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UV-enhanced cytotoxicity of thiol-capped CdTe quantum dots in human pancreatic carcinoma cells
Toxicology Letters 188 (2009) 104–111
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
UV-enhanced cytotoxicity of thiol-capped CdTe quantum dots in human pancreatic carcinoma cells Shu-quan Chang a , Yao-dong Dai a,∗ , Bin Kang a , Wei Han a , Ling Mao b , Da Chen a a b
College of Material Science and Technology, Nanjing University of Aeronautics and Astronautics, Nanjing 210016, PR China National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention, Beijing 100088, PR China
a r t i c l e
i n f o
Article history: Received 8 November 2008 Received in revised form 6 February 2009 Accepted 13 March 2009 Available online 25 March 2009 Keywords: Quantum dots Cytotoxicity UV illumination Reactive oxygen species
a b s t r a c t Quantum dots (QDs) have been gaining popularity due to their potential application in cellular imaging and diagnosis, but their cytotoxicity under light illumination has not been fully investigated. In this study, green and red mercaptopropionic acid capped CdTe quantum dots (MPA-CdTe QDs) were employed to investigate their cytotoxicity in human pancreatic carcinoma cells (PANC-1) under UV illumination. MPACdTe QDs exhibited excellent photostability under UV illumination and could be easily ingested by cells. The cytotoxicity of MPA-CdTe QDs was significantly enhanced under UV illumination, which was determined by changes in cell morphology as well as by decreases in the metabolic activity and cell counting. Our results indicated that green and red QDs had different cellular distribution and exhibited distinct UV-enhanced cytotoxicity. UV illumination enhanced the generation of reactive oxygen species (ROS) in cells containing QDs, and NAC antioxidant could reduce their damage to cells under UV illumination. Moreover, the influences of different UV illumination conditions on the viability of cells containing QDs were examined and discussed in detail. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction With the development of nanotechnology, numerous nanomaterials have been widely used in biology and medicine (Nel et al., 2006). The cytotoxicity of nanomaterials is considered to be very crucial to their wide applications in biomedical area and has received more and more attention in recent years (Chan and Shiao, 2008; Hardman, 2006; Monteiro-Riviere and Inman, 2006; Monteiro-Riviere et al., 2005; Nikitin et al., 2008; Sayes et al., 2006, 2007). Semiconductor nanoparticles, often referred as quantum dots (QDs), are usually 1–12 nm in diameter and have unique optical and electronic properties (Murray et al., 2000). QDs can absorb irradiated energy at any wavelength greater than that of their lowest energy transition and may then convert the irradiated energy to an extremely narrow bandwidth emission. QDs are emerging as alternative or complementary tools to the organic fluorescent dyes currently. Compared with the traditional organic fluorescence probes, QDs have many advantages, such as broadband excitation, narrow bandwidth emission, emission of high intensity light, resistance to quenching and good photochemical stability (Alivisatos, 2004; Gerion et al., 2001).
∗ Corresponding author. Tel.: +86 25 52112918; fax: +86 25 52119016. E-mail address: yd [email protected] (Y.d. Dai). 0378-4274/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2009.03.013
Recently, QDs have been gaining popularity due to their potential applications in cellular imaging and medical diagnosis (Medintz et al., 2005; Michalet et al., 2005). Meanwhile, the potential toxicity of QDs is a growing concern in spite of early studies in immortalized cell lines showed little or no deleterious effects of QDs in chronic treatment paradigms (Jaiswal et al., 2003; Parak et al., 2002; Wu et al., 2003). Hoshino et al. (2004) demonstrated that toxicity of QDs was not dependent on the nanocrystal itself but rather on the surface molecules. Another study suggested that in addition to the release of toxic Cd2+ ions from the particles also their surface chemistry, in particular their stability toward aggregation, played an important role for their cytotoxicity (Kirchner et al., 2005). Maysinger and co-workers also carried out many studies on the pathways of QDs-induced cytotoxicity and found that QDs-induced cell death involved Fas upregulation and lipid peroxidation in human neuroblastoma cells (Choi et al., 2007, 2008). Chan et al. (2006) demonstrated that QDs induced apoptosis in human neuroblastoma cells via mitochondrial-dependent pathways and inhibition of survival signals. Several studies reported that QDs could induce the generation of reactive oxygen species (ROS) and ROS was related to the cytotoxicity of QDs (Ipe et al., 2005; Tsay et al., 2007). As is mentioned above, QDs are mainly used as imaging and diagnosis agents in biomedicine area. So, they have to be exposed to illumination conditions usually. It will be meaningful to study the cytotoxicity of QDs under illumination conditions. Using
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primary hepatocytes as a liver model, Derfus et al. (2004) found that the toxicity of CdSe-core QDs could be enhanced under UV irradiation. Liang et al. (2007) investigated the calf thymus DNA damage induced by CdSe QDs under UV irradiation. Fujioka et al. (2008) compared the cytotoxicity of Si QDs and CdSe QDs in HeLa cells. Recently, Mortensen et al. (2008) examined the in vivo skin penetration of carboxylated QDs in the murine model under UV irradiation and concluded that UV irradiation could enhance the toxic effects of QDs in the skin of SKH-1 mice. However, the detailed cellular effects of the UV-enhanced toxicity were not fully investigated. In this paper, green and red mercaptopropionic acid capped CdTe quantum dots (MPA-CdTe QDs) were employed to investigate their cytotoxicity in human pancreatic carcinoma cells (PANC-1) under UV illumination. The different distribution and cytotoxicity of red and green QDs under UV irradiation were proposed. The generation of ROS in cells containing QDs under UV illumination and the influences of different UV illumination conditions (UV irradiation time, QDs concentration, incubation time with QDs, post-exposure time) on the UV-enhanced cytotoxicity of QDs were examined and discussed in detail. 2. Materials and methods 2.1. Preparation and characterization of MPA-CdTe QDs All the reagents used in this study were analytical grade and purchased from Sigma–Aldrich Company unless indicated otherwise. Green and red mercaptopropionic acid capped CdTe quantum dots (MPA-CdTe QDs) employed in this study were synthesized as the method described in previous literatures (Lovric et al., 2005b). In a typical synthesis: 1.65 g sodium borohydride was dissolved in 40 mL water at 0 ◦ C while stirring under N2 atmosphere protection, then 2.61 g tellurium powder was added into above solution portionwise, and the mixture was stirred at 0 ◦ C under N2 for 6 h, yielding a purple NaHTe solution. 0.5 mL cadmium perchlorate hydrate (1 M aqueous solution) and 0.3 mL 3-mercaptopropionic acid were dissolved in 200 mL water. The pH of the solution was adjusted to 11 with 1 M NaOH solution prior to addition of an aliquot of the NaHTe solution previously prepared (0.2 mL). The reaction mixture was heated to reflux under N2 protection. Green QDs formed after the reaction time of 10 min, while red QDs were generated after 7 h. The as prepared QDs solution was dialyzed against deionized water for 6 h and concentrated by using a rotary evaporator. QDs were collected by centrifugation and purified by size-selective precipitation. The photoluminescence properties of MPA-CdTe QDs were characterized subsequently. Ultraviolet–visible (UV–vis) spectroscopy was carried out at room temperature using a PerkinElmer -17 spectrophotometer. The photoluminescence (PL) spectrum was taken with a HITACHI 850 spectrofluorophotometer. The photostability of green and red QDs dissolved in water or PBS was measured after UV illumination. UV light (365 nm) was generated by ZF-20D Ultraviolet Analyzing Equipment with a power density of 19 mW cm−2 . After illumination, photoluminescence (PL) spectra were recorded to evaluate the photostability of QDs. 2.2. Cell culture, QDs treatment and UV illumination Human pancreatic carcinoma cells (PANC-1, ATCC # TIB-222) were cultured in DMEM medium containing 10% (v/v) fetal calf serum at 37 ◦ C in humidified air containing 5% CO2 . For fluorescence microscopic measurements, cells should be seeded onto a glass coverslip placed in 6-well plates. For MTT assay, cells were cultured in 96well plates. Cells were incubated up to about 24 h and grown to about 80% confluence before experiments. QDs dispersed in PBS were added to each well to achieve a final concentration. Cells were then incubated in these medium containing QDs at 37 ◦ C and in 5% CO2 atmosphere for different time periods. After incubation, all cells were washed with PBS to remove excess QDs and placed in fresh solutions before next experiments. UV irradiation was carried out on ZF-20D Ultraviolet Analyzing Equipment which generated 365 nm UV light with a power density of 19 mW cm−2 . All treatments were done in triplicates or quadruplicates in three or more independent experiments.
