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Preparation, cytotoxicity and in vivo bioimaging of highly luminescent water-soluble silicon quantum dots
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Nanotechnology Nanotechnology 26 (2015) 215703 (10pp)
doi:10.1088/0957-4484/26/21/215703
Preparation, cytotoxicity and in vivo bioimaging of highly luminescent watersoluble silicon quantum dots Jing-Wun Fan1, Raviraj Vankayala2, Chien-Liang Chang1, Chia-Hua Chang1, Chi-Shiun Chiang3 and Kuo Chu Hwang2 1
Chemical System Research Division, National Chung-Shan Institute of Science & Technology, PO Box 90008-17, Lung-Tan, Tao-Yuan 32599, Taiwan 2 Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan 3 Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu 30013, Taiwan E-mail: [email protected] Received 8 February 2015, revised 28 March 2015 Accepted for publication 2 April 2015 Published 6 May 2015 Abstract
Designing various inorganic nanomaterials that are cost effective, water soluble, optically photostable, highly fluorescent and biocompatible for bioimaging applications is a challenging task. Similar to semiconducting quantum dots (QDs), silicon QDs are another alternative and are highly fluorescent, but non-water soluble. Several surface modification strategies were adopted to make them water soluble. However, the photoluminescence of Si QDs was seriously quenched in the aqueous environment. In this report, highly luminescent, water-dispersible, blue- and green-emitting Si QDs were prepared with good photostability. In vitro studies in monocytes reveal that Si QDs exhibit good biocompatibility and excellent distribution throughout the cytoplasm region, along with the significant fraction translocated into the nucleus. The in vivo zebrafish studies also reveal that Si QDs can be evenly distributed in the yolk-sac region. Overall, our results demonstrate the applicability of water-soluble and highly fluorescent Si QDs as excellent in vitro and in vivo bioimaging probes. S Online supplementary data available from stacks.iop.org/NANO/26/215703/mmedia Keywords: silicon quantum dots, cytotoxicity, bioimaging, zebrafish, luminescence (Some figures may appear in colour only in the online journal) 1. Introduction
biochemical processes at the molecular level in living cells [8, 9]. Ideally, the fluorescent probes should be nontoxic, possess good photostability and resist enzymatic degradation in the physiologic medium [10]. To date, organic dyes were extensively used to label the intracellular organelles. However, they suffer from severe photobleaching upon continuous light exposure. Semiconducting quantum dots (QDs) are another alternative to organic dyes and attract significant attention because of their unique optical properties. They have been successfully demonstrated as in vitro and in vivo imaging probes in the field of nanobiomedicine [11]. Semiconducting QDs possess strong absorption, size-tunable
In recent years, extensive research efforts have been devoted to the utilization of inorganic nanomaterials for various biomedical applications, such as imaging, gene delivery and drug delivery [1–3]. Apart from the utility of nanoparticles as drug carriers for different treatments, they can also serve as multimodal imaging agents in clinics, which include magnetic resonance imaging (MRI), ultrasound (US), computed tomography (CT), photoacoustic (PA) and fluorescence imaging [4–7]. Among various imaging modalities, fluorescence imaging is widely adopted for investigation of various 0957-4484/15/215703+10$33.00
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© 2015 IOP Publishing Ltd Printed in the UK
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fluorescence emission, high quantum yields and good photostability [11]. However, a major concern that may limit their use in biology and medicine is the toxicity associated with the cadmium-containing QDs. Overall, the safety issues or concerns regarding the cytotoxicity of QDs severely hampered their use in bioimaging [12]. Some of the recent reports also suggest carbon/graphene QDs as an alternative to semiconductor QDs for bioimaging purposes. However, their structural formation, optical properties, and short-term and long-term cytotoxicity effects are still not well explored [1, 2]. Silicon is one of the most abundant materials, and it is widely used in the field of microelectronics. It is considered to be an indirect band-gap semiconductor material that is less interesting for light-emitting device applications in bulk form [13]. However, the nanosized silicon offers efficient fluorescence emission with excellent photoluminescence quantum yields due to the quantum confinement effect [14, 15]. Although Si QDs are expected to be far less toxic than semiconducting QDs, they have thus far not been widely used because of their limitation in making them highly luminescent, water dispersible, and compatible to the biological fluids and physiological medium. In general, most of the literature has reported that highly fluorescent Si QDs are non-water soluble. The photoluminescence of Si QDs was quenched in the aqueous medium. For example, it was reported that Si QDs attained water dispersibility by means of carboxylic acids or amine functional groups [16, 17]. It is often easier to functionalize nonpolar functional groups, such as styrene and octane, on the surface of Si QDs. However, this shows poor colloidal stability and weak photoluminescence in the aqueous environments. In another study, it was reported that Si QDs can be encapsulated into the nonpolar region of polyethylene glycol (PEG) grafted phospholipids to obtain water dispersibility [18]. However, the photoluminescence from Si QDs was quenched upon lipid encapsulation. Therefore, it is greatly challenging to prepare Si QDs that are highly fluorescent and also water soluble. A brief overview of the optical properties and bioimaging studies were summarized for various Si QDs reported in the literature (see table S1). To date, several reports have demonstrated the bioimaging applications of Si QDs, but their cellular fate in monocytes was never reported [14–16]. Moreover, the co-localization and biodistribution of Si QDs in the in vivo zebrafish animal model has not yet been investigated. Overall, it is necessary to develop a facile synthetic process to prepare highly fluorescent, water-soluble, biocompatible and optically stable colloidal Si QDs and make them useful for in vitro and in vivo biological labelling applications. In this study, we report on the preparation of watersoluble, highly fluorescent, optically photostable and biocompatible blue- and green-emitting Si QDs using an atmospheric plasma process. The etched silicon nanopowder was subjected to either thermal oxidation or hydrogen peroxide– ethanol treatment to produce superbright fluorescent Si QDs coated with a monolayer of SiOx. To achieve water dispersibility, silicon QDs were capped with a thin monolayer of polyacrylic acid (PAA) using ultraviolet (UV) light photo irradiation. After surface modification with PAA, the photoluminescence becomes very stable in the aqueous solution.
Figure 1. Schematic representation of the synthesis of blue- and
green-emitting Si QDs using the atmospheric plasma method, followed by either thermal oxidation or treatment with hydrogen peroxide and ethanol mixture.
Further, the cytotoxicity and uptake capabilities of blue- and green-emitting Si QDs were demonstrated in monocytes and also in the in vivo zebrafish system. To the best of our knowledge, this is the first report to demonstrate the direct visualization of the biodistribution of highly fluorescent Si QDs in the zebrafish animal model.
