Translocation of C60 from Aqueous Stable Colloidal Aggregates into

Nov 20, 2009 - J.B.H. e-mail: [email protected]; phone: 404-894-2201; fax: 404-894-2278. J.-H.K. ... Further evidence to support C60′s spo...
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Environ. Sci. Technol. 43, 9124–9129

Translocation of C60 from Aqueous Stable Colloidal Aggregates into Surfactant Micelles B O Z H A N G , 1,2,†,‡ M I N C H O , 1,† J O S E P H B . H U G H E S , 1,* ,† A N D J A E - H O N G K I M * ,1,† 1 School of Civil and Environmental Engineering, Georgia Institute of Technology, 200 Bobby Dodd Way, Atlanta, Georgia 30332 and 2 School of Environmental Science & Engineering, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai, China, 200240

Received August 31, 2009. Revised manuscript received November 4, 2009. Accepted November 6, 2009.

C60’s unique property of forming stable aggregates (nC60) in water, despite its exceedingly low aqueous solubility, has been linked to the potential for transport in the environment and exposure to biological receptors. The reversibility of aggregate formation could be an equally important parameter in understanding the ultimate fate of C60, including accumulation in nonaqueous environments such as biological membranes, micellular phases, and the organic fraction of soils. This study suggests that C60 molecules in nC60 readily translocate into nonionic surfactant micelles, a commonly used surrogate for biological membranes. Upon contact with surfactant micelles, the restoration of C60′s photochemical reactivity was observed; i.e., efficient production of reactive oxygen species (ROS) such as singlet oxygen upon UVA irradiation. Further evidence to support C60′s spontaneous translocation from colloidal aggregates into surfactant micelles is provided, including UV-vis spectral change, visual observation via transmission electron microscope, change in the fluorescence of surfactant micelles, and a reduction in the particle size of the parent nC60. Experiments performed with Escherichia coli also showed that singlet oxygen was produced when E. coli was in contact with nC60, resulting in peroxidation of lipids. These findings collectively suggest that micelle/lipid systems could be one of the receptors of C60 in the environment and provide insights into the previous observations of ROS production in biological systems exposed to nC60.

Introduction Despite its exceedingly low water solubility (1), C60 has been recently considered as a potential pollutant in water, largely due to the fact that it forms stable colloidal aggregates in water (often referred to as nC60) which allow facile transport in the environment and potential exposure to living receptors. The reversibility of aggregate formation might be an equally important parameter in understanding C60 transport and * Address correspondence to either author. J.B.H. e-mail: joseph. [email protected]; phone: 404-894-2201; fax: 404-894-2278. J.-H.K. e-mail: [email protected]; phone: 404-894-2216; fax: 404-385-7087. † Georgia Institute of Technology. ‡ Shanghai Jiaotong University. 9124

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impact on the environment. If C60 molecules can readily transfer from the aggregated form into a more dispersed form in nonaqueous environments such as biological membranes, micellular phases, and the organic fraction of soils, it is likely that nC60 will not be persistent in water, and that the fate of C60 may be consistent with other high-molecularweight, hydrophobic organic pollutants. A change from aggregate form into more dispersed state can be accompanied by the drastic change in photochemical reactivity of C60 (2–4). C60 is an efficient mediator for energy and electron transfer when it is molecularly dispersed in organic solvent, producing reacitve oxygen species (ROS) including singlet oxygen (1O2) in the presence of oxygen and under UVA irradiation. However, recent studies have demonstrated that nC60 in water is relatively photochemically inert (2, 3). Alternatively, C60 can be dispersed in water when exposed to encapsulating agents such as surfactants and polymers that encapsulate C60 within a micellular phase (3). C60 dissolved within surfactant micelle core has been demonstrated to exhibit photochemical reactivity leading to ROS production, even comparable to C60 dissolved in organic solvent (3). Recent studies have also reported that organic matter, including natural organic matter (NOM) and small molecular organic acids, also has an obvious effect on the dispersion status of C60 in the aqueous solution (5, 6). Dependence of C60′s photochemical reactivity on its dispersion status might explain discrepancies in the mechanisms of toxicity observed with a variety of organisms including bacteria (7, 8), aquatic organisms (9, 10) and even human tissue cells (11). It has been proposed that the toxicity of nC60 is ROS-independent and that exposure to nC60 results in protein oxidation and impedes the respiratory function of cell membranes (12, 13), consistent with abiotic studies indicating negligible ROS production by aggregate form of C60 (2, 3). In contrast, several past studies reported accumulation of nC60 inside cells and subsequent oxidative damage to cell constituents such as lipid peroxidation due to photochemically generated ROS (7, 9, 10, 14). Production of ROS in biological systems could be related to C60′s dispersion status change (i.e., from relatively inert nC60 to reactive molecular C60) as a result of the interaction with surrounding environment (e.g., biological membrane). This study tests the hypothesis that C60 in aggregated forms can transfer or “translocate” into micelle cores, employed herein as a model biological membrane. Translocation was observed and was correlated with a change in C60 photochemical reactivity with respect to 1O2 production. Additional experiments were performed with Escherichia coli to verify 1 O2 production by nC60 in a biological system. The results suggest that micellular and lipid structures may be one of the ultimate receptors of C60 in the environment, as C60 derived from C60 aggregates was found to readily translocate into micelle cores. C60 translocation and the resulting dramatic change in photochemical reactivity may explain observations of ROS production in biological systems exposed to nC60.

