CN113413463B - Preparation method and application of nano-cell-like personalized tumor vaccine - Google Patents

Preparation method and application of nano-cell-like personalized tumor vaccine Download PDF

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CN113413463B
CN113413463B CN202110705859.2A CN202110705859A CN113413463B CN 113413463 B CN113413463 B CN 113413463B CN 202110705859 A CN202110705859 A CN 202110705859A CN 113413463 B CN113413463 B CN 113413463B
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任雪玲
马超群
马梦雅
郭汝悦
张雪玲
张慧娟
张振中
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Zhengzhou University
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Abstract

The invention relates to a preparation method and application of a novel nano quasi-cell personalized tumor vaccine, which can effectively solve the problems of personalized application, immunogenicity and antigen delivery of the tumor vaccine and adopts the technical scheme that 12-aminododecanoic acid is grafted on hyaluronic acid at first and then the other end of a fatty chain is modifiedLCysteine and polyethyleneimine are prepared into a nano carrier, tumor cells are prepared into nano-scale tumor cell membrane-wrapped holoantigen vesicles by using omentum filtration, and a mucous membrane immune adjuvant holotrans-retinoic acid is loaded in a hydrophobic layer formed by a fatty chain when the tumor cells and the carrier are subjected to electrostatic compression to prepare the nano cell-mimetic personalized tumor vaccine with a 'core-shell' structure.

Description

Preparation method and application of nano-cell-like personalized tumor vaccine
Technical Field
The invention relates to the field of medicines, in particular to a preparation method and application of a nano cell-like personalized tumor vaccine.
Background
Tumors are always threatening the life safety of human beings as a worldwide public health problem. The tumor vaccine is a promising treatment scheme by activating the immune system of a patient to spontaneously kill and monitor the tumor for a long time. However, due to the strong specificity of tumors in their development, the universal therapeutic approach is limited by the antigen restriction of human leukocytes and the expression difference between tumor-specific and non-specific antigens, and it is often difficult to achieve satisfactory therapeutic effects(s) (reminars in Immunology, 2010, 22: 132). Therefore, an ideal tumor vaccine should provide personalized treatment strategies for different patients, and in order to prepare an effective personalized vaccine, how to select appropriate antigens and adjuvants to achieve both broad specific and non-specific immunity, and achieving efficient antigen delivery is the biggest problem.
Autologous tumor cells can be prepared into nanoscale full-antigen vesicles (tumor-like cells ITCs) coated by tumor cell membranes by a omentum filtration technology, so that the antigen vesicles cover wide T cell epitopes and have personalized characteristics, and sufficient specific anti-tumor immunity can be activated. Chemotactic cytokine receptor 9 (CCR 9) is an intestinal tract-specific homing receptor that specifically recognizes chemokine ligand 25 targeting small intestinal epithelial cells, which supports the body's gastrointestinal immunity, and is usually expressed on gastrointestinal Dendritic Cells (DCs) and confined to the mesenteric lymph nodes or Peyer's node of the gastrointestinal tract, and recent studies have found that the secretion of gastrointestinal DCs, all-trans Retinoic Acid (RA), is a receptor signaling-mediated in vitro CCR9 marker for immune cells, and thus, exogenous application of RA outside the intestinal tract may increase the distribution of immune cells in the gastrointestinal tract and thus promote systemic immunity (Advanced Materials, 2018, 30: 1801067), Hyaluronic Acid (HA) is a natural linear high-molecular polysaccharide, widely distributed in the human body, and according to previous studies, HA receptor CD44 is highly expressed on the DC surface, and plays an important role as a costimulatory factor for T cell activation (Journal of Immunotherapy, 2004, 27: 1), and the construction of the nano-vaccine based on HA is expected to promote internalization of antigen in DC and synergistically promote DC maturation, and activate CD8+The vaccine prepared based on the T cell immune response can deliver the personalized whole tumor antigen to dendritic cells in a synergistic and targeted manner in cooperation with a mucosal immune adjuvant, promote the maturation and gastrointestinal distribution of the dendritic cells, and activate specific and nonspecific immunity of an organism, so that long-term anti-tumor immunity is achieved, but similar published reports are not found so far.
Disclosure of Invention
In view of the above situation, in order to solve the defects of the prior art, the present invention aims to provide a preparation method and an application of a nano cell-like personalized tumor vaccine, which can effectively solve the problems of personalized application, immunogenicity and antigen delivery of the tumor vaccine.
The technical scheme for solving the problem is that the preparation method of the nano cell-like personalized tumor vaccine comprises the steps of grafting 12-aminododecanoic acid on hyaluronic acid, and then modifying the other end of a fatty chainLCysteine and polyethyleneimine are prepared into a nano carrier, tumor cells are prepared into a nano-scale tumor cell membrane-wrapped holoantigen vesicle by using omentum filtration, a mucosal immune adjuvant holotrans-retinoic acid is loaded in a hydrophobic layer formed by a fatty chain when the tumor cells and the carrier are compressed by static electricity, so that the nano cell-like personalized tumor vaccine with a 'core-shell' structure is prepared, a hyaluronic acid shell is targeted and positioned on dendritic cells, and the personalized tumor antigen and the mucosal adjuvant are efficiently delivered to form antitumor immunity.
