Fabrication of quantum dot/silica core–shell particles immobilizing Au nanoparticles and their dual imaging functions
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The present work proposes preparation methods for quantum dot/silica (QD/SiO2) core–shell particles that immobilize Au nanoparticles (QD/SiO2/Au). A colloid solution of QD/SiO2 core–shell particles with an average size of 47.0 ± 6.1 nm was prepared by a sol–gel reaction of tetraethyl orthosilicate in the presence of the QDs with an average size of 10.3 ± 2.1 nm. A colloid solution of Au nanoparticles with an average size of 17.9 ± 1.3 nm was prepared by reducing Au3+ ions with sodium citrate in water at 80 °C. Introduction of amino groups to QD/SiO2 particle surfaces was performed using (3-aminopropyl)-triethoxysilane (QD/SiO2-NH2). The QD/SiO2/Au particles were fabricated by mixing the Au particle colloid solution and the QD/SiO2-NH2 particle colloid solution. Values of radiant efficiency and computed tomography for the QD/SiO2/Au particle colloid solution were 2.23 × 107 (p/s/cm2/sr)/(μW/cm2) at a QD concentration of 8 × 10−7 M and 1180 ± 314 Hounsfield units and an Au concentration of 5.4 × 10−2 M. The QD/SiO2/Au particle colloid solution was injected into a mouse chest wall. Fluorescence emitted from the colloid solution could be detected on the skin covering the chest wall. The colloid solution could also be X-ray-imaged in the chest wall. Consequently, the QD/SiO2/Au particle colloid solution was found to have dual functions, i.e., fluorescence emission and X-ray absorption in vivo, which makes the colloid solution suitable to function as a contrast agent for dual imaging processes.
KeywordsQuantum dot Au Nanoparticle Silica coating Core–shell Fluorescence imaging X-ray imaging
Nanoparticles of semiconductor compounds such as CdS, CdSe, and CdTe exhibit unique fluorescent properties and are called quantum dots (QDs). QDs have often been used as a contrast agent for in vivo fluorescence imaging (Ozawa et al. 2013; Sapsford et al. 2013; Geißler et al. 2014).
Because these QDs contain cadmium, they might harm living tissues (Yang et al. 2012; Tassali et al. 2014; Bereza-Malcolm et al. 2015). Coating the QDs with materials inert to living organisms forms core–shell particles with a QD core; the inert shell prevents contact with living tissues because of the physical barrier of the shell. Accordingly, forming such core–shell structures is a candidate method for reducing toxicity. Shell materials should be chemically inert in a wide variety of solvents and nontoxic to living organisms. Core–shell particles with QD nanoparticle cores must flow without aggregating in the blood vessels of an organism after injection because blood flow is weakened or stopped by such aggregation.
Silica is chemically inert and nontoxic relative to many solid materials (Hu et al. 2013; Young and Santra 2014; Zeng et al. 2014). Because silica particles form stable colloids in various dispersions (Mondragon et al. 2012; Bitter et al. 2013; Chou et al. 2014), silica-coated particles become highly dispersed, which provides steady particle flow in blood vessels. Accordingly, silica is a promising shell material. Silica particles can be easily fabricated via the Stöber method (Bell et al. 2012; Goertz et al. 2012; Neville et al. 2012; Börner et al. 2013). Several researchers have extended the Stöber method to produce QDs coated with silica, or QD/SiO2 core–shell particles (Wang et al. 2012; Ma et al. 2014; Aubert et al. 2014). Their method uses a sol–gel reaction with silicone alkoxide and base catalyst in the presence of the QDs. Our research group has proposed an alternative method for producing QD/SiO2 particles (Kobayashi et al. 2010a, b, 2012a, 2013a, 2015) and performed fluorescence imaging of mice tissues, into which the colloid nanoparticle solutions were injected (Kobayashi et al. 2013a, 2015).
