiments, the tumor cells are derived from one or more of a cancer cell line cultured or propagated in vitro, a tumor tissue cell digested or propagated in vitro.

Preferably, the tumor cells or cell fragments are cancer cells or cancer cell fragments.

In a second aspect, an embodiment of the present invention further provides a preparation method of the quantum dot enhanced inactivated tumor vaccine, including: the quantum dots and the tumor cells are mixed, and the biocompatible functional groups on the surfaces of the quantum dots and the groups on the protein molecular chains on the tumor cells are interacted to form hydrogen bonds under the action of heat.

The quantum dot particles are mixed with tumor cells of a biological material, and under the action of external energy, the biocompatible functional groups on the surfaces of the quantum dot particles interact with groups on protein molecular chains on the surfaces of the tumor cells through the thermal action to form hydrogen bonds. The tumor cells mainly comprise one or more of cancer cell lines cultured or proliferated externally and tumor tissue cells digested or proliferated in vitro. Through the action of heat, the secondary structure of the antigen protein is opened, and the protein chain is loosened to form a gap, so that the quantum dot particles can enter to form a hydrogen bond conveniently.

In an alternative embodiment, the protein space structure on the tumor cell is opened under the action of heat, and the quantum dots with the biocompatible functional groups on the surface enter the secondary structure protein space structure on the tumor cell on the basis of keeping the primary structure of the tumor protein undamaged.

In alternative embodiments, the thermal action comprises any one of thermal conduction, thermal radiation, photo-thermal, magneto-thermal, or microwave heating;

preferably, the heat is applied at 50-90 deg.C for 1-30 min.

In an alternative embodiment, the mass ratio of the quantum dots to the tumor cells is 1: 0.1-10000. The reason why the mass ratio of the quantum dots to the tumor cells here is wide is that: since most of the cells are water, it is estimated that the solution proposed by the embodiment of the present invention can be implemented in a wide range of mass ratio.

In an alternative embodiment, the quantum dots are prepared as follows: mixing citric acid and urea, dissolving in dimethyl sulfoxide, placing in a high-pressure reaction kettle, heating and reacting for 2-10h at the temperature of 110-.

It should be noted that the above method for preparing quantum dots is only one specific method for preparing carbon quantum dots provided by the inventors, and the preparation of quantum dot particles in the present invention is not limited to the above method.

In a third aspect, the embodiment of the invention provides an application of the quantum dot enhanced fire-extinguishing tumor vaccine as a tumor immunity drug.

In an alternative embodiment, the quantum dot enhanced fire-fighting tumor vaccine is used as a tumor immunity drug.

The features and properties of the present invention are described in further detail below with reference to examples.

Unless otherwise specified, the devices used in this example are all conventional experimental devices, the materials and reagents used are commercially available, and the experimental method without specific description is also a conventional experimental method.

Example 1

Dissolving citric acid and urea in a DMSO solvent according to a mass ratio of 1:4 to obtain a transparent solution, placing the transparent solution in a 50ml polytetrafluoroethylene high-pressure reaction kettle, reacting for 4 hours at 160 ℃, adding a large amount of ethanol into the solution after reaction, washing to obtain a black solid, washing the solid with water, centrifuging (8000rpm, 5min), and drying to obtain dark blue powder. The diameter of the obtained carbon quantum dot particle is measured to be 3-5 nm. The mass ratios of C, N, O, S elements in the carbon quantum dot particles were 59.6%, 16.2%, 23.0%, and 1.2%, respectively.

Dissolving the obtained carbon quantum dot particles in deionized water, preparing a solution with the concentration of (0.1-1.5) mg/mL, mixing the carbon quantum dot particles and bovine serum albumin according to the mass ratio of 1:50, heating to 60 ℃ by adopting heating modes such as illumination heating, hot plate heating, water bath (oil bath) heating, oven heating or ultrasonic heating and the like, reacting for 10min, and stirring at the rotating speed of 100 rpm. Obtaining the bovine serum albumin (CQD-BSA) inactivated vaccine of the carbon quantum dot particles.

Example 2

The carbon quantum dot particles of example 1 were dissolved in deionized water to a concentration of (0.1-1.2) mg/mL, and 1X10 was added to each mL of the solution⁴-1x10¹⁰4T1 breast cancer cells. Heating to 60 ℃ by adopting an oven heating mode, reacting for 10min, and stirring at the rotating speed of 100 rpm. Obtaining the carbon quantum dot particle breast cancer cell (CQD-cell) inactivated vaccine.