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only two channels (CH1: excitation 488 nm, filter 500–600 nm for green QDs or 600–700 nm for red QDs; CH3: 488 nm, DIC) were set to record QDs and DIC signals. Cells were observed using 60× oil immersion objective. Images were acquired at a resolution of 800 × 800, and the scan size was 212 m × 212 m. 2.4. MTT assay and cell counting Colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assays were performed to assess the metabolic activity of cells treated with different conditions. After the treatment, the medium was removed and replaced with serum-free media (200 L/well). A total of 20 L stock MTT (5 mg mL−1 ) was added to each well, and the cells were then incubated for 1 h at 37 ◦ C. The medium was removed, and the cells were lysed with DMSO. The absorbance was measured at 595 nm. Cell number was determined by trypan blue exclusion assay (Hyunsoo et al., 2009). 2.5. ROS imaging and analysis Reactive oxygen species generation was imaged by using dihydroethidium (DHE) and Olympus FV-1000 laser scanning confocal microscope. After treatments, cells were washed and medium was replaced with serum-free medium. DHE was dissolved in DMSO (3 mM) and added to the culture medium at a final concentration of 10 M, and cells were incubated for 30 min at 37 ◦ C. Cells were washed with PBS before imaging. The oxidation product of DHE was ethidium, whose fluorescence was enhanced about 10-fold when bound to DNA. Three channels (CH1: excitation 488 nm, filter 500–550 nm; CH2: excitation 488 nm, filter 610–650 nm; CH3: 488 nm, DIC) in LSCM were set to record QDs, ethidium and DIC signals. Collected images were used to quantify the relative fluorescence intensity (RFI) of ethidium. N-acetylcysteine (NAC) was employed to study the effect of ROS on cell viability. NAC was dissolved in PBS (500 mM) and added to culture medium reaching a concentration of 5 mM. 2.6. Statistics analysis Results were expressed as mean ± standard deviation (SD). Significance was evaluated using Student’s t-test. Difference was considered significant if p < 0.05.
3. Results 3.1. Characterization and photoluminescence stability of MPA-CdTe QD MPA capped CdTe QDs were prepared following the general procedure previously reported. The size of QDs grew larger with the reaction proceeding, and consequently their emission wavelength shifted to longer wavelength. To assess the size-dependent effect of QDs on cells, we employed two size QDs with green-emitting and red-emitting respectively in this study. Green QDs formed after the reaction time of 10 min, while red QDs were generated after 7 h. The optical properties of MPA-CdTe QDs used in this study were shown in Fig. 1A. As it can be seen, green QDs had an absorption peak at around 510 nm and a fluorescence emission peak at 538 nm. While the absorption peak and emission peak of red QDs were about 569 and 629 nm respectively. As shown in the insets of Fig. 1A, both the green and red QDs exhibited excellent water-solubility and bright fluorescence under UV illumination. The photostability of QDs in cells under UV irradiation was considered to be very crucial in the following experiments, so the changes of their fluorescence intensity in water and PBS were examined after UV irradiation and the results were presented in Fig. 1B. It can be seen that the red QDs had better stability than the green QDs, and the QDs in water had better stability than in PBS. All the results revealed that decrease of fluorescence intensity of green and red QDs in water and PBS was less than 20% after UV illumination.
2.3. Laser scanning confocal microscopy Olympus FV-1000 laser scanning confocal microscope (LSCM) was used to acquire images. After the treatment, PANC-1 cells were fixed with 4% paraformaldehyde for 15 min, and then washed three times with PBS buffer. To clearly observe the distribution and positions of QDs in cells, nuclei were stained with 0.1 g mL−1 DAPI for 2 min. Before imaging, cells were washed with PBS again. Three channels (CH1: excitation 405 nm, filter 420–470 nm; CH2: excitation 488 nm, filter 500–600 nm for green QDs and 600–700 nm for red QDs; CH3: 405 nm/488 nm, DIC) were set to record DAPI, QDs and DIC signals. To study the changes of cellular morphology,
3.2. Cellular distribution of QDs and UV-induced morphological changes of PANC-1 cells containing QDs To study the cellular distribution of green and red MPA-CdTe QDs, fluorescence imaging examination of PANC-1 cells incubated with 50 g mL−1 QDs for 4 h were carried out. In order to determine and visualize the location of QDs within the cells, the nuclei
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of PANC-1 cells were stained by DAPI. The most striking difference between green and red QDs in their localization was observed in the nuclear vs. cytoplasmic compartments. The PANC-1 cells treated with green QDs showed that some QDs located in the cytoplasm and a few QDs appeared in the nuclei (Fig. 2A and C). In contrast, the cells treated with red QDs indicated that most of the QDs were distributed throughout the cytoplasm but no QDs were observed in the nuclei (Fig. 2D and F). These results were consistent with the previous reports (Lovric et al., 2005a). The size of QDs was considered very important to their distribution and metabolizability in cells, and it also might be closely related to their cytotoxicity. To assess the damage of cells containing QDs under UV illumination, PANC-1 cells incubated with green or red QDs (50 g mL−1 , 4 h) were treated with UV irradiation ( = 365 nm, 19 mW cm−2 , 1 h). After the treatments, the cells were characterized by LSCM. The cells without any treatments (no QDs and no UV illumination) were considered as control group in our experiments. Compared with the control group (Fig. 3A), the PANC-1 cells without QDs had no morphological changes after UV illumination (Fig. 3B). The cells only treated with QDs also showed no distinct changes in morphology (Fig. 3C and D). In contrast, the cells containing green or red QDs were badly damaged and the integrality of cells was affected seriously after UV illumination (Fig. 3E and F). It was also seen that the cells incubated with green QDs were damaged more seriously in morphology than the cells with red QDs after UV illumination. These results indicated that UV illumination significantly enhanced the damages of QDs to cells and QDs with different size exhibited different UV-enhanced damages to cells. Fig. 1. Photoluminescence properties of green and red MPA-CdTe QDs. (A) UV–vis (left) and PL (right) spectra of green (a) and red (b) MPA-CdTe QDs. The excitation wavelength for the PL spectrum was 430 nm. Insets showed the photographs of green (a) and red (b) QDs under UV illumination. (B) Time-dependent photoluminescence properties of green and red MPA-CdTe QDs under UV illumination (365 nm, 19 mW cm−2 ) in water or PBS.
