2.2.1.

Tissue samples

In the first step, the synergy effect between organic absorbers and microparticles was investigated in vitro using human split skin. In the second phase, split skin freshly obtained from pig ears was used. Approval was obtained for these experiments from the Veterinary Board of Control, Berlin Treptow-Ko¨penick.

All tissue samples had a thickness of 0.3 mm and were cut using a dermatome (GA 140, Aesculap AG, Tuttlingen, Germany). Samples of identical size were used in all experiments. Therefore, tissue samples with a diameter of 14 mm were punched from the split skin.

2.2.2.

Topically applied organic and inorganic substances

The inorganic particles (received by Merck KGaA, Darmstadt, Germany) used in the experiments are listed in Table 1.

Table 1

All microparticles, excluding type 5, have absorption bands in the UV range. Therefore, the effect of the increase in absorption efficacy caused by scattering microparticles was not investigated in the UV but in the visible range. Here no superposition of the absorption bands of the organic and inorganic components could be observed. Evans blue was selected as an absorbing organic component. The substance has a characteristic absorption band at 600 nm.

Six different types of formulation were prepared. The first formulation consisted of an oil and water (o/w) emulsion. The second type of the formulation was identical to formulation 1. Additionally, 10% of different types of microparticles listed in Table 1 were added to this formulation. The third and fourth formulations were identical to formulations 1 and 2. Additionally, 1% of Evans blue was added. The fifth formulation was based on formulation 1. Additionally, it contained 2% of Eusolex^® 9020 (BMDBM, Avobenzone), and 4% Eusolex^® 6300 (MBC). The sixth formulation was based on formulation 5. Additionally, it contained 10% of microparticles (spherical silica particles, type 5).

The compositions of the six formulations are summarized in Table 2.

Table 2

2.2.3.

Application of the formulation

The formulation was applied onto the split skin at a concentration of 2 mg/cm² following the COLIPA standard.¹² The homogeneous application of such a small amount on a small skin sample (Ø 14 mm) became difficult. Therefore, a special application system was developed (Fig. 1), which consists of an optical quartz cell formed like a “pot” with a flat bottom (optical window). The bottom has a diameter of 14 mm, identical to the diameter of the skin samples. The optical cell was put on a balance (BP211 D-OCE, Satorius GmbH, Go¨ttingen, Germany). 3.5 mg of the formulation (corresponding to 2 mg/cm²) were applied to this optical cell. Subsequently, the skin sample was placed onto the optical cell so that the skin surface was faced to the formulation. Using a small “stamp,” which fit exactly into the opening of the optical cell, the skin was gently pressed to the bottom of the pot. In this way the formulation was distributed homogeneously between the optical window and the skin surface. The stamp was removed and the opening was closed with a thin optical quartz window. After a penetration time of 6 h, the optical cell was put into the spectrometer and the spectrum was measured.

Figure 1

System for the homogeneous application of formulation on the skin samples.

2.2.4.

Absorption measurements

The absorption measurements were carried out in the spectral range of 280 nm up to 800 nm using a spectrometer M 80 (Perkin Elmer, Go¨ttingen, Germany). The measuring spot was 6×8 mm². Because of the scattering properties of the tissue and the microparticles, the light transmitted through the samples was collected by an Ulbricht sphere containing the detector. The absorption of Evans blue was detected by determining the difference spectra of formulations 1 and 3 (Evans blue absorption without microparticles) and of the formulations 2 and 4 (Evans blue absorption with microparticles). The principle of the determination of the absorption of Evans blue in the skin samples is demonstrated in Fig. 2, where the spectra of the formulations 1 and 3 are presented. The absorption band of Evans blue at 600 nm is seen in the spectrum obtained from formulation 3. The spectrum of formulation 1 was used for base line correction. In analogy, the Evans blue absorption applied in formulation 4 containing microparticles was determined using formulation 2 for base line correction.

Figure 2

Determination of absorption of Evans blue by calculation of the difference spectrum of the spectra obtained from formulation 1 (emulsion) and formulation 3 (emulsion+Evans blue).

The increase in the absorption caused by the microparticles was determined by comparison of the absorption of Evans blue in formulation 3 (without particles) and formulation 4 (with particles).

The formulation 3 and all types of formulation 4, containing different types of microparticles, were applied and analyzed on ten skin samples. The main value and the standard deviation were determined.

2.3.1.

Topically applied organic and inorganic substances

In the second part of the experiments, we checked whether the changes of the visible absorption of the samples containing Evans blue on split skin could be reproduced with the UV filters (2% BMDBM and 4% MBC) in the UV range. Microparticles of type 5 (silica microparticles) were used in these experiments, which have no absorption in the spectral range of the UV filters. Formulations 5 and 6 were applied on the back of six volunteers at a concentration of 2 mg/cm² following the COLIPA protocol.¹² The SPF was determined for formulations 5 (without microparticles) and 6 (with SiO ₂ microparticles, type 5) one hour after application.

