Cytotoxicity assessment of functionalized CdSe, CdTe and InP quantum dots in two human cancer cell models

Materials Science and Engineering C 57 (2015) 222–231

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Cytotoxicity assessment of functionalized CdSe, CdTe and InP quantum dots in two human cancer cell models Jing Liu a,1, Rui Hu b,1, Jianwei Liu a, Butian Zhang b, Yucheng Wang b, Xin Liu c, Wing-Cheung Law d, Liwei Liu e, Ling Ye a,⁎, Ken-Tye Yong b,⁎ a

Institute of Gerontology and Geriatrics & Beijing Key Lab of Aging and Geriatrics, Chinese PLA General Hospital, Beijing 100853, PR China School of Electrical and Electronic Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore Lawrence Berkeley National Laboratory, Berkeley, CA, United States d Department of Industrial and System Engineering, The Hang Kong Polytechnic University, Hung Hom, Hong Kong e School of Science, Changchun University of Science and Technology, Changchun 130022, PR China b c

a r t i c l e

i n f o

Article history: Received 2 September 2014 Received in revised form 9 June 2015 Accepted 23 July 2015 Available online 26 July 2015 Keywords: Cytotoxicity Quantum dots Cancer cell

a b s t r a c t The toxicity of quantum dots (QDs) has been extensively studied over the past decade. Some common factors that originate the QD toxicity include releasing of heavy metal ions from degraded QDs and the generation of reactive oxygen species on the QD surface. In addition to these factors, we should also carefully examine other potential QD toxicity causes that will play crucial roles in impacting the overall biological system. In this contribution, we have performed cytotoxicity assessment of four types of QD formulations in two different human cancer cell models. The four types of QD formulations, namely, mercaptopropionic acid modified CdSe/CdS/ZnS QDs (CdSe-MPA), PEGylated phospholipid encapsulated CdSe/CdS/ZnS QDs (CdSe-Phos), PEGylated phospholipid encapsulated InP/ZnS QDs (InP-Phos) and Pluronic F127 encapsulated CdTe/ZnS QDs (CdTe-F127), are representatives for the commonly used QD formulations in biomedical applications. Both the core materials and the surface modifications have been taken into consideration as the key factors for the cytotoxicity assessment. Through side-by-side comparison and careful evaluations, we have found that the toxicity of QDs does not solely depend on a single factor in initiating the toxicity in biological system but rather it depends on a combination of elements from the particle formulations. More importantly, our toxicity assessment shows different cytotoxicity trend for all the prepared formulations tested on gastric adenocarcinoma (BGC-823) and neuroblastoma (SH-SY5Y) cell lines. We have further proposed that the cellular uptake of these nanocrystals plays an important role in determining the final faith of the toxicity impact of the formulation. The result here suggests that the toxicity of QDs is rather complex and it cannot be generalized under a few assumptions reported previously. We suggest that one have to evaluate the QD toxicity on a case to case basis and this indicates that standard procedures and comprehensive protocols are urgently needed to be developed and employed for fully assessing and understanding the origins of the toxicity arising from different QD formulations. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Since 1998, the use of quantum dots (QDs) in biomedical research has received a burst of intensive investigation that significantly enhanced the understanding of using these particles for biological applications [1–4]. QDs exhibit several attractive advantages for biomedical imaging applications [5,6]. For example, they have rich surface chemistries that allow one to chemically modify and engineer their surface with multiple functional reaction groups for bioconjugation purpose [7–9]. As for their optical property, these nanocrystals have wide absorption spectra, narrow emission spectra, flexible emission peak ⁎ Corresponding authors. E-mail addresses: [email protected] (L. Ye), [email protected] (K.-T. Yong). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.msec.2015.07.044 0928-4931/© 2015 Elsevier B.V. All rights reserved.

tunability and high resistance to photobleaching. All these unique features have made QDs an important optical probe for imaging of cells and small animals [10,11]. Currently, they have been widely adopted for immunoassay [12,13], nucleic acid detection [14], fluorescence resonance energy transfer (FRET) sensing [15], cancer cell imaging [16] and in vivo tumor imaging [17]. In addition, some research groups have even demonstrated that it is possible to use QDs as a nanocarrier system for drug delivery [18–20]. Despite for all these attractive applications, many researchers have raised significant concerns about translating QDs for clinical practice because of their heavy metal contents and they may induce toxicity when they are interacting with the local biological environments [21,22]. Standard tests such as cell viability assay and intracellular reactive oxygen species (ROS) assay are commonly used to determine the cytotoxicity of QDs in vitro. It is clear that the release of the heavy metal ions from poorly passivated QDs has been

