2.1.

Tissue

2.2.

Topical Application of Emulsions

An oil in water (o/w) emulsion containing 0.2 of the fluorescent dye curcumin (Merck-Schuchardt, Hohenbrunn, Germany) was used in the laser scanning microscopy experiments. (An o/w emulsion is a dispersion of oil droplets into an aqueous medium using emulsifiers.) The penetration studies were performed applying the o/w emulsion and an oil containing 1.5 of the UV filter substance butyl methoxydibenzoylmethane (Merck, Darmstadt, Germany). The tape stripping was started 1 h after application of the emulsions.

The emulsions (2 mg/cm²) were homogeneously distributed on a skin area of 5×6 cm² on the flexor forearms of human volunteers or on pig ears, as described previously.⁸ The emulsion was applied with a stump syringe inside the marked area and distributed homogeneously with a gloved finger previously saturated with the emulsion.

2.3.

Tape Stripping Protocol

The tape strips were removed from the skin areas as described in detail by Weigmann et al.;⁸ The adhesive tape (tesa film No. 5529, Beiersdorf, Hamburg, Germany) was pressed onto the surface of the skin 10 times with a roller. A piece of paper was placed between the tape and the roller to avoid a transfer of emulsion from the adjacent skin part to the adhesive film. The tape strip was removed with one quick movement and fixed to a slide frame. The tape stripping procedure was repeated 10 to 30 times on the same skin area.

2.4.

Laser Scanning Microscopic Measurements

The thickness of the corneocyte aggregates removed by tape stripping was determined by confocal laser scanning microscopy (LSM 2000, Carl Zeiss, Jena, Germany) using an excitation at 488 nm. Therefore an o/w emulsion containing 0.2 of the fluorescent dye curcumin was applied on the skin in order to make the corneocytes visible. After a penetration time of 1 h, the tape strips were removed from different depths of the stratum corneum.

Figure 1 shows a laser scanning microscope (LSM) image of the typical distribution of the corneocyte aggregates on a tape strip removed from human skin. The contours of the corneocyte aggregates can be seen clearly. The average thickness of the corneocytes on the tape strips was determined by scanning the focus of the LSM through the tape strips. A typical image of the cross-sections of two corneocyte aggregates collected on the adhesive tape is presented in Fig. 2. The dimension of the corneocyte aggregates can be clearly recognized owing to the fluorescence of the dye covering the surface of the corneocytes.

Figure 1

Distribution of human corneocytes on a tape strip (tape 1) measured with a laser scanning microscope.

Figure 2

Cross-section of corneocytes on a removed tape strip. The corneocytes were covered with fluorescent dye.

The density of the corneocytes on the tape strips was also analyzed using the standard LSM software program. Figure 3 shows the density of the corneocytes on the removed tape strips determined by laser scanning microscopy. The contour of each corneocyte aggregate on different subareas of a tape strip was marked manually; the area A_i of the aggregate was calculated by the software program. The sum of all areas ∑ A_i was related to the total area of the complete image A _tape , representing the relative covering density D of the corneocytes.

Figure 3

Determination of the covering density of human corneocytes on a tape strip (No. 4) using laser scanning microscopy.

2.5.

Determination of the Covering Density Using Light Microscopy

The covering rate of human corneocytes on tape strips was determined using a light microscope with transmitted light (System microscope BX60, Olympus Optical Co., Hamburg, Germany) in the phase contrast mode (objective UPLFL4X/0.13, Olympus Optical Co., Hamburg, Germany). The microscope was equipped with a digital camera (SIS ColorView12, Soft Imaging System GmbH, Mu¨nster, Germany) and the appropriate software (analySIS AUTO 3.1, Soft Imaging System GmbH). The phase contrast mode used offers the possibility of evaluating the images of removed tape strips for the covering density of the corneocyte aggregates. These densities were determined by relating the dark areas (areas covered by corneocytes) to the total sizes of corresponding images [Fig. 4(a)]. The software analysis AUTO 3.1 was used to distinguish between the corneocyte aggregates and the background by an alteration in the contrast of the image [Fig. 4(b)]. A histogram of the image was calculated automatically. A threshold level easy to identify in this histogram was chosen to mark only the corneocytes. This calculation was performed on ten subareas of 3.68 mm² inside a total area of 4 cm² for each tape strip owing to the nonhomogeneous distribution of the corneocytes on the removed strips. An average value of the corneocyte density was calculated. This procedure was performed frequently image by image.

Figure 4

The distribution of corneocytes on a tape strip detected by light microscopy (No. 6). (a) Detected in the mode of phase contrast. (b) Evaluated by the contrast software program (resulting relative covering density of the horny layer particles, 59.7).

2.6.

UV/VIS Spectroscopic Measurements

UV/VIS spectroscopic measurements were performed with a modified double-beam spectrometer (Lambda 20, Perkin Elmer, U¨berlingen, Germany) with a quadratic beam of 1×1 cm². These measurements were used to determine the pseudoabsorption of the corneocytes as a standard value for evaluation of the microscopic methods. In these experiments, the pseudoabsorption of the corneocytes was determined at 430 nm. In principle, all wavelengths can be used that are not disturbed by absorption bands of exogenous and endogenous compounds.⁸ The removed tape strips were measured using an empty tape as reference. After determining the amount of corneocytes, the UV/VIS spectroscopic measurements were used to determine the amount of topically applied UV filter substance.

