Grantee Research Project Results
2007 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, 2006 through November 30, 2007
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: Safer Chemicals , Nanotechnology
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 this second year the group supported collaborations to expand studies of their libraries and extended structure-function relationships into evaluations of quantum dots. For 2007, specific research outputs in the form of 6 peer reviewed publications and a number of conference presentations, highlighting project progress, are attached as Appendix A.
Progress Summary:
Aim 1: Development of probes for the nanoparticle-biological interface in solution. For many emerging applications, nanocrystals are surface functionalized with polymers to control self-assembly, prevent aggregation, and promote incorporation into polymer matrices and biological systems. The hydrodynamic diameter of these nanoparticle–polymer complexes is a critical factor for many applications, and predicting this size is complicated by the fact that the structure of many grafted polymer at the nanocrystalline interface is not generally established. To address these issues we have evaluated the use size-exclusion chromatography (SEC) to determine the overall hydrodynamic diameter of model nanocrystals (Au, CdSe, d < 5 nm) surface coated with polystyrene of varying molecular weight. Not only is the surface curvature very high for these small nanocrystals, but for most molecular weights studied the polymer is approximately the same size as the nanocrystal. Such a situation is increasingly common in nanoscience where the most striking size dependent changes often occur inmaterials with extremely small diameters. These data show that polystyrene on gold nanocrystals adopts a brush conformation with a length that, at saturation coverage, is 45±6% longer than that of the unbound random polystyrene coil. The length of the bound polymer scales linearly withmolecular weight following the predictions of scaling and mean-field theory for polymer brushes on flat surfaces. Polystyrene on CdSe nanocrystals is also evaluated and found to extend 42 ± 11% over the unbound random coil. This shows the generality of polymer brush formation on nanoparticles and permits the hydrodynamic diameter of polymer coated nanocrystals to be estimated from HD = Dc + 2(1.44DRC), where DRC is the diameter of the unbound random polymer coil in solution and Dc is the diameter of the core nanocrystal (Kreuger et al. 2007).
Figure 1. Nanoparticle coating thickness is determined using a geometric model. (a) The hydrodynamic diameter (HD) is calculated from core diameter (Dc) and surface-coating thickness (Tshell) (HD = Dc + 2(Tshell)). (b) SEC detects the difference in capping agent length between CdSe nanoparticles coated with 1-hexane, 1-dodecane, and 1-octadecane thiol. (c) The hydrodynamic diameter for these coated nanoparticles determined from SEC is compared to expected values for 3.6 nm CdSe core plus literature values for the shell thickness. The line slope is set to 1. (Kreuger et al. 2007).
Aim 2: Continued development of nanoscale material libraries. To fully explore the structure and optimize amphiphilic polymers for nanocrystal stabilization requires simple methods to generate amphiphilic polymers through coupling of single component hydrophobic and hydrophilic chains (Yu et al., 2007). Of particular interest for biocompatibility is the incorporation of poly(ethylene glycol) (PEG) into the surface coatings. Nanocrystals that have PEG- functionalized exteriors exhibit generally much less toxicity and longer circulation time than water-soluble nanocrystals made with other agents. The few schemes for producing such materials as part of amphiphilic polymers require the use of derivatized PEG polymers such as mPEG-NH2 (primary amino group terminated poly(ethylene glycol) methyl ether) or PEG-SH. These starting products are usually expensive and available in only a few molecular weights and with limited additional functionality for coupling (e.g., -COOH moieties). To gain flexibility and economy, we developed a general method to form stable nanocrystals in water using amphiphilic polymers generated through simple and low cost protocols. Our amphiphilic coating agents are formed using a maleic anhydride coupling scheme that works both with mPEG-NH2 and underivatized mPEG-OH (poly-(ethylene glycol) methyl ether) as starting materials. Because these materials are available with a wide variety of chain lengths, we can compare how PEG size influences physiological stability as well as biocompatibility. The method is quite general, and we demonstrate the technique using our model system of quantum dots as well as nanoscale Fe3O4. Furthermore, the as-prepared water-soluble nanocrystals were very stable and preserved the same properties as the ones in organic solvents, such as the same absorption, the same photoluminescence, and the same quantum yield for QDs.
Figure 2. Top: one-step formation of PMAO-PEG amphiphilic polymers through reaction between maleic anhydride and amino groups. Bottom: schematic structure of water-soluble quantum dots (F stands for a functional group instead of -OCH3, such as -OH, -COOH, -NH2, etc.). Quantum dot particles were encapsulated by PMAO-PEG amphiphilic polymer hydrophobic interaction. Hydrophilic side chains of PEGs stayed exteriorly to make the whole structure soluble in water; carboxylic and F functional groups were used for bioconjugation.
Such amphiphilic polymer coatings have expanded the versatility of conjugation schemes, and we have recently reported methods to conjugate various biomolecules to make protease-activated quantum dot probes. Additionally, we characterized the molar concentration and hydrodynamic size of our solutions using multiple tools and also show that the PEG coatings produce materials that are nontoxic in cell culture (Watersoluble QDs exposed to Human Breast Cells SK-BR-3 at various concentrations and monitored using a commercial LIVE/DEAD stain assay (Invitrogen) at different exposure times (up to 48 h)). Our efforts on nanoparticle libraries have supported other EPA funded groups (e.g. Alvarez (Rice); Montiere-Rivere (NC-State) who needed controlled materials for their efforts.
