Effect of Encapsulating Agents on Dispersion Status and

Feb 6, 2008 - Ji Il Choi , Samuel D. Snow , Jae-Hong Kim , and Seung Soon Jang ... Min Cho , Jaesang Lee , Yuri Mackeyev , Lon J. Wilson , Pedro J. J...
0 downloads 0 Views 152KB Size
Environ. Sci. Technol. 2008, 42, 1552–1557

Effect of Encapsulating Agents on Dispersion Status and Photochemical Reactivity of C60 in the Aqueous Phase JAESANG LEE AND JAE-HONG KIM* School of Civil and Environmental Engineering, Georgia Institute of Technology, 200 Bobby Dodd Way, Atlanta, Georgia 30332-0373

Received October 9, 2007. Revised manuscript received December 10, 2007. Accepted December 13, 2007.

This study demonstrates that the degree of C60 clustering in the aqueous phase is strongly dependent on the type and concentration of encapsulating agents, such as surfactant, polymer, and natural organic matter that interact with C60. The degree of C60 clustering was quantitatively analyzed using ultraviolet-visible spectral characteristics. The dispersion status played a critical role in determining the photochemical reactivity of C60, in particular, its ability to mediate energy transfer and to produce singlet oxygen in the presence of oxygen. Consistent with findings in the organic phase, C60 in the aqueous phase lost its intrinsic photochemical reactivity when they formed aggregates. Experiments performed using a laser flash photolysis suggested that the loss of reactivity resulted from a drastic decrease in lifetime of a key reaction intermediate, that is, triplet-state C60. This study suggests that the photochemical reactivity of C60 in the aqueous phase, which has been linked to oxidative damage in biological systems in earlier studies, is strongly dependent on the media environment surrounding C60.

Introduction With unique physical and chemical properties, C60 has been widely recognized as a promising nanomaterial with various potential applications (1). One notable characteristic is its strong photochemical reactivity, that is, provided with photon energy above 2.3 eV (2), C60 is readily excited with very high quantum yield (nearly 1.0) to a triplet state (3C60*), which subsequently produces reactive oxygen species (ROS) such as singlet oxygen (1O2) and superoxide radical anion (O2•-) in the presence of oxygen (1, 3–9). The capability of C60 to generate ROS has been instrumental in biomedical applications such as enzyme inhibition, antiviral activity, and photodynamic therapy (9–12). Accordingly, various methods have been developed to effectively disperse extremely hydrophobic C60 in aqueous media and biological systems. These include 1) generation of water-stable fullerene colloidal aggregates (often termed as nano-C60 or nC60) via ultrasonication (13) or stepwise solvent exchange (14, 15), 2) incorporation of fullerene into micelle/polymer/vesicle structures (4, 6, 11, 16–18), and 3) surface modification by addition of hydrophilic functional moieties (10, 19–21). Strong photosensitizing activity and aqueous availability combined have been at the center of concerns in environ* Corresponding author e-mail: [email protected]; phone: (404) 894-2216; fax: (404) 385-7087. 1552

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 5, 2008

mental and ecological impacts of this material. The exact same property mentioned above (i.e., production of ROS) has been claimed to be responsible for oxidative damages and cell death in biological receptors (15, 22–24). In addition, following the similar mechanisms discussed above, dispersion of C60 in the natural aqueous environment might be facilitated by 1) formation of colloidal aggregates, which has been demonstrated to be possible after prolonged exposure to water (15, 25), 2) interaction with natural encapsulant analogues such as natural organic matter (NOM) and other anthropogenic contaminants such as surfactants that strongly interact with C60, and 3) functionalization of C60 by chemical reactions in natural environment or during water and wastewater treatment processes (e.g., chemical treatment). Therefore, it is possible that C60 can be widely dispersed in the natural waterways, potentially interacting with biological systems, due to unintended events of mass discharge to the environment. However, accurate assessment of environmental impact of this material should consider how these two properties are related to each other. Our recent study suggested that the photochemical reactivity of C60 (i.e., related to toxicological effect) would be dependent on its dispersion status in the aqueous phase (i.e., related to exposure route). Although C60 associated with encapsulating agents such as surfactants or polymers retained the above intrinsic photochemical property (size of C60 colloids 18.2 MΩ) produced by a Milli-Q water purification system (Millipore, Billerica, Massachusetts) was used for the preparation of all solutions. Preparation of Aqueous-stable C60. Ultrasound (50/60 Hz, 125 W) was applied to a heterogeneous mixture of 10 mL of toluene containing 5 mg of C60 and 90 mL of ultrapure water in a sealed bottle for 24 h using an ultrasonicator (Model 8845–40, Cole-Parmer, United States). The water phase gradually assumed a brownish-orange hue as C60 was dispersed. Ultrasound was further applied to the mixture open to atmosphere for an additional 24 h at 60 °C to evaporate toluene, and the solution was further filtered 10.1021/es702552a CCC: $40.75

