Imaging of Zinc Oxide Nanoparticle Penetration in Human Skin in Vitro and in Vivo

Journal of Biomedical Optics 13͑6͒, 064031 ͑November/December 2008͒ Imaging of zinc oxide nanoparticle penetration in human skin in vitro and in vivo Andrei V. Zvyagin Abstract. Zinc oxide ͑ZnO-nano͒ and titanium dioxide nanoparticles Xin Zhao ͑20 to 30 nm͒ are widely used in several topical skin care products, Macquarie University such as sunscreens. However, relatively few studies have addressed Department of Physics the subdermal absorption of these nanoparticles in vivo. We report on Centre of MQ Photonics Sydney, NSW 2109, Australia investigation of the distribution of topically applied ZnO in excised and in vivo human skin, using multiphoton microscopy ͑MPM͒ imaging with a combination of scanning electron microscopy ͑SEM͒ and Audrey Gierden an energy-dispersive x-ray ͑EDX͒ technique to determine the level of Université Claude Bernard Lyon penetration of nanoparticles into the sub-dermal layers of the skin. Institut des Sciences Pharmaceutiques et Biologiques The good visualization of ZnO in skin achieved appeared to result Lyon, France from two factors. First, the ZnO principal photoluminescence at 385 nm is in the “quiet” spectral band of skin autofluorescence domi- ͓ ͔ Washington Sanchez nated by the endogenous skin fluorophores, i.e., NAD P H and FAD. ͓␴͑TPEF͒ Justin A. Ross Second, the two-photon action cross section of ZnO-nano ZnO ϳ ͔ ϳ Michael S. Roberts 0.26 GM; diameter, 18 nm is high: 500-fold of that inferred ͓ ␹͑3͒ ͔ The University of Queensland from its bulk third-order nonlinear susceptibility Im ZnO , and is School of Medicine favorably compared to that of NAD͓P͔H and FAD. The overall out- Therapeutics Research Unit come from MPM, SEM, and EDX studies was that, in humans in vivo, Princess Alexandra Hospital ͑ ͒ Australia ZnO nanoparticles stayed in the stratum corneum SC and accumu- lated into skin folds and/or hair follicle roots of human skin. Given the lack of penetration of these nanoparticles past the SC and that the outermost layers of SC have a good turnover rate, these data suggest that the form of ZnO-nano studied here is unlikely to result in safety concerns. © 2008 Society of Photo-Optical Instrumentation Engineers. ͓DOI: 10.1117/1.3041492͔ Keywords: nonlinear optical microscopy; nanoparticle toxicity; zinc oxide; transdermal permeability; multiphoton microscopy. Paper 08185R received Jun. 13, 2008; revised manuscript received Sep. 5, 2008; accepted for publication Oct. 20, 2008; published online Dec. 23, 2008. 1 Introduction blocking creams. Transparency is achieved by reducing scattering efficiency of ZnO particles, which are normally white Nanomaterials including nanoemulsions, nanosomes, and and visible, by using a nanoparticle size of less than 30 nm, nanoparticles are employed as active components1,2 and deliv- 6 3,4 its transparency threshold. An inevitable question then is ery vehicles in cosmetics and medicine. One example of a whether there a potential toxicity of these nanoparticles whose nanotechnology application is the widespread use of zinc ox- size may be small enough to allow them to penetrate the skin. ͑ ͒ ͑ ͒ ide ZnO-nano and titanium dioxide TiO2 nanoparticles as The extent of nanoparticles penetration through the topmost ingredients in cosmetic and sun-blocking creams. These na- layer of the skin, stratum corneum ͑average thickness, less nomaterials are efficient absorbers of the ultraviolet ͑UV͒ ra- than 20 ␮m͒ into the viable epidermis, is hotly debated. As an diation: ZnO absorbs both UVA ͑400 to 320 nm͒ and UVB illustration, the studies of Alvarez-Roman et al. shows no pen- ͑320 to 290 nm͒ radiation and reemits them as less damaging etration through excised human skin.6 In contrast, UVA or as visible fluorescence ͑and is dictated by the need to Ryman-Rasmussen33 show extensive penetration of neutral, minimize skin damage due to UV light and potential conse- positively charged and negatively charged quantum dots quences such as skin cancer͒. The use of these sun-blocking creams is widespread in Australia, where over 250,000 people through pig skin. In vitro cytotoxicity of ZnO-nano to epider- ͑ ͒ are diagnosed with nonmelanoma skin cancer, and over 8000 mal and, especially, epithelial cells can occur and have been ͑ with melanoma annually.5 attributed to free radical generation mainly via hydroxyl radi- cals formed through oxidation͒, causing adverse effects in iso- The upper size limit for ZnO and TiO2 nanoparticles is 8,9 dictated by the aesthetic marketing value of transparent sun- lated cell experiments. Conventional scanning electron microscopy ͑SEM͒, trans- mission electron microscopy and Franz cell penetration in- Address all correspondence to: Andrei V. Zvyagin, Dept. of Physics, Centre of MQ Photonics, Macquarie Univ., Sydney, NSW 2109, Australia; Tel: +61 2 9850 7760; Fax: +61 2 9850 7760 8115; Email: [email protected] 1083-3668/2008/13͑6͒/064031/9/$25.00 © 2008 SPIE Journal of Biomedical Optics 064031-1 November/December 2008 b Vol. 13͑6͒ Downloaded From: http://biomedicaloptics.spiedigitallibrary.org/ on 10/15/2015 Terms of Use: