Fabrication of mono-dispersed silica-coated quantum dot-assembled magnetic nanoparticles

Abstract

Multifunctional nanoparticles (NPs) with magnetic and luminescent properties have garnered considerable attention in various fields of biomedical and physiological applications. In this study, we report the fabrication of QD-embedded silica NPs with an iron oxide NP core (Fe₃O₄@SiO₂@QDs NPs) that has dual functional properties. The Fe₃O₄@SiO₂@QDs NPs were mono-dispersed in size and exhibited super-paramagnetic and highly fluorescent properties. Most of the Fe₃O₄@SiO₂@QDs NPs were naturally internalized into MDA-MB-231 human breast cancer cells, and the NP containing cells were successfully sorted by utilizing both fluorescence flow cytometry and a magnetic field. Results indicate that the Fe₃O₄@SiO₂@QDs NPs have great potential for multimodal cell separation.

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Introduction

Multifunctional nanoparticles (NPs) with both magnetic and luminescent properties have great potential for applications in biomedical research fields. NPs have been exploited in numerous studies including rapid bioseparation,^1–5 biolabeling,⁶ targeted drug delivery,⁷ and in magnetic resonance imaging (MRI) as dual modal nanoprobes.⁸ Multifunctional NPs with enhanced luminescent brightness and magnetic behavior can drastically improve the performance of the aforementioned bioapplications.⁹

Semiconductor nanocrystals, known as quantum dots (QDs), have been considered as promising fluorophores for biomedical research because of their highly fluorescent quantum yield (QY), good photostability, wide absorption spectra, and narrow emission bandwidths.^10–12 However, because QDs exhibit hydrophobic properties, surface modification is essential before use in bioapplications.^13–15 Among the surface modification methods, silica encapsulation has been widely employed because of its numerous advantages such as compatibility with aqueous environment, easy functionalization, and good chemical stability.^3,16–18 However, significant decrease in the QY of the employed QDs is inevitable during the encapsulation process. Recently, several efforts to facilitate the silica encapsulation of QDs within magnetic NPs have been reported, thereby enabling the preparation of multimodal NPs without significant loss in QY.^19–21 However, the fabricated NPs revealed poly-dispersed size distribution. For reliable application to biological systems, preparation of nanomaterials that are mono-dispersed, highly fluorescent, and magnetic is crucial.

Recently, we reported on the fabrication of QDs-embedded silica NPs (SiO₂@QDs NPs) for biomedical applications.²² The fabrication process yielded an efficient assembly of approximately 500 QDs on a silica NP (approximately 150 nm), without significant loss in QY. The particle size of the SiO₂@QDs NPs was uniform and easily tunable by controlling the size of the silica core.

In this study, we describe the preparation of QDs-embedded silica NPs, whose core has an iron oxide NP. These multifunctional NPs, denoted as Fe₃O₄@SiO₂@QDs NPs, are uniform in size and super-paramagnetic with intense fluorescence. Fe₃O₄@SiO₂@QDs NPs were characterized using two kinds of cell separation methods: fluorescence-activated cell sorting (FACS) and magnetic field induced cell separation. These assessments are performed to determine the potential of Fe₃O₄@SiO₂@QDs NPs as a tool for biomedical applications, particularly multimodal bioseparation.

Experimental details

Chemicals

All chemicals were used as received without further purification. A dispersion of Fe₃O₄ NPs (18 nm in average diameter, oleate-stabilized in toluene) was purchased from Ocean Nanotech, USA. Polyvinylpyrrolidone (PVP-10k), tetraethyl orthosilicate (TEOS), and 3-mercaptopropyltriethoxysilane (MPTS) were purchased from Sigma-Aldrich, Korea. Diethyl ether (anhydrous), dimethylformamide (DMF), dichloromethane (DCM), ethanol (EtOH), and ammonium hydroxide (28 wt% in water) were obtained from Daejung Parm, Korea. A dispersion of QDs (core–multishell structure composed of CdSe@CdS@ZnS, ∼4.5 nm in average diameter, oleate-stabilized in toluene) was purchased from Nanosquare, Korea.

Ligand exchange of oleate-stabilized Fe₃O₄ NPs with PVP

A 0.2 mL mixture of oleate-stabilized Fe₃O₄ NPs in toluene was transferred to a 10 mL vial and diluted with 5 mL of DMF–DCM (1 : 1, v/v). Then, 60 mg of PVP was added and refluxed overnight at 100 °C. After the reflux, the reaction mixture was added to diethyl ether (10 mL), and subsequently precipitated to obtain PVP-stabilized Fe₃O₄ NPs. The precipitate was centrifuged at 4500 rpm for 5 min and then transferred to 6.5 mL of EtOH to facilitate the formation of a stable dispersion of Fe₃O₄ NPs.

