4D confocal microscopy method for drug localization in the skin Ulf Maeder*a, Thorsten Bergmanna, Jan Michael Burga, Sebastian Beera, Peggy Schluppb, Thomas Schmidtsb, Johannes T. Heverhagenc, Frank Runkelb, Martin Fiebicha 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 *[email protected]; 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 . Modern DCS like multiple emulsions  and solid lipid nanoparticles  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  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. Additionally, measurements of a skin permeation study are presented. In this study naphthofluorescein dissolved in water is applied topically onto a pig skin sample for several hours. The results show the location of the penetrated dye in the samples. 2. MATERIAL 2.1 Fluorescent dyes The fluorescent dyes fluorescein natrium and naphthofluorescein (both Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) are used. The excitation/emission maxima of fluorescein natrium and naphthofluorescein are 485 nm/514 nm and 594 nm/663 nm respectively. Both dyes are soluble in water and therefore suitable for incorporation in tissue phantoms. The fluorescence of naphthofluorescein is pH dependent and can be observed at a pH higher than 8. Therefore pH adjustment is necessary. 2.2 Fluorescein phantom The phantom consists of agarose (200 mg), aqua dest (19 ml), lipid emulsion (1 ml) and fluorescein natrium dissolved in aqua dest (30 µl, 10 mM solution). The ingredients are heated up to 90°C on a heating plate and stirred using a magnetic stirrer. Afterwards the fluid phantom is poured out into a petri dish and cooled down using a thermal pack. 2.3 Skin Samples Porcine skin  extracted from the ear is used in this study. The subcutis tissue is softly removed with a scalpel. For the permeation study skin samples (20mm diameter) are taken and stored in phosphate buffered saline at room-temperature. 2.4 Confocal Microscope A TCS SP5 II (Leica Microsystems, Mannheim, Germany) providing 496 nm, 514 nm and 561 nm excitation lines is used for the measurements. The image evaluation is performed with the accompanying software LAS AF Lite. 3. METHODS 3.1 Phantom permeation study For the permeation study 50 µl of a 1 mM naphthofluorescein solution (dissolved in aqua dest, adjusted to pH 9) is applied onto the fluorescein phantom for 4 hours. After removing the residue a 4D scan is performed recording the fluorescence intensity distribution in dependency of the excitation and emission wavelengths. Table 1 lists the spectral scanning parameters. The excitation wavelengths 496nm and 561nm are nearest to the excitation maximum of fluorescein and naphthofluorescein respectively. Table 1: 4D scanning parameters excitation [nm] 496 514 561 detection start/end [nm] 527 / 727 550 / 725 586 / 763 detection steps/width 10 / 20nm 10 / 20nm 10 / 20nm 3.2 Skin measurements In this preliminary study the skin is treaded with dimethyl sulfoxid (DMSO) dissolved in water. DMSO is known to degrade the skin barrier and therefore helps incorporating substances into the skin. Naphthofluorescein (50 µl of a 1mM solution) is applied topically onto the skin samples as a model drug to simulate a skin permeation study. After 5 minutes the sample is cleaned with a tissue to eliminate rough residues but to keep small amounts of the dye on the skin. Afterwards the 4D measurements are accomplished. 4. RESULTS Figure 1: Phantom penetration measurements with varying excitation wavelengths (496nm, 514nm, 561nm). Left: Overview. Right: Detailed view of the emission region of interest. 4.1 Phanton measurements Figure 1 shows spectra of selected ROIs recorded from the fluorescein phantom treaded with the naphthofluorescein solution excited with 496nm, 514nm and 561nm respectively. The ROIs are defined to cover the naphthofluorescein containing areas (see Figure 2 red box). A detailed view of the wavelengths of interest when identifying naphthofluorescein is given on the right. Figure 2 shows the images recorded of the phantom at different emission and excitation wavelengths. The inclusions of naphthofluorescein are clearly visible at the 561nm excitation line and 660nm emission wavelength. On the left side a circular structure can be identified (red circle) at the 590nm emission images that seems to be a naphthofluorescein inclusion as well. Taking the information of the 660nm emission images into account it becomes obvious that this structure does not contain naphthofluorescein and is possibly an air-inclusion. The spectral information is further used for the spectral unmixing technique to differentiate the naphthofluorescein inclusions from the phantom. The intensity depth profile of the phantom and an inclusion are shown in figure 3. It is possible to evaluate the area and the permeation depth of naphthofluorescein. 