CN113440607A - Full-target antigen presenting cell tumor vaccine and preparation method and application thereof - Google Patents

Full-target antigen presenting cell tumor vaccine and preparation method and application thereof Download PDF

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CN113440607A
CN113440607A CN202110731947.XA CN202110731947A CN113440607A CN 113440607 A CN113440607 A CN 113440607A CN 202110731947 A CN202110731947 A CN 202110731947A CN 113440607 A CN113440607 A CN 113440607A
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曲松楠
汤子康
周胤宁
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University of Macau
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Abstract

The invention discloses a whole target antigen presenting cell tumor vaccine and a preparation method and application thereof, wherein the preparation method of the whole target antigen presenting cell tumor vaccine comprises the following steps: and co-incubating the quantum dot modified tumor cells and immature antigen presenting cells, wherein the quantum dot modified tumor cells are obtained by connecting quantum dots and protein molecular chains of the tumor cells through hydrogen bonds. After the tumor cells modified by the quantum dots are incubated with immature Antigen Presenting Cells (APC), the immature APC (such as MP or DC) can be converted from an immature state to a super-activated state, so that the uptake of the tumor antigen is enhanced, the expression of MHC molecules is up-regulated on a large scale, and the antigen presentation efficiency is improved. Meanwhile, the preparation process is simple, the cost is low, and the application prospect is good.

Description

Full-target antigen presenting cell tumor vaccine and preparation method and application thereof
Technical Field
The invention relates to the technical field of biomedical engineering, in particular to a full-target antigen presenting cell tumor vaccine and a preparation method and application thereof.
Background
In recent years, with the development of technology and the increasing awareness of early cancer screening, the discovery rate of various cancers is increasing, but many tumors are scattered and easy to recur, and cannot be completely resected by the same operation or multiple operations, and at present, a standard treatment scheme is lacking. The existing tumor treatment methods, no matter radiotherapy, chemotherapy and surgery, can not radically cure tumor recurrence and metastasis. Antigen Presenting Cells (APCs) are known to be indispensable professional cells in the immune system, specifically activating T cells and precisely killing tumor cells. Upon receiving a specific activation stimulus, APCs (MP or DC) will migrate to lymphoid organs and present the captured antigen or antigens to T cells via the Major Histocompatibility Complex (MHC), thereby eliciting a precise specific immune response.
However, specific tumor cell antigens cannot be correctly recognized, captured and presented due to various complex reasons, such as inhibitory tumor microenvironment, tolerance of the body, tumor cell escape mechanism, and insufficient immunogenicity of the antigens. It has been reported that the use of nanoparticles of smaller size (<50nm) simultaneously transports tumor antigens and adjuvants into draining lymph nodes, enabling DC more direct uptake and extraction of antigen. This approach increases the efficiency of antigen uptake by the DC to some extent. However, all reported nano vaccines are subjected to antigen presentation after non-specific uptake by immature DC, and the efficiency of reaching lymph nodes by both nano vaccines and DC vaccines is still low. Therefore, the development of a new antigen peptide targeting transport carrier or an APC migration enhancement strategy to improve the antigen presentation efficiency in lymph nodes is still the key to improve the anti-tumor effect of the vaccine, and the development of the vaccine for cancer becomes a new trend. Research searches for effective treatment means for tumors, and the realization of effective presentation of specific antigens is a key problem which needs to be solved urgently at present.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a full-target antigen presenting cell tumor vaccine, a preparation method and application thereof, so as to improve the technical problem.
The invention is realized by the following steps:
in a first aspect, the present invention provides a method for preparing a full-target antigen presenting cell tumor vaccine, comprising: and co-incubating the quantum dot modified tumor cells and immature antigen presenting cells, wherein the quantum dot modified tumor cells are obtained by connecting quantum dots and protein molecular chains of the tumor cells through hydrogen bonds by ligands.
Optionally, the surface of the quantum dot has active sites comprising one or more of hydroxyl, carbonyl, carboxyl, and amino groups.
Alternatively, the particle size of the quantum dots is less than 80nm, more preferably less than 20 nm;
optionally, the quantum dots are carbon quantum dots, and further preferably have a spherical or cake shape, and the core structure is a multilayer graphene structure.
Alternatively, the tumor cell is selected from malignant tumor cells derived from epithelial cells of any one of lung cancer cells, breast cancer cells, ovarian cancer cells, kidney cancer cells, nasopharyngeal cancer cells, esophageal cancer cells, colorectal cancer cells, malignant glioma cells, and cerebellar medulloblastoma cells.
