ZnS Quantum Dots in

Feb 4, 2010 - Uptake, Translocation, and Transformation of Quantum Dots with Cationic versus Anionic Coatings by Populus deltoides × nigra Cuttings. ...
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Environ. Sci. Technol. 2010, 44, 1841–1846

Quantification of Water Solubilized CdSe/ZnS Quantum Dots in Daphnia magna NASTASSJA A. LEWINSKI,† HUIGUANG ZHU,‡ HUN-JE JO,§ DON PHAM,§ RASHMI R. KAMATH,† CLARE R. OUYANG,† CHRISTOPHER D. VULPE,§ VICKI L. COLVIN,‡ AND R E B E K A H A . D R E Z E K * ,† Department of Bioengineering, Rice University, Houston, Texas, Department of Chemistry, Rice University, Houston, Texas, Department of Nutritional Science and Toxicology, University of California at Berkeley, Berkeley, California

Received September 13, 2009. Revised manuscript received January 11, 2010. Accepted January 20, 2010.

The relative transparency of Daphnia magna (daphnia) and the unique optical properties of quantum dots (QDs) were paired to study the accumulation potential and surface coating effects on uptake of amphiphilic polymer coated CdSe/ZnS QDs. Fluorescence confocal laser scanning microscopy was used to visualize and spectrally distinguish QDs from competing autofluorescent signals arising from the daphnia themselves and their food sources. QDs were found to accumulate within the digestive tracts of daphnia, as well as, in some cases, adhere to the carapace, antennae, and thoracic appendages. After 48 h of gut clearance with and without feeding, QD fluorescence signal was still apparent in the digestive tracts of daphnia, and inductively coupled plasma mass spectrometry (ICP-MS) measurements confirmed that 36-53% of the initial uptake was retained. As surface charge and pegylation can influence the uptake of nanoparticles, uptake of QDs coatedwithtwodifferentamphililicpolymersandtheirpolyethylene glycol (PEG) coated counterparts was also examined. Fluorescence microscopy and ICP-MS measurements revealed differences in uptake after 24 h of exposure which were attributed to particle surface coating and stability.

Introduction Because of their distinct optical properties, quantum dots (QDs) are being extensively developed for biomedical use as imaging contrast agents and traceable therapeutic vectors and for energy applications including photovoltaic solar cells (1-3). QD optical properties include their size dependent band gap which enables emission wavelength tunability and resistance to photobleaching (4, 5). High photostability makes QDs advantageous over conventional fluorophores for longterm tracking; however, due to toxicity and degradation concerns, their use has been limited to in vitro and small animal models (6, 7). Several in vivo biodistribution studies using QDs in mouse models to understand nanoparticle * Corresponding author e-mail: [email protected]. † Department of Bioengineering, Rice University. ‡ Department of Chemistry, Rice University. § University of California at Berkeley. 10.1021/es902728a