The preparation method specifically comprises the following steps:
1) preparing hyaluronic acid grafted carbon twelve chains: weighing 0.1500-0.5000g of HA (monobasic weak acid) and dissolving in 15-40mL of formamide, adding 0.0690-0.2890g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 0.0420-0.1690g of N-hydroxysuccinimide (NHS), stirring and reacting at room temperature (23 +/-2 ℃) for 4-6h under the protection of nitrogen and in dark place, adding 0.1800-0.5970g of 12-aminododecanoic acid, stirring and reacting at room temperature and in dark place for 48h, transferring the reaction solution to 8 multiplied by 10 with molecular weight cutoff3Da-1.4×104Dialyzing in Da dialysis bag for 2-3d, and freeze drying to obtain hyaluronic acid (HA-C) grafted with carbon twelve chains12）；
2) Preparing hyaluronic acid carbon-dodecyl chain branch cysteine: 0.1510-0.5000g are weighedL-Cys (L-cysteine) is dissolved in 10-30mL of ultrapure water, pH is adjusted to 5, 0.1510-0.5892g EDC and 0.1400-0.3017g NHS are added, activation is carried out for 1-2h, 0.2500-0.8000g HA-C are added12，N2Performing protection reaction for 5-6h, dialyzing with deionized water for 2-3d, and freeze-drying to obtain hyaluronic acid (HA-C-Cys) grafted with carbon dodecachains and cysteine;
3) preparing a nano carrier: weighing 0.1500-0.5000g of HA-C-Cys, dissolving the HA-C-Cys in 10-30mL of deionized water and 5-10mL of boric acid buffer solution with pH of 9.18, weighing 0.5000-1.7640g of PEI, dissolving the PEI in 400 mu L of 3.7-4.0% hydrochloric acid, slowly dropwise adding the PEI into the solution containing the HA-C-Cys, stirring and reacting for 36-48h under the conditions of room temperature, nitrogen protection and light protection, and transferring the reaction solution to a solution with the molecular weight cut-off of 8 multiplied by 103Da-1.4×104Dialyzing in a dialysis bag of Da, dialyzing with deionized water for 2-3d, and freeze drying to obtain nano carrier (HTCP);
4) preparing a nano tumor cell antigen: 3-15mL of a solution with a density of 1.0X 107Irradiating the/mL tumor cell PBS suspension for 1-2h under an ultraviolet lamp, then passing the suspension through a 800nm polyethersulfone membrane for 2-3 times, and then passing through a 200nm polyethersulfone membrane for 3-4 times to obtain a pseudo-tumor cell antigen;
5) preparing a nano cell-like vaccine: weighing 0.1-1.0mg HTCP and dissolving in 0.5-1.0mL PBS, adding 2.5-12.5mL pseudo-tumor cell suspension, simultaneously dropwise adding 10.5-52.5 mu g RA solution, centrifuging for 15-20min at 10000-.
The nano cell-like personalized tumor vaccine prepared by the method is effectively used for preparing various personalized solid tumor vaccines, and the application of the nano cell-like personalized tumor vaccine in resisting cancers is realized.
The tumor cells are prepared into the nano-scale tumor-like cells by the omentum filtering technology, the preparation method is simple, the raw materials are wide, the specific immunity and the non-specific immunity can be stimulated, the effect of the tumor vaccine is greatly improved, and the preparation method is an innovation on personalized tumor vaccines.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
Example 1
In the specific implementation, the invention specifically comprises the following steps:
1) preparing hyaluronic acid grafted carbon twelve chains: HA 0.1500g was weighed out and dissolved in 15mL formamide, 0.0690g of 1- (3-dimethyl) formamide was addedAminopropyl) -3-ethyl carbodiimide hydrochloride (EDC) and 0.0420g of N-hydroxysuccinimide (NHS) are stirred and reacted for 4 hours at room temperature in a nitrogen protection and in a dark place, 0.1800g of 12-aminododecanoic acid is added, stirred and reacted for 48 hours at room temperature in a dark place, and the reaction solution is transferred to a reaction solution with the molecular weight cutoff of 8 multiplied by 103Da-1.4×104Dialyzing in Da dialysis bag for 2d, and lyophilizing to obtain hyaluronic acid (HA-C) grafted with carbon twelve chains12）；
2) Preparing hyaluronic acid carbon-dodecyl chain branch cysteine: weighing 0.1510gLCys was dissolved in 10mL of ultrapure water, pH was adjusted to 5, 0.1510g EDC and 0.1400g NHS were added, activation was performed for 1h, and 0.2500g HA-C was added12，N2Carrying out protection reaction for 5h, dialyzing with deionized water for 2d, and freeze-drying to obtain hyaluronic acid (HA-C-Cys) grafted with carbon dodecachains and cysteine;
3) preparing a nano carrier: weighing 0.1500g of HA-C-Cys, dissolving the HA-C-Cys in 10mL of deionized water and 5mL of boric acid buffer solution with pH of 9.18, weighing 0.5000g of PEI, dissolving the PEI in 400 mu L of 3.7% hydrochloric acid, slowly and dropwise adding the PEI into the HA-C-Cys-containing solution, stirring and reacting for 48 hours at room temperature under the conditions of nitrogen protection and light protection, and transferring the reaction solution to a solution with the molecular weight cutoff of 8 multiplied by 103Da-1.4×104Dialyzing in a dialysis bag of Da, dialyzing for 2d with deionized water, and freeze-drying to obtain a nano carrier (HTCP);
4) preparing a nano tumor cell antigen: 3mL of a solution having a density of 1.0X 107Irradiating the PBS suspension of the 4T1 mouse breast cancer tumor cells for 1h under an ultraviolet lamp, and then, firstly passing the PBS suspension through a 800nm polyethersulfone membrane for 3 times, and then passing the PBS suspension through a 200nm polyethersulfone membrane for 3 times to obtain a pseudo-tumor cell antigen;
5) preparing a nano cell-like vaccine: 0.2mg of HTCP is weighed and dissolved in 500. mu.L of PBS, 2.5mL of pseudo-tumor cell suspension is added, 10.5. mu.g of RA solution is added dropwise at the same time, centrifugation is carried out for 15min at 12000rpm, and the precipitate is collected to obtain the nano pseudo-cell vaccine (HTCP/ITCs/RA).