4.2 Skin measurements The same 4D scans are performed for the prepared skin samples. Figure 4 shows images and the spectra of a sample excited at 514 nm and detected at 562 nm and 659 nm (±10 nm bandwidth) respectively. The appearance of the sample surface changes depending on the regarded emission band. This is due to naphthofluorescein being present in the observed layer. The spectral data shows that naphthofluorescein clearly permeated into the sample at ROI 2 but does not show a strong influence on the signal detected at ROI 1. Figure 4 also depicts the spectral unmixing results (red: skin autofluorescence; green: naphthofluorescein; using the spectral data shown) indicating that naphthofluorescein has permeated through the sample except the boundary. This might be explained by the topographical surface of the skin sample. The topically applied naphthofluorescein does not reach the small skin folds. The structure described by ROI 2 in figure 4 can be interpreted as a local accumulation of naphthofluorescein. Excitation 496nm 514nm 561nm Recorded at 590 ± 10nm Recorded at 660 ± 10nm Figure 2: Images of the fluorescein phantom treaded with naphthofluorescein showing dye inclusions. Top row: Images recorded at 590 ± 10nm emission wavelength. Button row: Images recorded at 660 ± 10nm. Excitation wavelengths: 496nm (left), 514nm (middle), 561nm (right). Circle: possibly an air inclusion. Box: naphthofluorescein inclusion Figure 3: Depth profile of the sample and the naphthofluorescein inclusion (left) and the fluorescence image (right) after performing spectral unmixing. The inclusion is shown in red, the phantom is colored green Emission [nm] 562 (ROI 1) 659 (ROI 2) spectral unmixing Figure 4: Skin sample detected at 562 nm and 659 nm (±10 nm bandwidth) respectively and the spectral unmixing results (left). Spectral unmixing was performed using the spectral data (shown on the right) of ROI 1 and ROI 2. The skin autofluorescence is shown in red, naphthofluorescein is shown in green. 5. DISCUSSION The phantom measurements show the possibility to localize external dyes and to evaluate their permeation depth. Because of the broad autofluorescence spectrum of tissue that superimposes the signal of external dyes spectral information is important to differentiate the signal origins. It is possible to include dyes like melanin and riboflavin and to add oil-water emulsions to the phantoms to resemble the skins autofluorescence and to produce realistic scattering behavior . In this way standardized measurements can be performed to validate the method. The presented preliminary skin permeation study also shows results indicating that the method is applicable for comparing DCS. It has to be mentioned that the sample was only roughly cleaned to keep residues of the dye on the surface. In this way it was assured to detect the dye and to test the method on tissue. In the next studies DMSO as permeation enhancer that destroys the skin barrier will be replaced by a submicroemulsion that is specially designed to increase permeation rates of drugs into the skin. Naphthofluorescein will also be replaced because of the strong pH value dependence on the fluorescence intensity. It is suitable for phantom measurements where the pH value is easily adjusted, but as the skin has varying pH values in different skin layers [6,7] the dye is not adequate for these studies. Furthermore the following permeation studies will be performed for different time periods to investigate the influence on the dye uptake. Assuming that the skin autofluorescence is equally distributed in z-direction and therefore the signal decreases only due to the extinction coefficient of the sample, the autofluorescence intensity can be used as internal normalization reference for the dye intensity. In this way we can qualitatively describe the signal loss of the dye due to the decreasing dye concentration along the z-direction and it is therefore possible to evaluate the drug distribution in the sample. The main problem with depth dependent measurements is the low achievable imaging depth of confocal microscopes in tissue. For measuring depth profiles of structures in phantoms with low light scattering and absorption it is well suited, but skin in contrast is highly scattering and therefore strongly reduces the imaging depth. Two-photon microscopy as used by other groups [7,9] might solve this problem or at least enhance the imaging depth to get useful information of the dye distribution in deeper tissue layers. Accompanying these studies, Monte Carlo models are designed to calculate calibration factors for quantitative measurements . Using these factors it becomes possible to convert the detected signal intensities into the dye concentration. 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