 2010 American Chemical Society

Published on Web 02/04/2010

delivery and human health implications have been published (8-10). For environmental implications, the release of heavy metals into the environment due to increased QD use is also of concern, and there is a need for more studies examining the biodistribution of QDs in aquatic species. In both cases, the mechanisms behind tissue uptake and accumulation of QDs have yet to be determined, and it may be possible to gain insights into QD biodistribution through the study of simpler model systems. The freshwater crustacean Daphnia magna, also known as the water flea, is a standard model system for aquatic toxicity studies as they are sensitive to changes in their environment, such as the presence of hazardous chemicals (11, 12). Daphnia serve as a unique model organism since they are small (adult body length ranging between 2 and 5 mm) and relatively transparent, making them advantageous for whole organism microscope imaging. Although published studies have utilized daphnia in assessing aquatic toxicity of different nanomaterials, little is known of end points other than mortality for daphnia exposure to nanomaterials (13-15). For daphnia, routes of exposure to nanomaterials would include external contact with or ingestion by filter feeding of nanoparticles contained in the water. We anticipated QDs would be readily ingested and assuming the QDs are indigestible, hypothesized that they would be excreted with little accumulation. The aim of this study is to utilize the unique optical properties of QDs to study nanoparticle uptake and elimination in daphnia. Only a handful of studies have been published on QD uptake in daphnia, and although mostly qualitative, they demonstrate the value of fluorescence microscopy for QD localization in the organism (16-18). We first characterized the sources of competing fluorescent signals. A clear understanding of the endogenous fluorescence sources can provide insight valuable to improving the utility of fluorescence microscopy for fluorescent nanoparticle localization in daphnia. For simplicity, we chose QDs with a fluorescence emission peak that did not overlap with the endogenous signals to better distinguish the QD fluorescence when taking images. Fluorescence imaging paired with inductively coupled plasma mass spectrometry (ICPMS) Cd concentration measurements was used to study gut clearance of QDs after removal from exposure conditions. As algal feeding has been suggested to facilitate clearance of materials in the digestive tracts of daphnia, both feeding and no feeding clearance conditions were tested to determine their influence on the gut clearance of QDs (19). In addition, we hypothesized that the QD surface functionality would influence their distribution and examined the surface coating effects on uptake using four surface coatings, two amphiphilic polymer coatings (a poly(acrylic acid)-octylamine amphiphilic copolymer (PAA) and poly(maleic anhydride-alt1-octadecene) (PMAO)) with and without polyethylene glycol (PEG) conjugation.

Experimental Section Synthesis and Characterization of Water-Soluble Quantum Dots. The method for CdSe/ZnS QDs synthesis was adapted from previous work (20, 21). See Supporting Information for further synthesis details. Absorption spectra were used to calculate the core size and concentration of QDs (5). Two amphiphilic polymers, poly(acrylic acid)-octylamine copolymer (PAA, MW ) 1800) and poly(maleic anhydride-alt-1octadecene) (PMAO) (Mn ) 30,000-50,000), were used to transfer the QDs from organic to aqueous solution. MethylPEG-amine (PEG, MW ) 1000) was further conjugated to PAA VOL. 44, NO. 5, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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or PMAO coated QDs as previously reported (22). QD core diameter was determined via transmission electron microscopy. Hydrodynamic diameter and zeta potential were determined using dynamic and phase analysis light scattering, respectively (Malvern Zetasizer Nano-ZS). Daphnia magna Culture and QDs Exposures. Daphnia were cultured according to U.S. EPA standard operating procedure with a 16 h light, 8 h dark cycle in moderately hard synthetic freshwater (MHW) (11). Daphnia were fed daily and the culture water was changed every 2-3 days. Water temperatures were 22 ( 1 °C. Acute lethal concentration 50 (LC50) levels were determined following the standard EPA protocol (11). Briefly, triplicate runs using 10 neonate (10 days old) per group in 100 mL beakers after a 24 h starvation period. Daphnia were exposed for 24 h to MHW containing 7.7 nM watersolubilized CdSe/ZnS QDs (4.63 × 1012 particles/mL) with [Cd] ) 0.6 ppm. Four different surface coatings, PAA, PAAPEG, PMAO, and PMAO-PEG, were tested, and the pH of the exposure water varied between 7.2 and 7.6. QD Gut Clearance Experiments. After 24 h exposure to PAA coated QDs, daphnia were removed to clean MHW or clean MHW with algae, Pseudokirchneriella capricornutum, at 2.8 × 108 cells/L. Triplicate samples of 10 adult daphnia were removed and fixed with 4% formaldehyde at 0, 4, 8, 24, and 48 h time points. Fluorescence confocal laser-scanning microscopy was used to observe QD localization in whole, exposed daphnia and then the samples (10 daphnia each) were transferred to preweighed glass vials for ICP-MS preparation. Briefly, the samples were digested in 0.5 mL of 69% trace metal grade HNO3 for 30 min at 60-90 °C and then overnight at room temperature. The acid digests were diluted with 9 mL of Milli-Q water, collected using a syringe, and filtered through a 0.45 µm polyethersulfone (PES) filter into a 10 mL volumetric flask. The final HNO3 concentration after dilution to 10 mL was ∼3.5%. The Cd concentration in each sample was determined by ICP-MS. Average Cd concentrations for the 3 replicates were reported. Fluorescence Spectroscopy and Confocal Laser-Scanning Microscopy. Fluorescence excitation-emission matrixes were measured with a Horiba Jobin Yvon SPEX FL3-22 spectrofluorometer (slit width 5 mm, 450 W Xe lamp) using standard 1 cm path length quartz cuvettes. The emission intensity for each sample was measured for excitation wavelengths ranging from 250 to 600 nm. A background scan was performed using the synthetic freshwater in which the samples were suspended. Imaging was performed on a Zeiss LSM 510 laser scanning confocal microscope (LSM) utilizing a 4× objective with numerical aperture (NA) of 0.13. The samples were excited with 488 nm light from a 30-mW argon/ two laser set at 50% output, corresponding to a tube current of 5.5-5.7 A. 12-bit 512 × 512 images, corresponding to 5.1 mm2, were obtained with 50% laser transmission, and with a pixel dwell time of 1.60 µs. The emission signal was passed through a main dichroic to eliminate the excitation light and a secondary beamsplitter, which passed light above 490 nm. The emission signal was then directed through 500-550 nm, 565-615 nm, and 650-710 nm bandpass filters or directed to the META detector. The detector gain was set between 500 and 700 with an amplifier offset of -0.1 and an amplifier gain of 1. At these imaging settings, the detection limit of QDs in solution was around 130 nM QDs (7.7 × 1013 particles/ mL) with [Cd] ) 10 ppm. Postacquisition image processing, including false coloring, spectra extraction, and overlaying images, was carried out in LSM 510 Image Browser. 1842

