Synthesis and characterization of quantum dots designed for biomedical use.

G Model IJP 13962 No. of Pages 8

International Journal of Pharmaceutics xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical nanotechnology

Synthesis and characterization of quantum dots designed for biomedical use Weronika Kuzyniak a , Oluwasesan Adegoke b , Kutloano Sekhosana b , Sarah D’Souza b , Sesethu Charmaine Tshangana b , Björn Hoffmann a , Eugeny A. Ermilov a , Tebello Nyokong b , Michael Höpfner a, * a b

Institute of Physiology, Charité – Universitätsmedizin Berlin, Charitéplatz 1, Berlin 10117, Germany Department of Chemistry, Rhodes University, Grahamstown 6140, South Africa

A R T I C L E I N F O

A B S T R A C T

Article history: Received 6 January 2014 Received in revised form 17 March 2014 Accepted 18 March 2014 Available online xxx

Semiconductor quantum dots (QDs) have become promising nanoparticles for a wide variety of biomedical applications. However, the major drawback of QDs is their potential toxicity. Here, we determined possible cytotoxic effects of a set of QDs by systematic photophysical evaluation in vitro as well as in vivo. QDs were synthesized by the hydrothermal aqueous route with sizes in the range of 2.0– 3.5 nm. Cytotoxic effects of QDs were studied in the human pancreatic carcinoid cell line BON. Cadmium telluride QDs with or without zinc sulfide shell and coated with 3-mercaptopropionic acid (MPA) were highly cytotoxic even at nanomolar concentrations. Capping with L-glutathione (GSH) or thioglycolic acid (TGA) reduced the cytotoxicity of cadmium telluride QDs and cadmium selenide QDs. Determination of the toxicity of QDs revealed IC50 values in the micromolar range. In vivo studies showed good tolerability of CdSe QDs with ZnS shell and GSH capping. We could demonstrate that QDs with ZnS shell and GSH capping exhibit low toxicity and good tolerability in cell models and living organisms. These QDs appear to be promising candidates for biomedical applications such as drug delivery for enhanced chemotherapy or targeted delivery of light sensitive substances for photodynamic therapy. ã 2014 Elsevier B.V. All rights reserved.

Keywords: Nanoparticles Quantum dots Cytotoxicity Biocompatibility

1. Introduction Nanoparticles, previously associated only with physics or chemistry are gaining increasing interest in biomedical research. Among the many of newly synthesized nanoparticles of the last decades, semiconductor quantum dots (QDs) turned out to belong to the most promising ones in the field. The size of QDs can vary from 1 to 6 nm and absorption and luminescence properties can be precisely adjusted from the UV to the far infrared region by changing the QDs size and composition. Even more importantly, the surface coating of QDs can be functionalized to make them both water soluble and biocompatible. Last but not least, because of the ability to functionalize them, ODs have become interesting candidates for a variety of biomedical applications, such as

* Corresponding author. Tel.: +49 30450528515; fax: +49 30450528918. E-mail addresses: [email protected] (W. Kuzyniak), [email protected] (O. Adegoke), [email protected] (K. Sekhosana), [email protected] (S. D’Souza), [email protected] (S.C. Tshangana), [email protected] (Bjö. Hoffmann), [email protected] (E. A. Ermilov), [email protected] (T. Nyokong), [email protected] (M. Höpfner).

photodynamic therapy, where they are under extensive investigation as carriers for targeted delivery of photosensitizers (Iga et al., 2007; Jamieson et al., 2007; Medintz et al., 2008; Ku et al., 2011; Medintz et al., 2005; Paszko et al., 2011). Despite the exceptional properties of QDs, serious concerns about their potential long-term toxicity have limited their use in biomedical applications so far. Several critical studies have reported that the release of heavy metals (e.g., Cd) from the core of QDs (e.g., CdTe, CdSe or CdS) can be a reason for their toxicity (Chen et al., 2012; Su et al., 2010). Kirchner and co-workers have reported that any type of nanoparticle might reduce cell viability when applied at micromolar concentrations (Kirchner et al., 2005). Wang and colleagues have shown that even in nanomolar concentrations, QDs can exert toxic effects on microorganisms, leading to DNA damage and protein degradation (Wang et al., 2011). Nevertheless, a number of publications suggested that stabilization of the QD core with an inorganic shell such as ZnS and the type of capping agent on the surface of the QDs can reduce or entirely suppress their toxicity (Tiwari et al., 2011; Zhang et al., 2006; Jaiswal et al., 2003). Although the ZnS shell turned out as the most effective core protection, there is no certainty which capping (e.g., mercaptoacetic acid (MAA), tri-n-octylphosphine oxide

