Chemosphere 112 (2014) 92–99

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The interactions between CdSe quantum dots and yeast Saccharomyces cerevisiae: Adhesion of quantum dots to the cell surface and the protection effect of ZnS shell Jie Mei a, Li-Yun Yang a, Lu Lai a, Zi-Qiang Xu a, Can Wang b, Jie Zhao a, Jian-Cheng Jin a, Feng-Lei Jiang a,⇑, Yi Liu a,⇑ a State Key Laboratory of Virology & Key Laboratory of Analytical Chemistry for Biology and Medicine (MOE), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, PR China b College of Life Science and Chemistry, Wuhan Donghu University, Wuhan 430212, PR China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Detailed comparison of the biological

The biological effects of CdSe and CdSe/ZnS QDs (ZnS: protection shell) on yeast Saccharomyces cerevisiae were investigated via microcalorimetric, spectroscopic and microscopic methods, clearly demonstrating a toxic order CdSe > CdSe/ZnS QDs.

effect between CdSe QDs and CdSe/ ZnS QDs on yeast Saccharomyces cerevisiae.  Carboxyl-QDs adhere to the surface of S. cerevisiae.  Mechanism of toxicity induced by heavy metal ion was investigated.  Epitaxial coating of ZnS shell efficiently reduces the toxicity of Cdcontaining QDs.

a r t i c l e

i n f o

Article history: Received 20 January 2014 Received in revised form 13 March 2014 Accepted 16 March 2014

Handling Editor: S. Jobling Keywords: CdSe QDs Yeast Biological effect Toxicity Microcalorimetry

a b s t r a c t The interactions between quantum dots (QDs) and biological systems have attracted increasing attention due to concerns on possible toxicity of the nanoscale materials. The biological effects of CdSe QDs and CdSe/ZnS QDs with nearly identical hydrodynamic size on Saccharomyces cerevisiae were investigated via microcalorimetric, spectroscopic and microscopic methods, demonstrating a toxic order CdSe > CdSe/ZnS QDs. CdSe QDs damaged yeast cell wall and reduced the mitochondrial membrane potential. Noteworthy, adhesion of QDs to the yeast cell surface renders this work a good example of interaction site at cell surface, and the epitaxial coating of ZnS could greatly reduce the toxicity of Cd-containing QDs. These results will contribute to the safety evaluation of quantum dots, and provide valuable information for design of nanomaterials. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction ⇑ Corresponding authors. Tel.: +86 27 68756667; fax: +86 27 6854067. E-mail addresses: fl[email protected] (F.-L. Jiang), [email protected] (Y. Liu). http://dx.doi.org/10.1016/j.chemosphere.2014.03.071 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.

Quantum dots (QDs), namely semiconductor nanocrystals, have gained considerable attention owing to their unique size-dependent optical and electronic properties that make them attractive