2. Materials and methods 2.1. Synthesis and surface modification of Si QDs
In the first step, plasma silicon nanopowder was prepared using an atmospheric plasma-triggered arcing process. In a typical experiment, 0.1% SiH4 precursor gas and carrier gas (argon) were injected into the atmospheric plasma reactor (60 W) containing an organic solvent in the quartz vessel. The two electrodes were connected to the power supply (60 W), and the atmospheric plasma was then triggered to obtain the precipitate of the reddish-brown solid silicon nanopowder. In the second step, etching of as-prepared silicon powder was carried out by a thermal oxidation process upon mixing 0.01 g of silicon nanopowder in 5 mL HF and ultrasonicated for 30 min so that the large particles could break apart. During the etching process, the solution color turned from reddish brown to pale yellow. Further, the solution was filtered through a 0.2 μm PVDF membrane and washed thoroughly with methanol–water (1:3) mixture to remove impurities. The asprepared nanoparticles were further mixed with 10% acrylic acid in ethanol solution (1 mL) and toluene (4 mL) and then subjected to photoirradiation to undergo hydrosilylation under a UV lamp (254 nm) for 1 h to achieve water dispersibility. 2.2. Cell culture, materials and reagents
The monocytes used in the experiments were isolated from C57BL/6J mouse blood. The mouse was sacrificed and the monocytes were collected and purified from the blood using a standard ficoll-paque density gradient protocol (Invitrogen, USA). Briefly, 10 mL of blood were collected in a centrifuge 2
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Figure 2. (a) and (b) represent the transmission electron microscopy (TEM) images of green-emitting silicon quantum dots. The inset in (b)
shows the high-resolution TEM image of a single nanoparticle. (c) UV-visible absorption spectra for blue- and green-emitting quantum dots.
bottle and diluted with 4 mL of buffer solution with gentle mixing. In a separate centrifuge bottle, 3 mL of ficoll-paque media (Invitrogen, USA) was withdrawn, followed by the addition of 4 mL of the diluted blood sample and the contents were gently mixed. The contents were then centrifuged for 30 min at 1500 rpm. The upper layer containing the plasma and platelets was drawn off, leaving the cell layer undisturbed at the interface. The monocytes were transferred into a separate centrifuge tube, followed by washing with a buffer 2 times. The collected monocytes were then plated in 10 cm dish in the presence of RPMI-1640 (Sigma, USA) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO, USA) and 1% penicillin-streptomycin (GIBCO, USA) at 5% CO2 atmosphere and 37 °C temperature.
trypsinized and suspended in the PBS buffer solution and then subjected to flow cytometry (FACS Calibur) analysis equipped with a 488 nm laser. The fluorescence intensities obtained from the fluorescein isothiocyanate (FITC) channel were quantified and analysed using Win MDI software. 2.4. In vitro bioimaging of Si QDs using confocal laser scanning microscopy (CLSM)
The cell seeding procedure and the pretreatment of greenemitting Si QDs to the monocytes were similar to those mentioned in the section 2.3. The green-emitting Si QDs internalized monocytes were washed again with PBS, further fixed onto a glass slide using p-formaldehyde solution (4%) in PBS for 5 min, and washed with PBST (5% Tween 20 in PBS) solution three times; then Triton X-100 solution (1 mL) was added. After exposure to Triton X-100 (1 h), the monocytes were stained with DAPI (4′,6-diamidino-2-phenylindole) (1 ng mL−1 PBS, 30 min). The samples were examined under a confocal laser scanning microscope (LSM 700) equipped with a UV laser (λex = 405 nm; λem = 420 ∼ 450 nm) and Ar laser (λex = 488 nm; λem = 510 ∼ 540 nm).
2.3. In vitro cellular uptake of Si QDs using flow cytometry
Two millilitres of monocyte-containing solution (2.0 × 105 cells mL−1) in the presence of RPMI-1640 were added to each well of a 6-well plate and incubated for 1 day to allow the cells to stick on the surface of the plate. Aliquots of aqueous solution containing green-emitting Si QDs were added to the 6-well plate, and the cell solutions were incubated for an additional 6 h. After 6 h, the cells were washed twice with phosphate buffer solution (PBS) (pH 7.4) and then incubated for another 20 h in the presence of fresh RPMI-1640. Then the cells were
2.5. In vitro cytotoxicity assay
One millilitre of monocyte-containing solution (2.0 × 104 cells mL−1) in the presence of the RPMI-1640 medium were 3
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colorimetric detection at 490 nm, 1 M HCl was added to stop the enzymatic reaction. 2.7. In vitro Annexin V apoptosis assay
Two millilitres of monocyte-containing solution (2.0 × 105 cells mL−1) in the presence of RPMI-1640 were added to each well of a 6-well plate and incubated for 1 day to allow the cells to stick on the surface of the plate. Aliquots of an aqueous solution of green-emitting Si QDs were added to the 6-well plate, and the cell solutions were incubated for an additional 6 h. The cells were then trypsinized, aspirated and suspended in 2 mL PBS. The cells were then stained with FITC-Annexin V (5 μL) and PI (5 μL) from the BD Annexin V apoptosis kit and then kept for 15 min at room temperature in darkness, followed by flow cytometry analysis. 2.8. In vivo cytotoxicity and bioimaging of silicon QDs in zebrafish
Wild-type AB strains of Danio rerio (zebrafish) embryos obtained from the Taiwan Zebrafish Core Facility Center, National Tsing Hua University, were used in all the experiments. Fresh embryos were collected to the microinjection embryo tray just before the experiment. Blue- and greenemitting Si QDs were diluted at appropriate concentrations in double distilled water and sonicated up until microinjection. An approximately 10 nL volume was microinjected into the animal pole region of the embryos between stages 1 (one-cell embryo) and 3 (four-cell embryo) using a Drummond microinjector. Each experiment was performed on 50 embryos per condition. Following microinjection, the embryos were transferred onto a petri dish filled with the system water and incubated at 28 °C in the dark. For the in vivo cytotoxicity measurements, the live embryos were counted each day until 72 hpf (hours post fertilization). After the 72 hpf, all the developed embryos were hatched manually and the types of abnormalities undergone were examined through a fluorescence microscope (Nikon, E600). For in vivo imaging experiments, the zebrafish embryos (72 hpf) were hatched and fixed on cover slips (15 × 16 mm, GeneFrame) with 1% agarose gel (Sigma, USA) to monitor the fluorescence of Si QDs using CLSM (10×, LSM-700).
Figure 3. Photoluminescence emission spectra for (a) blue- and (b)
green-emitting silicon quantum dots at different excitation wavelengths. The inset represents the characteristic blue and green emissions originating from silicon quantum dots under the exposure of UV light with 320 nm excitation.
added to each well of a 24-well plate and incubated for 1 day to allow the cells to stick on the surface of the plate. Aliquots of an aqueous solution of blue- and green-emitting Si QDs were added to the 24-well plate, and the cell solutions were incubated for another 2 days. A 50 μL amount of an MTT aqueous solution (0.5 mg mL−1) was added to each well of the 24-well plate 4 h before termination of the 3 day incubation, and the cells were allowed to incubate for another 4 h. Then, the upper layer of the solutions in the 24-well plate was discarded and 1 mL of DMSO was added to each well to dissolve the violet-color formazon product by pipette stirring. The final solution in each well was centrifuged at 13 000 rpm for 5 min to remove any solid residues before measurements of the optical absorbance at 570 nm. The optical absorbances were converted to cell viabilities based on a standard curve (absorbance versus cell numbers) obtained from controlled experiments carried out under the same condition, except that no Si QDs were added during the cell culture processes.