Experimental Section Preparation of C60 Samples. Three different types of aqueous C60 samples were prepared. First, nC60 was prepared based on the method by Fortner et al. (7, 15). The details are provided in Supporting Information (SI; Text S1). The second C60 sample was prepared by first preparing nC60-containing aqueous solution and mixing it with a separately prepared aqueous solution of nonionic surfactants, Triton X100 (TX100, Sigma-Aldrich) or Triton X100-Reduced (TX100R, Sigma 10.1021/es9026369

 2009 American Chemical Society

Published on Web 11/20/2009

Aldrich; refer to SITable S1 for the chemical structures and properties) for 24 h in the dark. The concentration of surfactant was either above critical micelle concentration (cmc, applied concentration ) 100 g/L for TX100 and 20 g/L for TX100R) or below cmc (0.2 g/L for both TX100 and TX100R). The third C60 sample was prepared based on the method by Lee et al. (2). Briefly, 8 mg of C60 was dissolved in 20 mL of toluene and mixed with 20 mL of water containing 4 g of TX100 or TX100R under ultrasound (50/60 Hz, 125 W). In contrast to the sample prepared by mixing separately prepared aqueous nC60 solution and aqueous surfactant solution, C60 gradually transfers from organic solvent into aqueous phase in this preparation method. This specific sample is denoted as TX100/C60 or TX100R/C60. Photochemical Experiments. Photochemical experiments were performed using a photoreactor equipped with six 4-W black light blue lamps (BLB lamps, Philips TL4W) (2). The emission wavelength region of 350-400 nm was monitored by a Spectropro-500 spectrophotometer (Acton Research Co., USA). The incident light intensity in this active wavelength region, measured by ferrioxalate actinometry (16), was 6 × 10-6 Einstein · L-1 · s-1. Reaction solutions contained 5 mg/L nC60 (or TX100/C60 or TX100R/C60), 10 mM furfuryl alcohol (FFA, 1O2 indicator), and/or a varying amount of surfactant and were buffered at pH 7 using phosphate (10 mM). The mixture (30 mL) was placed in a 40-mL quartz cylinder under mixing. After UVA irradiation for a predetermined amount of time, sample aliquots of 1 mL were withdrawn using a syringe, filtered through a 0.45-µm PTFE filter (Millipore), and injected into a 2-mL amber glass vial for further analyses. The residual FFA concentration was measured using a HPLC (Agilent 1100) equipped with an Agilent Zorbax SB-C18 column (4.6 × 150 mm, 5 µm) and a diode-array detector. All the experiments were conducted in triplicate. Photochemical Experiments with E. coli Culture. Photochemical experiments were also performed with E. coli. The procedures used in this study for the preparation and enumeration for E. coli were similar to those used by Cho et al. (17, 18). Briefly, E. coli (ATCC 8739) was inoculated in tryptic soy broth (Difco Co.) and grown for 18 h at 37 °C. The bacteria were harvested by centrifugation at 1000g for 10 min and washed twice with phosphate buffered saline solution (PBS 10 mM) at pH 7.2. Stock solutions of E. coli were prepared by resuspending the final pellets in PBS. The initial population of E. coli was determined by diluting the prepared stock solution and enumerated by the spreading plate method, in which the produced colonies are counted after overnight incubation of the plates at 37 °C. The initial population of E. coli for the experiments was adjusted at approximately 1 × 108 cfu/mL. For selective experiments, L-histidine (10 mM) was used as singlet oxygen scavenger (k(L-histidine + 1O2) ≈ 108 M-1 s-1 2, 19). All the experiments were conducted in triplicate. Analysis. UV-vis spectra were obtained using a Varian Cary 50 UV-vis spectrophotometer. Fluorescence spectra were obtained using a Shimadzu RF 5301PC spectrofluorophotometer with λex ) 310 nm. Electron micrographs were obtained by a JEOL 100 CX-II transmission electron microscope (TEM) (New York). TEM specimens were prepared by placing a droplet of sample solution on a carbon/Formvar coated copper grid (Electron Microscopy Science, Hatfield, PA) and drying overnight at room temperature. Malondialdehyde (MDA), one of the major products from lipid peroxidation, was quantified using thiobarbituric acid (TBA) which forms a pink-colored MDA-TBA adduct (20). E. coli sample (0.5 mL) was mixed with 1 mL of 10% (wt/vol) trichloroacetic acid (TCA) and centrifuged at 11,000g for 30 min. Freshly prepared 1.5 mL of TBA reagent (0.67% (wt/ vol)) was added to the supernatant and incubated in a boiling