2.3.2.

SPF determination

The minimal erythema dose (MED) was determined on the back of volunteers using the sun simulator ETG 1 (Fa. A.L.T. Lichttherapietechnik GmbH, Zo¨rbig, Germany).¹¹ This device was equipped with a lamp TL 4 W/12 (Fa. Philips, Hamburg, Germany). The time of irradiation was calculated on the basis of the skin phototype, taking into consideration the Fitzpatrick scale.¹³ All MED measurements started 1 h after application of the emulsions to the skin. The erythema was determined 24 h later using the colorimeter “spectropen” (Fa. Dr. Lange, Berlin, Germany). The SPF of formulations 5 and 6 was determined using the results of the MED measurements.

The SPF determination was repeated for every formulation on six volunteers aged between 26 and 35 years. Ethical approval had been obtained for these experiments from the Ethics Commission of the Medical Faculty Charite´.

2.3.3.

Sunscreen distribution on the skin

Tape stripping can be used to determine the homogeneity of the distribution of topically applied substances on the skin.^{14 15 16} The homogeneity of the distribution of formulations 5 and 6 used for SPF determination on the skin was investigated by laser scanning microscopy (LSM 2000, Carl Zeiss, Germany). 0.1% of the fluorescent dye sodium fluorescein (MERCK-Schuchardt, Hohenbrunn, Germany) was added to both formulations.^{17 18}

The formulations were applied onto the skin following the COLIPA protocol.¹² After 1 h, the tape strips were removed from the treated skin areas following the previously described protocol² using tesa tapes (number 5529, Beiersdorf AG, Hamburg, Germany). The homogeneity of distribution of the dye on the removed corneocytes was analyzed by fluorescence measurements using the LSM excitation wavelength at 488 nm.

3.2.1.

Comparisons between human and porcine skin

The influence of coated TiO ₂ microparticles (type 6) on the absorption of Evans blue was analyzed using six samples of human and porcine split skin obtained from pig ears. The results are presented in Fig. 4. For better comparison, the absorption of Evans blue in the nonparticle-containing formulation 3 was standardized to a value of 1. The absorption of the same amount of Evans blue in the particle-containing formulation 4 was related to this standard. The absorption of Evans blue increased if the microparticles were added to the formulation. Identical results were obtained for human and porcine skin.

Figure 4

Comparison of the Evans blue absorption with and without addition of microparticles measured on human and porcine skin.

3.2.2.

Influence of different types of microparticles

The influence of different types of microparticles on the absorption of Evans blue is demonstrated in Fig. 5. The absorption of Evans blue without microparticles (formulation 3) was standardized to 1 (bar A in Fig. 5). The Evans blue absorption increased up to 150% if silica microparticles covered with TiO ₂ (type 1) were added (bar B in Fig. 5). TiO ₂ microparticles with a high reflection coefficient (type 2) increased the Evans blue absorption up to 174% (bar C in Fig. 5). On the other hand, the Evans blue absorption did not change if coated TiO ₂ microparticles with a different structure and a low reflection coefficient (type 3) were added (bar D in Fig. 5). ZnO microparticles in the configuration used (type 4) increased the Evans blue absorption up to 125% (bar E). Pure silica microparticles with a special high reflection coefficient (type 5) increased the Evans blue absorption up to 180% (bar F).

Figure 5

Influence of different types of microparticles on the absorption of Evans blue: A is formulation 3, B is formulation 4+microparticles type 1, C is formulation 4+microparticles type 2, D is formulation 4+microparticles type 3, E is formulation 4+microparticles type 4, and F is formulation 4+microparticles type 5.

3.3.1.

SPF determination

SPF measurements were carried out to confirm the in vitro results concerning the influence of microparticles on the absorption of a topically applied substance in vivo. The measurements were carried out using formulation 5 (emulsion+UV filters) and formulation 6 (emulsion+UV filters+microparticles). These silica microparticles (type 5) showed a high influence on the Evans blue absorption in the previous experiments, and have no absorption in the spectral region of the UV filter substances. In Fig. 6, the sun protection factors are presented, which were obtained for the UV filter containing formulations with and without silica microparticles. The SPF values are compared with the results of the in vitro Evans blue absorption measurements. Similar to the Evans blue absorption, the SPF increased if microparticles were added.

Figure 6

In vivo SPF and Evans blue absorption obtained with and without silica microparticles (type 5).

3.3.2.

Sunscreen distribution on the skin

It was checked whether the microparticles have an influence on the distribution of the formulations on the skin. The distribution of formulations 5 and 6 on the skin could be considered to be identical. The distribution of the fluorescent dye sodium fluorescein on the removed tape strips is presented in Figs. 7(a) and 7(b). No differences in the distribution of formulations 5 and 6 were observed.

Figure 7

Distribution of the fluorescent dye sodium fluorescein added to formulations 5 and 6 on the removed tape strips: (a) formulation 5 and (b) formulation 6.