J. Mei et al. / Chemosphere 112 (2014) 92–99

for a wide range of applications, such as biomedical imaging, DNA detection, drug delivery, solar energy generation, and electronics industries (Murray et al., 2000; Zhang et al., 2005; Portney and Ozkan, 2006; Kamat, 2008; Delehanty et al., 2009; Shirasaki et al., 2012). Nevertheless, the possible threats to human health and environment have also attracted increasing attention as the production and applications of QDs increase rapidly while standard evaluation of the safety is lack to some extent (Holbrook et al., 2008; Teow et al., 2011; Tsoi et al., 2012). Hence, clear elucidation of the relationship between biological effects and the unique properties of nanomaterials, e.g. size, shape, surface coatings, etc. for better design and syntheses with good biocompatibility has become a hot topic (Zrazhevskiy et al., 2010; Rivera-Gil et al., 2012). The diverse types of QDs bring certain difficulties for toxicological evaluation because each individual type of QDs owns its unique physicochemical properties, which will dominate its interactions with biological systems. Consequently, it is necessary to continuously study the biological effects of QDs on different organisms and the possible mechanisms. Previous studies indicated that the toxicity of QDs comes from the leakage of heavy metal ions (Derfus et al., 2004; Lewinski et al., 2008), the enhancement of reactive oxygen species (ROS) level (Slaveykova et al., 2009; Mahto et al., 2010), and other causes (Chang et al., 2006; Hardman, 2006). For heavy metal-containing QDs, such as cadmium selenide (CdSe) and cadmium telluride (CdTe), both of which bear excellent optical properties, e.g. really high quantum yields over 60%, the release of toxic metal ions causes cellular toxicity. Several studies have evidenced the inconspicuous toxicity of QDs in several cellular models (Voura et al., 2004; Chaves et al., 2008; Zhang et al., 2010). Pace et al. (2010) showed that QDs with thiol stabilizer induced toxicity on Daphnia magna caused by releasing Cd2+. Contradictory results obtained from Priester et al. (2009) demonstrated that QDs themselves were more toxic to planktonic Pseudomonas aeruginosa PG201 than cadmium ions, suggesting that the release of Cd2+ is not the exclusive factor. In addition, other studies pointed out that QDs themselves could also induce cellular damage besides the leakage of heavy metal ions (Male et al., 2008; Liu et al., 2011; Aye et al., 2012). Moreover, physicochemical properties of QDs such as chemical composition (Cho et al., 2007), size (Duan and Nie, 2007), dosage (Ryman-Rasmussen et al., 2007), surface charge (Geys et al., 2008) and surface coating (Nabiev et al., 2007) leaded to toxicity. Furthermore, the stability of QDs was a vital factor to their toxicity. Mahendra et al. (2008) found that QDs were potentially safe materials at near-neutral pH but exerted toxicity under acidic or alkaline conditions. This was proven to be the weathering effect in extreme condition that destabilized QDs followed by release of the cadmium and selenite ions rapidly. In our previous studies, we have found CdTe QDs were toxic due to the released cadmium ions (Li et al., 2010), while sizes and surface coatings of QDs could influence their biological effects (Li et al., 2011; Han et al., 2012; Lai et al., 2012a,b; Xu et al., 2013). It is widely accepted that epitaxial growth of another material with wider band gap can make the lattice more stable as well as reduce the toxicity (Bao et al., 2011; Chen et al., 2012). Aaron et al. studied the interactions between CdSe QDs and immune cells, suggesting that their particle geometry significantly affected their interactions with the plasma membrane, uptake into cells, and localization within intracellular vesicles (Aaron et al., 2011). Gosso et al. used neurosectrtory mouse chromaffin cells of the adrenal gland for testing the effect of CdSe/ZnS QDs on Ca2+ channels functionality and Ca2+-dependent neurosecretion, showing that exposure to CdSe/ZnS QDs impaired Ca2+-influx and severely interfered with the functionality of the exocytotic machinery, thus compromised the overall catecholamine supply from chromaffin cells (Gosso et al., 2011). However, detailed comparison of the biological effects

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between CdSe QDs and CdSe/ZnS QDs is thus far less documented. In fact, the results from the comparisons will give valuable information for safe use of CdSe QDs in future. To address this issue, herein, by using Saccharomyces cerevisiae (S. cerevisiae) as a model eukaryotic organism, we attempt to evaluate and compare the toxicity of CdSe and CdSe/ZnS QDs, both of which are orange-emitting with similar core sizes as well as hydrodynamic sizes, and coated with amphiphilic polymer octylamine-modified polyacrylic acid (OPA) to become water-soluble. The biological effects of QDs on the metabolism of S. cerevisiae were determined by microcalorimetry, spectroscopy, transmission electron microscopy (TEM), confocal laser scanning microscopy and flow cytometry. The mechanism of CdSe QDs toxicity towards S. cerevisiae was investigated via spectroscopy, microcalorimetry, inductively coupled plasma atomic emission spectroscopy (ICP-AES) so as to determine the main threats that brought by the applications of QDs. 2. Materials and methods 2.1. Reagents Cadmium oxide (99.99%), selenium powder (99.99%, about 100 mesh), trioctylphosphine oxide (TOPO, 90%), hexadecylamine (HDA, 90%), zinc acetate (99.99%), 5,50 ,6,60 -terachloro-1,10 ,3,30 tetraethylbenzimidazolylcarbocyanine iodide (JC-1), hexamethyldisilathiane (90%) and CdCl2 (99.99%) were obtained from Sigma–Aldrich. Trioctylphosphine (TOP, 90%) was purchased from Alfa Aesar. Poly (acrylic acid) (PAA, Mw 1800) and n-Octylamine (99%) were obtained from Aladdin. All reagents were used without further purification. Deionized (DI) distilled water was prepared from a Milli-Q-RO4 water purification system (Millipore). 2.2. Preparation and characterization of water-soluble CdSe QDs The preparation of highly luminescent CdSe and CdSe/ZnS nanocrystals were synthesized as previously reported (Qu and Peng, 2002; Xie et al., 2005). Experimental details for preparation and characterization of CdSe and CdSe/ZnS QDs are described in the supporting information. The water-soluble QDs were coated with amphiphilic polymer octylamine-modified poyacrylic acid (OPA), which was synthesized according to Zhou’s method (Zhou et al., 2007). QDs concentrations were determined using the extinction coefficients according to a literature (Yu et al., 2003). The quantum yield (QY) values were determined by the following equation:

