Imaging Inorganic Nanoparticles As Immune Adjuvants To Enhance Dendritic Cell-based Cancer Immunotherapy

Immune system can protect human body against various infectious diseases.In recent years,tumor immunotherapy,based on its own unique advantages and promising future development,has received extensive attention.In tumor immunotherapy,immune system is able to recognize and eliminate uncontrolled tumor cells,which is called immunological surveillance.Effective process of immune response to tumor needs three indispensable steps.Firstly,antigens expressed on the surface of tumor cells are phagocytized by antigen-presenting cells（APCs）.Secondly,activated APCs migrate to lymph node,and degrade antigen proteins into antigen peptides which will combined with major histocompatibility complex（MHC）,then present MHC-peptide to naive T cells and release cytokines at the same time,leading to the initiation and activation of effector T cells.Thirdly,activated effector T cells migrate and infiltrate into tumor area,then specifically target and kill tumor cells through T cell receptor and MHC-peptide.With the development of nanotechnology,nanomaterials have been widely used in tumor immunotherapy based on their numerous advantages,such as huge specific surface area,delivery function as carrier and controllable surface chemistry.Besides,tumor specific antigens（TSAs）and tumor associated antigens（TAAs）will be released from the residual of dead tumor cells after tumor cells are killed through tumor photothermal therapy（PTT）activated by near-infrared（NIR）laser.With the help of immune adjuvants,dendritic cells（DCs）near tumor microenvironment are stimulated into activation and induce the following immune response to tumor,achieving a better treatment effects by the combination of tumor immunotherapy and PTT.This thesis is about nanomaterials used in the application of tumor immunotherapy and the associated combination therapy,the main research work is summed up as follows:Firstly,nanocomposite system is established through the combination of fluorescent carbon dots（CDs）and ovalbumin（OVA）antigens,for the first time achieving that fluorescent CDs are used in the application of tumor immunotherapy.In this thesis,CDs with fluorescent imaging function act as carrier delivery system and immune adjuvant,and OVA act as tumor antigen model.CDs are successfully synthesized with the uniform size of 1-5 nm,and the excellent fluorescence performance with multiple emission wavelength after different excitation wavelength is studied.CDs-OVA nanocomposites are successfully synthesized through covalent binding of the surface functional groups of CDs and OVA.At the cellular level,obtained CDs-OVA nanocomposites are able to activate DCs from immaturation into maturation with the corresponding release of tumor necrosis factorα（TNF-α）cytokines.Then mature DCs are able to activate T cells to accordingly release interferonγ（IFN-γ）cytokines in vitro.Furthermore,spleen cells obtained from mice vaccinated with CDs-OVA can induce strong proliferation after re-stimulation of OVA antigens.At the animal level,this vaccine can induce strong immune response to tumor cells,leading to dramatic inhibition of the B16-OVA melanoma growth.Secondly,tumor vaccine system is established by the combination of ultra-small Fe₃O₄ nanoparticles（Fe₃O₄ NPs）and OVA antigens.It is for the first time that Fe₃O₄NPs act as nano-immunopotentiator to achieve effective immunotherapy and prevention of malignant melanoma and the metastatic melanoma of lung.In this thesis,obtained Fe₃O₄ NPs with the size of 5 nm are combined with OVA through the covalent binding of their surface functional groups,acquiring Fe₃O₄-OVA nanocomposites with the uniform size of 20-40 nm.At the cellular level,this nanocomposites can not only efficiently stimulate bone marrow derived DCs into maturation and induce T cells to release IFN-γcytokines but also potentially stimulate macrophages to largely secrete TNF-αcytokines.At the animal level,with the help of Fe₃O₄ NPs nano-immunopotentiator,Fe₃O₄-OVA vaccine can not only efficiently inhibit the growth of subcutaneous and metastatic B16-OVA melanoma but also successfully prevent the formation of subcutaneous and metastatic B16-OVA melanoma.More importantly,Fe₃O₄ NPs nano-immunopotentiator can not only be carrier to protect antigens against degradation and inactivation but also act as efficient potentiator participating in tumor immunotherapy to promote the immune response.Thirdly,nanoscale MnFeBi oxidation compound（MnFeBiO NPs）associated tumor diagnosis and treatment system is established for the application of magnetic resonance imaging（MRI）,thermal imaging and heating effect under the NIR with low power,achieving the combination of tumor imaging and treatment.In this thesis,MnFeBi compounds are synthesized through high-energy ball milling method and modified with PVP-58000 on the surface,obtaining MnFeBiO NPs with the size of 50nm and the function of T₁ and T₂ MR imaging.At the cellular level,this materials can be easily phagocytized by 4T1 mouse breast cancer cells.Under the 808 nm NIR laser（0.7 W/cm²,5 minutes）,MnFeBiO NPs can kill 80 percent of the 4T1 cells.At the animal level,MnFeBiO NPs are able to produce remarkable heating effect under 808nm NIR laser,curing the subcutaneous tumor through effective PTT method.Fourthly,the combination system of MnFeBiO NPs and immune adjuvant cytosine-phosphate-guanine-containing oligodeoxynucleotide（CpG ODN）is for the first time established,which is aimed at the combination therapy of in situ malignant breast cancer through combining PTT and tumor immunotherapy.In this thesis,electropositive MnFeBiO NPs are combined with electronegative CpG through electrostatic adsorption to form a new type of MnFeBiO NPs-CpG nanocomponds with great water solubility.At the cellular level,MnFeBiO NPs-CpG with FAM fluorophores are easily phagocytized by 4T1 tumor cells.At the animal level,MnFeBiO NPs-CpG injected into tumor can induce remarkable heating effect 808 nm NIR laser with low laser power density（0.7 W/cm²）,and induce immunotherapy at the same time,resulting in synergistic combination therapy of PTT and immunotherapy for the better treatment effect of orthotopic breath cancer.