Preparation Of Dendritic Cells Targeting Thermo-sensitive Delivery System For SiRNA

Background: Dendritic cells（DCs）, as professional antigen presenting cells （APC）,play an important role both in natural immunity and acquired immunity. But theirclinical applications were limited due to their short lifespan and small quantities in vivo.More and more research focused on the drug delivery system about DCs targeting andextended life. It has been shown that the expression of Bak can promote the apoptosis ofDCs. RNA interference is a specific and efficient approach to cause some gene silencing,which carrieris have been developed. Therefore the research of siRNA delivery systemtargeting Bak will be expected to increase its antigen presenting, promote the immuneresponse and enhance anti-tumor effects. Based on the surface expression of DEC-205receptor on dendritic cells, the DEC-205modified poloxamer-g-chitosan containingsiRNA targeting Bak was synthesized to prepare thermo-sensitive micelles, and thenano-carriers were then characterized with respect to the pharmaceutical properties,cytotoxicity, transfection efficiency and the survival rate of DCs in order to provide theevidence of the optimizing design and in vivo evaluation.Method: First, synthesis of DEC-205modified poloxamer-g-chitosan based on theprevious work in our laboratory and siRNA loaded PMs were prepared by directlydissolved method. The characteristics of PMs on size, Zeta potential, encapsulationefficiency and cytotoxicity were investigated. DCs were generated from murine bonemarrow cell by an established in vitro method. Lipofectamine2000as the positivecontrol, Subsequently, the uptaken of PMs by macrophage and DCs was measured usingfluorescence microscope and flow cytometry. The expression of Bak in DCs wasdetected with Western blot and the survival rate of DCs after transfection was measuredby cell death detection kit.Result: The structure of poloxamer-g-chitosan were confirmed by IR, NMR andthermogravimetric analysis. The value of CMC and LCST were （1.39±0.08）×10^-6mol·L-1and40℃. The concentrations of PMs and siRNA were0.5mg·mL^-1and120nM.The average size and Zeta potential of siRNA loaded PMs were （230±4.1） nm and+（13.98±0.8） mV, respectively. The entrapment efficiency of siRNA was （80.2±2.93）%. It showed that endocytosis of the PMs containing FAM-siRNA was more thanFAM-siRNA solution when checked by fluorescence microscope and flow cytometry.The modification of DEC-205can improve the uptaken of PMs by DCs but its impacton macrophage was small. The results of Western blot showed that siRNA could inhibitthe expression of Bak up to （80.17±6.90）%at48h after transfection with PMs. Theviable rate of DCs at48h after transfection with PMs was （64.12±1.82）%higher thanboth （41.08±1.99）%of negative control and （50.45±8.13）%of positive control.Conclusion: This paper prepared DEC-205modified poloxamer-g-chitosan. Thetoxicity of PMs was low. The results indicated that the nano-carrier can improve theuptake of FAM-siRNA by macrophage and DCs effectively. The expression of Bak wasblocked and the lifespan of DCs was prolonged by the RNA interference.