 2008 American Chemical Society

Published on Web 02/06/2008

TABLE 1. UV–vis Spectral Features and Photochemical Reactivity of C60 Associated with Surfactants surfactant type Above cmc anionic

cationic nonionic

Below cmc anionic

cationic nonionic

surfactanta

λmax (nm)

surfactant concn (g/L)

SDS SDES SOS SDBS CTAB DTAB OTAB Brij 78 Brij 35 TX 100 TX 100-R TX 405 Tween 65

5 10 50 10 1 10 50 10 10 50 10 50 0.05

218 220 218

SDS SDES SOS SDBS CTAB DTAB OTAB Brij 78 Brij 35 TX 100 TX 100-R TX 405 Tween 65

1 5 10 0.25 0.01 0.2 10 0.003 0.06 0.1 0.1 1 0.00017

A450/A330

A330/As

photoactivityb (µM/min)

262 265 263 261 263 261

340 343 340 336 340 338

0.27 0.31 0.27 0.23 0.28 0.27

1.88 1.96 2.11 1.80 2.10 1.75

N.D N.D N.D 2.04 N.D N.D

214

259 259

218

269

337 335 330 350 337 337

0.17 0.23 0.10 0.26 0.23 0.38

1.01 1.46 0.85 1.10 1.51 1.05

8.81 2.10 15.9 0.86 1.59 N.D

219 220 220 223

265 264 266 263 272

345 343 345 344 352

0.36 0.32 0.35 0.36 0.50

1.84 2.02 1.77 2.01 1.62

N.D N.D N.D N.D N.D

221 221 223 222 222 221

266 267 274 268 264 266

347 347 345 349 340 345

0.37 0.37 0.34 0.39 0.28 0.40

1.15 1.28 1.73 1.04 1.40 0.95

N.D N.D N.D N.D N.D N.D

a SDS, sodium dodecyl sulfate; SDES, sodium decyl sulfate; SOS, sodium octyl sulfate; SDBS, sodium dodecylbezene sulfonate; CTAB, cetyltrimethylammonium bromide; DTAB, dodecyltrimethylammonium bromide; OTAB, octyltrimethylammonium bromide; TX 100, Triton X100; TX 100-R, Triton X100 reduced; TX 405, Triton X405. b Initial (20 min) degradation rate of FFA as 1O indicator (duplicate). 2

through a 0.45-µm PTFE filter (Millipore Corp.). An aqueous suspension of aggregate form of C60 prepared according to this specific method is herein referred to as son/C60. An alternative method based on solvent exchange (15) was also used to prepare an aqueous suspension of C60 aggregate (termed separately as nC60) for comparison purpose. C60 associated with a target encapsulating agent, referred to as C60/encapsulant, was prepared following the same method as son/C60 except that varying concentration of encapsulating agent was dissolved in the aqueous phase prior to sonication. Note that the previously established preparation methods (4, 18) were not applicable in our study, because some target compounds (e.g., ionic surfactants) were negligibly soluble in organic solvents such as toluene and chloroform. However, C60/TX 100 (above critical micelle concentration [cmc]) and C60/γ-cyclodextrin prepared by our method were comparable to ones prepared according to the methods described in refs 4, 7, and 30, based on UV spectral analysis. Preliminary experiments also suggested that UV spectra of C60 suspensions prepared by this method (C60/SDS, C60/SDBS, C60/TX 100, and C60/CTAB) were reproducible (see Table 1 for abbreviations of chemical names). Photochemical Experiments. Photochemical experiments were performed following the method previously described by Lee et al. (2006). Details are provided in the Supporting Information. Laser Flash Photolysis. Prior to experiment, a solution (3 mL) containing 5 mg/L C60 was placed in a rectangular quartz reactor, purged with argon gas for 30 min, and sealed to inhibit the transfer of absorbed energy from 3C60* to oxygen. A laser pulse at 355 nm (5 mJ, pulse width ) 6 ns) generated from a Quanta Ray Nd:YAG laser system was used as an excitation source for C60. A monochromatic laser at 740 nm, produced by filtering the light from a xenon lamp using a monochromator and aligned perpendicular to the excitation

laser, was used to monitor the concentration of 3C60* at 740 nm. The spectrum of 3C60*, when produced, matched with that reported in literature (27, 28). Each laser flash photolytic experiment was duplicated.