Example 2
In the specific implementation, the invention specifically comprises the following steps:
1) preparing hyaluronic acid grafted carbon twelve chains: weighing HA 03000g of formamide dissolved in 30mL of water is added with 0.1440g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 0.0910g of N-hydroxysuccinimide (NHS), the mixture is stirred at room temperature and under nitrogen protection and light protection for 5h of reaction, 0.3600g of 12-aminododecanoic acid is added, the mixture is stirred at room temperature and light protection for 48h of reaction, and the reaction solution is transferred to the solution with the molecular weight cutoff of 8 multiplied by 103Da-1.4×104Dialyzing in Da dialysis bag for 2d, and freeze drying to obtain hyaluronic acid (HA-C) grafted with carbon twelve chains12）；
2) Preparing hyaluronic acid carbon-dodecyl chain branch cysteine: weighing 0.3011gLCys in 30mL ultrapure water, pH5 adjusted, 0.3020g EDC and 0.2807g NHS added, activated for 1h, 0.5000g HA-C added12，N2Carrying out protection reaction for 6h, dialyzing with deionized water for 2d, and freeze-drying to obtain hyaluronic acid (HA-C-Cys) grafted with carbon dodecachains and cysteine;
3) preparing a nano carrier: weighing 0.3000g of HA-C-Cys, dissolving in 10mL of deionized water and 5mL of boric acid buffer solution with pH of 9.18, weighing 1.1028g of PEI, dissolving in 400 mu L of 4.0% hydrochloric acid, slowly dropwise adding the solution containing the HA-C-Cys, stirring and reacting for 48 hours at room temperature under the protection of nitrogen and in the absence of light, and transferring the reaction solution to a solution with molecular weight cut-off of 8 multiplied by 103Da-1.4×104Dialyzing in a dialysis bag of Da, dialyzing for 3d with deionized water, and freeze-drying to obtain a nano carrier (HTCP);
4) preparing a nano tumor cell antigen: 3mL of a solution having a density of 1.0X 107Irradiating MCF-7 humanized breast cancer tumor cell PBS suspension for 1.5h under an ultraviolet lamp, and then, firstly passing through a 800nm polyethersulfone membrane for 3 times, and then passing through a 200nm polyethersulfone membrane for 3 times to obtain a pseudo-tumor cell antigen;
5) preparing a nano cell-like vaccine: weighing 0.2mg HTCP, dissolving in 500 μ L PBS, adding 2.5mL pseudo-tumor cell suspension, simultaneously adding 10.5 μ g RA solution dropwise, centrifuging at 10000rpm for 20min, and collecting precipitate to obtain nano pseudo-cell vaccine (HTCP/ITCs/RA).
