Study Of Simultaneous Silencing CD40 Genes In Macrophages And Dendritic Cells Inducing Immune Tolerance Using A Novel SiRNA Delivery System

Background and object:In recent years,the rapid development of nanotechnology results in the widely application of Nanoparticles in medicine and biology,and Nanoparticles play a very important role in the diagnosis and treatment of many diseases.Nanoparticles can achieve efficient delivery of drug molecules,including polypeptides,proteins,nucleic acids,small molecule drugs,etc.,by adsorbing drug molecules on the surface of the particles or encapsulating them inside.CD40 is a member of the tumor necrosis factor superfamily,mainly expressed in the antigen-presenting cells.CD40 / CD40 L pathway is an important costimulatory pathway in T lymphocyte activation.Tissue and organ transplantation is the most important treatment of end-stage organ failure.However,the transplant tissue,organ failure caused by transplanted immune rejection and side effects always plague patients.Nowadays,chronic transplantation immune rejection is the most important problem that needs to be solved to induce long-term transplant immune tolerance.RNA interference is a biological process in which RNA molecules inhibit gene expression or translation,by neutralizing targeted mRNA molecules.Because siRNA is unstable in the physiological environment and difficult to cross the cell membrane,so the in vivo treatment of siRNA requires a stable drug delivery system.The object of our study was to develop a novel siRNA delivery system to down-regulate the expression of CD40 in macrophages and DCs ex & in vivo and prolong the survival of allogeneic skin graft.Methods:In this study,PEG5k-PLGA10 k nanoparticles loaded with siRNA were prepared by double emulsion method.The particle size and zeta potential of nanoparticles were characterized by Zetasizer.The morphology of nanoparticles was observed by Cryo-TEM.PLGA/Cy5-siRNA NPs,PLGA/FAM-siRNA NPs were co-cultured with DC1.2,RAW264.7 cells and then analyzed by flow cytometry and laser confocal microscopy.Furthermore,DC1.2 and RAW264.7 cells were collected for analysis of CD40 expression by quantitative PCR(q PCR)after co-cultured with PLGA/si CD40 NPs.The C57BL/6 mice were sacrificed and PBMCs,splenocytes and bone marrow cells were collected for flow cytometry to analyze the PLGA/Cy5-siRNA NPs distribution in macrophages and DCs 16 h after PLGA/Cy5-siRNA NPs i.v.injection.The C57BL/6 mice were sacrificed and bone marrow cells were separated,bone marrow-derived dendritic cells were induced in vitro and LPS was added for the stimulation.The cell markers related to activation of BMDCs were analyzed by flow cytometry.Then,We evaluated the biological effects of PLGA/si CD40 NPs in vivo in Balb/c to C57BL/6 mice skin allogeneic transplantation model.Results:1.The particle size of PLGA/si CD40 NPs prepared by double emulsion method was about 103 nm,the zeta potential was 27.5m V,and the nanoparticles showed a regular spherical structure in the aqueous solution.2.PEG5k-PLGA10 k nanoparticles can deliver siRNA into DC1.2,RAW264.7 cytoplasm in vitro.3.PLGA/si CD40 NPs can down regulate of CD40 expression in DC1.2 and RAW264.7 cells.4.PLGA/Cy5-siRNA NPs can deliver siRNA into dendritic cells and macrophages in vivo.5.PLGA/si CD40 NPs can hinder the LPS stimulated activation of BMDCs.6.PLGA/si CD40 NPs can prolong the survival of allogeneic skin grafts.Conclusion:1.PLGA/si CD40 NPs can be successfully prepared by double emulsion method.2.PEG5k-PLGA10 k can deliver siRNA into cytoplasm ex & in vivo.3.PLGA/si CD40 NPs can deliver CD40 siRNA and significantly down regulate of CD40 expression ex & in vivo.4.PLGA/si CD40 NPs can significantly prolong the survival of allogeneic skin grafts in Balb/c to C57BL/6 mice skin allogeneic transplantation model.