Results and Discussion UV–vis Spectral Characteristics of C60 Associated with Surfactants. UV–vis analysis of C60 associated with selected surfactant suggests that spectral characteristics that reflect the dispersion status of C60 are strongly affected by the presence of surfactants (30) (Figure 1). Spectra of son/C60 and nC60 both showed a specific absorption peak at around 350 nm and broadband absorption in the wavelength region from 400 to 500 nm, consistent with the previous literature (14, 15). However, C60 associated with TX 100, a nonionic surfactant widely used as an artificial cell membrane (4, 6, 30), showed a sharp absorption peak at 330 nm (i.e., blue-shifted compared to that in son/C60 and nC60) and negligible absorption in the wavelength ranges from 400 to 500 nm. TX 100 and all the other surfactants used in this study had little or no absorption above 300 nm. The spectrum of C60/TX 100 is fairly similar with that of C60 molecularly dissolved in toluene or hexane (30). The spectrum of C60 further depended on the type and amount of surfactants applied. When anionic SDBS and nonionic Brij 35 were applied, relatively weak broadband absorption in 400–500 nm along with a moderate level of peak shifting at UV range were observed. In contrast, C60 associated with cationic CTAB exhibited higher absorption at the 400–500 nm region, and the position of the characteristic UV peak was comparable to son/C60. Quantitative analyses on UV–vis spectra of all the C60 suspensions are summarized in Table 1. Characteristic UV absorption peaks of C60 associated with surfactant applied below cmc were red-shifted compared to C60/surfactant above cmc. This red-shifting has been reported to occur when VOL. 42, NO. 5, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

1553

FIGURE 1. UV–visible spectra of 5 mg/L C60 in the aqueous phase: son/C60, nC60 and C60 associated with CTAB, SDBS, Brij35, and TX 100 ([CTAB]0 ) 0.01 g/L (below cmc); [SDBS]0 ) 20 g/L (above cmc); [Brij35]0 ) 10 g/L (above cmc); [TX 100]0 ) 50 g/L (above c.m.c.); [phosphate]0 ) 10 mM; pHi ) 7). C60 forms aggregates in binary organic solvent mixture or in an artificial model lipid membrane (30, 33). In the aqueous phase, this bathochromic shift indicates that the surfactant applied below cmc does not significantly suppress C60 aggregation. Consequently, the maximum bathochromic shift was observed with son/C60 (λmax ) 218, 269, and 347 nm), which was prepared in the absence of surfactant. In contrast, all C60 associated with surfactant applied above cmc showed significant hypsochromic shifts relative to son/C60, indicating that aggregation is inhibited and that C60 is exposed to a media environment closer to organic phase (i.e., inclusion within the hydrophobic micelle cores). As surfactant concentration was further increased beyond cmc and more micelles were available for C60 encapsulation, λmax became closer to that of individually dispersed C60. For example, when the concentration of SDBS was gradually increased as 0.25, 10, 20, and 50 g/L, λmax for the third specific peak gradually moved to shorter wavelength as 344, 336, 331, and 330 nm, respectively. λmax at the highest surfactant concentration is comparable to that of C60 associated with γ-cyclodextrin (λmax ) 214, 260, and 332 nm) where a single C60 molecule resides within the relatively hydrophobic (i.e., compared to water) cavity of each γ-cyclodextrin molecule (16). These values are also comparable to those of C60 dispersed in hexane (i.e. λmax ) 211, 257, and 328 nm in the absence of solvent interference). A450/A330 (Table 1) represents the absorbance at 450 nm normalized by that of the specific peak at 330–350 nm region (i.e., λmax varies depending on the sample). This broadband absorption in visible range (400–500 nm) results from solid state C60-C60 interactions (14, 15, 34). A450/A330 was as high as 0.41 in the absence of surfactant (i.e., nC60 and son/C60) and slightly lower when surfactants were applied below cmc. When applied above cmc, a substantial decrease in A450/A330 was observed. This observation is consistent with the peak shifting, which suggested that that C60 aggregation would be limited by applying surfactant above cmc. C60 clustering was more effectively prevented as more micelles were available in the solution. As surfactant concentration was gradually increased beyond cmc, A450/A330 also further decreased (Figure 2a). As the presence of micelle structure is critical for determining dispersion status of C60 as discussed above, it might be essential for promoting C60 transfer to and stabilization in the aqueous phase. The peak absorbance of the sample at the 330–350 nm region was compared to the 1554