Alternatively, the tumor cells are obtained by:
obtaining a tumor cell suspension by centrifuging sheared tumor tissues obtained from a patient body and carrying out enzyme digestion with a culture solution;
mixing the tumor cell suspension with the inactivated human serum, standing, taking out bottom cells, and further performing centrifugal purification to obtain the tumor cells.
Optionally, the tumor cell is a 4T1 breast cancer cell.
Optionally, the quantum dot modified tumor cell is prepared by a thermal compounding method. The conditions for the thermal recombination of the quantum dots and the tumor cells are as follows: the temperature is 10-100 ℃, preferably 50-90 ℃, more preferably 50-80 ℃, and the compounding time is 1-360 min, preferably 1-100 min, more preferably 2-30 min.
Alternatively, the thermal compounding process is carried out in a solution system.
Optionally, the ratio of the mass of the quantum dots to the number of tumor cells is 0.1mg:104-1010。
Optionally, the antigen presenting cells comprise at least one of macrophage MP and dendritic cell DC.
Optionally, the co-incubation conditions are: the concentration of the quantum dot modified and compounded tumor cells is 103-108The ratio of the cells/mL to the immature APC is 1-10: 1-10, the incubation time is 0.01-10 days, more preferably 1-7 days, and the incubation temperature is 20-40 ℃, preferably 25-37 ℃.
Optionally, the co-incubation process comprises the steps of: the tumor cells after quantum dot modification and compounding and the obtained immature antigen presenting cells are incubated in a culture solution, and a cell culture dish can be placed in a biological safety cabinet or a cell culture box to observe and supplement or replace the culture solution at any time.
Optionally, the tumor vaccine obtained after incubation is isolated, purified and harvested.
Alternatively, the preparation of immature antigen presenting cells comprises the steps of: monocytes are obtained from peripheral blood mononuclear cells isolated from a patient and induced to differentiate into immature antigen presenting cells, respectively.
In a second aspect, the invention also provides a whole-target antigen presenting cell tumor vaccine, which is prepared by the preparation method.
In a third aspect, the invention also provides an application of the whole-target antigen presenting cell tumor vaccine in preparing a medicament for treating or preventing tumors.
One of the technical schemes of the invention at least has the following beneficial effects: after the tumor cells modified by the quantum dots are incubated with immature Antigen Presenting Cells (APC), the immature APC (such as MP or DC) can be converted from an immature state to a super-activated state, so that the uptake of the tumor antigen is enhanced, the expression of MHC molecules is up-regulated on a large scale, and the antigen presentation efficiency is improved. Meanwhile, the quantum dot thermal composite tumor cells can enhance the immunogenicity of tumor antigens by full targets, promote activated and mature MP and DC to migrate to lymph nodes, and effectively induce organisms to generate strong whole-body anti-tumor immune response reaction. In addition, the whole-target antigen presenting cell tumor vaccine obtained by co-incubation can be applied to the tumor immunotherapy of individual whole-target cancer patients or the prevention of high-risk cancer people, and meanwhile, the preparation process is simple, the cost is low, and the whole-target antigen presenting cell tumor vaccine has a good application prospect.
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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 graph showing the activation of APC in co-incubation with pure quantum dot material, heat-treated tumor cells and quantum dot heat-complexed tumor cells under a confocal microscope in comparative examples 1 to 2 and examples 1 to 3 of the present invention;
FIG. 2 is a graph comparing the expression of MHC molecules after the co-incubation of APC with pure quantum dot material, heat-treated tumor cells and quantum dot heat-complexed tumor cells in comparative examples 1-2 and examples 1-3 of the present invention;
FIG. 3 is a graph comparing the tumor inhibition of mice after subcutaneous injection after co-incubation of APC with pure quantum dot material, heat-treated tumor cells and quantum dot heat-complexed tumor cells in comparative examples 1-2 and examples 1-3 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 whole target antigen presenting cell tumor vaccine provided by the invention, and the preparation method and the application thereof are specifically explained below.
The inventor finds that co-incubated MP or DC can be converted from an immature state to a super-activated state by adding quantum dot heat-compounded tumor cells in long-term research and practice processes, so that the uptake of tumor antigens by the co-incubated MP or DC is greatly enhanced, the expression of MHC molecules is up-regulated in a large scale, and the antigen presentation efficiency is improved. The quantum dot thermal composite tumor cell can enhance the immunogenicity of tumor antigens by a whole target spot, promote activated and mature MP and DC to migrate to lymph nodes, and effectively induce organisms to generate strong whole-body anti-tumor immune response reaction. Therefore, the following technical solutions are proposed.
