Yolanda Hedberg

Cell membrane damage and protein interaction induced by copper containing nanoparticles—Importance of the metal release process

Cu-containing nanoparticles are used in various applications in order to e.g. achieve antimicrobial activities and to increase the conductivity of fluids and polymers. Several studies have reported on toxic effects of such particles but the mechanisms are not completely clear. The aim of this study was to investigate the interactions between cell membranes and well-characterized nanoparticles of CuO, Cu metal, a binary Cu-Zn alloy and micron-sized Cu metal particles. This was conducted via in vitro investigations of the effects of the nanoparticles on (i) cell membrane damage on lung epithelial cells (A549), (ii) membrane rupture of red blood cells (hemolysis), complemented by (iii) nanoparticle interaction studies with a model lipid membrane using quartz crystal microbalance with dissipation monitoring (QCM-D). The results revealed that nanoparticles of the Cu metal and the Cu-Zn alloy were both highly membrane damaging and caused a rapid (within 1h) increase in membrane damage at a particle mass dose of 20 μg/mL, whereas the CuO nanoparticles and the micron-sized Cu metal particles showed no such effect. At similar nanoparticle surface area doses, the nano and micron-sized Cu particles showed more similar effects. The commonly used LDH (lactate dehydrogenase) assay for analysis of membrane damage was found impossible to use due to nanoparticle-assay interactions. None of the particles induced any hemolytic effects on red blood cells when investigated up to high particle concentrations (1mg/mL). However, both Cu and Cu-Zn nanoparticles caused hemoglobin aggregation/precipitation, a process that would conceal a possible hemolytic effect. Studies on interactions between the nanoparticles and a model membrane using QCM-D indicated a small difference between the investigated particles. Results of this study suggest that the observed membrane damage is caused by the metal release process at the cell membrane surface and highlight differences in reactivity between metallic nanoparticles of Cu and Cu-Zn and nanoparticles of CuO.

In vitro biocompatibility of CoCrMo dental alloys fabricated by selective laser melting

OBJECTIVE Selective laser melting (SLM) is increasingly used for the fabrication of customized dental components made of metal alloys such as CoCrMo. The main aim of the present study is to elucidate the influence of the non-equilibrium microstructure obtained by SLM on corrosion susceptibility and extent of metal release (measure of biocompatibility). METHODS A multi-analytical approach has been employed by combining microscopic and bulk compositional tools with electrochemical techniques and chemical analyses of metals in biologically relevant fluids for three differently SLM fabricated CoCrMo alloys and one cast CoCrMo alloy used for comparison. RESULTS Rapid cooling and strong temperature gradients during laser melting resulted in the formation of a fine cellular structure with cell boundaries enriched in Mo (Co depleted), and suppression of carbide precipitation and formation of a martensitic ɛ (hcp) phase at the surface. These features were shown to decrease the corrosion and metal release susceptibility of the SLM alloys compared with the cast alloy. Unique textures formed in the pattern of the melting pools of the three different laser melted CoCrMo alloys predominantly explain observed small, though significant, differences. The susceptibility for corrosion and metal release increased with an increased number (area) of laser melt pool boundaries. SIGNIFICANCE This study shows that integrative and interdisciplinary studies of microstructural characteristics, corrosion, and metal release are essential to assess and consider during the design and fabrication of CoCrMo dental components of optimal biocompatibility. The reason is that the extent of metal release from CoCrMo is dependent on fabrication procedures.

Surface-protein interactions on different stainless steel grades: effects of protein adsorption, surface changes and metal release

Implantation using stainless steels (SS) is an example where an understanding of protein-induced metal release from SS is important when assessing potential toxicological risks. Here, the protein-induced metal release was investigated for austenitic (AISI 304, 310, and 316L), ferritic (AISI 430), and duplex (AISI 2205) grades in a phosphate buffered saline (PBS, pH 7.4) solution containing either bovine serum albumin (BSA) or lysozyme (LSZ). The results show that both BSA and LSZ induce a significant enrichment of chromium in the surface oxide of all stainless steel grades. Both proteins induced an enhanced extent of released iron, chromium, nickel and manganese, very significant in the case of BSA (up to 40-fold increase), whereas both proteins reduced the corrosion resistance of SS, with the reverse situation for iron metal (reduced corrosion rates and reduced metal release in the presence of proteins). A full monolayer coverage is necessary to induce the effects observed.