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Results and Discussion Fluorescence Characterization. To determine the sources of endogenous fluorescence, fluorescence excitationemission matrices (EEMs) of daphnia, spirulina, algae, and amphiphilic polymer coated CdSe/ZnS QDs were collected and are presented in Figure 1A-D. The fluorescence EEM from starved Daphnia magna revealed three peaks, attributed to tryptophan (located at 280/350 nm excitation/emission) and NADH (located at both 270/460 nm and 360/460 nm excitation/emission) (23, 24). In addition to these peaks, Spirulina platensis fluorescence included peaks attributed to phycocyanin (located at 360/650 nm and 600/650 nm excitation/emission) (25). As the spirulina phycocyanin peak was not present in the daphnia EEM, we assume that the daphnia EEM represents the endogenous fluorescence from the daphnia only. For the green algae Pseudokirchneriella capricornutum, chlorophyll a serves as the endogenous fluorophore with an emission peak around 680 nm (26, 27). To ensure no overlap in fluorescence signal, QDs with emission peak at 600 nm were synthesized as the fluorescence signal near this emission wavelength was low in the daphnia, spirulina, and algae EEMs. Figure 2 presents a representative TEM image and UV-vis absorbance and fluorescence spectra from PAA coated QDs. Quantum Dot Localization. To confirm that the QDs could be distinguished from food present in the daphnia gut, initial fluorescence images were taken from daphnia that were starved, fed spirulina only, fed QD only, and fed spirulina + QD. Representative images of the fluorescence collected from the daphnia using three band-pass emission filters (500-550 nm, 565-615 nm, and 650-710 nm) are given in Figure S1 in the Supporting Information. Spirulina was chosen as the food source for this part of the experiment as spirulina exhibited more autofluorescence compared to the algae in the collected EEMs. Minimal fluorescence was observed from the starved daphnia (Figure S1A) while spirulina only (Figure S1B), QD only (Figure S1C), and spirulina + QD (Figure S1D) fed daphnia exhibited fluorescence, with stronger signal observed in the digestive tract region for emission wavelengths above 565 nm. Comparing the QD only (Figure S1C) and spirulina + QD (Figure S1D) fed daphnia, fluorescence signal was present in both the 565-615 nm band containing the QD signal (∼600 nm) and the 650-710 nm band containing the spirulina signal (∼650 nm); however, only the 565-615 nm band exhibited signal in the thoracic appendages. Similar findings have been reported for Ceriodaphnia dubia exposed to QDs (17). Since these organs are exposed to the surrounding media, the presence of QDs was expected due to their addition to the water; however, with rigorous pipetting during rinsing, strong fluorescence signal attributed to QD aggregates generally found coating the carapace, antennae, and thoracic appendages of the daphnia can be minimized in most cases. For the spirulina + QD fed daphnia (Figure S1D), the qualitative fluorescence intensity in the digestive tract region for emission wavelengths 565-615 nm and 650-710 nm were relatively similar. To further confirm the fluorescence signal observed in the digestive tract of spirulina + QD fed daphnia in these two emission bands were from two distinct fluorophores (QD and phycocyanin), lambda scans using the LSM META detector were acquired which allowed for generation of fluorescence spectra from the sample fluorescence image series. Mean region of interest (ROI) images from the lambda scans corresponding to the same daphnia are also given in Figures S1. As shown in Figure S1C-D, daphnia exposed to QDs had a relatively strong fluorescence peak at 600 nm present in the digestive tract and thoracic appendage regions with emission peaks at both 600 nm and around 660 nm found in the daphnia exposed to both QDs and spirulina.