Example 3

The preparation of the carbon quantum dot enhanced cell inactivated vaccine is the same as that of example 2.

0.05mL of the obtained carbon quantum dot enhanced cell inactivated vaccine (the concentration of the carbon quantum dot is 200ppm, the number of 4T1 cells is 1x 10)⁴-1x10¹⁰Single), pure carbon dots (carbon dots concentration of 200ppm), DiD-stained inactivated 4T1 breast cancer cells (4T1 cells number 1x 10)⁴-1x10¹⁰One, as in experimental group) and PBS were added to a 6cm macrophage RAW264.7 culture dish, and morphological changes of macrophages were observed with confocal after 24 h.

Example 4

The preparation of the carbon quantum dot enhanced cell inactivated vaccine is the same as that of example 2.

0.1mL of the obtained carbon quantum dot enhanced cell inactivated vaccine (the concentration of the carbon quantum dot is 200ppm, the number of 4T1 cells is 1x 10)⁴-1x10¹⁰Single), pure carbon dots (carbon dots concentration of 200ppm), DiD-stained inactivated 4T1 breast cancer cells (4T1 cells number 1x 10)⁴-1x10¹⁰As in experimental groups) and PBS were injected subcutaneously into balb/c mice, and their fourth pair of mammary glands were removed at 4h and 24h, respectively, imaged, and observed for fluorescence of the lymph.

Example 5

The carbon quantum dot particles were prepared as in example 1.

And removing the tumor of the breast cancer cell transgenic mouse, digesting and proliferating to obtain the cancer cell.

Dissolving the obtained carbon quantum dot particles in deionized water to prepare a solution with the concentration of (0.1-1.5) mg/mL, mixing the carbon quantum dot particles and the digested cancer cells according to the mass ratio of 1.0:10, heating to 60 ℃ by adopting a hot plate, reacting for 10min, and stirring at the rotating speed of 100 rpm. Thus obtaining the carbon quantum dot particle enhanced tumor cell vaccine.

Experimental example 1

The zeta potential of the carbon quantum dot particles (CQD) obtained in the examples and the bovine serum albumin (CQD-BSA) inactivated vaccines of the obtained carbon quantum dot particles was measured, and the results are shown in fig. 2. The CQD-BSA obtained after the compounding shows the enhancement of zeta potential.

The carbon quantum dot particles (CQD) obtained in the examples and bovine serum albumin (CQD-BSA) inactivated vaccines obtained therefrom were subjected to electrophoretic gel-running, and the results are shown in fig. 3. The CQD-BSA obtained after the compounding shows fluorescence when the protein corresponds to an electrophoresis band, which shows that the carbon dots are effectively bonded with the protein.

The infrared absorption spectra of the carbon quantum dot particles (CQD) obtained in the examples and bovine serum albumin (CQD-BSA) inactivated vaccines of the obtained carbon quantum dot particles were measured, and the results are shown in fig. 4. The CQD-BSA obtained after compounding is 3300cm^-1There is a sharp peak, which proves that the carbon quantum dots are hydrogen bonded with the protein on the tumor cells.

Experimental example 2

The fluorescence electron microscopy of the inactivated vaccine of the carbon quantum dot particles (CQD-cell) obtained in the example was performed, and the results are shown in fig. 5. The CQD-cell obtained after the compounding shows strong fluorescence, which indicates that the carbon dots can compound the protein on the cell without difference.

Experimental example 3

0.05mL of the obtained carbon quantum dot enhanced cell inactivated vaccine (the concentration of the carbon quantum dot is 200ppm, the number of 4T1 cells is 1x 10)⁴-1x10¹⁰Single), pure carbon dots (carbon dots concentration of 200ppm), DiD-stained inactivated 4T1 breast cancer cells (4T1 cells number 1x 10)⁴-1x10¹⁰One, as specified for the experimental group) and PBS were added to a 6cm macrophage RAW264.7 petri dish, respectively. After 24h, morphology change of macrophages is observed by confocal, and as shown in figure 6, macrophages cultured by carbon quantum dot particle tumor cell (CQD-cell) inactivated vaccine are obviousThe marked volume increase and dendritic cell differentiation indicate that CQD-cell can promote the maturation of macrophage and the ingestion of antigen.