Hemolytic properties of synthetic nano- and porous silica particles: The effect of surface properties and the protection by the plasma corona

Novel silica materials incorporating nanotechnology are promising materials for biomedical applications, but their novel properties may also bring unforeseen behavior in biological systems. Micro-size silica is well documented to induce hemolysis, but little is known about the hemolytic activities of nanostructured silica materials. In this study, the hemolytic properties of synthetic amorphous silica nanoparticles with primary sizes of 7-14 nm (hydrophilic vs. hydrophobic), 5-15 nm, 20 nm and 50 nm, and model meso/macroporous silica particles with pore diameters of 40 nm and 170 nm are investigated. A crystalline silica sample (0.5-10 μm) is included for benchmarking purposes. Special emphasis is given to investigations of how the temperature and solution complexity (solvent, plasma), as well as the physicochemical properties (such as size, surface charge, hydrophobicity and other surface properties), link to the hemolytic activities of these particles. Results suggests the potential importance of small size and large external surface area, as well as surface charge/structure, in the hemolysis of silica particles. Furthermore, a significant correlation is observed between the hemolytic profile of red blood cells and the cytotoxicity profile of human promyelocytic leukemia cells (HL-60) induced by nano- and porous silica particles, suggesting a potential universal mechanism of action. Importantly, the results generated suggest that the protective effect of plasma towards silica nanoparticle-induced hemolysis as well as cytotoxicity is primarily due to the protein/lipid layer shielding the silica particle surface. These results will assist the rational design of hemocompatible silica particles for biomedical applications.

Bioaccessibility, bioavailability and toxicity of commercially relevant iron- and chromium-based particles: in vitro studies with an inhalation perspective

BackgroundProduction of ferrochromium alloys (FeCr), master alloys for stainless steel manufacture, involves casting and crushing processes where particles inevitably become airborne and potentially inhaled. The aim of this study was to assess potential health hazards induced by inhalation of different well-characterized iron- and chromium-based particles, i.e. ferrochromium (FeCr), ferrosiliconchromium (FeSiCr), stainless steel (316L), iron (Fe), chromium (Cr), and chromium(III)oxide (Cr2O3), in different size fractions using in vitro methods. This was done by assessing the extent and speciation of released metals in synthetic biological medium and by analyzing particle reactivity and toxicity towards cultured human lung cells (A549).ResultsThe amount of released metals normalized to the particle surface area increased with decreasing particle size for all alloy particles, whereas the opposite situation was valid for particles of the pure metals. These effects were evident in artificial lysosomal fluid (ALF) of pH 4.5 containing complexing agents, but not in neutral or weakly alkaline biological media. Chromium, iron and nickel were released to very low extent from all alloy particles, and from particles of Cr due to the presence of a Cr(III)-rich protective surface oxide. Released elements were neither proportional to the bulk nor to the surface composition after the investigated 168 hours of exposure. Due to a surface oxide with less protective properties, significantly more iron was released from pure iron particles compared with the alloys. Cr was predominantly released as Cr(III) from all particles investigated and was strongly complexed by organic species of ALF. Cr2O3 particles showed hemolytic activity, but none of the alloy particles did. Fine-sized particles of stainless steel caused however DNA damage, measured with the comet assay after 4 h exposure. None of the particles revealed any significant cytotoxicity in terms of cell death after 24 h exposure.ConclusionIt is evident that particle and alloy characteristics such as particle size and surface composition are important aspects to consider when assessing particle toxicity and metal release from alloy particles compared to pure metal particles. Generated results clearly elucidate that neither the low released concentrations of metals primarily as a result of protective and poorly soluble surface oxides, nor non-bioavailable chromium complexes, nor the particles themselves of occupational relevance induced significant acute toxic response, with exception of DNA damage from stainless steel.