http://dx.doi.org/10.1016/j.ijpharm.2014.03.037 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: W. Kuzyniak, et al., Synthesis and characterization of quantum dots designed for biomedical use, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.03.037


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W. Kuzyniak et al. / International Journal of Pharmaceutics xxx (2014) xxx–xxx

(TOPO), polyethylene glycol (PEG) should be used to increase the stability of QDs and protect them from ion leakage. Therefore, the goal of this study was to evaluate the potential toxicity of seven different types of QDs with different structures (with or without shell) and with different capping agents (summarized in Fig. 1), in order to determine which QDs may be promising candidates for biological or medical use. 2. Materials and methods 2.1. Materials Cadmium chloride hemi (pentahydrate), L-cysteine and Lglutathione (GSH) were purchased from Fluka. Sodium borohydride, mercaptopropionic acid (MPA), thioglycolic acid (TGA), selenium powder and tellurium powder were purchased from Sigma–Aldrich (St. Louis, MO, USA). Zinc chloride, zinc acetate and sodium hydroxide pellets (98%) were purchased from Merck (Darmstadt, Germany). All chemicals were of analytical grade and used without prior purification. Aqueous solutions were prepared using Millipore water Milli-Q Water Systems (Millipore Corp., Bedford, MA, USA). 2.2. Synthesis of QDs The synthesis of the following QDs has been reported before: Lcysteine-CdTe (Idowu et al., 2008), TGA-CdTe (Idowu et al., 2008), MPA-CdTe (Idowu et al., 2008; Dong et al., 2006), MPA-CdTe/ZnS (Adegoke and Nyokong, 2013), GSH-CdTe/ZnS (Liu and Yu, 2010) and TGA-CdSe (Yu et al., 2003). 2.2.1. The synthesis of CdSe/ZnS-GSH QDs CdSe QDs were synthesized using methods described previously with some slight modifications (Peng et al., 2009). Briefly, 0.5 mmol/l of selenium powder and 1.3 mmol/l of sodium borohydride (molar ratio of Se to NaBH4 was 1:2.6) were loaded into a 50 ml 3-necked flask and 30 ml of absolute ethanol was added. The flask was fitted with a septum and the solution deaerated with argon at room temperature. After 1 h, the colorless ethanol solution of NaHSe was obtained and used as the Se precursor for CdSe QDs synthesis as follows: CdCl22.5H2O (0.4 mmol/l) was placed in a 500 ml 3-necked flask attached to a