QYsample ¼ ðF sample =F ref Þ  ðAref =Asample Þ  ðn2sample =n2ref Þ  QYref where F, A, and n are the measured fluorescence (area under the emission peak), absorbance at the excitation wavelength and refractive index of the solvent respectively. PL spectra were spectrally corrected and quantum yields were determined relative to Rhodamine 6G (QY = 94%) (Grabolle et al., 2009). 2.3. Microorganism culture Yeast S. cerevisiae (BY4742) was provided by the microbial genetics laboratory, Wuhan University, PR China. The yeast extract peptone dextrose (YPED) medium was consisted of yeast extract (1%), peptone (1%) and glucose (2%) at natural pH, which was sterilized under high-pressure steam at 120 °C for 30 min. A single colony of yeast S. cerevisiae grown on YPED agar plates was inoculated in fresh medium and grown in a shaking incubator at 30 °C for 12 h. Subsequently, 1‰ inoculated yeast and QDs were incubated together at the beginning.

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2.4. Methods

3. Results and discussion

2.4.1. Microcalorimetry A TAM air isothermal microcalorimeter (Thermometric AB, Sweden), equipped with eight twin calorimetric channels, was used to record the heat flow rate of yeast cells. The structure and operation of the instrument have been described previously (Li et al., 2010). The microcalorimetric measurements was performed using the ampule method that sealed glass ampoules contained 4.0 mL of YPED medium incubated with yeast and different amount of CdSe QDs or CdSe/ZnS QDs. All experimental procedures were conducted at 30 °C.

3.1. Characterization of CdSe and CdSe/ZnS QDs

2.4.2. Optical density studies After 12 h cocultivation at 30 °C, the yeast pellets were harvested by centrifugation and washed with freshly prepared PBS buffer (pH = 7.4) three times. Finally, optical density at 600 nm was measured by a UV–vis absorption spectrophotometer.

2.4.3. Cell staining Yeast S. cerevisiae was incubated with QDs at 30 °C for 18 h and washed with PBS buffer three times. Cells were stained with methylene blue (final concentration 0.05%) for several minutes and observed through a phase contrast fluorescence microscope. The amount of live and dead cells was counted after co-culture of 12 h and 24 h.

2.4.4. Transmission electron microscopy Treated and untreated yeast cells were fixed for 30 min at 4 °C using glutaraldehyde at a final concentration of 2.5% in PBS buffer, postfixed with 1% osmium tetroxide and dehydrated. The ultrastructure of yeast cells thin section was observed with a FEI Tencnai G2 20 TWIN transmission electron microscope.

2.4.5. Mitochondrial membrane potential After 18 h incubation, microbes were washed twice with sterilized PBS buffer and the concentration of suspension was adjusted to 106 cells mL1. Assays were conducted according to the JC-1 Mitochondrial Membrane Potential Assay Kit (MultiSciences Biotech, PR China). The loss of mitochondrial membrane potential was determined by flow cytometry (FACSAriaTM III, Becton Dickinson, USA).

2.4.6. Laser scanning confocal microscopy Images were acquired by Perkin–Elmer Ultraview Vox confocal microscope equipped with a spectral detection system. Yeast incubated with QDs for 18 h was washed and observed through confocal microscope. Diode laser at 488 nm was used for excitation of QDs.

2.4.7. Inductively coupled plasma atomic emission spectroscopy (ICPAES) An ICP-AES (Thermo, USA) with a concentric model nebulizer and a cinnabar model spray chamber was used for the determination of cadmium. The samples, S. cerevisiae incubated with QDs for 12 h and 24 h, were centrifuged to detect the amount of Cd in the supernatant and yeast pellet. To detect the free cadmium ions, yeast cells that incubated with QDs for 18 h were successively filtered with a 0.45 lm and a 0.22 lm PES Membrane. After that, a process of ultrafiltration (30K Millipore) separated free cadmium ions from QDs. Each experiment has been repeated at least three times.

