Grantee Research Project Results
2006 Progress Report: Structure-function Relationships in Engineered Nanomaterial Toxicity
EPA Grant Number: R832536Title: Structure-function Relationships in Engineered Nanomaterial Toxicity
Investigators: Colvin, Vicki L.
Institution: Rice University
EPA Project Officer: Hahn, Intaek
Project Period: December 1, 2005 through November 30, 2008
Project Period Covered by this Report: December 1, 2005 through November 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 objective of this research project is to establish correlations between nanoparticle structure and acute toxicity. Such information contributes to the overall sustainability of the emerging nano-material industry in that it will identify material modifications that may produce systems with minimal environmental and health impacts. Correspondingly, this information benefits regulators by not only indicating whether information on one nanoparticle type can be used to predict the properties of a related material, but also by setting a framework for evaluating newly developed nanoparticle variants. Finally, a correlation between biological effects and nanoparticle structure will enable the development of chemical methods to alter more toxic nanomaterial species into less toxic materials upon disposal. Towards these ends, specific aims include 1) the development of methods to examine nanoparticle structure (size/surface fouling) in solution media 2) the generation of well-controlled libraries of nanoparticles with varying structural features (e.g. size, surface functionality) and 3) the examination in relevant biological models of how these physio-chemical parameters influence biological properties relevant to acute toxicity as well as exposure. In project year one, the group was most active in developing material libraries as well as establishing structure-function relationships for carbon nanotubes and titania. For 2006, specific research outputs in the form of 6 peer reviewed publications and a number of conference proceedings, highlighting project progress, are attached in Appendix A.
Progress Summary:
Aim 1: Development of probes for the nanoparticle-biological interface in solution. In this first year we refined prior efforts to control and measure the association between nanoparticles and key biomolecules such as DNA. Calabretta et al. reports on the novel use of analytical ultracentrifugation among other techniques to evaluate how DNA interactions with repressor proteins (E. coli DNA binding protein lac repressor (LacI) and a derivative with a designed thiol (T334C)) change when gold nanoparticles are present. Both intentional and unintentional modifications of the biomolecules leads to very distinct responses in the protein activity. Specfically, results show that LacI does not retain significant DNA binding function when conjugated to gold nanocrystals, presumably because the basic DNA-binding domain is the site for nonspecific conjugation. T334C, with the potential for both directed and nonspecific conjugation, shows enhanced interaction with a DNA when conjugated. Interestingly, we found the order of component addition is a key factor in producing functional lac repressor conjugates.
Additionally, work was focused on the investigation of Polyethylene Glycol coatings (PEG), a common stabilizing agent for aqueous suspensions of nanoscale materials, and their capacities to passivate the surface of a three-dimensional nanoparticle (gold in this case) and the corresponding relationship with protein binding, structure and function. A 2-D matrix of several size gold nanoparticles and PEG chain lengths was constructed. It was hypothesized that PEG-nanoparticle complexes would be most effective when the PEG chains fully interacted, i.e. it was expected that shorter chain polymers might prove more effective because they could pack more effectively on highly curved surfaces. These hypotheses stem from the fact that a high degree of curvature at an interface leads to an inability to induce an intramolecular interaction; as a result, less dense mushroom like conformations could result even at maximum packing densities. Figure 1. shows sedimentation curves (Analytical ultracentrifugation as described in Calabretta et al.) for the particle size versus chain length and data is evaluated for effective coverage. In keeping with our expectations, the very smallest gold nanoparticle could not be passivated for any chain length; the 10-nm particle can be passivated by the 1000 mw and 2000 mw PEG chains, but protein association occurs at any length greater than 2000 mw (n45). 20-nm particles are passivated by PEG molecules up to 5000 mw (n113). For the 5-nm particles the chain lengths tested were not sufficiently short enough to be able to effectively cover the surface. Passivation can only be obtained on nanoparticle surfaces with the correct chain length to radius of curvature arrangements. To further prove that insufficient grafting density is not the culprit for protein association the PEG concentration on the 10-nm and 20-nm particles for PEGs 1000 mw and 2000 mw were lowered below saturation and then the particles were cleaned and interacted with protein. Each of the samples prepared below saturation grafting density showed protein association.
