Doping Method Determines Para- or Superparamagnetic Properties of

Article Cite This: Chem. Mater. 2018, 30, 4233−4241

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Doping Method Determines Para- or Superparamagnetic Properties of Photostable and Surface-Modifiable Quantum Dots for Multimodal Bioimaging Florian Part,†,◇ Christoph Zaba,†,◇ Oliver Bixner,† Tilman A. Grünewald,‡ Herwig Michor,§ Seta Küpcü,† Monika Debreczeny,∥ Elisabetta De Vito Francesco,† Andrea Lassenberger,⊥ Stefan Schrittwieser,# Stephan Hann,∇ Helga Lichtenegger,⊗ and Eva-Kathrin Ehmoser*,† Downloaded via UNIV OF CALIFORNIA SANTA BARBARA on July 17, 2018 at 18:34:34 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Department of Nanobiotechnology, Institute for Synthetic Bioarchitectures, University of Natural Resources and Life Sciences, Muthgasse 11/II, 1190 Vienna, Austria ‡ ESRFThe European Synchrotron, CS 40220, 38043 Grenoble Cedex 9, France § Institute of Solid State Physics, TU Wien, Wiedner Hauptstrasse 8-10, 1040 Vienna, Austria ∥ Imaging Center, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria ⊥ Department of Nanobiotechnology, Institute for Biologically Inspired Materials, University of Natural Resources and Life Sciences, Muthgasse 11/II, 1190 Vienna, Austria # Molecular Diagnostics, AIT Austrian Institute of Technology, Donau-City-Strasse 1, 1220 Vienna, Austria ∇ Department of Chemistry, Division of Analytical Chemistry, University of Natural Resources and Life Sciences, Muthgasse 118, 1190 Vienna, Austria ⊗ Department of Material Sciences and Process Engineering, Institute of Physics and Materials Science, University of Natural Resources and Life Sciences, Peter-Jordan-Straße 82, 1190 Vienna, Austria S Supporting Information *

ABSTRACT: Semiconductor quantum dots (QDs) are widely used for optical applications and bioimaging. In comparison to organic dyes used for fluorescent labeling, QDs exhibit very high photostability and can be further surface modified. Equipping QDs with magnetic properties (mQDs) makes it possible to combine fluorescence and magnetic resonance imaging analyses. For this purpose, we have prepared water-dispersible and magnetic CdTe/ ZnS mQDs, whereby ferrous ions are selectively incorporated in either their cores or their shells. This study aims at understanding the differences in optical, structural, and magnetic properties between these core- and shell-doped mQDs. Field-dependent isothermal magnetic susceptibility measurements show that shell-doped mQDs exhibit paramagnetic and their core-doped equivalents superparamagnetic behavior near room temperature. Shell doping results in about 1.7 times higher photoluminescence quantum yields and 1.4 times higher doping efficiency than core doping. X-ray diffraction patterns reveal that core doping leads to defects in the lattice and hence to a severe decrease in crystallinity, whereas shell doping has no significant impact on the crystal structure and consequently fewer disadvantages regarding the mQD’s quantum yield. These selective doping approaches, particularly shell doping, allow for the tailored design of paramagnetic QDs having modifiable and biocompatible particle surfaces. The organic ligandsin this study N-acetyl-L-cysteinesufficiently prevent leakage of toxic metal ions, as shown by cytotoxicity assays with HepG2 cells. Confocal laser scanning microscopy shows that mQDs are internalized by these cells and accumulated near their nuclei. This study shows that biocompatible, fluorescent, and paramagnetic QDs are promising photostable labels for multimodal bioimaging.

1. INTRODUCTION Fluorescence dye tracking and detection of specific targets in living systems such as subcellular components play important roles in biological investigations. Therefore, fluorescent semiconductor nanocrystals, known as quantum dots (QDs), are among the tracing agents with the fastest growing development in biomolecular imaging and diagnostic platforms.1−5 However, for complex applications in bioanalysis and bioimaging, such as © 2018 American Chemical Society

in vivo localization studies, optical tracing alone is insufficient. QDs doped with magnetic ions offer an elegant solution by merging fluorescent and magnetic properties in a single material. The production of water-dispersible and magnetic QDs (mQDs) Received: January 29, 2018 Revised: May 30, 2018 Published: May 30, 2018 4233

DOI: 10.1021/acs.chemmater.8b00431 Chem. Mater. 2018, 30, 4233−4241

Article

Chemistry of Materials

doping depends on three main factors: nanocrystal shape, surface morphology, and type of capping agents used in the growth solution.32,33 Our research aims at providing a facile aqueous synthesis route, where magnetic ions can be incorporated into either the QD’s core or shell (Figure 1), while preserving the QD’s spherical shape and small size. Furthermore, differences in the physicochemical properties of core- and shell-doped CdTe/ZnS magnetic quantum dots (mQDs) are pinpointed. We employed ferrous ions as magnetic doping agents and used N-acetyl-L-cysteine (NAC) as biocompatible capping agent to promote both high water dispersibility and colloidal stability. X-ray diffraction (XRD) was used to assess the impact on the crystallinity of core- or shelldoped mQDs. Differences in their magnetic and compositional properties as well as in their quantum yields were determined by superconducting quantum interference device (SQUID), highresolution inductively coupled plasma mass spectrometry (HR-ICP-MS), and UV/vis and fluorescence spectroscopy. The colloidal stability was assessed via time-dependent changes in water-dispersed mQDs by using dynamic light scattering (DLS), while QD internalization by cells and QD cytotoxicity was tested on the HepG2 cell line using confocal laser scanning microscopy (CLSM) and cytotoxicity assays.

