Impact of Surface Functionalization on Bacterial Cytotoxicity of Single

Article pubs.acs.org/est

Impact of Surface Functionalization on Bacterial Cytotoxicity of Single-Walled Carbon Nanotubes Leanne M. Pasquini,† Sara M. Hashmi,† Toby J. Sommer,‡ Menachem Elimelech,† and Julie B. Zimmerman*,†,§ †

Department of Chemical and Environmental Engineering, Yale University, New Haven, Connecticut 06520-8286, United States Department of Chemistry, Yale University, New Haven, Connecticut 06520-8107, United States § School of Forestry and Environmental Studies, Yale University, New Haven, Connecticut 06520, United States ‡

S Supporting Information *

ABSTRACT: The addition of surface functional groups to single-walled carbon nanotubes (SWNTs) is realized as an opportunity to achieve enhanced functionality in the intended application. At the same time, several functionalized SWNTs (fSWNTs), compared to SWNTs, have been shown to exhibit decreased cytotoxicity. Therefore, this unique class of emerging nanomaterials offers the potential enhancement of SWNT applications and potentially simultaneous reduction of their negative human health and environmental impacts depending on the specific functionalization. Here, the percent cell viability loss of Escherichia coli K12 resulting from the interaction with nine fSWNTs, n-propylamine, phenylhydrazine, hydroxyl, phenydicarboxy, phenyl, sulfonic acid, n-butyl, diphenylcyclopropyl, and hydrazine SWNT, is presented. The functional groups range in molecular size, chemical composition, and physicochemical properties. While physiochemical characteristics of the fSWNTs did not correlate, either singularly or in combination, with the observed trend in cell viability, results from combined light scattering techniques (both dynamic and static) elucidate that the percent loss of cell viability can be correlated to fSWNT aggregate size distribution, or dispersity, as well as morphology. Specifically, when the aggregate size polydispersity, quantified as the width of the distribution curve, and the aggregate compactness, quantified by the fractal dimension, are taken together, we find that highly compact and narrowly distributed aggregate size are characteristics of fSWNTs that result in reduced cytotoxicity. The results presented here suggest that surface functionalization has an indirect effect on the bacterial cytotoxicity of SWNTs through the impact on aggregation state, both dispersity and morphology.

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INTRODUCTION The study of single-walled carbon nanotubes (SWNTs) flourished after Iijima’s synthetic discovery in 1991.1 SWNTs are sought after for their unique physical, electronic, mechanical, thermal, and antimicrobial properties.2−8 These properties can be further enhanced for desired applications through functionalization of the SWNT surface. SWNTs and functionalized SWNTs (fSWNTs) have the potential to advance medical treatment, energy generation, and storage, among other sectors.8−10 However, the properties that make fSWNTs desirable may also contribute to observed adverse human health and environmental impacts.11−15 An increasing number of studies have reported the potential environmental and human health concerns of nanomaterials.16−23 Despite significant efforts to reach a definitive understanding of the toxicity mechanism, there still remains a lack of consensus and methods to quantify potential concerns associated with the use of and exposure to SWNTs. A primary reason for the lack of agreement about SWNT toxicity concerns is the inconsistency between reported studies including the testing of SWNTs with varying fundamental properties such as tube length, diameter, solubility, © 2012 American Chemical Society

aggregation tendency, and metal catalyst contamination, all of which complicate the exploitation of traditional toxicity assays.16,17,22 Additionally, the host of different end points and test organisms utilized in toxicity studies compounds the difficulty in interpreting and comparing reported results. With increasing manufacturing demand and incorporation of SWNTs into products likely,10,24,25 direct and indirect release to the environment and human exposure is of growing concern. As such, it is important to evaluate the potential for SWNTs to adversely impact human health and the environment in a systematic and controlled manner. Perhaps of even greater importance is to understand the relationship between the addition of specific functional groups of interest for performance enhancement in a variety of applications and the potential subsequent impacts on toxicity. Previous studies have shown that specific functional group density can lead to decreases in cytotoxic behavior as compared to the starting nanomateriReceived: Revised: Accepted: Published: 6297

February April 16, April 19, April 19,

7, 2012 2012 2012 2012

dx.doi.org/10.1021/es300514s | Environ. Sci. Technol. 2012, 46, 6297−6305

Environmental Science & Technology

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al.26,27 This presents potentially useful insights into the design of safer nanomaterials where the addition of surface functional groups at certain densities can lead to reduced toxicity while maintaining the intended performance of the SWNTs. As such, rather than the impact of surface functional group density, the focus here is on the addition of functional groups that yield a range of physiochemical properties. The objective of this study is to evaluate how the addition of a range of functional groups, with the potential for functional enhancement, impacts SWNT bacterial cytotoxicity and to correlate the observed toxicity trends with fSWNT physiochemical characteristics and aggregation state. Since physiochemical characteristics cannot explain the observed behavior alone, light scattering techniques were utilized to determine if fSWNT aggregate state, both dispersity and morphology, could be correlated to the observed cytotoxic behavior.