Some embodiments of the present invention provide a method of preparing a full-target antigen presenting cell tumor vaccine, comprising: and co-incubating the quantum dot modified tumor cells and immature antigen presenting cells, wherein the quantum dot modified tumor cells are obtained by connecting quantum dots and protein molecular chains of the tumor cells through hydrogen bonds by ligands.
In particular, in some embodiments, the quantum dot modified tumor cells are prepared by thermal complexation. The conditions for the thermal recombination of the quantum dots and the tumor cells are as follows: the temperature is 10-100 ℃, preferably 50-90 ℃, more preferably 50-80 ℃, and the compounding time is 1-360 min, preferably 1-100 min, more preferably 2-30 min. For example, thermal compounding at 20 ℃ for 200min, or at 40 ℃ for 100min, or at 80 ℃ for 10min, etc.
In some embodiments, the thermal compounding process is performed in a solution system. Wherein, the dosage ratio of the quantum dots to the tumor cells in the compounding process is that the ratio of the mass of the quantum dots to the number of the tumor cells is 0.1mg:104-1010。
Further, in some embodiments, the surface of the quantum dot for modifying tumor cells has active sites, and the active sites include one or more of hydroxyl, carbonyl, carboxyl and amino. Generally, the larger the number of active sites on the surface of the quantum dot undergoing recombination, the more favorable the recombination reaction.
It should be noted that, in order to enable the quantum dots to be better combined with tumor cells, in some embodiments, the particle size of the quantum dots is less than 20 nm; the carbon quantum dots are preferably selected as the quantum dots, and the carbon quantum dots are further preferably spherical or cake-shaped, and the core structure is a nano particle obtained by a multi-layer graphene structure or a graphite structure.
Wherein, the quantum dots are prepared by using citric acid as a main raw material and carrying out solvothermal reaction. Centrifuging and purifying the primary product for many times, and packaging with lyophilized cover to obtain lyophilized powder for injection. Specifically, the quantum dot can be prepared by the following steps:
and (3) carrying out solvothermal preparation on the organic matter or amino acid containing hydroxyl or carboxyl and urea to obtain the carbon nanodot.
In a preferred embodiment, the carbon nanodots are prepared by solvothermal preparation of citric acid and urea. Optionally, the mass ratio of citric acid to urea is 1: 1-3, the reaction temperature for preparing the carbon nanodots is 120-220 ℃, and the reaction time is 4-6 hours.
Alternatively, the solvent selected for preparing the carbon nanodots is an organic solvent, and in some embodiments the organic solvent is dimethyl sulfoxide. Further, in some embodiments, the reaction solution obtained after the thermal reaction of the citric acid and the urea solvent is added into an alcohol solvent to precipitate the carbon nanodots, and then the solid-liquid separation is performed. Alternatively, the alcohol solvent added to prepare the carbon nanodots is ethanol.
The carbon nanodots prepared by the method are spherical or cake-shaped (flat structures), the diameter of the carbon nanodots is less than 20nm, and the inner core structure is a multilayer graphene structure. The carbon nanodot edge is rich in a large number of functional groups (hydroxyl, carboxyl and the like), and provides a good active site for further dehydration condensation later.
The tumor cells complexed with the quantum dots include, but are not limited to, malignant tumor cells derived from epithelial cells of any one of lung cancer cells, breast cancer cells, ovarian cancer cells, renal cancer cells, nasopharyngeal cancer cells, esophageal cancer cells, colorectal cancer cells, malignant glioma cells, and cerebellar medulloblastoma cells.
Specifically, in some embodiments, the tumor cells are obtained by:
obtaining a tumor cell suspension by centrifuging sheared tumor tissues obtained from a patient body and carrying out enzyme digestion with a culture solution;
mixing the tumor cell suspension with the inactivated human serum, standing, taking out bottom cells, and further performing centrifugal purification to obtain the tumor cells.
In some preferred embodiments, the tumor cell can be a 4T1 breast cancer cell.
In light of the foregoing, the antigen presenting cells co-incubated with the quantum dot-modified tumor cells include at least one of macrophage MP and dendritic cell DC. In some embodiments, immature APCs can be obtained by:
isolating Peripheral Blood Mononuclear Cells (PBMCs) from the patient's blood;
the obtained PBMC is placed in a cell culture device again, a culture medium is added, the density is adjusted, and adherent culture is carried out;
and (4) sucking and removing the supernatant for multiple times, adding the culture medium, and repeating the operation to obtain the mononuclear cells.
Addition of the corresponding cytokine induces differentiation of monocytes to MP or DC, respectively, to obtain immature APC (MP or DC).