Example 3
In the specific implementation, the invention specifically comprises the following steps:
1) preparing hyaluronic acid grafted carbon twelve chains: weighing 0.2000g of HA, dissolving in 30mL of formamide, adding 0.0997g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 0.0670g of N-hydroxysuccinimide (NHS), stirring at room temperature and under the protection of nitrogen and in the dark for reaction for 4h, adding 0.1800g of 12-aminododecanoic acid, stirring at room temperature and in the dark for reaction for 48h, transferring the reaction solution to 8 x 10 molecular weight cut-off3Da-1.4×104Dialyzing in Da dialysis bag for 2d, and freeze drying to obtain hyaluronic acid (HA-C) grafted with carbon twelve chains12）；
2) Preparing hyaluronic acid carbon-dodecyl chain branch cysteine: weighing 0.2000gLCys was dissolved in 10mL of ultrapure water, pH was adjusted to 5, 0.2000g EDC and 0.1900g NHS were added, activation was performed for 1h, 0.2500g HA-C was added12，N2Carrying out protection reaction for 6h, dialyzing with deionized water for 2d, and freeze-drying to obtain hyaluronic acid (HA-C-Cys) grafted with carbon dodecachains and cysteine;
3) preparing a nano carrier: weighing 0.1510g of HA-C-Cys, dissolving in 10mL of deionized water and 5mL of pH9.18 boric acid buffer solution, weighing 0.4000g of PEI, dissolving in 400 mu L of 3.7% hydrochloric acid, slowly and dropwise adding the solution containing the HA-C-Cys, stirring and reacting for 36h at room temperature under the protection of nitrogen and in the absence of light, and transferring the reaction solution to 8 × 10 with the molecular weight cut-off3Da-1.4×104Dialyzing in a dialysis bag of Da, dialyzing for 2d with deionized water, and freeze-drying to obtain a nano carrier (HTCP);
4) preparing a nano tumor cell antigen: 2mL of a solution having a density of 1.0X 107Irradiating the PBS suspension of the 4T1 mouse breast cancer tumor cells for 1h under an ultraviolet lamp, and then passing the PBS suspension through a membrane for 2 times by using a polyether sulfone membrane with the wavelength of 800nm, and then passing through a membrane for 4 times by using a polyether sulfone membrane with the wavelength of 200nm to obtain a pseudo-tumor cell antigen;
5) preparing a nano cell-like vaccine: 0.1mg of HTCP is weighed and dissolved in 500. mu.L of PBS, 1.25mL of pseudo-tumor cell suspension is added, 5.3. mu.g of RA solution is added dropwise at the same time, centrifugation is carried out for 15min at 12000rpm, and the precipitate is collected to obtain the nano pseudo-cell vaccine (HTCP/ITCs/RA).
Example 4
In specific implementation, the method specifically comprises the following steps:
1) grafting carbon twelve chains on hyaluronic acid: weighing 0.5000g of HA, dissolving in 40mL of formamide, adding 0.2890g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 0.1690g of N-hydroxysuccinimide (NHS), stirring at room temperature and under nitrogen protection and in the dark for reaction for 6h, adding 0.5970g of 12-aminododecanoic acid, stirring at room temperature and in the dark for reaction for 48h, transferring the reaction solution to a solvent with the molecular weight cutoff of 8 multiplied by 103Da-1.4×104Dialyzing in Da dialysis bag, dialyzing for 3d, and freeze drying to obtain hyaluronic acid (HA-C) grafted with carbon twelve chains12）；
2) Hyaluronic acid carbon twelve-chain branch cysteine: weighing 0.5000gLCys in 30mL ultrapure water, pH5 adjusted, 0.5892g EDC and 0.3017g NHS added, activated for 2h, 0.8000g HA-C added12，N2And (4) carrying out protection reaction for 6 hours, dialyzing for 3 days by using deionized water, and carrying out freeze drying to obtain hyaluronic acid (HA-C-Cys) grafted with carbon dodecyl chains and cysteine.
3) Preparing a nano carrier: weighing 0.5000g of HA-C-Cys, dissolving in 30mL of deionized water and 10mL of boric acid buffer solution with pH of 9.18, weighing 1.7640g of PEI, dissolving in 600 mu L of 4.0% hydrochloric acid, slowly dropwise adding the solution containing the HA-C-Cys, stirring and reacting for 48 hours at room temperature under the protection of nitrogen and in the absence of light, and transferring the reaction solution to a solution with the molecular weight cut-off of 8 multiplied by 103Da-1.4×104Dialyzing in a dialysis bag of Da, dialyzing for 3d with deionized water, and freeze-drying to obtain a nano carrier (HTCP);
4) preparing a nano tumor cell antigen: 15mL of a solution having a density of 1.0X 107Irradiating MCF-7 humanized breast cancer tumor cell PBS suspension for 2h under an ultraviolet lamp, and then, firstly passing through a 800nm polyethersulfone membrane for 2 times, and then passing through a 200nm polyethersulfone membrane for 4 times to obtain a pseudo-tumor cell antigen;
5) preparing a nano cell-like vaccine: weighing 1.0mg HTCP and dissolving in 1mL PBS, adding 12.5mL pseudo-tumor cell suspension, simultaneously adding 52.5 μ g RA solution dropwise, centrifuging at 12000rpm for 20min, and collecting precipitate to obtain nano pseudo-cell vaccine (HTCP/ITCs/RA).
The invention adopts energyHyaluronic acid capable of binding with CD44 receptor highly expressed on the surface of dendritic cells is used as a material for targeting dendritic cells, and carbon dodecalong chain is grafted on the hyaluronic acid,LPreparing a nano carrier from cysteine and polyethyleneimine; tumor cells are prepared into nano-scale tumor-like cells by a net membrane filtration method to be used as personalized antigens for activating specific immunity, the antigens are compressed into inner cores by utilizing electrostatic action, hyaluronic acid is wrapped under the drive of a fatty chain to form a shell, and a hydrophobic layer formed by the fatty chain is used for loading mucosa immune adjuvant all-trans retinoic acid to obtain a nano-cell-like vaccine; the particle size of the prepared nano vaccine is between 200 and 300 nm; the vaccine has the following characteristics: 1) the hyaluronic acid shell of the vaccine has a dendritic cell targeting effect, so that the antigen delivery efficiency is improved; 2) the vaccine simultaneously and efficiently loads personalized antigens and mucosal immune adjuvants to stimulate specific and non-specific immunity of organisms; 3) the prepared vaccine has biocompatibility and wide application.