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identified as the main culprit in causing the cytotoxicity [23–25]. Some researchers even suggested that the QD toxicity should be dosage dependent since these particles will accumulate in the body and stay for a long period of time. Later, it was also found that photo-induced free radicals generated in the presence of QDs and the surface charge of QDs play significant roles in initiating the cytotoxicity [26–28]. Other than those, surface oxidation of cadmium based QDs has also been considered as a main cause of the cytotoxicity [29]. These findings have raised lots of safety concerns regarding the use of QDs in clinical practices. To date, there are four common factors that are known to generate or initiate the QD toxicity when these particles are interacting with biological systems, namely, core composition of the QDs, surface coating of QDs, hydrodynamic diameter of the QDs and the particle surface charge [22]. In recent years, tremendous efforts have been devoted in preparing heavy-metal free QDs for reducing their toxicity effects. For instance, our group has demonstrated the preparation of silicon (Si) QDs for bioimaging applications and these particles possess very low toxicity [30,31]. Such result suggests that this material will be a strong candidate in replacing heavy-metal based QDs for future clinical applications [32,33]. Applying surface coating on QDs is another useful strategy to prevent the breakdown of the nanocrystals. In general, many QDs are passivated with a thin layer of zinc sulfide (ZnS) shell whereby completely covering the heavy-metal based QD core [4,34]. The shell not only provides a strong protection to the core material against degradation but also greatly improves the overall optical property of the QDs such as their brightness and narrower emission spectrum width. Besides from the ZnS shell coating, additional functionalization steps are needed to prepare different types of bioconjugated QDs for specific biomedical applications and this step will generate significant complexity in understanding the overall QD toxicity since each and every surface coating formulation is made by unique ingredients. The ingredients may very likely be a critical factor that induces local cytotoxicity since their chemical structures have been altered during the surface modification process. Different types of materials have been used in this functionalization step. For example, silicon dioxide shell coating on the particle surface [35,36], anchoring the QDs surface with hetero-bifunctional molecules such as mercapto-acids [16], coating the QD surface with biocompatible polymer layer [37] and micelle encapsulation of QDs [38]. After the surface modification, the physical property of the resulting QDs will be quite different from their original form. Studies have shown that green emitting cadmium telluride (CdTe) QDs (2.2 nm) are more toxic than that of the larger ones (5.5 nm) which may be due to the small hydrodynamic size of the particles that enables them to enter the cell nucleus [39,40]. For biomedical application purpose, the overall surface charge and colloidal stability of QD formulations are crucial parameters to be considered for designing successful and achievable experiments. Based the complexity of the QD formulations, we hypothesized that each and every type of QD formulations will react differently towards the same cell type. In this contribution, we have performed cytotoxicity assessment of four types of QD formulations in two human cancer cell models, gastric adenocarcinoma (BGC-823) and neuroblastoma (SH-SY5Y). The four different QD formulations, namely, mercaptopropionic acid (MPA) modified CdSe/CdS/ZnS QDs (CdSe-MPA), PEGylated phospholipid encapsulated CdSe/CdS/ZnS QDs (CdSe-Phos), PEGylated phospholipid encapsulated InP/ZnS QDs (InP-Phos) and Pluronic F127 encapsulated CdTe/ZnS QDs (CdTe-F127) are typical representatives for the commonly used QD formulations in biomedical applications. We have taken both the core materials and the surface modifications into consideration as the key factors for the cytotoxicity assessment. Through side-byside comparison, we have performed an overall evaluation on the cytotoxicity-causing factors of QD formulations. We have found that the toxicity of QDs does not solely depend on a single factor in initiating the toxicity in biological system but rather it depends on a combination of elements from the particle formulations. When BGC-823 cells are