The two QDs, namely CdSe and CdSe/ZnS QDs, both exhibit bright orange fluorescence either in hexane or PBS (Fig. 1). The emission peaks of both QDs are at 595 nm (Fig. S1). The aqueous QDs have similar sizes: CdSe QDs (2r = 3.86 ± 0.17 nm, quantum yield, QY 20%) and CdSe/ZnS QDs (2r = 3.96 ± 0.14 nm, QY 35%), which are determined from empirical formula proposed by Peng et al. (Qu and Peng, 2002). Both samples exhibit wellresolved first electronic transition absorption maximum and narrow half-peak width, indicating they are of sufficiently narrow size distributions and nearly monodisperse (Zheng et al., 2007). The overview and high-resolution TEM images (Fig. S2) present similarly spherical shape of QDs and well-seperated nanocrystals with mean sizes of approximately 4 nm, well agreed with the sizes determined by empirical formula, while high-resolution TEM images also show distinctly resolved lattice fringes, indicating good quality of QDs prepared in organic solvents. After phase transfer from organic solvent to water, the hydrodynamic diameters of both QDs are about 11.7 nm determined by dynamic light scattering (DLS), which will render the comparison between the two QDs with no interference of the size of QDs. The powder Xray diffraction (XRD) spectra of QDs was given in Fig. S3. The Xray photoelectron spectra (XPS) obtained from the as-prepared CdSe and CdSe/ZnS QDs prove the compositions of the QDs (Fig. S4), while the Fourier transform infrared spectrum of surface coating polymer OPA is also measured (Fig. S5). 3.2. Metabolic thermogenic curves and thermodynamic parameters Microcalorimetry, a technique measuring the metabolism of living species by recording the growth power–time curves, has been proved to be efficient in evaluating the biological effects of nanomaterials. Modern isothermal microcalorimeters make it possible that the measurement is universal, integral, non-destructive, and highly sensitive. TAM air thermal activity monitor, detection limit less than a microwatt, was used to record the heat flow of yeast metabolism. The growth thermogenic curves of yeast S. cerevisiae affected by various amounts of CdSe and CdSe/ZnS QDs at 30 °C are shown in Fig. 2. The growth curves change regularly with the increasing concentration of CdSe QDs (Fig. 2A) while CdSe/ZnS QDs (Fig. 2B) just tenderly affect metabolic curves at a concentration as high as 1.3 lM. Microcalorimetric assays indicate that CdSe QDs inhibit yeast growth more severely than CdSe/ZnS QDs. The heat power (P) of growth is exponential in the exponential growth phase, corresponding to cells multiplying exponentially in this phase. The growth thermogenic curves of the log phase correspond to Eq. (1) (Hawkins and Hall, 1975):

Pt ¼ P 0 expðktÞ or ln Pt ¼ ln P0 þ kt

ð1Þ

Fig. 1. The photographs of CdSe (left) and CdSe/ZnS (right) QDs in hexane and PBS under room light and the radiation of a UV lamp (kex = 365 nm).

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where k represents the growth rate constant or metabolism rate constant, and P0 and Pt are the heat output power of microorganism at time 0 and t (min), respectively. The growth rate constant k, which is calculated from the slope of the power of exponential phase, provides an important quantitative index of yeast activity. Fig. 2A reveals that the multiplying metabolism of the microorganism is affected as QDs are added, which results in k changed. Therefore, the multiplying metabolism of microorganism affected by QDs could be analyzed through the change of k. The growth rate

2.0

A

a b

P / mW

1.5

c d 1.0

e f g

a b c d e f g h

0 33.0 nM 66.0 nM 92.4 nM 145.1 nM 263.9 nM 527.8 nM 1319.5 nM

0.0 0

500

1000

1500

2000

2500

t / min

B

P / mW

a b c d e f g h

a-g

1.5

h

1.0

0 33.9 nM 66.0 nM 92.3 nM 145.2 nM 263.7 nM 527.3 nM 1319.3 nM

0.5



 k0  kc  100% k0

ð2Þ

where k0 and kc represent the growth rate constants of the control and QDs-treated microorganism with the inhibitor concentration c, respectively. Therefore, I indicates the extent of the inhibition on the microbial metabolism. The larger the value of I, the stronger the inhibition effect of QDs is. When the concentration of CdSe QDs is increasing, the inhibitory ratio I becomes larger (Fig. S7). Furthermore, the half-inhibitory concentration (IC50) is defined as the concentration of inhibitor at 50% inhibition. IC50 could represent the inhibition capability of an inhibitor quantitatively. The inhibition effect of CdSe/ZnS QDs is not obvious, and the value of IC50 of CdSe/ZnS cannot be obtained directly by this method. S. cerevisiae has different tolerance towards two kinds of QDs (Fig. 2C). Under the same condition (304.0 nM, IC50 of CdSe QDs), the inhibition effect of CdSe on S. cerevisiae is much more remarkable than that of CdSe/ZnS QDs. It can be deduced that the IC50 of CdSe/ZnS QDs is much larger than that of CdSe QDs, i.e., CdSe/ZnS QDs have much lower toxicity than CdSe QDs when other related factors are kept the same. 3.3. Optical density (OD) studies

