Penetration of titanium dioxide nanoparticles through slightly damaged skin in vitro and in vivo
Abstract
Purpose
Titanium dioxide nanoparticles (TiO2-NPs) have been widely developed for versatile use, but the potential risk form their skin exposure is still unclear. To evaluate this risk, the skin penetration of TiO2-NPs is necessary to be understood first. The aims of this study are to investigated the penetration of TiO2-NPs through slightly damaged skin and intact skin in vitro and in vivo.
Methods
TiO2-NPs with a diameter of 20 nm was labeled with 125I.The skin of rat was treated with 2% SLS solution and obtained as slightly damaged skin. The 125I labeled TiO2-NPs (125I-TiO2-NPs)solution and 0.9% PS solution were added into the donor chamber and receptor chamber of static diffusion cells which clamped the skin at the middle of two half-cells, respectively. During 24 hours, samples were extracted from the receptor chamber and counted for 1 min using γ-counter to detect the radioactivity. The skin penetration of TiO2-NPs in vitro was expressed as the percentage of radioactivity of receptor chamber solution compared with total radioactivity in the donor chamber. Thereafter, the 125I-TiO2-NPs was exposed to the rats. After 1 day and 3 days, the blood and tissues of rats were harvested, weighed and counted for 1 min using γ-counter to detect the tissue radioactivity. The skin penetration of TiO2-NPs in vivo was expressed as the percentage dose per gram tissue (% dose/g).
Results
In the skin penetration experiment in vitro, the radioactivity of receptor chamber solution through damaged skin was higher than that of through intact skin and was about 2% radioactivity of donor chamber on 24 h. In the skin penetration experiment in vivo, the radioactivity of blood and tissues of rats after exposing to 125I-TiO2-NPs solution though damaged skin or intact skin were less than 0.05% dose/g on 1 d and quickly declined on 3 d. The skin penetration rates of TiO2-NPs through slightly damaged skin and intact skin in vitro and vivo were lower than the rate of free 125I in the TiO2-NPs solution.
Conclusions
The TiO2-NPs could not penetrate through the damaged skin or intact skin both in vitro and in vivo. It suggested that the TiO2-NPs should be safe when it was applied and contacted with skin.
J Appl Biomater Funct Mater 2015; 13(4): e356 - e361
Article Type: ORIGINAL RESEARCH ARTICLE
DOI:10.5301/jabfm.5000243
Authors
Guangping Xie, Weixin Lu, Dongmin Lu
Article History
• Accepted on 16/07/2015
• Available online on 27/11/2015
• Published online on 18/12/2015
Disclosures
Financial support: This work was supported by grants from Natural Science Foundation of China (no. 31300795), Zhejiang Provincial Natural Science Foundation of China (no. Y4110665) and Zhejiang Province Public welfare Technology Applied Research Project (no. 2012C33115).
Conflict of interest: The authors declare that there are no conflicts of interest.
Titanium dioxide nanoparticles (TiO2-NPs) is a fine white powder, often used as a pigment or additive for paints, paper, ceramics, plastics, foods and other products (1, 2). The well absorption and reflection with ultraviolet radiation (UVR) also promote TiO2-NPs widely used in the variety of cosmetics and sunscreens (3). Most recently, TiO2-NPs is being tested in environmental protection because the TiO2-NPs activated by sunlight can convert ambient nitrogen dioxide gas into less toxic nitrates (4). These wide applications mean that TiO2-NPs come into close and frequent contacted with us.
