Free radicals are molecular species with an unpaired electron on the external orbital and of high chemical reactivity. All radicals existing in a human organism can be divided into two kinds, natural and alien. The former are those which are inherently produced in the organism during chemical reactions: radical oxygen species (ROS) such as superoxide , singlet oxygen , and hydroxyl radical (OH), as well as nitric oxide (NO), etc. They play an important role as regulatory mediators in signalling processes such as regulation of vascular tone, monitoring of oxygen tension in the control of ventilation and erythropoietin production and signal transduction from membrane receptors in various physiological processes.1, 2 Under normal conditions, the amount of free radicals is balanced by enzymes and antioxidants. An excessive increase in ROS production is involved, for example, in the pathogenesis of cancer, diabetes mellitus, atherosclerosis, neurodegenerative diseases, rheumatoid arthritis, and ischemia/reperfusion injury.1 The alien free radicals appear as a consequence of the effect of ionizing radiation, UV light, xenobiotics, etc. on human tissue and are harmful.
Studying of free radicals can be carried out by direct and indirect methods. The direct methods implement the effects of either electron paramagnetic resonance (EPR) or chemoluminescence. The indirect methods include investigations of the end products of the reactions with free radicals involved or application of inhibitors. The EPR (also called electron spin resonance) technique is based on the absorption of microwave radiation by an unpaired electron of a molecule located in a magnetic field.
Direct detection of free short-living radicals by the EPR method is possible only at a quite low temperature ( , liquid nitrogen) due to their sufficient steady-state concentrations only under such conditions.3 In order to achieve suitable concentrations at room temperature, spin traps and spin markers are used (e.g., PCA, DPPH, Tempol, TEMPO, DMPO, 4-POBN). Spin traps are molecules which bind to short-lived free radicals and form detectable stable forms of radicals (spin adducts). Spin markers are stabilized radicals themselves contributing to the EPR signal; however, being in contact with short-lived free radicals, they loose or add an electron and become undetectable. The decrease in the EPR signal in this case quantifies the free radicals under investigation. The EPR methodology is a useful tool for the noninvasive in vivo measurements of skin barrier function, drug/skin interaction and cutaneous oxygen tension.3
Skin is a tissue protecting deeper-located organs from various hazards of the environment, such as chemical, biological, and physical hazards, in particular, from UV light. Exceeding doses of UV radiation can cause direct or indirect (via formation of free radicals) DNA damage, leading to carcinogenesis.4 Skin is a suitable object for EPR investigations due to its surface location and relatively small thickness. Using the microwave radiation of , which penetrates as deep as into skin, it is possible to monitor penetration of spin traps and spin markers inside skin5, 6 and in-depth appearance of generated radicals by means of EPR imaging.7 According to investigations, UV-induced radicals include ROS and lipid radicals8, 9, 10 as well as melanin radicals.11 Formation of free radicals under UV irradiation and affect of various substances on this process in lipid-model systems12, 13 of different complexity as in vitro counterparts of the intercellular lipid matrix of the stratum corneum was investigated by Trommer, in particular, using the EPR technique.12 As shown by experiments with human skin in vivo,14 UVA part of the UV spectrum, is mainly responsible for the generation of free radicals (80–90% of the total amount) because of higher penetration depth, in contrast to UVB light, which contributes to radical generation only in the epidermis (up to a depth of ).
Titanium dioxide nanoparticles are extensively used today in cosmetics, paints, air and water waste purification.15 Among three existing crystal modifications of (anatase, rutile, and brookite), anatase is the most photoactive, if irradiated by UV light, according to some authors.16 However, its photoactivity strongly depends on particle size, dopants,17 and coating and correlates with the reflectance spectra.18 Coatings are introduced to suppress photocatalytic properties of the particles.19 It is reported that both anatase and rutile particles used in sunscreens oxidize DNA and RNA in vitro and in human cell culture20, 21, 22 under UV irradiation. However, it is important to know whether the amount of radicals generated by the particles on skin exceeds that produced by the skin itself.
In this paper, we experimentally investigate the ability of titanium dioxide particles (anatase) of two average sizes (25 and in diam) to produce free radicals under UV irradiation when applied to two different surfaces, glass and porcine skin in vitro.
Materials and Methods
Two types of commercially available samples were used in the experiments. A placebo (sunscreen o/w emulsion without any filters, Creme Sante soleil SPF 8 F148-006, RoC S.A., France) with coated titanium dioxide nanoparticles embedded was applied either onto glass slides or onto porcine skin in vitro. The surface density of the substance was , corresponding to the recommendations of COLIPA23 for sunscreen applications. The placebo was weighted on precise-balance BP 211D (Sartorius, Göttingen, Germany) with an accuracy of using a syringe. One type of spin marker, 3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCA, Sigma-Aldrich Chemie GmbH, Munich, Germany), was used to detect short-lived free radicals emerging under UV irradiation. PCA was dissolved from powder in a water–ethanol (1:1) solution at a concentration of . It was chosen from other available spin markers due to satisfactory stability for our applications: after -long UV irradiation, the EPR signal decrease was , and after -long irradiation it was only 6%.24 Additionally, in porcine skin there is a low amount of antioxidants, thus prolonging the existence on PCA. As a source of UV radiation , a device TH-1E (Cosmedico Medizintechnik GmbH, Schwenningen, Germany) with the spectral maximum located at about was used. The radiation intensity was , measured by the powermeter HBM-1 (Hydrosun Medizintechnik GmbH, Mullheim, Germany), which corresponds to the solar UV intensity . The EPR system LBM MT 03 (Magnettech GmbH, Berlin, Germany) operating in L-band was used for detecting the EPR signal. The generated short-lived radicals react with the nitrogen oxide PCA, which is thereby reduced to the corresponding hydroxylamine. PCA looses its free electron and the signal intensity in the EPR signal decreases.
