Controlled Release Of SiRNA Nanoparticles Targeting Tissue Factor Attenuates Neointima Hyperplasia In Vein Grafts

Part 1 Inhibition of Tissue Factor gene expression with chemically synthesized siRNA in Rat Vascular Smooth Muscle CellsObjective To investigate the feasibility and efficacy of TF gene silencing in rat vascular smooth muscle cells using small interfering RNA.Methods Double-stranded 25bp-RNA molecules targeted at sequences within the rat TF gene were designed and constructed.Primary cultures of rat vascular smooth muscle cells were either transfected with TF siRNA, with a control siRNA or cultured without transfection.Transfection efficacy was determined using fluorescence microscopy.The mRNA expression of TF was analyzed by RT-PCR,and the protein expression was analyzed by Western blot and immunochemistry after stimulation with PDGF-BB.Results Successful siRNA transfection of VSMC was confirmed by fluorescence microscopy,and the efficiency was about 90±2.3%.Cells transfected with the highest effective siRNA of three candidate sequences showed a 86±0.6% decreased TF mRNA expression at 24h after transfection, 92±1.0% decreased protein expression at 48h after transfection compared with untransfected cells,at the same time TF staining was markedly decreased after transfection.Conclusion TF expression can be effectively silenced by RNAi on vascular smooth muscle cells from rat aorta. Part 2 Development and Characterisation of Chitosan Nanoparticles for Tissue Factor siRNA DeliveryObjective To explore a novel chitosan-based siRNA nanoparticle delivery system for targeted silencing of tissue factor gene via RNA interference.Methods Chitosan nanoparticles were prepared by ionic gelation using sodium tripolyphosphate.Mean particle diameter,zeta potential,siRNA loading efficiency,controlled release ability in vitro of the nanoparticles were determined.Serum stability and biological activity of chitosan–siRNA nanoparticles were assayed.The effect of chitosan on cytotoxicity was measured by determining cell viability of chitosan-siRNA nanoparticles.Results Nanosize particles could be obtained by ionic gelation, and the mean diameters were less than 400 nm depending on molecular weight as well as concentration of chitosan.In the case of ionic gelation, two further factors, namely chitosan to TPP weight ratio and pH, affected the particle size.100% siRNA loading efficiency was obtained for all the entrapped siRNA chitosan–TPP nanoparticles.Nanoparticles from higher molecular weight had more release rate in vitro.SiRNA recovered from chitosan–TPP nanoparticles started to degrade after 24h incubation and full degradation was only observed after 60h incubation in 5% serum,and siRNA recovered from chitosan–siRNA nanoparticles was intact up to 6h and fully degraded after 48h incubation in 50% serum.The silencing effect of certain chitosan–siRNA nanoparticles had a comparable effect compared to Lipofectamine 2000 at 24h post-transfection: 79±1.5%,62±5.9%of gene knockdown for siRNA entrapment and siRNA adsorption,respectively.Conclusion Chitosan–TPP nanoparticles show much potential as viable vector candidates for safer and cost-effective siRNA delivery.Part 3 Significance of Tissue Factor Protein Expression in Rat Experimental Venous Bypass GraftsObjective To examine changes in tissue factor(TF) protein expression in response to venous bypass grafting.Methods Sprague-Dawley rats underwent interposition bypass grafting of the common carotid artery via the ipsilateral external jugular vein.The vein grafts were harvested in 1,3,7,14,28d after surgery,respectively.The contralateral external jugular veins were also harvested as the control group.The expression of TF and proliferating cell nuclear antigen (PCNA) were identified by immunochemistry methods.TF activity in vessel protein extracts was determined with TF activity assay kit.The thickness of neointima and media of the vein grafts were calculated by computer imaging analysis system.Results The adventitia of all vessels showed abundant TF staining.In early vein grafts (1,3,7 d after grafting), TF staining was markedly increased in the intima and media.However,intimal and media TF staining was absent in the contralateral control jugular veins and late vein grafts (14,28 d after grafting).The number of PCNA positive cells increased in the vein grafts from the third day after grafting and reached the peak at 14d postoperatively.TF activity in whole-vessel protein extracts was similar in control veins and early and late vein grafts (mean±SD:control vein, 0.28±0.02 U/mg;early