Materials with high X-ray absorption properties can be applied for X-ray imaging techniques. In imaging, iodinated contrast agents are usually used to obtain clear images. Typical X-ray contrast agents that are commercially available are iodine compounds dissolved homogeneously in solvents at the molecular level. However, iodine compound X-ray contrast agents face a problem; they may provoke adverse events such as allergic reactions in patients (Lusic and Grinstaff 2013; Scoditti et al. 2013; Wendel et al. 2014), preventing their administration to such people. In addition to iodine compounds, Au is also promising for imaging because Au also highly absorbs X-rays and is less toxic relative to the iodine compounds. From this viewpoint, Au nanoparticles have been examined with respect to their use as contrast agents for imaging tissues in living bodies at the nanometer level (Lusic and Grinstaff 2013; Cole et al. 2014; Betzer et al. 2014).
Materials composed of components that have different properties should have multiple functions. Considering the multi-functionalization of materials, particles containing QDs and Au will act as both a fluorescent contrast agent and an X-ray contrast agent. The present work proposes a method for fabricating composite particles composed of QD/SiO2 core–shell particles and Au nanoparticles, or QD/SiO2 core–shell particles on which Au nanoparticles are immobilized (QD/SiO2/Au). QD/SiO2 core–shell particles were fabricated according to the method outlined in our previous works (Kobayashi et al. 2010a, b, 2012a, 2013a, 2015), and amino groups were introduced onto their surface using silicone alkoxide with a terminal amino group. Au nanoparticles were prepared with a conventional method using citrate as a reducing reagent. The QD/SiO2/Au particles were fabricated by simply mixing the QD/SiO2 core–shell particle colloid solution and the Au nanoparticle colloid solution. Imaging abilities based on both fluorescence and X-ray absorption of the composite particle colloid solution were also studied in the present work.
The QD nanoparticles were silica-coated via the sol–gel method using TEOS, similarly to the method outlined in our previous works (Kobayashi et al. 2010a, b, 2012a, 2013a, 2015). Solutions of 38.0 mL of 18.4/81.6 (v/v) water/ethanol and 11.8 mL of 4.24 × 10−3 M TEOS/ethanol were added to 0.004 mL of the 8 × 10−6 M QD colloid solution in turn. The sol–gel reaction was then initiated by rapidly injecting 0.2 mL of 0.1 M aqueous NaOH into 49.8 mL of the QD/TEOS colloid solution, which resulted in the (0.1 M aqueous NaOH)/(QD/TEOS colloid solution) volume ratio of 0.402 %. The above-mentioned amounts of solutions gave the initial concentrations of 6.4 × 10−10 M QD, 1.0 × 10−3 M TEOS, 8 M H2O, and 4.0 × 10−4 M NaOH. The reaction proceeded for 24 h at room temperature. Our previous work indicated no serious effect of the silica coating on emission wavelength and fluorescence intensity of QDs (Kobayashi et al. 2010b). The QD/SiO2 colloid solution prepared in the present work was also considered to have the same fluorescence properties as the QD colloid solution. The QD/SiO2 colloid solution was concentrated to a QD concentration of 3.2 × 10−9 M by evaporating the solvent, centrifuging, removing the supernatant, adding water, and redispersing the concentrated QD/SiO2 colloid solution.
A freshly prepared 0.118 mL of 0.339 M Na-cit aqueous solution was added to 24.9 mL of 2.41 × 10−4 M HAuCl4 aqueous solution at a constant temperature of 80 °C under vigorous stirring, which resulted in initial concentrations of 2.4 × 10−4 M Au and 1.6 × 10−3 M Na-cit.