The hyaluronic acid used in the invention is a human endogenous polysaccharide, has good biological safety, has dendritic cell targeting and activating effects, is a good vaccine preparation material, the prepared pseudotumor cell antigen can be well compressed and loaded by a carrier, the preparation is simple and convenient, the preparation comprises all personalized tumor antigen epitopes, the all-trans retinoic acid can promote gastrointestinal tract migration of dendritic cells and enhance the anti-tumor immunity of the vaccine, and the prepared nano pseudocell vaccine delivers the personalized antigen and adjuvant after reaching the dendritic cells in a targeting manner and stimulates the dendritic cells to mature, so that effective anti-tumor immunity is achieved, and relevant experimental data are as follows (taking example 1 as an example):
experiment 1: in vitro antigen delivery assay
Bone marrow-derived dendritic cells (BMDCs) are extracted and differentiated from mouse bone marrow, the capacity of taking up HTCP/ITCs/RA by the BMDCs is examined, only 26.8% of independent ITCs are taken up by the BMDCs after 24 hours of treatment, and the taking efficiency reaches 59.3% after HTCP entrapment, which shows that CD44 on the surface of a vaccine system can be combined with receptors on the DCs to play the roles of targeting and increasing the taking.
Experiment 2: in vitro dendritic cell maturation assay
The effect of HTCP/ITCs/RA on the curing of BMDCs is investigated through antibody fluorescence labeling flow, the surface CD86 expression level of the BMDCs treated by the vaccine system is obviously increased to 54.7 percent, even higher than that of a positive control group treated by lipopolysaccharide, and the independent ITCs and the carrier control HTCP/Lipo have certain effect but are obviously lower than that of the vaccine system group, and the result shows that the vaccine delivery system HTCP/ITCs/RA can promote the curing of the BMDCs.
Experiment 3: mouse tumor model
30 female BALB/c mice of 4-6 weeks old were randomly divided into 6 groups, namely, a normal saline group NC, a carrier group HTCP/Lipo, a pseudo-tumor cell group ITCs, a carrier adjuvant group HTCP/Lipo/RA, a pseudo-tumor cell delivery group HTCP/ITCs and a final vaccine group HTCP/ITCs/RA, and subcutaneous vaccination (W) was performed at the right groin of the mice (W)pITCs=100 μ g/cup, WHTCP: WpITCs=1:5，WRA=0.42 μ g/lane), 3 inoculations were made every 7 days. 3 days after the last inoculation, the 1X 10 growth phase of mouse breast cancer 4T1 cells6One/one mouse was subcutaneously transplanted to the left groin of the mouse. The length (L, mm)) and width (W, mm) of the mouse subcutaneous tumor were measured every 2 days during tumor monitoring, and the tumor volume (V =1/2 × L × W) was calculated (V =1/2 × L × W)2). After 14 days of tumor monitoring, the mice were decapped, tumor tissues were dissected, weighed, and tumor inhibition rates were calculated. The tumor tissue was deparaffinized, stained, dehydrated, cleared, mounted and histopathologically analyzed. The lungs of mice were dissected and stained by Bouin's stain to count the number of lung metastases.
The results show that the tumor volume of mice in the nano-vaccine HTCP/ITCs/RA group is always lower than that of the control group, and the tumor volume difference is obvious in the last day, while the HTCP/ITCs group also shows obvious tumor volume reduction but is obviously higher than that of the vaccine system group, which indicates that gastrointestinal tract immune activation caused by RA plays a role in tumor inhibition, while the HTCP/Lipo/RA group probably does not show obvious tumor inhibition due to lack of effective antigen, and the tumor inhibition rates of the HTCP/ITCs and the HTCP/ITCs/RA group reach 66.81% and 80.11% respectively, thus showing strong tumor inhibition; the number of lung metastases of the HTCP/ITCs/RA group is obviously lower than that of other groups, which indicates that the systemic anti-tumor immunity caused by the vaccine system greatly inhibits the tumor metastasis capability, while the HTCP/Lipo/RA group has the same obvious effect, and further indicates the significance of the gastrointestinal tract immunity caused by RA on anti-metastasis.
Experiment 4: immune activation in mice
Selecting each group of mice in experiment 3, after tumor monitoring for 14 days, treating the mice, dissecting and removing right inguinal lymph nodes, weighing, grinding and filtering by a cell screen to prepare single cell suspension, carrying out flow cytometry analysis after staining by CD11c-PE and CD86-APC antibodies, dissecting and removing intestinal tract Peyer's patches of the mice to prepare single cell suspension, carrying out flow cytometry analysis after staining by CD11c-PE and CCR9-FITC antibodies, dissecting and weighing spleen and thymus, preparing single cell suspension from spleen, separating lymphocytes by using a lymph separation liquid, co-culturing with 4T1 cells according to an effective target ratio of 40:1, and measuring the inhibition rate of the spleen lymphocytes to 4T1 cells by an MTT method after 6 hours.