However, the versatile use of TiO2-NPs could cause new side effects. As the biosafety of nanotechnology becomes a growing concern, attention has been paid to the toxicity of nanoparticles (5, 6). The majority of toxicologists believe that size effects of nanoparticles may cause higher toxicity because of their larger surface area, enhanced chemical reactivity, and easier cell penetration (7, 8). Moreover, as pointed out by Dunford et al (9), once it absorbs UV light, TiO2, as a well-known photo-catalyst, also catalyzes the generation of reaction oxygen species (ROS) (10). This photo-catalyst property can make TiO2-NPs a potentially hazardous material since ROS products in turn are known to cause genetic damage and other adverse effects in living tissues (11). Most toxicological researches have expressed the in vivo toxicity of nanoparticles coming from their inhalation, intravenous injection, intranasal instilment, oral administration (4, 12-13-14). But, with increasing skin application and contact of TiO2-NPs, the potential risk from their skin exposure is still unclear. To evaluate this risk, the skin penetration of TiO2-NPs is necessary to be understood first. We need to know whether the TiO2-NPs contacting with skin should penetrate into or through the skin.
Mammalian skin is structured in several layers, the stratum corneum (SC), epidermis, dermis and the subcutaneous layer. For most substances, the SC is the rate-limiting barrier against absorption/percutaneous penetration of topically applied substances (15). The intercellular space between the cells composing SC measures approximately 100 nm (16). In theory, only small (<600 Da) and lipophilic molecules can easily penetrate the skin passively (17), but nanoparticles penetration into intact skin is impossible (18). Some studies have also indicated that TiO2 and other inorganic particles, even on a nano-grade scale, do not penetrate intact skin in vitro (15, 19). However, the SC of skin should be slightly damaged by topical application of various products sometimes and its intercellular space should be widened (20). In this pathological condition, TiO2-NPs may penetrate the skin. Moreover, the living skin in vivo has many viable conditions and these conditions would help TiO2-NPs penetrate into or through the damaged skin. Currently, it is unknown whether TiO2-NPs could penetrate into or through slightly damaged skin caused by applied products in vitro and in vivo.
Herein, this study investigated the penetration of TiO2-NPs through the damaged skin in vitro and in vivo. We used sodium lauryl sulphate (SLS) to cause slight skin damage according to previous reports. In order to evaluate the penetration exactly, the used TiO2-NPs with a diameter of approximately 20 nm were modified with aminopropyltriethoxysilane (APTS) to introduce amino groups on the surface and labeled with 125I. A γ-counter was used to assess quantitatively the 125I labeled TiO2-NPs, which penetrated through the skin.
Materials and methods
Modification and characterization of TiO2-NPs
The rutile-type TiO2-NPs were obtained from Nano-Science&Technology Research Center of Shanghai University (China) and modified with APTS as necessary. Briefly, APTS in EtOH [2% (v%)] was adjusted to pH 3.5 using oxalic acid and stirred for 1 h at room temperature. Thereafter, TiO2-NPs were added at a concentration of 10% and incubated with stirring for 6 h at 60°C. After vacuum drying, washing with alcohol and vacuum drying again, the modified TiO2-NPs were collected. After modification, the physicochemical properties of TiO2-NPs were characterized. The TiO2-NPs structure was confirmed by TEM (JEM-2010, JEOL Ltd, Japan). The size distribution was analyzed by laser scattering (ELS-Z, Otsuka Electronics, Japan). X-ray diffraction (Bruker-AXS, Bruker Co, Germany) was used to analyze the crystallinity of TiO2-NPs.
Radiolabeling of TiO2-NPs with 125I
A test tube was coated with 25 µl Bolton-Hunter reagent (10 mg/mL in benzene) and was subsequently dried with a gentle stream of nitrogen for 30-60 min. Dimethylformamide (DMF, 2 µl) and Na125I (60 MBq, 1.5 µl) were incubated at room temperature with 1.5 µl chloramine (4 mg/mL) in the coated test tube. The modified TiO2-NPs [100 mg in 50 µl 0.05 M borate buffer (pH 8.4)] were added, stirred, and incubated on ice for 30 min. The reaction was terminated by addition of 200 µl borate buffer supplemented with 0.2 M glycine. After the separation of 125I labeled TiO2-NPs (125I -TiO2-NPs) by column chromatography, 2 μl of the resulting solution was extracted and subjected to paper chromatography using instant thin layer chromatography–silica gel (ITLC-SG), with 2.5% BSA (w/w) in 0.01 M PBS (pH 7.4) as the solvent, and a γ-counter (Shanghai Institute of Nuclear Instrument Factory, China) to identify the radioactive substance. Before the skin experiments, the stability of 125I -TiO2-NPs was analyzed in vitro. The 125I-TiO2-NPs were dissolved in physiological saline (PS) containing 10% (v%) mouse serum and incubated at 37°C. Each day during 7 days, 2 µl of 125I-TiO2-NPs suspension were extracted and analyzed by paper chromatography using ITLC-SG with 2.5% BSA (w/w) in 0.01 M PBS. The purity of 125I-TiO2-NPs was expressed as the percentage of radioactivity at the site of 125I-TiO2-NPs compared with the total radioactivity on the paper and its change was expressed as the stability of 125I-TiO2-NPs. After vacuum drying, 125I -TiO2-NPs were collected.