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used for the cytotoxicity evaluation test, majority of the formulations have shown high biocompatibility except for the CdSe-MPA formulation. On the other hand, the SH-SY5Y cells have shown high tolerance to CdSe-MPA formulation but are quite vulnerable to the other three QD formulations. Our studies show that the difference of cytotoxicity trend is mainly due to the dissimilar cellular uptake rate of QDs from the two cell lines. These results suggest that the toxicity of QDs is rather complex and it cannot be generalized under a few simple assumptions such as shape, size and surface charge of the nanoparticles, indicating that standard procedures and comprehensive protocols are required to be developed for fully assessing and understanding the origins of the toxicity arising from each and individual QD formulations. 2. Materials and methods 2.1. Materials Cadmium oxide (CdO), cadmium chloride, Selenium, tellurium, Zinc acetate, sulfur, tetradecylphosphonic acid (TDPA), tri-n-octylphosphine oxide (TOPO), tri-n-octylphosphine (TOP), sodium borohydride, oleic acid, L-cysteine, sodium hydroxide, 1-dodecanethiol, ammonium hydroxide and mercaptopropionic acid (MPA) were purchased from Sigma-Aldrich. PEGylated phospholipid mPEG-DSPE (MW 5000) was purchased from Laysan Bio Inc. Pluronic F127 was obtained as a gift from BASF. Solvents such as acetone, chloroform, dimethyl sulfoxide (DMSO) and toluene were obtained from Sigma-Aldrich. Ethanol was obtained from Best Chemical CO. Pte Ltd. (Singapore). All chemicals were used as received without any further purification. InP/ZnS QDs were obtained from NN Labs LLC (Fayetteville, AR). Deionized water (18.2 M Ω cm) was obtained from a Milli-Q system (Millipore, Molsheln, France). 2.2. Synthesis of quantum dots (QDs) 2.2.1. CdSe/CdS/ZnS core/shell/shell structure of QDs The synthesis of CdSe/CdS/ZnS core/shell/shell QDs was adapted from our previous reported protocol [41]. Briefly, CdO (1.6 mmol), TDPA (3 mmol) and TOPO (3 g) were heated in a three-neck flask to 290–300 °C under argon. After a clear solution was obtained and maintained at 300 °C for about 10 min, TOP-Se (0.8 ml, 1 M as of Se concentration) was rapidly injected. The heating mantle was removed after 2–3 min to stop the reaction. The resultant CdSe QD cores were washed with ethanol and dispersed in toluene. To grow the CdS/ZnS graded shell, CdO (1 mmol), zinc acetate (4 mmol) and TOPO (7 g) were dissolved in 10 ml of oleic acid and heated to 180 °C for 30 min under argon, to which the CdSe cores dispersion was slowly injected and the reaction temperature was kept at 180 °C to evaporate toluene through a needle outlet. The needle was removed after 15–20 min and the temperature was raised to 210 °C for shell coating, where TOP-S (2 ml, 1 M as of S concentration) was added drop wise. The mixture was held at 210 °C for another 10–15 min and then transferred into excess toluene by syringe to quench further reaction. The final product CdSe/CdS/ZnS QDs were washed with ethanol and dispersed in toluene for future use. For long term storage, the solvent was evaporated and the bottle was filled with nitrogen gas and properly sealed. 2.3. CdTe/ZnS core/shell QDs The synthesis of CdTe QDs was adapted from our previous reported protocol [42]. Briefly, 2 mmol of tellurium powder and 2 mmol of sodium borohydride (in excess) were mixed with 10 ml of nitrogen saturated water in a triangular flask and stirred for 1–2 h. The flask was sealed with a needle outlet to release the hydrogen gas pressure. A light-pink colored solution is formed in the end and referred as the Te precursor. In parallel, 6 mmol of cadmium chloride and 15 mmol of L-cysteine were dissolved in 300 ml of nitrogen saturated water in a 500 ml

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three-neck round-bottom flask. Under stirring, the pH was adjusted to 11–12 by sodium hydroxide (1 M). The flask was then sealed and the Te precursor was injected into the mixture under nitrogen atmosphere. The reaction mixture was heated to 98.5 °C. The reaction was kept going until the proper emission wavelength was reached. The resultant CdTe QDs were washed with ethanol and re-dispersed in water with high concentration. To coat the ZnS shell, the water dispersible CdTe QDs were transferred to organic phase. 6 ml of 1-dodecanethiol, 6 ml of butanol and 2 ml of acetone were mixed with the as prepared high concentration CdTe QDs (in water). Under vigorous stirring, 0.4 ml of ammonium hydroxide was added to the mixture and the reaction was kept running for 3–4 h. After phase separation, the CdTe QDs in the oil phase were collected, washed with ethanol and dispersed in chloroform. For the ZnS shell coating, 0.5 g of zinc acetate was dissolved in 20 ml of oleic acid at 150 °C in a three-neck round-bottom flask. The temperature was cooled down to 40 °C and the CdTe chloroform dispersion was injected. The temperature was brought to 65 °C to evaporate the chloroform and then 120 °C when 1 ml of sulfur TOPO solution (1 mmol) was added drop wise. The mixture was kept at 120 °C for 5 h and cooled down to room temperature. Chloroform was added to dissolve the resultant and the CdTe/ZnS QDs were washed with ethanol. 2.4. Surface modification of QDs For the ligand exchange with MPA, 5 ml of CdSe/CdS/ZnS QDs (weighted and re-dispersed in chloroform at a concentration of 0.2 mg/ml) was mixed with 3 ml of MPA (0.2 mmol in water). Under vigorous stirring, 1.5 ml of ammonium hydroxide was then added and the mixture was stirred for 4 h. After phase separation, the MPA capped QDs in aqueous solution were washed with ethanol and dispersed in water for future use. For amphiphilic polymer encapsulation, the oilphase QDs were washed with ethanol to remove excess surfactant and dispersed in chloroform at a concentration of 1 mg/ml. Amphiphilic polymers were dissolved in chloroform and mixed with QDs at a weight ratio of 5:1 and 10:1 for PEGylated phospholipid and Pluronic F127, respectively. The chloroform was then evaporated using a vacuum rotary evaporator in water bath at room temperature. The resulting lipidic film was collected and then dispersed with water assisted by ultrasound sonication. The solution is then filtered with a 0.2 μm syringe filter to remove possible aggregation. 2.5. Characterization of QDs The absorption and photoluminescence (PL) spectra of the QDs were collected using a Shimadzu model UV-2450 spectrophotometer and a Fluorolog-3 Spectrofluorometer (Edison, NJ USA), respectively. QD samples were loaded into a quartz cuvette for measurement while the corresponding solvents were used as blank for reference. The hydrodynamic size distribution of the nanoparticle formulations were analyzed by dynamic light scattering (DLS) method using the particle size analyzer (Brookhaven Instruments 90Plus). Transmission electron microscopy (TEM) images were obtained using a JEOL model JEM2010 microscope at an acceleration voltage of 200 kV. The specimens were prepared by drop-casting the sample dispersions onto formvar/carbon coated 300 mesh copper grids. The excess solvent was absorbed by a filter paper underneath. All measurements were carried out at room temperature. 2.6. PL intensity stability study All the four QD formulations were dispersed in three different media (DI water, DMEM and DMEM supplemented with 10% FBS) with the same concentration of 50 μg/ml and loaded into different wells of a 96-well flat-bottom Microtiter plate. The peak values of the PL for each formulation was monitored by a multimode microplate reader SpectraMax M5 (Molecular Devices LLC, CA, USA) for over 72 h at a

