Polydopamine Nanoparticles Loaded With Tumor Antigens For The Treatment Of Colorectal Cancer

With the development of surgical technique and chemotherapy,the treatment strategy of colorectal cancer(CRC)continues to improve,but it still faces severe challenges in terms of treatment efficacy and side effects.Recently,immunotherapy has been widely accepted,and has led to the breakthroughs of cancer treatment in some types of cancer,including colorectal cancer.At the same time,novel vaccines(NVs)are being developed with the potential to surpass traditional vaccines.Polypeptide vaccine that is based on recombinant antigen or polypeptide is expected to become a substitute for traditional vaccine because of its good specificity and biosafety.On the other hand,polymer nanoparticles have outstanding advantages in tumor immunotherapy,because they possess capabilities of the high antigen loading,good biocompatibility and flexible modifiability.Thus nanoparticle vaccine,a type of NVs,is attracting more and more attention.However,how to effectively activate anti-cancer immunity in vivo to achieve the purpose of tumor treatment still remains as a major challenge in the development of vaccines.In this thesis,poly dopamine nanoparticles(PDANPs)with good biocompatibility and high loading capacity were used to load tumor model antigen OVA or tumor lysate.We investigated their immunological evaluation in vitro and in vivo and tested their anti-tumor effect.The effect of PDA NPs loaded with single antigen and complex antigens on the anti-tumor immunity was evaluated with the colorectal cancer bearing mice,which provided basic experimental data and references for the application of PDANPs in the tumor immunity field.The thesis is divided into the following three parts.The first part describes the construction of OVA-GMC38 colon cancer cell line carrying tumor model antigen OVA and green fluorescent protein(GFP).Firstly,the lentivirus vector carrying double genes,OVA and GFP genes,was designed and constructed.Then puromycin.was used to screen for positive OVA-GMC38 cells among the lentivirus infected MC38 cells.The stable expression of GFP and OVA genes in MC38 cells was detected by laser confocal microscope,flow cytometry,Western Blot and quantitative reverse transcriptase polymerase chain reaction(qRT-PCR).The effects on the establishment of tumor-bearing mice models with OVA-GMC38 subcutaneous implantation in C57BL/6 mice were investigated.Compared with MC38,the results showed that the transgene had no effect on the tumorigenic ability of OVA-GMC38.Using OVA to immunize tumor-bearing mice,the preliminarily result showed that the OVA-GMC38 can be applied to evaluate the immunotherapy efficacy against OVA antigen in colorectal cancer.The second part investigated the characteristics and efficacy of PDA NPs loaded with model antigen OVA(OVA@PDANPs).OVA@PDANPs was prepared as a tumor vaccine.Observed under the particle size analyzer and scanning electron microscope,the nanoparticles appeared to be uniform and monodisperse spheres.Cytotoxicity experiments showed that nanoparticles had good biocompatibility.By observation of the cellular phagocytosis of nanoparticles by confocal microscope,it was found that nanoparticles could enhance the phagocytosis of antigens,and a part of the nanoparticles that were internalized by the cells may escape from the lysosomes.In vivo imaging showed that OVA@PDANPs promoted the accumulation of antigens in lymph nodes and prolonged the retention time of antigens.The flow cytometry analysis showed that OVA@PDA NPs could promote the expression of mature markers on the surface of DCs in lymph nodes.The tumor-bearing mouse model with OVA-GMC38 colon cancer was established.The immunization experiment on tumor-bearing mice showed that OVA@PDANPs could significantly inhibit the tumor growth and increase the proportion of CD4+ and CD8+ T cells in spleen,lymph nodes and tumor tissues.OVA@PDA NPs promoted T cell response and T cell proliferation.At the same time,OVA@PDA NPs improved the microenvironment of intratumoral immunosuppression.The results showed that OVA@PDA NPs has a good immunostimulatory effect and has the potential to be used as a tumor vaccine.The third part focused on the investigation of polydopamine nanoparticles(TCL@PDA NPs)loaded with tumor lysate.Polydopamine nanoparticles were prepared by dopamine self-polymerization,and then the tumor lysate(TCL)was covalently connected to the surface of PDA NPs by Michael addition and Schiff base reaction.The average size of TCL@PDANPs was 241.9 nm and the polydispersion index was 0.200.It had good storage stability and low cytotoxicity to bone marrow-derived dendritic cells(BMDC).In addition,TCL@PDANPs promoted antigen uptake and DC maturation,resulting in increased expression of markers related to antigen presentation on the surface of DC cells and secretion of cytokines.After immunizing tumor-bearing mice with TCL@PDANPs,the tumor progress was significantly delayed and the cytotoxic CD8+ T cells and memory T cell subsets were significantly increased,thus providing long-term protection against tumor.In addition to a significant increase in CTL,we observed a significant decrease in intratumoral Ml TAM and bone marrow-derived immunosuppressive cells,which suggests that TCL@PDA NPs has outstanding anti-tumor ability.In summary,as an antigen delivery carrier,PDA NPs is a promising candidate for antigen delivery because of its simple and rapid preparation,effective activation of immunity and outstanding anti-tumor ability.