Grantee Research Project Results
2006 Progress Report: Assessment of the Environmental Impacts of Nanotechnology on Organisms and Ecosystems
EPA Grant Number: R832635Title: Assessment of the Environmental Impacts of Nanotechnology on Organisms and Ecosystems
Investigators: Bonzongo, Jean-Claude J. , Kopelevich, Dmity , Bitton, Gabriel
Institution: University of Florida
EPA Project Officer: Aja, Hayley
Project Period: October 1, 2005 through September 30, 2008
Project Period Covered by this Report: October 1, 2005 through September 30, 2006
Project Amount: $375,000
RFA: Exploratory Research: Nanotechnology Research Grants Investigating Environmental and Human Health Effects of Manufactured Nanomaterials: A Joint Research Solicitation - EPA, NSF, NIOSH (2005) RFA Text | Recipients Lists
Research Category: Nanotechnology , Safer Chemicals
Objective:
The goal of this project is to develop an understanding of the potentially complex interplay between manufactured nanomaterials (MNs) and the health of organisms and ecosystems. The driving research hypothesis is that chemical elements used in the production of MNs could lead to environmental dysfunctions due to: (1) the potential toxicity of these elements and their derivatives; (2) the nanometer size that make MNs prone to bio-uptake/bioaccumulation; and (3) the large surface area which might lead MNs to act as carriers/deliverers of pollutants adsorbed onto them. To address this broad hypothesis, toxicity studies using microbiotests in combination with experimental investigations at the system level are used. These experimental studies are complemented by molecular dynamic (MD) simulations to investigate events on very small time- and length-scales and to assess the contributions of different types of intermolecular interactions (e.g., electrostatic, van der Waals, hydrogen bonding, etc.), as well as chemical reactions to the transport and toxicity of nanomaterials at the cell level.
Progress Summary:
Laboratory studies conducted during year 1 of the project have focused primarily on: (1) adapting the selected microbiotests for use in laboratory studies of the toxicity of MNs; and (2) the use of these fine-tuned toxicity tests as screening tools for the identification of toxic MNs that will be used later in experiments assessing the impacts of MNs on ecosystem functions. In parallel, the modeling component has focused on the study of mechanisms of permeation of carbonaceous nanomaterials, namely C60, into the cell interior and the assessment of the possibility of this specific MN to cause lethal damage to cell membranes. Our preliminary findings and their significance to this growing research field, as well as their relationship to the goals of the project and their relevance to the environment and living organisms, are summarized below.
Toxicity and Environmental Fate of Nanomaterials
Toxicity Screening Using Microbiotests. In this first year of the project, our research program has focused primarily on fine-tuning the three preselected microbiotests to improve their use and efficiency in testing the toxicity of different types of nanomaterials. The Ceriodaphnia dubia (for determination of the acute toxicity of nanomaterials [U.S. EPA, 1994]), MetPLATE (which uses a mutant strain of E. coli to selectively sense the toxicity of metals [Bitton and Koopman, 1992; Bitton, et al., 1994]), and the Selenastrum capricornutum or Pseudokirchneriella subcapitata (a chronic toxicity test used in standard 96-hour chronic algal assays [Lewis, et al., 1994]) were used. While the first two mentioned tests have been easy to adapt and are now up and running, the algal test took longer, and preliminary assays with this latter test are now ongoing. Accordingly, presented preliminary data will be limited to those obtained using the C. dubia and MetPLATE tests. Also, pending the confirmation of the toxicity or non-toxicity of tested MN by the three methods, we opt in this first report to present the toxicity data as simple trends instead of listing the actual determined EC50 or LC50. This is because we intend to repeat several of these tests due to a few inconclusive test results, and there is potential for some of the numbers we currently have to change. The initial toxicity experiments have been limited so far to the assessment of the toxicity of metal oxide nanoparticles (e.g., TiO2, Al2O3, Fe2O3, SiO2-TiO2), a few metal nanoparticles (Ag, Cu, and Ni), and carbonaceous nanomaterials (i.e., C60, single-walled carbon nanotubes [SWCNTs]). Obtained results are summarized in Table 1.