SWNT Characterization. Schrodinger’s QikProp was used to obtain 49 physicochemical properties of the functional groups. The 49 descriptors and a detailed description of each are presented in the SI. Raman spectra for fSWNTs were obtained using a JASCO NRS-3100 Laser Raman Spectrophotometer with 785 nm incident wavelength. Spectra were collected from five different locations on a bulk powder sample, normalized and averaged. The compiled average spectra were then normalized to the Gband. For each sample, the G:D ratios were calculated. Tube diameters, dt (in nm), were estimated from the radial breathing mode (RBM) peak shifts (ωRBM = 100−300 cm−1) using dt = A/(ωRBM − B), where A = 234 cm−1 and B = 10 cm−1.38 The thermal properties of the fSWNTs were characterized by thermogravimetric analysis (TGA) (Setsys 16/18 system) in the temperature range of 200−1000 °C and ramp rate of 10 °C/min under flowing air. TGA was used to determine the success of the acid pretreatment and the relative thermal properties of the fSWNTs, using the percent mass loss and rate of change of mass curves, respectively. All curves were normalized per milligram of sample. X-ray photoelectron spectroscopy (XPS) was used to determine elemental composition and percent surface functionalization. Data were collected using a ThermoScientific ESCALAB 250 instrument with a monochronized Al X-ray source (150 eV pass energy for survey scans, 20 eV for composition scans, 500 μm spot size) at the University of Oregon CAMCOR facility. The samples were prepared by sonication in ethanol, then drop cast onto a silicon coupon. Both HF treated and nontreated coupons were used. The additional HF pretreatment eliminated the need to account for O as SiO2 in the final composition results. The electrophoretic mobility (EPM) (Zeta PALS analyzer, Brookhaven Instruments) of pristine and fSWNTs was determined in 0.9% NaCl solution at room temperature to gain insight into the SWNT charge properties. Samples were probe sonicated (Branson Sonifier 450, Duty Cycle 50%, Output control 5) for 15 min then diluted appropriately depending on the extent of dispersion. The samples remained stable during the duration of the measurement (∼45 min) and did not sediment. Transmission electron microscopy (TEM) images were obtained to provide information on the relative structural morphology and tube diameter estimates of the fSWNTs. Images were collected using a FEI Titan FEG-TEM (80−300 Kv, 0.8 Å resolution) at the University of Oregon CAMCOR facility. Samples were sonicated in ethanol, drop cast onto a TEM grid, and allowed to dry prior to imaging. Average diameters were determined using Image J 1.43u software (National Institutes of Health). Analysis of SWNT Aggregation State via Light Scattering. To determine the structural morphology and aggregation state of fSWNTs, static light scattering (SLS, ALVGmbH, Germany) was used to obtain the fractal dimension, Df. The scattered light intensity, I, scales as I/I0 ∼ q−Df, where I0 is the incident intensity and q is the wave vector which depends on the scattering angle.39 Measurements were taken every 1° using eight detectors and a 20 s collection time over the range of 0.00516 < q < 0.03397 nm−1, corresponding to scattering angles 17−153°. Multiple iterations were performed on each sample. Samples were prepared in the same manner as for the filter-based toxicity assay followed by a 10× dilution. The dispersions remained stable against sedimentation throughout