Further, by adding the tumor cells thermally compounded with the carbon quantum dots, differentiated immature MP or DC is induced to become mature activated MP or DC, and the whole target Antigen Presenting Cell (APC) tumor vaccine is obtained after co-incubation under appropriate conditions.
In some embodiments, the co-incubation conditions are: the concentration of the quantum dot modified and compounded tumor cells is 0.001-100 mg/mL, the ratio of the quantum dot modified and compounded tumor cells to immature APC (adenosine monophosphate) is 1-10: 1-10, the incubation time is 0.01-10 days, the more preferable time is 1-7 days, and the incubation temperature is 20-40 ℃, and the preferable time is 25-37 ℃. For example, the concentration of the quantum dot modified and compounded tumor cells is 0.1mg/mL, the ratio of the quantum dot modified and compounded tumor cells to immature APC is 5:1, and the cells are incubated for 5 days at 30 ℃; or 1mg/mL, at a ratio of 8:3 to immature APC, and incubating at 37 deg.C for 7 days; or 10mg/mL, at a 1:1 ratio to immature APC, and incubating at 33 ℃ for 3 days; or 100mg/mL, at a 2:1 ratio to immature APC, incubated at 37 ℃ for 6 days, etc.
Specifically, the co-incubation process comprises the following steps: and (3) standing the tumor cells subjected to quantum dot modification and compounding and the obtained differentiated immature antigen presenting cells in a culture solution according to the concentration and the proportion for incubating for 3-7 days, wherein the cell culture dish can be placed in a biological safety cabinet or a cell culture box during the period, the mature state of the antigen presenting cells can be observed at any time, and the culture solution is supplemented or replaced.
In some embodiments, the tumor vaccine obtained after incubation is isolated, purified and harvested. Specifically, the purification process of the obtained whole target Antigen Presenting Cell (APC) tumor vaccine comprises the following steps: and (3) sucking and removing the supernatant, replacing the culture solution, repeatedly obtaining a full-target Antigen Presenting Cell (APC) tumor vaccine (the centrifugation speed is 600-1500 rpm and 2-15 mins), and detecting and recording the appearance, the number and the survival rate of cells so as to wait for clinical use.
In the process of incubating the quantum dot thermal-complexed tumor cells and the APCs, samples are collected and the antigen capture amount, the MHC expression amount and the factors (co-stimulatory molecules and the like) related to the activation process are detected. And samples were also collected and tested for safety (sterility, endotoxin, mycoplasma, etc.) and efficacy (vaccine purity and maturity).
Some embodiments of the present invention also provide a whole-target antigen presenting cell tumor vaccine, which is prepared by the preparation method.
Some embodiments of the invention also provide application of the whole-target antigen presenting cell tumor vaccine in preparing a medicament for treating or preventing tumors.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a preparation method of a whole-target antigen presenting cell tumor vaccine, which specifically comprises the following steps:
(1) and preparing the carbon quantum dots.
Dissolving 1g of citric acid and 3g of urea in 30mL of mixed solution of sodium chloride and sodium chloride to obtain a transparent solution, placing the transparent solution in a 50mL of polytetrafluoroethylene high-pressure reaction kettle, reacting for 4 hours at 160 ℃, adding 60mL of ethanol into the reacted solution to obtain a black solid, washing the black solid with water, centrifuging (8000rpm, 5min), and freeze-drying to obtain dark blue powder, namely the carbon nanodots.
(2) And thermally compounding the carbon quantum dots with the 4T1 breast cancer cells to obtain the quantum dot modified tumor cells.
Obtaining a tumor cell suspension by centrifuging sheared tumor tissues obtained from a mouse with 4T1 breast cancer and carrying out enzyme digestion and culture solution auxiliary; mixing the tumor cell suspension with inactivated fetal calf serum, standing, taking out bottom cells, and further performing centrifugal purification to obtain the tumor cells. The carbon quantum dots are further added at a ratio of 0.1mg:106The mixture is mixed with breast cancer cells in culture solution uniformly and thermally compounded for 20min at 60 ℃ to obtain quantum dot modified tumor cells.
(3) And co-incubating the quantum dot modified tumor cells and immature APC (APC) to obtain the whole-target antigen presenting cell tumor vaccine.