The result shows that the maturation degree of dendritic cells in inguinal lymph nodes of mice inoculated with HTCP/ITCs/RA in a subcutaneous way, the expression rate of CCR9 of the dendritic cells in intestinal tract Pe's nodules and the killing capacity of spleen lymphocytes to 4T1 are far higher than those of an NC group, and the nano vaccine can effectively activate the anti-tumor immunity of organisms.
The same experiment was performed on the other examples, which are not exemplified herein, but the experiment was performed on example 1, and the same experiment was performed on the other examples, which are consistent with or similar to the result.
According to the invention, HA and cationic polymer Polyethyleneimine (PEI) are connected through carbon twelve long chains, individual pseudo-tumor cells are compressed into an inner core through electrostatic action, HA is wrapped around by being driven by a fat long chain to form an outer shell, a hydrophobic layer formed by the fat long chain is used for loading a mucosal adjuvant RA, and the nano pseudo-cell personalized tumor vaccine with a core-shell structure is prepared, and experiments show that compared with the prior art, the nano pseudo-cell personalized tumor vaccine HAs the following outstanding characteristics and beneficial effects:
1. dendritic cells can be targeted, and the delivery efficiency of the antigen is improved;
2. can deliver personalized antigen and mucosal immune adjuvant at the same time, stimulate specific and nonspecific immunity of the organism;
3. has good biocompatibility and safety and wide application range, can be effectively used for preparing personalized vaccines aiming at various solid tumors, develops new application of personalized treatment in the field of tumor vaccines, and has remarkable economic and social benefits.

Claims (6)

1. A process for preparing the personalized nm-class pseudo-cell tumor vaccine includes such steps as grafting 12-aminododecanoic acid to hyaluronic acid, and modifying the other end of fatty chainLCysteine and polyethyleneimine are prepared into a nano carrier, then tumor cells are prepared into nano-scale tumor cell membrane-wrapped holoantigen vesicles by using omentum filtration, and a mucosal immune adjuvant holo-transretinoic acid is loaded in a hydrophobic layer formed by a fatty chain when the tumor cells and the carrier are subjected to electrostatic compression to prepare a nano cell-mimetic personalized tumor vaccine with a 'core-shell' structure; the method specifically comprises the following steps:
1) preparing hyaluronic acid grafted carbon twelve chains: weighing 0.1500-0.5000g of HA, dissolving in 15-40mL of formamide, adding 0.0690-0.2890g of EDC and 0.0420-0.1690g of NHS, stirring at room temperature and under the protection of nitrogen and in the dark for reaction for 4-6h, adding 0.1800-0.5970g of 12-aminododecanoic acid, stirring at room temperature and in the dark for reaction for 48h, transferring the reaction solution to 8 x 10 molecular weight cutoff3Da-1.4×104Dialyzing in Da dialysis bag for 2-3d, and freeze drying to obtain hyaluronic acid HA-C grafted with carbon twelve chains12；
2) Preparing hyaluronic acid carbon-dodecyl chain branch cysteine: 0.1510-0.5000g are weighedL-Cys is dissolved in 10-30mL of ultrapure water, pH is adjusted to 5, 0.1510-0.5892g EDC and 0.1400-0.3017g NHS are added, activation is carried out for 1-2h, 0.2500-0.8000g HA-C is added12，N2Carrying out protection reaction for 5-6h, dialyzing with deionized water for 2-3d, and freeze-drying to obtain hyaluronic acid HA-C-Cys grafted with carbon dodecachains and cysteine;
3) preparing a nano carrier: weighing 0.1500-0.5000g of HA-C-Cys, dissolving in 10-30mL of deionized water and 5-10mL of boric acid buffer solution with pH of 9.18, weighing 0.5000-1.7640g of PEI is dissolved in 400-600 mu L of 3.7-4.0% hydrochloric acid, then the PEI is slowly and dropwise added into the solution containing the HA-C-Cys, the reaction is stirred for 36-48h under the conditions of room temperature, nitrogen protection and light protection, and the reaction solution is transferred to the solution with the molecular weight cut-off of 8 multiplied by 103Da-1.4×104Dialyzing in a dialysis bag of Da, dialyzing with deionized water for 2-3d, and freeze-drying to obtain a nano-carrier HTCP;
4) preparing a nano tumor cell antigen: 3-15mL of a solution with a density of 1.0X 107Irradiating the/mL tumor cell PBS suspension for 1-2h under an ultraviolet lamp, then passing the suspension through a 800nm polyethersulfone membrane for 2-3 times, and then passing through a 200nm polyethersulfone membrane for 3-4 times to obtain a pseudo-tumor cell antigen;
5) preparing a nano cell-like vaccine: weighing 0.1-1.0mg HTCP, dissolving in 0.5-1.0mL PBS, adding 2.5-12.5mL pseudo-tumor cell suspension, simultaneously dropwise adding 10.5-52.5 mu g all-trans retinoic acid solution, centrifuging for 15-20min at 10000-12000rpm, and collecting precipitate to obtain the nano pseudo-cell vaccine.