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MATERIALS AND METHODS Pristine and Functionalized Single-Walled Carbon Nanotube (SWNT) Preparation. SWNTs synthesized by CVD method were purchased from Nanostructured & Amorphous Materials Inc., Houston TX (90% SWNTs, 95% CNTs, Lot: 1284-091009). To remove residual amorphous carbon, the nanotubes were placed in an open glass Petri dish and heated at 350 °C for 3 h (Thermolyne 48000 Furnace). Once cooled, the tubes were placed in 12 M HCl (200 mL/g SWNTs), bath sonicated under ambient conditions for one hour to remove residual metal catalyst, and then filtered through a 5.0 μm PTFE membrane (Millipore, JMWP04700). The tubes were then repeatedly resuspended in DI water and filtered until neutral to pH paper. The final solution was filtered and the resulting acid treated SWNTs were dried in an oven overnight in an open glass Petri dish at 60 °C. These purified SWNTs were used as the starting material in the preparation of functionalized SWNTs. All functional groups are covalently bound to the surface of the prepared fSWNTs. With the exception of hydrazine and diphenylcyclopropyl SWNTs, functionalization was carried out according to literature methods.26,28−33 The preparation of hydrazine SWNTs closely follows the method of Yokoi et al.32 The preparation of diphenylcyclopropyl SWNTs was adapted from literature procedures for the carbene derivatization of other recalcitrant polymers.34,35 Synthesis details that deviate from the literature methods are described in the Supporting Information (SI). When indicated, dry powder starting material SWNT was physically altered using a ball mill (Heavy-duty Wig-L-Bug grinder/mixer analog) for specified intervals of time. The fSWNT samples were prepared for the cytotoxicity assay by probe sonication for 15 min (0.5 mg fSWNT in 20 mL dimethylsulfoxide (DMSO), Sigma-Aldrich, [67−68−5]). An additional 10-fold dilution was completed prior to light scattering experiments. Bacterial Cytotoxicity Assay. Chemical and material toxicity is measured in a variety of ways and includes a range of end points. In this particular study, the relative cytotoxicity of fSWNTs to Escherichia coli K12 (MG 1655) is measured and identified by the loss of cell wall integrity, referred to here as cell viability. A live-dead fluorescent cytotoxicity assay, similar to that used in previous studies,14,36,37 was utilized to obtain relative percent cell viability loss of E. coli K12 exposed to pristine and fSWNT deposit layers. Assay details are described and representative fluorescent images can be found in the SI. 6298



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the normalized elemental composition, as percent of the element present, and the known structure of the functional groups (Table 1). These calculations were limited to functional groups containing elements other than carbon and hydrogen, as they are identifiable from the carbon nanotube itself. Details on these calculations can be found in the SI. Proton NMR confirmed the presence of diphenylcyclopropyl, n-propylamine, phenylhydrazine, phenyl, and n-butyl groups, albeit without any quantitative measure of the degree of functionalization (SI Figures S1−S5). It is clear from these results that the intended surface modifications were successful and that the percent surface functionalization varies greatly depending on the functional group. There are several anomalies that arose from the XPS analysis. Although concentrated hydrochloric acid was used for the acid pretreatment of the starting material to avoid surface oxidation, XPS results indicate the presence of oxygen in the starting material. This oxygen is likely in the form of hydroxyl and carboxyl groups and results from the nitric-sulfuric acid treatment performed by the manufacturer (NanoAmor, personal communication, June 9, 2011). Further, XPS results also indicate the presence of residual nitrogen and sulfur in the phenyldicarboxy sample (Figure 1i). Closer inspection of the nitrogen N1s envelope in the XPS spectra confirms the presence of covalently bound nitrogen (detailed discussion of potential byproducts can be found in the SI). The sulfur in this same sample is likely residual oleum reagent. The presence of nitrogen in the diphenylcyclopropyl SWNT is discussed in the SI. Bacterial Cytotoxicity of Functionalized SWNTs. The results of cytotoxicity testing indicate that various fSWNTs differentially affect cell viability (Figure 2). The percent cell viability loss of the pristine SWNTs (starting material) is consistent with previously reported studies.26,27 Three of the nine fSWNTs tested in this study resulted in a statistically significant decrease in percent cell viability loss compared with the starting material. Of the remaining six fSWNTs, one resulted in approximately equal cell viability loss and five resulted in a statistically significant increase in cell viability loss compared to that of the starting material. The percent cell viability loss of the control (no SWNTs) remained consistent at 3.3 ± 1.1%. Further details on the statistical analysis are given in the SI. The compiled results suggest that bacterial cytotoxicity is affected by surface functionalization of the SWNT, either directly or indirectly. Observed Cytotoxicity of Functionalized SWNTs is not Correlated to Physiochemical Properties. There are several proposed mechanisms of SWNT cytotoxicity, including production of and cell interaction with reactive oxygen species (ROS), direct cell surface oxidation, and physical perturbation of the cell wall.11,14,21,37 It has also been shown that reduced cytotoxicity of carbon nanomaterials is achievable by the addition of surface functional groups27 and dependent upon the functional group density.26 As such, several physiochemical properties of pristine and fSWNTs were characterized and the results systematically correlated with the observed variability in cytotoxic response. Regression analysis of the combined results suggests that there is no direct correlation between bacterial cytotoxicity and physiochemical, structural, or thermal properties of functionalized SWNTs. Therefore, we are unable to isolate specific chemical properties of the individual functional groups that correlate directly with the observed trend in cell viability loss.

