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Vol. 26, No. 2, 2013
Issue release date: April 2013
Section title: Original Paper
Skin Pharmacol Physiol 2013;26:76–84
(DOI:10.1159/000345976)

Photoprotective Properties of the Fluorescent Europium Complex in UV-Irradiated Skin

Vogt O.a · Lademann J.a · Rancan F.a · Meinke M.C.a · Schanzer S.a · Stockfleth E.b · Sterry W.a, b · Lange-Asschenfeldt B.a, b
aCenter of Experimental and Applied Cutaneous Physiology, Department of Dermatology, Venereology and Allergology, and bSkin Cancer Center Charité, Department of Dermatology and Allergy, Charité, Universitätsmedizin Berlin, Berlin, Germany
email Corresponding Author

Abstract

In this study, we compared the UV-protective abilities of the europium complex compared to titanium dioxide, which represents the most common physical filter for ultraviolet light in the broad-band spectral range. The UV absorption and light transformative capacities of the europium complex were evaluated using a spectrometer with a double-integrating sphere showing that the europium complex does not only absorb and reflect UV light, but transforms it into red and infrared light. It was found that the europium complex binds to the surface of Jurkat cells in vitro. Cells incubated with the europium complex showed a significantly higher viability after UVA and UVB irradiation as compared to untreated cells and cells incubated with titanium dioxide pointing out its photoprotective properties. The europium complex and titanium dioxide show similar penetration capacities into the stratum corneum as tested in human and porcine skin using tape stripping analysis. The europium complex has proved to be an efficient UV filter with a low cyto- and phototoxic profile and therefore represents a potential candidate for use in sunscreen formulations.

© 2013 S. Karger AG, Basel


  

Key Words

  • Ultraviolet filter
  • Sunscreen
  • Titanium dioxide
  • Europium complex

 Introduction

In the course of the expansion of sun-related diseases, the demand for effective UV protection has increased tremendously over the last few years [1,2,3]. Sun exposure of humans in early years is currently discussed as a major risk factor for the development of melanoma [4], basal cell carcinoma and squamous cell carcinoma [5,6]. The increase in immunosuppressive treatments predisposes the patient to sun damage [6]. Overall, there is a high demand for efficient UV protection.

Modern sunscreens are composed of several physical and chemical filters that either reflect or absorb the UV irradiation. The development of an ideal broad-band UV filter comprises its ability to efficiently protect the skin from UVB (280–320 nm) as well as UVA (320–400 nm), its chemical stability and the lack of photo- and cytotoxicity [7]. In addition, all UV filters as well as the variety of ingredients in sunscreen formulations can induce side effects such as allergic or photo-allergic reactions. Since particular UVA filters can be unstable after irradiation, these substances and their metabolites can represent a special risk for sensitization [8]. Some UV filters, i.e. titanium dioxide, are not described as carcinogenic agents [9,10], but tissue damage has been reported depending on the size of the nanoparticles [11,12]. In contrast, physical sun-protective agents are known for their stability and the broad filter spectrum [13,14]. Coated titanium dioxide nanoparticles represent efficient physical UV filters covering a broad UV spectrum, although a variety of side effects are discussed [15]. Due to their scattering properties, these nanoparticles provide not only protection from UV light, but also from visible light and infrared irradiation of the solar radiation spectrum. These protective properties are of special importance, because Zastrow et al. [2] recently demonstrated that approximately 50% of the free radicals are produced by sunlight in the visible and infrared irradiation spectral ranges.

Current chemical filters mainly absorb either in the UVA (e.g. dibenzoylmethane and benzophenone) or in the UVB spectral range (e.g. p-aminobenzoic acid, camphor derivates and cinnamic acid esters) [7]. Moreover, only a few filter substances provide UV broad-band protection (i.e. Tinosorb®). Usually, chemical filters do not have scattering properties, as they absorb the UV radiation and thereby reach an electronically excited state. During the relaxation of the filter molecules from the excited to the ground state, the excitation energy is transformed into thermal energy. As a result, radicals are produced. Vergou et al. [16 ] demonstrated that the solar protection efficacy of UV filter substances could be strongly increased, if the excitation energy was transformed into visible fluorescent light and not into heat.

In the present study, a europium complex was investigated, which possesses absorption properties in addition to highly efficient scattering properties, thus transforming the excitation energy into fluorescent light in the infrared irradiation spectral range. Inorganic europium (III)-containing compounds dispersed in polymer films are sold under the name Ksanta® by Ranita (Regensburg, Germany). The element europium is a typical member of the lanthanide series. Most applications of the rare element europium utilize the phosphorescence of europium compounds. The polymer films incorporating the Ksanta additive are commercially available under the name Redlight®. The incorporation of luminophores into the films stabilizes the polymer films leading to an extended service life and a more efficient use of solar energy [17]. The accelerated growth and development of plants under such films is known as the ‘Polysvetan effect’, which is thought to have been caused by the effect of low-intensity red luminescent radiation on the plant hormonal balance [17]. Europium is frequently used in medicine as a component of contrast media for medical imaging [18,19].