condenser and 200 ml of Millipore water was added, 1 mmol/l of Lcysteine was then injected into the mixture under vigorous stirring. The pH of the solution was adjusted to 11 using 0.1 M NaOH and the mixture was deaerated with argon gas. Freshly prepared oxygen-free NaHSe (0.2 mmol/l) was then injected into the solution using a syringe and the reaction was subjected to reflux at a constant temperature of 90 C under N2 atmosphere. In our experiment, the molar ratio of Cd2+: NaHSe:L-cysteine was 1:0.6:2.0. A yellowish L-cysteine-CdSe QDs solution was obtained. Aliquots of the reaction mixtures were taken at different time intervals for emission and absorption measurements. The resulting product was precipitated with ethanol and unreacted precursors were removed via centrifugation at 3000 rpm for 10 min. The resulting precipitate was re-precipitated with ethanol more than 3 times and dried under vacuum and kept in the dark for further use. Purified L-cysteine-CdSe QDs (100 mg) were added to 100 ml aqueous solution (pH 8) containing 2 mmol/l ZnCl2 and the capping ligand, 4 mmol GSH. GSH was used as both the capping agent and sulfur source for the growth of ZnS shell on the respective CdSe cores. The solutions was heated to 100 C in open-air and refluxed with time to control the sizes of the core-shell QDs. Aliquots of the reaction mixture were taken at different time intervals for emission and absorption measurements. The core-shell GSHCdSe/ZnS QDs were precipitated with ethanol, centrifuged and dried under vacuum. 2.3. Determination of photophysical properties Using Cu-Ka radiation (l = 1.5405 Å, nickel filter), X-ray powder diffraction (XRD) patterns were recorded on a Bruker D8 Discover equipped with a proportional counter. Data were collected in the range 2u = 15 to 60 , scanning at 1 min1. The filter time-constant and the slit width were 2.5 s per step and 6.0 mm, respectively. Samples were placed on a silicon wafer slide. The X-ray diffraction data were processed using the Eva (evaluation curve fitting) software (Bruker AXS, WI, USA). Baseline correction was performed on each diffraction pattern by subtracting a spline fitted to the curved background. The QD diameters were calculated using the Scherrer equation (Sapra and Sarma, 2005): d¼

0:9l bcos u

(1)

Fig. 1. 2D structures and names of quantum dots.




where d is the mean diameter of a quantum dot in nanometers (nm), l is the X-ray source wavelength (1.5405 Å), b is the full width at half maximum of the diffraction peak, and u is the angle of the peak. Ground state absorption spectra were recorded using a Shimadzu UV–Vis 2550 spectrophotometer. The emission spectra were recorded on a Varian Cary Eclipse fluorescence spectrophotometer in 1 1 cm quartz cells. To obtain luminescence quantum yields of the QDs, a reference solution of rhodamine 6G in ethanol R6G was used (Ff l ¼ 0:95 (Kubin and Fletcher, 1982)) and the values were calculated using the following Eq. (2): R6G FQD ¼ Ff l l

SQD nwater 2 l ODR6G ðlexc Þ R6t OD QD ðlexc Þ nethanol Sf l

(2)

where Fl , SQD , ODQD(lexc) and Ff l , SR6G f l , ODR6G(lexc) are the l luminescence quantum yield, the integrated luminescence intensity and the optical density at the excitation wavelength, lexc, of the sample and the reference, respectively. nwater and nethanol are the refractive indices of the solvent used for the sample (water, nwater = 1.333) and the reference (ethanol, nethanol = 1.361), respectively. QD

R6G

2.4. Cell culture

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Table 1 Size, luminescence maxima and luminescence quantum yields of QDs in solubilized in water. QDs

Size, nm

llum, nm

Flum

GSH-CdSe/ZnS GSH-CdTe/ZnS MPA-CdTe/ZnS MPA-CdTe TGA-CdSe TGA-CdTe L-cysteine-CdTe

2.8 3.1 3.1 2.7 2.0 3.2 3.5

580 600 557 563 539 609 633

0.61 0.41 0.72 0.47 0.016 0.14 0.44

2.6. Determination of cytotoxicity Cells were seeded at a density of 4000 cell/well into a 96-well microtiter plate and incubated with increasing concentrations of GSH-CdSe/ZnS QD for 6 h or 24 h. Release of the cytoplasmic enzyme (LDH), indicating cytotoxicity, was measured in supernatant samples by using a colorimetric kit (Roche Diagnostics, Penzberg, Germany) as described in literature (Decker and Lohmann-Matthes, 1988). Maximum LDH release was measured after adding 2% Triton X-100 to untreated cells. The absorbance of the samples was measured at 490 nm, using an ELISA-Reader. 2.7. Uptake of QDs

Human pancreatic carcinoid BON cells (Gloesenkamp et al., 2012) were cultured in DMEM/Ham’s F-12 (1:1) medium (Biochrom AG, Berlin, Germany) supplemented with 10% fetal calf serum (FCS, Biochrom AG), 100 U/ml penicillin and 100 mg/ml streptomycin (Biochrom AG). Cells were maintained under standard conditions (37 C in a humidified atmosphere of 5% CO2). The culture media was changed every second day and once a week the cells were passaged using 1% Trypsin/EDTA (Biochrom AG).