Animals and pretreatments
Male Wistar rats, 10 weeks of age and weighing approximately 250 g, were purchased from SLACCAS Laboratory Animal Co, Ltd (Shanghai, China), and allowed access to food and water ad libitum with a 12-hour light/12-hour dark cycle. The hair was clipped with a No. 40 surgical blade on the back. A piece of rubber with 1 mm thickness and a 2 cm × 4 cm hole was bonded to the back skin of the rats using medical glue. The margin of rubber was watertight seal with the skin. The 2% SLS solution or 0.9% PS solution as a control was evenly dropped onto the skin surface exposed in the hole of rubber with the ratio of 100 μl/cm2. In order to limit the SLS solution on the skin surface, a piece of polyethylene film was bonded and covered on the rubber. After 4 hours, the polyethylene film was moved and the SLS or PS solution was extracted out. The skin exposed in the hole of rubber was washed with PS solution and dried. All the animal experiments were approved by the Animal Ethical Committee of Shanghai Jiaotong University (China).
Histopathological analysis of pretreated skin
After pretreatments, the animals were sacrificed and the back skin exposed in the hole of rubber was excised with full-thickness. The obtained skin was fixed in 10% buffered formalin-saline at 4°C overnight and then embedded in paraffin blocks. Tissue sections with 7 μm thickness were prepared and stained with hematoxylin and eosin (H&E). The tissue morphology was observed under a microscope at 400 × magnification.
Skin penetration experiment in vitro
After pretreatments, the treated skin was excised with 1 mm thickness from the animals and biopsied using a 20 mm steel circular punch that provided a dosing area of 0.84 cm2. The in vitro static diffusion cells described in previous experimental model were used (21). The skin sample was placed at the middle of two half-cells and held in place by a clamp, which at the same time kept the half-cells together. The 125I-TiO2-NPs solution (1 mg/mL) and 0.9% PS solution were added into the donor chamber and the receptor chamber with the same volume, respectively. The cells were kept at a constant temperature (32°C) in a water bath with magnetic stirring. This temperature assured a temperature on the skin surface close to 32°C. At seven time points (0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h), samples (200 μl) were extracted from the receptor chamber and counted for 1 min in a γ-counter to detect the radioactivity. After detection, the samples were returned to the receptor chamber to keep the constant volume. The skin penetration was expressed as the percentage of radioactivity of receptor chamber solution compared with total radioactivity in the donor chamber.
Skin penetration experiment in vivo
After pretreatments, the 125I-TiO2-NPs solution (1 mg/mL) was dropped onto the animal back skin surface exposed in the hole of rubber with the ratio of 100 μl/cm2 and a piece of polyethylene film was bonded and covered on the rubber. After 1 day and 3 days, five animals were sacrificed. Their blood and tissues, including subcutaneous fat, lung, heart, liver, spleen, kidney, stomach and intestine, were harvested, weighed, and counted for 1 min in a γ-counter to detect the tissue radioactivity. The skin penetration was expressed as the percentage dose per gram tissue (% dose/g).
Statistical analysis
The radioactivity of the materials was determined from following equation: C = C0 * e-(0.693159t/T), where C was the actual radioactivity, C0 was the measured radioactivity, T was the half-life of 125I (59.6 days), and t was the time interval between C and C0. Compiled data were presented as mean ± standard deviation. Where feasible, the data were analyzed for statistical significance by the student’s t-test. For all tests, significance was set at the 95% confidence level.