0.0 0

500

1000

1500

2000

2500

t / min 2.0

C 1.5

control CdSe/ZnS

P / mW

I¼

h

0.5

2.0

constant k is steadily on decrease when the concentration of CdSe QDs increases (Fig. S6), representing the inhibition of yeast cell growth by CdSe QDs. In addition, representing the ability of the microorganism to grow under specific conditions, the total heat output Qtotal and the maximum heat power Pm of the growth phase are important parameters for the metabolism of microorganism. By analyzing the power–time curves for yeast S. cerevisiae in Fig. 2, the thermodynamic parameters of S. cerevisiae growth at different concentration of CdSe QDs can be obtained (Table 1). From the thermodynamic and kinetic information about multiplying metabolism in Table 1, it is apparent that k and Pm clearly decreases as the QDs concentration increases, suggesting that CdSe QDs could inhibit the metabolism of S. cerevisiae. The change of the total heat output (Qtotal) is not distinct because the existence of QDs does not alter the metabolic way of S. cerevisiae. To evaluate the toxicological effect of QDs, the inhibitory ratio I can be calculated by Eq. (2):

1.0

The antimicrobial activity of CdSe and CdSe/ZnS QDs were determined by comparing the colony-forming capability of QDstreated yeast cells with that of untreated ones. The CdSe QDs exhibit much more severe toxic effect on S. cerevisiae than CdSe/ZnS QDs (Fig. S8). The viability of S. cerevisiae decreases dramatically with the increase of the concentration of CdSe QDs, while CdSe/ ZnS QDs affect cells negligibly. The results demonstrate that CdSe QDs could effectively inhibit the metabolism of S. cerevisiae in a concentration-dependent manner, which is corroborated with microcalorimetric results.

CdSe Table 1 Thermodyanamic parameters of S. cerevisiae growth at different concentration of CdSe QDs.

0.5

0.0 0

500

1000

1500

2000

2500

t / min Fig. 2. Growth thermogenic curves of S. cerevisiae affected by different concentrations of CdSe (A) and CdSe/ZnS (B) QDs at 30 °C. Thermogenic curves of S. cerevisiae affected by CdSe and CdSe/ZnS QDs with identical concentration (C). The concentration of QDs is 304.0 nM, the IC50 of CdSe QDs.

QDs

c (nM)

k (103 min1)

Pm (mW)

Qtotal (J)

I (%)

IC50 (nM)

