The Effect And Mechanism Of Nanoparticles Co-delivering VD3 And Lkb1-siRNA On Experimental Autoimmune Encephalomyelitis

BackgroundMultiple sclerosis(MS)is a chronic autoimmune disease with chronic inflammatory demyelinating of white matter as the main pathological manifestation,and there is no effective clinical treatment.Autoimmune diseases are caused by the imbalance of the body’s immune system and the immune response to its own antigens.Autoreactive CD4+T cells are the main pathogenic cells of autoimmune diseases.Regulatory T cells(Treg)have the effect of suppressing immune response and regulating immune tolerance,which can effectively reduce the onset and progression of autoimmune diseases.In vitro expansion and reinfused Treg has become a research hotspot for the treatment of autoimmune diseases,however,in vitro expansion of Treg has the limitations of low efficiency,long cycle,poor cell function and stability.Therefore,induction of amplification of Treg under in vivo conditions becomes key to improving the efficacy of autoimmune diseases.DCs cells,as the most powerful group of antigen presenting cells in vivo,play a crucial role in the pathogenesis of autoimmune diseases.DCs can induce the differentiation of primitive CD4+T cells into autoreactive T cells,while tolerant DCs,as a special type of DCs,can induce proliferation of Tregs and inhibit the function and proliferation of effector T cells.VD3(1α,25-hydroxyvitaminD3)is a steroid derivative and active form of vitamin D.At the same time,as an immunomodulator,it can also induce DCs differentiation to tolerance phenotype by regulating DCs costimulatory signal level,so as to further induce Treg proliferation.Lkb1 is a central regulator of cell metabolism and growth.Deletion of Lkb1 leads to increased glycolytic levels and enhanced immunogenicity of peripheral dendritic cells,but this phenotype with the ability to enhance maturation also allows DCs to increase thymus Treg function and amplification under homeostasis conditions.Both different mechanisms of treatment can affect the state of DCs cells,but how to achieve the regulation of DCs co-stimulation signal and metabolic level at the same time has become a problem to be solved in this topic.RNA interfere(RNAi)provides a solution for targeting the expression of target genes.However,due to the limitations of nucleic acid delivery,biodegradable polymer nanoparticles can be used as a multifunctional platform for transmitting signals to the immune system.It can be loaded with mutiple drugs,while being easily surface-modified to target specific receptors and designed to release encapsulated drugs in a precise,sustained manner,improving drug solubility and extending drug half-life.And it also has the functions of reducing toxic side effects and synergistic drugs to penetrate biological barriers.ObjectiveNanomedicine technology and RNA interference technology were used to develop a nanodrug particle carrying the immunomodulator VD3 and small interfering RNA and delivered it to DCs,so as to achieve the purpose of regulating dendritic cell phenotypic differentiation and amplifying Treg.Moreover,the experimental autoimmune encephalomyelitis(EAE)animal model was used to investigate the therapeutic effect of nanodrug particles on this disease and explore the mechanism.Methods(1)Synthesis of NPs/VD3/siLkb1 nanodrug particles containing VD3 and Lkb1-siRNA;(2)Real-time quantitative PCR was used to detect the effect of nanodrug particles on Lkb1 expression in dendritic cells;(3)Flow cytometry(FCM)and laser confocal microscopy(CLSM)were used to observe and detect the distribution of nanodrug particles in dendritic cells;(4)Small animal in vivo imager,laser confocal microscopy and flow cytometry were used to detect the distribution of nanodrug particles in vivo and tissues of mice after intradermal inguinal injection;(5)FCM was used to detect the influence of nanodrug particles on BMDC phenotype;(6)FCM was used to investigate the effect of intradermal inguinal injection of nanodrug particles on dendritic cell phenotype and Treg cell proliferation in normal C57BL/6J mice draining lymph nodes.(7)The EAE model was established by MOG35-55 and Freund’s complete adjuvant(FCA)mixed emulsion,subcutaneous injection of C57BL/6J mice,and intraperitoneal injection of pertussis toxin(PTX);(8)The effect of intradermal inguinal injection of nanodrug particles on the incidence of EAE mice was observed,and the effects of dendritic cell phenotype and Treg cell proliferation in mice draining lymph nodes were detected by flow cytometry.H&E and LFB staining were used to observe the infiltration of inflammatory cells and demyelinating changes in the spinal cord of mice.Result(1)Nanodrug particles containing VD3 and Lkb1-siRNA were successfully prepared,and the expression of Lkb1 in dendritic cells could be down-regulated;(2)Nanodrug particles can deliver drugs into DCs in vitro,and can be enriched in draining lymph nodes and engulfed by DCs in vivo;(3)NPs/VD3/siLkb1 nanodrug particles could affect the change of BMDC phenotype;(4)NPs/VD3/siLkb1 nanodrug particles could affect the DCs phenotype in the draining lymph nodes of normal mice and induce the proliferation of Treg cells;(5)Subcutaneous injection of MOG35-55 antigen and FCA emulsion,PTX intraperitoneal injection can effectively induce EAE model;(6)NPs/VD3/siLkb1 nanodrug particles can delay the onset time and reduce the pathogenesis of EAE mice,and reduce the inflammatory cell infiltration and demyelination changes in the spinal cord.ConclusionIn this study,nanodrug particles co-encapsulated VD3 and Lkb1-siRNA were successfully prepared.The particles can be phagocytosed by the DC cells in the draining lymph nodes,and deliver hydrophobic and hydrophilic drugs into the cells to function in the cell matrix.NPs/VD3/siLkb1 can effectively induce and maintain DCs differentiation to the To1DC phenotype,and can increase the level of Treg to a certain extent.The combination of VD3 and Lkb1-siRNA delayed and reduced the onset of EAE in mice,and alleviated the inflammatory infiltration and demyelination in the spinal cord.The mechanism may be related to differentiation of tolerance phenotypes through induction and maintenance of DCs in draining lymph node.