FIGURE 1. Degradation of FFA by nC60 aggregates after mixing with solution containing TX100 and TX100R micelles under UVA irradiation ([nC60]0 ) 5 mg/L; [TX100]0 ) 100 g/L; [TX100R]0 ) 20 g/L; [C153]0 ) 160 µM; [phosphate (pH 7)]0 ) 10 mM; temperature: 22 ( 1 °C; light intensity ) 6 × 10-6 Einstein · L-1 s-1). Note that nC60 + TX 100 (or TX100R) indicates that aqueous solutions containing nC60 and TX100 (or TX100R) were individually prepared and mixed for 24 h before UVA irradiation. water bath for 10 min. After cooling to a room temperature, the absorbance of solution at 532 nm was measured using an Agilent 8453 UV-vis spectrophotometer. The calibration was performed using a standard MDA complex with TBA (extinction coefficient at 532 nm ) 49.5 mM-1 cm-1).

Results and Discussion Restoration of nC60′s Photochemical Reactivity. Consistent with the findings by Lee et al. (2), nC60 alone (Figure 1) or nC60 associated with TX100 or TX100R applied below cmc (i.e., TX100/C60 or TX100R/C60) (results not shown) did not produce a detectable amount of 1O2 under the experimental conditions employed. Surfactants alone also did not produce any 1O2 (Figure S1). When the nC60 solution was mixed with separately prepared solution containing surfactants applied below cmc for 24 h and subject to UVA irradiation, 1O2 production was still not detected (Figure S1). However, when nC60 was brought in contact with surfactants above their cmc, the rapid formation of 1O2 was observed (Figure 1). This result indicates a restoration of C60′s photochemical reactivity due to contact of nC60 with surfactant micelles. When TX100 micelles were pre-exposed to Coumarin 153 (C153) for 24 h before they were in contact with nC60, 1O2 production was significantly decreased (Figure 1). Water-insoluble C153 readily occupies TX100 micelle cores, thus impeding further transport of other weakly interacting molecules into micelle cores (21–23). Further experiments showed that 1O2 production rate increased as TX100 concentration increased (beyond cmc and hence higher concentration of micelles) (Figure S2) and the contact time between TX100 micelles and nC60 prior to photochemical test increased (Figure S3). Production of 1O2 by the mixture of nC60 and surfactant micelles, which are individually not capable of producing 1 O2, could be related to the change in dispersion status of C60 during this physical interaction process. Based on this observation, we hypothesize that C60 in aggregated forms VOL. 43, NO. 24, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Hypothetical mechanism for the recovery of photochemical reactivity of C60 due to C60 translocation from colloidal aggregates into micelle cores. can transfer or translocate into TX100 micelle cores during physical contact (Figure 2). Once C60 is within the surfactant micelles, C60 is virtually under organic phase and dispersed as either individual molecule or small clusters. It is wellknown that TX100/C60, C60 encapsulated in TX100 micelles prepared via a different route (i.e., the third method of preparation discussed above), is also capable of producing 1 O2 (2, 3). Therefore, during this hypothesized C60 translocation, C60 might be forming the similar surfactant/C60 system which is capable of 1O2 production. Evidence for C60 Translocation. UV-vis spectra were found to change after nC60 was in contact with surfactant micelles (Figure 3a). UV-vis spectrum of nC60 showed a specific absorption peak at around 350 nm and broadband absorption in the wavelength region from 400 to 500 nm, consistent with earlier reports (7). While there was no spectral change when nC60 was in contact with TX100 or TX100R applied below cmc (results not shown), hypsochromic shift of the characteristic peak at 350 nm was observed when TX100 and TX100R micelles were present. The blue shift to 330 nm in the presence of TX100 micelle was comparable to that in TX100/nC60 (2) as well as C60 molecularly dissolved in organic solvents such as toluene and hexane (24). Broadband absorption in the wavelength region from 400 to 500 nm, which is indicative of solid state C60-C60 interaction, also decreased. Colloidal aggregates of C60 could be readily removed by either ultracentrifugation (9300g for 60 min) or membrane filtration (Whatman Anotop 25 plus 0.02 µm filter), resulting in disappearance of C60′s characteristic UV spectra (e.g., 350 nm) in the supernatant or filtrate (Figure 3b and c). After contact with surfactant micelles, C60′s colloidal dispersion status was modified such that a significant amount remained stable even after ultracentrifugation or became smaller enough to pass through the filter with the nominal pore size of 20 nm, as evidenced by the appearance of characteristic peak at 330 nm. TEM analyses provided direct evidence on size reduction of nC60 due to interaction with surfactant micelles (Figure 4). According to TEM images, the average size of nC60 was approximately 100 nm. After nC60 was in contact with surfactant micelles, many small particles near or below 5 nm were observed in TEM images (Figure 4). These particles are most likely nC60 that are not within the micelle cores since the hydrodynamic radius of TX100 micelles was reported to be approximately 4.4 nm at 25 °C (21, 23) with the micellar palisade layer of 2.5 nm (25, 26). Therefore, the physical dimensions of the micelle cores are most likely too small to encapsulate these particles, although no direct evidence is currently available. This dramatic size reduction is presumably due to the relocation of C60 from colloidal aggregates to inside the surfactant micelles. Another evidence was provided by fluorescence analysis. TX100 micelles show fluorescence emission at 348 nm when 9126