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 5, 2008

FIGURE 2. Effect of surfactant concentration on (a) A450/A330 and (b) 1O2 production rate ([C60]0 ) 5 mg/L; [phosphate]0 ) 10 mM; pHi ) 7). Average of duplicate experiments plotted. absorbance of son/C60 at 347 nm (As) and was designated as A330/As in Table 1. This analysis suggests that the presence of surfactants accelerated the dispersion of C60 (A330/As > 1.0) in most cases, even when concentration was below cmc. This enhanced transfer might be related to surfactants arranged at the interface between organic and aqueous phases, acting as phase transfer agent (i.e., facilitating the transport of C60 through toluene/water interphase), although no definite answer was available at the current stage. UV–vis spectra provide critical information regarding dispersion status (i.e., aggregate vs molecule), especially when a large amount of surfactants are present. Dynamic light scattering (DLS) of C60 associated with surfactant above cmc suggests that sizes were below the detection limit of DLS (ca. 5 nm) in our past study (7), but no further insights as to exact number of C60 inside the micelle cores could be gained. However, based on the above UV analysis performed with varying surfactant concentration, it is very plausible that C60 is molecularly dispersed inside surfactant micelles. Microscopic imaging using transmission electron microscope (TEM) was not possible because excessive surfactant deposited on carbon grid prevented clear images. In the following discussions, spectral shifting and A450/A330 were used as criteria to conjuncture dispersion status of C60 in the aqueous phase. Effect of Surfactant types. Effect of surfactant on the dispersion status of C60 depended not only on the surfactant concentration, but also on its type. Among nonionic surfactants (i.e., in the absence of any potential Coulombic interaction between C60 and surfactant) with the same hydrophilic moieties (Brij and TX series), TX 100 was the most effective in suppressing aggregate formation. This resulted because the aromatic moiety in the hydrophobic tail of TX 100 would interact with the conjugated π system

TABLE 2. UV–vis Spectral Features and Photoactivities of Aqueous C60 Colloids Associated with Other Encapsulants type carbohydrate polymer natural organic matter

encapsulating agenta

concn (g/L)

γ-cyclodextrin PA PVP PEG SRH SRF

3 10 10 10 0.05b 0.05b

λmax (nm) 214

260 264

218 214 215

269 262 262

332 344 345 347 338 339

A450/A330

A330/As

photoactivity (mM/min)

0.12 0.34 0.44 0.36 0.30 0.28

1.28 0.37 0.22 1.47 1.94 (1.20)c 2.17 (0.51)c

16.3 N.D N.D N.D 1.22 (1.18)d 1.32 (1.29)d

a PA, polyacrylate; PVP, polyvinylpyrrolidone; PEG, polyethylene glycol; SRH, Suwannee River humic; SRF, Suwannee River fulvic. b Carbon g/L measured by TOC (total organic carbon) analyzer. c Absorbance ratio of SRH and SRF to son/C60 at 330 nm regions. d Photoactivity (initial 1O2 generation rate) of SRH and SRF.

of C60 more favorably as compared to n-alkyl (Brij 78 and 35) or cycloalkyl (TX 100-R) groups (6). Functional groups in hydrophilic head also seemed to affect the dispersion characteristics. Even though TX 405 has the same hydrophobic moiety as TX 100, it was much less effective in suppressing aggregate formation. This difference should have resulted from the difference in hydrophilic chain length (-(OCH2CH2)n, n ) 40 on average for TX 405 and n ) 9∼10 for TX 100). An attractive interaction has been reported between polyethylene glycol ((OCH2CH2)n) of TX series surfactant and the π system of C60 (17). It is also consistent with the observation that polyethylene glycol moderately facilitated the dispersion of C60 in water (i.e., greater A330/As value), whereas other water-soluble polymers did not (Table 2). Therefore, the presence of a longer ethylene glycol chain in TX 405 might have hindered C60 from being incorporated into the hydrophobic inner phase. Aliphatic anionic surfactants (e.g., SDS, SDES, and SOS) were less effective in inhibiting the clustering of C60 than their aliphatic nonionic counterparts (e.g., Brij 78 and 35), suggesting that Coulombic interaction also played a role in aggregate formation. During ultrasonication, it is unlikely that C60 as an individual molecule directly transfers from organic phase into the micelles dispersed in the aqueous phase, because molecular C60 is extremely hydrophobic. A transient formation of water-stable aggregates with a negatively charged surface (15, 25) and subsequent translocation into the micelle are more likely during phase transition. Therefore, a repulsive interaction between C60 aggregates with a negatively charged hydrophilic group might have limited the encapsulation of C60 by anionic surfactants, which caused higher A450/A350 values, compared to C60 associated with nonionic surfactants. SDBS was more effective than other anionic surfactants (with the same hydrophilic functionality) in suppressing aggregate formation, following the similar mechanism that TX 100 was more efficient that other nonionic surfactants. That is, SDBS contains a benzene ring in the hydrophobic tail, which has the more favorable solvent–solute interaction, whereas other surfactants (SOS, SDES, and SDS) contained n-alkyl groups (6). Accordingly, attractive electrostatic interaction is possible between negatively charged C60 aggregates and the quaternary ammonium group of cationic surfactants (CTAB, DTAB, and OTAB). In this case, therefore, it would be feasible that this attractive interaction between cationic surfactant and aqueous C60 colloid might allow C60 particles to favorably merge into larger clusters by neutralizing negative charge on colloidal surface. When OTAB and DTAB were applied at concentrations below cmc, yellowish aqueous C60 suspension resulted but quickly settled down and completely filtered through a 0.45-µm PTFE filter. C60 colloid prepared in the presence of CTAB below cmc did not settle down. However, it exhibited the greatest level of red shift in characteristic peaks and the largest A450/A330 value, indicating formation of larger particles. These should result from agglomeration and floc formation of C60 aggregates due to charge neutralization