time interval of 1 h. Assays were performed in triplicate and the results were averaged and normalized, assigning the starting PL intensity in DI water as 100%. 2.7. Cell viability measurement and imaging BGC-823 cells were obtained from Shanghai Cell bank of Chinese Academy of Sciences (Shanghai, China) and SH-SY5Y cells were from ATCC. Both cells were maintained in Dulbecco's Modified Eagle's medium (DMEM, Invitrogen) with 10% fetal bovine serum (FBS, GIBCO) at 37 °C in a humidified atmosphere with 5% CO2. For cell viability study, cell counting kit-8 (CCK-8) assay was used [43,44]. Briefly, in each assay, 5000 cells were loaded into each well of a 96-well flat-bottom Microtiter plate and cultured overnight. Six sets of the samples were treated with different concentrations of QD formulations and one set was treated with PBS buffer (control). Assays were performed in triplicate and the results were averaged. The cells were subsequently incubated for 48 h or 72 h before performing the viability study. After that, 10 μl of the CCK-8 solution was added to each well of the plate and the cells were incubated for another 2 h at 37 °C with 5% CO2. The absorbance of the mixture in each well was measured at 450 nm by a multiwell plate reader. The cell viability was obtained by normalizing the absorbance of the sample against the control set and expressed as percentage, assigning the viability of non-treated cells as 100%. 3. Results and discussion Three types of QDs were used in this study, namely, CdSe/CdS/ZnS core/shell/shell QDs, core/shell structured CdTe/ZnS and InP/ZnS QDs. Fig. 1a shows the UV–vis absorption and the photoluminescence (PL) spectra of the QDs before and after surface modifications. The absorption spectra of CdSe/CdS/ZnS, CdTe/ZnS, and InP/ZnS QDs feature excitonic peaks around 595, 565 and 600 nm, respectively. The PL spectra of the CdSe/CdS/ZnS, CdTe/ZnS, and InP/ZnS QDs show band edge emissions at 616, 607 and 634 nm, respectively. Due to the differences in making the QDs, the emission bandwidth of the three types of QDs varies among each other, with the CdSe/CdS/ZnS QDs preserving the narrowest. After the surface modifications, the absorption and PL spectra are slightly different from the organic dispersions yet did not show noticeable difference in the peaks, indicating that the surface modification did not induce significant change in the structures of the QDs. Fig. 1b–d shows the transmission electron microscopy (TEM) images of the QDs before surface modifications and they have demonstrated relatively monodispersed size distributions with averaged sizes of ~ 6.5 ± 0.3 nm, ~ 5.5 ± 0.6 nm and ~ 5 ± 0.5 nm, for CdSe/CdS/ZnS QDs, CdTe/ZnS QDs and InP/ZnS QDs respectively. The as-synthesized QDs are passivated with hydrophobic moieties and they can be dispersed in various organic solvents such as toluene and hexane. To enable aqueous dispersion, further surface modification steps are needed. In this work, we have employed two types of approaches for preparing water-dispersible QDs. The first approach involves replacing the hydrophobic moieties with hetero-bifunctional ligands such as mercaptopropionic acid (MPA). Basically, this molecule possesses both thiol and carboxylic acid groups. The thiol group from the molecule will bind strongly to the ZnS surface and the carboxylic acid groups will enable the modified QDs to be dispersible in aqueous buffer solution. The second approach employs amphiphilic polymers to encapsulate the QDs and making them water-dispersible. Two different FDA approved amphiphilic polymers, PEGylated phospholipids and tri-block copolymer pluronic F127, are used in our study, respectively. The major difference between these two surface modification methods is that, the surfactant exchange method involves a replacement of hydrophobic moieties from the QD surface with hetero-bifunctional molecules, which usually bears a high risk in creating substantial surface defects, while the polymer encapsulation method only relying on physical hydrophobic interactions between the oily parts of phospholipids