Results
Physicochemical properties of TiO2-NPs
The physicochemical properties of modified TiO2-NPs were expressed in Figure 1. It can be observed that TiO2-NPs were rod-shape with the size about 20 nm and exhibited a XRD pattern of rutile-type.
TEM image (A) size distribution (B) and XRD pattern (C) of TiO2-NPs modified with APTS.
Identification and stability of 125I-TiO2-NPs
Figure 2 shows the identification and stability of radioactive materials after the radiolabeling. It can be observed that the 125I moved toward the solvent front (Rf = 0.9) faster than 125I-BH (Rf = 0.7), but the 125I-TiO2-NPs remained at the point of spotting (Rf = 0). Figure 2 also reveals the purity of 125I-TiO2-NPs was more than 96% and was stable for 7 days.
Identification (A) and in vitro stability (B) of 125I-TiO2-NPs. After radiolabeling with 125I, the identification and stability of 125I-TiO2-NPs were analyzed by paper chromatography using ITLC-SG with 2.5% BSA (w/w) in 0.01 M PBS (pH 7.4) as the solvent. The purity of 125I-TiO2-NPs was expressed as the percentage of radioactivity at the site of 125I-TiO2-NPs compared with the total radioactivity on the paper and its change was expressed as the stability of 125I-TiO2-NPs.
Morphology of pretreated skin
The results of histopathological analysis, expressed in Figure 3, show that the SC layer of SLS treated skin was thinner and weaker than that of PS treated skin (intact skin). These results indicated the SLS treatment had caused a slight SC damage of the skin.
Morphology of 0.9% PS solution treated skin (A) and 2% SLS solution treated skin (B). The skin of animals was treated with 2% SLS solution or 0.9% PS solution for 4 hours in vivo and excised with full-thickness. Tissue sections with 7 μm thickness were prepared, stained with H&E and observed under a microscope at 400 × magnification. The white arrow and black arrow denote epidermis and SC layer of skin respectively.
Penetration of skin in vitro
The in vitro penetration through damaged skin or intact skin was evaluated during 24 h on 7 time-points. The results showed that the radioactivity of receptor chamber solution through damaged skin was higher than that of through intact skin and was about 2% radioactivity of donor chamber on 24 h (Fig. 4).
Penetration of TiO2-NPs through damaged skin or intact skin in vitro. The 125I-TiO2-NPs solution (1 mg/mL) and 0.9 % PS solution were added into the donor chamber and the receptor chamber of static diffusion cells with the same volume respectively. At seven time-points (0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h), samples (200 μl) were extracted from the receptor chamber and counted for 1 min in a γ-counter to detect the radioactivity. The skin penetration was expressed as the percentage of radioactivity of receptor chamber solution compared with total radioactivity in the donor chamber *P<0.01 vs intact skin (n = 5).
Penetration of skin in vivo
Figure 5 shows the radioactivity of blood and tissues of rats after exposing to 125I-TiO2-NPs solution though damaged skin or intact skin on day 1 and day 3. It can be found that the radioactivity of blood and tissues were less than 0.05% dose/g on day 1 and quickly declined on day 3.
Penetration of TiO2-NPs through damaged skin or intact skin in vivo on 1 d (A) and 3 d (B). The 125I-TiO2-NPs solution (1 mg/mL) was dropped onto the damaged skin or intact skin surface. After 1 day and 3 days, animals were sacrificed and their blood and tissues were harvested, weighed, and counted for 1 min in a γ-counter to detect the tissue radioactivity. The skin penetration was expressed as the percentage injected dose per gram tissue (% ID/g) (n = 5).
