CN112158826B

CN112158826B - Carbon dot nano preparation and preparation method and application thereof

Info

Publication number: CN112158826B
Application number: CN202011086796.9A
Authority: CN
Inventors: 梁萍; 于杰; 梁兴杰; 宫宁强; 周群芳; 张东云
Original assignee: Fifth Medical Center of PLA General Hospital
Current assignee: Fifth Medical Center of PLA General Hospital
Priority date: 2020-10-12
Filing date: 2020-10-12
Publication date: 2023-03-21
Anticipated expiration: 2040-10-12
Also published as: CN112158826A

Abstract

The invention belongs to the technical field of nano-medicines and preparation thereof, and discloses a carbon dot nano-preparation, and a preparation method and application thereof. According to the preparation, mannose is used as a carbon dot substrate, ethylenediamine is used as a heat source to provide energy for glucose reaction, and then after the target product is enriched by an extracting agent, dialysis is completed by utilizing the concentration difference between a high-molecular separation membrane and ultrapure water, so that a finished product with uniform particle size is obtained. After the product is injected into tumors, mannose-derived carbon dot finished products (Man-CDs) can effectively capture various histones and other tumor-associated antigens, the activation of Dendritic Cells (DCs) is enhanced by the various histones and the like, the dendritic cells present the antigens to T cells, and the T cells finish the killing of the tumor cells. The preparation process is simple and convenient, the yield is high, the energy is saved, the environment is protected, the reaction among the raw materials is promoted to be fully fused on the basis of reducing the cost and reducing the complex process, and then the finished product is promoted to have stronger immune reaction and can be used for the aspect of anti-tumor immune reaction.

Description

Carbon dot nano preparation and preparation method and application thereof

Technical Field

The invention belongs to the technical field of nano-medicines and preparation thereof, and particularly relates to a carbon dot nano-preparation as well as a preparation method and application thereof.

Background

Microwave ablation (MWA) is of great interest in hepatocellular carcinoma ablation, but recurrence and metastasis following microwave ablation therapy remains a challenge. The related technology shows that the recurrence rate of 3 years is as high as 25.8%, and the recurrence rate of 5 years is higher. Conventional cancer immunotherapy, which attacks tumor cells by modulating the immune system, is expected to reduce recurrence and metastasis after MWA treatment, since MWA can lyse tumor cells, releasing tumor cell fragments containing tumor-associated antigens, which can be used for cancer vaccination. However, the related art suggests that the immune response induced by MWA treatment is not sufficient to effectively inhibit tumor growth and metastasis because of insufficient internalization and presentation of antigen in Antigen Presenting Cells (APCs), such as Dendritic Cells (DCs). Therefore, following MWA treatment, new strategies that enhance APCs antigen presentation are urgently needed to obtain a robust immune response that suppresses HCC recurrence.

The nano carbon is a new member of a carbon nano material family, and has attracted much attention at home and abroad in recent years. Compared with the traditional fluorescent dye and semiconductor quantum dot luminescent material, the carbon dot not only has excellent optical performance and size effect, but also has the advantages of low preparation cost, good biocompatibility, easy functionalization, adjustable energy band structure and the like.

Recently, there is a related art showing that in situ immunization using nanoparticle-based photodynamic therapy/photothermal therapy/radiotherapy in combination with an immunoadjuvant can induce an effective anti-tumor immune response.

The related art discloses a core-shell structure gold cluster-carbon dot nano-particle and a preparation method and application thereof. According to the technical scheme, a product is obtained by utilizing the catalytic performance of the noble metal nanocluster to be combined with the carbon dot nanoparticles, through the combination of chemical bonds among various chemical components and various redox reactions, and the product can be used for treating tumors. Firstly, the product is used for playing a therapeutic role, secondly, various chemical preparation methods in the technical scheme are complex, the selected raw materials are noble metals, the preparation cost is high, and the process flow is not simple.

The carbon dot surface is rich in functional groups such as hydroxyl groups and carboxyl groups, and these functional groups provide specific regions and can effectively bind biomolecules such as RNA and proteins. Furthermore, the carbon dots can specifically bind to the large neutral amino acid transporter 1 and induce effective cancer inhibition in vivo. Carbon dots are typically synthesized from a variety of precursors, including a variety of sugars.

Carbon Dots (CDs) are receiving increasing attention in biomedical applications due to their ease of synthesis, chemical inertness, biocompatibility, and fluorescence. Many studies in recent years have shown that sugar modifications on the surface of nanoparticles can specifically target antigen-presenting cells.

