2.1.

Volunteers

The investigations were performed using healthy male and female volunteers of skin phototypes 1 through 3,¹⁴ aged 26±9 years (protocol 1) and 23±1 year (protocol 2). Approval was obtained for these experiments from the Ethics Committee of the Charite´ Hospital, Berlin.

2.2.

Materials

A sunscreen consisting of an o/w emulsion containing the UV filters octylmethoxycinnamate (7) and butyl methoxydibenzoylmethane (1.5) with a declared SPF of 8 was used in the experiments. The same o/w emulsion without UV filters was used as a control in the experiments.

The sunscreen was dissolved in a mixture of chloroform/methanol=2:1 (both UVASOL, Merck, Darmstadt, Germany) for the application in optical cells. In addition, the sunscreen was mixed with the emulsion at a ratio of 1:1 (sunscreen/emulsion mixture) for the application in an optical cell.

In the case of laser scanning microscopy (LSM), the fluorescent dye curcumin (Merck-Schuchardt, Hohenbrunn, Germany) was added at a concentration of 0.2 to the sunscreen to visualize the substance distribution on the skin.

2.3.

Tape Stripping

Tape strips were removed from the skin treated with the sunscreen using tesa film (number 5529, Beiersdorf, Hamburg, Germany). The tapes were pressed onto the skin using a roller and removed with one quick movement, as described previously.^{15 16}

2.4.

Laser Scanning Microscopy

The distribution of the sunscreen containing curcumin on the removed tape strips was investigated by fluorescence measurements (excitation wavelength 488 nm, detection of the fluorescence signal at 600 nm) using the LSM 2000 (Carl Zeiss, Jena, Germany).

The in vivo measurements were performed using a dermatological LSM system (Optiscan Limited, Melbourne, Australia), which can be applied directly to the skin.

2.5.

Application Protocol and Determination of the SPF

The SPF determination was carried out in accordance with the COLIPA protocol¹ based on the ratio of the minimal erythema dose (MED) with and without sunscreen application. The MED was determined on the back of the volunteers using the sun simulator ETG 1 (Fa. A.L.T. Lichttherapietechnik GmbH, Zo¨rbig, Germany). The device was equipped with a lamp TL 4 W/12 to 8.09 W/m² (Fa. Philips, Hamburg, Germany). The correlation of the UV spectrum to the sun spectrum of this lamp was evaluated by Meffert¹⁷ and Piazena.¹⁸ The spectral characteristics of the used UV lamp do not influence the SPF ratio if the same sunscreen is applied in different forms.¹⁹ The sun simulator emits a parallel light beam. The time of irradiation was calculated on the basis of the skin phototype in accordance with the scale by Fitzpatrick.¹⁴

Erythema were determined 24 h after irradiation using the colorimeter Spectropen (Fa. Dr. Lange, Berlin, Germany). Then, the length of the erythema was measured. The MED was determined on the basis of this erythema length in dependence on the UV dose applied. The MED was determined for untreated skin on the back, and for skin after the direct application of the sunscreen, as well as for skin covered by optical cells containing the sunscreen incorporated in the emulsion or solved form.

Irradiation started 1 h after application of the formulations, to make sure that the sunscreen had achieved the maximal homogeneity of distribution on the skin. The measurements were realized with an experimental arrangement as illustrated in Fig. 1. Two different protocols were used.

Figure 1

Arrangement for the determination of the SPF. (a) Protocol 1: direct application, cell length d=1 mm. (b) Protocol 2: dissolved form, cell length d=1 cm.

The first protocol (protocol 1, 6 volunteers) was established to compare the sunscreen applied topically onto the skin, with the emulsioned form applied into the optical cell [Fig. 1(a)]. The optical cell consisted of two quartz plates with a distance holder in-between. Because it was difficult to distribute the small amount of 2 mg/cm² sunscreen homogeneously in the optical cell, the sunscreen was diluted with the corresponding emulsion in the ratio 1:1. Before the irradiation experiments started, ten samples of this mixture were analyzed using a spectrometer to ensure that the UV filter substances were distributed homogeneously in the mixture.

In case A, the sunscreen was applied directly onto the skin (2 mg/cm²) and the optical cell was filled with the emulsion (4 mg/cm²). Due to this amount, the thickness of the optical cell was 1 mm. The optical cell was positioned 1 cm above the skin surface. Case B was realized by applying the emulsion onto the skin (2 mg/cm²) and filling the mixture of sunscreen and emulsion into the cell (4 mg/cm²). In this way, identical optical parameters of the skin were obtained. The distance of the radiation source to the skin was 2 cm.

