CN113293137B - Modification method of dendritic cells based on cell membrane surface modification technology and application of modification method - Google Patents

Modification method of dendritic cells based on cell membrane surface modification technology and application of modification method Download PDF

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CN113293137B
CN113293137B CN202010107008.3A CN202010107008A CN113293137B CN 113293137 B CN113293137 B CN 113293137B CN 202010107008 A CN202010107008 A CN 202010107008A CN 113293137 B CN113293137 B CN 113293137B
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CN113293137A (en
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陈红
王蕾
郁李胤
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Bmd Biotechnology Suzhou Co ltd
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    • C12N2510/00—Genetically modified cells

Abstract

The invention discloses a modification method of dendritic cells based on a cell membrane surface modification technology and application thereof. The modification method of the dendritic cells based on the cell membrane surface modification technology provided by the invention comprises the following steps: 1) Synthesizing a substrate for photothermal transfection; 2) Coating a plasmid containing a gene sequence encoding HTP with PEI and placing the plasmid on the surface of the substrate; 3) Preparing modified dendritic cells; 4) Synthesizing a functional sugar polymer molecule; 5) Mixing and incubating the functional glycomer molecule and the modified dendritic cell. The modification method of the dendritic cells based on the cell membrane surface modification technology can efficiently modify the sugar polymer with the specific recognition function to the surface of the DC cells, so that the interaction between the DC and the T cells is improved, the activation efficiency of the T cells is finally improved, and the cell transfection efficiency, the activity of the transfected cells and the safety of the whole transfection system are improved.

Description

Modification method of dendritic cells based on cell membrane surface modification technology and application of modification method
Technical Field
The invention relates to the fields of molecular biology and vaccines, in particular to a modification method of dendritic cells based on a cell membrane surface modification technology and application thereof.
Background
The immunotherapy strategy provides a new thought and development direction for the prevention and treatment of tumors, and is considered as the most promising tumor treatment strategy. Current strategies for tumor treatment mainly include: cancer vaccine, CAR-T (Chimeric Antigen Receptor T-Cell, chimeric antigen receptor T Cell), etc. adoptive immunotherapy, and immune checkpoint blockers, etc. Among these strategies, cancer vaccines are considered as the most promising tumor treatment strategies because they activate the regulatory functions of the human autoimmune system, and they overcome the toxic and side effects of adoptive immunotherapy, immune checkpoint blockers, and the like.
In cancer vaccines, strategies to target dendritic cells, T cells, macrophages and bone marrow derived immunosuppressive cells are widely used. Among them, dendritic Cell (DC) vaccines have the ability to modulate the autoimmune system in vivo, and are considered the most safe and effective vaccine strategies. After surgical excision and chemotherapy, a large amount of tumor antigen is generated in situ, DC passes phagocytic antigen and presents antigen information to T cells in the form of major histocompatibility complex (Major histocompatibility complex, MHC), and after the T cells are stimulated by a first signal of MHC, activation procedures are started, and then co-stimulatory molecules CD80, CD40 and the like on the DC surface are combined with T cell surface receptors (TCR) to further activate proliferation and differentiation of the T cells. In the process of interaction between DC and T cells, cytokines such as Interleukin-2 (IL-2) secreted by DC and T cells, interferon-gamma (IFN-gamma) and the like further promote differentiation and maturation of T cells. Therefore, the activation efficiency of the DC to the T cells can be effectively improved by enhancing the interaction between the DC and the T cells. This is an effective means of improving the efficiency of DC vaccines. Prior art studies on DC vaccines include:
Patent document 1 (CN 102787097B) discloses that a virus vector is used to transfect a SYK gene into a DC, and the DC is modified to be used for the preparation of a vaccine for the DC. In the patent document, SYK gene transfection is carried out on DC by adopting lentivirus, so that the DC can efficiently express a co-stimulatory molecule for promoting T cell activation. However, this method is not efficient in transfection. The transfected cells need to be accessible through further screening and culturing methods, and the method is long in period, high in cost and unfavorable for practical use. The reason for this may be that virus transfected cells are tolerant to some extent and expression is lost to some extent during the metabolism of the cells. Thereby affecting the final expression. Patent document 2 (CN 107286247B) discloses a chimeric antigen receptor and its gene and recombinant expression vector, engineered mesothelin-targeted dendritic cells (mesoCAR-DCs), and uses thereof. The method disclosed in the patent expresses a DC cell of a chimeric antigen receptor, and the modification method of the gene recombination has higher requirements on the structural sequence and the arrangement sequence of the genes. The dendritic cells constructed by the method are dendritic cells which can only target mesothelin, have single functions and have no wide application. Patent document 3 (CN 100548377C) discloses a recombinant adenovirus carrying a gene encoding epstein barr virus latent membrane protein 2, an antigen presenting cell transduced or modified by the recombinant adenovirus, and a vaccine composition containing the transduced or modified antigen presenting cell. The method disclosed in this patent loads adenovirus vectors carrying EB virus into DC cells. The method has the following defects: first, the adenovirus used in this method is at risk for transfection, and the transfection efficiency can only reach around 50%. Secondly, the DC cells obtained by the transfection method are only opposite to EB virus and have no universal applicability. Patent document 4 (CN 102181407B) discloses a recombinant adenovirus and modified dendritic cells thereof and their use; the invention provides a recombinant adenovirus containing a specific protein expression sequence, which is used for infecting dendritic cells to obtain recombinant cells. The recombinant cells and the recombinant adenovirus can be used for preparing cervical cancer prevention and/or treatment vaccines. The method adopts recombinant adenovirus to infect cells, firstly has higher cost for preparing the recombinant adenovirus, and secondly has high cost and relatively low transfection efficiency by utilizing the virus to transfect.