Table 1. Toxicity of Tested MNs Using Microbiotests. Observed toxic effects are shown with a positive sign (+), while the lack of toxicity is represented by a negative sign (-). The corresponding EC50 and LC50 are not given for reasons stated in the text. nd = not determined
Chemical composition |
Ceriodaphnia dubia |
MetPLATE |
Pseudokirchneriella subcapitata |
|
Metal oxides |
TiO2 |
- |
- |
nd |
TiO2-SiO2 |
+ |
- |
nd |
|
Fe0 |
- |
- |
nd |
|
Al/Fe-nanocomposite |
- |
- |
nd |
|
SiO2 |
- |
- |
nd |
|
Metal nanoparticles |
Ag |
+ |
+ |
nd |
Cu |
+ |
+ |
nd |
|
Ni |
- |
- |
nd |
|
Carbonaceous nanomaterials |
C60 |
+ |
- |
nd |
SWCNTs |
+ |
- |
nd |
Overall, tested metal oxide nanoparticles do not exhibit toxicity (even at concentrations exceeding 100 ppm) based on the first two microbiotests, except for the nanocomposite TiO2-SiO2 which resulted in rather acidic solutions (pH ~ 4 when dissolved in Nanopure water). The negative response of MetPLATE suggests that metals forming these oxides are not bioavailable or are not toxic at all. For tested metal nanoparticles, Ag and Cu exhibited toxicity, while Ni did not show toxicity, even at a concentration of > 100 mg/L. It is worth noting that the algal test has not been used to test the toxicity of the above nano-oxide particles yet. These experiments are ongoing and will be available in the year 2 report. Unlike the metal-based nanomaterials, the carbon-based ones showed a lethal effect when the C. dubia test was used, and no toxicity was detected with the same nanomaterials when using MetPLATE. This result confirms the non-sensitivity of MetPLATE to organic compounds.
Environmental Fate. Experiments testing the bioaccumulation and the environmental fate of MN have been initiated and results are anticipated in year 2 of the project.
Molecular Dynamic (MD) Simulations
In year 1 of the project, much effort was devoted to the computational study, with a limited focus on carbonaceous nanomaterials. Obtained results are summarized under two different task categories and presented below.
Task 1. In the current computational study, the cellular membrane is modeled as a lipid bilayer, and the presence of membrane proteins is neglected. Therefore, the main focus of this work is on the direct diffusion of nanoparticles through the cellular membranes, and the protein-mediated transport of nanoparticles into cells is neglected. Recent experiments by Rothen-Rutishauser, et al. (2006) have shown that MNs can permeate some cells (such as red blood cells) by a currently unknown mechanism, different from phagocytosis and endocytosis. It is expected that the direct diffusion mechanism investigated in this work is dominant in these systems, and therefore, the current study is expected to be relevant to a significant number of nanoparticle-cell membrane systems.
In this work, the coarse-grained molecular model proposed by Marrink, et al. (2004) is employed. This model maps groups of several atoms onto a single “united atom” and is known to increase the computational efficiency by several orders of magnitude while yielding good agreement with more detailed models as well as with experimental data for several systems, including lipid membranes. The simulations are performed using the GROMACS molecular dynamics package (http://www.gromacs.org Exit ).
The transport of hydrophobic nanoparticles of spherical and nearly spherical shapes through the cellular membrane was investigated. The parameters of these nanoparticles were chosen to mimic fullerene properties. Since the transport timescales can be quite large (see Figure 1) and are not directly accessible to MD simulations, an indirect simulation technique of constrained simulations (Marrink and Berendsen, 1994) is applied to obtain the free energy of the system as a function of the particle position within the bilayer and therefore to predict the timescales of the MN transport. The obtained free energy profiles for spherical nanoparticles with radii ranging from σ = 0.47 nm to σ = 1.1 nm, as well as for a non-spherical (tetrahedron-shaped) nanoparticle, are shown in Figure 2. The Ζ coordinate in this plot represents the direction perpendicular to the membrane surface; the bilayer width is approximately 4-6 nm. Therefore, the regions with |Ζ| > 4 nm correspond to bulk water.