Sequential Studies of Silver Released from Silver Nanoparticles in Aqueous Media Simulating Sweat, Laundry Detergent Solutions and Surface Water

From an increased use of silver nanoparticles (Ag NPs) as an antibacterial in consumer products follows a need to assess the environmental interaction and fate of their possible dispersion and release of silver. This study aims to elucidate an exposure scenario of the Ag NPs potentially released from, for example, impregnated clothing by assessing the release of silver and changes in particle properties in sequential contact with synthetic sweat, laundry detergent solutions, and freshwater, simulating a possible transport path through different aquatic media. The release of ionic silver is addressed from a water chemical perspective, compared with important particle and surface characteristics. Released amounts of silver in the sequential exposures were significantly lower, approximately a factor of 2, than the sum of each separate exposure. Particle characteristics such as speciation (both of Ag ionic species and at the Ag NP surface) influenced the release of soluble silver species present on the surface, thereby increasing the total silver release in the separate exposures compared with sequential immersions. The particle stability had no drastic impact on the silver release as most of the Ag NPs were unstable in solution. The silver release was also influenced by a lower pH (increased release of silver), and cotransported zeolites (reduced silver in solution).

Adsorption and protein-induced metal release from chromium metal and stainless steel.

A research effort is undertaken to understand the mechanism of metal release from, e.g., inhaled metal particles or metal implants in the presence of proteins. The effect of protein adsorption on the metal release process from oxidized chromium metal surfaces and stainless steel surfaces was therefore examined by quartz crystal microbalance with energy dissipation monitoring (QCM-D) and graphite furnace atomic absorption spectroscopy (GFAAS). Differently charged and sized proteins, relevant for the inhalation and dermal exposure route were chosen including human and bovine serum albumin (HSA, BSA), mucin (BSM), and lysozyme (LYS). The results show that all proteins have high affinities for chromium and stainless steel (AISI 316) when deposited from solutions at pH 4 and at pH 7.4 where the protein adsorbed amount was very similar. Adsorption of albumin and mucin was substantially higher at pH 4 compared to pH 7.4 with approximately monolayer coverage at pH 7.4, whereas lysozyme adsorbed in multilayers at both investigated pH. The protein-surface interaction was strong since proteins were irreversibly adsorbed with respect to rinsing. Due to the passive nature of chromium and stainless steel (AISI 316) surfaces, very low metal release concentrations from the QCM metal surfaces in the presence of proteins were obtained on the time scale of the adsorption experiment. Therefore, metal release studies from massive metal sheets in contact with protein solutions were carried out in parallel. The presence of proteins increased the extent of metals released for chromium metal and stainless steel grades of different microstructure and alloy content, all with passive chromium(III)-rich surface oxides, such as QCM (AISI 316), ferritic (AISI 430), austentic (AISI 304, 316L), and duplex (LDX 2205).

Bioaccessibility studies of ferro-chromium alloy particles for a simulated inhalation scenario: A comparative study with the pure metals and stainless steel

The European product safety legislation, REACH, requires that companies that manufacture, import, or use chemicals demonstrate safe use and high level of protection of their products placed on the market from a human health and environmental perspective. This process involves detailed assessment of potential hazards for various toxicity endpoints induced by the use of chemicals with a minimum use of animal testing. Such an assessment requires thorough understanding of relevant exposure scenarios including material characteristics and intrinsic properties and how, for instance, physical and chemical properties change from the manufacturing phase, throughout use, to final disposal. Temporary or permanent adverse health effects induced by particles depend either on their shape or physical characteristics, and/or on chemical interactions with the particle surface upon human exposure. Potential adverse effects caused by the exposure of metal particles through the gastrointestinal system, the pulmonary system, or the skin, and their subsequent potential for particle dissolution and metal release in contact with biological media, show significant gaps of knowledge. In vitro bioaccessibility testing at conditions of relevance for different exposure scenarios, combined with the generation of a detailed understanding of intrinsic material properties and surface characteristics, are in this context a useful approach to address aspects of relevance for accurate risk and hazard assessment of chemicals, including metals and alloys and to avoid the use of in vivo testing. Alloys are essential engineering materials in all kinds of applications in society, but their potential adverse effects on human health and the environment are very seldom assessed. Alloys are treated in REACH as mixtures of their constituent elements, an approach highly inappropriate because intrinsic properties of alloys generally are totally different compared with their pure metal components. A large research effort was therefore conducted to generate quantitative bioaccessibility data for particles of ferro-chromium alloys compared with particles of the pure metals and stainless steel exposed at in vitro conditions in synthetic biological media of relevance for particle inhalation and ingestion. All results are presented combining bioaccessibility data with aspects of particle characteristics, surface composition, and barrier properties of surface oxides. Iron and chromium were the main elements released from ferro-chromium alloys upon exposure in synthetic biological media. Both elements revealed time-dependent release processes. One week exposures resulted in very small released particle fractions being less than 0.3% of the particle mass at acidic conditions and less than 0.001% in near pH-neutral media. The extent of Fe released from ferro-chromium alloy particles was significantly lower compared with particles of pure Fe, whereas Cr was released to a very low and similar extent as from particles of pure Cr and stainless steel. Low release rates are a result of a surface oxide with passive properties predominantly composed of chromium(III)-rich oxides and silica and, to a lesser extent, of iron(II,III)oxides. Neither the relative bulk alloy composition nor the surface composition can be used to predict or assess the extent of metals released in different synthetic biological media. Ferro-chromium alloys cannot be assessed from the behavior of their pure metal constituents.