A second protocol (protocol 2, 6 volunteers) was established to compare the sunscreen applied topically onto the skin, with the solved one applied into the optical cell [Fig. 1(b)]. Therefore, eight quartz cells with a thickness of 10 mm (Type 100-QS, Fa. Hellma, Jena, Germany) were fitted together to form one large cell (total area: 4×8 cm²) using a frame. In case A, this setup was used to determine the SPF obtained after the application of the sunscreen directly onto the skin (2 mg/cm²) and filling the solved emulsion into the optical cell. The concentration of the solved emulsion was 2 mg/ml, which corresponds to 2 mg/cm². Due to this amount, the thickness of the optical cell was 1 cm. In case B, vice versa, the sunscreen was filled as a homogeneous solution into the optical cell (2 mg/cm²), while the emulsion was applied onto the skin (2 mg/cm²). The distance of the radiation source to the skin was 2 cm.

Thus, in both protocols, identical optical conditions were realized in cases A and B. Only the positions of the UV filter substances were different. The application protocols are summarized in Table 1. The substances were applied on the back of volunteers on an area of 8×10 cm², which was marked with a permanent marker. The formulations were applied with a syringe and distributed homogeneously with a saturated gloved finger. Formulations were rubbed in for approx. 30 s. Thereafter, the volunteers rested for 1 h without sweating and without covering the test area with textiles until the irradiation started. Cases A and B were always compared on the same volunteer.

Table 1

2.6.

Statistics

Statistical analysis was performed with the software program SPSS^®. The Wilcoxon test was utilized to analyze the SPF obtained with protocols 1 or 2 depending on the case (A or B). The SPF of both groups of volunteers, differing in the protocol (case A, protocols 1 and 2), were compared using the Mann-Whitney test.

3.1.

In Vivo Measurements Using Laser Scanning Microscopy

The penetration kinetics of the o/w sunscreen emulsion containing a fluorescent dye (curcumin) were investigated on the same skin area of the forearm at different times after application. The fluorescence images of the stratum corneum of a volunteer, obtained 5 and 20 min after application, are presented in Fig. 2. The fluorescent dye was mainly located in the furrows and in the lipid layer around the first layer of the stratum corneum, 5 min after application. Later, it could also be detected around deeper corneocyte layers. After 20 min, four layers of the stratum corneum became visible. The shifted structure of the stratum corneum could be clearly distinguished.

Figure 2

Deeper cell layers of the stratum corneum become visible during penetration of the fluorescent dye into the horny layer (a) 5 min after application and (b) 20 min after application.

20 min after application, the distribution of the formulation within the skin was very nearly constant. Only slight changes were observed up to 45 min after dye application (data not shown). Therefore, the following measurements were carried out 1 h after the topical application of the sunscreen when the maximal homogeneity of the distribution of the sunscreen on the skin was certain.

3.2.

Comparison of In Vivo and In Vitro Measurements Using Laser Scanning Microscopy

Tape strips were removed from the skin 1 h after treatment with the o/w sunscreen emulsion containing curcumin. The distribution of the dye on the tape strips was immediately analyzed by LSM measurements after removal. In addition, the distribution of the dye was determined in vivo to make sure that the in vitro measurements reflect the real distribution of substances on the skin. In Fig. 3, typical results of these measurements are shown. A similar nonhomogeneous distribution was observed on the tape strips and on the living skin. In both cases, the dye was mainly located in the lipid layers around the corneocytes. The highest amount was detected inside the furrows of the skin in vivo [Fig. 3(b)].

Figure 3

Distribution of topically applied curcumin (white fluorescence signal) determined by laser scanning microscopy: (a) in vitro, measured on a removed tape strip and (b) in vivo, measured on the forearm.

3.3.

Measurements of the Sun Protection Factor

In primary experiments, it was established that the solvents used in the experiments did not change the position and intensity of the characteristic absorption bands of the filter substances, in comparison to the original sunscreen. The results of the SPF determination are presented in Fig. 4 for cases A and B of both protocols, after direct application of the UV filters onto the skin and into the optical cell above the skin. The results were compared to the SPF, as declared by the company producing the sunscreen. The SPF of 8 was reproduced with both protocols (10.3±2.2 and 8.8±2.3, respectively) taking into consideration case A (p>0.05). Significantly higher SPF values were obtained in both protocols for case B (p<0.05 for both protocols).