Experiments with Nanoparticles Applied onto Glass Slides
Glass slides had the following dimensions: width , length , thickness . There were three types of prepared slide samples (five couples of samples of each type): (i) placebo with small particles, (ii) placebo with large particles, and (iii) placebo only. Slides were covered homogeneously with a mixture of the corresponding substances and PCA . In each couple, one sample was irradiated with the UV lamp ( particles for , particles and placebo only for ); the other was used for control (was not irradiated). The samples were put into the EPR system and measured before and after the irradiation. The signals were displayed in real time on the computer screen in the software environment supplied with the system. They were stored as files and then analyzed.
Experiments with Nanoparticles Applied onto Porcine Skin
Native ears of domestic pigs were delivered from a nearby farm (Gut Hesterberg, Neurupin, Germany) on the day following the slaughter. Before the experiments, the ears were washed with cold running water and gently wiped with paper napkins. All hairs on surfaces of the ears were completely removed with medical scissors with curved edges to avoid injuring the skin. Then, tesa adhesive tape (Beiersdorf AG, Hamburg, Germany) was used for tape stripping according to Weigmann 25 Twenty tape strips were taken from the same area to remove the stratum corneum (horny layer, superficial layer of skin) in part. Corresponding to Ref. 26, such an amount of strips means that about 50% of stratum corneum was removed. A roller was used to press the tape to the skin. This procedure was a prerequisite to ease penetration of PCA into the skin.6 PCA served as a marker for revealing the production of free radicals. In the investigation of free radicals with the EPR technique, the deeper the PCA penetrated, the higher the amount of skin volume was involved. The surface of each porcine ear area, in size, was marked with a permanent marker. The areas were divided into couples of small areas of or . One area in the couple was used immediately after the preparation procedure was completed; the other area was used after the application of substances. The borders of all areas were covered by a color glue to build barriers between neighbouring areas. As in experiments with application of particles onto glass slides, three different substances were investigated: placebo with particles, placebo with particles, and placebo without particles. PCA was added to all substances ( per each small area). The substances were topically applied onto the skin with a surface density of . During the application of the corresponding (according to the area) amount of the substances, the skin was massaged with a massage device (Petra PC 60, petra-electric GmbH Co.KG) covered with a latex glove finger saturated in advance with the substance. From each small area, two punch biopsies were taken with a round-edged sharp cutter; one was later irradiated with the UV lamp (stratum corneum side), while the other was used for control. The diameter of the skin samples was ; the thickness was (all skin layers from the surface to the cartilage in the middle of the ear). The biopsies were removed from the ear using a scalpel and pincers and were fixed onto a glass slide with acryl glue. Afterward, certain samples were irradiated by UV light for . The irradiated samples were measured in the EPR system before, immediately after and after the irradiation. Those serving as controls were measured at 0, 3, 7, 10, and (zero time corresponds to the start-time point of sample irradiation). Four to six samples were used for each measurement to collect statistical data.
Transmission Electron Microscopy (TEM)
TEM photos of particles were obtained using a transmission electron microscope Philips Morgagni 280 D (Eindhoven, the Netherlands).
The Raman spectroscopy system (LMTB, Berlin, Germany)27 was based on a CW laser operating at the wavelength of (power ). Its radiation was focused into an optical fiber connected to an optical imaging system, where the light was filtered and focused onto samples. The Raman signal reflected from samples was collected by a lens system and transferred into another fiber bundle connected to a spectrograph. The spectrum was recorded by a charge-coupled device camera and transferred to a PC for processing. Because of flexibility of light delivering and collecting fibers, samples ( particles as powder and imbedded in placebo) in cuvettes or applied onto glass slides were just put in contact with them. One measurement took a few seconds.
For Mie calculations, MieTab 7.23 software28 was used. As input parameters, the radiation wavelength, refractive indices of the particles and the surrounding medium, as well as particle sizes were required.
Results and Discussion
TEM photographs of the particles in diluted placebo are represented in Fig. 1 . Magnification for the images of particles was 89, while for particles it was equal to 22. It is seen that the particles of both sizes form multipartilce structures. This process affects optical properties of the solutions.