Therefore, for the carbon dot nanometer combined microwave ablation to obtain a product, the product does not need to pay a large amount of cost, the preparation scheme is simple in process, and the probability of liver cancer recurrence can be effectively reduced.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a carbon dot nano preparation, and a preparation method and application thereof.

The technical scheme adopted by the invention is as follows: a carbon dot nano preparation is mainly prepared from the following raw materials in parts by mass:

50-200 parts of mannose, 8-30 parts of ethylenediamine, 4-15 parts of inorganic acid, 16-60 parts of an extracting agent and 100-400 parts of water.

Preferably, the preparation is mainly prepared from the following raw materials in parts by mass:

80-150 parts of mannose, 12-23 parts of ethylenediamine, 6-12 parts of inorganic acid, 24-46 parts of an extracting agent and 160-300 parts of water;

100 parts of mannose, 15 parts of ethylenediamine, 8 parts of inorganic acid, 30 parts of an extracting agent and 200 parts of water.

Preferably, the inorganic acid comprises one or more of phosphoric acid, hydrochloric acid and sulfuric acid.

Preferably, the extractant comprises an organic solvent or an aqueous organic solvent solution,

the organic solvent comprises one or more of methanol, ethanol, acetone and acetonitrile;

the organic solvent aqueous solution comprises one or more of methanol aqueous solution, ethanol aqueous solution, acetone aqueous solution and acetonitrile aqueous solution.

A preparation method of a carbon dot nano-preparation comprises the following steps:

preparation of supernatant a: selecting mannose according to a corresponding proportion, dissolving the mannose into ethylenediamine, adding inorganic acid, stirring, cooling, adding a solvent, uniformly mixing, centrifuging, and collecting supernatant A;

preparation of supernatant B: adding an extracting agent into the supernatant A for centrifugation, and collecting a supernatant B;

preparation of intermediate product i: removing residual ethylenediamine and extractant in the supernatant B to obtain an intermediate product I;

preparing a finished product: and loading the obtained intermediate product I by using a polymer separation membrane, putting the intermediate product I into water for dialysis, and obtaining a finished product after the reaction is finished.

The obtained product is a product coated in the polymer separation membrane.

Preferably, the stirring time for preparing the supernatant A is 1-5 minutes, preferably 2-4 minutes, and more preferably 3 minutes;

the solvent comprises deionized water;

the mass ratio of the using amount of the solvent to the using amount of the ethylenediamine is 4-6;

the centrifugation time is 10-30 minutes, preferably 12-25 minutes, and further preferably 15-20 minutes;

the temperature during centrifugation is 15-30 ℃, preferably 20-28 ℃, and further preferably 25 ℃.

Preferably, when the supernatant B is prepared, the mass ratio of the using amount of the extracting agent to the using amount of the ethylenediamine is 2-5;

Preferably, when preparing the standby I, the polymer separation membrane comprises one or more of a reverse osmosis membrane, an ultrafiltration membrane, a microfiltration membrane, an ion exchange membrane, an organic liquid permeation evaporation membrane, a power forming membrane, a mosaic charged membrane, a liquid membrane, a dialysis membrane and a biomedical membrane;

preferably, the polymeric separation membrane comprises a microfiltration membrane.

Preferably, when the finished product is prepared, the reaction is completed for 10 to 18 hours, preferably 11 to 15 hours, and further preferably 12 hours;

the drying temperature is minus 50 ℃ to minus 100 ℃, preferably minus 60 ℃ to minus 80 ℃, and further preferably minus 70 ℃;

the drying time is 1 to 3 hours, preferably 1.5 to 2.5 hours, and more preferably 2 hours.

The use of a carbon dot nano-preparation, the use of a finished product of a carbon dot nano-preparation according to any one of claims 1-2 in an anti-tumor immunoreaction medicine.

The invention has the beneficial effects that:

the invention provides a carbon dot nano preparation, which is prepared by taking mannose as a carbon dot substrate and ethylenediamine as a heat source to provide energy for glucose reaction, enriching a target product by an extractant, and completing dialysis by utilizing the concentration difference between a high-molecular separation membrane and ultrapure water to obtain a finished product with uniform particle size. After the product is injected into tumors, mannose-derived carbon dot products (Man-CDs) can effectively capture damage-related molecular patterns, calreticulin, various histones and other tumor-related antigens, and can transmit signals to Dendritic Cells (DCs) in a targeted manner. The injury-associated molecular patterns, calreticulin and various histones, among others, enhance the activation of Dendritic Cells (DCs) that present antigens to T cells that accomplish the killing of tumor cells, enhancing their ability to process and present tumor antigens. The carbon dot nano preparation has the advantages of simple preparation process, high yield, energy conservation and environmental protection, and can promote the reactions among all the raw materials to be fully fused on the basis of reducing the cost and reducing the complex processes, so as to promote the carbon dot nano preparation to have stronger immune reaction and be used for the aspect of anti-tumor immune reaction.