Silica coating on PVP-stabilized Fe₃O₄ NPs

The silica coating procedure on PVP-stabilized Fe₃O₄ NPs was based on a modified Stöber method.^23,24 A 0.28 mL solution of ammonium hydroxide (28 wt% in water) was added to the EtOH dispersion of PVP-stabilized Fe₃O₄ NPs (6.5 mL), followed by the addition of TEOS (6.5 μL). The reaction mixture was stirred overnight at 25 °C. The silica-coated Fe₃O₄ NPs were then centrifuged at 10 000 rpm for 1 h, washed repeatedly with EtOH, and re-dispersed in distilled water. A 4 mL solution of TEOS in EtOH (3 vol%) was added to water containing the silica-coated Fe₃O₄ NPs, and then stirred for one day. The resulting Fe₃O₄@SiO₂ NPs were centrifuged at 7000 rpm for 10 min and re-dispersed in EtOH.

Preparation of QDs-embedded Fe₃O₄@SiO₂ NPs

Thiol groups were introduced to the surface of Fe₃O₄@SiO₂ NPs (5 mL, 0.6 mg mL⁻¹ suspension in EtOH) by adding MPTS (25 μL) and ammonium hydroxide (25 μL, 28 wt% in water), and by stirring overnight at 25 °C. These thiol-functionalized Fe₃O₄@SiO₂ NPs were then washed three times with EtOH and re-dispersed in EtOH. The resulting NPs (1 mL, 10 mg mL⁻¹ in EtOH) were subsequently added to QDs containing DCM solution (4 mL, 1.75 mg mL⁻¹), and vigorously stirred for 3 min. Subsequently, MPTS (55 μL) and ammonium hydroxide (55 μL, 28 wt% in water) were added to the Fe₃O₄@SiO₂@QDs NPs mixture, and was shaken for 1 h at 25 °C. The resulting mixtures were then washed with EtOH, dispersed in EtOH (5 mL) containing TEOS (55 μL) and ammonium hydroxide (55 μL), and stirred overnight. The obtained Fe₃O₄@SiO₂@QDs NPs were washed three times with EtOH and subsequently dispersed in EtOH.

Characterization of the prepared NPs

Transmission electron microscopy (TEM) images were obtained with a JEOL JEM1010. The hydrodynamic size and the degree of dispersion of the NPs were determined using a Nanosight LM10. Ultraviolet-visible (UV-vis) absorption spectra were obtained with an Optigen 2120UV. Photoluminescence (PL) spectra were obtained with a Perkin-Elmer LS55. The QY of the prepared NPs was obtained with a JASCO FP-6500 equipped with an integrating sphere and a QY calculation program. The fluorescence decay of the prepared NPs was obtained by a time-correlated single photon counting (TCSPC) technique using a 405 nm diode laser at a repetition rate of 10 MHz. Field-dependent magnetization of the Fe₃O₄-based NPs was performed with a Quantum Design PPMS-14.

Confocal laser microscopy and flow cytometry analysis

For confocal microscopy analysis, MDA-MB-231 cells (1 × 10⁵) were seeded on clean coverslips in a 6-well plate, followed by incubation for 24 h at 37 °C. The cells were rinsed using phosphate buffered saline (PBS), treated with Fe₃O₄@SiO₂@QDs NPs (40 μg mL⁻¹ in PBS), and incubated for 1 h at 37 °C. The cells treated with Fe₃O₄@SiO₂@QDs NPs were fixed by treating with 4% paraformaldehyde solution (Affymetrix, USA) for 15 min. The cells were thoroughly washed three times with PBS and attached to the coverslip using a mounting solution containing 4′,6-diamidino-2-phenylindole (DAPI) solution (Vector Laboratories, USA). Confocal microscopy images were obtained using a Zeiss LSM 510. For magnetic separation of NP-treated cells, each of the two types of NPs, namely, non-magnetic SiO₂@red-QDs NPs and magnetic Fe₃O₄@SiO₂@green-QDs NPs (40 μg mL⁻¹ in PBS), were incubated with MDA-MB-231 cells at 37 °C. After 1 h, the NPs-treated cells were harvested and combined. A magnet was located on one side of the well plate, and the floating cells were incubated with the magnet at room temperature for 4 h. After an additional 24 h of incubation at 37 °C in the absence of the magnet, the cells were fixed and analyzed with confocal microscopy. Flow cytometric analysis (BD FACS Calibur-2, USA) was performed with a cell mixture composed of the same numbers of SiO₂@red-QDs NP-internalized and SiO₂@green-QDs NP-internalized cells. The mixture was analyzed by laser excitation at 488 nm, with 530/30 nm and 661/16 nm emission filters.