The aim of this study was to compare the optical characteristics of these UV-protective agents, the europium complex and titanium dioxide, their adherence to the cell membrane and potential uptake by cells. Moreover, the cytotoxic and phototoxic properties of these UV filters were compared in a cell culture model, where cells are incubated with these UV filters followed by exposure to UV radiation. Finally, the penetration features of the europium complex and titanium dioxide nanoparticles into the stratum corneum were described using the porcine ear skin model.

 

 Materials and Methods


 Investigated UV-Protective Agents

The UV-protective agents titanium dioxide and the europium complex were both tested in vitro for cytotoxicity and the cellular uptake and adsorption pattern. The size of the titanium dioxide particles was 100 nm, while the size of the europium complex ranged between 100 and 300 nm. For these experiments, the UV filters had to be successfully brought into solution. Because of the hydrophobic silica coating of the inorganic titanium dioxide, Cremophor (Sigma-Aldrich, Taufkirchen, Germany) had to be added to the solution, and in order to maintain comparable conditions in all tests, the same solubilizer was used for the preparation of the europium complex solution. For the in vitro tests, cells were incubated in a phosphate-buffered saline (PBS) solution containing either 4.86 µg titanium dioxide (T805, Degussa, Essen, Germany)/ml or 4.86 µg europium complex (Ranita®, Regensburg, Germany)/ml as well as 10 mg/ml Cremaphor. The europium complex has an average diameter of approximately 15 nm. The chemical structure is shown in figure 1. The cells serving as control were incubated in a PBS solution containing 10 mg/ml Cremaphor only.

FIG01
Fig. 1. The centrepiece of the investigated UV filter is the europium complex as shown in the chemical structure.

For the investigation of the penetration characteristics in porcine and human skin, titanium dioxide was applied in the form of the commercially available sunscreen Anthelios S (La Roche-Posay, Düsseldorf, Germany). The applied sunscreen emulsion contains the stable UV filters Mexoryl® XL, Mexoryl® SX, Parsol® 1798 and Eusolex® in combination with the nanoparticle titanium dioxide filter. The europium complex was incorporated in basis cream (Deutscher Arzneimittel Codex) both containing the UV-protective agent at a concentration of 2 mg/ml.

 Quantification of the Total Transmission, the Diffuse Backscattering, and the Non-Scattered Transmission of the Europium Complex and Titanium Dioxide

The europium complex transforms UV radiation into visible red light. The absorption and the emission properties of the europium complex were analysed using the spectrophotometer (Lambda 20/Perkin Elmer) with a UV double-integrating sphere (double-Ulbricht sphere).

This technique allows the quantification of the diffuse backscattering, the total transmission and the non-scattered transmission. The sample (either the europium complex or titanium dioxide) is placed between two integrating spheres. The inside of each of the integrating spheres is coated with highly reflecting barium sulphate (BaSO4) in order to measure the radiation within the sphere. Diffuse reflectance standards Hg high-pressure lamps serve as a light source in combination with a monochromator. The light beam passes the first integrating sphere, hits the sample and enters the second integrating sphere. The reflection is measured by a detector connected to the first integrating sphere, whilst the transmission is measured by a detector connected to the second integrating sphere. The calibration of the integrating spheres is performed using reflection standards (Spectralon®, BFi OPTiLAS GmbH, Gröbenzell, Germany). The measuring range of this set-up is between 300 and 2,500 nm at a wavelength resolution of 8–16 nm. Silicon photodiodes (300–2,500 nm) or lead sulphide photodiodes (1,100–2,500 nm) are used as detectors. Samples are placed in cylindrical quartz cuvettes (diameter of 15 mm). The fluorescence spectrum of the europium complex was measured after excitation with an argon laser (488 nm).

 Cell Culture

The phototoxicity of the europium complex was investigated using Jurkat cells (clone E 6-1, ATCC, UK), which are transformed immortalized human T lymphocytes originating from an acute lymphatic leukaemia cell line. Cells were cultured in RPMI medium (Life Technologies GmbH, Darmstadt, Germany) containing 2 mML-glutamine, 25 mM HEPES and supplemented with 10% foetal calf serum, 100 µg/ml streptomycin, 100 IU/ml penicillin and 25 IU/ml nystatin. All supplements were purchased from Invitrogen (Karlsruhe, Germany). Cells were cultivated at 37°C in 100% humidity and 5% CO2 and were split every 2–3 days.

