Target regulation of both VECs and VSMCs by dual‐loading miRNA‐126 and miRNA‐145 in the bilayered electrospun membrane for small‐diameter vascular regeneration

Abstract

Clinical utility of small‐diameter vascular grafts is still challenging in blood vessel regeneration owing to thrombosis and intimal hyperplasia. To cope with the issues, modulation of gene expression via microRNAs (miRNAs) could be a feasible approach by rational regulating physiological activities of both vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs). Our previous studies demonstrated that individually loaded miRNA‐126 (miR‐126) or miRNA‐145 (miR‐145) in the electrospun membranes showed the tendency to promote vascular regeneration. In this work, the bilayered electrospun graft in 1.5‐mm diameter was developed by emulsion electrospinning to dual‐load miR‐126 and miR‐145 for target regulation of both VECs and VSMCs, respectively. Accelerated release of miR‐126 was achieved by introducing poly(ethylene glycol) in the inner electrospun poly(ethylene glycol)‐b‐poly(l‐lactide‐co‐caprolactone) ultrafine fibrous membrane, reaching 61.3 ± 1.2% of the cumulative release in the initial 10 days, whereas the outer electrospun poly(l‐lactide‐co‐glycolide) membrane composed of microfibers fulfilled prolonged release of miR‐145 for about 56 days. In vivo tests suggested that dual‐loading with miR‐126 and miR‐145 in the bilayered electrospun membranes could modulate both VECs and VSMCs for rapid endothelialization and hyperplasia inhibition as well. It is reasonably expected that dual target‐delivery of miR‐126 and miR‐145 in the electrospun vascular grafts has effective potential for small‐diameter vascular regeneration. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 371–382, 2019.

Local Delivery of Dual MicroRNAs in Trilayered Electrospun Grafts for Vascular Regeneration

“…The trilayered electrospun grafts encapsulating both miR-126 and miR-145 in the fibrous inner and middle layers, respectively, were prepared by sequential three-step electrospinning (Figure A). First, the inner layer was prepared by emulsion electrospinning of PELCL (76 kDa) and PELCL-REDV (about 20 kDa) with TPR/miR-126 complexes. , The electrospun membranes or grafts were collected on a grounded rotating stainless steel mandrel or a thin stainless steel rod covered by aluminum foil. After 3 h, the middle layer was prepared by emulsion electrospinning of PELCL (190 kDa) solution containing TPV/miR-145 complexes for another 3 h. The outer layer was further electrospun from the PCL solution.…”

“…Electrospun membranes can serve as suitable TEVGs because of their ultrafine fibrous structure which can closely resemble the natural extracellular matrix (ECM). ,− Moreover, the electrospun ultrafine fibers can be easily functionalized by encapsulating bioactive macromolecules, such as growth factors, , plasmid, siRNA, and microRNAs (miRNAs) − and modified with fusion protein or peptides. − Additionally, mimicking the native artery tissues by electrospinning through multilayered structures makes full use of biomimetic strategies for fabrication of bioactive TEVGs with functional lumen and annular support. ,, Many efforts have been dedicated to develop multiple layered vascular grafts, striving to match the native vascular function for blood vessel regeneration. ,,,,− ,− Previous outcomes have suggested that bilayered electrospun vascular grafts are feasible for loading two kinds of bioactive substances in fibers with diverse diameters for regulating ECs and SMCs, respectively, ,,, while trilayered electrospun grafts can provide better structural configuration with proper biological and biomechanical properties. ,,− However, overall characterizations of the bioactive TEVGs with in vivo regenerative activities are still challenging. − …”

“…miRNAs, small noncoding RNAs consisting of about 22 nucleotides in length, could suppress the relative translation of proteins by binding to their target message RNAs . In the cardiovascular system, miRNA-126 (miR-126) can accelerate synthesis of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) by downregulating Sprouty-related EVH1 domain-containing protein 1 (SPRED1) and phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2) genes. − In addition, miRNA-145 (miR-145) has an inhibitory effect on Krüppel-like factor 4/5 (KLF4/5) gene to modulate the contractile SMC phenotype. − In order to decrease the side effects of miRNAs, local delivery of miRNA mimics to the specific vascular tissues is preferred. − Our previous research demonstrated that the bioactivity and functional efficacy of the encapsulated miRNAs in the electrospun membranes or grafts via peptide and poly­(ethylene glycol) (PEG)-modified trimethyl chitosan (TMC) could be preserved perfectly with negligible cytotoxicity. ,− …”

“…Synthetic polymers including poly­( l -lactic acid), poly­(ε-caprolactone) (PCL), and their related copolymers are often applied to prepare TEVGs with sufficient mechanical properties. ,,− ,− Poly­(ethylene glycol)- b -poly­( l -lactide- co -ε-caprolactone) (PELCL) was particularly synthesized in our laboratory for preparing small-caliber vascular grafts by electrospinning because of its biocompatibility, biodegradability, and flexibility. , With the aid of Arg-Glu-Asp-Val (REDV) peptide, the electrospun membranes could significantly improve EC adhesion and proliferation by cell-specific binding of ECs over SMCs via the integrin α4β1 subunit. ,− The electrospun membranes of PELCL loaded with miR-126 complexes in REDV peptide-modified TMC- g -PEG (TPR) could locally deliver miR-126 to ECs, ,, while Val-Ala-Pro-Gly peptide-linked TMC- g -PEG (TPV) could be applied for local delivery of miR-145 to SMCs. ,, Based on the abovementioned studies, in this work, a deep investigation is performed for examination of the effect of trilayered electrospun grafts encapsulating both miR-126 and miR-145 in the fibrous inner and middle layers, respectively, on regulating vascular cells and macrophages for blood vessel regeneration. The bioactive trilayered electrospun grafts were developed specifically consisting of the respective PELCL/REDV-modified PELCL inner layer, PELCL middle layer, and PCL as the outer layer with suitable biomechanical properties for fast or slow release of miRNAs.…”

Challenges and strategies for in situ endothelialization and long-term lumen patency of vascular grafts

“…The miRNA complex was incorporated into electrospun polymers utilizing emulsion electrospinning to construct vascular scaffold, and miRNA was released sustainedly for enhanced EC performance. Cui et al [ 188 ] loaded miRNA-126 in inner electrospun fibers, and miRNA-145 in outer fibers, respectively to regulate biological behavior of ECs and SMCs. Moreover, some other miRNAs are also found and explored their effects on vascularization.…”