gives access to multimodal contrast agents and photostable labels that can be used for various applications in nanomedicine.6−9 Merging these two powerful functionalities in a single particle allows for multimodal probing and QD tracking across a wide application range: from organs and tissues by magnetic resonance imaging (MRI), to cells and organelles as well as individual molecules (e.g., proteins, polymers) by fluorescence imaging or super-resolution microscopy.10−15 However, the traceability and optical properties of QDs highly depend on the extent of their surface defects.16 These can be reduced via surface modification with inorganic or organic capping agents, resulting in an enhancement of the QDs’ photoluminescence quantum yields.17 Additionally, surface modifications prevent the leakage of toxic metals from the QDs’ core or shell materials. Overcoating of a Cd-containing QD core with a higher band gap semiconductor, such as ZnS, results in the formation of an outer shell that additionally passivates the QD’s surface and significantly reduces leaching of toxic Cd ions into the microenvironment.18−20 Further surface modifications by using organic surface coatings can be conducted in order to control dispersibility and colloidal stability and to allow for surface functionalization.21−24 In the past decade, a lot of effort has been dedicated to optimizing the optical and interfacial properties of QDs that consist of a CdTe core, a ZnS shell, and organic ligands for steric stabilization.25−27 Moreover, various protocols have been established in order to dope QDs with specific impurities to extend their functional repertoire.28−31 Dopingthe intentional introduction of impurities into a materialis a versatile strategy to tune the properties of semiconductors. The underlying mechanisms of doping are based on co-adsorption of impurities onto the growing nanocrystal structure or on replacement of constituent ions from lattice sites.32,33 In summary, the efficiency of

2. RESULTS AND DISCUSSION 2.1. Elemental Composition. HR-ICP-MS was used to quantify the doping efficiency of ferrous ions into core- or shelldoped mQDs (Table 1). The measurements show that the mass fraction of inorganic core−shell materials for undoped QDs (NAC-CdTe/ZnS) is about 44% (436 mg/g), for shell-doped mQDs (NAC-CdTe/30%Fe:ZnS) about 43% (433 mg/g), and for core-doped mQDs (NAC-30%Fe:CdTe/ZnS) about 46% (459 mg/g). Table 1 shows the elemental compositions of irondoped mQDs and undoped QDs. The latter, hereafter used as reference sample, exhibit a Zn:Cd molar ratio of 0.4 that is in good agreement with literature.28 The amount of iron present in core-doped QDs related to the total metal/metalloid concentration is 14.9% (n/n), whereas shell-doped QDs exhibit an irondoping level of 20.7% (n/n), indicating different incorporation efficiencies. These differences can be explained with the fact that the surface morphology and shape of nanocrystals as well as capping agents in growth solutions are key factors for the incorporation efficiency of dopants during the reaction.32,33 In our case, doping was conducted on mQDs in different growth phases with same initial Fe:Cd ratios via admixture of Fe dopants either during core or shell growth. In the case of core doping, our results indicate that CdTe nanocrystals are primarily formed during the initial growth phase, followed by the accumulation of an increasing number of dopants in the growing core with time as

Figure 1. Architecture of water-dispersible shell-doped mQDs (NACCdTe/30%Fe:ZnS) (A) and core-doped mQDs (NAC-30%Fe-CdTe/ ZnS (B).

Table 1. Elemental Composition of the QDs Prepared in This Studya Cd NAC-capped QD type bare QDsb core−shell QDsc core-doped mQDsd shell-doped mQDse

Te

[mg/g]

[%] w/w

[%] n/n

256 ± 4.9% 203 ± 4.3% 169 ± 2.1% 171 ± 2.4%

50.5 46.5 36.9 39.5

53.7 45.3 31.7 34.2

Fe

[mg/g]

[%] w/w

[%] n/n

251 ± 0.9% 187 ± 7.5% 172 ± 1.9% 163 ± 2.7%

49.5 42.7 37.5 37.6

46.3 36.7 28.4 28.7

Zn

[mg/g]

[%] w/w

[%] n/n

[mg/g]

[%] w/w

[%] n/n

40 ± 8.7% 51 ± 3.8%

8.6 11.9

14.9 20.7

47 ± 12.9% 78 ± 62.5% 48 ± 21.2%

10.8 17.0 11.1

18.1 25.0 16.5

a

All QDs are NAC-capped. Percentages in weight (w/w) or in amount of substance (n/n) are related to total metal/metalloid concentrations, which were determined using microwave-assisted acid digestion and HR-ICP-MS. Total metal/metalloid concentrations of core- and shell-doped mQDs were 459 and 432 mg/g, respectively. Metal/metalloid concentration of the undoped equivalents was 436 mg/g. bCdTe QDs. cCdTe/ZnS QDs, which are referred to as “undoped QDs” and used as reference sample. d30%Fe:CdTe/ZnS mQDs. eCdTe/30%Fe:ZnS mQDs. 4234