3.3. Metabolic activity and cell counting of PANC-1 cells containing QDs under UV illumination Based on above observed changes in cell morphology, MTT assays and cell counting were carried out to investigate the UV-
Fig. 2. Cellular distribution of green and red MPA-CdTe QDs. (A) Fluorescence image of cells incubated with green QDs for 4 h. (B) The DIC image of (A). (C) The overlay image of (A and B). (D) Fluorescence image of cells incubated with red QDs for 4 h. (E) The DIC image of (D). (F) The overlay image of (D and E). Insets in (C and F) showed the magnified image of a certain cell. The final concentration of MPA-CdTe QDs in medium was 50 g mL−1 . The cellular nuclei were stained by DAPI (0.1 g mL−1 , 2 min). The scale bar represents 30 m.
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enhanced cytotoxicity of QDs and the results were shown in Fig. 4. The cell counting and metabolic activity of cells without QDs showed no distinct changes after UV illumination compared with the control group. In contrast, the metabolic activity of cells with red QDs decreased from 75% to 27%, and metabolic activity of cells with green QDs decreased from 59% to 11% after UV illumination. That was to say, UV illumination reduced the viability of cells containing red and green QDs by 65.7% and 81.4% respectively, comparing with their values before UV illumination. The loss of cell number is also very notable. UV illumination reduced the cell number of cells containing red and green QDs by 55.2% and 85.1% respectively, comparing with their values before UV illumination. These results indicated that UV illumination could distinctly enhance the cytotoxicity of QDs, which was consistent
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with the results of LSCM. In addition, the increase of cytotoxicity of green QDs was more notable than red QDs after UV illumination. 3.4. Influences of different UV illumination conditions on the cell number and metabolic activity of cells containing QDs To determine the effects of UV illumination on the cytotoxicity of QDs, different parameters were examined and discussed in detail. These parameters included: UV illumination time (10, 30, 60, 120 min); concentration of QDs (1, 10, 50, 500 g mL−1 ) for cells incubating; incubation time (1, 2, 4, 8 h) with QDs before UV illumination; post-exposure time (0, 1, 2, 4 h). Green MPA-CdTe QDs were used in this section. Cells assays were used to study each parame-
Fig. 3. Combined confocal micrographs of PANC-1 cells under different treatment. (A) Cells were incubated under usual conditions (no QDs and no UV). (B) Cells were irradiated by UV only. (C) Cells were incubated with green QDs only. (D) Cells were incubated with red QDs only. (E) Cells were incubated with green QDs and irradiated by UV. (F) Cells were incubated with red QDs and irradiated by UV. Insets in (E and F) showed the magnified image of a certain cell. The final concentration of MPA-CdTe QDs in medium was 50 g mL−1 and incubation time was 4 h. The power density of UV ( = 365 nm) was 19 mW cm−2 and UV irradiation time was 1 h. The scale bar represents 30 m.
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of cells decreased sharply with the increase of post-exposure time. The cell number and metabolic activity of cells decreased to below 5% when post-exposure time reached 4 h. The loss of cell numbers was not more notable than the decrease of metabolic activity when the post-exposure time is very short. All results revealed that the cytotoxicity of QDs was significantly enhanced by UV illumination and the UV-enhanced cytotoxicity could be influenced by many conditions. 3.5. UV-enhanced generation of ROS in cells containing QDs
Fig. 4. The metabolic activity (A) and cell counting (B) of PANC-1 cells exposed to different conditions. The final concentration of MPA-CdTe QDs in medium was 50 g mL−1 and incubation time was 4 h. The power density of UV ( = 365 nm) was 19 mW cm−2 and UV illumination time was 1 h. MTT assay and cell counting were carried out after UV irradiation for 2 h. The (+) or (−) in above legends mean treating cells with or without corresponding condition. Data represents mean ± SD for five independent experiments (n = 5) (* p < 0.01 and ** p < 0.001).
ter by cell counting and MTT assay. All results were summarized in Fig. 5. Fig. 5A showed that the metabolic activity of cells containing QDs decreased significantly with the increase of UV illumination time, while the metabolic activity of cells treated without QDs kept slight changes under UV illumination. Under UV illumination for 2 h, the metabolic activity of cells without QDs also remained about 80% but that of cells containing green QDs decreased to below 10%. The loss of cell numbers was more notable than the decrease of metabolic activity. The cell number decreased to 2% after 2 h UV irradiation. By increasing the concentration of QDs for cells incubation, the cytotoxicity of QDs without and with UV treatments were both distinctly enhanced, while cell number and metabolic activity of cells under UV illumination were reduced more rapidly than the cells without UV illumination (Fig. 5B). The position of QDs in cells might be closely related to their cytotoxicity. So we examined the influences of incubation time with QDs before UV illumination. Fig. 5C indicated that the cytotoxicity of QDs under UV illumination was significantly enhanced as the increase of incubation time with QDs before UV illumination. The cell number and metabolic activity of cells decreased to below 5% when the incubation time with QDs before UV illumination was 8 h. The loss of cell numbers was more notable than the decrease of metabolic activity. The effects of post-exposure time (the incubation time after UV illumination) were examined and the results were shown in Fig. 5D. It can be seen that the cell number and metabolic activity
The generation of excess reactive oxygen species can cause the modification and damage of cellular proteins, lipids and DNA, and it would subsequently lead to the cell death or apoptosis. To investigate the oxidative stress induced by QDs under UV irradiation, DHE was used in this section. DHE could be oxidized by ROS to ethidium, which intercalated with cellular DNA, yielding bright red fluorescent. Using laser scanning confocal microscope, the oxidative stress was studied and the results were given in Fig. 6. Very weak fluorescence from ethidium was detected in cells without any treatments (Fig. 6A-a) or cells only treated by UV irradiation (Fig. 6A-b). Cells with QDs produced more fluorescence (Fig. 6A-c) than control cells. Meanwhile, a marked increase of fluorescence was detected in QDtreated cells after UV illumination (Fig. 6A-d), compared with the cells only treated with QDs or UV illumination. The quantitative results were shown in Fig. 6B. It can be concluded that the existence of QDs significantly promoted the generation of ROS in cells under UV illumination. N-acetylcysteine (NAC), a strong antioxidant containing a mercapto group, was employed to examine the effect of ROS on the decrease of cell viability caused by QDs under UV illumination. As shown in Fig. 7A, NAC prevented the generation of ROS in cells containing QDs under UV illumination. Fig. 7B also showed that the quantity of ROS in cells after QDs and UV treatments was reduced more than 50% when NAC was added. In addition, NAC could reduce the damage of cells containing QDs under illumination in morphology (Fig. 7A) and improved their viability (Fig. 7C). These results revealed that ROS played an important role in the damage to cells caused by QDs under UV illumination. 4. Discussion In the present study, we mainly demonstrate that: (a) the cytotoxicity of MPA-CdTe QDs in PANC-1 cells was significantly enhanced under UV illumination; (b) green and red QDs had different cellular distribution and exhibited distinct UV-enhanced cytotoxicity; (c) UV illumination significantly enhanced the generation of ROS in cells containing QDs, and NAC antioxidant could reduce these damages to cells under UV illumination; (d) the UVenhanced cytotoxicity could be influenced by many factors, such as the UV illumination time, the concentration of QDs, the incubation time with QDs before UV illumination and post-exposure time. MPA-CdTe QDs exhibit excellent photostability under UV illumination in water or PBS (Fig. 1), which provides the basis for our following study. They also can be easily ingested by PANC-1 cells and exhibit different distribution when their size is different. Red QDs are distributed throughout the cytoplasm but no QDs are observed in the nuclei (Fig. 2D and F). In contrast, some green QDs locate in the cytoplasm and a few QDs appear in the nuclei (Fig. 2A and C). Judging from the changes in cell morphology as well as the decreases in the metabolic activity and cell counting, MPA-CdTe QDs have lesser toxicity to PANC-1 cells without UV irradiation and this cytotoxicity can be distinctly enhanced under UV irradiation (Figs. 3 and 4). In addition, green and red QDs exhibit distinct UV-enhanced cytotoxicity according to our study. The increase of cytotoxicity of green QDs
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Fig. 5. The influences of different UV illumination conditions on the metabolic activity and cell counting of PANC-1 cells. (A) Effect of UV illumination time on PANC-1 cells viability. Cells were incubated with QDs for 4 h and the final concentration of QDs in medium was 50 g mL−1 . Cells were irradiated for different time. MTT assay and cell counting were carried out after UV illumination for 2 h. Controls were cells incubated with no QDs. (B) Effect of concentration of QDs. Cells were incubated with QDs for 4 h under different QDs concentration. UV illumination time was 1 h. MTT assay and cell counting were carried out after UV illumination for 2 h. Controls were cells which were not irradiated by UV. (C) Effect of incubation time before UV illumination. Cells were incubated with QDs for different time and the concentration of QDs was 50 g mL−1 . UV illumination time was 1 h. MTT assay and cell counting were carried out after UV irradiation for 2 h. Controls were cells which were not irradiated by UV. (D) The metabolic activity of cells and cell counting at different post-exposure time. Cells were incubated with QDs for 4 h and the concentration of QDs was 50 g mL−1 . UV illumination time was 1 h. MTT assay and cell counting were carried out after UV irradiation for different time. Controls were cells incubated with no QDs. Green MPA-CdTe QDs were used in this study. The power density of UV ( = 365 nm) was 19 mW cm−2 . Data represents mean ± SD for five independent experiments (n = 5) (* p < 0.01 and ** p < 0.001 vs. control group).