the duration of the experimental collection (∼30 min). UV−vis measurements of the sample solution confirmed negligible absorption of the sample at λ = 532 nm used for SLS measurements. Dynamic light scattering (DLS) data was collected using both a ZetaPALS analyzer (Brookhaven Instruments) and the ALV-GmbH instrument at an angle of 90°. Samples were prepared in the same manner as for SLS experiments. Measurements on the ZetaPALS instrument were taken every 5 s for 1000 cycles, and every 10 s for 500 cycles on the ALV instrument. The scattered light intensity was monitored to confirm the absence of sedimentation. UV−vis measurements of the sample solution confirmed negligible absorption at the ZetaPALS and ALV-GmbH incident wavelength (658 and 532 nm respectively). The raw correlation functions were exported and analyzed in MATLAB and Fortran, using the CONTIN algorithm.40,41

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RESULTS AND DISCUSSION Molecular Structure of Functionalized SWNTs. Characterization of carbon nanotubes offers a unique challenge due to their inherent size, shape, electronic properties, incongruence between manufactured batches, and tendency to agglomerate and interact with other materials.42−44 The inconsistency of these physiochemical properties among manufactured batches makes it even more critical to fully characterize the material when evaluating the toxic effects.45 Previous studies have established a precedent for characterization techniques to determine physiochemical properties contributing to MWNTs bacterial cytotoxicity,46 and this study utilizes a similar approach to the study of pristine and fSWNTs. The range of functional groups used to derivatize the SWNTs represents varying molecular size and chemical composition (Figure 1). XPS (Table 1) and NMR (SI Figures S1−S5) confirmed success of surface functionalization. The percent functionalization for each sample was calculated using

Figure 1. Molecular structures illustrating the surface functional groups covalently bound to the acid treated SWNTs. (a) npropylamine, (b) hydrazine, (c) phenylhydrazine, (d) phenyl, (e) diphenylcyclopropane, (f), n-butyl, (g) sulfonic acid, (h) hydroxy, (i) phenyldicarboxy. The functional groups are covalently bound to defect sites on both the tube surface and ends. The starting material contains oxygen in the form of hydroxyl groups and carboxyl groups; both resulting from the acid treatment performed by the manufacturer. 6299



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Table 1. Elemental Composition of the Starting Material and Each Functionalized SWNT (fSWNT) Sample, As Determined by XPSa

a

sample

%C

starting material n-propylamine phenylhydrazine hydroxy phenyldicarboxy phenyl sulfonic acid n-butyl diphenylcyclopropane hydrazine

95.9 95.2 95.7 84.8 77.8 96.6 78.1 97 95.9 95.9

%N 0.6 0.8 1.92

0.53 0.4

%O

%S

4.1 3.3 3.4 11.6 19.9 3.45 18.4 2.99 3.4 3.7

≤4.1

0.3 3.45

% functionalization 0.6 0.4 13.7 8.6 N/A 6.0 N/A N/A 0.2

These results combined with the known functional group composition, were used to approximate the percent functionalization.

at 166, 216, 244, and 278 cm−1 and correspond with tube diameters of 1.41, 1.08, 0.96, and 0.84 nm, respectively. While the shape of these peaks changes slightly in the spectra of the fSWNTs, their location remains the same. The ratio of the Gand D-band intensities is typically used to determine the relative presence of surface defects. The G:D ratios calculated from the normalized spectra of fSWNTs are all lower than that of the starting material (SI Figure S7), indicating an increased disruption of the ordered conjugated tube structure. Yet, there is no correlation between the extent of disruption and the estimated percent surface functionalization or the observed trend in cell viability loss. TGA measures the change in mass of a sample with increasing temperature. The percent mass loss curves of the purchased tubes before and after acid treatment is shown in SI Figure S8. The temperatures at which significant sample mass is lost is represented by peaks in the rate of change of mass curves (SI Table S1). Since they are held to the tube surface by weaker bonds, the evolution of surface impurities and functional groups takes place at lower temperatures than the main SWNT peak, which occurs ∼600 °C for unfunctionalized SWNTs. As expected, the appearance of mass loss peaks at lower temperatures than that of the starting material is observed for all samples due to the disruption of the ordered tube system and evolution of surface functional groups. This provides further evidence of successful surface functionalization as the addition of covalent functional groups effectively weakens structural integrity via the addition of tube defects.52 Yet, we were unable to correlate the magnitude of change in thermal properties of the fSWNTs with the calculated percent surface functionalization or the observed cytotoxicity behavior. TEM images were collected for pristine and fSWNTs. Measurements of tube diameter indicate that average outer wall diameters are between 0.35 and 0.6 nm for bundled tubes and 0.4 and 1.1 nm for isolated SWNTs (SI Figure S9). Based on the images collected, we estimate tube lengths of pristine and all fSWNTs to be greater than 1 μm. Furthermore, tube diameters and lengths are consistent across all fSWNT samples suggesting that structure is not responsible for the trend in cell viability loss. Surface Charge of Functionalized SWNTs. When bacterial cells deposit onto the SWNT coated membrane, they contact tube ends, tube surfaces, and the covalently bound functional groups, all of which have the potential to carry a charge in an aqueous solution. Due to the sensitivity of bacterial cells to changes in the charge of their environment, the potential influence of surface charge on cytotoxicity must be considered.

