Research Article

Nanocomposites of Silicon Nanocrystals and Magnetic Nanoparticles for Biomedical Imaging

Dr. Doan Thi Kim Dung,
Doan Thi Kim Dung*
Ho Chi Minh City Institute of Physics, Ho Chi Minh City, Vietnam
*Corresponding author:

Doan Thi Kim Dung, Ho Chi Minh City Institute of Physics, 1 Mac Dinh Chi, 1st District, Ho Chi Minh City, Vietnam.
Email: dkdung@hcmip.vast.vn

Keywords:

Silicon quantum dots, Magnetic nanoparticles, Magnetic resonance imaging

Semiconductor Quantum Dots (QDs) are of great interest by researcher in bioimaging because they have many advantages over conventional organic fluorescent dyes. However, a cadmium component of casual QDs has negative effect on cellular activity and biological environment. Silicon QDs are recommended as a alternative. Si QDs (size 2 ± 1.25 nm) emit blue light have been used as a biological probe for cell imaging. In order to combine with other imaging technique such as Magnetic Resonance Imaging (MRI), a bimodal imaging probe composed of silicon QDs and magnetic nanoparticles (Fe3O4) is synthesized which can emit blue violet light and enhance negative contrast in magnetic resonance imaging. The silicon QDs are synthesized via microemulsion, while superparamagnetic Fe3O4 nanoparticles are obtained by co-precipitation method. A coating is conducted by using SiO2. The characterization of composites and the bimodal properties are studied. Cellular imaging is also conducted.

Biomedical imaging including magnetic, radiation, or optical approaches are important techniques used in prognosis and treatment of the disease. However, while these techniques utilize a characteristic of “energy-matter” interaction to provide details of biological distinctions or processes, each technique pertains to its own merits and demerits in terms of spatial and temporal resolution, sensitivity, anatomical and molecular details. The combination of different imaging techniques, multimodal imaging, is believed that it can exploit the advantages and compensate the disadvantages of each technique to offer a comprehensive image of biological specimens. Multimodalities of imaging can be obtained by construction of a hardware platform of different imaging techniques (two or more techniques) as well as design favorable multimodal probes which can be used by all techniques.

MRI is a non-ionizing technique which can discriminate tiny change in soft tissues as well as anatomical reconstruction. However, one of significant disadvantage of MRI is its poor sensitivity in contrast to the sensitive ability of fluorescent imaging. The combination between fluorescent and MRI are preferable because MRI can provide an image of whole body, while the fluorescent image has higher sensitivity and spatial resolution than MRI which can elucidate the details at cellular scale. The potential of an opto-magnetic imaging can be done by utilizing fluorescent-magnetic hybrid nanostructures.

Synthesis of a multimodal bioimaging probe, which has high efficient emissions and non-toxicity, is emerging as a versatile tool for actualizing multimodal imaging in diagnosis and treatment. Among the fluorescent probes, semiconductor nanocrystals such as Quantum Dots (QDs) are of great interest because they have numerous characteristics promising for applications in biomedical imaging including wide excitation wavelengths, sharp emissions, and high quantum efficiency [1–6]. However, these nanocrystals are hindered in biomedical applications because they composed of extremely toxic components such as Cd3+ ions [7–9]. These toxic ions leaked into the surrounding, and damaged the biological environment when the oxidation of the surface of nanocrystals occurs.

 Silicon (Si) nanocrystal or Si QD is a potential alternative for QDs because of its safety when using in the body which has been proved through the numerous applications of Si in medical. Moreover, Si is cheap, non-toxic and environmentally friendly. Si QDs also show biocompatibility comparing to traditional semiconductor nanocrystals made of Cd3+ ions [10]. Therefore, Si QDs show advantages over other QD materials. Moreover, silicon is a semiconductor material commonly used in opto-electrics. At the bulk size, Si particles show no luminescence due to its indirect band-gap. As the size of crystals decrease to smaller than Bohr radius, the optical band gap of Si nanocrystals can be tuned by size control based on the quantum confinement effect in various QDs materials [10–14] resulting in the emission of fluorescence.

With respect to MRI, there are few materials which shown the contrast enhancement effect on negative and positive relaxation time T1 and T2. In brief, T1 agents enhance the brightness of the contrast, while T2 agents improve the darkness of the MRI images. Among T2 agents, magnetic nanoparticles from Fe3O4 iron oxides are one of the most popular one with advantages in biocompatibility and super-paramagnetic behavior, which are capable to use as a T2 contrast enhancer for MRI [15,16]. They are also can be used in drug delivery, hyperthermia or magnetic targeting, cell labeling and sorting applications [17–19], etc.

In this work, a bimodal imaging probe (Fe3O4+Si-QDs)@SiO2 is developed from the encapsulation of Si QDs as a fluorescent probe and magnetic nanoparticles (Fe3O4) as a contrast agent for MRI. It is worth to emphasize that the combination between Si QDs and Fe3O4 nanoparticles have not been conducted so far.