3. Results and discussion 3.1. Synthesis and characterization of blue- and green-emitting silicon QDs
2.6. Lactate dehydrogenase release (LDH) assay
The blue- and green-emitting Si QDs were synthesized by a modified literature method (see figure 1) [16]. The as-obtained Si QDs were subjected to either thermal oxidation or hydrogen peroxide–ethanol treatment to form a thin layer of SiOx on the surface of Si QDs. Further, Si QDs were mixed with 10% acrylic acid in an ethanol solution (1 mL) and toluene (4 mL) and then subjected to photoirradiation to undergo hydrosilylation under a UV lamp (254 nm) for 1 h. This resulted in a thin monolayer of PAA on the surface of silicon QDs and that
The cell seeding procedure and the pretreatment of greenemitting Si QDs to the monocytes were similar to those mentioned in the section 2.5. The cells were washed with PBS, trypsinized and centrifuged at 13 000 rpm for 5 min. 100 μL of the supernatant were transferred into another 96-well plate. To this, 100 μL of LDH reaction solution (Clontech Cytotoxicity Detection Kit, USA) was added and incubated for 30 min in the dark at room temperature. Before 4
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Figure 4. (a) Confocal laser scanning microscopic images of green Si QDs’ internalized monocytes. The nucleus was stained with DAPI dye (λex = 404 nm) and the green fluorescence of the Si QDs was obtained from the FITC channel (λex = 488 nm). The scale bars are 20 μm (upper panel) and 40 μm (lower panel) images. (b) Flow cytometry histograms for green-Si QDs’ internalized monocytes. The black line indicates the fluorescence intensity of control cells, and the filled red line indicates the fluorescence intensity of the green-Si QDs’ internalized cell population. (c) Mean fluorescence intensities obtained from green-Si QDs’ internalized monocytes using flow cytometry.
subsequently became water soluble. The structure of the asprepared green-emitting Si QDs was examined under transmission electron microscopy (TEM), which revealed an average particle size of 5 ∼ 10 nm (see figure 2(a)). The highmagnification TEM image in figure 2(b) clearly shows the clustered silicon particles are of average size ∼5 nm. The inset in figure 2(b) represents the high-resolution TEM image of silicon particles, which are crystalline in nature. The UV-visible–near-infrared (NIR) absorption spectra of both blue and green Si QDs reveal an absorption maxima at ∼230 nm (with an energy band-gap of ∼5.38 eV) and negligible absorption was shown at the visible and NIR regions (see figure 2(c)) [15]. The powder x-ray diffraction (PXRD) patterns of the atmospheric plasma–induced, as-prepared silicon powder exhibit three characteristic peaks at 28.5, 47.2 and 56.1 degrees, which represents the (111), (220) and (311) planes, respectively (see figure S1) [19]. The Fourier transform infrared spectroscopy (FTIR) spectra shown in figure S2 confirm the successful functionalization of acrylic acid moieties on Si QDs. The
Figure 5. Photostabilities of blue- and green-emitting silicon
quantum dots upon comparison with fluorescein isothiocyanate (FITC) dye under exposure of 100 W Hg lamps for 2 h. 5
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Figure 6. (a) MTT and (b) LDH assays for blue and green Si QDs’ internalized monocytes at 48 h incubation time. Apoptosis detection by Annexin-V assay in monocytes. (c) Dot plots between the fluorescence intensities of FITC versus PI. (d) Percentage of apoptotic and necrotic cells treated with various concentrations of blue- and green-emitting Si QDs by flow cytometry.
atmospheric plasma silicon nanoparticles and etched silicon nanoparticles, with and without surface grafting, exhibit the characteristic vibrational stretching of Si-O-Si at 1080 and 806 cm−1, respectively [20]. However, upon surface grafting, a weak vibrational stretching of the Si-CH2 characteristic peak was observed at 1460 cm−1 and also a strong peak at 2900 cm−1 which corresponds to the C-H stretching. In addition, a characteristic peak of C=O stretching at 1696 cm−1 from the acrylic moieties was observed. This confirms the successful surface grafting of acrylic moieties. The photoluminescence (PL) properties of Si QDs were evaluated in figure 3. As shown in figures 3(a) and (b), both blue- and green-emitting Si QDs exhibit excitation-wavelength-dependent emission. As the excitation wavelength of Si QDs increases, the emission maximum shifts to a longer wavelength [19]. The observation of the excitationwavelength-dependent photoluminescence emission was well documented in the literature, and the phenomenon is very similar to that of the carbon dots and graphene quantum dots [21, 22].
effective cellular uptake of nanomaterials. Without examining the cellular uptake efficiencies, there is no common ground to compare the cytotoxicities of different nanomaterials. To this end, we first evaluated the cellular uptake efficiencies of green Si QDs in monocytes (isolated from the C57BL/6J mouse blood) using CLSM and flow cytometry. The confocal images in figure 4(a) reveal that the green-emitting Si QDs were efficiently internalized by the monocytes in the cytoplasm along with the significant fraction being entered into the nucleus (see figure S3). The mean fluorescence intensities of green Si QDs’ internalized monocytes were also evaluated using flow cytometry (see figures 4(b) and (c)). It is believed that the uptake of nanoparticles into monocytes occurs via endocytosis. In the literature, it was reported that intracellular uptake of alkyl functionalized Si QDs was significantly faster in the malignant cells than in the normal cells. This indicates that the Si QDs can be effectively internalized via an endocytosis process and can act as very efficient in vitro imaging probes or cellular markers. Usually, the size, surface charge and surface functional group of nanomaterials play a key role in dictating cellular uptake and cytotoxicity. Conventionally, organic dyes were used to label the intracellular organelles. Most of the organic dyes suffer from severe photobleaching
3.2. In vitro cellular uptake and cytotoxicity assays of Si QDs
In order to evaluate the biocompatibility or to achieve good gene/drug delivery efficiencies, it is essential for cells to have 6
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Figure 7. Biocompatibility of blue- and green-emitting silicon QDs in zebrafish. (a) Survival rates were monitored after microinjection of
nanoparticles at different hours post fertilization (hpf). Abnormalities observed in zebrafish after microinjection of blue- and green-emitting Si QDs at 120 hpf. (b) No microinjection and zebrafish developed under normal development. (c) Blue Si QDs (i-ii) represents yolk-sac edema. (d) Blue Si QDs (i-ii) represents head edema. (e) Green Si QDs (i-ii) represents tail flexure and truncation.
problems. However, the Si QDs exhibit good photostabilities even after exposed to 100 W Hg lamps for 1 h, and no significant changes in the emission intensities were observed (see figure 5). The cell viabilities of blue- and green-emitting Si QDs examined using MTT assay are comparable and exhibit a concentration-dependent behaviour (see figure 6(a)). It is clearly evident that at higher concentrations, the cell viability percentage of both blue- and green-emitting Si QDs dropped to ∼69%, which could be attributed to the grafting of negatively charged PAA ligands on the surface of Si QDs. However, the cell viability values may slightly differ in the cancer cells, as the cytotoxicity behaviour is usually cell type dependent. LDH is a characteristic assay to investigate the cell membrane integrity. After exposure of blue- and greenemitting Si QDs to monocytes, the LDH levels of the cell culture medium were increased slightly when compared to that of the controls (see figure 6(b)). In addition to MTT and LDH assays, we determined the percentage of apoptotic and necrotic cells using Annexin-V dye by flow cytometry (see figure 6(c)). From figure 6(d), it is clearly evident that higher concentrations of Si QDs induce a slightly higher percentage (∼11%) of apoptotic and necrotic cells. The slight induction of apoptotic and necrotic cells by
the exposure of Si QDs could be due to the repulsive interactions of the PAA monolayer to the cell membrane, which might cause membrane disruption. In the literature, semiconducting QDs (such as CdSe or CdTe) were successfully demonstrated as in vitro cellular imaging probes without any photobleaching problems and good photostability [15]. But, the inherent limitation of semiconducting QDs is that the core nanomaterial (for example, Cd) itself can induce long-term cytotoxic effects. Therefore, in this regard, Si QDs can simply overcome all the drawbacks of conventional organic dyes and stand as robust, biocompatible and nontoxic cellular imaging probes.