Drawings

FIG. 1 is a schematic representation of a transmission electron microscope of the product prepared in example 2 of the present invention;

FIG. 2 is a schematic diagram showing the measurement of dynamic light scattering of a product prepared in example 2 of the present invention;

FIG. 3 is a schematic representation of the infrared spectroscopic examination of the product prepared in example 2 of the present invention;

FIG. 4 is a schematic representation of the flow cytometry assay of co-culture with dendritic cells after pre-incubation of the product prepared in example 2 of the present invention with carbon dots and tumor lysate in protocol 1 prepared according to the preparation method of example 2;

FIG. 5 is a schematic representation of the flow cytometry assay of co-culture with dendritic cells after pre-incubation of the product prepared in example 2 of the present invention with carbon dots and tumor lysate in protocol 1 prepared according to the preparation method of example 2;

FIG. 6 is a schematic diagram of ELISA detection of the expression level of IL-6 in co-culture with dendritic cells after pre-incubation of the product prepared in example 2 of the present invention with carbon dots and tumor lysate in Experimental scheme 2 prepared according to the preparation method of example 2;

FIG. 7 is a schematic representation of ELISA detection of TNF- α expression levels in co-culture with dendritic cells after pre-incubation of the product prepared in example 2 of the present invention with carbon dots and tumor lysate in protocol 2 prepared according to the method of preparation in example 2;

FIG. 8 is a schematic representation of ELISA detection of the expression level of IL-1. Beta. In co-culture with dendritic cells after pre-incubation of the product prepared in example 2 of the present invention with carbon dots and tumor lysate in protocol 2 prepared according to the preparation method of example 2;

FIG. 9 is a schematic flow contour diagram of a control group for detecting the expression level of mature dendritic cells;

FIG. 10 is a schematic flow contour diagram of detection of expression levels of mature dendritic cells by a glucose-derived carbon dot in combination with microwave ablation treatment group;

FIG. 11 is a schematic flow contour line of sucrose-derived carbon dot combined with microwave ablation treatment group for detecting the expression level of mature dendritic cells;

FIG. 12 is a schematic flow contour line of galactose-derived carbon dot combined with microwave ablation treatment group for detecting the expression level of mature dendritic cells;

FIG. 13 is a schematic flow contour line of expression level of mature dendritic cells detected by lactose derived carbon dots combined with microwave ablation treatment group;

FIG. 14 is a schematic flow contour diagram of mannose-derived carbon dots combined with microwave ablation treatment group for detecting the expression level of mature dendritic cells;

FIG. 15 is a graph showing the growth curve of the primary tumor of the subject mouse in the experimental example;

FIG. 16 is a graph showing the growth curve of the distal tumor of the subject mouse in the experimental example;

FIG. 17 is a graph showing the change in body weight of a subject mouse in an experimental example.

Detailed Description

The present invention is further illustrated below with reference to specific examples. It will be appreciated by those skilled in the art that the following examples, which are set forth to illustrate the present invention, are some, but not all, of the examples of the present invention and should not be construed as limiting the scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples were carried out under the conventional conditions, unless otherwise specified. The reagents used are all conventional products which are commercially available.

Example 1:

a preparation method of a carbon dot nano preparation comprises the following steps:

preparation of supernatant a: selecting 50mg of mannose (Man) according to a corresponding proportion, dissolving the mannose (Man) in 8mL of ethylenediamine, adding 4mL of phosphoric acid, stirring for 1 minute, cooling, adding 11mL of deionized water, uniformly mixing, centrifuging for 10 minutes at 11000rpm and 15 ℃, and collecting supernatant A;

preparation of supernatant B: adding 16ml of methanol into the supernatant A, centrifuging for 10 minutes at 11000rpm and 15 ℃, and collecting a supernatant B;

preparation of intermediate product i: removing residual ethylenediamine and methanol in the supernatant B by using a rotary evaporation mode to obtain an intermediate product I;

preparing a finished product: and (3) loading the obtained intermediate product I by using a reverse osmosis membrane, putting the intermediate product I into 100ml of ultrapure water for dialysis, and reacting for 10 hours to obtain a finished product.