CdSe

0.0 33.0 66.0 92.4 145.1 263.9 527.8 1319.5

4.91 4.52 4.34 4.13 3.65 2.63 1.52 0.48

1.68 1.55 1.24 1.13 1.02 0.92 0.73 0.55

39.44 37.02 40.63 41.60 41.61 40.85 39.14 40.46

0 9.6 13.2 17.4 27.0 47.4 69.6 90.4

304.0

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3.4. Cell staining assay The live/dead analysis of cell viability was performed to confirm the QDs-induced cytotoxicity. Methylene blue, a classical dye for yeast cell staining, can stain dead yeast cells in blue, the color distinct from that reduced by live cells to leuco. The completely unstained cells are living yeast cells, and blue ones are senior cells or nonviable cells (Fig. S9). By counting the amount of cells in blue and the total number, the death rate can be calculated (Fig. S10). The experimental result reveals that the percentage of dead cells increases as the concentration of CdSe QDs increases. Long-time incubation will critically enhance the death rate and CdSe QDs exhibit more severe toxic effect on S. cerevisiae compared with CdSe/ZnS QDs. This result confirms the microcalorimetric and optical density studies. 3.5. Microscopic studies Transmission electron microscope was used to observe the external morphology and inner structure change of QDs-treated S. cerevisiae. By comparison with untreated yeast cells, both of the QDs do not induce the change in morphology of yeast cells even at 2 lM and yeast could bud normally (Fig. S11). The inner structure change in yeast cells was examined using ultrathin section for electron microscopy. Fig. S12A–C shows the TEM images of S. cerevisiae cells grown in the absence of QDs. Yeast cells are in good condition with intact cell walls. When treated with CdSe QDs (Fig. S12D–F), the cell walls are impaired at a low concentration (200 nM). The thickness and density of cell walls are reduced further at a relatively high concentration (2 lM, Fig. S12G–I). In contrast, CdSe/ZnS QDs do not obviously influence the yeast cell walls at the concentration of 200 nM, while the cell walls thicken slightly at the concentration of 2 lM (Fig. S13). This demonstrates that CdSe QDs impair S. cerevisiae more seriously than CdSe/ZnS QDs, and the impairment effect is resulted from the damage of yeast cell walls, which may contribute to the mechanism of the inhibition effect of CdSe QDs on yeast cells. 3.6. Mitochondrial membrane potential The dissipation of mitochondrial membrane potential (DWm) is a key cellular event (Krysko et al., 2001), which leads to the opening of the mitochondria permeability transition pore and further cellular damage (Edlich et al., 2011). The lipophilic cationic dye, JC-1, was used to detect DWm for its selectivity in entering the mitochondria. It spontaneously forms complexes known as

J-aggregates (red fluorescence) in healthy cells with high DWm, and remains in the monomeric form (green fluorescence) in apoptotic or unhealthy cells. As studied by flow cytometry, significant loss of DWm is observed in the yeast cells incubated with CdSe QDs for 18 h, while CdSe/ZnS QDs scarcely affect DWm (Fig. 3 and Table S1). This suggests that CdSe QDs also cause inner cellular injury besides the damage of the yeast cell surface. The distinguishing injury induced by CdSe QDs of different concentrations is in a concentration-dependent manner between CdSe QDs and mitochondrial membrane potential (Fig. S14 and Table S2). Noteworthy, CdSe/ZnS QDs does not exhibit obvious injury to S. cerevisiae. 3.7. Adhesion of QDs to the surface of S. cerevisiae Based on the changes in yeast cell morphology, further studies were attempted to clarify the behavior of QDs. After 18 h of co-culture, the morphology of S. cerevisiae and the fluorescence of QDs were observed (Fig. S15). The fluorescence of CdSe/ZnS QDs can be observed clearly while CdSe QDs exhibit fluorescence quenching. Fig. S15D shows that QDs were likely to adhere to the surface of yeast cells. Meanwhile, yeast cells with QDs adhesion could be easily stained blue by methylene blue. It is likely that the metabolism of yeast cells may be remarkably affected after QDs adhesion. Confocal microscopy was used to further confirm the adhesion of QDs (Fig. 4). Apparently, the surface physicochemical properties of CdSe QDs are almost the same as those of CdSe/ZnS QDs, so that CdSe QDs may adhere to yeast cells by the same way but with fluorescence quenched. The fluorescence queching of CdSe QDs is a result of the lower fluorescence quantum yield and the interactions with biological systems like proteins, etc., which further supports good stability of CdSe/ZnS QDs in biological applications. By analyzing the location, QDs tend to get adsorbed at the position cells concentrated or at the budding position. QDs are extremely reactive and possess high surface energy because of their large surface to volume ratios (Lai et al., 2012a), which may lead to the adhesion of QDs on the surface of S. cerevisiae. The Deltavision OMX imaging system offers super-resolution observation for S. cerevisiae and CdSe/ZnS QDs (Fig. S16). CdSe/ ZnS QDs tend to be adsorbed tightly on the surface of yeast cells as graininess in the 360° rotating figures composed by the fluorescence images of different focal plane (Fig. 5). ICP-AES was used to quantify the amount of Cd bound to yeast cells after different incubation times. The blue and green columns in Fig. S17 represent the absolute amount of Cd in supernatant and those bound to the yeast cells, respectively. Overwhelming

Fig. 3. The changes in mitochondrial membrane potential (DWm) of yeast cells incubated with CdSe and CdSe/ZnS QDs for 18 h studied by flow cytometry. Green fluorescence in P2 gate represents cells with low DWm (apoptotic or unhealthy cells), while red fluorescence represents cells with high DWm (healthy cells). c(QDs) = 1 lM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. Confocal microscopic images of CdSe (upper) and CdSe/ZnS (lower) QDs. c(QDs) = 1 lM.

