Acceleration of Diabetic Wound Healing with PHD2- and miR-210-Targeting Oligonucleotides.

In diabetes-associated chronic wounds, the normal response to hypoxia is impaired and many cellular processes involved in wound healing are hindered. Central to the hypoxia response is hypoxia-inducible factor-1α (HIF-1α), which activates multiple factors that enhance wound healing by promoting cellular motility and proliferation, new vessel formation, and re-epithelialization. Prolyl hydroxylase domain-containing protein 2 (PHD2) regulates HIF-1α activity by targeting it for degradation under normoxia. HIF-1α also upregulates microRNA miR-210, which in turn regulates proteins involved in cell cycle control, DNA repair, and mitochondrial respiration in ways that are antagonistic to wound repair. We have identified a highly potent short synthetic hairpin RNA (sshRNA) that inhibits expression of PHD2 and an antisense oligonucleotide (antimiR) that inhibits miR-210. Both oligonucleotides were chemically modified for improved biostability and to mitigate potential immunostimulatory effects. Using the sshRNA to silence PHD2 transcripts stabilizes HIF-1α and, in combination with the antimiR targeting miR-210, increases proliferation and migration of keratinocytes in vitro. To assess activity and delivery in an impaired wound healing model in diabetic mice, PHD2-targeting sshRNAs and miR-210 antimiRs both alone and in combination were formulated for local delivery to wounds using layer-by-layer (LbL) technology. LbL nanofabrication was applied to incorporate sshRNA into a thin polymer coating on a Tegaderm mesh. This coating gradually degrades under physiological conditions, releasing sshRNA and antimiR for sustained cellular uptake. Formulated treatments were applied directly to splinted full-thickness excisional wounds in db/db mice. Cellular uptake was confirmed using fluorescent sshRNA. Wounds treated with a single application of PHD2 sshRNA or antimiR-210 closed 4 days faster than untreated wounds, and wounds treated with both oligonucleotides closed on average 4.75 days faster. Markers for neovascularization and cell proliferation (CD31 and Ki67, respectively) were increased in the wound area following treatment, and vascular endothelial growth factor (VEGF) was increased in sshRNA-treated wounds. Our results suggest that silencing of PHD2 and miR-210 either together or separately by localized delivery of sshRNAs and antimiRs is a promising approach for the treatment of chronic wounds, with the potential for rapid clinical translation.

Biocompatibility assessment of insulating silicone polymer coatings using an in vitro glial scar assay.

Vapor-deposited silicone coatings are attractive candidates for providing insulation in neuroprosthetic devices owing to their excellent resistivity, adhesion, chemical inertness and flexibility. A biocompatibility assessment of these coatings is an essential part of the materials design process, but current techniques are limited to rudimentary cell viability assays or animal muscle implantation tests. This article describes how a recently developed in vitro model of glial scar formation can be utilized to assess the biocompatibility of vapor-deposited silicone coatings on micron-scale wires. A multi-cellular monolayer comprising mixed glial cells was obtained by culturing primary rat midbrain cells on poly(D-lysine)-coated well plates. Stainless steel microwires were coated with two novel insulating thin film silicone polymers, namely poly(trivinyltrimethylcyclotrisiloxane) (polyV(3)D(3)) and poly(trivinyltrimethylcyclotrisiloxane-hexavinyldisiloxane) (polyV(3)D(3)-HVDS) by initiated chemical vapor deposition (iCVD). The monolayer of midbrain cells was disrupted by placing segments of coated microwires into the culture followed by immunocytochemical analysis after 7 d of implantation. Microglial proximity to the microwires was observed to correlate with the amount of fibronectin adsorbed on the coating surface; polyV(3)D(3)-HVDS adsorbed the least amount of fibronectin compared to both stainless steel and polyV(3)D(3). Consequently, the relative number of microglia within 100 µm of the microwires was least on polyV(3)D(3)-HVDS coatings compared to steel and polyV(3)D(3). In addition, the astrocyte reactivity on polyV(3)D(3)-HVDS coatings was lower compared to stainless steel and polyV(3)D(3). The polyV(3)D(3)-HVDS coating was therefore deemed to be most biocompatible, least reactive and most preferable insulating coating for neural prosthetic devices.

Incorporation of Linear Spacer Molecules in Vapor Deposited Silicone Polymer Thin Films.

Poly (trivinyl-trimethyl-cyclotrisiloxane) or polyV(3)D(3) is a promising insulating thin film known for its potential application in neural probe fabrication. However, its time-consuming synthesis rate renders it impractical for manufacturing standards. Previously, the growth mechanism of polyV(3)D(3) was shown to be affected by significant steric barriers. This article describes the synthesis of a copolymer of polyV(3)D(3) via initiated chemical vapor deposition (iCVD) using V(3)D(3) as the monomer, hexavinyl disiloxane (HVDS) as a spacer, and tert-butyl peroxide (TBP) as the initiator to obtain nearly a 4-fold increase in deposition rate. The film formation kinetics is limited by the adsorption of the reactive species on the surface of the substrate with an activation energy of -41.5 kJ/mol with respect to substrate temperature. The films deposited are insoluble in polar and non polar solvents due to their extremely crosslinked structure. They have excellent adhesion to silicon substrates and their adhesion properties are retained after soaking in a variety of solvents. Spectroscopic evidence shows that the films do not vary in structure after boiling in DI water for 1 hour, illustrating hydrolytic stability. PolyV(3)D(3)-HVDS has a bulk resistivity of 5.6 (±1) × 10(14) Ω-cm, which is comparable to that of parylene-C; the insulating thin film currently used in neuroprosthetic devices.