Isolating Peripheral Blood Mononuclear Cells (PBMC) from 50mL donor peripheral venous blood; the obtained PBMC is placed in a T-75 cell culture device again, a culture medium is added, the density is adjusted, and adherent culture is carried out; and (4) sucking and removing the supernatant and adding the culture medium for multiple times, and repeating the operation within several days to obtain the mononuclear cells. Adding cell factor to induce monocyte to differentiate to DC to obtain immature DC. The tumor cells after quantum dot modification and compounding are uniformly mixed with differentiated immature DCs in a culture solution according to the proportion of 3:1 at the concentration of 0.1mg/mL, and the mixture is placed in a T-75 cell culture bottle. The cell density can be adjusted to 5 × 106Per mL, 5% CO at 37 ℃2The incubator was allowed to stand for 3 hours. The supernatant was gently aspirated and the culture broth was replaced every two days. On day 6 of co-incubation culture, the DC cell status and purity were observed. And (3) absorbing and removing the supernatant, replacing the culture solution, repeatedly obtaining the whole target Antigen Presenting Cell (APC) tumor vaccine (the centrifugation speed is 1200rpm, 10mins), detecting and recording the appearance, the quantity and the survival rate of the cells, and obtaining the high-purity and high-survival-rate whole target antigen presenting cell tumor vaccine.
Example 2
The embodiment provides a preparation method of a whole-target antigen presenting cell tumor vaccine, which specifically comprises the following steps:
(1) and preparing the carbon quantum dots.
Dissolving 2g of citric acid and 5g of urea in 30mL of mixed solution of sodium chloride and sodium chloride to obtain a transparent solution, placing the transparent solution in a 50mL of polytetrafluoroethylene high-pressure reaction kettle, reacting for 6 hours at 180 ℃, adding 40mL of ethanol into the reacted solution to obtain a black solid, washing the black solid with water, centrifuging (5000rpm for 10min), and freeze-drying to obtain dark blue powder, namely the carbon nanodots.
(2) And thermally compounding the carbon quantum dots with the 4T1 breast cancer cells to obtain the quantum dot modified tumor cells.
Obtaining a tumor cell suspension by centrifuging sheared tumor tissues obtained from a mouse with 4T1 breast cancer and carrying out enzyme digestion and culture solution auxiliary; mixing the tumor cell suspension with inactivated fetal calf serum, standing, taking out bottom cells, and further performing centrifugal purification to obtain the tumor cells. The carbon quantum dots are further mixed at a ratio of 1mg:1010The mixture is mixed with breast cancer cells in culture solution uniformly and thermally compounded for 60min at 50 ℃ to obtain the quantum dot modified tumor cells.
(3) And co-incubating the quantum dot modified tumor cells and immature APC (APC) to obtain the whole-target antigen presenting cell tumor vaccine.
Isolating Peripheral Blood Mononuclear Cells (PBMC) from 50mL donor peripheral venous blood; the obtained PBMC is placed in a T-75 cell culture device again, a culture medium is added, the density is adjusted, and adherent culture is carried out; and (4) sucking and removing the supernatant and adding the culture medium for multiple times, and repeating the operation within several days to obtain the mononuclear cells. Adding cell factor to induce the mononuclear cell to differentiate to MP to obtain immature MP. The tumor cells modified and compounded by the quantum dots and the differentiated immature MP are uniformly mixed in a culture solution at a concentration of 1mg/mL in a ratio of 5:1, and the mixture is placed in a T-75 cell culture bottle. The cell density can be adjusted to 5 × 106Per mL, 5% CO at 37 ℃2The incubator was allowed to stand for 2 hours. The supernatant was gently aspirated and the culture broth was replaced every two days. On day 7 of co-incubation culture, the DC cell status and purity were observed. And (3) absorbing and removing the supernatant, replacing the culture solution, repeatedly operating to obtain the whole target Antigen Presenting Cell (APC) tumor vaccine (the centrifugation speed is 1000rpm, 5mins), detecting and recording the appearance, the quantity and the survival rate of the cells, and obtaining the high-purity and high-survival-rate whole target antigen presenting cell tumor vaccine.
Example 3
The embodiment provides a preparation method of a whole-target antigen presenting cell tumor vaccine, which specifically comprises the following steps:
(1) and preparing the carbon quantum dots.
Dissolving 1g of citric acid and 3g of urea in 30mL of mixed solution of sodium chloride and sodium chloride to obtain a transparent solution, placing the transparent solution in a 50mL of polytetrafluoroethylene high-pressure reaction kettle, reacting for 2 hours at 200 ℃, adding 50mL of ethanol into the reacted solution to obtain a black solid, washing the black solid with water, centrifuging (8000rpm, 20min), and freeze-drying to obtain dark blue powder, namely the carbon nanodots.
