Chitosan Nanoparticle-based Delivery Of Fused Nkg2d-il-21 Gene Suppresses Colon Cancer Growth In Mice

IL-21 is a new type I cytokine found in 2000,belonging to the IL-2 family and shares a common yc chain with others 1L-2 family members.IL-21 is mainly produced by activated CD4+T cells(including Th17 cells,follicular helper T cells and NKT cells),while IL-21 can induce cell activation,promote cell proliferation or stimulate cells to play cytotoxic effects and so on.IL-21 receptor(IL-21R)is a heterodimer consisting of IL-21 Ra and common yc chains,widely expressed not only on the surface of lymphocytes(such as NK cells,T cells,B cells and NKT cells)and bone marrow cells(such as macrophages and dendritic cells),but also non-immune cells(such as enteric fibroblasts and keratinocytes).NKG2D is a C-type lectin-like activated receptor,widely expressed by NK,NKT,CD8+ T and γδT cells.The mouse NKG2D ligands are composed of retinoic acid early transcription factors(RAE-1),H60 and murine UL-16 binding protein-like transcript 1(MULT1),and human NKG2D ligands are the major histocompatibility complex class I proteins A and B(MICA and MICB)and ULBP6-1.NKG2D ligands are not expressed on most normal tissues but virus-infected cells or tumor cells,so that NKG2D ligands can act as tumor targeting antigens in immunotherapy.In the present study,we construct the pcDNA3.1(-)plasmid vectors harboring NKG2D and IL-21 expression gene units based on the ability of NKG2D ligands targeting to tumors,in order to study the antitumor activity of dsNKG2D-IL-21 protein.The recombinant plasmid pcDNA3.1-dsNKG2D-IL21 is delivered by chitosan,which is a strong ionomer compound modified by chemical methods(HTCC),and then they form into a positive charge nanoparticle(NP)which size about 200-400nm.Nanoparticles are low toxicity to CT-26 cell,NIH-3T3 cell and RAW264.7 cell tested by MTS/PMS method.The pretransfected CT-26 cells can express the target protein dsNKG2D-IL-21 confirmed by Elisa and western blot methods,meanwhile,Elisa tests the concentration of IL-21 expressed in the serum of normal Balb/c mice injected with nanoparticles for three days.It is proved that the nanoparticles can express the target protein dsNKG2D-IL-21 at the cell level or in the host.When the tumor-bearing mice are injected with nanoparticles,their tomor volume are smaller than other groups,and they have a longer life.At the same time,the frequency of CD4+,CD8+,DX5+ cell subgroups in spleen and tumor tissues are significantly higher than other mice,and DX5 +CD69+,DX5+ CD44+,DX5+ CD44+ cells frequencies are still higher suggesting that the nanoparticles can activate NK and T Cells so that inhibit tumor growth.The twice inculated-tumor mice hardly grow tumor at the last time due to injected nanoparticles,indicate that the target protein--dsNKG2D-IL21 induce a memory immune response in the mice and produce tumor-associated antibodies.In this study,the fusion of NKG2D and IL-21 gene fragments delivered by chitosan nanoparticles,are able to express target dsNKG2D-IL-21 protein and activate CD4+,CD8+ and NK Cells.Notably,the nanoparticles can accumulate in tumor tissue and inhibit tumor growth by activating murine T cells and NK cells.Therefore,the NKG2D-IL-21 gene nanoparticles can play an effective anti-tumor activity,so it is highly possible to be used in the treatment of cancer.