The edges of the tape strips were cut in order to achieve defined areas of 1.5×4.3 cm². Then the strips were put into tubes filled with 6.45 ml ethanol (Uvasol, Merck, Darmstadt, Germany) and treated for 5 min in an ultrasonic bath (Sonorex Super RK 102H, Bandelin Electronic, Berlin, Germany). The solutions were purified by centrifugation (5 min at 4000 cycles/s, centrifuge MR 1812, Jouan GmbH, Unterfaching, Germany) to separate small horny-layer particles. The uppermost part was decanted with a pipette into a quartz cell with a length of 10 mm or 1 mm (Hellma, Jena, Germany).

The UV/VIS spectra of the solutions were measured between 240 and 500 nm using the pure solvent as a reference. The concentration of butyl methoxydibenzoylmethane was calculated on the basis of a calibration curve of UV filter substance in ethanol using the determined absorption maximum at 358 nm.

2.7.

Calculation of Penetration Profiles

The penetration profiles were calculated based on the determination of the concentration of butyl methoxydibenzoylmethane and the amount of the removed corneocyte aggregates on each tape strip (Fig. 5). The strips were removed after topical application and penetration of the o/w emulsion or the oil containing the UV filter.

Figure 5

Penetration profiles of butyl methoxydibenzoylmethane; based on the calculated horny layer profile determined via the relative covering density and on the concentration of butyl methoxydibenzoylmethane, 1 h after application to the forearm. (a) Applied with an o/w emulsion. (b) Applied with an oil.

The amount of corneocytes was determined for each removed tape strip using light microscopy. The amount of butyl methoxydibenzoylmethane on each strip was determined spectroscopically. Since sunscreens are usually localized in the upper part of the horny layer,^{6 17} only fifteen tape strips were removed. The relative thickness of the removed horny layer is characterized by the sum of the covering densities of corneocytes on these fifteen tape strips. Every strip was related to its position in the removed part of the horny layer. The horizontal lines in Fig. 5 correspond to the tape strip number. The distance between these lines is determined by the amount of corneocytes on the corresponding tape strips. The horny layer profile represents a cut through the removed part of the horny layer, which allows the determination of the local position of each tape inside this profile.⁸

3.1.

Determination of the Thickness of the Corneocytes

The average thickness of the corneocytes on the tape strips was determined by scanning the focus of the LSM through the tape strips. A typical image of the cross-sections of two corneocyte aggregates attached on the adhesive tape is presented in Fig. 2. The dimension of the corneocyte aggregates can be clearly recognized owing to the fluorescence of the dye covering the surface of the corneocytes. The average thickness of the corneocyte aggregates was nearly identical on all thirty tape strips removed from different depths of the horny layer. The average thickness determined on six volunteers was ca. 0.5 μm. A stack of two or more corneocytes was not observed.

3.2.

Determination of the Covering Density Using Laser Scanning Microscopy

The density of the corneocytes on the removed tape strips was determined by laser scanning microscopy (see Fig. 3). The resulting relative covering densities (D) were compared with the pseudoabsorption of the corneocytes measured with a modified UV/VIS spectrometer at 430 nm. The correlation of both quantities is shown in Fig. 6. A high correlation coefficient of R²=0.95 was obtained.

Figure 6

Correlation between the pseudoabsorption at 430 nm and the tape area covered by human corneocytes, determined by laser scanning microscopy (tape strips 1 to 10).

3.3.

Determination of the Covering Density Using Light Microscopy

The covering rate of human corneocytes on tape strips was determined using a light microscope in the phase contrast mode. The average density of the corneocytes on each tape strip was correlated with the pseudoabsorption at 430 nm. The results are presented in Fig. 7 for a series of twenty tapes removed from human skin. A correlation coefficient R² of 0.93 was obtained. The procedure was repeated and evaluated statistically for eight series of twenty tape strips each. These experiments were carried out on porcine skin. The mean correlation coefficient and the standard deviation from the eight experiments were calculated as R²±S.D.=0.95±0.02.

Figure 7

Correlation between the pseudoabsorption at 430 nm and the tape area covered by corneocytes using light microscopy (tapes 1 to 20).

3.4.

Calculation of Penetration Profiles

The penetration of the UV filter butyl methoxydibenzoylmethane applied in two different formulations was investigated. The penetration profiles are presented in Fig. 5. The concentration of butyl methoxydibenzoylmethane is depicted by the length of the black bars. In the case of the oil [Fig. 5(b)], the main amount of the UV filter is localized on the skin surface and in the uppermost part of the stratum corneum, whereas the o/w emulsion [Fig. 5(a)] causes a deeper penetration of the UV filter. In the case of this emulsion, a higher amount of corneocytes was removed by the tape strips than after application of the oil.