by positively charged surfactants. In a separate test, we observed the similar spectral change when 50 µM of ferric ion was added to son/C60 (results not shown). When CTAB and DTAB were applied above cmc, the charge neutralization effect becomes less important and provides a similar environment to C60 as compared to their anionic counterpart (i.e. anionic aliphatic surfactants such as SDS). C60/OTAB, even over cmc, did not produce stable suspensions. Overall, a longer aliphatic moiety facilitated encapsulation of C60 in the aqueous phase and prevented aggregation. Photochemical Activities of Aqueous C60 Associated with Surfactants. The photochemical activity of C60 in the aqueous phase, measured herein as an ability to produce 1O2 during UV irradiation, was strongly affected by the dispersion status of C60 and was consistent with UV–vis spectral characteristics. When surfactants were applied at concentrations below cmc and when C60 forms aggregates similar to son/C60 and nC60, no photoactivity was observed. Only when surfactants were applied above cmc to form micelles that can encapsulate C60, 1O2 production was observed. The rate of 1O2 generation was inversely proportional to A450/A330, which is indicative of the degree of C60 aggregation (i.e., 1O2 generation rate: TX 100 > Brij 78 > Brij 35 ≈ SDBS > TX 405 > TX 100-R; A450/A330: TX 100-R > Brij 35 ) SDBS ) TX 405 > Brij 78 > TX 100). Note that surfactant involvement in 1O2 production was highly unlikely, as most exhibited no absorption in the active wavelength region, except SDBS, and no 1O2 production was observed in control tests performed using surfactants only (TX100 and SDBS). When surfactant concentration was further increased above cmc, 1O2 production rate also increased (Figure 2b). C60 associated with surfactants containing aromatic moieties (SDBS and TX 100) applied at the highest concentration (50 g/L) produced 1O2 at extremely high rates, which were comparable to the 16.3 µM/min measured with molecularly dispersed C60 associated with γ-cyclodextrin (16) (Table 1). At such a high concentration, even relatively ineffective encapsulant, that is, aliphatic SDS, also rendered C60 photochemically active. For cationic surfactant CTAB, no 1O2 production was observed even when concentration was increased as high as 50 g/L due to floc formation and settling of colloids. Decay Kinetics of Triplet-state C60. In our past study (7), we hypothesized that “photoexcited triplet C60 (3C60*) at the aggregate surface may be effectively quenched by surrounding ground state C60 (self-quenching) and another triplet C60 (triplet-triplet annihilation) within the aggregate structure and dissipated as heat”. As a result, the pathway for energy transfer to oxygen and subsequent production of 1O2 might be prohibited when C60 aggregates in the aqueous phase. This hypothesis was verified in this study by tracing the fate of 3C60*, which is the key transient intermediate for the energy transfer process, using nanosecond transient spectroscopy under anoxic, Ar-saturated conditions. First, the lifetime of 3C60* in toluene/acetonitrile cosolvent (i.e., aggregates of sizes approximately 100–200 nm (36, 37)) VOL. 42, NO. 5, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