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Chemistry of Materials predicted by the theoretical doping model of Erwin et al.32 In the case of shell doping, the incorporation of ferrous ions is accelerated, presumably due to the similar radii of Zn2+ and Fe2+, which facilitates the integration of the latter into the host lattice. Hence, iron incorporation seems to be more efficient during shell growth than during core growth. This tendency was confirmed by additional EDX analyses on different synthesis batches (Figure S1 in the Supporting Information). 2.2. Influence of Iron Doping on Optical Properties. In order to study the impact of core or shell doping with Fe2+ on the optical properties of mQDs, absorption and emission spectra were recorded and the results compared with the undoped equivalent (i.e., NAC-CdTe/ZnS vs NAC-30%Fe:CdTe/ZnS or NAC-CdTe/30%Fe:ZnS). The optical properties of QDs are summarized in Table 2. It is important to note that the reaction

likely due to the incorporation of dopant ions. The UV/vis measurements show that the hypsochromic shift increased with increasing doping level as presented in the Supporting Information (Figure S3). On the contrary, these results indicate that shell doping had comparatively little impact on the nanocrystal growth regardless of the doping level. In the case of shell doping, it might be possible that Fe2+ dopants are incorporated either through the formation of additional shell material (FeS) or via replacement of Zn2+. With regard to the similar optical characteristics like their undoped equivalents and to the decrease in the Zn content upon shell doping evinced by the ICP-MS data, we conclude that substitution of Zn2+ is more likely than the formation of FeS. The employed concentration of shell dopants showed no major impacts on the first excitonic absorption and emission peaks (presented in Figures S3 in the Supporting Information). In this study, shell-doped mQDs exhibited a PL QY of 5% that is about 38% lower than for their undoped equivalents. Similar results were obtained by Jing et al.28 for mQDs with Mn2+-doped shells, where the PL QY was found to be highly dependent on impurity concentration (higher Mn/Cd molar ratios resulted in lower PL QYs). In summary, our results show that core doping has a higher impact on mQD’s photoluminescence properties than shell doping despite the higher iron dopant content in the shell. These findings confirm that fluorescence arises almost exclusively from excitation of the nanocrystal’s core, in which iron doping leads to structural defects and subsequently to fluorescence quenching.31,39 In general, shells composed of wider band gap materials predominantly confine the electron−hole pair and reduce surface reactions, which enhances quantum efficiency.8,38 As a consequence, structural defects within the nanocrystal’s core caused by iron impurities influence the optical properties of mQDs much more adversely than defects within the shell. 2.3. Influence of Iron Doping on Structural Properties. The XRD curves of the undoped QDs show characteristic reflections of CdTe and ZnS at q = 16.99 nm−1 (CdTe 111), 19.55 nm−1 (Wurtzite 002), 28.05 nm−1 (Wurtzite 101 or CdTe 311), and 32.43 nm−1 (Wurtzite 100) (Figure 2). Doping of the shell has only a marginal impact on the crystal structure of the

Table 2. Optical Properties of the Prepared mQDs Using UV/ Vis and Fluorescence Spectroscopy in Order To Compare the Effects of the Two Doping Approachesa NAC-capped QD type

λAb,max [nm]

undoped QDs 537 ± 1 core-doped 521 ± 1 mQDs shell-doped 540 ± 1 mQDs

λEm,max [nm]

Stokes fwhm shift [nm] [nm]

PL QY [%]

Δ PL QY [%]

568 ± 2 562 ± 2

31 41

51 54

8 ± 0.5 3 ± 0.1

−63

573 ± 2

33

53

5 ± 0.2

−38

a Core doping has a higher impact on the mQD’s photoluminescence quantum yield (PL QY) than shell doping. Both doping approaches show minor effects on the full width half maximum (fwhm).

time for each preparation step and for all samples was kept constant for 30 min to grow the QD’s core and 45 min to deposit a ZnS shell to ensure comparability. The corresponding absorption and emission spectra are presented in the Supporting Information (Figures S2 and S3). The undoped NAC-CdTe/ZnS QDs have a photoluminescence quantum yield (PL QY) of 8% that is in good agreement with literature, illustrating that the PL QY is highly dependent on the precursors’ pH and molar ratios as well as on the reaction temperature and QD size.34,35 In general, CdTe QDs can be overcoated with higher band gap materials, such as ZnS, to enhance their quantum yields and photostability and to further passivate the QD’s core.8 In our study, ZnS deposition onto the surface of NAC-CdTe QDs results in an emission red shift of about 40 nm (Figure S2 in the Supporting Information). This bathochromic shift of the QD’s characteristic absorption and emission maxima is expected upon formation of an additional ZnS layer.36−38 Our study shows that, in comparison with the undoped NAC-CdTe/ZnS, core and shell doping of QDs lead to significant fluorescence quenching: the PL QYs is reduced by about 63 and 38%, respectively. Iron-core doping lead to a higher Stokes shift, accompanied by a slight increase of the full width at half-maximum (fwhm) (Table 2). The same effects were observed by Saha et al.31 who showed that the Stokes shift and fwhm are highly dependent on reaction time during mQD synthesis. Erwin et al.32 and Norris et al.33 proposed that doping is generally governed by growth kinetics, where the dopant adsorbs and binds on the nanocrystal’s surface that is subsequently overcoated with additional material. In agreement to that, our experimental data show that nanocrystal growth is decelerated by the presence of dopants. In particular, core doping results in a hypsochromic shift of the first excitonic absorption and emission peaks, indicating a deceleration of nanocrystal growth