Experimental example 4

The carbon quantum dot enhanced cell inactivated vaccine (carbon quantum dot concentration 200ppm, number of 4T1 cells 1X 10) obtained in 0.05mL of example 1⁴-1x10¹⁰Single), pure carbon dots (carbon dots concentration of 200ppm), DiD-stained inactivated 4T1 breast cancer cells (4T1 cells number 1x 10)⁴-1x10¹⁰One, as specified for the experimental group) and PBS, respectively, were injected subcutaneously into mice. The lymphatic conditions were observed at 4h and 24h and the results are shown in FIG. 7. The carbon quantum dot particle-containing inactivated vaccine for tumor cells (CQD-cells) is shown to increase the lymphatic presentation of antigens.

Experimental example 5

The carbon quantum dot particles were prepared as in example 1.

And removing the tumor of the breast cancer cell transgenic mouse, digesting and proliferating to obtain the cancer cell. Dissolving the obtained carbon quantum dot particles in deionized water to prepare a solution with the concentration of (0.1-1.5) mg/mL, mixing the carbon quantum dot particles and the digested cancer cells according to the mass ratio of 1.0:10, heating to 60 ℃ by adopting a hot plate heating mode, reacting for 10min, and stirring at the rotating speed of 100 rpm. Thus obtaining the carbon quantum dot particle enhanced tumor cell vaccine.

A metered amount of the carbon quantum dot particle-enhanced tumor cell vaccine was injected back into the transgenic mice. Preferably, the injection is metered to 200ul and the method of injection is one of intravenous injection, subcutaneous injection and ascites injection, preferably subcutaneous injection.

Tumor growth curves of the mice were measured, and the results are shown in fig. 8. Fig. 8 shows that the tumor growth of the experimental group a is significantly inhibited and cured, while the tumor of the control group b is gradually increased. The carbon quantum dot particle enhanced tumor cell vaccine preparation effectively activates the tumor immunity function of the mouse, and inhibits and kills the tumor in the mouse body.

Comparative example 1

Similar to the procedure of example 2, except that: the temperature of the heat application was 25 ℃ and it was found that the fluorescence was not enhanced, and no fluorescence was shown in one band corresponding to the protein, as shown in FIG. 9.

Comparative example 2

Similar to the procedure of example 1, except that: the temperature of the heat application was 120 ℃ and the fluorescence was found to be greatly reduced, as shown in FIG. 10.

From the above experimental results of comparative examples 1-2, it can be seen that the thermal recombination temperature is the key for the recombination of the quantum dots and the tumor cells, the thermal recombination temperature cannot be too low, the protein structure cannot be opened when the temperature is too low, and the two cannot be recombined. The temperature of thermal compounding cannot be too high, is higher than 100 ℃, and the vaccine cannot be obtained because the protein is completely denatured and the fluorescence cannot be enhanced after long-time heating. Namely, under the condition of not denaturing protein, the quantum dot is combined with the protein, so that the quantum dot enhanced cancer cell inactivated vaccine provided by the embodiment of the invention can be successfully prepared.

To sum up, the embodiment of the invention provides a quantum dot enhanced cancer cell inactivated vaccine, a preparation method and an application thereof, and the quantum dot enhanced cancer cell inactivated vaccine has the following characteristics: the particle size of the quantum dot particles is less than 15nm, the surface of the quantum dot particles contains a plurality of biocompatible functional groups, and the functional groups are one or more of amino, imino, carbon-nitrogen double bond, pyridine nitrogen, pyrrole nitrogen, graphene nitrogen, carboxyl, hydroxyl and carbonyl; under the action of heat, the space structure of a protein molecular chain on the cancer cell is opened, the protein molecular chain is loosened to form a gap, the small-size quantum dots enter the space conformation of the protein, and hydrogen bonds are formed between the functional groups on the surfaces of the quantum dots and the groups on the protein molecular chain, so that the inactivated cancer cell compound modified by the quantum dots is formed. The quantum dot material can reduce the function of the cancer cell surface immunosuppressive protein (the ' don't eat me ' signal protein) by changing the conformation of the protein molecules on the cancer cells, enhance the immunogenicity of the normal protein (the ' eat me ' signal protein), and improve the capture and presentation of the cancer cell antigen protein by an immune system and induce specific anti-tumor immune response on the basis of keeping the primary structure of the cancer cell antigen, namely, an amino acid sequence is not damaged. The quantum dot enhanced cancer cell inactivated vaccine can be applied to personalized cancer immunotherapy.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.