| Ovarian cancer is the most lethal gynecological malignancy.Conventional chemotherapy has limited efficacy,while most patients will eventually relapse and become resistant to chemotherapy.In recent years,with the improvement of people's understanding of the molecular mechanism of immune recognition and the regulation of tumor immunity,immunotherapy is expected to become an effective alternative therapy.Dendritic cell-based cancer vaccine has achieved certain efficacy in ovarian cancer,showing good application prospects.However,dendritic cell vaccine has difficulties in storage,low lymph node enrichment,poor anti-apoptotic ability and low antigen presentation efficiency.The artificial biomimetic antigen-presenting cell vaccine prepared by bioengineering method may solve the above problems and may open up a new way for the prevention and treatment of ovarian cancer.ObjectiveThe purpose of this study is to prepare the biomimetic tumor nanovaccine(denoted“mini DC”)specific for ovarian cancer through the cell membrane coating nanotechnology and to explore the its ability of immune stimulation and the feasibility of its application in the prevention and treatment of ovarian cancer as well as the associated mechanism.Content1,To preparation,optimization and characterization of the b iomemetic tumor nanovaccine(denoted“mini DC”)specific for ovarian cancer.2,To investigate the effects of mini dc on the activation,proliferation and cytokine secretion of T cells in vitro and in vivo.3,To explore the therapeutic and preventive effects of mini DC on ovarian cancer in mouse ovarian cancer model and the mechanism behind it.4,To assess the biosafety of mini DC both in vitro and in vivo.MethodsPoly(lactic-co-glycolic acid)nanoparticle(PLGA-NP)loaded with IL-2 was prepared by double emulsion method and the preparation procedure was optimized by adjusting the volume ratio of water phase and oil phase.The mouse ovarian cancer ID8cells were treated with hypochlorite solution,and the dead tumor cells were ground into cell lysates with a Dounce homogenizer.Bone marrow derived dendritic cells were extracted,and the dendritic cells were stimulated by the above tumor lysates.Then mature dendritic cells loaded with tumor specific antigens were obtained.Ultracentrifugation was used to extract the cell membranes of the above-mentioned tumor antigen-pulsed dendritic cells.For the prepration of mini DC,DC membranes were mixed with PLGA-NP at a certain ratio and then the mixture was physically extruded through a miniextruder.The particle size and surface potential of mini DC were determined by dynamic light scattering(DLS).The morphology of mini DC was observed by transmission electron microscope.Western Blot and BCA assay were used to detect the composition and content of proteins on the surface of mini DC.And the direction of proteins on the surface of mini DC was determined by fluorescence quantitation.The encapsulation efficiency of IL-2 in PLGA-NP and release of IL-2 by PLGA-NP and mini DC were measured by ELISA.The effect of mini DC on the proliferation of normal cells in vitro was detected by CCK8 method.The adhesion of mini DC to T cells and NIH-3T3 cells was determined by confocal laser scanning microscope and flow cytometry.The activation and expansion of mouse primary T cells after co-culture with DC was determined by flow cytometry.The ability of mini DC to stimulate T cells to produce cytokines IFN-?and TNF-?in vitro was detected by ELISA.The biological distribution of mini DC was observed by in vivo imaging system after mice were immunized with mini DC subcutaneously.The proportion of T cell subsets in draining lymph nodes and spleen was detected by flow cytometry,and the levels of IFN-?and TNF-?in peripheral blood were detected by ELISA.T cells from spleens of immunized mice were isolated and their specific cytotoxicity for ovarian cancer cells was detected by calcein-AM retention assay.Mouse subcutaneous tumor model and peritoneal metastasis model of ovarian cancer were constructed.The therapeutic effect of mini DC on subcutaneous tumor and its preventive effect on metastasis were assessed by measuring the size of tumor,the weight of mice and the number of abdominal metastasis.The peripheral blood serum of tumor bearing mice was collected to detect the liver and kidney function.The tumor masses were collected and sectioned.The infiltration of T cells in tumor tissue