after UV irradiation is more notable than that of red QDs. That might due to their different cellular distribution. The position of QDs in cells is considered to be closely related to their cytotoxicity. Compared with the red QDs which are only distributed in cytoplasm, the green QDs can enter the nuclei partially and might induce more directly and seriously damage to DNA or other targeting positions in the nuclei under UV irradiation. Based on our results, QDs with larger size may be safer under UV irradiation conditions. According to our LSCM results, UV illumination significantly enhances the generation of ROS in cells containing QDs (Fig. 6). The UV-enhanced generation of ROS is attenuated in the presence of the antioxidant NAC (Fig. 7). Judging from the cellular morphology, cell counting and metabolic activity, the damage of QDs to cells under UV is reduced notably when NAC is added (Fig. 7). Above results reveal that ROS play an important role in QDs-induced damage of cells under UV illumination. That is because ROS can let cells undergo oxidative stress. Results of this stress include modification and damage of cellular components, such as lipids, proteins, and DNA (Finkel and Holbrook, 2000; Prestwich et al., 2005). All above direct or indirect damages to cells can lead to the apoptosis or death of cells in the end (Lovric et al., 2005b; Maysinger, 2007). Proper antioxidant should be employed to reduce the UV-enhanced toxicity of QDs in clinical applications.
Our results also demonstrate that the UV-enhanced cytotoxicity could be influenced by many conditions (Fig. 5). Firstly, the cytotoxicity of QDs under UV irradiation is enhanced more and more notably as the increase of UV irradiation time or QDs concentration (Fig. 5A and B). The combination of UV irradiation and QDs induces more serious damage to cells. Above results also indicate the synergistic effect between QDs and UV irradiation on the damage of cells. The UV-enhanced cytotoxicity of QDs is less significantly than the QDs-enhanced cytotoxicity of UV irradiation. The incubation time of cells with QDs before UV illumination can also evidently affect the UV-enhanced cytotoxicity of QDs (Fig. 5C). That is because the uptake and distribution of QDs in cells need a period of time and should undergo a complex process. The position of QDs in cells is considered to be closely related to their cytotoxicity. So, QDs incubated with QDs for different period of time before UV illumination exhibit different UV-enhanced cytotoxicity. Moreover, the cytotoxicity of QDs under UV irradiation is enhanced as the increase of post-exposure time. The initial damages to cells could be repaired or enlarged with the cellular metabolism, which also need a period of time. As the increase of post-exposure time, the initial damages to cells are enlarged. As a result, more serious toxicity is observed. Now that the UV-enhanced toxicity of QDs can be influenced by above conditions, we should be able
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Fig. 6. QDs and UV illumination induced the generation of ROS in cells. (A) Confocal micrographs of DHE-stained cells treated under different conditions: (a) with no QDs and no UV, (b) with no QDs and under UV, (c) with QDs and no UV, (d) with QDs and under UV. (B) Relative fluorescence intensity of DHE-stained cells corresponding to (A). Data represents mean ± SD for three independent experiments (n = 5) (** p < 0.001). Cells were incubated with green MPA-CdTe QDs for 4 h and the concentration of QDs was 50 g mL−1 . The power density of UV ( = 365 nm) was 19 mW cm−2 and UV illumination time was 1 h. PNAC-1 cells were stained with DHE (10 M) after treatment.
Fig. 7. NAC reduced the generation of ROS in cells and enhanced the viability of cells under UV treatment. (A) Confocal micrographs of DHE-stained cells treated with QDs and UV. (a) Fluorescence image of cells treated with QDs only. (b) Overlay image of (a) and DIC image. (c) Fluorescence image of cells treated with QDs and NAC (10 mM). (d) Overlay image of (c) and DIC image. (B) Relative fluorescence intensity of DHE-stained cells corresponding to (A). Controls were cells stained with DHE and incubated with no QDs and no UV irradiation. Data represents mean ± SD for three independent experiments (n = 5) (* p < 0.01 and ** p < 0.001). (C) Metabolic activity of cells corresponding to (A). Controls were cells stained with DHE and incubated with no QDs and no UV irradiation. Data represents mean ± SD for three independent experiments (n = 5) (* p < 0.01). Cells were incubated with green MPA-CdTe QDs for 4 h and the concentration of QDs was 50 g mL−1 . The power density of UV ( = 365 nm) was 19 mW cm−2 and UV illumination time was 1 h. PNAC-1 cells were stained with DHE (10 M) after treatment.
