Non-invasive iontophoretic delivery of enzymatically active ribonuclease A (13.6 kDa) across intact porcine and human skins

The purpose of the study was to demonstrate the feasibility of using transdermal iontophoresis to deliver a functional protein, ribonuclease A (RNAse; 13.6 kDa), non-invasively across the skin. Iontophoretic transport experiments were conducted using porcine skin in vitro and established the effect of current density and protein concentration on delivery kinetics. A methylene blue-based assay was used to quantify RNAse transport and to simultaneously demonstrate that protein functionality was retained post-iontophoresis. The results confirmed that intact functional RNAse was indeed delivered across the skin; cumulative permeation and steady state flux after 8 h iontophoresis at 0.3 mA/cm2 were 224.37 ± 72.34 µg/cm2 and 68.28 ± 23.87 µg/cm2 h, respectively. Significant amounts of protein were also deposited within the membrane (e.g., 1425.13 ± 312.09 µg/cm2 at 0.3 mA/cm2). In addition to the evidence provided by the enzymatic assay with regards to RNAse integrity and functionality, SDS-PAGE gels and MALDI-TOF spectra were also used to characterize RNAse present in the receiver phase (MALDI-TOF spectra: RNAse control, 13.690 kDa cf. RNAse from permeation samples, 13.692 kDa). Co-iontophoresis of acetaminophen showed that, despite its molecular weight, electromigration was the predominant electrotransport mechanism, accounting for > 80% of RNAse total flux. Increasing RNAse concentration from 0.35 to 0.7 mM in the formulation did not result in a statistically significant increase in delivery. Iontophoretic transport of RNAse across human skin was statistically equivalent to that seen with porcine skin under the same conditions; cumulative permeation across human and porcine skin was 241.48 ± 60.01 and 170.71 ± 92.13 µg/cm2, respectively. Laser scanning confocal microscopy was used to visualize the distribution of rhodamine B-labelled RNAse in the epidermis and dermis as a function of depth following 8 h iontophoresis (results were compared to control experiments involving passive administration of the same formulation for 8 h). Although fluorescence was localized at the skin surface following passive administration, it was visible throughout the membrane after current application. In conclusion, the results demonstrate that non-invasive transdermal iontophoresis can be used to deliver significant amounts of a structurally intact, functional protein across skin.

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