Wound Healing Society

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Raman Spectroscopy And Hplc: In Vivo And Ex-vivo Validation Of A Combi-approach For Testing Transdermal Delivery Of Compounds In Wounds And Scars
Rubinder Basson1, Martin Isabelle2, Weiping Li1, Mohamed Baguneid3, David Reece2, Ardeshir Bayat1.
1University of Manchester, Manchester, United Kingdom, 2Renishaw, Gloucestershire, United Kingdom, 3Manchester University NHS Foundation Trust, Manchester, United Kingdom.

BACKGROUND The goal of any topical formulation is efficient transdermal delivery of its actives. However, delivery of compounds can be problematic with penetration through layers of dermal scar tissue. Testing of the presence and depth of penetration of compounds can prove challenging. We propose a new combined approach to assessment of transdermal delivery of topicals; initially using an ex vivo human skin culture with high performance liquid chromatography (HPLC), and subsequently validated by Raman Spectroscopy (RS) of in-vivo human normal and scarred skin. METHODS Topicals were applied to ex-vivo skin organ culture, which were quantified by an optimised HPLC system. Longitudinal sections were analysed to differentiate presence of the topical between skin layers. In-vivo normal and scarred skin from sequential biopsies with application of both topicals were analysed with RS by acquiring static spectral point measurements from cross sections. RESULTS HPLC isolated peaks for 2 actives. One compound was identified in ex vivo ‘whole skin’ and within the papillary and reticular dermis. The presence of the topical was confirmed by RS in the epidermis and reticular dermis (98% and 99% specificity, 89% and 93% sensitivity, 96%, and 97% accuracy respectively). RS also demonstrated presence of this topical over sequential time points (day 0 to week 8 and 12). Alterations in the secondary structure conformation of protein peaks (α-helix to β-sheet) accounted for changes during remodelling. CONCLUSIONS This unique approach enables successful detection as well as measurement of exact depth of penetration of compounds following application of a topical formulation in cutaneous scar tissue in both ex-vivo and in-vivo models. Where there is uncertainty regarding discrimination of skin layers using longitudinal sections, the cross-sectional approach in RS can validate findings, allowing for the simultaneous evaluation of the effects of the compounds in healing wounds and scar maturation over time.


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