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to reduce or enhance this toxicity to serve us by adopting proper conditions. The UV-enhanced cytotoxicity of QDs is harmful for normal cells and will limit their wide application in imaging and diagnosis, but it might be beneficial to kill cancer cells and can be used as a means of cancer therapy (Bakalova et al., 2004; Samia et al., 2006). A deeper understanding of the mechanism of QDs-induced cell damage under UV illumination might be very meaningful to promote their clinical applications. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgement We acknowledge financial support from the Natural Science Foundation of Jiangsu Province (BK2008400). References Alivisatos, P., 2004. The use of nanocrystals in biological detection. Nat. Biotechnol. 22, 47–52. Bakalova, R., Ohba, H., Zhelev, Z., Ishikawa, M., Baba, Y., 2004. Quantum dots as photosensitizers? Nat. Biotechnol. 22, 1360–1361. Chan, W.H., Shiao, N.H., 2008. Cytotoxic effect of CdSe quantum dots on mouse embryonic development. Acta Pharmacol. Sin. 29, 259–266. Chan, W.H., Shiao, N.H., Lu, P.Z., 2006. CdSe quantum dots induce apoptosis in human neuroblastoma cells via mitochondrial-dependent pathways and inhibition of survival signals. Toxicol. Lett. 167, 191–200. Choi, A.O., Brown, S.E., Szyf, M., Maysinger, D., 2008. Quantum dot-induced epigenetic and genotoxic changes in human breast cancer cells. J. Mol. Med. 86, 291–302. Choi, A.O., Cho, S.J., Desbarats, J., Lovric, J., Maysinger, D., 2007. Quantum dot-induced cell death involves Fas upregulation and lipid peroxidation in human neuroblastoma cells. J. Nanobiotechnol. 5, 1–13. Derfus, A.M., Chan, W.C.W., Bhatia, S.N., 2004. Probing the cytotoxicity of semiconductor quantum dots. Nano Lett. 4, 11–18. Finkel, T., Holbrook, N.J., 2000. Oxidants, oxidative stress and the biology of ageing. Nature 408, 239–247. Fujioka, K., Hiruoka, M., Sato, K., Manabe, N., Miyasaka, R., Hanada, S., Hoshino, A., Tilley, R.D., Manome, Y., Hirakuri, K., Yamamoto, K., 2008. Luminescent passiveoxidized silicon quantum dots as biological staining labels and their cytotoxicity effects at high concentration. Nanotechnology 19, 415102. Gerion, D., Pinaud, F., Williams, S.C., Parak, W.J., Zanchet, D., Weiss, S., Alivisatos, A.P., 2001. Synthesis and properties of biocompatible water-soluble silica-coated CdSe/ZnS semiconductor quantum dots. J. Phys. Chem. B 105, 8861–8871. Hardman, R., 2006. A toxicologic review of quantum dots: toxicity depends on physicochemical and environmental factors. Environ. Health. Perspect. 114, 165–172. Hoshino, A., Fujioka, K., Oku, T., Suga, M., Sasaki, Y.F., Ohta, T., Yasuhara, M., Suzuki, K., Yamamoto, K., 2004. Physicochemical properties and cellular toxicity of nanocrystal quantum dots depend on their surface modification. Nano Lett. 4, 2163–2169. Hyunsoo, K., Sung, C.Y., Tae, Y.L., Daewon, J., 2009. Discriminative cytotoxicity assessment based on various cellular damages. Toxicol. Lett. 184, 13–17.
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
UV-enhanced cytotoxicity of thiol-capped CdTe quantum dots in human pancreatic carcinoma cells Shu-quan Chang a , Yao-dong Dai a,∗ , Bin Kang a , Wei Han a , Ling Mao b , Da Chen a a b
College of Material Science and Technology, Nanjing University of Aeronautics and Astronautics, Nanjing 210016, PR China National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention, Beijing 100088, PR China
a r t i c l e
i n f o
Article history: Received 8 November 2008 Received in revised form 6 February 2009 Accepted 13 March 2009 Available online 25 March 2009 Keywords: Quantum dots Cytotoxicity UV illumination Reactive oxygen species
a b s t r a c t Quantum dots (QDs) have been gaining popularity due to their potential application in cellular imaging and diagnosis, but their cytotoxicity under light illumination has not been fully investigated. In this study, green and red mercaptopropionic acid capped CdTe quantum dots (MPA-CdTe QDs) were employed to investigate their cytotoxicity in human pancreatic carcinoma cells (PANC-1) under UV illumination. MPACdTe QDs exhibited excellent photostability under UV illumination and could be easily ingested by cells. The cytotoxicity of MPA-CdTe QDs was significantly enhanced under UV illumination, which was determined by changes in cell morphology as well as by decreases in the metabolic activity and cell counting. Our results indicated that green and red QDs had different cellular distribution and exhibited distinct UV-enhanced cytotoxicity. UV illumination enhanced the generation of reactive oxygen species (ROS) in cells containing QDs, and NAC antioxidant could reduce their damage to cells under UV illumination. Moreover, the influences of different UV illumination conditions on the viability of cells containing QDs were examined and discussed in detail. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction With the development of nanotechnology, numerous nanomaterials have been widely used in biology and medicine (Nel et al., 2006). The cytotoxicity of nanomaterials is considered to be very crucial to their wide applications in biomedical area and has received more and more attention in recent years (Chan and Shiao, 2008; Hardman, 2006; Monteiro-Riviere and Inman, 2006; Monteiro-Riviere et al., 2005; Nikitin et al., 2008; Sayes et al., 2006, 2007). Semiconductor nanoparticles, often referred as quantum dots (QDs), are usually 1–12 nm in diameter and have unique optical and electronic properties (Murray et al., 2000). QDs can absorb irradiated energy at any wavelength greater than that of their lowest energy transition and may then convert the irradiated energy to an extremely narrow bandwidth emission. QDs are emerging as alternative or complementary tools to the organic fluorescent dyes currently. Compared with the traditional organic fluorescence probes, QDs have many advantages, such as broadband excitation, narrow bandwidth emission, emission of high intensity light, resistance to quenching and good photochemical stability (Alivisatos, 2004; Gerion et al., 2001).
∗ Corresponding author. Tel.: +86 25 52112918; fax: +86 25 52119016. E-mail address: yd [email protected] (Y.d. Dai). 0378-4274/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2009.03.013
Recently, QDs have been gaining popularity due to their potential applications in cellular imaging and medical diagnosis (Medintz et al., 2005; Michalet et al., 2005). Meanwhile, the potential toxicity of QDs is a growing concern in spite of early studies in immortalized cell lines showed little or no deleterious effects of QDs in chronic treatment paradigms (Jaiswal et al., 2003; Parak et al., 2002; Wu et al., 2003). Hoshino et al. (2004) demonstrated that toxicity of QDs was not dependent on the nanocrystal itself but rather on the surface molecules. Another study suggested that in addition to the release of toxic Cd2+ ions from the particles also their surface chemistry, in particular their stability toward aggregation, played an important role for their cytotoxicity (Kirchner et al., 2005). Maysinger and co-workers also carried out many studies on the pathways of QDs-induced cytotoxicity and found that QDs-induced cell death involved Fas upregulation and lipid peroxidation in human neuroblastoma cells (Choi et al., 2007, 2008). Chan et al. (2006) demonstrated that QDs induced apoptosis in human neuroblastoma cells via mitochondrial-dependent pathways and inhibition of survival signals. Several studies reported that QDs could induce the generation of reactive oxygen species (ROS) and ROS was related to the cytotoxicity of QDs (Ipe et al., 2005; Tsay et al., 2007). As is mentioned above, QDs are mainly used as imaging and diagnosis agents in biomedicine area. So, they have to be exposed to illumination conditions usually. It will be meaningful to study the cytotoxicity of QDs under illumination conditions. Using
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primary hepatocytes as a liver model, Derfus et al. (2004) found that the toxicity of CdSe-core QDs could be enhanced under UV irradiation. Liang et al. (2007) investigated the calf thymus DNA damage induced by CdSe QDs under UV irradiation. Fujioka et al. (2008) compared the cytotoxicity of Si QDs and CdSe QDs in HeLa cells. Recently, Mortensen et al. (2008) examined the in vivo skin penetration of carboxylated QDs in the murine model under UV irradiation and concluded that UV irradiation could enhance the toxic effects of QDs in the skin of SKH-1 mice. However, the detailed cellular effects of the UV-enhanced toxicity were not fully investigated. In this paper, green and red mercaptopropionic acid capped CdTe quantum dots (MPA-CdTe QDs) were employed to investigate their cytotoxicity in human pancreatic carcinoma cells (PANC-1) under UV illumination. The different distribution and cytotoxicity of red and green QDs under UV irradiation were proposed. The generation of ROS in cells containing QDs under UV illumination and the influences of different UV illumination conditions (UV irradiation time, QDs concentration, incubation time with QDs, post-exposure time) on the UV-enhanced cytotoxicity of QDs were examined and discussed in detail. 