Effect of release kinetics from anti-miR-eluting wound dressings on the promotion of wound angiogenesis

Introduction:: Non-healing diabetic wounds, or ulcers, continue to plague many patients, yet there has been little meaningful improvement in standard-of-care treatment. More recently, it has been discovered that non-coding RNAs, particularly microRNAs (miRs) are dysregulated in the diabetic wound, driving the impaired healing state. Strategies using inhibitory nucleic acid therapies to correct expression of the aberrantly overexpressed RNAs are challenged by their delivery to the wound bed. This strategy enables regulation of hundreds of genes involved in key pathways with a single oligonucleotide sequence. Several biomaterial strategies have been employed to package and control the release of these delicate therapies while also enhancing their cellular internalization and sub-cellular localization. An additional challenge is the dynamic nature of wound healing, which requires timing the therapy effectively to the underlying process. We have previously demonstrated efficacy of a fast release formulation in vitro and in vivo. It is currently unknown how the release kinetics of pro-angiogenic anti-miR therapy locally to the wound bed may affect its efficacy.

Materials and Methods:: We leverage electrostatic assembly of anti-miR towards anti-angiogenic miR-92a (anti-miR-92a, 92ai) with transfection polymers and biocompatible polymeric excipients through the layer-by-layer process. Through this, we can package the anti-miR and control its release kinetics. The anti-miR therapy, which is itself a polyanion due to the phosphate backbone of RNA, and polymeric coating components are conformally coated on commercial woven nylon wound dressings and elute to the wound bed in the inverse order of their deposition. The fast release formulation contains dextran sulfate as an anionic polymer excipient (Fig. 1A). The slow-release formulation contains a nanoclay, laponite, as the anionic polymer excipient (Fig. 1A). Laponite is discoid and adsorbs to the coating such that its high aspect ratio forms a barrier to diffusion. The release kinetics of these two formulations were demonstrated by releasing dressings containing fluorescent RNA into 1% BSA in PBS. The release kinetics were then confirmed in the wounds of C57Bl6/J mice by IVIS imaging. We investigated anti-miR bioactivity when released from these dressings by incubating directly with human umbilical vein endothelial cells (HUVECs) and through local delivery in the wounds of C57Bl6/J mice. The effect of release kinetics on efficacy (target miRNA expression, downstream mRNA target expression, gross wound area, granulation tissue thickness, and CD31⁺ blood vessel formation) can be investigated in a murine genetically diabetic (db/db) full-thickness wound model.

Results, Conclusions, and Discussions:: Results: While fast-release dressings release about 60% of their loaded RNA therapy over the first day with sustained release over the next week in vitro, a slow-release formulation releases this therapy only about 20% in the first day with sustained release that has not completed over the next 9 days (Fig. 1B). Measurement of the RNA therapy remaining on the dressing over time in the wounds of C57Bl6/J mice confirms sustained release in the slow-release formulations and rapid release over 12-24 hours in the slow-release formulation (Fig. 1C). These experiments demonstrate the ability of laponite to act as a diffusion barrier that slows the release of RNA therapy from the thin coatings on dressing surfaces. We then demonstrate that both fast- and slow-release dressings maintain anti-miR bioactivity in inhibition miR-92a when released over 24 hours into HUVECs (Fig. 1D). We furthermore demonstrate that when placed on the wounds of C57Bl6/J mice, the slow-release dressings containing 92ai inhibit miR-92a relative to those containing Scr by about 30% at day 4. At this time point, they also de-repress Itga5 and Pten by 2 and 1.5-fold, respectively (Fig. 1E). By day 7, the inhibition of miR-92a and de-repression of downstream targets has normalized (data not shown).

Discussion: It remains unknown how release kinetics of pro-angiogenic anti-miRs may impact downstream angiogenic signaling and effects on wound healing. This is of particular importance since wound healing is a dynamic process with changing signaling over the course of the healing process. An appropriately-powered full-scale study in db/db mice is needed to study these differences.

Conclusions: This study suggests that we can tune the release kinetics of pro-angiogenic anti-miR from wound dressings. Although we have previously demonstrated bioactivity of the fast-release dressings, we confirmed that anti-miR released from slow-release dressings maintains bioactivity in vitro in HUVECs and in vivo in a pilot-scale wound study. Further full-scale studies in db/db mice will enable us to understand if release kinetics impact the promotion of angiogenesis and closure in diabetic ulcers.


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