Figure 1. Mean Time of MN Transport (τtm) Through and Escape (τesc) From the Membrane
Figure 2. Free Energy Profiles for Model Nanoparticles Inside a Lipid Membrane
The obtained profiles show several important features that are expected to play an important role in MN toxicity (Figure 1):
- There is a very small (~ 1 κBT) energy barrier to enter the membrane, even for relatively large MNs.
- There is a significant barrier for an MN to leave the membrane. This barrier increases as the MN size increases. Therefore, an MN can easily enter the membrane but will spend a significant amount of time inside the bilayer before leaving it. It is expected that the interactions between the nanoparticle and the surrounding lipids will lead to membrane instability and possibly to leakage.
- The general features of the free energy profile obtained for the non-spherical particle are the same. Specifically, there is a very small energy barrier to enter the membrane and a very large barrier to leave the membrane. However, there is a significant qualitative difference in the free energy profile inside the membrane: for the spherical particles, the minima of the free energy (and hence, the preferred location of the particle) are located away from the membrane center, whereas for the non-spherical MN, the minimum of the free energy is directly at the membrane center. This implies that the MNs will be localized in different parts of the membrane depending on their shape and therefore will act differently on different groups of the lipid molecules. This in turn may lead to different nanoparticle toxicity.
In order to make the above observations more quantitative, the transport time (τtm) of MNs across the membrane and the escape time (τesc) of MNs from the center of the bilayer to the bulk aqueous medium were computed using the escape rate theory (Gardiner, 1983). The results of these calculations for the spherical particles of various radii are shown in Figure 2. There is essentially no difference between the transport time across the entire membrane and the escape time from the membrane center, which implies that the average time of entrance into the membrane is very small as suggested by the small free energy barrier for the particle entry. However, the MNs of relatively large radii reside inside the membrane for a very long time. Therefore, the diffusion transport mechanism across the cell membrane is expected to be insignificant for a hydrophobic nanoparticle with a radius > 1 nm. However, such a particle can easily diffuse into the cell membrane interior. During its long residence time within the membrane, the MN may significantly disrupt the integrity of the membrane, either through chemical reactions, or through inducing a local instability of a lipid bilayer by physical effects such as a lipid phase transition in the MN neighborhood.
Task 2. The aim of Task 2 of this project is to investigate effects of MN-membrane interaction on the membrane stability. Here, results of a preliminary study of effects of lipid peroxidation caused by MNs are reported. Recent experiments by Sayes, et al. (2004, 2005) demonstrated that fullerenes lead to lipid peroxidation that, in turn, leads to leaky membranes that cause cell death. The results described below demonstrate the capability of MD simulations to predict membrane instability caused by lipid peroxidation. The details of this process will be investigated in the planned future study.
In order to perform an initial assessment of the effects of lipid peroxidation on membrane stability, the following simple model for the peroxidation reaction was implemented: one of the hydrophobic groups of the lipid tails is replaced by a hydrophilic (oxide) group. A fraction of lipids within the membrane are selected at random to undergo this reaction. MD simulations of such a model of a peroxidized membrane show that when the concentration of the reacted lipids is extremely high (50% and 100%), the membrane becomes very unstable and is essentially destroyed within 100 ns. However, for more realistic lower concentrations of reacted lipids (18% and 38%), it is observed that the membrane remains intact on the timescale of 300 ns. Nevertheless, significant structural changes within the membrane are observed. Specifically, the peroxidized lipids tend to fold inside the membrane so that their tails point to the exterior of the membrane, rather than to the membrane interior. It is observed that this folding takes place very quickly (within 100 ns) after the reaction has taken place and is very likely to lead to membrane instability. In fact, although the membrane remains intact on the scale of MD simulations, it is observed visually that it becomes softer, since the amplitude of its fluctuations increases with increased concentration of reacted lipids. A more detailed quantification of this result, by computing the membrane bending modulus, is currently in progress. It is expected that this analysis will yield a limit for the fraction of reacted peroxidized lipids that does not lead to membrane instability (and hence safe exposure to MN).