2. Materials and methods 2.1. Preparation and characterization of MPA-CdTe QDs All the reagents used in this study were analytical grade and purchased from Sigma–Aldrich Company unless indicated otherwise. Green and red mercaptopropionic acid capped CdTe quantum dots (MPA-CdTe QDs) employed in this study were synthesized as the method described in previous literatures (Lovric et al., 2005b). In a typical synthesis: 1.65 g sodium borohydride was dissolved in 40 mL water at 0 ◦ C while stirring under N2 atmosphere protection, then 2.61 g tellurium powder was added into above solution portionwise, and the mixture was stirred at 0 ◦ C under N2 for 6 h, yielding a purple NaHTe solution. 0.5 mL cadmium perchlorate hydrate (1 M aqueous solution) and 0.3 mL 3-mercaptopropionic acid were dissolved in 200 mL water. The pH of the solution was adjusted to 11 with 1 M NaOH solution prior to addition of an aliquot of the NaHTe solution previously prepared (0.2 mL). The reaction mixture was heated to reflux under N2 protection. Green QDs formed after the reaction time of 10 min, while red QDs were generated after 7 h. The as prepared QDs solution was dialyzed against deionized water for 6 h and concentrated by using a rotary evaporator. QDs were collected by centrifugation and purified by size-selective precipitation. The photoluminescence properties of MPA-CdTe QDs were characterized subsequently. Ultraviolet–visible (UV–vis) spectroscopy was carried out at room temperature using a PerkinElmer -17 spectrophotometer. The photoluminescence (PL) spectrum was taken with a HITACHI 850 spectrofluorophotometer. The photostability of green and red QDs dissolved in water or PBS was measured after UV illumination. UV light (365 nm) was generated by ZF-20D Ultraviolet Analyzing Equipment with a power density of 19 mW cm−2 . After illumination, photoluminescence (PL) spectra were recorded to evaluate the photostability of QDs. 2.2. Cell culture, QDs treatment and UV illumination Human pancreatic carcinoma cells (PANC-1, ATCC # TIB-222) were cultured in DMEM medium containing 10% (v/v) fetal calf serum at 37 ◦ C in humidified air containing 5% CO2 . For fluorescence microscopic measurements, cells should be seeded onto a glass coverslip placed in 6-well plates. For MTT assay, cells were cultured in 96well plates. Cells were incubated up to about 24 h and grown to about 80% confluence before experiments. QDs dispersed in PBS were added to each well to achieve a final concentration. Cells were then incubated in these medium containing QDs at 37 ◦ C and in 5% CO2 atmosphere for different time periods. After incubation, all cells were washed with PBS to remove excess QDs and placed in fresh solutions before next experiments. UV irradiation was carried out on ZF-20D Ultraviolet Analyzing Equipment which generated 365 nm UV light with a power density of 19 mW cm−2 . All treatments were done in triplicates or quadruplicates in three or more independent experiments.
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only two channels (CH1: excitation 488 nm, filter 500–600 nm for green QDs or 600–700 nm for red QDs; CH3: 488 nm, DIC) were set to record QDs and DIC signals. Cells were observed using 60× oil immersion objective. Images were acquired at a resolution of 800 × 800, and the scan size was 212 m × 212 m. 2.4. MTT assay and cell counting Colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assays were performed to assess the metabolic activity of cells treated with different conditions. After the treatment, the medium was removed and replaced with serum-free media (200 L/well). A total of 20 L stock MTT (5 mg mL−1 ) was added to each well, and the cells were then incubated for 1 h at 37 ◦ C. The medium was removed, and the cells were lysed with DMSO. The absorbance was measured at 595 nm. Cell number was determined by trypan blue exclusion assay (Hyunsoo et al., 2009). 2.5. ROS imaging and analysis Reactive oxygen species generation was imaged by using dihydroethidium (DHE) and Olympus FV-1000 laser scanning confocal microscope. After treatments, cells were washed and medium was replaced with serum-free medium. DHE was dissolved in DMSO (3 mM) and added to the culture medium at a final concentration of 10 M, and cells were incubated for 30 min at 37 ◦ C. Cells were washed with PBS before imaging. The oxidation product of DHE was ethidium, whose fluorescence was enhanced about 10-fold when bound to DNA. Three channels (CH1: excitation 488 nm, filter 500–550 nm; CH2: excitation 488 nm, filter 610–650 nm; CH3: 488 nm, DIC) in LSCM were set to record QDs, ethidium and DIC signals. Collected images were used to quantify the relative fluorescence intensity (RFI) of ethidium. N-acetylcysteine (NAC) was employed to study the effect of ROS on cell viability. NAC was dissolved in PBS (500 mM) and added to culture medium reaching a concentration of 5 mM. 2.6. Statistics analysis Results were expressed as mean ± standard deviation (SD). Significance was evaluated using Student’s t-test. Difference was considered significant if p < 0.05.
3. Results 3.1. Characterization and photoluminescence stability of MPA-CdTe QD MPA capped CdTe QDs were prepared following the general procedure previously reported. The size of QDs grew larger with the reaction proceeding, and consequently their emission wavelength shifted to longer wavelength. To assess the size-dependent effect of QDs on cells, we employed two size QDs with green-emitting and red-emitting respectively in this study. Green QDs formed after the reaction time of 10 min, while red QDs were generated after 7 h. The optical properties of MPA-CdTe QDs used in this study were shown in Fig. 1A. As it can be seen, green QDs had an absorption peak at around 510 nm and a fluorescence emission peak at 538 nm. While the absorption peak and emission peak of red QDs were about 569 and 629 nm respectively. As shown in the insets of Fig. 1A, both the green and red QDs exhibited excellent water-solubility and bright fluorescence under UV illumination. The photostability of QDs in cells under UV irradiation was considered to be very crucial in the following experiments, so the changes of their fluorescence intensity in water and PBS were examined after UV irradiation and the results were presented in Fig. 1B. It can be seen that the red QDs had better stability than the green QDs, and the QDs in water had better stability than in PBS. All the results revealed that decrease of fluorescence intensity of green and red QDs in water and PBS was less than 20% after UV illumination.
2.3. Laser scanning confocal microscopy Olympus FV-1000 laser scanning confocal microscope (LSCM) was used to acquire images. After the treatment, PANC-1 cells were fixed with 4% paraformaldehyde for 15 min, and then washed three times with PBS buffer. To clearly observe the distribution and positions of QDs in cells, nuclei were stained with 0.1 g mL−1 DAPI for 2 min. Before imaging, cells were washed with PBS again. Three channels (CH1: excitation 405 nm, filter 420–470 nm; CH2: excitation 488 nm, filter 500–600 nm for green QDs and 600–700 nm for red QDs; CH3: 405 nm/488 nm, DIC) were set to record DAPI, QDs and DIC signals. To study the changes of cellular morphology,
3.2. Cellular distribution of QDs and UV-induced morphological changes of PANC-1 cells containing QDs To study the cellular distribution of green and red MPA-CdTe QDs, fluorescence imaging examination of PANC-1 cells incubated with 50 g mL−1 QDs for 4 h were carried out. In order to determine and visualize the location of QDs within the cells, the nuclei
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of PANC-1 cells were stained by DAPI. The most striking difference between green and red QDs in their localization was observed in the nuclear vs. cytoplasmic compartments. The PANC-1 cells treated with green QDs showed that some QDs located in the cytoplasm and a few QDs appeared in the nuclei (Fig. 2A and C). In contrast, the cells treated with red QDs indicated that most of the QDs were distributed throughout the cytoplasm but no QDs were observed in the nuclei (Fig. 2D and F). These results were consistent with the previous reports (Lovric et al., 2005a). The size of QDs was considered very important to their distribution and metabolizability in cells, and it also might be closely related to their cytotoxicity. To assess the damage of cells containing QDs under UV illumination, PANC-1 cells incubated with green or red QDs (50 g mL−1 , 4 h) were treated with UV irradiation ( = 365 nm, 19 mW cm−2 , 1 h). After the treatments, the cells were characterized by LSCM. The cells without any treatments (no QDs and no UV illumination) were considered as control group in our experiments. Compared with the control group (Fig. 3A), the PANC-1 cells without QDs had no morphological changes after UV illumination (Fig. 3B). The cells only treated with QDs also showed no distinct changes in morphology (Fig. 3C and D). In contrast, the cells containing green or red QDs were badly damaged and the integrality of cells was affected seriously after UV illumination (Fig. 3E and F). It was also seen that the cells incubated with green QDs were damaged more seriously in morphology than the cells with red QDs after UV illumination. These results indicated that UV illumination significantly enhanced the damages of QDs to cells and QDs with different size exhibited different UV-enhanced damages to cells. Fig. 1. Photoluminescence properties of green and red MPA-CdTe QDs. (A) UV–vis (left) and PL (right) spectra of green (a) and red (b) MPA-CdTe QDs. The excitation wavelength for the PL spectrum was 430 nm. Insets showed the photographs of green (a) and red (b) QDs under UV illumination. (B) Time-dependent photoluminescence properties of green and red MPA-CdTe QDs under UV illumination (365 nm, 19 mW cm−2 ) in water or PBS.