Uptake of GSH-CdSe/ZnS QDs in BON cells was determined using a fluorescence microscope (Zeiss Axioskop 40, Jena, Germany) at ex/em 546 12/575–640 nm. Cells grown on glass coverslips were incubated with QDs in concentration 0.2 and 0.4 mM for 4, 8 or 24 h. After the incubation period, samples were washed twice with PBS, then fixed with methanol (99%) in 20 C for 20 min and again washed with PBS. All investigated areas contained at least n = 10 cells.

2.5. Measurement of growth inhibition

2.8. Chicken chorioallantoic membrane (CAM) assay

Evaluation of the changes in cell numbers induced by the QDs was performed by crystal violet staining, as described previously (Nitzsche et al., 2010). Briefly, the cells were seeded in 96-well plates (1500 cell/well) with 100 ml growth medium and cultured at 37 C, 5% CO2 for 24 h and subsequently fixed with 1% glutaraldehyde after incubation with the respective QDs at different concentrations. The cells were then stained with 0.1% crystal violet in phosphate buffered solution (PBS). The unbound dye was removed by rinsing with water. Bound crystal violet was solubilized with 0.2% Triton X-100 in PBS. Light extinction, which increases linearly with the cell number, was analyzed at 570 nm using an ELISA-Reader.

Embryotoxicity in terms of lethality and vein network degeneration was evaluated by CAM assay. Fertilized chicken eggs were incubated at 37 C in constant humidity for 15 days (the first day of incubation was considered as the first day of chicken embryonic development). On day 7 of incubation, a window was cut into the shell of each egg, then sealed with clear tape and bred in the incubator for an additional 4 days. On day 11, the tape was removed and the QDs were administered either topically or intravenously. In the case of topical application, a small silicone ring (5 mm in diameter) was placed onto the CAM and QDs-PBS or PBS were added (100 ml per egg, each day until day 15); in the case of intravenous application a superficial CAM vein was injected with

Fig. 2. Powder XRD spectra of the L-cysteine-CdSe and GSH-CdSe/ZnS QDs.

Fig. 3. UV/vis absorption (dotted line) and fluorescence emission (spectra solid line) of GSH-CdSe/ZnS. Excitation wavelength = 300 nm.



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10–15 ml of QDs-NaCl or NaCl 0.9% using a 301/2G needle. The concentration of injected compound was determined assuming the total blood volume of 11 day-old embryo (Kind, 1975). Chicken embryo development was observed until day 15. 3. Results 3.1. Photophysical properties of QDs 3.1.1. XRD spectroscopy The synthesis of QDs described in this work has been reported before (Adegoke et al., 2013; Liu et al., 2010; Idowa et al., 2008;

Dong et al., 2006; Yu et al., 2003). All QDs were synthesized in an aqueus media using several thiol ligands (Fig. 1) for stabilization. The synthesis of GSH-CdSe/ZnS was reported here in aqueous media. Fig. 2 shows the X-ray diffraction patterns of L-cysteineCdSe and GSH-CdSe/ZnS QDs for comparison. A typical zinc blend crystal structure (Sapra et al., 2005) with planes at 111, 2 2 0, and 3 11 was obtained for CdSe QDs with peaks at 26.8 , 44.0 and 52.1. The peaks for CdSe/ZnS QDs were at 28.6 , 47.6 and 56.2 . Following the growth of ZnS shells on the core CdSe, the peak position shifted to higher angles and thus, confirmed the formation of CdSe/ZnS core shell QDs. All QDs showed similar behavior to that shown in Fig. 2.