For efficient immobilization of Au nanoparticles on the QD/SiO2 particle surface, or production of QD/SiO2/Au particles, amino groups were first introduced on the QD/SiO2 particle surface using APES (QD/SiO2-NH2), because the alkoxide groups of the APES were expected to react with the OH groups on the silica surface of the QD/SiO2 particles. APES (0.035 mL) was added to 25 mL of the concentrated QD/SiO2 colloid solution with the QD concentration of 3.2 × 10−9 M at room temperature, which resulted in an initial APES concentration of 6 × 10−3 M. In a preliminary experiment, an iso-electric point of the QD/SiO2-NH2 particles shifted to high pH with the introduction of amino groups, and the shift was the largest at the initial APES concentration of 6 × 10−3 M in an initial APES concentration range of 6 × 10−5 to 6 × 10−2 M, which indicated that efficient introduction of amino groups was performed at 6 × 10−3 M. Thus, the initial APES concentration was adjusted to 6 × 10−3 M in the present work. The reaction time was 24 h. The QD/SiO2-NH2 colloid solution was concentrated to a QD concentration of 1.28 × 10−7 M by centrifuging, removing the supernatant, adding ethanol, and redispersing the concentrated QD/SiO2-NH2 colloid solution. For the Au nanoparticles’ immobilization, ethanol, water, and the Au nanoparticle colloid solution were added in turn to the QD/SiO2-NH2 particle colloid solution at 35 °C, as the amino groups on the particle surface were expected to coordinate with the surface of Au nanoparticles. The initial concentrations of QD, Au, and H2O were 7.4 × 10−10, 5 × 10−5, and 12.4 M, respectively. The QD/SiO2/Au colloid solution was concentrated by evaporating the solvent, centrifuging, removing the supernatant, adding water, and redispersing the colloid solution, which produced a QD/SiO2/Au colloid solution with a QD concentration of 8.0 × 10−7 M and an Au concentration of 5.4 × 10−2 M
The samples were characterized by ultra-visible (UV–VIS) spectroscopy, electrophoretic light scattering (ELS), and TEM. VIS extinction of the particle colloid solution was measured with a Shimadzu UV-3101PC (Kyoto, Japan) spectrophotometer. ζ-potentials of the particles were measured by ELS to obtain information on the state of the particles. The ELS was performed with a Malvern Zetasizer Nano ZS90 (Worcestershire, UK). Either an HCl aqueous solution or an NaOH aqueous solution was added to the solution to vary the pH of the solution for the ELS measurement. The TEM imaging was performed using a JEOL JEM-2100 microscope operating at 200 kV. The TEM samples were prepared by dropping the nanoparticle suspensions onto a collodion-coated copper grid and evaporating them. Tens of particle diameters were measured using the TEM images to determine the volume-averaged particle size and its standard deviation.
Fluorescence imaging and X-ray imaging with the particle colloid solutions were performed using a Xenogen IVIS 100 fluorometer in vivo imaging system (IVIS) and an Aloka La theta LCT-200 CT system, Japan), respectively, which were also used in our previous works (Ayame et al. 2011; Kobayashi et al. 2011, 2012b, 2013a, b, c, d, 2014, 2015). CT values were estimated on the basis of CT values of −1000 and 0 for air and water, respectively. For mouse imaging, the colloid solution (50 μL) was injected into the chest wall of an anesthetized mouse. Fluorescence emitted from inside the colloid-injected mouse was detected with the IVIS. X-ray images of the colloid-injected mouse were taken by transmitting X-rays through the mouse with the CT system. The mice used were ICR mice, aged 5–6 weeks.
Results and discussion
The QD/SiO2-NH2 nanoparticles dispersed well in water, or they were colloidally stable, which indicated no serious effect of the APES addition on their colloidal stability in water. Figure 1c shows a TEM micrograph of the QD/SiO2-NH2 nanoparticles. Some particles appeared to form aggregates composed of several particles that were likely to precipitate. However, no precipitates were found in the particle colloid solution. Accordingly, the aggregates were thought to be produced during the preparation of TEM samples accompanied by the evaporation of solvent on the TEM grid. Similarly to the QD/SiO2 nanoparticles, particles containing a few QD cores were observed. Their average particle size was 42.4 ± 4.3 nm, which was considered to be almost the same as that of the QD/SiO2 nanoparticles within the statistical error. This similarity indicated that their core–shell structure was chemically stable, even after the amination process. Figure 2b shows the ζ-potential of the QD/SiO2-NH2 particles as a function of pH. Similarly to the case of QD/SiO2 nanoparticles, the ζ-potential decreased and passed through an IEP with an increase in pH. The IEP was 9.5, which was higher than that of the QD/SiO2 nanoparticles. This higher IEP was within a range of 9–11, which is typical of acid dissociation constants for amino groups in many types of amines, such as ammonia, alkylamine, and dialkylamine. Accordingly, this result confirmed that the amino groups were successfully introduced onto the particle surface.