(2) And thermally compounding the carbon quantum dots with EMT6 breast cancer cells to obtain the quantum dot modified tumor cells.
Obtaining a tumor cell suspension by centrifuging sheared tumor tissues obtained from a mouse with EMT6 breast cancer and carrying out enzyme digestion and culture solution auxiliary; mixing the tumor cell suspension with inactivated fetal calf serum, standing, taking out bottom cells, and further performing centrifugal purification to obtain the tumor cells. The carbon quantum dots are further added at a ratio of 0.05mg:108The mixture is mixed with breast cancer cells in culture solution uniformly and thermally compounded for 30min at 70 ℃ to obtain quantum dot modified tumor cells.
(3) And co-incubating the quantum dot modified tumor cells and immature APC (APC) to obtain the whole-target antigen presenting cell tumor vaccine.
Isolating Peripheral Blood Mononuclear Cells (PBMC) from 50mL donor peripheral venous blood; the obtained PBMC is placed in a T-75 cell culture device again, a culture medium is added, the density is adjusted, and adherent culture is carried out; and (4) sucking and removing the supernatant and adding the culture medium for multiple times, and repeating the operation within several days to obtain the mononuclear cells. Adding cell factor to induce monocyte to differentiate to DC to obtain immature DC. The tumor cells modified and compounded by the quantum dots and the differentiated immature MP are uniformly mixed in a culture solution at a concentration of 5mg/mL and a ratio of 5:2, and the mixture is placed in a T-75 cell culture bottle. The cell density can be adjusted to 5 × 106Per mL, 5% CO at 37 ℃2The incubator was allowed to stand for 2 hours. The supernatant was gently aspirated and the culture broth was replaced every two days. On day 5 of co-incubation culture, the DC cell status and purity were observed. Absorbing and removing supernatant, replacing culture solution, repeatedly harvesting whole target Antigen Presenting Cell (APC) tumor vaccine (centrifugation rate is 1500rpm, 5mins), detecting and recording cell appearance, quantity and survival rate, and obtaining high-purity and high-survival rateA full-target antigen presenting cell tumor vaccine.
Comparative example 1
This comparative example the pure carbon quantum dot material prepared in example 1 was co-incubated with the immature APC in example 1 under the same co-incubation conditions as in example 1.
Comparative example 2
This comparative example co-incubates the heat-treated only tumor cells prepared in example 1 with the immature APC of example 1 under the same co-incubation conditions as in example 1, wherein the heat-treatment of the heat-treated only tumor cells is performed by treating the tumor cells of example 1 at a thermal recombination temperature for the same time.
Comparing the activation state of APC activation during the co-incubation of comparative examples 1-2 and example 1, the result is shown in FIG. 1, and the left panel shows that APC in comparative example 1 was incubated with pure quantum dot material, and no APC maturation activation reaction was observed. The middle panel shows that the APC in comparative example 2 was incubated with tumor cells (4T1 breast cancer cells) that were heat-treated only, and no significant APC maturation activation reaction was observed. The right panel shows that the APC in example 1 is incubated with quantum dot heat-complexed tumor cells (4T1 breast cancer cells), and a significant APC maturation activation reaction can be seen.
Further comparing the expression state of MHC molecules during the co-incubation of comparative examples 1-2 and example 1, as shown in FIG. 2, the left column shows that APC in comparative example 1 was co-incubated with pure quantum dot material, and no high expression of MHC was observed. In the middle panel, the APCs of comparative example 2 were incubated with tumor cells that were heat-treated only (4T1 breast cancer cells), and no significant upregulation of MHC expression was seen. The right column shows that the APC in example 1 is incubated with quantum dot heat-complexed tumor cells (4T1 breast cancer cells), and obvious MHC up-regulation and high expression are observed, which marks successful maturation and activation of the APC.
Test examples
The APC obtained after co-incubation of the APC in comparative examples 1-2 and examples 1-3 of the present invention with pure quantum dot material, heat-treated tumor cells and quantum dot heat-complexed tumor cells was subcutaneously injected into mice with 4T1 breast cancer, and the tumor inhibition of the mice is shown in FIG. 3. All animal experiments were performed on animals as provided by the animal Care and use Committee of the university of AustraliaThe study was conducted as a guideline. The subcutaneous injection amount of the APC obtained after the co-incubation of the APC and the pure quantum dot material, the heat-treated tumor cell and the quantum dot heat-compounded tumor cell is 5 multiplied by 106500 μ L. During operation, the skin at the injection site is disinfected conventionally and then lifted, the injection needle penetrates into the skin at an obtuse angle, and the needle slightly swings left and right. If the swing is easy, the subcutaneous injection is punctured, then the suction is performed gently, and the drug can be injected into the subcutaneous injection slowly without blood return. The tumor volume status of the treated group and the control group is continuously observed within 30 days, and it is obvious that the tumor volume increase can not be obviously controlled after the APC and the pure quantum dot material are incubated together and the APC and the tumor cell which is only heat treated (4T1 breast cancer cell) are injected together. And after the APC and the quantum dot thermal-compounded tumor cells (4T1 breast cancer cells) are incubated and injected, obvious tumor volume inhibition can be seen.