3.3. In vivo biocompatibility studies and imaging of ailicon QDs in zebrafish
Zebrafish (Danio rerio) is an excellent animal model for in vivo imaging and biocompatibility evaluations of nanomaterials, due to its high transparency, faster embryonic development in 120 h, easy maintenance and similarity with mammals such as mice, rats and humans. These unique features can offer the direct detection of abnormalities or malformations. To study the biocompatibility of blue- and green-emitting Si QDs on the embryonic development of 7
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Figure 8. Confocal laser scanning optical microscopy images of zebrafish microinjected with (a) blue- and (b) green-emitting silicon quantum dots. The images were recorded at 10× magnification for 72 hpf. The blue (λex = 400 nm, λem = 410–440 nm) and green (λex = 488 nm, λem = 510–550 nm) fluorescence were originated from the Si QDs.
zebrafish, Si QDs were microinjected during the 1 ∼ 4 cell stage of the embryo development. From figure 7(a), it is clear that blue- and green-emitting Si QDs exhibit a concentrationdependent behavior on the survival rates of the zebrafish. Control experiments were also performed by microinjecting deionized water and a set of embryos without microinjection to compare the survival rate of zebrafish under identical experimental conditions. Yolk-sac edema, head edema and tail truncation were the most frequently observed abnormalities in zebrafish treated with blue and green Si QDs (see figures 7(b) ∼ (e)). The percentages of abnormalities induced by both blue- and green-emitting QDs are summarized in table S2 [28–30]. The abnormalities such as yolk-sac edema, head edema and tail truncation are presumably due to the up/
down regulation of certain genes, which are very similar to that observed in the zebrafish treated with silver nanoparticles (Ag NPs), iron oxide NPs, core/shell iron/carbon NPs (Fe@CNPs) and nanodiamonds (NDs) [23–26]. For in vivo applications, silicon can serve as an exciting alternative because it is nontoxic in its bulk form and can be readily degraded and excreted in the urine. It should also be noted that silicon is used as a trace nutrient or food additive [15]. The primary challenge with any in vivo luminescent probe is to prevent the enzymatic degradation in the physiological medium, achieve high photostability and possess low cytotoxicity. In this regard, organic dyes and semiconducting QDs were extensively studied as in vivo bioimaging probes. However, the inherent limitations of organic dyes include 8
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poor photostabilities prone to enzymatic degradation and exhibit long-term cytotoxicity. Figure 8 shows the CLSM images of zebrafish treated with 0.772 ppm of blue- and green-emitting Si QDs. The blue- and green-emitting Si QDs were microinjected into the embryos of zebrafish at the 4-cell stage and the images were recorded at 72 hpf. The blue and green fluorescence from Si QDs were mainly observed in the yolk-sac region. This could be attributed to the interactions of Si QDs with lipid-rich yolk cells during embryonic development [14]. To date, in the literature, efforts have been devoted to designing nanomaterials as cargoes to carry various molecular anticancer drugs for successful destruction of cancer cells. In order to fulfill the criterion of the theranostic reagent, it is mandatory to develop nanocarriers to impart diagnostic and therapeutic functions in a single nanomedicine platform. Ideally, the so-called theranostic nanocarrier itself has to be able to selectively target, diagnose and kill the cancer cells by leaving the normal cells healthy [31]. In the present study, the blue- and green-emitting Si QDs exhibit good biocompatibility and imaging capabilities both in the in vitro and in vivo models and are expected to act as a theranostic reagent [31]. Another major concern regarding the bioimaging property of blue-emitting Si QDs is their practical usage of low excitation wavelengths for activation. However, green-emitting Si QDs may overcome this problem to some extent. The in vivo bioimaging of green-emitting Si QDs are excitable using even longer wavelengths (488–550 nm), as they exhibit excitationwavelength-dependent fluorescence emission properties (see figure 3(b)). In addition, the problem with low excitation wavelengths can be overcome by using two photon excitation techniques, where the particles can be excited by the two photons of half the energy [27].
were proved successful in vitro and in vivo bioimaging probes. Upon comparison with the conventional semiconducting QDs, Si QDs are easy to produce in large quantities with uniform size; are cost effective; have tuneable fluorescence properties, no blinking problems and good photostability; and have superior in vitro and in vivo biocompatibilities. In the near future, it is expected that Si QDs can replace the conventional semiconducting QDs and organic dyes as a superior bioimaging probe and are also expected to be utilized as a theranostic reagent for personalized biomedicine.
Acknowledgments The authors are grateful to the financial support from National Chung-Shan Institute of Science & Technology, the Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University and the Ministry of Science and Technology, Taiwan.