Figure 2. XRD pattern of undoped QDs (NAC-CdTe/ZnS, in gray), shell-doped mQDs (NAC-CdTe/30%Fe:ZnS, in red) and core-doped mQDs (NAC-30%Fe-CdTe/ZnS, in blue). The structure of undoped QDs displays the typical features of the CdTe/Wurzite crystal structure at 16.99, 19.55, and 32.43 nm−1. Fe doping of the shell has only a minor impact on the crystal structure, whereas core doping destroys the structure and only the peak at 16.99 nm−1 is retained. 4235


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Chemistry of Materials nanocrystal and hence these peaks are retained. The Scherrer width of the peak, which can provide a rough estimate of grain dimensions, gives crystal sizes of 2.02 nm (peak position 28.05 nm−1) and 2.17 nm (peak position 32.43 nm−1) for the nanocrystal’s core of undoped mQDs, and slightly different values, 2.23 and 1.52 nm, for that of shell-doped mQDs. Core doping has rather dramatic effects on the QD crystal structure. The overall scattered intensity of the sample decreases and the peaks at q = 28.05 nm−1 and q = 32.43 nm−1 disappear in a region of amorphous scatter. Only the peak at q = 16.99 nm−1 remains, although it is significantly broadened and thus discarded for fitting. The disappearance of the ZnS signal in the shell is interpreted as a strong disturbance of the crystal structure that may render the shell either amorphous or largely irregular. The strong broadening of the CdTe peak indicates that the iron doping creates defects in the lattice and hence decreases the coherence length of the crystal, which correlates with the low quantum yields of these nanocrystals. It is obvious that core doping has also a strong impact on the shell, even stronger than that caused by shell doping. One way to interpret this is a likely disruption of the crystal lattice of the core by Fe dopants that results in the creation of an amorphous layer, which in turn affects the ordering of Zn and S. 2.4. Influence of Iron Doping on Magnetic Properties. Temperature- and field-dependent magnetization measurements, M(T,H), were performed in order to analyze the differences in magnetic properties between core- and shell-doped mQDs. The temperature-dependent magnetic susceptibility (χ ≡ M/H) data, displayed in Figure 3, reveal that χ of core-doped mQDs at low

temperatures is almost 4 times higher than that of shell-doped mQDs. For the undoped QDs, a temperature independent, purely diamagnetic susceptibility of χ ≈ −2.5 × 10−8 m3/kg was observed. Although the Fe mass fraction in shell-doped mQDs is about 1.4 times larger than that in core-doped QDs (20.7% and 14.9% n/n, respectively) (Table 1), the difference in magnetic properties seems to be reflected by the disparate composition of the host materials for Fe dopants rather than by the disparate content of Fe in shell- and core-doped mQDs. Regarding the yielded doping content it needs to be noted that a Fe-dopant content of 5.5% n/n in similar core-doped mQDs was shown to be sufficient to produce MR contrast in vitro and in vivo, which was predominantly dependent on the deployed particle concentration.31 The field-dependent isothermal magnetization measurements, M(H), presented in Figure 4 reveal an approximately linear

Figure 4. Field-dependent isothermal magnetization of shell-doped (A) and core-doped QDs (B) measured at temperatures as indicated in the legend.

paramagnetic behavior in shell-doped mQDs near room temperature and up to 2 T, whereas core-doped mQDs show a nonlinear superparamagnetic behavior even near room temperature. In both cases, the coupling of Fe moments and/or clusters increases with decreasing temperature and thus tends to block the paramagnetic response. Finally, below about 10 K, i.e., below the respective maxima of M/H(T), a clearly hysteretic behavior occurs (Figure 4). Saha et al.31 reported magnetization values of 71 emu/gFe for core-doped CdTeS-QDs, which closely matched those of Feridex (72 emu/gFe), a regularly employed MRI contrast agent. For comparison, we obtained room temperature magnetization values of 23 emu/gFe for core-doped mQDs, and 4.4 emu/gFe for

Figure 3. Temperature-dependent dc magnetic susceptibility, M/H(T), of shell-doped (A) and core-doped QDs (B) measured at magnetic fields as labeled. 4236


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QD aggregation in aqueous media was investigated by monitoring the changes in the hydrodynamic diameter (HDDs) of water-dispersed mQDs over time using DLS. The recorded time evolution of the mQD’s HDDs over 18 days is shown in Figure S5 in the Supporting Information. Initially on day 0, shell-doped mQDs formed small aggregates in the size range of 17.4 ± 6.7 nm (polydispersity index, PDI = 0.22). In case of core-doped mQDs, the aggregated clusters were initially larger (HDD = 37.0 ± 13.0 nm, PDI = 0.20), whereas the undoped equivalent showed the smallest HDD of about 8.3 ± 2.5 nm (PDI = 0.24). After 18 days the HDDs increased to 31.9 ± 13.5 nm (PDI = 0.21) in the case of shell-doped QDs. The HDDs of core-doped mQDs increased to 39.9 ± 13.3 nm (PDI = 0.19), and to 24.5 ± 9.5 nm (PDI = 0.29) in the case of their undoped equivalents. Thus, undoped QDs and iron-doped mQD types exhibited similar tendency to aggregate. The results show that after 18 days no clear differences were observed between shell- or core-doped mQDs. The small differences in HDDs between doped mQDs and undoped QDs can be attributed to differences in densities of the inorganic core−shell materials. 2.6. Cellular Uptake by HepG2 Cells and mQD Localization. QDs have been established as fluorescent labels for bioimaging and are promising complements to organic dyes, which are deficient in photostability.44 Additionally, QDs have another unique optical property, called photoluminescence blinking, which enables distinctive localization of (bioconjugated) QDs in living cells by super-resolution imaging.45−47 Overcoating the QD core material with additional passivation layers, such as ZnS, causes a considerable increase in quantum yield and simultaneously reduces leakage of toxic components.48 An additional organic layer, i.e., of NAC, improves colloidal stability of the QDs and acts as a matrix that mediates the interaction with biomolecules and additionally allows for further surface modification and bioconjugation. In this study, the half-maximal inhibitory concentrations (IC50) were used in order to compare the differences in cell viability affected by undoped QDs or doped mQDs. The IC50 values are found to be 20.8 μg/mL for undoped QDs, 5.2 μg/mL for shell-doped mQDs, and 0.2 μg/mL for core-doped mQDs. Note that, for the sake of comparison, the IC50 value for dissolved Cd ions (as CdCl2) was approximately 300 times lower than that for undoped QDs. At QD concentrations ≤10 μg/mL (in QD-containing culture medium), the cell viability of HepG2

