C60 oxide as a key component of aqueous C60 colloidal suspensions.

Stable aqueous fullerene colloidal suspensions (nC(60)) are demonstrated to rely on the [6,6]-closed epoxide derivative of the fullerene (C(60)O) for stability. This derivative is present, though often unrecognized, in small quantities in nearly all C(60) starting materials due to a reaction with air. The low-yield formation of nC(60) from organic solvent solutions results from a preferential partitioning and thus enrichment of C(60)O in the colloidal particles. This partitioning is significantly retarded in the nC(60) synthesis method that does not involve organic solvent solutions: long-term stirring in water. Instead, this method relies on trace levels of ozone in the ambient atmosphere to produce sufficient C(60)O at the surfaces of the nC(60) particles to allow stable suspension in water. Controlled-atmosphere syntheses, deliberate C(60)O enrichment, light scattering measurements, and extraction followed by HPLC analysis and UV-visible absorption spectroscopy support the above model of nC(60) formation and stabilization.

Correlating nanoscale titania structure with toxicity: a cytotoxicity and inflammatory response study with human dermal fibroblasts and human lung epithelial cells.

Nanocrystalline titanium dioxide (nano-TiO(2)) is an important material used in commerce today. When designed appropriately it can generate reactive species (RS) quite efficiently, particularly under ultraviolet (UV) illumination; this feature is exploited in applications ranging from self-cleaning glass to low-cost solar cells. In this study, we characterize the toxicity of this important class of nanomaterials under ambient (e.g., no significant light illumination) conditions in cell culture. Only at relatively high concentrations (100 microg/ml) of nanoscale titania did we observe cytotoxicity and inflammation; these cellular responses exhibited classic dose-response behavior, and the effects increased with time of exposure. The extent to which nanoscale titania affected cellular behavior was not dependent on sample surface area in this study; smaller nanoparticlulate materials had effects comparable to larger nanoparticle materials. What did correlate strongly to cytotoxicity, however, was the phase composition of the nanoscale titania. Anatase TiO(2), for example, was 100 times more toxic than an equivalent sample of rutile TiO(2). The most cytotoxic nanoparticle samples were also the most effective at generating reactive oxygen species; ex vivo RS species generation under UV illumination correlated well with the observed biological response. These data suggest that nano-TiO(2) samples optimized for RS production in photocatalysis are also more likely to generate damaging RS species in cell culture. The result highlights the important role that ex vivo measures of RS production can play in developing screens for cytotoxicity.

Functionalization density dependence of single-walled carbon nanotubes cytotoxicity in vitro.

The cytotoxic response of cells in culture is dependant on the degree of functionalization of the single-walled carbon nanotube (SWNT). After characterizing a set of water-dispersible SWNTs, we performed in vitro cytotoxicity screens on cultured human dermal fibroblasts (HDF). The SWNT samples used in this exposure include SWNT-phenyl-SO(3)H and SWNT-phenyl-SO(3)Na (six samples with carbon/-phenyl-SO(3)X ratios of 18, 41, and 80), SWNT-phenyl-(COOH)(2) (one sample with carbon/-phenyl-(COOH)(2) ratio of 23), and underivatized SWNT stabilized in 1% Pluronic F108. We have found that as the degree of sidewall functionalization increases, the SWNT sample becomes less cytotoxic. Further, sidewall functionalized SWNT samples are substantially less cytotoxic than surfactant stabilized SWNTs. Even though cell death did not exceed 50% for cells dosed with sidewall functionalized SWNTs, optical and atomic force microscopies show direct contact between cellular membranes and water-dispersible SWNTs; i.e. the SWNTs in aqueous suspension precipitate out and selectively deposit on the membrane.

Nano-C60 cytotoxicity is due to lipid peroxidation.