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4. Conclusions We have developed a facile strategy to prepare water-soluble and highly fluorescent Si QDs using an atmospheric plasma process followed by thermal oxidation and H2O2-ethanol treatment. The as-synthesized luminescent Si QDs were subjected to UV light–initiated polymerization of an acrylic acid monomer to achieve surface grafting of PAA and thus water dispersibility. The photostability studies reveal that blue- and green-emitting Si QDs are highly photostable as compared to the organic dyes. Further, the toxicological studies have revealed that blue- and green-emitting Si QDs were considered to be biocompatible to monocytes. In vitro cellular imaging of green-emitting silicon QDs in monocytes exhibits excellent co-localization and distribution in the cytoplasmic region, along with the significant fraction being entered into the cell nucleus. The in vivo toxicity studies show that higher concentrations (⩾0.772 ppm) of Si QDs can induce very few abnormalities in the development of zebrafish and also slightly affect the survival rate. In vivo imaging of Si QDs in zebrafish revealed even distribution of blue and green fluorescence, especially in the yolk-sac region. Overall, water-dispersible and highly photostable luminescent Si QDs 9
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biocompatibility of single silver nanoparticles in early development of zebrafish embryos ACS Nano 1 133–43 Williams F E, Sickelbaugh T J and Hassoun E 2006 Modulation by ellagic acid of DCA-induced developmental toxicity in the zebrafish (Danio rerio) J. Biochem. Mol. Toxicol. 20 183–90 Hallare A V, Schirling M, Luckenbach T, Köhler H R and Triebskorn R 2005 Combined effects of temperature and cadmium on developmental parameters and biomarker responses in zebrafish (Danio rerio) embryos J. Therm. Biol. 30 7–17 Vankayala R, Kalluru P, Tsai H-H, Chiang C-S and Hwang K C 2014 Effects of surface functionality of carbon nanomaterials on short-term cytotoxicity and embryonic development in zebrafish J. Mater. Chem. B 2 1038–47 Tu C, Ma X, House A, Kauzlarich S M and Louie A Y 2011 PET imaging and biodistribution of silicon quantum dots in mice ACS Med. Chem. Lett. 2 285–8 Kouki F et al 2008 Luminescent passive-oxidized silicon quantum dots as biological staining labels and their cytotoxicity effects at high concentration Nanotechnology 19 415102 Erogbogbo F, Tien C-A, Chang C-W, Yong K-T, Law W-C, Ding H, Roy I, Swihart M T and Prasad P N 2011 Bioconjugation of luminescent silicon quantum dots for selective uptake by cancer cells Bioconjugate Chem. 22 1081–8 Cheng X, Lowe S B, Ciampi S, Magenau A, Gaus K, Reece P J and Gooding J J 2014 Versatile ‘click chemistry’ approach to functionalizing silicon quantum dots: applications toward fluorescent cellular imaging Langmuir 30 5209–16 Chinnathambi S, Chen S, Ganesan S and Hanagata N 2014 Silicon quantum dots for biological applications Adv. Healthc. Mater. 3 10–29
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Preparation, cytotoxicity and in vivo bioimaging of highly luminescent water-soluble silicon quantum dots
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Nanotechnology 26 215703 (http://iopscience.iop.org/0957-4484/26/21/215703) View the table of contents for this issue, or go to the journal homepage for more
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Nanotechnology Nanotechnology 26 (2015) 215703 (10pp)
doi:10.1088/0957-4484/26/21/215703
Preparation, cytotoxicity and in vivo bioimaging of highly luminescent watersoluble silicon quantum dots Jing-Wun Fan1, Raviraj Vankayala2, Chien-Liang Chang1, Chia-Hua Chang1, Chi-Shiun Chiang3 and Kuo Chu Hwang2 1
Chemical System Research Division, National Chung-Shan Institute of Science & Technology, PO Box 90008-17, Lung-Tan, Tao-Yuan 32599, Taiwan 2 Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan 3 Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu 30013, Taiwan E-mail: [email protected] Received 8 February 2015, revised 28 March 2015 Accepted for publication 2 April 2015 Published 6 May 2015 Abstract
Designing various inorganic nanomaterials that are cost effective, water soluble, optically photostable, highly fluorescent and biocompatible for bioimaging applications is a challenging task. Similar to semiconducting quantum dots (QDs), silicon QDs are another alternative and are highly fluorescent, but non-water soluble. Several surface modification strategies were adopted to make them water soluble. However, the photoluminescence of Si QDs was seriously quenched in the aqueous environment. In this report, highly luminescent, water-dispersible, blue- and green-emitting Si QDs were prepared with good photostability. In vitro studies in monocytes reveal that Si QDs exhibit good biocompatibility and excellent distribution throughout the cytoplasm region, along with the significant fraction translocated into the nucleus. The in vivo zebrafish studies also reveal that Si QDs can be evenly distributed in the yolk-sac region. Overall, our results demonstrate the applicability of water-soluble and highly fluorescent Si QDs as excellent in vitro and in vivo bioimaging probes. S Online supplementary data available from stacks.iop.org/NANO/26/215703/mmedia Keywords: silicon quantum dots, cytotoxicity, bioimaging, zebrafish, luminescence (Some figures may appear in colour only in the online journal) 1. Introduction
biochemical processes at the molecular level in living cells [8, 9]. Ideally, the fluorescent probes should be nontoxic, possess good photostability and resist enzymatic degradation in the physiologic medium [10]. To date, organic dyes were extensively used to label the intracellular organelles. However, they suffer from severe photobleaching upon continuous light exposure. Semiconducting quantum dots (QDs) are another alternative to organic dyes and attract significant attention because of their unique optical properties. They have been successfully demonstrated as in vitro and in vivo imaging probes in the field of nanobiomedicine [11]. Semiconducting QDs possess strong absorption, size-tunable
In recent years, extensive research efforts have been devoted to the utilization of inorganic nanomaterials for various biomedical applications, such as imaging, gene delivery and drug delivery [1–3]. Apart from the utility of nanoparticles as drug carriers for different treatments, they can also serve as multimodal imaging agents in clinics, which include magnetic resonance imaging (MRI), ultrasound (US), computed tomography (CT), photoacoustic (PA) and fluorescence imaging [4–7]. Among various imaging modalities, fluorescence imaging is widely adopted for investigation of various 0957-4484/15/215703+10$33.00
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© 2015 IOP Publishing Ltd Printed in the UK
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fluorescence emission, high quantum yields and good photostability [11]. However, a major concern that may limit their use in biology and medicine is the toxicity associated with the cadmium-containing QDs. Overall, the safety issues or concerns regarding the cytotoxicity of QDs severely hampered their use in bioimaging [12]. Some of the recent reports also suggest carbon/graphene QDs as an alternative to semiconductor QDs for bioimaging purposes. However, their structural formation, optical properties, and short-term and long-term cytotoxicity effects are still not well explored [1, 2]. Silicon is one of the most abundant materials, and it is widely used in the field of microelectronics. It is considered to be an indirect band-gap semiconductor material that is less interesting for light-emitting device applications in bulk form [13]. However, the nanosized silicon offers efficient fluorescence emission with excellent photoluminescence quantum yields due to the quantum confinement effect [14, 15]. Although Si QDs are expected to be far less toxic than semiconducting QDs, they have thus far not been widely used because of their limitation in making them highly luminescent, water dispersible, and compatible to the biological fluids and physiological medium. In general, most of the literature has reported that highly fluorescent Si QDs are non-water soluble. The photoluminescence of Si QDs was quenched in the aqueous medium. For example, it was reported that Si QDs attained water dispersibility by means of carboxylic acids or amine functional groups [16, 17]. It is often easier to functionalize nonpolar functional groups, such as styrene and octane, on the surface of Si QDs. However, this shows poor colloidal stability and weak photoluminescence in the aqueous environments. In another study, it was reported that Si QDs can be encapsulated into the nonpolar region of polyethylene glycol (PEG) grafted phospholipids to obtain water dispersibility [18]. However, the photoluminescence from Si QDs was quenched upon lipid encapsulation. Therefore, it is greatly challenging to prepare Si QDs that are highly fluorescent and also water soluble. A brief overview of the optical properties and bioimaging studies were summarized for various Si QDs reported in the literature (see table S1). To date, several reports have demonstrated the bioimaging applications of Si QDs, but their cellular fate in monocytes was never reported [14–16]. Moreover, the co-localization and biodistribution of Si QDs in the in vivo zebrafish animal model has not yet been investigated. Overall, it is necessary to develop a facile synthetic process to prepare highly fluorescent, water-soluble, biocompatible and optically stable colloidal Si QDs and make them useful for in vitro and in vivo biological labelling applications. In this study, we report on the preparation of watersoluble, highly fluorescent, optically photostable and biocompatible blue- and green-emitting Si QDs using an atmospheric plasma process. The etched silicon nanopowder was subjected to either thermal oxidation or hydrogen peroxide– ethanol treatment to produce superbright fluorescent Si QDs coated with a monolayer of SiOx. To achieve water dispersibility, silicon QDs were capped with a thin monolayer of polyacrylic acid (PAA) using ultraviolet (UV) light photo irradiation. After surface modification with PAA, the photoluminescence becomes very stable in the aqueous solution.
Figure 1. Schematic representation of the synthesis of blue- and
green-emitting Si QDs using the atmospheric plasma method, followed by either thermal oxidation or treatment with hydrogen peroxide and ethanol mixture.