Figure 2. Box plot for the percent loss of viability for bacteria incubated with the various functionalized SWNT samples. Asterisk (*) indicates samples resulting in a statistically significant difference in percent loss of cell viability compared with the starting material as determined by two-sample t tests (95% CI, α = 0.05).

Functional Group Physiochemical Properties. QikProp (Version 3.4, Schrodinger, LLC, New York, NY, 2011) was used to identify 49 physiochemical and molecular properties of the nine functional groups. Properties such as molecular weight, molecular dipole, octanol−water partition coefficient, electron affinity, aqueous solubility and polar surface area are hypothesized to be of particular interest in relation to cytotoxicity.47,48 The QikProp output for these particular properties are tabulated (Table S1) for each of the nine functional groups in the SI. These properties were included in a regression analysis to determine whether specific properties correlate with the trend in cytotoxicity. The results from this statistical analysis indicate that individual and combinations of these physiochemical properties of the functional groups do not correlate with the observed trend in cytotoxic behavior. Structural and Thermal Properties of Functionalized SWNTs. The presence of surface defect sites as well as tube diameter and length are hypothesized to influence the loss of cell viability.37,46 Raman spectroscopy, electron microscopy, and thermogravimetric analysis are conventional characterization techniques to probe these particular structural and thermal properties of fSWNTs.38,49−51 The Raman spectrum (785 nm) of SWNTs consists of three identifiable regions: the radial breathing mode (RBM) between 100 and 300 cm−1, the defect or D-band at ∼1300 cm−1, and the G-band at ∼1600 cm−1.38,50 The RBM peaks of the starting material are observed 6300



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measurements. The dependence of the scattered light intensity, I, on the angle, θ, reveals morphological features of the sample. For spherical aggregates of characteristic size a, such that aq > 1, their fractal dimension, Df, can be extracted from

The pKa values of the functional groups are estimated based on tabulated values (SI Table S2).53,54 Protonation or deprotonation can affect the charge of the particular group and potentially, the overall surface charge of the tubes. Therefore, the pH of fSWNT dispersions in 0.9% saline is important to understanding the charge environment in which the SWNTs and bacteria interact. The cytotoxicity assay is conducted in 0.9% NaCl; therefore, electrophoretic mobility (EPM) measurements were collected in this saline solution to determine the surface charge properties of all fSWNTs. While the charge of the functional groups varies due to differences in pKa values, the EPM of all samples in 0.9% NaCl is negative (SI Figure S10). Functional groups that are predicted to carry a positive charge are not abundant enough to neutralize the inherent negatively charged SWNT surface, whereas the negatively charged groups contribute to the overall negative surface charge. Since there is no significant distinction in the EPM of the fSWNTs under experimental conditions, surface charge cannot be used to explain the observed trend in cytotoxicity. Surface Functionalization has an Indirect Effect on fSWNT Cytotoxicity Through Changes in SWNT Aggregate State. One proposed mechanism of SWNT cytotoxicity is the perturbation of the cell wall upon direct contact with nanotubes.14,37,55 Therefore, one might expect that the more surface area and tube ends available for interaction with bacteria, the more cytotoxic the SWNT sample. Yet, the extreme hydrophobic nature of SWNTs and their natural tendency to bundle and aggregate pose challenges to quantification of the surface area in contact with bacterial cells. Utilization of various surfactants and organic solvents as well as the addition of surface functional groups is known to enhance the dispersity of SWNTs.55−57 Due to the range of functional groups examined in this study, the extent to which the fSWNTs disperse is of utmost importance to understanding the relationship to the observed cytotoxic behavior. Gentle filtration, as utilized in sample preparation for the cytotoxicity assay, allows the SWNTs to settle onto the membrane and form a coated layer of SWNT aggregates. This process does not significantly alter the aggregate structure of the sample as additional mechanical pressure, hydraulic pressure, or mixing is not applied to system. The BET method and techniques such as atomic force microscopy (AFM) are commonly used to characterize the surface area and surface characteristics of solid materials. Yet, it is impossible to quantify the available contact area of the sample to bacteria that are micrometers in length and width. Determination of the SWNT surface area in contact with the cell is further complicated by the heterogeneous morphology of SWNT aggregates and the bacterial cell surface. Due to the difficulties in quantifying the precise available contact area of each fSWNT sample to the bacterial cells, the results from the combined light scattering experiments, described below, provide a valuable first approximation of the comparative aggregate state of the fSWNTs under experimental conditions. Structural Morphology and Aggregation State of Dispersed fSWNTs. Static light scattering (SLS) was utilized to determine the fractal dimension (Df) and investigate morphological differences of the dispersed fSWNTs. SLS can be used to track colloidal aggregation and the evolution of fractal dimension in suspensions.58,59 However, given our sample preparation, the fSWNTs are already in a globular fractal aggregation state at the onset and throughout the SLS

