Jürgen Lademann

The potential risks of nanomaterials: a review carried out for ECETOC

During the last few years, research on toxicologically relevant properties of engineered nanoparticles has increased tremendously. A number of international research projects and additional activities are ongoing in the EU and the US, nourishing the expectation that more relevant technical and toxicological data will be published. Their widespread use allows for potential exposure to engineered nanoparticles during the whole lifecycle of a variety of products. When looking at possible exposure routes for manufactured Nanoparticles, inhalation, dermal and oral exposure are the most obvious, depending on the type of product in which Nanoparticles are used. This review shows that (1) Nanoparticles can deposit in the respiratory tract after inhalation. For a number of nanoparticles, oxidative stress-related inflammatory reactions have been observed. Tumour-related effects have only been observed in rats, and might be related to overload conditions. There are also a few reports that indicate uptake of nanoparticles in the brain via the olfactory epithelium. Nanoparticle translocation into the systemic circulation may occur after inhalation but conflicting evidence is present on the extent of translocation. These findings urge the need for additional studies to further elucidate these findings and to characterize the physiological impact. (2) There is currently little evidence from skin penetration studies that dermal applications of metal oxide nanoparticles used in sunscreens lead to systemic exposure. However, the question has been raised whether the usual testing with healthy, intact skin will be sufficient. (3) Uptake of nanoparticles in the gastrointestinal tract after oral uptake is a known phenomenon, of which use is intentionally made in the design of food and pharmacological components. Finally, this review indicates that only few specific nanoparticles have been investigated in a limited number of test systems and extrapolation of this data to other materials is not possible. Air pollution studies have generated indirect evidence for the role of combustion derived nanoparticles (CDNP) in driving adverse health effects in susceptible groups. Experimental studies with some bulk nanoparticles (carbon black, titanium dioxide, iron oxides) that have been used for decades suggest various adverse effects. However, engineered nanomaterials with new chemical and physical properties are being produced constantly and the toxicity of these is unknown. Therefore, despite the existing database on nanoparticles, no blanket statements about human toxicity can be given at this time. In addition, limited ecotoxicological data for nanomaterials precludes a systematic assessment of the impact of Nanoparticles on ecosystems.

Grey Goo on the Skin? Nanotechnology, Cosmetic and Sunscreen Safety

Many modern cosmetic or sunscreen products contain nano-sized components. Nanoemulsions are transparent and have unique tactile and texture properties; nanocapsule, nanosome, noisome, or liposome formulations contain small vesicles (range: 50 to 5000 nm) consisting of traditional cosmetic materials that protect light-or oxygen-sensitive cosmetic ingredients. Transdermal delivery and cosmetic research suggests that vesicle materials may penetrate the stratum corneum (SC) of the human skin, but not into living skin. Depending on the physical/chemical properties of the ingredient and the formulation, nano-sized formulations may enhance or reduce skin penetration, albeit at a limited rate. Modern sunscreens contain insoluble titanium dioxide (TiO2) or zinc oxide (ZnO) nanoparticles (NP), which are colorless and reflect/scatter ultraviolet (UV) more efficiently than larger particles. Most available theoretical and experimental evidence suggests that insoluble NP do not penetrate into or through normal as well as compromised human skin. Oral and topical toxicity data suggest that TiO2 and ZnO NP have low systemic toxicity and are well tolerated on the skin. In vitro cytotoxicity, genotoxicity, and photogenotoxicity studies on TiO2 or other insoluble NP reporting uptake by cells, oxidative cell damage, or genotoxicity should be interpreted with caution, since such toxicities may be secondary to phagocytosis of mammalian cells exposed to high concentrations of insoluble particles. Caution needs to be exercised concerning topical exposure to other NP that either have characteristics enabling some skin penetration and/or have inherently toxic constituents. Studies on wear debris particles from surgical implants and other toxicity studies on insoluble particles support the traditional toxicology view that the hazard of small particles is mainly defined by the intrinsic toxicity of particles, as distinct from their particle size. There is little evidence supporting the principle that smaller particles have greater effects on the skin or other tissues or produce novel toxicities relative to micro-sized materials. Overall, the current weight of evidence suggests that nano-materials such as nano-sized vesicles or TiO2 and ZnO nanoparticles currently used in cosmetic preparations or sunscreens pose no risk to human skin or human health, although other NP may have properties that warrant safety evaluation on a case-by-case basis before human use.