Further, the cytotoxicity and uptake capabilities of blue- and green-emitting Si QDs were demonstrated in monocytes and also in the in vivo zebrafish system. To the best of our knowledge, this is the first report to demonstrate the direct visualization of the biodistribution of highly fluorescent Si QDs in the zebrafish animal model.
2. Materials and methods 2.1. Synthesis and surface modification of Si QDs
In the first step, plasma silicon nanopowder was prepared using an atmospheric plasma-triggered arcing process. In a typical experiment, 0.1% SiH4 precursor gas and carrier gas (argon) were injected into the atmospheric plasma reactor (60 W) containing an organic solvent in the quartz vessel. The two electrodes were connected to the power supply (60 W), and the atmospheric plasma was then triggered to obtain the precipitate of the reddish-brown solid silicon nanopowder. In the second step, etching of as-prepared silicon powder was carried out by a thermal oxidation process upon mixing 0.01 g of silicon nanopowder in 5 mL HF and ultrasonicated for 30 min so that the large particles could break apart. During the etching process, the solution color turned from reddish brown to pale yellow. Further, the solution was filtered through a 0.2 μm PVDF membrane and washed thoroughly with methanol–water (1:3) mixture to remove impurities. The asprepared nanoparticles were further mixed with 10% acrylic acid in ethanol solution (1 mL) and toluene (4 mL) and then subjected to photoirradiation to undergo hydrosilylation under a UV lamp (254 nm) for 1 h to achieve water dispersibility. 2.2. Cell culture, materials and reagents
The monocytes used in the experiments were isolated from C57BL/6J mouse blood. The mouse was sacrificed and the monocytes were collected and purified from the blood using a standard ficoll-paque density gradient protocol (Invitrogen, USA). Briefly, 10 mL of blood were collected in a centrifuge 2
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Figure 2. (a) and (b) represent the transmission electron microscopy (TEM) images of green-emitting silicon quantum dots. The inset in (b)
shows the high-resolution TEM image of a single nanoparticle. (c) UV-visible absorption spectra for blue- and green-emitting quantum dots.
bottle and diluted with 4 mL of buffer solution with gentle mixing. In a separate centrifuge bottle, 3 mL of ficoll-paque media (Invitrogen, USA) was withdrawn, followed by the addition of 4 mL of the diluted blood sample and the contents were gently mixed. The contents were then centrifuged for 30 min at 1500 rpm. The upper layer containing the plasma and platelets was drawn off, leaving the cell layer undisturbed at the interface. The monocytes were transferred into a separate centrifuge tube, followed by washing with a buffer 2 times. The collected monocytes were then plated in 10 cm dish in the presence of RPMI-1640 (Sigma, USA) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO, USA) and 1% penicillin-streptomycin (GIBCO, USA) at 5% CO2 atmosphere and 37 °C temperature.
trypsinized and suspended in the PBS buffer solution and then subjected to flow cytometry (FACS Calibur) analysis equipped with a 488 nm laser. The fluorescence intensities obtained from the fluorescein isothiocyanate (FITC) channel were quantified and analysed using Win MDI software. 2.4. In vitro bioimaging of Si QDs using confocal laser scanning microscopy (CLSM)
The cell seeding procedure and the pretreatment of greenemitting Si QDs to the monocytes were similar to those mentioned in the section 2.3. The green-emitting Si QDs internalized monocytes were washed again with PBS, further fixed onto a glass slide using p-formaldehyde solution (4%) in PBS for 5 min, and washed with PBST (5% Tween 20 in PBS) solution three times; then Triton X-100 solution (1 mL) was added. After exposure to Triton X-100 (1 h), the monocytes were stained with DAPI (4′,6-diamidino-2-phenylindole) (1 ng mL−1 PBS, 30 min). The samples were examined under a confocal laser scanning microscope (LSM 700) equipped with a UV laser (λex = 405 nm; λem = 420 ∼ 450 nm) and Ar laser (λex = 488 nm; λem = 510 ∼ 540 nm).
2.3. In vitro cellular uptake of Si QDs using flow cytometry
Two millilitres of monocyte-containing solution (2.0 × 105 cells mL−1) in the presence of RPMI-1640 were added to each well of a 6-well plate and incubated for 1 day to allow the cells to stick on the surface of the plate. Aliquots of aqueous solution containing green-emitting Si QDs were added to the 6-well plate, and the cell solutions were incubated for an additional 6 h. After 6 h, the cells were washed twice with phosphate buffer solution (PBS) (pH 7.4) and then incubated for another 20 h in the presence of fresh RPMI-1640. Then the cells were
2.5. In vitro cytotoxicity assay
One millilitre of monocyte-containing solution (2.0 × 104 cells mL−1) in the presence of the RPMI-1640 medium were 3
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colorimetric detection at 490 nm, 1 M HCl was added to stop the enzymatic reaction. 2.7. In vitro Annexin V apoptosis assay
Two millilitres of monocyte-containing solution (2.0 × 105 cells mL−1) in the presence of RPMI-1640 were added to each well of a 6-well plate and incubated for 1 day to allow the cells to stick on the surface of the plate. Aliquots of an aqueous solution of green-emitting Si QDs were added to the 6-well plate, and the cell solutions were incubated for an additional 6 h. The cells were then trypsinized, aspirated and suspended in 2 mL PBS. The cells were then stained with FITC-Annexin V (5 μL) and PI (5 μL) from the BD Annexin V apoptosis kit and then kept for 15 min at room temperature in darkness, followed by flow cytometry analysis. 2.8. In vivo cytotoxicity and bioimaging of silicon QDs in zebrafish
Wild-type AB strains of Danio rerio (zebrafish) embryos obtained from the Taiwan Zebrafish Core Facility Center, National Tsing Hua University, were used in all the experiments. Fresh embryos were collected to the microinjection embryo tray just before the experiment. Blue- and greenemitting Si QDs were diluted at appropriate concentrations in double distilled water and sonicated up until microinjection. An approximately 10 nL volume was microinjected into the animal pole region of the embryos between stages 1 (one-cell embryo) and 3 (four-cell embryo) using a Drummond microinjector. Each experiment was performed on 50 embryos per condition. Following microinjection, the embryos were transferred onto a petri dish filled with the system water and incubated at 28 °C in the dark. For the in vivo cytotoxicity measurements, the live embryos were counted each day until 72 hpf (hours post fertilization). After the 72 hpf, all the developed embryos were hatched manually and the types of abnormalities undergone were examined through a fluorescence microscope (Nikon, E600). For in vivo imaging experiments, the zebrafish embryos (72 hpf) were hatched and fixed on cover slips (15 × 16 mm, GeneFrame) with 1% agarose gel (Sigma, USA) to monitor the fluorescence of Si QDs using CLSM (10×, LSM-700).
