PLGA-fasudil/PLA-everolimus/PCL-simvastatin Vascular External Sheath Loaded Drugs Inhibits Long-term Vein Graft Failure

In china,CAD(coronary artery disease)has superior morbidity and mortality.CABG(coronary artery bypass graft) is an effective therapeutic method for CAD which can relieve angina pectoris and promote patients' survival rate.The saphenous vein as the most widely used autologous blood vessel,has advantages of easily acquisition,sufficient length and suitable diameter matching with coronary artery.However,the major drawback of the saphenous vein in CABG is the development of SVGF(saphenous vein graft failure).According to the statistics,SVG(saphenous vein graft) patency rate is 89% 1 year after CABG.It decreases to 61% at 10 years after CABG.The reduction in SVG patency restricts therapeutic effect of CABG remarkably.Therefore,an effective therapy to solve SVGF and improve long-term SVG patency rate needs to be developed.SVGF pathological mechanisms on the basis of time are divided into early,middle and late period.In early period(within 30 days after CABG),main pathological mechanism is thrombosis.In middle period(30 days to 1 year after CABG),main pathological mechanism is IH(intimal hyperplasia).In late period(more than 1year after CABG) is atherosclerosis,respectively.In the current study,according to SVGF pathological mechanisms change over time,three drugs were chosen to target three different pathological mechanisms.Applying electrospinning to load drugs into biodegradable vascular external sheath as drug delivery system with good mechanical property inhibits long-term vein graft failure.Part one: Exploring the influence of electrospinning parameters on the morphology of PLGA,PLA and PCL nano-fibers and the optimal parameters of electrospinningObjective: Electrospinning was wildly used in medical materials,tissue engineering,catalysis,protection and defense because of electrospinning nano-fibers have advantages of nanoscale diameter,high specific surface and porosity,uniformity,regulatability and mechanical tensile properties.In the current study,we explore the influence of solvent formulation,polymer concentration,voltage,distance and flow rate on the morphology of PLGA,PLA and PCL nano-fibers and the optimal electrospinning parameters.Methods: we identify the optimal parameters of solvent formulation,voltage,distance,flow rate,high molecular polymer concentration by SEM,observing coaxial structure by LSCM.Results: optimal solvent formulation is chloroform: DMF=4:1.optimal voltage is 17 KV.optimal distance is 15 cm.Tranditional electrospining and coxial optimal outer rate is0.9ml/h.optimal coxial inner rate is 0.18ml/h.optimal concentration of PLA and PCL are 10%(W/W).optimal concentration of PLGA is 20%(W/W).We found Rhodamine B labeled water emitting green fluorescence warpped by gray PLGA nanofibers,indicating that coaxial structure was successfully electrospinning in the current study.Part two: PLGA-fasudil/PLA-everolimus/PCL-simvastain vascular sheath loaded drugs was prepared by the combination of mixing and coaxial electrospiningObjective: we choose Rho A inhibitor fasudil,mTOR inhibitor everolimus and simvastatin as delivery drugs,using PLGA,PLA and PCL as material,applying the optimal parameters to prepared vascular sheath releasing drugs with different rates,studying the vascular sheath's characterizations.Methods: PLGA fasudil / PLA-everolimus / PCL-simvastatin nanofiber loaded drugs vascular sheath was prepared using the optimal electrospinning parameters obtained in part two.The morphology of the vascular sheath was observed by SEM.The histocompatibility of the vascular sheath was evaluated by the contact angle.The mechanical properties of the vascular sheath were measured by electronic universal testing machine.Encapsulation efficiency was evaluated by HPLC.The vascular sheath's degradation and release rate were evaluated in vitro.Results: PLGA-fasudil/PLA-everolimus/PCL-Simvastatin nanofibers' diameter were539±139.81 nm.Contact angle was 64.95±2.80°.Maximum tension and tensile strength in transverse and longitudinal direction were 22.98±1.94 N,5.41±0.54 MPa and31.59±2.39 N,7.41±1.03 MPa.At 20 weeks after incubation,PLGA patches degraded completely,PLA patches degraded 36.16±3.70%,PCL patches degraded 20.17±2.72%and the vascular external sheath degraded 51.28±3.16%.The vascular sheath encapsulation of fasudil dihydrochloride,everolimus and simvastatin were15.33±0.76 mg,127.67±14.50?g and 33.6±2.55 mg.Encapsulation efficiencies of drugs were 38.32±1.9%,23.95± 2.72% and 42±3.18%.At 63 days after incubation,fasudil released completely,Everolimus released completely at 84 days,As for simvastatin it acumulatively released 10.71±0.52 mg account for 31.87±1.55% of encapsulated.Conclusion: PLGA-fasudil/PLA-everolimus/PCL-Simvastatin vascular external sheath loaded drugs has good histocompatibility,mechanical properties to restrict vein graft dilatation,loaded fasudil,everolimus