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

A new method was developed to determine the horny layer profile of volunteers using tape stripping in combination with UV/visible spectroscopy. The optical absorbance and the weight of corneocyte aggregates were compared as parameters for the determination of the mass of the horny layer particles fixed to the individual tapes. It was shown that the potential disturbances influencing both parameters must be considered critically before calculating the correlation factor, found as R2mean = 0.93 ± 0.05. It was proven that the absorbance in the visible range is better suited than the weight to quantify the amount of corneocyte aggregates removed by a single strip. The new method allows an exact anatomical localization of the individual tapes and all data obtained within the depth profile of the stratum corneum. This was exemplified by the determination of the penetration of chemical and physical UV filters into the horny layer.

Follicular transport route--research progress and future perspectives.

The important role of hair follicles as penetration pathways and reservoir structures for topically applied compounds has been validated in numerous animal models as well as in humans. Follicular penetration rates are modulated by regional variations in size and proportions and the functional status. Advances have especially been made in the targeting of hair follicle-associated cell populations including antigen-presenting cells and stem cells. Improved investigative methods based on differential stripping, spectrophotometry and confocal laser scanning microscopy have led to the determination of the penetration profiles and kinetics for a multiplicity of drugs and drug delivery systems. The observation that particulate delivery systems aggregate and remain in hair follicle openings and their penetration along the follicular duct occurs in a size-dependent manner, which has led to advanced concepts of targeted drug delivery of bioactive compounds in the field of solid particles, as well as semi-solid particles, such as liposomes. This review summarizes the recent progress in this field, and underlines the necessity for pilot studies in human volunteers to further the development of clinical applications for follicular targeting.

Investigation of follicular penetration of topically applied substances.

The influence of specific follicle properties, sebum production and hair growth on the follicular penetration of topically applied substances was investigated. The behavior of follicles identified in selected skin areas of volunteers was analyzed by various tape stripping and staining methods in combination with laser scanning microscopy. Furthermore hair growth in the selected skin areas was determined. A correlation between sebum production, hair growth activity and follicular penetration was observed.

Skin Penetration and Cellular Uptake of Amorphous Silica Nanoparticles with Variable Size, Surface Functionalization, and Colloidal Stability

In this study, the skin penetration and cellular uptake of amorphous silica particles with positive and negative surface charge and sizes ranging from 291 ± 9 to 42 ± 3 nm were investigated. Dynamic light scattering measurements and statistical analyses of transmission electron microscopy images were used to estimate the degree of particle aggregation, which was a key aspect to understanding the results of the in vitro cellular uptake experiments. Despite partial particle aggregation occurring after transfer in physiological media, particles were taken up by skin cells in a size-dependent manner. Functionalization of the particle surface with positively charged groups enhanced the in vitro cellular uptake. However, this positive effect was contrasted by the tendency of particles to form aggregates, leading to lower internalization ratios especially by primary skin cells. After topical application of nanoparticles on human skin explants with partially disrupted stratum corneum, only the 42 ± 3 nm particles were found to be associated with epidermal cells and especially dendritic cells, independent of their surface functionalization. Considering the wide use of nanomaterials in industries and the increasing interest for applications in pharmaceutics and cosmetics versus the large number of individuals with local or spread impairment of the skin barrier, e.g., patients with atopic dermatitis and chronic eczema, a careful dissection of nanoparticle-skin surface interactions is of high relevance to assess possible risks and potentials of intended and unintended particle exposure.