Fig. 5. The 360° rotating images of CdSe/ZnS QDs adhered to yeast. The rotating image is decomposed by fluorescence of different focal planes of the image in Fig. S16. c(QDs) = 1 lM. The scale bar is 5 lm.

majority of the QDs is found in the supernatant, whereas much less QDs adhere to the yeast surface. Prolonging the incubation time would add the amount of adhered QDs. 3.8. Toxicity mechanism 3.8.1. Fluorescence spectroscopy Selenium (Se), as the trace element, plays an important role in the health of humans and animals (Xia et al., 2007), while free heavy metal ion Cd2+ is well known to be highly toxic. Cd2+ and reactive oxygen species (ROS) generated from QDs, by influencing the apoptosis gene express and inducing the injury of cytomembrane and organelle, are supposed to be the possible reasons for the antimicrobial effect of Cd-containing QDs (Ipe et al., 2005; Kirchner et al., 2005; Chan et al., 2006). The decomposition process of QDs, accompanied with release of heavy metal ions and oxides, would lead to the blue-shift of the emission peak (Derfus et al., 2004; Zhelev et al., 2006). Herein, the fluorescence spectroscopy of the QDs incubated with yeast cells was monitored. Yeast cells alone and pure YPED culture medium both could not affect the fluorescence of QDs even after long time, so that the mixture suspension containing cells and YPED was taken into consideration. Fluorescence spectra of QDs – S. cerevisiae mixture in

growth medium at different time intervals were measured (Fig. S18). The emission peak intensities of CdSe and CdSe/ZnS QDs gradually decline as the incubation time increases, along with the slightly blue-shift of the emission maxima for 3 nm. Thus, the growth of S. cerevisiae may cause degradation of QDs accompanied with fluorescence quenching, and the influence will strengthen as the incubation time increases. The blue-shift occurs at the beginning when QDs add into the freshly inoculation yeast cells followed by nonspecific binding with protein or other nutriments in the growth medium. After co-culture for a longer time, the fluorescence peak of CdSe QDs is hardly observed but that of CdSe/ZnS QDs is still superior. The distinction explains why the fluorescence of CdSe QDs cannot be observed in the confocal microscopic studies. This also confirms the epitaxial growth of the ZnS shell can promote the fluorescence quantum yield and make the CdSe core much more stable. The YPED medium before incubation is close to neutral (pH = 6.83). However, the pH reaches 5.09 after 24 h incubation due to the metabolism of yeast cells. This low pH may contribute to fluorescence quenching of QDs. QDs degradation, followed by fluorescence quenching and release of Cd2+, is one of the possible reasons triggering the cytotoxicity to microorganism (Kirchner et al., 2005). Besides the release of heavy metal ions upon the degradation

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of QDs, other factors also could strengthen the cytotoxicity of QDs to S. cerevisiae, such as the interaction between QDs and cells, the accumulation of QDs on the surface of cells, etc., which may affect the morphology of cells and initiate dysfunction of cell metabolism.

3.8.3. Detection of free cadmium ions Free cadmium ions released due to the degradation of CdSe QDs were separated from yeast cells and detected by ICP-AES to further explain the cytotoxicity mechanism (Su et al., 2010). The amount of free cadmium ions has a positive correlation to the concentration of QDs (Table S4). Although free Cd2+ released from CdSe/ ZnS QDs is also concentration-dependent, the variation (0.1– 0.5 lM) is far smaller than that of CdSe QDs (0.1–4.8 lM). Hence the epitaxial growth of ZnS shell can coat CdSe efficiently and prevent the release of Cd2+. Without yeast, weakly acidic YPED medium could decompose QDs to some degree after long-term incubation. The metabolism of yeast cells makes the environment more acidic, thus to deteriorate QDs more obviously. This can be also evidenced by the fluorescence quenching effect caused by the combined effect of yeast cells and YPED medium. 3.8.4. Protection effect by EDTA Ethylene diamine tetraacetic acid (EDTA), a classical protective agent versus Cd2+, can protect yeast cells by coordination with Cd2+. To confirm the protection capability of EDTA, a preliminary experiment was conducted, e.g. yeast cells was inhibited by Cd2+ at IC50 and mixed with EDTA of different concentrations. Thermogenic curve gradually returns to the control with the increase of EDTA, and can coincide with the control curve when the concentration of EDTA is five folds of Cd2+ (Fig. S20). Thus, EDTA could efficiently alleviate the inhibitory effect of Cd2+. Based on the above protection effect of EDTA, CdSe QDs was first loaded to inhibit the metabolism of yeast cells to half-inhibition and EDTA was added. By analyzing the power–time relationship (Fig. 6), EDTA has the same protection effect in the presence of CdSe QDs, and can neutralize the inhibitory effect of QDs when the concentration of EDTA is 33.0 lM. This further confirms the evidence that Cd2+ contributes to the cytotoxicity of CdSe QDs indeed, but its inhibitory effect can be weakened by addition of EDTA.