Fig. 4. Determination of IC50 values: BON cells were treated for 48 h with increasing concentrations of GSH- CdSe/Zn (A), TGA-CdSe (B), GSH-CdTe/ZnS (C), MPA-CdTe or MPACdTe/ZnS (D). Data are given as percentage of untreated controls, which were set 100% (mean S.D. of 3 independent experiments). IC50 value determined after 48 h of incubation with the respective QD (E).




Using Eq. (1) and the position of XRD peaks, the size of QDs was calculated and the values are summarized in Table 1. It was observed that the QDs size varied in the range between 2.0 and 3.5 nm. 3.1.2. UV/Vis absorption and luminescence spectroscopy To further characterize the synthesized QDs, absorption and luminescence spectra of QDs were recorded in water. Fig. 3 shows a representative UV/Vis absorption spectrum of GSH-CdSe/ZnS QDs as well as a luminescence spectrum recorded upon excitation at 300 nm (for other QDs in this study, 400 nm excitation wavelength was used, and the corresponding luminescence spectra are shown in ESI). The spectral position of luminescence maxima are compiled in Table 1. All QDs exhibited broad absorption and well-resolved luminescence spectra. The emission maxima lay between 560 and 630 nm, depending on the size and structure of the QDs. Using rhodamine 6G in ethanol as the reference (with FR6G ¼ 0:9520 ), luminescence quantum yields of the synthesized fl QDs were calculated using Eq. (2) and the values were found to be 0.61, 0.41, 0.72, 0.47, 0.016, 0.14 and 0.44 for GSH-CdSe/ZnS, GSHCdTe/ZnS, MPA-CdTe/ZnS, MPA-CdTe, TGA-CdSe, TGA-CdTe and Lcysteine-CdTe, respectively (Table 1). 3.2. Growth inhibitory effect of QDs To determine growth inhibitory effect of the QDs on BON cancerous cells, crystal violet assays were performed after 48 h of continuous incubation with increasing concentrations of the respective QDs. TGA-CdSe, GSH-CdTe/ZnS and GSH-CdSe/ZnS inhibited the growth of BON cells dose-dependently. MPA-CdTe/ZnS exhibited an even more pronounced growth inhibitory effect, leading to a decrease in cell numbers of almost 100% even at low nanomolar concentrations (20 nM). MPA-CdTe, which was investigated as the ZnS-lacking counterpart of MPA-CdTe/ZnS, was less toxic than the ZnS-bearing QD, but still inhibited the growth of BON cells at nanomolar concentrations. The IC50 value of MPA-CdTe was calculated to be 0.054 mM (Fig. 4). TGA-CdTe and L-cysteine-CdTe QDs were not tested in vitro because they precipitated from the medium, which was an unexpected finding as both QDs were water soluble.

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When administering 0.2 mM of GSH-CdSe/ZnS QDs, a timedependent increase was observed during the first 4 h. Higher concentrations (i.e., 0.4 mM) increased the intracellular accumulation within the first 4 h, reflecting the dose-dependency of the QDuptake. Increasing the incubation time to 8 and 24 h led to an additional increase in QD-uptake only at the concentration of 0.2 mM, whereas at 0.4 mM a slight loss of intracellular QD fluorescence was observed. Moreover, the distribution pattern changed from even and diffuse (0.2 mM) to an uneven and more punctate intracellular fluorescence signal at 0.4 mM. We suggest that the drop of fluorescence intensity in combination with the changed intracellular distribution pattern may occur due to an accumulation of the QDs in subcellular compartments (e.g., organelles, vacuoles). However, further experiments will have to determine the exact intracellular localization and distribution, since this may be of great importance for the suitability and mode of action of QD-based biomedical applications. 3.5. Embryotoxicity Evaluation of the toxicity of QDs in vivo was determined by performing CAM assays. QDs were either injected into the CAMvein or applied topically to 11-days old fertilized chicken eggs (Table 2). Initially, the TGA-capped CdSe QDs were investigated. Topical application of QDs of the IC50 (i.e., 2.9 mM) led to death in 40% of investigated embryos, whereas injection of QDs at 1 mM resulted in embryo death in 60% of cases. Next, we evaluated GSH-capped QDs. GSH-CdTe/ZnS QDs applied topically at 1 mM showed no influence on embryo vitality, while administered intravenously at 0.6 mM embryo death in almost 90% was observed. Finally, GSH-CdSe/ZnS QDs did neither affect the vascular network and microvessel structure of the developing CAM, nor did these QDs influence chicken embryo vitality at theIC50 of 0.4 mM. These observations were regardless of the mode of application (i.e., intravenously or topically) All chicken embryos were sacrificed at the end of the study and examined for developmental defects such as encephalic hernia or cleft beak (Peterka and Klepácek, 2001). However, no such defects were found in GSH-CdSe/ZnS QDs injected embryos (data not shown), further supporting the finding that these QDs do not cause embryotoxicity.