and simvastatin releaseing with different rate.Its releasing time match the time window of SVGF pathological mechanisms.Part three: PLGA-fasudil/PLA-everolimus/PCL-simvastatin vascular external sheath loaded drugs inhibits long-term vein graft failureObjective: Studies have pointed out after CABG,IH is the main pathological mechanism of vain graft stenosis introduced by intima injury,VSMC(vascular smooth muscle cell)proliferation and migration.After SVG was transplant to artery system,hypoxia in SVG promote Rho A expression,mediated AKT signal pathway to influence endothelial function,VSMC proliferation and migration.We establish hypoxia model in ECs(endothelial cells)and VSMCs to explore the influence of using Rho A inhibitor and mTOR inhibitor to ECs' function,VSMC proliferation and migration.The therapy efficacy of PLGA-fasudil/PLA-everolimus/ PCL-simvastatin vascular external sheath loaded drugs in the prevention and treatment of long-term vein graft failure was evaluated in animal experiments.Methods: Human umbilical vein ECs were divided into 6 group,group 1 is ECs without any handle.Group 2 is hypoxia for 3h.Group 3 is hypoxia for 3h+1?mol/L fasudil.Group 4 is hypoxia for 3h + 10?mol/l fasudil.Group 5 is hypoxia for 3h + 100?mol/L fasudil.Group 6 is hypoxia for 3h + 100?mol/L fasudil + 20mmol/L L-NAME.Then we extract total protein conventional after incubated 24 hours,detecting expression of Rho A,AKT,e NOS,,MMP-9 by western blot.Human VSMCs were divided in to 9 groups.Group 1 is VSMCs without any handle.Group 2 is hypoxia for 3h.Group 3 is hypoxia for 3h +0.1nmol/l everolimus.Group 4 is hypoxia for 3h +1nmol/l everolimus.Group 5 is hypoxia for 3h +10nmol/l everolimus.Group 6 is hypoxia for 3h + 10?mol/L fasudil.Group 7 is hypoxia for 3h + 100?mol/l fasudil.Group 8 is hypoxia for 3h + 10nmol/l everolimus + 100?mol/l fasudil.Then we extract total protein conventional after incubated 24 hours,detecting expression of Rho A,AKT,CDK1,PCNA and count VSMCs numbers in cell cycle by flow cytometry.We establish left carotid-jugular bypass graft model in rabbits,grouping the rabbits into three groups randomly.Group C(carotid-jugular bypass graft).Group S(artery bypass graft + vascular sheath without drugs).Group D(artery bypass graft + vascular sheath loaded drugs).At immediately after surgery,4 weeks,8 weeks,12 weeks,16 weeks,we evaluate vein graft inner diameter and peak flow rate by vascular ultrasound.R value is identified as ratio of peak flow rate in vein graft to contralateral artery.At 4 weeks,8weeks,12 weeks,16 weeks after operation,we sacrifice rabbits to evaluate degree of IH by HE and Masson staining.I/M is identified as ratio of intima thickness to media thickness.Immunohistochemical staining of ?-SMA and PCNA were preformed.Expression of Rho A,?-SMA,PCNA,AKT,ABCA1,CDK1,MMP-9 were evaluated by western blot.Results: Expression of Rho A,AKT and MMP-9 were up-regulated after hypoxia in ECs.Expression of e NOS were down-regulated.Cultured with different concentration fasudil can alleviate the effect of hypoxia,L-NAME can inhibits protection effect of fasudil.In VSMC,expression of Rho A,AKT,CDK1 and PCNA were regulated after hypoxia,both fasudil and everolimus can inhibit expression of Rho A,AKT,CDK1,PCNA and suppressing VSMC mitosis.The effect of verminous was superior than fastidious(P<0.01),and the combined effect of everolimus and fasudil was better than that of alone(P <0.01).At preset interval,diameters in group C were 44.25±0.14 mm,4.5±0.26 mm,4.8±0.05 mm,5.2±0.1mm and 6.1±0.07 mm.In group S diameters were 3.8±0.25 mm,3±0.07 mm,2.8±0.2mm,2.6±0.07 mm and 2.4±0.07 mm.In group D diameters were 3.8mm±0.07,3.3±0.07 mm,3.0±0.28 mm,3.3±0.07 mm and 3.3±0.07 mm.The R value in group C were0.77±0.03,0.53±0.03,0.44±0.04,0.47±0.05 and 0.38±0.03.The R value in group D were 0.84±0.05,0.95±0.06,1.35±0.04,1.44±0.06 and 1.55±0.05.The R value in the group S were 0.86±0.03,0.88±0.06,0.92±0.06,1.09±0.09 and 1.14±0.05.I/M in group C were 3.01±0.33,3.96±1.03,4.013±1.07 and 4.06±0.50.I/M in group D were2.12±0.38,2.24±0.20,2.29±0.44 and 2.36±0.39.I/M in group D were 0.61±0.20,0.60±0.23,0.99±0.19 and 0.78±0.28.PCNA indexes in group C were 0.55±0.14,0.64±0.21,0.71±0.18 and 0.83±0.09.PCNA indexes in group S were 0.51±0.11,0.58±0.13 and 0.37±0.10.PCNA indexes in the group D were 0.45±0.06,0.31±0.31,0.18±0.16 and 0.07±0.01.The vascular diameter,R value,I/M and PCNA index of C group,S group and D group were significantly different at the same time point(P <0.01).Conculsion: Using fastidious can improve epithelial function mediated by hypoxia.Combinating using fasudil and everolimus can up-regulate endothelial function,down-regulating VSMCs proliferation and migration by down-regulating Rho A/mTOR signal pathway synergistically.The PLGA fasudil/PLA-everolimus/PCL-simvastatin nanofibrous vascular sheath loaded drugs can effectively inhibit the long-term vein graft failure through limiting expansion and graded releasing drugs.