a control b +QDs (304 nM) c +QDs+EDTA (3.3µM)

a h g f

1.5

P / mW

3.8.2. Microcalorimetric study Release of Cd2+ was considered to be significantly responsible for the cytotoxicity in Cd-containing QDs (Kirchner et al., 2005; Li et al., 2010; Han et al., 2012). Fig. S19 shows the metabolism thermogenic curves of S. cerevisiae growth affected by Cd2+ of different concentrations at 30 °C. The inhibitory effect is correlated to the concentration of Cd2+. As the concentration of Cd2+ increases, the intensity of metabolism curve declines and time of the total heat output is prolonged. Thermodynamic and kinetic parameters (Table S3) are obtained by analyzing the power–time curves for S. cerevisiae (Fig. S19). The growth rate constant k decreases significantly as Cd2+ concentration increases, while inhibitory ratio I increases with Cd2+ concentration increases (IC50 = 3.3 lM). Both the functions of k–c and I–c have similar patterns with those for CdSe QDs. Pm is also on decrease while Qtotal remains unchanged, indicating that cadmium gradually damages the physiological function of S. cerevisiae but does not alter the metabolic pathway. This observation is also similar with that found in the effect of CdSe QDs on S. cerevisiae. It meets our previous work (Han et al., 2012). The investigation in the possible effect of the surface coating OPA was conducted. Thermogenic curves affected by OPA of different mass concentrations almost coincide with the control. Thus, the influence of surface coating polymer can be excluded.

2.0

d +QDs+EDTA (6.6 µM)

e d

1.0

e +QDs+EDTA (13.2µM) f +QDs+EDTA (19.8µM) g +QDs+EDTA (26.4µM) h +QDs+EDTA (33.0µM)

0.5

c b

0.0 0

500

1000

1500

2000

2500

3000

t / min Fig. 6. Thermogenic curves of S. cerevisiae in the presence of CdSe QDs and different concentrations of EDTA.

4. Conclusion In summary, the toxicity of CdSe and CdSe/ZnS QDs to S. cerevisiae was investigated via microcalorimetric, spectroscopic, and microscopic methods. The distinction between two kinds of QDs in microcalorimetric studies indicates that CdSe QDs inhibits the metabolism of S. cerevisiae while CdSe/ZnS QDs shows negligible toxicity, which is ascertained by optical density measurement and cell staining assay. Microscopic studies reveal that addition of QDs will not influence the external cell morphology but CdSe QDs damages the cell wall of S. cerevisiae. The existence of CdSe QDs could also affect inner structure and reduce the mitochondrial membrane potential. Phase contrast fluorescence and confocal microscopic studies show that QDs adhere to the surface of yeast cells, and the adhesion amount increase with the incubation time prolonged. By ICP-AES, free cadmium ions released from QDs are detected, which implies that QDs can be decomposed in the presence of both of yeast cells and YPED medium. In brief, the released cadmium ions contribute to the cytotoxicity of CdSe QDs. Notably, the epitaxial coating of ZnS can efficiently reduce the toxicity of Cd-containing QDs, which will be instructive in the future design and applications. Acknowledgments The Chinese 973 Program (2011CB933600), the National Natural Science Foundation of China (21225313, 21303126), Program for Changjiang Scholars and Innovative Research Team in University (IRT1030), the Fundamental Research Funds for Central Universities (2042014kf0287), and the Project sponsored by SRF for ROCS are acknowledged for financial support. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2014.03.071. References Aaron, J.S., Greene, A.C., Kotula, P.G., Bachand, G.D., Timlin, J.A., 2011. Advanced optical imaging reveals the dependence of particle geometry on interactions between CdSe quantum dots and immune cells. Small 7, 334–341. Aye, M., Di Giorgio, C., Berque-Bestel, I., Aime, A., Pichon, B.P., Jammes, Y., Barthélémy, P., De Méo, M., 2012. Genotoxic and mutagenic effects of lipidcoated CdSe/ZnS quantum dots. Mutat. Res.-Gen. Toxicol. Environ. 750, 129– 138.

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