Abstract
To investigate a nuclear factor-kappa B decoy oligonucleotides strategy on the inhibition of tissue factor (TF) expression in cultured rat brain microvascular endothelial cells (BMECs) by polylactic acid (PLA) nanoparticles delivery system and to evaluate this new vector for in vitro gene therapy. Nanoparticles were formulated using poly D,L-polylactic acid with surface modifying by polysorbates 80. 3-[4,5-Dimethylthiazol-2,5-diphenyl-2H-tetrazolium bromide] (MTT) assays showed that PLA nanoparticles were not toxic to the cultured BMECs.The decoy oligonuceotides (ODNs) loaded within nanoparticles was 6 μg/mg, encapsulation efficacy was (60.5±1.5)%. It was observed by flow cytometry that the cellular uptake of nanoparticles depended on the time of incubation and the concentration of nanoparticles in the medium. And confocal microscopy demonstrated that nanoparticles localized mostly in the BMECs cytoplasm. The released decoy oligonuceotides (ODNs) uptaked by BMECs retained their biologic activity and led to reduced level of tissue factor expression as compared to control cultures. These findings offer a potential therapeutic strategy in the control of TF expression in BMECs in vitro and suggest that PLA nanoparticles may be appropriate as delivery vehicles for decoy strategy in the gene therapy of cerebral thrombosis.
Highlights
Thrombosis plays a major role in the pathogenesis of ischemic stroke
Expression in brain microvascular endothelial cells (BMECs) in vitro and suggest that polylactic acid (PLA) nanoparticles may be appropriate as delivery vehicles for decoy strategy in the gene therapy of cerebral thrombosis
We have identified NF-κB transcription factor decoy (TFD) strategy, which is believed to be a powerful tool for anti-gene strategies and transcription regulation [10,11,12], and can inhibit tissue factor (TF) over-expression in stimulated human umbilical vein endothelial cells (HUVEC) by disruption of NF-κB activation [13]
Summary
Thrombosis plays a major role in the pathogenesis of ischemic stroke. some limited progress has been made in the treatment of cerebral thrombosis, current therapeutic options are still controversial because of a discouragingly high incidence of intracerebral hemorrhage and restenosis in treated patients [1,2,3,4]. We have identified NF-κB transcription factor decoy (TFD) strategy, which is believed to be a powerful tool for anti-gene strategies and transcription regulation [10,11,12], and can inhibit TF over-expression in stimulated human umbilical vein endothelial cells (HUVEC) by disruption of NF-κB activation [13]. Brain microvascular endothelial cells (BMECs) were important targets in gene therapies for cerebral thrombosis. The mechanism suggests that drug-loaded NPs may be endocytosised by BMECs. the presence of this mechanism may provide an efficient way in the brain vascular endothelial cells targeting. NPs to suppress TF gene expression in primary cultures of rat BMECs. The data offered a potential therapeutic strategy in the regulation of TF expression in BMECs in vitro and suggest that PLA NPs may be an appropriate delivery vehicle for decoy strategy in the gene therapy of cerebral thrombosis
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