3.3. Cytotoxicity of QDs The cytotoxicity of QDs displaying the lowest growth inhibitory potential and no embryotoxicity was determined by measuring the release of LDH into the supernatant of BON cells. Cells were exposed to GSH-CdSe/ZnS QDs in concentrations ranging from 0.1 to 5 mM for 6 or 24 h. In BON cells treated with 0.1 and 0.2 mM of the respective QDs, only slight increases in LDH release (10% after 6 h and >20% after 24 h. At 5 mM, the release of LDH into the supernatant increased by more than 30% compared to untreated cells after 6 h and 24 h. The results indicated that GSH-CdSe/ZnS did not directly affect cell membrane integrity and did not have immediate cytotoxic effects in BON cells at concentrations below 0.5 mM (Fig. 5). 3.4. Cellular uptake of QDs Incubating BON cells with GSH-CdSe/ZnS QDs (0.2 and 0.4 mM) for 4–24 h led to a time- and dose-dependent intracellular accumulation of QDs (Fig. 6).

Fig. 5. Determination of QD-induced cytotoxicity. LDH release into the supernatant of BON cells was determined after 6 and 24 h of continuous incubation with increasing concentrations of GSH-CdSe/ZnS. Data are given as percentage of untreated controls, which were set 100 % (mean SEM of 3 independent experiments).



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Fig. 6. Time- and dose-dependent uptake of GSH-CdSe/ZnS QD in BON cells. QD uptake was determined by fluorescence microscopy after 4–24 h of incubation with 0.2 mM or 0.4 mM GSH-CdSe/ZnS. Representative findings of three independent experiments with n = 10–50 cells per concentration and incubation time. Upper panel: phase-contrast images; lower panel: corresponding fluorescence micrographs. Scale bar, 20 mm.

4. Discussion A multitude of nanoparticles has been developed in the last decades. Of those, QDs have gained much interest in many areas of science. QDs, which barely exceed the size of atoms, possess unique physicochemical properties and offer great potential for numerous biomedical applications (Salata, 2004; McNeil, 2005; Ferrari, 2005; Mansur, 2010; Cormode et al., 2009; Paszko et al., 2011). Nevertheless, their potential toxicity is alarming and limits their use for therapeutic applications. Here, we investigated different types of QDs and screened their photobiological properties as well as their potential toxicity with the aim to better estimate their suitability as carriers for targeted delivery of photosensitizers for photodynamic therapy (PDT). Despite a number of long-term studies, there is no certainty on the underlying mechanisms of the toxicity that has been observed for some QDs. Many studies suggest ion leakage from the core of

QDs, especially Cd2+ (Wang et al., 2011; Derfus et al., 2004), which has become a significant problem because the cores of QDs are mostly synthesized from heavy metals. However, stable coating with a shell and capping may effectively prevent ion leakage and additionally protect the core from air oxidation (Su et al., 2009). In our study, we could demonstrate that GSH-CdSe/ZnS QDs were stable structurally and potentially safe for biomedical use as shown in respective in vitro and in vivo investigations. Capping of QDs plays an important role in preventing degradation and subsequent release of cytotoxic heavy metal ions. Thus, we examined QDs with the same core and shell but with different cappings. CdTe/ZnS QDs with GSH capping showed decidedly lower cytotoxicity than those with MPA capping. Accordingly, MPA-CdTe/ ZnS QDs killed almost 100% of incubated cells even at low nanomolar concentrations (20 nM), while GSH-capped QDs such as GSH-CdTe/ ZnS exhibited comparatively low cytotoxicity in BON cells, even at 25 times higher doses. Our data are somewhat in discordance to