Fusion of antigen and DCs in vitro, stimulation of DC maturation followed by reinfusion back into the patient is one of the earliest strategies used [ J Immunother 34,382-389 (2011) ]. However, in vivo tumor microenvironments are complex, and the interaction of the purely activated DC and T cells is significantly restricted. Even if DC is purely mature, the activation efficiency of T cells can be improved in vitro, and the strategy has very little curative effect. To further enhance the effect of the interaction between DC and T cells, the method of enhancing T cell activation signal by transfecting DC with genetic engineering techniques to allow efficient expression of costimulatory molecules favorable to antigen presentation is currently the most commonly used method [ Cancer ImmunolImmunother, 831-842 (2015) ]. However, there are two major problems with this type of approach: 1. the vaccine has high sample variability, and the activation signal of the T cells firstly needs the mutual identification of the T cells and the DC cells, and secondly, the chain reaction of the costimulatory molecules can be induced. However, recognition between DC and T cells is weak, which results in the enhanced signal from modification not being effective; 2. the use of viral transfection presents serious safety risks, the introduction of exogenous genetic material may lead to unpredictable risks of gene mutation, and the viral transfection cycle is long and costly, which is not beneficial for practical applications [ Genes,65,8 (2017) ]. Although electrotransfection can avoid the safety problem caused by virus transfection, the efficiency of electrotransfection is very unstable, and the electrotransfection method can cause serious damage to cells, so that the cell viability is very low [ Cellular and Molecular Bioengineering,4,538-545 (2016) ]. Patent document 5 (CN 104906567B) discloses a ligand-modified nano-preparation for activating antigen presenting ability of DCs to T cells by entrapping a tumor antigen peptide or a complex formed by a tumor antigen peptide and a heat shock protein. The method adopts nano particles as a carrier, and uses a DC endocytosis method to transfer functional plasmids. This approach only enhances the maturation efficiency of DCs, and is not helpful in enhancing DC interactions with T cells. Therefore, this method has extremely limited efficiency in the activation of the T cells. And the gold nanoparticles are ingested without effective metabolic degradation pathways, which can cause certain damage to DC cells.
In the human immune system, T cells are the final effector cells, and have tumor-specific killing functions after being activated by Dendritic Cells (DCs). Thus, the use of dendritic cells as a vaccine to activate the tumor immune activity of T cells is an effective strategy for treating tumors. Activation of T cells depends on the antigen presenting capacity of DCs. During antigen presentation, first MHC signaling molecules on the DC surface bind to T cell receptors. Subsequently, the co-stimulatory molecules on the DC surface promote T cell activation upon binding to the corresponding receptors on the T cell surface. In order to increase the efficiency of activation, it is an effective strategy to make DCs efficient in expressing costimulatory molecules that assist in antigen presentation.
At present, the most common means is to transfer a plasmid capable of expressing co-stimulatory molecules efficiently into DC by gene transfection techniques. However, this strategy has the following problems. First, the transfection methods currently used are mostly virus-dependent, which may lead to unpredictable risks of gene mutation, and the virus transfection cycle is long and costly, which is not advantageous for practical use. Although the method of electrotransformation can avoid these problems, the efficiency of electrotransformation is extremely unstable and can cause massive cell death. The chemical modification method can simply control the expression of cell membrane surface receptors and ligands, but needs to do a large amount of polymerization reaction on the cell surface, which can cause serious cytotoxicity and can not maintain the long-time modification effect. Secondly, the binding of MHC molecules of DCs to T-cells, which is first required to bind to T-cell receptors in order to initiate the co-stimulatory pathway, is less strongly associated with the early stages of DC and T-cell action, and this strategy ignores this problem. For non-contacting DC and T cells, simply enhancing the second signal in the absence of the first signal does not increase the antigen presentation efficiency of DC to T cells. Thus, by modifying DCs to increase the ability of the DCs to interact with T cells, the premise of DC antigen presentation efficiency can be increased.
Many activated groups exist on the surface of the cell membrane, such as amino groups, biotin, etc., so that modification strategies can be designed by the groups on the surface of the cell membrane. Modification strategies such as DNA hybridization, liposome-aptamer, liposome-peptide, biotin-avidin, sugar and lectin [ angelwcheminted engl53,5112-5116 (2014) ] have been developed. Among them, sugar and lectin strategies possess stability and high biocompatibility, and have been widely used for modification of cell membrane surfaces. The method of phospholipid intercalation is a very mature means at present to modify sugar molecules to the surface of cell membranes. However, the membrane insertion mode makes the residence time of the modified functional molecules on the cell membrane extremely short, and the modified functional molecules can only be maintained for a plurality of hours, so that the biological functions of the modified cells cannot be maintained for a long time in the practical application process. Therefore, there is a need to develop a method capable of maintaining the modifying function for a long period of time.
The above problems can be effectively solved by using Halotag transmembrane proteins (Halotag Transmembrane protein, HTP) as anchoring functional groups on the cell membrane surface [ ACS biomater. Sci. Eng.4,11,3658-3677 (2018) ]. The method can solve the problem that the modified functional molecules stay on the surface of the cell membrane for a long time, and the modification mode does not need to carry out polymerization reaction in a cell solution, so that the cytotoxicity to modified cells is greatly reduced. HTP modification techniques first require HTP plasmid transfection of cells, again requiring technical challenges of transfection.
In the human immune system, T cells are the final effector cells, and have tumor-specific killing functions after being activated by Dendritic Cells (DCs). Thus, the use of dendritic cells as a vaccine to activate the tumor immune activity of T cells is an effective strategy for treating tumors. Activation of T cells depends on the antigen presenting capacity of DCs. During antigen presentation, first MHC signaling molecules on the DC surface bind to T cell receptors. Subsequently, the co-stimulatory molecules on the DC surface promote T cell activation upon binding to the corresponding receptors on the T cell surface. In order to increase the efficiency of activation, it is an effective strategy to make DCs efficient in expressing costimulatory molecules that assist in antigen presentation. The DC modification strategy adopted by the invention has wider universality.
Disclosure of Invention
Problems to be solved by the invention
Aiming at the problems in the prior art, the invention hopes to modify the surface of a DC cell membrane by a chemical synthesis method so as to enhance the interaction between DC and T cells and further improve the activation efficiency of the DC to the T cells.
Solution for solving the problem
The invention adopts a gene transfection method of photoinduced perforation, which can effectively overcome the safety problem caused by virus transfection, and the method is efficient, quick and low in cost. The invention develops a modified DC method by utilizing the set of photothermal transfection system: firstly, transfecting the HTP functional plasmid into DC through photo-thermal transfection, leading the surface of the DC to have chloralkane acceptor groups, simultaneously carrying out chloralkane modification on functional molecules for modifying the DC, and then anchoring the modified functional molecules to the surface of the DC through simple incubation. The invention can effectively solve the problems of weak interaction and weak recognition capability of DC and T cells, and the DC modification method used by the invention is safe, efficient and low in cost, and can obtain a large amount of modified DC in a short time.
The DC modification method adopted by the invention is as follows: halotag protein (HTP) is transfected into DC by using a photothermal transfection method to form HTP-DC with an anchoring functional group. Meanwhile, synthesizing a chain transfer agent containing the HTP ligand, and polymerizing functional molecules by taking the chain transfer agent as a framework to form a modified polymer. Finally, DC modification can be completed through simple incubation. Specifically, the method for modifying DC provided by the invention is as follows:
first, substrates that can be used for photothermal transfection are synthesized. Polydimethylsiloxane (PDMS) with good biocompatibility was selected as a transfection substrate, and MCNT was incorporated into PDMS using the photothermal effect of Multi-walled carbon nanotubes (Multi-walled Carbon Nanotube, MCNT). Specifically, PDMS is mixed with a cross-linking agent, MCNT is added, and the mixture is heated and cured to form a film, so that the substrate for photo-thermal transfection can be prepared. The vacuumizing operation can be performed simultaneously in the heating process, so that bubbles can be eliminated better.