2. The method for preparing the nano cell-like personalized tumor vaccine according to claim 1, specifically comprising the following steps:
1) preparing hyaluronic acid grafted carbon twelve chains: weighing HA 0.1500g, dissolving in 15mL formamide, adding 0.0690g EDC and 0.0420g NHS, stirring at room temperature under nitrogen protection and in dark place for 4h, adding 0.1800g 12-aminododecanoic acid, stirring at room temperature and in dark place for 48h, transferring the reaction solution to 8 × 10 molecular weight cut-off3Da-1.4×104Dialyzing in Da dialysis bag for 2d, and freeze drying to obtain hyaluronic acid HA-C grafted with carbon twelve chains12；
2) Preparation of hyaluronic acid carbododecalinyl cysteine: weighing 0.1510gLCys was dissolved in 10mL of ultrapure water, pH was adjusted to 5, 0.1510g EDC and 0.1400g NHS were added, activation was performed for 1h, and 0.2500g HA-C was added12，N2Carrying out protection reaction for 5h, dialyzing with deionized water for 2d, and freeze-drying to obtain hyaluronic acid HA-C-Cys grafted with carbon dodecachains and cysteine;
3) preparing a nano carrier: weighing HA-C-Cys 0.1500g dissolved in 10mL deionized water andweighing 0.5000g of PEI in 5mL of boric acid buffer solution with pH of 9.18, dissolving the PEI in 400 mu L of 3.7% hydrochloric acid, slowly dropwise adding the PEI into the solution containing HA-C-Cys, stirring and reacting for 48 hours at room temperature under the protection of nitrogen and in the dark, and transferring the reaction solution to a molecular weight cut-off of 8 x 103Da-1.4×104Dialyzing in a dialysis bag of Da, dialyzing for 2d by using deionized water, and freeze-drying to obtain a nano-carrier HTCP;
4) preparing a nano tumor cell antigen: 3mL of a solution having a density of 1.0X 107Irradiating the PBS suspension of the 4T1 mouse breast cancer tumor cells for 1h under an ultraviolet lamp, and then, firstly passing the PBS suspension through a 800nm polyethersulfone membrane for 3 times, and then passing the PBS suspension through a 200nm polyethersulfone membrane for 3 times to obtain a pseudo-tumor cell antigen;
5) preparing a nano cell-like vaccine: weighing 0.2mg HTCP, dissolving in 500 μ L PBS, adding 2.5mL of pseudotumor cell suspension, simultaneously dropwise adding 10.5 μ g of all-trans retinoic acid solution, centrifuging at 12000rpm for 15min, and collecting precipitate to obtain the nano-pseudocell vaccine.
3. The method for preparing the nano cell-like personalized tumor vaccine according to claim 1, which is characterized by comprising the following steps:
1) preparing hyaluronic acid grafted carbon twelve chains: weighing 0.3000g of HA, dissolving in 30mL of formamide, adding 0.1440g of EDC and 0.0910g of NHS, stirring and reacting for 5 hours at room temperature under nitrogen protection and in the absence of light, adding 0.3600g of 12-aminododecanoic acid, stirring and reacting for 48 hours at room temperature and in the absence of light, transferring the reaction solution to a solution with the molecular weight cutoff of 8 multiplied by 103Da-1.4×104Dialyzing in Da dialysis bag for 2d, and freeze drying to obtain hyaluronic acid HA-C grafted with carbon twelve chains12；
2) Preparing hyaluronic acid carbon-dodecyl chain branch cysteine: weighing 0.3011gLCys in 30mL ultrapure water, pH5 adjusted, 0.3020g EDC and 0.2807g NHS added, activated for 1h, 0.5000g HA-C added12，N2Carrying out protection reaction for 6h, dialyzing with deionized water for 2d, and freeze-drying to obtain hyaluronic acid HA-C-Cys grafted with carbon dodecachains and cysteine;
3) preparing a nano carrier: weighing HA-C-Cys 0.3000g and dissolving in 10mL of deionized water and 5mL of pH9.18 boric acid buffer solution, 1.1028g of PEI is weighed and dissolved in 400 μ L of 4.0% hydrochloric acid, then the PEI is slowly and dropwise added into the solution containing HA-C-Cys, the mixture is stirred and reacted for 48 hours at room temperature under the condition of nitrogen protection and light protection, and the reaction solution is transferred to the solution with the molecular weight cutoff of 8 multiplied by 103Da-1.4×104Dialyzing in a dialysis bag of Da, dialyzing for 3d by using deionized water, and freeze-drying to obtain a nano-carrier HTCP;
4) preparing a nano tumor cell antigen: 3mL of a solution having a density of 1.0X 107Irradiating MCF-7 humanized breast cancer tumor cell PBS suspension for 1.5h under an ultraviolet lamp, and then, firstly passing through a 800nm polyethersulfone membrane for 3 times, and then passing through a 200nm polyethersulfone membrane for 3 times to obtain a pseudo-tumor cell antigen;
5) preparing a nano cell-like vaccine: weighing 0.2mg HTCP, dissolving in 500 μ L PBS, adding 2.5mL pseudo-tumor cell suspension, simultaneously dropwise adding 10.5 μ g all-trans retinoic acid solution, centrifuging at 10000rpm for 20min, and collecting precipitate to obtain the nano pseudo-cell vaccine.
