Matthew C. Duch

Minimizing Oxidation and Stable Nanoscale Dispersion Improves the Biocompatibility of Graphene in the Lung

To facilitate the proposed use of graphene and its derivative graphene oxide (GO) in widespread applications, we explored strategies that improve the biocompatibility of graphene nanomaterials in the lung. In particular, solutions of aggregated graphene, Pluronic dispersed graphene, and GO were administered directly into the lungs of mice. The introduction of GO resulted in severe and persistent lung injury. Furthermore, in cells GO increased the rate of mitochondrial respiration and the generation of reactive oxygen species, activating inflammatory and apoptotic pathways. In contrast, this toxicity was significantly reduced in the case of pristine graphene after liquid phase exfoliation and was further minimized when the unoxidized graphene was well-dispersed with the block copolymer Pluronic. Our results demonstrate that the covalent oxidation of graphene is a major contributor to its pulmonary toxicity and suggest that dispersion of pristine graphene in Pluronic provides a pathway for the safe handling and potential biomedical application of two-dimensional carbon nanomaterials.

Colloidal Properties and Stability of Graphene Oxide Nanomaterials in the Aquatic Environment

While graphene oxide (GO) has been found to be the most toxic graphene-based nanomaterial, its environmental fate is still unexplored. In this study, the aggregation kinetics and stability of GO were investigated using time-resolved dynamic light scattering over a wide range of aquatic chemistries (pH, salt types (NaCl, MgCl2, CaCl2), ionic strength) relevant to natural and engineered systems. Although pH did not have a notable influence on GO stability from pH 4 to 10, salt type and ionic strength had significant effects on GO stability due to electrical double layer compression, similar to other colloidal particles. The critical coagulation concentration (CCC) values of GO were determined to be 44 mM NaCl, 0.9 mM CaCl2, and 1.3 mM MgCl2. Aggregation and stability of GO in the aquatic environment followed colloidal theory (DLVO and Schulze-Hardy rule), even though GOs shape is not spherical. CCC values of GO were lower than reported fullerene CCC values and higher than reported carbon nanotube CCC values. CaCl2 destabilized GO more aggressively than MgCl2 and NaCl due to the binding capacity of Ca(2+) ions with hydroxyl and carbonyl functional groups of GO. Natural organic matter significantly improved the stability of GO in water primarily due to steric repulsion. Long-term stability studies demonstrated that GO was highly stable in both natural and synthetic surface waters, although it settled quickly in synthetic groundwater. While GO remained stable in synthetic influent wastewater, effluent wastewater collected from a treatment plant rapidly destabilized GO, indicating GO will settle out during the wastewater treatment process and likely accumulate in biosolids and sludge. Overall, our findings indicate that GO nanomaterials will be stable in the natural aquatic environment and that significant aqueous transport of GO is possible.

Pluronic F108 Coating Decreases the Lung Fibrosis Potential of Multiwall Carbon Nanotubes by Reducing Lysosomal Injury

We compared the use of bovine serum albumin (BSA) and pluronic F108 (PF108) as dispersants for multiwalled carbon nanotubes (MWCNTs) in terms of tube stability as well as profibrogenic effects in vitro and in vivo. While BSA-dispersed tubes were a potent inducer of pulmonary fibrosis, PF108 coating protected the tubes from damaging the lysosomal membrane and initiating a sequence of cooperative cellular events that play a role in the pathogenesis of pulmonary fibrosis. Our results suggest that PF108 coating could serve as a safer design approach for MWCNTs.

Deposition and release of graphene oxide nanomaterials using a quartz crystal microbalance

Interactions of graphene oxide (GO) with silica surfaces were investigated using a quartz crystal microbalance with dissipation monitoring (QCM-D). Both GO deposition and release were monitored on silica- and poly-l-lysine (PLL) coated surfaces as a function of GO concentration and in NaCl, CaCl2, and MgCl2 as a function of ionic strength (IS). Under favorable conditions (PLL-coated positive surface), GO deposition rates increased with GO concentration, as expected from colloidal theory. Increased NaCl concentration resulted in a greater deposition attachment efficiency of GO on the silica surface, indicating that deposition of GO follows Derjaguin-Landau-Verwey-Overbeek (DLVO) theory; GO deposition rates decreased at high IS, however, due to large aggregate formation. GO critical deposition concentration (CDC) on the silica surface is determined to be 40 mM NaCl which is higher than the reported CDC values of fullerenes and lower than carbon nanotubes. A similar trend is observed for MgCl2 which has a CDC value of 1.2 mM MgCl2. Only a minimal amount of GO (frequency shift <2 Hz) was deposited on the silica surface in CaCl2 due to the bridging ability of Ca(2+) ions with GO functional groups. Significant GO release from silica surface was observed after adding deionized water, indicating that GO deposition is reversible. The release rates of GO were at least 10-fold higher than the deposition rates under similar conditions indicating potential high release and mobility of GO in the environment. Under favorable conditions, a significant amount of GO was released which indicates potential multilayer GO deposition. However, a negligible amount of deposited GO was released in CaCl2 under favorable conditions due to the binding of GO layers with Ca(2+) ions. Release of GO was significantly dependent on salt type with an overall trend of NaCl > MgCl2 > CaCl2.