Subsequently, the prepared substrate was sterilized at high temperature and high pressure, and placed in a cell culture plate to be dried. The plasmid containing HTP gene is coated by small molecule polyether imide (PEI) (plasmid is purchased from Jinweisu state technology Co., ltd.) and DC cells are inoculated on the surface of the substrate according to a certain density after the plasmid is stably adsorbed on the surface of the substrate, after the cells are attached to the wall, the plasmid containing HTP gene coated by PEI is added into the solution again, and the transfection is carried out under the laser with specific power. After transfection is completed, cells are continuously cultured for 48 hours, transfected cells are harvested, the transfection efficiency can be verified by adopting immunofluorescent staining and RT-PCR methods, and the activity of the cells can be verified by CCK-8 detection.
Simultaneously, anhydrous Tetrahydrofuran (THF), anhydrous N, N-Dimethylformamide (DMF), sodium hydride (NaH) and 6-chloro-1-iodohexane (6-chloro-1-iodohexane) are used as raw materials for reaction, separation and purification are carried out, deprotection is carried out, and an intermediate product C is synthesized:
and then taking a chain transfer agent (Chain transfer agent, CTA), N-hydroxysuccinimide (NHS) and Dicyclohexylcarbodiimide (DCC) as raw materials, tracking the synthesis of NHS-RAFT by using a TLC (thin layer chromatography) plate, adding the deprotected intermediate product C in situ for reaction after the raw material point disappears, and purifying by a silica gel column to obtain the chain transfer agent A with chlorinated alkane ligand:
subsequently, the sugar molecule monomer with double bonds is synthesized by referring to a synthesis method of the sugar molecule monomer with double bonds in the patent number of CN2015102032. X, and the chain transfer agent A obtained by the synthesis is utilized to obtain the needed modified polysaccharide molecule through polymerization reaction.
And finally, mixing the modified DC and the modified glycan molecules according to a certain concentration, and incubating under the conventional cell culture condition to obtain the functional DC modified by the sugar polymer molecules.
In a first aspect, the present invention provides a method for modifying dendritic cells based on a cell membrane surface modification technique, the method comprising the steps of:
1) Synthesizing a substrate for photothermal transfection;
2) Coating a plasmid containing a gene sequence encoding a Halotag protein (HTP) with Polyetherimide (PEI) and placing said plasmid on the surface of said substrate in step 1);
3) Preparing modified dendritic cells;
4) Synthesizing a functional sugar polymer molecule;
5) Mixing and incubating the functional sugar polymer molecule in the step 4) and the modified dendritic cell in the step 3) to obtain the functional sugar polymer molecule modified dendritic cell.
Wherein, the preparation method of the base material in the step 1) comprises the following steps:
the flexible substrate and crosslinking agent were combined at 50: uniformly mixing the materials according to the mass ratio of 1-5:1, adding nano particles of 0.5-10 per mill of the flexible substrate, uniformly mixing the materials again, and heating, curing and forming a film to obtain the substrate for photo-thermal transfection;
preferably, the mass ratio of the flexible substrate to the crosslinking agent is 10:1, adding 10 per mill of nano particles of a flexible substrate, wherein the nano particles are more than one of a group consisting of multi-wall carbon nano tubes (MCNTs), graphene, gold nano particles and polydopamine nano particles, and the flexible substrate is more than one of a group consisting of thermosetting plastic and hydrogel; more preferably, the nanoparticle is a Multiwall Carbon Nanotube (MCNT) and the flexible substrate is Polydimethylsiloxane (PDMS).
The concentration of the plasmid in step 2) is 0.5-2. Mu.g/10 4 The plasmid is placed on the surface of the substrate in the step 1) for 1 to 3 hours; preferably, the plasmid concentration is 1.5. Mu.g/10 4 Individual cells.
The specific operation of the step 3) is as follows: inoculating dendritic cells on the surface of the substrate containing the plasmid in the step 2), adding the plasmid coated with PEI and containing the gene sequence for encoding HTP again after the dendritic cells are attached, transfecting under laser, and continuously culturing the cells for 36-60 h;
wherein the dendritic cells have an seeding density of 10 4 ～10 5 Individual/cm 2 The laser intensity is 1-10W/cm 2 The irradiation time of the laser is 5-60 s; preferably, the dendritic cells are seeded at a density of 5X 10 4 Individual/cm 2 The laser intensity is 1-4W/cm 2 The irradiation time of the laser is 20-60 s; more preferably, the laser intensity is 2W/cm 2 The irradiation time of the laser was 30s.
The preparation method of the functional sugar polymer molecule in the step 4) comprises the following steps:
(i) Synthesis of intermediate products: 2- (2-Boc-aminoethoxy) ethanol, anhydrous Tetrahydrofuran (THF), anhydrous N, N-Dimethylformamide (DMF), sodium hydride (NaH) and 6-chloro-1-iodohexane (6-chloro-1-iodohexane) are used as raw materials for reaction, separation and purification, deprotection and intermediate product synthesis are carried out;
(ii) Synthesizing a chain transfer agent containing a chloralkane structure: dissolving a chain transfer agent, NHS and DCC in anhydrous DMF, placing the anhydrous DMF for reaction for 10 to 15 hours at the temperature of 28 to 32 ℃, adding the intermediate product synthesized in the step (i) in situ after the raw material point of the chain transfer agent in a reaction system disappears, reacting overnight at the temperature of 25 to 35 ℃, and washing, concentrating and purifying the solution obtained after the reaction to obtain the chain transfer agent containing chloralkane structure;
(iii) Placing the chain transfer agent containing the chloralkane structure, the sugar monomer and the initiator in the step (ii) at 60-80 ℃ for reaction for 10-12 hours to carry out polymerization reaction, transferring the reaction solution into a dialysis bag with the molecular weight cutoff of 3500Da for dialysis after the reaction is finished, and freeze-drying to obtain the functional sugar polymer;
preferably, the chain transfer agent is one or more selected from the group consisting of aliphatic mercaptans, dodecyl mercaptan, water, ethers, acids, esters and quinone carbon dioxide, oxygen, alcohols and organic acids; the sugar monomer is more than one selected from mannose (amine), glucose (amine), galactose (amine), fructose (amine), arabinose (amine), ribose (amine), xylose (amine), lyxose (amine) and glyceraldehyde modified by double bonds; the initiator is azo initiator;
More preferably, the chain transfer agent is 4-cyanovaleric acid dithiobenzoic acid (CPADB); the sugar monomer is N-methacryloyl mannosamine (MAM); the initiator is Azobisisobutyronitrile (AIBN).