 Laser Scanning Microscopy

Laser scanning microscopy was performed in order to study the uptake and adsorption pattern of the europium complex within the Jurkat cells. Cells were washed with Dulbecco’s PBS (Sigma-Aldrich) and incubated with a formulation of the investigated substances as described above. Images were taken from cells incubated for 1 h with TiO2, europium complex and control solution.

The cells were visualized using a laser scanning microscope 410 invert (Zeiss, Wetzlar, Germany). The transmitted light, the fluorescence and reflection of the probe were visualized. For the quantification of the fluorescence of the UV-protective agents, the samples were excited by a 488-nm argon laser.

 Cytotoxocity and Phototoxicity Assays

The cytotoxic and phototoxic effects of the europium complex and titanium dioxide were tested by measuring the viability of Jurkat cells incubated with both UV filters before and after irradiation over time up to 180 min. For the UV irradiation, a UVA light source (R-UVA TL09 40W, Philips, Hamburg, Germany) and a UVB light source (TL 40W/01, Philips) were used. The emission spectrum of the UVA lamp was between 315 and 400 nm with a maximum at 350 nm. The irradiation intensity was 10.7 W/m2. The UVB lamp was emitting a small spectrum within 280 and 315 nm, the emission maximum being at 311 nm. The irradiation intensity was 8.2 W/m2. After 10 min of incubation, cells were irradiated by the UVA and UVB light sources, which were positioned at a distance of 4 cm from the vials, using UVA irradiation doses between 0 and 15 J/cm2 and UVB irradiation doses between 0 and 10 J/cm2. The irradiation intensity within the samples was measured by a temperature-stabilized double monochromator-spectroradiometer (Optronic Inc., Orlando, Fla., USA) with a cosine-corrected integrating sphere serving as an optical head.

For the irradiation experiments, a 100-µl cell suspension (1,000 cells/µl) was placed in each vial. The cell solutions contained either titanium dioxide or europium complex. Cells serving as a control group were incubated without any UV-protective agent. The final concentration of both UV filters within the test medium was 4.86 µg/ml. The phototoxicity was tested by detection of the cell membrane disruption after irradiation (trypan blue assay, Sigma, Germany). The cytotoxicity of the solutions containing either titanium dioxide or europium complex was tested after incubation (180 min) with the cells in the dark by means of the trypan blue test. Data are expressed as percentages of living cells with respect to the overall cell number and are given as mean values of at least 3 experiments ± standard deviation. For the statistical evaluation of the data the non-parametric U test (Mann-Whitney) was used.

 Tape-Stripping Technique on Human Skin

In order to determine the penetration profile of titanium dioxide within the epidermis the tape-stripping technique was utilized. The female 26-year-old volunteer was not allowed to use soap or cosmetics 24 h before the start of the experiments. The skin was washed with cold water only. All experiments had been performed at a room temperature of 23°C and a relative humidity of 60%. On the right volar forearm of the volunteer, an 8 × 10 cm test site was marked and 160 mg of the sunscreen-containing emulsion Anthelios S (SPF 30, La Roche-Posay) was applied corresponding to 2 mg per square centimetre. During a 60-min penetration time, movements and sweating of the volunteers were avoided.

Tape stripping was performed using a 5-cm-long Tesa® strip (No. 5529, clear, 1.9 cm wide, Beiersdorf, Hamburg, Germany) which was pressed onto the treated test site 10 times by a roller to ensure complete contact between the tape and the skin [20]. Each stripping procedure was performed unidirectionally, and each strip was glued afterwards onto a glass slide frame. In order to remove the entire human corneal layer, about 60–100 stripping procedures had to be undertaken. Measuring 100% transmission of visible light in the tested strips would theoretically correspond to the complete removal of all corneocytes. In our studies the stripping procedure was continued until 97% of the light transmission had been measured. As a control, the stripping procedure was undertaken on untreated skin of the other left forearm of each study participant.

 In vitro Penetration Profile of the Europium Complex and Titanium Dioxide on Pig Ears

In order to investigate the penetration profile of titanium dioxide and the europium complex, fresh, cleaned and otherwise untreated porcine ears were used for the experiments since the europium complex has not yet been approved for in vivo investigations in humans. Approval for the experiments had been obtained from the Veterinary Inspection Office Berlin-Treptow. The pig ear was fixed to the surface of a styrofoam board. On one pig ear a 5 × 6 cm test site was marked and treated with Anthelios S (2 mg/cm2). On another pig ear another test site was marked and treated with the europium complex containing the same concentration of 2 mg/cm2. After 1 h of incubation sequential tape stripping was undertaken from the surface down.

