Novel metal allergy patch test using metal nanoballs
© Sugiyama et al.; licensee BioMed Central Ltd. 2014
Received: 15 September 2014
Accepted: 6 November 2014
Published: 3 December 2014
Patch tests are often used in the clinical diagnosis of metal allergies. In currently available patch tests, high concentrations of metal salt solutions are used. However, diagnosis accuracy can be influenced not only by acute skin reactions to high concentrations of metal salt, but also by skin reactions to other components present in the patch or to pH changes. In this study, we developed Ni nanoparticles (termed “nanoballs”) for use in patch-test solutions.
Highly soluble, spherical Ni nanoballs were prepared using plasma electrolysis. The Ni released from the nanoballs permeated through a dialysis membrane, and the nanoball-containing solution’s pH was maintained constant. Ni ions were released slowly at low concentrations in a time-dependent manner, which contrasted the rapid release observed in the case of a commercial patch test. Consequently, in the new test system, reactions caused by high concentrations of metal salts were avoided.
By exploiting the high specific surface area of Ni nanoballs, we obtained an effective dissolution of Ni ions that triggered Ni allergy in the absence of direct contact between the nanoballs and mouse skin. This novel patch system can be applied to other metals and alloys for diagnosing various types of metal-induced contact dermatitis.
KeywordsNanoparticle Nickel Metal allergy Patch test Elemental distribution
An ideal patch test must reproduce the metal erosion that occurs in metallic equipment (Figure 1b). Therefore, historically, large metal particles have been used as the metal source in the patch test . However, their dissolution rates are inadequate for triggering an allergic reaction because of their low specific surface area ,.
To address the aforementioned limitations of patch tests, in this study, we prepared 40–50-nm-diameter Ni nanoballs (Figure 1c) through plasma electrolysis at the surface of electrodes . Because of their size, we expected the Ni nanoballs to exhibit a high rate of Ni dissolution, which is suitable for a patch test. In our novel patch system, we used a Ni-nanoball suspension enclosed in a dialysis tube, which we call a “Ni-nanoball pack.” Ni ions released from the nanoball pack permeated through the dialysis membrane into the skin of mice, and thus direct contact between the Ni nanoballs and the skin was avoided. We evaluated this Ni-permeation behavior in vitro and also in vivo by using mouse skin. The Ni distribution in the skin was measured using synchrotron radiation-excited X-ray fluorescence (SR-XRF) analysis and particle-induced X-ray emission (PIXE), which are highly sensitive analysis methods. Furthermore, we examined the chemical state of the Ni nanoballs and the permeated Ni in the skin by performing X-ray absorption fine structure (XAFS) analysis.
Preparation of Ni-nanoball suspensions and an in vitro test of Ni-ion release
In vivo Ni-permeation test designed for mouse skin
C57BL/6 mice (45–75 weeks old) were used in this study. All animal protocols were approved by the Animal Ethics Review Board of the Dental Hospital of Nihon University School of Dentistry, Tokyo, Japan, and conformed to the guidelines of the National Institutes of Health. The skin on the back of mice was depilated under general anesthesia. Approximately 20 mL of the Ni-nanoball suspension was placed into the dialysis tube and both ends were clamped. The Ni-nanoball pack was fixed onto the depilated back skin of the mice by using film dressing. The application period ranged from 30 min to 24 h, after which the mice were sacrificed. The Ni-nanoball pack was then removed and the skin was gently wiped, and the part of the skin that contacted the pack was excised and used for preparing frozen sections (20-μm thick) according to the method reported by Kubo et al. . The sections were placed on Kapton film, dried and subjected to elemental-distribution analysis. As a control, a commercial patch test for Ni allergy (5% w/v NiSO4 aq.) was applied to mice using the same method. Adjacent specimen slices were stained with hematoxylin and eosin (H-E) and used for histopathological diagnosis. Detailed information on the chemicals, materials, and equipments used are presented in Additional file 1: Table S1.
Elemental-distribution and chemical-state analyses
Elemental distribution analysis of the entire specimen was performed using SR-XRF. The specimen was irradiated with micro-focused X-ray and the specimen stage was scanned two- dimensionally. The fluorescence X-ray was detected at each point and the elemental distribution images were processed. The high-resolution elemental distribution was analyzed using micro-PIXE analysis, which involved exposure to the micro-focused proton beam with raster scanning over the target area of the specimen. The characteristic X-rays were detected, and the elemental distribution image was processed. The chemical states of the Ni contained in Ni nanoballs and the Ni in mouse skin were examined using XAFS analysis. Detailed conditions of the elemental analyses are shown in Additional file 1: Table S2.
Results and discussion
The time-dependent release of Ni in vitro is shown in Figure 2b. We observed a continuous release of Ni. The Ni-dissolution rate of Ni nanoballs dispersed in PBS (pH 5.8) was higher than that of nanoballs dispersed in DW. This result agrees with the previous findings of previous studies showing that Ni solubility is increased in acidic solutions ,. The pH of the commercial Ni patch-test solution was 3.8, which is lower than that of natural human skin, where the pH is typically below 5.0 . Thus, we do not expect the Ni-nanoball suspension at pH 5.8 to irritate human skin. Furthermore, the Ni-release rate in our test system could be controlled by adjusting the pH.
Figure 4 also shows the Ni K-edge XAFS spectra of Ni nanoballs (Figure 4c) and the permeated Ni in skin (Ni released from the nanoballs) (Figure 4d). The spectrum of the Ni nanoballs closely fits that of a mixture of metallic Ni (20%) and NiO (80%). This suggests that the Ni nanoballs were almost oxidized during storage. Conversely, the spectrum of the permeated Ni in skin (derived from the Ni-nanoball pack) was similar to that of NiSO4 aq., but distinct from that of NiO, a major component of Ni nanoballs. Therefore, the dissolved Ni ions were successfully released from the Ni nanoballs and they permeated the skin through the dialysis membrane.
During a genuine allergic reaction to metals, metal erosion and skin permeation occur in a low and continuous dose. We have shown that our Ni-nanoball pack releases Ni ions slowly from the suspension under mildly acidic conditions. Recently, the health risks posed by nanomaterials have become a growing concern -. Thus, in this study, we exploited the high specific surface area of Ni nanoballs in order to obtain an effective dissolution of Ni ions and used this to trigger Ni allergy without direct contact between the nanoballs and the skin.
A high dose of metals and counter-ions and an acidic pH are factors that lower the accuracy of currently available metal-allergy patch tests. These factors were addressed effectively in this study, and the test system presented here could potentially serve as a state-of-the-art patch test. Another limitation of current patch tests is that they are available only for a few metal species, and the accuracy of these tests is still in question. The facile process used in this study for preparing Ni nanoballs can be applied to most pure metals and alloys, and then the method can be readily used for diagnosing other metal allergies. By conducting further studies on the ability to induce allergy in animal and human skin, the accuracy of the diagnosis obtained using our novel patch test designed for metal allergy could be potentially enhanced.
Synchrotron radiation-excited X-ray fluorescence
Particle-induced X-ray emission
X-ray absorption fine structure
Phosphate buffer solution
2-(5-Nitro-2-pyridylazo)-5-[N-n-propyl-N-(3-sulfopropyl)-amino]phenol disodium salt dehydrate
The SR-XRF measurements were performed with the approval of the Photon Factory Program Advisory Committee (Proposal Nos. 2011G011 and 2014G017). This work was financially supported by the Japan Society for the Promotion of Science KAKENHI (Grant No. 23390438 to M. Uo). T. Sugiyama is supported by a research fellowship for young scientists from the Japan Society for the Promotion of Science.
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