Electrospun PLGA Membranes With Dextran-g-Poly(L-Lysine)-VAPG/microRNA-145 To Specially Modulate Vascular SMCs

Within the field of vascular engineering,miRNA-145 is considered to regulate SMC phenotype from a synthetic type to a contractile state and inhibit the excessive proliferation via targeting a network of Krüppel-like factor 4(KLF4)and myocardin.Incorporation of miRNA-145 into tissue engineered vascular grafts could be a promising approach to inhibit intimal hyperplasia and maintain long-term patency of small vascular grafts.However,naked miRNAs are susceptible to degrade in serum by nucleases due to the instability.Meanwhile,off-target often brings about high cytotoxicity.Therefore,a targeted local delivery system with low cytotoxicity is of great importance.Dextran,a FDA-approved natural polysaccharide,was considered as an alternative of PEGylation to hinder the positive charges and nonspecific adsorption as well as improve biocompatibility and biodegradability in the presence of a large amount of hydroxyl groups.In this work,Dextran was activated to initiate ring-opening of L-lysine N-carboxyanhydride(Dex-g-PLL),and then a specific adhesive peptide to vascular smooth muscle cells(SMCs),Val-Ala-Pro-Gly(VAPG)was introduced to prepare the polymer(Dex-g-PLL-VAPG)in order to enhance transfection efficiency.Meanwhile,functional electrospun poly(lactide-co-glycolide)(PLGA)membranes were developed by loading Dex-g-PLL-VAPG/miRNA-145(DPVm)complexes to modulate the phenotype and proliferation of SMCs.The polymer/miRNA-145 complexes exhibited trivial cytotoxicity,excellent serum stability,enhanced cellular uptake by SMCs over vascular endothelial cells and higher transfection efficiency than Lipo2000.When encapsulated into the electrospun PLGA membrane,miRNA-145 presented a controlled release as well as good biological activity.A burst release of 15% was detected and the overall released amount was up to 80%.It was found that the electrospun membranes were able to support the adhesion and spreading of SMCs.Additionally,significant down-regulation of Krüppel-like factor 4 expression and up-regulation of myocardin and ?-smooth muscle actin expression at both gene and protein levels were detected on day 3 owing to Dex-g-PLL-VAPG.Moreover,due to the sustained release of miRNA-145,SMCs maintained the contractile phenotype at a slow proliferation rate in comparison with the negative control.The functional bilayer electrospun scaffold with poly(ethylene glycol)-b-poly(L-lactide-co-?-caprolactone)as the inner layer and PLGA containing DPVm complexes as the outer layer was developed.Subsequently,the replacement of rabbit carotid artery by the functional scaffolds was performed for 2 months.H&E,Masson trichrome,Verho?eff and immunofluorescene staining demonstrated that the vascular graft containing miRNA-145 could also be efficient to modulate SMC phenotype in vivo and establish endothelial monolayer as well as extracellular matrix.It was suggested that a stable,targeted local delivery system with trivial cytotoxicity was successfully established.In addition,encapsulation of miRNA-145 complexes in small-diameter scaffolds could serve as an effective approach to prevent intimal hyperplasia during vascular tissue regeneration.