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Fig. 1. Characterization of the QDs in this study. (a) Extinction (full line) and photoluminescence (PL, dashed line) spectra of the QD formulations before (CdSe-Org, CdTe-Org and InP-Org) and after (CdSe-MPA, CdSe-Phos, CdTe-F127 and InP-Phos) surface modifications. (b)–(d) are the TEM images of the CdSe/CdS/ZnS (b), CdTe/ZnS (c) and InP/ZnS (d) QDs dispersed in chloroform. Scale bars represent 20 nm.

and TOPO/TOP surfactants, which will not disrupt the QD surface but will generate larger particles by encapsulating multiple QDs within a micelle [38,41]. In this study, four types of aqueous dispersible QD

formulations have been prepared by using the two methods mentioned above. MPA-functionalized CdSe/CdS/ZnS QDs (CdSe-MPA) were synthesized by using surfactants exchange process. As a comparison,

Fig. 2. Hydrodynamic size distributions of various functionalized quantum dots with different surface coatings. (a) CdSe-MPA: MPA modified CdSe/CdS/ZnS QDs through surfactant exchange. (b) CdSe-Phos: CdSe/CdS/ZnS QDs encapsulated with PEGylated phospholipids. (c) CdTe-F127: CdTe/ZnS QDs encapsulated it with Pluronic F127. (d) InP-Phos: InP/ZnS QDs encapsulated with PEGylated phospholipids.

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the same QD material, CdSe/CdS/ZnS QDs were encapsulated with PEGylated phospholipids to obtain micelle-encapsulated QDs (CdSePhos). Similarly, InP/ZnS QDs and CdTe/ZnS QDs are encapsulated PEGylated phospholipids and Pluronic F127 micelles (InP-Phos and CdTe-F127). The hydrodynamic diameter distributions of the prepared formulations were measured using dynamic light scattering (DLS) technique as shown in Fig. 2. As expected, CdSe-MPA displayed the smallest hydrodynamic diameter of around 11 nm when compared to the other three formulations of 97.2 nm, 58.5 nm and 90.9 nm for CdSe-Phos, InP-Phos and CdTe-F127, respectively. A schematic illustration of the nanoparticle formulations was shown in Fig. 3, where the surface charge profile has also been investigated. Because of the carboxylic acid groups, the CdSe-MPA formulation exhibits a zeta potential value of −22.51 mV, while the other three formulations are slightly negatively charged with values of − 13.01, − 8.63 and − 8.9 mV for CdSe-Phos, InP-Phos and CdTe-F127, respectively, which are in good agreements with previous reports [45]. When QDs are used in the in vitro or in vivo experiments, these biological environments are complex and usually will directly or indirectly impacting the colloidal and optical stability of the QD formulation. Thus, the aqueous colloidal stability of QDs is of utmost concern for using them in long term in vivo imaging applications such as tumor detection and labeling. In this study, we have monitored the photoluminescence (PL) intensity of the as-prepared QD formulations in various cell culture media for over three days. The change in the PL intensity is used as an indicator for evaluating the overall colloidal stability of the QD formulations. In addition, there is great possibility of proteins absorption onto the QD surface when these particles are interacting in the blood stream and this might cause QDs to agglomerate and be taken away from the blood circulation. To simulate such environment, cell culture medium supplemented with fetal bovine serum (FBS, 10%) is used in this investigation. Fig. 4 shows the PL intensity profiles of QDs dispersed in various biological buffers (e.g. DMEM and DMEM with FBS) monitored against time at room temperature. From Fig. 4, we can observe that for

