| Background and objective: Autoimmune diseases are chronic disorders of the immune system that affect millions of people worldwide and,in severe cases,can be fatal.Current clinical treatments are often difficult to cure and long-term use can reduce the body’s immune response,increasing the risk of infection and cancer.Immune tolerance is necessary to prevent the immune system from responding against itself,and co-stimulatory molecules targeting antigen-presenting cells and T cells have potential in the treatment of autoimmune diseases by modulating T cell function to induce immune tolerance.CD40 is widely expressed on the surface of antigen presenting cells and its costimulatory pathway with its ligand CD40 L is essential for T cell activation in autoimmune diseases.The development of antibodies against CD40 L has been halted due to the development of thrombosis in clinical trials.The development of alternative therapeutic approaches against the CD40 pathway is critical.RNA interference(RNAi)refers to the silencing of gene expression in an organism by targeting the m RNA of a target gene in a cell.The introduction of exogenous synthetic small interfering RNA(siRNA),which can induce RNAi through specific complementary base pairing,has specific and efficient knockdown effects on disease-associated genes.However,siRNA face challenges in vivo,such as low efficiency of cellular endocytosis and susceptibility to degradation by nucleases,and a carrier is needed to protect them and deliver them efficiently to target cells.Nano transport systems have emerged as an ideal solution.Nanoparticles show great potential for siRNA delivery due to their flexible size,structure and modifiable surface modifications.The use of PEG-PLGA nanoparticles as carriers of CD40 siRNA has been shown to target antigen-presenting cells and induce immune tolerance,which has been therapeutically effective in allogeneic skin graft rejection assays and experimental allergic encephalomyelitis(EAE)models.However,the therapeutic efficacy of NPs/siCD40 alone is limited and the combination of immunosuppressive agents or other siRNAs is required to achieve better therapeutic efficacy.It has been shown that modification of the maleimide functional group at the end of PEG can improve the circulation time and targeting ability of nanoparticles in vivo through the maleimide-mercapto reaction,therefore we are investigating whether the efficiency of the nanoparticle delivery system can be improved by maleimide functional group modification of PEG-PLGA nanoparticles to enhance the therapeutic effect in autoimmune diseases.The aim of this study was to optimize nanoparticles by maleimide functional group modification to increase the efficiency of siRNA delivery by nanotransport systems and the ability to reduce CD40 expression levels in dendritic cells at in vivo and in vitro levels to improve therapeutic efficacy for EAE.Methods:In this study,Mal-PEG-PLGA nanoparticles and siRNA-encapsulated PEG-PLGA nanoparticles were first synthesized by double emulsification method,and then the particle size and surface potential of the nanoparticles were characterized using dynamic light scattering(DLS).Mal-NPs/Cy5-siNC and NPs/Cy5-siNC were cocultured with bone marrow-derived dendritic cells,and the ability of both nanoparticles to deliver siRNA in vitro was determined by flow cytometry.Mal-NPs/siCD40 and NPs/siCD40 were co-cultured with DC1.2 and RAW264.7 cells,and CD40 expression on the cells was detected by flow cytometry and q PCR.C57BL/6 mice were injected with Mal-NPs/Cy5-siNC and NPs/Cy5-siNC in the tail vein,and small animal live imager was applied to detect the nanoparticles.C57BL/6 mice were injected with MalNPs/siCD40 and NPs/siCD40 in the tail vein,and the bone marrow cells were isolated,and the bone marrow-derived dendritic cells were induced in vitro and stimulated with LPS.The ratio of dendritic cells and CD40 expression levels were determined by flow cytometry.The EAE mouse model was used to verify the biological effects of MalNPs/siCD40.Results:1.Mal-NPs/siCD40 and NPs/siCD40 were prepared by double emulsification with particle sizes of 149.33±3.57 nm and 95.34±3.75 nm,respectively,and surface potentials of 37.4±0.79 m V and 37.7±0.70 m V,respectively.2.Mal-NPs/siCD40 was able to deliver siRNA to bone marrow-induced dendritic cells in vitro and had a greater ability to silence cellular CD40 expression in vitro compared to NPs/siCD40.3.Compared with NPs/Cy5-siNC,Mal-NPs/Cy5-siNC was more enriched in major mouse organs,as well as in mouse DCs and macrophages.4.Compared to NPs/siCD40,Mal-NPs/siCD40 inhibited myeloid differentiation and silenced CD40 expression more.5.Compared with NPs/siCD40,Mal-NPs/siCD40 was more effective for the treatment of EAE models.Conclusion:In this study,maleimide-modified PEG-PLGA nanoparticles(Mal-NPs/siCD40)encapsulated with siRNA were successfully prepared using a double emulsification method.The maleimide modification enhanced the ability of NPs to deliver siRNA to major organs and peripheral blood,spleen,bone marrow DC and macrophages in mice;and enhanced the ability of NPs/siCD40 to silence CD40 expression on bone marrow DC.Maleimide modification in a mouse model of multiple sclerosis enhanced the disease-modifying effect of NPs/siCD40.This study provides a new strategy for the optimization of nano-delivery systems and thus new research ideas for future intervention and treatment of autoimmune diseases. |
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