Shape-Dependent Dissolution and Cellular Uptake of Silver Nanoparticles.

The cellular uptake and dissolution of trigonal silver nanoprisms (edge length 42 ± 15 nm, thickness 8 ± 1 nm) and mostly spherical silver nanoparticles (diameter 70 ± 25 nm) in human mesenchymal stem cells (hMSC's) and human keratinocytes (HaCaT cells) were investigated. Both particles are stabilized by polyvinylpyrrolidone (PVP), with the prisms additionally stabilized by citrate. The nanoprisms dissolved slightly in pure water but strongly in isotonic saline or at pH 4, corresponding to the lowest limit for the pH during cellular uptake. The tips of the prisms became rounded within minutes due to their high surface energy. Afterward, the dissolution process slowed down due to the presence of both PVP stabilizing Ag{100} sites and citrate blocking Ag{111} sites. On the contrary, nanospheres, solely stabilized by PVP, dissolved within 24 h. These results correlate with the finding that particles in both cell types have lost >90% of their volume within 24 h. hMSC's took up significantly more Ag from nanoprisms than from nanospheres, whereas HaCaT cells showed no preference for one particle shape. This can be rationalized by the large cellular interaction area of the plateletlike nanoprisms and the bending stiffness of the cell membranes. hMSC's have a highly flexible cell membrane, resulting in an increased uptake of plateletlike particles. HaCaT cells have a membrane with a 3 orders of magnitude higher Young's modulus than for hMSC. Hence, the energy gain due to the larger interaction area of the nanoprisms is compensated for by the higher energy needed for cell membrane deformation compared to that for spheres, leading to no shape preference.

Effects on detection of radical formation in skin due to solar irradiation measured by EPR spectroscopy.

In various research projects, oxidative stress in irradiated skin was investigated by measuring the production of free radical using EPR spectroscopy. However, comparison of the obtained measuring results proved to be difficult as different preparation parameters were used for those measurements. In the present study the influence of the preparation parameters on the detected radical production was methodically investigated. For this purpose, porcine skin was exposed in situ to UV and VIS-NIR radiation, respectively, while being measured in an X band EPR spectrometer. Prior to the measurements, the skin had been treated with the spin trap N-tert-Butyl-α-phenylnitrone (PBN) and the spin marker 3-(Carboxyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA). The two methods were investigated for quantitative comparability, for advantages and disadvantages and for errors potentially affecting the evaluation of the results. A significant influence of the preparation parameters (concentration and amount of substance) on the detected radical formations could be found. This influence had a nonlinear effect on the detected radical production. 120µl of incubated amount for 1M PBN and for PCA at a concentration of 0.6 and 1.5mM were determined to be the optimum parameters. The incubated skin samples were 1cm in diameter and 300µm thick. Between 22 and 37°C the incubation temperature showed no significant influence on the detected radical production. For the first time it could be demonstrated for PCA-incubated skin that the radiation-induced radical production depends exclusively on the irradiation dose, provided the preparation parameters and the spectral region are kept constant. In addition, the radical production in the UVB-UVA and VIS-NIR spectral regions was measured in PCA- and PBN-treated excised porcine skin. It was found that PBN and PCA provide comparable results for the relative quantity and kinetics of radical production.

Comparison of different methods to study effects of silver nanoparticles on the pro- and antioxidant status of human keratinocytes and fibroblasts.

In times of antibiotic-resistant bacteria new strategies to avoid the septic-inducing threat of dangerous microorganisms are needed. Silver ions (Ag+) in the forms of silver nitrate or silver sulfadiazine have been used as antimicrobial agents for years. A step further was the development of micro and silver particles (AgNP). In contrast to other Ag+ ion sources, AgNP allow a sustained release of Ag+ ions, due to their high surface to volume ratio. However, AgNP are also toxic to eukaryotic cells and the mechanisms of cytotoxicity have not yet been fully elucidated. In this study, the impact of different AgNP preparations on a human keratinocyte cell line was investigated. The intracellular radical formation was confirmed by the 2',7'-dichlorodihydrofluorescein di-acetate (H2DCF-DA) assay on two cell types (HaCaT cells and normal human dermal fibroblasts) as well as by electron paramagnetic resonance (EPR) spectroscopy, which showed comparable results. EPR spectroscopy was performed for the first time for 24h in experiments using keratinocytes. Drastic changes in the mitochondrial activity were induced in cells incubated with AgNP containing high concentrations of Ag+ ions. It was also possible to show that the quantitative uptake of AgNP was dependent on the AgNP concentration. In addition, the effects of AgNP on the GSH/GSSG system were elucidated. The results showed a batch- and concentration-dependent decrease of the total glutathione concentration which correlated well with the decrease of cell viability. Furthermore, the results suggest a direct reaction of GSH molecules with Ag+ ions. In conclusion, this study proves the efficacy of the H2DCF-DA assay and the EPR spectroscopy. The investigations show that AgNP formulations containing high amounts of released Ag+ ions induce radicals in human keratinocytes and deplete them of their natural anti-oxidative molecules. On the contrary, nanoparticles prepared and stored under argon did not induce significant adverse effects, suggesting that slowing down the release of Ag+ may help to reduce AgNP-related side effects without affecting the antibacterial impact.