Figure 1. Sedimentation coefficient distributions results for radius of curvature versus PEG chain length. The gold 5nm, 10nm and 20nm were coated with PEG molecular weights: 1000 (n≈20), 2000 (n≈45), 5000 (n≈115), 10000 (n≈225), 20000 (n≈455), then allowed to interact with HSA. The black curves show sa distributions for PEGylated nanoparticle alone and the red curves show PEGylated nanoparticles in the presence of protein. Particles that interact with the protein show peak position shifts ≥5% and are presented in a red box otherwise, the protein is repelled from the surface and is presented in a green box. PEG coverage at the lower molecular weights showed great protein resistance on nanoparticle surfaces than higher molecular weight PEGs.
Aim 2: Development of nanomaterial libraries - Structure function relationships can only be discovered and delineated if nanoparticle libraries can be made with well-controlled, reproducible variations in size, phase composition and shape. Thus, an essential element of this work is to develop synthetic routes for these libraries especially for those systems in which this control is not already established.
Figure 2. TEM images of solvothermal anatase prepared at (a) 150 °C and (b) 300 °C.
In this first year, we completed and refined efforts to make nanoscale titania that were essential for follow-on toxicity studies. The paper by Wahi, Colvin et al. reports the hydrothermal synthesis of nanoscale titania. As shown in the figure X it is possible to engineer not just the size of the material, but also its phase composition. Phase-pure, ultrafine nanocrystalline anatase with high specific surface area (up to 250 m2 g−1) was obtained upon injection of a titanium alkoxide precursor into ethanol with designed volume of water under mild solvothermal conditions (<200 °C, 2 h) (Figure 2). Primary particle sizes were tuned by adjusting various reaction parameters, with the smallest grain sizes occurring at low temperatures (140–150 °C), low initial alkoxide concentrations, and intermediate hydrolysis ratios (r ≡[H2O]/[Ti(OR)4] = 5–10). Additionally, variations in the reaction temperature result in changes in particle morphology and distribution, with high-temperature samples exhibiting bimodal distributions of small spherical and larger cubic particles that suggest grain growth via Ostwald ripening. A crystalline product with high thermal stability and specific surface area up to 5 times that of commercial nano-titania can be obtained at a relatively low temperature of 150 °C. The physical properties of the titania samples obtained in this study suggest they might be well suited for catalytic applications.
For quantum dots, our focus was on specific surface engineering using block co-polymers. Surface functionality is thought to be a major factor in dictating quantum dot toxicity; however, systematic evaluation of the role of the surface has been challenging due to the lack of available controlled surface quantum dots. While commercial materials are available with varying surface charge; perhaps, more critical for this area is the role of the length of the poly(ethylene) glycol or PEG groups attached to prevent surface fouling by proteins. Using a novel ethylene diamine coupling scheme (EDC = N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride) we created amphiphilic PEG co-polymers with a hydrophobic tether and PEG block with variable length and branching character (Yu et al. 2006)
Aim 3: Develop structure-function relationships. High levels of material control(s) enables the detailed examination of how the size, shape, and phase-dependent properties of nanoparticles affect biological systems. A fundamental understanding of these relationships will permit nanoscale scientists and engineers to create environmentally friendly and biologically relevant nanomaterials. In addition, if chemical properties can be established as a strong predictor for toxicological behavior, then ex vivo tests become powerful initial screens for the design of low-toxicity nanomaterials. In these studies (Sayes et al. 2006 Tox. Sci.; Sayes et al.2006 Tox. Lett.), we exploit state-of-the-art methods in nanochemistry to generate a set of nanocrystalline titanium dioxide (nano-TiO2) samples of controlled phase composition and surface modified (derivatized) single wall carbon nanotube (SWNT) relying on in vitro cytotoxicity assays to compare their biological effects.
Nanocrystalline titanium dioxide (nano-TiO2) 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 class of nanomaterials under ambient (e.g., no significant light illumination) conditions in cell culture (Human Dermal Fibroblasts and Human Lung Epithelial Cells). Only at relatively high concentrations (100 mu g/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 TiO2, for example, was 100 times more toxic than an equivalent sample of rutile TiO2. 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-TiO2 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.
