Improving Wound Healing with DICER1-Modified Keratinocytes

Wound healing is a complex, orchestrated process, and impaired wound closure, as seen in diverse pathological states, occurs due to interference during this dynamic process. Here, we show that DICER1 levels that are downregulated in stalled wounds during diabetes promote effective wound closure when they are upregulated. Using Lipofectamine 2000, HaCaT cells were transfected with either the empty vector or the Dicer1 overexpression plasmid, and after 24 and 48 h, there was increased wound closure by Dicer1 overexpression as compared to only vector-transfected cells, and this was accompanied by the upregulation of wound-healing genes. Since Lipofectamine 2000 exerts significant cell toxicity, we employed a more specific and comparatively less toxic in-house-developed cell-penetrating peptide gene nanocarrier, M9-DICER1-CS-A. Dicer1-encapsulated coated nanocomplexes exhibited a size of ∼200 nm and a polydispersity index of ∼0.25, suggesting toward the formation of homogeneous nanocomplexes. The surface charge was around 18-20 mV, and the nanocomplexes showed effective nucleic acid condensation and release after a heparin challenge. As compared to Lipofectamine 2000, M9-DICER1-CS-A-transfected cells depicted increased cellular viability and comparable wound closure, together with the upregulation of transcript levels of wound-healing genes, suggesting a potentially advantageous utilization of this peptide-based nonviral vector. Such M9-DICER1-CS-A re-engineered HaCaT cells, when grown in a polysaccharide-based functional hydrogel, depicted persistent cell viability and a time-dependent increase in cell proliferation. These suggest that Dicer1 is a critical promoter of wound closure, and M9-DICER1-CS-A-engineered cells, when embedded within the hydrogel matrix, have future prospects to be used as a viable therapeutic strategy to address the challenges of impaired wound closure.