shell-doped mQDs at 6 T, which are considerably smaller than those obtained by Saha et al.31 In addition to that, the magnetic characteristics of our samples appear to be quite different. Saha’s doped CdTeS mQDs were reported to saturate at much smaller field strengths (1 T) and only at low temperatures, characteristic for a weak superparamagnetic material. On the other hand, shelldoped mQDs show paramagnetic behavior at room temperature. This disparate behavior can be explained by the different composition of our QDs, as variations in the composition of the host material have been reported to affect the magnetic couplings upon variation of the phase content via subtle changes in the crystal structure.40 In this regard, Saha et al.31 suggests that a higher S-content in the CdTe host material, which is about 1.5 times higher than that of our core-doped samples, may be beneficial and needs to be taken into account in future studies in order to obtain higher saturation magnetizations through ferrous doping. 2.5. Morphology and Colloidal Stability of QDs. QDs with hydrophilic capping agents are commonly dispersed in ultrapure water or aqueous stock solutions prior to their further use for cell uptake and imaging studies. For these purposes, QD dispersions have to be colloidally stable as possible aggregation adversely affects their behavior and performance during imaging. In particular, aggregated particle markers could potentially modify subcellular localizations when conjugated to small mobile analytes and/or compromise MRI contrast, as QDs may change their magnetic relaxation characteristics.41,42 In this study, valuable information about the QD’s morphology was obtained via TEM analysis (Figure 5). It is important to note

Figure 5. Representative TEM image of shell-doped mQDs (A) and the corresponding diffraction pattern (inset), indicating that the mQDs are crystalline. (B) The corresponding size distribution, where the average particle diameter was determined to be 2.9 ± 0.3 nm.

that TEM analyses give limited information about the aggregation state of nanoparticles, as vacuum conditions and preparation procedures can cause significant deviations from their solution structure.43 STEM micrographs (Figure S4 in the Supporting Information) indicate a strong tendency of mQDs to aggregate at relatively high concentrations, where capillary forces during drying as well as interparticle interactions in vacuum lead to significant clustering of mQDs. However, when the dispersions are sufficiently diluted and drop-casted, mQDs remain dispersed on the grid as shown by the TEM micrograph (Figure 5A). Thus, TEM analyses were conducted only in order to determine the nanocrystal’s size and shape. Figure 5 reveals that mQDs have spherical shapes and a diameter of 2.9 ± 0.3 nm, which is in good agreement with XRD data, where the crystallite size was slightly smaller as expected and was measured to be at 2.2 nm.

Figure 6. Dose-dependent, in vitro cytotoxicity of QDs on HepG2 cells after 24h incubation. 4237


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Figure 7. Representative CLSM image of HepG2 cells after 3h incubation with shell-doped mQDs (20 μg/mL). Bright field image of adherent cells depicting the cell membrane (white arrows) and the nucleus (A). The corresponding fluorescence image confirmed that QDs were internalized within 3h and accumulated near the cell nuclei (B) (red arrows). (C) shows the overlay of the micrographs (A) and (B).

the core or the shell of the mQD were doped with ferrous ions at high molar fractions (14.9% and 20.7% n/n, respectively) which lead to differences in structural, optical and magnetic properties of the resulting mQDs. Core doping reduced the particle growth rate compared to that of undoped QDs. The iron incorporation efficiency was found to be higher for shell doping than for core doping. Dopant incorporation into the mQD’s core disrupts the lattice structure, leading to a significant decrease in photoluminescence quantum yield and to a higher magnetic susceptibility near room temperature. Contrarily, the incorporation of iron into the mQD’s shell resulted in mQDs with lower magnetic susceptibility but had no noticeable effect on the mQD’s core crystal structure. Consequently, shell doping has a smaller adverse impact on the mQD’s quantum yield than core doping. We found that iron doping of the shell and the core influenced the magnetic behavior of the mQDs: core-doped mQDs exhibited superparamagnetic behavior while shell-doped mQDs were paramagnetic near room temperature. Our results also indicate that photoluminescence properties of mQDs are predominantly influenced by the core’s crystallinity. The higher dopant incorporation efficiency in shell-doped mQDs is attributed to the similar size of divalent Zn and Fe ions, allowing for efficient substitution without disrupting the host lattice structure. However, it remains unclear whether the doping mechanism is predominantly based upon exchange of divalent metal ions or upon formation of another layer. In conclusion, we favor shell-doped over core-doped QDs for their comparatively higher quantum yields and crystallinity (i.e., crystal stability) as well as for their lower in vitro cytotoxicity on Hep2G cells in comparison to core-doped mQDs. With respect to future applications, it needs to be considered that the performance of mQDs in biological media is strongly dependent on the mQD’s aggregation behavior, which in turn highly depends on environmental conditions as well as on the used capping agents and surface properties of (bioconjugated) QDs. For future studies, the presented N-acetyl-L-cysteine-capped mQDs can be used for further surface modifications and for cross-linking peptides, antibodies and proteins of particular interest, which allows then for targeting of specific cells or organelles. In terms of nanosafety, such functionalized “mQD platforms” may then be used to elucidate and visualize the unknown kinetics of internationalization of similar nanomaterials.