To facilitate detection, biotin monomer is added in the polymerization reaction in (iii).
In a second aspect, the invention provides a dendritic cell prepared by the method of the first aspect of the invention.
In a third aspect the invention provides a vaccine composition comprising a dendritic cell according to the second aspect of the invention and one or more pharmaceutically acceptable carriers, adjuvants and/or excipients.
In a fourth aspect, the present invention provides the use of a dendritic cell according to the third aspect of the present invention in the manufacture of a medicament for the treatment and prophylaxis of malignant neoplasms. Wherein the malignant tumor is any one of lung cancer, liver cancer, colon cancer, melanoma, prostate cancer, gastric cancer, breast cancer, chronic Lymphocytic Leukemia (CLL) or chronic granulocytic leukemia; preferably, the malignancy is colon cancer.
ADVANTAGEOUS EFFECTS OF INVENTION
The DC cell transformation technology combining gene transfection and chemical modification can efficiently modify the sugar polymer with the specific recognition function to the surface of the DC cell, thereby improving the interaction between the DC and the T cell and finally improving the activation efficiency of the T cell. The method combines the gene transfection technology of photo-induced perforation, avoids the defect of DC caused by virus transfection and electroporation technology, and improves the transfection efficiency, the activity of transfected cells and the safety of the whole transfection system. Specifically, compared with the prior art, the technical scheme of the invention has the following beneficial effects:
(1) The photothermal transfection system adopted by the invention has higher universality. The photothermal transfection system can transfect a large number of cells in a short time and can transfect 10 in 10min 7 Individual cells, but the viral transfection efficiency was not yet observable at the same time.
(2) The raw materials required for synthesis are all chemicals and substrates commonly used in laboratories, and the transfected substrates can be reused for transfection of 10 7 The cost of the substrate required for individual cells is lower, about 1/5 of the cost of viral transfection under equivalent conditions.
(3) The photothermal transfection method adopted by the invention can effectively improve the transfection efficiency of DC cells. Compared with the virus transfection method, the photo-thermal transfection efficiency can reach more than 95% in a short time under the same transfection time condition, and the virus transfection efficiency is only below 10%. And the photothermal transfection system has higher safety coefficient. In addition, the effect of photothermal transfection on cell activity was smaller than that of viral transfection, wherein the cell activity of photothermal transfection was reduced by about 20% and viral transfection was reduced by about 30%.
(4) The activity of the T cells is detected by co-incubation of the DC and the T cells, and the influence of the strategy of the modified DC adopted by the invention on the induction of the immune activity of the T cells is verified. The result shows that: the modified DC adopted by the invention can obviously promote the proliferation of T cells, which is about 2.5 times of that of unmodified DC. Compared with the virus transfection method, the proliferation efficiency of T cells is improved by about 1.5 times under the action of the method.
(5) The induction efficiency of the modified DCs of the present invention on T cell differentiation was verified by detecting the expression of the T cell activating factors TNF- α and IFN- γ. The results show that: the expression rate of the induced T cells TNF-alpha and IFN-gamma is about 10 times and 8 times of that of unmodified DC by adopting the modification method; the expression rate of TNF-alpha and IFN-gamma is improved by about 3-4 times compared with unmodified DC.
(6) T cells and tumor cells were incubated together and the killing effect of T cells on tumors was examined. The results show that: the apoptosis rate of T cell tumor induced by the modified DC reaches more than 95 percent; the T cell tumor apoptosis rate induced by unmodified DC is only about 45%. The apoptosis rate of T cell tumor induced by virus transfection modified DC is about 60%. The results show that the modified DC cells adopted by the invention can induce T cells to generate stronger tumor killing capability.
(7) The modified functional molecule of the DC cell modification method not only can improve the induction efficiency of DC on T cell immune activation, but also can not influence the specificity of antigen presented by DC to T cells, so the method has higher universality and can be suitable for the treatment of various malignant tumors, and the method comprises the following steps: lung cancer, liver cancer, colon cancer, melanoma, prostate cancer, stomach cancer, breast cancer, chronic lymphocytic leukemia and chronic myelogenous leukemia. The invention in a specific example performs functional verification on colon cancer cells therein.
In order to make the above and other objects, features and advantages of the present invention more comprehensible, preferred embodiments accompanied with figures are described in detail below:
drawings
FIG. 1 shows a graph of the photo-thermal intensity of a photo-thermally transfected substrate over time under different power irradiation.
FIG. 2 shows the optimization of DC transfection conditions. Wherein a in fig. 2 shows the effect of different plasmid concentrations on transfection efficiency; b in fig. 2 shows the effect of different laser intensities on transfection efficiency; c in fig. 2 shows the effect of different laser intensities on transfected cell activity; d in fig. 2 shows the effect of different irradiation times on transfection efficiency.
FIG. 3 shows a comparison of transfection efficiency and cell activity for different transfection methods. Wherein a in fig. 3 shows transfection efficiency of viral transfection and photothermal transfection; FIG. 3B shows the effect of different transfection methods on cell activity; the results of DAPI and FITC staining for the different transfection methods are shown at C in fig. 3.
Figure 4 shows the molecular structure of polymannan containing chlorinated alkane ligands.
FIG. 5 shows fluorescence characterization of modified dendritic cells.
Figure 6 shows the proliferative activity of T cells under induction of different DC vaccines. Wherein a in fig. 6 shows the effect of different DC induction patterns on T cell proliferation rate; FIG. 6B shows the effect of different DC induction patterns on TNF- α expression levels; FIG. 6C shows the effect of different DC induction patterns on IFN-gamma expression levels.
Figure 7 shows the tumor toxicity of different kinds of DC vaccines for colon cancer cell CT 26. Wherein a in fig. 7 shows the effect of different kinds of DC vaccine on tumor cell growth after acting on colon cancer cells CT 26; b in fig. 7 shows the effect of different kinds of DC vaccine on tumor cell viability after acting on colon cancer cells CT 26.
Detailed Description
The specific embodiments of the present invention are listed only as examples of the present invention, and the present invention is not limited to the specific embodiments described below. Any equivalent modifications and substitutions of the embodiments described below will be apparent to those skilled in the art, and are intended to be within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.
The test materials, test reagents and instruments used in the examples of the present invention are all commercially available, and specific information thereof is shown in table 1 below:
table 1: commercial information of test materials, test reagents
Reagent name Product commercial information
4-Cyanovaleric acid dithiobenzoic acid (CPADB) Sigma Aldrich trade Co Ltd
2- (2-BOC-Aminoethoxy) ethanol Sigma Aldrich trade Co Ltd
N-hydroxysuccinimide (NHS) Shanghai TCI
sodium hydride Shanghai TCI
Dicyclohexylcarbodiimide (DCC) Shanghai TCI
Anhydrous Tetrahydrofuran (THF) Shanghai TCI
Azobisisobutyronitrile (AIBN) Sinopharm Group Chemical Reagent Co., Ltd.