W. Kuzyniak et al. / International Journal of Pharmaceutics xxx (2014) xxx–xxx Table 2 QDs were applied topically (A) or injected into the CAM-vein of 11-days old fertilized chicken eggs (B). TGA-CdSe caused embryos death in 40% applied topically and 60% applied intravenously. GSH-capped QDs showed no influence on chicken embryos vitality applied topically. However GSH-CdTe/ZnS applied intravenously leads to embryos death in almost 90%, while GSH-CdSe/ZnS has showed no influence on their vitality. A. QD

Concentration

Topical application, % of survival

TGA-CdSe GSH-CdTe/ZnS GSH-CdSe/ZnS

2.9 mM 1 mM 0.4 mM

0.660 1 1100

B. QD

Concentration

Intravenous application, % of survival

TGA-CdSe GSH-CdTe/ZnS GSH-CdSe/ZnS

1 mM 0.6 mM 0.4 mM

40 11 100

findings reported by Lee et al. who observed that MPA-capped CdSe/ ZnS QDs did not cause toxicity in living organisms even at concentration 2500 mg/l after 48 h of exposure to the systems (Lee et al., 2010). As the study of Lee and co-workers employed the so called "daphnia test” to estimate QD toxicity in vivo, it is not surprising that the IC50-values determined with this method differ from those of the in vitro tests on human cells used in our investigation. Compared to the direct measurement of growth inhibition and cytotoxicity in our study, the daphnia test does not directly measure cytotoxicity or death but uses the treatmentinduced loss of mobility of water-living small crustacean (Daphnia magna) as a read-out for the toxicity of a compound. However, the exact reasons for the discrepancy between our findings and those of Lee et al. remain to be elucidated. Nevertheless, in the context of our investigation MPA-capped CdSe-QDs were found to be too toxic to be favorable for biomedical applications and were thus excluded from further in vivo examinations. Changes in the core materials of QDs can have an impact on their toxicity. It has been previously been shown that not all of the QD cores were completely coated with a ZnS shell (using the onepot synthesis also employed in this work) due to the lattice mismatch between ZnS and CdTe (Ithurria et al., 2007). This mismatch is estimated to be 16%, which makes QDs susceptible to degradation. For CdSe and ZnS, the lattice mismatch is 12% (Yong et al., 2011), which means less degradation of the QDs will occur. As a result, CdSe/ZnS QDs should be less toxic and more stable than CdTe/ZnS QDs. Additionally, we investigated QDs without shell but with TGA capping. As mentioned above, well-matched capping and proper structure of the core are very important. We found that TGA-CdSe exhibited lower cytotoxic potency than GSH-CdTe/ZnS. This is an unexpected finding, since QDs with a shell are supposed to be less toxic than those without shell (Su et al., 2009). It can be speculated that TGA somehow stabilized the structure of the core and prevented oxidation and/or ion leakage. However, such a hypothesis requires further examination and will be addressed in further studies. GSH-CdTe/ZnS and TGA-CdSe QDs showed marked toxicity in vivo which rejected them from further investigations. Two other QD types (i.e., L-cysteine-CdTe and TGA-CdTe) were not soluble in the experimental medium, and thus, excluded from further studies. When QDs become unstable, they precipitate out of solution. This can be caused by the capping materials or by interactions with medium components (e.g., serum, salts and proteins). It has been reported that instability of surface ligands can allow QD cores to degrade or induce air oxidation, which in