Penetration of silver nanoparticles into porcine skin ex vivo using fluorescence lifetime imaging microscopy, Raman microscopy, and surface-enhanced Raman scattering microscopy.

In order to investigate the penetration depth of silver nanoparticles (Ag NPs) inside the skin, porcine ears treated with Ag NPs are measured by two-photon tomography with a fluorescence lifetime imaging microscopy (TPT-FLIM) technique, confocal Raman microscopy (CRM), and surface-enhanced Raman scattering (SERS) microscopy. Ag NPs are coated with poly-N-vinylpyrrolidone and dispersed in pure water solutions. After the application of Ag NPs, porcine ears are stored in the incubator for 24 h at a temperature of 37°C. The TPT-FLIM measurement results show a dramatic decrease of the Ag NPs' signal intensity from the skin surface to a depth of 4 µm. Below 4 µm, the Ag NPs' signal continues to decline, having completely disappeared at 12 to 14 µm depth. CRM shows that the penetration depth of Ag NPs is 11.1 ± 2.1 µm. The penetration depth measured with a highly sensitive SERS microscopy reaches 15.6 ± 8.3 µm. Several results obtained with SERS show that the penetration depth of Ag NPs can exceed the stratum corneum (SC) thickness, which can be explained by both penetration of trace amounts of Ag NPs through the SC barrier and by the measurements inside the hair follicle, which cannot be excluded in the experiment.

Ultra-small lipid nanoparticles promote the penetration of coenzyme Q10 in skin cells and counteract oxidative stress.

UV irradiation leads to the formation of reactive oxygen species (ROS). An imbalance between the antioxidant system and ROS can lead to cell damage, premature skin aging or skin cancer. To counteract these processes, antioxidants such as coenzyme Q10 (CoQ10) are contained in many cosmetics. To improve and optimize cell/tissue penetration properties of the lipophilic CoQ10, ultra-small lipid nanoparticles (usNLC) were developed. The antioxidant effectiveness of CoQ10-loaded usNLC compared to conventional nanocarriers was investigated in the human keratinocyte cell line HaCaT. Using confocal laser scanning microscopy investigations of the carriers additionally loaded with nile red showed a clear uptake into cells and their distribution within the cytoplasm. By use of the XTT cell viability test, CoQ10 concentrations of 10-50 µg/ml were shown to be non-toxic, and the antioxidant potential of 10 µg/ml CoQ10 loaded usNLC in the HaCaT cells was analyzed via electron paramagnetic resonance spectroscopy after cellular exposure to UVA (1J/cm(2)) and UVB (18 mJ/cm(2)) irradiation. In comparison with the CoQ10-loaded conventional carriers, usNLC-CoQ10 demonstrated the strongest reduction of the radical formation; reaching up to 23% compared to control cells without nanocarrier treatment. Therefore, usNLC-CoQ10 are very suitable to increase the antioxidant potential of skin.

Influence of sun exposure on the cutaneous collagen/elastin fibers and carotenoids: negative effects can be reduced by application of sunscreen.

Resonance Raman spectroscopy and multi-photon tomography were used in vivo to analyse the influence of sun exposure on the cutaneous carotenoids and collagen/elastin fibers. Comparing Berlin (low sun exposure) and Monegasque (high sun exposure) volunteers, it could be demonstrated that extended sun exposure significantly reduces the cutaneous carotenoids and collagen/elastin concentration (p < 0.05). The tendency towards correlation (R(2) = 0.41) between the dermal collagen/elastin (SAAID) and carotenoids confirms the important role of antioxidants in the protection against sun-induced negative effects. The application of sunscreen was shown to be effective, protecting cutaneous carotenoids and collagen/elastin from being damaged subsequent to sun exposure.

Comparison of silver nanoparticles stored under air or argon with respect to the induction of intracellular free radicals and toxic effects toward keratinocytes.