Anhydrous N, N-Dimethylformamide (DMF) Ara Ding Keji Co Ltd
Recombinant HTP plasmids Suzhou Jin Wei Intelligent technologies and biology Co., ltd
6-diamidino-2-phenylindole (DAPI) Siemens Feilier
FITC (fluorescein isothiocyanate) Sigma Aldrich trade Co Ltd
DC2.4 cell line Shanghai enzyme research Biotech Co.Ltd
CT-26 cell line Shanghai enzyme research Biotech Co.Ltd
TNF-alpha and IFN-gamma cytokine detection kit Suzhou family biotechnology Co., ltd
Sylgard 184 silicon rubber curing agent Daokanning medicine for curing coronary heart disease
Multiwall Carbon Nanotubes (MCNT) Scott pharmaceutical technology Co Ltd
Example 1: preparation of photothermal transfection substrate capable of desorbing cells
The transfection system used in the present invention is a photo-thermal transfection system independent of viruses. Thus, a substrate for photothermal transfection needs to be synthesized by a chemical synthesis method.
The photothermal transfection substrate provided by the invention is a composite material, which comprises: (1) nanoparticles, and (2) a flexible substrate; the nano particles comprise one or more of multi-wall carbon nano tubes, graphene, gold nano particles and polydopamine nano particles, the flexible substrate comprises one or more of thermosetting plastics such as polydimethylsiloxane and hydrogel, and the mass percentage of the nano particles in the composite material is 0-60 per mill.
Multiwall Carbon Nanotubes (MCNTs) MCNTs were chosen as nanoparticles in this example because of their good dispersibility; polydimethylsiloxane (PDMS) has a smooth surface topology and is easy for cell desorption due to good biocompatibility, so PDMS is selected as a flexible substrate in this example.
In the preparation process of the photo-thermal material, firstly, a PDMS precursor and a Sylgard 184 silicon rubber curing agent, namely a cross-linking agent, are mixed according to the mass ratio of 50: mixing the materials according to the ratio of 1-5:1. The higher the proportion of the crosslinking agent, the higher the hardness of the obtained photothermal material. In order to obtain proper hardness, after a PDMS precursor and a cross-linking agent are mixed according to a mass ratio of 10:1, the mixture is poured into an ultrasonic cleaner for ultrasonic dispersion, and after uniform mixing, MCNT is doped and fully and uniformly stirred. The mass ratio of MCNT in PDMS can be controlled within the range of 0.5-10 per mill, and MCNT with the mass ratio of 10 per mill is selected as the final concentration in order to facilitate the observation of the cell state and maintain good photo-thermal effect. Pouring the mixture into a culture dish, fully and uniformly stirring, and curing for 1h at 65 ℃. After demoulding, the sample is cut into round samples with the diameter of one centimeter, and the samples are respectively washed three times by using 95% ethanol and water and then dried by nitrogen. Then at 1W/cm 2 、2W/cm 2 、3W/cm 2 And 4W/cm 2 The material was tested for photo-thermal effects at different times under different laser intensities.
As can be seen from fig. 1, the photo-thermal intensity of the material prepared in this example was changed with the change of the laser intensity, the photo-thermal effect of the material prepared in this example was increased with the increase of the laser intensity before 40s, and the difference was not significant after 40 s.
Example 2: construction of a Halotag Transmembrane Protein (HTP) expressing DC cell line
1 construction of functional plasmids for transfection of DCs
Functional plasmids designed for transfection of DCs. The concrete design mode is as follows: recombinant HTP plasmid (purchased from Jin Weizhi su technology limited) was integrated into pcdna3.1 vector by conventional molecular biology means, and integrated with the insert antibiotic neomycin gene for screening at the time of post-transfection cells, and heme (Hemagglutinin A epitope) gene was contained in the recombinant pcdna3.1 vector as a marker gene, which can be combined with fluorescein-labeled avidin (FITC-avidin) for verification of transfection efficiency.
2 optimization of transfection conditions and acquisition of HTP modified DC cells
The photothermal material prepared in example 1 above was sterilized, and then the sterilized substrate was immersed in a PBS solution containing 1% fetal bovine serum at a mass concentration of 20%, incubated overnight at 37℃and the supernatant was discarded. The HTP-containing plasmid DNA prepared in section 1 of this example was then coated with a small molecule Polyetherimide (PEI) at a plasmid concentration of 0.5-5. Mu.g/10 4 The individual cells are adjusted in the range of 1.5. Mu.g/10 was finally selected in order to achieve optimal transfection efficiency and minimal plasmid consumption 4 The individual cell plasmid concentration was the experimental concentration.
Spread on the surface of the substrate treated as described in example 1 for 1 to 3 hours, followed by planting dendritic cells on the plasmid-adsorbed substrate at a density which may be 10 4 ～10 5 Individual/cm 2 In-range regulation, in order to achieve better transfection effect, the optimization conditions are as follows 5×10 4 Individual/cm 2 Is planted with dendritic cells. After the dendritic cells are attached, the plasmid DNA which is wrapped by the PEI and contains HTP is added again, and transfection is carried out under specific laser conditions. Specifically, the intensity of the laser is set to be 1-10W/cm 2 The irradiation time of the laser light is set to 5 to 60 seconds. The laser intensity was set to 1 to 4W/cm due to the negative effect of the excessively high laser intensity on the cell activity 2 The method comprises the steps of carrying out a first treatment on the surface of the Considering transfection efficiency, the irradiation time of the laser is selected to be 20-60 s. In order to optimize transfection efficiency and cell activity, optimal condition screening was performed under the above conditions. Finally, it was determined that the concentration was 2W/cm 2 Is the optimal reaction condition for cell transfection for 30s under the laser intensity. After transfection, the cells were placed in an incubator at 37℃for further culturing for 48 hours to obtain HTP modified DC cells.
As can be seen from the effect data of fig. 2: (1) The concentration of the plasmid has a more remarkable effect on the transfection efficiency compared with 2W/cm 2 Under the laser irradiation intensity, the different plasmid concentrations (0.5. Mu.g/10 were compared 4 Individual cells, 1.0. Mu.g/10 4 Individual cells, 1.5. Mu.g/10 4 Individual cells and 2ug/10 4 Individual cells) on transfection, indicating 1.5 μg/10 4 The individual cells can achieve the optimal transfection effect (see in particular A of FIG. 2). (2) There are also different effects of laser intensity on transfection efficiency and cell activity, either too large or too small laser intensity is detrimental to transfection, too small laser intensity is not efficient for cell transfection (see B in fig. 2), and too high laser intensity affects cell activity (C in fig. 2). (3) In addition, the time of laser irradiation was screened, and it was found that a good transfection result could be obtained for 30 seconds, and the prolonged time did not significantly improve the transfection efficiency (see D in fig. 2).