The finished product obtained in the above embodiment is a liquid preparation, and in the actual implementation process, the finished product is freeze-dried at minus 50 ℃ for 1 hour, so that the product convenient to package and transport is obtained.

Example 2:

preparation of supernatant a: selecting 200mg of mannose (Man) according to a corresponding proportion, dissolving the mannose (Man) in 30mL of ethylenediamine, adding 15mL of hydrochloric acid, stirring for 5 minutes, cooling, adding 28mL of deionized water, uniformly mixing, centrifuging for 30 minutes at 11000rpm and 30 ℃, and collecting supernatant A;

preparation of supernatant B: adding 60ml of ethanol into the supernatant A, centrifuging for 30 minutes at 11000rpm and 30 ℃, and collecting a supernatant B;

preparation of intermediate product i: removing residual ethylenediamine and ethanol in the supernatant B by using a rotary evaporation mode to obtain an intermediate product I;

preparing a finished product: the intermediate product I obtained was loaded by an ultrafiltration membrane and dialyzed in 400ml of ultrapure water, and the reaction was completed after 18 hours to obtain a finished product.

The finished product obtained in the above embodiment is a liquid preparation, and in the actual implementation process, the finished product is freeze-dried at-100 ℃ for 3 hours to obtain a product convenient for packaging and transportation.

Example 3:

preparation of supernatant a: selecting 80mg of mannose (Man) according to a corresponding proportion, dissolving the mannose (Man) in 12mL of ethylenediamine, adding 6mL of sulfuric acid, stirring for 2 minutes, cooling, adding 16mL of deionized water, uniformly mixing, centrifuging for 12 minutes at 11000rpm and 20 ℃, and collecting supernatant A;

preparation of supernatant B: adding 24ml of acetone into the supernatant A, centrifuging for 12 minutes at 11000rpm and 20 ℃, and collecting a supernatant B;

preparation of intermediate I: removing residual ethylenediamine and acetone in the supernatant B by a rotary evaporation mode to obtain an intermediate product I;

preparing a finished product: the intermediate product I obtained was loaded by a microfiltration membrane and dialyzed in 160ml of ultrapure water, and the reaction was completed after 18 hours to obtain a finished product.

The finished product obtained in the above embodiment is a liquid preparation, and in the actual implementation process, the finished product is freeze-dried at-60 ℃ for 1.5 hours, so that the product convenient to package and transport is obtained.

Example 4:

preparation of supernatant a: selecting 150mg of mannose (Man) according to a corresponding proportion, dissolving the mannose (Man) in 23mL of ethylenediamine, adding 12mL of phosphoric acid, stirring for 4 minutes, cooling, adding 27mL of deionized water, uniformly mixing, centrifuging for 25 minutes at 11000rpm and 28 ℃, and collecting supernatant A;

preparation of supernatant B: adding 46ml of acetonitrile into the supernatant A, centrifuging for 25 minutes at 11000rpm and 28 ℃, and collecting a supernatant B;

preparation of intermediate product i: removing residual ethylenediamine and acetonitrile in the supernatant B by using a rotary evaporation mode to obtain an intermediate product I;

preparing a finished product: the obtained intermediate product I was loaded by an ion exchange membrane and dialyzed in 300ml of ultrapure water, and the reaction was completed after 15 hours to obtain a finished product.

The finished product obtained in the above embodiment is a liquid preparation, and in the actual implementation process, the finished product is freeze-dried at-80 ℃ for 2.5 hours, so that the product convenient to package and transport is obtained.

Example 5:

preparation of supernatant a: selecting 100mg of mannose (Man) according to a corresponding proportion, dissolving the mannose (Man) in 15mL of ethylenediamine, adding 8mL of phosphoric acid, stirring for 4 minutes, cooling, adding 27mL of deionized water, uniformly mixing, centrifuging for 25 minutes at 11000rpm and 28 ℃, and collecting supernatant A;

preparation of supernatant B: adding 30ml of methanol aqueous solution into the supernatant A, centrifuging for 25 minutes at 11000rpm and 28 ℃, and collecting the supernatant B;

preparation of intermediate product i: removing residual ethylenediamine and methanol aqueous solution in the supernatant B by using a rotary evaporation mode to obtain an intermediate product I;

preparing a finished product: the obtained intermediate product I was loaded by an ion exchange membrane and dialyzed in 200ml of ultrapure water, and the reaction was completed after 15 hours to obtain a finished product.