I = q−Df

(1)

where I is the scattered light intensity and q is the wave vector defined as

q=

4πnDsin

θ 2

(2)

λ

with nD being the solvent index of refraction, λ the wavelength of incident light, and θ the scattering angle. In general, Df values of colloidal aggregates fall between 1 and 3, with more compact morphological structures associated with higher fractal dimensions.44,60,61 The compiled plot of the scattered intensity, I/Io, as a function of q is shown in SI Figure S11.39 The solid lines represent fits to the data in the range of q, where I(q) (eq 1) is linear on a log−log scale. Fractal dimensions were extracted from the slope of the fitted lines (SI Table S3). The average Df of 2.65 ± 0.16 is indicative of relatively compact carbon nanotube aggregates.44,60 There are three samples having Df values one standard deviation below the average: hydroxy, phenyl, and sulfonic acid SWNTs. Fractal dimensions between 2 and 3 suggest morphologies between plate-like and sphere-like structures. The lower Df values of these three samples imply less tightly bound fSWNT aggregate morphology than in the other fSWNT systems.44,60 While these three fSWNTs exhibit increased cell viability losses, n-propylamine, phenylhydrazine, and phenyldicarboxy SWNTs result in equivalent cell viability loss, yet have average or greater than average Df values. As such, the fractal structure of the samples alone cannot explain the trend in cell viability loss. fSWNT Dispersed Aggregate Size Distribution. To compare the extent of dispersion of the fSWNTs, dynamic light scattering (DLS) measurements were collected on all of the samples immediately following the same sample preparation used for SLS analysis. DLS provides a measure of g(Δt), the autocorrelation of the scattered light intensity at a fixed angle, θ. Diffusion of particles in the sample causes fluctuations in the scattered light intensity, and g(Δt) decays exponentially, in the general form: g(Δt ) = exp( − Δt/τ )

(3)

where Δt is the time lag and τ is the diffusive time scale in the system. The diffusion coefficient, D, is determined from the measured diffusion time, τ, through

τ = 1/2q 2D

(4)

The particle size can be determined if the particle shape is known. For instance, the diffusion of spheres is given by the Stokes−Einstein equation: D = kBT /6πηa

(5)

where a is the particle radius, kB is the Boltzmann constant, T is the absolute temperature, and η is the solvent viscosity. In this way, DLS is typically used to determine size of spherical particles. DLS can also be used to determine the relative size of rod-shaped materials, through an established relationship between the aspect ratio and the diffusion 6301



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coefficient.62,63 While the diffusion coefficient can be obtained directly from eqs 4 and 5 for systems of nearly monodispersed particles, the heterogeneity of our SWNT dispersions required use of CONTIN analysis40,41 to fit g(Δt) to a Laplace transform: g(Δt ) =