As can be seen from FIG. 2, both the plasmid concentration and the irradiation time of the laser, the intensity of the laser, have an influence on the final transfection effect. Specifically, when the plasmid concentration was 1.5. Mu.g/10 4 The transfection efficiency at individual cells could reach around 95%, while continuing to increase plasmid concentration did not significantly increase the transfection efficiency (see a in fig. 2). The laser intensity is 2W/cm 2 The transfection efficiency reached around 95% at this time, after which the transfection efficiency was in a decreasing trend with increasing laser intensity (see B in fig. 2). The cell activity after laser irradiation is another factor to be considered, and the laser intensity is 1W/cm 2 When the cell activity was close to 100%,2W/cm 2 At about 90%, the cell activity tends to decrease with increasing laser intensity (see C in FIG. 2). Comprehensive comparison of cell Activity and transfection efficiency, 2W/cm 2 Is the most suitable laser intensity. Finally, comparing the irradiation time of the laser, the transfection efficiency under the condition is only 50% when the irradiation is performed for 10s, and the efficiency reaches more than 95% after 30s, and the transfection efficiency is not significantly improved by continuously increasing the irradiation time (see D in fig. 2).
Thus, the final plasmid concentration was selected to be 1.5. Mu.g/10 4 Individual cells, 2W/cm 2 Laser intensityThe laser irradiation time period was 30s as the final transfection condition.
3 comparison of transfection efficiency and cell Activity by different transfection systems
Based on the above test, the photo-thermal transfection system was compared with the viral vector transfection system to verify the superiority of the transfection efficiency of the photo-thermal transfection system.
The experimental group adopts a photothermal transfection system, the transfection is carried out according to the optimized transfection conditions, the negative control group adopts the combination of slow virus and recombinant HTP plasmid purchased from the technology Co-Ltd. The specific procedure was to fix transfected cells with 5% paraformaldehyde for 10min, to break Triton membranes for 5min, to incubate HA primary antibody overnight, and to observe it after staining with DAPI and FITC, respectively.
The results are shown in FIG. 3: the transfection efficiency of the photo-thermal transfection system reaches more than 90%, and the transfection efficiency of the virus transfection system is only less than 20% within the same time. As can be seen by comparing the two transfection systems, the activity of the photo-thermal transfection cells is reduced by less than 20%, and the activity of the virus transfection cells is reduced by about 30%, which shows that the photo-thermal transfection system has more advantages than the virus transfection system.
EXAMPLE 3 Synthesis of functional sugar Polymer molecules for modification of DC cells
The synthesis of functional glycomer molecules for modifying DC cells requires a chloroalkane end-modified chain transfer agent, sugar monomer and initiator. The synthesis of the sugar molecules for modifying the DC cells firstly carries out chloralkane modification on the chain transfer agent, and then carries out polymerization reaction on the chloralkane modified chain transfer agent, sugar monomers and initiator under certain conditions, thus obtaining the sugar polymer for modifying the DC. The obtained sugar polymer has molecular weight in the range of 2000-9000Da and can be used for subsequent research. The specific experimental process is as follows:
(1) First, a chain transfer agent containing a chloroalkane structure is synthesized.
2- (2-Boc-aminoethoxy) ethanol (4.30 g,20.95 mmol), anhydrous THF (40 mL) and no Water DMF (20 mL) was added sequentially at 0deg.C NaH (60% dispersion in paraffin liquid, 1.12g, 28.00 mmol) and mixed, after stirring at 0deg.C for 30min 1-chloro-6-iodohexane (4.80 mL,31.60 mmol) was added to the above mixture at 0deg.C. The mixture was stirred at 0deg.C for 20min, at room temperature for 16h, then saturated with NH 4 The reaction was quenched with aqueous Cl, the mixture was extracted twice with ethyl acetate, washed with brine, dried over sodium sulfate and collected by filtration to give deprotected intermediate C.
0.5g of the chain transfer agent 4-cyanovaleric dithiobenzoic acid (CPADB), 0.33g of N-hydroxysuccinimide (NHS) and 0.74g of DCC were weighed out and reacted in 10ml of anhydrous N, N-Dimethylformamide (DMF) at 30℃for 12 hours, followed by thin layer chromatography. After the CPADB starting point had disappeared in the reaction system, 0.387g of deprotected intermediate C was added in situ. Dropping the deprotected intermediate C, reacting at 30 deg.C overnight, washing with water, concentrating, and purifying with silica gel chromatographic column. And (3) purifying to obtain the chloroalkane terminal modified RAFT chain transfer agent A.
(2) Synthesis of chloroalkane end-functionalized glycomers.
0.1240g N-Methacrylmannosamine (MAM) (0.5 mmol) was weighed, 0.0024g of the chloroalkane-modified RAFT chain transfer agent A prepared in the above step (1) and 0.0004g of AIBN were dissolved in 3mL of anhydrous DMF, the above solution was purged with nitrogen for 30min, and then the reaction flask was transferred to a glove box to react at 70℃for 12h. After the reaction, the reaction solution was transferred to a dialysis bag having a molecular weight cut-off of 3500 and dialyzed for 2 days, and the polymer was collected after lyophilization.
FIG. 4 shows the molecular structure of a composition, i.e., a polymannan containing chlorinated alkane ligands; the molecular weight of the sugar polymer is about 5000 Da. Indicating successful synthesis of polymannan with chlorinated alkylated ligands. Table 2 shows the molecular weight and Polymer Dispersion Index (PDI) distribution of the polymannans:
table 2: polymannan molecular weight and PDI distribution
(3) Preparation of modified DC with sugar Polymer function
Dissolving the polymer (namely, the sugar polymer) synthesized in the step (2) by using a sterile PBS solution, adding the sugar polymer into the culture solution of the HTP-DC cells in the embodiment 2, adjusting the final concentration to be 0.1mg/mL, sucking the culture solution after 1h, washing for 2 times by using PBS, and adding the normal culture solution to obtain the modified DC with the sugar polymer function.
Example 4: verification of binding of glycomers to HTP-DC cells
To demonstrate the binding of the glycomers to HTP-DC cells, the biotinylated glycomers functionalized with chloroalkane ends were used to act on dendritic cells and the antigen-antibody fluorescence method was used to determine whether the effect was produced.