Example 6:

preparation of supernatant B: adding 30ml of ethanol aqueous solution into the supernatant A, centrifuging for 25 minutes at 11000rpm and 28 ℃, and collecting a supernatant B;

preparation of intermediate product i: removing residual ethylenediamine and ethanol aqueous solution in the supernatant B by using a rotary evaporation mode to obtain an intermediate product I;

preparing a finished product: and loading the obtained intermediate product I by using a dialysis membrane, dialyzing the intermediate product I in 200ml of ultrapure water, and reacting for 15 hours to obtain a finished product.

Example 7:

preparation of supernatant B: adding 30ml of acetone aqueous solution into the supernatant A, centrifuging for 25 minutes at 11000rpm and 28 ℃, and collecting a supernatant B;

preparation of intermediate product i: removing residual ethylenediamine and acetone aqueous solution in the supernatant B by using a rotary evaporation mode to obtain an intermediate product I;

preparing a finished product: the obtained intermediate product I was loaded on a dialysis membrane and dialyzed in 200ml of ultrapure water, and the reaction was completed after 15 hours to obtain a finished product.

Example 8:

preparation of supernatant B: adding 30ml of acetonitrile aqueous solution into the supernatant A, centrifuging for 25 minutes at 11000rpm and 28 ℃, and collecting a supernatant B;

preparation of intermediate product i: removing residual ethylenediamine and acetonitrile aqueous solution in the supernatant B by using a rotary evaporation mode to obtain an intermediate product I;

preparing a finished product: and loading the obtained intermediate product I by using a microporous filtering membrane, putting the intermediate product I into 200ml of ultrapure water for dialysis, and reacting for 15 hours to obtain a finished product.

In the above embodiments, in the specific implementation process, when preparing the supernatant a, the mannose is dissolved in the ethylenediamine, and the phosphoric acid is added, and when the mannose carbide (yellow precipitate) is obtained by stirring, the cooling and the subsequent deionized water adding processes may be performed.

In the concrete implementation process of the above embodiments, when the residual ethylenediamine and the extractant in the supernatant B are removed in the preparation of the stock product i, the removal is performed by using a rotary evaporation device under vacuum.

In the practical operation process of the above embodiment, the selected rotary steaming device comprises a vacuum concentration rotary steaming instrument, and the specification and model are as follows: TQG, manufacturer: wenzhou worker big light industry machinery, inc. The selection of the model of the rotary steaming equipment and the selection of manufacturers and the like can be correspondingly set according to the actual environment and the actual requirements in the specific implementation process. All the products which can be steamed in a rotary manner and have no influence on the components of the finished products belong to the protection scope of the invention.

In the actual operation process of all the embodiments, the selected stirring device comprises a three-dimensional motion mixer, stainless steel materials with specification models of SYH-30 and SYH-1000, and manufacturers: jiangyin and Rong mechanical Co. The selection of the stirring device is not limited to the above, and all devices which can complete the corresponding stirring work and have no influence on raw materials belong to the protection scope of the invention.

In the practical operation process of the embodiment, the selected freeze drying device comprises a vacuum freeze dryer which is made of stainless steel and has the specification model of FD-1A-50, and a manufacturer: feathering instruments ltd, jiangsu. The selection of the freeze drying device is not limited to the above description, and all devices capable of performing the corresponding freeze drying operation are within the scope of the present invention.

In the practical operation process of the above embodiment, the selected centrifugal device comprises a high-speed centrifuge made of stainless steel, the specification and model number of the centrifuge is H3-18K, the maximum capacity is 100ml × 4, and the maximum rotation speed is 18500rpm. The manufacturer: well laboratory products, suzhou, inc. The selection of the centrifugal device is not limited to the above-mentioned ones, and all devices capable of performing corresponding centrifugal work are within the scope of the present invention.

Examples of the experiments

Dendritic Cells (DCs) play an important role in the initiation and regulation of innate and adaptive immunity. And the mature dendritic cells jointly stimulate the up-expression of the receptor and enhance the secretion of cytokines, and show superior capability in the aspects of antigen processing and expression.

Purpose of the experiment: simulated microwave ablation treatment conditions were used to see if various carbon spots could absorb tumor lysates and induce Dendritic Cell (DCs) maturation.

The experimental method comprises the following steps: the measurement method of transmission electron microscope is that sample powder is mixed with ultrapure water, the concentration is 1mg/ml, ultrasonic vibration is carried out to form suspension, the suspension is dropped on a copper net attached with a supporting film, and the liquid can be observed after being volatilized.

The dynamic light scattering was measured by mixing a sample powder with ultrapure water at a concentration of 1mg/ml, vibrating the mixture with ultrasonic waves to form a suspension, and placing the suspension in a sample bottle for observation.

