The purpose of this present study is to prepare NF-κB/p65 antisense oligonucleotide loaded chitosan nanoparticles (NPs) and evaluate their physicochemical characterization and antisense effects in RAW264.7 macrophages. Condensed nanoparticles with mean particle size of 128±16nm, average Zeta potential of 19.6±6.3mV and high entrapment efficiency (EE) of 98.6±0.11% were formed between NF-κB/p65 antisense gene (NAG) and chitosan by complex coacervation method. Trypan blue staining and MTT tests showed that NAG chitosan NPs had no toxic effect on RAW264.7 macrophages when the dose was no more than 20μg/mL. Confocal microscopy images showed that NAG chitosan NPs were capable to deliver NAG into cytoplasm of RAW264.7 macrophages and finally into nucleus. Real-time PCR tests verified that NAG chitosan NPs could significantly decrease the mRNA expression level of NF-κB/p65 and inflammatory cytokines including TNF-ɑ, IL-1 and IL-6. Accordingly, western blot study showed that NAG NPs uptaken in the cells could efficiently reversed the expression of NF-κB/p65 protein induced by LPS. At last, downstream release level of inflammatory factors including TNF-ɑ, IL-1 and IL-6 in LPS stimulated RAW264.7 macrophages was significantly decreased after treated by NAG chitosan NPs. It could be concluded that chitosan NPs were excellent delivery vectors to ferry the NAG into the cytoplasm and nucleus of macrophages. The NAG chitosan NPs might be a novel therapeutic apparatus for the treatment of LPS induced sepsis by inhibiting NF-κB-related pro-inflammatory cytokines secretion.
Keywords: Anti-sense gene; Chitosan nanoparticles; Macrophages; Nuclear factor-kappa B; Sepsis.
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