Figure 4

Comparison of the SPF obtained with the different protocols [Protocol 1: sunscreen applied onto the skin (case A) and into the optical cell (case B); Protocol 2: sunscreen applied onto the skin (case A) and dissolved in a solvent into the optical cell (case B)]. ^*p=0.035 (Wilcoxon test), ^#p=0.035 (Wilcoxon test), ^§p=0.337 (Whitney-Mann test).

In Table 2, the ratio of the SPF values measured for cases A and B following identical protocols is summarized. A drastic increase in the SPF, by a factor of approximately 5, was observed when the emulsion was located inside the optical cell above the skin surface. This difference was increased to 10 when the optical cell was filled with the solved sunscreen emulsion.

Table 2

4.1.

Nonhomogeneity of Distribution

Both in vivo as well as in vitro, the dye applied in a sunscreen was mainly located around the corneocytes and inside the furrows (see Fig. 3). These results are in agreement with the reported observations, which illustrate that topically applied substances were inhomogeneously distributed on the skin in vivo ^{9 18} and in vitro.²⁰ In vivo measurements using laser scanning microscopy demonstrated that the sunscreen penetrated into the upper part of the stratum corneum. After 20 min it was located around the upper layers of the corneocytes without further changes of the distribution in the skin.

These results demonstrate that the sunscreen should be applied approximately 20 min earlier onto the skin before going into the sun, as recommended by most of the producers. Only in this case can maximum sun protection of the formulation be obtained. Therefore, the investigations in the present study were started 1 h after application of the sunscreen, thus the maximum protection efficacy of the sunscreen was achieved.

In addition, it has been shown that the distribution of the sunscreen on the removed tape strips reflects the real (actual) situation on living skin (see Fig. 3). This effect can be used for the in vitro determination of absorption properties of sunscreens applied topically when taking the actual distribution on the skin into consideration. An application of this effect for SPF determination will be an object of further investigation. This method may be more precise than model calculations, if an artificial nonhomogeneous distribution of sunscreens is given to simulate the skin surface structure.^{6 21}

It is well-known in spectroscopy that a disturbance of the homogeneous distribution reduces the intensity of the absorption. Indeed, an influence of the in vivo distribution of the UV filter substances on their protection properties has been reported.^{8 9} These results were quantitatively tested in vivo by SPF measurements in the present study.

4.2.

Measurements of the Sun Protection Factor

The results given in Fig. 4 and Table 2 describe the difference of the SPF up to 1 order of magnitude, depending on the distribution of the UV filters. In these experiments, only the homogeneity of the distribution of the identical concentrations of UV filter substances per cm² varied. All other experimental conditions used for the SPF determination were constant, even the changes in the optical properties of the skin after application of an emulsion as reported by Tuchin et al.;¹⁰ and Lademann et al.;¹¹ This can be realized only by in vivo measurements using the erythema in both experiments as an indicator.

The SPF increased by 1 order of magnitude if the sunscreen was solved in chloroform/methanol and put into the optical cell above the skin (protocol 2). These significant changes were caused by the nonhomogeneous distribution of the filter substances on the human skin after topical application, compared to the homogeneous distribution in the optical cells.

The results obtained using protocol 1 demonstrate that the UV filter substances were not only distributed on the skin nonhomogeneously, but also to a much lower extent in the sunscreen sample used. The ratio of the SPF (case B) to the SPF (case A) given in Table 2 is decreased to 5.3, reflecting a lower degree of homogeneity of the sunscreen in the emulsion in comparison to the solution.

This means that in normal use, the nonhomogeneous distribution of the UV filter substances within the stratum corneum, after application of a sunscreen, reduced the protection potential by a factor of 10 in comparison to the optimum. These results quantitatively confirm the findings of Brown,⁹ O’Neill,⁶ and Sayre et al.;,⁸ who found less SPF values in vivo than in in vitro experiments. The slight difference between the measured SPF (case A, both protocols) and the declared SPF might be the result of a higher degree of homogeneity in distribution achieved during the 1 h after application compared to the 15 min as recommended by the COLIPA.¹ The COLIPA protocol¹ is usually used by companies to determine the SPF of commercial products.

The in vivo method, described herein, allows the qualitative and quantitative determination of the influence of the nonhomogeneity of the distribution of a sunscreen onto the SPF for defined formulations. Therefore, it is a future challenge for pharmaceutical and cosmetic research to increase the SPF of a sunscreen by optimizing the formulation in terms of homogeneity to significantly increase the efficacy of sunscreens after topical application.