The infrared spectrum detection method comprises the steps of mixing sample powder with methanol, enabling the concentration to be 1mg/ml, dropping suspension on a KBr film base, paving, and generating a uniform film after a solvent is volatilized.

FIG. 1 is a schematic transmission electron microscope image of the product prepared in example 2 of the present invention, where Man-CDs have a clear spherical morphology as determined by transmission electron microscopy. The scale of the diagram is 20nm.

FIG. 2 is a schematic diagram of measurement of Dynamic Light Scattering (DLS) of the product prepared in example 2 of the present invention, which shows uniform particle size distribution according to infrared spectroscopy detection, and Man-CDs has many functional groups. A

FIG. 3 is a schematic representation of the IR spectroscopy measurements of the product prepared in example 2 of the present invention, based on which Man-CDs have many functional groups

Experimental protocol 1:

different nanocarbon point particles (Glu-CDs: glucose-derived carbon points, sug-CDs: sucrose-derived carbon points, gla-CDs: galactose-derived carbon points, lac-CDs: lactose-derived carbon points, man-CDs: mannose-derived carbon points) were incubated with the tumor cell lysate for 10 minutes, and then the carbon points were washed with PBS (phosphate buffered saline) not less than 5 times. The antigen-adsorbed carbon spots were then incubated with dendritic cells for 24 hours and the dendritic cell maturation markers (CD 80, CD 86) were detected by flow cytometry.

FIGS. 4 and 5 are flow charts showing that mannose carbon dots that have absorbed tumor antigens stimulate the maturation of Dendritic Cells (DCs).

FIG. 4 is a schematic representation of flow cytometry detection of the dendritic cell maturation marker CD80, showing that mannose carbon dots that have taken up tumor antigens are capable of stimulating the maturation of Dendritic Cells (DCs).

FIG. 5 is a schematic representation of the flow cytometry detection of the dendritic cell maturation marker CD86, showing that mannose carbon sites, which have absorbed tumor antigens, are capable of stimulating the maturation of Dendritic Cells (DCs).

Experimental protocol 2:

fig. 6, 7 and 8, the production levels of the nanoparticle-treated Dendritic Cell (DCs) cytokines were further investigated using ELISA. Man-CDs (tumor lysate preincubation) treated DCs secreted higher levels of TNF-. Alpha.IL-6, IL-1. Beta. Than other carbon spots. These results demonstrate that pre-cultured Man-CDs with tumor lysate can stimulate Dendritic Cell (DCs) maturation and enhance antigen processing and presentation in DCs.

The various carbon spots were observed to absorb tumor lysate and induce maturation of DCs by using simulated microwave ablation treatment conditions. We incubated different carbon nanodot particles of 100ug/ml (Glu-CDs, sug-CDs, gla-CDs, lac-CDs, man-CDs) with tumor cell lysates for 10 min, and then washed the carbon spots more than 5 times with PBS. The carbon spots with the adsorbed antigen were then incubated with dendritic cells for 24 hours and the levels of TNF-. Alpha.IL-6, IL-1. Beta. Secretion from DCs were measured by ELISA.

TNF- α is a type of tumor necrosis factor. The effect is a cytokine which can directly kill tumor cells and has no obvious toxicity to normal cells.

IL-6, a pleiotropic cytokine with a wide range of functions. IL-6 can regulate the growth and differentiation of various cells, has the functions of regulating immune response, acute phase reaction and hematopoiesis, and plays an important role in the anti-infection immune response of the organism.

IL-1 beta has important regulatory effects on a variety of immunocompetent cells.

The results of the graphs of fig. 6, 7 and 8 demonstrate that pre-cultured Man-CDs in tumor lysate can stimulate DC maturation and enhance antigen processing and presentation in DCs.

FIGS. 9, 10, 11, 12, 13, and 14 are performed by injecting Hepa-6 tumor cells (1X 106) into the right side of the mice, dividing the mice into 6 groups (Control, MWA + Man-CDs, MWA + glu-CDs, MWA + sug-CDs, MWA + glal-CDs, MWA + Lac-CDs) when the tumor size is about 10X10mm, injecting carbon spots into the Control group and microwave ablation group, respectively, taking lymph nodes in the groin near the tumor three days after treatment, and performing flow cytometry to detect the expression level of mature dendritic cells (CD 80, CD 86).