Figure 1d shows a TEM micrograph of the QD/SiO2/Au nanoparticles. All the Au nanoparticles were immobilized on the particle surface, indicating that the Au nanoparticles’ immobilization was successfully performed with the present method. Several Au nanoparticles-free particles were also observed, since a number-ratio of Au nanoparticles/QD/SiO2-NH2 nanoparticles was small compared to 1/1. Accordingly, optimization of fabrication conditions such as concentrations of Au nanoparticles and QD/SiO2-NH2 nanoparticles in the final colloid solution, reaction temperature and stirring rate is required to improve the efficiency of Au nanoparticles’ immobilization. This improvement may also improve dual imaging ability of the QD/SiO2/Au particle colloid solution.
Imaging of the QD/SiO2/Au particle colloid solution
Figure 5c shows an IVIS image of a mouse after the injection of the QD/SiO2/Au particle colloid solution. Fluorescence was clearly observed on the chest of the mouse after injection into its chest wall, which meant that fluorescence penetrated through the chest skin from inside the mouse to outside. Its radiant efficiency was 3.30 × 107 (p/s/cm2/sr)/(μW/cm2). This value was ca. 1.5 times larger than the value of 2.23 × 107 (p/s/cm2/sr)/(μW/cm2) for the QD/SiO2/Au particle colloid solution, which indicated that the QD/SiO2/Au particle colloid solution could emit strong fluorescence even inside living bodies without quenching. The detection of large radiant efficiency implied that the QD/SiO2/Au particles were accumulated in the chest wall, though the reason for the large radiant efficiency is still unclear.
Figure 5c shows an X-ray image of the mouse after it was injected with the QD/SiO2/Au particle colloid solution. The location of the particle colloid solution could be recognized clearly at its chest wall because of its light contrast. Its CT value was 1060 ± 374 HU, which was as high as the value of 1180 ± 314 HU for the QD/SiO2/Au particle colloid solution. Similarly to the fluorescence imaging, it was found that the QD/SiO2/Au particle colloid solution could be clearly observed even inside living bodies without quenching.
Another result of note is that when the mouse that was injected with the QD/SiO2/Au particle colloid solution was imaged simultaneously with the IVIS and X-ray, the position imaged by the IVIS was the same as that from X-ray imaging. This result suggests that the colloid solution can function as a contrast agent for dual imaging processes.
QD/SiO2 particles averaging 47.0 ± 6.1 nm in size were produced via the sol–gel method using TEOS and NaOH in a water/ethanol solution containing QDs. The QD/SiO2 particles were aminated by reacting silanol groups on the QD/SiO2 particle surface and alkoxide groups in APES. Au nanoparticles averaging 17.9 ± 1.3 nm in size were produced by reducing HAuCl4 with Na-cit in water. The Au nanoparticles were immobilized on particles via the reaction of amino groups on the QD/SiO2 particle surface and the Au particle surface. A QD/SiO2/Au particle colloid solution with a QD concentration of 8 × 10−7 M and an Au concentration of 5.4 × 10−2 M had a radiant efficiency of 2.23 × 107 (p/s/cm2/sr)/(μW/cm2) and a CT value of 1180 ± 314 HU. After injection of the QD/SiO2/Au particle colloid solution into the chest wall of a mouse, the colloid solution could be detected simultaneously with IVIS and CT on the skin covering the chest wall and in the chest wall, respectively. Accordingly, the QD/SiO2/Au particle colloid solution has the potential to be used as a contrast agent with dual functions for techniques such as fluorescence emission and X-ray absorption in vivo.
This work was supported by JSPS KAKENHI Grant Number 24310085, and by a Grant-in-Aid for Scientific Research on Innovative Areas “Nanomedicine Molecular Science” (No. 2306) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. We express our thanks to Prof. T. Noguchi at the College of Science of Ibaraki University, Japan (current affiliation: Faculty of Arts and Science of Kyusyu University, Japan) for his help with the TEM observations.
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