Downregulation of microRNA-31 (miR-31) and microRNA-132 (miR-132) has been associated with delayed wound healing. Therefore, it was hypothesised that intracellular delivery of miR-31 and miR-132, both as individual and blend formulations, could promote tissue repair. The use of a blend could minimise potential toxicity and achieve synergistic effects, thus maximising the therapeutic effect. miR-31 and miR-132 were condensed with a 30-mer positively charged amphipathic peptide, RALA, to form nanocomplexes with an average size <200 nm and zeta-potential ≥10 designed to facilitate cellular internalisation. This enabled a fold increase in miR-31 and miR-132 expression of ≥100,000 in a murine fibroblast cell line (NCTC-929) and a skin human keratinocyte cell line (HaCaT), with intracellular delivery >70% for individual and blend formulations. Moreover, incubation with the nanocomplexes increased the migration of HaCaT cells ≥25% at 4 and 8 h post-incubation, as well as downregulation of EMP-1 and RASA1 and HB-EGF and RASA1, target genes for miR-31 and miR-132, respectively. Electrospinning was then employed to produce an alginate/polyvinyl alcohol/ciprofloxacin nanofibrous wound patch to facilitate the controlled delivery of the nanocomplexes. Nanofibres were crosslinked with glutaraldehyde to improve stability in aqueous solvents, and they were proven to be biocompatible with antimicrobial activity without cellular attachment to avoid injury upon removal. RALA/miR nanoparticles were incorporated to the nanofibrous wound dressing and in vivo wound healing studies using C57BL/6J mice demonstrated a >60% acceleration in the wound closure rate at Day 7 post-wounding, a ≥1.5 increase in epidermal thickness, and a ≥2 increase in blood vessel count with respect to commercial and untreated controls. Taken together, this data proves that delivery of RALA/miR-31 and RALA/miR-132 from an alginate/polyvinyl alcohol/ciprofloxacin nanofibrous wound dressing constitutes an advanced therapy for wound healing, by accelerating wound closure and improving healed tissue quality. STATEMENT OF SIGNIFICANCE: In this study, we report for the first time the use of the RALA peptide to deliver two miRNA 31 & 132 simultaneously from an electrospun patch. Both miRs have been shown to be downregulated in wounds and this study endeavoured to deliver a blend of the miRs from a nanofibre patch. Electrospinning was used to produce an alginate/polyvinyl alcohol/ciprofloxacin wound patch to enable controlled delivery of the miRs without cellular attachment to the wound with the added benefit of anti-microbial activity. Application of the nanofibre patch loaded with the blended RALA/miR nanoparticles demonstrated a synergistic effect with acceleration of wound closure rate, a significant increase in epidermal thickness and blood vessel count with respect to commercial and untreated controls.
Keywords: Electrospinning; Nanoparticles; Nucleic acid delivery; Peptide; Polymer patch; RALA; Wound healing; miR; miR132; miR31.
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