vein grafts, 0.29±0.04U/mg; and late vein grafts, 0.28±0.04U/mg). The thickness of neointima of the vein graft increased significantly at 7,14,28d,and the thickness of media increased significantly at 14,28d.Conclusion Marked changes in TF expression occur in vein grafts and these findings may have important implications for the development of therapeutic strategies to limit vein graft failure.Part 4 Atelocollagen-mediated Small Interfering RNA Delivery for Effective Gene Silencing in Rat Vein GraftsObjective To evaluate the efficacy of using small interfering RNA targeting TF as a therapy for vein graft failure.Methods Jugular vein-to-carotid artery interposition vein grafts, which were applied to a low flow condition, were made in Sprague-Dawley rats.Small interfering RNA mixed with atelocollagen was administrated to the external wall of grafted veins.BLOCK-iTTM fluorescent oligo was used to confirm its stability and successful transfer into the vein graft wall.The TF protein expression of vein grafts was analyzed by Western blot and immunochemistry.The proliferation index was determined.Neointimal hyperplasia was evaluated at 28d after the operation.Results Fluorescence of BLOCK-iTTM fluorescent oligo could be detected in the graft wall even at 7d after operation.Knockdown of the TF expression was achieved by perivascular application of siRNA using atelocollagen.Compared with controls,the intima thickness at 28d after grafting was reduced 68±3.2%.This phenomenon was preceded by significant reductions of cell proliferation in siRNA-treated grafts.Conclusions The expression of TF in vein grafts can be effectively inhibited by specific siRNAs using a atelocollagen-based nonviral delivery approach in vivo.This promising strategy may be a useful and practical form of gene therapy against human vein graft failure.Part 5 Controlled Release of siRNA Nanoparticles Loaded in a Novel External Stent Prepared by Electrospinning Attenuates Neointima Hyperplasia In Vein GraftsObjective To evaluate a strategy of using TF siRNA loaded in a novel external stent prepared by hybrid ultrafine fibrous membrane consisting of PLGA/Chitosan nannoparticles as a therapy for vein graft disease.Methods Hybrid ultrafine fibrous membranes consisting of PLGA/Chitosan nannoparticles were fabricated via a specially designed electrospinning setup.After soaking in chloroform to dissolve PLGA, the amount of chitosan in the hybrid membranes was determined.The water uptake of the hybrid ultrafine fibrous membranes was investigated by incubation in phosphate buffer solution.Right jugular vein-carotid artery interposition grafting models in Sprague-Dawley rats were randomly divided into five groups:Group A(external stent consisting of PLGA/CS-TFsiRNA nanoparticles),Group B(external stent consisting of PLGA/CS-StealthTM RNAi negative control nanoparticles),Group C(external stent consisting of PLGA/CS blank nanoparticles),Group D(external stent consisting of PLGA),GroupE (without perivenous external stent).BLOCK-iTTM Fluorescent Oligo was used to confirm its stability and successful transfer into the vein graft wall. The vein grafts were harvested at 1,3,7,14,28d after operation,respectively.The TF protein expression of vein grafts was analyzed by Western blot and immunochemistry at 1,3,7 d after operation,respectively.The expression of proliferating cell nuclear antigen (PCNA) was identified by immunochemistry methods.The thickness of neointima at 28 d was calculated by computer imaging analysis system.Results The PLGA and CS amount in PLGA/Chitosan nannoparticles membranes could be well controlled by adjusting the flow rate for electrospinning of PLGA and chitosan nannoparticles, respectively.Because of the introduction of chitosan,which is a naturally hydrophilic polymer, the hybrid membranes exhibited good water absorption properties.BLOCK-iTTM Fluorescent Oligo could be detected in the graft wall even 12days after operation.The expression of TF protein in Group A was significantly less than that in control groups at 3,7d after operation(P<0.05).The expression of PCNA in Group A decreased significantly in comparison with control groups at 14d after operation (P<0.01).The thickness of neointima at 28d after grafting in Group A was significantly less than the untreated group(P<0.01).Conclusion The novel external stent prepared by hybrid ultrafine fibrous membrane consisting of PLGA/Chitosan nannoparticles inhibits early neointima formation in rat vein grafts.This strategy may be a practicable and promising form of gene delivery against vein graft failure.