cells was approximately 77%, 66% and at 34% for undoped, shelldoped, and core-doped QDs, respectively. In the case of undoped QDs and shell-doped mQDs, cell viability apparently decreased above a concentration of 10 μg/mL. In contrast to that, coredoped QDs did not show a clear concentration-dependent cytotoxicity (Figure 6). This difference is likely to be due to the presence of crystal defects in the core-doped QDs, as shown by the XRD measurements (indicated in Figure 2). These defects may lead to unstable core−shell structures that ease the leaching of toxic Cd ions into the culture medium. mQD uptake by HepG2 cells was observed by confocal laser scanning microscopy (CLSM). Cell visualization was conducted at different time intervals after the addition of QDs into the growth medium. After 3 h, there was a clear accumulation of shell-doped QDs near the cell nuclei, as shown in Figure 7. Z-stacks of the same area revealed that all three QD types do not penetrate the cell nucleus, not even after a long incubation period. It remains unclear whether mQDs aggregate somewhere within the endoplasmic reticulum, or whether mQDs accumulate in specific organelles like the mitochondria. However, not only does the incubation time of mQDs play a critical role in imaging, but also the concentration of the mQDs in the growth medium. Prolonged cell incubation in growth medium with mQDs at concentrations higher than 20 μg/mL results in cell detachment, which consequently hinders accurate QD localization. In summary, the fate and toxicity of Cd-based QDs is generally dependent on their size and specific surface properties (including further surface modifications, such as cell-penetrating peptides), as well as on assay type and exposure time.48 For future localization studies, it should be taken into account that further surface modifications, which are often needed for functionalization and bioconjugation, affect QD uptake, toxicity and their ultimate fate, that is shown exemplarily in Tan et al.49

3. CONCLUSIONS This study gives insights in the differences in crystallinity, optical properties, composition and colloidal behavior as well as cytotoxicity of water-dispersible magnetic quantum dots (mQDs), which were doped with ferrous ions in either the CdTe core or ZnS shell of the QDs. The advantages of such multimodal imaging agents over conventional organic fluorophores are the very high photostability and thermal stability. In addition, the mQD’s intrinsic hydrophilicity does not require further surface modification to become water-dispersible, a requirement for efficient cell administration and bioimaging. In our study, either

4. EXPERIMENTAL SECTION 4.1. Materials and Reagents. Cadmium chloride (Fluka, ≥99.0%), tellurium powder (Aldrich, 99.997%), sodium borohydride (Aldrich, 4238