Fig. 9, 10, 11, 12, 13, 14 are schematic diagrams showing significant improvement of expression of the DCs mutation marker after microwave ablation treatment and intratumoral carbon spot injection. The schematic diagram shows that after mice are treated by MWA + Man-CDs, the proportion of mature DCs (CD 80+ CD86 +) is remarkably improved from 9.87 percent to 43.06 percent, and the Control group (Control) and other carbon dots (glu-CDs, sug-CDs, gla-CDs and Lac-CDs) have limited promotion effect on the maturation of the DCs.

In conclusion, MWA + Man-CDs can remarkably induce DCs to mature in vitro and in vivo.

Experimental protocol 3: a bilateral mouse tumor model was constructed.

Hepa-6 tumor cells (1X 106) were first injected into the right side of the mice to form primary tumors. After 4 days, 1 × 106 tumor cells were injected into the left side of the mouse as distant tumors. When the primary tumor size reached 10X10mm, 2 MWA (microwave power 5W, heating to 60 ℃ C., preservation for 1 min) were administered on days 0 and 3. After each MWA treatment, 100. Mu.L of Man-CDs (100. Mu.g/mL) were injected intratumorally. Growth of primary and distant tumors was measured every two days.

Tumor-bearing mice were monitored for body weight every two days.

As shown in fig. 15, the graph of the growth of the primary tumor in mice: control group, without any treatment, the tumor grew rapidly; the microwave ablation group (MWA) does not completely ablate and kill the tumor, and the tumor has a growth trend after gradually reducing; carbon-point treated (Man-CDs) tumors grew second only to the control group; the tumors of the combination treatment group (MWA-Man-CDs) were gradually reduced. The combined treatment group (MWA-Man-CDs) has the best effect on treating tumors.

The primary tumor size of the microwave ablation group (MWA) was significantly smaller than the control group, but distal tumor growth was not affected compared to the control group. The microwave ablation group is a local treatment, and although it also causes the release of tumor-associated antigens and "danger signals", these proteins may not be efficiently taken up by antigen-presenting cells, resulting in limited anti-tumor immune responses.

"danger signals" are meant to include injury-associated molecular patterns, calreticulin, and various histones, among others.

As shown in fig. 15, the carbon-dotting group alone (Man-CDs) significantly inhibited tumor growth, and the antitumor effect of the carbon-dotting group alone (Man-CDs) was probably due to the enhanced treatment of tumor antigens at the injection site, since intratumoral injection also induced mechanical damage of tumor cells and release of antigens.

As shown in fig. 16, the distal tumor growth plot: distal tumors (mock metastases) of each group were not treated and tumor growth was observed to be inhibited in the combination treatment group (MWA-Man-CDs) and faster in the other groups.

The carbon-dot treated group (Man-CDs) alone did not effectively inhibit the growth of distant tumors, probably because mechanical damage to the syringe needle induced only limited release of antigen and "danger signals" and the immune response was insufficient to inhibit the growth of distant tumors.

The combination of microwave ablation group (MWA) treatment and carbon-point treatment group (Man-CDs) injection significantly inhibited tumor growth on both primary and distal tumors. MWA + human-cds treatment almost completely inhibited the growth of the primary tumor, with some mice having no tumor even in distant tumors.

As in fig. 17, no significant weight loss was observed in each group of mice.

The invention provides a carbon dot nano preparation, which is prepared by taking mannose as a carbon dot substrate and ethylenediamine as a heat source to provide energy for glucose reaction, enriching a target product by an extractant, and completing dialysis by utilizing the concentration difference between a high-molecular separation membrane and ultrapure water to obtain a finished product with uniform particle size. After the product is injected into tumors, mannose-derived carbon dot products (Man-CDs) can effectively capture damage-related molecular patterns, calreticulin, various histones and other tumor-related antigens, and can transmit signals to Dendritic Cells (DCs) in a targeted manner. The injury-associated molecular patterns, calreticulin and various histones, among others, enhance the activation of Dendritic Cells (DCs), which present antigens to T cells that accomplish the killing of tumor cells, and their ability to process and present tumor antigens. The carbon dot nano preparation has the advantages of simple preparation process, high yield, energy conservation and environmental protection, and can promote the reactions among all the raw materials to be fully fused on the basis of reducing the cost and reducing the complex processes, so as to promote the carbon dot nano preparation to have stronger immune reaction and be used for the aspect of anti-tumor immune reaction.

While particular embodiments of the present invention have been illustrated and described, it will be appreciated that the present invention is not limited to the above-described alternative embodiments, and that various other forms of product may be devised by anyone in light of the present invention. The foregoing detailed description should not be construed as limiting the scope of the invention, and it will be understood by those skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or that equivalent substitutions may be made to some or all of the technical features thereof, without departing from the spirit and scope of the invention, and that these modifications or substitutions may not substantially depart from the essence of the corresponding technical solutions.

