Three-Dimensional Oral Mucosal Equivalents as Models for Transmucosal Drug Permeation Studies.

Oral transmucosal administration, where drugs are absorbed directly through the non-keratinized, lining mucosa of the mouth, represents a solution to drug delivery with several advantages. Oral mucosal equivalents (OME) developed as 3D in vitro models are of great interest since they express the correct cell differentiation and tissue architecture, simulating the in vivo conditions better than monolayer cultures or animal tissues. The aim of this work was to develop OME to be used as a membrane for drug permeation studies. We developed both full-thickness (i.e., connective plus epithelial tissue) and split-thickness (i.e., only epithelial tissue) OME using non-tumor-derived human keratinocytes OKF6 TERT-2 obtained from the floor of the mouth. All the OME developed here presented similar transepithelial electrical resistance (TEER) values, comparable to the commercial EpiOral™. Using eletriptan hydrobromide as a model drug, we found that the full-thickness OME had similar drug flux to EpiOral™ (28.8 vs. 29.6 µg/cm2/h), suggesting that the model had the same permeation barrier properties. Furthermore, full-thickness OME showed an increase in ceramide content together with a decrease in phospholipids in comparison to the monolayer culture, indicating that lipid differentiation occurred due to the tissue-engineering protocols. The split-thickness mucosal model resulted in 4-5 cell layers with basal cells still undergoing mitosis. The optimum period at the air-liquid interface for this model was twenty-one days; after longer times, signs of apoptosis appeared. Following the 3R principles, we found that the addition of Ca2+, retinoic acid, linoleic acid, epidermal growth factor and bovine pituitary extract was important but not sufficient to fully replace the fetal bovine serum. Finally, the OME models presented here offer a longer shelf-life than the pre-existing models, which paves the way for the further investigation of broader pharmaceutical applications (i.e., long-term drug exposure, effect on the keratinocytes' differentiation and inflammatory conditions, etc.).

New concepts for transdermal delivery of oxygen based on catalase biochemical reactions studied by oxygen electrode amperometry.

The development of formulation concepts for improved skin tissue oxygenation, including methods for measuring oxygen (O2) transport across biological barriers, are important research topics with respect to all processes that are affected by the O2 concentration, such as radiation therapy in oncology treatments, wound healing, and the general health status of skin. In this work we approach this topic by a novel strategy based on the antioxidative enzyme catalase, which is naturally present in the skin organ where it enables conversion of the reactive oxygen species hydrogen peroxide (H2O2) into O2. We introduce various applications of the skin covered oxygen electrode (SCOE) as an in-vitro tool for studies of catalase activity and function. The SCOE is constructed by placing an excised skin membrane directly on an O2 electrode and the methodology is based on measurements of the electrical current generated by reduction of O2 as a function of time (i.e. chronoamperometry). The results confirm that a high amount of native catalase is present in the skin organ, even in the outermost stratum corneum (SC) barrier, and we conclude that excised pig skin (irrespective of freeze-thaw treatment) represents a valid model for ex vivo human skin for studying catalase function by the SCOE setup. The activity of native catalase in skin is sufficient to generate considerable amounts of O2 by conversion from H2O2 and proof-of-concept is presented for catalase-based transdermal O2 delivery from topical formulations containing H2O2. In addition, we show that this concept can be further improved by topical application of external catalase on the skin surface, which enables transdermal O2 delivery from 50 times lower concentrations of H2O2. These important results are promising for development of novel topical or transdermal formulations containing low and safe concentrations of H2O2 for skin tissue oxygenation. Further, our results indicate that the O2 production by catalase, derived from topically applied S. epidermidis (a simple model for skin microbiota) is relatively low as compared to the O2 produced by the catalase naturally present in skin. Still, the catalase activity derived from S. epidermidis is measurable. Taken together, this work illustrates the benefits and versatility of the SCOE as an in vitro skin research tool and introduces new and promising strategies for transdermal oxygen delivery, with simultaneous detoxification of H2O2, based on native or topically applied catalase.