This study examines the biological effects of water-soluble fullerene aggregates in an effort to evaluate the fundamental mechanisms that contribute to the cytotoxicity of a classic engineered nanomaterial. For this work we used a water-soluble fullerene species, nano-C60, a fullerene aggregate that readily forms when pristine C60 is added to water. Nano-C60 was cytotoxic to human dermal fibroblasts, human liver carcinoma cells (HepG2), and neuronal human astrocytes at doses>or= 50 ppb (LC50=2-50 ppb, depending on cell type) after 48 h exposure. This water-soluble nano-C60 colloidal suspension disrupts normal cellular function through lipid peroxidation; reactive oxygen species are responsible for the membrane damage. Cellular viability was determined through live/dead staining and LDH release. DNA concentration and mitochondrial activity were not affected by the nano-C60 inoculations to cells in culture. The integrity of cellular membrane was examined by monitoring the peroxy-radicals on the lipid bilayer. Subsequently, glutathione production was measured to assess the cell's reaction to membrane oxidation. The damage to cell membranes was observed both with chemical assays, and confirmed physically by visualizing membrane permeability with high molecular weight dyes. With the addition of an antioxidant, L-ascorbic acid, the oxidative damage and resultant toxicity of nano-C60 was completely prevented.

Principles for characterizing the potential human health effects from exposure to nanomaterials: elements of a screening strategy.

The rapid proliferation of many different engineered nanomaterials (defined as materials designed and produced to have structural features with at least one dimension of 100 nanometers or less) presents a dilemma to regulators regarding hazard identification. The International Life Sciences Institute Research Foundation/Risk Science Institute convened an expert working group to develop a screening strategy for the hazard identification of engineered nanomaterials. The working group report presents the elements of a screening strategy rather than a detailed testing protocol. Based on an evaluation of the limited data currently available, the report presents a broad data gathering strategy applicable to this early stage in the development of a risk assessment process for nanomaterials. Oral, dermal, inhalation, and injection routes of exposure are included recognizing that, depending on use patterns, exposure to nanomaterials may occur by any of these routes. The three key elements of the toxicity screening strategy are: Physicochemical Characteristics, In Vitro Assays (cellular and non-cellular), and In Vivo Assays. There is a strong likelihood that biological activity of nanoparticles will depend on physicochemical parameters not routinely considered in toxicity screening studies. Physicochemical properties that may be important in understanding the toxic effects of test materials include particle size and size distribution, agglomeration state, shape, crystal structure, chemical composition, surface area, surface chemistry, surface charge, and porosity. In vitro techniques allow specific biological and mechanistic pathways to be isolated and tested under controlled conditions, in ways that are not feasible in in vivo tests. Tests are suggested for portal-of-entry toxicity for lungs, skin, and the mucosal membranes, and target organ toxicity for endothelium, blood, spleen, liver, nervous system, heart, and kidney. Non-cellular assessment of nanoparticle durability, protein interactions, complement activation, and pro-oxidant activity is also considered. Tier 1 in vivo assays are proposed for pulmonary, oral, skin and injection exposures, and Tier 2 evaluations for pulmonary exposures are also proposed. Tier 1 evaluations include markers of inflammation, oxidant stress, and cell proliferation in portal-of-entry and selected remote organs and tissues. Tier 2 evaluations for pulmonary exposures could include deposition, translocation, and toxicokinetics and biopersistence studies; effects of multiple exposures; potential effects on the reproductive system, placenta, and fetus; alternative animal models; and mechanistic studies.

Optimized solvent-exchange synthesis method for C60 colloidal dispersions.

An existing solvent exchange method used to produce aqueous suspensions of fullerene C(60) aggregates (nC(60)) using the solvents toluene, tetrahydrofuran, acetone, and water, has been optimized for producing 75 nm diameter particles. Numerous synthesis parameters were evaluated for their effects on colloid yield and particle size distribution. Varying the relative volumes used of the intermediate solvents relative to the initial toluene volume allowed the controlled tuning of the resulting particle size up to a diameter of 210 nm. The resulting suspensions produced 10-20 ppm concentrations and reduced the residual organic solvents to less than the detection limit of 1 ppm.