Discussion
SLS is commonly used in cosmetics, soap, body wash and other products as a surfactant and vesicant, and also can cause slight skin impairment. This impairment has previously been attributed to the fluidization of the lipid bilayers in the SC layer and the removal of intercellular hydrophobic lipids (22). These two effects should increase percutaneous penetration of primarily hydrophilic compounds, but not affect the percutaneous penetration of the most lipophilic compounds (23). The increasing concentration of SLS is required to enhance percutaneous penetration as the degree of lipophilicity of the model compounds increase. Nielsen (24) demonstrated that a concentration of 2% SLS destabilizes the integrity of the skin and probably removes much of the barrier function. But Borrás-Blasco et al (23) found that increasing the concentration of SLS above 1% would not further enhance percutaneous penetration of lipophilic model substances. For establishment of a model with a slightly damaged skin, the present study used SLS solution with a concentration of 2%, which was a common concentration in its applied products and could cause a constant and repeatable change in barrier properties, to pretreat the skin before the penetration experiment. The results of histopathological analysis in this study showed the SC layer was thin and weak after the pretreatment of 2% SLS solution and indicated the skin damaged was slight.
Although there were some studies that had reported that TiO2-NPs could not penetrate through the intact skin, currently, it was unclear whether TiO2-NPs could penetrate the damaged skin. The present study investigated the penetration of 125I labeled TiO2-NPs with a diameter of approximately 20 nm through damaged and intact skin in vitro and vivo. The result of in vitro experiment showed that the radioactivity of receptor chamber solution through damaged skin was higher than that of through intact skin. It indicated that the SLS treatment would promote the penetration of radioactive substance through the damaged skin. However, this penetration rate was only about 2%, which was lower than the rate of free 125I in the TiO2-NPs solution in the donor chamber. The penetrated radioactive substance might be the free 125I because 125I could easily penetrate the skin due to its small molecular weight. These results suggested the TiO2-NPs could not penetrate the SLS damaged skin in vitro. As known, the intercellular space between the SC cells, which was an important factor that determined the penetration of materials into the skin, should be widened with topical application of various products, such as SLS. But if the materials should penetrate into and through the skin, another barrier, the epidermis of skin, must be passed in this process. Larese et al (25) found polyvinylpirrolidone coated silver nanoparticles with a diameter of about 25 nm was able to penetrate the epidermis damaged skin in vitro. Thus, both SC and epidermis damage would promote the nanoparticles penetrating into and through skin.
We then considered whether TiO2-NPs could penetrate the viable and slightly damaged skin. The penetration of 125I labeled TiO2-NPs through the SC damaged skin caused by 2% SLS solution in vivo was then investigated and the results showed that the radioactivity of in vivo tissues was very low and quickly declined. These results also indicated the penetrated radioactive substance might be the free 125I and TiO2-NPs could not penetrate the SLS damaged skin in vivo. Mavon et al (26) also suggested the mineral nanoparticles in sunscreen remain on the skin surface or the outer layers of the SC and do not penetrate into or through the living skin. Zhang and Monteiro-Riviere (27) investigated the skin penetration in vivo of Quantum dot (QD) with diameter of 6-12 nm and found barrier perturbation by tape stripping did not cause penetration, but epidermis abrasion allowed QD to penetrate deeper into the dermal layers. These researches and our results indicated that 20 nm TiO2-NPs could not penetrate skin in vivo even if the SC layer of skin was damaged.
Conclusions
In conclusion, this study investigated the penetration of 125I labeled TiO2-NPs with a diameter of 20 nm through slightly damaged skin caused by 2% SLS solution in vitro and in vivo. The results indicated that the TiO2-NPs could not penetrate through the damaged skin both in vitro and in vivo. It suggested that the TiO2-NPs should be safe when it was applied and contacted with skin.
Disclosures
Financial support: This work was supported by grants from Natural Science Foundation of China (no. 31300795), Zhejiang Provincial Natural Science Foundation of China (no. Y4110665) and Zhejiang Province Public welfare Technology Applied Research Project (no. 2012C33115).
Conflict of interest: The authors declare that there are no conflicts of interest.
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Department of Stomatology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai - China
Department of Clinical Medicine, Medical School, Huzhou University, Huzhou - China
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