| Malignant tumor is a great challenge to human health and safety.Tumor vaccines are considered to be one of the most promising new cancer treatments by activating the human immune system to eliminate tumor cells.Tumor-associated carbohydrate antigen（TACA）,which is abundantly expressed on the surface of tumor cells,has high specificity and conservative chemical structure,making it an ideal target for tumor vaccine development.Among them,tumor-associated mucin 1（Mucin 1,MUC1）with specific glycosylation is overexpressed in a variety of malignancies and has been widely used to develop tumor diagnosis and treatment options.However,MUC1 has some problems such as weak immunogenicity and T cell-independent antigen.How to design an effective vaccine structure has become a key issue in vaccine development.In addition to the traditional protein carrier vaccine design strategy,some novel biomaterials and nanoparticles were developed for constructing MUC1-based vaccines and showed promising results.In this paper,starting from chitosan with immunostimulatory activity,we designed and synthesizedβ-cyclodextrin-grafted chitosan（CS-g-CD）as a carrier to assemble a polypeptide vaccine of MUC1 by using host-guest interaction.A nanoparticle vaccine was further prepared,and the immunological activity of the vaccine was evaluated by in vitro cell experiments and in vivo immune activity studies in mice,which proved that the vaccine design effectively improved the immunogenicity of the MUC1 vaccine.The main results are as follows:（1）Construction and characterization of CS-g-CD/MUC1 nanoparticle vaccine.Four polypeptides,MUC1,ada-ACP-MUC1,ada-ACP-MUC1-Tn and ada-ACP-MUC1-FITC,were synthesized by solid-phase synthesis with yields of 21.21%,38.9%,27.1%,and 23.51%,respectively.After purification,both high-performance liquid chromatography and mass spectrometry identifications were correct.Three kinds of CS-g-CD with different cyclodextrin grafting ratios were synthesized by chemical method,and the grafting ratios were 2.60%,9.47%and 15.60%respectively.Among them,CS-g-CD with a grafting ratio of 15.60%has the best solubility.The adamantane-modified MUC1 polypeptide was self-assembled with CS-g-CD through host-guest interaction,and the antigen loading was detected by high performance liquid chromatography.The antigen loading was above 38%;and the vaccine was further prepared into nanoparticles by ion gel method.The particle size was characterized by transmission electron microscopy and dynamic light scattering,and the size was about 100 nm.The surface of the nanoparticles encapsulating the polypeptide was positively charged.（2）In vitro safety evaluation and in vitro cellular uptake activity evaluation of CS-g-CD/MUC1 nanoparticle vaccine.Through in vitro cell experiments,it was found that the concentration of the nanoparticle vaccine from 20-80μg·m L-1 had no obvious toxic effect on the RAW264.7 and HEK-293 cells,which proved that the constructed nanoparticle vaccine had good biological safety.And through the uptake experiment of the fluorescent nanoparticle vaccine by antigen-presenting cells,it was found that dendritic cells showed stronger phagocytosis to the vaccine in the form of nanoparticles,which proved that the nanoparticle form can promote the uptake of antigens.（3）Evaluation of in vivo immune activity of CS-g-CD/MUC1 nanoparticle vaccine.Through in vivo immunization experiments in mice,it was found that all three groups of vaccines could produce specific antibodies,among which CS-g-CD/ada-ACP-MUC1 vaccine produced the highest antibody titer,proving that the vaccine can produce high titer specificity Antibody.Through flow cytometry and complement-dependent cytotoxicity experiments,it was found that the antibodies produced by the immunized mouse sera can specifically recognize and bind to tumor cells compared with the sera of the unimmunized mice,which proves that the immunized antibodies can specifically recognize antigen and activate the complement system to kill tumor cells. |
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