Results and discussion

Fe₃O₄@SiO₂@QDs NPs (Fig. 1) were prepared by first synthesizing Fe₃O₄@SiO₂ NPs using the method described previously.²⁴ Briefly, the Fe₃O₄@SiO₂ NPs were prepared by subjecting Fe₃O₄ NPs to silica encapsulation by a modified Stöber method.²³ First, amphiphilic PVP was used to replace oleic acid as the surface ligand of the Fe₃O₄ NPs (18 nm in average diameter) to obtain a hydrophilic surface. As shown in Fig. 2a and b, the size and shape of the Fe₃O₄ NPs remained unchanged during the ligand exchange process. The Fe₃O₄@SiO₂ NPs were formed after reacting with TEOS and ammonium hydroxide, as shown in the TEM image of Fig. 2c. The resulting NPs have a magnetic core and silica shell, exhibiting uniform average particle size (75 ± 2.6 nm) and hydrodynamic size (Fig. S1a†). The average size and standard deviation of the prepared NPs were determined by analyzing fifty randomly selected sample images from the obtained TEM micrographs.

Fig. 1 Illustration of the synthesis of Fe₃O₄@SiO₂@QDs NPs.

Fig. 2 TEM images of NPs at each step of the synthesis. (a) Oleate-coated Fe₃O₄ NPs, (b) PVP-coated Fe₃O₄ NPs, (c) Fe₃O₄@SiO₂ NPs, (d) Fe₃O₄@SiO₂@QDs NPs.

The Fe₃O₄@SiO₂ NPs were then functionalized with a thiol silicate monomer, MPTS, to facilitate the immobilization of the CdSe@CdS@ZnS QDs. The QDs immobilization and silica shell addition were performed using the same method described previously.²² First, controlled amounts of an EtOH solution containing the thiol-functionalized Fe₃O₄@SiO₂ NPs and a DCM solution containing the QDs were mixed. The thiol groups of the Fe₃O₄@SiO₂ NPs bound to the Zn atoms on the surface of CdSe@CdS@ZnS QDs, replacing the organic ligands.¹³ Subsequently, MPTS was added to the QDs-immobilized Fe₃O₄@SiO₂ NPs to replace the remaining organic ligands, and hydrolyzed into silanol groups, forming a thin silica-shell precursor on the surface of the NPs. The NPs were then encapsulated with a silica shell by TEOS. As shown in Fig. 2d, the size of Fe₃O₄@SiO₂@QDs NPs was ∼100 nm, which is slightly larger than that of the bare Fe₃O₄@SiO₂ NPs; this is because of the addition of QDs and a thin silica shell layer on the surface. Significant aggregation of NPs was not detected by dynamic light scattering analysis (Fig. S1b†), and the obtained NPs were uniform in size as determined by TEM analysis (97 ± 4.8 nm).

First, the fluorescence properties of the NPs were analyzed. The Fe₃O₄@SiO₂@red-QDs NPs and the Fe₃O₄@SiO₂@green-QDs NPs were both excited at the wavelength of 350 nm using a xenon lamp, and exhibited bright photoluminescence with emission maxima at 620 and 540 nm (Fig. 3a), respectively, which correspond to those observed in the case of the original single QDs (Fig. S2†). The prepared NPs exhibited overlapping UV absorption spectra of the Fe₃O₄@SiO₂ NPs and original single QDs with small knolls at 600 and 510 nm, indicating Stokes shifts of the red and green QDs. The original red QDs in the DCM solution had a QY of 86%. The QY of the Fe₃O₄@SiO₂@red-QDs NPs was determined to be 57%, which is slightly less than that (71%) measured for the bare SiO₂@red-QDs NPs. The QY of the original green QDs in the DCM solution was determined to be 70%, and the QYs of the SiO₂@green-QDs NPs and Fe₃O₄@SiO₂@green-QDs NPs were 63% and 40%, respectively. To clarify the QY difference between the Fe₃O₄@SiO₂@QDs NPs and SiO₂@QDs NPs, TCSPC detection was performed to clarify the QY difference between the Fe₃O₄@SiO₂@QDs NPs and SiO₂@QDs NPs. As shown in Fig. S3,† the Fe₃O₄@SiO₂@QDs NPs show a short fluorescence lifetime compared to the SiO₂@QDs NPs, indicating that the inserted Fe₃O₄ NPs could quench the QD fluorescence by electronic coupling and energy transfer.²⁵ The magnetic properties of the Fe₃O₄@SiO₂@QDs NPs were also measured to demonstrate that they can be employed for cell separation. The field-dependent magnetism of the Fe₃O₄@SiO₂@QDs NPs at 300 K exhibited super-paramagnetic characteristics (Fig. 3b), with a saturated magnetization of ∼1.25 emu g⁻¹. Furthermore, the NPs could be collected using a NdFeB magnet (Fig. 3c) and then clearly re-dispersed under gentle agitation. After agitation, the Fe₃O₄@SiO₂@QDs NPs were found to be well-dispersed in various hydrophilic solvents (Fig. 3d) owing to their hydrophilic silica surface.