In order to investigate the distribution profile of both UV filters within the corneal layers, the transmission of the tape after each stripping was measured using the UV/VIS spectrophotometer (Lambda 20/Perkin Elmer) at a wavelength of 900 nm. The concentration of the titanium dioxide nanoparticles sticking to each tape was quantified by the X-ray fluorescence analysis. For this procedure the nanoparticles were harvested from a 4.48-cm2 tape area and dissolved from the tape with 5 ml HNO3 at 7 bar using a microwave. Thereafter, the nanoparticles were quantified using an EX 1000 X-ray spectrometer (IUT GmbH, Berlin, Germany) [21,22]. The penetration of the tested substances is represented in a penetration profile graphic. The concentration of the test agent is correlated with the number of tape strips, which were necessary to remove all test agents. The tape-stripping procedure can be correlated with the thickness of the corneal layer [20,23,24]. Thereby, this technique allows the quantification of the concentration of the test agent within each corneocyte layer.

 

 Results


 Analysis of Optical Characteristics of the Europium Complex and the Titanium Dioxide-Coated Nanoparticles

In order to investigate the absorption, transmission and fluorescence properties of the tested UV filters, studies were undertaken on the double-integrating sphere.

Transmission of either the europium complex or titanium dioxide nanoparticles was measured at 250 µm layer thickness using a wavelength of 200–700 nm. The absorption maximum of the titanium dioxide was found at 200–380 nm wavelength covering almost the entire UV spectrum (fig. 2). Transmission of the nanoparticles was 5% in the wavelength spectrum of 200–380 nm, whereas 75% was measured at the spectrum of visible light.

FIG02
Fig. 2. Transmission of titanium dioxide nanoparticles and the europium complex was measured at 250 µm layer thickness using a spectrum of 200–700 nm wavelength. The absorption maximum of the titanium dioxide and the europium complex was found at 200–380 nm wavelength covering almost the entire UV spectrum.

The absorption maximum of the europium complex was also found to be between 200 and 380 nm. The transmission in the UV spectrum was 35%, and 88% within the spectrum of visible light (fig. 2). The fluorescence of the europium complex was determined using an argon laser (488 nm) for excitation. Two fluorescence maxima (595 and 618 nm) were seen after stimulation of the europium complex using the argon 488-nm laser light source (data are not shown).

 Analysis of Membrane Adsorption and Uptake of the Europium Complex by Jurkat Cells

In order to study the uptake of the europium complex by living cells or its adsorption to the cell membrane, cell suspensions were incubated with and without the europium complex, and the fluorescence pattern of the europium complex associated with the cells was studied by laser scanning microscopy. The surface and cross-sections of the cells were visualized, and the transmission of light as well as the fluorescence was analysed.

The Jurkat cells are roughly 12 µm in diameter and the images in transmission mode show the typically increased nuclei in relation to the cytoplasm of these neoplastic cells. In order to evaluate the autofluorescence of Jurkat cells, untreated cells were excited at 488 nm using an argon laser. The images showed no fluorescence pattern. The potential uptake of the europium complex by the cells or the adsorption of the complex to the cell membrane was studied after the incubation of these cells with the europium complex followed by excitation (488 nm). Fluorescence was detected on the surface of the cells (fig. 3, overlay of the images obtained by transmission and fluorescence mode). Therefore, based on the confocal imaging results, we concluded that the europium complex adsorbs on cell membrane surface (fig. 3a). Measurement of light reflection showed cross-section images of these cells and additional fluorescence around the membrane of the nucleus, which was probably resulting from an uptake of the europium complex by means of endocytosis and accumulation of the complex in endosomes and lysosomes (fig. 3b).

FIG03
Fig. 3. Representative laser scanning microscope images of untreated Jurkat cell suspensions, in transmission and fluorescence mode. a Untreated cells showed no fluorescence when excited with a 488-nm argon laser. b Fluorescence of the europium complex within the Jurkat cells. After incubation of the Jurkat cells with the europium complex and excitation with an argon laser at 488 nm, fluorescence was detected on the surface of the cells and within the cells (upper left corner) as shown by imaging in the reflection mode.

 Investigation of the Cytotoxicity and Phototoxicity of the Europium Complex and Titanium Dioxide Nanoparticles

The cytotoxicity of the investigated substances towards Jurkat cells was evaluated in the dark after cell incubation with and without titanium dioxide or the europium complex. The viability rate of cells was determined at the beginning of the experiment, after 60, 120 and 180 min of incubation. Figure 4 shows that the incubation with both the investigated substances at concentrations of 4.86 µg/ml did not significantly affect the viability rate of the cells over the time under investigation (180 min).