Figure 3. Photoluminescence emission spectra for (a) blue- and (b)
green-emitting silicon quantum dots at different excitation wavelengths. The inset represents the characteristic blue and green emissions originating from silicon quantum dots under the exposure of UV light with 320 nm excitation.
added to each well of a 24-well plate and incubated for 1 day to allow the cells to stick on the surface of the plate. Aliquots of an aqueous solution of blue- and green-emitting Si QDs were added to the 24-well plate, and the cell solutions were incubated for another 2 days. A 50 μL amount of an MTT aqueous solution (0.5 mg mL−1) was added to each well of the 24-well plate 4 h before termination of the 3 day incubation, and the cells were allowed to incubate for another 4 h. Then, the upper layer of the solutions in the 24-well plate was discarded and 1 mL of DMSO was added to each well to dissolve the violet-color formazon product by pipette stirring. The final solution in each well was centrifuged at 13 000 rpm for 5 min to remove any solid residues before measurements of the optical absorbance at 570 nm. The optical absorbances were converted to cell viabilities based on a standard curve (absorbance versus cell numbers) obtained from controlled experiments carried out under the same condition, except that no Si QDs were added during the cell culture processes.
3. Results and discussion 3.1. Synthesis and characterization of blue- and green-emitting silicon QDs
2.6. Lactate dehydrogenase release (LDH) assay
The blue- and green-emitting Si QDs were synthesized by a modified literature method (see figure 1) [16]. The as-obtained Si QDs were subjected to either thermal oxidation or hydrogen peroxide–ethanol treatment to form a thin layer of SiOx on the surface of Si QDs. Further, Si QDs were mixed with 10% acrylic acid in an ethanol solution (1 mL) and toluene (4 mL) and then subjected to photoirradiation to undergo hydrosilylation under a UV lamp (254 nm) for 1 h. This resulted in a thin monolayer of PAA on the surface of silicon QDs and that
The cell seeding procedure and the pretreatment of greenemitting Si QDs to the monocytes were similar to those mentioned in the section 2.5. The cells were washed with PBS, trypsinized and centrifuged at 13 000 rpm for 5 min. 100 μL of the supernatant were transferred into another 96-well plate. To this, 100 μL of LDH reaction solution (Clontech Cytotoxicity Detection Kit, USA) was added and incubated for 30 min in the dark at room temperature. Before 4
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Figure 4. (a) Confocal laser scanning microscopic images of green Si QDs’ internalized monocytes. The nucleus was stained with DAPI dye (λex = 404 nm) and the green fluorescence of the Si QDs was obtained from the FITC channel (λex = 488 nm). The scale bars are 20 μm (upper panel) and 40 μm (lower panel) images. (b) Flow cytometry histograms for green-Si QDs’ internalized monocytes. The black line indicates the fluorescence intensity of control cells, and the filled red line indicates the fluorescence intensity of the green-Si QDs’ internalized cell population. (c) Mean fluorescence intensities obtained from green-Si QDs’ internalized monocytes using flow cytometry.
subsequently became water soluble. The structure of the asprepared green-emitting Si QDs was examined under transmission electron microscopy (TEM), which revealed an average particle size of 5 ∼ 10 nm (see figure 2(a)). The highmagnification TEM image in figure 2(b) clearly shows the clustered silicon particles are of average size ∼5 nm. The inset in figure 2(b) represents the high-resolution TEM image of silicon particles, which are crystalline in nature. The UV-visible–near-infrared (NIR) absorption spectra of both blue and green Si QDs reveal an absorption maxima at ∼230 nm (with an energy band-gap of ∼5.38 eV) and negligible absorption was shown at the visible and NIR regions (see figure 2(c)) [15]. The powder x-ray diffraction (PXRD) patterns of the atmospheric plasma–induced, as-prepared silicon powder exhibit three characteristic peaks at 28.5, 47.2 and 56.1 degrees, which represents the (111), (220) and (311) planes, respectively (see figure S1) [19]. The Fourier transform infrared spectroscopy (FTIR) spectra shown in figure S2 confirm the successful functionalization of acrylic acid moieties on Si QDs. The
Figure 5. Photostabilities of blue- and green-emitting silicon
quantum dots upon comparison with fluorescein isothiocyanate (FITC) dye under exposure of 100 W Hg lamps for 2 h. 5
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Figure 6. (a) MTT and (b) LDH assays for blue and green Si QDs’ internalized monocytes at 48 h incubation time. Apoptosis detection by Annexin-V assay in monocytes. (c) Dot plots between the fluorescence intensities of FITC versus PI. (d) Percentage of apoptotic and necrotic cells treated with various concentrations of blue- and green-emitting Si QDs by flow cytometry.
atmospheric plasma silicon nanoparticles and etched silicon nanoparticles, with and without surface grafting, exhibit the characteristic vibrational stretching of Si-O-Si at 1080 and 806 cm−1, respectively [20]. However, upon surface grafting, a weak vibrational stretching of the Si-CH2 characteristic peak was observed at 1460 cm−1 and also a strong peak at 2900 cm−1 which corresponds to the C-H stretching. In addition, a characteristic peak of C=O stretching at 1696 cm−1 from the acrylic moieties was observed. This confirms the successful surface grafting of acrylic moieties. The photoluminescence (PL) properties of Si QDs were evaluated in figure 3. As shown in figures 3(a) and (b), both blue- and green-emitting Si QDs exhibit excitation-wavelength-dependent emission. As the excitation wavelength of Si QDs increases, the emission maximum shifts to a longer wavelength [19]. The observation of the excitationwavelength-dependent photoluminescence emission was well documented in the literature, and the phenomenon is very similar to that of the carbon dots and graphene quantum dots [21, 22].
effective cellular uptake of nanomaterials. Without examining the cellular uptake efficiencies, there is no common ground to compare the cytotoxicities of different nanomaterials. To this end, we first evaluated the cellular uptake efficiencies of green Si QDs in monocytes (isolated from the C57BL/6J mouse blood) using CLSM and flow cytometry. The confocal images in figure 4(a) reveal that the green-emitting Si QDs were efficiently internalized by the monocytes in the cytoplasm along with the significant fraction being entered into the nucleus (see figure S3). The mean fluorescence intensities of green Si QDs’ internalized monocytes were also evaluated using flow cytometry (see figures 4(b) and (c)). It is believed that the uptake of nanoparticles into monocytes occurs via endocytosis. In the literature, it was reported that intracellular uptake of alkyl functionalized Si QDs was significantly faster in the malignant cells than in the normal cells. This indicates that the Si QDs can be effectively internalized via an endocytosis process and can act as very efficient in vitro imaging probes or cellular markers. Usually, the size, surface charge and surface functional group of nanomaterials play a key role in dictating cellular uptake and cytotoxicity. Conventionally, organic dyes were used to label the intracellular organelles. Most of the organic dyes suffer from severe photobleaching
3.2. In vitro cellular uptake and cytotoxicity assays of Si QDs
In order to evaluate the biocompatibility or to achieve good gene/drug delivery efficiencies, it is essential for cells to have 6
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Figure 7. Biocompatibility of blue- and green-emitting silicon QDs in zebrafish. (a) Survival rates were monitored after microinjection of
nanoparticles at different hours post fertilization (hpf). Abnormalities observed in zebrafish after microinjection of blue- and green-emitting Si QDs at 120 hpf. (b) No microinjection and zebrafish developed under normal development. (c) Blue Si QDs (i-ii) represents yolk-sac edema. (d) Blue Si QDs (i-ii) represents head edema. (e) Green Si QDs (i-ii) represents tail flexure and truncation.