1555

FIGURE 3. Absorption time profile of 3C60* recorded at 740 nm in aqueous (a) son/C60, (b) nC60, (c) C60/TX 100, and (d) C60/SDBS suspensions ([C60]0 ) 5 mg/L; [SDBS]0 ) 50 g/L; [TX 100]0 ) 50 g/L; [phosphate]0 ) 10 mM; pHi ) 7; Ar-saturated condition). was measured to be approximately 3 orders of magnitude shorter than that in toluene (i.e., molecularly dispersed) (Supporting Information Figure S1). This result was consistent with the previous report that the lifetime of 3C60* in aggregated C60 derivatives was within 0.1 µs, and that in surfactantencapsulated or molecular C60 in organic phase was typically measured as several tens to a hundred of microseconds (21). Figure 3 shows the transient absorption of 3C60* in the aqueous phase: son/C60, nC6,0 and C60 encapsulated with TX 100 and SDBS (i.e., applied above cmc to prevent C60 clustering). Similar to the observations made for organic solvents obtained in this study and reported in the literature, 3C60* in son/C60 and nC60 decayed within 100 ns, with the kinetics that was too fast to be accurately characterized using nanosecond transient spectroscopy, whereas the lifetime of 3C * in C associated with TX 100 and SDBS extended to the 60 60 several hundred microsecond scale. This result demonstrates that C60 clustering in the aqueous phase prevents the formation of 1O2 by limiting the availability of key intermediate for energy transfer process, that is, 3C60*. In contrast, when C60 is dispersed in the aqueous phase associated with TX 100 and SDBS, 3C60* lifetime was comparable to that in toluene, indicating that C60 should probably be dispersed as individual molecules within hydrophobic micelle cores. However, not all surfactants function in the same way; when C60 was associated with other surfactants such as SDS (aliphatic anionic) and DTAB (aliphatic cationic), 3C60* lifetime was also too short to be measured on the nanosecond scale (Supporting Information Figure S2). This was consistent with the fact that they exhibited UV–vis spectral features indicating aggregate formation (i.e., red-shifted characteristic peak and appearance of broadband absorption in the visible regions), and 1O production was negligible. 2 C60 Associated with NOM. C60 associated with SRH and SRF exhibited red-shifted characteristic UV peaks and lower A450/A330 values compared to son/C60. A330/As values for C60/ SRH and C60/SRF significantly increased, although they were 1556

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 5, 2008

slightly overestimated by the absorbance of SRH and SRF at this wavelength. These spectral features suggest that the presence of SRH and SRF contributed to inhibiting C60 aggregation to some degree and to enhancing dispersion in water. Portions of heterogeneous mixtures of SRH and SRF might interact closely with C60, providing sites for C60 to be present closer to molecular state. Based on the above findings, organic matter with greater aromatic moieties might be more efficient in providing sites for C60 encapsulation. Production of 1O2 production was observed, albeit at an extremely slow rate after considering 1O2 production by SRH and SRF only, suggesting that ROS production in this case should have negligible toxicological effects. As C60 is released to natural aqueous environments, C60 will interact with various natural and anthropogenic macromolecules. This study suggests that association with these encapsulating agents determines the dispersion status of C60, that is, the degree of clustering, which is critical for accurate estimation of C60 transport in natural waterways and exposure route to human. Surfactants used in this study represent anthropogenic pollutants that strongly interact with C60, but more importantly, the findings obtained with surfactant as surrogate for lipid (31) and NOM (32) suggest that functionalities and ionic charge of these macromolecule would play a critical role in interaction with C60 in the aqueous phase. Some of these encapsulants prohibit clustering of C60 in water, leading to production of ROS and, consequently, toxicological effects. Diverse scenarios seem possible, for example, C60 encapsulation by surfactant-like molecules that will facilitate transport not only in the subsurface media but across the cell membrane and induce ROS production. Future study should further focus on evaluation of C60 interaction with diverse organic molecules both in natural waters and in biological systems.

Acknowledgments This work was funded by the U.S. Environmental Protection Agency (USEPA) STAR Grant No. D832526. Mention of trade

names or commercial products does not constitute endorsement or recommendation for use. The scientific views expressed are solely those of the authors and do not necessarily reflect those of the USEPA. The authors would like to thank Dr. Prashant Kamat, Dr. Anusorn Kongkanand, and Dr. Yoichiro Matsunaga for their assistance during laser flash photolysis, which was performed at Notre Dame Radiation Laboratory, South Bend, Indiana.

Supporting Information Available Additional figures showing the absorption time profile of the excited triplet state of molecular C60 in toluene and that of a C60 cluster in a toluene-acetonitrile mixture, and decay kinetics of triplet C60 in aqueous suspension of C60 associated with SDS and DTAB are also provided. This material is available free of charge via the Internet at http://pubs.acs.org.