Use of a pro-fibrogenic mechanism-based predictive toxicological approach for tiered testing and decision analysis of carbonaceous nanomaterials

Engineered carbonaceous nanomaterials (ECNs), including single-wall carbon nanotubes (SWCNTs), multiwall carbon nanotubes (MWCNTs), graphene, and graphene oxide (GO), are potentially hazardous to the lung. With incremental experience in the use of predictive toxicological approaches, seeking to relate ECN physicochemical properties to adverse outcome pathways (AOPs), it is logical to explore the existence of a common AOP that allows comparative analysis of broad ECN categories. We established an ECN library comprising three different types of SWCNTs, graphene, and graphene oxide (two sizes) for comparative analysis according to a cell-based AOP that also plays a role in the pathogenesis of pulmonary fibrosis. SWCNTs synthesized by Hipco, arc discharge and Co-Mo catalyst (CoMoCAT) methods were obtained in their as-prepared (AP) state, following which they were further purified (PD) or coated with Pluronic F108 (PF108) or bovine serum albumin (BSA) to improve dispersal and colloidal stability. GO was prepared as two sizes, GO-small (S) and GO-large (L), while the graphene samples were coated with BSA and PF108 to enable dispersion in aqueous solution. In vitro screening showed that AP- and PD-SWCNTs, irrespective of the method of synthesis, as well as graphene (BSA) and GO (S and L) could trigger interleukin-1β (IL-1β) and transforming growth factor-β1 (TGF-β1) production in myeloid (THP-1) and epithelial (BEAS-2B) cell lines, respectively. Oropharyngeal aspiration in mice confirmed that AP-Hipco tubes, graphene (BSA-dispersed), GO-S and GO-L could induce IL-1β and TGF-β1 production in the lung in parallel with lung fibrosis. Notably, GO-L was the most pro-fibrogenic material based on rapid kinetics of pulmonary injury. In contrast, PF108-dispersed SWCNTs and -graphene failed to exert fibrogenic effects. Collectively, these data indicate that the dispersal state and surface reactivity of ECNs play key roles in triggering a pro-fibrogenic AOP, which could prove helpful for hazard ranking and a proposed tiered testing approach for large ECN categories.

Interactions of Graphene Oxide Nanomaterials with Natural Organic Matter and Metal Oxide Surfaces

Interactions of graphene oxide (GO) nanomaterials with natural organic matter (NOM) and metal oxide surfaces were investigated using a quartz crystal microbalance with dissipation monitoring (QCM-D). Three different types of NOM were studied: Suwannee River humic and fulvic acids (SRHA and SRFA) and alginate. Aluminum oxide surface was used as a model metal oxide surface. Deposition trends show that GO has the highest attachment on alginate, followed by SRFA, SRHA, and aluminum oxide surfaces, and that GO displayed higher interactions with all investigated surfaces than with silica. Deposition and release behavior of GO on aluminum oxide surface is very similar to positively charged poly-L-lysine-coated surface. Higher interactions of GO with NOM-coated surfaces are attributed to the hydroxyl, epoxy, and carboxyl functional groups of GO; higher deposition on alginate-coated surfaces is attributed to the rougher surface created by the extended conformation of the larger alginate macromolecules. Both ionic strength (IS) and ion valence (Na(+) vs Ca(2+)) had notable impact on interactions of GO with different environmental surfaces. Due to charge screening, increased IS resulted in greater deposition for NOM-coated surfaces. Release behavior of deposited GO varied significantly between different environmental surfaces. All surfaces showed significant release of deposited GO upon introduction of low IS water, indicating that deposition of GO on these surfaces is reversible. Release of GO from NOM-coated surfaces decreased with IS due to charge screening. Release rates of deposited GO from alginate-coated surface were significantly lower than from SRHA and SRFA-coated surfaces due to trapping of GO within the rough surface of the alginate layer.

Graphene Oxide Enhances Cellular Delivery of Hydrophilic Small Molecules by Co-incubation

The delivery of bioactive molecules into cells has broad applications in biology and medicine. Polymer-modified graphene oxide (GO) has recently emerged as a de facto noncovalent vehicle for hydrophobic drugs. Here, we investigate a different approach using native GO to deliver hydrophilic molecules by co-incubation in culture. GO adsorption and delivery were systematically studied with a library of 15 molecules synthesized with Gd(III) labels to enable quantitation. Amines were revealed to be a key chemical group for adsorption, while delivery was shown to be quantitatively predictable by molecular adsorption, GO sedimentation, and GO size. GO co-incubation was shown to enhance delivery by up to 13-fold and allowed for a 100-fold increase in molecular incubation concentration compared to the alternative of nanoconjugation. When tested in the application of Gd(III) cellular MRI, these advantages led to a nearly 10-fold improvement in sensitivity over the state-of-the-art. GO co-incubation is an effective method of cellular delivery that is easily adoptable by researchers across all fields.