FIG04
Fig. 4. The cytotoxicity of the europium complex and the titanium dioxide particles towards Jurkat cells was evaluated in the dark. Jurkat cells incubated without titanium dioxide or europium complex served as control. The viability of cells was determined over time with the trypan blue test. The incubation with both sunscreens at a concentration of 4.86 µg/ml did not significantly affect the vitality rate of the cells over the investigated period of 180 min.

In order to study the extent of phototoxicity of titanium dioxide and the europium complex, the viability of Jurkat cells was determined after incubation with both substances followed by radiation with UVA using doses between 5, 10 and 15 J/cm2. Jurkat cells without any addition of the UV filters served as control and were irradiated with the same UV doses. While the cell viability at a UVA dose of 5 J/cm2 was comparable between all groups, at a dose of 10 J/cm2 the cells incubated with the europium complex showed a higher viability (86.0%) when compared to the control group (72.8%), whereas the titanium dioxide-incubated cells had the lowest viability (46.3%; p < 0.001; fig. 5a). At a UVA irradiation dose of 15 J/cm2 the viability of all groups started to drop. However, the cells incubated with the europium complex showed again a higher viability (60.0%) compared to the titanium dioxide-incubated cells (38.1%) and to the control group (50.4%; p < 0.001; fig. 5a). Consistent with the UVA irradiation, comparable experiments have been carried out using UVB light. Jurkat cells incubated with either the titanium dioxide or the europium complex, as well as untreated control cells, were irradiated with UVB doses of 1, 5 and 10 J/cm2 and the viability of the cells was determined by trypan blue staining. The cells incubated with the europium complex were significantly more resistant to the UVB irradiation at all doses applied as compared to the titanium dioxide-incubated cells and the control cells (fig. 5b). While the viability of the cells remained quite high (85%) at a UVB dose of 1 J/cm2, at 5 J/cm2 UVB the viability of europium-incubated cells was 76.6%, significantly higher when compared to the vitality of the control (53.1%) and the titanium dioxide groups (50.9%; p < 0.001). At 10 J/cm2 UVB, the viability of europium-incubated cells was 36.8%, while those of the control group (22.1%) and the titanium dioxide group (18.7%) were clearly lower (fig. 5b).

FIG05
Fig. 5. The phototoxicity of titanium dioxide and the europium complex was determined after the incubation of Jurkat cells with both substances followed by irradiation with UVA using doses of 5, 10 and 15 J/cm2 or UVB using (1, 5 and 10 J/cm2). Jurkat cells without any addition of the UV filters served as a control and were irradiated with the same doses. a At 10 J/cm2 UVA, the europium complex-incubated cells showed the highest viability (86.0%) when compared to the control group. At a UVA irradiation dose of 15 J/cm2, the viability of all groups dropped to 40–60%. b The cells incubated with the europium complex were significantly more resistant to all used UVB irradiation doses as compared to cells incubated with titanium dioxide and control cells.

 Penetration Profile of the Titanium Dioxide Particles in Human Skin in vivo

Seventy-eight stripping procedures were undertaken in order to reach a transmission value of almost 100%, thereby ensuring the entire removal of the stratum corneum. Titanium dioxide penetrated into 85% of the corneal layers, but most of the nanoparticles were found on the surface and the upper 5 layers (5 µg/cm2), whereas the concentration in deeper layers was very small (data not shown).

 Penetration Profile of Titanium Dioxide and the Europium Complex in Porcine Skin in vitro

After the 30th stripping procedure the corneocytes had been almost completely removed. Titanium dioxide was found in nearly 90% of the corneal layers. The highest concentration of titanium dioxide was found within the first 8 layers (7.8 µg/cm2). From the 6th layer up to the 30th layer, the concentration of titanium dioxide remained at a constant level (fig. 6a).

FIG06
Fig. 6.a Titanium dioxide penetrates into almost 90% of the corneal layers of porcine skin in vitro. The highest concentration of titanium dioxide was found within the first 8 layers. From the 6th layer on, the concentration of titanium dioxide remained at a constant level. b The europium complex penetrated into 85% of the relative corneocyte layer thickness. The penetration profile into the epidermis is comparable to that of titanium dioxide. The highest concentration of the europium complex was found on the surface (8 µg/cm2). Below the upper 25% of the corneal layer thickness, only low concentrations were found.