all the QD formulations, the PL intensity of QDs generally drops when the particles are dispersed in DMEM medium upon comparing to those dispersed in water. The CdSe-MPA and CdTe-F127 formulations are very stable in water as no changes in the PL intensity are observed during the 70 h monitoring experiments. However, both CdSe-Phos and InP-Phos formulations behave quite differently in water. We observed a gradual decrease in the PL intensity for the first 20 to 30 h and subsequently the PL intensity reach a relatively plateau stage and maintained. Some common factors that could affect the PL intensity of QD dispersion include pH, ions, oxygen and etc. [46–48] For example, Liu et al. reported that the fluorescence intensity of intracellular mercaptoacetic acid-capped CdSe/ZnSe/ZnS QDs increases monotonically with pH [49]. Avellini et al. have shown that protons affected the surface ligands of QDs to induce QD aggregation and caused luminescence quenching of the system [50]. Ions in the nanoparticles dispersion were also reported to have great impact on the PL intensity of QDs where these ions were able to penetrate the surface ligands and bonded to the QD surface [51]. In general, all these reports show that these factors will lead to the generation of surface defects on the QDs and such traps will prevent the excitons to participate in the recombination process which is an important step for producing photon emission [3]. In our case, the decrease of the PL intensity of the QD formulations might be resulted from multiple factors. The main contribution for weakening in PL intensity is likely coming from the surface defects produced by photoinduced oxidation process [52,53]. Since the average size of the CdSe-Phos formulation is slightly greater than the InP-Phos formulation, the former is having a relatively higher absorption cross section [54]. Such high absorption cross section will certainly speed up the weakening of the QD PL intensity. Also, it is worth noting that all the samples were dispersed into different biological buffers and they were left undisturbed for an hour to stabilize before taking the measurement of their PL intensities. During this one hour, the ions, amino acids and proteins in the DMEM media will interact with the surface coating of QDs and impact their PL intensities. For the amphiphilic polymer

Fig. 3. Schematic illustration and zeta potential values of the four different QD formulations: CdSe-MPA, CdSe-Phos, InP-Phos and CdTe-F127.

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Fig. 4. Normalized PL intensity profile monitored against time at room temperature for different QD formulations of (a) CdSe-MPA, (b) CdSe-Phos, (c) InP-Phos and (d) CdTe-F127.

encapsulated QD formulations, small molecules and ions may have penetrated into the micelle particles and induce surface defects on the multiple QDs encapsulated within the micelle. As a result, a decrease in the PL intensity was observed. Similar decrease of PL intensity was also observed for the CdSe-MPA formulation. This may be due to the surface defects generated in the ligand exchange process between the MPA molecules and the small proteins or amino acid molecules in the biological medium. In addition to the evaluation performed at room temperature, the same experiments were carried out at 37 °C, where the PL intensity profiles follow the similar trends for those samples evaluated under the room temperature, despite the fact that higher temperature accelerated the weakening of the PL intensity of the QD formulations (data not shown). In general, the photostability of the QD type determines the faith of their PL intensity at different temperatures. The core/shell/shell structured CdSe/CdS/ZnS QDs generally preserve a much better photostability than that of single shell coated QDs. Such shell coating has offered the CdSe/CdS/ZnS QDs to have higher stability against photo-oxidation than that of the core/shell structured QD system [55,56]. The increase in the temperature will escalate the penetration rate of the ions and small molecules into the phospholipid micelle and subsequently causing the photoinduced oxidation process to take place rapidly. The QD toxicity has always been a great concern in the community and many of researchers would like to identify the main causes and subsequently develop solutions to resolve these challenges. Some reports have shown that the surface coating of QDs plays an important role in developing the toxicity within the biological system [21,57–59]. Cell viability assay is commonly used as a quantitative way to evaluate the toxicity of a substance [60,61]. In this study, a mitochondrial activity associated assay Cell Counting Kit-8 (CCK-8) was used for the evaluation of QD toxicity [62]. CCK-8 assay uses water soluble WST-8 as the tetrazolium salt which will reduce to orange colored formazan in the presence of dehydrogenases in cells. It was also reported to have a better sensitivity than the well-known MTT assay [63]. The amount of the formazan dye generated within the sample solution is directly proportional to the numbers of living cells in the assay. As a result, the relative viability of cells can be obtained from the absorption of the total concentration of formazan dye generated within the cells. In this study, human gastric carcinoma cell line, BGC-823, was treated with the CdSe-MPA, CdSe-Phos, InP-Phos and CdTe-F127 formulations, and the cell viabilities were measured for different concentrations of QD formulations. It is important to note that, instead of using the weight of QD formulations