http://spiedigitallibrary.org/ss/TermsOfUse.aspx Zvyagin et al.: Imaging of zinc oxide nanoparticle penetration in human skin… volve indirect or/and destructive sampling of skin that limits the experimental methods to in vitro observations. Optical microscopy of nanoparticle distribution in skin in vivo is pre- cluded due to high scattering efficiency of the superficial layers of skin, especially human skin. Fluorescence confocal microscopy has enabled production of clear subsurface im- ages of skin at depths down to ϳ100 ␮m at the micrometer resolution by way of “optical sectioning” thin layers of the specimen. The skin morphology contrast is derived from the skin autofluorescence, although often requires additional staining. The visibility of nanoparticles depends on their fluorescent properties, which limits the choice to the following materials: micro- and nanobeads impregnated with fluorescence dye molecules, photoluminescent nanomaterials, quantum dot ͑Qdot͒ structures,10 and emerging nanocrystals with color centers.11 Fabrication of nanobeads impregnated with fluorescent dye is difficult and they are prone to photobleach- ing. Qdots are cytotoxic, thus, posing hazard for in vivo ap- plications on humans. Although silica Qdots are less cytotoxic, their photoluminescence ͑PL͒ degradation and complex passivation chemistry makes their application cumbersome and expensive.12 PL materials, such as semiconductor metal oxides, are either toxic, radiation-inefficient ͑due to the indirect band gap structure͒, or rare. In contrast, ZnO-nano repre- sents a promising nanomaterial that is widespread, inexpen- sive, and has proven to be suitable for cosmetic care and pharmacy. Its production is straightforward, yielding nanoparticle of various sizes and shapes, starting from 3nm and larger.7,13 ZnO material properties, including a direct wide bandgap ͑3.4 eV͒ electronic structure, and high exciton energy of 60 meV ͑compare to that of 25 meV of cadmium selenide͒, are very attractive for a new generation of electro- optic devices, e.g., light-emitting diodes and lasers in the UV spectral range. The wide bandgap of ZnO requires excitation Fig. 1 Emission spectrum of ZnO-nano at an excitation wavelength of at a wavelength of 320 nm in the UV spectral range, with 320 nm ͑scatter plot, squares͒ superimposed on TPEF spectra of ͑a͒ principle emission at 385 nm, as shown in Fig. 1 ͑scattered epidermal cells ͑circles͒, similar to a spectrum of NAD͓P͔H; FAD ͑tri- squares graph͒. This excitation requirement is problematic for angles͒; melanin ͑inverted triangles͒; keratin ͑diamonds͒, adapted from Ref. 14;and͑b͒ elastin ͑diamonds͒, similar to collagen, adapted application of ZnO-nano in optical biomedical imaging. First, ͑ ͒ ͑ from Ref. 14; and the second-harmonic generation SHG spectrum of UV photons efficiently excite a number of intrinsic endog- collagen calculated at a 740-nm fundamental wavelength. A dashed enous͒ fluorophores in skin that produce a broadband autof- area designates FWHM of the bandpass filter BP380 used to discrimi- luorescence background. Among these, nicotinamide adenine nate ZnO PL signal against the skin autofluorescence. dinucleotide/nicotinamide adenine dinucleotide phosphate ͑NAD͓P͔H͒, flavin adenine dinucleotide ͑FAD͒, and porphy- rins contribute 75, 25, and 2% to the skin autofluorescence, range, and the water transparency window; and, second, to the with their respective fluorescence bands centered at 425, 520, reduced scattering efficiency of skin tissue at longer wave- and 625 nm ͑Ref. 15͓͒also see Fig. 1͑a͔͒. Second, UV radia- lengths. tion is heavily absorbed and scattered by the skin tissue, se- In this paper, we communicate our results on application of verely limiting imaging penetration depth. Hence, optical im- multiphoton microscopy to study of ZnO nanoparticles pen- aging of thick skin tissue using UV light is not practical. etration in human skin in vitro and in vivo. This is the first It has been recently found that the zinc oxide structure is time, to the best of our knowledge, that the in vivo observa- efficiently excited via a nonlinear optical process of simulta- tion of inorganic PL nanoparticles in the morphology context neous absorption of two or three photons under illumination of live cells and tissue has been reported. This will enable by an ultrashort-pulse laser.16 This shifts the ZnO excitation imaging of a new class of engineered nanostructures as band to the infrared ͑IR͒ range, where the ultrashort-pulse applied to transdermal delivery and toxicology. laser sources predominantly operate, falling into the so-called therapeutic window, 600 to 1400 nm, wherein the maximum 2 Materials and Methods imaging penetration depth in tissue of ϳ200 ␮m is attainable. The increased penetration depth is due, first, to the markedly 2.1 Materials reduced absorption of the major tissue constituents, including Dr. Lewinns’ private formula ͑19% w/w͒͑Advanced Nano- proteins, hemoglobin, and melanin in the UV/visible spectral technology Ltd., WA, Australia͒ is comprised of 26 to 30-nm Journal of Biomedical Optics 064031-2 November/December 2008 b Vol.

Load more