To label a functional glycomer, a functional glycomer with a biotin label is first synthesized. The specific implementation process is as follows: 0.1112g of N-methacrylomannosamine (MAM), 0.0257g of biotin monomer and 0.0004g of Azobisisobutyronitrile (AIBN) were weighed out in 3mL of anhydrous DMF, and 0.0024 g of the chloroalkane end modified RAFT chain transfer agent A described in example 3 was weighed out in 0.5mL of anhydrous DMF. Mixing the above solutions, and then drum N 2 Deoxygenation was performed for 30 minutes. The reaction flask was transferred to a glove box and reacted at 70℃for 10 to 12 hours. After the reaction is finished, the reaction solution is transferred into a dialysis bag with 3500 molecular weight cut-off for dialysis for 2 days, and the polymer is obtained after freeze drying.
According to 5-10 ten thousand/cm 2 HTP-DC cells were seeded in culture dishes at a cell density of (C). Culturing in 5% carbon dioxide and 37 deg.C incubator, adhering overnight, washing cells twice with sterile PBS, adding the above sugar polymer without passing through cells, and collecting sugarThe concentration of the polymer solution is set to be 0.1 mg/mL-0.5 mg/mL, the polymer solution is incubated for 1h at 37 ℃, 200 mu l of sterile PPS is added after incubation to clean cells, FITC solution is added into an orifice plate after unbound polymer is removed, wherein the FITC solution is obtained by mixing FITC dye and PBS (1% fetal bovine serum) according to any proportion of 1:20-1:50, and the specific operation is that the solution obtained by mixing the FITC dye and the PBS (1% fetal bovine serum) according to the proportion of 1:36. Cells were incubated at 4℃for 1h. And (3) carrying out marking imaging on the cells by utilizing the combination of FITC and biotin, cleaning the marked cells by using sterile PBS, and observing the green fluorescence distribution condition on the cell membrane surface by using a laser confocal microscope to determine the grafting efficiency of the cells.
Fig. 5 shows fluorescence characterization of modified DCs. The sugar polymers were attached with fluorescent labels, and the grafting of the sugar polymers was observed under a fluorescent microscope. The cell membrane surface fluorescence is a labeled glycomer.
As can be seen from fig. 5, after co-incubation of the glycomer and HTP-DC, bright fluorescence was observed on the cell surface by FITC staining, whereas after blending the glycomer and unmodified DC, FITC staining failed to detect fluorescence, indicating that the glycomer molecules could be effectively anchored to the surface of the cell membrane by incubation.
Example 5: effect of sugar Polymer modified DCs on T cell immune activation Induction efficiency
To verify the immune activation efficiency of T cells under different DC induction conditions, the proliferation efficiency of T cells was first verified.
The CT26 cells are lysed to obtain whole cell antigens, and then the whole cell antigens are added into 1640 culture medium to culture the DC cells, and the DC cells are induced to mature after 48h of culture. T cells were obtained from the spleen of mice, and DC cells after staining the markers with CSFE and induction of maturation were prepared according to 1:10 were co-cultured for 48 hours, T cells were collected, and proliferation efficiency of T cells was analyzed based on the distribution of fluorescence intensity and cell number detected by flow cytometry.
The experimental groups were set as: (1) an unmodified DC block (DC); (2) an unmodified dc+cpg adjuvant group (DC-CpG); (3) lentivirus transfected CD28 modified DC group (CD 28-DC); (4) polymannan modified DC group (pMAM-DC).
The results show that the proliferation efficiency of the polymannan pMAM modified DC induced T cells is far higher than that of the traditional adjuvant and virus transfection system. About twice as much as the conventional method (see a in fig. 6).
To further verify induction of T cell differentiation capacity, the supernatants after the above culture were used for detection of cytokines TNF- α and IFN- γ (see instructions for ELIZA kit for TNF- α and IFN- γ for specific methods).
Detection method of TNF-alpha and IFN-gamma: cell culture supernatants were first assayed using TNF- α and IFN- γ ELIZA, using standard standards to make standard curves, and samples were added to the well plates where antibodies were incubated.
The results indicate that the secretion of TNF- α by pMAM-modified DC is 10-fold higher than that of unmodified DC, about 2-3-fold higher than that of conventional CpG and virus transfection systems (see B in FIG. 6). Meanwhile, the results show that IFN-gamma expression can be more efficiently expressed in the DC induced by the sugar polymer, about 8 times of that of the unmodified DC, and more than 2 times of that of the traditional CpG and virus modification (see C in FIG. 6).
As can be seen from fig. 6, the proliferation induction efficiency of mature DCs on T cells can be improved by about 80% with the aid of adjuvant CpG, while the proliferation induction efficiency of DCs modified with CD28 on T cells can be improved by about 1-fold, while the proliferation induction efficiency of DCs modified with sugar polymers on T cells can be improved by about 4-fold. In addition, modified DCs significantly promoted expression of T cell activating factors. Under the action of the adjuvant, the expression level of TNF-alpha and IFN-gamma can be improved by about 4 times, the expression level of TNF-alpha and IFN-gamma of the CD28 modified DC cell can be improved by about 4 times, and the expression level of TNF-alpha and IFN-gamma of the sugar polymer modified DC cell can be improved by about 8 times. The sugar polymer modified DC can obviously promote proliferation and activation of T cells.
Example 6: influence of glycomer-modified DC-induced T cells on specific tumor cell killing Activity
The DCs harvested in example 5 were co-cultured with T cells and with colon cancer cells, CT26 cells. After 24h, the growth of tumor cells, including morphology, number and size, was observed, and the culture supernatant was subjected to Lactate Dehydrogenase (LDH) detection, followed by analysis of tumor cell apoptosis.
The experimental components were as follows: (1) colon cancer cell group: i.e. CT26 cells not mixed with vaccine; (2) Cpg-mature DC vaccine group: namely, the CT26 cells are added with the DC vaccine mixed by the adjuvant Cpg; (3) viral transfected DC vaccine group: namely, adding a lentivirus transfected CD28 modified DC vaccine into CT26 cells, (4) modified DC vaccine group: i.e. CT26 cells were added with the polymannan modified DC vaccine.
Specifically, each group of activated T cells and CT26 cells were isolated according to 2:1, simultaneously setting independent T cells and CT26 cells as blank control, placing the blank control in a cell culture box for continuous culture for 24 hours, detecting the light absorption value at 450nm in each group by using an LDH detection kit, and comparing the killing rates of the T cells induced by different methods to tumors.
Killing = (CT 26 cell OD + T cell OD-experimental OD)/CD 26 cell OD x 100%.
As can be seen from fig. 7, the adjuvant-assisted DC vaccine has a certain killing effect on CT26 cells, the apoptosis rate of tumor cells is close to 40%, and the DC vaccine obtained by virus modification has similar results. And the killing rate of T cells obtained by the sugar polymer modified DC to tumor cells is as high as 80%. The modified DC induced T cells have higher tumor killing capacity.
