title: The effects of nanofiber diameter and orientation on siRNA uptake and gene silencing.
abstract: While substrate topography influences cell behavior, RNA interference (RNAi) has also emerged as a potent method for understanding and directing cell fate. However, the effects of substrate topography on RNAi remain poorly understood. Here, we report the influence of nanofiber architecture on siRNA-mediated gene-silencing in human somatic and stem cells. The respective model cells, human dermal fibroblasts (HDFs) and mesenchymal stem cells (MSCs), were cultured onto aligned or randomly oriented electrospun poly(ε-caprolactone) fibers of different average diameters (300 nm, 700 nm and 1.3 μm). In HDFs, decreasing fiber diameter from 1.3 μm to 300 nm improved Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Collagen-I silencing efficiencies by ∼ 3.8 and ∼4.4 folds respectively (p < 0.05) while the effective siRNA uptake pathway was altered from clathrin-dependent endocytosis to macropinocytosis. In MSCs, aligned fibers generated significantly higher level of gene silencing of RE-1 silencing transcription factor (REST) and green fluorescent protein (GFP) (∼1.6 and ∼1.5 folds respectively, p < 0.05), than randomly-oriented fibers. Aligned fiber topography facilitated functional siRNA uptake through clathrin-mediated endocytosis and membrane fusion. Taken together, our results demonstrated a promising role of three-dimensional fibrous scaffolds in modulating siRNA-mediated gene-silencing and established the critical synergistic role of these substrates in modulating cellular behavior by RNAi.
authors.affiliation: Division of Chemical and Biomolecular Engineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637459, Singapore.
authors.forename: Winifred Wing Yiu
authors.initials: WW
authors.lastname: Yau
authors.affiliation: Division of Chemical and Biomolecular Engineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637459, Singapore.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Long
authors.affiliation: Mechanobiology Institute, National University of Singapore, 5A Engineering Drive 1, Singapore 117411, Singapore.
authors.forename: Nils C
authors.initials: NC
authors.lastname: Gauthier
authors.affiliation: Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119074, Singapore; Department of Reproductive Medicine, KK Women's and Children's Hospital, Singapore 229899, Singapore; Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore 169857, Singapore.
authors.forename: Jerry Kok Yen
authors.initials: JK
authors.lastname: Chan
authors.affiliation: Division of Chemical and Biomolecular Engineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637459, Singapore; Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 308232, Singapore. Electronic address: sychew@ntu.edu.sg.
authors.forename: Sing Yian
authors.initials: SY
authors.lastname: Chew
mesh_terms: D002232:Carbocyanines / Q000378:metabolism; D048430:Cell Shape / Q000187:drug effects; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D024042:Collagen Type I / Q000378:metabolism; D020405:Dermis / Q000166:cytology; D004734:Energy Metabolism / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D055785:Gene Knockdown Techniques; D020868:Gene Silencing / Q000187:drug effects; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000378:metabolism; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects; D057139:Nanofibers / Q000737:chemistry* / Q000648:ultrastructure; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010316:Particle Size; D010873:Pinocytosis / Q000187:drug effects; D011091:Polyesters / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000378:metabolism*
chemical_list: D002232:Carbocyanines; D002966:Clathrin; D024042:Collagen Type I; D009838:Oligodeoxyribonucleotides; D011091:Polyesters; D034741:RNA, Small Interfering; C085321:cyanine dye 5; C016240:polycaprolactone; D002784:Cholesterol; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)
keywords: Electrospinning; Endocytosis pathway; Gene knockdown; Mesenchymal stem cells; Nanotopography; RNA interference