After 55 stripping procedures nearly all corneocytes had been removed from the porcine skin. The europium complex penetrated into 85% of the relative corneocyte layer thickness, which was comparable to the penetration properties of titanium dioxide. The highest concentration of the europium complex was found on the surface (8 µg/cm2). Only low concentrations of the complex were found below the upper 25% of the corneal layer thickness (fig. 6b).

 

 Discussion

In our study, we compared a europium complex, currently used in transparent sheets covering greenhouses, with the established UV filter titanium dioxide. In contrast to the already known UV broad-spectrum filters, the europium complex cannot only absorb or reflect UV irradiation, but transforms the UV light into red and infrared light [17]. The europium complex shows a similar but slightly increased transmission as compared to titanium dioxide within the spectrum of 200–380 nm, covering the harmful UV spectrum. As already shown by Vergou et al. [16], the protection efficacy of broad-band UV filters can be increased if the absorbed energy is released by fluorescence and not by heat. The europium complex possesses these important properties [16]. However, the fluorescence of the europium complex itself might be responsible for the increased total transmission measured when compared to titanium dioxide. Since the fluorescent light adds to the total transmission and is not harmful, the transmission of harmful UV irradiation might be even smaller than the total transmission value measured.

The uptake of the europium complex by an experimental cell culture and its adsorption to the cell membrane was investigated using a laser scanning microscope. These experiments allowed the localization of the europium complex on the cell membrane and within the membranous organelles surrounding the cell nucleus. This intracellular binding affinity of the europium complex to inner cell membranes could potentially explain the increased photoprotective effect of the europium complex as observed in the phototoxicity assays [16]. At this point the uptake mechanism of the europium complex by the cells has not been investigated, yet. It remains to be elucidated whether the europium complex enters the cells by endocytosis as it has been shown for titanium dioxide which is taken up by macrophages [25]. Moreover, it is to be investigated whether or not the presence of the UV filter on the cell membrane plays an additional photoprotective role. As cytotoxicity experiments in the dark have shown, neither the europium complex nor the titanium dioxide nanoparticles had significant toxic effects on Jurkat cells when incubated with both substances for a period of up to 3 h at UV-protective doses. Based on this knowledge, the UV exposure in the phototoxicity experiments can be mainly held accountable for the cytotoxic effects. Cells that have been incubated with titanium dioxide followed by UVB irradiation show similar viability rates as the control cells, independent of the irradiation doses applied. Therefore, at least in the cell suspension, titanium dioxide does neither provide protection against the harmful effects nor does it enhance the damaging effects of UVB irradiation. However, UVA irradiation leads to a significantly decreased viability percentage of the cells incubated with titanium dioxide. This effect indicates a phototoxic reaction, most probably caused by the photocatalytical formation of hydroxide radicals as it already has been suggested for titanium dioxide [26]. In contrast, adding the europium complex to the cell suspension leads to a significantly increased cell viability percentage with respect to control cells, which can be partly explained by the absorption efficiency of the complex, as well as the transformation of UV irradiation into red and infrared light. The europium complex seems to have photoprotective properties both in the UVA and in the UVB spectrum, since the viability percentages of the cells are significantly higher than that of untreated control cells after both UVA and UVB treatment. It remains unclear to what extent the binding of the europium complex on the cell membrane surface, as shown by laser scanning microscopy, might account for the decreased phototoxicity of the substance.

Sufficient penetration of the UV filter into the corneal layers of the epidermis is required in order to prolong the time of protection. The penetration characteristics of the europium complex were compared with the penetration profile of titanium dioxide using the tape-stripping procedure [27]. Both substances were shown to penetrate the stratum corneum, without accessing the living cells of the deeper epidermis. Former experiments have shown that titanium dioxide does indeed penetrate into the hair follicle canals but cannot be detected in the dermis [22]. Therefore, titanium dioxide cannot penetrate into living tissue unless the skin is damaged. The penetration profile of the europium complex has been shown to be comparable to the penetration profile of titanium dioxide as studied on porcine skin in vitro. Since the europium complex is not allowed to be studied on human skin, the penetration profile has only been established for titanium dioxide particles in human skin. Compared to human skin, significantly more titanium dioxide (56%) was found on the skin surface of pigs, most probably due to the skin structure of porcine ear skin, which is characterized by very broad furrows and wide follicular orifices. Nevertheless, porcine skin shows most of the anatomical, physiological and biochemical similarities with human skin compared to the skin of other species [28,29]. The small amount of titanium dioxide found in the deeper epidermal layers might be explained by the hair follicles serving as a reservoir for the UV filter [22].