3.3. Metabolic activity and cell counting of PANC-1 cells containing QDs under UV illumination Based on above observed changes in cell morphology, MTT assays and cell counting were carried out to investigate the UV-
Fig. 2. Cellular distribution of green and red MPA-CdTe QDs. (A) Fluorescence image of cells incubated with green QDs for 4 h. (B) The DIC image of (A). (C) The overlay image of (A and B). (D) Fluorescence image of cells incubated with red QDs for 4 h. (E) The DIC image of (D). (F) The overlay image of (D and E). Insets in (C and F) showed the magnified image of a certain cell. The final concentration of MPA-CdTe QDs in medium was 50 g mL−1 . The cellular nuclei were stained by DAPI (0.1 g mL−1 , 2 min). The scale bar represents 30 m.
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enhanced cytotoxicity of QDs and the results were shown in Fig. 4. The cell counting and metabolic activity of cells without QDs showed no distinct changes after UV illumination compared with the control group. In contrast, the metabolic activity of cells with red QDs decreased from 75% to 27%, and metabolic activity of cells with green QDs decreased from 59% to 11% after UV illumination. That was to say, UV illumination reduced the viability of cells containing red and green QDs by 65.7% and 81.4% respectively, comparing with their values before UV illumination. The loss of cell number is also very notable. UV illumination reduced the cell number of cells containing red and green QDs by 55.2% and 85.1% respectively, comparing with their values before UV illumination. These results indicated that UV illumination could distinctly enhance the cytotoxicity of QDs, which was consistent
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with the results of LSCM. In addition, the increase of cytotoxicity of green QDs was more notable than red QDs after UV illumination. 3.4. Influences of different UV illumination conditions on the cell number and metabolic activity of cells containing QDs To determine the effects of UV illumination on the cytotoxicity of QDs, different parameters were examined and discussed in detail. These parameters included: UV illumination time (10, 30, 60, 120 min); concentration of QDs (1, 10, 50, 500 g mL−1 ) for cells incubating; incubation time (1, 2, 4, 8 h) with QDs before UV illumination; post-exposure time (0, 1, 2, 4 h). Green MPA-CdTe QDs were used in this section. Cells assays were used to study each parame-
Fig. 3. Combined confocal micrographs of PANC-1 cells under different treatment. (A) Cells were incubated under usual conditions (no QDs and no UV). (B) Cells were irradiated by UV only. (C) Cells were incubated with green QDs only. (D) Cells were incubated with red QDs only. (E) Cells were incubated with green QDs and irradiated by UV. (F) Cells were incubated with red QDs and irradiated by UV. Insets in (E and F) showed the magnified image of a certain cell. The final concentration of MPA-CdTe QDs in medium was 50 g mL−1 and incubation time was 4 h. The power density of UV ( = 365 nm) was 19 mW cm−2 and UV irradiation time was 1 h. The scale bar represents 30 m.
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of cells decreased sharply with the increase of post-exposure time. The cell number and metabolic activity of cells decreased to below 5% when post-exposure time reached 4 h. The loss of cell numbers was not more notable than the decrease of metabolic activity when the post-exposure time is very short. All results revealed that the cytotoxicity of QDs was significantly enhanced by UV illumination and the UV-enhanced cytotoxicity could be influenced by many conditions. 3.5. UV-enhanced generation of ROS in cells containing QDs
Fig. 4. The metabolic activity (A) and cell counting (B) of PANC-1 cells exposed to different conditions. The final concentration of MPA-CdTe QDs in medium was 50 g mL−1 and incubation time was 4 h. The power density of UV ( = 365 nm) was 19 mW cm−2 and UV illumination time was 1 h. MTT assay and cell counting were carried out after UV irradiation for 2 h. The (+) or (−) in above legends mean treating cells with or without corresponding condition. Data represents mean ± SD for five independent experiments (n = 5) (* p < 0.01 and ** p < 0.001).
ter by cell counting and MTT assay. All results were summarized in Fig. 5. Fig. 5A showed that the metabolic activity of cells containing QDs decreased significantly with the increase of UV illumination time, while the metabolic activity of cells treated without QDs kept slight changes under UV illumination. Under UV illumination for 2 h, the metabolic activity of cells without QDs also remained about 80% but that of cells containing green QDs decreased to below 10%. The loss of cell numbers was more notable than the decrease of metabolic activity. The cell number decreased to 2% after 2 h UV irradiation. By increasing the concentration of QDs for cells incubation, the cytotoxicity of QDs without and with UV treatments were both distinctly enhanced, while cell number and metabolic activity of cells under UV illumination were reduced more rapidly than the cells without UV illumination (Fig. 5B). The position of QDs in cells might be closely related to their cytotoxicity. So we examined the influences of incubation time with QDs before UV illumination. Fig. 5C indicated that the cytotoxicity of QDs under UV illumination was significantly enhanced as the increase of incubation time with QDs before UV illumination. The cell number and metabolic activity of cells decreased to below 5% when the incubation time with QDs before UV illumination was 8 h. The loss of cell numbers was more notable than the decrease of metabolic activity. The effects of post-exposure time (the incubation time after UV illumination) were examined and the results were shown in Fig. 5D. It can be seen that the cell number and metabolic activity
The generation of excess reactive oxygen species can cause the modification and damage of cellular proteins, lipids and DNA, and it would subsequently lead to the cell death or apoptosis. To investigate the oxidative stress induced by QDs under UV irradiation, DHE was used in this section. DHE could be oxidized by ROS to ethidium, which intercalated with cellular DNA, yielding bright red fluorescent. Using laser scanning confocal microscope, the oxidative stress was studied and the results were given in Fig. 6. Very weak fluorescence from ethidium was detected in cells without any treatments (Fig. 6A-a) or cells only treated by UV irradiation (Fig. 6A-b). Cells with QDs produced more fluorescence (Fig. 6A-c) than control cells. Meanwhile, a marked increase of fluorescence was detected in QDtreated cells after UV illumination (Fig. 6A-d), compared with the cells only treated with QDs or UV illumination. The quantitative results were shown in Fig. 6B. It can be concluded that the existence of QDs significantly promoted the generation of ROS in cells under UV illumination. N-acetylcysteine (NAC), a strong antioxidant containing a mercapto group, was employed to examine the effect of ROS on the decrease of cell viability caused by QDs under UV illumination. As shown in Fig. 7A, NAC prevented the generation of ROS in cells containing QDs under UV illumination. Fig. 7B also showed that the quantity of ROS in cells after QDs and UV treatments was reduced more than 50% when NAC was added. In addition, NAC could reduce the damage of cells containing QDs under illumination in morphology (Fig. 7A) and improved their viability (Fig. 7C). These results revealed that ROS played an important role in the damage to cells caused by QDs under UV illumination. 