The Polyethyleneimine Grafted Konjac Glucomannan For Anti-inflammatory SiRNA Delivery

Polyethyleneimine(PEI)is suitable for effective delivery of nucleic acid due to its high density of amino group and outstanding buffer capacity.However,its cytotoxicity and lack of targeting ability restrict its application.Chemical modification of PEI can make it more biocompatible and targeted.Konjac glucomannan(KGM)is a kind of neutral polysaccharide with good biocompatibility and it can specifically recognize the mannose receptor(MR)on the surface of macrophage.Therefore,KGM was introduced to PEI to produce KGM-grafting-PEI(KGM-PEI)for combining both of advantages.In this paper,KGM-PEI was used for targeted delivery of small interfering RNA(si RNA)to macrophages,and its biocompatibility and transfection efficiency of this delivery system were evaluated,and then the effect of KGM-PEI/si TNF-?complexes-mediated silencing of inflammatory gene was proved on the repair of diabetic wound skin.KGM-PEI was prepared by reacting KGM with PEI with CDI as coupling reagent.The structure of KGM-PEI was confirmed by FT-IR,~1H-NMR and element analysis.CCK-8 assay proved that KGM-PEI had good biocompatibility.KGM-PEI could effectively encapsulate si TNF-?to form nanocomplexes.The complexes were assessed by DLS and agarose gel electrophoresis.The mean hydrodynamic size decreased and zeta potential of the complexes slowly increased with the increasing of N/P ratio and both reached a plateau,197.2 nm and 13.8 m V respectively,when N/P ratio reaches 20.CCK-8 assay results showed that the KGM-PEI and its loading si TNF-?had good biocompatibility.The cellular uptake experiments by Raw264.7 cells and L929 cells showed KGM-PEI could achieve targeted si RNA delivery to macrophages.Lysosomal escape test showed that KGM-PEI had strong buffering ability and could effectively deliver si RNA into cytoplasm for gene silencing.The results of RT-q PCR and ELISA showed that KGM-PEI/si TNF-?complexes could down-regulate the expression of TNF-?in Raw264.7 cells,and the efficiency of gene silencing was closely related to the N/P ratio.When the N/P ratio reached 20 or more,the silence efficiency of the system tends to be stable.The collagen sponge containing KGM-PEI/si TNF-?complexes was used for the repair of mouse diabetic wound skin.The m RNA relative expression of TNF-?in wounds was detected by RT-q PCR.The results showed KGM-PEI/si TNF-?complexes could effectively silence the expression of inflammatory cytokines in wounds.Gross observation and H&E staining analysis proved that KGM-PEI/si TNF-?complexes could relieve wound inflammation and promote the healing of mouse diabetic skin wounds.In conclusion,KGM-PEI showed excellent performances as a si RNA vector for targeted delivery to macrophages.In addition,the collagen sponge containing KGM-PEI/si TNF-?complexes reduced the local inflammation of mouse diabetic skin wounds and effectively promoted the healing process.Therefore,KGM-PEI had the potential for macrophage-targeting vector.