In summary, in the embodiments of the present invention, the co-incubated MP or DC is converted from an immature state to a super-activated state by adding the carbon quantum dot thermal-complexed tumor cell, so that the uptake of the tumor antigen by the co-incubated MP or DC is greatly enhanced, the expression of MHC molecules is up-regulated on a large scale, and the antigen presentation efficiency is improved. The tumor cells thermally compounded by the carbon quantum dots can enhance the immunogenicity of tumor antigens by a whole target spot, promote activated and mature MP and DC to migrate to lymph nodes, and effectively induce organisms to generate strong whole-body anti-tumor immune response reaction. The vaccine prepared by the invention is mainly applied to the tumor immunotherapy of individual full-target cancer patients or the prevention of cancer high risk groups, and has simple preparation process, low cost and excellent application prospect.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by 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.

Claims (10)

1. A method for preparing a whole-target antigen presenting cell tumor vaccine, which is characterized by comprising the following steps: and co-incubating the quantum dot modified tumor cells and immature antigen presenting cells, wherein the quantum dot modified tumor cells are obtained by connecting the quantum dots and protein molecular chains of the tumor cells in a ligand connection mode through hydrogen bonds.
2. The preparation method of claim 1, wherein the surface of the quantum dot has active sites, and the active sites comprise one or more of hydroxyl, carbonyl, carboxyl and amino;
preferably, the particle size of the quantum dots is less than 80nm, more preferably less than 20 nm;
preferably, the quantum dots are carbon quantum dots, further preferably spherical or cake-shaped, and the core structure is a multilayer graphene structure.
3. The method according to claim 1, wherein the tumor cell is selected from malignant tumor cells derived from epithelial cells of any one of lung cancer cells, breast cancer cells, ovarian cancer cells, kidney cancer cells, nasopharyngeal cancer cells, esophageal cancer cells, colorectal cancer cells, malignant glioma cells, and cerebellar medulloblastoma cells;
preferably, the tumor cells are obtained by:
obtaining a tumor cell suspension by centrifuging sheared tumor tissues obtained from a patient body and carrying out enzyme digestion with a culture solution;
mixing the tumor cell suspension with the inactivated human serum, standing, taking out bottom cells, and further performing centrifugal purification to obtain the tumor cells;
preferably, the tumor cell is a 4T1 breast cancer cell.
4. The preparation method according to any one of claims 1 to 3, wherein the quantum dot modified tumor cells are prepared by thermal recombination;
the conditions of the quantum dots and the tumor cells are as follows: the temperature is 30-100 ℃, preferably 50-90 ℃, more preferably 50-80 ℃, and the compounding time is 1-360 min, preferably 1-100 min, more preferably 2-30 min;
preferably, the thermal compounding process is carried out in a solution system;
preferably, the ratio of the mass of the quantum dots to the number of the tumor cells is 0.1mg:104-1010。
5. The method of any one of claims 1 to 3, wherein the antigen-presenting cells comprise at least one of macrophage MP and dendritic cell DC.
6. The method according to any one of claims 1 to 3, wherein the co-incubation is carried out under the following conditions: the concentration of the quantum dot modified and compounded tumor cells is 103-108The ratio of the cells/mL to the immature APC is 1-10: 1-10, the incubation time is 0.01-10 days, more preferably 1-7 days, and the incubation temperature is 20-40 ℃, preferably 25-37 ℃.
7. The method of claim 6, wherein the co-incubation process comprises the steps of: carrying out co-incubation on the tumor cells subjected to quantum dot modification and compounding and the obtained immature antigen presenting cells in a culture solution, and observing and supplementing or replacing the culture solution at any time by a cell culture dish which can be placed in a biological safety cabinet or a cell culture box;
preferably, the tumor vaccine obtained after incubation is isolated, purified and harvested.
8. The method for preparing an antigen-presenting cell according to claim 7, wherein the preparation of an immature antigen-presenting cell comprises the steps of: monocytes are obtained from peripheral blood mononuclear cells isolated from a patient and induced to differentiate into immature antigen presenting cells, respectively.