problems. However, the Si QDs exhibit good photostabilities even after exposed to 100 W Hg lamps for 1 h, and no significant changes in the emission intensities were observed (see figure 5). The cell viabilities of blue- and green-emitting Si QDs examined using MTT assay are comparable and exhibit a concentration-dependent behaviour (see figure 6(a)). It is clearly evident that at higher concentrations, the cell viability percentage of both blue- and green-emitting Si QDs dropped to ∼69%, which could be attributed to the grafting of negatively charged PAA ligands on the surface of Si QDs. However, the cell viability values may slightly differ in the cancer cells, as the cytotoxicity behaviour is usually cell type dependent. LDH is a characteristic assay to investigate the cell membrane integrity. After exposure of blue- and greenemitting Si QDs to monocytes, the LDH levels of the cell culture medium were increased slightly when compared to that of the controls (see figure 6(b)). In addition to MTT and LDH assays, we determined the percentage of apoptotic and necrotic cells using Annexin-V dye by flow cytometry (see figure 6(c)). From figure 6(d), it is clearly evident that higher concentrations of Si QDs induce a slightly higher percentage (∼11%) of apoptotic and necrotic cells. The slight induction of apoptotic and necrotic cells by
the exposure of Si QDs could be due to the repulsive interactions of the PAA monolayer to the cell membrane, which might cause membrane disruption. In the literature, semiconducting QDs (such as CdSe or CdTe) were successfully demonstrated as in vitro cellular imaging probes without any photobleaching problems and good photostability [15]. But, the inherent limitation of semiconducting QDs is that the core nanomaterial (for example, Cd) itself can induce long-term cytotoxic effects. Therefore, in this regard, Si QDs can simply overcome all the drawbacks of conventional organic dyes and stand as robust, biocompatible and nontoxic cellular imaging probes.
3.3. In vivo biocompatibility studies and imaging of ailicon QDs in zebrafish
Zebrafish (Danio rerio) is an excellent animal model for in vivo imaging and biocompatibility evaluations of nanomaterials, due to its high transparency, faster embryonic development in 120 h, easy maintenance and similarity with mammals such as mice, rats and humans. These unique features can offer the direct detection of abnormalities or malformations. To study the biocompatibility of blue- and green-emitting Si QDs on the embryonic development of 7
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Figure 8. Confocal laser scanning optical microscopy images of zebrafish microinjected with (a) blue- and (b) green-emitting silicon quantum dots. The images were recorded at 10× magnification for 72 hpf. The blue (λex = 400 nm, λem = 410–440 nm) and green (λex = 488 nm, λem = 510–550 nm) fluorescence were originated from the Si QDs.
zebrafish, Si QDs were microinjected during the 1 ∼ 4 cell stage of the embryo development. From figure 7(a), it is clear that blue- and green-emitting Si QDs exhibit a concentrationdependent behavior on the survival rates of the zebrafish. Control experiments were also performed by microinjecting deionized water and a set of embryos without microinjection to compare the survival rate of zebrafish under identical experimental conditions. Yolk-sac edema, head edema and tail truncation were the most frequently observed abnormalities in zebrafish treated with blue and green Si QDs (see figures 7(b) ∼ (e)). The percentages of abnormalities induced by both blue- and green-emitting QDs are summarized in table S2 [28–30]. The abnormalities such as yolk-sac edema, head edema and tail truncation are presumably due to the up/
down regulation of certain genes, which are very similar to that observed in the zebrafish treated with silver nanoparticles (Ag NPs), iron oxide NPs, core/shell iron/carbon NPs (Fe@CNPs) and nanodiamonds (NDs) [23–26]. For in vivo applications, silicon can serve as an exciting alternative because it is nontoxic in its bulk form and can be readily degraded and excreted in the urine. It should also be noted that silicon is used as a trace nutrient or food additive [15]. The primary challenge with any in vivo luminescent probe is to prevent the enzymatic degradation in the physiological medium, achieve high photostability and possess low cytotoxicity. In this regard, organic dyes and semiconducting QDs were extensively studied as in vivo bioimaging probes. However, the inherent limitations of organic dyes include 8
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poor photostabilities prone to enzymatic degradation and exhibit long-term cytotoxicity. Figure 8 shows the CLSM images of zebrafish treated with 0.772 ppm of blue- and green-emitting Si QDs. The blue- and green-emitting Si QDs were microinjected into the embryos of zebrafish at the 4-cell stage and the images were recorded at 72 hpf. The blue and green fluorescence from Si QDs were mainly observed in the yolk-sac region. This could be attributed to the interactions of Si QDs with lipid-rich yolk cells during embryonic development [14]. To date, in the literature, efforts have been devoted to designing nanomaterials as cargoes to carry various molecular anticancer drugs for successful destruction of cancer cells. In order to fulfill the criterion of the theranostic reagent, it is mandatory to develop nanocarriers to impart diagnostic and therapeutic functions in a single nanomedicine platform. Ideally, the so-called theranostic nanocarrier itself has to be able to selectively target, diagnose and kill the cancer cells by leaving the normal cells healthy [31]. In the present study, the blue- and green-emitting Si QDs exhibit good biocompatibility and imaging capabilities both in the in vitro and in vivo models and are expected to act as a theranostic reagent [31]. Another major concern regarding the bioimaging property of blue-emitting Si QDs is their practical usage of low excitation wavelengths for activation. However, green-emitting Si QDs may overcome this problem to some extent. The in vivo bioimaging of green-emitting Si QDs are excitable using even longer wavelengths (488–550 nm), as they exhibit excitationwavelength-dependent fluorescence emission properties (see figure 3(b)). In addition, the problem with low excitation wavelengths can be overcome by using two photon excitation techniques, where the particles can be excited by the two photons of half the energy [27].
were proved successful in vitro and in vivo bioimaging probes. Upon comparison with the conventional semiconducting QDs, Si QDs are easy to produce in large quantities with uniform size; are cost effective; have tuneable fluorescence properties, no blinking problems and good photostability; and have superior in vitro and in vivo biocompatibilities. In the near future, it is expected that Si QDs can replace the conventional semiconducting QDs and organic dyes as a superior bioimaging probe and are also expected to be utilized as a theranostic reagent for personalized biomedicine.
Acknowledgments The authors are grateful to the financial support from National Chung-Shan Institute of Science & Technology, the Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University and the Ministry of Science and Technology, Taiwan.
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4. Conclusions We have developed a facile strategy to prepare water-soluble and highly fluorescent Si QDs using an atmospheric plasma process followed by thermal oxidation and H2O2-ethanol treatment. The as-synthesized luminescent Si QDs were subjected to UV light–initiated polymerization of an acrylic acid monomer to achieve surface grafting of PAA and thus water dispersibility. The photostability studies reveal that blue- and green-emitting Si QDs are highly photostable as compared to the organic dyes. Further, the toxicological studies have revealed that blue- and green-emitting Si QDs were considered to be biocompatible to monocytes. In vitro cellular imaging of green-emitting silicon QDs in monocytes exhibits excellent co-localization and distribution in the cytoplasmic region, along with the significant fraction being entered into the cell nucleus. The in vivo toxicity studies show that higher concentrations (⩾0.772 ppm) of Si QDs can induce very few abnormalities in the development of zebrafish and also slightly affect the survival rate. In vivo imaging of Si QDs in zebrafish revealed even distribution of blue and green fluorescence, especially in the yolk-sac region. Overall, water-dispersible and highly photostable luminescent Si QDs 9
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