Controlled release of siRNA nanoparticles loaded in a novel external stent prepared by emulsion electrospinning attenuates neointima hyperplasia in vein grafts

OBJECTIVE: To evaluate a strategy of using TF siRNA loaded in a novel external stent prepared by hybrid ultrafine fibrous membrane consisting of PLGA/Chitosan nanoparticles as a therapy for vein graft disease. METHODS: Hybrid ultrafine fibrous membranes consisting of PLGA/Chitosan nanoparticles 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-Stealth(TM) RNAi negative control nanoparticles), Group C (external stent consisting of PLGA/CS blank nanoparticles), Group D (external stent consisting of PLGA), Group E (without perivenous external stent). BLOCK-iT(TM) 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, 28 d 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 nanoparticles membranes could be well controlled by adjusting the flow rate for electrospinning of PLGA and chitosan nanoparticles, respectively. Because of the introduction of chitosan, which is a naturally hydrophilic polymer, the hybrid membranes exhibited good water absorption properties. BLOCK-iT(TM) Fluorescent Oligo could be detected in the graft wall even 12 days after operation. The expression of TF protein in Group A was significantly less than that in control groups at 3 d after operation (P < 0.05, 0.40 +/- 0.03 vs 0.75 +/- 0.01, 0.75 +/- 0.05, 0.77 +/- 0.07) and at 7 d after operation (P < 0.05, 0.30 +/- 0.03 vs 0.84 +/- 0.05, 0.86 +/- 0.06, 0.85 +/- 0.06). The expression of PCNA in Group A decreased significantly in comparison with control groups at 14 d after operation (P < 0.01, 13.0% +/- 2.6% vs 25.0% +/- 2.8%, 24.2% +/- 3.9%, 24.0% +/- 4.1%, 44.8% +/- 3.7%). The thickness of neointima at 28 d after grafting in Group A was significantly less than the untreated group (P < 0.01, 18.8 microm +/- 2.9 microm vs 38.7 microm +/- 5.0 microm, 37.3 microm +/- 3.6 microm, 37.2 microm +/- 2.6 microm, 67.5 microm +/- 4.8 microm). CONCLUSION: The novel external stent prepared by hybrid ultrafine fibrous membrane consisting of PLGA/Chitosan nanoparticles inhibits early neointima formation in rat vein grafts. This strategy may be a practicable and promising form of gene delivery against vein graft failure.