In summary, the europium complex shows significant UV-protective properties by scattering, reflection, absorption and transformation of the absorbed radiation into fluorescence light. These experiments have proven that in particular with high UV doses, the viability of the cells that were incubated with europium was significantly higher compared to cells incubated with titanium dioxide. The europium complex shows a similar penetration profile into the stratum corneum of the epidermis as required for efficient use as a sunscreen. Regardless of the fact that europium is used for different applications in medicine (e.g. contrast media), the europium complex should be tested in sunscreen formulations in clinical studies to examine its efficiency and safety.

 

 Acknowledgements

We would like to thank Dr. Robert Khramov from the Institute of Theoretical and Experimental Biophysics of the Russian Academy in Poshino, Russia, and Polysvetan Inc., Russia, for kindly providing the europium complex and for the helpful discussions.


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  21. Weigmann HJ, Jacobi U, Antoniou C, Tsikrikas GN, Wendel V, Rapp C, Gers-Barlag H, Sterry W, Lademann J: Determination of penetration profiles of topically applied substances by means of tape stripping and optical spectroscopy: UV filter substance in sunscreens. J Biomed Opt 2005;10:14009.
  22. Lademann J, Weigmann H, Rickmeyer C, Barthelmes H, Schaefer H, Mueller G, Sterry W: Penetration of titanium dioxide microparticles in a sunscreen formulation into the horny layer and the follicular orifice. Skin Pharmacol Appl Skin Physiol 1999;12:247–256.
  23. Lademann J, Jacobi U, Surber C, Weigmann HJ, Fluhr JW: The tape stripping procedure – evaluation of some critical parameters. Eur J Pharm Biopharm 2009;72:317–323.
  24. Teichmann A, Jacobi U, Ossadnik M, Richter H, Koch S, Sterry W, Lademann J: Differential stripping: determination of the amount of topically applied substances penetrated into the hair follicles. J Invest Dermatol 2005;125:264–269.
  25. Scherbart AM, Langer J, Bushmelev A, van Berlo D, Haberzettl P, van Schooten FJ, Schmidt AM, Rose CR, Schins RP, Albrecht C: Contrasting macrophage activation by fine and ultrafine titanium dioxide particles is associated with different uptake mechanisms. Particle Fibre Toxicol 2011;8:31.
  26. Sanders K, Degn LL, Mundy WR, Zucker R, Dreher K, Zhao B, Roberts JE, Boyes WK: In vitro phototoxicity and hazard identification of nano-scale titanium dioxide. Toxicol Appl Pharmacol 2012;258:226–236.
  27. Weigmann H, Lademann J, Meffert H, Schaefer H, Sterry W: Determination of the horny layer profile by tape stripping in combination with optical spectroscopy in the visible range as a prerequisite to quantify percutaneous absorption. Skin Pharmacol Appl Skin Physiol 1999;12:34–45.
  28. Simon GA, Maibach HI: The pig as an experimental animal model of percutaneous permeation in man: qualitative and quantitative observations – an overview. Skin Pharmacol Appl Skin Physiol 2000;13:229–234.
  29. Lademann J, Richter H, Meinke M, Sterry W, Patzelt A: Which skin model is the most appropriate for the investigation of topically applied substances into the hair follicles? Skin Pharmacol Physiol 2010;23:47–52.

  

Author Contacts

PD Dr. B. Lange-Asschenfeldt
Skin Cancer Center Charité, Department of Dermatology, Venereology and Allergology
Charité, Universitätsmedizin Berlin
Charitéplatz 1, DE–10117 Berlin (Germany)
E-Mail bernhard.lange-asschenfeldt@charite.de

  

Article Information

Received: April 26, 2012
Accepted after revision: November 19, 2012
Published online: January 10, 2013
Number of Print Pages : 9
Number of Figures : 6, Number of Tables : 0, Number of References : 29

  

Publication Details

Skin Pharmacology and Physiology (Journal of Pharmacological and Biophysical Research)

Vol. 26, No. 2, Year 2013 (Cover Date: April 2013)

Journal Editor: Lademann J. (Berlin)
ISSN: 1660-5527 (Print), eISSN: 1660-5535 (Online)

For additional information: http://www.karger.com/SPP


Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Abstract

In this study, we compared the UV-protective abilities of the europium complex compared to titanium dioxide, which represents the most common physical filter for ultraviolet light in the broad-band spectral range. The UV absorption and light transformative capacities of the europium complex were evaluated using a spectrometer with a double-integrating sphere showing that the europium complex does not only absorb and reflect UV light, but transforms it into red and infrared light. It was found that the europium complex binds to the surface of Jurkat cells in vitro. Cells incubated with the europium complex showed a significantly higher viability after UVA and UVB irradiation as compared to untreated cells and cells incubated with titanium dioxide pointing out its photoprotective properties. The europium complex and titanium dioxide show similar penetration capacities into the stratum corneum as tested in human and porcine skin using tape stripping analysis. The europium complex has proved to be an efficient UV filter with a low cyto- and phototoxic profile and therefore represents a potential candidate for use in sunscreen formulations.