∫ P(τ)exp(−Δt /τ)dt

The fSWNT suspensions exhibit diffusive time scale distributions P(τ) falling mainly between τ = 103 and 104 μs. Of the fSWNT samples resulting in increased cell viability loss as compared to the starting SWNT material, n-propylamine, phenylhydrazine, and phenyldicarboxy samples are correlated with broader P(τ) distributions (an order of magnitude greater than other fSWNTs), indicating a large range of aggregate sizes. While sample aggregate size distribution, or polydispersity, is a promising explanation for the observed trend in cell viability loss, it cannot be generalized to explain the observed trend in cytotoxicity. While neither fractal dimension nor aggregate size distribution of the fSWNT samples by themselves can adequately explain the observed trend in cell viability loss, analysis of the combination of these measurements can be correlated to the observed cytotoxic behavior. Both Df and P(τ) point to features of the fSWNT aggregates: Df indicates the compactness of the aggregate, while P(τ) indicates both the relative size and polydispersity of the aggregates. The polydispersity of the aggregate size distribution is quantified in terms of the width of the P(τ) curves. The value is obtained by calculating the width of the curve falling between 102 and 105 μs and above a threshold y-value of 0.002 × 10−4. Figure 4

(6)

to obtain a distribution of diffusion times, P(τ), for each sample. Figure 3 shows the P(τ) distributions for the starting SWNT material and fSWNTs. The range of τ is restricted by the

Figure 4. Plot of the width of the P(τ) curves versus the fractal dimension (Df) for the starting material and fSWNTs. Higher values of curve width are associated with greater polydispersity of aggregate size and higher values of Df are associated with more compact aggregate morphology. Cluster (iii) comprises the three fSWNTs (n-butyl, diphenylcyclopropyl, and hydrazine) associated with significant decrease in cell viability loss compared with the starting material. Df values of cluster (iii) fall between 2.6 and 2.8, indicative of compact aggregates, while the width values are associated with narrow aggregate size distributions. The other two groups contain samples associated with equal or increased loss in cell viability compared with the starting material. The values associated with the samples in cluster (i) are indicative highly compact and polydisersed aggregates, while those associated with cluster(ii) are indicative of less tightly bound monodispersed aggregates.

Figure 3. Normalized probability distributions of diffusion times (τ) for the starting material and (a) functionalized SWNTs exhibiting equal or increased cell viability loss and (b) functionalized SWNTs with decreased cell viability loss compared to the starting material. Diffusion times were determined by dynamic light scattering (DLS) and the probability distributions result from CONTIN analysis of the compiled correlation functions.

instrument resolution, in this case 102 < τ < 105 μs. The distribution of τ is presented here as a proxy to particle size for the fSWNTs due to their aforementioned unique attributes. Since larger particles and aggregates have smaller diffusion constants, as seen in eq 5, comparatively larger values of τ indicate comparatively larger aggregates. Assuming spherical particles, points of reference can be calculated. A measurement of τ = 103 μs corresponds with a = 43.6 nm and τ = 104 μs to a = 436.0 nm. Furthermore, broad distributions of τ indicate more polydispersed samples, while narrow distributions indicate more uniformly dispersed SWNT samples.

shows the curve width plotted against Df for each fSWNT. Three distinct clusters emerge: (i) high Df and high polydispersity, (ii) low Df and low polydispersity, and (iii) high Df and low polydispersity. Cluster (iii) comprises the three fSWNTs (n-butyl, diphenylcyclopropyl, and hydrazine) associated with significant decrease in cell viability loss compared with the starting material. The associated Df and width values of 6302



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result in similar impacts on aggregate morphology and dispersity. Implications. The results presented here suggest that both the addition of covalent surface functional groups and mechanical perturbation indirectly impact the cytotoxicity of SWNTs. We conclude that the indirect impact results from the extent of dispersion, which is quantified by both the fractal dimension, Df, and aggregate size distribution. The compiled results are the first to systematically investigate the relationship between fSWNT physiochemical properties and bacterial cytotoxicity. While the results are limited to nine functional groups and E. coli cytotoxicity, they do suggest that there is an indirect relationship between surface functionalization and cytotoxicity through aggregate morphology and dispersity. This indicates that these SWNT aggregate characteristics are more important than individual physiochemical properties of the functional groups. As such, it appears to be important to consider aggregation characteristics first and foremost when predicting the likelihood for fSWNTs to adversely impact cytotoxicity, an indicator of potential greater impacts for human health and the environment. It is important to understand that further studies are necessary to fully realize the potential of this knowledge and its application to controlled fSWNT design. Additional toxicity testing involving other organisms will further elucidate the material interactions within a variety of biological systems and should include measuring additional toxic end points. While the results presented here do not conclude with a clear set of design rules for reduced hazard, they do support the notion that there may be critical minimum properties that could define such a set.