Literature Cited (1) Kadish, K. M.; Ruoff, R. S. Fullerenes: Chemistry, Physics, and Technology. Wiley-Interscience: New York, 2000; p 1–916. (2) Lof, R. W.; van Veenendaal, M. A. K., B.; Jonkman, H. T.; Sawatzky, G. A. Band gap, excitons, and Coulomb interaction in solid C60. Phys. Rev. Lett. 1992, 68, 3924–3927. (3) Arbogast, J. W.; Darmanyan, A. P.; Foote, C. S.; Rubin, Y.; Diederich, F. N.; Alvarez, M. M.; Anz, S. J.; Whetten, R. L. Photophysical properties of C60. J. Phys. Chem. 1991, 95, 11–12. (4) Beeby, A.; Eastoe, J.; Heenan, R. K. Solubilization of C60 in aqueous micellar solution. J. Chem. Soc. Chem. Communs. 1994, 173–175. (5) Belousov, V. P.; Belousova, I. M.; Krisko, A. V.; Krisko, T. K.; Muraveva, T. D.; Sirotkin, A. K. Aqueous micellar solution C60: Preparation, properties, and capability for generation of singlet oxygen. Russ. J. Gen. Chem. 2006, 76, 251–257. (6) Eastoe, J.; Crooks, E. R.; Beeby, A.; Heenan, R. K. Structure and photophysics in C60 micellar solutions. Chem. Phys. Lett. 1995, 245, 571–577. (7) Lee, J.; Fortner, J. D.; Hughes, J. B.; Kim, J.-H. Photochemical production of reactive oxygen species by C60 in the aqueous phase during UV irradiation. Environ. Sci. Technol. 2007, 41, 2529–2535. (8) Yamakoshi, Y.; Sueyoshi, S.; Fukuhara, K.; Miyata, N. OH and O2- generation in aqueous C60 and C70 solutions by photoirradiation: An EPR study. J. Am. Chem. Soc. 1998, 120, 12363– 12364. (9) Yamakoshi, Y.; Umezawa, N.; Ryu, A.; Arakane, K.; Miyata, N.; Goda, Y.; Masumizu, T.; Nagano, T. Active oxygen species generated from photoexcited fullerene C60 as potential medicines: O2- versus 1O2. J. Am. Chem. Soc. 2003, 125, 12803–12809. (10) Bosi, S.; Da Ros, T.; Spalluto, G.; Prato, M. Fullerene derivatives: an attractive tool for biological applications. Eur. J. Med. Chem. 2003, 38, 913–923. (11) Iwamoto, Y.; Yamakoshi, Y. A highly water-soluble C60-NVP copolymer: a potential material for photodynamic therapy. Chem. Commun. 2006, 4805–4807. (12) Kasermann, F.; Kempf, C. Buckminsterfullerene and photodynamic inactivation of viruses. Rev. Med. Virol. 1998, 8, 143–151. (13) Andrievsky, G. V.; Kosevich, M. V.; Vovk, O. M.; Shelkovsky, V. S.; Vashchenko, L. A. On the production of an aqueous colloidal solution of fullerenes. J. Chem. Soc. Chem. Communs. 1995, 1281–1282. (14) Deguchi, S.; Alargova, R. G.; Tsujii, K. Stable dispersions of fullerenes, C60 and C70, in water. Preparation and characterization. Langmuir 2001, 17, 6013–6017. (15) Fortner, J. D.; Lyon, D. Y.; Sayes, C. M.; Boyd, A. M.; Falkner, J. C.; Hotze, E. M.; Alemany, L. B.; Tao, Y. J.; Guo, W.; Ausman, K. D.; Colvin, V. L.; Hughes, J. B. C60 in water: nanocrystal formation and microbial response. Environ. Sci. Technol. 2005, 39, 4307–4316.