Materials

Iron(II) chloride tetrahydrate FeCl2·4H2O ≥99%, iron(III) chloride hexahydrate FeCl3·6H2O ≥99.0%, ammonium hydroxide solution NH4OH (28% NH3 in H2O), silicon tetra chloride SiCl4 99%, tetraoctylammonium bromide (TOAB), toluene, lithium aluminum hydride LiBH4 in tetrahydrofuran (THF) 99%, chloroplatinic acid solution H2PtCl6, anhydrous isopropanol, hexadiene, N, N-dimethylformamide, Tetraethylorthosilicate (TEOS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All of the chemicals were used without any further purification.

Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Sigma-Aldrich. A 10% (vol/vol) fetal bovine serum from Nichirei Bioscience Inc. (Tokyo, Japan), 1% (vol/vol) penicillin streptomycin from Wako Ltd., and 25 µg/mL amphotericin B from Sigma-Aldrich were added to the DMEM. Phosphate buffered saline (PBS (−)) and 4% paraformaldehyde were purchased from Wako Ltd.


Synthesis methods

Synthesis of magnetic nanoparticles Fe3O4 is conducted via co-precipitation method. A mixture of FeCl2·4H2O and FeCl3·6H2O were dissolved in 50 mL of distilled water as the ratio of Fe2+: Fe3+=1: 2. 100 mL of distilled water in a three-necked flask was heated to 70°C and agitated by an ultrasonic irradiator while bubbling by N2 gas. The iron solution and NH4OH solution were dropped simultaneously into the flask in about 60 min. The pH was kept at 8. The chemical reaction of Fe3O4 precipitation is expected as follows:

FeCl2 + 2 FeCl3 + 8NH4OH ® Fe3O4 + 8NH4Cl                  (1)

The precipitated black Fe3O4 particles were collected by permanent magnet and cleaned several times by re-deionized water.

Silicon nanocrystals were then prepared by reduction of SiCl4 (Sigma-Aldrich, ≥99.0%). At first, 96 mL anhydrous toluene and 1.5 g Tetraoctylammonium Bromide (TOAB) were poured in a three-necked flask. The flask was purged with nitrogen. The solution was stirred in 1 h. Then, 0.5 g SiCl4 dissolved in 2 mL toluene and 2 mL of reducing agent including of 2 M LiBH4 in Tetrahydrofuran (THF) were simultaneously dropped in to reaction flask and keep stirring within 3 h. A sufficient amount of methanol was then added into the flask and stirred in 20 min to terminate the reaction. A total of 100 µL of H2PtCl6 (Sigma-Aldrich, 99.99%) in anhydrous isopropanol (Sigma-Aldrich, 99.5%) as catalyst and 4 mL allylamine were added into the reactor to modify the surface of nanocrystal. To remove remain solvent, the product continue to wash several times by N, N-dimethylformamide. The product was dissolved in hexane and evaporated in vacuum at 50°C, repeat 2 times.

(Fe3O4+Si-QDs)@SiO2 was prepared as following: 1 mL aqueous solution of magnetic particles and 1 mL silicon nanocrystals were shaken with 7.5 mL of anhydrous ethanol. 50 µL of tetraethylorthosilicate (TEOS, Sigma-Aldrich) and 200 mL of ammonia solution (Sigma-Aldrich) were added to the reaction solution while shaking at 300 rpm in 24 h at room temperature. The reaction was terminated by adding methanol. The (Fe3O4+Si-QDs)@SiO2 nanoparticles were collected by centrifugation at 12,000 rpm for 5 min and washed with distilled water several times to remove excess reactants.

Silicon nanocrystals were synthesized by micro-emulsion method. Silicon precursor from silicon tetrachloride was reduced by strong hydride reducing agents. Hydrogen terminated silicon nanoparticles were then capped with organic molecules by using a Pt-based catalyst. Superparamagnetic iron oxide nanocrystals were obtained via a co-precipitation route. The fluorescent-magnetic encapsulate was obtained by using SiO2 capping agent.

Figure 1. Morphology of Si QDs (a) and magnetic nanoparticles (b); crystalline structure in magnetic nanoparticles (c and d) morphology of composite of Si QDs, and magnetic nanoparticles. Size distribution of Si QDs (e) and magnetic nanoparticles (f)

Cellular preparation: In order to prepare the cellular samples, HeLa cells were cultured on two glass-base dishes in DMEM with 10% fetal bovine serum and 1% penicillin streptomycin at 37°C and incubated in 5% CO2. Bio-modal nanoparticles were sterilized and re-dispersed in DMEM. The nanoparticles medium was transferred into a cell dish and keep for 24 h in incubator. The cell uptake of nanoparticles is enabled via the endocytosis pathway. Another HeLa cell dish without particles was used as the control. Both samples were washed several times with PBS (−) and then fixed by 4% paraformaldehyde for 20 min.

Si nanocrystals were synthesized by micro-emulsion method. Si precursor from Si tetrachloride was reduced by strong hydride reducing agents. Hydrogen terminated Si nanoparticles were then capped with organic molecules by using a Pt-based catalyst. Superparamagnetic iron oxide nanocrystals were obtained via co-precipitation. The fluorescent-magnetic encapsulate was obtained by using SiO2 capping agent.