4. Discussion In the present study, we mainly demonstrate that: (a) the cytotoxicity of MPA-CdTe QDs in PANC-1 cells was significantly enhanced under UV illumination; (b) green and red QDs had different cellular distribution and exhibited distinct UV-enhanced cytotoxicity; (c) UV illumination significantly enhanced the generation of ROS in cells containing QDs, and NAC antioxidant could reduce these damages to cells under UV illumination; (d) the UVenhanced cytotoxicity could be influenced by many factors, such as the UV illumination time, the concentration of QDs, the incubation time with QDs before UV illumination and post-exposure time. MPA-CdTe QDs exhibit excellent photostability under UV illumination in water or PBS (Fig. 1), which provides the basis for our following study. They also can be easily ingested by PANC-1 cells and exhibit different distribution when their size is different. Red QDs are distributed throughout the cytoplasm but no QDs are observed in the nuclei (Fig. 2D and F). In contrast, some green QDs locate in the cytoplasm and a few QDs appear in the nuclei (Fig. 2A and C). Judging from the changes in cell morphology as well as the decreases in the metabolic activity and cell counting, MPA-CdTe QDs have lesser toxicity to PANC-1 cells without UV irradiation and this cytotoxicity can be distinctly enhanced under UV irradiation (Figs. 3 and 4). In addition, green and red QDs exhibit distinct UV-enhanced cytotoxicity according to our study. The increase of cytotoxicity of green QDs
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Fig. 5. The influences of different UV illumination conditions on the metabolic activity and cell counting of PANC-1 cells. (A) Effect of UV illumination time on PANC-1 cells viability. Cells were incubated with QDs for 4 h and the final concentration of QDs in medium was 50 g mL−1 . Cells were irradiated for different time. MTT assay and cell counting were carried out after UV illumination for 2 h. Controls were cells incubated with no QDs. (B) Effect of concentration of QDs. Cells were incubated with QDs for 4 h under different QDs concentration. UV illumination time was 1 h. MTT assay and cell counting were carried out after UV illumination for 2 h. Controls were cells which were not irradiated by UV. (C) Effect of incubation time before UV illumination. Cells were incubated with QDs for different time and the concentration of QDs was 50 g mL−1 . UV illumination time was 1 h. MTT assay and cell counting were carried out after UV irradiation for 2 h. Controls were cells which were not irradiated by UV. (D) The metabolic activity of cells and cell counting at different post-exposure time. Cells were incubated with QDs for 4 h and the concentration of QDs was 50 g mL−1 . UV illumination time was 1 h. MTT assay and cell counting were carried out after UV irradiation for different time. Controls were cells incubated with no QDs. Green MPA-CdTe QDs were used in this study. The power density of UV ( = 365 nm) was 19 mW cm−2 . Data represents mean ± SD for five independent experiments (n = 5) (* p < 0.01 and ** p < 0.001 vs. control group).
after UV irradiation is more notable than that of red QDs. That might due to their different cellular distribution. The position of QDs in cells is considered to be closely related to their cytotoxicity. Compared with the red QDs which are only distributed in cytoplasm, the green QDs can enter the nuclei partially and might induce more directly and seriously damage to DNA or other targeting positions in the nuclei under UV irradiation. Based on our results, QDs with larger size may be safer under UV irradiation conditions. According to our LSCM results, UV illumination significantly enhances the generation of ROS in cells containing QDs (Fig. 6). The UV-enhanced generation of ROS is attenuated in the presence of the antioxidant NAC (Fig. 7). Judging from the cellular morphology, cell counting and metabolic activity, the damage of QDs to cells under UV is reduced notably when NAC is added (Fig. 7). Above results reveal that ROS play an important role in QDs-induced damage of cells under UV illumination. That is because ROS can let cells undergo oxidative stress. Results of this stress include modification and damage of cellular components, such as lipids, proteins, and DNA (Finkel and Holbrook, 2000; Prestwich et al., 2005). All above direct or indirect damages to cells can lead to the apoptosis or death of cells in the end (Lovric et al., 2005b; Maysinger, 2007). Proper antioxidant should be employed to reduce the UV-enhanced toxicity of QDs in clinical applications.
Our results also demonstrate that the UV-enhanced cytotoxicity could be influenced by many conditions (Fig. 5). Firstly, the cytotoxicity of QDs under UV irradiation is enhanced more and more notably as the increase of UV irradiation time or QDs concentration (Fig. 5A and B). The combination of UV irradiation and QDs induces more serious damage to cells. Above results also indicate the synergistic effect between QDs and UV irradiation on the damage of cells. The UV-enhanced cytotoxicity of QDs is less significantly than the QDs-enhanced cytotoxicity of UV irradiation. The incubation time of cells with QDs before UV illumination can also evidently affect the UV-enhanced cytotoxicity of QDs (Fig. 5C). That is because the uptake and distribution of QDs in cells need a period of time and should undergo a complex process. The position of QDs in cells is considered to be closely related to their cytotoxicity. So, QDs incubated with QDs for different period of time before UV illumination exhibit different UV-enhanced cytotoxicity. Moreover, the cytotoxicity of QDs under UV irradiation is enhanced as the increase of post-exposure time. The initial damages to cells could be repaired or enlarged with the cellular metabolism, which also need a period of time. As the increase of post-exposure time, the initial damages to cells are enlarged. As a result, more serious toxicity is observed. Now that the UV-enhanced toxicity of QDs can be influenced by above conditions, we should be able
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Fig. 6. QDs and UV illumination induced the generation of ROS in cells. (A) Confocal micrographs of DHE-stained cells treated under different conditions: (a) with no QDs and no UV, (b) with no QDs and under UV, (c) with QDs and no UV, (d) with QDs and under UV. (B) Relative fluorescence intensity of DHE-stained cells corresponding to (A). Data represents mean ± SD for three independent experiments (n = 5) (** p < 0.001). Cells were incubated with green MPA-CdTe QDs for 4 h and the concentration of QDs was 50 g mL−1 . The power density of UV ( = 365 nm) was 19 mW cm−2 and UV illumination time was 1 h. PNAC-1 cells were stained with DHE (10 M) after treatment.
Fig. 7. NAC reduced the generation of ROS in cells and enhanced the viability of cells under UV treatment. (A) Confocal micrographs of DHE-stained cells treated with QDs and UV. (a) Fluorescence image of cells treated with QDs only. (b) Overlay image of (a) and DIC image. (c) Fluorescence image of cells treated with QDs and NAC (10 mM). (d) Overlay image of (c) and DIC image. (B) Relative fluorescence intensity of DHE-stained cells corresponding to (A). Controls were cells stained with DHE and incubated with no QDs and no UV irradiation. Data represents mean ± SD for three independent experiments (n = 5) (* p < 0.01 and ** p < 0.001). (C) Metabolic activity of cells corresponding to (A). Controls were cells stained with DHE and incubated with no QDs and no UV irradiation. Data represents mean ± SD for three independent experiments (n = 5) (* p < 0.01). Cells were incubated with green MPA-CdTe QDs for 4 h and the concentration of QDs was 50 g mL−1 . The power density of UV ( = 365 nm) was 19 mW cm−2 and UV illumination time was 1 h. PNAC-1 cells were stained with DHE (10 M) after treatment.
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