(16) Andersson, T.; Nilsson, K.; Sundahl, M.; Westman, G.; Wennerstrom, O. C60 embedded in r-cyclodextrin—a water-soluble fullerene. J. Chem. Soc. Chem. Communs 1992, 604–606. (17) Hungerbuhler, H.; Guldi, D. M.; Asmus, K. D. Incorporation of C60 into artificial lipid membranes. J. Am. Chem. Soc. 1993, 115, 3386–3387. (18) Yamakoshi, Y. N.; Yagami, T.; Fukuhara, K.; Sueyoshi, S.; Miyata, N. Solubilization of fullerenes into water with polyvinylpyrrolidone applicable to biological tests. J. Chem. Soc. Chem. Communs. 1994, 517–518. (19) Chiang, L. Y.; Swirczewski, J. W.; Hsu, C. S.; Chowdhury, S. K.; Cameron, S.; Creegan, K. Multihydroxy additions onto C60 fullerene molecules. J. Chem. Soc. Chem. Communs. 1992, 1791– 1793. (20) Tokuyama, H.; Yamago, S.; Nakamura, E.; Shiraki, T.; Sugiura, Y. Photoinduced biochemical activity of fullerene carboxylic acid. J. Am. Chem. Soc. 1993, 115, 7918–7919. (21) Guldi, D. M.; Prato, M. Excited-state properties of C60 fullerene derivatives. Acc. Chem. Res. 2000, 33, 695–703. (22) Irie, K.; Nakamura, Y.; Ohigashi, H.; Tokuyama, H.; Yamago, S.; Nakamura, E. Photocytotoxicity of water-soluble fullerene derivatives. Biosci. Biotechnol. Biochem. 1996, 60, 1359–1361. (23) Kamat, J. P.; Devasagayam, T. P. A.; Priyadarsini, K. I.; Mohan, H.; Mittal, J. P. Oxidative Damage Induced by the Fullerene C60 on Photosensitization in Rat Liver Microsomes. Chem. Biol. Interact. 1998, 114, 145–159. (24) Sayes, C. M.; Fortner, J. D.; Guo, W.; Lyon, D.; Boyd, A. M.; Ausman, K. D.; Tao, Y. J.; Sitharaman, B.; Wilson, L. J.; Hughes, J. B.; West, J. L.; Colvin, V. L. The differential cytotoxicity of water-soluble fullerenes. Nano Lett. 2004, 4, 1881–1887. (25) Brant, J.; Lecoanet, H.; Hotze, M.; Wiesner, M. Comparison of electrokinetic properties of colloidal fullerenes (nC60) formed using two procedures. Environ. Sci. Technol. 2005, 39, 6343– 6351. (26) Dhawan, A.; Taurozzi, J. S.; Pandey, A. K.; Shan, W. Q.; Miller, S. M.; Hashsham, S. A.; Tarabara, V. V. Stable colloidal dispersions of C60 fullerenes in water: Evidence for genotoxicity. Environ. Sci. Technol. 2006, 40, 7394–7401. (27) Dimitrijevic, N. M.; Kamat, P. V. Triplet excited state behavior of fullerenes—pulse radiolysis and laser flash photolysis of C60 and C70 in benzene. J. Phys. Chem. 1992, 96, 4811–4814. (28) Ebbesen, T. W.; Tanigaki, K.; Kuroshima, S. Excited-state properties of C60. Chem. Phys. Lett. 1991, 181, 501–504. (29) Fraelich, M. R.; Weisman, R. B. Triplet-states of C60 and C70 in solution—long intrinsic lifetimes and energy pooling. J. Phys. Chem. 1993, 97, 11145–11147. (30) Bensasson, R. V.; Bienvenue, E.; Dellinger, M.; Leach, S.; Seta, P. C60 in model biological systems—a Visible-UV absorption study of solvent-dependent parameters and solute aggregation. J. Phys. Chem. 1994, 98, 3492–3500. (31) Qiao, R.; Ke, P. C. Lipid-carbon nanotube self-assembly in aqueous solution. J. Am. Chem. Soc. 2006, 128, 13656–13657. (32) Conte, P.; Agretto, A.; Spaccini, R.; Piccolo, A. Soil remediation: humic acids as natural surfactants in the washings of highly contaminated soils. Environ. Pollut. 2005, 135, 515–522. (33) Gayathri, S. S.; Agarwal, A. K.; Suresh, K. A.; Patnaik, A. Structure and dynamics in solvent-polarity-induced aggregates from a C60 fullerene-based dyad. Langmuir 2005, 21, 12139–12145. (34) Larsson, S.; Volosov, A.; Rosen, A. Optical-Spectrum of the Icosahedral C60—Follene-60. Chem. Phys. Lett. 1987, 137, 501– 504. (35) Mukerjee, P.; Mysels, K. J., Critical Micelle Concentrations of Aqueous Surfactant Systems. In National Bureau of Standards: Washington DC, 1971; pp 51–185. (36) Barazzouk, S.; Hotchandani, S.; Kamat, P. V. Nanostructured fullerene films. Adv. Mater. 2001, 13, 1614-+. (37) Beck, M. T. Solubility and molecular state of C60 and C70 in solvents and solvent mixtures. Pure Appl. Chem. 1998, 70, 1881–1887.

ES702552A

VOL. 42, NO. 5, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

1557