In the case of single wall carbon nanotubes, the cytotoxic response of human dermal cells in culture is dependant on the degree of functionalization of the single-walled carbon nanotube. 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-SO3H and SWNT-phenyl-SO3Na (six samples with carbon/-phenyl-SO3X 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 observed that as the degree of sidewall functionalization increases, the SWNT sample becomes less cytotoxic (Figure 3). 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.
Figure 3. Similarities in cellular activity of SWNT-phenyl-SO3H and SWNT-phenyl-SO3Na. The former is a precursor to the later. Both samples were tested because different SWNT applications requires different starting materials, i.e. SWNT-phenyl-SO3H or SWNTphenyl-SO3Na. Results are combined from three independent exposures. Groups significantly different from the control group (by ANOVA P < 0.403 followed by Dunnett’s test) are shown by (*P < 0.05) or (**P < 0.01).
Future Activities:
Future studies will be in line with proposed hypotheses testing, Specific inquires include the evaluation of how nanoparticle-protein interactions may affect the protein structure. For this purpose we will apply isothermal titration calorimetry (ITC) and circular dichroism (CD). To further develop an understanding of nanoparticle role in protein denaturation and fibrillation, 2m and RNase A proteins will be studied as they interact with various nanoparticle surfaces. Since the absorbance of gold interferes with wavelengths monitored in CD silica particles similar in size to the current gold between 5 nm and 20 nm will be used. The silica nanoparticles will be allowed to associate with the protein while the percent alpha-helix and the percent beta-sheet are monitored for changes. From this study we will be able to determine the effects of nanoparticle dimensions on protein structure and if proteins are able associate to PEG. Furthermore, the work proposed here could lead to valuable information about protecting against some of the possible risks nanomaterials pose in biological systems.
Journal Articles on this Report : 6 Displayed | Download in RIS Format
| Other project views: | All 31 publications | 12 publications in selected types | All 12 journal articles |
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Calabretta MK, Matthews KS, Colvin VL. DNA binding to protein-gold nanocrystal conjugates. Bioconjugate Chemistry 2006;17(5):1156-1161. |
R832536 (2006) R832536 (2007) |
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Sayes CM, Wahi R, Kurian PA, Liu Y, West JL, Ausman KD, Warheit DB, Colvin VL. Correlating nanoscale titania structure with toxicity: a cytotoxicity and inflammatory response study with human dermal fibroblasts and human lung epithelial cells. Toxicological Sciences 2006;92(1):174-185. |
R832536 (2006) R832536 (2007) |
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Sayes CM, Liang F, Hudson JL, Mendez J, Guo W, Beach JM, Moore VC, Doyle CD, West JL, Billups WE, Ausman KD, Colvin VL. Functionalization density dependence of single-walled carbon nanotubes cytotoxicity in vitro. Toxicology Letters 2006;161(2):135-142. |
R832536 (2006) R832536 (2007) |
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Wahi RK, Liu Y, Falkner JC, Colvin VL. Solvothermal synthesis and characterization of anatase TiO2 nanocrystals with ultrahigh surface area. Journal of Colloid and Interface Science 2006;302(2):530-536. |
R832536 (2006) R832536 (2007) |
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Warheit DB, Webb TR, Sayes CM, Colvin VL, Reed KL. Pulmonary instillation studies with nanoscale TiO2 rods and dots in rats: toxicity is not dependent upon particle size and surface area. Toxicological Sciences 2006;91(1):227-236. |
R832536 (2006) R832536 (2007) |
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Yu WW, Chang E, Drezek R, Colvin VL. Water-soluble quantum dots for biomedical applications. Biochemical and Biophysical Research Communications 2006;348(3):781-786. |
R832536 (2006) R832536 (2007) |
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Supplemental Keywords:
Nanotechnology, environmental impact, bioconjugation, nanoscience, structure-function relationship,, Health, Scientific Discipline, ENVIRONMENTAL MANAGEMENT, Health Risk Assessment, Risk Assessments, Biochemistry, Risk Assessment, structure function relationship, bioaccumulation, fate and transport, nanochemistry, toxicology, human health risk, biochemical researchProgress 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.