Claims

1. An application of a carbon dot nanometer preparation in preparing anti-tumor immunoreaction medicines;

the carbon dot nano preparation is prepared from the following raw materials in parts by mass:

50-200 parts of mannose, 8-30 parts of ethylenediamine, 4-15 parts of inorganic acid, 16-60 parts of an extracting agent and 100-400 parts of water;

the preparation method of the carbon dot nano preparation comprises the following steps:

preparation of supernatant a: selecting mannose according to a corresponding proportion, dissolving the mannose in ethylenediamine, adding inorganic acid, stirring, cooling, adding a solvent, uniformly mixing, centrifuging, and collecting supernatant A;

2. The application of the carbon dot nano preparation in preparing an anti-tumor immunoreaction medicine according to claim 1, wherein the carbon dot nano preparation is prepared from the following raw materials in parts by mass:

80-150 parts of mannose, 12-23 parts of ethylenediamine, 6-12 parts of inorganic acid, 24-46 parts of an extracting agent and 160-300 parts of water.

3. The application of the carbon dot nano preparation in preparing an anti-tumor immunoreaction medicine according to claim 2 is characterized in that the carbon dot nano preparation is prepared from the following raw materials in parts by mass:

4. The use of the carbon dot nano-preparation according to any one of claims 1 to 3, wherein the inorganic acid comprises one or more of phosphoric acid, hydrochloric acid and sulfuric acid for preparing a drug for anti-tumor immune reaction.

5. The use of the carbon dot nano-preparation according to any one of claims 1 to 3 in the preparation of a drug for anti-tumor immune reaction, wherein the extraction agent comprises an organic solvent or an aqueous solution of an organic solvent.

6. The use of the carbon dot nano-formulation according to claim 5 for preparing an anti-tumor immune reaction medicine, wherein the organic solvent comprises one or more of methanol, ethanol, acetone and acetonitrile.

7. The use of the carbon dot nano-formulation according to claim 6 for preparing an anti-tumor immune reaction medicine, wherein the organic solvent aqueous solution comprises one or more of methanol aqueous solution, ethanol aqueous solution, acetone aqueous solution and acetonitrile aqueous solution.

8. The use of the carbon dot nano-preparation according to claim 1 in the preparation of an anti-tumor immunoreaction medicine, wherein in the preparation method, the stirring time is 1-5min when the supernatant A is prepared;

the solvent comprises deionized water;

the centrifugation time is 10-30min;

the temperature during centrifugation is 15-30 ℃.

9. The use of the carbon dot nano-preparation according to claim 8 in the preparation of an anti-tumor immunoreaction medicine, wherein in the preparation method, the stirring time is 2-4min when the supernatant A is prepared; the centrifugation time is 12-25min; the temperature during centrifugation is 20-28 ℃.

10. The use of the carbon dot nano-preparation according to claim 9 for preparing an anti-tumor immunoreaction medicine, wherein in the preparation method, the stirring time is 3min when the supernatant A is prepared; the centrifugation time is 15-20min; the temperature during the centrifugation was 25 ℃.

11. The application of the carbon dot nano-preparation in the preparation of an anti-tumor immunoreaction medicine according to claim 1, wherein in the preparation method, when the supernatant B is prepared, the mass ratio of the dosage of the extracting agent to the dosage of the ethylenediamine is 2-5; the centrifugation time is 10-30min; the temperature during centrifugation is 15-30 ℃.

12. The use of the carbon dot nano-preparation according to claim 11 for preparing an anti-tumor immunoreaction medicine, wherein the centrifugation time is 12-25min; the temperature during centrifugation is 20-28 ℃.

13. The use of the carbon dot nano-preparation according to claim 12 for preparing an anti-tumor immunoreaction medicine, wherein the centrifugation time is 15-20min; the temperature during the centrifugation was 25 ℃.

14. The use of the carbon dot nano-preparation according to claim 1 in the preparation of an anti-tumor immunoreaction medicine, wherein the reaction completion time is 10-18h when the finished product is prepared.

15. The use of the carbon dot nano-preparation according to claim 14 for preparing an anti-tumor immunoreaction medicine, wherein the reaction completion time for preparing a finished product is 11-15h.

16. The use of the carbon dot nano-preparation according to claim 15 for preparing an anti-tumor immunoreaction medicine, wherein the reaction completion time for preparing a finished product is 12 hours.