The Si QDs and magnetic nanoparticles are synthesized separately and the morphology is shown in Figure 1. Figure 1a shows the morphology of Si QDs and Figure 1b shows the morphology of Fe3O4 nanoparticles by TEM on carbon grids. Figure 1c shows the enlarge view of bare Fe3O4 particles and the crystal lattice pattern can be clearly seen. Two kinds of nanocrystals are encapsulated by using a coating made from SiO2. According to the contrast difference in TEM images in Figure 1d, image of a single encapsulation of Si nanocrystals and Fe3O4 nanoparticles presenting for dark core and grey SiO2 shell. The mean diameter of Si nanocrystals was 2 ± 1.25 nm and iron oxide was 18 ± 2.50 nm as in Figure 1e and 1f, respectively. The average diameter of the encapsulated particle is estimated about 100 nm.

Figure 2. Emission spectrum of Si QDs under 325 and 350 nm excitation (a); bright-field image of HeLa cells (b); fluorescence from bi-modal probe (c); and overlap image (d)

After the nanocomposite obtained, the bi-modal properties are following investigated. The photoluminescence spectrum of the composite (Fe3O4+Si-QDs)@SiO2 is shown in Figure 2a. The photoluminescence of sample was measured with excitation wavelengths from 325 and 350 nm. The emission peaks appear around 368 and 400 nm as the excitation wavelengths are 325 and 350 nm, respectively. As the excitation changes, a shift of emission peaks are clearly observed. The potential of Si-magnetic nanocrystals as a chromophore for biological imaging is demonstrated in Figure 2b-2d. The fluorescence from the bio-modal nanoparticles uptake in HeLa cells was obtained with fluorescent microscopy and overlap image of fluorescence in the cells is shown in Figure 2d. For imaging, an excitation wavelength of 365 nm was used. The emission at 420 and 480 nm was monitored. The fluorescence can be observed, which corresponds to the silicon quantum dots. These findings indicated the high possibility of (Fe3O4+Si-QDs) as a fluorescent imaging probe.

The saturation magnetization values of the core magnetic nanoparticles and the bimodal encapsulation from magnetic and Si nanocrystals are 70 and 40 emu/g, respectively (Figure 3a). The magnetization curves of both samples represent superparamagnetic properties as the forward and backward curves overlap each other. The loss of magnetization in bimodal encapsulated nanoparticles may be due to the presence of the coating surrounding the magnetic cores.

Figure 3. Hysteresis loop of bare magnetic nanoparticles and encapsulated nanocomposites (a); T2-weight image (upper) and relaxivity of bi-modal nanocomposites (below) versus iron concentration (b)

The relaxivity of encapsulated nanocrystals was measured at 1.5 T. The upper part of Figure 3b shows sensitive and concentration-dependent dark-blue areas in MR images obtained from the aqueous medium with magnetic-fluorescent encapsulated nanocrystals. The significant reduction of brightness even at low iron concentrations implies that the magnetic-fluorescent encapsulated nanocrystals induce negative contrast enhancement in MRI. The effect of iron on negative contrast in MRI is evaluated in terms of the negative or transverse relaxivity (r2), which represents the efficiency of the magnetic moments of nanocrystals to enhance the relaxation time T2. The relaxation rates (1/T2) of magnetic-fluorescent encapsulated nanocrystals vary linearly with iron concentrations. The transverse relaxivity (r2) is calculated from the slope of the graph is 42.942 mM-1s-1. These results imply a high possibility of (Fe3O4+Si-QDs) for using as a MRI imaging probe.

In this work, we demonstrated a simple and robust method for synthesis of bimodal opto-magnetic nanocomposites by the encapsulation of Si QDs and iron oxide nanoparticles with SiO2 coating. The particles emit luminescence within HeLa cells under 365 nm excitation where they acted as a fluorophore. The nanocomposites also display a MRI contrast enhancement effect under external magnetic field. Superparamagnetic properties and MRI enhancement were confirmed and the r2 relaxivity of the nanocomposites is 42.942 mM-1s-1.

Because Si QDs have size-dependent emissions, it is possible to prepare Si QDs with multicolor emission. Besides, Si is safety as using in biological system comparing with other QDs, the applications of Si QDs in in vivo imaging is applicable. Recently, the trend of using Near Infrared (NIR) fluorescent imaging probes for in vivo imaging is attracted because the deep penetration of the NIR light in the body. The use of Si QDs is still applicable as tuning their emission wavelengths. The combination of MRI and fluorescent imaging in the body can be done by utilizing the (Fe3O4+Si-QDs)@SiO2 nanocomposites.

This research was supported by the Research Grants for Independent Young Researchers of Vietnamese Academy of Science and Technology (VAST), Hanoi, Vietnam.

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Citation: Dung DTK (2017) Nanocomposites of Silicon Nanocrystals and Magnetic Nanoparticles for Biomedical Imaging. Int J Nanotechnol Nanomed Res 1:008

Published: 26 September 2017

Reviewed By : Dr. Filiz Keles,

Copyright:

Copyright: © 2017 Dung. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.