© 2013 S. Karger AG, Basel


  

Author Contacts

PD Dr. B. Lange-Asschenfeldt
Skin Cancer Center Charité, Department of Dermatology, Venereology and Allergology
Charité, Universitätsmedizin Berlin
Charitéplatz 1, DE–10117 Berlin (Germany)
E-Mail bernhard.lange-asschenfeldt@charite.de

  

Article Information

Received: April 26, 2012
Accepted after revision: November 19, 2012
Published online: January 10, 2013
Number of Print Pages : 9
Number of Figures : 6, Number of Tables : 0, Number of References : 29

  

Publication Details

Skin Pharmacology and Physiology (Journal of Pharmacological and Biophysical Research)

Vol. 26, No. 2, Year 2013 (Cover Date: April 2013)

Journal Editor: Lademann J. (Berlin)
ISSN: 1660-5527 (Print), eISSN: 1660-5535 (Online)

For additional information: http://www.karger.com/SPP


Article / Publication Details

First-Page Preview
Abstract of Original Paper

Received: 4/26/2012 11:24:54 AM
Accepted: 11/19/2012
Published online: 1/10/2013
Issue release date: April 2013

Number of Print Pages: 9
Number of Figures: 6
Number of Tables: 0

ISSN: 1660-5527 (Print)
eISSN: 1660-5535 (Online)

For additional information: http://www.karger.com/SPP


Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

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    External Resources

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  19. Bottrill M, Kwok L, Long NJ: Lanthanides in magnetic resonance imaging. Chem Soc Rev 2006;35:557–571.
  20. Lange-Asschenfeldt B, Marenbach D, Lang C, Patzelt A, Ulrich M, Maltusch A, Terhorst D, Stockfleth E, Sterry W, Lademann J: Distribution of bacteria in the epidermal layers and hair follicles of the human skin. Skin Pharmacol Physiol 2011;24:305–311.
  21. Weigmann HJ, Jacobi U, Antoniou C, Tsikrikas GN, Wendel V, Rapp C, Gers-Barlag H, Sterry W, Lademann J: Determination of penetration profiles of topically applied substances by means of tape stripping and optical spectroscopy: UV filter substance in sunscreens. J Biomed Opt 2005;10:14009.
  22. Lademann J, Weigmann H, Rickmeyer C, Barthelmes H, Schaefer H, Mueller G, Sterry W: Penetration of titanium dioxide microparticles in a sunscreen formulation into the horny layer and the follicular orifice. Skin Pharmacol Appl Skin Physiol 1999;12:247–256.
  23. Lademann J, Jacobi U, Surber C, Weigmann HJ, Fluhr JW: The tape stripping procedure – evaluation of some critical parameters. Eur J Pharm Biopharm 2009;72:317–323.
  24. Teichmann A, Jacobi U, Ossadnik M, Richter H, Koch S, Sterry W, Lademann J: Differential stripping: determination of the amount of topically applied substances penetrated into the hair follicles. J Invest Dermatol 2005;125:264–269.
  25. Scherbart AM, Langer J, Bushmelev A, van Berlo D, Haberzettl P, van Schooten FJ, Schmidt AM, Rose CR, Schins RP, Albrecht C: Contrasting macrophage activation by fine and ultrafine titanium dioxide particles is associated with different uptake mechanisms. Particle Fibre Toxicol 2011;8:31.
  26. Sanders K, Degn LL, Mundy WR, Zucker R, Dreher K, Zhao B, Roberts JE, Boyes WK: In vitro phototoxicity and hazard identification of nano-scale titanium dioxide. Toxicol Appl Pharmacol 2012;258:226–236.
  27. Weigmann H, Lademann J, Meffert H, Schaefer H, Sterry W: Determination of the horny layer profile by tape stripping in combination with optical spectroscopy in the visible range as a prerequisite to quantify percutaneous absorption. Skin Pharmacol Appl Skin Physiol 1999;12:34–45.
  28. Simon GA, Maibach HI: The pig as an experimental animal model of percutaneous permeation in man: qualitative and quantitative observations – an overview. Skin Pharmacol Appl Skin Physiol 2000;13:229–234.
  29. Lademann J, Richter H, Meinke M, Sterry W, Patzelt A: Which skin model is the most appropriate for the investigation of topically applied substances into the hair follicles? Skin Pharmacol Physiol 2010;23:47–52.