Aim 3: Continued development of material structure-(biological) function relationships.
Knowledge of the disposition and pharmacokinetics of nanomaterials in tissues is important for both developing nanotechnology-based drug delivery systems as well as conducting realistic risk assessments. There is minimal literature on the pharmacokinetics of nanomaterials, with that available being focused on therapeutic applications. The few reported studies have employed indirect measures of concentration (IR, PET) or particles functionalized with specific tracers. Literature exists on nanomaterial deposition after inhalational exposure, primarily on local deposition within the respiratory tract. Knowledge of how absorbed particles distribute in other tissues is lacking. Quantum Dots are semiconductor nanocrystals whose properties make them good candidates for application ranging from diagnostic imaging to solar cells. QD are heterogeneous nanoparticles that consist of a colloidal core surrounded by one or more surface coatings. The surface coating can prevent agglomeration, encapsulate toxic metals, affect absorption and transport, modulate immunological responses, modify or prevent toxicity, and assist in tissue elimination. Coatings are frequently applied to customize QD for specific applications. QD are readily detected because of their intense and photostable fluorescence, making them a useful model for assessing nanomaterial interactions with biological systems. In 2007 our work focused on studies assessing the biodistribution of model QD systems with Porcine skin, which is widely used for skin absorption and biodistribution studies because it is anatomically, physiologically, and biochemically similar to human skin. For these studies CD materials with two surface stabilizing were synthesized, one with amphiphilic PEG the other with terminal carboxylic acid moeties, rendering both systems stable in water. Arterial extraction of quantum dots (QD) assayed by fluorescence or inductively coupled plasma (ICP) emission spectrometry were studied after infusion into isolated perfused skin (Figure 3). Extraction was mathematically modeled using three linear differential equations. COOH-coated QD had greater tissue deposition, assessed both by model prediction and laser confocal scanning microscopy, than did QD-PEG. Both QD had a unique periodicity in arterial extraction never observed with drug infusions, suggesting a potentially important nanomaterial behavior that could affect systemic disposition (Lee et al. 2007).
Figure 3. Laser scanning confocal microscopy: (a) confocal fluorescence image of QD deposition, (b) differential interference contrast (DIC) overlay depicting localization of QD in the capillary in the skin, (c) light micrograph of same area stained with hematoxylin and eosin for anatomical orientation. QD-PEG and QD-COOH infused in the IPPSF at a concentration of 3.33 nM. Arrows denote QD. Bars equal 100μm.
Future Activities:
In our final year, we will continue to write review papers detailing the structure function relationships we have found for carbon nanoparticles, Nanoscale titania and quantum dots. These papers, which have been outlined in talks to date, provide the first examples of trends that could be captured by more quantitative and expanded structure-function studies. We will add to our collection of nanoparticles systematic studies of Nanoscale iron oxides. These systems are interesting because their oxidation states can be tuned (varying levels of Fe(II)/Fe(III) and we will explore this impact on their reactivity through Fenton chemistry in biological systems. We will return in all of our materials to issues of purity, particularly in the carbon systems and consider the role of small organic molecules which are present in most samples.
Journal Articles on this Report : 12 Displayed | Download in RIS Format
Other project views: | All 23 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 (2007) |
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Colvin VL, Kulinowski KM. Nanoparticles as catalysts for protein fibrillation. Proceedings of the National Academy of Sciences of the United States of America 2007;104(21):8679-8680. |
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Gopee NV, Roberts DW, Webb P, Cozart CR, Siitonen PH, Warbritton AR, Yu WW, Colvin VL, Walker NJ, Howard PC. Migration of intradermally injected quantum dots to sentinel organs in mice. Toxicological Sciences 2007;98(1):249-257. |
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Krueger KM, Al-Somali AM, Mejia M, Colvin VL. The hydrodynamic size of polymer stabilized nanocrystals. Nanotechnology 2007;18:475709. |
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Lee HA, Imran M, Monteiro-Riviere NA, Colvin VL, Yu WW, Riviere JE. Biodistribution of quantum dot nanoparticles in perfused skin: evidence of coating dependency and periodicity in arterial extraction. Nano Letters 2007;7(9):2865-2870. |
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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. |
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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. |
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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. |
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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. |
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Warheit DB, Webb TR, Colvin VL, Reed KL, Sayes CM. Pulmonary bioassay studies with nanoscale and fine-quartz particles in rats: toxicity is not dependent upon particle size but on surface characteristics. Toxicological Sciences 2007;95(1):270-280. |
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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. |
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Yu WW, Chang E, Falkner JC, Zhang J, Al-Somali AM, Sayes CM, Johns J, Drezek R, Colvin VL. Forming biocompatible and nonaggregated nanocrystals in water using amphiphilic polymers. Journal of the American Chemical Society 2007;129(10):2871-2879. |
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, fate and transport, nanochemistry, toxicology, bioaccumulation, biochemical research, exposure assessment, human health riskProgress 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.