Future Activities:
Microbiotests and Toxicity of Nanomaterials
In year 2 of the project, we will use the now well-established microbiotests to assess the toxicity of: (1) metal nanoparticles (e.g., Ag, Cu, Ni, Au, etc.); (2) quantum dots (e.g., Cd/Se); and (3) single and multi WCNTs. In this second year, the emphasis will also be on the fate and environmental impacts of toxic nanomaterials as determined from sediments and river/lake water studies. We will use biogeochemical approaches to investigate the impacts of these nanoparticles on ecological functions. Studies on the mobility of these nanomaterials in sedimentary environments are scheduled for year 3.
Molecular Dynamic Simulations
- We will investigate effects of particle shape and other physical properties on its transport across the cellular membrane and preferred localization within the membrane. The preliminary data show that relatively small deviations from the spherical shape of MNs lead to significant changes in the preferred MN location within the membrane. These effects will be investigated further. In particular, rod-shaped hydrophobic MNs mimicking carbon nanotubes will be investigated in detail. Moreover, effects of electrostatic charges of MNs on their transport through the membrane will be investigated. It is possible that there exists a cooperative effect between charged lipid headgroups and MNs that facilitates the MN transport.
- A more realistic membrane model based on an inhomogeneous mixture of different lipids (instead of a single lipid) will be considered. It is possible that MNs have preferred interactions with certain types of lipids (depending on their headgroup structures and tail lengths) that may lead to transport properties different from those observed in the preliminary data reported above, which were obtained for a homogeneous dipalmitoylphosphatidylcholine (DPPC) lipid bilayer.
- Obtain critical levels of lipid peroxidation that lead to membrane instability.
- Assess possible physical mechanisms of membrane instability due to its interaction with MNs. For example, the presence of MNs inside a membrane can lead to local phase transition to a non-bilayer phase in the area surrounding the MNs.
- Estimate limits of safe exposure of membranes to MNs, i.e., the conditions under which the membrane remains stable.
References:
Bitton G, Koopman B. Bacterial and enzymatic bioassays for toxicity testing in the environment. Review of Environmental Contamination and Toxicology 1992;125:1-22.
Bitton G, Jung K, Koopman B. Evaluation of a microplate assay specific for heavy metal toxicity. Archives of Environmental Contamination and Toxicology 1994;27:25-28.
Gardiner GW. Handbook of Stochastic Methods for Physics, Chemistry, and the Natural Sciences. Berlin: Springer-Verlag, 1983.
Marrink SJ, Berendsen HJC. Simulation of water transport through a lipid membrane. Journal of Chemical Physics 1994;98:4155.
Marrink SJ, de Vries AH, Mark AE. Coarse grained model for semi-quantitative lipid simulations. Journal of Physical Chemistry B 2004;108:750-760.
Rothen-Rutishauser BM, Schurch S, Haenni B, Kapp N, Gehr P. Interaction of fine particles with red blood cells visualized with advanced microscopic techniques. Environmental Science & Technology 2006;40:4353-4359.
Sayes CM, Fortner JD, Guo W, Lyon D, Boyd AM, Ausman KD, Tao YJ, Sitharaman B, Wilson LJ, Hughes JB, West JL, Colvin VL. The differential cytotoxicity of water-soluble fullerenes. Nano Letters 2004;4:1881-1887.
Sayes CM, Gobin AM, Ausman KD, Mendez J, West JL, Colvin VL. Nano-C60 cytotoxicity is due to lipid peroxidation. Biomaterials 2005;26:7587-7595.
U.S. EPA. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms. 3rd Edition, EPA/600/4-91/002, Cincinnati, OH.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 14 publications | 5 publications in selected types | All 4 journal articles |
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Type | Citation | ||
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Tasseff RA, Kopelevich DI. Molecular modeling of nanoparticle transport across lipid bilayers. University of Florida. Journal of Undergraduate Research 2006;7(4). |
R832635 (2006) R832635 (2007) R832635 (Final) |
not available |
Supplemental Keywords:
nanomaterials, toxicity, microbiotests, environmental fate and transport, molecular dynamic simulations,, Health, Scientific Discipline, Health Risk Assessment, Risk Assessments, Biochemistry, biological pathways, nanochemistry, bioavailability, nanotechnology, manufactured nanomaterials, nanomaterials, toxicologic assessment, biogeochemistry, cellular response to nanoparticles, nanoparticle toxicity, bioaccumulation, biochemical researchRelevant Websites:
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.