FIGURE 1. Fluorescence excitation emission matrixes for (A) Daphnia magna, (B) Spirulina platensis, (C) Pseudokirchneriella capricorntum, (D) water-solublized CdSe/ZnS quantum dots with normalized intensities. Peaks include tryptophan at 280/350 nm (excitation/emission), NADH at both 270/460 nm and 360/460 nm, phycocyanin (in spirulina) at 360/650 nm and 600/650 nm, chlorophyll a (in green algae) at 680 nm emission, and CdSe/ZnS QDs at 600 nm emission.

FIGURE 2. Representative TEM image of PAA coated CdSe/ZnS QDs (left, scale bar ) 50 nm) with a measured core diameter of 5.2 ( 0.4 nm and UV-vis absorbance and fluorescence spectra (right) on normalized scale. Peak absorbance wavelength ) 584 nm and peak emission wavelength ) 600 nm. QD Gut Clearance and Accumulation. With the QDs clearly distinguishable from the food, gut clearance was examined from daphnia under both feeding and nonfeeding conditions. Feeding was hypothesized to facilitate QD clearance from the digestive tracts of the daphnia as suggested in the literature (19). The exposure concentration used, 7.7 nM QD (4.63 × 1012 particles/mL) with [Cd] ) 0.6 ppm, was chosen to be high enough to ensure QD uptake but also be a sublethal concentration. For the PAA coated QDs used, a LC50 value could not be determined for the concentration range tested, with the maximum concentration tested being 25.6 nM QD (1.54 × 1013 particles/mL) with [Cd] ) 2 ppm. Excretion of QDs from exposed daphnia was the focus of this