as the sample concentration unit, we have measured the heavy metal content of each formulations using inductively coupled plasma mass spectrometry (ICP-MS) to obtain the precise concentration of each prepared QD formulations. As shown in Fig. 5, the viability of BGC-823 cells treated with CdSe-MPA remained above 80% after 48 h of treatment over a broad range of QD concentrations. However, for cells of 72 h post-treatment, the viability started to decrease at a concentration of 1.4 μg/ml of Cd content. Previous studies have shown that ZnS capped CdSe QDs functionalized with MPA molecules were highly biocompatible. In an early study conducted by Cho et al., MPA modified CdSe/ZnS QDs were reported to be nontoxic to MCF-7 cells [64]. The intracellular Cd2+ concentration was measured to be below the assay detection limit (b5 nM). In another study, Mahto et al. have shown that the MPA functionalized CdSe/ZnSe QDs did not cause any obvious cytotoxicity to fibroblast cells [65]. However, these results were obtained within a short period of time ranging from 12 h to 24 h and it may not reflect the actual potential cytotoxicity of the QD formulations. We speculate that as time progresses, the QDs internalized by cells will be likely ended up in the lysosome and the acidic condition within the lysosome will prompt the degradation of QDs and subsequently causing the release of Cd2 + from the particle surface. In our study, we observe that the viability of the BGC-823 cells is dependent on the dosage of the QDs and this suggests that the gradual decomposition of high concentration of CdSe-MPA within cells might be the main leading cause in generating the toxicity above 1 μg/ml of Cd (from the CdSe-MPA formulation). For the micelle-encapsulated QD formulations, the viability of the cells maintained above 80% for all the concentrations considered in this test within 48 and 72 h. The result here strongly suggests that the amphiphilic polymers coating provide a much better protection on QDs against degradation when they are exposed to the BGC-823 cells. However, a different trend was observed when we perform the same set of experiments using the SH-SY5Y cell line. SH-SY5Y is a human derived cell line of neuroblastoma [66,67]. As shown in Fig. 6a, the viability of SH-SY5Y cells remains above 80% for all concentrations considered for CdSe-MPA formulation at 48 and 72 h. To our surprise, the micelleencapsulated QD formulations have shown a considerable toxicity to SH-SY5Y cells. As shown in Fig. 6b and c, the phospholipid-micelle encapsulated CdSe/CdS/ZnS and InP/ZnS QD formulations have both shown a concentration dependent cytotoxicity pattern in the cell viability, especially for cells evaluated at 72 h. The half maximal inhibitory concentration values (IC50) for 72 h post treatment are estimated to be 2.0 μg/ml (Cd content) and 2.5 μg/ml (indium content) for CdSe-

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Fig. 5. Relative cell viability studies of a human gastric carcinoma cell line BGC-823 treated with different QD formulations of (a) CdSe-MPA, (b) CdSe-Phos, (c) InP-Phos and (d) CdTe-F127. Concentrations of each formulation were quantified through inductively coupled plasma mass spectrometry (ICP-MS). Results were measured for both 48 and 72 h of post treatments.

Phos and InP-Phos, respectively. The result here also indicated that the toxicity impact of the CdSe-Phos and InP-Phos formulations is time dependent. For the InP-Phos formulation, it did not show any obvious toxicity for 48 h but toxicity impact from the QDs take place at 72 h. In the case of CdSe-Phos, the viability trends share very similar pattern when compared to InP-Phos formulation. For the case of CdTe-F127 formulation, the viability of SH-SY5Y cells has shown a weak tolerance with these functionalized nanocrystals. The IC50 values were estimated to be 0.1 μg/ml and 0.3 μg/ml, respectively for 48 h and 72 h. This suggests that the CdSe based QDs possess lower cytotoxicity than that of CdTe QDs at similar concentration. This is consistent with a previous study demonstrated by Cho et al. They have shown that the reactive oxygen species (ROS) generated by the QDs causes significant lysosomal

damage via Cd2 +-specific cellular pathways and/or CdTe-triggered photo-oxidative processes [64]. Some studies have reported that the nanoparticle toxicity varies with different cell lines [68,69]. But, such fact has not been fully understood. In our study, we hypothesize that the different uptake rate of QDs between the two cell types might be the driving force that causes the variation of toxicity in vitro. It is well known that the cellular uptake of nanoparticles by non-phagocytic cells is affected by factors such as particles size, surface charge, particles concentration, and incubation time [70–72]. A two-step process is used to describe the nanoparticle uptake process, namely, membrane adhesion and internalization [73]. Specifically, the membrane adhesion is initiated by the interaction between the ligand associated to the nanoparticle surface and the

Fig. 6. Relative cell viability studies of a human neuroblastoma cell line SH-SY5Y treated with different QD formulations of (a) CdSe-MPA, (b) CdSe-Phos, (c) InP-Phos and (d) CdTe-F127. Concentrations of each formulation were determined by ICP-MS and expressed as the heavy metal content.