Translocation of Single-Wall Carbon Nanotubes Through Solid-State Nanopores

We report the translocation of individual single-wall carbon nanotubes (SWNTs) through solid-state nanopores. Single-strand DNA oligomers are used to both disperse the SWNTs in aqueous solution and to provide them with a net charge, allowing them to be driven through the nanopores by an applied electric field. The resulting temporary interruptions in the measured nanopore conductance provide quantitative information on the diameter and length of the translocated nanotubes at a single-molecule level. Furthermore, we demonstrate that the technique can be utilized to monitor bundling of SWNT in solution by using complementary nucleotides to induce tube-tube agglomeration.

Mechanisms of Gadographene-Mediated Proton Spin Relaxation

Gd(III) associated with carbon nanomaterials relaxes water proton spins at an effectiveness that approaches or exceeds the theoretical limit for a single bound water molecule. These Gd(III)-labeled materials represent a potential breakthrough in sensitivity for Gd(III)-based contrast agents used for magnetic resonance imaging (MRI). However, their mechanism of action remains unclear. A gadographene library encompassing GdCl3, two different Gd(III)-complexes, graphene oxide (GO), and graphene suspended by two different surfactants and subjected to varying degrees of sonication was prepared and characterized for their relaxometric properties. Gadographene was found to perform comparably to other Gd(III)-carbon nanomaterials; its longitudinal (r1) and transverse (r2) relaxivity is modulated between 12-85 mM-1s-1 and 24-115 mM-1s-1, respectively, depending on the Gd(III)-carbon backbone combination. The unusually large relaxivity and its variance can be understood under the modified Florence model incorporating the Lipari-Szabo approach. Changes in hydration number (q), water residence time (τM), molecular tumbling rate (τR), and local motion (τfast) sufficiently explain most of the measured relaxivities. Furthermore, results implicated the coupling between graphene and Gd(III) as a minor contributor to proton spin relaxation.

Response to comment on "colloidal properties and stability of graphene oxide nanomaterials in the aquatic environment"

Graphene Oxide Nanomaterials in the Aquatic Environment” W appreciate the opportunity to clarify issues raised in the comment of Zhang et al. on our paper describing the colloidal properties and stability of graphene oxide (GO) nanomaterials in the aquatic environment. Zhang et al. commented on our findings regarding the pH effect on the physicochemical properties of GO. Zhang et al. conducted similar GO stability experiments using similar solution chemistry which yielded results (unpublished) that were very close to those we observed for NaCl solution chemistry. Our comment on pH in our paper is based on NaCl conditions, and we made this clear in our paper by stating “Because we did not observe notable changes in electrokinetic or hydrodynamic properties of GO over this range, it is quite likely that pH will have minor effects on fate and transport of GO in the aquatic environment” (page 6291). The natural aquatic environment is complex and consists of various ions and organic matter. Zhang et al. investigated the pH effect in a mix of CaCl2 and NaCl and found that hydrodynamic diameter of GO increased with pH. They observed similar effects for aged tap water. They attributed this increased diameter to Ca ions, which neutralize GO surface charge by adsorbing on GO. Since Zhang et al. did not provide any surface charge data and detailed information on aged tap water (filtered or not, other ions, organic matter content) in the comment, it is difficult to assess the veracity of this conclusion. However, other studies have shown that pH can affect the protonation and deprotonation properties of GO. Whitby et al. found that acidic groups on GO can be protonated increasing the hydrophobicity of GO at low pH, while acidic groups become deprotonated at high pH resulting higher hydrophilicity. Szabo ́ et al. delineated the protonation and deprotonation mechanisms of graphite oxide because of pH change. This study found that increased pH and salt concentration can enhance the dissociation of acidic functional groups on graphite oxide. Another study investigated the pHdependent behavior of GO using experimental and molecular dynamics simulation study and determined the aggregate structure at different pH. These surface functionality changes on GO due to pH can influence the interactions of GO with divalent ions (Ca) and organic matter. Since pH effect on GO is well investigated, we primarily focused our study on the other environmental parameters including ionic strength, ion valence, and organic matter. Our comment on pH in our paper is based on monovalent salt (NaCl) condition, which will be appropriate where monovalent salts are dominating in the aquatic environment. For complex environmental conditions, interactions of divalent ions and organic matter with GO can be pH dependent. Indranil Chowdhury†,§ Matthew C. Duch‡ Nikhita D. Mansukhani‡ Mark C. Hersam‡ Dermont Bouchard* †National Research Council Research Associate, Athens, Georgia 30605, United States ‡Departments of Material Science and Engineering, Chemistry, and Medicine, Northwestern University, Evanston, Illinois 60208, United States National Exposure Research Laboratory, Ecosystem Research Division, United States Environmental Protection Agency, Athens, Georgia 30605, United States