Bacterial infections decreased considerably after the discovery of antibiotics. Nevertheless, because of the rising rate of infections caused by antibiotic-resistant bacteria strains, the search for new bactericidal agents has again become a crucial topic in clinical medicine. Silver nanoparticles (AgNP) have a huge potential in dermatology and wound care management because of their ability to release silver ions (Ag(+) ions) in a prolonged and sustained way. However, negative effects of silver on the patient's cells should not be underestimated. Furthermore, it has been controversially discussed whether AgNP are responsible for nanoparticle-specific outcomes or not. In this study, we investigated the effects of AgNP on human skin keratinocytes (HaCaT) in order to better understand the mechanisms of cytotoxicity and to improve the use of this highly reactive biocide in wound healing. We found that most of the cells with internalized AgNP displayed the typical morphological signs of apoptosis. The cell viability assay (XTT) showed concentration-dependent toxic effects of the AgNP toward HaCaT cells. The generation of reactive oxygen species (ROS) induced by AgNP was investigated in cell suspensions by means of electron paramagnetic resonance (EPR) spectroscopy. In order to distinguish between the effects of Ag(+) ions released during AgNP storage and those of Ag(+) ions released after nanoparticle application, we compared AgNP stored under air (O2) with AgNP stored under argon (Ar). Dispersions of AgNP stored under Ar have a low content of Ag(+) ions because of the absence of oxygen which is needed for oxidative dissolution. The results show that Ag(+) ions released during particle storage are responsible for most of the ROS produced during 1h incubation with the cells. AgNP (Ar) also induced intracellular ROS but to a much smaller extent compared to AgNP (O2). These findings highlight the complexity of experiments to assess the toxicity of AgNP and suggest the possibility of reducing AgNP toxic effects by storing AgNP formulations and even silver-containing wound dressing under an inert gas atmosphere.

Interaction of dermatologically relevant nanoparticles with skin cells and skin.

The investigation of nanoparticle interactions with tissues is complex. High levels of standardization, ideally testing of different material types in the same biological model, and combinations of sensitive imaging and detection methods are required. Here, we present our studies on nanoparticle interactions with skin, skin cells, and biological media. Silica, titanium dioxide and silver particles were chosen as representative examples for different types of skin exposure to nanomaterials, e.g., unintended environmental exposure (silica) versus intended exposure through application of sunscreen (titanium dioxide) or antiseptics (silver). Because each particle type exhibits specific physicochemical properties, we were able to apply different combinations of methods to examine skin penetration and cellular uptake, including optical microscopy, electron microscopy, X-ray microscopy on cells and tissue sections, flow cytometry of isolated skin cells as well as Raman microscopy on whole tissue blocks. In order to assess the biological relevance of such findings, cell viability and free radical production were monitored on cells and in whole tissue samples. The combination of technologies and the joint discussion of results enabled us to look at nanoparticle-skin interactions and the biological relevance of our findings from different angles.

PVP-coated, negatively charged silver nanoparticles: A multi-center study of their physicochemical characteristics, cell culture and in vivo experiments.

PVP-capped silver nanoparticles with a diameter of the metallic core of 70 nm, a hydrodynamic diameter of 120 nm and a zeta potential of -20 mV were prepared and investigated with regard to their biological activity. This review summarizes the physicochemical properties (dissolution, protein adsorption, dispersability) of these nanoparticles and the cellular consequences of the exposure of a broad range of biological test systems to this defined type of silver nanoparticles. Silver nanoparticles dissolve in water in the presence of oxygen. In addition, in biological media (i.e., in the presence of proteins) the surface of silver nanoparticles is rapidly coated by a protein corona that influences their physicochemical and biological properties including cellular uptake. Silver nanoparticles are taken up by cell-type specific endocytosis pathways as demonstrated for hMSC, primary T-cells, primary monocytes, and astrocytes. A visualization of particles inside cells is possible by X-ray microscopy, fluorescence microscopy, and combined FIB/SEM analysis. By staining organelles, their localization inside the cell can be additionally determined. While primary brain astrocytes are shown to be fairly tolerant toward silver nanoparticles, silver nanoparticles induce the formation of DNA double-strand-breaks (DSB) and lead to chromosomal aberrations and sister-chromatid exchanges in Chinese hamster fibroblast cell lines (CHO9, K1, V79B). An exposure of rats to silver nanoparticles in vivo induced a moderate pulmonary toxicity, however, only at rather high concentrations. The same was found in precision-cut lung slices of rats in which silver nanoparticles remained mainly at the tissue surface. In a human 3D triple-cell culture model consisting of three cell types (alveolar epithelial cells, macrophages, and dendritic cells), adverse effects were also only found at high silver concentrations. The silver ions that are released from silver nanoparticles may be harmful to skin with disrupted barrier (e.g., wounds) and induce oxidative stress in skin cells (HaCaT). In conclusion, the data obtained on the effects of this well-defined type of silver nanoparticles on various biological systems clearly demonstrate that cell-type specific properties as well as experimental conditions determine the biocompatibility of and the cellular responses to an exposure with silver nanoparticles.