As can also be seen from fig. 7, the modified DC-induced T cells had higher toxicity to tumor cells, with about 80% decrease in tumor cell viability, while adjuvant-helper DCs and virus-transfected DC-induced T cells had some toxicity to tumor cells, with about 50% decrease in tumor cell viability. Demonstrating that the glycomer modified DC-induced T cells have higher tumor toxicity than previous methods. It can be seen from a in fig. 7 that the T cells induced by the glycomer modified DC have a strong tumor killing effect, but the T cell tumor killing effect induced by the methods of virus transfection and adjuvant assistance is not very remarkable. These results indicate that the use of the glycomer modified DCs as vaccine formulations will exert a greater tumor killing effect than conventional methods.

Claims (17)

1. A method for modifying dendritic cells based on a cell membrane surface modification technique, the method comprising the steps of:
1) Synthesizing a substrate for photothermal transfection;
2) Coating a plasmid containing a gene sequence encoding a Halotag protein with Polyetherimide (PEI) and placing said plasmid on the surface of said substrate in step 1);
3) Performing transfection under laser to prepare modified dendritic cells;
4) Synthesizing a functional sugar polymer molecule;
5) Mixing and incubating the functional glycomer molecule of step 4) with the modified dendritic cell of step 3) to obtain a functional glycomer molecule modified dendritic cell;
the photothermal transfected substrate comprises nano particles and a flexible substrate, wherein the nano particles are multi-wall carbon nanotubes, and the flexible substrate is Polydimethylsiloxane (PDMS);
wherein, the preparation method of the base material in the step 1) comprises the following steps:
the flexible substrate and crosslinking agent were combined at 50: mixing uniformly in a mass ratio of 1-5:1, adding nano particles of 0.5-10 per mill of the flexible substrate, mixing uniformly again, heating, curing and forming a film, thereby obtaining the substrate for photo-thermal transfection.
2. The method for modifying dendritic cells based on a cell membrane surface modification technique according to claim 1, wherein the mass ratio of the flexible substrate and the crosslinking agent is 10:1, 10 per mill of nanoparticles are added to the flexible substrate.
3. The method for modifying dendritic cells based on cell membrane surface modification technique according to claim 1 or 2, wherein the concentration of the plasmid in step 2) is 0.5 to 2. Mu.g/10 4 The number of cells, and the plasmid is placed on the surface of the substrate in step 1) for 1 to 3 hours.
4. The method for modifying dendritic cells according to claim 3, wherein the method is based on a cell membrane surface modification techniqueThe method, wherein the concentration of the plasmid is 1.5. Mu.g/10 4 Individual cells.
5. The method for modifying dendritic cells based on cell membrane surface modification technique according to claim 1 or 2, wherein the specific operation of step 3) is: inoculating dendritic cells on the surface of the substrate containing the plasmid in the step 2), adding the plasmid coated with PEI and containing a gene sequence for encoding Halotag protein again after the dendritic cells are attached, transfecting under laser, and continuously culturing the cells for 36-60 h;
wherein the dendritic cells have an seeding density of 10 4 ～10 5 Individual/cm 2 The laser intensity is 1-10W/cm 2 The irradiation time of the laser is 5 to 60 seconds.
6. The method for modifying dendritic cells based on a cell membrane surface modification technique according to claim 5, wherein the seeding density of the dendritic cells is 5X 10 4 Individual/cm 2 The laser intensity is 1-4W/cm 2 The irradiation time of the laser is 20 to 60 seconds.
7. The method for modifying dendritic cells based on a cell membrane surface modification technique according to claim 6, wherein the laser intensity is 2W/cm 2 The irradiation time of the laser was 30s.
8. The method for modifying dendritic cells based on cell membrane surface modification technique according to claim 1 or 2, wherein the preparation method of the functional glycomer molecule in step 4) is as follows:
(i) Synthesis of intermediate products: 2- (2-Boc-aminoethoxy) ethanol, anhydrous Tetrahydrofuran (THF), anhydrous N, N-Dimethylformamide (DMF), sodium hydride (NaH) and 6-chloro-1-iodohexane (6-chloro-1-iodohexane) are used as raw materials for reaction, separation and purification, deprotection and intermediate product synthesis are carried out;
(ii) Synthesizing a chain transfer agent containing a chloralkane structure: dissolving a chain transfer agent, NHS and DCC in anhydrous DMF, placing the anhydrous DMF for reaction for 10 to 15 hours at the temperature of 28 to 32 ℃, adding the intermediate product synthesized in the step (i) in situ after the raw material point of the chain transfer agent in a reaction system disappears, reacting overnight at the temperature of 25 to 35 ℃, and washing, concentrating and purifying the solution obtained after the reaction to obtain the chain transfer agent containing chloralkane structure;
(iii) And (3) placing the chain transfer agent containing the chloralkane structure, the sugar monomer and the initiator in the step (ii) at 60-80 ℃ for reaction for 10-12 hours to carry out polymerization reaction, transferring the reaction solution into a dialysis bag with the molecular weight cutoff of 3500Da for dialysis after the reaction is finished, and freeze-drying to obtain the functional sugar polymer.
9. The method for modifying dendritic cells based on a cell membrane surface modification technique according to claim 8, wherein the chain transfer agent is one or more selected from the group consisting of aliphatic thiol, dodecyl thiol, water, ether, acid, ester, quinone, carbon dioxide, oxygen and alcohol; the sugar monomer is selected from more than one of mannose modified by double bonds, mannosamine, glucose, glucosamine, galactose, galactosamine, fructose, fructosamine, arabinose, arabinamine, ribose, ribosamine, xylose, xylitol, lyxose, ly Su Tangan and glyceraldehyde; the initiator is azo initiator.
10. The method for modifying dendritic cells based on a cell membrane surface modification technique according to claim 9, wherein the acid is an organic acid.
11. The method for modifying dendritic cells based on a cell membrane surface modification technique according to claim 9 or 10, wherein the chain transfer agent is 4-cyanovaleric dithiobenzoic acid (CPADB); the sugar monomer is N-methacryloyl mannosamine; the initiator is Azobisisobutyronitrile (AIBN).
12. The method for modifying dendritic cells based on the cell membrane surface modification technique according to claim 8, wherein a biotin monomer is added in the polymerization reaction in (iii).
13. A dendritic cell produced by the method of any one of claims 1 to 12.
14. A vaccine composition comprising the dendritic cells of claim 13 and one or more pharmaceutically acceptable carriers, adjuvants and/or excipients.
15. Use of the dendritic cells of claim 13 in the manufacture of a medicament for the treatment and prevention of malignancy.
16. The use of claim 15, wherein the malignancy is any one of lung cancer, liver cancer, colon cancer, melanoma, prostate cancer, stomach cancer, breast cancer, chronic Lymphocytic Leukemia (CLL), or chronic myelogenous leukemia.
17. The use of claim 16, wherein the malignancy is colon cancer.
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