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Chemistry of Materials ≥98.0%), N-acetyl-L-cysteine (NAC) (Sigma-Aldrich, ≥99%), iron(II) chloride·4H2O (Sigma-Aldrich, ≥99.0%), sodium sulfide·9H2O (Fluka, ≥98.0%), zinc(II) chloride (Sigma-Aldrich, 99.999%), and fluorescein (Sigma) were employed for the synthesis of QDs. All chemicals were used as received. Ultrapure water (18.2 MΩ·cm, Milli-Q Integral10 water purification system, Millipore) was degassed and purged with nitrogen (N2) for 30 min before use. 4.2. Preparation of Undoped and Iron-Doped NAC-CdTe QDs. First a 62.5 mM NaHTe solution was prepared by mixing tellurium powder (0.63 mmol) with NaBH4 (1.32 mmol) in 10 mL of N2-purged ultrapure water under inert atmosphere. The mixture was stirred in an ice bath for about 2 h under constant N2 purging. Subsequently, the ice bath was removed and stirring was maintained at room temperature until the reaction was completed (5−6 h). The freshly prepared purple NaHTe solution was kept under N2-atmosphere at 4 °C until further use. CdCl2 (0.5 mmol) and NAC (1.25 mmol) were dissolved in 200 mL of N2-purged ultrapure water. In the case of core-doped QDs, FeCl2· 4H2O (0.08 mmol) was added. The precursor solution was adjusted to pH 8.3 by addition of 1.0 M NaOH and stored at room temperature. Prior to crystal growth, 80 mL of the precursor solution was transferred into a three-neck, round-bottom flask and purged with N2 for 30 min in an ice bath. The purple NaHTe solution was added to the CdCl2/NAC precursor solution at a constant Cd:Te:NAC molar ratio of 2:1:5 under vigorous stirring and N2 atmosphere. Crystal growth was conducted at 140 °C in a preheated oil bath for 45 min and cooled down to room temperature in an ice−water bath immediately after. The obtained crude reaction mixture was lyophilized and redispersed in 3 mL of ultrapure water. Subsequently, the crude QD dispersion was precipitated in cold 2-propanol, centrifuged, and redispersed in ultrapure water. This purification step was repeated twice. Finally, purified pristine QDs and core-doped mQDs, NAC-CdTe and NAC-30% Fe:CdTe, respectively, were lyophilized and stored at room temperature for further experiments. The resulting undoped and iron-doped CdTe QDs were used for further surface modification. 4.3. Preparation of Undoped and Core-Doped NAC-CdTe/ ZnS QDs. The as-prepared NAC-CdTe and NAC-30%Fe:CdTe QD cores were overcoated with ZnS shells. The shell coating solution was prepared according to Xiao et al.50 with some modifications regarding the purification process. Briefly, 20 mg of as-prepared QD cores was redispersed in 20 mL of an aqueous solution containing 0.50 mmol of ZnCl2, 0.10 mmol of Na2S, and 2.50 mmol of NAC. ZnS deposition was conducted in a preheated oil bath at 65 °C under N2 atmosphere for 45 min. The crude reaction mixture was then cooled down to room temperature in an ice bath, and the resulting products were purified by repeated precipitation in 2-propanol as described in section 2.2. 4.4. Preparation of Undoped and Shell-Doped NAC-CdTe/ ZnS QDs. For the preparation of shell-doped mQDs (NAC-CdTe/30% Fe:ZnS), 20 mg of NAC-CdTe QDs was redispersed in 20 mL of an aqueous solution containing 0.50 mmol of ZnCl2, 0.03 mmol of Na2S· 9H2O, and 2.50 mmol of NAC. The Cd concentration in NAC-CdTe QD cores was determined by HR-ICP-MS, allowing us to fix a Cd:Fe ratio of 1:0.3. Consequently, we additionally added 0.03 mmol of FeCl2· 4H2O. The mixture was placed under N2 atmosphere in a preheated oil bath at 65 °C for 45 min. After the crude reaction mixture was cooled to room temperature in an ice bath, samples were purified as previously described in Section 2.2. 4.5. Characterization. X-ray Diffraction (XRD). XRD measurements were carried out with a Rigaku S-Max 3000 SAXS/WAXS instrument equipped with a copper-target X-ray tube MicroMax002+, operated at 45 kV, and 0.88 mA. The beam was collimated with a threepinhole collimation system (400, 200, and 700 μm diameter of the pinholes), resulting in a beam size of 210 μm fwhm at the sample position. The scattered photons were detected with an image plate situated 30 mm behind the sample, giving access to scattering vectors from q = 12 to 45 nm−1, where q is related to the crystal lattice spacing d by q = 2π/d and to the scattering angle 2θ by q = 4π/λ sin θ (λ is the wavelength of the incident beam). The samples were prepared as thin powder film on cellophane adhesive tape and exposed for 300 s. The measured intensities were normalized by means of a photodiode measuring the

transmitted photons and the background was subtracted. The diffraction peaks were fitted individually to Gaussian profiles with a home-written code in Mathematica 9.0.1. The peak width was used to determine the crystal size L according to the Scherrer formula Δ(2θ) = (Kλ)/(L cos θ), where Δ(2θ) is the full width at half-maximum (fwhm) in 2θ units, and K is a constant close to 1. Magnetic Measurements. A 6 T Cryogenic S600 SQUID magnetometer was employed for temperature-dependent DC magnetic susceptibility measurements as well as for isothermal field-dependent magnetization studies in the temperature range from 3 K to room temperature and in magnetic fields up to 6 T. Prior to measurements, approximately 10 mg of sample was filled into a diamagnetic gelatin capsule ( χ ≈ −5 × 10−9 m3/kg). QD Chemical Analysis. Elemental and compositional analyses of the prepared QDs were conducted by using microwave-assisted acid digestion (Anton Paar, Graz, Austria) and high resolution, double focusing sector field inductively coupled plasma mass spectrometry (HR-ICP-MS) (ELEMENT 2 ICP-SFMS, ThermoFisher, Bremen, Germany). As described in Popp et al.,51 ultrapure water and double-distilled 2% (v/v) and 60% (v/v) nitric acid (p.a. grade, Merck) were used for the preparation of QD dispersions and serial dilutions of standards (ICP multielement standard solution VI, Merck KGaA, Darmstadt, Germany). For digestion, 6 mL of 60% nitric acid was added to 1 mL of a QDs aqueous dispersion (100 mg/L). After digestion, the samples were filled up to 10 mL with ultrapure water and, prior to ICP analysis, diluted by a factor of 1000 using 2% (v/v) nitric acid. A low-level fortified standard TM-27.2 of the National Water Research Institute NWRI (Burlington, Ontario, Canada) and indium as internal standard were employed for quality assurance. All laboratory materials and conditions were similar to those described by Popp et al.51 Energy-dispersive X-ray spectroscopy (EDX) was additionally used to perform faster quality controls of different synthesis batches. UV/Vis Spectroscopy. UV/vis spectra were recorded using a UV/vis spectrophotometer (Hitachi UV-2900, Japan) at a scan speed of 400 nm/min. Fluorescence spectra (LS 55 fluorescence spectrometer, 230 V, PerkinElmer, UK) were recorded at a scan speed of 400 nm/min. The excitation and emission slit-width was fixed at 4 nm, while the emission spectrum was recorded between 400 and 700 nm. Absorbance of each sample was adjusted to optical density values below 0.1 in order to avoid self-quenching and homo-aggregation effects.52 The photoluminescence quantum yield (PL QY) of QD dispersions was determined by using fluorescein (in 0.1 M NaOH) as fluorescence standard at an excitation wavelength of 470 nm. It is important to note that fluorescein in 0.1 M NaOH exhibits a PL QY of 91%.53 The PL QY was determined according to Würth et al.54 All optical measurements were performed at room temperature and under atmospheric conditions. Transmission Electron Microscopy (TEM). TEM images were recorded on a FEI Tecnai G2 20 transmission electron microscope operating at 160 kV. Samples were prepared by drop-casting of diluted QD dispersions onto 300-mesh carbon-coated copper grids and subsequent solvent evaporation at ambient conditions. Nanoparticle diameter and size distributions were evaluated using the Pebbles software package with a local intensity model fitting algorithm.55 Scanning transmission electron microscopy (STEM) images were recorded on a Zeiss Supra 40 electron microscope at 20 kV and a STEM detector. Samples were prepared by drop-casting of concentrated dispersions on 400-mesh carbon-coated copper grids. Dynamic Light Scattering (DLS). Changes in the aggregation behavior were monitored with a Zetasizer Nano Z, beam wavelength = 633 nm, 173° backscatter, Malvern Instruments Ltd., UK, over a period of 18 days. Freeze-dried, doped- and undoped QDs were redispersed in ultrapure water (18.2 MΩ·cm at 25 °C, Milli-Q, USA) at a concentration of 100 μg/mL and sonicated for about 30 s. These dispersions were subsequently filtered through a poly(vinylidene fluoride) membrane (PVDF, 0.20 μm, Graphic Controls Ltd., UK) prior to storage at 37 °C. DLS measurements were conducted at the same temperature. Hydrodynamic diameters (HDDs) were acquired on day 0 as well as on days 1, 2, 3, 6, and 18 using a refractive index of 1.338 for waterdispersible QDs56 and 1.330 for water. All samples were measured in triplicate and all particle size distributions are number-weighted. 4239