Fig. 3 Various properties of Fe₃O₄@SiO₂@QDs NPs. (a) UV absorption (dashed line) and fluorescence emission (straight line) spectrum of red and green QDs-embedded Fe₃O₄@SiO₂@QDs NPs and Fe₃O₄@SiO₂ NPs (inset: photographs of red and green QDs-embedded Fe₃O₄@SiO₂@QDs NPs), (b) hysteresis loop of Fe₃O₄@SiO₂@QDs NPs, (c) NP separation using NdFeB magnet, (d) dispersed Fe₃O₄@SiO₂@QDs NPs in various solvents.

In vitro analysis was subsequently performed using both fluorescence and magnetic field to show that the NPs could be exploited for cell separation. The natural cell uptake of the prepared NPs was confirmed by incubating MDA-MB-231 human breast cancer cells with Fe₃O₄@SiO₂@QDs NPs for 1 h. Confocal microscopy images (Fig. 4a) confirmed that most of the Fe₃O₄@SiO₂@QDs NPs were accumulated in the cytoplasm of the cells by natural endocytosis. In order to verify the applicability of Fe₃O₄@SiO₂@QDs NPs in FACS experiments, cells containing red or green QDs-embedded NPs were then analyzed using flow cytometry. Each cell was excited by a 488 nm laser source. As shown in the scatter plots of Fig. 4b, the cells can be clearly distinguished from the 661/16 nm and 530/30 nm emission filters. These results indicate that cells containing Fe₃O₄@SiO₂@QDs NPs are suitable for use with flow cytometry.

Fig. 4 Cellular internalization assessment of Fe₃O₄@SiO₂@QDs NPs. (a) Fluorescence images of MDA-MB-231 cells that internalized Fe₃O₄@SiO₂@red-QDs NPs (upper panel) and the cells that internalized Fe₃O₄@SiO₂@green-QDs NPs (lower panel). (b) Scatter plot of the fluorescence-activated cell sorting (FACS) analysis of cells that had internalized either Fe₃O₄@SiO₂@red-QDs NPs or Fe₃O₄@SiO₂@green-QDs NPs.

Furthermore, the ability of the Fe₃O₄@SiO₂@QDs NPs to undergo magnetic separation was confirmed. Thus, cells that had internalized non-magnetic SiO₂@red-QDs NPs or Fe₃O₄@SiO₂@green-QDs NPs were harvested separately, mixed in equal amounts, and then placed on a 6-well plate near a NdFeB magnet for 4 h. The regions of the plate adjacent to and distant from the magnet were simultaneously excited by a 405 nm laser and examined using confocal microscopy. As shown in Fig. 5, the cells that internalized Fe₃O₄@SiO₂@green-QDs NPs were mostly observed in regions near the magnet, while the cells that internalized non-magnetic SiO₂@red-QDs NPs were located at the region distant from the magnet; i.e., most of the magnetized cells were accumulated close to the magnet. These results demonstrate the potential use of the Fe₃O₄@SiO₂@QDs NPs in magnetic field-assisted cell separation.

Fig. 5 Illustration of magnetic field-assisted cell separation using Fe₃O₄@SiO₂@QDs NPs and microscopic images of the separated cells containing either magnetic Fe₃O₄@SiO₂@green-QDs NPs or nonmagnetic SiO₂@red-QDs NPs. Sample plate regions (a) adjacent to and (b) distant from the magnet.

Conclusion

Multifunctional NPs of uniform size that have strong fluorescence and super-paramagnetic properties were fabricated by immobilizing QDs onto the surface of silica-coated iron oxide NPs. The applicability of Fe₃O₄@SiO₂@QDs NPs in flow cytometry and magnetic-assisted cell separation was demonstrated. Our results prove that Fe₃O₄@SiO₂@QDs NPs have a great potential for use in biomedical applications, particularly for multimodal cell separation.

Acknowledgements

The authors are very grateful to Prof. Seong Keun Kim and Jooyoun Kang for help in fluorescence lifetime analysis. This work was supported by the Israel-Korea Scientific Research Cooperation Program (Grant number 2012K1A3A1A31055183) through the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning, and the Korean Health Technology R&D Project (Grant number HI13C-1299-0100), funded by the Ministry of Health & Welfare, by the Ministry of Science, ICT & Future Planning (2014-A002-0065).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra03139g

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