In order to reveal the crystal form of the used titanium dioxide particles, we measured Raman spectra of the powder of particles and of the placebo with embedded particles. The obtained spectra are presented in Fig. 2a . The signal produced by the placebo was much smaller than that of the powder due to a smaller concentration of particles (not shown). It is clearly seen that three peaks are present on the graph. This indicates, according to Ref. 29, that the crystal form of the particles under investigation is anatase.
The Mie theory was used to describe the interaction between the particles and UV radiation. The spectrum of the used UV light source is depicted in Fig. 3 . For the calculations, two wavelengths were chosen: 310 and . The first was chosen because it corresponds both to the maximum of the source spectrum and to the maximum of the product of the solar spectral irradiance over wavelength with the erythemal action spectrum.30 The second wavelength was taken for comparison; the anatase particles do not absorb light with a wavelength longer than .31
Titanium dioxide is a birefringent crystal, with different refractive indices for light polarized perpendicular or parallel to the optic axis. In the “average index” approximation, the particles are supposed to be isotropic, with real and imaginary parts of the refractive index equal to and , where and ( and ) are the ordinary (extraordinary) real and imaginary parts of the refractive index, respectively.32 For the 310- and UV radiation, these constants taken from Ref. 31, result in: 0.83 (for ) and 0.25 (for ). The refractive index of the surrounding medium (placebo) was 1.4, and the diameters of the particles were considered to be with steps. The result of the calculation is shown in Fig. 2b. Efficiency factor is the ratio of the absorption and the geometrical cross sections of a particle. The value , where is a particle diameter, is proportional to the absorption coefficient of a particle suspension30, 33, 34, 35 and therefore takes into account the presence of other particles of the same type in the sample.
Figure 4 illustrates typical signals obtained with the EPR system for the two investigated types of samples with particles: located on glass and on porcine skin. The signals are normalized to the highest amplitude of the signal from the sample before irradiation. It is seen that after -long exposure to UV light the signal decreases. This means that short-lived free radicals appear.
Figure 5 shows the mean values with standard deviations of the results obtained from five samples on glass with or without particles. Standard deviations vary between 0.02 and 0.24 for nonirradiated samples and between 0.10 and 0.26 for the irradiated samples. Dependencies of the EPR signal on time for the irradiated and nonirradiated samples are depicted. Statistically, there is no effect of UV radiation in the presence of large particles and placebo without particles. However, the effect is distinctly seen in the case of small particles. This phenomenon can be explained in frames of the Mie theory. Considering Fig. 2b representing the absorption efficiency curves for 310- and radiation, we can conclude that particles absorb UV light much more efficiently than the particles for or at the same level for at the same volume concentrations. As shown in Fig. 1, the particles form aggregates and agglomerates although ultrasonic stirring was used during the process of embedding particles into placebo. Formation of the above-mentioned structures causes an increase in the average size of particles, leading to the increased absorption efficacy of the particles and decreasing that of the particles. The larger absorption means more active production of free short-lived radicals. The curve corresponding to the nonirradiated large particles looks very similar to that of placebo and different from that of small particles.
The other series of experiments was carried out with porcine ear skin. One group of skin samples was irradiated immediately after preparation and the other with -long delay. The results of the measurements of the first group with standard deviations are presented in Fig. 6 . Standard deviations vary between 0.04 and 0.25 for nonirradiated samples and between 0.08 and 0.30 for the irradiated samples. Taking into account the statistical errors, the effect of UV irradiation is clearly seen in all cases (the points corresponding to such samples are located lower than those of nonirradiated samples), and the magnitude of the effect is almost the same for the samples with the particles, with placebo only, and without placebo and particles. It means that the amounts of short-lived free radicals appearing under UV irradiation are comparable and do not depend on the presence of the particles on the skin surface. In other words, the contribution of skin to free-radical generation under UV irradiation exceeds that of the particles.
The effect of penetration time (0 versus ) of the substances before UV irradiation is depicted in Fig. 7 . There is no significant difference, but in the samples corresponding to large particles [Fig. 7b] as well as to the placebo [Fig. 7c] and to skin without placebo and particles [Fig. 7d], the lines connecting the average values relative to measurements without any delay are located above the lines corresponding to the delayed experiments. However, the situation is reverse for the samples with small particles [Fig. 7a].
As we proved experimentally by means of EPR spectroscopy, small ( in diam) nanoparticles of titanium dioxide (anatase form) are more photoactive than large ( in diam) particles. This effect is clearly seen if the particles embedded into the placebo are applied on glass and is in agreement with the Mie theory. However, if the particles are applied onto the porcine skin in vitro, no distinct difference is observed. This is caused by high skin contribution to the generation of radicals. In comparison to the skin’s ability to produce radicals, the nanoparticles do not play a significant role in the concentrations used .
The authors thank Dr. Maxim Darvin for his assistance during the experiments and Dr. Elena Zagainova from Nizhny Nov gorod Medical Academy (Russia) for the TEM photos of the particles. A.P.P. thanks Infotech Oulu and the Tauno Tönning Foundation (both Finland) and DAAD (Germany) for support of this study. This work was partially supported by the RFBR Grant No. 07-02-01000.