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4.6. In Vitro Cytotoxicity Assay and Fluorescence Imaging. Cytotoxicity of QDs was evaluated on the human liver hepatocellular carcinoma cell line HepG2 (ATTC HB-6085) by AlamarBlue Assay (Thermofisher, Austria). HepG2 cells were cultured in Eagle’s minimum essential medium (EMEM) containing 0.1% glutamine, and supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/ antimycotic at 37 °C in a humidified atmosphere of 5% CO2 and 95% H2O. For the assay, HepG2 cells were seeded in 96-well microtiter plates at a density of 10 000 cells in 200 μL of culture medium per well. After reaching approximately 60% confluence, the HepG2 cells were exposed to QDs in 200 μL of medium containing 10% alamarBlue reagent. The cells were exposed to shell-doped, core-doped, and undoped QDs in different concentrations (100, 75, 50, 25, 10, 1, and 0.1 μg/mL). After 24 h incubation at 37 °C the emission was acquired at a wavelength of 590 nm (λex = 560 nm) using an Infinite F200 plate reader (TECAN, Austria). Untreated cells were used as negative control. Wide-field fluorescence and confocal microscopy were employed to investigate the internalization of QDs by the cells. HepG2 cells were grown to 60% confluence in μ-Slide 4 wells (Ibidi, Munich, Germany). After reaching the desirable confluence (usually after 48 h) cells were rinsed with medium, and incubated for 3 h or overnight with different QD dispersions at concentrations of 2, 20, and 100 μg/mL, in modified culture medium with 2% FBS and without phenol red. For wide-field fluorescence, the microscope (DMI600B Leica Microsystem, Wetzlar, Germany) was equipped with a metal-halide lamp and a triple-band filter cube (VBG, cat. no.11523031) to excite the QDs and collect the emitted fluorescence. For confocal measurements, the confocal microscope (TCS SP8 Leica Microsystems, Mannheim, Germany) was equipped with a pulsed white light laser (WLL 2) and a 405 nm diode laser as excitation sources, and a HyD spectral detector in the range of 520−560 nm, for collecting the fluorescence emission.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.8b00431. Energy-dispersive X-ray spectra of updoped QDs and shell-/core-doped mQDs; absorption and emission spectra; absorption spectra of QDs with different dopant levels; batch-dependent variability of the characteristic first excitonic absorption peaks; STEM images; timedependent particle number-weighted size distributions of QD dispersed in ultrapure water; CLSM images of untreated and QD-treated U937 cells (PDF)

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Florian Part: 0000-0003-1301-1502 Stefan Schrittwieser: 0000-0001-5807-3331 Stephan Hann: 0000-0001-5045-7293 Helga Lichtenegger: 0000-0002-6624-1419 Eva-Kathrin Ehmoser: 0000-0001-9201-268X Author Contributions ◇

F.P. and C.Z. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was partially supported by the European Commission FP7 NAMDIATREAM project (EU NMP4-LA2010−246479). Our special thanks to Dr. Susana Moreno-Flores for editing and proofreading the manuscript. 4240


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Doping Method Determines Para- or Superparamagnetic Properties of

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