study as literature suggests that gut filling occurs within 30 min of exposure (28, 29). For all purging time points, 10 daphnia, with 9 in a few cases, were alive at collection. Fluorescence microscopy revealed the presence of QD fluorescence signal in the digestive tract for all daphnia regardless of the purging duration. Representative images are presented in Figure 3A-B. After 48 h in clean media without QDs, QD fluorescence signal was still observed in the digestive tracts of the daphnia under both feeding conditions. This suggests that the QDs were not completely eliminated from the digestive tract after 48 h. No apparent shift in the emission peak was observed in the lambda scan spectra. This suggests that the QD cores were still intact. VOL. 44, NO. 5, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Brightfield and fluorescence (488 nm excitation, 565-615 nm emission, false color) images obtained from adult daphnia exposed for 24 h to 7.7 nM QD after purging in (A) MHW with algae, (B) MHW without algae. Magnification at 4×. (C) Average total amount of Cd measured using ICP-MS from adult daphnia after removal from exposure; red ) algae fed daphnia, blue ) unfed daphnia. Although Figure 3 only presents images at t ) 0, 24, and 48 h, the images at t ) 4 and 8 also revealed consistent fluorescence signal within the gut, with stronger intensity visible in the tail end. The reduced fluorescence in the fore gut and the higher fluorescence in the hind gut suggests that excretion occurred. These results align with studies on C60 and gold nanoparticles. For C60, Baun et al. reported that the lower section of the digestive tract contained C60 after a 48 h depuration period in clean media (30). For gold nanoparticles, Lovern et al. reported a shift in high numbers of gold nanoparticles from the mouth to tail region after a 24 h purging duration (31). However, while some retention of QDs in the exposed daphnia after 48 h in clean media was expected for the unfed daphnia, this was not expected in the fed case. Barata et al. found that Cd contaminated algae would clear from the gut of daphnia in 3-6 h and Gilles et al. recommends only 8 h for gut clearance (19, 32). Since QD fluorescence signal was still present after 48 h in the daphnia fed algae during depuration, QDs may have an increased gut passage time or may be adhering to the intestinal wall. Containment of observed fluorescence suggests QDs are not being assimilated; however, further study is needed to confirm this hypothesis. As shown in Figure 3C, ICP-MS measurements revealed a decrease in the total Cd content with longer purging times. Cadmium was measured since our hypothesis was that Cd release was a primary source of toxicity from quantum dot exposure. In addition, Cd is a nonessential metal, and the levels observed were expected to be due to exposure to QDs only. The average total Cd for the algae fed and unfed daphnia controls was low with algae fed containing 3 ( 1 ng Cd/ daphnia and unfed containing 2 ( 0.2 ng Cd/daphnia. For the daphnia exposed to QDs for 24 h, the average total Cd 1844

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was elevated compared to the controls. On average, the Cd present was 15 times higher than the unfed daphnia control and 9 times higher than the fed daphnia control immediately (t ) 0 h) after removing them from exposure. The average total Cd at t ) 48 h was also elevated compared to controls, further corroborating some retention of QDs. Throughout the gut clearance duration, the differences between the average total Cd levels measured from the algae fed daphnia and the respective unfed cases were not statistically significant (p > 0.05, Wilcoxon rank sum). This suggests that under both feeding and nonfeeding conditions, the daphnia eliminated the QDs at similar rates. In both cases, QDs were not completely eliminated from the digestive tract after 48 h. Comparing the average total Cd amounts at t ) 48 h to t ) 0 h, 53% of the initial Cd was retained in the algae fed case while 36% was retained in the unfed case. Petersen et al. reported similar depuration findings for carbon nanotubes in daphnia; carbon nanotubes were not completely purged in daphnia after removal to clean media and 48 h of algae feeding (33). Further study is needed to determine if complete clearance would occur after longer purging durations. Surface Coating Effect on Uptake. Daphnia were exposed to three additional surface coatings (PAA-PEG, PMAO, PMAOPEG) to explore surface coating effects on uptake. Characterization data are presented in Figure 4. For all coatings, QD fluorescence was located primarily in the intestines with some fluorescence observed in the thoracic appendage area. Dissecting the intestines from the daphnia and measuring the Cd from the intestines and remaining carcass separately with ICP-MS revealed 90% or more of the total Cd reported in Figure 4 was associated with the intestines. In addition, the fluorescence images suggest the PMAO and PMAO-PEG