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FIGURE 3. UV-vis spectra of nC60 aggregates (a) after interaction with TX100 and TX100R micelles and after (b) centrifugation and (c) membrane filtration ([nC60]0 ) 5 mg/L; [TX100]0 ) 100 g/L; [TX100R]0 ) 20 g/L; [phosphate (pH 7)]0 ) 10 mM; temperature: 22 ( 1 °C). excited at 310 nm due to phenyl groups in TX100 (27) (Figure 5). When C153 was added to micelles, it occupied the micelle cores and reduced the fluorescence emission. This fluorescence reduction results from the formation of electron donor (TX100) and acceptor (C153) pair and subsequent energy transfer between this pair and has been regarded as evidence for the molecule’s intrusion into the micelle core structure

FIGURE 4. TEM images of (a) nC60 and nC60 after contact with (b) TX100 micelles and (c) TX100R micelles. Bars represent 20 nm.

FIGURE 5. Fluorescence spectra of nC60, TX100 micelles, and TX100 micelles added with C153 and nC60 ([nC60]0 ) 10 mg/L; [TX100]0 ) 100 g/L; [phosphate (pH 7)]0 ) 10 mM; temperature: 22 ( 1 °C). (10, 28, 29). The fluorescence reduction was more pronounced as the concentration of C153 increased (Figure 5). Interestingly, when nC60 was added to micelle solution, the similar fluorescence decrease was observed after prolonged contact. Greater fluorescence decrease was observed when more nC60 was added (Figure S4). Although the detailed mechanism remains unclear regarding the role of C60 on fluorescence reduction in this case (27, 28, 30), this observation provides another indirect evidence for C60′s insertion into surfactant micelles. If C60 translocated from colloidal aggregates into TX100 micelles, C60′s aqueous stability would be determined by that of the micelles, not nC60. It is known that nC60 with negative surface becomes destabilized by the addition of Mg(ClO4)2 (7). Consistently, when the solution containing nC60 and 0.1 M Mg(ClO4)2 was filtered using a 0.22-µm filter (a Whatman Anotop 25 plus), most C60 in the aqueous solution was removed (Figure 6a). However, the same Mg(ClO4)2 addition had much less impact on nC60 mixed with surfactant micelles. Note that TX100 is a nonionic surfactant and therefore will not be electrostatically destabilized, unlike nC60. Approximately 25% and 22% of characteristic peak absorption might have resulted since not all C60 translocated into the micelle cores of TX100 and TX100R, respectively. While there is currently no available technique to accurately quantify the amount of C60 translocated from nC60 into surfactant micelles, this result indicates that approximately 75% and 78% of C60