Metal release and speciation of released chromium from a biomedical CoCrMo alloy into simulated physiologically relevant solutions

The objective of this study was to investigate the extent of released Co, Cr(III), Cr(VI), and Mo from a biomedical high-carbon CoCrMo alloy exposed in phosphate-buffered saline (PBS), without and with the addition of 10 µM H2 O2 (PBS + H2 O2 ), and 10 g L(-1) bovine serum albumin (PBS + BSA) for time periods up to 28 days. Comparative studies were made on AISI 316L for the longest time period. No Cr(VI) release was observed for any of the alloys in either PBS or PBS + H2 O2 at open-circuit potential (no applied potential). However, at applied potentials (0.7 V vs. Ag/AgCl), Cr was primarily released as Cr(VI). Co was preferentially released from the CoCrMo alloy at no applied potential. As a consequence, Cr was enriched in the utmost surface oxide reducing the extent of metal release over time. This passivation effect was accelerated in PBS + H2 O2 . As previously reported for 316L, BSA may also enhance metal release from CoCrMo. However, this was not possible to verify due to the precipitation of metal-protein complexes with reduced metal concentrations in solution as a consequence. This was particularly important for Co-BSA complexes after sufficient time and resulted in an underestimation of metals in solution.

Particles, sweat, and tears: A comparative study on bioaccessibility of ferrochromium alloy and stainless steel particles, the pure metals and their metal oxides, in simulated skin and eye contact

Ferrochromium alloys are manufactured in large quantities and placed on the global market for use as master alloys (secondary raw materials), primarily for stainless steel production. Any potential human exposure to ferrochromium alloy particles is related to occupational activities during production and use, with 2 main exposure routes, dermal contact and inhalation and subsequent digestion. Alloy and reference particles exposed in vitro in synthetic biological fluids relevant for these main exposure routes have been investigated in a large research effort combining bioaccessibility; chemical speciation; and material, surface, and particle characteristics. In this paper, data for the dermal exposure route, including skin and eye contact, will be presented and discussed. Bioaccessibility data have been generated for particles of a ferrochromium alloy, stainless steel grade AISI 316L, pure Fe, pure Cr, iron(II,III)oxide, and chromium(III)oxide, upon immersion in artificial sweat (pH 6.5) and artificial tear (pH 8.0) fluids for various time periods. Measured released amounts of Fe, Cr, and Ni are presented in terms of average Fe and Cr release rates and amounts released per amount of particles loaded. The results are discussed in relation to bulk and surface composition of the particles. Additional information, essential to assess the bioavailability of Cr released, was generated by determining its chemical speciation and by providing information on its complexation and oxidation states in both media investigated. The effect of differences in experimental temperature, 30 degrees C and 37 degrees C, on the extent of metal release in artificial sweat is demonstrated. Iron was the preferentially released element in all test media and for all time periods and iron-containing particles investigated. The extent of metal release was highly pH dependent and was also dependent on the medium composition. Released amounts of Cr and Fe were very low (close to the limit of detection, <0.008% of particles released or dissolved as iron or chromium) for the alloy particles (ferrochromium alloy and stainless steel), the pure Cr particles, and the metal oxide particles. The released fraction of Cr (Cr/[Cr + Fe]) varied with the material investigated, the test medium, and the exposure time and cannot be predicted from either the bulk or the surface composition. Chromium was released as noncomplexed Cr(III) and in addition in very low concentrations (<3 microg/L). Nickel released was under the limit of detection (0.5 microg/L), except for ultrafine stainless steel particles (<10 microg/L). It is evident that media chemistry and material properties from a bulk and surface perspective, as well as other particle characteristics, and the chemical speciation of released metals have to be considered when assessing any potential hazard or risk induced by sparingly soluble metal or alloy particles.