9. A whole-target antigen presenting cell tumor vaccine, which is prepared by the preparation method of any one of claims 1 to 8.
10. Use of the whole-target antigen presenting cell tumor vaccine of claim 9 in the preparation of a medicament for treating or preventing a tumor.
CN202110731947.XA 2021-06-30 2021-06-30 Full-target antigen presenting cell tumor vaccine and preparation method and application thereof Pending CN113440607A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023274297A1 (en) * 2021-06-30 2023-01-05 澳门大学 Quantum dot-enhanced tumor inactivated vaccine, preparation method, and application
WO2023274299A1 (en) * 2021-06-30 2023-01-05 澳门大学 Full-target antigen-presenting cell tumor vaccine, and preparation method therefor and application thereof
WO2023274298A1 (en) * 2021-06-30 2023-01-05 澳门大学 Quantum dot modified protein vaccine and preparation method therefor and application thereof

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130195963A1 (en) * 2011-12-07 2013-08-01 The Methodist Hospital Research Institute Mesoporous silicon particles for the presentation of tumor antigens and adjuvant for anti-cancer immunity
CN105079806A (en) * 2014-05-13 2015-11-25 中国科学院深圳先进技术研究院 Carbon nanomaterial directly modified by polypeptide as well as preparation method and application thereof
CN105219383A (en) * 2014-07-02 2016-01-06 中国人民解放军军事医学科学院毒物药物研究所 Oligonucleotide functional fluorescence nano-sized carbon point, Preparation Method And The Use
CN105502341A (en) * 2015-12-30 2016-04-20 中国科学院长春光学精密机械与物理研究所 Carbon nanodots with effective wide absorption and preparation method thereof
US20200163899A1 (en) * 2016-03-08 2020-05-28 Children's National Medical Center Functionalized prussian blue nanoparticles, combination prussian blue nanoparticle-based nano-immunotherapy and applications thereof
CN111375054A (en) * 2018-12-27 2020-07-07 复旦大学附属肿瘤医院 Tumor vaccine composition, and preparation method and application thereof
CN111606319A (en) * 2020-06-18 2020-09-01 澳门大学 Carbon nano coil, preparation method and application thereof, and carbon nano belt

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113797356A (en) * 2020-06-17 2021-12-17 广东量子墨滴生物科技有限公司 Carbon nano composite biological preparation, preparation method and application
CN113440607A (en) * 2021-06-30 2021-09-28 澳门大学 Full-target antigen presenting cell tumor vaccine and preparation method and application thereof

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130195963A1 (en) * 2011-12-07 2013-08-01 The Methodist Hospital Research Institute Mesoporous silicon particles for the presentation of tumor antigens and adjuvant for anti-cancer immunity
CN105079806A (en) * 2014-05-13 2015-11-25 中国科学院深圳先进技术研究院 Carbon nanomaterial directly modified by polypeptide as well as preparation method and application thereof
CN105219383A (en) * 2014-07-02 2016-01-06 中国人民解放军军事医学科学院毒物药物研究所 Oligonucleotide functional fluorescence nano-sized carbon point, Preparation Method And The Use
CN105502341A (en) * 2015-12-30 2016-04-20 中国科学院长春光学精密机械与物理研究所 Carbon nanodots with effective wide absorption and preparation method thereof
US20200163899A1 (en) * 2016-03-08 2020-05-28 Children's National Medical Center Functionalized prussian blue nanoparticles, combination prussian blue nanoparticle-based nano-immunotherapy and applications thereof
CN111375054A (en) * 2018-12-27 2020-07-07 复旦大学附属肿瘤医院 Tumor vaccine composition, and preparation method and application thereof
CN111606319A (en) * 2020-06-18 2020-09-01 澳门大学 Carbon nano coil, preparation method and application thereof, and carbon nano belt

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ZIKANG TANG等: "Cancer Immunotherapy Based on Carbon-Quantum- Dot Modified Cancer Cells" *
王薇等: "碳纳米管增强树突细胞对肿瘤抗原的吞噬功能" *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023274297A1 (en) * 2021-06-30 2023-01-05 澳门大学 Quantum dot-enhanced tumor inactivated vaccine, preparation method, and application
WO2023274299A1 (en) * 2021-06-30 2023-01-05 澳门大学 Full-target antigen-presenting cell tumor vaccine, and preparation method therefor and application thereof
WO2023274298A1 (en) * 2021-06-30 2023-01-05 澳门大学 Quantum dot modified protein vaccine and preparation method therefor and application thereof

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