this cluster are indicative of compact aggregates with a narrow size distribution, respectively. The other two groups contain samples associated with equal or increased loss in cell viability compared with the starting material. To further investigate the relationship of fSWNT aggregate morphology and size distribution to cytotoxicity, an additional experiment was conducted in which aggregation state was affected by physical means rather than chemical surface functionalization. Since sample preparation by ball milling is known to affect carbon nanotube size and morphology,64 the pristine SWNT was subjected to mechanical perturbation using the ball mill (Heavy-duty Wig-L-Bug grinder/mixer analog), after which SLS (ALV-GmbH), DLS, and cytotoxicity analyses were performed. The ball milling preparation allowed for alteration of SWNT morphology and dispersity with minimal alteration of surface chemistry. A starting material SWNT sample was collected after 30 min exposure to the ball mill. This sample resulted in statistically significant increase in cell viability loss (SI Figure S12a). Furthermore, the aggregate size distribution curve shifted toward shorter diffusive time, τ, (i.e., smaller aggregate size). Static light scattering measurements were completed as before to determine the sample fractal dimensions, which decreased from 2.70 to 2.24 after ball milling. This indicates that smaller less tightly bound aggregates correlate with an increase in cell viability loss. The impact of fractal dimension and dispersity on loss of cell viability is further exemplified by the comparison of diphenylcyclopropyl SWNT before and after ball milling. In the same way, the aggregate size distribution shifts to lower τ, the fractal dimension decreases from 2.73 to 2.32, and the loss of cell viability significantly increases after ball milling (from 62.2 ± 5.3 to 92.4 ± 2.7% cell viability loss) (SI Figure S12). The P(τ) widths and Df associated with the SWNT samples before and after treatment in the ball mill are shown in Figure 5. Their

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ASSOCIATED CONTENT

S Supporting Information *

Supporting Information includes further details on novel aspects of the hydrazine and diphenylcyclopropyl SWNT functionalization syntheses, the cytotoxicity assay, including representative fluorescent images, the proposed azo coupling byproduct (Figure S6), a list of QikProp descriptors, percent functionalization calculations, and statistical analysis. Additional characterization data including NMR data for diphenylcyclopropyl, n-propylamine, phenylhydrazine, phenyl, and butyl SWNT (Figure S1−S5), QikProp output data of six descriptors for fSWNTs (Table S1), Raman spectra (Figure S7), percent mass loss curves from TGA (Figure S8), tabulated burn off temperatures from DTG curves (Table S1), representative TEM images (Figure S9), tabulated pKa values of the functional groups and proposed dominant species (Table S2), electrophoretic mobilities in 0.9% NaCl (Figure S10), I/Io vs q plots for determining Df (Figure S11), tabulated Df for all fSWNTs (Table S3) and cytotoxicity and aggregate size distributions of ball milled samples (Figure S12). This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 5. Plot of the width of the P(τ) curves versus the fractal dimension (Df) for the starting material and diphenylcyclopropyl SWNTs before and after ball milling. Higher values of curve width are associated with greater polydispersity of aggregate size and higher values of Df are associated with more compact aggregate morphology.

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location on the plot supports the hypothesis that both aggregate morphology and dispersivity impact the delineation between increased and decreased SWNT cytotoxicity. This demonstrates that cytotoxicity can be modified via physical alteration analogous to chemical modification since both can

*Phone: (203) 432-9703; fax: (203) 432-4837; e-mail: julie. [email protected]. Notes

The authors declare no competing financial interest. 6303



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ACKNOWLEDGMENTS We acknowledge the generous support of the National Science Foundation under the Research Grant CBET-0854373. LMP recognizes support from the U.S. Environmental Protection Agency (EPA) STAR Fellowship Assistance Agreement No. FP91716701-0 and the NSF Graduate Research Fellowship Program (GRFP). This publication has not been formally reviewed by the EPA. The views expressed in this publication are solely those of the authors, and the EPA does not endorse any products or commercial services mentioned in this publication. The CAMCOR TEM facility is supported by grants from the W. M. Keck Foundation, the M. J. Murdock Charitable Trust, the Oregon Nanoscience and Microtechnologies Institute, the Air Force Research Laboratory (under agreement number FA8650-05-1-5041), at the University of Oregon. We also thank Dr. Steve Kang, Dr. Chad Vecitis, Dr. Codruta Zoican, and Dr. Stephen Golledge for their assistance with methods and instrument training, and technical support throughout the analysis of the characterization results.

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