Understanding the poor iontophoretic transport of lysozyme across the skin: When high charge and high electrophoretic mobility are not enough

The original aim of the study was to investigate the transdermal iontophoretic delivery of lysozyme and to gain further insight into the factors controlling protein electrotransport. Initial experiments were done using porcine skin. Lysozyme transport was quantified by using an activity assay based on the lysis of Micrococcus lysodeikticus and was corrected for the release of endogenous enzyme from the skin during current application. Cumulative iontophoretic permeation of lysozyme during 8 h at 0.5mA/cm(2) (0.7 mM; pH 6) was surprisingly low (5.37 +/- 3.46 mu g/cm(2) in 8 h) as compared to electrotransport of cytochrome c (Cyt c) and ribonuclease A (RNase A) under similar conditions (923.0 +/- 496.1 and 170.71 +/- 92.13 mu g/cm(2) respectively) - despite its having a higher electrophoretic mobility. The focus of the study then became to understand and explain the causes of its poor iontophoretic transport. Lowering formulation pH to 5 increased histidine protonation in the protein and decreased the ionisation of fixed negative charges in the skin (pI similar to 4.5) and resulted in a small but statistically significant increase in permeation. Co-iontophoresis of acetaminophen revealed a significant inhibition of electroosmosis; inhibition factors of 12-16 were indicative of strong lysozyme binding to skin. Intriguingly, lidocaine electrotransport, which is due almost exclusively to electromigration, was also decreased (approximately 2.7-fold) following skin pre-treatment by lysozyme iontophoresis (cf. iontophoresis of buffer solution) - suggesting that lysozyme was also able to influence subsequent cation electromigration. In order to elucidate the site of skin binding, different porcine skin models were tested (dermatomed skin with thicknesses of 250 and 750 mu m, tape-stripped skin and heat- separated dermis). Although no difference was seen between permeation across 250 and 750 mu m dermatomed skin (13.57 +/- 12.20 and 5.37 +/- 3.46 mu g/cm(2),respectively), there was a statistically significant increase across tape-stripped skin and heat- separated dermis (36.86 +/- 7.48 and 43.42 +/- 13.11 mu g/cm(2),respectively) - although transport was still much less than that seen across intact skin for Cyt c or RNase A. Furthermore, electroosmotic inhibition factors fell to 2.2 and 1.0 for tape-stripped skin and heat- separated dermis-indicating that lysozyme affected convective solvent flow through interactions with the epidermis and predominantly the stratum corneum. Finally, cation exchange and hydrophobic interaction chromatography confirmed that although lysozyme had greater positive charge than Cyt c or RNase A under the conditions used for iontophoresis, it also possessed the highest surface hydrophobicity, which may have facilitated the interactions with the transport pathways and encouraged aggregation in the skinmicroenvironment. Thus, high charge and electrophoretic mobility seem to be inadequate descriptors to predict the transdermal iontophoretic transport of proteins whose complex three dimensional structures can facilitate interactions with cutaneous transport pathways. (C) 2014 Elsevier B.V. All rights reserved.