4. The method for preparing the nano cell-like personalized tumor vaccine according to claim 1, which is characterized by comprising the following steps:
1) preparation of hyaluronic acid grafted carbon twelve chains: weighing HA 0.2000g, dissolving in formamide 30mL, adding EDC 0.0997g and NHS 0.0670g, stirring at room temperature under nitrogen protection and in dark place for 4h, adding 12-aminododecanoic acid 0.1800g, stirring at room temperature and in dark place for 48h, transferring the reaction solution to 8 × 10 molecular weight cut-off3Da-1.4×104Dialyzing in Da dialysis bag for 2d, and freeze drying to obtain hyaluronic acid HA-C grafted with carbon twelve chains12；
2) Preparation of hyaluronic acid carbododecalinyl cysteine: weighing 0.2000gLCys was dissolved in 10mL of ultrapure water, pH5 adjusted, 0.2000g EDC and 0.1900g NHS added, activated for 1h, 0.2500g HA-C added12，N2Carrying out protection reaction for 6h, dialyzing with deionized water for 2d, and freeze-drying to obtain hyaluronic acid HA-C-Cys grafted with carbon dodecachains and cysteine;
3) preparing a nano carrier: weighing HA-Cys 0.1510g is dissolved in 10mL deionized water and 5mL boric acid buffer solution with pH of 9.18, 0.4000g PEI is weighed and dissolved in 400 μ L3.7% hydrochloric acid, then the solution containing HA-C-Cys is slowly added dropwise, the reaction is stirred for 36 hours under the conditions of room temperature, nitrogen protection and light protection, and the reaction solution is transferred to the molecular weight cut-off of 8 × 103Da-1.4×104Dialyzing in a dialysis bag of Da, dialyzing for 2d by using deionized water, and freeze-drying to obtain a nano-carrier HTCP;
4) preparing a nano tumor cell antigen: 2mL of a solution having a density of 1.0X 107Irradiating the PBS suspension of the 4T1 mouse breast cancer tumor cells for 1h under an ultraviolet lamp, and then passing the PBS suspension through a membrane for 2 times by using a polyether sulfone membrane with the wavelength of 800nm, and then passing through a membrane for 4 times by using a polyether sulfone membrane with the wavelength of 200nm to obtain a pseudo-tumor cell antigen;
5) preparing a nano cell-like vaccine: weighing 0.1mg HTCP, dissolving in 500 μ L PBS, adding 1.25mL of pseudotumor cell suspension, simultaneously dropwise adding 5.3 μ g of all-trans retinoic acid solution, centrifuging at 12000rpm for 15min, and collecting precipitate to obtain the nano-pseudocell vaccine.
5. The method for preparing the nano cell-like personalized tumor vaccine according to claim 1, which is characterized by comprising the following steps:
1) grafting carbon twelve chains on hyaluronic acid: weighing HA 0.5000g, dissolving in 40mL formamide, adding 0.2890g EDC and 0.1690g NHS, stirring at room temperature under nitrogen protection and in dark place for 6h, adding 0.5970g 12-aminododecanoic acid, stirring at room temperature and in dark place for 48h, transferring the reaction solution to 8 × 10 molecular weight cut-off3Da-1.4×104Dialyzing in Da dialysis bag, dialyzing for 3d, and freeze drying to obtain hyaluronic acid HA-C grafted with carbon twelve chains12；
2) Hyaluronic acid carbododecalinyl cysteine: weighing 0.5000gLCys in 30mL ultrapure water, pH5 adjusted, 0.5892g EDC and 0.3017g NHS added, activated for 2h, 0.8000g HA-C added12，N2Carrying out protection reaction for 6h, dialyzing with deionized water for 3d, and freeze-drying to obtain hyaluronic acid HA-C-Cys grafted with carbon dodecachains and cysteine;
3) preparing a nano carrier: weighing HA-C-Cys 0.5000g is dissolved in 30mL deionized water and 10mL pH9.18 boric acid buffer solution, 1.7640g of PEI is weighed and dissolved in 600 μ L of 4.0% hydrochloric acid, then slowly and dropwise added into the solution containing HA-C-Cys, the reaction is stirred and reacted for 48h under the conditions of room temperature, nitrogen protection and light protection, and the reaction solution is transferred to the molecular weight cut-off of 8 × 103Da-1.4×104Dialyzing in a dialysis bag of Da, dialyzing for 3d by using deionized water, and freeze-drying to obtain a nano-carrier HTCP;
4) preparing a nano tumor cell antigen: 15mL of a solution having a density of 1.0X 107Irradiating MCF-7 humanized breast cancer tumor cell PBS suspension for 2h under an ultraviolet lamp, and then, firstly passing through a 800nm polyethersulfone membrane for 2 times, and then passing through a 200nm polyethersulfone membrane for 4 times to obtain a pseudo-tumor cell antigen;
5) preparing a nano cell-like vaccine: weighing 1.0mg HTCP, dissolving in 1mL PBS, adding 12.5mL pseudo-tumor cell suspension, simultaneously dropwise adding 52.5 mu g all-trans retinoic acid solution, centrifuging at 12000rpm for 20min, and collecting precipitate to obtain the nano pseudo-cell vaccine.
6. Use of the nano-sized pseudocellular vaccine prepared by the preparation method of claim 1 in the preparation of personalized solid anti-tumor vaccine.
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