FIGURE 6. (a) Spectral change by addition of Mg(ClO4)2 and (b) quantitative analysis of C60 incorporated into the TX100 and TX100R micelles. ([nC60]0 ) 50 mg/L; [TX100]0 ) 100 g/L; [TX100 R]0 ) 20 g/L; [phosphate (pH 7)]0 ) 10 mM; temperature: 22 ( 1 °C). might have entered TX100 and TX100R micelles during this physical contact period. ROS Production by nC60 in the Presence of E. coli. A similar phenomenon was observed when E. coli was added to nC60 instead of surfactant micelles. While E. coli alone (results not shown) or nC60 alone (Figure 1) did not produce a measurable amount of 1O2 upon UVA irradiation, the mixture exhibited a significant photochemical reactivity as shown in Figure 7. The FFA decrease was directly related to 1 O2 production since it was eliminated by the addition of excess (10 mM) L-histidine but was not affected by the addition of excess (2 µM) p-chlorobenzoic acid, a •OH radical scavenger. It was estimated that approximately 6.9 × 10-12 M of 1O2 was produced by the solution containing approximately 108 cfu/mL E. coli and 10 mg/L C60. Such a production of 1O2 was found to be accompanied by peroxidation of lipid, abundant in E. coli cell membranes (31, 32). Figure 8 shows that slight but apparent MDA formation compared to the control test result obtained with TiO2 (Figure VOL. 43, NO. 24, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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The results also indicate that nC60 could be involved in photochemical 1O2 production in the presence of surfactant micelles as well as E. coli cells. Consistent with past studies that reported 1O2-induced lipid peroxidation in biological systems (35, 36), lipid peroxidation was observed in E. coli by nC60, which would not be expected considering a negligible photoactivity of nC60 in the aqueous phase. Although lipid peroxidation has been related to cell toxicity (37, 38), it is presently unclear to what degree the observed lipid peroxidation is responsible for toxicological effects of this material on the microorganism. Nevertheless, the results indicate that nC60 could undergo a dramatic change in dispersion status via interaction with micellar structures, both artificial and biological, significantly affecting their fundamental photochemical reactivity. The proposed translocation process might provide an explanation on one of the potential mechanisms of toxicological effects of nC60 in the aqueous phase.

FIGURE 7. 1O2 production in the solution containing nC60 and E. coli ([nC60]0 ) 10 mg/L; [FFA]0 ) 0.5 mM; [L-histidine]0 ) 10 mM; [E. coli]0 ) 1 × 108 cfu/mL; [phosphate (pH 7)]0 ) 10 mM; light intensity ) 6 × 10-6 Einstein L-1 s-1; temperature: 20 ( 1 °C).

Acknowledgments This study was supported by the U.S. Environmental Protection Agency (STAR Grant D832526) and Georgia Institute of Technology. We thank Dr. Jim Millette and Whitney Hill at MVA Scientific Consultants (Duluth, Georgia) for their help with TEM analysis.

Supporting Information Available This material is available free of charge via the Internet at http://pubs.acs.org.

Literature Cited

FIGURE 8. Formation of MDA in the solution containng E. coli and nC60 ([nC60]0 ) 10 mg/L; [L-histidine]0 ) 10 mM; [E. coli]0 ) 1 × 108 cfu/mL; [phosphate (pH 7)]0 ) 10 mM; light intensity ) 6 × 10-6 Einstein L-1 s-1; temperature: 20 ( 1 °C). S5). When E. coli was in contact with nC60 and TiO2 (20 mg/ L) for 90 min UVA irradiation, 0.87 and 8.9 µmol/(L · 1010cfu/ mL) of MDA formations were observed, respectively. The MDA formation rate decreased beyond 100 min in Figure S5 due to the degradation of MDA by radical species (33). Formation of MDA and hence degradation of phospholipids in E. coli virtually stopped when 1O2 scavenger was added to the mixture. Significance of Findings. The results present above provide a line of evidence regarding translocation of C60 from the colloidal aggregates into surfactant micelles. It is noteworthy that TX100 micelles have been widely used as artificial, model biological membranes (4, 34). Therefore, the above results might suggest one of the important routes of ultimate fate of water stable C60 aggregates, i.e., potential accumulation of C60 in lipid membranes in biological systems and perhaps other organic phases in the environment. Although no direct measurements were attempted in this study due to difficulties associated with C60 analysis in biological matrix and further study needs to be performed, the results also implied that the proposed C60 translocation occurred with E. coli and perhaps other biological systems. 9128

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