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turn releases cadmium ions to the environment (Yong et al., 2011). Biocompatibilities as well as solubility are essential requirements for biological uses of QDs. A number of studies have been published that described QDs to be biocompatible and without impact on embryonic development or vitality (Smith et al., 2007; Dubertret et al., 2002). Our in vivo study, also found no evidence teratogenic effects after injection with QDs. Particularly, GSH-CdSe/ZnS QDs induced no embryotoxicity, which suggests that this QD in can be safely used in biomedical research in the reported doses (i.e., 0.1– 0.4 mM). Moreover, QDs have become widely used probes for in vitro and in vivo imaging as well as diagnostic agents for tumor monitoring or photodynamic therapy (Gao et al., 2004; Michalet et al., 2005). Here, we have shown that nontoxic concentrations of GSH-CdSe/ ZnS QDs of 0.2 mM and 0.4 mM, respectively, achieve sufficient cellular uptake to obtain distinct fluorescent signals (Fig. 5). This suggests that in addition to their properties as a carrier of photosensitizers for PDT, GSH-CdSe/ZnS QDs, may also be interesting for tumor monitoring and diagnosis. 5. Conclusions We conclude that suitable capping and encapsulation can remarkably reduce the toxicity of QDs. CdSe QDs with ZnS shell and GSH capping appear to be promising candidates for biological uses and merit further investigations. Acknowledgements We thank the Bundesministerium für Bildung und Forschung (BMBF) and the National Research Foundation (NRF) for grant support (SUA 10/006). This work was additionally supported through the DST/NRF South African Research Chairs Initiative for Professor of Medicinal Chemistry and Nanotechnology, the NRF Knowledge Field Development (KFD) Competitive Support for Unrated Researchers (CSUR) grant, as well as Rhodes University and DST/Mintek Nanotechnology Innovation Centre (NIC) – Sensors, South Africa. We are deeply grateful to Dr. C. Eberhardt (School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Ireland) for carefully revising spelling and grammar. References Adegoke, O., Nyokong, T., 2013. Probing the sensitive and selective luminescent detection of peroxynitrite using thiol-capped CdTe and CdTe/ZnS quantum dots. Journal of Luminescence 134, 448–455. Chen, N., He, Y., Su, Y., Li, X., Huang, Q., Wang, H., Zhang, X. Tai, Fan, C., 2012. The cytotoxicity of cadmium-based quantum dots. Biomaterials 1238–1244. Cormode, D.P., Skajaa, T., Fayad, Z.A., Mulder, W., 2009. Nanotechnology in medical imaging: probe design and applications. Arteriosclerosis, Thrombosis, and Vascular Biology 29, 992–1000. Decker, T., Lohmann-Matthes, M.L., 1988. A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. Journal of Immunological Methods 115, 61–69. Derfus, A.M., Chan, W.C.W., Bhatia, S.N., 2004. Probing the cytotoxicity of semiconductor quantum dots. Nano Letters 4, 11–18. Dong, C., Qian, H., Fang, N., Ren, J., 2006. Study of fluorescence quenching and dialysis process of CdTe quantum dots, using ensemble techniques and fluorescence correlation spectroscopy. The Journal of Physical Chemistry B 110, 11069–11075. Dubertret, B., Skourides, P., Norris, D.J., Noireaux, V., Brivanlou, A.H., Libchaber, A., 2002. In vivo imaging of quantum dots encapsulated in phospholipid micelles. Science 298, 1759–1762. Ferrari, M., 2005. Cancer nanotechnology: opportunities and challenges. Nature Reviews. Cancer 5, 161–171. Gao, X., Cui, Y., Levenson, R.M., Chung, L.W.K., Nie, S., 2004. In vivo cancer targeting and imaging with semiconductor quantum dots. Nature Biotechnology 22, 969– 976. Gloesenkamp, C., Nitzsche, B., Lim, A.R., Normant, E., Vosburgh, E., Schrader, M., Ocker, M., Scherübl, H., Höpfner, M., 2012. Heat shock protein 90 is a promising target for effective growth inhibition of gastrointestinal neuroendocrine tumors. International Journal of Oncology 40, 1659–1667.



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