In vivo determination of skin surface topography using an optical 3D device

Background/purpose: Topography of the skin surface is a mirror of the functional skin status. Therefore, its evaluation is of great interest for dermatological research. In the present study, a new optical measuring system was applied to evaluate human skin surface topography in vivo and non‐invasively, in order to determine elastotic and mimic wrinkles dependent on age, body site and therapy.

TiO2 nanoparticles as an effective UV-B radiation skin-protective compound in sunscreens

Protecting human skin against harmful UV-B radiation coming from the sun is currently a problem. Due to the decreased thickness of the ozone layer, a more dangerous amount of UV-B light reaches the surface of our planet. This causes increased frequency of skin diseases. Titanium dioxide (TiO2) fine particles are embedded with sunscreens into the skin to effectively attenuate UV-B radiation. This study evaluates the most appropriate size of such particles assuming they are spheres. The distribution of TiO2 particles within the skin, achieved with topically applied sunscreens, is determined experimentally by the tape-stripping technique. Computer code implementing the Monte Carlo method is used to simulate photon migration within the plain 20??m thick horny layer matrix partially filled with nano-sized TiO2 particles. Dependences of harmful UV-B radiation of 307?311?nm absorbed by, backscattered from and transmitted through the horny layer on the concentration of TiO2 particles are obtained and analysed. As a result, particles of 62?nm are found to be the most effective in protecting skin against UV-B light.

Surface Functionalization of Silica Nanoparticles Supports Colloidal Stability in Physiological Media and Facilitates Internalization in Cells

The influence of the surface functionalization of silica particles on their colloidal stability in physiological media is studied and correlated with their uptake in cells. The surface of 55 ± 2 nm diameter silica particles is functionalized by amino acids or amino- or poly(ethylene glycol) (PEG)-terminated alkoxysilanes to adjust the zeta potential from highly negative to positive values in ethanol. A transfer of the particles into water, physiological buffers, and cell culture media reduces the absolute value of the zeta potential and changes the colloidal stability. Particles stabilized by L-arginine, L-lysine, and amino silanes with short alkyl chains are only moderately stable in water and partially in PBS or TRIS buffer, but aggregate in cell culture media. Nonfunctionalized, N-(6-aminohexyl)-3-aminopropyltrimethoxy silane (AHAPS), and PEG-functionalized particles are stable in all media under study. The high colloidal stability of positively charged AHAPS-functionalized particles scales with the ionic strength of the media, indicating a mainly electrostatical stabilization. PEG-functionalized particles show, independently from the ionic strength, no or only minor aggregation due to additional steric stabilization. AHAPS stabilized particles are readily taken up by HeLa cells, likely as the positive zeta potential enhances the association with the negatively charged cell membrane. Positively charged particles stabilized by short alkyl chain aminosilanes adsorb on the cell membrane, but are weakly taken up, since aggregation inhibits their transport. Nonfunctionalized particles are barely taken up and PEG-stabilized particles are not taken up at all into HeLa cells, despite their high colloidal stability. The results indicate that a high colloidal stability of nanoparticles combined with an initial charge-driven adsorption on the cell membrane is essential for efficient cellular uptake.

The Role of Follicular Penetration

Textbooks describe percutaneous absorption through normal skin as diffusion through the stratum corneum barrier, i.e., in a simplified view, as a physiochemical interaction of a penetrating compound with the horizontal multilayer lipid structures and the underlying corneocytes within the horny layer. Hair follicles and sweat glands are vertical structures and are considered of little importance since ‘they represent a small and insignificant percentage of the skin surface’. To this integral view, a differentiated hypothesis is proposed: the more perfect the barrier function of the horny layer, the greater the relative role of the imperfections within the skin as a defence organ. Three kinds of imperfections should be taken into account: (1) the follicular apparatus of hair follicles; (2) the sweat glands, and (3) microlesions in the interfollicular horny layer. Each of these can theoretically represent a vertical pathway for percutaneous absorption, i.e. a ‘shunt’.