FIGURE 4. Comparison of surface coating effect on uptake from 4 different surface coatings. QD characterization data (left) was measured from stock solution at pH ) 10. Diameter values represent number average hydrodynamic diameter. Representative fluorescence images (right) of the exposed daphnia with excitation wavelength ) 488 nm, band-pass emission filters, false color, magnification at 4×. In coded overlay images, green ) 500-550 nm emission, red ) 565-615 nm emission, blue ) 650-710 nm emission. coated QDs had significantly higher uptake after 24 h of exposure compared to the PAA and PAA-PEG coated QDs. ICP-MS measurements confirmed the higher uptake observed and revealed a difference in average total Cd amounts present in daphnia exposed to PMAO coated QDs (128 ( 20 ng Cd/ daphnia) compared to daphnia exposed to PMAO-PEG coated QDs (41 ( 7 ng Cd/daphnia). The PEG coated particles were anticipated to exhibit less uptake and toxicity due to the reduction in the negative surface charge, inhibiting the electrostatic attraction between QDs and biological particulates (34). In addition to lower uptake, the effect on toxicity was also apparent with a LC50 value of 3.1 nM QD (1.89 × 1012 particles/mL) with [Cd] ) 0.244 ppm determined for QDs coated with PMAO alone while LC50 values were not reached up to 25.6 nM QD (1.54 × 1013 particles/mL) with [Cd] ) 2 ppm for the PEG conjugated PMAO coated QDs. The stability of QD suspensions in daphnia media was also assessed using dynamic light scattering. Aggregates greater than 100 nm were measured in water samples taken from QD contaminated daphnia media before exposure with larger aggregates reaching micrometer size measured for the PMAO coated QDs compared to the other three coatings. The observed increased uptake for PMAO coated QDs could be due to reduced particle stability and aggregation. While daphnia filter particulates from the water indiscriminately, it has been suggested that daphnia tend to retain larger particles while smaller particles are retained less efficiently (35). One theory of particle movement by daphnia proposes that particulates are filtered from water via mechanical sieving, with particle uptake correlated with filter mesh size (36). Daphnia have been identified as fine mesh filter feeders with their filter mesh size remaining the same throughout their growth (37). Food particles are captured by setae, or filter combs, on the thoracic appendages, removed by longer bristles and transported to the food groove. The size range of particles filtered by Daphnia magna includes 0.6-40 µm particles, and particles smaller than 5 µm have lower uptake compared to larger particles (37). While nanoparticles could be captured by “direct interception, inertial impaction,

gravitational deposition and diffusion deposition,” they could be lost during transport to the food groove (38). Therefore the increased uptake observed could be correlated to aggregation, with better dispersed QDs, such as the ones coated with PEG, having lower retention. Ingestion by the daphnia could also influence particle stability as suggested by Roberts et al. who hypothesized that daphnia altered the solubility of SWNTs by digesting the LPC coating and excreting insoluble SWNTs (39). In the presence of daphnia, visible QD sediments were observed in the PMAO coated QD solution within an hour. No visible QD sediments were observed for the other three coatings. Sedimentation also occurred in solutions with PMAO coated QDs only but were observed later, after 24 h. This accelerated sedimentation induced by the daphnia could be due to adsorption to organic matter from the daphnia (e.g., carapace molts, fecal material), ingestion with concentration of the particles in the digestive tract, or potential alteration by digestive enzymes. If any biodegradation occurred, the fluorescence images collected suggest that the QD cores were unaffected as fluorescence signal was still detected and the emission peak was stable. It is important to note that the daphnia exposed to PMAO coated QDs were the only ones that exhibited mortality, with an average of 20% survival, after the 24 h exposure period. The dead daphnia were often found buried in the QD sediments, and it is believed that the aggregation of the QDs on the carapace, antennae, and thoracic appendages inhibited movement and contributed to the mortality observed. The average total Cd value presented in Figure 4 represents a mixture of live and dead daphnia at collection. Separate measurements from 10 live and 10 dead daphnia after PMAO coated QD exposure confirmed that mortality did not influence the Cd measurements. Real-life exposure conditions will most likely involve significantly lower QD concentrations; therefore, further investigation is needed to evaluate the ability of confocal fluorescence microscopy and ICP-MS to detect environmentally relevant concentrations of QD exposure in freshVOL. 44, NO. 5, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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water organisms such as D. magna. Since strong fluorescence signal was collected from the digestive tracts of the exposed daphnia, this indicates that the QDs are concentrated or accumulated within the daphnia and suggests that this may occur at lower QD concentrations as well. Since the QDs were not completely eliminated after 48 h of purging, this suggests some QD retention and potential accumulation in the organisms over time. Further investigation is needed to determine if the QDs are being absorbed through the intestinal wall and entering the organism.

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Acknowledgments We thank Robert Langsner for his editorial assistance. This work was supported in part by the Center for Biological and Environmental Nanotechnology (NSF Award EEC-0118007) and in part by a NSF Graduate Research Fellowship to N.A.L.

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Supporting Information Available Details of experimental methods and additional fluorescence images. This material is available free of charge via the Internet at http://pubs.acs.org.

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