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receptors on the cell membrane. During this interaction, the local density of receptors on the membrane will match accordingly to the ligand density on the nanoparticle surface. Throughout this ligand–receptor binding event, the cell membrane will start to wrap the nanoparticle surface and such wrapping process will form a vesicle (endosome) where the nanoparticle is located in the core. This process is called internalization. The surface coating of the particles play a major role in determining the adhesion process [74]. Recently, He et al. have shown that the cellular uptake of nanoparticles is cell line-dependent [75]. The authors suggested that such occurrence was directly related to the distinct cell surface property and their corresponding specific endocytosis process [75,76]. The QD formulations prepared in our work are not conjugated with targeting moieties such as antibodies for targeted delivery to the cells. As the nanoparticles are dispersed in the cell culture media, proteins and other biomolecules in the media might rapidly be absorbed onto the nanoparticle surface to form a protein corona and such additional “bio-coating” will greatly affect the physicochemical property of the particle [77–79]. These nanoparticles were then internalized by the two cell lines at different uptake rate due to the difference in the cell membrane surface property. The SH-SY5Y cell line is thrice cloned subline of SK-N-SH cells which is established from a bone marrow biopsy of a neuroblastoma patient [66]. It has been widely used as a model of dopaminergic neurons as the cells possess similar biochemical functionalities of neurons [80]. Specifically, they are able to synthesize dopamine and also express dopamine transporter on the cell membrane [80]. The dopamine transporter is a membranespanning protein which regulates dopamine homeostasis through specific uptake and sequestration of dopamine [81,82]. We speculate that this hormone may have absorbed onto the nanoparticle surface as part of the coating and may have partially promoted the uptake of nanoparticles through dopamine transporters pathway. Another possible reason that has caused the differences of the cell viability in the two cell lines may come from the variation of their sensitivity towards oxidative stress, which is highly related to the expression level of the redox protein thioredoxin. Thioredoxin acts as a growth factor and is found to be elevated in many human primary cancers including gastric carcinoma when compared to normal tissue [83,84]. Zhao et al. have shown that the overexpression of thioredoxin in five different cancer cell lines and one of the cell lines includes BCG-823 cells, which is the cell line used in this study [85]. As a comparison, Andoh et al. have shown

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that SH-SY5Y cell has a low thioredoxin expression level (2.3 ng/μg of protein) and thus is highly sensitive to oxidative stress [86]. It is well documented that QDs cause oxidative stress, disrupting the activities of antioxidant defenses and activate protein kinases in the cells, causing the apoptosis process to take place [22,23,87]. As the two cell line models have different sensitivity towards the oxidative stress level, this may help to explain why different cell viability trends are observed for the same QD formulation. To further investigate on this issue, BGC823 and SH-SY5Y cells treated with different QD formulations were imaged and analyzed using fluorescent microscopy. Fig. 7 shows fluorescence images of cells treated identically with the CdTe-F127 formulation with cadmium concentration equivalent to 0.656 μg/ml. From the red fluorescent signals of QDs, it can be clearly seen that SH-SY5Y cells have a relatively higher uptake of the QDs than that of the BGC-823 cells at both 4 h and 48 h post treatment. It is worth noting that the phase-contrast images reveal that the SH-SY5Y cells experienced an abnormal morphology change after 48 h of nanoparticles treatment upon comparing to the blank control sample in Fig. 7d. This indicates that the cells are damaged after exposing them with excessive QDs. Such observation is consistent with the cell viability results. 4. Conclusions In conclusion, we have evaluated the cytotoxicity of four different QD formulations, namely, CdSe-MPA, CdSe-Phos, InP-Phos and CdTeF127, respectively. To make QDs dispersible in water, two strategies are used, the surface ligand-exchange approach and coating QDs with biocompatible polymer layer. The PL intensities of the four QD formulations were monitored to evaluate their stability over time. Our result shows that the PL intensity of the CdSe-MPA formulation is more stable than that of the micelle-encapsulated QD formulations. In addition, the PL intensity of QDs decreases when they are dispersed in the cell culture medium. On the other hand, increasing the temperature of the QD dispersion generally weakens the PL intensity as time progresses. To assess the cytotoxicity of the prepared QD formulations, gastric carcinoma and neuroblastoma cell lines were used for the evaluation. We found that the surface coating and the core material of QDs play important roles in determining the cytotoxicity level in these cell lines. More interestingly, we even observed that for the same QD formulation with identical dosage, the cell viability pattern varies across the cell lines. Upon

Fig. 7. Fluorescence and the corresponding phase-contrast images of BGC-823 (a–c) and SY5Y (d–f) cells. (a) and (d) are blank controls while others are treated with CdTe-F127 QDs at a cadmium equivalent concentration of 0.656 μg/ml. Fluorescence and the phase-contrast images of cells at 4 h and 48 h post treatment are provided. Cell nucleuses are stained with DAPI (blue) and the QD signals are color-coded in red.

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