was detected by immunofluorescence staining and the apoptosis of tumor cells was detected by TUNEL staining.HE staining of major organs from tumor bearing mice was used to detect the biosafety of mini DC.ResultsThe particle size of PLGA-NP was 184.3±0.82 nm,and the surface potential was-14.4±0.57 m V.The particle size of mini DC was 204.5±0.85 nm,the surface potential was-22.7±0.66 m V.Under transmission electron microscope,it was found that mini DC was round,well dispersed and had obvious core-shell structure.2.The loading amount of IL-2 in PLGA-NP was about 1.66ng/mg PLGA-NP,and PLGA-NP and mini DC displayed similar release pattern and total release of IL-2 was not different in 5 days,while mini DC released IL-2 in a relatively more sustained way.The results of Western Blot showed that there were important surface proteins CD11c,CD86 and CD40 of dendritic cells on the surface of mini DC.According to the gray value of protein bands,about 2×10~6 dendritic cells and mini DC with 25?g protein content has the same amount of these surface proteins.Additionally,once lyophilized and preserved at-20?,mini DC showed good stability as the protein content remined over 80%within 4 weeks and over 65%within 8 weeks.Compared with PLGA-NP,mini DC has higher adhesion ability to primary T cells,but their adhesion abilities to NIH-3T3 cells were same.After co-culture of mini DC with 293T cells and NIH-3T3cells in vitro,the proliferation of the two cell lines was not affected by mini DC.After co-culture of mini dc with T cells in vitro for 24 hours,the proportion of activated T cells(CD8~+CD69~+)was 16.2±0.87%,which was higher than that of BMDC(4.5±0.40%).After 72 hours of co-culture in vitro,mini DC has a higher promoting effect on T cell proliferation than BMDC(49.55±0.87%VS 34.70±3.6%).T cells co-cultured with mini DC could produce more IFN-?and TNF-?.Three days after subcutaneous injection of fluorescence labeled mini DC into the base of tail,in vivo imaging results showed that mini DC mainly accumulated in the draining lymph nodes,while there was no obvious fluorescence signal in the organs such as heart,liver,spleen,lung and kidney.In the spleen of mice immunized with mini DC,the percentage of effector T cells(IFN-?~+CD8~+)is higher and the percentage of regulatory T cells(Foxp3~+CD25~+CD4~+)is lower.In the draining lymph nodes,the percentage of CD8~+T cells in the mini DC-immunized group is higher than that of other groups.T cells extracted from the spleen of mini DC-immunized mice had higher cytotoxic effect on ovarian cancer cells in vitro.Mini DC could effectively inhibit the growth of subcutaneous tumor of mice,and the tumor size at the end of the experiment was 31.2±12.99 mm~2.Compared with other groups,mini DC had the best therapeutic effect,and the percentage of effective T cells in spleen is the highest,while the percentage of regulatory T cells is the lowest.Immunofluorescence staining of tumor section shows that the most infiltrated CD8~+T cells and apoptosis cells were observed in mini DC group.Mini DC could effectively prevent the formation of ovarian cancer metastasis in mice abdominal cavity.The number of metastatic tumors on the abdominal wall and gastrointestinal tract of mini DC-immunized mice were 14.4±4.8 and 2.6±1.1,respectively,both of which were the least in all groups.The liver and kidney function of mice treated with mini DC had no significant difference compared with that of PBS group,and the HE staining of main organ sections showed no significant organ damage.ConclusionsThe biomimetic nanovaccine specific for ovarian cancer,mini DC,was successfully prepared.The resulting nanovaccine was coated with membrane protein of dendritic cells,possessing the same antigen presentation and T cell activation function as dendritic cells.Meanwhile,mini DC can also release IL-2 in the form of paracrine.Because of its nano size,mini DC can rapidly accumulate in lymph nodes and interact with T cells more frequently.Therefore,compared with DC,mini DC has higher T cell activation efficiency in vitro and in vivo.It shows better therapeutic and prophylactic effect in mouse subcutaneous tumor model and peritoneal metastasis model of ovarian cancer,and has no obvious toxic and side effects.It has potential application and is expected to open up a new way for prevention and treatment of ovarian cancer. |
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