4D confocal microscopy method for drug localization in the skin Ulf Maeder* a , Thorsten Bergmann a , Jan Michael Burg a , Sebastian Beer a , Peggy Schlupp b , Thomas Schmidts b , Johannes T. Heverhagen c , Frank Runkel b , Martin Fiebich a a Institute of Medical Physics and Radiation Protection, Technische Hochschule Mittelhessen - University of Applied Sciences, Germany b Institute of Biopharmaceutical Technology, Technische Hochschule Mittelhessen - University of Applied Sciences, Germany c Department of Diagnostic Radiology, Philips University Marburg, Germany *ulf.maeder@kmub.th-mittelhessen.de; phone ++49 641 3092645; imps.th-mittelhessen.de ABSTRACT A 4D confocal microscopy (xyzλ) method for measuring the drug distribution in skin samples after a permeation study is investigated. This approach can be applied to compare different drug carrier systems in pharmaceutical research studies. For the development of this detection scheme phantom permeation studies and preliminary skin measurements are carried out. The phantom studies are used to detect the permeation depth and the localization of the external applied fluorescent dye naphthofluorescein that is used as a model agent. The skin study shows the feasibility of the method for real tissue. For the differentiation of tissue/phantom and the dye, spectral unmixing is performed using the spectral information detected by a confocal microscope. The results show that it is possible to identify and localize external dyes in the phantoms as well as in the skin samples. Keywords: transdermal drug transport, skin permeation study, confocal microscopy, spectral unmixing, drug permeation depth, local drug distribution 1. INTRODUCTION The development of drug carrier systems (DCS) for the treatment of skin diseases is emerging in recent years [1]. Modern DCS like multiple emulsions [2] and solid lipid nanoparticles [3] are developed to increase the drug-uptake of the skin. Regarding the very poor uptake-rate compared to the amount administered to the skin for conventional DCS, improved systems to reduce costs and dose are needed. The evaluation of the developed DCS is an important step in designing new systems. A common method to investigate the performance of a DCS is a permeation study using Franz diffusion cells. This method measures the total amount of a drug that permeates through and the total amount that is located within a skin sample using high-performance liquid chromatography. Unfortunately, no information about the local drug distribution in the skin is available. Furthermore the method is error-prone and time-consuming. In this study we introduce a method for measuring the drug distribution after a permeation study using 4D confocal microscopy (xyzλ). We use fluorescent dyes as model agents to evaluate the drug transport and the local distribution. Measuring the emission spectra allows the separation into skin auto-fluorescence and the dye. For validating the method a tissue phantom that resembles the optical properties of the dermis skin layer of human and porcine skin [4] is used to perform phantom permeation studies. Inclusions of the fluorescent dye naphthofluorescein are detected inside a fluorescein phantom and the depth of these inclusions is determined.